key: cord-0280845-zpbxa7bh
authors: Gonzales, S. Jake; Clarke, Kathleen N.; Batugedara, Gayani; Braddom, Ashley E.; Garza, Rolando; Reyes, Raphael A.; Ssewanyana, Isaac; Garrison, Kendra C.; Ippolito, Gregory C.; Greenhouse, Bryan; Bol, Sebastiaan; Bunnik, Evelien M.
title: A comparative analysis of memory B cell and antibody responses against Plasmodium falciparum merozoite surface protein 1 in children and adults from Uganda
date: 2021-11-08
journal: bioRxiv
DOI: 10.1101/2021.11.04.467302
sha: 4959bbc04d606440c4fd3c6e5d0d1952cb97b9e1
doc_id: 280845
cord_uid: zpbxa7bh

Memory B cells (MBCs) and plasma antibodies against Plasmodium falciparum merozoite antigens are important components of the protective immune response against malaria. To gain understanding of how responses against P. falciparum develop in these two arms of the humoral immune system, we evaluated MBC and antibody responses against the most abundant merozoite antigen, merozoite surface protein 1 (MSP1), in individuals from a region in Uganda with high P. falciparum transmission. Our results showed that MSP1-specific B cells in adults with immunological protection against malaria were predominantly IgG+ classical MBCs, while children with incomplete protection mainly harbored IgM+ MSP1-specific classical MBCs. In contrast, anti-MSP1 plasma IgM reactivity was minimal in both children and adults. Instead, both groups showed high plasma IgG reactivity against MSP1 and whole merozoites, with broadening of the response against non-3D7 strains in adults. The antibodies encoded by MSP1-specific IgG+ MBCs carried high levels of amino acid substitutions and recognized relatively conserved epitopes on the highly variable MSP1 protein. Proteomics analysis of MSP119-specific IgG in plasma of an adult revealed a limited repertoire of anti-MSP1 antibodies, most of which were IgG1 or IgG3. Similar to MSP1- specific MBCs, anti-MSP1 IgGs had relatively high levels of amino acid substitutions and their sequences were predominantly found in classical MBCs, not atypical MBCs. Collectively, these results showed evolution of the MSP1-specific humoral immune response with cumulative P. falciparum exposure, with a shift from IgM+ to IgG+ B cell memory, diversification of B cells from germline, and stronger recognition of MSP1 variants by the plasma IgG repertoire.

Malaria, caused by the parasite Plasmodium falciparum, is responsible for 54 approximately half a million deaths every year, of which two-thirds occur in children 55 under the age of five (1) . A much larger number of people experience non-fatal malaria, 56 amounting to an estimated 230 million cases of disease annually. Although the mortality 57 rate for malaria has slowly but consistently declined over the past two decades, the 58 decrease in malaria incidence has plateaued in the past five years. Current 59 interventions are thus insufficient for malaria elimination and novel tools, such as a 60 highly efficacious malaria vaccine, are urgently needed in the fight against this 61 devastating disease. RTS,S, the only malaria vaccine that has elicited protection 62 against (severe) malaria in a phase III clinical trial, had an efficacy against clinical 63 malaria of 30 -40% in infants and young children (2). Vaccine efficacy was high shortly 64 after vaccination but declined rapidly, and was lower against parasites that were 65 genetically different from strain 3D7 that the subunit vaccine RTS,S was based on (3). 66

Compared to vaccination, repeated natural P. falciparum infections eventually elicit 67 superior immunity, consisting of relatively long-lived antibody responses (~2 -4 years) 68

with cross-strain reactivity (4, 5). This naturally acquired humoral immunity against 69 malaria is associated with the presence of circulating immunoglobulin G (IgG) against 70 with the exception of two Ugandan adults who were anonymous blood donors with high 122 levels of plasma antibodies against various malaria antigens, suggesting frequent 123 exposure to P. falciparum (Supplementary figure 1) . Cryopreserved PBMCs were 124 used to first isolate bulk B cells, followed by staining with fluorescently labeled tetramers 125 of full-length MSP1 from P. falciparum strain 3D7 (MSP13D7) and decoy tetramers for 126 analysis by flow cytometry, a strategy developed and used by others (26, 27) ( Figure  127 1A). In both children and adults, MSP1-specific B cells (defined as 128 CD19 + CD20 + MSP1 + decoy -) were predominantly found among CD21 + CD27 + classical 129 memory B cells (MBCs) ( Figure 1B ). In addition, in both groups, very few MSP1-130 specific B cells were found among CD21 -CD27atypical MBCs ( Figure 1B) . To further 131 define the phenotype of MSP1-specific B cells, we determined the isotype usage of 132 MSP1-specific classical MBCs. Children showed much larger percentages of IgM + 133 (median, ~70%) than IgG + (median, ~10%) classical MBCs in the total repertoire, which 134 was reflected in the percentage of IgM + and IgG + MSP1-specific classical MBCs ( Figure  135 1C). In contrast, IgM + and IgG + classical MBCs were equally abundant in adults in the 136 total repertoire, while IgM + MSP1-specific classical MBCs were depleted and IgG + 137 MSP1-specific classical MBCs were slightly enriched ( Figure 1C) . The ratio of IgG + to 138

IgM + MSP1-specific classical MBCs was higher in all adults as compared to the children 139 ( Figure 1D) . Collectively, these results suggest that the B cell response against MSP1 We therefore cloned a multimerization domain into the C-terminus of the IgG1 heavy 164 chain to express IgM-derived antibodies as pentameric IgG. In contrast to results 165 reported by Thouvenel et al. (15) , we were unable to rescue the MSP1-reactivity of IgM-166

IgG reactivity against MSP1HB3 than children. The average reactivity of adult samples 236 against MSP1Dd2 was also higher but not statistically significantly different from that in 237 children (P = 0.06). These results suggest that while children already have plasma IgG 238 against different variants of MSP1, this response continues to broaden with age. 239

To determine whether the differential IgM and IgG reactivity of plasma is specific for 240 anti-MSP1 antibodies or can be extrapolated to anti-merozoite antibody responses in 241 general, we also tested reactivity of plasma IgM and IgG to whole merozoites from P. 242 falciparum strain 3D7. In this assay, we observed anti-IgM and anti-IgG reactivity in both 243 groups, with adults showing higher reactivity for both IgM and IgG as compared to 244 children ( Figure 4D ). IgM and IgG anti-merozoite reactivity were not directly compared 245

since the values measured could have been influenced by the binding affinity of the 246 secondary antibody. Collectively, these results suggest that in contrast to our 247 observation that the MBC response in children is enriched for IgM + MBCs, the plasma 248 antibody response against MSP1 in both children and adults is dominated by IgG. 249

However, IgM responses against other merozoite antigens may be better developed, in 250 particular in adults. 251

The anti-MSP1 plasma IgG repertoire has limited diversity, high levels of amino 253 acid substitutions, and mainly overlaps with sequences found in classical 254

To further explore the connection between the plasma cell and MBC compartments of 256 the humoral immune response, we performed an integrative analysis of the plasma anti-257 MSP1 IgG and B cell receptor repertoires in adult donor 2, who was selected based on 258 the availability of additional PBMCs and plasma. Although the results of this experiment 259 will require confirmation in additional individuals, this is the first analysis of its kind in a 260 malaria-experienced person and will provide valuable insight into the molecular 261 characteristics of the anti-MSP1 plasma antibody repertoire after life-long exposure to 262 P. falciparum. In a previous study, we generated B cell receptor sequencing (BCR-seq) 263 data of antibody heavy chain variable regions of naïve B cells, classical MBCs, and 264 atypical MBCs (30). The full BCR-seq data set and all sequences obtained from MSP1-265 specific MBCs were used to construct a personalized, donor-specific reference heavy 266 chain antibody variable region sequence database. We then isolated anti-MSP1 IgG 267 from plasma using commercially available 19 kDa C-terminal fragment of MSP1 268 (MSP119), which limited our analysis to the most conserved domain of MSP1 (31, 32). 269

We analyzed the anti-MSP119 IgG preparation by high-resolution liquid chromatography 270 with tandem mass spectrometry and searched the obtained spectra against the donor-271 specific antibody variable region database ( Figure 5A ). This step allowed us to identify 272 the full-length antibody sequences that these short peptide spectra were derived from. 273

Eighteen anti-MSP119 IgG lineages were identified, of which four lineages made up over 274 75% of all plasma anti-MSP119 IgG ( Figure 5B, Supplementary table 3 

To further analyze antibody characteristics that may influence their selection and and lower-affinity B cells to MBCs (33). For mAb10 and mAb22, it would therefore be 319 expected that mAb10 has higher antigen-binding affinity. We determined binding affinity 320 of mAb10 and mAb22 to MSP1 using a chaotropic ELISA with urea and observed that 321 mAb10 indeed showed higher binding affinity to MSP1 than mAb22 ( Figure 6A) . The 322 observation that mAb10 and mAb22 had different light chains suggests that antigen 323 binding by these mAbs is dominated by their heavy chain. To test this, we expressed 324 mAb22 with light chains from unrelated non-MSP1-binding antibodies and observed that 325 it was still reactive with MSP13D7, albeit with lower binding affinity ( Figure 6B) . These 326 results suggest that the heavy chain of mAb22 (and presumably mAb10) is sufficient for 327 binding to MSP1, but that its light chain is important for optimal binding to antigen. This 328 raised the question whether the difference in binding affinity between mAb10 and 329 mAb22 is caused by the different light chains used by the two mAbs. For each mAb, we 330 therefore compared binding affinity between the antibody expressed with its own light 331 chain and a chimeric antibody in which the light chain was swapped. Expression of 332 mAb10 with the mAb22 light chain resulted in a reduction of binding affinity, while the 333 binding affinity of mAb22 was unchanged when expressed with the mAb10 light chain 334 ( Figure 6C ). These results suggest that the light chain of mAb10 may play a role in the 335 increased binding affinity of mAb10 over mAb22, but is dependent on the mAb10 heavy 336 chain for this effect. Interestingly, despite higher binding affinity, mAb10 showed 337 reduced activity in a growth inhibition assay as compared to mAb22 ( Figure 2C) . Our results showed that children harbored a larger fraction of MSP1-specific IgM + MBCs 356 than adults, in line with a recent report (16). Interestingly, based on these observations, 357 one might expect that the plasma anti-MSP1 antibody response would also be 358 dominated by IgM, but this was not the case. In both adults and children, the level of 359 anti-MSP1 in plasma was much lower than that of anti-MSP1 IgG. We recognize that it 360 is difficult to directly compare IgM and IgG measurements due to potential differences 361 between secondary antibodies used in these assays. However, our control samples 362 from recovered COVID-19 patients demonstrate that we were able to measure IgM 363

reactivity, yet we detected plasma IgM reactivity against MSP1 in only a small 364 percentage of adults. These observations suggest that malaria-experienced children 365 develop a strong IgG response against MSP1 that is not reflected in the MBC 366 compartment. One explanation for the relative lack of IgG + MBCs in children could be 367 that the B cell response predominantly gives rise to short-lived IgG + plasmablasts, had equal opsonic phagocytosis activity and two-fold lower fixation capacity of C1q, the 418 primary component of the classical complement pathway, as compared to plasma IgG, 419 but induced nine-fold higher deposition of components of the membrane attack complex 420 (14, 16). While these results suggest that IgM can indeed contribute to parasite 421 inhibition, the concentration of IgM in plasma is almost one order of magnitude lower 422 than that of IgG (average, 1.5 g/l for IgM versus 11 g/l for IgG, (47, 48)). Therefore, the 423 relative contributions of IgM and IgG to parasite inhibition remain to be determined. 424

Finally, a passive immunization study showed that IgG-depleted plasma from malaria-425 experienced adults had no effect on parasitemia in children with P. falciparum malaria, 426

whereas treatment with IgG from the same individuals resulted in a dramatic decrease 427 of parasite counts (49), suggesting that IgG is the main effector antibody responsible for 428

The acquisition of high levels of amino acid substitutions in IgG + MBCs from both 431 children and adults suggests that these cells are the product of multiple rounds of 432 affinity selection. We also detected several B cells with long HCDR3s, similar to broadly 433 neutralizing antibodies against HIV (50), although this was not a universal characteristic 434 of anti-MSP1 mAbs. In addition, we observed that children and adults had equally high 435

IgG reactivity against MSP1 3D7, but that reactivity against MSP1 HB3 was lower in 436 children. This suggests that the IgG response continues to broaden with subsequent P. Culture supernatants were collected 5 -7 days post-transfection by centrifuging the 525 culture at 4,000 × g for 25 min. at RT. A 10 kDa cutoff Protein Concentrator PES 526 (Thermo #88527) was used (5,000 × g at 4⁰C) to exchange culture medium containing 527 free biotin for PBS (pH 7.2) (> 100,000 dilution) and to concentrate the protein to a final 528 volume of 0.5 -1 ml. The 3D7 MSP1 protein was mixed with 6 -12 volumes of PBS 529 (pH 5.5) in a final volume of six ml and was subsequently loaded onto gravity flow 530 columns (Thermo #29924) containing CaptAvidin agarose (Thermo #C21386) for 531 purification. After three washes with PBS (pH 5.5) and five 6 ml elutions with PBS (pH 532 10.5), the elutions were pooled (30 ml) and the pH was immediately neutralized by 533 adding 12 ml PBS (pH 5.5). After concentrating, the protein was quantified using the 534 Coomassie Plus (Bradford) Assay Kit (Thermo #23236) on a NanoDrop One 535 spectrophotometer, according to the manufacturer's instructions, visualized by SDS-536 PAGE (Supplementary Figure 3A) , diluted to 1 mg/ml, aliquoted, and stored at -70°C. 537

Since each streptavidin molecule has the ability to bind four biotinylated MSP1 539 molecules, MSP1 tetramers were made by incubating MSP1 in a tube revolver (Thermo 540 #88881001) at 40 rpm and RT for 30 min. with streptavidin-PE (Thermo #S866) at a 6:1 541 molar ratio. After this incubation, the tetramers were washed with PBS (pH 7.2) using a 542

Vivaspin centrifugal concentrator (Sartorius #VS0141) three times for 5 min. at 15,000 × 543 g at RT. To make decoy tetramers, streptavidin-PE was first conjugated to Alexa-fluor 544 647 (Thermo #A20186) per manufacturer's instructions. This double-conjugated 545 streptavidin was then coupled to R. norvegicus CD200 (Addgene #36152 (53)) as 546 min. in the dark on ice), followed by a wash with 1 ml of cold PBS/1% BSA (5 min. at 577

Human PE Positive Selection Kit (#17664) and subsequently stained on ice for 30 min. 579

with an antibody panel against B cell surface markers (Supplementary table 4) . 580

UltraComp eBeads (Thermo #01222242) were used to prepare compensation controls 581 for each fluorophore per manufacturer's instructions. Before acquisition on a BD 582

FACSAria II cell sorter, the cells were washed with 3 ml of cold PBS with 1% BSA (5 583 min. at 250 × g and 4°C), diluted to 20 -30 million cells/ml in PBS with 1% BSA, and 584 filtered into a FACS tube with filter cap. Lymphocytes were gated using forward and 585 After incubation at 37°C and 8% CO2 for two weeks, the wells were screened for the 596 production of IgM or IgG by enzyme-linked immunosorbent assay (ELISA). 597 598

To detect IgG and IgM, 96-well ELISA plates (Corning #3361) were coated with either 600 goat anti-human IgG (Sigma #I2136) or IgM (Sigma #I1636) antibody at a concentration 601 of 4 and 8 µg/ml, respectively, diluted in PBS, at a total volume of 100 µl per well. After 602 a one hour incubation at 37°C or O/N at 4°C, each well was washed once using slowly 603 running (approximately 900 ml / min.) deionized water. This washing method resulted in 604 significantly higher specificity than other methods tested in the lab (using a plate washer 605 with water or PBS containing 0.1% tween-20, or a squeeze bottle filled with PBS 606 containing 0.1% tween-20). All subsequent washes were performed this way. 150 µl 607 blocking buffer (one-third Non-Animal Protein (NAP)-Blocker (G-Biosciences #786-608 190P) and two-thirds PBS) was added to each well to prevent non-specific binding. Reverse primer #67 was added for the heavy chain variable region PCR to allow for 694 amplification of variable regions originating from IgG2, IgG3 and IgG4 mRNA, in addition 695 to #30 which was specific for IgG1. Cycling conditions were as described above, except 696 for the extension step (shortened to 30 sec.) and the annealing step, which was 30 sec. Heavy and light chain antibody expression plasmids were used at a molar ratio of 1:2 to 753 transfect 5 ml cultures. The antibodies were purified from the culture supernatant 4 -6 754 days later using protein G magnetic beads (Promega #G7472). Purified antibodies and 755 antibody elution buffer (5 parts elution buffer (100 µM glycine-HCl, pH 2.7) and 1 part 756 neutralization buffer (2M Tris buffer, pH 7.5)) were buffer exchanged to PBS using 100 757 kDa cutoff Protein Concentrators (Thermo #88523). The samples were diluted > 50,000 758 × in PBS by repeated centrifugation at 4,000 × g and 4⁰C. Purified antibodies were 759 quantified using the Coomassie Plus (Bradford) Assay Kit (Thermo #23236) on a 760

NanoDrop One spectrophotometer, according to the manufacturer's instructions, and 761 visualized on SDS-PAGE gel with a standard amount of BSA to confirm protein size and 762 purity (Supplementary figure 3B) . Parasites were cultured at 37°C in an atmosphere of 5% O2, 5% CO2, and 90% N2. 774

Before use in cultures, 12.5 ml packed erythrocytes were washed twice with 10 ml cold 775 incomplete medium (complete culture medium without human serum) and pelleted 776 between each wash by centrifugation at 500 × g for 8 min. at 4°C (max. acceleration 777 and weakest break). Washed erythrocytes were resuspended in 2 volumes of complete 778 medium and stored at 4°C. Growth inhibition assay 871 P. falciparum isolate 3D7 parasites were pre-synchronized at the ring stage with a 5% 872 D-sorbitol (Fisher #S459-500) treatment as described above, followed four days later by 873 two additional 5% D-sorbitol treatments 14 hours apart (60). At the late trophozoite / 874 early schizont stage (24 hours after the third D-sorbitol treatment), parasitemia was 875 determined by inspection of a Giemsa-stained blood smear. The smear was also used 876 to confirm correct parasite staging. Immediately after, 20 µl of each antibody (1 mg/ml in 877 PBS) was added to wells containing 30 µl complete medium in a black clear bottom 96-878 well plate (Corning #3603). A monoclonal antibody specific for apical membrane antigen 879 1 (AMA1) was used as a positive control (BEI #MRA-481A). Antibody elution buffer (100 880 mM glycine-HCl, pH 2.7) that was buffer exchanged to PBS alongside purified 881 antibodies (see "Antibody expression and purification" above) was used as a negative 882 control. Fifty µl parasite culture (1% parasitemia and 2% hematocrit) was then added to 883 wells containing antibody or negative control. Uninfected erythrocytes (2% hematocrit) 884 were used to determine the background signal. The plate was then incubated at 885 standard parasite culture conditions (described above) for 48 hours before being 886 transferred to a -70°C freezer. After overnight incubation of the plate at -70°C, SYBR 887 green dye (Invitrogen #S7585) was added to lysis buffer (20 mM Tris-HCl (pH7.5), 5 888 mM EDTA, 0.008% saponin (Sigma # 558255100GM), 0.08% Triton X-100 in MQ 889 water) at 0.2 µl dye per ml of lysis buffer. One hundred µl SYBR green lysis buffer was 890 added to each well and the plate was incubated in the dark at 37°C for 3 -6 hours. fractions of 1% formic acid. IgG-containing elution fractions were concentrated to 908 dryness in a speed-vac, resuspended in ddH2O, combined, neutralized with 1M Tris / 909 3M NaOH, and prepared for liquid chromatography-tandem mass spectrometry (LC-910 MS/MS) as described previously (64, 65) with the modifications that (i) peptide 911 separation using acetonitrile gradient was run for 120 min and (ii) data was collected on 912 an Orbitrap Fusion (Thermo Fisher Scientific) operated at 120,000 resolution using HCD 913 

Flow cytometry data were analyzed and plotted using FlowJo (v10.7.1). Dot plots were 926 generated using the package ggplot2 in RStudio (v1.4.1103) using R (v4.0.4). All other 927 plots were generated in GraphPad Prism 9, which was also used for statistical analyses. 928

The statistical test used for each analysis is indicated in the figure legends. 929 930

The BCR-seq data set analyzed in the current study is available in the NCBI SRA 932 repository under accession numbers SAMN17497575-7. 933 934

The authors declare that the research was conducted in the absence of any commercial 936 or financial relationships that could be construed as a potential conflict of interest. (n = 10) and chimeric antibodies (n = 9, three replicates for each of three unrelated light 1284 chains that were combined for analysis) were tested for statistical significance using the 1285 Mann Whitney test. C) The binding strength of mAb10 and mAb22 (n = 10 replicates 1286 each) expressed with their own light chains or chimeric mAbs in which the light chains 1287 were swapped (n = 6 replicates each). In all panels, data points are the average of three 1288 technical replicates. Differences between antibodies with the same heavy chain but a 1289 different light chain were tested for statistical significance using a Kruskal-Wallis test, 1290 followed by comparisons between select groups using Dunn's post-hoc test, corrected 1291 for multiple comparisons. **** P < 0.0001; ** P < 0.01; * P < 0.05

Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a 982 booster dose in infants and children in Africa: final results of a phase 3, individually 983 randomised, controlled trial

Genetic 985 Diversity and Protective Efficacy of the RTS,S/AS01 Malaria Vaccine

Antibody 988 responses to merozoite antigens after natural Plasmodium falciparum infection: 989 kinetics and longevity in absence of re-exposure

The relationship between 994 anti-merozoite antibodies and incidence of Plasmodium falciparum malaria: A 995 systematic review and meta-analysis

Human 997 antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes 998 and are associated with protection against malaria

Breadth and 1000 magnitude of antibody responses to multiple Plasmodium falciparum merozoite 1001 antigens are associated with protection from clinical malaria

Acquisition of 1007 antibodies against Plasmodium falciparum merozoites and malaria immunity in 1008 young children and the influence of age, force of infection, and magnitude of 1009 response

Identification and prioritization of merozoite antigens as targets of protective human 1012 immunity to Plasmodium falciparum malaria for vaccine and biomarker 1013 development

Breadth of 1015 Functional Antibodies Is Associated With Plasmodium falciparum Merozoite 1016 Phagocytosis and Protection Against Febrile Malaria

Targets of 1019 complement-fixing antibodies in protective immunity against malaria in children

IgM in human 1022 immunity to Plasmodium falciparum malaria

Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict 1025 Plasmodium parasites

Plasmodium 1027 falciparum-specific IgM B cells dominate in children, expand with malaria, and 1028 produce functional IgM

B cell memory: building two walls of protection 1030 against pathogens

A 1032 prospective analysis of the Ab response to Plasmodium falciparum before and after 1033 a malaria season by protein microarray

The 1036 Plasmodium falciparum-specific human memory B cell compartment expands 1037 gradually with repeated malaria infections

Holder AA. The carboxy-terminus of merozoite surface protein 1: structure, specific 1039 antibodies and immunity to malaria

Diversity 1041 analysis of MSP1 identifies conserved epitope organization in block 2 amidst high 1042 sequence variability in Indian Plasmodium falciparum isolates

Plasmodium falciparum Protein Microarray Antibody Profiles Correlate With 1046

Protection From Symptomatic Malaria in Kenya

Blood 1048 stage malaria vaccine eliciting high antigen-specific antibody concentrations confers 1049 no protection to young children in Western Kenya

ChAd63-MVA-vectored blood-stage malaria vaccines targeting MSP1 and AMA1: 1052 assessment of efficacy against mosquito bite challenge in humans

1055 Malaria transmission, infection, and disease at three sites with varied transmission 1056 intensity in Uganda: implications for malaria control

Deletion and anergy of polyclonal B cells specific for ubiquitous membrane-bound 1060 self-antigen

Somatically Hypermutated Plasmodium-Specific IgM(+) Memory B Cells Are Rapid

Early Responders upon Malaria Rechallenge

IMGT((R)) tools for the 1065 nucleotide analysis of immunoglobulin (IG) and T cell receptor (TR) V-(D)-J 1066 repertoires, polymorphisms, and IG mutations: IMGT/V-QUEST and IMGT/HighV-1067 QUEST for NGS

Activation Dynamics and Immunoglobulin Evolution of Pre-existing and Newly 1070 Generated Human Memory B cell Responses to Influenza Hemagglutinin

Receptor Repertoire Analysis in Malaria-Naive and Malaria-Experienced Individuals 1074 Reveals Unique Characteristics of Atypical Memory B Cells. mSphere

Analysis of sequence 1077 diversity in the Plasmodium falciparum merozoite surface protein-1 (MSP-1)

Allelic dimorphism in a surface 1080 antigen gene of the malaria parasite Plasmodium falciparum

Atypical 1086 activation of dendritic cells by Plasmodium falciparum

Hemozoin-mediated inflammasome activation limits long-lived anti-malarial 1090 immunity

Atypical memory 1092 B cells in human chronic infectious diseases: An interim report

Potential functions of atypical 1095 memory B cells in Plasmodium-exposed individuals

Infection-induced plasmablasts are a nutrient sink that impairs humoral immunity to 1099 malaria

T-Bet(+) IgM Memory Cells Generate Multi-lineage Effector B Cells. 1102

Heritability 1104 of antibody isotype and subclass responses to Plasmodium falciparum antigens

Association between naturally acquired antibodies to erythrocyte-binding antigens 1108 of Plasmodium falciparum and protection from malaria and high-density 1109 parasitemia

Stage-1111 specific Plasmodium falciparum immune responses in afebrile adults and children 1112 living in the Greater Accra Region of Ghana

Acquisition and decay of IgM and IgG responses to merozoite antigens after 1115 Plasmodium falciparum malaria in Ghanaian children

Cohort 1118 study of the association of antibody levels to AMA1, MSP119, MSP3 and GLURP 1119 with protection from clinical malaria in Ghanaian children

Antibody 1121 levels against GLURP R2, MSP1 block 2 hybrid and AS202.11 and the risk of 1122 malaria in children living in hyperendemic (Burkina Faso) and hypo-endemic 1123 (Ghana) areas

Acquired antibodies to merozoite antigens in children from Uganda with 1126 uncomplicated or severe Plasmodium falciparum malaria

IgA, IgM) in a general adult population and 1130 their relationship with alcohol consumption, smoking and common metabolic 1131 abnormalities

Gamma-globulin and acquired immunity to 1136 human malaria

Aberrant B 1138 cell repertoire selection associated with HIV neutralizing antibody breadth

Estimating the 1141 annual entomological inoculation rate for Plasmodium

Anopheles gambiae s.l. using three sampling methods in three sites in Uganda

A library 1145 of functional recombinant cell-surface and secreted P. falciparum merozoite 1146 proteins

Large-1148 scale screening for novel low-affinity extracellular protein interactions

Isolation of 1151 human monoclonal antibodies from peripheral blood B cells

Memory B Cells Harbor Diverse Cross-Neutralizing Antibodies against BK and JC 1155 Polyomaviruses

Base 1157 preferences in non-templated nucleotide incorporation by MMLV-derived reverse 1158 transcriptases

Incorporation of non-natural 1160 nucleotides into template-switching oligonucleotides reduces background and 1161 improves cDNA synthesis from very small RNA samples

High-1164 throughput isolation of immunoglobulin genes from single human B cells and 1165 expression as monoclonal antibodies

Human malaria parasites in continuous culture

Synchronization of Plasmodium falciparum 1169 erythrocytic stages in culture

Detection of antibodies 1171 to variant antigens on Plasmodium falciparum-infected erythrocytes by flow 1172 cytometry

1174 Isolation of viable Plasmodium falciparum merozoites to define erythrocyte invasion 1175 events and advance vaccine and drug development

Separation of malaria-1178 infected erythrocytes from whole blood: use of a selective high-gradient magnetic 1179 separation technique

1181 Identification and characterization of the constituent human serum antibodies 1182 elicited by vaccination

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD 1185 spike epitopes

High-quality full-length immunoglobulin profiling with unique molecular barcoding

Search and clustering orders of magnitude faster than BLAST

CD21 -CD27 + ; and atypical memory B cells (atMBC)

Data are shown as pseudocolor plots, in which overlapping cells in the plots 1199 showing MSP1-specific B cells and cMBCs are shown in orange and red. B) Relative 1200 abundance of major B cell subsets among the total B cell repertoire and among

specific B cells in partially immune children (n = 4) and immune adults (n = 4). C) The 1202 percentage of IgM + and IgG + B cells among the total repertoire of cMBCs and among 1203

MSP1-specific cMBCs in malaria-experienced children (n = 4) and adults (n = 4). In 1204 panels B and C, differences were tested for statistical significance

Student's t-test, even though a non-parametric test would be more appropriate given the 1206 small sample size. However, the paired non-parametric alternative (Wilcoxon signed-1207 rank test) does not return a P value lower than 0.13 when using groups of 4. D) The 1208 ratio of the percentage of IgG + over IgM + MSP1-specific cMBCs in malaria-experienced 1209 children and adults. A data point with value 0 was plotted at 0.1 for visualization 1210 purposes

05 1212 experiments are shown. Scale bar is 5 µm. C) P. falciparum growth inhibition by six 1224 select mAbs isolated from adult donor 2. Inhibition was calculated relative to a negative 1225 control

mAb 481A served as a positive control

Parasites were synchronized to the ring stage by treatment with 5% D-sorbitol (60) 781 (Fisher #S459-500). Cultures containing high percentages of ring-stage parasites were 782 centrifuged at 250 × g for 5 min. at RT. Pelleted erythrocytes were resuspended in 10 783 volumes of 5% D-sorbitol in MQ water, vortexed for 30 sec. and incubated for 8 min. at 784 37°C. The cells were then washed with 5 volumes of complete culture medium (250 × g 785 for 5 min. at RT) and resuspended in complete culture medium at 5% hematocrit and 786 cultured as described above. To obtain tightly synchronized parasites, sorbitol 787 treatments were performed twice, 14 hours apart. 788Infected erythrocytes containing parasites in the late-trophozoite and schizont stages 789