Myeloid cell tropism enables MHC-E-restricted CD8+ T cell priming and vaccine efficacy by the RhCMV/SIV vaccine.

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.

Characterization of a live-attenuated HCMV-based vaccine platform.

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 (TEM) 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 TEM 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 TEM 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.

Enhancing safety of cytomegalovirus-based vaccine vectors by engaging host intrinsic immunity.

Rhesus cytomegalovirus (RhCMV)-based vaccines maintain effector memory T cell responses (TEM) that protect ~50% of rhesus monkeys (RMs) challenged with simian immunodeficiency virus (SIV). Because human CMV (HCMV) causes disease in immunodeficient subjects, clinical translation will depend upon attenuation strategies that reduce pathogenic potential without sacrificing CMV's unique immunological properties. We demonstrate that "intrinsic" immunity can be used to attenuate strain 68-1 RhCMV vectors without impairment of immunogenicity. The tegument proteins pp71 and UL35 encoded by UL82 and UL35 of HCMV counteract cell-intrinsic restriction via degradation of host transcriptional repressors. When the corresponding RhCMV genes, Rh110 and Rh59, were deleted from 68-1 RhCMV (ΔRh110 and ΔRh59), we observed only a modest growth defect in vitro, but in vivo, these modified vectors manifested little to no amplification at the injection site and dissemination to distant sites, in contrast to parental 68-1 RhCMV. ΔRh110 was not shed at any time after infection and was not transmitted to naïve hosts either by close contact (mother to infant) or by leukocyte transfusion. In contrast, ΔRh59 was both shed and transmitted by leukocyte transfusion, indicating less effective attenuation than pp71 deletion. The T cell immunogenicity of ΔRh110 was essentially identical to 68-1 RhCMV with respect to magnitude, TEM phenotype, epitope targeting, and durability. Thus, pp71 deletion preserves CMV vector immunogenicity while stringently limiting vector spread, making pp71 deletion an attractive attenuation strategy for HCMV vectors.

A live-attenuated RhCMV/SIV vaccine shows long-term efficacy against heterologous SIV challenge.

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) SIVmac239 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.

Prevention of tuberculosis in rhesus macaques by a cytomegalovirus-based vaccine.

Despite widespread use of the bacille Calmette-Guérin (BCG) vaccine, tuberculosis (TB) remains a leading cause of global mortality from a single infectious agent (Mycobacterium tuberculosis or Mtb). Here, over two independent Mtb challenge studies, we demonstrate that subcutaneous vaccination of rhesus macaques (RMs) with rhesus cytomegalovirus vectors encoding Mtb antigen inserts (hereafter referred to as RhCMV/TB)-which elicit and maintain highly effector-differentiated, circulating and tissue-resident Mtb-specific CD4+ and CD8+ memory T cell responses-can reduce the overall (pulmonary and extrapulmonary) extent of Mtb infection and disease by 68%, as compared to that in unvaccinated controls, after intrabronchial challenge with the Erdman strain of Mtb at ∼1 year after the first vaccination. Fourteen of 34 RhCMV/TB-vaccinated RMs (41%) across both studies showed no TB disease by computed tomography scans or at necropsy after challenge (as compared to 0 of 17 unvaccinated controls), and ten of these RMs were Mtb-culture-negative for all tissues, an exceptional long-term vaccine effect in the RM challenge model with the Erdman strain of Mtb. These results suggest that complete vaccine-mediated immune control of highly pathogenic Mtb is possible if immune effector responses can intercept Mtb infection at its earliest stages.

Cytomegalovirus pp65 limits dissemination but is dispensable for persistence.

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.

Multifaceted evasion of the interferon response by cytomegalovirus.

Human cytomegalovirus (HCMV), which infects the majority of the population worldwide, causes few, if any, symptoms in otherwise healthy people but is responsible for considerable morbidity and mortality in immunocompromised patients and in congenitally infected newborns. The evolutionary success of HCMV depends in part on its ability to evade host defense systems. Here we review recent progress in elucidating the remarkable assortment of mechanisms employed by HCMV and the related beta-herpesviruses, murine cytomegaloviruses (MCMV) and rhesus cytomegaloviruses (RhCMV), for counteracting the host interferon (IFN) response. Very early after infection, cellular membrane sensors such as the lymphotoxin beta receptor initiate the production of antiviral cytokines including type I IFNs. However, virion factors, such as pp65 (ppUL83) and viral proteins made soon after infection including the immediate early gene 2 protein (pUL122), repress this response by interfering with steps in the activation of IFN regulatory factor 3 and NF-kappaB. CMVs then exert a multi-pronged attack on downstream IFN signaling. HCMV infection results in decreased accumulation and phosphorylation of the IFN signaling kinases Jak1 and Stat2, and the MCMV protein pM27 mediates Stat2 down-regulation, blocking both type I and type II IFN signaling. The HCMV immediate early gene 1 protein (pUL123) interacts with Stat2 and inhibits transcriptional activation of IFN-regulated genes. Infection also causes reduction in the abundance of p48/IRF9, a component of the ISGF3 transcription factor complex. Furthermore, CMVs have multiple genes involved in blocking the function of IFN-induced effectors. For example, viral double-stranded RNA-binding proteins are required to prevent the shutoff of protein synthesis by protein kinase R, further demonstrating the vital importance of evading the IFN response at multiple levels during infection.

Essential role for either TRS1 or IRS1 in human cytomegalovirus replication.

Viral infections often produce double-stranded RNA (dsRNA), which in turn triggers potent antiviral responses, including the global repression of protein synthesis mediated by protein kinase R (PKR) and 2'-5' oligoadenylate synthetase (OAS). As a consequence, many viruses have evolved genes, such as those encoding dsRNA-binding proteins, which counteract these pathways. Human cytomegalovirus (HCMV) encodes two related proteins, pTRS1 and pIRS1, which bind dsRNA and can prevent activation of the PKR and OAS pathways. HCMV mutants lacking either IRS1 or TRS1 replicate at least moderately well in cell culture. However, as we demonstrate in the present study, an HCMV mutant lacking both IRS1 and TRS1 (HCMV[DeltaI/DeltaT]) has a severe replication defect. Infection with HCMV[DeltaI/DeltaT] results in a profound inhibition of overall and viral protein synthesis, as well as increased phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). The vaccinia virus E3L gene can substitute for IRS1 or TRS1, enabling HCMV replication. Despite the accumulation of dsRNA in HCMV-infected cells, the OAS pathway remains inactive, even in HCMV[DeltaI/DeltaT]-infected cells. These results suggest that PKR-mediated phosphorylation of eIF2alpha is the dominant dsRNA-activated pathway responsible for inhibition of protein synthesis and HCMV replication in the absence of both IRS1 and TRS1 and that the requirement for evasion of the PKR pathway likely explains the necessity for IRS1 or TRS1 for productive infection.

Binding and nuclear relocalization of protein kinase R by human cytomegalovirus TRS1.

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VVDeltaE3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2alpha kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VVDeltaE3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VVDeltaE3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VVDeltaE3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VVDeltaE3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2alpha.