Hepatitis C Virus E2 Research Articles

Minicircle-based vaccine induces potent T-cell and antibody responses against hepatitis C virus

An effective vaccine against hepatitis C virus (HCV) should elicit both humoral and cellular immune responses. Previously, we characterized a bivalent vaccine candidate against hepatitis B (HBV) and HCV using chimeric HBV-HCV virus-like particles (VLP), in which the highly conserved epitope of HCV E2 glycoprotein (residues 412–425) was inserted into the hydrophilic loop of HBV small surface antigen (sHBsAg). While sHBsAg_412–425 elicited cross-neutralizing antibodies, it did not trigger a T-cell response against HCV. Thus, this study aimed to develop a vaccine candidate engaging both arms of adaptive immune response, potentially offering stronger protection against HCV. We evaluated the immunogenicity of minicircle (MC) DNA vaccines encoding sHBsAg_412–425 and HCV nonstructural (NS) proteins in BALB/c mice. Co-administration of sHBsAg_412–425 and NS induced a potent T-cell response, especially against NS3 and high titers of antibodies specific to HCV E2. Additionally, these antibodies recognized native HCV envelope glycoprotein heterodimers (E1E2) across multiple HCV genotypes and showed binding profiles to E1E2 alanine mutants comparable to the broadly neutralizing AP33 antibody. Overall, the findings demonstrate that MC DNA vaccine incorporating both sHBsAg_412–425 and HCV NS protein sequences induces robust, T-cell and AP33-like antibody responses, highlighting its potential as pan-genotypic prophylactic vaccine against HCV.

The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein

Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.

Alterations in N-glycosylation of HCV E2 Protein in Children Patients with IFN-RBV Therapy Failure.

The glycosylation of viral envelope proteins plays an important role in virus biology and the immune response of the host to infection. Hepatitis C virus (HCV) envelope proteins E1 and E2, key players in virus entry and spread, are highly N-glycosylated and possess 4 (5 in certain genotypes) to 11 conserved glycosylation sites, respectively. Many published results based on recombinant proteins indicate that the glycan shield can mask the epitopes targeted by neutralizing antibodies. Glycan shifting within the conserved linear E2 region (412-423) could be one of the escape strategies used by HCV. In the present report, we isolated E2 genes from samples (collected before the IFN-RBV therapy) originating from pediatric patients infected with HCV gt 1a. We analyzed the biochemical properties of cloned E2 glycoprotein variants and investigated their glycosylation status. The sequencing of E2 genes isolated from patients who did not respond to therapy revealed mutations at N-glycosylation sites, thus leading to a lower molecular weight and a low affinity to both linear and conformational neutralizing antibodies. The loss of the glycosylation site within the conserved epitope (amino acid 417) impaired the binding with AP33, an antibody that potently neutralizes all genotypes of HCV. Our findings, based on clinical samples, confirm the influence of N-glycosylation aberrations on the antigenic and conformational properties of HCV E1/E2, which may possibly correlate with the outcome of therapy in patients.

The conformational dynamics of Hepatitis C Virus E2 glycoprotein with the increasing number of N-glycosylation unraveled by molecular dynamics simulations

The Hepatitis C Virus (HCV), responsible for causing hepatitis and a significant contributor to liver disorders, presents a challenge for treatment due to its high genetic variability. Despite efforts, there is still no effective medication available for this virus. One of the promising targets for drug development involves targeting glycoprotein E2. However, our understanding of the dynamic behavior of E2 and its associated glycans remains limited. In this study, we investigated the dynamic characteristics of E2 with varying degrees of glycosylation using all-atom molecular dynamics simulations. We also explored glycan’s interactions with the protein and among themselves. An overall increase in correlation between the vital protein regions was observed with an increase in glycan number. The protein dynamics is followed by the analysis of glycan dynamics, where the flexibility of the individual glycans was analyzed in their free and bound state, which revealed a decrease in their fluctuation in some cases. Furthermore, we generated the free energy landscape of individual N-glycan linkages in both free and bound states and observed both increases and decreases in flexibility, which can be attributed to the formation and breakage of hydrogen bonds with amino acids. Finally, we found that for a high glycosylation system, glycans interact with glycoprotein and form hydrogen bonds among themselves. Moreover, the hydrogen bond profiles of a given glycan can vary when influenced by other glycans. In summary, our study provides valuable insights into the dynamics of the core region of HCV E2 glycoprotein and its associated glycans. Communicated by Ramaswamy H. Sarma

Establishment and methodological evaluation of a chemiluminescence assay for detection of anti-envelope protein (E1, E2) antibodies in the serum of hepatitis C virus-infected patients.

To establish a chemiluminescence method for detecting anti-E1 and anti-E2 antibodies in the serum of patients with hepatitis C virus (HCV) infection. The microplate was coated with recombinant envelope proteins E1 and E2 by indirect method, respectively, and the kits for detecting anti-E1 and anti-E2 antibodies were prepared. The methodological indexes were evaluated. The methodological indexes of the kits were as follows: precision test (the variation coefficient of anti-E1 antibody 6.71%-8.95% for within run and 9.91%-12.16% for between run, the variation coefficient of anti-E2 antibody 6.06%-8.44% for within run and 10.77%-13.98% for between run, respectively). The blank limit and detection limit were 1.18 RLIR and 3.16 RLIR for the anti-E1 antibody, and 1.26 RLIR and 3.32 RLIR for the anti-E2 antibody, respectively. The correlation coefficients (r) of anti-E1 and anti-E2 were 0.9963 and 0.9828, the analysis and measurement ranges (AMR) were 1.66-41.28 RLIR and 1.55-19.46 RLIR, and the average recovery was 96.4% and 93.7%, respectively. The rheumatoid factor and other positive serum samples had no interference or cross-reaction to the test, and the kits were stable within 15 months. The positive rates of anti-E1 and anti-E2 antibodies in 45 patients with HCV infection were 35.6% (16/45) and 44.4% (20/45), respectively. The kits for detecting anti-E1 and anti-E2 meet the requirements of methodology, and can be used in screening diagnosis, disease monitoring, prognosis evaluation, disease mechanism, and epidemiological studies of HCV infection. The HCV envelope proteins E1 and E2 have an immune response in HCV-infected patients.

Characterization of antibody-dependent cellular phagocytosis in patients infected with hepatitis C virus with different clinical outcomes.

Early neutralizing antibodies against hepatitis C virus (HCV) and CD8 + T cell effector responses can lead to viral clearance. However, these functions alone are not sufficient to protect patients against HCV infection, thus undefined additional antiviral immune mechanisms are required. In recent years, Fc-receptor-dependent antibody effector functions, particularly, antibody-dependent cellular phagocytosis (ADCP) were shown to offer immune protection against several RNA viruses. However, its development and clinical role in patients with HCV infection remain unknown. In this study, we found that patients with chronic GT1a or GT3a HCV infection had significantly higher concentrations of anti-envelope 2 (E2) antibodies, predominantly IgG1 subclass, than patients that cleared the viruses while the latter had antibodies with higher affinities. 97% of the patients with HCV had measurable ADCP of whom patients with chronic disease showed significantly higher ADCP than those who naturally cleared the virus. Epitope mapping studies showed that patients with antibodies that target antigenic domains on the HCV E2 protein that are known to associate with neutralization function are also strongly associated with ADCP, suggesting antibodies with overlapping/dual functions. Correlation studies showed that ADCP significantly correlated with plasma anti-E2 antibody levels and neutralization function regardless of clinical outcome and genotype of infecting virus, while a significant correlation between ADCP and affinity was only evident in patients that cleared the virus. These results suggest ADCP was mostly driven by antibody titer in patients with chronic disease while maintained in clearers due to the quality (affinity) of their anti-E2 antibodies despite having lower antibody titers.

Hepatitis C virus modified sE2F442NYT as an antigen in candidate vaccine facilitates human immune cell activation.

The rational selection of hepatitis C virus (HCV) vaccine antigen will aid in the prevention of future chronic liver disease burden and associated healthcare costs. We have previously shown that HCV E2 glycoprotein is not highly immunogenic, and the modification of E2 reduced CD81 binding and displayed altered cytokine and protective immune responses in vitro and in a surrogate mouse model. Here, we compared the influence of a parental and a modified sE2F442NYT glycoprotein region from HCV genotype 1a for the activation of peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), CD4+T cells, and B cells. Modified sE2F442NYT, when incubated with DCs, induced a higher number of CD86-positive cells. The sE2F442NYT or parental sE2 encapsulated as mRNA-lipid nanoparticle (sE2F442NYT mRNA-LNP) primed DCs co-cultured with autologous CD4+T cells did not induce CD25 or forkhead box P3 expression. PBMC-derived CD4+T cells treated with sE2F442NYT exhibited enhanced signal transducer and activator of transcription (Stat)1/Stat4 phosphorylation in response to anti-CD3/CD28 stimulation in comparison to parental sE2 treatment and facilitated isotype switching in B cells, leading to the generation of a broader subclass of antibodies. Cells treated with modified sE2F442NYT displayed an increase in activated Stat3 and extracellular signal-regulated kinase (ERK). Likewise, PBMC-derived naïve B cells upon in vitro stimulation with sE2F442NYT induced an increased proliferation, Stat3 and ERK activation, and protein kinase B (Akt) suppression. Thus, the modified sE2F442NYT antigen from HCV facilitates improved DC, CD4+T, and B cell activation compared to parental sE2 to better induce a robust protective immune response, supporting its selection as an HCV candidate vaccine antigen for preclinical and clinical HCV vaccine trials.IMPORTANCEThe nature of an enhanced immune response induced by sE2F442NYT will help in the selection of a broad cross-protective antigen from hepatitis C virus genotypes, and the inclusion of relatively conserved sE1 with sE2F442NYT may further strengthen the efficacy of the candidate vaccine in evaluating it for human use.

HCV E1 influences the fitness landscape ofE2 andmay enhance escape from E2-specific antibodies.

The Hepatitis C virus (HCV) envelope glycoprotein E1 forms a non-covalent heterodimer with E2, the main target of neutralizing antibodies. How E1-E2 interactions influence viral fitness and contribute to resistance to E2-specific antibodies remain largely unknown. We investigate this problem using a combination of fitness landscape and evolutionary modeling. Our analysis indicates that E1 and E2 proteins collectively mediate viral fitness and suggests that fitness-compensating E1 mutations may accelerate escape from E2-targeting antibodies. Our analysis also identifies a set of E2-specific human monoclonal antibodies that are predicted to be especially resilient to escape via genetic variation in both E1 and E2, providing directions for robust HCV vaccine development.

Effect of Glycan Shift on Antibodies against Hepatitis C Virus E2 412-425 Epitope Elicited by Chimeric sHBsAg-Based Virus-Like Particles.

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.

Structure of the hepatitis C virus E1E2 glycoprotein complex.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma in humans and afflicts more than 58 million people worldwide. The HCV envelope E1 and E2 glycoproteins are essential for viral entry and comprise the primary antigenic target for neutralizing antibody responses. The molecular mechanisms of E1E2 assembly, as well as how the E1E2 heterodimer binds broadly neutralizing antibodies, remain elusive. Here, we present the cryo-electron microscopy structure of the membrane-extracted full-length E1E2 heterodimer in complex with three broadly neutralizing antibodies-AR4A, AT1209, and IGH505-at ~3.5-angstrom resolution. We resolve the interface between the E1 and E2 ectodomains and deliver a blueprint for the rational design of vaccine immunogens and antiviral drugs.

Immunity of two novel hepatitis C virus polyepitope vaccines

Hepatitis C virus (HCV) infection remains a serious public health burden around the world. So far there is no effective vaccine against this virus. Neutralizing antibody (NAb) responses to the epitopes within HCV E1 and E2 proteins are related to the resolution of hepatitis C infection. E. coli heat-labile enterotoxin B subunit (LTB) has been described as potent immunity adjuvants. In this study, we constructed recombinant pET vectors: pET-R9-Bp (B cell polyepitopes) expressing 7 epitopes from HCV E1 and E2 proteins including R9 (E2384-411aa)-Bp (E1313-327aa-E2396-424aa-E2436-447aa-E2523-540aa-E2610-627aa-E2631-648aa) and pET-LTB-R9-Bp expressing LTB adjuvant in combination with R9-Bp. Recombinant proteins R9-Bp and LTB-R9-Bp were expressed successfully in E. coli and purified by the Ni-NTA column. Both R9-Bp and LTB-R9-Bp in BALB/c mice induced robust humoral immune response in the context of intraperitoneal or intramuscular immunization but not oral immunization. Intraperitoneal administration of LTB-R9-Bp induced a higher antibody titer (peak titer: 1:341000) than that of R9-Bp (peak titer: 1:85000) after the second boost (P = 0.0036 or 0.0002). However, comparable antibody peak titers were elicited for both R9-Bp and LTB-R9-Bp in intramuscular immunization albeit with significant difference (P = 0.0032) a week after the second boost. In addition, both R9-Bp and LTB-R9-Bp induced the secretion of cytokines including IFN-γ and IL-4 at similar levels. anti-sera induced by both R9-Bp and LTB-R9-Bp recognized native HCV E1 and E2 proteins. Moreover, these HCV-specific antisera inhibited significantly the entry of HCV (P < 0.0001). Taken together, these findings showed that E. coli-based both R9-Bp and LTB-R9-Bp could become promising HCV vaccines.

Developing an Epitope-Based Peptide Vaccine for the Hepatitis C Virus Using an in Silico Approach

A vaccine has still not been found for the hepatitis C virus (HCV) and the number of global cases of HCV is still high. The development of HCV vaccines is challenging due to the complex genetic diversity of the virus. Epitope-based vaccine design using in silico computational methods is an effective strategy that could lead to the development of vaccines with the ability to induce the required immunogenicity without the emergence of cytokine storms or immune tolerance. This study aimed to find an epitope candidate from the HCV E2 protein which has potential as a peptide vaccine. The research was observational descriptive and was carried out in silico on an HCV vaccine candidate. The software used was MEGA X, IEDB, VaxiJen 2.0, BLASTp. 3 conserved regions were obtained from 10 countries, namely VCGPVYCFTPSPVVVGTTD, CPTDCFRK, and YRLWHYPCT. The sequences between these countries still have phylogenetic relationships, even though they are in different branches, showing the evolution of the HCV subtypes. The VYCFTPSPVVVGTTD epitope became the candidate for the development of a peptide vaccine because of its antigenicity score and its ability to be used for effective epitope-based vaccines. This HCV vaccine candidate epitope does not cause an autoimmune response because it has been confirmed to be using BLASTp with the result that it shares no similarity with human cell surface proteins.&#x0D; Keywords: vaccines, peptides, epitope, hepatitis C virus, in silico

The Glycodiversity of HCV E2 Glycoprotein-Specific Antibodies as a Signature of Hepatic Damage and Virotherapy Efficacy

The HCV E2 glycoprotein-specific Abs (E2-Ab) is an important factor in the host resistance to hepatitis C virus (HCV) infection. There is evidence that the E2-Ab sialylation is associated with liver damage and virotherapy efficacy. The aim of this study was to further profile the E2-Ab glycosylation. The fucosylation and sialylation of E2-Ab in one hundred six (HCV)-infected patients were tested using the lectin-based ELISA platform. Data were analyzed by the stage of hepatic fibrosis, HCV genotype and the response to IF-RBV virotherapy. The changes in the E2–Ab glycosylation were also evaluated by the receiver operator characteristic curve (ROC) and multiple regression analysis. The E2-Ab reactivity to fucose-specific Aleuria aurantia lectin (AAL) and sialo-specific Sambucus nigra agglutinin (SNA) was decreased in the advanced stages of liver fibrosis. The SNA binding analysis was more informative in the discrimination of patients with advanced fibrosis compared to those with earlier fibrosis stages or no fibrosis group. No significant correlation between the reactivities of SNA and AAL lectins was established irrespective of HCV genotype. The patients infected with HCV 3a genotype showed an increased E2-Ab fucosylation, a lower E2-SNA/E2-AAL ratio and a better response to virotherapy. The association of the E2-SNA/E2-AAL ratio with virotherapy outcome was observed in patients infected with HCV 1b genotype. A better response to IF-RBV therapy was found in patients with a higher fucosylated E2 Abs and a lower E2-SNA/E2-AAL ratio. Thus the significance of E2 antibodies in the course of HCV hepatitis was demonstrated to be dependent on their glycosylation, the sialylation/fucosylation ratio, as well as on HCV genotype.

Induction of neutralizing antibodies against hepatitis C virus by a subviral particle-based DNA vaccine

Direct-acting antivirals (DAAs) have been introduced for the treatment of hepatitis C virus (HCV); however, there is still no available vaccine for preventing HCV infection. We previously reported on a Japanese encephalitis virus (JEV) subviral particle (SVP)-based vaccine with insertion of the HCV E2 neutralization epitope at three positions (SVP-E2/Tri). In this study, we utilized this SVP platform for DNA immunization. In addition, we explored further sites permitting the insertion of HCV epitopes without impairing viral assembly and secretion to elicit higher titers of neutralizing antibodies, and we identified three new positions for foreign epitope insertion. Successful secretion of SVPs with the insertion of HCV epitopes at five positions (SVP-E2/Pent) was seen from transfected cells. Compared to SVP-E2/Tri, sera from mice immunized with the plasmid expressing SVP-E2/Pent showed more neutralization activity against HCV, and less neutralization activity against JEV, suggesting that the additional insertion of HCV epitopes contributed to the induction of antibodies against the inserted peptide, whereas the neutralizing epitopes against JEV were disrupted. This study provides a potentially effective novel DNA vaccine platform.

Design, Synthesis, and Biological Evaluation of 2-((4-Bisarylmethyl-piperazin-1-yl)methyl)benzonitrile Derivatives as HCV Entry Inhibitors.

Viral entry inhibitors are absent in hepatitis C virus (HCV) treatment regimens although a dozen direct-acting antiviral (DAA) drugs are available now. Based on a previously identified HCV entry inhibitor L0909, chemical space exploration and structure-activity relationship (SAR) studies led to the discovery of a new derived scaffold 2-((4-bisarylmethyl-piperazin-1-yl)methyl)benzonitrile. Several new scaffold derivatives exhibited higher in vitro anti-HCV activity at low nanomolar concentrations compared to L0909. A biological study indicated that the high potency of active derivatives 3d, 3h, and 3i was primarily driven by the inhibitory effect on the virus entry stage. Moreover, an SPR experiment confirmed that this class of derivatives might target the HCV E1 protein. Pharmacokinetic studies indicated that compounds 3d and 3i are orally available and long-lasting in rat plasma after oral administration to rats by a single dose of 15 mg/kg. In conclusion, this work provided a novel 2-((4-bisarylmethyl-piperazin-1-yl)methyl)benzonitrile chemotype deserving further investigation into its antiviral therapeutic potential.

BacMam virus-based surface display for HCV E2 glycoprotein induces strong cross-neutralizing antibodies and cellular immune responses in vaccinated mice

BackgroundDespite recent advancements, limitations in the treatment and control of hepatitis C virus (HCV) infection reprioritized the studies for invention of an efficient HCV vaccine to elicit strong neutralizing antibodies (NAbs) and cellular responses.MethodsHerein, we report molecular construction of a BacMam virus-based surface display for a subtype-1a HCV gpE2 (Bac-CMV-E2-gp64; Bac) that both expressed and displayed gpE2 in mammalian cells and bacouloviral envelope, respectively.ResultsAssessments by western blotting, Immunofluorescence and Immunogold-electron microscopy indicated the proper expression and incorporation in insect cell and baculovirus envelope, respectively. Mice immunized in three different prime-boost immunization groups of: Bac/Bac, Bac/Pro (bacoulovirus-derived gpE2) and Bac/DNA (plasmid DNA (pCDNA)-encoding gpE2) developed high levels of IgG and IFN-γ (highest for Bac/Bac group) indicating the induction of both humeral and cellular immune responses. Calculation of the IgG2a/IgG1 and IFN-γ/IL-4 ratios indicated a Th1 polarization of immune responses in the Bac/Bac and Bac/DNA groups but a balanced Th1-Th2 phenotype in the Bac/Pro group. Sera of the mice in the Bac/Bac group provided the highest percentage of cross-NAbs against a subtype-2a HCVcc (JFH1) compared to Bac/Pro and Bac/DNA groups (62% versus 41% and 6%).ConclusionsResults indicated that BacMam virus-based surface display for gpE2 might act as both subunit and DNA vaccine and offers a promising strategy for development of HCV vaccine for concurrent induction of strong humoral and cellular immune responses.

Structural insights into hepatitis C virus receptor binding and entry.

Hepatitis C virus (HCV) infection is a causal agent of chronic liver disease, cirrhosis and hepatocellular carcinoma in humans, and afflicts more than 70 million people worldwide. The HCV envelope glycoproteins E1 and E2 are responsible for the binding of the virus to the host cell, but the exact entry process remains undetermined1. The majority of broadly neutralizing antibodies block interaction between HCV E2 and the large extracellular loop (LEL) of the cellular receptor CD81 (CD81-LEL)2. Here we show that low pH enhances the binding of CD81-LEL to E2, and we determine the crystal structure of E2 in complex with an antigen-binding fragment (2A12) and CD81-LEL (E2-2A12-CD81-LEL); E2 in complex with 2A12 (E2-2A12); and CD81-LEL alone. After binding CD81, residues 418-422 in E2 are displaced, which allows for the extension of an internal loop consisting of residues 520-539. Docking of the E2-CD81-LEL complex onto a membrane-embedded, full-length CD81 places the residues Tyr529 and Trp531 of E2 proximal to the membrane. Liposome flotation assays show that low pH and CD81-LEL increase the interaction of E2 with membranes, whereas structure-based mutants of Tyr529, Trp531 and Ile422 in the amino terminus of E2 abolish membrane binding. These data support a model in which acidification and receptor binding result in a conformational change in E2 in preparation for membrane fusion.

Hepatitis C virus E2 envelope glycoprotein produced in Nicotiana benthamiana triggers humoral response with virus-neutralizing activity in vaccinated mice.

SummaryChronic infection with hepatitis C virus (HCV) remains a leading cause of liver‐related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus‐neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N‐glycosylation pattern. Here we report the first evidence of successful production of the full‐length HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium‐mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N‐glycan analysis of HCV‐E2 produced in N. benthamiana and mammalian cells indicated host‐specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant‐derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV‐neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost‐effective production platforms for the development of HCV vaccines.

Sialylation of HCV E2 Glycoprotein-Specific and Natural Anti-Glycan (TF, αGal) Antibodies as Signatures of Liver Damage

The E2 glycoprotein is the target of broadly neutralizing antibodies against Hepatitis C Virus (HCV). There is evidence that the HCV E2-specific antibody glycosylation profile is associated with hepatic fibrosis progression. The main aim of this study was to compare the sialylation of E2-specific and naturally occurring antiglycan Abs to determine whether their combination could be beneficial for the non-invasive evaluation of hepatic damage. Fifty-eight patients with various stages of hepatic fibrosis or without were tested. The sialylation of HCV E2 glycoprotein-specific antibodies (E2-Abs), the Thomsen-Friedenreich antigen- and αGal glycotope-specific antibodies (TF-Abs, αGal-Abs) was analysed using the ELISA platform. The level of IgG Abs and their reactivity to Sialospecific Sambucus Nigra Lectin (SNA) were determined and changes in Abs sialylation were analysed based on the stage of liver fibrosis, HCV genotype and antiviral therapy efficacy. The late stage of liver Fibrosis (F4) was characterized by dramatically decreased E2-Ab SNA reactivity unlike stages with no fibrosis (P=0.003) and stages F1–F3 (P=0.0007). In contrast, antiglycan Abs showed an increased sialylation. In multiple regression analysis, the combination of E2 and TF-Abs sialylation patterns gave a significant advantage in assessing liver damage. A high rate of discrimination between F0 and F4 stages of fibrosis as well as between F1–F3 and F4 was obtained (ACC=0.948 and ACC=0.90, respectively). Thus, the combined analysis of disease-specific and natural Abs sialylation can remarkably enhance the clinical value of the approach in the non-invasive evaluation of hepatic damage.

From Structural Studies to HCV Vaccine Design.

Hepatitis C virus (HCV) is a serious and growing public health problem despite recent developments of antiviral therapeutics. To achieve global elimination of HCV, an effective cross-genotype vaccine is needed. The failure of previous vaccination trials to elicit an effective cross-reactive immune response demands better vaccine antigens to induce a potent cross-neutralizing response to improve vaccine efficacy. HCV E1 and E2 envelope (Env) glycoproteins are the main targets for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. Therefore, a molecular-level understanding of the nAb responses against HCV is imperative for the rational design of cross-genotype vaccine antigens. Here we summarize the recent advances in structural studies of HCV Env and Env-nAb complexes and how they improve our understanding of immune recognition of HCV. We review the structural data defining HCV neutralization epitopes and conformational plasticity of the Env proteins, and the knowledge applicable to rational vaccine design.