| Avian influenza virus (AIV) is type A influenza virus of the family Orthomyxoviridae, and in particular the highly pathogenic H5N1 AIV has crossed the species barrier and caused a direct result of human infection and death of the infected persons. It was first found in 1997 that H5N1 AIV could infect humans and cause deaths and disseminated infections continued to occur. Since the end of 2003, highly pathogenic H5N1 AIV has spreaded rapidly from Southeast Asia to more than 60 countries and regions including China, Africa, Turkey and Siberia, et al. 15 countries and regions have reported human cases infected by the virus. Totally 423 persons were infected and 258 of them died,with the mortality rate as high as 61%. Although the human cases were mainly distributed in Asia, but Europe and Africa also had accasional cases. The epidemic caused a large scale poultry deaths and particularly, caused human infection and death,resulting in huge economic losses and great threatening to public health and social security.Two glycoprotein spikes, hemagglutinin (HA) and neuraminidase (NA), located on surface of influenza A virus. Based on the differences of HA and NA, influenza virus was further divided into many subtypes. HA and NA are not only the major surface antigens of AIV and can induce protective immunity, but also the main reason for antigenic drift and antigenic shift to cause new subtypes and variants by their mutations. The research of their antigenicity, especially HA, would not only disclose the regulations of antigenic variation, but also provide theoretical guidance for the vaccine development. Research has proven that there are at least five neutralizing epitopes (A, B, C, D and E) distributed on HA protein of H5N1, all located in the heavy chain HA1. The eptitopes are overlapped in some extent. However, the size and detailed structure of the epitopes has not yet been determined. It will be helpful for understand the pathogenesis of the virus by exploring the detaied structure of these epitopes.Based on a human strain of AIV A/Beijing/01/03(H5N1) isolated in China, mutiple specific monoclonal antibodies(McAb) and polyclonal serums were utilized to determine the antigenic epitopes (in particular, the neutralizing epitopes) on hemagglutinin of human H5N1 AIV, and to ascertain the amino acid sequences recognized by those McAbs. The detailed analyze of the epitope structures by specialized McAbs could be applied as the first step for further developing of novel influenza epitope vaccines. At the same time, in order to explore the molecular basis of H5N1 virus crossing the species barrier to infect human directly from birds, another 10 strains of H5N1 isolates from human and wild birds in Qinghai, China were employed to explore the differences of the replication capacity in human target cells and the antagonism against human cytokines.Firstly, 17 primers (a total of 16 pairs) were designed by Oligo6.0 software according to 984bp sequences encoding HA1 of H5N1 strain A/Beijing/01/03. 16 overlapped HA1 gene fragments in different length were amplified by RT-PCR and cloned into the mammalian expression vector pDisplay. Then the positive clones were screened and sequenced for subsequent construction of 16 recombinant plasmids pDisplay-HA1-1~10 and pDisplay-HA1-1'~6'. The concentration and purity of the 16 recombinant plasmids were determined and then recombinant plasmids were prepared and transfected into HeLa cells by lipidosome lipofetaineTM2000. The HA1 overlapped peptides were expreed successfully in vitro. Indirect immunofluorescence was established for epitope analysis.Secondly, anti-H5N1 (A/Beijing/01/03 strain) McAbs and polyclonal antibodies were prepared and their properties were analyzed. The BALB/c mice were immunized three times with chick embryo allantoic fluid inactivated by formaldehyde, then the mouse with positive serum antibodies was selected and their spleens were taken for cell fusion experiments. McAbs were screened by limiting dilution cloning assay and 12 hybridoma cell lines which secret anti-H5N1 McAbs were obtained. Totally, 30 McAbs were included in this study. Among them, 18 were kindly supplied by professor Che Xiaoyan. The immunological properties, such as antibody titers, hemagglutination inhibition activity, neutralizing activity, specific recognization of these McAbs to HA protein were investigated and identified. The results showed that the titers of all 30 McAbs were between 400-20480; 13 McAbs exhibited higher hemagglutination inhibition activities; 18 McAbs were specific against HA protein; 6 fo 18 anti-HA McAbs showed neutralizing activities, with titers between 1:52-1:2032. The anti-H5N1 polyclonal serum antibodies were collected from the peripheral blood of those mice after three immunizations.Thirdly, overlapped HA1 peptides expressed on the transfected cell surface and the prepared McAbs and polyclonal antibodies were utilized to analyze the neutralizing epitopes and dominating epitopes on HA by indirect immunological assay (IFA). Each McAb reacted with every peptide expressed on HeLa cells transfected with 16 recombinant plasmids respectively. Similarly, the major immunodominant regions on HA protein were determined according to the reactions of polyclonal antibodies with different HA1 protein fragments. The antigenic epitopes recognized by 3B5,3D1,3B2 and M6 McAbs were located in the region of amino acid residues 133-143,155-165,166-196 and 166-176 of HA1 protein, respectively, and all of them seemed to be linear epitopes. The antigenic epitopes recognized by M3 and M7 were mapped to the regions of amino acid residues 34-66 and 296-328 of HA1 protein, which presumed to be conformational epitopes. The major immunodominant epitope of HA1 protein was located in the region of amino acid residues 133-165 with the sequence of IPKSSWSNHEASSGVSSACPYLGRPSFFRNVVW. These results disclosed the major immunodominant region and epitopes on HA protein recognized by antibodies, and the amino acid sequence recognized by McAb. Through the analysis of the relationship between the key amino acid sites and the epitopes, the detailed structure of epitopes were demonstrated and could be utilized for further study of the function of HA protein. These results provided important data for further understanding of the relationship between the structure and function of HA epitopes, and would be helpful for exploring the regulation of antigenic variations and for development of new vaccines.Fourthly, the molecular basis of the different replicating abilities of A/Beijing/01/03 H5N1 virus and other 9 human and wild birds strains of H5N1 isolates on human target cells were investigated. Plaque titers of all viruses were determined. Then the replication capacities of these isolates in human A549 cells and their sensitivity against human IFN and TNF cytokines were evaluated. The genome sequences of these isolates were analyzed and aligned. The results indicated that the ability of H5N1 virus crossing the species barrier from birds to humans might be closely related to their strong replication capacities in human target cells. Three amino acid mutations on NS1 protein may be associated with the ability of these strains to adapt to and reproduce in human target cells. A/Vietnam/1194/2004 (H5N1) NS1 gene mutant strain was constructed for further molecular basis analysis of H5N1 virus to adapt to and cause serious infections in humans.In summary, by using the prepared McAbs and expressed overlapped HA1 peptides, several linear and conformational epitopes and major immunodominant regions on hemagglutinin protein of H5N1 virus were primarily determined in this study. The epitopes can be divided into hemagglutination, non-hemagglutination-related and neutralizing epitopes, and hemagglutination-related epitopes were mainly linear epitopes, and the neutralizing-related epitopes were mainly conformational epitopes. The McAbs and pepetides prepared were important for further developing of influenza A virus-specific diagnostic reagents. These results would provide theoretical evidence for further developing of novel vaccines and exploring the mechanism of viral antigenic variation. At the same time, it was presumed that the strong replication capacity on human target cells of human-derived H5N1 virus may be a prerequisite for its ability to adapt to and infect humans. It was also presumed that the three amino acid mutations on NS1 protein may be associated with its adaptation to humans and it had great significance for further exploring the pathopoiesis of H5N1 virus.
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