Compartmentalized Immune Response in Leishmaniasis: Changing Patterns throughout the Disease.

Visceral leishmaniasis (VL) is characterized by loss of T-cell responsiveness and absence of Leishmania-specific IFN-γ production by peripheral blood mononuclear cells. However, the expressions of IFN-γ and TNF-α are up-regulated in the tissues and plasma of VL patients. There is a paucity of information regarding the cytokine profile expressed by different target tissues in the same individual and the changes it undergoes throughout the course of infection. In this work we evaluated IFN-γ, TNF-α, IL-10, and TGF-ß mRNA expression using real-time RT-PCR in 5 target tissues at 6 months and 16 months post-infection (PI) in a canine experimental model which mimics many aspects of human VL. The spleen and liver of Leishmania infantum experimentally-infected dogs elicited a pro- and anti- inflammatory response and high parasite density at 6 and 16 months PI. The popliteal lymph node, however, showed an up-regulation of IFN-γ cytokin at commencement of the study and was at the chronic phase when the IL-10 and TGF-ß expression appeared. In spite of skin parasite invasion, local cytokine response was absent at 6 months PI. Parasite growth and onset of clinical disease both correlated with dermal up-regulation of all the studied cytokines. Our VL model suggests that central target organs, such as the spleen and liver, present a mixed cytokine immune response early on infection. In contrast, an anti-inflammatory/regulatory immune response in peripheral tissues is activated in the later chronic-patent stages of the disease.

Performance of commercially available serological diagnostic tests to detect Leishmania infantum infection on experimentally infected dogs.

Leishmania infantum (syn. Leishmania chagasi) is the etiological agent of a widespread serious zoonotic disease that affects both humans and dogs. Prevalence and incidence of the canine infection are important parameters to determine the risk and the ways to control this reemergent zoonosis. Unfortunately, there is not a gold standard test for Leishmania infection. Our aim was to assess the operative validity of commercial tests used to detect antibodies to Leishmania in serum samples from experimental infections. Three ELISA tests (LEISCAN(®) Leishmania ELISA Test, INGEZIM(®) LEISHMANIA, and INGEZIM(®) LEISHMANIA VET), three immunochromatographic tests (INGEZIM(®) LEISHMACROM, SNAP(®) Leishmania, and WITNESS(®) Leishmania), and one IFAT were evaluated. LEISCAN(®) Leishmania ELISA test achieved the highest sensitivity and accuracy (both 0.98). Specificity was 1 for all tests except for IFAT. All tests but IFAT obtained a positive predictive value of 1, while the maximum negative predictive value was achieved by LEISCAN(®) Leishmania ELISA Test (0.93). The best positive likelihood ratio was obtained by INGEZIM(®) LEISHMANIA VET (30.26), while the best negative likelihood ratio was obtained by LEISCAN(®) Leishmania ELISA Test (0.02). The highest diagnostic odds ratio was achieved by LEISCAN(®) Leishmania ELISA Test (729.00). The largest area under the ROC curve was obtained by LEISCAN(®) Leishmania ELISA Test (0.981). Quantitative ELISA based tests performmed better than qualitative tests ("Rapid Tests"), and the test best suited to detect Leishmania in infected dogs and to provide clinically useful information was LEISCAN(®) Leishmania ELISA Test. This and other results point also to the need of revising the status of IFAT as a gold standard for the diagnosis of leishmaniasis.

Head-to-head comparison of three vaccination strategies based on DNA and raw insect-derived recombinant proteins against Leishmania.

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.

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.

Dynamics and predictive potential of antibodies against insect-derived recombinant Leishmania infantum proteins during chemotherapy of naturally infected dogs.

A predictive marker for the success treatment of canine leishmaniasis is required for the application of a more rational therapy protocol, which must improve the probability of cure and reduce Leishmania resistance to drugs. We investigated the dynamics and predictive value of antibodies against insect-derived recombinant L. infantum proteins rKMPII and rTRYP by using an enzyme-linked immunosorbent assay with retrospective serum samples from 36 dogs during treatment of canine leishmaniasis. In the entire group of dogs, concentrations of antibodies against rKMPII and rTRYP significantly decreased earlier than concentrations of antibodies against crude total Leishmania antigen (one versus six months), which suggested that the dynamics of antibodies against recombinant proteins may be useful for assessing clinical improvement after treatment. Interestingly, decreases in antibody concentrations against rKMPII occurred earlier in disease-free dogs than in dogs that remain clinically ill one year after beginning of treatment, which suggested that these antibodies may be useful for predicting disease-free survival one year after the beginning of therapy against canine leishmaniasis.

Seroreactivity against raw insect-derived recombinant KMPII, TRYP, and LACK Leishmania infantum proteins in infected dogs.

The recombinant proteins KMPII, TRYP, and LACK of Leishmania infantum were produced in baculovirus-infected Trichoplusia ni larvae and used to analyze the seroreactivity of 165 dog serum samples by the multiple-well ELISA technique (57 infected dogs with clinical signs, 46 naturally infected and 11 experimentally infected; and 108 non-infected dogs, 76 from non-endemic areas and 32 from endemic areas). Recombinant (r) KMPII was the most recognized antigen, as the majority of infected dogs seroreacted against it (0.75). This is the first report of seroreactivity against rTRYP (0.51) and rLACK (0.42) in L. infantum-infected dogs, since previous studies using recombinant TRYP and LACK proteins produced in prokaryotic systems failed to detect specific seroreactivity. All non-infected dogs were negative for rTRYP and rLACK, and only one of the 32 from endemic areas seroreacted against rKMPII. The results demonstrate that L. infantum-infected dogs develop humoral immunity against rKMPII, rTRYP, and rLACK antigens. There was substantial agreement between crude total L. infantum antigen (CTLA)-based ELISA and rKMPII ELISA (kappa=0.664), although this was higher than that found between the CTLA-based ELISA and rTRYP (kappa=0.427) or rLACK (kappa=0.343) ELISA, which can be interpreted as fair and moderate agreement, respectively. Ninety-three percent of the infected dogs analyzed developed specific antibodies against at least one of these three recombinant antigens. When the three recombinant antigen-based ELISA techniques were evaluated in parallel, almost perfect agreement (kappa=0.880) with CTLA-based ELISA was observed, with a specificity of 0.97 and a sensitivity of 0.93 in relation to CTLA-based ELISA. Further studies using purified recombinant antigens in a single-well test or individually, depending on the objective of the study, are warranted.

Anti-Leishmania IgA in urine samples from dogs with clinical leishmaniasis.

Recently, anti-Leishmania IgG has been detected in urine samples from Leishmania-infected dogs and its concentrations have been correlated with impairment of renal function. The presence and relationship with other anti-Leishmania Ig isotypes in urine have not yet been investigated. The current study analyzed the concentrations of anti-Leishmania IgA and IgG in sera (Ig-S) and urine (Ig-U) samples by ELISA in 64 untreated dogs with clinical leishmaniasis. All 64 serum samples tested were positive for anti-Leishmania IgG. Fifty of them (78.1%) were also positive for anti-Leishmania IgA. The results showed the presence of anti-Leishmania IgA-U in 38% of the 50 dogs that were positive for specific IgA-S. Thirty-eight of the 64 dogs positive for Leishmania-specific IgG-S (59.4%) were also positive for Leishmania-specific IgG in urine (IgG-U). The concentrations of anti-Leishmania IgA-U were significantly correlated with urine protein/creatinine (uP/C) ratio (rho=0.542; P<0.001) and with serum biochemical parameters, such as gamma-globulins, urea and creatinine. Goldmann-Witmer coefficient (C value) indicated that detection of specific IgA in urine samples from dogs with leishmaniasis might not only be due to impairment of filtration of the glomerular barrier but also be due to local production of this isotype, which might reflect a local immunological response to the presence of the parasite in the genitourinary tract. Anti-Leishmania IgG-U concentrations were highly correlated with uP/C ratio (rho=0.779; P<0.001) and C value did not support in any case local production of this isotype. IgG isotype might be a more suitable and specific tool to evaluate renal damage due to the lower IgA-U sensitivity and correlation coefficients and evidence of IgA local production. However, dogs found positive for both Ig isotypes in urine presented significantly higher specific IgG-U concentrations and higher uP/C ratios than dogs found positive only for IgG-U, thus suggesting that the first group suffered more severe renal damage. This fact makes it necessary to evaluate the prognosis of dogs showing both anti-Leishmania IgA-U and IgG-U in future studies.