Heat shock protein 70 fused to or complexed with Hantavirus nucleocapsid protein significantly enhances specific humoral and cellular immune responses in C57BL/6 mice

Abstract

Heat shock proteins (HSPs) are known to act as an effective molecular adjuvant to enhance the induction of antigen peptide-specific cellular immunity, when coupled with the antigen or peptide. Hantaan virus (HTNV) nucleocapsid protein (NP) is relatively conserved among hantaviruses and highly immunogenic in both animals and humans. To analyze the influence of HSP70 on NP vaccine potency, and evaluate the possibility of developing a novel effective vaccine against hantaviruses, we constructed prokaryotic expression plasmids, and expressed three recombinant proteins, namely, HTNV NP, HSP70 and HSP70-NP fusion protein. As an alternative to fusion protein, we also generated HSP70 and HTNV NP complexes (HSP70+NP) in vitro. C57BL/6 mice were immunized with those recombinant proteins, the humoral and cellular responses elicited against NP were measured by ELISA, fluorescence flow cytometry, cytotoxicity assays, and IFN-gamma ELISPOT assay. We found that immunization of mice with HSP70-NP fusion protein, or HSP70+NP complexes elicited significantly higher NP-specific antibody titers, frequencies of IFN-gamma-producing cells and cytotoxic T lymphocyte (CTL) activities in vivo than conventional HTNV NP vaccination. Antibody isotype analysis showed that the antibody response was characterized by a higher HTNV NP-specific titer of IgG2a than IgG1 antibodies, resulting in a significant higher IgG2a/IgG1 ratio. By comparison, HSP70-NP fusion protein is significantly superior to HSP70+NP complexes in enhancement of NP antigenicity. These results indicated that HSP70, when fused to or complexed with HTNV NP, greatly enhance NP vaccine potency by preferential induction of a predominant Th1 immune response in a NP-specific, HSP70-dependent manner.