Strain 68-1 rhesus cytomegalovirus (RhCMV) vaccine vectors expressing simian immunodeficiency virus (SIV) antigens elicit immune responses that stringently control and ultimately clear highly pathogenic SIV challenge in more than half of rhesus monkeys (RMs). The high frequency of effector-differentiated CD8⁺ T cells elicited by this vaccine contribute to this efficacy, mediating SIV replication arrest before the establishment of long-lived SIV reservoirs. However, we unexpectedly found that SIV peptides targeted by these CD8⁺ T cells are presented by major histocompatibility complex-E (MHC-E) or MHC-II instead of MHC-Ia. This raised the question of how these unconventional T cell responses arise and whether they are necessary for vaccine efficacy.

MHC-E binds the conserved VL9 peptide embedded in the leader sequence of MHC-Ia proteins. This MHC-E–VL9 complex predominantly engages inhibitory receptors on natural killer (NK) cells, thus serving as a protective “self” signal for healthy cells. To evade NK cells, both rhesus and human CMVs encode viral glycoproteins (Rh67 and UL40, respectively) that contain a VL9 sequence providing the MHC-E/VL9 complex to NK cells when cellular VL9 is not available because of viral MHC-Ia downregulation. We hypothesized that viral-encoded VL9 also controls the ability of 68-1 RhCMV to elicit MHC-E–restricted CD8⁺ T cells. This allowed us to examine the role of these cells in RhCMV/SIV vaccine efficacy.

We show that Rh67-embedded VL9 is required for intracellular transport of MHC-E in RhCMV-infected fibroblasts and for their recognition by MHC-E–targeting CD8⁺ T cells. Rh67 deletion did not substantially affect the character of RhCMV/SIV–elicited T cell responses, but such deletion, or VL9 mutation within Rh67, completely abrogated MHC-E–restricted CD8⁺ T cell priming, leaving vaccine-elicited T cell responses entirely MHC-II restricted. In contrast to RMs vaccinated with the parent 68-1 RhCMV/SIV vaccine, post-SIV challenge replication arrest was not observed among RMs vaccinated with the Rh67-deleted vaccine. Thus, 68-1 RhCMV/SIV vaccine efficacy requires Rh67-induced, MHC-E–restricted CD8⁺ T cell priming, implicating this response in the mechanism of protection.

RhCMV appears to elicit MHC-E–restricted T cells as a consequence of viral NK cell evasion. However, as separately reported, these responses are only manifest in 68-1 RhCMV because of a genetic rearrangement that abrogates the function of eight independent viral inhibitors of these responses. Thus, RhCMV has evolved to evade both NK cells and the MHC-E–restricted CD8⁺ T cells that result from this evasion. By contrast, SIV neither elicits nor seems to evade these responses. Our results strongly suggest that CD8⁺ T cells targeting MHC-E–presented HIV peptides will be necessary for an effective CMV-based HIV vaccine Fortunately, both the positive and negative RhCMV regulators of MHC-E–restricted responses are conserved in human CMV, suggesting that human CMV and/or HIV vaccines might also be programmed to elicit this unusual type of protective immunity.

Fibroblasts infected in vitro with 68-1 RhCMV can stimulate MHC-E–restricted CD8⁺ T cells only if the MHC-E VL9 peptide ligand (in red) embedded within the Rh67 gene is expressed. This peptide is loaded onto MHC-E in the endoplasmic reticulum, thus enabling the …