Patent Application
20080274113
A protein, referred to herein as PfMTI-1, and orthologs thereof have now been identified as providing a key molecular link for malaria parasites between the red blood cell, which is to be invaded, and the actin myosin based motor machinery of the parasite that drives the invasion process. Inhibition of the function of this protein or a homolog or ortholog thereof or a nucleic acid encoding this protein or a homolog or ortholog thereof prevents the parasite from invading red blood cells and is expected to prevent or lessen the clinical severity of malaria infection. Accordingly, the present invention provides methods for development of compositions for use in immunologic and chemotherapeutic therapies for treatment and prevention of malaria. Compositions and methods for use of these compositions in the treatment and prevention of malaria are also provided.
Presently, malaria is a significant burden on humans. The parasite responsible for this disease continues to develop resistance to antimalarial drugs and there is no suitable vaccine to control the disease. These two major features ensure that malaria remains a major global health problem. Plasmodium falciparum causes the most severe form of the disease in humans and is responsible for 200-300 million infections per year. Greater than two million people die as a result of the disease annually. The development of an effective vaccine and the development of new and inexpensive antimalarial drugs remain top priorities in the world health community.
A key process to target for both vaccine and drug development research is the process of parasite invasion. Of the various forms of the parasite, three stages must have the ability to invade host cells: the ookinete, the sporozoite and the merozoite. There are both common features as well as unique features during the invasion process by each form. For example, although the invasive stages of the parasite are morphologically and biochemically different from one another, they share a highly conserved structural organization and special organelles called micronemes and rhopteries (for review see Sinnis and Sini, Trends in Microb. 1997 5:52-58; Pinder et al, J. Cell Sci. 2000 111:1831-1839; Chitnis, C. E. Curr. Op. in Hemat. 2001 8:85-91; and Mota and Rodriquez, Bioessays 2002 24:149-156). While function of these conserved structures has not been fully clarified, it is known that they are required for host cell invasion. All of these common structures confer some similarity to target cell invasion; the specific details in each case, however, differ. Each invasive stage of the parasite has a different target cell specificity, which is believed to be governed by a specific receptor-ligand type interaction. Furthermore, the invasion of the host cell appears to be an active process, which requires an actin-myosin based motility system to enter the host cell (Pinder et al, J. Cell Sci. 2000 111:1831-1839 and Morrissette and Sibley Microb. Mol. Bio. Rev. 2002 66:21-38).
In the case of hepatocyte invasion by sporozoites, injection of a very small number of Plasmodium sporozoites is sufficient to initiate infection, suggesting that the invasion process is extremely efficient. Furthermore, the process of hepatocyte invasion is extremely rapid, occurring within minutes after the infectious bite of the mosquito. The efficiency and rapidity of sporozoite invasion suggests that it involves specific interactions between parasite-encoded surface proteins and host molecules. Several lines of evidence suggest that the circumsporozoite protein (CS) plays a key role in this process (Stewart et al. Infect. Immunol. 1986 51:859-864; Stewart and Vanderberg J. Protozol. 1988 35:389-393; Menard, R. Cell Microb. 2001 3:63-73; Mota and Rodriquez Bioessays 2002 24:149-156). The CS protein of all Plasmodium species contains a highly conserved region, called region H, that is also found in the type I repeats of thrombospondin and some other adhesion molecules. It is believed that region H of CS binds to glycosaminoglycan chains of heparin sulfate proteoglycans (HSPGS) that are found on the surface of hepatocytes (Sinnis et al. J. Exp. Med. 1994 180:297-306). This binding is required for sporozoite attachment to hepatocytes, but is not necessary for invasion. Subsequent invasion with formation of a parasitophoros vacuole is tightly associated with exocytosis of the apical organelles. The attachment of the parasite to the host cell also triggers a transient cytosolic Ca2+ increase that is required for invasion. This increase in the Ca2+ concentration seems to induce the exocytosis of micronemes, a process required for the formation of the moving junction and the internalization of the parasite. Whether this increase in Ca2+ concentration is also required for activation of the actin-myosin based motility system, as seen with other non-Apicomplexan motility systems, is not known. The exocytosis of the apical organelles results in the release of the TRAP (Thrombospondin-related anonymous protein) molecule and its distribution along the surface of the sporozoite. TRAP is a transmembrane protein that contains two well-characterized adhesive modules, an A domain of Von Willebrand factor and a type 1 repeat of thrombospondin, in its extracellular domain. TRAP (−) parasites, created by gene-targeting technology in Plasmodium, are unable to invade mosquito salivary glands or infect liver cells in vivo after intravenous injection, suggesting that TRAP plays a key role in the invasion process (Sultan et al. Mol. Biochem. Parasitol. 1997 113:151-156). A structurally related molecule, Plasmodium CTRP, produced by the ookinete stage, probably plays a similar role as inactivation of CTRP in Plasmodium berghei and Plasmodium falciparum showed that CTRP is necessary for ookinete transformation into an oocyst, a stage of the life cycle that requires ookinete migration through the midget epithelium of the mosquito (Dessens et al. EMBO J. 1999 18:6221-6227; Yuda et al. J. Exp. Med. 1999 190:1711-1715; Templeton et al. Mol. Microb. 2000 36:1-9).
The merozoite form of the asexual life cycle in the blood stage attaches to the surface of the red blood cell (RBC) which initiates the invasion process of this host cell. Many of the surface proteins of the merozoite are thus exposed to the immune system and consequently are potential vaccine candidates. Many of these proteins are thought to play a role in merozoite invasion of RBCs but the details of their function and interactions remain unclear at best. Merozoite invasion takes place following initial interaction with the RBC surface followed by re-orientation to allow the apical end of the parasite to interact with the membrane of the host cell. This re-orientation allows the contents of the apical organelles to be released and a tight junction is formed between the merozoite surface and the RBC membrane. The tight junction moves along the surface of the merozoite, possibly via force generated by the actin-myosin motor, until the membrane fuses at the posterior end of the parasite, resulting in the formation of the parasitophorous vacuole contain the newly invaded parasite (Pinder et al. Parasit. Today 2000 16:240-245). While multiple proteins appear to be involved in this complex process of RBC invasion, very little is known about the particular role of any individual protein in the process. One of the surface proteins, merozoite surface protein I (MSPI) has been postulated to be involved in the initial interaction of the merozoite with the RBC surface (Holder and Freeman, J. Exp. Med. 1984 160:624-629; Holder et al. Nature 1985 317:270-273; Perkins and Rocco J. Immunol. 1988 141:3190-3196). The localization of this GPI-anchored protein is shared with a number of other GPI-linked proteins including MSP2, MSP4 and MSP5 (Smythe et al. Proc. Natl Acad. Sci. USA 1988 85:5195-5199; Marshall et al. Infect. Immun. 1997 65:4460-4467; Marshall et al. Mol. Biochem. Parasitol. 1998 94:13-25). Apical membrane antigen I (AMA I) is an integral membrane protein that is initially localized to the neck of the rhoptries although it re-distributes onto the surface of the merozoite (Deans et al. Mol. Biochem. Parasitol. 1984 11:189-204; Peterson et al. Mol. Cell Biol. 1989 9:3151-3154; Narum and Thomas Mol. Biochem. 1994 67:59-68; Marshall et al. Mol. Biochem. Parasitol. 1989 27:281-284; Peterson et al. Mol. Cell Biol. 1990 9:3151-3154). The function of AMAI is presently unknown. Within the rhoptries, a number of proteins have been identified that may be involved in the invasion process. These include the rhoptry-associated proteins 1 and 2 (RAP I and RAP2) (Ridley et al. Mol. Biochem. Parasitol. 1990 41:125-134; and Perrin et al. J. Clin. Invest. 1985 75:1718-1721). The soluble complex of these two molecules is delivered out to the rhoptries during invasion and is carried through into the parasitophorous vacuole (Baldi et al. EMBO J. 2000 19:1-9). The erythrocyte-binding antigen 175 (EBA 175) of Plasmodium falciparum is located in the micronemes and has been shown to bind to the RBC surface molecule glycophorin A in a sialic acid-dependent manner (Sim et al. Exp. Parasit. 1992 78:259-268; Wu et al. Proc. Natl Acad. Sci. USA 1996 93:1130-1134; Crabb et al. Cell 1997 89:287-296). Disruption of the gene encoding EBA175 to investigate the role of the conserved carboxy-terminal cysteine-rich domain, the transmembrane domain and the cytoplasmic domain suggested these regions were not essential for merozoite invasion (Reed et al. Proc. Natl Acad. Sci USA 2000 97:7509-7514). However, analysis of RBC invasion with these mutants suggested that the EBA175/glycophorinA pathway was disrupted. It appears that the mutant parasites now invaded using a sialic-acid independent pathway suggesting that the parasite has the ability to utilize alternative pathways for invasion of RBCs.
The involvement of an actin-myosin-based motor in invasion was suggested by Miller et al. based upon studies of the effect of cytochalasin B on Plasmodium invasion (Miller et al. J. Exp. Med. 1979 149:172-184). In Plasmodium, there are two genes for actin. Actin I is intronless and is expressed throughout the parasite life cycle, wherein actin II has an intron and is transcribed only in the sexual stages (Wesseling et al. Mol. Biochem. Parasitol. 1988 27:313-320; Wesseling et al. Mol. Biochem. Parasit. 1988 27:313-320, Wesseling et al. Mol. Biochem. Parasitol. 1989 35:167-176). The amino acid sequence of actin II diverges from previously characterized actins exhibiting only 79% sequence similarity to the sequence of actin I (Wesseling et al. Mol. Biochem. Parasit. 1988 27:313-320). The majority of actin in Apicomplexan parasites appears to be monomeric rather than being in the polymerized form. Localization studies using anti-actin antibodies have indicated that actin is present in the region between the plasma membrane and the inner membrane complex (IMC), which is composed of two closely aligned membranes. The involvement of myosin in Apicomplexan invasion was suggested by Dobrowski et al. and Pinder et al. based upon studies where invasion was reversibly inhibited by the myosin ATPase inhibitor butane-2,3-monoxime (Dobrowski et al. Mol. Microbiol. 1997 26:163-173; Pinder et al. J. Cell Sci. 1998 111:1831-1839). As in other species, Apicomplexa contains several myosin genes. Of these, MyoA has been disclosed as rather unique (Heintzehnan and Schwartzman J. Parasitol. 1997 87:429-432). This myosin is expressed in all Plasmodium invasive stages. The molecule is very small (approximately 90 kD) but the head domain displays the universally conserved ATP and actin binding sites. MyoA binds actin and actin is released in an ATP-dependent fashion. However, MyoA has several unique features, including virtually no recognizable neck domain (which is normally the binding site for a canonical myosin light chain) and a very short carboxy terminal tail. In the mature merozoite, MyoA is peripherally located with most staining occurring at the apical end and electron microscopy indicates that it is also located between the plasma membrane and the RAC (Pinder et al. J. Cell Sci. 1998 111:1831-1839).
Using a yeast two-hybrid system (Fields and Song Nature 1989 340:245-248; Luban and Goff Curr. Opin. Biotech. 1995 6:59-65), a molecule termed MTIP (MyoA Tail Interacting Protein) was isolated. This molecule binds to the short carboxy terminal tail of MyoA. Studies on the localization of this molecule have led to the proposal of a new model for the organization of the actin-myosin based machinery located between the plasma membrane and the IMC (Bergman et al. J. Cell Sci. 2003 116:39-49). It is believed that the “moving junction” which forms during invasion is a circumferential zone of attachment at the opening of the host cell invagination. This zone is characterized by a markedly thickened host cell membrane with increased electron density and is frequently accompanied by a constriction in the parasite body. The parasite enters the newly forming parasitophorous vacuole by capping the moving junction down its body. Eventually, the parasite becomes enclosed within a cavity delimited by the invaginated host cell membrane (Aikawa et al. J. Cell Bio. 1978 77:72-82; Michel et al. Int. J. Parasitol. 1980 10:309-313). The moving junction is a highly specialized interface of the parasite with the host cell, presumably utilizing cytoskeletal proteins, signaling molecules and receptors. However, very little is known about this interface. The protein MCP-I (Merozoite Capping Protein-1) was localized initially at the attachment site formed between the merozoite apical region and the erythrocyte (Klotz et al. Mol. Biochem. Parasitol. 1989 36:177-185). During the invasion process, MCP-1 migrates around merozoites in an anterior-to-posterior movement to finally persist at the posterior end of the newly invaded parasite (at the end nearest the erythrocyte membrane). MCP-1 is a 415 amino acid protein but interestingly is detected as a 60 kD protein in extracts from blood stage parasites. The amino terminal third of the molecule is an oxido-reductase domain but the function of this domain is not known. Thus, it appears that MCP-1 is localized to the moving junction in invading parasites but its role and potential interactions with other molecules remains unclear,
A new protein, Plasmodium falciparum Merozoite TRAP-like Invasin 1, referred to herein as PfMTI-1, has now been isolated and characterized. Blockage or inhibition of this protein is believed to prevent the malaria parasite from invading red blood cells, thus preventing or lessening the clinical severity of the disease. This protein and homologs or orthologs thereof serve as prime targets for immunologic and chemotherapeutic intervention.
An aspect of the present invention relates to an isolated protein referred to herein as PfMTI-1, of the malaria parasite Plasmodium falciparum and homologs or orthologs thereof that interact with a red blood cell to be invaded by the parasite and link with a component of the actin-myosin based machinery of the malaria parasite.
Another aspect of the present invention relates to isolated nucleic acid sequences encoding the protein PfMTI-1 of the malaria parasite Plasmodium falciparum and homologs or orthologs thereof that interact with a red blood cell to be invaded by the parasite and link with a component of the actin-myosin based machinery of the malaria parasite.
Another aspect of the present invention relates to isolated antibodies which specifically bind the protein PfMTI-1 or a homolog or ortholog thereof, or a fragment thereof.
Another aspect of the present invention relates to a method for identifying potential therapeutic agents for malaria which comprises assessing the ability of a potential therapeutic agent to inhibit the function of the PfMTI-1 protein or a homolog or ortholog thereof thereby inhibiting the interaction of this protein with a red blood cell to be invaded by the malaria parasite and/or its linkage to a component of the actin-myosin based machinery of the malaria parasite.
Another aspect of the present invention relates to compositions and methods for inhibiting or preventing invasion of red blood cells by a malaria parasite. Compositions of the present invention comprise an agent which inhibits interaction of PfMTI-1 or an ortholog thereof with a host red blood cell and/or linkage to a component of actin-myosin based machinery of the malaria parasite. Methods of the present invention comprise administering this composition as a chemotherapeutic agent to a subject infected with a malaria parasite or as an immunologic agent to prevent infection in subjects at risk for infection by a malaria parasite.
SEQ ID NO: 1 and SEQ ID NO:2 are the amino acid sequence and the nucleic acid sequence, respectively, of PfMTI-1 isolated from Plasmodium falciparum strain 3D7.
SEQ ID NO:3 and SEQ ID NO:4 are the amino acid sequence and the nucleic acid sequence, respectively, of PfMTI-1 isolated from Plasmodium falciparum strain FVO.
SEQ ID NO:5 and SEQ ID NO:6 are the amino acid sequence and the nucleic acid sequence, respectively, of PfMTI-1 from Plasmodium falciparum strain D10.
SEQ ID NO:7 and SEQ ID NO:8 are the amino acid sequence and the nucleic acid sequence, respectively, of PfMTI-1 isolated from Plasmodium falciparum strain HB3.
SEQ ID NO:9 and SEQ ID NO:10 are the amino acid sequence and the nucleic acid sequence, respectively, of PfMTI-1 isolated from Plasmodium falciparum strain M24.
SEQ ID NO: 11 and SEQ ID NO: 12 are the amino acid sequence and the nucleic acid sequence, respectively, of PfMTI-1 isolated from Plasmodium falciparum strain MCAMP.
SEQ ID NO: 13 and SEQ ID NO: 14 are the amino acid sequence and the nucleic acid sequence, respectively, of PfMTI-1 isolated from Plasmodium falciparum strain C2A.
SEQ ID NO: 15 is an orthologous amino acid sequence to PfMTI-1 isolated from Plasmodium vivax.
SEQ ID NO: 16 an orthologous amino acid sequence to PfMTI-1 isolated from Plasmodium knowlesi.
All invasive forms of the human malaria parasite (Plasmodium falciparum) must have a protein that serves as a molecular bridge or link between the host cell that is being invaded and the motor machinery of the parasites that drives the invasion process. The bridging or linking protein of the merozoite, the invasive form of the asexual or blood stage of the parasite life cycle that interacts with the red blood cell to be invaded and links with a component of the actin-myosin based motor machinery of the parasite has now been identified. Exemplary amino acid sequences of this protein, Plasmodium falciparum Merozoite TRAP-like Invasion 1, referred to herein as PfMTI-1, are depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11 and 13. SEQ ID NO: 1 is the amino acid sequence of the protein isolated from P. falciparum strain 3D7. SEQ ID NO:3 is the amino acid sequence of the protein isolated from P. falciparum strain FVO. SEQ ID NO:5 is the amino acid sequence of the protein isolated from P. falciparum strain D10. SEQ ID NO:7 is the amino acid sequence of the protein isolated from P. falciparum strain HB3. SEQ ID NO:9 is the amino acid sequence of the protein isolated from P. falciparum strain M24. SEQ ID NO: 11 is the amino acid sequence of the protein isolated from P. falciparum strain MCAMP. SEQ ID NO: 13 is the amino acid sequence of the protein isolated from P. falciparum strain C2A. Nucleic acid sequences encoding the PfMTI-1 protein of strains 3D7, FVO, D10, HB3, M24, MCAMP and C2A are depicted in SEQ ID NO:2, 4, 6, 8, 10, 12 and 14, respectively.
This protein was identified using a bioinformatic approach to search for blood stage receptors in a P. falciparum predicted gene product database. Search criteria used allowed for the presence of an additional trans-membrane domain (signal sequence and two trans-membrane domains) as the computer can predict a trans-membrane domain from merely a stretch of hydrophobic domains. Only 6 proteins of P. falciparum met all criteria, one of which is PfMTI-1.
PfMTI-1 of the various strains comprises 488 to 503 amino acids and contains a signal sequence, a single transmembrane domain, a thrombospondin domain and an acidic cytoplasmic domain with a tryptophan residue near the carboxy-terminus (residue 495 of 3D7). Using microarray data, PfMTI-1 was identified as being expressed in late stage parasites. Further, examination of other genes with a similar pattern of expression revealed that PfMTI-1 is expressed at the same approximate time during the parasite cell cycle as other molecules involved in the invasion process, such as PfMYOA (Plasmodium falciparum Myosin A, Pinder et al, J. Cell Sci. 2000 111:1831-1839), PfMTIP (Plasmodium falciparum Myosin A Tail Interacting Protein, Bergman et al. J. Cell Sci. 2003 116:39-49) and PfAMA1 (Plasmodium falciparum Apical Membrane Antigen 1, Hodder et al, Infect. Immun. 2001 69:3286-3294). It is believed that this protein serves as the molecular bridge or link between a host erythrocyte being invaded by the malaria parasite and the motor machinery of the malaria parasite that drives the infection process.
The carboxy-terminal 44 amino acids of PfMTI-1 are expressed as a histidine-tagged recombinant protein and are incubated in an in vitro binding assay with recombinant Pf Aldolase (expressed as a glutathione-S-transferase fusion protein). The binding of the GST-aldolase fusion to the PfMTI-1 protein is specifically detected by incubation with an antibody directed against the GST moiety of the fusion protein and subsequent incubation with a conjugation secondary antibody. Furthermore, the specificity of the binding to Pf aldolase has been demonstrated using a similar histidine-tagged PfMTI-1 recombinant protein containing a tryptophan→alanine change at the fourth position from the carboxy-terminus of PfMTI-1. This result implicates PfMTI-1 as being a link between the plasma membrane of the parasite and the actin-myosin motor required for the invasion of red blood cells. This link between PfMTI-1 and Pf actin is indirect via the bridging molecule, Pf aldolase. Thus, inhibition of the function of the extracellular domain of PfMTI-1 or the interaction between PfMTI-1 and Pf aldolase may block the ability of the parasite to invade red blood cells.
Thus, the present invention provides an isolated malaria protein, PfMTI-1, and isolated homologs or orthologs thereof, now characterized as functioning as the molecular bridge or link between a host red blood cell that is being invaded and the motor machinery of the parasite that drives the invasion process. The amino acid sequence of exemplary PfMTI-1 proteins of the present invention which function as the molecular bridge or link between a host red blood cell that is being invaded and the motor machinery of the parasite that drives the invasion process are depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11 and 13.
Based upon these exemplary amino acid sequences and the activity now taught herein for this protein, those skilled in the art can now routinely identify variant amino acid sequences, homologous or orthologous amino acid sequences and fragments of this exemplary amino acid sequence as well as fragments of variant and homologous or orthologous amino acid sequences exhibiting similar binding capabilities and/or activities with respect to host red blood cells and/or a component of the actin-myosin based motor machinery of the malaria parasite. Using such techniques, the inventors have identified orthologous proteins in P. vivax (SEQ ID NO:15) and P. knowlesi (SEQ ID NO:16).
The present invention also provides isolated nucleic acid sequences encoding PfMTI-1 and isolated homologs or orthologs, or fragments thereof. Exemplary nucleic acid sequences encoding PfMTI-1 are depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12 and 14. As will be understood by those skilled in the art, however, due to degeneracy in the genetic code, additional nucleic acid sequences may encode this protein. Identification of these degenerative sequences can be performed routinely by those skilled in the art based upon the teachings herein. Accordingly, these additional degenerate nucleic acid sequences as well as isolated nucleic acid sequences encoding homologs or orthologs of PfMTI-1 are also encompassed within the scope of this invention.
By the term “isolated”, as used herein, it is a meant a protein or fragment thereof, or a nucleic acid sequence that is (1) no longer associated with naturally associated components that accompany it in its native state, (2) is free of other proteins or nucleic acid sequences from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a protein or fragment or nucleic acid sequence that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein or fragment thereof or a nucleic acid sequence may also be rendered substantially free of naturally associated components by isolation, using protein or nucleic acid sequence purification techniques well-known in the art.
By “homolog” or “homologous” when referring to a protein of the present invention it is meant proteins from different organisms with a similar sequence to the encoded amino acid sequence of PfMTI-1 and a similar biological activity or function. Although two proteins are said to be “homologous,” this does not imply that there is necessarily an evolutionary relationship between the proteins. Instead, the term “homologous” is defined to mean that the two proteins have similar amino acid sequences and similar biological activities or functions. In a preferred embodiment, a homologous protein is one that exhibits 50% sequence similarity to PfMTI-1, preferred is 60% sequence similarity, more preferred is 70% sequence similarity. Even more preferred are homologous proteins that exhibit 80%, 85% or 90% sequence similarity to PfMTI-1. In a yet more preferred embodiment, a homologous protein exhibits 95%, 97%, 98% or 99% sequence similarity.
Most preferred is an ortholog or orthologous protein, meaning that the homologous proteins have a common ancestral species.
The term “fragment” as used herein with respect to proteins of the present invention refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion compared to the full-length PfMTI-1 protein or a homolog or ortholog thereof. In a preferred embodiment, the fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring PfMTI-1 protein, or homolog or ortholog thereof. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long. Most preferably, the fragment is of sufficient length to mimic the binding capability and/or activities of the full length protein.
The present invention also relates to derivatives of the PfVN-291 protein and homologues, orthologues and fragments thereof
A “derivative” when used herein with respect to proteins of the present invention refers to a modified protein substantially similar in primary structural sequence to PfMTI-1 or a homolog or ortholog thereof but which includes, e.g., in vivo or in vitro chemical and biochemical modifications that are not found in the naturally occurring proteins. Examples of such modifications include, but are in no way limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Other modifications include, for example, labeling with a detectable label such as a radionuclide, a fluorophore or an enzyme.
The present invention also provides isolated antibodies which specifically bind PfMTI-1 or a homolog or ortholog thereof or a fragment of PfMTI-1 or a homolog or ortholog thereof. The term “antibody” as used herein refers to an intact immunoglobulin, or to an antigen-binding portion of an immunoglobulin that competes with the intact antibody for specific binding to a protein or fragment of a protein of the present invention. Antigen-binding portions of an immunoglobulin of the present invention can be produced by various techniques including, but not limited to recombinant DNA techniques and enzymatic or chemical cleavage of intact antibodies. Exemplary antigen-binding portions include Fab, Fab′, F(ab′)2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Fab fragments are monovalent fragments consisting of VL, VH, CL and CH1 domains. F(ab′)2 fragments are bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region. Fd fragments comprise a VH and CH1 domain. Fv fragment comprise a VL and VH domain of a single arm of an antibody. dAb fragments comprise a VH domain (Ward et al., Nature 1989 341: 544-546).
Antibodies of the present invention may be single-chain antibodies (scFv), meaning an antibody in which VL and VH regions are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain (Bird et al., Science 1988 242: 423-426; Huston et al., Proc. Natl. Acad. Sci. USA 1988 85: 5879-5883). Antibodies of the present invention may also be diabodies, meaning bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (Holliger et al., Proc. Natl. Acad. Sci. USA 1993 90: 6444-6448; Poljak et al. 1994 Structure 2: 1121-1123). Further, one or more CDRs can be incorporated into a molecule either covalently or noncovalently to produce an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest, in this case PfMTI-1 or a homolog or ortholog thereof. A chimeric antibody is an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.
An “isolated antibody” as used herein is an antibody that (1) is not associated with naturally-associated components, including other naturally-associated antibodies, that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. It is known that purified proteins, including purified antibodies, may be stabilized with non-naturally-associated components. The non-naturally-associated component may be a protein, such as albumin (e.g., BSA) or a chemical such as polyethylene glycol (PEG).
By “bind specifically” and “specific binding” as used herein it is meant the ability of an antibody of the present invention to bind to a first molecular species in preference to binding to other molecular species with which the antibody and first molecular species are admixed. An antibody is said specifically to “recognize” a first molecular species when it can bind specifically to that first molecular species. In the present invention the first molecular species comprises PfMTI-1 or a homolog or ortholog thereof or a fragment of PfMTI-1 or a homolog or ortholog thereof.
Identification of PfMTI-1 and homologs and orthologs thereof can be used to design and/or identify therapeutic agents which prevent infection by malaria parasites and/or decrease the severity of clinical.
For example, the structure of these proteins can be used in computer modeling programs to design mimetics of PfMTI-1 and homologs or orthologs thereof. By “mimetic” it is meant to include peptide based compounds, also referred to as peptidomimetics, as well as small organic molecules. Preferably mimetics of the present invention are designed to bind to either the erythrocyte binding site of PfMTI-1 or the component of the actin-myosin based motor machinery of the malaria parasite to which PfMTI-1 binds, thereby inhibiting the ability of PfMTI-1 or homologs or orthologs thereof to form a molecular bridge between red blood cells and the parasite.
Identification of PfMTI-1 and its function can also be used in the development of high throughput screening assays to identify potential chemotherapeutic agents which inhibit the function of this protein and orthologs or homologs thereof, thereby decreasing severity of the clinical symptoms of malaria infections. In vitro binding assays, in a high throughput format could be developed to screen for agents that disrupt or prevent the binding of PfMTI-1 to parasite aldolase. These agents would then be screen in culture for their effect on the in vivo growth or invasion of Plasmodium falciparum. Alternatively, an agent may be isolated that blocks the presumptive interaction between PfMTI-1 and a component on the surface of the red cell. Again, similar assay would be utilized to screen the efficacy of the agent in vivo.
Further, this protein and homologs or orthologs thereof serve as prime targets for vaccine development against malaria infection. A useful vaccine will comprise an antigen capable of invoking an immune response against the PfMTI-1 protein or an ortholog or homolog thereof. Exemplary antigens include, but are not limited to the entire PfMTI-1 sequence, the exocellular domain of PfMTI-1, or regions thereof.
Thus, the present invention also provides methods for identifying potential therapeutic agents for malaria which comprises assessing the ability of a potential therapeutic to inhibit the function of the PfMTI-1 protein or an ortholog or homolog thereof thereby inhibiting the interaction of this protein with a red blood cell to be invaded by the malaria parasite and/or its linkage to a component of the actin-myosin based machinery of the malaria parasite. Agents may inhibit the function directly by acting on the protein or protein's binding site or indirectly by invoking an immune response against the protein.
Further, agents identified as inhibiting the function of the PfMTI-1 protein or an ortholog or homolog thereof are expected to be useful in inhibiting and/or preventing malaria infections. Thus, the present invention also provides compositions and methods for inhibiting or preventing invasion of red blood cells by a malaria parasite.
Compositions of the present invention comprise an agent, which inhibits interaction of PfMTI-1 or an ortholog or homolog thereof with a host red blood cell and/or linkage to a component of actin-myosin based machinery of the malaria parasite in a pharmaceutically acceptable carrier. Selection of a pharmaceutically acceptable carrier for use in these compositions can be performed routinely by those of skill in the art based upon the solubility characteristics of the agent, its mode of action and the mode of administration desired for the composition. For example, if the agent is a vaccine typically administered via intramuscular injection, the carrier may comprise a sterile aqueous vehicle such as sterile saline or sterile phosphate buffered saline and may further comprise an adjuvant to enhance immunogenicity such as alum adjuvant or other adjuvants approved for human use.
Compositions of the present may be administered as a chemotherapeutic agent to a subject infected with a malaria parasite to prevent or decrease clinical severity of the infection. Alternatively, compositions of the present invention may be administered as immunologic agents to prevent or inhibit infection in subjects at risk for infection by a malaria parasite.
This patent application claims the benefit of priority from U.S. Provisional Application Ser. No. 60/628,572 filed Nov. 17, 2004, teachings of which are herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/41509 | 11/17/2005 | WO | 00 | 7/20/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60628572 | Nov 2004 | US |
