This invention was made with government support under an award by the Departmenc of Veteran Affairs. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention The invention relates to the field of immunology and, more particularly, to human monoclonal antibodies specific for parvovirus.

Description of the Background Art B19 parvovirus is the causative agent of such human illnesses as erythemia infectiosum (fifth disease, Anderson, .J. et al. ,

Lancet, ii:695 (1983)), aplastic crisis in sickle cell anemia

(Sergeant, et al.. Lancet, ii:695 (1981)), other chronic anemias

(Young, Semin He atol . 25:159 (1988)) and acute symmetrical peripheral polyarthropathy (Woolf, Behring Inst. Mitt. 85:64 (1990)). In addition it may result in fetal hydrops and some neuropathies, vasculitides and myocarditides (Torok, T.J. Ann. Intern . Med. 37:431 (1992)).

The neutralizing activity of human polyclonal antibodies (Abs) to B19 (anti-B19) has been demonstrated in vitro (Sato, et al., J. Virol . 65:5485 (1991)) and successful treatment of patients suffering from acute persistent B19 parvovirus infections with commercial immunoglobulins containing high titre of anti-B19 has been reported (Schwartz, et al., J. Infect . Dis. 162:1214 (1990); Takahashi, et al. , Am. J. Hemat. 37:68 (1991)). The use of polyclonal anti-B19 in patients, however, sometimes leads to complications due to the formation of antigen-Ab complexes as a consequence of the infusion of specific Abs into patients with extreme viremia (Kurtzman, et al., J. Clin. Invest. 84:1114 (1989); Frickhofen, et ai., Ann . Intern. Med. 113:926 (1990)). The use of monoclonal antibodies (mAbs) in this context may possibly avert this complication.

Until now, only mouse mAbs to B19 parvovirus have been described (Yaegό3hi, et al., Microbiol . Immunol . 13:561 (1989); Sato, et al., J. Virol . 65:1667 (1991); Yoshimoto, et al. , J.

Virol . 65:7056 (1991)). Production of human mAbs required the identification of patients with circulating B cells producing specific anti-B19. Antibody-producing cells are only present for limited periods of time after antigenic stimulation (Lane, et al. , J. Exp. Med. 154:1043 (1981)). Such cells would not normally be demonstrable after the convalescent period of an infection unless chronic infection exists and continually stimulates the immune system. Thus, despite the wide seroprevalence of anti-B19 in the normal population, circulating B cells producing anti-B19 have not been found, in individuals without recent or chronic B19 infection, in sufficient quantities to allow culturing of such cells to provide a source of recoverable and/or commercially useful amounts of an anti-B19 parvovirus antibody.

Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior arc. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

SUMMARY OF THE INVENTION It is an object of the present invention to overcome one or more of the deficiencies of the related art.

The present invention also provides diagnostic and therapeutic methods for diagnosing and/or treating human parvoviral infections in humans, such as B19 parvoviral infections, including, but not limited to erythemia infectiosum (fifth disease) , aplastic crisis in sickle cell anemia, other chronic anemias, acute symmetrical peripheral polyarthropathy, fetal hydrops, neuropathies, vasculitides and myocarditides, using anti-human parvoviral monoclonal antibodies, and derivatives or fragments thereof.

The present invention also provides methods for producing human mAbs against human parvovirus, and cell lines, such as hybridomas and/or heterohybridomas, which produce anti-human parvoviral human monoclonal antibodies in commercially useful amounts, which antibodies, or fragments thereof, can be used in methods of the present invention.

Other objects of the invention will be apparent to skilled practitioners from the following detailed description and examples relating to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a representation of radioimmunoprecipitation of parvovirus B19 intact capsids and denatured polypeptides with human monoclonal antibody 712-55-D. Radiolabeled intact capsids containing VP1 and VP2 (VP1 + 2 capsids; track 1-4), or VP2 alone (VP2 capsids; tracks 5-8) , or denatured unassembled polypeptides

( [VP1 + 2 (SDS) ] were prepared as described in the Examples below.

Aliquots were immunoprecipitated with pre-immune rabbit serum

(tracks 1, 5, and 9) , hyperim unized rabbit serum (tracks 2, 6 and

10), an unrelated human monoclonal antibody (tracks 3, 7 and 11), and human monoclonal antibody 712-55-D (tracks 4, 8 and 12) . The immunoprecipitated, radiolabeled proteins were resolved by electrophoresis in SDS-containing 10% polyacrylrmide gel and were detected by fluorography. The positions of VPl and VP2 proteins are indicated at the left. Figure 2 is a graphical representation of neutralization of the parvovirus B19 infection by human anti-B19 monoclonal antibodies. The "rescue" of CFU-E from human bone marrow from B19 infection by mAb 712-55D and/or mAb 814-55D is shown on the ordinate as a function of mAb concentration on the abscissa.

DESCRIPTION OF THE PREFERRED EMBODIMENTS It has now been discovered that human monoclonal antibodies specific for human parvovirus, such as B19 parvovirus, can be obtained from human patients, such as HIV infected humans, with circulating B cells in sufficient quantities to isolate and culture such cells to produce human cell lines or human-human or human mouse hybridomas for commercial useful proc'uction of human monoclonal antibodies specific for human parvovirus, such as B19 parvovirus, in recoverable amounts. Since such parvovirus specific B cells are in insufficient quantities to provide isolated and/or cultured B cells producing anti-human parvovirus, human mAbs, found circulating in humans, the present invention provides a method for obtaining commercially useful amounts of monoclonal antibodies specific for human

parvovirus, such as B19 parvovirus, which human monoclonal antiboides can be used for therapeutic or diagnostic purposes.

During the study of immune responses of individuals infected with the human immunodeficiency virus (HIV) , it was unexpectedly discovered that the prevalence of anti-human parvovirus, such as anti-B19 parvovirus antibodies, was increased despite the absence of evidence of current or chronic human parvovirus infection. Subsequent experiments demonstrated the presence of anti-human parvovirus producing B cells in the blood of these individuals, such as anti-B19. Immortalization of these cells led to the establishment of cell lines producing human mAbs to human parvovirus, the characteristics of which are described herein.

The present invention is also directed to an antibody which binds an epitope specific for a human parvovirus, such as B19 parvovirus, of the present invention and the use of such an antibody as a therapeutic agent for the treatment of a human parvovirus and/or to detect the presence of, or measure the quanti¬ ty or concentration of (including use as a positive control) , a human parvovirus in a cell, a cell or tissue extract, a biological fluid (e.g., blood, plasma, lymph, urine, or CNS) , an extract thereof, a solution, or sample, in vitro, in situ, or in vivo.

The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs) , human-human chimeric (hereinafter "human chimeric") antibodies, and anti-idiotypic (anti-Id) antibodies to antibodies specific for a human parvovirus, such as B19 parvovirus, as well as fragments and/or derivatives thereof.

The present invention thus provides antibodies that are specific for, and capable of binding to, immunogenic human parvovirus antigens, such as B19 parvovirus antigen, which proteins may be found in nature. Such anti-human parovirus mAbs are useful for diagnostic and therapeutic purposes in subjects having, developing parvovirus infections, including, but not limited to, erythemia infectiosum (fifth disease) , aplastic crisis in sickle cell anemia, other chronic anemias, acute symmetrical peripheral polyarthropathy, fetal hydrops, neuropathies, vasculitides and myocarditides.

The present invention provides not only human, human-human and human-mouse mAbs, but also human chimeric antibodies which are constructed from human V regions derived from the mAbs of the

present invention and human constant or C regions from alternative subclasses of Ig's to provide anti-human parovirus, human mAbs having alternative Ig subclasses, as described herein. Thus, human parvovirus human chimeric antibodies maintain the ability to recognize the same parvovirus epitopes as the mAbs.

The term "epitope" in the context of the present invention refers to that portion of any molecule capable of being recognized by, and/or bound by, an antibody. In general, epitopes consist of chemically active surface groupings of molecules, for example, amino acids, lipid or sugar side chains, or any combination thereof, which provide specific three dimensional structural characteristics, as well as specific charge characteristics, unique to the particular antigen, such as a human parvoviral antigen, preferably a B19 parvoviral antigen. Preferred parvoviral epitopes of the present invention are epitopes comprising amino acids corresponding to native parvoviral antigens.

An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal, preferably a human, to produce antibody capable of binding to an epitope of that antigen. An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will bind, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens. A "hapten" is a molecule having a lower molecular weight than an antigen, which can react specifically with an Ab, but which is unable to induce Ab formation unless attached to another molecule, usual?.-"- a protein, such as KLH. Preferred hapten are peptide fragments of B19 parvovirus, such as VP1 and/or VP2 capsids which can be used to generate anti-peptide antibodies to human parvovirus according to known method steps. See, e.g. Ausubel et al, eds. , CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Wiley Interscience, N.Y., (1987, 1992); and Harlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory (1988) , the contents of which references are incorporated entirely herein by reference.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.

A monoclonal antibody contains a substantially homogeneous population of antibodies specific to a particular antigen, which population contains substantially similar epitope binding sites. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent No. 4,376,110. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The term "antibody" is also meant to include both intact immunoglobulin molecules as well as fragments and derivatives thereof, such as, for example, Fab, Fab', F(ab') ₂ and Fv, which are capable of binding antigen. These fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl . Med. 24;316-325 (1983)). These fragments are produced from intact antibodies using methods well known in the art, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') ₂ fragments). See Harlow and Lane, supra; and Ausubel et al, vipra . A hybridoma producing an anti-human parvovirus mAb of the present invention may be cultivated in vi tro, in si tu or in vivo. Production of high titers of mAbs in vivo or in situ makes mAbs the presently preferred method of production. Preferably, immortalization of human B cells, producing human anti-B19 mAbs, with Epstein-Barr virus and generation of heterohybridomas by fusion with a mouse x human heteromyeloma may be used to generate heterohybridoma cell lines producing IgG, such as IgG ₃ or IgG _! K , or other Ig subclass monoclonal antibodies (mAbs) . Such mAbs are found to be specific for conformational epitopes on the VP 1 and/or VP 2 capsid protein of B19 parvovirus and are capable of neutralizing at least 50%, such as 55, 60, 65, 70, 75, 80 or 90%, of the virus input, such as the non-limiting example of neutralization of virus input at 0.9 and 0.6 μg mAb/ml in a human erythroid colony forming assay. A "derivative" of an antibody of the present invention contains additional chemical moieties not normally a part of the protein. Covalent modifications of the protein are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the

antibody with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. For example, derivatization with bifunctional agents, well-known in the art, is useful for cross-linking the antibody or fragment to a water-insoluble support matrix or to other macromolecular carriers. As used herein, the term "antigen binding region" refers to that portion of an antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity ynd affinity for the antigen. The antibody region includes the "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues.

As used herein, the term "human chimeric antibody" includes monovalent, divalent or polyvalent immunoglobulins which are preferably substantially all derived from human Ig portions. A monovalent, chimeric antibody is a dimer (HL) ) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain. A divalent chimeric antibody is tetramer (H ₂ L ₂ ) formed by two HL dimers associated through at least one disulfide bridge. A polyvalent chimeric antibody can also be produced, for example, by employing a C _H region that aggregates (e.g., from an IgM H chain, or μ chain) . The invention also provides for "derivatives" of monoclonal or human chimeric antibodies according to the present invention, which term includes those proteins encoded by truncated or modified genes to yield molecular species functionally resembling the immunoglobulin fragments. The modifications include, but are not limited to, addition of genetic sequences coding for cytotoxic proteins such as plant and bacterial toxins. The fragments and derivatives can be produced from prokaryotic or eukaryotic hosts, as described herein by recombinant means. Alternatively, the fragments and derivatives may be produced by chemical means, such as proteolytic cleavage of intact immunoglobulin molecules, or other chemical modifications or derivatizations known in the art. See Harlow, supra, and Ausubel, supra . Such derivatized moieties may improve the solubility, absorption, biological half life, or other in vivo biological or therapeutic or diagnostic properties of a human, anti-parvovirus mAb of the present invention. The moieties may alternatively eliminate or attenuate one or more undesirable <.- Λde effects of an

anti- uman mAb of the present invention. Moieties capable of mediating such effects are disclosed, for example, in Remington ' s Pharmaceutical Sciences, 16th ed. , Mack Publishing Co., Easton, PA (1980) ; Harlow, supra, and references cited therein. Antibodies, fragments or derivatives having chimeric H chains and L chains of the same or different V region binding specificity, can be prepared by appropriate association of the individual polypeptide chains, as taught, for example by Sears et al., Proc. Natl . Acad. Sci . USA 72:353-357 (1975). With this approach, hosts expressing human chimeric H chains (or their derivatives) are separately cultured from hosts expressing chimeric L chains (or their derivatives) , and the immunoglobulin chains are separately recovered and then associated. Alternatively, the hosts can be co- cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled immunoglobulin, fragment or derivative.

Preferably, B cells which produce anti-human parvoviral antibodies, such as anti-B19 parvovirus antibodies, are provided according to the present invention by isolation of such B cell from a human, preferably from a human having an HIV infection, which cells are determined by screening methods as described herein, and as would be clear to one skilled in the art, bas^d on the teaching and guidance presented herein. Preferably, the isolated B cell producing an anti-human parvovirus, isolated according to the present invention, is then fused with a suitable fusion partner cell lines, such as a human-mouse, or human myeloma cell, in order to produce fusion partner cell lines, such as hybridomas, which fused cells produce recoverable amounts of anti-human parvovirus antibodies, as farther described herein. The cell fusions are accomplished by standard procedures well known to those skilled in the field of immunology (Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110; Hartlow, E. et al., supra; Campbell, "Monoclonal Antibody Technology, " In: Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon, R. , et al., eds.), Elsevier,

Amsterdam (1984); Kennett et al., Monoclonal Antibodies eds. pp.

365-367, Plenum Press, NY, 1980); de St. Groth, et al. , J ^" . Immunol .

Meth. 35: 1-21 (1980); Galfre, et al., Method Enzymol . 73:3-46

(1981) ; Goding, J.W. 1987. Monoclonal Antibodies : Principles and

Practice. 2nd ed. Academic Press, London, 1987) ; Ausubel et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Wiley Interscience, N.Y., (1987, 1992); and Harlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory (1988) , the contents of which references are entirely incorporated herein by reference.

Fusion partner cell lines and methods for fusing and selecting hybridomas and screening for mAbs are well known in the art

(Hartlow, et al. , supra; Kawamoto, et al. , Meth.. Enzymol 121:266-

277 (1986); Kearney, et al. , J. Immunol . 123:1548-1550 (1979); Kilmartin, et al., J. Cell Biol . 93:576-582 (1982); Kohler, et al.,

Eur. J. Immunol . 6:292-295 (1976) ; Lane, et al., J. Immunol . Meth .

47:303-307 (1981); Mueller, et al., J. Immunol . Meth. 87:193-196

(1986); Pontecorvo, Somatic Cell Genet. 1:397-400 (1975); Sharo, et al., Proc . Natl . Acad. Sci . USA 76:1420-1424 (1979); Shulman, et al., Nature 276:269-270 (1978); Springer, (ed) , Hybridoma Technology in the Biosciences and Medicine, Plenum Press, New York, 1985; and Taggart, et al., Science 219:1228-1230 (1982)), the contents of which references are entirely incorporated herein by reference. By the term "heteromyeloma" is intended a hybrid cell produced by fusion of a non-human myeloma cell line and a human myeloma cell line. Typically, a mouse myeloma or plasmacytoma cell is the fusion partner of the human myeloma cell. Such non-human and human myeloma and heteromyeloma cell lines are well-known in the art and are exemplified by cell lines reported in Teng, et al., Proc. Natl . Acad. Sci . USA 80:7308 (1983); Kozbor, et al. , Hybridoma 2:7 (1983); and Grunow, et al., J. Immunol . Meth. 106:257-265 (1988).

As intended in the present invention, heteromyeloma cells are used as fusion partners for selected EBV-transformed human cells producing anti-human parvovirus, human mAbs, to produce the heterohybridomas of this invention.

In a preferred embodiment, the heteromyeloma SHM-D33 is used as a fusion partner. This cell line is available from the ATCC, under accession number ATCC CRL1668. The term "heterohybridoma", as used herein, refers to a hybrid cell line produced by fusion of an antibody-producing cell of one species with a heteromyeloma. The term "heterohybridoma" has also been used elsewhere to refer to any interspecies hybridoma, such as one resulting from the fusion of an antibody-producing human

ly phocytoid cell line cell and a murine myeloma cell. In the context of the present invention, a human-mou_e or human-human heteromyeloma is preferred.

In one embodiment of this invention, a human antibody- producing cell is fused with a mouse-human heteromyeloma. In a preferred embodiment, the heterohybridoma is the result of fusing an EBV-transformed human lymphocyte which is producing an antibody to a neutralizing epitope of a human parvovirus, such as a B19 parvovirus, with a human-mouse heteromyeloma. In a more preferred embodiment, the human-mouse heteromyeloma is the cell line designated as 712-55D (ATCC # ) and 814-55D (ATCC # ), with 712-55D most preferred.

By the term "neutralizing epitope" is intended an epitope which, when bound by an antibody specific for this epitope, results in neutralization of a human parvovirus, such as B19 parvivirus. Neutralization of any biological activity of the virus, such as, for example, syncytium formation, falls within the scope of "neutralization", as used herein.

To generate human mAbs against a neutralizing epitope of a human parvovirus, such as B19 parvovirus, human peripheral blood B cells producing such antibodies, determined or screened as provided herein, are transformed by EBV, as described, for example in Gorny, et al. , Proc . Nat ' l . Acad. Sci . USA 86:1624-1628 (1989), which is hereby incorporated by reference. Preferably, the cells to be transformed are derived from the blood of an individual infected with HIV or producing anti-HIV-1 antibodies, wherein the individual also produces recoverable amounts of circulating B cells which produce anti-human parvovirus antibodies, such as B19 parvovirus mAbs. The cultures of EBV-transformed cells are screened for antibody to the epitope of interest. In one embodiment the epitope is a neutralizing epitope of the B19 parvovirus and the screening is performed by titrating supernatants from cultures of lymphocytes from patients by an immunoassay for the presence of IgG anti-B19 using recombinant B19 particles, such as those containing VP1 and/or VP2 capsid B19 proteins, a fragment thereof, or a synthetic peptide representing a portion thereof.

Any other of a number of immunoassays well known in the art can be used for chis screening process. A prer^rred immunoassay

is an Enzyme Linked Immunosorbent Assay, or ELISA. Using such an assay, the culture supernatants are tested for the presence of lymphocytes or B cells producing antibodies of desired specificity and isotype. Positive EBV-transformed cultures are cloned repeatedly by any of a number of cloning methods known in the art, such as, for example, by doubling dilution. Cells from cultures found to be positive for the desired antibody specificity are also fused with cells of the heteromyeloma line to produce a heterohybridoma. Fused cells are subsequently cloned by culturing at a density of about 0.3 to 100 cells per well. See Harlow, supra; Ausubel, supra .

Specificity of the antibody produced by the heterohybridoma is determined by immunoassay methods which are well known in the art. In a preferred embodiment, ELISA and radioiiruunoprecipitation (RIP) procedures are used. The antigen preparation comprises human parvovirus virions, such as B19 parvovirus lysates of viruses or of infected cells, viral proteins such as VP1 or VP2, or recombinant or synthetic viral peptides thereof. See Harlow, supra; Ausubel, supra. mAbs of the present invention may be of the IgG isotypes and/or suclasses, and may be recovered from the supernatants of the heterohybridoma cell cultures and purified by conventional methods known in the art for purification of IgG. Such methods include, but are not limited to, protein-A Sepharose affinity chromatography, a combination of Affigel Blue (BioRad, Richmond, CA) and Protein-A Sepharose chromatography, or High Performance Liquid Chromatography. See Harlow, supra; Ausubel, supra .

Anti-human parvovirus mAbs of the present invention may be produced in large quantities by injecting hybridoma cells secreting the antibody into the peritoneal cavity of immunocomprised mice and, after appropriate time, harvesting the ascites fluid which contains a high titer of the mAb, and isolating the mAb therefrom. Alternatively, the mAbs may be produced by culturing hybridoma cells in vitro and isolating the secreted mAb from the cell culture medium, according to known method steps. See, e.g. Harlow, supra.

Human genes which encode the C regions of antibodies, such as human chimeric antibodies, of the present invention are derived from cells which express, and preferably, produce, human

immunoglobulins. The human C _H region can be derived from any of the known classes or isotypes of human H chains, including gamma, μ , a, δ or e . Since the H chain isotype is responsible for the various effector functions of an antibody, the choice of C _H region will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity (ADCC) . Preferably, the C _H region is derived from at least one of gamma 1 (IgGl) , gamma 3 (IgG3) , gamma 4 (IgG4) , and μ (IgM) . The human C _L region can be derived from either human L chain isotype, kappa or lambda.

Genes encoding human immunoglobulin C regions are obtained from human cells by standard cloning techniques (Sambrook, et al. (Molecular Cloning: A Laboratory Manual , 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) ) . Human C region genes are readily available from known clones containing genes representing the two classes of L chains and the five classes of H chains. Human chimeric antibody fragments, such as F(ab') ₂ and Fab, can be prepared by designing a chimeric H chain gene which is appropriately trnncated. For example, a chimer: -; gene encoding a the H chain portion of an F(ab') ₂ fragment would include DNA sequences encoding the CHj domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule. The DNA encoding the antibody-binding region may be genomic DNA or cDNA. A convenient alternative to the use of human chromo¬ somal gene fragments as the source of DNA encoding a human parvovirus V region antigen-binding segment is the use of human cDNA, such as made from RNA encoding anti-B19 parvovirus antibodies produced from B cells isolated form HIV infected patients, for the construction of chimeric immunoglobulin genes, as reported for murine variable region by Liu et al. (Proc. Natl . Acad. Sci . , USA 84:3439 (1987) and J. Jirππunology 139:3521 (1987) , which references are hereby entirely incorporated by reference. The use of cDNA requires that g,-.ιe expression elements appropriate for the host cell be combined with the gene in order to achieve synthesis of the desired protein. The use of cDNA sequences is advantageous over genomic sequences (which contain introns) , in that cDNA sequences

can be expressed in bacteria or other hosts which lack appropriate RNA splicing systems.

Therefore, in an embodiment utilizing cDNA encoding the antibody V region, the method for changing the subclass of the antibody involves several steps, outlined below:.

1. Isolation of messenger RNA (mRNA) from the human cell line producing the monoclonal antibody, cloning and cDNA production therefrom;

2. Preparation of a full length cDNA library from purified mRNA from which the appropriate V region gene segments of the L and

H chain genes can be: (i) identified with appropriate probes, (ii) sequenced, and (iii) made compatible with a C gene segment;

3. Preparation of C region gene segments by cDNA preparation and cloning;

4. Construction of complete H or L chain coding sequences by linkage of the cloned specific V region gene segments to cloned human C region gene, as described above;

5. Expression and production of the newly constructed L and H chains in selected hosts, including prokaryotic and eukaryotic cells.

C region cDNA vectors prepared from human cells can be modified by site-directed mutagenesis to place a restriction site at the analogous position in the human sequence. For example, one can clone the complete human kappa chain C (C.) region and the complete human gamma-1 C region (C _gamma _.ι) . In this case, the alternative method based upon genomic C region clones as the source for C region vectors would not allow these genes to be expressed in bacterial systems where enzymes needed to remove intervening sequences are absent. Cloned V region segments are excised and ligated to L or H chain C region vectors. Alternatively, the human ^c _amma -ι region can be modified by introducing a termination codon thereby generating a gene sequence which encodes the H chain portion of an Fab molecule. The coding sequences with linked V and C regions are then transferred into appropriate expression vehicles for expression in appropriate hosts, prokaryotic or eukaryotic, according to known method steps. See Harlow, supra; Ausubel, supra .

Two coding DNA sequences are said to be "operably linked" if the linkage results in a continuously translatable sequence without alteration or interruption of the triplet reading frame. A DNA coding sequence is operably linked to a gene expression element if the linkage results in the proper function of that gene expression element to result in expression of the coding sequence.

Expression vehicles include plasmids or other vectors. Preferred among these are vehicles carrying a functionally complete human C _H or C _L chain sequence having appropriate restriction sites engineered so that any V _H or V _L chain sequence with appropriate cohesive ends can be easily inserted therein. Human C _H or C _L chain sequence-containing vehicles thus serve as intermediates for the expression of any desired complete H or L chain in any appropriate host. See, e.g., Harlow, supra . A human-human or human chimeric antibody Lll typically be synthesized from genes driven by the chromosomal gene promoters native to the human H and L chain V regions used in the constructs; splicing usually occurs between the splice donor site in the human J region and the splice acceptor site preceding the human C region and also at the splice regions that occur within the human C _H region; polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. Preferably, the J and/or C region is for a different subclass Ig than that of the V region. Gene expression elements useful for the expression of cDNA genes are well known and may include, but are not limited to: (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama, et al., Mol . Cell . Biol . 3:280 (1983)), Rous sarcoma virus LTR

(Gorman, et al., Proc. Natl . Acad. Sci . , USA 79:6777 (1982)), and Moloney murine leukemia virus LTR (Grosschedl, e; al. , Cell 41:885

(1985) ) ; (b) splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayama et al., supra) ; and (c) polyadenylation sites such as in SV40 (Okayama et al., supra) , See, also, e.g., Ausubel et al., supra, and Sambrook et al., supra. Immunoglobulin cDNA genes may be expressed as described by Liu supra, and Weidle et al.., Gene 51:21 (1987), using as expression elements the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancers, SV40 late region mRNA splicing, rabbit /3-globin intervening sequence, immunoglobulin and

rabbit β-globin polyadenylation sites, and SV40 polyadenylation elements. For immunoglobulin genes comprised of part cDNA, part genomic DNA (Whittle et al. , Protein Engineering 1:499 (1987) ) , the transcriptional promoter may be any suitable promoter (see Ausubel, supra; Harlow, supra) such as, human cytomegalovirus (CMV) , the promoter enhanc -s derived from CMV and mouse/hua n or human/human or human immunoglobulin, and mRNA splicing and polyadenylation regions derived from the native chromosomal immunoglobulin sequences. In one embodiment, for expression of cDNA genes in human or mammalian cells, the transcriptional promoter is a viral LTR sequence, the transcriptional promoter enhancers are either or both the human immunoglobulin heavy chain enhancer and the viral LTR enhancer, the splice region contains an intron of greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the human immunoglobulin chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the above-recited expression elements to achieve expression of the proteins in mammalian cells.

Each fused gene is assembled in, or inserted into, an expression vector, according to known method steps. Recipient cells capable of expressing the chimeric immunoglobulin chain gene product are then transfected singly with a human chimeric H or chimeric L chain-encoding gene, or are co-transfected with a chimeric H and a chimeric L chain gene. The transfected recipient cells are cultured under conditions that permit expression of the incorporated genes and the expressed immunoglobulin chains or intact antibodies or fragments are recovered the culture. In one embodiment, the fused genes encoding the chimeric H and L chains, or portions thereof, are assembled in separate expression vectors that are then used to co-transfect a recipient cell. See, e.g., Ausubel et al, supra, and Sambrook et al, supra.

For transfection of the expression vectors and production of the human chimeric antibody, the preferred recipient cell line is a human/mouse h-teromyeloma or a human myelo:.αn cell. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess the mechanism for glycosylation of the immunoglobulin. A particularly preferred

recipient cell is the Ig-non-producing myeloma cell SP2/0 (ATCC #CRL 8287, ATCC, Rockville, Md. ) . SP2/0 cells produce only immunoglobulin encoded by the transfected genes. Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, where secreted immunoglobulin can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells such as B lymphocytes of human or non-human origin, hybridoma cells of human or non-human origin, or interspecies heterohybridoma cells, wherein human derived cells are preferred. The expression vector carrying human-chimeric antibody construct of the present invention may be introduced into an appropriate host- cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment (Johnston et al. , Science 240:1538 (1988)) . A preferred way of introducing DNA into lymphoid cells is by electroporation (Potter et al. , Proc. Natl . Acad. Sci . USA 81:7161 (1984); Yoshikawa, et al., Jpn. J. Cancer Res . 77:1122-1133); Sambrook, supra; Ausubel, supra.

Antibody immunoglobulin genes of the present invention can also be expressed in nonlymphoid mammalian cells or in other eukaryotic cells, such as yeast, or in prokaryotic cells, in particular bacteria.

Yeast provides substantial advantages for the production of immunoglobulin H and L chains. Yeasts carry out p st-translational peptide modifications including glycosylation. A number of recombinant DNA strategies now exist which utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides) (See, e.g., Hitzman, et al., llfch international Conference on Yeast, Genetics and Molecular Biology, Montpelier, France, September 13-17, 1982) ; Ausubel, supra; Sambrook, supra.

Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and the stability of human or human chimeric H and L chain proteins and assembled human chimeric

antibodies. Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized. Known glycolytic genes can also provide very efficient transcription control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. A number of approaches may be taken for evaluating optimal expression plasmids for the expression of cloned immunoglobulin cDNAs in yeast (see, e.g., Glover, ed., DNA Cloning, Vol . II, pp45-66, IRL Press, 1985; Ausubel, supra; Sambrook, supra) .

Bacterial strains may also be utilized as hosts for the production of antibody molecules or antibody fragments described by this invention, which include, but are not limited to, E. coli K12 strains such as E. coli W3110 (ATCC 27325) , and other enterobacteria uch as Salmonella typhimur ium or Serratia marcescens, and various Pseudomonas species may be used.

Plasmid vectors containing replicon and control sequences which are derived from species compatible with a host cell are used in connection with these bacterial hosts. The vector carries a replication site, as well as specific genes which are capable of providing phenotypic selection in transformed cells. A number of approaches may be taken for evaluating the expression plasmids for the production of chimeric antibodies or antibody chains encoded by the cloned immunoglobulin cDNAs in bacteria (see Glover, ed., DNA Cloning, Vol . I, IRL Press, 1985); Harlow, supra; Ausubel, supra; Sambrook, supra . )

Other preferred hosts are mammalian cells, grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules including leader peptide removal, folding and assembly of H and L chains, glycosylation of the antibody molecules, and secretion of functional antibody protein. Mammalian cells which may be useful as hosts for the production of antibody proteins, in addition to the cells of lymphoid origin described above, include, but are not limited to, cells of fibroblast origin, such as Vero (ATCC CRL 81) or CH0-K1

(ATCC CRL 61), or other human or mammalian cells. See, e.g., Ausubel, supra; Sambrook, supra .

Many vector systems are available for the expression of cloned H and L chain genes in mammalian cells (see Glover, supra) . Different approaches can be followed to obtain complete H ₂ L ₂ antibodies. As discussed above, it is possible to co-express H and L chains in the ε'ume cells to achieve intracellular association and linkage of H and L chains into complete tetrameric H ₂ L ₂ antibodies. The co-expression can occur by using either the same or different plasmids in the same host. Genes for both H and L chains can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains. Alternatively, cells may be transfected first with a plasmid encoding one chain, for example the L chain, followed by transfection of the resulting cell line with an H chain plasmid containing a second selectable marker. Cell lines producing H ₂ L ₂ molecules via either route could be transfected with plasmids encoding additional copies of H, L, or H plus L chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled H ₂ L ₂ antibody molecules or enhanced stability of the transfected cell lines.

In addition to mAbs or human chimeric antibodies, the present invention is also directed to an anti-idiotypic (anti-Id) antibody specific for V region epitopes of the mAb antibody of the invention. An anti-Id antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding region of another antibody. The antibody specific for an anti- human parvovirus, such as B19, antigen binding or epitope binding region, is termed the anti-idiotypic or anti-Id antibody. The anti-Id can be prepared by immunizing an animal with an anti-human parvovirus or the antigen-binding region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id antibody. The anti- Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The ό.uti-anti-Id may be epitopically identical to the original antibody which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible

to identify other clones expressing antibodies of identical specificity.

Accordingly, the mAbs of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice or humans. Spleen cells from such immunized mice or humans can be used to produce anti-Id hybridomas secretinc anti-Id mAbs. Further, the anti-Id mAbs can be used to immunize additional BALB/c mice or humans. Sera from these mice or humans will contain anti- anti-Id antibodies that have the binding properties of the original mAb specific for a human parvovirus epitope.

The antibodies of the present invention, including their antige -binding Iragments and derivatives, have a multitude of uses relating to the diagnosis, monitoring and therapy of human parvoviral infection. In diagnosis, the antibodies may be used in immunoassays

(described below) to screen body fluids, such as serum, sputum, effusions, urine, cerebrospinal fluid, and the like, for the presence of a human parvovirus, such as B19 parvovirus, according to known method steps. See, e.g., Harlow and Lane, supra. Additionally, the antibodies may be used as positive controls for methods and kits used for detection of humar parvovirus, such as B19 parvovirus in a biological sample, derived or prepared from an animal, preferably a human.

In addition to their diagnostic utility, the antibodies of the present invention are useful for monitoring the progression of disease by screening body fluids for a human parvovirus, such as B19 parvovirus, according to known method steps, as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes direct neutralization of the virus, direct cytotoxicity by the antibody, either mediated by complement (CDC) or by effector cells (ADCC) , conjugated to anti-parvoviral drugs, toxins, radionuclides.

Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation, based on the teaching and guidance presented herein.

The preferred animal subject of the present invention is a mammal, preferably a human. By the term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects. By the term "treating" is intended the administering to subjects of the antibodies of the present invention or a fragment or derivative thereof for purposes which may include prevention, amelioration, or cure of parvovirus infection.

The pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose. Amounts and regimens for the administration of human parvovirus antibodies, their fragments or derivatives can be determined readily by those with ordinary skill in the clinical art of treating infectious diseases. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, trans- dermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

Compositions within the scope of this invention include all compositions wherein the antibody, fragment or derivative is contained in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. The effective dose is a function of the individual human chimeric or monoclonal antibody, the presence and nature of a conjugated therapeutic agent (see below) , the patient and his clinical status, and can vary from about 10 ng/kg body weight to about 100 mg/kg body weight such as 1 mg/kg to 10 mg/kg, 10 μg/kg to 1 mg/kg or 100 μg /kg to 10 mg/kg. The preferred dosages comprise 0.1 to 10 mg/kg body wt.

In addition to the pharmacologically active compounds, the new pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferably, the preparations, contain from about 0.01 to 99 percent, such as 1, 5, 10, 20, 30,

40, 50, 60%, preferably from about 20 to 75 percent of active com¬ pound^), such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70%, together with the excipient.

Preparations of the antibody, fragment or derivative of the present invention for parenteral administration, such as in detectably labeled form for imaging or in a free or conjugated form for therapy, include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aq-..sous solvents are propyleneglycol, polyethyleneglycol, vegetable oil such as olive oil, and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media, parenteral vehicles including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. See, generally, Remington ' s Pharmaceutical Science, 16th ed., Mack Publishing Co., Easton, PA, 1980.

In particular, the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing a human parvovirus infection. Such t? ^atment comprises parenterally administering a single or multiple doses of the antibody, fragment or derivative, or a conjugate thereof.

The antibodies of this invention can be adapted for therapeutic efficacy by virtue of their ability to neutralize the virus and/or to mediate ADCC and/or CDC against cells or tissues having a human parvovirus associated with their surface. For these cellular activities, either an endogenous source or an exogenous source of effector cells (for ADCC) or complement components (for CDC) can be utilized.

The antibodies of this invention, their fragments, and derivatives can be used therapeutically as immunoconjugates (see for review: Dillman, Ann . Int . Med. 111:592-603 (1989)). They can be coupled to antiviral compounds, cytotoxic proteins, including, but not limited to gamma globulin, amantadine, guanidine, hydroxybenzimidc -:ole, interferon-α, interferon - 3, interferon-γ, thiosemicarbarzones, methisazone, rifampin, ribvirin, a pyrimidine

analog, a purine analog, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, ganciclovir, ricin-A, Pseudomonas toxin, Diphtheria toxin, and tumor necrosis factor. Toxins conjugated to antibodies or other ligands, are known in the art. See, for example, Olsnes, S. et al., Immunol . Today 10:291-295 (1989) ; Katzung, supra, and the references cited therein on pages 680-681, respectively, which references are herein entirely incorporated by reference. Plant and bacteria toxins typically kill a virus-infected cell by disrupting the protein synthetic machinery.

The antibodies of this invention can be conjugated to additional types of therapeutic moieties including, but not limited to diagnostic radionuclides and cytotoxic agents such as cytotoxic radionuclides, ά-ugs and proteins. Examples of --iionuclides which can be coupled to antibodies and delivered in vivo to sites of antigen include, but note limited to, ²¹² Bi, ¹³¹ I, ¹⁸⁶ Re, and ⁹⁰ Y. Such radionuclides exert their cytotoxic effect by locally irradiating the human parvovirus infected cells or parvovirus leading to various intracellular lesions or viral death as is known in the art of radiotherapy.

Cytotoxic drugs which can be conjugated to antibodies and subsequently used for in vivo therapy include, b t are not limited to, daunorubicin, doxorubicin, methotrexate, end Mitomycin C. Cytotoxic drugs interfere with critical cellular processes including DNA, RNA, and protein synthesis. For a fuller exposition of these classes of drugs which are known in the art, and their mechanisms of action, see Goodman, et al., Goodman and Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 8th Ed., Macmillan Publishing Co., 1990. The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hemopoietic growth factors, etc., which serve to increase the number or activity of effector cells which interact with the antibodies. The antibodies, fragments, or derivatives of this invention, attached to a solid support, can be used to remove soluble human parvovirus associated antigens from fluids or tissue or cell extracts. In a preferred embodiment, they ars used to remove soluble B19 parvoviral antigens from blood or blood plasma

products. In another preferred embodiment, the antibodies are advantageously used in extracorporeal immunoadsorbent devices, which are known in the art (see, for examole, Seminars in Hematology, 26 (2 Suppl. 1) (1989)) . Patient blood or other body fluid is exposed to the attached antibody, resulting in partial or complete removal of circulating human parvovirus (free or in immune complexes) , or human parvovirus-bearing cells, following which the fluid is returned to the body. This immunoadsorption can be implemented in a continuous flow arrangement, with or without interposing a cell centrifugation step. See, for example, Terman, et al., J ^" . Immunol . 117:1971-1975 (1976).

Additionally, anti-human parvovirus antibodies of the present invention may be bound to a solid support for commercial scale or less purification of parvovirus antigens to be used in diagnosis or vaccine production, according to known method steps. See Harlow, supra .

The present invention also provides the above antibodies, fragments and derivatives, detectably labeled, as described below.

The antibodies of the present invention are useful for immunoassays which detect or quantitate human parvovirus, preferably B19 parvovirus, or cells bearing a human parvovirus or epitope thereof, in a sample. Such an immunoassay typically comprises incubating a biological sample in the presence of a detectably labeled antibody of the present invention capable of identifying a human parvovirus antigen, and detecting the labeled antibody which is bound in a sample.

Thus, in this aspect of the invention, a biological sample may be treated with nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins or glycoproteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled antibody of the present invention. The solid phase support may then be washed with the buffer a second time ^; :o remove unbound antibody. The amount of bound label on said solid support may then be detected by conventional means. Additionally, such Abs may be used as positive controls for such assays.

By "solid phase support" or "carrier" is intended any support capable of binding antigen or antibodies. Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene,

dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a human parvovirus or the antibody specific for a human parvovirus. Thus, the support configuration may be spherical, as in a bead, rr cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation. The binding activity of a given lot of antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experi¬ mentation. One of the ways in which the antibody of the present invention can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) , or enzyme-linked immunosorbent assay (ELISA). This enzyme, when subsequent]7 exposed to its substrate, will react with the substrate generating a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. In an alternate embodiment, the enzyme is used to label a binding partner for the antibody of the invention. Such a binding partner may be an antibody against the constant or variable region of the antibody of the invention, such as a heterologous anti-human or anti-mouse immunoglobulin antibody. Alternatively, the binding partner may be a non-antibody protein capable of binding to the antibody of the present invention, such as Staphylococcal protein A, or Streptococcal protein G.

Enzymes which can be used to detectably label a human parvovirus-specific antibody of present invention, or the binding partners for such an antibody, include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeas alcohol dehydrogenase, alphε -glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase,

alkaline phosphatase, asparaginase, glucose oxidase, beta-galac- tosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

By radioactively labeling the antibody of the present invention or the binding partner, it is possible to detect human parvovirus through the use of a radioimmunoassay (RIA) (see, for example, Work, et al.. Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, N.Y. (1978) . The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. Isotopes which are particularly useful for the purpose of the present invention are well known in the art, such as radio-isotopes of H, C, N, P, S, I, and other elements.

It is als possible to label the antibodies or binding partners with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocy- anin, o-phthaldehyde and fluorescamine.

The antibodies can also be detectably labeled using fluorescence-emitting metals such as ¹⁵² Eu, or others of the lan- thanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamin«pentaacetic acid (DTPA) or ethylenediamine-tetraacetic acid (EDTA) .

The antibodies of the present invention also can be detectably labeled by coupling to a chemiluminescent compound. The presence of the chemiluminescently labeled antibody is then determined by detecting the presence of luminescence that --rises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, imidazole, acridinium salt and oxalate ester.

Likewise, a biolu inescent compound may be used to label the antibody, fragment or derivative of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.

Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

Detection of the antibody, fragment or derivative may be accomplished by a scintillation counter, for example, if the detectable labe . is a radioactive gamma ea'.tter, or by a fluorometer, for example, if the label is a fluorescent material. In the case of an enzyme label, the detection can be accomplished by colorimetric methods which employ a substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

In situ detection may be accomplished by removing a histological specimen from a patient, and providing the labeled antibody, or the unlabeled antibody plus a labeled binding partner to such a specimen. Through the use of such a procedure, it is possible to determine not only the presence of the antigen but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achi ve such in situ detection.

The antibody, fragment or derivative of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support that is insoluble in the fluid being tested and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitatior. of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the human parvovirus antigen from the sample by formation of a binary solid phase antibody--vuman parvovirus complex. . ^τ ter a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted human parvovirus antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a

"reporter molecule") . After a second incubation period to permit the labeled antibody to complex with the human parvovirus bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody. This type of forward sandwich assay may be a simple "yes/no" assay to determine whether a human parvovirus, preferably B19 parvovirus, is present or may be made quantitative by comparing the measure of labeled antibody with that obtained for a standard sample containing known quantities of the antigen. Such "two-site" or "sandwich" assays are described by Wide (Radioimπane Assay Method,

Kirkha , ed. , E. & S. Livingstone, Edinburgh, 1970, pp. 199-206).

Other type of "sandwich" assays, which may also be useful with a human parvovirus, are the so-called "simultaneous" and "reverse" assays. A simultaneous assay involves a single incubation step wherein the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antiboly associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period, is utilized. After a seconc incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays. In one embodiment, a combination of antibodies of the present invention specific for separate epitopes may be used to construct a sensitive three-site immunoradiometric assay. Specifically exemplified mAbs for human parvovirus may also be used to facilitate the production of add_tional mAbs which bind the same or immunologically cross-reactive parvovirus- associated antigens. First, these antibodies may be conjugated to a chromatographic support, and used to immunopurify parvovirus- associated antigens. These purified antigens, in turn, may be used to stimulate an immune response in suitable animals. Secondly,

spleen cells from the responsive animals may be fused to immortalizing cells, and the resulting hybridomas screened for secretion of antibodies which bind to the purified antigen and/or whose binding to a human parvovirus-associated antigen is competitively inhibited by such specific anti-human parvovirus antibody.

Having now generally described the invention, the same will be further understood by reference to certain specific examples which are included herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

EXAMPLE I: ISOLATION OF CELL LINES PRODUCING HUMAN ANTI_HUMAN PARVOVIRUS AND GENERATION OF HETERHYBRIDOMAS PRODUCING ANTI_HUMAN PARVOVIRUS ANTIBODY

MATERIALS AND METHODS

Subjects used for the detection of serum Ab to B-19 Parvovirus. Blood was obtained from subjects seen an in-patient or in the Infectious Disease Clinic of the New York Veterans Affairs Medical Center or volunteers for these studies referred by private physicians in the New York area. Each specimen was tested for Abs to HIV by ELISA and Western blot kits (Genetic Systems, Seattle, WA. ; BioRad, Hercules, CA) . The control group was derived from this same population and were all HIV-negative. The disease status of the HIV-positive patients was established on the basis of the following immunological staging system (Table A) previously described by S. Zolla-Pazner, et al. 84:5404 (1987)).

TABLE A: STAGING SYSTEM Scale Score T4/T8 cell T4 Cells Lymphocytes ratio ./mm ³ No./mm ³

ELISA assays for IgM and IgG anti-B19 antibodies: All serum samples were kept frozen (-70°C) before analysis and were then tested simultaneously. All sera were tested for IgG anti-B19 with an indirect ELISA method, using recombinant B19 parvovirus obtained from insect cells infected with B19-containing baculovirus constructs (see below) . Two different types .f self-assembled recombinant empty capsids were used: One contained B19 virus structural proteins, VP1 and VP2, in a ratio of 1:3; the other capsids contained VP2 only. These antigens, at 1 μg/ml in PBS, were absorbed onto Immulon 496-well plates (Dynatech, Alexandria,

VA) for 16-18h, at 4°C. Serum samples were tested at various dilutions in PBS-Tween 20 (0.05%) containing 10% normal goat-serum

(PBST-GS) . Sera in antigen-coated plates were incubated for 1.5h at 37°C after which peroxidase-labeled goat affinity-purified anti- human IgG, gamma-chain specific (Sigma, Redmor.nt, WA) , diluted 1:2000 in PBST-GS was added. After an incubation of 1 hour at 37°C, ABTS-substrate solution was added for 30 minutes, and the color reaction was detected using an MR780 Microplate Reader (Dynatech) at 405 nm. Each washing procedure was performed three times with PBS-Tween 20 (0.05%) . Specificity of the positive results was confirmed by testing each sample n two wells, one coated with B19 recombinant antigen (P) , and the other coated with an antigen unrelated to B19, such as bovine serum albumin or the lysate of insect cells infected with baculovirus lacking the B19 insert (N) . Criteria for specificity and positivity included serum reactivity with the B19 antigen more than twice that of the control antigen (P/N ≥ 2) and a difference in O.D. of at least 0.2.

Detection of IgM anti-B19 in sera was performed using an IgM-capture assay previously described (Cohen, B.J., et al., J. Hygiene 91:113 (1983)). Thus, plates were coattd with goat anti- human IgM (μ-chain) F(ab ^! ) ₂ fragments (Cappel) at 2 μg/ml in PbS for 2 hours, at 37°C. Then, serum samples, diluted in 1:100 in PBST- GS, were incubated overnight. After washing, recombinant B19, diluted to 10 μg/ml in PBST-GS, was added and incubated for 2 hours at 37°C. B19 antigen, bound to IgM anti-B19, was reacted with mouse monoclonal anti-B19 (Chemicon, Inc.) diluted 1:1000 in PBST- GS for 1 hour at 37°C. The reaction was detected using goat anti- mouse IgG labeled with alkaline phosphatase (Sigma Co) , diluted 1:2000 in PBST-GS, during 1 hour at 37°C. After the final wash

para-nitrophenal phosphate substrate solution was added and the results were read after 30 minutes on an ELISA reader at 405 nm. The positive control used was a sample of serum from a patient with Fifth Disease. Negative serum was obtained f^om anti-B19 IgG- negative healthy individuals. The "cut off" le-"-el was calculated as twice the mean of the negative control, run in duplicate.

Production of cell, lines synthesizing human mAbs to B19 parvovirus: Blood of patients with IgG anti-B19 titres of ≥ 1:1000 was used for the isolation of peripheral blood lymphocytes (PBL) and the production of lymphoblastoid cells lines. Standard procedures for isolation PBL with Ficoll-Hi itopaque and for transformation of B cells with Epstein-Barr virus were used (Gorny, et al., Proc . Natl . Acad. Sci . (USA) 86:1624 (1989)). After 3-4 weeks of cultivation of the transformed cell in 96-well plates (Costar, Cambridge, MA.), supernatants were tested for anti-B19 by the same ELISA as described above for sera. The cells from positive wells were grown in 1 cc of medium for two weeks. Those wells containing cells which continued to produce anti-B19 and give a P/N ≥ 5 were fused with the human X mouse heteromyeloma cell lines SHM-D33 (Teng, et al. , Proc . Natl . Acad. Fci . (USA) 80:7308

(1983)) . Wells containing heterohybrides and making IgG anti-B19 were expanded and cloned sequentially at 100, 10 and 1 cell per well, as previously described (Gorny, et al., Proc . Natl . Acad.

Sci . (USA) 88:3238 (1991)). Quantitation and characterization of anti-B19 human mAbs:

The concentration of IgG was detected by ELISA using goat anti- human IgG (gamma-chain specific) (Cappel Products, Westchester, PA.) -coated plates. After incubation with undiluted culture supernatants, bound IgG was detected with alkaline phosphatase- labeled goat anti-human IgG (gamma-chain specific) . Affinity purified human IgG (Cappel) was used for standard curves.

Detection of the light chain type and IgG subclass of the anti-B19 human mAbs was performed with anti-human IgG-coated plates, using alkaline phosphatase-conjugates of antibodies specific for human kappa and lambda chains (Caυpel) or the four subclasses of human IgG (Zymed Laboratories, San Francisco, CA. ) .

To study whether the two human mAbS reacted with identical or overlapping epitopes, a blocking assay was performed in which ELISA plates were coated with a human mAb at 1 μg/ml in

PBS overnight at 4°C and the washed. The second human Ab was mixed at a final concentration of 10 μg/ml and incuba .ϊd for 30 minutes at 37°C and the mixture was then added to the coated plates. After 2 hours at 37°C and the mixture was then added to the coated plates. After 2 hours at 37°C and washing, a mouse IgG anti-Bl9 mAb (anti-VPl or -VP2, Chemicon, Inc.) was added at a dilution of 1:1000 PBST-GS for one hour at 37°C. After washing, alkaline phosphatase conjugated-goat anti-mouse Ig (Sigma) at 1:2000 in PBST-GS was added and incubated for 1 hour at 37°C, followed by addition of paranitrophenol phosphate substrata solution. The results were read after 30 minutes in an ELISA reader at 405 nm. As a positive control of blocking, the same human mAb was used on the solid phase and the mixture with antigen (100% inhibition) . As a negative control (0% inhibition) , an anti-CMV human mAb at 10 μg/ml was used in the mixture with the B19 antigen. Percent of inhibition was calculated as:

Radioimmunoprecipitation Assay: Radiolabeled parvovirus

B19 empty capsids were prepared from Spodoptera Frugiperda Sf9 cells infected with recombinant baculoviruses (Kajigaya, et al., Proc. Natl . Acad. Sci . (USA) 88:4646 (1991)) . Separate cultures of Sf9 cells were either infected with baculovirus bacVP2 alone or coinfected with baculoviruses bacVPl and bacVP2 at the same multiplicity of infection (moi = 3) . The cultures were incubated with methionine-free Grace's medium (Gibco, Grand Island, NY) for 1 h at 40 h pos -infection, and then radiolabeled for 6 h in a methionine-free :r.edium, supplemented with 100 uCi/ml of 35S-labeled nethionine (ICN) . Cells were harvested and prepared for immunoprecipitation by two procedures. Intact, radiolabeled capsids were obtained by lysis of cells in RIPA buffer at 4°C (Collett, et al. , Virol . , 165:200 (1988)) . To obtain denatured unassembled parvovirus polypeptides, cells were lysed by boiling

in SDS lysis buffer, followed by dilution in o an appropriate buffer (Keegan, et al, J. Virol . 58:263 (1986)). In both cases, lysates were cleared by centrifugation at 90,000 x g for 20 minutes. Immunoprecipitations and electrophoresis were carried out as described elsewhere (Collett, supra) .

Virus Neutralization Assay: Measurement of pravovirus B19 virus neutralizing antibody employed a human erythroid colony- forming assay (Sambrook, supra) . Positive and negative sera were heated to 56°C for 30 minutes to inactivate complement. Dilutions of control sera or mAb-containing cell culture fluids were incubated with a 1:20 dilution of infectious paravovirus-containing human serum for 2 hours at 4°C. Bone marrow cells were harvested from normal volunteers under a protocol approved by the National Heart, Lung, and Blood Institute Institutional Review Board, NIH. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation and were then incubated for 1 hour at 4°C with the virus/antibody mixtures at a final concentration of 2 x 10 ⁶ cells per ml. Cells were then diluted into cell culture medium specific for erythroid progenitor colony formation (Iscove's modified Dulbecco's medium supplemented with 30% fetal bovine serum, 1% bovine serum albumin, 0.8% methylcellulose, 1 mM 2-mercaptoethanol, and 1 unit recombinant erythropoietin per ml) . Cultures were incubated at 37°C, 95% humidity for 7 days, at which time erythroid colony forming units (CFU-E) were visually counted. All assays were performed in duplicate, and controls included virus without added mAb or serum, virus with known neutralizing rabbit serum, cells with serum or cell culture fluid negative an_;i-B19, and cells alone.

RESULTS

Detection of anti-B19 antibodies in human sera: The sera of 160 HIV-positive patients (scale score 0-3) and of 88 HIV- negative individuals were titrated by ELISA for the presence of IgG anti-B19 with recombinant B19 particles containing VP1 and VP2 (Table 1) . Sera of 98 of 104 HIV-infected subjects (94%) with a scale score of 3 were positive for anti-B19 at a dilution of 1:100 compared to 61 to 88 (69%) HIV-seronegative individuals (p<0.00001). Patients of all other scale scor? groups showed a prevalence of IgG anti-B19 intermediate between the scale score 3 group and HIV-negative controls) . [See Table I] The prevalence of IgG anti-B19 in HIV-infected individuals was higher than that in our control group and higher than that reported in the literature for the normal population (Fridell, et al. , Scand. J. Infect. Dis . 21:597 (1989); Schwarz, et al. , J. Virol . 66:1273 (1992)). Statistical significance was achieved between the: scale score 3 and HIV-negative groups when compared at a serum dilution of 1:100 and 1:1000 The assay for IgM anti-B19 showed none positive from 248 tested sera, including 160 HIV-positive and 88 HIV-negative individuals. At the same time, absorbance of the positive control samples run in duplicate on each plate > 1.0 was at least five times higher than the mean negative control.

Production of anti-B19 in vitro by EBV-transformed B cells: Blood from 22 HIV-positive patients at different stages of HIV infection (scale scores 0 to 3) and with an IgG anti-B19 serum titre of ≥ 1:1000 was used for isolation of PBL and transformation with EBV. After in vitro cultivation in 96-well plates and screening for IgG anti-B19 production, positive v/ells (defined as P/N > 5) were identified, the cells therein were grown in larger

wells, and tested again for IgG anti-B19 after two weeks. The results, shown i.- Table 2, demonstrate the presence of significant numbers of anti-B19-producing B cells in the circulation of 19 of 22 (86%) of patients tested.

Generation of human mAbs against B19 parvovirus: Cells positive for anti-B19 after expansion were fused with the human x mouse heteromyeloma SHM-D33 to stabilize antibody production. Fused cells were subsequently cloned sequentially at 100, 10 and 1 cell per wel ^"1 . Cultures from the 19 patients whose cells continued to produce anti-B19 on expansion (Table 2) were fused with the heteromyeloma. After 2-3 weeks of growth, 5.6% of wells contained hybrids; generally 5-10 clumps of hybrid cells were observed in each of these wells. Upon expansion, 69% of wells with hybrids continued to secrete specific antibody, and after sequential cloning, only two monoclonal heterohybridomas synthesizing anti-B19 mAbs were produced. These two lines were designated 712-55D and 814-55D, respectively, each was obtained from two separate individuals, one whose scale score was 2 and one whose scale score was 3.

Characterization of the anti-B19 human mAbs 712-55D and 814-55D: Both cells were found to secrete IgG, mAbs. Spent media in which 712-55D or 812-55D were grown for 5 days contained 19.4 and 84.5 μg mAb/ml, respectively. Both mAbs reacted with ELISA with recombinant B119 capsids containing VPl and VP2 and with capsids composed of VP2 only (data not shown) .

Further studies of the epitopes recognized by these mAbs were performed using RIP assay. As shown in Fig. 1, both mAbs react with capsids composed of VPl + 2 and with VP2, only, but fail

to react with VPl + 2 capsids denatured prior to immunoprecipitation with sodium dodecyl sulfat . These studies suggest that both mAbs recognize a conformational epiope (epitopes) on VP2. In order to study the competition between human mAbs 712-

55D and 814-55D, blocking assays were performed. Both HuMabs blocked themselves and one another. Monoclonal antibodies 712-55D blocked the binding of B19 to 814-55D by 100%, but 814-55D blocked B19 binding to 712-55D by only 60%. The ability of these mAbs to neutralize B19 was tested using a human erythroid colony-forming assay as described above. Fifty percent neutralization of B19 was achieved with 0.9 μg/ml of mAb 712-55D and with 0.6 μg/ml of 814-55D (Fig. 2). At 4.0 μg/ml, both mAbs were able to neutralize 100% of the virus. The potency of those Mabs are thus similar to mAbs previously described (Sato, et al., J ^" . Virol . 65:1667 (1991)) which neutralizes parvovirus and to other human and rodent mAbs that neutralize other viruses (Gorny, et al. , J. Virol . (1992); Ho, et al. , Iroc. Natl . Acad. Sci . (USA) , 88:8949 (1991); Umino, et al. , (1990); Cheung, et al. , J. Virol . 63:2445 (1989)). Discussion

There have been some reports of the detection of B19 DNA in the bone marrow and in the peripheral blood of AIDS patients with severe red cell aplasia (Kurtzman, et al., J ^" . Clin . Invest . 84:1114 (1989); Frickhofen, et al. , Ann. Intern . Med. 113:926

(1990)). This suggests that B19 infection is involved in some cases of anemia in AIDS patients. To date, however, the presence of specific markers for B19 infection, such as antibody, antigen and DNA in HiV-infected patients has not been described

systematically. Thus, in one study, no evidence of current B19 infection was found in 50 AIDS patients (Anderson, et al. , Ann. Intern . Med. 102:275 (1985)), while a second study reported one case of an HIV-infected patient who tested positively for B19 DNA and eight cases of 55 in which significant IgM titres were detected in mostly asymptomatic patients (Frickhofen, et al. , Ann . Intern. Med. 113:926 (1990)). In our study, however, none of the sera tested was positive for IgM anti-B19. These negative results do not exclude the possibility that a current B19 infection occurs in some HIV-infected individuals, since IgM anti-B19 may be undetectable in persistent B19 infection (Kurt;man, et al., J. Clin . Invest. 84:1114 (1989)), and the immune response may be depressed in these immunocompromised patients (Frickhofen, et al., Ann . Intern. Med. 113:926 (1990)) . Having observed the augmented IgG response, attempts were made to capture and immortalize the B cells making IgG anti-B19. Using the blood of HIV-infected patients with high titres of IgG anti-B19, two cell lines were produced synthe&izing monoclonal anti-B19 Abs with neutralizing activity. Both human mAbs were directed to conformational epitopes of VP2 and failed to react with denatured capsid proteins.

Cross competition of obtained HuMabs i i'icate whether the same epitope for both or overlapping epitopes are in the same cluster. However, only exact epitope mapping can confirm this suggestion. Moreover, the conformational structure of the epitopes can lead to stuchiometric interaction, including the inhibition due to Ab binding, even in cases of separate epitope localization, as it was described for HIV discrepancies in the inhibition percent between 712-55D and 814-55D may also be based n this mechanism,

but another explanation especially in cases of the same epitope, could be the different affinity of these HuMabs.

Conformational epitopes of B19 parvovirus appear to be more immunogenic than linear epitopes. Thus, an analysis of human sera with synthetic peptides covering the entire length of the B19 structural proteins of B19 parvovirus showed only a single linear epitope (aa292-301 of VP2) which was recognized by all six convalescent sera tested from patient with Fifth Disease (Fridell, et al., Scand. J. Infect . Dis . 21:597 (1989)). In another report, less than 10% of sera with high titres of anti-B19 reacted with limited numbers of synthetic peptides which cover the hydrophilic regions of VP2 which were predicted to have high antigenicity (Sato, et al., J. Virol . 65:5485 (1991)). The same authors demonstrated that affinity-purified human polyclonal antibodies directed against the linear epitope of VP2 were neutralizing.

In the absence of any chemotherapy against B19 infection, the only successful treatment described is based upon immunotherapy using preparations of human IgG containing a high titre anti-B19 (Schwartz, et al. ^' , J. Infect . Dis . 162:1214 (1990)). As been shown in cases of other infections, such as CMV, substitution of hyperi mune gamma-globin with human mAbs provides some advantages, based on at least 100-fold higher activity of the human mAb

(Masuho, et al., J. Gen. Virol . 68:1457 (1987); Mashoko, et al.,

Develop. Biol . Standard. 71:127 (1990)). Thus, there is a potential therapeutic application for the anti-B19 human mAbs described above. They could be administered ^~ gι ^r .phylactically to prevent the spread of infection during outbreaks of Fifth Disease. This would be particularly important for pregnant B19-seronegative women exposed during such an outbreak (Public Health Laboratory

Service, Bri t . Med. J. 300:1166 (1990) ; Rodis, et al. , Am. J. Obstet . Gynecoi . 163:1168 (1990); Kinney, et al. , J ^" . Infect . Dis . 157:663 (1988)) .

Moreover, in accord with results obtained with non¬ specific gamma-globulin therapy, passive immunization using human mAbs could also be an effective treatment for patients with acute and persistent E-19. Again, this might prove to be particularly critical for the 1-2% of pregnant women in this country with active B19 infection. (Kinney, et al. , J. Infect . Dis . 157:663 (1988)) .

TABLE 1 TITRATION OF ANTI-B19 ANTIBODIES IN HUMAN SERA

Total No. Number Positive (%) at Dilution of:

* corrected ch -squared test for trend

Table 2 CHARACTERISTICS OF CELL CULTURE SCREENED FOR ANTI-B19 ANTIBODIES DERIVED FROM PATIENTS AT DIFFERENT STAGES OF HIV INFECTION

* Patients used all had ant -B19 titres ≥ 1:1,000 ** From these 22 patients, 19 gave positive wells.

TABLE 3

CHARACTERIZATION OF 712 - 55D AND 814 - 55D ANTI -B19 HUMANS

^♦ Concentration of IgG (μg/ml) in spent medium.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art

(including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

REFERENCES

1. Anderson, et al. , Lancet. ii:695 (1983) .

2. Sergeant, et al . , Lancet. ii:695 (1983) .

3. Young, Semin Hematol. 25:159 (1988) .

4. Woolf, Behring Inst. Mitt. 85:64 (1990) . 5. Torok, Ann. Intern. Med. 37:431 (1992) .

6. Sato, et al., J. Virol. 65:5485 (1990) .

7. Schwartz, et al . , J. Infect. Dis. 162:1214 (1990) .

8. Takahashi, et al., Am. J. Hemat. 37:68 (1991) .

9. Kurtzman, et al . , J. Clin. Invest. 84:1114 (1989) . 10. Frickhofen, et al . , Ann. Intern. Med. 113:926 (1990) .

11. Yaegashi, et al. , Microbiol. Immunol. 33:561 (1989) .

12. Sato, et al., J. Virol. 65:1667 (1991) .

13. Yoshimoto, et al. , J. Virol. 65:7056 (1991) .

14. Lane, et al., J.Exp. Med. 154:1043 (1981) . 15. Zolla-Pazner, et al. , Proc. Natl. Acad. Sci. (USA).

84:5404 (1987) .

16. Cohen, et al . , J. Hygiene. 91:113 (1983) . 17. Gorny, et al. , Proc. Natl. Acad. Sci. (USA). 86:1624

(1989) .

18. Teng, et al . , Proc. Natl. Acad. Sci. (USA). 80:7308

(1983) .

19. Gorny, et al., Proc. Natl. Acad. Sci. (USA). 88:3238

(1991) .

20. Kajigaya, et al., Proc. Natl. Acad. Sci. (USA). 88:4646 (1991) .

21. Collett, et al. , Virol. 165:200 (1988) .

22. Keegan, et al., J. Virol. 58:263 (1986) .

23. Sambrook, et al., Molecular cloning: A laboratory Manual, second edition Cold Spring Harbor Laboratory Press, New York. (1989) 24. Fridell, et al., Scand. J. Infect. Dis. 21:597 (1989) .

25. Schwarz, et al . , J. Virol. 66:1273 (1992) .

26. Gorny, et al . , J. Virol. (1992)

27. Ho, et al., Proc. Natl. Acad. Sci. (USA). 88:8949

(1991) .

28. Umino, et al., Kohama, T.S. Sato, A. Sugiura, H.D. Klenk, and R. Rott. 1990. Monoclonal antibodies to three structural proteins of Newcastle disease virus: biological characterization with particular reference to the conformational change of envelope glycoproteins associated with proteolytic cleavage. 1189.

29. Cheung, et al. , J ^" . Virol. 63:2445 (1989). 30. Anderson, et al. , Ann. Intern. Med. 102:275 (1985).

31. Masuho, et al. , J. Gen. Virol. 68:1457 (1987).

32. Mashoko, et al. , Develop. Biol . Standard. 71:127 (1990).

33. Public Health Laboratory Service Working Party on Fifth Disease 1990, Prospective Study of human parovirus (B19) infection in pregnancy. Brit. Med. J. 300:1166 (1990). 34. Rodis, et al. , Am. J. Obstet. Gynecol. 163:1168 (1990).

35. Kinney, J ^" . Infect. Dis. 157:663 (1988).