Antibody-Mediated Entry Renders Non-Susceptible Macrophages Permissive to SARS-CoV-2 Replication 2307324

Abstract Introduction SARS-CoV-2 entry into epithelial cells via ACE2 is well characterised, but its interactions with alveolar macrophages remain to be fully defined. Macrophages are among the first immune cells to detect infection and initiate protective responses, however, in severe COVID-19 the response is hyperinflammatory. Whether they express ACE2, support viral replication, or directly drive COVID-19 pathology is unresolved. Defining SARS-CoV-2 entry routes and replication competence in macrophages is essential to understanding their dysfunction in severe disease. Methods iPSC-derived macrophages were used as an in vitro model of tissue-resident macrophages. Viral internalisation and intracellular localisation were analysed by imaging flow cytometry. Viral RNA was quantified by smFISH and qRT-PCR, and infectious output virus measured by focus-formation assay. Antigen presentation was assessed in macrophage-T cell co-cultures using intracellular cytokine staining. Results Macrophages efficiently internalise SARS-CoV-2 through a non-specific mechanism and do not express ACE2 or spike-binding receptors. Internalised viral RNA does not replicate and is degraded. Macrophages subsequently present viral antigens and activate CD4+ T cells. Notably, macrophages become both susceptible and permissive to productive infection, when provided with a surrogate entry receptor, such as transgenic ACE2 or specific anti-RBD antibodies. Replication is enhanced by JAK1/2 inhibition, consistent with interferon-mediated restriction. Conclusion These findings show that macrophages are not susceptible to SARS-CoV-2 infection since they do not express ACE2, despite efficiently internalising and degrading the virus. Specific anti-RBD antibodies can act as surrogate receptors that permit productive macrophage infection, suggesting a potential in vivo pathway that may contribute to macrophage dysfunction and hyperinflammation in severe COVID-19. This raises considerations for the therapeutic use of certain monoclonal antibodies. Funding Source University of Oxford, Dunn School of Pathology, EPA Studentship Topic Categories Viral Immunology (VIR)