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.

Molecular basis for antiviral activity of pediatric neutralizing antibodies targeting SARS-CoV-2 Spike receptor binding domain

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV- 2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC-evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

Humoral immune responses against SARS-CoV-2 Spike variants after mRNA vaccination in solid organ transplant recipients

While SARS-CoV-2 mRNA vaccination has been shown to be safe and effective in the general population, immunocompromised solid organ transplant recipients (SOTR) were reported to have impaired immune responses after one or two doses of vaccine. In this study, we examined humoral responses induced after the second and the third dose of mRNA vaccine in different SOTR (kidney, liver, lung and heart). Compared to a cohort of SARS-CoV-2 naive immunocompetent health care workers (HCW), the second dose induced weak humoral responses in SOTR, except for the liver recipients. The third dose boosted these responses but they did not reach the same level as in HCW. Interestingly, while the neutralizing activity against Delta and Omicron variants remained very low after the third dose, Fc-mediated effector functions in SOTR reached similar levels as in the HCW cohort. Whether these responses will suffice to protect SOTR from severe outcome remains to be determined.

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.

Highly divergent white-tailed deer SARS-CoV-2 with potential deer-to-human transmission

Wildlife reservoirs of SARS-CoV-2 may enable viral adaptation and spillback from animals to humans. In North America, there is evidence of unsustained spillover of SARS-CoV-2 from humans to white-tailed deer (Odocoileus virginianus), but no evidence of transmission from deer to humans. Through a biosurveillance program in Ontario, Canada we identified a new and highly divergent lineage of SARS-CoV-2 in white-tailed deer. This lineage is the most divergent SARS-CoV-2 lineage identified to date, with 76 consensus mutations (including 37 previously associated with non-human animal hosts) and signatures of considerable evolution and transmission within wildlife. Phylogenetic analysis also revealed an epidemiologically linked human case. Together, our findings represent the first clear evidence of sustained evolution of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.

VE607 Stabilizes SARS-CoV-2 Spike In the "RBD-up" Conformation and Inhibits Viral Entry

SARS-CoV-2 infection of host cells starts by binding of the Spike glycoprotein (S) to the ACE2 receptor. The S-ACE2 interaction is a potential target for therapies against COVID-19 as demonstrated by the development of immunotherapies blocking this interaction. Here, we present the commercially available VE607, comprised of three stereoisomers, that was originally described as an inhibitor of SARS-CoV-1. We show that VE607 specifically inhibits infection of SARS-CoV-1 and SARS-CoV-2 S-expressing pseudoviral particles as well as authentic SARS-CoV-2. VE607 stabilizes the receptor binding domain (RBD) in its "up" conformation. In silico docking and mutational analysis map the VE607 binding site at the RBD-ACE2 interface. The IC50 values are in the low micromolar range for pseudoparticles derived from SARS-CoV-2 Wuhan/D614G as well as from variants of concern (Alpha, Beta, Gamma, Delta and Omicron), suggesting that VE607 has potential for the development of drugs against SARS-CoV-2 infections.

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.

An anti-SARS-CoV-2 non-neutralizing antibody with Fc-effector function defines a new NTD epitope and delays neuroinvasion and death in K18-hACE2 mice

Emerging evidence in animal models indicate that both neutralizing activity and Fc- mediated effector functions of neutralizing antibodies contribute to protection against SARS-CoV-2. It is unclear if antibody effector functions alone could protect against SARS-CoV-2. Here we isolated CV3-13, a non-neutralizing antibody from a convalescent individual with potent Fc-mediated effector functions that targeted the N- terminal domain (NTD) of SARS-CoV-2 Spike. The cryo-EM structure of CV3-13 in complex with SAR-CoV-2 spike revealed that the antibody bound from a distinct angle of approach to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, an Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Thus, we demonstrate that efficient Fc-mediated effector functions can contribute to the in vivo efficacy of anti-SARS-CoV-2 monoclonal antibodies in the absence of neutralization.

Covid-19 vaccine immunogenicity in people living with HIV-1

IntroductionCOVID-19 vaccine efficacy has been evaluated in large clinical trials and in real-world situation. Although they have proven to be very effective in the general population, little is known about their efficacy in immunocompromised patients. HIV-infected individuals response to vaccine may vary according to the type of vaccine and their level of immunosuppression. We evaluated immunogenicity of an mRNA anti-SARS CoV-2 vaccine in HIV-positive individuals. MethodsHIV-positive individuals (n=121) were recruited from HIV clinics in Montreal and stratified according to their CD4 counts. A control group of 20 health care workers naive to SARS CoV-2 was used. The participants Anti-RBD IgG responses were measured by ELISA at baseline and 3 to 4 weeks after receiving the first dose of an mRNA vaccine). ResultsEleven of 121 participants had anti-COVID-19 antibodies at baseline, and a further 4 had incomplete data for the analysis. Mean anti-RBD IgG responses were similar between between the HIV negative control group (n=20) and the combined HIV+ group (n=106) (p = 0.72). However, these responses were significantly lower in the group with <250 CD4 cells/mm3. (p<0.0001). Increasing age was independently associated with decreased immunogenicity. ConclusionHIV-positive individuals with CD4 counts over 250 cells/mm3 have an anti-RBD IgG response similar to the general population. However, HIV-positive individuals with the lowest CD4 counts (<250 cells/mm3) have a weaker response. These data would support the hypothesis that a booster dose might be needed in this subgroup of HIV-positive individuals, depending on their response to the second dose.

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.

Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among {beta}-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

Impact of temperature on the affinity of SARS-CoV-2 Spike for ACE2

The seasonal nature in the outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. The current COVID-19 pandemic makes no exception, and temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2. The receptor binding domain (RBD) of the Spike glycoprotein binds to the angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Studying the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike to ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide, bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

Short-term antibody response and tolerability of one dose of BNT162b2 vaccine in patients receiving hemodialysis

BackgroundPatients receiving in-center hemodialysis (HD) are at high risk of exposure to SARS-CoV-2 with high mortality, and response to vaccination is uncertain. MethodsWe obtained serial plasma from 58 HD patients and 32 health-care workers (HCW) before and after vaccination with one dose of the BNT162b2 mRNA vaccine; as well as convalescent plasma from 11 HD patients who survived COVID-19. Anti-RBD (region binding domain of the SARS-CoV-2 Spike protein) IgG and IgM levels were measured by ELISA. Groups were stratified by evidence of prior SARS-CoV-2 infection. ResultsIn HD patients without prior SARS-CoV-2, antiRBD levels were significantly lower at 4 and 8 weeks after vaccination, compared to in HCWs after 3 weeks (p<0.001), and to convalescent plasma (p=0.002). Anti-RBD IgG was non-detectable in 29/46 (63%) of HD, compared to 1/16 (6%) of HCWs (p<0.0001). No patient with non-detectable levels at 4 weeks developed antiRBD by 8 weeks. In HD patients with prior SARS-CoV-2, mean 8-week anti-RBD IgG levels were similar to controls at 3 weeks (p=0.16), and to convalescent plasma (p=0.45). No patients reported symptoms 7 days after vaccination on a standardized questionnaire. InterpretationWhile the BNT162b2 vaccine was well-tolerated in hemodialysis patients, a single dose failed to elicit a humoral immune response in the majority of SARS-CoV-2 naive patients even after prolonged observation. In those with prior SARS-CoV-2 infection, the humoral response after vaccination was delayed. Whether HD patients develop T-cell responses requires further study. Until then, we advise the second dose be administered to all HD patients at the recommended 3-week time interval, and that rigorous SARS-CoV-2 infection prevention and control measures be continued in dialysis units until vaccine efficacy is proven.

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.

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.