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Summary Cytomegalovirus (CMV) induction of large frequencies of highly functional memory T cells has attracted much interest in the utility of CMV‐based vaccine vectors, with exciting preclinical data obtained in models of infectious diseases and cancer. However, pathogenesis of human CMV (HCMV) remains a concern. Attenuated CMV‐based vectors, such as replication‐ or spread‐deficient viruses, potentially offer an alternative to fully replicating vectors. However, it is not well understood how CMV attenuation impacts vector immunogenicity, particularly when administered via relevant routes of immunization such as the skin. Herein, we used the murine cytomegalovirus (MCMV) model to investigate the impact of vector attenuation on T‐cell memory formation following subcutaneous administration. We found that the spread‐deficient virus (ΔgL‐MCMV) was impaired in its ability to induce memory CD8+ T cells reactive to some (M38, IE1) but not all (IE3) viral antigens. Impaired‐memory T‐cell development was associated with a preferential and pronounced loss of polyfunctional (IFN‐γ+ TNF‐α+) T cells and also reduced accumulation of TCF1+ T cells, and was not rescued by increasing the dose of replication‐defective MCMV. Finally, whilst vector attenuation reduced dendritic cell (DC) recruitment to skin‐draining lymph nodes, systematic depletion of multiple DC subsets during acute subcutaneous MCMV infection had a negligible impact on T‐cell memory formation, implying that attenuated responses induced by replication‐deficient vectors were likely not a consequence of impaired initial DC activation. Thus, overall, these data imply that the choice of antigen and/or cloning strategy of exogenous antigen in combination with the route of immunization may influence the ability of attenuated CMV vectors to induce robust functional T‐cell memory., Cytomegalovirus (CMV) is a potentially exciting vaccine vector, but pathogenicity may preclude the use of replicating viruses. Here, we show that a spread‐deficient CMV vector is attenuated in the ability to induce T‐cell memory formation after subcutaneous immunization. This results in pronounced loss of polyfunctional T cells, possibly linked to reduced development of TCF1‐expressing T cells.

Candidate vaccines designed to generate T cell–based immunity are typically vectored by nonpersistent viruses, which largely fail to elicit durable effector memory T cell responses. This limitation can be overcome using recombinant strains of CMV. Proof-of-principle studies have demonstrated the potential benefits of this approach, most notably in the SIV model, but safety concerns require the development of nonreplicating alternatives with comparable immunogenicity. In this study, we show that IL-33 promotes the accumulation and recall kinetics of circulating and tissue-resident memory T cells in mice infected with murine CMV. Using a replication-deficient murine CMV vector, we further show that exogenous IL-33 boosts vaccine-induced memory T cell responses, which protect against subsequent heterologous viral challenge. These data suggest that IL-33 could serve as a useful adjuvant to improve the efficacy of vaccines based on attenuated derivatives of CMV.

The HIRA histone chaperone complex deposits histone H3.3 into nucleosomes in a DNA replication- and sequence-independent manner. As herpesvirus genomes enter the nucleus as naked DNA, we asked whether the HIRA chaperone complex affects herpesvirus infection. After infection of primary cells with HSV or CMV, or transient transfection with naked plasmid DNA, HIRA re-localizes to PML bodies, sites of cellular anti-viral activity. HIRA co-localizes with viral genomes, binds to incoming viral and plasmid DNAs and deposits histone H3.3 onto these. Anti-viral interferons (IFN) specifically induce HIRA/PML co-localization at PML nuclear bodies and HIRA recruitment to IFN target genes, although HIRA is not required for IFN-inducible expression of these genes. HIRA is, however, required for suppression of viral gene expression, virus replication and lytic infection and restricts murine CMV replication in vivo. We propose that the HIRA chaperone complex represses incoming naked viral DNAs through chromatinization as part of intrinsic cellular immunity.

The antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) inhibits cell entry of a number of viruses, and genetic diversity within IFITM3 determines susceptibility to viral disease in humans. Here, we used the murine CMV (MCMV) model of infection to determine that IFITM3 limits herpesvirus-associated pathogenesis without directly preventing virus replication. Instead, IFITM3 promoted antiviral cellular immunity through the restriction of virus-induced lymphopenia, apoptosis-independent NK cell death, and loss of T cells. Viral disease in Ifitm3–/– mice was accompanied by elevated production of cytokines, most notably IL-6. IFITM3 inhibited IL-6 production by myeloid cells in response to replicating and nonreplicating virus as well as following stimulation with the TLR ligands Poly(I:C) and CpG. Although IL-6 promoted virus-specific T cell responses, uncontrolled IL-6 expression in Ifitm3–/– mice triggered the loss of NK cells and subsequently impaired control of MCMV replication. Thus, IFITM3 represents a checkpoint regulator of antiviral immunity that controls cytokine production to restrict viral pathogenesis. These data suggest the utility of cytokine-targeting strategies in the treatment of virus-infected individuals with impaired IFITM3 activity.

Summary During primary infection, murine cytomegalovirus (MCMV) spreads systemically, resulting in virus replication and pathology in multiple organs. This disseminated infection is ultimately controlled, but the underlying immune defense mechanisms are unclear. Investigating the role of the cytokine IL-22 in MCMV infection, we discovered an unanticipated function for neutrophils as potent antiviral effector cells that restrict viral replication and associated pathogenesis in peripheral organs. NK-, NKT-, and T cell-secreted IL-22 orchestrated antiviral neutrophil-mediated responses via induction in stromal nonhematopoietic tissue of the neutrophil-recruiting chemokine CXCL1. The antiviral effector properties of infiltrating neutrophils were directly linked to the expression of TNF-related apoptosis-inducing ligand (TRAIL). Our data identify a role for neutrophils in antiviral defense, and establish a functional link between IL-22 and the control of antiviral neutrophil responses that prevents pathogenic herpesvirus infection in peripheral organs., Graphical Abstract, Highlights • Neutrophils are critical antiviral effector cells during MCMV virus infection • Neutrophils directly inhibit virus replication in a TRAIL-dependent manner • IL-22 inhibits virus replication in peripheral but not secondary lymphoid tissues • IL-22 orchestrates CXCL1-dependent neutrophil recruitment, Murine cytomegalovirus (MCMV) targets multiple peripheral organs during infection. Stacey et al. report that in response to MCMV infection, NK, NKT, and T cells secrete the cytokine IL-22, which recruits antiviral neutrophils to infected peripheral organs in a CXCL1-dependent manner. Neutrophils exert antiviral effector functions via proapoptotic TRAIL expression.

Interleukin-10 (IL-10) is an immunomodulatory cytokine that acts to antagonize T cell responses elicited during acute and chronic infections. The IL-10 receptor (IL-10R) signalling pathway thus provides a potential therapeutic target in strategies aimed at combating infectious diseases. In this study, we set out to investigate whether IL-10 expression impacted on Natural Killer (NK) cells. Murine cytomegalovirus (MCMV) infection provides the best characterized in vivo system to evaluate the NK cell response, with NK cells being critical in the early control of acute infection. Blockade of IL-10 receptor (IL-10R) during acute MCMV infection markedly reduced the accumulation of cytotoxic NK cells in the spleen and lung; a phenotype associated with a transient elevation of virus DNA load. Impaired NK cell responsiveness after IL-10R blockade was attributed to elevated levels of apoptosis observed in NK cells exhibiting an activated phenotype. We therefore conclude that IL-10 contributes to antiviral innate immunity during acute infection by restricting activation-induced death in NK cells.