WO1991002536A1 - Compositions and methods for detection and treatment of epstein-barr virus infection and immune disorders - Google Patents

Abstract

Synthetic polypeptides corresponding to the extracellular domain of the B lymphocyte membrane receptor CR2 are disclosed together with polypeptide aggregates, compositions and methods for the treatment of Epstein-Barr virus infection and immunological disorders, as well as testing for the presence of said virus.

Description

COMPOSITIONS AND METHODS FOR DETECTION

AND TREATMENT OF EPSTEIN-BARR VIRUS

INFECTION AND IMMUNE DISORDERS

Description

Technical Field

The present invention relates to synthetic polypeptides, and polypeptide aggregates, that correspond to a B lymphocyte membrane receptor protein (CR2) for Epstein-Barr virus. Background of the Invention

A 145 kilodalton (kDa) B lymphocyte membrane glycoprotein, designated CR2, serves as a receptor for Epstein-Barr virus (EBV) and the C3d and C3dg fragments of the third component of complement.

Epstein-Barr virus (EBV) , a human herpesvirus, is the causative agent of infectious mononucleosis (Henle, G., et al, Proc. Natl. Acad. Sci.. 59:94-101, 1968), a benign lymphoproliferative disease. Under certain conditions, EBV infection may facilitate the development of malignant diseases including nasopharyngeal carcinoma (Henle, W. , Science 157:1064-1065, 1967 and Raab-Traub, N. , et al, Int. J. Cancer 39:25-29, 1987), Burkitt's lymphoma (De-The, G. , et al, Advances in Comparative Leukemia Research. Eds., D.S. Yohn and J.R. Blakeslee, Elsevier, NY, 1981) and the X-linked lymphoproliferative syndrome (Harada, S., et al, J. Immunol. 129:2532-2535, 1982). 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. EXP. Med. 164:2049-2060, 1986; Yarchoan, R. , et al, J. Clin. Invest. 78:439- 447, 1986; and Montagnier, L. , et al, Science 225:63- 66, 1984). EBV is unique among the human herpes viruses 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 CR2 glycoprotein which binds the C3d/C3dg complement fragments (Fingeroth, J.D., et al, Proc. Natl Acad. Sci.. USA 81:4510-4516, 1984; Nemerow, G.R. , et al, J. Virol. 55:347-351, 1985; Frade, R. , et al, Proc. Natl. Acad. Sci.. USA 82:1490-1493, 1985; and Mold, C, et al, J. Immunol. 136:4140-4145, 1986). Molecular cloning of CR2 has revealed that the entire extracellular domain of this receptor is comprised of tandemly arranged repeating elements which share significant sequence similarity with a number of other complement- and noncomplement- binding plasma and membrane proteins (Moore, M.P., et al, Proc. Natl. Acad. Sci.. USA 84:9194-9198, 1987; and Weis, J.J., et al, J. Exp. Med. 167:1047-1066. 1988) .

In addition to its dual ligand binding function, CR2 is involved in a pathway of B cell activation (Nemerow, G.R. , et al, supra, 1985; Chagelian & Fearnon, J. EXP. Med 163:101-115, 1986; Wilson, et al. Blood 66:824-829, 1985; Frade, R. , et al, Proc. Natl. Acad. Sci.. USA 82:1490-1493, 1985; and Melchers, et al, Nature 317:264-267, 1985). This property may play a crucial role in preparing the cell for transformation by EBV.

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, Proc. Natl. Acad. Sci. U.S.A. 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 binding of EBV to CR2 is specifically mediated by a 9 amino acid residue epitope located near the N-terminus of gp350/220 (Nemerow, G.R., et al., Cell 56:369-377, 1989) . Binding of EBV, via gp350/220, to CR2 is followed by endocytosis of the virus, de-envelopment and transformation (Nemerow, G.R. , et al, Virology 132:186-198, 1984; Tedder, T.F., et al, J. Immunol. 137:1387-1391, 1986; and Tanner, et al, supra, 1987). Purified CR2 (Nemerow, G.R. et al., Virol. 61:1416-1420, 1987 and Nemerow, G.R. et al. , J. Virol. 58:709-712, 1986) and cells transfected with cDNA encoding CR2 (Ahem, J.M. et al., Proc. Natl. Acad. Sci.. U.S.A. 85:9307-9311, 1988) bind EBV, and gp350/220 selectively absorbs CR2 from B lymphocyte membrane extracts (Fingeroth, J.D. et al., supra, 1984 and Tanner, J. et al.. Cell 50:203-213, 1987) . Molecular cloning and sequencing of cDNA encoding CR2 has revealed that the extracellular domain of the receptor contains 16 tandemly arranged short repeating regions of about 60 to 75 amino acids, followed by a hydrophobic transmembrane domain and a short, 34 amino acid residue cytoplasmic tail (Moore, M.D. et al., supra 1987, Weis, J.J. et al., J. Exp. Med. 167:1047-1066, 1988, and Fujisaku, A. et al., J. Biol. Chem. 264:2118-2125, 1989).

It has now been discovered that regions of the extracellular portion of CR2 located at the amino terminus of the CR2 protein participate in the binding interaction of CR2 with EBV, gp35/220 or C3d/C3dg complement fragments Summary of the Invention The present invention is directed to diagnostic and treatment methods that utilize synthetic polypeptides that have amino acid residue sequences which correspond to EBV-binding regions of the extracellular domain of CR2, polypeptide aggregates that contain amino acid residue sequences corresponding to the EBV-binding regions of CR2 and pharmacological compositions.

Methods for the inhibition and treatment of EBV infections are provided by the present invention. In these methods the aqueous medium, such as blood, in contact with cells at risk for infection is admixed with a CR2 polypeptide of the present invention. The CR2 polypeptide-containing admixture so formed is maintained for a time period sufficient to allow any Epstein-Barr virus present in the suspension to bind to the polypeptide. In one embodiment a pharmacological composition containing CR2 polypeptides is preferably injected into the blood of a patient at risk for Epstein-Barr virus infection.

A method of treatment for immune disorders is also provided by the present invention. This method is directed to the administration to a patient of a therapeutically effective amount of a pharmacological composition of the present invention to bind to either a C3dg fragment of complement or antibodies directed against CR2.

In the present invention, a CR2 polypeptide is defined by the presence of an amino acid residue sequence corresponding to CR2 amino acid residues 1- 256 as shown in Figure 9. In one embodiment, a polypeptide aggregate is contemplated by the present invention that contains at least two CR2 polypeptides of the present invention operatively linked together. A pharmacological composition is provided by the present invention that contains a physiologically tolerable carrier together with a substantially purified CR2 polypeptide and/or CR2 polypeptide aggregate as described herein as an active ingredient.

Methods for the detection of Epstein-Barr virus and/or anti-CR2 antibodies present in a fluid sample and diagnostic kits are provided by the present invention.

A method for the detection of Epstein-Barr virus in a fluid sample is provided in which a fluid sample is contacted with a solid matrix that has a CR2 polypeptide attached thereto for a time period sufficient to allow any Epstein-Barr virus present in the sample to specifically bind to the polypeptide. The solid matrix is then separated from the liquid sample, and washed (rinsed) to remove any unbound material. The washed matrix is then contacted with a label that is capable of indicating the presence of any Epstein-Barr virus that is specifically bound to the matrix.

A method of detecting the presence of antibodies directed against CR2 in a fluid sample is contemplated. In this method a fluid sample is contacted with a solid matrix having a CR2 polypeptide of the present invention linked thereto for a time period sufficient to allow the formation of an immunoreaction product between the CR2 polypeptide and any anti-CR2 antibodies present in the sample. The solid matrix is separated from the fluid sample and washed (rinsed) to remove any unbound material. The washed matrix is then contacted with a label capable of indicating the presence of any antibodies bound to the matrix. A diagnostic system in kit form is provided that contains a package containing a polypeptide of the present invention, preferably attached to a solid matrix and a label for indicating the presence of either an Epstein-Barr virus or an anti-CR2 antibody that binds to the polypeptide. Brief Description of the Drawings

Figure 1 illustrates the circular dichroism spectral analysis of rCR2 performed as described in Example 3.

Figure 2 illustrates the binding of rCR2 polypeptide to gp350/220 (triangles) , C3dg (circles) and ovalbumin (squares) coated onto 96 well plates and determined as described in Example 4. Figure 3 illustrates an ultracentrifugation analysis of rCR2 binding to gp350/220 (Panel A) and C3dg (Panel B) as described in Example 5. In Panel -~_r [¹²⁵I]-gp350/220 was applied to linear sucrose gradients containing no rCR2 (open circles) , 3 nM rCR2 (closed circles) , 30 nM rCR2 (closed squares) , or 100 nM rCR2 (open squares) . In Panel B, [¹⁵I]- rCR2 was applied to linear sucrose gradients containing either 1 uM C3dg (open circles) or no C3dg (closed circles) . Figure 4 illustrates gp350/220 binding to rCR2 as determined in Example 5. In Panel A, The relative amount (r) of rCR2 bound per gp350/220 was plotted versus the concentration of rCR2. The data were derived from two experiments. Panel B shows a Scatchard plot analysis of the same rCR2-gp350/220 binding data. Panel C shows a Hill plot analysis of the same rCR2-gp350/220 data.

Figure 5 illustrates C3dg binding to rCR2 at low ionic strength as determined in Example 5. Panel A shows the results of ultracentrifugation of C3dg and rCR2 admixtures in 5-15% sucrose density gradients with 150 mM NaCl (open circles) , 25 mM NaCl (closed circles) , and 10 mM NaCl (closed squares) , and the insert shows the relative amount (r) of C3dg bound per CR2 plotted against NaCl concentration. Panel B shows a determination of the dissociation constant (K_D) of rCR2 for C3dg by extrapolation from lower salt concentrations. In Panel B the open circles represent the data from Panel A and the closed circles represent the data from a similar study performed using 1 uM C3dg.

Figure 6 illustrates rCR2 inhibition of binding of gp350/220 to B-lymphoblastoid cells as determined in Example 6. Fluorescent microspheres coated with purified gp350/220 were reacted with CR2- negative SF9 cells (Panel A) or with CR2-positive Raji cells (Panels B, C and D) . Gp350/220 coated microspheres were reacted with 2.0 ug (Panel C) or 10 ug (Panel D) of purified rCR2 prior to incubation with Raji cells. Figure 7 illustrates the inhibition of EBV infectivity of human peripheral blood mononuclear cells (B cells) by rCR2 as determined in Example 7. The outgrowth of transformed B cell colonies (closed circles) and stimulation of ³H-thymidine (closed triangles) incorporation were each measured to indicate the extent of EBV infection. In control experiments EBV was incubated with cells prior to the addition of rCR2 (open circles and triangles) .

Figure 8 illustrates the inhibition of EBV infectivity by rCR2 for increasing amounts of EBV present as determined in Example 7. Varying amounts of EBV supernate were incubated with media alone (solid lines) or with 10 ug of purified rCR2 (broken lines) . Samples were then reacted with peripheral blood lymphocytes as described in FIGURE 7 and assessed for B cell transformation by colony outgrowth and stimulation of DNA synthesis.

Figure 9 illustrates the nucleotide sequence of a cDNA that codes for CR2, shown from left to right and in the direction of 5' terminus to 3' terminus using the single letter nucleotide base code. The mature, leaderless, structural gene for CR2 begins at base 123 and ends at base 3320, with the numbers for base residue positions indicated in the right margin.

The amino acid residue sequence for CR2 is indicated by the single letter code above the nucleotide base sequence, with the numbers for each residue position indicated in the left margin. The reading frame is indicated by placement of the deduced amino acid residue sequence above the nucleotide sequence such that the single letter that represents each amino acid is located above the middle base in the corresponding codon. Figure 10 illustrates in schematic form the construction and resulting structure of the CR2 polypeptide expression vectors for producing rCR2-4, rCR2 and rCR2-10, where the vectors are labeled as pAC373 CR2(l-4), pAC373 CR2(1-16) and pAC373 CR2(1- 10) , respectively. The expression vectors manipulated and produced by the construction process are indicated in the figure by the circles. The construction proceeds by a series of steps as indicated by the arrows connecting the circles and the intermediate nucleic acid fragments in the figure, and the steps are described in detail in Example 1. Landmark and utilized restriction enzyme recognition sites are indicated on the circles and fragments by words or by labeled lines intersecting the circles. Individual genes and their direction of transcription are indicated by the arrows in the circles.

Detailed Description of the Invention A. CR2 Polypeptides Substantially purified polypeptides that correspond in amino acid residue sequence to regions of the extracellular domain of CR2 have been synthesized in the present invention, and are designated herein as CR2 polypeptides. A CR2 polypeptide of the present invention includes an amino acid residue sequence that corresponds to the amino acid residue sequence shown in Figure 9 from residue 1 to residue 70.

In preferred embodiments a CR2 polypeptide includes an amino acid residue sequence that corresponds to the amino acid residue sequence shown in Figure 9 from residue 1 to residue 133, from residue 1 to residue 256, from residue 1 to residue 632, or from residue 1 to residue 1005. Exemplary of two of the preferred embodiments are the CR2 polypeptides rCR2 and rCR2-4 that are described in Example 1.

In one embodiment, the CR2 polypeptide can be produced as a fusion protein wherein one portion of the polypeptide has a sequence that corresponds to CR2 and another portion corresponds to the amino acid sequence of a protein other than CR2. Representative of this embodiment is the CR2 polypeptide rCR2-10 which includes as its σarboxy terminus a portion of the polyhedrin gene as described in Example 1.

Compositions and diagnostic kits containing the CR2 polypeptides of the present invention are provided, together with methods for the detection and treatment of EBV infections and immune disorders. All amino acid residues identified herein are in the natural L-conformation. In keeping with standard polypeptide nomenclature, J. Biol. Chem. 243:3557-59 (1969), abbreviations for amino acid residues are as shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

CR2 polypeptides are preferably produced by recombinant expression in the present invention. For example, a baculovirus expression system is used to generate milligram amounts of CR2 polypeptides. In the baculovirus expression system a plasmid containing a cDNA that encodes a portion of the extracellular domain of CR2 was prepared and utilized to produce recombinant CR2 polypeptides as described in Example 1.

In the baculovirus expression system, a recombinant DNA plasmid, containing a cDNA that encodes CR2, is truncated to encode a CR2 polypeptide that comprises a region of the extracellular domain of CR2. The CR2 polypeptide construct was then inserted downstream of an appropriate promoter in a transfer vector and integrated into a baculovirus. The recombinant baculovirus is used to infect host insect cells which then secrete recombinantly produced CR2 polypeptides. Examples of the baculovirus-expression system can be found in Example 1. As used herein, the term "CR2 polypeptide aggregate" refers to a polymer containing at least two CR2 polypeptides corresponding to regions of the extracellular domain of CR2 operatively linked either in tandem or to a polypeptide carrier. As used herein, in reference to polypeptide aggregates, the term "operatively linked" refers to the covalent attachment of a CR2 polypeptide to another CR2 polypeptide or to a peptide, polypeptide or protein carrier. The CR2 polypeptide aggregates of the present invention can contain a plurality of the same or different CR2 polypeptides, wherein the CR2 polypeptides are bound to each other or to a peptide, polypeptide or protein carrier. B. Pharmacological Compositions

Pharmacological compositions of the present invention contain a physiologically tolerable carrier together with a substantially purified CR2 polypeptide, or CR2 polypeptide aggregate, as described above, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the pharmacological composition is not immunogenic when administered to a mammal or human patient.

As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The pharmacological composition of the present invention can include pharmaceutically acceptable salts of the components therein. 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.

Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose and other solutes.

Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.

In a method of treatment, the pharmacological compositions are conventionally administered intravenously, as by injection of a unit dose, for example.

The term "unit dose" and its grammatical equivalents, as used herein, refers to physically discrete units suitable as unitary dosages for human patients and other warm-blooded animals, each unit containing a predetermined effective and potentiating amount of active material calculated to produce the desired therapeutic effect in association with the required physiologically tolerable carrier. The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, and the capacity of the subject to utilize the active ingredient. Precise amounts of active ingredients required to be administered depend on the judgment of the practitioner and are peculiar to each individual, and usually are in a range of about 100 ug to about 100 mg of CR2 polypeptide per ml of patient blood. Alternatively, continuous intravenous infusion sufficient to maintain therapeutically effective concentrations in the blood are contemplated.

C. Methods of Inhibition and Treatment of

Epstein-Barr Virus Infection Epstein-Barr virus infection of mammalian cells in an aqueous medium, such as blood, is inhibited by methods of the present invention.

An aqueous medium containing mammalian cells, such as B lymphocytes, is admixed with a therapeutically effective amount of a pharmacological composition of the present invention and maintained for a time period sufficient to allow the CR2 polypeptides of the composition to specifically bind any Epstein-Barr virus present in the aqueous medium. When the admixture contains a concentration of Epstein-Barr virus that is sufficient to infect the mammalian cells under normal physiological conditions when no pharmacological intervention or treatment is undertaken, the therapeutically effective amount of the CR2 polypeptide-containing composition utilized in this method is that which produces a concentration of CR2 polypeptide in the aqueous medium sufficient to bind essentially all of the EBV present, and usually is at a concentration of about 100 ug to about 1 mg per ml. When the method of treatment of the present invention is utilized to inhibit EBV infection in vivo the therapeutically effective amount of the pharmacological composition administered is that which produces a blood concentration of CR2 polypeptides sufficient to specifically bind the circulating EBV present. Such a blood concentration is usually about 100 ug to about 1 mg of CR2 polypeptide per ml. It is contemplated that multiple administrations of the pharmacological composition of this invention over an appropriate time period and at a dosage level determined by a medical practitioner for the patient will be undertaken for the inhibition of EBV infection in a human patient.

As used herein, the terms "specifically bind", and "specifically attach", and grammatical forms thereof are used interchangeably and refer to non-random ligand binding, such as that which occurs between gp350/220 and CR2.

In the method of treatment of the present invention, a pharmacological composition, as described above, is administered to a patient in any manner that will efficaciously inhibit the infection of mammalian cells, such as B lymphocytes, by EBV. Preferably, the composition is administered by either intravenous injection of a unit dosage or continuous intravenous infusion of a predetermined concentration of CR2 polypeptides or CR2 polypeptide aggregates to a patient.

D. Methods of Treatment for Immune Disorders Methods for the treatment of immunological conditions in a patient are contemplated by the present invention.

In one embodiment, a method for treating an immune disorder characterized by the presence in a patient of circulating antibodies directed against CR2 present on B lymphocytes is provided. In this method CR2 polypeptide or CR2 polypeptide aggregate is administered to the patient in a therapeutically effective amount to effectively bind to the antibodies and thus inhibit or prevent the immunoreaction of the antibodies with B lymphocytes. It is preferred that a pharmacological composition administered in the present invention is not immunogenic.

In another embodiment, a method for treating an immunological disorder or condition in a patient, in which an immunoreaction product containing a C3d or C3dg fragment of complement is present, is provided. In this method, a patient is administered a therapeutically effective amount of a CR2 polypeptide or polypeptide aggregate of the present invention to bind to the C3d or C3dg fragment of complement present in the immunoreaction product, and thus facilitate the alleviation of the symptoms of the disorder. E. Diagnostic Systems and Methods

A diagnostic system in kit form of the present invention includes, in an amount sufficient for at least one assay, a CR2 polypeptide and/or CR2 polypeptide aggregate and/or 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. In one embodiment, a diagnostic system for assaying for the presence of or to quantitate anti- CR2 antibodies in a sample, such as blood, plasma or serum, comprises a package containing at least one CR2 polypeptide of this invention. In another embodiment, a diagnostic system of the present invention for assaying for the presence or amount of EBV comprises a package containing a CR2 polypeptide, CR2 polypeptide aggregate or pharmacological composition of this invention.

In preferred embodiments, a diagnostic system of the present invention further includes a label or indicating means capable of signaling the formation of a specifically bound complex containing a CR2 polypeptide or polypeptide aggregate of the present invention. As used herein, the terms "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. Illustrative labels include radionuclides, such as ¹²⁵I, and fluorescent radicals such as fluorescein isothiocyanate and the like.

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 to Nestor et al., which are incorporated herein by reference.

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 polypeptide aggregate of the present invention. Exemplary specific binding agents are antibody molecules, complement proteins or fragments thereof, protein A and the like. For detecting EBV, the specific binding agent can bind the CR2 polypeptide of this invention when it is present as part of a complex. When detecting patient anti-CR2 antibodies, anti-human Fc antibodies are conveniently used. In preferred embodiments the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent. In these embodiments, 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-CR2 antibodies in a body fluid sample such as blood 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. 4,016,043, which are all incorporated herein by reference. Thus, in preferred embodiments, the CR2 polypeptide, or polypeptide aggregate, 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. Where 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, vials, plastic and plastic- foil laminated envelopes and the like. The present invention contemplates any method that results in detecting EBV or anti-CR2 antibodies in a body fluid sample using CR2 polypeptides or polypeptide aggregates of this invention. Thus, while exemplary methods are described herein, the invention is not so limited.

The presence of anti-CR2 antibodies in an antibody-containing bodily fluid is indicative of an autoimmune disorder in the host animal. To detect either EBV infection or the presence of anti-CR2 antibodies in a patient, a bodily fluid such as blood, plasma or serum from the patient is contacted under biological assay conditions with a CR2 polypeptide or aggregate thereof for a period of time sufficient to form a polypeptide containing complex. The presence of the complex is indicative of infection by EBV or of a CR2 autoimmune disorder, respectively. The complex can be detected as described herein. Biological assay conditions are those that maintain the biological activity of the CR2 polypeptide molecules of this invention and the EBV or anti-CR2 antibodies 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.

F. Expression Constructs In living organisms, the 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. Thus, a nucleotide sequence can be defined in terms of the amino acid residue sequence, i.e., protein or polypeptide, for which it codes. 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 polypeptide sequence of this invention. Representative nucleotide sequences that encode a CR2 polypeptide of the present invention can include nucleotide sequences that correspond to the nucleotide base sequence shown in Figure 9. Thus, a preferred CR2 polypeptide-encoding nucleotide sequence includes a sequence that corresponds to the sequence shown in Figure 9 from nucleotide base 123 to base 332, from base 123 to base 521, from base 123 to base 2018, or from base 123 to base 3137.

A DNA segment of the present invention that encodes an extracellular CR2 amino acid residue sequence and preferably an amino terminal sequence, can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al, J. Am. Chem. Soc.. 103:3185 (1981). Of course. by chemically synthesizing the coding sequence, 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 CR2 extracellular domain can be obtained by digesting B lymphocytes. 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, preferably in a baculovirus such as the Autographa californica nuclear polyhedrosis virus.

As used herein, the term "operatively linked", in reference to cDNA integration, describes 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. As used herein, 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. Vectors capable of directing the expression of genes encoding CR2 amino acid residue sequences are referred to herein as "expression vectors". Thus, a recombinant DNA molecule (rDNA) is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature.

The choice of vector to which a 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 can be used to induce an immune response to an expressed foreign polypeptide included in the vector. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are vaccinia virus vectors and baculovirus vectors.

A variety of methods have 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 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. Thus, 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. Also contemplated by the present invention are RNA equivalents of the above described recombinant DNA molecules. The CR2 polypeptides are thus readily synthesized and expressed by an appropriate host cell such as the Spodoptera frugiperda (SF9) insect cells. Examples

The following examples are given for illustrative purposes only and do not in any way limit the scope of the invention.

1. Preparation of CR2 and CR2 Polypeptides a. Isolation and Purification of CR2

CR2 was isolated from detergent lysates of Raji B lymphoblastoid cells by immunoaffinity chromatography.

Raji cells (2 x 10¹⁰) were washed twice in cold (about 4 C) phosphate buffered saline (PBS, pH 7.4) and suspended in 400 ml of 10 mM phosphate buffer (pH 8.0) containing 3.3% Brij 96 (Sigma Chemical Co. , St. Louis, MO) , 3 mM sodium azide, 50 mM iodoacetamide, 5 mM phenylmethylsulfonyl fluoride, 5 mM EDTA and 0.2 trypsin inhibitory units of Aprotinin/ml (Sigma Chemical Co.).

The suspension was thoroughly mixed at 4 C for 30 min followed by high speed centrifugation to remove the nuclei and cell debris. Sodium deoxycholate (DOC) in 10 mM Tris hydrochloride (pH 8.0) was added to the supernatant to obtain a final concentration of 1%. This mixture was stirred at 4 C for 60 minutes, and then maintained, with constant mixing, for 18 hours with 2 ml of packed fixed Staphylococcus aureus (Pansorbin, Calbiochem-Behring, La Jolla, CA) . The mixture was centrifuged and the supernatant was saved to be applied to an immunomatrix column having anti-CR2 monoclonal antibody HB-5 attached (HB-5 immunomatrix column) . The immunomatrix was batch-washed four times with 200 ml of 10 mM Tris hydrochloride (pH 8.0) containing 0.5% DOC and four times with 200 ml of 10 mM Tris hydrochloride (pH 8.0) containing 150 mM NaCl and 0.5% DOC. The immunomatrix was poured into a column.

The CR2-containing supernatant was applied to the HB-5 immunomatrix column and eluted with 7 to 10 ml of 50 mM diethylamine (pH 11.5) (Kodak, Rochester, NY) containing 0.5% DOC. The pH of the CR2-containing eluate was adjusted to pH 8.0 by the addition of solid glycine. The protein was rechromatographed on the immunomatrix column to remove minor contaminants.

Purification of CR2 was monitored by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions, and the gels were silver stained.

The protein concentration of CR2 was estimated by comparing densitometric scans of CR2 with known amounts of standard markers (Bio-Rad

Laboratories, Richmond, CA) run in adjacent lanes on the gels.

An ELISA was used to monitor purified CR2. Various amounts of the purified CR2 (0.25 to 4 ng) in 100 uL aliquots in 0.1 M bicarbonate buffer (pH 9.5) were coated onto 96 well microtiter plates (Immulon II, Dynatech Laboratories Inc., Alexandria, VA) for 18 hours at 4 C. The wells were then blocked with 0.5% nonfat dry milk in PBS (pH 7.4) containing 0.05% antifoam A (Sigma Chemical Co.) as blotto buffer for 1 hour at 37 C. Anti-CR2 antibodies (1 ug) in 100 ul of blotto buffer were added to the wells and maintained for one hour at 22 C.

The wells were then washed three times with blotto buffer, 100 ul of a 1:500 dilution of biotinlyated anti-mouse immunoglobulin (Vector

Laboratories, Burlingame, CA) in blotto buffer was added, and the plates incubated at 22 C for 1 hour. The wells were again washed and then incubated for 30 minutes at 22 C with 100 ul of a 1:250 dilution of avidin-glucose oxidase (Vector Laboratories) . The plates were washed and the reaction was developed by the addition of chromogenic substrate (140 ug/ml 2,2'-azino-bis-3-ethylbenzthiazoline sulfonate, 7 ug/ml peroxidase, 19 mg/ml glucose in 0.1 M phosphage buffer (pH 6.0)). The reaction was read at 405 nm wavelength in a Titertak Multiskan MC ELISA plate reader (Flow Laboratories, Inc., McLean, VA) . b. Isolation of cDNA for Expression of CR2 Polypeptides A cDNA clone of about 4.2 kilobases

(Kb) , representing the entire CR2 coding sequence was prepared as described by Moore et al. , Proc. Natl. Acad. Sci. USA. 84:9194-98, (1987). A cDNA library was first constructed in lambda gtll (Stratagene, San Diego, CA) using mRNA isolated from Raji B lymphoblastoid cells. Complementary DNA was synthesized with a commercially available synthesis kit (cDNA synthesis system, Amersham, Arlington Heights, IL) . Packaging of recombinant cDNA clones with phage coat proteins was done as recommended by the manufacturer (Gigapack, Stratagene) . The library contains 2.5 x 10⁶ recombinants.

Candidate CR2 cDNA clones were isolated from 85 mm plate lysates using affinity chromatography with anti-lambda phage antibody (LambdaSorb, Promega Biotec, Madison, WI) . Large scale preparation of lambda phage was carried out, digested with EcoR I and the cDNA inserts were isolated by preparative gel electrophoresis. Approximately 5 x 10⁵ recombinant lambda phage were screened with a CR2 oligonucleotide probe in order to isolate a clone containing a 1.2 kilobase insert encoding CR2. The CR2 cDNA insert isolated from the clone was nick-translated and used to reprobe the Raji library to obtain additional cDNA inserts encoding CR2.

The isolated CR2 inserts were ligated into the EcoRI site of plasmid Bluescript (Stratagene) , designated plasmid Bluescript/CR2. c. Construction of CR2/Baculovirus

Expression Vectors and Production of CR2 Polypeptides The preparation of CR2 polypeptide expression vectors is described hereinbelow and is represented schematically in Figure 10. rCR2: CR2 polypeptide rCR2 has an amino acid residue sequence that corresponds to the sequence shown in Figure 9 from residue 1 to residue 1005. rCR2 is expressed by the baculovirus expression vector pAC373 CR2(1-16) shown in the middle portion of Figure 10. For the preparation of pAC373 CR2(1- 16) plasmid Bluescript/CR2 (pBS/CR2) was digested with Bgl 1 and Pst 1 to release the cDNA insert encoding the entire extracellular domain of CR2. Following subcloning of the CR2 fragment into pBS, a synthetic oligonucleotide BamH I linker (5¹- dCGGATCCG-3• ; Pharmacia/LKB Biotechnology, La Jolla, California) was ligated to the 5' end of the CR2 insert that had been previously digested with Bgll and blunt-ended with T4 DNA polymerase. The construct was further modified by two successive oligonucleotide-based site-directed mutagenesis reactions according to the method of Kunkel, Proc. Natl. Acad. Sci.. USA. 82:488-492 (1985) . The first mutagenesis step involved ablation of the BamH I site at nucleotide position No. 2017 (using the nucleotide base pair numbering system shown in Figure 9) by substitution of a single nucleotide (GGATCC to GGACCC) that preserved the amino acid coding sequence of CR2. Verification of the mutation was carried out by sequence analysis and restriction mapping.

The second mutagenesis step was then carried out by incorporating a termination codon and BamH I site (TGAGGATCC) following nucleotide position No 3137, immediately preceding the transmembrane domain of CR2.

Subsequently, the truncated CR2 fragment encoding the extracellular domain of CR2 was inserted into the BamH I cloning site of pAC373. Recombinant plasmids containing a single copy of truncated CR2 in the correct orientation was identified by restriction mapping. Transfer of the rCR2 sequence from the plasmid to the Auto rapha californica nuclear polyhedrosis virus (AcNPV) genome was carried out by cotransfection of Spodoptera fruigiperda (SF9) cells with 10 ug of rCR2 plasmid and 1 ug of AcNPV DNA using calcium phosphate. Recombinant viruses were selected and characterized.

The transfected SF9 cells were cultured and the cell supernatant was subjected to immunoprecipitation with anti-CR2 monoclonal antibodies (HB5) attached to a resin. The resin was washed and the rCR2 was eluted with 50 mM diethylamine (pH 11.5) containing 0.5% sodium deoxycholate, a mild chaotropic agent, to produce purified rCR2 which was greater than 90% homogenous. rCR2-4: CR2 polypeptide rCR2-4 has an amino acid residue sequence that corresponds to the sequence shown in Figure 9 from residue 1 to residue 756. rCR2-4 is expressed by the baculovirus expression vector pAC373 CR2(l-4) shown in the left portion of Figure 10. The preparation of pAC373 CR2(l-4) differed from the preparation of pAC373 CR2(1-16) in that the first mutagenesis was omitted, and the second mutagenesis step was conducted so as to incorporate a termination codon and BamH I site (TGAGGATCC) following nucleotide position No. 887. Subsequently the mutagenized and truncated CR2 polypeptide encoding fragment was inserted into the BamH I cloning site of pAC373 as shown for pAC373 CR2(1-16), the vector was manipulated as before to produce a recombinant virus. The virus was introduced into SF9 cells as before, and the expressed rCR2-4 protein was harvested from the transfected SF9 cell culture to and purified as before to produce purified rCR2-4 which was greater than 90% homogenous. rCR2-10: CR2 polypeptide rCR2-10 is a fusion protein that includes at its carboxy terminus a portion of the polyhedrin protein, and has at its amino terminus an amino acid residue sequence that corresponds to the sequence shown in Figure 9 from residue 1 to residue 632. rCR2-10 is expressed by the baculovirus expression vector pAC373 CR2(1-10) shown in the right portion of Figure 10. The preparation of pAC373 CR2(1-16) in that no mutagenesis was conducted. Rather, the BamH I linker modified plasmid pBS/CR2 was digested with BamH I and inserted into the BamH I cloning site of pAC373 that contained the polyhedrin structural gene and promoter. Thus, the polyhedrin gene was fused to the carboxyl terminus of the CR2 polypeptide coding gene. The resulting vector pAC373 CR2(1-10) was manipulated as before to produce recombinant virus. The virus was transfected into SF9 cells and rCR2-10 protein was expressed therefrom, harvested and purified as before.

2. Preparation of gp350/220 and C3dg

The Epstein-Barr virus membrane glycoprotein gp350/220 was isolated from GH3 19 rat pituitary tumor cells transfected with gp350/220 cDNA as described by Whang et al. , J. Virol. 61:1795-1807

(1987) and purified by immunoaffinity chromatography using anti-gp350/220 monoclonal antibodies.

The C3dg fragment of the third component of complement was purified from Mg⁺²EGTA-activated aged human serum as described by Vik et al., J. Immunol. , 138:254-258 (1987).

3. Physical Characterization of rCR2 Soluble recombinant CR2 polypeptide (rCR2) corresponding to the extracellular region of CR2 was produced and purified as described in Example 1. The molecular weight of rCR2 was determined by SDS gel electrophoresis to be 125 kDa. This value is higher than the molecular weight of 110 kDa predicted from the cDNA sequence for rCR2. The difference is presumed to be due to post-translational modification, such as glycosylation, of the soluble rCR2.

Soluble rCR2 (2 milliliters (ml) ) was applied to a 2.6 x 95 cm column of Sephacryl S-300 (Pharmacia, Piscataway, NJ) that had been equilibrated with 300 mM NaCl, 20 mM imidazole-HCl pH 7.3 at 4 C. Fractions (2.5 ml each) were collected at a flow rate of 17 ml/hour. [¹²⁵I]-labelled proteins were detected by gamma counting in a Micromedic Systems 4/600 gamma counter, and unlabelled protein in the fractions were detected by absorbance at 280 nm. The Sephacryl S-300 column was calibrated with the following standards, where the diffusion coefficient (D_20>w) is listed in parenthesis: thyroglobulin (2.54 x 10^"7cm²/s) , Factor H (2.54 x 10^"7cm²/s) , C3 (4.53 x 10^"7cm²/s) , bovine serum albumin (5.94 x 10^"7cm²/s) , and myoglobulin (1.13 xl0^"6cm²/s) . The partition coefficients were determined as Kd = (V_e - V₀)/ (V_t - V₀) , where V_e is the elution volume of the sample, V₀ is the void volume for Blue Dextan and V_t is the included volume plus the void volume.

The diffusion coefficient (D₂₀.„) of rCR2 was determined to be 3.1 x 10^"7cm²/s.

The Stokes radius for rCR2 was calculated from the D₂₀.„ to be 69.3 Angstrom, and the frictional coefficient (f/fo) was determined as 2.1.

Soluble rCR2 (100 ul) was centrifuged through a 5-16% linear sucrose gradient (4 ml) prepared in phosphate-buffered saline (PBS) at 45,000 rpm in an SW-60 rotor (Beckman Instruments, Palo Alto, CA) for 18-24 hours at 4 C. Gradient fractions (160 ul) were collected and the sedimentation constant (s_20w) for rCR2 was determined to be 4.5S.

The values of f/fo and S_20w obtained for rCR2 are consistent with it being an extended molecule.

Circular dichroism (CD) spectral analysis was carried out in an Aviv CD spectropolarimeter model 61 DS, standardized with d-10-camphosulfonic acid. A sample of rCR2 (0.12 mg/ml) in 20 mM Tris HCl, pH 8.2 was scanned for CD between 187 and 260 nm in a 0.1 cm path length cuvette. The CD spectrum for rCR2 (Figure 1) is unlike that of most globular proteins because it is dominated by a maximum positive ellipticity at 229 nm and a maximum negative ellipticity between 200 and 205 nm. The CD spectrum for rCR2 also indicates the absence of alpha-helix secondary structure. This spectrum is similar to that for the complement regulatory protein, factor H, that also contains repeating regions.

Transmission electron microscopic analysis of negatively stained samples of rCR2 were prepared by the "pleated sheet" technique using 2% uranyl acetate according to the method described in Smith et al. , J. Ultrastructure Res. 89:111-122 (1983). The samples were examined in Hitachi 12A and Hitachi 600 transmission electron microscopes, calibrated using a ' diffraction grating for low magnification (2-48,000 x) and a catalase crystal for high magnifications (28,000 - 200,000 x) with an objective aperature of 50 urn and accelerated voltage of 75 kilovolts. The anchorless form of rCR2 was determined to be an elongated flexible molecule with an estimated contour length of 386*35 angstroms. When rCR2 was imaged at a high primary magnification of 150,000x, the ultrastructure of the polypeptide chain was discernable to be composed of a chain of ringlets, each of which presumably represents a single repeating region. Since there are 16 tandem repeating regions in the rCR2 used in this study, each region is calculated to be 24.1*2 angstroms. TABLE 1 summarizes the physical data obtained. TABLE 1 Molecular Weight Apoprotein, predicted from sequence Calculated from D_20jW and S_20w SDS gel electrophoresis

Partial specific volume, v (cm³/g) Diffusion coefficient,D₂₀ „(cm²/sec) Strokes radius (angstroms) S₂₀,v(S)^b Frictional ratio, f/f₀

"Assuming the difference in molecular weight between the apoprotein and the mature glycoprotein is entirely due to carbohydrate, and without knowing the carbohydrate composition a range was calculated for the most and least dense sugars for rCR2. The values for both ends of the range were the same for CR2 to two significant digits. Values given are average of six runs. 4. Binding of CR2 Polypeptide to gp350/220 or C3dg

Purified gp350/220 and C3dg obtained as described in Example 1, and ovalbumin were independently coated and immobilized onto separate wells of 96 well plates. Aliquots of rCR2 prepared in Example 1 were then added to each well at varied concentrations, maintained for 2 hrs to allow binding of rCR2 to the immobilized ligand, followed by rinsing to remove any unbound rCR2. The presence of bound rCR2 was determined by the CR2-specific ELISA described in Example la. The results are illustrated in Figure 2. The rCR2 bound to both gp350/220 and C3dg in a dose-dependent manner, while only background binding was observed with ovalbumin. Similarly, CR2 polypeptides rCR2-4 and rCR2-

10, prepared in Example 1, were analyzed by ELISA for their ability to bind ligand. rCR2-4 and rCR2-10 both exhibited binding to immobilized ligands comprised of EBV virus particles purified gp350/220 or complement C3dg. Therefore the exemplary CR2 polypeptides rCR2, rCR2-4 and rCR2-10 were shown to have the capacity to specifically bind to the target ligands for CR2.

5. Ligand Binding to rCR2 The binding of rCR2 to ligands was measured by mobility shift of the receptor in sucrose density gradients in which rCR2 was distributed throughout the gradient prior to centrifugation. The apparent increase in sedimentation rate of rCR2- ligand complexes was measured, and the fractional occupancy of the binding sites was calculated from the relative increase in sedimentation rate of the complex as a function of increasing rCR2. The sedimentation rate of the complex was calculated as described by Ziccardi et al., J. Biol. Chem.

259:13674-13679 (1984) as S_c = S₀(M_C/M₀)°^*68 where S_c is the S_20jW of the complex, M_c is the molecular weight of the complex, S₀ is the S_20jW of the rCR2 and M₀ is the molecular weight of rCR2. A value r represents the increase in S_20w normalized to the S_20w of a 1:1 complex of rCR2 to ligand where r=(S_app-S₀) /(S_c-S₀) where S_app is the measured value for a given sample and is equivalent to the number of ligands bound per rCR2. For the binding of gp350/220 to rCR2, gp350/220 was centrifuged through a sucrose gradient in which rCR2 was evenly distributed throughout. Saturable binding of gp350/220 to rCR2 was exhibited at physiological pH and ionic strength that is univalent, forming a 1:1 complex with rCR2. Results are shown in Figures 3A, 4A, 4B and 4C. A Scatchard plot of the binding data (Figure 4B) indicated a single high affinity (K_D = 3.2 x 10^"9M) binding site on gp350/220 for rCR2. Linear regression analysis of the data gives a slope of 0.311, and a linear correlation coefficient of -0.95. A further examination of the data by a Hill plot (Figure 4C) revealed a binding slope of 0.75 with a linear correlation coefficient of 0.98, indicating that multiple binding sites involved in cooperative rCR2- gp350/220 binding are not present.

For the binding of C3dg to rCR2, C3dg prepared as described in Example 1 was used in a sucrose gradient, as described above. Under conditions of physiologic pH and ionic strength, no binding of monomeric C3dg to rCR2 was detected (Figure 3B) .

In order to maximize the potential binding of the low affinity interaction of C3dg with rCR2, studies were carried out at reduced ionic strength using two concentrations of C3dg (1 or 2 uM) , as illustrated in Figure 5.

Ultracentrifugation of rCR2 in 5-15% sucrose gradients was carried out with 2 uM [¹²⁵I-]-C3dg in 10, 25 and 150 mM NaCl, respectively. Unlabeled CR2 (50mg) was detected in gradient factors by an ELISA.

A dose-dependent increase in C3dg binding to rCR2 was observed with decreasing ionic strength (Figure 5A) . The binding data obtained indicated that the C3dg intersection with rCR2 is univalent. A dissociation constant of 2.75 x 10^"5 M for rCR2 binding to C3dg at physiologic ionic strength and pH was obtained by extrapolation of the binding data (Figure 5B) . K_D was calculated for each of the binding experiments from the fractional saturation (1-r/r) : K_D = [CR2] x ((1 - r)/r). The negative log of K_D was plotted against the salt concentration and 36 used to extrapolate K_D for 150 mM NaCl, indicated by the arrow. Least squares analysis was used to determine the best fit of the data to a line, from this the predicted value for K_D at 150 mM NaCl is 27.5 uM. The linear correlation coefficient for this line is 0.91. The slight increase of sedimentation rates with 2 uM C3dg in 10 mM NaCl indicates that small amounts of C3dg aggregation may have occurred.

6. Inhibition of gp350/220 Binding to Cells Fluorescent microspheres coated with purified gp350/220 were preincubated with rCR2 purified as described in Example 1, or with buffer alone prior to addition to cells to determine the ability of rCR2 to inhibit the binding of gp350/220 to the cells. The binding of gp350/220 was determined by fluorescent activated cell sorting (FACS) . The results of the FACS analysis are illustrated in Figure 6. The gp350/220 coated microspheres that were preincubated in buffer alone were reacted with CR2 negative SF9 cells (Panel A) and CR2 positive Raji cells (Panel B) . Micropheres preincubated with 2 ug rCR2 (Panel C) or 10 ug rCR2 (Panel D) were reacted with Raji cells, and show that rCR2 produced a dose-dependent inhibition of gp350/220 fluorescent microsphere binding to Raji cells, while no inhibition was observed with microspheres incubated with buffer alone.

7. Inhibition of Epstein-Barr Virus Infection The ability of rCR2 to alter EBV infection of peripheral blood B cells was measured by outgrowth of transformed B cells and by virus-induced stimulation of DNA synthesis.

EBV was preincubated with varying amounts of purified rCR2 prior to addition of unseparated peripheral blood mononuclear cells (6 x 10⁵ cells) . The mixture of cells with EBV and rCR2 were maintained for 1 hour at 4 C, washed and the cells (2 x 10⁵/plate, in triplicate) were then cultured for 14 to 21 days in complete RPMI medium with 0.1 ug/ml cyclosporine A and then assessed for transformation. As illustrated in Figure 7, a preincubation of EBV with rCR2 resulted in dose-dependent inhibition of EBV infection as measured by both outgrowth of transformed colonies and by ³H-thymidine incorporation. The ability of rCR2 to block infection was not due to toxic effects of rCR2 upon the cells because the highest dose of rCR2 used (7.5 ug) did not abolish infection of B cells if EBV was allowed to bind to B cells prior to exposure to rCR2. When varying amounts of EBV were preincubated with a constant amount of CR2 (10 ug) prior to addition of B cells, EBV induction of B cells transformation was reduced by nearly 90% as illustrated in Figure 8. These results show that rCR2 blocks EBV infection in vitro and indicates that CR2 is the primary EBV receptor on B cells.

The foregoing specification, including the specific embodiments and EXAMPLES, is intended to be illustrative of the present invention and is not to be taken as limiting. Numerous other variations and modifications can be effected without departing from the true spirit and scope of the present invention.

Claims

What Is Claimed Is:

1. A method of inhibiting Epstein-Barr virus infection of mammalian cells in contact with an aqueous medium, which method comprises: a) admixing said aqueous medium with a therapeutically effective amount of a CR2 polypeptide consisting of an amino acid residue sequence that corresponds to the sequence shown in Figure 9 from residue 1 to residue 256.

2. The method of claim 1, wherein said aqueous medium comprises mammalian blood.

3. The method of claim 1 wherein said polypeptide is synthesized by recombinant expression.

4. The method of claim 2, wherein said polypeptide is admixed with said blood by injection of said polypeptide into a human patient.

5. A method of inhibiting the infection of human B lymphocytes by Epstein-Barr virus, which method comprises injecting into a patient a therapeutically effective amount of a CR2 polypeptide consisting of an amino acid residue sequence corresponding to the sequence shown in Figure 9 from residue 1 to residue 256.

6. A CR2 polypeptide consisting of an amino acid residue sequence corresponding to the amino acid residue sequence shown in Figure 9 from residue 1 to residue 256, from residue 1 to residue 632, or from residue 1 to residue 1005.

7. A pharmacological composition comprising a physiologically tolerable carrier together with a

CR2 polypeptide as an active ingredient dissolved or dispersed therein, said polypeptide consisting of the polypeptide of claim 6.

8. The composition of claim 7, wherein said polypeptide is in aggregate form.

9. A substantially purified CR2 polypeptide aggregate comprising at least two CR2 polypeptides operatively linked, said CR2 polypeptides each comprising a polypeptide of claim 9.

10. The polypeptide aggregate of claim 9, wherein said polypeptides are synthesized by recombinant expression.

11. The polypeptide aggregate of claim 10, wherein said recombinant expression is in a baculovirus expression system.

12. A diagnostic system in kit form for detecting the presence of Epstein-Barr virus in an aqueous sample, comprising: a) a package containing a polypeptide according to claim 6; and b) a label for indicating the presence of any Epstein-Barr virus that specifically binds to said polypeptide.

13. The diagnostic system of claim 12, wherein said polypeptide is attached to a solid matrix.

14. A method of detecting the presence of Epstein-Barr virus in a fluid sample, comprising a) contacting said fluid sample with a polypeptide according to claim 6 for a time period sufficient to allow any Epstein-Barr virus present in said sample to specifically bind to said polypeptide to form a complex; and b) determining the presence of any complex formed in step a) , thereby detecting the presence of Epstein-Barr virus in said sample.

15. A diagnostic system in kit form for detecting antibodies directed against CR2, comprising a) a package containing a polypeptide according to claim 6; and b) a label for indicating the presence of any specifically bound antibody.

16. A method of detecting the presence of antibody molecules against CR2 in a body fluid sample, which method comprises: a) contacting said fluid sample with a polypeptide according to claim 6 for a time period sufficient to allow any antibody molecules directed against CR2 to specifically bind to said polypeptide to form a complex; and b) determining the presence of any complex formed in step a) , and thereby the presence of said antibody molecules in said sample.