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. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9569 |
| Date | 03 July 2018 |
| Creators | Tolson, Douglas Leonard |
| Contributors | Pearson, Terry W. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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