CMV Replication Research Articles

The Value of Persistent Cytomegalovirus Infection in the Formation of Groups of Children with Recurrent Acute Respiratory Diseases

So far not defined the value of CMV persistence in frequently and chronically ill children, the impact of active CMV-infection on the severity of the condition and the incidence of viral-bacterial complications. 149 surveyed frequently and long ill children with lymphadenopathy (58), obstructive bronchitis (40), the defeat of the ENT organs (18), meningoencephalitis (13), pleuropneumonia (12), thrombocytopenia (8), were in the offices MDKB. The age of the children ranged from 1 month. up to 14 years. Structural identification of CMV pp65 antigen is recommended in severe patients and in children with frequent and prolonged acute respiratory infections, complications of purulent-inflammatory diseases. To confirm active CMV replication method should be used in real time PCR in saliva and urine (positive at 48.6%).

Timing of CMV-specific effector memory T cells predicts viral replication and survival after allogeneic hematopoietic stem cell transplantation.

The aim of this study was to characterize timing, kinetic, and magnitude of CMV-specific immune response after hematopoietic stem cell transplantation (HSCT) and its ability to predict CMV replication and clinical outcomes. Using cell surface and intracellular cytokine staining by flow cytometry, CMV-specific T-cell response was measured in blood, while CMV viral load and chimerism were determined by real-time PCR. Patients that reconstituted CMV-specific T-cell response within 6weeks after Allo-SCT showed a more robust immune response (CD8(+) : 0.7cells/μl vs. 0.3/μl; P-value=0.01), less incidence of CMV replication (33% vs. 89.5%; P-value=0.007), reduced viral loads (1.81 log copies/ml vs. 0 copies/ml; P-value=0.04), and better overall survival (72%; CI: 0.53-0.96 vs. 42% CI: 0.24-0.71; P-value=0.07) than patients with a delayed immune reconstitution. Viremic patients had significantly higher transplant-related mortality than nonviremic patients after 1year (33% CI: 0.15-0.52 vs. 0% CI: 0.05-0.34; P-value=0.01). Risk factors independently associated with viral replication were receptor pretransplant CMV-positive serostatus (P-value=0.02) and acquiring CMV-specific T-cell response after 6weeks post-transplantation (P-value=0.009). In conclusion, timing of acquiring a positive CMV-specific T-cell immune response after transplantation may identify patients with different risk for viral replication and different clinical outcomes, including survival.

Reversible Silencing of Cytomegalovirus Genomes by Type I Interferon Governs Virus Latency

Herpesviruses establish a lifelong latent infection posing the risk for virus reactivation and disease. In cytomegalovirus infection, expression of the major immediate early (IE) genes is a critical checkpoint, driving the lytic replication cycle upon primary infection or reactivation from latency. While it is known that type I interferon (IFN) limits lytic CMV replication, its role in latency and reactivation has not been explored. In the model of mouse CMV infection, we show here that IFNβ blocks mouse CMV replication at the level of IE transcription in IFN-responding endothelial cells and fibroblasts. The IFN-mediated inhibition of IE genes was entirely reversible, arguing that the IFN-effect may be consistent with viral latency. Importantly, the response to IFNβ is stochastic, and MCMV IE transcription and replication were repressed only in IFN-responsive cells, while the IFN-unresponsive cells remained permissive for lytic MCMV infection. IFN blocked the viral lytic replication cycle by upregulating the nuclear domain 10 (ND10) components, PML, Sp100 and Daxx, and their knockdown by shRNA rescued viral replication in the presence of IFNβ. Finally, IFNβ prevented MCMV reactivation from endothelial cells derived from latently infected mice, validating our results in a biologically relevant setting. Therefore, our data do not only define for the first time the molecular mechanism of IFN-mediated control of CMV infection, but also indicate that the reversible inhibition of the virus lytic cycle by IFNβ is consistent with the establishment of CMV latency.

Digitoxin analogues with improved anticytomegalovirus activity.

Cardiac glycosides are potent inhibitors of cancer cell growth and possess antiviral activities at nanomolar concentrations. In this study we evaluated the anticytomegalovirus (CMV) activity of digitoxin and several of its analogues. We show that sugar type and sugar length attached to the steroid core structure affects its anticytomegalovirus activity. Structure-activity relationship (SAR) studies identified the l-sugar containing cardiac glycosides as having improved anti-CMV activity and may lead to better understanding of how these compounds inhibit CMV replication.

P3.188 Cytomegalovirus and HIV Co-Infection Among Patients Accessing Care in a Tertiary Care Centre in Nigeria

BackgroundCytomegalovirus (CMV) is one of the commonest viral opportunistic infections in persons with Acquired Immune Deficiency Syndrome (AIDS). In AIDS patients, progressive loss of immune function, and in particular, loss...

CMV Replication After Allogeneic Hematopoietic Cell Transplantation and Relapse Risk: Evidence for Early Protection Against Relapse in Acute Leukemia and Lymphoma

CMV Replication After Allogeneic Hematopoietic Cell Transplantation and Relapse Risk: Evidence for Early Protection Against Relapse in Acute Leukemia and Lymphoma

Treatment of Cytomegalovirus infection after renal transplantation: Experience from a Single Center in China

We compared the efficacy and safety of 2 different treatments of CMV infection, including asymptomatic CMV replication and CMV disease. 852 renal transplantation recipients, including asymptomatic CMV replication and CMV disease, received antiviral therapies of intravenous acyclovir or comprehensive anti-infection solution, mainly with intravenous ganciclovir. Effect, time, acute allograft rejection, and safety were analyzed during the antiviral therapy The total effective rates were higher with ganciclovir in both asymptomatic CMV replication (98.96% vs. 84.90%) and CMV disease (96.29% vs. 50.36%). Ganciclovir significantly shortened antiviral therapy duration in both asymptomatic CMV replication (15.0 ± 2.3 days vs. 16.0 ± 3.4 days) and CMV disease (19.7 ± 3.1 days vs. 21.5 ± 4.0 days). The acute allograft rejection incidences were significantly lower with ganciclovir in both asymptomatic CMV replication (8% vs. 14%) and CMV disease (11% vs. 22%). CMV-IEA was detected in renal grafts of patients with acute rejection. There was more CMV-associated acute rejection using acyclovir than using ganciclovir. Except for the higher incidence of anemia leucopenia and anemia with ganciclovir, the safety profiles of both drugs were similar. Comprehensive anti-infection solution, mainly with intravenous ganciclovir, can effectively treat CMV infection, shorten duration of therapy, and decrease acute rejection. The few adverse effects had negligible effects on use of ganciclovir.

CMV-Replication After Allogeneic Stem Cell Transplantation Is Associated with a Gvhd-Independent Reduced Relapse Risk in Lymphoma

Abstract 1635 Background:We have previously showed that a CMV- reactivation after allogeneic HSCT is associated with a reduced risk for leukemic relapse and improved overall survival in patients with AML (Elmaagacli et al Blood 2011). Further, experimental data in a lymphoma mice model reported from Erlach et al. showed that coinfection with murine CMV revealed a strong anti-lymphoma effect by induction of apoptosis in lymphoma cells and improving rate of overall survival in mice after transplant. Methods:This prompted us to investigate the influence of early replicative CMV infection in 94 (median age [years]: 45, 18 – 70) patients with lymphoma, who received transplants from unrelated (n=67) or related (n=27) donors. Patients were transplanted from HLA-identical (n=74), HLA-mismatched (n=20). 13 patients were transplanted for indolent lymphoma (FL n=11, CLL n=2), 67 patients for aggressive lymphoma (B-lineage n=35, T-lineage n=27, transformed n=5), 11 patients for MCL and 3 patients for HD. The disease stages of patients at HSCT were CR in 20 patients, PR in 40 patients, refractory in 30 patients and untested in 2 patients. 55 patients (59%) received previous autograft and 82 patients (87%) were treated prior to transplant with at least 3 chemotherapy lines. The hematopoietic cell transplantation specific comorbidity index (HCT-CI) were 0–2 in 76 patients (81%) and 3+ in 18 patients (19%). Myeloablative preparative regimen was applied in 60 patients (64%) while 34 patients (36%) received a RIC. Sixty-eight % of patients (n=48) were at risk for CMV reactivation based on either patient or donor pretransplant CMV serostatus. CMV replication as detected by pp65 antigenemia assay occurred in 34 patients (36%). Results:Taking all competing risks into account, the cumulative incidence of progressive free survival (PFS) at 5 years after alloSCT was 38 % (95 % confidence limit [95 % CL]: 31 – 45) in patients without as compared to 20 % (95 % CL: 9 – 31) in patients with early pp65 antigenemia (p<0.018). In multivariate analysis including parameters as grades II-IV acute graft-versus-host disease (GvHD), chronic GvHD, disease stage, chemorefractory, previous chemotherapy lines and pp65 antigenemia, CMV replicative status was confirmed as a strong independent predictor of PFS (hazard ratio [HR]: 0.29, 95 % CL: 0.08 – 1.00, p<0.049) together with chronic GvHD (HR: 0.32, 95 % CL: 0.13 – 0.80, p<0.016), and chemorefractory HR: 3.3, 95 % CL: 1.28 – 8.4, p<0.013). The anti-lymphoma effect was detectable across all lymphoma subsets and was most pronounced in patients with chemotherapy refractory lymphoma or refractory disease of lymphoma. However, Overall survival rate did not differ in both groups (51.9% for patients with CMV-replication versus 50.7% without n.s.) Conclusions:This is the first report which demonstrates a strong and independent effect of early CMV replication on the PFS in patients with lymphoma. This effect deserves further and more comprehensive studies with regard to its clinical relevance and the underlying anti-lymphoma mechanisms.Cumulative incidence of progression-free survival (PFS) stratified by posttransplant HCMV pp65-antigenemia and cell type of aggressive lymphoma. Disclosures:No relevant conflicts of interest to declare.

Donor CMV Status Influence On the Outcome of Allogeneic Stem Cell Transplantation (HSCT); A Study by the Infectious Diseases Working Party (IDWP) of the EBMT

AbstractAbstract ▪465▪This icon denotes a clinically relevant abstractA previous study from the IDWP of the EBMT showed that CMV seropositive patients undergoing unrelated donor HSCT had increased NRM and decreased survival if they were grafted from a CMV seronegative donor. This finding has been controversial and we therefore decided to revisit the question with a larger number of patients and including new factors such as conditioning intensity.Patients were selected from the EBMT database who had received an allogeneic HSCT from 1992 – 2008 and for whom both the donor and recipient CMV status were known. Patients receiving cord blood grafts were excluded. 54660 patients were identified and included in the study; 32320 seropositive and 22340 seronegative patients. The different donor categories (sibling, mis-matched family, unrelated) were analyzed separately. Cox multivariate models were fitted to estimate the effect of donor serological status (positive versus negative) on outcome both in CMV seropositive and CMV seronegative patients adjusted for year of HSCT, donor and patient gender, recipient age, stem cell source, diagnosis, use of alemtuzumab or ATG, country, and conditioning intensity (RIC vs. MAC).Seronegative patients receiving grafts from seropositive unrelated donors had an decreased overall survival (OS; HR 1.13; p<.01); relapse free survival (RFS; HR 1.10; <p<.01) and increased non-relapse mortality (NRM; HR 1.13; p<;.01) while no significant effect was seen on relapse incidence (RI; HR 1.06). There were no significant effects in patients receiving HLA-identical or mis-matched family donors. Seropositive patients receiving grafts from seropositive unrelated donors had improved OS (HR 0.91; p<.01), RFS (HR 0.94; p<.05), and decreased NRM (HR 0.87; p<.01) if they had received MAC. No effect of donor serostatus was seen in patients receiving unrelated donor grafts after RIC. No effect of donor status was seen on RI. There were no significant effects in patients receiving HLA-identical or mis-matched family donors. Based on the recent study of Elmaagacli et al showing a decreased relapse risk in patients transplanted for AML of early CMV replication, a separate analysis regarding RI was performed only in AML patients in first CR. However, no effect of donor serostatus on RI was found in either seropositive or seronegative patients.In this analysis we confirm the negative effect on OS, RFS, and NRM if a CMV seropositive unrelated donor is selected for a CMV seronegative patient. It therefore remains important to select a seronegative donor when the patient is seronegative. For a CMV seropositive patient our data support the choice of a CMV seropositive donor if the patient is planned to receive a myeloablative conditioning regimen. Disclosures:No relevant conflicts of interest to declare.

Extremely Higher Incidence of CMV-pp65 Antigenemia After Allogeneic Hematopoietic Stem Cell Transplantation in Adult T-Cell Leukemia/Lymphoma (ATL) Compared with Other Hematological Diseases

Abstract 4477Cytomegalovirus (CMV) infection is one of significant factors contribute to morbidity and mortality in recipients of allogeneic hematopoietic stem cell transplantation (SCT). Prevention and treatment of active CMV infection based on monitoring CMV-pp65 antigenemia test or CMV-DNA PCR assay have been done in this field. As the limitation of insurance, only CMV-pp65 antigenemia test can be clinically carried out in Japan. Adult T-cell leukemia/lymphoma (ATL), HTLV-1 related hematological malignancy, is known as poor prognostic disease and only allogeneic SCT had been prolonged survival outcomes. ATL cells are morphologically characterized as having complicated nuclei and their surface markers are usually shown as CD3+CD4+CD25+. Some reports suggested that ATL patients have tendency to be in immune compromised status. However, in our knowledge, there was no report about incidence of CMV-replication in ATL patients following allgeneic SCT so far. Here we analyze the incidence of CMV-pp65 antigenemia and related clinical issues in 85 allogeneic SCT recipients of single center located in HTLV-1 endemic area. There were consecutively 97 patients (106 times) undergone allogeneic SCT from November 2006 to May 2012 in Imamura Bun-in Hospital, Kagoshima, Japan. Totally, 12 patients (21 times), 9 times of deaths earlier than 30 days after SCT, 3 times of deaths earlier than engraftment, 9 times of second or more chances of SCTs, were excluded from this study. Rest of 85 (male: 52, female: 33) patients (85 times) were analyzed. We have first picked up patients’ clinical data from medical charts including result of CMV-pp65 antigenemia tests and related data. Statistical analysis had carried out using SPSS 11.0J (SPSS Japan Inc., Japan). Cumulative incidences of CMV-pp65 antigenemia were analyzed by Kaplan-Meir method in total 85 patients. Statistical significance defined P<=0.05 in Log-Rank test. Cox regression analysis about incidence of CMV-pp65 antigenemia were also done in 68 patients who had available data including positivity of CMV-IgG before SCT. Median age was 52 (range 19–69) yrs. Thirty-seven ATL patients and 48 non-ATL (AML 21, MDS 14, NHL 7, ALL 3, AA 2, MM 1) patients were included. In disease status, 49 patients were categorized as high risk and 36 standard risk. Donor sources were 19 of related donors (BM 7, PB 12), 66 unrelated (UBM 56, UCB 10) respectively. Sixty four patients carried out SCT with myeloablative conditioning regimens (MAC) and 21 reduced intensive conditioning regimens (RIC). Sixty-eight recipients (ATL: 27, non-ATL: 41) had available data of their CMV-IgG antibody before SCT and 79.4% (ATL/non-ATL: 96.3%/70.7%, P<0.001) of them were shown antibody positive. Although there were no significant differences about positivity of CMV-IgG antibody before SCT between age >=50 and <50 in ATL patients, there was significant difference between age >=50 and <50 in non-ATL patients. There were no significant differences about positivity of donors’ CMV-IgG antibody before SCT between ATL and non-ATL. Among patients shown CMV-pp65 antigenemia, 45/68 patients had administrated iv steroids before or after CMV-pp65 antigenemia. Twenty-four out of 45 were administrated iv steroids as treatment of GVHD or engraftment syndrome, on the other hand, 21 as premedication of blood transfusion. Absolute WBC counts, lymphocyte counts and CMV-pp65 antigen positive WBCs counts at the incidence of CMV antigenemia, duration from SCT to CMV-pp65 antigenemia and duration of treatments with anti-viral agents didn’t have statistically significant differences between ATL and non-ATL respectively. CMV disease occurred in 7 (ATL 4, non-ATL 3) patients, however, there was also no significant difference between ATL and non-ATL. The cumulative incidence of CMV-pp65 antigenemia in ATL and non-ATL patients were 91.9% and 60.4% respectively (P<0.001). Interestingly, recipients whose ages were >=50 showed significantly high incidence of incidence of CMV-pp65 antigenemia compared with <50 (P=0.002). However, there were no difference between age >=50 and <50 (92% vs 91.7%) in ATL recipients. In multivariate analysis, ATL (HR: 2.625, P=0.008) and MAC (HR: 2.532, P=0.014) were positively contributed to incidence of CMV antigenemia by Cox regression analyses. Our study suggests that ATL could be one of independent risk factors of CMV replication after allogeneic hematopoietic stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.

Profilaxis de citomegalovirus en niños y adultos sometidos a trasplante de órganos sólidos y de precursores hematopoyéticos

CMV is one of the main infectious problems for SOT and HSCT. The severity of the complications are mainly associated with the type of transplant and immune status against the virus of the transplant donor and the transplant recipient. It is important to prevent exposure, using safe blood transfusion CMV seronegative donors (B1) and/or use of blood leucocytes-depleted by filtration (Al). In addition to preventing exposure, there are two widely used prevention strategies: universal prophylaxis with antiviral therapy or "pre-emptive" strategy based on the use of antivirals only to the early detection of CMV replication in blood. The first option is most used in the SOT management, especially for those identified as the high risk group of CMV disease: R (+), with D (+) or D (-) (Al), where the recommended drug is ganciclovir or valganciclovir . The second approach is preferable for HSCT, which recommends weekly monitoring for CMV viral load from day 10 to 100 post transplant (A3). This strategy requires having a viral laboratory support (A2). The selected antiviral in the case of pre emptive therapy is intravenous ganciclovir (A1).

Immune response to CMV in solid organ transplant recipients: current concepts and future directions

Despite advances in immunosuppression and antiviral therapy, CMV continues to be a significant opportunistic pathogen adversely affecting the outcome of solid organ transplantation (SOT) recipients. While a significant proportion of CMV disease is caused by reactivation of latent virus, the risk is highest among CMV donor+ and recipient- SOT patients. CMV is responsible for both direct (e.g., pneumonitis, colitis) and indirect (e.g., rejection, atherosclerosis) morbidity and mortality. Healthy CMV-seropositive individuals have a high frequency of CMV-specific CD4+ and CD8+ T cells that provide immune protection by limiting CMV reactivation and replication. Changes to the innate and adaptive immune system from immunosuppressive therapy following SOT contribute to CMV disease pathogenesis. CMV disease after SOT is associated with poorer outcomes, thus novel strategies to prevent it are an area of active research. In this article, we review the current state of knowledge on the immune response to CMV following SOT.

Endothelial Cells and CMV Dissemination

Using the murine CMV animal model and the well-established model of Cre-lox-P-mediated green-fluorescence tagging of endothelial cell (EC)-derived mouse CMV to quantify the role of infected ECs in transplantation-associated CMV dissemination (in mice expressing Cre recombinase under the control of either the Tie2 or the Tek promoter selectively expressed in vascular EC-Tie-Cre and Tek-Cre mice), it was shown that EC-derived virus contributed to 50% of the total viral load during primary infection, and there was no preference for dissemination of EC-derived viruses over viruses produced by other cell types. In addition, during secondary viremia, there was only a negligible contribution of EC-derived virus to dissemination to other organs. These results are novel in the methodology employed and are somewhat interesting. However, the data are limited to the mouse model with a short-term follow-up, and the immunodeficient host has not yet been studied. In humans, these conclusions must be taken with caution. First, in primary infection occurring through natural routes, epithelial cells are infected first, then ECs, unless primary infection occurs through blood transfusion, in which case endothelial vascular cells may become infected first. In both cases, the virus transport occurs through the intervention of leukocytes, namely monocytes and polymorphonuclear leukocytes. As monocytes differentiate to macrophages, they become highly susceptible to human CMV replication inside organ tissues, while polymorphonuclear leukocytes are active in virus capturing from infected endothelial vascular cells and transporting to distant sites.

Profilaxis de la infección por citomegalovirus en el trasplante hepático

CMV prevention strategies in liver transplant recipients should be stratified according to serological status. In donor (D)-/recipient (R)- combinations, no prophylaxis or preemptive therapy is recommended. In the remaining combinations, the most widely used strategies are universal prophylaxis and preemptive therapy. Both strategies are effective compared with placebo but have not been compared with each other in well-designed clinical trials. Preemptive therapy is the preferred strategy in low-risk patients while prophylaxis is the most widely used option in those at high-risk. Delayed CMV disease is an adverse consequence of universal prophylaxis. Prolongation of prophylaxis from 100 to 200 days does not reduce the incidence of CMV disease. CMV-specific cell mediated immunity, facilitated by preemptive therapy and delayed by prophylaxis, has a therapeutic effect by reducing CMV replication. The drug of choice in both strategies is valganciclovir but the duration and dose differ. When preemptive therapy is used, viremia monitoring is required for the first 4 months. The technique of choice is quantitative polymerase chain reaction. Given the lack of conclusive data, the choice of one or other strategy in these patients should be individualized in each patient and center according to the available resources and possibilities of follow-up.

Cytomegalovirus-Specific T Cell Immunity Is Maintained in Immunosenescent Rhesus Macaques

Although CMV infection is largely benign in immunocompetent people, the specific T cell responses associated with control of this persistent virus are enormous and must be maintained for life. These responses may increase with advanced age and have been linked to an "immune risk profile" that is associated with poor immune responsiveness and increased mortality in aged individuals. Based on this association, it has been suggested that CMV-specific T cell responses might become dysfunctional with age and thereby contribute to the development of immune senescence by homeostatic disruption of other T cell populations, diminished control of CMV replication, and/or excess chronic inflammation. In this study, we use the rhesus macaque (RM) model of aging to ask whether the quantity and quality of CMV-specific T cell responses differ between healthy adult RMs and elderly RMs that manifest hallmarks of immune aging. We demonstrate that the size of the CD4(+) and CD8(+) CMV-specific T cell pools are similar in adult versus old RMs and show essentially identical phenotypic and functional characteristics, including a dominant effector memory phenotype, identical patterns of IFN-γ, TNF-α, and IL-2 production and cytotoxic degranulation, and comparable functional avidities of optimal epitope-specific CD8(+) T cells. Most importantly, the response to and protection against an in vivo CMV challenge were identical in adult and aged RMs. These data indicate that CMV-specific T cell immunity is well maintained in old RMs and argue against a primary role for progressive dysfunction of these responses in the development of immune senescence.

Sustained elite suppression of replication competent HIV-1 in a patient treated with rituximab based chemotherapy

The mechanism of elite control of HIV-1 replication is not fully understood. While immunosuppression due to rituximab based chemotherapy has been associated with increased replication of HBV, CMV, and HIV-1, control of replication-competent HIV-1 was maintained in an elite controller/suppressor treated with a regimen that included vincristine, cyclophosphamide, prednisone, four rounds of plasmapheresis and ten cycles of rituximab. The data suggests that de-novo antibody responses do not play a significant role in the control of viral replication in these patients.

Noncytotoxic Inhibition of Cytomegalovirus Replication through NK Cell Protease Granzyme M-Mediated Cleavage of Viral Phosphoprotein 71

Granzyme M (GrM) is highly expressed in cytotoxic granules of NK cells, which provide the first line of defense against viral pathogens. GrM knockout mice show increased susceptibility toward murine CMV infection. Although GrM is a potent inducer of cell death, the mechanism by which GrM eliminates viruses remains elusive. In this paper, we show that purified human GrM in combination with the perforin-analog streptolysin O (SLO) strongly inhibited human CMV (HCMV) replication in fibroblasts in the absence of host cell death. In a proteomic approach, GrM was highly specific toward the HCMV proteome and most efficiently cleaved phosphoprotein 71 (pp71), an HCMV tegument protein that is critical for viral replication. Cleavage of pp71 occurred when viral lysates were incubated with purified GrM, when intact cells expressing recombinant pp71 were challenged with living cytotoxic effector cells, and when HCMV-infected fibroblasts were incubated with SLO and purified GrM. GrM directly cleaved pp71 after Leu(439), which coincided with aberrant cellular localization of both pp71 cleavage fragments as determined by confocal immunofluorescence. In a luciferase reporter assay, cleavage of pp71 after Leu(439) by GrM completely abolished the ability of pp71 to transactivate the HCMV major immediate-early promoter, which is indispensable for effective HCMV replication. Finally, GrM decreased immediate-early 1 protein expression in HCMV-infected fibroblasts. These results indicate that the NK cell protease GrM mediates cell death-independent antiviral activity by direct cleavage of a viral substrate.

Peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate

Dendrimers are hyperbranched synthetic well-defined molecules with a number of potential applications, especially in relation to the need for new antiviral agents. One subclass of dendrimers are peptide-derivatized dendrimers which consist of a peptidyl branching core and covalently attached surface peptide functional units. Few studies have addressed the potential uses of peptide dendrimers as direct-acting antiviral agents. Here, we report on the ability of two peptide dendrimers, SB105 and SB105_A10, to directly and almost completely inhibit human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells; the agents were also found to inhibit murine CMV replication, whereas they were not able to inhibit adenovirus or vesicular stomatitis virus. The peptide dendrimers prevented adsorption of the HCMV to cells at 4 °C, whereas SB104, a dendrimer with a different amino acid sequence within the functional group and minimal anticytomegaloviral activity, was ineffective in blocking HCMV attachment. In effect, SB105_A10 bound to human cells through an interaction with cell surface heparan sulfate and thereby blocked virion attachment to target cells. These results indicate that the SB105 and SB105_A10 dendrimers could provide a useful starting point for the development of novel molecules to block HCMV infection.

The Bidirectional Relationship Between Cytomegalovirus Replication and Graft-Versus-Host Disease - a Retrospective Single Center Study.

Abstract 2236Poster Board II-213Cytomegalovirus (CMV) infection and graft-versus host disease (GVHD) are important complications after allogeneic HSCT with a clear link. Multiple studies show that GVHD and its treatment put patients at risk for CMV reactivation. Data on CMV infection as a cause of GVHD, in contrast, are controversial. The association of pre-transplant CMV serology with GVHD development and reduced rates of chronic GVHD after preemptive CMV treatment are indicative of such an association. However, analyses of the direct impact of CMV infection on GVHD are rare; a recent small study found no effect of CMV replication on subsequent development of acute GVHD (Wang et al, BMT 2008). We analyzed in a single centre study the association of CMV reactivation with acute GVHD in 517 patients treated between 1993 and 2008. 59% of patients were male, median age was 42 years (range 16 to 70). Diagnoses were AML (31%), ALL (16%), CML (15%), MDS/MPN (13%), lymphoma (21%), and other (4%). Conditioning regimens were Cy/TBI ±/- etoposide (49%), Cy/Bu (17%), fludarabine and TBI (16%), or others (18%). GVHD prophylaxis consisted of CyA/MTX (78%) or CyA/MMF (21%). Donors were HLA-identical siblings (65%), other family members (4%), or unrelated donors (31%). We made use of a standardized CMV policy over the last decades. CMV reactivation was monitored using real-time polymerase chain reaction or pp65 antigenemia assay weekly in patients without infection, twice weekly in patients with CMV replication. CMV was preemptively treated with gancyclovir or foscarnet. To determine the correlation of CMV infection with acute GVHD, we used a stringent Cox regression model, in which CMV replication was modeled as a time-dependent covariate becoming positive on the day of the first detection of CMV and negative on the first negative assay thereafter. Multiple episodes of CMV replication were considered. Acute GVHD was modeled as a time-dependent covariate in models with CMV infection as endpoint. Hazard ratios (HR) were adjusted for patient age, disease, disease stage, donor type, stem cell source, conditioning regimen and GVHD prophylaxis. The analysis was restricted to the time from transplant to day 100. CMV reactivation was detected at least once in 16% (84/517) of patients at a median of 33 (range 1-95) days after HSCT. Median duration of CMV reactivation was 8.5 days (range 2-62). 19 patients showed multiple episodes of CMV replication. Donor and recipient serostatus significantly influenced the day 100 cumulative incidence of CMV infection: D-/R- (N=173) 6%; D±/R- (N=61) 10%; D±/R± (N=128) 25%; and D-/R± (N=99) 37%, p<0.001. The cumulative incidence for any acute GVHD (grade I-IV) was 67% (95% CI 56-78%) with a median onset time at day 14 (range day 5-94); the cumulative incidence for severe acute GVHD (grade II-IV) was 48% (95% CI 40-56%). When both endpoints (CMV, GVHD) were combined, 150 patients (29%) experienced neither GVHD nor CMV reactivation, 281 (54%) GVHD only, 19 (4%) CMV reactivation only, and 67 (13%) both CMV reactivation and GVHD. Of the 67 patients with both GVHD and CMV, 46 (69%) developed GVHD prior to CMV reactivation, 17 (25%) developed GVHD during CMV reactivation, and 4 (6%) developed GVHD after CMV reactivation. Cox modeling revealed that presence of GVHD grade II-IV increased the risk of CMV infection (HR 1.61, 95% CI 1.03-2.52, p=0.04). Similarly, patients were at increased risk of developing acute GVHD during phases of CMV replication (grades I-IV: HR 2.23, 95% CI 1.39-3.81, p=0.001; grades II-IV: HR 2.00, 95% CI 1.08-3.72, p=0.03). GVHD grade was not influenced by concomitant CMV reactivation (median grade II, in patients with or without CMV reactivation). The overall proportion of GVHD that occurred during phases of CMV replication was small (3% versus 64% occurring in CMV non replicating patients). Even if GVHD occurring after resolution of CMV reactivation was additionally taken into account, the majority of GVHD occurred without preceding CMV infection (63% versus 4%). These data describe the complex relationship between CMV infection and GVHD. We confirm previous studies that GVHD (and GVHD therapy) can induce CMV infection. We describe as well that patients with active CMV replication have a significantly higher risk of developing GVHD compared to patients without CMV replication. However, the proportion of GVHD that could be linked to CMV reactivation was small in this population with a low overall incidence of CMV reactivation. Disclosures:No relevant conflicts of interest to declare.

Adoptive Transfer of CMV-Specific Donor T Cells Following Allogeneic Transplantation Leads to Rapid and Safe Systemic Reconstitution.

Abstract 44Reactivation of CMV remains a significant problem following allogeneic hematopoietic stem cell transplantation. Antiviral drug therapy is effective but toxic, and resistant strains of CMV are increasingly being reported. Virus-specific T lymphocytes are necessary for the control of viral reactivation. Adoptive transfer of donor derived CMV-specific T cells has been reported previously but most methods to produce such cells have involved several weeks of in vitro culture or have produced a therapeutic product restricted to CD8 T cells. The current method involves a short incubation of donor peripheral blood mononuclear cells with either CMV-pp65 protein (20 hours) or a pool of peptides from pp65 (6 hours) with subsequent isolation of interferon-gamma secreting cells by CliniMACS using IFNψ capture microbeads (Miltenyi Biotec). This technique permits rapid isolation of an enriched IFNψ secreting T cell product, manufactured to clinical grade, which is then cryopreserved in dosed aliquots for subsequent infusion. Here we report the outcome of a single arm phase I/II in which CMV-T cells given pre-emptively at first detection (qPCR) of CMV DNA in peripheral blood, or at day +40-60 as prophylaxis. CMV replication was monitored by weekly PCR and reconstitution of CMV-specific T cells by pentamer labelling and/or IFNψ secretion assay. Conventional antiviral drug therapy was instituted if the viral load rose above institutional threshold. 30 recipients of T cell depleted low intensity transplants from HLA-matched CMV-seropositive related donors were enrolled between 2006 and 2008. Donors underwent a second, short apheresis procedure approximately 15 days after collection of the mobilised HPC-A for the collection of CMV-T cells. 26 clinical-grade products were produced to full cGMP standards; four donors were unsuitable or withdrew. The mean yield of cells following enrichment was 41.7% with a median purity of 43.9% (range 1.4-81.8). Adequate CMV-T cells were isolated from all donors. Both pp65 and peptide stimulated products contained both CD4 and CD8 reactive T cells. Median dose of CMV-specific CD4 T cells was 2840/kg and of CMV-specific CD8 was 630/kg. Eighteen patients received a single dose of 1×10^4 CD3+/kg; 13 were CMV seropositive; 11 were treated pre-emptively and 7 prophylactically. 83% had received T cell deplete regimens. Within 2 weeks of infusion in vivo expansion of CMV-T cells was observed in 17 of 18 patients. One patient required 4 weeks to generate detectable CMV-T cell in his peripheral blood. TCR-BV usage of the CMV-T cells post infusion matched that of the cells which had been infused. The 7 patients who had cells infused prophylactically all showed expansions of CMV-T cells in the absence of detectable viral DNA in peripheral blood. Subsequent low level CMV-reactivation was seen in one of these and was associated with rapid CMV-T cell expansion with clearance of virus without anti-viral drug therapy. One developed subsequent extensive chronic GvHD and required antiviral treatment for multiple reactivation episodes following introduction of steroids. Of the 11 patients treated pre-emptively, 9 received antiviral therapy for the initial reactivation, although in 7 patients this was required for only 7-15 days. (compared to a median of 21 days in historical controls). Three patients had a further CMV reactivation event. One followed prednisolone therapy for acute grade II GvHD. The second was the patient who had shown poor T cell expansion post infusion and had required prolonged anti-viral therapy (33 days) for the initial CMV reactivation. The third patient received no treatment and cleared virus following a further in vivo expansion of CMV-reactive T cells, suggesting the presence of a functional memory population. GVHD incidence and severity was no worse than seen in comparable historical controls. 3 patients suffered grade 2-3 acute GvHD. 3/17 evaluable patients developed extensive chronic GvHD (2 were recipients of T replete grafts). 16/18 patients were alive at the end of the 6 month monitoring period and CMV-reactive T cells were detectable in all 16. CMV-specific donor T cells can be readily produced to cGMP compliance which can be safely infused and lead to early immune reconstitution in at-risk patients. Cells expand in response to subsequent CMV-reactivation and patients appear to require fewer anti-viral treatment episodes which is being tested in an ongoing phase III trial. Disclosures:Lowdell:Cell Medica Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.