The Investigation of the Prospects for Using NS4A Antigen of Hepatitis C Virus for the Development of Recombinant Mosaic Vaccines with Self-Adjuvant Properties

Abstract

The aim of this study was to design promising variants of recombinant proteins based on NS4A antigen of hepatitis C virus (HCV) for subsequent work on the creation of a mosaic recombinant vaccine against hepatitis C. Methods. The recombinant proteins, containing different fragments of NS4A (belong to HCV subtype 1b) and murine interleukin-2, were prepared by genetic engineering approaches, using vectors pQE30 and pQE60 for E. coli. The size of the recombinant protein particles were evaluated by atomic force microscopy. Immunogenicity of these recombinant proteins was tested for Balb/c mice. The murine sera were analyzed by enzyme immunoassay. The recombinant proteins were also tested by immunoblotting with human sera specific to HCV antigens. Results. Six variants of recombinant genetic engineering constructions based on NS4A antigen of hepatitis C virus were designed. In the first variant amino acid sequence of NS4A was inserted using vector pQE60 into the immunodominant loop of HBc protein (core protein of hepatitis B virus). However, further analysis of the product showed the absence of virus-like particles in it. The following three constructs (with glycine linker 19s), without it and N-truncated NS4A) were done using vector pQE30. Only N-truncated NS4а product had a high expression level. Then new protein, consisted of NS4A and N-truncated murine interleukin-2 (IL-2), was obtained to enhance immunogenicity. It is known that IL-2 has adjuvant property. The new product (NS4a-IL-2) is well expressed, but it is accumulated in inclusion bodies. It was extracted with 7M guanidine chloride, purified on a Ni-sorbent and dialyzed in PBS. A shortened version of NS4A (ANS4a-IL-2) was also obtained with a high expression level. Taking in account that increasing the repetition of antigenic regions in recombinant constructs can enhance their immunogenicity, we obtained a recombinant protein comprising three repeat of NS4A. But its efficiency of expression was low. The construction NS4a had very poor immunogenicity, but NS4a-IL-2 (which contains the full length NS4A) displayed the best one for Balb/c mice. As it was shown earlier the immunogenicity of the protein preparation is dependent on the presence of aggregates, so we investigated our recombinant proteins for the presence of protein aggregates by atomic force microscopy.The presence of the particles with size of 6 - 8 nm was revealed in solution of NS4a-IL-2. Conclution. Only ANS4a-IL-2 and ANS4a-IL-2 of the six constructs had high expression and antigenic properties. And only NS4a-IL-2 possessed the high immunogenic property. So, this construction can be used for subsequent work on the creation of a mosaic recombinant vaccine against hepatitis C.