Investigating The Effect Of Levamisole On The Immune Enhancement Of Staphylococcus Aureus Clumping Factor A Subunit Vaccines

Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of diseases. Due to the emergence of serious antibiotic resistance to S.aureus, it is urgently needed to develop vaccine of S.aureus. And subunit protein vaccine occupies an important position in the treatment of S. aureus disease. However, protein vaccine has the limit of activating the cellular immune responses, choosing a suitable adjuvant which could enhance the immune effect of the vaccine is a novel immunization strategy. Levamisole (LMS), a synthetic low molecular weight compound, could stimulate the functions of cellular immunity in normal. ClfA is an important adhesion factor during early phases S.aureus infection. In this study, LMS combined with the recombinant ClfA were performed to subcutaneous injection in mice to investigate whether co-administration injection of LMS as the adjuvant could enhance protein vaccine immune effect in animals.In our study, ClfA fragment of S. aureus was amplificated by PCR at frist. Then the DNA fragments were introduced to construct recombinant expression vector pGEX-4T-1-ClfA. The recombinant plasmid was transformed into E. coli BL21 (DE3) and the recombinant protein expected was properly expressed subsequently. And the recombinant proteins were analyzed by SDS-PAGE and Western Blot. The recombinant protein was purified and immunized with adjuvant LMS in BALB/c mouse subsequently. ELISAs were performed to detect the specific antibodies IgG titers against ClfA and IgGl, as well as IgG2a subtypes; CFSE staining was used to detect T cell proliferation responses; cell division of CD4+-IFN-y, CD4+-IL-17 and CD8+-IFN-y cells were determined by flow cytometry; and the cytokines secretion of IFN-y, IL-4 and IL-17A were tested by ELISA in the supernatants of spleen cells.The result showed that LMS plus protein vaccine group stimulated much higher IgG level than ClfA alone after immunization (P<0.05), and the subtype of IgG induced by ClfA plus LMS group was predominantly IgG2a, nevertheless, the protein vaccine ClfA was mainly IgGl. Flow cytometry result indicated that the cell division of CD4+-IFN-y, CD4+-IL-17 and CD8+-IFN-y cells was significantly higher in ClfA plus LMS group than ClfA alone. Futhermore, levels of both IL-17A and IFN-y in the supernatants of spleen cells in LMS+ClfA group were higher than those in ClfA group (P<0.05).These result demonstrated that LMS as an adjuvant could not only strongly enhance the humoral immune responses induced by the subunit protein vaccine ClfA, but also increase the level of Thl-type cellular immune response.To some extent, LMS could trigger Thl7-type cellular immune responses.