Studies on the Cellular Immune Response to Feline Immunodeficiency Virus

Veterinary science, Animal diseases, Virology

Beatty, Julia A

University:University of Glasgow

关键词: Veterinary science, Animal diseases, Virology;   

Others  :  http://theses.gla.ac.uk/75670/1/13833797.pdf

来源: University of Glasgow

【 摘 要 】

The aim of this study was to investigate cellular immunity to FIV. The main part of the project involved the development of an assay system to detect FIV-specific cytotoxic T lymphocytes (CTL). In Chapter 1 the general features of the retrovirus family are described. The epidemiology, clinical signs and pathogenesis of human immunodeficiency virus (HIV) infection in man and of feline immxmodeficiency virus (FIV) infection in the cat are reviewed in detail. The current knowledge on the host imm\me response to both of these viruses is summarised. Chapter 2 covers the materials and methods which have been used routinely throughout these studies. Chapter 3 describes the development of an autologous, FIV-GAG expressing, Cr labelled target cell system for detecting feline cytotoxic effector cells. Peripheral blood mononuclear cells (PBMC), renal fibroblasts, bone marrow fibroblasts and skin fibroblasts were compared on the basis of ease of harvesting, in vitro growth characteristics and target antigen expression. Virus antigen expression in fibroblasts was achieved using a recombinant FIV/vaccinia virus, vFIV/GL14-gag, as fibroblasts are not normally infectable by FIV. Skin fibroblasts were selected as the most suitable target cell for the assay. A convenient and effective method of Cr labelling of target cells was developed using overnight incubation of target cell monolayers in situ.It was intended to use the prototype cytotoxicity assay to detect secondary cytotoxic responses since these are generally of greater magnitude than primary responses. The conditions necessary for the generation of secondary FIV-specific effector cells from circulating precursor cells were unknown. In Chapter 4, potential sources of endogenously processed viral antigen for the in vitro restimulation of circulating lymphocytes primed by FIV infection in vivo were investigated. Mitogen-activated PBMC from FIV-infected cats, lymphoblasts infected with FIV in vitro followed by fixation and vFIV/GL14-gag infected, fixed lymphoblasts were compared for efficiency of preparation and antigen expression. The latter were found to be the most suitable and batches of vFIV/GL14-gag infected, fixed lymphoblasts were prepared from the experimental cats and used as stimulator cells in subsequent experiments. In Chapter 5, the capacity of a prototype assay to measure feline cytotoxic responses was determined. From 3 experimental cats, skin fibroblasts were harvested and stimulator cells were prepared. Two of these cats (E3 and F23) were then experimentally infected with FTV and the third cat remained iminfected as a control (F22). PBMC were isolated from these cats prior to, and at intervals post-infection or mock-infection. Following a 6-7 day in vitro restimulation period, these bulk cultures were assayed for secondary cytotoxic effector cell activity. The assay was successful in detecting antigen-specific cytotoxic effector cells in both nV-infected cats. Cytotoxic cells specific for autologous targets expressing FIV- GAG were detected from 2 weeks post-infection in 1 FIV-infected cat and at 14 weeks post-infection in the other. Killing of heterologous targets cells was also detected from FIV-infected cats although to a lesser extent than autologous targets. No target cell lysis above background was detected using effector cells from the uninfected control cat. In a separate set of experiments (Chapter 6) the capacity of a recombinant FIV-ENV fusion protein to stimulate PBMC from naive cats to proliferate in vitro was investigated. A region of FIV/GL8 env, incorporating the third and fourth variable regions (V3A 4), was cloned, inserted into the pGEX vector and expressed in Escherichia coli to yield milligram quantities of the recombinant polypeptide as a fusion protein with glutathione S-transferase (GST). The fusion protein, V3A4GST, was used in lymphocyte proliferation assays, where it consistently caused PBMC from naive cats to proliferate in a dose-dependent manner. Other FIV fusion proteins produced under identical conditions (V5GST and p24GST) and GST alone did not cause proliferation in this system. The monoclonal antibody vpgl5, which has been shown to block infection of susceptible cells in vitro, did not decrease the response to V3/V4GST. Human PBMC did not proliferate in response to V3/V4GST. In Chapter 7 the results of this thesis are brought together, conclusions drawn and implications discussed. Suggestions for the future direction of this work are included.