A Conserved Domain of HCV E2 Glycoprotein Interacts with Human CD81 and Induces Interferon-Gamma Secretion from Peripheral Blood Mononuclear Cells.

BACKGROUND: Hepatitis C virus (HCV) infection is a global health threat to the public, and vaccines against it are not yet available. The HCV envelope glycoprotein E2 is a key target for anti-HCV vaccines. The majority of previous studies have focused on the hypervariable region and the glycosylation sites of the_HCV structural protein. This study aims to investigate a conserved domain of HCV E2 glycoprotein and explore its potential to induce an immune response against HCV. METHODS: HCV E2 conserved domain (encompassing amino acids 505-702) was prepared in Escherichia coli (E. coli). Peripheral blood mononuclear cells (PBMCs) were isolated from patients with HCV or healthy controls. Interferon-gamma (IFN-γ) enzyme-linked immunosorbent spot assay was conducted to examine the HCV E2-specific immune response as reflected by IFN-γ-secreting cells/106 PBMCs. RESULTS: HCV E2 conserved domain was highly conserved among 25 HCV subtypes, and its recombinant soluble production in E. coli was recognized by anti-HCV E2 monoclonal antibodies. This study characterized in vitro direct interaction between bacterially expressed HCV E2 conserved domain and human CD81 (hCD81). Furthermore, the recombinant HCV E2_conserved domain markedly induced the production of IFN-γ by PBMCs from patients with HCV. Its stimulated specific immune response was significantly different from non-specific peptide controls or PBMCs isolated from healthy controls. CONCLUSIONS: HCV E2 conserved domain directly binds hCD81 and activates the production of IFN-γ in the PBMCs of patients with HCV. Therefore, the conserved domain of HCV E2 glycoprotein may be a new candidate for developing an HCV vaccine.

Performance Evaluation of Protein Chip Assay for Rapid Diagnosis of Hepatitis C Virus Infection in Injection Drug Abusers.

BACKGROUND: We evaluated the performance of a protein chip assay for the detection of antibodies to hepatitis C virus (HCV) peptides among injection drug abusers (IDAs) by comparing the assay with existing methods, including enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and recombinant immunoblot assay (RIBA). METHODS: Seventy serum samples collected from IDAs were analyzed by protein chip assay. ELISA, RT-PCR, and RIBA assay were used to validate the results. RESULTS: The protein chips could detect different peptides' antibodies against HCV-C, HCV-NS3, HCV-NS4, HCV-NS5, and HCV-mix antigen; no cross reactivity between antigens was observed. Results of the protein-chip assay were compared with those of ELISA. Any inconsistency in results was validated by both RT-PCR and RIBA. Concordance between the results of the protein-chip assay and ELISA was 96.3% for positive samples and 100% for negative samples. The protein chip had a higher specificity than ELISA, a higher sensitivity than RTPCR, and similar specificity and sensitivity as compared to RIBA. The limit of detection of HCV antibodies in the protein chip assay was examined and calculated by incubation of model array with different dilutions. The protein chip assay required smaller amounts of both samples and reagents; it detected serum antibody in sample quantity as low as 3 ng/mL and at antibody dilution as low as 1:1000; its cost was low as well. The positive rates in the antiC, anti-NS3, anti-NS4, and anti-NS5 groups were significantly associated with levels of HCV RNA and the viral load. The HCV RNA and protein chip positive rate in the injection equipment-sharing group was higher than that in the non-injection equipment-sharing group. CONCLUSIONS: The protein chip assay is a faster and simpler approach to simultaneously screen for all HCV peptide antibodies accurately and to provide a rapid diagnosis of HCV infection in IDAs. The dominant positive HCV peptide antibodies were significantly associated with HCV RNA load, especially in the injection equipment-sharing group.

Adenovirus-based vaccines generate cytotoxic T lymphocytes to epitopes of NS1 from dengue virus that are present in all major serotypes.

Dengue virus (DENV) infection is an expanding global threat to public health. Effective vaccine and treatment approaches remain elusive. B cell-directed vaccines may be complicated by an antibody-dependent enhancement (ADE) phenomenon based on cross-serotype, nonneutralizing antibodies. We hypothesized that a CD8(+) T cell-directed genetic vaccine that targets a DENV nonstructural protein, NS1, could be a potential strategy to overcome the ADE barrier and accomplish cross-serotype protection. We selected an adenovirus-based vector as the dengue vaccine carrier. To bypass preexisting immunity to human adenoviruses and to improve vaccine efficacy, we created vaccine vectors, based on simian adenoviruses SAdV22 and SAdV25 as well as human adenovirus serotype 5, expressing the NS1 antigen of the Hainan strain, DENV serotype 2. An NS1 peptide library was screened to identify the immunodominant and functional epitope within the NS1 protein for H-2(d)-restricted CD8(+) T cells in BALB/c mice, using interferon-gamma enzyme-linked immunospot and intracellular cytokine-staining assays. Our study identified the 9-mer AGPWHLGKL (NS1(265273)) as the H-2(d)-restricted T cell epitope whose sequence is highly conserved among 26 strains of DENV serotypes 1, 2, 3, and 4, suggesting potential cross-serotype protection of NS1-directed genetic vaccines in the BALB/c model of DENV infection. Importantly, we characterized the cytokine profile of CD8(+) NS1-specific T cells in BALB/c mice vaccinated with HAdV-5-NS1, SAdV-22-NS1, and SAdV-25 NS1 and demonstrated the effective in vivo cytolytic killing capacity of CD8(+) T cells from SAdV-25-NS1-vaccinated mice.

Bxz1, a new generalized transducing phage for mycobacteria.

We have isolated and characterized a new generalized transducing phage, Bxz1, from soil sampling at a neighboring Wildlife Preservation Park. The hosts of the phage, measured by the formation of plaques, include fast growing Mycobacterium smegmatis and Mycobacterium vaccae. Bxz1 is capable of transducing chromosomal markers, point mutations, and plasmids at frequencies ranging from 10(-8) to 10(-6) per plaque forming unit between strains of M. smegmatis. We also demonstrated cotransduction of a transposon insertion linked to a point mutation of the ndh gene.