Adjuvant effect of recombinant 4-1BB ligand on therapeutic recombinant human papillomavirus-16 protein vaccine

Abstract

Objective To study the adjuvant effect of recombinant 4-1BB ligand (r4-1BBL) on therapeutic recombinant human papillomavirus-16 (HPV16) protein vaccine. Methods The coding sequence of 4-1BBL extracellular functional domain was optimized and cloned into the pET28a vector at NdeⅠand XhoⅠsites directionally. The recombinant plasmid pET28a-4-1BBL was transformed into E. coli BL21(DE3), and protein expression was induced with IPTG. Expression product was identified by western blotting, and protein structure was analyzed by non-reducing SDS-PAGE and electron microscopy. CCK-8 kit was used to detect the effect of r4-1BBL on mouse T cell proliferation in vitro. C57BL/6 mice were immunized with a combination of r4-1BBL and HPV16 protein vaccine, and the level of cellular immune responses in mice were detected by ELISPOT, and the effect of r4-1BBL on tumor growth inhibition by HPV16 protein vaccine was observed. Results The codon optimized 4-1BBL extracellular functional domain could be expressed in recombinant plasmid pET28a-4-1BBL. The expressed proteins existed as inclusion body and soluble form simultaneously. The structure of soluble proteins was mainly dimer and subunit-like structure was observed under electron microscope. The purified r4-1BBL enhanced the proliferation of mice spleen lymphocytes in vitro. In comparison with HPV16 protein vaccine alone, HPV16 protein vaccine combined with r4-1BBL induced stronger T cell immune responses (t=3.525, P=0.024) and higher tumor growth inhibition (t=2.534, P=0.021). Conclusion r4-1BBL can improve the immune effect of HPV16 protein vaccine in mice, and has the potential as an adjuvant for HPV16 protein vaccine. Key words: Recombinant proteins; Adjuvants, immunlolgic; Human papillomavirus 16; Papillomavirus vaccines