Peptide-based T cell vaccines targeting the conserved epitopes of influenza virus can provide cross-protection against distantly related strains, but they are generally not immunogenic. Foreign antigen-specific regulatory T (Treg) cells are induced under subimmunogenic conditions peripherally, although their development and role in vaccine-mediated antiviral immunity is unclear. Here, we demonstrated primary vaccination with peptides alone significantly induced antigen-specific Foxp3 Treg cells, which were further expanded by repeated vaccination with unadjuvanted peptides. Certain adjuvants, including CpG, suppressed the induction and expansion of antigen-specific Treg cells by peptide vaccination. Interestingly, secondary influenza virus infection significantly increased the frequency of preexisting antigen-specific Treg cells, although primary infection barely induced them. Importantly, specific depletion of vaccine-induced antigen-specific Treg cells promoted influenza viral clearance, indicating their inhibitory role in vivo. Immunization with CpG-adjuvanted peptides by the subcutaneous prime-intranasal-boost strategy restricted the recruitment and accumulation of antigen-specific Treg cells in lung, and stimulated robust T cell immunity. Finally, subcutaneous prime-intranasal-boost immunization with CpG-adjuvanted peptides or whole-inactivated influenza vaccines protected mice from heterosubtypic influenza virus infection. In conclusion, antigen-specific Treg cells induced by peptide vaccines attenuate the antiviral immunity against influenza virus infection. CpG-adjuvanted peptide vaccines provide heterosubtypic influenza protection probably by inhibiting Treg development and enhancing T cell immunity.

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T cell immunity targeting the conserved epitopes of influenza virus has the potential to provide cross-protection against distinctly-related strains. Peptide vaccines are an ideal strategy to induce specific antiviral T cell immunity, but subimmunogenic stimulation by peptide vaccines tend to induce regulatory T (Treg) cells. However, it remains unclear about the roles of vaccine-induced antigen-specific Treg cells in vivo. Here, we aimed to investigate how vaccine-induced antigen-specific Treg cells respond during acute influenza virus infection, and the effects of adjuvants on them. By adoptive transfer experiments with OT-II TCR transgenic T cells, we found that the OVA OT-II peptide vaccine induced Treg cells and secondary vaccination further expanded them. Infection with influenza virus containing the OVA OT-II peptide also drove the expansion of pre-existing vaccine-induced Treg cells. Importantly, specific depletion of vaccine-induced antigen-specific Treg cells enhanced the antiviral immunity. Vaccination combined with adjuvants, especially with CpG, suppressed the development of vaccine-induced antigen-specific Treg cells. Finally, CpG-adjuvanted OVA protein or whole inactivated influenza vaccine by the subcutaneous-prime-intranasal-boost strategy reduced the ratio of antigen-specific Treg cells in lung and protected mice from the heterosubtypic influenza infection. In conclusion, unadjuvanted peptide vaccines promoted antigen-specific Treg cells, which were further expanded upon acute influenza infection. The CpG adjuvant restricted the development of vaccine-induced antigen-specific Treg cells, and enhanced the antiviral T cell immunity against heterosubtypic influenza infection.

Peptide vaccines are an ideal strategy to induce cross-protective T cell immunity against a broad range of influenza virus strains, but they are subimmunogenic and tend to induce regulatory T (Treg) cells. Our previous study has shown that adjuvanted peptide vaccines can suppress the development of antigen-specific Treg cells. MF59 is the first oil-in-water adjuvant approved for protein-based influenza vaccine, which enhances humoral and Th2 cellular immune responses. However, the role of MF59 in peptide-based vaccines regarding the induction of T cell immunity is unknown. We thus investigated the effects of MF59-adjuvanted peptide vaccines on antigen-specific T cell immunity against influenza virus infection. Using OT-I and OT-II TCR transgenic T cells, we found that the MF59-adjuvanted cognate OVA peptide vaccines induced a significant portion of OT-II Treg cells in primary immunization. However, MF59-adjuvanted peptide vaccine could prevent the expansion of pre-existing antigen-specific Treg cells. Of note, primary influenza virus infection induced a negligible portion of antigen-specific Treg cells, but secondary influenza virus infection drove the expansion of pre-existing Treg cells. Interestingly, swapping of the sequential immunizations with MF59-peptide vaccination and influenza virus infection resulted in very different levels of antigen-specific Treg cells, suggesting that the first immunization experience affects the development of antigen-specific Treg cells. Finally, MF59-peptide vaccines stimulated the production of IFN-γ and TNF-α of CD4 and CD8 T cells. In conclusion, MF59-peptide vaccine exhibits a unique feature in stimulation of antigen-specific T cell immunity.