key: cord-0289740-z7kuekp0 authors: Meyer-Arndt, L.; Schwarz, T.; Loyal, L.; Henze, L.; Kruse, B.; Dingeldey, M.; Guercan, K.; Uyar-Aydin, Z.; Mueller, M. A.; Drosten, C.; Paul, F.; Sander, L. E.; Demuth, I.; Lauster, R.; Giesecke-Thiel, C.; Braun, J.; Corman, V. M.; Thiel, A. title: Serum but not mucosal antibody responses are predicted by pre-existing SARS-CoV-2 spike cross-reactive CD4+ T cells following BNT162b2 vaccination in the elderly date: 2021-10-07 journal: nan DOI: 10.1101/2021.10.05.21264545 sha: e7a11ebe36fc0d7cb25945abaac9cb1d9a48eae5 doc_id: 289740 cord_uid: z7kuekp0 Advanced age is a main risk factor for severe COVID-19 and thus elderly were often prioritized for vaccination. However, low vaccination efficacy and accelerated waning immunity have been reported in this age group. To elucidate age-related differences in immunogenicity, we analysed cellular, serological and salivary SARS-CoV-2 spike glycoprotein-specific immune responses to BNT162b2 COVID-19 vaccine in old (69-92 years) and middle-aged (24-57 years) vaccinees compared to natural infection (COVID-19 convalescents of 21-55 years). Serological humoral responses to vaccination exceeded those of convalescents but salivary anti-spike subunit 1 (S1) IgA and neutralizing capacity were less durable in vaccinees. In old vaccinees, we observed that pre-existing spike-specific CD4+ T cells correlated with efficient induction of serological anti-S1 IgG and neutralizing capacity after vaccination. Our results highlight the role of pre-existing cross-reactive CD4+ T cells with respect to SARS-CoV-2 vaccination particularly in old individuals, in whom their presence predicted efficient COVID-19-vaccine-induced humoral immune responses. BNT162b2 mRNA vaccine (Tozinameran™, Comirnaty™ 8,9 ) in old and comorbid nursing home 79 residents (n=18; mean age 83±6) and their middle-aged caregivers (n=14; mean age 47±10) 80 at baseline (prior to first vaccination), at day 28 (d28, 7 days after booster vaccination) and at 81 day 49 (d49, 28 days after booster vaccination). For comparison with naturally acquired 82 immunity, we additionally analysed COVID-19 convalescents (age-matched to the middle-83 aged cohort; mean age 36±11) after mild natural SARS-CoV-2 infection at ~d28 (n=10), ~d49 84 (n=16) or ~d94 (n=11) after symptom onset (Table 1) . We first analysed anti-SARS-CoV-2 spike glycoprotein subunit 1 (S1) immunoglobulin G (IgG) 87 and anti-S1 immunoglobulin A (IgA) antibody levels in serum ( Fig. 1a and b) . At d28, eight old 88 donors (44%) failed to seroconvert for anti-S1 IgA and five of them (28%) also for anti-S1 IgG. In contrast, all middle-aged donors exhibited high anti-S1 IgG and anti-S1 IgA levels at d28 90 indicating a fast and robust systemic humoral response to vaccination. Anti-S1 IgG and anti-91 S1 IgA levels were more heterogeneous and significantly lower in the old compared to the 92 middle-aged cohort at d28 ( Fig. 1a and b) . At d49, anti-S1 IgG levels were still significantly 93 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.05.21264545 doi: medRxiv preprint 4 lower in the old vaccinees (Fig. 1a) , but no significant differences were present in anti-S1 IgA at 94 d49 comparing old and middle-aged vaccinees (Fig. 1b) . Assessing S1-specific functional 95 neutralization capacity in serum using a surrogate viral neutralization assay (sVNT), all middle-96 aged vaccinees demonstrated a prompt and uniform increase of virus neutralization at 97 d28, which remained stable at d49 (Fig. 1c) . In the old vaccinees, this response was 98 significantly delayed and remained lower than in middle-aged vaccinees at d49 with one non-99 responder (inhibition < 30%). Compared to COVID-19 convalescents, anti-S1 IgG levels and 100 median S1 neutralizing capacity in serum were more homogenously distributed and, moreover, To evaluate salivary humoral responses as potential correlate of first-line adaptive protection 105 against SARS-CoV-2 infection, we furthermore analysed the presence of anti-S1 secretory IgA 106 (sIgA) in the saliva. Following vaccination, we observed increased levels of anti-S1 sIgA 107 in both the middle-aged and old cohort (Fig. 1d) . At d28, anti-S1 sIgA levels were significantly 108 higher in the middle-aged donors as compared to the old individuals. All 14 middle-aged 109 donors showed anti-S1 sIgA levels above their age group's maximum pre-vaccination level 110 (0.45 optical density (OD) ratio) while only six of the old vaccinees (60%) reached above their 111 age group's maximum pre-vaccination level (0.31 OD ratio). At d49, we observed anti-S1 112 sIgA levels comparable to pre-vaccination in all vaccinees (Fig. 1d ). By contrast, COVID-19 113 convalescents exhibited significantly higher anti-S1 sIgA levels at ~d49 and even ~d94 114 compared to age-matched middle-aged vaccinees at d49 (Fig. 1d ). S1 neutralizing activity in 115 saliva developed more heterogeneously after vaccination (Fig. 1e ). An increase in S1 116 neutralizing activity was detected in most vaccinees at d28 but dropped back to pre-vaccination 117 levels at d49 mirroring anti-S1 sIgA kinetics ( Fig. 1d and e) . In some responsive old donors, a 118 delayed anti-S1 sIgA response was reflected by a similarly delayed increase of salivary S1 119 neutralizing activity, which caused some old donors to show increased anti-S1 sIgA and S1 120 neutralizing activity at d49 (Fig. 1d und e) . COVID-19 convalescents demonstrated higher S1 121 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in In contrast to anti-S1 sIgA, S1 salivary neutralizing capacity did not remain significantly 123 increased in convalescents at ~d94 compared to d49 in age-matched middle-aged vaccinees 124 indicating that anti-S1 sIgA does not correspond to S1 neutralizing activity following infection 125 at this time point (Fig. 1d und e) . Finally, we analysed frequencies of antigen-reactive CD40L + 4-1BB + CD4 + T cells after in vitro 128 stimulation with the N-terminal part (S1, covered by peptide mix S-I) and the C-terminal part 129 (S2, peptide mix S-II) of the spike glycoprotein ( Fig. 2a and b, Suppl. Fig. 2) . We have recently 130 demonstrated that, in contrast to the N-terminal part, the C-terminal part of the spike 131 glycoprotein contains highly conserved domains and triggers CD4 + T cell cross-reactivity to 132 SARS-CoV-2 10 . One week after the second vaccination (d28), all middle-aged and 89% (S-I; 133 n=16/18) and 94% (S-II; n=17/18) respectively of the old donors acquired T cell reactivity to S-134 I and S-II. However, S-I-and S-II-reactive T cell frequencies increased more quickly 135 and peaked at a higher level in the middle-aged than in the old cohort. At d49, S-I-and S-II-136 reactive CD4 + T cell frequencies of the middle-aged vaccinees dropped slightly so that both 137 age groups reached comparable levels. In convalescents, we observed a significantly lower T 138 cell reactivity to S-I and S-II at ~d49 after symptom onset compared to both vaccinated cohorts 139 ( Fig. 2a and b ). Pre-existing T cell reactivity to S-II at baseline was significantly lower in old 140 individuals than in the middle-aged cohort ( Fig. 2b and c) . Remarkably though, in these old 141 individuals, high levels of S-II-specific, but not S-I-specific, CD4 + T cells at baseline correlated 142 with higher anti-S1 IgG and consistently with elevated S1 neutralizing capacity in serum at d28 143 ( Fig. 2d and e, Suppl. Fig. 1 ). These correlations did not exist for anti-S1 sIgA or salivary S1 144 neutralizing capacity ( Fig. 2f and g) . Hence, in old vaccinees, increased pre-existing 145 frequencies of S-II-specific CD4 + T cells predicted the efficiency of neutralizing and anti-S1 IgG humoral vaccination responses. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in immune responses, a significantly decreased anti-S1 IgG response was observed in the old 153 vaccinees after two doses of BNT162b2, which was intra-individually associated with an 154 equally reduced anti-S1 IgA response (Suppl. Fig. 3 ). However, compared to COVID-19 155 convalescents with a mild disease course, COVID-19 vaccination induced more pronounced 156 systemic immunity with significantly higher levels of spike-reactive CD4 + T cells, S1-specific 157 antibodies and serum S1 neutralizing capacity regardless of age. Reduced humoral and cellular vaccination responses in old individuals have been described 159 for vaccines against influenza, yellow fever, pneumococcal disease, and tetanus as well as for We additionally investigated anti-S1 sIgA in the saliva as a potential correlate of local mucosal 171 protection from SARS-CoV-2 infection following COVID-19 vaccination as compared to natural 172 infection. We assume that the presence of S1-specific antibodies and S1 neutralizing capacity 173 in the saliva may contribute to protection against SARS-CoV-2 infection and reduce local 174 replication 21, 22 . However, within four weeks (d49) after booster vaccination, anti-S1 sIgA levels 175 and S1 neutralizing activity in the saliva returned to low levels. By contrast, COVID-19 176 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.05.21264545 doi: medRxiv preprint 7 convalescents exhibited significantly higher salivary anti-S1 sIgA levels and higher salivary S1 177 neutralizing activity at ~d49 after symptom onset compared to age-matched vaccinees. We 178 found a correlation between anti-S1 sIgA levels and salivary S1 neutralization in convalescents 179 at ~d28 and at ~d49, which was not observed in vaccinees (Suppl. Fig. 4 ). This indicates that 180 neutralizing capacity in the saliva following vaccination may not only rely on anti-S1 sIgA but 181 possibly anti-S1 IgG, which is consistent with reports on detectable anti-S1 IgG in the saliva of 182 vaccinated individuals 23, 24 . In COVID-19 convalescents, anti-S1 sIgA secretion in salivary 183 glands (and salivary S1 neutralizing activity) is likely induced by locally primed B and T cells in Taken together, the presence of anti-S1 sIgA and S1 neutralizing capacity in the saliva after perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 7, 2021. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 7, 2021. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 7, 2021. d e c Serum S1 IgA Serum S1 IgG Saliva S1 IgA Serum sVNT All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 7, 2021. Suppl. Fig. 1 : Correlation of pre-existing T cell reactivity to S-I with anti-S1 IgG response. Anti-S1 serum IgG OD ratios in serum in the old (red dots) and middle-aged (white dots) 439 vaccinees at d28 grouped according to S-I-specific CD4 + T cell reactivity at BL (StimIndex > All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.05.21264545 doi: medRxiv preprint Suppl. Fig. 3 : Correlation of anti-S1 IgG and anti-S1 IgA in serum after vaccination. Anti-450 S1 IgG OD ratios in serum correlated with anti-S1 IgA in serum at d28 and d49, respectively, perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.05.21264545 doi: medRxiv preprint SARS-CoV-2 vaccines in development Risk factors of critical & mortal COVID-19 cases: A systemic literature review 322 and meta-analysis Efficacy and effectiveness of 324 influenza vaccines: a systematic review and meta-analysis B-cell responses to vaccination at the extremes of age Delayed Antibody and T-Cell Responses to BNT162b2 Vaccination in the 329 Cross-reactive CD4 + T cells enhance SARS-CoV-2 immune responses upon 331 infection and vaccination Older adults lack SARS CoV-2 cross-reactive T lymphocytes directed to human 333 coronaviruses OC43 and NL63 Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine Impact and effectiveness of mRNA BNT162b2 vaccine against SARS-CoV-2 337 infections and COVID-19 cases, hospitalisations, and deaths following a nationwide vaccination 338 campaign in Israel: an observational study using national surveillance data SARS-CoV-2-reative T cells in healthy donors and patients with COVID-19 Immunosenescence and its hallmarks: How to oppose aging strategically? 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