Clinical Validation of a Novel ELISpot-based in Vitro Diagnostic Assay to Monitor CMV-specific Cell-Mediated Immunity in Kidney Transplant Recipients
Bernhard Banas1, Dominik Steubl2, Lutz Renders2, Dominik Chittka1, Miriam C. Banas1, Thomas Wekerle3, Martina Koch4, Oliver Witzke5, Anja Mühlfeld6, Claudia Sommerer7, Antje Habicht8, Christian Hugo9, Thomas Huenig10, Monika Lindemann11, Traudel Schmidt12, Anne Rascle12, Sascha Barabas12, Ludwig Deml12, Ralf Wagner12,13, Bernhard K. Kraemer14, Bernd Krueger14.
1Nephrology, University Medical Center Regensburg, Regensburg, Germany; 2Nephrology, Klinikum rechts der Isar, Technical University Munich, Munich, Germany; 3Surgery, Medical University of Vienna, Vienna, Germany; 4Transplantation Immunology Research Group, UKE University Medical Center Hamburg-Eppendorf, Hamburg-Eppendorf, Germany; 5Nephrology and Infectious Diseases, University Hospital Essen, Essen, Germany; 6Uniklinik RWTH Aachen, Aachen, Germany; 7Nephrology, University Hospital Heidelberg, Heidelberg, Germany; 8Transplantation Center, Ludwig-Maximilians-University Medical Center Munich, Munich, Germany; 9Nephrology, Carl Gustav Carus University Medical Center Dresden, Dresden, Germany; 10Institute of Virology and Immunobiology, University Medical Center Würzburg, Würzburg, Germany; 11Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany; 12Lophius Biosciences, Regensburg, Germany; 13Institute of Clinical Microbiology and Hygiene, University Medical Center Regensburg, Regensburg, Germany; 14Vth Department of Medicine, UMM University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany
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). .
