Angel M. Padilla

CD8+ T cells in Trypanosoma cruzi infection

CD8(+) T cells have emerged as crucial players in the control of a number of protozoan pathogens, including Trypanosoma cruzi, the agent of human Chagas disease. The recent identification of the dominant targets of T. cruzi-specific T cells has allowed investigators to follow the generation of and document the functionality of T cell responses in both mice and humans. Although slow to develop in the early stages of the infection, T. cruzi-specific CD8(+) T cells reach prodigious levels and remain highly functional throughout chronic infections in mice. Following drug-induced cure during either the acute or chronic stage, these immunodominant T cells persist as stable, antigen-independent memory populations. T. cruzi-specific CD8(+) T cells in humans are less-well-studied but appear to lose functionality and decline in numbers in these decades-long infections. Changes in the frequency of parasite-specific T cell upon therapeutic treatment in humans may provide a new metric for determining treatment efficacy.

In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds.

Background The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential. Methods In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC50 lower than that of BZ. Findings This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course. Conclusion These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

Insufficient TLR activation contributes to the slow development of CD8+ T cell responses in Trypanosoma cruzi infection.

During experimental infection with Trypanosoma cruzi, mice develop a strong CD8+ T cell response focused mainly on a few immunodominant peptides encoded in trans-sialidase family genes. Despite the potency of this response, the initial emergence and peak of parasite-specific CD8+ T cells has been noted to be relatively slow. In this study, we further document this delayed onset of T cell responses to T. cruzi as measured by the increase in frequency of parasite-specific T cells, the effector function of these cells, T cell proliferation in general, and the recruitment of cells into the draining lymph nodes. This delay does not appear to be the result of general immunosuppressive effects of the infection, a limitation in parasite numbers, or parasite trafficking to lymph nodes or to the specific epitope. Increasing the initial infecting dose or the density of parasite epitopes on APCs can modestly speed the generation of anti-T. cruzi T cell responses. Given these characteristics of the response, we propose that T. cruzi is a stealth invader, largely avoiding recognition by components of the innate immune system until the infection is well established. This conclusion is supported by the ability to accelerate the induction of T cell responses to T. cruzi by administration of ligands for TLR2 and TLR9 at the time of infection. These studies highlight a previously unappreciated mechanism of immune evasion, the surreptitious establishment of infection, by the protozoan T. cruzi.

Limited Role for CD4+ T-Cell Help in the Initial Priming of Trypanosoma cruzi-Specific CD8+ T Cells

ABSTRACT Immune control of the protozoan parasite Trypanosoma cruzi requires the activation of both CD4+ and CD8+ T cells. We recently identified two T. cruzi trans-sialidase peptides that are targets of approximately 30% of all CD8+ T cells during acute T. cruzi infection in mice. To determine whether CD4+ T cells are required for generation of these dominant CD8+ T-cell responses, major histocompatibility complex class II (MHC II)-deficient mice were infected with the Brazil strain of T. cruzi and examined for the generation of antigen-specific CD8+ T cells. Strong trans-sialidase TSKB18- and TSKB20-specific CD8+ T-cell responses were generated in both the presence and the absence of CD4+ help. However, the magnitudes of the immunodominant TSKB20-specific CD8+ T-cell responses detectable using class I MHC-peptide tetramers were consistently lower in the blood and spleens of MHC II-deficient mice. Spleen cells from infected MHC II-deficient mice produced gamma interferon after in vitro stimulation with T. cruzi peptides at levels similar to those in wild-type mice, and MHC II-deficient mice displayed strong T. cruzi peptide-specific cytotoxic T-lymphocyte activity in vivo. Thus, primary CD8+ T-cell responses in experimental T. cruzi infection are generated in the absence of CD4+ T cells, providing further evidence that T. cruzi directly activates and licenses antigen-presenting cells. Nevertheless, unhelped CD8+ T cells in T. cruzi-infected mice fail to reach the frequencies achieved in the presence of CD4 T-cell help and are unable to prevent acute-phase death of these mice.

Targeted deletion of the gp72 gene decreases the infectivity of Trypanosoma cruzi for mice and insect vectors.

The infective behavior of a mutant Trypanosoma cruzi clone, carrying a targeted deletion of the gp72 gene, was studied in the insect vector Triatoma infestans and in mice. After feeding T. infestans with complement-resistant forms (CRF) of Ynull and wild-type clones, it was observed that the number of parasites released in the bugs feces was reduced to less than 1% in the mutant clone. Both gp72-null and wild-type clones had a low infectivity for mice in comparison with other T. cruzi isolates, probably as a consequence of prolonged in vitro culture. Therefore, the behavior of both clones was tested in highly susceptible BALB suckling mice and immunodeficient athymic mice. After infecting the animals with 105 CRF, wild-type parasites could be detected in fresh blood mounts of most mice, but mutants were never found by this method. However, in 4 of 22 hemocultures from 11 athymic mice, gp72-null epimastigotes carrying the mutant phenotype were reisolated by day 29 of infection. Serological and polymerase chain reaction determinations performed on the blood of animals inoculated with the mutants indicated the possibility of temporary infections, which were extinguished after 90 days. The intact GP72 gene seems essential for sustaining latent infections in immunocompetent animals.

Biological behavior of different Trypanosoma cruzi isolates circulating in an endemic area for Chagas disease in the Gran Chaco region of Argentina

The biological behavior of the different Trypanosoma cruzi strains is still unclear and the importance of exploring the relevance of these differences in natural isolates is of great significance. Herein we describe the biological behavior of four T. cruzi isolates circulating sympatrically in a restricted geographic area in Argentina endemic for Chagas Disease. These isolates were characterized as belonging to the Discrete Typing Units (DTUs) TcI, TcIII, TcV and TcVI as shown by Multilocus Enzyme Electrophoresis and Multilocus Sequence Typing. In order to study the natural behavior of the different isolates and to preserve their natural properties, we developed a vector transmission model that allows their maintenance in the laboratory. The model consisted of serial passages of these parasites between insect vectors and mice. Vector-derived parasite forms were then inoculated in C57BL/6J mice and number of parasite in peripheral blood, serological response and histological damage in acute and chronic phases of the infection were measured. Parasites from DTUs TcI, TcIII and TcVI were detected by direct fresh blood examination, while TcV parasites could only be detected by Polimerase Chain Reaction. No significant difference in the anti-T. cruzi antibody response was found during the chronic phase of infection, except for mice infected with TcV parasites where no antibodies could be detected. Histological sections showed that TcI isolate produced more damage in skeletal muscle while TcVI induced more inflammation in the heart. This work shows differential biological behavior among different parasite isolates obtained from the same cycle of transmission, permitting the opportunity to formulate future hypotheses of clinical and epidemiological importance.

Force of infection and evolution of lesions of canine tegumentary leishmaniasis in Northwestern Argentina

A clinical-serological follow-up was carried out in a canine population in endemic foci of Leishmania braziliensis spread in northwestern Argentina. Each dog was studied in at least two visits, 309+/-15 days (X+/-SE) apart. Some initially healthy dogs (n=52) developed seroconversion or lesions. The clinical evolution of the disease in dogs resembles in many aspects the human disease. Similarities include the long duration of most ulcers with occasional healing or appearance of new ones and the late appearance of erosive snout lesions in some animals. Yearly incidence rates of 22.7% for seroconversion and of 13.5% for disease were calculated as indicators of the force of infection by this parasite upon the canine population.

Protective immunity against Trypanosoma cruzi.

Upon infection, Trypanosoma cruzi triggers a strong immune response that has both protective and pathological consequences. In this work, several important questions regarding protective immunity are reviewed. Emphasis is placed on recent studies of the important protective role of CD8+ T cells and on previous studies of immunisation of domestic T. cruzi reservoirs that sought to address practical vaccination problems. Research on the maturation of memory cells and studies indicating that the prevalence of T. cruzi-specific T-cell responses and a high frequency of committed CD8+ T cells are associated with better clinical outcomes are also reviewed. Additionally, animal models in which protection was achieved without immunopathological consequences are discussed.

Knockout of the dhfr-ts Gene in Trypanosoma cruzi Generates Attenuated Parasites Able to Confer Protection against a Virulent Challenge

Background Trypanosoma cruzi is a protozoan parasite that causes severe disease in millions of habitants of developing countries. Currently there is no vaccine to prevent this disease and the available drugs have the consequences of side effects. Live vaccines are likely to be more effective in inducing protection than recombinant proteins or DNA vaccines; however, safety problems associated to their use have been pointed out. In recent years, increasing knowledge on the molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may protect against such reversion to virulent genotypes. Methods and Findings By targeted gene disruption we generated monoallelic mutant parasites for the dhfr-ts gene in a T. cruzi strain that has been shown to be naturally attenuated. In comparison to T. cruzi wild type epimastigotes, impairment in growth of dhfr-ts+/− mutant parasites was observed and mutant clones displayed decreased virulence in mice. Also, a lower number of T. cruzi-specific CD8+ T cells, in comparison to those induced by wild type parasites, was detected in mice infected with mutant parasites. However, no remarkable differences in the protective effect of TCC wild type versus TCC mutant parasites were observed. Mice challenged with virulent parasites a year after the original infection with the mutant parasites still displayed a significant control over the secondary infection. Conclusion This study indicates that it is possible to generate genetically attenuated T. cruzi parasites able to confer protection against further T. cruzi infections.

Engineered trivalent immunogen adjuvanted with a STING agonist confers protection against Trypanosoma cruzi infection

The parasite Trypanosoma cruzi is the causative agent of Chagas disease, a potentially life-threatening infection that represents a major health problem in Latin America. Several characteristics of this protozoan contribute to the lack of an effective vaccine, among them: its silent invasion mechanism, T. cruzi antigen redundancy and immunodominance without protection. Taking into account these issues, we engineered Traspain, a chimeric antigen tailored to present a multivalent display of domains from key parasitic molecules, combined with stimulation of the STING pathway by c-di-AMP as a novel prophylactic strategy. This formulation proved to be effective for the priming of functional humoral responses and pathogen-specific CD8+ and CD4+ T cells, compatible with a Th1/Th17 bias. Interestingly, vaccine effectiveness assessed across the course of infection, showed a reduction in parasite load and chronic inflammation in different proof of concept assays. In conclusion, this approach represents a promising tool against parasitic chronic infections.Chagas disease: protecting from chronic parasitic diseaseAn amalgamation of parasitic proteins may be the first effective vaccine against the as yet untreatable chronic phase of Chagas disease. The infliction, caused by the parasite Trypanosoma cruzi (T. cruzi), is the world’s leading cause of infectious cardiac inflammation and puts one-sixth of the population of Latin America at risk of infection. International collaborators led by Emilio Malchiodi, of the University of Buenos Aires, Argentina, constructed a vaccine (dubbed ‘Traspain’) comprised of key T. cruzi proteins alongside a novel ‘adjuvant’—designed to promote the efficacy of a vaccine by activating inflammatory responses. The chimera and adjuvant combination elicited a promising immune response and also showed the capacity to prevent tissue damage caused by chronic infection. Multi-part vaccines such as Traspain offer an attractive direction for research into vaccines against chronic parasitic infections.