Non‐organ‐specific and anti‐endothelial antibodies in relation to CMV infection and acute rejection in renal transplant recipients

Costa C, Touscoz GA, Bergallo M, Terlizzi ME, Astegiano S, Sidoti F, Sinesi F, Segoloni GP, Cavallo R. Non‐organ‐specific and anti‐endothelial antibodies in relation to CMV infection and acute rejection in renal transplant recipients.
Clin Transplant 2010: 24: 488–492.
© 2009 John Wiley & Sons A/S. Abstract: The presence of non‐organ‐specific (NOSA) and anti‐endothelial antibodies (AECAs) and the onset of rejection in relation to cytomegalovirus (CMV) infection was investigated in 96 renal transplant recipients: 48 CMV pp65‐antigenemia‐negative (group 1) and 48 positive (group 2). The presence of autoantibodies (autoAbs) was evaluated before and following renal transplantation (first three months) by indirect immunofluoresce. Before transplantation, none of the patients was positive to AECAs, while eight (8.3%) were positive to NOSAs. Post‐transplantation, AECA were found in none of patients from group 1 vs. 15/48 (31.2%) from group 2 (p < 0.05); NOSAs were detected in 9/48 (18.8%) and 9/48 patients from group 1 and 2, respectively. An acute rejection was diagnosed in ten cases: six of interstitial type (antigenemia‐, and AECA‐negative; two NOSA‐positive); four of vascular type (all of them NOSA‐negative, 3/4 antigenemia‐, and AECA‐positive). CMV infection did not seem to be significantly associated with the appearance of NOSAs, while there was a significant correlation with the occurrence of AECAs. No significant correlation was found between acute rejection and the occurrence of NOSAs, while 75% of the cases of vascular rejection was associated to CMV infection and AECA‐positivity, suggesting the pathogenic role of CMV‐mediated endothelial damage.     

First Report of Successful Treatment of Multidrug‐Resistant Cytomegalovirus Disease with the Novel Anti‐CMV Compound AIC246

We report the first case of cytomegalovirus (CMV) disease treated with AIC246, a novel anti‐CMV compound which targets the viral terminase complex and remains active against virus resistant to DNA polymerase inhibitors. A lung transplant recipient developed refractory multidrug‐resistant CMV disease involving the lungs, gastrointestinal tract and retina. His disease progressed despite treatment with all DNA polymerase inhibitors; multiple agents reported to have activity against CMV in case series, and reduction in his immunosuppressive medications. AIC246 which is in clinical development was obtained for emergency use, and combined with additional reduction in immunosuppression resulted in rapid clinical, virological and radiological resolution of disease. The patient has remained free of CMV disease or viremia off treatment for greater than 3 months. In summary AIC246, while still in development, may be a promising alternative to current therapies.     

HHV‐6 reactivation is often associated with CMV infection in liver transplant patients

Abstract Human herpesvirus 6 (HHV‐6) infection has been recently reported in liver transplant patients. HHV‐6 is closely related to cytomegalo‐virus (CMV), and some interaction between the viruses has been suggested. In this study, the post‐transplant HHV‐6 antigenemia was investigated in relation to symptomatic CMV infections in adult liver transplant patients. CMV infections were diagnosed by the pp65 antigenemia test and by viral cultures. HHV‐6 infections were demonstrated by the HHV‐6 antigenemia test and by serology. Significant symptomatic CMV infection was diagnosed in 42 of 75 patients during the first 6 months after transplantation. All CMV infections were successfully treated with ganciclovir. Concurrent HHV‐6 antigenemia was detected in 21 (50%) of 42 patients with CMV infection. All HHV‐6 infections were reactivations. HHV‐6 also responded to the antiviral treatment, but with less clear effect. In conclusion, HHV‐6 reactivation is often associated with CMV infection in liver transplant patients. The results support the suggestion that CMV and HHV‐6 may have interactions.     

Pretransplant Immediately Early‐1‐Specific T Cell Responses Provide Protection for CMV Infection After Kidney Transplantation

Cytomegalovirus (CMV) infection is still a major complication after kidney transplantation. Although cytotoxic CMV‐specific T cells play a crucial role controlling CMV survival and replication, current pretransplant risk assessment for CMV infection is only based on donor/recipient (IgG)‐serostatus. Here, we evaluated the usefulness of monitoring pre‐ and 6‐month CMV‐specific T cell responses against two dominant CMV antigens (IE‐1 and pp65) and a CMV lysate, using an IFN‐γ Elispot, for predicting the advent of CMV infection in two cohorts of 137 kidney transplant recipients either receiving routine prophylaxis (n = 39) or preemptive treatment (n = 98). Incidence of CMV antigenemia/disease within the prophylaxis and preemptive group was 28%/20% and 22%/12%, respectively. Patients developing CMV infection showed significantly lower anti‐IE‐1‐specific T cell responses than those that did not in both groups (p < 0.05). In a ROC curve analysis, low pretransplant anti‐IE‐1‐specific T cell responses predicted the risk of both primary and late‐onset CMV infection with high sensitivity and specificity (AUC > 0.70). Furthermore, when using most sensitive and specific Elispot cut‐off values, a higher than 80% and 90% sensitivity and negative predictive value was obtained, respectively. Monitoring IE‐1‐specific T cell responses before transplantation may be useful for predicting posttransplant risk of CMV infection, thus potentially guiding decision‐making regarding CMV preventive treatment.     

Cytomegalovirus‐Responsive CD8+ T Cells Expand After Solid Organ Transplantation in the Absence of CMV Disease

Cytomegalovirus (CMV) is a major cause of morbidity and mortality in solid organ transplant recipients. Approximately 60% of adults are CMV seropositive, indicating previous exposure. Following resolution of the primary infection, CMV remains in a latent state. Reactivation is controlled by memory T cells in healthy individuals; transplant recipients have reduced memory T cell function due to chronic immunosuppressive therapies. In this study, CD8⁺ T cell responses to CMV polypeptides immediate‐early‐1 and pp65 were analyzed in 16 CMV‐seropositive kidney and heart transplant recipients longitudinally pretransplantation and posttransplantation. All patients received standard of care maintenance immunosuppression, antiviral prophylaxis, and CMV viral load monitoring, with approximately half receiving T cell–depleting induction therapy. The frequency of CMV‐responsive CD8⁺ T cells, defined by the production of effector molecules in response to CMV peptides, increased during the course of 1 year posttransplantation. The increase commenced after the completion of antiviral prophylaxis, and these T cells tended to be terminally differentiated effector cells. Based on this small cohort, these data suggest that even in the absence of disease, antigenic exposure may continually shape the CMV‐responsive T cell population posttransplantation.     

Pretransplant Interferon‐γ Secretion by CMV‐Specific CD8+ T Cells Informs the Risk of CMV Replication After Transplantation

In this prospective study we analyzed pretransplant interferon‐γ secretion by cytomegalovirus (CMV)‐specific CD8+ T cells to assess its possible utility in determining the risk of CMV replication after solid organ transplantation. A total of 113 lung and kidney transplant patients were enrolled in the study but only 55 were evaluable. All CMV‐seronegative recipients were pretransplant “nonreactive” (IFNγ <0.2 IU/mL) (11/11), whereas 30/44 (68.2%) CMV‐seropositive (R+) recipients were “reactive” (IFNγ ≥0.2 IU/mL) and 14/44 (31.8%) were “nonreactive”. In the R(+) “nonreactive” group, 7/14 (50%) developed posttransplant CMV replication, whereas the virus replicated only in 4/30 (13.3%) of the R(+) “reactive” patients (p = 0.021). According to the best multivariate model, pretransplant “nonreactive” recipients receiving an organ from a CMV‐seropositive donor had a 10‐fold increased risk of CMV replication compared to pretransplant “reactive” recipients (adjusted OR 10.49, 95% CI 1.88–58.46). This model displayed good discrimination ability (AUC 0.80) and calibration (Hosmer–Lemeshow test, p = 0.92). Negative and positive predictive values were 83.7% and 75%, respectively. The accuracy of the model was 82%. Therefore, assessment of interferon‐γ secretion by cytomegalovirus (CMV)‐specific CD8+ T cells prior to transplantation is useful in informing the risk of posttransplant CMV replication in solid organ transplant patients.     

Clinical Utility of Cytomegalovirus (CMV) Serology Testing in High-risk CMV D+/R− Transplant Recipients

Late-onset cytomegalovirus (CMV) disease is a significant problem in D+/R- solid organ transplant (SOT) patients who receive antiviral prophylaxis. We assessed the clinical utility of CMV IgG and IgM serology testing for predicting late-onset CMV disease. We evaluated 352 D+/R- transplant recipients who participated in a trial comparing 100 days of ganciclovir versus valganciclovir prophylaxis. CMV serology was assessed on day 28, 56, 100, and 6 and 12 months post-transplant. IgG seroconversion occurred in 26.9% of patients by day 100, and in 63.4% and 75.3% by 6 and 12 months, respectively. IgM seroconversion occurred in 8.3%, 41.8% and 54.9% by day 100, month 6 and month 12, respectively. Seroconversion by day 100 (end of prophylaxis) was not predictive of subsequent CMV disease (CMV disease 13.3% if seropositive vs. 17.8% if seronegative; p = NS). However, at 6 months post-transplant, IgG serostatus was predictive of subsequent CMV disease between month 6 and 12 (CMV disease 1.3% if seropositive vs. 10.0% if seronegative; p = 0.002). In D+/R- patients, CMV serology testing is for the most part not clinically useful for predicting subsequent disease. However, seroconversion by 6 months may be useful for identifying patients at risk of late-onset CMV disease.     

Virus‐Specific CD8+ T Cells Cross‐Reactive to Donor‐Alloantigen Are Transiently Present in the Circulation of Kidney Transplant Recipients Infected With CMV and/or EBV

T cells play a dual role in transplantation: They mediate transplant rejection and are crucial for virus control. Memory T cells generated in response to pathogens can cross‐react to alloantigen, a phenomenon called heterologous immunity. Virus‐specific CD8⁺ T cells cross‐reacting to donor‐alloantigen might affect alloimmune responses and hamper tolerance induction following transplantation. Here, we longitudinally studied these cross‐reactive cells in peripheral blood of 25 kidney transplant recipients with a cytomegalovirus and/or Epstein‐Barr virus infection. Cross‐reactive T cells were identified by flow cytometry as virus‐specific T cells that proliferate in response to donor cells in a mixed‐lymphocyte reaction. In 13 of 25 patients, we found cross‐reactivity to donor cells for at least 1 viral epitope before (n = 7) and/or after transplantation (n = 8). Cross‐reactive T cells were transiently present in the circulation, and their precursor frequency did not increase following transplantation or viral infection. Cross‐reactive T cells expressed interferon‐γ and CD107a in response to both alloantigen and viral peptide and resembled virus‐specific T cells in phenotype and function. Their presence was not associated with impaired renal function, proteinuria, or rejection. In conclusion, virus‐specific T cells that cross‐react to donor‐alloantigen are transiently detectable in the circulation of kidney transplant recipients.     

巨细胞病毒抗原检测对巨细胞病毒病预防的意义

目的：提高对肾移植术后巨细胞病毒（CMV）病的防治水平,方法：应用免疫组织化学方法（LSAB法）对1997年度肾移植患者进行CMV抗原检测,对检查阳性的患者采用丙氧鸟苷预防性治疗,并与1996年度未预防治疗组进行对照.结果：1997年预防性治疗后,CMV病发病率为3．1％（5／160）,病死率为20．0（1／5）,而1996年未预防治疗组,CMV病发病率为11．7％（16／136）,病死率为43．8％（7／16）,结论：对肾移植术后CMV抗原检测阳性患者预防性应用丙氧鸟式能显著降低CMV病的发生率和病死率。     

PD‐1 Analysis on CD28−CD27− CD4 T Cells Allows Stimulation‐Independent Assessment of CMV Viremic Episodes in Transplant Recipients

Expression of the inhibitory receptor programmed death 1 (PD‐1) on cytomegalovirus (CMV)‐specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28^?CD27^? double negativity is known as a typical phenotype of CMV‐specific CD4 T cells. Therefore, the co‐expression of inhibitory receptors on CD28^?CD27^? CD4 T cells was assessed as a rapid, stimulation‐independent parameter for monitoring CMV complications after transplantation. Ninety‐three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross‐sectional and longitudinal manner. CMV‐specific CD4 T cell levels quantified after stimulation were compared to levels of CD28^?CD27^? CD4 T cells. PD‐1 and cytotoxic T lymphocyte–associated antigen 4 (CTLA‐4) expression on CD28^?CD27^? CD4 T cells were related to viremia. A percentage of ≥0.44% CD28^?CD27^? CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV‐specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD‐1 expression levels on CD28^?CD27^? CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA‐4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD‐1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD‐1 expression on CD28^?CD27^? CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation‐independent parameter to guide duration of antiviral therapy.

     

A prospective longitudinal analysis of cytomegalovirus (CMV)‐specific CD4+ and CD8+ T cells in kidney allograft recipients at risk of CMV infection

Cytomegalovirus (CMV)‐specific cellular immunity is essential in controlling CMV infection after transplantation. We investigated whether CMV‐specific T cell levels predict CMV DNAemia after kidney transplantation. Using cytokine‐flow cytometry, we enumerated interferon‐γ producing CMV‐specific CD4+ and CD8+ T cells at serial time points among CMV‐mismatched (D+/R?) and seropositive (R+) kidney recipients who received 3 months of valganciclovir prophylaxis. Among 44 patients, eight (18%) developed CMV DNAemia at a mean (±SD) time of 151 (±33) days after transplantation, including two (5%) with CMV syndrome and three (7%) with tissue‐invasive CMV disease. Cox proportional hazards regression analysis showed that CMV mismatch (D+/R?) status (HR: 13, 95% CI: 1.6–106.4; P = 0.02) and diabetes mellitus (HR: 5.6; 95%CI: 1.1–27.9; P = 0.03) were significantly associated with CMV DNAemia. In contrast, the percentage or change‐over‐time in CMV‐specific CD4+ [pp65 (P = 0.45), or CMV lysate (P = 0.22)] and CD8+ [pp65 (P = 0.43), or IE‐1 (P = 0.37)] T cells were not significantly associated with CMV DNAemia. CMV‐specific T cell assays have limited clinical utility among CMV R+ kidney recipients who received valganciclovir prophylaxis. On the other hand, the clinical utility of CMV‐specific T cell assays will need to be assessed in a larger cohort of CMV D+/R? kidney recipients who remain at high‐risk of delayed‐onset CMV disease.     

Prophylactic CMV therapy does not improve three‐yr patient and graft survival compared to preemptive therapy

Despite increasing evidence in favor of prophylactic valganciclovir treatment in kidney transplant recipients for the prevention of cytomegalovirus (CMV) infection, the impact of preemptive vs. prophylactic treatment on long‐term clinical outcomes is unclear. In this retrospective study, 187 kidney transplant recipients with serologic intermediate‐risk constellation (recipient CMV IgG positive) received either preemptive or prophylactic treatment with valganciclovir. Patient survival (primary endpoint), graft survival, viremia rates, and other CMV‐related outcomes were analyzed. Prophylactic therapy reduced the rates for CMV viremia during the first year (hazard ratio: 0.48, 95% confidence interval [CI] 0.30–0.75; p < 0.001). There was a trend for higher three‐yr patient mortality in the prophylactic group (hazard ratio: 5.08, 95% CI 0.62–41.3; p = 0.091), and the rate of graft loss was not reduced (hazard ratio: 0.93, 95% CI 0.32–2.68; p = 0.894). Estimated glomerular filtration rate over three yr was on average 6.8 mL/min/1.73 m² lower in the prophylactic group (95% CI −11.68 to −1.81; p = 0.007) using a multivariate random effects model but showed more improvement over time. Prophylactic valganciclovir treatment reduced the rate of CMV infections during the first year post‐transplant but no effects of prophylactic treatment on patient and graft survival or kidney function over three yr were observed.     

Cytomegalovirus (CMV) infection and risk of mortality in allogeneic hematopoietic stem cell transplantation (Allo‐HSCT): A systematic review,meta‐analysis,and meta‐regression analysis

Controversy surrounds the potential association between cytomegalovirus (CMV) infection and increased risk of mortality after allogeneic hematopoietic stem cell transplantation (Allo‐HSCT). A systematic literature search was conducted using the PubMed, EMBASE, and Web of Science databases, assessing the association between CMV infection, as documented by the pp65 antigenemia assay or by polymerase chain reaction (PCR) using blood specimens, and overall mortality (OM) and nonrelapse mortality (NRM) in the allo‐HSCT setting. Pooled effects were estimated using the generic inverse variance random effects model. Heterogeneity was evaluated by Cochrane's Q test and I² statistics. The source of heterogeneity was investigated by meta‐regression and subgroup analyses. Twenty‐six of 1367 studies fulfilled eligibility criteria. CMV infection identified by PCR monitoring was significantly associated with an increased risk of OM and NRM (hazard ratio 1.47, 95% confidence interval [1.20‐1.81], P ≤ .001; hazard ratio 1.68, 95% confidence interval [1.14‐2.49], P = .05, respectively). In this setting, the use of preemptive antiviral therapy (PET) resulted in a twofold increased risk of OM and NRM. The estimated effect sizes were associated with allo‐HSCT modalities. Although our analyses point to an association between CMV infection and an increased risk of OM and NRM in allo‐HSCT recipients, the high heterogeneity across studies prevented drawing of robust conclusions on this matter.     

Evaluation of CMV viral load using TaqMan CMV quantitative PCR and comparison with CMV antigenemia in heart and lung transplant recipients.

BACKGROUND: Quantitative assessment of cytomegalovirus (CMV) infection using the antigenemia test has been used to monitor CMV infection in heart and lung transplant patients enabling a preemptive treatment strategy. However, the method is labour intensive, samples have to be processed within a few hours and requires skilled interpretation. A comparative prospective evaluation of a real-time TaqMan CMV quantitative PCR (QPCR) with the CMV antigenemia was undertaken. METHODS: A real-time quantitative TaqMan CMV PCR from EDTA bloods was developed. In this study 25 heart transplant and single-lung transplant patients were monitored posttransplantation by antigenemia and TaqMan CMV QPCR. CMV DNA extracted from EDTA blood was amplified by TaqMan QPCR using primers and probe designed from the CMV glycoprotein B (gB) gene. Quantification of the genome copies is extrapolated from a standard curve generated from amplification of quantified standards. RESULTS: Antigenaemia levels and TaqMan CMV QPCR genome copies showed a linear correlation between the two assays (R=0.843, P=0.001). A clinically significant threshold of 50 CMV pp65 antigen positive polymorphonuclear leucocytes (PMNLs) per 200 000 cells previously reported was used to extrapolate an equivalent value of 40 000 (log 4.6) genome copies per ml of blood for the TaqMan CMV QPCR. CONCLUSIONS: The TaqMan system enables a rapid high-throughput of samples. The TaqMan CMV QPCR can be used as an accurate and robust alternative to the antigenemia test to predict CMV disease and to monitor effectiveness of treatment.     

CMV Infection in the Donor and Increased Kidney Graft Loss: Impact of Full HLA‐I Mismatch and Posttransplantation CD8+ Cell Reduction

Despite a large body of literature, the impact of chronic cytomegalovirus (CMV) infection in donor on long‐term graft survival remains unclear, and factors modulating the effect of CMV infection on graft survival are presently unknown. In this retrospective study of 1279 kidney transplant patients, we analyzed long‐term graft survival and evolution of CD8⁺ cell population in donors and recipients by CMV serology and antigenemia status. A positive CMV serology in the donor was an independent risk factor for graft loss, especially among CMV‐positive recipients (R⁺). Antigenemia was not a risk factor for graft loss and kidneys from CMV‐positive donors remained associated with poor graft survival among antigenemia‐free recipients. Detrimental impact of donor's CMV seropositivity on graft survival was restricted to patients with full HLA‐I mismatch, suggesting a role of CD8⁺ cells. In R⁺ patients with positive CMV antigenemia during the first year, CD8⁺ cell count did not increase at 2 years posttransplantation, in contrast to R^? recipients. In addition, marked CD8⁺‐cell decrease was a risk factor of graft failure in these patients. This study identifies HLA‐I full mismatch and a decrease of CD8⁺ cell count at 2 years as important determinants of CMV‐associated graft loss.