A simple method to purify recombinant HCV core protein expressed in Pichia pastoris for obtaining virus-like particles and producing monoclonal antibodies.

In this study, we describe an optimized method of obtaining virus-like particles (VLPs) of the recombinant hepatitis C virus (HCV) core protein (HCcAg) expressed in yeast cells (Pichia pastoris), which can be used for the construction of diagnostic test systems and vaccine engineering. The described simplified procedure was developed to enable in vitro self-assembly of HCcAg molecules into VLPs during protein purification. In brief, the HCcAg protein was precipitated from yeast cell lysates with ammonium sulfate and renatured by gel filtration on Sephadex G-25 under reducing conditions. VLPs were self-assembled after the removal of the reducing agent by gel filtration on Sephadex G-25. Protein purity and specificity were evaluated by SDS-PAGE and immunoblotting analysis. The molecular mass of VLPs and their relative quantity were measured by HPLC, followed by confirmation of VLPs production and estimation of their shape and size by transmission electron microscopy. As a result, we obtained recombinant HCcAg preparation (with ~90% purity) in the form of VLPs and monomers, which has been used to produce hybridomas secreting monoclonal antibodies (mAbs) against HCcAg.

Development of a downstream process for the production of an inactivated whole hepatitis C virus vaccine.

There is a large unmet need for a prophylactic hepatitis C virus (HCV) vaccine to control the ongoing epidemic with this deadly pathogen. Many antiviral vaccines employ whole viruses as antigens. For HCV, this approach became feasible following the development of infectious cell culture systems for virus production. However, the lack of efficient downstream processes (DSP) for HCV purification poses a roadblock for the development of a whole virus vaccine. Using cell culture-derived genotype 1a HCV we developed a scalable and efficient DSP train, employing commonly used clarification and ultrafiltration techniques, followed by two membrane-based chromatography steps. For virus capture, steric exclusion chromatography using cellulose membranes was established, resulting in a virtually complete virus recovery with > 99% protein and 84% DNA depletion. Virus polishing was achieved by sulphated cellulose membrane adsorbers with ~ 50% virus recovery and > 99% protein and 90% DNA depletion. Additional nuclease digestion resulted in 99% overall DNA depletion with final DNA concentrations of 2 ng/mL. Process results were comparable for cell culture-derived HCV of another major genotype (5a). This study provides proof-of-concept for establishment of an efficient and economically attractive DSP with potential application for production of an inactivated whole virus vaccine against HCV for human use.

Incompatible Translation Drives a Convergent Evolution and Viral Attenuation During the Development of Live Attenuated Vaccine.

Live attenuated vaccines are widely used to protect humans or animals from pathogen infections. We have previously developed a chicken embryo-attenuated Duck Hepatitis A Virus genotype 1 (DHAV-1) vaccine (CH60 strain). This study aims to understand the mechanisms that drive a virulent strain to an attenuated virus. Here, we systematically compared five DHAV-1 chicken embryo attenuated strains and 68 virulent strains. Phylogenetic analysis indicated that duck virulent strains isolated from different geographic regions of China undergo a convergent evolution in the chicken embryos. Comparative analysis indicated that the codon usage bias of the attenuated strains were shaped by chicken codons usage bias, which essentially contributed to viral adaption in the unsuitable host driven by incompatible translation. Of note, the missense mutations in coding region and mutations in untranslated regions may also contribute to viral attenuation of DHAV-1 to some extent. Importantly, we have experimentally confirmed that the expression levels of four viral proteins (2A3pro, 2A3pro, 3Cpro, and 3Dpro) in the liver and kidney of ducks infected with an attenuated strain are significantly lower than that infected with a virulent strain, despite with similar virus load. Thus, the key mechanisms of viral attenuation revealed by this study may lead to innovative and easy approaches in designing live attenuated vaccines.

Development of new hepatitis E vaccines.

Hepatitis E virus (HEV) infection is an emerging zoonotic disease posing a severe threat to public health in the world, especially to pregnant women. Currently, no specific treatments are available for HEV infection. Therefore, it is crucial to develop vaccine to prevent this infection. Although several potential candidate vaccines against HEV have been studied for their immunogenicity and efficacy, only Hecolin® which is developed by Xiamen Innovax Biotech Co., Ltd. and approved by China Food and Drug Administration (CFDA) in 2012, is the licensed HEV vaccine in the world so far. Extensive studies on safety, immunogenicity and efficacy in phase III clinical trials have shown that Hecolin® is a promising vaccine for HEV prevention and control. In this article, the advances on HEV vaccine development and research are briefly reviewed.

Future landscape of hepatitis C research - Basic, translational and clinical perspectives.

With the latest all-oral interferon- and ribavirin-free regimens based on direct acting antivirals against the hepatitis C virus (HCV), sustained virological response rates of >90% are achieved, which is equivalent to cure. This has become possible for all genotypes and all subgroups of patients, including many of the most difficult-to-treat populations so far. Since a prophylactic HCV vaccine is not yet available, control of HCV infection will for the time being have to rely on the use of effective and safe antiviral treatments as well as their accessibility and affordability. Different approaches may apply to different parts of the world, eradication of HCV representing a major long-term goal. Whether hepatitis C becomes the first chronic viral infection to be eradicated without a prophylactic vaccine remains to be shown. Here, we briefly summarize advances in the molecular virology of hepatitis C, highlight lessons of biological relevance that were learned through the study of HCV, and its translational and clinical implications. We have also listed selected unsolved challenges, emphasizing that HCV is a unique model and that advances in this direction may yield knowledge of broad biological significance, novel technologies and insights into related important human pathogens.

The history of hepatitis C virus (HCV): Basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control.

The discovery of hepatitis C virus (HCV) in 1989 permitted basic research to unravel critical components of a complex life cycle for this important human pathogen. HCV is a highly divergent group of viruses classified in 7 major genotypes and a great number of subtypes, and circulating in infected individuals as a continuously evolving quasispecies destined to escape host immune responses and applied antivirals. Despite the inability to culture patient viruses directly in the laboratory, efforts to define the infectious genome of HCV resulted in development of experimental recombinant in vivo and in vitro systems, including replicons and infectious cultures in human hepatoma cell lines. And HCV has become a model virus defining new paradigms in virology, immunology and biology. For example, HCV research discovered that a virus could be completely dependent on microRNA for its replication since microRNA-122 is critical for the HCV life cycle. A number of other host molecules critical for HCV entry and replication have been identified. Thus, basic HCV research revealed important molecules for development of host targeting agents (HTA). The identification and characterization of HCV encoded proteins and their functional units contributed to the development of highly effective direct acting antivirals (DAA) against the NS3 protease, NS5A and the NS5B polymerase. In combination, these inhibitors have since 2014 permitted interferon-free therapy with cure rates above 90% among patients with chronic HCV infection; however, viral resistance represents a challenge. Worldwide control of HCV will most likely require the development of a prophylactic vaccine, and numerous candidates have been pursued. Research characterizing features critical for antibody-based virus neutralization and T cell based virus elimination from infected cells is essential for this effort. If the world community promotes an ambitious approach by applying current DAA broadly, continues to develop alternative viral- and host- targeted antivirals to combat resistant variants, and invests in the development of a vaccine, it would be possible to eradicate HCV. This would prevent about 500 thousand deaths annually. However, given the nature of HCV, the millions of new infections annually, a high chronicity rate, and with over 150 million individuals with chronic infection (which are frequently unidentified), this effort remains a major challenge for basic researchers, clinicians and communities.

Establishment and characterization of duck embryo epithelial (DEE) cell line and its use as a new approach toward DHAV-1 propagation and vaccine development.

The primary cell culture was derived from duck embryonic tissue, digested with collagenase type I. The existence of cell colonies with epithelial-like morphology, named duck embryo epithelial (DEE), were purified and optimally maintained at 37°C in M199 medium supplemented with 5% fetal bovine serum. The purified cells were identified as epithelial cell line by detecting Keratin-18 expression using immunofluorescence assay. Our findings demonstrated that DEE cell line can be propagated in culture with (i) a great capacity to adhere, (ii) a great proliferation activity, and (iii) a population doubling time of approximately 18h. Chromosomal features of the DEE cell line were remained constant after the 50th passage. Further characterizations of DEE cell line showed that cell line can normally be grown even after several passages and never converted to tumorigenic cells either in vitro or in vivo study. Susceptibility of DEE cell line was determined for transfection and duck hepatitis A type 1 virus (DHAV-1)-infection. Interestingly, the 50% egg lethal dose (ELD50) of the propagated virus in DEE cell line was higher than ELD50 of the propagated virus in embryonated eggs. Finally, DEE cell line was evaluated to be used as a candidate for DHAV-1 vaccine development. Our results showed that the propagated DHAV-1 vaccine strain SDE in DEE cell line was able to protect ducklings against DHAV-1 challenge. Taken together, our findings suggest that the DEE cell line can serve as a valuable tool for DHAV-1 propagation and vaccine production.

Hepatitis C virus infection treatment: An era of game changer direct acting antivirals and novel treatment strategies.

Chronic hepatitis C virus infection and associated liver diseases represent a major health care burden all over the world. The current standard of care, i.e. peginterferon-alfa (PEG-IFNα) plus ribavirin (RBV) are associated with frequent and sometimes serious adverse effects and contraindications, which further limit their therapeutic efficacy. The approval of first and second generation HCV protease inhibitors represents a major breakthrough in the development of novel direct acting antivirals (DAAs) against different HCV genotypes and establishes a new standard of care for chronically infected HCV genotypes 1 patients. Similarly, next generation protease inhibitors and HCV RNA polymerase inhibitors have shown better pharmacokinetics and pharmacodynamics in terms of broader HCV genotypes coverage, better safety profile, fewer drug interactions and possible once daily administration than first generation direct acting antivirals. The testing of adenovirus-based vector vaccines, which escalates the innate and acquired immune responses against the most conserved regions of the HCV genome in chimpanzees and humans, may be a promising therapeutic approach against HCV infection in coming future. This review article presents up-to-date knowledge and recent developments in HCV therapeutics, insights the shortcomings of current HCV therapies and key lessons from the therapeutic potential of improved anti-HCV treatment strategies.

A Novel Adeno-Associated Virus-Based Genetic Vaccine Encoding the Hepatitis C Virus NS3/4 Protein Exhibits Immunogenic Properties in Mice Superior to Those of an NS3-Protein-Based Vaccine.

More than 170 million individuals worldwide are infected with hepatitis C virus (HCV), and up to an estimated 30% of chronically infected individuals will go on to develop progressive liver disease. Despite the recent advances in antiviral treatment of HCV infection, it remains a major public health problem. Thus, development of an effective vaccine is urgently required. In this study, we constructed novel adeno-associated virus (AAV) vectors expressing the full-length NS3 or NS3/4 protein of HCV genotype 1b. The expression of the NS3 or NS3/4 protein in HepG2 cells was confirmed by western blotting. C57BL/6 mice were intramuscularly immunised with a single injection of AAV vectors, and the resultant immune response was investigated. The AAV2/rh32.33.NS3/4 vaccine induced stronger humoral and cellular responses than did the AAV2/rh32.33.NS3 vaccine. Our results demonstrate that AAV-based vaccines exhibit considerable potential for the development of an effective anti-HCV vaccine.

Lessons from hepatitis E vaccine design.

Acute hepatitis E is still a major public health issue, especially in developing countries, and hepatitis E virus (HEV) infection will likely only be preventable through prophylactic vaccines. In this review, we describe the lessons learnt from developing the first commercial hepatitis E vaccine (Hecolin), launched to market in China in 2012. The antigenicity and immunogenicity of VLP immunogens concomitant with the scalable Escherichia coli system and our large-scale clinical verification resulted in the success of our vaccine. The structures of the HEV capsid protein in complex with different antibodies provide important molecular insights into capsid assembly and antibody neutralization of the virus, providing a paradigm for B-cell epitope-based vaccine design.

Virus-like particle-based human vaccines: quality assessment based on structural and functional properties.

Human vaccines against three viruses use recombinant virus-like particles (VLPs) as the antigen: hepatitis B virus, human papillomavirus, and hepatitis E virus. VLPs are excellent prophylactic vaccine antigens because they are self-assembling bionanoparticles (20 to 60 nm in diameter) that expose multiple epitopes on their surface and faithfully mimic the native virions. Here we summarize the long journey of these vaccines from bench to patients. The physical properties and structural features of each recombinant VLP vaccine are described. With the recent licensure of Hecolin against hepatitis E virus adding a third disease indication to prophylactic VLP-based vaccines, we review how the crucial quality attributes of VLP-based human vaccines against all three disease indications were assessed, controlled, and improved during bioprocessing through an array of structural and functional analyses.

Progress in the development of preventive and therapeutic vaccines for hepatitis C virus.

Hepatitis C virus (HCV) is a blood borne disease estimated to chronically infect 3% of the worlds' population causing significant morbidity and mortality. Current medical therapy is curative in approximately 50% of patients. While recent treatment advances of genotype 1 infection using directly acting antiviral agents (DAAs) are encouraging, there is still a need to develop vaccine strategies capable of preventing infection. Moreover, vaccines may also be used in future in combination with DAAs enabling interferon-free treatment regimens. Viral and host specific factors contribute to viral evasion and present important impediments to vaccine development. Both, innate and adaptive immune responses are of major importance for the control of HCV infection. However, HCV has evolved ways of evading the host's immune response in order to establish persistent infection. For example, HCV inhibits intracellular interferon signalling pathways, impairs the activation of dendritic cells, CD8(+) and CD4(+) T cell responses, induces a state of T-cell exhaustion and selects escape variants with mutations CD8(+) T cell epitopes. An effective vaccine will need to produce strong and broadly cross-reactive CD4(+), CD8(+) T cell and neutralising antibody (NAb) responses to be successful in preventing or clearing HCV. Vaccines in clinical trials now include recombinant proteins, synthetic peptides, virosome based vaccines, tarmogens, modified vaccinia Ankara based vaccines, and DNA based vaccines. Several preclinical vaccine strategies are also under development and include recombinant adenoviral vaccines, virus like particles, and synthetic peptide vaccines. This paper will review the vaccines strategies employed, their success to date and future directions of vaccine design.

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope.

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

Induction of hepatitis C virus-specific humoral and cellular immune responses in mice and rhesus by artificial multiple epitopes sequence.

The investigation of antigenic epitopes in hepatitis C virus (HCV) protein suggests that a central sequence combined with multiple antigenic epitopes of HCV might be significant as a potential vaccine candidate. This artificial sequence of combined and modified multiple antigenic epitopic peptides (Hc-B2), containing three B and four T cell epitopes, was constructed and expressed in E. coli. Antigen analysis indicated that this peptide antigen was capable of interacting with anti-sera collected from hepatitis C patients infected by three genotypes of HCV from three different geographic areas of China, respectively. The immunological analysis of this peptide antigen in mice and rhesus suggested that its immunogenicity was effective. However, a complete evaluation of this peptide could not be made as an effective animal model for HCV infection (such as in the chimpanzee) was not available for this study.

Immunogenicity and protective efficacy of a vaccine prepared from 53 kDa truncated hepatitis E virus capsid protein expressed in insect cells.

Hepatitis E virus (HEV) is an enterically transmitted virus that causes acute hepatitis. Expression of recombinant HEV capsid protein in insect cells results in two major proteolytically-processed products of 56 and 53kDa which consist of amino acids (aa) 112-607 and 112-578, respectively. The only neutralization epitope identified to date is located at least partially between amino acids 578 and 607 meaning it should be present only in the 56 and not in the 53kDa protein. Previously, it was shown that vaccination with the 56kDa protein greatly reduced virus shedding and protected Rhesus monkeys from hepatitis E when challenged with a high intravenous dose of homologous or heterologous HEV. To evaluate the immunogenicity and protective efficacy of the 53kDa protein, we vaccinated Rhesus monkeys with this protein and challenged them with a high or low dose of homologous virus. Vaccination with the 53kDa protein greatly reduced virus shedding but did not protect against hepatitis following the high dose challenge. Virus was not detected in the vaccinated animals following the low dose challenge, suggesting that sterilizing immunity may have been achieved.

Approaches to improve engineered vaccines for human immunodeficiency virus and other viruses that cause chronic infections.

We used several approaches to develop enhanced vaccines for chronic viral infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). 1) Selected epitopes were used to avoid potentially harmful immune responses. 2) Linkage between helper and cytotoxic T-lymphocyte (CTL) epitopes was found to be important. 3) We developed an "epitope enhancement" approach modifying the sequences of epitopes to make more potent vaccines, including examples for HIV and HCV epitopes presented by murine class II and human class I major histocompatibility complex (MHC) molecules. 4) CTL avidity was found to be important for clearing viral infections in vivo, and the mechanism was examined. High-avidity CTLs, however, were found to undergo apoptosis when confronted with high-density antigen, through a mechanism involving tumor necrosis factor (TNF), TNF-RII, and a permissive state induced through the T-cell receptor. 5) We employed cytokines in the adjuvant to steer immune responses toward desired phenotypes, and showed synergy between cytokines. 6) Intrarectal immunization with peptide vaccine induced mucosal and systemic CTL. Local mucosal CTL were found to be critical for resistance to mucosal viral transmission and this resistance was enhanced with mucosally delivered interleukin-12. 7) We used an asymmetry in induction of mucosal and systemic immune responses to circumvent pre-existing vaccinia immunity for use of recombinant vaccinia vaccines.

Evaluation of the purity of a purified, inactivated hepatitis A vaccine (VAQTA).

Manufacture of VAQTA, an inactivated hepatitis A virus vaccine, includes extensive purification of the intact virus particle to remove endogenous components from the host cell culture lysate as well as compounds introduced in the upstream purification process. Analysis of the final purified hepatitis A virus product by SDS-PAGE prior to inactivation shows that greater than 95% of the protein in the preparation is found in four protein bands, which have been confirmed to be hepatitis A virus capsid proteins VP0, VP1, VP2 and VP3 based on Western blot and mass spectrometry analyses. Validation of the manufacturing process and direct analysis of the final product were used to demonstrate that no other specific host cell-derived components are detected and that process residuals are all below the limits of detection of the assays used. Establishment of a rigorous standard of high purity for this product was pursued to minimize the impact of impurities during clinical development of this product and will facilitate the incorporation of this product into combination vaccines.

Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells.

The hepatitis E virus (HEV) capsid antigen has been proposed as a candidate subunit vaccine for the prevention of hepatitis E. The full-length HEV ORF2 protein product is predicted to contain 660 amino acids and to weigh 72,000 daltons. Expression of the HEV ORF2 capsid gene from recombinant baculoviruses in insect cells produced multiple immunoreactive proteins ranging in size from 30 to 100 kDa. The most abundant HEV proteins had molecular weights of 72, 63, 56, and 53 kDa. Temporal expression kinetics of these viral polypeptides indicated that the 72- and 63-kDa polypeptides were produced abundantly within the initial 36 h. postinfection but were replaced by 56- and 53-kDa polypeptides in the cell and medium, respectively, by 48 h postinfection. The 53-kDa protein was secreted as early as 24 h. postinfection, and accumulation in the medium peaked by 72 h postinfection. Purification of the 53-, 56-, and 63-kDa viral polypeptides was accomplished by anion-exchange and subsequent gel filtration chromatography. Sequence analysis of the 53-, 56-, and 63-kDa HEV polypeptides indicated that the amino terminus was amino acid residue 112 of the predicted full-length protein product. The results of carboxy terminal amino acid sequencing indicated that the carboxy terminus of the 53-, 56-, and 63-kDa HEV proteins was located at amino acid residues 578, 607, and 660, respectively. The molecular masses of the 53- and 56-kDa HEV polypeptides were 53,872 and 56,144 as determined by mass spectroscopy.

Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry.

A protein with a molecular mass of approximately 62.10(3), derived from open reading frame 2 (ORF-2) of the hepatitis E virus (HEV: Burma strain), was expressed in a baculovirus expression vector and purified to homogeneity. The recombinant 62 kDa protein appeared to be a doublet, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tryptic digestion in conjunction with laser desorption mass spectrometry (LD-MS) and sequence analysis of the tryptic peptides indicated that the amino terminus was blocked, although no proteolytic degradation occurred. The determined internal sequences of peptides were in agreement with the predicted ORF-2 protein. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS) resolved the doublet proteins into two major components with molecular masses of 56548.5 and 58161.4. Confirmation of the amino terminus of the molecule by LD-MS post-ion decay enabled us to tentatively assign the carboxyl terminus of each species at residues 540 and 525. Sequencing of the intact protein by automated carboxyl terminal sequencing confirmed that the carboxyl terminus was truncated and that the sequence assignment predicted by LC-MS was correct.