* Purification of a defined parasite antigen, dp72, for use in laboratory vaccine trials against canine leishmaniasis.
* Development of molecular biological methods, specifically reverse transcriptase - polymerase chain reaction (RT-PCR), to measure cytokine responses, Interleukin (IL-4, IL-10), Tumor necrosis factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma), in vaccinated dogs following infection.
* Examination of Th1 and Th2 responses to vaccine antigens in the experimental model for canine leishmaniasis.
* Identification of a suitable site in Western Turkey where a vaccine field can be carried out.
Attempts to develop an IL-4 RT-PCR based on sequence information from other IL-4s have not been successful. We have postponed IL-4 detection for the assessment of Th2 response, and continued with IL-10 RT-PCR for monitoring TH2 response. We have developed a competitive RT-PCR using in vitro internal control (IC) mRNA which is added to the sample in order to be able to quantify the amount of RNA present in the sample.
Peripheral blood mononuclear cells (PBMC) from several dogs were tested in this way: one dog was parasitological positive for VL, two dogs were asymptomatic but serologically positive and one dog was parasitologically and clinically negative at time of sampling but became symptomatic over time.
PBMCs were incubated either in the absence of antigen or in the presence of Leishmania LV9 antigen or ConA. Ten-fold serial dilutions of IC-RNA were added to the RNA isolates. In general, pPBMCs which have been stimulated with either LV9 antigen or Con1 showed a rise in the production of IFN-gamma; no significant differences could be observed for IM-10 and TNF-alpha mRNA levels.
Dogs were vaccinated with Leishmania antigen dp72 plus BCG as an adjuvant. As negative controls they used dogs vaccinated with BCG or saline or nothing at all. PBMCs from these dogs were isolated at five different time points before, during and after vaccination. These samples were stimulated with nothing (negative control), ConA (positive control) or different amounts of antigen.
Preliminary experiments indicate that, in a qualitative RT-PCR using beta-actin, IL-10, TNF-alpha and IFN-gamma primers, the only significant difference that could be observed was an increase in IFN-gamma levels upon stimulation with ConA. Further quantitation revealed a 100- to 400- fold increase in IFN-gamma levels. However, beta-actin levels were also increased up to 100-fold after stimulation with ConA.
We are presently testing more samples and working on the interpretation of the data.%
Reverse transcriptase polymerase chain reactions (RP-PCR) for the detection in canine peripheral blood mononuclear cells (PMBC) of lymphokines known to be important in protective and exacerbating responses to Leishmania infections. This will include the measurement of IL-4, IL-10, TNF-alpha and IFN-gamma. PMBC will be developed by standard Ficoll-Hypaque technique using whole dog blood and mRNA isolated by the guanidinium/silica method.
* The antigen dp72 from L. d. infantum will be produced for use in vaccination and immunological studies in dogs. Enough protein to vaccinate about 25 dogs (approximately 900 ug) will be purified. The protein dp72 will be purified from a crude membrane fraction of L.d. infantum (LV9) by affinity chromatography with a monoclonal antibody (mAb) D13 against the antigen.
* Protection studies in an experimental model for canine visceral leishmaniasis.
* In parallel, dogs with clinical VL will be identified in the field by positive serological diagnosis and tissue biopsies or cultures. Th1 and Th2 T-cell lymphokine expression will be examined by RT-PCR following antigen-specific and mitogen stimulation of PMBL taken from these dogs. This will be compared with the vaccinated dogs as described above.
* Field sites will be identified for vaccines trials. The prevalence of canine leishmaniasis will be examined longitudinally in all the potential field test sites, as described above, during the period preceding the vaccine trial. In addition, a survey of the human population in these areas will be carried out and includes a medical check-up, the DAT and leismanin skin test. This will be used to evaluate the immune status of the dog and human populations at the field trial site.
Funding SchemeCSC - Cost-sharing contracts