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A prophylactic vaccine is required to achieve the World Health Organization’s objective for hepatitis C virus elimination as a serious public health threat. However, the lack of robust immunocompetent animal models supporting hepatitis C virus infection impedes vaccine development as well as studies of immune responses and viral evasion.

Two of the most important mechanisms of hepatitis C virus (HCV) immune evasion are the high variability of the amino acid sequence and epitope shielding via heavy glycosylation of the envelope (E) proteins. Previously, we showed that chimeric sHBsAg (hepatitis B virus [HBV] small surface antigen)-based virus-like particles (VLPs) carrying highly conserved epitope I from the HCV E2 glycoprotein (sHBsAg_412–425) elicit broadly neutralizing antibodies (bnAbs). However, many reports have identified escape mutations for such bnAbs that shift the N-glycosylation site from N417 to N415. This shift effectively masks the recognition of epitope I by antibodies raised against the wild-type glycoprotein. To investigate if glycan-shift-mediated immune evasion could be overcome by targeted vaccination strategies, we designed sHBsAg-based VLPs carrying epitope I with an N417S change (sHBsAg_N417S). Studies in BALB/c mice revealed that both sHBsAg_412–425 and sHBsAg_N417S VLPs were immunogenic, eliciting antibodies that recognized peptides encompassing epitope I regardless of the N417S change. However, we observed substantial differences in E1E2 glycoprotein binding and cell culture-derived HCV (HCVcc) neutralization between the sera elicited by sHBsAg_412–425 and those elicited by sHBsAg_N417S VLPs. Our results suggest a complex interplay among antibodies targeting epitope I, the E1E2 glycosylation status, and the epitope or global E1E2 conformation. Additionally, we observed striking similarities in the E1E2 glycoprotein binding patterns and HCVcc neutralization between sHBsAg_412–425 sera and AP33, suggesting that the immunization of mice with sHBsAg_412–425 VLPs can elicit AP33-like antibodies. This study emphasizes the role of antibodies against epitope I and represents an initial effort toward designing an antigen that elicits an immune response against epitope I with a glycan shift change. IMPORTANCE Epitope I, located within amino acids 412 to 423 of the HCV E2 glycoprotein, is an important target for an epitope-based HCV vaccine. One interesting feature of epitope I is the N417 glycosylation site, where a single change to S417 or T417 can shift the glycosylation site to position N415. This shift can effectively prevent the binding of broadly neutralizing antibodies targeting epitope I. Aiming to overcome glycan-shift-mediated immune evasion, we constructed sHBsAg_N417S VLPs carrying E2 epitope I, with N417S, and compared them with VLPs carrying wild-type epitope I. We show that antibodies elicited by the sHBsAg-based VLPs presenting two variants of the 412–425 epitope targeted two distinct glycan variants of the HCV E1E2 heterodimer. Our study suggests that due to the conformational flexibility of the E2 glycoprotein and epitope I, future vaccine antigens should elicit antibodies targeting more than one conformation and glycosylation variant of the 412–423 epitope.

Efforts to mitigate the coronavirus disease 2019 (COVID-19) pandemic include the screening of existing antiviral molecules that could be repurposed to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Although SARS-CoV-2 replicates and propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (NUCs) often show decreased activity in these cells due to inefficient metabolization. SARS-CoV-2 exhibits low viability in human cells in culture. Here, serial passages of a SARS-CoV-2 isolate (original-SARS2) in the human hepatoma cell clone Huh7.5 led to the selection of a variant (adapted-SARS2) with significantly improved infectivity in human liver (Huh7 and Huh7.5) and lung cancer (unmodified Calu-1 and A549) cells. The adapted virus exhibited mutations in the spike protein, including a 9-amino-acid deletion and 3 amino acid changes (E484D, P812R, and Q954H). E484D also emerged in Vero E6-cultured viruses that became viable in A549 cells. Original and adapted viruses were susceptible to scavenger receptor class B type 1 (SR-B1) receptor blocking, and adapted-SARS2 exhibited significantly less dependence on ACE2. Both variants were similarly neutralized by COVID-19 convalescent-phase plasma, but adapted-SARS2 exhibited increased susceptibility to exogenous type I interferon. Remdesivir inhibited original- and adapted-SARS2 similarly, demonstrating the utility of the system for the screening of NUCs. Among the tested NUCs, only remdesivir, molnupiravir, and, to a limited extent, galidesivir showed antiviral effects across human cell lines, whereas sofosbuvir, ribavirin, and favipiravir had no apparent activity. Analogously to the emergence of spike mutations in vivo, the spike protein is under intense adaptive selection pressure in cell culture. Our results indicate that the emergence of spike mutations will most likely not affect the activity of remdesivir.

Ribavirin has been used for 25 years to treat patients with chronic hepatitis C virus (HCV) infection; however, its antiviral mechanism of action remains unclear. Here, we studied virus evolution in a subset of samples from a randomized 24-week trial of ribavirin monotherapy versus placebo in chronic HCV patients, as well as the viral resistance mechanisms of the observed ribavirin-associated mutations in cell culture. Thus, we performed next-generation sequencing of the full-length coding sequences of HCV recovered from patients at weeks 0, 12, 20, 32 and 40 and analyzed novel single nucleotide polymorphisms (SNPs), diversity, and mutation-linkage. At week 20, increased genetic diversity was observed in 5 ribavirin-treated compared to 4 placebo-treated HCV patients due to new synonymous SNPs, particularly G-to-A and C-to-U ribavirin-associated transitions. Moreover, emergence of 14 nonsynonymous SNPs in HCV nonstructural 5B (NS5B) occurred in treated patients, but not in placebo controls. Most substitutions located close to the NS5B polymerase nucleotide entry site. Linkage analysis showed that putative resistance mutations were found in the majority of genomes in ribavirin-treated patients. Identified NS5B mutations from genotype 3a patients were further introduced into the genotype 3a cell-culture-adapted DBN strain for studies in Huh7.5 cells. Specific NS5B substitutions, including DBN-D148N+I363V, DBN-A150V+I363V, and DBN-T227S+S183P, conferred resistance to ribavirin in long-term cell culture treatment, possibly by reducing the HCV polymerase error rate. In conclusion, prolonged exposure of HCV to ribavirin in chronic hepatitis C patients induces NS5B resistance mutations leading to increased polymerase fidelity, which could be one mechanism for ribavirin resistance.

Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that differs between strains of different virulence. In the highly virulent CSFV strain Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV strain Koslov (here called vKos_SL) to generate modified CSFVs with substitutions at residues 763 and/or 764 (vKos_LL, vKos_SP, and vKos_LP). The properties of these mutant viruses, in comparison to those of vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF, but the vKos_LP strain produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3 kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single-nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high-frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue-dependent quasispecies differentiation occurred within inoculated pigs; however, clear evidence for transmission bottlenecks to contact animals was observed, with subsequent loss of sequence diversity. IMPORTANCE The surface-exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent, but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.

The introduction of highly efficient therapies with direct-acting antivirals (DAA) for patients with chronic hepatitis C virus (HCV) infection offers exceptional opportunities to globally control this deadly disease. For achieving this ambitious goal, it is essential to prevent antiviral resistance against the most optimal first-line and retreatment DAA choices. We performed independent comparisons of the efficacy and barrier to resistance of pangenotypic DAA regimens for HCV genotype 2 infections, using previously and newly developed efficient cell culture-adapted strains of subtypes 2a, 2b, and 2c. With the applied experimental cell culture conditions, combination treatment with the sofosbuvir-velpatasvir or glecaprevir-pibrentasvir DAA regimen was efficient in eradicating HCV infections; in contrast, single-drug treatments frequently led to viral escape. Sequence analysis of drug targets from recovered viruses revealed known resistance-associated substitutions (RAS) emerging in the NS3 protease or NS5A after treatment failure. These RAS were genetically stable after viral passage, and viruses with these RAS exhibited significant phenotypic resistance. After sofosbuvir treatment failure, only a genotype 2a virus harbored NS5B RAS S282T and thus had decreased susceptibility to nucleotide analogs (nucs). However, in most cases, viral escape from sofosbuvir led to other NS5B substitutions but drug susceptibility was maintained, and in one case, no changes in NS5B were detected. For a genotype 2b virus, after treatment failure with sofosbuvir-velpatasvir, the efficacy of retreatment with glecaprevir-pibrentasvir was maintained due to the high barrier to resistance and low cross-resistance of pibrentasvir. Our findings suggest the slight superiority of glecaprevir-pibrentasvir against genotype 2b in culture, which could have potential therapeutic interest meriting more definitive investigations in the clinic.

The surveillance and correct subtyping of hepatitis C virus strains require available and up-to-date publicly available reference genomes. Here, we present the complete open reading frame sequence of a hepatitis C virus genotype 6 strain of an unknown subtype that was discovered during routine subtyping of patients in the clinic.

Animal hepaciviruses represent promising surrogate models for hepatitis C virus (HCV), for which there are no efficient immunocompetent animal models. Experimental infection of laboratory rats with rodent hepacivirus isolated from feral Rattus norvegicus (RHV-rn1) mirrors key aspects of HCV infection in humans, including chronicity, hepatitis, and steatosis. Moreover, RHV has been adapted to infect immunocompetent laboratory mice. RHV in vitro systems have not been developed but would enable detailed studies of the virus life cycle crucial for designing animal experiments to model HCV infection. Here, we established efficient RHV-rn1 selectable subgenomic replicons with and without reporter genes. Rat and mouse liver-derived cells did not readily support the complete RHV life cycle, but replicon-containing cell clones could be selected with and without acquired mutations. Replication was significantly enhanced by mutations in NS4B and NS5A and in cell clones cured of replicon RNA. These mutations increased RHV replication of both mono- and bicistronic constructs, and CpG/UpA-dinucleotide optimization of reporter genes allowed replication. Using the replicon system, we show that the RHV-rn1 NS3-4A protease cleaves a human mitochondrial antiviral signaling protein reporter, providing a sensitive readout for virus replication. RHV-rn1 replication was inhibited by the HCV polymerase inhibitor sofosbuvir and high concentrations of HCV NS5A antivirals but not by NS3 protease inhibitors. The microRNA-122 antagonist miravirsen inhibited RHV-rn1 replication, demonstrating the importance of this HCV host factor for RHV. These novel RHV in vitro systems will be useful for studies of tropism, molecular virology, and characterization of virus-host interactions, thereby providing important complements to in vivo systems. IMPORTANCE A vaccine against hepatitis C virus (HCV) is crucial for global control of this important pathogen, which induces fatal human liver diseases. Vaccine development has been hampered by the lack of immunocompetent animal models. Discovery of rodent hepacivirus (RHV) enabled establishment of novel surrogate animal models. These allow robust infection and reverse genetic and immunization studies of laboratory animals, which develop HCV-like chronicity. Currently, there are no RHV in vitro systems available to study tropism and molecular virology. Here, we established the first culture systems for RHV, recapitulating the intracellular phase of the virus life cycle in vitro. These replicon systems enabled identification of replication-enhancing mutations and selection of cells highly permissive to RHV replication, which allow study of virus-host interactions. HCV antivirals targeting NS5A, NS5B, and microRNA-122 efficiently inhibited RHV replication. Hence, several important aspects of HCV replication are shared by the rodent virus system, reinforcing its utility as an HCV model.

Yearly, ∼2 million people become hepatitis C virus (HCV) infected, resulting in an elevated lifetime risk for severe liver-related chronic illnesses. Characterizing epitopes of broadly neutralizing antibodies (NAbs), such as AR3A, is critical to guide vaccine development. Previously identified alanine substitutions that can reduce AR3A binding to expressed H77 envelope were introduced into chimeric cell culture-infectious HCV recombinants (HCVcc) H77(core-NS2)/JFH1. Substitutions G523A, G530A, and D535A greatly reduced fitness, and S424A, P525A, and N540A, although viable, conferred only low-level AR3A resistance. Using highly NAb-sensitive hypervariable region 1 (HVR1)-deleted HCVcc, H77/JFH1(ΔHVR1) and J6(core-NS2)/JFH1(ΔHVR1), we previously reported a low barrier to developing AR5A NAb resistance substitutions. Here, we cultured Huh7.5 cells infected with H77/JFH1, H77/JFH1(ΔHVR1), or J6/JFH1(ΔHVR1) with AR3A. We identified the resistance envelope substitutions M345T in H77/JFH1, L438S and F442Y in H77/JFH1(ΔHVR1), and D431G in J6/JFH1(ΔHVR1). M345T increased infectivity and conferred low-level AR3A resistance to H77/JFH1 but not H77/JFH1(ΔHVR1). L438S and F442Y conferred high-level AR3A resistance to H77/JFH1(ΔHVR1) but abrogated the infectivity of H77/JFH1. D431G conferred AR3A resistance to J6/JFH1(ΔHVR1) but not J6/JFH1. This was possibly because D431G conferred broadly increased neutralization sensitivity to J6/JFH1(D431G) but not J6/JFH1(ΔHVR1/D431G) while decreasing scavenger receptor class B type I coreceptor dependency. Common substitutions at positions 431 and 442 did not confer high-level resistance in other genotype 2a recombinants [JFH1 or T9(core-NS2)/JFH1]. Although the data indicate that AR3A has a high barrier to resistance, our approach permitted identification of low-level resistance substitutions. Also, the HVR1-dependent effects on AR3A resistance substitutions suggest a complex role of HVR1 in virus escape and receptor usage, with important implications for HCV vaccine development. IMPORTANCE Hepatitis C virus (HCV) is a leading cause of liver-related mortality, and limited treatment accessibility makes vaccine development a high priority. The vaccine-relevant cross-genotype-reactive antibody AR3A has shown high potency, but the ability of the virus to rapidly escape by mutating the AR3A epitope (barrier to resistance) remains unexplored. Here, we succeeded in inducing only low-level AR3A resistance, indicating a higher barrier to resistance than what we have previously reported for AR5A. Furthermore, we identify AR3A resistance substitutions that have hypervariable region 1 (HVR1)-dependent effects on HCV viability and on broad neutralization sensitivity. One of these substitutions increased envelope breathing and decreased scavenger receptor class B type I HCV coreceptor dependency, both in an HVR1-dependent fashion. Thus, we identify novel AR3A-specific resistance substitutions and the role of HVR1 in protecting HCV from AR3-targeting antibodies. These viral escape mechanisms should be taken into consideration in future HCV vaccine development.

Various protease inhibitors (PIs) currently are becoming available for treatment of hepatitis C virus (HCV). For genotype 1, substitutions at NS3 protease positions 155, 156, and 168 are the main determinants of PI resistance. For other genotypes, similar substitutions were selected during PI treatment but were not characterized systematically. To elucidate the impact of key PI resistance substitutions on genotypes 2 to 6, we engineered the substitutions R155A/E/G/H/K/Q/T, A156G/S/T/V, and D/Q168A/E/G/H/N/V into HCV recombinants expressing genotype 2 to 6 proteases. We evaluated viral fitness and sensitivity to nine PIs (telaprevir, boceprevir, simeprevir, asunaprevir, vaniprevir, faldaprevir, paritaprevir, deldeprevir, and grazoprevir) in Huh7.5 cells. We found that most variants showed decreased fitness compared to that of the original viruses. Overall, R155K, A156G/S, and D/Q168A/E/H/N/V variants showed the highest fitness; however, genotype 4 position 168 variants showed strong fitness impairment. Most variants tested were resistant to several PIs. Resistance levels varied significantly depending on the specific substitution, genotype, and PI. For telaprevir and boceprevir, specific 155 and 156, but not 168, variants proved resistant. For the remaining PIs, most genotype 2, 4, 5, and 6, but not genotype 3, variants showed various resistance levels. Overall, grazoprevir (MK-5172) had the highest efficacy against original viruses and variants. This is the first comprehensive study revealing the impact of described key PI resistance substitutions on fitness and PI resistance of HCV genotypes 2 to 6. In conclusion, the studied substitutions induced resistance to a panel of clinically relevant PIs, including the newer PIs paritaprevir, deldeprevir, and grazoprevir. We discovered complex patterns of resistance, with the impact of substitutions varying from increased sensitivity to high resistance.

Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4 + and CD8 + T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4 + T cells, whereas the NS3 pepmix induced a far more vigorous CD4 + T cell response and was as potent a CD8 + T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-γ + ) tumor necrosis factor alpha-positive (TNF-α + ) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity. IMPORTANCE With at least 700,000 annual deaths, development of a vaccine against hepatitis C virus (HCV) has high priority, but the tremendous ability of the virus to dodge the human immune system poses great challenges. Furthermore, many chronic infections, including HCV infection, have a remarkable ability to drive initially strong CD4 + and CD8 + T cell responses against dominant epitopes toward an exhausted, dysfunctional state. Thus, new and innovative vaccine approaches to control HCV should be evaluated. Here, we report on a novel peptide-based nanoparticle vaccine strategy (NS3 pepmix) aimed at generating T cell immunity against potential subdominant T cell epitopes that are not efficiently targeted by vaccination with full-length recombinant protein (rNS3) or infection with HCV. As proof of concept, we found that NS3 pepmix excels in broadening the repertoire of epitope-specific, multifunctional, and cytotoxic CD4 + and CD8 + T cells compared to vaccination with rNS3, which generated only CD4 + T cell responses.

Hepatitis C virus (HCV) strains belong to seven genotypes with numerous subtypes that respond differently to antiviral therapies. Genotype 1, and primarily subtype 1b, is the most prevalent genotype worldwide. The development of recombinant HCV infectious cell culture systems for different variants, permitted by the high replication capacity of strain JFH1 (genotype 2a), has advanced efficacy and resistance testing of antivirals. However, efficient infectious JFH1-based cell cultures of subtype 1b are limited and comprise only the 5′ untranslated region (5′UTR)-NS2, NS4A, or NS5A regions. Importantly, it has not been possible to develop efficient 1b infectious systems expressing the NS3/4A protease, an important target of direct-acting antivirals. We developed efficient infectious JFH1-based cultures with genotype 1b core-NS5A sequences of strains DH1, Con1, and J4 by using previously identified HCV cell culture adaptive substitutions A1226G, R1496L, and Q1773H. These viruses spread efficiently in Huh7.5 cells by acquiring additional adaptive substitutions, and final recombinants yielded peak supernatant infectivity titers of 4 to 5 log 10 focus-forming units (FFU)/ml. We subsequently succeeded in adapting a JFH1-based 5′UTR-NS5A DH1 recombinant to efficient growth in cell culture. We evaluated the efficacy of clinically relevant NS3/4A protease and NS5A inhibitors against the novel genotype 1b viruses, as well as against previously developed 1a viruses. The inhibitors were efficient against all tested genotype 1 viruses, with NS5A inhibitors showing half-maximal effective concentrations several orders of magnitude lower than NS3/4A protease inhibitors. In summary, the developed HCV genotype 1b culture systems represent valuable tools for assessing the efficacy of various classes of antivirals and for other virological studies requiring genotype 1b infectious viruses.

The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro . Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ∼100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles.

Protective immunity after resolved hepatitis C virus (HCV) infection has been reported. However, the breadth of this immunity has remained controversial, and the role of neutralizing antibodies has not been well-defined. In the present study, two chimpanzees (CH96A008 and CH1494) with resolved monoclonal H77C (genotype 1a) infection were rechallenged with low-dose homologous H77C virus about 12 months after viral clearance; CH96A008 became persistently infected, and CH1494 had transient viremia lasting 2 weeks. CH1494 was subsequently either partially or completely protected following five homologous rechallenges with monoclonal H77C or polyclonal H77 and after six heterologous rechallenges with HC-J4 (genotype 1b) or HC-J6 (genotype 2a) viruses. Subsequently, a final challenge with H77C resulted in persistent HCV infection. In both chimpanzees, serum neutralizing antibodies against retroviral pseudoparticles bearing the H77C envelope proteins were not detected during the initial infection or during rechallenge. However, anamnestic cellular immune responses developed during the initial homologous rechallenge, in particular in CH96A008, which developed a persistent infection. Polyprotein sequences of viruses recovered from CH1494 after the two homologous rechallenges that resulted in transient viremia were identical with the H77C virus. In contrast, the polyprotein sequences of viruses recovered from both chimpanzees after homologous rechallenge resulting in persistent infection had numerous changes. These findings have important implications for our understanding of immunity against HCV; even in the best-case scenario with autologous rechallenge, low-level viral persistence was seen in the presence of primed T-cell responses.

Using both a mass spectrometry-based method and the classical method of cloning and sequencing, we demonstrated weekly changes in the hypervariable region 1 quasispecies of a chimpanzee infected with an infectious clone, coinciding with neutralizing antibody emergence. We also used the mass spectrometry method in the clinical follow-up of a chronically infected patient over a 5-year period.

The importance of neutralizing antibodies (NAbs) in protection against hepatitis C virus (HCV) remains controversial. We infused a chimpanzee with H06 immunoglobulin from a genotype 1a HCV-infected patient and challenged with genotype strains efficiently neutralized by H06 in vitro . Genotype 1a NAbs afforded no protection against genotype 4a or 5a. Protection against homologous 1a lasted 18 weeks, but infection emerged when NAb titers waned. However, 6a infection was prevented. The differential in vivo neutralization patterns have implications for HCV vaccine development.

The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10 4.0 focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10 3.5 and 10 4.4 FFU/ml, respectively. IMPORTANCE Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro . HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3′ untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ∼4 log 10 focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reported in vivo . In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low concentrations.

A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While polyclonal antibodies to aa 412 to 423 from HCV-infected individuals confirmed broad neutralization, conflicting findings have been reported on polyclonal antibodies to an adjacent region, aa 434 to 446, that may or may not interfere with neutralization by antibodies to aa 412 to 423. To define the interplay between these antibodies, we isolated human monoclonal antibodies (HMAbs) to aa 412 to 423, designated HC33-related HMAbs (HC33 HMAbs), and characterized their interactions with other HMAbs to aa 434 to 446. A subset of the HC33 HMAbs neutralized genotype 1 to 6 infectious cell culture-derived HCV virions (HCVcc) with various activities. Although nonneutralizing HC33 HMAbs were isolated, they had lower binding affinities than neutralizing HC33 HMAbs. These antibodies could be converted to neutralizing antibodies by affinity maturation. Unidirectional competition for binding to E2 was observed between HC33 HMAbs and HMAbs to aa 434 to 446. When HMAbs to aa 434 to 446, which mediated neutralization, were combined with neutralizing HC33 HMAbs, biphasic patterns in neutralization were observed. A modest degree of antagonism was observed at lower concentrations, and a modest degree of synergism was observed at higher concentrations. However, the overall effect was additive neutralization. A similar pattern was observed when these antibodies were combined to block E2 binding to the HCV coreceptor, CD81. These findings demonstrate that both of these E2 regions participate in epitopes mediating virus neutralization and that the antibodies to aa 412 to 423 and aa 434 to 446 do not hinder their respective virus-neutralizing activities.

The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8 + T cells is a dominant force driving early amino acid replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8 + T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute-phase nonsynonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome. Moreover, the ratio of nonsynonymous to synonymous mutations, a measure of positive selection pressure, was increased 50-fold in class I epitopes compared with the rest of the HCV genome. Finally, some mutation of the clonal H77C genome toward a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the majority of these amino acid substitutions occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8 + T cells. This influence of immune pressure on viral evolution appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.

To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (Δ40) or 25-aa (Δ25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. Δ40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log 10 focus-forming units (FFU)/ml. 2a(J6) with Δ40 or Δ25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFPΔ40 acquired various deletions in EGFP, while 2a(J6)Δ40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5AΔ40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log 10 FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.

The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77.39, neutralized infection of strains from five of these genotypes. The three most potent neutralizing MAbs in our panel, H77.16, H77.39, and J6.36, inhibited infection at an early postattachment step. Receptor binding studies demonstrated that H77.39 inhibited binding of soluble E2 protein to both CD81 and SR-B1, J6.36 blocked attachment to SR-B1 and modestly reduced binding to CD81, and H77.16 blocked attachment to SR-B1 only. Using yeast surface display, we localized epitopes for the neutralizing MAbs on the E2 protein. Two of the strongly inhibitory MAbs, H77.16 and J6.36, showed markedly reduced binding when amino acids within hypervariable region 1 (HVR1) and at sites ∼100 to 200 residues away were changed, suggesting binding to a discontinuous epitope. Collectively, these studies help to define the structural and functional complexity of antibodies against HCV E2 protein with neutralizing potential.

GB virus B (GBV-B), the virus most closely related to hepatitis C virus (HCV), infects tamarins and causes acute hepatitis. The 3′ untranslated region (UTR) of an infectious GBV-B clone (pGBB) has a proximal short sequence followed by a poly(U) tract and a 3′ terminal sequence. Our investigators previously demonstrated that the 3′ terminal sequence was critical for in vivo infectivity. Here, we tested the effect of deleting the short sequence and/or the poly(U) tract from pGBB; infectivity of each mutant was tested by intrahepatic transfection of two tamarins with transcribed RNA. A mutant lacking both regions was not viable. However, mutants lacking either the short sequence or the poly(U) tract were viable. All four tamarins had a wild-type-like acute infection and developed acute hepatitis. Whereas we found that five tamarins transfected with the wild-type clone pGBB had acute resolving infection, one tamarin transfected with the poly(U) deletion mutant became persistently infected. This animal had viremia and hepatitis until its death at week 90. The genomes recovered at weeks 2, 7, 15, 20, 60, and 90 lacked the poly(U) stretch. Eight amino acid changes were identified at week 90. One change, in the putative p7 protein, was dominant at week 15. Thus, persistence of GBV-B, like persistence of HCV, was associated with the emergence of virus variants. Four tamarins inoculated with serum collected at weeks 2 and 90 from the tamarin with persistent infection had an acute resolving infection. Nonetheless, the demonstration that GBV-B can persist in tamarins strengthens its relevance as a surrogate model for the study of HCV.