Leishmaniasis, caused by an infection of the Leishmania protozoa, is a neglected tropical disease and a major health problem in tropical and subtropical regions of the world, with approximately 350 million people worldwide at risk and 2 million new cases occurring annually. Current treatments for leishmaniasis are not highly efficacious and are associated with high costs, especially in low- and middle-income endemic countries, and high toxicity. Due to a surge in the incidence of leishmaniases worldwide, the development of new strategies such as a prophylactic vaccine has become a high priority. However, the ability of Leishmania to undermine immune recognition has limited our efforts to design safe and efficacious vaccines against leishmaniasis. Numerous antileishmanial vaccine preparations based on DNA, subunit, and heat-killed parasites with or without adjuvants have been tried in several animal models but very few have progressed beyond the experimental stage. However, it is known that people who recover from Leishmania infection can be protected lifelong against future infection, suggesting that a successful vaccine requires a controlled infection to develop immunologic memory and subsequent long-term immunity. Live attenuated Leishmania parasites that are non-pathogenic and provide a complete range of antigens similarly to their wild-type counterparts could evoke such memory and, thus, would be effective vaccine candidates. Our laboratory has developed several live attenuated Leishmania vaccines by targeted centrin gene disruptions either by homologous recombination or, more recently, by using genome editing technologies involving CRISPR-Cas9. In this review, we focused on the sequential history of centrin gene-deleted Leishmania vaccine development, along with the characterization of its safety and efficacy. Further, we discussed other major considerations regarding the transition of dermotropic live attenuated centrin gene-deleted parasites from the laboratory to human clinical trials.

Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania donovani causes severe disease. Age appears to be critical in determining the clinical outcome of VL and at present there is no effective vaccine available against VL for any age group. Previously, we showed that genetically modified live attenuated L. donovani parasites (LdCen-/-) induced a strong protective innate and adaptive immune response in young mice. In this study we analyzed LdCen-/- parasite mediated modulation of innate and adaptive immune response in aged mice (18 months) and compared to young (2 months) mice. Analysis of innate immune response in bone marrow derived dendritic cells (BMDCs) from both young and aged mice upon infection with LdCen-/- parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4⁺ Th1 cell effector function compared to LdWT infected BMDCs in vitro. Similarly, parasitized splenic dendritic cells from LdCen-/- infected young and aged mice also revealed induction of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) and subsequent down regulation of anti-inflammatory cytokine (IL-10) genes compared to LdWT infected mice. We also evaluated in vivo protection of the LdCen-/- immunized young and aged mice against virulent L. donovani challenge. Immunization with LdCen-/- induced higher IgG2a antibodies, lymphoproliferative response, pro- and anti-inflammatory cytokine responses and stimulated splenocytes for heightened leishmanicidal activity associated with nitric oxide production in young and aged mice. Furthermore, upon virulent L. donovani challenge, LdCen-/- immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells correlating to a significantly reduced parasite burden in the spleen and liver compared to naïve mice. It is interesting to note that even though there was no difference in the LdCen-/- induced innate response in dendritic cells between aged and young mice; the adaptive response specifically in terms of T cell and B cell activation in aged animals was reduced compared to young mice which correlated with less protection in old mice compared to young mice. Taken together, LdCen-/- immunization induced a significant but diminished host protective response in aged mice after challenge with virulent L. donovani parasites compared to young mice. Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani. There is no effective vaccine available against VL for any age group and importantly, there are no previous studies regarding immune responses against experimental Leishmania vaccines tested in aged animals. We have reported earlier that immunization with a live attenuated L. donovani parasites (LdCen-/-) induced protective immune response in young animals viz, mice, hamsters and dogs. In this study we analyzed LdCen-/- mediated modulation of innate and adaptive responses in aged mice and compared to young mice. We observed that LdCen-/- infected dendritic cells from young and aged mice resulted in enhanced innate effector functions compared to LdWT parasites both in vitro and in vivo. Further, upon virulent challenge, LdCen-/- immunized young and aged mice displayed protective Th1 immune response which correlated with a significantly reduced parasite burden in the visceral organs compared with naïve challenged mice. Although there was no difference in the LdCen-/- induced dendritic cell response between aged and young mice; adaptive response in aged was reduced, compared to young which correlated with less protection in aged compared to young mice. This study supports the potential use of LdCen-/- as vaccine candidate across all age groups against VL.

No licensed vaccine exists against visceral leishmaniasis (VL), a disease caused by the Leishmania donovani parasite. We have previously reported both macrophages and dendritic cells play important role in the protection induced by a live attenuated centrin gene–deleted L. donovani (LdCen−/−) parasite vaccine. The role of neutrophils in orchestrating the initial innate response to pathogens is widely recognized. To investigate the early interaction of LdCen−/− with neutrophils, we immunized mice intradermally in the ear pinna with LdCen−/−. Compared with LdWT infection, LdCen−/− parasites induced higher recruitment of neutrophils to the ear dermis and ear draining lymph nodes (dLN) as early as 6–18 h after immunization, which were predominantly proinflammatory in nature. Neutrophils from ear dLN of LdCen−/−-immunized mice exhibited heightened expression of costimulatory molecules and attenuated expression of coinhibitory molecules necessary for higher T cell activation. Further phenotypic characterization revealed heterogeneous neutrophil populations containing Nα and Nβ subtypes in the ear dLN. Of the two, the parasitized Nα subset from LdCen−/−-immunized mice exhibited much stronger Ag-specific CD4+ T cell proliferation ex vivo. Adoptive transfer of neutrophils bearing LdCen−/− parasites induced an increased Th1 response in naive mice. Importantly, neutrophil depletion significantly abrogated Ag-specific CD4+ T cell proliferation in LdCen−/−-immunized mice and impaired protection against virulent challenge. Conversely, replenishing of neutrophils significantly restored the LdCen−/− -induced host-protective response. These results suggest that neutrophils are indispensable for protective immunity induced by LdCen−/− parasite vaccine.

Background: Neutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuatedLeishmania donovanicentrin deleted parasite vaccine (LdCen^-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs duringLdCen^-/-infection and compared with wild type parasite (LdWT)bothin vitroandin vivo.Methodology/findings: LdCen^-/-parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared toLdWTinfection. To further illustrate neutrophil-DCs interactionsin vivo, we infected LYS-eGFP mice with red fluorescentLdWT/LdCen^-/-parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containingLdCen^-/-parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4⁺T cell primingex-vivo. Notably, potent DC activation occurred whenLdCen^-/-parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment ofLdCen^-/-parasites by DCs. Neutrophil depletion inLdCen^-/-infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4⁺Th1 cell primingex vivothat correlated with attenuated Tbet expression in ear dLN derived CD4⁺T cellsin vivo.Conclusions: Collectively,LdCen^-/-containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response.

No human vaccine is available for visceral leishmaniasis (VL). Live attenuated centrin gene-deleted L. donovani (LdCen^−/−) parasite vaccine has been shown to induce robust innate immunity and provide protection in animal models. Toll-like receptors (TLRs) are expressed in innate immune cells and are essential for the early stages of Leishmania infection. Among TLRs, TLR-9 signaling has been reported to induce host protection during Leishmania infection. Importantly, TLR-9 ligands have been used as immune enhancers for non-live vaccination strategies against leishmaniasis. However, the function of TLR-9 in the generation of a protective immune response in live attenuated Leishmania vaccines remains unknown. In this study, we investigated the function of TLR-9 during LdCen^−/− infection and found that it increased the expression of TLR-9 on DCs and macrophages from ear-draining lymph nodes and spleen. The increase in TLR-9 expression resulted in changes in downstream signaling in DCs mediated through signaling protein myeloid differentiation primary response 88 (MyD88), resulting in activation and nuclear translocation of nuclear factor-κB (NF-κB). This process resulted in an increase in the DC’s proinflammatory response, activation, and DC-mediated CD4⁺T cell proliferation. Further, LdCen^−/− immunization in TLR-9^−/− mice resulted in a significant loss of protective immunity. Thus, LdCen^−/− vaccine naturally activates the TLR-9 signaling pathway to elicit protective immunity against virulent L. donovani challenge.