Prokaryotic Expression and Immunogenicity of Dominant Epitope Region of Goose Parvovirus Structural Protein VP3

[Objective] The paper was to develop a subunit vaccine candidate for prevention and control of goose parvovirus infection. [Method]Based on the prokaryotic expression system, the antigenic epitopes and locations of the structural protein VP3 were predicted by software analysis,and the region displaying a large portion of antigenic epitopes was amplified by PCR. The target VP3 DNA fragment was inserted into pET-30 a-VP3 vector, was transformed into Escherichia coli BL21 competent cells for protein expression and animal test. The SPF chickens were immunized with the recombinant protein and the antisera were collected for neutralization test by using a goose embryo fibroblast. [Result] The recombinant plasmid was constructed, and the target region of VP3 protein was expressed efficiently in a soluble form. The neutralizing titers of antisera could reach up to-2.608. [Conclusion] The target region displaying a large portion of antigenic epitopes of the structural protein VP3 could be expressed efficiently in soluble form, and the expressed protein could induce neutralizing antibodies in SFP chicken.

PRODUCTION IN PICHIAPASTORISAND 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). MethodsThe 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 Pichiapastorisunder 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 visualiza-tion. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoriswith 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 immuno-genicity 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.

The Leu477 and Leu613 of ORF2-Encoded Protein Are Critical in Forming Neutralization Antigenic Epitope of Hepatitis E Virus Genotype 4

Hepatitis E virus （HEV） genotype 4 was originally identified in China. Its neutralization antigenic epitopes have not been characterized. Recently, we identified a neutralizing monoclonal antibody （mAb） IG10, which was generated following immunization of mice with p166Chn, a recombinant protein comprising 464-629 amino acids （aa） of the HEV genotype 4 capsid protein. In this study, a panel of 22 N- and/or C-terminal truncated and 6 site-directed mutated p166Chn proteins were prepared. Only those N- or C-terminal truncated proteins containing the region 477-613 aa could react with the mAb 1G10, suggesting the neutralization epitope of HEV genotype 4 is located between aa477 and aa613. However, a both N- and C-terminal truncated protein, pN477-C613, neither reacted to 1G10 nor elicited neutralizing antibodies in mice, while another both terminal truncated protein, pN472-C617, did, suggesting the flanking regions of the pN477-C613 could help to stabilize and allow presentation of the neutralization epitope to the immune system. Substituting Leu477 and/or Leu613 with the polar, uncharged threonine （Thr） caused 〉 50% reduction of the mutants＇ immunoreactivity to IG10, whereas replacement by hydrophobic phenylalanine （Phe） made little impact on the immunoreactivity, revealing functional associations between hydrophobicity of aa at positions 477 and 613 and the antigenicity of p166Chn. These data suggested Leu477 and Leu613 are critical in forming the neutralization epitope of HEV genotype 4. Cellular ＆ Molecular Immunology. 2008;5（6）：447-456.

Association of variations of NAb 2F5 and 4E10 epitopes and disease progression in Chinese antiretroviral treatment-na（i）ve patients infected with HIV-1 clade B＇

Background Studies on human immunodeficiency virus type 1 （HIV-1） vaccines have recently focused on targeting the conserved neutralizing epitopes 2F5 and 4E10, and hence it is important to understand the extent of mutations in these two viral epitopes. Here, we investigated the amino acid mutations in epitopes of 2F5 （ELDKWA, HIV-1 HXB2 env 662-667 aa） and 4E10 （NWFDIT, HIV-1 HXB2 env 671-676 aa） in the membrane proximal-external region of gp41 from clade B＇ HIV-1-infected individuals living in Henan province, China. We also examined the frequency of a mutation and its relation to disease progression.Methods A cohort of 54 treatment-na（i）ve HIV-1-infected individuals was recruited in this study, and 16 individuals were selected for a short-term longitudinal study on sequence evolution. The HIV-1 env gp41 gene was amplified, cloned, and sequenced, and predicted amino acid sequences were aligned for analysis.Results The mutations E662A and K665E on the 2F5 epitope and N671S and T676S on the 4E10 epitope were seen.Simultaneous RNA sequencing showed some discrepancies with proviral DNA sequences. In our longitudinal study,mutation levels of these two neutralizing epitopes were low but diverse and persistent. The frequencies of mutations within the 4E10 peptide NWFDIT in slow progressors were noticeably lower than those in AIDS patients （P ＜0.05）.Conclusions Antigenic variation of the neutralizing epitopes 2F5 and 4E10 is limited in subtype B＇ infection, and that 4E10 peptide mutation is correlated with disease progression. Monitoring epitope mutations will offer useful data for development of the candidate 2F5-like and 4E10-like antibodies to prevent and treat AIDS.