Sascha Barabas

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

ABSTRACT Predominant T-helper 1 (Th1) responses with increased gamma interferon (IFN-γ) 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-γ, 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-γ 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.

CD4+ T Helper Cell Responses against Human Bocavirus Viral Protein 2 Viruslike Particles in Healthy Adults

Abstract 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 immunsorbent 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/cm3) was demonstrated. A significant increase in secretion of VP2-specific interferon-γ 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.

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.

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

BackgroundUncontrolled 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.ResultsPositive 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.ConclusionT-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.

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

BackgroundIn 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.MethodsObjective 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.ResultsOptimized 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.ConclusionThe 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.

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.

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-&ggr; 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-&ggr; 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-&ggr; 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).