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1.  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.
doi:10.4269/ajtmh.2010.09-0147
PMCID: PMC2861386  PMID: 20439957
2.  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
3.  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
4.  Cytokine Responses to Novel Antigens in a Peri-Urban Population in Brazil Exposed to Leishmania infantum chagasi 
Visceral leishmaniasis (VL) is fatal if untreated, and there are no vaccines for this disease. High levels of CD4-derived interferon-γ (IFN-γ) in the presence of low levels of interleukin-10 (IL-10) predicts vaccine success. Tumor necrosis factor-α (TNF-α) is also important in this process. We characterized human immune responses in three groups exposed to Leishmania infantum chagasi in Brazil: 1) drug-cured VL patients (recovered VL); 2) asymptomatic persons with positive Leishmania-specific delayed-type hypersensitivity skin reactions (DTH+); and 3) DTH-negative household contacts. Magnitude of DTH correlated with crude Leishmania antigen–driven IFN-γ, TNF-α, and IL-5, but not IL-10. DTH+ persons showed equivalent levels of IFN-γ, but higher levels of IL-10, to tryparedoxin peroxidase and Leishmania homolog of receptor for activated C kinase compared with recovered VL patients. The IFN-γ:IL-10 and TNF-α:IL-10 ratios were higher in recovered VL patients than in DTH+ persons. Seven of 11 novel candidates (R71, L37, N52, L302.06, M18, J41, and M22) elicited cytokine responses (36–71% of responders) in recovered VL patients and DTH+ persons. This result confirmed their putative status as cross-species vaccine/immunotherapeutic candidates.
doi:10.4269/ajtmh.2012.12-0180
PMCID: PMC3516316  PMID: 22826477
5.  TLR1/2 Activation during Heterologous Prime-Boost Vaccination (DNA-MVA) Enhances CD8+ T Cell Responses Providing Protection against Leishmania (Viannia) 
Background
Leishmania (Viannia) parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.
Methodology
Using a newly developed mouse model of chronic L. (Viannia) panamensis infection and the heterologous DNA prime – modified vaccinia virus Ankara (MVA) boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP) could provide protection against infection/disease.
Results
Heterologous prime – boost (DNA/MVA) vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V.) panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.
Conclusions
Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia) to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that CD8 T cells should be targeted for the development of a vaccine against infection caused by Leishmania (Viannia) parasites. Further, TLR1/2 modulation may be useful in vaccines where CD8 T cell responses are critical.
Author Summary
Leishmania (Viannia) are the predominant agents of leishmaniasis in Latin America. Given the fact that leishmaniasis is a zoonosis, eradication is unlikely; a vaccine could provide effective prevention of disease. However, these parasites present a challenge and we do not fully understand what elements of the host immune defense prevent disease. We examined the ability of vaccination to protect against L. (Viannia) infection using the highly immunogenic heterologous prime-boost (DNA-modified vaccinia virus) modality and a single Leishmania antigen (TRYP). Although this mode of vaccination can induce protection against other leishmaniases (cutaneous, visceral), no protection was observed against L. (V.) panamensis. However, we found that if the vaccination was modified and the innate immune response was activated through Toll-like receptor1/2(TLR1/2) during the DNA priming, vaccinated mice were protected. Protection was dependent on CD8 T cells. Vaccinated mice had higher CD8 T cell responses and decreased levels of cytokines known to promote infection. Given the long-term persistence of CD8 T cell memory, these findings are encouraging for vaccine development. Further, these results suggest that modulation of TLR1/2 signaling could improve the efficacy of DNA-based vaccines, especially where CD8 T cell activation is critical, thereby contributing to effective and affordable anti parasitic vaccines.
doi:10.1371/journal.pntd.0001204
PMCID: PMC3114751  PMID: 21695103
6.  Characterization of the Immunostimulatory Properties of Leishmania infantum HSP70 by Fusion to the Escherichia coli Maltose-Binding Protein in Normal and nu/nu BALB/c Mice 
Infection and Immunity  1998;66(1):347-352.
Leishmania infantum HSP70 has been described as an immunodominant antigen in both humans and dogs suffering from visceral leishmaniasis. In this study, we used L. infantum HSP70 fused to Escherichia coli maltose-binding protein (MBP), as the reporter protein, to analyze the influence of HSP70 on the immunogenicity of MBP in BALB/c mice. Plasmids were constructed to produce the three recombinant proteins used in this study, namely, MBP, L. infantum HSP70, and MBP-HSP70, which consists of MBP fused to the L. infantum HSP70 amino terminus. Immunization of BALB/c mice with the MBP-HSP70 fusion protein elicited humoral and cellular responses against MBP that were higher by an order of magnitude than those elicited by immunization with MBP alone or with a mixture of MBP and HSP70. Covalent linkage of MBP to HSP70 was essential for eliciting a strong anti-MBP immune response. Cytokine secretion and immunoglobulin G isotype analyses indicated that immunization with the MBP-HSP70 fusion protein preferentially induces a Th1 immune response. Immunization of athymic nu/nu mice with the MBP-HSP70 fusion protein unexpectedly gave rise to an anti-MBP humoral response showing features of a T-cell-dependent response. Thus, we present evidence that L. infantum HSP70 demonstrates an adjuvant effect in the immune response against a covalently linked reporter protein.
PMCID: PMC107897  PMID: 9423878
7.  Leish-111f, a Recombinant Polyprotein Vaccine That Protects against Visceral Leishmaniasis by Elicitation of CD4+ T Cells▿  
Infection and Immunity  2007;75(9):4648-4654.
The Leishmania-derived recombinant polyprotein Leish-111f or its three component proteins, thiol-specific antioxidant (TSA), Leishmania major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF), have previously been demonstrated to be efficacious against cutaneous or mucosal leishmaniasis in mice, nonhuman primates, and humans. In this study we demonstrate that Leish-111f is also a vaccine antigen candidate against visceral leishmaniasis (VL) caused by Leishmania infantum. We evaluated the immune response and protection induced by Leish-111f formulated with monophosphoryl lipid A in a stable emulsion (Leish-111f+MPL-SE) and demonstrated that mice developed strong humoral and T-cell responses to the vaccine antigen. Analysis of the cellular immune responses of immunized, uninfected mice demonstrated that the vaccine induced a significant increase in CD4+ T cells producing gamma interferon, interleukin 2, and tumor necrosis factor cytokines, indicating a Th1-type immune response. Experimental infection of immunized mice and hamsters demonstrated that Leish-111f+MPL-SE induced significant protection against L. infantum infection, with reductions in parasite loads of 99.6%, a level of protection greater than that reported for other vaccine candidates in animal models of VL. Taken together, our results suggest that this vaccine represents a good candidate for use against several Leishmania species. The Leish-111f+MPL-SE product we report here is the first defined vaccine for leishmaniasis in human clinical trials and has completed phase 1 and 2 safety and immunogenicity testing in normal, healthy human subjects.
doi:10.1128/IAI.00394-07
PMCID: PMC1951162  PMID: 17606603
8.  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
9.  The Leishmania HSP20 Is Antigenic during Natural Infections, but, as DNA Vaccine, It does not Protect BALB/c Mice against Experimental L. amazonensis Infection 
Protozoa of the genus Leishmania are causative agents of leishmaniasis, an important health problem in both human and veterinary medicine. Here, we describe a new heat shock protein (HSP) in Leishmania, belonging to the small HSP (sHSP) family in kinetoplastids. The protein is highly conserved in different Leishmania species, showing instead significant divergence with sHSP's from other organisms. The humoral response elicited against this protein during Leishmania infection has been investigated in natural infected humans and dogs, and in experimentally infected hamsters. Leishmania HSP20 is a prominent antigen for canine hosts; on the contrary, the protein seems to be a poor antigen for human immune system. Time-course analysis of appearance of anti-HSP20 antibodies in golden hamsters indicated that these antibodies are produced at late stages of the infection, when clinical symptoms of disease are patent. Finally, the protective efficacy of HSP20 was assessed in mice using a DNA vaccine approach prior to challenge with Leishmania amazonensis.
doi:10.1155/2008/695432
PMCID: PMC2288687  PMID: 18401455
10.  Immunologic Indicators of Clinical Progression during Canine Leishmania infantum Infection▿  
In both dogs and humans Leishmania infantum infection is more prevalent than disease, as infection often does not equate with clinical disease. Previous studies additively indicate that advanced clinical visceral leishmaniasis is characterized by increased production of anti-Leishmania antibodies, Leishmania-specific lymphoproliferative unresponsiveness, and decreased production of gamma interferon (IFN-γ) with a concomitant increase of interleukin-10 (IL-10). In order to differentiate infection versus progressive disease for better disease prognostication, we temporally evaluated humoral and cellular immunologic parameters of naturally infected dogs. The work presented here describes for the first time the temporal immune response to natural autochthonous L. infantum infection in foxhounds within the United States. Several key changes in immunological parameters should be considered when differentiating infection versus clinical disease, including a dramatic rise in IgG production, progressive increases in antigen-specific peripheral blood mononuclear cell proliferation, and IFN-γ production. Polysymptomatic disease is precluded by increased IL-10 production and consistent detection of parasite kinetoplast DNA in whole blood. This clinical presentation and the immuno-dysregulation mirror those observed in human patients, indicating that this animal model will be very useful for testing immunomodulatory anti-IL-10 and other therapies.
doi:10.1128/CVI.00456-09
PMCID: PMC2815526  PMID: 20032217
11.  TriTrypDB: a functional genomic resource for the Trypanosomatidae 
Nucleic Acids Research  2009;38(Database issue):D457-D462.
TriTrypDB (http://tritrypdb.org) is an integrated database providing access to genome-scale datasets for kinetoplastid parasites, and supporting a variety of complex queries driven by research and development needs. TriTrypDB is a collaborative project, utilizing the GUS/WDK computational infrastructure developed by the Eukaryotic Pathogen Bioinformatics Resource Center (EuPathDB.org) to integrate genome annotation and analyses from GeneDB and elsewhere with a wide variety of functional genomics datasets made available by members of the global research community, often pre-publication. Currently, TriTrypDB integrates datasets from Leishmania braziliensis, L. infantum, L. major, L. tarentolae, Trypanosoma brucei and T. cruzi. Users may examine individual genes or chromosomal spans in their genomic context, including syntenic alignments with other kinetoplastid organisms. Data within TriTrypDB can be interrogated utilizing a sophisticated search strategy system that enables a user to construct complex queries combining multiple data types. All search strategies are stored, allowing future access and integrated searches. ‘User Comments’ may be added to any gene page, enhancing available annotation; such comments become immediately searchable via the text search, and are forwarded to curators for incorporation into the reference annotation when appropriate.
doi:10.1093/nar/gkp851
PMCID: PMC2808979  PMID: 19843604
12.  Compensation for Decreased Expression of B7 Molecules on Leishmania infantum-Infected Canine Macrophages Results in Restoration of Parasite-Specific T-Cell Proliferation and Gamma Interferon Production 
Infection and Immunity  1999;67(1):237-243.
Infection of humans and dogs by Leishmania infantum may result in visceral leishmaniasis, which is characterized by impaired T-cell-mediated immune responses to parasite antigens. Dogs are natural hosts of Leishmania parasites and play an important role in the transmission of the parasites to humans. In an effort to characterize the immune response in dogs infected with this intracellular pathogen, we examined how infection with L. infantum affects canine macrophages and the consequences for T-cell activation in vitro. We showed that the proliferation of T-cell lines to cognate antigen decreases to background levels when infected autologous monocyte-derived macrophages are used as antigen-presenting cells (APC). The observed reduction of antigen-specific T-cell proliferation was shown to be dependent on the parasite load and to require cell-to-cell interaction of T cells with the infected APC. In addition, we observed a decreased expression of costimulatory B7 molecules on infected monocyte-derived macrophages. The expression of other surface molecules involved in T-cell activation, such as major histocompatibility complex class I and class II, on these cells did not change upon infection, whereas the expression of intracellular adhesion molecule 1 was marginally increased. Compensation for the decreased expression of B7 molecules by the addition of B7-transfected cells resulted in the restoration of cell proliferation and gamma interferon (IFN-γ) production by a Leishmania-specific T-cell line. These results showed that for the activation of parasite-specific canine T cells producing IFN-γ, which are most likely involved in protective immunity, sufficient expression of B7 molecules on infected macrophages is required. Provision of costimulatory molecules may be an approach for immunotherapy of leishmaniaisis as well as for vaccine development.
PMCID: PMC96302  PMID: 9864221
13.  A Novel Leishmania infantum Recombinant Antigen Which Elicits Interleukin 10 Production by Peripheral Blood Mononuclear Cells of Patients with Visceral Leishmaniasis 
Infection and Immunity  2000;68(2):630-636.
We report here the characterization of a novel Leishmania infantum protein termed papLe22 (22-kDa potentially aggravating protein of Leishmania). A positive clone from a cDNA library was identified by serum of a visceral leishmaniasis (VL) patient. Full-length cDNA obtained using rapid amplification of cDNA ends-PCR codes for a 22-kDa protein. In L. infantum promastigotes an endogenous nuclear protein of 14-kDa electrophoretic mobility was found by using an antiserum prepared against the fusion protein glutathione S-transferase–papLe22. Its expression was also shown in L. infantum amastigotes and in Leishmania major and Leishmania guyanensis promastigotes. VL patients' sera showed anti-papLe22 immunoglobulin M (IgM) and IgG reactivities, indicating that a primary response against the leishmanial protein papLe22 accompanied acute VL manifestations. Specific IgG levels were correlated with patients' clinical status. The presence of IgG1, IgG2, and IgG3 subclasses suggested a mixed Th1- and Th2-type response; there was no correlation between subclass reactivity and the disease course. The recombinant papLe22 specifically activated interleukin-10 production by VL patients' peripheral blood mononuclear cells collected at diagnosis and after treatment-induced cure, indicating its contribution to VL pathogenesis and concomitant immunosuppression and its potential role in the reactivation of latent parasites. As a dominant immunogen, papLe22 might be used as a vaccine component, provided that the vaccination protocol directs the response toward the Th1 pattern.
PMCID: PMC97185  PMID: 10639426
14.  Cytokine Responses to Novel Antigens in an Indian Population Living in an Area Endemic for Visceral Leishmaniasis 
Background
There are no effective vaccines for visceral leishmaniasis (VL), a neglected parasitic disease second only to malaria in global mortality. We previously identified 14 protective candidates in a screen of 100 Leishmania antigens as DNA vaccines in mice. Here we employ whole blood assays to evaluate human cytokine responses to 11 of these antigens, in comparison to known defined and crude antigen preparations.
Methods
Whole blood assays were employed to measure IFN-γ, TNF-α and IL-10 responses to peptide pools of the novel antigens R71, Q51, L37, N52, L302.06, J89, M18, J41, M22, M63, M57, as well as to recombinant proteins of tryparedoxin peroxidase (TRYP), Leishmania homolog of the receptor for activated C kinase (LACK) and to crude soluble Leishmania antigen (SLA), in Indian patients with active (n = 8) or cured (n = 16) VL, and in modified Quantiferon positive (EHC+ve, n = 20) or modified Quantiferon negative (EHC−ve, n = 9) endemic healthy controls (EHC).
Results
Active VL, cured VL and EHC+ve groups showed elevated SLA-specific IFN-γ, but only active VL patients produced IL-10 and EHC+ve did not make TNF-α. IFN-γ to IL-10 and TNF-α to IL-10 ratios in response to TRYP and LACK antigens were higher in cured VL and EHC+ve exposed individuals compared to active VL. Five of the eleven novel candidates (R71, L37, N52, J41, and M22) elicited IFN-γ and TNF-α, but not IL-10, responses in cured VL (55–87.5% responders) and EHC+ve (40–65% responders) subjects.
Conclusions
Our results are consistent with an important balance between pro-inflammatory IFNγ and TNFγ cytokine responses and anti-inflammatory IL-10 in determining outcome of VL in India, as highlighted by response to both crude and defined protein antigens. Importantly, cured VL patients and endemic Quantiferon positive individuals recognise 5 novel vaccine candidate antigens, confirming our recent data for L. chagasi in Brazil, and their potential as cross-species vaccine candidates.
Author Summary
Visceral leishmaniasis is a parasitic infection that results in death in susceptible people unless they are treated. Current drugs are expensive and toxic, and there are no vaccines in use in humans. We know that it is possible to become immune to infection with this parasite because people who have been cured using drug treatment are resistant to further infection. In addition, a large percentage of people infected with the parasite remain asymptomatic and develop a specific immune response that can be measured using crude leishmanial antigens. We hypothesized that these resistant people might hold the key to understanding the kind of immune response required for protection. In this paper we compared the immune response to a series of novel vaccine candidates in people with active disease, in those drug-cured from the disease, and in the naturally resistant individuals. We show that immune individuals make strong cytokine responses to five of eleven novel vaccine candidates that were tested, making them ideal candidates to take forward in the development of a defined vaccine against leishmaniasis.
doi:10.1371/journal.pntd.0001874
PMCID: PMC3493615  PMID: 23150744
15.  Heterologous Priming-Boosting with DNA and Modified Vaccinia Virus Ankara Expressing Tryparedoxin Peroxidase Promotes Long-Term Memory against Leishmania major in Susceptible BALB/c Mice▿  
Infection and Immunity  2006;75(2):852-860.
Leishmaniasis affects 12 million people, but there are no vaccines in routine clinical use. Th1 polarizing vaccines that elicit long-term protection are required to prevent disease in susceptible populations. We recently showed that heterologous priming-boosting with tryparedoxin peroxidase (TRYP) DNA followed by TRYP-modified vaccinia virus Ankara (TRYP MVA) protected susceptible BALB/c mice from Leishmania major. Here we compared treatment with TRYP DNA with treatment with TRYP DNA/TRYP MVA. We found that equivalent levels of protection during the postvaccination effector phase correlated with equivalent levels of serum immunoglobulin G2a and gamma interferon (IFN-γ) in draining lymph nodes. In contrast, challenge infection during the memory phase revealed that there was enhanced clinical efficacy with TRYP DNA/TRYP MVA. This correlated with higher levels of effector phase splenic IFN-γ, sustained prechallenge levels of memory phase IFN-γ, and a more polarized post-L. major challenge Th1 response compared to the Th2/Treg response. Thus, TRYP DNA/TRYP MVA, but not TRYP DNA alone, provides long-term protection against murine leishmaniasis.
doi:10.1128/IAI.01490-06
PMCID: PMC1828487  PMID: 17101647
16.  Specific Serodiagnosis of Canine Visceral Leishmaniasis Using Leishmania Species Ribosomal Protein Extracts▿  
Clinical and Vaccine Immunology : CVI  2009;16(12):1774-1780.
In the present work, we have analyzed the antigenicity of Leishmania species ribosomal proteins (LRPs). To accomplish this, Leishmania infantum ribosomes were biochemically purified from promastigote cytosolic extracts, and their reactivities were analyzed by using the sera from dogs naturally infected with L. infantum. Since antibodies reacting against different ribosomal proteins were observed in all the serum samples obtained from dogs with symptomatic visceral leishmaniasis tested, we have analyzed the potential usefulness of the LRP extracts in the development of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of canine visceral leishmaniasis (CVL) in an area of Brazil where visceral leishmaniasis is endemic due to infection by Leishmania chagasi. A comparative ELISA with crude soluble Leishmania chagasi antigen (SLA) and L. infantum LRPs was performed. LRP- and SLA-based ELISAs gave similar sensitivities for the diagnosis of symptomatic CVL, but the LRP extract provided a very high sensitivity for the detection of oligosymptomatic and asymptomatic dogs. In addition, an LRP-based ELISA showed a higher specificity when the sera from dogs harboring other infections were included in the analysis. The LRP antigen displayed no cross-reactivity with sera from dogs that had any of the other diseases tested, notably, Chagas' disease. Our findings suggest that LRPs are a potential tool for the diagnosis of CVL and will be particularly useful for the diagnosis of asymptomatic CVL.
doi:10.1128/CVI.00295-09
PMCID: PMC2786384  PMID: 19812259
17.  Immune Responses Induced by the Leishmania (Leishmania) donovani A2 Antigen, but Not by the LACK Antigen, Are Protective against Experimental Leishmania (Leishmania) amazonensis Infection  
Infection and Immunity  2003;71(7):3988-3994.
Leishmania amazonensis is one of the major etiologic agents of a broad spectrum of clinical forms of leishmaniasis and has a wide geographical distribution in the Americas, which overlaps with the areas of transmission of many other Leishmania species. The LACK and A2 antigens are shared by various Leishmania species. A2 was previously shown to induce a potent Th1 immune response and protection against L. donovani infection in BALB/c mice. LACK is effective against L. major infection, but no significant protection against L. donovani infection was observed, in spite of the induction of a potent Th1 immune response. In an attempt to select candidate antigens for an American leishmaniasis vaccine, we investigated the protective effect of these recombinant antigens (rLACK and rA2) and recombinant interleukin-12 (rIL-12) against L. amazonensis infection in BALB/c mice. As expected, immunization with either rA2-rIL-12 or rLACK-rIL-12 induced a robust Th1 response prior to infection. However, only the BALB/c mice immunized with rA2-rIL-12 were protected against infection. Sustained gamma interferon (IFN-γ) production, high levels of anti-A2 antibodies, and low levels of parasite-specific antibodies were detected in these mice after infection. In contrast, mice immunized with rLACK-rIL-12 displayed decreased levels of IFN-γ and high levels of both anti-LACK and parasite-specific antibodies. Curiously, the association between rA2 and rLACK antigens in the same vaccine completely inhibited the rA2-specific IFN-γ and humoral responses and, consequently, the protective effect of the rA2 antigen against L. amazonensis infection. We concluded that A2, but not LACK, fits the requirements for a safe vaccine against American leishmaniasis.
doi:10.1128/IAI.71.7.3988-3994.2003
PMCID: PMC162020  PMID: 12819086
18.  Heme oxygenase-1 promotes the persistence of Leishmania chagasi infection 
Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection in order to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. Herein, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan (LPG) isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX (CoPP), a HO-1 inductor, increased the parasite burden in both mouse and human derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared to those from wild type mice. Furthermore, upregulation of HO-1 by CoPP diminished the production of TNF-α and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach.
doi:10.4049/jimmunol.1103072
PMCID: PMC3331931  PMID: 22461696
Leishmania infantum chagasi; visceral leishmaniasis; macrophage heme oxygenase
19.  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
20.  LBSapSal-vaccinated dogs exhibit increased circulating T-lymphocyte subsets (CD4+ and CD8+) as well as a reduction of parasitism after challenge with Leishmania infantum plus salivary gland of Lutzomyia longipalpis 
Parasites & Vectors  2014;7:61.
Background
The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8+ T cells, and high NO production.
Methods
We investigated the immunogenicity and protective effect of LBSapSal vaccination after intradermal challenge with 1 × 107 late-log-phase L. infantum promastigotes in the presence of sand fly saliva of Lutzomyia longipalpis. The dogs were followed for up to 885 days after challenge.
Results
The LBSapSal vaccine presents extensive antigenic diversity with persistent humoral and cellular immune responses, indicating resistance against CVL is triggered by high levels of total IgG and its subtypes (IgG1 and IgG2); expansion of circulating CD5+, CD4+, and CD8+ T lymphocytes and is Leishmania-specific; and reduction of splenic parasite load.
Conclusions
These results encourage further study of vaccine strategies addressing Leishmania antigens in combination with proteins present in the saliva of the vector.
doi:10.1186/1756-3305-7-61
PMCID: PMC3943450  PMID: 24507702
LBSapSal vaccine; Canine visceral leishmaniasis; Immunogenicity; Experimental challenge; Leishmania infantum; Saliva of Lutzomyia longipalpis
21.  KSAC, the First Defined Polyprotein Vaccine Candidate for Visceral Leishmaniasis▿ 
A subunit vaccine using a defined antigen(s) may be one effective solution for controlling leishmaniasis. Because of genetic diversity in target populations, including both dogs and humans, a multiple-antigen vaccine will likely be essential. However, the cost of a vaccine to be used in developing countries must be considered. We describe herein a multiantigen vaccine candidate comprised of antigens known to be protective in animal models, including dogs, and to be recognized by humans immune to visceral leishmaniasis. The polyprotein (KSAC) formulated with monophosphoryl lipid A, a widely used adjuvant in human vaccines, was found to be immunogenic and capable of inducing protection against Leishmania infantum, responsible for human and canine visceral leishmaniasis, and against L. major, responsible for cutaneous leishmaniasis. The results demonstrate the feasibility of producing a practical, cost-effective leishmaniasis vaccine capable of protecting both humans and dogs against multiple Leishmania species.
doi:10.1128/CVI.05024-11
PMCID: PMC3147330  PMID: 21632891
22.  Resolution of tongue lesions caused by Leishmania infantum in a dog treated with the association miltefosine-allopurinol 
Parasites & Vectors  2009;2(Suppl 1):S6.
Canine leishmaniosis is a severe systemic disease caused by the kinetoplastid protozoan Leishmania infantum, an obligatory intracellular parasite of mammalian macrophages, transmitted by the bite of phlebotomine sandflies. The infection in dogs might occur without any clinical signs or might be characterised by chronic viscerocutaneous signs, such as lymphadenopathy, skin lesions, splenomegaly, onychogryphosis, and renal as well as ocular damage due to immunocomplex deposition. In atypical cases the parasites can be found in the striated musculature, the central nervous system, the endocrine glands or gonads, with or without functional damage. Leishmania infection might seldom induce oral lesions, particularly on the tongue. The authors describe the clinical case of a four-year old mongrel dog with tongue lesions caused by L. infantum. The dog was presented due to diarrhoea, lack of appetite and hypersalivation. Examination of the oral cavity revealed the presence of multiple red, nodular lesions on the dorsal and lateral surfaces of the tongue. Definite diagnosis of an infection with L. infantum was obtained by an indirect immunofluorescence antibody test (IFAT) and by the cytological identification of the parasite in nodular, lingual lesions and bone marrow aspirates. The dog was treated with a combination of miltefosine (Milteforan®, Virbac), 2 mg/kg orally once a day for four weeks and allopurinol (Ziloric®, GlaxoSmithKline), 10 mg/kg orally twice a day for six months. At the end of the treatment, the animal showed full remission of clinical signs. The authors outline the atypical manifestations in the oral cavity in combination with a L. infantum infection and discuss the therapeutic potential of the combination treatment of miltefosine and allopurinol in canine leishmaniosis.
doi:10.1186/1756-3305-2-S1-S6
PMCID: PMC2679398  PMID: 19426445
23.  Performance of LBSap Vaccine after Intradermal Challenge with L. infantum and Saliva of Lu. longipalpis: Immunogenicity and Parasitological Evaluation 
PLoS ONE  2012;7(11):e49780.
In the last decade, the search for new vaccines against canine visceral leishmaniasis has intensified. However, the pattern related to immune protection during long periods after experimental infection in vaccine trials is still not fully understood. Herein, we investigated the immunogenicity and parasitological levels after intradermal challenge with Leishmania infantum plus salivary gland extract in dogs immunized with a vaccine composed of L. braziliensis antigens plus saponin as an adjuvant (LBSap vaccine). The LBSap vaccine elicited higher levels of total anti-Leishmania IgG as well as both IgG1 and IgG2. Furthermore, dogs vaccinated had increased levels of lymphocytes, particularly circulating B cells (CD21+) and both CD4+ and CD8+ T lymphocytes. LBSap also elicited an intense in vitro cell proliferation associated with higher levels of CD4+ T lymphocytes specific for vaccine soluble antigen and soluble lysate of L. infantum antigen even 885 days after experimental challenge. Furthermore, LBSap vaccinated dogs presented high IFN-γ and low IL-10 and TGF-β1 expression in spleen with significant reduction of parasite load in this tissue. Overall, our results validate the potential of LBSap vaccine to protect against L. infantum experimental infection and strongly support further evaluation of efficiency of LBSap against CVL in natural infection conditions.
doi:10.1371/journal.pone.0049780
PMCID: PMC3506642  PMID: 23189161
24.  Immunobiology of visceral leishmaniasis 
Visceral leishmaniasis (VL), commonly known as kala-azar, is caused by Leishmania donovani and Leishmania infantum (Leishmania chagasi in the Americas). These Leishmania species infect macrophages throughout the viscera, and parasites are typically found in the spleen, liver, and bone marrow. Patients with active disease typically exhibit marked immunosuppression, lack reactivity to the Leishmania skin test (LST), a delayed type hypersensitivity test, and their peripheral blood mononuclear cells (PBMC) fail to respond when stimulated with leishmanial antigens in vitro. However, most people infected with visceralizing species of Leishmania never develop disease. Understanding immune failure and the underlying immune mechanism that lead to disease as well as control of infection are key questions for research in this field. In this review, we discuss immunological events described in human and experimental VL and how these can affect the outcome of infection.
doi:10.3389/fimmu.2012.00251
PMCID: PMC3418610  PMID: 22912637
visceral leishmaniasis; immune regulation; Leishmania donovani; IL-10; T cells
25.  Validity and Reliability of Enzyme Immunoassays Using Leishmania major or L. infantum Antigens for the Diagnosis of Canine Visceral Leishmaniasis in Brazil 
PLoS ONE  2013;8(7):e69988.
Background
American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity.
Methods
A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories.
Results
The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used.
Interpretation
ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil.
doi:10.1371/journal.pone.0069988
PMCID: PMC3724735  PMID: 23922884

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