After SIVmac239 challenge, all these animals showed efficient in-vitro anti-SIV-G64723mt efficacy (more than two-fold reduction in viral production) of CD8+ cells, sooner or later, in the chronic phase. The levels of in-vitro anti-SIV-G64723mt efficacy of CD8+ cells tended to become higher in the chronic phase. Anti-SIVmac239 efficacy of CD8+ cells was not associated with anti-SIV-G64723mt efficacy. For instance, some CD8+ cells efficiently suppressed SIV-G64723mt but not SIVmac239 replication. After all, all SIV controllers acquired CD8+ cells able to suppress the mutant SIV-G64723mt replication in vitro in the chronic phase.
These animals were superchallenged with a mutant SIV, SIV-G64723mt, that has five gag mutations resulting in escape from recognition by Gag206-216-specific, Gag241-249-specific, and Gag367-381-specific CTLs around 1 year (R06-015, R03-014, and R03-012), 2 years (R04-015, R04-016, and R06-007), 3 years (R02-002), or 4 years (R02-003) after SIVmac239 challenge. The replicative ability of SIV-G64723mt is significantly lower than that of wild-type SIVmac239, but SIV-G64723mt challenge of naive 90-120-Ia-negative rhesus macaques can result in persistent viral replication and AIDS progression [23,28]. It has previously been shown that 90-120-Ia-positive macaques vaccinated with DNA-prime/SeV-Gag-boost are unable to contain a SIV-G64723mt challenge, whereas they can control replication of wild-type SIVmac239 . Indeed, we confirmed that CD8+ cells obtained from these 90-120-Ia-positive vaccinees before challenge efficiently suppressed wild-type SIVmac239 but not SIV-G64723mt replication in vitro. In the present study, however, all eight wild-type SIV controllers contained the SIV-G64723mt superchallenge without detectable viremia (Fig. 1b). SIVmac239-specific neutralizing antibody responses were undetectable around the superchallenge in any of these controllers (Fig. 1a). These results indicate that, after SIVmac239 challenge, the SIV controllers acquired the potential to control SIV-G64723mt replication in the absence of neutralizing antibody responses, although to what extent CD8+ cell responses may contribute to this containment of SIV-G64723mt superchallenge remains unclear. Postsuperchallenge CD8+ cells suppressed both SIVmac239 and SIV-G64723mt replication in vitro efficiently (Figs. 2 and 3).
We also examined Gag-specific CTL responses in SIV controllers at several time points by using a panel of overlapping peptides (Gag peptide pools 1–10) spanning the entire SIVmac239 Gag (Fig. 4b). Group I macaques vaccinated with DNA/SeV-Gag elicited CTL responses directed against not only Gag peptide pool 5 (including Gag206-216 and Gag241-249) and 7 (including Gag367-381) but also other Gag peptide pools after boost and after challenge; some peptide pool-specific CTLs were diminished, whereas others appeared in the chronic phase. In contrast, group II macaques eliciting CTL responses directed against single Gag206-216 (R04-015) or Gag241-249 (R04-016 and R06-007) epitope after boost showed predominant Gag peptide pool 5-specific CTL responses after challenge and accumulated multiple Gag epitope-specific CTL responses in the chronic phase. These results indicate dynamics of postchallenge Gag-specific CTL responses in vaccine-based SIV controllers. After SIV-G64723mt superchallenge, changes in the pattern of Gag-specific CTL responses were observed in some animals.
Next, in SIV controllers, we examined CTL responses directed against SIV non-Gag antigens by using panels of overlapping peptides spanning the entire SIVmac239 antigens other than Gag (Fig. 5a). These SIV controllers showed SIV non-Gag-specific CTL responses from the early phase after challenge. After SIV-G64723mt superchallenge, broadening or changes in the pattern of these CTL responses were observed in some animals; Vif-specific or Nef-specific CTL responses were detected predominantly, although we did not find common CTL epitopes in Vif or Nef.
Finally, we analyzed correlation of antigen-specific CTL levels with in-vitro anti-SIVmac239 or anti-SIV-G64723mt efficacy levels of CD8+ cells (Fig. 5b). We found a correlation of anti-SIVmac239 efficacy levels with Gag206-216-specific and Gag241-249-specific CTL levels but not with total Gag-specific CTL levels. The anti-SIVmac239 efficacy levels did not correlate with either Gag206-216-specific or Gag241-249-specific CTL levels alone (data not shown), although our previous study  indicated inverse correlation between peak plasma viral loads and the levels of Gag241-249-specific CTLs dominantly induced in DNA/SeV-Gag236-250-EGFP-vaccinated animals in the acute phase after challenge. Correlations of anti-SIVmac239 efficacy levels after challenge with Vif-specific CTL levels and with Nef-specific CTL levels were indicated. On the contrary, anti-SIV-G64723mt efficacy levels after challenge strongly correlated with Vif-specific CTL levels, although any correlation of these levels with other SIV antigen-specific CTL levels was not indicated. These results suggest that Vif-specific CTL induction may contribute in part to acquisition of the potential to suppress SIV-G64723mt replication efficiently.
The group I animals induced multiple Gag epitope-specific CTL responses after boost (before challenge) and after challenge, whereas the group II animals elicited only Gag206-216-specific or Gag241-249-specific CTL responses before challenge and showed induction of additional CTL responses directed against Gag epitopes other than Gag206-216 and Gag241-249 after challenge. Furthermore, both groups elicited SIV non-Gag-specific CTL responses after challenge. These results indicate postchallenge accumulation of broader CTL responses. The in-vitro anti-SIVmac239 efficacy levels correlated with Vif-specific and Nef-specific CTL as well as Gag206-216-specific and Gag241-249-specific CTL levels but not with total Gag-specific or total SIV-specific CTL levels, suggesting that not all but some particular epitope-specific CTL responses were involved in suppression of SIVmac239 replication. Nef-specific CTL responses were detected more frequently than Vif-specific ones, whereas the latter showed stronger correlation with antiviral efficacy levels (Fig. 5). We did not find common CTL epitopes in Vif or Nef. These may imply higher frequencies of effective CTLs in Vif-specific ones; conversely, Nef-specific CTLs may include effective ones but with higher frequencies of ineffective ones.
We found dynamics of cellular immune responses during viral control in vaccine-based SIV controllers, but the exact mechanism for broadening or changes in dominance patterns of CTL responses remains unclear. All the group I animals and macaque R04-015 showed rapid selection of a CTL escape gag mutation, L216S, at week 5 after challenge, whereas no gag mutations were selected at week 5 in macaques R04-016 or R06-007 (data not shown). We failed to recover viral genome cDNAs for sequencing from plasma after week 5 due to undetectable viral loads, but selection of viral CTL escape mutations and reversions [23,28,52–57] under undetectable levels of viral replication may contribute to induction of broader CTL responses in SIV controllers.
It is difficult to directly compare anti-SIVmac239 and anti-SIV-G64723mt efficacy of CD8+ cells because of difference in their replicative ability, but the ratios of the latter level to the former 1 year after challenge were higher than those after boost in all animals. Indeed, CD8+ cells 1 year after challenge in macaques R03-012 and R02-003 showed suppressive effect on SIV-G64723mt but not on wild-type SIVmac239 replication, although R03-012 CD8+ cells at 5 months and 1 year after challenge efficiently suppressed SIVmac239 replication at higher E/T ratio of 1: 1 (R02-003 CD8+ cells in the chronic phase for this analysis were unavailable). Because no SIV controllers elicited CTL responses specific for peptides with mutated amino acid sequences (data not shown), all CTLs specific for SIV-G64723mt antigens in SIV controllers are expected to recognize SIVmac239 antigens also. Thus, our observation that some postchallenge CD8+ cells showed efficient suppressive effect on SIV-G64723mt but not on SIVmac239 replication in vitro may be explained by higher replicative ability of SIVmac239 compared with SIV-G64723mt; it could be more difficult for CD8+ cells to suppress replication of the wild-type SIVmac239 than the mutant SIV-G64723mt, implying a possible requirement of more potent CTL responses for SIVmac239 control than for SIV-G64723mt control.
In summary, this study showed dynamics of postchallenge cellular immune responses in vaccine-based SIV controllers. Our results suggest that, during persistent viral control, vaccine-based SIV controllers can acquire CD8+ cells with the potential to suppress replication of SIV variants carrying CTL escape mutations. Elucidation of the mechanism for induction of broader responses in these controllers may contribute to development of a vaccine effective against highly diversified HIV infection.
This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, a grant-in-aid from the Japan Society for the Promotion of Science, grants-in-aid from the Ministry of Health, Labor, and Welfare, and a grant from Takeda Science Foundation in Japan. N.I. is a Research Fellow of the Japan Society for the Promotion of Science. The animal experiments were conducted through the Cooperative Research Program in the Tsukuba Primate Research Center (TPRC), National Institute of Biomedical Innovation, with the help of the Corporation for Production and Research of Laboratory Primates (CPRLP). We thank Dr H. Akari, A. Saito, Y. Yasutomi, A. Hiyaoka, K. Komatsuzaki, K. Oto, and F. Ono for their assistance in animal experiments, T. Naruse and A. Kimura for MHC-I haplotyping, and DNAVEC Corporation for providing SeV vectors.
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