Mimotope-Based Vaccines of Leishmania infantum Antigens and Their Protective Efficacy against Visceral Leishmaniasis

Background

The development of cost-effective prophylactic strategies to prevent leishmaniasis has become a high-priority. The present study has used the phage display technology to identify new immunogens, which were evaluated as vaccines in the murine model of visceral leishmaniasis (VL). Epitope-based immunogens, represented by phage-fused peptides that mimic Leishmania infantum antigens, were selected according to their affinity to antibodies from asymptomatic and symptomatic VL dogs'' sera.

Methodology/Main Findings

Twenty phage clones were selected after three selection cycles, and were evaluated by means of in vitro assays of the immune stimulation of spleen cells derived from naive and chronically infected with L. infantum BALB/c mice. Clones that were able to induce specific Th1 immune response, represented by high levels of IFN-γ and low levels of IL-4 were selected, and based on their selectivity and specificity, two clones, namely B10 and C01, were further employed in the vaccination protocols. BALB/c mice vaccinated with clones plus saponin showed both a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with individual clones or L. infantum extracts. Additionally, these animals, when compared to control groups (saline, saponin, wild-type phage plus saponin, or non-relevant phage clone plus saponin), showed significant reductions in the parasite burden in the liver, spleen, bone marrow, and paws'' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, mainly by CD8⁺ T cells, against parasite proteins. These animals also presented decreased parasite-mediated IL-4 and IL-10 responses, and increased levels of parasite-specific IgG2a antibodies.

Conclusions/Significance

This study describes two phage clones that mimic L. infantum antigens, which were directly used as immunogens in vaccines and presented Th1-type immune responses, and that significantly reduced the parasite burden. This is the first study that describes phage-displayed peptides as successful immunogens in vaccine formulations against VL.     

Antigenicity,Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis

In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4⁺ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite-specific IgG2a antibodies. The polyproteins vaccine administration induced a more pronounced Th1 response before and after challenge infection than individual vaccines, which was correlated to a higher control of parasite dissemination to internal organs.     

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.     

UCP2 Deficiency Helps to Restrict the Pathogenesis of Experimental Cutaneous and Visceral Leishmaniosis in Mice

Background

Uncoupling protein 2 (UCP2) is a mitochondrial transporter that has been shown to lower the production of reactive oxygen species (ROS). Intracellular pathogens such as Leishmania upregulate UCP2 and thereby suppress ROS production in infected host tissues, allowing the multiplication of parasites within murine phagocytes. This makes host UCP2 and ROS production potential targets in the development of antileishmanial therapies. Here we explore how UCP2 affects the outcome of cutaneous leishmaniosis (CL) and visceral leishmaniosis (VL) in wild-type (WT) C57BL/6 mice and in C57BL/6 mice lacking the UCP2 gene (UCP2KO).

Methodology and Findings

To investigate the effects of host UCP2 deficiency on Leishmania infection, we evaluated parasite loads and cytokine production in target organs. Parasite loads were significantly lower in infected UCP2KO mice than in infected WT mice. We also found that UCP2KO mice produced significantly more interferon-γ (IFN-γ), IL-17 and IL-13 than WT mice (P<0.05), suggesting that UCP2KO mice are resistant to Leishmania infection.

Conclusions

In this way, UCP2KO mice were better able than their WT counterparts to overcome L. major and L. infantum infections. These findings suggest that upregulating host ROS levels, perhaps by inhibiting UPC2, may be an effective approach to preventing leishmaniosis.     

Inaccuracy of Enzyme-Linked Immunosorbent Assay Using Soluble and Recombinant Antigens to Detect Asymptomatic Infection by Leishmania infantum

Background

One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period.

Methodology

Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve.

Findings

The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10).

Conclusions

Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias in population studies. Therefore, studies which have used serological assays to estimate prevalence, to evaluate intervention programs or to identify risk factors for Leishmania infection, may have had their results compromised.     

The Severity of Visceral Leishmaniasis Correlates with Elevated Levels of Serum IL-6, IL-27 and sCD14

Background

Visceral leishmaniasis (VL) is a severe disease caused by infection with protozoa of the genus Leishmania. Classic VL is characterized by a systemic infection of phagocytic cells and an intense activation of the inflammatory response. It is unclear why 90% of infected individuals do not develop the disease while a minority develop the classical form. Furthermore, among those that develop disease, a small group progresses to more severe form that is unresponsive to treatment. The presence of inflammatory mediators in serum could theoretically help to control the infection. However, there is also a release of anti-inflammatory mediators that could interfere with the control of parasite multiplication. In this study, we took advantage of the spectrum of outcomes to test the hypothesis that the immune profile of individuals infected with Leishmania (L.) infantum is associated with the development and severity of disease.

Methodology/Principal Findings

Sera from patients with confirmed diagnosis of VL were evaluated for the presence of numerous molecules, and levels compared with healthy control and asymptomatic infected individuals.

Conclusions/Principal Findings

Although differences were not observed in LPS levels, higher levels of sCD14 were detected in VL patients. Our data suggest that L. infantum may activate the inflammatory response via CD14, stimulating a generalized inflammatory response with production of several cytokines and soluble molecules, including IFN-γ, IL-27, IL-10, IL-6 and sCD14. These molecules were strongly associated with hepatosplenomegaly, neutropenia and thrombocytopenia. We also observed that IL-6 levels greater than 200 pg/ml were strongly associated with death. Together our data reinforce the close relationship of IFN-γ, IL-10, IL-6, TNF-α and IL-27 in the immune dynamics of VL and suggest the direct participation of sCD14 in the activation of the immune response against L. infantum.     

Experimental Infection of Dogs with Leishmania and Saliva as a Model to Study Canine Visceral Leishmaniasis

Background

Canine Visceral Leishmaniasis (CVL) is a zoonotic disease caused by Leishmania infantum, transmitted by the bite of Lutzomyia longipalpis sand flies. Dogs are the main domestic reservoir of the parasite. The establishment of an experimental model that partially reproduces natural infection in dogs is very important to test vaccine candidates, mainly regarding those that use salivary proteins from the vector and new therapeutical approaches.

Methodology/Principal Findings

In this report, we describe an experimental infection in dogs, using intradermal injection of Leishmania infantum plus salivary gland homogenate (SGH) of Lutzomyia longipalpis. Thirty-five dogs were infected with 1×10⁷ parasites combined with five pairs of Lutzomyia longipalpis salivary glands and followed for 450 days after infection and clinical, immunological and parasitological parameters were evaluated. Two hundred and ten days after infection we observed that 31,4% of dogs did not display detectable levels of anti-Leishmania antibodies but all presented different numbers of parasites in the lymph nodes. Animals with a positive xenodiagnosis had at least 3,35×10⁵ parasites in their lymph nodes. An increase of IFN-γ and IL-10 levels was detected during infection. Twenty two percent of dogs developed symptoms of CVL during infection.

Conclusion

The infection model described here shows some degree of similarity when compared with naturally infected dogs opening new perspectives for the study of CVL using an experimental model that employs the combination of parasites and sand fly saliva both present during natural transmission.     

Significantly Lower Anti-Leishmania IgG Responses in Sudanese versus Indian Visceral Leishmaniasis

Background

Visceral leishmaniasis (VL), a widely distributed systemic disease caused by infection with the Leishmania donovani complex (L. donovani and L. infantum), is almost always fatal if symptomatic and untreated. A rapid point-of-care diagnostic test for anti-Leishmania antibodies, the rK39-immunochromatographic test (rK39-ICT), has high sensitivity and specificity in South Asia but is less sensitive in East Africa. One of the underlying reasons may be continent-specific molecular diversity in the rK39 antigen within the L. donovani complex. However, a second reason may be differences in specific IgG anti-Leishmania levels in patients from different geographical regions, either due to variable antigenicity or immunological response.

Methodology/Principal Findings

We determined IgG titres of Indian and Sudanese VL patients against whole cell lysates of Indian and Sudanese L. donovani strains. Indian VL patients had significantly higher IgG titres against both L. donovani strains compared to Sudanese VL patients (p<0.0001). Mean reciprocal log₁₀ 50% end-point titres (1/log₁₀t₅₀) were i) 3.80 and 3.88 for Indian plasma and ii) 2.13 and 2.09 for Sudanese plasma against Indian and Sudanese antigen respectively (p<0.0001). Overall, the Indian VL patients therefore showed a 46.8–61.7 -fold higher mean ELISA titre than the Sudanese VL patients. The higher IgG titres occurred in children (<16 years old) and adults of either sex from India (mean 1/log₁₀t₅₀: 3.60–4.15) versus Sudan (mean 1/log₁₀t₅₀: 1.88–2.54). The greatest difference in IgG responses was between male Indian and Sudanese VL patients of ≥ 16 years old (mean 1/log₁₀t₅₀: 4.15 versus 1.99 = 144-fold (p<0.0001).

Conclusions/Significance

Anti-Leishmania IgG responses among VL patients in Sudan were significantly lower than in India; this may be due to chronic malnutrition with Zn²⁺ deficiency, or variable antigenicity and capacity to generate IgG responses to Leishmania antigens. Such differential anti-Leishmania IgG levels may contribute to lower sensitivity of the rK39-ICT in East Africa.     

A genomic-based approach combining in vivo selection in mice to identify a novel virulence gene in Leishmania

Background

Infection with Leishmania results in a broad spectrum of pathologies where L. infantum and L. donovani cause fatal visceral leishmaniasis and L. major causes destructive cutaneous lesions. The identification and characterization of Leishmania virulence genes may define the genetic basis for these different pathologies.

Methods and Findings

Comparison of the recently completed L. major and L. infantum genomes revealed a relatively small number of genes that are absent or present as pseudogenes in L. major and potentially encode proteins in L. infantum. To investigate the potential role of genetic differences between species in visceral infection, seven genes initially classified as absent in L. major but present in L. infantum were cloned from the closely related L. donovani genome and introduced into L. major. The transgenic L. major expressing the L. donovani genes were then introduced into BALB/c mice to select for parasites with increased virulence in the spleen to determine whether any of the L. donovani genes increased visceral infection levels. During the course of these experiments, one of the selected genes (LinJ32_V3.1040 (Li1040)) was reclassified as also present in the L. major genome. Interestingly, only the Li1040 gene significantly increased visceral infection in the L. major transfectants. The Li1040 gene encodes a protein containing a putative component of an endosomal protein sorting complex involved with protein transport.

Conclusions

These observations demonstrate that the levels of expression and sequence variations in genes ubiquitously shared between Leishmania species have the potential to significantly influence virulence and tissue tropism.     

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.     

Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine visceral leishmaniasis

Background

The immune response in the skin of dogs infected with Leishmania infantum is poorly understood, and limited studies have described the immunopathological profile with regard to distinct levels of tissue parasitism and the clinical progression of canine visceral leishmaniasis (CVL).

Methodology/Principal Findings

A detailed analysis of inflammatory cells (neutrophils, eosinophils, mast cells, lymphocytes, and macrophages) as well as the expression of chemokines (CCL2, CCL4, CCL5, CCL13, CCL17, CCL21, CCL24, and CXCL8) was carried out in dermis skin samples from 35 dogs that were naturally infected with L. infantum. The analysis was based on real-time polymerase chain reaction (PCR) in the context of skin parasitism and the clinical status of CVL. We demonstrated increased inflammatory infiltrate composed mainly of mononuclear cells in the skin of animals with severe forms of CVL and high parasite density. Analysis of the inflammatory cell profile of the skin revealed an increase in the number of macrophages and reductions in lymphocytes, eosinophils, and mast cells that correlated with clinical progression of the disease. Additionally, enhanced parasite density was correlated with an increase in macrophages and decreases in eosinophils and mast cells. The chemokine mRNA expression demonstrated that enhanced parasite density was positively correlated with the expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. In contrast, there was a negative correlation between parasite density and CCL24 expression.

Conclusions/Significance

These findings represent an advance in the knowledge about skin inflammatory infiltrates in CVL and the systemic consequences. Additionally, the findings may contribute to the design of new and more efficient prophylactic tools and immunological therapies against CVL.     

Leishmania infantum Amastigotes Enhance HIV-1 Production in Cocultures of Human Dendritic Cells and CD4+ T Cells by Inducing Secretion of IL-6 and TNF-α

Background

Visceral leishmaniasis has emerged as an important opportunistic disease among patients infected with HIV-1. Both HIV-1 and the protozoan parasite Leishmania can productively infect cells of the macrophage-dendritic cell lineage.

Methodology/Principal Findings

Here we demonstrate that Leishmania infantum amastigotes increase HIV-1 production when human primary dendritic cells (DCs) are cocultured together with autologous CD4⁺ T cells. Interestingly, the promastigote form of the parasite does not modulate virus replication. Moreover, we report that amastigotes promote virus replication in both cell types. Our results indicate that this process is due to secretion of parasite-induced soluble factors by DCs. Luminex micro-beads array system analyses indicate that Leishmania infantum amastigotes induce a higher secretion of several cytokines (i.e. IL-1α, IL-2, IL-6, IL-10 and TNF-α) and chemokines (i.e. MIP-1α, MIP-1β and RANTES) in these cells. Studies conducted with pentoxifylline and neutralizing antibodies revealed that the Leishmania-dependent augmentation in HIV-1 replication is due to a higher secretion of IL-6 and TNF-α.

Conclusions/Significance

Altogether these findings suggest that the presence of Leishmania within DC/T-cell conjugates leads to an enhancement of virus production and demonstrate that HIV-1 and Leishmania can establish complex interactions in such a cellular microenvironment.     

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.     

Cytokine Release Assays as Tests for Exposure to Leishmania,and for Confirming Cure from Leishmaniasis,in Solid Organ Transplant Recipients

Spain has one of the world’s largest pools of organ donors and is a global leader in terms of the number of transplants it performs. The current outbreak of leishmaniasis in Fuenlabrada (in the southwest of the region of Madrid, Spain) has involved 600 clinical cases since late 2009 (prevalence 0.2%). It may therefore be wise to monitor the town’s transplanted population for Leishmania infantum; its members are immunosuppressed and at greater risk of infection and relapse following treatment. The present work examines the use of cytokine release assays to determine the prevalence of Leishmania infection in this population, and to confirm recovery following treatment for visceral leishmaniasis (VL). The humoral and cellular immune responses to L. infantum were characterized in 63 solid organ transplant (SOT) recipients from Fuenlabrada, 57 of whom reported no previous episode of VL (NVL subjects), and six of whom had been cured of VL (CVL subjects). Seventeen subjects (12 NVL and 5 CVL) showed a patent lymphoproliferative response to soluble Leishmania antigen (SLA). Stimulation of peripheral blood mononuclear cell cultures and of whole blood with SLA led to the production of different combinations of cytokines that might serve to confirm Leishmania infection or recovery from VL and help prevent cured patients from relapsing into this serious condition.     

rKLO8, a Novel Leishmania donovani – Derived Recombinant Immunodominant Protein for Sensitive Detection of Visceral Leishmaniasis in Sudan

Background

For effective control of visceral leishmaniasis (VL) in East Africa, new rapid diagnostic tests are required to replace current tests with low sensitivity. The aim of this study is to improve diagnosis of VL in East Africa by testing a new antigen from an autochthonous L. donovani strain in Sudan.

Methodology and Principle Findings

We cloned, expressed and purified a novel recombinant protein antigen of L. donovani from Sudan, designated rKLO8, that contains putative conserved domains with significant similarity to the immunodominant kinesin proteins of Leishmania. rKLO8 exhibited 93% and 88% amino acid identity with cloned kinesin proteins of L. infantum (synonymous L. chagasi) (K39) and L. donovani (KE16), respectively. We evaluated the diagnostic efficiency of the recombinant protein in ELISA for specific detection of VL patients from Sudan. Data were compared with a rK39 ELISA and two commercial kits, the rK39 strip test and the direct agglutination test (DAT). Of 106 parasitologically confirmed VL sera, 104 (98.1%) were tested positive by rKLO8 as compared to 102 (96.2%) by rK39. Importantly, the patients'' sera showed increased reactivity with rKLO8 than rK39. Specificity was 96.1% and 94.8% for rKLO8- and rK39 ELISAs, respectively. DAT showed 100% specificity and 94.3% sensitivity while rK39 strip test performed with 81.1% sensitivity and 98.7% specificity.

Conclusion

The increased reactivity of Sudanese VL sera with the rKLO8 makes this antigen a potential candidate for diagnosis of visceral leishmaniasis in Sudan. However, the suitability at the field level will depend on its performance in a rapid test format.     

Prevalence and factors associated with Leishmania infantum infection of dogs from an urban area of Brazil as identified by molecular methods

Background

Various factors contribute to the urbanization of the visceral leishmaniasis (VL), including the difficulties of implementing control measures relating to the domestic reservoir. The aim of this study was to determine the prevalence of canine visceral leishmaniasis in an urban endemic area in Brazil and the factors associated with Leishmania infantum infection among seronegative and PCR-positive dogs.

Methodology

A cross-sectional study was conducted in Belo Horizonte, Minas Gerais, Brazil. Blood samples were collected from 1,443 dogs. Serology was carried out by using two enzyme-linked immunosorbent assays (Biomanguinhos/FIOCRUZ/RJ and “in house”), and molecular methods were developed, including PCR-RFLP. To identify the factors associated with early stages of infection, only seronegative (n = 1,213) animals were evaluated. These animals were divided into two groups: PCR-positive (n = 296) and PCR-negative (n = 917) for L. infantum DNA. A comparison of these two groups of dogs taking into consideration the characteristics of the animals and their owners was performed. A mixed logistic regression model was used to identify factors associated with L. infantum infection.

Principal Findings

Of the 1,443 dogs examined, 230 (15.9%) were seropositive in at least one ELISA, whereas PCR-RFLP revealed that 356 animals (24.7%) were positive for L. infantum DNA. Results indicated that the associated factors with infection were family incomeConclusionsPCR detected a high prevalence of L. infantum infection in dogs in an area under the Control Program of VL intervention. Socioeconomic variables, dog behavior and the knowledge of the owner regarding the vector were factors associated with canine visceral leishmaniasis (CVL). The absence of previous serological examination conducted by the control program was also associated with L. infantum infection. It is necessary to identify the risk factors associated with CVL to understand the expansion and urbanization of VL.     

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.     

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.     

Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from Leishmania major Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

Background

There are several reports demonstrating the role of CD8 T cells against Leishmania species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II.

Methods and Findings

HLA-A*0201 restricted epitopes from LPG-3, LmSTI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to ex-vivo and in-vitro evaluation of specific IFN-γ production and in-vitro cytotoxicity against individual peptides by ELISpot and standard chromium-51(⁵¹Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, LmSTI-1-P3 and LmSTI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, LmSTI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential.

Conclusions

Here we described a new DNA-polytope construct for Leishmania vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine the efficacy of this polytope construct protecting against infectious challenge of Leishmania. Fortunately HLA transgenic mice are promising preclinical models helping to speed up immunogenicity analysis in a human related mouse model.