Post-transplant Cytomegalovirus Infection Research Articles

An Evaluation of the Benefit of Cytomegalovirus Prophylaxis with Acyclovir on Post-Transplant Cytomegalovirus Infection Prevention in a Population of Renal Transplant Recipients in Nigeria

Background: Cytomegalovirus (CMV) is an important infection in renal transplant recipients and may significantly impact recipients’ long-term outcome and graft survival. Objective: This study aimed to evaluate the benefit of prophylaxis with acyclovir on post-transplant CMV infection prevention in a population of renal transplant recipients in Lagos, Nigeria. Subjects and Methods: The study was a cross-sectional design involving renal transplant recipients attending post-transplant follow-up clinics in Lagos, Nigeria between October 2004 and July 2005. Data on the use of CMV prophylaxis were obtained from the hospital case records of the study subjects. Enzyme-Linked Immunosorbent Assay (ELISA) was employed to detect CMV IgM antibodies for the diagnosis of post-transplant CMV infection and Microsoft Excel and EPI-Info 2002 statistical software were used for data entry and analysis. Results: Forty (40) renal transplant recipients were studied, 32 recipients were males and 8 were females with M:F ratio of 4:1. The mean age of the recipients was 39 ± 11.6 years old. The recipients’ post-transplant duration ranged from 2 to 80 months (Mean 17.6 ± 18.6 months). Fifteen (37.5%) of the transplant recipients received acyclovir prophylaxis for six months, one recipient (2.5%) received ganciclovir prophylaxis for three weeks while 24 recipients (60%) received no prophylactic therapy. There was no significant difference in the prevalence of seropositive CMV-IgM between transplant recipients who used CMV prophylaxis and those who did not (Fisher exact p = 0.45). Conclusion: Prophylaxis with acyclovir for six months showed no significant benefit on post-transplant CMV infection prevention in renal transplant recipients.

Cytomegalovirus infection and kidney transplantation- A retrospective study of risk factors and long-term clinical outcome

Aim: The aim was to study the clinical characteristics of postrenal transplant cytomegalovirus (CMV) infection and analyze its risk factors and its impact on graft and patient survival. Materials and Methods: We reviewed medical records of 739 renal transplant patients over 17 years (2002–2018). The demographic characteristics of patients were collected and compared with and without CMV infection. Multiple logistic regression analysis was done to identify risk factors for posttransplant CMV infection. Kaplan–Meier survival curve analysis was performed to analyze graft and patient survival by CMV infection. Results: The prevalence of CMV infection in our center was 12.4%. The most common presentation of CMV infection posttransplant is CMV syndrome. The use of antirejection therapy (hazard ratio [HR] 4.2, 95% confidence interval [CI] 2.6–6.9, P = 0.00), and new-onset diabetes after transplantation (NODAT) (HR 5.95, 95% CI 3.4–10, P = 0.00) was independently associated with postrenal transplant CMV infection. In Kaplan–Meier survival analysis, death-censored graft survival was significantly superior in patients without CMV infection/disease (CMV group: 55.4% vs. non-CMV group: 70.6% at 140 months P = 0.046). Patient survival was also significantly superior in patients without CMV infection (CMV group :59.8% vs. non-CMV group: 75.9% at 140 months P = 0.016). Conclusions: The use of antirejection therapy and NODAT are strong risk factors for developing CMV infection. Posttransplant CMV infection has a significant negative impact on graft and patient survival.

1198. Measurement of Pre-transplant Anti-cytomegalovirus (CMV) Immunoglobulin G Titer to Predict Risk of CMV Infection in CMV-seropositive Kidney Transplant Recipients

BackgroundAlthough cytomegalovirus (CMV)-seropositive solid organ transplant recipients have a lower risk of CMV infection compared with CMV-seronegative recipients, some patients remain at risk of CMV infection after transplant. Low pre-transplant anti-CMV immunoglobulin G (IgG) titer has been reported as a predictor of CMV infection in CMV-seropositive liver and heart transplant recipients, but this association in CMV-seropositive kidney transplant (KT) recipients has not been explored. We investigated the pre-transplant anti-CMV IgG titer and other CMV infection risk factors in CMV-seropositive KT recipients.MethodsThis retrospective study was conducted on CMV-seropositive KT recipients aged >18 years old at Ramathibodi Hospital during 2017 and 2018. The cumulative incidence of CMV infection was estimated with Kaplan–Meier methodology. The pre-transplant anti-CMV IgG titer was measured with an enzyme-linked fluorescent immunoassay. Risk factors for CMV infection were analyzed with Cox proportional hazards models.ResultsOf the 340 included CMV-seropositive KT recipients (37% female; age [mean±SD]: 43±11 years), 69% and 64% received deceased-donor allograft and induction therapy, respectively. The anti-CMV IgG titer was < 20 and >20 AU/ml in 7.1% and 92.9% of patients, respectively. During a mean follow-up of 14 months, the cumulative incidence of CMV infection was 14.8%, including both asymptomatic CMV infection (69%) and tissue-invasive disease (31%). A pre-transplant anti-CMV IgG titer of < 20 AU/ml was significantly associated with CMV infection in both the univariate analysis (HR, 2.70; 95%CI, 1.21–6.05, [p=0.02]) and the multivariate analysis (HR, 2.98; 95% CI, 1.31–6.77, [p=0.009]). Other significant risk factors of CMV infection included older donor age (HR, 1.03; 95% CI, 1.01–1.06, [p=0.005]), anti-thymocyte induction therapy (HR, 2.90; 95% CI 1.09–7.74, [p=0.033]), and prolonged cold ischemic time (HR, 1.06; 95% CI, 1.02–1.10, [p=0.002]).ConclusionA low pre-transplant CMV-specific humoral immunity is independently associated with post-transplant CMV infection in CMV-seropositive KT recipients. The universally available anti-CMV IgG titer test could potentially stratify those at risk and target preventive strategy appropriately.Disclosures All Authors: No reported disclosures

1075. Absolute Lymphocyte Count as a Predictor of Cytomegalovirus (CMV) Infection and Recurrence in Hematopoietic Stem Cell Transplant (HSCT) Recipients

BackgroundCytomegalovirus (CMV) is a serious complication following Hematopoietic Stem Cell Transplant (HSCT) and can lead to serious organ disease and mortality. This study aimed to investigate the association between absolute lymphocyte count (ALC) and CMV to determine whether ALC could help to identify those at an increased risk of CMV infection and recurrenceMethodsAdults undergoing HSCT between 2011 and 2016 at Rigshospitalet, Denmark were included. Cox proportional hazards models investigated risk factors, including ALC, for CMV infection in the first year post-transplant and recurrent CMV infection 6 months after clearance and stopping CMV treatment for the first infection. For the primary outcome ALC was investigated as a time-updated risk factor lagged by 7 days, and for recurrent CMV, ALC measured at the time at the time of stopping treatment for the first CMV infection was investigated (+/- 7 days).ResultsOf the 352 HSCT recipients included, 57% were male, 40% received myeloablative conditioning, 42% had high risk (D-R+) CMV IgG serostatus at transplant and the median age was 56 (IQR 43-63). 143 (40.6%) patients had an episode of CMV DNAemia a median of 47 days after transplant (IQR 35-62). A lower current ALC (≤ 0.3 x109/L) was associated with a higher risk of CMV infection in univariate analysis compared to a high current ALC (> 1 x109/L). However, this association was attenuated after adjustment, particularly for acute graft versus host disease (Figure). 102 HSCT recipients were investigated for risk of recurrent CMV of which 41 (40.2%) had a recurrent CMV episode a median of 27 days (IQR 16-50) after stopping CMV treatment for the first infection. A lower ALC (≤ 0.3 x109/L) at the time of stopping CMV treatment was associated with a significantly higher risk of recurrent CMV after adjustment (Figure). A higher peak viral load (> 1500 IU/ml) during the first episode of CMV infection was also associated with an increased risk of recurrent CMV (aHR 2.47, 95%CI 1.00-6.10 compared to < 750 IU/ml).Association between absolute lymphocyte count (ALC) and risk of CMV infection and recurrent CMV within 6 months. **First CMV infection multivariable model also adjusted for sex, CMV serostatus, age, year of transplant, Charlson Comorbidity Index, Anti-thymocyte globulin (ATG) given, HLA donor-recipient matching, and acute graft versus host disease (time-updated) *Recurrent CMV infection multivariable model also adjusted for conditioning regimen, sex, CMV serostatus, age, year of transplant Anti-thymocyte globulin (ATG) given, HLA donor-recipient matching, and acute graft versus host disease and peak CMV viral load during the first CMV infectionConclusionA lower ALC at the time of stopping treatment for the first CMV infection was associated with an increased risk of recurrent CMV and could be used to help guide decisions for augmented CMV surveillance and clinical awareness of CMV disease symptoms in these patients. Disclosures All Authors: No reported disclosures

Pretransplant adaptive NKG2C+ NK cells protect against cytomegalovirus infection in kidney transplant recipients.

Cytomegalovirus (CMV) infection constitutes a complication for kidney transplant recipients (KTR) and CMV-specific T cells reduce the risk of viral replication in seropositive patients. CMV promotes the adaptive differentiation and expansion of an NK cell subset, hallmarked by expression of the CD94/NKG2C receptor with additional characteristic features. We previously reported an association of pretransplant NKG2C+ NK cells with a reduced incidence of CMV infection. We have strengthened the analysis in cryopreserved peripheral blood mononuclear cells from an enlarged KTR cohort (n=145) with homogeneous immunosuppression, excluding cases at low risk of infection (ie, CMV D-R-) or receiving antiviral prophylaxis. Moreover, adaptive NKG2C+ NK cell-associated markers (ie, NKG2A, CD57, Immunoglobulin-like transcript 2 [LIR1 or LILRB1], FcεRI γ chain, and Prolymphocytic Leukemia Zinc Finger transcription factor) as well as T lymphocyte subsets were assessed by multicolor flow cytometry. The relation of NKG2C+ NK cells with T cells specific for CMV antigens was analyzed in pretransplant patients (n=29) and healthy controls (n=28). Multivariate Cox regression and Kaplan-Meier analyses supported that NKG2C+ NK cells bearing adaptive markers were specifically associated with a reduced incidence of posttransplant symptomatic CMV infection; no correlation between NKG2C+ NK cells and CMV-specific T cells was observed. These results support that adaptive NKG2C+ NK cells contribute to control CMV infection in KTR.

Killer Immunoglobulin-Like Receptor 2DS2 (KIR2DS2), KIR2DL2-HLA-C1, and KIR2DL3 as Genetic Markers for Stratifying the Risk of Cytomegalovirus Infection in Kidney Transplant Recipients

Infection with cytomegalovirus (CMV) remains a major problem in kidney transplant recipients, resulting in serious infectious complications and occasionally mortality. Accumulating evidence indicates that natural killer cell immunoglobulin-like receptors (KIRs) and their ligands affect the susceptibility to various diseases, including viral infections (e.g., CMV infection). We investigated whether KIR genes and their ligands affect the occurrence of CMV infection in a group of 138 kidney transplant recipients who were observed for 720 days posttransplantation. We typed the recipients for the presence of KIR genes (human leukocyte antigen C1 [HLA-C1], HLA-C2, HLA-A, HLA-B, and HLA-DR1) by polymerase chain reaction with sequence-specific primers. The multivariate analysis revealed that the lack of KIR2DS2 (p = 0.035), the presence of KIR2DL3 (p = 0.075), and the presence of KIR2DL2–HLA-C1 (p = 0.044) were risk factors for posttransplant CMV infection. We also found that a lower estimated glomerular filtration rate (p = 0.036), an earlier time of antiviral prophylaxis initiation (p = 0.025), lymphocytopenia (p = 0.012), and pretransplant serostatus (donor-positive/recipient-negative; p = 0.042) were independent risk factors for posttransplant CMV infection. In conclusion, our findings confirm that the KIR/HLA genotype plays a significant role in anti-CMV immunity and suggest the contribution of both environmental and genetic factors to the incidence of CMV infection after kidney transplantation.

Going beyond serology for stratifying the risk of CMV infection in transplant recipients.

Knowledge of donor and recipient (D/R) cytomegalovirus (CMV) serostatus is critical for risk stratification of CMV infection and disease in transplant recipients, particularly in the solid organ transplantation (SOT) setting. Despite its broad availability and the success of it use, the risk stratification based on the D/R serostatus is not free of limitations since there are a nondepreciable number of patients that are not accurately categorized by this approach. In fact, up to 20% of seropositive SOT recipients, classically considered at intermediate risk, develop episodes of CMV infection and disease after transplantation. Here, we provide an overview of additional donor and recipient factors that may have utility in identifying patients at risk for post-transplant CMV infection. Specifically, we summarize our current understanding regarding the potential use of use CMV-specific T-cell-mediated immunity, neutralizing antibodies and host genetics that may influence the risk of CMV infection and disease. We provide an overview of the benefits and limitations associated with using these immunological factors in risk stratification and propose specific variables that could be analyzed at the pretransplant evaluation to improve the identification of patients with increased individual susceptibility.

Optimizing the Management of Cytomegalovirus Infection in Transplant Patients

The European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) is the world's leading congress in infectious diseases, attracting nearly 13,000 delegates in 2018. Cytomegalovirus was a major topic for discussion, since it remains an important cause of morbidity and mortality in transplant recipients. In the future, novel diagnostic technologies and new antiviral drugs may help to reduce the risk of post-transplant cytomegalovirus infection and disease, and so improve outcomes for patients. Report from the 28th European Congress of Clinical Microbiology and Infectious Diseases (European Congress of Clinical Microbiology and Infectious Diseases 2018), Madrid, Spain, 21–24 April 2018.

Effect of Low-Dose Vs Standard-Dose Valganciclovir in the Prevention of Cytomegalovirus Disease in Kidney Transplantation Recipients: A Systemic Review and Meta-Analysis

BackgroundValganciclovir is widely used to prevent post-transplant cytomegalovirus (CMV) infection in kidney transplant patients. However, the currently used dose remains controversial because the continuous use of this drug decreases kidney function and can induce leukopenia. ObjectiveThe purpose of this study was to measure the appropriate dose of valganciclovir required to prevent CMV infection. MethodsA systematic review and meta-analysis were performed by using a random effects model. The Cochrane Central Register, MEDLINE, EMBASE, and PubMed databases were searched up to April 15, 2017. We conducted analysis on low-dose (450 mg) and standard-dose (900 mg) valganciclovir groups. ResultsAfter completion of the research, the analysis revealed that the glomerular filtration rate, graft loss, tacrolimus level, antibody-mediated rejection, and fungal and Candida infection rates did not differ between the 2 groups. However, the incidence of CMV tended to decrease in the low-dose group (0.584 [95% confidence interval [CI], 0.352–0.967]; P = .036). The biopsy-proven rejection rate decreased by 0.427 times in the low-dose group compared with the standard-dose group (95% CI, 0.274–0.667; P = .002). Furthermore, the incidence of leukopenia decreased by 0.371 times in the low-dose group compared with the standard-dose group (95% CI, 0.264–0.523; P = .001). ConclusionsThe 450-mg dose of valganciclovir effectively prevented post-transplantation CMV infection and decreased drug-induced side effects such as leukopenia. In the future, the lower dose of valganciclovir should be considered to prevent CMV infection and enhance cost-effectiveness.

Earlier and higher rates of cytomegalovirus infection in pediatric liver transplant recipients as compared to adults: An observational study.

AIM:To study and compare the incidence and time of occurrence of cytomegalovirus (CMV) infection in the posttransplant period in adult and pediatric liver transplant recipients.MATERIALS AND METHODS:Consecutive live donor liver transplant recipients not on CMV prophylaxis, were prospectively enrolled from March 2012 to September 2015 and followed up for 1 year post transplant to look for CMV infection. For analysis, patients were divided into pediatric (up to 18 years) and adult (>18 years) age groups.RESULTS:The study population of 146 patients consisted of 132 adult and 14 pediatric patients. Overall CMV infection posttransplant was seen in 54/146 (36.98%) patients, and 16/54 (29.6%) patients developed CMV disease. Post-transplant CMV infection rate was significantly higher in pediatric patients(10/14 [71.4%]) as compared to adults (44/132 [33.4%]) (P = 0.004). Among adults, CMV infection was seen in 22 (50%) patients in the 1st month, 13 (29.5%) patients in the 2nd month, 5 (11.4%) patients in the 3rd month, 2 (4.5%) patients in the 4th month, and 1 (2.3%) patient each in the 5th and 6th month. However, in pediatric patients, all the patients having CMV infection had it in the 1st-month posttransplant (P = 0.003). The median time of occurrence of CMV infection was 11.5 (7.75–19.00) days in pediatric patients versus 30 (18.5–54.5) days in adult patients (P = 0.001).CONCLUSIONS:The results of this study show a clear difference in the incidence and timeline of posttransplant CMV infection in pediatric patients as compared to adults.

Pre-Transplant Cytomegalovirus Immunoglobulin G Antibody Levels Could Prevent Severe Cytomegalovirus Infections Post-Transplant in Liver Transplant Recipients: Experience from a Tertiary Care Liver Centre

Background: Humoral immune responses in cytomegalovirus (CMV) are not studied well. Pre-transplant CMV immunoglobulin G (IgG) antibody levels (Pre-Tx IgG) could influence the occurrence of post-transplant CMV infections. Objective: Correlation between pre-Tx IgG and post-Tx risk of acquiring CMV infection was investigated. Materials and Methods: A total of 146 liver Tx recipients, not on CMV prophylaxis, were included. Pre-Tx IgG in donor (D) and recipient (R) were estimated and all the recipients were followed up for 1 year for CMV infections. Results: D+ R+ serostatus was seen in 142 (97.3%) and D− R+ in 4 (2.7%). A total of 113 (77.4%) recipients had pre-Tx IgG of ≥250 AU/ml. Overall, post-Tx CMV infections were seen in 54 (36.9%) recipients. In 32 (59.2%) patients, CMV infection was seen during the 1st month after TX. Incidences of post-Tx CMV infection in recipients with pre-Tx IgG <250 AU/mL and ≥250 AU/mL were 42.4% and 34.5%, respectively (P = 0.99). Median viral load was significantly higher in patients with pre-Tx IgG <250 AU/ml: 4log10 (R: 2.8–6.6 log 10) copies/ml than those with ≥250 AU/ml: 2.2 log10 (R: 1.6–3.8 log10) copies/ml, P = 0.04. There was no difference in the time of occurrence of CMV infection in both the groups. Concurrent occurrence of rejection and CMV infection was seen in significantly more patients 18/54 (32.7%) than in patients without CMV infection (12/99, 12%, P = 0.002). Conclusions: Higher pre-Tx CMV IgG levels might prevent severe CMV infections post-Tx. Recipients with low pre-Tx CMV titre might be benefitted by CMV prophylaxis or aggressive pre-emptive treatment.

Cytomegalovirus infection after kidney transplantation and long-term graft loss

BackgroundDespite the use of prevention strategies, cytomegalovirus (CMV) infection is the most common viral complication after renal transplant and its impact on long-term outcomes is still open to debate. ObjectiveTo evaluate the incidence of CMV infection and disease during the use of prevention strategies in our centre and to analyse the association between CMV infection and long-term patient and graft survival and other potentially clinical events related with CMV. MethodsWe reviewed the medical records of 377 recipients of kidney transplants performed between January 1998 and December 2008. Kaplain-Meier survival curve analysis was performed to analyse graft and patient survival by CMV infection/disease and Cox proportional hazards regression was used to identify factors associated with CMV infection/disease, graft loss and mortality. ResultsThe incidence of CMV infection was 34.7% and CMV disease was 9.5%. Patient and graft survival was significantly lower in patients with CMV infection/disease. CMV infection/disease was associated with a higher risk of graft loss (HR 1.91, 95% CI 1.09–3.36, p=0.023), but not with a higher mortality (HR 1.29, 95% CI 0.7–2.38, p=0.4). ConclusionCMV replication after renal transplant is a risk factor for long-term graft loss but not mortality. Prevention strategies decrease post-transplant CMV infection and disease.

La infección por citomegalovirus postrasplante renal y pérdida del injerto a largo plazo

BackgroundDespite the use of prevention strategies, cytomegalovirus (CMV) infection is the most common viral complication after renal transplant and its impact on long-term outcomes is still open to debate. ObjectiveTo evaluate the incidence of CMV infection and disease during the use of prevention strategies in our centre and to analyse the association between CMV infection and long-term patient and graft survival and other potentially clinical events related with CMV. MethodsWe reviewed the medical records of 377 recipients of kidney transplants performed between January 1998 and December 2008. Kaplain-Meier survival curve analysis was performed to analyse graft and patient survival by CMV infection/disease and Cox proportional hazards regression was used to identify factors associated with CMV infection/disease, graft loss and mortality. ResultsThe incidence of CMV infection was 34.7% and CMV disease was 9.5%. Patient and graft survival was significantly lower in patients with CMV infection/disease. CMV infection/disease was associated with a higher risk of graft loss (HR 1.91, 95% CI 1.09-3.36, p=0.023), but not with a higher mortality (HR 1.29, 95% CI 0.7-2.38, p=0.4). ConclusionCMV replication after renal transplant is a risk factor for long-term graft loss but not mortality. Prevention strategies decrease post-transplant CMV infection and disease.

Cytomegalovirus infection in living-donor and cadaveric lung transplantations.

Cytomegalovirus (CMV) infection remains a major cause of morbidity after lung transplantation. Some studies have reported prognostic factors for the postoperative development of CMV infection in cadaveric lung transplantation (CLT), but no research has been performed in living-donor lobar lung transplantation (LDLLT). Therefore, we analysed the possible risk factors of post-transplant CMV infection and the differences between LDLLT and CLT. The development of CMV disease and viraemia in 110 patients undergoing lung transplantation at Kyoto University Hospital in 2008-2015 were retrospectively assessed. The prognostic factors in the development of CMV infection and the differences between LDLLT and CLT were analysed. Among 110 patients, 58 LDLLTs and 52 CLTs were performed. The 3-year freedom rates from CMV disease and viraemia were 92.0% and 58.5%, respectively. There was no difference in the development of CMV infection between LDLLT and CLT (disease: 94.6% vs 91.0%, P = 0.58 and viraemia: 59.3% vs 57.2%, P = 0.76). In preoperative anti-CMV immunoglobulin status, R-D+ recipients (recipient: negative, donor: positive) and R-D- recipients (recipient: negative, donor: negative) tended to have higher and lower cumulative incidences, respectively, of CMV infection (disease: P = 0.34 and viraemia: P = 0.24) than that with R+ recipients (recipient: seropositive). Significantly lower cumulative incidence of CMV viraemia was observed in patients receiving 12-month prophylactic medication (70.6% vs 36.8%, P < 0.001). Twenty-eight patients (25.5%) had early cessation of anti-CMV prophylaxis due to toxicity; however, the extended prophylaxis duration did not increase the incidence of early cessation (P = 0.88). These trends were seen in both LDLLT and CLT. We found that there was no difference in the development of CMV infection between LDLLT and CLT. Twelve-month prophylaxis protocol provides beneficial effect without increased toxicity also in LDLLT.

Substantial Variation in Nlv-Specific T Cells Phenotype and Activation Ability upon Antigen Stimulation May Have Implications for Virus-Specific Adoptive Therapy

IntroductionInfusion of viral antigen-specific donor T lymphocytes can be used to treat post-transplant cytomegalovirus infection and has several advantages over the highly toxic conventional antiviral therapy. Most of the protocols for adoptive transfer rely on in vitro expansion of lymphocytes. Besides being time consuming laborious and expensive in vitro cell culture can potentially alter the phenotype of T cells diminishing their proliferative capacity. In the meantime if the donor has high concentration of antigen specific cells in circulation expansion step can be avoided and cells can be transferred immediately after selection. Infusion of relatively low (103 - 104/kg) amounts of unmanipulated antigen-specific CD8+ cells can result in rapid clearance of the viral infection (Uhlin, 2012, Clin Infect Dis). Due to high antigen specificity anti-viral T cells from the third party donor could be used without causing alloreactivity.Immediate transfer of antigen-specific lymphocytes relies on the proliferative capacity of intact donor lymphocytes, thus it expected to be dependent on their in vivo phenotype. Here we describe the variability of immunophenotype of virus-specific cells in healthy donors and their ability to activate upon antigen stimulation in vivo.Materials and methodsWe have measured cellular immune response to NLV viral antigen in 11 healthy CMV seropositive HLA-A*02:01 allele carriers using in house prepared NLV-A*02:01 tetramer and CD3/CD8 antibodies. In 4 donors cell activation was measured by HLA-DR and granzyme B expression, T cell immunophenotype was determined using antibodies to CCR7, CD45RO, CD95 and CD28 surface antigens. NLV-specific population was enriched using magnetic cell-sorting and cultivated with autologous or allogeneic feeder cells with or without NLV peptide. Cell proliferation was measured by CFSE staining and cells were analyzed for the ability to produce TNF and IFNγ.ResultsHLA-A*02:01 is present in around 30% of individuals in Russian population. Vast majority (90%) of healthy Russian donors is CMV seropositive. We found that 7 of 11 analyzed donors have a strong (³1% of CD8+ cells) response to the immunodominant epitope of CMV pp65 protein (NLV). This makes T cell adoptive transfer of directly enriched and unmanipulated NLV-specific T cells highly applicable for Russian patients. We also found significant variability in both the immunophenotype of NLV-specific T cells and their response to antigenic stimulation. Numbers of activated HLA-DRhigh T cells differ substantially between the donors (Fig.1). To the lesser extent this is true regarding granzyme B expression in tetramer-positive cells. Most of the NLV-specific cells have memory phenotype but different donor vary widely in the percentages TTM (CCR7-,CD45RO+,CD28+) TEM (CCR7-,CD45RO+,CD28-) and TTE (CCR7-,CD45RO-,CD28-) cells (Fig.2).In response to specific stimuli magnetically enriched NLV-specific T cells from the donors with more HLA-DRhigh T cells produced more TNF and proliferated more rapidly, though produced less IFNγ (Fig. 3) though we don’t yet have enough data to see if it is statistically significant. The numbers of dividing cells and cell producing inflammatory cytokines upon stimulation was widely distributed in different donors. Cells from all donors reacted only when specific antigen was added. Stimulation with both unrelated antigen or allogeneic (MHC mismatched) feeder cells failed to produce immune response, confirming that these cells are highly specific and are unlikely to cause alloreactivity even in MHC-mismatched setting.ConclusionAdoptive transfer of unmanipulated cell product from donors with high titers of antigen-specific T cells is a promising therapeutic strategy. The level of T cells activation and their ability to proliferate and produce cytokines upon antigen encounter from different healthy donor are widely variable. Further studies are needed to determine to what extent the phenotype and activation status of donor lymphocytes influences the outcome of the adoptive cell therapy. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Recombinant MHC Tetramers for Isolation of Virus-Specific CD8+ Cells from Healthy Donors: Potential Approach for Cell Therapy of Posttransplant Cytomegalovirus Infection.

Patients undergoing allogeneic hematopoietic stem cell transplantation have a high risk of cytomegalovirus reactivation, which in the absence of T-cell immunity can result in the development of an acute inflammatory reaction and damage of internal organs. Transfusion of the virus-specific donor T-lymphocytes represents an alternative to a highly toxic and often ineffective antiviral therapy. Potentially promising cell therapy approach comprises transfusion of cytotoxic T-lymphocytes, specific to the viral antigens, immediately after their isolation from the donor's blood circulation without any in vitro expansion. Specific T-cells could be separated from potentially alloreactive lymphocytes using recombinant major histocompatibility complex (MHC) multimers, carrying synthetic viral peptides. Rapid transfusion of virus-specific T-cells to patients has several crucial advantages in comparison with methods based on the in vitro expansion of the cells. About 30% of hematopoietic stem cell donors and 46% of transplant recipients at the National Research Center for Hematology were carriers of the HLA-A*02 allele. Moreover, 94% of Russian donors have an immune response against the cytomegalovirus (CMV). Using recombinant HLA-A*02 multimers carrying an immunodominant cytomegalovirus peptide (NLV), we have shown that the majority of healthy donors have pronounced T-cell immunity against this antigen, whereas shortly after the transplantation the patients do not have specific T-lymphocytes. The donor cells have the immune phenotype of memory cells and can be activated and proliferate after stimulation with the specific antigen. Donor lymphocytes can be substantially enriched to significant purity by magnetic separation with recombinant MHC multimers and are not activated upon cocultivation with the antigen-presenting cells from HLA-incompatible donors without addition of the specific antigen. This study demonstrated that strong immune response to CMV of healthy donors and prevalence of HLA-A*02 allele in the Russian population make it possible to isolate a significant number of virus-specific cells using HLA-A*02-NLV multimers. After the transfusion, these cells should protect patients from CMV without development of allogeneic immune response.

Assessment of a cytomegalovirus serology dual-testing strategy in hematopoietic stem cell transplant recipients.

Accurate determination of recipient cytomegalovirus (CMV) serostatus before allogeneic hematopoietic stem cell transplantation (HSCT) is critical, as it is the most important predictor of post-transplant CMV infection and remains associated with non-relapse mortality. The purpose of this study was to assess a recipient dual-testing strategy before HSCT. CMV serologic testing was performed before allogeneic HSCT using 2 different assays: reference laboratory (RL) and American Red Cross (ARC). In all cases, blood samples were obtained for RL testing either before ARC testing (median 130 days before HSCT [range 12-2594]) or at the same time (median 25 days before HSCT [range 8-129]). The results of serologic testing were correlated with CMV viremia post HSCT. Of 287 recipients evaluated, 76 (26.5%) had discordant results, of which 74 (97.4%) tested RL-/ARC+. Ten had RL and ARC testing performed on simultaneously obtained samples, 3 of which (30%) were discordant (3 [100%] RL-/ARC+). Acute myeloid leukemia and receipt of blood product transfusion in the interval between testing were associated with RL-/ARC+ discordance. Correlation with viremia after HSCT suggested that RL-/ARC+ discordance was caused by detection of anti-CMV immunoglobulin transferred in transfused blood products and reduced specificity of the ARC assay. CMV-seronegative hematopoietic stem cell transplant recipients may be misclassified as seropositive if testing is performed after receipt of blood products or when using assays optimized for sensitivity at the expense of specificity. This misclassification may negatively affect post-HSCT outcomes for individual patients and studies that rely on accurate CMV serology reporting.

Posttransplant CMV infection and the role of immunosuppression (Sponsor: Novartis Pharma GmbH, Nürnberg)

AbstractImmunosuppression permits graft survival after transplantation and consequently a longer and better life. On the other hand, it increases the risk of infection, for instance with cytomegalovirus (CMV). However, the various available immunosuppressive therapies differ in this regard. One of the first clinical trials using de novo everolimus after kidney transplantation [1] already revealed a considerably lower incidence of CMV infection in the everolimus arms than in the mycophenolate mofetil (MMF) arm. This result was repeatedly confirmed in later studies [2–4]. Everolimus is now considered a substance with antiviral properties. This article is based on the expert meeting “Posttransplant CMV infection and the role of immunosuppression”. The expert panel called for a paradigm shift: In a CMV prevention strategy the targeted selection of the immunosuppressive therapy is also a key element. For patients with elevated risk of CMV, mTOR inhibitor-based immunosuppression is advantageous as it is associated with a significantly lower incidence of CMV events.

Monitoring the Cytomegalovirus-specific cellular immunity in lung transplant recipients: A comparative analysis of two assay systems

Abstract Objective Monitoring the cellular immunity for Cytomegalovirus (CMV) in organ transplant recipients is a promising tool to support prevention strategies for posttransplant CMV infection or reactivation. Commercially available in vitro test systems differ substantially in their capacity to stimulate subpopulations of T-lymphocytes. We compared two assays for CMV immune monitoring in respect of their clinical practicability and significance. Methods Blood samples of 30 lung transplant recipients (LuTRs) were examined before transplantation and over a period of six months afterwards with T-Track® CMV (Lophius Biosciences GmbH, Regensburg) and QuantiFERON®-CMV (Cellestis GmbH, Darmstadt). The T-Track® CMV is based on ELISpot-technology, allowing quantification of interferon-gamma (IFNγ) secreting CD4+ and CD8+ T-cells after specific stimulation. Contrarily, the QuantiFERON®-CMV assay is restricted to detection of IFNγ secreted by CD8+ T-lymphocytes with ELISA. The data are evaluated in the context of transplant outcome and determination of viral load in plasma by qPCR. Results and Conclusion Both approaches provide similar results while exhibiting certain advantages and limitations. Early during immune suppressive therapy, QuantiFERON®-CMV generates often indeterminate results as depletion of T-cells is not taken into account. Although a comparatively large volume of blood is required, T-Track® CMV circumvents this drawback. In addition, T-Track® CMV reaches a higher sensitivity prior to LuTx and after onset of immunosuppression.

Cytomegalovirus Infection After Intestinal/Multivisceral Transplantation: A Single-Center Experience With 210 Cases.

Cytomegalovirus (CMV) infection is the most prevalent infectious complication after solid organ transplantation, and recipients of isolated intestinal transplantation (IIT)/multivisceral transplantation (MVT) are among those at the highest risk. Limited clinical data exist regarding CMV infection after IIT/MVT. The aim of this study is to analyze risk factors for posttransplant CMV infection and to assess the efficacy and validity of our prophylaxis and treatment regimens in intestinal transplantation. Medical records of 210 IIT/MVT patients were retrospectively reviewed. Posttransplant CMV prophylaxis regimen consisted of ganciclovir followed by 1 year of valganciclovir. The addition of CMV immunoglobulin (CMVIG) was decided according to donor/recipient CMV serostatus (D/R). All results of CMV PCR and/or pp65 antigenemia, and pathological reports were reviewed. Time to the incidence of CMV infection (viremia and/or tissue invasive disease) and risk factors for CMV infection were investigated. CMV infection was observed in 34 of 210 (16%) with a median onset of 347 days. Rejection was significantly associated with CMV infection (P = 0.01, odds ratio = 2.61). In the high-risk serostatus group (D+/R-), prophylactic CMVIG and induction with high-dose rabbit antithymocyte globulin (>10 mg/kg) were associated with a lower CMV infection rate on univariate analysis. The CMVIG remained to be an independent factor on multivariate analysis (P = 0.04, hazard ratio = 0.93/dose). Mortality associated with CMV infection occurred in 4, and CMV infection adversely affected patient survival (P = 0.001, hazard ratio = 2.71). Prophylaxis with CMVIG and appropriate induction with rabbit antithymocyte globulin may be important to reduce CMV infection in high-risk serostatus group (D+/R-).