An alternative neutralization assay for H5N1 influenza A virus using retroviral pseudotypes

The main protection against infection by influenza viruses is the presence of virus neutralizing antibodies in the blood. People who have recovered from flu have neutralizing antibodies to that strain of influenza virus, and effective flu jabs elicit similar protective antibodies. To aid the development and testing of vaccines against the new, deadly H5N1 influenza virus, we shall refine a novel neutralization test that uses an artificially constructed, harmless virus-like particle in place of the harmful influenza virus itself. Our test should be simple to use and will not require a high containment laboratory. It should offer a means to compare variation between sub-strains of H5N1 influenza viruses from different locations, and to evaluate whether candidate vaccines protect against all sub-strains. It may also offer a method to test candidate drugs that block influenza virus getting into cells.

The MRC/UCL Centre for Medical Molecular Virology includes a strong research platform on retrovirus neutralization and on retroviral vectors. We propose to apply this expertise to address H5N1 influenza neutralization. Immunity to influenza A virus infection is closely correlated to titres of neutralizing antibodies (NAb) that recognise haemagglutinin (HA). Conventional neutralization assays require titration of live influenza virus, and haemagglutination inhibition (HI) represents a less sensitive surrogate marker for neutralization. This project will develop and improve a neutralization test based on retroviral vectors that bear HA envelope antigens and carry an easily quantifiable reporter gene. The objective is to construct retroviral pseudotypes carrying a variety of H5N1 influenza HA antigens and to test their sensitivity to neutralization by natural human and avian sera, and by antisera raised against candidate H5N1 vaccines. The WHO has recommended the development of more sensitive and specific neutralization assays, and our approach could accelerate the testing of vaccines. Pseudotypes could also be applied to functional testing of the switch from avian to human sialic acid receptors, and to screening candidate drugs that inhibit HA-receptor interactions.