Long-Term Exposure to Air Pollution and COVID-19 Vaccine Antibody Response in a General Population Cohort (COVICAT Study, Catalonia).

BACKGROUND: Ambient air pollution has been associated with COVID-19 disease severity and antibody response induced by infection. OBJECTIVES: We examined the association between long-term exposure to air pollution and vaccine-induced antibody response. METHODS: This study was nested in an ongoing population-based cohort, COVICAT, the GCAT-Genomes for Life cohort, in Catalonia, Spain, with multiple follow-ups. We drew blood samples in 2021 from 1,090 participants of 2,404 who provided samples in 2020, and we included 927 participants in this analysis. We measured immunoglobulin M (IgM), IgG, and IgA antibodies against five viral-target antigens, including receptor-binding domain (RBD), spike-protein (S), and segment spike-protein (S2) triggered by vaccines available in Spain. We estimated prepandemic (2018-2019) exposure to fine particulate matter [PM ≤2.5µm in aerodynamic diameter (PM2.5)], nitrogen dioxide (NO2), black carbon (BC), and ozone (O3) using Effects of Low-Level Air Pollution: A Study in Europe (ELAPSE) models. We adjusted estimates for individual- and area-level covariates, time since vaccination, and vaccine doses and type and stratified by infection status. We used generalized additive models to explore the relationship between air pollution and antibodies according to days since vaccination. RESULTS: Among vaccinated persons not infected by SARS-CoV-2 (n=632), higher prepandemic air pollution levels were associated with a lower vaccine antibody response for IgM (1 month post vaccination) and IgG. Percentage change in geometric mean IgG levels per interquartile range of PM2.5 (1.7 µg/m3) were -8.1 (95% CI: -15.9, 0.4) for RBD, -9.9 (-16.2, -3.1) for S, and -8.4 (-13.5, -3.0) for S2. We observed a similar pattern for NO2 and BC and an inverse pattern for O3. Differences in IgG levels by air pollution levels persisted with time since vaccination. We did not observe an association of air pollution with vaccine antibody response among participants with prior infection (n=295). DISCUSSION: Exposure to air pollution was associated with lower COVID-19 vaccine antibody response. The implications of this association on the risk of breakthrough infections require further investigation. https://doi.org/10.1289/EHP11989.

RBD-Based ELISA and Luminex Predict Anti-SARS-CoV-2 Surrogate-Neutralizing Activity in Two Longitudinal Cohorts of German and Spanish Health Care Workers.

The ability of antibodies to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important correlate of protection. For routine evaluation of protection, however, a simple and cost-efficient anti-SARS-CoV-2 serological assay predictive of serum neutralizing activity is needed. We analyzed clinical epidemiological data and blood samples from two cohorts of health care workers in Barcelona and Munich to compare several immunological readouts for evaluating antibody levels that could be surrogates of neutralizing activity. We measured IgG levels against SARS-CoV-2 spike protein (S), its S2 subunit, the S1 receptor binding domain (RBD), and the full length and C terminus of nucleocapsid (N) protein by Luminex, and against RBD by enzyme-linked immunosorbent assay (ELISA), and assessed those as predictors of plasma surrogate-neutralizing activity measured by a flow cytometry assay. In addition, we determined the clinical and demographic factors affecting plasma surrogate-neutralizing capacity. Both cohorts showed a high positive correlation between IgG levels to S antigen, especially to RBD, and the levels of plasma surrogate-neutralizing activity, suggesting RBD IgG as a good correlate of plasma neutralizing activity. Symptomatic infection, with symptoms such as loss of taste, dyspnea, rigors, fever and fatigue, was positively associated with anti-RBD IgG positivity by ELISA and Luminex, and with plasma surrogate-neutralizing activity. Our serological assays allow for the prediction of serum neutralization activity without the cost, hazards, time, and expertise needed for surrogate or conventional neutralization assays. Once a cutoff is established, these relatively simple high-throughput antibody assays will provide a fast and cost-effective method of assessing levels of protection from SARS-CoV-2 infection. IMPORTANCE Neutralizing antibody titers are the best correlate of protection against SARS-CoV-2. However, current tests to measure plasma or serum neutralizing activity do not allow high-throughput screening at the population level. Serological tests could be an alternative if they are proved to be good predictors of plasma neutralizing activity. In this study, we analyzed the SARS-CoV-2 serological profiles of two cohorts of health care workers by applying Luminex and ELISA in-house serological assays. Correlations of both serological tests were assessed between them and with a flow cytometry assay to determine plasma surrogate-neutralizing activity. Both assays showed a high positive correlation between IgG levels to S antigens, especially RBD, and the levels of plasma surrogate-neutralizing activity. This result suggests IgG to RBD as a good correlate of plasma surrogate-neutralizing activity and indicates that serology of IgG to RBD could be used to assess levels of protection from SARS-CoV-2 infection.

Maternal and neonatal immune response to SARS-CoV-2, IgG transplacental transfer and cytokine profile.

SARS-CoV-2 infected pregnant women are at increased risk of severe COVID-19 than non-pregnant women and have a higher risk of adverse pregnancy outcomes like intrauterine/fetal distress and preterm birth. However, little is known about the impact of SARS-CoV-2 infection on maternal and neonatal immunological profiles. In this study, we investigated the inflammatory and humoral responses to SARS-CoV-2 in maternal and cord blood paired samples. Thirty-six pregnant women were recruited at delivery at Hospital Sant Joan de Déu, Barcelona, Spain, between April-August 2020, before having COVID-19 available vaccines. Maternal and pregnancy variables, as well as perinatal outcomes, were recorded in questionnaires. Nasopharyngeal swabs and maternal and cord blood samples were collected for SARS-CoV-2 detection by rRT-PCR and serology, respectively. We measured IgM, IgG and IgA levels to 6 SARS-CoV-2 antigens (spike [S], S1, S2, receptor-binding domain [RBD], nucleocapsid [N] full-length and C-terminus), IgG to N from 4 human coronaviruses (OC43, HKU1, 229E and NL63), and the concentrations of 30 cytokines, chemokines and growth factors by Luminex. Mothers were classified as infected or non-infected based on the rRT-PCR and serology results. Sixty-four % of pregnant women were infected with SARS-CoV-2 (positive by rRT-PCR during the third trimester and/or serology just after delivery). None of the newborns tested positive for rRT-PCR. SARS-CoV-2 infected mothers had increased levels of virus-specific antibodies and several cytokines. Those with symptoms had higher cytokine levels. IFN-α was increased in cord blood from infected mothers, and in cord blood of symptomatic mothers, EGF, FGF, IL-17 and IL-15 were increased, whereas RANTES was decreased. Maternal IgG and cytokine levels showed positive correlations with their counterparts in cord blood. rRT-PCR positive mothers showed lower transfer of SARS-CoV-2-specific IgGs, with a stronger effect when infection was closer to delivery. SARS-CoV-2 infected mothers carrying a male fetus had higher antibody levels and higher EGF, IL-15 and IL-7 concentrations. Our results show that SARS-CoV-2 infection during the third trimester of pregnancy induces a robust antibody and cytokine response at delivery and causes a significant reduction of the SARS-CoV-2-specific IgGs transplacental transfer, with a stronger negative effect when the infection is closer to delivery.

Sustained seropositivity up to 20.5 months after COVID-19.

This study evaluated the persistence of IgM, IgA, and IgG to SARS-CoV-2 spike and nucleocapsid antigens up to 616 days since the onset of symptoms in a longitudinal cohort of 247 primary health care workers from Barcelona, Spain, followed up since the start of the pandemic. The study also assesses factors affecting antibody levels, including comorbidities and the responses to variants of concern as well as the frequency of reinfections. Despite a gradual and significant decline in antibody levels with time, seropositivity to five SARS-CoV-2 antigens combined was always higher than 90% over the whole study period. In a subset of 23 participants who had not yet been vaccinated by November 2021, seropositivity remained at 95.65% (47.83% IgM, 95.65% IgA, 95.65% IgG). IgG seropositivity against Alpha and Delta predominant variants was comparable to that against the Wuhan variant, while it was lower for Gamma and Beta (minority) variants and for IgA and IgM. Antibody levels at the time point closest to infection were associated with age, smoking, obesity, hospitalization, fever, anosmia/hypogeusia, chest pain, and hypertension in multivariable regression models. Up to 1 year later, just before the massive roll out of vaccination, antibody levels were associated with age, occupation, hospitalization, duration of symptoms, anosmia/hypogeusia, fever, and headache. In addition, tachycardia and cutaneous symptoms associated with slower antibody decay, and oxygen supply with faster antibody decay. Eight reinfections (3.23%) were detected in low responders, which is consistent with a sustained protective role for anti-spike naturally acquired antibodies. Stable persistence of IgG and IgA responses and cross-recognition of the predominant variants circulating in the 2020-2021 period indicate long-lasting and largely variant-transcending humoral immunity in the initial 20.5 months of the pandemic, in the absence of vaccination.

Maternal and neonatal immune response to SARS-CoV-2, IgG transplacental transfer and cytokine profile

SARS-CoV-2 infected pregnant women are at increased risk of severe COVID-19 than non-pregnant women and have a higher risk of adverse pregnancy outcomes like intrauterine/fetal distress and preterm birth. However, little is known about the impact of SARS-CoV-2 infection on maternal and neonatal immunological profiles. In this study, we investigated the inflammatory and humoral responses to SARS-CoV-2 in maternal and cord blood paired samples. Thirty-six pregnant women were recruited at delivery at Hospital Sant Joan de Déu, Barcelona, Spain, between April-August 2020, before having COVID-19 available vaccines. Maternal and pregnancy variables, as well as perinatal outcomes, were recorded in questionnaires. Nasopharyngeal swabs and maternal and cord blood samples were collected for SARS-CoV-2 detection by rRT-PCR and serology, respectively. We measured IgM, IgG and IgA levels to 6 SARS-CoV-2 antigens (spike [S], S1, S2, receptor-binding domain [RBD], nucleocapsid [N] full-length and C-terminus), IgG to N from 4 human coronaviruses (OC43, HKU1, 229E and NL63), and the concentrations of 30 cytokines, chemokines and growth factors by Luminex. Mothers were classified as infected or non-infected based on the rRT-PCR and serology results. Sixty-four % of pregnant women were infected with SARS-CoV-2 (positive by rRT-PCR during the third trimester and/or serology just after delivery). None of the newborns tested positive for rRT-PCR. SARS-CoV-2 infected mothers had increased levels of virus-specific antibodies and several cytokines. Those with symptoms had higher cytokine levels. IFN-α was increased in cord blood from infected mothers, and in cord blood of symptomatic mothers, EGF, FGF, IL-17 and IL-15 were increased, whereas RANTES was decreased. Maternal IgG and cytokine levels showed positive correlations with their counterparts in cord blood. rRT-PCR positive mothers showed lower transfer of SARS-CoV-2-specific IgGs, with a stronger effect when infection was closer to delivery. SARS-CoV-2 infected mothers carrying a male fetus had higher antibody levels and higher EGF, IL-15 and IL-7 concentrations. Our results show that SARS-CoV-2 infection during the third trimester of pregnancy induces a robust antibody and cytokine response at delivery and causes a significant reduction of the SARS-CoV-2-specific IgGs transplacental transfer, with a stronger negative effect when the infection is closer to delivery.

SARS-CoV-2 Seroprevalence Study in Pediatric Patients and Health Care Workers Using Multiplex Antibody Immunoassays.

SARS-CoV-2 infection has become a global health problem specially exacerbated with the continuous appearance of new variants. Healthcare workers (HCW) have been one of the most affected sectors. Children have also been affected, and although infection generally presents as a mild disease, some have developed the Pediatric Inflammatory Multisystem Syndrome Temporally Associated with SARS-CoV-2 (PIMS-TS). We recruited 190 adults (HCW and cohabitants, April to June 2020) and 57 children (April 2020 to September 2021), of whom 12 developed PIMS-TS, in a hospital-based study in Spain. Using an in-house Luminex assay previously validated, antibody levels were measured against different spike and nucleocapsid SARS-CoV-2 proteins, including the receptor-binding domain (RBD) of the Alpha, Beta, Gamma, and Delta variants of concern (VoC). Seropositivity rates obtained from children and adults, respectively, were: 49.1% and 11% for IgG, 45.6% and 5.8% for IgA, and 35.1% and 7.3% for IgM. Higher antibody levels were detected in children who developed PIMS-TS compared to those who did not. Using the COVID-19 IgM/IgA ELISA (Vircell, S.L.) kit, widely implemented in Spanish hospitals, a high number of false positives and lower seroprevalences compared with the Luminex estimates were found, indicating a significantly lower specificity and sensitivity. Comparison of antibody levels against RBD-Wuhan versus RBD-VoCs indicated that the strongest positive correlations for all three isotypes were with RBD-Alpha, while the lowest correlations were with RBD-Delta for IgG, RBD-Gamma for IgM, and RBD-Beta for IgA. This study highlights the differences in antibody levels between groups with different demographic and clinical characteristics, as well as reporting the IgG, IgM, and IgA response to RBD VoC circulating at the study period.

SARS-CoV-2 infection, vaccination, and antibody response trajectories in adults: a cohort study in Catalonia.

BACKGROUND: Heterogeneity of the population in relation to infection, COVID-19 vaccination, and host characteristics is likely reflected in the underlying SARS-CoV-2 antibody responses. METHODS: We measured IgM, IgA, and IgG levels against SARS-CoV-2 spike and nucleocapsid antigens in 1076 adults of a cohort study in Catalonia between June and November 2020 and a second time between May and July 2021. Questionnaire data and electronic health records on vaccination and COVID-19 testing were available in both periods. Data on several lifestyle, health-related, and sociodemographic characteristics were also available. RESULTS: Antibody seroreversion occurred in 35.8% of the 64 participants non-vaccinated and infected almost a year ago and was related to asymptomatic infection, age above 60 years, and smoking. Moreover, the analysis on kinetics revealed that among all responses, IgG RBD, IgA RBD, and IgG S2 decreased less within 1 year after infection. Among vaccinated, 2.1% did not present antibodies at the time of testing and approximately 1% had breakthrough infections post-vaccination. In the post-vaccination era, IgM responses and those against nucleoprotein were much less prevalent. In previously infected individuals, vaccination boosted the immune response and there was a slight but statistically significant increase in responses after a 2nd compared to the 1st dose. Infected vaccinated participants had superior antibody levels across time compared to naïve-vaccinated people. mRNA vaccines and, particularly the Spikevax, induced higher antibodies after 1st and 2nd doses compared to Vaxzevria or Janssen COVID-19 vaccines. In multivariable regression analyses, antibody responses after vaccination were predicted by the type of vaccine, infection age, sex, smoking, and mental and cardiovascular diseases. CONCLUSIONS: Our data support that infected people would benefit from vaccination. Results also indicate that hybrid immunity results in superior antibody responses and infection-naïve people would need a booster dose earlier than previously infected people. Mental diseases are associated with less efficient responses to vaccination.

Eleven-month longitudinal study of antibodies in SARS-CoV-2 exposed and naïve primary health care workers upon COVID-19 vaccination.

We evaluated the kinetics of antibody responses to Two years into the COVID-19 pandemic and 1 year after the start of vaccination rollout, the world faced a peak of cases associated with the highly contagious Omicron variant of concern (VoC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) and nucleocapsid (N) antigens over five cross-sectional visits (January-November 2021), and the determinants of pre-booster immunoglobulin levels, in a prospective cohort of vaccinated primary health care workers in Catalonia, Spain. Antibodies against S antigens after a full primary vaccination course, mostly with BNT162b2, decreased steadily over time and were higher in pre-exposed (n = 247) than naïve (n = 200) individuals, but seropositivity was maintained at 100% (100% IgG, 95.5% IgA, 30.6% IgM) up to 319 days after the first dose. Antibody binding to variants of concern was highly maintained for IgG compared to wild type but significantly reduced for IgA and IgM, particularly for Beta and Gamma. Factors significantly associated with longer-term antibodies included age, sex, occupation, smoking, adverse reaction to vaccination, levels of pre-vaccination SARS-CoV-2 antibodies, interval between disease onset and vaccination, hospitalization, oxygen supply, post COVID and symptomatology. Earlier morning vaccination hours were associated with higher IgG responses in pre-exposed participants. Symptomatic breakthroughs occurred in 9/447 (2.01%) individuals, all among naïve (9/200, 4.5%) and generally boosted antibody responses. Additionally, an increase in IgA and/or IgM seropositivity to variants, and N seroconversion at later time points (6.54%), indicated asymptomatic breakthrough infections, even among pre-exposed. Seropositivity remained highly stable over almost a year after vaccination. However, gradually waning of anti-S IgGs that correlate with neutralizing activity, coupled to evidence of an increase in breakthrough infections during the Delta and Omicron predominance, provides a rationale for booster immunization.

Determinants of early antibody responses to COVID-19 mRNA vaccines in a cohort of exposed and naïve healthcare workers.

BACKGROUND: Two doses of mRNA vaccination have shown >94% efficacy at preventing COVID-19 mostly in naïve adults, but it is not clear if the second dose is needed to maximize effectiveness in those previously exposed to SARS-CoV-2 and what other factors affect responsiveness. METHODS: We measured IgA, IgG and IgM levels against SARS-CoV-2 spike (S) and nucleocapsid (N) antigens from the wild-type and S from the Alpha, Beta and Gamma variants of concern, after BNT162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna) vaccination in a cohort of health care workers (N=578). Neutralizing capacity and antibody avidity were evaluated. Data were analyzed in relation to COVID-19 history, comorbidities, vaccine doses, brand and adverse events. FINDINGS: Vaccination induced robust IgA and IgG levels against all S antigens. Neutralization capacity and S IgA and IgG levels were higher in mRNA-1273 vaccinees, previously SARS-CoV-2 exposed, particularly if symptomatic, and in those experiencing systemic adverse effects (p<0·05). A second dose in pre-exposed did not increase antibody levels. Smoking and comorbidities were associated with 43% (95% CI, 19-59) and 45% (95% CI, 63-18) lower neutralization, respectively, and 35% (95% CI, 3-57%) and 55% (95% CI, 33-70%) lower antibody levels, respectively. Among fully vaccinated, 6·3% breakthroughs were detected up to 189 days post-vaccination. Among pre-exposed non-vaccinated, 90% were IgG seropositive more than 300 days post-infection. INTERPRETATION: Our data support administering a single-dose in pre-exposed healthy individuals as primary vaccination. However, heterogeneity of responses suggests that personalized recommendations may be necessary depending on COVID-19 history and life-style. Higher mRNA-1273 immunogenicity would be beneficial for those expected to respond worse to vaccination and in face of variants that escape immunity such as Omicron. Persistence of antibody levels in pre-exposed unvaccinated indicates maintenance of immunity up to one year. FUNDING: This work was supported by Institut de Salut Global de Barcelona (ISGlobal) internal funds, in-kind contributions from Hospital Clínic de Barcelona, the Fundació Privada Daniel Bravo Andreu, and European Institute of Innovation and Technology (EIT) Health (grant number 20877), supported by the European Institute of Innovation and Technology, a body of the European Union receiving support from the H2020 Research and Innovation Programme. We acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the "Centro de Excelencia Severo Ochoa 2019-2023" Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. L. I. work was supported by PID2019-110810RB-I00 grant from the Spanish Ministry of Science & Innovation. Development of SARS-CoV-2 reagents was partially supported by the National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Surveillance (contract number HHSN272201400008C). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

Multiplex Antibody Analysis of IgM, IgA and IgG to SARS-CoV-2 in Saliva and Serum From Infected Children and Their Close Contacts.

COVID-19 affects children to a lesser extent than adults but they can still get infected and transmit SARS-CoV-2 to their contacts. Field deployable non-invasive sensitive diagnostic techniques are needed to evaluate the infectivity dynamics of SARS-CoV-2 in pediatric populations and guide public health interventions, particularly if this population is not fully vaccinated. We evaluated the utility of high-throughput Luminex assays to quantify saliva IgM, IgA and IgG antibodies against five SARS-CoV-2 spike (S) and nucleocapsid (N) antigens in a contacts and infectivity longitudinal study in 122 individuals (52 children and 70 adults). We compared saliva versus serum/plasma samples in infected children and adults diagnosed by weekly RT-PCR over 35 days (n=62), and those who consistently tested negative over the same follow up period (n=60), in the Summer of 2020 in Barcelona, Spain. Saliva antibody levels in SARS-CoV-2 RT-PCR positive individuals were significantly higher than in negative individuals and correlated with those measured in sera/plasmas. Asymptomatic infected individuals had higher levels of anti-S IgG than symptomatic individuals, suggesting a protective anti-disease role for antibodies. Higher anti-S IgG and IgM levels in serum/plasma and saliva, respectively, in infected children compared to infected adults could also be related to stronger clinical immunity in them. Among infected children, males had higher levels of saliva IgG to N and RBD than females. Despite overall correlation, individual clustering analysis suggested that responses that may not be detected in blood could be patent in saliva, and vice versa. In conclusion, measurement of SARS-CoV-2-specific saliva antibodies should be considered as a complementary non-invasive assay to serum/plasma to determine COVID-19 prevalence and transmission in pediatric populations before and after vaccination campaigns.

Antibody conversion rates to SARS-CoV-2 in saliva from children attending summer schools in Barcelona, Spain.

BACKGROUND: Surveillance tools to estimate viral transmission dynamics in young populations are essential to guide recommendations for school opening and management during viral epidemics. Ideally, sensitive techniques are required to detect low viral load exposures among asymptomatic children. We aimed to estimate SARS-CoV-2 infection rates in children and adult populations in a school-like environment during the initial COVID-19 pandemic waves using an antibody-based field-deployable and non-invasive approach. METHODS: Saliva antibody conversion defined as ≥ 4-fold increase in IgM, IgA, and/or IgG levels to five SARS-CoV-2 antigens including spike and nucleocapsid constructs was evaluated in 1509 children and 396 adults by high-throughput Luminex assays in samples collected weekly in 22 summer schools and 2 pre-schools in 27 venues in Barcelona, Spain, from June 29th to July 31st, 2020. RESULTS: Saliva antibody conversion between two visits over a 5-week period was 3.22% (49/1518) or 2.36% if accounting for potentially cross-reactive antibodies, six times higher than the cumulative infection rate (0.53%) assessed by weekly saliva RT-PCR screening. IgG conversion was higher in adults (2.94%, 11/374) than children (1.31%, 15/1144) (p=0.035), IgG and IgA levels moderately increased with age, and antibodies were higher in females. Most antibody converters increased both IgG and IgA antibodies but some augmented either IgG or IgA, with a faster decay over time for IgA than IgG. Nucleocapsid rather than spike was the main antigen target. Anti-spike antibodies were significantly higher in individuals not reporting symptoms than symptomatic individuals, suggesting a protective role against COVID-19. CONCLUSION: Saliva antibody profiling including three isotypes and multiplexing antigens is a useful and user-friendlier tool for screening pediatric populations to detect low viral load exposures among children, particularly while they are not vaccinated and vulnerable to highly contagious variants, and to recommend public health policies during pandemics.

Ambient Air Pollution in Relation to SARS-CoV-2 Infection, Antibody Response, and COVID-19 Disease: A Cohort Study in Catalonia, Spain (COVICAT Study).

BACKGROUND: Emerging evidence links ambient air pollution with coronavirus 2019 (COVID-19) disease, an association that is methodologically challenging to investigate. OBJECTIVES: We examined the association between long-term exposure to air pollution with SARS-CoV-2 infection measured through antibody response, level of antibody response among those infected, and COVID-19 disease. METHODS: We contacted 9,605 adult participants from a population-based cohort study in Catalonia between June and November 2020; most participants were between 40 and 65 years of age. We drew blood samples from 4,103 participants and measured immunoglobulin M (IgM), IgA, and IgG antibodies against five viral target antigens to establish infection to the virus and levels of antibody response among those infected. We defined COVID-19 disease using self-reported hospital admission, prior positive diagnostic test, or more than three self-reported COVID-19 symptoms after contact with a COVID-19 case. We estimated prepandemic (2018-2019) exposure to fine particulate matter [PM with an aerodynamic diameter of ≤2.5µm (PM2.5)], nitrogen dioxide (NO2), black carbon (BC), and ozone (O3) at the residential address using hybrid land-use regression models. We calculated log-binomial risk ratios (RRs), adjusting for individual- and area-level covariates. RESULTS: Among those tested for SARS-CoV-2 antibodies, 743 (18.1%) were seropositive. Air pollution levels were not statistically significantly associated with SARS-CoV-2 infection: Adjusted RRs per interquartile range were 1.07 (95% CI: 0.97, 1.18) for NO2, 1.04 (95% CI: 0.94, 1.14) for PM2.5, 1.00 (95% CI: 0.92, 1.09) for BC, and 0.97 (95% CI: 0.89, 1.06) for O3. Among infected participants, exposure to NO2 and PM2.5 were positively associated with IgG levels for all viral target antigens. Among all participants, 481 (5.0%) had COVID-19 disease. Air pollution levels were associated with COVID-19 disease: adjusted RRs=1.14 (95% CI: 1.00, 1.29) for NO2 and 1.17 (95% CI: 1.03, 1.32) for PM2.5. Exposure to O3 was associated with a slightly decreased risk (RR=0.92; 95% CI: 0.83, 1.03). Associations of air pollution with COVID-19 disease were more pronounced for severe COVID-19, with RRs=1.26 (95% CI: 0.89, 1.79) for NO2 and 1.51 (95% CI: 1.06, 2.16) for PM2.5. DISCUSSION: Exposure to air pollution was associated with a higher risk of COVID-19 disease and level of antibody response among infected but not with SARS-CoV-2 infection. https://doi.org/10.1289/EHP9726.

Infection induced SARS-CoV-2 seroprevalence and heterogeneity of antibody responses in a general population cohort study in Catalonia Spain.

Sparse data exist on the complex natural immunity to SARS-CoV-2 at the population level. We applied a well-validated multiplex serology test in 5000 participants of a general population study in Catalonia in blood samples collected from end June to mid November 2020. Based on responses to fifteen isotype-antigen combinations, we detected a seroprevalence of 18.1% in adults (n = 4740), and modeled extrapolation to the general population of Catalonia indicated a 15.3% seroprevalence. Antibodies persisted up to 9 months after infection. Immune profiling of infected individuals revealed that with increasing severity of infection (asymptomatic, 1-3 symptoms, ≥ 4 symptoms, admitted to hospital/ICU), seroresponses were more robust and rich with a shift towards IgG over IgA and anti-spike over anti-nucleocapsid responses. Among seropositive participants, lower antibody levels were observed for those ≥ 60 years vs < 60 years old and smokers vs non-smokers. Overweight/obese participants vs normal weight had higher antibody levels. Adolescents (13-15 years old) (n = 260) showed a seroprevalence of 11.5%, were less likely to be tested seropositive compared to their parents and had dominant anti-spike rather than anti-nucleocapsid IgG responses. Our study provides an unbiased estimate of SARS-CoV-2 seroprevalence in Catalonia and new evidence on the durability and heterogeneity of post-infection immunity.

Spike-based COVID-19 immunization increases antibodies to nucleocapsid antigen.

Antibodies to the nucleocapsid (N) antigen are suggested to be used to monitor infections after COVID-19 vaccination, as first generation subunit vaccines are based on the spike (S) protein. We used multiplex immunoassays to simultaneously measure antibody responses to different fragments of the SARS-CoV-2 S and N antigens for evaluating the immunogenicity of the mRNA-1273 (Spykevax) and the BNT162b2 (Comirnaty) vaccines in 445 health care workers. We report a >4-fold increase post-vaccination of IgG levels to the full length (N FL) and C-terminus of N (N CT) in 5.2% and 18.0% of individuals, respectively, and of IgA in 3.6% (N FL) and 9.0% (N CT) of them. The increase in IgG levels and avidity was more pronounced after Spykevax than Comirnaty vaccination (36.2% vs 13.1% for N CT, and 10.6% vs 3.7% for N FL). Data suggest the induction of cross-reactive antibodies against the N CT region after administering these S-based vaccines, and this should be taken into account when using N seropositivity to detect breakthroughs.

Agreement between commercially available ELISA and in-house Luminex SARS-CoV-2 antibody immunoassays.

Serological diagnostic of the severe respiratory distress syndrome coronavirus 2 (SARS-CoV-2) is a valuable tool for the determination of immunity and surveillance of exposure to the virus. In the context of an ongoing pandemic, it is essential to externally validate widely used tests to assure correct diagnostics and epidemiological estimations. We evaluated the performance of the COVID-19 ELISA IgG and the COVID-19 ELISA IgM/A (Vircell, S.L.) against a highly specific and sensitive in-house Luminex immunoassay in a set of samples from pregnant women and cord blood. The agreement between both assays was moderate to high for IgG but low for IgM/A. Considering seropositivity by either IgG and/or IgM/A, the technical performance of the ELISA was highly imbalanced, with 96% sensitivity at the expense of 22% specificity. As for the clinical performance, the negative predictive value reached 87% while the positive predictive value was 51%. Our results stress the need for highly specific and sensitive assays and external validation of diagnostic tests with different sets of samples to avoid the clinical, epidemiological and personal disturbances derived from serological misdiagnosis.

Seven-month kinetics of SARS-CoV-2 antibodies and role of pre-existing antibodies to human coronaviruses.

Unraveling the long-term kinetics of antibodies to SARS-CoV-2 and the individual characteristics influencing it, including the impact of pre-existing antibodies to human coronaviruses causing common cold (HCoVs), is essential to understand protective immunity to COVID-19 and devise effective surveillance strategies. IgM, IgA and IgG levels against six SARS-CoV-2 antigens and the nucleocapsid antigen of the four HCoV (229E, NL63, OC43 and HKU1) were quantified by Luminex, and antibody neutralization capacity was assessed by flow cytometry, in a cohort of health care workers followed up to 7 months (N = 578). Seroprevalence increases over time from 13.5% (month 0) and 15.6% (month 1) to 16.4% (month 6). Levels of antibodies, including those with neutralizing capacity, are stable over time, except IgG to nucleocapsid antigen and IgM levels that wane. After the peak response, anti-spike antibody levels increase from ~150 days post-symptom onset in all individuals (73% for IgG), in the absence of any evidence of re-exposure. IgG and IgA to HCoV are significantly higher in asymptomatic than symptomatic seropositive individuals. Thus, pre-existing cross-reactive HCoVs antibodies could have a protective effect against SARS-CoV-2 infection and COVID-19 disease.

Immunogenicity and crossreactivity of antibodies to the nucleocapsid protein of SARS-CoV-2: utility and limitations in seroprevalence and immunity studies.

COVID-19 patients elicit strong responses to the nucleocapsid (N) protein of SARS-CoV-2 but binding antibodies are also detected in prepandemic individuals, indicating potential crossreactivity with common cold human coronaviruses (HCoV) and questioning its utility in seroprevalence studies. We investigated the immunogenicity of the full-length and shorter fragments of the SARS-CoV-2 N protein, and the crossreactivity of antibodies with HCoV. We identified a C-terminus region in SARS-CoV2 N of minimal sequence homology with HCoV that was more specific for SARS-CoV-2 and highly immunogenic. IgGs to the full-length SARS-CoV-2 N also recognized N229E N, and IgGs to HKU1 N recognized SARS-CoV-2 N. Crossreactivity with SARS-CoV-2 was stronger for alpha- rather than beta-HCoV despite having less sequence identity, revealing the importance of conformational recognition. Higher preexisting IgG to OC43 N correlated with lower IgG to SARS-CoV-2 N in rRT-PCR negative individuals, reflecting less exposure and indicating a potential protective association. Antibodies to SARS-CoV-2 N were higher in patients with more severe and longer duration of symptoms and in females. IgGs remained stable for at least 3 months, while IgAs and IgMs declined faster. In conclusion, N protein is a primary target of SARS-CoV-2-specific and HCoV crossreactive antibodies, both of which may affect the acquisition of immunity to COVID-19.

Highly Sensitive and Specific Multiplex Antibody Assays To Quantify Immunoglobulins M, A, and G against SARS-CoV-2 Antigens.

Reliable serological tests are required to determine the prevalence of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to characterize immunity to the disease in order to address key knowledge gaps in the coronavirus disease 2019 (COVID-19) pandemic. Quantitative suspension array technology (qSAT) assays based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and enzyme-linked immunosorbent assays (ELISAs) with their higher precision, dynamic range, throughput, miniaturization, cost-efficiency, and multiplexing capacity. We developed three qSAT assays for IgM, IgA, and IgG against a panel of eight SARS-CoV-2 antigens, including spike protein (S), nucleocapsid protein (N), and membrane protein (M) constructs. The assays were optimized to minimize the processing time and maximize the signal-to-noise ratio. We evaluated their performances using 128 prepandemic plasma samples (negative controls) and 104 plasma samples from individuals with SARS-CoV-2 diagnosis (positive controls), of whom 5 were asymptomatic, 51 had mild symptoms, and 48 were hospitalized. Preexisting IgG antibodies recognizing N, M, and S proteins were detected in negative controls, which is suggestive of cross-reactivity to common-cold coronaviruses. The best-performing antibody/antigen signatures had specificities of 100% and sensitivities of 95.78% at ≥14 days and 95.65% at ≥21 days since the onset of symptoms, with areas under the curve (AUCs) of 0.977 and 0.999, respectively. Combining multiple markers as assessed by qSAT assays has the highest efficiency, breadth, and versatility to accurately detect low-level antibody responses for obtaining reliable data on the prevalence of exposure to novel pathogens in a population. Our assays will allow gaining insights into antibody correlates of immunity and their kinetics, required for vaccine development to combat the COVID-19 pandemic.

RTS,S/AS01E malaria vaccine induces IgA responses against CSP and vaccine-unrelated antigens in African children in the phase 3 trial.

BACKGROUND: The evaluation of immune responses to RTS,S/AS01 has traditionally focused on immunoglobulin (Ig) G antibodies that are only moderately associated with protection. The role of other antibody isotypes that could also contribute to vaccine efficacy remains unclear. Here we investigated whether RTS,S/AS01E elicits antigen-specific serum IgA antibodies to the vaccine and other malaria antigens, and we explored their association with protection. METHODS: Ninety-five children (age 5-17 months old at first vaccination) from the RTS,S/AS01E phase 3 clinical trial who received 3 doses of RTS,S/AS01E or a comparator vaccine were selected for IgA quantification 1 month post primary immunization. Two sites with different malaria transmission intensities (MTI) and clinical malaria cases and controls, were included. Measurements of IgA against different constructs of the circumsporozoite protein (CSP) vaccine antigen and 16 vaccine-unrelated Plasmodium falciparum antigens were performed using a quantitative suspension array assay. RESULTS: RTS,S vaccination induced a 1.2 to 2-fold increase in levels of serum/plasma IgA antibodies to all CSP constructs, which was not observed upon immunization with a comparator vaccine. The IgA response against 13 out of 16 vaccine-unrelated P. falciparum antigens also increased after vaccination, and levels were higher in recipients of RTS,S than in comparators. IgA levels to malaria antigens before vaccination were more elevated in the high MTI than the low MTI site. No statistically significant association of IgA with protection was found in exploratory analyses. CONCLUSIONS: RTS,S/AS01E induces IgA responses in peripheral blood against CSP vaccine antigens and other P. falciparum vaccine-unrelated antigens, similar to what we previously showed for IgG responses. Collectively, data warrant further investigation of the potential contribution of vaccine-induced IgA responses to efficacy and any possible interplay, either synergistic or antagonistic, with protective IgG, as identifying mediators of protection by RTS,S/AS01E immunization is necessary for the design of improved second-generation vaccines. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT008666191.