Longitudinal Humoral and Cell-Mediated Immune Responses in a Population-Based Cohort in Zurich, Switzerland between March and June 2022 - Evidence for Protection against Omicron SARS-CoV-2 Infection by Neutralizing Antibodies and Spike-specific T cell responses

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The correlate(s) of protection against SARS-CoV-2 remain incompletely defined. 3 Additional information regarding the combinations of antibody and T cell-mediated immunity 4 which can protect against (re)infection are needed. 5 6

We conducted a population-based, longitudinal cohort study including 1044 individ-7 uals of varying SARS-CoV-2 vaccination and infection statuses. We assessed Spike (S)- and 8 Nucleocapsid (N)-IgG and wildtype, delta, and omicron neutralizing antibodies. In a subset of 9 328 individuals, we evaluated S, Membrane (M) and N -specific T cells . 3 months later, w e 10 reassessed antibody (n=964) and T cell (n=141) responses and evaluated factors associated 11 with protection from (re)infection. 12 13

At study start, >98% of participants were S-IgG seropositive. N-IgG and M/N-T cell 14 responses increased over time, indicating viral (re)exposure, despite existing S-IgG. Com-15 pared to N-IgG, M/N-T cells were a more sensitive measure of viral exposure. N-IgG titers in 16 the top 33% of participants, omicron neutralizing antibodies in the top 25%, and S-specific T 17 cell responses were all associated with reduced likelihood of (re)infection over time. 18 19

Population-level SARS-CoV-2 immunity is S-IgG-dominated, but heterogenous. 20 M/N T cell responses can distinguish previous infection from vaccination, and monitoring a 21 combination of N-IgG, omicron neutralizing antibodies and S-T cell responses may help esti-22 mate protection against SARS-CoV-2 (re)infection. 23 24 25 KEY WORDS 26 27 SARS-CoV-2 28 Hybrid Immunity 29 Seroprevalence 30 Neutralizing Antibodies 31 T cell Responses 32 Interferon-gamma Release Assay 33 34 35 36 37 . CC-BY-NC 4.0 International licenseIt 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 is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 3

(total word count 3454) 1 2 It is now well -understood that exposure to SARS -CoV-2 elicits robust antibody and T cell-3 mediated immune responses to multiple viral proteins – in particular spike (S), nucleocapsid 4 (N) and membrane (M) proteins1-5. In contrast to infection, Covid-19 vaccination elicits re-5 sponses to the viral S protein; the only antigenic component of the vaccines most widely-used 6 in the United States and Europe 6,7. As the correlate(s) of protection, and level of said corre-7 late(s), needed to prevent infection or severe illness have yet to be clearly defined8, data on 8 population-level humoral and cellular immune responsiveness to SARS -CoV-2 remain im-9 portant for understanding 1) the scope of viral exposure and 2) what proportion of the popula-10 tion possesses some degree of virus-specific immunity. 11 12 While much is now known regarding population-level antibody responses to SARS-CoV-2 in-13 fection, our understanding of T cell-mediated immunity is much less comprehensive. T cell 14 responses have been described following both vaccination9-13 and infection; including mild or 15 asymptomatic cases even without seroconversion 1-3,5,13-16. However, extensive studies of T 16 cell responses, particularly at the population level, are lacking, partially due to the labor-inten-17 sive and relatively low -throughput nature of assays designed to evaluate them, such as 18 ELISpot and flow cytometry -based assays. To address this, adaptation of interferon (IFN) -19 gamma release assays (IGRAs), such as those used in Mycobacterium tuberculosis and Cy-20 tomegalovirus screening17,18, may aid in the detection of SARS -CoV-2-specific T cells in a 21 larger number of samples. Importantly, as both humoral and cellular responses contribute to 22 immunity against SARS-CoV-2, a better understanding of the heterogenous combinations of 23 immune memory which can protect against disease may help to inform vaccination strategies, 24 including the administration of additional booster vaccine doses. 25 26 Here, we conducted a population-based cohort study evaluating antibody and T cell responses 27 to SARS-CoV-2 among individuals aged 16+ in Zurich, Switzerland, including individuals of 28 varying vaccination and infection statuses. In March 2022, for all study participants (n=1044) 29 we evaluated total SARS-CoV-2 S- and N-IgG antibody levels, as well as neutralizing antibody 30 activity to wildtype (WT) virus and delta and omicron variants. In a randomly selected subset 31 of individuals (n=328), we further assessed T cell responses to S, M and N proteins by IGRA. 32 To investigate longitudinal changes in immune responses over time we reassessed antibody 33 (n=964) and T cell (n=141) responses 3 months later, in June 2022. Overall, we found distinct 34 immune response patterns among participants depending on reported infection and vaccina-35 tion statuses. Already at the beginning of the study, n early all participants had detectable S-36 IgG responses. In contrast, N-IgG and M/N-specific T cell responses increased significantly 37 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 4 over time, in spite of existing S-IgG, indicating viral (re)exposure. Importantly, participants with 1 N-IgG titers in the to 33% , omicron neutralizing antibody activity in the top 25%, and those 2 with IFN-gamma-producing S-reactive T cells all had significantly reduced likelihood of (re)in-3 fection between March and June 2022 . Together, our results indicate that population -level 4 immune responses to SARS-CoV-2 are S-IgG-dominated, but heterogenous. They suggest a 5 role for assessing M/N-specific T cells in estimating previous viral exposure, and further sug-6 gest that monitoring a combination of N-IgG, omicron neutralizing antibodies and S-reactive T 7 cell responses may help to predict population -level protection against omicron SARS-CoV-2 8 (re)infection. Our findings are consistent with SARS-CoV-2-specific immunity which is medi-9 ated by co-correlates, rather than a single correlate, of protection. 10 11 12 ABBREVIATED METHODS 13 14 Detailed methods and information on statistical analyses can be found in the supplementary 15 materials. 16 17 Participant Recruitment and Sample Collection 18 19 Individuals aged 16+ residing in the canton of Zurich, Switzerland were randomly selected by 20 age-stratified intervals from a population registry and invited to participate. In total, 4875 indi-21 viduals were contacted and 1044 enrolled (21.4% participation, Supplementary Fig.1). Initial 22 study visits were conducted from March 1st through 31st, 2022 and second study visits 23 (964/1044, 92.3% participation, Supplementary Fig.1) were conducted from June 7th through 24 July 11th, 2022. At each visit, participants provided information regarding previous Covid-19 25 vaccination and positive SARS-CoV-2 tests. From each participant, 10mL of venous blood 26 were collected and plasma cryo preserved prior to analysis of S -Ig and N-Ig levels and WT, 27 delta, and omicron SARS-CoV-2 neutralizing antibody activity. For participants selected for T 28 cell assessment, an additional 5mL of venous blood were collected and immediately used for 29 IFN-gamma Release Assay analysis. 30 31 Spike- and Nucleocapsid-Specific IgG and SARS-CoV-2 Neutralizing Antibody Activity 32 33 Cryopreserved plasma samples were thawed and analyzed for S - and N-specific IgG by Lu-34 minex assay as described19. Mean fluorescence intensity (MFI) values for each sample were 35 divided by the mean value of negative control samples to yield an MFI ratio. Individuals were 36 considered seropositive if the MFI ratio exceeded a lower limit o f detection (LOD) of 6.0 19. 37 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 5 Plasma samples were further evaluated for WT, delta, and omicron SARS-CoV-2 neutralizing 1 antibodies using a cell- and virus-free assay as described20. Half maximal inhibitory concen-2 tration (IC50) values of 50.0 and 2430.0 were set as lower and LODs, respectively. 3 4 Interferon-gamma Release Assay (IGRA) 5 6 T cell responses were assessed by IGRA from whole blood stimulated overnight with overlap-7 ping 15mer peptide pools spanning the entire M and N proteins (M/N pool) or the S1 domain 8 of the spike protein and a mix of the predicted immunodominant peptides from spike contain-9 ing the majority of the S2 domain (S pool) (M, N, S1 and S PepTivator peptide pools, respec-10 tively; Miltenyi Biotec). After incubation, stimulated plasma was collected and IFN-gamma as-11 sessed using the Human IFN -gamma ELISA assay (Human IFN -gamma DuoSet ELISA kit, 12 R&D Systems, Catalog DY285B, and DuoSet ELISA Ancillary Reagent Kit 2, R&D Systems, 13 Catalog DY008) according to manufacturer’s instructions. 14 15 16

17 18 Participant Demographics and Overall Antibody and T cell Immune Responses 19 20 Of March 2022 study participants (n=1044, Supplementary Fig.1, Supplementary Table 1), 21 45.5% were male and 54.3% were female. 73.7% were aged 16-64 and 26.3% were 65+. 22 93.5% reported previous SARS-CoV-2 vaccination; 90.8% were fully vaccinated (2+ vaccine 23 doses) and 72.1% had received at least one booster (3+ vaccine doses)21,22. 32.6% of partic-24 ipants reported a previous SARS-CoV-2 infection (defined as having received a positive PCR 25 or antigen test result) at some point from the p andemic start up to the study visit. Older par-26 ticipants (65+) were more likely to report being immunized against Covid-19 (OR 2.87, 95% 27 CI 1. 36-6.09, p=0.0 06) and less likely to report previous infection (OR 0.44, 0.3 2-0.61, 28 p<0.0001) compared to participants aged 16-64, possibly reflecting both the emphasis on vac-29 cination for those 65+, as well as the preventative effect of vaccination on subsequent infec-30 tion. 31 32 98.4% of participants were S -IgG seropositive and 23.2% were N-IgG seropositive 33 (Fig.1A)21,22. 96.8%, 93.7% and 89.5% of participants had detectable neutralization IC50 val-34 ues to WT, delta and omicron viral variants, respectively. Geometric mean IC50 values, how-35 ever, differed significantly between variants , being highest for WT and lowest for omicron 36 (p<0.0001 for all comparisons, Friedman test with Dunn’s multiple comparisons, Fig.1B). In a 37 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 6 subset of study participants (n=328), circulating T cell responses to S, or a combination of M 1 and N proteins 14, were assessed . 89.6% had detectable S -specific T cell responses, while 2 57.3% had detectable M/N-specific T cell responses; geometric mean IFN-gamma production 3 was greater for S-, as compared to M/N-stimulation (Fig.1C). Taken together, these data indi-4 cate that, as of March 2022, nearly 99% of the population had previous SARS-CoV-2 antigen 5 exposure (either through vaccination, infection, or both). As M/N proteins are not present in 6 available vaccines6,7 but are generated in response to infection, and, as M- and N-T cell re-7 sponses are longer-lasting than N-IgG1,3,14, these findings further suggest that at least 57% of 8 the population had been previously infected by this time. 9 10 Impacts of Infection and Vaccination on Antibody and T cell Responses 11 12 We next assessed the impacts of infection and vaccination on antibody titers and T cell re-13 sponses by multivariable linear regression . Increasing age (65+ vs. 16 -64) was significantly 14 and independently associated with lower S- and N-IgG titers, lower anti-WT, -delta, and -omi-15 cron neutralization IC50 values and lower S-T cell responses (Fig.2). Previous SARS-CoV-2 16 infection and receiving an increasing number of vaccine doses were both associated with sig-17 nificantly increased S-IgG titers, anti-WT, -delta, and -omicron neutralization IC50 values, and 18 S-T cell responses (Fig.2). Previous infection was further associated with increased N-IgG 19 titers and M/N -T cell responses (Fig.2). Participants were stratified into four groups : in-20 fected/vaccinated (n=285 antibody-, 80 T cell-tested), uninfected/vaccinated (n=686 antibody, 21 229 T cell-tested), infected/unvaccinated (n=53 antibody, 14 T cell-tested), and uninfected/un-22 vaccinated (n=15 antibody, 3 T cell -tested) (Fig.3A and B) . S-IgG and neutralizing activity 23 tended to be higher in vaccinated individuals (both previously infected and uninfected, Fig.3A), 24 and these did not correlate with N-IgG responses (Fig.3B). In contrast, N-IgG and M/N-T cell 25 responses were higher among previously infected individuals (both vaccinated and unvac-26 cinated, Fig.3A and B). Unsurprisingly, the lowest overall responses were observed in unin-27 fected/unvaccinated individuals and, due to low sample number, there were insufficient data 28 to assess T cell correlation patterns for this group (Fig.3A and B). In general, however, anti-29 body and T cell respons e patterns appeared more similar between vaccinated participants 30 compared to infected participants, with robust S-IgG and neutralizing antibody responses fol-31 lowing vaccination and greater N-IgG and M/N-T cell responses among infected individuals. 32 33 Longitudinal Responses and Protection from (Re)infection 34 35 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 7 964 participants returned for a second study visit, 3 months later, in June 2022 (Supplemen-1 tary Fig.1, Supplementary Table 1). 141 were assessed for T cell responses (118 longitudi-2 nally from March and an additional 23 not evaluated for T cell responses in the March round; 3 Supplementary Table 1). At this time, 6. 4% of participants were unvaccinated, 2.4% had re-4 ceived a single vaccine dose, 17.0% had received 2 doses, and 74.2% had received 3 or more 5 doses. 19 individuals (2.0% of the study population) received an additional vaccination be-6 tween March and June, all of which were 2nd or booster doses. 16.0% reported a positive 7 SARS-CoV-2 PCR or antigen test (infection) between March and June. Of these, 14.3% also 8 reported previous infection in March (repeated infections ; 85.7% new infections). In total, 9 45.3% of the population reported at least one SARS-CoV-2 infection. 10 11 98.8% of participants were S-IgG seropositive (similar to March) and 36.7% were N-IgG sero-12 positive (increasing from March; p<0.0001, Two-sample test of proportions). Geometric mean 13 MFI ratio titers for both S-IgG and N-IgG increased between March and June (Fig.4A; S-IgG 14 p<0.0001, N-IgG p<0.0001, Wilcoxon matched-pairs signed rank test ). 97.2% and 72.3% of 15 participants had detectable S- and M/N-T cell responses, respectively; significantly more than 16 in March (Fig.4A and B, S p=0.043, M/N p=0.001, Fisher’s exact test) and geometric mean 17 IFN-gamma production among the overall population was higher for both in June (Fig.4B, S 18 p=0.05, M/N p=0.053, Mann-Whitney test). M/N-T cell responses tended also to be higher in 19 the longitudinal subset, though this was not statistically significant (p =0.109, Wilcoxon 20 matched-pairs signed rank test). Between March and June, 0.4% (4/948) of those who were 21 seropositive for S-IgG became seronegative while 50% (8/16) of those who were seronegative 22 became seropositive. For N-IgG, 32.3% (72/223) of those who were seropositive became ser-23 onegative and 27.4% (203/741) of those who were seronegative became seropositive. Of in-24 dividuals tested longitudinally for T cell responses, 1.9% (2/107) of those S-T cell positive in 25 March were negative in June, while 81.8% (9/11) of those negative in March were positive in 26 June. 13.3% (8/60) of those who were M/N -T cell positive in March were negative in June, 27 while 58.6% (34/58) of those negative in March were positive in June. Of those that became 28 N-IgG seropositive or M/N-T cell reactive, only 59.1% (120/203) and 29.4% (10/34), respec-29 tively, reported infection, highlighting the importance of immune monitoring efforts in under-30 standing SARS-CoV-2 exposures. 31 32 Overall, we observed three dominant immune response patterns among participants , which 33 were similar in both March and June. In March we observed 17.7% group1 (S-IgG+/N-IgG+/S-34 T cell+/M/N-T cell+ -> positive for all factors), 36.3% group2 (S-IgG+/N-IgG-/S-T cell+/M/N-T 35 cell+ -> positive for everything except N -IgG), and 34.2% group3 ( S-IgG+/N-IgG-/S-T 36 cell+/M/N-T cell- -> S-IgG and S -T cell positive only ). In June, we observed 30.5% group1, 37 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 8 39.0% group2, and 23.4% group3 (Supplementary Fig.2A and B). Interestingly, only 5.2% of 1 those in group1 (positive of all factors) reported an infection between March and June com-2 pared to 26.6% of those in group2 (positive for everything except N-IgG) and 21.9% of those 3 in group3 (S-IgG and S-T cell positive only), potentially suggesting superior protection by the 4 group1 combination of immune responses. In order to evaluate which immune response com-5 ponents might be capable of providing protection against (re)infection, we assessed whether 6 an individual’s levels of S- and N-IgG and S- and M/N-T cells in March were associated with 7 infection between March and June (Fig.5). As vaccination is expected to influence SARS-CoV-8 2-specific immune responses, individuals vaccinated between March and June (n=19) were 9 excluded from the analysis. Having an N-IgG MFI ratio titer above 10 (titers in the top 33% of 10 the population) was associated with an 84% reduced odds of infection between March and 11 June (OR 0.16, 95% 0.03-0.85, p=0.031; compared to the lowest 33%). Having omicron neu-12 tralizing IC50 titers above 360 (titers in the top 25% of the population) was associated with a 13 94% reduced odds of infection (OR 0.06, 0.006-0.60, p=0.017; compared to the lowest 25%), 14 while having S-T cells was associated with a 60% reduced likelihood of infection (production 15 of >=25 to <65pg/mL IFN-gamma, OR 0.39, 0.17-0.92, p=0.030; compared to the lowest 25%). 16 Therefore, N-IgG, omicron neutralizing antibodies, and S-specific T cells are associated with 17 protection from omicron (re)infection, and monitoring a combination of these responses may 18 aid in the assessment of population-level immunity against omicron SARS-CoV-2 (re)infection. 19 20 21

22 23 While antibody responses among individuals in the Zurich area, and throughout Switzerland 24 have been well-described21-24, much less is kn own regarding population-level T cell respon-25 siveness to SARS-CoV-2. Here, we utilized an interferon (IFN)-gamma release assay (IGRA) 26 based on short-term culture of whole blood with SARS -CoV-2-specific peptides to assess T 27 cell responses, which demonstrated good concordance with an ELISpot assay which we used 28 previously14. We found that , by June 2022, 97% and 72%, of study participants had S - and 29 M/N-specific T cells, respectively. In comparison, 99% of participants were S-IgG seropositive 30 and slightly less than 40 % were N-IgG seropositive. That S-specific antibody and T cell re-31 sponses were higher, in general, than N-specific antibody and M/N-specific T cell responses 32 is consistent with the high vaccination coverage in the population (>90% fully vaccinated), as 33 available vaccines contain S, but not M or N antigens6,7. Furthermore, the half-life of S-IgG is 34 substantially longer than that of N-IgG14,25,26, consistent with our observation that the fraction 35 of participants who were initially seropositive in March but became seronegative by June was 36 greater for N -IgG (14.1%) compared to S -IgG (1.2%). The higher percentage of M/N -T cell 37 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 9 positivity compared to N-IgG positivity is worth noting and is possibly due to 1) the use of both 1 M- and N-specific peptides in the IGRA, 2) that the half-lives of circulating M- and N-specific 2 T cells are longer than that of N -IgG1,3,14, 3) that some individuals develop only M - and N-3 specific T cell responses after infection 14, and, 4) that previous exposure to endemic human 4 coronaviruses (HCoV)-229E, -NL63, -OC43, and -HKU1) can generate low levels of T cells 5 cross-reactive to SARS-CoV-22,4,5. Our findings, however, indicate that assessing SARS-CoV-6 2 M/N-T cells is likely a more sensitive method for evaluating exposure to SARS-CoV-2 com-7 pared to N-IgG and that monitoring M/N-T cells may help to assess population-level “hybrid 8 immunity”. 9 10 An additional takeaway from our findings is that the majority of individuals had more than one 11 type of virus-specific memory response and that protection was not clearly mediated only by 12 a single subset. The most common patterns, representing over 90% of t he study population 13 were group1 (S-IgG+/N-IgG+/S-T cell+/M/N-T cell+), group2 (S-IgG+/N-IgG+/S-T cell+/M/N-14 T cell-), and group3 (S-IgG+/N-IgG-/S-T cell+/M/N-T cell-). All of these patterns included S -15 IgG, but nearly 95% of those in group1 did not report an infection between March and June 16 compared to 73-78% of those in groups 2 and 3 . Furthermore, despite high S-IgG seroposi-17 tivity already in March 2022, the percentage of participants with detectable N-IgG titers and 18 M/N-T cells increased significantly by June 2022, indicating continued viral (re)infections, even 19 among individuals with some level of S-specific immunity. In assessing potential mediators of 20 protective immunity, we found that having a high N-IgG titer and/or high omicron neutralizing 21 antibody activity were both protective against omicron SARS-CoV-2 infection. As the half-life 22 of N-IgG is relatively short at approximately 60-90 days14,25,26, individuals with high titers were 23 likely recently infected – perhaps in the 3-6 months prior to March. It would also make sense 24 that these individuals were infected with the omicron variant, which was responsible for >99% 25 of reported Covid -19 cases in late January 2022 in Switzerland27. As recent infection may 26 contribute to a state of “trained immunity” 28 with enhanced baseline activation of the innate 27 immune system, we speculate that N-IgG is likely not a sole mediator of protection in and of 28 itself, but may serve as a marker for a persisting “antiviral” state which, in turn, limits reinfec-29 tion. We additionally found that S-T cells were associated with reduced likelihood of infection. 30 S-reactive T cells are known to be generated following SARS-CoV-2 infection and SARS-CoV-31 2 vaccination1-3,9-15, and individuals can also possess pre-existing memory T cell responses 32 generated from previous endemic human coronavirus exposure2,4,5. Though a role for S-T cell 33 responses as a corre late of protection against SARS -CoV-2 is not completely clear8, it is 34 known that T cell-mediated immunity is more cross-reactive than corresponding antibody re-35 sponses29. Furthermore, it has been observed in animal models that, in the absence of anti-36 body responses, protection from SARS-CoV-2 can be mediated solely by T cell immunity 30, 37 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 10 and, similarly, we recently observed that individuals can clear SARS -CoV-2 infection in the 1 absence of detectable antibody responses14, highlighting the importance of this subset in pro-2 tection from infection. 3 4 Some limitations to our study include, first, that we relied on self-reported SARS-CoV-2 infec-5 tions based on receiving a positive PCR or antigen test result. While false positive results are 6 possible, it is also likely that, as many individuals use self-tests, which have limited sensitivity 7 especially early in infection, that true infections are under -reported. Similarly, we observed 8 that a substantial fraction - 20% (3/15) - of participants that reported being uninfected/unvac-9 cinated had detectable S- or N-IgG titers, which we would expect only in responses to SARS-10 CoV-2 antigen exposure. An additional limitation was t he low number of uninfected/unvac-11 cinated individuals, and as we collected T cell data only from a subset of individuals, we did 12 not have sufficient data to thoroughly assess this group, which represents an interesting im-13 mune “baseline”. Furthermore, in terms of the assays used, we assessed only IFN-gamma as 14 a measure of T cell activity. Studies have demonstrated that IL-2-producing T cell responses 15 are also generated in SARS-CoV-2 infection and vaccination5,16. It would be valuable to test 16 the IGRA approach in evaluating IL -2 responses in further studies as well. In addition, we 17 limited our T cell analysis to the three dominant antigens for cellular immune responses (S, M 18 and N), and furthermore, for experimental feasibility, S1 and S2 domains as well as M and N 19 responses were pooled, although they have been shown in other studies to exhibit some dis-20 tinct behaviors1,2,14,16. We cannot exclude the importance of subdominant T cell responses 21 against other viral antigens in some of the participants, which may have led to an underesti-22 mation of T cell responses. Another limitation is that, although the IGRA results had a high 23 degree of concordance with ELISpot assay (Supplementary Methods, Supplementary Fig3A 24 and B), they did not strongly correlate. T his is not unexpected , though, as IGRA measures 25 total IFN -gamma output in pg/mL which could be produced by few specific T cells, while 26 ELISpot assesses only the number of IFN-gamma-producing cells without taking the amount 27 of IFN-gamma produced by individual cells into account, making it difficult to compare values 28 from these two assays directly. Furthermore, we used a surrogate assay to indirectly quantify 29 neutralizing activity by measuring competitive inhibition of trimeric SARS -CoV-2 S protein 30 binding to the Angiotensin Converting Enzyme 2 (ACE2) receptor. However, this assay 31 showed high sensitivity compared to live virus assays during validation20 and permitted simul-32 taneous assessment of neutralization against WT-SARS-CoV-2, delta and omicron variants. 33 34 Nevertheless, we provide here population-level estimates of cellular immunity as well as fac-35 tors which may be associated with protection from omicron SARS-CoV-2 infection. Our results 36 suggest that, while the majority of individuals possess anti -S-IgG, these responses in and of 37 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 11 themselves are likely not a good predictor of protection from (re)infection. In terms of estimat-1 ing what fraction of the population has been infected with SARS-CoV-2, monitoring M/N-reac-2 tive T cells responses may be helpful. However, to assess what fraction of the population 3 might be protected from (re)infection with omicron SARS-CoV-2, our data suggest that moni-4 toring a combination of anti -N-IgG responses, omicron -specific neutralizing antibody re-5 sponses, and S-reactive T cell responses may be beneficial. Our findings indicate a pattern 6 where co-correlates of protection, rather than simply S-IgG, are likely important for mediating 7 long-term protective immunity against SARS-CoV-2 and future variants and provide important 8 information for policy makers regarding vaccination strategy in the case of changing disease 9 epidemiology. 10 11 12 CONFLICT OF INTEREST 13 14 The authors declare no competing interests. 15 16 17 FUNDING 18 19 The Corona Immunitas research network is coordinated by the Swiss School of Public Health 20 (SSPH+) and funded by fundraising of SSPH+ including funds of the Swiss Federal Office of 21 Public Health and private funders (ethical guidelines for funding stated by SSPH+ were re-22 spected), by funds of the cantons of Switzerland (Vaud, Zurich, and Basel), and by institutional 23 funds of the Universities. 24 25 26 ETHICAL APPROVAL 27 28 We obtained written, informed consent from all participants upon study enrollment. Partici-29 pants were compensated with a flat fee for any travel expenses related to study visits, but 30 otherwise did not receive any compensation for their participation. The study protocol was 31 approved by the Cantonal Ethics Committee of Zurich (BASEC Registration No. 2020-01247) 32 and registered (ISRCTN registry 18181860, date of registration 13 July 2020, retrospectively 33 registered). 34 35 36

37 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 12 1 The authors would like to thank Danusia Vanoaica and Osman Yoztekin for their support with 2 the T cell assays, and the study administration team and the study participants for their dedi-3 cated contribution to this research project. 4 5 6

7 8 1 Dan, J. M. et al. Immunological memory to SARS-CoV-2 assessed for up to 8 9 months after infection. Science 371, eabf4063 (2021). 10 https://doi.org:doi:10.1126/science.abf4063 11 2 Grifoni, A. et al. Targets of T Cell Responses to SARS-CoV-2 Coronavirus in 12 Humans with COVID-19 Disease and Unexposed Individuals. Cell 181, 1489-13 1501.e1415 (2020). https://doi.org:https://doi.org/10.1016/j.cell.2020.05.015 14 3 Cohen, K. W. et al. Longitudinal analysis shows durable and broad immune memory 15 after SARS-CoV-2 infection with persisting antibody responses and memory B and 16 T cells. Cell Reports Medicine 2, 100354 (2021). 17 https://doi.org:https://doi.org/10.1016/j.xcrm.2021.100354 18 4 Le Bert, N. et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and 19 SARS, and uninfected controls. Nature 584, 457-462 (2020). 20 https://doi.org:10.1038/s41586-020-2550-z 21 5 Sekine, T. et al. Robust T Cell Immunity in Convalescent Individuals with 22 Asymptomatic or Mild COVID-19. Cell 183, 158-168.e114 (2020). 23 https://doi.org:10.1016/j.cell.2020.08.017 24 6 Creech, C. B., Walker, S. C. & Samuels, R. J. SARS-CoV-2 Vaccines. JAMA 325, 25 1318-1320 (2021). https://doi.org:10.1001/jama.2021.3199 26 7 Amanat, F. & Krammer, F. SARS-CoV-2 Vaccines: Status Report. Immunity 52, 583-27 589 (2020). https://doi.org:https://doi.org/10.1016/j.immuni.2020.03.007 28 8 Goldblatt, D., Alter, G., Crotty, S. & Plotkin, S. A. Correlates of protection against 29 SARS-CoV-2 infection and COVID-19 disease. Immunol Rev 310, 6-26 (2022). 30 https://doi.org:10.1111/imr.13091 31 9 Goel, R. R. et al. mRNA vaccines induce durable immune memory to SARS-CoV-2 32 and variants of concern. Science 374, abm0829 (2021). 33 https://doi.org:doi:10.1126/science.abm0829 34 10 Zhang, Z. et al. Humoral and cellular immune memory to four COVID-19 vaccines. 35 Cell 185, 2434-2451.e2417 (2022). 36 https://doi.org:https://doi.org/10.1016/j.cell.2022.05.022 37 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 13 11 Mateus, J. et al. Low-dose mRNA-1273 COVID-19 vaccine generates durable 1 memory enhanced by cross-reactive T cells. Science 374, eabj9853 (2021). 2 https://doi.org:doi:10.1126/science.abj9853 3 12 Tarke, A. et al. SARS-CoV-2 vaccination induces immunological T cell memory able 4 to cross-recognize variants from Alpha to Omicron. Cell 185, 847-859.e811 (2022). 5 https://doi.org:https://doi.org/10.1016/j.cell.2022.01.015 6 13 Sette, A. & Crotty, S. Immunological memory to SARS-CoV-2 infection and COVID-7 19 vaccines. Immunological Reviews 310, 27-46 (2022). 8 https://doi.org:https://doi.org/10.1111/imr.13089 9 14 Menges, D. et al. Heterogenous humoral and cellular immune responses with distinct 10 trajectories post-SARS-CoV-2 infection in a population-based cohort. Nature 11 Communications 13, 4855 (2022). https://doi.org:10.1038/s41467-022-32573-w 12 15 Rodda, L. B. et al. Functional SARS-CoV-2-Specific Immune Memory Persists after 13 Mild COVID-19. Cell 184, 169-183.e117 (2021). 14 https://doi.org:https://doi.org/10.1016/j.cell.2020.11.029 15 16 Thieme, C. J. et al. Robust T Cell Response Toward Spike, Membrane, and 16 Nucleocapsid SARS-CoV-2 Proteins Is Not Associated with Recovery in Critical 17 COVID-19 Patients. Cell Reports Medicine 1, 100092 (2020). 18 https://doi.org:https://doi.org/10.1016/j.xcrm.2020.100092 19 17 Pai, M. et al. Gamma Interferon Release Assays for Detection of Mycobacterium 20 tuberculosis Infection. Clinical Microbiology Reviews 27, 3-20 (2014). 21 https://doi.org:doi:10.1128/CMR.00034-13 22 18 Giulieri, S. & Manuel, O. QuantiFERON®-CMV assay for the assessment of 23 cytomegalovirus cell-mediated immunity. Expert Review of Molecular Diagnostics 11, 24 17-25 (2011). https://doi.org:10.1586/erm.10.109 25 19 Fenwick, C. et al. Changes in SARS-CoV-2 Spike versus Nucleoprotein Antibody 26 Responses Impact the Estimates of Infections in Population-Based Seroprevalence 27 Studies. J Virol 95 (2021). https://doi.org:10.1128/jvi.01828-20 28 20 Fenwick, C. et al. A high-throughput cell- and virus-free assay shows reduced 29 neutralization of SARS-CoV-2 variants by COVID-19 convalescent plasma. Science 30 Translational Medicine 13, eabi8452 (2021). 31 https://doi.org:doi:10.1126/scitranslmed.abi8452 32 21 Amati, R. et al. Functional immunity against SARS-CoV-2 in the general population 33 after a booster campaign and the Delta and Omicron waves, Switzerland, March 34 2022. Euro Surveill 27 (2022). https://doi.org:10.2807/1560-35 7917.Es.2022.27.31.2200561 36 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 14 22 Frei, A. et al. Development of hybrid immunity during a period of high incidence of 1 infections with Omicron subvariants: A prospective population based multi-region 2 cohort study. medRxiv, 2022.2010.2014.22281076 (2022). 3 https://doi.org:10.1101/2022.10.14.22281076 4 23 West, E. A. et al. Seroprevalence of SARS-CoV-2 antibodies, associated factors, 5 experiences and attitudes of nursing home and home healthcare employees in 6 Switzerland. BMC Infectious Diseases 22, 259 (2022). 7 https://doi.org:10.1186/s12879-022-07222-8 8 24 Stringhini, S. et al. Seroprevalence of anti-SARS-CoV-2 antibodies 6 months into the 9 vaccination campaign in Geneva, Switzerland, 1 June to 7 July 2021. Euro Surveill 10 26 (2021). https://doi.org:10.2807/1560-7917.Es.2021.26.43.2100830 11 25 Lumley, S. F. et al. The Duration, Dynamics, and Determinants of Severe Acute 12 Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Responses in 13 Individual Healthcare Workers. Clin Infect Dis 73, e699-e709 (2021). 14 https://doi.org:10.1093/cid/ciab004 15 26 Wheatley, A. K. et al. Evolution of immune responses to SARS-CoV-2 in mild-16 moderate COVID-19. Nature Communications 12, 1162 (2021). 17 https://doi.org:10.1038/s41467-021-21444-5 18 27 COVID-⁠19 Switzerland Information on the current situation, Epidemiological 19 course,Switzerland and Liechtenstein, 20 (2022). 21 28 Netea, M. G. et al. Defining trained immunity and its role in health and disease. 22 Nature Reviews Immunology 20, 375-388 (2020). https://doi.org:10.1038/s41577-23 020-0285-6 24 29 Sewell, A. K. Why must T cells be cross-reactive? Nature Reviews Immunology 12, 25 669-677 (2012). https://doi.org:10.1038/nri3279 26 30 Kingstad-Bakke, B. et al. Vaccine-induced systemic and mucosal T cell immunity to 27 SARS-CoV-2 viral variants. Proceedings of the National Academy of Sciences 119, 28 e2118312119 (2022). https://doi.org:doi:10.1073/pnas.2118312119 29 30 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint A B C S-IgG N-IgG 100 101 102 103 104 Log(MFI Titer Ratio) LOD S-TC M/N-TC 100 101 102 103 104 Log(IFNg pg/mL) WT N-Ab Delt. N-Ab Omic. N-Ab 100 101 102 103 104 Log(MFI IC50 Ratio) LOD LOD . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint Figure 1. Quantitative Antibody and T cell Responses among Participants, March 2022. A) anti-S- and N-IgG geometric mean MFI titer ratios (n=1044), assay Limit of Detection (LOD)=6.0. B) anti-wildtype, delta and omicron geometric mean neutralizing antibody titers (n=1044), assay LOD 50.0-2430.0. C) Geometric mean IFN-gamma production following S or M/N peptide stimulation of whole blood (n=328). . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint -2 -1 0 1 2 3 4 Intercept 3+ Doses 2 Doses 1 Dose Positive Test 65+ Female Intercept 3+ Doses 2 Doses 1 Dose Positive Test 65+ Female Intercept 3+ Doses 2 Doses 1 Dose Positive Test 65+ Female Intercept 3+ Doses 2 Doses 1 Dose Positive Test 65+ Female Intercept 3+ Doses 2 Doses 1 Dose Positive Test 65+ Female Intercept 3+ Doses 2 Doses 1 Dose Positive Test 65+ Female Intercept 3+ Doses 2 Doses 1 Dose Positive Test 65+ Female S-IgGN-IgGWT N-Ab 0.12 (0.01-0.23)* -0.07 (-0.13- -0.01)* 0.06 (0.001-0.11)* 0.22 (0.15-0.28)*** 1.83 (1.71-1.95)*** 1.98 (1.86-2.09)*** 1.68 (1.49-1.88)*** Delt. N-AbOmic. N-AbS-TCM/N-TC -0.26 (-0.68-0.17) 0.78 (0.28-1.28)** 2.96 (2.69-3.22)*** 2.47 (2.19-2.74)*** 1.21 (0.80-1.61)*** -0.20 (-0.33- -0.07)** 0.12 (0.01-0.23)* 1.85 (1.44-2.26)*** -0.22 (-0.35- -0.09)*** 0.85 (0.72-1.00)*** 1.76 (1.49-2.03)*** 2.24 (1.98-2.50)*** 2.03 (1.15-2.92)*** 1.99 (1.08-2.91)*** 1.03 (0.89-1.16)*** 0.01 (-0.38-0.41) 1.84 (0.26-3.42)* 0.22 (-0.74-1.17) 1.90 (1.38-2.43)*** -0.60 (-1.00- -0.19)** -0.24 (-0.62-0.13) -0.07 (-0.99-0.86) 0.67 (-0.99-2.33) Estimate (95% CI) -0.16 (-0.26- -0.05)** 0.08 (-0.01-0.18) 1.42 (1.31-1.54)*** -0.44 (-0.65- -0.22)*** -0.44 (-0.65- -0.23)*** -0.29 (-0.64-0.05) -0.20 (-0.32- -0.08)*** 0.10 (-0.01-0.21) 0.71 (0.58-0.84)*** 2.73 (2.47-2.99)*** 3.29 (3.04-3.54)*** 2.15 (1.76-2.53)*** . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint Figure 2. Factors Associated with March 2022 S- or N-IgG, N-Ab Titers, or S- or M/N-T cell IFN-gamma Levels. Multivariable linear regression modeling was used to assess the relationship between gender (female vs. male), age group (65+ vs. 16-64 years), reporting a previous SARS-CoV-2 infection (positive PCR or antigen test) (yes vs. no), and the number of Covid-19 vaccine doses received (1, 2, 3+ vs. 0), and S- or N-IgG, N-Ab MFI ratio titers, or S- or M/N-T cell IFN-gamma levels (natural logarithm- transformed). *p>0.05, **p>0.01, ***p>0.005. . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint A B 1.00 0.03 0.01 -0.01 -0.04 0.24 0.32 0.03 1.00 0.28 0.29 0.37 -0.19 -0.07 0.01 0.28 1.00 0.95 0.84 -0.05 -0.04 -0.01 0.29 0.95 1.00 0.88 -0.05 -0.05 -0.04 0.37 0.84 0.88 1.00 -0.11 -0.17 0.24 -0.19 -0.05 -0.05 -0.11 1.00 0.55 0.32 -0.07 -0.04 -0.05 -0.17 0.55 1.00 S-IgG N-IgG Neut. WT Neut. Delt. Neut. Omic. S1/S2 IFNg M/N IFNg S-IgG N-IgG Neut. WT Neut. Delt. Neut. Omic. S1/S2 IFNg M/N IFNg -0.2 0 0.2 0.4 0.6 0.8 1.0 1.0 0.8 0.6 0.4 0.2 -0.20.0 Spearman Coefficient VaccinatedUnvaccinated InfectedUninfected S-IgG N-IgG WT N-Ab Delt. N-Ab Omic. N-Ab S-TC M/N-TC Infected/Vaccinated Uninfected/Vaccinated Infected/Unvaccinated Uninfected/Unvaccinated 0 Ln(Units) 2 4 6 1.00 0.71 0.67 0.52 0.67 0.71 1.00 0.82 0.70 0.82 0.67 0.82 1.00 0.83 0.77 0.52 0.70 0.83 1.00 0.94 0.67 0.82 0.77 0.94 1.00 1.00 1.00 S-IgG N-IgG WT N-Ab Delt. N-Ab Omic. N-Ab S-TC M/N-TC S-IgG N-IgG WT N-Ab Delt. N-Ab Omic. N-Ab S-TC M/N-TC -0.2 0 0.2 0.4 0.6 0.8 1.0 1.00 -0.01 0.04 0.05 0.01 -0.08 0.10 -0.01 1.00 0.21 0.25 0.29 0.16 0.29 0.04 0.21 1.00 0.96 0.87 0.20 0.06 0.05 0.25 0.96 1.00 0.90 0.19 0.08 0.01 0.29 0.87 0.90 1.00 0.15 0.08 -0.08 0.16 0.20 0.19 0.15 1.00 0.17 0.10 0.29 0.06 0.08 0.08 0.17 1.00 S-IgG N-IgG WT N-Ab Delt. N-Ab Omic. N-Ab S-TC M/N-TC S-IgG N-IgG WT N-Ab Delt. N-Ab Omic. N-Ab S-TC M/N-TC -0.2 0 0.2 0.4 0.6 0.8 1.0 1.00 0.53 0.92 0.87 0.59 0.51 0.41 0.53 1.00 0.39 0.48 0.60 0.08 0.29 0.92 0.39 1.00 0.91 0.58 0.48 -0.02 0.87 0.48 0.91 1.00 0.63 0.43 -0.10 0.59 0.60 0.58 0.63 1.00 0.09 0.35 0.51 0.08 0.48 0.43 0.09 1.00 0.05 0.41 0.29 -0.02 -0.10 0.35 0.05 1.00 S-IgG N-IgG WT N-Ab Delt. N-Ab Omic. N-Ab S-TC M/N-TC S-IgG N-IgG WT N-Ab Delt. N-Ab Omic. N-Ab S-TC M/N-TC -0.2 0 0.2 0.4 0.6 0.8 1.0 1.00 0.04 0.01 -0.04 0.09 0.16 0.04 1.00 0.28 0.29 0.37 0.11 0.31 0.01 0.28 1.00 0.95 0.84 0.07 -0.06 0.29 0.95 1.00 0.89 0.02 -0.02 -0.04 0.37 0.84 0.89 1.00 -0.03 -0.03 0.09 0.11 0.07 0.02 -0.03 1.00 0.40 0.16 0.31 -0.06 -0.02 -0.03 0.40 1.00 S-IgG N-IgG WT N-Ab Delt. N-Ab Omic. N-Ab S-TC M/N-TC S-IgG N-IgG WT N-Ab Delt. N-Ab Omic. N-Ab S-TC M/N-TC -0.2 0 0.2 0.4 0.6 0.8 1.0 0.005 0.005 . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint Figure 3. Antibody and T cell Responses among Participants by Infection and Vaccination Status, March 2022. A) Quantitative Antibody and T cell Responses. Log10 anti-S- and N-IgG geometric mean MFI titer ratios, anti-wildtype, delta and omicron geometric mean neutralizing antibody titers and geometric mean IFN-gamma production following S or M/N peptide stimulation of whole blood. B) Correlation between Antibody and T cell Responses. Top left: Infected and Unvaccinated individuals (n=53 antibody tested, 14 T cell tested), Top right: Infected and Vaccinated (n=285 antibody tested, 80 T cell tested), Bottom left: Uninfected and Unvaccinated (n=15 antibody tested, 3 T cell tested), Bottom right: Uninfected and Vaccinated (n=686 antibody tested, 229 T cell tested). Values represent Spearman correlation coefficients for indicated antibody and T cell response pairs. Crosses indicate pairs with insufficient data for analysis. . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint A B S-IgG N-IgG 100 101 102 103 104 Log(MFI Titer Ratio) LOD S-TC M/N-TC 100 101 102 103 104 Log(IFNg pg/mL) . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint Figure 4. Quantitative Antibody and T cell Responses, June 2022 . A) S- and N-IgG geometric mean MFI ratio titers (n=964). B) Geometric mean IFN-gamma production following S or M/N peptide stimulation of whole blood (n=141). . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint 0 1 2 3 4 5 10 15 20 25 Q4 (>=95) Q3 (>=30:=10:=155) Q3 (>=65:=25:=360) Q3 (>=175:=100:=695) Q3 (>=350:=185:=1535) Q3 (>=795:=395:=10) Q2 (>=5:=105) Q2 (>=95:<105) 65+ Female S-IgGN-IgGWT N-AbDelt. N-AbOmic. N-AbS-TCM/N-TC 2.91 (1.41-6.00)*** 0.08 (0.45-1.77) 1.27 (0.52-3.13) 1.21 (0.55-2.67) Odds Ratio (95% CI) 0.16 (0.03-0.85)* 0.18 (0.02-1.82) 0.69 (0.12-3.87) 1.22 (0.35-4.26) 0.26 (0.02-2.94) 0.55 (0.10-2.87) 0.44 (0.13-1.44) 2.02 (0.17-23.5) 0.78 (0.21-2.91) 0.91 (0.30-2.72) 0.06 (0.01-0.60)* 1.15 (0.44-3.00) 0.40 (0.17-0.92)* 0.52 (0.17-1.57) 0.72 (0.22-2.42) 2.37 (0.91-6.10) 0.96 (0.31-3.00) . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint Figure 5. Factors Associated with Reporting a SARS-CoV-2 Infection between March and June 2022. Multivariable logistic regression modeling was used to assess the relationship between gender (female vs. male), age group (65+ vs. 16-64 years), quantiles of S- or N-IgG, N-Ab Titers, or S- or M/N-T cell IFN-gamma levels in March 2022, and reporting a previous SARS-CoV-2 infection (positive PCR or antigen test) (yes vs. no) in June/July 2022. For S- and N-IgG MFI ratio titers, individuals were assigned to one of three expression quantiles (<33%, 33-67%, 67+% of all participants); for N-Ab IC50 values, and S- and M/N-T cell IFN-gamma levels, individuals were assigned to one of four expression quantiles (50%, 50->75%, 75+% of all participants). Corresponding MFI ratios/IC50 titers/IFN-gamma levels are listed next to each variable. *p>0.05, ***p>0.005. . CC-BY-NC 4.0 International licenseIt 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 thisthis version posted February 23, 2023. ; https://doi.org/10.1101/2023.02.20.23286166doi: medRxiv preprint