EBV inSIGHT™ T Cell Immunity Testing

The Epstein-Barr Virus (EBV) inSIGHT™ T Cell Immunity test measures the strength of T cell responses to Epstein-Barr (EBV) specific antigens , BZLF1 and EBNA3. It evaluates and reports the activity of CD4 and CD8 T cell responses independently.

Background

Epstein-Barr Virus is a member of the herpes virus family, also known as human herpesvirus 4 (HHV-4). Approximately 90-95% of people have been infected with the virus by the time they reach adulthood. The virus is predominantly spread through bodily fluids, like saliva.

A primary infection with EBV can cause infectious mononucleosis (mono), as well as, other illnesses. EBV can affect patients with weakened immune systems and is a common risk factor in patients following solid organ (SOT) or hematopoietic stem cell transplant (HSCT). In the transplant setting, post-transplant lymphoproliferative disorders (PTLD) can arise as a consequence of the immunosuppression used and the loss of T cells that typically control reactivation of EBV. Rapidly rising EBV viral loads can indicate patients at risk for PTLD, a critical cause of morbidity and mortality in , SOT and HSCT patients. The overall incidence of PTLD ranges from 1 – 20% depending on the type of transplant. There is evidence which suggests there is higher incidence associated with multi-organ transplants, high intensity immunosuppression and in EBV naïve hosts.¹ For this reason PTLD is a serious concern in children, due to the lower EBV infection rate at an early age.

Often, reduction in immunosuppression (RIS) is used to allow immune recovery to occur as part of treatment for reactivated EBV to help prevent progression to PTLD. When RIS is ineffective or fails to resolve the PTLD, EBV specific immune cell therapy or Rituximab (Brand Name: RITUXAN®), or chemotherapy have been found to be effective treatment for PTLD.²

Cell mediated immunity is a component of EBV immune control. Monitoring EBV-specific T cell responses utilizing intracellular cytokine staining may aid in the detection of patients with an increased risk of EBV DNAemia and may be useful for identifying patients that could benefit from reduced immunosuppression if their viral load should start to increase. T cell immunity testing may be useful for monitoring immunity to EBV pre- and post-transplant and may be useful when used with monitoring low level viral load while reducing -immunosuppression (RIS), or in administration of preemptive therapy in tandem with RIS.

This assay was developed to include reporting for Interferon-γ (IFN-gamma) producing T cells, as well as polyfunctional CD4 and CD8 T cells. The presence of virus specific polyfunctional T cells has been found to be an indicator of highly functional T cells. The Polyfunctional Index (PFI) represents the proportion of IFN-gamma cells that also express Tumor Necrosis Factor alpha and/or Interleukin.²The lytic phase antigen (BZLF1) and latent phase antigens (EBNA3) were selected to detect CD8 T cell responses to both phases of a virus infection. An EBV cell lysate was selected to contain multiple proteins to better stimulate a CD4 T cell response (which is often reported to be much lower than the CD8 counterparts). Research has shown that healthy individuals who carry EBV mount a significant polyfunctional T cell response to EBV lytic and latent proteins; conversely patients with PTLD have shown impaired T cell immunity and a low proportion of polyfunctional T Cells. Additionally, in the case of EBV induced PTLD, research has also found that highly functional EBV Specific CD8 T Cells are required to control an infection.^1,2,3,4

“…Results inferred that EBV-specific PFCs were functionally superior to their single functional counterparts because they have greater production of cytokines and degranulation capacity.⁴”

Learn more about the assay design here.

Quantitative EBV DNA PCR can also be used to monitor the course of the disease, aid in the early diagnosis of PTLD , and monitor response to treatment.

Whole blood is stimulated with the positive control SEB, EBV specific antigens (EBV lysate, BZLF1 and EBNA 3) or left unstimulated as a negative control and incubated to 37ºC. During the incubation Brefeldin A is added causing the cytokines (Interferon – gamma (IFN-γ) Tumor Necrosis Factor – alpha (TNF-α) and/or Interleukin 2 (IL-2)) to be retained inside the cell. Following the stimulation phase, cells are recovered, stained for cellular markers (CD3, CD4, CD8, CD69), the cytokines above and analyzed by flow cytometry. This test was developed and its performance characteristics determined by Eurofins Viracor. This test has not been cleared or approved for diagnostic use by the U.S. Food and Drug Administration.

Test will measure the % Interferon-gamma and polyfunctional index responses to the EBV lytic antigen BZLF1, EBV latent antigens EBNA 3A/3C and an EBV infected cell lysate. Polyfunctional index is the proportion of interferon -gamma expressing cells that also express Tumor necrosis factor-alpha and/or interleukin-2. All EBV seronegative donors tested did not have T cell responses to any of the EBV antigens.