The study of heterosubtypic neutralising antibody responses against HPAI influenza viruses in human subjects using a highly adaptable multiplex neutralisation assay

Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardised influenza assays for sero-surveillance and vaccine evaluation. We describe a multiplex assay that can be used for the study of neutralising antibodies that are directed against both influenza H5 and H7 HA in human serum samples. This assay permits the measurement of neutralising antibody responses against two antigenically distinct HA envelope glycoproteins in the same human serum sample thus increasing the amount and quality of serological data that can be acquired from valuable sera, and potentially reducing interassay variability. A panel of serum samples collected from the Italian population between 1992 and 2007 and previously found to be positive for antibodies against H5N1 as determined by SRH, were evaluated in this multiplex assay and anti-H5 and –H7 neutralising antibody responses were studied. From the results obtained it can be concluded that this assay is effective for the measurement of functional heterosubtypic crossreactive responses in human sera. This assay is useful as an adjunct to the EMA approved SRH and HI assays as it measures antibodies with different (functional) specificities contributing to comprehensive analyses of humoral immunity to influenza viruses