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

Abstract Studies investigating the cumulative incidence of and immune status against SARS‐CoV‐2 infection provide valuable information for shaping public health decision‐making. A cross‐sectional study on 935 participants, conducted in the Valencian Community (VC), measuring anti‐SARS‐CoV‐2‐receptor binding domain‐RBD‐total antibodies and anti‐Nucleocapsid (N)‐IgGs via electrochemiluminescence assays. Quantitation of neutralizing antibodies (NtAb) against ancestral and Omicron BA.1 and BA.2 variants and enumeration of SARS‐CoV‐2‐S specific‐IFNγ‐producing CD4+ and CD8+ T cells was performed in 100 and 137 participants, respectively. The weighted cumulative incidence was 51.9% (95% confidence interval [CI]: 48.7–55.1) and was inversely related to age. Anti‐RBD total antibodies were detected in 97% of participants; vaccinated and SARS‐CoV‐2‐experienced (VAC‐ex; n = 442) presented higher levels (p < 0.001) than vaccinated/naïve (VAC‐n; n = 472) and nonvaccinated/experienced (UNVAC‐ex; n = 63) subjects. Antibody levels correlated inversely with time elapsed since last vaccine dose in VAC‐n (Rho, −0.52; p < 0.001) but not in VAC‐ex (rho −0.02; p = 0.57). Heterologous booster shots resulted in increased anti‐RBD antibody levels compared with homologous schedules in VAC‐n, 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. For Omicron BA.2, the figures were 97%, 84%, and 40%, respectively. SARS‐CoV‐2‐S‐reactive IFN‐γ T cells were detected in 73%, 75%, and 64% of VAC‐ex, VAC‐n and UNVAC‐ex, respectively. Median frequencies for both T‐cell subsets were comparable across groups. In summary, by April 2022, around half of the VC population had been infected with SARS‐CoV‐2 and, due to extensive vaccination, displayed hybrid immunity.


| INTRODUCTION
Spain has been severely affected by the SARS-CoV-2 pandemic, with more than 12.4 million microbiologically confirmed cases as of June 8th, 2022. 1 The Valencian Community (VC) is the fourth most populous Spanish autonomous community with more than five million inhabitants and has reported around 1.5 million SARS-CoV-2 infections and almost 10,000 attributed deaths to date. 1 Importantly, a large percentage of the VC population has received either a primary full vaccination series (93.9%) or a booster vaccination schedule (57.3%). 2 Registries of confirmed COVID-19 cases tend to underestimate the cumulative incidence of SARS-CoV-2 infection, as previously shown in the ENE-COVID study, a nationwide, population-based, seroepidemiological study conducted in Spain during 2020. 3 Some serosurvey studies in the general population and extending into the Omicron wave have been reported. 4

| Study design and setting
The current cross-sectional, region-wide study was conducted in the primary care zones (

| Variables and definitions
Registered, prior SARS-CoV-2 infection was established by consulting the VC microbiology registry (RedMiVa) and taking into account the results of SARS-CoV-2 antibody assays performed, as described below. The vaccination status of participants was obtained from the VC vaccination registry. Participants were grouped into the following categories: vaccinated/experienced (VAC-ex, vaccinated individuals with a record of a positive AIDT-active infection diagnostic testresult and/or serological evidence of past infection), vaccinated/naïve (VAC-n, vaccinated participants with no record or serological evidence of previous SARS-CoV-2 infection), unvaccinated/experienced (UNVAC-ex, unvaccinated population with history or serological evidence of prior SARS-CoV-2 infection) and unvaccinated/naïve (UNVAC-n, unvaccinated population with no history or serological evidence of prior SARS-CoV-2 infection). AIDTs included rapid antigen-detection assays targeting the N protein or commerciallyavailable RT-PCRs.

| Cumulative incidence of SARS-CoV-2 infection
Blood volume was insufficient for analysis in four participants, who were accordingly excluded, leaving 931 individuals for the analyses detailed in continuation. Past SARS-CoV-2 infection was documented in a total of 442 (47.4%) participants (Table 2 and Supporting   Information: Table S3)
In turn, VAC-ex and VAC-n individuals displayed significantly higher Overall, the number of vaccine doses received had a direct impact on the level of anti-RBD total antibodies, irrespective of SARS-CoV-2 infection status ( Figure 1B). Interestingly, among participants who had received a third vaccine dose, heterologous booster shots resulted in increased anti-RBD antibody levels compared with homologous schedules in SARS-CoV-2-naïve, but not SARS-CoV-2-experienced participants, regardless of age and sex ( Figure 1C).  Finally, subtle age-related differences in anti-RBD total antibody levels were noticed in both VAC-ex and VAC-n subgroups (Supporting Information: Figure S1), which were likely related to dissimilarities across age groups regarding the number of vaccine shots received and time elapsed since administration of the last vaccine dose.

| Neutralizing antibodies against the ancestral Wuhan-Hu-1 and Omicron subvariants
A total of 100 randomly selected participants across all age groups were screened for the presence of NtAb against SARS-CoV-2 (sub) variants. The main characteristics of the subjects evaluated are shown in Supporting Information: Table S4.

| DISCUSSION
To date, only a handful of seroepidemiological studies extending into the Omicron wave have been performed in the general population. [4][5][6] Here, we conducted a cross-sectional serosurvey to estimate the prevalence of SARS-CoV-2 antibodies in the general VC population after the Omicron BA.1 variant surge and retrieved prior documented SARS-CoV-2 infection data for the recruited population from the RedMiVa VC registry. Our results can probably be extrapolated to other autonomous communities in Spain, as previously suggested. 3 We estimated an age/sex weighted cumulative incidence of prior In this setting, 97% of participants had detectable anti-RBD total antibodies, whose levels were increased in VAC-ex individuals compared with VAC-n participants and, especially, with UNVAC-ex individuals matched for time of SARS-CoV-2 diagnosis, suggesting that antibody waning may occur more rapidly in these latter individuals. VAC-ex participants displayed higher levels of anti-RBD total antibodies than Vac-n across virtually all age groups. This Taken collectively, this data suggested that hybrid immunity provided stronger and more durable anti-RBD antibody responses than vaccination alone, which concurs with previous observations. 12 vaccination. [27][28][29] Our data supported this notion in that SARS-CoV-