Evaluation of the Association Between Congenital Cytomegalovirus Infection and Pediatric Acute Lymphoblastic Leukemia.

Importance: Acute lymphoblastic leukemia (ALL) is the most common form of pediatric cancer, and a leading cause of death in children. Understanding the causes of pediatric ALL is necessary to enable early detection and prevention; congenital cytomegalovirus (cCMV) has recently been identified as a potential moderate-to-strong factor associated with risk for ALL. Objective: To compare the prevalence of cCMV infection between ALL cases and matched controls. Design, Setting, and Participants: In this population-based case-control study of ALL cases and matched controls, cases consisted of children aged 0 to 14 years between 1987 and 2014 with an ALL diagnosis identified through the Michigan Cancer Surveillance Program and born in Michigan on or after October 1, 1987. Cancer-free controls were identified by the Michigan BioTrust for Health and matched on age, sex, and mother's race and ethnicity. Data were analyzed from November to May 2022. Exposures: cCMV infection measured by quantitative polymerase chain reaction in newborn dried blood spots. Main Outcomes and Measures: ALL diagnosed in children aged 0 to 14 years. Results: A total of 1189 ALL cases and 4756 matched controls were included in the study. Bloodspots were collected from participants at birth, and 3425 (57.6%) participants were male. cCMV was detected in 6 ALL cases (0.5%) and 21 controls (0.4%). There was no difference in the odds of cCMV infection comparing ALL cases with controls (odds ratio, 1.30; 95% CI, 0.52-3.24). Immunophenotype was available for 536 cases (45.1%) and cytogenetic data for 127 (27%). When stratified by subtype characteristics, hyperdiploid ALL (74 cases) was associated with 6.26 times greater odds of cCMV infection compared with unmatched controls (95% CI, 1.44-27.19). Conclusions and Relevance: In this case-control study of cCMV and pediatric ALL, cCMV was associated with increased risk of hyperdiploid ALL. These findings encourage continued research.

Cytomegalovirus viremia as a risk factor for mortality in HIV-associated cryptococcal and tuberculous meningitis.

OBJECTIVES: CMV viremia is associated with increased mortality in persons with HIV. We previously demonstrated that CMV viremia was a risk factor for 10-week mortality in antiretroviral therapy (ART)-naïve persons with cryptococcal meningitis. We investigated whether similar observations existed over a broader cohort of patients with HIV-associated meningitis at 18 weeks. METHODS: We prospectively enrolled Ugandans with cryptococcal or TB meningitis into clinical trials in 2015-2019. We quantified CMV DNA concentrations from stored baseline plasma or serum samples from 340 participants. We compared 18-week survival between those with and without CMV viremia. RESULTS: We included 308 persons with cryptococcal meningitis and 32 with TB meningitis, of whom 121 (36%) had detectable CMV DNA. Baseline CD4+ T-cell counts (14 vs. 24 cells/µl; P = 0.07) and antiretroviral exposure (47% vs. 45%; P = 0.68) did not differ between persons with and without CMV viremia. The 18-week mortality was 50% (61/121) in those with CMV viremia versus 34% (74/219) in those without (P = 0.003). Detectable CMV viremia (adjusted hazard ratio [aHR] 1.60; 95% confidence interval [CI] 1.13-2.25; P = 0.008) and greater viral load (aHR 1.22 per log10 IU/ml increase; 95% CI 1.09-1.35; P <0.001) were positively associated with all-cause mortality through 18 weeks. CONCLUSION: CMV viremia at baseline was associated with a higher risk of death at 18 weeks among persons with HIV-associated cryptococcal or TB meningitis, and the risk increased as the CMV viral load increased. Further investigation is warranted to determine whether CMV is a modifiable risk contributing to deaths in HIV-associated meningitis or is a biomarker of immune dysfunction.

The transcription factor GLI1 cooperates with the chromatin remodeler SMARCA2 to regulate chromatin accessibility at distal DNA regulatory elements.

The transcription factor GLI1 (GLI family zinc finger 1) plays a key role in the development and progression of multiple malignancies. To date, regulation of transcriptional activity at target gene promoters is the only molecular event known to underlie the oncogenic function of GLI1. Here, we provide evidence that GLI1 controls chromatin accessibility at distal regulatory regions by modulating the recruitment of SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2) to these elements. We demonstrate that SMARCA2 endogenously interacts with GLI1 and enhances its transcriptional activity. Mapping experiments indicated that the C-terminal transcriptional activation domain of GLI1 and SMARCA2's central domains, including its ATPase motif, are required for this interaction. Interestingly, similar to SMARCA2, GLI1 overexpression increased chromatin accessibility, as indicated by results of the micrococcal nuclease assay. Further, results of assays for transposase-accessible chromatin with sequencing (ATAC-seq) after GLI1 knockdown supported these findings, revealing that GLI1 regulates chromatin accessibility at several regions distal to gene promoters. Integrated RNA-seq and ATAC-seq data analyses identified a subset of differentially expressed genes located in cis to these regulated chromatin sites. Finally, using the GLI1-regulated gene HHIP (Hedgehog-interacting protein) as a model, we demonstrate that GLI1 and SMARCA2 co-occupy a distal chromatin peak and that SMARCA2 recruitment to this HHIP putative enhancer requires intact GLI1. These findings provide insights into how GLI1 controls gene expression in cancer cells and may inform approaches targeting this oncogenic transcription factor to manage malignancies.

Cytomegalovirus Viremia Associated With Increased Mortality in Cryptococcal Meningitis in Sub-Saharan Africa.

BACKGROUND: Cryptococcal meningitis and tuberculosis are both important causes of death in persons with advanced human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). Cytomegalovirus (CMV) viremia may be associated with increased mortality in persons living with HIV who have tuberculosis. It is unknown whether concurrent CMV viremia is associated with mortality in other AIDS-related opportunistic infections. METHODS: We prospectively enrolled Ugandans living with HIV who had cryptococcal meningitis from 2010-2012. Subsequently, we analyzed stored baseline plasma samples from 111 subjects for CMV DNA. We compared 10-week survival rates among those with and without CMV viremia. RESULTS: Of 111 participants, 52% (58/111) had detectable CMV DNA (median plasma viral load 498 IU/mL, interquartile range [IQR] 259-2390). All samples tested were positive on immunoglobin G serology. The median CD4+ T cell count was 19 cells/µL (IQR 9-70) and did not differ by the presence of CMV viremia (P = .47). The 10-week mortality rates were 40% (23/58) in those with CMV viremia and 21% (11/53) in those without CMV viremia (hazard ratio 2.19, 95% confidence interval [CI] 1.07-4.49; P = .03), which remained significant after a multivariate adjustment for known risk factors of mortality (adjusted hazard ratio 3.25, 95% CI 1.49-7.10; P = .003). Serum and cerebrospinal fluid cytokine levels were generally similar and cryptococcal antigen-specific immune stimulation responses did not differ between groups. CONCLUSIONS: Half of persons with advanced AIDS and cryptococcal meningitis had detectable CMV viremia. CMV viremia was associated with an over 2-fold higher mortality rate. It remains unclear whether CMV viremia in severely immunocompromised persons with cryptococcal meningitis contributes directly to this mortality or may reflect an underlying immune dysfunction (ie, cause vs effect). CLINICAL TRIALS REGISTRATION: NCT01075152.

Additive Protection against Congenital Cytomegalovirus Conferred by Combined Glycoprotein B/pp65 Vaccination Using a Lymphocytic Choriomeningitis Virus Vector.

Subunit vaccines for prevention of congenital cytomegalovirus (CMV) infection based on glycoprotein B (gB) and pp65 are in clinical trials, but it is unclear whether simultaneous vaccination with both antigens enhances protection. We undertook evaluation of a novel bivalent vaccine based on nonreplicating lymphocytic choriomeningitis virus (rLCMV) vectors expressing a cytoplasmic tail-deleted gB [gB(dCt)] and full-length pp65 from human CMV in mice. Immunization with the gB(dCt) vector alone elicited a comparable gB-binding antibody response and a superior neutralizing response to that elicited by adjuvanted subunit gB. Immunization with the pp65 vector alone elicited robust T cell responses. Comparable immunogenicity of the combined gB(dCt) and pp65 vectors with the individual monovalent formulations was demonstrated. To demonstrate proof of principle for a bivalent rLCMV-based HCMV vaccine, the congenital guinea pig cytomegalovirus (GPCMV) infection model was used to compare rLCMV vectors encoding homologs of pp65 (GP83) and gB(dCt), alone and in combination versus Freund's adjuvanted recombinant gB. Both vectors elicited significant immune responses, and no loss of gB immunogenicity was noted with the bivalent formulation. Combined vaccination with rLCMV-vectored GPCMV gB(dCt) and pp65 (GP83) conferred better protection against maternal viremia than subunit or either monovalent rLCMV vaccine. The bivalent vaccine also was significantly more effective in reducing pup mortality than the monovalent vaccines. In summary, bivalent vaccines with rLCMV vectors expressing gB and pp65 elicited potent humoral and cellular responses and conferred protection in the GPCMV model. Further clinical trials of LCMV-vectored HCMV vaccines are warranted.

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection.

UNLABELLED: Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Nonpathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome [BAC]) has a 4-bp deletion that disrupts GP129, which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130, an overlapping open reading frame (ORF) of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in Escherichia coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to that of N13R10. However, following experimental challenge of immunocompromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 × 10(6)-fold) viral loads in blood and end organs compared to N13R10. In pregnant guinea pigs, challenge with doses of r129 virus of ≥5 × 10(6) PFU resulted in levels of maternal viremia, congenital transmission, pup viral loads, intrauterine growth restriction, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129-GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC. IMPORTANCE: Tissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions, and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of human cytomegalovirus (HCMV) proteins UL128, UL130, and UL131A. These mutations can attenuate the course of infection when the virus is reintroduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frameshift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection.

Comparison of monovalent glycoprotein B with bivalent gB/pp65 (GP83) vaccine for congenital cytomegalovirus infection in a guinea pig model: Inclusion of GP83 reduces gB antibody response but both vaccine approaches provide equivalent protection against pup mortality.

Cytomegalovirus (CMV) subunit vaccine candidates include glycoprotein B (gB), and phosphoprotein ppUL83 (pp65). Using a guinea pig cytomegalovirus (GPCMV) model, this study compared immunogenicity, pregnancy outcome, and congenital viral infection following pre-pregnancy immunization with a three-dose series of modified vaccinia virus Ankara (MVA)-vectored vaccines consisting either of gB administered alone, or simultaneously with a pp65 homolog (GP83)-expressing vaccine. Vaccinated and control dams were challenged at midgestation with salivary gland-adapted GPCMV. Comparisons included ELISA and neutralizing antibody responses, maternal viral load, pup mortality, and congenital infection rates. Strikingly, ELISA and neutralization titers were significantly lower in the gB/GP83 combined vaccine group than in the gB group. However, both vaccines protected against pup mortality (63.2% in controls vs. 11.4% and 13.9% in gB and gB/GP83 combination groups, respectively; p<0.0001). Reductions in pup viral load were noted for both vaccine groups compared to control, but preconception vaccination resulted in a significant reduction in GPCMV transmission only in the monovalent gB group (26/44, 59% v. 27/34, 79% in controls; p<0.05). We conclude that, using the MVA platform, the addition of GP83 to a gB subunit vaccine interferes with antibody responses and diminishes protection against congenital GPCMV infection, but does not decrease protection against pup mortality.

Identification by mass spectrometry and immune response analysis of guinea pig cytomegalovirus (GPCMV) pentameric complex proteins GP129, 131 and 133.

Development of a vaccine against congenital infection with human cytomegalovirus (HCMV) is a major public health priority. A potential vaccine target receiving considerable recent attention is the pentameric complex (PC) of HCMV proteins consisting of gL, gH, UL128, UL130, and UL131, since some antibodies against these target proteins are capable of potently neutralizing virus at epithelial and endothelial cell surfaces. Recently, homologous proteins have been described for guinea pig cytomegalovirus (GPCMV), consisting of gH, gL, and the GPCMV proteins GP129, GP131, and GP133. To investigate these proteins as potential vaccine targets, expression of GP129-GP133 transcripts was confirmed by reverse-transcriptase PCR. Mass spectrometry combined with western blot assays demonstrated the presence of GP129, GP131, and GP133 proteins in virus particles. Recombinant proteins corresponding to these PC proteins were generated in baculovirus, and as GST fusion proteins. Recombinant proteins were noted to be immunoreactive with convalescent sera from infected animals, suggesting that these proteins are recognized in the humoral immune response to GPCMV infection. These analyses support the study of PC-based recombinant vaccines in the GPCMV congenital infection model.