Davide Abate

Evaluation of Cytomegalovirus (CMV)-Specific T Cell Immune Reconstitution Revealed That Baseline Antiviral Immunity, Prophylaxis, or Preemptive Therapy but not Antithymocyte Globulin Treatment Contribute to CMV-Specific T Cell Reconstitution in Kidney Transplant Recipients

BACKGROUND The ultimate goal of organ transplantation is the reestablishment of organ function and the restoration of a solid immunity to prevent the assault of potentially deadly pathogens. T cell immunity is crucial in controlling cytomegalovirus (CMV) infection. It is still unknown how preexisting antiviral T cell levels, prophylaxis, or preemptive antiviral strategies and pharmacological conditioning affect immune reconstitution. METHODS Seventy preemptively treated CMV-seropositive recipients, 13 prophylaxis-treated CMV-seronegative recipients of seropositive donor transplants, 2 seropositive recipients of seronegative donor kidneys, and 27 pretransplant subjects were enrolled in a cross-sectional study and analyzed for CMV viremia (DNAemia) and CMV-specific T cell response (interferon-gamma enzyme-linked immunospot assay) before transplantation and at 30, 60, 90, 180, and 360 days after transplantation. RESULTS CMV-seropositive transplant recipients displayed a progressive but heterogeneous pattern of immune reconstitution starting from day 60 after transplantation. CMV-seronegative recipients did not mount a detectable T cell response throughout the prophylaxis regimen. A single episode of CMV viremia (CMV copy number, 7000-170,000 copies/mL) was sufficient to prime a protective T cell immune response in CMV-seronegative recipients. Antithymocyte globulin treatment did not significantly affect CMV-specific T cell response. CONCLUSIONS Baseline immunity, antiviral therapy but not antithymocyte globulin treatments profoundly influence T cell reconstitution in kidney transplant recipients.

Comparison of Cytomegalovirus (CMV) Enzyme-Linked Immunosorbent Spot and CMV Quantiferon Gamma Interferon-Releasing Assays in Assessing Risk of CMV Infection in Kidney Transplant Recipients

ABSTRACT Assessing cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) represents an appealing strategy for identifying transplant recipients at risk of infection. In this study, we compared two gamma interferon-releasing assays (IGRAs), Quantiferon-CMV and CMV enzyme-linked immunosorbent spot (ELISPOT), to determine the ability of each test to predict protective CMV-specific T-cell responses. Two hundred twenty-one Quantiferon-CMV and ELISPOT tests were conducted on 120 adult kidney transplant recipients (KTRs), including 100 CMV-seropositive transplant recipients (R+) and 20 CMV-seronegative transplant recipients of a CMV-positive donor (D+/R−). As a control cohort, 39 healthy adult subjects (including 33 CMV-seropositive and 6 CMV-seronegative subjects) were enrolled. CMV IgG serology was used as a reference for both tests. In the CMV-seropositive individuals, the ELISPOT and Quantiferon-CMV assays provided 46% concordance with the serology, 12% discordance, 18% disagreement between ELISPOT or Quantiferon-CMV and the serology, and 24% gray areas when one or both tests resulted in weak positives. None of the CMV-seronegative subjects showed detectable responses in the ELISPOT or the Quantiferon-CMV test. In transplant recipients, both the ELISPOT and Quantiferon-CMV assays positively correlated with each other and negatively correlated with CMV DNAemia in a significant way (P < 0.05). During the antiviral prophylaxis, all 20 D+/R− KTRs we examined displayed undetectable Quantiferon-CMV and ELISPOT results, and there was no evidence of CMV seroconversion. The receiving operator curve (ROC) statistical analysis revealed similar specificities and sensitivities in predicting detectable viremia (areas under the curve [AUC], 0.66 and 0.62 for Quantiferon-CMV and ELISPOT, respectively). ELISPOT and Quantiferon-CMV values of >150 spots/200,000 peripheral blood mononuclear cells (PBMCs) and >1 to 6 IU gamma interferon (IFN-γ) were associated with protection from CMV infection (odds ratios [OR], 5 and 8.75, respectively). In transplant recipients, the two tests displayed similar abilities for predicting CMV infection. Both the ELISPOT and Quantiferon-CMV assays require several ameliorations to avoid false-negative results.

Human cytomegalovirus-specific T-cell immune reconstitution in preemptively treated heart transplant recipients identifies subjects at critical risk for infection.

ABSTRACT Human cytomegalovirus (CMV) infection represents a major threat for heart transplant recipients (HTXs). CMV-specific T cells effectively control virus infection, and thus, assessment of antiviral immune recovery may have clinical utility in identifying HTXs at risk of infection. In this study, 10 CMV-seropositive (R+) pretransplant patients and 48 preemptively treated R+ HTXs were examined before and after 100 days posttransplant. Preemptive treatment is supposed to favor the immune recovery. CMV DNAemia and gamma interferon enzyme-linked immunosorbent spot (ELISPOT) assay were employed to assess the viremia and immune reconstitution. HTXs could be categorized into three groups characterized by high (>100), medium (50 to 100), and low (<50) spot levels. Early-identified high responders efficiently controlled the infection and also maintained high immunity levels after 100 days after transplant. No episodes of grade ≥2R rejection occurred in the high responders. Midresponders were identified as a group with heterogeneous trends of immune reconstitution. Low responders were 41% and 21% of HTXs before and after 100 days posttransplant, respectively. Low responders were associated with a higher incidence of infection. The effect of viremia on immune recovery was investigated: a statistically significant inverse correlation between magnitude of viremia and immune recovery emerged; in particular, each 10-fold increase in viremia (>4 log10 DNAemia/ml) was associated with a 36% decrease of the ELISPOT assay spot levels. All episodes of high viremia (>4 log10 DNAemia/ml) occurred from 1 to 60 days after transplant. Thus, the concomitant evaluation of viremia and CMV immune reconstitution has clinical utility in identifying HTXs at risk of infection and may represent a helpful guide in making therapeutic choices.

High Cytomegalovirus (CMV) DNAemia Predicts CMV Sequelae in Asymptomatic Congenitally Infected Newborns Born to Women With Primary Infection During Pregnancy

OBJECTIVE We investigated the kinetics of cytomegalovirus (CMV) clearance in blood and urine and the relationship between the viral load in blood at birth and the development of late-onset sequelae in asymptomatic congenital CMV infection. METHODS Thirty-three newborns with congenital asymptomatic CMV infection born to women with primary CMV infection during pregnancy were enrolled. CMV infection was monitored by polymerase chain reaction analysis of blood and urine. The follow-up examination was concluded at 6 years of age. RESULTS Ten infants developed postnatal sequelae, whereas twenty-three infants remained asymptomatic. Fifty percent of babies cleared CMV in blood and urine within 3 and 36 months, respectively. Logistic multivariate regression revealed that the risk of neonatal clinical disease crossed the level of 50% with a DNAemia at birth of ≥ 12,000 copies/mL (P = .0002). The risk of hearing deficit crossed the level of 50% with a DNAemia at birth of ≥ 17,000 copies/mL (P = .0001). No significant difference was found between the kinetics of CMV clearance in asymptomatic children as compared to babies with late-onset disease. CONCLUSIONS Asymptomatic newborns with a CMV DNAemia at birth of ≥ 12,000 copies/mL were more likely to experience CMV-related sequelae. The risk of hearing deficit increased with a viral load in blood of ≥ 17,000 copies/mL.

A Prospective Study on Modulation of Immunosuppression for Epstein-Barr Virus Reactivation in Pediatric Patients Who Underwent Unrelated Hematopoietic Stem-Cell Transplantation

Background. Posttransplant lymphoproliferative disease caused by Epstein-Barr virus (EBV-PTLD) is a severe complication after allogeneic hematopoietic stem-cell transplantation (HSCT). We evaluated whether the modulation of immunosuppression (IS) guided by quantitative polymerase chain reaction for EBV (EBV-PCR) was effective as a first-line therapeutic approach for EBV reactivation. Methods. Eighty-nine pediatric patients who received an HSCT from an unrelated donor were prospectively assessed by quantitative EBV-PCR. The EBV-PCR threshold to modulate IS was set to more than 300 genomic copies (gc)/105 peripheral blood mononuclear cells. Results. EBV-PCR positivity was observed in 56 (63%) of 89 patients at a median time of 44 days after HSCT. The variables associated with EBV-PCR positivity were bone marrow stem cells (P=0.047) and a lower total dose of nuclear cells reinfused (P=0.03). Thirty-one patients (35%) had more than or equal to 300 gc. IS was withdrawn or reduced in 18 (58%) and 13 (42%) of the 31 patients, respectively. EBV viral load (EBV-VL) less than 300 gc was achieved in 30 of these 31 patients at a median of 25 days. Only 1 (1%) of the 89 patients progressed to EBV-PTLD. The patients with EBV-VL more than 300 gc had a lower incidence of acute graft versus host disease III-IV than patients with EBV-VL less than 300 gc: 13% vs. 36%, P=0.02. No differences in terms of chronic graft versus host disease, overall survival, event-free survival and transplant-related mortality were observed between the two groups. Conclusions. We conclude that PCR-guided modulation of IS may play a role in early intervention for EBV-PTLD and a prospective, randomized study is needed.

Delayed Acquisition of High-Avidity Anti-Cytomegalovirus Antibody Is Correlated with Prolonged Antigenemia in Solid Organ Transplant Recipients

Previous studies have demonstrated that maturation of cytomegalovirus (CMV)-specific antibodies in solid organ transplant recipients is delayed after primary CMV infection. To investigate the clinical significance of this finding, the avidity indices of anti-CMV antibody were determined in parallel with other serologic and virologic parameters in serial serum samples from 24 solid organ transplant recipients who had primary CMV infection that began during the first 3 months after transplantation. The data obtained show that a delay in antibody maturation is significantly correlated with a long persistence of positive antigenemia.

Diagnostic utility of human cytomegalovirus-specific T-cell response monitoring in predicting viremia in pediatric allogeneic stem-cell transplant patients

Background. Several studies proved that virus-specific T-cells play a pivotal role in controlling cytomegalovirus (CMV) infection in adult allogeneic hematopoietic stem-cell transplant (HSCT) patients. Fewer data are available in pediatric HSCT settings, when immature and inexperienced immune system may affect antiviral immune reconstitution. Methods. We analyzed prospectively the CMV-specific T-cell reconstitution in a cohort of 31 pediatric allogeneic HSCT recipients at 30, 60, 90, 120, 180, and 360 days after HSCT. Results. Depending on donor-recipient CMV serostatus, we observed distinct patterns and kinetics of CMV-specific T-cell immune reconstitution: during the early time-points, patients displayed a severe reduction in CMV-specific T-cell recovery in both CMV seropositive donor (D+) group and CMV seronegative donor (D−) on CMV seropositive recipients (R+). From day 90 onward, statistical significant differences in the profile of T-cell immune reconstitution emerged between D+ and D−. The pattern of immune reconstitution was characterized by heterogeneous kinetics and efficiencies: we report cases of: (1) spontaneous antiviral T-cell recovery with no previous viremia, (2) immune T-cell recovery anticipated by CMV viremia, and (3) no T-cell immune reconstitution despite previous viremia episodes. Conclusions. Given the heterogeneous scenarios of antiviral T-cell immune recovery in pediatric allogeneic HSCT, we conclude that the evaluation of the antiviral immune reconstitution is a promising and appealing system for identifying patients at higher risk of CMV infection. The use of interferon-&ggr; ELISPOT test is a valid tool for immunological monitoring and predicting CMV viremia in pediatric HSCT.

Strong Cell-Mediated Immune Response to Human Cytomegalovirus Is Associated With Increased Risk of Fetal Infection in Primarily Infected Pregnant Women

BACKGROUND Human cytomegalovirus (CMV) represents one of the leading causes of congenital infections worldwide. Early diagnosis of fetal infection and consequent rapid therapeutic intervention with immunoglobulin treatment may prevent fetal transmission and virus-related sequelae. In this study, the cell-mediated immunity and immunoglobulin avidity were evaluated as potential predictors of congenital transmission of the infection. METHODS CMV immunoglobulin G (IgG) avidity and CMV enzyme-linked immunospot (ELISpot) assays were employed in 80 pregnant women including 57 primary and 23 nonprimary CMV infections. Congenital infection was assessed using CMV DNA quantitative polymerase chain reaction on amniotic fluid or offspring urine. Logistic regression and receiver operating characteristic statistical methods were employed to determine the association with congenital infection. RESULTS Low CMV IgG avidity (25%) alone correlated with a probability of congenital transmission of 18.2% (95% confidence interval, 7.7%-28.8%). In contrast to the expectations, an increase in CMV ELISpot levels was statistically associated with congenital transmission (P = .006). The combined use of CMV ELISpot and low CMV IgG avidity resulted in a higher level of association than either method alone with the incidence of fetal transmission (area under the curve, 0.8685). CONCLUSIONS CMV-specific cell-mediated immunity represents a relevant marker in assessing the likelihood of congenital CMV transmission, particularly in combination with CMV IgG avidity.

Optimization of interferon gamma ELISPOT assay to detect human cytomegalovirus specific T-cell responses in solid organ transplants

Assessing the CMV specific CMI in transplant subjects represents a promising strategy to determine the risk of infection on individual basis. In this study 61 adult CMV IgG seropositive solid organ transplant recipients were examined in order to improve the efficacy of CMI detection. For this purpose, pair-wise comparisons were conducted comparing positive control stimuli PWM and PMA/iono and CMV stimuli, pp65 peptide pool and whole CMV particle. Rosette pre-depletion of blood was also investigated for detecting CD4+ or CD8+ T-cell responses using the IFN-g ELISPOT assay. In the time-points 30-180 days after transplantation, PMA/iono produced statistically significant higher responses compared to PWM, probably because PMA/iono activation pathway is independent from the effect of immunosuppressive drugs. The data showed that 11% of transplant patients displayed very low or undetectable responses to pp65 peptide pool antigen while having sustained high responses to whole CMV particle. In addition, in all the subjects analyzed, CMI responses to CMV particle produced a statistically significant higher number of spots compared to pp65 peptide pool antigen. Rosette pre-depletion of whole blood proved to be effective in detecting CD4+ or CD8+ T-cell responses similarly to flow cytometry. Taken together, the following recommendations are suggested to optimize the CMV-ELISPOT for transplantation settings: (1) use PMA/iono as positive control; (2) whole virus particle should be used to avoid peptide-related false negative responses; (3) a rosette pre-depletion step may be useful to detect CD4+ or CD8+ T-cell responses.

Comparison of the Cytomegalovirus (CMV) Enzyme-Linked Immunosorbent Spot and CMV QuantiFERON Cell-Mediated Immune Assays in CMV-Seropositive and -Seronegative Pregnant and Nonpregnant Women.

ABSTRACT Human cytomegalovirus (CMV) infection is a major cause of congenital infection leading to birth defects and sensorineural anomalies, including deafness. Recently, cell-mediated immunity (CMI) in pregnant women has been shown to correlate with congenital CMV transmission. In this study, two interferon gamma release assays (IGRA), the CMV enzyme-linked immunosorbent spot (ELISPOT) and CMV QuantiFERON assays, detecting CMV-specific CMI were compared. These assays were performed for 80 CMV-infected (57 primarily and 23 nonprimarily) pregnant women and 115 controls, including 89 healthy CMV-seropositive pregnant women without active CMV infection, 15 CMV-seronegative pregnant women, and 11 seropositive or seronegative nonpregnant women. Statistical tests, including frequency distribution analysis, nonparametric Kruskal-Wallis equality-of-populations rank test, Wilcoxon rank sum test for equality on unmatched data, and lowess smoothing local regression, were employed to determine statistical differences between groups and correlation between the assays. The CMV ELISPOT and CMV QuantiFERON assay data were not normally distributed and did not display equal variance. The CMV ELISPOT but not CMV QuantiFERON assay displayed significant higher values for primarily CMV-infected women than for the healthy seropositive pregnant and nonpregnant groups (P = 0.0057 and 0.0379, respectively) and those with nonprimary infections (P = 0.0104). The lowess local regression model comparing the assays on an individual basis showed a value bandwidth of 0.8. Both assays were highly accurate in discriminating CMV-seronegative pregnant women. The CMV ELISPOT assay was more effective than CMV-QuantiFERON in differentiating primary from the nonprimary infections. A substantial degree of variability exists between CMV ELISPOT and CMV QuantiFERON assay results for CMV-seropositive pregnant women.