The genome of Hepatitis C Virus (HCV) consists of 5' and 3' untranslated regions that flank a single open reading frame (ORF) encoding structural and non-structural proteins [1] The structural proteins of HCV include the capsid (core) protein and two envelope glycoproteins (E1 and E2). Around 80% of infected individuals develop chronic infections, more than 2% of the globe is chronically infected and infection is the main etiological agent of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma [2] Among the mechanisms the virus exerts to persist infection are, down regulating expression of its glycoproteins on cell surface, thus reducing the possibility of antibody recognition and destruction of infected cells, interference with the correct expression of major histocompatibility complexes (MHCs) on the cell surface or blocking antiviral immune responses such as the release of interferons (IFNs) and complement-mediated lysis [3,4], however, it is still not clear whether liver damage is directly caused by infection or results from host's immune reactions. Accumulation of mutations during viral replication results in a significant genetic heterogeneity and this may be attributed to lack of RNA-dependent RNA polymerase proofreading activity [1]. These mutations result in production of closely related, yet heterogeneous sequences so called quasispecies [[5-7] and [8]]. Such mutations within either the envelop or the core proteins may allow blocking of the viral infectivity or increase its aggression, respectively [9]. It is accepted that each quasispecies functions independently different from other mutants in terms of its response to neutralizing antibodies (escape mutants) and induction of liver damage. This was clearly demonstrated in chimpanzee where antibodies against HVR1 sequence of a quasispecies could neutralize its infection but did not function with other quasispecies [10] and also in patients who develop chronic HCV infection where the immune system is not entirely capable of controlling the infection because of the emergence of multiple escape mutants [11]. Viral mutants differ not only in their infectivity but also in their intracellular pathogenesis. Several lines of evidence showed that the viral core protein plays a key role in development of hepatic steatosis [12], fibrosis [13] and hepatocellular carcinoma [14]. Interestingly, patients were reported to have mild or absent hepatic changes even though viral loads are extremely high [15]. These findings suggest that genomic structure of the quasispecies complex and hence expression of viral proteins in each case are more influential factors on the disease morbidity than the more viral loads. When ALT was used as a marker for liver damage among HCV patients its level showed positive correlation with viral load measured by PCR and branched DNA assays [16]. The same correlation was obtained for both ALT and AST with viral load [17]. Contradictory results were reported [18-20] where significant inverse association between HCV-RNA and ALT levels was observed. From those reports it is evident that the available biochemical, molecular and serological tools, although sensitive and specific, cannot allow clear conclusions with respect to HCV induced pathogenesis in patients infected with different genotypes. On the other hand for a developing country like Egypt where genotype 4 is prevailing [21], there is an increasing need for a supplemental assay besides the costly molecular quantification of viral RNA load for sensitive evaluation of the severity of HCV disease before treatment or during post therapeutic follow ups. This would help better assessment of early response to the expensive peg-interferon/ribavirin combined therapy. The aim of the present work was to evaluate the performance of home made polyclonal mono-specific antibodies raised in rabbits against synthetic peptides of conserved core and envelope sequences as quantitative assays for HCV core and envelope antigens. Measurements of circulating viral structural proteins were tested as markers for the degree of liver damage using ALT and AST levels as indicators. The long term objective was to enhance the cost effectiveness of therapy via better monitoring of patients' response.

Monitoring the pathogenesis of human HCV disease has been a conflicting issue. Several investigators have looked for laboratory tools that can monitor progress in liver pathogenesis and response to antiviral treatments, yet, the obtained results did not allow clear conclusions and dramatically varied among different HCV genotypes [34,35]. These inter-individual variations in diagnosing active infection, responsiveness to treatment and development of symptoms could be due to the extensive mutational rates in HCV genome followed by emergence of several quasispecies in a single patient [[36-38] and [8]]. Thus the present study was designed to demonstrate the complexity in diagnosing HCV and to discuss whether testing the levels of viral antigens correlates with the severity of the disease and whether the available tools including the quantification of viral load are reliable. Measurements of anti-HCV core and E1 antibodies were expected to be more useful than viral load in better diagnosis of active HCV infection since they will detect viral proteins which reflect the ability of assembling viral particles and subsequent intracellular inflammatory reactions, not only the existence of viral RNA. The anti-synthetic peptide antibodies used in the present study have recently been shown sensitive to detect viral core and E1 antigens in PBMCs derived from HCV infected patients [39]. Furthermore, the conserved viral core and E1 peptides that were used to develop the polyclonal antibodies in rabbits were recognized by human anti- HCV IgG antibodies in more than 90 % of infected patients [30] thus suggesting that these two peptides are immunogenic in humans and rabbits. The results presented herein clearly demonstrate that our polyclonal antibodies are 100% sensitive since they were able to detect viral antigens in all HCV RNA positive cases. Viral genotyping revealed that all cases included contain HCV genotype 4, probably explaining disagreement with previous reports on genotypes 1 and 2 that displayed positive correlation between core levels and viral loads [[40,41] and [42]]. This disagreement could likely be due to differences in the nature and antigenicity of the detected core epitopes in our study than in previous reports. Noteworthy, our anti E1 antibodies were raised specifically against conserved peptides [30] among nearly all the reported viral genotypes. The observed high degree of correlation between core and E1 levels (r = 0.98) supports the earlier hypothesis that both proteins are cleaved simultaneously during processing of viral polyprotein precursor [43]. In addition, since bDNA is an assay testing genomic copy number based on untranslated sequence of the virus, thus from our particular point of view it has nothing to do with expressed viral proteins (whether core or E1), and hence, one can very much accept absence of correlation between circulating viral antigens and viral loads. Also, the over sensitivity of the bDNA can come at the expense of its specificity and may question its value as a proper and realistic tool for monitoring the existence of the virus especially in following up the response to treatment Our hypotheses are not only speculative but were also based on our frequent observations among HCV genotype 4 infected patients with high copy number who neither had pathogenic symptoms, abnormal ultrasound findings nor abnormal profile of liver enzymes (our unpublished data). Noteworthy, immune complexes precipitated from sera of those subjects also showed undetectable levels of HCV core and E1 antigens. Our data clearly demonstrate the absence of correlation between viral copy number as measured by bDNA with either levels of ALT or AST and this was in agreement with McCormick et al., [18]; Yeo et al., [20]; Anand and Velez, [44] and contradictory to results obtained by Beld et al., [45]; Sabin et al., [17] and Hassan et al., [16]. Although it seems conflicting that results of Hassan et al., [16] and ours are contradicting while the study populations in both reports were Egyptians, this is not surprising particularly under the random available treatments in Egypt (natural products, liver supportive drugs, antiviral drugs and interferon) which may lead to lowering liver enzymes while having no effect on different viral quasispecies that may exist in infected patients or may result in emergence of new quasispecies with various degrees of aggression. To avoid such conflicts, one has to recruit properly characterized patients who have been certainly receiving no treatment before getting enrolled into such studies. A major scientific advantage of this study is the development of a serological system that can be automated for high throughput analysis. Once this system is available, one can pick up early infected subjects via massive screening programs in heavily infected areas.

HCV antigens were detected in serum, blood mononuclear cells and liver tissues from infected patients [[46,40] and [47]]. In the present study, sera of HCV infected patients were used to provide direct proof on the specificity of the antibodies used in the current study by showing single and discrete bands in western blotting analysis. The immunogenic protein bands around ~38 kDa for core and ~88 kDa for E1 were only found in sera of HCV patients but not in sera from the healthy controls. Zhang et al., [48] found a fusion protein of 32 kDa of HCV core protein exhibiting specific reaction with HCV-positive serum. Also, Choukhi et al., [49] found a 90 kDa band of E1/E2 heterodimer in infected but not in healthy subjects. Further investigations on the structure of the target HCV core and E1 serum antigens are required. In conclusion we present a new combination of serological tools correlating with liver enzyme levels that could be utilized as supplemental tests to viral load testing. This new combination of tests would probably allow clinicians to get to better conclusions about not only the severity of liver pathogenesis in viremic patients but also for following up longitudinally the responsiveness of those patients to whichever antiviral therapy by tracing the titers of core or E1 together with viral loads and liver enzyme levels. Such combination of tools should provide significant data on the selection criteria for the initiation of antiviral therapy and also for deciding whether to continue or terminate the selected antiviral treatment. Furthermore, the development of direct immunoassay test for viral activity should allow cheap and efficient screening for acute and early infections in areas where the viral burden is high.