Live imaging of SARS-CoV-2 infection in mice reveals that 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 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.

Detection of the HIV-1 Accessory Proteins Nef and Vpu by Flow Cytometry Represents a New Tool to Study Their Functional Interplay within a Single Infected CD4+ T Cell.

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. IMPORTANCE HIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release, and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate for the lack of a functional Nef, which has implications for studies that use Nef-defective viruses to study ADCC responses.

Mechanisms of HIV-1 evasion to the antiviral activity of chemokine CXCL12 indicate potential links with pathogenesis.

HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses' receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART.

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

The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect 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 glycoprotein with the 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 (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

NKNK: a New Essential Motif in the C-Terminal Domain of HIV-1 Group M Integrases.

Using coevolution network interference based on comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four noncontiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3' processing and inefficient nuclear import of reverse-transcribed genomes. Solution of the crystal structures of wild-type (wt) and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K's could be permutated or additional K's could be inserted in the motif, generally without affecting integration per se Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions that integrase exerts. We propose that the existence of several amino acid arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution.IMPORTANCE Intensive studies of HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatment escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens up the possibility of adapting to the optimization of further functionalities exerted by the same protein. Such a property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein.

CD4 Incorporation into HIV-1 Viral Particles Exposes Envelope Epitopes Recognized by CD4-Induced Antibodies.

CD4 downregulation on infected cells is a highly conserved function of primate lentiviruses. It has been shown to positively impact viral replication by a variety of mechanisms, including enhanced viral release and infectivity, decrease of cell reinfection, and protection from antibody-dependent cellular cytotoxicity (ADCC), which is often mediated by antibodies that require CD4 to change envelope (Env) conformation. Here, we report that incorporation of CD4 into HIV-1 viral particles affects Env conformation resulting in the exposure of occluded epitopes recognized by CD4-induced antibodies. This translates into enhanced neutralization susceptibility by these otherwise nonneutralizing antibodies but is prevented by the HIV-1 Nef accessory protein. Altogether, these findings suggest that another functional consequence of Nef-mediated CD4 downregulation is the protection of viral particles from neutralization by commonly elicited CD4-induced antibodies.IMPORTANCE It has been well established that Env-CD4 complexes expose epitopes recognized by commonly elicited CD4-induced antibodies at the surface of HIV-1-infected cells, rendering them vulnerable to ADCC responses. Here, we show that CD4 incorporation has a profound impact on Env conformation at the surface of viral particles. Incorporated CD4 exposes CD4-induced epitopes on Env, rendering HIV-1 susceptible to neutralization by otherwise nonneutralizing antibodies.

CCR5 structural plasticity shapes HIV-1 phenotypic properties.

CCR5 plays immune functions and is the coreceptor for R5 HIV-1 strains. It exists in diverse conformations and oligomerization states. We interrogated the significance of the CCR5 structural diversity on HIV-1 infection. We show that envelope glycoproteins (gp120s) from different HIV-1 strains exhibit divergent binding levels to CCR5 on cell lines and primary cells, but not to CD4 or the CD4i monoclonal antibody E51. This owed to differential binding of the gp120s to different CCR5 populations, which exist in varying quantities at the cell surface and are differentially expressed between different cell types. Some, but not all, of these populations are antigenically distinct conformations of the coreceptor. The different binding levels of gp120s also correspond to differences in their capacity to bind CCR5 dimers/oligomers. Mutating the CCR5 dimerization interface changed conformation of the CCR5 homodimers and modulated differentially the binding of distinct gp120s. Env-pseudotyped viruses also use particular CCR5 conformations for entry, which may differ between different viruses and represent a subset of those binding gp120s. In particular, even if gp120s can bind both CCR5 monomers and oligomers, impairment of CCR5 oligomerization improved viral entry, suggesting that HIV-1 prefers monomers for entry. From a functional standpoint, we illustrate that the nature of the CCR5 molecules to which gp120/HIV-1 binds shapes sensitivity to inhibition by CCR5 ligands and cellular tropism. Differences exist in the CCR5 populations between T-cells and macrophages, and this is associated with differential capacity to bind gp120s and to support viral entry. In macrophages, CCR5 structural plasticity is critical for entry of blood-derived R5 isolates, which, in contrast to prototypical M-tropic strains from brain tissues, cannot benefit from enhanced affinity for CD4. Collectively, our results support a role for CCR5 heterogeneity in diversifying the phenotypic properties of HIV-1 isolates and provide new clues for development of CCR5-targeting drugs.

Buffering deleterious polymorphisms in highly constrained parts of HIV-1 envelope by flexible regions.

BACKGROUND: Covariation is an essential process that leads to coevolution of parts of proteins and genomes. In organisms subject to strong selective pressure, coevolution is central to keep the balance between the opposite requirements of antigenic variation and retention of functionality. Being the viral component most exposed to the external environment, the HIV-1 glycoprotein gp120 constitutes the main target of the immune response. Accordingly its more external portions are characterised by extensive sequence heterogeneity fostering constant antigenic variation. RESULTS: We report that a single polymorphism, present at the level of the viral population in the conserved internal region C2, was sufficient to totally abolish Env functionality when introduced in an exogenous genetic context. The prominent defect of the non-functional protein is a block occurring after recognition of the co-receptor CCR5, likely due to an interference with the subsequent conformational changes that lead to membrane fusion. We also report that the presence of compensatory polymorphisms at the level of the external and hypervariable region V3 fully restored the functionality of the protein. The functional revertant presents different antigenic profiles and sensitivity to the entry inhibitor TAK 779. CONCLUSIONS: Our data suggest that variable regions, besides harbouring intrinsic extensive antigenic diversity, can also contribute to sequence diversification in more structurally constrained parts of the gp120 by buffering the deleterious effect of polymorphisms, further increasing the genetic flexibility of the protein and the antigenic repertoire of the viral population.

Genetic diversity of the highly variable V1 region interferes with Human Immunodeficiency Virus type 1 envelope functionality.

BACKGROUND: The HIV envelope (Env) promotes viral entry in the host cell. During this process, Env undergoes several conformational changes to ensure its function. At the same time, the gp120 component of Env is the protein of the virus presenting the largest genetic diversity. Understanding how the virus maintains the balance between the competing requirements for maintenance of functionality and antigenic variation of this protein is central for the comprehension of its strategies of evolution and can highlight vulnerable aspects of its replication cycle. We focused on the variable domains V1 and V2 of the HIV-1 gp120 that are involved in conformational changes and are critical for viral escape from antibody neutralization. RESULTS: Despite the extensive sequence diversity found in the epidemic for these regions and their location on the external face of the protein, we observed that replacing V1V2 of one primary isolate with that of another severely interferes with Env functionality in more than half of the cases studied. Similar results were obtained for intra- and intersubtype chimeras. These observations are indicative of an interference of genetic diversity in these regions with Env functionality. Therefore, despite the extensive sequence diversity that characterizes these regions in the epidemic, our results show that functional constraints seem to limit their genetic variation. Defects in the V1V2 chimeras were not relieved by the insertion of the V3 region from the same isolate, suggesting that the decrease in functionality is not due to perturbation of potential coevolution networks between V1V2 and V3. Within the V1V2 domain, the sequence of the hypervariable loop of the V1 domain seems to be crucial for the functionality of the protein. CONCLUSIONS: Besides the well-documented role of V1V2 in the interplay with the immune response, this work shows that V1 is also involved in the selection of functional envelopes. By documenting a compromise between the opposing forces of sequence diversification and retention of functionality, these observations improve our understanding of the evolutionary trajectories of the HIV-1 envelope gene.

Structure-function analyses reveal key molecular determinants of HIV-1 CRF01_AE resistance to the entry inhibitor temsavir.

The HIV-1 entry inhibitor temsavir prevents the viral receptor CD4 (cluster of differentiation 4) from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this, temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveals that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad antiviral activity.

Structure-function Analyses Reveal Key Molecular Determinants of HIV-1 CRF01_AE Resistance to the Entry Inhibitor Temsavir.

The HIV-1 entry inhibitor temsavir prevents CD4 from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir-resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveal that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad-antiviral activity.

Small CD4 mimetics sensitize HIV-1-infected macrophages to antibody-dependent cellular cytotoxicity.

HIV-1 envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to antibody-dependent cellular cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu, and Env. Limited data exist, however, of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu, and Env are all required to efficiently downregulate CD4 on infected CD4+ T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared with CD4+ T cells. However, treatment of infected macrophages with small CD4 mimetics exposes vulnerable CD4-induced Env epitopes and sensitizes them to ADCC.

Molecular basis for antiviral activity of two pediatric neutralizing antibodies targeting SARS-CoV-2 Spike RBD.

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.

A Fc-enhanced NTD-binding non-neutralizing antibody delays virus spread and synergizes with a nAb to protect mice from lethal SARS-CoV-2 infection.

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.

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, which remain effective against emerging variants of concern in vitro and in vivo. CV3-1 binds to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggers potent shedding of the S1 subunit. In contrast, CV3-25 inhibits membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among ß-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in the RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

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

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

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

Spacing of BNT162b2 mRNA doses beyond 3 weeks raises concerns about vaccine efficacy. We longitudinally analyze 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 elicits robust RBD-specific B cell responses whose kinetics differs between cohorts, the second dose leading to increased magnitude in naive participants only. While boosting does not increase magnitude of CD4+ T cell responses further compared with the first dose, unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. Integrated analysis shows longitudinal immune component-specific associations, with early T helper responses post first dose correlating with B cell responses after the second dose, and memory T helper 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.

Evaluation of the performance of multiple immunoassay diagnostic platforms on the National Microbiology Laboratory SARS-CoV-2 National Serology Panel.

BACKGROUND: Serological assays designed to detect SARS-CoV-2 antibodies are being used in serological surveys and other specialized applications. As a result, and to ensure that the outcomes of serological testing meet high quality standards, evaluations are required to assess the performance of these assays and the proficiency of laboratories performing them. METHODS: A panel of 60 plasma/serum samples from blood donors who had reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 infections and 21 SARS-CoV-2 negative samples were secured and distributed to interested laboratories within Canada (n = 30) and the United States (n = 1). Participating laboratories were asked to provide details on the diagnostic assays used, the platforms the assays were performed on, and the results obtained for each panel sample. Laboratories were blinded with respect to the expected outcomes. RESULTS: The performance of the different assays evaluated was excellent, with the high-throughput platforms of Roche, Ortho, and Siemens demonstrating 100% sensitivity. Most other high-throughput platforms had sensitivities of >93%, with the exception of the IgG assay using the Abbott ARCHITECT which had an average sensitivity of only 87%. The majority of the high-throughput platforms also demonstrated very good specificities (>97%). CONCLUSION: This proficiency study demonstrates that most of the SARS-CoV-2 serological assays utilized by provincial public health or hospital laboratories in Canada have acceptable sensitivity and excellent specificity.

HISTORIQUE: Les dosages sérologiques conçus pour dépister les anticorps anti-SRAS-CoV-2 sont utilisés dans les études sérologiques et d'autres applications spécialisées. Par conséquent, et pour s'assurer que leurs résultats respectent des normes de qualité, il faut procéder à des évaluations de leur performance et de la compétence des laboratoires à les effectuer. MÉTHODOLOGIE: Les chercheurs ont obtenu une batterie de 60 prélèvements de plasma et de sérum chez des donneurs dont l'amplification en chaîne par polymérase après transcription inverse (RT-PCR) avait confirmé des infections par le SRAS-CoV-2 et de 21 prélèvements dont les résultats étaient négatifs au SRAS-CoV-2 et les ont distribués aux laboratoires intéressés du Canada (n = 30) et des États-Unis (n = 1). Ils ont invité les laboratoires participants à fournir de l'information détaillée sur les dosages diagnostiques utilisés, les plateformes sur lesquelles les dosages étaient exécutés et les résultats obtenus pour chaque échantillon. Les chercheurs ont demandé aux laboratoires participants de fournir de l'information détaillée sur les dosages diagnostiques utilisés, les plateformes sur lesquelles les dosages ont été effectués, et les résultats obtenus à l'égard de chaque échantillon. Les laboratoires ont mené les études à l'insu des résultats escomptés. RÉSULTATS: Les divers dosages avaient une excellente exécution, les plateformes à haut débit de Roche, d'Ortho et de Siemens démontrant une sensibilité de 100 %. La plupart des autres plateformes à haut débit avaient des sensibilités de plus de 93 %, à l'exception des dosages des IgG faisant appel à l'analyseur ARCHITECT d'Abbott, dont la sensibilité moyenne était de seulement 87 %. La majorité des plateformes à haut débit avaient également une très bonne spécificité (plus de 97 %). CONCLUSION: La présente étude de compétence démontre que la plupart des dosages sérologiques du SRAS-CoV-2 évalués dans des laboratoires sanitaires provinciaux ou les laboratoires hospitaliers du Canada possèdent une sensibilité acceptable et une excellente spécificité.

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

Across Functional Boundaries: Making Nonneutralizing Antibodies To Neutralize HIV-1 and Mediate Fc-Mediated Effector Killing of Infected Cells.

In HIV-1 infection, many antibodies (Abs) are elicited to Envelope (Env) epitopes that are conformationally masked in the native trimer and are only available for antibody recognition after the trimer binds host cell CD4. Among these are epitopes within the Co-Receptor Binding Site (CoRBS) and the constant region 1 and 2 (C1-C2 or cluster A region). In particular, C1-C2 epitopes map to the gp120 face interacting with gp41 in the native, "closed" Env trimer present on HIV-1 virions or expressed on HIV-1-infected cells. Antibodies targeting this region are therefore nonneutralizing and their potential as mediators of antibody-dependent cellular cytotoxicity (ADCC) of HIV-1-infected cells diminished by a lack of available binding targets. Here, we present the design of Ab-CD4 chimeric proteins that consist of the Ab-IgG1 of a CoRBS or cluster A specificity to the extracellular domains 1 and 2 of human CD4. Our Ab-CD4 hybrids induce potent ADCC against infected primary CD4+ T cells and neutralize tier 1 and 2 HIV-1 viruses. Furthermore, competition binding experiments reveal that the observed biological activities rely on both the antibody and CD4 moieties, confirming their cooperativity in triggering conformational rearrangements of Env. Our data indicate the utility of these Ab-CD4 hybrids as antibody therapeutics that are effective in eliminating HIV-1 through the combined mechanisms of neutralization and ADCC. This is also the first report of single-chain-Ab-based molecules capable of opening "closed" Env trimers on HIV-1 particles/infected cells to expose the cluster A region and activate ADCC and neutralization against these nonneutralizing targets. IMPORTANCE Highly conserved epitopes within the coreceptor binding site (CoRBS) and constant region 1 and 2 (C1-C2 or cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4; therefore, they are not accessible on virions and infected cells, where the expression of CD4 is downregulated. Here, we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or cluster A specificity. We optimized the conjugation sites and linker lengths to allow each of these novel bispecific fusion molecules to recognize native "closed" Env trimers and induce the structural rearrangements required for exposure of the epitopes for antibody binding. Our in vitro functional testing shows that our Ab-CD4 molecules can efficiently target and eliminate HIV-1-infected cells through antibody-dependent cellular cytotoxicity and inactivate HIV-1 virus through neutralization.