Delayed Activation of T Cells at the Site of Infection Facilitates the Establishment of Trypanosoma cruzi in Both Naive and Immune Hosts.

Although parasite entry through breaks in the skin or mucosa is one of the main routes of natural transmission of Trypanosoma cruzi, little is known about the host cell types initially invaded nor the ability of those host cells to initiate immune responses at the site of infection. To gain insights into these early events, we studied the fate of fluorescently tagged T. cruzi delivered subcutaneously in mouse footpads or ears. We demonstrate that the majority of parasites introduced into the skin initially proliferate there until 8 to 10 days postinfection, when the parasite load decreases. This decline in parasite numbers is dependent on the presence of an intact T cell compartment and on the ability of hosts to produce gamma interferon (IFN-γ). Many of the parasite-containing cells at the initial infection site display a macrophage/monocyte phenotype but with low expression of activation markers, suggesting that these cells provide an early niche for T. cruzi proliferation, rather than being active in parasite control. It is only after the first round of T. cruzi replication and release from host cells that signs of immune activation and control of parasites become apparent. The delay in the activation and failure to rapidly control parasite replication are observed even when T. cruzi-primed T cells are present, such as in chronically infected mice. This failure of a primed immune system to recognize and react prior to extensive parasite expansion at the infection site likely poses a significant challenge for the development of vaccines aiming to prevent T. cruzi infection. IMPORTANCE Trypanosoma cruzi, the parasite causing Chagas disease, usually infects through the mucosa or breaks in the skin, but little is known about the parasite's fate at the site of entry or the early events involving immune control there. Here, we track the local proliferation and subsequent dissemination of fluorescently tagged T. cruzi and the initial immune response at the point of entry. We show that T. cruzi preferentially infects innate immune cells in the skin and that the stimulation of an adaptive T cell response does not occur until after the release of parasites from this first round of infected host cells. This first immunologically "silent" proliferation occurs even in the presence of a strong immune T cell memory generated by previous infection. This capacity of T. cruzi to establish infections while avoiding initial immune recognition has important implications for the potential to develop vaccines to prevent T. cruzi infection.

Prophylactic low-dose, bi-weekly benznidazole treatment fails to prevent Trypanosoma cruzi infection in dogs under intense transmission pressure.

Trypanosoma cruzi naturally infects a wide variety of wild and domesticated mammals, in addition to humans. Depending on the infection dose and other factors, the acute infection can be life-threatening, and in all cases, the risk of chagasic heart disease is high in persistently infected hosts. Domestic, working, and semi-feral dogs in the Americas are at significant risk of T. cruzi infection and in certain settings in the southern United States, the risk of new infections can exceed 30% per year, even with the use of vector control protocols. In this study, we explored whether intermittent low-dose treatment with the trypanocidal compound benznidazole (BNZ) during the transmission season, could alter the number of new infections in dogs in an area of known, intense transmission pressure. Preliminary studies in mice suggested that twice-weekly administration of BNZ could prevent or truncate infections when parasites were delivered at the mid-point between BNZ doses. Pre-transmission season screening of 126 dogs identified 53 dogs (42.1%) as T. cruzi infection positive, based upon blood PCR and Luminex-based serology. Serial monitoring of the 67 uninfected dogs during the high transmission season (May to October) revealed 15 (22.4%) new infections, 6 in the untreated control group and 9 in the group receiving BNZ prophylaxis, indicating no impact of this prophylaxis regimen on the incidence of new infections. Although these studies suggest that rigorously timed and more potent dosing regimen may be needed to achieve an immediate benefit of prophylaxis, additional studies would be needed to determine if drug prophylaxis reduced disease severity despite this failure to prevent new infections.

Discovery of an orally active benzoxaborole prodrug effective in the treatment of Chagas disease in non-human primates.

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs.

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease's complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA.

Trypanosoma cruzi, the causative agent of human Chagas disease, is endemic to the southern region of the United States where it routinely infects many host species. The indoor/outdoor housing configuration used in many non-human primate research and breeding facilities in the southern of the USA provides the opportunity for infection by T. cruzi and thus provides source material for in-depth investigation of host and parasite dynamics in a natural host species under highly controlled and restricted conditions. For cynomolgus macaques housed at such a facility, we used a combination of serial blood quantitative PCR (qPCR) and hemoculture to confirm infection in >92% of seropositive animals, although each method alone failed to detect infection in >20% of cases. Parasite isolates obtained from 43 of the 64 seropositive macaques were of 2 broad genetic types (discrete typing units, (DTU's) I and IV); both within and between these DTU groupings, isolates displayed a wide variation in growth characteristics and virulence, elicited host immune responses, and susceptibility to drug treatment in a mouse model. Likewise, the macaques displayed a diversity in T cell and antibody response profiles that rarely correlated with parasite DTU type, minimum length of infection, or age of the primate. This study reveals the complexity of infection dynamics, parasite phenotypes, and immune response patterns that can occur in a primate group, despite being housed in a uniform environment at a single location, and the limited time period over which the T. cruzi infections were established.

A modified drug regimen clears active and dormant trypanosomes in mouse models of Chagas disease.

A major contributor to treatment failure in Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is that current treatment regimens do not address the drug insensitivity of transiently dormant T. cruzi amastigotes. Here, we demonstrated that use of a currently available drug in a modified treatment regimen of higher individual doses, given less frequently over an extended treatment period, could consistently extinguish T. cruzi infection in three mouse models of Chagas disease. Once per week administration of benznidazole at a dose 2.5 to 5 times the standard daily dose rapidly eliminated actively replicating parasites and ultimately eradicated the residual, transiently dormant parasite population in mice. This outcome was initially confirmed in "difficult to cure" mouse infection models using immunological, parasitological, and molecular biological approaches and ultimately corroborated by whole organ analysis of optically clarified tissues using light sheet fluorescence microscopy (LSFM). This tool was effective for monitoring pathogen load in intact organs, including detection of individual dormant parasites, and for assessing treatment outcomes. LSFM-based analysis also suggested that dormant amastigotes of T. cruzi may not be fully resistant to trypanocidal compounds such as benznidazole. Collectively, these studies provide important information on the phenomenon of dormancy in T. cruzi infection in mice, demonstrate methods to therapeutically override dormancy using a currently available drug, and provide methods to monitor alternative therapeutic approaches for this, and possibly other, low-density infectious agents.

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.

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.

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.

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 IC(50) 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.

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.

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.

Multilocus enzyme electrophoresis analysis of Trypanosoma cruzi isolates from a geographically restricted endemic area for Chagas' disease in Argentina.

A set of 65 Trypanosoma cruzi stocks from dogs, opossums, insect vectors and humans was isolated in a geographically restricted endemic area for Chagas' disease in Argentina and was analysed by multilocus enzyme electrophoresis for 15 loci. The results show that at least five multilocus genotypes (clonets) circulate in the study area, one belonging to T. cruzi IIe, one to T. cruzi IId and three clonets belonging to T. cruzi I; and they confirm the presence of these lineages in the country. The three clonets attributed to T. cruzi I were identical to each other for all loci except for Sod-2, where three different patterns were identified. These patterns suggest the presence of two homozygous genotypes and one heterozygous genotype. Our results also suggest association of clonet IIe with dogs, clonet IId with humans and the three T. cruzi I clonets with Didelphis albiventris. On the other hand, there was no significant association between Triatoma infestans and any particular clonet circulating in the area. These findings are consistent with the hypothesis of natural selection, from mixed populations of T. cruzi in vectors, toward more restricted populations in mammals. The epidemiological implications of the possible selection of different clonets by different mammal hosts and the significance of two homozygous genotypes and one heterozygous genotype for the Sod-2 locus are discussed.

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 bug's 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 10(5) 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.