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.

Activation of double-stranded RNA (dsRNA)-mediated pathways contributes to the innate immune response to viral infection. Among the genes involved in this antiviral response are several interferon-induced genes, including those encoding protein kinase R (PKR) and 2Ј-5Ј oligoadenylate synthetase (OAS). After binding to dsRNA, PKR dimerizes, autophosphorylates, and then phosphorylates the translation initiation factor eukaryotic initiation factor 2␣ (eIF2␣). Phosphorylated eIF2␣ inhibits guanine nucleotide exchange factor eIF2B, preventing restoration of the eIF2␣-tRNA Met -GTP ternary complex and thus halting protein synthesis at the level of initiation (reviewed in reference 20). OAS catalyzes the synthesis of 2Ј-5Ј oligoadenylates, which activate latent RNase L, resulting in degradation of single-stranded mRNA and rRNA (reviewed in reference 44). Together, activation of the PKR and OAS pathways results in an antiviral environment by causing a shutdown of protein synthesis.Since viral replication requires protein synthesis, many viruses have had to evolve strategies for evading these dsRNAmediated antiviral response pathways (32, 36). For example, the vaccinia virus (VV) E3L protein binds to dsRNA and prevents activation of both PKR and OAS (14,28,50). VV lacking E3L (VV⌬E3L) is sensitive to interferon, has a limited cellular host range, and is avirulent in mice (5, 9, 51). Replication of VV⌬E3L in cell culture can be rescued by dsRNAbinding proteins (dsRBPs) from other viruses (30). For example, the dsRBPs pIRS1 and pTRS1 of human cytomegalovirus (HCMV) can inhibit eIF2␣ phosphorylation and RNase L activation, prevent the shutoff of protein synthesis, and rescue viral replication in VV⌬E3L-infected human cells (17). In murine CMV (MCMV), two US22 family members, pm142 and pm143, have functions similar to those of pIRS1 and pTRS1 in that they rescue the replication of VV⌬E3L in otherwise nonpermissive cells, and together they bind to dsRNA and block PKR activation (11,16,18,48).Deletions of individual dsRNA-binding genes have differing consequences in MCMV and HCMV systems. Deletion of either m142 or m143 from the MCMV genome eliminates viral replication and results in the activation of PKR and the inhibition of protein synthesis (48). Thus, both genes are essential and likely act together as a complex (16,18). In contrast, neither TRS1 nor IRS1 is essential for HCMV replication. Several viruses with deletions of IRS1 have been reported, and each replicates as well as wild-type virus (6, 21, 31). Deletion of TRS1 inhibits viral replication, but only by approximately 2 log units and primarily after low multiplicity of infection (MOI) (6). TRS1 appears to have a role in viral assembly late in infection that accounts for the modest replication defect of the TRS1 deletion mutant (1). Notably, deletion of TRS1 does not appear to cause a defect in viral protein synthesis (6). Therefore, unlike MCMV, neither of the two PKR evasion genes of HCMV is essential.To determine whether HCMV relies on having at least one of the...

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.

Through major histocompatibility complex class Ia leader sequence-derived (VL9) peptide binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccinations revealed contexts where HLA-E and the rhesus homologue, Mamu-E, presented diverse pathogen-derived peptides to CD8+ T cells, respectively. Here we present crystal structures of HLA-E in complex with HIV and Mtb-derived peptides. We show that despite the presence of preferred primary anchor residues, HLA-E-bound peptides can adopt alternative conformations within the peptide binding groove. Furthermore, combined structural and mutagenesis analyses illustrate a greater tolerance for hydrophobic and polar residues in the primary pockets than previously appreciated. Finally, biochemical studies reveal HLA-E peptide binding and exchange characteristics with potential relevance to its alternative antigen presenting function in vivo.

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2␣) 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 (VV⌬E3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2␣ kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VV⌬E3L 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 VV⌬E3L 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 VV⌬E3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VV⌬E3L 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, eIF2␣.Double-stranded RNA (dsRNA) activates the host cell response to viral infection in numerous ways, one of which involves the interferon-induced, dsRNA-dependent protein kinase R (PKR) (reviewed in reference 43). After binding to dsRNA, PKR dimerizes, autophosphorylates, and then phosphorylates the eukaryotic initiation factor 2 (eIF2) on its ␣ subunit. Phosphorylated eIF2␣ sequesters the guanine nucleotide exchange factor eIF2B, resulting in the inhibition of protein synthesis at the level of translation initiation. Since viruses depend on the cellular translational machinery, the shutoff of host cell protein synthesis inhibits viral replication and spread.Many viruses have mechanisms for inhibiting the PKR-mediated phosphorylation of eIF2␣ (56). The vaccinia virus (VV) E3L protein prevents the activation of PKR by binding to and sequestering dsRNA via a carboxy-terminal double-stranded RNA binding domain (dsRBD) (39). VV from which the E3L gene has been deleted (VV⌬E3L) exhibits a dsRNA-dependent restriction of host cell range, and this phenotype can be reversed by expression of the dsRBD from pE3L or dsRNAbinding proteins from other viruses (4, 47, 62). We previously found that the products of the human cytomegalovirus (HCMV) genes TRS1 and IRS1 restore the host cell range of VV⌬E3L, inhibit the phosphorylation of eIF2␣, and prevent the shutoff...

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.

Human cytomegalovirus (HCMV), which infects the majority of the population world-wide, 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 β-herpesviruses, murine (MCMV) and rhesus (RhCMV) cytomegaloviruses, for counteracting the host interferon response. Very early after infection, cellular membrane sensors such as the lymphotoxin β receptor initiate the production of antiviral cytokines including type I interferons. 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 interferon regulatory factor 3 and NF-κB. CMVs then exert a multi-pronged attack on downstream interferon signaling. HCMV infection results in decreased accumulation and phosphorylation of the interferon signaling kinases Jak1 and Stat2, and the MCMV protein pM27 mediates Stat2 down-regulation, blocking both type I and type II interferon signaling. The HCMV immediate early gene 1 protein (pUL123) interacts with Stat2 and inhibits transcriptional activation of interferon-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 interferon-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 interferon response at multiple levels during infection.

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.