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Currently a lot of effort is being invested in the development of a vaccine that could protect humans against a pandemic influenza A (H5N1) virus. In order to study the immunogenicity of novel influenza vaccine candidates in the mice, we have chosen the recombinant vaccine approach in which the HA and NA of A/Vietnam/1194/2004 virus are cloned and expressed under the control of a mammalian expression vector. The recombinant virus, designated VN1194, has been shown to be highly infectious in the mouse following intranasal inoculation. It is currently being tested for efficacy in a preliminary study using mice. To evaluate the immunogenicity of the recombinant vaccine in mice, we have produced a panel of monoclonal antibodies (MAbs) against VN1194 by using the recombinant HA expressed in a baculovirus expression system.
Methods
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The recombinant HA gene was amplified from the VN1194 virus and cloned into the eukaryotic expression plasmid pFastBac HT A. Recombinant baculovirus expressing the HA gene (Bac-rHA) was produced using the baculovirus system. A total of 100 *μ*l of the supernatant of the Bac-rHA virus was used for immunizing mice. The mice were injected with 50 *μ*l of a 1:50 diluted pool of antisera against the recombinant HA proteins. Following three immunizations, mice were boosted with 100 *μ*l of a 1:50 dilution of rabbit anti-sera and blood samples were collected to generate the monoclonal antibodies. The MAbs were first selected by ELISA using the purified recombinant HA proteins. Those that bound to HA were then titrated in a hemagglutination inhibition (HI) test. The antigenic specificity of the MAbs was identified using a panel of monoclonal antibodies that were described previously. HI test results showed that the MAbs were able to recognize various influenza strains including those of the H5 and H7 subtypes.
Results
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In this study, we have produced MAbs to the recomb
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