title: Novel Approaches for Vaccination against Equine Viral Arteritis
Creators: name: Castillo-Olivares, Javier
Publisher: name: The Open University
Year: 2004

description: This thesis explores the potential of two prototype (‘marker’) vaccines with differential diagnostic capability for improved control of equine viral arteritis. The rational for using a sub-unit adjuvanted vaccine and a recombinant live virus vaccine, analysis of immune responses they induce and assessment of the protection against infection in experimental conditions is described and discussed in this thesis. Also, the development of new methods for measuring cytotoxic T-lymphocyte responses to EAV is described, increasing the repertoire of immunological assays for EAV infections. Finally, attempts to determine the antigenic specificity of the cytotoxic T-lymphocyte response have been taken. <br><br> The sub-unit vaccination approach developed in this project derives from the findings of Dr. Ewan Chirnside <i>et al</i> (1995a). This strategy has been developed further. A recombinant protein expressed in E.coli comprising the putative ectodomain of the large envelope glycoprotein (G<sub>L</sub>) of equine arteritis virus (EAV) was used as a vaccine in ponies resulting in the induction of virus neutralising antibody (VNAb) responses which are comparable in titre to those induced by inactivated whole virus vaccines. The protection afforded by this vaccine against virulent challenge, which correlated with the pre-challenge VNAh titres, was characterised by reduction of nasal virus excretion and viraemia. <br><br> A second ‘marker’ vaccination strategy for EAV was explored. A recombinant live virus containing a deletion of a neutralisation domain on G<sub>L</sub> was constructed by manipulations of the cDNA full length clone of EAV. A synthetic peptide (Peptide-1) whose sequence is contained within the deleted fragment is the basis of an already developed antibody capture diagnostic ELISA test for EAV. The deletion mutant virus (EAV-GlA) showed <i>in vitro</i> slightly slower growth kinetics than wild type EAV and when administered by the nasopharyngeal route to ponies these experienced an asymptomatic infection, although virus was recovered from blood and nasal secretions. Both inoculated ponies developed a discriminating antibody response which lacked the Peptide-1 antigen specificity. The serum VNAb response was of high titre against EAV-GlA but of low titre against wild type EAV. Yet, the EAV-AGl inoculated ponies were highly protected against virulent challenge. This novel approach shows great potential as a marker vaccine. Safety and duration of immunity need to be investigated. The structure of VNAb epitopes and effector mechanisms of immunity stimulated by administration of EAV-AGl deserve also attention. <br><br> The serum VNAb response that invariably develops in the infected animal lasts for many months or years and is believed to play an important role in virus clearance. However, very little was known about the cellular immunity against EAV due the lack of methods to evaluate these immune responses. Methods to detect cytotoxic T-lymphocyte (CTL) precursors in the peripheral blood of EAV convalescent ponies are described using a <sup>51</sup>Cr release cytolysis assay. Primary equine dermal cells, used as CTL targets, were shown to express MHC-I but not MHC-II, retain <sup>51</sup>Cr efficiently and support EAV replication. Peripheral blood mononuclear cells (PBMC) collected from EAV convalescent ponies that have been incubated with or without live EAV were used as effectors. EAV induced PBMC cultures showed evidence for expansion and activation of lymphoblasts, with an increase in the CD8+/CD4+ ratio in comparison to mock-induced PBMC. The cytotoxicity induced by EAV stimulated PBMC was virus specific, showed genetic restriction, was mediated by CD8+ T lymphocytes and could be detected for periods between 4 months to more than 1 year post-infection. These findings and methods will hopefully contribute to understanding of virus-host interactions in horses, in particular the mechanisms of virus clearance occurring during EAV infection. <br><br> The last part of this project described efforts to try to identify proteins serving as targets for EAV specific CTL’s. Target cells transiently expressing EAV0RF7 were used in the standard CTL assay developed during the course of these studies (Chapter 4). Cloning of EAV0RF7 into the mammalian expression vector pcDNA3 for subsequent expression in different cell lines is described. Whilst, expression of EAV N protein in C0S7 and RK-13 cells using plasmid transfection techniques was achieved, EDC’s failed to express N efficiently. Unlike plasmid transfection, baculovirus transduction appeared to be a suitable method to achieve protein expression in a high proportion of EDC. The levels of expression of EAV N antigen in transduced EDC, which occurred without the loss of their original morphology, suggested <i>a priori</i> that this method was adequate to identify CTL targets. Time and resources constraints limited the number of experiments performed but sufficient progress was made to indicate that this expression technique has good potential for examining EAV CTL targets. <br><br> In this research project two novel approaches have been used for the generation of marker vaccines against EAV and methods to evaluate cytotoxic T lymphocyte responses developed. The essential findings of the thesis provide new and interesting information about EAV vaccinology/immunology that now provide the basis for rational approaches to be undertaken to disease prevention.