Biological and immunological characterization of recombinant Yellow Fever 17D Viruses expressing a Trypanosoma cruzi Amastigote Surface Protein-2 CD8+ T cell epitope at two distinct regions of the genome

<p>Abstract</p> <p>Background</p> <p>The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a <it>Trypanosoma cruzi </it>CD8⁺T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8⁺T cell epitope was cloned and expressed based on two different genomic insertion sites: in the <it>fg </it>loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope.</p> <p>Results</p> <p>Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by <it>T. cruzi </it>with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8⁺T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of <it>T. cruzi</it>.</p> <p>Conclusions</p> <p>We conclude that the YF 17D platform is useful to express <it>T. cruzi </it>(Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model <it>T. cruzi </it>epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response.</p

4-aminopyridyl-based lead compounds targeting CYP51 prevent spontaneous parasite relapse in a chronic model and improve cardiac pathology in an acute model of Trypanosoma cruzi infection.

BackgroundChagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice.Methodology/principal findingsBoth acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart.Conclusions/significanceThe positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life

Recombinant Yellow Fever Viruses Elicit CD8(+) T Cell Responses and Protective Immunity against Trypanosoma cruzi

Chagas' disease is a major public health problem affecting nearly 10 million in Latin America. Despite several experimental vaccines have shown to be immunogenic and protective in mouse models, there is not a current vaccine being licensed for humans or in clinical trial against T. cruzi infection. Towards this goal, we used the backbone of Yellow Fever (YF) 17D virus, one of the most effective and well-established human vaccines, to express an immunogenic fragment derived from T. cruzi Amastigote Surface Protein 2 (ASP-2). the cDNA sequence of an ASP-2 fragment was inserted between E and NS1 genes of YF 17D virus through the construction of a recombinant heterologous cassette. the replication ability and genetic stability of recombinant YF virus (YF17D/ENS1/Tc) was confirmed for at least six passages in Vero cells. Immunogenicity studies showed that YF17D/ENS1/Tc virus elicited neutralizing antibodies and gamma interferon (IFN-gamma) producing-cells against the YF virus. Also, it was able to prime a CD8(+) T cell directed against the transgenic T. cruzi epitope (TEWETGQI) which expanded significantly as measured by T cell-specific production of IFN-gamma before and after T. cruzi challenge. However, most important for the purposes of vaccine development was the fact that a more efficient protective response could be seen in mice challenged after vaccination with the YF viral formulation consisting of YF17D/ENS1/Tc and a YF17D recombinant virus expressing the TEWETGQI epitope at the NS2B-3 junction. the superior protective immunity observed might be due to an earlier priming of epitope-specific IFN-gamma-producing T CD8(+) cells induced by vaccination with this viral formulation. Our results suggest that the use of viral formulations consisting of a mixture of recombinant YF 17D viruses may be a promising strategy to elicit protective immune responses against pathogens, in general

The involvement of FAK and Src in the invasion of cardiomyocytes by Trypanosoma cruzi

The activation of signaling pathways involving protein tyrosine kinases (PTKs) has been demonstrated during Trypanosoma cruzi invasion. Herein, we describe the participation of FAK/Src in the invasion of cardiomyocytes by T. cruzi. The treatment of cardiomyocytes with genistein, a PTK inhibitor, significantly reduced T. cruzi invasion. Also, PP1, a potent Src-family protein inhibitor, and PF573228, a specific FAK inhibitor, also inhibited T. cruzi entry; maximal inhibition was achieved at concentrations of 25 lM PP1 (53% inhibition) and 40 lM PF573228 (50% inhibition). The suppression of FAK expression in siRNA- treated cells and tetracycline-uninduced Tet-FAK(WT)-46 cells significantly reduced T. cruzi invasion. The entry of T. cruzi is accompanied by changes in FAK and c-Src expression and phosphorylation. An enhancement of FAK activation occurs during the initial stages of T. cruzi-cardiomyocyte interaction (30 and 60 min), with a concomitant increase in the level of c-Src expression and phosphorylation, suggesting that FAK/Src act as an integrated signaling pathway that coordinates parasite entry. These data provide novel insights into the signaling pathways that are involved in cardiomyocyte invasion by T. cruzi. A better understanding of the signal transduction networks involved in T. cruzi invasion may contribute to the development of more effective therapies for the treatment of Chagas’ disease

Trypomastigote-induced mortality and parasitemia in immunized A/J mice.

<p>A) mouse survival after <i>T. cruzi</i> challenge. Kaplan-Meier curves of (7 to 12 mice per group) of experimental groups which were immunized with the recombinant viruses YF17D/NS2B3/Tc (•), YF17D/ENS1/Tc (▾), Formulation 1 (50% of YF17D/ENS1/Tc and 50% of YF17D/NS2B3/Tc viruses) (♦) or vaccine YF 17DD control virus (□). Differences between the groups immunized with the recombinant 17D viruses and that one which received the YF 17DD vaccine were statistically significant (***<i>P</i><0.0001, logrank test). B) Average value of trypomastigote-induced parasitemias of immunized A/J mice and intraperitoneally challenged with 250 bloodstream trypomastigotes. The parasitemia for each individual mouse is represented in the graphs for: (C) mice immunized with YF 17DD; (D) mice immunized with YF 17D/NS2B3/Tc; (E) mice immunized with YF 17D/ENS1/Tc; (E) immunized with Formulation 1. The parasitemic peak of mice immunized with Formulation 1 were lower than the parasitemias of mice immunized with YF 17DD or YF17D/NS2B3/Tc (*<i>P</i><0.05, Tukey’s test).</p

Induction of IFN-γ secreting splenocytes in vaccinated mice before and after <i>T. cruzi</i> challenge.

<p>Groups of A/J mice were immunized once or twice with medium (mock) (○), YF 17DD vaccine (□), YF17D/NS2B3/Tc virus (•) and YF17D/ENS1/Tc virus (▾) or alternatively with Formulation 1 (50,000 PFU of YF17D/NS2B3/Tc and 50,000 PFU of YF17D/ENS1/Tc) (♦) and challenged or not with 250 <i>T. cruzi</i> trypomastigotes. Spleen cells of each mouse were obtained one week after the first dose (A and C), one week after the second dose (B and D) or two weeks after challenge (E) and after that, were stimulated <i>in vitro</i> with YF 17DD inactivated virus (A and B) or TEWETGQI-peptide (C, D and E) to assess cellular responses to YF peptides or to the <i>T. cruzi</i> peptide, respectively. Results represent IFN-γ producing cells (SFC) per 10⁶ spleen cells. Asterisks indicate statistically significant differences between groups of immunization in comparison to Mock (A and B) or in comparison to Mock and YF 17DD group (C, D and E) and were done by ANOVA Tukeýs test (***<i>P</i><0.0001; **<i>P</i><0.01; * <i>P</i><0.05).</p

Expression of the <i>T. cruzi</i> TEWETGQI epitope by the recombinant YF17D/ENS1/Tc virus.

<p>Indirect immunofluorescence assay of Vero cells infected with YF 17DD vaccine virus (A and D), recombinant YF17D/ENS1/Tc viral (B and E), and recombinant YF17D/NS2B3/Tc virus (C and F). Cells were stained with a mouse polyclonal hyperimmune serum to YF 17D (panels A, B and C) or a polyclonal antibodies directed to the TEWETGQI epitope (panels D, E and F). The employed secondary antibodies were labeled with Alexa Fluor 488 (A, B and C) and Alexa Fluor 546 (D, E and F).</p