Previous studies have established that strain 68-1–derived rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) proteins (RhCMV/SIV) are able to elicit and maintain cellular immune responses that provide protection against mucosal challenge of highly pathogenic SIV in rhesus monkeys (RMs). However, these efficacious RhCMV/SIV vectors were replication and spread competent and therefore have the potential to cause disease in immunocompromised subjects. To develop a safer CMV-based vaccine for clinical use, we attenuated 68-1 RhCMV/SIV vectors by deletion of the Rh110 gene encoding the pp71 tegument protein (ΔRh110), allowing for suppression of lytic gene expression. ΔRh110 RhCMV/SIV vectors are highly spread deficient in vivo (~1000-fold compared to the parent vector) yet are still able to superinfect RhCMV⁺ RMs and generate high-frequency effector-memory–biased T cell responses. Here, we demonstrate that ΔRh110 68-1 RhCMV/SIV–expressing homologous or heterologous SIV antigens are highly efficacious against intravaginal (IVag) SIV_mac239 challenge, providing control and progressive clearance of SIV infection in 59% of vaccinated RMs. Moreover, among 12 ΔRh110 RhCMV/SIV–vaccinated RMs that controlled and progressively cleared an initial SIV challenge, 9 were able to stringently control a second SIV challenge ~3 years after last vaccination, demonstrating the durability of this vaccine. Thus, ΔRh110 RhCMV/SIV vectors have a safety and efficacy profile that warrants adaptation and clinical evaluation of corresponding HCMV vectors as a prophylactic HIV/AIDS vaccine.

Viral peptide is key to T cell priming: Simian immunodeficiency virus (SIV) vaccines containing a strain 68-1 rhesus cytomegalovirus (RhCMV) vector elicit strong CD8 ⁺ T cell responses that can control and clear SIV infections. The SIV peptides targeted by these T cells are presented on major histocompatibility complex (MHC) II and the nonclassical MHC-Ib molecule MHC-E rather than the more typical MHC-Ia. Verweij et al. show that the 68-1 RhCMV–encoded peptide VL9 drives intracellular transport of MHC-E and recognition of RhCMV-infected targets by MHC-E–restricted CD8 ⁺ T cells. Rhesus macaques vaccinated with a mutant 68-1 RhCMV lacking VL9 showed no priming of MHC-E–restricted CD8 ⁺ T cells and no protection against SIV. This work strongly suggests that future effective CMV-based HIV vaccines in humans will also require MHC-E–restricted CD8 ⁺ T cell priming. Science , this issue p. eabe9233

Eight RhCMV gene products control induction of unconventionally restricted CD8 ⁺ T cells and RhCMV/SIV vaccine efficacy.

The strain 68-1 rhesus cytomegalovirus (RhCMV)–based vaccine for simian immunodeficiency virus (SIV) can stringently protect rhesus macaques (RMs) from SIV challenge by arresting viral replication early in primary infection. This vaccine elicits unconventional SIV-specific CD8 ⁺ T cells that recognize epitopes presented by major histocompatibility complex (MHC)–II and MHC-E instead of MHC-Ia. Although RhCMV/SIV vaccines based on strains that only elicit MHC-II– and/or MHC-Ia–restricted CD8 ⁺ T cells do not protect against SIV, it remains unclear whether MHC-E–restricted T cells are directly responsible for protection and whether these responses can be separated from the MHC-II–restricted component. Using host microRNA (miR)–mediated vector tropism restriction, we show that the priming of MHC-II and MHC-E epitope–targeted responses depended on vector infection of different nonoverlapping cell types in RMs. Selective inhibition of RhCMV infection in myeloid cells with miR-142–mediated tropism restriction eliminated MHC-E epitope–targeted CD8 ⁺ T cell priming, yielding an exclusively MHC-II epitope–targeted response. Inhibition with the endothelial cell–selective miR-126 eliminated MHC-II epitope–targeted CD8 ⁺ T cell priming, yielding an exclusively MHC-E epitope–targeted response. Dual miR-142 + miR-126–mediated tropism restriction reverted CD8 ⁺ T cell responses back to conventional MHC-Ia epitope targeting. Although the magnitude and differentiation state of these CD8 ⁺ T cell responses were generally similar, only the vectors programmed to elicit MHC-E–restricted CD8 ⁺ T cell responses provided protection against SIV challenge, directly demonstrating the essential role of these responses in RhCMV/SIV vaccine efficacy.

Vaccines based on cytomegalovirus (CMV) demonstrate protection in animal models of infectious disease and cancer. Vaccine efficacy is associated with the ability of CMV to elicit and indefinitely maintain high frequencies of circulating effector memory T cells (T_EM) providing continuous, life-long anti-pathogen immune activity. To allow for the clinical testing of human CMV (HCMV)-based vaccines we constructed and characterized as a vector backbone the recombinant molecular clone TR3 representing a wildtype genome. We demonstrate that TR3 can be stably propagated in vitro and that, despite species incompatibility, recombinant TR3 vectors elicit high frequencies of T_EM to inserted antigens in rhesus macaques (RM). Live-attenuated versions of TR3 were generated by deleting viral genes required to counteract intrinsic and innate immune responses. In addition, we eliminated subunits of a viral pentameric glycoprotein complex thus limiting cell tropism. We show in a humanized mouse model that such modified vectors were able to establish persistent infection but lost their ability to reactivate from latency. Nevertheless, attenuated TR3 vectors preserved the ability to elicit and maintain T_EM to inserted antigens in RM. We further demonstrate that attenuated TR3 can be grown in approved cell lines upon elimination of an anti-viral host factor using small interfering RNA, thus obviating the need for a complementing cell line. In sum, we have established a versatile platform for the clinical development of live attenuated HCMV-vectored vaccines and immunotherapies.

Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68–1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells in vitro, RhCMV 68–1 failed to productively infect MCM in vivo, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68–1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68–1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68–1 and 68–1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics. Nonhuman primates are excellent models of HCMV infection and immunity, and their CMVs closely resemble HCMV in genomic organization and coding potential. Here, we expand the existing models by isolating new full-length, non-human primate CMV strains that more closely resemble primary HCMV isolates. In addition, we identify determinants needed for recombinant clones of RhCMV to infect MCM both in vitro and in vivo. Given their simplified immunogenetics, these results enable the use of MCM to deepen our understanding of CMV immunology.