The present invention relates to the identification of a subunit vaccine to prevent or treat infection of Epstein Barr Virus. In particular, EBNA-1 was identified as a vaccine antigen. In a specific embodiment, a purified protein corresponding to EBNA-1 elicited a strong CD4.sup.+ T cell response. The responsive CD4.sup.+ T cell are primarily T.sub.H1 in function. EBNA-1 is an attractive candidate for a protective vaccine against EBV, and for immunotherapy of EBV infection and neoplasms, particularly with dendritic cells charged with EBNA-1.

Description of the Invention

The present invention is based, in part on unexpected discoveries concerning a protective antigen of Epstein Barr Virus (EBV). In most EBV seropositive adults, strong CD8.sup.+ cytotoxic T lymphocytes (CTL) responses have been demonstrated (Murray, et al., J. Exp. Med., 1992, 176:157-168). However, these are preferentially directed toward the nuclear antigens, EBNA3A, 3B and 3C (Kieff, E., Epstein-Barr Virus and Its Replication. In Fields Virology. B. N. Fields, D. M. Knipe, and P. M. Howley, editors. 1996, Lippincott-Raven Publishers, Philadelphia. 2343-2396; Khanna, et al. J. Exp. Med., 1992, 176:169-176), which are not expressed in many EBV-associated malignancies. EBV-transformed cells exhibit one of three latency phenotypes distinguished from each other by the panel of expressed EBV antigens (Murray, et al., 1992, supra). In latency I, e.g., Burkitt's lymphoma, EBNA-1 alone is expressed. In latency II, exemplified by Hodgkin's lymphoma, LMP1 and LMP2 as well as EBNA-1 are expressed. Only in latency m immunoblastic lymphomas are the highly immunogenic EBNA3 genes expressed. Therefore, many EBV-associated malignancies do not seem to provide good targets for the human CD8.sup.+ T cell response to EBV latency gene products.

The evidence in the present invention suggests that, unexpectedly, the Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA-1) is an effective antigen for developing an EBV vaccine, particularly an anti-tumor vaccine. EBNA-1 immunity greatly reduces viral replication because this antigen is crucial for the persistence of the EBV episome in replicating EBV-transformed human B cells (Yates, et al., Nature, 313:812-5, 1985). Therefore, all EBV-induced tumors express this foreign antigen. However, EBNA-1 protein is invisible to CD8.sup.+ cytotoxic T lymphocytes (CTLs). The gly-ala repeat domain prevents proteasome dependent processing and thus presentation on MHC class I (Levitskaya, et al., supra). It has now been found that CD4.sup.+ T cells from most individuals do respond to EBNA-1. In fact, among EBV latent antigens that stimulate CD4.sup.+ cells, EBNA-1 is preferentially recognized. Recognition can occur via endogenous and exogenous processing of EBNA-1 onto MHC class II molecules of dendritic cells (DCs). The CD4.sup.+ response includes direct cytolysis of transformed B lymphocyte cell lines (B-LCL). Therefore, the immune system can recognize the EBNA-1 protein that is crucial for EBV persistence.

The type of CD4.sup.+ T cell also influences the response (reviewed in O'Garra, and Murphy, Curr. Opin. Immunol., 1994, 6:458-66. 20). T.sub.H1 CD4.sup.+ cells secrete IFN.gamma. and help in the development of cellular immunity, including the activation of macrophages. T.sub.H2 CD4.sup.+ cells secrete IL-4 and IL-5, thereby stimulating eosinophils and antibody production. The expression of specific chemokine receptors on the T.sub.H1 cells results in the migration of the T.sub.H1 cells to the normal cites of inflammation. T.sub.H2 cells migrate to cites more closely associated with allergic responses. CD4.sup.+ T cells also can kill targets (reviewed in Hahn, et al., Immunol Rev., 1995., 146:57-79), primarily through Fas-FasL interactions. Cytotoxicity mainly has been found with T.sub.H1 CD4.sup.+ cells (Erb, et al., Cell Immunol., 1991, 135:232-44; Erb, et al., J. Immunol., 1990, 144:790-795; Del Prete, et al., J. Exp. Med., 1991, 174:809-13; Nishimura, et al., J. Exp. Med., 1999, 190:617-628.), but a limited number of cytotoxic T.sub.H2 CD4.sup.+ clones have been reported (Lancki, et al., J. Immunol., 1991, 146:3242-9).

Evidence suggests that the EBNA-1-specific, CD4.sup.+ T cell response in cells that are directly isolated from blood is predominantly a T.sub.H1 response. Furthermore, the isotype of the EBNA-1 antibody response is skewed to the IgG1 subclass, reflecting T.sub.H1 polarization in vivo. This result has important implications for a therapeutic vaccine for EBNA-1 based on the emerging evidence that T.sub.H1 cells are important for resistance to viruses and tumors, and thus are key to long term immunity.

Immunotherapy of EBV with EBNA-1 provides significant advantages of safety and efficacy. Subunit vaccines ensure the greatest degree of safety because there is no opportunity for infection by the pathogen. This is always of some concern when immunizing with killed or attenuated virus. Furthermore, a single component minimizes adverse side effects, such as anaphylaxis or antigen cross reactivity, that may result from an unrelated antigen in a whole virus vaccine. Immunotherapy with dendritic cells charged with EBNA-1 has substantial therapeutic potential. Furthermore, because EBNA-1 is involved in EBV infection and tumorigenesis, it will elicit the most protective immune response to prevent or treat EBV infection and associated diseases or disorders. Another alternative vaccine approach is to define the DR-specific peptides in the EBNA-1 protein. Pulsing dendritic cells with these peptides, and then immunizing subjects with the pulsed dendritic cells would establish the protective effect of the vaccine strategy.

The Immuno-Protective Antigen of Epstein Barr Virus

The present invention provides an immunoprotective antigen of Epstein Barr Virus, a protective or therapeutic protein or DNA vaccine, and immunotherapy using EBNA-1 charged dendritic cells to prevent or treat EBV infection. The immunoprotective antigen is an immunogenic EBNA-1 polypeptide. As discussed in greater detail below, an EBNA-1 polypeptide can be an EBNA-1 protein, a fusion protein comprising an amino acid sequence, or a fragment of EBNA-1 that includes the immunoprotective epitope.

The term "immunogenic EBNA-1-polypeptide" refers to the EBNA-1 protein, or a portion thereof, that is immunogenic and elicits a protective immune response when administered to an animal. Thus, an EBNA-1 immunoprotective antigen need not be the entire protein. The protective immune response generally involves cellular immunity at the CD4.sup.+ T cell level.

The immunogenic polypeptide can comprise an immuno-protective EBNA-1 antigen from any strain of Epstein Barr Virus, or sequence variants of EBNA-1, as found in nasopharyngeal carcinoma and infected individuals (Chen et al., J. Gen. Virol., 80:447, 1999; Gutierrez et al., J. Gen. Virol., 78:1663, 1997).

As used herein, the term "immunogenic" means that the polypeptide is capable of eliciting a humoral or cellular immune response, and preferably both. An immunogenic polypeptide is also antigenic. A molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. An antigenic polypeptide contains an epitope of at least about five, and preferably at least about 10, amino acids. An antigenic portion of a polypeptide, also called herein the epitope, can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier polypeptide for immunization. A molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier.

The term "carrier polypeptide" as used herein refers to a protein or immunogenic fragment thereof that can be conjugated or joined with the immunogenic EBNA-1 to enhance immunogenicity of the polypeptide. Examples of carrier proteins include, but are by no means limited to, keyhole limpet hemocyanin (KLY), albumin, cholera toxin (discussed in greater detail below), heat labile enterotoxin (LT), and the like. While chemical cross-linking of a peptide comprising the immuno-protective epitope of EBNA-1 with the carrier polypeptide can be used to prepare an immunogenic polypeptide, preferably the two components are prepared as a chimeric construct for expression as a fusion polypeptide.

In addition, chimeric fusion polypeptides of the immunogenic polypeptide with a purification handle, such as FLAG or GST (for immunopurification), or a HIS-tag (for Ni-chelation purification), are contemplated.

Where the full length recombinant EBNA-1 is used as the immunogenic polypeptide, preferably it is free from viral components, e.g., in distinction to vaccines comprising whole killed or attenuated virus. EBNA-1 polypeptide can be purified after recombinant expression, or it can be delivered by expression in situ, i.e., by expression from a vector (a DNA vaccine).

In addition, the present invention permits use of various mutants, sequence conservative variants, and functional conservative variants of EBNA-1, provided that all such variants retain the required immuno-protective effect.

The terms "mutant" and "mutation" mean any detectable change in genetic material, e.g. DNA, or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g. DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g. protein) expressed by a modified gene or DNA sequence. The term "variant" may also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant.

"Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. Allelic variants can be sequence-conservative variants.

"Function-conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Some allelic variations result in functional-conservative variants, such that an amino acid substitution does not dramatically affect protein function. Similarly, homologous proteins can be function-conservative variants. Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A "function-conservative variant" also includes a polypeptide or enzyme which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90%, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared.

As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin," including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck, et al., Cell 50:667, 1987). Such proteins (and their encoding genes) have sequence homology, as reflected by their sequence similarity, whether in terms of percent similarity or the presence of specific residues or motifs.

Accordingly, the term "sequence similarity" in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck, et al., supra). However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and may or may not relate to a common evolutionary origin.

In a specific embodiment, two DNA sequences are "substantially homologous" or "substantially similar" when a sufficient number of the nucleotides match over the defined length of the DNA sequences to differentiate the sequences from other sequences, as determined by sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, etc. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.

Similarly, in a particular embodiment, two amino acid sequences are "substantially homologous" or "substantially similar" when enough of the amino acids are identical or similar (functionally identical) over a defined length to differentiate the sequences from other sequences. Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program, or any of the programs described above (BLAST, FASTA, etc.).

Furthermore, it should be noted that depending on the expression system employed, the expressed protein can differ from the predicted amino acid sequence encoded by a coding sequence. For example, a construct for expression of the immunogenic polypeptide can express a protein comprising a signal sequence, which may be cleaved or not during cellular processing. In addition, other proteolytic cleavages may occur during expression. If the polypeptide is expressed in eukaryotic cells, it may be glycosylated if it contains a glycosylation site. Other possible changes include N-methylation, and the like.

As used herein, the term "isolated" means that the referenced material is removed from its native environment, e.g., a cell. Thus, an isolated biological material can be free of some or all cellular components, i.e., components of the cells in which the native material occurs naturally (e.g., cytoplasmic or membrane component). A material shall be deemed isolated if it is present in a cell extract or if it is present in a heterologous cell or cell extract. In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined or proximal to non-coding regions (but may be joined to its native regulatory regions or portions thereof), or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like, i.e., when it forms part of a chimeric recombinant nucleic acid construct. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism. An isolated material may be, but need not be, purified.

The term "purified" as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.

Methods for purification are well-known in the art. For example, nucleic acids can be purified by precipitation, chromatography (including without limitation preparative solid phase chromatography, oligonucleotide hybridization, and triple helix chromatography), ultracentrifugation, and other means. Polypeptides and proteins can be purified by various methods including, without limitation, preparative disc-gel electrophoresis and isoelectric focusing; affinity, HPLC, reversed-phase HPLC, gel filtration or size exclusion, ion exchange and partition chromatography; precipitation and salting-out chromatography; extraction; and countercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence, or a sequence that specifically binds to an antibody, such as FLAG and GST. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents. Cells can be purified by various techniques, including centrifugation, matrix separation (e.g., nylon wool separation), panning and other immunoselection techniques, depletion (e.g., complement depletion of contaminating cells), and cell sorting (e.g., fluorescence activated cell sorting (FACS)). Other purification methods are possible and contemplated herein. A purified material may contain less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components, media, proteins, or other nondesirable components or impurities (as context requires), with which it was originally associated. The term "substantially pure" indicates the highest degree of purity which can be achieved using conventional purification techniques known in the art.

In a specific embodiment, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. Alternatively, logarithmic terms used in biology, the term "about" can mean within an order of magnitude of a given value, and preferably within one-half an order of magnitude of the value.

Recombinant Expression Systems

The present invention contemplates various cloning and expression vectors for expression of the immunogenic polypeptides described herein. Such expression vectors can be used to transform cells in vitro to produce immunogenic polypeptides for protein vaccines, or in vivo to express the immunogenic polypeptide for a DNA vaccine.

The coding sequence for an immunogenic polypeptide may, and preferably does, include a signal sequence, which can be a heterologous signal sequence, e.g., for optimized signal sequence processing in a bacterial, yeast, insect, or mammalian cell. The term "signal sequence" is used herein to refer to the N-terminal, hydrophobic sequence found on most secreted proteins that identifies it for processing for secretion from the cell. Generally, the signal sequence is cleaved during processing. However, various constructs of the invention can include a partial signal sequence. It is not necessarily the case that the partial signal sequence is processed normally, or that it even provides for translocation during expression, e.g., to the bacterial periplasm.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

Molecular Biology Definitions

A "nucleic acid molecule" (or alternatively "nucleic acid") refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. This term includes double-stranded DNA found, inter alia, in linear (e.g., restriction fragments) or circular DNA molecules, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.

A "coding sequence" or a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

The coding sequences herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'-non-coding regions, and the like.

The term "gene", also called a "structural gene" means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.

The introduced gene or coding sequence may also be called a "cloned", "foreign", or "heterologous" gene or sequence, and may include regulatory or control sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function.

The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. A host cell has been "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. A cell has been "transformed" by exongenous or heterologous DNA when the transfected DNA is expressed and effects a function or phenotype on the cell in which it is expressed. The term "expression system" means a host cell transformed by a compatible expression vector and cultured under suitable conditions e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

Proteins and polypeptides can be made in the host cell by expression of recombinant DNA. As used herein, the term "polypeptide" refers to an amino acid-based polymer, which can be encoded by a nucleic acid or prepared synthetically. Polypeptides can be proteins, protein fragments, chimeric proteins, etc. Generally, the term "protein" refers to a polypeptide expressed endogenously in a cell, e.g., the naturally occurring form (or forms) of the amino acid-based polymer. Generally, a DNA sequence having instructions for a particular protein or enzyme is "transcribed" into a corresponding sequence of RNA. The RNA sequence in turn is "translated" into the sequence of amino acids which form the protein or enzyme. An "amino acid sequence" is any chain of two or more amino acids.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

A coding sequence is "under the control" or "operatively associated with" of transcriptional and translational (i.e., expression) control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if it contains introns) and translated into the protein encoded by the coding sequence.

The terms "express" and "expression" mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself , e.g. the resulting protein, may also be said to be "expressed" by the cell. An expression product can be characterized as intracellular, extracellular, or secreted. The term "intracellular" means something that is inside a cell. The term "extracellular" means something that is outside a cell, either in the cell membrane or secreted from the cell. A substance is "secreted" by a cell if it appears in significant measure in the external medium outside the cell, from somewhere on or inside the cell.

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc. A "cassette" refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct." A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes.

The term "expression system" means a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Expression systems include bacterial, insect, or mammalian host cells and vectors. Bacterial and insect cell expression is exemplified infra. Suitable mammalian cells include C12 cells, CHO cells, HeLa cells, 293 and 293T (human kidney cells), COS cells, mouse primary myoblasts, and NIH 3T3 cells.

The term "heterologous" refers to a combination of elements not naturally occurring. For example, heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell. A heterologous expression regulatory element is a such an element operatively associated with a different gene than the one it is operatively associated with in nature. In the context of the present invention, an gene is heterologous to the recombinant vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed, e.g., a CHO cell.

A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook, et al., supra). For hybrids of greater than 100 nucleotides in length, equations for calculating T.sub.m have been derived (see Sambrook, et al., supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook, et al., supra, 11.7-11.8). A minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.

Expression Vectors

A wide variety of host/expression vector combinations (i.e., expression systems) may be employed in expressing the immunogenic polypeptides of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, SV40 and pMal-C2, pET, pGEX (Smith, et al., Gene 67:31-40, 1988), pMB9 and their derivatives, plasmids such as RP4; gram positive vectors such as Strep. gardonii; phage DNAS, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2m plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Expression of the protein or polypeptide may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist and Chambon, Nature 290:304-310, 1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 22:787-797, 1980), the herpes thymidine kinase promoter (Wagner, et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981), the regulatory sequences of the metallothionein gene (Brinster, et al., Nature 296:39-42, 1982); prokaryotic expression vectors such as the b-lactamase promoter (Villa-Komaroff, et al., Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731, 1978), or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A. 80:21-25, 1983); see also "Useful proteins from recombinant bacteria" in Scientific American, 242:74-94, 1980; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and control regions that exhibit hematopoietic tissue specificity, in particular: immunoglobin gene control region, which is active in lymphoid cells (Grosschedl et al., Cell, 38:647, 1984; Adames et al., Nature, 318:533, 1985; Alexander et al., Mol. Cell Biol., 7:1436, 1987); beta-globin gene control region which is active in myeloid cells (Mogram, et al., Nature 315:338-340, 1985; Kollias, et al., Cell 46:89-94, 1986), hematopoietic stem cell differentiation factor promoters; erythropoietin receptor promoter (Maouche, et al., Blood, 15:2557, 1991), etc.; and control regions that exhibit mucosal epithelial cell specificity.

Preferred vectors, particularly for cellular assays in vitro and vaccination in vivo or ex vivo, are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses, Fowl pox, AV-pox, modified vaccinia Ankara (MVA) and other recombinant viruses with desirable cellular tropism. In a specific embodiment, a vaccinia virus vector is used to infect dendritic cells. In another specific embodiment, a baculovirus vector that expresses EBNA-1 is prepared. Thus, a vector encoding an immunogenic polypeptide can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.

Viral vectors commonly used for in vivo or ex vivo targeting and vaccination procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques, 7:980-990, 1992). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. Preferably, the replication defective virus is a minimal virus, i.e., it retains only the sequences of its genome which are necessary for encapsidating the genome to produce viral particles.

DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), vaccinia virus, and the like. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt, et al., Molec. Cell. Neurosci. 2:320-330, 1991; International Patent Publication No. WO 94/21807, published Sep. 29, 1994; International Patent Publication No. WO 92/05263, published Apr. 2, 1994); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet, et al. (J. Clin. Invest. 90:626-630, 1992; see also La Salle, et al., Science 259:988-990, 1993); and a defective adeno-associated virus vector (Samulski, et al., J. Virol. 61:3096-3101, 1987; Samulski, et al., J. Virol. 63:3822-3828, 1989; Lebkowski, et al., Mol. Cell. Biol. 8:3988-3996, 1988).

Various companies produce viral vectors commercially, including but by no means limited to Avigen, Inc. (Alameda, Calif.; AAV vectors), Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).

Adenovirus Vectors.

Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Various serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see WO94/26914). Those adenoviruses of animal origin which can be used within the scope of the present invention include adenoviruses of canine, bovine, murine (example: Mav1, Beard, et al., Virology 75 (1990) 81), ovine, porcine, avian, and simian (example: SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800), for example). Various replication defective adenovirus and minimum adenovirus vectors have been described (WO94/26914, WO95/02697, WO94/28938, WO94/28152, WO94/12649, WO95/02697 WO96/22378). The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (Levrero, et al., Gene 101:195 1991; EP 185 573; Graham, EMBO J. 3:2917, 1984; Graham, et al., J. Gen. Virol. 36:59 1977). Recombinant adenovirus is an efficient and non-perturbing vector for human dendritic cells (Zhong et al., Eur. J. Immunol., 29:964, 1999; DiNicola et al., Cancer Gene Ther., 5:350-6, 1998). Recombinant adenoviruses are recovered and purified using standard molecular biological techniques, which are well known to one of ordinary skill in the art.

Adeno-Associated Viruses.

The adeno-associated viruses (AAV) are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see WO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941, EP 488 528). The replication defective recombinant AAVs according to the invention can be prepared by cotransfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line which is infected with a human helper virus (for example an adenovirus). The AAV recombinants which are produced are then purified by standard techniques. These viral vectors are also effective for gene transfer into human dendritic cells (DiNicola et al., supra).

Retrovirus Vectors.

In another embodiment the gene can be introduced in a retroviral vector, e.g., as described in Anderson, et al., U.S. Pat. No. 5,399,346; Mann, et al., 1983, Cell 33:153; Temin, et al., U.S. Pat. No. 4,650,764; Temin, et al., U.S. Pat. No. 4,980,289; Markowitz, et al., 1988, J. Virol. 62:1120; Temin, et al., U.S. Pat. No. 5,124,263; EP 453242, EP178220; Bernstein, et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689; International Patent Publication No. WO 95/07358, published Mar. 16, 1995, by Dougherty, et al.; and Kuo, et al., 1993, Blood 82:845. The retroviruses are integrating viruses which infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In recombinant retroviral vectors, the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukaemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Suitable packaging cell lines have been described in the prior art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line (WO 90/02806) and the GP+envAm-12 cell line (WO 89/07150). In addition, the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene (Bender, et al., J. Virol. 61:1639, 1987). Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

Retrovirus vectors can also be introduced by DNA viruses, which permits one cycle of retroviral replication and amplifies tranfection efficiency (see WO 95/22617, WO 95/26411, WO 96/39036, WO 97/19182).

Lentivirus Vectors.

In another embodiment, lentiviral vectors are can be used as agents for the direct delivery and sustained expression of a transgene in several tissue types, including brain, retina, muscle, liver and blood. The vectors can efficiently transduce dividing and nondividing cells in these tissues, and maintain long-term expression of the gene of interest. For a review, see, Naldini, Curr. Opin. Biotechnol., 9:457-63, 1998; see also Zufferey, et al., J. Virol., 72:9873-80, 1998). Lentiviral packaging cell lines are available and known generally in the art. They facilitate the production of high-titer lentivirus vectors for gene therapy. An example is a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 106 IU/ml for at least 3 to 4 days (Kafri, et al., J. Virol., 73: 576-584, 1999). The vector produced by the inducible cell line can be concentrated as needed for efficiently transducing nondividing cells in vitro and in vivo.

Vaccinia Virus Vectors.

Vaccinia virus is a member of the pox virus family and is characterized by its large size and complexity. Vaccinia virus DNA is double-stranded and terminally crosslinked so that a single stranded circle is formed upon denaturation of the DNA. The virus has been used for approximately 200 years in a vaccine against smallpox and the properties of the virus when used in a vaccine are known (Paoletti, Proc. Natl. Acad. Sci. U.S.A., 93:11349-53, 1996; and Ellner, Infection, 26:263-9,1998). The risks of vaccination with vaccinia virus are well known and well defined and the virus is considered relatively benign. Vaccinia virus vectors can be used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into the vaccinia vector and creating synthetic recombinants of the vaccinia virus has been described (see U.S. Pat. No. 4,603,112, U.S. Pat. No. 4,722,848, U.S. Pat. No. 4,769,330 and U.S. Pat. No. 5,364,773). A large number of foreign (i.e. non-vaccinia) genes have been expressed in vaccinia, often resulting in protective immunity (reviewed by Yamanouchi, Barrett, and Kai, Rev. Sci. Tech., 17:641-53, 1998; Yokoyama, et al., J. Vet. Med. Sci., 59:311-22, 1997; and see Osterhaus, et al., Vaccine, 16:1479-81 1998: and Gherardi et al., J. Immunol., 162:6724-33, 1999). Vaccinia virus may be inappropriate for administration to immunocompromised or immunosuppressed individuals. Alternative pox viruses which may be used in the invention include Fowl pox, AV-pox, and modified vaccinia Ankara (MVA) virus. The preferred embodiment to improve the immunogenic potential of these alternative viruses is to deliver the viruses containing EBNA-1 directly to dendritic cells, and then induce the dendritic cells to mature.

Nonviral Vectors.

In another embodiment, the vector can be introduced in vivo by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner, et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417, 1987; Felgner and Ringold, Science 337:387-388, 1989; see Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031, 1988; Ulmer, et al., Science 259:1745-1748, 1993). Useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127. Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et al., supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication WO95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO96/25508), or a cationic polymer (e.g., International Patent Publication WO95/21931).

Alternatively, non-viral DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun (ballistic transfection; see, e.g., U.S. Pat. No. 5,204,253, U.S. Pat. No. 5,853,663, U.S. Pat. No. 5,885,795, and U.S. Pat. No. 5,702,384 and see Sanford, TIB-TECH, 6:299-302, 1988; Fynan et al., Proc. Natl. Acad. Sci. U.S.A., 90:11478-11482, 1993; and Yang et al., Proc. Natl. Acad. Sci. U.S.A., 87:1568-9572, 1990), or use of a DNA vector transporter (see, e.g., Wu, et al., J. Biol. Chem. 267:963-967, 1992; Wu and Wu, J. Biol. Chem. 263:14621-14624, 1988; Hartmut, et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams, et al., Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991). Receptor-mediated DNA delivery approaches can also be used (Curiel, et al., Hum. Gene Ther. 3:147-154, 1992; Wu and Wu, J. Biol. Chem. 262:4429-4432, 1987). U.S. Pat. Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Mir, et al., C. P. Acad. Sci., 321:893, 1998; WO 99/01157; WO 99/01158; WO 99/01175).

Vaccine Technology and Immunotherapy

As noted above, the present invention contemplates polypeptide vaccines, and DNA vaccines to deliver an immunogenic EBNA-1 polypeptide to prevent or treat an Epstein Barr Virus infection, or an associated disease (e.g., infectious mononucleosis, endemic Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, T cell lymphoma, gastric carcinoma, and uterine leiomyosarcoma, and possibly chronic diseases such as chronic fatigue syndrome).

The vaccines of the invention are broadly applicable to protect an animal from infection by Epstein Barr Virus. The term "protect" is used herein to mean for the treatment or prevention of Epstein Barr Virus infection. Thus, any animal susceptible to this type of infection can be vaccinated. EBV shows a greater similarity phylogenetically to gammaherpesviruses, herpesvirus saimiri, and bovine herpesvirus 4 than to other classes of herpesvirus (Karlin, et al., J. Virol., 68:1886;902, 1994; and Bublot, et al., Virology, 190:654-65, 1992). Animal models for EBV occur in some species of New World monkeys (Franken, et al., J. Virol., 69:8011-9, 1995) as well as in mice (Mistrikova and Mrmusova, Acta. Virol., 42:79-82, 1998, Weck, et al., J. Virol., 73:4651-61, 1999; and Simas and Efstathiou, Trends Microbiol., 6:276-82, 1998) and rabbits (Wutzler, et al., Arch. Virol., 140:1979-95, 1995; and Handley, et al., Vet. Microbiol., 47:167-81, 1995). At least one EBV-like herpesvirus that infects monkeys contains a gene with homology to EBNA-1 (Li, et al., Int. J. Cancer, 59:287-95, 1994). Animals infected with EBV-like viruses could be treated with a vaccine of the invention to prevent or treat disease.

Polypeptide Vaccines

As used herein, the term "polypeptide vaccine" refers to a vaccine comprising an immunogenic polypeptide and, generally, an adjuvant. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood, et al., Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p. 384). Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response. Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. An example of a preferred synthetic adjuvant is QS-21. Alternatively, or in addition, immunostimulatory proteins, as described below, can be provided as an adjuvant or to increase the immune response to a vaccine. Preferably, the adjuvant is pharmaceutically acceptable.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Sterile water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

Certain adjuvants mentioned above, particularly mineral oils and adjuvants containing mineral oils (e.g., Freund's adjuvant) are not acceptable for use in humans.

"DNA" Vaccines

The term "DNA vaccines" is an informal term of art, and is used herein to refer to vaccines delivered by means of a recombinant vector. An alternative, and more descriptive term used herein is "vector vaccine" (since some potential vectors, such as retroviruses and lentiviruses are RNA viruses, and since in some instances non-viral RNA instead of DNA can be delivered to cells). Generally, the vector is administered in vivo, but ex vivo transduction of appropriate antigen presenting cells, such as dendritic cells, with administration of the transduced cells in vivo, is also contemplated. The vector systems described above are ideal for delivery of a vector for expression of an immunogenic polypeptide of the invention.

Vaccination and Immunotherapy Strategies

Various strategies can be employed to vaccinate subjects against Epstein Barr Virus infection. The polypeptide vaccine formulations can be delivered by subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.), subdermal (s.d.), intradermal (i.d.), or by administration to antigen presenting cells ex vivo followed by administration of the cells to the subject. Prior to administration to the subject, the antigen presenting cells may be induced to mature.

Similarly, any of the gene delivery methods described above can be used to administer a vector vaccine to a subject, such as naked DNA and RNA delivery, e.g., by gene gun or direct injection.

Vaccination effectiveness may be enhanced by co-administration of an immunostimulatory molecule (Salgaller and Lodge, J. Surg. Oncol., 68:122, 1998), such as an immunostimulatory, immunopotentiating, or pro-inflammatory cytokine, lymphokine, or chemokine with the vaccine, particularly with a vector vaccine. For example, cytokines or cytokine genes such as interleukin (IL)-1, IL-2, IL-3, IL-4, IL-12, IL-13, granulocyte-macrophage (GM)-colony stimulating factor (CSF) and other colony stimulating factors, macrophage inflammatory factor, Flt3 ligand (Lyman, Curr. Opin. Hematol., 5:192, 1998), as well as some key costimulatory molecules or their genes (e.g., B7.1, B7.2) can be used. These immunostimulatory molecules can be delivered systemically or locally as proteins or by expression of a vector that codes for expression of the molecule. The techniques described above for delivery of the immunogenic polypeptide can also be employed for the immunostimulatory molecules.

Dendritic Cell Targeting.

Vaccination and particularly immunotherapy may be accomplished through the targeting of dendritic cells (Steinman, J. Lab. Clin. Med., 128:531, 1996; Steinman, Exp. Hematol., 24:859, 1996; Taite et al., Leukemia, 13:653, 1999; Avigan, Blood Rev., 13:51, 1999; DiNicola et al., Cytokines Cell. Mol. Ther., 4:265, 1998). Dendritic cells play a crucial role in the activation of T-cell dependent immunity. Proliferating dendritic cells can be used to capture protein antigens in an immunogenic form in situ and then present these antigens in a form that can be recognized by and stimulates T cells (see, e.g., Steinman, Exper. Hematol. 24:859-862, 1996; Inaba, et al., J. Exp. Med., 188:2163-73, 1998 and U.S. Pat. No. 5,851,756). For ex vivo stimulation, dendritic cells are plated in culture dishes and exposed to (pulsed with) antigen in a sufficient amount and for a sufficient period of time to allow the antigen to bind to the dendritic cells. Additionally, dendritic cells may be transfected with DNA using a variety of physical or chemical as described by Zhong et al., Eur. J. Immunol., 29:964-72, 1999; Van Tendeloo, et al., Gene Ther., 5:700-7, 1998; Diebold et al., Hum. Gene Ther., 10:775-86, 1999; Francotte and Urbain, Proc. Natl. Acad. Sci. USA, 82:8149, 1985 and U.S. Pat. No. 5,891,432 (Casares et al., J. Exp. Med., 186:1481-6, 1997). The pulsed cells can then be transplanted back to the subject undergoing treatment, e.g., by intravenous injection. Preferably autologous dendritic cells, i.e., dendritic cells obtained from the subject undergoing treatment, are used, although it may be possible to use MHC-Class II-matched dendritic cells, which may be obtained from a type-matched donor or by genetic engineering of dendritic cells to express the desired MHC molecules (and preferably suppress expression of undesirable MHC molecules.)

Preferably, the dendritic cells are specifically targeted in vivo for expression of EBNA-1. Various strategies are available for targeting dendritic cells in vivo by taking advantage of receptors that mediate antigen presentation, such as DEC-205 (Swiggard et al., Cell. Immunol., 165:302-11, 1995; Steinman, Exp. Hematol., 24:859, 1996) and Fc receptors. Targeted viral vectors, discussed above, can also be used. Additionally, dendritic cells may be induced to mature in vitro after infection by the viral vector, prior to transplantation in vivo.

Mucosal Vaccination.

Mucosal vaccine strategies are particularly effective for many pathogenic viruses, since infection often occurs via the mucosa. Additionally, mucosal delivery of recombinant vaccinia virus vaccines may be able to overcome a pre-existing immunity to poxviruses due to previous smallpox vaccination (Belyakov, et al., Proc. Natl. Acad. Sci. U.S.A., 96:4512-7, 1999). The mucosa harbors dendritic cells, which are important targets for EBNA-1 vaccines and immunotherapy. Thus, mucosal vaccination strategies for both polypeptide and DNA vaccines are contemplated. While the mucosa can be targeted by local delivery of a vaccine, various strategies have been employed to deliver immunogenic proteins to the mucosa (these strategies include delivery of DNA vaccines as well, e.g., by using the specific mucosal targeting proteins as vector targeting proteins, or by delivering the vaccine vector in an admixture with the mucosal targeting protein).

For example, in a specific embodiment, the immunogenic polypeptide or vector vaccine can be administered in an admixture with, or as a conjugate or chimeric fusion protein with, cholera toxin, such as cholera toxin B or a cholera toxin A/B chimera (Hajishengallis, J Immunol., 154:4322-32, 1995; Jobling and Holmes, Infect Immun., 60:4915-24, 1992). Mucosal vaccines based on use of the cholera toxin B subunit have been described (Lebens and Holmgren, Dev Biol Stand 82:215-27, 1994). In another embodiment, an admixture with heat labile enterotoxin (LT) can be prepared for mucosal vaccination.

Other mucosal immunization strategies include encapsulating the immunogen in microcapsules (U.S. Pat. No. 5,075,109, No. 5,820,883, and No. 5,853,763) and using an immunopotentiating membranous carrier (WO 98/0558). Immunogenicity of orally administered immunogens can be enhanced by using red blood cells (rbc) or rbc ghosts (U.S. Pat. No. 5,643,577), or by using blue tongue antigen (U.S. Pat. No. 5,690,938). Systemic administration of a targeted immunogen can also produce mucosal immunization (see, U.S. Pat. No. 5,518,725).

Various strategies can be used to deliver genes for expression in mucosal tissues, such as using chimeric rhinoviruses (U.S. Pat. No. 5,714,374), adenoviruses, vaccinia viruses, or specific targeting of a nucleic acid (WO 97/05267).
 

Claim 1 of 6 Claims

1. A method for making a human dendritic cell capable of eliciting an immune response to EBV-infected cells, which method comprises contacting a human dendritic cell with an isolated nucleic acid encoding an EBNA-1 polypeptide in humans; wherein the dendritic cell is capable of eliciting an immune response to an EBV-infected cell.

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