Reduced pathogenicity of fructose-1,6-bisphosphatase deficient Leishmania donovani and its use as an attenuated strain to induce protective immunogenicity

Currently, there is no approved vaccine for visceral leishmaniasis (VL) caused by L. donovani. The ability to manipulate Leishmania genome by eliminating or introducing genes necessary for parasites' survival considered as the powerful strategy to generate the live attenuated vaccine. In the present study fructose-1,6-bisphosphatase (LdFBPase) gene deleted L. donovani (Δfbpase) was generated using homologous gene replacement strategy. Though LdFBPase gene deletion (Δfbpase) does not affect the growth of parasite in the promastigote form but axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Though Δfbpase L. donovani parasite persisted in BALB/c mice up to 12 weeks but was unable to cause infection, we tested its ability to protect against a virulent L. donovani challenge. Notably, intraperitoneal immunisation with live Δfbpase parasites displayed the reduction of parasites load in mice spleen and liver post challenge. Moreover, immunised BALB/c mice showed a reversal of T cell anergy and high levels of NO production that result in the killing of the parasite. A significant, correlation was found between parasite clearance and elevated IFNγ, IL12, and IFNγ/IL10 ratio compared to IL10 and TGFβ in immunised and challenged mice. Results suggested the generation of protective Th1 type immune response which induced significant parasite clearance at 12-week, as well as 16 weeks post, challenged immunised mice, signifying sustained immunity. Therefore, we propose that Δfbpase L. donovani parasites can be a live attenuated vaccine candidate for VL and a good model to understand the correlatives of protection in visceral leishmaniasis.