Construction and Immunological Evaluation of Multivalent Hepatitis B Virus (HBV) Core Virus-Like Particles Carrying HBV and HCV Epitopes

A multivalent vaccine candidate against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was constructed on the basis of HBV core (HBc) virus-like particles (VLPs) as carriers. Chimeric VLPs that carried a virus-neutralizing HBV pre-S1 epitope corresponding to amino acids (aa) 20 to 47 in the major immunodominant region (MIR) and a highly conserved N-terminal HCV core epitope corresponding to aa 1 to 60 at the C terminus of the truncated HBcΔ protein (N-terminal aa 1 to 144 of full-length HBc) were produced in Escherichia coli cells and examined for their antigenicity and immunogenicity. The presence of two different foreign epitopes within the HBc molecule did not interfere with its VLP-forming ability, with the HBV pre-S1 epitope exposed on the surface and the HCV core epitope buried within the VLPs. After immunization of BALB/c mice, specific T-cell activation by both foreign epitopes and a high-titer antibody response against the pre-S1 epitope were found, whereas an antibody response against the HBc carrier was notably suppressed. Both inserted epitopes also induced a specific cytotoxic-T-lymphocyte (CTL) response, as shown by the gamma interferon (IFN-γ) production profile.Genetically engineered virus-like particle (VLP)-based vaccines are one of the most promising tools in modern vaccinology. VLPs from almost all classes of viruses are being evaluated now or have just been adopted to use as carriers for presentation of foreign immunological epitopes (for a review, see references 29 and 31). VLP technologies possess obvious advantages for the generation of safe and efficacious vaccines. First, the repetitive antigenic structure of VLPs makes them highly immunogenic. Second, VLPs lack viral genomes or genes and are noninfectious, although they mimic infectious viruses in their structural and immunological features. Third, VLPs are generated by highly efficient heterologous expression of the cloned viral structural genes with subsequent quantitative in vivo or in vitro self-assembly of their products. Fourth, VLPs can be obtained by simple and efficient purification procedures. VLPs can be used for a broad range of applications, but the generation of vaccines against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is of special interest.The HBV core (HBc) protein was first reported as a promising VLP carrier in 1986 and was published in 1987 (6, 10, 24). In many ways, HBc occupies a unique position among the VLP carriers because of its high-level synthesis and efficient self-assembly in virtually all known homologous and heterologous expression systems, including bacteria (for a review, see references 29 to 31). The major HBc B-cell epitopes (c and e1) are localized within the major immunodominant region (MIR), whereas the next important epitope, e2, is localized around amino acid position 130, close to the C-terminal histone-like region (for a review, see reference 30).The high-resolution spatial structure of HBc icosahedrons (11, 43) shows that the MIR is located on the tip of the spike, around the most protruding region between amino acids (aa) 78 and 82. For this reason, the MIR is generally accepted as the target site of choice for insertion of foreign epitopes (30). The other widely accepted site for insertions is C-terminal position 144, a short stretch after the e2 epitope. For C-terminal insertions, so-called HBcΔ vectors lacking a 39-aa-long positively charged C-terminal histone-like fragment are preferred for their high insertion capacities (up to 741 amino acid residues) (30).Here, we present the construction and preliminary immunological characterization of a first multivalent HBV and HCV vaccine candidate. As an HBV epitope, we chose the pre-S1 sequence aa 20 to 47, which alone is able to elicit HBV-neutralizing and protective antibodies (23), for insertion into the HBc MIR. Concurrently, we inserted at the C terminus of the HBcΔ vector the N-terminal 60-aa fragment of the HCV core, which is highly conserved among various HCV genotypes with amino acid homology exceeding 95% (12, 14) and therefore is an attractive target for the generation of an HCV vaccine (19, 41). Such a combination of foreign epitopes did not prevent correct self-assembly of chimeric HBc-based particles and provided them with specific HBV and HCV antigenicity and immunogenicity in mice.