The immunochemistry and immunobiology of Leishmania Donovani lipophosphoglycan

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dc.contributor.author Tolson, Douglas Leonard
dc.date.accessioned 2018-07-03T18:46:50Z
dc.date.available 2018-07-03T18:46:50Z
dc.date.copyright 1991 en_US
dc.date.issued 2018-07-03
dc.identifier.uri https://dspace.library.uvic.ca//handle/1828/9569
dc.description.abstract Using intact Leishmania donovani promastigotes or purified L. donovani lipophosphoglycan (LPG) as immunogens, four LPG-specific monoclonal antibodies (mAbs) were derived. Two of these mAbs (CA7AE and BF9CC) specifically bound to an epitope consisting of the repeating phosphorylated Gal-(β1,4)-Man disaccharide portion of the LPG molecule. MAb CA7AE also bound antigens in L. donovani promastigoteconditioned culture medium; specifically, the secreted forms of LPG (phosphoglycan) and acid phosphatase, demonstrating that major secreted glycoconjugates of L. donovani share phosphorylated carbohydrate epitope(s). The two other mAbs (L157 and L98) bound to a parasite-derived protein component that was discovered to be tightly associated with the phosphocarbohydrate core region of the LPG molecule (LPG-associated protein; LPGAP). These are the first defined epitopes of LPG. Immunochemical assays were used to analyze the distribution of the LPG repeat and LPGAP epitopes over a wide sampling of Leishmania species and strains. MAb CA7AE recognized, to varying extents, epitopes from most of the Leishmania species examined; both as parasite surface-exposed, membrane-bound molecules and as antigens released into parasite-conditioned culture medium. The anti-LPGAP mAbs bound to all twenty Leishmania and Trypanosoma strains assayed but not to the surface of living parasites. None of the anti-LPG mAbs bound the amastigote form of the parasites. Experiments involving amastigote-to-promastigote in vitro transformation showed that the CA7AE epitope was expressed on the surface of transforming cells within 5 hours of culture at 26°C. The epitope was excreted into the culture supernatant within 15 hours. In addition, the mAb CA7AE “epitope was detected in 50% of sera tested from L. donovani infected (Kala-azar-positive) patients. Murine macrophages, infected with L. donovani promastigotes, were examined by immunofluorescence for the expression of LPG epitopes. The CA7AE epitope, detected as early as 5-10 minutes post infection (p.i.), was initially localized to the immediate area of internalization of the promastigote into the macrophage with even distribution over the entire macrophage surface by 25 minutes p.i. These epitopes remained on the macrophage surface until approximately 88 hours p.i Acetone permeabilization of the macrophages, prior to mAb probing, exposed LPG epitopes present within the macrophages to at least 160 hours p.i. Treatments which inhibited macrophage phagolysosomal degradation processes had no effect on epitope expression whereas reagents that affected macrophage membrane flow, and thus phagocytosis, drastically reduced or abolished expression. Purified LPG or de-lipidated LPG were also shown to bind to a variety of different cell types but in a temperature-independent manner. The early and continued expression of LPG epitopes on the macrophage surface suggests the possibility that LPG epitopes may be involved in the immune response which is directed to Leishmania-infected macrophages. Lymph node cells from mice primed with L. donovani LPG or LPGAP or with living, virulent L. donovani promastigotes were specifically stimulated to proliferate in vitro by the LPGAP moiety of the LPG molecule. The T cell response was antigen-specific, dose-dependent, and non-mitogenic. In addition, purified T lymphocytes primed with purified LPG or LPGAP were not stimulated by LPG or LPGAP in vitro unless promastigote-infected or LPG-pulsed or LPGAP-pulsed macrophages were added. Recognition of LPGAP epitopes was an MHC-restricted event. LPGAP epitopes specifically stimulated CD4+CD8-, IL-2 secreting T lymphocytes and that active antigen processing by macrophages was required for T cell stimulation. Both L. donovani LPG and L. tropica LPG which have antigenically different repeat epitopes but which share LPGAP epitopes stimulated lymphocyte proliferation independent of the LPG source used for priming. The T cell stimulation caused by LPGAP was not species-specific and since the responding T cells were of the Th 1 phenotype and recognized epitopes in amastigotes, the LPGAP epitopes are very likely of considerable importance in Leisimania-specific immunity. The data suggests that Leishmania LPGAP is a potential vaccine candidate for leishmaniasis. en_US
dc.language English eng
dc.language.iso en en_US
dc.rights Available to the World Wide Web en_US
dc.subject Leishmania en_US
dc.subject Immunological aspects en_US
dc.title The immunochemistry and immunobiology of Leishmania Donovani lipophosphoglycan en_US
dc.type Thesis en_US
dc.contributor.supervisor Pearson, Terry W.
dc.degree.department Department of Biochemistry and Microbiology en_US
dc.degree.level Doctor of Philosophy Ph.D. en_US
dc.description.scholarlevel Graduate en_US

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