Immune and behavioral correlates of protection against symptomatic post-vaccination SARS-CoV-2 infection.

Introduction: We sought to determine pre-infection correlates of protection against SARS-CoV-2 post-vaccine inzfections (PVI) acquired during the first Omicron wave in the United States. Methods: Serum and saliva samples from 176 vaccinated adults were collected from October to December of 2021, immediately before the Omicron wave, and assessed for SARS-CoV-2 Spike-specific IgG and IgA binding antibodies (bAb). Sera were also assessed for bAb using commercial assays, and for neutralization activity against several SARS-CoV-2 variants. PVI duration and severity, as well as risk and precautionary behaviors, were assessed by questionnaires. Results: Serum anti-Spike IgG levels assessed by research assay, neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against PVIs after multivariable regression analysis. Commercial assays did not perform as well as research assay, likely due to their lower dynamic range. Discussion: In the 32 participants that developed PVI, anti-Spike IgG bAbs correlated with lower disease severity and shorter duration of illness.

Understanding "Hybrid Immunity": Comparison and Predictors of Humoral Immune Responses to Severe Acute Respiratory Syndrome Coronavirus 2 Infection (SARS-CoV-2) and Coronavirus Disease 2019 (COVID-19) Vaccines.

BACKGROUND: Comparison of humoral responses in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinees, those with SARS-CoV-2 infection, or combinations of vaccine/ infection ("hybrid immunity") may clarify predictors of vaccine immunogenicity. METHODS: We studied 2660 US Military Health System beneficiaries with a history of SARS-CoV-2 infection-alone (n = 705), vaccination-alone (n = 932), vaccine-after-infection (n = 869), and vaccine-breakthrough-infection (n = 154). Peak anti-spike-immunoglobulin G (IgG) responses through 183 days were compared, with adjustment for vaccine product, demography, and comorbidities. We excluded those with evidence of clinical or subclinical SARS-CoV-2 reinfection from all groups. RESULTS: Multivariable regression results indicated that vaccine-after-infection anti-spike-IgG responses were higher than infection-alone (P < .01), regardless of prior infection severity. An increased time between infection and vaccination was associated with greater post-vaccination IgG response (P < .01). Vaccination-alone elicited a greater IgG response but more rapid waning of IgG (P < .01) compared with infection-alone (P < .01). BNT162b2 and mRNA-1273 vaccine-receipt was associated with greater IgG responses compared with JNJ-78436735 vaccine-receipt (P < .01), regardless of infection history. Those with vaccine-after-infection or vaccine-breakthrough-infection had a more durable anti-spike-IgG response compared to infection-alone (P < .01). CONCLUSIONS: Vaccine-receipt elicited higher anti-spike-IgG responses than infection-alone, although IgG levels waned faster in those vaccinated (compared to infection-alone). Vaccine-after-infection elicits a greater humoral response compared with vaccine or infection alone; and the timing, but not disease severity, of prior infection predicted these post-vaccination IgG responses. While differences between groups were small in magnitude, these results offer insights into vaccine immunogenicity variations that may help inform vaccination timing strategies.

Durability of Antibody Response and Frequency of SARS-CoV-2 Infection 6 Months after COVID-19 Vaccination in Healthcare Workers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies decay but persist 6 months postvaccination; lower levels of neutralizing titers persist against Delta than wild-type virus. Of 227 vaccinated healthcare workers tested, only 2 experienced outpatient symptomatic breakthrough infections, despite 59/227 exhibiting serologic evidence of SARS-CoV-2 infection, defined as presence of nucleocapsid protein antibodies.

Prospective Assessment of Symptoms to Evaluate Asymptomatic SARS-CoV-2 Infections in a Cohort of Health Care Workers.

BACKGROUND: The frequency of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unclear and may be influenced by how symptoms are evaluated. In this study, we sought to determine the frequency of asymptomatic SARS-CoV-2 infections in a prospective cohort of health care workers (HCWs). METHODS: A prospective cohort of HCWs, confirmed negative for SARS-CoV-2 exposure upon enrollment, were evaluated for SARS-CoV-2 infection by monthly analysis of SARS-CoV-2 antibodies as well as referral for polymerase chain reaction testing whenever they exhibited symptoms of coronavirus disease 2019 (COVID-19). Participants completed the standardized and validated FLU-PRO Plus symptom questionnaire scoring viral respiratory disease symptom intensity and frequency at least twice monthly during baseline periods of health and each day they had any symptoms that were different from their baseline. RESULTS: Two hundred sixty-three participants were enrolled between August 25 and December 31, 2020. Through February 28, 2021, 12 participants were diagnosed with SARS-CoV-2 infection. Symptom analysis demonstrated that all 12 had at least mild symptoms of COVID-19, compared with baseline health, near or at time of infection. CONCLUSIONS: These results suggest that asymptomatic SARS-CoV-2 infection in unvaccinated, immunocompetent adults is less common than previously reported. While infectious inoculum doses and patient factors may have played a role in the clinical manifestations of SARS-CoV-2 infections in this cohort, we suspect that the high rate of symptomatic disease was due primarily to participant attentiveness to symptoms and collection of symptoms in a standardized, prospective fashion. These results have implications for studies that estimate SARS-CoV-2 infection prevalence and for public health measures to control the spread of this virus.

Adverse Effects and Antibody Titers in Response to the BNT162b2 mRNA COVID-19 Vaccine in a Prospective Study of Healthcare Workers.

BACKGROUND: The relationship between postvaccination symptoms and strength of antibody responses is unclear. The goal of this study was to determine whether adverse effects caused by vaccination with the Pfizer/BioNTech BNT162b2 vaccine are associated with the magnitude of vaccine-induced antibody levels. METHODS: We conducted a single-center, observational cohort study consisting of generally healthy adult participants that were not severely immunocompromised, had no history of coronavirus disease 2019, and were seronegative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein before vaccination. Severity of vaccine-associated symptoms was obtained through participant-completed questionnaires. Testing for immunoglobulin G antibodies against SARS-CoV-2 spike protein and receptor-binding domain was conducted using microsphere-based multiplex immunoassays performed on serum samples collected at monthly visits. Neutralizing antibody titers were determined by microneutralization assays. RESULTS: Two hundred six participants were evaluated (69.4% female, median age 41.5 years old). We found no correlation between vaccine-associated symptom severity scores and vaccine-induced antibody titers 1 month after vaccination. We also observed that (1) postvaccination symptoms were inversely correlated with age and weight and more common in women, (2) systemic symptoms were more frequent after the second vaccination, (3) high symptom scores after first vaccination were predictive of high symptom scores after second vaccination, and (4) older age was associated with lower titers. CONCLUSIONS: Lack of postvaccination symptoms after receipt of the BNT162b2 vaccine does not equate to lack of vaccine-induced antibodies 1 month after vaccination.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mRNA Vaccine-Breakthrough Infection Phenotype Includes Significant Symptoms, Live Virus Shedding, and Viral Genetic Diversity.

Little is known about severe acute respiratory syndrome coronavirus 2 "vaccine-breakthrough" infections (VBIs). Here we characterize 24 VBIs in predominantly young healthy persons. While none required hospitalization, a proportion endorsed severe symptoms and shed live virus as high as 4.13 × 103 plaque-forming units/mL. Infecting genotypes included both variant-of-concern (VOC) and non-VOC strains.

Durability of SARS-CoV-2-Specific T-Cell Responses at 12 Months Postinfection.

BACKGROUND: Characterizing the longevity and quality of cellular immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enhances understanding of coronavirus disease 2019 (COVID-19) immunity that influences clinical outcomes. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Analysis of the function, durability, and diversity of cellular response long after natural infection, over a range of ages and disease phenotypes, is needed to identify preventative and therapeutic interventions. METHODS: We identified participants in our multisite longitudinal, prospective cohort study 12 months after SARS-CoV-2 infection representing a range of disease severity. We investigated function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry, and compared magnitude of SARS-CoV-2-specific antibodies. RESULTS: SARS-CoV-2-specific antibodies and T cells were detected 12 months postinfection. Severe acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12 months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. CONCLUSIONS: SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12 months postinfection, with higher frequency noted in those who experienced severe disease.

Adverse effects and antibody titers in response to the BNT162b2 mRNA COVID-19 vaccine in a prospective study of healthcare workers.

BACKGROUND: mRNA COVID-19 vaccines are playing a key role in controlling the COVID-19 pandemic. The relationship between post-vaccination symptoms and strength of antibody responses is unclear. OBJECTIVE: To determine whether adverse effects caused by vaccination with the Pfizer/BioNTech BNT162b2 vaccine are associated with the magnitude of vaccine-induced antibody levels. DESIGN: Single center, prospective, observational cohort study. SETTING: Participants worked at Walter Reed National Military Medical Center and were seen monthly at the Naval Medical Research Center Clinical Trials Center. PARTICIPANTS: Generally healthy adults that were not severely immunocompromised, had no history of COVID-19, and were seronegative for SARS-CoV-2 spike protein prior to vaccination. MEASURES: Severity of vaccine-associated symptoms was obtained through participant completed questionnaires. Testing for IgG antibodies against SARS-CoV-2 spike protein and receptor binding domain was conducted using microsphere-based multiplex immunoassays. RESULTS: 206 participants were evaluated (69.4% female, median age 41.5 years old). We found no correlation between vaccine-associated symptom severity scores and vaccine-induced antibody titers one month after vaccination. We also observed that 1) post-vaccination symptoms were inversely correlated with age and weight and more common in women, 2) systemic symptoms were more frequent after the second vaccination, 3) high symptom scores after first vaccination were predictive of high symptom scores after second vaccination, and 4) older age was associated with lower titers. LIMITATIONS: Study only observes antibody responses and consists of healthy participants. CONCLUSIONS: Lack of post-vaccination symptoms following receipt of the BNT162b2 vaccine does not equate to lack of vaccine-induced antibodies one month after vaccination. This study also suggests that it may be possible to design future mRNA vaccines that confer robust antibody responses with lower frequencies of vaccine-associated symptoms. FUNDING: This study was executed by the Infectious Disease Clinical Research Program (IDCRP), a Department of Defense (DoD) program executed by the Uniformed Services University of the Health Sciences (USUHS) through a cooperative agreement by the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF). This project has been funded by the Defense Health Program, U.S. DoD, under award HU00012120067. Project funding for JHP was in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The funding bodies have had no role in the study design or the decision to submit the manuscript for publication.

Antibody Responses to SARS-CoV-2 Following an Outbreak Among Marine Recruits With Asymptomatic or Mild Infection.

We investigated serological responses following a SARS-CoV-2 outbreak in spring 2020 on a US Marine recruit training base. 147 participants that were isolated during an outbreak of respiratory illness were enrolled in this study, with visits approximately 6 and 10 weeks post-outbreak (PO). This cohort is comprised of young healthy adults, ages 18-26, with a high rate of asymptomatic infection or mild symptoms, and therefore differs from previously reported longitudinal studies on humoral responses to SARS-CoV-2, which often focus on more diverse age populations and worse clinical presentation. 80.9% (119/147) of the participants presented with circulating IgG antibodies against SARS-CoV-2 spike (S) receptor-binding domain (RBD) at 6 weeks PO, of whom 97.3% (111/114) remained positive, with significantly decreased levels, at 10 weeks PO. Neutralizing activity was detected in all sera from SARS-CoV-2 IgG positive participants tested (n=38) at 6 and 10 weeks PO, without significant loss between time points. IgG and IgA antibodies against SARS-CoV-2 RBD, S1, S2, and the nucleocapsid (N) protein, as well neutralization activity, were generally comparable between those participants that had asymptomatic infection or mild disease. A multiplex assay including S proteins from SARS-CoV-2 and related zoonotic and human endemic betacoronaviruses revealed a positive correlation for polyclonal cross-reactivity to S after SARS-CoV-2 infection. Overall, young adults that experienced asymptomatic or mild SARS-CoV-2 infection developed comparable humoral responses, with no decrease in neutralizing activity at least up to 10 weeks after infection.

Antigen-based multiplex strategies to discriminate SARS-CoV-2 natural and vaccine induced immunity from seasonal human coronavirus humoral responses.

Sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 sero-surveillance. With the rollout of SARS-CoV-2 vaccines, such assays must be able to distinguish vaccine from natural immunity to SARS-CoV-2 and related human coronaviruses. Here, we developed and implemented multiplex microsphere-based immunoassay strategies for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2 and the endemic seasonal human coronaviruses (HCoV), enabling high throughout measurement of pre-existing cross-reactive antibodies. We varied SARS-CoV-2 antigen compositions within the multiplex assay, allowing direct comparisons of the effects of spike protein, receptor-binding domain protein (RBD) and nucleocapsid protein (NP) based SARS-CoV-2 antibody detection. Multiplex immunoassay performance characteristics are antigen-dependent, and sensitivities and specificities range 92-99% and 94-100%, respectively, for human subject samples collected as early as 7-10 days from symptom onset. SARS-CoV-2 spike and RBD had a strong correlative relationship for the detection of IgG. Correlation between detectable IgG reactive with spike and NP also had strong relationship, however, several PCR-positive and spike IgG-positive serum samples were NP IgG-negative. This spike and NP multiplex immunoassay has the potential to be useful for differentiation between vaccination and natural infection induced antibody responses. We also assessed the induction of de novo SARS-CoV-2 IgG cross reactions with SARS-CoV and MERS-CoV spike proteins. Furthermore, multiplex immunoassays that incorporate spike proteins of SARS-CoV-2 and HCoVs will permit investigations into the influence of HCoV antibodies on COVID-19 clinical outcomes and SARS-CoV-2 antibody durability.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and controls for pre-existing seasonal human coronavirus antibody cross-reactivity.

With growing concern of persistent or multiple waves of SARS-CoV-2 in the United States, sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we describe the development and application of a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies, utilizing serum samples from non-human primate SARS-CoV-2 infection models, an archived human sera bank and subjects enrolled at five U.S. military hospitals. The MMIA incorporates prefusion stabilized spike glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human coronaviruses HCoV-HKU1 and HCoV-OC43, into a multiplexing system that enables simultaneous measurement of off-target pre-existing cross-reactive antibodies. We report the sensitivity and specificity performances for this assay strategy at 98% sensitivity and 100% specificity for subject samples collected as early as 10 days after the onset of symptoms. In archival sera collected prior to 2019 and serum samples from subjects PCR negative for SARS-CoV-2, we detected seroprevalence of 72% and 98% for HCoV-HKU1 and HCoV-0C43, respectively. Requiring only 1.25 µL of sera, this approach permitted the simultaneous identification of SARS-CoV-2 seroconversion and polyclonal SARS-CoV-2 IgG antibody responses to SARS-CoV-1 and MERS-CoV, further demonstrating the presence of conserved epitopes in the spike glycoprotein of zoonotic betacoronaviruses. Application of this serology assay in observational studies with serum samples collected from subjects before and after SARS-CoV-2 infection will permit an investigation of the influences of HCoV-induced antibodies on COVID-19 clinical outcomes.