In the present work, we have expressed recombinant HCV core, E1, E2, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins by baculovirus system in insect cells. This expression system was chosen in order for the recombinant proteins to undergo all possible posttranslational modifications such as glycosylation and phosphorylation. In addition, in contrast to proteins expressed in E. coli, humans are likely to have very low or nonexisting antibodies against insect cell proteins that might be contaminating the recombinant protein preparations. The expression levels of individual HCV proteins were relatively high and they could be purified by preparative SDS-PAGE (Fig. ). Some, but not all HCV genomes are also encoding protein F from an alternative reading frame of the core sequence. For unknown reasons we were not able to produce the F protein by baculovirus expression and therefore we could not include this protein in our analyses. We also did not express the small ion channel protein p7.
We used full-length recombinant HCV proteins from HCV genotype 1b to analyze antibody responses against individual viral proteins in patients suffering from chronic infection caused by HCV genotypes 1, 2, 3 or 4. Our analyzes revealed that all recombinant HCV proteins except that of NS2 were immunogenic in humans and there were no major differences in the magnitude of immune responses at least against the core and NS proteins between the different genotype infections. It was of interest that NS2 protein appears to completely lack immunogenicity in humans. This was unexpected, but yet we are confident with the results, since the sequence of NS2 expression construct was correct and monoclonal anti-NS2 antibodies readily detected the transiently expressed protein [24
]. This may indicate that in humans there may be proteases or other molecules homologous to NS2 leading to an inability of the host to recognize the NS2 protein as foreign. Further evidence that baculovirus expressed recombinant proteins of HCV genotype 1b are suitable for immunological analyses was obtained from the comparison of our Western blot analysis with the commercial INNO-LIA Score test, which is able to detect antibodies from genotypes 1–5. These methods showed a very good correlation in the case of anti-core, NS4A+B and NS5A antibody responses. This suggests that the baculovirus produced HCV proteins provide valuable and very specific research reagents for analyzing HCV-specific immune responses against HCV. However, the INNO-LIA Score test was more sensitive than the Western blot method in the case of anti-NS3 and E2 antibodies. The reason for this discrepancy is not known, but it may be that the relative amount of HCV antigens used in the INNO-LIA assay was higher that what we used in the Western blot analysis. In addition, the conformation of the recombinant proteins may also contribute to the results, since it is known that many antigenic epitopes in viral envelope glycoproteins like the E2 of HCV are likely conformational and thus these sites are not necessarily detected by antibodies in denatured proteins. By increasing the amount of viral antigens in Western blot analysis we could theoretically have been able to enhance the sensitivity of our analysis. However, the idea in our analysis was to systematically study the immunogenicity of various HCV proteins in order to reveal which viral proteins are the targets of humoral anti-HCV immune responses in humans. In order to detect the relative immunogenicity of different HCV proteins we used a similar amount of each purified protein in the assay. Based on this analysis we were able to determine qualitative and quantitative differences in host antibody responses to different HCV proteins, which had not been systematically studied before.
Previously, anti-HCV antibody responses have been analysed in acute and chronic phases of HCV infection [25
]. In the present study we focused on patients suffering from a chronic HCV infection and we found remarkable differences in the frequency of anti-HCV antibody responses as well as there was a lot of variation in antibody titers against individual HCV proteins (Fig. ). We found out that 97% of the sera studied recognized the core protein in very high levels, whereas the other proteins such as the NS4B, NS3, NS5A and E2 were found to be immunogenic in 85% to 31% of the cases, respectively (Fig ). A study carried out by Chen and coworkers among 60 chronic HCV patients, revealed E2 antibodies in 98%, core in 97%, NS3 in 88%, NS5 in 68% and NS4 in 48% of the cases [27
]. As analyzed by EIA the highest antibody levels were observed against the core protein (ca. 1:5000), while the antibody responses against other viral proteins or peptides derived of them remained at a lower level [27
]. As a whole the results of the above study are concordant with the observations of the present study, except that our Western blot analysis gave up to 10-fold higher titers against the core proteins and several fold higher levels of specific antibody responses against other HCV proteins. Also Nikolaeva and coworkers observed the core protein to be highly immunogenic (antibody titers up to 1:40 000) while other HCV proteins were less important immunogens in chronic HCV patients [25
]. Direct comparisons of the frequencies and antibody levels to individual HCV proteins in different studies is very difficult, since the methods to produce and purify viral antigens vary and also the form of the assay to detect anti-HCV antibodies varies from one study to another. In our analysis we decided to use the full-length baculovirus-expressed HCV proteins and Western blot analysis in order to be sure of the specificity of the antibody responses to a given protein. One of the drawbacks of the assay is, however, that only antibodies against linear antigenic epitopes within the denatured proteins are being detected in Western blotting.
The prevalence of anti-HCV antibodies have been followed during the chronic phase of infection [25
]. When we analysed sera from five HCV RNA and antibody positive patients during a period of 18 to 25 month, the antibody levels against the major immunogenic proteins were found to remain relatively constant. However, in three patients there were some changes in anti-HCV antibody levels, namely a weak decrease in the core and NS-specific antibody levels during the follow-up was seen. Similar analysis by others [27
] revealed very similar results with highly persistent antibody patterns. While in most cases anti-HCV antibodies remain at a constant level, there were some individuals whose antibody levels showed some fluctuation [27