Evaluation of anti-nucleocapsid level variation to assess SARS-CoV-2 seroprevalence in a vaccinated population.

BACKGROUND: Serosurveys have been key to public health decision-making since the beginning of the SARS-CoV-2 pandemic. However, several studies have uncovered that vaccination blunts the anti-nucleocapsid (N) response to a subsequent infection, which hinders the ability of serologic assays (including commercial ones) to detect recent infections. We therefore developed a new analytical approach to increase the sensitivity of detection of infection in vaccinated individuals. METHODS: Two samples were obtained from 248 SARS-CoV-2-positive (PCR-confirmed), vaccinated donors: one before the infection (reference sample) and one after (test sample). All samples were tested using an in-house, anti-N enzyme-linked immunosorbent assay (ELISA) which had a sensitivity of 98.1% before the mass vaccination campaign. Instead of applying a seropositivity threshold based on a single absorbance value (i.e. conventional approach), seropositivity was determined based on the ratio between the anti-N absorbance of the test and reference samples. RESULTS: The sensitivity of the new approach to detect infection in vaccinated individuals was 95.2% using a cut-off of 1.5 for the anti-N ratio, whereas that of the conventional approach was 63.3%. CONCLUSION: The new analytical approach described herein captured a significantly greater proportion of vaccinated individuals with a known history of SARS-CoV-2 infection than the conventional approach used in most serosurveys.

Cohort profile: A Québec-based plasma donor biobank to study COVID-19 immunity (PlasCoV).

PURPOSE: The long-term humoral immunity to COVID-19 is not well understood owing to the continuous emergence of new variants of concern, the evolving vaccine-induced and infection-induced immunity, and the limited duration of follow-up in previous studies. As the sole blood service in Québec (Canada), Héma-Québec established a COVID-19-focused biobank ('PlasCoV') in April 2021. PARTICIPANTS: As of January 2022, the biobank included 86 483 plasma samples from 15 502 regular donors (age range=18-84 years, females=49.7%), for an average of 5.6 donations per donor. Nearly two-thirds (65.6%) of biobank donors made at least two donations, with many donors having provided samples prevaccination and postvaccination (3061 (19.7%)) or preinfection and postinfection (131 (0.8%)), thus allowing for longitudinal studies on vaccine-induced and infection-induced immunity. FINDINGS TO DATE: A study that used PlasCoV samples revealed that previously infected individuals who received a single dose of the BNT162b2 COVID-19 vaccine exhibited the strongest immune response. By contrast, SARS-CoV-2-naïve individuals required two vaccine doses to produce a maximal immune response. Furthermore, the results of a four-phase seroprevalence study indicated that the antinucleocapsid (N) response wanes rapidly, so that up to one-third of previously infected donors were seronegative for anti-N. FUTURE PLANS: Donations from individuals who consented to participate before 1 October 2022 will be collected up until 31 March 2023. This plasma biobank will facilitate the conduct of longitudinal studies on COVID-19 immunity, thus helping to provide valuable insights into the anti-SARS-CoV-2 immune response and its persistence, and the effects of vaccination and variants on the specificity of the anti-SARS-CoV-2 immune response.

HLA and red blood cell antigen genotyping in SARS-CoV-2 convalescent plasma donors.

Aim: More data is required regarding the association between HLA allele and red blood cell (RBC) antigen expression in regard to SARS-CoV-2 infection and COVID-19 susceptibility. Methods: ABO, RhD, 37 other RBC antigens and HLA-A, B, C, DRB1, DQB1 and DPB1 were determined using high throughput platforms in 90 Caucasian convalescent plasma donors. Results: The AB group was significantly increased (1.5×, p = 0.018) and some HLA alleles were found to be significantly overrepresented (HLA-B*44:02, C*05:01, DPB1*04:01, DRB1*04:01 and DRB1*07:01) or underrepresented (A*01:01, B51:01 and DPB1*04:02) in convalescent individuals compared with the local bone marrow registry population. Conclusion: Our study of infection-susceptible but non-hospitalized Caucasian COVID-19 patients contributes to the global understanding of host genetic factors associated with SARS-CoV-2 infection and severity.

Spike recognition and neutralization of SARS-CoV-2 Omicron subvariants elicited after the third dose of mRNA vaccine.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have recently emerged, becoming the dominant circulating strains in many countries. These variants contain a large number of mutations in their spike glycoprotein, raising concerns about vaccine efficacy. In this study, we evaluate the ability of plasma from a cohort of individuals that received three doses of mRNA vaccine to recognize and neutralize these Omicron subvariant spikes. We observed that BA.4/5 and BQ.1.1 spikes are markedly less recognized and neutralized compared with the D614G and other Omicron subvariant spikes tested. Also, individuals who have been infected before or after vaccination present better humoral responses than SARS-CoV-2-naive vaccinated individuals, thus indicating that hybrid immunity generates better humoral responses against these subvariants.

Humoral Responses against BQ.1.1 Elicited after Breakthrough Infection and SARS-CoV-2 mRNA Vaccination.

The Omicron BQ.1.1 variant is now the major SARS-CoV-2 circulating strain in many countries. Because of the many mutations present in its Spike glycoprotein, this variant is resistant to humoral responses elicited by monovalent mRNA vaccines. With the goal to improve immune responses against Omicron subvariants, bivalent mRNA vaccines have recently been approved in several countries. In this study, we measure the capacity of plasma from vaccinated individuals, before and after a fourth dose of mono- or bivalent mRNA vaccine, to recognize and neutralize the ancestral (D614G) and the BQ.1.1 Spikes. Before and after the fourth dose, we observe a significantly better recognition and neutralization of the ancestral Spike. We also observe that fourth-dose vaccinated individuals who have been recently infected better recognize and neutralize the BQ.1.1 Spike, independently of the mRNA vaccine used, than donors who have never been infected or have an older infection. Our study supports that hybrid immunity, generated by vaccination and a recent infection, induces higher humoral responses than vaccination alone, independently of the mRNA vaccine used.

COVID-19 vaccine humoral response in frequent platelet donors with plateletpheresis-associated lymphopenia.

BACKGROUND: Plateletpheresis involves platelet separation and collection from whole blood while other blood cells are returned to the donor. Because platelets are replaced faster than red blood cells, as many as 24 donations can be done annually. However, some frequent apheresis platelet donors (>20 donations annually) display severe plateletpheresis-associated lymphopenia; in particular, CD4+ T but not B cell numbers are decreased. COVID-19 vaccination thereby provides a model to assess whether lymphopenic platelet donors present compromised humoral immune responses. STUDY DESIGN AND METHODS: We assessed vaccine responses following 2 doses of COVID-19 vaccination in a cohort of 43 plateletpheresis donors with a range of pre-vaccination CD4+ T cell counts (76-1537 cells/µl). In addition to baseline T cell measurements, antibody binding assays to full-length Spike and the Receptor Binding Domain (RBD) were performed pre- and post-vaccination. Furthermore, pseudo-particle neutralization and antibody-dependent cellular cytotoxicity assays were conducted to measure antibody functionality. RESULTS: Participants were stratified into two groups: <400 CD4/µl (n = 27) and ≥ 400 CD4/µl (n = 16). Following the first dose, 79% seroconverted within the <400 CD4/µl group compared to 87% in the ≥400 CD4/µl group; all donors were seropositive post-second dose with significant increases in antibody levels. Importantly differences in CD4+ T cell levels minimally impacted neutralization, Spike recognition, and IgG Fc-mediated effector functions. DISCUSSION: Overall, our results indicate that lymphopenic plateletpheresis donors do not exhibit significant immune dysfunction; they have retained the T and B cell functionality necessary for potent antibody responses after vaccination.

Seroprevalence of SARS-CoV-2 antibodies among blood donors in Québec: an update from a serial cross-sectional study.

OBJECTIVES: We previously estimated the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies following the first pandemic wave at 2.23% in Québec, Canada. Following the much bigger second wave in fall 2020 and early 2021, we estimated the seroprevalence of anti-SARS-CoV-2 in Québec during the first months of 2021. METHODS: Blood samples from regular, asymptomatic (for ≥ 14 days) donors were collected between January 25, 2021 and March 11, 2021. Anti-SARS-CoV-2 seropositivity was assessed using an enzyme-linked immunosorbent assay that captures antibodies directed against the receptor binding domain of the SARS-CoV-2 spike (and hence cannot discriminate between infection- and vaccine-induced seropositivity). Seroprevalence estimates were adjusted for regional distribution, age, and sex. RESULTS: Samples from 7924 eligible donors were analyzed, including 620 (7.8%) vaccinated donors and 7046 (88.9%) unvaccinated donors (vaccination status unknown for 258 (3.3%) donors). Overall, median age was 51 years; 46.4% of donors were female. The adjusted seroprevalence was 10.5% (95% CI = 9.7-11.3) in the unvaccinated population and 14.7% (95% CI = 13.8-15.6) in the overall population. Seroprevalence gradually decreased with age and was higher among donors who self-identified as having a racial/ethnic background other than white, both in the overall and in the unvaccinated populations. CONCLUSION: The seroprevalence of SARS-CoV-2 antibodies significantly increased in Québec since spring 2020, with younger persons and ethnic minorities being disproportionately affected. When compared with the cumulative incidence rate reported by public health authorities (i.e., 3.3% as of March 11, 2021), these results suggest that a substantial proportion of infections remain undetected despite improvements in access to COVID-19 testing.

RéSUMé: OBJECTIFS: Lors d'une première étude, nous avons estimé la séroprévalence des anticorps contre le syndrome respiratoire aigu sévère coronavirus 2 (SRAS-CoV-2) après la première vague pandémique à 2,23 % au Québec, Canada. Cette seconde étude estime la séroprévalence de l'anti-SRAS-CoV-2 au Québec lors de la deuxième vague pandémique. MéTHODES: Des échantillons de donneurs de sang asymptomatiques (≥ 14 jours) ont été prélevés entre le 25 janvier et le 11 mars 2021. La séropositivité a été évaluée à l'aide d'un dosage immuno-enzymatique qui capture les anticorps dirigés contre la protéine Spike du récepteur de domaine de liaison du SARS-CoV-2 (et ne peut donc distinguer l'immunité induite par l'infection et la vaccination). La séroprévalence a été ajustée en fonction de l'âge et du sexe par région. RéSULTATS: Des échantillons de 7 924 donneurs ont été analysés, dont 620 (7,8 %) étaient vaccinés et 7 046 (88,9 %) étaient non vaccinés (statut vaccinal inconnu pour 258 (3,3 %) donneurs). Dans l'ensemble, l'âge médian était de 51 ans et 46,4 % des donneurs étaient des femmes. La séroprévalence ajustée était de 10,5 % (IC 95 % = 9,7 à 11,3) dans la population non vaccinée et de 14,7 % (IC 95 % = 13,8 à 15,6) dans la population globale. La séroprévalence diminuait progressivement avec l'âge et était plus élevée chez les donneurs d'origine ethnique autre que blanche. CONCLUSION: La séroprévalence anti-SRAS-CoV-2 a considérablement augmenté au Québec depuis le printemps 2020, les personnes plus jeunes et les minorités ethniques étant plus touchées. Comparés au taux d'incidence cumulatif signalé par la santé publique (c.-à-d. 3,3 % au 11 mars 2021), ces résultats suggèrent qu'une proportion importante d'infections reste non détectée.

SARS-CoV-2 Omicron Spike recognition by plasma from individuals receiving BNT162b2 mRNA vaccination with a 16-week interval between doses.

Continuous emergence of SARS-CoV-2 variants of concern (VOCs) is fueling the COVID-19 pandemic. Omicron (B.1.1.529) rapidly spread worldwide. The large number of mutations in its Spike raise concerns about a major antigenic drift that could significantly decrease vaccine efficacy and infection-induced immunity. A long interval between BNT162b2 mRNA doses elicits antibodies that efficiently recognize Spikes from different VOCs. Here, we evaluate the recognition of Omicron Spike by plasma from a cohort of SARS-CoV-2 naive and previously infected individuals who received their BNT162b2 mRNA vaccine 16 weeks apart. Omicron Spike is recognized less efficiently than D614G, Alpha, Beta, Gamma, and Delta Spikes. We compare with plasma activity from participants receiving a short (4 weeks) interval regimen. Plasma from individuals of the long-interval cohort recognize and neutralize better the Omicron Spike compared with those who received a short interval. Whether this difference confers any clinical benefit against Omicron remains unknown.

Strong humoral immune responses against SARS-CoV-2 Spike after BNT162b2 mRNA vaccination with a 16-week interval between doses.

The standard regimen of the BNT162b2 mRNA vaccine for SARS-CoV-2 includes two doses administered three weeks apart. However, some public health authorities spaced these doses, raising questions about efficacy. We analyzed longitudinal humoral responses against the D614G strain and variants of concern for SARS-CoV-2 in a cohort of SARS-CoV-2-naive and previously infected individuals who received the BNT162b2 mRNA vaccine with sixteen weeks between doses. While administering a second dose to previously infected individuals did not significantly improve humoral responses, these responses significantly increased in naive individuals after a 16-week spaced second dose, achieving similar levels as in previously infected individuals. Comparing these responses to those elicited in individuals receiving a short (4-week) dose interval showed that a 16-week interval induced more robust responses among naive vaccinees. These findings suggest that a longer interval between vaccine doses does not compromise efficacy and may allow greater flexibility in vaccine administration.

SARS-CoV-2 seroprevalence among blood donors in Québec, and analysis of symptoms associated with seropositivity: a nested case-control study.

OBJECTIVES: A substantial proportion of individuals infected with SARS-CoV-2 do not experience noticeable symptoms typical of COVID-19. Our objectives were to evaluate the impact of the first wave of the pandemic in Québec by measuring SARS-CoV-2 antibody seroprevalence in a convenience sample of healthy blood donors and to study the association between seropositivity and the occurrence of COVID-19 symptoms. METHODS: The study design was a cross-sectional serological survey with a nested case-control study. Residual blood samples from donations collected between May 25 and July 9, 2020 (well before vaccination rollout) in the province of Québec were tested for anti-Spike RBD antibodies by ELISA. Seropositive donors and a control group of seronegative donors were questioned about prior COVID-19 symptoms. All qualified blood donors were eligible for participation. RESULTS: A total of 7691 blood donors were included in the study. After adjustments, the seroprevalence rate was 2.2% (95% CI 1.9-2.6). Seropositive donors reported one or more symptoms in a proportion of 52.2% (95% CI 44.2-60.1); this proportion was 19.1% (95% CI 13.4-26.1) among seronegative donors, suggesting that approximately 50-66% of all infections were asymptomatic. Univariate analysis of associations between symptoms and seropositivity revealed that except for rhinorrhea, all symptoms were significantly associated with seropositivity. CONCLUSION: Assuming that blood donors are fairly representative of the general adult population, this study shows that less than 3% of 18-69-year-olds have been infected during the first wave of the pandemic in the province of Québec. Our data also confirm that many infections escaped detection, including a substantial proportion that were asymptomatic.

RéSUMé: OBJECTIFS: Une proportion substantielle de personnes infectées par le SRAS-CoV-2 ne présentent pas de symptômes visibles typiques de la COVID-19. Nos objectifs étaient d'évaluer l'impact de la première vague de la pandémie au Québec en mesurant la séroprévalence des anticorps anti-SRAS-CoV-2 chez les donneurs de sang en bonne santé, et d'étudier l'association entre la séropositivité et la survenue des symptômes de la COVID-19. MéTHODES: Le design de l'étude était une enquête de sérologie transversale avec une étude cas-témoins nichée dans la cohorte. Des échantillons de sang provenant de dons recueillis entre le 25 mai et le 9 juillet 2020 (bien avant le déploiement de la vaccination) dans la province de Québec ont été testés pour les anticorps anti-spicule RBD (Receptor Binding Domain) par ELISA. Les donneurs séropositifs et un groupe témoin de donneurs séronégatifs ont été interrogés sur les symptômes spécifiques à la COVID-19. Tous les donneurs qualifiés pour le don de sang étaient éligibles à participer à l'étude. RéSULTATS: Au total, 7 691 donneurs de sang ont été inclus dans l'étude. Après ajustements, le taux de séroprévalence était de 2,2 % (IC à 95% 1,9­2,6). Les donneurs séropositifs ont signalé un ou plusieurs symptômes dans une proportion de 52,2 % (IC à 95% 44,2­60,1); cette proportion était de 19,1 % (IC à 95% 13,4­26,1) parmi les donneurs séronégatifs, ce qui suggère qu'entre 50 % et 66 % de toutes les infections étaient asymptomatiques. Une analyse univariée des associations entre les symptômes et la séropositivité a révélé qu'à l'exception de la rhinorrhée, tous les symptômes étaient significativement associés à la séropositivité. CONCLUSION: En supposant que les donneurs de sang sont assez représentatifs de la population adulte générale, cette étude montre que moins de 3 % des 18­69 ans ont été infectés lors de la première vague de la pandémie dans la province du Québec. Nos données confirment également que de nombreuses infections n'ont pas fait l'objet d'un test moléculaire de dépistage, y compris une proportion importante qui était asymptomatique.

High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing more than two million deaths. The SARS-CoV-2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID-19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike declines rapidly after the resolution of the infection. STUDY DESIGN AND METHODS: To extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full-length SARS-CoV-2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS-CoV-2 Spike and used it to develop a high-throughput flow cytometry-based assay to detect SARS-CoV-2 Spike-specific antibodies in the plasma of convalescent donors. RESULTS AND CONCLUSION: We found that the level of antibodies targeting the full-length SARS-CoV-2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations but strongly correlated with the decline of RBD-specific antibodies and the number of days post-symptom onset. These findings help to better understand the decline of humoral responses against the SARS-CoV-2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.

Decline of Humoral Responses against SARS-CoV-2 Spike in Convalescent Individuals.

In the absence of effective vaccines and with limited therapeutic options, convalescent plasma is being collected across the globe for potential transfusion to coronavirus disease 2019 (COVID-19) patients. The therapy has been deemed safe, and several clinical trials assessing its efficacy are ongoing. While it remains to be formally proven, the presence of neutralizing antibodies is thought to play a positive role in the efficacy of this treatment. Indeed, neutralizing titers of ≥1:160 have been recommended in some convalescent plasma trials for inclusion. Here, we performed repeated analyses at 1-month intervals on 31 convalescent individuals to evaluate how the humoral responses against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein, including neutralization, evolve over time. We observed that the levels of receptor-binding-domain (RBD)-specific IgG and IgA slightly decreased between 6 and 10 weeks after the onset of symptoms but that RBD-specific IgM levels decreased much more abruptly. Similarly, we observed a significant decrease in the capacity of convalescent plasma to neutralize pseudoparticles bearing wild-type SARS-CoV-2 S or its D614G variant. If neutralization activity proves to be an important factor in the clinical efficacy of convalescent plasma transfer, our results suggest that plasma from convalescent donors should be recovered rapidly after resolution of symptoms.IMPORTANCE While waiting for an efficient vaccine to protect against SARS-CoV-2 infection, alternative approaches to treat or prevent acute COVID-19 are urgently needed. Transfusion of convalescent plasma to treat COVID-19 patients is currently being explored; neutralizing activity in convalescent plasma is thought to play a central role in the efficacy of this treatment. Here, we observed that plasma neutralization activity decreased a few weeks after the onset of the symptoms. If neutralizing activity is required for the efficacy of convalescent plasma transfer, our results suggest that convalescent plasma should be recovered rapidly after the donor recovers from active infection.

Cross-Sectional Evaluation of Humoral Responses against SARS-CoV-2 Spike.

SARS-CoV-2 is responsible for the coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoproteins of SARS-CoV-2 mediate viral entry and are the main targets for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic, and public health interventions. Here, we perform a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against SARS-CoV-2 Spike. Most infected individuals elicit anti-Spike antibodies within 2 weeks of the onset of symptoms. The levels of receptor binding domain (RBD)-specific immunoglobulin G (IgG) persist over time, and the levels of anti-RBD IgM decrease after symptom resolution. Although most individuals develop neutralizing antibodies within 2 weeks of infection, the level of neutralizing activity is significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.