Immunization with recombinant HPV16-E7d in fusion with Flagellin as a cancer vaccine: Effect of antigen-adjuvant orientation on the immune response pattern.

Human papillomavirus (HPV) is the leading cause of cervical cancer worldwide. The pathogenesis of HPV is mainly dependent on its E7 and E6 proteins. Up to now, different adjuvants have been used to enhance the efficacy of the immune response against these two proteins. In this study, Flagellin (FLA) was used as adjuvant to test adjuvant activity and also see whether its orientation of attachment can affect the immune response pattern. The E7d-FLA and FLA-E7d in pET28a vector were constructed and then the recombinant proteins were expressed in E. coli BL21 (DE3) bacteria under IPTG induction. The expression of recombinant E7d-FLA and FLA-E7d proteins is confirmed by SDS-PAGE and western blot. Then, recombinant fusion proteins were purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. The recombinant proteins were checked for endotoxin contamination and then quantified by Bradford. Eight-to-ten-week-old male Balb/C mice were immunized subcutaneously with 10 µg recombinant E7d-FLA, FLA-E7d and HPV16E7d vaccine on days 0, 14 and 28. In addition, PBS and FLA groups were considered as control group. Then, spleen cells were harvested to assess lymphocyte proliferation and IFN-γ, IL-4 and IL-17 cytokines. In addition, mice sera were used for specific total IgG and IgG1, IgG2a, IgG2b and IgM antibodies assessment by ELISA. The results show that E7d-FLA is more potent in the induction of lymphocyte proliferation, CTL response and specific total IgG, IgG2a and IgG2b response, while the FLA-E7d vaccine was associated with more IFN-γ, and IL-17 cytokine response. The results of this study proved the ability of FLA as an adjuvant in fusion with E7d in the induction of cellular and humoral immune responses. In addition, it also emphasizes that antigen-adjuvant orientation can affect the immune response strength and polarization against HPV E7d vaccine candidate. HIGHLIGHTS: Flagellin is attached to HPV-16 E7d at the C- or N-terminus to create E7d-FLA and FLA-E7d candidate vaccines. The E7d-FLA vaccine showed a significant increase in lymphocyte proliferation, CTL response and IgG response versus FLA-E7d vaccine. The FLA-E7d vaccine is associated with a significant increase in IFN-γ and IL-17 cytokines response versus E7d-FLA vaccine. It seems that that antigen-adjuvant orientation is an important parameter in the strength and polarization of immune response in HPV E7d vaccine candidate.