# ent

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type: CELL_LINE
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  FreeStyle HEK 293 F cells Life Technologies using 293fectin transfection reagent according to manufacturer s instructions After transfection cells


id: cord-103940-a2cqw8kg
type: PROTEIN
entity:
  receptor hAPN 670 C Structural model of HCoV 229E S NTD Magenta RBD green SD1 cyan 671 SD2 D Dose dependent binding


id: cord-253438-k8iqv1jb
type: GGP
entity:
  SARS CoV 2 WHU01 GenBank accession no MN988668 1 SARS CoV BJ01 GenBank AY278488 2 Pangolin CoV National


id: cord-133453-23rfdkuw
type: PROTEIN
entity:
  R457K K458Q K458N S459F S459Y N460T I468F I468V I468T S469P T470N E471Q E471G E471D I472V A475V G476S S477G S477T S477I


id: cord-133453-23rfdkuw
type: DISEASE
entity:
  HIV 1 gp120 SARS CoV 2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor COVID 19 immunopathology and its implications for therapy Convalescent


id: cord-254735-8reu45yz
type: CHEMICAL
entity:
  N terminal peptide trimming Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1 Structure of aminopeptidase N from Escherichia coli


id: cord-256156-mywhe6w9
type: CHEMICAL
entity:
  GlcNAc N J o u r n a l P r e p r o o f 11 deacetylase N sulfotransferase that N deacetylates and N sulfates N acetylglucosamine


id: cord-256156-mywhe6w9
type: DISEASE
entity:
  herpes simplex virus 1 entry Nidovirus sialate O acetylesterases evolution and substrate specificity of coronaviral and toroviral receptor destroying enzymes The sweet spot defining virus sialic acid


id: cord-262043-66qle52a
type: CHEBI
entity:
  protein ion channel NetNGlyc 1 0 Server Center for Biological Sequence Analysis A review on the cleavage priming of the spike protein


id: cord-273893-3nd6ptrg
type: DNA
entity:
  JX869059 spike residues 367 606 SARS CoV RBD accession number NC_004718 spike residues 306 527 human CD26 accession


id: cord-273893-3nd6ptrg
type: PROTEIN
entity:
  human CD26 0 5 10 20 40 80 160 320 640 and 1 280 nM or human ACE2 0 10 20 40 80 160 320 640 and 1 280


id: cord-273893-3nd6ptrg
type: ORG
entity:
  World Health Organization Cumulative Number of Reported Probable Cases of Severe Acute Respiratory Syndrome SARS Dipeptidyl


id: cord-280941-ds6x0yym
type: PROTEIN
entity:
  recombinant NPs hRID WT RBD FR hRID 2 m RBD FR hRID 9 m RBD FR hRID WT RBD SSG FR and FR and MERS CoV RBD protein


id: cord-276493-hoaxv5e0
type: PROTEIN
entity:
  K417 V445 G446 Y449 Y453 L455 F456 Y473 A475 G476 E484 F486 N487 Y489 Q493 G496 Q489 T500 N501 G502 V503


id: cord-276493-hoaxv5e0
type: DNA
entity:
  N terminal β hairpin aa 31 50 extended nidovirus RdRpassociated nucleotidyl transferase domain NiRAN aa 117 250 interface domain aa 251 365 and


id: cord-308310-wtmjt3hf
type: GGP
entity:
  horses 9 10 11 To generate a COVID 19 vaccine candidate we therefore attempted to display the RBD domain


id: cord-278869-7zr1118b
type: DNA
entity:
  NTD and RBD RBM regions Fig 2C and Suppl 155 Table 1 The recombinant RBD induced high titer antibodies


id: cord-278869-7zr1118b
type: PROTEIN
entity:
  Sino Biologicals S1 S2 302 ectodomain 40589 V08B1 S1 40591 V08H RBD 40592 V08H or S2 40590 V08B Recombinant purified proteins


id: cord-262145-i29e3fge
type: DISEASE
entity:
  H7N9 769 influenza from naturally infected humans Optimisation of a micro neutralisation assay and its application in 771 antigenic characterisation of influenza viruses Influenza Other Respir Viruses 9


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type: CELL_LINE
entity:
  human non human primate porcine and bat de Wit et al 2013 Traditional small laboratory animals such as mice Coleman et al 2014 hamsters de Wit et al 2013 and ferrets Raj


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type: WORK_OF_ART
entity:
  Tracking Changes in SARS CoV 2 Spike Evidence that D614G Increases Infectivity of the COVID 19 Virus Potent Neutralizing Antibodies against SARS CoV 2 Identified by


id: cord-261877-4y37676n
type: DNA
entity:
  4 Å Y453 H34 L455 K31 F456 T27 D30 K31 Y473 T27 A475 S19 T27 G476 S19 Q24 F486 L79 M82 Y83 N487 Q24 Y83 Y489 T27 F28 K31 F490 K31 Q493 K31 H34 S494 H34 Y495 H34 G496 K353 Q498 Y41 T500 Y41 D355 R357 N501 K353 G502 K353 G354 Y505 K353 G354 A386 R393 PHENIX


id: cord-261877-4y37676n
type: PROTEIN
entity:
  membrane fusion Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion Structural


id: cord-310230-9wfb43gt
type: ORG
entity:
  Vivo Fitness of Severe Acute Respiratory Syndrome Coronavirus Disease and Diplomacy GISAID s Innovative Contribution to Global Health Structural Basis for Receptor Recognition


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type: WORK_OF_ART
entity:
  The SARS Coronavirus S Glycoprotein Receptor Binding Domain Fine Mapping and Functional Characterization Structure of the SARS CoV 2 Spike Receptor Binding Domain Bound


id: cord-308428-zw26usmh
type: DNA
entity:
  forward primers 1 ATATATGCTCTTCTTCTCAAGTCCAGCTGGTGG 2 ATATATGCTCTTCTTCTGGTGGTGGCGGTAGCGGCGGTGGCGGTAGTCAAGTCCAGCTGGTGG or 3


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type: WORK_OF_ART
entity:
  Molecular Diagnosis of a Novel Coronavirus 2019 nCoV Causing an Outbreak of Pneumonia Positive RT PCR Test Results in Patients Recovered From COVID 19


id: cord-259185-qg4jwbes
type: ORG
entity:
  Biochemistry Dominant Protein Electrochemistry Application in Medicine A Review Detection of Food Decay Products Using Functionalized One


id: cord-274280-x5s4l0pp
type: PROTEIN
entity:
  ACE2 receptor N 1490 data points f The error bar indicates s d of the mean value for a single interaction 0 200 pN The solid line


id: cord-274280-x5s4l0pp
type: GGP
entity:
  surface b Retraction part of four force distance curves showing either nonadhesive or specific adhesive curves c Box


id: cord-267001-csgmc155
type: PROTEIN
entity:
  labeled antibodies CD45 AF700 CD11b PE Cy5 CD11c BV711 Ly6C PerCP CD40 APC CCR7 PE CD80 BV650 CD86 BV421 Ly6G PE Cy7


id: cord-267001-csgmc155
type: ORG
entity:
  Adjuvant Enhances the Expansion of Memory B Cell Compartment Within the Draining Lymph Node Evaluation


id: cord-297747-kifqgskc
type: PROTEIN
entity:
  Y505 S19 Q24 T27 F28 D30 K31 H34 E35 D38 Y41 Q42 L45 M82 Y83 N330 K353 G354 D355 R357 K417 G446 Y449 Y453 L455 F456 A475 F486 N487 Y489 Q493 Y495 G496 Q498 T500


id: cord-297747-kifqgskc
type: CELL_LINE
entity:
  K353 G354 D355 R357 K417 G446 Y449 Y453 L455 F456 A475 F486 N487 Y489 Q493 Y495 G496 Q498 T500 N501 G502 Y505 Table


id: cord-297747-kifqgskc
type: CELL_LINE
entity:
  R426 Y436 Y440 Y442 L443 P462 D463 G464 L472 N473 Y475 N479 G482 Y484 T486 T487 G488 I489 Y491 Q24 T27 K31


id: cord-297747-kifqgskc
type: GGP
entity:
  bioRxiv DOI 10 1101 2020 03 24 005561 sha 5e31a311e160a1814381ec2660e59bd6e7065f47 doc_id 297747 cord_uid kifqgskc A novel coronavirus


id: cord-310636-y7n22ykt
type: GGP
entity:
  cytomegalovirus n 10 varicella zoster virus n 25 hepatitis B virus n 25 hepatitis C virus n 24 HIV n 37 syphilis n 16 Toxoplasma n 1


id: cord-310636-y7n22ykt
type: WORK_OF_ART
entity:
  the Treatment of Covid 19 Preliminary Report A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid 19 Replication Competent Vesicular Stomatitis Virus Vaccine Vector Protects


id: cord-310636-y7n22ykt
type: WORK_OF_ART
entity:
  Tracking Changes in SARS CoV 2 Spike Evidence that D614G Increases Infectivity of the COVID 19 Virus Phylogenetic


id: cord-281536-8y7yxcp4
type: CELL_LINE
entity:
  R426 Y53 T433 W226 N437 R162 Y440 D182 P470 D202 N479 D182 D480 R162 D480 S163 D480 N164 D480 R223 Y481 R223 Y484 Y102 T486 Y53 T487 Y53 T487 D99 G488 A33


id: cord-281536-8y7yxcp4
type: GGP
entity:
  RepoRtS 2020 10 16862 https doi org 10 1038 s41598 020 73820 8 www nature com scientificreports www nature com scientificreports correlation with site


id: cord-320238-qbjrlog1
type: PROTEIN
entity:
  HR2 containing region amino acid residues 1215 1287 Figure 1B Supplementary Figure S1 Chimeric nanoparticles


id: cord-326337-s0fp5z1q
type: WORK_OF_ART
entity:
  Tracking Changes in SARS CoV 2 Spike Evidence that D614G Increases 456 Infectivity of the COVID 19 Virus


id: cord-290290-wyx9ib7s
type: ORG
entity:
  the Shared Access Equipment Centre Industrial Biotechnology the Research Center of Biotechnology of the Russian Academy of Sciences


id: cord-291790-z5rwznmv
type: WORK_OF_ART
entity:
  N463 Glycosylation Site on V5 Loop of a Mutant gp120 Regulates the Sensitivity of 632 HIV 1 to Neutralizing Monoclonal Antibodies


id: cord-326282-uxn64olw
type: PROTEIN
entity:
  RCSB Protein Data Bank accessories 6VSB Down S1 S2 protomer S1 light cyan S2 dark blue and 15 6VYB 6M0J Up protomer S1 S2


id: cord-280939-d478p8u6
type: DNA
entity:
  AviTag biotinylation motif 29 Supplemental Figure 1 A woodchuck hepatitis virus posttranscriptional regulatory element WPRE


id: cord-280939-d478p8u6
type: ORG
entity:
  Structural Basis for Potent Neutralization of Betacoronaviruses by Single Domain Camelid Antibodies Clinical


id: cord-280939-d478p8u6
type: ORG
entity:
  Science Human IgG Neutralizing Monoclonal Antibodies Block the SARS CoV 2 Infection Evaluation of inactivation


id: cord-309898-sju15hev
type: DNA
entity:
  COVID 19 Virus COVID 19 and cardiovascular disease from basic mechanisms to clinical perspectives We acknowledge support from the MIT IBM AI lab ONR N000141612333 and N000141912375 AFOSR FATE MURI FA9550 15 1 0514 NIH U01


id: cord-287205-k64svq6n
type: PROTEIN
entity:
  N terminal asparagine residue reduced 408 hyperglycosylation thus allowing for easier purification of the antigen


id: cord-285900-3rr0j5tk
type: DISEASE
entity:
  HIV influenza virus and Ebola virus Cell lines HEK293T human embryonic kidney and Vero E6 monkey kidney


id: cord-329392-fufattj8
type: ORG
entity:
  SARS CoV 2 Specific Antibody Detection for Seroepidemiology A Multiplex Analysis Approach Accounting for Accurate Seroprevalence


id: cord-318444-sgm24q1i
type: PROTEIN
entity:
  GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRVFVGWHYFGQGTQVTVS Sb 2 QVQLVESGGGLVQAGGSLRLSCATSGFPVYQANMHWYRQAPGKEREWVAAIQSYGDGTHYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRAVYVGMHYFGQGTQVTVS Sb 3 QVQLVESGGGLVQAGGSLRLSCAASGFPVNYKTMWWYRQAPGKEREWVAAIWSYGHTTHYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVVWVGHNYEGQGTQVTVS Sb 4


id: cord-318444-sgm24q1i
type: PROTEIN
entity:
  QVQLVESGGGLVQAGGSLRLSCAASGFPVKNFEMEWYRKAPGKEREWVAAIQSGGVETYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCFVYVGRSYIGQGTQVTVS Sb 16


id: cord-318444-sgm24q1i
type: PROTEIN
entity:
  KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCIVWVGAQYHGQGTQVTVS Sb 17 QVQLVESGGGLVQAGGSLRLSCAASGFPVAGRNMWWYRQAPGKEREWVAAIYSSGTYTEYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCHVWVGSLYKGQGTQVTVS Sb 18


id: cord-318444-sgm24q1i
type: PROTEIN
entity:
  QVQLDESGGGLVQAGGSLRLSCAASGFPVYHAWMEWYRQAPGKEREWVAAITSSGRHTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVKDAGRVYNSYDYWGQGTQVTVS Sb 33


id: cord-318444-sgm24q1i
type: PROTEIN
entity:
  QVQLVESGGGLVQAGGSLRLSCAASGFPVYHAYMEWYRQAPGKEREWVAAIYSNGEHTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVKDSGSFNQAYDYWGQGTQVTVS Sb 38


id: cord-318444-sgm24q1i
type: PROTEIN
entity:
  QVQLVESGGGSVQAGGSLRLSCAASGSIGQIEYLGWFRQAPGKEREGVAALNTWTGRTYYADSVK GRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAARWGRTKPLNTYYYSYWGQGTPVTVS Sb 50


id: cord-318444-sgm24q1i
type: PROTEIN
entity:
  QVQLVESGGGSVQAGGSLRLSCAASGNISQIHYLGWFRQAPGKEREGVAALNTDYGYTYYADSVK GRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAAYYFGDDIPLWWEAYSYWGQGTQVTVS Sb 58


id: cord-318444-sgm24q1i
type: PROTEIN
entity:
  GRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAASYGAAHPLSIMRYYYWGQGTQVTVS Sb 65 QVQLVESGGGSVQAGGSLRLSCAASGTIAHIKYLGWFRQAPGKEREGVAALMTKWGQTYYADSVK GRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAASYGANFPLKASDYSYWGQGTQVTVS Sb 66 QVQLVESGGGSVQAGGSLRLSCAASGSIQAITYLGWFRQAPGKEREGVAALVTWNGQTYYADSVK GRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAADWGYDWPLWDEWYWYWGQGTQVTVS Sb 67 QVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREGVAALVTYSGNTYYADSVK GRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAATWGHSWPLYNDEYWYWGQGSQVTVS Sb 68


id: cord-318444-sgm24q1i
type: GGP
entity:
  bioRxiv DOI 10 1101 2020 04 16 045419 sha e2c783e2fa7ae3ddd7eb4666b63c051e52345cd0 doc_id 318444 cord_uid sgm24q1i


id: cord-318444-sgm24q1i
type: PERSON
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  QVQLVESGGGLVQAGGSLRLSCAASGFPVAHAWMEWYRQAPGKEREWVAAITSYGYKTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVKDTGTYRFYYDYWGQGTQVTVS


id: cord-303868-aes92l6s
type: PROTEIN
entity:
  residues 788 806 and heptad repeat 1 and 2 HR1 residues 920 970 HR2 residues 1163 1202 the transmembrane domain


id: cord-314676-ndke9agh
type: PROTEIN
entity:
  À20 85 2 74 kcal mol À26 89 3 31 kcal mol and À26 06 1 33 kcal mol respectively supplementary material Table 9


id: cord-314676-ndke9agh
type: GGP
entity:
  delta d viniferin myricitrin chrysanthemin myristicin taiwanhomoflavone A lactucopicrin 15 oxalate nympholide A


id: cord-314676-ndke9agh
type: CHEMICAL
entity:
  ethyl 1 3 2 4 dichlorobenzyl carbamoyl 1 ethyl 6 fluoro 4 oxo 1 4 dihydro 7 quinolinyl 4 piperazine carboxylate


id: cord-314676-ndke9agh
type: CHEMICAL
entity:
  ethyl 1 f3 2 4 dichlorobenzyl carbamoyl 1 ethyl 6 fluoro 4 oxo 1 4 dihydro 7 quinolinylg 4 piperazine carboxylate


id: cord-319571-fspmgg4s
type: ORG
entity:
  the Directorate General of Scientific Research and Technological Development of the Ministry of High Education and Scientific Research


id: cord-315437-h6xjudm0
type: CELL_LINE
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  Huh 7 cell lysates 5 Â 10 7 mL in 1 mL lysis buffer containing 0 3 N decyl D maltopyranoside phosphate


id: cord-315437-h6xjudm0
type: PROTEIN
entity:
  Ron A Fouchier Erasmus Medical Center Rotterdam The Netherlands followed by daily monitoring for the onset of clinical manifestations e g weight


id: cord-296187-nnv2e7gr
type: DNA
entity:
  EcoRI and BglII sites pVSV eGFP dG 31842 pMD2 G 12259 pCAG VSV P 64088 pCAG VSV L 64085 pCAG VSV N 64087


id: cord-292883-7hvq9qaj
type: DNA
entity:
  human alphacoronavirus 229E and betacoronavirus OC43 H1 influenza virus hemagglutinin and tetanus toxoid TTd


id: cord-309182-t9ywnshj
type: ORG
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  Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease COVID 19 Severe Acute Respiratory Syndrome Coronavirus 2 Specific Antibody Responses


id: cord-323967-2mo915u1
type: PROTEIN
entity:
  Streptomyces enzyme endo beta N acetylglucosaminidase H 726 Phaser 730 crystallographic software Features and development of Coot Towards 735 automated crystallographic structure


id: cord-323967-2mo915u1
type: DATE
entity:
  15045 15046 15047 15048 15049 15050 15051 15052 15053 15054 15055 15056 15057 15058 15059 15060 15061 15062 15063 15064


id: cord-323967-2mo915u1
type: CHEMICAL
entity:
  15047 15048 15049 15050 15051 15052 15053 15054 15055 15056 15057 15058 15059 15060 15061 15062 15063


id: cord-306438-db2rqz4d
type: DNA
entity:
  human coronavirus OC43 HCoV OC43 human coronavirus HKU1 HCoV HKU1 SARS CoV human coronavirus 229E HCoV 229E human coronavirus NL63 HCoV NL63 and human coronavirus NL63 HCoV NL63 1 2 3 4 5 6 Coronaviruses CoVs


id: cord-265697-bbvlowyo
type: PROTEIN
entity:
  N terminal truncations 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 3


id: cord-265697-bbvlowyo
type: GGP
entity:
  short 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 ACE2 isoforms


id: cord-265697-bbvlowyo
type: GGP
entity:
  ccg epfl ch cgi bin pwmtools 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67


id: cord-265697-bbvlowyo
type: CHEMICAL
entity:
  N terminal truncations 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 3 Results and discussions 3 1 Vertebrate ACE2 orthologs share an functional constraint


id: cord-265697-bbvlowyo
type: CHEMICAL
entity:
  N terminal truncations Figure 4B 4D This indicates that these short ACE2 isoforms in domestic animals have little chance to


id: cord-339093-mwxkvwaz
type: ORG
entity:
  EM Structural Basis for Potent Neutralization of Betacoronaviruses by Single Domain Camelid Antibodies Identification of Human Single Domain Antibodies


id: cord-335118-oa9jfots
type: GGP
entity:
  kinases c Kit and Abl Unbinding of nicotine from the acetylcholine binding protein steered molecular


id: cord-335118-oa9jfots
type: WORK_OF_ART
entity:
  Critical Differences Between the Binding Features of the Spike Proteins of SARS CoV 2 and SARS CoV Effect of mutation


id: cord-321918-9jwma2y6
type: GGP
entity:
  N 2hydroxypropyl 3 trimethylammonium chitosan chloride HTCC 10 and hydrophobically modified HTCC HM HTCC 11


id: cord-321918-9jwma2y6
type: CHEMICAL
entity:
  N cluster 1 central cluster 2 and Cterminal cluster 3 portion Figure 5 purple box Additionally helix α2 and loop 3 4 connecting


id: cord-332948-h297ukuu
type: ORG
entity:
  the Fusion Peptide Role of the Spike Glycoprotein of Human Middle East Respiratory Syndrome Coronavirus


id: cord-332948-h297ukuu
type: WORK_OF_ART
entity:
  Can We Rely on Computational Predictions To Correctly Identify Ligand Binding Sites on Novel Protein Drug Targets Assessment of Binding Site Prediction Methods and a Protocol for Validation of Predicted Binding Sites Identifying


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type: PROTEIN
entity:
  secondary antibody anti mouse Alexa Fluor 488 1 2000 Thermo Fisher Scientific A 21202 Hoechst 1 1000 Invitrogen and Ty1 AS635P 0 5


id: cord-333264-jdvb8px4
type: GGP
entity:
  Evilfit 12 13 14 The shown titration data were processed applying reference sensor subtraction and Savitzky Golay filter operations For ITC proteins


id: cord-338517-1mxcssjj
type: WORK_OF_ART
entity:
  Lambrecht BN Hammad H Lung Dendritic Cells in Respiratory Viral Infection and Asthma From Protection to Immunopathology Dendritic


id: cord-338517-1mxcssjj
type: WORK_OF_ART
entity:
  Neutralization of Virus Infectivity by Antibodies Old Problems in New Perspectives Neutralizing Antibodies against SARS CoV 2 and Other Human Coronaviruses MERS CoV


id: cord-338517-1mxcssjj
type: WORK_OF_ART
entity:
  Severe Acute Respiratory Syndrome Coronavirus 2 Specific Antibody Responses in Coronavirus Disease Longitudinal


id: cord-338517-1mxcssjj
type: ORG
entity:
  Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti


id: cord-338517-1mxcssjj
type: ORG
entity:
  The Immunology of Posttransplant CMV Infection Potential Effect of CMV Immunoglobulins on Distinct Components


id: cord-338517-1mxcssjj
type: WORK_OF_ART
entity:
  A Personalized Signature and Chronotherapy Based Platform for Improving the Efficacy of Sepsis Treatment


id: cord-328003-yovp8squ
type: WORK_OF_ART
entity:
  Structure Function and Antigenicity of the SARS CoV 2 Spike Glycoprotein SARS CoV 2 Vaccines Status Report Composition


id: cord-328003-yovp8squ
type: WORK_OF_ART
entity:
  Structural Modeling of SARS CoV 2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop


id: cord-328003-yovp8squ
type: WORK_OF_ART
entity:
  Tracking Changes in SARS CoV 2 Spike Evidence that D614G Increases Infectivity of the COVID 19 Virus


id: cord-356264-q0yqnlyl
type: WORK_OF_ART
entity:
  The Effect of Species Representation on the Detection of Positive Selection in Primate Gene Data Sets UCSF Chimera


id: cord-332855-u0amf1oh
type: FAC
entity:
  the Veterinary Pathologic Diagnostic Center Department of Pathobiology Faculty of Veterinary Medicine Utrecht University


id: cord-335316-x2t5h5gu
type: ORG
entity:
  aguesia anosmia fatigue myalgia headache co morbidities respiratory cardiovascular renal diabetes autoimmune


id: cord-342557-a7q8vp8m
type: ORG
entity:
  the de Novo and Consensus Based Prediction of Protein Structure Structural Basis of Receptor Recognition


id: cord-342557-a7q8vp8m
type: WORK_OF_ART
entity:
  Ewald Sums in Large Systems PRODIGY A Web Server for Predicting the Binding Affinity of Protein Protein Complexes A Molecular Modeling Approach to Identify Effective Antiviral Phytochemicals


id: cord-353748-y1a52z8e
type: PROTEIN
entity:
  hACE2 SARS CoV 2 hACE2 nisin A hACE2 nisin Z hACE2 nisin H hACE2 nisin Q hACE2 nisin U hACE2 nisin U2 hACE2nisin F


id: cord-353748-y1a52z8e
type: PROTEIN
entity:
  T13 K31 C19 K31 K12 K31 T8 K31 P9 K31 K12 E35 K22 D38 N20 E35 C26 D38 H27 D38 C28 K353 T23 K353 Fig 2


id: cord-353748-y1a52z8e
type: GGP
entity:
  hACE2 SARS CoV 2 hACE2 nisin A hACE2 nisin Z hACE2 nisin H hACE2 nisin Q hACE2 nisin U hACE2 nisin U2


id: cord-346670-34wfy52f
type: WORK_OF_ART
entity:
  A Bayesian View on Cryo EM Structure Determination Processing of Structurally Heterogeneous Cryo EM Data


id: cord-328189-jpkxjn6e
type: GGP
entity:
  bioRxiv DOI 10 1101 2020 03 10 986398 sha cb19a324508f3bf37f1c708f263d2e0d6224ad64 doc_id 328189 cord_uid jpkxjn6e


id: cord-353161-mtq6yh25
type: PROTEIN
entity:
  viral spike protein 10 11 12 13 These studies also identified key differences between the sequences of the receptor binding domains RBD


id: cord-329011-spiuqngp
type: GGP
entity:
  IFN α recombinant human IFN α1b IFN α2a remdesivir chloroquine favipiravir and lopinavir ritonavir Aluvia


id: cord-354868-pqn59ojj
type: CELL_LINE
entity:
  disulfide pairs Cys379 Cys432 and Cys336 Cys361 and 383 388 bridges Cys379 Cys432 and Cys 391 Cys525 Fig 4D


id: cord-347587-auook38y
type: PROTEIN
entity:
  HRP conjugated anti goat IgG 1 3 000 or HRP conjugated anti mouse IgG 1 5 000 antibody GE Healthcare


id: cord-347587-auook38y
type: CHEMICAL
entity:
  N terminal IgG heavy chain fragment ϳ700 bp including the VHH gene while the latter was used as the template


id: cord-347587-auook38y
type: CHEMICAL
entity:
  N terminal IgG heavy chain fragment ϳ700 bp using the forward primer VHH L F 5 GGTGGTCCTGGCTGC 3 and the reverse primer CH2 R 5 GGTACGTGCTGTTGAACT GTTCC 3 The VHH gene ϳ300 to 450 bp was further amplified using the above DNA fragment as the template


id: cord-347587-auook38y
type: CHEMICAL
entity:
  D F 5 TTTCTATTACTAGGCCCAGCCGGCCGAGTCTGGAGGRR GCTTGGTGCA 3 and the reverse primer VHH FR4 D R 5 AAACCGTTGGCCATAATGGCCTGAGGAGACGR TGACSTSGGTC


id: cord-343107-oj1re34k
type: PROTEIN
entity:
  MERS CoV spike glycoprotein date 2019 07 11 journal Nat Commun DOI 10 1038 s41467 019 10897 4 sha 1fb860b35fac03bc319a2d12de832b65ca392c55 doc_id 343107


id: cord-343107-oj1re34k
type: DNA
entity:
  Trp18 Asp20 Pro23 Asp24 Val26 Ser28 Glu188 Ser191 Asn222 Asn226 Lue234 Arg235 and Asn236 We first examined the effects of these NTD mutations


id: cord-343107-oj1re34k
type: ORG
entity:
  Dr Changfa Fan Division of Animal Model Research Institute for Laboratory Animal Resources National Institutes for Food and Drug Control


id: cord-339724-roj8ksvc
type: ORG
entity:
  the Committee on the Ethics of Animal Experiments of the Chinese Centre for Diseases Control and Prevention


id: cord-355728-wivk0bm0
type: PROTEIN
entity:
  HEK293T I 9 0x10 5 5 3x10 1 5 9x10 7 1 0x10 6 9 9x10 1 9 7x10 7 8 3x10 6 1 7x10 6 NP NP Nb3 II 1 8x10 6 1 1x10 1 6 1x10 8 NB NC 3 9x10 6 7 9x10 7 3 0x10 6 3 2x10 7 Nb6 I 2 7x10 5 5 6x10 2 2 1x10 7 2 1x10 6 8 7x10 2 4 1x10 8 3 7x10 7 4 9x10 8 2 0x10 6 3 5x10 7 3 3x10 6 7 2x10 7 Nb8 I 1 4x10 5 8 1x10 1 5 8x10 6 6 6x10 5 3 3x10 1 5 1x10 7 4 8x10 6 4 9x10 7 NP NP Nb11 I 1 2x10 6 1 6x10 1 1 4x10 7 3 2x10 6 2 4x10 1 7 6x10 8 5 4x10 7 1 2x10 7 2 4x10 6 5 4x10 7 NP Nb12 I 1 2x10 2 2 0x10 4 1 6x10 6 Biphasic Biphasic Biphasic 2 5x10 7 5 5x10 8 1 2x10 6 9 0x10 7 NP Nb15 I 1 7x10 5 2 3x10 1 1 3x10 6 6 0x10 5 2 2x10 1 3 6x10 7 2 2x10 6 2 5x10 7 6 7x10 6 3 6x10 6 NP Nb16 I 1 1x10 5 1 3x10 1 1 3x10 6 NP 9 5x10 7 1 1x10 7 NP NP Nb17 II 7 3x10 5 2 0x10 1 2 7x10 7 NB NC 7 6x10 6 1 0x10 6 NP Nb18 II 1 4x10 5 6 4x10 3 4 5x10 8 NB 5 2x10 5 1 5x10 5 NP NP Nb19 I 2 4x10 4 1 1x10 1 4 5x10 6 1 0x10 5 8 9x10 2 8 8x10 7 4 1x10 6 4 9x10 7 2 4x10 5 7 7x10 6 NP Nb24 I 9 3x10 5 2 7x10 1 2 9x10 7 2 4x10 6 3 5x10 1 1 5x10 7 7 5x10 7 1 0x10 7 NP NP ACE2 N A 2 7x10 5 1 2x10 2 4 4x10 8 NP NP NP 1 7x10 7 6 6x10 8 6 2x10 7 1 7x10 7 NP mNb6 I 1 0x10 6 4 5x10 4 4 5x10 10 1 1x10 6 6 4x10 4 5 6x10 10 1 3x10 9 4 1x10 10 6 3x10 9 1 6x10 9


id: cord-355728-wivk0bm0
type: GGP
entity:
  T I 9 0x10 5 5 3x10 1 5 9x10 7 1 0x10 6 9 9x10 1 9 7x10 7 8 3x10 6 1 7x10 6 NP NP Nb3 II 1 8x10 6 1 1x10 1 6 1x10 8 NB NC 3 9x10 6 7 9x10 7 3 0x10 6 3 2x10 7 Nb6 I 2 7x10 5 5 6x10 2 2 1x10 7 2 1x10 6 8 7x10 2 4 1x10 8 3 7x10 7 4 9x10 8 2 0x10 6 3 5x10 7 3 3x10 6 7 2x10 7


id: cord-355728-wivk0bm0
type: GGP
entity:
  Nb11 I 1 2x10 6 1 6x10 1 1 4x10 7 3 2x10 6 2 4x10 1 7 6x10 8 5 4x10 7 1 2x10 7 2 4x10 6 5 4x10 7 NP Nb12 I 1 2x10 2 2 0x10 4 1 6x10 6 Biphasic Biphasic Biphasic 2 5x10 7 5 5x10 8 1 2x10 6 9 0x10 7


id: cord-355728-wivk0bm0
type: GGP
entity:
  ACE2 N A 2 7x10 5 1 2x10 2 4 4x10 8 NP NP NP 1 7x10 7 6 6x10 8 6 2x10 7 1 7x10 7 NP mNb6 I 1 0x10 6 4 5x10 4 4 5x10 10 1 1x10 6 6 4x10 4 5 6x10 10 1 3x10 9 4 1x10 10 6 3x10 9 1 6x10 9


id: cord-333703-1ku3jc9s
type: ORG
entity:
  SOX Transcription Factors in Endothelial 1358 Differentiation and Endothelial Mesenchymal Transitions


id: cord-333703-1ku3jc9s
type: DISEASE
entity:
  olfactory and taste loss cardiac 75 dysfunction renal pathologies neurological damage muscle and joint pain gastrointestinal 76 symptoms clotting disorders


id: cord-344180-v8xs5ej8
type: ORG
entity:
  the Electrochemical Detection of Tuberculosis Biomarker Vapors Detection of Food Decay Products Using Functionalized One Dimensional


# bib

id: cord-015235-lv8mll28
author: Kim, Hyun
date: 2014-04-16
txt: ./txt/cord-015235-lv8mll28.txt
cache: ./cache/cord-015235-lv8mll28.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7097624/
doi: 10.1007/s13258-014-0186-9
summary:
  The receptor-binding domain (RBD) positioned in S1 can specifically bind to angiotensin-converting enzyme 2 (ACE2) on target cells, and ACE2 regulates the balance between vasoconstrictors and vasodilators within the heart and kidneys. Infection of SARS-CoV is initiated by binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) functional receptor expressed on target cells (Li et al. Our cellular enzyme-linked immunosorbent assay (ELISA) and competitive binding assay using a polyclonal ACE2 antibody indicated that our prepared recombinant RBD fusion protein binds to various tissues as well as NIH3T3 and HEK293 cells through ACE2. After washing with PBS, the RBD fusion protein was incubated for 1 h at room temperature to bind with the ACE2 molecules on the cell membranes. Next, we examined whether RBD binding was blocked by the ACE2 antibody in a Western blot using mouse tissue cell lysates with a pair of membranes.

title:
  Functional analysis of the receptor binding domain of SARS coronavirus S1 region and its monoclonal antibody

abstract:
  Severe acute respiratory syndrome (SARS) is caused by the SARS coronavirus (CoV). The spike protein of SARS-CoV consists of S1 and S2 domains, which are responsible for virus binding and fusion, respectively. The receptor-binding domain (RBD) positioned in S1 can specifically bind to angiotensin-converting enzyme 2 (ACE2) on target cells, and ACE2 regulates the balance between vasoconstrictors and vasodilators within the heart and kidneys. Here, a recombinant fusion protein containing 193-amino acid RBD (residues 318–510) and glutathione S-transferase were prepared for binding to target cells. Additionally, monoclonal RBD antibodies were prepared to confirm RBD binding to target cells through ACE2. We first confirmed that ACE2 was expressed in various mouse cells such as heart, lungs, spleen, liver, intestine, and kidneys using a commercial ACE2 polyclonal antibody. We also confirmed that the mouse fibroblast (NIH3T3) and human embryonic kidney cell lines (HEK293) expressed ACE2. We finally demonstrated that recombinant RBD bound to ACE2 on these cells using a cellular enzyme-linked immunosorbent assay and immunoassay. These results can be applied for future research to treat ACE2-related diseases and SARS.


id: cord-024319-isbqs7hg
author: Zeng, Xin
date: 2020-04-30
txt: ./txt/cord-024319-isbqs7hg.txt
cache: ./cache/cord-024319-isbqs7hg.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197610/
doi: 10.1093/abt/tbaa008
summary:
  title: Isolation of a human monoclonal antibody specific for the receptor binding domain of SARS-CoV-2 using a competitive phage biopanning strategy SARS-CoV-2 relies on its spike protein, in particular the receptor binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. It was proved to competitively block the binding of RBD to ACE2 and potently inhibit SARS-CoV-2 pseudovirus infection with IC(50) values of 12 nM. Several high-affinity antibodies targeting SARS-CoV-2 RBD and blocking its binding to ACE2 were isolated, and the top 1 lead exhibited a neutralization activity of SARS-CoV-2 pseudotyped VSV infection. These antibodies all compete with ACE2 to bind SARS-CoV RBD, but their epitopes only have limited overlaps of the Two SARS-CoV-2 RBD-specific antibodies selected from different strategies showed different neutralization activities.

title:
  Isolation of a human monoclonal antibody specific for the receptor binding domain of SARS-CoV-2 using a competitive phage biopanning strategy

abstract:
  The infection of the novel coronavirus SARS-CoV-2 has caused more than 200,000 deaths, but no vaccine or therapeutic monoclonal antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a “sandwich” complex. Only antibodies competed with ACE2 can bind to the free RBD-His in the supernatant and be subsequently separated by the Ni-NTA magnetic beads. Top 1 lead from the competitive biopanning of our synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 and potently inhibit SARS-CoV-2 pseudovirus infection with IC(50) values of 12 nM. Nevertheless, top 1 lead from the standard biopanning can only bind to RBD in vitro, but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.


id: cord-102920-z5q3wo7v
author: Sang, Eric R.
date: 2020-06-28
txt: ./txt/cord-102920-z5q3wo7v.txt
cache: ./cache/cord-102920-z5q3wo7v.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.06.27.174961
doi: 10.1101/2020.06.27.174961
summary:
  Previous reports using structural analysis of the viral spike protein (S) binding its cell receptor of angiotensin-converting enzyme 2 (ACE2), indicate a broad SARS-CoV2 susceptibility in wild and particularly domestic animals. In addition to showing a broad susceptibility potential across mammalian species based on structural analysis, our results also reveal that domestic animals including dogs, pigs, cattle and goats may evolve ACE2-related immunogenetic diversity to restrict SARS-CoV2 infections. Along with showing a broad susceptibility potential across mammalian species based on structural analysis [26] [27] [28] , our results further reveal that domestic animals including dogs, pigs, cattle and goats may evolve previously unexamined immunogenetic diversity to restrict SARS-CoV2 infections. In addition to structural analysis of simulated S-RBD-ACE2 interaction, we propose that several immunogenetic factors, including the evolution of S-binding-void ACE2 isoforms in some domestic animals, the species-specific IFN system, and epigenetic regulation of IFN-stimulated property of host ACE2 genes, contribute to the viral susceptibility and the development of COVID-19-like symptoms in certain animal species [15, 38, 39, 49] .

title:
  Integrate Structural Analysis, Isoform Diversity, and Interferon-Inductive Propensity of ACE2 to Refine SARS-CoV2 Susceptibility Prediction in Vertebrates

abstract:
  The current new coronavirus disease (COVID-19) has caused globally near 0.4/6 million confirmed deaths/infected cases across more than 200 countries. As the etiological coronavirus (a.k.a. SARS-CoV2) may putatively have a bat origin, our understanding about its intermediate reservoir between bats and humans, especially its tropism in wild and domestic animals, are mostly unknown. This constitutes major concerns in public health for the current pandemics and potential zoonosis. Previous reports using structural analysis of the viral spike protein (S) binding its cell receptor of angiotensin-converting enzyme 2 (ACE2), indicate a broad SARS-CoV2 susceptibility in wild and particularly domestic animals. Through integration of key immunogenetic factors, including the existence of S-binding-void ACE2 isoforms and the disparity of ACE2 expression upon early innate immune response, we further refine the SARS-CoV2 susceptibility prediction to fit recent experimental validation. In addition to showing a broad susceptibility potential across mammalian species based on structural analysis, our results also reveal that domestic animals including dogs, pigs, cattle and goats may evolve ACE2-related immunogenetic diversity to restrict SARS-CoV2 infections. Thus, we propose that domestic animals may be unlikely to play a role as amplifying hosts unless the virus has further species-specific adaptation. These findings may relieve relevant public concerns regarding COVID-19-like risk in domestic animals, highlight virus-host coevolution, and evoke disease intervention through targeting ACE2 molecular diversity and interferon optimization.


id: cord-103940-a2cqw8kg
author: Shi, Yuejun
date: 2020-06-09
txt: ./txt/cord-103940-a2cqw8kg.txt
cache: ./cache/cord-103940-a2cqw8kg.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.06.09.141580
doi: 10.1101/2020.06.09.141580
summary:
  Currently, structural studies have shown that Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) RBDs are in lying and standing state, respectively. In this study, 130 we selected SARS-CoV, SARS-CoV-2, and HCoV-229E as models, which adopt the 131 two RBD states, and evaluated and compared immune responses to the S trimers and 132 7 RBDs of these coronaviruses through immunological and bioinformatics approaches. 133 We also investigated the mechanism through which the HCoV-229E S trimer 134 produced effective nAbs. Finally, we provide possible vaccine strategies for alphaTo address this issue, we performed B-cell epitope predictions for the S trimers 152 and RBDs of alpha-CoV (HCoV-229E) and beta-CoVs (SARS-CoV and 153 SARS-CoV-2). Taken together, these results showed that the intact and stable S1 subunit of 240 HCoV-229E is a prerequisite for the production of effective nAbs. Furthermore, our experimental results show that RBD has a higher ability to bind 242 12 to the receptor hAPN (Fig. 4B) , which indicates that the characteristics of RBD itself 243 may lead to the generation of less neutralizing antibodies.

title:
  Insight into vaccine development for Alpha-coronaviruses based on structural and immunological analyses of spike proteins

abstract:
  Coronaviruses that infect humans belong to the Alpha-coronavirus (including HCoV-229E) and Beta-coronavirus (including SARS-CoV and SARS-CoV-2) genera. In particular, SARS-CoV-2 is currently a major threat to public health worldwide. However, no commercial vaccines against the coronaviruses that can infect humans are available. The spike (S) homotrimers bind to their receptors through the receptor-binding domain (RBD), which is believed to be a major target to block viral entry. In this study, we selected Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) as models. Their RBDs were observed to adopt two different conformational states (lying or standing). Then, structural and immunological analyses were used to explore differences in the immune response with RBDs among these coronaviruses. Our results showed that more RBD-specific antibodies were induced by the S trimer with the RBD in the “standing” state (SARS-CoV and SARS-CoV-2) than the S trimer with the RBD in the “lying” state (HCoV-229E), and the affinity between the RBD-specific antibodies and S trimer was also higher in the SARS-CoV and SARS-CoV-2. In addition, we found that the ability of the HCoV-229E RBD to induce neutralizing antibodies was much lower and the intact and stable S1 subunit was essential for producing efficient neutralizing antibodies against HCoV-229E. Importantly, our results reveal different vaccine strategies for coronaviruses, and S-trimer is better than RBD as a target for vaccine development in Alpha-coronavirus. Our findings will provide important implications for future development of coronavirus vaccines. Importance Outbreak of coronaviruses, especially SARS-CoV-2, poses a serious threat to global public health. Development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority. Coronavirus spike (S) protein is considered as a major target for vaccine development. Currently, structural studies have shown that Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) RBDs are in lying and standing state, respectively. Here, we tested the ability of S-trimer and RBD to induce neutralizing antibodies among these coronaviruses. Our results showed that Beta-CoVs RBDs are in a standing state, and their S proteins can induce more neutralizing antibodies targeting RBD. However, HCoV-229E RBD is in a lying state, and its S protein induces a low level of neutralizing antibody targeting RBD. Our results indicate that Alpha-coronavirus is more conducive to escape host immune recognition, and also provide novel ideas for the development of vaccines targeting S protein.


id: cord-133453-23rfdkuw
title: Prediction and mitigation of mutation threats to COVID-19 vaccines and antibody therapies
author: Chen, Jiahui
date: 2020-10-13
txt: ./txt/cord-133453-23rfdkuw.txt
cache: ./cache/cord-133453-23rfdkuw.txt
extension: .txt
mime: text/plain
url: https://arxiv.org/pdf/2010.06357v1.pdf
doi: nan
summary:
  By integrating genetics, biophysics, deep learning, and algebraic topology, we deduce that some of the mutations such as M153I, S254F, and S255F may weaken the binding of S protein and antibodies, and potentially disrupt the efficacy and reliability of antibody therapies and vaccines in the development. The vaccination mechanism is to stimulate the primary immune response of the human body, which will activate T cells and B cells to generate the antibodies and long-lived memory cells that prevent infectious diseases, which is one of the most effective and economical means for combating with COVID-19 at this stage. Notably, understanding how mutations have changed the SARS-CoV-2 structure, function, infectivity, activity, and virulence is of great importance for coming up with life-saving strategies in virus control, containment, prevention, and medication, especially in the antibodies and vaccines development. Next, we study the BFE changes ∆∆G induced by 39 mutations on the SARS-CoV-2 S protein RBD for the antibody Fab 2-4 (PDB: 6XEY) in Figure 6 .

abstract:
  Antibody therapeutics and vaccines are among our last resort to end the raging COVID-19 pandemic.They, however, are prone to over 1,800 mutations uncovered by a Mutation Tracker. It is urgent to understand how vaccines and antibodies in the development would be impacted by mutations. In this work, we first study the mechanism, frequency, and ratio of mutations on the spike (S) protein, which is the common target of most COVID-19 vaccines and antibody therapies. Additionally, we build a library of antibody structures and analyze their 2D and 3D characteristics. Moreover, we predict the mutation-induced binding free energy (BFE) changes for the complexes of S protein and antibodies or ACE2. By integrating genetics, biophysics, deep learning, and algebraic topology, we deduce that some of the mutations such as M153I, S254F, and S255F may weaken the binding of S protein and antibodies, and potentially disrupt the efficacy and reliability of antibody therapies and vaccines in the development. We provide a strategy to prioritize the selection of mutations for designing vaccines or antibody cocktails.


id: cord-183197-dxmto1tu
title: Tetracycline as an inhibitor to the coronavirus SARS-CoV-2
author: Zhao, Tom Y.
date: 2020-08-13
txt: ./txt/cord-183197-dxmto1tu.txt
cache: ./cache/cord-183197-dxmto1tu.txt
extension: .txt
mime: text/plain
url: https://arxiv.org/pdf/2008.06034v1.pdf
doi: nan
summary:
  Tetracycline appears to target viral residues that are usually involved in significant hydrogen bonding with ACE2; this inhibition of cellular infection complements the anti-inflammatory and cytokine suppressing capability of Tetracycline, and may further reduce the duration of ICU stays and mechanical ventilation induced by the coronavirus SARS-CoV-2. The amino acid residues of the RBD involved in hydrogen bonding with the Tetracycline molecule are Tyr 449, Asn 501, Gly 496, and Tyr 505 (Fig. 1) , which have been shown to be crucial for the SARS-CoV 2 RBD in binding to ACE2 for cellular access 8 . To verify this statement, steered molecular dynamics simulations were carried out to find the potential of mean force (PMF) along a singular dissociation pathway for the inhibited and uninhibited RBD-ACE2 complexes. The tetracycline class of antibiotics, including Tetracycline, Oxytetracycline, and Doxycycline may be helpful in the fight against the coronavirus SARS-CoV-2, due to its preferential association with the important residues in the viral receptor binding domain and the resulting strong inhibition of the RBD-ACE2 complex.

abstract:
  The coronavirus SARS-CoV-2 remains an extant threat against public health on a global scale. Cell infection begins when the spike protein of SARS-CoV-2 binds with the cell receptor, angiotensin-converting enzyme 2 (ACE2). Here, we address the role of Tetracycline as an inhibitor for the receptor-binding domain (RBD) of the spike protein. Targeted molecular investigation show that Tetracycline binds more favorably to the RBD (-9.40 kcal/mol) compared to Chloroquine (-6.31 kcal/mol) or Doxycycline (-8.08 kcal/mol) and inhibits attachment to ACE2 to a greater degree (binding efficiency of 2.98 $frac{text{kcal}}{text{mol}cdot text{nm}^2}$ for Tetracycline-RBD, 5.59 $frac{text{kcal}}{text{mol}cdot text{nm}^2}$ for Chloroquine-RBD, 5.16 $frac{text{kcal}}{text{mol}cdot text{nm}^2}$ for Doxycycline-RBD). Stronger Tetracycline inhibition is verified with nonequilibrium PMF calculations, for which the Tetracycline-RBD complex exhibits the lowest free energy profile along the dissociation pathway from ACE2. Tetracycline appears to target viral residues that are usually involved in significant hydrogen bonding with ACE2; this inhibition of cellular infection complements the anti-inflammatory and cytokine suppressing capability of Tetracycline, and may further reduce the duration of ICU stays and mechanical ventilation induced by the coronavirus SARS-CoV-2.


id: cord-252919-647zcjgu
title: Structure analysis of the receptor binding of 2019-nCoV
author: Chen, Yun
date: 2020-02-17
txt: ./txt/cord-252919-647zcjgu.txt
cache: ./cache/cord-252919-647zcjgu.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.bbrc.2020.02.071
doi: 10.1016/j.bbrc.2020.02.071
summary:
  We performed a structural analysis of the receptor binding domain (RBD) of spike glycoprotein responsible for entry of coronaviruses into host cells. Structural analysis suggests that ACE2 from these animals can potentially bind RBD of 2019-nCoV, making them all possible natural hosts for the virus. In this study, we analyzed the structure of spike glycoprotein RBD of 2019-nCoV and identified a unique feature that potentially allows a high affinity binding to ACE2 in human cells. There are 16 amino acid residues in SARS-CoV RBD that are directly in contact with ACE2, of which 8 are conserved in 2019-nCoV (see Fig. 1B ). Among the 16 amino acid residues in RBD of SARS that are in contact with ACE2, 14, 14, 7, and 8 are shared by SARSv, civet, bat, and 2019-nCoV, respectively (Fig. 1B) . Our study suggests unique structural features of the spike glycoprotein RBD of 2019-nCoV that confers potentially higher affinity binding for its receptor than found with SARS-CoV.

abstract:
  Abstract 2019-nCoV is a newly identified coronavirus with high similarity to SARS-CoV. We performed a structural analysis of the receptor binding domain (RBD) of spike glycoprotein responsible for entry of coronaviruses into host cells. The RBDs from the two viruses share 72% identity in amino acid sequences, and molecular simulation reveals highly similar ternary structures. However, 2019-nCoV has a distinct loop with flexible glycyl residues replacing rigid prolyl residues in SARS-CoV. Molecular modeling revealed that 2019-nCoV RBD has a stronger interaction with angiotensin converting enzyme 2 (ACE2). A unique phenylalanine F486 in the flexible loop likely plays a major role because its penetration into a deep hydrophobic pocket in ACE2. ACE2 is widely expressed with conserved primary structures throughout the animal kingdom from fish, amphibians, reptiles, birds, to mammals. Structural analysis suggests that ACE2 from these animals can potentially bind RBD of 2019-nCoV, making them all possible natural hosts for the virus. 2019-nCoV is thought to be transmitted through respiratory droplets. However, since ACE2 is predominantly expressed in intestines, testis, and kidney, fecal-oral and other routes of transmission are also possible. Finally, antibodies and small molecular inhibitors that can block the interaction of ACE2 with RBD should be developed to combat the virus.


id: cord-253438-k8iqv1jb
author: Li, Yujun
date: 2020-10-27
txt: ./txt/cord-253438-k8iqv1jb.txt
cache: ./cache/cord-253438-k8iqv1jb.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32847856/
doi: 10.1128/jvi.01283-20
summary:
  We found that ACE2 orthologs of a wide range of domestic and wild mammals, including camels, cattle, horses, goats, sheep, cats, rabbits, and pangolins, were able to support cell entry of SARS-CoV-2, suggesting that these species might be able to harbor and spread this virus. In this study, we found that ACE2 orthologs of a wide range of domestic and wild animals can support cell entry of SARS-CoV-2 and three related coronaviruses, providing insights into identifying animal hosts of these viruses. The RBD of Bat-CoV RaTG13 then showed a binding profile significantly different and narrower than the other three RBDs. Note that human ACE2 and ACE2 orthologs of some domestic animals, including camels, cattle, horses, goats, sheep, cats, and rabbits, support efficient binding to all the four tested RBDs, suggesting that these ACE2 orthologs might be generally functional for supporting cell entry of the four tested viruses.

title:
  SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

abstract:
  The ongoing coronavirus disease 2019 (COVID-19) pandemic has caused >20 million infections and >750,000 deaths. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, has been found closely related to the bat coronavirus strain RaTG13 (Bat-CoV RaTG13) and a recently identified pangolin coronavirus (Pangolin-CoV-2020). Here, we first investigated the ability of SARS-CoV-2 and three related coronaviruses to utilize animal orthologs of angiotensin-converting enzyme 2 (ACE2) for cell entry. We found that ACE2 orthologs of a wide range of domestic and wild mammals, including camels, cattle, horses, goats, sheep, cats, rabbits, and pangolins, were able to support cell entry of SARS-CoV-2, suggesting that these species might be able to harbor and spread this virus. In addition, the pangolin and bat coronaviruses, Pangolin-CoV-2020 and Bat-CoV RaTG13, were also found able to utilize human ACE2 and a number of animal-ACE2 orthologs for cell entry, indicating risks of spillover of these viruses into humans in the future. We then developed potently anticoronavirus ACE2-Ig proteins that are broadly effective against the four distinct coronaviruses. In particular, through truncating ACE2 at its residue 740 but not 615, introducing a D30E mutation, and adopting an antibody-like tetrameric-ACE2 configuration, we generated an ACE2-Ig variant that neutralizes SARS-CoV-2 at picomolar range. These data demonstrate that the improved ACE2-Ig variants developed in this study could potentially be developed to protect from SARS-CoV-2 and some other SARS-like viruses that might spillover into humans in the future. IMPORTANCE The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the currently uncontrolled coronavirus disease 2019 (COVID-19) pandemic. It is important to study the host range of SARS-CoV-2, because some domestic species might harbor the virus and transmit it back to humans. In addition, insight into the ability of SARS-CoV-2 and SARS-like viruses to utilize animal orthologs of the SARS-CoV-2 receptor ACE2 might provide structural insight into improving ACE2-based viral entry inhibitors. In this study, we found that ACE2 orthologs of a wide range of domestic and wild animals can support cell entry of SARS-CoV-2 and three related coronaviruses, providing insights into identifying animal hosts of these viruses. We also developed recombinant ACE2-Ig proteins that are able to potently block these viral infections, providing a promising approach to developing antiviral proteins broadly effective against these distinct coronaviruses.


id: cord-253447-4w6caxwu
author: Zeng, Xin
date: 2020-04-20
txt: ./txt/cord-253447-4w6caxwu.txt
cache: ./cache/cord-253447-4w6caxwu.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.04.19.049643
doi: 10.1101/2020.04.19.049643
summary:
  title: Blocking antibodies against SARS-CoV-2 RBD isolated from a phage display antibody library using a competitive biopanning strategy SARS-CoV-2 relies on its spike protein, in particular the receptor binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. It was proved to competitively block the binding of RBD to ACE2 protein, and potently inhibit SARS-CoV-2 pseudovirus infection of ACE2-overexpressing Hela cells with IC50 values of 12nM. Several high-affinity antibodies targeting SARS-CoV-2 RBD and blocking its binding to ACE2 were isolated, and the top 1 lead exhibited a neutralization activity of SARS-CoV-2 pseudotyped VSV infection. A high-affinity antibody against the target protein can be screened from a phage display antibody library using the standard biopanning process, but its binding epitopes are identified by some extra steps, such as epitope mapping and competitive ELISA.

title:
  Blocking antibodies against SARS-CoV-2 RBD isolated from a phage display antibody library using a competitive biopanning strategy

abstract:
  The infection of the novel coronavirus SARS-CoV-2 have caused more than 150,000 deaths, but no vaccine or specific therapeutic antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His recombinant protein was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His proteins were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a “sandwich” complex. Only antibodies competed with ACE2 for recognizing RBD at the same or similar epitopes can bind to the free RBD-His in the supernatant and be subsequently separated by the Ni-NTA magnetic beads. Top 1 lead from the competitive biopanning of a synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 protein, and potently inhibit SARS-CoV-2 pseudovirus infection of ACE2-overexpressing Hela cells with IC50 values of 12nM. Nevertheless, top 1 lead from the standard biopanning of Lib AB1, can only bind to RBD in vitro but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.


id: cord-254735-8reu45yz
author: Reguera, Juan
date: 2012-08-02
txt: ./txt/cord-254735-8reu45yz.txt
cache: ./cache/cord-254735-8reu45yz.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1371/journal.ppat.1002859
doi: 10.1371/journal.ppat.1002859
summary:
  Here we describe the crystal structures of the receptor-binding domains (RBDs) of two closely related CoV strains, transmissible gastroenteritis virus (TGEV) and porcine respiratory CoV (PRCV), in complex with their receptor, porcine APN (pAPN), or with a neutralizing antibody. The report uncovers a unique virus-receptor recognition mode that engages a glycan N-linked to the pAPN ectodomain, revealing structural determinants of the receptor-binding specificity in CoVs. Neutralizing antibodies target viral residues used for binding to the APN receptor and entry into host cells, showing that efficient CoV neutralization requires immune responses focused toward key receptor binding motifs in the virus envelope. The RBD tip, shown here as the pAPN-binding edge of the domain (Figure 3) , is the main S protein determinant of antigenic site A, recognized by the most effective neutralizing antibodies of TGEV and related CoV infections [25, 26] .

title:
  Structural Bases of Coronavirus Attachment to Host Aminopeptidase N and Its Inhibition by Neutralizing Antibodies

abstract:
  The coronaviruses (CoVs) are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10–20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN), a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S) mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs) of two closely related CoV strains, transmissible gastroenteritis virus (TGEV) and porcine respiratory CoV (PRCV), in complex with their receptor, porcine APN (pAPN), or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.


id: cord-256156-mywhe6w9
title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2
author: Clausen, Thomas Mandel
date: 2020-09-14
txt: ./txt/cord-256156-mywhe6w9.txt
cache: ./cache/cord-256156-mywhe6w9.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32970989/
doi: 10.1016/j.cell.2020.09.033
summary:
  We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain (RBD). Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. In this report, we show that the ectodomain of the SARS-CoV-2 spike (S) protein interacts with cell surface HS through the Receptor Binding Domain (RBD) in the S1 subunit. Adjacent to the ACE2 binding site and exposed in the RBD lies a group of positively-charged amino acid residues that represents a potential site that could interact with heparin or heparan sulfate ( Fig. 1A and Suppl. The SARS-CoV-2 spike protein depends on cellular heparan sulfate for cell binding. Heparin inhibits cellular invasion by SARS-CoV-2: structural dependence of the interaction of the surface protein (spike) S1 receptor binding domain with heparin

abstract:
  We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2 binding site. Both ACE2 and heparin can bind independently to spike protein in vitro and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.


id: cord-256572-sqz8yc7b
title: Neutralization of SARS-CoV-2 by destruction of the prefusion Spike
author: Huo, Jiandong
date: 2020-05-06
txt: ./txt/cord-256572-sqz8yc7b.txt
cache: ./cache/cord-256572-sqz8yc7b.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.05.05.079202
doi: 10.1101/2020.05.05.079202
summary:
  The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 Å of the Fab/RBD complex. Potent nanomolar affinity neutralising human monoclonal antibodies against the SARS-CoV RBD have been identified that attach at the ACE2 receptor binding site (including M396, CR3014 and 80R (Ter Meulen et al., 2006; Sui et al., 2004; Zhu et al., 2007) ). We determined the crystal structure of the SARS-CoV-2 RBD-CR3022 Fab complex (see Methods and Table S3 ) to investigate the relationship between the binding epitopes of ACE2 and CR3022. Full interpretation of the detailed interactions between CR3022 and the RBD was enabled by the second crystal form which diffracted to high resolution, 2.4 Å, and the structure of which was refined to give an R-work/R-free of 0.213/0.239 and good stereochemistry (Methods, Table S3, Figure S5 ).

abstract:
  There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment. Highlights CR3022 neutralises SARS-CoV-2 Neutralisation is by destroying the prefusion SPIKE conformation This antibody may have therapeutic potential alone or with one blocking receptor attachment


id: cord-258902-h0wrs01h
author: Liu, Xianglei
date: 2020-09-22
txt: ./txt/cord-258902-h0wrs01h.txt
cache: ./cache/cord-258902-h0wrs01h.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0264410X20312299?v=s5
doi: 10.1016/j.vaccine.2020.09.058
summary:
  title: Enhanced Elicitation of Potent Neutralizing Antibodies by the SARS-CoV-2 Spike Receptor Binding Domain Fc Fusion Protein in Mice The cell-cell fusion assay results correlated well with the virus neutralization potency and could be used for high-throughput screening of large panels of anti-SARS-CoV-2 antibodies and vaccines without the requirement of live virus infection in BSL3 containment. Based on its highly homology to SARS-CoV, SARS-CoV-2 RBD is corroborated to contain immune dominant epitopes capable of eliciting antibodies that can neutralize viral infection and block viral entry by competing hACE2 Pseudovirus neutralization assay was then performed by incubation of SARS-CoV-2 pseudovirus with serially diluted mice serum for 1h at 37 °C, followed by addition of the mixture into pre-seeded 293T-ACE2 cells. On day 0 (pre-immunization), day 13 and day 27, mouse sera were collected and analyzed for RBD binding, pseudovirus and live virus neutralization, and cell-cell fusion inhibition.

title:
  Enhanced Elicitation of Potent Neutralizing Antibodies by the SARS-CoV-2 Spike Receptor Binding Domain Fc Fusion Protein in Mice

abstract:
  The development of an effective vaccine against SARS-CoV-2 is urgently needed. We generated SARS-CoV-2 RBD-Fc fusion protein and evaluated its potency to elicit neutralizing antibody response in mice. RBD-Fc elicited a higher neutralizing antibodies titer than RBD as evaluated by a pseudovirus neutralization assay and a live virus based microneutralization assay. Furthermore, RBD-Fc immunized sera better inhibited cell-cell fusion, as evaluated by a quantitative cell-cell fusion assay. The cell-cell fusion assay results correlated well with the virus neutralization potency and could be used for high-throughput screening of large panels of anti-SARS-CoV-2 antibodies and vaccines without the requirement of live virus infection in BSL3 containment. Moreover, the anti-RBD sera did not enhance the pseudotyped SARS-CoV-2 infection of K562 cells. These results demonstrate that Fc fusion can significantly improve the humoral immune response to recombinant RBD immunogen, and suggest that RBD-Fc could serve as a useful component of effective vaccines against SARS-CoV-2.


id: cord-259185-qg4jwbes
title: Functionalized TiO2 nanotube-based Electrochemical Biosensor for Rapid Detection of SARS-CoV-2
author: Vadlamani, B. S.
date: 2020-09-09
txt: ./txt/cord-259185-qg4jwbes.txt
cache: ./cache/cord-259185-qg4jwbes.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.09.07.20190173
doi: 10.1101/2020.09.07.20190173
summary:
  In this work, we report the synthesis of a cheap yet highly sensitive cobalt-functionalized TiO2 nanotubes (Co-TNTs)-based electrochemical biosensor and its efficacy for rapid detection of spike glycoprotein of SARS-CoV-2 by examining S-RBD protein as the reference material. Our manuscript reports the synthesis of a cheap yet highly sensitive cobalt-functionalized TiO2 nanotubes (Co-TNTs)-based electrochemical biosensor for rapid detection of spike glycoprotein of SARS-CoV-2. . https://doi.org/10.1101/2020.09.07.20190173 doi: medRxiv preprint asymptomatic individuals are needed, which is feasible only after the development of a simple, portable and rapid point-of-use sensor for the detection of SARS-CoV-2. In the current work, we have determined the potential of Co-functionalized TiO2 nanotubes (Co-TNTs) for the electrochemical detection of S-RBD protein of SARS-CoV-2. Our data showed that cobalt functionalized TNTs could selectively detect the S-RBD protein of SARS-CoV-2 using the amperometry electrochemical technique in ~ 30 secs.

abstract:
  The coronavirus disease (COVID-19) is a newly emerging viral disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Rapid increase in the number of COVID-19 cases worldwide led the WHO declare pandemic within a few month after the first case of infection. Due to the lack of a prophylactic measure to control the virus infection and spread, early diagnosis and quarantining of infected as well as the asymptomatic individuals are necessary for the containment of this pandemic. However, the current methods for SARS-CoV-2 diagnosis are expensive and time consuming although some promising and inexpensive technologies are coming out for emergency use. In this work, we report the synthesis of a cheap yet highly sensitive cobalt-functionalized TiO2 nanotubes (Co-TNTs)-based electrochemical biosensor and its efficacy for rapid detection of spike glycoprotein of SARS-CoV-2 by examining S-RBD protein as the reference material. A simple, low-cost, and one-step electrochemical anodization route was used to synthesize TNTs, followed by an incipient wetting method for cobalt functionalization of the TNTs platform, which is connected to a potentiostat for data collection. The sensor specifically detected the S-RBD protein of SARS-CoV-2 even at very low concentration (range of 14 nM to 1400 nM). Additionally, our sensor showed linear response in the detection of viral protein with concentration. In summary, our Co-TNT sensor is highly effective in detecting SARS-CoV-2 S-RBD protein in approximately 30 seconds, which can be explored for developing a point of care diagnostics for rapid detection of SARS-CoV-2 in nasal secretions or saliva samples.


id: cord-260334-xo8ruswo
title: Antibody-mediated protection against MERS-CoV in the murine model()
author: New, R.R.C.
date: 2019-07-09
txt: ./txt/cord-260334-xo8ruswo.txt
cache: ./cache/cord-260334-xo8ruswo.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0264410X1930711X
doi: 10.1016/j.vaccine.2019.05.074
summary:
  Murine antisera with neutralising activity for the coronavirus causative of Middle East respiratory syndrome (MERS) were induced by immunisation of Balb/c mice with the receptor binding domain (RBD) of the viral Spike protein. To test the neutralising capacity of these antisera in vivo, susceptibility to MERS-CoV was induced in naive recipient Balb/c mice by the administration of an adenovirus vector expressing the human DPP4 receptor (Ad5-hDPP4) for MERS-CoV, prior to the passive transfer of the RBD-specific murine antisera to the transduced mice. The data gained indicate that this dual-route vaccination with novel formulations of the RBD-Fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of MERS-CoV. We have used this transduced mouse model to test the capacity of the antiserum derived from the dual route immunisation to neutralise MERS-CoV in vivo, by passive transfer prior to challenge with the EMC2012 strain and we have demonstrated a significant reduction in viral load in lung tissue in transduced mice.

abstract:
  Murine antisera with neutralising activity for the coronavirus causative of Middle East respiratory syndrome (MERS) were induced by immunisation of Balb/c mice with the receptor binding domain (RBD) of the viral Spike protein. The murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the MERS coronavirus (MERS-CoV). To test the neutralising capacity of these antisera in vivo, susceptibility to MERS-CoV was induced in naive recipient Balb/c mice by the administration of an adenovirus vector expressing the human DPP4 receptor (Ad5-hDPP4) for MERS-CoV, prior to the passive transfer of the RBD-specific murine antisera to the transduced mice. Subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the MERS-CoV resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. The murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of RBD with a human IgG Fc tag (RBD-Fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. The data gained indicate that this dual-route vaccination with novel formulations of the RBD-Fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of MERS-CoV.


id: cord-260406-qvc2fb0c
author: Chen, Long
date: 2020-10-26
url: https://doi.org/10.1002/smll.202004237
doi: 10.1002/smll.202004237
title:
  Severe Acute Respiratory Syndrome Coronavirus‐2 Spike Protein Nanogel as a Pro‐Antigen Strategy with Enhanced Protective Immune Responses

abstract:
  Prevention and intervention methods are urgently needed to curb the global pandemic of coronavirus disease‐19 caused by severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). Herein, a general pro‐antigen strategy for subunit vaccine development based on the reversibly formulated receptor binding domain of SARS‐CoV‐2 spike protein (S‐RBD) is reported. Since the poor lymph node targeting and uptake of S‐RBD by antigen‐presenting cells prevent effective immune responses, S‐RBD protein is formulated into a reversible nanogel (S‐RBD‐NG), which serves as a pro‐antigen with enhanced lymph node targeting and dendritic cell and macrophage accumulation. Synchronized release of S‐RBD monomers from the internalized S‐RBD‐NG pro‐antigen triggers more potent immune responses in vivo. In addition, by optimizing the adjuvant used, the potency of S‐RBD‐NG is further improved, which may provide a generally applicable, safer, and more effective strategy for subunit vaccine development against SARS‐CoV‐2 as well as other viruses.


id: cord-260412-yjr83ef6
title: Developing a low-cost and accessible COVID-19 vaccine for global health
author: Hotez, Peter J.
date: 2020-07-29
txt: ./txt/cord-260412-yjr83ef6.txt
cache: ./cache/cord-260412-yjr83ef6.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1371/journal.pntd.0008548
doi: 10.1371/journal.pntd.0008548
abstract: nan
summary:
  Our group is developing a two-pronged approach to advance recombinant protein-based vaccines to prevent COVID-19 caused by SARS-CoV-2 and other coronavirus infections. One vaccine is based on a yeast-derived (Pichia pastoris) recombinant protein comprised of the receptor-binding domain (RBD) of the SARS-CoV formulated on alum and referred to as the CoV RBD219-N1 Vaccine. In addition to their low cost and suitability for use in public immunization programs in lowand middle-income countries, we pursued RBD recombinant protein-based vaccines as a technology to maximize safety relative to other platforms, such as virus vectors that have previously been found to induce immune enhancement. Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate Potential for developing a SARS-CoV receptor-binding domain (RBD) recombinant protein as a heterologous human vaccine against coronavirus infectious disease (COVID)-19 Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Alum Induces Protective Immunity and Reduces Immune Enhancement


id: cord-261877-4y37676n
author: Xu, Cong
date: 2020-06-30
txt: ./txt/cord-261877-4y37676n.txt
cache: ./cache/cord-261877-4y37676n.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.06.30.177097
doi: 10.1101/2020.06.30.177097
summary:
  Recent cryoelectron microscopy (cryo-EM) studies on the stabilized ectodomain of SARS-CoV-2 S protein revealed a closed state of S trimer with three RBD domains in "down" conformation (Walls et al., 2020) , as well as an open state with one RBD in the "up" conformation, corresponding to the receptor-accessible state (Walls et al., 2020; Wrapp et al., 2020) . To gain a thorough picture on how the receptor ACE2 binding induces conformational dynamics of the SARS-CoV-2 S trimer and triggers transition towards the postfusion state, we determine the cryo-EM structure of SARS-CoV-2 S trimer in complex with human ACE2 PD domain to 3.8 Å resolution (termed SARS-CoV-2 S-ACE2, Figs. Based on the data, we put forward a mechanism of ACE2 binding-induced conformational transitions of SARS-CoV-2 S trimer from the tightly closed ground prefusion state transforming towards the postfusion state (Fig. 6) . Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding

title:
  Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

abstract:
  The recent outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid international spread pose a global health emergency. The trimeric spike (S) glycoprotein interacts with its receptor human ACE2 to mediate viral entry into host-cells. Here we present cryo-EM structures of an uncharacterized tightly closed SARS-CoV-2 S-trimer and the ACE2-bound-S-trimer at 2.7-Å and 3.8-Å-resolution, respectively. The tightly closed S-trimer with inactivated fusion peptide may represent the ground prefusion state. ACE2 binding to the up receptor-binding domain (RBD) within S-trimer triggers continuous swing-motions of ACE2-RBD, resulting in conformational dynamics of S1 subunits. Noteworthy, SARS-CoV-2 S-trimer appears much more sensitive to ACE2-receptor than SARS-CoV S-trimer in terms of receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and residue Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2, and provided structural basis of the spike D614G-mutation induced enhanced infectivity. Our findings offer a thorough picture on the mechanism of ACE2-induced conformational transitions of S-trimer from ground prefusion state towards postfusion state, thereby providing important information for development of vaccines and therapeutics aimed to block receptor binding.


id: cord-262043-66qle52a
author: Basit, Abdul
date: 2020-05-20
txt: ./txt/cord-262043-66qle52a.txt
cache: ./cache/cord-262043-66qle52a.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1080/07391102.2020.1768150
doi: 10.1080/07391102.2020.1768150
summary:
  Spike (S) glycoprotein is the structural protein of SARS-CoV-2 located on the envelope surface, involve in interaction with angiotensin converting enzyme 2 (ACE2), a cell surface receptor, followed by entry into the host cell. The protein-protein docking and molecular dynamic simulation showed that tACE2 has higher binding affinity for RBD and form more stabilized complex with RBD than the intact ACE2. We designed a truncated version (tACE2) of ACE2 receptor covering the binding residues and performed protein-protein docking and molecular dynamic simulations to analyze its binding affinity for RBD and complex stability. Based on the HADDOCK score and the docking RMSD value, the docked complexes of ACE2 and tACE2 with RBD were analyzed for binding affinity DG (kcal mol À1 ) and stability using protein binding energy prediction (PRODIGY) server (Xue et al., 2016) .

title:
  Truncated human angiotensin converting enzyme 2; a potential inhibitor of SARS-CoV-2 spike glycoprotein and potent COVID-19 therapeutic agent

abstract:
  The current pandemic of Covid-19 caused by SARS-CoV-2 is continued to spread globally and no potential drug or vaccine against it is available. Spike (S) glycoprotein is the structural protein of SARS-CoV-2 located on the envelope surface, involve in interaction with angiotensin converting enzyme 2 (ACE2), a cell surface receptor, followed by entry into the host cell. Thereby, blocking the S glycoprotein through potential inhibitor may interfere its interaction with ACE2 and impede its entry into the host cell. Here, we present a truncated version of human ACE2 (tACE2), comprising the N terminus region of the intact ACE2 from amino acid position 21-119, involved in binding with receptor binding domain (RBD) of SARS-CoV-2. We analyzed the in-silico potential of tACE2 to compete with intact ACE2 for binding with RBD. The protein-protein docking and molecular dynamic simulation showed that tACE2 has higher binding affinity for RBD and form more stabilized complex with RBD than the intact ACE2. Furthermore, prediction of tACE2 soluble expression in E. coli makes it a suitable candidate to be targeted for Covid-19 therapeutics. This is the first MD simulation based findings to provide a high affinity protein inhibitor for SARS-CoV-2 S glycoprotein, an important target for drug designing against this unprecedented challenge. Communicated by Ramaswamy H. Sarma


id: cord-262145-i29e3fge
title: Breadth and function of antibody response to acute SARS-CoV-2 infection in humans
author: Huang, Kuan-Ying A.
date: 2020-10-19
txt: ./txt/cord-262145-i29e3fge.txt
cache: ./cache/cord-262145-i29e3fge.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.08.28.267526
doi: 10.1101/2020.08.28.267526
summary:
  A subset of anti-spike (10 of 32) and over half of anti-nucleocapsid (19 of 35) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. The MAbs with 161 strong anti-RBD binding have a relatively long heavy chain CDR3 length (50% 162 binding concentration <0.5 µg/ml versus >0.5 µg/ml, p=0.03, two-tailed Mann-163 Whitney test; Supplemental Figure 3 The 32 anti-spike glycoprotein MAbs were systematically examined by plaque 173 reduction neutralisation (PRNT) assay for neutralisation of wild type SARS-CoV-2 174 virus (see methods; summarised in Table 1 ). Potent neutralising antibodies to the RBD of SARS-CoV-2 spike glycoprotein were 188 identified and we thus analyse the blockade of the ACE2-RBD interaction by anti-189 RBD antibodies in two assays ( Figure 3 , Table 1 The structure of VHH72-Fc bound to RBD is known (17) and its footprint on the 198 RBD does not overlap that of ACE2, so inhibition is thought to occur by steric 199 hindrance.

abstract:
  Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 13.0% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) and over half of anti-nucleocapsid (19 of 35) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-RBD, three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. At last, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.


id: cord-263042-qdmunb9l
author: Zhao, Yongkun
date: 2016-11-24
txt: ./txt/cord-263042-qdmunb9l.txt
cache: ./cache/cord-263042-qdmunb9l.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166354216303928
doi: 10.1016/j.antiviral.2016.11.016
summary:
  Passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of MERS-CoV infected mice. Our data show that horses immunized with MERS-CoV VLPs can serve as a primary source of protective F(ab'')(2) for potential use in the prophylactic or therapeutic treatment of exposed or infected patients. Several research groups have developed and produced anti-MERS patientderived or humanized monoclonal neutralizing antibodies in vitro that were able to protect MERS-CoV infected mice (Corti et al., 2015; Li et al., 2015; Zhao et al., 2014) . Prophylactic or therapeutic treatment of MERS-CoV infected mice with either IgG or F(ab'') 2 significantly decreased the virus load in their lungs. In both prophylactic and therapeutic settings, passive transfer of equine immune antibodies resulted in a 2e4 log reduction of virus titers in the lungs of MERS-CoV infected mice, and accelerated virus clearance in the serum treated group (Fig. 5A, B) .

title:
  Passive immunotherapy for Middle East Respiratory Syndrome coronavirus infection with equine immunoglobulin or immunoglobulin fragments in a mouse model

abstract:
  Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a coronavirus (CoV), MERS-CoV. With the continuing spread of MERS-CoV, prophylactic and therapeutic treatments are urgently needed. In this study, we prepared purified equine F(ab’)(2) from horses immunized with MERS-CoV virus-like particles (VLPs) expressing MERS-CoV S, M and E proteins. Both IgG and F(ab’)(2) efficiently neutralized MERS-CoV replication in tissue culture. Passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of MERS-CoV infected mice. Our data show that horses immunized with MERS-CoV VLPs can serve as a primary source of protective F(ab’)(2) for potential use in the prophylactic or therapeutic treatment of exposed or infected patients.


id: cord-263090-29n9tsk9
title: Dynamical asymmetry exposes 2019-nCoV prefusion spike
author: Roy, Susmita
date: 2020-04-21
txt: ./txt/cord-263090-29n9tsk9.txt
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extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.04.20.052290
doi: 10.1101/2020.04.20.052290
summary:
  In this study, a structural-topology based model Hamiltonian of C3 symmetric trimeric spike is developed to explore its complete conformational energy landscape using molecular dynamic simulations. B. Side and top views of the homo-trimeric structure of SARS-CoV-2 spike protein with one RBD of the S1 subunit head rotated in the up conformation. A number of Cryo-EM structures captured the ''up'' and ''down'' conformations of the RBD domain of spike proteins of other coronaviruses including SARS-CoV-2 where the S1 subunit undergoes a hinge-like conformational movement prerequisite for receptor binding (Fig. 2C) (7, 8, 10, 17) . Analysis of all the simulations yields the 2-D free energy landscape of the trimeric spike protein of SARS-CoV-2 ( Fig 3B) with its all possible conformations. This generates a homo-trimeric SARS-CoV-2 spike where this initial structure has important components in terms of intra and inter-chain contacts (interaction) leading to an ''S1-head-up'' and an ''S1-head-down'' conformation for each protomer.

abstract:
  The novel coronavirus (2019-nCoV) spike protein is a smart molecular machine that instigates the entry of coronavirus to the host cell causing the COVID-19 pandemic. In this study, a structural-topology based model Hamiltonian of C3 symmetric trimeric spike is developed to explore its complete conformational energy landscape using molecular dynamic simulations. The study finds 2019-nCoV to adopt a unique strategy by undertaking a dynamic conformational asymmetry induced by a few unique inter-chain interactions. This results in two prevalent asymmetric structures of spike where one or two spike heads lifted up undergoing a dynamic transition likely to enhance rapid recognition of the host-cell receptor turning on its high-infectivity. The crucial interactions identified in this study are anticipated to potentially affect the efficacy of therapeutic targets. One Sentence Summary Inter-chain-interaction driven rapid symmetry breaking strategy adopted by the prefusion trimeric spike protein likely to make 2019-nCoV highly infective.


id: cord-263167-es806qhz
author: Rogers, Thomas F.
date: 2020-06-15
txt: ./txt/cord-263167-es806qhz.txt
cache: ./cache/cord-263167-es806qhz.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1126/science.abc7520
doi: 10.1126/science.abc7520
summary:
  We showed that passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters, as revealed by maintained weight and low lung viral titers in treated animals. Donor plasma were tested for binding to recombinant SARS-CoV-2 and SARS-CoV-1 S and receptor binding domain (RBD) proteins, for binding to cell surface expressed spikes and for neutralization in both live replicating virus and pseudovirus assays (Fig. 2, B to D, and fig. The bulk-transformed ligation products for both the heavy chain and light chain were transfected and tested for binding to RBD and S protein, and for neutralization in the SARS-CoV-2 pseudovirus assay using HeLa-ACE2 target cells ( fig. To investigate the relationship between in vitro neutralization and protection in vivo against SARS-CoV-2, we selected two mAbs for passive transfer/challenge experiments in a Syrian hamster animal model based on a summary of the nAb data (table S3 and fig.

title:
  Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model

abstract:
  Countermeasures to prevent and treat COVID-19 are a global health priority. We enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen over 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. We showed that passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters, as revealed by maintained weight and low lung viral titers in treated animals. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.


id: cord-263481-w5ytp1q7
author: Lokman, Syed Mohammad
date: 2020-06-02
txt: ./txt/cord-263481-w5ytp1q7.txt
cache: ./cache/cord-263481-w5ytp1q7.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S1567134820302203?v=s5
doi: 10.1016/j.meegid.2020.104389
summary:
  MERS-CoV uses dipeptidyl peptidase-4 (DPP4) as entry receptor [11] whereas SARS-CoV and SARS-CoV-2 utilize ACE-2 (angiotensin converting enzyme-2) [12] , abundantly available in lung alveolar epithelial cells and enterocytes, suggesting S glycoprotein as a potential drug target to halt the entry of SARS-with remarkable properties like glutamine-rich 42 aa long exclusive molecular signature (DSQQTVGQQDGSEDNQTTTIQTIVEVQPQLEMELTPVVQTIE) in position 983-1024 of polyprotein 1ab (pp1ab) [16] , diversified receptor-binding domain (RBD), unique furin cleavage site (PRRAR↓SV) at S1/S2 boundary in S glycoprotein which could play roles in viral pathogenesis, diagnosis and treatment [17] . There is growing evidence that spike protein, a 1273 amino acid long glycoprotein having multiple domains, possibly plays a major role in SARS-CoV-2 pathogenesis. In this study, we have analyzed 320 genomic sequences of SARS-CoV-2 to identify mutations between the available genomes followed by the amino acid variations in the glycoprotein S to foresee their impact on the viral entry to host cell from structural biology viewpoint.

title:
  Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: A computational biology approach

abstract:
  The newly identified SARS-CoV-2 has now been reported from around 185 countries with more than a million confirmed human cases including more than 120,000 deaths. The genomes of SARS-COV-2 strains isolated from different parts of the world are now available and the unique features of constituent genes and proteins need to be explored to understand the biology of the virus. Spike glycoprotein is one of the major targets to be explored because of its role during the entry of coronaviruses into host cells. We analyzed 320 whole-genome sequences and 320 spike protein sequences of SARS-CoV-2 using multiple sequence alignment. In this study, 483 unique variations have been identified among the genomes of SARS-CoV-2 including 25 nonsynonymous mutations and one deletion in the spike (S) protein. Among the 26 variations detected, 12 variations were located at the N-terminal domain and 6 variations at the receptor-binding domain (RBD) which might alter the interaction of S protein with the host receptor angiotensin converting enzyme-2 (ACE2). Besides, 22 amino acid insertions were identified in the spike protein of SARS-CoV-2 in comparison with that of SARS-CoV. Phylogenetic analyses of spike protein revealed that Bat coronavirus have a close evolutionary relationship with circulating SARS-CoV-2. The genetic variation analysis data presented in this study can help a better understanding of SARS-CoV-2 pathogenesis. Based on results reported herein, potential inhibitors against S protein can be designed by considering these variations and their impact on protein structure.


id: cord-265697-bbvlowyo
author: Sang, Eric R.
date: 2020-08-31
url: https://www.ncbi.nlm.nih.gov/pubmed/32904785/
doi: 10.1016/j.heliyon.2020.e04818
title:
  Integrate structural analysis, isoform diversity, and interferon-inductive propensity of ACE2 to predict SARS-CoV2 susceptibility in vertebrates

abstract:
  The current new coronavirus disease (COVID-19) has caused globally near 0.4/6 million confirmed deaths/infected cases across more than 200 countries. As the etiological coronavirus (a.k.a. SARS-CoV2) may putatively have a bat origin, our understanding about its intermediate reservoir between bats and humans, especially its tropism in wild and domestic animals are mostly unknown. This constitutes major concerns in public health for the current pandemics and potential zoonosis. Previous reports using structural analysis of the viral spike protein (S) binding its cell receptor of angiotensin-converting enzyme 2 (ACE2), indicate a broad potential of SARS-CoV2 susceptibility in wild and particularly domestic animals. Through integration of key immunogenetic factors, including the existence of S-binding-void ACE2 isoforms and the disparity of ACE2 expression upon early innate immune response, we further refine the SARS-CoV2 susceptibility prediction to fit recent experimental validation. In addition to showing a broad susceptibility potential across mammalian species based on structural analysis, our results also reveal that domestic animals including dogs, pigs, cattle and goats may evolve ACE2-related immunogenetic diversity to restrict SARS-CoV2 infections. Thus, we propose that domestic animals may be unlikely to play a role as amplifying hosts unless the virus has further species-specific adaptation. Findings may relieve relevant public concerns regarding COVID-19-like risk in domestic animals, highlight virus-host coevolution, and evoke disease intervention through targeting ACE2 molecular diversity and interferon optimization.


id: cord-267001-csgmc155
author: George, Parakkal Jovvian
date: 2020-05-27
txt: ./txt/cord-267001-csgmc155.txt
cache: ./cache/cord-267001-csgmc155.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.3390/vaccines8020251
doi: 10.3390/vaccines8020251
summary:
  Moreover, immunization with rASP-1 co-administered with the alum-adjuvanted RBD vaccine in separate sites resulted in an enhanced frequency of TfH and GC B cells within the draining lymph nodes, both of which were positively associated with the titers of the neutralizing antibody response related to anti-MERS-CoV protective immunity. When the total IgG response to the MERS-RBD antigen was studied using the MERS-CoV S1 protein as the target protein, we found that although the RBD-specific total IgG antibody titers were ~8 times higher in mice that were vaccinated by co-administrating rASP-1 and the alum-adjuvanted RBD vaccine in separate sites (G6-142,525 end point titer), they were not significantly different from those elicited by immunization with RBD + rASP-1 + alum administered in a single inoculum (G5-18,149 end point titer), or with the alum-adjuvanted RBD vaccine (G4-53,104 end point titer; Figure S1A ).

title:
  The Potency of an Anti-MERS Coronavirus Subunit Vaccine Depends on a Unique Combinatorial Adjuvant Formulation

abstract:
  Vaccination is one of the most successful strategies to prevent human infectious diseases. Combinatorial adjuvants have gained increasing interest as they can stimulate multiple immune pathways and enhance the vaccine efficacy of subunit vaccines. We investigated the adjuvanticity of Aluminum (alum) in combination with rASP-1, a protein adjuvant, using the Middle East respiratory syndrome coronavirus MERS-CoV receptor-binding-domain (RBD) vaccine antigen. A highly enhanced anti-MERS-CoV neutralizing antibody response was induced when mice were immunized with rASP-1 and the alum-adjuvanted RBD vaccine in two separate injection sites as compared to mice immunized with RBD + rASP-1 + alum formulated into a single inoculum. The antibodies produced also significantly inhibited the binding of RBD to its cell-associated receptor. Moreover, immunization with rASP-1 co-administered with the alum-adjuvanted RBD vaccine in separate sites resulted in an enhanced frequency of TfH and GC B cells within the draining lymph nodes, both of which were positively associated with the titers of the neutralizing antibody response related to anti-MERS-CoV protective immunity. Our findings not only indicate that this unique combinatorial adjuvanted RBD vaccine regimen improved the immunogenicity of RBD, but also point to the importance of utilizing combinatorial adjuvants for the induction of synergistic protective immune responses.


id: cord-268144-maa8c4a4
author: Zhang, Yuan
date: 2007-02-17
txt: ./txt/cord-268144-maa8c4a4.txt
cache: ./cache/cord-268144-maa8c4a4.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S1476927107000266
doi: 10.1016/j.compbiolchem.2007.02.005
summary:
  The sequential determination of crystal structures of the SARS coronavirus spike receptor-binding domain (RBD) in complex with its cellular receptor or neutralizing antibody opened a door for the design and development of antiviral competitive inhibitors. As an envelope glycoprotein, the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV) plays a key role in the viral entry and neutralization (Bartlam et al., 2005; Denison, 2004; Lau and Peiris, 2005; Xu and Gao, 2004; Zhu, 2004) . Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine Receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies Structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association

title:
  Computational characterization and design of SARS coronavirus receptor recognition and antibody neutralization

abstract:
  The sequential determination of crystal structures of the SARS coronavirus spike receptor-binding domain (RBD) in complex with its cellular receptor or neutralizing antibody opened a door for the design and development of antiviral competitive inhibitors. Based on those complex structures, we conduct computational characterization and design of RBD-mediated receptor recognition and antibody neutralization. The comparisons between computational predictions and experimental evidences validate our structural bioinformatics protocols. And the calculations predict a number of single substitutions on RBD, receptor or antibody that could remarkably elevate the binding affinities of those complexes. It is reasonable to anticipate our structure-based computation-derived hypotheses could be informative to the future biochemical and immunological tests.


id: cord-268894-amfv3z2y
author: Nguyen-Contant, Phuong
date: 2020-07-21
url: https://doi.org/10.1101/2020.07.20.213298
doi: 10.1101/2020.07.20.213298
title:
  S protein-reactive IgG and memory B cell production after human SARS-CoV-2 infection includes broad reactivity to the S2 subunit

abstract:
  The high susceptibility of humans to SARS-CoV-2 infection, the cause of COVID-19, reflects the novelty of the virus and limited preexisting B cell immunity. IgG against the SARS-CoV-2 spike (S) protein, which carries the novel receptor binding domain (RBD), is absent or at low levels in unexposed individuals. To better understand the B cell response to SARS-CoV-2 infection, we asked whether virus-reactive memory B cells (MBCs) were present in unexposed subjects and whether MBC generation accompanied virus-specific IgG production in infected subjects. We analyzed sera and PBMCs from non-SARS-CoV-2-exposed healthy donors and COVID-19 convalescent subjects. Serum IgG levels specific for SARS-CoV-2 proteins (S, including the RBD and S2 subunit, and nucleocapsid [N]) and non-SARS-CoV-2 proteins were related to measurements of circulating IgG MBCs. Anti-RBD IgG was absent in unexposed subjects. Most unexposed subjects had anti-S2 IgG and a minority had anti-N IgG, but IgG MBCs with these specificities were not detected, perhaps reflecting low frequencies. Convalescent subjects had high levels of IgG against the RBD, S2, and N, together with large populations of RBD- and S2-reactive IgG MBCs. Notably, IgG titers against the S protein of the human coronavirus OC43 in convalescent subjects were higher than in unexposed subjects and correlated strongly with anti-S2 titers. Our findings indicate cross-reactive B cell responses against the S2 subunit that might enhance broad coronavirus protection. Importantly, our demonstration of MBC induction by SARS-CoV-2 infection suggests that a durable form of B cell immunity is maintained even if circulating antibody levels wane. IMPORTANCE Recent rapid worldwide spread of SARS-CoV-2 has established a pandemic of potentially serious disease in the highly susceptible human population. Key questions are whether humans have preexisting immune memory that provides some protection against SARS-CoV-2 and whether SARS-CoV-2 infection generates lasting immune protection against reinfection. Our analysis focused on pre- and post-infection IgG and IgG memory B cells (MBCs) reactive to SARS-CoV-2 proteins. Most importantly, we demonstrate that infection generates both IgG and IgG MBCs against the novel receptor binding domain and the conserved S2 subunit of the SARS-CoV-2 spike protein. Thus, even if antibody levels wane, long-lived MBCs remain to mediate rapid antibody production. Our study also suggests that SARS-CoV-2 infection strengthens preexisting broad coronavirus protection through S2-reactive antibody and MBC formation.


id: cord-273891-7w334xgt
title: Receptor binding and proteolysis do not induce large conformational changes in the SARS-CoV spike
author: Kirchdoerfer, Robert N.
date: 2018-03-31
txt: ./txt/cord-273891-7w334xgt.txt
cache: ./cache/cord-273891-7w334xgt.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/292672
doi: 10.1101/292672
summary:
  The viral spike glycoprotein (S) utilizes angiotensin-converting enzyme 2 (ACE2) as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. Subsequent studies of the highly pathogenic human coronavirus S proteins of SARS-64 CoV 15,22 and MERS-CoV 17,22 showed that these viral S1 RBD do indeed sample an ''up'' 65 conformation where the receptor-binding site is accessible. 70 To examine the hypothesized conformational transitions induced by proteolysis and 71 receptor binding, we used single-particle cryo-EM to determine structures of S in uncleaved, 72 S1/S2 cleaved and ACE2-bound states. Three-dimensional classification of the S1 RBD 73 positions and corresponding atomic protein models revealed that neither ACE2-binding nor 74 trypsin cleavage at the S1/S2 boundary induced substantial conformational changes in the CoV may use a distinct mechanism of FP2 membrane insertion. Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein 381 reveal a prerequisite conformational state for receptor binding

abstract:
  Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as a highly transmissible pathogenic human betacoronavirus. The viral spike glycoprotein (S) utilizes angiotensin-converting enzyme 2 (ACE2) as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. As SARS-CoV enters host cells, the viral S undergoes two proteolytic cleavages at S1/S2 and S2’ sites necessary for efficient membrane fusion. Here, we present a cryo-EM analysis of the trimeric SARS-CoV S interactions with ACE2 and of the trypsin-cleaved S. Surprisingly, neither binding to ACE2 nor cleavage by trypsin at the S1/S2 cleavage site impart large conformational changes within S or expose the secondary cleavage site, S2’. These observations suggest that S2’ cleavage does not occur in the S prefusion conformation and that additional triggers may be required.


id: cord-273893-3nd6ptrg
title: Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26
author: Lu, Guangwen
date: 2013-07-07
txt: ./txt/cord-273893-3nd6ptrg.txt
cache: ./cache/cord-273893-3nd6ptrg.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1038/nature12328
doi: 10.1038/nature12328
summary:
  Here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. Here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. Sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the MERS-CoV RBD core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition. Sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the MERS-CoV RBD core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition.

abstract:
  The newly emergent Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pulmonary disease in humans(1,2), representing the second example of a highly pathogenic coronavirus, the first being SARS-CoV(3). CD26 (also known as dipeptidyl peptidase 4, DPP4) was recently identified as the cellular receptor for MERS-CoV(4). The engagement of the MERS-CoV spike protein with CD26 mediates viral attachment to host cells and virus–cell fusion, thereby initiating infection. Here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. Furthermore, binding between the RBD and CD26 is measured using real-time surface plasmon resonance with a dissociation constant of 16.7 nM. The viral RBD is composed of a core subdomain homologous to that of the SARS-CoV spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades IV and V of the CD26 β-propeller. The atomic details at the interface between the two binding entities reveal a surprising protein–protein contact mediated mainly by hydrophilic residues. Sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the MERS-CoV RBD core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/nature12328) contains supplementary material, which is available to authorized users.


id: cord-274280-x5s4l0pp
title: Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor
author: Yang, Jinsung
date: 2020-09-11
url: https://www.ncbi.nlm.nih.gov/pubmed/32917884/
doi: 10.1038/s41467-020-18319-6
abstract:
  Study of the interactions established between the viral glycoproteins and their host receptors is of critical importance for a better understanding of virus entry into cells. The novel coronavirus SARS-CoV-2 entry into host cells is mediated by its spike glycoprotein (S-glycoprotein), and the angiotensin-converting enzyme 2 (ACE2) has been identified as a cellular receptor. Here, we use atomic force microscopy to investigate the mechanisms by which the S-glycoprotein binds to the ACE2 receptor. We demonstrate, both on model surfaces and on living cells, that the receptor binding domain (RBD) serves as the binding interface within the S-glycoprotein with the ACE2 receptor and extract the kinetic and thermodynamic properties of this binding pocket. Altogether, these results provide a picture of the established interaction on living cells. Finally, we test several binding inhibitor peptides targeting the virus early attachment stages, offering new perspectives in the treatment of the SARS-CoV-2 infection.


id: cord-274480-aywdmj6o
title: Identification of residues on human receptor DPP4 critical for MERS-CoV binding and entry
author: Song, Wenfei
date: 2014-10-21
txt: ./txt/cord-274480-aywdmj6o.txt
cache: ./cache/cord-274480-aywdmj6o.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S004268221400453X
doi: 10.1016/j.virol.2014.10.006
summary:
  Middle East respiratory syndrome coronavirus (MERS-CoV) infects host cells through binding the receptor binding domain (RBD) on its spike glycoprotein to human receptor dipeptidyl peptidase 4 (hDPP4). Previously, we have generated a panel of MERS-CoV mutant RBD proteins at the residues D539, Y499, D510, E513, L506, W553 and V555 to characterize their impacts on binding activity to hDPP4 and the entry efficiency into target cells. To study the impacts of the substitutions of the critical residues on hDPP4 described above on the interaction between MERS-CoV RBD and hDDP4, we determined the binding efficiency between these two proteins by employing SPR technique. To further study the importance of the critical residues on hDPP4 on viral entry, we measured the entry efficiency of pseudovirus into COS7 cells expressing the wide-type and mutant forms of hDPP4. These results are consistent with our findings and suggest these residues play an important role in RBD binding and viral entry, and determining the tropism to MERS-CoV infection.

abstract:
  Middle East respiratory syndrome coronavirus (MERS-CoV) infects host cells through binding the receptor binding domain (RBD) on its spike glycoprotein to human receptor dipeptidyl peptidase 4 (hDPP4). Here, we report identification of critical residues on hDPP4 for RBD binding and virus entry through analysis of a panel of hDPP4 mutants. Based on the RBD–hDPP4 crystal structure we reported, the mutated residues were located at the interface between RBD and hDPP4, which potentially changed the polarity, hydrophobic or hydrophilic properties of hDPP4, thereby interfering or disrupting their interaction with RBD. Using surface plasmon resonance (SPR) binding analysis and pseudovirus infection assay, we showed that several residues in hDPP4–RBD binding interface were important on hDPP4–RBD binding and viral entry. These results provide atomic insights into the features of interactions between hDPP4 and MERS-CoV RBD, and also provide potential explanation for cellular and species tropism of MERS-CoV infection.


id: cord-275185-9br8lwma
author: Zeng, Hao
date: 2020-10-06
txt: ./txt/cord-275185-9br8lwma.txt
cache: ./cache/cord-275185-9br8lwma.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1038/s41392-020-00329-x
doi: 10.1038/s41392-020-00329-x
summary:
  Following CP transfusion, six out of eight patients showed improved oxygen support status; chest CT indicated varying degrees of absorption of pulmonary lesions in six patients within 8 days; the viral load was decreased to a negative level in five patients who had the previous viremia; other laboratory parameters also tended to improve, including increased lymphocyte counts, decreased C-reactive protein, procalcitonin, and indicators for liver function. Herein, we performed a retrospective observational study involving eight critical or severe patients with COVID-19 from four designated hospitals in the southwest region of China, aiming to explore the potential efficacy and safety of CP therapy, and to provide more evidence for the quality control of donated plasma and reasonable clinical application of CP transfusion. 23 Assessing the effects of neutralizing activity of CP on the patients'' clinical efficacy, we found that patients treated by CP with high NAT50 (>1:640) had more obvious improvement than patients receiving low NAT50 value (≤1:640) of CP, including shorter negative conservation time of viral RNA, and higher increment of IgG level after CP transfusion.

title:
  The efficacy assessment of convalescent plasma therapy for COVID-19 patients: a multi-center case series

abstract:
  Convalescent plasma (CP) transfusion has been indicated as a promising therapy in the treatment for other emerging viral infections. However, the quality control of CP and individual variation in patients in different studies make it rather difficult to evaluate the efficacy and risk of CP therapy for coronavirus disease 2019 (COVID-19). We aimed to explore the potential efficacy of CP therapy, and to assess the possible factors associated with its efficacy. We enrolled eight critical or severe COVID-19 patients from four centers. Each patient was transfused with 200–400 mL of CP from seven recovered donors. The primary indicators for clinical efficacy assessment were the changes of clinical symptoms, laboratory parameters, and radiological image after CP transfusion. CP donors had a wide range of antibody levels measured by serology tests which were to some degree correlated with the neutralizing antibody (NAb) level. No adverse events were observed during and after CP transfusion. Following CP transfusion, six out of eight patients showed improved oxygen support status; chest CT indicated varying degrees of absorption of pulmonary lesions in six patients within 8 days; the viral load was decreased to a negative level in five patients who had the previous viremia; other laboratory parameters also tended to improve, including increased lymphocyte counts, decreased C-reactive protein, procalcitonin, and indicators for liver function. The clinical efficacy might be associated with CP transfusion time, transfused dose, and the NAb levels of CP. This study indicated that CP might be a potential therapy for severe patients with COVID-19.


id: cord-276493-hoaxv5e0
title: Therapeutic Strategies Against COVID-19 and Structural Characterization of SARS-CoV-2: A Review
author: Jeong, Gi Uk
date: 2020-07-14
txt: ./txt/cord-276493-hoaxv5e0.txt
cache: ./cache/cord-276493-hoaxv5e0.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.3389/fmicb.2020.01723
doi: 10.3389/fmicb.2020.01723
summary:
  With increasing structural data of key proteins in both SARS-CoV-2 and the host, such as the spike glycoprotein (S), the main protease (M pro ), RNA-dependent RNA polymerase (RdRp), and human angiotensin-converting enzyme 2 (hACE2), the structure-based design of new drugs has emerged as the most promising antiviral strategy. Several structure-based drug discovery studies have investigated the interaction of inhibitors in the substrate-binding pockets of SARS-CoV-2 M pro ( Figure 3C ) (Dai et al., 2020; Jin et al., 2020; Zhang et al., 2020b) . Because most inhibitors occupy the substrate binding pocket of SARS-CoV-2 FIGURE 4 | CryoEM structure of RdRp in complex with cofactors (nsp7 and nsp8), RNA template, and remdesivir. In addition, we provided structural insights into the mechanism of action of well-characterized drugs targeting the interaction between hACE2 and the spike protein of SARS-CoV-2 for viral entry, as well as M pro and RdRp for viral replication.

abstract:
  The novel coronavirus, SARS-CoV-2, or 2019-nCoV, which originated in Wuhan, Hubei province, China in December 2019, is a grave threat to public health worldwide. A total of 3,672,238 confirmed cases of coronavirus disease 2019 (COVID-19) and 254,045 deaths were reported globally up to May 7, 2020. However, approved antiviral agents for the treatment of patients with COVID-19 remain unavailable. Drug repurposing of approved antivirals against other viruses such as HIV or Ebola virus is one of the most practical strategies to develop effective antiviral agents against SARS-CoV-2. A combination of repurposed drugs can improve the efficacy of treatment, and structure-based drug design can be employed to specifically target SARS-CoV-2. This review discusses therapeutic strategies using promising antiviral agents against SARS-CoV-2. In addition, structural characterization of potentially therapeutic viral or host cellular targets associated with COVID-19 have been discussed to refine structure-based drug design strategies.


id: cord-276833-haci44cy
author: Kim, Ju
date: 2019-05-18
txt: ./txt/cord-276833-haci44cy.txt
cache: ./cache/cord-276833-haci44cy.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.imbio.2019.05.004
doi: 10.1016/j.imbio.2019.05.004
summary:
  We analyzed the mechanisms by which HBD 2 primes innate antiviral immunity and polarized activation of macrophage-like THP-1 cells using the receptor-binding domain (RBD) of Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein (S RBD) as a model antigen. We previously reported that HBD 2 promotes an antiviral innate immune response in macrophage-like THP-1 cells and elicits an enhanced Ag-specific and virus-neutralizing antibody (Ab) response in vivo using the receptor binding domain (RBD) of MERS-CoV spike protein (S RBD) as a model Ag (Kim et al., 2018) . In this study, we investigated the mechanism by which HBD 2 enhances the type I IFN immune response in THP-1 cells by modulating Nod2 signaling pathways using HBD 2-conjugated S RBD of MERS-CoV. HBD 2-conjugated Ag stimulates the Nod2 signaling procedure, which leads to type I IFN production in macrophage-like THP-1 cells MERS-CoV infection inhibits the production of IFN-α/β and the host antiviral immune response .

title:
  Human β-defensin 2 is involved in CCR2-mediated Nod2 signal transduction, leading to activation of the innate immune response in macrophages

abstract:
  Beta-defensins contribute to host innate defense against various pathogens, including viruses, although the details of their roles in innate immune cells are unclear. We previously reported that human β-defensin 2 (HBD 2) activates primary innate immunity against viral infection and suggested that it plays a role in the induction of the adaptive immune response. We analyzed the mechanisms by which HBD 2 primes innate antiviral immunity and polarized activation of macrophage-like THP-1 cells using the receptor-binding domain (RBD) of Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein (S RBD) as a model antigen. The expression of nucleotide-binding oligomerization domain containing 2 (Nod2), type I interferons, (IFNs), and proinflammatory mediators was enhanced in S RBD-HBD 2-treated THP-1 cells. S RBD-HBD 2 treatment also enhanced phosphorylation and activation of receptor-interacting serine/threonine-protein kinase 2 and IFN regulatory factor 3 compared to S RBD alone. Finally, HBD 2-conjugated S RBD interacted with C-C chemokine receptor 2 (CCR2), and Nod2 was involved in HBD 2-mediated CCR2 signaling, which was associated with the activation and M1 polarization of THP-1 cells. Therefore, HBD 2 promotes CCR2-mediated Nod2 signaling, which induces production of type I IFNs and an inflammatory response, and enhances primary innate immunity leading to an effective adaptive immune response to HBD 2-conjugated antigen.


id: cord-278869-7zr1118b
title: Antibody repertoire induced by SARS-CoV-2 spike protein immunogens
author: Ravichandran, Supriya
date: 2020-05-13
txt: ./txt/cord-278869-7zr1118b.txt
cache: ./cache/cord-278869-7zr1118b.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.05.12.091918
doi: 10.1101/2020.05.12.091918
summary:
  To better understand antibody response induced by spike protein-based vaccines, we immunized rabbits with various SARS-CoV-2 spike protein antigens: S-ectodomain (S1+S2) (aa 16-1213), which lacks the cytoplasmic and transmembrane domains (CT-TM), the S1 domain (aa 16-685), the receptor-binding domain (RBD) (aa 319-541), and the S2 domain (aa 686-1213 as control). The spike ectodomain (S1+S2) generated antibodies that predominantly bound to S1+S2 108 6 (black bar), followed by the S1 protein (blue bar), and 3-fold lower antibody binding to the RBD 109 and the S2 domain (red and green bars, respectively) (Fig. 1D ). Antibody off-rate constants, which describe the fraction of antigen-antibody complexes 119 that decay per second, were determined directly from the serum sample interaction with SARS-120 CoV-2 spike ectodomain (S1+S2), S1, S2, and RBD using SPR in the dissociation phase only for 121 sensorgrams with Max RU in the range of 20-100 RU (Suppl.

abstract:
  Multiple vaccine candidates against SARS-CoV-2 based on viral spike protein are under development. However, there is limited information on the quality of antibody response generated following vaccination by these vaccine modalities. To better understand antibody response induced by spike protein-based vaccines, we immunized rabbits with various SARS-CoV-2 spike protein antigens: S-ectodomain (S1+S2) (aa 16-1213), which lacks the cytoplasmic and transmembrane domains (CT-TM), the S1 domain (aa 16-685), the receptor-binding domain (RBD) (aa 319-541), and the S2 domain (aa 686-1213 as control). Antibody response was analyzed by ELISA, Surface Plasmon Resonance (SPR) against different Spike proteins in native conformation, and a pseudovirion neutralization assay to measure the quality and function of the antibodies elicited by the different Spike antigens. All three antigens (S1+S2 ectodomain, S1 domain, and RBD) generated strong neutralizing antibodies against SARS-CoV-2. Vaccination induced antibody repertoire was analyzed by SARS-CoV-2 spike Genome Fragment Phage Display Libraries (SARS-CoV-2 GFPDL), which identified immunodominant epitopes in the S1, S1-RBD and S2 domains. Furthermore, these analyses demonstrated that surprisingly the RBD immunogen elicited a higher antibody titer with 5-fold higher affinity antibodies to native spike antigens compared with other spike antigens. These findings may help guide rational vaccine design and facilitate development and evaluation of effective therapeutics and vaccines against COVID-19 disease. One Sentence Summary SARS-CoV-2 Spike induced immune response


id: cord-280939-d478p8u6
title: A simple protein-based surrogate neutralization assay for SARS-CoV-2
author: Abe, Kento T.
date: 2020-10-02
txt: ./txt/cord-280939-d478p8u6.txt
cache: ./cache/cord-280939-d478p8u6.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32870820/
doi: 10.1172/jci.insight.142362
summary:
  Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). SARS-CoV-2 ELISAs are performed by immobilizing a recombinantly produced viral antigen (such as the spike trimer or RBD) ( Figure 1B and Supplemental Figures 1 and 2; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.142362DS1) (see Methods) onto multiwell plastic plates that are then incubated with diluted patient serum or plasma samples.

abstract:
  Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector–based assay.


id: cord-280941-ds6x0yym
author: Kim, Young-Seok
date: 2018-05-17
txt: ./txt/cord-280941-ds6x0yym.txt
cache: ./cache/cord-280941-ds6x0yym.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.3389/fimmu.2018.01093
doi: 10.3389/fimmu.2018.01093
summary:
  The receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) was fused with the RNA-interaction domain (RID) and bacterioferritin, and expressed in Escherichia coli in a soluble form. The concentration of the ion Fe(2+), salt, and fusion linker also contributed to the assembly in vitro, and the stability of the NPs. The kinetic "pace-keeping" role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of NPs and virus-like particles as recombinant vaccines and for serological detection of viral infections. Taken together, the results confirmed the immunologically relevant conformation of the MERS-CoV RBD displayed on the hybrid ferritin particles, and the crucial role of RNA in controlling the kinetic pathway for the assembly of viral antigen monomers into stable NPs. To evaluate the immunogenicity of ferritin-based NPs, BALB/c mice (n = 5) were immunized with RBD, RBD-FR, and RBD-[SSG]-FR NPs antigens.

title:
  Chaperna-Mediated Assembly of Ferritin-Based Middle East Respiratory Syndrome-Coronavirus Nanoparticles

abstract:
  The folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (NP) vaccines in a timely and reproducible manner. Despite significant advances in in silico design and structure-based assembly, most engineered NPs are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. The failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. Capitalizing on a novel function of RNA as a molecular chaperone (chaperna: chaperone + RNA), we provide a robust protein-folding vehicle that may be implemented to NP assembly in bacterial hosts. The receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) was fused with the RNA-interaction domain (RID) and bacterioferritin, and expressed in Escherichia coli in a soluble form. Site-specific proteolytic removal of the RID prompted the assemblage of monomers into NPs, which was confirmed by electron microscopy and dynamic light scattering. The mutations that affected the RNA binding to RBD significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of NPs of a defined size. This underscored the RNA-antigen interactions during NP assembly. The sera after mouse immunization effectively interfered with the binding of MERS-CoV RBD to the cellular receptor hDPP4. The results suggest that RNA-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of NPs into highly regular and immunologically relevant conformations. The concentration of the ion Fe(2+), salt, and fusion linker also contributed to the assembly in vitro, and the stability of the NPs. The kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of NPs and virus-like particles as recombinant vaccines and for serological detection of viral infections.


id: cord-281005-6gi18vka
author: Singh, Praveen Kumar
date: 2020-09-01
txt: ./txt/cord-281005-6gi18vka.txt
cache: ./cache/cord-281005-6gi18vka.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1055/s-0040-1715790
doi: 10.1055/s-0040-1715790
summary:
  title: Mutations in SARS-CoV-2 Leading to Antigenic Variations in Spike Protein: A Challenge in Vaccine Development Therefore, we aimed to predict the mutations in the spike protein (S) of the SARS-CoV-2 genomes available worldwide and analyze its impact on the antigenicity. A total of 1,604 spike proteins were extracted from 1,325 complete genome and 279 partial spike coding sequences of SARS-CoV-2 available in NCBI till May 1, 2020 and subjected to multiple sequence alignment to find the mutations corresponding to the reported single nucleotide polymorphisms (SNPs) in the genomic study. In this study, we aimed to predict the mutations in the spike protein (S) of SARS-CoV-2 genomes available in the database (whole genome sequences as well as partial coding sequences of spike protein) and analyze the effect of each mutation on the antigenicity of the predicted epitopes.

title:
  Mutations in SARS-CoV-2 Leading to Antigenic Variations in Spike Protein: A Challenge in Vaccine Development

abstract:
  Objectives The spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus has been unprecedentedly fast, spreading to more than 180 countries within 3 months with variable severity. One of the major reasons attributed to this variation is genetic mutation. Therefore, we aimed to predict the mutations in the spike protein (S) of the SARS-CoV-2 genomes available worldwide and analyze its impact on the antigenicity. Materials and Methods Several research groups have generated whole genome sequencing data which are available in the public repositories. A total of 1,604 spike proteins were extracted from 1,325 complete genome and 279 partial spike coding sequences of SARS-CoV-2 available in NCBI till May 1, 2020 and subjected to multiple sequence alignment to find the mutations corresponding to the reported single nucleotide polymorphisms (SNPs) in the genomic study. Further, the antigenicity of the predicted mutations inferred, and the epitopes were superimposed on the structure of the spike protein. Results The sequence analysis resulted in high SNPs frequency. The significant variations in the predicted epitopes showing high antigenicity were A348V, V367F and A419S in receptor binding domain (RBD). Other mutations observed within RBD exhibiting low antigenicity were T323I, A344S, R408I, G476S, V483A, H519Q, A520S, A522S and K529E. The RBD T323I, A344S, V367F, A419S, A522S and K529E are novel mutations reported first time in this study. Moreover, A930V and D936Y mutations were observed in the heptad repeat domain and one mutation D1168H was noted in heptad repeat domain 2. Conclusion S protein is the major target for vaccine development, but several mutations were predicted in the antigenic epitopes of S protein across all genomes available globally. The emergence of various mutations within a short period might result in the conformational changes of the protein structure, which suggests that developing a universal vaccine may be a challenging task.


id: cord-281536-8y7yxcp4
author: Lim, Hocheol
date: 2020-10-08
url: https://www.ncbi.nlm.nih.gov/pubmed/33033344/
doi: 10.1038/s41598-020-73820-8
title:
  Hot spot profiles of SARS-CoV-2 and human ACE2 receptor protein protein interaction obtained by density functional tight binding fragment molecular orbital method

abstract:
  The prevalence of a novel β-coronavirus (SARS-CoV-2) was declared as a public health emergency of international concern on 30 January 2020 and a global pandemic on 11 March 2020 by WHO. The spike glycoprotein of SARS-CoV-2 is regarded as a key target for the development of vaccines and therapeutic antibodies. In order to develop anti-viral therapeutics for SARS-CoV-2, it is crucial to find amino acid pairs that strongly attract each other at the interface of the spike glycoprotein and the human angiotensin-converting enzyme 2 (hACE2) complex. In order to find hot spot residues, the strongly attracting amino acid pairs at the protein–protein interaction (PPI) interface, we introduce a reliable inter-residue interaction energy calculation method, FMO-DFTB3/D/PCM/3D-SPIEs. In addition to the SARS-CoV-2 spike glycoprotein/hACE2 complex, the hot spot residues of SARS-CoV-1 spike glycoprotein/hACE2 complex, SARS-CoV-1 spike glycoprotein/antibody complex, and HCoV-NL63 spike glycoprotein/hACE2 complex were obtained using the same FMO method. Following this, a 3D-SPIEs-based interaction map was constructed with hot spot residues for the hACE2/SARS-CoV-1 spike glycoprotein, hACE2/HCoV-NL63 spike glycoprotein, and hACE2/SARS-CoV-2 spike glycoprotein complexes. Finally, the three 3D-SPIEs-based interaction maps were combined and analyzed to find the consensus hot spots among the three complexes. As a result of the analysis, two hot spots were identified between hACE2 and the three spike proteins. In particular, E37, K353, G354, and D355 of the hACE2 receptor strongly interact with the spike proteins of coronaviruses. The 3D-SPIEs-based map would provide valuable information to develop anti-viral therapeutics that inhibit PPIs between the spike protein of SARS-CoV-2 and hACE2.


id: cord-281793-tj4m01s4
title: Perspectives on the development of neutralizing antibodies against SARS-CoV-2
author: Ho, Mitchell
date: 2020-05-20
txt: ./txt/cord-281793-tj4m01s4.txt
cache: ./cache/cord-281793-tj4m01s4.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1093/abt/tbaa009
doi: 10.1093/abt/tbaa009
summary:
  Crossreactive antibodies (e.g., 47D11, S309, and VHH-72) that bind highly conserved epitopes on the RBDs of SARS-CoV and SARS-CoV-2 could have broad neutralization activities against viral infection. The receptor binding domain (RBD) of the SARS-CoV-2 S protein contains several novel residues that might be introduced through recombination with the pangolin coronavirus, indicating a possible critical step in the evolution of the ability of SARS-CoV-2 to infect humans [10] . isolated a human monoclonal antibody (named "rRBD-15") that inhibits the interaction of the RBD of SARS-CoV-2 and the ACE2 and neutralizes the pseudovirus infection [5] . The structure complex of 47D11 and the RBD (or the S1/S protein) would reveal a novel conserved site on the RBD for broad-neutralizing antibodies against SARSr-CoVs. In addition to 47D11, another human antibody (S309) isolated from memory B cells of a SARS survivor infected in 2003 neutralizes SARS-CoV-2 [18] .

abstract:
  SARS-CoV-2 gains entry to human cells through its spike (S) protein binding to angiotensin-converting enzyme 2 (ACE2). Therefore, the receptor binding domain (RBD) of the S protein is the primary target for neutralizing antibodies. Selection of broad-neutralizing antibodies against SARS-CoV-2 and SARS-CoV is attractive and might be useful for treating not only COVID-19 but also future SARS-related CoV infections. Broad-neutralizing antibodies, such as 47D11, S309, and VHH-72, have been reported to target a conserved region in the RBD of the S1 subunit. The S2 subunit required for viral membrane fusion might be another target. Due to their small size and high stability, single-domain antibodies might have the ability to be administered by an inhaler making them potentially attractive therapeutics for respiratory infections. A cocktail strategy combining two (or more) antibodies that recognize different parts of the viral surface that interact with human cells might be the most effective.


id: cord-284102-rovyvv45
title: NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response
author: Wagner, Teresa R.
date: 2020-09-28
txt: ./txt/cord-284102-rovyvv45.txt
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extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.09.22.308338
doi: 10.1101/2020.09.22.308338
summary:
  Here we identified 11 unique nanobodies (Nbs) with high binding affinities to the SARS-CoV-2 spike receptor domain (RBD). Considering that Nbs targeting diverse epitopes within the RBD:ACE2 interface are beneficial 201 in both reducing viral infectivity and preventing mutational escape, we next combined the most 202 potent inhibitory and neutralizing candidates derived from Nb-Set1 (NM1226, NM1228) and 203 We incubated our previously generated color-coded beads 232 comprising RBD, S1 domain or homotrimeric spike with serum samples from patients or non-233 infected individuals, in addition to dilution series of the combinations NM1226/ NM1230 or 234 NM1228/ NM1230 and used this to detect patient-derived IgGs bound to the respective 235 antigens. As a result, we modified our previously described multiplex immunoassay 303 (MULTICOV-AB, 20 ) and developed a novel diagnostic test called NeutrobodyPlex to monitor 304 the presence and the emergence of neutralizing antibodies in serum samples of SARS-CoV-2 305 infected individuals. Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block 681 interaction with ACE2

abstract:
  As the COVID-19 pandemic escalates, the need for effective vaccination programs, diagnosis tools and therapeutic intervention ever increases. Neutralizing binding molecules have become important tools for acute treatment of COVID-19 and also provide a unique possibility to monitor the emergence and presence of a neutralizing immune response in infected or vaccinated individuals. Here we identified 11 unique nanobodies (Nbs) with high binding affinities to the SARS-CoV-2 spike receptor domain (RBD). Of these, 8 effectively block the RBD:ACE2 interface. Via competitive binding analysis and detailed epitope mapping, we grouped all Nbs into 3 sets and demonstrated their neutralizing effect. Combinations from different sets showed a profound synergistic effect by simultaneously targeting different epitopes within the RBD. Finally, we established a competitive multiplex binding assay (“NeutrobodyPlex”) enabling the detection of neutralizing antibodies in serum of infected patients. Overall, our Nbs have high potential for prophylactic and therapeutic options and provide a novel approach to screen for a neutralizing immune response in infected or vaccinated individuals, helping to monitor immune status or guide vaccine design.


id: cord-285039-9piio754
author: Zhou, Haixia
date: 2019-09-14
txt: ./txt/cord-285039-9piio754.txt
cache: ./cache/cord-285039-9piio754.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1007/978-1-0716-0211-9_4
doi: 10.1007/978-1-0716-0211-9_4
summary:
  Three-dimensional structures of the receptor-binding domain (RBD) of MERS-CoV spike glycoprotein bound to cellular receptor and monoclonal antibodies (mAbs) have been determined by X-ray crystallography, providing structural information about receptor recognition and neutralizing mechanisms of mAbs at the atomic level. The first three-dimensional structure of the MERS-CoV spike glycoprotein receptor-binding domain (RBD), providing the molecular basis of viral attachment to host cells, was determined in the complex with it cellular receptor dipeptidyl peptidase 4 (DPP4, also called CD26) by X-ray crystallography [1] . After obtaining the phases of these diffracted X-rays by heavy-atom derivative, anomalous scattering or molecular replacement methods, a protein crystallographer then calculates the density of electrons with the protein crystal and builds a structural model based on the density map. MER-CoV RBD can be expressed using the Bac-to-Bac baculovirus expression system (Fig. 1 ), collected and captured using NTA Sepharose (GE Healthcare) and then further purified by gel filtration chromatography using a Superdex 200 High Performance column (GE Healthcare).

title:
  Crystallization and Structural Determination of the Receptor-Binding Domain of MERS-CoV Spike Glycoprotein

abstract:
  Three-dimensional structures of the receptor-binding domain (RBD) of MERS-CoV spike glycoprotein bound to cellular receptor and monoclonal antibodies (mAbs) have been determined by X-ray crystallography, providing structural information about receptor recognition and neutralizing mechanisms of mAbs at the atomic level. In this chapter, we describe the purification, crystallization, and structure determination of the MERS-CoV RBD.


id: cord-285758-c18arb6s
title: SARS Vaccine Development
author: Jiang, Shibo
date: 2005-07-17
txt: ./txt/cord-285758-c18arb6s.txt
cache: ./cache/cord-285758-c18arb6s.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16022774/
doi: 10.3201/eid1107.050219
summary:
  The spike (S) protein of SARS-CoV is the major inducer of neutralizing antibodies, and the receptor-binding domain (RBD) in the S1 subunit of S protein contains multiple conformational neutralizing epitopes. (30) reported that mucosal immunization of African green monkeys with an attenuated parainfluenza virus expressing S protein resulted in production of neutralizing antibodies and protected animals from infection by challenge with SARS-CoV. These findings suggest that RBD contains the major neutralizing epitopes in the S protein and is an ideal SARS vaccine candidate because RBD contains the receptor-binding site, which is critical for virus attachment to the target cell for infection (15, (17) (18) (19) . Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

abstract:
  Developing effective and safe vaccines is urgently needed to prevent infection by severe acute respiratory syndrome (SARS)–associated coronavirus (SARS-CoV). The inactivated SARS-CoV vaccine may be the first one available for clinical use because it is easy to generate; however, safety is the main concern. The spike (S) protein of SARS-CoV is the major inducer of neutralizing antibodies, and the receptor-binding domain (RBD) in the S1 subunit of S protein contains multiple conformational neutralizing epitopes. This suggests that recombinant proteins containing RBD and vectors encoding the RBD sequence can be used to develop safe and effective SARS vaccines.


id: cord-285900-3rr0j5tk
author: Du, Lanying
date: 2016-11-22
url: https://doi.org/10.1038/ncomms13473
doi: 10.1038/ncomms13473
title:
  Introduction of neutralizing immunogenicity index to the rational design of MERS coronavirus subunit vaccines

abstract:
  Viral subunit vaccines often contain immunodominant non-neutralizing epitopes that divert host immune responses. These epitopes should be eliminated in vaccine design, but there is no reliable method for evaluating an epitope's capacity to elicit neutralizing immune responses. Here we introduce a new concept ‘neutralizing immunogenicity index' (NII) to evaluate an epitope's neutralizing immunogenicity. To determine the NII, we mask the epitope with a glycan probe and then assess the epitope's contribution to the vaccine's overall neutralizing immunogenicity. As proof-of-concept, we measure the NII for different epitopes on an immunogen comprised of the receptor-binding domain from MERS coronavirus (MERS-CoV). Further, we design a variant form of this vaccine by masking an epitope that has a negative NII score. This engineered vaccine demonstrates significantly enhanced efficacy in protecting transgenic mice from lethal MERS-CoV challenge. Our study may guide the rational design of highly effective subunit vaccines to combat MERS-CoV and other life-threatening viruses.


id: cord-287205-k64svq6n
author: Pollet, Jeroen
date: 2020-11-05
url: https://doi.org/10.1101/2020.11.04.367359
doi: 10.1101/2020.11.04.367359
title:
  SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice

abstract:
  There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel® containing formulation and possibly in combination with other immunostimulants.


id: cord-288761-fyvr0tc1
title: Allosteric inhibition of aminopeptidase N functions related to tumor growth and virus infection
author: Santiago, César
date: 2017-04-10
txt: ./txt/cord-288761-fyvr0tc1.txt
cache: ./cache/cord-288761-fyvr0tc1.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/28393915/
doi: 10.1038/srep46045
summary:
  These structures identified three distinct APN conformations, based on active site accessibility, which we termed closed, intermediate and open forms (Fig. 1a) . The phenylalanine was located in the loop that connects α 26 and α 27 in the single domain IV ARM repeat of human and pig APN (Fig. 2a) ; it penetrated the active site groove in the closed conformation and locked the peptide, ready for hydrolysis. CoV binding to APN would lock the protein in its open conformation (Fig. 2b) , preventing the ectodomain movement probably necessary for peptide hydrolysis (Fig. 2a) . In flow cytometry, we determined the binding of an RBD-Fc fusion protein to cells that expressed pAPN or an active site mutant (pAPN-HH/AA), alone or with various drugs (Fig. 4a,b) . Disulfide bonds that lock the APN closed conformation or drugs that prevent opening of the ectodomain inhibited CoV protein binding and cell infection, whereas porcine CoV S proteins probably hinder APN transition to the closed form and peptide hydrolysis.

abstract:
  Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.


id: cord-290290-wyx9ib7s
author: Sinegubova, Maria V.
date: 2020-11-05
url: https://doi.org/10.1101/2020.11.04.368092
doi: 10.1101/2020.11.04.368092
title:
  High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector

abstract:
  The spike (S) protein is one of the three proteins forming the coronaviruses’ viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests – the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 – human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and similar applications.


id: cord-291420-40xsypzt
author: Nelson-Sathi, Shijulal
date: 2020-10-01
txt: ./txt/cord-291420-40xsypzt.txt
cache: ./cache/cord-291420-40xsypzt.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.05.02.071811
doi: 10.1101/2020.05.02.071811
summary:
  title: Mutational landscape and in silico structure models of SARS-CoV-2 Spike Receptor Binding Domain reveal key molecular determinants for virus-host interaction Formation of a stable binding interface between the Spike (S) protein Receptor Binding Domain (RBD) of SARS-CoV-2 and Angiotensin-Converting Enzyme 2 (ACE2) of host actuates viral entry. In silico structure modelling of interfaces induced by mutations on residues which directly engage ACE2 or lie in the near vicinity revealed molecular rearrangements and binding energies unique to each RBD mutant. The structural analysis of the mutated spike glycoprotein of SARS-CoV-2 RBD domain was done to assess the impact of interface amino acid residue mutations on binding affinity towards the human ACE2 (hACE2) receptor. Comparative analysis of structures showed key differences in all three binding clusters of SARS-CoV-2 RBD wild type and mutant interfaces with human or mouse ACE2 (Figure 2C, 2D and Table S1 ).

title:
  Mutational landscape and in silico structure models of SARS-CoV-2 Spike Receptor Binding Domain reveal key molecular determinants for virus-host interaction

abstract:
  Protein-protein interactions between virus and host are crucial for infection. SARS-CoV-2, the causative agent of COVID-19 pandemic is an RNA virus prone to mutations. Formation of a stable binding interface between the Spike (S) protein Receptor Binding Domain (RBD) of SARS-CoV-2 and Angiotensin-Converting Enzyme 2 (ACE2) of host actuates viral entry. Yet, how this binding interface evolves as virus acquires mutations during pandemic remains elusive. Here, using a high fidelity bioinformatics pipeline, we analysed 31,403 SARS-CoV-2 genomes across the globe, and identified 444 non-synonymous mutations that cause 49 distinct amino acid substitutions in the RBD. Molecular phylogenetic analysis suggested independent emergence of these RBD mutants during pandemic. In silico structure modelling of interfaces induced by mutations on residues which directly engage ACE2 or lie in the near vicinity revealed molecular rearrangements and binding energies unique to each RBD mutant. Comparative structure analysis using binding interface from mouse that prevents SARS-CoV-2 entry uncovered minimal molecular determinants in RBD necessary for the formation of stable interface. We identified that interfacial interaction involving amino acid residues N487 and G496 on either ends of the binding scaffold are indispensable to anchor RBD and are well conserved in all SARS-like corona viruses. All other interactions appear to be required to locally remodel binding interface with varying affinities and thus may decide extent of viral transmission and disease outcome. Together, our findings propose the modalities and variations in RBD-ACE2 interface formation exploited by SARS-CoV-2 for endurance. Importance COVID-19, so far the worst hit pandemic to mankind, started in January 2020 and is still prevailing globally. Our study identified key molecular arrangements in RBD-ACE2 interface that help virus to tolerate mutations and prevail. In addition, RBD mutations identified in this study can serve as a molecular directory for experimental biologists to perform functional validation experiments. The minimal molecular requirements for the formation of RBD-ACE2 interface predicted using in silico structure models may help precisely design neutralizing antibodies, vaccines and therapeutics. Our study also proposes the significance of understanding evolution of protein interfaces during pandemic.


id: cord-291790-z5rwznmv
title: The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity
author: Li, Qianqian
date: 2020-07-17
url: https://www.sciencedirect.com/science/article/pii/S0092867420308771?v=s5
doi: 10.1016/j.cell.2020.07.012
abstract:
  Summary The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious but variants including A475V, L452R, V483A and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious whilst deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.


id: cord-292578-co5essuw
author: Johnson, Marina
date: 2020-08-02
url: https://api.elsevier.com/content/article/pii/S1386653220303140
doi: 10.1016/j.jcv.2020.104572
title:
  Evaluation of a novel multiplexed assay for determining IgG levels and functional activity to SARS-CoV-2

abstract:
  BACKGROUND: The emergence of SARS-CoV-2 has led to the development of serological assays that could aid in an understanding of the burden of COVID-19 disease. Many available tests lack rigorous evaluation and therefore results may be misleading. OBJECTIVES: The aim of this study was to assess the performance of a novel multiplexed immunoassay for the simultaneous detection of antibodies against SARS-CoV-2 trimeric spike (S), spike receptor binding domain (RBD), spike N terminal domain and nucleocapsid antigen and a novel pseudo-neutralisation assay. METHODS: A multiplexed solid-phase chemiluminescence assay (Meso Scale Discovery) was evaluated for the simultaneous detection of IgG binding to four SARS-CoV-2 antigens and the quantification of antibody-induced ACE-2 binding inhibition (pseudo-neutralisation assay). Sensitivity was evaluated with a total of 196 COVID-19 serum samples (169 confirmed PCR positive and 27 anti-nucleocapsid IgG positive) from individuals with mild symptomatic or asymptomatic disease. Specificity was evaluated with 194 control serum samples collected from adults prior to December 2019. RESULTS: The specificity and sensitivity of the binding IgG assay was highest for S protein with a specificity of 97.4 % and sensitivity of 96.2 % for samples taken 14 days and 97.9 % for samples taken 21 days following the onset of symptoms. IgG concentration to S and RBD correlated strongly with percentage inhibition measured by the pseudo-neutralisation assay. CONCLUSION: Excellent sensitivity for IgG detection was obtained over 14 days since onset of symptoms for three SARS-CoV-2 antigens (S, RBD and N) in this multiplexed assay which can also measure antibody functionality.


id: cord-292883-7hvq9qaj
author: Nguyen-Contant, Phuong
date: 2020-09-25
url: https://www.ncbi.nlm.nih.gov/pubmed/32978311/
doi: 10.1128/mbio.01991-20
title:
  S Protein-Reactive IgG and Memory B Cell Production after Human SARS-CoV-2 Infection Includes Broad Reactivity to the S2 Subunit

abstract:
  The high susceptibility of humans to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the cause of coronavirus disease 2019 (COVID-19), reflects the novelty of the virus and limited preexisting B cell immunity. IgG against the SARS-CoV-2 spike (S) protein, which carries the novel receptor binding domain (RBD), is absent or at low levels in unexposed individuals. To better understand the B cell response to SARS-CoV-2 infection, we asked whether virus-reactive memory B cells (MBCs) were present in unexposed subjects and whether MBC generation accompanied virus-specific IgG production in infected subjects. We analyzed sera and peripheral blood mononuclear cells (PBMCs) from non-SARS-CoV-2-exposed healthy donors and COVID-19 convalescent subjects. Serum IgG levels specific for SARS-CoV-2 proteins (S, including the RBD and S2 subunit, and nucleocapsid [N]) and non-SARS-CoV-2 proteins were related to measurements of circulating IgG MBC levels. Anti-RBD IgG was absent in unexposed subjects. Most unexposed subjects had anti-S2 IgG, and a minority had anti-N IgG, but IgG MBCs with these specificities were not detected, perhaps reflecting low frequencies. Convalescent subjects had high levels of IgG against the RBD, S2, and N, together with large populations of RBD- and S2-reactive IgG MBCs. Notably, IgG titers against the S protein of the human coronavirus OC43 were higher in convalescent subjects than in unexposed subjects and correlated strongly with anti-S2 titers. Our findings indicate cross-reactive B cell responses against the S2 subunit that might enhance broad coronavirus protection. Importantly, our demonstration of MBC induction by SARS-CoV-2 infection suggests that a durable form of B cell immunity is maintained even if circulating antibody levels wane.


id: cord-295482-qffg6r91
title: Receptor-binding loops in alphacoronavirus adaptation and evolution
author: Wong, Alan H. M.
date: 2017-11-23
txt: ./txt/cord-295482-qffg6r91.txt
cache: ./cache/cord-295482-qffg6r91.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1038/s41467-017-01706-x
doi: 10.1038/s41467-017-01706-x
summary:
  Here we report the X-ray crystal structure of the receptor-binding domain (RBD) of the human coronavirus, HCoV-229E, in complex with the ectodomain of its receptor, aminopeptidase N (APN). Phylogenetic analysis shows that the natural HCoV-229E receptor-binding loop variation observed defines six RBD classes whose viruses have successively replaced each other in the human population over the past 50 years. The structure shows that receptor binding is mediated solely by three extended loops, a feature shared by HCoV-NL63 and the closely related porcine respiratory coronavirus, PRCoV. The six RBDs differ in their receptor-binding affinity and their ability to be bound by a neutralizing antibody (9.8E12) and taken together, our findings suggest that the HCoV-229E sequence variation observed arose through adaptation and selection. HCoV-229E infection in humans does not provide protection against different isolates 37 , and viruses that contain a new RBD class that cannot be bound by the existing repertoire of loop-binding neutralizing antibodies provide an explanation for this observation.

abstract:
  RNA viruses are characterized by a high mutation rate, a buffer against environmental change. Nevertheless, the means by which random mutation improves viral fitness is not well characterized. Here we report the X-ray crystal structure of the receptor-binding domain (RBD) of the human coronavirus, HCoV-229E, in complex with the ectodomain of its receptor, aminopeptidase N (APN). Three extended loops are solely responsible for receptor binding and the evolution of HCoV-229E and its close relatives is accompanied by changing loop–receptor interactions. Phylogenetic analysis shows that the natural HCoV-229E receptor-binding loop variation observed defines six RBD classes whose viruses have successively replaced each other in the human population over the past 50 years. These RBD classes differ in their affinity for APN and their ability to bind an HCoV-229E neutralizing antibody. Together, our results provide a model for alphacoronavirus adaptation and evolution based on the use of extended loops for receptor binding.


id: cord-296187-nnv2e7gr
title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19
author: Mulgaonkar, Nirmitee
date: 2020-08-18
url: https://doi.org/10.1101/2020.06.18.158196
doi: 10.1101/2020.06.18.158196
abstract:
  The rapid geographic expansion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infectious agent of Coronavirus Disease 2019 (COVID-19) pandemic, poses an immediate need for potent drugs. Enveloped viruses infect the host cell by cellular membrane fusion, a crucial mechanism required for virus replication. The SARS-CoV-2 spike glycoprotein, due to its primary interaction with the human angiotensin-converting enzyme 2 (ACE2) cell-surface receptor, is considered as a potential target for drug development. Based on in silico screening followed by in vitro studies, here we report that the existing FDA-approved Bcr-Abl tyrosine kinase inhibitor, imatinib, inhibits SARS-CoV-2 with an IC50 of 130 nM. We provide evidence that although imatinib binds to the receptor-binding domain (RBD) of SARS-CoV-2 spike protein with an affinity at micromolar, i.e., 2.32 ± 0.9 μM levels, imatinib does not directly inhibit the spike RBD:ACE2 interaction – suggesting a Bcr-Abl kinase-mediated fusion inhibition mechanism is responsible for the inhibitory action. We also show that imatinib inhibits other coronaviruses, SARS-CoV, and MERS-CoV via fusion inhibition. Based on promising in vitro results, we propose the Abl tyrosine kinase inhibitor (ATKI), imatinib, to be a viable repurposable drug against COVID-19.


id: cord-296319-fwn97wds
title: Immunogenic profile of SARS-CoV-2 spike in individuals recovered from COVID-19
author: Juno, J. A.
date: 2020-05-21
url: http://medrxiv.org/cgi/content/short/2020.05.17.20104869v1?rss=1
doi: 10.1101/2020.05.17.20104869
abstract:
  The rapid global spread of SARS-CoV-2 and resultant mortality and social disruption have highlighted the need to better understand coronavirus immunity to expedite vaccine development efforts. Multiple candidate vaccines, designed to elicit protective neutralising antibodies targeting the viral spike glycoprotein, are rapidly advancing to clinical trial. However, the immunogenic properties of the spike protein in humans are unresolved. To address this, we undertook an in-depth characterisation of humoral and cellular immunity against SARS-CoV-2 spike in humans following mild to moderate SARS-CoV-2 infection. We find serological antibody responses against spike are routinely elicited by infection and correlate with plasma neutralising activity and capacity to block ACE2/RBD interaction. Expanded populations of spike-specific memory B cells and circulating T follicular helper cells (cTFH) were detected within convalescent donors, while responses to the receptor binding domain (RBD) constitute a minor fraction. Using regression analysis, we find high plasma neutralisation activity was associated with increased spike-specific antibody, but notably also with the relative distribution of spike-specific cTFH subsets. Thus both qualitative and quantitative features of B and T cell immunity to spike constitute informative biomarkers of the protective potential of novel SARS-CoV-2 vaccines.


id: cord-296657-mymndjvd
title: High affinity modified ACE2 receptors prevent SARS-CoV-2 infection
author: Higuchi, Yusuke
date: 2020-09-16
txt: ./txt/cord-296657-mymndjvd.txt
cache: ./cache/cord-296657-mymndjvd.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.09.16.299891
doi: 10.1101/2020.09.16.299891
summary:
  The extracellular domain of modified ACE2 fused to the Fc region of the human immunoglobulin IgG1 had stable structure and neutralized SARS-CoV-2 pseudotyped lentivirus and authentic virus with more than 100-fold lower concentration than wild-type. Engineering ACE2 decoy receptors with directed evolution is a promising approach to develop a SARS-CoV-2 neutralizing drug that has affinity comparable to monoclonal antibodies yet displaying resistance to escape mutations of virus. Three cycles of screening resulted in an identification of mutant ACE2 clones with more than 100-fold higher binding affinity to the RBD and lower half-maximal inhibitory concentration (IC50) for SARS-CoV-2 pseudotyped lentivirus as well as authentic virus. We engineered ACE2 to bind the RBD of the SARS-CoV-2 spike protein with the combination of surface display of mutagenized library and fluorescence-activated cell sorting (FACS) to perform the evolution in 293T human cells.

abstract:
  The SARS-CoV-2 spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor via receptor binding domain (RBD) to enter into the cell. Inhibiting this interaction is a main approach to block SARS-CoV-2 infection and it is required to have high affinity to RBD independently of viral mutation for effective protection. To this end, we engineered ACE2 to enhance the affinity with directed evolution in human cells. Three cycles of random mutation and cell sorting achieved more than 100-fold higher affinity to RBD than wild-type ACE2. The extracellular domain of modified ACE2 fused to the Fc region of the human immunoglobulin IgG1 had stable structure and neutralized SARS-CoV-2 pseudotyped lentivirus and authentic virus with more than 100-fold lower concentration than wild-type. Engineering ACE2 decoy receptors with directed evolution is a promising approach to develop a SARS-CoV-2 neutralizing drug that has affinity comparable to monoclonal antibodies yet displaying resistance to escape mutations of virus.


id: cord-297072-f5lmstyn
author: Struck, Anna-Winona
date: 2012-01-16
txt: ./txt/cord-297072-f5lmstyn.txt
cache: ./cache/cord-297072-f5lmstyn.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166354211005481
doi: 10.1016/j.antiviral.2011.12.012
summary:
  title: A hexapeptide of the receptor-binding domain of SARS corona virus spike protein blocks viral entry into host cells via the human receptor ACE2 Peptide (438)YKYRYL(443) is part of the receptor-binding domain (RBD) of the spike protein of SARS-CoV. The interaction of SARS-CoV with its receptor ACE2 is an attractive drug target as epitopes of the RBD on the spike protein may serve as leads for the design of effective entry inhibitors (Du et al., 2009) . This method allows the determination of the binding specificity, as Table 2 Synthetic peptide library of fourteen 6mer peptides comprising RBD-residues N435-E452 and A471-S500 of SARS-CoV spike protein. We found a hexapeptide in the receptor-binding domain (RBD) of the S protein of SARS-CoV that carries a significant portion of the binding affinity of the virus to the human cell. Inhibition of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infectivity by peptides analogous to the viral spike protein

title:
  A hexapeptide of the receptor-binding domain of SARS corona virus spike protein blocks viral entry into host cells via the human receptor ACE2

abstract:
  In vitro infection of Vero E6 cells by SARS coronavirus (SARS-CoV) is blocked by hexapeptide Tyr-Lys-Tyr-Arg-Tyr-Leu. The peptide also inhibits proliferation of coronavirus NL63. On human cells both viruses utilize angiotensin-converting enzyme 2 (ACE2) as entry receptor. Blocking the viral entry is specific as alpha virus Sindbis shows no reduction in infectivity. Peptide (438)YKYRYL(443) is part of the receptor-binding domain (RBD) of the spike protein of SARS-CoV. Peptide libraries were screened by surface plasmon resonance (SPR) to identify RBD binding epitopes. (438)YKYRYL(443) carries the dominant binding epitope and binds to ACE2 with K(D) = 46 μM. The binding mode was further characterized by saturation transfer difference (STD) NMR spectroscopy and molecular dynamic simulations. Based on this information the peptide can be used as lead structure to design potential entry inhibitors against SARS-CoV and related viruses.


id: cord-297747-kifqgskc
author: Lupala, Cecylia S.
date: 2020-03-27
txt: ./txt/cord-297747-kifqgskc.txt
cache: ./cache/cord-297747-kifqgskc.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.03.24.005561
doi: 10.1101/2020.03.24.005561
summary:
  Using homology modeling and molecular dynamics (MD) simulation methods, we report here the detailed structure of the ACE2 in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The simulation data further revealed critical residues at the complex interface and provided more details about the interactions between the SARS-CoV-2 RBD and human ACE2. When this study was started, neither the crystal structure of the SARS-CoV-2 spike protein nor the RBD segment were determined, so the homology modeling approach was applied to construct the model of the SARS-CoV-2 spike RBD in complex with the human ACE2 binding domain (denoted as CoV2-RBD/ACE2 in the following). Although the crystal structure and the predicted model of the CoV2-RBD/ACE2 complex provide important information about the binding interactions at the molecular interfaces, MD simulations can extend the knowledge to a dynamics regime in a fully solvated environment.

title:
  Computational simulations reveal the binding dynamics between human ACE2 and the receptor binding domain of SARS-CoV-2 spike protein

abstract:
  A novel coronavirus (the SARS-CoV-2) has been identified in January 2020 as the causal pathogen for COVID-19 pneumonia, an outbreak started near the end of 2019 in Wuhan, China. The SARS-CoV-2 was found to be closely related to the SARS-CoV, based on the genomic analysis. The Angiotensin converting enzyme 2 protein (ACE2) utilized by the SARS-CoV as a receptor was found to facilitate the infection of SARS-CoV-2 as well, initiated by the binding of the spike protein to the human ACE2. Using homology modeling and molecular dynamics (MD) simulation methods, we report here the detailed structure of the ACE2 in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The predicted model is highly consistent with the experimentally determined complex structures. Plausible binding modes between human ACE2 and the RBD were revealed from all-atom MD simulations. The simulation data further revealed critical residues at the complex interface and provided more details about the interactions between the SARS-CoV-2 RBD and human ACE2. Two mutants mimicking rat ACE2 were modeled to study the mutation effects on RBD binding to ACE2. The simulations showed that the N-terminal helix and the K353 of the human ACE2 alter the binding modes of the CoV2-RBD to the ACE2.


id: cord-299783-8ti6r0eh
author: Bruni, M.
date: 2020-08-01
url: https://doi.org/10.1101/2020.07.30.20164368
doi: 10.1101/2020.07.30.20164368
title:
  Persistence of anti-SARS-CoV-2 antibodies in non-hospitalized COVID-19 convalescent health care workers

abstract:
  Background. Coronavirus disease-19 (COVID-19) is a respiratory illness caused by the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), a novel beta-coronavirus. Although antibody response to SARS-CoV-2 can be detected early during the infection, several outstanding questions remain to be addressed regarding magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response, as measured by serum levels of pro-inflammatory mediators. Methods. An ELISA assay has been developed by expressing and purifying the recombinant SARS-CoV-2 Spike Receptor Binding Domain (RBD), Soluble Ectodomain (Spike), and full length nucleocapsid protein (N protein). Sera from healthcare workers affected by non-severe COVID-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in Intensive Care Units (ICU) and SARS-CoV-2-negative subjects for the presence of IgM, IgG and IgA antibodies as well as soluble pro-inflammatory mediators in the sera. Results. Specificity and sensitivity of the ELISA assays were high for anti-RBD IgG and IgA (92-97%) and slightly lower for IgM and the Spike and N proteins (70-85%). The ELISA allowed quantification of IgM, IgG and IgA antibody responses against all the viral antigens tested and showed a correlation between magnitude of the antibody response and disease severity. Non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles as compared to patients in Intensive Care Units (ICU), irrespective of the antibodies tested. Noteworthy, in non-severe COVID-19 infections, antibody titers against RBD and Spike, but not against the N protein, as well as pro-inflammatory cytokines decreased within a month after viral clearance. Conclusions. Rapid decline in antibody titers and in pro-inflammatory cytokines may be a common feature of non-severe SARS-CoV-2 infection, suggesting that antibody-mediated protection against re-infection with SARS-CoV-2 is of short duration. These results suggest caution in use serological testing to estimate the prevalence of SARS-CoV-2 infection in the general population.


id: cord-300707-k9uk14b3
title: Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins
author: Bouwman, Kim M.
date: 2020-09-04
url: https://doi.org/10.1101/2020.09.04.282558
doi: 10.1101/2020.09.04.282558
abstract:
  Receptor binding studies using recombinant SARS-CoV proteins have been hampered due to challenges in approaches creating spike protein or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric RBD proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric fully glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that fully glycosylated trimeric RBD proteins are attractive to analyze receptor binding and explore ACE2 expression profiles in tissues.


id: cord-300784-4jeaqqn9
author: Ma, Huan
date: 2020-04-22
url: https://doi.org/10.1101/2020.04.17.20064907
doi: 10.1101/2020.04.17.20064907
title:
  COVID-19 diagnosis and study of serum SARS-CoV-2 specific IgA, IgM and IgG by a quantitative and sensitive immunoassay

abstract:
  Background The current pandemic of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a great loss in lives and economy. Detecting viral RNAs on nasopharyngeal and throat swabs is the standard approach for SARS-CoV-2 diagnosis with variable success. Currently, there are only a few studies describing the serological diagnostic methods that involve the detection of SARS-CoV-2-specific IgM and IgG. Here, we aimed to develop a more quantitative and sensitive serological test for COVID-19 diagnosis, monitoring and clinical investigation, based on the detection of antigen-specific IgA as well as IgM and IgG in blood in response to SARS-CoV-2 infection. Methods In this investigation, we report the development of a set of validated diagnostic kits for detecting serum IgA, IgM, and IgG specific to SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein by chemi-luminescence immuno-analysis. The kits were tested with a cohort of 216 sera from 87 laboratory-confirmed COVID-19 patients, and 483 sera from SARS-CoV-2 negative or healthy individuals as negative controls. A standard receiver operating characteristic (ROC) analysis was conducted to evaluate the diagnostic accuracy. Using the kits, serum levels of IgA, IgM, and IgG were analyzed, in response to SARS-CoV-2 infection and COVID-19 pathogenesis. Findings The diagnostic kits based on the RBD antigen outperformed those based on the NP. RBD-specific IgA, IgM, and IgG detection kits showed sensitivities of 98.6%, 96.8%, and 96.8%, and specificities of 98.1%, 92.3%, and 99.8%, respectively. In addition, using purified RBD-specific immunoglobulins from a serum pool of COVID-19 patients as standards, the serum concentrations of RBD-specific IgA, IgM, and IgG proteins were determined. The concentrations varied widely among different patients. Median concentration of IgA and IgM reached peaks at 16-20 days after illness onset at 8.84 μg/mL and 7.25 μg/mL, respectively, while median concentration of IgG peaked during 21-25 days after illness onset at 16.47 μg/mL. Furthermore, the serum IgA level positively correlates with COVID-19 severity. Interpretation Our immunoassay of measuring SARS-CoV-2 specific antibodies IgA, IgM, and IgG in serum provides a better serological testing with improved sensitivity and specificity. Data of IgA, IgM, and IgG responses in blood of COVID-19 patients may provide novel insight for the monitoring and treatments of COVID-19. The kits are also suitable for epidemiological studies and vaccine validations.


id: cord-300847-ycuiso0g
author: Li, Wei
date: 2020-06-02
txt: ./txt/cord-300847-ycuiso0g.txt
cache: ./cache/cord-300847-ycuiso0g.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32511413/
doi: 10.1101/2020.05.13.093088
summary:
  We identified panels of fully human monoclonal antibodies (mAbs) from eight large phage-displayed Fab, scFv and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. By using phage display we have previously identified a number of potent fully human mAbs (m396, m336, m102.4) against emerging viruses including severe acute respiratory syndrome coronavirus (SARS-CoV) (4) , Middle East respiratory syndrome coronavirus (MERS-CoV) (5) and henipaviruses (6, 7) , respectively, which are also highly effective in animal models of infection (8) (9) (10) (11) ; one of them was administered on a compassionate basis to humans exposed to henipaviruses and successfully evaluated in a clinical trial (12) . Thus, to generate high affinity and safe mAbs we used eight very large (size ~ 10 11 clones each) naive human antibody libraries in Fab, scFv or VH format using PBMCs from 490 individuals total obtained before the SARS-CoV-2 outbreak.

title:
  Rapid selection of a human monoclonal antibody that potently neutralizes SARS-CoV-2 in two animal models

abstract:
  Effective therapies are urgently needed for the SARS-CoV-2/COVID19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from eight large phage-displayed Fab, scFv and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. One high affinity mAb, IgG1 ab1, specifically neutralized replication competent SARS-CoV-2 with exceptional potency as measured by two different assays. There was no enhancement of pseudovirus infection in cells expressing Fcγ receptors at any concentration. It competed with human angiotensin-converting enzyme 2 (hACE2) for binding to RBD suggesting a competitive mechanism of virus neutralization. IgG1 ab1 potently neutralized mouse ACE2 adapted SARS-CoV-2 in wild type BALB/c mice and native virus in hACE2 expressing transgenic mice. The ab1 sequence has relatively low number of somatic mutations indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 does not have developability liabilities, and thus has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 days) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.


id: cord-301347-22lt6h40
author: Jarvis, Matthew C.
date: 2016-01-01
txt: ./txt/cord-301347-22lt6h40.txt
cache: ./cache/cord-301347-22lt6h40.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0167587715300416
doi: 10.1016/j.prevetmed.2015.10.020
summary:
  Phylogenetic analysis of the complete genome sequence data revealed high rates of recombination, resulting in differing evolutionary patterns in phylogenies inferred for the spike region versus whole genomes. Despite excising a large portion of the genome prior to analysis, the Bayesian trees illustrate two distinct entries of PEDV into the US and characterize the evolution of PEDV compared to other CoVs. Modeling of the pAPN RBD region has revealed that Asian strains have increasing diversity compared to previously developed vaccines, and the variability in both the American and Asian strains needs to be considered for future vaccine development. Phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in central China based on the ORF3 gene and the main neutralization epitopes Complete genome sequence of porcine epidemic diarrhea virus strain USA/Colorado/2013 from the United States Cell culture isolation and sequence analysis of genetically diverse US porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene

title:
  Genomic and evolutionary inferences between American and global strains of porcine epidemic diarrhea virus

abstract:
  Porcine epidemic diarrhea virus (PEDV) has caused severe economic losses both recently in the United States (US) and historically throughout Europe and Asia. Traditionally, analysis of the spike gene has been used to determine phylogenetic relationships between PEDV strains. We determined the complete genomes of 93 PEDV field samples from US swine and analyzed the data in conjunction with complete genome sequences available from GenBank (n = 126) to determine the most variable genomic areas. Our results indicate high levels of variation within the ORF1 and spike regions while the C-terminal domains of structural genes were highly conserved. Analysis of the Receptor Binding Domains in the spike gene revealed a limited number of amino acid substitutions in US strains compared to Asian strains. Phylogenetic analysis of the complete genome sequence data revealed high rates of recombination, resulting in differing evolutionary patterns in phylogenies inferred for the spike region versus whole genomes. These finding suggest that significant genetic events outside of the spike region have contributed to the evolution of PEDV.


id: cord-303868-aes92l6s
author: Steffen, Tara L.
date: 2020-08-22
url: https://doi.org/10.1101/2020.08.21.261727
doi: 10.1101/2020.08.21.261727
title:
  The receptor binding domain of SARS-CoV-2 spike is the key target of neutralizing antibody in human polyclonal sera

abstract:
  Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. Here, we functionally define the role SARS-CoV-2 spike plays as a target of the human neutralizing antibody response. In this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qRT-PCR-positive for SARS-CoV-2. Using an ELISA format, we assessed binding of human sera to spike subunit 1 (S1), spike subunit 2 (S2) and the receptor binding domain (RBD) of spike. To functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. We show that epitopes within RBD are the target of a majority of the neutralizing antibodies in the human polyclonal antibody response. These data provide critical information for vaccine development and development of sensitive and specific serological testing.


id: cord-305742-wf6qxplf
title: Binding of SARS–CoV–2 to cell receptors: a tale of molecular evolution
author: Gomez, Santiago A.
date: 2020-09-28
url: https://doi.org/10.1002/cbic.202000618
doi: 10.1002/cbic.202000618
abstract:
  The magnified infectious power of the SARS–CoV–2 virus compared to its precursor SARS–CoV is intimately linked to an enhanced ability in the mutated virus to find available hydrogen bond sites in the host cells. This characteristic is acquired during virus evolution because of the selective pressure exerted at the molecular level. We pinpoint the specific residue (in the virus) to residue (in the cell) contacts during the initial recognition and binding and show that the virus· · · cell interaction is mainly due to an extensive network of hydrogen bonds and to a large surface of non–covalent interactions. In addition to the formal quantum characterization of bonding interactions, computation of absorption spectra for the specific virus· · · cell interacting residues yields significant shifts of ∆λ max = 47 and 66 nm in the wavelength for maximum absorption in the complex with respect to the isolated host and virus, respectively.


id: cord-306438-db2rqz4d
author: Kalathiya, Umesh
date: 2020-05-14
url: https://www.ncbi.nlm.nih.gov/pubmed/32422996/
doi: 10.3390/jcm9051473
title:
  Highly Conserved Homotrimer Cavity Formed by the SARS-CoV-2 Spike Glycoprotein: A Novel Binding Site

abstract:
  An important stage in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) life cycle is the binding of the spike (S) protein to the angiotensin converting enzyme-2 (ACE2) host cell receptor. Therefore, to explore conserved features in spike protein dynamics and to identify potentially novel regions for drugging, we measured spike protein variability derived from 791 viral genomes and studied its properties by molecular dynamics (MD) simulation. The findings indicated that S2 subunit (heptad-repeat 1 (HR1), central helix (CH), and connector domain (CD) domains) showed low variability, low fluctuations in MD, and displayed a trimer cavity. By contrast, the receptor binding domain (RBD) domain, which is typically targeted in drug discovery programs, exhibits more sequence variability and flexibility. Interpretations from MD simulations suggest that the monomer form of spike protein is in constant motion showing transitions between an “up” and “down” state. In addition, the trimer cavity may function as a “bouncing spring” that may facilitate the homotrimer spike protein interactions with the ACE2 receptor. The feasibility of the trimer cavity as a potential drug target was examined by structure based virtual screening. Several hits were identified that have already been validated or suggested to inhibit the SARS-CoV-2 virus in published cell models. In particular, the data suggest an action mechanism for molecules including Chitosan and macrolides such as the mTOR (mammalian target of Rapamycin) pathway inhibitor Rapamycin. These findings identify a novel small molecule binding-site formed by the spike protein oligomer, that might assist in future drug discovery programs aimed at targeting the coronavirus (CoV) family of viruses.


id: cord-308310-wtmjt3hf
author: Zha, Lisha
date: 2020-05-14
txt: ./txt/cord-308310-wtmjt3hf.txt
cache: ./cache/cord-308310-wtmjt3hf.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.05.06.079830
doi: 10.1101/2020.05.06.079830
summary:
  Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus resulted in a vaccine candidate (RBD-CuMVTT) that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized the SARS-CoV-2 virus in vitro. Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus resulted in a vaccine candidate (RBD-CuMVTT) that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized the SARS-CoV-2 virus in vitro. The receptor binding domain (RBD) of the SARS spike protein binds to ACE2 and is an important target for neutralizing antibodies [5] [6] [7] . Hence, the RBD-CuMVTT vaccine candidate is able to induce high levels of SARS-CoV-2 neutralizing antibodies. Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

title:
  Development of a COVID-19 vaccine based on the receptor binding domain displayed on virus-like particles

abstract:
  The recently ermerging disease COVID-19 is caused by the new SARS-CoV-2 virus first detected in the city of Wuhan, China. From there it has been rapidly spreading inside and outside China. With initial death rates around 4%, COVID-19 patients at longer distances from Wuhan showed reduced mortality as was previously observed for the SARS coronavirus. However, the new coronavirus spreads more strongly, as it sheds long before onset of symptoms or may be transmitted by people without symptoms. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here we demonstrate that recombinantly expressed receptor binding domain (RBD) of the spike protein homologous to SARS binds to ACE2, the viral receptor. Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus resulted in a vaccine candidate (RBD-CuMVTT) that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized the SARS-CoV-2 virus in vitro.


id: cord-308428-zw26usmh
title: Highly potent bispecific sybodies neutralize SARS-CoV-2
author: Walter, Justin D.
date: 2020-11-10
txt: ./txt/cord-308428-zw26usmh.txt
cache: ./cache/cord-308428-zw26usmh.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.11.10.376822
doi: 10.1101/2020.11.10.376822
summary:
  Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. We identified a sybody pair (Sb#15 and Sb#68) that can bind simultaneously to the RBD, and block ACE2 binding, thereby neutralizing pseudotyped and live SARS-CoV-2 viruses. However, binders of the isolated RBD may not effectively engage the aforementioned pre-fusion conformation of the spike protein, which could account for the poor neutralization ability of recently described single-domain antibodies that were raised against the RBD of SARS-CoV-2 spike protein [29] . Since Sb#15 and Sb#68 can bind simultaneously to the RBD and the full-length spike protein, we mixed Sb#15 and Sb#68 together to investigate potential additive or synergistic neutralizing activity of these two independent sybodies. To gain structural insights into how Sb#15 and Sb#68 recognize the RBD, we performed single particle cryo-EM analysis of the spike protein in complex with the sybodies. Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2

abstract:
  The COVID-19 pandemic has resulted in a global crisis. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. We identified a sybody pair (Sb#15 and Sb#68) that can bind simultaneously to the RBD, and block ACE2 binding, thereby neutralizing pseudotyped and live SARS-CoV-2 viruses. Cryo-EM analyses of the spike protein in complex with both sybodies revealed symmetrical and asymmetrical conformational states. In the symmetric complex each of the three RBDs were bound by both sybodies, and adopted the up conformation. The asymmetric conformation, with three Sb#15 and two Sb#68 bound, contained one down RBD, one up-out RBD and one up RBD. Bispecific fusions of the sybodies increased the neutralization potency 100-fold, as compared to the single binders. Our work demonstrates that linking two binders that recognize spatially-discrete binding sites result in highly potent SARS-CoV-2 inhibitors for potential therapeutic applications.


id: cord-309182-t9ywnshj
author: Premkumar, Lakshmanane
date: 2020-06-11
url: https://www.ncbi.nlm.nih.gov/pubmed/32527802/
doi: 10.1126/sciimmunol.abc8413
title:
  The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients

abstract:
  The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by PCR testing of symptomatic patients for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the spike protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV-specific antibodies in people. Here we use a large panel of human sera (63 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate RBD's performance as an antigen for reliable detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) for antibodies induced by SARS-CoVs. We observed a strong correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, support using the RBD antigen in serological diagnostic assays and RBD-specific antibody levels as a correlate of SARS-CoV-2 neutralizing antibodies in people.


id: cord-309411-2dfiwo65
title: Loss of pH switch unique to SARS-CoV2 supports unfamiliar virus pathology
author: Paris, Kristina A.
date: 2020-06-23
url: https://doi.org/10.1101/2020.06.16.155457
doi: 10.1101/2020.06.16.155457
abstract:
  Cell surface receptor engagement is a critical aspect of viral infection. This paper compares the dynamics of virus-receptor interactions for SARS-CoV (CoV1) and CoV2. At low (endosomal) pH, the binding free energy landscape of CoV1 and CoV2 interactions with the angiotensin-converting enzyme 2 (ACE2) receptor is almost the same. However, at neutral pH the landscape is different due to the loss of a pH-switch (His445Lys) in the receptor binding domain (RBD) of CoV2 relative to CoV1. Namely, CoV1 stabilizes a transition state above the bound state. In situations where small external strains are applied by, say, shear flow in the respiratory system, the off rate of the viral particle is enhanced. As a result, CoV1 virions are expected to detach from cell surfaces in time scales that are much faster than the time needed for other receptors to reach out and stabilize virus attachment. On the other hand, the loss of this pH-switch, which sequence alignments show is unique to CoV2, eliminates the transition state and allows the virus to stay bound to the ACE2 receptor for time scales compatible with the recruitment of additional ACE2 receptors diffusing in the cell membrane. This has important implications for viral infection and its pathology. CoV1 does not trigger high infectivity in the nasal area because it either rapidly drifts down the respiratory tract or is exhaled. By contrast, this novel mutation in CoV2 should not only retain the infection in the nasal cavity until ACE2-rich cells are sufficiently depleted, but also require fewer particles for infection. This mechanism explains observed longer incubation times, extended period of viral shedding, and higher rate of transmission. These considerations governing viral entry suggest that number of ACE2-rich cells in human nasal mucosa, which should be significantly smaller for children (and females relative to males), should also correlate with onset of viral load that could be a determinant of higher virus susceptibility. Critical implications for the development of new vaccines to combat current and future pandemics that, like SARS-CoV2, export evolutionarily successful strains via higher transmission rates by viral retention in nasal epithelium are also discussed.


id: cord-309898-sju15hev
author: Hu, Yiwen
date: 2020-11-02
url: https://www.ncbi.nlm.nih.gov/pubmed/33163958/
doi: 10.1016/j.matt.2020.10.032
title:
  Comparative analysis of nanomechanical features of coronavirus spike proteins and correlation with lethality and infection rate

abstract:
  The novel coronavirus disease, COVID-19, has spread rapidly around the world. Its causative virus, SARS-CoV-2, enters human cells through the physical interaction between the receptor-binding domain (RBD) of its spike protein and the human cell receptor ACE2. Here, we provide a novel way in understanding coronavirus spike proteins, connecting their nanomechanical features – specifically its vibrational spectrum and quantitative measures of mobility – with virus lethality and infection rate. The key result of our work is that both, the overall flexibility of upward RBD and the mobility ratio of RBDs in different conformations, represent two significant factors that show a positive scaling with virus lethality and an inverse correlation with the infection rate. Our analysis shows that epidemiological virus properties can be linked directly to pure nanomechanical, vibrational aspects, offering an alternative way of screening new viruses and mutations, and potentially exploring novel ways to prevent infections from occurring.


id: cord-310230-9wfb43gt
title: Critical Sequence Hot-spots for Binding of nCOV-2019 to ACE2 as Evaluated by Molecular Simulations
author: Ghorbani, Mahdi
date: 2020-06-27
txt: ./txt/cord-310230-9wfb43gt.txt
cache: ./cache/cord-310230-9wfb43gt.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32637962/
doi: 10.1101/2020.06.27.175448
summary:
  Our goal is to provide a detailed structural mechanism of how nCOV-2019 recognizes and establishes contacts with ACE2 and its difference with an earlier coronavirus SARS-COV in 2002 via extensive molecular dynamics (MD) simulations. 7 Based on the sequence similarity between RBD of nCOV-2019 and SARS-COV and also the tight binding between RBD of nCOV-2019 and ACE2, it is most probable that nCOV-2019 uses this receptor on human cells to gain entry into the body. The focus of this article is to elucidate the differences between the interface of SARS-COV and nCOV-2019 with ACE2 to understand with atomic resolution the interaction mechanism and hotspot residues at the RBD/ACE2 interface using long-timescale molecular dynamics (MD) simulation. The binding energetics between ACE2 and the RBD of SARS-COV, nCOV-2019 and all its mutant complexes were investigated by the MMPBSA method. Computational Simulations Reveal the Binding Dynamics between Human ACE2 and the Receptor Binding Domain of SARS-CoV-2 Spike Protein

abstract:
  The novel coronavirus (nCOV-2019) outbreak has put the world on edge, causing millions of cases and hundreds of thousands of deaths all around the world, as of June 2020, let alone the societal and economic impacts of the crisis. The spike protein of nCOV-2019 resides on the virion’s surface mediating coronavirus entry into host cells by binding its receptor binding domain (RBD) to the host cell surface receptor protein, angiotensin converter enzyme (ACE2). Our goal is to provide a detailed structural mechanism of how nCOV-2019 recognizes and establishes contacts with ACE2 and its difference with an earlier coronavirus SARS-COV in 2002 via extensive molecular dynamics (MD) simulations. Numerous mutations have been identified in the RBD of nCOV-2019 strains isolated from humans in different parts of the world. In this study, we investigated the effect of these mutations as well as other Ala-scanning mutations on the stability of RBD/ACE2 complex. It is found that most of the naturally-occurring mutations to the RBD either strengthen or have the same binding affinity to ACE2 as the wild-type nCOV-2019. This may have implications for high human-to-human transmission of coronavirus in regions where these mutations have been found as well as any vaccine design endeavors since these mutations could act as antibody escape mutants. Furthermore, in-silico Ala-scanning and long-timescale MD simulations, highlight the crucial role of the residues at the interface of RBD and ACE2 that may be used as potential pharmacophores for any drug development endeavors. From an evolutional perspective, this study also identifies how the virus has evolved from its predecessor SARS-COV and how it could further evolve to become more infectious.


id: cord-310636-y7n22ykt
title: COVID-19 neutralizing antibodies predict disease severity and survival
author: Garcia-Beltran, W. F.
date: 2020-10-20
url: https://www.ncbi.nlm.nih.gov/pubmed/33106822/
doi: 10.1101/2020.10.15.20213512
abstract:
  COVID-19 exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, and high anti-RBD antibody levels. While anti-RBD IgG levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting protection from reinfection by this strain. However, SARS-CoV-2 sera was unable to cross-neutralize a highly-homologous pre-emergent bat coronavirus, WIV1-CoV, that has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.


id: cord-311035-s3tkbh9r
title: Deep mutagenesis in the study of COVID-19: a technical overview for the proteomics community
author: Procko, Erik
date: 2020-10-21
txt: ./txt/cord-311035-s3tkbh9r.txt
cache: ./cache/cord-311035-s3tkbh9r.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1080/14789450.2020.1833721
doi: 10.1080/14789450.2020.1833721
summary:
  A deep mutational scan of ACE2 expressed on human cells identified mutations that increase S affinity and guided the engineering of a potent and broad soluble receptor decoy. • The experimental mutational landscape of ACE2 for binding the RBD of SARS-CoV-2 provides a blueprint for engineering high affinity decoy receptors. Following FACS selection of the human culture to enrich a cell population with high binding activity for SARS-CoV-2 protein S, RNA transcripts were isolated and Illumina sequenced. The deep mutational scan of ACE2 revealed that mutations can indeed be found to enhance binding toward SARS-CoV-2 RBD (Figure 2) , suitable for engineering high affinity soluble decoy receptors [15] . A soluble ACE2 variant that combines three mutations, called sACE2 2 .v2.4, was found to be highly expressed, is a stable monodisperse dimer, binds SARS-CoV-2 S with picomolar affinity and potently neutralizes infection of a susceptible cell line by authentic virus.

abstract:
  Introduction The spike (S) of SARS coronavirus 2 (SARS-CoV-2) engages angiotensin-converting enzyme 2 (ACE2) on a host cell to trigger viral-cell membrane fusion and infection. The extracellular region of ACE2 can be administered as a soluble decoy to compete for binding sites on the receptor-binding domain (RBD) of S, but it has only moderate affinity and efficacy. The RBD, which is targeted by neutralizing antibodies, may also change and adapt through mutation as SARS-CoV-2 becomes endemic, posing challenges for therapeutic and vaccine development. Areas Covered Deep mutagenesis is a Big Data approach to characterizing sequence variants. A deep mutational scan of ACE2 expressed on human cells identified mutations that increase S affinity and guided the engineering of a potent and broad soluble receptor decoy. A deep mutational scan of the RBD displayed on the surface of yeast has revealed residues tolerant of mutational changes that may act as a source for drug resistance and antigenic drift. Expert Opinion Deep mutagenesis requires a selection of diverse sequence variants; an in vitro evolution experiment that is tracked with next-generation sequencing. The choice of expression system, diversity of the variant library and selection strategy have important consequences for data quality and interpretation.


id: cord-312560-onfabcfv
title: Role of IgM and IgA Antibodies to the Neutralization of SARS-CoV-2
author: Klingler, J.
date: 2020-08-21
txt: ./txt/cord-312560-onfabcfv.txt
cache: ./cache/cord-312560-onfabcfv.txt
extension: .txt
mime: text/plain
url: http://medrxiv.org/cgi/content/short/2020.08.18.20177303v1?rss=1
doi: 10.1101/2020.08.18.20177303
summary:
  The data demonstrate high prevalence of spike-and RBD-specific IgM and IgA, similar to that of IgG1, in plasma/serum from COVID-19 patients and their significant contributions to virusneutralizing activities. In Fig. 3 , comparing levels of total Ig with the different Ig isotypes showed a highly significant correlation with IgG1 for both Abs specific for spike and RBD indicating that IgG1 is the major isotype induced by SARS-CoV-2 infection. To ask directly to what extent Abs of different isotypes mediate neutralization, we evaluated the neutralization activities of IgM, IgG, and IgA fractions purified from plasma from five COVID-19-convalescent individuals (RP#1-5). Several SARS-CoV-2 vaccine candidates tested in animal models and humans were shown to induce IgG responses against spike and RBD as well as virus neutralizing activities, but in many of these studies, the induction of other Ig isotypes was not evaluated 46-49 .

abstract:
  SARS-CoV-2 has infected millions of people and is on a trajectory to kill more than one million globally. Virus entry depends on the receptor-binding domain (RBD) of the spike protein. Although previous studies demonstrated anti-spike and -RBD antibodies as essential for protection and convalescent plasma as a promising therapeutic option, little is known about the immunoglobulin (Ig) isotypes capable of blocking virus entry. Here, we studied spike- and RBD-specific Ig isotypes in plasma/sera from two acutely infected and 29 convalescent individuals. Spike- and RBD-specific IgM, IgG1, and IgA1 antibodies were produced by all or nearly all subjects at varying levels and detected at 7-8 days post-disease onset. IgG2, IgG3, IgG4, and IgA2 were also present but at much lower levels. All samples also displayed neutralizing activity. IgM, IgG, and IgA were capable of mediating neutralization, but neutralization titers correlated better with binding levels of IgM and IgA1 than IgG.


id: cord-314574-3e6u4aza
author: Tian, Xiaolong
date: 2020-02-17
txt: ./txt/cord-314574-3e6u4aza.txt
cache: ./cache/cord-314574-3e6u4aza.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1080/22221751.2020.1729069
doi: 10.1080/22221751.2020.1729069
summary:
  Considering the relatively high identity of receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. Interestingly, some of the most potent SARS-CoV-specific neutralizing antibodies (e.g. m396, CR3014) that target the ACE2 binding site of SARS-CoV failed to bind 2019-nCoV spike protein, implying that the difference in the RBD of SARS-CoV and 2019-nCoV has a critical impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD. Next, we expressed and purified several representative SARS-CoV-specific antibodies which have been reported to target RBD and possess potent neutralizing activities, including m396 [3] , CR3014 [4] , CR3022 [5] , as well as a MERS-CoV-specific human monoclonal antibody m336 developed by our laboratory [15] , and measured their binding ability to 2019-nCoV RBD by ELISA (Figure 1(e)) .

title:
  Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody

abstract:
  The newly identified 2019 novel coronavirus (2019-nCoV) has caused more than 11,900 laboratory-confirmed human infections, including 259 deaths, posing a serious threat to human health. Currently, however, there is no specific antiviral treatment or vaccine. Considering the relatively high identity of receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. Here, we report for the first time that a SARS-CoV-specific human monoclonal antibody, CR3022, could bind potently with 2019-nCoV RBD (KD of 6.3 nM). The epitope of CR3022 does not overlap with the ACE2 binding site within 2019-nCoV RBD. These results suggest that CR3022 may have the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-nCoV infections. Interestingly, some of the most potent SARS-CoV-specific neutralizing antibodies (e.g. m396, CR3014) that target the ACE2 binding site of SARS-CoV failed to bind 2019-nCoV spike protein, implying that the difference in the RBD of SARS-CoV and 2019-nCoV has a critical impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD.


id: cord-314676-ndke9agh
author: Gollapalli, Pavan
date: 2020-11-04
url: https://www.ncbi.nlm.nih.gov/pubmed/33146070/
doi: 10.1080/07391102.2020.1841681
title:
  Pathway enrichment analysis of virus-host interactome and prioritization of novel compounds targeting the spike glycoprotein receptor binding domain–human angiotensin-converting enzyme 2 interface to combat SARS-CoV-2

abstract:
  SARS-CoV-2 has become a pandemic causing a serious global health concern. The absence of effective drugs for treatment of the disease has caused its rapid spread on a global scale. Similarly to the SARS-CoV, the SARS-CoV-2 is also involved in a complex interplay with the host cells. This infection is characterized by a diffused alveolar damage consistent with the Acute Respiratory Disease Syndrome (ARDS). To explore the complex mechanisms of the disease at the system level, we used a network medicine tools approach. The protein-protein interactions (PPIs) between the SARS-CoV and the associated human cell proteins are crucial for the viral pathogenesis. Since the cellular entry of SARS-CoV-2 is accomplished by binding of the spike glycoprotein binding domain (RBD) to the human angiotensin-converting enzyme 2 (hACE2), a molecule that can bind to the spike RDB-hACE2 interface could block the virus entry. Here, we performed a virtual screening of 55 compounds to identify potential molecules that can bind to the spike glycoprotein and spike-ACE2 complex interface. It was found that the compound ethyl 1-{3-[(2,4-dichlorobenzyl) carbamoyl]-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-7-quinolinyl}-4-piperidine carboxylate (the S54 ligand) and ethyl 1-{3-[(2,4-dichlorobenzyl) carbamoyl]-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-7-quinolinyl}-4 piperazine carboxylate (the S55 ligand) forms hydrophobic interactions with Tyr41A, Tyr505B and Tyr553B, Leu29A, Phe495B, respectively of the spike glycoprotein, the hotspot residues in the spike glycoprotein RBD-hACE2 binding interface. Furthermore, molecular dynamics simulations and free energy calculations using the MM-GBSA method showed that the S54 ligand is a stronger binder than a known SARS-CoV spike inhibitor SSAA09E3 (N-(9,10-dioxo-9, 10-dihydroanthracen-2-yl) benzamide). Communicated by Ramaswamy H. Sarma


id: cord-315415-3aotsb2g
title: Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2
author: Dong, Jianbo
date: 2020-10-20
url: https://www.ncbi.nlm.nih.gov/pubmed/33082473/
doi: 10.1038/s41598-020-74761-y
abstract:
  SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction with the human ACE2 receptor. In this study we used computer-aided design to construct multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19.


id: cord-315437-h6xjudm0
title: Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen
author: Nyon, Mun Peak
date: 2018-03-27
url: https://api.elsevier.com/content/article/pii/S0264410X18302524
doi: 10.1016/j.vaccine.2018.02.065
abstract:
  Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377–588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.


id: cord-318018-ybdkp398
author: Bruni, Margherita
date: 2020-10-01
txt: ./txt/cord-318018-ybdkp398.txt
cache: ./cache/cord-318018-ybdkp398.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.3390/jcm9103188
doi: 10.3390/jcm9103188
summary:
  Sera from healthcare workers affected by non-severe COVID-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in Intensive Care Units (ICU) and SARS-CoV-2-negative subjects for the presence of IgM, IgG and IgA antibodies as well as soluble pro-inflammatory mediators in the sera. Our data show that humoral immune responses against SARS-CoV-2 correlated with disease severity in terms of both antibody titers, persistence over time and serum levels of pro-inflammatory cytokines. Here we show that humoral immune responses against SARS-CoV-2 correlated with disease severity in terms of both antibody titers, persistence over time and serum levels of pro-inflammatory mediators. Moreover, we showed that the vast majority of COVID-19 mildly symptomatic patients analyzed in the study halved their anti-RBD antibody titers after 4 weeks from viral negativization, thus confirming the short lifespan of humoral immune responses against SARS-CoV-2.

title:
  Persistence of Anti-SARS-CoV-2 Antibodies in Non-Hospitalized COVID-19 Convalescent Health Care Workers

abstract:
  Although antibody response to SARS-CoV-2 can be detected early during the infection, several outstanding questions remain to be addressed regarding the magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response. An ELISA assay has been developed by expressing and purifying the recombinant SARS-CoV-2 Spike Receptor Binding Domain (RBD), Soluble Ectodomain (Spike), and full length Nucleocapsid protein (N). Sera from healthcare workers affected by non-severe COVID-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in Intensive Care Units (ICU) and SARS-CoV-2-negative subjects for the presence of IgM, IgG and IgA antibodies as well as soluble pro-inflammatory mediators in the sera. Non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles as compared to patients in Intensive Care Units (ICU), irrespective of the antibodies tested. Noteworthy, in non-severe COVID-19 infections, antibody titers against RBD and Spike, but not against the N protein, as well as pro-inflammatory cytokines decreased within a month after viral clearance. Thus, rapid decline in antibody titers and in pro-inflammatory cytokines may be a common feature of non-severe SARS-CoV-2 infection, suggesting that antibody-mediated protection against re-infection with SARS-CoV-2 is of short duration. These results suggest caution in using serological testing to estimate the prevalence of SARS-CoV-2 infection in the general population.


id: cord-318444-sgm24q1i
title: Sybodies targeting the SARS-CoV-2 receptor-binding domain
author: Walter, Justin D.
date: 2020-05-16
url: https://doi.org/10.1101/2020.04.16.045419
doi: 10.1101/2020.04.16.045419
abstract:
  The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has resulted in a global health and economic crisis of unprecedented scale. The high transmissibility of SARS-CoV-2, combined with a lack of population immunity and prevalence of severe clinical outcomes, urges the rapid development of effective therapeutic countermeasures. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2. In an expeditious process taking only twelve working days, sybodies were selected entirely in vitro from three large combinatorial libraries, using ribosome and phage display. We obtained six strongly enriched sybody pools against the isolated RBD and identified 63 unique anti-RBD sybodies which also interact in the context of the full-length SARS-CoV-2 spike ectodomain. Among the selected sybodies, six were found to bind to the viral spike with double-digit nanomolar affinity, and five of these also showed substantial inhibition of RBD interaction with human angiotensin-converting enzyme 2 (ACE2). Additionally, we identified a pair of anti-RBD sybodies that can simultaneously bind to the RBD. It is anticipated that compact binders such as these sybodies could feasibly be developed into an inhalable drug that can be used as a convenient prophylaxis against COVID-19. Moreover, generation of polyvalent antivirals, via fusion of anti-RBD sybodies to additional small binders recognizing secondary epitopes, could enhance the therapeutic potential and guard against escape mutants. We present full sequence information and detailed protocols for the identified sybodies, as a freely accessible resource.


id: cord-319571-fspmgg4s
author: Sehailia, Moussa
date: 2020-07-22
url: https://www.ncbi.nlm.nih.gov/pubmed/32696720/
doi: 10.1080/07391102.2020.1796809
title:
  Antimalarial-agent artemisinin and derivatives portray more potent binding to Lys353 and Lys31-binding hotspots of SARS-CoV-2 spike protein than hydroxychloroquine: potential repurposing of artenimol for COVID-19

abstract:
  Medicinal herbs have proved along history to be a source of multiple cures. In this paper, we demonstrate how hydroxychloroquine can act as a good inhibitor of SARS-CoV-2 Spike protein receptor-binding-domain using molecular docking studies. We also unveil how hydroxychloroquine can interfere in the prevention of Lys353 in hACE2 from interacting with the corresponding binding hotspot present on the Spike protein. Further screening of artemisinin & derived compounds produced better Vina docking score than hydroxychloroquine (-7.1 kcal mol(−1) for artelinic acid vs. −5.5 kcal mol(−1) for hydroxychloroquine). Artesunate, artemisinin and artenimol, showed two mode of interactions with Lys353 and Lys31 binding hotspots of the Spike protein. Molecular dynamics analysis confirmed that the formed complexes are able to interact and remain stable in the active site of their respective targets. Given that these molecules are effective antivirals with excellent safety track records in humans against various ailment, we recommend their potential repurposing for the treatment of SARS-CoV-2 patients after successful clinical studies. In addition, an extraction protocol for artemisinin from Artemisia annua L. is proposed in order to cope with the potential urgent global demand. Communicated by Ramaswamy H. Sarma


id: cord-319590-f9qcabcx
title: Computational Design of ACE2-Based Peptide Inhibitors of SARS-CoV-2
author: Han, Yanxiao
date: 2020-04-14
url: https://www.ncbi.nlm.nih.gov/pubmed/32286790/
doi: 10.1021/acsnano.0c02857
abstract:
  [Image: see text] Peptide inhibitors against the SARS-CoV-2 coronavirus, currently causing a worldwide pandemic, are designed and simulated. The inhibitors are mostly formed by two sequential self-supporting α-helices (bundle) extracted from the protease domain (PD) of angiotensin-converting enzyme 2 (ACE2), which bind to the SARS-CoV-2 receptor binding domains. Molecular dynamics simulations revealed that the α-helical peptides maintain their secondary structure and provide a highly specific and stable binding (blocking) to SARS-CoV-2. To provide a multivalent binding to the SARS-CoV-2 receptors, many such peptides could be attached to the surfaces of nanoparticle carriers. The proposed peptide inhibitors could provide simple and efficient therapeutics against the COVID-19 disease.


id: cord-319855-78xmxymu
author: BR, Bharath
date: 2020-07-01
url: https://doi.org/10.12688/f1000research.24143.1
doi: 10.12688/f1000research.24143.1
title:
  In silico screening of known small molecules to bind ACE2 specific RBD on Spike glycoprotein of SARS-CoV-2 for repurposing against COVID-19

abstract:
  Background: Human coronavirus (SARS-CoV-2) is causing a pandemic with significant morbidity and mortality. As no effective novel drugs are available currently, drug repurposing is an alternative intervention strategy. Here we present an in silico drug repurposing study that implements successful concepts of computer-aided drug design (CADD) technology for repurposing known drugs to interfere with viral cellular entry via the spike glycoprotein (SARS-CoV-2-S), which mediates host cell entry via the hACE2 receptor. Methods: A total of 4015 known and approved small molecules were screened for interaction with SARS-CoV-2-S through docking studies and 15 lead molecules were shortlisted. Additionally, streptomycin, ciprofloxacin, and glycyrrhizic acid (GA) were selected based on their reported anti-viral activity, safety, availability and affordability. The 18 molecules were subjected to molecular dynamics (MD) simulation. Results: The MD simulation results indicate that GA of plant origin may be repurposed for SARS-CoV-2 intervention, pending further studies. Conclusions: Repurposing is a beneficial strategy for treating COVID-19 with existing drugs. It is aimed at using docking studies to screen molecules for clinical application and investigating their efficacy in inhibiting SARS-CoV-2-S. SARS-CoV-2-S is a key pathogenic protein that mediates pathogen-host interaction. Hence, the molecules screened for inhibitory properties against SARS-CoV-2-S can be clinically used to treat COVID-19 since the safety profile is already known.


id: cord-320238-qbjrlog1
author: Okba, Nisreen M. A.
date: 2020-05-29
url: https://doi.org/10.1080/22221751.2020.1760735
doi: 10.1080/22221751.2020.1760735
title:
  Particulate multivalent presentation of the receptor binding domain induces protective immune responses against MERS-CoV

abstract:
  Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (RBD) ─ and tested their immunogenicity and protective capacity in rabbits. Using a “plug-and-display” SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS-CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses such as SARS-CoV-2.


id: cord-321166-nvphu1fm
author: Thomson, Emma C.
date: 2020-11-05
url: https://doi.org/10.1101/2020.11.04.355842
doi: 10.1101/2020.11.04.355842
title:
  The circulating SARS-CoV-2 spike variant N439K maintains fitness while evading antibody-mediated immunity

abstract:
  SARS-CoV-2 can mutate to evade immunity, with consequences for the efficacy of emerging vaccines and antibody therapeutics. Herein we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is the most divergent region of S, and provide epidemiological, clinical, and molecular characterization of a prevalent RBM variant, N439K. We demonstrate that N439K S protein has enhanced binding affinity to the hACE2 receptor, and that N439K virus has similar clinical outcomes and in vitro replication fitness as compared to wild- type. We observed that the N439K mutation resulted in immune escape from a panel of neutralizing monoclonal antibodies, including one in clinical trials, as well as from polyclonal sera from a sizeable fraction of persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.


id: cord-321854-cy8vyb6j
author: Ripperger, Tyler J.
date: 2020-10-14
url: https://www.ncbi.nlm.nih.gov/pubmed/33129373/
doi: 10.1016/j.immuni.2020.10.004
title:
  Orthogonal SARS-CoV-2 Serological Assays Enable Surveillance of Low Prevalence Communities and Reveal Durable Humoral Immunity.

abstract:
  We conducted a serological study to define correlates of immunity against SARS-CoV-2. Relative to mild COVID-19 cases, individuals with severe disease exhibited elevated virus-neutralizing titers and antibodies against nucleocapsid (N) and the receptor binding domain (RBD) of spike protein. Age and sex played lesser roles. All cases, including asymptomatic individuals, seroconverted by 2 weeks post-PCR confirmation. Spike RBD and S2 and neutralizing antibodies remained detectable through 5-7 months post-onset, whereas α-N titers diminished. Testing of 5882 members of the local community revealed only 1 sample with seroreactivity to both RBD and S2 that lacked neutralizing antibodies. This fidelity could not be achieved with either RBD or S2 alone. Thus, inclusion of multiple independent assays improved the accuracy of antibody tests in low seroprevalence communities and revealed differences in antibody kinetics depending on the antigen. We conclude that neutralizing antibodies are stably produced for at least 5-7 months after SARS-CoV-2 infection.


id: cord-321918-9jwma2y6
title: Inhibitors of SARS-CoV-2 Entry: Current and Future Opportunities
author: Xiu, Siyu
date: 2020-06-15
txt: ./txt/cord-321918-9jwma2y6.txt
cache: ./cache/cord-321918-9jwma2y6.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1021/acs.jmedchem.0c00502
doi: 10.1021/acs.jmedchem.0c00502
summary:
  The spike protein can be divided into two domains; S1 is responsible for angiotensin-converting enzyme II(ACE2) recognition, the recently identified host cell receptor, and S2 mediates membrane fusion (Figure 2 ). 98 99 On the basis of this approach, they identified two small molecules, TGG (12, Table 4 ) and luteolin (13) , that can bind avidly to the SARS-CoV S2 protein and inhibit viral entry of SARS-CoV into Vero E6 cells with IC 50 values of 4.5 and 10.6 μM, respectively. 113 A high-throughput screen (HTS) of a 1000-compound library that resulted in the identification of MDL28170 (17 , Table 4 ) by Bates et al., and in an antiviral activity assay, 17 specifically inhibited cathepsin L-mediated substrate cleavage and blocked SARS-CoV viral entry, with an IC 50 value of 2.5 nM and EC 50 value in the range of 100 nM.

abstract:
  [Image: see text] Recently, a novel coronavirus initially designated 2019-nCoV but now termed SARS-CoV-2 has emerged and raised global concerns due to its virulence. SARS-CoV-2 is the etiological agent of “coronavirus disease 2019”, abbreviated to COVID-19, which despite only being identified at the very end of 2019, has now been classified as a pandemic by the World Health Organization (WHO). At this time, no specific prophylactic or postexposure therapy for COVID-19 are currently available. Viral entry is the first step in the SARS-CoV-2 lifecycle and is mediated by the trimeric spike protein. Being the first stage in infection, entry of SARS-CoV-2 into host cells is an extremely attractive therapeutic intervention point. Within this review, we highlight therapeutic intervention strategies for anti-SARS-CoV, MERS-CoV, and other coronaviruses and speculate upon future directions for SARS-CoV-2 entry inhibitor designs.


id: cord-323324-h2a25xym
author: Armijos‐Jaramillo, Vinicio
date: 2020-05-07
url: https://www.ncbi.nlm.nih.gov/pubmed/32837536/
doi: 10.1111/eva.12980
title:
  SARS‐CoV‐2, an evolutionary perspective of interaction with human ACE2 reveals undiscovered amino acids necessary for complex stability

abstract:
  The emergence of SARS‐CoV‐2 has resulted in nearly 1,280,000 infections and 73,000 deaths globally so far. This novel virus acquired the ability to infect human cells using the SARS‐CoV cell receptor hACE2. Because of this, it is essential to improve our understanding of the evolutionary dynamics surrounding the SARS‐CoV‐2 hACE2 interaction. One way theory predicts selection pressures should shape viral evolution is to enhance binding with host cells. We first assessed evolutionary dynamics in select betacoronavirus spike protein genes to predict whether these genomic regions are under directional or purifying selection between divergent viral lineages, at various scales of relatedness. With this analysis, we determine a region inside the receptor‐binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor ACE2. Next, to gain further insights into factors associated with recognition of the human host receptor, we performed modeling studies of five different betacoronaviruses and their potential binding to hACE2. Modeling results indicate that interfering with the salt bridges at hot spot 353 could be an effective strategy for inhibiting binding, and hence for the prevention of SARS‐CoV‐2 infections. We also propose that a glycine residue at the receptor‐binding domain of the spike glycoprotein can have a critical role in permitting bat SARS‐related coronaviruses to infect human cells.


id: cord-323514-jaom3p6s
author: He, Yuxian
date: 2006-05-26
txt: ./txt/cord-323514-jaom3p6s.txt
cache: ./cache/cord-323514-jaom3p6s.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16615996/
doi: 10.1016/j.bbrc.2006.03.139
summary:
  Abstract The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318–510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. In this study, we used the RBD-Fc as a model to study how a single residue mutation in the RBD can abolish the major function of full-length S protein, since this molecule can efficiently bind to the receptor ACE2 and contains multiple conformation-dependent epitopes (Conf I-VI) capable of inducing highly potent neutralizing antibodies [29] .

title:
  A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity

abstract:
  Abstract The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318–510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.


id: cord-323967-2mo915u1
author: Miersch, Shane
date: 2020-11-01
url: https://doi.org/10.1101/2020.10.31.362848
doi: 10.1101/2020.10.31.362848
title:
  Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations

abstract:
  Recombinant neutralizing antibodies (nAbs) derived from recovered patients have proven to be effective therapeutics for COVID-19. Here, we describe the use of advanced protein engineering and modular design principles to develop tetravalent synthetic nAbs that mimic the multi-valency exhibited by IgA molecules, which are especially effective natural inhibitors of viral disease. At the same time, these nAbs display high affinity and modularity typical of IgG molecules, which are the preferred format for drugs. We show that highly specific tetravalent nAbs can be produced at large scale and possess stability and specificity comparable to approved antibody drugs. Moreover, structural studies reveal that the best nAb targets the host receptor binding site of the virus spike protein, and thus, its tetravalent version can block virus infection with a potency that exceeds that of the bivalent IgG by an order of magnitude. Design principles defined here can be readily applied to any antibody drug, including IgGs that are showing efficacy in clinical trials. Thus, our results present a general framework to develop potent antiviral therapies against COVID-19, and the strategy can be readily deployed in response to future pathogenic threats.


id: cord-326282-uxn64olw
title: Real-time Conformational Dynamics of SARS-CoV-2 Spikes on Virus Particles
author: Lu, Maolin
date: 2020-09-13
url: https://doi.org/10.1101/2020.09.10.286948
doi: 10.1101/2020.09.10.286948
abstract:
  SARS-CoV-2 spike (S) mediates entry into cells and is critical for vaccine development against COVID-19. Structural studies have revealed distinct conformations of S, but real-time information that connects these structures, is lacking. Here we apply single-molecule Förster Resonance Energy Transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to hACE2, S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences of convalescent plasma and antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to RBD or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.


id: cord-326337-s0fp5z1q
title: An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants
author: Chan, Kui K.
date: 2020-10-19
txt: ./txt/cord-326337-s0fp5z1q.txt
cache: ./cache/cord-326337-s0fp5z1q.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.10.18.344622
doi: 10.1101/2020.10.18.344622
summary:
  Deep mutagenesis of the isolated receptor-binding domain (RBD) by yeast surface display 44 has easily identified mutations in S that retain high expression and ACE2 affinity, yet are no longer bound 45 by monoclonal antibodies and confer resistance (19) . An alternative protein-based antiviral to monoclonal antibodies is to use soluble ACE2 (sACE2) as a 56 decoy to compete for receptor-binding sites on the viral spike (6, (22) (23) (24) (25) of diverse SARS-associated betacoronaviruses that use ACE2 for entry. The sequence 162 diversity observed among natural betacoronaviruses, which display high diversity at the ACE2 binding 163 site, is therefore replicated in the deep mutational scan, which predicts the SARS-CoV-2 spike tolerates 164 substantial genetic diversity at the receptor-binding site for function. From this accessible sequence 165 diversity SARS-CoV-2 might feasibly mutate to acquire resistance to monoclonal antibodies or 166 engineered decoy receptors targeting the ACE2-binding site.

abstract:
  The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, due to close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find an engineered decoy receptor, sACE22.v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild type receptor. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.


id: cord-327654-9g8zcxaa
author: Chi, Xiaojing
date: 2020-09-10
url: https://doi.org/10.1038/s41467-020-18387-8
doi: 10.1038/s41467-020-18387-8
title:
  Humanized single domain antibodies neutralize SARS-CoV-2 by targeting the spike receptor binding domain

abstract:
  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads worldwide and leads to an unprecedented medical burden and lives lost. Neutralizing antibodies provide efficient blockade for viral infection and are a promising category of biological therapies. Here, using SARS-CoV-2 spike receptor-binding domain (RBD) as a bait, we generate a panel of humanized single domain antibodies (sdAbs) from a synthetic library. These sdAbs reveal binding kinetics with the equilibrium dissociation constant (K(D)) of 0.99–35.5 nM. The monomeric sdAbs show half maximal neutralization concentration (EC(50)) of 0.0009–0.07 µg/mL and 0.13–0.51 µg/mL against SARS-CoV-2 pseudotypes, and authentic SARS-CoV-2, respectively. Competitive ligand-binding experiments suggest that the sdAbs either completely block or significantly inhibit the association between SARS-CoV-2 RBD and viral entry receptor ACE2. Fusion of the human IgG1 Fc to sdAbs improve their neutralization activity by up to ten times. These results support neutralizing sdAbs as a potential alternative for antiviral therapies.


id: cord-327711-welf0eb1
title: Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient
author: Zhou, Daming
date: 2020-06-13
txt: ./txt/cord-327711-welf0eb1.txt
cache: ./cache/cord-327711-welf0eb1.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.06.12.148387
doi: 10.1101/2020.06.12.148387
summary:
  Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19. A neutralisation test for EY6A based on quantitative PCR detection of virus in the supernatant bathing infected Vero E6 cells after 5 days of culture, showed a ~1000-fold reduction in virus signal (Methods, Extended Data Fig. 3 ) indicating that it is highly neutralising. To elucidate the epitope of EY6A, we determined the crystal structures of the deglycosylated SARS-CoV-2 RBD in complex with EY6A Fab alone and in a ternary complex incorporating a nanobody (Nb) which has been shown to compete with ACE2 (for data on a closely related Nb see Huo 2020, submitted), as a crystallisation chaperone.

abstract:
  The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6Å crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.


id: cord-328003-yovp8squ
author: Duan, Liangwei
date: 2020-10-07
txt: ./txt/cord-328003-yovp8squ.txt
cache: ./cache/cord-328003-yovp8squ.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/33117378/
doi: 10.3389/fimmu.2020.576622
summary:
  Here, we provide a comprehensive overview of the wealth of research related to the SARS-CoV-2 S glycoprotein biosynthesis, structure, function, and antigenicity, aiming to provide useful insights into the design and development of the S protein-based vaccines as well as therapeutics to prevent or treat the ongoing global spread of SARS-CoV-2/COVID-19. Prefusion structures of human coronavirus HKU1 (HCoV-HKU1) and mouse hepatitis virus S protein ectodomains without two consecutive proline mutations reveal only fully closed conformation (37, 42) , similar to that observed for a full-length, wild-type prefusion form of the SARS-CoV-2 S glycoprotein (41) . Therefore, SARS-CoV-2 evades immune surveillance also through conformational masking, which is well-documented for HIV-1 (43, 44) ; while at the same time, the S protein could transiently sample the functional state to engage ACE2, consistent with the notion that the fusion glycoprotein of highly pathogenic viruses have evolved to perform its functions while evading host neutralizing antibody responses.

title:
  The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens

abstract:
  The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a grave threat to global public health and imposes a severe burden on the entire human society. Like other coronaviruses, the SARS-CoV-2 genome encodes spike (S) glycoproteins, which protrude from the surface of mature virions. The S glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. Surface location of the S glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. In the light of its crucial roles in viral infection and adaptive immunity, the S protein is the focus of most vaccine strategies as well as therapeutic interventions. In this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the SARS-CoV-2 S glycoprotein, aiming to provide valuable insights into the design and development of the S protein-based vaccines as well as therapeutics.


id: cord-328189-jpkxjn6e
title: The SARS-CoV-2 exerts a distinctive strategy for interacting with the ACE2 human receptor
author: Brielle, Esther S.
date: 2020-03-12
txt: ./txt/cord-328189-jpkxjn6e.txt
cache: ./cache/cord-328189-jpkxjn6e.txt
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url: https://doi.org/10.1101/2020.03.10.986398
doi: 10.1101/2020.03.10.986398
summary:
  We compare the interaction between the human ACE2 receptor and the SARS-CoV-2 spike protein with that of other pathogenic coronaviruses using molecular dynamics simulations. Herein, we analyze the binding of several CoV RBDs to ACE2 with molecular dynamics (MD) simulations and compare the stability, relative interaction strength, and dynamics of the interaction between the viral spike protein and the human ACE2 receptor. While the sequence identity between the RBDs of COVID-19 and SARS-2002 is 73% (Table 1) , we observe a significantly higher residue substitution rate at the interaction interface with the ACE2 receptor. Our MD simulation analysis reveals that the SARS-des has a substantially lower interaction scores with ACE2 (median of -2199.2, Fig. S2) , as expected for an optimized human ACE2-binding RBD design. We relied on the crystal structure of the spike protein receptor-binding domain from a SARS coronavirus designed human strain complexed with the human receptor ACE2 (PDB 3SCI, resolution 2.9Å) as a template for comparative modeling.

abstract:
  The COVID-19 disease has plagued over 110 countries and has resulted in over 4,000 deaths within 10 weeks. We compare the interaction between the human ACE2 receptor and the SARS-CoV-2 spike protein with that of other pathogenic coronaviruses using molecular dynamics simulations. SARS-CoV, SARS-CoV-2, and HCoV-NL63 recognize ACE2 as the natural receptor but present a distinct binding interface to ACE2 and a different network of residue-residue contacts. SARS-CoV and SARS-CoV-2 have comparable binding affinities achieved by balancing energetics and dynamics. The SARS-CoV-2–ACE2 complex contains a higher number of contacts, a larger interface area, and decreased interface residue fluctuations relative to SARS-CoV. These findings expose an exceptional evolutionary exploration exerted by coronaviruses toward host recognition. We postulate that the versatility of cell receptor binding strategies has immediate implications on therapeutic strategies. One Sentence Summary Molecular dynamics simulations reveal a temporal dimension of coronaviruses interactions with the host receptor.


id: cord-328578-9qzo18v3
title: SARS‐CoV‐2 S1 is superior to the RBD as a COVID‐19 subunit vaccine antigen
author: Wang, Yunfei
date: 2020-07-21
txt: ./txt/cord-328578-9qzo18v3.txt
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url: https://doi.org/10.1002/jmv.26320
doi: 10.1002/jmv.26320
summary:
  When immunized in mice, the S1 domain induced much higher IgG and IgA antibody levels than the RBD and more efficiently neutralized SARS‐CoV‐2 when adjuvanted with alum. Article SARS-CoV-2 S1 induces higher IgG and IgA titers than RBD 2 weeks after the third intramuscular immunization (Figure 2A) , both S1-specific (S1-coated plate in Figure 2 ) and RBD-specific (RBD-coated plate in Figure 2 ) antibodies were analyzed. HEK293K cell-expressed recombinant S1 (S1 immunized) and E.coli-expressed norovirus shell domain-S1 fusion protein (S-S1 immunized) induced similar S1-specific IgG titers (64 000) and similar RBD-specific titers (8 000) ( Figure 2B ). HEK293K cell-expressed recombinant RBD (RBD immunized) and E.coli-expressed norovirus shell domain-RBD fusion proteins (S-RBD immunized) induced low levels of IgG1 and IgG2a titers specific to both S1 (S1 coated in Figure 3A&B ) and the RBD (RBD coated in Figure 3A&B ).

abstract:
  Since its emergence in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has developed into a global pandemic within a matter of months. While subunit vaccines are one of the prominent options for combating coronavirus disease 2019 (COVID‐19), the immunogenicity of spike protein‐based antigens remains unknown. When immunized in mice, the S1 domain induced much higher IgG and IgA antibody levels than the RBD and more efficiently neutralized SARS‐CoV‐2 when adjuvanted with alum. It is inferred that a large proportion of these neutralization epitopes are located in the S1 domain but outside the RBD and that some of these are spatial epitopes. This finding indicates that expression systems with posttranslational modification abilities are important to maintain the natural configurations of recombinant spike protein antigens and are critical for effective COVID‐19 vaccines. Further, adjuvants prone to a Th1 response should be considered for S1‐based subunit COVID‐19 vaccines to reduce the potential risk of antibody‐dependent enhancement (ADE) of infection. This article is protected by copyright. All rights reserved.


id: cord-329011-spiuqngp
author: Huang, Yuan
date: 2020-08-03
txt: ./txt/cord-329011-spiuqngp.txt
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url: https://doi.org/10.1038/s41401-020-0485-4
doi: 10.1038/s41401-020-0485-4
summary:
  The spike (S) protein of SARS-CoV-2, which plays a key role in the receptor recognition and cell membrane fusion process, is composed of two subunits, S1 and S2. A large number of glycosylated S proteins cover the surface of SARS-CoV-2 and bind to the host cell receptor angiotensinconverting enzyme 2 (ACE2), mediating viral cell entry [8] . The SARS-CoV-2 S protein is highly conserved among all human coronaviruses (HCoVs) and is involved in receptor recognition, viral attachment, and entry into host cells. Structure of the S1 subunit The binding of virus particles to cell receptors on the surface of the host cell is the initiation of virus infection; therefore, receptor recognition is an important determinant of viral entry and a drug design target. Therefore, the development of antibodies targeting this functional motif may cross-bind and neutralize these two viruses and related CoVs. Antiviral peptides prevent SARS-CoV-2 membrane fusion and can potentially be used for the prevention and treatment of infection.

title:
  Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19

abstract:
  Coronavirus disease 2019 is a newly emerging infectious disease currently spreading across the world. It is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein of SARS-CoV-2, which plays a key role in the receptor recognition and cell membrane fusion process, is composed of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme 2, while the S2 subunit mediates viral cell membrane fusion by forming a six-helical bundle via the two-heptad repeat domain. In this review, we highlight recent research advance in the structure, function and development of antivirus drugs targeting the S protein.


id: cord-329392-fufattj8
author: den Hartog, Gerco
date: 2020-08-08
url: https://www.ncbi.nlm.nih.gov/pubmed/32766833/
doi: 10.1093/infdis/jiaa479
title:
  SARS-CoV-2–Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence

abstract:
  BACKGROUND: The COVID-19 pandemic necessitates better understanding of the kinetics of antibody production induced by infection with SARS-CoV-2. We aimed to develop a high-throughput multiplex assay to detect antibodies to SARS-CoV-2 to assess immunity to the virus in the general population. METHODS: Spike protein subunits S1 and receptor binding domain, and nucleoprotein were coupled to microspheres. Sera collected before emergence of SARS-CoV-2 (n = 224) and of non-SARS-CoV-2 influenza-like illness (n = 184), and laboratory-confirmed cases of SARS-CoV-2 infection (n = 115) with various severities of COVID-19 were tested for SARS-CoV-2–specific IgG concentrations. RESULTS: Our assay discriminated SARS-CoV-2–induced antibodies and those induced by other viruses. The assay specificity was 95.1%–99.0% with sensitivity 83.6%–95.7%. By merging the test results for all 3 antigens a specificity of 100% was achieved with a sensitivity of at least 90%. Hospitalized COVID-19 patients developed higher IgG concentrations and the rate of IgG production increased faster compared to nonhospitalized cases. CONCLUSIONS: The bead-based serological assay for quantitation of SARS-CoV-2–specific antibodies proved to be robust and can be conducted in many laboratories. We demonstrated that testing of antibodies against multiple antigens increases sensitivity and specificity compared to single-antigen–specific IgG determination.


id: cord-331786-wgt7kg6f
author: Diego-Martin, Borja
date: 2020-10-13
txt: ./txt/cord-331786-wgt7kg6f.txt
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url: https://doi.org/10.1101/2020.10.13.331306
doi: 10.1101/2020.10.13.331306
summary:
  For this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against SARS-CoV-2 in Nicotiana benthamiana. In parallel, we also produced the recombinant SARS-CoV-2 N protein and its Receptor Binding Domain (RBD) in planta and used them to test the binding specificity of the recombinant mAbs. Finally, for two of the antibodies we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. Finally, we performed sandwich ELISA tests of sybody17 and nanobody72 ( Fig 5E and Fig 5F, respectively) using the total and concentrated apoplastic fluid as detection reagent against serial dilutions of crude plant extracts from RBD-producing plants, showing that this simple antibody preparation can be directly employed in detection procedures without the need of additional purification steps.

title:
  Pilot production of SARS-CoV-2 related proteins in plants: a proof of concept for rapid repurposing of indoors farms into biomanufacturing facilities

abstract:
  The current CoVid-19 crisis is revealing the strengths and the weaknesses of the world’s capacity to respond to a global health crisis. A critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. On the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions. In this work we explore how, in a context of rapid exchange of scientific information, plant biofactories can serve as a rapid and easily adaptable solution for local manufacturing of bioreagents, more specifically recombinant antibodies. For this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against SARS-CoV-2 in Nicotiana benthamiana. For the design of the antibodies we took advantage, among other data sources, of the DNA sequence information made rapidly available by other groups in preprint publications. mAbs were all engineered as single-chain fragments fused to a human gamma Fc and transiently expressed using a viral vector. In parallel, we also produced the recombinant SARS-CoV-2 N protein and its Receptor Binding Domain (RBD) in planta and used them to test the binding specificity of the recombinant mAbs. Finally, for two of the antibodies we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. Our results indicate that gram amounts of anti-SARS-CoV-2 antibodies could be easily produced in little more than 6 weeks in repurposed greenhouses with little infrastructure requirements using N. benthamiana as production platform. Similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid therapeutic responses.


id: cord-332134-88wfcc3y
title: A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection
author: Li, Tingting
date: 2020-09-24
url: https://doi.org/10.1101/2020.06.09.143438
doi: 10.1101/2020.06.09.143438
abstract:
  SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22–7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8–18. Llama-derived single-domain antibodies (nanobodies, ∼15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL−1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL−1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.


id: cord-332185-a96r1k7a
author: Zhang, Shuyuan
date: 2020-09-22
url: https://doi.org/10.1101/2020.09.21.307439
doi: 10.1101/2020.09.21.307439
title:
  Bat and pangolin coronavirus spike glycoprotein structures provide insights into SARS-CoV-2 evolution

abstract:
  In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the “down” conformation, indicating they are more prone to adopt this receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. Through structure and sequence comparisons, we identified critical residues in the RBD that underlie the different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes and propose that N-linked glycans serve as conformational control elements of the RBD. These results collectively indicate that strong RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.


id: cord-332855-u0amf1oh
author: Parsons, Lisa M.
date: 2019-03-22
txt: ./txt/cord-332855-u0amf1oh.txt
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url: https://doi.org/10.1074/jbc.ra119.007532
doi: 10.1074/jbc.ra119.007532
summary:
  In silico docking experiments with the recently published cryo-EM structure of the M41 IBV spike protein and our glycosylation results revealed a potential ligand receptor site that is ringed by four glycosylation sites that dramatically impact ligand binding. However, the Beaudette strain is a cell culture-adapted strain, is nonvirulent in chickens (15) , and does not bind chicken tissues known to be important for infectivity (11) , making it difficult to extrapolate these results to clinically relevant IBVs. To characterize and assess the role that glycosylation plays when interacting with host tissues through the RBD of pathogenic IBV strain M41, we used a combination of molecular and analytical techniques, including histochemistry, ELISA, circular dichroism (CD), MS, and docking analyses as listed in Table 1 . Six of the 10 glycosylation sites in the RBD domain of IBV M41 were essential for binding to chicken trachea tissue and an ELISA-presented sialylated oligosaccharide ligand.

title:
  Glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding

abstract:
  Avian coronaviruses, including infectious bronchitis virus (IBV), are important respiratory pathogens of poultry. The heavily glycosylated IBV spike protein is responsible for binding to host tissues. Glycosylation sites in the spike protein are highly conserved across viral genotypes, suggesting an important role for this modification in the virus life cycle. Here, we analyzed the N-glycosylation of the receptor-binding domain (RBD) of IBV strain M41 spike protein and assessed the role of this modification in host receptor binding. Ten single Asn–to–Ala substitutions at the predicted N-glycosylation sites of the M41–RBD were evaluated along with two control Val–to–Ala substitutions. CD analysis revealed that the secondary structure of all variants was retained compared with the unmodified M41–RBD construct. Six of the 10 glycosylation variants lost binding to chicken trachea tissue and an ELISA-presented α2,3-linked sialic acid oligosaccharide ligand. LC/MS(E) glycomics analysis revealed that glycosylation sites have specific proportions of N-glycan subtypes. Overall, the glycosylation patterns of most variant RBDs were highly similar to those of the unmodified M41–RBD construct. In silico docking experiments with the recently published cryo-EM structure of the M41 IBV spike protein and our glycosylation results revealed a potential ligand receptor site that is ringed by four glycosylation sites that dramatically impact ligand binding. Combined with the results of previous array studies, the glycosylation and mutational analyses presented here suggest a unique glycosylation-dependent binding modality for the M41 spike protein.


id: cord-332948-h297ukuu
author: Olotu, Fisayo A.
date: 2020-10-16
txt: ./txt/cord-332948-h297ukuu.txt
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url: https://api.elsevier.com/content/article/pii/S2352914820306018
doi: 10.1016/j.imu.2020.100451
summary:
  authors: Olotu, Fisayo A.; Omolabi, Kehinde F.; Soliman, Mahmoud E.S. title: Leaving no stone unturned: Allosteric targeting of SARS-CoV-2 Spike protein at putative druggable sites disrupts human angiotensin-converting enzyme interactions at the receptor binding domain. 30 Identification of other functional (allosteric) sites on the prefusion S protein could present another dynamic and effective approach of preventing SARS-CoV-2 infectivity relative to its interaction with the host cell ACE2 and proteases. 53 Relatively, this study was implemented to (i) identify potential druggable sites across the S1 and S2 domains of the SARS-CoV-2 S protein other than the RBD-hACE2 interface (ii) perform high-throughput (virtual) screening of ~1500 FDA approved drugs against the most druggable site(s) (iii) investigate the binding dynamics and interaction mechanisms of the compounds and their consequential effects on the S-protein RBD-ACE2 complex. We believe this systematic study will be able to provide structural and molecular insights into possible allosteric sites on SARS-CoV-2 S protein suitable for selective targeting and structureComputational methodologies

title:
  Leaving no stone unturned: Allosteric targeting of SARS-CoV-2 Spike protein at putative druggable sites disrupts human angiotensin-converting enzyme interactions at the receptor binding domain.

abstract:
  The systematic entry of SARS-CoV-2 into host cells, as mediated by its Spike (S) protein, is highly essential for pathogenicity in humans. Hence, targeting the viral entry mechanisms remains a major strategy for COVID-19 treatment. Although recent efforts have focused on the direct inhibition of S-protein receptor-binding domain (RBD) interactions with human angiotensin-converting enzyme 2 (hACE2), allosteric targeting remains an unexplored possibility. Therefore, in this study, for the first time, we employed an integrative meta-analytical approach to investigate the allosteric inhibitory mechanisms of SARS-CoV-2 S-protein and its association with hACE2. Findings revealed two druggable sites (Sites 1 and 2) located at the N-terminal domain (NTD) and S2 regions of the protein. Two high-affinity binders; ZINC3939013 (Fosaprepitant – Site 1) and ZINC27990463 (Lomitapide – Site 2) were discovered via site-directed high-throughput screening against a library of ∼1500 FDA approved drugs. Interestingly, we observed that allosteric binding of both compounds perturbed the prefusion S-protein conformations, which in turn, resulted in unprecedented hACE2 displacement from the RBD. Estimated ΔG(binds) for both compounds were highly favorable due to high-affinity interactions at the target sites. In addition, Site 1 residues; R190, H207, K206 and K187, I101, R102, I119, F192, L226, V126 and W104 were identified for their crucial involvement in the binding and stability of ZINC3939013. Likewise, energy contributions of Q957, N953, Q954, L303, Y313, Q314, L858, V952, N953, and A956 corroborated their importance to ZINC27990463 binding at the predicted Site 2. We believe these findings would pave way for the structure-based discovery of allosteric SARS-CoV-2 S-protein inhibitors for COVID-19 treatment.


id: cord-333089-ufyzqgqk
title: Structural and functional analysis of female sex hormones against SARS-Cov2 cell entry
author: Aguilar-Pineda, Jorge Alberto
date: 2020-07-29
txt: ./txt/cord-333089-ufyzqgqk.txt
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mime: text/plain
url: https://doi.org/10.1101/2020.07.29.227249
doi: 10.1101/2020.07.29.227249
summary:
  Based on the structural complementarity and steric impediments between the S protein and human ACE2 (hACE2) protein membranes, we mapped the glycosylation sites of both models [21] [22] [23] [24] and performed molecular dynamics simulations (MDS) by 250 ns to stabilize the glycosylated SARS-CoV2 spike (S) and hACE2 complex (suppl. Given the possibility that occupancy at glycosylated residues or S-RBD binding sites by estrogens could modify the affinity of the SARS-CoV2 virus and alter entry into the cell thereby reducing infectivity, we sought to further examine these interactions using a range of complementary experimental approaches (see Table S1 ). In an effort to explore the potential protective effects of female sex hormones against SARS-CoV-2 infection, we examined the impact of estradiol (17β-diol) and a dietary-derived phytoestrogen (S-equol) on hACE2 structure and protein expression by a combination of in silico modeling, in vitro, and in vivo analysis.

abstract:
  Emerging evidence suggests that males are more susceptible to severe infection by the SARS-CoV-2 virus than females. A variety of mechanisms may underlie the observed gender-related disparities including differences in sex hormones. However, the precise mechanisms by which female sex hormones may provide protection against SARS-CoV-2 infectivity remains unknown. Here we report new insights into the molecular basis of the interactions between the SARS-CoV-2 spike (S) protein and the human ACE2 receptor. We further observed that glycosylation of the ACE2 receptor enhances SARS-CoV-2 infectivity. Importantly estrogens can disrupt glycan-glycan interactions and glycan-protein interactions between the human ACE2 and the SARS-CoV2 thereby blocking its entry into cells. In a mouse model, estrogens reduced ACE2 glycosylation and thereby alveolar uptake of the SARS-CoV-2 spike protein. These results shed light on a putative mechanism whereby female sex hormones may provide protection from developing severe infection and could inform the development of future therapies against COVID-19.


id: cord-333264-jdvb8px4
title: An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction
author: Hanke, Leo
date: 2020-09-04
txt: ./txt/cord-333264-jdvb8px4.txt
cache: ./cache/cord-333264-jdvb8px4.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32887876/
doi: 10.1038/s41467-020-18174-5
summary:
  Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. The S ectodomain was purified from filtered supernatant on Streptactin XT resin (IBA Lifesciences), followed by size-exclusion chromatography on a Superdex 200 in 5 mM Tris pH 8, 200 mM NaCl. The RBD domain (RVQ-VNF) of SARS-CoV-2 was cloned upstream of an enterokinase cleavage site and a human IgG1 Fc. This plasmid was used to transiently transfect FreeStyle 293F cells using the FreeStyle MAX reagent. Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2

abstract:
  SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the ‘up’ and ‘down’ conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.


id: cord-333465-cha7ndv5
author: Horspool, A. M.
date: 2020-08-31
txt: ./txt/cord-333465-cha7ndv5.txt
cache: ./cache/cord-333465-cha7ndv5.txt
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mime: text/plain
url: https://doi.org/10.1101/2020.08.24.20180877
doi: 10.1101/2020.08.24.20180877
summary:
  Patient mortality, sex, blood type, and age were all associated with differences in antibody production to SARS-CoV-2 antigens which may help explain variation in immunity between these populations. We evaluated anti-193 SARS-CoV-2 antibody production to 3 antigens (RBD, N, and S1) in 82 in-patients 194 Table 1 ) by developing a novel rapid-ELISA technique. Our survey of SARS-CoV-2 positive patients demonstrated that antibody (IgG) 198 production to RBD, N, and S1 proteins developed over the first 10 to 20 days post-199 symptom onset (Figure 1a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. To accurately assess 223 differences in antibody production independently of disease outcome, we quantified anti-224 SARS-CoV-2 IgG production in patients who survived infection grouped by biological sex, 225 . . https://doi.org/10.1101/2020.08.24.20180877 doi: medRxiv preprint significantly increased in patients that did not survive SARS-CoV-2 infection compared to 272 those that did (Figure 4d ).

title:
  Interplay of antibody and cytokine production reveals CXCL-13 as a potential novel biomarker of lethal SARS-CoV-2 infection

abstract:
  The SARS-CoV-2 pandemic is continuing to impact the global population. This study was designed to assess the interplay of antibodies with the systemic cytokine response in SARS-CoV-2 patients. We demonstrate that significant anti-SARS-CoV-2 antibody production to Receptor Binding Domain (RBD), Nucleocapsid (N), and Spike S1 subunit (S1) of SARS-CoV-2 develops over the first 10 to 20 days of infection. The majority of patients produced antibodies against all three antigens (219/255 SARS-CoV-2 positive patient specimens, 86%) suggesting a broad response to viral proteins. Patient mortality, sex, blood type, and age were all associated with differences in antibody production to SARS-CoV-2 antigens which may help explain variation in immunity between these populations. To better understand the systemic immune response, we analyzed the production of 20 cytokines by SARS-CoV-2 patients over the course of infection. Cytokine analysis of SARS-CoV-2 positive patients exhibited increases in proinflammatory markers (IL-6, IL-8, IL-18) and chemotactic markers (IP-10, SDF-1, MIP-1{beta}, MCP-1, and eotaxin) relative to healthy individuals. Patients who succumbed to infection produced decreased IL-2, IL-4, IL-12, IL-13, RANTES, TNF-, GRO-, and MIP-1 relative to patients who survived infection. We also observed that the chemokine CXCL13 was particularly elevated in patients that succumbed to infection. CXCL13 is involved in B cell activation, germinal center development, and antibody maturation, and we observed that CXCL13 levels in blood trended with anti-SARS-CoV-2 antibody production. Furthermore, patients that succumbed to infection produced high CXCL13 and also tended to have high ratio of nucleocapsid to RBD antibodies. This study provides insights into SARS-CoV-2 immunity implicating the magnitude and specificity of response in relation to patient outcomes.


id: cord-333703-1ku3jc9s
author: Kraus, Aurora
date: 2020-11-08
txt: ./txt/cord-333703-1ku3jc9s.txt
cache: ./cache/cord-333703-1ku3jc9s.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.11.06.368191
doi: 10.1101/2020.11.06.368191
summary:
  Exposure of larvae to SARS-CoV-2 Spike (S) receptor binding domain (RBD) recombinant protein was sufficient to elevate larval heart rate and treatment with captopril, an ACE inhibitor, reverted this effect. In mice and humans, ace2 expression is detected in 121 sustentacular cells, olfactory stem cells known as horizontal and globose basal cells in the 122 olfactory epithelium, and vascular cells (pericytes) in the olfactory bulb (Brann et al., 2020 The present study reports for the first time that zebrafish larvae exposed to SARS-CoV-2 appear 134 to mount innate immune responses that resemble cytokine responses of mild COVID-19 patients. There are copious amounts of immune cells in the teleost olfactory organ ( Intranasal delivery of SARS-CoV-2 S RBD induces inflammatory responses and 318 widespread loss of olfactory receptor expression in adult zebrafish olfactory organ 319 320

title:
  A zebrafish model for COVID-19 recapitulates olfactory and cardiovascular pathophysiologies caused by SARS-CoV-2

abstract:
  The COVID-19 pandemic has prompted the search for animal models that recapitulate the pathophysiology observed in humans infected with SARS-CoV-2 and allow rapid and high throughput testing of drugs and vaccines. Exposure of larvae to SARS-CoV-2 Spike (S) receptor binding domain (RBD) recombinant protein was sufficient to elevate larval heart rate and treatment with captopril, an ACE inhibitor, reverted this effect. Intranasal administration of SARS-CoV-2 S RBD in adult zebrafish recombinant protein caused severe olfactory and mild renal histopathology. Zebrafish intranasally treated with SARS-CoV-2 S RBD became hyposmic within minutes and completely anosmic by 1 day to a broad-spectrum of odorants including bile acids and food. Single cell RNA-Seq of the adult zebrafish olfactory organ indicated widespread loss of expression of olfactory receptors as well as inflammatory responses in sustentacular, endothelial, and myeloid cell clusters. Exposure of wildtype zebrafish larvae to SARS-CoV-2 in water did not support active viral replication but caused a sustained inhibition of ace2 expression, triggered type 1 cytokine responses and inhibited type 2 cytokine responses. Combined, our results establish adult and larval zebrafish as useful models to investigate pathophysiological effects of SARS-CoV-2 and perform pre-clinical drug testing and validation in an inexpensive, high throughput vertebrate model.


id: cord-335118-oa9jfots
title: Critical Interactions Between the SARS-CoV-2 Spike Glycoprotein and the Human ACE2 Receptor
author: Taka, E.
date: 2020-09-21
txt: ./txt/cord-335118-oa9jfots.txt
cache: ./cache/cord-335118-oa9jfots.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.09.21.305490
doi: 10.1101/2020.09.21.305490
summary:
  By performing all-atom Molecular Dynamics (MD) simulations, we identified an extended network of salt bridges, hydrophobic and electrostatic interactions, and hydrogen bonding between the receptor-binding domain (RBD) of the S protein and ACE2. Initial studies have constructed a homology model of SARS-CoV-2 RBD in complex with ACE2, based on the SARS-CoV crystal structure (8, 14) and performed conventional MD (cMD) simulations totaling 10 ns (15, 16) and 100 ns (17, 18) in length to estimate binding free energies (15, 16) and interaction scores (18) . In this study, we performed a comprehensive set of all-atom MD simulations totaling 16.5 µs in length using the recently-solved structure of the RBD of the SARS-CoV-2 S protein in complex with the PD of ACE2 (7) . In 20 SMD simulations (each 15 ns, totaling 300 ns in length, table S1), the average work applied to unbind RBD from PD was 71.1 ± 12.7 kcal/mol (mean ± s.d.), demonstrating that the S protein binds stably to ACE2 (Fig. 3B) .

abstract:
  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human cells upon binding of its spike (S) glycoproteins to ACE2 receptors and causes the Coronavirus disease 2019 (COVID-19). Therapeutic approaches to prevent SARS-CoV-2 infection are mostly focused on blocking S-ACE2 binding, but critical residues that stabilize this interaction are not well understood. By performing all-atom Molecular Dynamics (MD) simulations, we identified an extended network of salt bridges, hydrophobic and electrostatic interactions, and hydrogen bonding between the receptor-binding domain (RBD) of the S protein and ACE2. Mutagenesis of these residues on the RBD was not sufficient to destabilize binding but reduced the average work to unbind the S protein from ACE2. In particular, the hydrophobic end of RBD serves as the main anchor site and unbinds last from ACE2 under force. We propose that blocking this site via neutralizing antibody or nanobody could prove an effective strategy to inhibit S-ACE2 interactions.


id: cord-335316-x2t5h5gu
author: Madariaga, M. L. L.
date: 2020-06-23
txt: ./txt/cord-335316-x2t5h5gu.txt
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mime: text/plain
url: http://medrxiv.org/cgi/content/short/2020.06.21.20132944v1?rss=1
doi: 10.1101/2020.06.21.20132944
summary:
  This was a prospective open label clinical study to assess the feasibility, safety and immunological impact of delivering anti-SARS-CoV-2 convalescent plasma to hospitalized patients aged 18 years or older with severe or life-threatening COVID-19 disease within 21 days from the onset of their illness. Univariate regression analysis for antibody titer (anti-RBD and anti-spike) was conducted against age, sex, body mass index (BMI), previous pregnancy, previous blood donation, blood type, symptoms (fever, cough, sore throat, dyspnea, abdominal pain, aguesia, anosmia, fatigue, myalgia, headache), co-morbidities (respiratory, cardiovascular, renal, diabetes, autoimmune disease, cancer, liver disease), smoking history, travel in the past 3 months to the United States, Asia or Europe, symptom duration, interval from symptoms resolution to plasma donation, and hospitalization. To determine predictors of anti-RBD and anti-spike antibody titer, we performed best subset multivariable analysis including age, sex, blood type, history of previous blood donation, fever, cough, fatigue, myalgia, symptom duration, hospitalization and travel in the United States within the past 3 months.

title:
  Clinical predictors of donor antibody titer and correlation with recipient antibody response in a COVID-19 convalescent plasma clinical trial

abstract:
  Background: Convalescent plasma therapy for COVID-19 relies on the transfer of anti-viral antibody from donors to recipients via plasma transfusion. The relationship between clinical characteristics and antibody response to COVID-19 is not well defined. We investigated predictors of convalescent antibody production and quantified recipient antibody response in a convalescent plasma therapy clinical trial. Methods: Multivariable analysis of clinical and serological parameters in 103 confirmed COVID-19 convalescent plasma donors 28 days or more following symptom resolution was performed. Mixed effects regression models with piecewise linear trends were used to characterize serial antibody responses in 10 convalescent plasma recipients with severe COVID-19. Results: Mean symptom duration of plasma donors was 11.9 and 7.8% (8/103) had been hospitalized. Antibody titers ranged from 0 to 1:3,892 (anti-receptor binding domain (RBD)) and 0 to 1:3,289 (anti-spike). Multivariable analysis demonstrated that higher anti-RBD and anti-spike titer were associated with increased age, hospitalization for COVID-19, fever, and absence of myalgia (all p<0.05). Fatigue was significantly associated with anti-RBD (p=0.03) but not anti-spike antibody titer (p=0.11). In pairwise comparison among ABO blood types, AB donors had higher anti-RBD titer than O negative donors (p=0.048) and higher anti-spike titer than O negative (p=0.015) or O positive (p=0.037) donors. Eight of the ten recipients were discharged, one remains on ECMO and one died on ECMO. No toxicity was associated with plasma transfusion. After excluding two ECMO patients and adjusting for donor antibody titer, recipient anti-RBD antibody titer increased on average 31% per day during the first three days post-transfusion (p=0.01) and anti-spike antibody titer by 40.3% (p=0.02). Conclusion: Advanced age, fever, absence of myalgia, fatigue, blood type and hospitalization were associated with higher convalescent antibody titer to COVID-19. Despite variability in donor titer, 80% of convalescent plasma recipients showed significant increase in antibody levels post-transfusion. A more complete understanding of the dose-response effect of plasma transfusion among COVID-19 patients is needed to determine the clinical efficacy of this therapy.


id: cord-336150-l8w7xk0b
title: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a newly emerged pathogen: an overview
author: Rathore, Jitendra Singh
date: 2020-08-25
txt: ./txt/cord-336150-l8w7xk0b.txt
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mime: text/plain
url: https://doi.org/10.1093/femspd/ftaa042
doi: 10.1093/femspd/ftaa042
summary:
  The essential surface glycoprotein of SARS-CoV-2 known as spike (S) protein, essential for host cell receptor binding, showed only 72% similarity with SARS-CoV at the nucleotide level. Comparative genome analysis of RaTG13, a virus from a Rhinolophusaffinis (i.e. horseshoe) bat sampled from Yunnan province in China in 2013, with SARS-CoV-2, showed that SARS-CoV-2 has 96% similarity at the nucleotide sequence level . Later, it was found that the disease was caused by a virus designated as a novel human coronavirus, MERS-CoV, phylogenetic data showed that it belonged to lineage C of the Betacoronavirusgenus and was highly similar to bat coronaviruses HKU4 (Tylonycterispachypus) and HKU5 (Pipistrelluspipistrellus; Lau et al. When cell lines over-expressed the transmembrane protein ''angiotensin-converting enzyme 2'' (ACE2) from humans, bats, pig or civet cats and were infected with SARS-CoV-2, results showed that they became hypersensitized to infection, thus indicating that ACE2 is a SARS-CoV-2 receptor . Recently, neutralizing monoclonal antibodies and nanobodies against the RBD domain of S protein showed protection against SARS-CoV and MERS-CoV (Du et al.

abstract:
  Coronavirus disease 2019 (COVID-19) is a viral pneumonia, responsible for the recent pandemic, and originated from Wuhan, China, in December 2019. The causative agent of the outbreak was identified as coronavirus and designated as severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2). Few years back, the severe acute respiratory syndrome coronavirus (SARS- CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) were reported to be highly pathogenic and caused severe infections in humans. In the current situation SARS-CoV-2 has become the third highly pathogenic coronavirus that is responsible for the present outbreak in human population. At the time of this review, there were more than 14 007 791 confirmed COVID-19 patients which associated with over 597 105 deaths in more then 216 countries across the globe (as reported by World Health Organization). In this review we have discussed about SARS-CoV, MERS-CoV and SARC-CoV-2, their reservoirs, role of spike proteins and immunogenicity. We have also covered the diagnosis, therapeutics and vaccine status of SARS-CoV-2.


id: cord-338517-1mxcssjj
title: Antibody response to SARS‐Co‐V‐2, diagnostic and therapeutic implications
author: Ishay, Yuval
date: 2020-08-26
txt: ./txt/cord-338517-1mxcssjj.txt
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mime: text/plain
url: https://doi.org/10.1002/hep4.1600
doi: 10.1002/hep4.1600
summary:
  The phage display method, allowing rapid and wide display of proteins directly correlated to their associated genes, can detect NAbs against SARS-CoV from both naïve and immune antibody libraries, capable of blocking the binding of S1 domain, thereby showing virus neutralization and prophylaxis capability either in vitro or in the animal models (31, 33, 36) . Another method, possibly allowing the production and utilization of existing NAbs, may include the use of Epstein-Barr virus (EBV) transformation of human B cells to improve the isolation of NAbs from the memory B cells harvested from the SARS-CoV infected patients (11) . Experimental and clinical data on the use of convalescent plasma products and humanized monoclonal antibodies for H5N1 influenza infection have also shown positive outcomes, and this treatment was proposed as a mean for overcoming anti-viral drug resistance (62, 79, 80) . In a study involving 20 patients with severe pandemic influenza A (H1N1) 2009 virus infection, administration of convalescent plasma reduced respiratory tract viral load, serum cytokine response, and mortality (81) .

abstract:
  The immune response against SARS‐CoV‐2 is comprised of both cellular and humoral arms. While current diagnostic methods are mainly based on PCR, they suffer from insensitivity. Therefore, antibody‐based serological tests are being developed to achieve higher sensitivity and specificity. Current efforts in treating SARS‐CoV‐2 infection include blocking of viral entry into the host cells, prohibiting viral replication and survival in the host cells, or reducing the exaggerated host immune response. Administration of convalescent plasma containing anti‐viral antibodies was proposed to improve the outcome in severe cases. In this paper, we review some of the aspects associated with the development of antibodies against SARS‐CoV‐2 and their potential use for improved diagnosis and therapy.


id: cord-338538-uea9kwge
title: Bacterial Outer Membrane Vesicles (OMVs)-Based Dual Vaccine for Influenza A H1N1 Virus and MERS-CoV
author: Shehata, Mahmoud M.
date: 2019-05-28
txt: ./txt/cord-338538-uea9kwge.txt
cache: ./cache/cord-338538-uea9kwge.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.3390/vaccines7020046
doi: 10.3390/vaccines7020046
summary:
  Herein, we generated a bacterial outer membrane vesicles (OMVs)-based vaccine presenting the antigenic stable chimeric fusion protein of the H1-type haemagglutinin (HA) of the pandemic influenza A virus (H1N1) strain from 2009 (H1N1pdm09) and the receptor binding domain (RBD) of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) (OMVs-H1/RBD). In addition, the OMVs-H1/RBD vaccinated mice showed a significant increase of neutralizing antibodies against the MERS-CoV strain HKU-NRCE-270 at week 2 and reached the highest neutralizing titer 160 (7.3 log2) at week eight compared to the control group (p < 0.001) (Figures 2c and 3a) . Based on these observations we engineered the expression of antigenically-stable and immunogenic (OMVs)-based bivalent vaccine that elicits protective antibodies (Abs) following immunization to control infections with H1N1pdm09 and MERS-CoV. In summary, the results show that the generated (OMVs-H1/RBD)-based vaccine presenting the antigenic stable chimeric fusion protein of H1-type HA of the pandemic influenza A virus (H1N1) strain and RBD of MERS-CoV induces specific neutralizing antibodies against H1N1pdm09 and MERS-CoV leading to protection of immunized mice against both viruses.

abstract:
  Vaccination is the most functional medical intervention to prophylactically control severe diseases caused by human-to-human or animal-to-human transmissible viral pathogens. Annually, seasonal influenza epidemics attack human populations leading to 290–650 thousand deaths/year worldwide. Recently, a novel Middle East Respiratory Syndrome Coronavirus emerged. Together, those two viruses present a significant public health burden in areas where they circulate. Herein, we generated a bacterial outer membrane vesicles (OMVs)-based vaccine presenting the antigenic stable chimeric fusion protein of the H1-type haemagglutinin (HA) of the pandemic influenza A virus (H1N1) strain from 2009 (H1N1pdm09) and the receptor binding domain (RBD) of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) (OMVs-H1/RBD). Our results showed that the chimeric antigen could induce specific neutralizing antibodies against both strains leading to protection of immunized mice against H1N1pdm09 and efficient neutralization of MERS-CoV. This study demonstrate that OMVs-based vaccines presenting viral antigens provide a safe and reliable approach to protect against two different viral infections.


id: cord-339093-mwxkvwaz
title: High potency of a bivalent human VH domain in SARS-CoV-2 animal models
author: Li, Wei
date: 2020-09-04
txt: ./txt/cord-339093-mwxkvwaz.txt
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extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S009286742031148X
doi: 10.1016/j.cell.2020.09.007
summary:
  It potently neutralized mouse adapted SARS-CoV-2 in wild type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. To identify potent neutralizing V H s against SARS-CoV-2, we panned our large (10 11 clones) and diverse phage-displayed human V H antibody library against recombinant RBD. One of those V H s, ab8, in an Fc (human IgG1, crystallizable fragment) fusion format, showed potent neutralization activity and specificity against SARS-CoV-2 both in vitro and in two animal models. They also suggest that the double mutations Q498T/P499Y on RBD did not influence V H -Fc ab8 binding and contribute to the validation of the mouse adapted SARS-CoV-2 model for evaluation of neutralizing antibody efficacy. In conclusion, we identified a fully human antibody V H domain that shows strong competition with ACE2 for binding to RBD and potent neutralization of SARS-CoV-2 in vitro and in two animal models.

abstract:
  Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8 bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse adapted SARS-CoV-2 in wild type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VH-Fc ab8 did not aggregate and did not bind to 5300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic.


id: cord-339724-roj8ksvc
author: Lan, Jiaming
date: 2014-11-18
txt: ./txt/cord-339724-roj8ksvc.txt
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extension: .txt
mime: text/plain
url: https://doi.org/10.1371/journal.pone.0112602
doi: 10.1371/journal.pone.0112602
summary:
  title: Tailoring Subunit Vaccine Immunity with Adjuvant Combinations and Delivery Routes Using the Middle East Respiratory Coronavirus (MERS-CoV) Receptor-Binding Domain as an Antigen Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. In this study, different adjuvants combination regimens including alum, IFA, CpG and poly(I:C) were compared in an effort to promote balance between Th1 and Th2 immune response to bystander rRBD antigen spanning residues 367-606 of MERS-CoV S in a murine model to develop an effective vaccine against MERS-CoV infection. The results indicated that rRBD protein combined with any adjuvant, including alum, IFA, CpG or poly(I:C), could induce a RBD-specific IgG antibody response in the majority of mice after the second immunisation.

title:
  Tailoring Subunit Vaccine Immunity with Adjuvant Combinations and Delivery Routes Using the Middle East Respiratory Coronavirus (MERS-CoV) Receptor-Binding Domain as an Antigen

abstract:
  The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV) pandemic. Some studies have indicated the receptor-binding domain (RBD) protein of MERS-CoV spike (S) is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly I:C) or alum and cysteine-phosphate-guanine (CpG) oligodeoxynucleotides (ODN). The immune responses of mice vaccinated with RBD, incomplete Freund’s adjuvant (IFA) and CpG ODN by a subcutaneous (s.c.) route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies) and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production). Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting effective humoral and cellular immune responses.


id: cord-340472-9ijlj4so
title: Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2
author: Li, Wenhui
date: 2005-03-24
txt: ./txt/cord-340472-9ijlj4so.txt
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mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15791205/
doi: 10.1038/sj.emboj.7600640
summary:
  Figure 3B -D shows three views of the crystal structure of human ACE2, in which residues that convert rat ACE2 to an efficient SARS-CoV receptor are shown in red, and additional residues whose alteration interferes with S1-Ig association are shown in yellow. (C) Murine leukemia viruses (MLV) expressing green fluorescent protein (GFP), lacking its endogenous envelope glycoprotein (MLV-GFP), and pseudotyped with the S protein of SARS-CoV (TOR2 isolate) were incubated with HEK293T cells transfected with plasmids encoding the indicated human or rat ACE2 variants. We have shown that entry is the primary barrier to SARS-CoV infection of murine Surface plasmon resonance experiments in which the indicated RBD-Ig TOR2 variants shown in Figure 6B bound to immobilized anti-human antibody were assayed for association with soluble human ACE2. S-protein alterations at residues 479 and 487 are important for high-affinity association with human ACE2, and for efficient infection of cells expressing this receptor.

abstract:
  Human angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS-CoV). Here we identify the SARS-CoV spike (S)-protein-binding site on ACE2. We also compare S proteins of SARS-CoV isolated during the 2002–2003 SARS outbreak and during the much less severe 2003–2004 outbreak, and from palm civets, a possible source of SARS-CoV found in humans. All three S proteins bound to and utilized palm-civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S-protein-binding site of human ACE2 to those of civet ACE2, or by altering S-protein residues 479 and 487 to residues conserved during the 2002–2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS-CoV to human cells, and provide insight into the severity of the 2002–2003 SARS epidemic.


id: cord-340960-abanr641
author: Brigger, D.
date: 2020-09-30
txt: ./txt/cord-340960-abanr641.txt
cache: ./cache/cord-340960-abanr641.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1111/all.14608
doi: 10.1111/all.14608
summary:
  In a mixed‐design evaluation study, we compared the diagnostic accuracy of serological immunoassays that are based on various SARS‐CoV‐2 proteins and assessed the neutralizing activity of antibodies in patient sera. A total of 54 randomly selected sera from individuals who were tested positive in either of the three ELISA immunoassays as well as 6 negative controls were assessed in a live SARS-CoV-2 neutralization assay (all collected in April 2020). Recombinantly expressed RBD has been used to establish an in-house ELISA for the detection of IgM and IgG anti-SARS-CoV-2 antibodies in human serum samples (supplementary Fig. 1a,b) . A total of 54 randomly selected sera from individuals who were tested positive in either of the three ELISA immunoassays as well as 6 negative controls were assessed in a live SARS-CoV-2 neutralization assay using ACE2-expressing Vero-E6 cells (34 inpatient samples, and 26 samples of medical personnel).

title:
  Accuracy of serological testing for SARS‐CoV‐2 antibodies: first results of a large mixed‐method evaluation study

abstract:
  BACKGROUND: Serological immunoassays that can identify protective immunity against SARS‐CoV‐2 are needed to adapt quarantine measures, assess vaccination responses, and evaluate donor plasma. To date, however, the utility of such immunoassays remains unclear. In a mixed‐design evaluation study, we compared the diagnostic accuracy of serological immunoassays that are based on various SARS‐CoV‐2 proteins and assessed the neutralizing activity of antibodies in patient sera. METHODS: Consecutive patients admitted with confirmed SARS‐CoV‐2 infection were prospectively followed alongside medical staff and biobank samples from winter 2018/2019. An in‐house enzyme‐linked immunosorbent assay utilizing recombinant receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike protein was developed and compared to three commercially available enzyme‐linked immunosorbent assays (ELISAs) targeting the nucleoprotein (N), the S1 domain of the spike protein (S1) and a lateral flow immunoassay (LFI) based on full‐length spike protein. Neutralization assays with live SARS‐CoV‐2 were performed. RESULTS: One‐thousand four‐hundred and seventy‐seven individuals were included comprising 112 SARS‐CoV‐2 positives (defined as a positive real‐time PCR result; prevalence 7.6%). IgG seroconversion occurred between day 0 and day 21. While the ELISAs showed sensitivities of 88.4% for RBD, 89.3% for S1, and 72.9% for N protein, the specificity was above 94% for all tests. Out of 54 SARS‐CoV‐2 positive individuals, 96.3% showed full neutralization of live SARS‐CoV‐2 at serum dilutions ≥1:16, while none of the 6 SARS‐CoV‐2 negative sera revealed neutralizing activity. CONCLUSIONS: ELISAs targeting RBD and S1 protein of SARS‐CoV‐2 are promising immunoassays which shall be further evaluated in studies verifying diagnostic accuracy and protective immunity against SARS‐CoV‐2.


id: cord-341396-0tn06al2
title: Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals
author: Ni, Ling
date: 2020-05-03
txt: ./txt/cord-341396-0tn06al2.txt
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mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S1074761320301813?v=s5
doi: 10.1016/j.immuni.2020.04.023
summary:
  In this study, we collected blood from COVID-19 patients who have recently become 5 virus-free and therefore were discharged, and analyzed their SARS-CoV-2-specific antibody 6 and T cell responses. NP-and S-RBD-specific 9 IgM and IgG antibodies were both detected in the sera of newly discharged patients, 10 compared with healthy donor groups. Anti-SARS-CoV-2 IgG antibodies were also more 11 obviously observed than IgM in the follow-up patients (#9-14), when compared with healthy 12 donors ( Figure 1B ). As shown in Figure 3C , compared with healthy donors, 25 the numbers of IFN-γ-secreting NP-specific T cells in patients #1, 2, 4, 5 and 8 were much 26 higher than other patients, suggesting that they had developed SARS-CoV-2-specific T cell responses. More interestingly, when combining all 14 patients in our analysis, there 9 was a significant correlation between the neutralizing antibody titers and the numbers of NPIn this study, we characterized SARS-CoV-2-specific humoral and cellular immunity in 2 recovered patients.

abstract:
  Summary The World Health Organization has declared SARS-CoV-2 virus outbreak a world-wide pandemic. However, there is very limited understanding on the immune responses, especially adaptive immune responses to SARS-CoV-2 infection. Here, we collected blood from COVID-19 patients who have recently become virus-free and therefore were discharged, and detected SARS-CoV-2-specific humoral and cellular immunity in 8 newly discharged patients. Follow-up analysis on another cohort of 6 patients 2 weeks post discharge also revealed high titers of IgG antibodies. In all 14 patients tested, 13 displayed serum neutralizing activities in a pseudotype entry assay. Notably, there was a strong correlation between neutralization antibody titers and the numbers of virus-specific T cells. Our work provides a basis for further analysis of protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It has also implications in developing an effective vaccine to SARS-CoV-2 infection.


id: cord-342312-rnq1hfsj
title: Recovered COVID-19 patients with recurrent viral RNA exhibit lower levels of anti-RBD antibodies
author: Liu, Bingfeng
date: 2020-09-16
txt: ./txt/cord-342312-rnq1hfsj.txt
cache: ./cache/cord-342312-rnq1hfsj.txt
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mime: text/plain
url: https://doi.org/10.1038/s41423-020-00528-0
doi: 10.1038/s41423-020-00528-0
abstract: nan
summary:
  To evaluate the effect of specific antibodies on RP status, we compared the levels of anti-SARS-CoV-2 IgG to the S, RBD, HR1-HR2, N, and M proteins in these patients during their convalescent period ( Fig. 1c; Fig. S3 ). The P value was calculated using a two-tailed Mann-Whitney U test the level of IgM to the RBD protein among these recovered patients also correlated with the S, HR1-HR2, and N proteins (r = 0.67, p < 0.0001; r = 0.56, p < 0.0001; and r = 0.60, p < 0.0001, respectively) (Fig. S6b) . In addition, a positive correlation was also observed between age and IgG level to the RBD, S, HR1-HR2, and N proteins (r = 0.38, P = 0.0077; r = 0.40, P = 0.0055; r = 0.45, P = 0.0017; and r = 0.44, P = 0.0021, respectively; Fig. S7 ), indicating the important role of age in the generation of specific binding antibodies. The online version of this article (https://doi.org/10.1038/s41423-020-00528-0) contains supplementary material.Competing interests: The authors declare no competing interests.


id: cord-342557-a7q8vp8m
title: Antiviral Peptides as Promising Therapeutics against SARS-CoV-2
author: Chowdhury, Surid Mohammad
date: 2020-10-23
txt: ./txt/cord-342557-a7q8vp8m.txt
cache: ./cache/cord-342557-a7q8vp8m.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/33095007/
doi: 10.1021/acs.jpcb.0c05621
summary:
  [Image: see text] Over 50 peptides, which were known to inhibit SARS-CoV-1, were computationally screened against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Peptides that showed higher S protein-binding affinity compared to the α-helix (AH) of the ACE2 peptidase were further analyzed with molecular dynamics (MD) simulation and the structure− activity relationship (SAR) in order to achieve a high-affinity binder for the S protein. 30 Initially, stepwise multiple linear regression (MLR) was performed considering these properties as variables to predict the calculated binding affinity of the test peptides with the RBD of the SARS CoV-2 spike protein. All 51 peptides were docked to the RBD of the SARS CoV-2 spike protein using PatchDock. Various residues including Glu484, Tyr449, and Tyr505 present in the ACE2 binding site of the RBD were involved in noncovalent interaction with the antiviral peptides ( Figure 1a) .

abstract:
  [Image: see text] Over 50 peptides, which were known to inhibit SARS-CoV-1, were computationally screened against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Based on the binding affinity and interaction, 15 peptides were selected, which showed higher affinity compared to the α-helix of the human ACE2 receptor. Molecular dynamics simulation demonstrated that two peptides, S2P25 and S2P26, were the most promising candidates, which could potentially block the entry of SARS-CoV-2. Tyr489 and Tyr505 residues present in the “finger-like” projections of the RBD were found to be critical for peptide interaction. Hydrogen bonding and hydrophobic interactions played important roles in prompting peptide–protein binding and interaction. Structure–activity relationship indicated that peptides containing aromatic (Tyr and Phe), nonpolar (Pro, Gly, Leu, and Ala), and polar (Asn, Gln, and Cys) residues were the most significant contributors. These findings can facilitate the rational design of selective peptide inhibitors targeting the spike protein of SARS-CoV-2.


id: cord-343107-oj1re34k
author: Zhou, Haixia
date: 2019-07-11
txt: ./txt/cord-343107-oj1re34k.txt
cache: ./cache/cord-343107-oj1re34k.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/31296843/
doi: 10.1038/s41467-019-10897-4
summary:
  Most neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) target the receptor-binding domain (RBD) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). Here we report the monoclonal antibody 7D10 that binds to the N-terminal domain (NTD) of the spike glycoprotein and inhibits the cell entry of MERS-CoV with high potency. The 7D10 antibody recognizes the NTD of MERS-CoV S glycoprotein and neutralizes the infectivity of pseudotyped and live virus with a potency comparable to those of the most active RBD-targeting antibodies. The NTD N222Q mutation also dramatically reduced the binding and neutralization by 7D10, but did not dramatically affect the cell infection of pseudotyped MERS-CoV ( Supplementary Fig. 11) . A conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndrome coronavirus spike protein A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein

title:
  Structural definition of a neutralization epitope on the N-terminal domain of MERS-CoV spike glycoprotein

abstract:
  Most neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) target the receptor-binding domain (RBD) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). The epitopes and mechanisms of mAbs targeting non-RBD regions have not been well characterized yet. Here we report the monoclonal antibody 7D10 that binds to the N-terminal domain (NTD) of the spike glycoprotein and inhibits the cell entry of MERS-CoV with high potency. Structure determination and mutagenesis experiments reveal the epitope and critical residues on the NTD for 7D10 binding and neutralization. Further experiments indicate that the neutralization by 7D10 is not solely dependent on the inhibition of DPP4 binding, but also acts after viral cell attachment, inhibiting the pre-fusion to post-fusion conformational change of the spike. These properties give 7D10 a wide neutralization breadth and help explain its synergistic effects with several RBD-targeting antibodies.


id: cord-343185-lbmbp9ca
title: SARS-CoV-2 antibody responses determine disease severity in COVID-19 infected individuals
author: Hansen, C. B.
date: 2020-07-29
txt: ./txt/cord-343185-lbmbp9ca.txt
cache: ./cache/cord-343185-lbmbp9ca.txt
extension: .txt
mime: text/plain
url: http://medrxiv.org/cgi/content/short/2020.07.27.20162321v1?rss=1
doi: 10.1101/2020.07.27.20162321
summary:
  Here we have developed novel flexible ELISA-based assays for specific detection of SARS-CoV-2 antibodies against the receptor-binding domain (RBD): An antigen sandwich-ELISA relevant for large population screening and three isotype-specific assays for in-depth diagnostics. Detection of IgM, IgA and IgG antibodies against SARS-CoV-2 protein N was evaluated by analyzing 136 positive samples and 174 negative controls and ROC curve analyses were assessed to estimate the assay performance . To provide a better insight into antibody seroconversion during SARS-CoV-2 infection and reactivity against different locations on protein S and protein N, we conducted IgM, IgA and IgG detection in 90 positive samples against 14 protein fragments and short peptides located on the protein S and protein N structures, full-length RBD, protein S and protein N (Figure 2A ). We have developed an ELISA-based platform for detection SARS-CoV-2 antibodies comprising an indirect RBD S-ELISA for pan Ig detection and direct ELISAs for in-depth analyses of the IgM, IgA and IgG isotype responses towards RBD and protein N.

abstract:
  Globally, the COVID-19 pandemic has had extreme consequences for the healthcare system and calls for diagnostic tools to monitor and understand the transmission, pathogenesis and epidemiology, as well as to evaluate future vaccination strategies. Here we have developed novel flexible ELISA-based assays for specific detection of SARS-CoV-2 antibodies against the receptor-binding domain (RBD): An antigen sandwich-ELISA relevant for large population screening and three isotype-specific assays for in-depth diagnostics. Their performance was evaluated in a cohort of 350 convalescent participants with previous COVID-19 infection, ranging from asymptomatic to critical cases. We mapped the antibody responses to different areas on protein N and S and showed that the IgM, A and G antibody responses against RBD are significantly correlated to the disease severity. These assays-and the data generated from them-are highly relevant for diagnostics and prognostics and contribute to the understanding of long-term COVID-19 immunity.


id: cord-344180-v8xs5ej8
title: Functionalized TiO(2) Nanotube-Based Electrochemical Biosensor for Rapid Detection of SARS-CoV-2
author: Vadlamani, Bhaskar S.
date: 2020-10-17
txt: ./txt/cord-344180-v8xs5ej8.txt
cache: ./cache/cord-344180-v8xs5ej8.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.3390/s20205871
doi: 10.3390/s20205871
summary:
  In this work, we report the synthesis of a cheap, yet highly sensitive, cobalt-functionalized TiO(2) nanotubes (Co-TNTs)-based electrochemical sensor for rapid detection of SARS-CoV-2 through sensing the spike (receptor binding domain (RBD)) present on the surface of the virus. In the current work, we have determined the potential of Co-functionalized TiO2 nanotubes (Co-TNTs) for the electrochemical detection of S-RBD protein of SARS-CoV-2. In the current work, we have determined the potential of Co-functionalized TiO2 nanotubes (Co-TNTs) for the electrochemical detection of S-RBD protein of SARS-CoV-2. Our data shows that cobalt functionalized TNTs can selectively detect the S-RBD protein of SARS-CoV-2 using the amperometry electrochemical technique in ~30 s. Our data shows that cobalt functionalized TNTs can selectively detect the S-RBD protein of SARS-CoV-2 using the amperometry electrochemical technique in ~30 s. In this study, we developed a Co-metal functionalized TNT as a sensing material for electrochemical detection of SARS-CoV-2 infection through the detection of the receptor binding domain (RBD) of spike glycoprotein.

abstract:
  The COronaVIrus Disease (COVID-19) is a newly emerging viral disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Rapid increase in the number of COVID-19 cases worldwide led the WHO to declare a pandemic within a few months after the first case of infection. Due to the lack of a prophylactic measure to control the virus infection and spread, early diagnosis and quarantining of infected as well as the asymptomatic individuals are necessary for the containment of this pandemic. However, the current methods for SARS-CoV-2 diagnosis are expensive and time consuming, although some promising and inexpensive technologies are becoming available for emergency use. In this work, we report the synthesis of a cheap, yet highly sensitive, cobalt-functionalized TiO(2) nanotubes (Co-TNTs)-based electrochemical sensor for rapid detection of SARS-CoV-2 through sensing the spike (receptor binding domain (RBD)) present on the surface of the virus. A simple, low-cost, and one-step electrochemical anodization route was used for synthesizing TNTs, followed by an incipient wetting method for cobalt functionalization of the TNTs platform, which was connected to a potentiostat for data collection. This sensor specifically detected the S-RBD protein of SARS-CoV-2 even at very low concentration (range of 14 to 1400 nM (nano molar)). Additionally, our sensor showed a linear response in the detection of viral protein over the concentration range. Thus, our Co-TNT sensor is highly effective in detecting SARS-CoV-2 S-RBD protein in approximately 30 s, which can be explored for developing a point of care diagnostics for rapid detection of SARS-CoV-2 in nasal secretions and saliva samples.


id: cord-344871-486sk4wc
author: Wu, Jianping
date: 2016-09-16
txt: ./txt/cord-344871-486sk4wc.txt
cache: ./cache/cord-344871-486sk4wc.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1038/srep33382
doi: 10.1038/srep33382
summary:
  We have previously shown that a non-structural protein 1 (NS1)-binding monoclonal antibody, termed as 2H6, can significantly reduce influenza A virus (IAV) replication when expressed intracellularly. As comparative ELISA in this and previous studies 29 showed that residues N48 and T49 in NS1(RBD) are important for the interaction with mAb 2H6, they were defined as active residues involved in the binding interaction to generate a series of models of the NS1(RBD) and 2H6-Fab complex. Overall, the predicted model from cluster 2 is consistent with our comparative ELISA data and suggests that residues N48 and T49 are important for the binding between NS1(RBD) and 2H6-Fab because their side-chains could make hydrogen bonds with residues in the VH-CDR2 of the Fab. In addition, R44 of NS1(RBD) was distal from the antibody-antigen interface, which is consistent with the results from comparative ELISA ( Figure S1 ) showing that substitution of R44 of NS1(RBD) with K did not affect its interaction with mAb 2H6.

title:
  Biochemical and structural characterization of the interface mediating interaction between the influenza A virus non-structural protein-1 and a monoclonal antibody

abstract:
  We have previously shown that a non-structural protein 1 (NS1)-binding monoclonal antibody, termed as 2H6, can significantly reduce influenza A virus (IAV) replication when expressed intracellularly. In this study, we further showed that 2H6 binds stronger to the NS1 of H5N1 than A/Puerto Rico/8/1934(H1N1) because of an amino acid difference at residue 48. A crystal structure of 2H6 fragment antigen-binding (Fab) has also been solved and docked onto the NS1 structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. In one of the models, the predicted molecular contacts between residues in NS1 and 2H6-Fab correlate well with biochemical results. Taken together, residues N48 and T49 in H5N1 NS1 act cooperatively to maintain a strong interaction with mAb 2H6 by forming hydrogen bonds with residues found in the heavy chain of the antibody. Interestingly, the pandemic H1N1-2009 and the majority of seasonal H3N2 circulating in humans since 1968 has N48 in NS1, suggesting that mAb 2H6 could bind to most of the currently circulating seasonal influenza A virus strains. Consistent with the involvement of residue T49, which is well-conserved, in RNA binding, mAb 2H6 was also found to inhibit the interaction between NS1 and double-stranded RNA.


id: cord-346670-34wfy52f
author: Gobeil, Sophie M-C.
date: 2020-10-12
txt: ./txt/cord-346670-34wfy52f.txt
cache: ./cache/cord-346670-34wfy52f.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.10.11.335299
doi: 10.1101/2020.10.11.335299
summary:
  Most structures of the SARS-CoV-2 S ectodomain currently available include two mutations, one to disrupt the furin cleavage site (RRAR to GSAS = S-GSAS), and a double proline mutation (PP) of residues 986-987, designed to prevent conformational change to the post-fusion state (Wrapp et al., 2020) . While the SARS-CoV-2 S ectodomain construct that includes mutations of residues K986 and V987, between the HR1 and CH subdomains (S2 domain), to prolines (PP) (named S-GSAS/PP in this study) (Figure 1 ) is widely used in the field, the origin of this PP construct was based upon the stabilization of the pre-fusion conformation of other coronavirus spikes (Pallesen et al., 2017; Walls et al., 2020; Wrapp et al., 2020) . Similar to observations made with the S-GSAS/D614G S ectodomain structure, the RBD up/down motion in the furin-cleaved G614 S ectodomain was associated with a movement in the SD1 domain and in the region of the RBD-to-NTD linker that joined the SD1 b sheet ( Figure 7C, S8B) .

title:
  D614G mutation alters SARS-CoV-2 spike conformational dynamics and protease cleavage susceptibility at the S1/S2 junction

abstract:
  The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we analyze the D614G mutation in the context of a soluble S ectodomain construct. Cryo-EM structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage altered the conformational dynamics of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on the RBD dynamics triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational dynamics and allostery, and have implications for vaccine design. Highlights SARS-CoV-2 S ectodomains with or without the K986P, V987P mutations have similar structures, antigenicity and stability. The D614G mutation alters S protein conformational dynamics. D614G enhances protease cleavage susceptibility at the S protein furin cleavage site. Cryo-EM structures reveal allosteric effect of changes at the S1/S2 junction on RBD dynamics.


id: cord-347587-auook38y
author: Zhao, Guangyu
date: 2018-08-29
txt: ./txt/cord-347587-auook38y.txt
cache: ./cache/cord-347587-auook38y.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1128/jvi.00837-18
doi: 10.1128/jvi.00837-18
summary:
  title: A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). Identification and characterization of MERS-CoV-RBD-specific Nbs. To construct the Nb (i.e., VHH) library, we immunized llama with recombinant MERS-CoV RBD (residues 377 to 588, EMC2012 strain) containing a C-terminal human IgG1 Fc tag (i.e., RBD-Fc) and isolated peripheral blood mononuclear cells (PBMCs) from the immunized llama. To examine of the role of the D539A mutation in DPP4 binding, we carried out an ELISA to detect the binding between DPP4 and The plates were coated with RBD-Fd protein (2 g/ml) and treated with or without DTT, followed by sequential incubation with serial dilutions of NbMS10 or NbMS10-Fc and goat anti-llama and HRP-conjugated anti-goat IgG antibodies.

title:
  A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV

abstract:
  The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life in vivo; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics. IMPORTANCE Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the in vivo half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.


id: cord-348455-vcxalkeo
author: Graham, N. R.
date: 2020-07-22
txt: ./txt/cord-348455-vcxalkeo.txt
cache: ./cache/cord-348455-vcxalkeo.txt
extension: .txt
mime: text/plain
url: http://medrxiv.org/cgi/content/short/2020.07.15.20154443v1?rss=1
doi: 10.1101/2020.07.15.20154443
summary:
  title: Kinetics and Isotype Assessment of Antibodies Targeting the Spike Protein Receptor Binding Domain of SARS-CoV-2 In COVID-19 Patients as a function of Age and Biological Sex. date: 2020-07-22 The receptor-binding domain of the CoV spike (RBD-S) protein is important in host cell recognition and infection and antibodies targeting this domain are often neutralizing. We first piloted our antigen preps for the RBD-S IgG screening assay using serum 81 samples from a PCR-confirmed severe COVID-19 patient (defined as admission to the Intensive 82 Care Unit, ICU) who was admitted to the hospital 10 days following symptom onset and based 83 on an early report suggesting that SARS-CoV-2 could trigger antibody responses in this 84 timeframe (24). Anti-S titers in patients with a negative RBD-S test were 138 generally low and in RBD-positive samples, followed the same trends as RBD-reactivity, 139 providing further confirmation of robust serological responses to SARS-CoV-2 during acute 140 COVID-19.

title:
  Kinetics and Isotype Assessment of Antibodies Targeting the Spike Protein Receptor Binding Domain of SARS-CoV-2 In COVID-19 Patients as a function of Age and Biological Sex.

abstract:
  SARS-CoV-2 is the newly emerged virus responsible for the global COVID-19 pandemic. There is an incomplete understanding of the host humoral immune response to SARS-CoV-2 during acute infection. Host factors such as age and sex as well the kinetics and functionality of antibody responses are important factors to consider as vaccine development proceeds. The receptor-binding domain of the CoV spike (RBD-S) protein is important in host cell recognition and infection and antibodies targeting this domain are often neutralizing. In a cross-sectional study of anti-RBD-S antibodies in COVID-19 patients we found equivalent levels in male and female patients and no age-related deficiencies even out to 93 years of age. The anti-RBD-S response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after symptom onset. Anti-RBD-S IgG, IgM, and IgA responses were simultaneously induced within 10 days after onset, but isotype-specific kinetics differed such that anti-RBD-S IgG was most sustained over a 5-week period. The kinetics and magnitude of neutralizing antibody formation strongly correlated with that seen for anti-RBD-S antibodies. Our results suggest age- and sex- related disparities in COVID-19 fatalities are not explained by anti-RBD-S responses. The multi-isotype anti-RBD-S response induced by live virus infection could serve as a potential marker by which to monitor vaccine-induced responses.


id: cord-351760-698voi9y
author: Han, Hui-Ju
date: 2018-11-30
txt: ./txt/cord-351760-698voi9y.txt
cache: ./cache/cord-351760-698voi9y.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/30513619/
doi: 10.3390/v10120680
summary:
  The receptor-binding domain (RBD) in the spike protein of MERS-CoV is a major target, and mouse, camel, or human-derived neutralizing mAbs targeting RBD have been developed. In vivo study demonstrated that prophylaxis with m336 reduced virus titers in the lung of rabbits infected with MERS-CoV [15] , and m336 also provided transgenic mice expressing human DPP4 with full prophylactic and therapeutic protection from MERS-CoV [16] . A Conformation-Dependent Neutralizing Monoclonal Antibody Specifically Targeting Receptor-Binding Domain in Middle East Respiratory Syndrome Coronavirus Spike Protein Prophylaxis with a Middle East Respiratory Syndrome Coronavirus (MERS-CoV)-Specific Human Monoclonal Antibody Protects Rabbits From MERS-CoV Infection Passive Transfer of a Germline-like Neutralizing Human Monoclonal Antibody Protects Transgenic Mice Against Lethal Middle East Respiratory Syndrome Coronavirus Infection Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV

title:
  Neutralizing Monoclonal Antibodies as Promising Therapeutics against Middle East Respiratory Syndrome Coronavirus Infection

abstract:
  Since emerging in 2012, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has been a global public health threat with a high fatality rate and worldwide distribution. There are no approved vaccines or therapies for MERS until now. Passive immunotherapy with neutralizing monoclonal antibodies (mAbs) is an effective prophylactic and therapeutic reagent against emerging viruses. In this article, we review current advances in neutralizing mAbs against MERS-CoV. The receptor-binding domain (RBD) in the spike protein of MERS-CoV is a major target, and mouse, camel, or human-derived neutralizing mAbs targeting RBD have been developed. A major problem with neutralizing mAb therapy is mutant escape under selective pressure, which can be solved by combination of neutralizing mAbs targeting different epitopes. Neutralizing mAbs are currently under preclinical evaluation, and they are promising candidate therapeutic agents against MERS-CoV infection.


id: cord-352527-eeyqh9nc
title: Advances in MERS-CoV Vaccines and Therapeutics Based on the Receptor-Binding Domain
author: Zhou, Yusen
date: 2019-01-14
txt: ./txt/cord-352527-eeyqh9nc.txt
cache: ./cache/cord-352527-eeyqh9nc.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/30646569/
doi: 10.3390/v11010060
summary:
  A number of MERS vaccines have been developed based on viral RBD, including nanoparticles, virus-like particles (VLPs), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus 5 (Ad5)-directed expression of human DPP4 (Ad5/hDPP4), hDPP4-transgenic (hDPP4-Tg) mice, and non-human primates (NHPs) [88] [89] [90] [91] [92] [93] [94] . Receptor usage of a novel bat lineage C Betacoronavirus reveals evolution of Middle East respiratory syndrome-related coronavirus spike proteins for human dipeptidyl peptidase 4 binding Recombinant receptor-binding domains of multiple Middle East respiratory syndrome coronaviruses (MERS-CoVs) induce cross-neutralizing antibodies against divergent human and camel MERS-CoVs and antibody escape mutants A conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndrome coronavirus spike protein A novel nanobody targeting Middle East respiratory syndrome coronavirus (MERS-CoV) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against MERS-CoV

abstract:
  Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is an infectious virus that was first reported in 2012. The MERS-CoV genome encodes four major structural proteins, among which the spike (S) protein has a key role in viral infection and pathogenesis. The receptor-binding domain (RBD) of the S protein contains a critical neutralizing domain and is an important target for development of MERS vaccines and therapeutics. In this review, we describe the relevant features of the MERS-CoV S-protein RBD, summarize recent advances in the development of MERS-CoV RBD-based vaccines and therapeutic antibodies, and illustrate potential challenges and strategies to further improve their efficacy.


id: cord-352934-ypls4zau
title: Human IgG neutralizing monoclonal antibodies block SARS-CoV-2 infection
author: Wan, Jinkai
date: 2020-07-03
txt: ./txt/cord-352934-ypls4zau.txt
cache: ./cache/cord-352934-ypls4zau.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S2211124720308998
doi: 10.1016/j.celrep.2020.107918
summary:
  title: Human IgG neutralizing monoclonal antibodies block SARS-CoV-2 infection We screened sera samples from 11 patients recently recovered from COVID-19, and 119 found all individuals showed certain levels of serological responses, with #507 and 120 #501 being the weakest, to SARS-CoV-2 Spike RBD and S1 proteins ( Figure 1A ). We 121 also found that 10 sera, except for 507, showed neutralization abilities against 122 SARS-CoV-2 pseudoviral infection of HEK293T cells stably expressing human ACE2 123 ( Figure 1B ). In order to screen for SARS-CoV-2 spike antigen specific monoclonal antibodies, we 143 used two primary assays based on ELISA (enzyme linked immunosorbent assay) and 144 FCA (flow cytometry assay), respectively. Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein to angiotensin 561 converting enzyme 2 receptor A potent neutralizing human antibody reveals the N-terminal domain of the 564 Spike protein of SARS-CoV-2 as a site of vulnerability Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific 612 human monoclonal antibody

abstract:
  Summary COVID-19 has become a worldwide threat to humans, and neutralizing antibodies have therapeutic potential. We have purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain), and obtain 729 paired heavy and light chain fragments. Among these, 178 antibodies test positive for antigen binding, and the majority of the top 17 binders with EC50 below 1 nM are RBD binders. Furthermore, we identify 11 neutralizing antibodies, 8 of which show an IC50 within 10 nM, and the best one, 414-1, with IC50 of 1.75 nM. Through epitope mapping, we find 3 main epitopes in RBD recognized by these antibodies, and epitope B antibody 553-15 could substantially enhance the neutralizing abilities of most of the other antibodies. We also find that 515-5 could cross-neutralize the SARS-CoV pseudovirus. Altogether, our study provides 11 potent human neutralizing antibodies for COVID-19 as therapeutic candidates.


id: cord-353161-mtq6yh25
title: Insights on cross-species transmission of SARS-CoV-2 from structural modeling
author: Rodrigues, João PGLM
date: 2020-06-05
txt: ./txt/cord-353161-mtq6yh25.txt
cache: ./cache/cord-353161-mtq6yh25.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32577636/
doi: 10.1101/2020.06.05.136861
summary:
  We found that species known not to be susceptible to SARS-CoV-2 infection have non-conservative mutations in several ACE2 amino acid residues that disrupt key polar and charged contacts with the viral spike protein. Collectively, our results provide a structural framework that explains why certain animal species are not susceptible to SARS-CoV-2 infection, and also suggests potential mutations that can enhance binding to the viral RBD. Although it is well-known that docking scores do not quantitatively correlate with experimental binding affinities [19] , these scores suggest that SARS-CoV-2 neg species lack one or more key ACE2 residues that contribute significantly to the interaction with RBD. Models of SARS-CoV-2 neg species -chicken, duck, guinea pig, mouse, and rat -generally have higher (worse) HADDOCK scores than average (Figure 2 ), suggesting that these species'' non-susceptibility to infection could stem from deficient RBD binding to ACE2.

abstract:
  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global pandemic that has infected more than 6 million people in more than 180 countries worldwide. Like other coronaviruses, SARS-CoV-2 is thought to have been transmitted to humans from wild animals. Given the scale and widespread geographical distribution of the current pandemic, the question emerges whether human-to-animal transmission is possible and if so, which animal species are most at risk. Here, we investigated the structural properties of several ACE2 orthologs bound to the SARS-CoV-2 spike protein. We found that species known not to be susceptible to SARS-CoV-2 infection have non-conservative mutations in several ACE2 amino acid residues that disrupt key polar and charged contacts with the viral spike protein. Our models also predict affinity-enhancing mutations that could be used to design ACE2 variants for therapeutic purposes. Finally, our study provides a blueprint for modeling viral-host protein interactions and highlights several important considerations when designing these computational studies and analyzing their results.


id: cord-353748-y1a52z8e
author: Bhattacharya, Rajarshi
date: 2021-01-02
txt: ./txt/cord-353748-y1a52z8e.txt
cache: ./cache/cord-353748-y1a52z8e.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S004268222030204X
doi: 10.1016/j.virol.2020.10.002
summary:
  title: A natural food preservative peptide nisin can interact with the SARS-CoV-2 spike protein receptor human ACE2 Nisin, a food-grade antimicrobial peptide produced by lactic acid bacteria has been examined for its probable interaction with the human ACE2 (hACE2) receptor, the site where spike protein of SARS-CoV-2 binds. Among the eight nisin variants examined, nisin H, nisin Z, nisin U and nisin A showed a significant binding affinity towards hACE2, higher than that of the RBD (receptor binding domain) of the SARS-CoV-2 spike protein. The present study attempts to investigate the ability of food-grade nisin A and its natural variants to block the interaction between hACE2 and the spike protein of SARS-CoV-2, a key step of COVID-19 disease initiation. The binding affinity of docked structures of all eight variants of nisin in complex with hACE2 was calculated as ΔG derived from analysis with Prodigy for each complex in comparison with the RBD of spike protein of SARS-CoV-2.

title:
  A natural food preservative peptide nisin can interact with the SARS-CoV-2 spike protein receptor human ACE2

abstract:
  Nisin, a food-grade antimicrobial peptide produced by lactic acid bacteria has been examined for its probable interaction with the human ACE2 (hACE2) receptor, the site where spike protein of SARS-CoV-2 binds. Among the eight nisin variants examined, nisin H, nisin Z, nisin U and nisin A showed a significant binding affinity towards hACE2, higher than that of the RBD (receptor binding domain) of the SARS-CoV-2 spike protein. The molecular interaction of nisin with hACE2 was investigated by homology modeling and docking studies. Further, binding efficiency of the most potent nisin H was evaluated through the interaction of hACE2:nisin H complex with RBD (receptor-binding domain) of SARS-CoV-2 and that of hACE2:RBD complex with nisin H. Here, nisin H acted as a potential competitor of RBD to access the hACE2 receptor. The study unravels for the first time that a globally used food preservative, nisin has the potential to bind to hACE2.


id: cord-354868-pqn59ojj
title: A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2
author: Yao, Hebang
date: 2020-09-25
txt: ./txt/cord-354868-pqn59ojj.txt
cache: ./cache/cord-354868-pqn59ojj.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.09.24.312595
doi: 10.1101/2020.09.24.312595
summary:
  title: A high-affinity RBD-targeting nanobody improves fusion partner''s potency against SARS-CoV-2 Considerable research have been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. A high-affinity RBD binder without neutralizing activity 85 Previously, we generated 99 sybodies from three highly diverse synthetic libraries by ribosome and phage display with in vitro selections against the SARS-CoV-2 RBD. Consistent with its inability to neutralize SARS-CoV-2 pseudovirus, SR31 did not affect RBD-ACE2 binding (Fig. 1C) . Most RBD-targeting neutralizing antibodies, including neutralizing nanobodies characterized so far (8, 13-15, 19, 20, 22-24, 26-28, 34, 35, 37) , engage the RBD at the receptor-binding motif (RBM) (Fig. 3A) , thus competing off ACE2 and preventing viral entry. Taken together, the structural data rationalize the high-affinity binding between SR31 and RBD, and its inability to neutralize SARS-CoV-2. Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2

abstract:
  A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research have been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.


id: cord-355728-wivk0bm0
author: Schoof, Michael
date: 2020-08-17
txt: ./txt/cord-355728-wivk0bm0.txt
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extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.08.08.238469
doi: 10.1101/2020.08.08.238469
summary:
  Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Class I nanobodies emerged with highly 144 variable activity in this assay with Nb6 and Nb11 as two of the most potent clones with IC50 145 values of 370 and 540 nM, respectively (Table 1) To define the binding sites of Nb6 and Nb11, we determined their cryogenic electron 156 microscopy (cryo-EM) structures bound to Spike* ( Fig. 2A state RBDs only contacts a single RBD (Fig. 2D) . 277 278 mNb6-tri displays further gains in potency in both pseudovirus and live SARS-CoV-2 infection 279 assays with IC50 values of 120 pM (5.0 ng/mL) and 54 pM (2.3 ng/mL), respectively (Fig. 4H-I, 280 Table 1).

title:
  An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

abstract:
  Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.


id: cord-355807-q3bngari
author: Yepes-Pérez, Andres F.
date: 2020-10-29
txt: ./txt/cord-355807-q3bngari.txt
cache: ./cache/cord-355807-q3bngari.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1080/07391102.2020.1837676
doi: 10.1080/07391102.2020.1837676
summary:
  Molecular modeling was carried out to evaluate the potential antiviral properties of the components of the medicinal herb Uncaria tomentosa (cat''s claw) focusing on the binding interface of the RBD–ACE-2 and the viral spike protein. tomentosa against focusing both on the binding interface of the RBD-ACE-2 and inside SARS-CoV-2 RBD spike protein, (2) simulations of ligand pathway of the best predicted compounds from step 1 to evaluate convenient entrance mechanism of the compounds to the binding site, (3) MD simulation to assess the stability of the best protein-ligand complexes from 1, (4) calculation of pharmacokinetics parameters for the most qualified compounds resulting from the previous parts of the docking protocol. Next, we used the cryo-EM structure of SARS-CoV-2 spike protein (PDB code: 6VYB) in their open state (Lipinski et al., 2012) to explore the potential inhibition of components of the cat''s claw, selecting ACE-2-binding pocket to this study.

title:
  Uncaria tomentosa (cat’s claw): a promising herbal medicine against SARS-CoV-2/ACE-2 junction and SARS-CoV-2 spike protein based on molecular modeling

abstract:
  COVID-19 is a novel severe acute respiratory syndrome coronavirus. Currently, there is no effective treatment and vaccines seem to be the solution in the future. Virtual screening of potential drugs against the S protein of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) has provided small molecular compounds with a high binding affinity. Unfortunately, most of these drugs do not attach with the binding interface of the receptor-binding domain (RBD)–angiotensin-converting enzyme-2 (ACE-2) complex in host cells. Molecular modeling was carried out to evaluate the potential antiviral properties of the components of the medicinal herb Uncaria tomentosa (cat’s claw) focusing on the binding interface of the RBD–ACE-2 and the viral spike protein. The in silico approach starts with protein–ligand docking of 26 Cat’s claw key components followed by molecular dynamics simulations and re-docked calculations. Finally, we carried out drug-likeness calculations for the most qualified cat’s claw components. The structural bioinformatics approaches led to the identification of several bioactive compounds of U. tomentosa with potential therapeutic effect by dual strong interaction with interface of the RBD–ACE-2 and the ACE-2 binding site on SARS-CoV-2 RBD viral spike. In addition, in silico drug-likeness indices for these components were calculated and showed good predicted therapeutic profiles of these phytochemicals found in U. tomentosa (cat’s claw). Our findings suggest the potential effectiveness of cat’s claw as complementary and/or alternative medicine for COVID-19 treatment. Communicated by Ramaswamy H. Sarma


id: cord-356264-q0yqnlyl
author: Armijos-Jaramillo, Vinicio
date: 2020-03-23
txt: ./txt/cord-356264-q0yqnlyl.txt
cache: ./cache/cord-356264-q0yqnlyl.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1101/2020.03.21.001933
doi: 10.1101/2020.03.21.001933
summary:
  With this analysis, we determine a region inside the receptor-binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor hACE2. We employ a multidisciplinary approach to look for evidence of diversifying selection on the S-protein gene, and model the interactions between human ACE2 (hACE2) and the RBD of selected coronavirus strains, which ultimately afforded us novel insights detailing virus and host cell interactions. All these experiments were performed again using the S-protein genes of a shorter list of accessions and more distantly related (broad dataset) to SARS-COV-2 (AY304488, AY395003, DQ412043, FJ882957, KY417144, MG772933, MG772934, MN908947, NC_004718) to test the reproducibility of the predicted branches and sites under positive selection. Modeling results suggest that interference with the hot spot 353 could be and effective strategy for inhibiting the recognition of the RBD of the SARS-COV-2 spike protein by its human host receptor ACE2 and hence prevent infections.

title:
  SARS-CoV-2, an evolutionary perspective of interaction with human ACE2 reveals undiscovered amino acids necessary for complex stability

abstract:
  The emergence of SARS-CoV-2 has resulted in more than 200,000 infections and nearly 9,000 deaths globally so far. This novel virus is thought to have originated from an animal reservoir, and acquired the ability to infect human cells using the SARS-CoV cell receptor hACE2. In the wake of a global pandemic it is essential to improve our understanding of the evolutionary dynamics surrounding the origin and spread of a novel infectious disease. One way theory predicts selection pressures should shape viral evolution is to enhance binding with host cells. We first assessed evolutionary dynamics in select betacoronavirus spike protein genes to predict where these genomic regions are under directional or purifying selection between divergent viral lineages at various scales of relatedness. With this analysis, we determine a region inside the receptor-binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor hACE2. Next, to gain further insights into factors associated with coronaviruses recognition of the human host receptor, we performed modeling studies of five different coronaviruses and their potential binding to hACE2. Modeling results indicate that interfering with the salt bridges at hot spot 353 could be an effective strategy for inhibiting binding, and hence for the prevention of coronavirus infections. We also propose that a glycine residue at the receptor binding domain of the spike glycoprotein can have a critical role in permitting bat variants of the coronaviruses to infect human cells.


