EFFECTS OF THREE NIGERIAN PLANTS ON THE HISTOPATHOLOGY OF CUTANEOUS LEISHMANIASIS (Leishmania major) AND IMMUNOTHERAPY OF LEISHMANIASIS USING DNA/PROTEIN VACCINE IN BALB/c MICE

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Department of Medicine

ABSTRACT
Three Nigerian plants (Annona senegalensis, Azadirachta indica and Vernonia amygdalina) were evaluated for antileishmanial effects in vitro and in vivo using the Leishmania major parasites. It involved the preliminary testing of the hexane, methanol and aqueous extracts of these plants on infected murine macrophages and other cell lines. This was to determine the leishmanicidal activity and cytotoxicity on normal cell lines (L929 fibroblasts and macrophages), cancer cell lines (Jurkat and SH-5YSY) and Trypanosome brucei brucei. The leishmanicidal activity was determined using the microscopic counting method while the cytotoxicity involved the Alamar Blue colorimetric technique. Furthermore, doseresponse sensitivity tests were carried out on bone marrow macrophages infected with Leishmania major parasites and only extracts with positive indications from the preliminary tests were used. Also, the efficacy of the extracts which gave positive indications was tested in vivo in BALB/c mice treated orally for five days at a dose of 50 mg/kg per day. The aqueous extract of Vernonia amygdalina significantly (p<0.001) suppressed infection rate of intracellular amastigotes (>60%) and significantly (p<0.001) reduced parasite levels to almost zero in the infected macrophages. In the dose-response sensitivity test, the methanol extract of Vernonia amygdalina suppressed infection rates (>50%) at a lower concentration (50 ?g/ml) than that which caused toxicity to the macrophages. It also significantly reduced the lesion sizes by about 50% as well as minimised histopathological changes in the skin of infected mice. The hexane extract of Annona senegalensis, showed good trypanocidal effect against Trypanosome brucei brucei at a concentration of 3.13 ?g/ml. This was over 15 fold more potent than the reference drug, cylemersan used at 50 ?g/ml. viii Also, the potentials of a DNA based vaccine and its recombinant protein were evaluated as vaccine candidates. Leishmania donovani gene encoding gamma glutamylcysteine synthetase and its soluble recombinant ?GCS antigen in the presence of a non-ionic vesicular surfactant (NIVS) as an adjuvant were tested. Susceptible BALB/c mice were immunised with the plasmid encoding the full sequence for ?GCS (pVAX?GCS), recombinant protein in presence of adjuvant (r?GCS-NIV) or plasmid alone (pVAX control) prior to challenge with a high dose of Leishmania major promastigotes. Mice immunised with the recombinant protein (r?GCS-NIV), demonstrated an enhanced production of specific IgG1 and IgG2a antibodies indicating that it was immunogenic. Also, it resulted in significantly lower lesion sizes (p<0.05) as compared to the controls. This partial protection corresponded to a significant increase (p<0.05) in gamma interferon production suggesting an enhanced Th1 response. These results showed that the protein vaccine from Leishmania donovani induced a strong immuno protection against cutaneous leishmaniasis, suggesting that it represents a very good candidate for use as a vaccine against several leishmania species.