Innate and T-cellular immune responses to sequential vaccination with chimeric hemagglutinin split influenza virus...

Chimeric hemagglutinin (cHA)-based vaccines represent a promising strategy toward the development of a broadly protective universal influenza virus vaccine. Previous studies show that sequential administration of cHA split influenza vaccines stimulates broadly cross-reactive antibodies targeting the HA stalk and induces systemic and localized CD4⁺ T-cell responses to the HA, neuraminidase (NA), and nucleoprotein (NP). However, the exact role of the T-cellular immune response in the protection after vaccination with cHA constructs remains underinvestigated. Here we provide an in-depth characterization of the innate immune response and antigen-specific T-cellular immune response in mice after sequential immunization with the group 2 cH15/3_HK14N2_HK14 and cH4/3_HK14N2_HK14 virus vaccine candidates with or without CpG 1018 adjuvant and during a heterologous virus challenge. The non-adjuvanted split vaccine induced mixed Th1/Th2 T-cellular immune response associated with interferon-γ, interleukin (IL)-2, tumor necrosis factor α, IL-4, IL-5, IL-13, and IL-10 production whereas the adjuvanted vaccine (split/CpG) induced a Th1-polarized T-cellular immune response mediated by IL2⁺TNFα⁺ and IFNγ⁺IL2⁺TNFα⁺ polyfunctional CD4 effector memory T-lymphocytes. An increase in the Th2-associated cytokines (IL-4, IL-5, IL-13) and the eosinophil accumulation was shown in the lungs of mice immunized with a non-adjuvanted split vaccine after a challenge with a phylogenetically distant influenza virus. We also investigated the ability of T-cells to induce protection against the heterologous virus challenge in passive-transfer and T-cell depletion experiments. Passive transfer of sera from mice immunized with split/CpG vaccine reduced weight loss, inflammatory cytokine production, and viral replication in lungs after challenge. Passive transfer of T-cells from immunized to naïve mice did not reduce weight loss and viral replication but affected the innate immune cell population dynamics and cytokine response in mouse lungs after the challenge. The depletion of CD4⁺ T-cells did not affect viral replication in mouse lungs but influenced cytokine production and innate immune cell population dynamics.