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SARS-CoV-2 variants with undetermined properties have emerged intermittently throughout the COVID-19 pandemic. Some variants possess unique phenotypes and mutations which allow further characterization of viral evolution and Spike functions. Around 1,100 cases of the B.1.640.1 variant were reported in Africa and Europe between 2021 and 2022, before the expansion of Omicron. Here, we analyzed the biological properties of a B.1.640.1 isolate and its Spike. Compared to the ancestral Spike, B.1.640.1 carried 14 amino acid substitutions and deletions. B.1.640.1 escaped binding by some anti-N-terminal domain and anti-receptor-binding domain monoclonal antibodies, and neutralization by sera from convalescent and vaccinated individuals. In cell lines, infection generated large syncytia and a high cytopathic effect. In primary airway cells, B.1.640.1 replicated less than Omicron BA.1 and triggered more syncytia and cell death than other variants. The B.1.640.1 Spike was highly fusogenic when expressed alone. This was mediated by two poorly characterized and infrequent mutations located in the Spike S2 domain, T859N and D936H. Altogether, our results highlight the cytopathy of a hyper-fusogenic SARS-CoV-2 variant, supplanted upon the emergence of Omicron BA.1. (This study has been registered at ClinicalTrials.gov under registration no. NCT04750720.)IMPORTANCEOur results highlight the plasticity of SARS-CoV-2 Spike to generate highly fusogenic and cytopathic strains with the causative mutations being uncharacterized in previous variants. We describe mechanisms regulating the formation of syncytia and the subsequent consequences in a primary culture model, which are poorly understood., Competing Interests: The authors declare no conflict of interest.

The HIV-1 Env protein is exposed at the surface of virions and infected cells. Env fluctuates between different closed and open structural states and these conformations influence both viral infectivity and sensitivity to antibody binding and neutralization. We established a flow virometry assay to visualize Env proteins at the surface of human immunodeficiency virus type 1 (HIV-1) virions. The assay is performed on ultracentrifuged fluorescent viral particles that are stained with a panel of broadly neutralizing antibodies (bNAbs) and nonneutralizing antibodies (nnAbs) that probe different epitopes of Env. We used this assay to compare Env at the surface of producer cells and viral particles and to analyze the effect of Nef, CD4, and SERINC5 on Env accessibility to antibodies. We studied the laboratory-adapted strain NL4-3 and two transmitted/founder viruses, THRO and CH058. We confirm that antibody accessibility varies between viral strains and show that Nef, CD4, and SERINC5 additively impact Env conformations. We further demonstrate that the Env accessibility profile on virions is globally similar to that observed on HIV-1-infected cells, with some noticeable differences. For instance, nnAbs bind to virions more efficiently than to producer cells, likely reflecting changes in Env conformational states on mature viral particles. This test complements other techniques and provides a convenient and simple tool for quantifying and probing the structure of Env at the virion surface and to analyze the impact of viral and cellular proteins on these parameters. IMPORTANCE HIV-1 Env conformation is one of the key parameters determining viral infectivity. The flow virometry-based assay developed in this study allows for the characterization of proteins incorporated in HIV-1 particles. We studied the conformation of HIV-1 Env and the impact that the viral protein Nef and the cellular proteins CD4 and SERINC5 have on Env accessibility to antibodies. Our assay permitted us to highlight some noticeable differences in the conformation of Env between producer cells and viral particles. It contributes to a better understanding of the actual composition of HIV-1 particles., (Copyright © 2020 American Society for Microbiology.)

HIV-1 poorly infects monocyte-derived dendritic cells (MDDCs). This is in large part due to SAMHD1, which restricts viral reverse transcription. Pseudotyping HIV-1 with vesicular stomatitis virus G protein (VSV-G) strongly enhances infection, suggesting that earlier steps of viral replication, including fusion, are also inefficient in MDDCs. The site of HIV-1 fusion remains controversial and may depend on the cell type, with reports indicating that it occurs at the plasma membrane or, conversely, in an endocytic compartment. Here, we examined the pathways of HIV-1 entry in MDDCs. Using a combination of temperature shift and fusion inhibitors, we show that HIV-1 fusion mainly occurs at the cell surface. We then asked whether surface levels or intracellular localization of CD4 modulates HIV-1 entry. Increasing CD4 levels strongly enhanced fusion and infection with various HIV-1 isolates, including reference and transmitted/founder strains, but not with BaL, which uses low CD4 levels for entry. Overexpressing coreceptors did not facilitate viral infection. To further study the localization of fusion events, we generated CD4 mutants carrying heterologous cytoplasmic tails (LAMP1 or Toll-like receptor 7 [TLR7]) to redirect the molecule to intracellular compartments. The intracellular CD4 mutants did not facilitate HIV-1 fusion and replication in MDDCs. Fusion of an HIV-2 isolate with MDDCs was also enhanced by increasing surface CD4 levels. Our results demonstrate that MDDCs are inefficiently infected by various HIV-1 and HIV-2 strains, in part because of low CD4 levels. In these cells, viral fusion occurs mainly at the surface, and probably not after internalization. IMPORTANCE Dendritic cells (DCs) are professional antigen-presenting cells inducing innate and adaptive immune responses. DCs express the HIV receptor CD4 and are potential target cells for HIV. There is debate about the sensitivity of DCs to productive HIV-1 and HIV-2 infection. The fusion step of the viral replication cycle is inefficient in DCs, and the underlying mechanisms are poorly characterized. We show that increasing the levels of CD4 at the plasma membrane allows more HIV fusion and productive infection in DCs. We further demonstrate that HIV fusion occurs mainly at the cell surface and not in an intracellular compartment. Our results help us understand why DCs are poorly sensitive to HIV infection., (Copyright © 2017 American Society for Microbiology.)

Anti-human immunodeficiency virus type 1 (HIV-1) nonneutralizing antibodies (nnAbs) capable of antibody-dependent cellular cytotoxicity (ADCC) have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. Broadly neutralizing antibodies (bNAbs) also mediate ADCC in cell culture and rely on their Fc region for optimal efficacy in animal models. Here, we selected 9 monoclonal nnAbs and 5 potent bNAbs targeting various epitopes and conformations of the gp120/41 complex and analyzed the potency of the two types of antibodies to bind and eliminate HIV-1-infected cells in culture. Regardless of their neutralizing activity, most of the selected antibodies recognized and killed cells infected with two laboratory-adapted HIV-1 strains. Some nnAbs also bound bystander cells that may have captured viral proteins. However, in contrast to the bNAbs, the nnAbs bound poorly to reactivated infected cells from 8 HIV-positive individuals and did not mediate effective ADCC against these cells. The nnAbs also inefficiently recognize cells infected with 8 different transmitted-founder (T/F) isolates. The addition of a synthetic CD4 mimetic enhanced the binding and killing efficacy of some of the nnAbs in an epitope-dependent manner without reaching the levels achieved by the most potent bNAbs. Overall, our data reveal important qualitative and quantitative differences between nnAbs and bNAbs in their ADCC capacity and strongly suggest that the breadth of recognition of HIV-1 by nnAbs is narrow. IMPORTANCE Most of the anti-HIV antibodies generated by infected individuals do not display potent neutralizing activities. These nonneutralizing antibodies (nnAbs) with antibody-dependent cellular cytotoxicity (ADCC) have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. However, in primate models, the nnAbs do not protect against simian-human immunodeficiency virus (SHIV) acquisition. Thus, the role of nnAbs with ADCC activity in protection from infection remains debatable. In contrast, broadly neutralizing antibodies (bNAbs) neutralize a large array of viral strains and mediate ADCC in cell culture. We analyzed the capacities of 9 nnAbs and 5 bNAbs to eliminate infected cells. We selected 18 HIV-1 strains, including virus reactivated from the reservoir of HIV-positive individuals and transmitted-founder isolates. We report that the nnAbs bind poorly to cells infected with primary HIV-1 strains and do not mediate potent ADCC. Overall, our data show that the breadth of recognition of HIV-1 by nnAbs is narrow., (Copyright © 2017 American Society for Microbiology.)

The existence of HIV reservoirs in infected individuals under combined antiretroviral therapy (cART) represents a major obstacle toward cure. Viral reservoirs are assessed by quantification of HIV nucleic acids, a method which does not discriminate between infectious and defective viruses, or by viral outgrowth assays, which require large numbers of cells and long-term cultures. Here, we used an ultrasensitive p24 digital assay, which we report to be 1,000-fold more sensitive than classical enzyme-linked immunosorbent assays (ELISAs) in the quantification of HIV-1 Gag p24 production in samples from HIV-infected individuals. Results from ultrasensitive p24 assays were compared to those from conventional viral RNA reverse transcription-quantitative PCR (RT-qPCR)-based assays and from outgrowth assay readout by flow cytometry. Using serial dilutions and flow-based single-cell sorting, we show that viral proteins produced by a single infected cell can be detected by the ultrasensitive p24 assay. This unique sensitivity allowed the early (as soon as day 1 in 43% of cases) and more efficient detection and quantification of p24 in phytohemagglutinin-L (PHA)-stimulated CD4 + T cells from individuals under effective cART. When seven different classes of latency reversal agents (LRA) in resting CD4 + T cells from HIV-infected individuals were tested, the ultrasensitive p24 assay revealed differences in the extent of HIV reactivation. Of note, HIV RNA production was infrequently accompanied by p24 protein production (19%). Among the drugs tested, prostratin showed a superior capacity in inducing viral protein production. In summary, the ultrasensitive p24 assay allows the detection and quantification of p24 produced by single infected CD4 + T cells and provides a unique tool to assess early reactivation of infectious virus from reservoirs in HIV-infected individuals. IMPORTANCE The persistence of HIV reservoirs in infected individuals under effective antiretroviral treatment represents a major obstacle toward cure. Different methods to estimate HIV reservoirs exist, but there is currently no optimal assay to measure HIV reservoirs in HIV eradication interventions. In the present study, we report an ultrasensitive digital ELISA platform for quantification of the HIV-1 protein p24. This method was employed to assess the early reactivation of infectious virus from reservoirs in HIV-1-infected individuals. We found that viral proteins produced by a single infected cell can be detected by an ultrasensitive p24 assay. This unprecedented resolution showed major advantages in comparison to other techniques currently used to assess viral replication in reactivation studies. In addition, such a highly sensitive assay allows discrimination of drug-induced reactivation of productive HIV based on protein expression. The present study heralds new opportunities to evaluate the HIV reservoir and the efficacy of drugs used to target it., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: Monocyte-derived dendritic cells (MDDC) stimulate CD8 cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses., Importance: Upon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early postentry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates the presentation of viral antigens derived from newly produced viral proteins, leading to the activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication but also impacts antigen presentation by DC. They highlight the link that exists between restriction factors and adaptive immune responses.

Unlabelled: The spontaneous control of human and simian immunodeficiency viruses (HIV/SIV) is typically associated with specific major histocompatibility complex (MHC) class I alleles and efficient CD8(+) T-cell responses, but many controllers maintain viral control despite a nonprotective MHC background and weak CD8(+) T-cell responses. Therefore, the contribution of this response to maintaining long-term viral control remains unclear. To address this question, we transiently depleted CD8(+) T cells from five SIV-infected cynomolgus macaques with long-term viral control and weak CD8(+) T-cell responses. Among them, only one carried the protective MHC allele H6. After depletion, four of five controllers experienced a transient rebound of viremia. The return to undetectable viremia was accompanied by only modest expansion of SIV-specific CD8(+) T cells that lacked efficient SIV suppression capacity ex vivo. In contrast, the depletion was associated with homeostatic activation/expansion of CD4(+) T cells that correlated with viral rebound. In one macaque, viremia remained undetectable despite efficient CD8(+) cell depletion and inducible SIV replication from its CD4(+) T cells in vitro. Altogether, our results suggest that CD8(+) T cells are not unique contributors to the long-term maintenance of low viremia in this SIV controller model and that other mechanisms, such as weak viral reservoirs or control of activation, may be important players in control., Importance: Spontaneous control of HIV-1 to undetectable levels is associated with efficient anti-HIV CD8(+) T-cell responses. However, in some cases, this response fades over time, although viral control is maintained, and many HIV controllers (weak responders) have very low frequencies of HIV-specific CD8(+) T cells. In these cases, the importance of CD8 T cells in the maintenance of HIV-1 control is questionable. We developed a nonhuman primate model of durable SIV control with an immune profile resembling that of weak responders. Transient depletion of CD8(+) cells induced a rise in the viral load. However, viremia was correlated with CD4(+) T-cell activation subsequent to CD8(+) cell depletion. Regain of viral control to predepletion levels was not associated with restoration of the anti-SIV capacities of CD8(+) T cells. Our results suggest that CD8(+) T cells may not be involved in maintenance of viral control in weak responders and highlight the fact that additional mechanisms should not be underestimated., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)