key: cord-0304109-7yhal6n3 authors: Haralambieva, I. H.; Monroe, J. M.; Grill, D. E.; Ovsyannikova, I. G.; Poland, G. A.; Kennedy, R. B. title: Homologous and Variant-Specific Memory B-Cell and Antibody Responses after SARS-CoV-2 mRNA Vaccination date: 2021-07-16 journal: nan DOI: 10.1101/2021.07.12.21260386 sha: f1daadf075a0637d6e7c4d5f5100c436889e21ba doc_id: 304109 cord_uid: 7yhal6n3 Abstract. Importance. A better understanding of the immune memory and functional humoral immunity directed at the emerging Variants of Concern (VoC) strains after SARS-CoV-2 vaccination is essential for predicting the longevity of heterotypic protection. Objective. The aim of our study was to characterize functional humoral immunity (including memory B cell response) after COVID-19 mRNA vaccination and to determine/compare the reactivity of COVID-19 vaccine-induced memory B cells to the emerging SARS-CoV-2 Variants of Concern (VoC). Design, setting, participants and interventions. We designed an exploratory longitudinal observational (convenience sample-based) study at Mayo Clinic, Rochester, MN that enrolled and followed naive subjects and recovered COVID-19 subjects from Olmsted County, MN and surrounding areas after COVID-19 vaccination in January-June 2021. The study enrolled 17 relatively healthy subjects, 59% females and 94% White/Non-Hispanic or Latino with median age at enrollment 41 years. The subjects received either the BNT162b2 (Pfizer/BioNtech) or mRNA-1273 (Moderna) vaccine (n=3) and provided a blood sample at baseline, at;3 weeks after their first vaccine dose/before the second dose, and at;2 weeks after the receipt of their second vaccine dose. Main outcomes and measures. Spike-specific humoral and memory B cells responses were assessed over time after vaccination against the original Wuhan-Hu-1/vaccine and against emerging VoC strains/antigens. Results. We observed a robust neutralizing antibody response after COVID-19 mRNA vaccination, but a reduction in the functional antibody activity to several of the emerging SARS-CoV-2 VoC. Consistent with this, we also found differences in the number of isotype-switched/IgG+ MBCs responding to homologous and variant receptor-binding domain/RBDs after vaccination. We found a reduction of MBCs reactive to RBDs of Beta, Gamma and Delta SARS-CoV-2 VoC strains. Conclusion and relevance. In this exploratory study in subjects following receipt of COVID-19 mRNA vaccine, we found differences in antibody titers observed for VoCs after vaccination that are accompanied with, and can partially be explained by, decreased MBC reactivity against the VoCs. This can further attenuate the generated recall humoral immune response upon exposure to these variants. The longevity of protective humoral immunity after SARS-CoV-2 infection and vaccination is largely dependent on the generation and persistence of antigen-specific isotype-switched memory B cells (MBCs), as well as long-lived plasma cells residing in the bone marrow. IgG is considered the dominant isotype of MBCs after COVID-19 infection 1 , and the frequency of receptor binding domain/RBD-specific IgG+ MBCs is considered to represent a marker of immune memory following SARS-CoV-2 infection/vaccination. 2, 3 The reactivity of mRNA vaccine-induced MBCs to emerging clinically significant SARS-CoV-2 variant of concern (VoC) strains is unknown and was assessed in this study in concert with functional antibody measures. The study sample consisted of 17 healthy subjects (15 naïve and 2 recovered from COVID-19 infection) who provided a blood sample prior to vaccination (baseline) with BNT162b2 (Pfizer/BioNtech) or mRNA-1273 (Moderna) vaccine, then at ∼3 weeks after their first vaccine dose and before the second dose (first vaccine dose timepoint), and at ∼2 weeks after the receipt of their second vaccine dose (second vaccine dose timepoint). All study participants provided written informed consent, and all study procedures were approved by the Mayo Clinic Institutional Review Board. The neutralizing antibody response was assessed using a high-throughput, fluorescence/GFPbased pseudovirus/VSV microneutralization assay 4 , further developed in our laboratory to allow All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2021. ; https://doi.org/10.1101/2021.07.12.21260386 doi: medRxiv preprint rapid imaging/quantification of cell infection with the ImageXpress® platform/software (Molecular Devices). Wuhan-Hu-1 Spike-specific 50% end-point titer (Neutralizing Dose, ND 50 ) for each sample was calculated using Karber's formula. The capacity of sera (post-second vaccination) to impair virus attachment/block the interaction between hACE2 and different RBDs was determined using the SARS-CoV-2 Surrogate Virus Neutralization Test/sVNT Kit (RUO.3.0, GenScript® USA). The dilution at which a sample crosses 50% inhibition is designated as the 50% inhibition dose (ID 50 ). The frequencies of SARS-CoV-2-specific MBCs were quantified using the Mabtech ELIspot PLUS kit for human IgG (Mabtech Inc.; Cincinnati, OH) using recombinant SARS-CoV-2 antigens (Sino Biologicals, Beijing, China) coated at 0.2 µg/well. Responses are presented in spotforming units (SFUs) per 2×10 5 cells. The Wilcoxon signed-rank test was used to assess differences for all comparisons. The ID 50 value was calculated for each subject and RBD by fitting a quadratic regression of the percent inhibition on the log 3 inverse dilution. The quadratic equation utilizing the regression coefficients was used to calculate the inverse dilution from the regression equation at 50% inhibition. Our study cohort consisted of 59% females and 94% White/Non-Hispanic or Latino. Their median age at enrollment was 41 years (IQR 24, 54). The majority of subjects received the Pfizer All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2021. ; https://doi.org/10.1101/2021.07.12.21260386 doi: medRxiv preprint BNT162b2 and three subjects received the Moderna mRNA-1273 vaccine series, both according to the standard schedule. As expected, SARS-CoV-2 vaccination elicited robust anti-Spike neutralizing antibody response with significant inter-individual variations. Titers increased significantly after each vaccine dose We profiled the frequencies and specificities of isotype-switched IgG+ MBC after SARS-CoV-2 mRNA vaccination, as an indication of mature, highly specific and functional immune memory. As expected, the antigen-specific MBC changed significantly over time during mRNA vaccination ( Fig. 2A , p-value<0.006 for all comparisons between timepoints for S1 and RBD MBC). The prime vaccination elicited significant SARS-CoV-2-specific MBC response directed mainly to the S1 portion of the SARS-CoV-2 Spike of Wuhan-Hu-1and particularly to its RBD All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2021. ; https://doi.org/10.1101/2021.07.12.21260386 doi: medRxiv preprint ( Fig. 2A) . This response was further boosted after the second vaccine dose to a median S1 MBC response of 45 SFUs per 2×10 5 cells and a median RBD response of 27 SFUs per 2×10 5 cells ( Fig. 2A) . Interestingly, IgG+ MBC directed to the S2 potion of the Spike, as well as to the Nterminal domain/NTD portion of the S1 were readily detectable for some of the subjects, but at a much lower frequency (Fig. 2B) . We also noted moderate, but consistent correlations between the ND 50 titers and the frequency of S1-and RBD-specific MBCs after first vaccine dose (r=0.82 for both, p-value=0.00019 and p=0.0002, respectively). The correlations were less robust with marginal statistical significance after the second vaccine dose (r=0.49/r=0.43, p-value=0.046 and p=0.08, respectively); likely due to the small sample size and inter-individual variability in vaccine response. We then probed the reactivity/ability of the IgG+ RBD-specific MBCs to target RBDs of Fig. 3B ). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We and others have demonstrated the dynamics of SARS-CoV-2-specific antibody response after mRNA vaccination, and have demonstrated a reduced functional antibody response to emerging VoC. 2, 5, 6 These findings corroborate the current understanding that COVID-19 vaccine-induced neutralizing antibody response may be less effective against newly arising VoC. Similar to recent findings, we found a robust S1/RBD-dominated MBC response after vaccination which benefits from a booster dose in COVID-19 naïve individuals and is correlated to a certain degree with antibody response. 2 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2021. ; https://doi.org/10.1101/2021.07.12.21260386 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2021. ; https://doi.org/10.1101/2021.07.12.21260386 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2021. ; https://doi.org/10.1101/2021.07.12.21260386 doi: medRxiv preprint but the scales of the axis reflect the untransformed values for easier interpretation. Each box was plotted using the 25% to 75% interquartile range and the median is represented by the bold line in the box. The "whiskers" extend up to 1.5 times the interquartile range above or below the 75th or 25th percentiles, respectively. Black dots represent naïve (at baseline) subjects, while white dots represent COVID-19-recovered (at baseline) subjects. The numbers below the x-axis represent the median response for all subjects (25% and 75% interquartile range). Titers The frequencies of IgG+ MBCs were measured using the Mabtech ELIspot PLUS kit for human IgG using the indicated antigens (dark grey and white boxes), after in vitro PBMC three-day stimulation with human recombinant IL-2 and R848. Detected responses are presented in spot-All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2021. ; https://doi.org/10.1101/2021.07.12.21260386 doi: medRxiv preprint forming units (SFUs) per 2×10 5 cells, as subjects' antigen-specific medians (from three replicates with subtracted subject-specific no-antigen background measure). The values are plotted in log2 scale, but the scales of the axis reflect the untransformed values for easier interpretation. Each box was plotted using the 25% to 75% interquartile range and the median was represented by the bold line in the box. The "whiskers" extend up to 1.5 times the interquartile range above or below the 75th or 25th percentiles, respectively. Black circles represent naïve subjects (at baseline), while white circles represent COVID-19-recovered subjects (at baseline). The numbers below the x-axis represent the median response for all subjects (25% and 75% interquartile range). The MBC responses and changes over time of were measured using ELISPOT assay and assessed after the first mRNA vaccine dose (A) and after the second mRNA vaccine dose (B). Each box was plotted using the 25% to 75% interquartile range and the median was represented by the bold line in the box. The "whiskers" extend up to 1.5 times the interquartile range above or below the 75th or 25th percentiles respectively. Black circles represent naïve (at baseline) subjects, while white circles represent COVID-19-recovered (at baseline) subjects. The numbers below the x-axis represent the median All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2021. ; https://doi.org/10.1101/2021.07.12.21260386 Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection Distinct antibody and memory B cell responses in SARS-CoV-2 naïve and recovered individuals following mRNA vaccination SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2 mRNA vaccination compared to infection elicits an IgG-predominant response with greater SARS-CoV-2 specificity and similar decrease in variant spike recognition mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants SARS-CoV-2 specific memory B-cells from individuals with diverse disease severities recognize SARS-CoV-2 variants of concern The need for broadly protective COVID-19 vaccines: Beyond S-only approaches