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

Subunit vaccines that are poorly immunogenic are often combined with adjuvants for immunization. Our previous research identified a pneumolysin variant (∆A146Ply), a Toll - like receptor 4 agonist, that was an effective adjuvant in the protection of fusion protein DnaJ-∆A146Ply against mucosal Streptococcus pneumoniae infections. For pneumcoccal 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- ∆A146Ply. We found that mice immunized subcutaneously with fusion proteins ∆A146Ply-DnaJ and DnaJ-∆A146Ply produced a higher anti-DnaJ IgG titer than when DnaJ alone was administered. DnaJ-∆A146Ply 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-∆A146Ply immunized mice. However, all these effects were negated in TLR4-/- mice compared to WT mice immunized with DnaJ-∆A146Ply. B-cell-deficient μMT mice, nude mice, IFN-γ-/- and IL-4-/- mice immunized with DnaJ-∆A146Ply could not resist infection with pneumococci. IL-17A-/- and TLR4-/- mice did not benefit from DnaJ-∆Ply immunization in colonization experiments although their survival was not impaired compared with WT mice. Collectively, our data indicated that ∆A146Ply can be a potential subcutaneous adjuvant, and the DnaJ-∆A146Ply fusion protein induces both humoral and cellular immune response to resist S. pneumoniae infection. The protective effect of colonization also depends on TLR4.