Epstein-Barr virus latency in transplant patients and healthy carriers

In this thesis, I studied the latency situation of Epstein-Barr virus (EBV) in bone marrow transplanted (BMT) patients and healthy virus carriers.

Paper 1: Epstein-Barr virus genomes are found predominantly in lgA-positive B cells in the blood of healthy carriers. B lymphocytes have been identified as the main reservoir of latent Epstein-Barr virus (EBV) in healthy virus carriers. We have established a semi-quantitative PCR method (SQ-PCR) to estimate the EBV genome load in the blood B-cell subpopulation in healthy individuals. EBV DNA was detected in subfractionated IgM-, IgG- and IgA-positive B cells. Between 80% and 90% of the viral DNA was found in the lgA-positive compared with the IgA-negative fraction.

Paper 2: Epstein-Barr virus (EBV) load in bone marrow transplant (BMT) recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells. We used same SQ-PCR as reported in paper 1 to monitor the blood levels of EBVDNA in 9 patients receiving allogeneic BMT. Four of 5 recipients of HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 107 EBV-specific cytotoxic T-lymphocytes (CTLS)/M2 starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. One patient, who received a T-cell culture lacking a major EBV-specific component, progressed to fatal EBV-positive lymphoma. Administration of EBVCTLs before the onset of the EBV-DNA peak resulted in stabilization of the virus titers within 2 to 3 logs above the normal levels in the fifth patient.

A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas one patient with Wiskott-Aldrich syndrome (WAS) reached a 5-log increase of EBV-DNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease.

Paper 3: Circulating Epstein-Barr virus infected "resting" B lymphocytes in bone marrow transplant recipients at risk to develop post-transplant lymphoproliferative disease. EBV establishes a life long infection of humans where the proliferative potential of latently infected B blasts is kept in cheek by strong T-cell mediated rejection responses. A key feature of this virus host relationship is the capacity of the virus to establish a restricted latent infection in resting B cells that are insensitive to rejection and provide a reservoir for reactivation and spread to susceptible hosts. In immunosuppressed individuals the EBV-infected blasts may give rise to lymphoproliferative disorders that are preceded by a dramatic increase of virus load in blood. We have used SQ-PCR assays and reverse transcriptase assisted (RT)-PCRs to investigate the EBV-DNA load and the pattern of viral gene expression in peripheral blood of immunosuppressed patients receiving T cell depleted or unmanipulated bone marrow grafts from healthy EBV carriers.

Patients in both groups showed a significant increase of EBV-DNA load compared to healthy controls. Virus titers exceeding the normal levels by more than 4 logs were detected in recipients of T-cell depleted marrow and in one patient with Wiskott-Aldrich syndrome. Measurement of EBV-DNA in serum and limiting dilution analysis of EBV-DNA in PBMC demonstrated that the increased virus load is due to expansion of a latently infected cell compartment that contains less than 10 EBV genomes copies per cell and expresses EBERs, LMP-2A and occasionally LMP 1 and EBNA 1 but not other latency associated viral proteins that serve as targets for virus-specific immune responses. This is compatible with latency forms I-II in non proliferating B-cells, but not latency Ill.

Administration of EBV-specific CTLs correlated with a slow decrease of EBV-DNA load followed by stabilization at levels significantly higher then in healthy controls. The results suggest that suppression of EBV-specific T cell responses allows the proliferation infected B blasts in lymphoid tissues and that these cells retain the capacity to differentiate into resting B cells that enter the circulation. Reconstitution of T cell immunity results in the establishment of a new virus host-balance characterised by a significant expansion of the latent viral reservoir.

The following main conclusions could be drawn from our results: · EBV can infect IgM-, IgG- and IgA-positive B cells. Between 80% and 90% of the viral DNA was found in the IgA-positive subfraction; · HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT; · Administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease; · In BMT patients, EBV is in latency forms I-II in non proliferating B-cells, neither latency 111 nor infected B cells in lytic cycle .

List of scientific papers

I. Ehlin-Henriksson B, Zou JZ, Klein G, Ernberg I (1999). Epstein-Barr virus genomes are found predominantly in IgA-positive B cells in the blood of healthy carriers. Int J Cancer. 83(1): 50-4.
https://pubmed.ncbi.nlm.nih.gov/10449607

II. Gustafsson A, Levitsky V, Zou JZ, Frisan T, Dalianis T, Ljungman P, Ringden O, Winiarski J, Ernberg I, Masucci MG (2000). Epstein-Barr virus (EBV) load in bone marrow transplant recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells. Blood. 95(3): 807-14.
https://pubmed.ncbi.nlm.nih.gov/10648390

III. Zou JZ, Levitsky V, Gustafss A, Frisan T, Dalianis T, Ljungman P, Ringden O, Winiaski J, Masucci MG, Ernberg I (2005). Circulating Epstein-Barr virus infected "resting" B lymphocytes in bone marrow transplant recipients at risk to develop post-transplant lymphoproliferative disease. [Manuscript]