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

Abstract

Pseudotyped viruses (PVs) produced by co-transfecting cells with plasmids expressing lentiviral core proteins and viral envelope proteins are potentially powerful tools for studying various aspects of equine influenza virus (EIV) biology. The aim of this study was to optimise production of equine influenza PVs. Co-transfection of the HAT protease to activate the haemagglutinin (HA) yielded a higher titre PV than TMPRSS2 with the HA from A/equine/Richmond/1/2007 (H3N8), whereas for A/equine/Newmarket/79 (H3N8), both proteases resulted in equivalent titres. TMPRSS4 was ineffective with the HA of either strain. There was also an inverse relationship between the amount of protease-expression plasmids and the PV titre obtained. Interestingly, the PV titre obtained by co-transfection of a plasmid encoding the cognate N8 NA was not as high as that generated by the addition of exogenous neuraminidase (NA) from Clostridium perfringens to allow the release of nascent PV particles. Finally, initial characterisation of the reliability of PV neutralisation tests (PVNTs) demonstrated good intra-laboratory repeatability. In conclusion, we have demonstrated that equine influenza PV production can be readily optimised to provide a flexible tool for studying EIV.

Evaluation of a Pseudotyped Virus Neutralisation Test for the Measurement of Equine Influenza Virus-Neutralising Antibody Responses Induced by Vaccination and Infection

“…Lentivirus particles pseudotyped with the A/equine/Richmond/1/2007 HA were produced as described in Scott et al, 2016 [ 10 ]. Briefly, four plasmids expressing the EIV HA gene, an HA-cleaving protease (i.e., TMPRSS2), the HIV gag-pol genes, and a manipulated HIV genome containing the luc reporter gene replacing endogenous genes, were co-transfected into HEK293/17 (PV producer) cells (see Figure 1 C).…”

“…In the pilot study from Scott et al [10], a threshold of 1.9 logIC50 was considered as the positivity threshold for the PVNT, using a PV displaying the HA of A/equine/Sussex/1989 (H3N8) [9]. This was redefined in the current study using pre-vaccination serum samples and serum samples from unvaccinated ponies as 2.21 logIC50.…”

“…The SRH assay measures antibodies against the NA also, which will contribute to the reduction of viral load during infection. However, PVs displaying both the HA and NA proteins of EIV have been successfully generated [10].…”

“…Measurement of SRH antibody levels provides a well-established correlate of protection against EIV infection, with defined thresholds associated with virus shedding reduction and clinical protection [6][7][8]. More recently, pseudotyped viruses (PVs) based on lentivirus vectors, have been generated to measure equine influenza neutralising antibodies in horse serum and have demonstrated reasonable correlation (correlation coefficient r = 0.65) with the SRH assay for detecting antibody levels [9,10]. PVs are non-replicating, hybrid viruses with the disabled core of a vector virus and the envelope protein (or proteins) of the study virus (e.g., influenza virus haemagglutinin).…”

Current status on the development of pseudoviruses for enveloped viruses

“…Some pseudoviruses need special enzymes/reagents during production to optimize titer. Scott et al optimized the production of equine influenza‐HA‐pseudotyped viruses via the addition of exogenous neuraminidase from Clostridium perfringens to allow the release of nascent pseudovirus particles . These observations indicate that the packaging conditions should be optimized to improve the pseudovirus yield on a case‐by‐case basis.…”