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Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro, but their role in infection and pathogenesis is unknown. To examine their in vivo function in an animal model evolutionarily closely related to humans, we identified and characterized Rh05, Rh152/151 and Rh173 as the complete set of vFcγRs encoded by rhesus CMV (RhCMV). Each one of these proteins displays functional similarities to their prospective HCMV orthologs with respect to antagonizing host FcγR activation in vitro. When RhCMV-naïve male rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma DNAemia levels and anti-RhCMV antibody responses were comparable to wildtype infections of both male and female animals. However, the duration of plasma DNAemia was significantly shortened in immunocompetent, but not in CD4 + T cell-depleted animals. Since vFcγRs were not required for superinfection of rhesus macaques, we conclude that these proteins can prolong lytic replication during primary infection by evading virus-specific adaptive immune responses, particularly antibodies., Competing Interests: Competing interests: S.R.P. has served as a consultant to Merck, Moderna, Pfizer, GSK and Dynavax and has led sponsored programs with Moderna and Merck. P.K. has received funding from Biotest AG. None of these activities have impacted this work. The remaining authors declare no competing interests., (© 2025. The Author(s).)

The initial development of cytomegalovirus (CMV) as a vaccine vector for HIV/simian immunodeficiency virus (SIV) was predicated on its potential to pre-position high-frequency, effector-differentiated, CD8 + T cells in tissues for immediate immune interception of nascent primary infection. This goal was achieved and also led to the unexpected discoveries that non-human primate (NHP) CMVs can be programmed to differentially elicit CD8 + T cell responses that recognize viral peptides via classical MHC-Ia, and/or MHC-II, and/or MHC-E, and that MHC-E-restricted CD8 + T cell responses can uniquely mediate stringent arrest and subsequent clearance of highly pathogenic SIV, an unprecedented type of vaccine-mediated protection. These discoveries delineate CMV vector-elicited MHC-E-restricted CD8 + T cells as a functionally distinct T cell response with the potential for superior efficacy against HIV-1, and possibly other infectious agents or cancers., Competing Interests: Declaration of interests L.J.P., S.G.H., and K.F. have a substantial financial interest in Vir Biotechnology, Inc., a company that may have a commercial interest in the results of this research and technology. L.J.P., S.G.H., and K.F. are also consultants to Vir Biotechnology, Inc. These potential individual and institutional conflicts of interest have been reviewed and managed by Oregon Health and Science University (OHSU)., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Strain 68-1 rhesus cytomegalovirus expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) primes MHC-E-restricted CD8+ T cells that control SIV replication in 50%-60% of the vaccinated rhesus macaques. Whether this unconventional SIV-specific immunity and protection is unique to rhesus macaques or RhCMV or is intrinsic to CMV remains unknown. Here, using cynomolgus CMV vectors expressing SIV antigens (CyCMV/SIV) and Mauritian cynomolgus macaques, we demonstrate that the induction of MHC-E-restricted CD8+ T cells requires matching CMV to its host species. RhCMV does not elicit MHC-E-restricted CD8+ T cells in cynomolgus macaques. However, cynomolgus macaques vaccinated with species-matched 68-1-like CyCMV/SIV mounted MHC-E-restricted CD8+ T cells, and half of the vaccinees stringently controlled SIV post-challenge. Protected animals manifested a vaccine-induced IL-15 transcriptomic signature that is associated with efficacy in rhesus macaques. These findings demonstrate that the ability of species-matched CMV vectors to elicit MHC-E-restricted CD8+ T cells that are required for anti-SIV efficacy is conserved in nonhuman primates, and these data support the development of HCMV/HIV for a prophylactic HIV vaccine., Competing Interests: Declaration of interests OHSU and Drs. Malouli, Früh, Picker, Hansen, and Sacha have a significant financial interest in Vir Biotechnology, Inc., a company that may have a financial interest in the results of this research and technology. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Simian immunodeficiency virus (SIV) insert-expressing, 68-1 rhesus cytomegalovirus (RhCMV/SIV) vectors elicit major histocompatibility complex E (MHC-E)- and MHC-II-restricted, SIV-specific CD8 + T cell responses, but the basis of these unconventional responses and their contribution to demonstrated vaccine efficacy against SIV challenge in the rhesus monkeys (RMs) have not been characterized. We show that these unconventional responses resulted from a chance genetic rearrangement in 68-1 RhCMV that abrogated the function of eight distinct immunomodulatory gene products encoded in two RhCMV genomic regions ( Rh157.5/Rh157.4 and Rh158-161 ), revealing three patterns of unconventional response inhibition. Differential repair of these genes with either RhCMV-derived or orthologous human CMV (HCMV)-derived sequences ( UL128/UL130 ; UL146/UL147 ) leads to either of two distinct CD8 + T cell response types-MHC-Ia-restricted only or a mix of MHC-II- and MHC-Ia-restricted CD8 + T cells. Response magnitude and functional differentiation are similar to RhCMV 68-1, but neither alternative response type mediated protection against SIV challenge. These findings implicate MHC-E-restricted CD8 + T cell responses as mediators of anti-SIV efficacy and indicate that translation of RhCMV/SIV vector efficacy to humans will likely require deletion of all genes that inhibit these responses from the HCMV/HIV vector., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques., Competing Interests: OHSU and Drs. S.G.H., L.J.P., K.F. and D.M. have a significant financial interest in VirBiotechnology, Inc., a company that may have a commercial interest in the results of this research and technology. The potential individual and institutional conflicts of interest have been reviewed and managed by OHSU.

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., Competing Interests: Oregon Health and Science University (OHSU), KF, LJP, JAN, and SGH have a financial interest in TomegaVax Inc., a company that may have a commercial interest in the results of this research and technology. JBS and DM are inventors on OHSU patents that have been licensed by TomegaVax, and therefore may have future financial interests in TomegaVax Inc. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU.

The natural killer cell receptor NKG2D activates NK cells by engaging one of several ligands (NKG2DLs) belonging to either the MIC or ULBP families. Human cytomegalovirus (HCMV) UL16 and UL142 counteract this activation by retaining NKG2DLs and US18 and US20 act via lysomal degradation but the importance of NK cell evasion for infection is unknown. Since NKG2DLs are highly conserved in rhesus macaques, we characterized how NKG2DL interception by rhesus cytomegalovirus (RhCMV) impacts infection in vivo. Interestingly, RhCMV lacks homologs of UL16 and UL142 but instead employs Rh159, the homolog of UL148, to prevent NKG2DL surface expression. Rh159 resides in the endoplasmic reticulum and retains several NKG2DLs whereas UL148 does not interfere with NKG2DL expression. Deletion of Rh159 releases human and rhesus MIC proteins, but not ULBPs, from retention while increasing NK cell stimulation by infected cells. Importantly, RhCMV lacking Rh159 cannot infect CMV-naïve animals unless CD8+ cells, including NK cells, are depleted. However, infection can be rescued by replacing Rh159 with HCMV UL16 suggesting that Rh159 and UL16 perform similar functions in vivo. We therefore conclude that cytomegaloviral interference with NK cell activation is essential to establish but not to maintain chronic infection., Competing Interests: The authors have declared that no competing interests exist.

The most abundantly produced virion protein in human cytomegalovirus (HCMV) is the immunodominant phosphoprotein 65 (pp65), which is frequently included in CMV vaccines. Although it is nonessential for in vitro CMV growth, pp65 displays immunomodulatory functions that support a potential role in primary and/or persistent infection. To determine the contribution of pp65 to CMV infection and immunity, we generated a rhesus CMV lacking both pp65 orthologs (RhCMVΔpp65ab). While deletion of pp65ab slightly reduced growth in vitro and increased defective particle formation, the protein composition of secreted virions was largely unchanged. Interestingly, pp65 was not required for primary and persistent infection in animals. Immune responses induced by RhCMVΔpp65ab did not prevent reinfection with rhesus CMV; however, reinfection with RhCMVΔUS2-11, which lacks viral-encoded MHC-I antigen presentation inhibitors, was prevented. Unexpectedly, induction of pp65b-specific T cells alone did not protect against RhCMVΔUS2-11 challenge, suggesting that T cells targeting multiple CMV antigens are required for protection. However, pp65-specific immunity was crucial for controlling viral dissemination during primary infection, as indicated by the marked increase of RhCMVΔpp65ab genome copies in CMV-naive, but not CMV-immune, animals. Our data provide rationale for inclusion of pp65 into CMV vaccines but also demonstrate that pp65-induced T cell responses alone do not recapitulate the protective effect of natural infection.

Cytomegalovirus (CMV) can superinfect persistently infected hosts despite CMV-specific humoral and cellular immunity; however, how it does so remains undefined. We have demonstrated that superinfection of rhesus CMV-infected rhesus macaques (RM) requires evasion of CD8+ T cell immunity by virally encoded inhibitors of major histocompatibility complex class I (MHC-I) antigen presentation, particularly the homologs of human CMV US2, 3, 6, and 11. In contrast, MHC-I interference was dispensable for primary infection of RM, or for the establishment of a persistent secondary infection in CMV-infected RM transiently depleted of CD8+ lymphocytes. These findings demonstrate that US2-11 glycoproteins promote evasion of CD8+ T cells in vivo, thus supporting viral replication and dissemination during superinfection, a process that complicates the development of preventive CMV vaccines but that can be exploited for CMV-based vector development.

Viral infection activates interferon regulatory factor 3 (IRF3), a cofactor for the induction of interferon-stimulated genes (ISGs). The role of IRF3 in the activation of ISGs by human cytomegalovirus (HCMV) is controversial despite the fact that HCMV has consistently been shown to induce ISGs during infection of fibroblasts. To address the function of IRF3 in HCMV-mediated ISG induction, we monitored ISG expression and global gene expression in HCMV-infected cells in which IRF3 function had been depleted by small interfering RNA or blocked by dominant negative IRF3. A specific reduction of ISG induction was observed, whereas other transcripts were unaffected. We therefore conclude that IRF3 specifically regulates ISG induction during the initial phase of HCMV infection.