Prior SARS‐CoV‐2 infection and COVID‐19 vaccine effectiveness against outpatient illness during widespread circulation of SARS‐CoV‐2 Omicron variant, US Flu VE network

Abstract Background We estimated combined protection conferred by prior SARS‐CoV‐2 infection and COVID‐19 vaccination against COVID‐19‐associated acute respiratory illness (ARI). Methods During SARS‐CoV‐2 Delta (B.1.617.2) and Omicron (B.1.1.529) variant circulation between October 2021 and April 2022, prospectively enrolled adult patients with outpatient ARI had respiratory and filter paper blood specimens collected for SARS‐CoV‐2 molecular testing and serology. Dried blood spots were tested for immunoglobulin‐G antibodies against SARS‐CoV‐2 nucleocapsid (NP) and spike protein receptor binding domain antigen using a validated multiplex bead assay. Evidence of prior SARS‐CoV‐2 infection also included documented or self‐reported laboratory‐confirmed COVID‐19. We used documented COVID‐19 vaccination status to estimate vaccine effectiveness (VE) by multivariable logistic regression by prior infection status. Results Four hundred fifty‐five (29%) of 1577 participants tested positive for SARS‐CoV‐2 infection at enrollment; 209 (46%) case‐patients and 637 (57%) test‐negative patients were NP seropositive, had documented previous laboratory‐confirmed COVID‐19, or self‐reported prior infection. Among previously uninfected patients, three‐dose VE was 97% (95% confidence interval [CI], 60%–99%) against Delta, but not statistically significant against Omicron. Among previously infected patients, three‐dose VE was 57% (CI, 20%–76%) against Omicron; VE against Delta could not be estimated. Conclusions Three mRNA COVID‐19 vaccine doses provided additional protection against SARS‐CoV‐2 Omicron variant‐associated illness among previously infected participants.

Seroprevalence studies indicate that many SARS-CoV-2 infections go undetected. 16,17 These include asymptomatic infections and those for which testing is not sought or produces false-negative results. Additionally, COVID-19 testing access and preferences vary by COVID-19 immunization status, clinical, and sociodemographic factors, likely contributing to misclassification of prior infection when relying solely on healthcare encounters data. 18,19 Further, the rapid influx of self-administered home testing in early 2022 reduced PCR-based testing at healthcare sites. 20 Together, these factors con-

| Study population
This study was conducted by participating institutions in the US Influenza Vaccine Effectiveness (Flu VE) Network with study sites in seven states (CA, MI, PA, TN, TX, WA, and WI) as previously described. 21 Briefly, active surveillance was conducted at outpatient healthcare facilities or COVID-19 testing sites for patients aged ≥6 months with acute illness with a symptom duration of ≤10 days. Ill individuals were screened using a standard case definition for COVID-19-like illness (CLI) that included at least one of fever, cough, or loss of taste/ smell. 22 Participants completed standardized enrollment questionnaires.
Data collected included participant age, gender, race, ethnicity, date of illness onset, symptoms, self-reported chronic medical condition (heart disease, lung disease, diabetes, cancer, liver/kidney disease, immune suppression, or high blood pressure), the highest level of education, and high-risk exposure. High-risk exposures in the 14 days prior to illness were captured with three questions related to healthcare work and close contact with a person with laboratory-confirmed SARS-CoV-2 or CLI (Supplemental Methods).
Participants were asked about their history of positive SARS-

| Specimen collection and laboratory testing
Participants had an oropharyngeal and nasal swab specimen collected for SARS-CoV-2 molecular testing and finger prick for collection of DBS on filter paper. Participants opting out of finger-stick blood collection were excluded from this analysis. Preparation of DBS is described in Supplemental Methods.
Briefly, research staff collected whole blood by finger stick and absorbed drops on up to five half-inch circles on Whatman 903 filter paper cards, which were dried at room temperature, packed with desiccant, and sent to CDC. DBS specimens were tested for immunoglobulin G (IgG) antibodies against three SARS-CoV-2 recombinant antigens representing the receptor binding domain (RBD) of the SARS-CoV-2 spike-1 protein, nucleocapsid protein (NP), and an RBD-NP hybrid antigen using a validated multiplex bead assay (FlexImmArray™ SARS-CoV-2 Human IgG Antibody Test, Tetracore, Rockville, MD) on a Luminex MAGPIX instrument with LX200 flow analyzer (Luminex Corporation, Austin, TX). Positive results for the presence of IgG antibodies against SARS-CoV-2 NP protein were defined according to the manufacturer's instructions as median fluorescence intensity (MFI) of sample greater than 1.2-fold the MFI of the human IgG calibrator serum for NP antigen. Samples with equivocal ratios (between ≥0.9 and ≤1.2) were repeated; specimens with final ratios < 0.9-fold that of NP antigen calibrator serum were defined as anti-NP IgG seronegative. Anti-NP IgG seropositivity was considered evidence of previous SARS-CoV-2 infection.

| COVID-19 vaccination status
Vaccination receipt was verified in electronic immunization records as previously described. 21 All vaccine doses during the study period were monovalent mRNA products. Participants were considered vaccinated with two doses if they received two doses of either mRNA vaccine with the second administered ≥14 days before illness onset. We required at least a 16-day interval between the first and second doses for Pfizer-BioNTech vaccine and at least a 23-day interval for Moderna vaccine. If illness onset occurred <7 days after a third dose of mRNA vaccine, the participant was considered to have received two doses.
Participants were considered vaccinated with three doses if they received three mRNA vaccine doses with at least a 16-day interval between the second and third doses for Pfizer-BioNTech vaccine and at least a 23-day interval for Moderna vaccine with the most recent dose received ≥7 days before illness onset. 23 Those with no EHRdocumented COVID-19 vaccination before illness onset were considered unvaccinated. Participants who had received one or four doses of mRNA vaccine or any non-mRNA COVID-19 vaccine were excluded ( Figure S1).

| Analyses
We limited all analyses to adults aged ≥18 years for whom a DBS was obtained. Characteristics were compared between those who were SARS-CoV-2-positive at enrollment versus those who tested negative.
We determined the distribution of characteristics for those with To investigate whether COVID-19 vaccination provided additional protection beyond that conferred by prior infection, we compared odds of confirmed/possible prior infection among those with SARS-CoV-2 positive results at enrollment (i.e., cases) to those who tested negative at enrollment (i.e., controls), stratified by vaccination status (unvaccinated, two doses, three doses). Adjusted odds were estimated using logistic regression models including age, sex, race and ethnicity, site, illness onset week, self-reported chronic medical condition, and high-risk SARS-CoV-2 exposure.  with CLI were enrolled and tested prospectively for SARS-CoV-2 infection using molecular assays. Of these, 1883 provided DBS specimens.
Of those, 1577 met the inclusion criteria for this analysis (Table 1).  (Table 3)

| Prior SARS-CoV-2 infection and risk of SARS-CoV-2 at enrollment
The adjusted odds of having SARS-CoV-2 at enrollment tended to be reduced for those with prior infection, regardless of vaccination status (  (Table S1). c Conditions included heart disease, lung disease, diabetes, cancer, liver/kidney disease, immune suppression, or high blood pressure. d High-risk SARS-CoV-2 exposures included healthcare work in close contact with patients, close contact in the 14 days before illness onset with either a person with laboratory-confirmed SARS-CoV-2 or a household member with laboratory-confirmed SARS-CoV-2 or symptoms consistent with COVID-19-like illness. e Unknown for four SARS-CoV-2-negative participants.
T A B L E 2 Characteristics of participants with prior SARS-CoV-2 infection category.

Most persons with SARS-CoV-2 infection generate detectable
anti-SARS-CoV-2 antibodies, with studies reporting seroconversion rates >90%. 29,30 The consistency in antibody response allows for highly sensitive serologically based detection methods for prior infections. Here, we found that EHR determination of prior infection alone was not very informative; supplementing those data with NP serology resulted in a fivefold increase in evidence of prior infection. Although EHR-documented evidence of prior infection is highly specific, it is not sensitive and likely misses a large proportion of those not tested, tested outside the home healthcare system, or self-tested with antigen-based kits. 20  behavior between our comparison groups and minimizes potential bias by vaccination status. Participants who did not provide a DBS specimen were similar to those who did with respect to vaccination status (data not shown). Finally, our analyses are based on the monovalent mRNA COVID-19 vaccine formulations and may not correspond to findings derived from bivalent formulations.
An improved understanding of cross-protection elicited by infection and vaccination is needed to inform future vaccine formulations and vaccination recommendations. The optimal vaccination strategy for previously infected individuals would boost protective immunity from natural infection. If natural immunity fosters cross-protection against emerging variants, formulations that include more crossreactive antigens may be necessary to improve VE. As new variants continue to emerge, ongoing analyses of cross-protection between strains will be important to inform vaccine programs.