The HCV core protein may represent a valuable component in the development of a vaccine, as it is the most conserved viral antigen. However, earlier studies using a full-length core sequence have revealed a rather limited immunogenicity of the HCV core when administered to a host in the form of naked DNA (18
). In this study, we found that genetic vaccination with plasmids expressing the full-length HCV core protein was ineffective for inducing specific antibody and T-cell responses. Truncating the HCV core protein could improve its immunogenicity and evoke stronger immune responses in DNA-vaccinated mice. These findings are largely consistent with those of previous studies (27
). We demonstrated that DNA immunization using the truncated HCV core protein induced specific T- and B-cell responses in a dose-dependent manner, while no significant improvement of immune responses could be reached by administration of increased doses of plasmids expressing the full-length HCV core protein. Strikingly, our study again demonstrated that the full-length HCV core protein was apparently able to actively interfere with the priming of specific immune responses. Plasmid DNA expressing the full-length HCV core protein inhibited the priming of T- and B-cell immune responses by a truncated HCV core protein.
It will be of great importance to understand the molecular mechanisms leading to the inhibitory action of the full-length HCV core protein on the priming of specific immune responses. It has been recently proposed that HCV core proteins may be the “core” of the immune deception (48
). The innate immune pathway, through engagement of pattern recognition receptors such as Toll-like receptors (TLR) and helicases, expressed in cells of the classical immune system and in hepatocytes, is affected by the HCV core protein (47
). The immunomodulatory role of the HCV core protein in the adaptive immune system has also been characterized. The HCV core protein is able to inhibit T-cell activation through its interaction with gC1qR (51
). Upon stimulation of human peripheral blood mononuclear cells (PBMC) with either ConA or anti-CD3/CD28, it was found that the HCV core protein inhibited the proliferation of T lymphocytes in a dose-dependent manner (23
). In addition, the production of interleukin-2 (IL-2) and IFN-γ in cells treated with the HCV core protein was markedly diminished compared to that in control cells (12
). Finally, several studies demonstrated that the HCV core protein may cause dysfunction in human DCs and mouse myeloid DCs in vitro
The observed interference of HCV core proteins with the priming of immune responses in the course of DNA vaccination may be explained by the dysfunction of dendritic cells in vivo
). The interference of the HCV core protein was not limited to HCV core protein-specific effector cells but extended to other antigens. This mechanism may be important for the establishment of chronic HCV infection, since the action of the HCV core protein would at least attenuate HCV-specific immune responses.
The ability of the HCV core protein to interfere with the priming of immune responses explained the failed attempts to develop HCV core protein-based vaccines. A high level of expression of the HCV core protein would not induce a better immune response but, rather, would more strongly inhibit the priming of immune responses. Previous studies of murine models consistently showed that immunization of mice with plasmids expressing the HCV core gene could reduce the number of CD4+
T cells; additionally, expression of the HCV core protein in transgenic mice can interfere with the host immune response (21
). However, an adenovirus vector expressing the HCV core gene did not induce obvious immunosuppression in the immunized mice (29
). The mechanisms involved have not yet been clarified.
Manipulation of the HCV core protein may influence its expression and subcellular localization. There is considerable evidence that HCV core protein maturation involves cleavage by signal peptidase (at position 191) and a second cellular protease at the signal peptide to give a product of about 173 aa in length (19
). Other in vitro
studies reported that the HCV core protein could also be truncated at aa 151 or 121, but a detailed processing mechanism was not clear (16
). The processing of the HCV core protein after expression is related to many factors, including the presence of the downstream sequence, expression conditions, or methods to detect HCV core protein. In our experiment using Huh7 hepatoma cells, three cleavage sites (at positions 191, 173, and 151) were apparently used in the processing of the HCV core protein, consistent with results from previous reports using different in vitro
expression systems (16
). We also showed that the HCV core protein, truncated at aa positions 145 or 151, displayed low expression levels, which was likely due to the lower stability levels of these truncated proteins. It has been reported that an HCV core mutant bearing a deletion of aa 125 to 166 is rapidly degraded by the proteasome (17
). The subcellular localization of the HCV core protein is related to the ER targeting and nuclear localization signaling (NLS) sequences of its N terminus. The effect of ER anchoring can obstruct the functions of NLS sequences. Our data were in accordance with those of these previous studies (30
). The proteins 2HA-191-S23′2HA-177-S23 and 2HA-N49-S23 were largely located in the cytoplasm, which is due mainly to the effect of ER anchoring. In contrast, 2HA-C145-S23 and 2HA-N20-S23 displayed the tendency of shifting to the nucleus, which is due to the functional NLS sequences after the deletion of the ER anchor.
Truncated HCV core proteins may lead to a rational vaccine design. DNA-based immunization with a C-terminally truncated HCV core construct induced enhanced host immune responses. A C-terminal 15-aa truncation of the HCV core protein allowed the generation of a slow but potent immune response (9
), while induction of immune responses by an HCV core protein with the C-terminal 37 amino acids removed was more immediate and vigorous (27
). A detailed mechanism of the relationship between immune induction and the length of C-terminal truncation is worthy of further investigation. In addition, a recombinant HCV core protein (aa 1 to 164) was also a good immunogen (18
). Furthermore, the induction of immune responses by truncated HCV core proteins may be enhanced by increasing their doses.