Multiplex evaluation of influenza neutralizing antibodies with potential applicability to in-field serological studies

Purpose: The increased number of outbreaks of H5 and H7 LPAI and

HPAI viruses in poultry has major public and animal health implications.

The continuous rapid evolution of these subtypes and the emergence

of new variants influence the ability to undertake effective surveillance.

Retroviral pseudotypes bearing influenza haemagglutinin (HA) and

neuraminidase (NA) envelope glycoproteins represent a flexible platform

for sensitive, readily standardized influenza serological assays.

We describe a multiplex assay for the study of neutralizing antibodies

that are directed against both influenza H5 and H7 HA. This assay

permits the measurement of neutralizing antibody responses against

two antigenically distinct HAs in the same serum/plasma sample thus

increasing the amount and quality of serological data that can be

acquired from valuable sera.

Methods & Materials: Sera obtained from chickens vaccinated

with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent

H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus

were evaluated in the pseudotype assay (simple and multiplex format).

Results: The results obtained using the pseudotype-based assays

correlated strongly with data obtained by HI assay. We show that pseudotypes

are highly stable under basic cold-chain storage conditions

and following multiple rounds of freeze-thaw.

Conclusion: We propose that this robust assay may have practical

utility for in-field serosurveillance and vaccine studies in resource-limited

regions worldwide.