Enhancing the antigenicity and immunogenicity of monomeric forms of hepatitis C virus E2 for use as a preventive vaccine.

The E2 glycoprotein of hepatitis C virus (HCV) is the major target of broadly neutralizing antibodies (bNAbs) that are critical for the efficacy of a prophylactic HCV vaccine. We previously showed that a cell culture-derived, disulfide-linked high-molecular-weight (HMW) form of the E2 receptor-binding domain lacking three variable regions, Δ123-HMW, elicits broad neutralizing activity against the seven major genotypes of HCV. A limitation to the use of this antigen is that it is produced only at low yields and does not have a homogeneous composition. Here, we employed a sequential reduction and oxidation strategy to efficiently refold two high-yielding monomeric E2 species, D123 and a disulfide-minimized version (D123A7), into disulfide-linked HMW-like species (Δ123r and Δ123A7r). These proteins exhibited normal reactivity to bNAbs with continuous epitopes on the neutralizing face of E2, but reduced reactivity to conformation-dependent bNAbs and nonneutralizing antibodies (non-NAbs) compared with the corresponding monomeric species. Δ123r and Δ123A7r recapitulated the immunogenic properties of cell culture-derived D123-HMW in guinea pigs. The refolded antigens elicited antibodies that neutralized homologous and heterologous HCV genotypes, blocked the interaction between E2 and its cellular receptor CD81, and targeted the AS412, AS434, and AR3 domains. Of note, antibodies directed to epitopes overlapping with those of non-NAbs were absent. The approach to E2 antigen engineering outlined here provides an avenue for the development of preventive HCV vaccine candidates that induce bNAbs at higher yield and lower cost.

Establishment of a Highly Sensitive Assay for Detection of Hepatitis E Virus-Specific Immunoglobulins.

Hepatitis E, a liver disease caused by infection with the hepatitis E virus (HEV), is a worldwide emerging disease. The diagnosis is based on the detection of viral RNA and of HEV-specific immunoglobulins (Ig). For the latter, various assays are commercially available but still lack harmonization. In this study, a Luminex-based multiplex serological assay was established that measures the presence of total IgG, IgA, and IgM antibodies, targeting a short peptide derived from the viral E2 protein. For the validation, 160 serum samples with a known HEV serostatus were used to determine the assay cutoff and accuracy. Thereby, HEV IgG- and RNA-positive sera were identified with a sensitivity of 100% and a specificity of 98% (95% confidence interval [CI], 94% to 100%). Application of the assay by retesting 514 serum samples previously characterized with different HEV-IgG or total antibody tests revealed a high level of agreement between the assays (Cohen's kappa, 0.58 to 0.99). The established method is highly sensitive and specific and can be easily implemented in a multiplex format to facilitate rapid differential diagnostics with a few microliters of sample input.

Evaluation of a Hepatitis C Virus Core Antigen Assay in Plasma and Dried Blood Spot Samples.

Simplified, affordable tools to diagnose active hepatitis C virus (HCV) infection are needed to scale up treatment. This study evaluated the analytical performance of HCV core antigen (HCVcAg) detection in samples of plasma and dried venous blood spots (DBSs). Paired plasma and DBS samples were prepared from remnant diagnostic samples, and plasma HCV RNA and HCVcAg were quantified. Sensitivity and specificity for HCVcAg (>3 fmol/L) at two HCV RNA thresholds (≥15 and ≥3000 IU/mL) were calculated. Of 120 paired samples tested, 25 had nonquantifiable HCV RNA and 95 had quantifiable HCV RNA. The median HCV RNA level in plasma was 5.6 log10 IU/mL (interquartile range: 5.2 to 6.2). The median HCVcAg levels in plasma and DBS samples were 2.3 log10 fmol/L (interquartile range: 0.1 to 3.1) and 1.1 log10 fmol/L (interquartile range: 0.0 to 1.9), respectively. For diagnosing HCV RNA ≥3000 IU/mL, the sensitivity and specificity of HCVcAg in plasma were 97.7% (95% CI, 91%-100%) and 100% (95% CI, 87%-100%), respectively. The sensitivity and specificity of HCVcAg in DBS were 88.6% (95% CI, 80%-94%) and 97% (95% CI, 82%-100%), respectively. The data from this study demonstrate good sensitivity and specificity of HCVcAg in plasma at an HCV RNA threshold of ≥3000 IU/mL. The level of HCVcAg quantified in plasma was higher than that in DBS.

Origin, antigenicity, and function of a secreted form of ORF2 in hepatitis E virus infection.

The enterically transmitted hepatitis E virus (HEV) adopts a unique strategy to exit cells by cloaking its capsid (encoded by the viral ORF2 gene) and circulating in the blood as "quasi-enveloped" particles. However, recent evidence suggests that the majority of the ORF2 protein present in the patient serum and supernatants of HEV-infected cell culture exists in a free form and is not associated with virus particles. The origin and biological functions of this secreted form of ORF2 (ORF2S) are unknown. Here we show that production of ORF2S results from translation initiated at the previously presumed AUG start codon for the capsid protein, whereas translation of the actual capsid protein (ORF2C) is initiated at a previously unrecognized internal AUG codon (15 codons downstream of the first AUG). The addition of 15 amino acids to the N terminus of the capsid protein creates a signal sequence that drives ORF2S secretion via the secretory pathway. Unlike ORF2C, ORF2S is glycosylated and exists as a dimer. Nonetheless, ORF2S exhibits substantial antigenic overlap with the capsid, but the epitopes predicted to bind the putative cell receptor are lost. Consistent with this, ORF2S does not block HEV cell entry but inhibits antibody-mediated neutralization. These results reveal a previously unrecognized aspect in HEV biology and shed new light on the immune evasion mechanisms and pathogenesis of this virus.

Targeting naturally occurring epitope variants of hepatitis C virus with high-affinity T-cell receptors.

Hepatitis C virus (HCV) readily establishes chronic infection, which is characterized by failure of virus-specific CD8+ T cells. HCV uses epitope mutation and T-cell exhaustion to escape from the host immune response. Previously, we engineered high-affinity T-cell receptors (HATs) targeting human immunodeficiency virus escape mutants. In this study, the affinity of a T-cell receptor specific for the HLA-A2-restricted HCV immunodominant epitope NS3 1406-1415 (KLVALGINAV) was improved from a KD of 6.6 µM to 40 pM. These HATs could also target HCV NS3 naturally occurring variants, including an escape variant vrt1 (KLVVLGINAV), with high affinities. The HATs can be used as high-affinity targeting molecules at the centre of the immune synapse for the HLA-restricted NS3 antigen. By fusing the HAT with a T-cell activation molecule, an anti-CD3 single-chain variable fragment, we constructed a molecule called high-affinity T-cell activation core (HATac), which can redirect functional CTLs possessing any specificity to recognize and kill cells presenting HCV NS3 antigens. This capability was verified with T2 cells loaded with prototype or variant peptides and HepG2 cells expressing the truncated NS3 prototype or variant proteins. The results indicate that HATac targeting the HLA-restricted NS3 antigen may provide a useful tool for circumventing immune escape mutants and T-cell exhaustion caused by HCV infection.

Rules of co-occurring mutations characterize the antigenic evolution of human influenza A/H3N2, A/H1N1 and B viruses.

BACKGROUND: The human influenza viruses undergo rapid evolution (especially in hemagglutinin (HA), a glycoprotein on the surface of the virus), which enables the virus population to constantly evade the human immune system. Therefore, the vaccine has to be updated every year to stay effective. There is a need to characterize the evolution of influenza viruses for better selection of vaccine candidates and the prediction of pandemic strains. Studies have shown that the influenza hemagglutinin evolution is driven by the simultaneous mutations at antigenic sites. Here, we analyze simultaneous or co-occurring mutations in the HA protein of human influenza A/H3N2, A/H1N1 and B viruses to predict potential mutations, characterizing the antigenic evolution. METHODS: We obtain the rules of mutation co-occurrence using association rule mining after extracting HA1 sequences and detect co-mutation sites under strong selective pressure. Then we predict the potential drifts with specific mutations of the viruses based on the rules and compare the results with the "observed" mutations in different years. RESULTS: The sites under frequent mutations are in antigenic regions (epitopes) or receptor binding sites. CONCLUSIONS: Our study demonstrates the co-occurring site mutations obtained by rule mining can capture the evolution of influenza viruses, and confirms that cooperative interactions among sites of HA1 protein drive the influenza antigenic evolution.

Transmitted/Founder Viruses Rapidly Escape from CD8+ T Cell Responses in Acute Hepatitis C Virus Infection.

UNLABELLED: The interaction between hepatitis C virus (HCV) and cellular immune responses during very early infection is critical for disease outcome. To date, the impact of antigen-specific cellular immune responses on the evolution of the viral population establishing infection and on potential escape has not been studied. Understanding these early host-virus dynamics is important for the development of a preventative vaccine. Three subjects who were followed longitudinally from the detection of viremia preseroconversion until disease outcome were analyzed. The evolution of transmitted/founder (T/F) viruses was undertaken using deep sequencing. CD8(+) T cell responses were measured via enzyme-linked immunosorbent spot (ELISpot) assay using HLA class I-restricted T/F epitopes. T/F viruses were rapidly extinguished in all subjects associated with either viral clearance (n = 1) or replacement with viral variants leading to establishment of chronic infection (n = 2). CD8(+) T cell responses against 11 T/F epitopes were detectable by 33 to 44 days postinfection, and 5 of these epitopes had not previously been reported. These responses declined rapidly in those who became chronically infected and were maintained in the subject who cleared infection. Higher-magnitude CD8(+) T cell responses were associated with rapid development of immune escape variants at a rate of up to 0.1 per day. Rapid escape from CD8(+) T cell responses has been quantified for the first time in the early phase of primary HCV infection. These rapid escape dynamics were associated with higher-magnitude CD8(+) T cell responses. These findings raise questions regarding optimal selection of immunogens for HCV vaccine development and suggest that detailed analysis of individual epitopes may be required. IMPORTANCE: A major limitation in our detailed understanding of the role of immune response in HCV clearance has been the lack of data on very early primary infection when the transmitted viral variants successfully establish the acute infection. This study was made possible through the availability of specimens from a unique cohort of asymptomatic primary infection cases in whom the first available viremic samples were collected approximately 3 weeks postinfection and at regular intervals thereafter. The study included detailed examination of both the evolution of the viral population and the host cellular immune responses against the T/F viruses. The findings here provide the first evidence of host cellular responses targeting T/F variants and imposing a strong selective force toward viral escape. The results of this study provide useful insight on how virus escapes the host response and consequently on future analysis of vaccine-induced immunity.

Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Protein of Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and Human HEVs.

UNLABELLED: Antisera raised against the avian hepatitis E virus (HEV) capsid protein are cross-reactive with human and swine HEV capsid proteins. In this study, two monoclonal antibodies (MAbs) against the avian HEV capsid protein, namely, 3E8 and 1B5, were shown to cross-react with the swine HEV capsid protein. The motifs involved in binding both MAbs were identified and characterized using phage display biopanning, peptide synthesis, and truncated or mutated protein expression, along with indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting. The results showed that the I/VPHD motif is a necessary core sequence and that P and H are two key amino acids for recognition by MAb 3E8. The VKLYM/TS motif is the minimal amino acid sequence necessary for recognition by MAb 1B5. Cross-reactivity between the two epitopes and antibodies against avian, swine, and human HEVs in sera showed that both epitopes are common to avian, swine, and human HEVs. In addition, amino acid sequence alignment of the capsid proteins revealed that the key motifs of both novel epitopes are the same in HEVs from different animal species, predicting that they may be common to HEV isolates from boars, rabbits, rats, ferrets, mongooses, deer, and camels as well. Protein modeling analysis showed that both epitopes are at least partially exposed on the surface of the HEV capsid protein. Protective capacity analysis demonstrated that the two epitopes are nonprotective against avian HEV infection in chickens. Collectively, these studies characterize two novel linear B-cell epitopes common to avian, swine, and human HEVs, which furthers the understanding of HEV capsid protein antigenic structure. IMPORTANCE: More and more evidence indicates that the host range diversity of hepatitis E virus (HEV) is a global public health concern. A better understanding of the antigenic structure of the HEV capsid protein may improve disease diagnosis and prevention. In this study, binding site mapping and localization as well as the antigenic biology of two novel linear B-cell epitopes common to several different species of HEV were characterized. These findings partially reveal the antigenic structure of the HEV capsid protein and provide potential applications for the development of diagnostics and interventions for HEV infection.

Rat hepatitis E virus: geographical clustering within Germany and serological detection in wild Norway rats (Rattus norvegicus).

Zoonotic hepatitis E virus (HEV) infection in industrialised countries is thought to be caused by transmission from wild boar, domestic pig and deer as reservoir hosts. The detection of HEV-specific antibodies in rats and other rodents has suggested that these animals may represent an additional source for HEV transmission to human. Recently, a novel HEV (ratHEV) was detected in Norway rats from Hamburg, Germany, showing the typical genome organisation but a high nucleotide and amino acid sequence divergence to other mammalian and to avian HEV strains. Here we describe the multiple detection of ratHEV RNA and HEV-specific antibodies in Norway rats from additional cities in north-east and south-west Germany. The complete genome analysis of two novel strains from Berlin and Stuttgart confirmed the association of ratHEV to Norway rats. The present data indicated a continuing existence of this virus in the rat populations from Berlin and Hamburg. The phylogenetic analysis of a short segment of the open reading frame 1 confirmed a geographical clustering of the corresponding sequences. Serological investigations using recombinant ratHEV and genotype 3 capsid protein derivatives demonstrated antigenic differences which might be caused by the high amino acid sequence divergence in the immunodominant region. The high amount of animals showing exclusively ratHEV RNA or anti-ratHEV antibodies suggested a non-persistent infection in the Norway rat. Future studies have to prove the transmission routes of the virus in rat populations and its zoonotic potential. The recombinant ratHEV antigen generated here will allow future seroepidemiological studies to differentiate ratHEV and genotype 3 infections in humans and animals.

Toxicology and biodistribution study of CIGB-230, a DNA vaccine against hepatitis C virus.

CIGB-230, a mixture of a DNA plasmid expressing hepatitis C virus (HCV) structural antigens and a HCV recombinant capsid protein, has demonstrated to elicit strong immune responses in animals. The present study evaluated the plasmid biodistribution after the administration of CIGB-230 in mice, as well as toxicity of this vaccine candidate in rats. In the biodistribution study, mice received single or repeated intramuscular injections of CIGB-230, 50 microg of plasmid DNA mixed with 5 microg of Co.120 protein. Plasmid presence was assessed in ovaries, kidney, liver, pancreas, mesenteric ganglion, blood, and muscle of the injection site by a qualitative polymerase chain reaction. The toxicology evaluation included treatment groups receiving doses 5, 15, or 50 times higher, according to the body weight, than the expected therapeutic clinical dose. During the first hour after repeated inoculation, a promiscuous distribution was observed. However, 3 months later, plasmid could not be detected in any tissue. There was an absence of detectable adverse effects on key toxicology parameters and no damage evidenced in inspected organs and tissues. These results indicate that CIGB-230 is nontoxic at local and systemic levels and no concerns about persistence are observed, which support clinical testing of this vaccine candidate against HCV.

A new artificial antigen of the hepatitis E virus.

An artificial antigen composed of 12 small antigenic regions derived from the ORF2 and ORF3 HEV proteins was designed. The gene encoding for this artificial antigen was assembled from synthetic oligonucleotides by a new method called Restriction Enzyme-Assisted Ligation (REAL). The diagnostic relevance of this second generation HEV mosaic protein (HEV MA-II) was demonstrated by testing this antigen against a panel of 142 well defined anti-HEV positive and anti-HEV negative serum samples. The data obtained in this study support the substantial diagnostic potential of this HEV mosaic antigen.

Immunogenicity of CIGB-230, a therapeutic DNA vaccine preparation, in HCV-chronically infected individuals in a Phase I clinical trial.

SUMMARY: Hepatitis C virus (HCV) is a worldwide health problem. No vaccine is available against this pathogen and therapeutic treatments currently in use are of limited efficacy. In the present study, the immunogenicity of the therapeutic vaccine candidate CIGB-230, based on the mixture of pIDKE2, a plasmid expressing HCV structural antigens, with a recombinant HCV core protein, Co.120, was evaluated. CIGB-230 was administered by intramuscular injection on weeks 0, 4, 8, 12, 16 and 20 to 15 HCV-chronically infected individuals, non-responders to previous treatment with interferon (IFN) plus ribavirin. Interestingly, following the final immunization, neutralizing antibody responses against heterologous viral pseudoparticles were modified in eight individuals, including six de novo responders. In addition, 73% of vaccinees exhibited specific T cell proliferative response and T cell IFN-gamma secretory response 24 weeks after primary immunization with CIGB-230. Furthermore, 33.3% of individuals developed de novo cellular immune response against HCV core and the number of patients (46.7% at the end of treatment) with cellular immune response against more than one HCV structural antigen increased during vaccination (P = 0.046). In addition, despite persistent detection of HCV RNA, more than 40% percent of vaccinated individuals improved or stabilized liver histology, particularly reducing fibrosis, which correlated with cellular immune response against more than one HCV antigen (P = 0.0053). In conclusion, CIGB-230 is a promising candidate for effective therapeutic interventions based on its ability for enhancing the immune response in HCV chronically infected individuals.

A mechanism to explain the selection of the hepatitis e antigen-negative mutant during chronic hepatitis B virus infection.

Hepatitis B virus (HBV) expresses two structural forms of the nucleoprotein, the intracellular nucleocapsid (hepatitis core antigen [HBcAg]) and the secreted nonparticulate form (hepatitis e antigen [HBeAg]). The aim of this study was to evaluate the ability of HBcAg- and HBeAg-specific genetic immunogens to induce HBc/HBeAg-specific CD4(+)/CD8(+) T-cell immune responses and the potential to induce liver injury in HBV-transgenic (Tg) mice. Both the HBcAg- and HBeAg-specific plasmids primed comparable immune responses. Both CD4(+) and CD8(+) T cells were important for priming/effector functions of HBc/HBeAg-specific cytotoxic T-lymphocyte (CTL) responses. However, a unique two-step immunization protocol was necessary to elicit maximal CTL priming. Genetic vaccination did not prime CTLs in HBe- or HBc/HBeAg-dbl-Tg mice but elicited a weak CTL response in HBcAg-Tg mice. When HBc/HBeAg-specific CTLs were adoptively transferred into HBc-, HBe-, and HBc/HBeAg-dbl-Tg mice, the durations of the liver injury and inflammation were significantly greater in HBeAg-Tg recipient mice than in HBcAg-Tg mice. Importantly, liver injury in HBc/HBeAg-dbl-Tg mice was similar to the injury observed in HBeAg-Tg mice. Loss of HBeAg synthesis commonly occurs during chronic HBV infection; however, the mechanism of selection of HBeAg-negative variants is unknown. The finding that hepatocytes expressing wild-type HBV (containing both HBcAg and HBeAg) are more susceptible to CTL-mediated clearance than hepatocytes expressing only HBcAg suggest that the HBeAg-negative variant may have a selective advantage over wild-type HBV within the livers of patients with chronic infection during an immune response and may represent a CTL escape mutant.

Antiviral therapy with entecavir combined with post-exposure "prime-boost" vaccination eliminates duck hepatitis B virus-infected hepatocytes and prevents the development of persistent infection.

Short-term antiviral therapy with the nucleoside analogue entecavir (ETV), given at an early stage of duck hepatitis B virus (DHBV) infection, restricts virus spread and leads to clearance of DHBV-infected hepatocytes in approximately 50% of ETV-treated ducks, whereas widespread and persistent DHBV infection develops in 100% of untreated ducks. To increase the treatment response rate, ETV treatment was combined in the current study with a post-exposure "prime-boost" vaccination protocol. Four groups of 14-day-old ducks were inoculated intravenously with a dose of DHBV previously shown to induce persistent DHBV infection. One hour post-infection (p.i.), ducks were primed with DNA vaccines that expressed DHBV core (DHBc) and surface (pre-S/S and S) antigens (Groups A, B) or the DNA vector alone (Groups C, D). ETV (Groups A, C) or water (Groups B, D) was simultaneously administered by gavage and continued for 14 days. Ducks were boosted 7 days p.i. with recombinant fowlpoxvirus (rFPV) strains also expressing DHBc and pre-S/S antigens (Groups A, B) or the FPV-M3 vector (Groups C, D). DHBV-infected hepatocytes were observed in the liver of all ducks at day 4 p.i. with reduced numbers in the ETV-treated ducks. Ducks treated with ETV plus the control vectors showed restricted spread of DHBV infection during ETV treatment, but in 60% of cases, infection became widespread after ETV was stopped. In contrast, at 14 and 67 days p.i., 100% of ducks treated with ETV and "prime-boost" vaccination had no detectable DHBV-infected hepatocytes and had cleared the DHBV infection. These findings suggest that ETV treatment combined with post-exposure "prime-boost" vaccination induced immune responses that eliminated DHBV-infected hepatocytes and prevented the development of persistent DHBV infection.

HCV-specific T-cell response in relation to viral kinetics and treatment outcome (DITTO-HCV project).

BACKGROUND & AIMS: The second slope of viral decline induced by interferon treatment has been suggested to be influenced mainly by the hepatitis C virus (HCV)-specific T-cell response; however, this hypothesis needs to be validated by results derived from experimental studies. METHODS: To address this issue, the HCV-specific T-cell response of 32 genotype-1-infected patients of the 270 patients enrolled in the dynamically individualized treatment of hepatitis C infection and correlates of viral/host dynamics phase III, open-label, randomized, multicenter trial was studied in relation to viral kinetics and treatment outcome. RESULTS: Greater proliferative responses by HCV-specific CD8 cells were found before treatment in patients with a fast viral decline and with a sustained viral response. However, no significant improvement of HCV-specific CD8 responses was observed in the first weeks of therapy in both rapid viral responder and non-rapid viral responder patients. A mild enhancement of proliferative T-cell responses and a partial restoration of the cytotoxic T-cell potential was expressed only late during treatment, likely favored by HCV clearance. CONCLUSIONS: Early restoration of an efficient T-cell response does not seem to be an essential requirement for a rapid viral decline in the first weeks of treatment. However, patients presenting a better HCV-specific CD8 cell proliferative potential at baseline are more likely to present a rapid and sustained viral response. Therefore, future treatment protocols should consider the development of strategies aimed at improving HCV-specific T-cell responses.

[Gene similarity between hepatitis C virus and human proteins--a blood transfusion problem].

INTRODUCTION: Hepatitis C is a post-transfusion hepatitis which causes serious problems in blood transfusion. Blood testing requires highly sensitive and specific assays with high predictive value. GENOMIC CHARACTERISTICS OF HEPATITIS C VIRUS: According to recommendations of International Association for the study of Liver Diseases etiological diagnosis of hepatitis is based on highly sensitive third generation assays: epitopes in the NS5 region comprising noncoding sequence UTR with 324-341 well conserved pair of homologous basis in 92% HCV genomes, therefore appropriate for virus RNA detection. DEVELOPMENT OF ASSAYS FOR HEPATITIS VIRUS: The first generation of immunoenzyme tests (IET) were based on detection of antibodies on antigen c 100-3, which is a part of the NS4 region of HCV genome. The second generation of tests with two recombinant proteins--c22-3 and c200, achieved higher sensitivity of assays. The third generation included epitopes from NS5 region, and removed the antigen c100-3. DEVELOPMENT OF AUTOIMMUNITY: Autoimmunity is a pathophysiological mechanism that's leads to chronic inflammatory diseases. Autoimunity is characterized by loss of tolerance towards self-antigens. Viral hepatitis C is associated with development of autoimmune phenomena. MOLECULAR MIMICRY: Molecular mimicry, as a mechanism of autoimmunity, was investigated to establish cross-Reactive immune reactions between HCV antigen and human nitrogen-oxide synthase, Tyrosine kinase Lck and hepatic growth factor activator. CROSS REACTIVITY BETWEEN HCV PROTEINS AND HUMAN PROTEINS: HCV capsid proteins initiate the autoimmune process in the liver because of cross reaction of antibodies with human Gor protein 19-27, which causes autoimmune chronic hepatitis. However, analysis of human protein from protein basis Swiss-prot shows homology between NS5 region and 3 human protein nitrogen oxide synthases, tyrosine kinase-Lck, proto-oncogene and hepatic growth factor activator. According to protein data analysis and competitive in vitro experiments, it was concluded that presence of auto-antibodies is probably the consequence of cross reactive immune response. CONCLUSION: Homology of amino acid sequences in the NS5 region of the HCV genome with nitrogen-oxide synthase, tyrosine kinase-Lck, and hepatic growth factor activator, causes auto-immune phenomena in HC, and can be a model for researching autoimmunity and human virus-induced autoimmune diseases.

Production in Pichia pastoris and characterization of genetic engineered chimeric HBV/HEV virus-like particles.

OBJECTIVE: To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). METHODS: The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization. RESULTS: The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg. CONCLUSION: The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

Identification of a glycosylation site in the woodchuck hepatitis virus preS2 protein and its role in protein trafficking.

The middle surface antigen (M-sAg) of hepadnaviruses is one of three envelope proteins that share a common C-terminal S domain. M-sAg contains the preS2 domain in addition to the S region. The preS2 region of woodchuck hepatitis virus (WHV) contains a potential glycosylation site Asn-Gln-Thr at amino acid (aa) positions 3-5. In this study, we mutated this site by site-directed mutagenesis and confirmed that glycosylation occurs here. In in vitro translation assays, the mutation Thr to Asn at aa 5 significantly impaired glycosylation of M-sAg. The mutated M-sAg formed abnormal clustered structures in transfected cells as determined by immunofluorescent staining. Confocal microscopic analysis showed that an enrichment of this glycosylation-deficient protein in the Golgi apparatus occurred, which is not typical for the wild-type protein. These results are consistent with earlier findings that incorrect glycosylation of viral proteins may interfere with virus assembly.

[Construction of a recombinant human adenovirus expressing the ORF2 antigen of HEV and immunization of mice by mucosal system].

OBJECTIVE: To construct a replication-defective recombinant adenovirus expressing the ORF2 (112-660aa) antigen of hepatitis E virus (HEV) and evaluate its immunization effect in BALB/c mice by mucosal inoculation. METHODS: The HEV ORF2 gene encoding for 112-660aa was amplified from plasmid pUC-HEV and inserted into the transfer vector pTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E. coli strain BJ5183. Taking the advantage of the high efficient homologous recombination machinery presented in bacteria, the recombinant adenovirus backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant Adenoviruses were propagated in 293 cells with high titers. 8-week-old BALB/c mice were inoculated intraperitoneally and intranasally with 10(7) pfu recombinant adenovirus each on weeks 0, 3, 5, 7, 10. RESULTS: Both groups of mice induced humoral IgG immune response with the highest titers 1:1,000 and 1:10,000 each. Only the group inoculated intranasally could induce mucosal IgA immune response. CONCLUSIONS: The adenoviral recombinant can stimulate specific humoral and mucosal immune response in mice and is potentially to be used as a candidate vaccine for the treatment of HEV infection.

Antigen-expressed recombinant Salmonella typhimurium driven by an in vivo-activated promoter is capable of inducing cellular immune response in transgenic mice.

To explore the approaches and mechanisms for reversing the immune tolerance in transgenic mouse, and the pathogenicity of hepatitis G virus (HGV), the promoter of phoP-activated gene (P(pagC)) of Salmonella typhimurium was used as a transcriptionally regulating element to construct an attenuated S. typhimurium expressing HGV NS3. The recombinant S. typhimurium was orally administered to HGV transgenic mice. As the results, HGV antigen in serum and liver as well as HGV mRNA in liver were decreased significantly, although the serum anti-HGV NS3 remained undetectable as the control transgenic mice. The spleen cell proliferation, in vitro HGV NS3 specific CTL, and IFN-gamma assays with the primed cultured splenocytes indicated the induction of Th1 immune responses in those administered transgenic mice. Adoptive transfer of fractionated primed spleen cells to the transgenic mice showed that T lymphocytes were responsible for, maybe through IFN-gamma, the down-regulation of HGV mRNA transcription. Histological examination found no significant inflammatory changes in liver of the transgenic mice. These findings suggested that the oral inoculation of the HGV NS3-expressed attenuated S. typhimurium driven by an in vivo-activated promoter should be a simple and effective approach for potential treatment of chronic viral infection.