The order of infection shapes disease outcomes in influenza and herpes simplex virus coinfection by modulating immune responses.

BACKGROUND: Coinfections of influenza A virus (IAV) and herpes simplex virus type 1 (HSV-1) have been increasingly reported in patients with severe pneumonia, yet their pathogenesis remains poorly understood. METHODS: We established murine models to investigate the effects of coinfection with HSV-1 (strain KOS) and IAV (A/Aichi/2/1968 (H3N2)) under different orders of infection. Mice were assigned to one of five groups: (1) HSV-1 monoinfection, (2) H3N2 monoinfection, (3) simultaneous coinfection (H3N2 + HSV-1), (4) sequential coinfection with H3N2 administered three days prior to HSV-1 (H3N2-HSV-1), and (5) sequential coinfection with HSV-1 administered three days prior to H3N2 (HSV-1-H3N2). We then compared disease severity, viral replication, lung injury, cytokine profiles along with innate and adaptive immune responses. RESULTS: Overall, all coinfection groups developed more severe disease than HSV-1 monoinfection. However, when compared to H3N2 monoinfection, the order of coinfection resulted in distinct differences in disease severity and immune response patterns. Specifically, simultaneous H3N2 + HSV-1 and sequential H3N2-HSV-1 coinfections led to increased mortality, higher H3N2 viral loads, and more pronounced pulmonary inflammation. These groups exhibited elevated cytokine levels and dysregulated immune responses, with the H3N2 + HSV-1 group displaying reduced proportions of natural killer cells (NK), plasmacytoid dendritic cells (pDCs) and interferon-producing killer dendritic cells (IKDCs) in bronchoalveolar lavage fluid (BALF), while the H3N2-HSV-1 group showed a robust expansion of these innate immune cells. Additionally, these two coinfection strategies were associated with increased H3N2-specific IFN-γ⁺CD8⁺ T cells, reflecting an exacerbated adaptive response. In contrast, sequential HSV-1-H3N2 coinfection resulted in milder disease manifestations, characterized by lower mortality, decreased clinical severity, reduced cytokine levels, and diminished proportions of NK cells, pDCs, IKDCs, and CD8⁺ T cells in BALF. Moreover, H3N2-specific IFN-γ⁺CD8⁺ T cells were reduced in both lung and spleen tissues, indicating a more controlled immune activation during HSV-1-H3N2 coinfection. CONCLUSION: Our study demonstrates that the infection order of H3N2 and HSV-1 coinfection critically shapes disease outcomes. Specifically, sequential H3N2 infection preceding HSV-1 or simultaneous coinfection with H3N2 and HSV-1 exacerbated immunopathology. Conversely, prior HSV-1 exposure attenuated H3N2-driven inflammation via reduced cytokine levels and immune cell recruitment. This study provides novel insights into immune dysregulation in coinfection models, with potential translational implications for managing influenza and herpesvirus coinfections.