Inclusion of the Guinea Pig Cytomegalovirus Pentameric Complex in a Live Virus Vaccine Aids Efficacy against Congenital Infection but Is Not Essential for Improving Maternal and Neonatal Outcomes.

The development of a vaccine against congenital human cytomegalovirus (HCMV) infection is a major priority. The pentameric complex (PC) of virion envelope proteins gH, gL, UL128, UL130, and UL131A is a key vaccine target. To determine the importance of immunity to the homologous PC encoded by guinea pig cytomegalovirus (GPCMV) in preventing congenital CMV, PC-intact and PC-deficient live-attenuated vaccines were generated and directly compared for immunogenicity and efficacy against vertical transmission in a vertical transmission model. A virulent PC-intact GPCMV (PC/intact) was modified by galK mutagenesis either to abrogate PC expression (PC/null; containing a frame-shift mutation in GP129, homolog of UL128) or to delete genes encoding three MHC Class I homologs and a protein kinase R (PKR) evasin while retaining the PC (3DX/Δ145). Attenuated vaccines were compared to sham immunization in a two-dose preconception subcutaneous inoculation regimen in GPCMV seronegative Hartley guinea pigs. Vaccines induced transient, low-grade viremia in 5/12 PC/intact-, 2/12 PC/null-, and 1/11 3DX/Δ145-vaccinated animals. Upon completion of the two-dose vaccine series, ELISA titers for the PC/intact group (geometic mean titer (GMT) 13,669) were not significantly different from PC/null (GMT 8127) but were significantly higher than for the 3DX/Δ145 group (GMT 6185; p < 0.01). Dams were challenged with salivary gland-adapted GPCMV in the second trimester. All vaccines conferred protection against maternal viremia. Newborn weights were significantly lower in sham-immunized controls (84.5 ± 2.4 g) compared to PC/intact (96 ± 2.3 g), PC/null (97.6 ± 1.9 g), or 3DX/Δ145 (93 ± 1.7) pups (p < 0.01). Pup mortality in sham-immunized controls was 29/40 (73%) and decreased to 1/44 (2.3%), 2/46 (4.3%), or 4/40 (10%) in PC/intact, PC/null, or 3DX/Δ145 groups, respectively (all p < 0.001 compared to control). Congenital GPCMV transmission occurred in 5/44 (11%), 16/46 (35%), or 29/38 (76%) of pups in PC/intact, PC/null, or 3DX/Δ145 groups, versus 36/40 (90%) in controls. For infected pups, viral loads were lower in pups born to vaccinated dams compared to controls. Sequence analysis demonstrated that infected pups in the vaccine groups had salivary gland-adapted GPCMV and not vaccine strain-specific sequences, indicating that congenital transmission was due to the challenge virus and not vaccine virus. We conclude that inclusion of the PC in a live, attenuated preconception vaccine improves immunogenicity and reduces vertical transmission, but PC-null vaccines are equal to PC-intact vaccines in reducing maternal viremia and protecting against GPCMV-related pup mortality.

A Fully Protective Congenital CMV Vaccine Requires Neutralizing Antibodies to Viral Pentamer and gB Glycoprotein Complexes but a pp65 T-Cell Response Is Not Necessary.

A vaccine against congenital cytomegalovirus infection is a high priority. Guinea pig cytomegalovirus (GPCMV) is the only congenital CMV small animal model. GPCMV encodes essential glycoprotein complexes for virus entry (gB, gH/gL/gO, gM/gN) including a pentamer complex (gH/gL/GP129/GP131/GP133 or PC) for endocytic cell entry. The cohorts for protection against congenital CMV are poorly defined. Neutralizing antibodies to the viral glycoprotein complexes are potentially more important than an immunodominant T-cell response to the pp65 protein. In GPCMV, GP83 (pp65 homolog) is an evasion factor, and the GP83 mutant GPCMV has increased sensitivity to type I interferon. Although GP83 induces a cell-mediated response, a GP83-only-based vaccine strategy has limited efficacy. GPCMV attenuation via GP83 null deletion mutant in glycoprotein PC positive or negative virus was evaluated as live-attenuated vaccine strains (GP83dPC+/PC-). Vaccinated animals induced antibodies to viral glycoprotein complexes, and PC+ vaccinated animals had sterilizing immunity against wtGPCMV challenge. In a pre-conception vaccine (GP83dPC+) study, dams challenged mid-2nd trimester with wtGPCMV had complete protection against congenital CMV infection without detectable virus in pups. An unvaccinated control group had 80% pup transmission rate. Overall, gB and PC antibodies are key for protection against congenital CMV infection, but a response to pp65 is not strictly necessary.

Analysis of the origin of inherited chromosomally integrated human herpesvirus 6 in the Japanese population.

Integration of the complete human herpesvirus 6 (HHV-6) genome into the telomere of a chromosome has been reported in some individuals (inherited chromosomally integrated HHV-6; iciHHV-6). Since the proportion of iciHHV-6-positive individuals with integration in chromosome 22 is high in Japan, we hypothesized a founder effect. In this study, we sought to elucidate the reason for the high proportion of viral integrations into chromosome 22. We analyzed six cases of iciHHV-6A and two cases of iciHHV-6B, including one iciHHV-6A case with a matched sample from a father and one iciHHV-6B case with a matched sample from a mother. In iciHHV-6A, the same copy numbers of viral telomeric repeat sequences (TRS) and the same five microsatellite markers were detected in both the index case and paternal sample. Moreover, the same five microsatellite markers were demonstrated in four cases and the same copy numbers of viral TRS were demonstrated in two pairs of two cases. The present microsatellite analysis suggested that the viral genomes detected in some iciHHV-6A patients were derived from a common ancestral integration.

A Novel Non-Replication-Competent Cytomegalovirus Capsid Mutant Vaccine Strategy Is Effective in Reducing Congenital Infection.

UNLABELLED: Congenital cytomegalovirus (CMV) infection is a leading cause of mental retardation and deafness in newborns. The guinea pig is the only small animal model for congenital CMV infection. A novel CMV vaccine was investigated as an intervention strategy against congenital guinea pig cytomegalovirus (GPCMV) infection. In this disabled infectious single-cycle (DISC) vaccine strategy, a GPCMV mutant virus was used that lacked the ability to express an essential capsid gene (the UL85 homolog GP85) except when grown on a complementing cell line. In vaccinated animals, the GP85 mutant virus (GP85 DISC) induced an antibody response to important glycoprotein complexes considered neutralizing target antigens (gB, gH/gL/gO, and gM/gN). The vaccine also generated a T cell response to the pp65 homolog (GP83), determined via a newly established guinea pig gamma interferon enzyme-linked immunosorbent spot assay. In a congenital infection protection study, GP85 DISC-vaccinated animals and a nonvaccinated control group were challenged during pregnancy with wild-type GPCMV (10(5) PFU). The pregnant animals carried the pups to term, and viral loads in target organs of pups were analyzed. Based on live pup births in the vaccinated and control groups (94.1% versus 63.6%), the vaccine was successful in reducing mortality (P = 0.0002). Additionally, pups from the vaccinated group had reduced CMV transmission, with 23.5% infected target organs versus 75.9% in the control group. Overall, these preliminary studies indicate that a DISC CMV vaccine strategy has the ability to induce an immune response similar to that of natural virus infection but has the increased safety of a non-replication-competent virus, which makes this approach attractive as a CMV vaccine strategy. IMPORTANCE: Congenital CMV infection is a leading cause of mental retardation and deafness in newborns. An effective vaccine against CMV remains an elusive goal despite over 50 years of CMV research. The guinea pig, with a placenta structure similar to that in humans, is the only small animal model for congenital CMV infection and recapitulates disease symptoms (e.g., deafness) in newborn pups. In this report, a novel vaccine strategy against congenital guinea pig cytomegalovirus (GPCMV) infection was developed, characterized, and tested for efficacy. This disabled infectious single-cycle (DISC) vaccine strategy induced a neutralizing antibody or a T cell response to important target antigens. In a congenital infection protection study, animals were protected against CMV in comparison to the nonvaccinated group (52% reduction of transmission). This novel vaccine was more effective than previously tested gB-based vaccines and most other strategies involving live virus vaccines. Overall, the DISC vaccine is a safe and promising approach against congenital CMV infection.

Clinical and laboratory features of human herpesvirus 6 chromosomal integration.

Human herpesvirus 6 (HHV-6) comprises two separate viruses, HHV-6A and HHV-6B, although this distinction is not commonly made. HHV-6B is ubiquitous in the population with primary infection usually occurring in early childhood, and often resulting in febrile illness. HHV-6B is also recognized as a pathogen in the immunocompromised host, particularly in transplant recipients. HHV-6A is less well characterized and may have a more restricted prevalence. Both viruses are unique among the human herpesviruses in that the entire viral genome can be found integrated into the telomeric regions of host cell chromosomes. Approximately 1% of persons have inherited integrated viral sequences through the germline, and these individuals characteristically have very high viral loads in blood and other sample types. Emerging evidence suggests that HHV-6A and HHV-6B chromosomal integration may not just be an uncommon biological observation, but more likely a characteristic of the replication properties of these viruses. The integrated viral genome appears capable of excision from the chromosomal site and potentially allows viral replication. The clinical consequences of inherited chromosomally integrated HHV-6 have yet to be fully appreciated.

Early developmental outcomes of children with congenital HHV-6 infection.

OBJECTIVE: The goal of this study was to determine if congenital human herpesvirus-6 (HHV-6) infection influences early neurodevelopment. METHODS: We enrolled 57 newborns with HHV-6 congenital infection and 242 control newborns without congenital infection into a prospective, double-blind study with 4 visits between 4 and 30 months of age. Assessments included the Fagan Test of Infant Intelligence, the Visual Expectation Paradigm, and the Mental Development Index (MDI) of the Bayley Scales of Infant Development II. Newborn audiology screening and follow-up audiology examinations were completed at 12 to 24 months. RESULTS: No differences were noted in baseline characteristics between infants with HHV-6 congenital infection and control infants. No clinical syndrome due to congenital infection with HHV-6 was evident at birth. No differences were identified on the Fagan Test of Infant Intelligence or the Visual Expectation Paradigm between the two groups. In 39 infants with HHV-6 congenital infection, the mean ± SD Bayley Scale of Infant Development II MDI score was 103.4 ± 8.9 at 12 months of age. The matched control infants had a mean score of 105.4 ± 12.4. After controlling for covariates, HHV-6 congenital infection was associated with lower scores on the Bayley Scale of Infant Development II MDI at 12 months of age (mean difference: 4.3 [95% confidence interval: 0.4 to 8.1]; P = .03) compared with infants without HHV-6 congenital infection. CONCLUSIONS: Congenital HHV-6 infection may have a detrimental effect on neurodevelopment at 12 months of age and requires further study given that congenital infection with HHV-6 is present in ∼1 in every 101 births.

Infección congenital por herpervirus humano 6 asociado a convulsión neonatal. Comunicación de un nuevo caso / Congenital infection due to human herpes virus 6 associated to neonatal seizure. Report of a new case

Introducción. La infección congénita por HHV-6 se produce en el 1% de los recién nacidos, como consecuencia de una reactivación del virus durante la gestación a través de la placenta. Los recién nacidos afectos pueden estar asintomáticos o presentar convulsiones afebriles y encefalitis así como hepatitis fulminante. Caso clínico. Se presenta un caso de convulsión neonatal debida a una infección congénita por HHV-6 por hallazgo de DNA viral mediante PCR específica en LCR durante las primeras 24 horas de vida, que se confirmó posteriormente en sangre materna y del neonato. Las convulsiones se controlaron con fenobarbital, siendo favorable hasta el momento la evolución sin desarrollo de secuelas neurológicas. Comentarios. Se debe realizar PCR para herpesvirus en LCR ante todo neonato con convulsiones afebriles y/o alteraciones neurológicas posteriores por ser de gran utilidad para el diagnóstico de estas afecciones (AU)

Introduction. The congenital infection with HHV-6 is produced in 1% of the newborns, because of a virus reactivation during the pregnancy by the placenta. The affected newborns can be asymptomatics or they can present afebrile seizures and encephalitis or fulminant hepatitis. Clinic case. We present a case of neonatal seizure due to an congenital infection with HHV-6 for the find of HHV-6 DNA by specific PCR in CSF in the first twenty hours of life, then it was confirmed in mother and baby bloods. The seizures were controlled with phenobarbital and the progress is favourable in the present moment without neurological sequelae. Discussion. Herpesvirus PCR in CSF must be made in all newborn with afebrile seizures and/or following neurological alterations and it can be useful to the diagnosis of these disease (AU)

Transplacental congenital human herpesvirus 6 infection caused by maternal chromosomally integrated virus.

Congenital human herpesvirus 6 (HHV-6) infection results from germline passage of chromosomally integrated HHV-6 (CI-HHV-6) and from transplacental passage of maternal HHV-6 infection. We aimed to determine whether CI-HHV-6 could replicate and cause transplacentally acquired HHV-6 infection. HHV-6 DNA, variant type, and viral loads were determined with samples (cord blood, peripheral blood, saliva, urine, and hair) obtained from 6 infants with transplacentally acquired HHV-6 and with samples of their parents' hair. No fathers but all mothers of infants with transplacentally acquired HHV-6 had CI-HHV-6, and the mother's CI-HHV-6 variant was the same variant causing the transplacentally acquired congenital HHV-6 infection. This suggests the possibility that CI-HHV-6 replicates and may cause most, if not all, congenital HHV-6 infections.

Chromosomal integration of human herpesvirus 6 is the major mode of congenital human herpesvirus 6 infection.

OBJECTIVE: We examined the frequency and characteristics of chromosomally integrated human herpesvirus 6 among congenitally infected children. METHODS: Infants with and without congenital human herpesvirus 6 infection were prospectively monitored. Cord blood mononuclear cell, peripheral blood mononuclear cell, saliva, urine, and hair follicle samples were examined for human herpesvirus 6 DNA. Human herpesvirus 6 RNA, serum antibody, and chromosomally integrated human herpesvirus 6 levels were also assessed. RESULTS: Among 85 infants, 43 had congenital infections and 42 had postnatal infections. Most congenital infections (86%) resulted from chromosomally integrated human herpesvirus 6; 6 infants (14%) had transplacental infections. Children with chromosomally integrated human herpesvirus 6 had high viral loads in all sites (mean: 5-6 log(10) genomic copies per mug of cellular DNA); among children with transplacental infection or postnatal infection, human herpesvirus 6 DNA was absent in hair samples and inconsistent in other samples, and viral loads were significantly lower. One parent of each child with chromosomally integrated human herpesvirus 6 who had parental hair samples tested had hair containing human herpesvirus 6 DNA. Variant A caused 32% of chromosomally integrated human herpesvirus 6 infections, compared with 2% of postnatal infections. Replicating human herpesvirus 6 was detected only among chromosomally integrated human herpesvirus 6 samples (8% of cord blood mononuclear cells and peripheral blood mononuclear cells). Cord blood human herpesvirus 6 antibody levels were similar among children with chromosomally integrated human herpesvirus 6, transplacental infection, and postnatal infection and between children with maternal and paternal chromosomally integrated human herpesvirus 6 transmission. CONCLUSIONS: Human herpesvirus 6 congenital infection results primarily from chromosomally integrated virus which is passed through the germ-line. Infants with chromosomally integrated human herpesvirus 6 had high viral loads in all specimens, produced human herpesvirus 6 antibody, and mRNA. The clinical relevance needs study as 1 of 116 newborns may have chromosomally integrated human herpesvirus 6 blood specimens.

Cyclic cidofovir (cHPMPC) prevents congenital cytomegalovirus infection in a guinea pig model.

BACKGROUND: Congenital cytomegalovirus (CMV) infection is a major public health problem. Antiviral therapies administered during pregnancy might prevent vertical CMV transmission and disease in newborns, but these agents have not been evaluated in clinical trials. The guinea pig model of congenital CMV infection was therefore used to test the hypothesis that antiviral therapy, using the agent agent cyclic cidofovir (cHPMPC), could prevent congenital CMV infection. RESULTS: Pregnant outbred Hartley guinea pigs were challenged in the early-third trimester with guinea pig CMV (GPCMV) and treated with placebo, or the antiviral agent, cyclic cidofovir. To optimize detection of vertical infection, an enhanced green fluorescent protein (eGFP)-tagged virus was employed. Compared to placebo, cyclic cidofovir-treated dams and pups had reduced mortality following GPCMV challenge. The magnitude of GPCMV-induced maternal and fetal mortality in this study was reduced from 5/25 animals in the placebo group to 0/21 animals in the treatment group (p = 0.05, Fisher's exact test). By viral culture assay, antiviral therapy was found to completely prevent GPCMV transmission to the fetus. In control pups, 5/19 (26%) were culture-positive for GPCMV, compared to 0/16 of pups in the cyclic cidofovir treatment group (p < 0.05, Fisher's exact test). CONCLUSION: Antiviral therapy with cyclic cidofovir improves pregnancy outcomes in guinea pigs, and eliminates congenital CMV infection, following viral challenge in the third trimester. This study also demonstrated that an eGFP-tagged recombinant virus, with the reporter gene inserted into a dispensable region of the viral genome, retained virulence, including the potential for congenital transmission, facilitating tissue culture-based detection of congenital infection. These observations provide support for clinical trials of antivirals for reduction of congenital CMV infection.

Human herpesviruses-6 and -7 infections.

PURPOSE OF REVIEW: To summarize the biology and clinical consequences of infection with the closely related human herpesviruses-6 and -7 (HHV-6/7) in children. RECENT FINDINGS: Over the last year there has been a paucity of paediatric publications on HHV-6 and only two studies focused on HHV-7. Steady progress has been made regarding the biology and clinical consequences of HHV-6 infection whereas the effect of HHV-7 infection remains a neglected topic. However, both viruses have been shown to contribute significantly and equally to the burden of disease in young children with suspected encephalitis or severe convulsions with fever. There continues to be uncertainty as to the effects of HHV-6 infection after stem cell transplant, although there is general agreement that it contributes to encephalitis. In contrast, HHV-7 seems to have little clinical impact after stem cell transplant, although central nervous system infection and disease have recently been reported in children. Understanding the contribution of chromosomal integration and inheritance of both HHV-6 variants A and B (HHV-6A/B) and their effect on diagnosis is emerging. SUMMARY: There is an urgent need for more research on HHV-6 and -7 in children, particularly in relation to chromosomal integration of HHV-6A and B, and clinical consequences of HHV-7 infection.

Congenital infections with human herpesvirus 6 (HHV6) and human herpesvirus 7 (HHV7).

OBJECTIVE: To examine whether: (1) congenital human herpesvirus 6 (HHV6) and human herpesvirus 7 (HHV7) infections occur; whether (2) their manifestations differ from postnatal infections; and whether (3) HHV6 and HHV7 infections differ despite their close relatedness. STUDY DESIGN: HHV6 and HHV7 infections acquired congenitally and postnatally in normal children were compared using viral isolation, serology, reverse-transcription polymerase chain reaction (RT-PCR) and nested DNA-PCR for HHV6 variant A (HHV6A), HHV6 variant B (HHV6B), and HHV7. RESULTS: HHV6 DNA was detected in 57 (1%) of 5638 cord bloods. HHV7 DNA, however, was not detected in 2129 cord bloods. Congenital HHV6 infections differed from postnatal infections, which were acute febrile illnesses. Congenital infections were asymptomatic, 10% demonstrated reactivation at birth, and HHV6 DNA persistence in follow-up blood samples was significantly more frequent. One-third of congenital infections were HHV6A, whereas all postnatal infections were HHV6B. CONCLUSIONS: Congenital HHV6 infections occurred in 1% of births, similar to the rate for cytomegalovirus infection. Congenital infections were clinically and virologically distinct from postnatal infections. Congenital HHV7 infections, however, were not detected, suggesting considerable differences in transmission and pathogenesis in these closely related beta-herpesviruses.