title: Biological properties of H5 hemagglutinin expressed by vaccinia virus vector and its immunological reactivity with human sera.
abstract: A recombinant vaccinia virus harboring the full length hemagglutinin (HA) gene derived from a highly pathogenic avian influenza A/Thailand/1(KAN-1)/2004 (H5N1) virus (rVac-H5 HA virus) was constructed. The immunogenicity of the expressed HA protein was characterized using goat antiserum, mouse monoclonal antibody, and human sera. The expressed HA protein localized both in the cytoplasm and on the cytoplasmic membrane of the thymidine kinase negative cells infected with the rVac-H5 HA virus, as determined by immunofluorescence assay. Western blot analysis demonstrated that the rVac-H5 HA protein was post-translationally processed by proteolytic cleavage of the HA0 precursor into HA1 and HA2 domains; and all of these HA forms were immunogenic in BALB/c mice. The molecular weight (MW) of each HA domain was the same as the wild-type H5 HA produced in Madin-Darby canine kidney cells infected with the H5N1 virus, but was higher than that expressed by a baculovirus-insect cell system. Sera from all H5N1 survivors reacted to HA0, HA1, and HA2 domains; whereas sera from H5N1-uninfected subjects reacted to the HA2 domain only, but not to HA0 or HA1, indicating that some cross-subtypic immunity exists in the general population. There was a lot-to-lot variation of the recombinant HA produced in the baculovirus-insect cell system that might affect the detection rate of antibody directed against certain HA domains.
authors: Department of Microbiology, Mahidol University, Bangkok-noi, Bangkok, Thailand.
authors: Pirom
authors: P
authors: Noisumdaeng
authors: Phisanu
authors: P
authors: Pooruk
authors: Alita
authors: A
authors: Kongchanagul
authors: Susan
authors: S
authors: Assanasen
authors: Rungrueng
authors: R
authors: Kitphati
authors: Prasert
authors: P
authors: Auewarakul
authors: Pilaipan
authors: P
authors: Puthavathana
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000097:blood* / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D004337:Drug Carriers; D005822:Genetic Vectors; D006041:Goats; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D014616:Vaccinia virus / Q000235:genetics*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D004337:Drug Carriers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; C109963:hemagglutinin, avian influenza A virus