Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with HCV envelope glycoproteins.

Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.

JFH1-based Core-NS2 genotype variants of HCV with genetic stability in vivo and in vitro: Important tools in the evaluation of virus neutralization.

BACKGROUND AND AIMS: HCV infection continues to be a major global health burden despite effective antiviral treatments. The urgent need for a protective vaccine is hindered by the scarcity of suitable HCV-permissive animal models tractable in vaccination and challenge studies. Currently, only antibody neutralization studies in infectious cell culture systems or studies of protection by passive immunization of human liver chimeric mice offer the possibility to evaluate the effect of vaccine-induced antibodies. However, differences between culture-permissive and in vivo-permissive viruses make it a challenge to compare analyses between platforms. To address this problem, we aimed at developing genotype-specific virus variants with genetic stability both in vitro and in vivo. APPROACH AND RESULTS: We demonstrated infection of human liver chimeric mice with cell culture-adapted HCV JFH1-based Core-NS2 recombinants of genotype 1-6, with a panel of 10 virus strains used extensively in neutralization and receptor studies. Clonal re-engineering of mouse-selected mutations resulted in virus variants with robust replication both in Huh7.5 cells and human liver chimeric mice, with genetic stability. Furthermore, we showed that, overall, these virus variants have similar in vitro neutralization profiles as their parent strains and demonstrated their use for in vivo neutralization studies. CONCLUSIONS: These mouse-selected HCV recombinants enable the triage of new vaccine-relevant antibodies in vitro and further allow characterization of protection from infection in vivo using identical viruses in human liver chimeric mice. As such, these viruses will serve as important resources in testing novel antibodies and can thus guide strategies to develop an efficient protective vaccine against HCV infection.

Identification of novel neutralizing determinants for protection against HCV.

BACKGROUND AND AIMS: HCV evasion of neutralizing antibodies (nAb) results in viral persistence and poses challenges to the development of an urgently needed vaccine. N-linked glycosylation of viral envelope proteins is a key mechanism for such evasion. To facilitate rational vaccine design, we aimed to identify determinants of protection of conserved neutralizing epitopes. APPROACH AND RESULTS: Using a reverse evolutionary approach, we passaged genotype 1a, 1b, 2a, 3a, and 4a HCV with envelope proteins (E1 and E2) derived from chronically infected patients without selective pressure by nAb in cell culture. Compared with the original viruses, HCV recombinants, engineered to harbor substitutions identified in polyclonal cell culture-passaged viruses, showed highly increased fitness and exposure of conserved neutralizing epitopes in antigenic regions 3 and 4, associated with protection from chronic infection. Further reverse genetic studies of acquired E1/E2 substitutions identified positions 418 and 532 in the N1 and N6 glycosylation motifs, localizing to adjacent E2 areas, as key regulators of changes of the E1/E2 conformational state, which governed viral sensitivity to nAb. These effects were independent of predicted glycan occupancy. CONCLUSIONS: We show how N-linked glycosylation motifs can trigger dramatic changes in HCV sensitivity to nAb, independent of glycan occupancy. These findings aid in the understanding of HCV nAb evasion and rational vaccine design, as they can be exploited to stabilize the structurally flexible envelope proteins in an open conformation, exposing important neutralizing epitopes. Finally, this work resulted in a panel of highly fit cell culture infectious HCV recombinants.

Hypervariable region 1 and N-linked glycans of hepatitis C regulate virion neutralization by modulating envelope conformations.

About two million new cases of hepatitis C virus (HCV) infections annually underscore the urgent need for a vaccine. However, this effort has proven challenging because HCV evades neutralizing antibodies (NAbs) through molecular features of viral envelope glycoprotein E2, including hypervariable region 1 (HVR1) and N-linked glycans. Here, we observe large variation in the effects of removing individual E2 glycans across HCV strains H77(genotype 1a), J6(2a), and S52(3a) in Huh7.5 cell infections. Also, glycan-mediated effects on neutralization sensitivity were completely HVR1-dependent, and neutralization data were consistent with indirect protection of epitopes, as opposed to direct steric shielding. Indeed, the effect of removing each glycan was similar both in type (protective or sensitizing) and relative strength across four nonoverlapping neutralization epitopes. Temperature-dependent neutralization (e.g., virus breathing) assays indicated that both HVR1 and protective glycans stabilized a closed, difficult to neutralize, envelope conformation. This stabilizing effect was hierarchical as removal of HVR1 fully destabilized closed conformations, irrespective of glycan status, consistent with increased instability at acidic pH and high temperatures. Finally, we observed a strong correlation between neutralization sensitivity and scavenger receptor BI dependency during viral entry. In conclusion, our study indicates that HVR1 and glycans regulate HCV neutralization by shifting the equilibrium between open and closed envelope conformations. This regulation appears tightly linked with scavenger receptor BI dependency, suggesting a role of this receptor in transitions from closed to open conformations during entry. This importance of structural dynamics of HCV envelope glycoproteins has critical implications for vaccine development and suggests that similar phenomena could contribute to immune evasion of other viruses.

Broadly neutralizing antibodies from an individual that naturally cleared multiple hepatitis C virus infections uncover molecular determinants for E2 targeting and vaccine design.

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1-6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1-6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.

Hepatitis C Virus Escape Studies of Human Antibody AR3A Reveal a High Barrier to Resistance and Novel Insights on Viral Antibody Evasion Mechanisms.

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/JFH1ΔHVR1 and J6(core-NS2)/JFH1ΔHVR1, we previously reported a low barrier to developing AR5A NAb resistance substitutions. Here, we cultured Huh7.5 cells infected with H77/JFH1, H77/JFH1ΔHVR1, or J6/JFH1ΔHVR1 with AR3A. We identified the resistance envelope substitutions M345T in H77/JFH1, L438S and F442Y in H77/JFH1ΔHVR1, and D431G in J6/JFH1ΔHVR1 M345T increased infectivity and conferred low-level AR3A resistance to H77/JFH1 but not H77/JFH1ΔHVR1 L438S and F442Y conferred high-level AR3A resistance to H77/JFH1ΔHVR1 but abrogated the infectivity of H77/JFH1. D431G conferred AR3A resistance to J6/JFH1ΔHVR1 but not J6/JFH1. This was possibly because D431G conferred broadly increased neutralization sensitivity to J6/JFH1D431G but not J6/JFH1ΔHVR1/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.

Hepatitis C Virus-Escape Studies for Human Monoclonal Antibody AR4A Reveal Isolate-Specific Resistance and a High Barrier to Resistance.

Global control of hepatitis C virus (HCV) depends on development of a prophylactic vaccine. We studied escape for cross-genotype-reactive neutralizing antibody AR4A, providing valuable information for HCV vaccine design. We cultured HCV core-NS2 recombinants H77 (genotype 1a)/JFH1 or the highly antibody-susceptible hypervariable region 1 (HVR1)-deleted variants H77/JFH1∆HVR1 and J6(genotype 2a)/JFH1∆HVR1 in Huh7.5 cells with AR4A. Long-term AR4A exposure of H77/JFH1 and H77/JFH1∆HVR1 did not yield resistance. However, J6/JFH1∆HVR1 developed the envelope-E2 substitutions I696T or I696N, which reduced AR4A binding (I696N > I696T). I696N conferred greater AR4A resistance than I696T in J6/JFH1∆HVR1, whereas the reverse was observed in J6/JFH1. This was because I696N but not I696T conferred broadly increased antibody neutralization susceptibility to J6/JFH1. I696N and I696T abrogated infectivity of H77/JFH1 and broadly increased neutralization susceptibility of S52 (genotype 3a)/JFH1. In conclusion, I696 is in the AR4A epitope, which has a high barrier to resistance, thus strengthening the rationale for its inclusion in rational HCV vaccine designs.

Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A.

Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3-4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare cross-genotype conserved epitope. By analyzing the genotype 1a envelope proteins (E1/E2) of recovered Core-NS2 recombinant H77/JFH1ΔHVR1 and performing reverse genetic studies we found that resistance to AR5A was caused by substitution L665W, also conferring resistance to the parental H77/JFH1. The mutation did not induce viral fitness loss, but abrogated AR5A binding to HCV particles and intracellular E1/E2 complexes. Culturing J6/JFH1ΔHVR1 (genotype 2a), for which fitness was decreased by L665W, with AR5A generated AR5A-resistant viruses with the substitutions I345V, L665S, and S680T, which we introduced into J6/JFH1 and J6/JFH1ΔHVR1. I345V increased fitness but had no effect on AR5A resistance. L665S impaired fitness and decreased AR5A sensitivity, while S680T combined with L665S compensated for fitness loss and decreased AR5A sensitivity even further. Interestingly, S680T alone had no fitness effect but sensitized the virus to AR5A. Of note, H77/JFH1L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2-6 parental and HVR1-deleted variants (not available for genotype 4a) we observed diverse effects on viral fitness and a universally pronounced reduction in AR5A sensitivity. Thus, we were able to take advantage of the neutralization-sensitive HVR1-deleted viruses to rapidly generate escape viruses aiding our understanding of the divergent escape pathways used by HCV to evade AR5A.

Hypervariable region 1 shielding of hepatitis C virus is a main contributor to genotypic differences in neutralization sensitivity.

There are 3-4 million new hepatitis C virus (HCV) infections yearly. The extensive intergenotypic sequence diversity of envelope proteins E1 and E2 of HCV and shielding of important epitopes by hypervariable region 1 (HVR1) of E2 are believed to be major hindrances to developing universally protective HCV vaccines. Using cultured viruses expressing the E1/E2 complex of isolates H77 (genotype 1a), J6 (2a), or S52 (3a), with and without HVR1, we tested HVR1-mediated neutralization occlusion in vitro against a panel of 12 well-characterized human monoclonal antibodies (HMAbs) targeting diverse E1, E2, and E1/E2 epitopes. Surprisingly, HVR1-mediated protection was greatest for S52, followed by J6 and then H77. HCV pulldown experiments showed that this phenomenon was caused by epitope shielding. Moreover, by regression analysis of HMAb binding and neutralization titer of HCV we found a strong correlation for HVR1-deleted viruses but not for parental viruses retaining HVR1. The intergenotype neutralization sensitivity of the parental viruses to HMAb antigenic region (AR) 2A, AR3A, AR4A, AR5A, HC84.26, and HC33.4 varied greatly (>24-fold to >130-fold differences in 50% inhibitory concentration values). However, except for AR5A, these differences decreased to less than 6.0-fold when comparing the corresponding HVR1-deleted viruses. Importantly, this simplified pattern of neutralization sensitivity in the absence of HVR1 was also demonstrated in a panel of HVR1-deleted viruses of genotypes 1a, 2a, 2b, 3a, 5a, and 6a, although for all HMAbs, except AR4A, an outlier was observed. Finally, unique amino acid residues in HCV E2 could explain these outliers in the tested cases of AR5A and HC84.26. CONCLUSION: HVR1 adds complexity to HCV neutralization by shielding a diverse array of unexpectedly cross-genotype-conserved E1/E2 epitopes. Thus, an HVR1-deleted antigen could be a better HCV vaccine immunogen. (Hepatology 2016;64:1881-1892).

HVR1-mediated antibody evasion of highly infectious in vivo adapted HCV in humanised mice.

OBJECTIVE: HCV is a major cause of chronic liver disease worldwide, but the role of neutralising antibodies (nAbs) in its natural history remains poorly defined. We analysed the in vivo role of hypervariable region 1 (HVR1) for HCV virion properties, including nAb susceptibility. DESIGN: Analysis of HCV from human liver chimeric mice infected with cell-culture-derived prototype genotype 2a recombinant J6/JFH1 or HVR1-deleted variant J6/JFH1ΔHVR1 identified adaptive mutations, which were analysed by reverse genetics in Huh7.5 and CD81-deficient S29 cells. The increased in vivo genomic stability of the adapted viruses facilitated ex vivo density analysis by ultracentrifugation and in vivo neutralisation experiments addressing the role of HVR1. RESULTS: In vivo, J6/JFH1 and J6/JFH1ΔHVR1 depended on single substitutions within amino acids 867-876 in non-structural protein, NS2. The identified A876P-substitution resulted in a 4.7-fold increase in genomic stability. In vitro, NS2 substitutions enhanced infectivity 5-10-fold by increasing virus assembly. Mouse-derived mJ6/JFH1A876P and mJ6/JFH1ΔHVR1/A876P viruses displayed similar heterogeneous densities of 1.02-1.1 g/mL. Human liver chimeric mice loaded with heterologous patient H (genotype 1a) immunoglobulin had partial protection against mJ6/JFH1A876P and complete protection against mJ6/JFH1ΔHVR1/A876P. Interestingly, we identified a putative escape mutation, D476G, in mJ6/JFH1A876P. This mutation in hypervariable region 2 conferred 6.6-fold resistance against H06 IgG in vitro. CONCLUSIONS: The A876P-substitution bridges in vitro and in vivo studies using J6/JFH1-based recombinants. We provide the first in vivo evidence that HVR1 protects cross-genotype conserved HCV neutralisation epitopes, which advocates the possibility of using HVR1-deleted viruses as vaccine antigens to boost broadly reactive protective nAb responses.

Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization.

Hepatitis C virus (HCV) genotype 4 is highly prevalent in the Middle East and parts of Africa. Subtype 4d has recently spread among high-risk groups in Europe. However, 4d infectious culture systems are not available, hampering studies of drugs, as well as neutralizing antibodies relevant for HCV vaccine development. We determined the consensus 4d sequence from a chronic hepatitis C patient by next-generation sequencing, generated a full-length clone thereof (pDH13), and demonstrated that pDH13 RNA-transcripts were viable in the human-liver chimeric mouse model, but not in Huh7.5 cells. However, a JFH1-based DH13 Core-NS5A 4d clone encoding A1671S, T1785V, and D2411G was viable in Huh7.5 cells, with efficient growth after inclusion of 10 additional substitutions [4d(C5A)-13m]. The efficacies of NS3/4A protease- and NS5A- inhibitors against genotypes 4a and 4d were similar, except for ledipasvir, which is less potent against 4d. Compared to 4a, the 4d(C5A)-13m virus was more sensitive to neutralizing monoclonal antibodies AR3A and AR5A, as well as 4a and 4d patient plasma antibodies. In conclusion, we developed the first genotype 4d infectious culture system enabling DAA efficacy testing and antibody neutralization assessment critical to optimization of DAA treatments in the clinic and for vaccine design to combat the HCV epidemic.

Two-component vaccine consisting of virus-like particles displaying hepatitis C virus envelope protein 2 oligomers.

Development of B-cell-based hepatitis C virus (HCV) vaccines that induce broadly neutralizing antibodies (bNAbs) is hindered by extensive sequence diversity and low immunogenicity of envelope glycoprotein vaccine candidates, most notably soluble E2 (sE2). To overcome this, we employed two-component approaches using self-assembling virus-like particles (cVLPs; component 1), displaying monomeric or oligomeric forms of HCV sE2 (sE2mono or sE2oligo; component 2). Immunization studies were performed in BALB/c mice and the neutralizing capacity of vaccine-induced antibodies was tested in cultured-virus-neutralizations, using HCV of genotypes 1-6. sE2-cVLP vaccines induced significantly higher levels of NAbs (p = 0.0065) compared to corresponding sE2 vaccines. Additionally, sE2oligo-cVLP was superior to sE2mono-cVLP in inducing bNAbs. Interestingly, human monoclonal antibody AR2A had reduced binding in ELISA to sE2oligo-cVLP compared with sE2mono-cVLP and competition ELISA using mouse sera from vaccinated animals indicated that sE2oligo-cVLP induced significantly less non-bNAbs AR2A (p = 0.0043) and AR1B (p = 0.017). Thus, cVLP-displayed oligomeric sE2 shows promise as an HCV vaccine candidate.

Mechanisms of Hepatitis C Virus Escape from Vaccine-Relevant Neutralizing Antibodies.

Hepatitis C virus (HCV) is a major causative agent of acute and chronic hepatitis. It is estimated that 400,000 people die every year from chronic HCV infection, mostly from severe liver-related diseases such as cirrhosis and liver cancer. Although HCV was discovered more than 30 years ago, an efficient prophylactic vaccine is still missing. The HCV glycoprotein complex, E1/E2, is the principal target of neutralizing antibodies (NAbs) and, thus, is an attractive antigen for B-cell vaccine design. However, the high genetic variability of the virus necessitates the identification of conserved epitopes. Moreover, the high intrinsic mutational capacity of HCV allows the virus to continually escape broadly NAbs (bNAbs), which is likely to cause issues with vaccine-resistant variants. Several studies have assessed the barrier-to-resistance of vaccine-relevant bNAbs in vivo and in vitro. Interestingly, recent studies have suggested that escape substitutions can confer antibody resistance not only by direct modification of the epitope but indirectly through allosteric effects, which can be grouped based on the breadth of these effects on antibody susceptibility. In this review, we summarize the current understanding of HCV-specific NAbs, with a special focus on vaccine-relevant bNAbs and their targets. We highlight antibody escape studies pointing out the different methodologies and the escape mutations identified thus far. Finally, we analyze the antibody escape mechanisms of envelope protein escape substitutions and polymorphisms according to the most recent evidence in the HCV field. The accumulated knowledge in identifying bNAb epitopes as well as assessing barriers to resistance and elucidating relevant escape mechanisms may prove critical in the successful development of an HCV B-cell vaccine.

Antigenic and immunogenic evaluation of permutations of soluble hepatitis C virus envelope protein E2 and E1 antigens.

Yearly, about 1.5 million people become chronically infected with hepatitis C virus (HCV) and for the 71 million with chronic HCV infection about 400,000 die from related morbidities, including liver cirrhosis and cancer. Effective treatments exist, but challenges including cost-of-treatment and wide-spread undiagnosed infection, necessitates the development of vaccines. Vaccines should induce neutralizing antibodies (NAbs) against the HCV envelope (E) transmembrane glycoprotein 2, E2, which partly depends on its interaction partner, E1, for folding. Here, we generated three soluble HCV envelope protein antigens with the transmembrane regions deleted (i.e., fused peptide backbones), termed sE1E2 (E1 followed by E2), sE2E1 (E2 followed by E1), and sE21E (E2 followed by inverted E1). The E1 inversion for sE21E positions C-terminal residues of E1 near C-terminal residues of E2, which is in analogy to how they likely interact in native E1/E2 complexes. Probing conformational E2 epitope binding using HCV patient-derived human monoclonal antibodies, we show that sE21E was superior to sE2E1, which was consistently superior to sE1E2. This correlated with improved induction of NAbs by sE21E compared with sE2E1 and especially compared with sE1E2 in female BALB/c mouse immunizations. The deletion of the 27 N-terminal amino acids of E2, termed hypervariable region 1 (HVR1), conferred slight increases in antigenicity for sE2E1 and sE21E, but severely impaired induction of antibodies able to neutralize in vitro viruses retaining HVR1. Finally, comparing sE21E with sE2 in mouse immunizations, we show similar induction of heterologous NAbs. In summary, we find that C-terminal E2 fusion of E1 or 1E is superior to N-terminal fusion, both in terms of antigenicity and the induction of heterologous NAbs. This has relevance when designing HCV E1E2 vaccine antigens.

An alternate conformation of HCV E2 neutralizing face as an additional vaccine target.

To achieve global elimination of hepatitis C virus (HCV), an effective cross-genotype vaccine is needed. The HCV envelope glycoprotein E2 is the main target for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. E2 is structurally flexible and functions in engaging host receptors. Many nAbs bind to the "neutralizing face" on E2, including several broadly nAbs encoded by the VH1-69 germline gene family that bind to a similar conformation (A) of this face. Here, a previously unknown conformation (B) of the neutralizing face is revealed in crystal structures of two of four additional E2-VH1-69 nAb complexes. In this conformation, the E2 front-layer region is displaced upon antibody binding, exposing residues in the back layer for direct antibody interaction. This E2 B structure may represent another conformational state in the viral entry process that is susceptible to antibody neutralization and thus provide a new target for rational vaccine development.

Alternative cell lines to improve the rescue of infectious human astrovirus from a cDNA clone.

A reverse genetics system for human astrovirus (HAstV) was established previously; however, it has not been exploited mainly because cells used for virus packaging are not permissive, requiring several rounds of replication to obtain acceptable infectious virus. In this work, in the search for alternative permissive cell lines to be used as packaging cells, Hek-293 and Huh7.5.1 were tested. Given that HAstV infection in Hek-293 showed differences with that in Caco-2, the gold standard for HAstV growth but scarcely transfectable, and it was more similar to that observed in the hepatoma Huh7.5.1 cell line, these last cells were further used to transfect viral RNA. Virus titers near to 10(8) infectious particles per ml (ffu/ml) were obtained at 16-20 h after transfection with RNA from infected cells. However, virus titers close to 10(6) ffu/ml were obtained by using in vitro transcribed RNA from a cDNA HAstV-1 clone. In contrast, virus recovery in BHK-21, reported previously as the packaging cells, from this RNA was of about 10(4) ffu/ml, two logarithms less than in Huh7.5.1. Apparently, the 5'-end modification of the viral RNA determined its specific infectivity, since virus recovery was abolished when the total RNA was treated with proteinase-K, probably by removing a protein-linked genome protein, but it increased when capping of the in vitro transcribed RNA was more efficient. Thus, an alternative and more efficient reverse genetics system for HAstV was established by using Huh7.5.1 cells.