Rusli, Nor Dini
(2013)
Interactions between equine lymphocytes and equine herpesvirus-1 (EHV-1).
MPhil thesis, University of Nottingham.
Abstract
Equine herpesvirus-1 (EHV-1) infects and causes contagious respiratory disease, epidemic abortion and sporadic neurological disease. In general, EHV-1 infection elicits the stimulation of both humoral and cellular immune responses in horses. As EHV-1 is a persistent virus, its infection leads to latent infection during the first two weeks with chronic reactivations. Virus neutralising antibodies combat EHV-1 infection by reducing nasal virus shedding, particularly in the nasopharynx, but they play insignificant roles in controlling systemic spread by cell-associated viraemia to the respiratory tract and lymphatic tissues. The cytotoxic T lymphocyte (CTL) response is the most crucial aspect of EHV-1 specific cell mediated immunity and its function is well described to protect against clinical disease, viraemia and nasopharyngeal virus shedding. EHV-1 immediate early gene products (gene 64) have been shown to be targets for EHV-1 specific CTL. Moreover, vaccination of ponies with the EHV-1 gene 64 protein induces cell mediated immune responses resulting in the reduction of clinical and virological disease. This important finding underlines the substantial role of viral gene 64 in cell mediated immunity and highlights its potential for improved vaccination strategies. Therefore, the overall aim of this study was to identify peptide sequences of CTL epitopes within EHV-1 immediate early protein’s Fragment D (encoded by gene 64) in ponies expressing the MHC class I A3/B2 allele. In order to achieve this, in vitro cellular techniques were established and T lymphocyte responses to EHV-1 were quantified as a prerequisite for identifying CTL target peptides.
This first investigation aimed to measure activation of equine peripheral blood mononuclear cells (PBMC) with either phorbol 12-myristate 13-acetate (PMA) or infectious EHV-1 by assessing interferon gamma (IFNγ) synthesis detected by flow cytometry as a prelude to stimulating PBMC with gene 64 protein or peptides. The intra-assay and inter-assay variations of IFNγ synthesis were characterised and the coefficients of variation of both assays were 8.42% and 5.8%. The phenotype of medium-stimulated and PMA-ionomycin stimulated PBMC comprised principally T cells as defined by antibodies specific for equine leukocyte markers. Intracellular IFNγ expression after in vitro EHV-1 stimulation of primed PBMC was assessed and revealed a very low percentage of IFNγ secreting cells in the responding population.
In order to induce cells to use for the preparation of equine T lymphocyte and CTL lines/clones, in vitro EHV-1 stimulation of PBMC in bulk culture was characterised. Phenotypic analyses and viability of EHV-1 stimulated PBMC showed that 5% NHS in culture medium at 7 days stimulation was more reliable to induce CD8+ cells compared with 10% FCS, indicating the preferred use of 5%NHS in culture media to generate and assay equine CTLs.
The next aim was to develop EHV-1 specific T cell lines and clones from whole virus (EHV-1). Several attempts were made to generate these using mouse anti equine CD3 monoclonal antibody (mAb) (clone UCF6G), autologous rhuIL-2 and mitomycin C treated PBMC as APC, but these failed. Using two different approaches; 5-ethynyl-2’-deoxyuridine (EdU) and Cu (I)-catalysed cycloaddition ‘click’ reaction assay and tritiated thymidine [3H] uptake to show lymphocyte activation in response to anti-CD3 monoclonal antibody, it was demonstrated that the anti-CD3 mAb was able to activate cell proliferation and allowed cell division and population expansion.
To further assist the establishment of CTL lines and clones, the peptides of the EHV-1 immediate early protein’s fragment D (amino acids 750-1143 in the IE protein) were investigated using an IFNγ ELISPOT assay. This approach was optimised and characterised using medium, mitogen and EHV-1 as the negative and positive controls. A library of 128 synthetic peptides was obtained comprising molecules of 14 amino acids in length, overlapping by 11 amino acids. All peptides were constructed into pool arrays linearly and overlapping peptides were not contained within the same pool. The optimal concentration of the peptide pool was determined and chosen as 0.048μg per well. This concentration was also used for screening the individual peptides. The results showed that several peptide pools produced high number of spot-forming-cells significantly greater than the threshold for positivity and most of individual peptides stimulated an IFNγ response from PBMC, suggesting that IFNγ synthesis is too insensitive to detect CD8+ IFNγ + responses.
In summary, this study has demonstrated the potential of new and relevant in vitro techniques that can be applied to the investigation of CTL target peptide(s) of EHV-1 as presented by the A3/B2 allele in horses.
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