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 the 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, ELISA, and neutralization assays, we observed a strong correlation between these 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 pseudoviral particles is required but not sufficient to mediate neutralization. Altogether, our results highlight the importance of better understanding the inactivation of S by plasma and neutralizing antibodies.

HIV-1 envelope glycoproteins (Envs) mediate viral entry and represent a target of choice for small molecule inhibitors. One of them, temsavir (BMS-626529) prevents the interaction of the host cell receptor CD4 with Env by binding the pocket under the β20-β21 loop of the Env subunit gp120. Along with its capacity to prevent viral entry, temsavir stabilizes Env in its "closed" conformation. We recently reported that temsavir affects glycosylation, proteolytic processing, and overall conformation of Env. Here, we extend these results to a panel of primary Envs and infectious molecular clones (IMCs), where we observe a heterogeneous impact on Env cleavage and conformation. Our results suggest that the effect of temsavir on Env conformation is associated with its capacity to decrease Env processing. Indeed, we found that the effect of temsavir on Env processing affects the recognition of HIV-1-infected cells by broadly neutralizing antibodies and correlates with their capacity to mediate antibody-dependent cellular cytotoxicity (ADCC).

Direct acting antivirals (DAAs) represent critical tools for combating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that have escaped vaccine-elicited spike-based immunity and future coronaviruses with pandemic potential. Here, we used bioluminescence imaging to evaluate therapeutic efficacy of DAAs that target SARS-CoV-2 RNA-dependent RNA polymerase (favipiravir, molnupiravir) or main protease (nirmatrelvir) against Delta or Omicron VOCs in K18-hACE2 mice. Nirmatrelvir displayed the best efficacy followed by molnupiravir and favipiravir in suppressing viral loads in the lung. Unlike neutralizing antibody treatment, DAA monotherapy regimens did not eradicate SARS-CoV-2 in mice, but combining molnupiravir with nirmatrelvir exhibited superior additive efficacy and led to virus clearance. Furthermore, combining molnupiravir with caspase-1/4 inhibitor mitigated inflammation and lung pathology whereas combining molnupiravir with COVID-19 convalescent plasma demonstrated synergy, rapid virus clearance, and 100% survival. Thus, our study provides insights into in vivo treatment efficacies of DAAs and other effective combinations to bolster COVID-19 therapeutic arsenal.

Small CD4-mimetic compounds (CD4mc) sensitize HIV-1-infected cells to antibody-dependent cellular cytotoxicity (ADCC) by facilitating antibody recognition of epitopes that are otherwise occluded on the unliganded viral envelope (Env). Combining CD4mc with two families of CD4-induced (CD4i) antibodies, which are frequently found in plasma of HIV-1-infected individuals, stabilizes Env in a conformation that is vulnerable to ADCC. We employed new-generation SRG-15 humanized mice, supporting natural killer (NK) cell and Fc-effector functions to demonstrate that brief treatment with CD4mc and CD4i-Abs significantly decreases HIV-1 replication, the virus reservoir and viral rebound after ART interruption. These effects required Fc-effector functions and NK cells, highlighting the importance of ADCC. Viral rebound was also suppressed in HIV-1+-donor cell-derived humanized mice supplemented with autologous HIV-1+-donor-derived plasma and CD4mc. These results indicate that CD4mc could have therapeutic utility in infected individuals for decreasing the size of the HIV-1 reservoir and/or achieving a functional cure.

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P Envs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) harbor mutations in the spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses, with structural studies of soluble S variants revealing an increased propensity toward conformations accessible to the human angiotensin-converting enzyme 2 (hACE2) receptor. However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Here, we report single-molecule Förster resonance energy transfer (smFRET) studies of critical mutations observed in VOCs, including D614G and E484K, in the context of virus particles. Investigated variants predominately occupied more open hACE2-accessible conformations, agreeing with previous structures of soluble trimers. Additionally, these S variants exhibited slower transitions in hACE2-accessible/bound states. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population. IMPORTANCE SARS-CoV-2 surface S glycoprotein-the target of antibodies and vaccines-is responsible for binding to the cellular receptor hACE2. The interactions between S and hACE2 trigger structural rearrangements of S from closed to open conformations prerequisite for virus entry. Under the selection pressure imposed by adaptation to the human host and increasing vaccinations and convalescent patients, SARS-CoV-2 is evolving and has adopted numerous mutations on S variants. These promote virus spreading and immune evasion, partially by increasing the propensity of S to adopt receptor-binding competent open conformations. Here, we determined a time dimension, using smFRET to delineate the temporal prevalence of distinct structures of S in the context of virus particles. We present the first experimental evidence of decelerated transition dynamics from the open state, revealing increased stability of S open conformations to be part of the SARS-CoV-2 adaption strategies.

HIV-1-infected individuals raise a polyclonal antibody response targeting multiple envelope glycoprotein (Env) epitopes. Interestingly, two classes of non-neutralizing CD4-induced (CD4i) antibodies, present in the majority of HIV-1-infected individuals have been described to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of small CD4 mimetic compounds (CD4mc). These antibodies recognize the coreceptor binding site (CoRBS) and the constant region one and two (C1C2 or inner domain cluster A) of the gp120. In combination with CD4mc they have been shown to stabilize an antibody-vulnerable Env conformation, known as State 2A. Here we evaluated the importance of these two families of Abs in ADCC responses by immunizing guinea pigs with gp120 immunogens that have been modified to elicit or not these types of antibodies. Underlying the importance of anti-CoRBS and anti-cluster A Abs in stabilizing State 2A, ADCC responses were only observed in the presence of these two types of CD4i antibodies. Altogether, our results suggest that these two families of CD4i antibodies must be taken into account when considering future strategies relying on the use of CD4mc to eliminate HIV-1-infected cells in vivo.

Since the beginning of the SARS-CoV-2 pandemic, several variants of concern (VOCs), such as the Alpha, Beta, Gamma, Delta and Omicron variants, have arisen and spread worldwide. Today, the predominant circulating subvariants are sublineages of the Omicron variant, which have more than 30 mutations in their Spike glycoprotein compared to the ancestral strain. The Omicron subvariants were significantly less recognized and neutralized by antibodies from vaccinated individuals. This resulted in a surge in the number of infections, and booster shots were recommended to improve responses against these variants. While most studies mainly measured the neutralizing activity against variants, we and others previously reported that Fc-effector functions, including antibody-dependent cellular cytotoxicity (ADCC), play an important role in humoral responses against SARS-CoV-2. In this study, we analyzed Spike recognition and ADCC activity against several Omicron subvariants by generating cell lines expressing different Omicron subvariant Spikes. We tested these responses in a cohort of donors, who were recently infected or not, before and after a fourth dose of mRNA vaccine. We showed that ADCC activity is less affected than neutralization by the antigenic shift of the tested Omicron subvariant Spikes. Moreover, we found that individuals with a history of recent infection have higher antibody binding and ADCC activity against all Omicron subvariants than people who were not recently infected. With an increase in the number of reinfections, this study helps better understand Fc-effector responses in the context of hybrid immunity.

Emerging evidence indicates that both neutralizing and Fc-mediated effector functions of antibodies contribute to protection against SARS-CoV-2. It is unclear whether Fc-effector functions alone can protect against SARS-CoV-2. Here, we isolated CV3-13, a non-neutralizing antibody, from a convalescent individual with potent Fc-mediated effector functions. The cryoelectron microscopy structure of CV3-13 in complex with the SARS-CoV-2 spike reveals that the antibody binds from a distinct angle of approach to an N-terminal domain (NTD) epitope that only partially overlaps with the NTD supersite recognized by neutralizing antibodies. CV3-13 does not alter the replication dynamics of SARS-CoV-2 in K18-hACE2 mice, but its Fc-enhanced version significantly delays virus spread, neuroinvasion, and death in prophylactic settings. Interestingly, the combination of Fc-enhanced non-neutralizing CV3-13 with Fc-compromised neutralizing CV3-25 completely protects mice from lethal SARS-CoV-2 infection. Altogether, our data demonstrate that efficient Fc-mediated effector functions can potently contribute to the in vivo efficacy of anti-SARS-CoV-2 antibodies.

The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.

Non-neutralizing antibodies (nnAbs) can eliminate HIV-1-infected cells via antibody-dependent cellular cytotoxicity (ADCC) and were identified as a correlate of protection in the RV144 vaccine trial. Fc-mediated effector functions of nnAbs were recently shown to alter the course of HIV-1 infection in vivo using a vpu-defective virus. Since Vpu is known to downregulate cell-surface CD4, which triggers conformational changes in the viral envelope glycoprotein (Env), we ask whether the lack of Vpu expression was linked to the observed nnAbs activity. We find that restoring Vpu expression greatly reduces nnAb recognition of infected cells, rendering them resistant to ADCC. Moreover, administration of nnAbs in humanized mice reduces viral loads only in animals infected with a vpu-defective but not with a wild-type virus. CD4-mimetics administration, known to "open" Env and expose nnAb epitopes, renders wild-type viruses sensitive to nnAbs Fc-effector functions. This work highlights the importance of Vpu-mediated evasion of humoral responses.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) mediates viral entry into cells and is critical for vaccine development against coronavirus disease 2019 (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 fluorescence (Förster) 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 human receptor angiotensin-converting enzyme 2 (hACE2), S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences observed upon exposure to convalescent plasma or antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to the receptor-binding domain (RBD) or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.

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 naïve 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 naïve 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.

While an important part of the world's population is vaccinated against SARS-CoV-2, new variants continue to emerge. We observe that even after a fifth dose of the mRNA bivalent vaccine, most vaccinated individuals have antibodies that poorly neutralize several Omicron subvariants, including BQ.1.1, XBB, XBB.1.5, FD.1.1, and CH.1.1. However, Fc-effector functions remain strong and stable over time against new variants, which may partially explain why vaccines continue to be effective. We also observe that donors who have been recently infected have stronger antibody functional activities, including neutralization and Fc-effector functions, supporting the observations that hybrid immunity leads to better humoral responses.

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 during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating 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 neutralization, depletion studies indicated that 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.

Human natural history and vaccine studies support a protective role of antibody dependent cellular cytotoxicity (ADCC) activity against many infectious diseases. One setting where this has consistently been observed is in HIV-1 vertical transmission, where passively acquired ADCC activity in HIV-exposed infants has correlated with reduced acquisition risk and reduced pathogenesis in HIV+ infants. However, the characteristics of HIV-specific antibodies comprising a maternal plasma ADCC response are not well understood. Here, we reconstructed monoclonal antibodies (mAbs) from memory B cells from late pregnancy in mother MG540, who did not transmit HIV to her infant despite several high-risk factors. Twenty mAbs representing 14 clonal families were reconstructed, which mediated ADCC and recognized multiple HIV Envelope epitopes. In experiments using Fc-defective variants, only combinations of several mAbs accounted for the majority of plasma ADCC of MG540 and her infant. We present these mAbs as evidence of a polyclonal repertoire with potent HIV-directed ADCC activity.

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.

To minimize immune responses against infected cells, HIV-1 has evolved different mechanisms to limit the surface expression of its envelope glycoproteins (Env). Recent observations suggest that the binding of certain broadly neutralizing antibodies (bNAbs) targeting the 'closed' conformation of Env induces its internalization. On the other hand, non-neutralizing antibodies (nNAbs) that preferentially target Env in its 'open' conformation, remain bound to Env on the cell surface for longer periods of time. In this study, we attempt to better understand the underlying mechanisms behind the differential rates of antibody-mediated Env internalization. We demonstrate that 'forcing' open Env using CD4 mimetics allows for nNAb binding and results in similar rates of Env internalization as those observed upon the bNAb binding. Moreover, we can identify distinct populations of Env that are differentially targeted by Abs that mediate faster rates of internalization, suggesting that the mechanism of antibody-induced Env internalization partially depends on the localization of Env on the cell surface.

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°C compared to the D614G variant. Whether improved ACE2 binding at higher temperature facilitates emerging variants transmission remain to be demonstrated.

The HIV-1 envelope glycoprotein (Env) (gp120-gp41)3 is the target for neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC). HIV-1 Env is flexible, sampling different conformational states. Before engaging CD4, Env adopts a closed conformation (State 1) that is largely antibody resistant. CD4 binding induces an intermediate state (State 2), followed by an open conformation (State 3) that is susceptible to engagement by antibodies that recognize otherwise occluded epitopes. We investigate conformational changes in Env that induce ADCC in the presence of a small-molecule CD4-mimetic compound (CD4mc). We uncover an asymmetric Env conformation (State 2A) recognized by antibodies targeting the conserved gp120 inner domain and mediating ADCC. Sera from HIV+ individuals contain these antibodies, which can stabilize Env State 2A in combination with CD4mc. Additionally, triggering State 2A on HIV-infected primary CD4+ T cells exposes epitopes that induce ADCC. Strategies that induce this Env conformation may represent approaches to fight HIV-1 infection.