Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. when purified from the core-glycosylated intracellular fraction whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells the antigen of choice is a mammalian cell-expressed monomeric E2 protein purified from the intracellular fraction. Hepatitis C virus (HCV) is the major cause of chronic hepatitis which can evolve into cirrhosis liver failure or hepatocellular carcinoma (2 4 There is no vaccine for HCV and the only available treatment a combination of alpha interferon and ribavirin is efficacious in only a minority of patients (33). Given that approximately 200 million chronic HCV infections have been estimated worldwide (52) there is a pressing need to develop new therapies and vaccination strategies. The development of such strategies will be aided greatly by a more complete picture of the structure-function features of HCV proteins. HCV JTT-705 (Dalcetrapib) is an enveloped plus-strand RNA virus of the family (24). Its genome is 9.5 kb in length with one open reading frame that is translated as a single polyprotein which is processed by host and virus proteases into at least three structural and seven presumptive nonstructural proteins with various enzymatic activities (5 22 47 Two glycoproteins E1 and E2 are probably virion envelope proteins containing multiple N-linked glycosylation sites and form heterodimers in vitro (23 32 35 45 The coexpressed E1-E2 complex localizes to the endoplasmic reticulum (ER) and lacks complex N-linked glycans (7 8 13 JTT-705 (Dalcetrapib) 15 45 49 Neutralizing antibodies often play a pivotal role in defeating viral infections including prominent human pathogens such as influenza virus and hepatitis B virus (9 28 The assessment of neutralizing antibody responses to HCV has been Mouse monoclonal to SNCA difficult because HCV does not grow efficiently in cell cultures. To overcome this obstacle we developed a surrogate assay which measures the ability of antibodies to inhibit the binding of recombinant E2 to its putative cellular receptor CD81 on human cells (neutralization-of-binding [NOB] assay) (44 46 CD81 is a membrane-associated protein belonging to the family of tetraspanins (30). Its large extracellular loop (LEL) binds E2 with a of 1 1.8 nM (42) and this interaction appears necessary and sufficient for binding of JTT-705 (Dalcetrapib) bona fide HCV particles (44). Importantly chimpanzee sera containing antienvelope antibodies which are capable of preventing HCV infection in vivo inhibit the binding of HCV to CD81 in vitro suggesting that this interaction is relevant to infection (44). Our research has focused primarily on comparing vaccine formulations of HCV E2 which is an obvious candidate for inclusion in a subunit vaccine because of its potential role in HCV attachment. Thus targeting antibodies to HCV E2 could be a viable strategy for disrupting the JTT-705 (Dalcetrapib) HCV-CD81 interaction. Despite the inherent difficulties in studying HCV infection and the lack of a clear correlate of protection there is evidence that neutralizing antibodies can be protective. Studies performed with human immunoglobulin (Ig) preparations have suggested some degree of efficacy in preventing the transmission of HCV in the transfusion setting after liver transplants and in sexual transmission (17 27 43 Relevant JTT-705 (Dalcetrapib) data on the existence of HCV-specific neutralizing antibodies also come from experimental vaccination studies in chimpanzees the only species besides humans susceptible to infection. When vaccinated with recombinant envelope proteins (E1-E2 heterodimer) chimpanzees developed high titers of anti-E2 antibodies in.