CMV-specific Cell-mediated Immunity Research Articles

CMV-Specific Cell-Mediated Immunity in Immunocompetent Adults with Primary CMV Infection: A Case Series and Review of the Literature.

Cytomegalovirus-specific cell-mediated immunity (CMV-CMI) in actively infected healthy immunocompetent hosts has been poorly investigated. Conversely, correlates of maternal protective immunity for the fetus after primary infection in pregnancy continue to be studied. The kinetics and magnitude of CMV-specific CMI in immunocompetent primary CMV-infected adults are described. A literature review on CMV-CMI in primarily infected pregnant women and its correlation to the risk of vertical virus transmission is included. Immunological measurements after infection were performed by enzyme-linked ImmunoSPOT assay enumerating IFN-γ secreting CMV-specific T cells, at a single cell level, upon in vitro stimulation with viral antigens. Simultaneously, serological and virological profiles of infected patients were investigated. Patients displayed mild-to-moderate clinical and laboratory profiles for infection, and all showed positive EliSpot results in the early stage of infection (<20 days after onset). The virus-CMI was strong in the majority of patients (58.8%) in which the lowest CMV-DNAemia levels (<300 copies/mL) were detected. Significantly higher viral loads were observed in patients with weak CMV-CMI at the same time-point post-infection (up to 15,104 copies/mL; p < 0.001). T cell response magnitudes to IE-1 and pp65-UL83 peptides were overlapping and stable over time. In these case series, the early presence of CMV-CMI was probably pivotal in controlling viral replication and led to spontaneous viral clearance.

Association of CMV-specific T-cell immunity and risk of CMV infection in lung transplant recipients.

Protecting against CMV infection and maintaining CMV in latent state are largely provided by CMV-specific T-cells in lung transplant recipients. The aim of the study was to assess whether a specific T-cell response is associated with the risk for CMV infection in seronegative patients who are at high risk for delayed CMV infection. All CMV-seronegative recipients (R-) from CMV-seropositive donors (D+) between January 2018 and April 2019 were included and retrospectively screened for CMV infection before and after assessment of CMV-specific cell-mediated immunity. Thirty-one of the 50 patients (62%) developed early-onset CMV infection. Lower absolute neutrophil counts were significantly associated with early-onset CMV infection. Antiviral prophylaxis was ceased after 137.2±42.8days. CMV-CMI were measured at a median of 5.5months after LTx. 19 patients experienced early and late-onset CMV infection after prophylaxis withdrawal within 15months post transplantation. Positive CMV-CMI was significantly associated with lower risk of late-onset CMV infection after transplantation in logistic and cox-regression analysis (OR=0.05, p=.01; OR=2,369, p=.026). D+/R- lung transplant recipients are at high risk of developing early and late-onset CMV infection. Measurement of CMV-CMI soon after transplantation might further define the CMV infection prediction risk in LTx recipients being at high risk for CMV viremia.

1102. Reconstitution of CMV-specific cell-mediated immunity during letermovir prophylaxis in hematopoietic stem cell recipients

BackgroundPatients who are cytomegalovirus (CMV) seropositive (R+) prior to hematopoietic cell transplant (HCT), have 30% incidence of clinically significant CMV reactivation in the absence of prophylaxis. At our institution, letermovir prophylaxis through Day 100 is used in CMV R+ high-risk (HR) (cord blood, haplocord, haploidentical) HCT recipients. We hypothesized that clinically nonsignificant CMV reactivation during letermovir prophylaxis may lead to reconstitution of CMV specific cell mediated immunity (CMV CMI), which may protect the host against CMV disease after letermovir discontinuation.MethodsBlood samples from CMV R+ HR HCT recipients on letermovir were tested by dual color CMV specific IL2/IFNg FLUOROSpot pre-transplant and on Days 100, 182 and 360 post-transplant. Clinical and virologic information were obtained from medical records.ResultsAmong 35 participants enrolled to date, 19 were eligible for this analysis, which included only participants with CMV CMI defined as ≥20 spot-forming cells/106 PBMC pre-transplantation and follow up ≥180 post-transplantation. Median age was 51.5 years (range 22-75), 9 were women, 9 were white non-Hispanic, 8 were Hispanic and the most common underlying malignancy was acute myeloid leukemia (n=10). 14 participants had CMV CMI reconstitution at Day 100; including 5 with and 9 without low level CMV DNAemia, defined as <5000 international units/ml in whole blood quantitative polymerase chain reaction assay, while on letermovir prophylaxis. Among the 14 participants, 11 remained free of clinically significant CMV reactivation for a median (range) of 260 (80; 260) days post-letermovir discontinuation, while 3 developed acute graft vs. host disease (aGvHD) followed by clinically significant CMV reactivation. 5 participants did not reconstitute CMV CMI at Day 100 and none of them had DNAemia while on letermovir. 1 of 5 participants without CMV CMI reconstitution or aGvHD developed CMV disease after letermovir discontinuation.ConclusionHigh-risk patient populations can reconstitute CMV CMI while on letermovir. Ongoing investigation will help establish predictive parameters for CMV CMI that may allow risk stratification for CMV monitoring and letermovir usage.Disclosures Maheen Abidi, MD, Merck (Research Grant or Support) Jonathan Gutman, MD, Merck (Research Grant or Support) Adriana Weinberg, MD, GSK (Grant/Research Support)merck (Grant/Research Support)

CMV-Specific Cell-Mediated Immunity Predicts a High Level of CMV Replication After Prophylaxis Withdrawal in Lung Transplant Recipients.

Monitoring cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) is useful in predicting late-onset CMV infection after solid organ transplantation, but few data have been reported after lung transplantation (LT). CMV CMI against 2 CMV antigens (IE-1, pp65) was evaluated in 60 seropositive LT at 6-month prophylaxis withdrawal. LT with late-onset CMV infection showed significantly lower (IE-1)CMV CMI than patients without (P = .045), and was more evident in patients developing high viral loads (P = .010). (IE-1)CMV CMI independently predicted high first late-onset viral replication (odds ratio, 4.358; 95% confidence interval, 1.043-18.215). CMV-specific CMI may be useful in CMV preventive strategies after LT.

Pretransplant CMV-Specific T-Cell Immunity But Not Dose of Antithymocyte Globulin Is Associated With Recovery of Specific Immunity After Kidney Transplantation.

This is a prospective, multicenter, observational study in cytomegalovirus (CMV)-seropositive kidney transplant recipients with pretransplant CMV-specific cell-mediated immunity (CMV-CMI) receiving antithymocyte globulin (ATG). We aimed to investigate posttransplant CMV-CMI over time and the impact of the dose-dependent ATG. CMV-CMI was assessed at days +30, +45, +60, and +90 after transplantation with the QuantiFERON-CMV assay. A reactive result (interferon-γ [IFN-γ] ≥ 0.2 IU/mL) indicated a positive CMV-CMI. A total of 78 positive CMV-CMI patients were enrolled in the study, of which 59.5% had a positive CMV-CMI at day +30 and 82.7% at day +90. Multivariate logistic regression analysis showed that ATG dose was not associated with positive CMV-CMI at any point. However, pretransplant IFN-γ level (>12 IU/mL vs ≤12 IU/mL) was associated with positive CMV-CMI at day +30 (odds ratio, 12.9; 95% confidence interval, 3.1-53.3; P < .001). In addition, all the patients who did not recover CMV-CMI at day +90 had a pretransplant IFN-γ level ≤12 IU/mL. More than half of CMV-seropositive kidney transplant recipients receiving ATG recover (or maintain) CMV-CMI by the first month after transplantation. The pretransplant IFN-γ level, but not the ATG dose, shows a strong association with the kinetics of this recovery.

Monitoring of CMV-specific cell-mediated immunity with a commercial ELISA-based interferon-γ release assay in kidney transplant recipients treated with antithymocyte globulin.

Monitoring for cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) may be useful for individualizing valganciclovir (VGCV) prophylaxis after kidney transplantation (KT). We performed a commercial ELISA-based interferon (IFN)-γ release assay (QTF-CMV) from posttransplant months 2-5 (362 points) in 120 CMV-seropositive KT recipients that received antithymocyte globulin as induction therapy and VGCV prophylaxis (median of 92days). Forty-seven patients (39.3%) had CMV infection after discontinuation of prophylaxis. The QTF-CMV assay was reactive, nonreactive, and indeterminate in 264 (72.9%), 90 (24.9%), and 8 points (2.2%). The QTF-CMV assay at prophylaxis discontinuation exhibited suboptimal accuracy for predicting protective CMV-CMI (sensitivity: 77.4%; specificity: 34.3%; positive predictive value [PPV]: 64.1%; negative predictive value [NPV]: 50.0%), with no differences in 1-year CMV infection rates between patients with negative (nonreactive or indeterminate) or reactive results (45.8% vs 36.1%; P=.244). Specificity and PPV to predict protective CMV-CMI improved by elevating the IFN-γ cutoff value to 1.13IU/mL (65.7% and 71.4%) and 7.0IU/mL (85.7% and 76.2%), although NPVs decreased. The QTF-CMV assay as per manufacturer's interpretative criteria performed poorly to predict protection from CMV infection following discontinuation of VGCV prophylaxis among ATG-treated CMV-seropositive KT recipients. This performance is slightly improved by modifying the IFN-γ positivity threshold.

Cellular Immunity to Predict the Risk of Cytomegalovirus Infection in Kidney Transplantation: A Prospective, Interventional, Multicenter Clinical Trial.

Improving cytomegalovirus (CMV) immune-risk stratification in kidney transplantation is highly needed to establish guided preventive strategies. This prospective, interventional, multicenter clinical trial assessed the value of monitoring pretransplant CMV-specific cell-mediated immunity (CMI) using an interferon-γ release assay to predict CMV infection in kidney transplantation. One hundred sixty donor/recipient CMV-seropositive (D+/R+) patients, stratified by their baseline CMV (immediate-early protein 1)-specific CMI risk, were randomized to receive either preemptive or 3-month antiviral prophylaxis. Also, 15-day posttransplant CMI risk stratification and CMI specific to the 65 kDa phosphoprotein (pp65) CMV antigen were investigated. Immunosuppression consisted of basiliximab, tacrolimus, mycophenolate mofetil, and corticosteroids in 80% of patients, whereas 20% received thymoglobulin induction therapy. Patients at high risk for CMV based on pretransplant CMI developed significantly higher CMV infection rates than those deemed to be at low risk with both preemptive (73.3% vs 44.4%; odds ratio [OR], 3.44 [95% confidence interval {CI}, 1.30-9.08]) and prophylaxis (33.3% vs 4.1%; OR, 11.75 [95% CI, 2.31-59.71]) approaches. The predictive capacity for CMV-specific CMI was only found in basiliximab-treated patients for both preemptive and prophylaxis therapy. Fifteen-day CMI risk stratification better predicted CMV infection (81.3% vs 9.1%; OR, 43.33 [95% CI, 7.89-237.96]). Pretransplant CMV-specific CMI identifies D+/R+ kidney recipients at high risk of developing CMV infection if not receiving T-cell-depleting antibodies. Monitoring CMV-specific CMI soon after transplantation further defines the CMV infection prediction risk. Monitoring CMV-specific CMI may guide decision making regarding the type of CMV preventive strategy in kidney transplantation. NCT02550639.

Monitoring of CMV-Specific Cell-Mediated Immunity in Kidney Transplant Recipients With a High Risk of CMV Disease (D+/R−): A Case Series

Cytomegalovirus (CMV) is the most common viral pathogen in kidney transplant recipients (KTRs), and CMV disease impacts patient and graft survivals. CMV-specific CD8 T cell mediated–immunity (CMI) may help to assess the risk of CMV disease and to adapt preventive treatment strategies.High-risk KTRs with CMV seropositive donors/seronegative recipients (D+/R−) were prospectively monitored after CMV prophylaxis discontinuation and during the first year post transplant for CMV viremia (World Health Organization standardization) and CMI (QuantiFERON-CMV). We analyzed the ability of CMI test to predict either subsequent spontaneous viral clearance or CMV disease after prophylaxis discontinuation in patients with asymptomatic viremia.We enrolled 12 consecutive (D+/R−) KTRs. Eleven patients developed a viremia during follow-up, but 7 of them (64%) exhibited a spontaneous viral clearance. At viremia onset, 6 of 11 patients (55%) had a positive CMI test, and all of them (6 of 6, 100%) had subsequent spontaneous viral clearance, compared with only 1 of 5 patients (20%) displaying a nonreactive CMI (P = .02). This latter patient exhibited a positive CMI test 15 days after viremia onset. Four of the 11 patients (36%) developed a CMV disease, and their CMI either remained nonreactive or became positive only after antiviral treatment.We conclude that D+/R− KTRs with asymptomatic viremia after prophylaxis discontinuation may benefit from QuantiFERON-CMV to predict when positive for the spontaneous viral clearance or when persistently negative or the development of a CMV disease.

Different impact of rATG induction on CMV infection risk in D+R- and R+ KTRs.

Rabbit antithymocyte globulin (rATG) induction is associated with profound immunosuppression, leading to a higher risk of cytomegalovirus (CMV) infection compared with anti-interleukin 2 receptor antibody (anti-IL-2RA). However, this risk, depending on the baseline CMV serological recipient/donor status, is still controversial. The CMV DNAemia-free survival between rATG- and anti-IL-2RA-treated patients was analyzed in donor-positive/recipient-negative (D+R-) and recipient-positive (R+) patients in 1 discovery cohort of 559 kidney transplant recipients (KTRs) and 2 independent cohorts (351 and 135 kidney KTRs). The CMV-specific cell-mediated immunity (CMI) at baseline and at different time points after transplantation was assessed using an interferon γ enzyme-linked immunosorbent spot assay. rATG increased the risk of CMV DNAemia in R+ but not in D+R- KTRs. In R+ CMI-positive (CMI+) patients, the CMV DNAemia rate was higher in rATG-treated than in anti-IL-2RA-treated patients; no difference was observed among R+ CMI-negative (CMI-) patients. Longitudinal follow-up demonstrated a deeper depletion of preformed CMV CMI in R+ rATG-treated patients. D+R- KTRs have the highest risk of CMV DNAemia, but rATG adds no further risk. Among R+ KTRs, we described 3 groups, the least prone being R+CMI+ KTRs without rATG, then R+CMI+ KTRs with rATG, and finally R+CMI- KTRs. CMV serostatus, baseline CMV-specific CMI, and induction therapy may lead to personalized preventive therapy in further studies.

Clinical Validation of a Novel Elispot-Based in Vitro Diagnostic Assay: Monitoring Cytomegalovirus-Specific Cell-Mediated Immunity and Risk Stratification in Hematopoietic Stem Cell Transplant Recipients

Increasing evidence suggests that impaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of uncontrolled CMV reactivations and associated complications in hematopoietic stem cell transplantation (HSCT). No reliable test exists to predict patients at risk of primary and/or recurrent CMV reactivations following HSCT. Accurately assessing CMV-CMI might therefore improve the risk stratification of patients and allow optimizing and individualizing patient care. This study aimed to evaluate the suitability of a novel IFN-γ ELISpot assay (T-Track® CMV), based on the stimulation of peripheral blood mononuclear cells with T-activated® pp65 and IE-1 CMV proteins, to predict protection from recurrent CMV reactivation following the resolution of a treatment-requiring primary CMV reactivation. A prospective, longitudinal, observational, multicenter study was conducted in 175 intermediate- and high-risk (Donor (D)+/Recipient (R)+, D+/R-, D-/R+) HSCT recipients (ClinicalTrials.gov ID: NCT02156479). Patients underwent preemptive antiviral therapy per institutional guidelines. CMV DNAemia was analyzed by quantitative PCR. CMV-CMI was measured at day 45, 60, 80, 100 and 120 post-transplantation, as well as at onset and following the end of preemptive treatment. Occurrence of recurrent CMV reactivation was monitored up to 7.5 months post-transplantation. 154/175 patients fulfilling the inclusion/exclusion criteria and having at least one valid T-Track® CMV test result were included in the final analysis. 101/154 (66%) patients experienced at least one (treatment-requiring) CMV reactivation during the observational period. Out of 74 patients (24 D+/R+, 3 D+/R-, 47 D-/R+) who experienced a first CMV reactivation and had a valid ELISpot test result at the end of this primary reactivation, 30 (41%) faced a recurrent CMV reactivation during the observational period. Interestingly, 41/44 patients free of recurrent reactivation had a positive test result (i.e. positive for at least one of pp65- and/or IE-1-specific result) after resolution of the primary CMV reactivation, resulting in a 93% specificity in diagnostic accuracy. Accordingly, a time-to-event analysis indicated a significantly lower incidence of recurrent CMV reactivation in patients with a positive test result (Figure 1; Hazard ratio=5.68; Log-Rank Test, p Altogether, this novel standardized IFN-γ ELISpot assay allows an improved risk stratification of CMV-related clinical complications, and can support clinicians in the identification and management of patients with increased risk of recurrent CMV reactivation following HSCT.

1575. Clinical Validation of a Novel ELISpot-based in vitro Diagnostic Assay to Monitor CMV-Specific Cell-Mediated Immunity in SOT and HSCT Immunocompromised Patients

BackgroundImpaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of uncontrolled CMV reactivation and associated complications in both solid-organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT). Reliably assessing CMV-CMI is desirable to individually adjust antiviral and immunosuppressive therapy. We demonstrate here the suitability of a novel IFN-γ ELISpot assay (T-Track® CMV), based on the stimulation of PBMC with pp65 and IE-1 CMV proteins, to monitor CMV-CMI in SOT and HSCT patients.MethodsTwo independent prospective, longitudinal, observational, multicenter studies were conducted: in 86 intermediate-risk (D−/R+, D+/R+) renal transplant recipients (completed), and in 175 intermediate- or high-risk (D+/R+, D+/R−, D−/R+) HSCT recipients (ongoing). In both studies, patients underwent pre-emptive antiviral therapy. CMV-CMI, CMV load and clinical complications were monitored over ~6 months post-transplantation.ResultsIn the kidney transplantation setting, 95% and 88–92% of IFN-γ ELISpot test results were positive pre- and post-transplantation, respectively. CMV-specific response was reduced following immunosuppressive therapy and increased in patients with graft rejection, indicating the ability of the assay to monitor the patients’ immunosuppressive state. Interestingly, median pp65-specific response was 9-fold higher in patients with self-clearing viral load compared with antivirally-treated patients prior to first detection of CMV (MWU; P < 0.001), suggesting that reactivity to pp65 is a potential immunocompetence marker. In HSCT patients, interim data analysis indicates that pp65-specific CMI measured after resolution of a primary CMV reactivation (requiring antiviral treatment) is a fair predictor of occurrence of recurrent CMV reactivation. Out of 71 patients (25 D+/R+, 3 D+/R−, 43 D−/R+) who experienced a primary CMV reactivation, 27 encountered a recurrent CMV reactivation. Interestingly, 39/44 (89%) patients free of recurrent reactivation had a positive pp65-specific test result following primary CMV reactivation.ConclusionAltogether, this novel IFN-γ ELISpot assay is a highly sensitive immune-monitoring tool with a potential use for the risk assessment of CMV-related clinical complications after SOT and HSCT.Disclosures All authors, Lophius Biosciences: Investigator, Research support.

CMV-specific Cell-mediated Immunity at 3-month Prophylaxis Withdrawal Discriminates D+/R+ Kidney Transplants at Risk of Late-onset CMV Infection Regardless the Type of Induction Therapy.

Whether cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) at prophylaxis cessation predicts D+/R+ kidney transplants at risk of late-onset CMV infection after receiving distinct induction therapies is still not well characterized. We prospectively assessed CMV-specific CMI predicting late-onset CMV infection at prophylaxis withdrawal and at earlier time-points, in 96 consecutive D+/R+ patients receiving either anti-interleukin 2-receptor antibody (anti-IL2RA; n = 50) or rabbit antithymoglobulin (n = 46). CMV-specific CMI was evaluated against CMV antigens (IE-1, pp65) using an IFN-γ ELISpot assay. Fourteen (14.6%) of 96 patients developed late-onset CMV infection and 2 (2.1%) of 96 displayed disease. At 3 months, CMV-specific CMI frequencies were significantly lower in patients developing late-onset CMV infection (P < 0.001 for IE-1, P = 0.030 for pp65), regardless the type of induction therapy. Receiver operating characteristic curve analyses showed accurate CMV-specific CMI cutoffs (25 and 130 IFN-γ spots for IE-1 and pp65, respectively) classifying patients into high risk, intermediate risk, or low risk (log-rank = 0.006; hazard ratio, 4.084; 95% confidence interval, 1.431-11.651; P = 0.009), being IE-1 CMI the strongest predictor (odds ratio, 5.554; 95% confidence interval, 1.486-20.766; P = 0.011). Although the profound posttransplant CMV-specific CMI inhibition among rabbit antithymocyte globulin-treated patients precludes its use for risk stratification both before and early after kidney transplant, a similar proportion of at-risk patients could be identified before month 3 within anti-interleukin 2-receptor antibody-treated patients. Monitoring CMV-specific CMI at 3-month prophylaxis cessation discriminates kidney transplant recipient at risk of late-onset CMV infection, regardless the type of induction therapy.

Migrating From Universal to Personalized Prevention: Predicting the Risk of Cytomegalovirus Infection After Organ Transplantation.

In Brief The use of CMV-specific cell-mediated immunity to predict the risk of infection after the end of prophylaxis in CMV seropositive recipients is now possible. The commentator suggests that this testing in prevention protocols is useful and might be incorporated by programs.

CMV specific T cell immunity predicts early viremia after liver transplantation

BackgroundCytomegalovirus (CMV) infection is one of the most important factors affecting liver transplant with direct and indirect effects. However, CMV disease after transplant remains poorly predicted. ObjectiveIn this study, preoperative CMV-specific cell-mediated immunity was evaluated in recipients of liver transplant in Korea, where most people are seropositive. MethodsA total of 32 patients were enrolled in a prospective study, and blood samples were collected before liver transplant to determine CMV-specific cell-mediated immunity. Testing using ELiSpot IFN-γ (CMVspot) and CMV serology were performed simultaneously. ResultsCMVspot results showed that 30 recipients had CMV-specific cell-mediated immunity, of which 29 were positive for phosphoprotein 65 and 14 for immediate early protein 1 (IE-1). All patients were positive for CMV IgG before transplantation, and 17 patients had a CMV viremia episode after transplantation. CMVspot showed 100% specificity and positive predictive value, and 11.76% sensitivity to predict CMV viremia. Patients with positive or borderline results for IE-1 did not show viremia two months after transplantation (p = .041). ConclusionCMVspot may be helpful in establishing a treatment strategy that includes regular monitoring for risk stratification of CMV reactivation.

Clinical Validation of a Novel ELISpot-based in Vitro Diagnostic Assay to Monitor CMV-specific Cell-Mediated Immunity in Kidney Transplant Recipients

Introduction Impaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of uncontrolled CMV reactivation and associated complications in solid-organ transplantation. Reliably assessing CMV-CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of a novel IFN-γ ELISpot assay (T-Track® CMV), based on the stimulation of peripheral blood mononuclear cells with pp65 and IE-I CMV proteins, to monitor CMV-CMI following kidney transplantation. Materials and Methods A prospective, longitudinal, observational, multicenter study was conducted in 86 intermediate risk (D-/R+, D+/R+) renal transplant recipients. Patients underwent pre-emptive antiviral therapy. CMV-CMI, CMV viral load and clinical complications (CMV disease, opportunistic infections and graft dysfunction) were monitored over six months post-transplantation. Results 95% and 88-92% of IFN-γ ELISpot test results were positive pre- and post-transplantation, respectively. CMV-specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection. Interestingly, median pp65-specific response was 9-fold higher in patients with self-clearing CMV viral load compared to antivirally-treated patients prior to first detection of viral load (p<0.001). Discussion The observation that the proportion of positive test results remains high (88-92%) post-transplantation, under immunosuppressive treatment, demonstrates the sensitivity of the assay and thus its suitability to measure CMV-CMI in immunocompromised patients. Similarly, the detection of reduced CMV-CMI following immunosuppression and of elevated CMV-CMI in association with graft rejection, indicates the ability of the ELISpot assay to monitor patients’ immunosuppressive state. Finally, the increased response to pp65 prior to first detection of viral load in patients with self-limiting viremia suggests that reactivity to pp65 is a potential marker of immunocompetence. Conclusion Altogether, this novel IFN-γ ELISpot assay (T-Track® CMV) is a highly sensitive immune-monitoring tool, suitable for the follow-up of renal transplant recipients, and with a potential use for the risk assessment of CMV-related clinical complications. This work was supported in part by the Bayerische Forschungsstiftung Grant AZ 924-10 (to LD) and by institutional funds of the University of Regensburg (ReForM C Project 03-082; to BKK, later transferred to BB).

The Efficacy of CMV Specific Immune Reconstitution is Highly Dependent on Donor/Recipient Serostatus and Type of Solid Organ Transplanted

Introduction The CMV specific immune recovery is known to control viral infection and disease. The CMV specific cell-mediated immunity (CMI) and CMV specific humoral immune response (IgG titer and IgG avidity) was explored as candidate biomarker predictive of CMV infection. Materials 653 observations from 297 SOT including 103 kidney (K), 60 liver (L), 47 heart (H), 87 Lung (Lu) transplants. The follow-up was 0-400 days after transplant. The following parameters were examined: 1) CMV serostatus before transplant 2) CMV IgG titer 3) CMV IgG avidity 4) CMV-ELISPOT 5) primary (D+/R-) or non-primary (D±/R+) CMV infection 6) CMV viremia. Data were statistically analyzed using ANOVA and linear regression analysis and spline model. Results The main findings of the study are: 1) CMV IgG titers and avidity are not predictive biomarker for CMV infection. The CMV IgG titer and IgG avidity levels are comparable either in infected and non-infected patients. 2) CMV viral load is statistically different between infected D±/R+ and D+/R-. The viral load in infected D+/R- is 1 Log higher compared to D±/R+ (10^5 vs 10^4 respectively) 4) The CMV immune reconstitution is more rapid and efficient in D±/R+ compared to D+/R- 5) D±/R+ and D+/R- Kidney and Lung transplants display a statistically significant lower CMI compared to heart and liver transplants; 6) Kidney and Lung transplants display a similar pattern of immune recovery 7) The CMV cell mediated immunity biomarker is predictive of infection in all organs with exception of lung transplants. Conclusion The study shows a marginal role of humoral immunity in controlling CMV infection. The pattern of CMI immune recovery is highly dependent upon pre-transplant CMV serostatus, type of organ transplant and immunosuppression therapy. High levels of CMV specific cell-mediated immunity correlate with a reduced CMV infection risk in heart, kidney and liver transplants. A different scenario emerged in lung transplants: high levels of circulating CMV specific T-cells can be found in lung transplants but cannot prevent CMV infection and clear the virus. In this view the lung may be considered a "sanctuary site" for CMV replication.

T-Track-CMV and QuantiFERON-CMV assays for prediction of protection from CMV reactivation in kidney transplant recipients

BackgroundAssays detecting CMV-specific cell-mediated immunity (CMI) may support the current management of CMV infection in solid-organ transplant (SOT) recipients, by allowing a better risk assessment and adjusting antiviral treatment. ObjectivesThe primary endpoint was the performance of two tests measuring CMV-specific interferon-gamma production, both approved for commercial use in clinical settings. Secondarily, we determined a cut-off for the cellular immune response, which protects against CMV reactivation/infection. Study designThirty kidney transplant (KTx) patients were stratified according to their CMV-IgG status pre-transplantation (Tx) and were divided into two groups: pre-emptive (donor-/recipient+, donor+/recipient+) and prophylaxis (donor+/recipient−). An ELISpot (T-Track-CMV) was performed at month 1 post-Tx (pre-emptive group) and end of prophylaxis and one month thereafter (prophylaxis group). An ELISA (QuantiFERON-CMV) was performed every 2–4 weeks (pre-emptive) or monthly (prophylaxis), in parallel to the CMV viral load (PCR). ResultsA good positive agreement was obtained between the QuantiFERON-CMV or T-Track-CMV and the CMV-IgG (kappa = 0.839 and 0.824, respectively). A cut-off of 19.5 spot forming units (SFU)/200,000 lymphocytes for the T-Track-CMV IE-1 (AUC = 0.802, sensitivity 45%, specificity 100%) and 495 SFU/200,000 lymphocytes for the T-Track-CMV pp65 (AUC = 0.617, sensitivity 11%, specificity 100%) was defined to assess protection against reactivation. The QuantiFERON-CMV performed modestly (AUC = 0.477, cut-off 85.1 IU/ml). ConclusionsThe QuantiFERON-CMV and T-Track-CMV enable the functional assessment of CMV-specific CMI in KTx recipients. In combination with CMV viral load monitoring, T-Track-CMV results could stratify patients at risk of CMV reactivation/infection.

Regular monitoring of cytomegalovirus-specific cell-mediated immunity in intermediate-risk kidney transplant recipients: predictive value of the immediate post-transplant assessment

ObjectivePrevious studies on monitoring of post-transplant cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) are limited by single-centre designs and disparate risk categories. We aimed to assess the clinical value of a regular monitoring strategy in a large multicentre cohort of intermediate-risk kidney transplant (KT) recipients. MethodsWe recruited 124 CMV-seropositive KT recipients with no T-cell-depleting induction pre-emptively managed at four Spanish institutions. CMV-specific interferon-γ-producing CD4+ and CD8+ T cells were counted through the first post-transplant year by intracellular cytokine staining after stimulation with pp65 and immediate early-1 peptides (mean of six measurements per patient). The primary outcome was the occurrence of any CMV event (asymptomatic infection and/or disease). Optimal cut-off values for CMV-specific T cells were calculated at baseline and day 15. ResultsTwelve-month cumulative incidence of CMV infection and/or disease was 47.6%. Patients with pre-transplant CMV-specific CD8+ T-cell count <1.0 cells/μL had greater risk of CMV events (adjusted hazard ratio (aHR) 2.84; p 0.054). When the CMI assessment was performed in the immediate post-transplant period (day 15), the presence of <2.0 CD8+ T cells/μL (aHR 2.18; p 0.034) or <1.0 CD4+ T cells/μL (aHR 2.43; p 0.016) also predicted the subsequent development of a CMV event. In addition, lower counts of CMV-specific CD4+ (but not CD8+) T cells at days 60 and 180 were associated with a higher incidence of late-onset events. ConclusionsMonitoring for CMV-specific CMI in intermediate-risk KT recipients must be regular to reflect dynamic changes in overall immunosuppression and individual susceptibility. The early assessment at post-transplant day 15 remains particularly informative.

Clinical validation of a novel enzyme-linked immunosorbent spot assay-based invitro diagnostic assay to monitor cytomegalovirus-specific cell-mediated immunity in kidney transplant recipients: a multicenter, longitudinal, prospective, observational study.

Impaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of CMV reactivation and associated complications in solid-organ transplantation. Reliably assessing CMV-CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of T-Track® CMV, a novel IFN-γ ELISpot assay based on the stimulation of peripheral blood mononuclear cells with pp65 and IE-I CMV proteins, to monitor CMV-CMI following kidney transplantation. A prospective longitudinal multicenter study was conducted in 86 intermediate-risk renal transplant recipients. CMV-CMI, CMV viral load, and clinical complications were monitored over 6months post-transplantation. Ninety-five percent and 88-92% ELISpot assays were positive pre- and post-transplantation, respectively. CMV-specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection, indicating the ability of the ELISpot assay to monitor patients' immunosuppressive state. Interestingly, median pp65-specific response was ninefold higher in patients with self-clearing viral load compared to antivirally treated patients prior to first viral load detection (P<0.001), suggesting that reactivity to pp65 represents a potential immunocompetence marker. Altogether, T-Track® CMV is a highly sensitive IFN-γ ELISpot assay, suitable for the immunomonitoring of CMV-seropositive renal transplant recipients, and with a potential use for the risk assessment of CMV-related clinical complications (ClinicalTrials.gov Identifier: NCT02083042).

P208 Comparison of two assays for CMV specific cell mediated immunity in the recipients of liver transplantation

Aim To prevent serious problem due to cytomegalovirus (CMV) infection, antiviral prophylaxis is commonly used in recipients of solid-organ transplants. To predict which patient will develop CMV disease, CMV-specific cell-mediated immunity can be measured by Quantiferon-CMV, ELISpot assay, cytokine profiling, intracellular cytokine staining and tetramer staining. We were comparing Quantiferon-CMV and ELISpot assay in the recipient of liver transplantation in Korea, in which most people are seropositive. Methods We prospectively included 35 patients who will receive antiviral prophylaxis for liver transplantation in a tertiary university hospital. We had tested interferon-gamma level following in vitro stimulation with CMV antigen using two assays – EliSpot IFN-γ CMVspot (CMVspot) (Autoimmun Diagnostika, Germany) and Quantiferon-CMV (QF-CMV) (Qiagen, Australia). Blood samples of recipient collected before liver transplantation and CMVspot, CF-CMV and CMV IgM/IgG assay (BioMerieux, Germany) were performed simultaneously. For detection of CMV viremia, real-time PCR was performed using CMV R-gene kit (BioMerieux, France). The testing and interpretation were performed according to the manufacturer’s recommendation. Results The agreement rate of two assays was overall 51.5%. Although CMVspot results showed 32 recipients has CMV-specific cell-mediated immunity, 17 patients show reactive results in the QF-CMV assay. The 28 patients show the reactive result of CMVspot pp65 and the 13 patients show reactive results of CMVspot IE-1. Two samples were failing during the process of CMVspot on the sample reception and interpretation step and seven samples were interpreted indeterminate because of low mitogen reactivity in QF-CMV. All patients had a positive result of CMV IgG before transplantation and 16 patients had CMV viremia episode after transplantation. Conclusions To choose the method for testing cell-mediated immunity for CMV, laboratory have to concern about the characteristics of assays and regional seropositive prevalence. The results of two commercial assays can be different at one time for testing. Further evaluation was proceeding for accurate prediction of CMV disease after solid-organ transplantation.