Broiler type chickens were immunized intramuscularly with a DNA vaccine encoding hemagglutinin (HA) from H5N1 avian influenza virus. The chickens were divided into four groups: control group which was not immunized, a group which obtained only one dose, and two groups which were immunized twice, one group with a boost two weeks after the priming and the other four weeks. Blood samples were collected at several time points and the dynamics of the humoral response to the vaccine was studied. High level of anti-HA antibodies was detected only in the last two groups, that is in chickens immunized according to the prime-boost strategy, regardless of the schedule. An additional interesting observation of this study was detection of the cross-reactivity of an anti-H5 HA positive serum with H5N2 and H1N1 viruses, suggesting that the DNA vaccine tested can induce antibodies of a broad specificity.
The A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. Recombinant HA protein was secreted into the culture medium reaching an approximately 15 mg/L (KM 71 strain). Fusion protein with a His6 tag was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated that the protein is cleaved into HA1 and HA2 domains linked by a disulfide bond. Analysis of the N-linked glycans revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. Immunological activity of the hemagglutinin protein was tested in mice, where rHA elicited a high immune response.
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