Standardized monitoring of cytomegalovirus-specific immunity can improve risk stratification of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation.

Recurrence of cytomegalovirus reactivation remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Monitoring cytomegalovirus-specific cellular immunity using a standardized assay might improve the risk stratification of patients. A prospective multicenter study was conducted in 175 intermediate- and high-risk allogeneic hematopoietic stem cell transplant recipients under preemptive antiviral therapy. Cytomegalovirus-specific cellular immunity was measured using a standardized IFN-γ ELISpot assay (T-Track® CMV). Primary aim was to evaluate the suitability of measuring cytomegalovirus-specific immunity after end of treatment for a first cytomegalovirus reactivation to predict recurrent reactivation. 40/101 (39.6%) patients with a first cytomegalovirus reactivation experienced recurrent reactivations, mainly in the high-risk group (cytomegalovirus-seronegative donor/cytomegalovirus-seropositive recipient). The positive predictive value of T-Track® CMV (patients with a negative test after the first reactivation experienced at least one recurrent reactivation) was 84.2% in high-risk patients. Kaplan-Meier analysis revealed a higher probability of recurrent cytomegalovirus reactivation in high-risk patients with a negative test after the first reactivation (hazard ratio 2.73; p=0.007). Interestingly, a post-hoc analysis considering T-Track® CMV measurements at day 100 post-transplantation, a time point highly relevant for outpatient care, showed a positive predictive value of 90.0% in high-risk patients. Our results indicate that standardized cytomegalovirus-specific cellular immunity monitoring may allow improved risk stratification and management of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation. This study was registered at www.clinicaltrials.gov as #NCT02156479.

Clinical validation of a novel enzyme-linked immunosorbent spot assay-based in vitro 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 6 months 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).

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity.

BACKGROUND: In healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions. METHODS: Objective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay. RESULTS: Optimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 104 and 2 × 105 PBMC per well upon stimulation with T-activated® IE-1 (R2 = 0.97) and pp65 (R2 = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3+CD4+ (Th), CD3+CD8+ (CTL), CD3-CD56+ (NK) and CD3+CD56+ (NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive. CONCLUSION: The combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.

Validation of T-Track® CMV to assess the functionality of cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients.

BACKGROUND: Uncontrolled cytomegalovirus (CMV) replication in immunocompromised solid-organ transplant recipients is a clinically relevant issue and an indication of impaired CMV-specific cell-mediated immunity (CMI). Primary aim of this study was to assess the suitability of the immune monitoring tool T-Track® CMV to determine CMV-reactive CMI in a cohort of hemodialysis patients representative of patients eligible for renal transplantation. Positive and negative agreement of T-Track® CMV with CMV serology was examined in 124 hemodialysis patients, of whom 67 (54%) revealed a positive CMV serostatus. Secondary aim of the study was to evaluate T-Track® CMV performance against two unrelated CMV-specific CMI monitoring assays, QuantiFERON®-CMV and a cocktail of six class I iTAg™ MHC Tetramers. RESULTS: Positive T-Track® CMV results were obtained in 90% (60/67) of CMV-seropositive hemodialysis patients. In comparison, 73% (45/62) and 77% (40/52) positive agreement with CMV serology was achieved using QuantiFERON®-CMV and iTAg™ MHC Tetramer. Positive T-Track® CMV responses in CMV-seropositive patients were dominated by pp65-reactive cells (58/67 [87%]), while IE-1-responsive cells contributed to an improved (87% to 90%) positive agreement of T-Track® CMV with CMV serology. Interestingly, T-Track® CMV, QuantiFERON®-CMV and iTAg™ MHC Tetramers showed 79% (45/57), 87% (48/55) and 93% (42/45) negative agreement with serology, respectively, and a strong inter-assay variability. Notably, T-Track® CMV was able to detect IE-1-reactive cells in blood samples of patients with a negative CMV serology, suggesting either a previous exposure to CMV that yielded a cellular but no humoral immune response, or TCR cross-reactivity with foreign antigens, both suggesting a possible protective immunity against CMV in these patients. CONCLUSION: T-Track® CMV is a highly sensitive assay, enabling the functional assessment of CMV-responsive cells in hemodialysis patients prior to renal transplantation. T-Track® CMV thus represents a valuable immune monitoring tool to identify candidate transplant recipients potentially at increased risk for CMV-related clinical complications.

Functional impairment of CMV-reactive cellular immunity during pregnancy.

Cytomegalovirus (CMV) is the most common congenital viral infection. Mother-to-child transmission can cause severe child disability. Intact CMV-specific cell-mediated immunity (CMI) was shown to prevent uncontrolled replication in healthy individuals. This study aimed to determine whether CMV-specific CMI is impaired in pregnant women, thus potentially increasing the overall risk for active CMV replication and transmission. CMV-specific CMI in peripheral blood of 60 pregnant women was determined using T-Track® CMV for detection of IE-1 and pp65-reactive effector cells by IFN-γ ELISpot, and compared to the CMV-IgG and -IgM serostatus. CMV-specific CMI was detected in 65% of CMV-seropositive pregnant women. Five percent of CMV-IgG seronegative women showed IE-1- but not pp65-reactive cells. The overall number of CMV-reactive cells in pregnant women was significantly lower compared to a matched non-pregnant control group (P < 0.001). No significant difference in CMV-specific CMI was detected in the course of the three trimesters of pregnancy of CMV-IgG seropositive women. Postpartum (median days postnatal = 123), IE-1- and pp65-specific CMI remained significantly lower than in the non-pregnant control group (P < 0.001 and 0.0032, respectively). Functional analysis of CMV-reactive immune cells using T-Track® CMV therefore suggests a systemic down-regulation of CMV-specific CMI in pregnant women. Further studies are needed to investigate whether this may be indicative of a higher susceptibility to CMV reactivation or transmission. J. Med. Virol. 89:324-331, 2017. © 2016 Wiley Periodicals, Inc.

Performance of T-Track® SARS-CoV-2, an Innovative Dual Marker RT-qPCR-Based Whole-Blood Assay for the Detection of SARS-CoV-2-Reactive T Cells.

T-cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a central role in the control of the virus. In this study, we evaluated the performance of T-Track® SARS-CoV-2, a novel CE-marked quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels in response to the S1 and NP SARS-CoV-2 antigens, in 335 participants with or without a history of SARS-CoV-2 infection and vaccination, respectively. Of the 62 convalescent donors, 100% responded to S1 and 88.7% to NP antigens. In comparison, of the 68 naïve donors, 4.4% were reactive to S1 and 19.1% to NP. Convalescent donors <50 and ≥50 years of age demonstrated a 100% S1 reactivity and an 89.1% and 87.5% NP reactivity, respectively. T-cell responses by T-Track® SARS-CoV-2 and IgG serology by recomLine SARS-CoV-2 IgG according to the time from the last immunisation (by vaccination or viral infection) were comparable. Both assays showed a persistent cellular and humoral response for at least 36 weeks post immunisation in vaccinated and convalescent donors. Our results demonstrate the very good performance of the T-Track® SARS-CoV-2 molecular assay and suggest that it might be suitable to monitor the SARS-CoV-2-specific T-cell response in COVID-19 vaccinations trials and cross-reactivity studies.

Performance of T-Track® TB, a Novel Dual Marker RT-qPCR-Based Whole-Blood Test for Improved Detection of Active Tuberculosis.

Tuberculosis (TB) is one of the leading causes of death by an infectious disease. It remains a major health burden worldwide, in part due to misdiagnosis. Therefore, improved diagnostic tests allowing the faster and more reliable diagnosis of patients with active TB are urgently needed. This prospective study examined the performance of the new molecular whole-blood test T-Track® TB, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels, and compared it to that of the QuantiFERON®-TB Gold Plus (QFT-Plus) enzyme-linked immunosorbent assay (ELISA). Diagnostic accuracy and agreement analyses were conducted on the whole blood of 181 active TB patients and 163 non-TB controls. T-Track® TB presented sensitivity of 94.9% and specificity of 93.8% for the detection of active TB vs. non-TB controls. In comparison, the QFT-Plus ELISA showed sensitivity of 84.3%. The sensitivity of T-Track® TB was significantly higher (p < 0.001) than that of QFT-Plus. The overall agreement of T-Track® TB with QFT-Plus to diagnose active TB was 87.9%. Out of 21 samples with discordant results, 19 were correctly classified by T-Track® TB while misclassified by QFT-Plus (T-Track® TB-positive/QFT-Plus-negative), and two samples were misclassified by T-Track® TB while correctly classified by QFT-Plus (T-Track® TB-negative/QFT-Plus-positive). Our results demonstrate the excellent performance of the T-Track® TB molecular assay and its suitability to accurately detect TB infection and discriminate active TB patients from non-infected controls.

Stimulatory Effect of CMV Immunoglobulin on Innate Immunity and on the Immunogenicity of CMV Antigens.

BACKGROUND: Cytomegalovirus (CMV) immunoglobulin (CMVIG) is used for the prophylaxis of CMV infection after transplantation. Beyond providing passive CMV-specific immunity, CMVIG exerts enhancing and suppressive immunomodulatory functions. Although the anti-inflammatory activities of CMVIG have been extensively documented, its immunostimulatory activities remain poorly characterized. METHODS: This exploratory study analyzed the capacity of CMVIG to modulate cell-mediated innate and adaptive immunities in vitro on freshly isolated peripheral blood mononuclear cells (PBMCs) of CMV-seropositive and -seronegative healthy individuals, using interferon-γ (IFN-γ) enzyme-linked immunospot and intracellular cytokine staining assays. RESULTS: We showed that CMVIG treatment increases the number of IFN-γ-secreting PBMCs of both CMV-seronegative and -seropositive individuals, indicating a global stimulatory effect on innate immune cells. Indeed, CMVIG significantly increased the frequency of natural killer cells producing the T helper cell 1-type cytokines tumor necrosis factor and IFN-γ. This was associated with the induction of interleukin-12-expressing monocytes and the activation of cluster of differentiation (CD) 4+ and CD8+ T cells, as measured by the expression of tumor necrosis factor and IFN-γ. Interestingly, stimulation of PBMCs from CMV-seropositive subjects with CMVIG-opsonized CMV antigens (phosphoprotein 65, CMV lysate) enhanced CD4+ and CD8+ T-cell activation, suggesting that CMVIG promotes the immunogenicity of CMV antigens. CONCLUSIONS: Our data demonstrate that CMVIG can stimulate effector cells of both innate and adaptive immunities and promote the immunogenicity of CMV antigens. These immunostimulatory properties might contribute to the protective effect against CMV infection mediated by CMVIG.

Comparison of Cytomegalovirus-Specific Immune Cell Response to Proteins versus Peptides Using an IFN-γ ELISpot Assay after Hematopoietic Stem Cell Transplantation.

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8+ counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients.

Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein.

Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I-dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8(+) cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

CD4+ T helper cell responses against human bocavirus viral protein 2 viruslike particles in healthy adults.

BACKGROUND: Human bocavirus (HBoV) was recently described as a new member of the Parvoviridae family, and its possible association with respiratory illness in infants has been discussed. To date, HBoV genomes have been detected worldwide in respiratory tract samples obtained from children with pulmonary diseases, whereas only limited data on virus-specific immunity are available, mainly because of the lack of recombinant viral antigens. METHODS: HBoV viruslike particles (VLPs) were produced in insect cells and characterized by electron microscopy and cesium chloride gradient centrifugation. HBoV viral protein 2 (VP2)-specific antibodies and CD4+ T helper cell responses were analyzed by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay. RESULTS: VP2 capsid proteins of HBoV were produced in insect cells infected with a recombinant baculovirus, and the formation of icosahedral VLPs (diameter, 21-25 nm; sedimentation density, 1.33 g/cm(3)) was demonstrated. A significant increase in secretion of VP2-specific interferon-gamma was detected in cultures of peripheral blood mononuclear cells obtained from 69 healthy adults found to be positive for HBoV-specific immunoglobulin G antibodies, compared with control stimulations. In parallel, T cell responses against identically expressed parvovirus B19 VP2 VLPs were frequently observed in the individuals studied, without there being obvious cross-reactions between HBoV and parvovirus B19. CONCLUSIONS: Data suggest the presence of HBoV-specific immune responses in adults and strongly support a high prevalence of HBoV among humans.

Characterization of the Helicobacter pylori cysteine-rich protein A as a T-helper cell type 1 polarizing agent.

Predominant T-helper 1 (Th1) responses with increased gamma interferon (IFN-gamma) levels have been proposed to play an important role in Helicobacter pylori-induced gastritis and peptic ulceration. However, bacterial factors contributing to the initiation of Th1 polarization of H. pylori-specific immune responses have not been characterized in detail thus far. We report here on the identification of Helicobacter cysteine-rich protein A (HcpA) as a novel proinflammatory and Th1-promoting protein. The capacity of HcpA to induce immune activation was studied in splenocyte cultures of naive H. pylori-negative mice. HcpA stimulated the release of high concentrations of the proinflammatory and Th1-promoting cytokines interleukin-6 (IL-6) and IFN-gamma, in addition to significant levels of IL-12, tumor necrosis factor alpha, and IL-10. The observed cytokine profile was comparable to that induced by lipopolysaccharide but differed in the kinetics and maximum levels of cytokine production. In addition, HcpA-induced cytokine release resembled that observed upon incubation with H. pylori except for IL-10, which was only moderately released upon HcpA stimulation. Both HcpA- and H. pylori-mediated IFN-gamma production was drastically reduced by a neutralizing antibody against IL-12 but not by an anti-IL-2 antibody. Thus, HcpA seems to represent a novel bacterial virulence factor triggering the release of a concerted set of cytokines to instruct the adaptive immune system for the initiation of proinflammatory and Th1-biased immunity.