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.

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.

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.

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.

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.

Integrated immunovirological profiling validates plasma SARS-CoV-2 RNA as an early predictor of COVID-19 mortality

Despite advances in COVID-19 management, it is unclear how to recognize patients who evolve towards death. This would allow for better risk stratification and targeting for early interventions. However, the explosive increase in correlates of COVID-19 severity complicates biomarker prioritisation. To identify early biological predictors of mortality, we performed an immunovirological assessment (SARS-CoV-2 viral RNA, cytokines and tissue injury markers, antibody responses) on plasma samples collected from 144 hospitalised COVID-19 patients 11 days after symptom onset and used to test models predicting mortality within 60 days of symptom onset. In the discovery cohort (n=61, 13 fatalities), high SARS-CoV-2 vRNA, low RBD-specific IgG levels, low SARS-CoV-2-specific antibody-dependent cellular cytotoxicity, and elevated levels of several cytokines and lung injury markers were strongly associated with increased mortality in the entire cohort and the subgroup on mechanical ventilation. Model selection revealed that a three-variable model of vRNA, age and sex was very robust at identifying patients who will succumb to COVID-19 (AUC=0.86, adjusted HR for log-transformed vRNA=3.5; 95% CI: 2.0-6.0). This model remained robust in an independent validation cohort (n=83, AUC=0.85). Quantification of plasma SARS-CoV-2 RNA can help understand the heterogeneity of disease trajectories and identify patients who may benefit from new therapies.

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.

Evaluation of a Commercial Culture-free Neutralization Antibody Detection Kit for Severe Acute Respiratory Syndrome-Related Coronavirus-2 and Comparison with an Anti-RBD ELISA Assay

BackgroundSARS-CoV-2 surrogate neutralization assays that obviate the need for viral culture offer substantial advantages regarding throughput and cost. The cPass SARS-CoV-2 Neutralization Antibody Detection Kit (Genscript) is the first such commercially available assay, detecting antibodies that block RBD/ACE-2 interaction. We aimed to evaluate cPass to inform its use and assess its added value compared to anti-RBD ELISA assays. MethodsSerum reference panels comprising 205 specimens were used to compare cPass to plaque-reduction neutralization test (PRNT) and a pseudotyped lentiviral neutralization (PLV) assay for detection of neutralizing antibodies. We assessed the correlation of cPass with an ELISA detecting anti-RBD IgG, IgM, and IgA antibodies at a single timepoint and across intervals from onset of symptoms of SARS-CoV-2 infection. ResultsCompared to PRNT-50, cPass sensitivity ranged from 77% - 100% and specificity was 95% - 100%. Sensitivity was also high compared to the pseudotyped lentiviral neutralization assay (93% [95%CI 85-97]), but specificity was lower (58% [95%CI 48-67]). Highest agreement between cPass and ELISA was for anti-RBD IgG (r=0.823). Against the pseudotyped lentiviral neutralization assay, anti-RBD IgG sensitivity (99% [95%CI 94-100]) was very similar to that of cPass, but overall specificity was lower (37% [95%CI 28-47]). Against PRNT-50, results of cPass and anti-RBD IgG were nearly identical. ConclusionsThe added value of cPass compared to an IgG anti-RBD ELISA was modest.

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.

Real-time conformational dynamics of SARS-CoV-2 spikes on virus particles

SARS-CoV-2 spike (S) mediates entry into cells and is critical for vaccine development against COVID-19. Structural studies have revealed distinct conformations of S, but real-time information that connects these structures, is lacking. Here we apply single-molecule Forster Resonance Energy Transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to hACE2, S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences of convalescent plasma and antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to RBD or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.

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.

Interaction of human ACE2 to membrane-bound SARS-CoV-1 and SARS-CoV-2 S glycoproteins

A novel severe acute respiratory (SARS)-like coronavirus (SARS-CoV-2) is responsible for the current global coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The viral entry of SARS-CoV-2 depends on an interaction between the receptor binding domain of its trimeric Spike glycoprotein and the human angiotensin converting enzyme 2 (ACE2) receptor. A better understanding of the Spike/ACE2 interaction is still required to design anti-SARS-CoV-2 therapeutics. Here, we investigated the degree of cooperativity of ACE2 within both the SARS-CoV-2 and the closely related SARS-CoV-1 membrane-bound S glycoproteins. We show that there exist differential inter-protomer conformational transitions between both Spike trimers. Interestingly, the SARS-CoV-2 spike exhibits a positive cooperativity for monomeric soluble ACE2 binding when compared to the SARS-CoV-1 spike, which might have more structural restrains. Our findings can be of importance in the development of therapeutics that block the Spike/ACE2 interaction.

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.