Comparison of the mucosal adjuvanticities of two Toll-like receptor ligands for recombinant adenovirus-delivered African swine fever virus fusion antigens

The mucosal immunity plays an important role against African swine fever virus (ASFV) infection and the efficacy of mucosal vaccination is highly dependent on the adjuvant. However, the mucosal adjuvant for ASFV vaccination is poorly studied. Toll-like receptor (TLR) ligands such as the FlaB flagellin from Vibrio vulnificus and the heat shock protein 70 from Mycobacterium tuberculosis (mHsp70) hold a great promise as novel vaccine adjuvant. However, the mucosal adjuvanticities of such TLR ligands have not been studied in pigs. In this study, three recombinant Adenovirus (rAd) vectors, namely rAd-F1, rAd-FlaB-F1 and rAd-F1-Hsp70, were constructed by fusing the FlaB or mHsp70 to ASFV CD2v-p30-p54 fusion antigen. Western blotting showed that the three fusion proteins expressed in rAd-infected cells reacted positively with ASFV antibodies. After intranasal immunization of pigs with the three rAd vectors, the antigen-specific IgG antibodies were detectable from day 7 after primary immunization, which were significantly boosted by the secondary immunization. Strong Th1/Th2 cytokine responses were detected in the peripheral blood mononuclear cells. Compared to immunization with the control rAd-F1, significantly higher levels of the antigen-specific IgA antibodies were detected in the nasal fluids, tracheal washes and lung lavages.1 Compared to immunization with rAd-Flab-F1, immunization with rAd-F1-Hsp70 induced significantly stronger mucosal IgA antibody response. Cytokine detection of the pig lung lavages showed that the elevated IgA antibody responses were correlated mainly with IL-4, IL-10 and IFN-α, which were confirmed by the significantly increased antigen-recall cytokine expression in the porcine alveolar macrophages. These data suggest that mHsp70 has potent mucosal adjuvanticity in pigs, and the fusion rAd vector can be used for ASFV mucosal vaccine development.