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

Abstract

BackgroundThe 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 Trypanosoma cruzi 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 fg 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.ResultsRecombinant 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 T. cruzi 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 T. cruzi.ConclusionsWe conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi 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.

Retention of a recombinant GFP protein expressed by the yellow fever 17D virus in the E/NS1 intergenic region in the endoplasmic reticulum

“…Initially, we performed the insertion of the GFP gene into the YF genome due to the ease of viral viability and heterologous protein expression monitoring. To date, several other genes have been expressed, such as the simian immuno deficiency virus Gag 45-269 fragment (Bonaldo et al 2010), the 261-380 fragment of the amastigote surface protein-2 of Trypanosoma cruzi (Nogueira et al 2011) and the 19 kDa domain of the Plasmodium falciparum merozoite surface protein-1 (MSP-1) (unpublished data). These different recombinant proteins expressed at the E/NS1 region with the described genomic structure are generally retained in the ER (unpublished observations).…”

The yellow fever 17D virus as a platform for new live attenuated vaccines

“…These viruses were stable through serial passages in cultured vertebrate cells and also retained attenuation for mice and monkeys. 58,60,62 In addition, the recombinant virus expressing a B-cell epitope from the malarial parasite Plasmodium falciparum induced antibodies against the CS protein after mouse and monkey vaccination. 60,62 The epitope expression in the E protein is an interesting approach, since it was recently reported that this protein elicits dominant immune responses corresponding either to MHC class I or class II antigens.…”

“…However, in recent studies for evaluating the capacity of distinct recombinant YF17D viral vectors to induce a protective immune response against the Trypanosoma cruzi protozoan parasite, the immunodominant CD8 + T cell epitope TEWETGQI of the ASP-2 (Amastigote Surface Protein -2) cloned and expressed between NS2B and NS3 led only to marginal protection after the T.cruzi challenge. 56,58 Another approach for the expression of defined epitopes by the YF17D recombinant virus was based on the use of the major envelope protein of flaviviruses, the E protein. Since this protein is the major target of the humoral immune response presenting 180 monomers at the surface of mature virions, the expression of heterologous humoral epitopes in this context would be appropriate to elicit antibodies specific to the foreign epitope.…”

Advances and challenges towards a vaccine against Chagas disease

“…However, such complicated immunization protocols may be difficult to translate into a field vaccine. New recombinant virus vectors have also recently be described, such as Yellow fever virus expressing ASP-2 45 and influenza virus NR, not reported. *parasite burden was actually measured in skeletal muscle rather than heart.…”