FIG. 1.

Design, immunogenicity, and efficacy of an rDNA/rYF17D/rRRV regimen encoding immunodominant classical CD8⁺ T cell epitopes. (A) Two Mamu-B*08⁺ (Group 1) and two Mamu-A*01⁺ (Group 2) Indian rhesus macaques were immunized with a heterologous prime/boost/boost vaccine regimen aimed at eliciting high frequency CD8⁺ T_EM responses against immunodominant SIV determinants. These animals were primed once with rDNA delivered by intramuscular electroporation followed by two viral vector boosts with rYF17D and then rRRV. Both the rDNA plasmids and the rYF17D encoded SIVmac239 minigenes corresponding to aa 45-210 of the Nef protein (Group 1) or aa 178-258 of the Gag polyprotein (Group 2). The rRRV vectors expressed a rev-tat-nef fusion (Group 1) or full-length gag (Group 2). Of note, all macaques in Groups 1 and 2 were seropositive for RRV at the time of the rRRV vaccinations. In keeping with the animals' expressed MHC class I genotypes, macaques in Group 1 received inserts encoding the Mamu-B*08-restricted Nef_137-146RL10 epitope, while those in Group 2 were vaccinated with gag sequences containing the Mamu-A*01-restricted Gag_181-189CM9 determinant. Nineteen weeks following the rRRV boost, we began challenging the Group 1 and Group 2 monkeys every week with IR inoculations of 200 TCID₅₀ of an in vivo-titered SIVmac239 stock.³² (B, C) Vaccine-induced SIV-specific CD8⁺ T cell responses in Group 1 (B) and Group 2 (C). We monitored the ontogeny of vaccine-elicited CD8⁺ T cell responses in Groups 1 and 2 by staining PBMC with fluorochrome-labeled Mamu-B*08/Nef_137-146RL10 and Mamu-A*01/Gag_181-189CM9 tetramers, respectively. Only time points following the rYF17D boost are displayed. The last measurement was performed at week 34 post rYF17D vaccination, 3 weeks before the first IR challenge with SIVmac239. (D, E) Memory phenotype of vaccine-induced CD8⁺ T cells in Group 1 (D) and Group 2 (E) at week 34 post rYF17D vaccination. Based on the expression of CD28 and CCR7, tetramer⁺CD8⁺ T cells in PBMC were classified as fully differentiated effector memory (T_EM2; CD28⁻CCR7⁻), transitional memory (T_EM1; CD28⁺CCR7⁻), or central memory (T_CM; CD28⁺CCR7⁺) subsets. (F) Rate at which macaques in Groups 1 and 2 became infected following repeated IR challenges with SIVmac239. The frequency of each animal's tetramer⁺CD8⁺ T cells at week 34 post rYF17D is shown as a reference. (G) Log-transformed VLs after SIVmac239 infection. The dashed line in the graph is for reference only and indicates a VL of 10⁶ vRNA copies/ml. The dotted line is also for reference only and denotes a VL of 10³ vRNA copies/ml. The limit of reliable quantitation of this VL assay was 15 vRNA copies/ml of plasma. To assess the extent to which vaccinees in Groups 1 and 2 decreased plasma viremia, geometric means of VLs measured in unvaccinated MHC class I-matched macaques that were rectally infected with SIVmac239 as part of current and recent studies conducted in our laboratory are also plotted (Martins et al., unpublished observations).^32,33,39 These geometric means were calculated based on VLs measured within weeks 1–20 PI in 12 Mamu-B*08⁺ and 8 Mamu-A*01⁺ macaques. IR, intrarectal; MHC, major histocompatibility complex; PI, postinfection; PBMC, peripheral blood mononuclear cell; RRV, rhadinovirus; SIV, simian immunodeficiency virus; T_EM, effector memory T cell; VL, viral load; vRNA, viral RNA.