Drug discovery for Chagas disease should consider Trypanosoma cruzi strain diversity.

This opinion piece presents an approach to standardisation of an important aspect of Chagas disease drug discovery and development: selecting Trypanosoma cruzi strains for in vitro screening. We discuss the rationale for strain selection representing T. cruzi diversity and provide recommendations on the preferred parasite stage for drug discovery, T. cruzi discrete typing units to include in the panel of strains and the number of strains/clones for primary screens and lead compounds. We also consider experimental approaches for in vitro drug assays. The Figure illustrates the current Chagas disease drug-discovery and development landscape

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

Chagas disease is the clinical manifestation of the infection produced by the flagellate parasite Trypanosoma cruzi and currently there is no vaccine to prevent this disease. Therefore, different approaches or alternatives are urgently needed. Vaccination with live attenuated parasites has been used effectively in mice to reduce parasitemia and histological damage. However, the use of live parasites as inmunogens is controversial due to the risk of reversion to a virulent phenotype. In this work we genetically manipulated a naturally attenuated strain of T. cruzi in order to produce parasites with impaired replication and infectivity, using the mutation as a safety device against reversion to virulence. We show that genetically modified parasites display a lower proliferation rate in vitro and induced almost undetectable levels of T. cruzi specific CD8+ T cells when injected in mice. Furthermore, the immune response induced by these live mutant parasites confers protection against a subsequent virulent infection even a year after the original immunization

The Acute Phase of Trypanosoma cruzi Infection Is Attenuated in 5-Lipoxygenase-Deficient Mice

In the present work we examine the contribution of 5-lipoxygenase- (5-LO-) derived lipid mediators to immune responses during the acute phase of Trypanosoma cruzi infection in 5-LO gene knockout (5-LO−/−) mice and wild-type (WT) mice. Compared with WT mice, the 5-LO−/− mice developed less parasitemia/tissue parasitism, less inflammatory cell infiltrates, and a lower mortality. This resistance of 5-LO−/− mice correlated with several differences in the immune response to infection, including reduced PGE2 synthesis; sustained capacity of splenocytes to produce high levels of interleukin (IL)-12 early in the infection; enhanced splenocyte production of IL-1β, IL-6, and IFN-γ; rapid T-cell polarization to secrete high quantities of IFN-γ and low quantities of IL-10; and greater numbers of CD8+CD44highCD62Llow memory effector T cells at the end of the acute phase of infection. The high mortality in WT mice was associated with increased production of LTB4/LTC4, T cell bias to produce IFN-γ, high levels of serum nitrite, and marked protein extravasation into the peritoneal cavity, although survival was improved by treatment with a cys-LT receptor 1 antagonist. These data also provide evidence that 5-LO-derived mediators negatively affect host survival during the acute phase of T. cruzi infection

Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis.

BackgroundThe present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL.Methodology/principal findingsThe LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed.Conclusions/significanceThe present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL

Correction: Antigenicity and Protective Efficacy of a Leishmania Amastigote-specific Protein, Member of the Super-oxygenase Family, against Visceral Leishmaniasis.

[This corrects the article on p. e2148 in vol. 7.]

Biomarkers of therapeutic responses in chronic Chagas disease: state of the art and future perspectives

The definition of a biomarker provided by the World Health Organization is any substance, structure, or process that can be measured in the body, or its products and influence, or predict the incidence or outcome of disease. Currently, the lack of prognosis and progression markers for chronic Chagas disease has posed limitations for testing new drugs to treat this neglected disease. Several molecules and techniques to detect biomarkers in Trypanosoma cruzi-infected patients have been proposed to assess whether specific treatment with benznidazole or nifurtimox is effective. Isolated proteins or protein groups from different T. cruzi stages and parasite-derived glycoproteins and synthetic neoglycoconjugates have been demonstrated to be useful for this purpose, as have nucleic acid amplification techniques. The amplification of T. cruzi DNA using the real-time polymerase chain reaction method is the leading test for assessing responses to treatment in a short period of time. Biochemical biomarkers have been tested early after specific treatment. Cytokines and surface markers represent promising molecules for the characterisation of host cellular responses, but need to be further assessed.RICET RD12/0018/0010. RICET RD12/0018/0021. AGAUR 2014SGR26. Plan Nacional de I+D+I SAF2012-35777. Plan Nacional de I+D+I SAF2013-48527-R. NIMHD/NIH 2G12MD007592. Financial support: CRESIB and IPBLN research members were partially supported by the RICET (RD12/0018/0010, RD12/0018/0021), M-JP and JG received research funds from AGAUR (2014SGR26) and Fundación Mundo Sano, M-CT and M-CL were supported by Plan Nacional de I+D+I (MINECO-Spain) (SAF2012-35777, SAF2013-48527-R and FEDER), ICA was partially supported by NIMHD/NIH (2G12MD007592). Financial support: CRESIB and IPBLN research members were partially supported by the RICET (RD12/0018/0010, RD12/0018/0021), M-JP and JG received research funds from AGAUR (2014SGR26) and Fundación Mundo Sano, M-CT and M-CL were supported by Plan Nacional de I+D+I (MINECO-Spain) (SAF2012-35777, SAF2013-48527-R and FEDER), ICA was partially supported by NIMHD/NIH (2G12MD007592).Peer reviewe

Novel 3-nitro-1H-1,2,4-triazole-based aliphatic and aromatic amines as anti-chagasic agents

A series of novel 2-nitro-1H-imidazole- and 3-nitro-1H-1,2,4-triazole-based aromatic and aliphatic amines were screened for anti-trypanosomal activity and mammalian cytotoxicity by the Drugs for Neglected Diseases initiative (DNDi). Out of 42 compounds tested, eighteen 3-nitro-1,2,4-triazoles and one 2-nitroimidazole displayed significant growth inhibitory properties against T. cruzi amastigotes (IC50 ranging from 40 nM to 1.97 microM) , without concomitant toxicity towards the host cells (L6 cells), having selectivity indices (SI) 44 to 1320. Most (16) of these active compounds were up to 33.8-fold more potent than the reference drug benznidazole, tested in parallel. Five novel 3-nitro-1,2,4-triazoles were active against bloodstream form (BSF) T. b. rhodesiense trypomastigotes (IC50 at nM levels and SI 220 to 993). An NADH-dependent nitroreductase (TbNTR) plays a role in the anti-parasitic activity, since BSF T. b. brucei trypomastigotes with elevated TbNTR levels were hypersensitive to tested compounds. Therefore, a novel class of affordable 3-nitro-1,2,4-triazole-based compounds with antitrypanosomal activity has been identifie

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