WO1999013903A1 - Methods and compositions for diagnosing and treating malaria - Google Patents

Abstract

Antibodies specifically reactive with lactate dehydrogenase (LDH) from the genus Plasmodium, such as Plasmodium falciparum, are disclosed. Also disclosed are methods of using the antibodies to specifically diagnose and treat malaria infections in an individual.

Description

METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING MALARIA

Background Malaria is a significant global health problem. It is widespread, and constitutes a growing health problem of major proportions, particularly in developing countries.

Malaria is caused by parasites of several species of the genus Plasmodium. the most virulent species being Plasmodium falciparum (P. falciparum). Parasites growing in erythrocytes are responsible for the pathological manifestations of the disease in man. During the blood stage of infection, P. falciparum parasites infect the cells and develop within the erythrocytes through three successive, morphologically distinct stages known as ring, trophozoites, and schizonts. A mature schizont eventually produces multiple infectious particles, known as merozoites, which are released upon rupture of the red blood cells. The merozoites invade new red blood cells after a short extracellular life in the blood.

The increasing resistance of P. falciparum to chemotherapy has stimulated molecular and biochemical analysis of parasite processes that may be selectively disrupted. Gene cloning combined with studies directed at expression of engineered genes encoding P. falciparum enzyme activities would permit more detailed structure and function studies of parasite enzymes and may help to eventually identify antimalarial drugs that can selectively inhibit certain parasite enzymes. One of the first malaria parasite enzymes shown to be electrophoretically and kinetically distinct from a corresponding host enzyme was lactate dehydrogenase (LDH; L-lactate: NAD⁺- oxidoreductase, EC 1.1.1.27) (Sherman, J. Exp. Med. Ji4: 1049- 1062 (1961); Sherman, Trans. N.Y. Acad. Sci Ser. 2 24:944-953 (1962); Sherman, in Proceedings of the First International Congress of Parasitology, (A. Cordetti, ed.), p. 73 (1966)). The kinetic properties of partially purified P. falciparum LDH differ significantly from those of two human LDH, LDH-H4 (B4) and LDH-M4 (A4), with respect to K_m (NADH) and K_m (pyruvate) (Vander Jagt et al., Mol. Biochem. Parasitol. 4:255-264 (1981)). While vertebrate LDH are markedly inhibited by pyruvate and the NAD⁺/pyruvate complex, P. falciparum LDH is totally insensitive to substrate inhibition (Vander Jagt et al., Mol. Biochem. Parasitol. 4:255-264 (1981)). Further, P. falciparum LDH is rapidly and completely inactivated by sodium deoxycholate at concentrations that do not inhibit LDH-H4 (B4), and which only slowly inactivate LDH-M4 (A4). Biochemical characterization of P. falciparum LDH from simian malaria parasites has also revealed significant differences in electrophoretic, biochemical, and kinetic characteristics from vertebrate LDH (Saxena et al., Mol. Biochem. Parasitol. 2]_: 199-202 (1986); Kaushal et al., Ind. J. Parasitol. 9:293-296 (1985)). While simian, mouse, avian, and human species of Plasmodium LDH are well conserved immunochemically, P. falciparum LDH is distinct from vertebrate LDH, helminth LDH, and other protozoan LDH (Watts et al., Biol. Mem. 13:161-164 (1987); Kaushal et a]., Infect. Immun. 17:507-516 (1988)). Glucose utilization in P. falciparum infected erythrocytes is as much as 100 times the rate observed in uninfected erythrocytes (Roth et al., Biochem. Biophys. Res. Commun. 109:355 (1982)). Lactate, which is produced from pyruvate by P. falciparum LDH activity, is the end product of glucose degradation in Plasmodium (Homewood and Neame, Ann. Trop. and Parasitol. 77:127-129 (1983)). P. falciparum LDH plays an important role in regulating glycolysis and in balancing the reduced/oxidized state of the malaria parasite (Sherman, in Antimalarial Drugs I (Peters and Richards, eds.), pp. 31 - 81 (1984); Fairlamb. Parasitol 99:93-1 12 (1989)). Many glycolytic enzymes have been observed in Plasmodium. and are distinct in some of their properties from corresponding host enzymes (Roth et al. Blood 72:1922-1925 (1988); Certa et al., Science 240:1036- 1038 (1988)). Several glycolytic enzyme genes of P. falciparum have been cloned, including fructose biphosphate aldolase (ALD) (Certa et al.. Science 240:1036-1038 (1988)), glucose phosphate isomerase (PGI) (Kaslow and Hill, J. Biol. Chem. 265:12337-12341 (1990)), AND 3 -phosphogly cerate kinase (PGK) (Hicks et al., Gene 100:123-129 (1991)). The malaria encoded LDH enzyme is unique among all known LDH in that it is the only LDH enzyme which efficiently uses 3-acetyl pyridine NAD (APAD) as a co-enzyme (Sherman et al. (1961) J Exp. Med. 114:1049-1062). This biochemical quirk of the parasite LDH has permitted a preliminary analysis of the feasibility of using LDH as a diagnostic marker for measuring the level of malaria infection (parasitemia). The data obtained using APAD as a marker of parasite LDH has demonstrated a strong correlation between the level of parasite LDH and the level of in vitro parasitemia for both P. falciparum and P. vivax (Makler et al. (1993) Am. J. Trp. Med. Hyg. 48:205-210; Makler et al. (1993) Am. J. Trp. Med. Hyg. 48:739-741; Eventoff et al. (1977) PNAS 74:2677-2681). The rapid assay using APAD was also used to measure in vitro drug sensitivity of parasite isolates (Makler et al. (1993). supra.). While the APAD-based assay is rapid it is not feasible for general use because it has poor sensitivity. The poor sensitivity of the APAD based assay is due to the poor discrimination of parasite LDH versus host LDH (only 25 to 50 fold) and the relatively high levels of host LDH present in erythrocytes and serum. A significant reservoir of active parasite LDH enzyme is found in the plasma and serum of malaria patients (Makler et al. (1993), supra.). Parasite LDH accumulates in plasma and serum in both P. falciparum and P. vivax infections. In fact, it is plausible that extra-parasite LDH represents much of the functional enzyme mass in human malaria. With respect to using LDH as a diagnostic marker of infection a positive clinical correlation was recently demonstrated between levels of active parasite LDH in serum and parasitemia in patients with cerebral malaria (Makler et al. (1993), supra.). Following chemotherapy for cerebral malaria, parasitemia and serum parasite LDH levels decreased. However, the rate of decrease of serum parasite LDH was slightly slower than the measured rate of decrease in parasitemia (Makler et al. (1993), supra.). This data suggests that parasite LDH in the serum retains activity for some time. The sources of serum parasite LDH likely arises from lysed parasitized erythrocytes, and/or by its release from currently infected erythrocytes. The exact ratio of serum to intracellular parasite LDH is not yet known. The level of serum parasite LDH was well correlated to parasitemia and suggested that LDH may provide a useful diagnostic marker for the level of malaria infection in vivo.

While the parasite LDH assay appears to provide an excellent marker for measuring parasite levels in vivo and in vitro, its application is currently limited by the absence of a diagnostic tool that is highly specific for the malaria LDH. Therefore, we will develop and evaluate monoclonal antibodies (mAb) specific to malaria LDH for malaria diagnostic applications.

Summary of the Invention

The present invention provides antibodies (e.g., monoclonal antibodies) which are specific for lactate dehydrogenase (LDH) from the genus Plasmodium. such as Plasmodium falciparum. The antibodies are specific in that they do not cross react with non-parasitic LDH proteins. Therefore, these antibodies can be used as sensitive tools to diagnose Plasmodium infections, such as malaria.

In one embodiment, the antibody is directed against all or a portion of a region of Plasmodium falciparum LDH selected from amino acids 1-20, amino acids 170-225, and amino acids 296-315. Amino acid numbering refers to the full length Plasmodium falciparum LDH amino acid sequence shown in SEQ ID NO:2. Particular epitopes within these regions encompassed by the invention include amino acids 170-205 (SEQ

ID NO: 16), amino acids 180-215 (SEQ ID NO: 17). and amino acids 190-225 (SEQ ID NO:18).

In another embodiment, the invention provides an antibody which is specific for Plasmodium falciparum LDH in that it does not cross react with other Plasmodium species. As a result, the antibody can be used in diagnostic assays to distinguish between malarial infections caused by Plasmodium falciparum specifically, and infections caused by other Plasmodium species. In a preferred embodiment, the antibody is a monoclonal antibody or fragment thereof.

The present invention further provides isolated peptides from Plasmodium falciparum lactate dehydrogenase which do not share substantial homology to other LDH proteins (e.g., human, porcine, rat, rabbit etc.). In one embodiment, the peptide comprises one of the following amino acid sequences: amino acids 1-20, 170-225 and 296-315 of SEQ ID NO:2. The invention also provides peptides comprising antigenic portions of these regions capable of generating an immune response (e.g., antibody production) when administered to a mammal.

The invention still further provides methods for diagnosing malaria in a subject using antibodies, such as monoclonal antibodies, specific for Plasmodium LDH. The antibodies are used to detect the presence of Plasmodium LDH in a biological sample taken from a subject, indicating infection by Plasmodium parasites and diagnosis of malaria. In one embodiment, antibody binding to Plasmodium LDH (e.g., P. falciparum LDH) is detected using an enzyme-linked immunosorbent assay (ELISA). In another embodiment, the method is specific for the diagnosis of a Plasmodium falciparum infection in a subject and uses antibodies which react with Plasmodium falciparum LDH, but not LDH from other Plasmodium species. Accordingly, the invention also provides an assay kit for diagnosing malaria in an individual which includes at least one antibody specific for Plasmodium LDH. In yet another embodiment, the invention provides methods for treating malaria in an individual by administering to the individual an antibody (e.g., a monoclonal antibody) which binds to and inhibits the function of Plasmodium LDH, particularly Plasmodium falciparum LDH.

The invention further provides methods for immunizing an individual against malaria by administering to the individual a peptide from Plasmodium LDH capable of generating an immune response (e.g., antibody production, T cell response, etc.).

Brief Description of the Drawings

Figure 1 is a comparison of the amino acid sequences of P. falciparum LDH and human LDH-A, LDH-B, and LDH-C isoforms. Position numbers are according to

Eventoff et al. (1977) PNAS 74:2677-2681. Amino acid positions shared between all four LDH proteins are designated (*). Positions shared by all human LDH isoforms but not in P. falciparum are designated (h). Positions unique to P. falciparum LDH are designated (p). Figure 2 is a secondary structure comparison of P. falciparum LDH to human LDH-A, LDH-B, and LDH-C isoforms. Predictions were made using MacVector programs (IBI). CfRg = combined predictions of Chow-Fausman and Robson-Garnier algorithms. Hlx = helix; sht = sheet; trn = turn. The comparison was made by adding 17 lysine residues to the N- terminus of P. falciparum LDH.

Figure 3 shows a comparison of the antigenic index of P. falciparum LDH to human LDH-A, LDH-B, and LDH-C isoforms. Predictions were made using the MacVector programs.

Detailed Description of the Invention

This invention is based on the discovery that Plasmodium falciparum lactate dehydrogenase (LDH), an enzyme which catalyzes the reduction of pyruvate to lactate, contains amino acid sequences which are highly non-conserved in LDH enzymes from other genuses. These sequences can therefor be used to develop highly specific antibodies against P. falciparum LDH and cross reacting LDH from other Plasmodium species. The full-length amino acid sequence of P. falciparum LDH is shown in SEQ ID NO: 2.

Accordingly, in one embodiment, the invention provides an antibody which binds to P. falciparum LDH within a region which shares no substantial homology to LDH proteins from other genuses (e.g., which is sufficiently non-homologous so that no antibody cross-reactivity is observed with LDH from other genuses). For example, anti- P. falciparum LDH antibodies of the invention preferably bind to regions sharing less than about 60%, more preferably less than about 50%, and most preferably not more than about 20% homology. In one embodiment, based on the primary and secondary structural analyses described in the Examples below, antibodies (e.g., preferably monoclonal antibodies ("mAbs")) specific for Plasmodium LDH bind to regions of P. falciparum LDH corresponding to amino acids 1-20, amino acids 170-225, and amino acids 296-315, referring to the amino acid sequence of P. falciparum LDH shown in SEQ ID NO:2. In a preferred embodiment, these highly non-conserved regions also constitute recognizable structural epitopes.

Generation of Antibodies

As used herein, the term "antibody" includes all known forms of antibodies such as polyclonal, monoclonal, single chain, chimeric, humanized, human etc., as well as functional fragments thereof which bind to an epitope of P. falciparum LDH. In a preferred embodiment, the antibody is a monoclonal antibody. Monoclonal antibodies capable of recognizing P. falciparum LDH (e.g., particular P. falciparum LDH peptide sequences of the invention) can be prepared using methods well known in the art. Such methods are described, for example, in detail in US 4,942,131 and US 5,583,053, the contents of which are incorporated by reference 5 herein. The term "monoclonal antibody," as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of P. falciparum LDH. A monoclonal antibody composition thus typically displays a single binding affinity for a particular region of P. falciparum LDH with which it immunoreacts. 0 Monoclonal antibodies useful in the methods of the invention are directed to an epitope of P. falciparum LDH such that complex formed between the antibody and the enzyme (or epitope-containing peptide thereof), also referred to herein as ligation, can be recognized in any immunoassay described herein. A monoclonal antibody to an epitope of P. falciparum LDH can be prepared using a technique which provides for the s production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), and the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy. Alan R. Liss. Inc., pp. 77-96), and o trioma techniques. Other methods which can effectively yield monoclonal antibodies useful in the present invention include phage display techniques (Marks et al. (1992) J Biol Chem 16007-16010).

In one embodiment, the antibody preparation applied in the subject method is a monoclonal antibody produced by a hybridoma cell line. Hybridoma fusion techniques 5 were first introduced by Kohler and Milstein (Kohler et al. Nature (1975) 256:495-97;

Brown et al. (1981) J. Immunol 127:539-46; Brown et al. (1980) J Biol Chem 255:4980- 83; Yeh et al. (1976) PNAS 76:2927-31 ; and Yeh et al. (1982) Int. J. Cancer 29:269-75). Thus, the monoclonal antibody compositions of the present invention can be produced by immunizing an animal with P. falciparum LDH or a peptide thereof. The o immunization is typically accomplished by administering P. falciparum LDH or a peptide thereof to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response. Preferably, the mammal is a rodent such as a rabbit, rat or mouse. The mammal is then maintained for a time period sufficient for the mammal to produce cells secreting antibody 5 molecules that immunoreact with the P. falciparum LDH immunogen. Such immunoreaction is detected by screening the antibody molecules so produced for immunoreactivity with a preparation of the immunogen protein. Optionally, it may be desired to screen the antibody molecules with a preparation of the protein in the form in which it is to be detected by the antibody molecules in an assay, e.g., a membrane- associated form of phosphoprotein. These screening methods are well known to those of skill in the art, e.g., ELISA and/or flow cytometry. A suspension of antibody-producing cells is then removed from each immunized mammal secreting the desired antibody is then prepared. After a sufficient time, the mouse is sacrificed and somatic antibody-producing lymphocytes are obtained. Antibody-producing cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals. Spleen cells are preferred, and can be mechanically separated into individual cells in a physiologically tolerable medium using methods well known in the art. Mouse lymphocytes give a higher percentage of stable fusions with the mouse myelomas described below. Rat, rabbit and frog somatic cells can also be used. The spleen cell chromosomes encoding desired immunoglobulins are immortalized by fusing the spleen cells with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol (PEG). Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques; for example, the P3-NSl/l-Ag4-l, P3- x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md.

The resulting cells, which include the desired hybridomas, are then grown in a selective medium, such as HAT medium, in which unfused parental myeloma or lymphocyte cells eventually die. Only the hybridoma cells survive and can be grown under limiting dilution conditions to obtain isolated clones. The supernatants of the hybridomas are screened for the presence of antibody of the desired specificity, e.g., by immunoassay techniques using the antigen that has been used for immunization. Positive clones can then be subcloned under limiting dilution conditions and the monoclonal antibody produced can be isolated. Various conventional methods exist for isolation and purification of the monoclonal antibodies so as to free them from other proteins and other contaminants. Commonly used methods for purifying monoclonal antibodies include ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (see, e.g., Zola et al. in Monoclonal Hybridoma Antibodies:

Techniques And Applications. Hurell (ed.) pp. 51-52 (CRC Press 1982)). Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques known in the art.

Generally, the individual cell line may be propagated in vitro, for example in laboratory culture vessels, and the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation. Alternatively, the yield of monoclonal antibody can be enhanced by injecting a sample of the hybridoma into a histocompatible animal of the type used to provide the somatic and myeloma cells for the original fusion. Tumors secreting the specific monoclonal antibody produced by the fused cell hybrid develop in the injected animal. The body fluids of the animal, such as ascites fluid or serum, provide monoclonal antibodies in high concentrations. When human hybridomas or EBV- hybridomas are used, it is necessary to avoid rejection of the xenograft injected into animals such as mice. Immunodeficient or nude mice may be used or the hybridoma may be passaged first into irradiated nude mice as a solid subcutaneous tumor, cultured in vitro and then injected intraperitoneally into pristane primed, irradiated nude mice which develop ascites tumors secreting large amounts of specific human monoclonal antibodies.

Media and animals useful for the preparation of these compositions are both well known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al. (1959) Virol. 8:396) supplemented with 4.5 gm/1 glucose, 20 mM glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

When antibodies produced in non-human subjects are used therapeutically in humans, they are recognized to varying degrees as foreign and an immune response may be generated in the patient. One approach for minimizing or eliminating this problem, which is preferable to general immunosuppression, is to produce chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region. Such antibodies are the equivalents of the monoclonal and polyclonal antibodies described above, but may be less immunogenic when administered to humans, and therefore more likely to be tolerated by the patient.

Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies) reactive with P. falciparum LDH can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the constant region of a murine (or other species) anti-P. falciparum LDH antibody molecule is substituted with a gene encoding a human constant region, (see Robinson et al., International Patent Publication

PCT/US86/02269; Akira, et al., European Patent Application 184,1 7; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al. (1987) J Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl Cancer Inst. 80:1553-1559).

A chimeric antibody can be further "humanized" by replacing portions of the variable region not involved in antigen binding with equivalent portions from human variable regions. General reviews of "humanized" chimeric antibodies are provided by Morrison, S. L. (1985) Science 229:1202-1207 and by Oi et al. (1986) BioTechniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of an immunoglobulin variable region from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from an anti-P. falciparum LDH antibody producing hybridoma. The cDNA encoding the chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector. Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution (see U.S. Patent 5,225,539 to Winter; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141 :4053-4060). As an alternative to humanizing a monoclonal antibody or fragment thereof from a mouse or other species, a human a monoclonal antibody (mAb) or fragment thereof directed against P. falciparum LDH can be generated. Transgenic mice carrying human antibody repertoires have been created which can be immunized with P. falciparum LDH or peptides thereof. Splenocytes from these immunized transgenic mice can then be used to create hybridomas that secrete human mAbs specifically reactive with P. falciparum LDH (see, e.g., Wood et al. PCT publication WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741 ; Lonberg et al. PCT publication WO 92/03918; Kay et al. PCT publication 92/03917; Lonberg, N. et al. (1994) Nature 368:856-859; Green. L.L. et al. (1994) Nature Genet. 7:13-21 ; Morrison. S.L. et al. (1994) Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. (1993) Year Immunol 7:33-40; Tuaillon et al. (1993) RN4S 90:3720-3724; Bruggeman et al. (1991) Eur J Immunol 21:1323-1326).

Monoclonal antibodies or fragments thereof of the present invention (i.e., which recognize and specifically bind to P. falciparum LDH) can also be produced by other methods well known to those skilled in the art of recombinant DΝA technology. Such alternative methods include the "combinatorial antibody display" method which identifies and isolates antibodies and antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal anti-P. falciparum LDH antibodies (for descriptions of combinatorial antibody display see e.g., Sastry et al.

(1989) PNAS 86:5728; Huse et al. (1989) Science 246:1275; and Orlandi et al. (1989) PNAS 86:3833). After immunizing an animal with a P. falciparum LDH immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned. Methods are generally known for directly obtaining the DΝA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR. For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies (Larrick et al. (1991) Biotechniques 11:152-156). A similar strategy can also been used to amplify human heavy and light chain variable regions from human antibodies (Larrick et al. (1991) Methods: Companion to Methods in Enzymology 2:106-110).

In an illustrative embodiment, RNA is isolated from activated B cells of, for example, peripheral blood cells, bone marrow, or spleen preparations, using standard protocols (e.g., U.S. Patent No. 4,683,202; Orlandi, et al. PNAS (1989) 86:3833-3837; Sastry et al., PNAS (\9S9) 86:5728-5732; and Huse et al. (1989) Science 246:1275- 1281.) First-strand cDΝA is synthesized using primers specific for the constant region of the heavy chain(s) and each of the K and λ light chains, as well as primers for the signal sequence. Using variable region PCR primers, the variable regions of both heavy and light chains are amplified, each alone or in combination, and ligated into appropriate vectors for further manipulation in generating the display packages. Oligonucleotide primers useful in amplification protocols may be unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease recognition sequences may also be incorporated into the primers to allow for the cloning of the amplified fragment into a vector in a predetermined reading frame for expression.

The V-gene library cloned from the immunization-derived antibody repertoire can be expressed by a population of display packages, preferably derived from filamentous phage, to form an antibody display library. Ideally, the display package comprises a system that allows the sampling of very large variegated antibody display libraries, rapid sorting after each affinity separation round, and easy isolation of the antibody gene from purified display packages. In addition to commercially available kits for generating phage display libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene S«r Z4RTM phage display kit, catalog no. 240612). examples of methods and reagents particularly amenable for use in generating a variegated anti-P. falciparum LDH antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J L2: 725-734; Hawkins et al. (1992) J Mol Biol 226:889-896: Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

In certain embodiments, the V region domains of heavy and light chains can be expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFV gene subsequently cloned into the desired expression vector or phage genome. As generally described in McCafferty et al., Nature (1990) 348:552- 554, complete V _j and VL domains of an antibody, joined by a flexible (Gly4>Ser)3 linker can be used to produce a single chain antibody which can render the display package separable based on antigen affinity. Isolated scFV antibodies immunoreactive with P. falciparum LDH can subsequently be formulated into a pharmaceutical preparation for use in the subject method.

Once displayed on the surface of a display package (e.g., filamentous phage), the antibody library is screened with a P. falciparum LDH protein, or peptide fragment thereof, to identify and isolate packages that express an antibody having specificity for P. falciparum LDH. Nucleic acid encoding the selected antibody can be recovered from the display package (e.g.. from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques.

An alternative method of obtaining an anti-P. falciparum LDH monoclonal antibody or fragment thereof for use in the present invention is to screen commercially available cell lines (e.g.. ATCC cell lines) which generate monoclonal antibodies directed against phosphate groups. Such antibodies can be screened for specific recognition of P. falciparum LDH. using standard assays such as Western blotting. In this assay, proteins in biological samples (e.g.. blood and blood-derived products) from P. falciparum infected patients are separated by PAGE, transferred to a blotting surface and then screened with radiolabeled antibody as will be familiar to one of ordinary skill in the art.

P. Falciparum LDH Peptides and Therapeutic Compositions

In another aspect, the present invention provides antigenic peptides of P. falciparum LDH useful, for example, as immunogens to raise antibodies specific for Plasmodium LDH. Preferred isolated peptides comprise an amino acid sequence which is not substantially homologous to sequences present in LDH of other genuses (e.g., preferred peptides are sufficiently non-homologous so that no antibody cross-reactivity is observed with LDH of other genuses). Homology refers to sequence similarity between two LDH proteins or peptides or between two nucleic acids encoding LDH proteins or peptides. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. In one particular embodiment, the isolated peptide comprises amino acids 1-20, 170-225 or 296-315 of P. falciparum LDH as shown in SEQ ID NO:2, or an antigenic portion of such a peptide capable of generating an immune response (e.g., humoral or cellular), such as the production of antibodies and/or stimulation of T cells. In another particular embodiment, the isolated peptide is 35 amino acids in length and has an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20. P. falciparum LDH peptides of the invention can be synthesized using known techniques, such as conventional Merrifield solid phase f-Moc or t-Boc chemistry or recombinant expression from the corresponding fragment of the nucleic acid of malaria LDH (SEQ ID NO:l). To increase immunogenicity, the peptides can be coupled to agents such as KLH (Harlow E. et al., Antibodies: A Laboratory Manual, ©1988) prior to immunization of mice (e.g., BALB/c mice). Alternatively, the peptides can be recombinantly produced in a host cell and then isolated from culture medium using known techniques. For example, gene fragmentation and recombinant expression methods can be applied using the P. falciparum LDH gene (full-length nucleotide sequence shown in SEQ ID NO:l). Alternatively, the peptides can be obtained by cleavage (e.g., digestion) of native P. falciparum LDH protein.

Isolated P. falciparum LDH peptides can be used to generate anti-Plasmodium LDH antibodies either in vitro as previously described, or in vivo as a vaccine or immunogen. Accordingly, the present invention further provides a method of immunizing or vaccinating an individual against Plasmodium (e.g., P. falciparum) infection. Immunizing a subject against infection by Plasmodium (e.g.. P. falciparum) can take two forms: active or passive. In passive immunization, antibodies of the invention against the infectious organism are administered directly to the subject to provide immediate protection. For example, monoclonal antibodies which are reactive to P. falciparum LDH can be administered to a subject who has been exposed to P. falciparum and may not have active immunity to P. falciparum. In active immunization, one of several types of vaccines may be administered. The vaccine can consist of recombinantly-expressed or synthetic P. falciparum LDH polypeptides. alone or attached to a carrier that enhances the immunogenic response. For example, in a recombinant antigen vaccine, recombinantly-expressed P. falciparum LDH peptides in a physiologically-acceptable carrier could be used to induce production of protective antibodies in a subject. This would enable the subject to mount a protective immune response to a subsequent challenge with the live pathogen. The term subject is intended to include living organisms which can raise an immune response, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

Vacines comprising P. falciparum peptides are preferably administered to a patient as a composition along with a pharmaceutically acceptable carrier or diluent. The term "pharmaceutically acceptable carrier or diluent" is intended to include any biologically compatible vehicle and which is physiologically tolerable to the patient. Such agents include a variety of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Administration of P. falciparum LDH peptides of the invention as described herein can be in any pharmacological form including a therapeutical ly active amount of the peptide alone or in combination with another therapeutic molecule (e.g.. an antiparasitic drug or agent). Administration of a therapeutical ly active amount of a therapeutic composition of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result (e.g.. reduction or prevention of infection). For example, a therapeufically active amount of a P. falciparum LDH peptide may vary according to factors such as the disease state, age. sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

Therapeutic P. falciparum LDH peptides of the invention may be administered in a convenient manner such as by injection (subcutaneous, intravenous, intraarticular etc.). oral administration, inhalation, or transdermal application. Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. For example, a P. falciparum LDH peptide may be administered to an individual in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil- in-water emulsions as well as conventional liposomes (Strejan et al., (1984) J. 5 Neuroimmunol 7:27). The kinase inhibitor may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use. these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterile aqueous 0 solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion 5 medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various o antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol. sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum 5 monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the P. falciparum LDH peptide in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which o contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (e.g., antibody) plus any additional desired ingredient from a previously sterile-filtered solution thereof. 5 It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the particular individual to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

The P. falciparum LDH peptide should be administered for a sufficient time period to alleviate the undesired malarial symptoms in a patient and/or to reduce parasitic load. The concentration of active compound in the drug composition will depend on absorption, inactivation, and other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time. A typical daily dose of a P. falciparum LDH peptide vaccine for all of the herein- described conditions is between 0.1 milligrams and 120 grams. The active compounds can be applied in any effective concentration, usually varying between 0.001 % and 100 % (all percentages are by weight). Alternatively, an effective concentration range is between 0.01 % and 50 %. Alternatively, an effective concentration range is between 1 % and 25 %.

Screening Assays Using Anti-P. falciparum LDH Antibodies

Antibodies (e.g., monoclonal antibodies or "mAbs") of the present invention can be used to specifically detect the presence of LDH from parasites of the genus Plasmodium (which cause malaria) in a sample (e.g.. biological sample, such as blood or blood serum, or a food sample). Accordingly, in yet another aspect, the present invention provides a method of diagnosing malaria in a subject (e.g., a mammal such as a human) by screening a biological sample taken from the subject with anti-P. falciparum LDH antibodies of the invention and detecting the presence of antibody binding in the sample. For example, a biological sample can be collected from an individual and contacted in vitro with an antibody of the invention under conditions which promote binding of the antibody to P. falciparum LDH present in the sample. Bound antibody (i.e., antibody complexed to antigen such as P. falciparum LDH) can then be detected using any of a variety of well known immunological screening assays.

For example, assays which can be employed include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and Western Blot Assay. Each assay generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. Accordingly, in the present invention, these assays are used to detect protein- antibody complexes formed between Plasmodium LDH contained in a biological sample such as blood or blood serum and anti-P. falciparum LDH antibodies of the invention. As will be described below, these protein-antibody complexes are preferably detected using an enzyme-linked antibody or antibody fragment (e.g., a monoclonal antibody or fragment thereof) which recognizes and specifically binds to the protein-antibody complexes.

In one embodiment, the present invention employs a sandwich ELISA assay to screen a blood sample of an individual having or suspected of having malaria (i.e., exhibiting symptoms indicative of malaria) for the presence of Plasmodium LDH or P. falciparum LDH, in particular. The assay is so named because it involves the use of an antibody-antigen-antibody sandwich on a solid phase. To perform the assay, a blood sample is first collected from the individual and is concentrated (e.g., centrifuged) to collect the serum. The serum is then adsorbed onto a solid support such as a microtiter plate (e.g., a 96 well ELISA plate) by incubating the serum and the plate for between 2- 20 hours at between about 1-24°C. The unbound components of the serum sample are then removed in a manner which leaves intact the immunoglobulms (e.g., IgG, IgA, IgM) adsorbed onto the plate. The removal is preferably carried out by washing the solid support with an eluent to which the immunoglobulms are inert (e.g., PBS-Tween).

In a next step, the plate is treated (i.e., contacted, e.g., incubated) with Plasmodium LDH (e.g., P. falciparum LDH) under conditions which permit antibody- protein binding between the immunoglobulms from the serum sample and proteins which they recognize from the biological sample. The incubation step can be carried out for a period of about 2-20 hours at a temperature of between 1 -24°C, higher temperatures being required for shorter incubation periods. This step results in the formation of protein-antibody complexes which are bound to the surface of the plate.

After the protein-antibody complexes have been allowed to form, unbound components (i.e., components which have not been recognized by antibodies in the patient's serum) are removed from the plate in a manner which leaves the complexes intact. The removal is preferably carried out by washing the plate with an eluent to which the complexes are inert (e.g.. PBS-Tween). The plate is then contacted with a solution containing an anti-P. falciparum LDH antibody of the invention.

The anti-P. falciparum LDH antibody (or fragment thereof) is preferably incubated on the plate under conditions which allow the antibody to recognize and bind to P. falciparum LDH which is itself bound onto the plate(s) via antibodies from the 5 patient's serum (e.g., immunoglobulms), thereby forming an antibody-protein-antibody sandwich. Again, the incubation step can be carried out for a period of about 2-20 hours at a temperature of between 1-24°C and any unbound anti-phosphoprotein antibody can be removed by washing with an appropriate eluent. After the anti-P. falciparum LDH antibody has been reacted with the plate, the presence of bound anti-P. falciparum LDH o antibody is determined by assays such as those described below and thus the presence of antibodies in the patient's blood serum specific for P. falciparum LDH or LDH from a cross reacting Plasmodium species can be both determined and quantified.

The assaying step may be carried out using any suitable procedure for detecting the binding of the anti-P. falciparum LDH antibody to the reaction plate. One preferred 5 means involves labeling the anti-P. falciparum LDH antibody via linkage to an enzyme which is then detected in an enzyme immunoassay (EIA) (Voller, "The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons 2:1-7, 1978, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller, et al., J. Clin. Pathol. 31 :507-520 (1978); Butler, Meth. Enzymol. 73:482-523 (1981 ); Maggio, (ed.) Enzyme 0 Immunoassay, CRC Press, Boca Raton, FL, 1980; Ishikawa, et al., (eds.) Enzyme

Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to 5 detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and o acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic 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.

As an alternative to the above-described sandwich ELISA assay, an ELISA assay 5 can similarly be employed to detect Plasmodium LDH in a biological sample by initially coating the plate with an anti-P. falciparum LDH antibody of the invention. The plate can then be incubated with the sample, rinsed and then screened for bound P. falciparum LDH again using labeled anti-P. falciparum LDH antibodies as described above.

Detection of Plasmodium LDH in a biological sample can also be accomplished using anti-Plasmodium LDH antibodies in a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography. It is also possible to label the anti-Plasmodium LDH antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The antibody can also be detectably labeled using fluorescence emitting metals such as

152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label the antibody. 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.

Inhibitory Antibodies

Antibodies generated as described herein which bind to Plasmodium LDH and peptides thereof can be tested for the ability to inhibit enzymatic activity (e.g.. catalysis of the reduction of pyruvate to lactate) by e.g., blocking an active site of the enzyme. This can be achieved using assays known in the art. such as those described in Gregenshchikova et al. (1992) Biokhimiia 57:758-66. the teachings of which are incorporated by reference herein. Such inhibitory antibodies can be administered to patients infected with parasites of the genus Plasmodium to inhibit, reduce or clear the infection.

Accordingly, in another aspect, the present invention provides a method of treating malaria in an individual by administering to the individual an inhibitory antibody which binds to and inhibits the function of Plasmodium LDH. As used herein, the term "inhibit" or "inhibitory" refers to partial or complete reduction in LDH activity compared to LDH not exposed to the antibody.

The invention is further illustrated by the following examples which should not be construed as further limiting the subject invention. The contents of all references and published patent applications cited throughout this application are hereby incorporated by reference.

EXEMPLIFICATION

EXAMPLE 1 - Cloning of Plasmodium falciparum LDH

Nucleic acids. P. falciparum strains Honduras- 1 and FCR3 were cultivated as described by Bzik et aL, Mol. Biochem. Parasitol. 30:279-288 (1988). Genomic DNA and poly (A)⁺ RNA were isolated as previously described by Bzik et al., Mol. Biochem. Parasitol. 30:279-288 (1988). Parasite chromosomes were prepared and separated by contour clamped homogeneous electric fields (CHEF) as previously described by Gu et al., Exp. Parasitol. 71:189-198 (1990). Honduras- 1 RNA and DNA were analyzed in Northern and Southern blots according to previously described conditions Bzik et al., Mol. Biochem. Parasitol. 30:279-288 (1988).

Construction of cDNA and mung bean nuclease libraries. Honduras- 1 genomic DNA was subjected to mung bean nuclease (MBN) cleavage in either 32.5% (Library 1), 35%o (Library 2), 37.5% (Library 3). or 40% (Library 4) formamide as previously described by Vernick et al., Nucleic Acids Res. 16:6883-6896 (1988). Digested DNA was size-fractionated in agarose gels and DNA sizes 0.05 to 0.2 kb (size fraction A), 0.2 to 0.5 kb (size fraction B), 0.5 to 1.5 kb (size fraction C), and greater than 1.5 kb (size fraction D) were purified by gene clean™ (BIO 101, Vista, CA). Separate master MBN

Libraries 1 A, IB, 1C, ID, 2A, 2B. 2C, 2D, 3A, 3B, 3C, 3D, 4A, 4B, 4C. and 4D were individually constructed by ligation of MBN digested and size fractionated DNA into vector M13 mp8, which was digested with Smal and then dephosphorylated (Bzik et al.. PNAS USA 84:8360-8364 (1987)). A Honduras-1 cDNA library was also constructed in the vector Lambda Zap II (Stratagene, La Jolla, CA) as previously described by Bzik et al., Mol. Biochem. Parasitol. 30:279-288 (1988). DNA sequence analysis. Individual Ml 3 mp8 MBN library clones were sequenced as previously described by Bzik et_al. PNAS USA 84:8360-8364 (1987). Each DNA sequence was analyzed for open reading frames, which were compared to the current NBRF protein database using the PAM250 scoring matrix of the Mac Vector™ programs. Similarity and identity scoring matrix values were determined by scoring 1 point for a perfect identity or chemical similarity match, 0 points for no match, and subtracting 1 point for each amino acid insertion or deletion. Percentage similarity and identity scores were then determined by dividing the total score by the number of amino acids contained in the smaller LDH for each comparison. Expression of P. falciparum LDH. Oligonucleotides for polymerase chain reaction (PCR) reconstruction of the P. falciparum LDH coding region were synthesized at the Dartmouth molecular genetics center. The sequence of oligonucleotide PI was 5'-CCTCGAATTCATGGCACCAAAAGCAAAAATCGT-3' (P. falciparum LDH initiation codon underlined) (SEQ ID NO: 3) and oligonucleotide P2 was 5'-CCCTGGATCCTTAAGCTAATGCCTTCATTCTCT-3' (P. falciparum LDH termination codon underlined) (SEQ ID NO: 4). PI and P2 were used in polymerase chain reaction (PCR) with Honduras- 1 genomic DNA as substrate as previously described by Fox and Bzik, Mol. Biochem. Parasitol. 49:289-296 (1991). The amplified 971 bp PCR product was digested with BamHI and was blunted by treatment with klenow polymerase and dNTP's. The resulting 966 bp fragment was digested with EcoRI and ligated with EcoRI and Smal digested pKK223-3 expression vector (Pharmacia, Piscataway, NJ). This strategy placed the P. falciparum LDH ATG initiation codon, 316 amino acid coding region, and termination codon in the correct orientation for expression of P. falciparum LDH relative to all of the transcriptional and translational expression signals carried by pKK223-3 (Pharmacia, Piscataway, NJ). Ligated DNA was transformed into E. coli strain JM105. A recombinant pKK223-3 clone, pLDH-Pl was isolated and it contained the P. falciparum LDH coding region based upon strong hybridization to MBN 2C93. The correct orientation and size of the P. falciparum LDH coding region in pLDH-Pl was confirmed by analyzing BamHI, EcoRI, and Hindlll digests.

Lactate Dehydrogenase Assays. Erythrocytes, parasite infected erythrocytes, and saponin released parasites were prepared for LDH assays as previously described by Vander Jagt et al., Mol. Biochem. Parasitol. 4:255-264 (1981) and Hicks et al., Gene 100:123-129 (1991). E. coli cells were grown aerobically in 5 ml overnight cultures at 37°C in LB broth lacking glucose and supplemented with 50 mg/ml ampicillin. Overnight cultures were diluted 1 to 5 in LB broth plus ampicillin and IPTG was added at mid-log phase. 2 ml of culture was harvested 3 hours after IPTG addition and cells were pelleted in a microfuge for 1 minute. The supernatant was removed and the pellet was resuspended in 0.8 ml of sonication buffer (20 mM potassium phosphate buffer, pH 7.2, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 M glycerol, and 0.5 mM 5 PMSF). Cells were sonicated at maximum power (Megason Ultrasonic Disintegrator with a microprobe) using four 15 second bursts at 4°C. The sonic extract was centrifuged at 4°C in a micro-centrifuge for 5 minutes, the supernatant was analyzed for LDH activity, and the pellet was saved for sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis (SDS-PAGE) (Laemmli, Nature 277:680-682 (1970)). Proteins 0 in SDS-PAGE gels were visualized by silver staining (Amersham, Arlington Heights, IL). Supernatants were subjected to protein concentration determination (Bradford assay kit. Bio-Rad, Cambridge, MA), LDH activity assay, LDH isoenzyme assay, and SDS- PAGE. Samples for LDH isoenzymes were electrophoresed at 30 volts for 5 hours in a 0.6% agarose gel, and were specifically stained by a formazan procedure (Nachlas et al., 5 Anal. Biochem 1 :317-326 (I960)). Kinetic studies were carried out at 25°C in 25 mM Tris, pH 7.5. LDH specific activities from E. coli extracts were measured with 0.1 mM N ADH and 10 mM pyruvate and are given in mmol/min per mg protein (Vander Jagt et al., Mol. Biochem. Parasitol. 4:255-264 (1981)).

o Results

Cloning, identification, DNA sequencing, and gene structure of P. falciparum LDH. DNA sequences derived from 200 Ml 3 mp8 clones from a Honduras- 1 mung bean nuclease (MBN) library (library 2C) were initially screened for introns and open reading frames (ORF). One MBN clone, MBN 2C93, exhibited an ORF with 5 statistically significant amino acid similarity (zeta score > 10 standard deviations above the mean score) to carboxyl-terminal regions of other LDH. The complete 1.1 kb MBN 2C93 insert DNA sequence was determined and was found to contain 110 bp of 5' flanking non-coding DNA. and probable P. falciparum LDH coding sequences to within approximately 20 amino acids of the probable carboxyl-terminus. To isolate the 0 carboxyl-terminal coding region, a cDNA clone. cLDH-1, was isolated by screening a cDNA library with MBN 2C93. The combined DNA sequences of clones MBN 2C93 and cLDH-1 revealed the complete coding region of the presumed P. falciparum LDH.

Because the MBN 2C93 ORF terminated 17 amino acids prior to the termination codon found in cLDH-1 the possibility of a carboxyl-terminal intron could not be ruled 5 out. Two oligonucleotides, 5'-CCTCGAATTCATGGCACCAAAAGCAAAAATCGT- 3' (P. falciparum LDH initiation codon underlined) (SEQ ID NO: 3), and 5'-CCCTGGATCCTTAAGCTAATGCCTTCATTCTCT-3' (P. falciparum LDH termination codon underlined) (SEQ ID NO: 4) were used to amplify the complete coding region from chromosomal DNA by PCR. The sequencing of the 1.1 kb PCR product confirmed the sequence shown in Figure 1 (SEQ ID NO:2) which demonstrated the absence of introns. The predicted molecular weight of the presumed P. falciparum LDH protein

(34.1 kDa) correlates well with the observed P. falciparum LDH apparent M_r of 31 to 36 kDa (Saxena et al, Mol. Biochem. Parasitol. 21 : 199-202 (1986) and Simmons et al., Mol. Biochem. Parasitol. ]_5:231-243 (1986). As is typical for other P. falciparum genes, the 5' and 3' flanking non-coding DNA has a higher A + T content (91% and 90%, respectively) than the coding region (67%).

Expression of P. falciparum LDH. To determine whether the presumed P. falciparum LDH gene identified encodes LDH activity, P. falciparum LDH was expressed in Escherichia coli. Due to the absence of useful restriction enzymes sites the P. falciparum LDH coding region was regenerated by PCR using primers PI (SEQ ID

NO:3) and P2 (SEQ ID NO:4), and inserted into the E. coli expression vector pKK223-3 to construct clone pLDH-Pl (a recombinant pKK223-3 clone). E. coli containing pKK223-3 or pLDH-Pl were assayed for total LDH activity (Table 1). The pLDH-Pl construct exhibited an approximately 20-fold increase in total LDH activity with, or without, IPTG induction in comparison to pKK223-3 alone (Table 1).

SDS-polyacrylamide gel electrophoresis of E. coli extracts demonstrated the presence of a new protein (apparent Mr of 33 kDa) in E. coli containing pLDH-Pl, which was not present in E. coli containing pKK223-3 alone. The majority of P. falciparum LDH synthesized was present in the pellet fraction following sonication. The major P. falciparum LDH band at 33 kDa co-migrated with a major E. coli protein in the supernatant and pellet fractions. Thus, it can be inferred that the 20-fold increase in total LDH activity in pLDH-Pl (Table 1 ) is due to P. falciparum LDH which comigrated with the major E. coli band at 33 kDa in the supernatant fraction. A second new protein band at an apparent Mr of approximately 21 kDa was also seen in pLDH-Pl pellet fractions and presumably this band represents a proteolytic product of the 33 kDa band. TABLE 1

^a E coli construct ^D Total LDH activity expressed as μmol min^~l (mg protein)" 1

EXAMPLE 2 - Recombinant Expression of Plasmodium falciparum LDH

Primers LDH1, 5'-CCGGATCCATGGCACCAAAAGCAAAAATCGT-3' (SEQ ID NO: 14), and LDH2, 5'-CCGAATTCTTAAGCTAATGCCTTCATTCTTCT-3' (SEQ ID NO: 15), were synthesized. LDH1 and LDH2 were used to PCR amplify P. falciparum LDH (PfLDH) from Honduras- 1 using the methods described above. The -970 bp PCR product was digested with BamHI and EcoRI and ligated to BamHI and EcoRI digested pGEX-2T vector (Pharmacia, Inc. Piscataway, NJ). This fused the PfLDH coding region to glutathione S-transferase (GST) where N-terminal GST- PfLDH-COOH was created.

E. coli strain DH5α was transformed with ligated DNA and four ampicillin- resistant colonies were selected for further study as described above. The manufacturer's instructions for the pGEX expression purification scheme were followed.

The mini -prep method was employed for the second culture. Cultures of the selected GST-PfLDH fusion clones (GST-PfLDH 1 to 4) were grown overnight and diluted to ODgQO^⁰-^{1 me} next morning. When ODgoo^⁰-⁷ was reached, IPTG was added to induce the pGEX-2T promoter. At two hours post-induction, cells were harvested and soluble protein extracts made as described above. Using the Pharmacia protocol for glutathione-SEPHAROSE™ (Pharmacia, Inc., Piscataway, NJ ), GST- PfLDH was purified from soluble extracts. In preliminary experiments, each clone had a fusion protein of correct size (55-60 kd). Purified GST-PfLDH fusion protein was also tested for specific activity of the LDH enzyme. LDH enzyme activity was measured in activity gels as described in Bzik et al. (Mol. Biochem. Parasitol. 59:155-166 (1993)). The activity seen in GST-PfLDH 1 represented an equivalent of approximately 25μl of E. coli GST-PfLDH culture. Surprisingly, GST-PfLDH protein retained PfLDH enzyme activity. EXAMPLE 3 - Preparation of Monoclonal Antibodies Against Plasmodium LDH

The following information and methods can be used to develop monoclonal antibodies (mAbs) against Plasmodium LDH. In particular, P. falciparum LDH and antigenic peptides thereof can be used to generate mABs which are either specific for falciparum LDH or which cross react with LDH from other Plasmodium species.

To develop mAbs, a two-pronged approach can be employed involving the use of active intact P. falciparum LDH (cloned and recombinantly expressed as described above), as well as defined peptides derived from selected regions of the enzyme. This approach favors the development of mAbs which are optimal for use in an ELISA based diagnostic kit for diagnosing malaria in infected subjects. Specifically, mAbs obtained using this two-pronged approach will recognize both linear and conformational epitopes of P. falciparum LDH.

I Peptides as Immunogens

Peptides suitable for use as immunogens in raising mAbs against P. falciparum LDH include those which correspond to regions of the enzyme which are relatively unique to P. falciparum LDH (e.g., substantially non-homologous to LDH proteins from other genuses, such as human (including the three human LDH-A, LDH-B, and LDH-C isoenzymes), porcine, rat, rabbit, helminth etc.), but which constitute recognizable structural epitopes when present in the naturally occurring P. falciparum LDH. For neutralizing mAbs, preferred regions are those which are further involved in catalytic activity of the enzyme, such as the region containing the active center histidine. Such preferred regions include, for example, peptide regions encompassing His 190 or His 195 (referring to the amino acid sequence shown in SEQ ID NO:2).

For example, as shown in Figure 1, the amino acid sequences of the three human LDH-A, LDH-B and LDH-C isoforms share identical amino acids at 65% of the residues, while P. falciparum and the three human LDH share identical residues in only 24% of the positions. In fact, 65% of the P. falciparum LDH amino acid residues are unique to P. falciparum LDH. They do not occur in any of the three human LDH enzymes at the corresponding position. In addition, while almost all of the secondary structures predicted in the three human LDH isoforms are conserved in all three human enzymes, P. falciparum LDH secondary structure predictions reveal the absence of some of the human features as well as the presence of some features not found in the human enzymes (Figure 2).

Similar observations can be made in predicting antigenicity of different regions of P. falciparum LDH using computer analyses. Figure 3 shows a comparison of the antigenic index of P. falciparum LDH to human LDH-A, LDH-B, and LDH-C isoforms. There are clearly detectable differences between P. falciparum LDH and human LDH in their predicted antigenic indexes. Accordingly, as part of the present invention, it was determined that amino acids

170-225 of malaria LDH are highly specific to the enzyme in that this region shares no notable homology to other LDH enzymes. For example, the region corresponding to amino acids 180-214 of malaria LDH shares only 13 amino acids with the corresponding region in porcine LDH (containing the active center histidine). In contrast, this region in porcine LDH shares at least 30 of 35 amino acids (-90% homology) with human LDH-A, LDH-B, and LDH-C isoenzymes. Thus, 22 of these 35 conserved human amino acid positions (180-214) are unique in malaria LDH.

Accordingly, peptides of suitable lengths (e.g., 35 amino acids) derived from unique regions in malaria LDH such as amino acids 170-225 (referring to the amino acid numbering shown in SEQ ID NO:2) can be used to develop mAbs specific for malaria LDH. In particular, peptides corresponding to amino acids 170-205 (peptide 1 , SEQ ID NO:16), 180-215 (peptide 2, SEQ ID NO:17), 190-225 (peptide 3, SEQ ID NO:18), and the N-terminal (aa 1-20) and C-terminal (aa 296-315) twenty amino acids (peptide 4 (SEQ ID NO: 19) and peptide 5 (SEQ ID NO: 20). respectively) of malaria LDH (SEQ ID NO:2) can be used. These peptides are of sufficient length to contain topographic immunogenic determinants of LDH in addition to linear determinants (Kaumaya et al.

(1992) J. Biol. Chem. 267:6338-6346).

Peptides suitable as immunogens for generating anti-malaria LDH mAbs, such as the peptides listed above, can be synthesized using known techniques, such as conventional Merrifield solid phase f-Moc or t-Boc chemistry or recombinant expression from the corresponding fragment of the nucleic acid of malaria LDH (SEQ ID NO:l). To increase immunogenicity, the peptides can be coupled to agents such as KLH (Harlow E. et al., Antibodies: A Laboratory Manual, ©1988) prior to immunization of mice (e.g., BALB/c mice).

II. Active Malaria LDH as Immunogen

Mice (e.g., BALB/c) can be immunized with partially purified recombinant parasite LDH obtained as described above in Examples 1 and 2 {see also, Bzik et al.

(1993) Molec. Biochem. Parasitol. 59:155-166), as well as with purified LDH fusion protein consisting of glutathione-S-transferase (GST) fused to malaria LDH. Both of these already purified recombinant proteins are in an active form. III. Immunization and Development of Monoclonal Antibody

To generate mAbs using the peptide and whole protein malaria LDH immunogens described above, any known method for raising mAbs can be used. For example, the following procedure can be employed: Approximately 50 μg of antigen can be emulsified in complete (primary immunization) or incomplete (boosting immunizations) Freund's adjuvant, and injected into the peritoneum of e.g., BALB/c mice. Mice can be bled, and the sera assayed for reactivity with purified active parasite LDH by ELISA. Once a high antibody titer is achieved, the spleen from a recently boosted animal can be removed, the splenocytes isolated and fused with the HAT-sensitive NS-1 parent myeloma cell line, and plated out in limiting dilution.

Supernatants can be analyzed for specific antibody to active malaria LDH by ELISA. Wells can be coated with parasite LDH, washed, incubated with supernatants, washed, incubated with GAM-alkaline-phosphatase (Cappel), and analyzed by a colorimetric assay of alkaline phosphatase activity. Specific positive clones can then be examined by ELISA to purified active human LDH (A, B, and C obtained from Sigma). Reactivity of these mAbs to human LDH is not to be expected, but this can be measured to determine which mAbs are the most specific to the malaria LDH. Specific malaria LDH positive clones can be expanded, frozen into permanent stocks, and analyzed again by ELISA.

EXAMPLE 4 - Characterization of Monoclonal Antibodies Specific for Plasmodium falciparum LDH

I. Reactivity to Malaria LDH MAbs generated as described in Example 3 specific for malaria LDH can be tested for reactivity using any conventional assay, such as an ELISA performed in vitro on extracts of cultured P. falciparum, or on parasite culture supernatants that contain released parasite LDH. A detailed discussion of a variety of art-employed immunological assays, including ELISA, is provided above in the Detailed Description of the invention. Suitable strains of P. falciparum for use in such studies include, for example, Honduras- 1 (South American) and FCR-3 (African) P. falciparum.

For clinical diagnosis of malaria it is necessary to measure parasite LDH within a crude preparation of blood, or serum. Thus, the medium for parasite culture can include plasma and red blood cells from human donors, in addition to other culture components such as antibiotics, mycotics, amino acids, DMEM medium, and fetal calf serum. II. Reactivity to Plasmodium Species Other than Falciparum

Specific mAbs generated against P. falciparum LDH (e.g.. the Honduras- 1 LDH isolate described by Bzik et al. (1993), supra.) using the procedures of Example 3 can be tested for cross reactivity with other species of Plasmodium as described below. In terms of conservation of LDH structures among Plasmodium species, it is known that polyclonal antibody made to any Plasmodium LDH will cross react to all other Plasmodium species. Further, P. falciparum LDH and the non-human malaria species P. knowlesi LDH are highly homologous at the amino acid sequence level (Bzik et al. (1993), supra.). Approximately 30 N-terminal amino acids of P. knowlesi LDH have been sequenced and 29 of those 30 are identical to P. falciparum LDH sequences (Bzik et al. {\992), supra.).

It is known that P. falciparum has two isoenzymes of LDH depending on the isolate (the organism is haploid and has only one isoenzyme in blood stages) (Carter et al. (1975) Trans, of the Royal Soc. Trop. Med. Hyg. 69: 371-376). It is likely that these alleles are only different in one amino acid residue, or just a few amino acids. Similarly, there may exist different alleles of P. vivax.

To examine whether cross-reactivity exists between mAbs raised against P. falciparum LDH isolates and LDH from other Plasmodium species (e.g., P. vivax), any standard protein-antibody binding assay (such as those described herein) can be employed (e.g., ELISA) using the various LDH proteins as substrates. MAbs specific for LDH from particular Plasmodium species can then be used as tools in malaria diagnostic assays to determine malaria type in infected patients (i.e.. to discriminate between Plasmodium species causing the infection, particularly human Plasmodium species). For example, these specific mAbs can be used to discriminate between P. falciparum and P. vivax malaria infections. This can significantly aid in decisions regarding choices of malaria treatment, particularly since P. falciparum is a severe, often drug resistant, infection.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

What is claimed is:

1. An antibody which binds to Plasmodium falciparum lactate dehydrogenase within a region selected from the group consisting of amino acids 1-20 of SEQ ID NO:2, amino acids 170-225 of SEQ ID NO:2, and amino acids 296-315 of SEQ ID NO:2.

2. The antibody of claim 1 wherein the antibody is monoclonal.

3. An antibody which binds to an amino acid sequence from Plasmodium falciparum lactate dehydrogenase selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17. SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20.

The antibody of claim 3 wherein the antibody is monoclonal.

5. An isolated peptide from Plasmodium falciparum lactate dehydrogenase comprising an amino acid sequence selected from the group consisting of amino acids 1 - 20 of SEQ ID NO:2, amino acids 170-225 of SEQ ID NO:2, and amino acids 296-315 of SEQ ID NO:2, or an antigenic portion of said peptide capable of generating an immune response when administered to a mammal.

6. The peptide of claim 5 wherein said immune response comprises the production of antibodies.

7. An isolated peptide from Plasmodium falciparum lactate dehydrogenase comprising an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:20.

8. A method of diagnosing malaria in a subject comprising: collecting a biological sample from the individual; contacting the sample with an antibody which binds to an amino acid sequence from Plasmodium falciparum lactate dehydrogenase selected from the group consisting of amino acids 1-20 of SEQ ID NO:2, amino acids 170-225 of SEQ ID NO:2 and amino acids 296-315 of SEQ ID NO :2 under conditions which promote binding of said antibody to said amino acid sequence; detecting the presence of antibody binding in the sample.

9. The method of claim 8 wherein the presence of antibody binding is detected by an enzyme-linked immunosorbent assay (ELISA).

5 10. The method of claim 8 wherein the biological sample is blood or blood serum.

1 1. The method of claim 8 wherein the antibody is a monoclonal antibody.

12. The method of claim 8 wherein the antibody binds to an amino acid sequence o selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,

SEQ ID NO: 19 and SEQ ID NO:20.

13. A method of diagnosing Plasmodium falciparum infection in a subject comprising: 5 collecting a biological sample from the individual; contacting the sample with an antibody which binds to a region of Plasmodium falciparum lactate dehydrogenase selected from the group consisting of amino acids 1 - 20 of SEQ ID NO:2, amino acids 170-225 of SEQ ID NO:2 and amino acids 296-315 of SEQ ID NO:2 under conditions which promote binding of said antibody to said lactate 0 dehydrogenase; detecting the presence of antibody binding in the sample as an indication of infection by Plasmodium falciparum.

14. The method of claim 8 wherein the presence of antibody binding is detected by 5 an enzyme-linked immunosorbent assay (ELISA).

15. The method of claim 8 wherein the antibody is a monoclonal antibody.

16. A method of treating malaria in an individual comprising administering to the o individual an antibody which binds to Plasmodium falciparum lactate dehydrogenase within a region selected from the group consisting of amino acids 1-20 of SEQ ID NO:2, amino acids 170-225 of SEQ ID NO:2, and amino acids 296-315 of SEQ ID NO:2.

17. The method of claim 16 wherein the antibody is a monoclonal antibody. 5

18. A method of immunizing an individual against malaria comprising administering to the individual a peptide comprising an amino acid sequence from Plasmodium falciparum lactate dehydrogenase selected from the group consisting of amino acids 1- 20 of SEQ ID NO:2, amino acids 170-225 of SEQ ID NO:2 and amino acids 296-315 of SEQ ID NO:2, or a portion of said peptide capable of generating an immune response in said individual.

19. An assay kit for diagnosing malaria in an individual comprising: a solid support capable of adsorbing an immunoglobulin; an antibody which binds to an amino acid sequence from Plasmodium falciparum lactate dehydrogenase selected from the group consisting of amino acids 1 - 20 of SEQ ID NO:2, amino acids 170-225 of SEQ ID NO:2 and amino acids 296-315 of SEQ ID NO:2.