H5 pseudotyped lentiviral particles: a new tool for H5N1 infection sero-diagnosis

Abstract

Highly pathogenic avian influenza (HPAI) H5N1 has spread globally in birds and infected over 393 humans with an apparently high mortality rate (248 deaths). To date, there has been an 88% mortality rate for the 8 patients in Cambodia known to have been infected by H5N1. Serologic studies to determine the extent of asymptomatic or pauci-symptomatic H5N1 infection in humans and other mammals and to investigate the immunogenicity of current H5N1 vaccine candidates have been hampered by the biosafety requirements needed for the ‘gold standard’ H5N1 micro-neutralization test. We have introduced a new technique in collaboration with the HKU-Pasteur Research Centre, where we can now produce lentiviral pseudotype particles which express the H5 glycoprotein of HPAI (H5pp) for use in a sero-neutralization assay. The advantages of using H5pp are that they are capable of a single-round infection, but do not produce progeny virus. Further advantages of this system are that these particles can be used under lower biosafety requirements than using the wild-type virus, and are adapted to high throughput testing. We will (i) describe this technique in detail, and (ii) present results from a comparative study using this technique together with the standard microneutralization assay used in a sero-prevalence study of over 1300 people living in the areas where the human infections occurred in Cambodia. Our results show that 21/1376 people had neutralizing antibodies to the H5 glycoprotein using this new method, which was confirmed using the microneutralization assay. We conclude that the use of H5pp pseudotype particle neutralization assay is an effective way of detecting neutralizing antibodies in sera from avian and human H5N1 cases with the advantage that it can be performed in a BSL-2 laboratory. The technique is also useful for future studies on correlation of the neutralizing antibody activity to prevalence of H5N1 in the population, correlates of immune protection, virus clearance and disease progression.