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.