The study of heterosubtypic antibody responses against influenza A viruses elicited by seasonal vaccination using a pseudotype neutralisation assay

Background: The study of heterosubtypic antibody responses directed against influenza A haemagglutinins in human populations is an important facet of pandemic preparedness. The evaluation of the ability of vaccines to increase heterosubtypic antibody responses to confer broad protection against different influenza subtypes is one approach to this. Classic serological assays, such as haemagglutination inhibition and microneutralisation, have demonstrated low sensitivity for the detection of cross-neutralising antibodies, especially those directed against epitopes in the haemagglutinin HA2 stalk region. For this reason there is a need for new assay formulations that are able to detect and quantify these heterosubtypic antibody responses. Influenza pseudotypes represent safe tools to study the neutralising antibody response since they are replication-defective viruses and they harbour on their envelope only the haemagglutinin that is the major target of this response. Materials and Methods: We have generated a panel of group 2 influenza A pseudotypes (H3 A/Udorn/307/1972, H4 A/duck/Czechoslovakia/1956, H7 A/chicken/Italy/1082/1999, H10 A/chicken/Germany/N49, H14 A/mallard/Astrakhan/263/1982, H15 A/shearwater/West Australia/2576/1979) and we have used them as surrogate antigens in neutralisation assays to study the presence and magnitude of heterosubtypic neutralising antibody responses in human sera collected before and after the 2007-2008 seasonal influenza vaccination. Results: In the human sera tested, neutralising antibody responses are detected against not only human influenza viruses, but also against influenza pseudotypes harbouring avian haemagglutinins belonging to group 2 viruses. After seasonal vaccination, the pseudotype neutralisation assays detect variation in the neutralising antibody titres against avian influenza pseudotypes. Conclusions: The increased sensitivity of the pseudotype neutralisation assay performed using a panel of influenza A pseudotypes permits the detection of heterosubtypic antibody responses before and after seasonal influenza vaccination. This has implications for the development of pandemic preparedness plans at the population level.