Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2001-08-01
2004-03-02
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S186100, C424S230100, C530S300000, C536S023720
Reexamination Certificate
active
06699477
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to cytotoxic T-cell (CTL) epitopes within Epstein-Barr virus. The present invention also relates to subunit vaccines including CTL epitopes.
BACKGROUND OF THE INVENTION
Epstein-Barr virus (EBV) is a gamma herpesvirus which establishes a latent lifelong infection in the host following acute infection (14,15). While primary infection generally occurs in childhood without significant morbidity, adolescents and young adults may present with the symptoms of acute infectious mononucleosis (IM). The main feature of IM is a self-limiting lymphoproliferation involving both T and B cells accompanied by clinical symptoms such as fever and lymphadenopathy (52,53). Occasionally, the clinical symptoms persist and recur for extended periods after the initial infection. Episodic IM such as this has been described as chronic active EBV infection or, in some cases, severe chronic active EBV infection (35). EBV DNA has been detected in both serum and peripheral blood lymphocytes (PBL) during acute IM with the levels of detectable DNA gradually decreasing as the illness abates (21.22.58).
Evidence for latent EBV infection includes the observation that spontaneous lymphoblastoid cell lines (LCLs), expressing latent proteins, can be regularly established from healthy immune individuals after explantation of either lymph node tissue (34) or fractionated B lymphocytes (59). Although latent EBV infection is usually asymptomatic, sequential studies have established that recrudescence of viral replication in the oral cavity may result in release of infectious virus (59). The exact site of persistence of the virus is uncertain, but the available evidence suggests that small lymphocytes in the circulation harbour the virus in a nonproductive episomal state (24). Accordingly, in asymptomatic donors, EBV DNA is detectable by sensitive PCR analysis in PBL expressing the B-cell marker CD19(29,55).
EBV is also involved in post transplant lymphoproliferative disease, which involves a polyclonal expansion of EBV infected B-cells which is a life threatening lymphoma especially in transplantation patients. EBV is also involved in nasopharyngeal carcinoma and Hodgkinson's disease.
Two types of EBV (types A and B or types 1 and 2) are distinguishable primarily on the basis of variation in the DNA and protein sequences of the latent EBV-associated nuclear antigens (referred to here as EBNAs-2A, -3A, -4A and -6A from the type A virus and EBNAs-2B, -3B, -4B and -6B from the type B virus) (9,46,48). Sequencing of the prototypical isolates of type A and type B EBV (B95-8 and Ag-876 respectively) in these regions revealed 53% amino acid homology between EBNA-2A and EBNA-2B (9) and 72-84% homology between EBNAs-3A and -3B. EBNAs-4A and -4B, and EBNAs-6A and -6B (46). Strain variation due to other DNA alterations or deletions as well as these A/B type differences have been defined at the protein (12) and the DNA level (16,25,26) and recombination between multiple infecting strains was found to occur frequently in oral hairy leukoplakia lesions (56). These variations offer an alternative means of categorising EBV isolates but the primary distinction of type A and type B is still useful. Type A EBV is more readily isolated from healthy donors: type B EBV infections or dual infections with both type A and type B have proven easier to detect in immunosuppressed or HIV infected individuals (5,47,51). A higher incidence of type B infection in some studies led to the suggestion that type B or dual infections are, in fact, relatively common and that resident type B virus levels increase during immunosuppression (3,20,51).
It appears that latent EBV infection is primarily controlled by HLA class I-restricted memory cytotoxic T cell (CTL) responses (reviewed in (18)). These CTL responses can be reactivated in vitro by stimulating lymphocytes from seropositive individuals with autologous lymphoblastoid cell lines (LCLs) which express and present MHC class I and class II restricted epitopes at the cell surface. Several of these epitopes have been identified using target cells infected by recombinant vaccinia constructs (17,19,32,33). Epitopes specific for type A EBV as well as cross-reactive epitopes encoded by both types A and B EBV have been defined (18) but no epitopes specific for type B EBV have been reported thus far. In addition co-pending International Patent Application No. WO 95/001400, the disclosure of which is incorporated herein by cross reference discloses a number of EBV CTL epitopes. In the present study, the response of a donor exposed to both type A and type B EBV was investigated and an epitope specific for type B EBV as well as a new cross-reactive epitope were identified.
SUMMARY OF THE INVENTION
In a first aspect the present invention consists in a cytotoxic Epstein-Barr virus T-cell epitope, the epitope being selected from the group consisting of QVKWRMTTL (SEQ ID NO: 31), VFSDGRVAC (SEQ ID NO: 32), VPAPAGPIV (SEQ ID NO: 33), TYSAGIVQI (SEQ ID NO: 34), LLDFVRFMGV (SEQ ID NO: 35), QNGALAINTF (SEQ ID NO: 36), VSSDGRVAC (SEQ ID NO: 37), VSSEGRVAC (SEQ ID NO: 38), VSSDGRVPC (SEQ ID NO: 39), VSSDGLVAC (SEQ ID NO: 40), VSSDGQVAC (SEQ ID NO: 41), VSSDGRVVC (SEQ ID NO: 42), VPAPPVGPIV (SEQ ID NO: 43), VEITPYEPTG (SEQ ID NO: 44), VEITPYEPTW (SEQ ID NO: 45), VELTPYKPTW (SEQ ID NO: 46), RRIYDLIKL (SEQ ID NO: 47), RKIYDLIEL (SEQ ID NO: 48) and PYLFWLAGI (SEQ ID NO: 49).
In a second aspect the present invention consists in a subunit vaccine, the vaccine including at least one T-cell epitope selected from the group consisting of QVKWRMTTL (SEQ ID NO: 31), VFSDGRVAC (SEQ ID NO: 32), VPAPAGPIV (SEQ ID NO: 33), TYSAGIVQI (SEQ ID NO: 34), LLDFVRFMGV (SEQ ID NO: 35), QNGALAINTF (SEQ ID NO: 36), VSSDGRVAC (SEQ ID NO: 37), VSSEGRVAC (SEQ ID NO: 38), VSSDGRVPC (SEQ ID NO: 39), VSSDGLVAC (SEQ ID NO: 40), VSSDGQVAC (SEQ ID NO: 41), VSSDGRVVC (SEQ ID NO: 42), VPAPPVGPIV (SEQ ID NO: 43), VEITPYEPTG (SEQ ID NO: 44), VEITPYEPTW (SEQ ID NO: 45), VELTPYKPTW (SEQ ID NO: 46), RRIYDLIKL (SEQ ID NO: 47), RKIYDLIEL (SEQ ID NO: 48) and PYLFWLAGI (SEQ ID NO: 49).
In a preferred embodiment of this aspect of the present invention the subunit vaccine includes at least one further epitope selected from the group consisting of TSLYNLRRGTALA (SEQ ID NO: 1), DTPLIPLTIF (SEQ ID NO: 2), TVFYNIPPMPL (SEQ ID NO: 3), VEITPYKPTW (SEQ ID NO: 4), VSFIEFVGW (SEQ ID NO: 5), FRKAQIQGL (SEQ ID NO: 6), FLRGRAYGL (SEQ ID NO:7), QAKWRLQTL (SEQ ID NO: 8), SVRDRLARL (SEQ ID NO: 9), YPLHEQHGM (SEQ ID NO: 10), HLAAQGMAY (SEQ ID NO: 11), RPPIFIRRL (SEQ ID NO: 12), RLRAEAGVK (SEQ ID NO: 13), IVTDFSVIK (SEQ ID NO: 14), AVFDRKSDAK (SEQ ID NO: 15), NPTQAPVIQLVHAVY (SEQ ID NO: 16), LPGPQVTAVLLHEES (SEQ ID NO: 17), DEPASTEPVHDQLL (SEQ ID NO: 18), RYSIFFDY (SEQ ID NO: 19), AVLLHEESM (SEQ ID NO: 20), RRARSLSAERY (SEQ ID NO: 21), EENLLDFVRF (SEQ ID NO: 22), KEHVIQNAF (SEQ ID NO: 23), RRIYDLIEL (SEQ ID NO: 24), QPRAPIRPI (SEQ ID NO: 25), EGGVGWRHW (SEQ ID NO: 26), CLGGLLTMV (SEQ ID NO: 27), RRRWRRLTV (SEQ ID NO: 28), RAKFKQLL (SEQ ID NO: 29) and RKCCRAKFKQLLQHYR (SEQ ID NO: 30).
In a further preferred embodiment of this aspect of the present invention the subunit vaccine includes the cytotoxic T-cell epitopes LLDFVRFMGV (SEQ ID NO: 35), QVKWRMTTL (SEQ ID NO: 31) and FLRGRAYGL (SEQ ID NO:7). An analysis of allele frequency in the HLA listings in the Queensland Institute of Medical Research data base shows that a vaccine including these epitopes would provide protection for 63.7% of the caucasian population.
In a yet further preferred embodiment of this aspect of the present invention the subunit vaccine includes the cytotoxic T-cell epitopes LLDFVRFMGV (SEQ ID NO: 35), QVKWRMTTL (SEQ ID NO: 31), FLRGRAYGL (SEQ ID NO:7) and EENLLDFVRF (SEQ ID NO: 22). An analysis of allele frequency in the HLA listings in the Queensland Institute of Medical Research data base shows that a vaccine including these epitopes would provide protection for 71.1% of the caucasian population.
In another preferred embodiment of this aspect of the present
Burrows Scott Renton
Kerr Beverley Mavis
Khanna Rajiv
Misko Ihor Stephan
Moss Denis James
Fulbright & Jaworski
Salimi Ali R.
The Council of the Queensland Institute of Medical Research
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