Cumulative incidence of SARS-CoV-2 infection in the general population of the Valencian Community (Spain) after the surge of the Omicron BA.1 variant

Background Studies investigating the cumulative incidence of and immune status against SARS-CoV-2 infection provide valuable information for shaping public health decision- making. Methods The current cross-sectional, population-based study, conducted in April 2022 in the Valencian Community (VC), recruited 935 participants of all ages. Anti-SARS-CoV-2-Receptor Binding Domain-RBD- total antibodies and anti-Nucleocapsid (N)- IgGs were measured by electrochemiluminescence assays. To account for past SARS-CoV-2 infection the VC microbiology registry (RedMiVa) was interrogated. Quantitation of neutralizing antibodies (NtAb) against the ancestral and Omicron BA.1 and BA.2 (sub)variants by an S-pseudotyped neutralization assay and for enumeration of SARS-CoV-2-S specific-IFNgamma;-producing CD4+ and CD8+ T cells by Intracellular Cytokine Staining assay was performed in a subset of participants (n=100 and 137, respectively). Findings The weighted cumulative incidence was 51.9% (95% CI, 48.7-55.1), and was inversely related to age. Anti-RBD total antibodies were detected in 906/931 (97.3%) participants, those vaccinated and SARS-CoV-2-experienced (VAC-ex;=442) displaying higher levels (P<0.001) than vaccinated/naive (VAC-n;(n=472) and non-vaccinated/experienced (UNVAC-ex; n(n=63). Antibody levels correlated inversely with the time elapsed since receipt of last vaccine dose in VAC-n (Rho, -0.52; 95% CI, -0.59 to -0.45; P<0.001) but not in VAC-ex. NtAbs against Omicron BA.1 were detected in 94%, 75% and 50% of VAC-ex, VAC-n and UNVAC-ex groups, respectively, while in 97%, 84% and 40%, against Omicron BA.2. SARS-CoV-2-S-reactive IFNgamma; T cells were detected in 73%, 75%, and 64% for VAC-ex, VAC-n, UNVAC-ex, respectively. Interpretation By April 2022 around half of the VC population had been infected with SARS-CoV-2 and due to extensive vaccination display hybrid immunity. The large percentage of participants with detectable functional antibody and T-cell responses against SARS-CoV-2, which may be cross-reactive to some extent, points towards lower expected severity than in previous waves.


Background 62
Studies investigating the cumulative incidence of and immune status against SARS-63 CoV-2 infection provide valuable information for shaping public health decision-64 making. 65

Methods 66
The current cross-sectional, population-based study, conducted in April 2022 in the 67 Valencian Community (VC), recruited 935 participants of all ages. Anti-SARS-CoV-2-68 Receptor Binding Domain-RBD-total antibodies and anti-Nucleocapsid (N)-IgGs were 69 measured by electrochemiluminescence assays. To account for past SARS-CoV-2 70 infection the VC microbiology registry (RedMiVa) was interrogated. |Quantitation of 71 neutralizing antibodies (NtAb) against the ancestral and Omicron BA.1 and BA.2 72 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted July 19, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 19, 2022. ; https://doi.org/10.1101/2022.07.19.22277747 doi: medRxiv preprint 6 estimating the cumulative incidence of SARS-CoV-2 infection in the general VC 121 population after the surge and spread of the Omicron BA.1 variant. Given that both 122 SARS-CoV-2-Spike (S)-binding functional antibodies and S-reactive T cells elicited 123 following vaccination or natural infection seemingly contribute to protection against 124 severe COVID-19, 7-9 we assessed as a secondary goal of the study the impact of 125 COVID-19 vaccination, SARS-CoV-2 infection and time elapsed since these two events 126 on anti-SARS-CoV-2-S(Spike)-Receptor Binding domain (RBD) total antibody levels 127 in all participants. In this context, we also measured SARS-CoV-2-S Wuhan-Hu-1 128 (G614), Omicron BA.1 and BA.2 neutralizing Ab titers (NtAb) and SARS-CoV-2-S-129 reactive IFN-γ-producing CD4 + and CD8 + T-cell levels in a subsample of randomly 130 selected subjects representative of the full recruited population. 131

Study design and setting 133
The current cross-sectional, region-wide, population-based study was conducted in the 134 primary care zones (PCZ) of the Valencia Health System ) in April 2022, after the 135 Omicron BA.1 variant had emerged and spread extensively within the VC. The sample 136 size was calculated to be 998 participants for a precision of ±1% (between ±3 and ±5% 137 in analyses by age and sex strata), assuming a prevalence of anti-RBD antibodies in the 138 general population of 95% and a confidence level of 90%. The sample was increased by 139 5% (up to 1,043 blood samples) to compensate for possible losses, and was age-and 140 sex-stratified proportionally to the distribution of the VC population, except for 141 oversampling of ≥ 80 age group and reduction of the 35-49 and 50-64 age groups. For 142 operational reasons, 100 PCZ were randomly selected to simplify sample transport 143 logistics. Each PCZ was given a specific date on which to collect between 8 and 17 144 All rights reserved. No reuse allowed without permission.
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Statistical analysis 191
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. shown for the sample and weighted by the proportion of VC population in each age/sex 196 stratum. The analyses were performed using SPSS version 20 0 (SPSS, Chicago, IL, 197 USA) and STATA 17 0 (StataCorp, College Station, Texas, USA). 198

Role of the funding source 199
The founder had no role in the design, analysis and interpretation of data or writing. 200

Cumulative incidence of SARS-CoV-2 infection 218
Blood volume was insufficient for analyses in four participants, who were accordingly 219 excluded, leaving 931 individuals included in the analyses detailed below. 220 Past SARS-CoV-2 infection was documented in a total of 442 (47 4%) participants 221 (Table 2 and Supplementary Table 3 the VC was thus 47 4% (95% CI, 44 1-50 6), with no differences across the three 226 VC provinces, whereas age/sex weighted cumulative incidence was 51 9% (95% CI, 227 48 7-55 1). The rate of past SARS-CoV-2 infection was inversely related to age; 228 evidence of prior SARS-CoV-2 infection was found in more than two-thirds of subjects 229 younger than 34 years old, in contrast to around one-fourth of those over 65 years old. and UNVAC-n (n=21). 239 All rights reserved. No reuse allowed without permission.
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Anti-SARS-CoV-2-RBD total antibodies 240
Overall, 906/931 (97 3%) had detectable anti-RBD total antibodies. Most individuals 241 (23/25) with undetectable levels were UNVAC-n children. As shown in Figure 1  Overall, the number of vaccine doses received had a direct impact on level of anti-RBD 260 total antibodies, irrespective of SARS-CoV-2 infection status ( Figure 1B). However, no 261 correlation was found between the time elapsed since receipt of the last vaccine dose 262 and anti-RBD total antibody levels (rho -0 02; 95% CI, -0 13 to 0 07; P=0 57) in 263 All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. BA.2 were comparable in participants across all groups (Figure 4). 293 Overall, comparable NtAb titers against all (sub)variants tested were seen across 294 participants either boosted or not with an additional vaccine dose (Supplementary Table  295 5). Nevertheless, a significant although weak inverse correlation between NtAb titers 296 against Wuhan-Hu-1 and Omicron BA.1 and time elapsed since the receipt of the last 297 vaccine dose was observed in VAC-n but not VAC-ex individuals (Supplementary 298 Table 6). 299
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  to other autonomous communities in Spain. 3 We estimated an age/sex weighed 331 cumulative incidence of prior SARS-CoV-2 infection of 51 9% (95% CI, 48 7-332 55 1). While this figure was comparable across males and females, age-related 333 differences were noticed, the highest rate occurring in individuals under 34 years old 334 and the lowest in those aged 65 or older. Such differences could be explained by 335 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted July 19, 2022. In this setting, 97% of participants had detectable anti-RBD total antibodies, whose 353 levels were increased in VAC-ex individuals compared to VAC-n participants across 354 virtually all age groups. This occurred irrespective of the time elapsed since diagnosis of 355 SARS-CoV-2 infection, and despite the fact that the VAC-n group had received the last 356 vaccine dose (in most cases a booster dose) more recently than the VAC-ex one. These 357 data can be interpreted as suggesting that hybrid immunity provides stronger functional 358 antibody responses than vaccination alone, as previously reported. [17][18][19] Moreover, such 359 responses may be more durable in the VAC-ex, as evidenced by an inverse correlation 360 All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 19, 2022. ; https://doi.org/10.1101/2022.07.19.22277747 doi: medRxiv preprint between the time elapsed since last vaccine dose and anti-RBD total antibody levels in 361 VAC-n but not VAC-ex participants. VAC-ex individuals also displayed greater anti-362 RBD antibody levels than UNVAC-ex individuals matched by time of SARS-CoV-2 363 diagnosis, suggesting that antibody waning may occur at a faster rate in these latter 364 individuals. 365 Our data on NtAb experiments seemed to support the assumption that hybrid immunity 366 provides a more robust and perhaps more durable functional antibody response against 367 Wuhan-Hu-1 and Omicron BA.1 than is elicited by either vaccination or natural 368 infection alone. A key observation of our study was the differential effect of a booster 369 dose on antibody levels across VAC-ex and VAC-n individuals. In effect, the data 370 indicated that following the vaccine booster, both anti-RBD antibody levels and NtAb 371 titers increased to a much lesser extent in VAC-ex than VAC-n, suggesting that in the 372 general population VAC-n may benefit more than VAC-ex from receiving additional 373 vaccine doses. This could have important implications in the design of vaccination 374 policies in the near future. Regarding the impact of booster vaccine doses on humoral 375 immunity, we showed that heterologous mRNA vaccine boosters elicited higher levels 376 of anti-RBD total antibodies than homologous schedules, as previously reported. 20,21 377 However, a similar analysis could not be performed for NtAb due to the small sample 378 size. 379 Our finding that a large percentage of vaccinated participants had either prior SARS-380 CoV-2 infection or detectable NtAb against Omicron BA.1 (94% and 75% respectively) 381 is compatible with the idea that both Wuhan-based vaccine platforms and breakthrough 382 infections elicit cross-reactive antibodies against SARS-CoV-2 variants of concern 22,23 383 and that Omicron BA.1 breakthrough infection may induce sublineage-specific NtAb in 384 addition to recall of memory B cells established by prior vaccination. 24-26 385 All rights reserved. No reuse allowed without permission.
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