WO1999060130A1

WO1999060130A1 - Expression of recombinant trypanosoma cruzi complement regulatory protein (crp)

Info

Publication number: WO1999060130A1
Application number: PCT/US1999/010977
Authority: WO
Inventors: Karen A. Norris
Original assignee: University Of Pittsburgh Of The Commonwealth System Of Higher Education
Priority date: 1998-05-21
Filing date: 1999-05-18
Publication date: 1999-11-25

Abstract

The present invention provides a recombinant cassette for producing a T. cruzi CRP within a defined eukaryotic cell. Concomitantly, the invention pertains to eukaryotic cells harboring the recombinant CRP expression cassette and a method for producing CRP by introducing the recombinant CRP expression cassette into a eukaryotic cell. The invention is useful for producing isolated and purified recombinant CRP from cells. Such protein can be employed as a vaccine to prime the immune system of an animal. The invention also concerns hybridomas secreting antibodies recognizing CRP and anti-CRP monoclonal antibodies produced therefrom. The availability of isolated CRP or antibodies recognizing CRP affords a rapid and reliable assay for detecting Chagas's disease-related proteins, and the invention provides kits for performing such assays.

Description

EXPRESSION OF RECOMBINANT TRYPANOSOMA CRUZ! COMPLEMENT REGULATORY PROTEIN (CRP)

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER 5 FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with support under Public Health Service Award AI32719 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. The United States Government may have certain rights in this invention. 0

TECHNICAL FDZLD OF THE INVENTION

The present invention relates to the expression of Trypanosoma cruzi complement regulatory protein within eukaryotic cells other than T. cruzi trypomastigotes. 5

BACKGROUND OF THE INVENTION

T. cruzi is the protozoan responsible for Chagas' disease, a lethal infection infecting roughly 20 million people. Initial T. cruzi infection in humans typically leads to acute myocarditis, which most patients survive. Following this, the disease 0 typically passes to an asymptomatic phase of indefinite duration. Approximately 30% of infected individuals progress to a chronic phase, most commonly with cardiovascular disturbances, including diffuse myocardial damage and conduction defects.

The life cycle of T. cruzi cycles between two hosts: insects and mammals. 5 During the insect phase (epimastigotes), the parasite survives from a bloodmeal taken from a paracitemic host. The epimastigotes divide within the midgut of the insect host and eventually convert to metcyclic trypanosomes, which are passed from the insect in feces. Through wounds or mucous membranes, metacyclic trypanosomes enter a mammalian host, shortly thereafter entering mammalian cells. 0 Within mammalian cells, the trypanosomes convert to a dividing amastigote stage. The amastigotes multiply within infected cells, ultimately leading to cell rupture. However, prior to rupture, the amastigotes convert to a pathogenic trypomastigote stage. Trypomastigotes survive extracellularly in the bloodstream and are disseminated thereby to various tissues of the host mammal. In addition to 5 transmission via insects, infectious trypomastigotes are transmittable through direct contact with bodily fluids of an infected individual. In this light, the transmission of Chagas' Disease through blood transfusion in the United States has been recognized for some time.

T. cruzi has evolved multiple defenses for evading host immune surveillance. For example, the conversion of the parasite from the insect stage to the infectious blood stage is characterized by a transition from a complement-sensitive form to a complement-resistant form (Anziano et al., Immunity, 6, 860-66 (1972); Kipnis et al, Proc. Nat. Acad. Sci. USA, 78, 602 (1981); Noguiera et al, J. Exp. Med., 142, 224-29 (1975)). Infectious T. cruzi resist complement lysis by producing a 160 kDa membrane-bound complement regulatory protein (CRP) that interferes with the activation and amplification of both classical and alternative complement attack (Norris et al.. J. Immunol, 147, 2240-47 (1991); Norris et al., Infect. Immun., 62, 236-43 (1994)). A nucleic acid for the 160 kDa CRP has been isolated and cloned in bacteria (Norris et al., Infect. Immun., 65, 349-57 (1997)). Additionally, roughly 700 putative CRP nucleic acids may be present in the T. cruzi genome (see, e.g., Id.; Van Voorhis et al., J. Exp. Med, 178, 681-94 (1993)).

Diagnosis of Chagas' disease commonly is accomplished by identification of parasites in the blood, cerebrospinal fluid, fixed tissue or lymph node during periods of high fever; however, the organisms may be difficult to detect during the latent (or so-called indeterminant) phase, or during chronic stages of infection. In xenodiagnosis. the intestinal contents of insect vectors are examined for T. cruzi several weeks after these parasites feed on the blood of a suspected patient. However, this procedure is laborious and lacks sensitivity (see E. L. Segura, Xenodiagnosis in Chagas ' Disease Vectors, R. R. Brenner et al., eds., 11, 41-45, Boca Raton, Fla.. CRC Press (1987)). There is little available treatment for Chagas' disease. Two chemotherapeutic agents (BENZNIDAZOLE (Roche) and NIFURTIMOX, (Bayer)) are somewhat effective to treat the acute phase of the disease; however, both of these have significant side effects. Neither of these agents are effective during the chronic phase. Some polyclonal antisera raised against the CRP have been demonstrated to neutralize the CRP, resulting in increased killing of trypomastigote stage T. cruzi (Norris et al, Infect. Immun., 57, 2372-77 (1989); Norris et al, 1991. supra). This finding suggests that the CRP might serve as the basis for a possible vaccine. However, because trypomastigotes are difficult to culture in high density, it is not feasible to isolate sufficient quantities of protein from infectious parasites for such purposes. As such, no vaccine against T. cruzi exists. In view of the foregoing problems, there exists a need for methods and systems for the expression of T. cruzi CRP within eukaryotic cells. Moreover, there is a need for a rapid and reliable assay with standardized components for diagnosing Chagas^* disease and/or for protecting the blood supply from T. cruzi.

SUMMARY OF THE INVENTION The present invention provides a recombinant cassette for producing a T. cruzi CRP within a defined eukaryotic cell. Concomitantly, the invention pertains to eukaryotic cells harboring the recombinant CRP expression cassette and a method for producing CRP by introducing the recombinant CRP expression cassette into a eukaryotic cell. The invention is useful for producing isolated and purified recombinant CRP from cells. Such protein can be employed as a vaccine to prime the immune system of an animal. The invention also concerns hybridomas secreting antibodies recognizing CRP and anti-CRP monoclonal antibodies produced therefrom. The availability of isolated CRP or antibodies recognizing CRP affords a rapid and reliable assay for detecting Chagas' disease-related proteins, and the invention provides kits for performing such assays. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the following detailed description.

DETAILED DESCRIPTION The present invention provides methods and systems for the expression of CRP within defined eukaryotic cellular environments other than trypomastigote- phase T. cruzi. In one aspect, there is provided a recombinant cassette for expressing the CRP nucleic acid within a eukaryotic cell to a produce a CRP in biologically active form. At a minimum, such an expression cassette includes a nucleic acid encoding a CRP operably linked to a promoter for expressing the nucleic acid in the eukaryotic cell of interest.

Nucleic acid sequences from which a CRP nucleic acid can be derived are set forth at SEQ ID NO: 1 and SEQ ID NO:2. In particular, SEQ ID NO: 1 is one full length CRP coding sequence from strain Y of T. cruzi (Norris et al., 1997, supra). By virtue of this disclosure, one of skill in the art is equipped to employ mutant forms of the disclosed exemplary nucleic acids to produce functional CRP. Alternatively, a CRP nucleic acid can be cloned anew from T. cruzi. Genetic sequences can vary between different strains of T. cruzi, and this natural scope of allelic variation is included within the scope of the invention. Additionally and alternatively, the sequence can include one or more point mutations from the exemplary sequences or a naturally occurring CRP nucleic acid. Thus, within the context of the present invention, a CRP nucleic acid is not limited to SEQ ID NO:l or SEQ ID NO:2, but can, in some contexts, encode or comprise an active fragment of these sequences or insertion, deletion, or substitution mutants. Preferably, any mutation is conservative in that it minimally disrupts the biochemical properties of the encoded CRP. Thus, where mutations are introduced to substitute amino acid residues, positively-charged residues (H, K, and R) preferably are substituted with positively-charged residues; negatively-charged residues (D and E) preferably are substituted with negatively-charged residues; neutral polar residues (C, G, N, Q, S, T. and Y) preferably are substituted with neutral polar residues; and neutral non-polar residues (A, F, I, L, M, P, V, and W) preferably are substituted with neutral non-polar residues.

Considering this range of variation, a nucleic acid encoding a CRP is any sequence that confers protection from complement lysis when expressed within insect phase T. cruzi epimastigotes (which, as mentioned, are normally sensitive to complement lysis). While any nucleic acid conferring this protection is within the scope of the present invention, such nucleic acids typically are homologues of SEQ ID NO: 1 (e.g., they will hybridize to at least a fragment of SEQ ID NO: 1 under at least mild stringency conditions, more preferably under moderate stringency conditions, and most preferably under high stringency conditions (employing the definitions of mild, moderate, and high stringency set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press (1989))). Thus, a CRP nucleic acid is typically at least about 75 % homologous to SEQ ID NO:l and preferably is at least about 80 % homologous to SEQ ID NO:l (e.g., at least about 85 % homologous to SEQ ID NO: 1 ); more preferably the CRP nucleic acid is at least about 90 % homologous to SEQ ID NO:l (such as at least about 95 % homologous to SEQ ID NO:l), and most preferably the CRP nucleic acid is at least about 97 % homologous to SEQ ID NO: 1.

Any promoter and/or enhancer sequence appropriate for controlling expression of nucleic acids within the desired eukaryotic cell can be used in the inventive recombinant CRP expression cassette. Many such promoter/enhancer elements are well known in the art. For example, the promoter can be a viral promoter, several of which are commonly employed in eukaryotic expression cassettes (e.g., retroviral ITRs, LTRs, immediate early viral promoters (IEp) (such as herpesvirus IEp, cytomegalovirus (CMV) IEp, etc.), Rous Sarcoma Virus (RSV) promoters, Murine Leukemia Virus (MLV) promoters, baculoviral promoters, etc.). Other suitable promoters are derived from eukaryotic DNA, such as yeast-derived promoters, eukaryotic enhancers (e.g., the rabbit β-globin regulatory elements), constitutively active promoters (e.g., the β-actin promoter, etc.), signal specific promoters (e.g., inducible promoters such as a promoter responsive to metallothionen, RU486, etc.), and tissue-specific promoters (e.g., those active in epidermal tissue, dermal tissue, tissue of the digestive organs (e.g., cells of the esophagus, stomach, intestines, colon, etc., or their related glands), smooth muscles, such as vascular smooth muscles, cardiac muscles, skeletal muscles, lung tissue, hepatocytes, lymphocytes, endothelial cells, sclerocytes, kidney cells, glandular cells (e.g., those in the thymus, ovaries, testicles, pancreas, adrenals, pituitary, etc.), tumor cells, cells in connective tissue, cells in the central nervous system (e.g., neurons, neuralgia, etc.), cells in the peripheral nervous system, and other cells of interest). The choice of promoter will depend largely on the eukaryotic cell type in which the CRP is to be produced. However, it is within the skill of the art to select a promoter appropriate for expressing nucleic acids in a given cell type. Aside from the CRP nucleic acid and the promoter, the expression cassette can contain other genetic elements. For example, the cassette can contain polyadenylation sequences, repressors, enhancers, splice signals, signals for secretion (see, e.g., U.S. Patent 4,845,046 and European Patent EP-B-319,641), etc. Moreover, the expression cassette can include more than one gene, (e.g., multiple genes separated by internal ribosome entry sites). Despite the presence of any such other genetic element, within the recombinant expression cassette, the CRP nucleic acid is operably linked to the promoter. Generally, such operable linkage is achieved when an event at the promoter (i.e., binding of cellular transcription factors and machinery) precipitates an event at the CRP nucleic acid (i.e., transcription). The recombinant expression cassette including the CRP nucleic acid and the promoter generally is constructed by standard molecular biological techniques. However, due to the complexities of T. cruzi genetics, achieving operable linkage between a CRP nucleic acid and a eukaryotic promoter typically requires some manipulation, particularly of the CRP nucleic acid. In particular, T. cruzi genes often contain multiple ATG codons, which hinder translation in contexts other than the T. cruzi chromosomes. For example, the CRP nucleic acid set forth at SEQ ID NO:l contains three putative start sites (at positions 121, 145, and 245, respectively). Considering this, an isolated CRP nucleic acid should be manipulated to contain an ATG translation initiation site conforming to the 24 amino acid eukaryotic signal sequence consensus and, desirably, also consistent with the amino acid sequence identifying the start of the mature CRP protein. While it has not yet been determined whether the Kozak consensus sequence is important in trypanosome genetic expression, the nucleotide sequence surrounding the ATG used as the translation initiation start site should be consistent with the Kozak consensus for translation in other cellular contexts (e.g., mammalian cells, insect cells, etc.). Additionally, as mentioned, functional CRP is a membrane-bound protein. However, the T. cruzi glycosylphosphatidyl inositol (GPI) anchor addition sequence (the GDS residues at 3163 in SEQ ID NO:l) is not recognized, or is poorly processed, in many eukaryotic cells. Accordingly, an appropriate anchor addition sequence (many of which are known in the art) desirably is engineered into the CRP nucleic acid for proper expression of the protein in a non- J¹. cruzi cell. To preserve proper function of the CRP protein, the anchor addition sequence preferably is a GPI anchor addition sequence. Manipulating DNA to include these sequence modifications is within the skill of the art.

To use the inventive recombinant expression cassette, it must be introduced into a eukaryotic cell in a manner suitable for the cell to express the CRP nucleic acid. Any suitable vector can be employed to deliver the recombinant CRP expression cassette into the desired cells, many of which are known in the art. Examples of such vectors include naked DNA vectors (such as oligonucleotides or plasmids), viral vectors such as adeno-associated viral vectors (Berns et al., Ann. N Y. Acad. Scl, 772, 95-104 (1995)), adenoviral vectors (Bain et al, Gene Therapy, 7, S68 (1994)), baculovirus vectors (see, e.g., Luckow et al., Bio/Technology, 6, 47 (1988)), herpesvirus vectors (Fink et al., Ann. Rev. Neurosci., 19, 265-87 (1996)), packaged amplicons (Federoff et al., Proc. Nat. Acad. Sci. USA, 89, 1636- 40 (1992)), papilloma virus vectors, picornavirus vectors, polyoma virus vectors, retroviral vectors, SV40 viral vectors, vaccinia virus vectors, and other vectors. In addition to the recombinant CRP expression cassette, the vector can also include other genetic elements, such as, for example, cassettes for expressing a selectable marker (e.g., β-gal or a marker conferring resistance to a toxin), a pharmacologically active protein, a transcription factor, or other biologically active substance. Once a given type of vector is selected, its genome must be manipulated for use as a background vector, after which it must be engineered to incorporate the inventive recombinant CRP expression cassette. Methods for manipulating the genomes of vectors are well known in the art (see e.g., Sambrook et al., supra)) and include direct cloning, site specific recombination using recombinases, such as the flp recombinase or the cre-lox recombinase system (reviewed in Kilby et al. Trends Genet., 9, 413-21 (1993)), homologous recombination, and other suitable methods of constructing a gene-transfer vector. In this manner, the recombinant CRP expression cassette can be inserted into any desirable locus of the vector. Such insertions can disrupt one or more genes present in the native vector, if desired, or the expression cassette can be inserted between genetic elements to minimize perturbation of the vector genome. Indeed, as certain promoters are already present in viral or plasmid vectors, a recombinant CRP expression cassette according to the present invention can comprise a native vector promoter operably linked to the CRP nucleic acid.

Where the vector is a viral vector, preferably it is replication incompetent. Thus, for example, an adenoviral vector preferably has an inactivating mutation in at least the El A region, and more preferably in region El (i.e., El A and/or E1B) in combination with inactivating mutations in region E2 (i.e., E2A, E2B, or both E2A and E2B). and/or E4 (see, e.g., International Patent Application WO 95/34671). An AAV vector can be deficient in AAV genes encoding proteins associated with DNA or RNA synthesis or processing or steps of viral replication (e.g., capsid formation) (see U.S. Patents 4,797,368, 5,354,768, 5,474,935,

5,436,146, and 5,681,731). Where the vector is a retroviral vector, the cis-acting encapsidation sequence (E) essential for virus production in helper cells can be deleted upon reverse transcription in the host cell to prevent subsequent spread of the virus (see, e.g., U.S. Patent 5,714,353). Where the vector is a herpesvirus, inactivation of the ICP4 locus and/or the ICP27 cassette renders the virus replication incompetent in any cell not complementing the proteins (see, e.g., U.S. Patent 5,658,724. see also DeLuca et al., J. Virol., 56, 558-70 (1985); Samaniego et al, J. Virol, 69(9), 5705-15 (1996)).

A vector harboring the recombinant CRP expression cassette is introduced into a eukaryotic cell by any method appropriate for the vector employed. Many such methods are well-known in the art (Sambrook et al., supra; see also Watson et al., Recombinant DNA, Chapter 12, 2d edition, Scientific American Books (1992)). Thus, plasmids are transferred by methods such as calcium phosphate precipitation, electroporation, liposome-mediated transfection, microinjection, viral capsid-mediated transfer, polybrene-mediated transfer, protoplast fusion, etc. Viral vectors are best transferred into the cells by infecting them.

Of course, the choice of vector, as well as the mode of delivery, depends to a large extent on the desired eukaryotic cells to be employed. Thus, for example, where it is desired to introduce the CRP expression cassette into insect cells, a baculovirus vector can be employed. Furthermore, certain methods for transfecting cells in vitro are less effective where cells are in vivo, and vice versa. Varying the vector choice and the mode of delivery in accordance with the desired cell type and location, however, is within the ordinary skill of the art.

As the vector is useful for transferring the CRP expression cassette to cells, the present invention provides a eukaryotic cell harboring a recombinant CRP expression cassette. Preferably, the eukaryotic cell presents a suitable microenvironment for the CRP nucleic acid within the expression cassette to be expressed such that the cell produces CRP. Any cell suitable for expressing the CRP nucleic acid can be employed in the context of the present invention. Depending on the use to which the cells are to be put, they can be either in vivo or in vitro. Where the cells are in vivo, they are typically cells of a mammal (e.g., human cells), and can be any type of cells (e.g., as described above with respect to tissue-specific promoters). Suitable cells for use in vitro include yeast, protozoa (e.g., T. cruzi epimastigotes). cells derived from any mammalian species (e.g., VERO, CV-1, COS-1, COS-7. CHO-K1, 3T3, NIH/3T3, HeLa, C1271, BS-C-1 MRC-5, etc.), insect cells (e.g., Drosophila Snyder cells), or other such cells.

Because the cells harboring the CRP expression cassette preferably express the CRP nucleic acid, the invention provides a method for producing CRP. In accordance with the inventive method, a recombinant CRP expression cassette is introduced into a eukaryotic cell, as described, such that the CRP nucleic acid is expressed and CRP is produced. Preferably, the cell processes the resulting CRP such that it is inserted into the cell membrane and present on the surface of the cell.

The ability to produce the T. cruzi CRP in novel eukaryotic cellular environments is useful in several respects. Where the eukaryotic cell harboring the recombinant CRP expression cassette is in vitro, the method permits the isolation and purification of CRP from the cells. In some protocols, where the expression cassette also includes a leader sequence, the recombinant protein is secreted from the cells into the culture medium, from where it can be recovered, for example, using substrates having ligands for CRP (e.g., human C3b (Norris et al., 1991, supra), an antibody molecule recognizing CRP (Norris et al., 1997, supra), etc.). In other applications, the CRP protein is not secreted, but is isolated from the membranes of producing cells. Such cells can be lysed, and the CRP produced from the cells purified. Because the CRP is a membrane-bound protein, to enrich the lysate for CRP the membrane fraction preferably is first purified from other lysate components and then exposed to such substrates. Any suitable protocol can be employed to purify the CRP from the cells, and many protein-purification protocols are known in the art. For example, the CRP- substrate particles can be immunoprecipited. In other protocols, the substrate is bound to a support, (e.g., those commonly employed for chromatographic or affinity assays, some of which are set forth below). Thus, for example, the cell lysate (or fraction thereof) can be passed through a column comprising the support onto which the substrate is bound, mixed with a slurry of such a support (e.g., beads or other preparation comprising the support-bound substrate), placed into a container (e.g., a tube, the well of a dish, etc.) which has been coated with the substrate, or otherwise exposed to the substrate. The CRP within the cell lysate selectively binds the ligands on the substrate, and thereby is separated from the remainder of the cell lysate not bound to the substrate. Subsequently, the CRP is eluted from the substrate. Repeated rounds of such a protocol produces isolated and substantially purified CRP.

Where the eukaryotic cell harboring the recombinant CRP expression cassette is in vivo, the method permits the production of CRP within the animal (particularly a mammal). The production of CRP within the animal will lead to a primary immune response directed against the cells expressing the CRP nucleic acid. An immunocompetent mammal will, thereby, produce antibodies recognizing the CRP. In one application, priming a mammalian immune system to recognize the CRP protein can guard against subsequent T. cruzi infection. Thus, a recombinant CRP expression cassette can be used as a DNA vaccine. Alternatively, the animal could be bled or milked to recover the anti-CRP antibodies for disparate uses. In still other applications, splenocytes from mammals producing anti-CRP antibodies can be harvested for the production of hybridomas secreting monoclonal antibodies directed against CRP by standard methods. The invention concerns such hybridoma and anti-CRP monoclonal antibodies produced therefrom (e.g., IgG, IgM, IgA, IgE, etc.).

In other applications, isolated and purified CRP (e.g., from cells in vitro described above), and or antibodies recognizing CRP, can be employed as diagnostic agents for detecting the presence of T. cruzi antigens or antibodies within a test fluid. While such a method can detect the presence of CRP and/or antibodies recognizing CRP in any test fluid, preferably the test fluid is drawn from a mammal (e.g., blood, milk, cerebrospinal fluid, lymph, mucus, saliva, semen, tears, sweat, etc.), or derived from such fluids (e.g., serum, packed red cells, whey, etc.). In such applications, a positive reaction is an indication that the individual from which the test fluid was drawn or derived has been infected with T. cruzi. Such an indication can prompt early intervention to treat the individual for Chagas' disease. Moreover, where the test fluid is blood product, a positive reaction can protect a potential recipient of that blood from Chagasic infection, thereby protecting the blood supply. In one protocol of the method, an isolated ligand recognizing CRP (e.g., an antibody molecule, human C3b, etc.) can be used in conjunction with other reagents for detecting the presence of CRP in a test fluid by contacting an aliquot of the test fluid with the ligand for a time and under conditions sufficient to form antigen/ligand complexes, contacting the antigen/ligand complexes with an indicator for a time and under conditions sufficient to form antigen/ligand/indicator complexes, and detecting the presence of CRP by measuring a signal generated by the indicator. In another protocol, isolated CRP (such as described herein) can be used in conjunction with other reagents, to detect the presence of an antibody recognizing CRP in a test fluid by contacting an aliquot of the test fluid with the isolated CRP for a time and under conditions sufficient to form antigen/antibody complexes, contacting the antigen/antibody complexes with an indicator for a time and under conditions sufficient to form antigen/antibody/indicator complexes, and detecting the presence of CRP by measuring the signal generated. In either protocol, the indicator includes a label conjugated to a specific binding member of either the isolated ligand or CRP. Thus, the indicator produces a detectable signal in the presence of the antibody or CRP, if any, present in the test fluid. In some embodiments, the signal can be relative to the amount of antibody or CRP, if any, present in the test fluid. The label component of the indicator is capable of generating a measurable signal detectable by external means. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances. Examples of commonly employed labels include chromagens; catalysts such as enzymes (e.g., horseradish peroxidase, alkaline phospatase, β-galactosidase, etc.); luminescent compounds (fluorescein and rhodamine); chemiluminescent compounds (e.g., acridinium compounds, phenanthridinium compounds, dioxetane compounds, etc.); radioactive elements: direct visual labels; and other similar labels. The specific binding member of the indicator can be a member of any specific binding pair, (e.g., biotin or avidin. carbohydrate or lectin, a complementary nucleotide sequence, effector or receptor molecule, enzyme or its cofactor or inhibitor, etc.). An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to the analyte as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay. If an antibody is used, it can be a monoclonal antibody, a polyclonal antibody, an antibody fragment (ScAbs, FaBs, etc.), a recombinant antibody, a mixture thereof, or a mixture of an antibody and other specific binding members. Considering the wide varieties of labels and specific binding members, the indicator will vary depending upon the type of assay performed. Thus, the inventive method can follow any protocol suitable for detecting the interaction of CRP with an antibody (or other ligand) recognizing CRP, and many such protocols are known in the art. Of course, to aid in any such protocol, other reagents can be employed. Thus, for example, for immunoprecipitation, Protein A can be employed to precipitate aggregates of antibody/CRP complexes. Where appropriate (e.g., for solid phase assays or ELISA), the isolated ligand or isolated CRP can be conjugated to a solid support, such as those described below or otherwise known in the art. To effectuate the inventive method, the invention provides a test kit for detecting the presence of CRP and or antibodies recognizing CRP in any test fluid. The kit includes, as a first element, either isolated CRP or an isolated ligand recognizing CRP. Additionally, the kit includes an indicator for producing a detectable signal at a level relative to the amount of the antibody or CRP, if any. present in the test fluid (e.g., those indicators described above), and reagents suitable for detecting the signal generated by the label. Additionally, the kit can include a negative control fluid (i.e., a fluid lacking either the CRP or antibody recognizing CRP to be detected) and/or a positive control fluid (i.e., a fluid having the CRP or antibody recognizing CRP to be detected). Where appropriate to the type of immunological assay to be employed, the kit can include a solid support for either the isolated antibody or isolated CRP. Examples of such supports include metals; natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and magnesium; aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass (these materials may be used as filters with the above polymeric materials); mixtures or copolymers of the above classes, such as graft copolymers; and other materials commonly employed in chromatographic or affinity separation. Such supports can be fashioned into beads, films, sheets, plates, etc., or coated onto, bonded, laminated, or otherwise joined to appropriate inert carriers, such as paper, glass, polymeric films, fabrics, etc. The kit also can include other suitable reagents (e.g., Protein A), where appropriate.

EXAMPLES

While it is believed that one of skill in the art is fully able to practice the invention after reading the foregoing detailed description, the following examples further illustrate some of its features. In particular, they demonstrate recombinant expression cassettes for producing CRP in eukaryotic cells other than infectious T. cruzi trypomastigotes, eukaryotic cells harboring recombinant CRP expression cassettes, and the production of functional CRP by such cells. As these examples are included for purely illustrative purposes, they should not be construed to limit the scope of the invention in any respect.

The procedures employed in these examples, such as affinity chromatography. Southern blots, PCR, DNA sequencing, vector construction (including DNA extraction, isolation, restriction digestion, ligation, etc.), cell culture (including G418 selection), transfection of cells, protein assays (Western blotting, immunoprecipitation, immunofluorescence), etc. are techniques routinely performed by those of skill in the art (see generally Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies: a Laboratory Manual, Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press (1989)). Accordingly, in the interest of brevity, the experimental protocols are not discussed in detail.

EXAMPLE 1

This example demonstrates the construction of a recombinant expression cassette for producing CRP in T. cruzi insect-phase epimastigotes, which, as mentioned above, do not normally express CRP.

For a first attempt at creating such a recombinant expression cassette, a 3 kb fragment containing the entire CRP- 10 coding region plus 53 bp upstream of the putative start site (SEQ ID NO:l) was cloned into the EαmHI and Xhol sites of pTEX a vector able to replicate in E. coli and T. cruzi (Kelly et al., Nucl. Acids Res., 20. 3963-69 (1992)). This vector confers resistance to G418, which can be used as an assay to detect successful transfection. The resulting plasmid produced G418- resistant cell lines when transfected into epimastigotes; however, no CRP mRNA or protein could be detected in these cells.

It was hypothesized that the failure of epimastigotes to successfully express the CRP expression cassette within the plasmid might be due to the presence of three putative start sites in the sequence. Therefore, the upstream sequence of SEQ ID NO:l was removed, and only the coding sequence from nucleotide 234 was cloned into pTEX in the BamHl dXhol sites. During cloning, restriction digests of several of the E. coli transformants revealed gross rearrangements of the insert, indicating instability within the E. coli host, possibly due to a prokaryotic promoter upstream of the T. cruzi expression cassette.

To derive a more stable construct, the entire expression cassette of pTEX from the SαcTI site through the Kpn\ site was inverted and recloned into the pTEX vector backbone. The CRP- 10 fragment from the putative translational start at nucleotide 235 through nucleotide 3253 of SEQ ID NO:l was cloned into this modified vector, and stable E. coli transformants were isolated. One isolate (pTEX- CRP) was verified by nucleic acid sequencing, and used for epimastigote transfection.

EXAMPLE 2 This example demonstrates the expression of recombinant CRP in non- infectious insect-phase T. cruzi epimastigotes.

The pTEX-CRP plasmid described in Example 1 was introduced into strain Y T. cruzi epimastigotes by electroporation, which were then assayed for sensitivity to G418 to determine successful transformation. As a control for G418 resistance, wild-type epimastigotes were tested for sensitivity to G418, and it was found that at a concentration of 50 mg/ml all the parasites were dead at approximately 10 days. Control cells electroporated with no DNA and cultured in the presence of G418 were dead by 10 to 14 days post-electroporation. Parasites transfected with either pTEX or pTEX-CRP yielded G418-resistant cell lines. After stable lines were established (approximately 4 weeks post-transfection), the G418 concentration was increased at 2 week intervals to 100 mg/ml, 200 mg/ml and 500 mg/ml. No detectable differences in growth rates of parasites were seen during the antibiotic increases. In addition, no detectable differences in growth rates were observed between epimastigotes transfected with the pTEX vector alone and those transfected with the pTEX-CRP construct. Two transfected cell lines (CRP 2.3 and CRP 2.14) were derived from separate transfections and maintained in supplemented culture medium with 500 mg/ml G418. Total RNA was isolated from the transfected cell lines, and RT-PCR was performed to determine whether the CRP nucleic acid was being expressed in the transfected epimastigotes. The expected product of the RT-PCR was a 1330 bp fragment covering the 5' end of CRP. A fragment of this size was detected by ethidium bromide staining of agarose gel electrophoresis of the RT-PCR product from the epimastigote cell lines, and by Southern blotting using a CRP-specific probe.

To verify that the signal observed was due to reverse transcription and amplification of the CRP nucleic acid, and not due to amplification of trace amounts of genomic CRP DNA in the RNA preparations, a mock RT-PCR was carried out on RNA from the cells without the addition of reverse transcriptase. No band was detected by ethidium bromide staining or Southern blotting, confirming that the transfected epimastigotes were transcribing the trypomastigote-specific CRP cDNA. Likewise, no band corresponding to CRP was detected in RT-PCR product of epimastigotes transfected with the vector alone, again confirming that expression of CRP is developmentally regulated and specific for the trypomastigote stage.

EXAMPLE 3

This example demonstrates the expression of a recombinant CRP nucleic acid in non-infectious insect-phase T. cruzi epimastigotes to produce a functional CRP protein.

Protein was isolated from the transfected cell lines described in Example 2 and subjected to Western blot analysis to determine whether CRP protein was produced within the transfected cells. Epimastigotes were grown to log phase, and detergent-solubilized. membrane enriched protein extracts were Western blotted. Polyclonal antiserum raised against the full length recombinant CRP expressed in E. coli (Norris et al.. 1997, supra) was used to detect CRP. A protein migrating at approximately 100 kDa was detected in the extracts derived from the CRP 2.3 and CRP 2.14 cell lines, but not in extracts prepared from the pTEX cell line. The difference in apparent molecular mass between the epimastigote-derived recombinant CRP and the native trypomastigote protein is approximately 60 kDa. To determine whether this 100 kDa recombinant protein was a functional CRP, metabolically labeled membrane proteins from pTEX and CRP 2.3 epimastigote cell lines were subjected to affinity chromatography on a human C3b-affinity matrix. Radiolabeled material in the membrane extracts was quantitated, and equal amounts of radiolabeled protein from pTEX and CRP 2.3 membrane preparations were loaded onto a C3b matrix. Proteins specifically bound to the C3b matrix were eluted and separated by SDS-PAGE followed by fluorography. Similar to the results of the Western blot, epimastigotes transfected with the pTEX-CRP construct produced a C3b-binding protein which migrated at approximately 100 kDa, and smaller proteins migrating in the 40 to 60 kDa range, possibly representing proteolytic degradation. Membrane preparations from CRP 2.14 cells gave similar results.

To test for functional activity of the recombinant epimastigote CRP in the intact cell, parasites were grown to log phase and treated with either normal guinea pig serum (as a source of complement) or heat- inactivated serum at a final dilution of 1 :2. As shown in Table 1, CRP 2.14 epimastigotes exhibited high levels of resistance to complement-mediated lysis compared to epimastigotes transfected with the vector alone. Decreasing the complement concentration by 50%, increased the survival of CRP-transfected epimastigotes to 100 %; however, 35 % to 40 % of the vector-transfected cells survived this treatment. Complement-mediated lysis assays with CRP 2.3 cells produced comparable results.

TABLE 1

^a Parasites were incubated for 60 min at 28°C in either fresh guinea pig complement (C) or heat-inactivated guinea pig complement (HIC), at a final dilution of 1 :2.

° Values are the mean numbers of motile parasites counted in a hemocytometer at a magnification of 400 X. Each value is the mean ± SD of quadruplicate samples. The results shown are representative of multiple experiments. ^c % Survival = 100 X the number of motile parasites treated with C/ number of motile parasites treated with HIC. Each treatment was carried out in duplicate, and duplicate counts were made of each sample. The results shown are representative of multiple individual experiments.

EXAMPLE 4

This example demonstrates the production of T. cruzi CRP in mammalian cells using a recombinant CRP expression cassette.

The initial attempts at expression of the T. cruzi CRP nucleic acid in a mammalian expression system were conducted with the expression plasmid pREP9 (INNITROGEN) which drives expression from the RS V-LTR promoter and has a SV40 poly-A addition signal sequence at the 3' end. Cloning of the full length CRP nucleic acid (SEQ ID NO:l) into this plasmid produced a construct which was transfected into COS cells. Cellular lysates were prepared and immunoprecipitated using polyclonal antiserum raised against the full length recombinant CRP expressed in E. coli (Norris et al., 1997, supra). The immunoprecipitation assays revealed the presence of no cross-reactive protein.

It was hypothesized that the failure of COS cells to successfully express the CRP expression cassette within the pREP9 vector might be due to the presence of three putative start sites in the sequence. Therefore, the CRP nucleic acid was modified such that nucleotides 1-270 were removed and BamHI linkers were added at the 5^" end to facilitate cloning into the BamHI site of pBC12BI, which provides the initiation codon and the first 5 codons of the rat preproinsulin gene upstream of the BamHI cloning site (Cullen, Meth. Enzymol, 152, 684-705 (1987)). This cloning strategy removed the first 8 amino acids of the signal sequence of CRP- 10 and replaced them with the upstream sequence and first 6 amino acids of preproinsulin II gene (plus a lysine codon) including the initiation signal.

The resulting construct (pBC12BI-271) was transfected into COS cells, and cellular lysates were prepared and immunoprecipitated using an antiserum recognizing full length recombinant CRP (Norris et al., 1997, supra). The immunoprecipitation assays revealed the presence of an approximately 140 kDa CRP cross-reactive protein. Transfected COS cells also were prepared for immunofluorescence staining using the CRP antiserum. No staining was detected on the surface of the transfected cells, although a strong signal was observed in the cytoplasm. The lower-than-expected molecular weight of the translated protein, and the lack of cell-surface staining indicated that the CRP expression cassette was transcribed, and protein was produced within the transfected COS cells. However, the protein was not properly processed for export to the cell surface.

Upon analysis of the protein sequence, it was hypothesized that the CRP protein contains a GPI membrane anchor addition sequence (including the GDS residues encoded by nucleotides 3163-3173 of SEQ ID NO:l) that was inefficiently recognized by the COS cells. Thus, the carboxy terminal end of the CRP nucleic acid was modified to promote surface production in mammalian cells. The predicted GPI anchor addition site of CRP was removed and replaced with the carboxy terminal sequence of the mammalian decay accelerating factor gene, which has a

GPI sequence recognized by mammalian cells (Davitz et al., J. Exp. Med. 163, 1150 (1986)). The sequence of the resulting nucleic acid is set forth at SEQ ID NO:2. This construct (pBC12BI-CRP/DAF) was transfected into COS cells which were assayed as described above.

Immunoprecipitation of cell lysates from COS cells transfected with pBC12BI-CRP/DAF revealed a CRP-cross-reacting recombinant protein of the proper molecular weight (160 kDa). Immunofluorescence assays using the CRP antiserum resulted in strong staining on the surface of COS cells transfected with pBC12BI-CRP/DAF. To determine that a GPI anchor was successfully incorporated into the recombinant CRP, the COS cells were treated with phosphatidylinositol specific phospholipase C, which specifically cleaves GPI anchors and releases the protein from the surface. Immunoprecipitation assays of the cell culture supernatant revealed that recombinant CRP was released by this treatment.

EXAMPLE 5

This example demonstrates the production of hybridomas secreting monoclonal antibodies directed against CRP.

Six week old, female BALB/c mice were immunized by intramuscular injection of the purified pBC12BI-CRP/DAF plasmid described in Example 4 (100 mg DNA in 50 ml PBS). After 4 injections, antibody titers were determined by ELISA using E. co//-derived recombinant CRP as the target antigen. Mice with serum titers in excess of 1 :3000 were boosted once more, and the spleens were harvested 5 days after the last boost. Fusion of spleen cells and selection of immunoglobulin-secreting hybridomas were carried out by standard methods. Hybridomas were screened by ELISA with either recombinant CRP or T. cruzi- derived CRP as the target antigens. Thirteen hybridomas secreting antibodies against CRP were subcloned by limiting dilution.

All of the references cited herein, including patents, patent applications, and publications, are hereby incorporated in their entireties by reference.

While this invention has been described with an emphasis upon preferred embodiments and illustrative examples, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

WHAT IS CLAIMED IS:

I . A recombinant T. cruzi complement regulatory protein (CRP) eukaryotic expression cassette comprising a promoter operably linked to a nucleic acid encoding said CRP.

2. The recombinant expression cassette of claim 1, wherein said nucleic acid further encodes a secretion leader sequence.

3. A gene transfer vector comprising the recombinant expression cassette of claim 1 or 2.

4. A eukaryotic cell harboring the recombinant expression cassette of claim 1 or 2.

5. The eukaryotic cell of claim 4, which produces said CRP.

6. The eukaryotic cell of claim 4 or 5, which secretes said CRP.

7. The eukaryotic cell of any of claims 4-6, which is selected from the group of eukaryotic cells consisting of T. cruzi epimastigotes, mammalian cells, insect cells, and yeast.

8. The eukaryotic cell of any of claims 4-6, which is a cell in vitro.

9. The eukaryotic cell of any of claims 4-6, which is a cell in vivo.

10. A method for producing CRP, said method comprising introducing a recombinant expression cassette comprising a promoter operably linked to a nucleic acid encoding said CRP into a eukaryotic cell, whereby said nucleic acid is expressed and said CRP is produced.

I I. The method of claim 10, wherein said eukaryotic cell is selected from the group of eukaryotic cells consisting of T. cruzi cells, mammalian cells, insect cells, and yeast.

12. The method of claim 10, wherein said eukaryotic cell is a cell in vitro.

13. The method of claim 10, wherein said eukaryotic cell is a cell in vivo.

14. A monoclonal antibody recognizing CRP.

15. A hybridoma secreting antibodies recognizing CRP.

16. A kit for detecting the presence of CRP within a test fluid, said kit comprising a ligand recognizing said CRP, an indicator that produces a detectable signal in the presence of said CRP, and a reagent for detecting said signal.

17. The kit of claim 16, wherein said ligand recognizing said CRP is an antibody.

18. A kit for detecting the presence of CRP antibodies within a test fluid, said kit comprising CRP. an indicator that produces a detectable signal in the presence of said CRP antibodies, and a reagent for detecting said signal.