Experimental leishmaniasis in humans: review

Experimental infection of humans with Leishmania parasites has contributed significantly to the understanding of the etiology, transmission, and pathogenesis of leishmaniasis and the immunity associated with it. Leishmania organisms recovered from human and animal tissue, insect vectors, and in vitro cultures have all produced cutaneous or visceral leishmaniasis in human subjects who were voluntarily inoculated with them. Volunteers bitten by infected Phlebotomine sandflies also developed cutaneous or visceral disease. In these experiments, it appeared that the parasite must undergo certain developmental changes within the sandfly for it to become infective and that the parasites in sandflies were far more efficient in causing full-blown infection than were cultured Leishmania organisms. The clinical manifestations of these experimental infections did not differ from infections that were acquired naturally. Natural or experimental infections appeared to confer resistance to subsequent leishmanial infection. This immunity was best documented to be a species-specific phenomenon; however, a small number of studies have demonstrated cross protection between some Leishmania species. In this review article, data from human experimental infections are summarized and discussed in light of recent advances in the field.     

Genetically modified live attenuated vaccine: A potential strategy to combat visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by a protozoan parasite Leishmania donovani mainly influencing the population of tropical and subtropical regions across the globe. The arsenal of drugs available is limited, and prolonged use of such drugs makes parasite to become resistant. Therefore, it is very imperative to develop a safe, cost-effective and inexpensive vaccine against VL. Although in recent years, many strategies have been pursued by researchers, so far only some of the vaccine candidates reached for clinical trial and more than half of them are still in pipeline. There is now a broad consent among Leishmania researchers that the perseverance of parasite is very essential for eliciting a protective immune response and may perhaps be attained by live attenuated parasite vaccination. For making a live attenuated parasite, it is very essential to ensure that the parasite is deficient of virulence and should further study genetically modified parasites to perceive the mechanism of pathogenesis. So it is believed that in the near future, a complete understanding of the Leishmania genome will explore clear strategies to discover a novel vaccine. This review describes the need for a genetically modified live attenuated vaccine against VL, and obstacles associated with its development.     

Cross‐protective efficacy from a immunogen firstly identified in Leishmania infantum against tegumentary leishmaniasis

Experimental vaccine candidates have been evaluated to prevent leishmaniasis, but no commercial vaccine has been proved to be effective against more than one parasite species. LiHyT is a Leishmania‐specific protein that was firstly identified as protective against Leishmania infantum. In this study, LiHyT was evaluated as a vaccine to against two Leishmania species causing tegumentary leishmaniasis (TL): Leishmania major and Leishmania braziliensis. BALB/c mice were immunized with rLiHyT plus saponin and lately challenged with promastigotes of the two parasite species. The immune response generated was evaluated before and 10 weeks after infection, as well as the parasite burden at this time after infection. The vaccination induced a Th1 response, which was characterized by the production of IFN‐γ, IL‐12 and GM‐CSF, as well as by high levels of IgG2a antibodies, after in vitro stimulation using both the protein and parasite extracts. After challenge, vaccinated mice showed significant reductions in their infected footpads, as well as in the parasite burden in the tissue and organs evaluated, when compared to the control groups. The anti‐Leishmania Th1 response was maintained after infection, being the IFN‐γ production based mainly on CD4⁺ T cells. We described one conserved Leishmania‐specific protein that could compose a pan‐Leishmania vaccine.     

Leishmania model for microbial virulence: the relevance of parasite multiplication and pathoantigenicity

Leishmanial mechanisms of virulence have been proposed previously to involve two different groups of parasite molecules. One group consists of largely surface and secretory products, and the second group includes intracellular molecules, referred to as 'pathoantigens'. In the first group are invasive/evasive determinants, which protect not only parasites themselves, but also infected host cells from premature cytolysis. These determinants help intracellular amastigotes maintain continuous infection by growing at a slow rate in the parasitophorous vacuoles of host macrophages. This is illustrated in closed in vitro systems, e.g. Leishmania amazonensis in macrophage cell lines. Although individual macrophages may become heavily parasitized at times, massive destruction of macrophages has not been observed to result from uncontrolled parasite replication. This is thus unlikely to be the direct cause of virulence manifested as the clinical symptoms seen in human leishmaniasis. Of relevance is likely the second group of immunopathology-causing parasite 'pathoantigens'. These are highly conserved cytoplasmic proteins, which have been found to contain Leishmania-unique epitopes immunologically active in leishmaniasis. How these intracellular parasite antigens become exposed to the host immune system is accounted for by periodic cytolysis of the parasites during natural infection. This event is notable with a small number of parasites, even as they grow in an infected culture. The cytolysis of these parasites to release 'pathoantigens' may be inadvertent or medicated by specific mechanisms. Information on the pathoantigenic epitopes is limited. T-cell epitopes have long been recognized, albeit ill-defined, as important in eliciting CD4+ cell development along either the Th1 or Th2 pathway. Their operational mechanisms in suppressing or exacerbating cutaneous disease are still under intensive investigation. However, immune response to B-cell epitopes of such 'pathoantigens' is clearly futile and counterproductive. Their intracellular location within the parasites renders them inaccessible to the specific antibodies generated. One example is the Leishmania K39 epitope, against which antibodies are produced in exceedingly high titers, especially in Indian kala-azar. Here, we consider the hypothetical emergence of this pathoantigenicity and its potential contributions to the virulent phenotype in the form of immunopathology. Microbial virulence may be similarly explained in other emerging and re-emerging infectious diseases. Attenuation of microbial virulence may be achieved by genetic elimination of pathoantigenicity, thereby providing mutants potentially useful as avirulent live vaccines for immunoprophylasis of infectious diseases.     

The immunobiology of leishmaniasis

Members of the genus Leishmania are important intracellular pathogens that produce either cutaneous, mucocutaneous, or visceral disease in many areas of the world. In humans as well as in other mammals, the parasite is inoculated through the skin as a flagellated, extracellular promastigote by its arthropod vector, the sandfly. Once in its mammalian host, the promastigote converts to its amastigote stage, which lacks an exteriorized flagellum and is found solely within mononuclear phagocytes during established infection. In vitro, human monocyte-derived macrophages and peritoneal macrophages from several species of rodents can ingest both promastigotes and amastigotes, and they can permit intracellular multiplication of amastigotes only. Although serum factors may play a role in the pathogenesis of the disease and in protection against reinfection, the resolution of leishmaniasis is dependent primarily on cell-mediated immune responses. There appears to be a complicated interplay between cell-mediated helper and suppressor activities. The outcome of infection in each type of leishmaniasis depends on the complex and intriguing interaction of virulence factors inherent in the parasite and genetically determined host defense mechanisms.     

Identification, sequencing and expression of peroxidoxin genes from Leishmania aethiopica

Cutaneous leishmaniasis (CL) is a painful, disfiguring and debilitating disease prevalent in Ethiopia and other countries around the world. In Ethiopia, CL is primarily caused by Leishmania aethiopica and less often by L. tropica and L. major. The intracellular survival mechanisms of Leishmania parasites are still not well understood. Recently a new family of antioxidant enzymes called peroxidoxins have been identified that play an important role in parasite survival. In this study, we have identified two distinct peroxidoxin genes (Pxn1 and Pxn2) that are part of a multi-gene family in L. aethiopica. Protein sequence analysis showed that Pxn1 and Pxn2 are highly homologous to peroxidoxins from other Leishmania species. We have found that L. aethiopica Pxn1 is predominantly expressed in amastigotes and stationary phase promastigotes, whereas Pxn2 is constitutively expressed in the different stages of the parasite. This pattern of RNA expression is consistent with patterns seen in some Leishmania species, but not all. Data from this study will be helpful in enhancing vaccine strategies and drug studies targeted towards peroxidoxins.     

Immunology of canine leishmaniasis

The role of dogs as the main reservoir of visceral leishmaniasis has led to an increased interest in the immune responses and in Leishmania antigens implicated in protective cellular immunity in canine visceral leishmaniasis. The primary goal is to control the prevalence of human disease. Immune responses in canine visceral leishmaniasis are reviewed. Cellular immune responses toward a Th1 subset mediated by IFN-gamma and TNF-alpha predominate in asymptomatic dogs exhibiting apparent resistance to visceral leishmaniasis. On the other hand, while the role of Th2 cytokines, such as IL-4 and IL-10, in symptomatic animals is still controversial, there is increasing evidence for a correlation of these cytokines with progressive disease. CD8+ cytotoxic T cells seem also likely to be involved in resistance to visceral leishmaniasis. Several Leishmania antigens implicated in protective immune responses are described and some pivotal points for development of an effective vaccine against canine visceral leishmaniasis are discussed.     

Refinement of techniques for the propagation of Leishmania donovani in hamsters

Improved animal models are urgently required for drug and vaccine development against visceral leishmaniasis. Here we report refinements to the hamster model of infection that reduce the severity of the disease as well as the number of animals required to maintain infection while improving parasite yields. A comparison between infection via the intracardiac and intraperitoneal routes showed that the less commonly used intraperitoneal route is the simpler and preferred method. The KAtex latex agglutination test for visceral leishmaniasis accurately detected Leishmania donovani antigen in hamster urine as early as 6 weeks post-inoculation. With modification, this assay could be an important tool in the evaluation of experimental drugs and vaccines.     

Recent developments in Leishmaniasis: Epidemiology,diagnosis, and treatment

The outbreaks of cutaneous disease caused by Leishmania tropica in Afghan refugees, visceral disease in Sudanese refugees, and cutaneous disease caused by Leishmania major in American forces in Iraq are examples of the large number of cases of leishmaniasis that can result when naive human populations intrude into regions where transmission is endemic. Injections of pentavalent antimony for 20 to 30 days have been the standard treatment for all forms of leishmaniasis, but resistance is growing and antimonials have moderate toxicity. Two major advances in the treatment of visceral leishmaniasis have been made in the past few years. Liposomal amphotericin B cures virtually all patients, with little side effects. Miltefosine is the first oral agent that is effective. For cutaneous disease, alternatives to antimony have been effective in certain regions but have not yet been generally evaluated.     

Characterization of Leishmania infantum thiol-dependent reductase 1 and evaluation of its potential to induce immune protection

The need to develop an effective vaccine against leishmaniasis to prevent the 2 million new cases each year led to the search for antigens able to elicit protection against infection with Leishmania. In this study, we have characterized a parasite-specific protein of Leishmania infantum named thiol-dependent reductase 1 (TDR1). The protein is present in both life cycle stages of L. infantum with a notable higher expression in the amastigote forms, suggesting a role in the interaction between the parasite and the mammalian host. Thiol-dependent reductase 1 is localized in the cytosol, although we were able to detect the protein in the culture medium of both promastigotes and axenic amastigotes, and consequently, TDR1 is considered an excreted/secreted molecule of the parasite. Therefore, we have evaluated the potential of TDR1 recombinant protein to protect against experimental challenge with L. infantum parasites using a murine model. Despite a reduction in spleen parasite load in the chronic phase of disease, TDR1 administration was not effective in the protection of Balb/c mice against visceral leishmaniasis and thus TDR1 do not have a crucial role in the modulation of mammalian host immune response, as observed with its protein counterpart Tc52 of Trypanosoma cruzi.     

The microcirculation in severe malaria

Severe malaria in humans and animals is initiated by interactions between malaria-infected cells, host blood cells (including monocytes, T cells and platelets) and endothelial cells of the microcirculation. Adhesion to vascular cells, and possible vascular obstruction in severe human disease, involves interaction between host receptors and parasite-derived proteins, such as the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Our understanding of how different PfEMP1 variants may target infected erythrocytes to specific sites, such as the placenta, is rapidly increasing. However, in most instances downstream immune-mediated inflammatory processes appear more central than parasite accumulation to development of severe malaria. Using genetically-manipulated animal models of severe malaria, key roles for CD8 T cells and mediators such as lymphotoxin in the pathogenesis of murine disease have been established. Experimental and human studies suggest vascular deposition of activated platelets may have a central role. Here, we review some recent advances in the understanding of severe malaria pathogenesis from human and animal studies, focusing on events at the level of the microcirculation, and highlight the role for activated host cells in initiating the pathology of the disease.     

Cutaneous leishmaniasis in Eastern Saudi Arabia. Epidemiological and clinical features in a nonimmune population living in an endemic area

Cutaneous leishmaniasis is endemic in Saudi-Arabia. Nonimmune Europeans and their families living temporarily in this area were affected by the disease in great number, resembling an epidemic outbreak. Clinical features and treatment of the disease, problems of re-infection and immunity, as well as epidemiological factors are discussed. Using biochemical methods the parasite was identified as Leishmania tropical major.     

CD8 cytotoxic T cells in cutaneous leishmaniasis

CD8 T cells are essential in the defence against viruses, yet little is known of their participation in the host defence against parasites, such as Leishmania, which can cause a variety of clinical diseases, such as localized cutaneous, diffuse cutaneous, mucocutaneous and visceral leishmaniasis. Murine models of leishmaniasis suggest that CD8 T cells participate through IFN-gamma production, yet their cytotoxic capacity also plays an important role, as has been found in patients infected with various Leishmania strains, where CD8 T cell cytotoxicity and apoptosis of autologous Leishmania-infected macrophages correlate with cure. Yet the mechanisms underlying the CD8 T activation in patients with leishmaniasis remain an enigma. It is possible that dendritic cells activate CD8 T cells through mechanisms that include antigen cross-presentation. Here we summarize the recent findings of CD8 T cells in cutaneous leishmaniasis and discuss their significance in the control of the disease. Further knowledge in this field will undoubtedly improve the design of therapeutic and vaccine strategies.     

Evaluation of In Vitro Production of IFN-gamma, IL-10, IL-12 and IL-13 by Blood Cells in Patients with Cutaneous Leishmaniasis Lesions

This study investigated the in vitro production of interferon-gamma, interleukin (IL)-10, IL-12, and IL-13, after antigenic stimulation of the cells (with Leishmania antigen and lipopolysaccharide) using whole blood from patients with cutaneous leishmaniasis lesions caused by Leishmania tropica and in normal volunteers with history of cutaneous leishmaniasis.ELISA results showed that the mean production of interferon-gamma by cells of whole blood in patients with lesions in response to Leishmania antigen was significantly lower than corresponding values in volunteers with history of cutaneous leishmaniasis (P< 0.05) and significantly higher levels of IL-10 production in patients with lesions were observed compared with cured volunteers of the disease (P<0.01). A similar level of IL-12, including p40 subunit of IL-12, was detected in both groups tested in this study in response to stimulation of parasite antigen. The levels of the IL-13 after stimulation with Leishmania antigen were significantly more in patients compared with volunteers with history of cutaneous leishmaniasis (P< 0.01). There was no significant difference in the mean production of IFN-gamma, IL-10, IL-12 and IL-13 by PHA or LPS stimulated cells from patients with lesions and volunteers with history of the disease, indicating that there was no qualitative defect in cytokine production in these patients.In this study, we have detected the decreased production of interferon- gamma by cells of patients with lesions of cutaneous leishmaniasis in response to parasite antigen and unbalanced production of regulatory cytokines such as IL-10 and IL-13 using the whole-blood stimulation assay technique. The required small volume of blood and the rapid set up time are the advantages in this assay technique. Using this assay for further immunodetection of cytokines may confirm its value for clinical investigation.     

DNA vaccine against visceral leishmaniasis: a promising approach for prevention and control

The visceral leishmaniasis (VL) caused by Leishmania donovani parasite severely affects large populations in tropical and subtropical regions of the world. The arsenal of drugs available is limited, and resistance is common in clinical field isolates. Therefore, vaccines could be an important alternative for prevention against VL. Recently, some investigators advocated the protective efficacy of DNA vaccines, which induces the T cell‐based immunity against VL. The vaccine antigens are selected as conserved in various Leishmania species and provide a viable strategy for DNA vaccine development. Our understanding for DNA vaccine development against VL is not enough and much technological advancement is required. Improved formulations and methods of delivery are required, which increase the uptake of DNA vaccine by cells; optimization of vaccine vectors/encoded antigens to augment and direct the host immune response in VL. Despite the many genes identified as vaccine candidates, the disappointing potency of the DNA vaccines in VL underscores the challenges encountered in the efforts to translate efficacy in preclinical models into clinical realities. This review will provide a brief background of DNA vaccines including the insights gained about the design, strategy, safety issues, varied candidates, progress and challenges that play a role in their ability against VL.     

Treatment of two patients with diffuse cutaneous leishmaniasis caused by Leishmania mexicana modifies the immunohistological profile but not the disease outcome

Two patients with diffuse cutaneous leishmaniasis caused by Leishmania mexicana were treated with two leishmanicidal drugs (pentamidine and allopurinol) combined with recombinant interferon-gamma restoring Th-1 favouring conditions in the patients. Parasites decreased dramatically in the lesions and macrophages diminished concomitantly, while IL-12-producing Langerhans cells and interferon-gamma- producing NK and CD8 + lymphocytes increased in a reciprocal manner. The CD4+/CD8 + ratio in the peripheral blood normalized. During exogenous administration of interferon-gamma the parasites' capacity to inhibit the oxidative burst of the patients' monocytes was abolished. Even though Th-1-favouring conditions were restored, both patients relapsed two months after therapy was discontinued. We conclude that the tendency to develop a disease-promoting Th-2 response in DCL patients is unaffected by, and independent of, parasite numbers. Even though intensive treatment in DCL patients induced Th-1 disease restricting conditions, the disease-promoting immunomodulation of few persistent Leishmania sufficed to revert the immune response.     

Advances in the immunopathogenesis of pulmonary tuberculosis

Tuberculosis remains a global emergency because of our lack of understanding of the details of its pathogenesis. In the last 12 months there have been striking advances in the molecular genetics of the organism. Mutated strains of Mycobacterium tuberculosis have been used to study the genetic requirements for virulence and establishment of latency, and the biology of the interaction with host cells. Genes involved in lipid metabolism seem particularly important. The probable sites of latency within the host lungs have been identified by in situ polymerase chain reaction. The complex control by M. tuberculosis of apoptosis of T cells and macrophages has been somewhat clarified, and the data may suggest that M. tuberculosis causes death of a subset of T cells, while preserving some macrophages as hiding places with reduced microbicidal and antigen-presenting function. Similarly the demonstration of a very large relative increase in interleukin (IL)-4 and IL-13 expression, (together with IL-4delta2, the IL-4 splice variant), that correlates with lung damage, has been supported by data from flow cytometry and in situ hybridization, and indicates that a subversive T helper-2 (Th2) component in the response to M. tuberculosis may undermine the efficacy of immunity and contribute to immunopathology. Recently defined changes in metabolism of cortisol within the lesions may contribute to the development of the Th2 component. These findings underline the need to start testing vaccine candidates in models that mimic the situations in which bacille Calmette-Guerin fails, such as in the presence of latent infection, pre-existing Th2 responses to cross-reactive organisms, and stress.     

Antibodies to human herpesvirus 8 in POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes) syndrome with multicentric Castleman's disease.

We describe a 15-month-old eutrophic immunocompetent male who presented with fever, hepatosplenomegaly, pancytopenia, and hypergammaglobulinemia. Leishmania amastigotes were identified in spleen and bone marrow specimens. In addition, tissue culture, animal inoculation, and isoenzyme analysis identified the parasite as Leishmania donovani infantum or Leishmania donovani chagasi. The infant was successfully treated with an antimonial drug. These findings represent the first case of visceral leishmaniasis reported in Costa Rica.     

Therapeutic vaccination with recombinant adenovirus reduces splenic parasite burden in experimental visceral leishmaniasis

Therapeutic vaccines, when used alone or in combination therapy with antileishmanial drugs, may have an important place in the control of a variety of forms of human leishmaniasis. Here, we describe the development of an adenovirus-based vaccine (Ad5-KH) comprising a synthetic haspb gene linked to a kmp11 gene via a viral 2A sequence. In nonvaccinated Leishmania donovani-infected BALB/c mice, HASPB- and KMP11-specific CD8(+) T cell responses were undetectable, although IgG1 and IgG2a antibodies were evident. After therapeutic vaccination, antibody responses were boosted, and IFNγ(+)CD8(+) T cell responses, particularly to HASPB, became apparent. A single vaccination with Ad5-KH inhibited splenic parasite growth by ～66%, a level of efficacy comparable to that observed in early stage testing of clinically approved antileishmanial drugs in this model. These studies indicate the usefulness of adenoviral vectors to deliver leishmanial antigens in a potent and host protective manner to animals with existing L. donovani infection.     

Studies on cocktails of 31‐kDa, 36‐kDa and 51‐kDa antigens of Leishmania donovani along with saponin against murine visceral leishmaniasis

A substantial number of antigens of Leishmania donovani have been described in the past. However, identifying candidate antigens is not enough. Appropriate antigen delivery to induce the right type of immune response against leishmaniasis (i.e. induction of a strong antigen‐specific Th1 type of immune response) is another crucial component of an effective vaccine. Therefore, ‘cocktail’ vaccines are proposed based on the assumption that such cocktails will show enhanced efficacy. Studies have been carried out on LD31 and LD51 polypeptides from L. donovani promastigotes, which have proven to be potential vaccine candidates. This study was designed to check the protective efficacy of various cocktails of low molecular weight antigens alone and along with saponin as adjuvant. Mice were sacrificed on different post‐challenge days for evaluation of parasite load and other immunological parameters. Protective efficacy of different vaccine formulations was revealed by significant decline in parasite burden and increased DTH Delayed Type Hypersenstivity responses. The antibody response was of IgG type with elevated IgG2a and decreased production of IgG1, whereas cytokine levels pointed towards the generation of protective Th1 type of immune response. Among all vaccine formulations, cocktail of 31+51+saponin was found to be highly immunogenic and imparted maximum protection.