Immunogenicity and efficacy of a bivalent DNA vaccine containing LeIF and TSA genes against murine cutaneous leishmaniasis

There is no effective vaccine for the prevention and elimination of leishmaniasis. For this reason, we assessed the protective effects of DNA vaccines containing LeIF, TSA genes alone, or LeIF–TSA fusion against cutaneous leishmaniasis pEGFP‐N1 plasmid (empty vector) and phosphate buffer saline (PBS) were used as control groups. Therefore, cellular and humoral immune responses were evaluated before and after the challenge with Leishmania major. Lesion diameter was also measured 3–12 weeks after challenge. All immunized mice with plasmid DNA encoding Leishmania antigens induced the partial immunity characterized by increased IFN‐γ and IgG2a levels compared with control groups (p < 0.001). Furthermore, the immunized mice showed significant reduction in mean lesion sizes compared with mice in empty vector and PBS groups (p < 0.05). The reduction in lesion diameter was 29.3%, 34.1%, and 46.2% less in groups vaccinated with LeIF, TSA, and LeIF‐TSA, respectively, than in PBS group at 12th week post infection. IFN/IL‐4 and IgG2a/IgG1 ratios indicated that group receiving LeIF–TSA fusion had the highest IFN‐γ and IgG2a levels. In this study, DNA immunization promoted Th1 immune response characterized by higher IFN‐γ and IgG2a levels and also reduction in lesion size. These results showed that a bivalent vaccine containing two distinct antigens may induce more potent immune responses against leishmaniasis.     

Prophylactic efficacy of high-molecular-weight antigenic fractions of a recent clinical isolate of Leishmania donovani against visceral leishmaniasis

T-cell mediated immune responses are key determinants to the natural course of infection caused by intracellular parasites such as Leishmania. Thus, T-cell activating proteins of these microbes continue to generate active interest particularly in view of their possible role in the design and development of newer and more effective vaccines. We have recently reported the presence of T-cell immunostimulatory antigens with the high-molecular-weight (MW) fractions (134-64.2 kDa) of whole Leishmania donovani antigen (strain 2001), which stimulated variable amounts of IFN-gamma, IL-12 and IL-10 in exposed immune individuals. The present study was undertaken to further evaluate these high-MW antigenic fractions (MW range >100-60 kDa) for potential protective efficacy. The high-MW region of the parasite was resolved into five antigenic fractions (Prep A-E) using continuous elution gel electrophoresis. Prior to in vivo protection studies in hamsters, these fractions were used to evaluate in vitro cellular responses in eight Leishmania-exposed individuals and treated cured hamsters. The protective efficacy of prep (A + B), C, D and E in combination with BCG was evaluated in inbred hamsters using standard immunization protocol. Proliferative responses were seen in all eight of eight exposed individuals to prep D [median stimulation index (SI): 5.2 (range 3.9-7.1)] and E [median SI: 5.6 (range 4.4-8.2)], five of eight individuals to prep B and prep C and three of eight to prep A [median SI: 0.2 (range 0.1-7.2)]. The median proliferative responses to prep D and prep E were significantly higher than to fraction prep A; (P < 0.05) but not to prep B and prep C. However, prep A-E induced equivalent levels of IFN-gamma, IL-10 and IL-12 cytokines. Fractions D and E also exhibited marked parasite inhibition in spleen (52.5% and 73.7%) and liver (65% and 80.2%) as compared with prep (A + B) (23% in spleen and 24% in liver) and prep C (38% in spleen and 24% in liver). Prep D and prep E vaccinated animals showed higher in vitro stimulatory responses (mean SI: 6.6 and 8.8) and nitric oxide (NO) induction (mean NO levels: 6.4 and 10.7 mug/ml) against whole cell extract as compared with other groups. The protection also correlated with presence of suppressed Leishmania-specific IgG levels in prep D and prep E immunized hamsters. These studies indicate the presence of immunostimulatory and protective molecules in 60-80 kDa region of L. donovani, which may be further exploited for developing a subunit vaccine.     

Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis.

The antibody response against an amastigote-specific protein (A2) from Leishmania donovani was investigated. Sera from patients with trypanosomiasis and various forms of leishmaniasis were screened for anti-A2 antibodies. Sera from patients infected only with L. donovani or Leishmania mexicana specifically recognized the A2 recombinant protein. These results were consistent with karyotype analyses which revealed that the A2 gene is conserved in L. donovani and L. mexicana strains. The potential of this antigen in diagnosis was further explored by screening a series of sera obtained from patients in regions of the Sudan and India where L. donovani is endemic. The prevalence of anti-A2 antibodies was determined by Western blotting for all samples. Enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay were also performed on some of the samples. Anti-A2 antibodies were detected by ELISA in 82 and 60% of the samples from individuals with active visceral leishmaniasis (kala-azar) from the Sudan and India, respectively, while the immunoprecipitation assay detected the antibodies in 92% of the samples from India. These data suggest that the A2 protein may be a useful diagnostic antigen for visceral leishmaniasis.     

Immunogenicity and protective efficacy of DNA vaccine against visceral leishmaniasis in BALB/c mice

The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis.Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1(+) encoding T cell epitopes of gp63 and Hsp70 individually and in combination.Animals were challenged intracardially with 10~7 promastigotes of Leishmania donovani 10 days post immunization and sacrificed 1,2 and 3 months post challenge.The immunized animals revealed a significant reduction(P < 0.05)in splenic and hepatic parasite burden as compared to the infected controls.Maximum reduction in parasite load(P < 0.05) was observed in animals treated with a combination of pcDNA/gp63 and pcDNA/Hsp70.These animals also showed heightened DTH response,increased IgG2a,elevated Th1 cytokines(IFN-γ and IL-2) and reduced IgG1 and IL-10 levels.Thus,mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens.     

Immune induction by adjuvanted Leishmania donovani vaccines against the visceral leishmaniasis in BALB/c mice

Visceral leishmaniasis (VL) is a neglected tropical disease caused by Leishmania donovani or Leishmania infantum. Currently, the patients are treated with chemotherapeutic drugs; however, their toxicity limits their use. It would be desirable to develop a vaccine against this infection. In this study, we assessed the efficacy of different vaccine formulations at variable time points. Heat-killed (HK) antigen of Leishmania donovani was adjuvanted with two adjuvants (AddaVax and Montanide ISA 201) and three immunizations at a gap of 2 weeks (wk) were given to BALB/c mice. After 2 weeks of the last booster, mice were given challenge infection and sacrificed before challenge and after 4wk, 8wk, and 12 wk post-challenge. Significant protective immunity was observed in all the immunized animals and it was indicated by the notable rise in delayed-type hypersensitivity (DTH) response, remarkably declined parasite burden, a significant increase in the levels of interferon-gamma (IFN-γ), interleukin-12, interleukin-17 (Th1 cytokines), and IgG2a in contrast to infected control mice. Montanide ISA 201 with HK antigen provided maximum protection followed by AddaVax with HK and then HK alone. These findings elaborate on the importance of the tested adjuvants in the vaccine formulations against murine visceral leishmaniasis.     

Cloning of Leishmania donovani genes encoding antigens recognized during human visceral leishmaniasis

In this report we describe the construction and analysis of a genomic library of Leishmania donovani gene segments in the bacteriophage vector lambda gt11. This cloning vector permits the expression of parasite polypeptides as fusion products with Escherichia coli beta-galactosidase. A group of 90 clones which express L. donovani antigens have been isolated from this library using various polyvalent antisera. Many of these clones appear to encode parasite membrane antigens some of which are recognized by sera from patients with visceral leishmaniasis (kala azar). Some clones reacted with specific subsets of kala azar sera while others reacted with all kala azar sera tested. In addition, some of the clones appear to encode conserved antigens that are recognized by sera from mice immunized with L. major. Preliminary characterization of five clones indicated that each one contains a distinct genetic element and that at least four encode different fusion peptides.     

Persistence of Leishmania donovani antibodies in past visceral leishmaniasis cases in India

The persistence of anti-Leishmania donovani antibodies in past visceral leishmaniasis (VL) cases was retrospectively assessed by means of the direct agglutination test (DAT) and the rK39 enzyme-linked immunosorbent assay (ELISA). Antibody titers remained high for an extended period of time in past cases of VL. These results highlight the need to carefully elicit the history of patients with VL symptoms.     

Specific serodiagnosis of visceral leishmaniasis using a Leishmania donovani antigen identified by expression cloning

A λgt11 expression library was constructed using mRNA from the promastigote form of Leishmania donovani (African isolate MHOM/ET/67/HU3). The library was screened with serum obtained from a patient who contracted visceral leishmaniasis in the Sudan. Several cDNA clones which expressed β-galactosidase/L. donovani antigen fusion proteins were isolated. One of these clones corresponded to a 60 kDa membrane-associated antigen. By a Western blot assay antibodies against the fusion protein were detected universally in the sera of visceral leishmaniasis patients. They were not detected in sera from patients with cutaneous leishmaniasis or other parasitic protozoan infections. The gene coding for this antigen was specific to the genus Leishmania as judged by DNA hybridisation, and its chromosomal location was conserved. A 20 kb mRNA was detected on Northern blots of promastigote RNA.     

Immuno stimulating glycophosphosphingolipid antigen from Leishmania donovani is recognized by visceral leishmaniasis patient sera

Surface antigens on Leishmania promastigotes and infected macrophages are obvious targets in immunoprophylaxis for leishmanial infection. The glycophosphosphingolipid (GSPL) antigen isolated from Leishmania donovani surface membrane was recognized by sera from patients with visceral leishmaniasis. GSPL was also expressed on the membrane of parasite-infected macrophages. The effect of GSPL on the production of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) was studied using the macrophage cell line J774.1. In addition, induction of IFNγ, IL4, IL10, IL12 secretion in presence of GSPL was investigated in PBMC from normal individuals. ROS and RNI in addition to IFNγ and IL12 were induced by GSPL. Though there was a moderate induction of IL10, there was very little induction of the Th2 cytokine IL4. GSPL also induced blood cells to proliferate. The data suggests that this functionally important antigen of L. donovani may be used as a candidate vaccine.     

Leishmania donovani whole cell antigen delivered with adjuvants protects against visceral leishmaniasis in vervet monkeys(Chlorocebus aethiops)

In a previous immunogenicity and efficacy study in mice,montanide ISA 720(MISA)was indicated to be a better adjuvant than bacillus calmette guerin vaccine(BCG)for a Leishmania vaccine.In the present study,we report the safety,immunogenicity and efficacy of Leishmania donovani(L.donovani)sonicated antigen delivered with alum-BCG(AlBCG),MISA or monophosphoryl lipid A(MPLA)in vervet monkeys following intradermal inoculums.Vaccinated and control animals were challenged with virulent L.donovani parasites and the parasitic burden was determined.Only animals vaccinated with alum-BCG adversely reacted to the inoculum by producing ulcerative erythematous skin indurations.Non-parametric ANOVA followed by a post test showed significantly higher IgG antibodies,and revealed the presence of lymphoproliferative and interferon gamma responses in both AlBCG+Ag and MISA+Ag as compared to the MPLA+Ag or other groups(P < 0.001).We conclude that L.donovani sonicated antigen containing MISA is safe and is associated with protective immune response against Leishmania donovani infection in the vervet monkey model.     

Identification and Characterization of a Novel, 37-Kilodalton Leishmania donovani antigen for diagnosis of Indian visceral leishmaniasis

The biggest challenge in the serological diagnosis of visceral leishmaniasis (VL) is to find a biomarker with a high specificity. This study was undertaken to identify novel Leishmania donovani antigens to solve the existing problem. The soluble L. donovani promastigote antigen was separated by SDS-PAGE, and a Western blot was probed with pooled sera of five subjects with confirmed VL before (n = 9 pools) and after (n = 9 pools) treatment and at the 6-month follow-up visit (n = 9 pools), healthy controls not from an area of endemicity (n = 9 pools), and healthy controls from an area of endemicity. The antibody response to the identified partially purified antigen was ascertained by an enzyme-linked immunosorbent assay (ELISA) with 70 sera from patients with parasitologically confirmed VL, 48 sera from healthy controls from an area where the disease is not endemic, 60 sera from healthy controls from an area of endemicity, and 42 sera from patients in different disease groups. The eluted protein was subjected to two-dimensional (2D) gel electrophoresis, Western blotted, and probed with sera from patients with confirmed VL and from healthy controls not from an area of endemicity. The antigenic protein was further characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The identified protein (BHUP2) corresponds to a cytochrome c-like synthesis protein of 37 kDa. ELISA results were 94% sensitive, whereas specificities with sera from healthy controls from an area of endemicity, healthy controls not from an area of endemicity, and disease controls were 98%, 100%, and 97%, respectively. The antigen identified via a proteomics-based approach has a strong potential for further development as a diagnostic tool for VL.     

Live nonpathogenic parasitic vector as a candidate vaccine against visceral leishmaniasis

To date, there are no proven vaccines against any form of leishmaniasis. The development of live attenuated vectors shows promise in the field of Leishmania vaccination because these organisms mimic more effectively the course of real infections and can elicit potent activation of the immune system. In the present study, we investigated the potential of a parasitic protozoan that is nonpathogenic to humans, Leishmania tarentolae, as a live candidate vaccine that efficiently targets dendritic cells and lymphoid organs, thus enhancing antigen presentation and consequently influencing the magnitude and quality of T-cell immune responses. We demonstrated that L. tarentolae activates the dendritic cell maturation process and induces T-cell proliferation and the production of gamma interferon, thus skewing CD4(+) T cells toward a Th1 cell phenotype. More importantly, we found that a single intraperitoneal injection of L. tarentolae could elicit a protective immune response against infectious challenge with Leishmania donovani in susceptible BALB/c mice. These results suggest that the use of L. tarentolae as a live vaccine vector may represent a promising approach for improving the effectiveness and safety of candidate live vaccines against Leishmania infections and possibly other intracellular pathogens for which T-cell mediated responses are critical for the development of protective immunity.     

Eco-epidemiology of visceral and cutaneous leishmaniasis in the Yemen Arab Republic. I. Presence, in sympatric condition, of Leishmania infantum and Leishmania donovani complexes

In complement to a previous survey, the authors proceed to the analysis of strains isolated from visceral human and canine leishmaniasis. Finally, among eight human strains isolated and identified with an enzymatic method, seven belong to the Leishmania donovani complex and one to the L. infantum complex. The L. donovani complex is represented by the MON-31 and MON-83 zymodem. The first one is also present in Saudi Arabia and Ethiopia. The second one, corresponding to a small variant, pleads for an intrafocal polymorphism phenomenon which was until now unknown in the L. donovani complex. The L. infantum complex is observed: 1) in sympatria with L. donovani in mountainous areas; 2) alone in the Tihama coastal plain. As for human cutaneous leishmaniasis present in the same focuses it is caused by L. tropica MON-71 and not by the above mentioned complexes.     

Resistance against Leishmania donovani induced with an aluminum hydroxide vaccine

Mice immunized with a subcutaneous protocol combining killed parasites and aluminum hydroxide gel exhibited significant resistance against subsequent challenge with Leishmania donovani promastigotes. Protection was greatest using 25 mg of aluminum hydroxide per injection. Resistance elicited by this killed parasite and aluminum hydroxide protocol was as effective on day 14 as that provided by immunization with a glucan and killed parasite preparation, and more effective in hepatic amastigote reduction at day 28. The effectiveness of aluminum hydroxide as an adjuvant appears to result, at least in part, from its ability to activate macrophages, thus aiding in the elimination of this intracellular parasite.     

Purification of two Leishmania donovani membrane proteins recognized by sera from patients with visceral leishmaniasis

Two Leishmania donovani membrane proteins recognized by sera from patients with visceral leishmaniasis were purified using species-specific monoclonal antibodies and characterized. The molecular weights of the proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were approximately 70,000 and approximately 72,000, respectively. The 70 kDa protein, which appears as a diffuse band on silver staining, was resolved into a doublet by Western blotting with monoclonal antibody. Though of similar molecular weight and amino acid composition, the two proteins were shown to be distinct by peptide mapping and Western blotting of the purified material. The two proteins are recognized specifically by human visceral leishmaniasis serum and not by serum from cutaneous leishmaniasis or Chagas' disease. These proteins will be useful in developing a direct serodiagnostic assay for visceral leishmaniasis.     

Vaccine prospects of killed but metabolically active Leishmania against visceral leishmaniasis

Leishmanization or live vaccination, the gold standard for immunoprophylactic success against cutaneous leishmaniasis, has been abandoned for safety reasons. Killed but metabolically active (KBMA) Leishmania, a new class of whole-cell vaccine, holds promise for safe vaccination. Amotosalen (S-59)-treated and UVA-irradiated Leishmania major and Leishmania infantum chagasi (KBMA-Lic) were rendered replication-incompetent and incapable of causing disease; this was demonstrated convincingly by sensitive techniques. However, the immune response and the level of protection elicited by vaccination with both live Lic and KBMA-Lic in BALB/c mice are less than optimal and warrant further studies to establish their potentiality as an effective vaccine strategy against visceral leishmaniasis.     

In vitro activity and in vivo efficacy of a combination therapy of diminazene and chloroquine against murine visceral leishmaniasis

The present study evaluated the in vitro activity and in vivo efficacy of diminazene combined with chloroquine as a potential drug against Leishmania donovani.Amphotericin B was used as a positive control drug.In vitro activity involved incubation of various drug concentrations with promastigotes or vero cells in culture before determination of parasite growth inhibition or cell death while in vivo evaluations involved infection of various mice groups with virulent L.donovani parasites and treatment with test drug compounds following disease establishment.Weight changes in experimental mice were also evaluated before infection and throughout the experiment.The results indicated that the diminazene-chloroquine combination was at least nine times more efficacious than individual drugs in killing promastigotes in culture.The diminazene-chloroquine combination was safer(Ld₅₀=0.03±0.04) than Amphotericin B(Ld₅₀=0.02±0.01).Body weight in infected mice increased significantly（P=0.0007） from day7 to day 37 following infection（P=0.026）.However,body weight remained comparable in all mice groups during treatment（P=0.16）.The diminazene-chloroquine combination significantly reduced splenic parasite numbers as compared to individual drug therapies（P=0.0001） although Amphotericin B was still more efficacious than any other treatment（P=0.0001）.Amongst the test compounds,the diminazene-chloroquine combination showed the lowest level of IgG antibody responses with results indicating significant negative correlation between antileishmanial antibody responses and protection against disease.These findings demonstrate the positive advantage and the potential use of a combined therapy of diminazene-chloroquine over the constituent drugs.Further evaluation is recommended to determine the most efficacious combination ratio of the two compounds.