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

Background: 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 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.

Results: 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 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.

Conclusions: We 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.

Figure 1

Schematic diagram of YF 17D virus genome organization and the corresponding insertion sites. The YF virus genome (A) is translated in a polyprotein of 3,411 amino acids (B), which is proteolytically processed by the viral protease complex NS2B-NS3. Open arrows indicate cleavage sites, releasing functional viral proteins in the cytosol of the cell. Dark arrows indicate host signalase cleavage sites. Open triangle indicates furin cleavage site and dark triangle indicates unknown host protease cleavage site. TEWETGQI epitope was inserted at two different positions (C): between f and g strands of the YF 17D virus E protein Domain II located at 482 amino acid of (left panel) or between duplicated flanking viral protease cleavage sites (GARR/S Glycine-Alanine-Arginine-Arginine-Serine) at the junction between NS2B and NS3 proteins (open arrows; right panel) located at 1486 amino acid of the YF 17D virus polyprotein.

Figure 2

Characterization of recombinant YF 17D viruses. A) Replicative kinetics in Vero cells of recombinant viruses. YF 17D/NS2B3/Tc (open triangles) and YF 17D/E200/Tc (black squares), control YF 17D/E200T3 (dark gray discontinuous circles) and YF 17DD (light gray complete circles). Each time point represents the average titer obtained from three separate experiments with the respective standard deviations. No statistically significant differences were found. B) Plaque morphology of YF 17DD, 17D/NS2B3/Tc and YF17D/E200/Tc.

Figure 3

Expression of the T. cruzi TEWETGQI epitope by recombinant YF17D/E200/Tc and YF17D/NS2B3/Tc viruses. Indirect immunofluorescence assay of Vero cells infected with recombinant viruses, YF 17DD vaccine virus or not infected (Earle's Medium 199) stained with a mouse polyclonal hyperimmune serum to YF17D (HIYF) or polyclonal antibody directed to the TEWETGQI epitope. Second antibodies were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 goat anti-mouse IgG for HIYF (green color) or anti-TEWETGQI (red color) preparations, respectively.

Figure 4

ELISPOT assay for TEWETGQI-specific IFN-γ secreting cells before and after a T. cruzi challenge. Groups of A/J mice were immunized with Mock (Earle's 199 Medium), YF17DD virus, recombinant viruses (YF17D/E200/Tc and YF17D/NS2B3/Tc) or with TEWETGQI emulsified in Freund's Adjuvant. Spleen cells were obtained one week after the last dose (before challenge) or two weeks after challenge. A) Spleen cells response to YF17DD stimulus before challenge. B) Spleen cells response to TEWETGQI stimulus before challenge. C) Spleen cells response to TEWETGQI stimulus after challenge. Results represent IFN-γ producing cells (SFC) per 10⁶ spleen cells. Statistically significant differences (ANOVA Tukey's test) are shown in the graphics.

Figure 5

Kaplan-Meier survival curves. Groups were immunized twice with medium (Mock), YF 17DD, the two recombinant viruses or with TEWETGQI emulsified in Freund's Adjuvant and infected or not intraperitoneally with 250 T. cruzi bloodstream trypomastigotes. Differences between the recombinant viruses and the YF17DD groups were statistically significant (*** P < 0.0001, logrank test). Results represents pooled data obtained from three independent experiments with 5 animals per group per experiment.