Evaluation Of Crude Leishmania Donovani Antigen Co-Administered With Th1 Adjuvants As A Potential Vaccine For Leishmaniasis In Vervet Monkey Model

Abstract

Visceral leishmaniasis (VL) or kala-azar is the most dreaded and devastating form of leishmaniasis, causing high mortality rate, mainly in children. A vaccine against different forms of leishmaniasis should be feasible considering the wealth of information on genetics and biology of the parasite, clinical and experimental immunology of leishmaniasis, and the availability of vaccines that can protect experimental animals against challenge with different Leishmania species. However, to date, there is no effective vaccine against any form of leishmaniasis for general human use. Efforts to develop an effective vaccine so far have been limited due to lack of an appropriate adjuvant. A mixture of safe Leishmania antigens and an adjuvant that preferentially stimulates cellular immune response presents a rational option for a vaccine against leishmaniasis. A vaccine for man needs to be tested in suitable primate models such as the vervet monkey due to their close phylogenetic relation to humans. This study used the vervet monkey model of visceral leishmaniasis to evaluate the safety, immunogenicity and efficacy of Leishmania donovani sonicate antigen (Ag) delivered alone or in conjunction with alum-BCG (AlBCG), monophosphoryl lipid A (MPL) or montanide ISA 720 (MISA) as adjuvants. Following vaccinations of groups of vervet monkeys at days 0, 28 and 42, safety was assessed by observation of indurations and erythema at sites of vaccination. Antibody responses and cytokines were quantified by enzyme linked immunosorbent assay (ELISA). Antigen recall lymphoproliferation was measured by blast assay while interferon gamma (IFN-γ)-producing CD4+ and CD8+ T cell responses were measured by intracellular cytokine staining and quantification using flow cytometer. Animals were challenged with L. donovani parasites and efficacy evaluated by parasite quantification in splenic impression smears. Data were analyzed using CellQuest, one way analysis of variance (ANOVA), Tukey-Kramer test, Spearman rank correlation analysis or Wilcoxon matched-paired sign-rank test. A P value < 0.05 was considered significant. Results indicated that vaccinations with MPL+Ag or MISA+Ag were safe while animals vaccinated with AlBCG+Ag developed erythematous ulcerative indurations at sites of vaccination. Delayedtype hypersensitivity (DTH) responses were significantly higher in the MISA+Ag group than in other vaccinated groups (P < 0.001) while both the AlBCG+Ag and MISA+Ag groups induced the highest total IgG and IgG2 subclass antibody responses as compared to vaccinations with MPL+Ag. Interleukin-4 and IL-10 cytokine responses were relatively higher in the MPL+Ag group than in other groups while vacinations with AlBCG+Ag and MISA+Ag induced comparable IFN-γ or TNF-α levels which were significantly higher than levels induced by MPL+Ag vaccination (P < 0.001). Interestingly, significantly higher IFN-γ producing CD4+ or CD8+ T cells were induced in AlBCG+Ag and MISA+Ag vaccinated animal groups as compared to other experimental groups (P < 0.001). There was a positive and significant correlation between IFN-γ producing CD4+ and CD8+ T cell populations in both experimental and control groups (r = 1.000; P = 0.0167). Positive significant correlations were also observed between either CD4+ or CD8+ T cells with IFN-γ, TNF-α cytokines or DTH responses (r = 1.000; P < 0.0167). Significant reductions in parasitic loads were associated with vaccinations with AlBCG+Ag or MISA+Ag as compared to other study groups (P < 0.001). All Th1 immune response parameters including DTH, IFN-γ, TNF-α, and IFN-γ-producing CD4+ or CD8+ T cells correlated negatively and significantly with parasitic loads. The findings from this study conclude that, MISA 720 is an appropriate adjuvant in terms of safety and immunogenicity and can be effectively used in the formulation of Leishmania vaccines for clinical applications. The study recommends the use of MISA 720 in the development of a Leishmania vaccine for clinical trials in humans. Data generated in this study is useful in Leishmania vaccine development.