NUDT15 polymorphism influences the metabolism and therapeutic effects of acyclovir and ganciclovir.

Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects.

The Status of Vaccine Development Against the Human Cytomegalovirus.

Numerous candidate vaccines against cytomegalovirus (CMV) infection and disease are in development. Whereas the previous article [1] provides background and opinions about the issues relating to vaccination, this article provides specifics about the vaccines in active development, as reported at a National Institutes of Health-sponsored meeting in Bethesda on September 4-6, 2018. Here, vaccine developers provide synopses of their candidate vaccines to immunize women to protect against congenital CMV disease and to prevent the consequences of CMV disease in recipients of transplanted organs or hematopoietic stem calls. The projects are presented here roughly in the descending order of their stage of development in the opinion of the first author.

Application of a New Paradigm for Cytomegalovirus Disease Prevention in Mayo Clinic's First Face Transplant.

The optimal approach to preventing cytomegalovirus (CMV) disease after face transplant is unknown. We report an individualized hybrid approach, initially using valganciclovir prophylaxis followed by surveillance and preemptive therapy guided by viral and immunologic markers. A 31-year-old man received a near-total face allotransplant for gunshot injury-related severe facial deformities. He was CMV seronegative, and the donor was CMV seropositive (D+/R- mismatch), and he received intravenous ganciclovir followed by oral valganciclovir prophylaxis. Monthly CD8+ T-cell immune competence assay revealed no CMV-specific CD8+ T-cell immunity development during valganciclovir prophylaxis. Because of severe leukopenia, valganciclovir was discontinued at 7 months after transplant when the patient had recovered and sustained normal global CD8+ T-cell function. Weekly CMV polymerase chain reaction testing detected asymptomatic CMV replication (plasma CMV, 549 IU/mL) 3 months later. Short-course preemptive valganciclovir treatment resulted in sustained virologic clearance. The patient had development of CMV-specific CD8+ T cells (12 cells/µL) together with CMV IgM and IgG. No rejection or infection relapse was detected 20 months after transplant. In conclusion, viral and immunologic monitoring allowed for an individualized approach to effective CMV disease prevention after face transplant. This case highlights the importance of understanding the interplay between the host immunity and the pathogen in determining the clinical course and outcome of CMV and other infectious disease syndromes.

Cytomegalovirus (CMV) infection and pregnancy-potential for improvements in Australasian maternity health providers' knowledge.

PURPOSE: To assess the knowledge, practice and attitudes of maternity clinicians regarding congenital cytomegalovirus (CMV). It is the most common congenital infection, and well-recognized cause of neurodevelopmental disability and hearing loss. New consensus recommendations state all pregnant women and health-care providers should be educated about congenital CMV infection and preventive measures. MATERIALS AND METHODS: An email questionnaire was distributed in October 2015 to specialists, diplomates (general practitioners), and trainees of the Royal Australian New Zealand College of Obstetricians and Gynaecologists (RANZCOG), and Victorian and New South Wales midwives. RESULTS: 774 responded: (37.3% specialists, 17.3% diplomates, 16.8% trainees, 28.6% midwives). Clinicians had variable knowledge of fetal sequelae, transmission routes and prevention. Overall, 30.2% felt confident about discussing CMV in pregnancy: less than 10% of midwives (7.4%) and less than half of specialists (47.1%, p < .0001). Only 8.8% of respondents routinely discussed CMV prevention with pregnant women. The majority (69.3%) responded that professional societies should make practice recommendations, and 88% thought more patient information was needed, preferably leaflets. CONCLUSIONS: Australasian maternity clinicians lack confidence and knowledge about congenital CMV. Few (<10%) routinely provide advice on prevention. There is urgent need for clinical guidance and patient information to reduce the burden of disease.

1,25 Dihydroxyvitamin D circulating levels, calcitriol administration, and incidence of acute rejection, CMV infection, and polyoma virus infection in renal transplant recipients.

Observation that 1,25-Dihydroxyvitamin-D3 has an immunomodulatory effect on innate and adaptive immunity raises the possible effect on clinical graft outcome. Aim of this study was to evaluate the correlation of biopsy-proven acute rejection, CMV infection, BKV infection, with 1,25-Dihydroxyvitamin-D3 deficiency and the benefit of calcitriol supplementation before and during the transplantation. Risk factors and kidney graft function were also evaluated. All RTRs received induction therapy with basiliximab, cyclosporine, mycophenolic acid, and steroids. During the first year, the incidence of BPAR (4% vs 11%, P=.04), CMV infection (3% vs 9%, P=.04), and BKV infection (6% vs 19%, P=.04) was significantly lower in users compared to controls. By multivariate Cox regression analysis, 1,25-Dihydroxyvitamin-D3 deficiency and no calcitriol exposure were independent risk factors for BPAR (HR=4.30, P<.005 and HR=3.25, P<.05), for CMV infection (HR=2.33, P<.05 and HR=2.31, P=.001), and for BKV infection (HR=2.41, P<.05 and HR=2.45, P=.001). After one year, users had a better renal function: eGFR was 62.5±6.7 mL/min vs 51.4±7.6 mL/min (P<.05). Only one user developed polyomavirus-associated nephropathy vs 15 controls. Two users lost their graft vs 11 controls. 1,25(OH)2-D3 deficiency circulating levels increased the risk of BPAR, CMV infection, BKV infection after kidney transplantation. Administration of calcitriol is a way to obtain adequate 1,25(OH)2-D3 circulating levels.

Infección sistémica por citomegalovirus: influencia del tratamiento con foscarnet en los niveles plasmáticos de calcio y magnesio / Systemic cytomegalovirus infection: Changes in serum calcium and magnesium levels with foscarnet treatment

La infección por citomegalovirus es frecuente en pacientes trasplantados cardiacos. Foscarnet se utiliza, con evidencia limitada, como tratamiento de segunda línea tras el fracaso de ganciclovir en estos pacientes. Presentamos un caso de alteraciones electrolíticas por foscarnet administrado para el tratamiento de infección por citomegalovirus en un paciente pediátrico trasplantado cardiaco. La infección se resolvió tras 6 semanas de tratamiento, apareciendo niveles de calcio iónico bajos durante la infusión del fármaco e hipomagnesemia mantenida tratada con suplementos, que revirtieron al retirar el fármaco

Cytomegalovirus infection is common in cardiac transplant patients. Foscarnet is used, with limited evidence, as second-line treatment after ganciclovir failure in these patients. We describe the case of a paediatric cardiac transplant patient who developed electrolyte disturbances during foscarnet treatment for cytomegalovirus infection. The infection resolved after 6 weeks of treatment. Low ionized calcium and magnesium levels were observed during the drug infusion, which were treated with supplements. The serum levels reverted to normal after drug withdrawal

Inhibition of cytomegalovirus infection and photothermolysis of infected cells using bioconjugated gold nanoparticles.

Human cytomegalovirus (CMV) is a herpesvirus that causes major health problems in neonates as well as in immunocompromised individuals. At present, a vaccine is not available for CMV infection and the available antiviral drugs suffer from toxicity, poor efficacy and resistance. Here, we chemically conjugated a monoclonal antibody raised against CMV surface glycoprotein (gB) with gold nanoparticles (GNP) and characterized the potential of this gB-GNP conjugate for antiviral activity against CMV. The gB-GNP blocks viral replication, virus-induced cytopathogenic effects and virus spread in cell culture without inducing cytotoxicity. High concentrations of gB-GNP that coat the surface of virus particles block virus entry, whereas lower concentrations block a later stage of virus life cycle. Also, cells treated with gB-GNP gain resistance to CMV infection. In addition, infected cells when bound to gB-GNP can be selectively lysed after exposing them to specific wavelength of laser (nanophotothermolysis). Thus, we have not only designed a potential antiviral strategy that specifically blocks CMV infection at multiple stages of virus life cycle, but we have also characterized a technique that can potentially be useful in eliminating CMV infected cells from donor tissue during transplant or transfusion.

Maternal cytomegalovirus infection prevention: the role of Dutch primary care midwives.

OBJECTIVE: to assess the knowledge of cytomegalovirus (CMV) infection among Dutch primary care midwives, and clinical approaches to informing women about CMV. DESIGN: cross-sectional study, using self-administered questionnaires. PARTICIPANTS: 330 Dutch primary care midwives. SETTING: primary midwifery care practices across the Netherlands. MAIN OUTCOME: Midwives' knowledge of CMV transmission routes and maternal symptoms, and clinical practice behaviours regarding CMV, the information typically provided or reasons for not informing pregnant women about CMV. FINDINGS: the overall median knowledge score was 8.0 out of a maximum possible score of 13.0. Of all participants, 10.6% reported always informing pregnant women about CMV infection prevention and 41.0% reported never informing pregnant women. The main reason indicated for not informing pregnant women was lack of knowledge about preventive methods (45.7%). CONCLUSION: Dutch primary care midwives have limited knowledge of CMV infection. Improvement in providing education to pregnant women about strategies to prevent CMV is necessary.

An email survey of midwives knowledge about CytoMegaloVirus (CMV) in Hannover and a skeletal framework for a proposed teaching program.

At present there is lack of information about CMV transmission given to midwives, general practitioners, neonatal pediatricians and nurses, with intrauterine transmission having profound consequences in terms of outcomes for the infected neonate. To identify one particular group of midwives knowledge about CMV, the research question surveyed midwives' knowledge of CMV. A quantitative electronic survey was the research method utilized in this study. To assess midwives knowledge about CMV, the first author emailed colleagues in Hannover and was in receipt of 40 completed questionnaires. Results showed that midwives have gaps in their knowledge about CMV and that an educational program is necessary to enlarge their understandings. Given the catastrophic consequences to the neonate of contracting congenital CMV, it is imperative that both health care professionals and women receive the educational message about prevention. In response an education program for lecturers has been proposed, which consists of eleven learning objectives.

Cytomegalovirus antivirals and development of improved animal models.

INTRODUCTION: Cytomegalovirus (CMV) is a ubiquitous pathogen that establishes a lifelong asymptomatic infection in healthy individuals. Infection of immunesuppressed individuals causes serious illness. Transplant and AIDS patients are highly susceptible to CMV leading to life-threatening end-organ disease. Another vulnerable population is the developing fetus in utero, where congenital infection can result in surviving newborns with long-term developmental problems. There is no vaccine licensed for CMV and current antivirals suffer from complications associated with prolonged treatment. These include drug toxicity and emergence of resistant strains. There is an obvious need for new antivirals. Candidate intervention strategies are tested in controlled preclinical animal models but species specificity of human CMV precludes the direct study of the virus in an animal model. AREAS COVERED: This review explores the current status of CMV antivirals and development of new drugs. This includes the use of animal models and the development of new improved models such as humanized animal CMV and bioluminescent imaging of virus in animals in real time. EXPERT OPINION: Various new CMV antivirals are in development, some with greater spectrum of activity against other viruses. Although the greatest need is in the setting of transplant patients, there remains an unmet need for a safe antiviral strategy against congenital CMV. This is especially important as an effective CMV vaccine remains an elusive goal. In this regard, greater emphasis should be placed on suitable preclinical animal models and greater collaboration between industry and academia.

VCL-CB01, an injectable bivalent plasmid DNA vaccine for potential protection against CMV disease and infection.

Vaccines for the prevention of human CMV (hCMV) infection and disease are a major public health priority. Immunization with DNA vaccines encoding key proteins involved in the immune response to hCMV has emerged as a major focus of hcmv vaccine research. Validation of the protective effect of DNA vaccination in animal models has provided support for clinical trials. VCL-CB01, under development by Vical Inc for the prevention of hCMV infection and disease, is a poloxamer-formulated, bivalent DNA vaccine that contains plasmids encoding hCMV tegument phosphoprotein 65 and the major hCMV surface glycoprotein B. In a phase I trial in healthy adults, VCL-CB01 was well tolerated. In interim results from a phase II trial in hCMV-seropositive hematopoietic cell transplant recipients, VCL-CB01 increased T-cell responses compared with placebo. The final results from the phase II trial will be of value for developing strategies to prevent hCMV disease in hCMV-seropositive transplant recipients, and may lead to other trials of VCL-CB01 or related vaccines for the prevention of congenital hCMV infection.

Effectiveness of ganciclovir against human herpesvirus-6 excreted in saliva in stem cell transplant recipients.

The aim of this study was to evaluate the effect of ganciclovir on human herpesvirus-6 (HHV)-6. Forty allogeneic stem cell transplant recipients were prospectively studied by repeated sampling of the saliva. The saliva samples were assayed for HHV-6 by quantitative polymerase chain reaction. HHV-6 was detected in 33 patients. Ganciclovir was given as preemptive therapy for cytomegalovirus infection during 15 episodes that were compared to 18 episodes without any concomitant antiviral therapy. The mean HHV-6 load decreased 0.49 (s.e. 0.31) log(10)/week in patients receiving ganciclovir whereas it increased 0.15 (s.e. 0.17) log(10)/week in episodes without antiviral therapy (P=0.04). We conclude that ganciclovir can decrease the HHV-6 viral load in saliva.

Valganciclovir is safe and effective as pre-emptive therapy for CMV infection in allogeneic hematopoietic stem cell transplantation.

Despite significant advances in prevention and therapy, cytomegalovirus (CMV) infection continues to be an important cause of morbidity and mortality in the hematopoietic stem cell transplant (HSCT) recipient. The standard drug for pre-emptive therapy is intravenous ganciclovir (GCV). Valganciclovir (VGC), the oral pro-drug of GCV, has excellent bioavailability and is ideal for oral therapy. Since March 2002, VGC was adopted in our center for outpatient pre-emptive therapy in all patients undergoing allogeneic HSCT. Fifty-two allogeneic HSCT recipients were followed weekly via Digene hybrid capture assay. Patients with a positive assay were treated with VGC 900 mg p.o. b.i.d. x 14 days followed by 900 mg p.o. QD until at least 7 days after a negative test. Eighteen patients (14 sib, four MUD) had 30 episodes of CMV DNA detection treated with oral VGC. Median duration of therapy was 21 days (range 10-21 days). The rate of response was 93% (28/30) as confirmed by a negative assay within 14 days. No significant toxicity was encountered. Two patients failed oral VGC. One case of CMV enteritis was diagnosed in a patient with acute GVHD. Pre-emptive therapy of CMV infection with oral VGC is safe and effective in allogeneic HSCT recipients.

I. Poloxamer-formulated plasmid DNA-based human cytomegalovirus vaccine: evaluation of plasmid DNA biodistribution/persistence and integration.

Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.

Progress toward an elusive goal: current status of cytomegalovirus vaccines.

Although infection with human cytomegalovirus (CMV) is ubiquitous and generally asymptomatic in most individuals, certain patient populations are at high risk for CMV-associated disease. These include HIV-infected individuals with AIDS, transplant patients, and newborn infants with congenital CMV infection. Immunity to CMV infection, both in the transplant setting and among women of childbearing age, plays a vital role in the control of CMV-induced injury and disease. Although immunity induced by CMV infection is not completely protective against reinfection, there is nevertheless a sound basis on which to believe that vaccination could help control CMV disease in high-risk patient populations. Evidence from several animal models of CMV infection indicates that a variety of vaccine strategies are capable of inducing immune responses sufficient to protect against CMV-associated illness following viral challenge. Vaccination has also proven effective in improving pregnancy outcomes following CMV challenge of pregnant guinea pigs, providing a 'proof-of-principle' relevant to human clinical trials of CMV vaccines. Although there are no licensed vaccines currently available for human CMV, progress toward this goal has been made, as evidenced by ongoing clinical trial testing of a number of immunization strategies. CMV vaccines currently in various stages of preclinical and clinical testing include: protein subunit vaccines; DNA vaccines; vectored vaccines using viral vectors, such as attenuated pox- and alphaviruses; peptide vaccines; and live attenuated vaccines. This review summarizes some of the obstacles that must be overcome in development of a CMV vaccine, and provides an overview of the current state of preclinical and clinical trial evaluation of vaccines for this important public health problem.

Gene transfer for activation of CMV specific T cells.

Cytomegalovirus (CMV) is responsible for significant morbidity and mortality in immunocompromised patients undergoing allogeneic hematopoietic stem cell transplantation. The limitations of antiviral drugs and a better understanding of the cellular immune response to CMV has lead to the development of alternative therapies that restore host cellular immunity to CMV. Infusion of donor T lymphocytes results in variable protection against CMV but a high incidence of graft-versus-host disease in the allogeneic setting. To prevent this complication and further improve anti-CMV immune response, several groups have developed new approaches, such as the introduction of a suicide gene to control alloreactivity against the host or the selective activation of CMV-specific T cells by antigen-presenting cells expressing CMV antigens introduced by gene transfer. Depending on the target cells and the strategy chosen, adenovirus, retrovirus or poxviruses derived vectors are used for gene transfer. The protocols as well as the preclinical and clinical results obtained in the field of anti-CMV immunotherapy using gene transfer are reported and discussed.

Randomized comparison of oral valacyclovir and intravenous ganciclovir for prevention of cytomegalovirus disease after allogeneic bone marrow transplantation.

In this multicenter, randomized study, cytomegalovirus (CMV)-seropositive patients who received an allogeneic bone marrow transplant were provided high-dose intravenous acyclovir (500 mg/m(2) q8h) from the day of transplantation until engraftment. The patients were then randomly assigned to receive either oral valacyclovir, 2 g q.i.d. (n=83), or intravenous ganciclovir, 5 mg/kg q12h for 1 week, then 6 mg/kg once daily for 5 days per week (n=85), until day 100 after transplantation. CMV infection occurred in 12% of the patients who received valacyclovir and in 19% of the patients who received ganciclovir (hazard ratio [HR], 1.042; 95% confidence interval [CI], 0.391-2.778; P=.934). CMV disease developed in only 2 patients who received valacyclovir and in 1 patient who received ganciclovir (HR, 1.943; 95% CI, 0.176-21.44; P=.588). Oral valacyclovir can be an effective alternative to intravenous ganciclovir for prophylaxis of CMV disease after bone marrow transplantation.

Bayesian estimation, simulation and uncertainty analysis: the cost-effectiveness of ganciclovir prophylaxis in liver transplantation.

This paper demonstrates the usefulness of combining simulation with Bayesian estimation methods in analysis of cost-effectiveness data collected alongside a clinical trial. Specifically, we use Markov Chain Monte Carlo (MCMC) to estimate a system of generalized linear models relating costs and outcomes to a disease process affected by treatment under alternative therapies. The MCMC draws are used as parameters in simulations which yield inference about the relative cost-effectiveness of the novel therapy under a variety of scenarios. Total parametric uncertainty is assessed directly by examining the joint distribution of simulated average incremental cost and effectiveness. The approach allows flexibility in assessing treatment in various counterfactual premises and quantifies the global effect of parametric uncertainty on a decision-maker's confidence in adopting one therapy over the other.

Protective effect of black seed oil from Nigella sativa against murine cytomegalovirus infection.

In this study, antiviral effect of black seed oil (BSO) from Nigella sativa was investigated using murine cytomegalovirus (MCMV) as a model. The viral load and innate immunity mediated by NK cells and Mφ during early stage of the infection were analyzed. Intraperitoneal (i.p.) administration of BSO to BALB/c mice, a susceptible strain of MCMV infection, strikingly inhibited the virus titers in spleen and liver on day 3 of infection with 1x10(5) PFU MCMV. This effect coincided with an increase in serum level of IFN-gamma. Although BSO treatment decreased both number and cytolytic function of NK cells on day 3 of infection, it increased numbers of Mφ and CD4(+) T cells. On day 10 of infection, the virus titer was undetectable in spleen and liver of BSO-treated mice, while it was detectable in control mice. Although spleen of both control and BSO-treated mice showed similar CTL activities on day 10 after infection, serum level of IFN-gamma in BSO-treated mice was higher. Furthermore, BSO treatment upregulated suppressor function of Mφ in spleen. These results show that BSO exhibited a striking antiviral effect against MCMV infection which may be mediated by increasing of Mφ number and function, and IFN-gamma production.