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Subunit vaccines that are poorly immunogenic are often combined with adjuvants for immunization. Our previous research identified a pneumolysin variant (ΔA146Ply), a Toll-like receptor 4 agonist, that was an effective adjuvant in the protection of fusion protein DnaJ-ΔA146Ply against mucosal  Streptococcus pneumoniae  infections. For pneumococcal vaccines, World Health Organization recommend injection as a regular vaccination approach. Subcutaneous immunization is a common and effective method of injection, so we explored the immunity mechanism of subcutaneous immunization with DnaJ-ΔA146Ply. We found that mice immunized subcutaneously with fusion proteins ΔA146Ply-DnaJ and DnaJ-ΔA146Ply produced a higher anti-DnaJ IgG titer than when DnaJ alone was administered. DnaJ-ΔA146Ply induced both B-cell and T-cell-dependent protection against both colonization and lethal pneumococcal infections. Levels of IFN-γ, IL-4, and IL-17A were also elevated in DnaJ-ΔA146Ply immunized mice. However, all these effects were negated in TLR4 −/−  mice compared to WT mice immunized with DnaJ-ΔA146Ply. B-cell-deficient μMT mice, nude mice, IFN-γ −/− , and IL-4 −/−  mice immunized with DnaJ-ΔA146Ply could not resist infection with pneumococci. IL-17A −/−  and TLR4 −/−  mice did not benefit from DnaJ-ΔPly immunization in colonization experiments although their survival was not impaired compared with WT mice. Collectively, our data indicated that ΔA146Ply can be a potential subcutaneous adjuvant, and the DnaJ-ΔA146Ply fusion protein induces both humoral and cellular immune response to resist  S. pneumoniae  infection. The protective effect of colonization also depends on TLR4.

frontiers in immunology

Subcutaneous Immunization with Fusion Protein DnaJ-ΔA146Ply without Additional Adjuvants Induces both Humoral and Cellular Immunity against Pneumococcal Infection Partially Depending on TLR4