A Novel Monoclonal Antibody Targeting a Large Surface of the Receptor Binding Motif Shows Pan-neutralizing SARS-CoV-2 Activity Including BQ.1.1 Variant (preprint)

In the present study we report the functional and structural characterization of 17T2, a new highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody (mAb) isolated from a convalescent COVID-19 individual infected during the first wave of the COVID-19 pandemic. 17T2 is a class 1 VH1-58/{kappa}3-20 antibody, derived from a receptor binding domain (RBD)-specific IgA memory B cell and developed as a human recombinant IgG1. Functional characterization revealed that 17T2 mAb has a high and exceptionally broad neutralizing activity against all SARS-CoV-2 spike variants tested, including BQ.1.1. Moreover, 17T2 mAb has in vivo prophylactic activity against Omicron BA.1.1 infection in K18-hACE2 transgenic mice. 3D reconstruction from cryogenic-electron microscopy (cryo-EM) showed that 17T2 binds the Omicron BA.1 spike protein with the RBD domains in up position and recognizes an epitope overlapping with the receptor binding motif, as it is the case for other structurally similar neutralizing mAbs, including S2E12. Yet, unlike S2E12, 17T2 retains its high neutralizing activity against all Omicron sublineages tested, probably due to a larger contact area with the RBD, which could confer a higher resilience to spike mutations. These results highlight the impact of small structural antibody changes on neutralizing performance and identify 17T2 mAb as a potential candidate for future therapeutic and prophylactic interventions.

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

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 1,076 adults of a cohort study in Catalonia between June-November 2020 and a second time between May-July 2021. Questionnaire data and electronic health records on vaccination and COVID-19 testing were available in both periods.Results Antibody seroreversion occurred in 35.8% of the 64 participants infected almost a year ago and non-vaccinated, and was related to asymptomatic infection, age above 60 years and smoking. Among vaccinated, 2.1% did not present antibodies at the time of testing. 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 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 type of vaccine, infection age, sex, smoking, 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 response to vaccination.

Determinants of early antibody responses to COVID-19 mRNA vaccines in exposed and naive healthcare workers (preprint)

BackgroundTwo doses of mRNA vaccination have shown >94% efficacy at preventing COVID-19 mostly in naive 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. MethodsWe 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. FindingsVaccination 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. A second dose in pre-exposed did not increase antibody levels. Smoking and comorbidities were associated with lower neutralization and antibody levels. 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. InterpretationOur data support administering a single-dose in pre-exposed healthy individuals. 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. Persistence of antibody levels in pre-exposed unvaccinated indicates maintenance of immunity up to one year. FundingThis work was supported by Institut de Salut Global de Barcelona (ISGlobal) internal funds, in-kind contributions from Hospital Clinic de Barcelona, the Fundacio 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.

SARS-CoV-2 naïve and recovered individuals show qualitatively different antibody responses following mRNA vaccination (preprint)

mRNA-based vaccines effectively induce protective neutralizing antibody responses against SARS-CoV-2, the etiological agent of COVID-19. The specific compositional patterns of these responses remain largely unknown. We found that SARS-CoV-2-naive individuals receiving the first dose of an mRNA vaccine developed a SARS-CoV-2-specific antibody response with a subclass profile comparable to that induced by the natural infection, except IgA2, which did not increase. SARS-CoV-2-naive subjects also mounted a robust virus-specific recall response after receiving the second dose. This response increased all IgG subclasses, but boosted neither IgM nor IgA1 and IgA2 subclasses. In contrast, individuals recovered from COVID-19 mounted peak virus-specific antibody responses upon primary immunization and did not further augment such responses following secondary immunization. Remarkably, compared to SARS-CoV-2-naive subjects, individuals with pre-existing immunity showed increased levels of all virus-specific antibodies but IgG3 following primary vaccination. By dissecting the heterogeneity of mRNA vaccine-induced humoral responses to SARS-CoV-2, our findings indicate that the induction of optimal immune protection may require the development of personalized vaccination programs.

SARS-CoV-2 sculpts the immune system to induce sustained virus-specific naïve-like and memory B cell responses (preprint)

SARS-CoV-2 infection induces virus-reactive memory B cells expressing unmutated antibodies, which hints at their emergence from naive B cells. Yet, the dynamics of virus-specific naive B cells and their impact on immunity and immunopathology remain unclear. Here, we longitudinally studied moderate to severe COVID-19 patients to dissect SARS-CoV-2-specific B cell responses overtime. We found a broad virus-specific antibody response during acute infection, which evolved into an IgG1-dominated response during convalescence. Acute infection was associated with increased mature B cell progenitors in the circulation and the unexpected expansion of virus-targeting naive-like B cells that further augmented during convalescence together with virus-specific memory B cells. In addition to a transitory increase in tissue-homing CXCR3+ plasmablasts and extrafollicular memory B cells, most COVID-19 patients showed persistent activation of CD4+ and CD8+ T cells along with transient or long-lasting changes of key innate immune cells. Remarkably, virus-specific antibodies and the frequency of naive B cells were among the major variables defining distinct immune signatures associated with disease severity and inflammation. Aside from providing new insights into the complexity of the immune response to SARS-CoV-2, our findings indicate that the de novo recruitment of mature B cell precursors into the periphery may be central to the induction of antiviral immunity.

Antibody Conversion rates to SARS-CoV-2 in Saliva from Children Attending Summer Schools in Barcelona, Spain (preprint)

Surveillance tools to estimate infection rates in young populations are essential to guide recommendations for school reopening and management during viral epidemics. Ideally, field-deployable non-invasive, sensitive techniques are required to detect low viral load exposures among asymptomatic children. We determined SARS-CoV-2 antibody conversion by high-throughput Luminex assays in saliva samples collected weekly in 1,509 children and 396 adults in 22 Summer schools and 2 pre-schools in 27 venues in Barcelona, Spain, from June 29th to July 31st 2020, between the first and second COVID-19 pandemic waves. Saliva antibody conversion defined as [≥]4-fold increase in IgM, IgA and/or IgG levels to SARS-CoV-2 antigens 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%) 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. To conclude, saliva antibody profiling including three isotypes and multiplexing antigens is a useful and more user-friendly tool for screening pediatric populations to determine SARS-CoV-2 exposure and guide public health policies during pandemics.

Multiplex Antibody Analysis of IgM, IgA and IgG to SARS-CoV-2 in Saliva and Serum from Infected Children and their Close Contacts (preprint)

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 the coronavirus in pediatric populations and guide public health interventions. We evaluated the utility of high-throughput Luminex-based assays applied to saliva samples to quantify IgM, IgA and IgG antibodies against five SARS-CoV-2 spike (S) and nucleocapsid (N) antigens in the context of a contacts and infectivity longitudinal study. We compared the antibody levels obtained in saliva versus serum/plasma samples from a group of children and adults tested weekly by RT-PCR over 35 days and diagnosed as positive (n=58), and a group of children and adults who consistently tested negative over the follow up period (n=61), in the Summer of 2020 in Barcelona, Spain. Antibody levels in saliva samples from individuals with confirmed RT-PCR diagnosis of SARS-CoV-2 infection were significantly higher than in negative individuals and correlated with those measured in sera/plasmas. Higher levels of anti-S IgG were found in asymptomatic individuals that could indicate protection against disease in infected individuals. 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, and therefore that both measurements are complementary. In addition to serum/plasma, measurement of SARS-CoV-2-specific saliva antibodies should be considered as a complementary non-invasive assay to better estimate the percentage of individuals who have experienced coronavirus infection. Saliva antibody detection could allow determining COVID-19 prevalence in pediatric populations, alternative to bleeding or nasal swab, and serological diagnosis following vaccination.

Seven-month kinetics of SARS-CoV-2 antibodies and protective role of pre-existing antibodies to seasonal human coronaviruses on COVID-19 (preprint)

Unraveling long-term kinetics of antibodies to SARS-CoV-2 and the factors influencing its course, like prior antibody levels to human coronaviruses causing common cold (HCoVs), is essential to understand protective immunity and effective surveillance strategies. Antibody levels against six SARS-CoV-2 and four HCoV antigens were quantified by Luminex, and antibody neutralization capacity was assessed by flow cytometry in a cohort of health care workers followed-up for 6 months. Seroprevalence increased over time from 13.5% (month 0) and 15.6% (month 1), to 16.4% (month 6). Levels of antibodies were stable over time, except IgG against nucleocapsid antigen and IgM levels that waned. After the peak response, anti-spike antibody levels increased from ~150 days post-symptom onset in all individuals (73% for IgG), in the absence of any evidence of re-exposure. The neutralizing capacity of antibodies was maintained. Pre-existing antibodies to alpha-HCoV were lower in individuals who subsequently seroconverted for SARS-CoV-2. IgG and IgA to HCoV were 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.

Persistence and baseline determinants of seropositivity and reinfection rates in health care workers up to 12.5 months after COVID-19 (preprint)

We assessed the duration and baseline determinants of antibody responses to SARS-CoV-2 spike antigens and the occurrence of reinfections in a prospective cohort of 173 Spanish primary health care worker patients followed up initially for nine months and subsequently up to 12.5 months after COVID-19 symptoms onset. Seropositivity to SARS-CoV-2 spike and receptor binding domain antigens up to 149-270 days was 92.49% (90.17% IgG, 76.3% IgA, 60.69% IgM). In a subset of 64 health care workers who had not yet been vaccinated by April 2021, seropositivity was 96.88% (95.31% IgG, 82.81% IgA) up to 322-379 days post symptoms onset. There were four suspected reinfections detected by passive case detection, two among seronegative individuals (five and seven months after the first episode), and one low antibody responder. Antibody levels significantly correlated with fever, hospitalization, anosmia/hypogeusia, allergies, smoking and occupation. Stable sustainment of IgG responses raises hope for long-lasting COVID-19 vaccine immunity.

Immunogenicity and crossreactivity of antibodies to SARS-CoV-2 nucleocapsid protein (preprint)

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 indentified a C-terminus region in SARS-CoV2 N of minimal sequence homology with HCoV that was more specific and highly immunogenic. IgGs to the full-length SARS-CoV-2 N also recognised N229E N, and IgGs to HKU1 N recognised 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 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 symptoms and in females. IgGs remained stable for at least 3 months, while IgAs and IgMs declined faster. In conclusion, N 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.

SARS-CoV-2-Specific Antibody Profiles Distinguish Patients with Moderate from Severe COVID-19 (preprint)

Summary The production of SARS-CoV-2-specific neutralizing antibodies is widely considered as a key mechanism for COVID-19 resolution and protection. However, beyond their protective function, antibodies to SARS-CoV-2 may also participate in disease pathogenesis. To explore the potential relationship between virus-specific humoral responses and COVID-19 immunopathology, we measured serum antibody classes and subclasses to the receptor-binding domain of the SARS-CoV-2 spike protein and the nucleoprotein in a cohort of hospitalized COVID-19 patients with moderate to severe disease. We found that RBD-specific IgG1 and IgG3 dominated the humoral response to SARS-CoV-2, were more abundant in severe patients, and positively correlated with several clinical parameters of inflammation. In contrast, a virus-specific IgA2 response skewed toward RBD rather than NP associated with a more favorable clinical course. Interestingly, RBD-dominant IgA2 responses were mostly detected in patients with gastrointestinal symptoms, suggesting the possible involvement of intrinsically tolerogenic gut immune pathways in the attenuation of virus-induced inflammation and disease resolution.

SARS-CoV-2 infections and antibody responses among health care workers in a Spanish hospital after a month of follow-up (preprint)

Background. At the peak of the COVID-19 pandemic in Spain, cumulative prevalence of SARS-CoV-2 infection in a cohort of 578 randomly selected health care workers (HCW) from Hospital Clinic de Barcelona was 11.2%. Methods. A follow-up survey one month after the baseline (April-May 2020) measured SARS-CoV-2 infection by real time reverse-transcriptase polymerase chain reaction (rRT-PCR) and IgM, IgA, IgG and subclasses to the receptor-binding domain of the SARS-CoV-2 spike protein by Luminex. Prevalence of infection was defined by a positive SARS-CoV-2 rRT-PCR and/or antibody seropositivity. Results. The cumulative prevalence of infection at month 1 was 14.9% (84/565) and the seroprevalence 14.5% (82/565) for IgM and/or IgG and/or IgA. We found 25 (5%) new infections in participants without previous evidence of infection at baseline (501) and two participants seroreverted for IgM and/or IgG and/or IgA. Among seropositive participants at baseline, IgM and IgA levels generally declined at month 1 (antibody decay rates of 0.49 (95% CI, 0.40-0.60) and 0.34 (95% CI, 0.26-0.44)), respectively. Eight percent of the participants seroreverted for IgM and 11% for IgA. Subjects reporting COVID-19-like symptoms and laboratory and other technicians had higher risk of infection. The most frequent subclass responses were IgG1 and IgG2, followed by IgG3, with higher levels of IgG1, and only IgA1 but no IgA2 was detected. Conclusions. Our findings highlight the importance of a continuous and improved surveillance of SARS-CoV-2 infections in HCW, particularly in high risk groups. The decay of IgA and IgM levels have implications for seroprevalence studies using these isotypes.

Highly sensitive and specific multiplex antibody assays to quantify immunoglobulins M, A and G against SARS-CoV-2 antigens (preprint)

Reliable serological tests are required to determine the prevalence of antibodies against SARS-CoV-2 antigens and to characterise immunity to the disease in order to address key knowledge gaps in the context of the COVID-19 pandemic. Quantitative suspension array technology (qSAT) assays based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and ELISA with their higher precision, dynamic range, throughput, miniaturization, cost-efficacy and multiplexing capacity. We developed three qSAT assays to detect IgM, IgA and IgG to a panel of eight SARS-CoV-2 antigens including spike (S), nucleoprotein (N) and membrane (M) protein constructs. The assays were optimized to minimize processing time and maximize signal to noise ratio. We evaluated the performance of the assays using 128 plasmas obtained before the COVID-19 pandemic (negative controls) and 115 plasmas from individuals with SARS-CoV-2 diagnosis (positive controls), of whom 8 were asymptomatic, 58 had mild symptoms and 49 were hospitalized. Pre-existing IgG antibodies recognizing N, M and S2 proteins were detected in negative controls suggestive of cross-reactive to common cold coronaviruses. The best performing antibody isotype/antigen signatures had specificities of 100% and sensitivities of 94.94% at [≥]14 days since the onset of symptoms and 96.08% at [≥]21 days since the onset of symptoms, with AUC of 0.992 and 0.999, respectively. Combining multiple antibody 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 prevalence of exposure to novel pathogens in a population. Our assays will allow gaining insights into antibody correlates of immunity required for vaccine development to combat pandemics like the COVID-19.

Seroprevalence of antibodies against SARS-CoV-2 among health care workers in a large Spanish reference hospital (preprint)

BackgroundHealth care workers (HCW) are a high-risk population to acquire SARS-CoV-2 infection from patients or other fellow HCW. At the same time, they can be contagious to highly vulnerable individuals seeking health care. This study aims at estimating the seroprevalence of antibodies against SARS-CoV-2 and associated factors in HCW from a large referral hospital in Barcelona, Spain, one of the countries hardest hit by COVID-19 in the world. MethodsFrom 28 March to 9 April 2020, we recruited a random sample of 578 HCW from the human resources database of Hospital Clinic in Barcelona. We collected a nasopharyngeal swab for direct SARS-CoV-2 detection through real time reverse-transcriptase polymerase chain reaction (rRT-PCR), as well as blood for plasma antibody quantification. IgM, IgG and IgA antibodies to the receptor-binding domain of the spike protein were measured by Luminex. The cumulative prevalence of infection (past or current) was defined by a positive SARS-CoV-2 rRT-PCR and/or antibody seropositivity. ResultsOf the 578 total participants, 39 (6.7%, 95% CI: 4.8-9.1) had been previously diagnosed with COVID-19 by rRT-PCR, 14 (2.4%, 95% CI: 1.4-4.3) had a positive rRT-PCR at recruitment, and 54 (9.3%, 95% CI: 7.2-12.0) were seropositive for IgM and/or IgG and/or IgA against SARS-CoV-2. Of the 54 seropositive HCW, 21 (38.9%) had not been previously diagnosed with COVID-19, although 10 of them (47.6%) reported past COVID-19-compatible symptoms. The cumulative prevalence of SARS-CoV-2 infection was 11.2% (65/578, 95% CI: 8.9-14.1). Among those with evidence of past or current infection, 40.0% (26/65) had not been previously diagnosed with COVID-19, of which 46.2% (12/26) had history of COVID-19-compatible symptoms. The odds of being seropositive was higher in participants who reported any COVID-19 symptom (OR: 8.84, 95% CI: 4.41-17.73). IgM levels positively correlated with age (rho=0.36, p-value=0.031) and were higher in participants with more than 10 days since onset of symptoms (p-value=0.022), and IgA levels were higher in symptomatic than asymptomatic subjects (p-value=0.041). ConclusionsThe seroprevalence of antibodies against SARS-CoV-2 among HCW was lower than expected. Thus, being a high-risk population, we anticipate these estimates to be an upper limit to the seroprevalence of the general population. Forty per cent of those with past or present infection had not been previously diagnosed with COVID-19, which calls for active periodic rRT-PCR testing among all HCW to minimize potential risk of hospital-acquired SARS-CoV-2 infections.