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Vaccine. 2009 Oct 19;27(44):6234-8. doi: 10.1016/j.vaccine.2009.07.107. Epub 2009 Aug 15.

Preparation, characterization, and immunogenicity in mice of a recombinant influenza H5 hemagglutinin vaccine against the avian H5N1 A/Vietnam/1203/2004 influenza virus.

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Program in Developmental and Molecular Immunity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2423, United States.


Production of influenza vaccines requires a minimum of 6 months after the circulating strain is isolated and the use of infectious viruses. The hemagglutinin (protective antigen) of circulating influenza viruses mutates rapidly requiring reformulation of the vaccines. Our goal is to eliminate the risk of working with infectious virus and reduce significantly the production time. A cDNA fragment encoding the influenza virus A/Vietnam/1203/2004 (H5N1) HA gene was prepared using RT-PCR with viral RNA as a template. Recombinant HA (rHA) protein was produced in Escherichia coli and purified from isolated inclusion bodies by urea solubilization and Ni(+)-ion column chromatography. Vaccine candidates were prepared by treating the rHA with formalin, adsorption onto alum or with both. Mice were injected subcutaneously with candidate vaccines two or three times 2 weeks apart. Sera were collected 1 week after the last injection and antibody measured by ELISA and hemagglutination inhibition (HI). The highest antibody response (GM 449EU) was elicited by three injections of 15microg alum-adsorbed rHA. Dosages of 5microg of rHA formulated with formalin and alum, and 5microg alum-adsorbed rHA elicited IgG anti-HA of GM 212 and 177EU, respectively. HI titers, >or=40 were obtained in >or=80% of mice with three doses of all formulations. We developed a method to produce rHA in a time-frame suitable for annual and pandemic influenza vaccination. Using this method, rHA vaccine can be produced in 3-4 weeks and when formulated with alum, induces HA antibody levels in young outbred mice consistent with the FDA guidelines for vaccines against epidemic and pandemic influenza.

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