RESUMEN
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.
Asunto(s)
Vacunas contra el SIDA , Infecciones por Citomegalovirus , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Linfocitos T CD8-positivos , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , CitomegalovirusRESUMEN
The signal sequences of the human cytomegalovirus (CMV) UL40 protein and its rhesus CMV (RhCMV) counterpart, Rh67, contain a peptide (VMAPRT[L/V][F/I/L/V]L, VL9) that is presented by major histocompatibility complex (MHC) antigen E (MHC-E). The CMV VL9 peptides replace VL9 peptides derived from classical MHC (Ia) signal sequences, which are lost when CMV disrupts antigen processing and presentation and MHC Ia expression. This allows infected cells to maintain MHC-E surface expression and escape killing by Natural Killer cells. We demonstrate that processing of the Rh67 VL9 peptide mirrors that of UL40, despite the lack of sequence conservation between the two proteins. Processing of both VL9 peptides is dependent on cleavage of their signal sequences by the host protease signal peptide peptidase. As previously shown for UL40, up-regulation of MHC-E expression by Rh67 requires only its signal sequence, with sequences upstream of VL9 critical for conferring independence from TAP, the transporter associated with antigen processing. Our results also suggest that the mature UL40 and Rh67 proteins contribute to CMV immune evasion by decreasing surface expression of MHC Ia. Unexpectedly, while the Rh67 VL9 peptide is resistant to the effects of Rh67, UL40 can partially counteract the up-regulation of MHC-E expression mediated by its own VL9 peptide. This suggests differences in the mechanisms by which the two VL9 peptides up-regulate MHC-E, and further work will be required to determine if any such differences have implications for translating a RhCMV-vectored simian immunodeficiency virus (SIV) vaccine to HIV-1 using human CMV as a vector. IMPORTANCE: The protective immune response induced by a rhesus cytomegalovirus (RhCMV)-vectored simian immunodeficiency virus (SIV) vaccine in rhesus macaques depends on the presence of the viral Rh67 gene in the vaccine. The Rh67 protein contains a peptide that allows the RhCMV-infected cells to maintain expression of major histocompatibility complex (MHC) antigen E at the cell surface. We show that production of this peptide, referred to as "VL9," mirrors that of the equivalent peptide present in the human cytomegalovirus (CMV) protein UL40, despite the little sequence similarity between the two CMV proteins. We also show that the mature UL40 and Rh67 proteins, which have no previously described function, also contribute to CMV immune evasion by reducing cell surface expression of MHC proteins important for the immune system to detect infected cells. Despite these similarities, our work also reveals possible differences between Rh67 and UL40, and these may have implications for the use of human CMV as the vector for a potential HIV-1 vaccine.
Asunto(s)
Citomegalovirus , Antígenos de Histocompatibilidad Clase I , Macaca mulatta , Proteínas Virales , Citomegalovirus/inmunología , Humanos , Animales , Proteínas Virales/metabolismo , Proteínas Virales/inmunología , Proteínas Virales/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Presentación de Antígeno , Señales de Clasificación de Proteína , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Evasión InmuneRESUMEN
Congenital cytomegalovirus (cCMV) is the leading infectious cause of neurologic defects in newborns with particularly severe sequelae in the setting of primary CMV infection in the first trimester of pregnancy. The majority of cCMV cases worldwide occur after non-primary infection in CMV-seropositive women; yet the extent to which pre-existing natural CMV-specific immunity protects against CMV reinfection or reactivation during pregnancy remains ill-defined. We previously reported on a novel nonhuman primate model of cCMV in rhesus macaques where 100% placental transmission and 83% fetal loss were seen in CD4+ T lymphocyte-depleted rhesus CMV (RhCMV)-seronegative dams after primary RhCMV infection. To investigate the protective effect of preconception maternal immunity, we performed reinfection studies in CD4+ T lymphocyte-depleted RhCMV-seropositive dams inoculated in late first / early second trimester gestation with RhCMV strains 180.92 (n = 2), or RhCMV UCD52 and FL-RhCMVΔRh13.1/SIVgag, a wild-type-like RhCMV clone with SIVgag inserted as an immunological marker, administered separately (n = 3). An early transient increase in circulating monocytes followed by boosting of the pre-existing RhCMV-specific CD8+ T lymphocyte and antibody response was observed in the reinfected dams but not in control CD4+ T lymphocyte-depleted dams. Emergence of SIV Gag-specific CD8+ T lymphocyte responses in macaques inoculated with the FL-RhCMVΔRh13.1/SIVgag virus confirmed reinfection. Placental transmission was detected in only one of five reinfected dams and there were no adverse fetal sequelae. Viral whole genome, short-read, deep sequencing analysis confirmed transmission of both reinfection RhCMV strains across the placenta with ~30% corresponding to FL-RhCMVΔRh13.1/SIVgag and ~70% to RhCMV UCD52, consistent with the mixed human CMV infections reported in infants with cCMV. Our data showing reduced placental transmission and absence of fetal loss after non-primary as opposed to primary infection in CD4+ T lymphocyte-depleted dams indicates that preconception maternal CMV-specific CD8+ T lymphocyte and/or humoral immunity can protect against cCMV infection.
Asunto(s)
Infecciones por Citomegalovirus , Citomegalovirus , Recién Nacido , Animales , Femenino , Embarazo , Humanos , Citomegalovirus/genética , Macaca mulatta , Reinfección , Placenta , Inmunidad InnataRESUMEN
Cytomegalovirus (CMV) is the most common congenital infection and cause of birth defects worldwide. Primary CMV infection during pregnancy leads to a higher frequency of congenital CMV (cCMV) than maternal re-infection, suggesting that maternal immunity confers partial protection. However, poorly understood immune correlates of protection against placental transmission contributes to the current lack of an approved vaccine to prevent cCMV. In this study, we characterized the kinetics of maternal plasma rhesus CMV (RhCMV) viral load (VL) and RhCMV-specific antibody binding and functional responses in a group of 12 immunocompetent dams with acute, primary RhCMV infection. We defined cCMV transmission as RhCMV detection in amniotic fluid (AF) by qPCR. We then leveraged a large group of past and current primary RhCMV infection studies in late-first/early-second trimester RhCMV-seronegative rhesus macaque dams, including immunocompetent (n = 15), CD4+ T cell-depleted with (n = 6) and without (n = 6) RhCMV-specific polyclonal IgG infusion before infection to evaluate differences between RhCMV AF-positive and AF-negative dams. During the first 3 weeks after infection, the magnitude of RhCMV VL in maternal plasma was higher in AF-positive dams in the combined cohort, while RhCMV glycoprotein B (gB)- and pentamer-specific binding IgG responses were lower magnitude compared to AF-negative dams. However, these observed differences were driven by the CD4+ T cell-depleted dams, as there were no differences in plasma VL or antibody responses between immunocompetent AF-positive vs AF-negative dams. Overall, these results suggest that levels of neither maternal plasma viremia nor humoral responses are associated with cCMV following primary maternal infection in healthy individuals. We speculate that other factors related to innate immunity are more important in this context as antibody responses to acute infection likely develop too late to influence vertical transmission. Yet, pre-existing CMV glycoprotein-specific and neutralizing IgG may provide protection against cCMV following primary maternal CMV infection even in high-risk, immunocompromised settings.
Asunto(s)
Infecciones por Citomegalovirus , Citomegalovirus , Animales , Femenino , Humanos , Embarazo , Citomegalovirus/fisiología , Macaca mulatta , Formación de Anticuerpos , Carga Viral , Placenta , Anticuerpos Antivirales , Glicoproteínas/metabolismo , Transmisión Vertical de Enfermedad Infecciosa , Inmunoglobulina G/metabolismoRESUMEN
The genome of cowpoxvirus (CPXV) could be considered prototypical for orthopoxviridae (OXPV) since it contains many open reading frames (ORFs) absent or lost in other OPXV, including vaccinia virus (VACV). These additional ORFs are non-essential for growth in vitro but are expected to contribute to the broad host range, virulence and immune evasion characteristics of CPXV. For instance, unlike VACV, CPXV encodes proteins that interfere with T cell stimulation, either directly or by preventing antigen presentation or co-stimulation. When studying the priming of naïve T cells, we discovered that CPXV, but not VACV, encodes a secreted factor that interferes with activation and proliferation of naïve CD8+ and CD4+ T cells, respectively, in response to anti-CD3 antibodies, but not to other stimuli. Deletion mapping revealed that the inhibitory protein is encoded by CPXV14, a small secreted glycoprotein belonging to the poxvirus immune evasion (PIE) family and containing a smallpoxvirus encoded chemokine receptor (SECRET) domain that mediates binding to chemokines. We demonstrate that CPXV14 inhibition of antibody-mediated T cell activation depends on the presence of Fc-gamma receptors (FcγRs) on bystander cells. In vitro, CPXV14 inhibits FcγR-activation by antigen/antibody complexes by binding to FcγRs with high affinity and immobilized CPXV14 can trigger signaling through FcγRs, particularly the inhibitory FcγRIIB. In vivo, CPXV14-deleted virus showed reduced viremia and virulence resulting in reduced weight loss and death compared to wildtype virus whereas both antibody and CD8+ T cell responses were increased in the absence of CPXV14. Furthermore, no impact of CPXV14-deletion on virulence was observed in mice lacking the inhibitory FcγRIIB. Taken together our results suggest that CPXV14 contributes to virulence and immune evasion by binding to host FcγRs.
Asunto(s)
Virus de la Viruela Vacuna , Evasión Inmune , Animales , Virus de la Viruela Vacuna/genética , Glicoproteínas , Ratones , Receptores de Quimiocina , Receptores de IgG , Virus Vaccinia , VirulenciaRESUMEN
CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and ß-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.
Asunto(s)
Linfocitos T CD8-positivos , Infecciones por VIH , Animales , Epítopos , Epítopos de Linfocito T , Macaca mulatta , Receptores de Antígenos de Linfocitos T , Factor de Necrosis Tumoral alfaRESUMEN
This corrects the article DOI: 10.1038/nature12519.
RESUMEN
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.
Asunto(s)
Infecciones por Citomegalovirus/virología , Citomegalovirus/genética , Genoma Viral/genética , Viremia , Animales , Línea Celular , Cromosomas Artificiales Bacterianos , Citomegalovirus/patogenicidad , ADN Recombinante , Modelos Animales de Enfermedad , Femenino , Fibroblastos/virología , Humanos , Macaca mulatta , Masculino , Mutación , Sistemas de Lectura Abierta/genética , Filogenia , Especificidad de la EspecieRESUMEN
Currently 247 million people are living with chronic hepatitis B virus infection (CHB), and the development of novel curative treatments is urgently needed. Immunotherapy is an attractive approach to treat CHB, yet therapeutic approaches to augment the endogenous hepatitis B virus (HBV)-specific T cell response in CHB patients have demonstrated little success. In this study, we show that strain 68-1 rhesus macaque (RM) CMV vaccine vectors expressing HBV Ags engender HBV-specific CD8+ T cells unconventionally restricted by MHC class II and the nonclassical MHC-E molecule in RM. Surface staining of human donor and RM primary hepatocytes (PH) ex vivo revealed the majority of PH expressed MHC-E but not MHC class II. HBV-specific, MHC-E-restricted CD8+ T cells from RM vaccinated with RM CMV vaccine vectors expressing HBV Ags recognized HBV-infected PH from both human donor and RM. These results provide proof-of-concept that MHC-E-restricted CD8+ T cells could be harnessed for the treatment of CHB, either through therapeutic vaccination or adoptive immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatocitos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Animales , Hepatitis B Crónica/virología , Hepatocitos/virología , Macaca mulattaRESUMEN
Most studies of T lymphocytes focus on recognition of classical major histocompatibility complex (MHC) class I or II molecules presenting oligopeptides, yet there are numerous variations and exceptions of biological significance based on recognition of a wide variety of nonclassical MHC molecules. These include αß and γδ T cells that recognize different class Ib molecules (CD1, MR-1, HLA-E, G, F, et al.) that are nearly monomorphic within a given species. Collectively, these T cells can be considered "unconventional," in part because they recognize lipids, metabolites, and modified peptides. Unlike classical MHC-specific cells, unconventional T cells generally exhibit limited T-cell antigen receptor (TCR) repertoires and often produce innate immune cell-like rapid effector responses. Exploiting this system in new generation vaccines for human immunodeficiency virus (HIV), tuberculosis (TB), other infectious agents, and cancer was the focus of a recent workshop, "Immune Surveillance by Non-classical MHC Molecules: Improving Diversity for Antigens," sponsored by the National Institute of Allergy and Infectious Diseases. Here, we summarize salient points presented regarding the basic immunobiology of unconventional T cells, recent advances in methodologies to measure unconventional T-cell activity in diseases, and approaches to harness their considerable clinical potential.
Asunto(s)
Vigilancia Inmunológica/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Animales , Antígenos , Antígenos HLA , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Receptores de Antígenos de Linfocitos T , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunologíaRESUMEN
Receptors recognizing the Fc part of immunoglobulin G (FcγRs) are key determinants in antibody-mediated immune responses. Members of the Herpesviridae interfere with this immune regulatory network by expressing viral FcγRs (vFcγRs). Human cytomegalovirus (HCMV) encodes four distinct vFcγRs that differ with respect to their IgG subtype specificity and their impact on antibody-mediated immune function in vitro The impact of vFcγRs on HCMV pathogenesis and immunomodulation in vivo is not known. The closest evolutionary animal model of HCMV is rhesus CMV (RhCMV) infection of rhesus macaques. To enable the characterization of vFcγR function in this model, we studied IgG binding by RhCMV. We show that lysates of RhCMV-infected cells contain an IgG-binding protein of 30 kDa encoded by the gene Rh05 that is a predicted type I glycoprotein belonging to the RL11 gene family. Upon deletion of Rh05, IgG-Fc binding by RhCMV strain 68-1 is lost, whereas ectopic expression of Rh05 results in IgG binding to transfected cells consistent with Rh05 being a vFcγR. Using a set of reporter cell lines stably expressing human and rhesus FcγRs, we further demonstrate that Rh05 antagonizes host FcγR activation. Compared to Rh05-intact RhCMV, RhCMVΔRh05 showed an increased activation of host FcγR upon exposure of infected cells to IgG from RhCMV-seropositive animals, suggesting that Rh05 protects infected cells from opsonization and IgG-dependent activation of host FcγRs. However, antagonizing host FcγR activation by Rh05 was not required for the establishment and maintenance of infection of RhCMV, even in a seropositive host, as shown by the induction of T cell responses to heterologous antigens expressed by RhCMV lacking the gene region encoding Rh05. In contrast to viral evasion of natural killer cells or T cell recognition, the evasion of antibody-mediated effects does not seem to be absolutely required for infection or reinfection. The identification of the first vFcγR that efficiently antagonizes host FcγR activation in the RhCMV genome will thus permit more detailed studies of this immunomodulatory mechanism in promoting viral dissemination in the presence of natural or vaccine-induced humoral immunity.IMPORTANCE Rhesus cytomegalovirus (RhCMV) offers a unique model for studying human cytomegalovirus (HCMV) pathogenesis and vaccine development. RhCMV infection of nonhuman primates greatly broadened the understanding of mechanisms by which CMVs evade or reprogram T cell and natural killer cell responses in vivo However, the role of humoral immunity and viral modulation of anti-CMV antibodies has not been studied in this model. There is evidence from in vitro studies that HCMVs can evade humoral immunity. By gene mapping and with the help of a novel cell-based reporter assay system we characterized the first RhCMV encoded IgG-Fcγ binding glycoprotein as a potent antagonist of rhesus FcγR activation. We further demonstrate that, unlike evasion of T cell immunity, this viral Fcγ receptor is not required to overcome anti-CMV immunity to establish secondary infections. These findings enable more detailed studies of the in vivo consequences of CMV evasion from IgG responses in nonhuman primate models.
Asunto(s)
Citomegalovirus/inmunología , Glicoproteínas/inmunología , Receptores de IgG/metabolismo , Animales , Anticuerpos Antivirales/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/virología , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunoglobulina G/metabolismo , Macaca mulatta/virología , Ratones , Unión Proteica/fisiología , Receptores de IgG/inmunología , Transducción de Señal , Proteínas Virales/metabolismoRESUMEN
Major histocompatibility complex E (MHC-E) is a highly conserved nonclassical MHC-Ib molecule that tightly binds peptides derived from leader sequences of classical MHC-Ia molecules for presentation to natural killer cells. However, MHC-E also binds diverse foreign and neoplastic self-peptide antigens for presentation to CD8+ T cells. Although the determinants of MHC-E-restricted T cell priming remain unknown, these cells are induced in humans infected with pathogens containing genes that inhibit the transporter associated with antigen processing (TAP). Indeed, mice vaccinated with TAP-inhibited autologous dendritic cells develop T cells restricted by the murine MHC-E homologue, Qa-1b. Here, we tested whether rhesus macaques (RM) vaccinated with viral constructs expressing a TAP inhibitor would develop insert-specific MHC-E-restricted CD8+ T cells. We generated viral constructs coexpressing SIVmac239 Gag in addition to one of three TAP inhibitors: herpes simplex virus 2 ICP47, bovine herpes virus 1 UL49.5, or rhesus cytomegalovirus Rh185. Each TAP inhibitor reduced surface expression of MHC-Ia molecules but did not reduce surface MHC-E expression. In agreement with modulation of surface MHC-Ia levels, TAP inhibition diminished presentation of MHC-Ia-restricted CD8+ T cell epitopes without impacting presentation of peptide antigen bound by MHC-E. Vaccination of macaques with vectors dually expressing SIVmac239 Gag with ICP47, UL49.5, or Rh185 generated Gag-specific CD8+ T cells classically restricted by MHC-Ia but not MHC-E. These data demonstrate that, in contrast to results in mice, TAP inhibition alone is insufficient for priming of MHC-E-restricted T cell responses in primates and suggest that additional unknown mechanisms govern the induction of CD8+ T cells recognizing MHC-E-bound antigen.IMPORTANCE Due to the near monomorphic nature of MHC-E in the human population and inability of many pathogens to inhibit MHC-E-mediated peptide presentation, MHC-E-restricted T cells have become an attractive vaccine target. However, little is known concerning how these cells are induced. Understanding the underlying mechanisms that induce these T cells would provide a powerful new vaccine strategy to an array of neoplasms and viral and bacterial pathogens. Recent studies have indicated a link between TAP inhibition and induction of MHC-E-restricted T cells. The significance of our research is in demonstrating that TAP inhibition alone does not prime MHC-E-restricted T cell generation and suggests that other, currently unknown mechanisms regulate their induction.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Vacunas contra el SIDAS/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Inhibidores Enzimáticos/metabolismo , Macaca mulatta , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacunas contra el SIDAS/administración & dosificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
While cytomegalovirus (CMV) infections are often limited in host range by lengthy coevolution with a single host species, a few CMVs are known to deviate from this rule. For example, rhesus macaque CMV (RhCMV), a model for human CMV (HCMV) pathogenesis and vaccine development, can replicate in human cells, as well as in rhesus cells. Both HCMV and RhCMV encode species-specific antagonists of the broadly acting host cell restriction factor protein kinase R (PKR). Although the RhCMV antagonist of PKR, rTRS1, has very limited activity against human PKR, here, we show it is essential for RhCMV replication in human cells because it prevents human PKR from phosphorylating the translation initiation factor eIF2α, thereby allowing continued translation and viral replication. Although rTRS1 is necessary for RhCMV replication, it is not sufficient to rescue replication of HCMV lacking its own PKR antagonists in human fibroblasts. However, overexpression of rTRS1 in human fibroblasts enabled HCMV expressing rTRS1 to replicate, indicating that elevated levels or early expression of a weak antagonist can counteract a resistant restriction factor like human PKR. Exploring potential mechanisms that might allow RhCMV to replicate in human cells revealed that RhCMV makes no less double-stranded RNA than HCMV. Rather, in human cells, RhCMV expresses rTRS1 at levels 2 to 3 times higher than those of the HCMV-encoded PKR antagonists during HCMV infection. These data suggest that even a modest increase in expression of this weak PKR antagonist is sufficient to enable RhCMV replication in human cells.IMPORTANCE Rhesus macaque cytomegalovirus (RhCMV) offers a valuable model for studying congenital human cytomegalovirus (HCMV) pathogenesis and vaccine development. Therefore, it is critical to understand variations in how each virus infects and affects its host species to be able to apply insights gained from the RhCMV model to HCMV. While HCMV is capable only of infecting cells from humans and very closely related species, RhCMV displays a wider host range, including human as well as rhesus cells. RhCMV expresses an antagonist of a broadly acting antiviral factor present in all mammalian cells, and its ability to counter both the rhesus and human versions of this host factor is a key component of RhCMV's ability to cross species barriers. Here, we examine the molecular mechanisms that allow this RhCMV antagonist to function against a human restriction factor.
Asunto(s)
Infecciones por Citomegalovirus/enzimología , Citomegalovirus/metabolismo , Fibroblastos/enzimología , Transducción de Señal , eIF-2 Quinasa/metabolismo , Línea Celular , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Fibroblastos/patología , Fibroblastos/virología , Humanos , Especificidad de la Especie , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/genéticaRESUMEN
Established infections with the human and simian immunodeficiency viruses (HIV and SIV, respectively) are thought to be permanent with even the most effective immune responses and antiretroviral therapies only able to control, but not clear, these infections. Whether the residual virus that maintains these infections is vulnerable to clearance is a question of central importance to the future management of millions of HIV-infected individuals. We recently reported that approximately 50% of rhesus macaques (RM; Macaca mulatta) vaccinated with SIV protein-expressing rhesus cytomegalovirus (RhCMV/SIV) vectors manifest durable, aviraemic control of infection with the highly pathogenic strain SIVmac239 (ref. 5). Here we show that regardless of the route of challenge, RhCMV/SIV vector-elicited immune responses control SIVmac239 after demonstrable lymphatic and haematogenous viral dissemination, and that replication-competent SIV persists in several sites for weeks to months. Over time, however, protected RM lost signs of SIV infection, showing a consistent lack of measurable plasma- or tissue-associated virus using ultrasensitive assays, and a loss of T-cell reactivity to SIV determinants not in the vaccine. Extensive ultrasensitive quantitative PCR and quantitative PCR with reverse transcription analyses of tissues from RhCMV/SIV vector-protected RM necropsied 69-172 weeks after challenge did not detect SIV RNA or DNA sequences above background levels, and replication-competent SIV was not detected in these RM by extensive co-culture analysis of tissues or by adoptive transfer of 60 million haematolymphoid cells to naive RM. These data provide compelling evidence for progressive clearance of a pathogenic lentiviral infection, and suggest that some lentiviral reservoirs may be susceptible to the continuous effector memory T-cell-mediated immune surveillance elicited and maintained by cytomegalovirus vectors.
Asunto(s)
Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Citomegalovirus/genética , Citomegalovirus/inmunología , Femenino , Macaca mulatta , Masculino , Datos de Secuencia Molecular , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Factores de Tiempo , Vacunas Atenuadas/inmunología , Carga Viral , Replicación Viral/fisiologíaRESUMEN
Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Herpesviridae/inmunología , Epítopos Inmunodominantes/inmunología , Vacunas Virales/inmunología , Animales , Antígenos Virales/metabolismo , Cromatografía Liquida , Epítopos de Linfocito T/inmunología , Citometría de Flujo , Memoria Inmunológica/inmunología , Activación de Linfocitos/inmunología , Espectrometría de Masas , Ratones , Muromegalovirus/inmunología , Mutagénesis Sitio-Dirigida , Péptidos , Vacunas Sintéticas/inmunología , Proteínas Virales/inmunología , Proteínas Virales/metabolismoRESUMEN
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.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Evasión Inmune , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Animales , Humanos , Células K562 , Macaca fascicularis , Glicoproteínas de Membrana/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Proteínas Virales/inmunologíaRESUMEN
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.
Asunto(s)
Infecciones por Citomegalovirus/transmisión , Citomegalovirus/genética , Modelos Animales de Enfermedad , Macaca fascicularis/virología , Macaca mulatta/virología , Animales , Infecciones por Citomegalovirus/genética , ADN Viral/análisis , ADN Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , Especificidad de la EspecieRESUMEN
Varicella zoster virus (VZV) causes chickenpox in humans and, subsequently, establishes latency in the sensory ganglia from where it reactivates to cause herpes zoster. Infection of rhesus macaques with simian varicella virus (SVV) recapitulates VZV pathogenesis in humans thus representing a suitable animal model for VZV infection. While the type I interferon (IFN) response has been shown to affect VZV replication, the virus employs counter mechanisms to prevent the induction of anti-viral IFN stimulated genes (ISG). Here, we demonstrate that SVV inhibits type I IFN-activated signal transduction via the JAK-STAT pathway. SVV-infected rhesus fibroblasts were refractory to IFN stimulation displaying reduced protein levels of IRF9 and lacking STAT2 phosphorylation. Since previous work implicated involvement of the VZV immediate early gene product ORF63 in preventing ISG-induction we studied the role of SVV ORF63 in generating resistance to IFN treatment. Interestingly, SVV ORF63 did not affect STAT2 phosphorylation but caused IRF9 degradation in a proteasome-dependent manner, suggesting that SVV employs multiple mechanisms to counteract the effect of IFN. Control of SVV ORF63 protein levels via fusion to a dihydrofolate reductase (DHFR)-degradation domain additionally confirmed its requirement for viral replication. Our results also show a prominent reduction of IRF9 and inhibition of STAT2 phosphorylation in VZV-infected cells. In addition, cells expressing VZV ORF63 blocked IFN-stimulation and displayed reduced levels of the IRF9 protein. Taken together, our data suggest that varicella ORF63 prevents ISG-induction both directly via IRF9 degradation and indirectly via transcriptional control of viral proteins that interfere with STAT2 phosphorylation. SVV and VZV thus encode multiple viral gene products that tightly control IFN-induced anti-viral responses.
Asunto(s)
Infecciones por Herpesviridae/metabolismo , Interacciones Huésped-Patógeno , Interferón Tipo I/metabolismo , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Varicellovirus/fisiología , Animales , Línea Celular , Cercopithecinae , Varicela/inmunología , Varicela/metabolismo , Varicela/patología , Varicela/virología , ADN Recombinante/metabolismo , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 3/inmunología , Herpesvirus Humano 3/fisiología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Inmunidad Innata , Interferón Tipo I/antagonistas & inhibidores , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/antagonistas & inhibidores , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal , Procesamiento Proteico-Postraduccional , Proteolisis , Proteínas Recombinantes/metabolismo , Factores de Transcripción STAT/genética , Varicellovirus/inmunologíaRESUMEN
UNLABELLED: Varicella-zoster virus (VZV) causes chickenpox upon primary infection and establishes latency in ganglia. Reactivation from latency causes herpes zoster, which may be complicated by postherpetic neuralgia. Innate immunity mediated by interferon and proinflammatory cytokines represents the first line of immune defense upon infection and reactivation. VZV is known to interfere with multiple innate immune signaling pathways, including the central transcription factor NF-κB. However, the role of these inhibitory mechanisms in vivo is unknown. Simian varicella virus (SVV) infection of rhesus macaques recapitulates key aspects of VZV pathogenesis, and this model thus permits examination of the role of immune evasion mechanisms in vivo. Here, we compare SVV and VZV with respect to interference with NF-κB activation. We demonstrate that both viruses prevent ubiquitination of the NF-κB inhibitor IκBα, whereas SVV additionally prevents IκBα phosphorylation. We show that the ORF61 proteins of VZV and SVV are sufficient to prevent IκBα ubiquitination upon ectopic expression. We further demonstrate that SVV ORF61 interacts with ß-TrCP, a subunit of the SCF ubiquitin ligase complex that mediates the degradation of IκBα. This interaction seems to inactivate SCF-mediated protein degradation in general, since the unrelated ß-TrCP target Snail is also stabilized by ORF61. In addition to ORF61, SVV seems to encode additional inhibitors of the NF-κB pathway, since SVV with ORF61 deleted still prevented IκBα phosphorylation and degradation. Taken together, our data demonstrate that SVV interferes with tumor necrosis factor alpha (TNF-α)-induced NF-κB activation at multiple levels, which is consistent with the importance of these countermechanisms for varicella virus infection. IMPORTANCE: The role of innate immunity during the establishment of primary infection, latency, and reactivation by varicella-zoster virus (VZV) is incompletely understood. Since infection of rhesus macaques by simian varicella virus (SVV) is used as an animal model of VZV infection, we characterized the molecular mechanism by which SVV interferes with innate immune activation. Specifically, we studied how SVV prevents activation of the transcription factor NF-κB, a central factor in eliciting proinflammatory responses. The identification of molecular mechanisms that counteract innate immunity might ultimately lead to better vaccines and treatments for VZV, since overcoming these mechanisms, either by small-molecule inhibition or by genetic modification of vaccine strains, is expected to reduce the pathogenic potential of VZV. Moreover, using SVV infection of rhesus macaques, it will be possible to study how increasing the vulnerability of varicella viruses to innate immunity will impact viral pathogenesis.