Due to the lack of a current chemotherapy to Chagas disease as other neglected disease, it is essential to develop new strategies for its prevention. For the past decade, several Trypanosoma cruzi antigens have been tested for protective immunity of mice. They have included plasmid DNA, recombinant proteins associated with several adjuvants and bacteria or virus vectors [24, 25, 29, 30]. However, DNA vaccine immunogenicity is not yet well established in man, despite the fact that recombinant protein or subunit vaccines may require the use of adjuvant formulations certified for human use .
The Yellow fever vaccine virus (YF 17D) has been successfully used as a viral platform to express antigenic sequences from other flaviviruses , non-related pathogens such as SIV [32, 33], Plasmodium spp. [13, 14, 16, 34] and influenza [35, 36]. The recombinant YF 17D viruses described here are replication-competent viruses with advantages over other viral vectors such as lifelong immunogenicity and no integration into the host genome. Importantly, in our studies, we demonstrated that pre-existing neutralizing antibodies to YF 17DD (elicited after the initial dose) did not hamper immune responses after a second dose of vaccination, as measured by antigen-specific IFN-γ producing cells and higher YF-specific neutralizing antibody titers.
Considering these favorable characteristics of the YF 17DD vaccine virus and based on the previous protection results reported with the antigens present in the intracellular forms of the parasite , we decided to express an epitope of T. cruzi Amastigote Surface Protein 2 (ASP-2). The selected TEWETGQI motif is an immunodominant CD8+ T cell epitope, which binds to the MHC class I H-2Kk molecule . To express this T.cruzi octapeptide, we employed two distinct methodologies. In the first, the insertion in the intergenic NS2B-NS3 region, it is expected to occur that, after the proteolytic cleavage of part of viral precursor polyprotein mediated by the viral NS2B-NS3 proteolytic complex, the foreign peptide will be released in cytoplasm and be presented by MHC class leading to CD8+ T cell activation. Using this approach, it was possible to demonstrate that mouse vaccination with single dose of different recombinant yellow fever viruses could promote partial or complete protection against a challenge with a rodent malaria parasite or a lethal melanoma, respectively. In both studies, the protection was associated to the induction of IFNγ-secreting CD8+ T cells [12, 13]. The second approach involved the epitope expression on the surface of viral E protein, the major flaviviral envelope protein. Besides being the main target of viral neutralizing antibodies, several CD8+ T cell epitopes have been mapped in YF 17D E protein in mice, rhesus macaques and humans [7, 17, 18]. Akondy and colleagues  verified that more than ten percent of total IFN-gamma producing CD8+ T cell in vaccines were specific to epitopes present in E protein. It is conceivable that processing of epitopes via MHC class I occurs through either virion endocytosed virus particles or ER-phagosome interaction .
Therefore, the expression of the TEWETGQI epitope at two different sites of a vector known to elicit strong CD8+ T cell immune response, with likely different pathways for processing and presentation, was approached in terms of its importance in protection against challenge with live trypanosomes. This is the first description of recombinant YF 17D viruses expressing an antigen of T. cruzi, the causative agent of Chagas disease. These recombinant viruses were characterized with regard to the foreign epitope expression, viral growth, attenuation and immunogenicity for mice. Both viruses replicated well in Vero cells, producing similar titers to YF 17DD at 72 hours post-infection and were capable to maintain the heterologous epitope insertions until at least the eighth serial passage in Vero cells. However, insertions clearly affected viral plaque morphology in Vero cells, when compared to the vaccine virus plaque phenotype. Both recombinants were also less virulent for mice after intracerebral inoculation than the YF 17DD vaccine virus. While YF 17DD killed most mice, only about one third of the animals died after the YF17D/NS2B3/Tc inoculation and a yet fewer died with YF 17D/E200/Tc. With regard to the induction of neutralizing antibodies after only one dose, the titers elicited by the recombinant viruses were significantly lower than those elicited by the YF 17DD. After the second dose, inferior titers are still evident for both recombinant viruses. Although we could not find any statistically difference between the recombinant viruses, YF17D/E200/Tc seemed to induce lower titers of neutralizing antibodies than YF17D/NS2B3/Tc. It is believed that this insertion in the fg loop of the E protein may affect the conformational change ocurring in the trimerization of this protein at the process of fusion to the endosome membrane which might result in a more attenuated phenotype or increased susceptibility to viral neutralization .
An unusual lineage of mice named A/J (H2-Kk) was adopted in this work to evaluate YF immune responses due to its high susceptibility to T. cruzi infection . We observed that immunization with the YF 17DD virus successfully protected mice of this lineage against a YF 17DD intracerebral challenge and also elicited titers of neutralizing antibodies against YF 17DD as described for BALB/c mice . It has already been demonstrated that every YF viral protein is immunogenic and possesses CD8+ T cell epitopes [6, 7]. As for A/J mice we also demonstrate that this lineage is capable of responding with specific IFN-γ SFC to YF 17DD, this result being comparable to those induced by the two recombinant viruses.
Immunity elicited by ASP-2 is associated with a type I response generated by IFN-γ producing CD4+ and CD8+ T cells [40, 41]. High levels of IFN-γ have been associated with protection for many infections by intracellular parasitic and viral agents [42, 43]. Former studies showed that IFN-γ secretion by CD8+ T cells was particularly important for survival of BALB/c and C57BL/6 mice after day 14 post infection with T. cruzi . The observed levels of protection elicited by both recombinants might be correlated with expansion of IFN-γ production by CD8+ T cells as demonstrated by ELISPOT on day 15 after challenge. We consider this result important as we know that any delay in CD8+ T cell expansion (and gamma interferon production) represents a major factor in T. cruzi infection outcome in mice and Chagas disease in man [40, 42, 44]. We also found that the period between 14 and 23 dpi is also critical for A/J mice survival as almost all naïve (mock) infected animals died by day 23 dpi as opposed to vaccinated animals. The immune response elicited by either recombinant virus controlled incipient infection, leading to partial protection afterwards in contrast to the naïve group. However, more experiments would be needed to determine whether this cellular response is able to persist for a longer period of time after immunization.
A plausible explanation for an incomplete protection may be related to the fact that immunization with this single peptide may not be sufficient to fully protect mice against T. cruzi  despite TEWETGQI-specific IFN-γ producing CD8+ T cells . Furthermore, low levels of protection afforded by vaccination with T. cruzi octapeptide correlated with a lower priming of memory peptide-specific T cells generation . In this regard, a broader repertoire of epitopes present in a larger ASP-2 insert may be required to increase the protection since larger portions of ASP-2, employed either as recombinant protein or expressed by plasmids or adenovirus vectors, in different vaccination protocols shown to induce protection against T. cruzi challenge in the range from 80% to 100% [25, 26, 30]. In addition, the presence of other CD4+ T cell epitopes may be essential in order to contribute to the enhancement of the CD8+ T cell response, as observed for Chagas disease  or malaria .
Recombinant YF 17D viruses have been considered as a promising approach for immunization against other diseases, particularly due to its polyfunctional immune response . Since a prophylactic strategy for Chagas disease remains to be established, our results on the expression and immunogenicity of T. cruzi CD8+ T cell epitope between the NS2B and NS3 proteins of the recombinant YF 17D virus warrant further development. Future viral formulations containing different YF constructs at the NS2B-3 site delivering distinct epitopes against the same pathogen might enhance protective responses. In addition, a YF construct that encompasses a larger ASP-2 segment inserted between the E protein and the nonstructural NS1 protein should also be tried as this site seems to tolerate larger fragments, while retaining fitness and immunogenicity [33, 39].