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

Trypanosoma cruzi isolates from Argentina and Chile grouped with the aid of DNA probes

Fifty-two isolates and several clones from Trypanosoma cruzi, the agent of Chagas' disease, were analyzed using cloned minicircles or total kinetoplast DNA as probes. Isolates were obtained from triatomines, guinea pigs and infected humans in the Central and Northern regions of Argentina and the North of Chile. 35% of all the randomly selected isolates could be identified with one cloned minicircle probe. This widely distributed T. cruzi group was detected on both sides of the Andes mountain range (Argentina and Chile) in Triatoma infestans as well as in human infections. Most of the other isolates could be grouped with four kinetoplast DNAs as probes, but their geographical distribution seems to be restricted as compared with the one mentioned above. These results confirm the heterogeneity of T. cruzi subspecies in nature and the usefulness of DNA probes to group them. © 1987

Short term protection in immunocompetent mice infected with TCC mutant parasites.

<p>(A) Lymphocytes were recovered from blood of B6 mice immunized with TCC wild type (grey bar) and TCC <i>dhfr-ts^+/−</i> (white bar) 14 days after the boost and were stained with the TSKB20 MHC I tetramer. Bars represent the mean frequencies of CD8⁺ tetramer-positive lymphocytes for four mice per group; error bars represent standard errors of the mean. (B) Parasitemia curve of B6 mice infected with 5×10⁵ TCC <i>dhfr-ts^+/−</i> metacyclic trypomastigotes, TCC wild type metacyclic trypomastigotes and PBS and challenge with 10⁴ virulent CL parasites. (C) Dispersion diagrams of antibody levels in either naive animals (non immunized) and those immunized with 5×10⁵ metacyclic trypomastigotes of TCC <i>dhfr-ts^+/−</i> or TCC wild type. The results are expressed as the ratio of the absorbance of each serum sample at a 490-nm optical density (OD) to the cutoff value. Dotted lines indicate the cutoff adopted for positivity, calculated as the mean of the values determined for the naive controls plus three times the standard deviation. Positive controls were infected with Tulahuen wild type parasites. (D) Parasitemia curve of Balb/c mice infected with TCC <i>dhfr-ts^+/−</i> metacyclic trypomastigotes, TCC wild type metacyclic trypomastigotes or PBS and challenge with 5×10³ virulent Tulahuen blood trypomastigotes. Values are given as means; error bars indicate standard errors of the mean.</p

<i>In vivo</i> infectivity of Tulahuen <i>dhfr-ts^+/−</i> and Tulahuen wild type metacyclic trypomatigotes.

<p>(A) Parasitemia curves of IFNγ^−/− mice inoculated with 5×10⁴ metacyclic trypomastigotes of Tulahuen wild type and <i>dhfr-ts^+/−</i> parasites. (B) Parasite load of nude mice inoculated with 5×10⁴ metacyclic trypomastigotes of Tulahuen wild type and <i>dhfr-ts^+/−</i> parasites at day 20 post-infection. (C) Parasitemia curves of Balb/c mice inoculated with 2×10⁴ metacyclic trypomastigotes of Tulahuen wild type and 2×10⁵ metacyclic trypomastigotes of Tulahuen <i>dhfr-ts^+/−</i> metacyclic trypomastigotes. Values are given as means; error bars indicate standard errors of the means.</p

<i>In vitro</i> growth for <i>dhfr-ts^+/−</i> and wild type epimastigotes.

<p>(A) Growth curve of TCC wild type versus TCC <i>dhfr-ts^+/−</i> clone and (B) growth curve of Tulahuen wild type versus Tulahuen <i>dhfr-ts^+/−</i> clone. These results are representative of 3 independent experiments.</p

Infectivity of TCC <i>dhfr-ts^+/−</i> and TCC wild type parasites in different mouse strains.

<p>*ND: not done.</p

Long-term protective immunization with TCC <i>dhfr-ts^+/−</i> metacyclic trypomastigotes against virulent challenge with <i>T. cruzi</i> CL-tdTomato.

<p>A) CD8⁺ T cells positive for TSKB20 at day 300 post infection in B6 mice inoculated with 5×10⁵ metacyclic trypomastigotes of mutant and wild type TCC parasites. B) Parasite load after challenge, at day 370 post infection, with 2.5×10⁵ bloodstream forms of the virulent CL-tdTomato strain. Fluorescence levels were measured during 13 days. Values are given as means; error bars indicate standard errors of the means.</p