First-Line Therapy with Donor Derived HCMV-Specific T Cells Reduce Persistent HCMV Infection after Allogenic Stem Cell Transplantation

Human cytomegalovirus (HCMV) infection, especially persistent HCMV infection, is an important cause of morbidity and mortality after allogenic stem cell transplantation (allo-SCT). Antiviral agents remain the first-line therapy but are limited by side effects and acquired resistance. We evaluated the safety and efficacy of donor-derived HCMV-specific cytotoxic T cells (CTLs) as a first-line therapy for HCMV infection after allo-SCT and investigated the underlying mechanisms. In humanized HCMV-infected mice, first-line therapy with CTLs effectively combated systemic HCMV infection by promoting the restoration of graft-derived endogenous HCMV-specific immunity in vivo. In a clinical trial, compared with the pair-matched, high-risk control cohort, first-line therapy with CTLs significantly reduced the rate of persistent (2.9% vs 20.0%, P = .018) and late (5.7% vs 20.0%, P = .01) HCMV infection and cumulative incidence of persistent HCMV infection (hazard ratio [HR], 0.13; 95% confidence interval [CI], 0.10-0.82; P = .02), lowered 1-year treatment-related mortality (HR, 0.15. 95% CI, 0.11-0.90. P = .03), and improved 1-year overall survival (HR, 6.35; 95% CI, 1.05-9.00; P = .04). Moreover, first-line therapy with CTLs promoted the quantitative and functional recovery of CTLs in patients, which was associated with HCMV clearance. We provide robust support for the benefits of CTLs combined with antiviral drugs as a first-line therapy for treating HCMV infection and suggest that adoptively infused CTLs may stimulate the recovery of endogenous HCMV-specific immunity. NCT02985775.

BackgroundThe seroprevalence of human cytomegalovirus (HCMV) infection ranges from 30 to 90 % in developed countries. Reliable estimates of HCMV seroprevalence are not available for Pakistan. This study determined the seroprevalence and sociodemographic factors associated with HCMV infection in adult populations of Karachi, Pakistan.MethodsA seroprevalence survey was conducted on 1000 adults, including residents of two semi-urban communities, and visitors to a government and a private hospital. Questionnaire-based interviews were conducted. Sera were analysed for HCMV-specific IgG and IgM. Chi-square or Fisher’s exact test was used for comparing sociodemographic variables against seropositivity of HCMV-IgG or IgM. Multiple logistic regression modeling was performed for IgG seroprevalence and adjusted odds ratios were computed.ResultsThe seroprevalence of HCMV-IgG and IgM was 93.2 and 4.3 % respectively. 95.3 % of individuals who were IgM seropositive were also seropositive for IgG. Around 6 % (15/250) of women of childbearing age remained uninfected and were therefore susceptible to primary infection. HCMV-IgG seroprevalence was associated with being female (p = 0.001), increasing age (p = 0.002) and crowding index (p = 0.003) and also with lower levels of both education (p < 0.001) and income (p = 0.008). Seroprevalence also differed significantly by marital status (p = 0.008) and sampling location (p < 0.001). A logistic regression model for HCMV-IgG seroprevalence showed associations with being female (OR = 1.89; 95 % CI: 1.10–3.25), increasing age (OR = 3.95; 95 % CI: 1.79–8.71) and decreasing income (OR = 0.72; 95 % CI: 0.54–0.96). A strong association was observed between increased seroprevalence of HCMV-IgM and decreasing household size (p = 0.008).ConclusionsSeroprevalence of HCMV is very high in Pakistan, although 6 % of women of childbearing age remain at risk of primary infection. The IgM seropositivity observed in some individuals living in small household size (1–3 individuals) with persistent HCMV infection could have resulted from a recurrent HCMV infection. Future longitudinal research in pregnant women and neonates is required to study the trends in HCMV seroprevalence over time in Pakistan for the development of a potential HCMV prevention and vaccination programme.

Comparing Costs Associated to Letermovir Prophylaxis Vs Ganciclovir Pre-Emptive Therapy in HCMV-Seropositive Patients Who Undergone to Hemopoietic Stem Cells Transplantation

Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Human cytomegalovirus (HCMV) infection reprograms metabolism, including lipid synthesis. While several metabolite-related pathways exhibit altered activity in infected cells, the alteration of lipid-related pathways by HCMV has not been examined beyond fatty acid synthesis and elongation. In this study, we addressed this lack of understanding by focusing on phosphatidylcholine (PC), a class of lipids we previously showed is increased by HCMV infection in human foreskin fibroblasts. Here, we expand upon this finding by demonstrating that HCMV infection increases the abundance of PCs in several different fibroblasts and, similarly, in endothelial and epithelial cells. Additionally, HCMV elevates PC levels regardless of the level of confluency, type of growth medium, and presence of serum. Next, we investigated if HCMV alters the activity in the three PC synthesis pathways. We demonstrate that HCMV infection promotes the activity in the de novo PC synthesis pathway using a 13C-choline isotopic tracer and liquid chromatography high-resolution tandem mass spectrometry. Infection did not alter the activity in the other two pathways. Moreover, we examined the kinetics of PC remodeling by HCMV and found that PC synthesis was promoted and the PC lipidome shifted after 24 h post-infection. Furthermore, we found that PC remodeling occurred when DNA synthesis and subsequent steps of virus replication were inhibited by phosphonoacetic acid. Overall, this work suggests that the early steps of HCMV replication promote the reprogramming of host lipid metabolism to ensure the synthesis of a lipidome necessary to support HCMV infection.IMPORTANCEHuman cytomegalovirus (HCMV) is a common herpesvirus that establishes a lifelong and persistent infection in its human host. HCMV infection in most people does not cause overt disease. However, in immunocompromised individuals, severe CMV-associated disease can lead to permanent disabilities and even death. Additionally, congenital CMV is the leading infectious cause of birth defects. Viruses have evolved to hijack host metabolic pathways to facilitate their replication cycle. In this study, we determine that HCMV promotes the activity in the de novo pathway of phosphatidylcholine (PC) synthesis. We demonstrate that the activity in the other PC synthesis pathways, the phosphatidylethanolamine N-methyltransferase and Lands cycles, is unaltered by HCMV infection. Moreover, we found that HCMV infection alters metabolic activity to increase the PC lipidome before 48 h post-infection. Additionally, our results suggest that immediate-early and early gene expression promotes changes in PC lipids. Together, our findings demonstrate that infection promotes the de novo PC pathway to increase PC lipids during the early stages of virus replication.

PDGFR-Mediated Cytomegalovirus Infection of Megakaryocytes Induces Thrombocytopenia Via Demethylation of AML1 and IEX-1 in the TPO/c-Mpl Signaling Pathway Following Allo-HSCT

Dendritic Cell Vaccination Induces HCMV Specific T-Cell Responses in Allogeneic Stem Cell Recipients.

Latent infection and reactivation of human cytomegalovirus

Oncomodulatory signals by regulatory proteins encoded by human cytomegalovirus: a novel role for viral infection in tumor progression

Evidence suggests that human cytomegalovirus (HCMV) infection may be implicated in the progression of colorectal cancer (CRC). However, the correlation between HCMV infection and survival outcomes in patients with CRC remains unclear. Here, we constructed a flow algorithm to identify HCMV sequences based on the RNA-seq data of patients with CRC derived from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The patients' clinical information matrix was used to calculate the Euclidean distance to filter out suitable patients not infected with HCMV, combined with patients' survival outcomes, to reveal how HCMV infection is involved in CRC progression. HCMV infection is widespread in patients with CRC, and the prevalence of HCMV infection ranges from 10 to 36% in four independent CRC datasets, with infection being concentrated in carcinoma tissue rather than in normal tissue. In addition, HCMV-positive patients had a poor survival prognosis, with three HCMV genes, UL82, UL42, and UL117, associated with poor patient survival outcomes. Most importantly, we suppose that the regulation of immune function by HCMV may be key to the poor prognosis of patients with CRC. We found that HCMV infection was associated with poor prognosis in CRC patients and identified three prognosis-associated HCMV genes. The regulation of immune function caused by HCMV infection was the key factor, while HCMV-positive patients with CRC mostly presented with a state of immunosuppression. This may provide new ideas for the personalized treatment of patients with CRC, especially with respect to immunotherapy.

Objective To observe the changes in the expression of pro-inflammatory cytokines IL-1β over time in THP-1 derived macrophages after human cytomegalovirus (HCMV) infection. Methods The model of HCMV infection in THP-1 derived macrophages was constructed, then HCMV infected group, the mock-infected control group, LPS + ATP group and poly (dA : dT) control group were established. ELISA analysis was performed to measure the levels of IL-1β in culture supernatant of THP-1 derived macrophages at 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 48 hours after infected with HCMV. Real-time PCR and western blot were performed to measure the expression levels of IL-1β gene and protein, respectively. Results The gene expression of pro-inflammatory cytokines IL-1β in HCMV infected group is 7.77 times to the mock-infected control group at 6 hours after HCMV infected THP-1 derived macrophages. IL-1β in supernatant of THP-1-derived macrophages increased significantly at 1 hour after HCMV infection, was continuously rising at 3 hours and 6 hours subsequently, and reached at peak at 12 hours and maintained till 48 hours. The protein expression of IL-1β in HCMV infected group was significantly higher than the mock-infected control group and poly (dA : dT) control group at 6 hours after HCMV infected THP-1 derived macrophages, while no significant difference between the LPS + ATP control group. Conclusions The expression of IL-1β is induced by HCMV infected THP-1 derived macrophages, and continue to increase over time. Key words: Human cytomegalovirus; THP-1 derived macrophages; Interleukine-1 beta

Human cytomegalovirus (HCMV) is a leading cause of birth defects in humans. These birth defects include microcephaly, sensorineural hearing loss, vision loss, and cognitive impairment. The process by which the developing fetus incurs these neurological defects is poorly understood. To elucidate some of these mechanisms, we have utilized HCMV-infected induced pluripotent stem cells (iPSCs) to generate in vitro brain organoids, modeling the first trimester of fetal brain development. Early during culturing, brain organoids generate neural rosettes. These structures are believed to model neural tube formation. Rosette formation was analyzed in HCMV-infected and mock-infected brain organoids at 17, 24, and 31 days postinfection. Histological analysis revealed fewer neural rosettes in HCMV-infected compared to mock-infected organoids. HCMV-infected organoid rosettes incurred multiple structural deficits, including increased lumen area, decreased ventricular zone depth, and decreased cell count. Immunofluorescent (IF) analysis found that nidogen-1 (NID1) protein expression in the basement membrane surrounding neural rosettes was greatly reduced by virus infection. IF analysis also identified a similar downregulation of laminin in basement membranes of HCMV-infected organoid rosettes. Knockdown of NID1 alone in brain organoids impaired their development, leading to the production of rosettes with increased lumen area, decreased structural integrity, and reduced laminin localization in the basement membrane, paralleling observations in HCMV-infected organoids. Our data strongly suggest that HCMV-induced downregulation of NID1 impairs neural rosette formation and integrity, likely contributing to many of HCMV's most severe birth defects. IMPORTANCE HCMV infection in pregnant women continues to be the leading cause of virus-induced neurologic birth defects. The mechanism through which congenital HCMV (cCMV) infection induces pathological changes to the developing fetal central nervous system (CNS) remains unclear. Our lab previously reproduced identified clinical defects in HCMV-infected infants using a three dimensional (3D) brain organoid model. In this new study, we have striven to discover very early HCMV-induced changes in developing brain organoids. We investigated the development of neural tube-like structures, neural rosettes. HCMV-infected rosettes displayed multiple structural abnormalities and cell loss. HCMV-infected rosettes displayed reduced expression of the key basement membrane protein, NID1. We previously found NID1 to be specifically targeted in HCMV-infected fibroblasts and endothelial cells. Brain organoids generated from NID1 knockdown iPSCs recapitulated the structural defects observed in HCMV-infected rosettes. Findings in this study revealed HCMV infection induced early and dramatic structural changes in 3D brain organoids. We believe our results suggest a major role for infection-induced NID1 downregulation in HCMV-induced CNS birth defects.

Human cytomegalovirus infection of cells of hematopoietic origin: HCMV-induced immunosuppression, immune evasion, and latency

Objective: To investigate the interaction between human cytomegalovirus infection and PAX9 gene polymorphisms in low birth weight (LBW) infants.Methods: Nested-PCR was used to detect human cytomegalovirus (HCMV) infection and restriction fragment length polymorphism PCR (RFLP-PCR) was then used to detect polymorphisms at rs2073244 and rs4904210 on the PAX9 gene in 65 HCMV-positive infant cases and 65 HCMV-negative infant controls. Cases and controls were compared for differences in the rates of LBW using the χ2 test. Genetic and environmental interactions were assessed by comparing rates of LBW between the cases and controls in stratified analyses and logistic regressions.Results: The study confirmed that HCMV infection is significantly associated with LBW (OR = 5.519, χ2 = 20.924, p < 0.05) and suggested that some PAX9 polymorphisms may promote the induction of LBW by HCMV infection. The AG and GG genotypes at rs2073244 and the CC genotype at rs4904210, with interaction coefficients greater than unity suggest that they may strengthen the association between HCMV infection and LBW.Conclusions: Congenital HCMV infection can cause LBW and some PAX9 polymorphisms can increase the risk of LBW in HCMV-infected infants. The results may provide new clues into the relationship among HCMV infection, PAX9 polymorphisms and LBW as well as a foundation for additional molecular epidemiological and biochemical research in the future.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs) and induces cell cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing the levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products-IE1, pp71, and UL26-were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS. Human cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.