The use of equine influenza pseudotypes for serological screening

Abstract

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.

Controlling equine influenza: Traditional to next generation serological assays

“…Thus far, it is thought that the assay demonstrates increased sensitivity as some serum samples were defined as negative by SRH but positive by PVNA. Overall 65% correlation was shown between the two assays (Scott et al, 2012).…”

The Application of Pseudotypes to Influenza Pandemic Preparedness

“…The use of pseudotypes as alternatives to wild-type influenza viruses, including HPAI strains and pandemic strains, has been implemented in neutralization assays, also referred to as pseudotype-based neutralization (pp-NT) assays. Previous studies have reported that results from HI, MN and SRH assays correlate well with those obtained by pp-NT assay [128,[137][138][139]. For the pp-NT assay, twofold serial dilutions of sera are incubated with a predetermined amount (quantification is done via the measurement of reporter gene expression) of pseudotypes and incubated for 1 h at 37°C in a 96-well plate.…”

The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus

“…Higher titres of the Richmond/07 PV were obtained using HAT than with TMPRSS2 while titres obtained with Newmarket/79 were equivalent using HAT or TMPRSS2. The use of TMPRSS2 to successfully generate PVs expressing HA from various subtypes has been previously reported [13,14], and TMPRSS2 was used to generate the first reported equine influenza PV [5]. The HAT protease has also been used previously to generate PVs expressing human H3, H1, and H5 [14].…”

“…The full-length HA genes of A/equine/Newmarket/1979 (H3N8) and A/equine/Richmond/2007 (H3N8) were kindly provided by Dr. Adam Rash and Dr. Debra Elton (Animal Health Trust, Newmarket, UK) who PCR-amplified the HA gene using the custom primers (Invitrogen) described in [5]. The PCR fragments were digested with restriction enzymes EcoRV/BamHI and BamHI/XhoI (Thermo Scientific) for A/equine/Newmarket/1979 and A/equine/Richmond/2007, respectively, and cloned into the pI.18 expression plasmid.…”

“…However, the single radial haemolysis (SRH) assay is more widely used to evaluate equine influenza vaccines as SRH levels correlate with protective efficacy of vaccines (reviewed in [4]). We previously demonstrated a 65% correlation between the results of a neutralisation test using an EIV PV and an SRH assay, with the pseudotyped virus neutralisation test (PVNT) appearing slightly more sensitive [5]. However, the PV used only expressed the EIV HA, and a recent study highlighted the importance of the antibody response to NA, measured by a neuraminidase inhibition test, in determining protection against infection for human influenza [6].…”