RESUMO
OBJECTIVE: To determine the positivity rate of congenital cytomegalovirus (cCMV) testing among universal, hearing-targeted CMV testing (HT-cCMV) and delayed targeted dried blood spot (DBS) testing newborn screening programs, and to examine the characteristics of successful HT-cCMV testing programs. STUDY DESIGN: Prospective survey of birth hospitals performing early CMV testing. SETTING: Multiple institutions. METHODS: Birth hospitals participating in the National Institutes of Health ValEAR clinical trial were surveyed to determine the rates of cCMV positivity associated with 3 different testing approaches: universal testing, HT-cCMV, and DBS testing. A mixed methods model was created to determine associations between successful HT-cCMV screening and specific screening protocols. RESULTS: Eighty-two birth hospitals were surveyed from February 2019 to December 2021. Seven thousand six hundred seventy infants underwent universal screening, 9017 infants HT-cCMV and 535 infants delayed DBS testing. The rates of cCMV positivity were 0.5%, 1.5%, and 7.3%, respectively. The positivity rate for universal CMV screening was less during the COVID-19 pandemic than that reported prior to the pandemic. There were no statistically significant drops in positivity for any approach during the pandemic. For HT-cCMV testing, unique order sets and rigorous posttesting protocols were associated with successful screening programs. CONCLUSION: Rates of cCMV positivity differed among the 3 approaches. The rates are comparable to cohort studies reported in the literature. Universal CMV prevalence decreased during the pandemic but not significantly. Institutions with specific order set for CMV testing where the primary care physician orders the test and the nurse facilitates the testing process exhibited higher rates of HT-cCMV testing.
Assuntos
Infecções por Citomegalovirus , Triagem Neonatal , Humanos , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/epidemiologia , Triagem Neonatal/métodos , Recém-Nascido , Estudos Prospectivos , COVID-19/epidemiologia , COVID-19/diagnóstico , Estados Unidos/epidemiologia , Teste em Amostras de Sangue Seco , Feminino , MasculinoRESUMO
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.
Assuntos
Infecções por Citomegalovirus , Leucemia-Linfoma Linfoblástico de Células Precursoras , Recém-Nascido , Criança , Humanos , Masculino , Feminino , Estudos de Casos e Controles , Infecções por Citomegalovirus/epidemiologia , Infecções por Citomegalovirus/complicações , Prevalência , Michigan , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiologiaRESUMO
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.
Assuntos
Cryptococcus , Infecções por Citomegalovirus , Infecções por HIV , Meningite Criptocócica , Tuberculose Meníngea , Contagem de Linfócito CD4 , Citomegalovirus , Infecções por Citomegalovirus/complicações , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Meningite Criptocócica/complicações , Meningite Criptocócica/tratamento farmacológico , Fatores de Risco , Tuberculose Meníngea/complicações , Tuberculose Meníngea/tratamento farmacológico , ViremiaRESUMO
BACKGROUND: Cerebral toxoplasmosis infection presents with non-specific neurologic symptoms in immunocompromised patients. With lack of measurable adaptive immune responses and reluctance to sample affected brain tissue, expedient diagnosis to guide directed treatment is often delayed. CASE PRESENTATION: We describe the use of cerebrospinal fluid polymerase chain reaction and plasma cell-free DNA technologies to supplement neuroimaging in the diagnosis of cerebral toxoplasmosis in an immunocompromised pediatric patient following allogeneic hematopoietic cell transplantation for idiopathic severe aplastic anemia. Successful cerebral toxoplasmosis treatment included antibiotic therapy for 1 year following restoration of cellular immunity with an allogeneic stem cell boost. CONCLUSIONS: Plasma cell-free DNA technology provides a non-invasive method of rapid diagnosis, improving the likelihood of survival from often lethal opportunistic infection in a high risk, immunocompromised patient population.
Assuntos
Anemia Aplástica , Transplante de Células-Tronco Hematopoéticas , Infecções Oportunistas , Toxoplasmose Cerebral , Anemia Aplástica/terapia , Criança , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Toxoplasmose Cerebral/diagnóstico , Toxoplasmose Cerebral/tratamento farmacológico , Transplante Homólogo/efeitos adversosRESUMO
Zika virus (ZIKV) exhibits a tropism for brain tumor cells and has been used as an oncolytic virus to target brain tumors in mice with modest effects on extending median survival. Recent studies have highlighted the potential for combining virotherapy and immunotherapy to target cancer. We postulated that ZIKV could be used as an adjuvant to enhance the long-term survival of mice with malignant glioblastoma and generate memory T-cells capable of providing long-term immunity against cancer remission. To test this hypothesis mice bearing malignant intracranial GL261 tumors were subcutaneously vaccinated with irradiated GL261 cells previously infected with the ZIKV. Mice also received intracranial injections of live ZIKV, irradiation attenuated ZIKV, or irradiated GL261 cells previously infected with ZIKV. Long-term survivors were rechallenged with a second intracranial tumor to examine their immune response and look for the establishment of protective memory T-cells. Mice with subcutaneous vaccination plus intracranial irradiation attenuated ZIKV or intracranial irradiated GL261 cells previously infected with ZIKV exhibited the greatest extensions to overall survival. Flow cytometry analysis of immune cells within the brains of long-term surviving mice after tumor rechallenge revealed an increase in the number of T-cells, including CD4+ and tissue-resident effector/ effector memory CD4+ T-cells, in comparison to long-term survivors that were mock-rechallenged, and in comparison to naïve untreated mice challenged with intracranial gliomas. These results suggest that ZIKV can serve as an adjuvant to subcutaneous tumor vaccines that enhance long-term survival and generate protective tissue-resident memory CD4+ T-cells.
Assuntos
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Terapia Viral Oncolítica , Linfócitos T/imunologia , Zika virus/imunologia , Adjuvantes Imunológicos , Animais , Neoplasias Encefálicas/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer , Glioblastoma/imunologia , Memória Imunológica , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Human cytomegalovirus (HCMV) infects the chorioamnion, but whether these infections cause fetal membrane dysfunction remains poorly understood. We sought to assess whether guinea pig cytomegalovirus (GPCMV) infects amnion-derived cells in vitro, compare the inflammatory response of amnion cells to GPCMV and HCMV, and determine if GPCMV infects the amnion in vivo. We found that GPCMV replicates in primary guinea pig amnion derived cells and HPV16 E6/E7-transduced amniotic epithelial cells (AEC[E6/E7]s). HCMV and GPCMV infection of amnion cells increased the transcription of the chemokines CCL5/Ccl5, CXCL8/Cxcl8, and CXCL10/Cxcl10. Myd88-knockdown decreased Ccl5 and Cxc8 transcription in GPCMV-infected AEC[E6/E7]s. GPCMV was detected in the guinea pig amnion after primary maternal infection, revealing that guinea pigs are an appropriate model to study fetal membrane physiology after cytomegalovirus infection. As inflammation is known to cause fetal membrane weakening, the amnion's response to cytomegalovirus infection may cause preterm birth and other adverse pregnancy outcomes.
Assuntos
Âmnio/imunologia , Quimiocinas/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Complicações na Gravidez/imunologia , Âmnio/virologia , Animais , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocinas/genética , Citomegalovirus/genética , Citomegalovirus/imunologia , Infecções por Citomegalovirus/genética , Feminino , Cobaias , Humanos , Interleucina-8/genética , Interleucina-8/imunologia , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/virologiaRESUMO
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.
Assuntos
Infecções por Citomegalovirus , Infecções por HIV , Meningite Criptocócica , África Subsaariana/epidemiologia , Contagem de Linfócito CD4 , Citomegalovirus , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/epidemiologia , Infecções por HIV/complicações , Humanos , Meningite Criptocócica/epidemiologia , Viremia/epidemiologiaRESUMO
Cytomegalovirus (CMV) causes sensorineural hearing loss and developmental disabilities in newborns when infections are acquired in utero Pregnant women may acquire CMV from oral exposure to CMV in urine or saliva from young children. Neutralizing antibodies in maternal saliva have the potential to prevent maternal infection and, in turn, fetal infection. As CMV uses different viral glycoprotein complexes to enter different cell types, the first cells to be infected in the oral cavity could determine the type of antibodies needed to disrupt oral transmission. Antibodies targeting the pentameric complex (PC) should block CMV entry into epithelial cells but not into fibroblasts or Langerhans cells (which do not require the PC for entry), while antibodies targeting glycoprotein complexes gB or gH/gL would be needed to block entry into fibroblasts, Langerhans cells, or other cell types. To assess the potential for antibodies to disrupt oral acquisition, CMV from culture-positive urine samples (uCMV) was used to study cell tropisms and sensitivity to antibody neutralization. uCMV entered epithelial cells poorly compared with the entry into fibroblasts. CMV-hyperimmune globulin or monoclonal antibodies targeting gB, gH/gL, or the PC were incapable of blocking the entry of uCMV into either fibroblasts or epithelial cells. Both phenotypes were lost after one passage in cultured fibroblasts, suggestive of a nongenetic mechanism. These results suggest that uCMV virions have a reversible block to epithelial cell entry. Antibodies may be ineffective in preventing maternal oral CMV acquisition but may limit viral spread in blood or tissues, thereby reducing or preventing fetal infection and disease.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/imunologia , Células Epiteliais/virologia , Tropismo Viral , Eliminação de Partículas Virais , Citomegalovirus/fisiologia , Fibroblastos/virologia , Humanos , Recém-Nascido , Vírion/imunologia , Vírion/fisiologia , Internalização do VírusRESUMO
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.
Assuntos
Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/imunologia , Portadores de Fármacos , Vírus da Coriomeningite Linfocítica/genética , Fosfoproteínas/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Infecções por Citomegalovirus/congênito , Vacinas contra Citomegalovirus/administração & dosagem , Modelos Animais de Doenças , Feminino , Cobaias , Camundongos Endogâmicos C57BL , Fosfoproteínas/genética , Linfócitos T/imunologia , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genéticaRESUMO
Congenital cytomegalovirus (CMV) infection is the most common infectious cause of disability in newborn infants. CMV also causes serious disease in solid organ (SOT) and haematopoietic stem cell transplant (HSCT) recipients. In otherwise healthy children and adults, primary CMV infection rarely causes illness. However, even asymptomatic CMV infections may predispose an individual towards an increased risk of atherosclerosis, cancer and immune senescence over the life course, although such associations remain controversial. Thus, although a vaccine against congenital CMV infection would have the greatest public health impact and cost-effectiveness, arguably all populations could benefit from an effective immunisation against this virus. Currently there are no licensed CMV vaccines, but there is increased interest in developing and testing potential candidates, driven by the demonstration that a recombinant CMV glycoprotein B (gB) vaccine has some efficacy in prevention of infection in young women and adolescents, and in CMV-seronegative SOT recipients. In this review, the recent and current status of candidate CMV vaccines is discussed. Evolving concepts about proposed correlates of protective immunity in different target populations for CMV vaccination, and how these differences impact current clinical trials, are also reviewed.
RESUMO
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.
Assuntos
Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/patologia , Fibroblastos/virologia , Mutação , Multimerização Proteica , Roseolovirus/genética , Roseolovirus/patogenicidade , Animais , Peso Corporal , Cromossomos Artificiais Bacterianos , Infecções por Citomegalovirus/virologia , Modelos Animais de Doenças , Escherichia coli/genética , Glicoproteínas/genética , Cobaias , Roseolovirus/crescimento & desenvolvimento , Deleção de Sequência , Inoculações Seriadas , Carga Viral , Proteínas Estruturais Virais/genética , Viremia , Virulência , Fatores de Virulência/genéticaRESUMO
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.
Assuntos
Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/imunologia , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Fosfoproteínas/imunologia , Roseolovirus/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Citomegalovirus/transmissão , Vacinas contra Citomegalovirus/administração & dosagem , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Cobaias , Testes de Neutralização , Gravidez , Análise de Sobrevida , Resultado do Tratamento , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Carga ViralRESUMO
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.
Assuntos
Substâncias Macromoleculares/química , Roseolovirus/química , Proteínas Estruturais Virais/análise , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Western Blotting , Cobaias , Espectrometria de Massas , Proteínas Recombinantes/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologiaRESUMO
The transmission of cytomegalovirus (CMV) from mother to fetus can give rise to severe neurodevelopment defects in newborns. One strategy to prevent these congenital defects is prophylactic vaccination in young women. A candidate vaccine antigen is glycoprotein B (gB). This antigen is abundant on the virion surface and is a major target of neutralization responses in human infections. Here, we have evaluated in a challenge model of congenital guinea pig CMV (GPCMV) infection, GPCMV-gB vaccines formulated with the clinically relevant Adjuvant Systems AS01B and AS02V, or with Freund's adjuvant (FA). Fifty-two GPCMV-seronegative female guinea pigs were administered three vaccine doses before being mated. GPCMV-challenge was performed at Day 45 of pregnancy (of an estimated 65 day gestation). Pup mortality rates in the gB/AS01B, gB/AS02V, and gB/FA groups were 24% (8/34), 10% (4/39) and 36% (12/33), respectively, and in the unvaccinated control group was 65% (37/57). Hence, efficacies against pup mortality were estimated at 64%, 84% and 44% for gB/AS01B (p<0.001), gB/AS02V (p<0.001) and gB/FA (p=0.014), respectively. Efficacies against GPCMV viremia (i.e. DNAemia, detected by PCR) were estimated at 88%, 68% and 25% for the same vaccines, respectively, but were only significant for gB/AS01B (p<0.001), and gB/AS02V (p=0.002). In dams with viremia, viral load was approximately 6-fold lower with vaccination than without. All vaccines were highly immunogenic after two and three doses. In light of these results and of other results of AS01-adjuvanted vaccines in clinical development, vaccine immunogenicity was further explored using human CMV-derived gB antigen adjuvanted with either AS01B or the related formulation AS01E. Both adjuvanted vaccines were highly immunogenic after two doses, in contrast to the lower immunogenicity of the unadjuvanted vaccine. In conclusion, the protective efficacy and immunogenicity of adjuvanted vaccines in this guinea pig model are supportive of investigating gB/AS01 and gB/AS02 in the clinic.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/imunologia , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Afinidade de Anticorpos , Relação Dose-Resposta Imunológica , Feminino , Cobaias , Testes de Neutralização , Gravidez , Roseolovirus , Viremia/prevenção & controleRESUMO
OBJECTIVE: To assess the validity of the guinea pig as a model for congenital cytomegalovirus (CMV) infection by comparing the effectiveness of detecting the virus by real-time polymerase chain reaction (PCR) in blood, urine, and saliva. DESIGN: Case-control study. SETTING: Academic research. SUBJECTS: Eleven pregnant Hartley guinea pigs. MAIN OUTCOME MEASURES: Blood, urine, and saliva samples were collected from guinea pig pups delivered from pregnant dams inoculated with guinea pig CMV. These samples were then evaluated for the presence of guinea pig CMV by real-time PCR assuming 100% transmission. RESULTS: Thirty-one pups delivered from 9 inoculated pregnant dams and 8 uninfected control pups underwent testing for guinea pig CMV and for auditory brainstem response hearing loss. Repeated-measures analysis of variance demonstrated no statistically significantly lower weight for the infected pups compared with the noninfected control pups. Six infected pups demonstrated auditory brainstem response hearing loss. The sensitivity and specificity of the real-time PCR assay on saliva samples were 74.2% and 100.0%, respectively. The sensitivity of the real-time PCR on blood and urine samples was significantly lower than that on saliva samples. CONCLUSIONS: Real-time PCR assays of blood, urine, and saliva revealed that saliva samples show high sensitivity and specificity for detecting congenital CMV infection in guinea pigs. This finding is consistent with recent screening studies in human newborns. The guinea pig may be a good animal model in which to compare different diagnostic assays for congenital CMV infection.
Assuntos
Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/diagnóstico , Análise de Variância , Animais , Animais Recém-Nascidos , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Cobaias , Testes Auditivos , Valor Preditivo dos Testes , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e EspecificidadeRESUMO
PURPOSE: : Bedside newborn hearing screening is highly successful in identifying deaf or hard-of-hearing infants. However, newborn hearing screening protocols have high loss to follow-up rates. We propose that bloodspot-based genetic testing for GJB2 alleles can provide a means for rapid confirmation in a subset of infants who fail bedside newborn hearing screening. METHODS: : We performed a case-control study comparing the prevalence of common GJB2 mutations from deidentified bloodspots designated as "refer" by newborn hearing screening and contemporaneously selected randomly chosen controls designated as "pass." Between March 2006 and December 2007, 2354 spots were analyzed for common alleles, c.35delG, c.167delT, c.235delC, and p.V37I in GJB2 with a subset reanalyzed by conventional Sanger sequencing to search for additional alleles. RESULTS: : The prevalence of biallelic GJB2 mutations in bloodspots from infants who referred by newborn hearing screening is approximately 1 in 50 (23/1177). In contrast, one bloodspot from an infant who passed newborn hearing screening was identified to harbor biallelic GJB2 mutations. CONCLUSIONS: : These findings show that when a newborn refers by newborn hearing screening, there is a significant chance that GJB2-related hearing loss is present. Bloodspot-based genetic testing for common GJB2 alleles should be considered as second tier testing for bedside newborn hearing screening.
Assuntos
Biomarcadores Tumorais/genética , Conexinas/genética , Testes Genéticos/métodos , Perda Auditiva/diagnóstico , Audição/fisiologia , Triagem Neonatal/métodos , Alelos , Biomarcadores Tumorais/sangue , Estudos de Casos e Controles , Conexina 26 , Conexinas/sangue , Análise Mutacional de DNA , Seguimentos , Genótipo , Perda Auditiva/sangue , Perda Auditiva/epidemiologia , Perda Auditiva/genética , Humanos , Recém-Nascido , Mutação , Prevalência , Estados Unidos/epidemiologiaRESUMO
Human cytomegalovirus (CMV) is responsible for approximately 40,000 congenital infections in the United States each year. Congenital CMV disease frequently produces serious neurodevelopmental disability, as well as vision impairment and sensorineural hearing loss. Development of a CMV vaccine is therefore considered to be a major public health priority. The mechanisms by which CMV injures the fetus are complex and likely include a combination of direct fetal injury induced by pathologic virally-encoded gene products, an inability of the maternal immune response to control infection, and the direct impact of infection on placental function. CMV encodes gene products that function, both at the RNA and the protein level, to interfere with many cellular processes. These include gene products that modify the cell cycle; interfere with apoptosis; induce an inflammatory response; mediate vascular injury; induce site-specific breakage of chromosomes; promote oncogenesis; dysregulate cellular proliferation; and facilitate evasion of host immune responses. This minireview summarizes current concepts regarding these aspects of the molecular virology of CMV and the potential pathogenic impact of viral gene expression on the developing fetus. Areas for potential development of novel therapeutic intervention are suggested for improving the outcome of this disabling congenital infection.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Complicações Infecciosas na Gravidez/virologia , Animais , Citomegalovirus/genética , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/imunologia , Feminino , Doenças Fetais/virologia , Regulação Viral da Expressão Gênica , Humanos , Transmissão Vertical de Doenças Infecciosas , Gravidez , Estados Unidos/epidemiologia , Proteínas ViraisAssuntos
Antivirais/uso terapêutico , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/administração & dosagem , Citomegalovirus/imunologia , Transplante de Órgãos , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Citomegalovirus/crescimento & desenvolvimento , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/etiologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoglobulinas/uso terapêutico , Imunossupressores/efeitos adversos , Interferon gama/biossíntese , Viremia/diagnósticoRESUMO
Congenital cytomegalovirus (CMV) infection is the leading cause of sensorineural hearing loss (SNHL) in children. During murine (M)CMV-induced encephalitis, the immune response is important for both the control of viral dissemination and the clearance of virus from the brain. While the importance of CMV-induced SNHL has been described, the mechanisms surrounding its pathogenesis and the role of inflammatory responses remain unclear. This study presents a neonatal mouse model of profound SNHL in which MCMV preferentially infected both cochlear perilymphatic epithelial cells and spiral ganglion neurons. Interestingly, MCMV infection induced cochlear hair cell death by 21 days post-infection, despite a clear lack of direct infection of hair cells and the complete clearance of the virus from the cochlea by 14 dpi. Flow cytometric, immunohistochemical, and quantitative PCR analysis of MCMV-infected cochlea revealed a robust and chronic inflammatory response, including a prolonged increase in reactive oxygen species production by infiltrating macrophages. These data support a pivotal role for inflammation during MCMV-induced SNHL.