Prion protein alters viral control and enhances pathology after perinatal cytomegalovirus infection.

Cytomegalovirus (CMV) infection poses risks to newborns, necessitating effective therapies. Given that the damage includes both viral infection of brain cells and immune system-related damage, here we investigate the involvement of cellular prion protein (PrP), which plays vital roles in neuroprotection and immune regulation. Using a murine model, we show the role of PrP in tempering neonatal T cell immunity during CMV infection. PrP-null mice exhibit enhanced viral control through elevated virus-specific CD8 T cell responses, leading to reduced viral titers and pathology. We further unravel the molecular mechanisms by showing CMV-induced upregulation followed by release of PrP via the metalloproteinase ADAM10, impairing CD8 T cell response specifically in neonates. Additionally, we confirm PrP downregulation in human CMV (HCMV)-infected fibroblasts, underscoring the broader relevance of our observations beyond the murine model. Furthermore, our study highlights how PrP, under the stress of viral pathogenesis, reveals its impact on neonatal immune modulation.

Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation.

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.

Comparison of Cytomegalovirus Reactivation in Children After Allogeneic Hematopoietic Cell Transplantation in 2 Transplant Eras.

BACKGROUND: Reactivation of cytomegalovirus (CMV) is typically considered harmless as long as the immune system remains unaffected by medications or other factors. CMV reactivation may occur as a result of acute graft-versus-host disease of Grades II to IV. One possible factor contributing to this risk is the rise in the number of donors who lack genetic similarities or relationships. We hypothesized that the anti-CMV IgG level before transplantation could potentially serve as an indicator of the likelihood of CMV reactivation following hematopoietic cell transplantation. METHODS: We examined a cohort of young individuals who underwent allogeneic HCT between 1998 and 2022 to evaluate the occurrence of CMV reactivation. The patients were divided into 2 time periods: 1998 to 2016 (comparison group) and 2017 to 2022 (intervention group). RESULTS: Between 1998 and 2016, 292 patients underwent hematopoietic HCT. Recipients from 2017 to 2022 experienced a slightly higher risk of CMV reactivation than those from 1998 to 2016. The comparison of prophylactic and preemptive medication showed no significant difference between the periods (P = .32). Patients treated from 1998 to 2016 experienced a 23% decrease in the risk of symptomatic CMV reactivation and related illnesses compared to those treated from 2017 to 2022 (P = .08 and .15, respectively). CONCLUSIONS: Our study showed that the intervention group had more symptomatic CMV reactivations. Various factors may contribute to this, including CD19-directed immunotherapy and the CMV status of the recipient before transplantation.

Neonatal and short-term outcome after late vertical transmission in congenital CMV-infected fetuses following primary first-trimester maternal seroconversion.

OBJECTIVE: To document the course of neonatal and short-term outcomes in pregnancies after first trimester CMV (cytomegalovirus) seroconversion and negative amniotic fluid (AF) CMV PCR. METHODS: We included 375 patients with a first-trimester CMV seroconversion and amniocentesis at ≥21 weeks. Termination of pregnancy (TOP) was offered in case antenatally severe CMV-related fetopathy was documented either by ultrasound or by MRI. AF CMV PCR-negative fetuses underwent a PCR CMV on neonatal urine (NU). Perinatal and short-term infant outcomes were investigated by a questionnaire, sent to parents. RESULTS: AF CMV PCR was positive in 118/375 cases (31.4%). TOP was performed in 46/118 (38.9%) and fetal demise occurred twice. Questionnaires were sent to 327 patients with an overall response rate of 77%. Three groups were considered: Group 1: the early infected group (AF CMV PCR positive; N=62), group 2: the late infected group (AF CMV PCR negative, NU CMV PCR positive; N=7) and group 3: the control group (AF+NU CMV PCR negative; N=160). Compared with group 3, group 1 was more frequently symptomatic at birth (6.2% vs 19.4%; p=0.006). In short-term follow-up, hearing impairment (23.5%; p<0.001), mild motor deficit - defined as abnormal early motor development or the need for physiotherapy in later life (21.6%; p=0.005) - and subnormal vision (15.7%; p=0.02) were significantly more frequent. Compared with group 3, group 2 showed more often jaundice (57.1%; p=0.04) and petechiae (28.6%; p=0.04) at birth, but other short-term symptoms were lacking. CONCLUSION: Although neonates may screen positive on urine for CMV after an AF CMV negative PCR, they show rarely and only mild sequelae in early life.

Dynamic monitoring of viral gene expression reveals rapid antiviral effects of CD8 T cells recognizing the HCMV-pp65 antigen.

Introduction: Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. Methods: We developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). Results: The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. Discussion: ARMATA does not only allow same day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection.

The adaptive and innate immune systems coordinate for successful control of CMV infection.

Previous studies of the immune control of cytomegalovirus infection have primarily focused on analysis of the traditional adaptive T-cell response. Donadeu et al. bring a new perspective through evaluation of multiple adaptive and innate immune subtypes in parallel with cytomegalovirus-specific cell-mediated immunity in a prospective cohort of kidney transplant recipients with findings validated in 2 independent studies. Identification of a natural killer T-cell subtype associated with cell-mediated immunity and freedom from cytomegalovirus infection demonstrates the importance of the coordinated innate and adaptive immune response for effective viral control.

Viral escape mutations do not account for non-protection from SIVmac239 challenge in RhCMV/SIV vaccinated rhesus macaques.

Simian immunodeficiency virus (SIV) vaccines based upon 68-1 Rhesus Cytomegalovirus (RhCMV) vectors show remarkable protection against pathogenic SIVmac239 challenge. Across multiple independent rhesus macaque (RM) challenge studies, nearly 60% of vaccinated RM show early, complete arrest of SIVmac239 replication after effective challenge, whereas the remainder show progressive infection similar to controls. Here, we performed viral sequencing to determine whether the failure to control viral replication in non-protected RMs is associated with the acquisition of viral escape mutations. While low level viral mutations accumulated in all animals by 28 days-post-challenge, which is after the establishment of viral control in protected animals, the dominant circulating virus in virtually all unprotected RMs was nearly identical to the challenge stock, and there was no difference in mutation patterns between this cohort and unvaccinated controls. These data definitively demonstrate that viral mutation does not explain lack of viral control in RMs not protected by RhCMV/SIV vaccination. We further demonstrate that during chronic infection RhCMV/SIV vaccinated RMs do not acquire escape mutation in epitopes targeted by RhCMV/SIV, but instead display mutation in canonical MHC-Ia epitopes similar to unvaccinated RMs. This suggests that after the initial failure of viral control, unconventional T cell responses induced by 68-1 RhCMV/SIV vaccination do not exert strong selective pressure on systemically replicating SIV.

Worse Wilms' Tumor Outcomes Associated With Chemical Complementarity for Multiple T-Cell Receptor CDR3-CMV Epitope Pairs.

BACKGROUND/AIM: Wilms' tumors are pediatric renal tumors that generally have a good prognosis and outcomes. Viral illnesses have been linked to development of neoplasms and should be considered as a factor that could modulate overall survival. MATERIALS AND METHODS: We considered recently developed adaptive immune receptor, genomics and bioinformatics approaches to assess the potential impact of cytomegalovirus (CMV) infections in Wilms' tumor. RESULTS: T-cell receptor (TCR) complementarity determining region-3 (CDR3) amino acid sequences from Wilms' tumor specimens represented by the Therapeutically Applicable Research to Generate Effective Treatments dataset were compared with known anti-CMV TCR CDR3s, indicating that cases representing the anti-CMV TCR CDR3s had worse outcomes. Then, a chemical complementarity scoring approach for the Wilms' tumor, TCR CDR3s and a series of CMV antigens further indicated that cases representing a higher chemical complementarity to the CMV antigens had worse outcomes. CONCLUSION: Overall, we present a potentially novel method to assess CMV infections and identify patients who could benefit from therapies that address such infections.

Cutting Edge: Retinoic Acid Promotes Brain-homing of CD8+ T Cells during Congenital Cytomegalovirus Infection.

The most common congenital viral infection is CMV, which leads to numerous neurologic disabilities. Using a mouse model of congenital CMV, we previously determined that Ag-specific CD8+ T cells traffic to the brain in a CCR9-dependent manner. The mechanism by which these CD8+ T cells acquire a CCR9-dependent "brain-tropic" phenotype remains unclear. In this study, we identify the key factor that imprints brain homing specificity on CD8+ T cells, the source of production, and the location where CCR9 expression is induced. Specifically, we discovered that CCR9 is induced on CD8+ T cells by retinoic acid-producing CD8α+ dendritic cells in the cervical lymph node postinfection. We found that retinoic acid is important for CD8+ T cells to establish tissue residency in the brain. Collectively, our data expand the role of retinoic acid during infection and mechanistically demonstrate how CD8+ T cells are primed to protect the brain during congenital viral infection.

Late-life Attenuation of Cytomegalovirus-mediated CD8 T Cell Memory Inflation: Shrinking of the Cytomegalovirus Latency Niche.

CMV drives the accumulation of virus-specific, highly differentiated CD8 memory T cells (memory inflation [MI]). In mice, MI was shown to directly correlate with the CMV infection dose, yet the CMV-associated CD8 MI plateaus over time. It is unclear how MI is regulated with aging. We infected young mice with 102, 104, and 106 PFU of murine CMV and confirmed that MI magnitude was directly proportional to the infectious dose, reaching a setpoint by midlife. By old age, MI subsided, most prominently in mice infected with 106 PFU, and reached statistical parity between groups in 26-mo-old mice. This corresponded to an age-related loss in lymphatic endothelial cells in lymph nodes, recently shown to be sufficient to drive MI in mice. We propose that MI size and persistence over the lifespan is controlled by the size of the lymphatic endothelial cell niche, whose shrinking leads to reduced MI with aging.

Immune surveillance of cytomegalovirus in tissues.

Cytomegalovirus (CMV), a representative member of the Betaherpesvirinae subfamily of herpesviruses, is common in the human population, but immunocompetent individuals are generally asymptomatic when infected with this virus. However, in immunocompromised individuals and immunologically immature fetuses and newborns, CMV can cause a wide range of often long-lasting morbidities and even death. CMV is not only widespread throughout the population but it is also widespread in its hosts, infecting and establishing latency in nearly all tissues and organs. Thus, understanding the pathogenesis of and immune responses to this virus is a prerequisite for developing effective prevention and treatment strategies. Multiple arms of the immune system are engaged to contain the infection, and general concepts of immune control of CMV are now reasonably well understood. Nonetheless, in recent years, tissue-specific immune responses have emerged as an essential factor for resolving CMV infection. As tissues differ in biology and function, so do immune responses to CMV and pathological processes during infection. This review discusses state-of-the-art knowledge of the immune response to CMV infection in tissues, with particular emphasis on several well-studied and most commonly affected organs.

Cytomegalovirus-Specific Hyperimmune Immunoglobulin Administration for Secondary Prevention after First-Trimester Maternal Primary Infection: A 13-Year Single-Center Cohort Study.

Primary cytomegalovirus infection during pregnancy has a high risk of vertical transmission, with severe fetal sequelae mainly associated with first-trimester infections. We conducted a retrospective analysis of 200 IU/kg cytomegalovirus-specific hyperimmune globulin (HIG), used in first-trimester maternal primary infections for congenital infection prevention. The primary outcome was vertical transmission, defined as neonatal viruria or positive amniocentesis if pregnancy was discontinued. HIG, initially administered monthly and since 2019 biweekly, was discontinued in negative amniocentesis cases. Women declining amniocentesis and positive amniocentesis cases with normal sonography were offered monthly HIG until delivery as a treatment strategy. The total transmission rate was 29.9% (32/107; 10 pregnancy terminations with positive amniocentesis, 18 completed pregnancies with positive amniocentesis and 4 declining amniocentesis). Maternal viremia was the only factor associated with fetal transmission (OR 4.62, 95% CI 1.55-13.74). The transmission rate was not significantly different whether HIG was started during the first or second trimester (28.2% vs. 33.3%; p = 0.58), or between monthly and biweekly subgroups (25.7% vs. 37.8%, p = 0.193). Pre-treatment maternal viremia could inform decisions as a predictor of congenital infection.

QuantiFERON CMV Test and CMV Serostatus in Lung Transplant: Stratification Risk for Infection, Chronic and Acute Allograft Rejection.

The QuantiFERON CMV (QCMV) test evaluates specific adaptive immune system activity against CMV by measuring IFN-γ released by activated CD8+ T lymphocytes. We aimed to evaluate the QCMV test as a predictive tool for CMV manifestations and acute or chronic lung allograft rejection (AR and CLAD) in lung transplant (LTx) patients. A total of 73 patients were divided into four groups based on donor and recipient (D/R) serology for CMV and QCMV assay: group A low-risk for CMV infection and disease (D-/R-); group B and C at intermediate-risk (R+), group B with non-reactive QCMV and group C with reactive QCMV; group D at high-risk (D+/R-). Group D patients experienced higher viral replication; no differences were observed among R+ patients of groups B and C. D+/R- patients had a higher number of AR events and group C presented a lower incidence of AR. Prevalence of CLAD at 24 months was higher in group B with a higher risk of CLAD development (OR 6.33). The QCMV test allows us to identify R+ non-reactive QCMV population as the most exposed to onset of CLAD. This population had a higher, although non-significant, susceptibility to AR compared to the R+ population with reactive QCMV.

Immune Control of Human Cytomegalovirus (HCMV) Infection in HCMV-Seropositive Solid Organ Transplant Recipients: The Predictive Role of Different Immunological Assays.

Human cytomegalovirus (HCMV) infection remains a major complication for solid organ transplant recipients (SOTRs). The aim of this study was to evaluate the role of HCMV-specific T cell immunity measured at the time of the HCMV-DNA peak in predicting the spontaneous clearance of infection. The performance of cytokine flow cytometry using infected dendritic cells (CFC-iDC), infected cell lysate (CFC-iCL) and pp65 peptide pool (CFC-pp65 pool) as stimuli, as well as ELISPOT assays using infected cell lysate (ELISPOT-iCL) and the pp65 peptide pool (ELISPOT-pp65 pool), was analysed. Among the 40 SOTRs enrolled, 16 patients (40%) required antiviral treatment for an HCMV infection (Non-Controllers), while the others spontaneously cleared the infection (Controllers). At the HCMV-DNA peak, the number of HCMV-specific CD4+ T cells detected by the CFC-iDC, CFC-iCL and CFC-pp65 pool assays in Controllers was higher than that detected in Non-Controllers, while no difference was observed in terms of HCMV-specific CD8+ T cell response. The same trend was observed when the HCMV-specific T cell response was measured by ELISPOT-iCL and ELISPOT-pp65 pool. We observed that the CD4+ CFC-pp65 pool assay was the best predictor of self-resolving HCMV infection at the time of the HCVM-DNA peak. The CFC-pp65 pool assay is able to discriminate between CD4+ and CD8+ T cell responses and could be used in daily clinical practice.

Antiviral Agents for Preventing Cytomegalovirus Disease in Recipients of Hematopoietic Cell Transplantation.

This systematic review discusses the use of prophylaxis to prevent cytomegalovirus (CMV) infection in recipients who have undergone hematopoietic cell transplantation. It highlights the need for new approaches to control and prevent CMV infection. The approval of the anti-CMV drug letermovir has made antiviral prophylaxis more popular. CMV-specific T cell-mediated immunity tests are effective in identifying patients who have undergone immune reconstitution and predicting disease progression. Maribavir (MBV) has been approved for the treatment of post-transplant CMV infection/disease in adolescents. Adoptive T-cell therapy and the PepVax CMV vaccine show promise in tackling refractory and resistant CMV. However, the effectiveness of PepVax in reducing CMV viremia/disease was not demonstrated in a phase II trial. Cell-mediated immunity assays are valuable for personalized management plans, but more interventional studies are needed. MBV and adoptive T-cell therapy are promising treatments, and trials for CMV vaccines are ongoing.

Predicting T cell receptor functionality against mutant epitopes.

Cancer cells and pathogens can evade T cell receptors (TCRs) via mutations in immunogenic epitopes. TCR cross-reactivity (i.e., recognition of multiple epitopes with sequence similarities) can counteract such escape but may cause severe side effects in cell-based immunotherapies through targeting self-antigens. To predict the effect of epitope point mutations on T cell functionality, we here present the random forest-based model Predicting T Cell Epitope-Specific Activation against Mutant Versions (P-TEAM). P-TEAM was trained and tested on three datasets with TCR responses to single-amino-acid mutations of the model epitope SIINFEKL, the tumor neo-epitope VPSVWRSSL, and the human cytomegalovirus antigen NLVPMVATV, totaling 9,690 unique TCR-epitope interactions. P-TEAM was able to accurately classify T cell reactivities and quantitatively predict T cell functionalities for unobserved single-point mutations and unseen TCRs. Overall, P-TEAM provides an effective computational tool to study T cell responses against mutated epitopes.

Regulation of the cell surface expression of classical and non-classical MHC proteins by the human cytomegalovirus UL40 and rhesus cytomegalovirus Rh67 proteins.

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.

The battle between host antiviral innate immunity and immune evasion by cytomegalovirus.

Cytomegalovirus (CMV) has successfully established a long-lasting latent infection in humans due to its ability to counteract the host antiviral innate immune response. During coevolution with the host, the virus has evolved various evasion techniques to evade the host's innate immune surveillance. At present, there is still no vaccine available for the prevention and treatment of CMV infection, and the interaction between CMV infection and host antiviral innate immunity is still not well understood. However, ongoing studies will offer new insights into how to treat and prevent CMV infection and its related diseases. Here, we update recent studies on how CMV evades antiviral innate immunity, with a focus on how CMV proteins target and disrupt critical adaptors of antiviral innate immune signaling pathways. This review also discusses some classic intrinsic cellular defences that are crucial to the fight against viral invasion. A comprehensive review of the evasion mechanisms of antiviral innate immunity by CMV will help investigators identify new therapeutic targets and develop vaccines against CMV infection.

Cytomegalovirus inhibitors of programmed cell death restrict antigen cross-presentation in the priming of antiviral CD8 T cells.

CD8 T cells are the predominant effector cells of adaptive immunity in preventing cytomegalovirus (CMV) multiple-organ disease caused by cytopathogenic tissue infection. The mechanism by which CMV-specific, naïve CD8 T cells become primed and clonally expand is of fundamental importance for our understanding of CMV immune control. For CD8 T-cell priming, two pathways have been identified: direct antigen presentation by infected professional antigen-presenting cells (pAPCs) and antigen cross-presentation by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Studies in mouse models using murine CMV (mCMV) and precluding either pathway genetically or experimentally have shown that, in principle, both pathways can congruently generate the mouse MHC/H-2 class-I-determined epitope-specificity repertoire of the CD8 T-cell response. Recent studies, however, have shown that direct antigen presentation is the canonical pathway when both are accessible. This raised the question of why antigen cross-presentation is ineffective even under conditions of high virus replication thought to provide high amounts of antigenic material for feeding cross-presenting pAPCs. As delivery of antigenic material for cross-presentation is associated with programmed cell death, and as CMVs encode inhibitors of different cell death pathways, we pursued the idea that these inhibitors restrict antigen delivery and thus CD8 T-cell priming by cross-presentation. To test this hypothesis, we compared the CD8 T-cell responses to recombinant mCMVs lacking expression of the apoptosis-inhibiting protein M36 or the necroptosis-inhibiting protein M45 with responses to wild-type mCMV and revertant viruses expressing the respective cell death inhibitors. The data reveal that increased programmed cell death improves CD8 T-cell priming in mice capable of antigen cross-presentation but not in a mutant mouse strain unable to cross-present. These findings strongly support the conclusion that CMV cell death inhibitors restrict the priming of CD8 T cells by antigen cross-presentation.