Development of an assay using real time cell analysis for the measurement of equid herpesvirus 1 specific neutralizing antibody in horses after experimental infection or field vaccination

Background: Equid herpesvirus-1 (EHV-1) is one of the most important horse pathogens, which induces respiratory disease, myeloencephalopathy, abortion or still-birth. The measurement of EHV-1 neutralising (EHV-1-VN) antibodies using a neutralisation assay (NA) gives an indication of the immune response of horses in the field. Objectives: To develop a real time neutralisation assay (RTNA) using the xCELLigence® technology for the measurement of EHV-1-VN antibodies in serums from EHV-1 experimentally infected or vaccinated horses. Study design: Assay development and field trial. Methods: The RTNA based on E. Derm cells was developed using serums (n=48) taken daily (from Days 0 to 18 post-infection) from ponies experimentally infected with a field EHV-1 strain. This method was further evaluated using field serums (n=63) taken during an EHV-1/-4 vaccination campaign. RTNA results were compared to those obtained with the conventional EHV-1 NA on RK13 cells. Two-fold serial dilutions of serums were incubated with a defined concentration of the KyD EHV-1 strain before infection of E. Derm cells. The normalised cell index was monitored pre- and post-infection using the xCELLigence® technology. Conventional NA was performed using the same experimental methodology on RK13 cells and cytopathic effects were measured 72 hours post-infection. Results: Serum titres which induced 50% of EHV-1 neutralisation (NT50) were determined using a dose-response approach. The increase of the EHV-1 NT50 was similar to the increase in titre measured with the conventional NA (R²=0.79). Seroconversion was detected between Days 0 and 18 post-infection for all infected ponies with both methods. The NT50 values measured for EHV-1/-4 vaccinated horse serums were also positively correlated with conventional NA titres (R²=0.83). Main limitations: EHV-1/-4 cross-reactivity will require further investigation. Each assay used different cells. Conclusion: While the conventional NA is time consuming, requires subjective measurements and provides semi-quantitative discrete ranked titres (e.g. 4, 8 etc.), the EHV-1-VN RTNA is an automated, sensitive and objective measurement that reports continuous titres.