Leishmaniasis Vaccine Research Articles

The development and clinical evaluation of second-generation leishmaniasis vaccines

Infection with Leishmania parasites results in a range of clinical manifestations and outcomes. Control of Leishmania parasite transmission is extremely difficult due to the large number of vectors and potential reservoirs, and none of the current treatments are ideal. Vaccination could be an effective strategy to provide sustained control. In this review, the current global situation with regard to leishmaniasis, the immunology of Leishmania infection and various efforts to identify second generation vaccine candidates are briefly discussed. The variety of clinical trials conducted using the only current second generation vaccine approved for clinical use, LEISH-F1+MPL-SE, are described. Given that epidemiological evidence suggests that reducing the canine reservoir also positively impacts human incidence, efforts at providing a vaccine for leishmaniasis in dogs are highlighted. Finally, potential refinements and surrogate markers that could expedite the introduction of a vaccine that can limit the severity and incidence of leishmaniasis are discussed.

C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

BackgroundWe have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects.Methodology/Principal FindingsThree pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b-CTE, pcDNA-cpa/b-CTE, cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b-CTE showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies.Conclusions/SignificanceThis report demonstrates cSLN's ability to boost immune response magnitude of cpa/cpb-CTE cocktail vaccination against leishmaniasis so that the average parasite inhibition percent could be increased significantly. Hence, cSLNs can be considered as suitable adjuvant and/or delivery systems for designing third generation cocktail vaccines.

Substrate Preferences and Catalytic Parameters Determined by Structural Characteristics of Sterol 14α-Demethylase (CYP51) from Leishmania infantum

Leishmaniasis is a major health problem that affects populations of ∼90 countries worldwide, with no vaccine and only a few moderately effective drugs. Here we report the structure/function characterization of sterol 14α-demethylase (CYP51) from Leishmania infantum. The enzyme catalyzes removal of the 14α-methyl group from sterol precursors. The reaction is essential for membrane biogenesis and therefore has great potential to become a target for antileishmanial chemotherapy. Although L. infantum CYP51 prefers C4-monomethylated sterol substrates such as C4-norlanosterol and obtusifoliol (V(max) of ∼10 and 8 min(-1), respectively), it is also found to 14α-demethylate C4-dimethylated lanosterol (V(max) = 0.9 min(-1)) and C4-desmethylated 14α-methylzymosterol (V(max) = 1.9 min(-1)). Binding parameters with six sterols were tested, with K(d) values ranging from 0.25 to 1.4 μM. Thus, L. infantum CYP51 is the first example of a plant-like sterol 14α-demethylase, where requirements toward the composition of the C4 atom substituents are not strict, indicative of possible branching in the postsqualene portion of sterol biosynthesis in the parasite. Comparative analysis of three CYP51 substrate binding cavities (Trypanosoma brucei, Trypanosoma cruzi, and L. infantum) suggests that substrate preferences of plant- and fungal-like protozoan CYP51s largely depend on the differences in the enzyme active site topology. These minor structural differences are also likely to underlie CYP51 catalytic rates and drug susceptibility and can be used to design potent and specific inhibitors.

KSAC, the First Defined Polyprotein Vaccine Candidate for Visceral Leishmaniasis

A subunit vaccine using a defined antigen(s) may be one effective solution for controlling leishmaniasis. Because of genetic diversity in target populations, including both dogs and humans, a multiple-antigen vaccine will likely be essential. However, the cost of a vaccine to be used in developing countries must be considered. We describe herein a multiantigen vaccine candidate comprised of antigens known to be protective in animal models, including dogs, and to be recognized by humans immune to visceral leishmaniasis. The polyprotein (KSAC) formulated with monophosphoryl lipid A, a widely used adjuvant in human vaccines, was found to be immunogenic and capable of inducing protection against Leishmania infantum, responsible for human and canine visceral leishmaniasis, and against L. major, responsible for cutaneous leishmaniasis. The results demonstrate the feasibility of producing a practical, cost-effective leishmaniasis vaccine capable of protecting both humans and dogs against multiple Leishmania species.

Vaccines for the leishmaniases: proposals for a research agenda.

The International Symposium on Leishmaniasis Vaccines, held in Olinda, Brazil, on March 9–11, 2009, congregated international experts who conduct research on vaccines against the leishmaniases. The questions that were raised during that meeting and the ensuing discussions are compiled in this report and may assist in guiding a research agenda. A group to further discussion on issues raised in this policy platform has been set up at http://groups.google.com/group/leishvaccines-l.

Novel strategy using live non-pathogenic Leishmania expressing selected parasite antigens as a candidate vaccine for leishmaniasis

Parasites of the genus Leishmania are intracellular protozoa which are transmitted to their mammalian host by the bite of infected sand flies and cause a group of diseases known as Leishmaniasis. Despite attempting different vaccination strategies, no human vaccine is yet available against this disease. There is increasing evidence that presence of a small number of live parasites is necessary to maintain durable immunity, and the only way to meet this requirement is by using attenuated live vaccines [1]. The main obstacle about attenuated live strains is the risk of reversion of the organism to its virulent state. Another approach to reach this strategy is to use nonpathogenic Leishmania such as L. tarentolae. This parasite is lizard parasite and has never been found associated with any leishmaniasis in humans and is considered as non-pathogenic to humans. Previous studies have shown that L. tarentolae can be used as a live vaccine against L. donovani and elicit a protective Th1 immune response [2]. Recently, by comparative genomic analysis and expression profiles of well-characterized virulence factors such as GP63, CPB, LPG3, Amastin and A2 between pathogenic Leishmania species (e.g. L. major, L. infantum and L. braziliensis) and non pathogenic L. tarentolae revealed that only A2 is absent at the level of DNA [3]. Ar ecombinantL. tarentolae expressing the A2 protein was generated and its potential as a live vaccine against L. infantum infection in BALB/c mice was examined [4]. The A2 expressing recombinant parasites showed higher macrophage infectivity in comparison to L. tarentolae used as a control. Immunization (i.v. and i.p.) of BALB/c mice with recombinant L. tarentolae A2 elicited a strong protective immunity against virulent L. infantum challenge, manifested by a dramatic decrease in parasite burdens in the liver and the spleen of immunized mice. IFN-g production upon antigen stimulation indicated that protection is associated with a Th1 cell-mediated immunity accompanied by reduced levels of IL-5 production (the Th2 type response). Interestingly, although IFN-g production is also induced in groups of mice immunized with wild type L. tarentolae, cytokine levels are increased in the group immunized with the recombinant L. tarentolae A2 and especially when the vaccine regimen is administered via the i.p. route [4]. In continuation of these promising results, we are expanded this idea against L. major infection as a novel vaccine regimen by including two immunogenic parasite proteins (cysteine proteinases A and B, CPA/CPB).

Carbohydrate Molecules: An Expanding Horizon in Drug Delivery and Biomedicine

This review presents successful applications of carbohydrate molecules in drug delivery, vaccine development, cancer, HIV and various other diseases based on advances in glycobiology and glycochemistry. Carbohydrate-mediated delivery could be site specific/cell specific. Carbohydrate-based delivery system has been successfully utilized for the delivery of macromolecular drugs, antigen, and potential therapeutic drug candidates. Lectin, the high affinity carbohydrate-binding nonimmune glycoproteins has specific and noncovalent binding sites for defined carbohydrates. Endogenous surface lectins of cancer cells participate in the regulation of tumor cell growth. The oligosaccharides constitute potential recognition sites for carbohydrate-mediated interactions between cells and drug carriers bearing suitable site directing molecules. The recognition of carbohydrate immunodeterminants has created great attention in the development of carbohydrate-based vaccines. Peptide mimotopes provide a strategy to augment human immunodeficiency virus 1 (HIV-1) specific carbohydrate reactive immune responses. Experimental cancer and HIV vaccines are being developed in attempts to overcome weak immunological responses to carbohydrate-rich surface antigens using carriers, adjuvants, and novel carbohydrate antigen constructs. Current carbohydrate-based vaccines are used for prostate cancer, typhus, pneumonia, and meningitis; vaccines for malaria, anthrax, and leishmaniasis are under development. This article discusses the current research involved in the role of carbohydrate molecules in targeted controlled drug delivery, immunology, and vaccine development.

Modifying Culture Media for Production of Plasmid DNA for Leishmaniasis Vaccine

Modifying Culture Media for Production of Plasmid DNA for Leishmaniasis Vaccine

Leishmaniasis vaccines: past, present and future

No vaccine exists against any form of leishmaniasis. Because recovery from infection is usually accompanied by a strong immunity and because it is possible to protect experimental animals against live challenge, hope for the development of a vaccine for humans has been high. However, leishmaniasis is a disease of the poor and the market for a vaccine is very limited. Until a few years ago, with minimal resources, only a pragmatic approach was possible for testing the first-generation vaccines (i.e. killed whole parasites). Recently, funding has become available for developing defined second-generation vaccines, including recombinant proteins and DNA constructs. With new adjuvants also being developed there is new hope, and several new vaccines are in development against leishmaniasis.

Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

Kinetoplastid membrane protein-11 (KMP-11), a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles). More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.

Evaluation of Ex Vivo Human Immune Response against Candidate Antigens for a Visceral Leishmaniasis Vaccine

People cured from visceral leishmaniasis (VL) develop protection mediated by Th1-type cellular responses against new infections. We evaluated cytokine responses against 6 defined candidate vaccine antigens in 15 cured VL subjects and 5 healthy endemic controls with no evidence of previous exposure to Leishmania parasites. Of the 6 cytokines examined, only interferon-gamma (IFN-gamma) differentiated cured VL patients from non-exposed individuals, with cured patients mounting a significantly higher IFN-gamma response to a crude parasite antigen preparation. Among candidate vaccine antigens tested, the largest number of cured subjects recognized cysteine proteinase B, leading to heightened IFN-gamma responses, followed by sterol 24-c-methyltransferase. These two antigens were the most immunogenic and protective antigens in a murine VL model, indicating a relationship between T cell recall responses of humans cured from VL and protective efficacy in an experimental model. Further studies may help prioritize antigens for clinical development of a subunit vaccine against VL.

Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis

For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

A current perspective on leishmaniasis

There are many challenges facing the successful control and eradication of cutaneous and visceral leishmaniasis. Leishmaniasis is still endemic in many poverty stricken and war torn areas. Through the use of an extensive literature review, this article examined the global disease burden of cutaneous and visceral leishmaniasis. Surveillance and control measures for leishmaniasis being used by the World Health Organization were also discussed in this article. Finally, potential new treatments and possible vaccines for leishmaniasis were reviewed in this article.

Leishmaniasis vaccine: Where are we today?

Leishmaniasis is a disease that ranges in severity from skin lesions to serious disfigurement and fatal systemic infection. WHO has classified the disease as emerging and uncontrolled and estimates that the infection results in two million new cases a year. There are 12 million people currently infected worldwide, and leishmaniasis threatens 350 million people in 88 countries. Current treatment is based on chemotherapy, which relies on a handful of drugs with serious limitations such as high cost, toxicity, difficult route of administration and lack of efficacy in endemic areas. Vaccination remains the best hope for control of all forms of the disease, and the development of a safe, effective and affordable antileishmanial vaccine is a critical global public-health priority. Extensive evidence from studies in animal models indicates that solid protection can be achieved by immunization with defined subunit vaccines or live-attenuated strains of Leishmania. However, to date, no such vaccine is available despite substantial efforts by many laboratories. The major impediment in vaccine design is the translation of data from animal models to human disease, and the transition from the laboratory to the field. Furthermore, a thorough understanding of protective immune responses and generation and maintenance of the immunological memory, the most important and least-studied aspect of antiparasitic vaccine development, during Leishmania infection is needed. This review focuses on recent findings in antileishmania vaccine field and highlights current difficulties facing vaccine development and implementation.

Safety and immunogenicity of a defined vaccine for the prevention of cutaneous leishmaniasis

Healthy Colombian adult volunteers with no history of leishmaniasis were evaluated for evidence of previous subclinical infection with Leishmania based on the Montenegro skin test (MST). Twelve MST-positive subjects were enrolled in an open-label, uncontrolled clinical trial (the “MST-positive trial”) and received three injections of the LEISH-F1 + MPL-SE vaccine (consisting of 10 μg recombinant Leishmania polyprotein LEISH-F1 antigen [TSA + LmSTI1 + LeIF] + 25 μg MPL ®-SE adjuvant). Sixty-eight MST-negative subjects were enrolled in a randomized, double-blind, controlled trial (the “MST-negative trial”) and were randomly assigned to receive three injections of either the vaccine ( n = 34), 10 μg LEISH-F1 protein alone ( n = 17), or saline placebo ( n = 17). In both trials, the study injections were given subcutaneously on Days 0, 28, and 56, and subjects were followed for safety and immunological endpoints. The LEISH-F1 + MPL-SE vaccine was safe and well tolerated in MST-positive and MST-negative subjects. In both trials, an IFN-γ response to the LEISH-F1 antigen at Day 84 was observed in more than half of the vaccine recipients. In the MST-negative trial, the IFN-γ response was significantly more frequent and of greater magnitude in vaccine recipients than in protein-alone or placebo recipients. An IgG antibody response to LEISH-F1 was observed in all vaccine recipients. In both trials, delayed-type hypersensitivity (DTH) to LEISH-F1 was observed in most of the vaccine recipients. In the MST-negative trial, DTH was significantly higher in vaccine than placebo recipients. These clinical trials of the first defined vaccine for leishmaniasis show that the LEISH-F1 + MPL-SE vaccine is safe and immunogenic in healthy subjects with and without evidence of previous subclinical infection with Leishmania.

Vaccination of dogs with six different candidate leishmaniasis vaccines composed of a chimerical recombinant protein containing ribosomal and histone protein epitopes in combination with different adjuvants

Chimerical protein “Q”, composed of antigenic ribosomal and histone sequences, in combination with live BCG is a promising canine leishmaniasis vaccine candidate; one of the few vaccine candidates that have been tested successfully in dogs. Unfortunately, live BCG is not an appropriate adjuvant for commercial application due to safety problems in dogs. In order to find a safe adjuvant with similar efficacy to live BCG, muramyl dipeptide, aluminium hydroxide, Matrix C and killed Propionibacterium acnes in combination with either E. coli- or baculovirus-produced recombinant JPCM5_Q protein were tested. Groups of five or seven dogs were vaccinated with six different adjuvant–antigen combinations and challenged with a high dose intravenous injection of Leishmania infantum JPC strain promastigotes. All candidate vaccines proved to be safe, and both humoral and cellular responses to the recombinant proteins were detected at the end of the prime-boost vaccination scheme. However, clinical and parasitological data obtained during the 10 month follow-up period indicated that protection was not induced by either of the six candidate vaccines. Although no direct evidence was obtained, our data suggest that live BCG may have a significant protective effect against challenge with L. infantum in dogs.

Aid Workers Face More Complex Hurdles

tious Disease Research Institute in Seattle, Wash, worked with the Corixa Corporation, now GlaxoSmithKline, to develop the first candidate vaccine for leishmaniasis, which sickens about 2 million a year, including 500 000 who contract the potentially fatal visceral form. Phase 1 clinical trials have been completed in Peru and Brazil; additional trials are under way in India, Sudan, and Venezuela. TheDNDI is anotherexampleof such partnerships.WithinternationalpharmaceuticalmakerSanofi-AventisandtheBraziliangovernment instituteFarmanguinhos, now Fiocruz, the DNDI in the last 2 years has ushered in 2 new artesunate combination treatments for malaria. With field support from MSF, the DNDI also supported clinical trials on the eflornithine-nifurtimox combination for sleeping sickness. Pecoul said the DNDI is beginning a phase 1 clinical trial of the antifungal fexinidazole, an oral treatment. “We need to have a very simple treatment to change the dynamic of control of sleeping sickness,” he noted. In addition, the DNDI is working on development of a pediatric formulation of benznidazole for Chagas disease and ways to combine existing medications for more effective treatment of visceral leishmaniasis. “The next step for us is to continue to strengthen the portfolio and make progress,” said Pecoul.

IgG1, but not IgG2a/c, is pathogenic in Leishmania mexicana infection (133.18)

Abstract FcγRs and IL-10 are both required for chronic disease caused by L. mexicana infection of C57BL/6 mice. We recently showed that IgG1 and IgG2a/c can both induce IL-10 from macrophages and dendritic cells when bound to Leishmania amastigotes, but through different FcγR types. IgG1 induces IL-10 through FcγRIII, whereas IgG2a/c induces IL-10 primarily through FcγRI. Given that FcγRIII KO mice are resistant to L. mexicana infection and because IgG1 responses occur much earlier than IgG2a/c responses, we examined whether IgG1 is specifically required for chronic L. mexicana disease. In fact, mice lacking IgG1 are more resistant to L. mexicana infection. Furthermore, we found that IgG1 KO mice had an earlier and stronger IgG2a/c response to L. mexicana infection. This demonstrates that IgG1 is pathogenic and that IgG2a/c is not, and may in fact be protective. Thus IgG2a/c does not appear to induce a suppressive IL-10 response in vivo. This opens up the question of whether macrophages are the source of suppressive IL-10 responsible for chronic disease in vivo, or whether another cell type is responsible. In addition it has important implications for vaccines for leishmaniasis and potentially diseases caused by other pathogens that share this FcγR/IL-10 mechanism, as vaccines must be engineered to minimize IgG1 responses to amastigote surface epitopes. Funded by VA Merit and the University of Pennsylvania.

Histological study of cell migration in the dermis of hamsters after immunisation with two different vaccines against visceral leishmaniasis

Vaccine candidates, including live and/or killed parasites, Leishmania-purified fractions, defined recombinant antigens and antigen-encoding DNA-plasmids have been proposed to use as vaccine anti- Leishmania. More recently, the hamsters have been used to pre-selection of antigens candidate to apply in further experiments using canine model. In this report we evaluated the kinetics of cell migration in dermal inflammatory infiltrate, circulating leukocytes and the presence of nitric oxide (NO)/induced nitric oxide synthase during the early (1–24 h) and late (48–168 h) periods following inoculation of hamsters with antigenic components of anti-canine visceral leishmaniasis vaccines Leishmune ® and Leishmania braziliensis antigen (LB) with and without saponin (Sap) adjuvant. Our results show that LB caused an early reduction of lymphocytes in the dermis while Sap and LBSap triggered a late recruitment, suggesting the role of the adjuvant in the traffic of antigen-presenting cells and the induction of lymphocyte migration. In that manner our results suggest that the kinetics of cell migration on hamster model may be of value in the selection of vaccine antigens prior the tests in dogs particularly in respect of the toxicity of the preparations.

UDP-Gal: N-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

As compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis (VL) has received limited attention. In this study, we demonstrate for the first time that an UDP-Galactose: N-acetylglucosamine beta 1-4 galactosyltransferase (GenBank Accession No. EF159943) expressing attenuated LD clonal population (A-LD) is able to confer protection against the experimental challenge with the virulent LD AG83 parasite. A-LD was also effective in established leishmania infection. The vaccinated animals showed both cell mediated (in vitro T-cell proliferation, and DTH response) and humoral responses (Th1 type). These results demonstrate the potential of the attenuated clones as an immunotherapeutic and immunoprophylactic agent against visceral leishmaniasis.