Although a cure for HCV is on the near horizon, emerging drug cocktails will be expensive, associated with side-effects and resistance making a global vaccine an urgent priority given the estimated high incidence of infection around the world. Due to the highly heterogeneous nature of HCV, an effective HCV vaccine which could elicit broadly cross-neutralizing antibodies has represented a major challenge. In this study, we tested for the presence of cross-neutralizing antibodies in human volunteers who were immunized with recombinant glycoproteins gpE1/gpE2 derived from a single HCV strain (HCV1 of genotype 1a). Cross neutralization was tested in Huh-7.5 human hepatoma cell cultures using infectious recombinant HCV (HCVcc) expressing structural proteins of heterologous HCV strains from all known major genotypes, 1–7. Vaccination induced significant neutralizing antibodies against heterologous HCV genotype 1a virus which represents the most common genotype in North America. Of the 16 vaccinees tested, 3 were selected on the basis of strong 1a virus neutralization for testing of broad cross-neutralizing responses. At least 1 vaccinee was shown to elicit broad cross-neutralization against all HCV genotypes. Although observed in only a minority of vaccinees, our results prove the key concept that a vaccine derived from a single strain of HCV can elicit broad cross-neutralizing antibodies against all known major genotypes of HCV and provide considerable encouragement for the further development of a human vaccine against this common, global pathogen.

Although effective hepatitis C virus (HCV) antivirals are on the horizon, a global prophylactic vaccine for HCV remains elusive. The diversity of the virus is a major concern for vaccine development; there are 7 major genotypes of HCV found globally. Therefore, a successful vaccine will need to protect against HCV infection by all genotypes. Despite the diversity, many monoclonal antibodies (MAbs) with broadly cross-neutralizing activity have been described, suggesting the presence of conserved epitopes that can be targeted to prevent infection. Similarly, a vaccine comprising recombinant envelope glycoproteins (rE1E2) derived from the genotype 1a HCV-1 strain has been shown to be capable of eliciting cross-neutralizing antibodies in guinea pigs, chimpanzees, and healthy human volunteers. In order to investigate the basis for this cross-neutralization, epitope mapping of anti-E1E2 antibodies present within antisera from goats and humans immunized with HCV-1 rE1E2 was conducted through peptide mapping and competition studies with a panel of cross-neutralizing MAbs targeting various epitopes within E1E2. The immunized-goat antiserum was shown to compete with the binding of all MAbs tested (AP33, HC33.4, HC84.26, 1:7, AR3B, AR4A, AR5A, IGH526, and A4). Antisera showed the best competition against HC84.26 and AR3B and the weakest competition against AR4A. Furthermore, antisera from five immunized human vaccinees were shown to compete with five preselected MAbs (AP33, AR3B, AR4A, AR5A, and IGH526). These data show that immunization with HCV-1 rE1E2 elicits antibodies targeting multiple cross-neutralizing epitopes. Our results further support the use of such a vaccine antigen to induce cross-genotype neutralization. IMPORTANCEAn effective prophylactic vaccine for HCV is needed for optimal control of the disease burden. The high diversity of HCV has posed a challenge for developing vaccines that elicit neutralizing antibodies for protection against infection. Despite this, we have previously shown that a vaccine comprising recombinant envelope glycoproteins derived from a single genotype 1a strain was capable of eliciting a cross-neutralizing antibody response in human volunteers. Here, we have used competition binding assays and peptide binding assays to show that antibodies present in the antisera from vaccinated goats and humans bind epitopes overlapping with those of a variety of well-characterized cross-neutralizing monoclonal antibodies. This provides a mechanism for the cross-neutralizing human antisera: antibodies present in the antisera bind to conserved regions associated with cross-neutralization. Importantly, this work provides further support for a vaccine comprising recombinant envelope glycoproteins, perhaps in a formulation with a vaccine component eliciting strong anti-HCV CD4 ؉ and CD8 ؉ T cell responses.

In this study, we differentiated the human hepatoma cell line Huh7.5 by supplementing tissue culture media with human serum (HS) and examined the production of hepatitis C virus (HCV) by these cells. We compared the standard tissue culture protocol, using media supplemented with 10% fetal bovine serum (FBS), to media supplemented with 2% HS. Cells cultured in HS undergo rapid growth arrest, have a hepatocyte-like morphology, and increase the expression of hepatocyte differentiation markers. In addition, expression of cell adhesion proteins claudin-1, occludin, and e-cadherin are also increased. The lipid droplet content of these cells is highly increased, as are key lipid metabolism regulators liver X receptor alpha, peroxisome proliferator-activated receptor (PPAR)-a, and PPAR-c. Very-low-density lipoprotein secretion, which is absent in FBSgrown cells, is restored in Huh7.5 cells that are cultured in HS. All these factors have been implicated in the life cycle of HCV. We show that viral production of Japanese fulminant hepatitis type 1 increases 1,000-fold when cells are grown in HS, compared to standard FBS culture conditions. The virus produced under these conditions is associated with apolipoprotein B, has a lower density, higher specific infectivity, and has a longer half-life than virus produced in media supplemented with FBS. Conclusion: We describe a convenient, cost-effective method to produce hepatocyte-like cells, which produce large amounts of virus that more closely resemble HCV present in serum of infected patients. (HEPATOLOGY 2013;58:1907-1917 H epatitis C virus (HCV) is an enveloped, positive-strand RNA virus of the family of Flaviviridae that causes acute and chronic hepatitis. HCV can cause cirrhosis, hepatocellular carcinoma, and steatosis in infected individuals.Replicon systems, both subgenomic and full length, and the Japanese fulminant hepatitis type 1 (JFH-1) tissue culture infection models have yielded important insight into the HCV life cycle. Most of these models make use of HuH-7 or HuH-7-derived cells, such as Huh7.5. HuH-7 or HuH-7-derived cells have many advantages for the in vitro study of HCV: they are readily available and rapidly dividing, and therefore enable large-scale experiments. However, these systems do not necessarily accurately represent events that occur during a natural HCV infection in vivo, because hepatocytes are normally nondividing and fully differentiated. To circumvent this, dimethyl sulfoxide

We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.

A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/ gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography.IMPORTANCE A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fctagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.