Professor Dorothy H. Crawford
MBBS, PhD, MD, DSc, FRCPath, FRSE, OBE
Robert Irvine Professor of Medical Microbiology
Assistant Principal, Public Understanding of Medicine
|Address||Centre for Infectious Diseases, Summerhall, Edinburgh EH9 1QH, UK|
|Telephone||+44 (0)131 650 3142|
|Fax||+44 (0)131 650 6511|
Epstein-Barr virus (EBV) is a herpesvirus which establishes a persistent life-long infection in over 90% of adults world-wide. Infection is usually asymptomatic but the virus is associated with a variety of diseases, including human tumours (see table).
Conditions associated with EBV infection in humans
|1. Clinically silent childhood primary infection||Universal in developing countries|
|2. Delayed primary infection (teenagers/ young adults)||Seen in developed countries:|
~75% are clinically silent, as in childhood
~25% have acute infectious mononucleosis (glandular fever) - usually resolves; may become chronic
|3. Primary infection with fatal outcome||Very rare familial x-linked lymphoproliferative disease (Duncan syndrome)|
|4. B lymphoproliferative disease in the immunocompromised host||In organ graft recipients|
In AIDS patients
|5. Oral hairy leucoplakia||In AIDS patients|
|6. Endemic Burkitt lymphoma||Common in children where falciparum malaria is hyperendemic. Areas of Equatorial Africa and Papua New Guinea|
|7. Nasopharyngeal carcinoma||Common in south Chinese and Inuit adults|
|8. Tumours with suspected links to EBV infection||Hodgkin's disease, some T cell lymphomas, leiomyosarcomas, salivary gland cancers, undifferentiated carcinoma of the stomach|
(From: Epstein and Crawford, Gammaherpesviruses: Epstein-Barr Virus. In: Topley and Wilson's Microbiology and Microbial Infections. Volume 1. Eds: BWJ Mahy, L Collier, Arnold 1998).
Our studies focus on EBV infection and persistence in the immunocompromised host, particularly organ transplant recipients, where EBV-associated post transplant lymphoproliferative disease (PTLD) occurs in 1-10% and is fatal in around 50% of cases despite treatment.
Immunotherapy for PTLD
Persistent EBV infection in healthy people is mainly controlled by cytotoxic T cells which recognise cells expressing viral antigens and eliminate them. However when the immune system is suppressed either because of a genetic disorder, an infection like HIV, or drugs, then EBV infected cells in the body are not controlled and the virus can drive them to proliferate uncontrollably forming a tumour. The drugs taken by transplant recipients to prevent rejection of their grafted organ suppress their immune system and often allows reactivation of the persistent EBV infection which may lead to PTLD.
Around 80% of PTLD are EBV associated with the tumour cells containing EBV DNA and expressing viral antigens. These tumour cells are therefore targets for EBV specific cytotoxic T lymphocytes (CTLs) and can only grow where these T cells are lacking. In a recent study we used EBV specific CTL grown in the laboratory to treat PTLD.
PTLD in bone marrow transplant recipients has been prevented and treated successfully using EBV specific CTL derived from the blood of the bone marrow donor (1). But in solid organ transplantation (liver, heart, bowel, lung) the donor is not generally available. We therefore established a bank of EBV-specific CTL lines generated from the blood of 100 HLA-typed healthy blood donors (2). We select CTL lines for infusion into PTLD patients with EBV-positive PTLD on the basis of the best HLA-match and highest specific cytotoxicity in in vitro assays (3).
In a phase 2, multicentre clinical trial, we treated 33 PTLD patients with weekly infusions (1 to 7 doses of 2x106/kgm body weight) of partially HLA-matched, allogeneic, EBV specific CTL. All patients had failed on conventional forms of therapy and had progressive disease. We saw no CTL related immediate or long-term adverse effects or graft-versus-host disease.
The results of the trail were reported by Haque et al (4). Overall there was a 52% response rate at 6 months with a significantly increased response rate in those who received CTLs with a high level of HLA matching and those whose infused CTLs contained high levels of CD4+ T cells.
A recent follow up study on 32 of the 33 CTL treated patients 4-9 years after the infusions showed that 19 (59%) were alive and 13 (41%) have died. Of 14 who achieved complete remission at 6 months, 12 survived and were still in remission. Of the two who had died, one had PTLD relapse and the other died of a chest infection while PTLD was in complete remission. Overall there was a significant increase in survival rate among the remission group compared to the no remission group (p=0.018).
We have also treated 4 non-transplant EBV-positive lymphoma patients (ulcerative colitis, chronic lymphocytic leukaemia, haemophagocytic syndrome and primary immunodeficiency) and CTL infusions were well tolerated in all. EBV-positive lymphoma in the brain of one patient with primary immunodeficiency regressed completely after 7 infusions of allogeneic CTL and the patient remains well and tumour-free after a bone marrow transplant to correct her immunodeficiency (5).
Allogeneic CTL banks bypass the need to grow CTL for individual patients and provide immediate access to fully characterised CTL lines for a large number of patients with EBV-positive lymphomas. We have shown that partially HLA matched allogeneic CTL are safe and effective and are presently producing a new CTL bank under GMP conditions to treat PTLD and other EBV associated conditions.
Genetic studies on infectious mononucleosis
Recently, we identified a significant association between Human Leukocyte Antigen (HLA) class I alleles and the development of IM (6). IM patients carrying these risk alleles (D6S265 allele 3 and D6S510 allele 1) show a distinct clinical picture with reduced lymphocyte numbers, increased EBV load and milder symptoms compared to non-carriers. Since EBV epitopes are presented to T-cells in the context of specific HLA class I and class II molecules, the association of HLA markers with IM suggests that the genetic makeup of an individual’s HLA locus dictates the efficiency of the EBV specific immune response. We are currently investigating this hypothesis in acute and convalescent IM cases by assessing EBV specific T-cell responses in the different genetic IM subsets.
EBV immune reconstitution after cord blood transplant
CBT has recently been developed for patients with haematological malignancies requiring stem cell transplantation but without a suitable sibling donor. Opportunistic infections - mainly viral - during the early post-transplant period are a major problem, causing over 50% of the deaths. In particular, drug induced immunosuppression required to prevent graft rejection suppresses EBV specific T-cell responses and may lead to EBV-associated post-transplant lymphoproliferative disease (PTLD). To prevent PTLD development short term protection by infusion of in vitro grown CTL is an attractive option. We are currently monitoring EBV infection and immunity post CBT to determine the period of vulnerability when infusions of CTLs may be beneficial.
- Rooney et al. Lancet 1995; 345: 9-13.
- Wilkie et al Immunotherapy 2004; 27: 309-316
- Haque et al Lancet 2002; 360: 436-442
- Haque et al Blood 2007; 110: 1123-1131
- Wynne et al Lancet Oncology 2005; 6:344-346
- McAulay et al, J Clin Invest, 2007
Haque T, Wilkie GM, Taylor C, Amlot PL, Murad P, Iley A, Dombagoda D, Britton KM, Swerdlow AJ & Crawford DH. Treatment of Epstein-Barr virus-positive post transplant lymphoproliferative disease using partially HLA-matched allogeneic cytotoxic T cells. Lancet 360 (9331), 436-442 (2002).
Williams H, Macsween K, McAulay K, Higgins C, Harrison N, Swerdlow A, Britton K & Crawford DH. Analysis of immune activation and clinical events in acute infectious mononucleosis. J Inf Dis 190 (1), 63-71 (2004).
Wilkie GM, Taylor C, Jones MM, Burns DM, Turner M, Kilpatrick D, Amlot PL, Crawford DH & Haque T. Establishment and characterisation of a bank of cytotoxic T lymphocytes for immunotherapy of EBV-associated diseases. J Immunotherapy 27 (4), 309-16 (2004).
Wynne RF, Arkwright PD, Haque T, Gharib MI, Wilkie G, Morton-Jones M, Crawford DH. Treatment of Epstein-Barr virus-associated primary CNS B-cell lymphoma with allogeneic T-cell immunotherapy and stem-cell transplantation. Lancet Oncology 6, 344-346 (2005).
Conacher M, Callard R, McAulay K, Chapel, H, Webster D, Kumararatne D, Chandra, A, Spickett G, Hopwood, PA, Crawford, DH. Epstein-Barr virus can establish infection in the absence of a classical memory B-cell population. J Virol 79, 11128-11134 (2005).
Thomas R, Macsween KF, McAulay K, Clutterbuck D, Anderson R, Reid S, Higgins CD, Swerdlow AJ, Harrison N, Williams H, Crawford DH. Evidence of shared Epstein-Barr virus isolates between sexual partners, and low level EBV in genital secretions. J Med Virol 78, 1204-1209 (2006).
Crawford DH, Macsween KF, Higgins CD, Thomas R, McAulay K, Williams H, Harrison N, reid S, Conacher M, Douglas J, Swerdlow SJ. A cohort study among university students: identification of risk factors for Epstein-Barr virus seroconversion and infectious mononucleosis. CID 43, 276-282 (2006).
Higgins CD, Swerdlow AJ, Macsween KF, Harrison N, Williams H, McAulay K, Thomas R, Reid S, Conacher M, Britton K, Crawford DH. A study of risk factors for acquisition of Epstein-Barr virus and its subtypes. JID 195, 469-470. (2007).
Gandi MK, Wilkie GM, Dua U, Mollee PN, Grimmett K, Williams T, Whitaker N, Gill D, Crawford DH. Immunity, homing and efficacy of allogeneic adoptive immunotherapy for posttransplant lymphoproliferative disorders. Am J Transplantation 7, 1-7. (2007).
Haque T, Wilkie GM, Jones MM, Higgins CD, Urquhart G, Wingate P, Burns D, McAulay K, Turner M, Bellamy C, Amlot PL, Kelly D, MacGilchrist A, Gandhi M, Swerdlow AJ, Crawford DH. Allogeneic cytotoxic T cell therapy for EBV positive post transplant lymphoproliferative disease: results of a phase II multicentre clinical trial. Blood 110, 1123-1131. (2007)
McAulay KA, Higgins CD, Macsween KF, lake A, Jarrett RF, Robertson FL, Williams H, Crawford DH. HLA class 1 polymprphisms are significantly associated with infectious mononucleosis. J Clin Invest 117: 3042-3048. (2007).
McAulay KA, Haque T, Urquhart G, Bellamy C, Guiretti D, Crawford DH. Epitope specificity and clonality of EBV-specific CTLs used to treat posttransplant lymphoproliferative disease. J Immunol 182, 3892-3901. (2009).
Wingate PJ, McAulay KA, Anthony IC, Crawford DH. Regulatory T cell activity in primary and persistent Epstein-Barr virus infection. J Med Virol. 81, 870-877. (2009).
Vanhoutte VJ, McAulay KA, McCarrell E, Turner M, Crawford DH, Haque T. Cytolytic mechanisms and T-cell receptor Vβ usage by ex vivo generated Epstein-Barr virus-specific cytotoxic T lymphocytes. Immunology 127, 577-586. (2009).
McAulay KA, Haque T, Crawford DH. Tumour necrosis factor gene polymorphism: a predictive factor for the development of post-transplant lymphoproliferative disease. Brit J Cancer. 101; 1019-1027. 2009.
KF Macsween1, CD Higgins2, KA McAulay1, H Williams1, N Harrison3*, AJ Swerdlow4, DH Crawford1 Infectious mononucleosis in university students: evaluation of the clinical features and consequences of the disease. CID 50: 699-706. 2010.
Crawford DH. The Invisible Enemy: A Natural History of Viruses (OUP 2000).
Crawford DH. Deadly Companions: How Microbes Shaped our History (OUP, 2007)
For further references, click here for PubMed.