The use of equine influenza pseudotypes for serological screening

Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and the necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralization assays. In this study we generated an equine influenza pseudotyped lentivirus for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of an H3N8 subtype equine influenza virus strain, HIV gag-pol and firefly luciferase reporter genes and harvesting virus from supernatant. In order to produce infective pseudotype particles it was necessary to additionally co-transfect a plasmid encoding the TMPRSS2 endoprotease to cleave the HA. High titre pseudotype virus (PV) was then used in PV antibody neutralization assays (PVNAs) to successfully distinguish between vaccinated and non-vaccinated equines. The sera were also screened by single radial haemolysis (SRH) assay. There was a 65% correlation between the results of the two assays, with the PVNA assay appearing slightly more sensitive. Future work will extend the testing of the PVNA with a larger number of serum samples to assess sensitivity/specificity, inter/intra-laboratory variability and to define a protective titre

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

Development of LPAI and HPAI H7 avian influenza pseudotypes for serological applications utilising a combination of protease cotransfection and site-directed mutagenesis

HPAI and LPAI H5 and H7 viruses have been disastrous for the economies of affected countries reliant on poultry production. In a situation similar to H5, H7 strains show adaptation to humans and therefore pose a serious public health concern. Applying knowledge acquired from study of H5 virus evolution and spread to the development of sensitive serological methods will improve our ability to understand and respond to the emergence of other HPAI and LPAI viruses with pandemic potential. We describe the production of pseudotypes bearing envelope glycoproteins of LPAI and HPAI H7 avian influenza for use as tools in pseudotype-based (pp-NT) neutralisation assays. A crucial feature of H7 pseudotype production is efficient intracellular cleavage of haemagglutinin. We show that the LPAI strain A/chicken/Italy/1082/1999 possesses a monobasic cleavage site and requires TMPRSS2 to effect cleavage of the HA. The HPAI strain A/ Pakistan/34668/95 possesses a sub-optimal furin cleavage site resulting in low yields. In order to circumvent this we have used site-directed mutagenesis to replace the polybasic cleavage site with a monobasic site that can subsequently be cleaved (during production) via the cotransfection of the TMPRSS2 protease. Sensitive pp-NT assays were then carried out on post-vaccination sera using these new surrogate viruses

The human transmembrane protease serine 2 is necessary for the production of Group 2 influenza A virus pseudotypes

The monomer of influenza haemagglutinin (HA) is synthesised as a single polypeptide precursor that during maturation is cleaved by proteases into two active subunits. Other studies have demonstrated that the human Transmembrane Protease Serine 2 (TMPRSS2) can cleave the HA of human seasonal influenza viruses and therefore has an important role in pathogenesis. As a model of this cleavage we have investigated the use of human TMPRSS2 to produce high-titre influenza HA lentiviral pseudotypes from Group 2 influenza viruses. Such pseudotypes represent powerful and safe tools to study viral entry mechanisms and immune responses. Influenza pseudotype particles are obtained by co-transfecting human embryonic kidney HEK293T/17 cells using plasmids coding for the influenza HA, HIV gag-pol and a retroviral vector incorporating firefly luciferase. However, in order to successfully produce Group 2 pseudotypes, it was necessary to co-transfect a plasmid expressing the TMPRSS2 endoprotease to achieve the necessary HA cleavage for infective particle generation. These lentiviral pseudotypes were shown to transduce HEK293T/17 cells with high efficiency. This demonstrates that TMPRSS2 can cleave, in vitro, both the HA of human seasonal influenza and also other Group 2 HA influenza strains. Pseudotypes represent an adjunct tool for predicting pandemic potential of emerging influenza viruses

The use of equine influenza pseudotypes for serological screening

Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralisation assays. In this study we have generated equine influenza pseudotyped lentiviruses for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of different H3N8 subtype equine influenza virus strains, HIV gag-pol and firefly luciferase reporter genes and harvesting virus from supernatant. To produce infective pseudotype particles it was also necessary to co-transfect a plasmid encoding the TMPRSS2 endoprotease to cleave HA. High titre pseudotype virus (PV) was then used in PV antibody neutralisation assays (PVNAs) to successfully distinguish between vaccinated and non-vaccinated equines. The sera were also screened by single radial haemolysis (SRH) assay. There was a 65% statistical correlation between the results of the two assays, with the PVNA assay appearing more slightly more sensitive. Future work will extend the testing of the PVNA with a larger cohort of serum samples to assess sensitivity/specificity, inter/intra-laboratory variability and to define a protective titre