Antibody response after a single dose of BBV152 vaccine negatively correlates with pre-existing antibodies and induces a significant but low levels of neutralizing antibodies to Omicron variant

24 Most adults in India have received at least one dose of COVID-19 vaccine and also been 25 infected naturally during the pandemic. As immunization of individuals continues under 26 this situation where the virus has attained endemicity, we assessed whether this hybrid 27 immunity is further boosted by a single dose of BBV152 , an inactivated SARS-CoV-2 28 vaccine, and, if these antibodies can neutralize SARS-CoV-2 delta and omicron variants. 29 We found that natural i nfection during the second wave in 2021 led to generation of 30 neutralizing antibodies against other lineages of SARS -CoV-2 including the omicron 31 variant, albeit at a significantly lower level for the latter. A single dose of BBV152 boosted 32 antibody titers against the delta and the omicron variants but the antibody levels remained 33 low for the omicron variant. Boosting of antibodies showed negative correlation with 34 baseline neutralizing antibody titers suggesting anergy of the immune system in 35 individuals with high levels of antibodies. 36 37

SARS-CoV-2; FRNT; ELISA; RBD; Delta; Omicron 38 39 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 3 3

40 Serosurveillance studies have showed that 69% of the Indian population had antibodies 41 for COVID-19 after the second wave 1 and a subsequent serosurvey from Delhi showed 42 seropositivity of over 90% 2 suggesting that India was/is a heterogenous mix of people 43 with immunity to COVID-19 due to vaccination or natural infection or both after the second 44 wave. As COVID-19 has attained a state of endemicity and majority of the population got 45 vaccinated after the second wave, there have been no studies to measure the impact of 46 pre-existing humoral immunity on subsequent vaccination. As antibody levels wane and 47 the risk of reinfection with the same variant and or a new variant warrants booster 48 vaccination, it is imperative to understand the efficiency of boosting vis-à-vis pre-existing 49 antibody levels. We enrolled subjects who got vaccinated with BBV152 as part of routine 50 vaccination program and assessed the effect of pre -existing antibody levels on boosting 51 after receiving a single dose of vaccine. We found that majority of the individuals had 52 neutralizing ant ibodies to delta variant and natural infection with delta variant led to 53 generation of neutralizing antibodies against other lineages of SARS-CoV-2 including the 54 omicron variant although the level of antibodies was significantly lower for this new variant 55 of concern as compared to delta and ancestral virus. 56 57

58 Human Ethics 59 The study was approved by the Institutional ethics committees for human research at 60 ESIC Hospital and Medical College and THSTI. Informed consent was obtained from all 61 the participants. 62 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 4 4 63 Human Samples 64 BBV152 cohort: An informed consent was obtained in person from each participant before 65 they were being recruited in this study at the ESIC Medical College & Hospital, Faridabad 66 between May – August 2021. All adults of > 18 years of age who came for the first dose 67 of vaccine were eligible to participate in this study including those who had recovered 68 from the COVID-19 in the recent past. In addition, participants were requested to provide 69 any history related to COVID -19 infection. Blood samples of the participants were 70 collected in anti-coagulant free vacutainers with the help of a professional phlebotomist 71 and stored at 2 - 8 ºC. First blood sample (5 ml) was collected on day 1 prior to the 72 administration of the first dose of the vaccine. Second blood sample (5 ml) was collected 73 during the follow-up visit before the administration of the second dose of the vaccine. 74 75 Cells 76 Vero E6 cells were obtained from European Collection of Authenticated Cell Cultures and 77 maintained in Minimal essential medium (MEM) (Gibco) supplemented with 10% heat -78 inactivated fetal bovine serum (FBS), 100U of penicillin and 100 µg of streptomycin and 79 L-glutamine (PSG) (Gibco), 1X non -essential amino acid mix (NEAA) (Gibco), 25 mM 80 HEPES in 5% CO2 incubator. Calu -3 cells (Human lung epitheli al cells derived from 81 adenocarcinoma: ATCC -HTB-55) were maintained in Dulbecco's minimal essential 82 medium (DMEM) (HiMedia) supplemented with 10% heat-inactivated fetal bovine serum 83 (FBS), 100U of penicillin and 100 µg of streptomycin and L-glutamine (PSG) (Gibco), 1X 84 non-essential amino acid (NEAA) (Gibco), 85 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 5 5 86 Viruses 87 SARS-CoV-2 to B.6 and delta lineage virus isolation has been described earlier3-5. SARS-88 CoV-2 Omicron isolate (sub-lineage BA.1) was obtained from Leo Poon 6. SARS-CoV-2 89 variants were propagated in Vero E6 cells or Calu-3 cells7 and virus passaging was limited 90 to four passages. All virus stocks used in this study was verified by who le genome 91 sequencing using total RNA sample of the culture on Nanopore sequencing platform as 92 described previously to confirm the variant8. 93 94 Quantitative Nucleoprotein ELISA 95 The bacterial expression plasmid pET -28a(+) containing the codon optimized 96 nucleocapsid (N) gene from severe acute respiratory syndrome -related coronavirus 2 97 (SARS-CoV-2), Wuhan -Hu-1 (GenBank: MN908947) having N -terminal hexa -histidine 98 affinity purificatio n tag was requested from BEI resources (NR -53507). His -tagged N 99 protein was purified by Ni -NTA chromatography as per previous report 9. 96 -well 100 MaxiSorp ELISA plates (Nunc) were coated with 1 µg/mL purified N protein diluted in 1X 101 PBS pH 7.4 and the plates were incubated for 1 h at room temperature (RT). Serum 102 samples were inactivated using Triton X -100 (Sigma) to a final concentration of 1 % at 103 RT for 1 h. The serum samples were two -fold serially diluted starting from 1:100 to 1: 104 6400 and 100 µl/well was added to the antigen -coated plate . After 30 min at room 105 temperature, the plate was washed using 1X PBST (phosphate -buffered saline with 0.1 106 % Tween 20). After washing, 50 µl/well HRP-conjugated anti-human IgG was added and 107 incubated at room temperature for 30 min. 100 µl/well of TMB subst rate was added for 108 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 6 6 10 min and the reaction was terminated using 1M H 2SO4 as stop solution. The intensity 109 of the color was quantified by measuring absorbance in a microplate reader at 450 nm 110 with 630 nm as reference wavelength. Antibody concentrations were calculated for each 111 sample dilution by interpolation of the OD values on the 4 -parameter logistic (4 -PL) 112 standard curve from in-house reference control (calibrated as a secondary standard using 113 WHO reference standard reagent (20/130)) and adjusted according to their corresponding 114 dilution factor using Gen5 software. The assay has a limit of quantitation of 3 binding 115 antibody units/mL (BAU/mL). This assay has been validated in -house and accredited 116 under ISO17025:2017 standard. 117 118 Quantitative RBD ELISA 119 Recombinant spike protein receptor binding domain (RBD) ELISA was performed as 120 described earlier4,10. Recombinant spike protein Receptor Binding Domain (RBD) antigen 121 of SARS-CoV-2 were coated onto 96-well polystyrene plate ( 0.1µg/well) and incubate 122 coated plate at 4oC for 18-22 hours. Antigen-coated plates were washed with wash buffer 123 and incubated by adding 200 µl of blocking buffer (5% non-fat dry milk powder in PBST). 124 Serum samples were inactivated by adding 10 µl of 10% Triton X -100 in 90 µl serum to 125 obtain a final concentration of 1% Triton X -100. Samples were gently mixed and 126 incubated at room temperature for 1 hour. After washing the plate with wash buffer, 100 127 µl of diluted serum (1:50 -1:6400 diluted in blocking buffer) was added to each well and 128 incubated at RT (23 2C) for 30 ± 10 min. Substrate was added and OD was recorded 129 and data was analyzed as described in the previous section. The assay has a limit of 130 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 7 7 quantitation of 12 binding antibody units/mL (BAU/mL). This assay has been validated in-131 house and accredited under ISO17025:2017 standard. 132 133 Virus microneutralization assay 134 Virus microneutralization assay by focus reduction neutralization titer assay using 135 indicating virus isolates was performed as described earlier with minor modifications in 136 omicron variant staining7. Briefly, serum samples were serially diluted from 1:20 to 1:640 137 and virus neutralization was tested in Vero E6 cells. Cells were incubated for 24 hours for 138 ancestral (B.6) and Delta (B.1.617.2) variants and for 32 hours for Omicron (B.1.1.529) 139 variant. After incubation, c ells were fixed with formaldehyde solution and then stained 140 with anti-spike RBD rabbit polyclonal antibody at 1:2000 dilution (Sino Biologicals , Cat. 141 No. 40592-T62) for 1 h, followed by HRP -conjugated anti -rabbit antibody at 1:4000 142 dilution (Invitrogen , C at. No. G-21234) for 1 h. For Omicron isolate, incubation was 143 extended to 32 h and a 1:1000 dilution of anti-nucleocapsid primary antibody (Genscript, 144 Cat. No. A02048-1) and 1:500 dilution of HRP-conjugated goat anti-mouse IgG secondary 145 antibody (Invitrogen, Cat. No. A16072) was used for staining. Cells were washed with 146 PBS and incubated with TrueBlue substrate ( KPL inc, USA, Cat. No. 5510-0030) for 10 147 minutes and washed with sterile MilliQ water. Microplaques developed after staining were 148 quantified by AID iSPOT reader (AID GmbH, Strassberg, Germany). The raw data 149 generated from the AID iSpot Analyser in a 96 -well format is pasted in a pre-defined 150 protocol template for calculation of FRNT50 by using log10 transformed dilution value and 151 neutralization percentages in an XY format . The Point -to-Point curve fit using a linear 152 equation to fit each pair of data points was used to calculate the FRNT50 value. 50% 153 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 8 8 neutralization values were calculated using SoftMax Pro GxP software v7.7.1 (Molecular 154 Devices). 155 156 Statistical analysis 157 Data was analysed and final graphs were prepared using GraphPad Prism (Version 9) 158 software. Statistical significance was estimated by two-tailed, non -parametric Mann-159 Whitney test or Wilcoxon signed rank test as indicated. 160 161

162 We collected baseline blood sample at the time of vaccination and a follow -up sample 163 four weeks later, before the second dose in the months of June -July 2021. A total of 94 164 (37 females) subjects were enrolled. Median age of the subjects was 31.5 yrs (range: 18-165 67 yrs). Sixty-seven of 94 (71.3%) subjects were positive in a quantitative RBD-ELISA in 166 the baseline sample. Four samples were indeterminate. We also measured the levels of 167 nucleocapsid (N) antibodies by quantitative ELISA. Fifty five of the 94 (58.5%) samples 168 were positive for N antibodies suggesting exposure in the second wave . After one dose 169 of vaccination, positivity in RBD -ELISA increased to Eighty four of 94 samples (89.4%) 170 and for N-ELISA 79 out of 94 samples (84%) were positive. The GMT of RBD antibodies 171 increased significantly from 109 (95% CI: 76, 156) to 206 (95% CI: 163, 260) in baseline 172 seropositive (Figure 1A) subjects, however, the increase in the GMT of N antibodies was 173 from 18 (95% CI: 12, 25) to 25 (95% CI: 20, 31) which was not significant (Figure 1B). 174 We next measured the virus neutralizing antibody titers against the SARS-CoV-2 175 B.6 lineage virus from 20204 (designated as WT) and B.1.617.2 (Delta variant) isolates in 176 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 9 9 both the baseline and post -single dose vaccination samples by focus reduction 177 neutralization titer (FRNT) assay. Overall GMT of neutralizing antibodies increased after 178 one dose of BBV152 vaccination for both WT virus and delta variant (Table 1). Both RBD 179 and N antibody levels correlated with neutralization titers against the delta variant after 180 the first dose (Supplementary Figure S1). The FRNT data were further analyzed based 181 on the seropositivity status at baseline in RBD-ELISA and we found that one dose of 182 BBV152 vaccine led to significant increase in GMT for neutralizing antibodies against the 183 WT virus (Table 1 and Figure 1C). However, the GMT for delta variant showed a marginal 184 and insignificant increase from 531.9 (95% CI: 371.6, 761.2) to 783.2 (95% CI: 624.8, 185 981.7) in the RBD-ELISA positive subgroup at baseline (Table 1 and Figure 1C). Twenty-186 two out of 23 baseline RBD-ELISA negative samples showed the presence of neutralizing 187 antibodies for WT and delta variant in FRNT assay , however, the levels of these 188 antibodies were about 7-8 fold lower than that in the ELISA-positive samples (Table 1) 189 suggesting that the antibody levels were below the level of detection of ELISA and these 190 are not seronegative subjects . In samples collected from baseline RBD-ELISA negative 191 participants, the GMT of antibodies against both the WT and B.1.617.2 lineage viruses 192 increased significantly after receiving the first dose of the vaccine (Table 1 and Figure 193 1D). Overall, we observed that samples with FRNT50 >1000 failed to show any induction 194 in antibodies after the first dose. We observed a clear negative correlation between the 195 baseline FRNT 50 titer for delta variant and the fold change in titer values after a single 196 dose of vaccination (Figure 1E). 197 While this study was in progress, omicron (B.1.1.529) emerged as a variant of 198 concern and antibodies from most vaccines showed reduced efficiency in neutralizing this 199 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 10 10 variant7,11,12. We randomly selected 55 paired samples (which had a FRNT50 value for the 200 delta variant) to test for their ability to neutralize the omicron variant. Only twenty out of 201 55 baseline samples had detectable levels of neutralizing antibodies against omicron. By 202 assigning a FRNT50 value of 10 for the samples which had no detectable levels of 203 antibodies in the starting dilution (1:20) of the assay, we obtained a GMT of 22 (95% CI: 204 16, 31) for these 55 samples. This value was 18-fold lower than the GMT of delta variant 205 which was 404 (95% CI: 248, 658). After a single dose of BBV152, the number of samples 206 positive for neutralizing antibodies against omicron increased to thirty six out of 55 207 subjects with a significant increase in GMT (p = 0.0011) to 52 (95% CI: 36, 75), however, 208 this was still 15-fold lower than the GMT for delta variant which was 784 (95% CI: 575, 209 1068) (Figure 1F and 1G ). Whether the modest but significant boosting observed for 210 omicron by BBV152 vaccination in seropositive individuals is protective or not warrants 211 further studies from the ongoing booster vaccination campaign across the country. 212 Vaccination in early convalescent individuals led to poorer boosting as observed 213 in other studies13-15. In naturally infected individuals, boosting after six months is predicted 214 to increase the vaccine effectiveness against variants of concern 16. We were not able to 215 ascertain the exact date of past infection in most of these individuals which is not unusual 216 as most COVID -19 infections are asymptomatic or mild and are not diagnosed . 217 Nucleocapsid antibodies are known to decay with a half-life of 68 days17 and 59% of our 218 study participants were positive for N antibodies suggesting that the y could have been 219 infected with SARS-CoV-2 within the past 6-8 months. Therefore, we cannot rule out the 220 possibility that some of the subjects with high antibody titers were in their early 221 convalescence and therefore, the boosting effect may not have been significant due to 222 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 11 11 anergy of the immune system as has been observed in other studies18. Antibody boosting 223 correlated negatively with the levels of pre -existing antibodies which indicates that a 224 strategy is needed to prioritize high -risk individuals based on their antibody levels for 225 booster vaccination. Nevertheless, the level of neutralizing antibodies for omicron variant 226 after boosting in both seropositive and seronegative individuals remained much lower 227 compared to the ancestral virus or the delta variant which is consistent with recent reports 228 for CoronaVac, an inactivated vaccine 19. However, the participants of our study were all 229 non-vaccinated individuals and we cannot rule out the possibility that vaccination of these 230 individuals after further reduction in antibody titers would have induced a better antibody 231 response. It has been estimated that neutralizing antibodies are a good correlate of 232 protection and contribute to about 60% of the protective efficacy of a vaccine which 233 indicates that the cellular responses play a critical synerg istic role along with antibodies 234 in mediating protection from SARS -CoV-2 VoCs4,5,16. Some of the recent studies have 235 also shown that heterologous boosting mounts a robust immune response to VoCs20. As 236 many new vaccines are likely to be licensed in the coming months in India, more studies 237 are required to measure the efficacy of homologous vs heterologous boosting against 238 new variants of concern. 239 240 Data availability 241 All the data are presented in this manuscript. Detailed Methods are available as online 242 methods. 243 244

245 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 12 12 We thank all the members of the bioassay lab for technical support. We thank Neha Garg 246 and Shamsher Singh for data management. We thank all the participants who consented 247 to enrol into the study. 248 249 AUTHOR CONTRIBUTIONS 250 JS, HS, BS, AA, and PS performed experiments and analyzed the data. SD and AKP 251 coordinated the study at clinical site , generated all the clinical site data and contributed 252 reagents. RP sequenced the virus isolates and analyzed the data. RL provided critical 253 inputs in experimental design, data analysis and writing the manuscript . GRM and AKP 254 conceived the study, designed the experiments and analyzed the data . GRM wrote the 255 manuscript. All authors have reviewed and approved the final version of the manuscript. 256 257 FUNDING INFORMATION 258 This work was supported by the Department of Biotechnology (DBT) through IndCEPI 259 Mission (BT/MB/CEPI/2016) , Translational Research Program 260 (BT/PR30159/MED/15/188/2018) and Global Immunology and Immune Sequencing for 261 Epidemic Response (INV-030592). The fund ers had no role in study design, data 262 collection and interpretation or the decision to submit the work for publication. 263 264 CONFLICT OF INTEREST STATEMENT 265 The authors have declared that no conflict of interest exists. 266 267 268 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 13 13 Table 1: Virus neutralization titers post single-dose of BBV152 vaccination based 269 on RBD-ELISA positivity 270 271 Comparison of titers at baseline and 3-4 weeks after 1st dose was performed using Wilcoxon 272 signed rank test. Comparison of titers between the groups (based on RBD ELISA results) was 273 done using Wilcoxon rank sum test. 274 275 Geometric Mean FRNT50 (95% CI) Sampling time Baseline WT (A) One dose WT (B) P value comparing the titers within each group; before and after 1 dose of vaccine (A vs B) (Wilcoxon signed rank test) Baseline B.1.617.2 (C) One dose B.1.617.2 (D) P value comparing the titers within each group; before and after 1 dose of vaccine (C vs D) (Wilcoxon signed rank test) All (n=94) 108.6 (81.3 – 145.1) 256.4 (196.1 – 335.3) <0.0001 291.4 (209.6 – 405.0) 508.1 (398.9 –647.2) 0.0126 Groups based on RBD ELISA

1. RBD- positive at baseline (n=67) 182.8 (133.6 – 250.0) 422.2 (340.5 – 523.5) 0.0001 531.9 (371.6 – 761.2) 783.2 (624.8 – 981.7) 0.448 2. RBD- Negative at baseline (n=23) 26.50 (19.8 – 35.5) 57.32 (33.0 – 99.6) 0.0033 63.94 (46.4 – 88.1) 146.6 (95.7 – 224.7) 0.0002 P value comparing the titers between 2 groups based on RBD ELISA (using Wilcoxon rank sum test) <0.0001 <0.0001 <0.0001 <0.0001 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 14 14

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Infection and Vaccine -Induced Neutralizing -Antibody 310 Responses to the SARS -CoV-2 B.1.617 Variants. N Engl J Med 385, 664 -666 311 (2021). 312 14. Stamatatos, L. , et al. mRNA vaccination boosts cross -variant neutralizing 313 antibodies elicited by SARS-CoV-2 infection. Science (2021). 314 15. Leier, H.C., et al. Previously infected vaccinees broadly neutralize SARS -CoV-2 315 variants. medRxiv, 2021.2004.2025.21256049 (2021). 316 16. Cromer, D., et al. Neutralising antibody titres as predictors of protection against 317 SARS-CoV-2 variants and the impact of boosting: a meta-analysis. Lancet Microbe 318 3, e52-e61 (2022). 319 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 16 16 17. Dan, J.M. , et al. Immunological memory to SARS -CoV-2 assessed for up to 8 320 months after infection. Science 371(2021). 321 18. Kannian, P., Mahanathi, P., Ashwini, V. & Kumarasamy, N. Booster and anergic 322 effects of the Covishield vaccine among healthcare workers in South India. 323 medRxiv, 2021.2008.2004.21261601 (2021). 324 19. Cheng, S.M.S., et al. Neutralizing antibodies against the SARS -CoV-2 Omicron 325 variant following homologous and heterologous CoronaVac or BNT162b2 326 vaccination. Nature Medicine (2022). 327 20. Angkasekwinai, N. , et al. The immunogenicity against variants of concern and 328 reactogenicity of four COVID -19 booster vaccinations following CoronaVac or 329 ChAdOx1 nCoV-19 primary series. medRxiv, 2021.2011.2029.21266947 (2022). 330 331 FIGURE LEGEND 332 Figure 1: Antibody boosting after a single dose of BBV152 vaccination. Serum levels 333 of antibodies against (A) SARS-CoV-2 RBD and (B) N protein was estimated by 334 quantitative ELISA after a single dose of BBV152 vaccine in baseline and post -335 vaccination samples collected 3-4 weeks later. Data was expressed as Binding Antibody 336 Units (BAU)/ml (international units). (C) FRNT50 titers in RBD ELISA-positive samples for 337 the ancestral B.6 lineage (WT) and the delta variant (B1.6 17.2) in baseline and post -338 single dose vaccination samples. (D) FRNT50 titers in RBD ELISA-negative samples for 339 the ancestral B.6 lineage (WT) and the delta variant (B1.617.2) in baseline and post -340 single dose vaccination samples. (E) Spearman correlation (r) between baseline FRNT50 341 titers and fold-change observed after single-dose of BBV152. (F and G) FRNT50 titers for 342 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint 17 17 the delta variant (B1.617.2) and omicron (1.1.529) variant in baseline and post -single 343 dose vaccination samples. LLOQ: Lower li mit of quantitation of the assay. Two -tailed t 344 test P values are indicated by - * p<0.05; ** p<0.01; *** p < 0.001; **** p < 0.00 01; ns - 345 not significant. 346 347 . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint . CC-BY-NC 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22270612doi: medRxiv preprint