Cytomegalovirus vaccine development.

Although infection with human cytomegalovirus (HCMV) is ubiquitous and usually asymptomatic, there are individuals at high risk for serious HCMV disease. These include solid organ and hematopoietic stem cell (HSC) transplant patients, individuals with HIV infection, and the fetus. Since immunity to HCMV ameliorates the severity of disease, there have been efforts made for over 30 years to develop vaccines for use in these high-risk settings. However, in spite of these efforts, no HCMV vaccine appears to be approaching imminent licensure. The reasons for the failure to achieve the goal of a licensed HCMV vaccine are complex, but several key problems stand out. First, the host immune correlates of protective immunity are not yet clear. Secondly, the viral proteins that should be included in a HCMV vaccine are uncertain. Third, clinical trials have largely focused on immunocompromised patients, a population that may not be relevant to the problem of protection of the fetus against congenital infection. Fourth, the ultimate target population for HCMV vaccination remains unclear. Finally, and most importantly, there has been insufficient education about the problem of HCMV infection, particularly among women of child-bearing age and in the lay public. This review considers the strategies that have been explored to date in development of HCMV vaccines, and summarizes both active clinical trials as well as novel technologies that merit future consideration toward the goal of prevention of this significant public health problem.

Cytomegalovirus Vaccines: Current Status and Future Prospects.

Congenital human cytomegalovirus (HCMV) infection can result in severe and permanent neurological injury in newborns, and vaccine development is accordingly a major public health priority. HCMV can also cause disease in solid organ transplant (SOT) and hematopoietic stem-cell transplant (HSCT) recipients, and a vaccine would be valuable in prevention of viremia and end-organ disease in these populations. Currently there is no licensed HCMV vaccine, but progress toward this goal has been made in recent clinical trials. A recombinant HCMV glycoprotein B (gB) vaccine has been shown to have some efficacy in prevention of infection in young women and adolescents, and has provided benefit to HCMV-seronegative SOT recipients. Similarly, DNA vaccines based on gB and the immunodominant T-cell target, pp65 (ppUL83), have been shown to reduce viremia in HSCT patients. This review provides an overview of HCMV vaccine candidates in various stages of development, as well as an update on the current status of ongoing clinical trials. Protective correlates of vaccine-induced immunity may be different for pregnant woman and transplant patients. As more knowledge emerges about correlates of protection, the ultimate licensure of HCMV vaccines may reflect the uniqueness of the target populations being immunized.

Immunogenicity evaluation of DNA vaccines that target guinea pig cytomegalovirus proteins glycoprotein B and UL83.

Vaccines are needed for control of congenital human cytomegalovirus (HCMV) infection. Although the species-specificity of cytomegaloviruses precludes preclinical evaluation of HCMV vaccines in animal models, the guinea pig cytomegalovirus (GPCMV), which causes disease in utero, is a relevant model for the study of vaccines against congenital infection. We investigated whether DNA vaccines that target two GPCMV proteins, glycoprotein B (gB) and UL83 (pp65), are capable of eliciting immune responses in vivo. After cloning each gene into an expression vector, DNA was delivered by intramuscular inoculation and by pneumatic epidermal delivery. In Swiss-Webster mice, anti-gB titers were significantly higher after epidermal delivery. After epidermal inoculation in guinea pigs, all gB-immunized animals (n = 6) had antibody responses comparable to those induced by natural infection. Viral neutralization titers ranged from 1:64 to greater than 1:128. A GPCMV UL83 DNA vaccine also elicited an antibody response in all immunized guinea pigs (n = 6) after epidermal administration. Immunoprecipitation and Western blot assays confirmed that immune sera were immunoreactive with virion-associated UL83 and gB proteins. We conclude that DNA vaccines against GPCMV structural proteins are immunogenic, and warrant further investigation in the guinea pig model of congenital CMV infection.

Non-immune hydrops fetalis caused by herpes simplex virus type 2 in the setting of recurrent maternal infection.

We report a case of non-immune hydrops fetalis (NIHF) caused by herpes simplex virus type 2 (HSV-2) in an infant whose mother had recurrent HSV-2 infection. In spite of prematurity, severe disseminated infection and hydrops, the infant survived and was neurologically intact. HSV-2-induced NIHF is extremely rare, particularly in the setting of recurrent maternal infection, and this case is, to our knowledge, the first report of a surviving infant. HSV-2 should be considered in the differential diagnosis of NIHF and early initiation of empiric acyclovir therapy is recommended in this setting, pending the results of virologic diagnostic tests.

Emergence of antiviral resistance during oral valganciclovir treatment of an infant with congenital cytomegalovirus (CMV) infection.

Congenital infection with human cytomegalovirus (CMV) is a major cause of morbidity, including sensorineural hearing loss (SNHL), in newborns. Antiviral therapy with ganciclovir (GCV) and its oral prodrug, valganciclovir (VAL-GCV) are increasingly being administered to infected infants, toward the goal of improving neurodevelopmental and auditory outcomes. In this case report, we describe a symptomatic congenitally infected infant treated with VAL-GCV in whom GCV resistance was suspected, based on a 50-fold increase in viral load after 6 weeks of oral therapy. Analyses of CMV sequences from both blood and urine demonstrated populations of viruses with M460V and L595F mutations in the UL97 phosphotransferase gene. In contrast, analysis of viral DNA retrieved from the newborn dried blood spot demonstrated wild-type UL97 sequences. DNAemia resolved after the discontinuation of VAL-GCV. Long-term VAL-GCV therapy in congenitally infected infants can select for resistant viral variants, and anticipatory virological monitoring may be warranted.

Guinea pig cytomegalovirus GP84 is a functional homolog of the human cytomegalovirus (HCMV) UL84 gene that can complement for the loss of UL84 in a chimeric HCMV.

The guinea pig cytomegalovirus (GPCMV) co-linear gene and potential functional homolog of HCMV UL84 (GP84) was investigated. The GP84 gene had delayed early transcription kinetics and transient expression studies of GP84 protein (pGP84) demonstrated that it targeted the nucleus and co-localized with the viral DNA polymerase accessory protein as described for HCMV pUL84. Additionally, pGP84 exhibited a transdominant inhibitory effect on viral growth as described for HCMV. The inhibitory domain could be localized to a minimal peptide sequence of 99 aa. Knockout of GP84 generated virus with greatly impaired growth kinetics. Lastly, the GP84 ORF was capable of complementing for the loss of the UL84 coding sequence in a chimeric HCMV. Based on this research and previous studies we conclude that GPCMV is similar to HCMV by encoding single copy co-linear functional homologs of HCMV UL82 (pp71), UL83 (pp65) and UL84 genes.

Sequence and transcriptional analysis of the guinea-pig cytomegalovirus DNA polymerase gene.

Although the guinea-pig cytomegalovirus (GPCMV) displays a similar pathogenesis to human cytomegalovirus (HCMV), there have unfortunately been few molecular analyses of the GPCMV genome to date. The guinea-pig has proved useful for the testing of drugs active against CMV infection, and insights derived from characterization of the specific virally encoded molecular targets of antiviral therapies would allow this model system to be more fully developed. Because the DNA polymerase serves as an important target for nucleoside antiviral agents active against herpesviruses, experiments were undertaken to identify, clone and sequence the GPCMV DNA polymerase gene (pol). A 3285 bp ORF capable of encoding a 1094 amino acid protein was identified spanning portions of the HindIII Q and P fragments of the genome. This ORF contained extensive homology to other herpesvirus DNA pol genes. Northern blot analyses identified two 3' coterminal pol-specific mRNAs of 3.9 and 1.9 kb at early times post-infection. Primer extension and nuclease protection analyses mapped the 5' end of the 3.9 kb transcript to a site 275 bases upstream of the pol initiation codon. Comparison of the GPCMV pol-encoded sequence to those of other herpesvirus polymerases identified non-conservative amino acid substitutions in a domain involved in substrate recognition.

Cloning and characterization of the guinea pig cytomegalovirus glycoprotein B gene.

Unlike other small animals, cytomegalovirus (CMV) infection of the guinea pig results in transplacental passage and intrauterine infection of the fetus. These features make the guinea pig model ideal for studying vaccine strategies designed to prevent congenital infection. Unfortunately, little is known about immunogenic guinea pig CMV gene products. In other animal cytomegaloviruses, a major target of the host immune response is the glycoprotein B (gB, gp UL 55) gene product. Using DNA probes containing human CMV gB sequences, the gB gene homolog of the guinea pig cytomegalovirus was identified, cloned, and sequenced. The gpCMV gB gene maps to a region spanning portions of the HindIII K, Q, and P fragments of the gpCMV genome. DNA sequence analysis identified an open reading frame of 2706 nucleotides capable of encoding a protein of 901 amino acids. Extensive similarity to the human and murine gB proteins was noted with 42% identity at the amino acid level. The predominant gpCMV gB mRNA is a 6.8-kb transcript with the expression kinetics of an early gene. RNase protection and primer extension analyses indicated that gB mRNAs were transcribed from two different initiation sites corresponding to distinct TATA elements. Polyclonal antisera prepared against a synthetic peptide derived from amino acid sequences within the ORF identified a 58-kDa virion-associated protein representing the cleaved COOH-terminus (gp 58) of the gpCMV gB molecule. The molecular characterization of gpCMV gB should facilitate studies of vaccine strategies in the guinea pig model of congenital CMV infection.

Recent advances in herpesvirus genetics using bacterial artificial chromosomes.

Although the members of the Herpesvirus family are responsible for a wide variety of human diseases, advances in the understanding of viral molecular mechanisms of pathogenesis have been hampered by the large size of herpesvirus genomes, rendering the viruses difficult to experimentally manipulate. Better techniques have been needed to facilitate mutagenesis of herpesvirus genomes, allowing for the assessment of the role of specific viral gene products in replication, immunity, and pathogenesis. Homologous recombination with plasmids containing genes of interest flanked by selectable markers has been a successful method for generating viral mutants, as has the generation of recombinant virus from transfection of cosmid clones. Although these efforts to generate recombinant viruses have met with modest success, the protocols have been cumbersome. More recently, a novel technique for the manipulation of herpesvirus genomes has been developed. This technology utilizes bacterial F plasmids, and allows for the stable cloning of herpesvirus genomes as bacterial artificial chromosomes (BACs) in Escherichia coli. Once cloned, such BACs are stable, and DNA purified from E. coli is infectious, fully capable of reproducing replication-competent virus. Manipulation of herpesvirus genomes is now feasible using the powerful techniques of bacterial genetics, and should facilitate a better understanding of the molecular pathogenesis of herpesvirus infections.

Molecular cloning of the guinea pig cytomegalovirus (GPCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli.

Since cytomegalovirus (CMV) infection is highly species-specific, it is necessary to study animal cytomegaloviruses to assess viral factors which contribute to pathogenesis. The generation of recombinant viruses carrying reporter genes would provide useful tools for studying the genetics of CMV pathogenicity in vivo. We evaluated whether the guinea pig cytomegalovirus (GPCMV) was amenable to such manipulation. Metabolic selection using the guanosylphosphoribosityl transferase (gpt) gene facilitated recovery of a recombinant virus, vAM403, containing a gpt/green fluorescent protein (eGFP) cassette introduced into the HindIII "N" region of the viral genome. This virus had replication kinetics identical to wild-type virus. We next attempted to clone the GPCMV genome as a bacterial artificial chromosome (BAC). A BAC plasmid containing a gpt/eGFP cassette and the chloramphenicol resistance marker was introduced into HindIII "N" to generate another GPCMV recombinant, vAMBGPCMV. Circular viral DNA isolated from vAMBGPCMV-infected cells was used to transform Escherichia coli. Restriction profiles revealed that the GPCMV genome had been cloned as a BAC plasmid, and transfection of BAC plasmid DNA confirmed that the BAC clone was infectious. A novel strategy based on a unique PmeI site was devised to quickly modify the BAC GPCMV plasmid. Recombinants retained the capability to replicate and express reporter genes in guinea pigs, suggesting that these viruses will be useful for in vivo pathogenesis studies.

Sequence and transcriptional analysis of the guinea pig cytomegalovirus UL97 homolog.

Guinea pig cytomegalovirus (GPCMV) displays a similar pathogenesis to human cytomegalovirus (HCMV), and the guinea pig has been used as a model system for testing anti-CMV therapies. However, not all agents active against HCMV share antiviral activity against GPCMV. For example, GPCMV appears resistant to the nucleoside analog, ganciclovir. The molecular basis for this discrepancy in antiviral susceptibility is unknown because to date there has been little analysis of the GPCMV genome. For HCMV, the antiviral effect of ganciclovir depends upon phosphorylation of the drug to its active form. This effect is mediated by the viral UL97 gene product. In order to begin to explore the molecular basis of the resistance of GPCMV to ganciclovir, experiments were undertaken to test whether the GPCMV genome encoded a homolog of the HCMV UL97 gene. Based on the prediction of co-linearity of UL97 homologs within the respective viral genomes, the EcoR I S and F fragments of the GPCMV genome were cloned and partially sequenced. A 1815 base pair open reading frame (ORF) capable of encoding a 604 amino acid (aa) protein was identified spanning portions of the EcoR I S and adjacent EcoR I F genome fragments. Computer-assisted matrix analyses revealed identity between this ORF and the HCMV UL97 gene. ORFs upstream of the GPCMV UL97 gene were identified which shared homology with the HCMV UL95 and 96 genes. Northern blot analyses identified a UL97-specific mRNA of 3.9 kb which was expressed at "early" times post-infection. RNA transcripts of 6.0 and 4.6 kb were identified which corresponded to the UL95 and UL96 homolog coding sequences, respectively. Comparison of the GPCMV UL97 sequence to that of other herpesvirus homologs as well as that of ganciclovir-resistant clinical isolates of HCMV identified nonconservative aa substitutions in two domains involved in catalysis and substrate recognition.

Identification and characterization of the guinea-pig cytomegalovirus glycoprotein H gene.

Subunit vaccines which target viral envelope glycoproteins offer promise for the prevention of congenital cytomegalovirus (CMV) infection. The guinea pig model of CMV infection is uniquely well suited to testing vaccines for prevention of congenital infection, since, in contrast to other animal cytomegaloviruses, the guinea pig CMV (GPCMV) crosses the placenta, producing intrauterine infection. Antibody to the CMV glycoproteins B (gB) and H (gH) appears to be important in conferring protective immunity. Unfortunately, little is known about specific GPCMV envelope glycoproteins. Sequencing of GPCMV genome fragments was therefore undertaken to test whether GPCMV encodes a gH homologue. Partial sequencing of the Hind III A fragment of the GPCMV genome revealed an open reading frame of 2,169 nucleotides capable of encoding a protein of 723 amino acids. Computer matrix analyses demonstrated identity between this ORF and the gH coding sequences of other herpesviruses. The GPCMV gH ORF encodes 12 highly conserved cysteine residues, contains 9 potential N-linked glycosylation sites, and has a predicted M(r) of 81.6 kDa. Northern blot hybridizations with gH-specific probes identified an abundant 5.1 kb mRNA with expression kinetics of an "early" gene. A polyclonal antiserum raised against a synthetic peptide derived from the deduced amino acid sequence of the gH ORF identified a virion-associated protein with an approximate M(r) of 85-kDa, the putative GPCMV gH, in immunoblot assays.

Molecular characterization of the guinea-pig cytomegalovirus glycoprotein L gene.

Although the guinea pig cytomegalovirus (GPCMV) model is well suited to the study of vaccines for prevention of congenital CMV infection, there has been limited molecular characterization of GPCMV glycoproteins. Since the in vivo co-expression of the human cytomegalovirus (HCMV) glycoprotein H (gH, gpUL75) with glycoprotein L (gL, gpUL115) may have relevance to CMV vaccine studies, these experiments were undertaken to test whether the GPCMV encodes a gL homolog. Sequencing of the EcoR I "G" fragment of the GPCMV genome identified an open reading frame (ORF) of 774 nucleotides capable of encoding a protein of 258 amino acids. Computer matrix analyses demonstrated identity between this ORF and the gL coding sequences of other betaherpesviruses. Sequence analysis also identified an ORF with identity to the HCMV uracil DNA glycosylase (UDG, UL114 gene). The GPCMV gL ORF encodes 6 cysteine residues, contains 3 potential N-linked glycosylation sites, and has a predicted Mr of 29.7 kDa. Northern blot studies identified an abundant 2.7 kb "early" transcript from infected cells, the putative gL message. In vitro translation of gL mRNA in reticulocyte lysate resulted in synthesis of 30 kDa polypeptide. A polyclonal antiserum was raised against a gL/glutathione-S-transferase fusion protein generated in E. coli using the pGEX expression system. This antibody identified a 40-kDa virion-associated protein, the putative GPCMV gL, in immunoblot assays.

Translational control of human cytomegalovirus gp48 expression.

Posttranscriptional controls modulate the expression of several human cytomegalovirus genes. Previous studies have shown that one cytomegalovirus gene transcript leader contains AUG codons which inhibit translation of a downstream reading frame. However, two other cytomegalovirus gene transcript leaders of similar structure do not inhibit translation. We have extended these studies to the analysis of the structural glycoprotein gp48, whose predominant transcript contains three upstream AUG codons. The 5' leader of this transcript strongly inhibits downstream translation in fibroblasts. Analyses of deletions and point mutations identify the second upstream AUG codon as an essential component of the inhibitory signal. Other leader sequences, but neither the first nor the third AUG codon, are also required. Intriguingly, the inhibitory signal appears also to depend on the amino acid coding information of the short reading frame associated with the second AUG codon. Insights derived from these studies are germane to understanding the translational regulation of other viral and cellular genes of similar structure.

Recurrent seizures following mucosal application of TAC.

A 5-year-old 20-kg boy developed grand mal seizures following application of 2 mL of tetracaine-adrenalin-cocaine to an oral mucosa laceration. Diazepam 6 mg IV followed by 195 mg phenobarbital was required to terminate the seizures. The patient was transferred to a pediatric intensive care unit for further evaluation and treatment. A toxicology screen obtained after transfer was positive only for diazepam and phenobarbital. The child remained lethargic for several hours but otherwise had a normal neurological examination. Brain computed tomography was normal. Anticonvulsant medication was discontinued prior to discharge and the child had no subsequent seizures.