Design, Synthesis & Testing Of Recombinant Proteins For

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The causative agent for human influenza or respiratory flu disease is Orthomyxovirus. This enveloped RNA virus from the Orthomyxoviridae family has a single strand, negative sense, segmented genome. Influenza-associated complications cause the death of an estimated half a million people every year including 36,000 deaths in the United States, primarily in the very young and elderly populations. During the 1918-1920 ?Spanish flu? pandemic, influenza killed 20 to 40 million of the world population. Influenza virus imposes a serious burden of disease. A highly mobile world population coupled to the threat of a human influenza pandemic emerging from the highly pathogenic avian H5N1 strain of influenza A virus has world health officials rethinking strategies for the best preventive approach should a pandemic become reality. In modern medical history, we have no precedent that can guide the difficult scientific decisions necessary to protect the entire world populations against such a formidable pathogen. Current influenza vaccines are produced in embryonated chicken eggs ? a manufacturing protocol that was developed over a half-century ago. In a world emergency, industrial surge capacity for the production of vaccines made in this manor cannot meet vaccine demand in time to stop the spread of a highly pathogenic influenza virus. By focusing on the primary protective immunogen of influenza vaccines, we have developed a protocol to produce an effective vaccine against any new strain of influenza virus within three to four weeks of receiving the virus strain and the nucleotide sequence of the gene encoding the hemagglutinin protein. As a demonstration of these protocols, we have produced a modified recombinant hemagglutinin (rHA) protein for use as a candidate vaccine against the highly pathogenic H5N1 avian influenza A/Vietnam/1203/2004 virus. Using molecular biology techniques, we cloned the gene encoding the HA protein from native influenza virus isolated from an individual who died of the viral infection. The rHA protein was produced in E. coli. Purified rHA protein antigen was formulated into several vaccine candidates using various combinations of aluminum hydroxide (AlOH3) and formaldehyde. These vaccine candidates are being tested in mice for their ability to produce anti-rHA antibody and the ability of the immune sera to inhibit hemagglutinin (HAI) of red blood cells (RBC) by the H5N1 influenza virus. A mini-neutralization assay using MDCK cells culture will be used to determine if the mouse immune sera can prevent replication of H5N1 influenza virus in cell culture. Preliminary test results are extremely encouraging.