Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
The data indicate that antibodies to the S protein alone are unlikely to support ADCC to coronavirally infected cells, and that depletion of anti-S from antisera from infected persons had negligible effect on ADCC.
Abstract
The presence of viral proteins in the plasma membranes of infected cells is essential for natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC). However, many enveloped viruses assemble inside cells rather than budding out through plasma membranes. Thus, the presence of viral proteins in plasma membranes of infected cells is uncertain, as is which of several viral structural proteins participates in ADCC. Their presence might represent leftover proteins after virion assembly.
To address external viral protein display and its sufficiency for antibody-dependent recognition, we utilized the endemic, cold-causing human coronavirus OC-43, a surrogate virus for SARS-CoV-2. We used NK-92-CD16A lymphocytes as killer cells, OC-43 from BEI, and infected, 51Cr-radiolabeled lung A549 cells as ADCC ‘targets’. Viral proteins were monitored by immunofluorescent microscopy. Antibodies to OC-43 included two monoclonal anti-spike S with human Fc’s, plasma from children who had OC-43 respiratory infections, rabbit affinity purified polyclonal anti-nucleocapsid N and S, and sera from unimmunized rabbits.
We found that all the antibody reagents reacted with OC-43 infected cells by immunofluorescent microscopy. Labeling of external viral proteins was punctate. However, only the immune plasma and the non-immunized rabbit sera supported ADCC. The finding with anti-S was surprising since the human mAb 1249A8 anti-S could support Fc-receptor dependent phagocytosis of beads with S protein [PMID35862475]. The finding is consistent with PMID35587364, that SARS CoV-2 S was under-expressed in plasma membranes of infected cells and that depletion of anti-S from antisera from infected persons had negligible effect on ADCC. The finding with anti-N was surprising in light of PMID41060789.
Conclusions are premature but the data indicate that antibodies to the S protein alone are unlikely to support ADCC to coronavirally infected cells.
University of Nevada, Reno Foundation Award.
Viral Immunology (VIR)
The expression of OC-43 coronaviral proteins in the plasma membranes of infected cells [that are needed to support antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) cells] occurs at the same time that virions begin to be released from infected cells. The viral production in the absence of ADCC or cytotoxic T cell death will continue for many days more. We queried the impact of early ADCC on unaffected total viral production.
We utilized the cold-causing, endemic human coronavirus OC-43, a virus in the same subfamily as SARS-CoV-2. We used NK-92-CD16A lymphocytes as killer cells, OC-43 from BEI Resources at an moi of 2, plasma antibodies from infected children, and infected lung A549 cells as ADCC ‘targets’. Viral production was monitored by PCR of virions released into the cell-free supernatants and by virions remaining inside cells. We used a Taqman probe from ThermoFisher for a propriety site within the positive RNA OC-43 virus, calibrating assays with genomic OC-43 RNA from BEI.
We found that PCR products began to appear in the cell-free supernatants on day 2 post infection (pi) and increased on subsequent days. These viral RNA signals coincided with our previous times for first susceptibility to ADCC and first detection of plasma membrane viral protein in the A549 cells. At day 3 pi, ADCC could eliminate most infected cells. OC-43 gene copies in the supernatants were 16.8 million on day 3 and 24.2 million on day 5 post infection, indicating that some virion production can be curtailed by early ADCC.
While substantial viral production before day 3 is observed, ADCC on day 3 reduced the subsequent production by over 75%. Additional conclusions are premature without assessment of infectious virions (by plaque forming units or other means) but the data indicate that early ADCC will substantially reduce the spread of coronaviral infections.
This poster was made possible by a grant from the National Institute of General Medical Sciences (GM103440) from the National Institutes of Health and by the T34 GM145539 grant awarded to the University of Nevada, Reno Institute for Neuroscience (MARC program), and the UNR foundation.
Viral Immunology (VIR)
Kendra Cook, J. C. Amaya, Nandini Naidu et al.· Journal of Immunology· 0 citations
Plasmacytoid dendritic cells (pDC) are major interferon (IFN)-α producing-cells in response to viruses. After maturation, they stimulate T cells. We found that they cross-present antigens from HIV-1-infected CD4+ T cells to specific cytotoxic T cells. During primary HIV infection, IFNs are essential to decrease viral loads, but during chronic infection, they induce immune suppression and metabolic syndrome. Free influenza or Sars-Cov2 viruses were shown by cytometry to induce pDC diversification. Because HIV is rarely free, we stimulated pDC by HIV-1 or 2-infected H9 CD4+ T cells to assess diversification.
Human pDC were purified from buffy coats by immunomagnetic depletion and CD304+ BD AriaIII sorting, stimulated for 16h by H9 cells, tested by flow cytometry (BD LSR2), multiparametric spectral cytometry (Cytek Aurora, Omics) and single-cell RNA sequencing (scRNAseq, Chromium Next Gem-X Flex 10x Genomics, Illumina NextSeq2000, Human genome GRCh38-2024-1, R v4.05, Seurat, DGE, UMAP, GOE, pseudo-trajectory).
HIV-infected CD4+ T cells induced diversification of pDC into IFN-α and IFN-γ-producing, or mature pDC with T-cell stimulatory potential (CD83), or cytotoxic cells (CD107a, target H9HIV cell apoptosis). Spectral cytometry showed diversification into 10 subpopulations with different functions. scRNAseq showed that H9 cells induced pDC with TLR7/9 signaling pathway, HIV-1 or-2-infected H9 cells induced additional subpopulations with IFN-α and -γ (more with HIV-2), IFN-Stimulated Genes (more with HIV-1), cytotoxicity, T cell activation and regulation genes.
H9 cells induced pDC primed for viral activation, HIV-infected H9 cells induced more populations with surprisingly contrasted functions and differences between HIV-1 and 2 infections which may explain HIV-2 lower pathogenicity. Understanding pDC diversification will enable development of targeted immunotherapeutic strategies to control HIV through adequate modulation of IFN production and cytotoxic T cell responses.
French Government’s Investissement d’Avenir program, Laboratoires d’Excellence “Integrative Biology of Emerging Infectious Diseases” (ANR-10-LABX-62-IBEID) ANRS
Viral Immunology (VIR)
A. Hosmalin, Daniela Apostol, S. Isnard et al.· Journal of Immunology· 0 citations
Influenza A virus can evade detection by the adaptive immune system, as evidenced by low vaccine efficacy and reinfections during human challenge studies. Like all successful viruses, influenza A virus regulates host antiviral responses, and it encodes multiple immunomodulatory proteins for this purpose. One such viral protein, PA-X, is an endoribonuclease that suppresses host gene expression during infection. Here we investigate the impact of PA-X on MHC I antigen presentation, a key process for host detection of intracellular pathogens.
To investigate PA-X dependent changes during infection, we infected primary human donor air liquid interface (ALI) cultures of airway epithelial cells with wild type (WT) or PA-X deficient H3N2 influenza A virus. Using single cell RNA sequencing, we detected changes in gene expression of antigen processing and presentation. We then investigated surface and intracellular MHC I protein levels using flow cytometry and MHC I trafficking to the cell surface using an acid strip time course.
The influenza A viral protein PA-X significantly reduced expression of antigen processing and presentation genes in infected epithelial cells. Both surface and intracellular MHC I levels were significantly reduced in WT influenza-infected ALI cultures compared to mock-infected cultures or cultures infected with PA-X deficient virus. Furthermore, PA-X activity halved the rate of MHC I trafficking to the surface during infection.
Through regulation of MHC I gene expression, PA-X decreases the rate of MHC I trafficking during infection. This likely delays detection by antigen-specific T cells, allowing the virus to replicate and spread, particularly in hosts with prior immunity to influenza A virus, and contributing to the continued success of this virus. Future work will investigate the functional impacts of PA-X disruption of MHC I using immunopeptidomics and in vivo studies.
n/a
Viral Immunology (VIR)
Alessandra C. Setaro, M. Gaglia· Journal of Immunology· 0 citations
The HIV-1 Env glycoprotein mediates both cell-free and cell-to-cell viral transmission and represents the primary target for protective humoral immune responses. Studies examining antibody neutralization of cell-free and cell-to-cell HIV transmission have found that cell-to-cell transmission is more resistant to neutralization. This resistance may be explained in part by antigenically distinct Env populations on virions and infected cells. Cell-surface Env may be more heterogeneous due to variations in cleavage, glycosylation, and conformational state. Nevertheless, the mechanisms that maintain antigenically distinct Env populations at the cell surface and on virions remain unclear, despite virion assembly occurring at the plasma membrane. In this focused review, we consider how Env endocytosis and recycling influence Env incorporation into virions and antibody recognition. We further consider how Env cleavage may influence trafficking and endocytic fate. Given the central role of Env’s cytoplasmic tail in engaging endosomal trafficking pathways, we review emerging structural models of the CT and discuss how its organization, symmetry, and conformational flexibility may contribute to Env trafficking and intracellular sorting. We also discuss how heterogeneous Env populations may influence antibody susceptibility. Finally, we review therapeutic strategies, including combinatorial antibodies and small-molecule Env modulators, that may enhance antibody recognition of infected cells and virions.
Dania M. Figueroa Acosta, Sara Khaleeq, S. Weiss et al.· Viruses· 0 citations
Antigen presenting cells (APC) can bind HIV-1 and subsequently trans infect CD4+ T cells. In comparison to direct (cis) infection of CD4+ T cells by free virus, APC-mediated HIV-1 trans infection is significantly more efficient and requires lower virus titers. As such, B cell-mediated HIV-1 trans infection of CD4+ T cells, particularly in secondary lymphoid organs (SLO) where B and CD4+ T cells interact frequently in and around B cell follicles, represents an efficient pathway for establishing and maintaining the latent HIV-1 reservoir. The molecular events involved in HIV-1 binding to B cells and transfer to CD4+ T cells are poorly understood. B cells are exposed to various activation signals in SLO including CD40 ligand (CD40L), interleukin-4 (IL-4), interferon-γ (IFN-γ), and B cell activating factor (BAFF). Here, we treated B cells with these different signals, or combination of signals, to identify those that facilitate HIV-1 binding to B cells and trans-infection of CD4+ T cells, and the mechanisms involved. We found that CD40L/IL-4 stimulated B cells are highly efficient mediators of HIV-1 trans infection of CD4+ T cells due to their enhanced capacity to bind HIV-1. Single cell RNA sequencing of differentially stimulated B cell populations revealed that CD40L/IL-4 stimulation significantly induced expression of the C-type lectin CD205. Confocal microscopy revealed that HIV-1 and CD205 co-localized on CD40L/IL-4 stimulated B cells, and antibody blocking of CD205 on these cells significantly reduced HIV-1 binding. Taken together, this study identifies CD205 as a critical receptor on B cells that facilitates HIV-1 binding and the transfer of virus to CD4+ T cells. Insight into the role of B cell mediated HIV-1 trans infection of CD4+ T cells is critical to optimizing the effectiveness of HIV-1 therapies in SLO. Author summary HIV-1 spreads in part by exploiting antigen presenting cells such as B cells, which bind and transfer virus to CD4+ T cells through a process called trans infection. The molecular events involved in HIV-1 binding to B cells and transfer to CD4+ T cells are undefined. B cells frequently interact with CD4+ T cells within B cell follicles in secondary lymphoid organs (SLO), which represent a major reservoir of HIV-1. Here, we treated B cells with different SLO-specific activation signals to identify those that facilitate B cell-mediated trans infection of CD4+ T cells, and the mechanisms involved. We found that B cells stimulated with CD40 ligand (CD40L) and interleukin-4 (IL-4) become highly efficient mediators of trans infection due to their increased ability to bind HIV-1. This enhanced binding is driven by upregulation of the C-type lectin receptor CD205, which acts as a critical receptor that facilitates HIV-1 binding and trans infection of CD4+ T cells. These findings may inform strategies for limiting the viral reservoir in people living with HIV.
A. Gerberick, Allison E. DePuyt, Peter E. J. Shoucair et al.· bioRxiv· 0 citations
Oncolytic herpes simplex virus type-1 (HSV1)–based therapies engage innate immune responses, including natural killer (NK) cells, which are regarded as antiviral effector cells that eliminate virus-infected tumor targets. In this study, we examined interactions between the HSV1–derived oncolytic virus HSV1716 and primary human NK cells. Co-culture experiments revealed increased activation and degranulation of NK cells in response to HSV1716-infected tumor cells, despite the downregulation of ligands for NK-cell activating receptors on infected targets. Following co-culture with infected tumor cells, but not after incubation with viral inoculum alone, viral gene expression and increased viral copy numbers were detected in NK cells, indicating enhanced viral acquisition and persistence associated with target-cell contact. HSV1716-infected NK cells displayed impaired tumor cell killing ability. Single-cell sequencing analysis revealed downregulation of key NK effector genes alongside alterations in stress-response pathways in HSV1716 infected NK cells. Together, these findings demonstrate that primary human NK cells are infected by HSV1716 and undergo functional and phenotypic changes, leading to a diminished cytotoxic capacity. Given the emerging role of NK cell–based therapies in cancer, these findings may be relevant for the design and timing of oncolytic virus–based strategies in the future.
K. Susek, D. Holla, C. Marsal et al.· Frontiers in Immunology· 0 citations
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