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

The monomer of influenza haemagglutinin is synthesized 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. Consequently, we have investigated the use of human Transmembrane Protease Serine 2 to produce high titre influenza haemmagglutinin (HA) lentiviral pseudotypes from Group 2 influenza viruses. Such pseudotypes represent powerful and safe tools to study viral entry 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 lentiviral vector incorporating firefly luciferase. However, in order to 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 is necessary for the functional activation, in vitro, of both the HA of human seasonal influenza and other Group 2 HA influenza strains. Additionally, we show that the Group 2 influenza pseudotype particles can be used as surrogate antigens in neutralization assays and are efficiently neutralized by corresponding influenza virus reference sera. These data demonstrate that the viral pseudotype system is a powerful method for serological surveillance of a wide range of influenza viruses

Targeting an Oncolytic Influenza A Virus to Tumor Tissue by Elastase

Oncolytic viruses are currently established as a novel type of immunotherapy. The challenge is to safely target oncolytic viruses to tumors. Previously, we have generated influenza A viruses (IAVs) containing deletions in the viral interferon antagonist. Those deletions have attenuated the virus in normal tissue but allowed replication in tumor cells. IAV entry is mediated by hemagglutinin (HA), which needs to be activated by a serine protease, for example, through trypsin. To further target the IAV to tumors, we have changed the trypsin cleavage site to an elastase cleavage site. We chose this cleavage site because elastase is expressed in the tumor microenvironment. Moreover, the exchange of the cleavage site previously has been shown to attenuate viral growth in lungs. Newly generated elastase-activated influenza viruses (AE viruses) grew to similar titers in tumor cells as the trypsin-activated counterparts (AT viruses). Intratumoral injection of AE viruses into syngeneic B16f1 melanoma-derived tumors in mice reduced tumor growth similar to AT viruses and had a better therapeutic effect in heterologous human PANC-1-derived tumors. Therefore, the introduction of the attenuation marker "elastase cleavage site" in viral HA allows for safe, effective oncolytic virus therapy

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

Haemagglutinin activation by human transmembrane protease serine 2 or by human airway trypsin-like protease is necessary for the production of high titre influenza A virus pseudotypes that can be employed for the evaluation of pandemic potential

Background: The monomer of influenza haemagglutinin (HA) is synthesized as a single polypeptide precursor that during maturation is cleaved by proteases into two active subunits. Recent studies have demonstrated that the human transmembrane protease serine 2 (TMPRSS2) and the human airway trypsin-like protease (HAT) can activate, by cleavage, the HA of human seasonal influenza viruses. As a model of activation of all influenza subtypes (human H1, H2, H3 viruses and other representative viruses from group 1 and group 2 avian subtypes), we have investigated the use of human TMPRSS2 and HAT to produce high titre influenza HA lentiviral pseudotypes. Such pseudotypes represent powerful and safe tools to study viral entry mechanisms and zoonotic potential. Materials and Methods: Influenza pseudotype particles are obtained by cotransfecting human embryonic kidney HEK293T/17 cells using plasmids coding for the influenza HA, HIV gag-pol and a retroviral vector incorporating firefly luciferase. To investigate the role of these proteases in the HA activation necessary for infective particle generation, a plasmid expressing TMPRSS2 or HAT was cotransfected during pseudotype production. The HA lentiviral pseudotypes produced were used to investigate cell entry potential via transduction of target cell lines and as surrogate antigens in neutralization assays. Results: Influenza pseudotype particles produced by cotransfection of these proteases can transduce HEK293T/17 cells with high efficiency compared with the pseudotypes produced in the absence of proteases. The high titre of these influenza pseudotypes permits their use as surrogate antigens in neutralization assays and subtype-specific sera can readily neutralize them. Conclusions: TMPRSS2 and/or HAT can activate, in vitro, both the HA of human seasonal influenza and also other avian HA influenza strains in a pseudotype particle production system. Furthermore, this panel of influenza pseudotypes can be used in neutralization assays to study heterosubtypic antibody responses and pandemic potential

The human Transmembrane Protease Serine 2 is necessary for the production of Group 2 influenza A virus pseudotypes

The monomer of influenza haemagglutinin is synthesized 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. Consequently, we have investigated the use of human Transmembrane Protease Serine 2 to produce high titre influenza haemmagglutinin (HA) lentiviral pseudotypes from Group 2 influenza viruses. Such pseudotypes represent powerful and safe tools to study viral entry 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 lentiviral vector incorporating firefly luciferase. However, in order to 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 is necessary for the functional activation, in vitro, of both the HA of human seasonal influenza and also other Group 2 HA influenza strains. Additionally, we show that the Group 2 influenza pseudotype particles could be used as surrogate antigens in neutralization assays and are efficiently neutralized by corresponding influenza reference sera. These data demonstrate that the pseudotype system is a powerful method for serological surveillance of a wide range of influenza viruses