Research Accomplishment Reports 2009

Immunologic Requirements for Vaccine Mediated Prevention of Equine Herpesvirus Neurologic Disease

D.W. Horohov
Department of Veterinary Sciences

 

Non-Technical Summary

Equine herpesvirus-1 (EHV-1) neurologic disease has been an infrequently observed disease of the central nervous system of horses that, in recent years, has emerged to become one characterized by a much higher incidence than in past decades. The emerging herpesvirus disease causes high mortality rates and results in a negative economic impact on the boarding and racing sectors of the industry.

Recent molecular characterization of EHV-1 isolates recovered from 52 outbreaks of neurologic disease has revealed that the majority of such disease outbreaks are caused by a mutant strain of the virus. A most unsettling property of these neuropathogenic mutants of EHV-1 is their inability to be curtailed by the level or type of immunity engendered by current-generation vaccines for EHV-1. It is precisely this challenge of the development of a new-generation vaccine for successful prevention of neurologic manifestations of EHV-1 infection that is addressed by the research activities proposed in this application.

This research project will focus on identifying the precise immune mechanisms of the horse that must be stimulated by vaccines in order to protect against neuropathogenic EHV-1 infection and subsequent neurologic disease. Additionally, the project will attempt to determine which vaccine types and which administration routes are most successful in eliciting protection of horses from EHV-1 neurologic disease. It is anticipated that results obtained from these studies will provide a more scientific basis for development of an improved vaccine for neurologic herpes.

2009 Project Description

The goal of this research was to identify the immunological underpinnings required for successful development of an efficacious vaccine for protection of horses against equine herpesvirus-1 (EHV-1) neurologic disease. Our objective was to determine if the production of EHV-1 specific cytotoxic T lymphocytes (CTLs) could be correlated with the expression of interferon-gamma, granzyme B or perforin mRNA in lymphocyte cultures stimulated with this virus. We could then use these indrect measures of CTL activity to determine if horses immunized with an intra-nasally administered, live-virus vaccine that developed CTL responses were protected from clinical neurologic disease following challenge-infection with a mutant, neuropathogenic strain of EHV-1.

2009 Impact

Surrogate markers (interferon-gamma, perforin and granzyme B) were developed for the identification and quantitation of EHV-1 specific CTL. Total RNA was isolated form lymphocyte cultures after in vitro stimulation with EHV-1 antigen. The RNA was reverse transcribed into cDNA and equine-specific primers and probes for the genes of interest were used to amplify the cDNA using real-time PCR. Beta-glucoronidase was used as the housekeeping gene.

Using this approach, we were able to demonstrate that the expression of these markers correlated with EHV-1 specific CTL activity in the cultures. In collaboration with another group (Soboll, CSU), we were also able to show that vaccination of horses with a live attenuated EHV-1 mutant induced the expression of these same markers following in vitro stimulation of lymphocytes with EHV-1 and that this in vitro expression correlated with protection observed in vivo post-challenge with EHV-1.