at 94?C, 1 min at 55?C and 1 min at 72?C for 30 cycles. as therapeutic Mouse monoclonal to SKP2 reagents. Keywords: Flow cytometry, Hepatitis C virus, E1 envelope, Therapeutic antibodies, Direct immuno-fluorescence, HepG2 cells INTRODUCTION Hepatitis C virus (HCV) is the major etiology of non-A, non-B hepatitis that infects 170 million people worldwide. Approximately 70% to 80% of HCV patients develop chronic hepatitis, 20% to 30% of which progress to liver cirrhosis[1]. At present, there is no vaccine available to prevent HCV infection, and current therapies are not optimal. The initial steps of HCV infection (binding and entry) that are critical for tissue tropism, and hence pathogenesis, are poorly understood. Studies to elucidate this process have been hampered by the lack of robust cell culture systems or convenient small animal models that can support HCV infection. HCV is an enveloped, positive-stranded RNA virus that belongs to the family. Based on the sequence heterogeneity of the genome, HCV is classified into six major genotypes and 100 subtypes[1]. The viral genome (9.6 kb) is translated into a single poly-protein of 3?000 amino acids (aa). A combination of host and viral proteases are involved in poly-protein processing to give at least nine different proteins[2]. Like other enveloped viruses, E1 and E2 proteins most likely play a pivotal role in the assembly of infectious particle and in the initiation of viral infection by binding to its cellular receptor(s). It has been suggested that the humoral and cellular immune responses to the E1 envelope protein are largely impaired in patients with chronic active hepatitis C, and that such responses may be important for clearance of HCV[3]. Leroux-Roels et al,[4] have previously reported APR-246 that cellular immune responses to the E1 envelope protein are almost absent in patients with chronic active hepatitis C, while long-term APR-246 responders to IFN- therapy, on average, show higher levels of E1 antibodies[5]. Depraetere et al,[6] suggesting that E1 antibodies contribute, at least partially, in viral elimination. Baumert et al[7] confirmed the presence of such higher antibody levels directed at the HCV envelope in sustained viral responders to IFN-based therapy. Maertens et al[8] have been able to show that therapeutic vaccination of chronically infected chimpanzees with the HCV E1 protein induces the appearance of T-helper immune responses and antibodies which are very rarely seen in patients[6,7] or chimpanzees[9] with chronic active hepatitis C. The use of a viral envelope protein has the advantage of potentially inducing not only T-cell responses, but also neutralizing antibodies and complement activation. The E1 protein was chosen as vaccine rather than the E2 protein not only because E2 has the disadvantage of displaying a very high strain-to-strain variation in the hypervariable region I (HVRI), but also because of the higher degree of inter-genotype cross-reactivity of E1 as compared to E2. The E2 hypervariable region is immunodominant and neutralizable[10]. However, strong anti-E2 vaccine responses directed against the HVR I do not cross-neutralize with the infecting strain[11,12]. Although the E1 antigen is also variable between genotypes, it shows a relatively high degree of conservation within the subtypes, such as subtype 1b[13], the APR-246 most widespread genotype worldwide. In the present study, we aimed to examine the neutralizing -related activity of an in house made antibody against the most conserved region of HCV E1 protein, for blocking the entry of HCV virion to HepG2 cells. MATERIALS AND METHODS Infected Serum samples We selected 28 serum samples which tested positive for HCV RNA at different viral loads (ranging from 615 to 3.2 million IU/ mL) for infection experiments. The presence of HCV RNA APR-246 was determined by nested RT-PCR and genotyped using Innolipa system (Bayer, Germany). Viral loads were determined by branched DNA method (Bayer, Germany). Design of E1 conserved synthetic peptides Sequence analysis of HCV quasi-species in local patients (Data not shown), revealed several conserved regions within the core and the E1 proteins. We designed 4 core and one E1-specific peptides and analyzed their ability to detect circulating antibodies in infected patients. The results of these studies showed that only one core-peptide (C1) had reasonable sensitivity and specificity. However the rest of peptides including E1 peptide had poor reactivity with circulating antibodies[14]. In the present study, we raised HCV specific polyclonal antibodies against the 4 core.