EP0436626A1 - REGION DE LIAISON DE LA gp350 DE L'EBV AU RECEPTEUR - Google Patents

REGION DE LIAISON DE LA gp350 DE L'EBV AU RECEPTEUR

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Publication number
EP0436626A1
EP0436626A1 EP19890911187 EP89911187A EP0436626A1 EP 0436626 A1 EP0436626 A1 EP 0436626A1 EP 19890911187 EP19890911187 EP 19890911187 EP 89911187 A EP89911187 A EP 89911187A EP 0436626 A1 EP0436626 A1 EP 0436626A1
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EP
European Patent Office
Prior art keywords
amino acid
ebv
residues
reactive
sequence
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EP19890911187
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German (de)
English (en)
Inventor
Glen R. Nemerow
Neil R. Cooper
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Scripps Research Institute
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Scripps Clinic and Research Foundation
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Publication of EP0436626A1 publication Critical patent/EP0436626A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • C07K16/087—Herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the CR2 binding region of Epstein-Barr virus (EBV) and to polypeptides which bind to CR2 and inhibit infection of a B cell by EBV.
  • EBV Epstein-Barr virus
  • Epstein-Barr virus a human herpesvirus
  • EBV infection may facilitate the development of malignant diseases including nasopharyngeal carcinoma (HENLE, W. , Science 157:1064- 1065, 1967, Raab-Traub, N. , et al. Int. J. Cancer 39:25- 29, 1987), Burkitt's lympho a (De-The, G. , et al.
  • EBV is also an opportunistic pathogen often associated with immunocompromised patients receiving allografts or with the acquired immune deficiency syndrome (AIDS) (Pelicci, P.G., et al, J. EXD. Med. 164:2049-2060, 1986 and Yarchoan, R. , et al, J. Clin. Invest. 78:439-447, 1986 and Montagnier, ⁇ L., et al. Science 225:63-66, 1984).
  • AIDS acquired immune deficiency syndrome
  • EBV is unique among the human herpesviruses in its selective tropism for B lymphocytes and epithelial cells. This is largely due to expression on the membrane of these cells of a specific EBV receptor which has shown to be antigenically, structurally and functionally identical to the glycoprotein which binds the C3d/C3dg complement fragments (Fingeroth, J.D., et al, Proc. Natl Acad. Sci.. USA 81:4510-4516, 1984 and Nemerow, G.R. , et al, J. Virol. 55:347-351, 1985 and Frade, R. , et al, Proc. Natl. Acad. Sci..
  • CR2 CD21
  • CR2 CD21
  • CR2 is involved in a pathway of B cell activation (Nemerow, G.R., et al, Supra, 1985 and Chagelian & Fearnon, J. Exp. Med 163:101-115, 1986 and
  • Gp350/220 The major envelope protein of EBV, designated gp350/220, is a primary target of neutralizing antibody in man and sub-human primates (Thorley-Lawson & Geilinger, PNAS 77:5307-5311. 1980). Gp350/220 is also the ligand which mediates attachment of the virus to CR2 on B cells (Nemerow, G. , et al, J. Virol. 61:1416-1420, 1987 and Tanner, J., et al. Cell 50:203-213, 1987) and as such represents one of the few human herpesvirus glycoproteins for which a receptor binding function has been identified.
  • the first region consisted of five identical amino acids, while the second encompassed a region in C3d (LYNVEA) which was reported (by Lambris, J.D., et al, Proc. Natl. Acad. Sci.. USA 82:4235-4239, 1985) to be involved in CR2 binding by C3d (Nemerow, G.R., et al. Supra, 1987).
  • the present invention provides CR2 reactive polypeptides that correspond to amino acid residues in the amino terminal region of gp350/220 and truncation and substitution analogues thereof.
  • a CR2 reactive polypeptide comprises not more than about 50 amino residues and includes an amino acid residue sequence that corresponds to at least about 5 contiguous residues of the linear sequence of EBV gp350/220 and includes at least one of residues 21-24 (EDPG) or 25-27 (FFN) .
  • CR2 reactive polypeptide aggregates comprising CR2 reactive polypeptides joined together or to a carrier, bind to CR2 at the EBV binding site, inhibiting infection of B cells by EBV. As a CR2 ligand, the aggregates also find use as immunopotentiators to increase B cell activation. Treatment methods and diagnostics are also provided. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGURE 1 is a drawing that illustrates the primary sequence and predicted secondary structure of the N-terminal region of gp350/220.
  • the boxed amino acid residues indicate amino acid which are similar to those in C3dg. Gaps in the gp350/220 sequence were inserted to maximize similarity.
  • the relative hydrophobicity and hydrophilicity of each residue is indicated by the length and direction of the vertical bars.
  • the predicted (Chou-Fasman) secondary structure of the N-terminal region of gp350/220 was obtained by a computer-generated program (UWGCG) .
  • FIGURE 2 is a graph that illustrates binding of purified CR2 to CR2 reactive peptides. Synthetic peptides having varying degrees of sequence similarity (underline residues) to C3dg were coated onto 96 well plates. CR2 binding of peptide-coated wells was determined by an ELISA.
  • FIGURE 3 are three graphs (FIGS. 3A-3C) that illustrate inhibition of CR2 binding to EBV by synthetic peptides. Purified EBV, or as controls, R-MuLV or BSA were coated onto ELISA wells. Binding of isolated CR2 alone or CR2 which had been preincubated with 500 nM of synthetic peptides was assessed by an ELISA.
  • FIGURE 4 is a series of six graphs (FIGS. 4A- 4F) which illustrate that fluorescent microspheres coated with the CR2 reactive peptides and gp350/220 bound to CR2 positive Raji cells but not to CR2 negative B95-8 cells. Binding of microspheres bearing
  • IHLTGEDPGFFNVE Panel A,B
  • recombinant gp350/220 Panel C,D
  • LTSGTPSGCENISGA Panel E,F
  • CR2 positive Raji B cells A,C,E
  • CR2 negative B95-8 cells B,D,F
  • FIGURE 5 is a series of two graphs (FIGS. 5A and 5B) which illustrate that the CR2 reactive synthetic peptide-albumin complex inhibits binding of fluorescent microspheres bearing gp350/220 or C3dg to Raji B Cells. Raji B cells were preincubated with varying amounts of
  • a A complexes prior to addition of gp350/220- coated fluorescent microsphere (Panel A) or C3dg-bearing microspheres (Panel B) . Cell binding was quantitated by flow cytometry.
  • FIGURE 6 are three graphs (FIGS. 6A-6C) that illustrate that the CR2 reactive peptide-albumin complex inhibited transformation of peripheral blood B cells (PBL) .
  • Human PBL's were preincubated with 0.1, 1.0 or 10.0 ⁇ g of the peptide-BSA conjugate before addition of EBV. The cells were then washed and cultured in the presence of cyclosporin A for 14 days before enumerating the number of transformed B cells.
  • Epstein Barr Virus The receptor binding region has been localized to residues 21-27 (EDGGFFN) .
  • polypeptides that correspond substantially to 5 contiguous residues in the linear sequence of gp350/220 and include at least one of residues 21-24 (EDPG) or residues 25-27 (FFN) , and analogs thereof also bind to the receptor.
  • CR2 reactive polypeptide aggregates comprising at least 2 CR2 reactive polypeptides joined together or to a carrier bind to CR2 at the EBV binding site, inhibiting infection of B cells by EBV. Compositions and methods based on the peptides 1 ability to bind to and stimulate CR2 and to mimic the EBV binding site are provided.
  • a CR2 reactive polypeptide of the present invention contains at least 5 amino acid residues and not more than about 50, more usually fewer than about 35 and preferably fewer than about 25 amino acid residues.
  • the upper size limitation of the polypeptides is important to achieve the functional properties of the polypeptide, the upper size limitation is not functional. Rather, the limit is a matter of convenience that provides for readily synthesizing the peptides by chemical techniques.
  • a polypeptide of the present invention is characterized by its amino acid residue sequence and novel functional properties.
  • a CR2 reactive polypeptide includes an amino acid residue sequence that substantially corresponds to at least about 5, more usually 7, contiguous residues of the linear sequence of EBV gp350/220 and includes at least one of residues 21-24 (EDPG) or 25-27 (FFN) of gp350/220.
  • the CR2 reactive polypeptide substantially corresponds to an amino acid residue sequence represented by a formula selected from the group consisting of: IHLTGEDPGFFNAE; IHLTGEDPGFFNVQ;
  • the CR2 reactive polypeptide substantially corresponds to an amino acid residue sequence represented by a formula selected from the group consisting of:
  • polypeptides correspond to a sequence represented by the following formulae:
  • IHLTGEDPGFFNVEIPE IHLTGEDPGFFNVE; IHLTGEDPGFFNAE; IHLTGEDPGFFNVQ; IHLTGEDPGFFN; IHLTGEDPG; EDPGFFN; EDPGFFNVE; EDPGFFNVEIPE; DPGFFNVEIPE; PGFFNVEIPE; PFFNVEIPE; GFFNVEIPE; and FNNVEIPE.
  • IHLTGEDPGFFNVEIPE A number of exemplary truncation and substitution analogs of residues 16-32 of gp350/220 (IHLTGEDPGFFNVEIPE) were found to bind to CR2 as described in detail in the examples. Those studies localized the receptor binding region of EBV gp350/220 to seven amino acids, residues 21-27 (EDPGFFN) .
  • Amino acid residues present in a polypeptide of the invention can be any residues that do not materially affect the basic and novel characteristics of a polypeptide as are discussed hereinafter. Such additional residues are usually added to one or both termini of a described polypeptide and can include repeats and partial repeats of a polypeptide sequence or contiguous residues of the gp350/220 protein sequence.
  • a CR2 reactive polypeptide sequence of the present invention has an amino acid residue sequence that corresponds to a portion of the amino acid residues sequence of gp350/220.
  • a CR2 reactive polypeptide of the present invention need not be identical to the amino acid residue sequence of gp350/220.
  • peptide analogs having substitutions which substantially reduce binding to CR2 may induce antibodies which are useful in diagnostic applications, usually the peptide analogues will retain CR2 binding activity. Therefore, a CR2 reactive polypeptide can be subject to various changes, such as insertions, deletions and substitutions, either conservative or non-conservative, where such changes provide for certain advantages in their use and maintain the important functional attributes for a particular use.
  • Conservative substitutions are those where one amino acid residue is replaced by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another such as between arginine and lysine, between glutamic and aspartic acids or between gluta ine and asparagine and the like.
  • conservative substitution also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that such a polypeptide also displays the requisite binding activity.
  • sequences which "substantially correspond" to a specified sequence are those having not more than 10% deletions, insertions or substitutions.
  • additional residues may be added at either terminus. As described previously, the additional residues may correspond to contiguous residues in the linear sequence of gp350/220, repeats of the sequence or may be unrelated to gp350/220.
  • One or more residues may be added, usually at the carboxy terminus, for the purpose of providing a "linker" by which the polypeptides of this invention can be conveniently operatively linked together or to another polypeptide, comprising a T cell epitope polypeptide or a carrier. Carriers and T cell epitope polypeptides which find use with the polypeptides of this invention are described hereinafter.
  • Amino acid residue linkers are usually at least one residue and can be 40 or more residues, more often 1 to 10 residues. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.
  • polypeptide sequence of this invention can differ from the natural sequence by the sequence being modified by terminal-NH 2 acylation, e.g., acetylation, carboxylamidation, e.g., ammonia, methylamine, etc.
  • terminal-NH 2 acylation e.g., acetylation, carboxylamidation, e.g., ammonia, methylamine, etc.
  • CR2 reactive synthetic peptides corresponding to regions of gp350/220 homologous to C3dg were used in several independent assay systems to determine their potential role in CR2 binding.
  • the studies demonstrated that the IHLTGEDPGFFNVE peptide bound to purified CR2 in an ELISA. In soluble form, the peptide partially inhibited the interaction of purified CR2 and EBV. Fluorescent beads bearing the peptide interacted with Raji B cells expressing CR2 with the same binding characteristics as recombinant gp350/220. Binding to CR2 negative cells including B95-8 cells and HSB-2 cells was not observed.
  • CR2 reactive polypeptide aggregates When two or more CR2 reactive peptides are joined together or to a carrier to form an aggregate of this invention, the peptides find use to bind to and stimulate CR2.
  • the peptide aggregates also inhibit infection of B lymphocytes by EBV.
  • CR2 Reactive Polypeptide Aggregates It has been found that CR2 reactive polypeptide aggregates, but not peptide monomers, are effective to block EBV infection of B cells. In addition to inhibiting EBV-induced B cell transformation, the aggregates act as immunopotentiators by stimulating B cells through the CR2 receptor.
  • a CR2 reactive polypeptide aggregate of this invention comprises at least 2, usually 3, more usually 4 and not more than about 50, usually fewer than 25, most usually 10 or fewer, operatively linked CR2 reactive polypeptides.
  • operatively linked it is meant that the CR2 reactive polypeptide is covalently bound, usually at its carboxy terminus, to another polypeptide. While some CR2 reactivity may be achieved by binding a peptide at its amino terminus, the orientation in which its bound has been found to be important to the activity of the peptide.
  • the CR2 reactive polypeptide aggregates can have a plurality of the same or different CR2 reactive polypeptides, wherein the peptides are bound to each other or to a carrier.
  • the CR2 reactive polypeptides are preferably linked to an immunogenic or nonim unogenic carrier, usually a nonimmunogenic carrier.
  • the peptide aggregate preferably comprises not more than about 20 CR2 reactive polypeptides, more preferably less than about 10 polypeptides, most preferably 6 to 8 polypeptides.
  • the carrier can be a host animal protein, conveniently a protein which is present in high quantities in the serum of the host. In this way, relatively large quantities of the aggregate can be administered without inducing an immune response.
  • a host animal protein conveniently a protein which is present in high quantities in the serum of the host.
  • relatively large quantities of the aggregate can be administered without inducing an immune response.
  • 6-8 CR2 polypeptides joined to human serum albumin (HSA) is preferred.
  • CR2 reactive polypeptide aggregates inhibited C3dg and gp350/220 binding to B cells in a dose-dependent manner and blocked EBV infection of peripheral and cord blood B cells in vitro.
  • the CR2 reactive polypeptide aggregates did not inhibit the binding of another CR2 ligand, demonstrating that the aggregate does not inhibit EBV and C3dg binding by steric hindrance, but rather competes for the same epitope.
  • the ability of peptide aggregates of this invention to inhibit 80% of gp350/220 binding and nearly 100% of EBV binding demonstrates that the region is the major epitope responsible for virus adsorption to B cells via CR2.
  • the CR2 reactive polypeptide aggregates find use for two therapeutic purposes, as CR2 ligands and to inhibit EBV infection.
  • the aggregates function as immunopotentiators.
  • a description of B cell activation without proliferation that occurs on ligand binding to CR2 is described in Masucci, et al, Eur. J. Immunol. 17:815-820 (1987) and includes stimulating antibody secretion and production of LIF.
  • Use of the aggregates to inhibit EBV infection by binding to the CR2 receptor is described hereinafter.
  • the polypeptide aggregate-containing compositions administered usually take the form of solutions or suspensions.
  • concentration of active ingredient can vary widely, depending on the purpose of the treatment. In particular, administration of an amount of aggregate that provides a concentration of at least about 100 ⁇ g of aggregate per ml of patient blood is sufficient to act as an immunopotentiator in immune suppressed patients such as those suffering from agammaglobulinemia or other immune deficiencies. Higher concentrations are preferred when using the aggregate for prevention of EBV infection. Usually a concentration of at least about 1.0 mg of aggregate per ml of patient blood is administered. For any therapeutic purpose, concentrations as high as 10 mg/ml, the high value of the normal concentration range for HSA, or somewhat' higher, is acceptable.
  • compositions that contains polypeptides as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient as are well known. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents which enhance the effectiveness of the active ingredient.
  • a polypeptide composition can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
  • the therapeutic polypeptide-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosages for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. While generally useful quantity ranges have been specified, it must be remembered that the quantity to be administered also depends on the subject to be treated, capacity of the subject to utilize the active ingredient, and degree of inhibition of receptor-ligand binding desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses administered every about 4 hours to 48 hours, preferably every 6 to 8 hours by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain therapeutically effective concentrations in the blood are contemplated.
  • a method of increasing antibody secretion in a patient comprises the following steps.
  • An amount of a CR2 reactive polypeptide aggregate effective to stimulate antibody secretion is administered to the patient.
  • the amount of aggregate will differ depending on whether the aggregate is used as an immunopotentiator in an immune suppressed patient or to inhibit EBV infection of B cells.
  • the aggregate is administered substantially concurrently with either recurrence of infection in a chronically infected patient or initial exposure to EBV, such as on receipt of tissue from an EBV-seropositive donor.
  • a polypeptide of this invention is operatively linked to a T cell epitope polypeptide and used in a pharmaceutically acceptable aqueous diluent to form an inoculum that, when administered in an effective amount, is capable of inducing antibodies that immunoreact with the CR2 binding site region of gp350/220.
  • the particular T cell epitope can vary widely depending on the purpose of antibody induction.
  • the T cell epitope polypeptide can be commonly used i ⁇ ffiiunogenic carriers, such as those used to immunize goats, rabbits, mice, marmosets or the like.
  • an EBV T cell epitope polypeptide is preferred.
  • Useful carriers for antibody induction are well known in the art, and are generally proteins themselves. Exemplary of such carriers are keyhole limpet hemocyanin (KLH) , edestin, thyroglobulin. albumins such as bovine serum albumin (BSA) or human serum albumin (HSA) , red blood cells such as sheep erythrocytes (SRBC) , tetanus toxoid, cholera toxoid as well as polyamino acids such as ply (D-lysine: D- glutamic acid) , and the like.
  • BSA bovine serum albumin
  • HSA human serum albumin
  • red blood cells such as sheep erythrocytes (SRBC)
  • tetanus toxoid cholera toxoid
  • polyamino acids such as ply (D-lysine: D- glutamic acid) , and the like.
  • Exemplary EBV T cell epitope polypeptides comprise EBNA2 and LMP.
  • Moss, et al. Nature 331:719-721 (1988) describes the T cell epitope-containing region of EBNA2 and Thorley-Lawson, et al, Proc. Natl. Acad. Sci.. USA 84:5384-5388 (1987) describes the location of the T cell epitope of LMP as residues 43 to 53 (VMSDWTGGALL) .
  • the EBV polypeptide or amino acid residue sequences corresponding to the T cell epitope-containing sequence can be used.
  • the T cell epitope is operatively linked to the CR2 reactive polypeptide.
  • the CR2 reactive peptide will be joined to the T cell epitope polypeptide so that T cell help in recognizing the CR2 reactive peptide will be provided.
  • the peptide will be covalently linked to the T cell epitope peptide.
  • encapsulating the CR2 reactive polypeptide in a liposome or other carrier may also find use.
  • the orientation of the CR2 reactive polypeptide in relation to the T cell polypeptide will depend on the particular T cell epitope used and will be designed to optimize induction of antibody to the CR2 binding site region.
  • one or more amino acid residues can be added to the amino- or carboxy-termini of the polypeptides to assist in operatively linking the polypeptides.
  • Cysteine residues added at the termini of the polypeptide are particularly useful for forming conjugates via disulfide bonds.
  • Exemplary additional linking procedures include the use of Michael addition reaction products, di-aldehydes such as glutaraldehyde, Klipstein, et al, J. Infect. Pis.. 147. 318-236 (1983) and the like, or the use of carbodiimide technology as in the use of a water-soluble carbodiimide to form amide links to the carrier.
  • inoculum in its various grammatical forms is used herein to describe a composition containing a polypeptide of this invention as an active ingredient used for the preparation of antibodies.
  • the present inoculum contains an effective, immunogenic amount of a composite polypeptide of this invention.
  • the effective amount of polypeptide or protein per unit dose depends, among other things, on the species of animal inoculated, the body weight of the animal and the chosen inoculation regimen as is well known in the art.
  • Inocula typically contain polypeptide or protein concentrations of about 10 micrograms to about 500 milligrams per inoculation (dose) , preferably about 50 micrograms to about 50 milligrams per dose.
  • Inocula are typically prepared from the dried solid polypeptide-conjugate by dispersing the polypeptide-conjugate in a physiologically tolerable (acceptable) diluent or vehicle such as water, saline or phosphate-buffered saline to form an aqueous composition.
  • a physiologically tolerable (acceptable) diluent or vehicle such as water, saline or phosphate-buffered saline to form an aqueous composition.
  • diluents are well known in the art and are discussed, for example, in Remington's Pharmaceutical Sciences f 16th Ed. , Mack Publishing Company, Easton, PA (1980) at pages 1465-1467.
  • Inocula can also include an adjuvant as part of the diluent.
  • Adjuvants such as complete Freund's adjuvant (CFA) , incomplete Freund's adjuvant (IFA) and alum are materials well known in the art, and are available commercially from
  • antibody in its various grammatical forms is used herein to refer to immunoglobulin molecules and immunol ⁇ gically active portions of immunoglobulin molecules, i.e., molecules ' that contain an antibody combining site or paratope.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab 1 , F(ab') 2 and F(v) .
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically bind (immunoreacts with) antigen.
  • the term “immunoreact” in its various forms means binding between an antigenic determinant- containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.
  • Antigenic determinant refers to the actual structural portion of the antigen that is immunologically bound by an antibody combining site.
  • An antibody composition of the present invention is characterized as containing antibody molecules that immunoreact with at least one specific polypeptide of this invention.
  • the antibodies are substantially free from reaction with amino acid residue sequences represented by the formulae:
  • An antibody composition of the present invention is typically produced by immunizing a mammal with an inoculum of the present invention and thereby induce in the mammal antibody molecules having the appropriate polypeptide immunospecificity.
  • the antibody molecules are then collected from the mammal and isolated to the extent desired by well known techniques such as, for example, by using DEAE Sephadex to obtain the IgG fraction.
  • the antibodies may be purified by immunoaffinity chromatography using solid phase-affixed CR2 reactive peptides.
  • the antibody composition is contacted with solid phase-affixed CR2 reactive polypeptides for a period of time sufficient for CR2 to react with the polypeptides to form a solid phase- affixed complex.
  • the bound antibodies are separated from the complex the complex by standard techniques. Since the antibodies recognize the CR2 binding site of EBV, the method can also be used to purify a CR2- containing composition.
  • a monoclonal antibody composition is also contemplated by the present invention.
  • the phrase "monoclonal antibody composition" in its various grammatical forms refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody composition thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody composition is typically composed of antibodies produced by clones of a single cell called a hybridoma that secretes (produces) but one kind of antibody molecule.
  • the hybridoma cell is formed by fusing an antibody-producing cell and a myeloma or other self-perpetuating cell line. The preparation of such antibodies were first described by Kohler and Milstein, Nature 256:495-497 (1975), which description is incorporated by reference. Other methods of producing monoclonal antibodies are also well known.
  • the hybridoma supernates can be screened for specificity for a CR2 reactive peptide or for inhibition of binding of EBV to CR2.
  • the antibody compositions of this invention can be used in, inter alia, the diagnostic methods and systems of the present invention to detect EBV or for passive immunization.
  • the preparation and concentration of the antibodies used do not differ from those used to treat or prevent other viral infections.
  • the immunogenicity of the antibody preparation will usually be minimized prior to administration.
  • Therapeutic formulations for administration of antibodies do not differ from those used to administer polypeptides and are described hereinbefore.
  • a diagnostic system in kit form of the present invention includes, in an amount sufficient for at least one assay, a polypeptide, antibody composition or monoclonal antibody composition of the present invention, as a separately packaged reagent. Instructions for use of the packaged reagent are also typically included.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
  • a diagnostic system for assaying for the presence of or to quantitate anti-EBV antibodies in a sample comprises a package containing at least one CR2 reactive peptide of this invention.
  • a diagnostic system of the present invention for assaying for the presence of or to quantitate EBV comprises a package containing an antibody composition of this invention.
  • a diagnostic system of the present invention further includes a label or indicating means capable of signaling the formation of a complex containing an antibody molecule or polypeptide of the present invention.
  • label and "indicating means” in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex.
  • the linking of labels i.e., labeling of, polypeptides and proteins is well known in the art.
  • the techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al. Scand. J. Immunol.. Vol. 8 Suppl. 7:7-23 (1978), Rodwell, et al, Biotech.. 3:889-894 (1984) and U.S. Pat. No. 4,493,795.
  • the diagnostic systems can also include, preferably as a separate package, a specific binding agent.
  • a “specific binding agent” is a molecular entity capable of selectively binding a reagent species of the present invention but is not itself a protein expression product, polypeptide, or antibody molecule of the present invention.
  • Exemplary specific binding agents are antibody molecules, complement proteins or fragments thereof, protein A and the like.
  • the specific binding agent can bind the antibody molecule of this invention when it is present as part of a complex.
  • anti-human Fc antibodies are conveniently used.
  • the specific binding agent is labeled.
  • the agent is typically used as an amplifying means or reagent.
  • the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.
  • the diagnostic kits of the present invention can be used in an "ELISA" format to detect the presence or quantity of EBV or anti-EBV antibodies in an antibody-containing body fluid sample such as serum, plasma or urine.
  • ELISA refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme- antibody conjugate to detect and quantify the amount of an antigen or antibody present in a sample.
  • a description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D.P. Sites, et al, published by Lange Medical Publications of Los Altos, CA in 1982, and in U.S. Patents No. 3,654,090; No. 3,850,752; and No.
  • the CR2 reactive polypeptide, or antibody molecule of the present invention can be affixed to a solid matrix to form a solid support that is separately packaged in the subject diagnostic systems.
  • the reagent is typically affixed to the solid matrix by adsorption from an aqueous medium although other modes of affixation, well known- to those skilled in the art can be used.
  • Useful solid matrices are well known in the art. Such materials include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ) ; agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, IL; polyvinyl chloride, polystyrene, cross- linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microliter plate such as those made from polystyrene or polyvinylchloride.
  • the reagent species, labeled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form.
  • the indicating means is an enzyme
  • the enzyme's substrate can also be provided in a separate package of a system.
  • a solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
  • the packages discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.
  • Such packages include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vial, plastic and plastic-foil laminated envelopes and the like.
  • the present invention contemplates any method that results in detecting EBV or anti-EBV antibodies using antibodies or peptides of this invention.
  • exemplary methods are described herein, the invention is not so limited.
  • anti-EBV antibodies in an antibody-containing bodily fluid is indicative of EBV infection in the host animal.
  • a four-fold rise in antibody titer between an acute sample and one taken about 4 to 6 weeks thereafter indicates infection by the particular virus.
  • detection of anti-EBV antibodies indicates EBV infection.
  • an antibody-containing bodily fluid from the patient is contacted with solid phase-affixed CR2 reactive polypeptides for a period of time sufficient to form a solid phase-affixed immune complex.
  • the presence of the solid phase-affixed immune complex is indicative of infection of by EBV, as stated previously.
  • the complex can be detected as described before.
  • the sample is contacted with solid phase-affixed antibodies of this invention for a period of time sufficient to form a solid phase-affixed immune complex.
  • the amount of the solid phase-affixed immune complex is indicative of the amount of EBV in the sample.
  • Biological assay conditions are those that maintain the biological activity of the antibody molecules and polypeptide molecules of this invention and the fibrinogen-bound platelets sought to be assayed. Those conditions include a temperature range of about 4*C to about 45*C, preferably about 37*C, a pH value range of about 5 to about 9, preferably about 7 and an ionic strength varying from that of distilled water to that of about one molar sodium chloride, preferably about that of physiological saline. Methods for optimizing such conditions are well known in the art.
  • amino acid residue sequence of a protein or polypeptide is directly related via the genetic code to the deoxyribonucleic acid (DNA) sequence of the structural gene that codes for the protein and the mRNA from which it is translated.
  • DNA deoxyribonucleic acid
  • a nucleotide sequence can be defined in terms of the amino acid residue sequence, i.e., protein or polypeptide, for which it codes.
  • nucleotide triplet An important and well known feature of the genetic code is its redundancy. That is, for most of the amino acids used to make proteins, more than one coding nucleotide triplet (codon) can code for or designate a particular amino acid residue. Therefore, a number of different nucleotide sequences can code for a particular amino acid residue sequence. Such nucleotide sequences are considered functionally equivalent since they can result in the production of the same amino acid residue sequence in all organisms. Occasionally, a methylated variant of a purine or pyrimidine may be incorporated into a given nucleotide sequence. However, such methylations do not affect the coding relationship in any way.
  • a nucleotide sequence of the present invention encodes a CR2 reactive polypeptide sequence of this invention.
  • the sequence will include nucleotides encoding at least about 10 amino acid residues, usually at least about 20 amino acid residues, most usually at least 50 amino acid residues.
  • the nucleotide sequence will additionally encode amino acid residues which substantially correspond to contiguous amino acid residues in the gp350/220 sequence.
  • the encoded sequence will usually include at least about one-quarter of the gp350/220 sequence, more usually about half of the gp350/220 sequence and less than the entire sequence.
  • the sequence can be homologous, preferably identical, to a portion of the amino acid residue sequence of gp350/220 to which the peptide corresponds.
  • a DNA segment of the present invention that encodes a CR2 reactive amino acid residue sequence can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al, J. Am. Che . Soc.. 103:3185 (1981).
  • any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence.
  • DNA segments consisting essentially of structural genes encoding the gp350/220 related proteins can be obtained by digesting EBV or from cell lines transformed with EBV.
  • the rat pituitary cell line, GH3 ⁇ 19 expresses the anchorless form of gp350/220 (Whang, et al, J. Virol. 61:1796-1807 1987).
  • a nucleic acid construction of the present invention can be produced by operatively linking an expression vector to the nucleic acid sequence of the present invention.
  • operatively linked is meant that the nucleotide sequence is joined to the vector so that the sequence is under the transcriptional and translation control of the expression vector and can be expressed in a suitable host cell.
  • the term "vector” refers to a DNA molecule capable of autonomous replication in a cell and to which another nucleotide sequence can be operatively linked so as to bring about replication of the attached segment.
  • a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature.
  • DNA segment of the present invention is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing recombinant DNA molecules.
  • Expression vectors which are relatively harmless in a host animal, including primates, are well known and are used to induce an immune response to the expressed foreign polypeptide included in the vector.
  • vectors are provided containing convenient restriction sites for insertion of the desired DNA segment.
  • vaccinia virus vectors are vaccinia virus vectors.
  • a variety of methods has been developed to operatively link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules. Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors.
  • the DNA segment generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E.
  • coli DNA polymerase I enzymes that remove protruding, 3• , single-stranded termini with their 3•-5' exonucleolytic activities and fill in recessed 3' ends with their polymerizing activities. The combination of these activities therefore generates blunt-ended DNA segments.
  • the blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules such as bacteriophage T4 DNA ligase.
  • the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies, Inc., New Haven, CT.
  • RNA equivalents of the above described recombinant DNA molecules are also contemplated by the present invention. Since the CR2 reactive polypeptides are readily synthesized, the expression constructs primarily find use to immunize a host animal rather than as a source of the peptides per se.
  • Example 1 CR2 Binds Specifically On An N-Terminal gp350/220 Synthetic Peptide.
  • CR2 and CR2 ligands were prepared as follows.
  • the EBV/C3d receptor (CR2) was isolated from detergent lysates of Raji B lymphoblastoid cells by affinity chromatography as previously described (Nemerow, G.R. , 'et al, J. Virol. 58:709-712, 1986).
  • C3dg was purified from Mg++EGTA activated (aged) human plasma (Vik, D.P., et al, J. Immunol. 134:2571-2579, 1985).
  • the rat pituitary cell line, CH3 ⁇ 19 expressing the anchorless form of gp350/220 (Whang, Y. , et al, J_j_ Virol. 61:1796-1807, 1987) was kindly provided by Dr. Elliott Kieff (Harvard Medical School, Boston, MA).
  • Recombinant gp350/220 was isolated from spent culture supernates by immunoaffinity chromatography (Nemerow, G.R., et al.
  • the monoclonal and polyclonal antibodies used were as follows. A monoclonal antibody to gp350/220, BOS-1, has been previously described (Nemerow, G.R. , et al. Supra, 1987). Anti-gp350/220 antibody 2L10 was kindly provided by Dr. Elliott Kieff. Anti-CR2 antibody HB5, from the ATCC (Rockville, MD) . Polyclonal anti-CR2 antisera was raised rabbits as follows. Approximately 2 ⁇ g of affinity purified receptor in complete Freund's adjuvant was injected subcutaneously into a NBZ rabbit. After 10 days, the rabbit was re-injected with 2 ⁇ g of CR2 in incomplete Freund's adjuvant and again thereafter at 2 week intervals.
  • Sera were obtained following the fourth through sixth immunizations and analyzed for the presence of anti-CR2 antibody by an ELISA and by immunoprecipitation.
  • the IgG F(ab') 2 fractions of polyclonal anti-CR2 and preimmune sera were provided by Dr. John Bohnsack (University of Utah College of Medicine, Salt Lake City, UT) .
  • Synthetic peptides and peptide-albumin conjugates were prepared as follows. Gp350/220 peptides were synthesized by the simultaneous multiple synthesis method (T-bag) (Houghten, R.A. , Proc. Natl. Acad. Sci., USA 82:5131-5135, 1985) or alternatively using an FMOC manual peptide synthesis system (RAMPS, E.I. dupont deNemours and Co., Boston, MA). All peptides were amidated at the C-terminus to mimic internal regions of native proteins. Peptides were characterized by reverse-phase HPLC on a C18 column (Vydac, Hesparia, CA) .
  • Synthetic peptides obtained by both synthesis methods were 80-90% homogeneous by this criteria. Amino acid composition analyses were also obtained on several of the peptides in order to confirm the purity of these reagents.
  • the gp350/220 peptides HLTGEDPGFFNVEC and KCKWTLTSGTPSGCE were coupled to bovine serum albumin (Fraction V, Sigma) with m-maleimidobenzoly-N- hydroxysuccinimide ester (Pierce, Rockford, IL) as previously described (Goldbard, D.S., et al, Nature 322;641-644, 1986).
  • cysteine- containing peptides were not deprotected with mercuric acetate prior to conjugation to BSA in order to assess the possible affect of the reagents used in the coupling procedure on the functional activities of the complex.
  • Peptide-albumin complexes were analyzed on 12.5% SDS- PAGE gels. These studies indicated that approximately 6-8 molecules of deprotected peptides were coupled per molecule of albumin while only 1-2 molecules of nondeprotected peptides were coupled per BSA molecule as determined by the difference in electrophoretic mobility of the peptide-albumin conjugate compared to underivatized albumin.
  • the inefficient coupling of nondeprotected peptide to BSA was likely due to inefficient deprotection of cysteine residues with TFA during cleavage of the peptide from the synthesis resin.
  • the ELISA for detecting synthetic gp350/220 peptide binding to CR2 was performed as follows.
  • Varying amounts of synthetic peptides were coupled to 96 well plates (Immulon II, Dynatech) in 0.1 M sodium bicarbonate buffer, pH 9.6. Non-specific binding was blocked by addition of 1% non-fat dry milk (Blotto) for 60 minutes at 22*C. Peptide- ⁇ oated wells were then incubated with 100-200 ng of purified CR2 for 60 minutes at 22*C followed by incubation with 1 ⁇ g/well of HB-5 monoclonal anti-CR2 for 60 minutes at 22"C.
  • Absorbance at A 405 was determined in a Titertek II ELISA reader (Flow Labs, Maclean, VA) .
  • Inhibition ELISA assays were carried out as follows: Purified EBV, approximately lxlO 6 , or R-MULV or 20 ⁇ g BSA were desiccated onto 96 well plates in bicarbonate buffer. After blocking nonspecific binding sites on the wells with Blotto, CR2 alone or CR2 which had been preincubated with 50 ⁇ M of synthetic peptides was added to the ligand-coated wells. After further incubation for 90 minutes at 22"C, detection of CR2 binding to virus-coated wells was determined as described above.
  • Example 2 The CR2 Reactive Peptides Inhibited CR2 Binding To EBV.
  • the ability of the IHLTGEDPGFFNVE peptide to block EBV binding to purified CR2 was examined.
  • Competition ELISA studies performed as described in Example 1 using immobilized purified EBV showed that the IHLTGEDPGFFNVE peptide blocked approximately 50% of the binding of purified CR2 to immobilized EBV while a closely related peptide lacking amino acid residues 28 and 29 had minimal inhibitory activity (Fig. 3).
  • lxlO 6 CR2 positive Raji B lymphoblastoid cells or CR2 negative B95-8 marmoset B cells or HSB-2 T lymphoblastoid cells as controls were incubated with 10 ⁇ l of ligand-coated microspheres in 100 ⁇ l of phosphate buffered saline (PBS), 2% BSA at 4'C for 60 minutes. Unbound microspheres were then separated from cell-associated microspheres by centrifugation through a cushion of 6% BSA-PBS. The cells were then fixed with 1% paraformaldehyde and examined by flow cyto etry using a FACS IV analyzer (Becton Dickinson) .
  • the cells were then washed by low speed centrifugation prior to incubation with peptide-coated microspheres.
  • Inhibition of gp350/220 binding or C3dg binding to Raji cells by peptides was carried out by preincubating lxlO 6 Raji cells with various amounts of peptide alone, peptide- albumin conjugates or albumin alone for 60 minutes at 4"C prior to the addition of the ligand-coated microspheres as described above.
  • fluorescent microspheres bearing the IHLTGEDPGFFNVE gp350/220 peptide showed significant binding to Raji cells (47.8%) but only minimal binding to CR2 negative B95-8 cells (10.8%).
  • the level of synthetic peptide binding to Raji cells (Panel A) was similar to that observed with recombinant gp350/220 (Panel C) .
  • Fluorescent microspheres bearing a synthetic peptide corresponding to the other C3d-similar region of gp350/220, LTSGTPSGCENISGA did not bind to Raji cells (Panel E) .
  • the specificity of peptide binding to CR2 positive cells was examined by preincubating Raji cells with either EBV or with polyclonal anti-CR2 prior to addition of peptide-coated microspheres. The results are illustrated in Table 1.
  • a 100 ⁇ g of peptide or 100 ng of purified recombinant gp350/220 were coupled to carboxylated or underivatized fluorescent microspheres, respectively.
  • the underlined residues indicate residues which are similar to those found it C3dg.
  • Ligand-coated fluorescent microspheres were reacted with either lxlO 6 Raji or B95-8 B cells alone or with cells which had been preincubated with viruses or anti-CR2 antibody prior to flow cytometric analysis (FACS) .
  • each peptide was coupled to carboxylated fluorescent microspheres prior to FACS analysis with Raji B or HSB-2 T lymphoblastoid cells.
  • IHLTGEDPGFFNVE coupled to fluorescent microspheres showed significant binding to Raji B cells but not to the CR2-negative T cell line HSB-2.
  • Deletions of N- terminal amino acid residues in this peptide up to but not including the C3dg-similar region resulted in only a slight loss in CR2 binding activity.
  • Deletion of the C- terminal valine and glutamic acid (VE) residues did not reduce CR2 binding.
  • further truncation of the C-terminal phenylalanme, phenylalanme, asparagine residues (FFN) abrogated approximately half of the cell binding activity.
  • Example 5 Multimeric CR2 Reactive Peptides Inhibit Recombinant gp350/220 Binding To CR2.
  • N-terminal gp350/220 peptide was synthesized with a C-terminal cysteine residue and it, as well as the KCKWTLTSGTPSGCE peptide which has a penultimate cysteine residue, were covalently coupled to albumin. The results are shown in Table 3. TABLE 3
  • Raji cells were preincubated with 10 mg of BSA, deprotected (D) peptide-BSA or nondeprotected (ND) peptide-BSA or peptide alone prior to incubation with CR2 ligand-coated microspheres.
  • HLTGEDPGFFNVE peptide coupled to albumin blocked 50-80% of the binding of fluorescent microspheres bearing recombinant gp350/220 to Raji B lymphoblastoid cells; neither monomeric peptide (3.3 nM) alone nor BSA reduced EBV binding to Raji cells.
  • a gp350/220 peptide duplicating the other C3dg-like domain, KCKWTLTSGTPSGCE complexed to albumin did not inhibit the binding of gp350/220 coated fluorescent microspheres to CR2 (Table 3).
  • the HLTGEDPGFFNVEC-BSA conjugate but not the KCKWTLTSGTPSGCE-BSA complex mediated dose-dependent inhibition of both recombinant gp350/220 and C3dg binding to Raji B lymphoblastoid cells (Fig. 5, panels A and B) .
  • approximately 0.3 nM and 0.6 nM of peptide coupled to albumin were required to block 50% of gp350/220 and C3dg binding to Raji cells, respectively.
  • the HLTGEDPGFFNVEC-BSA conjugate at comparable doses did not inhibit the binding of fluorescent beads bearing the anti-CR2 monoclonal antibody HB5 to Raji B lymphoblastoid cells (Table 3) .
  • BSA-synthetic peptide conjugates were analyzed for their ability to inhibit EBV infection of adult peripheral blood B cells. Unseparated peripheral blood mononuclear cells, 6xl0 5 , were incubated with varying amounts of BSA-peptide conjugates in RPMI 1640 containing 10% FCS for 60 minutes at 4 ⁇ C. Three hundred microliters of EBV-containing supernate derived from
  • B95-8 cells was then added and incubation was continued for 60 minutes at 4 ⁇ C.
  • the cells were then washed twice by centrifugation in media and plated at 2xl0 5 cells per well in triplicate wells in 96 well sterile tissue culture plates (Costar, Cambridge, MA) .
  • Cyclosporin A (Sigma, St. Louis, MO) at 0.1 ⁇ g/ml was added to each well to allow outgrowth of EBV infected B cells (Bird, A.G., et al. Nature 289:300-301, 1981).
  • Wells were scored for outgrowth of EBV transformed B cells and for stimulation of DNA synthesis 10-14 days after infection as previously described (Nemerow, G.R. , et al. Virology 132:186-198, 1984).
  • peptide-BSA complexes were also tested for their ability to block EBV-induced 3 H-thymidine incorporation in cord blood mononuclear cells as previously described (Nemerow, G.R. , et al. Supra, 1981) .
  • Cord blood cells (6xl0 5 ) were reacted with varying amounts of peptide-BSA followed by incubation with EBV as described above except that no cyclosporin A was used in these assays.
  • B cell proliferation was measured by 3 H-thymidine incorporation on day 4 as previously described (Nemerow, G.R. , et al, Supra, 1981) .
  • Peripheral blood mononuclear cells were preincubated with varying amounts of the HLTGEDPGFFNVEC- BSA or with BSA alone prior to incubation with EBV- containing supernates.
  • 1.0 and 10.0 ⁇ g (0.12 nM and 1.2 nM of peptide, respectively) of the deprotected N-terminal gp350/220 peptide-BSA conjugate abolished the outgrowth of EBV transformed B cells.
  • the KCKWTLTSGTPSGCE gp350/220 peptide representing the other C3dg-like domain, also conjugated to BSA did not inhibit EBV infectivity at comparable doses.
  • the nondeprotected N-terminal peptide-BSA complex inhibited EBV infectivity only at doses in excess of 100 ⁇ g.
  • Inhibitor 8 EBV 3 H-TdR Uptake b Inhibition
  • a 6x10 cord blood mononuclear cells were preincubated with varying amounts of peptide-BSA complexes prior to exposure to EBV.
  • the cells were cultured for 4 days and then pulsed with 3 H-thymidine.
  • b Values represent the mean ⁇ S.D.
  • Example 7 The CR2 Reactive Polypeptides Detected Human Anti-EBV Antibody In An ELISA.
  • Example 5 An ELISA was performed as described in Example 1 using gp350/220 and the IHLTGEDPGFFNVE polypeptide as solid phase-affixed antigens. Viral capsid antigen reactivity was tested for each serum as a control. The results are listed in Table 5.
  • the CR2 reactive polypeptides of this invention mimic the CR2 binding site region of EBV, acting as a CR2 ligand and stimulating the receptor while inhibiting infection of B cells by EBV.

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Abstract

L'invention concerne des polypeptides réagissant au CR2 et correspondant aux résidus d'acides aminés se trouvant dans la région amino terminale de la gp350/220 de l'EBV ainsi que des analogues de ceux-ci. Elle concerne également des séquences de polypeptides correspondant à au moins 5 résidus contigus de la séquence linéaire de la gp350/220 de l'EBV (virus Epstein-Barr) et comprenant au moins un des résidus 21 à 24 ou 25 à 27 de la liaison de la séquence linéaire au récepteur CR2. Elle concerne en outre des agrégats de polypeptides comprenant au moins deux polypeptides réagissant au CR2, et s'utilisant comme agents d'immuno-potentialisation stimulant les cellules B. Lesdits agrégats inhibent l'infection de cellules B par l'EBV. Des procédés de traitement et de diagnostic sont décrits.
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US6458360B1 (en) 1990-04-25 2002-10-01 The Johns Hopkins University Soluble complement regulatory molecules
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US6432679B1 (en) 1998-06-12 2002-08-13 Henry M. Jackson Foundation For The Advancement Of Military Medicine Enhancement of B cell activation and immunoglobulin secretion by co-stimulation of receptors for antigen and EBV Gp350/220
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FR2850384A1 (fr) * 2003-01-28 2004-07-30 Centre Nat Rech Scient Antigenes mimant les domaines extracellulaires de proteines membranaires de type iii issues de microorganismes intracellulaires pathogenes, anticorps conformationnels derives, et leurs applications
US20120108492A1 (en) * 2008-09-19 2012-05-03 The Regents Of The University Of Colorado Ligand-specific non-antibody compounds that inhibit cr2 activation and methods of use thereof
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