Hai Yu

Xiamen University, Amoy, Fujian, China

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Publications (10)46.67 Total impact

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    ABSTRACT: The protein encoded by ORF2 in hepatitis E virus (HEV) is the only capsid protein for this single-stranded RNA virus. It was previously shown that 148 aa (aa 459-606) was needed for dimer formation, whereas 239 aa (aa 368-606) was necessary to form virus-like particles (VLPs). The self-assembled VLPs of p239 were characterized with a series of methods including high performance size-exclusion chromatography to demonstrate the particulate nature of purified and properly refolded p239. A neutralizing and protective mouse monoclonal antibody (mAb) 8C11 was previously shown to bind three discontinuous peptide segments in the dimer. In addition to the good binding activity to recombinant dimeric form, E2s or E2, and VLP form p239, we demonstrated that 8C11 was able to capture the authentic HEV virions. The capability of virus capturing was demonstrated with a titration curve from 10(5) to 10(7) HEV genome copies, making binding activity to 8C11 a surrogate marker of virion-like epitopes on recombinant VLPs as well as vaccine efficacy in eliciting protective and neutralizing antibodies. Taken together, it was demonstrated that Escherichia coli expressed pORF2 proteins, p239 in particular, maintain the virion-like epitopes on VLP surface. This is consistent with the fact that p239 was demonstrated to be an effective prophylactic vaccine (recently licensed as Hecolin(®) in China) against HEV-induced hepatitis in a large scale clinical trial.
    Vaccine 03/2014; · 3.77 Impact Factor
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    ABSTRACT: The nucleoprotein (NP) of influenza A virus plays a critical role in the formation of viral ribonucleoprotein (vRNP) complex. However, it remains unclear which key residues in NP are associated with the assembly of vRNP and contribute to virus replication. Here, a highly conserved aspartic acid at residue 88 (D88) of NP was identified by molecular docking of NP with the Fv region of a broad-spectrum anti-NP mAb 19C10 and further demonstrated to be an important residue contributes to the RNP activity, virus growth in MDCK cells and replication in lungs of infected mice by comparing recombinant wild-type A/WSN/1933 virus to the mutant virus that contains an alanine instead of aspartic acid at NP residue 88. D88 was also predicted to interact with PB2 by molecular docking and further verified by immunoprecipitation. These findings provide new information for understanding the interaction between NP and other polymerase subunits in virus replication.
    Virology. 01/2014; s 464–465:11–20.
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    ABSTRACT: Human enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) and is associated with several severe neurological complications in the Asia-Pacific region. Here, we evaluated that while passive transfer of neutralizing monoclonal antibody (nMAb) against the VP2 protein protect against lethal EV71 infection in BALB/c mice. Protective nMAb were mapped to residues 141-155 of VP2 by peptide ELISA. High-resolution structural analysis showed that the epitope is part of the VP2 EF loop, which is the "puff" region that forms the "southern rim" of the canyon. Moreover, a three-dimensional structural characterization for the puff region with prior neutralizing epitopes and receptor-binding sites that can serve to inform vaccine strategies. Interestingly, using hepatitis B virus core protein (HBc) as a carrier, we demonstrated that the cross-neutralizing EV71 antibodies were induced, and the VP2 epitope immunized mice serum also conferred 100% in vivo passive protection. The mechanism of in vivo protection conferred by VP2 nMAb is in part attributed to the in vitro neutralizing titer and ability to bind authentic viral particles. Importantly, the anti-VP2(aa141-155) antibodies could inhibit the binding of human serum to EV71 virions showed that the VP2 epitope is immunodominant. Collectively, our results suggest that a broad-spectrum vaccine strategy targeting the high-affinity epitope of VP2 EF loop may elicits effective immune responses against EV71 infection.
    Theranostics 01/2014; 4(5):498-513. · 7.81 Impact Factor
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    ABSTRACT: Influenza virus still poses a major threat to human health worldwide. The nucleoprotein (NP) of influenza A virus plays an essential role in the viral replication and transcription and hence becomes a promising therapeutic target. NP forms a complicated conformation under native conditions and might denature when performing immunoassays such as western blot in the study of NP function. Therefore, it is useful to make an NP specific monoclonal antibody (mAb) that recognizes linear epitope instead of conformational epitope. In this study, a recombinant NP (rNP) of influenza A virus was over-expressed and used to generate a panel of anti-NP mAbs. These anti-NP mAbs were grouped into three classes based on their reactivity in Western blots. Only Class I mAb can react with linear rNP fragments. One of Class I mAb, 4D2, was characterized further by epitope mapping with a series of overlapping synthetic peptides, indicating that the 4D2 epitope is a surface exposed, linear epitope between amino acid residues 243 and 251. This epitope is highly conserved among different influenza A viruses with an identity of 98.4% (17,922/18,210). Western blot, co-immunoprecipitation, immunofluorescence, and immunohistochemistry experiments all indicated 4D2 is highly specific to NP of influenza A virus. The results demonstrated that 4D2 can be used as a research tool for functional study of NP in the replication cycle of influenza A virus. Further work is needed to understand the function and importance of this epitope. J. Med. Virol. © 2013 Wiley Periodicals, Inc.
    Journal of Medical Virology 10/2013; · 2.37 Impact Factor
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    ABSTRACT: 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 60nm 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.
    Trends in Biotechnology 10/2013; · 9.66 Impact Factor
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    ABSTRACT: This study aimed at investigating mutations in the hepatitis B surface protein (HBsAg) in occult hepatitis B virus (HBV) infection (OBI) and their influence on viral antigenicity and phenotype. The characteristics of 61 carriers with OBI (OBI group), 153 HBsAg(+) carriers with serum HBsAg ⩽100IU/ml (HBsAg-L group) and 54 carriers with serum HBsAg >100IU/ml (HBsAg-H group) from 38,499 blood donors were investigated. Mutations in the major hydrophilic region (MHR) of the viral sequences were determined. Thirteen representative MHR mutations observed in OBI sequences were antigenically characterized with a panel of monoclonal antibodies (MAbs) and commercial HBsAg immunoassays and functionally characterized in HuH7 cells and hydrodynamically injected mice. Of 61 OBI sequences, 34 (55.7%) harbored MHR mutations, which was significantly higher than the frequency in either the HBsAg-L (34.0%, p=0.003) or the HBsAg-H group (17.1%, p<0.001). Alterations in antigenicity induced by the 13 representative MHR mutations identified in the OBI group were assessed by reacting recombinant HBV mutants with 30 different MAbs targeting various epitopes. Four out of the 13 mutations (C124R, C124Y, K141E, and D144A) strongly decreased the analytical sensitivity of seven commercial HBsAg immunoassays, and 10 (G119R, C124Y, I126S, Q129R, S136P, C139R, T140I, K141E, D144A, and G145R) significantly impaired virion and/or S protein secretion in both HuH7 cells and mice. MHR mutations alter antigenicity and impair virion secretion, both of which may contribute to HBsAg detection failure in individuals with OBI.
    Journal of Hepatology 05/2012; 57(4):720-9. · 9.86 Impact Factor
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    ABSTRACT: P239, a truncated construct of the hepatitis E virus (HEV) ORF2 protein, has been proven able to bind with a chaperone, Grp78, in both an in vitro co-immune precipitation test and an in vivo cell model. We previously solved the crystal structure of E2s--the C-terminal domain of p239 involved in host interactions. In the present study, we built a 3D structure of Grp78 using homology modeling methods, and docked this molecule with E2s using the Zdockpro module of the InsightII software package. The modeled Grp78 structure was deemed feasible by profile 3D evaluation and molecular dynamic simulations. The docking result consists of six clusters of distinct complexes and C035 was selected as the most reasonable. The interacting interface of the predicted complex is comprised of the Grp78 linker region and nucleotide binding domain along with the E2s groove region and surrounding loops. Using energy, hydrogen bond and solvent accessible surface analyses, we identified a series of key residues that may be involved in the Grp78:E2s interaction. By comparing with the known structure of the Hsp70:J complex, we further concluded that the interaction of Grp78 and E2s could interrupt binding of Grp78 with the J domain, and in turn diminish or even eliminate the binding ability of the Grp78 substrate binding domain. The predicted series of key residues also provides clues for further research that should improve our understanding of the fundamental molecular mechanisms of HEV infection.
    Journal of Molecular Modeling 05/2011; 17(5):987-95. · 1.98 Impact Factor
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    ABSTRACT: Highly pathogenic H5N1 virus infection causes severe disease and a high rate of fatality in humans. Development of humanized monoclonal antibodies may provide an efficient therapeutic regime for H5N1 virus infection. In the present study, broadly cross-reactive monoclonal antibodies (MAbs) derived from mice were humanized to minimize immunogenicity. One chimeric antibody (cAb) and seven humanized antibodies (hAbs) were constructed. These antibodies retained broad-spectrum reactivity to H5N1 viruses, binding to recombinant H5-subtype HA1 molecules expressed in CHO cells in a dose-dependent manner and exhibiting similar reactivities against antigenically distinct H5N1 viruses in hemagglutination inhibition (HI) assays. One humanized antibody, 37 hAb, showed HI and neutralization activities comparable to that of the parental murine antibody, 13D4 MAb, while the other six antibodies were less reactive to H5N1 viruses. Analysis of amino acid sequences in the variable region frameworks of the seven humanized antibodies found that Q5 and Y27 in the VH region are highly conserved murine residues. Comparison of the three-dimensional structures derived from the variable regions of MAbs 37 hAb, H1202-34, and 13D4 revealed that residue substitutions at sites 70 and 46 may be the major cause for the observed differences in binding affinity. Examination of the chimeric antibody and one of the humanized antibodies, 37 hAb, showed that both antibodies offered postinfection protection against lethal challenge with antigenically diverse H5N1 viruses in the mouse model. Chimeric and humanized antibodies which retain the broadly reactive and protective properties of murine H5-specific monoclonal antibodies have great potential for use in the treatment of human H5N1 infection.
    Antimicrobial Agents and Chemotherapy 01/2011; 55(4):1349-57. · 4.57 Impact Factor
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    ABSTRACT: p239 is a virus-like particle constituted from hepatitis E virus (HEV) recombinant proteins. It can be used as a surrogate for HEV and as an investigative tool to study cellular interactions because of its ability to adsorb to and penetrate HepG2 cellular membranes. Our objective was to use p239 to define the role of HEV capsid proteins during the early stages of infection. Pull-down and MALDI-TOF MS experiments identified three host-cell proteins, Grp 78/Bip, alpha-tubulin and heat-shock protein 90 (HSP90), and the latter was investigated further. Antibodies to p239 alone or HSP90 alone could detect p239 or HSP90, suggesting the formation of a complex between p239 and HSP90. In the HepG2 cell, geldanamycin (GA), an HSP90-specific inhibitor, blocked intracellular transportation of p239, but had no effect on the binding and cellular entry of p239, suggesting that HSP90 was important for HEV capsid intracellular transportation. RT-PCR results showed that the efficiency of wild-type HEV infection was inhibited significantly by GA treatment, suggesting the importance of HSP90 in virus infectivity. It was concluded that HSP90 plays a crucial role in the intracellular transportation of viral capsids in the early stage of HEV infection.
    Journal of General Virology 03/2010; 91(Pt 7):1728-36. · 3.13 Impact Factor
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    ABSTRACT: A novel hepatitis B virus (HBV) strain (W29) was isolated from serum samples in the northwest of China. Phylogenetic and distance analyses indicate that this strain is grouped with a series of distinct strains discovered in Vietnam and Laos that have been proposed to be a new genotype I. TreeOrderScan and GroupScan methods were used to study the intergenotype recombination of this special group. Recombination plots and tree maps of W29 and these putative genotype I strains exhibit distinct characteristics that are unexpected in typical genotype C strains of HBV. The amino acids of P gene, S gene, X gene, and C gene of all genotypes (including subtypes) were compared, and eight unique sites were found in genotype I. In vitro and in vivo experiments were also conducted to determine phenotypic characteristics between W29 and other representative strains of different genotypes obtained from China. Secretion of HBsAg in Huh7 cells is uniformly abundant among genotypes A, B, C, and I (W29), but not genotype D. HBeAg secretion is low in genotype I (W29), whose level is close to genotype A and much lower than genotypes B, C, and D. Results from the acute hydrodynamic injection mouse model also exhibit a similar pattern. From an overview of the results, the viral markers of W29 (I1) in Huh7 cells and mice had a more similar level to genotype A than genotype C, although the latter was closer to W29 in distance analysis. All evidence suggests that W29, together with other related strains found in Vietnam and Laos, should be classified into a new genotype.
    PLoS ONE 01/2010; 5(2):e9297. · 3.53 Impact Factor

Publication Stats

87 Citations
46.67 Total Impact Points


  • 2010–2014
    • Xiamen University
      • National Institute of Diagnostics and Vaccine Development in Infectious Diseases
      Amoy, Fujian, China
  • 2011
    • The University of Hong Kong
      Hong Kong, Hong Kong