CTL epitopes from EBV

Details CTL epitopes from EBV CTL epitopes from EBV

2000-05-30

2004-04-20

Housel, James (Department: 1646)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S300000, C530S327000, C530S328000, C536S023100, C435S320100

Reexamination Certificate

active

06723695

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods of treating or preventing EBV infections. The present invention also relates to cytotoxic T-cell (CTL) epitopes within Epstein-Barr virus (EBV) structural and latent antigens, and to subunit vaccines and nucleic acid vaccines which include these epitopes.
BACKGROUND OF THE INVENTION
It is now well established that long-term protection from persistent viral infection requires the development of virus-specific memory T cells which recognize viral antigens in association with either class I or class II MHC molecules. Since immunization with whole viral proteins is unable to elicit an efficient CTL response, interest has been directed towards designing vaccines based on defined epitope sequences. This is particularly the case with oncogenic viruses, since individual viral genes introduced in recombinant vectors have the potential to initiate tumorgenic processes. Two broad approaches are currently being considered to design an effective vaccine for controlling Epstein-Barr virus (EBV) associated diseases (for review see ref. (7)). These include directing immune responses to either EBV structural antigens or latent antigens.
In the last few years, most of the vaccine development efforts have concentrated on the use of a subunit preparation of gp350 (recombinant and affinity purified) and have been directed towards blocking virus attachment to the target cell in the oropharyrix (31). The general approach has been to immunize cotton-top marmosets with gp350 and determine their ability to restrict the outgrowth of EBV-positive lymphomas in these animals. Indeed, highly purified gp350, when administered subcutaneously in conjunction with adjuvants (muramyl dipeptide or ISCONIS). induced high levels of serum neutralizing antibodies and inhibited tumor formation in cotton-top tamarins (32). A number of recombinant vectors including, vaccinia-gp350 and adenovirus 5-gp350 have also been successfully used in these animals to block tumor outgrowth (33). The precise mechanism by which gp350 affords protection from lymphomas in cotton-top tamarins remains unclear. The fact that development of neutralizing antibody titres in vaccinated animals does not always correlate with protection indicates that gp350-specific T cell-mediated immune responses may also have an effector role (34). Furthermore, Yao and colleagues (35) showed that very low levels of neutralizing anti-gp350 antibodies are present in the saliva of healthy EBV-immune donors, which suggests that such antibodies are unlikely to be the basis of long-term immunity in healthy seropositive individuals. It has been postulated that gp350 specific T cell -mediated immune responses may have an effector role in protection. There has been no identification to date, however, of CTL epitopes within the EBV structural antigens.
Post-transplant lymphoproliferative disease (PTLD) that arises in organ transplant patients is an increasingly important clinical problem. Histological analysis of PTLD shows a quite complex clonal diversity ranging from polymorphic B lymphocyte hyperplasia to malignant monoclonal lymphoma. This range of pathology encompasses the collective term PTLD while the lymphomas are frequently referred to as immunoblastic lymphomas(IL). This condition is clearly associated with the proliferation of Epstein-Barr virus (EBV) infected B cells which are carried for life in all previously infected individuals (about 80% of adults and 20% of children 7 years) (45, 46, 47, 49). These EBV-infected B cells are normally restricted in their growth in vitro and in vivo by virus-specific cytotoxic T cells (CTLs) which recognise epitopes included within the EBV latent proteins (see below) (48). Immunosuppression inhibits these specific CTL and results in an expansion of the pool of EBV-infected B cells and the emergence of the clinical problems associated with PTLD. It is known that the individuals at greatest risk of PTLD are EBV seronegative recipients who receive a transplant from a seropositive donor (Crawford and Thomas, 1993). Immunisation of EBV seronegative graft recipients prior to engraftment will greatly reduce the risk of PTLD.
The role of the immune system in the rejection of virus-associated cancers has also been the subject of intense study recently. The hypothesis under investigation is that many neoplasms express viral antigens that should potentially enable them to be recognized and destroyed by the immune system, including both T helper cells and cytotoxic T lymphocytes (CTL). There is now compelling evidence that most of the Epstein-Barr virus (EBV)-associated malignancies escape this potent virus-specific CTL response by restricting viral gene expression (7.20,21). For malignancies such as nasopharyngeal carcinoma (NTC) and Hodgkin's disease (HD). EBV nuclear antigen 1 (EBNA1) and latent membrane protein 1 (LMP1) are the only antigens consistently expressed and are therefore the potential target antigens for any future vaccine designed to control these tumors (3,28). Since it is well established that immunization with whole viral proteins does not elicit an efficient CTL response, interest has been directed towards developing peptide vaccines based on defined epitope sequences.
SUMMARY OF THE INVENTION
Results obtained by the present inventors indicate that CTL epitopes within EBV structural and latent proteins may be effective in providing antiviral immunity against EBV infection. In particular, the present inventors have analysed the latent antigen LMP1 sequence, using peptide stablization assays, and found that this antigen includes potential CTL epitopes. Following in vitro activation with these peptides, both polyclonal and clonal CTLs from HLA A2-positive donors showed strong reactivity against target cells expressing the LMP1 antigen. Moreover. lvmphoblastoid cell lines (LCL). expressing different HLA A2 supertypes were efficiently recognized by these CTLs, a result that has important implications for the design of an anti-viral vaccine aimed at protecting different ethnic populations.
The present inventors have also found that CTLs from acute infectious mononucleosis (IM) patients display strong reactivity against the EBV structural antigens gp85 and gp350. In addition, specific CTL epitopes within EBV structural antigens gp85 and gp350 have been identified for the first time. Importantly, prior immunisation of HLA A2/K
b
transgenic mice with these gp350 and gp85 CTL epitopes induced a strong epitope-specific CTL response and afforded protection against gp85- or gp350-expressing vaccinia virus challenge. These results provide evidence, for the first time, of the existence of CTL epitopes in EBV structural proteins and show that they may be used for establishing strong anti-viral immunity against EBV infection.
Accordingly, in a first aspect the present invention provides a cytotoxic Epstein-Barr virus (EBV) T-cell epitope, the epitope being derived from an EBV structural antigen.
In a preferred embodiment of the first aspect of the present invention, the EBV structural antigen is gp85 or gp350.
In a second aspect the present invention provides a cytotoxic Epstein-Barr virus T-cell epitope, the epitope being selected from the group consisting of YLLEMLWRL (SEQ ID NO:1), YFLEILWGL (SEQ ID NO:32), YLLEILWRL (SEQ ID NO:33), YLQQNWWTL (SEQ ID NO:6), LLLALLFWL (SEQ ID NO:2), LLVDLLWLL (SEQ ID NO:3), LLLIALWNL (SEQ ID NO:4), WLLLFLAIL (SEQ ID NO:5), TLLVDLLWL (SEQ ID NO:7), LLWLLLFLA (SEQ ID NO:8), ILLIIALYL (SEQ ID NO:9), VLFIFGCLL (SEQ ID NO:10), RLGATIWQL (SEQ ID NO:11), ILYFIAFAL (SEQ ID NO:15), SLVIVTTFV (SEQ ID NO:17), LMIIPLINV (SEQ ID NO:20), TLFIGSHVV (SEQ ID NO:24), LIPETVPYI (SEQ ID NO:26), VLQWASLAV (SEQ ID NO:27) and QLTPHTKAV (SEQ ID NO:29).
In a third aspect the present invention provides a subunit vaccine including a cytotoxic Epstein-Barr virus (EBV) T-cell epitope according to the first aspect of the present invention.
In a preferred embodiment, the subunit vaccine includes at least one T-cell epitope selected from the group consisting

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