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.

Assessing the potential of using telecommunication microwave links in urban drainage modelling.

The ability to predict the runoff response of an urban catchment to rainfall is crucial for managing drainage systems effectively and controlling discharges from urban areas. In this paper we assess the potential of commercial microwave links (MWL) to capture the spatio-temporal rainfall dynamics and thus improve urban rainfall-runoff modelling. Specifically, we perform numerical experiments with virtual rainfall fields and compare the results of MWL rainfall reconstructions to those of rain gauge (RG) observations. In a case study, we are able to show that MWL networks in urban areas are sufficiently dense to provide good information on spatio-temporal rainfall variability and can thus considerably improve pipe flow prediction, even in small subcatchments. In addition, the better spatial coverage also improves the control of discharges from urban areas. This is especially beneficial for heavy rainfall, which usually has a high spatial variability that cannot be accurately captured by RG point measurements.

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.

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.

Aerosol-derived airway morphometry (ADAM) in patients with lung emphysema diagnosed by computed tomography--reproducibility, diagnostic information and modelling.

Gravitational deposition of monodisperse particles can be used to determine effective airway diameter (EAD). The aim of our study was to assess intraindividual variability of EAD in healthy subjects and patients with emphysema, to compare EAD in patients with different degree and type of emphysema, and to evaluate whether parametric or model analysis would improve the results. EAD was measured vs volumetric lung depth (LD) in 11 healthy subjects (FEV subset1 107%pred) and 41 patients with emphysema (FEV subset1 60%pred; 8/9/24 mild/moderate/severe, 18/7/16 centriacinar/panacinar/bullous according to HRCT). Repeated measurements in LDs of 6-30% showed coefficients of variation of 7.0-10.4% in healthy subjects and 8.3-11.9% in emphysema. Average EAD in 10-16% LD was increased in emphysema, in particular moderate and severe (p<0.05, each). The slope of EAD in 10-16% LD differed between healthy subjects and emphysema, especially bullous and centriacinar. Patients with severe emphysema also showed a different slope compared to mild emphysema and controls. The parameters of the power function used for data fitting also showed differences between controls and emphysema, as well as between centriacinar vs panacinar and bullous emphysema. In a three-compartment lung model only the diameter of the intermediate compartment was enlarged in emphysema. We conclude that in using aerosol-derived airway morphometry, reproducibility of repeated measurements is acceptable. Average values and slopes of the EAD curve, as well as a power function for data fitting, were sensitive in the detection of type and severity of emphysema. In contrast, application of a lung model did not improve the results.

Dose reduction of inhaled corticosteroids under concomitant medication with montelukast in patients with asthma.

The present study aimed at comparing the effects of a dose reduction of inhaled corticosteroids on lung function, indirect measures of airway inflammation and clinical scores during treatment with a leucotriene receptor antagonist. In 50 patients (mean forced expiratory volume in one second (FEV1) 94% predicted), steroid doses (800 microg beclomethasone dipropionate) were first reduced to 50% and then to 25%, for 6 weeks each. One group received a placebo and the other group received montelukast (10 mg). The first reduction did not cause significant effects. During the second, FEV1 and peak expiratory flow decreased in both groups (p<0.001). Daytime symptoms were not altered with placebo but were reduced by montelukast (p<0.05). Night-time symptoms were slightly elevated with placebo (p<0.05) but not montelukast, as well as the use of supplemental salbutamol. Changes in provocative concentration of methacholine causing a 20% fall in FEV1 (PC20), sputum eosinophils and exhaled nitric oxide were mostly nonsignificant for both placebo and montelukast. These data demonstrate that a 75% reduction in the dose of steroid given to patients with asthma led to a deterioration in lung function not prevented by montelukast, whereas changes in clinical state seemed to favour montelukast treatment. It therefore appears that potential effects of montelukast, in the presence of low-dose steroids, could not be attributed to single indices of lung function or airway inflammation.

In patients with chronic bronchitis a four week trial with inhaled steroids does not attenuate airway inflammation.

Systemic corticosteroids have been recommended as a therapeutic option in patients with moderate to severe COPD. In an early stage of the disease, i.e. chronic bronchitis with mild or no airflow obstruction, a trial with inhaled steroids could reveal potential benefits, particularly in terms of a modulation of airway inflammation. We therefore investigated the effect of inhaled fluticasone (1000 microg day(-1)) on markers of airway inflammation in 19 patients with chronic bronchitis (mean+/-SEM FEV1, 83.4+/-3.0% predicted; FEV1/VC, 67.5+/-2.4%) in a double-blind, cross-over, placebo-controlled manner. Visits were performed before and after two 4-week treatment periods. separated by a 4-week washout period. Lung function, the concentration of exhaled nitric oxide, differential cell counts in induced sputum and the number of cells positive for iNOS, as well as the levels of LDH, ECP, neutrophil elastase and IL-8 in sputum supernatants were determined. Although the total cell number decreased significantly after fluticasone (geometric mean 12.3 vs. 7.7 x 10(6)/ml; P<0.05) it was not significantly different from the change observed after placebo (14.2 vs. 10.6 x 10(6)/ml; n.s.). None of the other parameters showed statistically significant changes after fluticasone or placebo and the results did not depend on the presence of airway hyperresponsiveness. We conclude that in patients with chronic bronchitis short-term treatment with inhaled corticosterids did not improve lung function or inflammatory parameters to an extent which was statistically significant as compared to spontaneous variability.

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.

Preconception immunization with a cytomegalovirus (CMV) glycoprotein vaccine improves pregnancy outcome in a guinea pig model of congenital CMV infection.

The guinea pig (gp) model of congenital cytomegalovirus (CMV) infection was used to evaluate a gpCMV glycoprotein vaccine. Hartley guinea pigs were immunized 3 times with 50 microg of lectin column-purified glycoproteins prepared from gpCMV-infected or -uninfected tissue culture. Immunization with the gpCMV vaccine produced seroconversion in all animals. Animals then were placed with gpCMV-seronegative male animals and were challenged late in pregnancy with virulent salivary gland-passaged gpCMV. Immunization with gpCMV glycoproteins significantly improved pregnancy outcome, with 54 of 63 pups live-born in immunized animals, compared with 21 of 48 in the controls (P<.001). In addition, virus was isolated from 24 of 54 live-born pups born to immunized mothers, compared with 16 of 20 live-born pups born to controls, indicating that immunization significantly reduced in utero transmission in surviving animals (P<.01).

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.

The concentration of hydrogen peroxide in exhaled air depends on expiratory flow rate.

Hydrogen peroxide (H2O2) is known to be detectable in exhaled air. The present study aimed to determine whether the concentration of exhaled H2O2 depends on expiratory flow rate in order to make inferences on the site of its production within the lung. Breath condensate was collected in cooled Teflon tubes, at three different expiratorv flow rates, in 15 healthy or mild asthmatic subjects. Tests were repeated 2-5 times to assess reproducibility. Mean+/-SEM concentrations of H2O2 at flow rates of 140, 69 and 48 mL.s(-1) were 0.12+/-0.02, 0.19+/-0.02 and 0.32+/-0.03 microM, respectively. These values differed significantly from each other (p<0.001). For comparison, average coefficients of variability within repeated measurements at each of the three flow rates were 68, 62 and 82%, respectively. These data demonstrate that the concentration of exhaled hydrogen peroxide depends on expiratory flow rate. Since flow dependence is an indicator of production within the airways, this result suggests that, to a large extent, the exhaled hydrogen peroxide originates within the airways. However, even under strictly controlled conditions, a high degree of variability persists, which may limit the usefulness of exhaled hydrogen peroxide as a marker of airway inflammation.