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Antibody-dependent cellular phagocytosis (ADCP) has been implicated in protection against HIV-1. However, methods for measuring ADCP currently rely on the phagocytosis of gp120- or gp41-coated beads that do not reflect physiologically relevant conformations of the viral envelope glycoprotein or the size of a virus-infected cell. We therefore developed a novel approach for measuring ADCP of HIV-infected cells expressing natural conformations of Env. A monocytic cell line (THP-1 cells) or primary human monocytes were incubated with a CD4+ T cell line that expresses eGFP upon HIV-1 infection in the presence of antibodies and ADCP was measured as the accumulation of eGFP+ material by flow cytometry. The internalization of HIV-infected cells by monocytes was confirmed visually by image-capture flow cytometry. Cytoskeletal remodeling, pseudopod formation and phagocytosis were also observed by confocal microscopy. We found that potent broadly neutralizing antibodies (bnAbs), but not non-neutralizing antibodies (nnAbs), mediate efficient phagocytosis of cells infected with either primary or lab-adapted HIV-1. A nnAb to a CD4-inducible epitope of gp120 (A32) failed to enable ADCP of HIV-infected cells but mediated efficient phagocytosis of gp120-coated beads. Conversely, a bnAb specific to intact Env trimers (PGT145) mediated potent ADCP of HIV-infected cells but did not facilitate the uptake of gp120-coated beads. These results underscore the importance of measuring ADCP of HIV-infected cells expressing physiologically relevant conformations of Env and show that most antibodies that are capable of binding to Env trimers on virions to neutralize virus infectivity are also capable of binding to Env on the surface of virus-infected cells to mediate ADCP., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Snow et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Virus-like particles (VLPs) are non-infectious and serve as promising vaccine platforms because they mimic the membrane-embedded conformations of fusion glycoproteins on native viruses. Here, we employed SARS-CoV-2 VLPs (SMEN) presenting ancestral, Beta, or Omicron spikes to identify the variant spike that elicits potent and cross-protective immune responses in the highly sensitive K18-hACE2 challenge mouse model. A combined intranasal and intramuscular SMEN vaccine regimen generated the most effective immune responses to significantly reduce disease burden. Protection was primarily mediated by antibodies, with minor but distinct contributions from T cells in reducing virus spread and inflammation. Immunization with SMEN carrying ancestral spike resulted in 100, 75, or 0% protection against ancestral, Delta, or Beta variant-induced mortality, respectively. However, SMEN with an Omicron spike provided only limited protection against ancestral (50%), Delta (0%), and Beta (25%) challenges. By contrast, SMEN with Beta spikes offered 100% protection against the variants used in this study. Thus, the Beta variant not only overcame the immunity produced by other variants, but the Beta spike also elicited diverse and effective humoral immune responses. Our findings suggest that leveraging the Beta variant spike protein can enhance SARS-CoV-2 immunity, potentially leading to a more comprehensive vaccine against emerging variants.

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds the receptor angiotensin converting enzyme 2 (ACE2) and drives virus-host membrane fusion through refolding of its S2 domain. Whereas the S1 domain contains high sequence variability, the S2 domain is conserved and is a promising pan-betacoronavirus vaccine target. We applied cryo-electron tomography to capture intermediates of S2 refolding and understand inhibition by antibodies to the S2 stem-helix. Subtomogram averaging revealed ACE2 dimers cross-linking spikes before transitioning into S2 intermediates, which were captured at various stages of refolding. Pan-betacoronavirus neutralizing antibodies targeting the S2 stem-helix bound to and inhibited refolding of spike prehairpin intermediates. Combined with molecular dynamics simulations, these structures elucidate the process of SARS-CoV-2 entry and reveal how pan-betacoronavirus S2-targeting antibodies neutralize infectivity by arresting prehairpin intermediates.

Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can contribute to the containment HIV-1 replication but whether such activities are sufficient for protection is unclear. We previously identified an antibody to the variable 2 (V2) apex of the HIV-1 Env trimer (PGT145) that potently directs the lysis of SIV-infected cells by NK cells but poorly neutralizes SIV infectivity. To determine if ADCC is sufficient for protection, separate groups of six rhesus macaques were treated with PGT145 or a control antibody (DEN3) by intravenous infusion followed five days later by intrarectal challenge with SIVmac239. Despite high concentrations of PGT145 and potent ADCC activity in plasma on the day of challenge, all animals became infected and viral loads did not differ between the PGT145- and DEN3-treated animals. To determine if PGT145 can protect against a neutralization-sensitive virus, two additional groups of six macaques were treated with PGT145 and DEN3 and challenged with an SIVmac239 variant with a single amino acid change in Env (K180S) that increases PGT145 binding and renders the virus susceptible to neutralization by this antibody. Although there was no difference in virus acquisition, peak and chronic phase viral loads were significantly lower and time to peak viremia was significantly delayed in the PGT145-treated animals compared to the DEN3-treated control animals. Env changes were also selected in the PGT145-treated animals that confer resistance to both neutralization and ADCC. These results show that ADCC is not sufficient for protection by this V2-specific antibody. However, protection may be achieved by increasing the affinity of antibody binding to Env above the threshold required for neutralization., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD4 1-4 . Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes 5-13 . Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design., (© 2023. The Author(s).)

Antibodies specific for diverse epitopes of the simian immunodeficiency virus envelope glycoprotein (SIV Env) have been isolated from rhesus macaques to provide physiologically relevant reagents for investigating antibody-mediated protection in this species as a nonhuman primate model for HIV/AIDS. With increasing interest in the contribution of Fc-mediated effector functions to protective immunity, we selected thirty antibodies representing different classes of SIV Env epitopes for a comparison of antibody-dependent cellular cytotoxicity (ADCC), binding to Env on the surface of infected cells and neutralization of viral infectivity. These activities were measured against cells infected with neutralization-sensitive (SIVmac316 and SIVsmE660-FL14) and neutralization-resistant (SIVmac239 and SIVsmE543-3) viruses representing genetically distinct isolates. Antibodies to the CD4-binding site and CD4-inducible epitopes were identified with especially potent ADCC against all four viruses. ADCC correlated well with antibody binding to virus-infected cells. ADCC also correlated with neutralization. However, several instances of ADCC without detectable neutralization or neutralization without detectable ADCC were observed. The incomplete correspondence between ADCC and neutralization shows that some antibody-Env interactions can uncouple these antiviral activities. Nevertheless, the overall correlation between neutralization and ADCC implies that most antibodies that are capable of binding to Env on the surface of virions to block infectivity are also capable of binding to Env on the surface of virus-infected cells to direct their elimination by ADCC., Competing Interests: There are no competing interests to declare., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

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., Competing Interests: A.F. has filed a provisional patent application on the following monoclonal antibodies: CV3-1 and CV3-25. A.F., J.P., V.L. M.A.S., V.-P.L., and E.H. have filed a provisional patent application on the following monoclonal antibodies: EH3 and EH8., (© 2022 The Author(s).)

COVID-19 convalescent plasmas (CCPs) are chosen for plasma therapy based on neutralizing titers and anti-Spike immunoglobulin levels. However, CCP characteristics that promote SARS-CoV-2 control are complex and incompletely defined. Using an in vivo imaging approach, we demonstrate that CCPs with low neutralizing (ID 50  ≤ 1:250), but moderate to high Fc-effector activity, in contrast to those with poor Fc function, delay mortality and/or improve survival of SARS-CoV-2-challenged K18-hACE2 mice. The impact of innate immune cells on CCP efficacy depended on their residual neutralizing activity. Fractionation of a selected CCP revealed that IgG and Ig(M + A) were required during therapy, but the IgG fraction alone sufficed during prophylaxis. Finally, despite reduced neutralization, ancestral SARS-CoV-2-elicited CCPs significantly delayed Delta and Beta-induced mortality suggesting that Fc-effector functions contribute to immunity against VOCs. Thus, Fc activity of CCPs provide a second line of defense when neutralization is compromised and can serve as an important criterion for CCP selection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

It is unclear whether SARS-CoV-2 VOCs differentially escape Fc effector functions of antibodies in addition to neutralization. In this issue of Cell Reports Medicine , Richardson et al. 1 show that VOCs differ both in their ability to evade as well as elicit cross-reactive Fc-effector functions., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

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 β-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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Early events in retrovirus transmission are determined by interactions between incoming viruses and frontline cells near entry sites. Despite their importance for retroviral pathogenesis, very little is known about these events. We developed a bioluminescence imaging (BLI)-guided multiscale imaging approach to study these events in vivo. Engineered murine leukemia reporter viruses allowed us to monitor individual stages of retrovirus life cycle including virus particle flow, virus entry into cells, infection and spread for retroorbital, subcutaneous, and oral routes. BLI permitted temporal tracking of orally administered retroviruses along the gastrointestinal tract as they traversed the lumen through Peyer's patches to reach the draining mesenteric sac. Importantly, capture and acquisition of lymph-, blood-, and milk-borne retroviruses spanning three routes was promoted by a common host factor, the I-type lectin CD169, expressed on sentinel macrophages. These results highlight how retroviruses co-opt the immune surveillance function of tissue-resident sentinel macrophages for establishing infection., Competing Interests: KH, ML, IU, HS, RP, AG, MG, PK, WM, PU No competing interests declared, PB Reviewing editor, eLife, (© 2021, Haugh et al.)

The rhesus macaque is an important animal model for AIDS and other infectious diseases. However, the investigation of Fc-mediated Ab responses in macaques is complicated by species-specific differences in FcγRs and IgG subclasses relative to humans. To assess the effects of these differences on FcγR-IgG interactions, reporter cell lines expressing common allotypes of human and rhesus macaque FcγR2A and FcγR3A were established. FcγR-mediated responses to B cells were measured in the presence of serial dilutions of anti-CD20 Abs with Fc domains corresponding to each of the four subclasses of human and rhesus IgG and with Fc variants of IgG1 that enhance binding to FcγR2A or FcγR3A. All of the FcγRs were functional and preferentially recognized either IgG1 or IgG2. Whereas allotypes of rhesus FcγR2A were identified with responses similar to variants of human FcγR2A with higher (H131) and lower (R131) affinity for IgG, all of the rhesus FcγR3A allotypes exhibited responses most similar to the higher affinity V158 variant of human FcγR3A. Unlike responses to human IgGs, there was little variation in FcγR-mediated responses to different subclasses of rhesus IgG. Phylogenetic comparisons suggest that this reflects limited sequence variation of macaque IgGs as a result of their relatively recent diversification from a common IGHG gene since humans and macaques last shared a common ancestor. These findings reveal species-specific differences in FcγR-IgG interactions with important implications for investigating Ab effector functions in macaques., (Copyright © 2020 by The American Association of Immunologists, Inc.)