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1.  Humoral and In Vivo Cellular Immunity against the Raw Insect-Derived Recombinant Leishmania infantum Antigens KMPII, TRYP, LACK, and papLe22 in Dogs from an Endemic Area 
Leishmania infantum causes visceral leishmaniasis, a severe zoonotic and systemic disease that is fatal if left untreated. Identification of the antigens involved in Leishmania-specific protective immune response is a research priority for the development of effective control measures. For this purpose, we evaluated, in 27 dogs from an enzootic zone, specific humoral and cellular immune response by delayed-type hypersensitivity (DTH) skin test both against total L. infantum antigen and the raw Trichoplusia ni insect-derived kinetoplastid membrane protein-11 (rKMPII), tryparedoxin peroxidase (rTRYP), Leishmania homologue of receptors for activated C kinase (rLACK), and 22-kDa potentially aggravating protein of Leishmania (rpapLe22) antigens from this parasite. rTRYP induced the highest number of positive DTH responses (55% of leishmanin skin test [LST]-positive dogs), showing that TRYP antigen is an important T cell immunogen, and it could be a promising vaccine candidate against this disease. When TRYP-DTH and KMPII-DTH tests were evaluated in parallel, 82% of LST-positive dogs were detected, suggesting that both antigens could be considered as components of a standardized DTH immunodiagnostic tool for dogs.
doi:10.4269/ajtmh.2010.09-0784
PMCID: PMC2990046  PMID: 21118936
2.  Use of a Recombinant Cysteine Proteinase from Leishmania (Leishmania) infantum chagasi for the Immunotherapy of Canine Visceral Leishmaniasis 
Background
A recombinant cysteine proteinase from Leishmania (Leishmania) infantum chagasi (rLdccys1) was previously shown to induce protective immune responses against murine and canine visceral leishmaniasis. These findings encouraged us to use rLdccys1 in the immunotherapy of naturally infected dogs from Teresina, Piauí, a region of high incidence of visceral leishmaniasis in Brazil.
Methodology/Principal Findings
Thirty naturally infected mongrel dogs displaying clinical signs of visceral leishmaniasis were randomly divided in three groups: one group received three doses of rLdccys1 in combination with the adjuvant Propionibacterium acnes at one month interval between each dose; a second group received three doses of P. acnes alone; a third group received saline. The main findings were: 1) dogs that received rLdccys1 with P. acnes did not display increase of the following clinical signs: weight loss, alopecia, onychogryphosis, cachexia, anorexia, apathy, skin lesions, hyperkeratosis, ocular secretion, and enlarged lymph nodes; they also exhibited a significant reduction in the spleen parasite load in comparison to the control dogs; 2) rLdccys1-treated dogs exhibited a significant delayed type cutaneous hypersensitivity elicited by the recombinant antigen, as well as high IgG2 serum titers and low IgG1 serum titers; sera from rLdccys1-treated dogs also contained high IFN-γ and low IL-10 concentrations; 3) control dogs exhibited all of the clinical signs of visceral leishmaniasis and had low serum IgG2 and IFN-γ levels and high concentrations of IgG1 and IL-10; 4) all of the dogs treated with rLdccys1 were alive 12 months after treatment, whereas dogs which received either saline or P. acnes alone died within 3 to 7 months.
Conclusions/Significance
These findings illustrate the potential use of rLdccys1 as an additional tool for the immunotherapy of canine visceral leishmaniasis and support further studies designed to improve the efficacy of this recombinant antigen for the treatment of this neglected disease.
Author Summary
Visceral leishmaniasis (VL) is an important public health problem and dogs are the main domestic reservoirs of zoonotic VL which has resulted in an annual incidence of 40,100–75,500 new human cases. Because canine VL chemotherapy is limited by the low efficacy of drugs currently used for human VL treatment, immunotherapy may provide a viable alternative. We used a recombinant cysteine proteinase from L. (L.) infantum chagasi, rLdccys1, in combination with the adjuvant P. acnes for the treatment of naturally infected mongrel dogs from Teresina, Pauí a state in Brazil that has a high incidence of VL. Dogs treated with rLdccys1 showed a significant delayed type hypersensitivity reaction against the recombinant antigen and displayed high serum concentrations of IgG2 and IFN-γ and low concentrations of IgG1 and IL-10. Immunotherapy with rLdccys1 resulted in no increase of the clinical signs of canine VL and an extensive reduction of spleen parasite load. Furthermore, all of the dogs treated with rLdccys1 survived for at least 12 months after treatment, whereas those that received either saline or P. acnes alone died within 3 to 7 months. These findings support the potential of rLdccys1 immunotherapy as an additional option for the treatment of canine VL.
doi:10.1371/journal.pntd.0002729
PMCID: PMC3953064  PMID: 24625516
3.  Canine Antibody Response to Phlebotomus perniciosus Bites Negatively Correlates with the Risk of Leishmania infantum Transmission 
Background
Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species.
Methodology/Principal Findings
Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins.
Conclusions
Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.
Author Summary
Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis in the Mediterranean Basin and Phlebotomus perniciosus serve as the major vector. In the endemic foci, Leishmania parasites are transmitted mostly to dogs, the main reservoir host, and to humans. We studied the canine humoral immune response to Phlebotomus perniciosus saliva and its potential use as a marker of sand fly exposure and consequently as a risk marker for Leishmania transmission. We also characterized major salivary antigens of P. perniciosus. We demonstrated that under laboratory conditions, the levels of anti-P. perniciosus saliva antibodies positively correlated with the number of blood-fed sand flies and therefore, may be used to evaluate the need for, and the effectiveness of, anti-vector campaigns. In parallel, we studied sera of dogs naturally exposed to P. perniciosus in highly active focus of canine leishmaniasis in Southern Italy. Specific antibodies against P. perniciosus saliva were significantly increased according to the ongoing sand fly season. Moreover, the levels of anti-P. perniciosus antibodies in naturally bitten dogs negatively correlated with anti-Leishmania seropositivity. Thus, for dogs living in endemic areas, specific antibody response against saliva of the vector is an important marker for estimating the risk of Leishmania transmission.
doi:10.1371/journal.pntd.0001344
PMCID: PMC3191129  PMID: 22022626
4.  Antigenicity and Protective Efficacy of a Leishmania Amastigote-specific Protein, Member of the Super-oxygenase Family, against Visceral Leishmaniasis 
Background
The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL.
Methodology/Principal Findings
The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed.
Conclusions/Significance
The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL.
Author Summary
Life-long immunity to leishmaniasis in recovered patients has inspired the development of vaccines against disease. The present study aimed to evaluate a non-described hypothetical Leishmania amastigote-specific protein, identified by an immunoproteomic approach in L. infantum, attempting to select a new candidate antigen for specific serodiagnosis and a vaccine against visceral leishmaniasis (VL). The recombinant protein (rLiHyp1) was recognized by antibodies from sera of asymptomatic and symptomatic canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of vaccinated dogs, those with Chagas' disease or healthy animals. In addition, the rLiHyp1 plus saponin was able to induce a Th1 response, which was based on the production of high levels of IFN-γ, IL-12, and GM-CSF after in vitro stimulation in BALB/c mice. The protective efficacy of rLiHyp1 plus saponin was evaluated in mice challenged with L. infantum promastigotes. Challenged and vaccinated mice showed significant reductions in the number of parasites in all evaluated organs, and the protection was associated with a Th1-type response. Therefore, the present study reveals a new potential candidate for the improvement of serodiagnosis of CVL, as well as an effective vaccine candidate against VL.
doi:10.1371/journal.pntd.0002148
PMCID: PMC3610918  PMID: 23573301
5.  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.
Author Summary
Globally, the number of new human cases of visceral leishmaniasis (VL) is estimated to be approximately 500,000 per year. This is the most severe of all forms of leishmaniasis, and the zoonotic form of VL, caused by Leishmania infantum (also known as Leishmania chagasi), represents 20% of human visceral leishmaniasis worldwide; additionally, its prevalence is increasing in urban and peri-urban areas of the tropics. In Brazil, the identification and elimination of infected dogs, which act as a reservoir for Leishmania parasites, is a control measure employed in addition to the use of insecticides against the vectors and the identification and treatment of infected humans. Currently, the diagnostic methods employed to identify infected animals are not able to detect all of these dogs, which compromises the effectiveness of control measures. Moreover, one of the most important issues in controlling VL is the difficulty of diagnosing asymptomatic dogs, which act as parasite reservoirs. Therefore, to contribute to the improvement of the diagnostic methods for CVL, we aimed to identify and characterize new antigens that were more sensitive and specific and could be applied in epidemiologic surveys.
doi:10.1371/journal.pntd.0001310
PMCID: PMC3172188  PMID: 21931874
6.  Head-to-Head Comparison of Three Vaccination Strategies Based on DNA and Raw Insect-Derived Recombinant Proteins against Leishmania 
PLoS ONE  2012;7(12):e51181.
Parasitic diseases plague billions of people among the poorest, killing millions annually, and causing additional millions of disability-adjusted life years lost. Leishmaniases affect more than 12 million people, with over 350 million people at risk. There is an urgent need for efficacious and cheap vaccines and treatments against visceral leishmaniasis (VL), its most severe form. Several vaccination strategies have been proposed but to date no head-to-head comparison was undertaken to assess which is the best in a clinical model of the disease. We simultaneously assayed three vaccination strategies against VL in the hamster model, using KMPII, TRYP, LACK, and PAPLE22 vaccine candidate antigens. Four groups of hamsters were immunized using the following approaches: 1) raw extracts of baculovirus-infected Trichoplusia ni larvae expressing individually one of the four recombinant proteins (PROT); 2) naked pVAX1 plasmids carrying the four genes individually (DNA); 3) a heterologous prime-boost (HPB) strategy involving DNA followed by PROT (DNA-PROT); and 4) a Control including empty pVAX1 plasmid followed by raw extract of wild-type baculovirus-infected T. ni larvae. Hamsters were challenged with L. infantum promastigotes and maintained for 20 weeks. While PROT vaccine was not protective, DNA vaccination achieved protection in spleen. Only DNA-PROT vaccination induced significant NO production by macrophages, accompanied by a significant parasitological protection in spleen and blood. Thus, the DNA-PROT strategy elicits strong immune responses and high parasitological protection in the clinical model of VL, better than its corresponding naked DNA or protein versions. Furthermore, we show that naked DNA coupled with raw recombinant proteins produced in insect larvae biofactories –the cheapest way of producing DNA-PROT vaccines– is a practical and cost-effective way for potential “off the shelf” supplying vaccines at very low prices for the protection against leishmaniases, and possibly against other parasitic diseases affecting the poorest of the poor.
doi:10.1371/journal.pone.0051181
PMCID: PMC3517401  PMID: 23236448
7.  Epidemiologic Aspects of an Emerging Focus of Visceral Leishmaniasis in Tbilisi, Georgia 
Background
Over the last 15 years, visceral leishmaniasis (VL) has emerged as a public health concern in Tbilisi, the capital of Georgia.
Methodology/Principal Findings
Seroepidemiological surveys were conducted to determine the prevalence and incidence of infection in children and dogs within the main focus of VL, and to identify risk factors associated with human infection. Of 4,250 children investigated, 7.3% were positive by direct agglutination test in a baseline survey; an apparent incidence rate of 6.0% was estimated by one year follow-up. None of the seropositive children progressed to VL during the survey. Increased seropositivity at one year was predicted by presence at baseline of clustered flying insects (OR = 1.49; P = 0.001), perceived satisfactory sanitation (OR = 1.65; P<0.001), stray dogs (OR = 1.33; P = 0.023), and by persistent fever during the 6 months prior to baseline survey (OR = 14.2; P<0.001). Overall, 18.2% (107/588) of domestic and 15.3% (110/718) of stray dogs were seropositive by the rk39 dipstick test. Clinical VL signs were found in 1.3% of domestic and 2.9% of stray, seropositive dogs. Parasites isolated from human and dog samples were identified by PCR and phylogenetic analysis of the Leishmania 70 kDa heat-shock protein (HSP70) gene as Leishmania infantum.
Conclusions/Significance
There is an active focus of L. infantum transmission in Tbilisi with a high prevalence of human and canine infections.
Author Summary
Visceral leishmaniasis (VL) has emerged as a public health problem in Tbilisi, the capital of Georgia. Dogs are the main infection reservoirs for transmission by sand flies of Leishmania infantum to humans, many of whom may become infected without developing disease. Since majority of cases are in children we were interested to know the rate of infection in children and in dogs living within the area where cases of VL have been found, and what factors may affect the risk of infection. Using a test that detects the presence of antibodies in blood as a marker of infection, 7.3% of 4,250 children examined were positive at the baseline survey, and 6% became positive after one year. Overall, 18.2% of domestic and 15.3% of stray dogs were seropositive. The infected children were more apt to live in areas where clustered flying insects and stray dogs were observed, and were far more likely to have experienced a persistent fever in the 6 months before the survey. We conclude that there is very active transmission of L. infantum to both humans and dogs in Tbilisi, and that children remain at high risk of developing clinical disease and sub-clinical infection.
doi:10.1371/journal.pntd.0001415
PMCID: PMC3236723  PMID: 22180796
8.  Recombinant Antigens from Phlebotomus perniciosus Saliva as Markers of Canine Exposure to Visceral Leishmaniases Vector 
Background
Phlebotomus perniciosus is the main vector in the western Mediterranean area of the protozoan parasite Leishmania infantum, the causative agent of canine and human visceral leishmaniases. Infected dogs serve as a reservoir of the disease, and therefore measuring the exposure of dogs to sand fly bites is important for estimating the risk of L. infantum transmission. In bitten hosts, sand fly saliva elicits a specific antibody response that reflects the intensity of sand fly exposure. As screening of specific anti-saliva antibodies is limited by the availability of salivary gland homogenates, utilization of recombinant salivary proteins is a promising alternative. In this manuscript we show for the first time the use of recombinant salivary proteins as a functional tool for detecting P. perniciosus bites in dogs.
Methodology/Principal Findings
The reactivity of six bacterially-expressed recombinant salivary proteins of P. perniciosus, yellow-related protein rSP03B, apyrases rSP01B and rSP01, antigen 5-related rSP07, ParSP25-like protein rSP08 and D7-related protein rSP04, were tested with sera of mice and dogs experimentally bitten by this sand fly using immunoblots and ELISA. In the immunoblots, both mice and canine sera gave positive reactions with yellow-related protein, both apyrases and ParSP25-like protein. A similar reaction for recombinant salivary proteins was observed by ELISA, with the reactivity of yellow-related protein and apyrases significantly correlated with the antibody response of mice and dogs against the whole salivary gland homogenate.
Conclusions/Significance
Three recombinant salivary antigens of P. perniciosus, yellow-related protein rSP03B and the apyrases rSP01B and rSP01, were identified as the best candidates for evaluating the exposure of mice and dogs to P. perniciosus bites. Utilization of these proteins, or their combination, would be beneficial for screening canine sera in endemic areas of visceral leishmaniases for vector exposure and for estimating the risk of L. infantum transmission in dogs.
Author Summary
The protozoan parasite Leishmania infantum is a causative agent of zoonotic visceral leishmaniasis, an important and potentially fatal human disease. The main reservoir hosts of this Leishmania species are dogs, and the only proven vectors are phlebotominae sand flies, Phlebotomus perniciosus being considered the major vector in the western Mediterranean area. During feeding on the host, sand flies spit saliva into the host skin; hosts develop a specific antibody response directed against sand fly salivary proteins and levels of these antibodies reflect the intensity of sand fly exposure. As the availability of salivary gland homogenate is limited, recombinant salivary proteins have been suggested as antigens suitable for measuring specific antibody levels. In the present work, we expressed six of the most-antigenic salivary proteins, and studied the mice and canine humoral immune responses to these recombinant proteins. We demonstrated that three proteins, a yellow-related protein and two apyrases, are suitable antigens for measuring anti-P. perniciosus antibody levels and estimating the host exposure to this sand fly species.
doi:10.1371/journal.pntd.0002597
PMCID: PMC3879210  PMID: 24392167
9.  Leishmania infantum Ecto-Nucleoside Triphosphate Diphosphohydrolase-2 is an Apyrase Involved in Macrophage Infection and Expressed in Infected Dogs 
Background
Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection.
Methodology/Principal Findings
We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis.
Conclusions/Significance
In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of NTPDases led to lower levels of in vitro adhesion and infection, suggesting that these proteins are possible targets for rational drug design.
Author Summary
Visceral leishmaniasis is a dangerous and important, but neglected, tropical disease that affects millions of people, mainly in underdeveloped and developing countries. Presently, there are no vaccines against Leishmaniasis, and the few drugs with which the disease is treated have low efficacy and high side effects. The pathogenic agent of this disease in the New World is Leishmania infantum chagasi. In this work, we studied a protein from this parasite named ENTPDase-2. We expressed it in a bacterial system, purified it and characterized it as a genuine nucleotidase of the ENTPDase family. This protein seems to be localized at the surface of the parasite and in other intracellular locations. ENTPDase seems to facilitate in vitro infection because its blockade leads to lower levels of infection of macrophages. In addition, the protein is found in naturally infected dogs. A previous study demonstrated that ENTPDase-2 from L. infantum chagasi is a good antigen for immunodiagnosis of canine visceral leishmaniasis. We have now studied this protein in greater depth and suggest that it may be a good target for drug development.
doi:10.1371/journal.pntd.0003309
PMCID: PMC4230930  PMID: 25393008
10.  Development of a Fluorescent Based Immunosensor for the Serodiagnosis of Canine Leishmaniasis Combining Immunomagnetic Separation and Flow Cytometry 
Background
An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce.
Methodology
Ni-NTA magnetic microspheres with 1,7 µm and 8,07 µm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry.
Principal Findings
A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%).
Conclusions
Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis.
Author Summary
Dogs are the most important domestic reservoirs of the parasite Leishmania infantum. Some infected animals develop a subclinical infection, without the classical symptoms characteristics of this disease. One of the major challenges in the serodiagnosis of canine leishmaniasis is the detection of actively infected animals that are already able to transmit the parasite to the vector, despite the fact they did not yet show external signs of the disease. In the present work, we have developed a new tool for the laboratorial diagnosis of canine leishmaniasis that clearly improves the performance of canine leishmaniasis serodiagnosis. An immunofluorescence assay was developed combining Leishmania recombinant protein-coated magnetic microspheres and flow cytometry. The antigen-coated microspheres were used to separate anti-Leishmania specific antibodies present in the serum of infected dogs. Flow cytometry allowed the specific quantification of the antibodies. The clinical evaluation of a panel of serum samples from natural infected dogs clearly demonstrates that this method detects with high specificity and sensitivity both clinical and subclinical forms of the disease.
doi:10.1371/journal.pntd.0002371
PMCID: PMC3749986  PMID: 23991232
11.  Gentamicin-Attenuated Leishmania infantum Vaccine: Protection of Dogs against Canine Visceral Leishmaniosis in Endemic Area of Southeast of Iran 
An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A vaccine trial was conducted using 103 naive dogs from a leishmaniosis non-endemic area (55 vaccinated and 48 unvaccinated) brought into an endemic area of southeast Iran. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination. The efficacy of the vaccine was evaluated after 24 months (4 sandfly transmission seasons) by serological, parasitological analyses and clinical examination. In western blot analysis of antibodies to L. infantum antigens, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs which were seropositive at >100, recognized the 21 kDa antigen of L. infantum wild-type (WT). Nine out of 31 (29%) unvaccinated dogs, but none of vaccinated dogs, were positive for the presence of Leishmania DNA. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. These results suggest that gentamicin-attenuated L. infantum induced a significant and strong protective effect against canine visceral leishmaniosis in the endemic area.
Author Summary
A 24 month vaccine trial was conducted using 103 leishmania free dogs in an area of southeast Iran endemic for visceral leishmaniosis. The dogs were vaccinated with gentamicin-attenuated line of Leishmania infantum. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination with an attenuated line of Leishmania infantum. Nine out of 31 (29%) unvaccinated dogs, but none of those vaccinated, were positive for presence of Leishmania DNA by PCR. In western blots, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs, recognized the 21 kDa antigen of Leishmania infantum wild-type. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. The attenuated Leishmania infantum induced a significant and strong protective effect against Leishmania infantum infection in the field.
doi:10.1371/journal.pntd.0002757
PMCID: PMC3990512  PMID: 24743691
12.  Improving Serodiagnosis of Human and Canine Leishmaniasis with Recombinant Leishmania braziliensis Cathepsin L-like Protein and a Synthetic Peptide Containing Its Linear B-cell Epitope 
Background
The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.
Methodology/Principal Findings
We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.
Conclusions/Significance
The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.
Author Summary
Leishmaniasis is one of the major diseases of importance in public health and its precise diagnosis may represent one of the most relevant challenges for the control and possible eradication of the disease. In this context, recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like (CatL) genes are more abundant in stationary promastigotes and amastigotes, and have less than 40% identity with human proteins and more than 60% identity with other Leishmania species. We mapped a linear B-cell epitope in the CatL protein sequence and compared its performance with the recombinant protein and current serology methodologies for the diagnosis of human tegumentary and visceral leishmaniasis as well as of canine visceral leishmaniasis (CVL). Both the recombinant protein and synthetic peptide showed higher specificity and sensitivity than crude preparations commonly used for other antigens, and thus, they are valuable targets to compose an antigen panel that could significantly improve leishmaniasis diagnosis.
doi:10.1371/journal.pntd.0003426
PMCID: PMC4287388  PMID: 25569432
13.  A prime/boost DNA/Modified vaccinia virus Ankara vaccine expressing recombinant Leishmania DNA encoding TRYP is safe and immunogenic in outbred dogs, the reservoir of zoonotic visceral leishmaniasis 
Vaccine  2009;27(7):1080-1086.
Previous studies demonstrated safety, immunogenicity and efficacy of DNA/modified vaccinia virus Ankara (MVA) prime/boost vaccines expressing tryparedoxin peroxidase (TRYP) and Leishmania homologue of the mammalian receptor for activated C kinase (LACK) against Leishmania major challenge in mice, which was consistent with results from TRYP protein/adjuvant combinations in non-human primates. This study aimed to conduct safety and immunogenicity trials of these DNA/MVA vaccines in dogs, the natural reservoir host of Leishmania infantum, followed-up for 4 months post-vaccination.
In a cohort of 22 uninfected outbred dogs, blinded randomised administration of 1000 μg (high dose) or 100 μg (low dose) DNA prime (day 0) and 1 × 108 pfu MVA boost (day 28) was shown to be safe and showed no clinical side effects. High dose DNA/MVA vaccinated TRYP dogs produced statistically higher mean levels of the type-1 pro-inflammatory cytokine IFN-γ than controls in whole blood assays (WBA) stimulated with the recombinant vaccine antigen TRYP, up to the final sampling at day 126, and in the absence of challenge with Leishmania. TRYP vaccinated dogs also demonstrated significantly higher TRYP-specific total IgG and IgG2 subtype titres than in controls, and positive in vivo intradermal reactions at day 156 in the absence of natural infection, observed in 6/8 TRYP vaccinated dogs. No significant increases in IFN-γ in LACK-stimulated WBA, or in LACK-specific IgG levels, were detected in LACK vaccinated dogs compared to controls, and only 2/9 LACK vaccinated dogs demonstrated DTH responses at day 156. In all groups, IgG1 subclass responses and antigen-specific stimulation of IL-10 were similar to controls demonstrating an absence of Th2/Treg response, as expected in the absence of in vivo restimulation or natural/experimental challenge with Leishmania.
These collective results indicate significant antigen-specific type-1 responses and in vivo memory phase cellular immune responses, consistent with superior potential for protective vaccine immunogenicity of DNA/MVA TRYP over LACK.
doi:10.1016/j.vaccine.2008.11.094
PMCID: PMC2663027  PMID: 19095029
Leishmania infantum; Tryparedoxin peroxidase; Prime/boost DNA/MVA vaccination
14.  Sand Fly Salivary Proteins Induce Strong Cellular Immunity in a Natural Reservoir of Visceral Leishmaniasis with Adverse Consequences for Leishmania 
PLoS Pathogens  2009;5(5):e1000441.
Immunity to a sand fly salivary protein protects against visceral leishmaniasis (VL) in hamsters. This protection was associated with the development of cellular immunity in the form of a delayed-type hypersensitivity response and the presence of IFN-γ at the site of sand fly bites. To date, there are no data available regarding the cellular immune response to sand fly saliva in dogs, the main reservoirs of VL in Latin America, and its role in protection from this fatal disease. Two of 35 salivary proteins from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG2 antibody levels and significant IFN-γ production following in vitro stimulation of PBMC with salivary gland homogenate (SGH). Upon challenge with uninfected or infected flies, immunized dogs developed a cellular response at the bite site characterized by lymphocytic infiltration and IFN-γ and IL-12 expression. Additionally, SGH-stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. Certain sand fly salivary proteins are potent immunogens obligatorily co-deposited with Leishmania parasites during transmission. Their inclusion in an anti-Leishmania vaccine would exploit anti-saliva immunity following an infective sand fly bite and set the stage for a protective anti-Leishmania immune response.
Author Summary
Leishmaniasis is a neglected infectious disease with a global distribution encompassing 88 countries, 350 million people at risk, and an annual incidence of 2 million cases. Leishmaniasis is a vector-borne disease transmitted by sand fly bites where parasites are co-deposited with saliva into the wound. Our group has demonstrated that distinct molecules in the saliva of various sand fly species drive an immune response that protects experimental rodent models from self-healing cutaneous and fatal visceral leishmaniasis. Here we show for the first time that dogs, natural reservoirs of visceral leishmaniasis, develop a strong immune response to two salivary proteins from the natural vector sand fly. Blood from immunized dogs contained immune cells that produced molecules (IFN-γ) typically associated with protection from Leishmania parasites. This response efficiently recruited appropriate immune cells to the site of sand fly bites in the skin and had an adverse effect on Leishmania parasites in an experimental assay. These findings suggest that inclusion of these salivary molecules in anti-Leishmania canine vaccines would enhance their efficiency in protecting dogs from visceral leishmaniasis. A successful anti-Leishmania canine vaccine would not only protect dogs from a fatal disease but could have a considerable effect on reducing human infections.
doi:10.1371/journal.ppat.1000441
PMCID: PMC2677456  PMID: 19461875
15.  Novel Recombinant Multiepitope Proteins for the Diagnosis of Asymptomatic Leishmania infantum-Infected Dogs 
Background
Visceral leishmaniasis is the most severe form of leishmaniasis. Worldwide, approximately 20% of zoonotic human visceral leishmaniasis is caused by Leishmania infantum, also known as Leishmania chagasi in Latin America. Current diagnostic methods are not accurate enough to identify Leishmania-infected animals and may compromise the effectiveness of disease control. Therefore, we aimed to produce and test two recombinant multiepitope proteins as a means to improve and increase accuracy in the diagnosis of canine visceral leishmaniasis (CVL).
Methodology/Principal Findings
Ten antigenic peptides were identified by CVL ELISA in previous work. In the current proposal, the coding sequences of these ten peptides were assembled into a synthetic gene. Furthermore, other twenty peptides were selected from work by our group where good B and T cell epitopes were mapped. The coding sequences of these peptides were also assembled into a synthetic gene. Both genes have been cloned and expressed in Escherichia coli, producing two multiepitope recombinant proteins, PQ10 and PQ20. These antigens have been used in CVL ELISA and were able to identify asymptomatic dogs (80%) more effectively than EIE-LVC kit, produced by Bio-Manguinhos (0%) and DPP kit (10%). Moreover, our recombinant proteins presented an early detection (before PCR) of infected dogs, with positivities ranging from 23% to 65%, depending on the phase of infection in which sera were acquired.
Conclusions/Significance
Our study shows that ELISA using the multiepitope proteins PQ10 and PQ20 has great potential in early CVL diagnosis. The use of these proteins in other methodologies, such as immunochromatographic tests, could be beneficial mainly for the detection of asymptomatic dogs.
Author Summary
Visceral leishmaniasis is the most severe form among leishmaniasis, being a neglected disease caused by a protozoan parasite. Its transmission through phlebotominae bites, between dogs and humans, classifies it as a zoonotic disease. It is caused by the specie Leishmania infantum ( = L. chagasi) and represents 20% of the world's human visceral leishmaniasis. Visceral leishmaniasis is a serious public health issue, fatal if untreated, and its incidence is increasing in urban areas of the tropics. In Brazil, one of the control measures is the identification and elimination of infected dogs, which act as reservoirs for Leishmania parasites. Diagnostic methods used to identify infection in these animals are still not accurate enough, which may compromise the effectiveness of this control measure. Thus, to contribute to the diagnosis of canine visceral leishmaniasis, we aimed to develop and test two new antigens that could be applied in early detection of infected dogs.
doi:10.1371/journal.pntd.0003429
PMCID: PMC4287523  PMID: 25569685
16.  Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell–Driven Protective Response 
Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199–314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5–88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.
Author Summary
The continued spread, morbidity and mortality of human leishmaniasis together with the emergence of drug-resistant variants, the failure of epidemiological control based on dog culling and insecticide vector control and the chemotherapy toxicity have spurred attempts to develop an effective vaccine. Leishmaniasis affects 12 million people and another 350 million live under risk worldwide. We developed the first licensed second generation vaccine against leishmaniasis, a canine vaccine that has already reduced the incidence of the human and canine disease in endemic areas. Its main component is the Nucleoside hydrolase of Leishmania donovani (NH36) which in its recombinant and DNA formulation is cross protective against agents of tegumentary leishmaniasis (TL). For this work we generated three recombinant peptides covering the NH36 sequence and identified the C-domain of the Nucleoside hydrolase as being responsible for its immunogenicity and vaccine-induced protective efficacy against VL and also for the reduction of lesion size and parasite load against TL. Since all Leishmanias species share high identity in their Nucleoside hydrolases amino acid sequences, our study represents a major step forward in the development of a bivalent synthetic vaccine against leishmaniasis and a potential future multivalent vaccine against pathogens that are dependent on NHs for replication.
doi:10.1371/journal.pntd.0000866
PMCID: PMC2976684  PMID: 21085470
17.  Application of an Improved Enzyme-Linked Immunosorbent Assay Method for Serological Diagnosis of Canine Leishmaniasis ▿  
Journal of Clinical Microbiology  2010;48(5):1866-1874.
Accurate diagnosis of canine leishmaniasis (CanL) is essential toward a more efficient control of this zoonosis, but it remains problematic due to the high incidence of asymptomatic infections. In this study, we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based techniques for the detection of antibodies against the recombinant protein Leishmania infantum cytosolic tryparedoxin peroxidase (LicTXNPx) and a comparison of the results with those employing soluble Leishmania antigens from promastigote or amastigote forms and the homologue recombinant protein L. infantum mitochondrial TXNPx (LimTXNPx). Moreover, we offer an evaluation of the diagnostic potential of rK39 for CanL in the Portuguese canine population and propose an improvement to the existing ELISA-based serological techniques by combining the LicTXNPx and rK39 antigens as a Leishmania antigen mixture (LAM). The data demonstrated that ELISAs based on soluble promastigote or amastigote antigens had generally higher levels of sensitivity for detection of antibodies in symptomatic or asymptomatic dogs than for detection of those against isolated recombinant proteins. Nevertheless, the specificities were found to be similar for all target antigens used. Importantly, the LAM-ELISA methodology improved the overall sensitivity, maintaining a high overall level of specificity. In addition, it was demonstrated that the detection of anti-LAM IgG2 can increase the accuracy of the serological diagnosis. Overall, the obtained results showed that the strategy of combining two well-defined Leishmania antigens, LicTXNPx and rK39, proved to be a sensitive and specific improvement to current serological diagnosis of CanL, being a useful tool for the detection of both clinical and subclinical forms of canine Leishmania infection.
doi:10.1128/JCM.02402-09
PMCID: PMC2863945  PMID: 20164286
18.  Use of a LiESP/QA-21 Vaccine (CaniLeish) Stimulates an Appropriate Th1-Dominated Cell-Mediated Immune Response in Dogs 
Canine leishmaniasis is an important zoonotic disease of dogs. The clinical outcome of infection is variable, with the efficiency of the immune response being the key determining factor. There is now a general consensus that a predominant Th1 immune profile in an overall mixed Th1/Th2 response is associated with resistance in dogs, and the absence of a strong Th1 influence is associated with a progression to clinical disease. As a result, there has been a growing demand for vaccines that can induce a specific, strong Th1 response. In this study, we measured the impact of a primary course of a newly available LiESP/QA-21 vaccine on selected humoral and cellular markers of the canine immune response during the onset of immunity. All vaccinated dogs developed a humoral response characterised by IgG2 production. More importantly, vaccinated dogs developed significantly stronger cell-mediated immunity responses than did control dogs. Vaccination induced specific cellular reactivity to soluble Leishmania antigens, with a Leishmania-specific lymphoproliferation (p = 0.0072), characterised by an increased population of T lymphocytes producing IFN-γ (p = 0.0021) and a significant ability of macrophages to reduce intracellular parasite burdens in vitro after co-culture with autologous lymphocytes (p = 0.0014). These responses were correlated with induction of the NOS pathway and production of NO derivatives, which has been shown to be an important leishmanicidal mechanism. These results confirm that vaccination with LiESP/QA-21 induces an appropriate Th1-profile cell-mediated response within three weeks of completing the primary course, and that this response effectively reduces the parasite load in pre-infected macrophages in vitro.
Author Summary
The dog is the principle reservoir of Leishmania infantum, a parasite spread from dog to dog by a sandfly vector. The reduction of canine leishmaniasis is therefore a key factor in the overall management of the epidemiology of this parasite. There is also a need for effective prevention on welfare grounds because of the clinical severity of this potentially fatal disease in dogs. The key factor determining the outcome of infection in dogs is the ability to mount a Th1-dominated immune response, because this is more effective against intracellular pathogens such as L. infantum. Until now, in Europe, only measures to reduce sandfly bites have been available, and for many years there has been a strong demand for a vaccine that provides specific and effective immunity. The recent launch of the first European canine leishmaniasis vaccine (CaniLeish) has provided a means to achieve this, but data on its impact on the dog's immune system are required. The data presented here demonstrate the specific stimulation of an effective Th1-dominated anti-L. infantum response within three weeks of the administration of the vaccine, and provides a basis for the understanding of the mode of action of this new tool.
doi:10.1371/journal.pntd.0001683
PMCID: PMC3378610  PMID: 22724031
19.  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.
Author Summary
Human visceral leishmaniasis causes significant morbidity and mortality, constituting an important global health problem. Absence of safe and cost effective anti-leishmanial drugs, together with emergence of drug resistance and HIV co-infection have posed a serious challenge to the disease containment. Given the urgent need to prevent approximately 0.2 to 0.4 million new VL cases annually worldwide, all reasonable efforts to achieve a safe and effective Leishmania vaccine should be made. We have previously reported the protective properties of the rA2 protein against experimental L. infantum infections both in mice and canines. To further evaluate the efficacy of A2 in a more relevant animal model to human disease, we used the primate Macaca mulatta. Primates vaccinated with different rA2-based prime-boost regimes displayed varying degrees of protective immunity, as indicated by a marked reduction of symptoms and parasite burden in the liver. In particular the vaccination approach with non-replicative adenovirus vector expressing A2 (rAd5-A2) and boosted with the rA2 protein resulted in a more efficient control of parasites as well as resolution of hepatic immune granulomas at 24 weeks post-infection. The clinical efficacy provided by A2 in an animal model that is evolutionary close to humans qualifies this antigen as a promising candidate vaccine against human VL.
doi:10.1371/journal.pntd.0002853
PMCID: PMC4063746  PMID: 24945284
20.  A Randomised, Double-Blind, Controlled Efficacy Trial of the LiESP/QA-21 Vaccine in Naïve Dogs Exposed to Two Leishmania infantum Transmission Seasons 
Canine leishmaniasis is an important zoonosis caused by uncontrolled infection with Leishmania infantum, where an inappropriate immune response is not only responsible for permitting this intracellular parasite to multiply, but is also responsible for several of the pathological processes seen in this disease. Effective canine vaccines are therefore a highly desirable prevention tool. In this randomised, double-blinded, controlled trial, the efficacy of the LiESP/QA-21 vaccine (CaniLeish, Virbac, France) was assessed by exposing 90 naïve dogs to natural L. infantum infection during 2 consecutive transmission seasons, in two highly endemic areas of the Mediterranean basin. Regular PCR, culture, serological and clinical examinations were performed, and the infection/disease status of the dogs was classified at each examination. The vaccine was well-tolerated, and provided a significant reduction in the risk of progressing to uncontrolled active infection (p = 0.025) or symptomatic disease (p = 0.046), with an efficacy of 68.4% and a protection rate of 92.7%. The probability of becoming PCR positive was similar between groups, but the probability of returning to a PCR negative condition was higher in the vaccinated group (p = 0.04). In conclusion, we confirmed the interest of using this vaccine as part of a comprehensive control program for canine leishmaniasis, and validated the use of a protocol based on regular in-depth assessments over time to assess the efficacy of a canine leishmaniasis vaccine.
Author Summary
Canine leishmaniasis is caused by uncontrolled infection with Leishmania infantum. The dog is also the principle reservoir of this parasite which can infect humans. The key determinant of the outcome of infection is the ability of the dog's immune system to respond appropriately. This should be mainly cell-mediated immunity; the antibody responses often desired for viral or bacterial infections are not protective. Therefore, vaccines capable of inducing this cell-mediated immune profile are a desirable prevention tool. Previous studies with CaniLeish (consisting of the parasite's excreted-secreted proteins with an adjuvant) have demonstrated the ability of this vaccine to induce an appropriate immune profile and to protect against an intravenous challenge, as well as to reduce the risk of transmission of the parasite to the sandfly vector. This paper reports a study where vaccinated dogs were exposed to natural parasite transmission over two full seasons in two Mediterranean locations (one in Italy and one in Spain). Data from the unvaccinated control group confirmed that the levels of exposure were high. We demonstrated that the vaccine significantly reduces the risk of progressive infections and disease confirming the interest of using vaccination as part of a comprehensive control program for this disease.
doi:10.1371/journal.pntd.0003213
PMCID: PMC4191955  PMID: 25299614
21.  The prevalence of canine Leishmania infantum infection in western China detected by PCR and serological tests 
Parasites & Vectors  2011;4:69.
Background
Canine leishmaniasis (CanL) is endemic in western China, resulting in important public health problem. It is essential to evaluate the prevalence of canine Leishmania infantum infection for designing control policy. In the present study we report for the first time prevalence of Leishmania infection in dogs living in Jiuzhaigou County (Sichuan Provence, China), which is not only an important endemic area of CanL but also a tourism scenic spot, detected by PCR, ELISA and dipstick test. The results could provide key information for designing control programs against canine and human leishmaniasis. In addition, the complete sequence of the Leishmania isolate from Sichuan Province has not been reported to date and we present the sequences of 116 base-pair (bp) fragment of the conserved region in the minicircle kinetoplast DNA (kDNA) and the results of phylogenetic analyses based on the sequence of the amplified fragment.
Results
The proportion of dogs infected with Leishmania in Jiuzhaigou County was 36.79%, 9.43%, and 51.88% detected by ELISA, dipstick test, and PCR, respectively. The ELISA and PCR tests were more sensitive than dipstick test. The PCR method is the most sensitive way to detect dogs infected with Leishmania parasites. The total positive rate for infected dogs in the area was 59.43% by the three methods. The PCR products of 116-bp fragment amplified from the kDNA conserved region of dog blood samples and laboratory maintained L. infantum were DNA sequenced and the variation of the sequences was observed. The phylogenetic tree based on the sequences of 116-bp fragment reveals that L. infantum is more genetically related to visceralizing species L. donovani than to the Leishmania species associated with cutaneous disease.
Conclusions
More than half of dogs living in the endemic Jiuzhaigou County were infected by L. infantum. Control measures, such as treatment or eradication of infected dogs, or prohibition of maintaining dogs, must be taken against these infected dogs due to their role in the transmission of the infection to vectors. The phylogenetic tree based on the sequences of conserved region in kDNA of Leishmania can effectively distinguish species of Leishmania.
doi:10.1186/1756-3305-4-69
PMCID: PMC3101648  PMID: 21554677
22.  Identification of Proteins in Promastigote and Amastigote-like Leishmania Using an Immunoproteomic Approach 
Background
The present study aims to identify antigens in protein extracts of promastigote and amastigote-like Leishmania (Leishmania) chagasi syn. L. (L.) infantum recognized by antibodies present in the sera of dogs with asymptomatic and symptomatic visceral leishmaniasis (VL).
Methodology/Principal Findings
Proteins recognized by sera samples were separated by two-dimensional electrophoresis (2DE) and identified by mass spectrometry. A total of 550 spots were observed in the 2DE gels, and approximately 104 proteins were identified. Several stage-specific proteins could be identified by either or both classes of sera, including, as expected, previously known proteins identified as diagnosis, virulence factors, drug targets, or vaccine candidates. Three, seven, and five hypothetical proteins could be identified in promastigote antigenic extracts; while two, eleven, and three hypothetical proteins could be identified in amastigote-like antigenic extracts by asymptomatic and symptomatic sera, as well as a combination of both, respectively.
Conclusions/Significance
The present study represents a significant contribution not only in identifying stage-specific L. infantum molecules, but also in revealing the expression of a large number of hypothetical proteins. Moreover, when combined, the identified proteins constitute a significant source of information for the improvement of diagnostic tools and/or vaccine development to VL.
Author Summary
Canine visceral leishmaniasis (CVL) is an important emerging zoonosis caused by Leishmania (Leishmania) infantum in the Mediterranean and Middle East and L. (L.) chagasi (syn. L. (L.) infantum) in Latin America. Due to their genotypic relationships, these species are considered identical. The present study focused on comparing the protein expression profiles of the promastigote and amastigote-like stages of L. infantum, by means of a protein separation by two-dimensional electrophoresis and identification by mass spectrometry. The present study attempted to identify proteins recognized by antibodies present in the sera of dogs with asymptomatic and symptomatic visceral leishmaniasis. A total of one hundred and four proteins were identified. Of these, several stage-specific proteins had been previously identified as diagnosis and/or vaccine candidates. In addition, antibodies from infected dogs recognized thirty-one proteins, which had been previously considered hypothetical, indicating that these proteins are expressed during active infection. Therefore, the present study reveals new potential candidates for the improvement of diagnosis of CVL.
doi:10.1371/journal.pntd.0001430
PMCID: PMC3260309  PMID: 22272364
23.  Canine Skin and Conjunctival Swab Samples for the Detection and Quantification of Leishmania infantum DNA in an Endemic Urban Area in Brazil 
Background
We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively.
Methodology/Principal Findings
Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti-Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups.
Conclusions
The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions.
Author Summary
Visceral leishmaniasis (VL) is considered the most lethal manifestation among the diseases caused by genus Leishmania. The main control measures for VL are: diagnosis and early treatment of human cases, insecticide vector control, and euthanasia of seropositive dogs. Although these strategies have been continuously applied in Brazil, the number of VL cases has increased and this disease still poses as a serious public health problem. Belo Horizonte is an endemic urban area in Brazil and it is considered by the Ministry of Health the third most affected Brazilian metropolitan region by VL. In the context of prophylaxis, the correct diagnosis of infected dogs is critical for the VL control because dogs represent the main domestic reservoir. However, the serological diagnosis techniques used in large-scale for canine screening in Brazil have important limitations including the difficulty of diagnosing asymptomatic dogs. Molecular methods based on Polymerase Chain Reaction (PCR) and real-time PCR (qPCR) have greatly improved VL diagnosis. Based on this, we aimed to evaluate different canine clinical samples for qualitative VL diagnosis by PCR and quantification of parasite load by qPCR in dogs from Belo Horizonte. The possible implications of results are discussed with emphasis on skin and conjunctival swab samples.
doi:10.1371/journal.pntd.0001596
PMCID: PMC3323509  PMID: 22506084
24.  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.
Author Summary
Visceral leishmaniasis is endemic in many areas of tropical and subtropical America where it constitutes a significant public health problem. It is usually diagnosed by enzyme-linked immunosorbent assays (ELISA) using crude Leishmania antigens, but a variety of other immunological methods may also be applied. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of antigens. In this context, the use of combinations of purified, well-characterized antigens appears preferable and may yield better results. In the present study, combinations of peptides derived from the previously described Leishmania diagnostic antigens A2, NH, LACK and K39 were used in ELISA against sera from 106 dogs and 44 human patients. Improved sensitivities and specificities, close to 100%, for both sera of patients and dogs was observed for ELISA using some combinations of the peptides, including the detection of VL in dogs with low anti-Leishmania antibody titers and asymptomatic infection. So, 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.
doi:10.1371/journal.pntd.0001622
PMCID: PMC3358334  PMID: 22629475
25.  Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes of Leishmania infantum for Serodiagnosis of Visceral Leishmaniasis 
Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3′-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts.
doi:10.1128/CDLI.12.5.647-653.2005
PMCID: PMC1112073  PMID: 15879027

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