Role of CD8(+) T cells in protection against Leishmania donovani infection in healed Visceral Leishmaniasis individuals.

BACKGROUND: Majority of individuals with history of visceral leishmaniasis (VL) exhibit strong immunity to re-infection, however, the mechanism of resistance is poorly understood. It is unclear whether CD8(+) T cells contribute to protection against Leishmania donovani infection through cytotoxic activity. The present study aims to evaluate immunological mechanism associated with resistance to the disease in healed VL (HVL) individuals and further, the contribution of CD8(+) T cells in the protective immunity. METHODS: Peripheral blood mononuclear cells (PBMCs) from VL, HVL and naive groups were exposed in vitro to total soluble Leishmania antigen (TSLA) from L. donovani. The proliferation index was determined by ELISA based lymphoproliferative assay. Cytokines and granzyme B levels were measured by CBA. Activated T-cell populations were estimated using flow cytometry. RESULTS: We observed significantly higher lymphoproliferation, cytokines and granzyme B levels in HVL group compared to naive or VL group. More strikingly, we found a strong association (rs = 0.895, P < 0.0001) between proliferation index (PI) and granzyme B level, with a significant proportion of activated CD8(+) T cells in HVL group. CONCLUSIONS: Leishmania immune group (HVL) exhibited durable and strong cellular immune response to TSLA in terms of lymphoproliferation as well as production of Th1 cytokines and granzyme B. Additionally, the elevated level of activated CD8(+) T cells and stimulation of cytotoxic activity through granzyme B production, indicated a possible role of CD8(+) T cells in resistance to L. donovani infection in the HVL group.

Peptide-based vaccine successfully induces protective immunity against canine visceral leishmaniasis.

Dogs are the main reservoir of zoonotic visceral leishmaniasis. Vaccination is a promising approach to help control leishmaniasis and to interrupt transmission of the Leishmania parasite. The promastigote surface antigen (PSA) is a highly immunogenic component of Leishmania excretory/secretory products. A vaccine based on three peptides derived from the carboxy-terminal part of Leishmania amazonensis PSA and conserved among Leishmania species, formulated with QA-21 as adjuvant, was tested on naive Beagle dogs in a preclinical trial. Four months after the full course of vaccination, dogs were experimentally infected with Leishmania infantum promastigotes. Immunization of dogs with peptide-based vaccine conferred immunity against experimental infection with L. infantum. Evidence for macrophage nitric oxide production and anti-leishmanial activity associated with IFN-γ production by lymphocytes was only found in the vaccinated group. An increase in specific IgG2 antibodies was also measured in vaccinated dogs from 2 months after immunization. Additionally, after challenge with L. infantum, the parasite burden was significantly lower in vaccinated dogs than in the control group. These data strongly suggest that this peptide-based vaccine candidate generated cross-protection against zoonotic leishmaniasis by inducing a Th1-type immune response associated with production of specific IgG2 antibodies. This preclinical trial including a peptide-based vaccine against leishmaniasis clearly demonstrates effective protection in a natural host. This approach deserves further investigation to enhance the immunogenicity of the peptides and to consider the possible engineering of a vaccine targeting several Leishmania species.

Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs.

Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates.

In vitro evaluation of a soluble Leishmania promastigote surface antigen as a potential vaccine candidate against human leishmaniasis.

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.

Long-lasting protection against canine visceral leishmaniasis using the LiESAp-MDP vaccine in endemic areas of France: double-blind randomised efficacy field trial.

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. Dogs constitute the major reservoir of Leishmania infantum/chagasi responsible for human visceral leishmaniasis. We have recently demonstrated that the combination of naturally excreted/secreted antigens, easily purified from culture supernatant of Leishmania infantum promastigotes (LiESAp) as vaccine antigen in formulation with muramyl dipeptide (MDP) as adjuvant, conferred 100% protection to dogs experimentally infected with L. infantum by inducing in vaccinees a significant, stable and long-lasting Th1-type cell response [Lemesre JL, Holzmuller P, Cavaleyra M, Bras Gonçalves R, Hottin G, Papierok G. Protection against experimental visceral leishmaniasis infection in dogs immunised with purified excreted secreted antigens of L. infantum promastigotes. Vaccine 2005; 23:2825-2840; Holzmuller P, Cavaleyra M, Moreaux J, Kovacic R, Vincendeau P, Papierok G, Lemesre JL. Lymphocytes of dogs immunised with purified excreted secreted antigens of L. infantum co-incubated with Leishmania-infected macrophages produce IFN-gamma resulting in nitric oxide-mediated amastigote apoptosis. Vet. Immunol. Immunopathol. 2005, 106:247-257]. In this report, protection against visceral leishmaniasis is investigated in naturally exposed dogs of endemic areas of the South of France vaccinated with LiESAp/MDP vaccine. A double-blind randomised efficacy field trial was developed on a large-scale dog population composed of vaccinees (n=205) and placebo-treated animals (n=209), which were prospectively studied for a 2-year period. 0f the initial 414 enrolled dogs, 340 (175 controls and 165 vaccinees) were analysed for clinical, serological and parasitological studies at 24 months post-vaccination, after two sand fly seasons. Strong seroconversion disclosed by an L. infantum indirect immunofluorescence antibody test (IFAT) associated with suspicious clinical symptoms, considered an indication that the animals had an established progressive infection, was only observed in the placebo group. The seropositive and/or symptomatic dogs were selected for further examination for possible Leishmania infection by culturing parasites from bone-marrow aspirate. The presence of leishmanial infection was also evaluated by means of the PCR analysis of bone marrow samples in all enrolled dogs prior to vaccination and in all evaluated animals (175 controls and 165 vaccinees) at 24 months post-vaccination. After two transmission cycles completed, the Leishmania infection rate was 0.61% (1/165) in vaccinated dogs and 6.86% (12/175) in the placebo group. The efficacy of the vaccine was calculated to be 92% (P=0.002). A clear difference between the dogs that received vaccine and those that received placebo was also established by the results of their immune status. Increased anti-LiESAp IgG2 reactivity and significant enhanced NO-mediated anti-leishmanial activity of canine macrophages in response to higher IFN-gamma production by T cells were almost exclusively revealed in vaccinees. The LiESAp-MDP vaccine induced a significant, long-lasting and strong protective effect against canine visceral leishmaniasis in the field.

Protection against experimental visceral leishmaniasis infection in dogs immunized with purified excreted secreted antigens of Leishmania infantum promastigotes.

The capacity of naturally excreted secreted antigens easily purified from culture supernatant of Leishmania infantum promastigotes (LiESAp), successfully cultivated in completely defined medium called CDM/LP [Lemesre JL. Methods for the culture in vitro of different stages of tissue parasites. International publication WO 94/26899, 1994; Merlen T, Sereno D, Brajon N, Rostand F, Lemesre JL. Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms. Am J Trop Med Hyg 1999;60(1):41-50] to protect dogs against experimental L. infantum infections is described. Eighteen healthy Beagle dogs were allocated into four groups that received at a 3-week interval either two subcutaneous injections of 50 microg (group 2, n = 3), 100 microg (group3, n = 6) and 200 microg (group 4, n = 3) LiESAp in formulation with muramyl dipeptide (MDP) or similar injections of placebo (group 1, n = 6). Dogs were intravenously infected with 10(8) metacyclic L. infantum promastigotes. Promastigotes of the MHOM/MA/67/ITMAP-263 and MHOM/FR/78/LEM75 strains were, respectively, administered 2 months (at day 84, homologous challenge 1) and 8 months post-immunization (at day 273, heterologous challenge 2). The data indicated that vaccine candidate confers total protection (100%) against challenges 1 and 2 in dogs from groups 3 and 4 and intermediate protection (66.7%) against challenge 1 in dogs from group 2 as determined by parasite detection in bone marrow aspirates during 14 months post-challenge follow-up. All placebo dogs of group 1 were found infected and failed to respond to LiESAp in cell-mediated assays before and after both challenges. Increased levels of total anti-leishmanial antibodies were exclusively detected in infected dogs from group 1. Vaccine-induced protection correlates with an early establishment of a long lasting predominantly Th1-type cellular immune response specifically directed against LiESAp before and after experimental infections, as demonstrated by: (i) anti-LiESAp IgG2 reactivity, and (ii) LiESAp-specific lymphocyte proliferation assays and enhanced NO-mediated anti-leishmanial activity of canine monocyte-derived macrophages (CM-DM) in response to higher IFNgamma production by T-cells, when L. infantum-infected CM-DM were co-cultured with autologous lymphocytes. Overall, our results support the view that a LiESAp vaccine might be useful in a promising vaccination approach against natural L. infantum infection.