Treatment Of Human Cytomegalovirus Infections Research Articles

A fully human neutralizing monoclonal antibody targeting a highly conserved epitope of the human cytomegalovirus glycoprotein B.

Human cytomegalovirus causes severe diseases in children (by congenital infection) and immunocompromised patients. Treatment with antiviral agents, such as ganciclovir, is limited by their toxicity. In this study, we investigated the effectiveness of a fully human neutralizing monoclonal antibody to inhibit human cytomegalovirus infection and viral cell-to-cell spread. We isolated a potent neutralizing antibody, EV2038 (IgG1 lambda), targeting human cytomegalovirus glycoprotein B using Epstein-Barr virus transformation. This antibody inhibited human cytomegalovirus infection by all four laboratory strains and 42 Japanese clinical isolates, including ganciclovir-resistant isolates, with a 50% inhibitory concentration (IC50) ranging from 0.013 to 0.105 μg/mL, and 90% inhibitory concentration (IC90) ranging from 0.208 to 1.026 μg/mL, in both human embryonic lung fibroblasts (MRC-5) and human retinal pigment epithelial (ARPE-19) cells. Additionally, EV2038 prevented cell-to-cell spread of eight clinical viral isolates, with IC50 values ranging from 1.0 to 3.1 μg/mL, and IC90 values ranging from 13 to 19 μg/mL, in ARPE-19 cells. EV2038 recognized three discontinuous sequences on antigenic domain 1 of glycoprotein B (amino acids 549-560, 569-576, and 625-632), which were highly conserved among 71 clinical isolates from Japan and the United States. Pharmacokinetics study in cynomolgus monkeys suggested the potential efficacy of EV2038 in vivo, the concentration of which in serum remained higher than the IC90 values of cell-to-cell spread until 28 days after intravenous injection of 10 mg/kg EV2038. Our data strongly support EV2038 as a promising candidate and novel alternative for the treatment of human cytomegalovirus infection.

Challenges, Recent Advances and Perspectives in the Treatment of Human Cytomegalovirus Infections.

Human cytomegalovirus (HCMV) is ubiquitous worldwide and elicits global health problems. The diseases associated with HCMV are a serious threat to humans, especially for the sick, infant, elderly and immunocompromised/immunodeficient individuals. Although traditional antiviral drugs (e.g., ganciclovir, valganciclovir, cidofovir, foscarnet) can be used to treat or prevent acute HCMV infections, their efficacy is limited because of toxicity, resistance issues, side effects and other problems. Fortunately, novel drugs (e.g., letermovir and maribavir) with less toxicity and drug/cross-resistance have been approved and put on the market in recent years. The nucleic acid-based gene-targeting approaches including the external guide sequences (EGSs)-RNase, the clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPRs-associated protein 9 (Cas9) system and transcription activator-like effector nucleases (TALENs) have been investigated to remove both lytic and latent CMV in vitro and/or in vivo. Cell therapy including the adoptive T cell therapy (ACT) and immunotherapy have been tried against drug-resistant and recurrent HCMV in patients receiving hematopoietic stem cell transplantation (HSCT) or solid organ transplant (SOT), and they have also been used to treat glioblastoma (GBM) associated with HCMV infections. These newly developed antiviral strategies are expected to yield fruitful results and make a significant contribution to the treatment of HCMV infections. Despite this progress, the nucleic acid-based gene-targeting approaches are still under study for basic research, and cell therapy is adopted in a small study population size or only successful in case reports. Additionally, no current drugs have been approved to be indicated for latent infections. Therefore, the next strategy is to develop antiviral strategies to elevate efficacy against acute and/or latent infections and overcome challenges such as toxicity, resistance issues, and side effects. In this review, we would explore the challenges, recent advances and perspectives in the treatment of HCMV infections. Furthermore, the suitable therapeutic strategies as well as the possibility for compassionate use would be evaluated.

Neutralization Epitopes in Trimer and Pentamer Complexes Recognized by Potent Cytomegalovirus-Neutralizing Human Monoclonal Antibodies.

Human cytomegalovirus (HCMV) infects 36% to almost 100% of adults and causes severe complications only in immunocompromised individuals. HCMV viral surface trimeric (gH/gL/gO) and pentameric (gH/gL/UL128/UL130/UL131A) complexes play important roles in HCMV infection and tropism. Here, we isolated and identified a total of four neutralizing monoclonal antibodies (MAbs) derived from HCMV-seropositive blood donors. Based on their reactivity to HCMV trimer and pentamer, these MAbs can be divided into two groups. MAbs PC0012, PC0014, and PC0035 in group 1 bind both trimer and pentamer and neutralize CMV by interfering with the postattachment steps of CMV entering into cells. These three antibodies recognize antigenic epitopes clustered in a similar area, which are overlapped by the epitope recognized by the known neutralizing antibody MSL-109. MAb PC0034 in group 2 binds only to pentamer and neutralizes CMV by blocking the binding of pentamer to cells. Epitope mapping using pentamer mutants showed that amino acid T94 of the subunit UL128 and K27 of UL131A on the pentamer are key epitope-associated residues recognized by PC0034. This study provides new evidence and insight information on the importance of the development of the CMV pentamer as a CMV vaccine. In addition, these newly identified potent CMV MAbs can be attractive candidates for development as antibody therapeutics for the prevention and treatment of HCMV infection. IMPORTANCE The majority of the global population is infected with HCMV, but severe complications occur only in immunocompromised individuals. In addition, CMV infection is a major cause of birth defects in newborns. Currently, there are still no approved prophylactic vaccines or therapeutic monoclonal antibodies (MAbs) for clinical use against HCMV infection. This study identified and characterized a panel of four neutralizing MAbs targeting the HCMV pentamer complex with specific aims to identify a key protein(s) and antigenic epitopes in the HCMV pentamer complex. The study also explored the mechanism by which these newly identified antibodies neutralize HCMV in order to design better HCMV vaccines focusing on the pentamer and to provide attractive candidates for the development of effective cocktail therapeutics for the prevention and treatment of HCMV infection.

Analysis of Novel Drug-Resistant Human Cytomegalovirus DNA Polymerase Mutations Reveals the Role of a DNA-Binding Loop in Phosphonoformic Acid Resistance.

The appearance of drug-resistant mutations in UL54 DNA polymerase and UL97 kinase genes is problematic for the treatment of human cytomegalovirus (HCMV) diseases. During treatment of HCMV infection in a pediatric hematopoietic cell transplant recipient, H600L and T700A mutations and E576G mutation were independently found in the UL54 gene. Foscarnet (FOS; phosphonoformic acid) resistance by T700A mutation is reported. Here, we investigated the role of novel mutations in drug resistance by producing recombinant viruses and a model polymerase structure. The H600L mutant virus showed an increase in resistance to ganciclovir (GCV) by 11-fold and to FOS and cidofovir (CDV) by 5-fold, compared to the wild type, while the E756G mutant virus showed an increase in resistance to FOS by 9-fold and modestly to CDV by 2-fold. With the FOS-resistant T700A mutation, only H600L produced increased FOS resistance up to 37-fold, indicating an additive effect of these mutations on FOS resistance. To gain insight into drug resistance mechanisms, a model structure for UL54 polymerase was constructed using the yeast DNA polymerase as a template. In this model, HCMV DNA polymerase contains a long palm loop domain of which H600 and T700 are located on each end and T700 interacts with the FOS binding pocket. Our results demonstrate that H600L and E756G mutations in UL54 polymerase are novel drug-resistant mutations and that the acquisition of both H600L and T700A mutations in the DNA-binding loop confers increased resistance to FOS treatment, providing novel insights for the mechanism acquiring foscarnet resistance.

The Endothelin Receptor Antagonist Macitentan Inhibits Human Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in immunocompromised patients and a major etiological factor for congenital birth defects in newborns. Ganciclovir and its pro-drug valganciclovir are the preferred drugs in use today for prophylaxis and treatment of viremic patients. Due to long treatment times, patients are at risk for developing viral resistance to ganciclovir and to other drugs with a similar mechanism of action. We earlier found that the endothelin receptor B (ETBR) is upregulated during HCMV infection and that it plays an important role in the life cycle of this virus. Here, we tested the hypothesis that ETBR blockade could be used in the treatment of HCMV infection. As HCMV infection is specific to humans, we tested our hypothesis in human cell types that are relevant for HCMV pathogenesis; i.e., endothelial cells, epithelial cells and fibroblasts. We infected these cells with HCMV and treated them with the ETBR specific antagonist BQ788 or ETR antagonists that are approved by the FDA for treatment of pulmonary hypertension; macitentan, its metabolite ACT-132577, bosentan and ambrisentan, and as an anti-viral control, we used ganciclovir or letermovir. At concentrations expected to be relevant in vivo, macitentan, ACT-132577 and BQ788 effectively inhibited productive infection of HCMV. Of importance, macitentan also inhibited productive infection of a ganciclovir-resistant HCMV isolate. Our results suggest that binding or signaling through ETBR is crucial for viral replication, and that selected ETBR blockers inhibit HCMV infection.

Strigolactone Analogs Are Promising Antiviral Agents for the Treatment of Human Cytomegalovirus Infection.

The human cytomegalovirus (HCMV) is a widespread pathogen and is associated with severe diseases in immunocompromised individuals. Moreover, HCMV infection is the most frequent cause of congenital malformation in developed countries. Although nucleoside analogs have been successfully employed against HCMV, their use is hampered by the occurrence of serious side effects. There is thus an urgent clinical need for less toxic, but highly effective, antiviral drugs. Strigolactones (SLs) are a novel class of plant hormones with a multifaceted activity. While their role in plant-related fields has been extensively explored, their effects on human cells and their potential applications in medicine are far from being fully exploited. In particular, their antiviral activity has never been investigated. In the present study, a panel of SL analogs has been assessed for antiviral activity against HCMV. We demonstrate that TH-EGO and EDOT-EGO significantly inhibit HCMV replication in vitro, impairing late protein expression. Moreover, we show that the SL-dependent induction of apoptosis in HCMV-infected cells is a contributing mechanism to SL antiviral properties. Overall, our results indicate that SLs may be a promising alternative to nucleoside analogs for the treatment of HCMV infections.

The trimeric artesunate derivative TF27 exerts strong anti-cytomegaloviral efficacy: Focus on prophylactic efficacy and oral treatment of immunocompetent mice

Human cytomegalovirus (HCMV) causes serious and even life-threatening diseases, particularly upon congenital or post-transplant infection. Treatment of HCMV infections with currently available drugs targeting viral enzymes is often limited by severe side effects and the emergence of drug-resistant viruses. To avoid this problem, novel therapeutic options directed to host proteins involved in virus replication are being investigated. Recently, we described the pronounced antiherpesviral activity of the trimeric artesunate derivative TF27 at low nanomolar concentrations in vitro and in vivo. In the present study, we report first data on the prophylactic efficacy of TF27 against human and murine CMV and the oncogenic avian alphaherpesvirus Marek's disease virus (MDV). The main findings of this study are (i) a pronounced activity of the experimental drug TF27 against alpha- and betaherpesviruses in vitro upon prophylactic treatment and (ii) a therapeutic and prophylactic efficacy upon oral treatment in an immunocompetent mouse model. Moreover, our data highlight (iii) the tolerability of orally administered TF27 free of compound-associated adverse events and further confirm (iv) the suitability of cellular factors as primary antiviral targets. Thus, we provide evidence for therapeutic and prophylactic antiherpesviral efficacy of TF27 upon oral treatment in immunocompetent hosts and thereby underline its potential for future antiviral drug development.

Current Diagnostic and Therapeutic Approaches to Cytomegalovirus Infections in Ulcerative Colitis Patients Based on Clinical and Basic Research Data.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus (the human herpesvirus 5) and an opportunistic pathogen that primarily infects HIV-positive and other immuno-compromised patients. Retrospective studies in the field of inflammatory bowel disease (IBD) have suggested a relationship between a concomitant colonic HCMV infection and poor outcomes in patients with an ulcerative colitis (UC) due to the presence of HCMV in surgical specimens of patients with a toxic megacolon or a steroid-resistant UC. Therefore, gastroenterologists have focused on the contribution of HCMV infections in the exacerbation of UC. Numerous studies have addressed the benefits of treating colonic HCMV reactivation in UC using an antiviral treatment. However, its clinical relevance remains uncertain as only a few prospective studies have assessed the direct relationship between clinical outcomes and the viral load of HCMV in colonic tissues. HCMV reactivation can be triggered by inflammation according to fundamental research studies. Thus, optimal control of intestinal inflammation is essential for preventing an HCMV reactivation in the intestinal mucosa. Indeed, several reports have indicated the effectiveness of an anti-tumor necrosis factor-alpha (TNFα) treatment in patients with an active UC and concomitant HCMV infections. In this review, we describe the mechanism of HCMV reactivation in UC cases and discuss the current issues regarding diagnosis and treatment of HCMV infections in UC patients.

An overview of letermovir: a cytomegalovirus prophylactic option

ABSTRACTIntroduction: Human cytomegalovirus (HCMV) or human herpesvirus 5 (HHV-5) is a β-herpesvirus that causes widespread infection in nearly all members of the human population worldwide. Its persistence in humans after primary infection in a latent phase as well as a partial non–protective immune response is the basis for repeated re-activation/re-infection episodes occurring both in immunocompetent and immunocompromised subjects. In the latter patient populations, which include hematopoietic stem cell transplant (HSCT) recipients, HCMV reactivation episodes may be particularly severe, leading to both systemic and end-organ diseases. Since the 90s, at least four antiviral drugs targeting the DNA polymerase complex have been developed for the prevention and treatment of HCMV infections in transplant recipients, used as first-line (ganciclovir and valganciclovir) and second-line therapy (foscarnet and cidofovir). However, due to their toxicity and drug-resistance induction, new drugs with different targets were needed.Areas covered: In 2017, a new drug named letermovir (LTV), which targets the HCMV DNA terminase complex, was licensed for prophylaxis of HCMV infections in HSCT recipients. This is the focus of this review.Expert opinion: LTV safety and efficacy are promising. However, long-term adverse events and the emergence of drug-resistant HCMV strains must be investigated in extended clinical trials prior to drawing final conclusions.

Possibilities Of Prevention And Treatment Of Human Cytomegalovirus Infections Including New Drugs And Compounds With Potential Application

Abstract Human Cytomegalovirus (hCMV) or human herpesvirus 5 (HHV5) is one of the most common pathogens. Studies indicate the presence of infection in 60–100% of individuals. The ability to cause asymptomatic, infection and a latency promotes the persistence and spread of the virus. hCMV infection is usually asymptomatic and does not require treatment, but in some cases especially in immunocompromised persons (e.g., transplant recipients, patients with hematological malignancies, untreated HIV infected individuals) symptoms can be serious and life-threatening. The paper presents drugs currently used for treatment or prevention of hCMV infection, as well as the prospect of new treatment options. Currently, ganciclovir or valganciclovir are used as the first-line drugs and foscarnet and cidofovir are used alternatively. These drugs usually allow to control hCMV infections, however, there are important limitations. These include the toxicity and the possibility of the development of resistance, including the cross-resistance to all four drugs because they have a common mechanism of action, inhibition of viral DNA polymerase. Therefore, the creation of new drugs, with different mechanisms of action, lower toxicity and better pharmacokinetic parameters is important. Recently, the new drug, letermovir have been registered. Letermovir acts as hCMV DNA terminase inhibitor and due to the different mechanism of action the drug is active against hCMV strains resistant to DNA polymerase inhibitors, and potentially can act synergistically with them. The other drugs that are in the research stage or clinical studies include: brincidofovir, a cidofovir derivative, maribavir, a competitive inhibitor of ATP, cyclopropavir, a guanosine analog and antiviral peptides. 1. Introduction – epidemiology of hCMV infections and prophylaxis schemes. 2. Drugs approved for use in the prevention and treatment of hCMV infections. 2.1. Nucleoside analogues: ganciclovir and valganciclovir. 2.2. Foscarnet. 2.3. Cidofovir. 2.4. Letermovir. 3. Compounds with potential use in the treatment of hCMV infections. 3.1. Brincidofovir. 3.2. Maribavir. 3.3. Cyclopropavir 3.4. Antiviral peptides. 4. Summary

In vivo proof-of-concept for two experimental antiviral drugs, both directed to cellular targets, using a murine cytomegalovirus model

Infections with the human cytomegalovirus (HCMV) cause serious medical problems including organ rejection and congenital infection. Treatment of HCMV infections with currently available medication targeting viral enzymes is often accompanied with severe side effects and the occurrence of drug-resistant viruses. This demands novel therapeutical approaches like targeting genetically stable host cell proteins that are crucial for virus replication. Although numerous experimental drugs with promising in vitro efficacy have been identified, the lack of available data in animal models limits their potential for further clinical development. Recently, we described the very strong in vitro antiherpesviral activity of the NF-κB inhibitor TF27 and the CDK7 inhibitor LDC4297 at low nanomolar concentrations. In the present study, we present first data for the in vivo efficacy of both experimental drugs using an established cytomegalovirus animal model (murine CMV replication in immunodefective Rag −/− mice). The main findings of this study are (i) a strong inhibitory potency against beta- and gamma-herpesviruses of both compounds in vitro, (ii) even more important, a pronounced anticytomegaloviral activity also exerted in vivo, that resulted from (iii) a restriction of viral replication to the site of infection, thus preventing organ dissemination, (iv) in the absence of major compound-associated adverse events. Thus, we provide evidence for a strong antiviral potency in vivo and proof-of-concept for both drugs, which may encourage their further drug development, possibly including pharmacologically optimized derivatives, for a potential use in future antiherpesviral treatment.

Hydroxypyridonecarboxylic Acids as Inhibitors of Human Cytomegalovirus pUL89 Endonuclease.

Human cytomegalovirus (HCMV) infection poses a major health threat to immunocompromised individuals. Until recently, treatment of HCMV infection has relied solely on polymerase inhibitors that have safety and resistance issues. pUL89 provides the enzymatic functions for the HCMV terminase complex in viral DNA packaging and is an attractive target for developing a new class of HCMV drugs. However, inhibitors of the endonuclease activity of the C terminus of pUL89 (pUL89-C) were unknown before our recently characterized hydroxypyridonecarboxylic acid (HPCA) hit 7 r (1-(3-chloro-4-fluorobenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid; numbered as 10 k in our previous publication: Y. Wang, L. Mao, J. Kankanala, Z. Wang, R. J. Geraghty, J. Virol. 2017, 91, e02152-16). Herein, we explored the structure-activity relationship (SAR) of the HPCA chemotype mainly with regard to the N1 site through the synthesis of 35 analogues. The SAR studies, along with molecular modeling, identified a possible pharmacophore model minimally consisting of a chelating triad and a hydrophobic phenyl or biphenyl methyl substituent at N1. Lastly, our best compounds consistently inhibited pUL89-C in the low micromolar range in biochemical assays and exhibited strong antiviral activity without cytotoxicity, laying a solid medicinal chemistry foundation for further HCMV drug discovery efforts targeting pUL89-C.

Human Cytomegalovirus UL54 and UL97 Mutations for Detection of Ganciclovir Resistance in Congenital Infection

Aim: Ganciclovir (GCV) is used as an antiviral drug for the treatment of human cytomegalovirus infection. The aim of this study was to demonstrate GCV-resistant human cytomegalovirus in congenitally infected neonates. Patients & methods: DNA of CMV positive newborn samples was extracted and UL97 and UL54 genes were amplified by PCR and real-time PCR. Sequencing of UL97 and UL54 genes were performed and analyzed. Results: UL97 GCV resistance mutation C603W was detected in one newborn. D605E was the most common polymorphism in UL97 observed in 7/13 (53.8%) of samples. N685S, A688V, A885T and N898D were four known common UL54 polymorphisms. Conclusion: The rate of GCV resistance in congenital CMV is low. Common polymorphisms in UL97 and UL54 genes are also reported as new mutations.

Single Chain Antibodies Against gp55 of Human Cytomegalovirus (HCMV) for Prophylaxis and Treatment of HCMV Infections.

Background:Immunotherapy is a promising prospective new treatment for cytomegalovirus (CMV) infections. Neutralizing effects have been reported using monoclonal antibodies. Recombinant single chain antibodies (scFvs) due to their advantages over monoclonal antibodies are potential alternatives and provide valuable clinical agents.Objectives:The aim of this study was to select specific single chain antibodies against gp55 of CMV and to evaluate their neutralizing effects. In the present study, we selected specific single chain antibodies against glycoprotein 55 (gp55) of CMV for their use in treatment and diagnosis.Materials and Methods:Single chain antibodies specific against an epitope located in the C-terminal part of gp55 were selected from a phage antibody display library. After four rounds of panning, twenty clones were amplified by the polymerase chain reaction (PCR) and fingerprinted by MvaI restriction enzyme. The reactivities of the specific clones were tested by the enzyme-linked immunosorbent assay (ELISA) and the neutralizing effects were evaluated by the plaque reduction assay.Results:Fingerprinting of selected clones revealed three specific single chain antibodies (scFv1, scFv2 and scFv3) with frequencies 25%, 20 and 20%. The clones produced positive ELISA with the corresponding peptide. The percentages of plaque reduction for scFv1, scFv2 and scFv3 were 23.7, 68.8 and 11.6, respectively.Conclusions:Gp55 of human CMV is considered as an important candidate for immunotherapy. In this study, we selected three specific clones against gp55. The scFvs reacted only with the corresponding peptide in a positive ELISA. The scFv2 with 68.8% neutralizing effect showed the potential to be considered for prophylaxis and treatment of CMV infections, especially in solid organ transplant recipients, for whom treatment of CMV is urgently needed. The scFv2 with neutralizing effect of 68.8%, has the potential to be considered for treatment of these patients. The specific scFv1 and scFv3 with lower neutralizing effects can be used for diagnostic purposes.

Diagnosis and treatment of ulcerative colitis with cytomegalovirus infection: importance of controlling mucosal inflammation to prevent cytomegalovirus reactivation.

Human cytomegalovirus (HCMV) is a member of the herpesvirus family. HCMV infection persists throughout the host lifespan in a latent state following primary infection. The ability of HCMV to escape control by the host immune system and its resulting reactivation suggests the importance of ongoing immune surveillance in the prevention of HCMV reactivation. HCMV is a common cause of opportunistic infection that causes severe and fatal disease in immune-compromised individuals. In inflammatory bowel disease patients, particularly those with ulcerative colitis (UC), HCMV is often reactivated because these patients are frequently treated with immunosuppressive agents. This reactivation exacerbates colitis. Additionally, HCMV infection can induce severe colitis, even in patients with UC who have never been treated with immunosuppressive agents. However, the role of HCMV in colonic inflammation in patients with UC remains unclear. Here, we present previous and current clinical data on the diagnosis and treatment of HCMV infection in UC. Additionally, our experimental data from a newly established mouse model mimicking UC with concomitant CMV infection clearly demonstrate that inflammation could result in the exacerbation of UC disease activity with induction of HCMV reactivation. In summary, optimal control of colonic inflammation should be achieved in UC patients who are refractory to conventional immunosuppressive therapies and are positive for HCMV.

Cytomegalovirus infection in pediatric rheumatic diseases: a review

Human cytomegalovirus (HCMV) is familiar to pediatric rheumatologists mainly as a cause of opportunistic disease in pharmacologically immune suppressed patients. However, HCMV also has a variety of immuno-modulatory effects, through which it may influence the course of rheumatic conditions. In this article we discuss the interplay between HCMV and the immune system, and review the clinical manifestations, diagnosis, and treatment of HCMV infection in children with rheumatic disease.

A HCMV pp65 polypeptide promotes the expansion of CD4+ and CD8+ T cells across a wide range of HLA specificities

Human cytomegalovirus (HCMV) can cause life-threatening disease in infected hosts. Immunization with human leukocyte antigen (HLA)-restricted immunodominant synthetic peptides and adoptive transfer of epitope-specific T cells have been envisaged to generate or boost HCMV-specific cellular immunity, thereby preventing HCMV infection or reactivation. However, induction or expansion of T cells effective against HCMV are limited by the need of utilizing peptides with defined HLA restrictions. We took advantage of a combination of seven predictive algorithms to identify immunogenic peptides of potential use in the prevention or treatment of HCMV infection or reactivation. Here we describe a pp65-derived peptide (pp65(340-355), RQYDPVAALFFFDIDL: RQY16-mer), characterized by peculiar features. First, RQY-16mer is able to stimulate HCMV pp65 specific responses in both CD4(+) and CD8(+) T cells, restricted by a wide range of HLA class I and II determinants. Second, RQY-16mer is able to induce an unusually wide range of effector functions in CD4(+) T cells, including proliferation, killing of autologous HCMV-infected target cells and cytokine production. Third, and most importantly, the RQY-16mer is able to stimulate CD4(+) and CD8(+) T-cell responses in pharmacologically immunosuppressed patients. These data suggest that a single reagent might qualify as synthetic immunogen for potentially large populations exposed to HCMV infection or reactivation.

Human cytomegalovirus load measurement and its applications for pre‐emptive therapy in patients undergoing hematopoietic stem cell transplantation

The hematopoietic stem cell transplantation (HSCT) setting is a rapidly evolving field, and procedures with a high degree of complexity are now entering into the clinic. The risk of developing opportunistic viral infections increases with the level of human leukocyte antigen disparity between the donor and the recipient of allogeneic HSCT and is enhanced by graft manipulations such as T-cell depletion or immune suppression required to control graft versus host disease (GVHD) or rejection. Among the opportunistic viral infections potentially hampering the outcome of HSCT, human cytomegalovirus (HCMV) infection plays a major role. In the early 1980s, before the introduction of effective antiviral treatment, HCMV disease accounted for the high mortality in recipients of allogeneic HSCT. In more recent years, the availability of HCMV-specific antiviral agents (ganciclovir, GCV, foscarnet, PFA and cidofovir, CDV) led to a dramatic decrease in HCMV-related morbidity and mortality. However, HCMV infection still remains a major concern in the HSCT setting. Historically, treatment of HCMV infection in HSCT recipients (HSCTR) evolved from treatment of overt HCMV disease to universal prophylaxis or pre-emptive treatment of active infection, in parallel with the improvement of techniques for diagnosing and monitoring HCMV infection in transplant recipients.

Residues of human cytomegalovirus DNA polymerase catalytic subunit UL54 that are necessary and sufficient for interaction with the accessory protein UL44.

The human cytomegalovirus DNA polymerase contains a catalytic subunit, UL54, and an accessory protein, UL44. Recent studies suggested that UL54 might interact via its extreme C terminus with UL44 (A. Loregian, R. Rigatti, M. Murphy, E. Schievano, G. Palu', and H. S. Marsden, J. Virol. 77:8336-8344, 2003). To address this hypothesis, we quantitatively measured the binding of peptides corresponding to the extreme C terminus of UL54 to UL44 by using isothermal titration calorimetry. A peptide corresponding to the last 22 residues of UL54 was sufficient to bind specifically to UL44 in a 1:1 complex with a dissociation constant of ca. 0.7 microM. To define individual residues in this segment that are crucial for interacting with UL44, we engineered a series of mutations in the C-terminal region of UL54. The UL54 mutants were tested for their ability to interact with UL44 by glutathione S-transferase pulldown assays, for basal DNA polymerase activity, and for long-chain DNA synthesis in the presence of UL44. We observed that deletion of the C-terminal segment or substitution of alanine for Leu1227 or Phe1231 in UL54 greatly impaired both the UL54-UL44 interaction in pulldown assays and long-chain DNA synthesis without affecting basal polymerase activity, identifying these residues as important for subunit interaction. Thus, like the herpes simplex virus UL30-UL42 interaction, a few specific side chains in the C terminus of UL54 are crucial for UL54-UL44 interaction. However, the UL54 residues important for interaction with UL44 are hydrophobic and not basic. This information might aid in the rational design of new drugs for the treatment of human cytomegalovirus infection.

In Vitro Model for the Study of the Dissociation of Increasing Antigenemia and Decreasing DNAemia and Viremia during Treatment of Human Cytomegalovirus Infection with Ganciclovir in Transplant Recipients

The paradox phenomenon (i.e., the dissociation of increasing antigenemia and decreasing DNAemia and viremia) that occurs during treatment of human cytomegalovirus (HCMV) infections with ganciclovir (Gcv), in transplant recipients, was investigated by use of an in vitro model for the study of interactions between polymorphonuclear leukocytes and endothelial cells. The paradox phenomenon was reproduced in vitro in the presence of Gcv and, to a much lesser extent, in the presence of cidofovir, but not in the presence of foscarnet. The pathogenetic basis for such a paradox response was found, by use of drug concentrations in the range of 90%-99% of the inhibitory dose, to rely on the partial synthesis of HCMV phosphoprotein 65. The opposite situation (i.e., the simultaneous increase of antigenemia, viremia, and DNAemia), which is observed in clinical conditions associated with inefficacy of treatment due to drug-resistant strains, was also reproduced in vitro by use of drug-resistant HCMV strains. The conclusion for clinicians is that antiviral therapy must be changed only in the latter case.