Combination of Liposomal CpG Oligodeoxynucleotide 2006 and Miltefosine Induces Strong Cell-Mediated Immunity during Experimental Visceral Leishmaniasis

Abstract

Immuno-modulators in combination with antileishmanial drug miltefosine is a better therapeutic approach for treatment of Visceral Leishmaniasis (VL) as it not only reduces the dose and toxicity of miltefosine but also shortens the treatment regimen. However, immunological mechanisms behind the perceived benefits of this combination therapy have not been investigated in detail. In the present study, we hypothesized that potential use of drugs that stimulates the host as well as targets the parasite might represent an alternative strategy for combination therapy. We investigated immune responses generated in Leishmania donovani infected hamsters treated with combination of CpG-ODN-2006 and miltefosine at short dose regimen. Infected hamsters were administered CpG-ODN-2006 (0.4 mg/kg, single dose), as free and liposomal form, either alone or in combination with miltefosine for 5 consecutive days and parasite clearance was evaluated in splenocytes at day 4 and 7 post treatment. Hamsters that received liposomal CpG-ODN-2006 (lipo-CPG-ODN-2006) and sub-curative miltefosine (5 mg/kg) showed the best inhibition of parasite multiplication (~ 97%) which was associated with a biased Th1 immune response in them. Moreover, compared to all the other treated groups, we observed increased mRNA expression levels of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-12) and significantly suppressed levels of Th2 cytokines (IL-10 and TGF-β) on day 4 post treatment in hamsters that underwent combination therapy with lipo-CPG-ODN-2006 and sub-curative miltefosine. Additionally, same therapy also induced heightened iNOS mRNA levels and NO generation, increased IgG2 antibody level and strong T-cell responses in these hamsters compared with all the other treated groups. Collectively, our results suggest that combination of lipo-CPG-ODN-2006 and sub-curative miltefosine generates protective T-cell responses in a hamster model of visceral leishmaniasis which is characterized by strong Th1 biased immune response thereby underlining our hypothesis that combination therapy, at short dose regimen can be used as a novel way of treating visceral leishmaniasis.