key: cord-0312918-nvqv99ak authors: Garcia-Valtanen, P.; Hope, C. M.; Masavuli, M. G.; Yeow, A. E. L.; Balachandran, H.; Mekonnen, Z. A.; Al-Delfi, Z.; Abayasingam, A.; Agapiou, D.; Ospina Stella, A.; Aggarwal, A.; Gummow, J.; Ferguson, C.; O'Connor, S.; McCartney, E. M.; Lynn, D. J.; Maddern, G.; Gowans, E. J.; Reddi, B. A.; Shaw, D.; Kok-Lim, C.; Turville, S. G.; Beard, M. R.; Weiskopf, D.; Sette, A.; Bull, R. A.; Barry, S. C.; Grubor-Bauk, B. title: COVID-19 convalescents exhibit deficient humoral and T cell responses to variant of concern Spike antigens at 12 month post-infection date: 2021-11-11 journal: nan DOI: 10.1101/2021.11.08.21266035 sha: b8c05ee8d8e7a21f19e0c0ee2e2af2ab907713d0 doc_id: 312918 cord_uid: nvqv99ak Background The duration and magnitude of SARS-CoV-2 immunity after infection, especially with regard to the emergence of new variants of concern (VoC), remains unclear. Here, immune memory to primary infection and immunity to VoC was assessed in mild-COVID-19 convalescents one year after infection and in the absence of viral re-exposure or COVID-19 vaccination. Methods Serum and PBMC were collected from mild-COVID-19 convalescents at ~6 and 12 months after a COVID-19 positive PCR (n=43) and from healthy SARS-CoV-2-seronegative controls (n=15-40). Serum titers of RBD and Spike-specific Ig were quantified by ELISA. Virus neutralisation was assessed against homologous, pseudotyped virus and homologous and VoC live viruses. Frequencies of Spike and RBD-specific memory B cells were quantified by flow cytometry. Magnitude of memory T cell responses was quantified and phenotyped by activation-induced marker assay, while T cell functionality was assessed by intracellular cytokine staining using peptides specific to homologous Spike virus antigen and four VoC Spike antigens. Findings At 12 months after mild-COVID-19, >90% of convalescents remained seropositive for RBD-IgG and 88.9% had circulating RBD-specific memory B cells. Despite this, only 51.2% convalescents had serum neutralising activity against homologous live-SARS-CoV-2 virus, which decreased to 44.2% when tested against live B.1.1.7, 4.6% against B.1.351, 11.6% against P.1 and 16.2%, against B.1.617.2 VoC. Spike and non-Spike-specific T cells were detected in >50% of convalescents with frequency values higher for Spike antigen (95% CI, 0.29-0.68% in CD4+ and 0.11-0.35% in CD8+ T cells), compared to non-Spike antigens. Despite the high prevalence and maintenance of Spike-specific T cells in Spike 'high-responder' convalescents at 12 months, T cell functionality, measured by cytokine expression after stimulation with Spike epitopes corresponding to VoC was severely affected. Interpretations SARS-CoV-2 immunity is retained in a significant proportion of mild COVID-19 convalescents 12 months post-infection in the absence of re-exposure to the virus. Despite this, changes in the amino acid sequence of the Spike antigen that are present in current VoC result in virus evasion of neutralising antibodies, as well as evasion of functional T cell responses. 37 Background 38 The duration and magnitude of SARS-CoV-2 immunity after infection, especially with regard 39 to the emergence of new variants of concern (VoC), remains unclear. Here, immune memory 40 to primary infection and immunity to VoC was assessed in mild-COVID-19 convalescents one 41 year after infection and in the absence of viral re-exposure or COVID-19 vaccination. At 12 months after mild-COVID-19, >90% of convalescents remained seropositive for RBD-IgG 54 and 88.9% had circulating RBD-specific memory B cells. Despite this, only 51.2% convalescents 55 had serum neutralising activity against homologous live-SARS-CoV-2 virus, which decreased 56 to 44.2% when tested against live B.1.1.7, 4.6% against B.1.351, 11.6% against P.1 and 16.2%, 57 against B.1.617.2 VoC. Spike and non-Spike-specific T cells were detected in >50% of 58 convalescents with frequency values higher for Spike antigen (95% CI, 0.29-0.68% in CD4 + and 59 0.11-0.35% in CD8 + T cells), compared to non-Spike antigens. Despite the high prevalence and 60 maintenance of Spike-specific T cells in Spike 'high-responder' convalescents at 12 months, T 61 cell functionality, measured by cytokine expression after stimulation with Spike epitopes 62 corresponding to VoC was severely affected. 63 Interpretations 64 SARS-CoV-2 immunity is retained in a significant proportion of mild COVID-19 convalescents Evidence before this study 81 We regularly searched on PubMed and Google Scholar in June-October 2021 using individual 82 or combinations of the terms "long-term immunity", "SARS-CoV-2", "antigenic breadth", 83 "variant of concern" and "COVID-19". We found studies that had assessed immune correlates 84 at multipe time points after COVID-19 disease onset in convalescents, but not the antigenic 85 breadth of T cells and antibodies and not in relation to VoC. Other immune studies in virus 86 naive vaccinees, or vaccinated convalescents evaluated VoC-specific immunity, but not in 87 convalescents that have not been vaccinated. In summary, we could not find long-term 88 studies providing and in-depth evaluation of functionality of humoral and cell-mediated 89 immunity, combined with addressing the adaptability of these immune players to VoC. 90 Added value of this study 91 The window of opportunity to conduct studies in COVID-19 convalescents (i.e. natural 92 immunity to SARS-CoV-2) is closing due to mass vaccination programs. Here, in a cohort of 93 unvaccinated mild-COVID-19 convalescents, we conducted a comprehensive, longitudinal, 94 long-term immune study, which included functional assays to assess immune fitness against 95 antigenically different VoC. Importantly, the cohort resided in a SARS-CoV-2-free community 96 for the duration of the study with no subsequent re-exposure or infection. Our findings reveal 97 a deeply weakened humoral response and functional vulnerability of T cell responses to VoC 98 Spike antigens. 99 Implications of all the available evidence 100 This study provides a valuable snapshot of the quality of SARS-CoV-2 natural immunity and its 101 durability in the context of a pandemic in which new variants continuously emerge and 102 challenge pre-existing immune responses in convalescents and vacinees. Our results serve as 103 a warning that delays in vaccination programs could lead to an increase in re-infection rates 104 of COVID-19 convalescents, caused by virus variants that escape humoral and cell-mediated 105 immune responses. Furthermore, they reinforce the potential benefit of booster vaccination 106 that is tuned to the active variants. (VoC). 125 After primary infection and in parallel with the antibody response, symptomatic COVID-126 19 convalescents generate a robust CD4 + and CD8 + memory T cell response which targets a 127 wider range of antigens and epitopes than that covered by antibodies. 20-24 Importantly, the 128 breath of SARS-CoV-2-specific T cell epitopes appears to be less sensitive to mutations present 129 in VoC. 25, 26 It is unclear to what extent T cells can protect from re-infection and progression 130 to severe COVID-19. However, it is likely that T cell responses in convalescents, which target 131 most SARS-CoV-2 antigens, 20 could afford some of level of protection for many months, even 132 years. In fact, SARS-CoV-specific T cells can be detected in convalescents for almost two 133 decades. 27 134 While current vaccines are highly effective in preventing severe disease and death, and 135 booster vaccinations may temporarily circumvent dwindling efficacy over time, 28 next 136 generation vaccines, that can prevent virus transmission, are likely needed to end the 137 pandemic. 29, 30 Long-term studies of the evolution of immune correlates in COVID-19 138 convalescents, where the immune system has encountered an active live virus infection in 139 the presence of all its antigens, are necessary to elucidate the fine specificities and immune 140 functionality of antibody and T cell responses. In particular, the adaptability of pre-existing 141 immunity to mutated Spike antigens, present in VoC, is the key piece of information that is 142 still unanswered. 143 Compared to the majority of the world, South Australia is in a unique position to 144 undertake studies on mid-to long-term immunity of COVID-19 due to 1) early and strict 145 border control measures with other countries, and other states within Australia, which were 146 enforced by health authorities in 2020-21, thus eliminating local transmission of the virus in 147 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 were coated overnight at 4C with 5g/mL of recombinant RBD protein and blocked with 5% 204 w/v skim milk in 0.05% Tween-20/PBS (PBST) at room temperature. Heat inactivated patient 205 sera were serially diluted in blocking buffer, added and incubated for 2h at room temperature, 206 followed by four washes in 0.05% PBST. Secondary antibodies were diluted in 5% skim milk in is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 included the key variants of concern; Alpha (B. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint was performed using FCS Express TM (DeNovo Software, Pasadena, CA, USA). All percentages 303 of activated cells were calculated subtracting unspecfic DMSO background for each cell 304 phenotype and individual patient. Variant of concern Spike-specific peptide pools. Table S1 . For the assays, lyophilised peptides were resuspended in 315 sterile miliQ water as per the manufacturer's instructions at 30 nM (50 µg/mL), aliquoted and 316 stored at -80 0 C until used. Spike high responder convalescents. 319 According to AIM assay results, convalescents with Spike-specific CD4 + and CD8 + T cell 320 frequencies above the mean plus 3*standard deviations in the healthy control group were 321 identified. Double responders (meeting criterion for both CD4 + and CD8 + T cells) and high CD4 + 322 with available PBMC samples were selected (n=15) for further analysis. Following methods 323 similar to other published prior to this study, 46 PBMCs were thawed and prepared for cell is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint surface IgG in 88.9% of individuals in the COVID-19 convalescent cohort (95% CI, 153-336 378 cells/ 10 6 B cells) compared to healthy controls (95% CI, 0.0-27.9 cells/10 6 B cells) (figure 1C), 379 indicative of the existence of long-term SARS-CoV-2-specific humoral immunity 12 months 380 post-infection. Spike-specific IgG + B cells were also elevated in COVID-19 convalescents, but 381 healthy control background frequencies were also higher (figure S2), likely due to cross-382 reactivity. RBD-and Spike-specific non-IgG + B cell frequencies were present a low rates (figure 383 S2). 384 Obtaining long-term serum neutralisation data from communities free of circulating SARS- 385 CoV-2, such as in this study, is difficult in other cohorts in the context of this pandemic. In our 386 cohort, sera in 64% of convalescents at 12 months yielded ID50 neutralisation titers 387 significantly above healthy background levels against the pseudotyped virus bearing a 388 Wuhan-like Spike protein, which is the same as the prevalent virus present in the community 389 when study participants were infected (figure 1D). 390 Since early 2020, when the study participants were recruited, four VoC, namely, Alpha is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint AIM assays revealed that the frequency of circulating Spike-specific CD4 + T cells did not is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint proportion of CD8 + CD69 + CD137 + cells at 6 months (>65%) and at 12 months (>60%), 460 irrespective of antigen specificity (figure 4B). 461 The presence of significant frequencies of SARS-CoV-2 antigen-specific CD4 + TEM and CD8 + 462 TEMRA at 12 months, has important implications, particularly in the absence of virus re-463 exposure, given that in principle these cells have a limited life-span. Importantly, a significant 464 decrease is these populations from 6 to 12 months suggests that in the absence of is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint p<0.001) while IFNγ, TNFα or IL-2 expressing cells were not significantly different ( figure 5D ). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint S2). To our knowledge, this is the first time that a loss of Spike-specific T cell functionality 534 against VoC, despite durable (12 months) maintainance in overall frequency, has been 535 reported in COVID-19 convalescents. These data suggest that suboptimal Spike-specific T is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 The presence of specific antibodies alone does not necessarily predict protection against 573 disease. Mathematical modelling of clinical data indicates that a minimum specific Ig titer is 574 needed for protection against COVID-19 disease. 57 This is also supported by clear evidence 575 showing that serum virus neutralisation activity, exerted by a portion, but not all antibodies, 576 is the best predictor of protection. 48, 55, 57, 58 Both live-virus and pseudovirus serum 577 neutralisation assays indicated that sera from 51% and 65%, respectively, of COVID19 578 convalescents can efficiently neutralise SARS-CoV-2 bearing the Spike protein homologous to 579 one which caused the original infection, 12 months post-infection. At 6-8 months post-COVID-580 19, the percentage of convalescents who retain serum neutralizing activity, estimated with 581 methods similar or identical to ours, varies considerably, from ~60% to >90%. 8, 22, 36, 43 It is 582 important to note that these studies included more heterogenous cohorts, which included 14, 16, 18, 57, [60] [61] [62] Our measurements at 12 598 months post-COVID-19 and in the absence of re-infection, reveal that although circulating 599 SARS-CoV-2 antibody responses persist up to 12 months, the antigenic drift of SARS-CoV-2 in 600 VoC outperforms humoral immune responses in mild-COVID-19 convalescents. 601 As such, our findings stress the necessity to reinforce the immune system to overcome these 602 insufficiencies in COVID-19 convalescents via vaccination. 603 Importantly, we report that SARS-CoV-2-specific circulating CD4 + and CD8 + T cell frequencies is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 in maintaining immunity as far as 12 months after infection. Interestingly, the maintenance 612 and large proportion of CD8 + TEMRA bears resemblance to the kinetics of equivalent cells in 613 long-term chronic infections by human CMV. 63 A significant decay of Spike-specific CD4 + TCM 614 cells was observed, however this was not reflected in their CD8 + T cell counterparts, perhaps 615 indicating that in the long term, cytotoxic immune function could be sustained more 616 efficiently thanks to cell self-renewal and proliferation. 51 Importantly, in our cohort of 'high is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint non-mutated epitopes. While this approach bears more resemblance to what might happen 651 during a SARS-CoV-2 infection, it potentially masks subtle differences in T cell reactivity and 652 the identification of individuals whose T cell responses are more severely affected by 653 antigenic changes in the Spike antigen of VoC. 25 Also, while the AIM assay is a powerful 654 method to detect and quatify T cell antigen specificity it does not measure function directly, is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Circles represent AUC individual patient values (n=43 at 6 months, orange, and 12 months, yellow, 706 n=15 for healthy controls, blue), with mean value denoted by a horizontal black line. Seronegative 707 samples were assigned a value of 0.001 data visualisation purposes. 708 (C) SARS-CoV-2 RBD-specific (n=28) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (A) Memory immunophenotype based on expression of surface CCR7 and CD45RA of SARS-CoV-2 738 Spike-, non-Spike-and cumulative (Spike + non-Spike)-specific CD4 + T cells detected in Figure 2A . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (A) Memory immunophenotype based on expression of surface CCR7 and CD45RA of SARS-CoV-2 752 Spike-, whole proteome pools A-and B-specific CD8 + T cells detected in Figure 2B . Immune phenotypes 753 were defined as in Figure 3 . Frequencies are indicated as percentage of total SARS-CoV-2 antigen-754 specific CD8 + T cells within the total pool of immune cells with same phenotype in the patient's PBMCs. (B) Doughnut charts indicating the proportion (%) of each immune phenotype in (A) of the total of 756 SARS-CoV-2-specific CD8 + T cells for each antigen. In (A) circles represent patient individual values. 757 Averages are denoted by a horizontal line and statistically significant differences between time points 758 indicated by asterisks. *** and **** denote P values < 0.05 and 0.01, respectively. ns = not significant. 759 760 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint Figure S3 . AIM assay gating strategy. 816 Live, CD14 -CD19 -CD3 + singlets were gated and further classified as either CD4 + or CD8 + T cells 817 using gating strategy on two top row panels. Antigen specific CD4 + and CD8 + T cells were 818 identified based on double expression of surface CD137 and OX40 or CD137 and CD69, 819 respectively (lower panels). Finally, memory phenotype of antigen-specific cells was further 820 determined according to expression of surface CCR7 and CD45RO. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint Figure S4 . T lymphocytes and CMV-specific CD4+ and CD8+ cells in study participants. (A) Left to right, percentage of CD3 + , CD3 + CD4 + and CD3 + CD8 + T cells in COVID-19 convalescents at 6 824 and 12 months after disease onset (n=43) and healthy controls (n=15) analysed in the AIM assay. (B) 825 CD4 + and CD8 + CMV-specific T cells in healthy controls and COVID-19 convalescents at 12 months after 826 SARS-CoV-2 infection. **** and ns denote statistically significantly differences at p <0.0001 and not 827 significant, respectively. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint Figure S5 . Flow cytometry data gating strategy for whole Spike (Wuhan)-specific intracellular 835 cytokine (ICS) assay and activated T Follicular Helpers. 836 Representative strategy to define CD4, CD8 and T Follicular helpers (CD4+ CXCR5+) for the 837 intracellular cytokine assay and activation induced CD154 (CD40L) expression. CD154 was measured 838 for activation of T Follicular helpers. 839 840 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.08.21266035 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 11, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 A pneumonia outbreak associated with a new coronavirus of 876 probable bat origin The impact of lockdown timing on COVID-19 878 transmission across US counties COVID-19 dynamics after a national 880 immunization program in Israel Impact of 882 scaling up SARS-CoV-2 vaccination on COVID-19 hospitalizations in Spain Impact of vaccination on new SARS-CoV-2 885 infections in the United Kingdom Vaccination reduces need for emergency care in 887 breakthrough COVID-19 infections: A multicenter cohort study The Combined Effect of Vaccination and Nonpharmaceutical Public Health 890 Interventions-Ending the COVID-19 Pandemic Neutralizing antibody titres in SARS-CoV-2 infections. 892 Twelve-month specific IgG response to SARS-CoV-2 receptor-binding 894 domain among COVID-19 convalescent plasma donors in Wuhan Dynamics of antibody response to BNT162b2 vaccine after 897 six months: a longitudinal prospective study. 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