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 recognize and neutralize better 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.

A third SARS-CoV-2 mRNA vaccine dose in people receiving hemodialysis overcomes B cell defects but elicits a skewed CD4+ T cell profile

Cellular immune defects associated with suboptimal responses to SARS-CoV-2 mRNA vaccination in people receiving hemodialysis (HD) are poorly understood. We longitudinally analyzed antibody, B cell, CD4+ and CD8+ T cell vaccine responses in 27 HD patients and 26 low-risk control individuals (CI). The first two doses elicit weaker B cell and CD8+ T cell responses in HD than in CI, while CD4+ T cell responses are quantitatively similar. In HD, a third dose robustly boosts B cell responses, leads to convergent CD8+ T cell responses and enhances comparatively more Thelper (TH) immunity. Unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. The third dose attenuates some features of TH cells in HD (TNF/IL-2 skewing), while others (CCR6, CXCR6, PD-1 and HLA-DR overexpression) persist. Therefore, a third vaccine dose is critical to achieve robust multifaceted immunity in hemodialysis patients, although some distinct TH characteristics endure.

SARS-CoV-2 BA.4/5 Spike recognition and neutralization elicited after the third dose of mRNA vaccine

Several 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 to the D614G and the 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.

A boost with SARS-CoV-2 BNT162b2 mRNA vaccine elicits strong humoral responses independently of the interval between the first two doses

Due to the recrudescence of SARS-CoV-2 infections worldwide, mainly caused by Omicron BA.1 and BA.2 variants of concern, several jurisdictions are administering a mRNA vaccine boost. Here, we analyzed humoral responses induced after the second and third doses of mRNA vaccine in naive and previously-infected donors who received their second dose with an extended 16-week interval. We observed that the extended interval elicited robust humoral responses against VOCs, but this response was significantly diminished 4 months after the second dose. Administering a boost to these individuals brought back the humoral responses to the same levels obtained after the extended second dose. Interestingly, we observed that administering a boost to individuals that initially received a short 3-4 weeks regimen elicited humoral responses similar to those elicited in the long interval regimen. Nevertheless, humoral responses elicited by the boost in naive individuals did not reach those present in previously-infected vaccinated individuals.

SARS-CoV-2 accessory protein ORF8 decreases antibody-dependent cellular cytotoxicity

SARS-CoV-2 Spike glycoprotein is the major target of host neutralizing antibodies and the most changing viral protein in the continuously emerging SARS-CoV-2 variants as a result of frequent viral evasion from host antibody responses. In addition, SARS-CoV-2 encodes multiple accessory proteins that modulate host antiviral immunity by different mechanisms. Among all SARS-CoV-2 accessory proteins, ORF8 is rapidly evolving and a deletion in this protein has been linked to milder disease. Here, we studied the effect of ORF8 on peripheral blood mononuclear cells (PBMC). Specifically, we found that ORF8 can bind monocytes as well as NK cells. Strikingly, ORF8 binds CD16a (Fc{gamma}RIIIA) with nanomolar affinity and decreases the overall level of CD16 at the surface of monocytes and, to a lesser extent, NK cells. Strikingly, this decrease significantly reduces the capacity of PBMCs and particularly monocytes to mediate antibody-dependent cellular cytotoxicity (ADCC). Overall, our data identifies a new immune-evasion activity used by SARS-CoV-2 to escape humoral responses.

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

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

Temporal associations of B and T cell immunity with robust vaccine responsiveness in a 16-week interval BNT162b2 regimen

Spacing of the BNT162b2 mRNA doses beyond 3 weeks raised concerns about vaccine efficacy. We longitudinally analyzed B cell, T cell and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 53 SARS-CoV-2 naive and previously-infected donors. This regimen elicited robust RBD-specific B cell responses whose kinetics differed between cohorts, the second dose leading to increased magnitude in naive participants only. While boosting did not increase magnitude of CD4+ T cell responses further compared to the first dose, unsupervised clustering analyses of single-cell features revealed phenotypic and functional shifts over time and between cohorts. Integrated analysis showed longitudinal immune component-specific associations, with early Thelper responses post-first dose correlating with B cell responses after the second dose, and memory Thelper generated between doses correlating with CD8 T cell responses after boosting. Therefore, boosting elicits a robust cellular recall response after the 16-week interval, indicating functional immune memory.

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

While the standard regimen of the BNT162b2 mRNA vaccine includes two doses administered three weeks apart, some public health authorities decided to space them, raising concerns about vaccine efficacy. Here, we analyzed longitudinal humoral responses including antibody binding, Fc-mediated effector functions and neutralizing activity against the D614G strain but also variants of concern and SARS-CoV-1 in a cohort of SARS-CoV-2 naive and previously infected individuals, with an interval of sixteen weeks between the two doses. While the administration of a second dose to previously infected individuals did not significantly improve humoral responses, we observed a significant increase of humoral responses in naive individuals after the 16-weeks delayed second shot, achieving similar levels as in previously infected individuals. We compared these responses to those elicited in individuals receiving a short (4-weeks) dose interval. For the naive donors, these responses were superior to those elicited by the short dose interval.

Contribution of single mutations to selected SARS-CoV-2 emerging variants Spike antigenicity

Towards the end of 2020, multiple variants of concern (VOCs) and variants of interest (VOIs) have arisen from the original SARS-CoV-2 Wuhan-Hu-1 strain. Mutations in the Spike protein are highly scrutinized for their impact on transmissibility, pathogenesis and vaccine efficacy. Here, we contribute to the growing body of literature on emerging variants by evaluating the impact of single mutations on the overall antigenicity of selected variants and their binding to the ACE2 receptor. We observe a differential contribution of single mutants to the global variants phenotype related to ACE2 interaction and antigenicity. Using biolayer interferometry, we observe that enhanced ACE2 interaction is mostly modulated by a decrease in off-rate. Finally, we made the interesting observation that the Spikes from tested emerging variants bind better to ACE2 at 37{degrees}C compared to the D614G variant. Whether improved ACE2 binding at higher temperature facilitates emerging variants transmission remain to be demonstrated.

Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We could visualize virus spread sequentially from the nasal cavity to the lungs and thereafter systemically to various organs including the brain, which culminated in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct Fab-mediated neutralization, Fc effector interactions of NAbs with monocytes, neutrophils and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

A single BNT162b2 mRNA dose elicits antibodies with Fc-mediated effector functions and boost pre-existing humoral and T cell responses

The standard dosing of the Pfizer/BioNTech BNT162b2 mRNA vaccine validated in clinical trials includes two doses administered three weeks apart. While the decision by some public health authorities to space the doses because of limiting supply has raised concerns about vaccine efficacy, data indicate that a single dose is up to 90% effective starting 14 days after its administration. We analyzed humoral and T cells responses three weeks after a single dose of this mRNA vaccine. Despite the proven efficacy of the vaccine at this time point, no neutralizing activity were elicited in SARS-CoV-2 naive individuals. However, we detected strong anti-receptor binding domain (RBD) and Spike antibodies with Fc-mediated effector functions and cellular responses dominated by the CD4+ T cell component. A single dose of this mRNA vaccine to individuals previously infected by SARS-CoV-2 boosted all humoral and T cell responses measured, with strong correlations between T helper and antibody immunity. Neutralizing responses were increased in both potency and breadth, with distinctive capacity to neutralize emerging variant strains. Our results highlight the importance of vaccinating uninfected and previously-infected individuals and shed new light into the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support to spacing the doses of two-vaccine regimens to vaccinate a larger pool of the population in the context of vaccine scarcity against SARS-CoV-2.

Longitudinal analysis of humoral immunity against SARS-CoV-2 Spike in convalescent individuals up to 8 months post-symptom onset

Functional and lasting immune responses to the novel coronavirus (SARS-CoV-2) are currently under intense investigation as antibody titers in plasma have been shown to decline during convalescence. Since the absence of antibodies does not equate to absence of immune memory, we sought to determine the presence of SARS-CoV-2-specific memory B cells in COVID-19 convalescent patients. In this study, we report on the evolution of the overall humoral immune responses on 101 blood samples obtained from 32 COVID-19 convalescent patients between 16 and 233 days post-symptom onset. Our observations indicate that anti-Spike and anti-RBD IgM in plasma decay rapidly, whereas the reduction of IgG is less prominent. Neutralizing activity in convalescent plasma declines rapidly compared to Fc-effector functions. Concomitantly, the frequencies of RBD-specific IgM+ B cells wane significantly when compared to RBD-specific IgG+ B cells, which increase over time, and the number of IgG+ memory B cells which remain stable thereafter for up to 8 months after symptoms onset. With the recent approval of highly effective vaccines for COVID-19, data on the persistence of immune responses are of central importance. Even though overall circulating SARS-CoV-2 Spike-specific antibodies contract over time during convalescence, we demonstrate that RBD-specific B cells increase and persist up to 8 months post symptom onset. We also observe modest increases in RBD-specific IgG+ memory B cells and importantly, detectable IgG and sustained Fc-effector activity in plasma over the 8-month period. Our results add to the current understanding of immune memory following SARS-CoV-2 infection, which is critical for the prevention of secondary infections, vaccine efficacy and herd immunity against COVID-19.

Major role of IgM in the neutralizing activity of convalescent plasma against SARS-CoV-2

Characterization of the humoral response to SARS-CoV-2, the etiological agent of Covid-19, is essential to help control the infection. In this regard, we and others recently reported that the neutralization activity of plasma from COVID-19 patients decreases rapidly during the first weeks after recovery. However, the specific role of each immunoglobulin isotype in the overall neutralizing capacity is still not well understood. In this study, we selected plasma from a cohort of Covid-19 convalescent patients and selectively depleted immunoglobulin A, M or G before testing the remaining neutralizing capacity of the depleted plasma. We found that depletion of immunoglobulin M was associated with the most substantial loss of virus neutralization, followed by immunoglobulin G. This observation may help design efficient antibody-based COVID-19 therapies and may also explain the increased susceptibility to SARS-CoV-2 of autoimmune patients receiving therapies that impair the production of IgM.

Antibody binding to SARS-CoV-2 S glycoprotein correlates with, but does not predict neutralization

Convalescent plasma from SARS-CoV-2 infected individuals and monoclonal antibodies were shown to potently neutralize viral and pseudoviral particles carrying the S glycoprotein. However, a non-negligent proportion of plasma samples from infected individuals as well as S-specific monoclonal antibodies were reported to be non-neutralizing despite efficient interaction with the S glycoprotein in different biochemical assays using soluble recombinant forms of S or when expressed at the cell surface. How neutralization relates to binding of S glycoprotein in the context of viral particles remains to be established. Here we developed a pseudovirus capture assay (VCA) to measure the capacity of plasma samples or antibodies immobilized on ELISA plates to bind to membrane-bound S glycoproteins from SARS-CoV-2 expressed at the surface of lentiviral particles. By performing VCA and neutralization assays we observed a strong correlation between these two parameters. However, while we found that plasma samples unable to capture viral particles did not neutralize, capture did not guarantee neutralization, indicating that the capacity of antibodies to bind to the S glycoprotein at the surface of viral particles is required but not sufficient to mediate neutralization. Altogether, our results highlights the importance of better understanding the inactivation of S by plasma and neutralizing antibodies.

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 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 one-month interval on 31 convalescent individuals to evaluate how the humoral responses against the SARS-CoV-2 Spike, including neutralization, evolve over time. We observed that receptor-binding domain (RBD)-specific IgG slightly decreased between six and ten weeks after symptoms onset but RBD-specific IgM decreased much more abruptly. Similarly, we observed a significant decrease in the capacity of convalescent plasma to neutralize pseudoparticles bearing SARS-CoV-2 S wild-type 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 symptoms resolution.

Cross-sectional evaluation of humoral responses against SARS-CoV-2 Spike

The SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoprotein of SARS-CoV-2 mediates viral entry and is the main target 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 performed a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against the SARS-CoV-2 Spike. The vast majority of infected individuals elicited anti-Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding domain (RBD)-specific IgG persisted overtime, while the levels of anti-RBD IgM decreased after symptoms resolution. Some of the elicited antibodies cross-reacted with other human coronaviruses in a genus-restrictive manner. While most of individuals developed neutralizing antibodies within the first two weeks of infection, the level of neutralizing activity was significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.