key: cord-311843-un6urdb1
authors: Baray, Juwel Chandra; Khan, Md. Maksudur Rahman; Mahmud, Asif; Islam, Md. Jikrul; Myti, Sanat; Ali, Md. Rostum; Sarker, Md. Enamul Haq; Kumar, Samir; Chowdhury, Md. Mobarak Hossain; Roy, Rony; Islam, Faqrul; Barman, Uttam; Khan, Habiba; Chakraborty, Sourav; Hossain, Md. Manik; Chowdhury, Md. Mashfiqur Rahman; Ghosh, Polash; Mohiuddin, Mohammad; Sultana, Naznin; Nag, Kakon
title: BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response
date: 2020-09-30
journal: bioRxiv
DOI: 10.1101/2020.09.29.319061
sha: 
doc_id: 311843
cord_uid: un6urdb1

Effective vaccine against SARS-CoV-2 is the utmost importance in the current world. More than 1 million deaths are accounted for relevant pandemic disease COVID-19. Recent data showed that D614G genotype of the virus is highly infectious and responsible for almost all infection for 2nd wave. Despite of multiple vaccine development initiatives, there are currently no report that has addressed this critical variant D614G as vaccine candidate. Here we report the development of an mRNA-LNP vaccine considering the D614G variant and characterization of the vaccine in preclinical trial. The surface plasmon resonance (SPR) data with spike protein as probe and competitive neutralization with RBD and S2 domain revealed that immunization generated specific antibody pools against the whole extracellular domain (RBD and S2) of the spike protein. The anti-sera and purified IgGs from immunized mice on day 7 and 14 neutralized SARS-CoV-2 pseudovirus in ACE2-expressing HEK293 cells in a dose dependent manner. Importantly, immunization protected mice lungs from pseudovirus entry and cytopathy. The immunologic responses have been implicated by a balanced and stable population of CD4+ cells with a Th1 bias. The IgG2a to IgG1 and (IgG2a+IgG2b) to (IgG1+IgG3) ratios were found 1±0.2 and 1.24±0.1, respectively. These values are comparatively higher than relevant values for other published SARS-CoV-2 vaccine in development,1, 2 and suggesting higher viral clearance capacity for our vaccine. The data suggested great promise for immediate translation of the technology to the clinic.

as dispersant. The formulation was concentrated using Ultra centrifugal filters (Merck, 181 Germany), filtered through 0.22 micron filter, and stored at 5±3 °C. 23 The formulation was 182 passed through the quality control for the particle size, encapsulation efficiency, endotoxin 183 limit and sterility.

A total number of 50 BALB/c swiss albino mice (male and female) of 6-8 weeks old, were 186 selected randomly and isolated 5 days before immunization. After careful observation and 187 conditioning, 30 mice (15 males and 15 females) were taken to the experiment room for 188 immunization and subsequent safety and efficacy analysis. 9 male mice were also separated for 189 local tolerance testing. The temperature in the experimental animal room was 26 °C (±2 °C) 190 and the relative humidity was 60±5%. The room was HVAC controlled ISO class 7 room with 191 70% fresh air intake and full exhaust. The mice were individually housed in polypropylene 192 cage with individual water bottle, provided with 5 g of in-house mouse feed daily and kept 193 under 12 hours of day-night cycle. 30 mice were separated into 5 different groups consisting 6 194 mice (3 males and 3 females) in each group. There were 3 different treatment groups such as 195 Treatment group 1, 2, and 3, 1 placebo group and 1 control group. Each mouse of treatment groups 1, 2, and 3 was immunized with sterile 0.1 µg/50 µL, 1.0 µg/50 µL and 3.0 µg/50 µL 197 of BANCOVID, respectively. Each mouse of the placebo group was injected with the vehicle 198 only and the control group mice were not injected with anything. Intramuscular (IM) injection 199 in the left quadriceps was done for immunization. The flow of the experimental design is The immunogenicity of BANCOVID was evaluated in BALB/c mice, post administration to 218 the quadriceps muscle. Approximately 200 µL blood was collected from facial vein and 219 centrifuged at 1500 X g for serum isolation (10 minutes at 4 CC). All serums were aliquot, frozen immediately and stored at -80 °C until analysis. The reactivity of the sera from each 221 group of mice immunized with BANCOVID was measured against SARS-CoV-2 S antigen 222 (SinoBiologicals, China). Analysis revealed IgG binding against SARS-CoV-2 S protein 223 antigens in the sera of the immunized mice. The serum IgG binding endpoint titers (EPTs) were 224 measured in mice immunized with BANCOVID. EPTs were observed in the sera of mice at 225 day 7 and day 14 after immunization with a single dose of the vaccine candidate.

Toxicity 227 Pre-immune whole blood (approximately 50 µL) from each mouse was collected for complete 228 blood count (CBC) in 2% EDTA at 3 days before immunization. Similarly, whole blood was 229 also collected after immunization at day 14 for CBC analysis using auto hematology analyzer Then co-transfection was performed using Lipofectamine 3000 (ThermoFisher, USA) reagent 244 according to manufacturer's protocol. Next day 1.25% low melting agarose in DMEM media 245 was spread on the well and incubated until plaques were formed. After formation of plaques, 246 multiple plaques were collected in DMEM media and titers were measured for plaque selection.

Then selected plaque was added on the fresh pre seeded viral production cell. After few days, For retro based neutralization assay, qPCR was used to analyzed the copy number of S gene 291 that integrated into cell. Copy number of S gene indicated the entry of pseudovirus into cell.

Magmax DNA multi-sample ultra-kit. (ThermoFisher, USA). These genomic DNA was used 294 for determination of S gene copy number by qPCR.

In-vivo neutralization 296 A total number of 18 albino male mice of 6-8 weeks were selected and isolated for the analysis. Ultimate 3000 (ThermoFisher, USA) system using 10 mM Disodium hydrogen phosphate 319 (Wako, Japan), 10 mM Sodium dihydrogen phosphate (Wako, Japan), 100 mM Sodium 320 chloride (Merck, Germany), pH 6.6 as mobile phase. Biobasic SEC-300 (300 x 7.8 mm, particle 321 size; 5 µm, ThermoFisher, USA) column was used with 1.0 mL/minute flow rate, 260 nm 322 wavelength, 10 µL sample injection volume for 20 minutes. For peptide identification, data dependent mass spectrometry was performed where full-MS 375 scan range was 350 m/z to 2200 m/z, resolution was 70,000, AGC target was 3E6, maximum 376 IT was 100 milliseconds (ms), and data dependent mass spectrometry resolution was 17,500, 377 AGC target was 1E5, maximum IT was100 ms. After getting raw data from mass spectrometry 378 system, data analysis was performed in BioPharma Finder (ThermoFisher, USA) using variable 379 parameters to get confident data, and then data were combined in one map to visualize complete IVT process was tuned to obtain desired mRNA with high yield and quality (Figure: 1C) . The 443 mRNA was encapsuled in lipid nano particle (LNP) ranging from 60 -140 nm with the final 444 pH of 7.2. We did a pilot study with limited numbers of mice to identify the suitable mRNA-445 LNP size for our formulation. mRNA-LNP either smaller than 70 nm or larger than 110 nm 446 did not generate considerable immunological response even with a dose of 10 ng/mice (data 447 not shown). To obtain the best process control for the dose production, we therefore, set our 448 mRNA-LNP size range at 85±10 nm. We used mRNA-LNP of this range throughout the rest whether the immunization have generated antibody pool spanning for the whole antigen or for 480 any specific domain (S1 or S2), we have chosen surface plasmon resonance (SPR) experiment.

The S protein chip recognized high-affinity antibody from the anti-sera (Figure: 3D) . The 482 response was attenuated significantly for S-protein(s) (S, S1 and S2) pretreated sera ( Figure   483 3D). S and S1 pretreatment showed similar and strong inhibitory response while S2 The pH (7.2) of our formulation buffer for mRNA-LNP is also lower than the other relevant 564 references (7.4~8.0). 2, 7, 8, 9 Lower pH helps quick release of the cargo from endosomal 565 compartment and protects mRNA from acid hydrolysis and lysosomal digestion in intracellular 566 milieu. 33 Together, numbers of minute changes in the design context likely playing in concert 567 and produced quick, balanced, stable Th1-IgG2-biased antibody response.

'BANCOVID' immunization did not produce any noticeable effect for local or systemic 569 toxicity as primarily evident by the absence of four cardinal signs of inflammation: redness 570 (Latin rubor), heat (calor), swelling (tumor), and pain (dolor). There was no erythema or 571 erythredema as well in any injection site. The CBC and blood chemistry data did not show significant changes in relevant profiles and has been suggesting that the vaccine behaves safely 573 in animal. A balanced response between Th1 and Th2 is desired to achieve safe and effective humoral 576 immunity performance. 35 'BANCOVID' has produced well-balanced IgG1 and IgG2 response 577 by 7 th day postimmunization and remained similar on 14 th day postimmunization sera, which 578 is suggesting a stable antibody response during the sampling period. Along with opsonizing 579 characteristics, IgG2 has higher affinity to its receptors and have superior complement system 580 activation potential over IgG1. 35,36 Accordingly, 'BANCOVID'-mediated higher ratio of IgG2 581 than IgG1 has suggested that higher capacity of the antibody pool to clear antigen from the 582 system. The ratio of IgG2a and IgG1, and cytokine-stained CD4 + and CD8 + T cell population 583 showed a Th1-bias response. Since mouse IgG2 is equivalent to human IgG1 35,36 therefore, it is plausible that 'BANCOVID' will elicit effective cellular and humoral response against 585 SARS-CoV-2 in human.

The early vaccine development initiatives were taken before the G614 variant became 

The roles of G614 mutation on constitutive infection have been attributed to its conformational 604 change. It has been proposed that the -COOH group of D614 forms hydrogen bond with the -

OH group of T859 across the S1/S2 interface, which cannot form in G614. 14 On the contrary, 606 structural modeling studies revealed that "the D614G substitution creates a sticky packing 607 defect in subunit S1, promoting its association with subunit S2 as a means to stabilize the structure of S1 within the S1/S2 complex. 30 In other words, the D614G mutation in fact 609 promotes the S1/S2 association and stabilize the spike. 30 The finding is in accordance with the 610 observation that G614 has a greater stability originating from less S1 domain shedding and 611 greater accumulation of the intact S protein into the pseudovirion. 29 It has also been reported A647 in the uncomplexed S1 and inhibits the S1/S2 association. G614 diminishes the salt 629 bridge formation and S1/S2 association resulting interaction with the RBD to facilitate higher 630 infection. 30 Therefore, blocking of G614 with a specific antibody would inhibit such acquired 631 fitness of SARS-CoV-2. 'BANCOVID' immunization has produced a pool of antibody that covers the whole length of the spike protein suggesting that highly likely relevant antibody- for facility and information management system. 

Flow cytometric analysis of total T cell (CD4 + ) populations producing TFN alpha on mouse splenocyte upon SARS-CoV-2 S protein stimulation. Cells were gated in an orderly manner, like singlets were gated, followed by lymphocytes, CD45 + , CD45 + CD4 + and CD45 + CD4 + TFNalpha + (A, B, C) 3 control panels where 0.48%, 0.43% and 0.37% CD45 + CD4 + TFNalpha + cells were identified respectively, (D, E, F) 3 treatment panels where 0.99%, 0.95% and 0.81% CD45 + CD4 + TFNalpha + cells were identified respectively.

Flow cytometric analysis of total T cell (CD4 + ) populations producing IL-2 on mouse splenocyte upon SARS-CoV-2 S protein stimulation. Cells were gated in an orderly manner, like singlets were gated, followed by lymphocytes, CD45 + , CD45 + CD4 + and CD45 + CD4 + IL2 + (A, B, C) 3 control panels where 0.26%, 0.26% and 0.13% CD45 + CD4 + IL2 + cells were identified respectively, (D, E, F) 3 treatment panels where 0.73%, 0.69% and 0.63% CD45 + CD4 + IL2 + cells were identified respectively. 

A Single Dose of Self-Transcribing and 701

Replicating RNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity 702 In Mice

Self-Amplifying RNA SARS-CoV-2 Lipid Nanoparticle Vaccine 706

Candidate Induces High Neutralizing Antibody Titers in Mice

Alnylam Launches Era of RNAi Drugs

Capping, and Proofreading Mechanisms of SARS-Coronavirus

Exoribonuclease Activity Are Susceptible to Lethal Mutagenesis: Evidence for 715 Proofreading and Potential Therapeutics

Molecular Architecture of Early Dissemination and 722

Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area

Tracking Changes in 731 SARS-CoV-2 Spike: Evidence That D614G Increases Infectivity of the COVID-19

D614G Spike Variant Does Not Alter IgG, IgM, 735 or IgA Spike Seroassay Performance

SARS-CoV-2 Viral Spike G614 Mutation Exhibits 785

Higher Case Fatality Rate

The Spike D614G Mutation Increases SARS-CoV-2 Infection of Multiple 789

The D614G Mutation in the SARS-CoV-2 Spike Protein Reduces S1 Shedding 793 and Increases Infectivity

Structural Impact of Mutation D614G in SARS-CoV-2 Spike Protein: 796 Enhanced Infectivity and Therapeutic Opportunity

Spike Mutation D614G Alters SARS-CoV-2 Fitness and Neutralization 802

The Effect 804 of Size and Charge of Lipid Nanoparticles Prepared by Microfluidic Mixing on Their 805

Lymph Node Transitivity and Distribution

Lipid-Based Nanoparticles in the Systemic 808 Delivery of SiRNA

Virus-like Particles Induces a Strong Antiviral-like Immune Response in Mice

Type 1/Type 2 Immunity in Infectious Diseases

IgG Subclass Co-Expression Brings Harmony to the Quartet Model of 818

Flow cytometric analysis of total T cell (CD4 + ) populations producing IL-6 on mouse splenocyte upon SARS-CoV-2 S protein stimulation. Cells were gated in an orderly manner, like singlets were gated

CD45 + CD4 + IL6 + cells were identified respectively

After dissolving, add 500 mM DTT (ThermoFisher Scientific, USA) to the solution to a final concentration of 20 mM (1:25 dilution) and mix briefly; incubate at 60 °C for 1 hour. For alkylation

To digest, add trypsin (ThermoFisher Scientific, USA) solution to the sample solution to a final trypsin to protein ratio of 1:23 (w/w). Incubate the sample tube at 37 °C for 16 -24 hours. After incubation, to stop digestion

Supplementary 30 µg of p20020 rDNA was restriction digested with SfoI (ThermoFisher, USA) for 16 hours, visualized using 0.8% agarose gel electrophoresis, gel excised and DNA extracted from gel using GeneJET Gel Extraction and DNA Cleanup Micro Kit, re-purification of DNA by phenol:chloroform:isoamyl alcohol, followed by phenol removal using chloroform (twice). Purified lyophilized DNA was reconstituted using nuclease-free water, quantified and store at -30 °C for future use.

Optimization step 1: Synthesis time factor 240 ng linear purified DNA was used for all 4 optimization reactions. Each reaction was performed in a 20 µL total volume. For every reaction, a DNase treatment reaction was also performed using 1 µL TURBO DNase (2 U/µL) at 37 °C for 15 minutes. For visualization, 1% agarose gel electrophoresis was performed after every step of reaction ( figure 1C) .In optimization step 1, where synthesis time dependency was observed, for that following components were mixed together apart from water and template, and reaction were run for 2, 4, 6, 8, 10 and 16 hours. 3 control reactions were also performed (1 µg control template pTRI-Xef for each reaction) for 2, 4 and 16 hours at 37 °C. Optimization step 3: RNase inhibitor and pyrophosphatase effectIn 3 rd step of optimization, murine RNase inhibitor and yeast pyrophosphatase effects were observed at a constant synthesis time (2 hours) and constant rNTPs at 37 °C. For that following components were mixed together apart from water and template. A higher concentration of rNTPs reaction was setup. As this point, last optimized condition was run as positive control. Optimization step 4: Temperature dependencyIn 4 th step of optimization, temperature dependency was observed at a constant synthesis time (2 hours), constant rNTPs, and constant RNase inhibitor and pyrophosphatase, and at 38, 37, 36, 35, 34 and 33 °C. For that following components were mixed together apart from water and template. A higher concentration of rNTPs reaction was also setup. As this point, last optimized condition was run as positive control. 

Tapping C18 spin column (ThermoFisher Scientific, USA) to settle resin. Place column into a receiver tube. To activate the column, add 200 µL 50% acetonitrile (Wako Pure Chemicals Industries Ltd., Japan) to wet resin. Centrifuge the column at 1500 × g for 1 minute. Repeat the step. To equilibrate, add 200 µL 0.5% formic acid (Wako Pure Chemicals Industries Ltd., Japan) in 5% acetonitrile (Wako Pure Chemicals Industries Ltd., Japan). Centrifuge the column at 1500 × g for 1 minute. Repeat the step. Load sample on top of resin bed. Place column into a receiver tube. Centrifuge the column at 1500 × g for 1 minute. To ensure complete binding, recover flowthrough and repeat the step 2 -3 times. To wash the column, Place column into a receiver tube. Add 200 µL 0.5% formic acid in 5% acetonitrile to column. Centrifuge the column at 1500 × g for 1 minute. Repeat the step. To recover sample, place column in a new receiver tube. Add 20 µL 70% acetonitrile to top of the resin bed. Centrifuge at 1500 × g for 1 minute. Repeat the step in same receiver tube.

RPMI complete media (RPMI + L-glutamine + penicillin streptomycin + mouse sera) was prepared first. Then a 100 mm petri dish was taken, 10 mL complete media was added and harvested spleen was taken into the dish. By using microscopic glass slides, spleen was smashed into pieces within the petri dish. Cells were washed out from slides using micropipette. A 10 ml pipette was used to draw the solution up and down, each time closing the end of the pipette against the bottom of the petri dishto forcefully expel the contents and break up the pieces. Cell solution was passed through a sterile 40 μm mesh strainer. Centrifugation was performed for 10 minutes at 250 xg, at 4 ºC. Supernatant discarded and cells were re-suspended in RBC(1X) lysing buffer (10X RBC lysis buffer: NH4Cl -4.01 gm, NaHCO3 -0.42 gm, EDTA -0.19 gm, pH adjusted to 7.4 using NaOH, volume adjusted to 50 ml with water. Filter sterilize and store at 4 °C for six months.) and incubated at room temp for 3-5 mins. Vigorous shaking was performed at 1 minute intervals. Again centrifugation was performed for 10 minutes at 250 xg, at 4 ºC. Supernatant discarded and cells were resuspended in PBS, following centrifugation and supernatant discard. PBS washing step was repeated again. Finally, re-suspension of cell pellet in 3 ml RPMI complete media, plating in a 6-well culture plate and incubate at 37°C, 5% CO2 as needed.