CA2156719A1

CA2156719A1 - Recombinant epstein-barr virus protein and its use in vaccine

Info

Publication number: CA2156719A1
Application number: CA002156719A
Authority: CA
Inventors: Ramesh Prakash
Original assignee: Individual
Current assignee: Actavis Laboratories UT Inc
Priority date: 1993-03-12
Filing date: 1994-03-11
Publication date: 1994-09-15
Also published as: AU6404594A; JPH08507442A; EP0690917A4; KR960700749A; AU681131B2; WO1994020138A1; EP0690917A1

Abstract

The present invention provides recombinant polypeptides based on the 66 kd serine- and threonine-rich portion of gp350/220 of EBV
and the recombinant polypeptide gp350/220. Such polypeptides are useful as vaccines effective against Epstein-Barr virus (EBV) infection.

Description

~ WO94/20138 t 2 1 ~ 6 ~ ~ 9 PCT~S94/02637 RECOMBINANT EPSTEIN-BARR VIRUS PROTEIN
AND ITS USE IN VACCINE

DESCRIPTION

TECHNICAL FIELD
The present invention relates generally to the production of vaccines, specifically to vaccines effective against Epstein-Barr virus (EBV) infection based on the gp350/220 of EBV and on a 66 kd serine- and threonine-rich portion of gp350/220 of EBV.

BACKGROUND ART
Epstein-Barr virus (EBV) is a member of the herpesvirus family which infects most hllm~nc The manifestations of primary infection in normal hllm~nc range from insignificant illness to infectious mononucleosis (Roberts, G.B., Diaqnosis and Clinical Testinq 27, 16-21, 1989). Persistent or latent EBV
infection is an important etiologic factor in Burkitt's lymphoma and nasopharyngeal carcinoma. EBV is an opportunistic pathogen often developing in immnnosuppressed patients who have allografts or in patients with acquired ;mmnne deficiency syndrome (AIDS).
EBV has also been implicated in rheumatoid arthritis and chronic fatigue syndrome. Because of the associated morbidity and mortality, prevention of EBV infection is desirable.
Because EBV does not replicate well in vitro, a systematic approach to selection and production of avirulent virus for preventive ;mmlln;zation is not WO94/20138 Z~S6~ ~ PCT~S94/02637 ~

feasible. Since EBV causes lymphocyte proliferation and is a presumed etiological agent of human malignancies, it is very important that virus preparations used for ;mml~nization be free of potential transforming genes.
For this reason, effective vaccines based on isolated proteins presented on the outer surface of the EBV virus and/or of EBV-infected cells are desirable.
Recombinant proteins provide several advantages over those purified from m~mm~lian tissues infected with the virus. For example, they can be readily produced in large quantities and at high purity. Especially valuable would be vaccines capable not only of neutralizing EBV
virus post-infection, but also to prevent viral binding and entry into B-cells.
Brief Description of the Background Art The principal components of the outer surface of EBV are glycoproteins of 350, 220 and 85 kilodaltons (Pearson, G.R. and Luka, J. In: M.A. Epstein and B.G.
Achong (Eds), The Epstein-Barr Virus. Recent Advances, pp. 48-73. William He;n~m~nn Medical Books, London.) These glycoproteins are also present on the outer surface of infected cell in which the virus is replicating.
Polyclonal and monoclonal antibodies directed against these proteins react with the surface of productively infected cells and neutralize the virus (ibid.).
However, target membrane antigens for antibody-dependent cellular cytotoxicity (ADCC) appeared to be associated with gp350 and gp220 but not with gp85 (ibid.). The EBV
gp350/220 has also been identified as the ligand that m~ tes attachment of the virus to receptor CR2 of human B lymphocytes (Nemerow, G.R., et al., J. Virol. 61, 1416-1420, 1987). The sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu, which corresponds to the N-t~rm;nllc region of gp350/220, has been found to be the epitope responsible W094/20138 ~ 71 9 PCT~S94/OZ~7 for attachment (Nemerow, G.R., et al., Cell 56, 369-377, 1989).
The gp350 and gp220 proteins are encoded by 3.2 and 2.5 kb RNAs which map to the same DNA fragment (~eisel, C., et al., J. Virol. 54, 665-674, 1985).
Antisera raised in rabbits against the gp350/220 protein expressed in E. coli specifically 1mml~noprecipitate gp350 and gp220, react with the plasma membrane of infected cells, and neutralize virus, particularly after the addition of complement. However, multiple injections of substantial quantities of protein are necessary to elicit an effective response. The poor 1mm~nogenicity of gp350/220 expressed in E. coli may be attributable to the absence of glycosylation and proper folding. When expressed in m~mm~lian cells, gp350/220 is highly immunogenic and elicits virus-neutralizing antibodies (Whang, Y., et al., J. Virol. 61, 1796-1807, 1987), indicating that glycosylation plays an important role in the immunogenicity of the glycoprotein. However, the expression of recombinant gp350/220 in m~mm~lian cells is generally very unstable.
Several features of the proteins have been predicted from the DNA and mRNA nucleotide sequence (Beisel, C., et al., J. Virol. 54, 665). The primary translation product of gp350 is a 907 amino acid polypeptide, while that of gp220 is a 710 amino acid polypeptide. At the amino t~rm;nllc of both proteins is an 18-amino acid region which is likely to be a cleavable signal peptide. A single hydrophobic domain which likely functions as a tr~nsm~mhrane anchor is located 26 amino acids from the carboxy terminus of both proteins. The 850 amino acids of gp350 and the 652 amino acids of gp220 between the putative signal and anchor sequences are serine- and threonine-rich and contain 35 (gp350) or 24 (gp220) asparagine-X-serine or -C-threonine sequences, WO94120138 21~ PCT~S94/02637 respectively, which serve as potential signals for N-linked glycosylation. These regions are therefore likely to be present on the outer surface of the membrane.

BRIEF SUMMARY OF THE lN V~N'l'lON
The present invention provides recombinant polypeptides based on the 66 kd serine- and threonine-rich portion of gp350/220 and fragments thereof which are useful as vaccines capable of eliciting EBV-neutralizing antibodies in m~mm~l ian subjects. Preferably, these polypeptides further incorporate a sequence encoding a nine amino acid peptide sequence, Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu, which is involved in mediating EBV
attachment to human B-cells. Vaccines based on polypeptides comprising both the 66 kd polypeptide and this nonamer peptide are also capable of inhibiting viral binding and entry into B-cells.
Accordingly, one aspect of the present invention is a substantially pure polypeptide consisting essentially of the 66 kd serine- and threonine- rich portion of gp350/220 of the Epstein-Barr virus or fragments thereof capable of eliciting EBV-neutralizing antibodies in a m~mm~ 1, Another aspect of the present invention is a substantially pure polypeptide comprising the 66 kd serine- and threonine- rich portion of gp350/220 of the Epstein-Barr virus or fragments thereof and the peptide sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu capable of eliciting EBV-neutralizing antibodieg in a m~mm~l, Such polypeptides are preferentially glycosylated.
Also embraced by the present invention are nucleic sequence encoding these polypeptides and expression vectors comprising such nucleic acids and WO94/20138 21 ~6 ~ PCT~S94/02637 expression control sequences that are operably linked to the nucleic acid and effective in directing expression of said nucleic acid in a cell. A further aspect of the invention is a cell containing such an expression vector as well as methods of producing the polypeptides of the present invention comprising growing such a cell under conditions that permit the production of the polypeptide.
Another aspect of the invention is a vaccine composition comprising an ;mmllnogenic amount of a polypeptide of the present invention admixed with a pharmaceutically acceptable carrier. The inventlon further embraces a method of protecting a m~mm~ 1 from EBV
infection comprising ~mintstering to said m~m~l an immunologically effective dose of such a vaccine composition.
Still another aspect of the invention is a method of producing a su~stantially pure EBV gp350/220 protein, said method comprising subcloning the complete EBV gp350/220 gene into the baculovirus expression vector pBlueBacHisC to form a plasmid pBac-TTI350 which comprises the EBV gp350/220 gene and a 6-histidine peptide gene. This plasmid is used to replace a viral gene encoding the EBV gp350/220 protein and the 6-histidine polypeptide. Then insect cells are transfected with the virus comprising these genes. ~ater the transfected insect cells are harvested and the recombinant EBV gp350/220 protein is ~eparated from the transfected insect cells. Then the EBV gp350/220 protein is puri~ied on a column whose resin has an affinity for a 6-histidine peptide. Another aspect of the present invention is the protein produced by this method.

DETAILED DESCRIPTION QF THE lNv~NllON
The present invention provides recombinant 35 polypcptides based on the EBV gp350/220 protein and on WO94/20138 21~ ~ ~ 19 PCT~S94/02637 ~

the 66 kd serine- and threonine-rich portion o~ gp350/220 which are 1mmllnoreactive with both the EBV imml~ne human serum and a monoclonal antibody that neutralizes EBV
virus. Also provided are polypeptides which further incorporate a sequence encoding a peptide sequence involved in mediating EBV attachment to human B-cells.
Vaccines based on such polypeptides are capable not only of eliciting the production of neutralizing antibodie~
against EBV but also of inhibiting viral binding and entry into B-cells.

Polype~tides The term "gp350/220" refers to the 350 kd and 220 kd outer membrane glycoproteins of Epstein-Barr virus (EBV) which are also present on the ouser surface of infected cells in which EBV is replica~ing. The term also embraces variants or fragments of gp350/220.
The term "66 kd protein" refers to the serine-and threonine- rich portion of gp350/220, encoded by the NcoI fragment of the DNA fragment cont~; n ing the complete EBV gp350/220 coding ~om~;n represented by base pairs numbered 1361-2292 from the initiation codon of the EBV
gp350/220 DNA coding ~om~ln. (Beisel, C., et al., J.
Virol. 54, 665) The term also embraces variants or fragments of the 66 kd protein. Preferably such polypeptides also comprise a peptide sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu, which is involved in mediating EBV attachment to human B-cell receptor CR2.
This nine-amino acid sequence is generally introduced at the amino- or carboxy-tprm;nl~s of the 66 kd protein, most preferably at the amino-t~rm;n-lc.
For the sake of convenience, the term "66 kd protein" as used herein al~o refers to polypeptides comprising the 66 kd polypeptide and this nine amino acid peptide. Only such polypeptides as are capable o~

-WO 94/20L~8 ,~ f~ 71~ PCTIUS94/02637 eliciting EBV-neutralizing antibodies in a human subject are considered to be within the scope of the present invention.
An "-mml~nologically effective dose" of such a 5 polypeptide is an amount capable of eliciting EBV-neutralizing antibodies in a human subject.
Ordinarily, the proteins of the present invention will be at least about 50~ homologous to the 66 kd protein sequence, preferably in excess of about 90~, 10 and, more preferably, at least about 95~ homologous.
Also included are proteins encoded by DNA which hybridize under high or low stringency conditions, to nucleic acids encoding the 66 kd protein, as well as closely related polypeptides or proteins retrieved by antisera to the 66 15 kd protein.
The length of polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 20 residues, and preferably more than about 35 residues.
~ ubstantial homoloqy or identity The tenn ~Isubstantial homology~ or llsubstantial identity", when referring to polypeptides, indicates that the polypeptide or protein in question exhibits at least about 30~
25 identity with an entire naturally occurring protein or a portion thereof, usually at least about 70~ identity, and preferably at least about 95~ identity.
Homology, for polypeptides, is typically measured using sequence analysis software. See, e.g., 30 Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705.
Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, 35 deletions, substitutions, and other modifications.

W094/20138 2 ~S~ PCT~S94/02637 Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glut~m'n~; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine.
A polypeptide "fragment," "portion," or "segment" is a stretch of amino acid residues of at least about 5 amino acids, often at least about 7 amino acids, typically at least about 9 to 13 amino acids, and, in various embodiments, at least about 17 or more amino acids.
The polypeptides of the present invention are generally soluble, but the polypeptide also can be coupled to a solid phase support. Many such supports are well known in the art.
"Isolated" The terms "isolated,"
"substantially pure," and "substantially homogenous" are used interchangeably to describe a protein or polypeptide which has been separated from components which naturally accompany it. A monom~ric protein is substantially pure when at least about 60 to 75~ of a sample exhibits a single polypeptide sequence. A substantially pure protein will typically comprise about 60 to 90~ W/W of a protein sample, more usually about 95~, and preferably will be over about 99~ pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis or a protein sample, followed by visualizing a single polypeptide band upon st~;ning the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.
A protein is considered to be isolated when it is separated from the cont~m;n~nts which accompany it in its natural state. Thus, a polypeptide which is ~ WO94/20138 ~ CT~S94/02637 ~9 g chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
Protein purification The present invention provides polypeptides which are typically purified from cells transformed with recombinant nucleic acids encoding these proteins. Such protein purification can be accomplished by various methods well known in the art. A
protein having six contiguous histidine residues at its N-tPrm~nll~ can be purified by binding to ProBond resin (Invitrogen, San Diego, CA), as described in Example 2 below. Other useful methods of protein purification are well known in the art, and include those described, e.g., in Guide to Protein Purification, ed. M. Deutscher, vol.
182 of Methods in Enzymoloqy (ACA~Pm; c Press, Inc.: San Diego, 1990) and R. Scopes, Protein Purification:
Princi~les and Practice, Springer-Verlag: New York, 1982.
If necessary, the amino acid sequence of the proteins of the present invention can be det~rm~ned by protein sequencing methods well known in the art.
Protein modifications: fragments: fusion proteins The present invention also provides for polypeptides or fragments thereof which are substantially homologous to the primary structural sequence of the 66 kd protein. The present invention also embraces n vivo or n vitro chemical and biochemical modifications or which incorporating unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labelling, e.g., with radionuclides, various enzymatic modifications, as will be readily appreciated by those well ~killed in the art. A variety of methods for labelling polypeptides and of substituents - 35 or labels useful for such purposes are well known in the 2~ PCT~S94/02637 ~

art and include radioactive isotopes such as 32p, ligands, which bind to labeled antiligands (e.g., antibodies), fluorophores, chemilllm~nescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods of labelling polypeptides are well known in the art. See, e.g., Molecular Cloning: A Laboratory ~m1~l, 2nd ed., Vol. 1-3, ed. Sambrook, et al., Cold Spring Harbor Laboratory Press (1989) or Current Protocols in Molecular Bioloqy, ed. F. Ausubel et ~1-, Greene Publishing and Wiley-Interscience: New York (1987 and periodic updates).
Besides substantially full-length polypeptides, the present invention provides for fragments of the polypeptides capable of eliciting EBV neutralizing antibodies. As used herein, the term fragment or segment, as applied to a polypeptide, will ordinarily be at least about 5 to 7 contiguous amino acids, typically at least about 9 to 13 contiguous amino acids, and most preferably at least about 20 to 30 or more contiguous amino acids.
For ;mm~lnological purposes, t~n~pm repeats of the polypeptides or polypeptide ~ragments of the present invention can be used as ;mm~lnogens, thereby producing more highly antigenic proteins.
The present invention also provides for fusion polypeptides comprising the 66 kd polypeptide or fragments thereof. Homologous polypeptides may be fusions between two or more sequences derived from the 66 kd protein or between the sequences of the 66 kd protein and a related protein. Likewise, heterologous ~usions may be constructed which would exhibit a combination of -WO 94/20L~8 ~ PCTIUS94/02637 ~ - 11-properties or activities of the derivative proteins.
See, e.g., Godowski et al. ~1988) Science 241:812-816.
Fusion proteins will typically be made by recombinant nucleic acid methods, but may be chemically 5 synthesized. Techniques for synthesis of polypeptides are described, for example, in Merrifield (1963) J. Amer.
Chem. Soc. 85:2149-2156.

Nucleic AcidS
The term present invention provides nucleic acids which encode a 66 kd polypeptide, fragment, homolog or variant, including, e.g., protein fusions or deletions. The nucleic acids of the present invention will possess a sequence which is either derived or substantially similar to a natural E~3V nucleic acid sequence encoding such a protein or one having substantial homology with this sequence or a portion thereof.
The nucleic acid compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
Recombinant nucleic acids comprising sequences otherwise not naturally occurring are also provided by this invention. Although the wild type sequence may be employed, the wild type sequence will often be altered, e.g., by deletion, substitution, or insertion.
The nucleic acid sequences used in this invention will usually comprise at least about 5 codons (15 nucleotides), more usually at least about 7 to 15 codons, and most preferably at least about 35 codons.
One or more introns may also be present. This number of nucleotides is usually about the m;ntm~l length required WO94120138 2~ 19 PCT~S94/02637 for a successful probe that would hybridize specifically with a such a sequence.
Techniques for nucleic acid manipulation are described generally, for example, in Sambrook et al., ibid~, or Ausubel et al., ibid.. Reagents useful in applying such techniques, such as restriction enzymes and the like, are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer ~Annheiml Amersham, Promega Biotec, U. S.
Biochemicals, New ~ngland Nuclear, and a number of other sources. The recombinant nucleic acid sequences used to produce fusion proteins of the present invention may be derived from natural or synthetic ~equences. Many natural gene sequences are obtA;n~hle from various cDNA
or from genomic libraries using appropriate probes. See, GPnR~nk, National Institutes of Health.
"Substantial homology" or "similaritY" A
nucleic acid or fragment thereof is "substantially homologous" (or "substantially simi~Arll) to another if, when optimally aligned (with appropriate nucleotide insertions or deletionc) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60~ of the nucleotide bases, usually at least about 70~, more usually at least about 80~, preferably at least about 90~, and more preferably at least about 95 to 98~ of the nucleotide bases.
Alternatively, a nucleic acid or fragment (or its complementary strand) is substantially homologous (or s;m-lAr) with a 66 kd polypeptide-encoding nucleic acid when they are capable of hybridizing under selective hybridization conditions. Selectivity of hybridization exists when hybridization occurs which is substantially more selective than total lack of specificity.
Typically, selecti~e hybridization will occur when there is at least about 55~ homology over a stretch of at least Og4/20~8 ~ PCT~S94/~7 about 14 nucleotides, preferably at least about 65~, more preferably at least about 75~, and most preferably at least about 90~. See, ~n~h~ sa (1984) Nuc. Acids Res.
12:203-213. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about 17 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.
Nucleic acid hybridization will be affected by such conditions as salt concentration (e.g., NaCl), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30 C, typically in excess of 37, and preferably in excess of 45. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the com~bination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370.
"Isolated" or "subs~antially pure" or "purified. An "isolated" or "substantially pure~ or ~purified~ nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a m~ xeA polymer, which is substantially separated from other DNA sequences which naturally accompany a native hllm~n sequence, e.g., ribosomes, polymerases, and many other human genome sequences. The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant WO94/20138 2~ PCT~S94/02637 *

or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
"Encodel' A nucleic acid is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide of a fragment thereof. The anti-sense strand of such a nucleic acid is also said to encode the sequence.
"O~erably linked" A nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Generally, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
~Recombinant~ The term "recombinant" nucleic acid is one which is not naturally occurring or is made by the artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
Such is usually done to replace a codon with a r~71n~nt codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions.
Preparation of re~ombinant or chemically synthesized nucleic acids: ~ectors. transformation. host cells Large amounts of the nucleic acids of the present WOg4/20138 !~ ~ S6 ~1 9 PCT~S94/02637 invention may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA
constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA
constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, m~mm~l ian, plant or other eukaryotic cell lines. The purification of nucleic acids produced by the methods of the present invention are describedj e.g., in Sambrook et al. (1989) or Ausubel et al. (1987 and periodic updates).
The nucleic acids of the present invention may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22:1859-1862 or the triester method according to Matteucci et al. (1981) J.
Am. Chem. Soc~ lQ3:3185, and may be performed on commercial automated oligonucleotide synthesizers. A
double-stranded fragment may be obt~;ne~ from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system recognized by the host, including the intended DNA fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory se~uences operably linked to the polypeptide encoding segment. Expression vectors may include, for example, an origin of WO94120138 2 ~ ~ 6 7 i 9 PCT~S94/02637 ~

replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information ~ite~, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional term-n~tor sequences, and mRNA
stabilizing sequences. Secretion signals from polypeptides secreted from the host cell of choice may also be included where appropriate, thus allowing the protein to cross and/or lodge in cell membranes, and thus attain its functional topology or be secreted from the cell. Such vectors may be prepared by means of st~n~rd recombinant techniques well known in the art and discussed, for example, in Sambrook et al. (1989) or Ausubel et al. (1987).
The selection of an appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host, and may, when appropriate, include those naturally associated with EBV genes.
Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al., 1989 or Ausubel et ~1., 1987); see also, e.g., Metzger et al.
1988), Nature 334:31-36. Many useful vectors are known in the art and may be obt~;ne~ such vendors as Stratagene, New England ~iolabs, Promega Biotech, and others. Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters include the promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible ~or maltose and galactose utilization, and others. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al. EP 73,657A. Appropriate nonnative m~mm~l ian promoters might include the early and f~
WO94/20138 ~'~5~ ~CT~S94/02637 late promoters from SV40 (Fiers et al. (1978) Nature 273:113) or promoters derived from murine molony leukemia virus, mouse ~mm~ry tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. In addition, the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made. For appropriate enhancer and other expression control sequences see also Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, N.Y. (1983).
While such expression vectors may replicate autonomously, they may less preferably replicate by being inserted into the genome of the host cell, by methods well known in the art.
Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures the growth of only those host cells which express the inserts. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media, e.g. the gene encoding D-alanine racemase for Bacilli. The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art.
The vectors cont~;n;ng the nucleic acids of interest can be transcribed in ~itro and the resulting RNA introduced into the host cell by well known methods (e.g., by injection. ~ee, T. Kubo et ~l., FE~S Lett.
241:119 (1988)), or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium W094l20~8 21~ ~ 7 ~ ~ PCT~S94/02637 ~

chloride, rubidium chloride calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome); and other methods. See generally, Sambrook et al. (1989) and Ausubel et al. (1987). The cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells.
~arge quantities of the nucleic acids and polypeptides of the present invention may be prepared by expressing the nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells. The most commonly used prokaryotic hosts are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or PseudomQnas may also be used.
~ mmA lian or other eukaryotic host cells, such as those of yeast, filamentous fungi, plant, insect, ~mph;hian or avian species, may also be useful for production of the proteins of the present invention.
Propagation of m~mm~lian cells in culture is per se well known. See, Tissue Culture, Kruse and Patterson, ed., Academic Press (1973). Examples of cnmm~ly used m~mm~ lian host cell lines are VERO and XeLa cells, Ch~n~se hamster ovary (CHO) cells, and WI38, BHK, and COS
cell lines, although it will be appreciated by the skilled practitioner that other cell lines may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns, or other features.
Clones are selected by using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule, prefera~ly the same DNA molecule. The transformant may be screened or, preferably, selected by any of the means well known Og4eOI38 ~ ~CT~S94/0~637 in the art, e.g., by resistance to such antibiotics as ampicillin, tetracycline.

Vaccines The present invention provides vaccines preparations comprising an imm-lnogenic amount of a polypeptide of the present invention. Each of these vaccines may comprise more than one of the proteins of the present invention, or a protein of the present invention in combination with another protein or other immunogen. By ~immllnogenic amount" is meant an amount capable of eliciting the production of antibodies directed against the polypeptide in an individual to which the vaccine has been ~mi n; ~tered.
Tmmllnogenic carriers may be used to enhance the immnnogenicity of the polypeptides of the present invention. Such carriers are soluble molecules, e.g., proteins and polysaccharides, or particles, e.g., liposomes and bacterial cells and membranes. Protein carriers may be joined to the 66 kd proteins of the present invention to form fusion proteins by recombinant means or by chemical coupling. Useful carriers and means of coupling such carriers to polypeptide antigens are well known in the art.
The vaccines of the present invention may be formulated by any of the means known to those skilled in the art. Such vaccines are typically prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes.
The active immllnogenic ingredients are often mi XP~ with excipients or carriers which are ph~rm~ceutically acceptable and compatible with the W094/20~8 ~ 2i5 ~ PCT~S94102637 active ingredient. Suitable excipients are, e.g., water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. The concentration of the immunogenic polypeptide in injectable formulation~ will usually be in the range of 0.2 to 5 mg/ml.
In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include: alnminl~m hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglut~m;ne (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglut~mtne (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alan`ine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE),and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2~ s~ualene/Tween 80 emulsion. The effectiveness of an adjuvant may be det~rm;nPd by measuring the amount of antibodies directed against the ;mmllnogen resulting from ~m;n;ctration of the ;mmllnogen in vaccines which are also comprised of the various adjuvants.
The vaccines are conventionally ~m; n; stered parenterally by intravenous, subcutaneous, or intramuscular injection. Additional formulations and modes of ~m; n;stration may also be used, including suppositories and, in some cases oral formulations and liposomes, or others well known in the art. For suppositories, traditional binders and carriers may include, e.g., polyalkaline glycols or triglycerides.
Such suppositories may be formed from mixtures cont~;n;ng the analog in the range of 0.5~ by weight, preferably 1 ~5 to 2~. Oral formulations typically include such W094/20138 ~1S6~ PCT~S94/02637 excipients as p~ rm~ ceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10 to 95~ of the polypeptides of the present invention, preferably 25~ to 70~.
The polypeptides of the present invention may be formulated into the ~accine as neutral or salt forms.
Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) which are formed with inorganic acids, e.g., hydrochloric acid or phosphoric acids, or organic acids, e.g., acetic, oxalic, tartaric, or maleic acid. Salts ~ormed with the free carboxyl groups may also be derived from inorganic bases, e.g., sodium, potassium, ~mmnn;um, calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.
The vaccines are ~m; n; stered in a m~nnPr compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be ~Am; n; ~tered, which i9 generally in the range of 5 to 250 mic oyldms of protein per dose, depends on the subject to be treated, capacity of the individual's ; mm~n~ system to synthesize antibodies, and the degree of protection desired.
Preciqe amounts of active ingredient required to be ~m; n; ~tered may depend on the judgment o~ the physician and may be peculiar to each individual, but such a determ;n~tion is within the skill of such a physician.
The vaccine may be given in a single dose or multiple dose schedule. A multiple dose s~heAl~le i9 one in which a primary course of vaccination may include 1 to 10 separate doses, followed by other doses ~Am; n; stered WO94/20138 215 6 719 PCT~S94/02~7 ~

at subsequent time intervals as required to maintain and or reinforce the imm~ne response, e.g., at 1 to 4 months for a second dose, and if needed, a subsequent dose(s) after several months.
The invention will be better understood by reference to the following examples, which are intended to merely illustrate the best mode now known for practicing the invention. The invention is not to be considered limited thereto, however.

Examples Example l: Identification of E~itope for EBV-Neutralizinq Antibody To identify the epitope for EBV-neutralizing antibody, the following recombinant plasmids were constructed. A 4.3 XhoII-~Ç~II DNA fragment cont~;n;ng the entire EBV gp350/220 coding ~nm~in (except the signal peptide) was subcloned from the pl~qm;~ pMA102 into the baculovirus expression vector pBlueBacHisC (Invitrogen), resulting in plasmid pBac-TTI350. This fragment contains the entire region of gp350/220 which i5 the protein present on the outer surface of EBV-infected cells. An NcoI fragment encoding the serine and threonine-rich sequences was deleted from pBac-TTI350 to generate plasmid pBac-TTI300. The NcoI fragment was recloned at the NcoI site of plasmid pBlue~ac~isC to generate pBac-TTI250. These manipulations employed E. coli strain Top 10 (Invitrogen). A synthetic DNA coding for the nine amino acids Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu was also introduced upstream of the se~uence coding for 66 kd protein. This peptide has been shown to be involved in me~i~ting EBV attachment to human B-cell receptor CR2.
Construction of such a protein will not only produce WO94/20138 ~ S67~ PCT~S94102637 neutralizing antibody but will also inhibit viral binding and entry into the B-cells.
A baculovirus transfer vector is used to replace the viral polyhedrin gene with the gene o~
interest by homologous recombination. Sequences which flank the polyhedrin gene in the wild-type genome are positioned 5' to 3' to the expression cassette on the transfer vector. Following cotransfection, homologous recombination between these sequences results in a recombinant ~irus with the gene of interest expressed under the control of the viral polyhedrin enhancer/promoter elements. The vector pBlueBacHisC used here also expresses galactosidase to provide a blue reporter for visual identification of blue recombinant plaques. The recombinant virus was produced and purified according to the method described in the Invitrogen m;3 n~
The expression of the gp350/220 sequence in insect cells derived from Spodoptera frugiperda ovarian cells (Sf9 Cells, Invitrogen) was confirmed by Western blot analysis in the following m~nn~r, Insect cells were infected with virus cont~ n ~ ng recombinant plasmid pBac-TTI350, pBac-TTI300 and pBac-TTI250, respectively. After three days, a lml sample of cells was L~mo~ed from each culture. As positive controls for molecular weights of 350 and 220 kd, l ml samples of Raji cells infected with EBV were also u~ed in the following test. The cells were pelleted and dissolved in lO0 ~l of T.~Qmm1 i buffer.
After boiling for 2 min, the samples were loaded on a lO~
SDS-polyacrylamide gel and electrophore~ed overnight at 70 volts. The proteins were transferred electrophoretically to a nitrocellulose membrane.
Nonspecific binding of the protein was blocked by treating the membrane with 5~ nonfat dry milk in TBST (lO
mM Tris, pH 8.0/l mM EDTA/0.05~ Tween-20). High-titer , WO94/20138 ~1 ~ 6 ~19 PCT~S94/02~7 EBV-;mm~ln~ human serum and a monoclonal EBV-neutralizing antibody to gp350/220 (DuPont, Boston, MA), respectively, were added to the blots and incubated for 1 hr, washed 3 times for 5 min each with TBST, and then incubated with anti-human IgG-alkaline phosphatase conjugate for 30 min.
After washing 3 times with TBST, the color was developed.
Cells infected with recombinant virus pBac-TTI350 showed two bands with molecular weights of 350 and 220 kd, with both EBV ;mmllne human serum and monoclonal EBV neutralizing antibody. The recombinant protein bands appeared in the same position as the protein bands from the Raji cells infected with EBV. No reaction was seen with cells infected with recombinant virus pBac-TTI300, from which the serine- and threonine-rich sequence of gp350/220 had been deleted. Because the TTI300 plasmid did not react, the serine- and threonine-rich sequence appears to be the likely epitope for neutralizing antibody. This result was further confirmed by ;mmllnoreaction of human serum and monoclonal antibody to cells infected with recombinant virus pBac-TTI250 which contains the serine- and threonine-rich sequences which has been deleted from pBac-TTI300. The size of the reactive protein from pBAC-TTI250 was 66 kd.

Example 2: Purification of Recombinant Proteins The vector pBl--~R~;8C contains the DNA
sequence below the translation start codon which produces a six-histidine peptide at the N-tPrm;n~l region of the expressed protein. This peptide has an affinity for the ProBond resin (Invitrogen) and thus allows one-step purification. For each of the two proteins, about 500 ml of insect cells Sf9 was grown at a density of 2 x 106 cells/ml in a 500 ml spinner ~lask. Cells were infected with high-titer viral stock of either the pBac-TTI250-infected virus (66 kd protein) or the pBac-TTI350-W094/20138 ~ S671~ PCT/US94/02637 infected virus (the 350/220 kd protein). Then the cells were grown for 3 days. The cells were pelleted by centrifugation and suspended in 20 mM sodium phosphate and 500 mM NaCl and lysed by sonication and centrifuged to remove cell debris. The recom~inant proteins were puri~ied from the supernatant through ProBond resin by the method described by Invitrogen. First, the 6-histidine end of the recombinant protein binds to the ProBond resin. Then the reco-mbinant protein is cleaved off by enterokinase applied to the column. This isolation method gave single protein bands (66 kd or 350/220 kd proteins) on SDS-polyacrylamide gel. The 350 and 220 kd bands were in the same position for recombinant and Raji-cell-derived proteins. About 5 mg of purified protein was obtained from each 500 ml of culture.
Because the 66 kd recombinant protein derived from gp350/220 is ;mml~noreactive with both the EBV lmml~n~
human serum and monoclonal antibody that neutralizes EBV
virus, this protein can be used to develop vaccine for EBV. In addition, because the 66 kd protein also has a nine-amino acid sequence for EBV attachment to B-cells, vaccines developed with this protein also should inhibit viral binding and entry into B-cells.
Expression in baculovirus provides several advantayes. While E. coli synthesize unglycosylated proteins, and yeast produce proteins which are differently glycosylated, proteins produced in baculovirus are glycosylated and have a glycosylation pattern similar to that resulting from expression in mAmm~lian cells. A further advantage i9 that much larger quantities of pure protein can be obtained from baculovirus than from mAmm~lian cells. Moreover, the expression of recombinant gp350/220 in m~mm~l ian cells is generally very unstable. Therefore, either the 66 kd or WO94/20138 21~ 6 ~ ~ 9 PCT~S94/02637 ~

the 350/220 kd protein produced in insect cells provides a more stable and efficient method of making purified vaccine protein.
Smaller proteins provide several advantages over large proteins for vaccine development. Smaller proteins permit the injection of more moles of epitope per weight or injection volume of vaccine. Smaller proteins may be processed more efficiently recombinantly and ;mmnnologically. Smaller proteins are less vulnerable to protease attack because they are less likely to have amino acid combinations for which the proteases are specific. Smaller proteins have fewer epitopes and elicit a more concentrated response.
Each of the publications and patents cited above are incorporated by reference.
While the invention has been described in connection with certain embo~;m~nts thereof, various modifications which may be apparent to one of skill in the art also fall within the scope of the invention as defined by the appended claims. The scope of the invention, therefore, should be det~rm; n~ not with reference to the above description, but should instead be det~rm;ne~ with reference to the appended claims, along with the full scope of e~uivalents to which such claims are entitled.

Claims

What is Claimed Is:

1. A substantially pure polypeptide consisting essentially of the 66 kd serine- and threonine-rich portion of gp350/220 of EBV or fragments thereof capable of eliciting EBV-neutralizing antibodies in a mammal.

2. A glycosylated polypeptide of claim 1.

3. A vaccine composition comprising an immunogenic amount of a polypeptide of claim 2 admixed with a pharmaceutically acceptable carrier.

4. A method of protecting a mammal from EBV
infection comprising administering to said mammal an immunologically effective dose of a vaccine composition comprising an immunogenic amount of a polypeptide of claim 2 admixed with a pharmaceutically acceptable carrier.

5. A nucleic sequence encoding the polypeptide of claim 1.

6. An expression vector comprising a nucleic acid sequence encoding a polypeptide of claim 1 and expression control sequences that are operably linked to said nucleic acid and effective in directing expression of said nucleic acid in a cell.

7. A cell containing an expression vector of claim 6.

8. A method of producing a polypeptide comprising growing a cell of claim 7 under conditions that permit the production of said polypeptide.

9. A vaccine composition comprising an immunogenic amount of the polypeptide of claim 1 admixed with a pharmaceutically acceptable carrier.

10. A method of protecting a mammal from EBV
infection comprising administering to said mammal an immunologically effective dose of a vaccine composition comprising an immunogenic amount of a polypeptide of claim 1 admixed with a pharmaceutically acceptable carrier.

11. A substantially pure polypeptide for eliciting EBV-neutralizing antibodies in a mammal and inhibiting EBV binding and entry into B-cells, the polypeptide consisting essentially of: a 66 kd serine-and threonine-rich portion of gp350/220 of the EBV or fragments thereof; and a peptide consisting of the sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu adjacent to the amino-terminus of the 66 kd portion.

12. A method of protecting a mammal from EBV
infection comprising administering to said mammal an immunologically effective dose of a vaccine composition comprising an immunogenic amount of a polypeptide of claim 11 admixed with a pharmaceutically acceptable carrier.

13. A glycosylated polypeptide of claim 11.

14. A method of protecting a mammal from EBV
infection comprising administering to said mammal an immunologically effective dose of a vaccine composition comprising an immunogenic amount of a polypeptide of claim 13 admixed with a pharmaceutically acceptable carrier.

15. A vaccine composition comprising an immunogenic amount of a polypeptide of claim 13 admixed with a pharmaceutically acceptable carrier.

16. A nucleic sequence encoding the polypeptide of claim 11.

17. An expression vector comprising a nucleic acid sequence encoding a polypeptide of claim 11 and expression control sequences that are operably linked to said nucleic acid and effective in directing expression of said nucleic acid in a cell.

18. A cell containing an expression vector of claim 17.

19. A method of producing a polypeptide comprising growing a cell of claim 18 under conditions that permit the production of said polypeptide.

20. A vaccine composition comprising an immunogenic amount of a polypeptide of claim 11 admixed with a pharmaceutically acceptable carrier.

21. A method of producing a substantially pure EBV gp350/220 protein, said method comprising subcloning the complete EBV gp350/220 gene into the baculovirus expression vector pBlueBacHisC to form a plasmid pBac-TTI350 which comprises the EBV gp350/220 gene and a 6-histidine peptide, using the plasmid pBac-TTI350 to replace a gene in a virus with DNA encoding at least the EBV gp350/220 protein and the 6-histidine peptide, transfecting insect cells with the virus containing the EBV gp350/220 gene, harvesting transfected insect cells, separating the EBV gp350/220 protein from the transfected insect cells, purifying the EBV gp350/220 protein on a column whose resin has an affinity for a 6-histidine peptide, and separating EBV gp350/220 protein from the 6-histidine peptide.

22. The protein produced by the method of claim 21.

23. The method of claim 21 wherein the EBV
gp350/220 gene lacks the amino terminus sequence encoding for the signal protein.

24. The protein produced by the method of claim 23.

25. A method of producing a substantially pure EBV gp350/220 protein, said method utilizing baculovirus comprising genes encoding EBV gp350/220 and a 6-histidine polypeptide, said method comprising:
transfecting insect cells with said baculovirus, harvesting transfected insect cells, separating the EBV gp350/220 protein and the 6-histidine polypeptide from the transfected insect cells, and purifying the EBV gp350/220 protein and the 6-histidine polypeptide on a column whose resin has an affinity for the 6-histidine peptide.

26. The EBV gp350/220 protein produced by the method of claim 25.

27. A method of protecting a mammal from EBV
infection comprising administering to said mammal an immunologically effective dose of a vaccine composition comprising an immunogenic amount of a polypeptide of claims 22, 24 or 26 admixed with a pharmaceutically acceptable carrier.