PTML Model for Proteome Mining of B-Cell Epitopes and Theoretical-Experimental Study of Bm86 Protein Sequences from Colima, Mexico.

In this work, we developed a general perturbation theory and machine learning method for data mining of proteomes to discover new B-cell epitopes useful for vaccine design. The method predicts the epitope activity εq(cqj) of one query peptide (q-peptide) under a set of experimental query conditions (cqj). The method uses as input the sequence of the q-peptide. The method also uses as input information about the sequence and epitope activity εr(crj) of a peptide of reference (r-peptide) assayed under similar experimental conditions (crj). The model proposed here is able to classify 1 048 190 pairs of query and reference peptide sequences from the proteome of many organisms reported on IEDB database. These pairs have variations (perturbations) under sequence or assay conditions. The model has accuracy, sensitivity, and specificity between 71 and 80% for training and external validation series. The retrieved information contains structural changes in 83 683 peptides sequences (Seq) determined in experimental assays with boundary conditions involving 1448 epitope organisms (Org), 323 host organisms (Host), 15 types of in vivo process (Proc), 28 experimental techniques (Tech), and 505 adjuvant additives (Adj). Afterward, we reported the experimental sampling, isolation, and sequencing of 15 complete sequences of Bm86 gene from state of Colima, Mexico. Last, we used the model to predict the epitope immunogenic scores under different experimental conditions for the 26 112 peptides obtained from these sequences. The model may become a useful tool for epitope selection toward vaccine design. The theoretical-experimental results on Bm86 protein may help the future design of a new vaccine based on this protein.

Immune protection against Trypanosoma cruzi induced by TcVac4 in a canine model.

Chagas disease, caused by Trypanosoma cruzi, is endemic in southern parts of the American continent. Herein, we have tested the protective efficacy of a DNA-prime/T. rangeli-boost (TcVac4) vaccine in a dog (Canis familiaris) model. Dogs were immunized with two-doses of DNA vaccine (pcDNA3.1 encoding TcG1, TcG2, and TcG4 antigens plus IL-12- and GM-CSF-encoding plasmids) followed by two doses of glutaraldehyde-inactivated T. rangeli epimastigotes (TrIE); and challenged with highly pathogenic T. cruzi (SylvioX10/4) isolate. Dogs given TrIE or empty pcDNA3.1 were used as controls. We monitored post-vaccination and post-challenge infection antibody response by an ELISA, parasitemia by blood analysis and xenodiagnosis, and heart function by electrocardiography. Post-mortem anatomic and pathologic evaluation of the heart was conducted. TcVac4 induced a strong IgG response (IgG2>IgG1) that was significantly expanded post-infection, and moved to a nearly balanced IgG2/IgG1 response in chronic phase. In comparison, dogs given TrIE or empty plasmid DNA only developed high IgG titers with IgG2 predominance in response to T. cruzi infection. Blood parasitemia, tissue parasite foci, parasite transmission to triatomines, electrocardiographic abnormalities were significantly lower in TcVac4-vaccinated dogs than was observed in dogs given TrIE or empty plasmid DNA only. Macroscopic and microscopic alterations, the hallmarks of chronic Chagas disease, were significantly decreased in the myocardium of TcVac4-vaccinated dogs. We conclude that TcVac4 induced immunity was beneficial in providing resistance to T. cruzi infection, evidenced by control of chronic pathology of the heart and preservation of cardiac function in dogs. Additionally, TcVac4 vaccination decreased the transmission of parasites from vaccinated/infected animals to triatomines.

Preventive and therapeutic DNA vaccination partially protect dogs against an infectious challenge with Trypanosoma cruzi.

American trypanosomiasis, or Chagas disease, is caused by Trypanosoma cruzi, and a vaccine would greatly improve disease control. While some studies in mice suggest that a vaccine is feasible, limited efficacy has been observed in dogs. We evaluated here the safety and efficacy of a DNA vaccine encoding TSA-1 and Tc24 antigens in a dog model of acute T. cruzi infection. Mongrel dogs were immunized with two doses of 500 µg of DNA vaccine, two weeks apart, and infected with T. cruzi (SylvioX10/4 strain) two weeks after the second vaccine dose. Another group of dogs was infected first and treated with the vaccine. Disease progression was monitored for up to 70 days post-infection. The vaccine did not induce any critical change in blood parameters, nor exacerbation of disease in vaccinated animals. On the contrary, it prevented anemia and a decrease in lymphocyte counts following T. cruzi infection in vaccinated dogs. Both preventive and therapeutic vaccination significantly reduced parasitemia, cardiac inflammation and cardiac parasite burden, and tended to reduce the development of cardiac arrhythmias. These results indicate that a preventive or therapeutic DNA vaccine encoding TSA-1 and Tc24 antigens is safe and may reduce both parasite transmission and the clinical progression of Chagas disease in vaccinated dogs. This DNA vaccine may thus be an excellent veterinary vaccine candidate. These data also further strengthen the feasibility of a Chagas disease vaccine for humans.

Antigenicity and diagnostic potential of vaccine candidates in human Chagas disease.

BACKGROUND: Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease. METHODS AND RESULTS: Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1(st)-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2(nd)-phase for antibody response to the recombinant antigens (individually or mixed) by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n=175). The IgG antibodies to TcG1, TcG2, and TcG4 (individually) and TcG(mix) were present in 62-71%, 65-78% and 72-82%, and 89-93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%), TcG2- (96%), TcG4- (94.6%) and TcG(mix)- (98%) based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8%) in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects. CONCLUSIONS: Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations.

Seroprevalence survey of American trypanosomiasis in Central Valley of Toluca.

American trypanosomiasis is a growing health issue in the Americas. México is an endemic country, where some locations such as in the State of México are considered highly prevalent. In the valley of Toluca city, the capital of the State of Mexico, there exists an apparent high prevalence in dogs. The absence of triatomine vectors suggests that dogs may not be infected. Therefore, we conducted a directed survey to domiciliated and nondomiciliated dogs to reassess dogs' T. cruzi seroprevalence status. HAI and ELISA serologic tests were applied to 124 and 167 serums of domiciliated and nondomiciliated dogs in the target city. Risk factors were estimated, but the results did not show any evidence to assess them. No domiciliated dogs tested positive to both tests, whereas only one non-domiciliated dog resulted positive. This animal may have acquired the infection in an endemic area and then migrated to Toluca. Research results indicate that T. cruzi infection is not actively transmitted among dogs, and it is pointed out that dogs are the main sentinel animal population to evaluate a possible expansion of the territory affected by Chagas' disease.

Evaluation of clinical and immunopathological features of different infective doses of Trypanosoma cruzi in dogs during the acute phase.

Infection with Trypanosoma cruzi is a major risk in Latin America, and dogs are believed to be good models for evaluating Chagas disease. Here, we evaluated the clinical and immunopathological alterations developed by mongrel dogs experimentally infected with different infective doses (2,000, 20,000, and 200,000 metacyclic trypomastigotes of Sylvio X10/4 strain kg(-1) via intraperitoneal). Clinical and electrocardiographic parameters, as well as antibody production and pathologic lesions were evaluated. All three doses of this strain of T. cruzi induced a similar pattern of infection characterized by cardiac arrhythmias and severe and diffuse myocarditis. Specific anti-T. cruzi IgG indicated seroconversion by day 14 after infection, and IgG levels increased during the period of evaluation. Mortality was observed only in dogs infected with the medium or high parasite doses, but not in the group infected with a low dose of 2,000 parasites kg(-1). Infection with a low dose of parasites provides an excellent nonlethal model to evaluate the immunopathology of the acute disease in dogs infected with the Sylvio X10/4 strain of T. cruzi.

Vaccine development against Trypanosoma cruzi and Chagas disease.

The pathology of Chagas disease presents a complicated and diverse picture in humans. The major complications and destructive evolutionary outcomes of chronic infection by Trypanosoma cruzi in humans include ventricular fibrillation, thromboembolism and congestive heart failure. Studies in animal models and human patients have revealed the pathogenic mechanisms during disease progression, pathology of disease and features of protective immunity. Accordingly, several antigens, antigen-delivery vehicles and adjuvants have been tested to elicit immune protection to T. cruzi in experimental animals. This review summarizes the research efforts in vaccine development against Chagas disease during the past decade.

Testing the efficacy of a multi-component DNA-prime/DNA-boost vaccine against Trypanosoma cruzi infection in dogs.

BACKGROUND: Trypanosoma cruzi, the etiologic agent of Chagas Disease, is a major vector borne health problem in Latin America and an emerging infectious disease in the United States. METHODS: We tested the efficacy of a multi-component DNA-prime/DNA-boost vaccine (TcVac1) against experimental T. cruzi infection in a canine model. Dogs were immunized with antigen-encoding plasmids and cytokine adjuvants, and two weeks after the last immunization, challenged with T. cruzi trypomastigotes. We measured antibody responses by ELISA and haemagglutination assay, parasitemia and infectivity to triatomines by xenodiagnosis, and performed electrocardiography and histology to assess myocardial damage and tissue pathology. RESULTS: Vaccination with TcVac1 elicited parasite-and antigen-specific IgM and IgG (IgG2>IgG1) responses. Upon challenge infection, TcVac1-vaccinated dogs, as compared to non-vaccinated controls dogs, responded to T. cruzi with a rapid expansion of antibody response, moderately enhanced CD8(+) T cell proliferation and IFN-γ production, and suppression of phagocytes' activity evidenced by decreased myeloperoxidase and nitrite levels. Subsequently, vaccinated dogs controlled the acute parasitemia by day 37 pi (44 dpi in non-vaccinated dogs), and exhibited a moderate decline in infectivity to triatomines. TcVac1-immunized dogs did not control the myocardial parasite burden and electrocardiographic and histopatholgic cardiac alterations that are the hallmarks of acute Chagas disease. During the chronic stage, TcVac1-vaccinated dogs exhibited a moderate decline in cardiac alterations determined by EKG and anatomo-/histo-pathological analysis while chronically-infected/non-vaccinated dogs continued to exhibit severe EKG alterations. CONCLUSIONS: Overall, these results demonstrated that TcVac1 provided a partial resistance to T. cruzi infection and Chagas disease, and provide an impetus to improve the vaccination strategy against Chagas disease.

Trypanosoma cruzi in dogs: electrocardiographic and echocardiographic evaluation, in Malinalco, State of Mexico.

Chagas disease caused by Trypanosoma cruzi is an important public health problem in Latin America. Dogs are considered a risk factor for human Chagas disease, a sentinel for T. cruzi infection in endemic regions and an animal model to study pathological aspects of the disease. The potential use of dogs as indicators of human cardiac pathogenicity of local T. cruzi strains has been studied insufficiently. We studied electrocardiographic (EKG) and echocardiographic (ECG) alteration frequencies observed in an open population of dogs in Malinalco, Mexico, and determined if such frequencies were statistically associated with T. cruzi infection in dogs. Animals (n = 139) were clinically examined and owners were asked to answer a questionnaire about dogs' living conditions. Two commercial serological tests (IHA, ELISA) were conducted to detect anti-T. cruzi serum antibodies. Significant differences between seropositive and seronegative animals in cardiomyopathic frequencies were detected through EKG and ECG (P < 0.05). Thirty dogs (21.58%) were serologically positive to anti-T. cruzi antibodies (to ELISA and IHA assays), of which nine (30%) had EKG and/or ECG alterations. From the remaining 104 (78.42%) seronegative animals, five (4.5%) had EKG and/or ECG abnormalities. Our data support the hypothesis that most EKG and ECG alterations found in dogs from Malinalco could be associated with T. cruzi infection. Considering the dog as a sentinel and as an animal model for Chagas disease in humans, our findings suggest that the T. cruzi strains circulating in Malinalco have the potential to produce cardiomyopathies in infected humans.

Risk factors associated with triatomines and its infection with Trypanosoma cruzi in rural communities from the southern region of the State of Mexico, Mexico.

Trypanosoma cruzi prevalence in triatomines and risk factors associated to the presence of the insect were studied in 990 rural houses in the southern region of the State of Mexico, Mexico. In each house, triatomines were collected, and information related to house construction material was obtained. T. cruzi infection was diagnosed in all triatomines. A primary screening was performed using 2 x 2 contingency tables of exposure variables. All variables with P

Trypanosoma cruzi circulating in the southern region of the State of Mexico (Zumpahuacan) are pathogenic: a dog model.

Here we describe clinical and pathologic evidence of Chagas disease caused in dogs by circulating Trypanosoma cruzi from a newly recognized endemic area in Mexico. We show that the Zumpahuacan isolate, although less virulent than the Sylvio-X10 reference strain that caused acute myocarditis and death, was pathogenic in dogs. Dogs infected with the Zumpahuacan isolate exhibited electrocardiographic alterations, left- and right-ventricle dilation, and hydropericardium. Histologically, diffused perimysial and endomysial lymphoplasmacytic cell infiltration, cardiomyocyte necrosis, and amastigote nests were noted in Zumpahuacan-infected dogs. These findings suggest that the risk of T. cruzi infection and Chagas disease is present in the State of Mexico, and further research is needed to identify the T. cruzi bio-types circulating in southern State of Mexico.

La lipofección incrementa la eficiencia de mutagénesis dirigida en células troncoembrionarias de ratón E14 TG2a / Lipofection improves gene targeting efficiency in E14 TG2a mouse embryonic stem cells

Electroporation has been the method of election for transfection of murine embryonic stem cells for over 15 years; however, it is a time consuming protocol because it requires large amounts of DNA and cells, as well as expensive and delicate equipment. Lipofection is a transfection method that requires lower amounts of cells and DNA than electroporation, and has proven to be efficient in a large number of cell lines. It has been shown that after lipofection, mouse embryonic stem cells remain pluripotent, capable of forming germ line chimeras and can be transfected with greater efficiency than with electroporation; however, gene targeting of mouse embryonic stem cells by lipofection has not been reported. The objective of this work was to find out if lipofection can be used as efficiently as electroporation for regular gene targeting protocols. This context compares gene targeting efficiency between these techniques in mouse embryonic stem cells E14TG2a, using a gene replacement type vector. No differences were found in gene targeting efficiency between groups; however, lipofection was three times more efficient than electroporation in transfection efficiency, which makes lipofection a less expensive alternative method to produce gene targeting in mouse embryonic stem cells.

Durante los últimos 15 años se ha demostrado que la electroporación representa el método ideal para la transfección de células troncoembrionarias de ratón; sin embargo, demanda grandes cantidades de ADN y células, así como equipo caro y delicado, ello hace que este proceso sea costoso y laborioso. La lipofección es un método de transfección que requiere menos de células y ADN que la electroporación; asimismo, ha probado ser eficiente en gran número de líneas celulares. Se ha demostrado que después de lipofectar células troncoembrionarias de ratón, éstas mantienen su pluripotencia y son capaces de formar quimeras de línea germinal y se transfectan con mayor eficiencia que con electroporación, pero no se ha notificado la mutagénesis dirigida mediante la lipofección de células troncoembrionarias de ratón. El objetivo del presente trabajo fue saber si la lipofección puede ser utilizada con la misma o mayor eficiencia que la electroporación para los protocolos regulares de mutagénesis dirigida; en este contexto, se compara la eficiencia en mutagénesis dirigida entre estas técnicas en células troncoembrionarias de ratón E14TG2a, utilizando un vector de reemplazo. Entre las células transfectadas no se hallan diferencias en la eficiencia en mutagénesis dirigida entre grupos; sin embargo, los resultados que aquí se ofrecen muestran que la lipofección es tres veces más eficiente en la transfección, lo cual indica que la lipofección es un método alternativo menos costoso para obtener mutagénesis dirigida en células troncoembrionarias de ratón.

Human Trypanosoma cruzi infection and seropositivity in dogs, Mexico.

We used 5 diagnostic tests in a cross-sectional investigation of the prevalence of Trypanosoma cruzi in Tejupilco municipality, State of Mexico, Mexico. Our findings showed a substantial prevalence of immunoglobulin G (IgG) and IgM antibodies to T. cruzi in human (n = 293, IgG 2.05%, IgM 5.5%, both 7.1%) and dog (n = 114, IgG 15.8%, IgM 11.4%, both 21%) populations. We also found antibodies to T. cruzi (n = 80, IgG 10%, IgM 15%, both 17.5%) in dogs from Toluca, an area previously considered free of T. cruzi. Our data demonstrate the need for active epidemiologic surveillance programs in these regions. A direct correlation (r2 = 0.955) of seropositivity between humans and dogs suggests that seroanalysis in dogs may help identify the human prevalence of T. cruzi infection in these areas.