Tumor-Suppressive Cross-Linking of Anti-T. cruzi Antibodies in Acute Lymphoblastic Leukemia.

Parasites have been associated with possible anticancer activity, including Trypanosoma cruzi, which has been linked to inhibiting the growth of solid tumors. To better understand this antitumor effect, we investigated the association of anti-T. cruzi antibodies with B cells of the acute lymphoblastic leukemia (ALL) SUPB15 cell line. The antibodies were generated in rabbits. IgGs were purified by affinity chromatography. Two procedures (flow cytometry (CF) and Western blot(WB)) were employed to recognize anti-T. cruzi antibodies on SUPB15 cells. We also used CF to determine whether the anti-T. cruzi antibodies could suppress SUPB15 cells. The anti-T. cruzi antibodies recognized 35.5% of the surface antigens of SUPB15. The complement-dependent cytotoxicity (CDC) results demonstrate the cross-suppression of anti-T. cruzi antibodies on up to 8.4% of SUPB15 cells. For the WB analysis, a band at 100 kDa with high intensity was sequenced using mass spectrometry, identifying the protein as nucleolin. This protein may play a role in the antitumor effect on T. cruzi. The anti-T. cruzi antibodies represent promising polyclonal antibodies that have the effect of tumor-suppressive cross-linking on cancer cells, which should be further investigated.

CD4+ and CD8+ T-cell multi-epitope chimeric protein associated with an MPLA adjuvant induce protective efficacy and long-term immunological memory for the immunoprophylaxis of American Tegumentary Leishmaniasis.

American Tegumentary Leishmaniasis (ATL) is a disease of high severity and incidence in Brazil, in addition to being a worldwide concern in public health. Leishmania amazonensis is one of the etiological agents of ATL, and the inefficiency of control measures, associated with the high toxicity of the treatment and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present study proposes to elaborate a chimeric protein (rChiP), based on the fusion of multiple epitopes of CD4+/CD8+ T cells, identified in the immunoproteome of the parasites L. amazonensis and L. braziliensis. The designed chimeric protein was tested in the L. amazonensis murine model of infection using the following formulations: 25 µg of the rChiP in saline (rChiP group) and 25 µg of the rChiP plus 25 µg of MPLA-PHAD® (rChiP+MPLA group). After completing immunization, CD4+ and CD8+ T cells, stimulated with SLa-Antigen or rChiP, showed an increased production of nitric oxide and intracytoplasmic pro-inflammatory cytokines, in addition to the generation of central and effector memory T cells. rChiP and rChiP+MPLA formulations were able to promote an effective protection against L. amazonensis infection determined by a reduction in the development of skin lesions and lower parasitic burden. Reduction in the development of skin lesions and lower parasitic burden in the vaccinated groups were associated with an increase of nitrite, CD4+/CD8+IFN-γ+TNF-α+ and CD4+/CD8+CD44highCD62Lhigh/low T cells, IgGTotal, IgG2a, and lower rates of IgG1 and CD4+/CD8+IL-10+. This data suggests that proposed formulations could be considered potential tools to prevent ATL.

Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model.

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

Identification and serological responses to a novel Plasmodium vivax merozoite surface protein 1 (PvMSP-1) derived synthetic peptide: a putative biomarker for malaria exposure.

Background: The integration of diagnostic methods holds promise for advancing the surveillance of malaria transmission in both endemic and non-endemic regions. Serological assays emerge as valuable tools to identify and delimit malaria transmission, serving as a complementary method to rapid diagnostic tests (RDT) and thick smear microscopy. Here, we evaluate the potential of antibodies directed against peptides encompassing the entire amino acid sequence of the PvMSP-1 Sal-I strain as viable serological biomarkers for P. vivax exposure. Methods: We screened peptides encompassing the complete amino acid sequence of the Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-1) Sal-I strain as potential biomarkers for P. vivax exposure. Here, immunodominant peptides specifically recognized by antibodies from individuals infected with P. vivax were identified using the SPOT-synthesis technique followed by immunoblotting. Two 15-mer peptides were selected based on their higher and specific reactivity in immunoblotting assays. Subsequently, peptides p70 and p314 were synthesized in soluble form using SPPS (Solid Phase Peptide Synthesis) and tested by ELISA (IgG, and subclasses). Results: This study unveils the presence of IgG antibodies against the peptide p314 in most P. vivax-infected individuals from the Brazilian Amazon region. In silico B-cell epitope prediction further supports the utilization of p314 as a potential biomarker for evaluating malaria transmission, strengthened by its amino acid sequence being part of a conserved block of PvMSP-1. Indeed, compared to patients infected with P. falciparum and uninfected individuals never exposed to malaria, P. vivax-infected patients have a notably higher recognition of p314 by IgG1 and IgG3.

ELISA with recombinant antigen Lb6H validated for the diagnosis of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.

Increased Natural Killer (NK)-cell cytotoxicity and Trypanosoma cruzi-specific memory B cells in subjects with discordant serology for Chagas disease.

The presence of memory T cell specific for Trypanosoma cruzi in subjects with discordant serology for Chagas disease supports a cleared infection in these subjects. Using high-dimensional flow cytometry, ELISPOT assays and quantitative PCR, antibody-secreting cells and memory B cells specific for T. cruzi, total B-cell phenotypes, innate immune responses and parasite DNA were evaluated in serodiscordant, seropositive and seronegative subjects for T. cruzi infection. T. cruzi-specific memory B cells but no antibody-secreting cells specific for T. cruzi, increased proportion of nonclassical monocytes and increased levels of polyfunctional NK cells were found in serodiscordant compared with seropositive subjects. None of the serodiscordant subjects evaluated showed detectable parasite DNA, most of them did not show cardiac abnormalities and a group of them had had confirmed positive serology for Chagas disease. The unique immune profiles in serodiscordant subjects support that T. cruzi infection was cleared or profoundly controlled in these subjects.

Diagnosis of Canine Visceral Leishmaniasis by Flow Cytometry Serology using the rMELEISH Multiepitope Antigen Coupled in a Functional Bead.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic disease, with dogs being the main reservoir of the Leishmania infantum parasite. OBJECTIVE: To develop a new flow cytometry test to diagnosis canine VL (CVL) diagnosis. METHODS: The current study addresses a new flow cytometry test using beads coupled to the multiepitope antigen rMELEISH. RESULTS: In the study set of samples a sensitivity (87.1%) and specificity (89.9%) was observed. Considering the dogs' clinical status, 20/20 (100.0%) of the symptomatic sera tested positive, while 19/22 (86.4%) of the oligosymptomatic and 16/20 (80.0%) of asymptomatic were positive. In the non-infected control, all samples (0/30) tested as negative. In the cross-reaction control, the test was more efficient in dogs infected with L. braziliensis (2/10) and Trypanosoma cruzi (0/10), than those with Babesia canis (4/10) and Ehrlichia canis (4/10). Dogs immunized with different vaccines (Leishmune, Leish-Tec®, or LBSap) did not present serological reactivity. CONCLUSION: The flow cytometry serology through coupling the antigen rMELEISH in functional beads showed high accuracy in diagnosing CVL.

Multiplexed Microsphere-Based Flow Cytometric Assay to Assess Strain Transcending Antibodies to Plasmodium vivax Duffy Binding Protein II Reveals an Efficient Tool to Identify Binding-Inhibitory Antibody Responders.

Malaria remains a major public health problem worldwide, and Plasmodium vivax is the most widely distributed malaria parasite. Naturally acquired binding inhibitory antibodies (BIAbs) to region II of the Duffy binding protein (DBPII), a P. vivax ligand that is critical for reticulocyte invasion, are associated with a reduced risk of clinical malaria. Owing to methodological issues in evaluating antibodies that inhibit the DBPII-DARC interaction, a limited number of studies have investigated DBPII BIAbs in P. vivax-exposed populations. Based on the assumption that individuals with a consistent BIAb response are characterized by strain-transcending immune responses, we hypothesized that detecting broadly reactive DBPII antibodies would indicate the presence of BIAb response. By taking advantage of an engineered DBPII immunogen targeting conserved DBPII neutralizing epitopes (DEKnull-2), we standardized a multiplex flow cytometry-based serological assay to detect broadly neutralizing IgG antibodies. For this study, a standard in vitro cytoadherence assay with COS-7 cells expressing DBPII was used to test for DBPII BIAb response in long-term P. vivax-exposed Amazonian individuals. Taken together, the results demonstrate that this DBPII-based multiplex assay facilitates identifying DBPII BIAb carriers. Of relevance, the ability of the multiplex assay to identify BIAb responders was highly accurate when the positivity for all antigens was considered. In conclusion, the standardized DBPII-based flow cytometric assay confirmed that DBPII-BIAb activity was associated with the breadth rather than the magnitude of anti-DBPII antibodies. Altogether, our results suggest that multiplex detection of broadly DBPII-reactive antibodies facilitates preliminary screening of BIAb responders.

HIV and Chagas Disease: An Evaluation of the Use of Real-Time Quantitative Polymerase Chain Reaction to Measure Levels of Trypanosoma cruzi Parasitemia in HIV Patients in Cochabamba, Bolivia.

This cross-sectional study evaluated epidemiologic characteristics of persons living with HIV (PWH) coinfected with Trypanosoma cruzi in Cochabamba, Bolivia, and estimated T. cruzi parasitemia by real-time quantitative polymerase chain reaction (qPCR) in patients with and without evidence of reactivation by direct microscopy. Thirty-two of the 116 HIV patients evaluated had positive serology for T. cruzi indicative of chronic Chagas disease (27.6%). Sixteen of the 32 (50%) patients with positive serology were positive by quantitative polymerase chain reaction (qPCR), and four of the 32 (12.5%) were positive by direct microscopy. The median parasite load by qPCR in those with CD4+ < 200 was 168 parasites/mL (73-9951) compared with 28.5 parasites/mL (15-1,528) in those with CD4+ ≥ 200 (P = 0.89). There was a significant inverse relationship between the degree of parasitemia estimated by qPCR from blood clot and CD4+ count on the logarithmic scale (rsBC= -0.70, P = 0.007). The correlation between T. cruzi estimated by qPCR+ blood clot and HIV viral load was statistically significant with rsBC = 0.61, P = 0.047. Given the significant mortality of PWH and Chagas reactivation and that 57% of our patients with CD4+ counts < 200 cells/mm3 showed evidence of reactivation, we propose that screening for chronic Chagas disease be considered in PWH in regions endemic for Chagas disease and in the immigrant populations in nonendemic regions. Additionally, our study showed that PWH with advancing immunosuppression have higher levels of estimated parasitemia measured by qPCR and suggests a role for active surveillance for Chagas reactivation with consideration of treatment with antitrypanosomal therapy until immune reconstitution can be achieved.

Assessment of Liaison XL Murex Chagas diagnostic performance in blood screening for Chagas disease using a reference array of chimeric antigens.

BACKGROUND: Chagas disease (CD) serological screening at blood banks is usually performed by a single highly sensitive serological assay, with chemiluminescent immunoassays (CLIAs) being the method of choice. CLIAs employ recombinant, fusion peptides and/or chimeric antigens that selectively capture anti-Trypanosoma cruzi antibodies. However, despite high sensitivity, the ability of these tests to identify CD-positive cases should be evaluated against T. cruzi strains circulating in specific locales. Herein, we used a latent class analysis (LCA) approach employing an array of four chimeric antigens to assess the diagnostic performance of the Liaison XL Murex Chagas CLIA for the detection of anti-T. cruzi IgG in serum samples. STUDY DESIGN AND METHODS: The study included a panel of 5014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia, submitted to anti-T. cruzi antibody detection using Liaison Chagas CLIA and LCA as a reference test in the absence of a gold standard. RESULTS: LCA classified 4993 samples as negative, while positivity for T. cruzi antibodies was predicted in 21 samples. Compared with LCA, CLIA demonstrated sensitivity and specificity of 76.2% and 99.5%, respectively, providing an overall accuracy of 99.4%. DISCUSSION: In blood banks lacking a de facto highly sensitive screening immunoassay, the low sensitivity offered by Liaison Chagas CLIA renders it unsuitable for standalone use in serological screening procedures for CD. Moreover, blood banks are encouraged to carefully assess the ability of diagnostic methods to identify local T. cruzi strains in circulation.

Assessment of a recombinant protein from Leishmania infantum as a novel tool for Visceral Leishmaniasis (VL) diagnosis in VL/HIV co-infection cases.

Visceral Leishmaniasis and HIV-AIDS coinfection (VL/HIV) is considered a life-threatening pathology when undiagnosed and untreated, due to the immunosuppression caused by both diseases. Serological tests largely used for the VL diagnosis include the direct agglutination test (DAT), ELISA and immunochromatographic (ICT) assays. For VL diagnosis in HIV infections, different studies have shown that the use of the DAT assay facilitates the VL diagnosis in co-infected patients, since the performance of the most widely used ELISA and ICT tests, based on the recombinant protein rK39, are much less efficient in HIV co-infections. In this scenario, alternative recombinant antigens may help the development of new serological diagnostic methods which may improve the VL diagnosis for the co-infection cases. This work aimed to evaluate the use of the recombinant Lci2 antigen, related to, but antigenically more diverse than rK39, for VL diagnosis in co-infected sera through ELISA assays. A direct comparison between recombinant Lci2 and rK39 was thus carried out. The two proteins were first tested using indirect ELISA with sera from VL afflicted individuals and healthy controls, with similar performances. They were then tested with two different sets of VL/HIV co-infected cases and a significant drop in performance, for one of these groups, was observed for rK39 (32% sensitivity), but not for Lci2 (98% sensitivity). In fact, an almost perfect agreement (Kappa: 0.93) between the Lci2 ELISA and DAT was observed for the coinfected VL/HIV patients. Lci2 then has the potential to be used as a new tool for the VL diagnosis of VL/HIV co-infections.

Autoantibodies against the immunodominant sCha epitope discriminate the risk of sudden death in chronic Chagas cardiomyopathy.

In Chagas disease (ChD) caused by Trypanosoma cruzi, new biomarkers to predict chronic cardiac pathology are urgently needed. Previous studies in chagasic patients with mild symptomatology showed that antibodies against the immunodominant R3 epitope of sCha, a fragment of the human basic helix-loop-helix transcription factor like 5, correlated with cardiac pathology. To validate sCha as a biomarker and to understand the origin of anti-sCha antibodies, we conducted a multicenter study with several cohorts of chagasic patients with severe cardiac symptomatology. We found that levels of antibodies against sCha discriminated the high risk of sudden death, indicating they could be useful for ChD prognosis. We investigated the origin of the antibodies and performed an alanine scan of the R3 epitope. We identified a minimal epitope MRQLD, and a BLAST search retrieved several T. cruzi antigens. Five of the hits had known or putative functions, of which phosphonopyruvate decarboxylase showed the highest cross-reactivity with sCha, confirming the role of molecular mimicry in the development of anti-sCha antibodies. Altogether, we demonstrate that the development of antibodies against sCha, which originated by molecular mimicry with T. cruzi antigens, could discriminate electrocardiographic alterations associated with a high risk of sudden death.

Innate and humoral immune parameters at delivery in colostrum and calves from heifers experimentally infected with Neospora caninum.

Neospora caninum is a leading cause of abortion in cattle worldwide. The study of the immune response against N. caninum is critical to understand its epidemiology, pathogenesis, diagnosis and, ultimately, in preventing and controlling bovine neosporosis. Herein, we determined the gene expression of innate immune components endosomal RNA-sensing TLRs, BMAP28 cathelicidin, TNF-α and IL-10 and characterized the variation in both IgG ratio and avidity at delivery in N. caninum-infected heifers challenged at day 210 of gestation, colostrum and their calves. Increased BMAP28 expression was observed not only in colostrum but also in peripheral blood mononuclear cells (PBMC) and umbilical cord of calves from N. caninum-infected heifers in comparison with mock-infected control group. In addition, statistically significant decrease of TLR7 and IL-10 expression levels were observed in umbilical cord, suggesting an attempt to avoid an exacerbated immune response against the parasite. At delivery, serum and colostrum samples from infected group evidenced specific IgG anti-N. caninum. Infected heifers showed IgG1/IgG2 ratios <1 and high avidity specific IgG. As expected, colostrum samples of these animals exhibited a high IgG1 concentration and elevated avidity values. Three out of four calves from N. caninum-infected heifers had specific IgG with IgG1/IgG2 ratios>1 and lower avidity values before colostrum intake. Interestingly, both IgG1/IgG2 ratios and avidity values increased in seropositive calves after colostrum intake. Overall, this study provides novel information on neonatal immunity in congenitally infected calves, which is essential to understand how the immune pathways could be manipulated or immune components could be employed in order to improve protection against neosporosis.

Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system.

BACKGROUND: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. METHODS: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications. FINDINGS: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. INTERPRETATION: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.

Cruzipain and Its Physiological Inhibitor, Chagasin, as a DNA-Based Therapeutic Vaccine Against Trypanosoma cruzi.

Chagas disease caused by the protozoan parasite Trypanosoma cruzi is endemic in 21 Latin American countries and the southern United States and now is spreading into several other countries due to migration. Despite the efforts to control the vector throughout the Americas, currently, there are almost seven million infected people worldwide, causing ~10,000 deaths per year, and 70 million people at risk to acquire the infection. Chagas disease treatment is restricted only to two parasiticidal drugs, benznidazole and nifurtimox, which are effective during the acute and early infections but have not been found to be as effective in chronic infection. No prophylactic or therapeutic vaccine for human use has been communicated at this moment. Here, we evaluate in a mouse model a therapeutic DNA vaccine combining Cruzipain (Cz), a T. cruzi cysteine protease that proved to be protective in several settings, and Chagasin (Chg), which is the natural Cz inhibitor. The DNAs of both antigens, as well as a plasmid encoding GM-CSF as adjuvant, were orally administrated and delivered by an attenuated Salmonella strain to treat mice during the acute phase of T. cruzi infection. The bicomponent vaccine based on Salmonella carrying Cz and Chg (SChg+SCz) was able to improve the protection obtained by each antigen as monocomponent therapeutic vaccine and significantly increased the titers of antigen- and parasite-specific antibodies. More importantly, the bicomponent vaccine triggered a robust cellular response with interferon gamma (IFN-γ) secretion that rapidly reduced the parasitemia during the acute phase and decreased the tissue damage in the chronic stage of the infection, suggesting it could be an effective tool to ameliorate the pathology associated to Chagas disease.

Early high avidity specific IgG production in experimental hamster visceral leishmaniasis.

Visceral leishmaniasis (VL) by Leishmania (Leishmania) infantum is epidemic in Brazil. Hypergammaglobulinemia appears early in patients with VL and is ineffective. Usually, high-affinity IgG B cells are selected during most infections, a critical step for an effective humoral response. The avidity of IgG antibodies in VL is unexplored due to the absence of temporal parameters in most patients, associated to low clinical significance. Experimental infection models overcome this fact, allowing the monitoring of the disease temporal evolution. In this study, the avidity of IgG antibodies was evaluated in experimental models, in infection in hamsters, and in immunization in rabbits. Specific IgG antibodies were detected by ELISA, using chaotropic solution to determine avidity, as reported for viral infections. The levels of IgG antibodies correlated with the progression of experimental infection in hamsters or antigenic stimulation in immunized rabbits. However, IgG avidity was high early in infected animals, even in early periods (> 80%), while in immunized rabbits, they had early antibodies of low avidity with progressive maturation, similar as other infections. These data suggest that the affinity maturation of the avidity of anti-Leishmania IgG antibodies promoted at an early stage, influencing the appropriate interaction between antigens and affecting the disease progression. This fact could be associated to monovalent immune complexes, as reported in human and experimental VL. This scenario may be related to an independent process of immune cell activation by the parasite but absent in antigen preparation used as immunogens.

A Leishmania amastigote-specific hypothetical protein evaluated as recombinant protein plus Th1 adjuvant or DNA plasmid-based vaccine to protect against visceral leishmaniasis.

Most studies evaluating vaccine candidates against visceral leishmaniasis (VL) have used parasite promastigote-expressed antigens; however, Leishmania proteins expressed in the amastigote forms should be considered, since few hours after infection this stage comes into contact with the host immune system and is responsible for the development of the disease. In this context, in the present study, a Leishmania amastigote-specific hypothetical protein, called LiHyJ, was evaluated as a recombinant protein plus saponin as an adjuvant or DNA vaccine to protect against VL. The vaccine effect was evaluated by means of the evaluation of immunological and parasitological analyses performed in BALB/c mice against Leishmania infantum infection. Results showed that rLiHyJ/saponin and DNA LiHyJ induced significantly higher levels of anti-protein and anti-parasite IFN-γ, IL-12, GM-CSF, and IgG2a isotype antibodies, which were associated with a low presence of IL-4 and IL-10. DNA vaccination induced higher IFN-γ production, mainly by CD8+ T cells, while rLiHyJ/saponin stimulated the production of this cytokine, mainly by CD4+ T cells. The parasite load evaluated in distinct organs showed that both immunization schedules significantly reduced organic parasitism, when compared to the controls. Similar results were found in the immunological and parasitological assays when using the recombinant protein or DNA, although the vaccination with rLiHyJ plus saponin induced a slightly higher Th1 response and lower parasite load, when compared to the use of DNA plasmid. The protein also proved to be immunogenic when peripheral blood mononuclear cells of treated VL patients and healthy subjects were in vitro stimulated, since higher IFN-γ and lower IL-4 and IL-10 levels were found in the culture supernatants. In conclusion, LiHyJ should be considered in future studies as a vaccine candidate to protect against VL.

Recombinant Plasmodium vivax circumsporozoite surface protein allelic variants: antibody recognition by individuals from three communities in the Brazilian Amazon.

Circumsporozoite protein (CSP) variants of P. vivax, besides having variations in the protein repetitive portion, can differ from each other in aspects such as geographical distribution, intensity of transmission, vectorial competence and immune response. Such aspects must be considered to P. vivax vaccine development. Therefore, we evaluated the immunogenicity of novel recombinant proteins corresponding to each of the three P. vivax allelic variants (VK210, VK247 and P. vivax-like) and of the C-terminal region (shared by all PvCSP variants) in naturally malaria-exposed populations of Brazilian Amazon. Our results demonstrated that PvCSP-VK210 was the major target of humoral immune response in studied population, presenting higher frequency and magnitude of IgG response. The IgG subclass profile showed a prevalence of cytophilic antibodies (IgG1 and IgG3), that seem to have an essential role in protective immune response. Differently of PvCSP allelic variants, antibodies elicited against C-terminal region of protein did not correlate with epidemiological parameters, bringing additional evidence that humoral response against this protein region is not essential to protective immunity. Taken together, these findings increase the knowledge on serological response to distinct PvCSP allelic variants and may contribute to the development of a global and effective P. vivax vaccine.

Delayed-type hypersensitivity skin test against Neospora caninum in heifers with undetectable specific antibodies.

Delayed-type hypersensitivity (DTH) has been used in human and veterinary medicine as a skin testing for evaluating in vivo cell-mediated immune responses (CMIR). Whereas CMIR is a key process to control intracellular pathogens, its value at identifying cattle exposed to the abortigenic intracellular coccidian parasite Neospora caninum is unknown. In this work, we have evaluated a DTH skin testing in cattle exposed to N. caninum and still seronegative. Female calves were experimentally sensitized by subcutaneous (SC) inoculation with live tachyzoites of N. caninum (NC-Argentina LP1) in sterile phosphate-buffered saline (PBS) (group A; n: 8) whereas other calveswere mock-sensitized with PBS (group B; n: 6). Two DTH skin tests were performed by intradermal inoculation with a soluble lysate of N. caninum tachyzoites (NC-Argentina LP1) in the neck region at 60d and 960 d after sensitization. Skinfold thickness at the intradermal inoculation site was measured at 0, 24, 48 h post each DTH skin test and skin biopsies taken for microscopic evaluation. Specific N. caninum antibodies kinetics was evaluated all throughthe experiment. We found that whereas N. caninum specific antibodies remained below the ELISA cut-off, a distinctive skinfold thickness increase was detected in sensitized animals (group A) at the DTH skin test site, showing induration, swelling and inflammatory infiltration. Mock sensitized animals (group B) showed no skinfold thickness growth and lacked specific antibody response. Thus, N. caninum DTH skin testing could be a useful diagnostic tool for the detection of CMIR during N. caninum infection in non-humoral responders.

Heterologous Chimeric Construct Comprising a Modified Bacterial Superantigen and a Cruzipain Domain Confers Protection Against Trypanosoma cruzi Infection.

Chagas disease is an endemic chronic parasitosis in Latin America affecting more than 7 million people. Around 100 million people are currently at risk of acquiring the infection; however, no effective vaccine has been developed yet. Trypanosoma cruzi is the etiological agent of this parasitosis and as an intracellular protozoan it can reside within different tissues, mainly muscle cells, evading host immunity and allowing progression towards the chronic stage of the disease. Considering this intracellular parasitism triggers strong cellular immunity that, besides being necessary to limit infection, is not sufficient to eradicate the parasite from tissues, a differential immune response is required and new strategies for vaccines against Chagas disease need to be explored. In this work, we designed, cloned and expressed a chimeric molecule, named NCz-SEGN24A, comprising a parasite antigen, the N-terminal domain of the major cysteine protease of T. cruzi, cruzipain (Nt-Cz), and a non-toxic form of the staphylococcal superantigen (SAg) G, SEG, with the residue Asn24 mutated to Ala (N24A). The mutant SAg SEGN24A, retains its ability to trigger classical activation of macrophages without inducing T cell apoptosis. To evaluate, as a proof of concept, the immunogenicity and efficacy of the chimeric immunogen vs. its individual antigens, C3H mice were immunized intramuscularly with NCz-SEGN24A co-adjuvanted with CpG-ODN, or the recombinant proteins Nt-Cz plus SEGN24A with the same adjuvant. Vaccinated mice significantly produced Nt-Cz-specific IgG titers after immunization and developed higher IgG2a than IgG1 titers. Specific cell-mediated immunity was assessed by in-vivo DTH and significant responses were obtained. To assess protection, mice were challenged with trypomastigotes of T. cruzi. Both schemes reduced the parasite load throughout the acute phase, but only mice immunized with NCz-SEGN24A showed significant differences against control; moreover, these mice maintained 100% survival. These results encourage testing mutated superantigens fused to specific antigens as immune modulators against pathogens.