Field trial of efficacy of the Leish-tec® vaccine against canine leishmaniasis caused by Leishmania infantum in an endemic area with high transmission rates.

BACKGROUND: Because domestic dogs are reservoir hosts for visceral leishmaniasis (VL) in Brazil, one of the approaches used to reduce human disease incidence is to cull infected dogs. However, the results of controlled intervention trials based on serological screening of dogs and killing of seropositive animals are equivocal. A prophylactic vaccine to protect dogs from being infectious to the sand fly vector could be an effective strategy to provide sustained control. Here, we investigated whether a currently licensed commercial subunit rA2 protein-saponin vaccine (Leish-tec®) had an additional effect to dog culling on reducing the canine infectious populations. METHODOLOGY/PRINCIPAL FINDINGS: This prospective study was conducted in an L. infantum highly endemic area of southeast Brazil. At the onset of the intervention, all of the eligible dogs received through subcutaneous route a three-dose vaccine course at 21-day intervals and a booster on month 12. For the purpose of comparison, newly recruited healthy dogs were included as the exposed control group. To ascertain vaccine-induced protection, dogs were screened on clinical and serological criteria every 6 months for a 2-year follow-up period. Antibody-based tests and histopathological examination of post-mortem tissue specimens from euthanized animals were used as a marker of infection. The standardized vaccine regime, apart from being safe, was immunogenic as immunized animals responded with a pronounced production of anti-A2-specific IgG antibodies. It should be noted the mean seroconversion time for infection obtained among immunized exposed dogs (~ 18 months), which was twice as high as that for unvaccinated ones (~ 9 months). After two transmission cycles completed, the cumulative incidence of infection did differ significantly (P = 0.016) between the vaccinated (27%) and unvaccinated (42%) dogs. However, the expected efficacy for the vaccine in inducing clinical protection was not evident since 43% of vaccine recipients developed disease over time. Our estimates also indicated that immunoprophylaxis by Leish-tec® vaccine in addition to dog culling might not have an impact on bringing down the incidence of canine infection with L. infantum in areas of high transmission rates. CONCLUSIONS/SIGNIFICANCE: Leish-tec® as a prophylactic vaccine showed promise but needs to be further optimized to be effective in dogs under field conditions, and thereby positively impacts human incidence.

Multi-antigen print immunoassay (MAPIA)-based evaluation of novel recombinant Leishmania infantum antigens for the serodiagnosis of canine visceral leishmaniasis.

BACKGROUND: Domestic dogs are the principal reservoir hosts of Leishmania infantum in regions where visceral leishmaniasis (VL) is endemic. Although serologic methods are frequently used for the screening of infected dogs, antibody-based tests require further assessment, due to lack of sensitivity and specificity. In this study, we employed a multi-antigen printing immunoassay (MAPIA) to compare the antibody responses to novel recombinant proteins of L. infantum with the potential for the detection of canine VL. FINDINGS: MAPIA strips were prepared employing 12 recombinant proteins. Antibody reactivity to these antigens was compared using a panel of sera collected from clinically asymptomatic (n = 16) and symptomatic (n = 41) culture-positive animals. Our findings showed that the canine immune response to antigen differs between dogs and depends on infection status. Using this screening assay, when five out of the 12 antigens were combined, an overall 81% detection rate of L. infantum-infected dogs was achieved. CONCLUSIONS: We conclude that MAPIA is an effective screening tool to rapidly select multiple antigens of diagnostic utility to be used in a more sensitive point of care diagnostic test such as the Dual-Path Platform (DPP) multiplex test for the rapid detection of infected dogs.

Clinical and parasitological protection in a Leishmania infantum-macaque model vaccinated with adenovirus and the recombinant A2 antigen.

BACKGROUND: Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs. METHODOLOGY/PRINCIPAL FINDINGS: Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. CONCLUSIONS/SIGNIFICANCE: The remarkable clinical protection induced by A2 in an animal model that is evolutionary close to humans qualifies this antigen as a suitable vaccine candidate against human VL.

Antibody responses induced by Leish-Tec®, an A2-based vaccine for visceral leishmaniasis, in a heterogeneous canine population.

Zoonotic visceral leishmaniasis (VL) is a widespread disease, and dogs are the main reservoirs for human parasite transmission. Hence, development of an effective vaccine that prevents disease and reduces the transmission of VL is required. As euthanasia of seropositive dogs is recommended in Brazil for VL epidemiological control, to include anti-VL canine vaccines as a mass control measure it is necessary to characterize the humoral responses induced by vaccination and if they interfere with the reactivity of vaccinated dogs in serological diagnostic tests. Leish-Tec(®) is an amastigote-specific A2 recombinant protein vaccine against canine visceral leishmaniasis (CVL) that is commercially available in Brazil. Here, we tested the immunogenicity of Leish-Tec(®) in a heterogeneous dog population by measuring A2-specific antibody responses. Healthy dogs (n=140) of various breeds were allocated to two groups: one group received Leish-Tec(®) (n=70), and the other group received a placebo (n=70). Anti-A2 or anti-Leishmania promastigote antigen (LPA) antibody levels were measured by ELISA in serum samples collected before and after vaccination. An immunochromatographic test (DPP) based on the recombinant K28 antigen was also used for serodiagnosis of CVL. Vaccinated animals, except one, remained seronegative for anti-LPA total IgG and anti-K28 antibodies. Conversely, seropositivity for anti-A2 total IgG antibodies was found in 98% of animals after vaccination. This value decreased to 81.13% at 6 months before rising again (98%), after the vaccination boost. Anti-A2 IgG2 and IgG1 titers were also increased in vaccinated animals relative to control animals. These data indicate that Leish-Tec(®) is immunogenic for dogs of different genetic backgrounds and that humoral responses induced by vaccination can be detected by A2-ELISA, but do not interfere with the LPA-ELISA and DPP diagnostic tests for CVL.

Ecological and epidemiological aspects of the sand fly (Diptera, Psychodidae) fauna of the National Monument of Pontões Capixabas, State of Espírito Santo, Southeastern Brazil.

We evaluated the ecological and epidemiological aspects of the sand fly fauna in an area of the Atlantic Forest biome with records of visceral and cutaneous leishmaniasis. Sand fly collections at three different localities at the National Monument of Pontões Capixabas, State of Espírito Santo, Southeastern Brazil, were conducted by using two Centers of Disease Control and Prevention automatic light traps in the peridomiciliary environment and eight Centers of Disease Control and Prevention automatic light traps in the forested environment. Collections occurred during four consecutive nights within each of the months and locations: São Luiz (December 2009, May 2010, July 2010, and December 2010), Córrego Palmital de Baixo (September 2010 and October 2010), and Córrego São Bento (February 2011 and May 2011). We collected 21,138 sand flies belonging to 31 species and 14 genera. Of this total, 12,412 sand flies were captured in the peridomiciliary environment and 8,726 in the forested environment. All of the vector species, Lutzomyia longipalpis (=Lutzomyia longipalpis, sensu; Young and Duncan), Migonemyia migonei (=Lutzomyia migonei, sensu; Young and Duncan), and Nyssomyia intermedia (=Lutzomyia intermedia, sensu; Young and Duncan), occurred in significantly higher numbers in the peridomiciliary environment than compared with the forested environment. Our results highlight the importance of conservation in the forest remains of the National Monument of Pontões Capixabas, because of higher species richness and diversity. Furthermore, they indicate the epidemiological role of Lu. longipalpis as the vector of Leishmania infantum within the study area, and the no evident role of Mg. migonei.

The effect of removing potentially infectious dogs on the numbers of canine Leishmania infantum infections in an endemic area with high transmission rates.

To assess the effect of the rapid removal of potentially infectious dogs on the prevalence and incidence of canine infections, a prospective study was undertaken in an area endemic for Leishmania infantum. We used serological testing based on the rapid DPP rK28 fusion protein chromatographic immunoassay for this dog screening-and-culling intervention trial. The outcome was evaluated by measuring seropositivity and sero-conversion/-reversion rates for canine infection. Our estimates indicated that concomitant detection and elimination of seropositive dogs with active disease may affect the numbers of canine infections and disease burden temporarily, although it is insufficient as a measure to interrupt the zoonotic L. infantum transmission. However, most of the asymptomatic, seropositive dogs continuously exhibit low levels of antibodies and/or reverted, remaining seronegative thereafter. In the process of waiting for an effective vaccine, one option for canine reservoir control may be to identify these possibly genetically resistant animals and promote their expansion in the population.

Making an anti-amastigote vaccine for visceral leishmaniasis: rational, update and perspectives.

Visceral leishmaniasis is a major health problem in Latina America, as well as the Mediterranean region of Europe and Asia. We aimed to develop a vaccine against visceral leishmaniasis targeting the intracellular amastigotes, which is the parasite stage that persists throughout infections with Leishmania parasites. With this in mind, we identified an amastigote specific antigen (A2) that contains an immunogenic epitope for CD4+ T helper (Th) cells and multiple repetitive units encoding CD8+ cytotoxic T lymphocyte (CTL) epitopes. Vaccine formulations containing the recombinant A2 associated with saponin, alum and IL-12 or expressed by attenuated adenovirus were shown to be protective in mice, dogs and nonhuman-primates. We are currently identifying novel amastigote specific immunogenic proteins that could be aggregated to A2 to further improve the level of vaccine-induced cell-mediated immunity and protection against visceral leishmaniasis.

Improved canine and human visceral leishmaniasis immunodiagnosis using combinations of synthetic peptides in enzyme-linked immunosorbent assay.

BACKGROUND: Zoonotic visceral leishmaniasis (VL) is a severe infectious disease caused by protozoan parasites of the genus Leishmania and the domestic dogs are the main urban parasite reservoir hosts. In Brazil, indirect fluorescence antibody tests (IFAT) and indirect enzyme linked immunosorbent assay (ELISA) using promastigote extracts are widely used in epidemiological surveys. However, their sensitivity and specificity have often been compromised by the use of complex mixtures of antigens, which reduces their accuracy allowing the maintenance of infected animals that favors transmission to humans. In this context, the use of combinations of defined peptides appears favorable. Therefore, they were tested by combinations of five peptides derived from the previously described Leishmania diagnostic antigens A2, NH, LACK and K39. METHODOLOGY/PRINCIPAL FINDINGS: Combinations of peptides derived A2, NH, LACK and K39 antigens were used in ELISA with sera from 44 human patients and 106 dogs. Improved sensitivities and specificities, close to 100%, were obtained for both sera of patients and dogs. Moreover, high sensitivity and specificity were observed even for canine sera presenting low IFAT anti-Leishmania antibody titers or from asymptomatic animals. CONCLUSIONS/SIGNIFICANCE: The use of combinations of B cell predicted synthetic peptides derived from antigens A2, NH, LACK and K39 may provide an alternative for improved sensitivities and specificities for immunodiagnostic assays of VL.

Evaluation of a novel chromatographic immunoassay based on Dual-Path Platform technology (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is the major source of human visceral leishmaniasis (VL) and is transmitted from dogs to sand flies to humans. To control the spread of this disease, early and accurate detection of infected dogs is critical but challenging. Here we demonstrate the potential of the Dual-Path Platform (DPP(®)) CVL rapid test for detecting K26/K39-reactive antibodies in sera from clinically symptomatic (n=60) and asymptomatic (n=60) Leishmania infantum-infected dogs. For the specificity evaluation, assays were performed using known negative diagnostic serum samples (n=59) and cross-reaction control sera (n=11) from animals born in a VL-free area of Brazil. The diagnostic kit displayed high specificity (96%) but low sensitivity (47%) in identifying parasite-positive dogs without signs of CVL. However, the test sensitivity was significantly higher (98%) in diseased cases, indicating that this convenient test may be useful to identify the most infectious dogs. Efforts should be pursued to obtain a more sensitive DPP-multiplexed test parameter (i.e. based on simultaneous yet separate antibody detection of carefully selected multiple antigens of diagnostic utility) for effective serodiagnosis of early-infected dogs, as this will likely allow more efficient canine removal regimens than those used in practice by public health services.

Disposition of antimony in rhesus monkeys infected with Leishmania braziliensis and treated with meglumine antimoniate.

Antimony (Sb) disposition and toxicity was evaluated in Leishmania braziliensis-infected monkeys (Macaca mulatta) treated with a 21-d course of low (LOW) or standard (STD) meglumine antimoniate (MA) dosage regimens (5 or 20 mg Sb(V)/kg body weight/d im). Antimony levels in biological matrices were determined by inductively coupled plasma mass spectrometry (ICPMS), while on-line ion chromatography coupled to ICPMS was used to separate and quantify Sb species in plasma. Nadir Sb levels rose steadily from 19.6 ± 4 and 65.1 ± 17.4 ng/g, 24 h after the first injection, up to 27.4 ± 5.8 and 95.7 ± 6.6 ng/g, 24 h after the 21st dose in LOW and SDT groups, respectively. Subsequently, Sb plasma levels gradually declined with a terminal elimination phase half-life of 35.8 d. Antimony speciation in plasma on posttreatment days 1-9 indicated that as total Sb levels declined, proportion of Sb(V) remained nearly constant (11-20%), while proportion of Sb(III) rose from 5% (d 1) to 50% (d 9). Plasma [Sb]/erythrocyte [Sb] ratio was >1 until 12 h after dosing and reversed thereafter. Tissue Sb concentrations (posttreatment days 55 and 95) were as follows: >1000 ng/g in thyroid, nails, liver, gall bladder and spleen; >200 and <1000 ng/g in lymph nodes, kidneys, adrenals, bones, skeletal muscles, heart and skin; and <200 ng/g in various brain structures, thymus, stomach, colon, pancreas. and teeth. Results from this study are therefore consistent with view that Sb(V) is reduced to Sb(III), the active form, within cells from where it is slowly eliminated. Localization of Sb active forms in the thyroid gland and liver and the pathophysiological consequences of marked Sb accumulation in these tissues warrant further studies.

High-throughput analysis of synthetic peptides for the immunodiagnosis of canine visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis. METHODOLOGY/PRINCIPAL FINDINGS: Immunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs. CONCLUSIONS/SIGNIFICANCE: Our study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.

Possible implication of the genetic composition of the Lutzomyia longipalpis (Diptera: Psychodidae) populations in the epidemiology of the visceral leishmaniasis.

Lutzomyia longipalpis (Diptera: Psychodidae) is the principal vector of American visceral leishmaniasis. Several studies have indicated that the Lu. longipalpis population structure is complex. It has been suggested that genetic divergence caused by genetic drift, selection, or both may affect the vectorial capacity of Lu. longipalpis. However, it remains unclear whether genetic differences among Lu. longipalpis populations are directly implicated in the transmission features of visceral leishmaniasis. We evaluated the genetic composition and the patterns of genetic differentiation among Lu. longipalpis populations collected from regions with different patterns of transmission of visceral leishmaniasis by analyzing the sequence variation in the mitochondrial cytochrome b gene. Furthermore, we investigated the temporal distribution of haplotypes and compared our results with those obtained in a previous study. Our data indicate that there are differences in the haplotype composition and that there has been significant differentiation between the analyzed populations. Our results reveal that measures used to control visceral leishmaniasis might have influenced the genetic composition of the vector population. This finding raises important questions concerning the epidemiology of visceral leishmaniasis, because these differences in the genetic structures among populations of Lu. longipalpis may have implications with respect to their efficiency as vectors for visceral leishmaniasis.

In situ characterization of the granulomatous immune response with time in nonhealing lesional skin of Leishmania braziliensis-infected rhesus macaques (Macaca mulatta).

We have recently introduced a macaque (Macaca mulatta) model of Leishmania braziliensis-induced self-healing cutaneous leishmaniasis in which the T cell-mediated inflammatory response effectively promotes parasite clearance and granuloma resolution. Here we show that macaques infected with a highly pathogenic L. braziliensis strain displayed longstanding granulomatous lesions which lasted until the end of the observation period (52 weeks). Immunoperoxidase staining of representative tissue sections indicated that distinct cell populations (CD3, CD4, CD8, CD20, Foxp3, CD20, CD68, HLA-DR, CCL2, and CXCL-10) change uniformly during infection, suggesting that the same components of the local immune response are working in unison. This model also confirmed that granuloma formation is orchestrated by diverse inflammatory mediators that are important for T helper type 1 (Th1) cell development and macrophage effector functions. Cytometry analysis of ex vivo granuloma-derived leukocytes revealed accumulation of distinct functional subsets of effector and regulatory T cells into the inflamed skin. We provide evidence that local interleukin (IL)-10 production by both Foxp3(+) and Foxp3(-) CD4(+) T subsets is likely important in promoting lesional granuloma maintenance. Further studying the immune suppression mechanisms that induces granulomas in L. braziliensis-infected macaques may reveal new opportunities for therapeutic control of this important human disease.

American visceral leishmaniasis dissociated from Lutzomyia longipalpis (Diptera, Psychodidae) in the State of Espírito Santo, Brazil.

The occurrence of American visceral leishmaniasis in the State of Espírito Santo, Brazil has always been associated with the presence of the Lutzomyia longipalpis vector. The geographic distribution of this vector in this state is related to the presence of specific geoclimatic characteristics, such as a dry climate, low elevations (< 450 m), steep slopes and rocky outcrops. The occurrence of human autochthonous cases of American visceral leishmaniasis in municipalities without these geoclimatic characteristics justifies the present study and our main goal was to test the association between the occurrence of American visceral leishmaniasis and the presence of the Lu. longipalpis. Sand flies were captured monthly from July 2006 to August 2007 using Shannon and CDC traps in two municipalities with records of autochthonous American visceral leishmaniasis and one with no record. We captured 13,112 sand flies, but no Lu. longipalpis was found. The absence of Lu. longipalpis and the possible role of another American visceral leishmaniasis vector in these localities were discussed.

Survey of natural infection by Leishmania in sand fly species collected in southeastern Brazil.

In this study, we sought to identify sand fly vectors of the Leishmania species that circulate in distinct eco-epidemiological disease-endemic rural areas within the Espírito Santo State in southeastern Brazil. PCR amplification of a conserved region of the minicircle kDNA was used to estimate infection rates in field-captured, peridomestic female sand flies. Only 13 of the 1689 female sand fly specimens (0.77%) actually contained Leishmania DNA. Leishmania braziliensis infections were found in Lutzomyia intermedia and Lu. whitmani, and, for the first time, in Lu. fischeri and Lu. ferreirana. Interestingly, the high rate of genetic polymorphism of the L. braziliensis parasites in one of the disease-endemic areas that were studied may reflect specific transmission cycles involving different sand fly vectors.

Cross-sectional and longitudinal epidemiologic surveys of human and canine Leishmania infantum visceral infections in an endemic rural area of southeast Brazil (Pancas, Espirito Santo).

In an endemic rural area of southeast Brazil, surveys confirmed that dogs serve as peridomestic reservoirs of Leishmania infantum. It is likely that the lack of efficient control is because presently used diagnostic tests miss positive dogs. Overall, 57% of the dogs had specific antibodies, but the canine infection was not uniformly fatal and many seropositive dogs remained asymptomatic or even spontaneously recovered. Furthermore, 42% of the human residents became leishmanin-positive reactors and 47% had positive serology at the initial survey, but our estimates also point at a high recovery rate among the infected population with time. The delayed-type hypersensitivity (DTH) reaction to Leishmania was a good indicator of resistance to infection in this particular epidemiologic situation. The lack of any significant differences in infection rates by gender or age indicate that all of the population was at an equal risk of infection and most people were infected in the peridomestic setting.

The utility of rhesus monkey (Macaca mulatta) and other non-human primate models for preclinical testing of Leishmania candidate vaccines.

Leishmaniasis causes significant morbidity and mortality, constituting an important global health problem for which there are few effective drugs. Given the urgent need to identify a safe and effective Leishmania vaccine to help prevent the two million new cases of human leishmaniasis worldwide each year, all reasonable efforts to achieve this goal should be made. This includes the use of animal models that are as close to leishmanial infection in humans as is practical and feasible. Old world monkey species (macaques, baboons, mandrills etc.) have the closest evolutionary relatedness to humans among the approachable animal models. The Asian rhesus macaques (Macaca mulatta) are quite susceptible to leishmanial infection, develop a human-like disease, exhibit antibodies to Leishmania and parasite-specific T-cell mediated immune responses both in vivo and in vitro, and can be protected effectively by vaccination. Results from macaque vaccine studies could also prove useful in guiding the design of human vaccine trials. This review summarizes our current knowledge on this topic and proposes potential approaches that may result in the more effective use of the macaque model to maximize its potential to help the development of an effective vaccine for human leishmaniasis.