Field of the Invention This invention relates to peptides derived from ankyrin, erythrocyte membrane protein 4.1, and nucleic acid molecules encoding the same. Each peptide inhibits function of malaria cysteine protease falcipain-2, such as the falcipain-2-mediated cleavage of ankyrin, cleavage of erythrocyte membrane protein 4.1, and cleavage of hemoglobin. The peptides and related nucleic acid molecules are useful in, inter alia, research, diagnostic and therapeutic contexts, particularly for treating malaria infection.

Background of the Invention The World Health Organization estimates that 300-500 million people are infected by malaria annually and that over 2 million people, mostly women and children under the age of five, die of the malaria disease each year. The disease has been classified as an"emerging infection"by many national and international health authorities in recent years, due to its dramatic comeback in regions where the disease was once eliminated or suppressed.

Conventional methods of control for malaria disease mainly rely on the use of antimalarial drugs. Due to a rapid rise and spread of drug resistance to most affordable and widely-used drugs in recent years, however, there is unfortunately limited means of treatment for the disease. At present, a malaria vaccine is not available.

Four species of parasite belonging to the genus Plasmodium cause nearly all malaria infections in humans. These are Plasniodiumfalciparum, Plasniodium vivax, Plasmodiuln ovale, and Plasmodium malariae. Human infection begins when a female Anopheles mosquito inoculates plasmodial sporozoites from its salivary gland during a blood meal.

Sporozoites travel via the bloodstream to the liver, where they undergo a period of asexual reproduction within hepatic parenchymal cells. Each hepatic sporozoite produces thousands of daughter merozoites, which are discharged into the bloodstream ready to invade erythrocytes in the symptomatic stage of the infection. During the erythrocytic cycle, intraerythrocytic merozoites become trophozoites and gametocytes over 48 hours (72 hours for P. malariae). Early during this stage the erythrocytes take on a characteristic ring form.

The trophozoites consume nearly all the hemoglobin within the erythrocyte and enlarge to occupy nearly the entire volume of the erythrocyte. Multiple nuclear divisions occur and the erythrocyte ruptures to release 6-30 daughter merozoites, each capable of infecting another red blood cell and repeating the erythrocytic cycle. To complete the transmission cycle, male and female gametocytes, ingested by a female Anopheles mosquito during a blood meal, form a zygote in the mosquito's gut, leading eventually to production of plasmodial sporozoites which migrate to the salivary gland of the mosquito.

Plasmodium falciparum causes a severe form of human malaria and is responsible for nearly all malaria-specific mortality. Resistance of Plasmodium to antimalarial drugs is an increasingly serious problem in fighting the disease. New chemotherapy approaches that block essential metabolic pathways of the parasite are therefore urgently needed.

In view of the foregoing, a need exists to develop new and improved methods and compositions for treating malaria infection. Preferably such methods and compositions are based upon inhibiting the erythrocytic growth of the malaria parasite and its release from red blood cells, thereby minimizing harmful side effects that may be due to non-specific therapeutic approaches.

Summary of the Invention The invention is based, in part, on the discovery by the Applicants that the malaria parasite-derived cysteine protease falcipain-2 (FP-2) cleaves erythrocyte membrane ankyrin and erythrocyte membrane protein 4.1. Falcipain-2 has also been described as an essential hemoglobinase of Plas7nodium falciparu7n. Shenai BR et al. (2000) JBiol Chem 275: 29000- 10. Although not wishing to be bound to a particular theory or mechanism, Applicants believe this cleavage is accompanied by destabilization of the erythrocyte membrane skeleton, facilitating malaria parasite release from erythrocytes. Findings of the Applicants suggest that falcipain-2 is a key malarial parasite enzyme with multiple roles, indicating its usefulness as a target for anti-malarial therapeutics.

Applicants have now identified two peptides, one each from ankyrin and from erythrocyte membrane protein 4.1, that inhibit falcipain-2-mediated cleavage of human erythrocyte ankyrin and/or human erythrocyte protein 4.1. The ankyrin-derived peptide, Ank Pi, is a 10 amino acid peptide corresponding to amino acid residues 1206-1215 of human erythrocyte ankyrin. The protein 4. 1-derived peptide, 4.1 Pl, is a 16 amino acid peptide corresponding to amino acid residues 428-443 of the spectrin/actin binding (SAB) domain of human erythrocyte protein 4.1 amino acid sequence. Each of these peptides, either alone or in combination with the other, inhibits falcipain-2-based cleavage of erythrocyte protein 4.1 and of erythrocyte membrane ankyrin, and is believed to inhibit host cell rupture and malarial release. Other protein 4.1 peptides within the spectrin/actin binding domain, for example Pii and Piii, do not appear to exhibit this inhibitory effect.

The discovery of the particular sequence region within ankyrin that is the site of cleavage by falcipain-2 protein permits the use of such sequences, and nucleic acids encoding the same, for the treatment of malaria infection and as inhibitors of falcipain-2 in vivo and in vitro. Likewise, the discovery of the particular sequence region within erythrocyte membrane protein 4.1 that is the site of cleavage by falcipain-2 protein permits the use of such sequences, and nucleic acids encoding same, for the treatment of malaria infection and as inhibitors of falcipain-2 ira vivo and in vitro. Thus, although not wishing to be bound to a particular theory or mechanism, Applicants believe that this cleavage is implicated in the breakdown of the erythrocyte membrane and may facilitate the release of malaria parasite from the host erythrocyte cell. Accordingly, the particular ankyrin-derived and erythrocyte protein 4. 1-derived sequences are useful for inhibiting falcipain-2 in vivo or in vitro and, more particularly, for further defining the nature of the interaction (s) between the parasite and the erythrocyte that result in parasite release from erythrocytes, as well as for developing diagnostic and therapeutic agents that are useful for detecting and treating malaria infection.

The knowledge of the particular sequences of ankyrin and of erythrocyte protein 4.1 (Ank Pi and 4.1 Pl) that inhibit falcipain-2 cleavage of protein 4.1 and erythrocyte membrane ankyrin also permits examination of additional therapeutic agents to inhibit cleavage activity of falcipain-2 and/or involvement of falcipain-2 in the release of malaria from erythrocytes.

The invention also is based, in part, on the discovery that particular sequences within protein 4. 1 selectively interact with falcipain-2 protein. Accordingly, these particular protein 4.1 sequences are useful as targets for developing diagnostic and therapeutic agents for detecting and treating malaria infection. These and other aspects of the invention are summarized below.

In view of the foregoing discoveries, the invention embraces methods for inhibiting the selective interaction between falcipain-2 and erythrocyte protein 4.1, between falcipain-2 and erythrocyte membrane ankyrin, and between falcipain-2 and hemoglobin, as well as related compositions. Such methods and compositions of the invention are useful for <BR> <BR> identifying compounds for therapeutic use (e. g. , screening assays), as well as for treating a malaria infection.

According to one aspect of the invention, an isolated falcipain-2 inhibitor peptide is provided. The peptide inhibits falcipain-2 cleavage of erythrocyte protein 4.1 and of erythrocyte membrane ankyrin, and has an amino acid sequence provided by one of the following: NVSARFWLSD (SEQ ID NO : 1 ; an ankyrin-derived peptide also referred to as Ank P1), DLDKSQEEIKKHHASI (SEQ ID NO : 5 ; an erythrocyte protein 4.1-derived peptide also referred to herein as 4.1 Pi), or an equivalent of 4.1 P1, for example: aLabSQaaIbbbbASI (SEQ ID NO : 14, wherein"a"can be either D or E and"b"can be either K or R or H) which shows conservative amino acid substitutions of the 4.1 Pi peptide.

In some embodiments, the peptide has an amino acid sequence that is one of the following: NVSARFWLSD (SEQ ID NO : 1 ; an ankyrin-derived peptide also referred to as Ank Pi), DLDKSQEEIKKHHASI (SEQ ID NO : 5 ; an erythrocyte protein 4.1-derived peptide also referred to herein as 4.1 Pi), or an equivalent of 4.1 Pl, for example: aLabSQaaIbbbbASI (SEQ ID NO : 14, wherein"a"can be either D or E and"b"can be either K or R or H) which shows conservative amino acid substitutions of the 4.1 Pl peptide.

In some embodiments of the invention, the falcipain-2 inhibitor peptide of the invention is a non-cleavable form of the peptide. Specifically, in some embodiments the falcipain-2 inhibitor peptide of the invention is non-cleavable by falcipain-2.

In important embodiments of the invention, the falcipain-2 inhibitor peptide of the invention is in a form which is capable of transfer into an erythrocyte, for example in a liposome or conjugated to a carrier such as the Aiiteniiapedia homeoprotein intemalization domain RQIKIWFQNRRMKWKK (SEQ ID NO : 15). Both types of compositions and methods for making the same are aspects of the invention. In some embodiments, the peptide molecules are transported into cells via liposomes. Thus, the invention also involves compositions in which the falcipain-2 inhibitor peptide is contained within liposomes and methods for making such medicaments in accordance with standard procedures for entrapping an active agent in a liposome. Such compositions optionally include pharmaceutically acceptable carrier (s).

According to another aspect of the invention, an isolated falcipain-2 inhibitor peptide is provided that is a unique fragment of the amino acid sequence of SEQ ID NO : 1, and inhibits falcipain-2. According to another aspect of the invention, an isolated falcipain-2 inhibitor peptide is provided that is a unique fragment of the amino acid sequence of SEQ ID NO : 5, and inhibits falcipain-2.

According to another aspect of the invention, isolated nucleic acid molecules that encode the foregoing peptides are provided. In certain embodiments, the nucleic acid comprises unique fragments of the nucleotide sequence encoding SEQ ID NO : 1 or SEQ ID NO : 5, and which preferably encode peptides that selectively inhibit falcipain-2. In one embodiment the isolated nucleic acid encoding SEQ ID NO : 1 has a nucleotide sequence provided as 5'-aatgtctctgccaggttttggctgtcggac-3' (SEQ ID NO : 4). In one embodiment the isolated nucleic acid encoding SEQ ID NO : 5 has a nucleotide sequence provided as 5'-gatttagacaagagtcaagaggagatcaaaaaacatcatgccagcatc-3' (SEQ ID NO : 10). An expression vector is also provided according to the invention. The expression vector includes the isolated foregoing nucleic acid operably linked to a promoter. In another aspect of the invention, isolated host cells transfected or transformed with the foregoing nucleic acids or expression vector are provided.

The nucleic acid molecules of the invention can be in a form which is capable of transport into an erythrocyte, for example in a liposome or conjugated to a carrier. Both types of compositions and methods for making the same are aspects of the invention. In some embodiments the transport is via liposomes. Thus, the invention also involves compositions in which the nucleic acids encoding the falcipain-2 inhibitor peptide are contained within liposomes and methods for making such medicaments in accordance with standard procedures for entrapping an active agent in a liposome. Such compositions optionally include pharmaceutically acceptable carrier (s). In these and other embodiments, membrane proteins are specifically targeted to deliver the nucleic acid molecules or peptides to erythrocytes, e. g. , via receptor-mediated delivery.

According to another aspect of the invention, therapeutic methods for treating malaria <BR> <BR> infection in a subject suspected of having malaria or a predisposition thereto (e. g. , exposure to malaria) are provided. The methods involve administering to the subject a therapeutically effective amount of isolated ankyrin molecules, for example Ank Pi, to inhibit malaria release from red blood cells of the subject. In some embodiments, the ankyrin molecules are peptides. In other embodiments, the ankyrin molecules are nucleic acids that encode ankyrin peptides of the invention.

According to another aspect of the invention, therapeutic methods for treating malaria infection in a subject suspected of having malaria or a predisposition thereto (e. g. , exposure to malaria) are provided. The methods involve administering to the subject a therapeutically effective amount of isolated erythrocyte protein 4.1 molecules, for example 4.1 Pl, to inhibit malaria release from red blood cells of the subject. In some embodiments, the erythrocyte protein 4.1 molecules are peptides. In other embodiments, the erythrocyte protein 4.1 molecules are nucleic acids that encode erythrocyte protein 4.1 peptides of the invention.

According to a related aspect, a method of making a medicament is provided. The method involves placing any one or combination of the foregoing isolated polypeptides or nucleic acids in a pharmaceutically acceptable carrier to form the medicament.

According to still another aspect, a method for identifying a candidate mimetic of an isolated falcipain-2 inhibitor peptide is provided. The method involves determining a control falcipain-2 activity in the absence of a falcipain-2 inhibitor peptide; and determining a test falcipain-2 activity in the presence of a test compound, wherein a test falcipain-2 activity that is less than (to a statistically significant degree) the control falcipain-2 activity indicates that the test compound is a falcipain-2 inhibitor peptide mimetic. In some embodiments, the method also involves determining a reference falcipain-2 activity in the presence of a reference falcipain-2 inhibitor peptide; and comparing the reference falcipain-2 activity and the test falcipain-2 activity; wherein a test falcipain-2 activity that is less than the reference falcipain-2 activity indicates that the falcipain-2 inhibitor peptide mimetic (i. e. , the test compound) has improved inhibitory falcipain-2 inhibitory activity compared to the reference falcipain-2 inhibitor peptide. The reference falcipain-2 inhibitor peptide in one embodiment has an amino acid sequence provided by SEQ ID NO : 1. In another embodiment the reference falcipain-2 inhibitor peptide has an amino acid sequence provided by SEQ ID NO : 5.

According to yet another aspect of the invention, a protein microarray comprising an isolated ankyrin peptide is provided. In one embodiment the ankyrin peptide has an amino acid sequence provided by SEQ ID NO : 1. Alternatively, a nucleic acid microarray comprising a nucleic acid encoding the foregoing ankyrin peptide is provided. In one embodiment the nucleic acid has a sequence provided by 5'-aatgtctctgccaggttttggctgtcggac-3' (SEQ ID NO : 4).

According to yet another aspect of the invention, a protein microarray comprising an isolated erythrocyte protein 4.1 peptide is provided. In one embodiment the protein 4.1 peptide has an amino acid sequence provided by SEQ ID NO : 5. In another embodiment the protein 4.1 peptide has an amino acid sequence provided by SEQ ID NO : 14. Alternatively, a nucleic acid microarray comprising a nucleic acid encoding the foregoing erythrocyte protein 4.1 peptide is provided. In one embodiment the nucleic acid has a sequence provided by 5'-gatttagacaagagtcaagaggagatcaaaaaacatcatgccagcatc-3' (SEQ ID NO : 10).

According to still another aspect of the invention, an antibody (referred to herein as an "anti-falcipain-2 inhibitor peptide antibody"), or a fragment thereof, that selectively binds to any one of the above-described falcipain-2 inhibitor peptides, particularly the Ank Pl peptide having the sequence SEQ ID NO : 1 or the 4.1 Pi peptide having the sequence SEQ ID NO : 5, is provided. In one embodiment, the antibody is a monoclonal antibody, and, in another embodiment, the antibody is a humanized monoclonal antibody. In accordance with the present invention, an anti-falcipain-2 inhibitor peptide antibody (or fragment thereof) inhibits falcipain-2 and/or blocks release of malaria parasite P. falciparum merozoite from human red blood cells.

According to still other aspects of the invention, therapeutic methods that utilize the anti-falcipain-2 inhibitor peptide antibodies of the invention for treating malaria are provided.

The methods for treating a malaria infection involve administering to a subject having a malaria infection an effective amount of an anti-falcipain-2 inhibitor peptide antibody to treat the malaria infection.

In some aspects of the invention, an isolated falcipain-2 inhibitor molecule of one of the foregoing embodiments of the invention is used for manufacture of a medicament for use in a method of treatment for malaria.

In some aspects of the invention, an isolated ankyrin molecule of one of the foregoing embodiments of the invention is used for manufacture of a medicament for use in a method of treatment for malaria.

These and other aspects of the invention will be more apparent in reference to the detailed description and the accompanying drawings.

Brief Description of the Figures The figures are illustrative only and are not required for enablement of the inventions disclosed herein. Figures 1 and 2 are described in the Examples section.

Fig. 3 is an image of an SDS-PAGE gel showing analysis of recombinant protein 4.1 digested with FP-2. A segment of human erythrocyte protein 4.1 (amino acids 294-588) was expressed as a His-tagged protein in E. coli and bound to Ni-NTA beads (lane 1). These beads were incubated with 0.2 LM FP-2 at pH 7.5 for 30 minutes at 37°C, and centrifuged to collect beads (lane 2). Bound protein was eluted in minimum volume of a buffer containing 1M imidazole (lane 3), which appeared to elute only the His-tagged proteins, namely undigested protein and the-26 kDa fragment (*).

Fig. 4 is a diagram of matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS) identification of a cleavage site in the protein 4.1 amino acid sequence. A segment of human erythrocyte protein 4.1, amino acids 294-588 (SEQ ID NO : 21), was expressed as a His-tagged protein (SEQ ID NO : 11), bound to Ni-NTA beads, and digested with FP-2. Cleavage site is indicated by an arrow, and the sequence of three peptides is underlined. Stretch of lower case amino acids represents the 21 amino acid alternative exon of the spectrin/actin binding domain of protein 4.1. HHHHHH (SEQ ID NO: 18), LGSKFRYSGRTQ (SEQ ID NO : 19), KKRERLDGENIYIRHSNLMEL (SEQ ID NO : 12), DLDKSQEEIKKHHASI (SEQ ID NO : 5), SELKKNFMESVPEPRPSEWDKRL (SEQ ID NO: 6), STHSPFRTLNINGQIPTGEGP (SEQ ID NO : 7), and VVVHQETEIADE (SEQ ID NO : 20).

Fig. 5 is an image of an SDS-PAGE gel showing peptide 4.1 Pi inhibition of FP-2- mediated cleavage of human erythrocyte membrane ankyrin and protein 4.1. 5 pi of human erythrocyte inside-out vesicles (IOVs ; 2 mg/ml) were added to 25 Ill reactions in 5 mM sodium phosphate buffer, pH 7.5, 1 mM DTT containing no enzyme (lane 1), or 0.1 pg recombinant falcipain-2 (rFP-2) in the absence (lane 2) or presence of peptides (lanes 3-9).

Lanes 3-7: 0.25 mM, 0.5 mM, 1.0 mM, 1.5 mM, or 2.0 mM, respectively of peptide Pi (corresponding to 4.1 Pi) ; Lane 8: 2.0 mM peptide Pii ; and Lane 9: 2.0 mM peptide Piii.

Fig. 6 is an image of an SDS-PAGE gel showing peptide 4.1 Pi inhibition of FP-2- mediated degradation of human hemoglobin (Hb). 3 pg of human hemoglobin was added to 25 pi reactions in 100 mM sodium acetate, pH 5.5, 1 mM DTT containing no enzyme (lane 1), or 100 nM recombinant FP-2 in the absence (lane 2) or presence of increasing amounts of the peptide 4.1 Pi : 100 pM, 200 pLM, and 500 ßM, respectively in lanes 3,4, and 5. The samples were run on a 15% SDS-PAGE gel under reducing conditions and stained with Coomassie Blue.

Description of the Sequences SEQ ID NO : 1 is the amino acid sequence of Ank P1, a human ankyrin peptide.

SEQ ID NO : 2 is the amino acid sequence of full-length human ankyrin.

SEQ ID NO : 3 is a nucleic acid sequence encoding human ankyrin.

SEQ ID NO : 4 is a nucleic acid sequence encoding the peptide of SEQ ID NO : 1.

SEQ ID NO : 5 is the amino acid sequence of 4.1 Pi, a human erythrocyte protein 4.1 peptide.

SEQ ID NO : 6 is the amino acid sequence of Pii, a human erythrocyte protein 4.1 peptide.

SEQ ID NO : 7 is the amino acid sequence of Piii, a human erythrocyte protein 4.1 peptide.

SEQ ID NO : 8 is the amino acid sequence of full-length human erythrocyte protein 4.1.

SEQ ID NO : 9 is a nucleic acid sequence encoding human erythrocyte protein 4.1.

SEQ ID NO : 10 is a nucleic acid sequence encoding the peptide of SEQ ID NO : 5.

SEQ ID NO: 11 is a partial amino acid sequence of erythrocyte protein 4.1.

SEQ ID NO : 12 is the amino acid sequence of SAB peptide antigen KKRERLDGENIYIRHSNLMLE.

SEQ ID NO : 13: is the amino acid sequence of C-term peptide antigen HPDMSVTKVVVHQETEIADE.

SEQ ID NO : 14 is the amino acid sequence of 4.1 Pi sequence with conservative amino acid substitutions.

SEQ ID NO : 15 is the amino acid sequence of Antennapedia homeoprotein internalization domain.

SEQ ZD NO: 16 is the amino acid sequence of an Antennapedia-Ank Pl fusion peptide.

SEQ ID NO : 17 is the amino acid sequence of a scrambled Antennapedia-Ank Pl fusion peptide.

Detailed Description of the Invention In view of the foregoing, the invention provides isolated falcipain-2 inhibitor molecules, including peptides, nucleic acids encoding such peptides, and antibodies that bind specifically to the peptides. As used herein, a"falcipain-2 inhibitor peptide"refers to a peptide of about 10 to about 20 amino acids that selectively inactivates falcipain-2 cleavage of erythrocyte protein 4.1 and ankyrin. The invention thus provides an ankyrin peptide NVSARFWLSD (Ank PI ; SEQ ID NO : 1) that selectively inactivates falcipain-2 cleavage of erythrocyte protein 4.1 and ankyrin. The complete amino acid and nucleic acid sequences for ankyrin are described in GenBank accession nos. AAA51732 and M28880 (SEQ ID NOs : 2 and 3, respectively. The open reading frame of SEQ ID N0 : 3 corresponds to nucleotides 85- 2724.

The invention also provides an isolated erythrocyte protein 4.1 peptide DLDKSQEEIKKHHASI (4.1 Pi, SEQ ID NO : 5) that selectively inactivates falcipain-2 cleavage of erythrocyte protein 4.1 and ankyrin. The complete amino acid and nucleic acid sequences for erythrocyte protein 4.1 are described in GenBank accession nos. AAA35795 and M14993 (SEQ ID NOs : 8 and 9, respectively. The open reading frame of SEQ ID NO : 9 corresponds to nucleotides 799-2562.

The results disclosed herein demonstrate that the particular ankyrin sequence identified herein alternatively as Ank PI Pi or SEQ ID NO : 1 selectively interacts with falcipain-2 and inhibits falcipain-2 cleavage of erythrocyte protein 4.1, erythrocyte membrane ankyrin, and hemoglobin. Although not wishing to be bound by any theory, it is believed that because the cleavage of the above-mentioned erythrocyte membrane proteins facilitates malaria parasite release from erythrocytes, the inhibition of the cleavage inhibits release of malaria parasites from erythrocytes. Hence, one aspect of the invention is an isolated peptide sequence (SEQ ID NO : 1), and unique fragments thereof that inactivate falcipain-2. The selection of this particular sequence and of the particular malaria protease with which it interacts, could not have been predicted based on the information presently known regarding the erythrocyte protein 4.1 structure.

The results disclosed herein also demonstrate that the particular erythrocyte protein 4.1 sequence identified herein alternatively as 4.1 Pi or SEQ ID NO : 5 selectively interacts with falcipain-2 and inhibits falcipain-2 cleavage of erythrocyte protein 4.1, erythrocyte membrane ankyrin, and hemoglobin. Again not wishing to be bound by any theory, it is believed that because the cleavage of the above-mentioned erythrocyte membrane proteins facilitates malaria parasite release from erythrocytes, the inhibition of the cleavage inhibits release of malaria parasites from erythrocytes. This peptide is alternatively referred to herein <BR> <BR> as"erythrocyte protein 4.1 peptide", "erythrocyte protein 4.1-derived falcipain-2 inhibitor<BR> peptide", "4.1 Pi"and the like. Hence, one aspect of the invention is an isolated peptide sequence (SEQ ID NO : 5), and unique fragments thereof that inactivate falcipain-2. The selection of this particular sequence and of the particular malaria protease with which it interacts, could not have been predicted based on the information presently known regarding the erythrocyte protein 4.1 structure.

Although not wishing to be bound to any particular theory or mechanism, it is believed that P. falciparum infection involves the cleavage of erythrocyte protein 4.1 and the cleavage of erythrocyte membrane ankyrin of the malarial-infected host erythrocyte by the malaria cysteine protease falcipain-2. The cleavage may destabilize the erythrocyte membrane, which ruptures, releasing the malaria parasites. This cleavage can be inhibited by <BR> <BR> Ank Pi molecules (e. g. , SEQ ID NO : 1) and by 4.1 Pi molecules (e. g. , SEQ ID NO : 5). Thus, administration of either or both the Ank Pi and the 4.1 Pi molecule of the invention is useful for reducing the release of infectious malarial parasites from erythrocytes.

Antibodies that recognize erythrocyte protein 4.1 regions such as 4.1 Pi can also be useful to block the cleavage of erythrocyte protein 4.1 and ankyrin and hence, can be useful to protect the host against the erythrocytic forms (merozoites) of malarial parasites, such as P. falciparum. Thus, the present invention also involves the use of monoclonal anti-erythrocyte <BR> <BR> protein 4.1 peptide antibodies (e. g. , anti-4.1 Pi antibodies) that are selective for the epitope used by P. falciparum to cleave the erythrocyte protein 4.1 and/or ankyrin and in the treatment of established infection.

Likewise, antibodies that recognize ankyrin regions such as Ank Pl can also be useful to block the cleavage of erythrocyte protein 4.1 and ankyrin and hence, can be useful to protect the host against the erythrocytic forms (merozoites) of malarial parasites, such as P. falciparum. Thus, the present invention also involves the use of monoclonal anti-alzlçyrin peptide antibodies (e. g., anti-Ank Pl antibodies) that are selective for the epitope used by P. falciparum to cleave the erythrocyte protein 4.1 and/or ankyrin and in the treatment of established infection.

As used herein, the terms"inhibits falcipain-2"or"inhibiting falcipain-2"means inhibits or inhibiting the falcipain-2 cleavage of erythrocyte protein 4.1 and/or erythrocyte membrane ankyrin. These terms also mean inhibits or inhibiting the falcipain-2 cleavage of hemoglobin.

A"subject"shall mean a human or vertebrate animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, non-human primate (e. g. , monkey), rabbit, rat, and mouse. The term"subject"also includes red blood cells collected from a human or animal, for example, blood collected for purposes such as, but not limited to, transfusions.

As used herein, the terms"malarial infection", "malarial parasite", and"malaria"refer to infection by all members of the genus Plasmodium. The application of the invention to Plasmodium falciparuna malarial parasites is described herein, and is intended to include application of the methods to all strains of P. falciparum, which include, but are not limited to: D6, Dd2, HB3, ItG, and W2. The methods of the invention are also envisioned to apply to treatment of malarial parasite infections that result from all other malarial species, which include, but are not limited to: Plasmodium vivax, Plasrnodiunn ovale, and Plasmodium malariae.

As used herein, the term"test compound"means any chemical or molecule to be evaluated, for example to be evaluated as a mimetic for the peptide of SEQ ID NO : 1 or as a mimetic for the peptide of SEQ ID NO : 5. Examples of test compounds include, but are not limited to: library molecules, natural and synthetic peptides other than SEQ ID NO : 1 and SEQ ID NO : 5, non-cleavable forms of peptides, small chemicals, species variants, fusion constructs involving any of the foregoing, etc. As used herein, the term"activity that is less than"means activity that is lower to a statistically significant degree.

As noted above, the invention provides isolated ankyrin peptides and isolated erythrocyte protein 4.1 peptides that inhibit falcipain-2. In important embodiments, the peptide is a non-cleavable peptide. Methods to render the peptides resistant to endopeptidases and/or non-cleavable will be known to those of skill in the art and include, but are not limited to, including individual non-cleavable non-peptidic bonds in points in the peptide sequence that are specially sensitive to enzymatic degradation. Meyer JP et al.

(1995) JMed Clie7iz 38: 3462-3468; Guichard G et al. (1994) Pept Res 7: 308-321. Reverse amide bonds Y [NHCO], reduced amide bonds Y [CH2 NH] or retro-reduced bonds Y [NHCH2 ] can be used as surrogates of the amide link [CONH] in peptides of the invention. Reduced amide links are preferred, since they result only in minor destabilization of oc-helices.

Dauber-Osguthorpe P et al. (1991) Int JPept Protein Res 38: 357-377. Alternatively, the peptides of the invention can be synthesized in all-D-conformations. All-D-peptides can be equally active as the original all-L-peptides (Merrifield B et al. (1994) Ciba Foundation Symposium 186: 5-20; Wade D et al. (1990) Proc Natl Acad Sci USA 87: 4761-4765), capable of successfully resisting enzymatic degradation and less immunogenic than their all-L- analogues (ICing TP et al. (1994) JImmunol 153 : 1124-1131). See U. S. Patent 6, 169, 074.

As noted above, the invention embraces functional variants, such as unique fragments, of the falcipain-2 inhibitor peptides. As used herein, a"functional variant"or"variant"of a falcipain-2 inhibitor peptide is a molecule that contains one or more modifications to the primary amino acid sequence of the ankyrin-derived falcipain-2 inhibitor peptide or of the erythrocyte protein 4. 1-derived falcipain-2 inhibitor peptide and retains the function of inhibiting falcipain-2 as disclosed herein. Modifications that create an ankyrin peptide functional variant can be made, for example, 1) to enhance a property of an ankyrin peptide, such as peptide stability in an expression system or the stability of protein inhibition such as inhibiting falcipain-2 (e. g. , a non-cleavable form of the peptide); or 2) to provide a novel activity or property to an ankyrin peptide, such as addition of an antigenic epitope or addition <BR> <BR> of a moiety to enhance transport across a cell membrane (e. g. , into a red blood cell) and/or to enhance detection. Similarly, modifications that create an erythrocyte protein 4.1 peptide functional variant can be made, for example, 1) to enhance a property of an erythrocyte protein 4.1 peptide, such as peptide stability in an expression system or the stability of protein <BR> <BR> inhibition such as inhibiting falcipain-2 (e. g. , a non-cleavable form of the peptide); or 2) to provide a novel activity or property to an erythrocyte protein 4.1 peptide, such as addition of an antigenic epitope or addition of a moiety to enhance transport across a cell membrane (e. g. , into a red blood cell) and/or to enhance detection. Modifications to a falcipain-2 inhibitor peptide can be made to a nucleic acid which encodes the peptide, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids.

Alternatively, modifications can be made directly to the peptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, substitution of one amino acid for another and the like. Modifications also embrace fusion proteins comprising all or part of the falcipain-2 inhibitor peptide amino acid sequence.

The amino acid sequence of falcipain-2 inhibitor peptides may be of natural or non-natural origin, that is, it may comprise a natural ankyrin peptide molecule or a natural erythrocyte protein 4.1 peptide molecule, or it may comprise a modified sequence as long as the amino acid sequence retains the property of inhibiting falcipain-2 cleavage of erythrocyte protein 4.1 and/or erythrocyte membrane ankyrin. For example, erythrocyte protein 4.1 peptides in this context may be fusion peptides or fusion proteins of an erythrocyte protein 4.1-derived falcipain-2 inhibitor peptide and unrelated amino acid sequences, synthetic peptides of amino acid sequences shown in SEQ ID NO : 5, peptides isolated from cultured cells that express erythrocyte protein 4.1, and peptides coupled to nonpeptide molecules (for <BR> <BR> example in certain drug delivery systems, e. g. , across a cell membrane, or detectable labels).

In one specific embodiment, discussed in the Examples section below, Ank Pl peptide is incorporated into a fusion peptide with Anteiinapedia homeoprotein internalization domain.

Nonpeptide analogs of the falcipain-2 inhibitor peptides of the invention, e. g. , those which provide a stabilized structure or lessened biodegradation, are also contemplated.

Peptide mimetic analogs can be prepared based on a selected ankyrin or erythrocyte protein 4.1 peptide by replacement of one or more residues by nonpeptide moieties. Preferably, the nonpeptide moieties permit the peptide to retain its natural conformation, or stabilize a preferred, e. g. , bioactive, conformation. One example of a method for preparation of nonpeptide mimetic analogs from peptides is described in Nachman RJ et al. (1995) Regul Pept 57: 359-370. Peptide mimetics also can be selected from libraries of synthetic compounds (e. g., combinatorial libraries of small organic molecules) or natural molecules <BR> <BR> according to the falcipain-2 inhibiting properties of such molecule (i. e. , ability to selectively inhibit falcipain-2 (isolated or expressed in a cell or organism)) and/or inhibit parasite release from human red blood cells. In general, the methods for selection involve determining whether the library's molecules inhibit falcipain-2 cleavage of erythrocyte protein 4.1 or ankyrin and/or block release of malaria parasite from red blood cells by for example inhibiting a natural process which merozoites use to be released from red blood cells.

If a variant involves a change to an amino acid of a falcipain-2 inhibitor peptide (e. g., SEQ ID NO : 5), functional variants of the a falcipain-2 inhibitor peptide having conservative <BR> <BR> amino acid substitutions typically will be preferred, i. e. , substitutions which retain a property of the original amino acid such as charge, hydrophobicity, conformation, etc. Examples of conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Other methods for identifying functional variants of the a falcipain-2 inhibitor peptides rely upon the development of amino acid sequence motifs to which potential epitopes may be compared. (See, e. g. , published PCT application US/96/03182 of Strominger and Wucherpfennig). In general, these methods rely upon the development of amino acid sequence motifs to which potential epitopes may be compared. Each motif describes a finite set of amino acid sequences in which the residues at each (relative) position may be (a) restricted to a single residue, (b) allowed to vary amongst a restricted set of residues, or (c) allowed to vary amongst all possible residues. For example, a motif might specify that the residue at an erythrocyte protein 4.1 peptide position may be any one of the residues valine, leucine, isoleucine, methionine, or phenylalanine; that the residue at the second position must be histidine; that the residue at the third position may be any amino acid residue; that the residue at the fourth position may be any one of the residues valine, leucine, isoleucine, methionine, phenylalanine, tyrosine or tryptophan; and that the residue at the fifth position must be lysine.

Sequence motifs for the falcipain-2 inhibitor peptide functional variants can be developed by analysis of the falcipain-2 contact points of the erythrocyte protein 4.1 peptide disclosed herein. By providing a detailed structural analysis of the residues involved in the binding of falcipain-2 to the erythrocyte protein 4.1 peptide disclosed herein, one of ordinary skill in the art is enabled to make predictions of sequence motifs for binding between these two proteins.

Using these sequence motifs as search, evaluation, or design criteria, one of ordinary skill in the art is enabled to identify classes of peptides (functional variants of the falcipain-2 inhibitor peptides disclosed herein) which have a reasonable likelihood of inhibiting falcipain-2 and inhibiting parasite release from erythrocytes. These peptides can be synthesized and tested for activity as described herein. Use of these motifs, as opposed to pure sequence homology (which excludes many peptides which are antigenically similar but quite distinct in sequence) or sequence homology with unlimited"conservative"substitutions (which admits many peptides which differ at critical highly conserved sites), represents a method by which one of ordinary skill in the art can evaluate peptides for potential application in the treatment of disease, such as malaria infection.

The ability of the variant falcipain-2 inhibitor peptides to inhibit falcipain-2 cleavage then is determined according to standard procedures. For example, the variant peptide can be contacted with falcipain-2, and standard procedures may be used to determine whether the falcipain-2 retains its function and cleaves erythrocyte protein 4.1 and/or ankyrin.

Variant falcipain-2 inhibitor peptides include unique fragments of the peptide having SEQ ID NO : 1 or of the peptide having SEQ ID NO : 5. As used herein, a"unique fragment" is one that is a signature for the larger amino acid or nucleic acid sequence. It, for example, is long enough to assure that its precise sequence is not found in molecules within the human genome outside of the erythrocyte protein 4.1 nucleic acid defined above (and human alleles).

Those of ordinary skill in the art may apply no more than routine procedures to determine if a fragment is unique within the human genome. Unique fragments, however, exclude fragments completely composed of the nucleotide sequences of any previously published sequences as of the filing date of the priority documents for sequences listed in a respective priority document or the filing date of this application for sequences listed for the first time in this application which overlap the sequences of the invention.

Inhibiting falcipain-2 cleavage of erythrocyte protein 4.1 and/or ankyrin by the variant peptide and/or blocking of the exit of the malaria parasite from erythrocytes (or other cells expressing erythrocyte protein 4.1 or ankyrin) indicates that the variant peptide is a functional variant. The methods also can include the step of comparing the Ank P1 peptide-or the 4.1 P1 peptide-mediated inhibition of falcipain-2 cleavage and/or the exit from erythrocytes by the malaria parasite as a determination of the effectiveness of the falcipain-2 inhibition by the functional variant. By comparing the functional variant with the Ank P1 peptide or 4.1 Pi peptide or other compositions of the invention disclosed herein, peptides with increased falcipain-2 cleavage inhibiting properties can be prepared.

Variants of the falcipain-2 inhibitor peptides prepared by any of the foregoing methods can be sequenced, if necessary, to determine the amino acid sequence and thus deduce the nucleotide sequence which encodes such variants. Thus, those nucleic acid sequences that code for ankyrin peptide Ank Pi or variants thereof, including allelic variants, are also a part of the invention. Likewise, those nucleic acid sequences that code for an erythrocyte protein 4.1 peptide or variants thereof, including allelic variants, are also a part of the invention. In screening for nucleic acids that encode a falcipain-2 inhibitor peptide of the invention, nucleic acid hybridization such as a Southern blot or a Northern blot may be performed under stringent conditions, together with a 32p or other suitable probe. The term "stringent conditions"as used herein refers to parameters with which the art is familiar.

Nucleic acid hybridization parameters may be found in references which compile such methods, e. g., Molecular Clonifag : A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or <BR> <BR> Current Protocols in Molecular Biology, F. M. Ausubel, et al. , eds. , John Wiley & Sons, Inc., New York. Exemplary stringent conditions include hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% Polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 25mM NaH2P04 (pH7), 0.5% SDS, 2mM EDTA). SSC is 0. 15M Sodium Chloride/0. 015M Sodium Citrate, pH 7; SDS is Sodium Dodecyl Sulfate; and EDTA is Ethylene diaminetetraacetic acid. After hybridization, the membrane upon which the DNA is transferred can be washed, for example, at 2xSSC at room temperature and then at 0.1-0. 5 x SSC/0.1 x SDS at temperatures up to 68°C. After washing the membrane to which DNA encoding a falcipain-2 inhibitor peptide is finally transferred, the membrane can be placed against X-ray film to detect the radioactive or other suitable signal.

There are other conditions, reagents, and so forth which can be used, which result in a similar degree of stringency. The skilled artisan will be familiar with such conditions, and thus they are not given here. It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of nucleic acids encoding the falcipain-2 inhibitor peptides of the invention. The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing.

The invention also includes the use of nucleic acid sequences which include alternative codons that encode the same amino acid residues of the falcipain-2 inhibitor peptides of the invention. For example, leucine (L) residues can be encoded by the codons CUA, CUC, CUG, CLOU, UUA and UUG. Each of the six codons is equivalent for the purposes of encoding a leucine residue. Thus, it will be apparent to one of ordinary skill in the art that any of the leucine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a leucine residue. Similarly, nucleotide sequence triplets which encode other amino acid codons comprising the falcipain-2 inhibitor peptide include: GCA, GCC, GCG, GCU (alanine codons, A); GAC, GAU (aspartic acid codons, D); GAA, GAG (glutamic acid codons, E); UUC, UUU (phenyalanine codons, F); CAC, CAU (histidine codons, H); AUA, AUC, AUU (isoleucine codons, 1) ; AAA, AAG (lysine codons, K); AAC, AAU (asparagine codons, N); CAA, CAG (glutamine codons, Q); AGA, AGG, CGA, CGC, CGG, CGU (arginine codons, R); AGC, AGU, UCA, UCC, UCG, UCU (serine codons, S) ; GUA, GUC, GUG, GUU (valine codons, V); and UGG (tryptophan codon, W). Other amino acid residues may be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the native ankyrin peptide-or erythrocyte protein 4.1 peptide-encoding nucleic acids in codon sequence due to the degeneracy of the genetic code.

Preferred nucleic acids encoding ankyrin-derived falcipain-2 inhibitor peptides are those which preferentially express the ankyrin peptide Ank Pj, such as those having SEQ ID NO : 1. The ankyrin nucleic acids of the invention do not encode the full-length ankyrin polypeptide of SEQ ID NO : 2, but do include nucleotide sequences encoding the ankyrin- derived falcipain-2 inhibitor peptide disclosed herein (e. g. , SEQ ID NO : 1), or functional equivalents thereof.

Preferred nucleic acids encoding erythrocyte protein 4.1-derived falcipain-2 inhibitor peptides are those which preferentially express the erythrocyte protein 4.1 peptide 4.1 P1, such as those having SEQ ID NO : 5. The erythrocyte protein 4.1 nucleic acids of the invention do not encode the full-length erythrocyte protein 4.1 polypeptide of SEQ ID NO : 8, but do include nucleotide sequences encoding the erythrocyte protein 4.1-derived falcipain-2 <BR> <BR> inhibitor peptide disclosed herein, or functional equivalents thereof, (e. g. , SEQ ID NO : 14).

The invention also provides modified nucleic acid molecules, which include additions, substitutions and deletions of one or more nucleotides. In preferred embodiments, these modified nucleic acid molecules and/or the polypeptides they encode retain at least one activity or function of the unmodified nucleic acid molecule and/or the polypeptides, such as antigenicity, enzyme binding, formation of complexes by binding of peptides to falcipain-2, inhibiting falcipain-2, etc. In certain embodiments, the modified nucleic acid molecules encode modified polypeptides, preferably polypeptides having conservative amino acid substitutions as are described elsewhere herein. The modified nucleic acid molecules are structurally related to the unmodified nucleic acid molecules and in preferred embodiments are sufficiently structurally related to the unmodified nucleic acid molecules so that the modified and unmodified nucleic acid molecules hybridize under stringent conditions known to one of skill in the art.

For example, modified nucleic acid molecules which encode polypeptides having single amino acid changes can be prepared. Each of these nucleic acid molecules can have one, two, or three nucleotide substitutions exclusive of nucleotide changes corresponding to the degeneracy of the genetic code as described herein. Likewise, modified nucleic acid molecules which encode polypeptides having two amino acid changes can be prepared which <BR> <BR> have, e. g. , 2-6 nucleotide changes. Numerous modified nucleic acid molecules like these will be readily envisioned by one of skill in the art, including for example, substitutions of nucleotides in codons encoding amino acids 2 and 3,2 and 4,2 and 5,2 and 6, and so on. In the foregoing example, each combination of two amino acids is included in the set of modified nucleic acid molecules, as well as all nucleotide substitutions which code for the amino acid substitutions. Additional nucleic acid molecules that encode polypeptides having additional substitutions (i. e. , 3 or more), additions or deletions (e. g. , by introduction of a stop<BR> codon or a splice site (s) ) also can be prepared and are embraced by the invention as readily envisioned by one of ordinary skill in the art. Any of the foregoing nucleic acids, peptides, or polypeptides can be tested by routine experimentation for retention of structural relation or activity to the nucleic acids, peptides, and/or polypeptides disclosed herein.

It will also be understood that the invention embraces the use of the sequences in expression vectors, as well as to transfect host cells and cell lines, be these prokaryotic (e. g., <BR> <BR> E. coli), or eukaryotic (e. g. , CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells). The expression vectors require that the pertinent sequence, i. e. , those described supra, be operably linked to a promoter.

Delivery of expression vectors encoding the falcipain-2 inhibitor peptide sequences in vivo and/or in vitro can be via the use of nucleic acid delivery systems known in the art. See, <BR> <BR> e. g. , Allsopp CE et al. (1996) Eur Jlmnuraol 26 : 1951-1959. Recombinant vectors including viruses selected from the group consisting of adenoviruses, adeno-associated viruses, poxviruses including vaccina viruses and attenuated poxviruses such as NYVAC, Semliki Forest virus, Venezuelan equine encephalitis virus, retroviruses, Sindbis virus, and Ty virus- like particle, plasmids (e. g. ,"naked"DNA), bacteria (e. g. , the bacterium bacille Calmette- Guérin, BCG), and the like can be used in such delivery, for example, for use as a vaccine.

Other viruses, expression vectors and the like which are useful in preparation of a vaccine are known to one of ordinary skill in the art. For example, one can test the erythrocyte protein 4.1 molecule delivery systems in standard model systems such as mice to determine efficacy of the delivery system. The systems also can be tested in human clinical trials.

As used herein, a"vector"can be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids, bacteria and virus genomes as disclosed herein, such as adenovirus, poxvirus and BCG. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e. g., P-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e. g. , green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said to be"operably" joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. As noted above, certain preferred nucleic acids express only fragments of ankyrin and erythrocyte protein 4.1 polypeptides that include the falcipain-2 inhibitor peptides described herein.

The precise nature of the regulatory sequences needed for gene expression can vary between species or cell types, but shall in general include, as necessary, 5'non-transcribed and 5'non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.

Especially, such 5'non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.

Regulatory sequences can also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention can optionally include 5'leader or signal sequences.

The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e. g. , Sambrook et al., Molecular Cloning : A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA) encoding a falcipain-2 inhibitor peptide of the invention. That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those such as pcDNA3. 1 (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences. Additionally, suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element. Another expression vector is the pEF- BOS plasmid containing the promoter of polypeptide Elongation Factor 1 oc, which stimulates efficiently transcription in vitro. The plasmid is described by Mizushima S et al. (1990) Nucleic Acids Res 18: 5322, and its use in transfection experiments is disclosed by, for example, Demoulin JB et al. (1996) Mol Cell Biol 16: 4710-4716. Still another preferred expression vector is an adenovirus which is defective for E1 and E3 proteins. Stratford- Perricaudet LD et al. (1992) J Clin Invest 90: 626-630. The use of the adenovirus to express proteins for immunization is disclosed by Warnier G et al. , in intradermal injection in mice for immunization against P1A (Int JCancer 67: 303-310,1996).

The invention also embraces so-called expression kits, which allow the artisan to prepare a desired expression vector or vectors. Such expression kits include at least separate portions of at least two of the previously discussed materials. Other components may be added, as desired.

The invention further includes nucleic acid or protein microarrays which include falcipain-2 inhibitor nucleic acids or peptides of the invention (preferably the isolated ankyrin peptide SEQ ID NO : 1 or the isolated erythrocyte protein 4.1 peptide SEQ ID NO : 5) or nucleic acids encoding such a peptide. In this aspect of the invention, standard techniques of microarray technology are utilized to assess expression of peptides which bind falcipain-2 inhibitor peptide (e. g. , anti-erythrocyte protein 4.1 antibodies; falcipain-2) and/or identify biological constituents that bind such peptides. The constituents of biological samples include antibodies, falcipain-2 molecules, and the like. Microarray technology, which is also known by other names including protein chip technology and solid-phase protein array technology, is well known to those of ordinary skill in the art and is based on, but not limited to, obtaining an array of identified peptides or proteins on a fixed substrate, binding target molecules or biological constituents to the peptides, and evaluating such binding. See, e. g., MacBeath G et al. (2000) Science 289: 1760-1763.

Microarray substrates include but are not limited to glass, silica, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, various clays, nitrocellulose, or nylon. The microarray substrates may be coated with a compound to enhance synthesis of a probe (peptide or nucleic acid) on the substrate. Coupling agents or groups on the substrate can be used to covalently link the first nucleotide or amino acid to the substrate. A variety of coupling agents or groups are known to those of skill in the art. Peptide or nucleic acid probes thus can be synthesized directly on the substrate in a predetermined grid.

Alternatively, peptide or nucleic acid probes can be spotted on the substrate, and in such cases the substrate may be coated with a compound to enhance binding of the probe to the substrate. In these embodiments, presynthesized probes are applied to the substrate in a precise, predetermined volume and grid pattern, preferably utilizing a computer-controlled robot to apply probe to the substrate in a contact-printing manner or in a non-contact manner such as ink jet or piezo-electric delivery. Probes may be covalently linked to the substrate.

In some embodiments, one or more control peptide or nucleic acid molecules are attached to the substrate. Preferably, control nucleic acid molecules allow determination of factors such as binding characteristics, reagent quality and effectiveness, hybridization success, and analysis thresholds and success.

The anti-falcipain-2 inhibitor peptide antibodies of the invention selectively bind to SEQ ID NO : 1 or to SEQ ID NO : 5 and inhibit falcipain-2 and inhibit release of P. falciparurra, or other malaria parasite from human red blood cells by virtue of effectively blocking the site on the erythrocytic molecule (ankyrin or erythrocyte protein 4.1) used as a target by the malaria parasite falcipain-2 protease. Accordingly, the invention provides monoclonal antibodies that have a combining site that has the same stereochemical configuration as the ligand site of the falcipain-2 of the malaria parasite. Such anti-ankyrin and anti-erythrocyte protein 4.1 antibodies are prepared by standard methods. The selected cloned hybridomas produce as large quantities of suitable monoclonal antibodies as desired.

In view of the foregoing, the invention also permits the artisan to treat a subject having or at risk of having a malaria infection. Treatments include administering to the subject a falcipain-2 inhibitor molecule of the invention, such as a falcipain-2 inhibitor peptide, nucleic acid, antibody, or other agent of the invention that inhibits falcipain-2 cleavage of erythrocyte protein 4.1 and/or ankyrin expressed by an erythrocyte, in an effective amount to treat (including prevent) a malaria infection of the subject. Agents useful in the foregoing treatments include ankyrin peptides and functional variants thereof, as well as the anti-ankyrin peptide antibodies of the invention disclosed herein. Additional agents useful in the foregoing treatments include erythrocyte protein 4.1 peptides and functional variants thereof, as well as the anti-erythrocyte protein 4.1 antibodies of the invention disclosed herein.

In certain embodiments, the falcipain-2 inhibitor peptides and anti-falcipain-2 inhibitor peptide antibodies of the invention are used to produce antibodies using standard techniques well known to the art. Antibodies raised against anti-falcipain-2 peptide antibodies (anti-antibody antibodies) include anti-idiotype antibodies. These anti-idiotype antibodies have binding sites which have the same stereochemical configuration as the falcipain-2 inhibitor peptide that is bound by the anti-falcipain-2 inhibitor peptide antibodies.

Standard reference works setting forth the general principles of antibody production include Catty, D., Antibodies, A Practical Approach, Vol. 1, IRL Press, Washington, D. C. (1988); Klein, J., Immuraology : The Science of Cell-Nora-Cell Discrimination, John Wiley and Sons, New York (1982); Kennett, R. , et al., Monoclonal Antibodies, Hybridoma, A New Dimension<BR> In Biological Analyses, Plenum Press, New York (1980); Campbell, A. , Monoclonal Antibody Technology, in Laboratory Techniques and Biochemistry and Molecular Biology, Vol. 13, Burdon, R. et al. , eds. , Elsevier Amsterdam (1984); and Eisen, H. N., Microbiology,<BR> third edition, Davis, B. D. et al. , eds. , Harper & Rowe, Philadelphia (1980). See also, U. S.<BR> <P>Patent 5,101, 017, issued March 31,1992 to Rubinstein, et al. , entitled, "Antibodies for<BR> providing protection against P. vivax malaria infection, "which also reports the preparation of anti-idiotypic antibodies for treating infectious disease. References that report vaccine approaches for malaria include: U. S. Patent 6,066, 623, issued to Hoffman, et al. , entitled "Polynucleotide vaccine protective against malaria, methods of protection and vector for delivering polynucleotide vaccines" ; and U. S. Patent 6,120, 770, issued to Adams et al., entitled"Plasmodiu77 proteins useful for preparing vaccine compositions." Thus, in one embodiment according to this aspect of the invention, an anti-ankyrin antibody (or fragment thereof) that selectively binds to a peptide having SEQ ID NO : 1 and which inhibits release of P. falciparum malaria parasite from human red blood cells is provided. These antibodies are prepared using any of a variety of methods, including administering the Ank Pl peptides of the invention, fragments of the foregoing, antibodies selective for the foregoing, and the like to an animal to induce monoclonal or polyclonal antibodies. The production of monoclonal antibodies is according to techniques well known in the art.

In another embodiment according to this aspect of the invention, an anti-erythrocyte protein 4.1 antibody (or fragment thereof) that selectively binds to a peptide having SEQ ID NO : 5 and which inhibits release of P. falciparuna malaria parasite from human red blood cells is provided. These antibodies are prepared using any of a variety of methods, including administering the erythrocyte protein 4.1 peptides of the invention, fragments of the foregoing, antibodies selective for the foregoing, and the like to an animal to induce monoclonal or polyclonal antibodies. The production of monoclonal antibodies is according to techniques well known in the art.

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) Tlae Experimental Foundations ofModern Immunology, Wiley & Sons, Inc., New York; Roitt, 1. (1991) Essential Immunology, 7th Ed. , Blackwell Scientific Publications, Oxford). The pFc'and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc'region has been enzymatically cleaved, or which has been produced without the pFc'region, designated an F (ab') 2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of nonspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of"humanized"antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc'regions to produce a functional antibody. See, e. g. , U. S. patents 4,816, 567,5, 225,539, 5,585, 089,5, 693,762 and 5,859, 205.

Accordingly, humanized anti-falcipain-2 inhibitor peptide antibodies, particularly those that selectively bind to SEQ ID NO : 1 or to SEQ ID NO : 5, and the use of such antibodies (e. g. , to provide passive immunity to a subject) are embraced with the inventions disclosed herein.

For example, PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric"antibodies.

Thus, the present invention also provides for F (ab') 2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F (ab') 2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies and human monoclonal antibodies, such as those produced by mice having functional human immunoglobulin gene loci.

Such antibodies also can be used to identify tissues expressing protein or to purify protein. Antibodies also can be coupled to specific labeling agents for imaging or to anti- infectious agents, toxins such as ricin, other cytostatic or cytolytic drugs, and so forth, for therapeutic purposes.

Non-limiting examples of supports for affinity-separation of antibodies, including monoclonals, include the following: activated Sepharose, activated cellulose and activated Sephadex. "Activated"refers to the creation, on the insoluble material, of reactive chemical groups that will form covalent linkages with the antibody molecules when incubated together under appropriate conditions. Typically, reactive groups are introduced into the insoluble substrate by the action of cyanogen bromide (CNBr) at high pH. Other examples of supports useful for separation of antibodies, including monoclonal antibodies, include immobilized protein A and protein G.

The compositions according to the invention may have the following characteristics and properties, as well as many others: (1) The antibodies can be used as the immunogenic agent in a vaccine and can be produced in virtually limitless quantity.

(2) The ankyrin peptides of the invention may be used for inhibiting the malaria parasite's specific cysteine proteases (e. g. , falcipain-2).

(3) The erythrocyte protein 4.1 peptides of the invention may be used for inhibiting the malaria parasite's specific cysteine proteases (e. g. , falcipain-2).

(4) The anti-falcipain-2 inhibitor peptide antibodies may be used directly in vivo to block the site of cleavage by the parasite falcipain-2. This is useful in the management of patients with particularly severe attacks of P. falciparum malaria, in whom the level of parasitemia may be very high.

The falcipain-2 inhibitor peptide, nucleic acid, and antibody compositions of the instant invention can be used to treat malaria infection in a subject. As used herein, the terms "treat"and"treating"refer to the reduction, slowing, halting, or cure of an established condition of a subject, as well as to preventing the development of a condition of a subject.

Thus the term"treat the malaria infection"as used herein refers to the reduction, slowing, halting, or cure of an established malaria infection of a subject, as well as to preventing a malaria infection of a subject. A"subject suspected of having malaria"is a subject that has or that is at risk of having a malaria infection. A subject that has a malaria infection is a subject with an objectively measurable manifestation of an existing malaria infection. A subject at risk of having a malaria infection includes a subject with a known exposure to <BR> <BR> malaria and a subject likely to be exposed to malaria, e. g. , through travel to an endemic area.

When administered, the therapeutic compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations can routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents. The term"pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The characteristics of the carrier will depend on the route of administration.

The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intranasal, intracavity, subcutaneous, intradermal, or transdermal.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The falcipain-2 inhibitor peptides or nucleic acids of the invention, as described herein, can be administered and delivered to a mammalian cell, examples of delivery methods include, but are not limited to delivery by: virus, liposomes (e. g. , see U. S. Patent No.

5,643, 599), or by any other suitable methods known in the art or later developed.

Methods of delivery of the peptides of the invention into erythrocytes may also include, but are not limited to, methods that facilitate the transport into a cell of a membrane- impermeable extracellular agent having an intracellular activity. For example, a carrier molecule for facilitating the transport of the peptides of the invention across the erythrocyte cell membrane. Such carrier molecules, which can be used to mediate transport of an extracellular agent across a cell or other lipid bilayer membrane, are described in U. S. Patent 5,846, 743.

Methods of delivery can be modified to target certain cells, and in particular, cell surface receptor molecules or antigens present on erythrocytes, for example by targeting receptors on erythrocytes, including, but not limited to, Band 3, glycophorin A, and/or glycophorin C. Methods of targeting cells to deliver nucleic acid constructs or other active agents (e. g. , peptides) are known in the art. The falcipain-2 inhibitor peptide can also be delivered into cells by expressing a recombinant protein fused with peptide carrier molecules, examples of which, though not intended to be limiting, are tat and Ante717lapedia homeoprotein internalization domain. These delivery methods are known to those of skill in the art and are described in U. S. Patent 6,080, 724 and U. S. Patent 5,783, 662, the entire contents of which are hereby incorporated by reference.

The preparations of the invention are administered in effective amounts. An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, stimulates the desired response. In the case of treating an infectious disease such as malaria, the desired response is inhibiting the onset, stage or progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently.

The falcipain-2 inhibitor peptide, nucleic acid, or antibody molecule dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days.

The absolute amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual patient parameters including age, physical condition, size, weight, and the stage of the disease. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

The therapeutically effective amount of the falcipain-2 inhibitor peptide, nucleic acid, or antibody molecule is that amount effective to inhibit malarial release from red blood cells.

Such inhibition can be determined using standard measurements of malarial infection, some <BR> <BR> of which are described in Harrison's Principles ofltiteriial Medicine, 14 th Ed. , McGraw Hill Companies, New York, 1998. For example, diagnosis of malaria can be made by microscopic identification of asexual forms of the parasite in peripheral blood smears stained with Romanovsky staining, or Giemsa at pH 7.2, Wright's, Field's, or Leishman's stain can be utilized. Both thin and thick blood smears should be examined. In addition, a finger-prick blood test is also available, in which the presence of P. falciparum histidine-rich protein 2 is determined. Additional methods of diagnosis and assessment of malarial infection are known to those of skill in the art. The level of parasitemia is important in the prognosis and can be determined with the above-identified diagnostic tests and by other means known in the art.

In addition to the diagnostic tests described above, clinical features of malarial infection can be monitored for assessment of infection. Theses features include, but are not limited to: normochromic, nomocytic anemia, erythrocyte sedimentation rate, plasma viscosity, and platelet count may be reduced. Subjects may also have metabolic acidosis, with low plasma concentrations of glucose, sodium, bicarbonate, calcium, phosphate, and albumin together with elevations in lactate, blood urea nitrogen, creatinine, urate, muscle and liver enzymes, and conjugated and unconjugated bilirubin. In adults and children with cerebral malaria, the mean opening pressure at lumbar puncture is about 160 mm cerebrospinal fluid; the cerebrospinal fluid usually is normal or has a slightly elevated total protein level (>100 mg/dL; see Harrison's Principles of Inte7nal Medicine, 14th Ed. , McGraw Hill Companies, New York, 1998).

These types of tests, as well as others known to those of ordinary skill in the medical arts, can be used to assess the malarial infection status of a subject and to evaluate a therapeutically effective amount of falcipain-2 inhibitor molecule administered. A first determination of malarial infection can be made using one of the methods described above, a subsequent determination of malarial infection can likewise be made, and a comparison of the infection levels can be used to assess the effectiveness of falcipain-2 inhibitor molecule administration as a prophylactic or a treatment of the malarial infection. Absence of a malarial infection can be an indication for prophylactic intervention by administering falcipain-2 inhibitor molecules to prevent malarial infection.

The falcipain-2 inhibitor molecules can be administered alone, in combination with each other, and/or in combination with other anti-malarial drug therapies. Antimalarial agents (for treatment and/or prophylaxis) that can be administered with erythrocyte protein 4.1 molecules may include, but are not limited to: mefloquine, doxycycline, chloroquine, aminoquinolines, dihydrofolate reductase inhibitors: pyrimethamine and proguanil (chloroguanide), dapsone, quinidine gluconate, quinine, artemisinin derivatives: artemether and artesunate, and primaquin.

The above-described drug therapies are well known to those of ordinary skill in the art and are administered by modes known to those of skill in the art. The drug therapies are administered in amounts that are effective to achieve the physiological goals (to reduce malarial infection, and/or reduce malarial titer in a subject), in combination with the falcipain-2 inhibitor molecules of the invention. Thus, it is contemplated that the drug therapies can be administered in amounts which are not capable of preventing or reducing the physiological consequences of the malarial infections when the drug therapies are administered alone, but which are capable of preventing or reducing the physiological consequences of malarial infection when administered in combination with the falcipain-2 inhibitor molecules of the invention.

In addition to the above-described inventions which are based, in part, on the discovery of the particular erythrocyte protein 4.1 sequence that interacts with falcipain-2, Applicants disclose herein related inventions which are based, in part, on the discovery of the particular portion of falcipain-2 that selectively binds to erythrocyte protein 4.1. It is to be understood that enablement of the falcipain-2-related inventions throughout a broad scope can be accomplished in an analogous manner to that described for the erythrocyte protein 4.1- related invention. Thus, the methods and definitions applied above in reference to the erythrocyte protein 4.1 molecule compositions and methods can be used in reference to the falcipain-2 molecule compositions and methods by substituting the falcipain-2 molecule for the erythrocyte protein 4.1 molecule. For example, vectors expressing a falcipain-2 protein can be prepared by substituting a falcipain-2 nucleic acid for an erythrocyte protein 4.1 nucleic acid and inserting into an expression vector as described above. Thus, the invention embraces various compositions containing such falcipain-2 peptides for use, e. g. , in screening assays to detect the specific interaction between falcipain-2 and erythrocyte protein 4.1, in screening assays to detect the specific interaction between falcipain-2 and ankyrin, in screening assays to detect the specific interaction between falcipain-2 and hemoglobin, as well as for use in diagnostic and therapeutic applications for detecting and treating, respectively, malaria infection. In general, such compositions contain components (or are contained in kits that contain additional components) which are selected to detect the specific interaction between falcipain-2 and erythrocyte protein 4.1 protein (particularly, the erythrocyte protein 4.1 peptides disclosed herein).

In summary, the various aspects of the invention include one or more of the following utilities: 1. Treatment and prevention of malaria disease in humans and animals: (a) Development of a peptide, peptidomimetic, and/or protein antimalarial drug partially or entirely based on the human ankyrin residues 1206-1215 (SEQ ID NO : 1).

(b) Development of a peptide, peptidomimetic, and/or protein antimalarial drug partially or entirely based on the human erythrocyte protein 4.1 protein residues 428-443 (SEQ ID NO : 5).

(c) Development of a peptide, peptidomimetic, and/or protein antimalarial drug using three-dimensional structure information of human ankyrin protein containing a partial or the entire amino acid sequence of residues 1206-1215 (SEQ ID NO : 1).

(d) Development of a peptide, peptidomimetic, and/or protein antimalarial drug using three-dimensional structure information of human erythrocyte protein 4.1 protein containing a partial or the entire amino acid sequence of residues 428-443 (SEQ ID NO : 5).

(e) Development of a non-peptide, non-protein, and/or non-peptidomimetic antimalarial drug derived from three-dimensional structure information of human erythrocyte ankyrin protein containing a partial or the entire amino acid sequence of residues 1206-1215 (SEQ ID NO : 1).

(f) Development of a non-peptide, non-protein, and/or non-peptidomimetic antimalarial drug derived from three-dimensional structure information of human erythrocyte protein 4.1 protein containing a partial or the entire amino acid sequence of residues 428-443 (SEQ ID NO : 5).

(g) Use of a non-human ankyrin gene and/or protein sequence corresponding to the human ankyrin protein residues 1206-1215 (SEQ ID NO : 1) for the purpose of developing drug or vaccine for human and/or animal malaria disease.

(h) Use of a non-human erythrocyte protein 4. 1 gene and/or protein sequence corresponding to the human erythrocyte protein 4.1 protein residues 428-443 (SEQ ID NO : 5) for the purpose of developing drug or vaccine for human and/or animal malaria disease.

2. Screening of the malaria parasite ligand (s) binding to the host erythrocyte protein 4.1 and/or ankyrin : (a) Use of a peptide and/or protein containing partial or entire sequence of human ankyrin protein residues 1206-1215 (SEQ ID NO : 1) in efforts to identify and/or develop a drug or vaccine for human and/or animal malaria disease. These include experiments including, but not limited to: protein or peptide binding experiments carried out in vitro and in vivo ; three- dimensional structure-based approaches; computer modeling ; combinatorial chemistry screening; and high throughput screening approaches. The malaria parasite ligand (s) identified and/or characterized by utilizing the inventions disclosed herein can be used as new targets for the development of a highly efficient malaria vaccine and/or drug.

(b) Use of nucleotide sequence encoding partial or entire amino acid sequence of human ankyrin protein residues 1206-1215 (SEQ ID NO : 1) to identify and/or functionally characterize the malaria parasite ligand binding to and/or cleaving the erythrocyte protein 4.1 and/or ankyrin protein.

(c) Use of a non-human ankyrin gene and/or protein sequence corresponding to the human ankyrin protein residues 1206-1215 (SEQ ID NO : 1) for the purpose of carrying out the screening of malaria parasite ligand as described in 2 (a) and 2 (b) above.

(d) Use of a peptide and/or protein containing partial or entire sequence of human erythrocyte protein 4.1 protein residues 428-443 (SEQ ID NO : 5) in efforts to identify and/or develop a drug or vaccine for human and/or animal malaria disease. These include experiments including, but not limited to: protein or peptide binding experiments carried out in vitro and in vivo ; three-dimensional structure-based approaches; computer modeling; combinatorial chemistry screening; and high throughput screening approaches. The malaria parasite ligand (s) identified and/or characterized by utilizing the inventions disclosed herein can be used as new targets for the development of a highly efficient malaria vaccine and/or drug.

(e) Use of nucleotide sequence encoding partial or entire amino acid sequence of human erythrocyte protein 4.1 protein residues 428-443 (SEQ ID NO : 5) to identify and/or functionally characterize the malaria parasite ligand binding to and/or cleaving the erythrocyte protein 4.1 and/or ankyrin protein.

(f) Use of a non-human erythrocyte protein 4.1 gene and/or protein sequence corresponding to the human erythrocyte protein 4.1 protein residues 428-443 (SEQ ID NO : 5) for the purpose of carrying out the screening of malaria parasite ligand as described in 2 (d) and 2 (e) above.

3. Screening assays to select agents which inhibit falcipain-2 binding to and/or cleavage of erythrocyte protein 4.1 and/or ankvrin protein: (a) Use of a peptide and/or protein containing partial or entire sequence of falcipain-2 protein in combination with a peptide and/or protein containing human ankyrin protein (e. g., ankyrin residues 1206-1215, SEQ ID NO : 1) to identify and/or develop a drug or vaccine for human and/or animal malaria disease. These include experiments including, but not limited to: protein or peptide binding experiments carried out in vitro and in vivo ; three-dimensional structure-based approaches; computer modeling ; combinatorial chemistry screening; and high throughput screening approaches. These screening assays can be used to detect lead molecules in mixtures (e. g. , libraries) of synthetic or naturally-occurnng molecules.

(b) Use of a peptide and/or protein containing partial or entire sequence of falcipain-2 protein in combination with a peptide and/or protein containing human erythrocyte protein <BR> <BR> 4.1 protein (e. g. , erythrocyte protein 4.1 residues 428-443, SEQ ID NO : 5) to identify and/or develop a drug or vaccine for human and/or animal malaria disease. These include experiments including, but not limited to: protein or peptide binding experiments carried out in vitro and in vivo ; three-dimensional structure-based approaches; computer modeling; combinatorial chemistry screening; and high throughput screening approaches. These screening assays can be used to detect lead molecules in mixtures (e. g., libraries) of synthetic or naturally-occurring molecules.

Examples Example 1. Digestion of erythrocyte membrane proteins with falcipain-2.

Methods. Referring to Fig. 1A, recombinant falcipain-2 was expressed in E. coli, purified and refolded as previously described (Shenai BR et al. (2000) JBiol Chem 275: 29000-10), and analyzed by SDS-PAGE under reducing conditions (lane 1) and by gelatin-substrate PAGE (performed as previously described (Rosenthal PJ et al. (1988) J Clin Invest 82: 1560-6); lane 2) before staining with Coomassie Blue. Five microliters of spectrin/actin-depleted inside-out vesicles (IOVs) (2 mg/ml) were incubated in 5 mM sodium phosphate buffer, pH 7.5, containing no enzyme (lane 3), 0. 1 J. g of recombinant falcipain-2 (rFP-2) (lane 4), or 1 ig of P. falciparum trophozoite extract (nFP-2; prepared as previously described (Raphael P et al. (2000) Mol Bioche7n Parasitol 110 : 259-72); lane 5) in a total volume of 25 Ill for 30 min at 37°C. Vesicles were collected by centrifugation at 38,000 x g for 20 min at 4°C, analyzed by reducing SDS-PAGE, and stained with Coomassie Blue. The size of the markers (kDa) is shown on the left.

Referring to Fig. 1B, IOVs incubated with no enzyme (lane 1), 0.1 pg (lane 2), and 0.2 jj. g (lane 3) of rFP-2 at pH 7.5, as above in Fig. 1A, were transferred to a nitrocellulose membrane and probed with specific antibodies against human erythrocyte spectrin, ankyrin, band 3, and protein 4.1. The immunoreactive bands were detected by ECL.

Referring to Fig. 1 C, the effect of pH on falcipain-2 activity was studied as follows.

The activity of nFP-2 (open symbols) and rFP-2 (closed symbols) was assayed against purified human erythrocyte ankyrin in 0.1 M citrate-phosphate buffer (pH 3.0-5. 5), 0.1 M PIPES (pH 6.0-6. 5), 0.1 M HEPES (pH 7.0-7. 5), 0.1 M Tris-HCl (pH 8.0-8. 5), and 0.1 M CAPS (pH 9.5-10. 0). The samples were analyzed by SDS-PAGE. The 155 kDa band was excised, eluted in 25% pyridine and absorbance measured at 605 nm. In a parallel experiment, activity of nFP-2 and rFP-2 was assayed against purified human erythrocyte protein 4.1 over the same range of pH conditions in the same set of buffers. Identical results were obtained with purified protein 4.1 as a substrate.

Referring to Fig. 1D, the effect of protease inhibitors on falcipain-2 activity was studied as follows. Both nFP-2 (striped bars) and rFP-2 (open bars) were incubated with E- 64 (10 uM), leupeptin (10 1M), MDL (60 1M), pepstatin A (10 1M), PMSF (1 mM), or EDTA (1 mM) in 5 mM sodium phosphate buffer, pH 7.5, for 10 min at room temperature, before incubating with ankyrin or protein 4.1. Activity was measured as above for Fig. 1C.

Results. Exposure of IOVs to rFP-2 and to nFP-2 resulted in two new protein bands migrating at 155 and 56 kDa (Fig. 1A). Recombinant falcipain-2 (rFP-2) produced a cleavage pattern identical to that produced with native enzyme (nFP-2) activity present in a trophozoite extract (Fig. 1A, lanes 4 and 5), indicating that falcipain-2 encodes the cysteine protease activity reported earlier. Raphael P et al. (2000) Mol Bioclaem Parasitol 110 : 259-72.

As shown in Fig. 1B, the 155 kDa band cross-reacted with anti-ankyrin antibodies, and the 56 kDa band cross-reacted with protein 4.1 antibodies, while spectrin and band 3 appeared to remain unaffected by incubation and falcipain-2. Both rFP-2 and nFP-2 exhibited maximal activity on ankyrin substrate (Fig. 1C) and on erythrocyte protein 4.1 substrate at pH 7.0-7. 5.

The cleavage of ankyrin and protein 4.1 by native and recombinant falcipain-2 was strongly inhibited by standard inhibitors of cysteine proteases (E-64, leupeptin and MDL 28,170), but not by inhibitors of aspartic proteases (pepstatin), serine proteases (PMSF), or metalloproteases (EDTA) (Fig. 1D).

Example 2. Initial identification of the cleavage sites of ankyrin and protein 4.1.

Introduction. To identify the cleavage site of ankyrin, we used two defined monoclonal antibodies, 8C3 and 2H1 with epitopes in different regions of the molecule. The epitope for 8C3 lies between residues 1 and 1012, and that for 2H1 lies between residues 1462 and 1798 in the regulatory domain of human erythrocyte ankyrin. Raphael P et al.

(2000) Mol Biochem Parasitol 110 : 259-72. IOVs incubated with or without recombinant falcipain-2 were probed with these antibodies.

Methods. Enzyme kinetics (see Fig. 2A). IOVs were incubated with rFP-2 for different time periods (0-60 min). Ten micrograms of IOVs and 0.2 u. g of recombinant falcipain-2 were used in a total volume of 25 p. l for each time period examined. Samples were analyzed by SDS-PAGE, followed by Western blot analysis using anti-ankyrin and anti- protein 4. 1 antibodies.

Cleavage of human erythrocyte ankyrin (see Fig. 2B). Top panel Fig. 2B shows a structure of ankyrin. Lux SE et al. (1990) Nature 344: 36-42. Epitopes for monoclonal antibodies 8C3 and 2H1 are shown. Bottom panel Fig. 2B shows 10 p. g ofIOVs digested with 0. 2 tM rFP-2 for 30 min at 37°C that were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with 8C3 and 2H1.

Cleavage of human erythrocyte protein 4.1 (see Fig. 2C). Top panel Fig. 2C shows a structure of protein 4.1. Krauss SW et al. (1997) J Cell Biol 137 : 275-89. The positions of peptide epitopes for antibodies SAB and C-term are shown. Bottom panel Fig. 2C shows 10 p. g ofIOVs alone, and 10 ug ofIOVs digested with 0.2 LM rFP-2 for 30 min at 37°C and then resolved by SDS-PAGE, transferred to nitrocellulose, and probed with SAB and C-term antibodies. The 78 kDa band in IOV lanes corresponds to intact protein 4.1.

Results. As shown in Fig. 2A, within 2 min of incubation at pH 7.5, more than 50% of both ankyrin (upper panel) and protein 4.1 (lower panel) were cleaved by rFP-2, and by 10 min of incubation, the hydrolysis was essentially complete.

As shown in Fig. 2B, the 155 kDa fragment of ankyrin reacted with 8C3 antibody and not with the 2H1, indicating that the 155 kDa truncated ankyrin obtained by falcipain-2 cleavage lacks the carboxy-terminal end of the molecule. Based on the size of the truncated fragment, it is likely that the enzymatic cleavage occurs at a site (s) near or within the regulatory domain of ankyrin. This result is identical to that obtained with native cysteine protease activity present in a parasite extract. Raphael P et al. (2000) Mol Biochem Parasitol 110: 259-72.

As shown in Fig. 1B and Fig. 2A, falcipain-2 cleavage of protein 4. 1 yields a major product of-56 kDa that appears to be somewhat resistant to further cleavage. To identify the initial cleavage site of protein 4.1, we used two peptide-specific antibodies: SAB, specific for a peptide within the spectrin/actin binding domain of protein 4.1 ; and C-term, specific for the 20 amino acid peptide at the very carboxy-terminus of protein 4.1. The sequences of the peptide antigens are as follows: SAB, KKRERLDGENIYIRHSNLMLE (amino acids 407- 427; SEQ ID NO : 12); and C-term, HPDMSVTKVWHQETEIADE (amino acids 569-588; SEQ ID N0 : 13). ConboyJetal. (1986) ProcNatlAcadSci USA 83: 9512-6. Both of these antibodies (Lawrence Berkley National Laboratory, CA) have been characterized. Krauss SW et al. (1997) J Cell Biol 137 : 275-89. Immunoblot analysis of IOVs digested with recombinant falcipain-2 showed that the 56 kDa fragment of protein 4.1 was recognized by SAB but not by C-term antibody (Fig. 2C). These results show that the protease-resistant 56 kDa truncated 4.1 lacks the very carboxy-terminal end of the molecule. Based on the size of the truncated fragment, it is likely that the enzymatic cleavage occurs at a site (s) near or within the 22-24 kDa carboxy-terminal domain (CTD) of protein 4.1. These results were further confirmed by the fact that falcipain-2 did not cleave a GST-fusion protein including the amino-terminal 30 kDa membrane-binding domain of human erythrocyte protein 4.1.

The function of CTD in erythrocytes is largely unknown. Falcipain-2-mediated cleavage of protein 4.1 near or within its CTD thus provides a unique model to study the function of this domain.

We have previously shown that digestion of ankyrin with native enzyme activity present in parasite extract substantially reduced its interaction with ankyrin-depleted membrane vesicles. Furthermore, ektacytometric measurements showed a dramatic increase in the rate of fragmentation of ghosts after treatment with native protease. Raphael P et al.

(2000) Mol Biochem Parasitol 110 : 259-72. Here, we have performed these experiments with recombinant falcipain-2 and have obtained identical results.

Taken together, these findings indicate that the P. falciparum cysteine protease falcipain-2 cleaves host membrane ankyrin and protein 4.1 near their carboxy-termini and that this cleavage is accompanied by membrane instability, as evidenced by an increased rate of membrane fragmentation. These findings suggest that falcipain-2 mediates the cleavage of ankyrin and protein 4.1, the cytoskeletal elements vital to the stability of the red cell membrane, thus modulating parasite release.

Example 3. Precise identification of the cleavage site of protein 4.1.

Iiitroduction. We have shown that the truncated protein 4.1 obtained after cleavage with falcipain-2 contains the 21 amino acid peptide within the 10 kDa spectrin/actin binding domain but it lacks the very C-terminus end of the molecule. These results suggested that FP-2 cleavage of erythrocyte protein 4.1 occurs in the region between amino acids 428-568 encoding parts of the 56 kDa domain and the 22-24 kDa C-terminal domain (CTD) of protein 4.1.

Methods. To further identify the precise cleavage site of protein 4. 1, we designed a cDNA construct pQ-4.1-2, containing nucleotides 1678-2565 encoding amino acids 294-588.

The insert was PCR amplified from a human reticulocyte cDNA library and ligated into the pQE-30 vector (which encodes an amino terminal 6-His tag; Qiagen) to produce expression construct pQ-4.1-2. After confirming the sequence, the construct was used to transform M15 (pREP4)-strain E. coli. Transformants were grown in the presence of isopropyl (3-D- thiogalactopyranoside (IPTG) and analyzed for expression of fusion protein by SDS-PAGE using standard methods. While the calculated molecular mass of expressed protein is 33.4 kDa, it migrated with an apparent molecular weight of approximately 55 kDa on SDS-PAGE (Fig. 3). Recombinant His-tagged protein was bound to Ni-NTA resin (Qiagen) by standard methods.

Recombinant protein 4.1 fragment bound to Ni-NTA beads was then incubated with 0.2 LM FP-2 at pH 7.5 for 30 minutes at 37°C. After incubation, the sample was centrifuged, and both the supernatant and the pelleted beads were analyzed by SDS-PAGE. Although no protein was detected in the supernatant, a major protein band with an apparent molecular weight of ca. 26 kDa was detected in the pellet (indicated by * in Fig. 3). In addition, a faint band corresponding to undigested recombinant protein was detected (Fig. 3). hnmunoblot analysis confirmed that the ca. 26 kDa band was a fragment of protein 4.1. This fragment remained bound to Ni-NTA beads, and it contained the N-terminal His tag, indicating that the cleavage took place from the C-terminus. Bound proteins were then eluted with 8M urea, 20 mM Tris-HCl, 1M imidazole, pH 8.0, and subjected to MALDI-MS measurements at Harvard Microchemistry Laboratory for the determination of precise molecular mass of the cleaved product.

Results. A major species with a molecular mass of 16,686 Daltons was detected. A small peak of 33,500 Daltons corresponding to undigested recombinant protein was also detected. Based on the amino acid sequence of recombinant His-tagged protein, cleavage after Lysine 437 (arrow in Fig. 4) is most consistent with the MALDI-MS data since this cleavage would yield an N-terminal fragment of 16,686. 50. This finding is further supported by a previous report, which showed that both native and recombinant falcipain-2 cleave synthetic peptide substrates containing arginine or lysine at the PI position, with a marked preference for a hydrophobic residue (isoleucine in our case) at the P2 position. Shenai BR et al. (2000) JBiol Chem 275: 29000-10.

Example 4. Identification of falcipain-2 inhibitor peptide 4.1 Pl.

Methods. To confirm specificity of cleavage, we designed three peptides, Pi (corresponding to 4.1 Pi, SEQ ID NO : 5), Pii, and Piii (Fig. 4). Synthetic peptides were prepared commercially. Screening of these peptides was done using the spectrin/actin depleted human erythrocyte inside-out membrane vesicles (IOVs) enzyme assay; 0.2 LM recombinant FP-2 was added to 25 pi reactions containing 10 pg ofIOVs in 5 mM sodium phosphate, pH 7.5, 1 mM DTT, and various amounts of peptides (dissolved in DMSO). The reactions were incubated at 37°C for 30 min, after which time the vesicles were collected by centrifugation at 38,000 x g for 20 min at 4°C and analyzed by SDS-PAGE. Intensities of the 155 kDa (truncated ankyrin) and 56 kDa (truncated protein 4.1) protein bands were monitored (Fig. 5). As shown in Figure 5, increasing amounts of the peptide Pi inhibited FP- 2-mediated cleavages of both ankyrin and protein 4.1, whereas peptides Pii and Piii had no significant effect as compared to the control sample. Studies are in progress using pure ankyrin and protein 4.1 as substrates.

Since FP-2 also cleaves hemoglobin (Shenai BR et al. (2000) JBiol Clzem 275: 29000- 10), the effect of peptide Pi on hemoglobin degradation was also tested. Recombinant FP-2 (100 nM) was added to 25 pi reactions containing 3 u, g of human hemoglobin (Sigma) in 100 mM sodium acetate, pH 5.5, 1 mM DTT, and different amounts of the peptide Pi. The reactions were incubated at 37°C for 60 min, and stopped by the addition of SDS-PAGE sample buffer. As shown in Figure 6, FP-2-mediated degradation of human hemoglobin was markedly inhibited with 500 M peptide Pi.

Results. We identified the cleavage site of protein 4.1, and identified a 16 amino acid peptide that inhibits all known functions of falcipain-2, namely, cleavage of human erythrocyte ankyrin, protein 4.1, and hemoglobin. The 16 amino acid peptide is located immediately following the 21 amino acid peptide of the spectrin/actin binding domain of human erythrocyte protein 4.1 (Figure 4). Previous studies have reported that the 21 amino acid alternative cassette plus the following 43 amino acids within the 10 kDa spectrin/actin binding domain is required for protein 4.1-spectrin binary interactions, which are important for erythrocyte membrane stability. Schischmanoff PO et al. (1995) JBiol Chem 270,: 21243- 50. Thus, identification of the cleavage site, as noted above, is consistent with our observation that incubation of human erythrocyte ghosts with FP-2 destabilizes the membrane. Raphael P et al. (2000) Mol Biochem Parasitol 110 : 259-72; Dua M et al. (2001) Mol Biochem Parasitol 116 : 95-9.

Example 5. Precise identification of the cleavage site of ankyrin.

Methods. To further identify the precise cleavage site of ankyrin, we designed a cDNA construct pQ-Ank-2 containing nucleotides 2998-4557 encoding amino acids 972- 1491. The insert was PCR amplified from a human reticulocyte cDNA library and ligated into the pQE-30 vector (which encodes an amino terminal 6-His tag; Qiagen) to produce expression construct pQ-Ank-2. After confirming the sequence, the construct was used to transform M15 (pREP4) -strain E. coli. Transformants were grown in the presence of IPTG and analyzed for expression of fusion protein by SDS-PAGE using standard methods. While the calculated molecular mass of expressed protein is 59.5 kDa, it migrated with an apparent molecular weight of approximately 60 kDa on SDS-PAGE. Recombinant His-tagged protein was bound to Ni-NTA resin (Qiagen) by standard methods.

Recombinant ankyrin fragment bound to Ni-NTA beads was then incubated with 0.2 p, M FP-2 at pH 7.5 for 30 minutes at 37°C. After incubation, the sample was centrifuged, and both the supernatant and the pelleted beads were analyzed by SDS-PAGE. Although no protein was detected in the supernatant, a major protein band with an apparent molecular weight of ca. 26 kDa was detected in the pellet. In addition, a faint band corresponding to undigested recombinant protein was detected. Immunoblot analysis confirmed that the ca. 26 kDa band was a fragment of ankyrin. This fragment remained bound to Ni-NTA beads, and it contained the N-terminal His tag, indicating that the cleavage took place from the C-terminus. Bound proteins were then eluted with 8M urea, 20 mM Tris-HCl, 1M imidazole, pH 8.0, and subjected to MALDI-MS measurements at Harvard Microchemistry Laboratory for the determination of precise molecular mass of the cleaved product.

Results. A major species with a molecular mass of 27,600 Daltons was detected.

Based on the amino acid sequence of recombinant His-tagged protein, cleavage after Arginine 1210 is most consistent with the MALDI-MS data since this cleavage would yield an N-terminal fragment of 27,600 Daltons.

Example 6. Identification of falcipain-2 inhibitor peptide Ank Pl.

Methods. To confirm specificity of cleavage, we designed a series of 10-mer to 20- mer peptides, including the 10-mer peptide Ank P, (SEQ ID NO : 1) corresponding to amino acid residues 1206-1215 of ankyrin (SEQ ID NO : 2). Synthetic peptides were prepared commercially. Screening of these peptides was done using the spectrin/actin depleted human erythrocyte inside-out membrane vesicles (IOVs) enzyme assay akin to the methods of Example 4.

The effect of peptide Ank Pi on hemoglobin degradation was also tested.

Recombinant FP-2 (100 nM) was added to 25 ; j. l reactions containing 3 llg of human hemoglobin (Sigma) in 100 mM sodium acetate, pH 5.5, 1 mM DTT, and different amounts of the peptide Ank Pi. The reactions were incubated at 37°C for 60 min, and stopped by the addition of SDS-PAGE sample buffer.

Results. Increasing amounts of the peptide Ank Pi inhibited FP-2-mediated cleavages of ankyrin, protein 4.1, and hemoglobin, whereas other ankyrin peptides had relatively little or no significant effect as compared to the control sample. Studies are in progress using pure ankyrin and protein 4.1 as substrates. We identified the cleavage site of ankyrin and identified a 10 amino acid peptide (SEQ ID NO : 1) that inhibits all known functions of falcipain-2, namely, cleavage of human erythrocyte ankyrin, protein 4.1, and hemoglobin.

The 10 amino acid peptide spans the specific site of cleavage, Arginine 1210.

Interestingly, the peptides Ank Pi and 4.1 Pi have no apparent sequence homology.

This surprising observation may be explained at least partially by the occurrence in both peptides of an apparent consensus falcipain-2 substrate cleavage site sequence of hydrophobic residue followed by arginine (R) or lysine (K).

Example 7. Effect of ankyrin peptide Ank P1 in vitro.

To determine the effect of Ank Pi peptide on the growth and development of P. falciparum, the peptide was delivered into intact parasite-infected erythrocytes by fusing it to the Antennapedia homeoprotein internalization domain. The internalization sequence of the Antennapedia protein was fused to the N-terminal end of ankyrin peptide Ank Pi.

Antennapedia-fused peptides (as well as unfused peptides) were synthesized by Tufts Core Facility, Physiology Dept. , Boston. Sequences of Ank Pi peptide, Antennapedia peptide (Ant), and fused peptides are as follows: Ant: RQIKIWFQNRRMKWKK (SEQ ID NO : 15); Ank PI : NVSARFWLSD (SEQ ID NO : 1); Ant-Ank Pi : RQIKIWFQNRRMKWKKNVSARFWLSD (SEQ ID NO : 16); and Scrambled Ant-Ank Pi : RQIKIWFQNRRMKWKKNSRSDVFWLA (SEQ ID NO : 17). All ankyrin peptides used in the study were biotin labeled at the amino terminus and amide-linked at the carboxyl terminus, and were HPLC purified. This whole procedure was performed at the Tufts Core Facility and purified peptides were delivered to us.

Human erythrocytes infected with stage-specific parasites were incubated with 100- 250 suM of the fused peptide Ant-Ank PI, Ank P1 peptide alone, Antennapedia peptide alone, or scrambled Ant-Ank Pi. Giemsa staining of thin smears after 6 and 24 hours of incubation showed that while the ring-stage parasites matured normally, the development of trophozoites and schizonts was markedly inhibited by the fused peptide Ant-Ank Pi. Less than 10% of new ring-stage parasites were detected compared to control cultures containing the same final concentrations of eitherAntennapedia peptide, Ank Pi peptide, or DMSO (Table 1).

Table 1: Effect of ankyrin peptide on the growth and development of P. falcipa7 u7n trophozoites in culture. Treatment Concentration (pM) % Ring Parasites DMSO-100 Ank Ank P, 250 100 Ant 250 95 50 73 Ant-Ank P1 100 40 250 <10 Scrambled Ant-Ank Pi250100 These results demonstrate the potential efficacy of the Ank Pi peptide in terms of its ability to inhibit growth and development of P. falciparutiz trophozoites in vivo, and they demonstrate the importance of delivery of the peptide into the erythrocyte.

Example 8. Antimalarial effects of Ant-Ank P, in vivo.

To assess the feasibility of using Ant-Ank Pi as an antimalarial in vivo, the effect of this peptide is studied in malaria-infected mice. Two species of rodent malaria are used: P. yoelii 17XL and P. yoelii NXL, which cause lethal and non-lethal infections, respectively.

Mice are infected intraperitoneally with 1 x 106 uncloned, infected erythrocytes of either species. The parasites are held in liquid nitrogen and passaged at least once in Swiss Webster (SW) mice before transfer to experimental mice. Parasitemia is monitored by taking blood films from the tail vein. Films are stained with Giemsa and at least 300 erythrocytes are counted. Toxicity of Ant-Ank Pi is first determined in infected and infected mice.

Increasing amounts of the inhibitor (dissolved in DMSO and diluted with sterile saline) are injected intravenously in uninfected and infected SW mice via tail vein 2-4 times per day for 3-4 days, and the mice are observed daily for evidence of toxicity. Control mice are injected with the same final concentration of emulphor.

Experimental mice with high parasitemia (generally 10-40%) are treated intravenously with Ant-Ank Pi (within the non-toxic dose level determined above) in DMSO as described above. Giemsa-stained peripheral blood films are made at periodic intervals of 4-6 hours. A possibility is tested that the inhibition is high within a short time after injection and then declines due to instability of the inhibitor in vivo.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references, including patent documents, disclosed herein are incorporated by reference in their entirety.

We claim: