Immunization with Live Attenuated Leishmania donovani Centrin−/− Parasites Is Efficacious in Asymptomatic Infection

Abstract

Currently, there is no vaccine against visceral leishmaniasis (VL). Toward developing an effective vaccine, we have reported extensively on the immunogenicity of live attenuated LdCentrin−/− mutants in naive animal models. In VL endemic areas, asymptomatic carriers outnumber symptomatic cases of VL and are considered to be a reservoir of infection. Vaccination of asymptomatic cases represents a viable strategy to eliminate VL. Immunological correlates of protection thus derived might have limited applicability in conditions where the immunized host has prior exposure to virulent infection. To examine whether LdCen−/− parasites can induce protective immunity in experimental hosts that have low-level parasitemia from a previous exposure mimicking an asymptomatic condition, we infected C57Bl/6 mice with wild-type Leishmania donovani parasites expressing LLO epitope (LdWTLLO 103, i.v.). After 3 weeks, the mice with low levels of parasitemia were immunized with LdCen−/− parasites expressing 2W epitope (LdCen−/−2W 3 × 106 i.v.) to characterize the immune responses in the same host. Antigen experienced CD4+ T cells from the asymptomatic (LdWTLLO infected) LdCen−/−2W immunized, and other control groups were enriched using LLO- and 2W-specific tetramers, followed by Flow cytometric analysis. Our analysis showed that comparable CD4+ T cell proliferation and CD4+ memory T cell responses (TCM) represented by CD62Lhi, CCR7+, and IL-7R+ T cell populations were induced with LdCen−/−2W in both asymptomatic and naive animals that received LdCen−/− immunization. Upon restimulation with peptide, TCM cells differentiated into effector T cells and there was no significant difference in the recall response in animals with asymptomatic infection. Following virulent challenge, comparable reduction in splenic parasite burden was observed in both asymptomatic and naive LdCen−/− immunized animals concomitant with the development of multifunctional CD4+ and CD8+ T cells. Further, LdCen−/−2W immunization resulted in complete clearance of the preexisting asymptomatic infection (LdWTLLO). Our results demonstrate that LdCen−/−2W immunization could be efficacious for use in asymptomatic VL individuals. Further, immunization with LdCen−/− could help in reducing the parasite burden in the asymptomatic cases and aid in controlling the VL in endemic areas.

miR-21 Expression Determines the Early Vaccine Immunity Induced by LdCen−/− Immunization

“…Although proliferation of the 2W specific CD4 + T cell population was observed in LNA treated LdWT infection, expression of other markers of early memory such as IL-7R were absent in this population. In contrast, a significantly higher IL-7R + 2W + CD4 + T cell population was observed LdCen −/ − infection similar to our previously published results (24) and no change in expression was observed upon LNA treatment (Figure 4G, * p < 0.05). To measure if blocking miR-21 expression impacts the parasite proliferation, we measured the splenic parasite burden following treatment with LNA treatment.…”

“…LdWT and LdCen −/− parasites secreting 2W epitope (EAWGALANWAVDSA) fused to L. donovani 3′ Nucleotidase/Nuclease protein lacking the N' terminus membrane anchoring domain, were used to enrich epitope specific CD4 + T cells. The recombinant LdWT and LdCen −/ − parasites expressing fluorescent or chimeric proteins have been described previously (11, 24). The 2W-PE tetramers (2W1S:I-A b -streptavidin-phycoerythrin) used to enrich the CD4T cells were obtained from NIH tetramer core facility in Emory Vaccine Center, Atlanta, GA.…”

MicroRNAs: Biological Regulators in Pathogen–Host Interactions

“…This mechanism occurs via the downregulation of the NF-κB mediating transcription of pro-inflammatory cytokine genes, such as TNF-α and IL-12, thereby altering the immune response to parasite survival inside the macrophages [83]. Interestingly, miR-21 is upregulated in peripheral blood mononuclear cells (PBMCs) infected with L. donovani, and miR-blocking increases the expression of IL-12 mRNA in murine-DCs infected with L. donovani, leading to a proliferation of CD4 + T cells [84]. Further, miRNA-361-3p, a regulator of TNF, was observed to be negatively correlated with localized cutaneous leishmaniasis (LCL) skin lesions caused by L. braziliensis, which is resistant to pentavalent antimonial and healing of parasites [85], while the expression of miR-193b and miR-671 was observed to be correlated with that of their respective target genes, CD40 and TNFR, in lesions of L. braziliensis-infected patients.…”

The potential impact of human visceral leishmaniasis vaccines on population incidence

“…Currently there are various VL vaccine candidates under study [ 27 ]: LEISH-F3+GLA-SE [ 28 , 29 ], and ChAd63-KH (ISRCTN07766359) [ 30 ] are currently in clinical development; Ad5-A2/rA2 Prime / Boost [ 31 ], genetically modified live attenuated whole parasites [ 25 , 26 , 32 ], and a LmCen -/- vaccine [ 33 ] are being developed for the clinic [ 34 ].…”