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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-017-0195-y) contains supplementary material, which is available to authorized users.

Prospective comparison of two enzyme-linked immunosorbent spot assays for the diagnosis of Lyme neuroborreliosis

“…Overall, the numbers of SFCs after stimulation with B. burgdorferi B31 were also relatively low. In our experience, as well as described by others -for tuberculosis or cytomegalovirus infections -the numbers of IFNγ-secreting T cells among exposed or infected individuals measured in an ELISpot assay using comparable amounts of PBMCs, ranging from 2.0 × 10 5 to 2.5 × 10 5 , are generally much higher [40][41][42]. The lack of T-cell activity among the active LNB patients could be explained by the choice of Borrelia antigens.…”

“…Thirdly, the centrifugation steps that were used to wash the PBMCs and the number of times the PBMCs were washed differed from the LymeSpot protocol. However, in the literature, various centrifugation speeds and times for washing the PBMCs are described, which range from 300 to 640 g for 7-10 min for the first wash step and from 300 to 470 g for 7-10 min for the second wash step [26,41,51,[53][54][55].…”

The Third International Consensus Guidelines on the Management of Cytomegalovirus in Solid-organ Transplantation

“…128 A commercial ELISpot assay (T-Track CMV, Lophius Biosciences, Germany) has recently received CE marking in Europe. 129,130 Another assay (T-SPOT.CMV, Oxford Immunotec, UK, CE marked in Europe) is used as a laboratory developed test (LDT) in the United States.…”

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

“…However, it has become clear that both IE1 and IE2 are highly immunogenic CD4+ and CD8+ T cell antigens adding to their complex roles in the immune response to HCMV infection [283][284][285]. Based on the stimulatory effect IE1 exerts on cellular adaptive immunity, the protein has been utilized in the development of both diagnostic assays as well as vaccine candidates [286][287][288].…”