WO2009073648A2 - Compositions and methods for regulating entamoeba histolytica function - Google Patents

Abstract

The present invention provides compositions and methods useful for preventing and inhibiting amoebiasis, as well as treating a subject with amoebiasis. The present invention encompasses inhibiting or preventing the function and levels of EEAK1 protein.

Description

COMPOSITIONS AND METHODS FOR REGULATING ENTAMOEBA HISTOLYTICA FUNCTION

CROSS REFERENCE TO RELATED APPLICATIONS This application is entitled to priority pursuant to 35 U. S. C. § 119(e) to U.S. provisional patent application no. 60/992,124, filed on December 4, 2007. The entire disclosure of the afore -mentioned patent application is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part from Grant Nos. AI26649, AI053678 and P20 RR021905-01 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

FIELD OF INVENTION

This invention relates generally to the fields of regulating Entamoeba histolytica function and treating and preventing amoebiasis.

BACKGROUND Entamoeba histolytica, the causative agent of amoebiasis, is the second leading cause of morbidity and mortality among protozoan parasites worldwide [I]. Phagocytosis has been one of the most recognized behaviors of E. histolytica, because of its importance in vivo [2, 3]. Erythrophagocytosis has been used as a diagnostic indicator of invasive Entamoeba histolytica infection by microscopy [4]. Still, little is known concerning why host cells are ingested and/or what affect this has on the course of disease.

Invasive infection with this ameba leads to dramatic tissue destruction [5-8], including hallmarks of both apoptotic and necrotic host cell death [3, 9, 10]. Apoptotic changes on host cells have been shown to encourage their clearance by E. histolytica [11], and exposed phosphatidylserine (PS) has been recognized as a ligand for this interaction [11, 12]. Although little is known concerning the role of this behavior in disease, phagocytosis has been suggested as a virulence determining factor [13]. Amebic clones [14], and engineered mutants by either expression of dominant negative constructs [15], or by chemical mutagenesis [16] which display defective in vitro phagocytosis are also less virulent in vivo. In addition, use of pan-caspase inhibitors to interfere with apoptotic induction in vivo has also reduced infection by this parasite [17]. Given these results, we hypothesized that apoptotic induction followed by clearance of PS exposing host cells allows deeper penetration into tissues leading to more severe disease.

There have been attempts in several organisms to identify essential proteins required for endocytosis [18-20]. The widespread availability of mass spectrometry sequencing has made this process an ideal target for proteomics, given that, beads of known densities could be interacted with cells and then purified from cell lysates by gradient centrifugation. These experiments have revealed many interesting things including possible endoplasmic reticulum involvement in endocytosis [21]. Recent publications have elucidated many of the proteins involved in amebic endocytosis [22, 23]. Both these proteomic screens and the recently published E. histolytica genome [24] have uncovered much more about this process. Many homologues of endosome maturation proteins were found, consistent with functions in metazoans. Rab7, Rabl 1, Rap2, PBK, Racl and Rho all appear, consistent with other systems. However, some metazoan proteins including EEAl, RINl, and LAMPs do not have discernable homologues in the E. histolytica genome. The most glaring omission of recent screens is the lack of surface proteins identified which could potentially act as receptors for apoptotic host cells. Among the surface proteins that were identified are HgI, IgI, ABC transporter, p-glycoprotein-2 and 6, and M 17. To date, only HgI and IgI have been confirmed as constituents of the phagosomes.

Exposed PS mediates ingestion by the ameba, yet there are still no credible candidates for PS receptors on the amebic surface.

There is a long felt need in the art for compositions and methods useful for preventing, inhibiting, and treating amoebiasis. The present invention satisfies these needs.

SUMMARY OF THE INVENTION

The present disclosure is the first to identify a function for EEAKl ("Early Endosomal Associated Kinase 1") in Entamoeba histolytica. The present application discloses that EEAKl plays a role in amoebiasis and the inhibition of EEAKl function and levels inhibits and prevents infection, phagocytosis, and virulence of E. histolytica.

EEAKl was previously called TMK (see 47 and 48), and has more recently been called PATMK. In one embodiment, the present invention provides compositions and methods useful for inhibiting or preventing amebiasis, comprising administering to a subject a pharmaceutical composition comprising an effective amount of at least one inhibitor of EEAKl function or EEAKl levels, a pharmaceutically-acceptable carrier, and optionally an antibiotic. In one aspect, the method inhibits or prevents amebic colitis. In one aspect, the method inhibits or prevents host cell phagocytosis by E. histolytica. In one aspect, the phagocytosis is erythrophagocytosis. In one aspect, the method inhibits virulence of E. histolytica. In one aspect, the method inhibits or prevents infection by E. histolytica. In one aspect, the method of the invention is useful for reducing the levels of EEAKl in E. histolytica. The present invention further provides compositions and methods useful for inhibiting EEAKl function or levels comprising the use of an EEAKl protein comprising SEQ ID NO: 1 or a fragment thereof, an antibody directed against an EEAKl protein comprising SEQ ID NO:1 or a fragment of SEQ ID NO:1, an RNAi complementary to an EEAKl nucleic acid sequence, an expression vector encoding an RNAi complementary to an EEAKl nucleic acid sequence, and an isolated nucleic acid comprising a nucleic acid sequence encoding a protein having SEQ ID NO:1 or encoding a fragment of SEQ ID NO:1.

Anti-EEAKl serum was used to illustrate that EEAKl was both associated with the membrane of the amoeba and co-localized to fluorescent erythrocytes on the surface of trophozoites. Pre-incubation of amoeba with this antibody inhibited amebic ingestion of PS-exposing erythrocytes. In addition, engulfment of PS-exposing erythrocytes was statistically significantly inhibited by the expression of either RNAi against EEAKl and/or a carboxy-truncation of EEAKl (EEAK1_Λ932)- Expression of EEAKIΛ932 (SEQ ID NO:2) also reduced the ability of these amebae to establish infection in the intestinal model of amebiasis although these amebae retained the ability to cause liver abscesses.

A compound of the invention can be further used with other regulators described herein, or known in the art, such as peptides, antisense oligonucleotides, nucleic acids encoding peptides described herein, as well as antibodies, kinase inhibitors, and drugs/agents/compounds. The present invention provides compositions and methods for preventing, treating, and inhibiting amoebiasis. In one aspect, the invention provides compositions and methods for inhibiting interactions of E. histolytica with host cells or cell membranes. In one aspect, the methods of the invention inhibit the interaction of E. histolytica with red blood cells.

The present invention encompasses methods and compositions useful for inhibiting or preventing E. histolytica infection in the intestine of a subject. In one aspect, the infection is invasive infection.

The present invention provides, inter alia, compositions and methods useful for inhibiting or preventing amebic ingestion of a host cell. In one aspect, the cell is an erythrocyte. In one aspect, the erythrocyte is a PS-exposing erythrocyte. In one aspect, ingestion is inhibited using an antibody or antiserum directed against full length EEAKl (SEQ ID NO: 1). In one aspect, the antibody is directed against a fragment of EEAKl . In one aspect, the fragment of EEAKl comprises the sequence EIQKQNPISTSLKISKISSD (SEQ ID NO: 7). In one aspect, ingestion is inhibited by interfering with the interaction of the amoeba with a host cell. In one aspect, the interaction is binding between the amoeba and the cell. Antibodies useful for the practice of the invention include a polyclonal antibody, a monoclonal antibody, a chimeric antibody, and a humanized antibody.

Various types of molecules are encompassed within the methods of the invention and are useful for inhibiting the effects of EEAKl . The inhibition may be direct or indirect. For example, useful molecules for inhibiting EEAKl include, but are not limited to, RNAi, antisense oligonucleotides, antibodies, aptamers, and other agents and compounds. The compounds of the invention may regulate EEAKl by regulating processes and functions including, but not limited to, gene expression of EEAKl, translation of EEAKl, protein levels of EEAKl, protein degradation of EEAKl, binding of EEAKl with other molecules, as well as both downstream and upstream pathways regulating EEAKl and its functions. By regulating EEAKl is meant regulating EEAKl synthesis, levels, function/activity, binding, and any upstream or downstream pathways regulating EEAKl and its functions.

Because targeting EEAKl is useful for inhibiting EEAKl functions and levels, the present invention is useful for inhibiting and preventing initial intestinal colonization by E. histolytica. Moreover, because the initial site of infection is the intestine, the present invention further encompasses inhibiting and preventing all forms of the amebic disease which occur subsequent to intestinal colonization, including amebic liver abscess. For these reasons, the compositions and methods of the present invention are useful for a vaccine against E. histolytica. In one aspect, the target is EEAKl, and homo logs, modifications, and fragments thereof. The evidence provided in this disclosure suggests that antibodies against this protein will interfere with ingestion of host cells, and further suggests this protein is essential for infection by E. histolytica. The data provided herein indicate that EEAKl or fragments thereof will be useful targets as an antigen for an E. histolytica protective vaccine and that they can elicit an immune response. Given the very recent description of EEAKl, there has been no prior suggestion of this protein as a vaccination target. In addition, there has been no prior demonstration of EEAKl function in amebiasis.

Vaccination strategies would include the use of full length, or partial amino acid sequences that are produced via recombinant protein expression or synthesized. This disclosure suggests that antibodies against EEAKl could prevent ingestion of apoptotic host cells in the intestine, and thereby provide protection from infection. The present invention therefore encompasses in one aspect inducing an immune response by administering a pharmaceutical composition an effective of EEAKl (SEQ ID NO:1) or a fragment thereof, as well as an isolated nucleic acid comprising a nucleic acid sequence encoding EEAKl or a fragment thereof.

In addition, the use of a multivalent vaccine using the Gal/GalNAc lectin (prior patents 5,004,608 and 6,187,310) could provide greater protection from amebiasis.

Not only could antibody raised against EEAKl, and homologs, modifications, and fragments thereof help opsonize the parasite, the present application further demonstrates that antibodies against the extracellular domain interfere with erythrophagocytosis, which is pathognomonic of amebiasis. In addition, expression of a dominant negative form of this protein has produced parasites lacking the ability to infect the murine model of amebiasis. This data and previous data suggesting a link between phagocytosis and virulence clearly indicates that this is an interesting vaccine target.

The present invention further provides for the use of interfering RNAs to inhibit or prevent amebiasis. In one aspect, an RNAi is complementary to a nucleic acid encoding EEAKl or a fragment of EEAKl. In one aspect, the RNAi has a sequence of SEQ ID NO:5 or SEQ ID NO:6. In one embodiment, the antisense oligonucleotides of the invention are directed against the same sequences as the RNAi molecules of the invention. The present invention further provides the proteins and nucleic acids described herein, and others useful to practice the invention, as well as pharmaceutical and therapeutic compositions comprising the proteins and nucleic acids of the invention.

The present invention encompasses the use of the following sequences, as well as homologs, modifications, and fragments thereof:

Full Length EEAKl- TMK96i₂₇₉ (SEO ID NO:!):

MSIIPFQWCYILLFLSIEIVGEYCDWNKNVFEECNTCSPCQFNYNKTGVFIIRNRET ETMKFIQPHDTYFIFKNVSLKTLVIQNQIQSTFDLKELNTSFIVLINDTTINGDLSVD KILSFQLNFCSPQIEIQKQNPISTSLKISKISSDYFISTCF APNLLRINEFISIHIDKLYSS FGYLYSTLIINNPIFNSPQYKFTLVKQNDLFHYYFYDPSVLTILKQEVVNSTLFTTL FLNQNNIDLYLFISSIKIYYSIPSLSLLIHSYTNAQFDIIINETIDSLTFHHNNDNFTFY SLTQGFLQFYESDVQDGIIYTIKTSSSSGILHITNLQSHIVFENLEESFHFQLNSFQYI LNAQNGCQSSIISSDLFQCLSDMKCPKQKFFDETKNICQNCSTYCSNCINSTYCIYC DDGYVNRNGICYDRSDICSSNSMSSCYFCDTKNSYFIESCRDCSSHCKLCQEKKV CLQCEDNYYLNQTRQCVKTDIHQEIMVQDHIISCISGYYLNNNQCISCSESYGLGC QICDSYQCISCSPTTVFLKGYCIIPEHCNKIINGVCVSCEGLFYTNFTSCIDFHEPIAT PSHCLLMHLGQCIVCNQNFFRLPNGTCSEYQPDYCVEPSQLGCFRCEDGFFLDLK GSCSPCIDNCRYCANSTYCLQCKPHHYLTNSHECLNASQLNDNCELITSFGNGCA VCQKGYYRYGLDCVPCNSSCLSCQNLDSCLECKDGYLMNKDAKCVALNETIGCI NITSEHIGCDRCAEGYYLEQYECVGCPFNCKECYKEKCALCFGNDIVISGVCYPM TAVNHCISVNNSQCSQCSIGYIVNYNGTRCILKIQISVHIFFLVAIVVTIIFIIMIICYL LRKIYFPTEQQKFAWNEFIFDIRKKKYKLLENFGGLLINTQKLIFDQFREGIPVNVS SEKTFIIASIRKNPLKIDFILNQKPQRYELTVSPITIILKKNEGCIFTFKVKPMCSCTIN DQFTILYEDVRTNKIFKKNISIEAQTILSTLIDYEEITLINKIAQGSFSIVFKGKFRGN TVAIKRMKDL WRKDDFEEFRKEIDLLSKFKCRYIIQFLGAVINKNNVSIITEYAPY GSCEQVMIKSNENPNLIPNIQLRIKISKDISKAIQYLHSNGILHRDIKL ANVLIISLED EMEINAKLSDFGSSRSLNFLVKNITFTKGIGSPIYMAPEILQRKNYTTSADIYSLAIS LFEIIEWKEAYPADDPKFKYQWQIAEYVCKGNRPKTTGISLKIKSLLDLMWCQNP ENRIPIDVVLKHLQLM

Amino terminal portion of carboxy truncated fragment of SEQ ID NO:1, designated EEAK_A932 or TMK96_Λcm (SEQ ID NO:2) herein: MSIIPFQWCYILLFLSIEIVGEYCDWNKNVFEECNTCSPCQFNYNKTGVFIIRNRET ETMKFIQPHDTYFIFKNVSLKTLVIQNQIQSTFDLKELNTSFIVLINDTTINGDLSVD KILSFQLNFCSPQIEIQKQNPISTSLKISKISSDYFISTCF APNLLRINEFISIHIDKLYSS FGYLYSTLIINNPIFNSPQYKFTLVKQNDLFHYYFYDPSVLTILKQEVVNSTLFTTL FLNQNNIDLYLFISSIKIYYSIPSLSLLIHSYTNAQFDIIINETIDSLTFHHNNDNFTFY SLTQGFLQFYESDVQDGIIYTIKTSSSSGILHITNLQSHIVFENLEESFHFQLNSFQYI LNAQNGCQSSIISSDLFQCLSDMKCPKQKFFDETKNICQNCSTYCSNCINSTYCIYC DDGYVNRNGICYDRSDICSSNSMSSCYFCDTKNSYFIESCRDCSSHCKLCQEKKV CLQCEDNYYLNQTRQCVKTDIHQEIMVQDHIISCISGYYLNNNQCISCSESYGLGC QICDSYQCISCSPTTVFLKGYCIIPEHCNKIINGVCVSCEGLFYTNFTSCIDFHEPIAT PSHCLLMHLGQCIVCNQNFFRLPNGTCSEYQPDYCVEPSQLGCFRCEDGFFLDLK GSCSPCIDNCRYCANSTYCLQCKPHHYLTNSHECLNASQLNDNCELITSFGNGCA VCQKGYYRYGLDCVPCNSSCLSCQNLDSCLECKDGYLMNKDAKCVALNETIGCI NITSEHIGCDRCAEGYYLEQYECVGCPFNCKECYKEKCALCFGNDIVISGVCYPM TAVNHCISVNNSQCSQCSIGYIVNYNGTRCILKIQISVHIFFLVAIVVTIIFIIMIICYL LRKIYFPTEQQKFAWNEFIFDIRKKKYKLLENFGGLLINTQKLIFDQFREGIPVNVS SEKTFIIASIRKNPLK

Carboxy terminal fragment of SEQ ID NO:1 deleted to form SEQ ID NO:2, designated (SEQ ID NO:3) herein:

IDFILNQKPQRYELTVSPITIILKKNEGCIFTFKVKPMCSCTINDQFTIL YEDVRTNKI FKKNISIEAQTILSTLIDYEEITLINKIAQGSFSIVFKGKFRGNTVAIKRMKDLWRKD DFEEFRKEIDLLSKFKCRYIIQFLGAVINKNNVSIITEYAPYGSCEQVMIKSNENPN LIPNIQLRIKISKDISKAIQYLHSNGILHRDIKLANVLIISLEDEMEINAKLSDFGSSR SLNFLVKNITFTKGIGSPIYMAPEILQRKNYTTSADIYSLAISLFEIIEWKEAYPADD PKFKYQWQIAEYVCKGNRPKTTGISLKIKSLLDLMWCQNPENRIPID VVLKHLQL M

RNAi 325 (SEQ ID NO:4): AATGGAGACTTATCAGTTGA RNAi 2273 (SEQ ID NO:5): AAGGGTATTATTTAGAACAA RNAi 3552 (SEQ ID NO:6): TTATATGGCTCCTGAAATAT

Fragment of SEQ ID NO:1 (SEQ ID NO:7): EIQKQNPISTSLKISKISSD 5' Primer for kinase region of EEAKl (SEQ ID NO:8):

CAATTTAGAGAAGGAATTCCT

3' Primer for kinase region of EEAKl (SEQ ID NO:9): TCACATTAATTGAAGATGTTTTAAAACAACA

5' Oligo for U6 driven RNAi hairpin constructs (SEQ ID NO:10):

CTACTGAAGCTTGTTTTTATGAAAAAGTGTATTTGC

325 first round 3' Oligo (SEQ ID NO:11):

TCTCTTGAAAGAATCTTATCAACTGATAAGTCTCCAGGGCCCAATTTTATTTTT CTTTTTATCC

325 second round 3' Oligo (SEQ ID NO: 12): GAATGCGGCCGCAAAAAATGGAGACTTATCAGTTGATAAGATTCTTCTCTTGA A

2273 first round 3' Oligo (SEQ ID NO: 13):

TCTCTTGAAACACTCATATTGTTCTAAATAATACCCTTGGGCCCAATTTTATTT TTCTTTTTATCC

2273 second round 3' Oligo (SEQ ID NO: 14):

GAATGCGGCCGCAAAAAAGGGTATTATTTAGAACAATATGAGTGTTCTCTTG AA

3552 first round 3' Oligo (SEQ ID NO:15):

TCTCTTGAAGCCATATAAATTGGACTTCCTATTCCCTTGGGCCCAATTTTATTT

TTCTTTTTATCC

3552 second round 3' Oligo (SEQ ID NO: 16):

GAATGCGGCCGCAAAAAAGGGAATAGGAAGTCCAATTTATATGGCTCTCTTG AA. 5' Oligo for carboxy FLAG and truncated EEAKl at residue 932 (SEQ ID NO: 17):

AAAGATCTTCAATGAGCATTATTCCATTTCAATGGTGCTAT

3' Oligo used to create EEAKIi₂₇₉ (SEQ ID NO: 18): AACTCGAGTTAGCCCTTGTCGTCGTCGTCCTTGTAGTCCATTAATTGAAGATG TTTTAAAACAACATCAATGGGTAT

3' Oligo used to create EEAKl _A932 (SEQ ID NO: 19):

AACTCGAGTTAGCCCTTGTCGTCGTCGTCCTTGTAGTCAATTTTTAATGGATTT TTCCTTATGCTTGCTAT

Full length EEAKl nucleic acid sequence- accession number XM 650501 (3840 bases) (SEQ ID NO:20)

(see Loftus et al.) atgagcattattccatttcaatggtgctatattcttttatttttgagtattgagatagtaggagaatattgtgattggaataaaaatgtgttt gaagaatgtaatacttgtagtccatgtcaatttaactataacaaaacaggtgtttttatcatccgtaatcgtgaaacagaaacaatgaa atttattcaacctcatgacacttatttcattttcaaaaacgtttctttaaaaactttagtaattcaaaatcaaattcaatcaacttttgacttaa aagaattaaacacttcattcattgttcttattaatgatacaacaattaatggagacttatcagttgataagattctttcttttcaactcaattt ttgttctccacaaattgaaattcagaaacaaaaccctatttctacttccttaaaaatttccaaaattagttctgattatttcatttccacttgt tttgcaccaaatcttctccgaattaatgaatttatttctatacatattgataagctttatagttcttttggctatttatattcaacattaatcatc aataatcctatttttaattctcctcaatataagtttacattagttaaacaaaatgatttgtttcattattatttttatgacccttcggtcttaacg attttaaaacaagaagttgttaattcgacattatttactactttatttctaaaccaaaataatatagatttatatttatttatttcttcaataaaa atttattattcaataccttctctttctcttcttattcattcatatacaaacgctcaatttgatattataataaatgaaacaattgactcattaac atttcatcacaataatgataattttactttttactctcttactcaaggatttcttcaattctatgaatctgatgtacaagatgggattatttata ctattaagacatcttcatcttctggtattcttcatataactaatcttcagtctcatattgtctttgaaaatttggaagaatcatttcattttcag ttaaattcttttcaatatattttaaatgctcaaaatggttgtcaatcatcaattatttcttctgatttatttcaatgtttatctgacatgaaatgtc ctaaacaaaagttttttgatgaaacaaaaaatatatgtcaaaattgttctacgtattgtagtaattgtataaattcaacatattgtatttatt gtgatgatggttacgtcaaccgaaatggtatatgttatgatagaagtgatatttgttcatctaattctatgtcaagttgttatttttgtgata caaaaaactcatatttcattgagtcatgtagagattgttcttctcattgtaaattatgtcaagaaaaaaaagtttgtcttcaatgtgaaga caattattatctaaaccaaactaggcaatgtgttaaaactgatattcatcaagagattatggtacaagatcatattatatcatgtatatct ggttattatttaaacaataatcaatgtatttcttgttcagaatcttatggtttaggttgtcaaatatgtgattcatatcaatgtatttcttgttca cctacgactgtatttctaaaaggatattgcattattccagaacattgtaacaaaattattaatggagtttgtgtaagttgtgaaggattat tttatactaattttacttcttgtatagactttcatgaaccaatcgctacaccaagtcattgtttattaatgcatttaggtcaatgtattgtttgt aatcaaaatttttttagattacctaatgggacttgttcagaataccaacctgactattgcgttgagccaagtcaacttggatgttttagat gtgaagatggattttttcttgatttaaaaggatcttgttcaccttgtatagataattgtaggtattgtgcaaactcaacatattgtcttcaat gtaaacctcaccactatcttacaaattcacacgaatgtcttaatgcgtctcagttaaatgataattgtgaattaattacaagttttggaaa tgggtgtgctgtttgtcaaaaaggatattatcgatatggtcttgattgtgttccatgtaatagttcgtgtttatcatgtcaaaacttagatt catgccttgaatgtaaggatggttatttaatgaataaagacgcgaagtgtgttgctttaaatgaaactattggttgtattaatattacctc agaacatattggatgtgacagatgtgctgaagggtattatttagaacaatatgagtgtgttgggtgtccatttaattgcaaagaatgtt ataaagagaaatgtgcattatgttttggtaatgatattgttatttcaggagtatgttatccaatgacagcagtcaatcattgtatatcagtt aataattcacagtgtagtcaatgtagtattggttatattgtaaattataatggaacacgatgtatattaaaaatacaaatatctgttcatat ttttttcttagtagcaatagttgttacaattatttttattattatgattatatgttatttattaagaaaaatatactttcctacagaacaacaaaa atttgcatggaatgaatttatatttgatataagaaagaaaaaatataagttgctagaaaattttggaggtttactcattaatacacaaaa gttaatttttgatcaatttagagaaggaattcctgttaatgtttcatcagagaaaacatttataatagcaagcataaggaaaaatccatt aaaaattgattttatattaaaccaaaaacctcaaagatatgaattaacagtaagtccaattactataatattaaaaaagaatgaaggat gtatttttacatttaaagttaaaccaatgtgttcttgtacgataaatgatcaatttactatattatatgaagatgttagaacaaataaaatat ttaaaaaaaatattagtattgaagcacaaacaattttatcaacattaattgattatgaagaaattactcttataaataaaatagcacaag gctcatttagtattgtattcaaaggaaaatttagaggaaatactgttgctataaaaaggatgaaagatttatggagaaaagatgatttt gaagaatttagaaaagaaattgatttattaagtaaattcaaatgtcgttatattattcaatttctaggtgctgtaataaataaaaataatgt tagtattattacagaatatgctccttatggaagttgtgaacaagtaatgattaaatcaaatgaaaatcctaatttaattccaaacattcaa cttagaataaaaataagtaaagacatttcaaaagcaattcaatatcttcattctaatggaatattacatcgtgatattaaattagcaaat gtattaattatttctttagaagatgaaatggaaattaatgctaagcttagtgattttggttcatctagaagtcttaattttttagtaaagaat attacttttactaagggaataggaagtccaatttatatggctcctgaaatattacaaagaaaaaattatacaacctctgctgatatttatt ctcttgcaatttctttatttgaaattattgaatggaaagaagcttatccagcagatgaccccaaatttaagtatcaatggcaaattgctg aatatgtttgtaaagggaatagacctaaaacaacaggaatttcattaaaaattaaatcgttattagatttaatgtggtgccaaaatcct gaaaatagaatacccattgatgttgttttaaaacatcttcaattaatgtga

The present invention further provides nucleic acids comprising nucleic acid sequences encoding the amino acid sequences of the invention as well as antisense oligonucleotides directed against the nucleic acid sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Sequencing of purified phagosomes identified known endocytic proteins as well as a novel protein, EEAKl (SEQ ID NO:1). A sample of identified proteins is listed. Shaded blocks indicate time points proteins were identified. Each time point is indicated by the time of the chase performed following a 5 minute spin of the beads onto the cells. Highlighted in red is the novel protein, EEAKl .

Figure 2. EEAKl (SEQ ID NO:1) is predicted to be a membrane protein, which was confirmed by surface staining. A. Illustration of bioinformatic information concerning EEAKl . This protein is predicted to be a receptor kinase containing a 15 hydrophobic residue signal peptide sequence (red), CXXC repeats in the extracellular domain, a membrane spanning region and a kinase domain, with no predicted specificity. B. Anti-EEAKl serum was generated by injection of New Zealand white rabbits with the EEAKl specific peptide: EIQKQNPISTSLKISKISSD (SEQ ID NO:7) and the resultant serum was affinity purified against this peptide. To assess the specificity of this anti- EEAKl serum 10⁷ ameba were separated on an SDS polyacrylamide gel, transferred to PVDF, cut into strips and blotted with anti-EEAKl serum in the absence (lane 1) or presence of the EIQKQNPISTSLKISKISSD peptide ranging from 10 μM to 500 nM. The final lane is the staining of pre-immune serum from the same rabbit. C. E. histolytica trophozoites were allowed to adhere to a coverslip (37°C), washed twice in PBS, then fixed and stained with pre-immune or anti-EEAKl serum and visualized with goat anti- rabbit Cy3 conjugated antibodies (where indicated cells were permeabilized in 0.2% Triton X-100).

Figure 3. Anti-EEAKl blocks erythrophagocytosis by E. histolytica. A. Phagocytosis of calcium-treated or healthy erythrocytes by amebae was assayed in the presence of PBS (black), 50 μg/ml anti-lgl (white), 10 μg/ml anti-EEAKl (dark grey), 50 μg/ml anti-EEAKl (vertical hatch), pre-immune (light grey) and 50 μg/ml anti-EEAKl serum pre-absorbed with 25 μM of peptide (diagonal hatch). Erythrocytes were stained with TAMRA, then spun onto amebae and incubated at 37° C for 15 minutes at a 10:1 erythrocyte to ameba ratio. Un-engulfed erythrocytes were lysed in water, and amebae containing ingested erythrocytes were counted by microscopy. Data are reported as means ± SD. P values were determined by a two-tailed t test for healthy and calcium- treated cells (* indicates p < 0.02 vs. PBS controls, n=6). B. E. histolytica trophozoites were allowed to adhere to a coverslip (37°C), before CFSE-labeled erythrocytes were spun into contact and allowed 10 minutes to interact at room temperature. Cells were washed twice in PBS, fixed and stained with pre-immune or anti-EEAKl serum and visualized with goat anti-rabbit Cy3 conjugated antibodies. Figure 4. Expression of small interfering RNAs against EEAKl reduced EEAKl protein levels as well as the rate of amebic ingestion of erythrocytes. A. The interfering RNAs corresponded to nucleotides 325-354 (325; SEQ ID NO:4), 2273-2302 (2273; SEQ ID NO:5), and 3552-3581 (3552; SEQ ID NO:6) of EEAKl as well as a control which contained the same nucleotide makeup of 3552 in random order (scrambled). B. Amebic cell lysate (10⁵ cells per lane) was separated on an 8% SDS polyacrylamide gel, transferred to PVDF and blotted with anti-EEAKl or anti-Lgl serum (as a loading control). C. Phagocytosis of calcium-treated erythrocytes by amebae, was assayed in M199S (black bars) or M199S competed with 55 mM D-galactose (hatched bars). Erythrocytes were stained with CFSE, then spun onto amebae and incubated at 37° C for 10 minutes at a 10:1 erythrocyte to ameba ratio. Un-engulfed erythrocytes were lysed in water, and amebae containing ingested erythrocytes were counted by microscopy. Data are reported as means ± SD. P values were determined by a two-tailed t test compared to scrambled controls (* indicates p < 0.039 n=6). Figure S. Expression of carboxy-truncated EEAKl yielded a significant reduction in ingestion of erythrocytes by E. histolytica. A. Amebic lysates (10⁷ cells of either EEAKl I₂₇₉ (SEQ ID NO:1), EEAKU₉₃₂ (SEQ ID NO:2), or empty vector) were subjected to immunoprecipitation using anti-Flag resin. Proteins from the IP were separated on an 8% polyacrylamide gel, transferred to PVDF, and blotted with anti- EEAKl or pre-immune serum. B. Phagocytosis of calcium-treated erythrocytes by amebae expressing EEAKI_A932, EEAKl I₂₇₉, and empty vector controls, were assayed in M199S (black bars) or M199S competed with 55 mM D-galactose (hatched bars). Erythrocytes were stained with CFSE, then spun onto amebae and incubated at 37°C for 10 minutes at a 10:1 erythrocyte to ameba ratio. Un-engulfed erythrocytes were lysed in water, and amebae containing ingested erythrocytes were counted by microscopy. Data are reported as means ± SD. P values were determined by a two-tailed t test compared to empty vector controls (* indicates p < 0.003 n=6). C. Amebic surface staining was performed on non-permeabilized fixed E. histolytica trophozoites using anti-Gal/GalNAc HgI specific serum and analyzed by flow cytometry. D. Anti-FLAG M2 antibody was used to stain permeabilized ameba EEAKI_A932 (green), EEAKl I₂₇₉ (black) and empty vector (red) as well as empty vector with secondary alone (blue).

DETAILED DESCRIPTION Abbreviations and Acronyms

BSA- Bovine Serum Albumin

CFSE- 5-carboxyfluorescein diacetate succinimidyl ester

CMTMR- 5-4-chloromethyl- benzoylaminotetramethylrhodamine EDTA- Ethylene Diaminetetraacetic Acid

EEAKl- Early Endosomal Associated Kinase 1 (also referred to as TMK or PATMK)

E. histolytica- Entamoeba histolytica

FLAG- epitope tag: DYKDDDDK

FITC- Fluorescein Gal/GalNAc lectin- Galactose/Nacetyl galactosamine binding lectin

HEPES- 4-2-hydroxyethyl-l-piperazineethanesulfonic acid

IgG- Immunoglobin G

MER- Mer tyrosine kinase

PATMK- Phagosome Associated Transmembrane Kinase PS- Phosphatidylserine

RNAi- RNA interference shRNA- short hairpin RNA siRNA- small interfering RNA

TIGR- The Institute for Genomic Research Definitions-

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. The term "about," as used herein, means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%. In one aspect, the term "about" means plus or minus 20% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

The terms "additional therapeutically active compound" or "additional therapeutic agent", as used in the context of the present invention, refers to the use or administration of a compound for an additional therapeutic use for a particular injury, disease, or disorder being treated. Such a compound, for example, could include one being used to treat an unrelated disease or disorder, or a disease or disorder which may not be responsive to the primary treatment for the injury, disease or disorder being treated.

As use herein, the terms "administration of and or "administering" a compound should be understood to mean providing a compound of the invention or a prodrug of a compound of the invention to a subject in need of treatment.

As used herein, an "agonist" is a composition of matter which, when administered to a mammal such as a human, enhances or extends a biological activity attributable to the level or presence of a target compound or molecule of interest in the mammal.

The term "amebiasis", as used herein, refers to the disease caused by E. histolytica. The term "amebiasis" is used interchangeably with the term "amoebiasis". The symptoms often are quite mild and can include loose stools, stomach pain, and stomach cramping. Amebic dysentery is a severe form of amebiasis associated with stomach pain, bloody stools, and fever. Rarely, E. histolytica invades the liver and forms an abscess. Even less commonly, it spreads to other parts of the body, such as the lungs or brain. An "antagonist" is a composition of matter which when administered to a mammal such as a human, inhibits a biological activity attributable to the level or presence of a compound or molecule of interest in the mammal.

As used herein, "alleviating a disease or disorder symptom," means reducing the severity of the symptom or the frequency with which such a symptom is experienced by a patient, or both.

As used herein, amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table: Full Name Three-Letter Code One-Letter Code

Aspartic Acid Asp D

Glutamic Acid GIu E

Lysine Lys K

Arginine Arg R

Histidine His H

Tyrosine Tyr Y

Cysteine Cys C

Asparagine Asn N

Glutamine GIn Q

Serine Ser S

Threonine Thr T

Glycine GIy G

Alanine Ala A

Valine VaI V

Leucine Leu L

Isoleucine lie I

Methionine Met M

Proline Pro P

Phenylalanine Phe F

Tryptophan Trp W

The term "amino acid" is used interchangeably with "amino acid residue," and may refer to a free amino acid and to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

Amino acids have the following general structure:

H R C COOH

NH₂

Amino acids may be classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.

The nomenclature used to describe the peptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified. The term "basic" or "positively charged" amino acid as used herein, refers to amino acids in which the R groups have a net positive charge at pH 7.0, and include, but are not limited to, the standard amino acids lysine, arginine, and histidine.

The term "antibody," as used herein, refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as single chain antibodies and humanized antibodies. An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules.

An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules.

By the term "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

The term "antigen" as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or Iy sates.

The term "antigenic determinant" as used herein refers to that portion of an antigen that makes contact with a particular antibody (i.e., an epitope). When a protein or fragment of a protein, or chemical moiety is used to immunize a host animal, numerous regions of the antigen may induce the production of antibodies that bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the "immunogen" used to elicit the immune response) for binding to an antibody.

The term "antimicrobial agents" as used herein refers to any naturally-occurring, synthetic, or semi-synthetic compound or composition or mixture thereof, which is safe for human or animal use as practiced in the methods of this invention, and is effective in killing or substantially inhibiting the growth of microbes. "Antimicrobial" as used herein, includes antibacterial, antifungal, and antiviral agents.

As used herein, the term "antisense oligonucleotide" or antisense nucleic acid means a nucleic acid polymer, at least a portion of which is complementary to a nucleic acid which is present in a normal cell or in an affected cell. "Antisense" refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded

DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences. The antisense oligonucleotides of the invention include, but are not limited to, phosphorothioate oligonucleotides and other modifications of oligonucleotides. The term "binding" refers to the adherence of molecules to one another, such as, but not limited to, enzymes to substrates, ligands to receptors, antibodies to antigens, DNA binding domains of proteins to DNA, and DNA or RNA strands to complementary strands. "Binding partner," as used herein, refers to a molecule capable of binding to another molecule.

As used herein, the term "biologically active fragments" or "bioactive fragment" of the polypeptides encompasses natural or synthetic portions of the full-length protein that are capable of specific binding to their natural ligand or of performing the function of the protein.

The term "biological sample," as used herein, refers to samples obtained from a subject, including, but not limited to, skin, hair, tissue, blood, plasma, cells, sweat and urine. As used herein, the term "carrier molecule" refers to any molecule that is chemically conjugated to the antigen of interest that enables an immune response resulting in antibodies specific to the native antigen.

As used herein, the term "chemically conjugated," or "conjugating chemically" refers to linking the antigen to the carrier molecule. This linking can occur on the genetic level using recombinant technology, wherein a hybrid protein may be produced containing the amino acid sequences, or portions thereof, of both the antigen and the carrier molecule. This hybrid protein is produced by an oligonucleotide sequence encoding both the antigen and the carrier molecule, or portions thereof. This linking also includes covalent bonds created between the antigen and the carrier protein using other chemical reactions, such as, but not limited to glutaraldehyde reactions. Covalent bonds may also be created using a third molecule bridging the antigen to the carrier molecule.

These cross-linkers are able to react with groups, such as but not limited to, primary amines, sulfhydryls, carbonyls, carbohydrates, or carboxylic acids, on the antigen and the carrier molecule. Chemical conjugation also includes non-covalent linkage between the antigen and the carrier molecule.

A "coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene. The term "competitive sequence" refers to a peptide or a modification, fragment, derivative, or homo log thereof that competes with another peptide for its cognate binding site. "Complementary" as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs). Thus, it is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

A "compound," as used herein, refers to any type of substance or agent that is commonly considered a drug, or a candidate for use as a drug, as well as combinations and mixtures of the above.

As used herein, the term "conservative amino acid substitution" is defined herein as an amino acid exchange within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, GIy;

II. Polar, negatively charged residues and their amides:

Asp, Asn, GIu, GIn;

III. Polar, positively charged residues: His, Arg, Lys;

IV. Large, aliphatic, nonpolar residues:

Met Leu, He, VaI, Cys

V. Large, aromatic residues: Phe, Tyr, Trp

A "control" cell is a cell having the same cell type as a test cell. The control cell may, for example, be examined at precisely or nearly the same time the test cell is examined. The control cell may also, for example, be examined at a time distant from the time at which the test cell is examined, and the results of the examination of the control cell may be recorded so that the recorded results may be compared with results obtained by examination of a test cell.

A "test" cell is a cell being examined.

"Cytokine," as used herein, refers to intercellular signaling molecules, the best known of which are involved in the regulation of mammalian somatic cells. A number of families of cytokines, both growth promoting and growth inhibitory in their effects, have been characterized including, for example, interleukins, interferons, and transforming growth factors. A number of other cytokines are known to those of skill in the art. The sources, characteristics, targets and effector activities of these cytokines have been described.

The use of the word "detect" and its grammatical variants refers to measurement of the species without quantification, whereas use of the word "determine" or "measure" with their grammatical variants are meant to refer to measurement of the species with quantification. The terms "detect" and "identify" are used interchangeably herein. As used herein, a "detectable marker" or a "reporter molecule" is an atom or a molecule that permits the specific detection of a compound comprising the marker in the presence of similar compounds without a marker. Detectable markers or reporter molecules include, e.g., radioactive isotopes, antigenic determinants, enzymes, nucleic acids available for hybridization, chromophores, fluorophores, chemiluminescent molecules, electrochemically detectable molecules, and molecules that provide for altered fluorescence-polarization or altered light-scattering. A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

As used herein, the term "domain" refers to a part of a molecule or structure that shares common physicochemical features, such as, but not limited to, hydrophobic, polar, globular and helical domains or properties such as ligand binding, signal transduction, cell penetration and the like. Specific examples of binding domains include, but are not limited to, DNA binding domains and ATP binding domains.

The term "EEAKl" is used interchangeably with the terms "TMK" and "PHAKl" herein. As used herein, an "effective amount" or "therapeutically effective amount" means an amount sufficient to produce a selected effect, such as alleviating symptoms of a disease or disorder. In the context of administering compounds in the form of a combination, such as multiple compounds, the amount of each compound, when administered in combination with another compound(s), may be different from when that compound is administered alone. Thus, an effective amount of a combination of compounds refers collectively to the combination as a whole, although the actual amounts of each compound may vary. The term "more effective" means that the selected effect is alleviated to a greater extent by one treatment relative to the second treatment to which it is being compared. As used herein, the term "effector domain" refers to a domain capable of directly interacting with an effector molecule, chemical, or structure in the cytoplasm which is capable of regulating a biochemical pathway.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. The term "epitope" as used herein is defined as small chemical groups on the antigen molecule that can elicit and react with an antibody. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly five amino acids or sugars in size. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity.

As used herein, an "essentially pure" preparation of a particular protein or peptide is a preparation wherein at least about 95%, and preferably at least about 99%, by weight, of the protein or peptide in the preparation is the particular protein or peptide.

A "fragment" or "segment" is a portion of an amino acid sequence, comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide. The terms "fragment" and "segment" are used interchangeably herein. As used herein, the term "fragment," as applied to a protein or peptide, can ordinarily be at least about 3-15 amino acids in length, at least about 15-25 amino acids, at least about 25-50 amino acids in length, at least about 50-75 amino acids in length, at least about 75-100 amino acids in length, and greater than 100 amino acids in length.

As used herein, the term "fragment" as applied to a nucleic acid, may ordinarily be at least about 20 nucleotides in length, typically, at least about 50 nucleotides, more typically, from about 50 to about 100 nucleotides, preferably, at least about 100 to about 200 nucleotides, even more preferably, at least about 200 nucleotides to about 300 nucleotides, yet even more preferably, at least about 300 to about 350, even more preferably, at least about 350 nucleotides to about 500 nucleotides, yet even more preferably, at least about 500 to about 600, even more preferably, at least about 600 nucleotides to about 620 nucleotides, yet even more preferably, at least about 620 to about 650, and most preferably, the nucleic acid fragment will be greater than about 650 nucleotides in length.

As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property by which it is characterized. A functional enzyme, for example, is one which exhibits the characteristic catalytic activity by which the enzyme is characterized.

"Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3ATTGCC5' and 3 'TATGGC share 50% homology. As used herein, "homology" is used synonymously with "identity." The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. MoI. Biol. 215:403-410), and can be accessed, for example at the National Center for Biotechnology Information (NCBI) world wide web site. BLAST nucleotide searches can be performed with the NBLAST program (designated "blastn" at the NCBI web site), using the following parameters: gap penalty = 5; gap extension penalty = 2; mismatch penalty = 3; match reward = 1; expectation value 10.0; and word size = 11 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated "blastn" at the NCBI web site) or the NCBI "blastp" program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

The term "inhibit," as used herein, refers to the ability of a compound, agent, or method to reduce or impede a described function, level, activity, rate, etc., based on the context in which the term "inhibit" is used. Preferably, inhibition is by at least 10%, more preferably by at least 25%, even more preferably by at least 50%, and most preferably, the function is inhibited by at least 75%. The term "inhibit" is used interchangeably with "reduce" and "block."

As used herein "injecting or applying" includes administration of a compound of the invention by any number of routes and means including, but not limited to, topical, oral, buccal, intravenous, intramuscular, intra arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ophthalmic, pulmonary, or rectal means.

By the term "immunizing a human against an antigen" is meant administering to the human a composition, a protein complex, a DNA encoding a protein complex, an antibody or a DNA encoding an antibody, which elicits an immune response in the human which immune response provides protection to the human against a disease caused by the antigen or an organism which expresses the antigen.

As used herein, the term "induction of apoptosis" means a process by which a cell is affected in such a way that it begins the process of programmed cell death, which is characterized by the fragmentation of the cell into membrane-bound particles that are subsequently eliminated by the process of phagocytosis.

"Inappropriate apoptosis" of cells refers to apoptosis (i.e. programmed cell death) which occurs in cells of an animal at a rate different from the range of normal rates of apoptosis in cells of the same type in an animal of the same type which is not afflicted with a disease or disorder.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the peptide of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the identified compound invention or be shipped together with a container which contains the identified compound.

Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

A "ligand" is a compound that specifically binds to a target receptor.

A "receptor" is a compound that specifically binds to a ligand.

A ligand or a receptor (e.g., an antibody) "specifically binds to" or "is specifically immunoreactive with" a compound when the ligand or receptor functions in a binding reaction which is determinative of the presence of the compound in a sample of heterogeneous compounds. Thus, under designated assay (e.g., immunoassay) conditions, the ligand or receptor binds preferentially to a particular compound and does not bind in a significant amount to other compounds present in the sample. For example, a polynucleotide specifically binds under hybridization conditions to a compound polynucleotide comprising a complementary sequence; an antibody specifically binds under immunoassay conditions to an antigen bearing an epitope against which the antibody was raised. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

As used herein, the term "linkage" refers to a connection between two groups. The connection can be either covalent or non-covalent, including but not limited to ionic bonds, hydrogen bonding, and hydrophobic/hydrophilic interactions.

As used herein, the term "linker" refers to a molecule that joins two other molecules either covalently or noncovalently, e.g., through ionic or hydrogen bonds or van der Waals interactions, e.g., a nucleic acid molecule that hybridizes to one complementary sequence at the 5' end and to another complementary sequence at the 3' end, thus joining two non-complementary sequences.

"Malexpression" of a gene means expression of a gene in a cell of a patient afflicted with a disease or disorder, wherein the level of expression (including non- expression), the portion of the gene expressed, or the timing of the expression of the gene with regard to the cell cycle, differs from expression of the same gene in a cell of a patient not afflicted with the disease or disorder. It is understood that malexpression may cause or contribute to the disease or disorder, be a symptom of the disease or disorder, or both.

The term "nucleic acid" typically refers to large polynucleotides. By "nucleic acid" is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

As used herein, the term "nucleic acid" encompasses RNA as well as single and double-stranded DNA and cDNA. Furthermore, the terms, "nucleic acid," "DNA," "RNA" and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. By "nucleic acid" is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil). Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5 '-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 '-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as "upstream sequences"; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences."

The term "nucleic acid construct," as used herein, encompasses DNA and RNA sequences encoding the particular gene or gene fragment desired, whether obtained by genomic or synthetic methods.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

The term "oligonucleotide" typically refers to short polynucleotides, generally, no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

By describing two polynucleotides as "operably linked" is meant that a single- stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other. By way of example, a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques. The term "pharmaceutical composition" shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

As used herein, the term "pharmaceutically-acceptable carrier" means a chemical composition with which an appropriate compound or derivative can be combined and which, following the combination, can be used to administer the appropriate compound to a subject. As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

"Pharmaceutically acceptable" means physiologically tolerable, for either human or veterinary application.

As used herein, "pharmaceutical compositions" include formulations for human and veterinary use.

"Plurality" means at least two. A "polynucleotide" means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.

"Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.

"Synthetic peptides or polypeptides" means a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known to those of skill in the art.

By "presensitization" is meant pre-administration of at least one innate immune system stimulator prior to challenge with a pathogenic agent. This is sometimes referred to as induction of tolerance. The term "prevent," as used herein, means to stop something from happening, or taking advance measures against something possible or probable from happening. In the context of medicine, "prevention" generally refers to action taken to decrease the chance of getting a disease or condition.

A "preventive" or "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs, or exhibits only early signs, of a disease or disorder. A prophylactic or preventative treatment is administered for the purpose of decreasing the risk of developing pathology associated with developing the disease or disorder.

"Primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. A "constitutive" promoter is a promoter which drives expression of a gene to which it is operably linked, in a constant manner in a cell. By way of example, promoters which drive expression of cellular housekeeping genes are considered to be constitutive promoters.

An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. This can be equated with "preventing".

As used herein, "protecting group" with respect to a terminal amino group refers to a terminal amino group of a peptide, which terminal amino group is coupled with any of various amino-terminal protecting groups traditionally employed in peptide synthesis.

Such protecting groups include, for example, acyl protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxycarbonyl; and aliphatic urethane protecting groups, for example, tert-butoxycarbonyl or adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides, vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitable protecting groups.

As used herein, "protecting group" with respect to a terminal carboxy group refers to a terminal carboxyl group of a peptide, which terminal carboxyl group is coupled with any of various carboxyl-terminal protecting groups. Such protecting groups include, for example, tert-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond.

The term "protein" typically refers to large polypeptides.

The term "peptide" typically refers to short polypeptides. "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.

A host cell that comprises a recombinant polynucleotide is referred to as a "recombinant host cell." A gene which is expressed in a recombinant host cell wherein the gene comprises a recombinant polynucleotide, produces a "recombinant polypeptide."

A "recombinant polypeptide" is one which is produced upon expression of a recombinant polynucleotide.

"Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.

The term "protein" typically refers to large polypeptides.

The term "peptide" typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences: the left- hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxy 1-terminus.

The term "protein regulatory pathway", as used herein, refers to both the upstream regulatory pathway which regulates a protein, as well as the downstream events which that protein regulates. Such regulation includes, but is not limited to, transcription, translation, levels, activity, posttranslational modification, and function of the protein of interest, as well as the downstream events which the protein regulates.

The terms "protein pathway" and "protein regulatory pathway" are used interchangeably herein. As used herein, the term "purified" and like terms relate to an enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in a native environment. The term "purified" does not necessarily indicate that complete purity of the particular molecule has been achieved during the process. A "highly purified" compound as used herein refers to a compound that is greater than 90% pure. In particular, purified sperm cell DNA refers to DNA that does not produce significant detectable levels of non- sperm cell DNA upon PCR amplification of the purified sperm cell DNA and subsequent analysis of that amplified DNA. A "significant detectable level" is an amount of contaminate that would be visible in the presented data and would need to be addressed/explained during analysis of the forensic evidence.

A "receptor" is a compound that specifically binds to a ligand.

A "ligand" is a compound that specifically binds to a target receptor.

A "recombinant cell" is a cell that comprises a transgene. Such a cell may be a eukaryotic or a prokaryotic cell. Also, the transgenic cell encompasses, but is not limited to, an embryonic stem cell comprising the transgene, a cell obtained from a chimeric mammal derived from a transgenic embryonic stem cell where the cell comprises the transgene, a cell obtained from a transgenic mammal, or fetal or placental tissue thereof, and a prokaryotic cell comprising the transgene.

The term "regulate" refers to either stimulating or inhibiting a function or activity of interest.

As used herein, the term "reporter gene" means a gene, the expression of which can be detected using a known method. By way of example, the Escherichia coli lacZ gene may be used as a reporter gene in a medium because expression of the lacZ gene can be detected using known methods by adding the chromogenic substrate o-nitrophenyl-β- galactoside to the medium (Gerhardt et al., eds., 1994, Methods for General and

Molecular Bacteriology, American Society for Microbiology, Washington, DC, p. 574).

As used herein, the term "secondary antibody" refers to an antibody that binds to the constant region of another antibody (the primary antibody). By the term "signal sequence" is meant a polynucleotide sequence which encodes a peptide that directs the path a polypeptide takes within a cell, i.e., it directs the cellular processing of a polypeptide in a cell, including, but not limited to, eventual secretion of a polypeptide from a cell. A signal sequence is a sequence of amino acids which are typically, but not exclusively, found at the amino terminus of a polypeptide which targets the synthesis of the polypeptide to the endoplasmic reticulum. In some instances, the signal peptide is proteo lyrically removed from the polypeptide and is thus absent from the mature protein.

By "small interfering RNAs (siRNAs)" is meant, inter alia, an isolated dsRNA molecule comprised of both a sense and an anti-sense strand. In one aspect, it is greater than 10 nucleotides in length. siRNA also refers to a single transcript which has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin. siRNA further includes any form of dsRNA (proteo lyrically cleaved products of larger dsRNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA) as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides.

As used herein, the term "solid support" relates to a solvent insoluble substrate that is capable of forming linkages (preferably covalent bonds) with various compounds. The support can be either biological in nature, such as, without limitation, a cell or bacteriophage particle, or synthetic, such as, without limitation, an acrylamide derivative, agarose, cellulose, nylon, silica, or magnetized particles.

By the term "specifically binds to", as used herein, is meant when a compound or ligand functions in a binding reaction or assay conditions which is determinative of the presence of the compound in a sample of heterogeneous compounds. The term "standard," as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered and used for comparing results when administering a test compound, or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or compound on a parameter or function. Standard can also refer to an "internal standard", such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured. Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker.

A "subject" of analysis, diagnosis, or treatment is an animal. Such animals include mammals, preferably a human. As used herein, a "subject in need thereof is a patient, animal, mammal, or human, who will benefit from the method of this invention.

As used herein, a "substantially homologous amino acid sequences" includes those amino acid sequences which have at least about 95% homology, preferably at least about 96% homology, more preferably at least about 97% homology, even more preferably at least about 98% homology, and most preferably at least about 99% or more homology to an amino acid sequence of a reference antibody chain. Amino acid sequence similarity or identity can be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0.14 algorithm. The default settings used for these programs are suitable for identifying substantially similar amino acid sequences for purposes of the present invention.

"Substantially homologous nucleic acid sequence" means a nucleic acid sequence corresponding to a reference nucleic acid sequence wherein the corresponding sequence encodes a peptide having substantially the same structure and function as the peptide encoded by the reference nucleic acid sequence; e.g., where only changes in amino acids not significantly affecting the peptide function occur. Preferably, the substantially identical nucleic acid sequence encodes the peptide encoded by the reference nucleic acid sequence. The percentage of identity between the substantially similar nucleic acid sequence and the reference nucleic acid sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more. Substantial identity of nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example by physical/chemical methods (i.e., hybridization) or by sequence alignment via computer algorithm. Suitable nucleic acid hybridization conditions to determine if a nucleotide sequence is substantially similar to a reference nucleotide sequence are: 7% sodium dodecyl sulfate SDS, 0.5 M NaPO₄, 1 mM EDTA at 50⁰C with washing in 2X standard saline citrate (SSC), 0.1% SDS at 50⁰C; preferably in 7% (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50⁰C. with washing in IX SSC, 0.1% SDS at 50⁰C; preferably 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50⁰C with washing in 0.5X SSC, 0.1% SDS at 50⁰C; and more preferably in 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50⁰C with washing in 0.1X SSC, 0.1% SDS at 65°C. Suitable computer algorithms to determine substantial similarity between two nucleic acid sequences include, GCS program package (Devereux et al, 1984 Nucl. Acids Res.

12:387), and the BLASTN or FASTA programs (Altschul et al., 1990 Proc. Natl. Acad.

Sci. USA. 1990 87:14:5509-13; Altschul et al., J. MoI. Biol. 1990 215:3:403-10; Altschul et al., 1997 Nucleic Acids Res. 25:3389-3402). The default settings provided with these programs are suitable for determining substantial similarity of nucleic acid sequences for purposes of the present invention.

The term "substantially pure" describes a compound, e.g., a protein or polypeptide that has been separated from components which naturally accompany it. Typically, a compound is substantially pure when at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, gel electrophoresis, or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.

The term "symptom," as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a "sign" is objective evidence of disease. For example, a bloody nose is a sign. It is evident to the patient, doctor, nurse and other observers.

A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

A "therapeutically effective amount" of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.

The term to "treat," as used herein, means reducing the frequency with which symptoms are experienced by a patient or subject or administering an agent or compound to reduce the frequency with which symptoms are experienced. Treating can be equated with, in the context of the present application, inhibiting EEAKl functions or levels as associated with amebiasis. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

By the term "vaccine," as used herein, is meant a composition which when inoculated into an animal has the effect of stimulating an immune response in the animal, which serves to fully or partially protect the animal against a disease or its symptoms. The term vaccine encompasses prophylactic as well as therapeutic vaccines. A combination vaccine is one which combines two or more vaccines.

A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer or delivery of nucleic acid to cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, recombinant viral vectors, and the like. Examples of non- viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA and the like. "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide. Embodiments

In one embodiment, the invention encompasses isolated nucleic acids. In one aspect, the isolated nucleic acids comprise nucleic acid sequences which encode antibodies or peptides of the invention, or homologs, fragments, derivatives, or modifications thereof. In another aspect, the nucleic acids comprise antisense oligonucleotides. It is not intended that the present invention be limited by the nature of the nucleic acid employed. The target nucleic acid may be native or synthesized nucleic acid. The nucleic acid may be from a viral, bacterial, animal or plant source. The nucleic acid may be DNA or RNA and may exist in a double-stranded, single-stranded or partially double- stranded form. Furthermore, the nucleic acid may be found as part of a virus or other macromolecule. See, e.g., Fasbender et al., 1996, J. Biol. Chem. 272:6479-89.

Nucleic acids useful in the present invention include, by way of example and not limitation, oligonucleotides and polynucleotides such as antisense DNAs and/or RNAs; ribozymes; interfering RNA; DNA for gene therapy; viral fragments including viral DNA and/or RNA; DNA and/or RNA chimeras; mRNA; plasmids; cosmids; genomic DNA; cDNA; gene fragments; various structural forms of DNA including single-stranded DNA, double-stranded DNA, supercoiled DNA and/or triple-helical DNA; Z-DNA; and the like. The nucleic acids may be prepared by any conventional means typically used to prepare nucleic acids in large quantity. For example, DNAs and RNAs may be chemically synthesized using commercially available reagents and synthesizers by methods that are well-known in the art (see, e.g., Gait, 1985, OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press, Oxford, England)). RNAs may be produce in high yield via in vitro transcription using plasmids such as SP65 (Promega Corporation, Madison, WI). In some circumstances, as where increased nuclease stability is desired, nucleic acids having modified internucleoside linkages may be preferred. Nucleic acids containing modified internucleoside linkages may also be synthesized using reagents and methods that are well known in the art. For example, methods for synthesizing nucleic acids containing phosphonate phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate, formacetal, thioformacetal, diisopropylsilyl, acetamidate, carbamate, dimethylene-sulfϊde (-CH₂-S-CH₂), diinethylene-sulfoxide (-CH₂-SO-CH₂), dimethylene-sulfone (-CH₂-SO₂-CH₂), 2'-O-alkyl, and 2'-deoxy2'-fluoro phosphorothioate internucleoside linkages are well known in the art (see Uhlmann et al., 1990, Chem. Rev. 90:543-584; Schneider et al., 1990, Tetrahedron Lett. 31 :335 and references cited therein). The nucleic acids may be purified by any suitable means, as are well known in the art. For example, the nucleic: acids can be purified by reverse phase or ion exchange HPLC, size exclusion chromatography or gel electrophoresis. Of course, the skilled artisan will recognize that the method of purification will depend in part on the size of the DNA to be purified.

The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

Modified gene sequences, i.e. genes having sequences that differ from the gene sequences encoding the naturally-occurring proteins, are also encompassed by the invention, so long as the modified gene still encodes a protein that functions to stimulate healing in any direct or indirect manner. These modified gene sequences include modifications caused by point mutations, modifications due to the degeneracy of the genetic code or naturally occurring allelic variants, and further modifications that have been introduced by genetic engineering, i.e., by the hand of man.

Techniques for introducing changes in nucleotide sequences that are designed to alter the functional properties of the encoded proteins or polypeptides are well known in the art. Such modifications include the deletion, insertion, or substitution of bases, and thus, changes in the amino acid sequence. Changes may be made to increase the activity of a protein, to increase its biological stability or half- life, to change its glycosylation pattern, and the like. All such modifications to the nucleotide sequences encoding such proteins are encompassed by this invention. Oligonucleotides which contain at least one phosphorothioate modification are known to confer upon the oligonucleotide enhanced resistance to nucleases. Specific examples of modified oligonucleotides include those which contain phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatomic or heterocyclic intersugar ("backbone") linkages. In addition, oligonucleotides having morpholino backbone structures (U.S. Patent No: 5,034,506) or polyamide backbone structures (Nielsen et al, 1991, Science 254: 1497) may also be used.

The examples of oligonucleotide modifications described herein are not exhaustive and it is understood that the invention includes additional modifications of the antisense oligonucleotides of the invention which modifications serve to enhance the therapeutic properties of the antisense oligonucleotide without appreciable alteration of the basic sequence of the antisense oligonucleotide.

In one embodiment, the invention encompasses peptides, proteins, and fragments, homo logs, derivatives, and modifications thereof. The peptides of the present invention may be readily prepared by standard, well-established techniques, such as solid-phase peptide synthesis (SPPS) as described by Stewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Illinois; and as described by Bodanszky and Bodanszky in The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York. At the outset, a suitably protected amino acid residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin. "Suitably protected" refers to the presence of protecting groups on both the α-amino group of the amino acid, and on any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions which will not affect the final peptide product. Stepwise synthesis of the oligopeptide is carried out by the removal of the N-protecting group from the initial amino acid, and couple thereto of the carboxyl end of the next amino acid in the sequence of the desired peptide. This amino acid is also suitably protected. The carboxyl of the incoming amino acid can be activated to react with the N-terminus of the support-bound amino acid by formation into a reactive group such as formation into a carbodiimide, a symmetric acid anhydride or an "active ester" group such as hydroxybenzotriazole or pentafluorophenly esters.

Examples of solid phase peptide synthesis methods include the BOC method which utilized tert-butyloxcarbonyl as the α-amino protecting group, and the FMOC method which utilizes 9-fluorenylmethyloxcarbonyl to protect the α-amino of the amino acid residues, both methods of which are well-known by those of skill in the art. Incorporation of N- and/or C- blocking groups can also be achieved using protocols conventional to solid phase peptide synthesis methods. For incorporation of C- terminal blocking groups, for example, synthesis of the desired peptide is typically performed using, as solid phase, a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C-terminal blocking group. To provide peptides in which the C-terminus bears a primary amino blocking group, for instance, synthesis is performed using a p-methylbenzhydrylamine (MBHA) resin so that, when peptide synthesis is completed, treatment with hydrofluoric acid releases the desired C-terminally amidated peptide. Similarly, incorporation of an N- methylamine blocking group at the C-terminus is achieved using N-methylaminoethyl- derivatized DVB, resin, which upon HF treatment releases a peptide bearing an N- methylamidated C-terminus. Blockage of the C-terminus by esterifϊcation can also be achieved using conventional procedures. This entails use of resin/blocking group combination that permits release of side-chain peptide from the resin, to allow for subsequent reaction with the desired alcohol, to form the ester function. FMOC protecting group, in combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this purpose, with cleavage from the support being effected by TFA in dicholoromethane. Esterifϊcation of the suitably activated carboxyl function e.g. with DCC, can then proceed by addition of the desired alcohol, followed by deprotection and isolation of the esterifϊed peptide product. Incorporation of N-terminal blocking groups can be achieved while the synthesized peptide is still attached to the resin, for instance by treatment with a suitable anhydride and nitrile. To incorporate an acetyl blocking group at the N-terminus, for instance, the resin-coupled peptide can be treated with 20% acetic anhydride in acetonitrile. The N-blocked peptide product can then be cleaved from the resin, deprotected and subsequently isolated.

To ensure that the peptide obtained from either chemical or biological synthetic techniques is the desired peptide, analysis of the peptide composition should be conducted. Such amino acid composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide. Alternatively, or additionally, the amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequenators, which sequentially degrade the peptide and identify the amino acids in order, may also be used to determine definitely the sequence of the peptide. Prior to its use, the peptide is purified to remove contaminants. In this regard, it will be appreciated that the peptide will be purified so as to meet the standards set out by the appropriate regulatory agencies. Any one of a number of a conventional purification procedures may be used to attain the required level of purity including, for example, reversed-phase high-pressure liquid chromatography (HPLC) using an alkylated silica column such as C₄ -,Cs- or C₁₈- silica. A gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid. Ion-exchange chromatography can be also used to separate peptides based on their charge. Substantially pure protein obtained as described herein may be purified by following known procedures for protein purification, wherein an immunological, enzymatic or other assay is used to monitor purification at each stage in the procedure. Protein purification methods are well known in the art, and are described, for example in Deutscher et al. (ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).

It will be appreciated, of course, that the peptides may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or stabilize the N- and C-termini from "undesirable degradation", a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N-terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal blocking groups, in which the carboxyl group of the C- terminus is either incorporated or not, include esters, ketones or amides. Ester or ketone- forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH₂), and mono- and di- alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also useful C-terminal blocking groups and can be either coupled to the peptide's C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the peptide to yield desamino and descarboxylated forms thereof without affect on peptide activity. Other modifications can also be incorporated without adversely affecting the activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may include one or more D-amino acid resides, or may comprise amino acids which are all in the D-form. Retro-inverso forms of peptides in accordance with the present invention are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.

Acid addition salts of the present invention are also contemplated as functional equivalents. Thus, a peptide in accordance with the present invention treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamie, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclic and the like, to provide a water soluble salt of the peptide is suitable for use in the invention. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.

Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine. Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.

The preparation and use of antibodies to inhibit protein synthesis or function or to inhibit other molecules or their synthesis is well known to those skilled in the art, and is described for example in Harlow et al. (Harlow et al., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY). Antibodies of the invention can also be used to detect proteins or other molecules which may be components of the EEAKl pathway. The generation of polyclonal antibodies is accomplished by inoculating the desired animal with the antigen and isolating antibodies which specifically bind the antigen therefrom.

Monoclonal antibodies can be used effectively intracellularly to avoid uptake problems by cloning the gene and then transfecting the gene encoding the antibody. Such a nucleic acid encoding the monoclonal antibody gene obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art.

Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well known monoclonal antibody preparation procedure. Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide or other molecules are generated from mice immunized with the peptide using standard procedures as referenced herein. A nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. Immunol. 12:125-168), and the references cited therein. Further, the antibody of the invention may be "humanized" using the existing technology described in, for example, Wright et al., id., and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77:755-759), and other methods of humanizing antibodies well-known in the art or to be developed. Techniques are also well known in the art which allow such an antibody to be modified to remain in the cell. The invention encompasses administering a nucleic acid encoding the antibody, wherein the molecule further comprises an intracellular retention sequence. Such antibodies, frequently referred to as "intrabodies", are well known in the art and are described in, for example, Marasco et al. (U.S. Patent No. 6,004,490) and Beerli et al. (1996, Breast Cancer Research and Treatment 38:11-17).

A nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4): 125-168) and the references cited therein. Further, the antibody of the invention may be "humanized" using the technology described in Wright et al., (supra) and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77(4):755- 759).

To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY).

Bacteriophage which encode the desired antibody, may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed. Thus, when bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophage which do not express the antibody will not bind to the cell. Such panning techniques are well known in the art and are described for example, in Wright et al, (supra).

Processes such as those described above, have been developed for the production of human antibodies using M 13 bacteriophage display (Burton et al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M 13 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage which encode the Fab portion of an antibody molecule. However, the invention should not be construed to be limited solely to the generation of phage encoding Fab antibodies. Rather, phage which encode single chain antibodies (scFv/phage antibody libraries) are also included in the invention. Fab molecules comprise the entire Ig light chain, that is, they comprise both the variable and constant region of the light chain, but include only the variable region and first constant region domain (CHl) of the heavy chain. Single chain antibody molecules comprise a single chain of protein comprising the Ig Fv fragment. An Ig Fv fragment includes only the variable regions of the heavy and light chains of the antibody, having no constant region contained therein. Phage libraries comprising scFv DNA may be generated following the procedures described in Marks et al. (1991, J. MoI. Biol. 222:581-597). Panning of phage so generated for the isolation of a desired antibody is conducted in a manner similar to that described for phage libraries comprising Fab DNA. The invention should also be construed to include synthetic phage display libraries in which the heavy and light chain variable regions may be synthesized such that they include nearly all possible specificities (Barbas, 1995, Nature Medicine 1 :837-839; de Kruif et al. 1995, J. MoI. Biol. 248:97-105).

By the term "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

In one embodiment, antisense nucleic acids complementary to EEAKl mRNA can be used to block the expression or translation of the corresponding mRNA. Antisense oligonucleotides as well as expression vectors comprising antisense nucleic acids complementary to nucleic acids encoding a EEAKl can be prepared and used based on techniques routinely performed by those of skill in the art, and described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York), and in Gerhardt et al. (eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC). The antisense oligonucleotides of the invention include, but are not limited to, phosphorothioate oligonucleotides and other modifications of oligonucleotides. Methods for synthesizing oligonucleotides, phosphorothioate oligonucleotides, and otherwise modified oligonucleotides are well known in the art (U.S. Patent No: 5,034,506; Nielsen et al., 1991, Science 254: 1497). Oligonucleotides which contain at least one phosphorothioate modification are known to confer upon the oligonucleotide enhanced resistance to nucleases. Specific examples of modified oligonucleotides include those which contain phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatomic or heterocyclic intersugar ("backbone") linkages. In addition, oligonucleotides having morpholino backbone structures (U.S. Patent No: 5,034,506) or polyamide backbone structures (Nielsen et al., 1991, Science 254: 1497) may also be used.

The examples of oligonucleotide modifications described herein are not exhaustive and it is understood that the invention includes additional modifications of the antisense oligonucleotides of the invention which modifications serve to enhance the therapeutic properties of the antisense oligonucleotide without appreciable alteration of the basic sequence of the antisense oligonucleotide. Phosphorothioate oligonucleotides, which have very low sensitivity to nuclease degradation, may be used. Some oligonucleotides may be prepared lacking CG motifs, which should help reduce toxicity for in vivo use.

In another aspect, antisense nucleic acids complementary to EEAKl mRNAs, can be used to block EEAKl synthesis, and subsequently EEAKl function and stimulated pathways. This can be done by transfecting an appropriate antisense sequence. Antisense nucleic acids may be readily prepared using techniques known to those skilled in the art.

The antisense oligonucleotide inhibitors of EEAKl may be used independently in the cell culture systems or administered to animals. In one embodiment of the invention, the inhibitor of EEAKl is an oligonucleotide, preferably from 5 to 25 nucleotides in length. In another embodiment, the oligonucleotide is from 25 to 50 nucleotides in length. In yet another embodiment, the oligonucleotide is from 50 to 100 nucleotides in length. In a further embodiment, the oligonucleotide is 100-400 nucleotides in length.

Phosphorothioate oligonucleotides enter cells readily without the need for transfection or electroporation, which avoids subjecting the cells to nonspecific inducers of a stress response that might confound the experiment. The oligonucleotides may be administered using several techniques known to those of skill in the art and described herein. Effective inhibitory concentrations for phosphorothioates range between 1 and 50 μM, so a titration curve for diminution of EEAKl signal in western blots can be done to establish effective concentrations for each oligonucleotide used. Once inside the cells, the phosphorothioate-oligonucleotides hybridize with the nascent mRNA very close to the transcriptional start site, a site having maximum effect for antisense oligonucleotide inhibition.

The ability to selectively inhibit transcription of EEAKl or other genes with specific antisense molecules is expected to also allow the inhibition of induction of

EEAKl synthesis in the diseases, disorders and conditions described herein. Thus, the invention provides methods for the use of antisense oligonucleotides that will be effective at diminishing steady- state levels of the protein of interest.

The invention should not be construed to include only EEAKl inhibition using antisense techniques, but should also be construed to include inhibition of other genes and their proteins which are involved in the EEAKl pathway. Furthermore, the invention should not be construed to include only these particular antisense methods described herein. Using Compounds to Inhibit EEAKl Synthesis

In one embodiment the invention includes a method of inhibiting EEAKl synthesis in a mammal, said method comprising administering to a mammal an effective amount of an inhibitor of EEAKl synthesis, or a derivative or modification thereof, thereby inhibiting EEAKl synthesis in a mammal. Preferably, the mammal is a human.

In one embodiment, the inhibitor comprises from about 0.0001% to about 15% by weight of the pharmaceutical composition. In one aspect, the inhibitor is administered as a controlled-release formulation.

Compounds and Methods Useful for Inhibiting EEAKl Function The invention, as disclosed herein, relates to the involvement of EEAKl as a key regulator in infection. The invention further relates to methods of inhibiting the function of EEAKl in order to alleviate or amoeba associated symptoms. The invention also relates to the involvement of EEAKl in other diseases and disorders. Inhibition of EEAKl function can be direct or indirect. Therefore, EEAKl function may be inhibited or caused to decrease using many approaches as described herein. Inhibition of EEAKl function may be assayed or monitored using techniques described herein as well as others known to those of skill in the art. Function can be measured directly or it can be estimated using techniques to measure parameters which are known to be correlative of EEAKl function. The invention should also be construed to include the use of compounds to modulate other EEAKl functions as well.

In one embodiment, the inhibitor comprises from about 0.0001% to about 15% by weight of the pharmaceutical composition.

The invention should be construed to include various methods of administration, including intravenous, intraperitoneal, topical, oral, intramuscular, intrathecal, vaginal, rectal, subcutaneous, and buccal. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human subject being treated, and the like.

By way of example, an inhibitor of EEAKl function may be an isolated nucleic acid encoding a nucleic acid sequence which is complementary to a EEAKl mRNA and in an antisense orientation. Other inhibitors include an antisense oligonucleotide, an antibody, or other compounds or agents such as small molecules. It should be understood that compositions and methods for inhibiting pathways, events, and precursors leading to the synthesis or production of EEAKl, may inhibit not only EEAKl synthesis, but also its accumulation, and ultimately its function. The invention should be construed to include compositions and methods to inhibit all pathways and precursors leading to EEAKl synthesis.

The invention provides methods for identifying inhibitors of amoeba infection by identifying inhibitors of EEAKl. In general, methods for the identification of a compound which effects the synthesis, production, accumulation or function of EEAKl, include the following general steps: The test compound is administered to a cell, tissue, sample, or subject, in which the measurements are to be taken. A control is a cell, tissue, sample, or subject in which the test compound has not been added. A higher or lower level of the indicator or parameter being tested, i.e., EEAKl levels, synthesis, function, degradation, etc., in the presence of the test compound, compared with the levels of the indicator or parameter in the sample which was not treated with the test compound, is an indication that the test compound has an effect on the indicator or parameter being measured, and as such, is a candidate for inhibition of the desired activity. Test compounds may be added at varying doses and frequencies to determine the effective amount of the compound which should be used and effective intervals in which it should be administered. In another aspect, a derivative or modification of the test compound may be used.

The invention relates to the administration of an identified compound in a pharmaceutical composition to practice the methods of the invention, the composition comprising the compound or an appropriate analog, homolog, derivative, modification, or fragment of the compound and a pharmaceutically-acceptable carrier. For example, a chemical composition with which an appropriate inhibitor of enzyme dependent production of EEAKl, or inhibitor of EEAKl accumulation or function, or stimulator of EEAKl removal, or degradation, is combined, is used to administer the appropriate compound to an animal. The invention should be construed to include the use of one, or simultaneous use of more than one, inhibitor of EEAKl or stimulator of EEAKl removal, and degradation. When more than one stimulator or inhibitor is used, they can be administered together or they can be administered separately.

In one embodiment, the present invention provides interfering RNAs useful for inhibiting EEAKl . The present invention is also directed to useful aptamers. In one embodiment, an aptamer is a compound that is selected in vitro to bind preferentially to another compound (in this case the identified proteins). In one aspect, aptamers are nucleic acids or peptides, because random sequences can be readily generated from nucleotides or amino acids (both naturally occurring or synthetically made) in large numbers but of course they need not be limited to these. In another aspect, the nucleic acid aptamers are short strands of DNA that bind protein targets. In one aspect, the aptamers are oligonucleotide aptamers. Oligonucleotide aptamers are oligonucleotides which can bind to a specific protein sequence of interest. A general method of identifying aptamers is to start with partially degenerate oligonucleotides, and then simultaneously screen the many thousands of oligonucleotides for the ability to bind to a desired protein. The bound oligonucleotide can be eluted from the protein and sequenced to identify the specific recognition sequence. Transfer of large amounts of a chemically stabilized aptamer into cells can result in specific binding to a polypeptide of interest, thereby blocking its function. [For example, see the following publications describing in vitro selection of aptamers: Klug et al., MoI. Biol. Reports 20:97-107 (1994); Wallis et al., Chem. Biol. 2:543-552 (1995); Ellington, Curr. Biol. 4:427-429 (1994); Lato et al., Chem. Biol. 2:291-303 (1995); Conrad et al., MoI. Div. 1 :69-78 (1995); and Uphoff et al., Curr. Opin. Struct. Biol. 6:281-287 (1996)]. The present invention further encompasses the use of phylomers which inhibit or prevent EEAKl function or levels.

As used herein, an antagonist or blocking agent may comprise, without limitation, an antibody, an antigen binding portion thereof or a biosynthetic antibody binding site that binds a particular target protein; an antisense molecule that hybridizes in vivo to a nucleic acid encoding a target protein or a regulatory element associated therewith, or a ribozyme, aptamer, or small molecule that binds to and/or inhibits a target protein, or that binds to and/or inhibits, reduces or otherwise modulates expression of nucleic acid encoding a target protein.

Aptamers offer advantages over other oligonucleotide-based approaches that artificially interfere with target gene function due to their ability to bind protein products of these genes with high affinity and specificity. However, RNA aptamers can be limited in their ability to target intracellular proteins since even nuclease-resistant aptamers do not efficiently enter the intracellular compartments. Moreover, attempts at expressing RNA aptamers within mammalian cells through vector-based approaches have been hampered by the presence of additional flanking sequences in expressed RNA aptamers, which may alter their functional conformation.

The idea of using single-stranded nucleic acids (DNA and RNA aptamers) to target protein molecules is based on the ability of short sequences (20 mers to 80 mers) to fold into unique 3D conformations that enable them to bind targeted proteins with high affinity and specificity. RNA aptamers have been expressed successfully inside eukaryotic cells, such as yeast and multicellular organisms, and have been shown to have inhibitory effects on their targeted proteins in the cellular environment.

The present application discloses compositions and methods for inhibiting the proteins described herein, and those not disclosed which are known in the art are encompassed within the invention. For example, various modulators/effectors are known, e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules, or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides. The present invention also encompasses pharmaceutical and therapeutic compositions comprising the compounds of the present invention.

The present invention is also directed to pharmaceutical compositions comprising the compounds of the present invention. More particularly, such compounds can be formulated as pharmaceutical compositions using standard pharmaceutically acceptable carriers, fillers, solublizing agents and stabilizers known to those skilled in the art.

Suitable preparations of vaccines include injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, suspension in, liquid prior to injection, may also be prepared. The preparation may also be emulsified, or the polypeptides encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.

The invention is also directed to methods of administering the compounds of the invention to a subject. In one embodiment, the invention provides a method of treating a subject by administering compounds identified using the methods of the invention description. Pharmaceutical compositions comprising the present compounds are administered to an individual in need thereof by any number of routes including, but not limited to, topical, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

In accordance with one embodiment, a method of treating and vaccinating a subject in need of such treatment is provided. The method comprises administering a pharmaceutical composition comprising at least one compound of the present invention to a subject in need thereof. Compounds identified by the methods of the invention can be administered with known compounds or other medications as well.

The invention also encompasses the use of pharmaceutical compositions of an appropriate compound, and homo logs, fragments, analogs, or derivatives thereof to practice the methods of the invention, the composition comprising at least one appropriate compound, and homo log, fragment, analog, or derivative thereof and a pharmaceutically- acceptable carrier.

In one embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In another embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 g/kg/day.

Pharmaceutically acceptable carriers which are useful include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

For in vivo applications, the peptides of the present invention may comprise a pharmaceutically acceptable salt. Suitable acids which are capable of forming such salts with the compounds of the present invention include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like. Pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents, excipients, solvents or adjuvants. The compositions are preferably sterile and nonpyrogenic. Examples of suitable carriers include, but are not limited to, water, normal saline, dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar-agar and tragacanth, or mixtures of these substances, and the like. The pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary pharmaceutical substances or excipients and/or additives, such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). Suitable additives include, but are not limited to, physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA or CaNaDTP A-bisamide), or, optionally, additions (e.g. 1 to 50 mole percent) of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) may be used. The compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Pharmaceutical compositions according to the present invention can be prepared in a manner fully within the skill of the art. The peptides of the invention, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising these compounds may be administered so that the compounds may have a physiological effect. Administration may occur enterally or parenterally; for example orally, rectally, intracisternally, intravaginally, intraperitoneally, locally (e.g., with powders, ointments or drops), or as a buccal or nasal spray or aerosol. Parenteral administration is preferred. Particularly preferred parenteral administration methods include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature), peri- and intra-target tissue injection (e.g. peri-tumoral and intra-tumoral injection), subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps), intramuscular injection, and direct application to the target area, for example by a catheter or other placement device.

Where the administration of the peptide is by injection or direct application, the injection or direct application may be in a single dose or in multiple doses. Where the administration of the compound is by infusion, the infusion may be a single sustained dose over a prolonged period of time or multiple infusions.

The present application encompasses the use of siRNA for blocking the pathways identified herein. In one aspect, the siRNA is directed against EEAKl or a fragment thereof. In a further aspect, a first siRNA can be used in combination with a second siRNA with a slightly different sequence than the first, or the second siRNA can be directed against a different sequence altogether. An siRNA of the invention can be further used with other regulators described herein, or known in the art, such as peptides, antisense oligonucleotides, nucleic acids encoding peptides described herein, aptamers, antibodies, kinase inhibitors, and drugs/agents/compounds.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

The source of active compound to be formulated will generally depend upon the particular form of the compound. Small organic molecules and peptidyl or oligo fragments can be chemically synthesized and provided in a pure form suitable for pharmaceutical/cosmetic usage. Products of natural extracts can be purified according to techniques known in the art. Recombinant sources of compounds are also available to those of ordinary skill in the art. Liquid derivatives and natural extracts made directly from biological sources may be employed in the compositions of this invention in a concentration (w/v) from about 1 to about 99%. Fractions of natural extracts and protease inhibitors may have a different preferred rage, from about 0.01% to about 20% and, more preferably, from about 1% to about 10% of the composition. Of course, mixtures of the active agents of this invention may be combined and used together in the same formulation, or in serial applications of different formulations.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

Typically, dosages of the compound of the invention which may be administered to an animal, preferably a human, will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. The compound can be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even lees frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the subject, etc.

It will be recognized by one of skill in the art that the various embodiments of the invention as described above relating to methods of preventing or treating amoeba infection and symptoms and inhibiting EEAKl or treating EEAKlpathway regulated diseases or conditions, includes other diseases and conditions not described herein. The compounds of the invention may be administered to, for example, a cell, a tissue, or a subject by any of several methods described herein and by others which are known to those of skill in the art.

The present invention further encompasses use of the yeast two-hybrid system to identify regulators of the proteins and pathways described herein. Such regulators can be drugs, compounds, peptides, nucleic acids, etc. Such regulators can include endogenous regulators.

Generally, the yeast two-hybrid assay can identify novel protein-protein interactions and compounds that alter those interactions. By using a number of different proteins as potential binding partners, it is possible to detect interactions that were previously uncharacterized. Secondly, the yeast two-hybrid assay can be used to characterize interactions already known to occur. Characterization could include determining which protein domains are responsible for the interaction, by using truncated proteins, or under what conditions interactions take place, by altering the intracellular environment. These assays can also be used to screen modulators of the interactions.

This invention encompasses methods of screening compounds to identify those compounds that act as agonists (stimulate) or antagonists (inhibit) of the protein interactions and pathways described herein. Screening assays for antagonist compound candidates are designed to identify compounds that bind or complex with the peptides described herein, or otherwise interfere with the interaction of the peptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.

All assays for antagonists are common in that they call for contacting the compound or drug candidate with a peptide identified herein under conditions and for a time sufficient to allow these two components to interact.

In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, one of the peptides of the complexes described herein, or the test compound or drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the peptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the peptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex. If the candidate compound interacts with, but does not bind to a particular peptide identified herein, its interaction with that peptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London), 340:245-246 (1989); Chien et al, Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Complete kits for identifying protein-protein interactions between two specific proteins using the two-hybrid technique are available. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a peptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding

(complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.

To assay for antagonists, the peptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the peptide indicates that the compound is an antagonist to the peptide. The peptide can be labeled, such as by radioactivity. Other assays and libraries are encompassed within the invention, such as the use of phylomers® and reverse yeast two-hybrid assays (see Watt, 2006, Nature Biotechnology, 24:177; Watt, U.S. Pat. No. 6,994,982; Watt, U.S. Pat. Pub. No. 2005/0287580; Watt, U.S. Pat. No. 6,510,495; Barr et al., 2004, J. Biol. Chem., 279:41 :43178-43189; the contents of each of these publications is hereby incorporated by reference herein in their entirety). Phylomers® are derived from sub domains of natural proteins, which makes them potentially more stable than conventional short random peptides. Phylomers® are sourced from biological genomes that are not human in origin. This feature significantly enhances the potency associated with Phylomers® against human protein targets. Phylogica's current Phylomer® library has a complexity of 50 million clones, which is comparable with the numerical complexity of random peptide or antibody Fab fragment libraries. An Interacting Peptide Library, consisting of 63 million peptides fused to the B42 activation domain, can be used to isolate peptides capable of binding to a target protein in a forward yeast two hybrid screen. The second is a Blocking Peptide Library made up of over 2 million peptides that can be used to screen for peptides capable of disrupting a specific protein interaction using the reverse two-hybrid system. The Phylomer® library consists of protein fragments, which have been sourced from a diverse range of bacterial genomes. The libraries are highly enriched for stable subdomains (15-50 amino acids long). This technology can be integrated with high throughput screening techniques such as phage display and reverse yeast two-hybrid traps. Vaccines and Immunogens

In one embodiment, the invention relates to methods and reagents for immunizing and treating a subject with an antigenic compound of the invention to elicit specific cellular and humoral immune-responses against specific antigens. The invention provides methods of using specifically prepared immunogen in fresh or lyophilized liposome, proper routes of administration of the immunogen, proper doses of the immunogen, and specific combinations of heterologous immunization including DNA priming in one administration route followed by liposome-mediated protein antigen boost in a different route to tailor the immune responses in respects of enhancing cell mediated immune response, cytokine secretion, humoral immune response, especially skewing T helper responses to be ThI or a balanced ThI and Th2 type.

For convenience, immune responses are often described in the present invention as being either "primary" or "secondary" immune responses. A primary immune response, which is also described as a "protective" immune response, refers to an immune response produced in an individual as a result of some initial exposure (e.g., the initial "immunization") to a particular antigen. Such an immunization can occur, for example, as the result of some natural exposure to the antigen (for example, from initial infection by some pathogen that exhibits or presents the antigen). Alternatively, the immunization can occur as a result of vaccinating the individual with a vaccine containing the antigen. For example, the vaccine can be a vaccine comprising one or more antigens from E. histolytica. The vaccine can also be modified to express other immune activators such as IL2, and costimulatory molecules, among others.

Another type of vaccine that can be combined with antibodies to an antigen is a vaccine prepared from a cell lysate of interest, in conjunction with an immunological adjuvant, or a mixture of lysates from cells of interest plus DETOX™ immunological adjuvant. Vaccine treatment can be boosted with anti-antigen antibodies, with or without additional chemotherapeutic treatment.

When used in vivo for therapy, the antibodies of the subject invention are administered to the patient in therapeutically effective amounts (i.e., amounts that have desired therapeutic effect). They will normally be administered parenterally. The dose and dosage regimen will depend upon the degree of the infection, the characteristics of the particular antibody or immunotoxin used, e.g., its therapeutic index, the patient, and the patient's history. Advantageously the antibody or immunotoxin is administered continuously over a period of 1-2 weeks. Optionally, the administration is made during the course of adjunct therapy such as antimicrobial treatment, or administration of tumor necrosis factor, interferon, or other cytoprotective or immunomodulatory agent.

For parenteral administration, the antibodies will be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic, and non-therapeutic. Examples of such vehicle are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes can be used as carriers. The vehicle can contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibodies will typically be formulated in such vehicles at concentrations of about 1.0 mg/ml to about 10 mg/ml. Use of IgM antibodies can be preferred for certain applications; however, IgG molecules by being smaller can be more able than IgM molecules to localize to certain types of infected cells. There is evidence that complement activation in vivo leads to a variety of biological effects, including the induction of an inflammatory response and the activation of macrophages (Unanue and Benecerraf, Textbook of Immunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)). The increased vasodilation accompanying inflammation can increase the ability of various agents to localize in infected cells.

Therefore, antigen-antibody combinations of the type specified by this invention can be used therapeutically in many ways. Additionally, purified antigens (Hakomori, Ann. Rev. Immunol. 2:103, 1984) or anti-idiotypic antibodies (Nepom et al., Proc. Natl. Acad. Sci. USA 81 : 2864, 1985; Koprowski et al., Proc. Natl. Acad. Sci. USA 81 : 216, 1984) relating to such antigens could be used to induce an active immune response in human patients. Such a response includes the formation of antibodies capable of activating human complement and mediating ADCC and by such mechanisms cause infected cell destruction.

Optionally, the antibodies of this invention are useful as antibody-cytotoxin conjugate molecules, as exemplified by the administration for treatment of neoplastic disease.

The antibody compositions used in therapy are formulated and dosages established in a fashion consistent with good medical practice taking into account the disorder to be treated, the condition of the individual patient, the site of delivery of the composition, the method of administration, and other factors known to practitioners. The antibody compositions are prepared for administration according to the description of preparation of polypeptides for administration, infra.

As is well understood in the art, biospecifϊc capture reagents include antibodies, binding fragments of antibodies which bind to activated integrin receptors on metastatic cells (e.g., single chain antibodies, Fab' fragments, F(ab)^T2 fragments, and scFv proteins and affibodies (Affibody, Teknikringen 30, floor 6, Box 700 04, Stockholm SE-10044, Sweden; See U.S. Pat. No. 5,831,012, incorporated herein by reference in its entirety and for all purposes)). Depending on intended use, they also can include receptors and other proteins that specifically bind another biomolecule. The hybrid antibodies and hybrid antibody fragments include complete antibody molecules having full length heavy and light chains, or any fragment thereof, such as Fab, Fab', F(ab')2, Fd, scFv, antibody light chains and antibody heavy chains. Chimeric antibodies which have variable regions as described herein and constant regions from various species are also suitable. See for example, U.S. Application No. 20030022244.

Initially, a predetermined target object is chosen to which an antibody can be raised. Techniques for generating monoclonal antibodies directed to target objects are well known to those skilled in the art. Examples of such techniques include, but are not limited to, those involving display libraries, xeno or humab mice, hybridomas, and the like. Target objects include any substance which is capable of exhibiting antigenicity and are usually proteins or protein polysaccharides. Examples include receptors, enzymes, hormones, growth factors, peptides and the like. It should be understood that not only are naturally occurring antibodies suitable for use in accordance with the present disclosure, but engineered antibodies and antibody fragments which are directed to a predetermined object are also suitable.

The present application discloses compositions and methods for inhibiting the proteins described herein, and those not disclosed which are known in the art are encompassed within the invention. For example, various modulators/effectors are known, e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules, or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides. Kits The present invention should be construed to include kits for treating, preventing, or inhibiting amoebic infection and for inhibiting or stimulating EEAKl, treating EEAKl associated diseases and disorders, and kits for measuring EEAKl and EEAKl related parameters. The invention includes a kit comprising an inhibitor of amoeba infection or EEAKl function or a compound identified in the invention, a standard, and an instructional material which describes administering the inhibitor or a composition comprising the inhibitor or compound to a cell or an animal. In one aspect, the inhibitor is an inhibitor of EEAKl. This should be construed to include other embodiments of kits that are known to those skilled in the art, such as a kit comprising a standard and a (preferably sterile) solvent suitable for dissolving or suspending the composition of the invention prior to administering the compound to a cell or an animal. Preferably the animal is a mammal. More preferably, the mammal is a human.

In accordance with the present invention, as described above or as discussed in the Examples below, there can be employed conventional clinical, chemical, cellular, histochemical, biochemical, molecular biology, microbiology and recombinant DNA techniques which are known to those of skill in the art. Such techniques are explained fully in the literature. See for example, Sambrook et al., 1989 Molecular Cloning - a Laboratory Manual, Cold Spring Harbor Press; Glover, (1985) DNA Cloning: a Practical Approach; Gait, (1984) Oligonucleotide Synthesis; Harlow et al., 1988 Antibodies - a Laboratory Manual, Cold Spring Harbor Press; Roe et al., 1996 DNA Isolation and Sequencing: Essential Techniques, John Wiley; and Ausubel et al., 1995 Current Protocols in Molecular Biology, Greene Publishing.

The invention is now described with reference to the following Examples. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, are provided for the purpose of illustration only and specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. Therefore, the examples should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Examples Materials and Methods

Culturing and storage of cells.

Entamoeba histolytica trophozoites (HM-I :IMSS) were grown axenically in TYI- S-33 (Trypticase-yeast extract iron serum) medium supplemented with 100 U of penicillin/ml and 100 μg of streptomycin sulfate/ml at 37° C [25]. Trophozoites were harvested during log-phase growth by incubation on ice for 10 min, centrifugation at 200 x g and 4° C for 5 minutes, and resuspension in either HEPES buffer or medium 199 (Gibco BRL, Grand Island, N.Y.) supplemented with 5.7 mM cysteine, 25 mM HEPES, and 0.5% bovine serum albumin at pH 6.8, (M199S) [26]. Human blood type B Rh+ was collected, heparinized and sedimented by centrifugation (1,000 x g; 4° C; 10 minutes) through Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, N.J.) to separate erythrocytes from other blood constituents. Pelleted erythrocytes were washed twice in HEPES buffer (1OmM hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.2, 140 mM NaCl and 0.1% bovine serum albumin with or without 2.5 mM CaCl₂), and resuspended at 1 x 10⁷ cells per ml in HEPES buffer and stored for up to 48 hours [27, 28]. Calcium-treated erythrocytes were prepared by incubation in HEPES buffer supplemented with 2.5 mM CaCl₂ at 37° C for 48 hours.

Preparation of amebic phagosomes using magnetic beads. Adherent amebae in log phase growth were washed three times with warm PBS and carboxylated 2.7 μm magnetic beads (M -270 Dynabeads, Dynal, Inc.) (2 x 10⁸ beads/ml in medium 199 (Gibco BRL) supplemented with 5.7 mM cysteine, 25 mM HEPES, and 0.5% BSA at pH 6.8 (M 199s)) were added to the cultures. Phagocytosis was then initiated by centrifugation (200 g, 5 min, rm. temp.) of the beads onto amebae using a plate spinner. After incubation at 37⁰C for 0, 5, or 10 min, uningested beads were removed by washing 3x with warm PBS followed immediately by addition of cold PBS and incubation on ice for 5 min to harvest the amebae. For longer time points, beads were removed after incubation for 10 min and a 'chase' incubation at 37⁰C was performed. Amebae were then resuspended in cold PBS supplemented with a protease inhibitor cocktail and mechanically disrupted using a Dounce homogenizer. Douncing was continued until approximately 90% of amebae were lysed, leaving the nuclei intact. Phagosomes containing ingested magnetic beads were isolated using a magnetic column, washed three times with PBS, and de-lipidated by methanol: chloroform precipitation in preparation for peptide sequencing [29]. Peptide Sequencing and analysis

The sample was dissolved in 10-20 μl of 1% SDS and then slowly diluted to a final concentration of 0.1% SDS with 10OmM ammonium bicarbonate pH=8.0. The sample was reduced with 10OmM DTT for 30 minutes at room temperature and alkylated with 50OmM iodoacetamide for 30 minutes at room temperature (RT) before addition of 1 μg of modified trypsin (Promega, Madison, WI) for 24 hours at RT. A second 1 μg of trypsin was added for an additional 24 hours at RT. The sample was acidified with acetic acid to 5% by volume. The resulting digest was desalted on a C18 column (20μm particle size - 10cm x 150μm id) and then SDS removed by strong cation ion exchange (lOμm particle size - 10cm x 150μm id). 25% of the sample was injected into the mass spectrometer. The LC-MS system consisted of a Finnigan LCQ ion trap mass spectrometer system with a Protana nanospray ion source interfaced to a self-packed 8 cm x 75 um id Phenomenex Jupiter lOμm C18 reversed-phase capillary column. The peptides were eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5μL per minute over 2 hours. The nanospray ion source was operated at 2.8 kV. The digest was analyzed using the double play capability of the instrument acquiring a full scan mass spectrum to determine peptide molecular weights and four product ion spectra to determine amino acid sequence in sequential scans. The data were analyzed by database searching using the Sequest search algorithm against the non- redundant database from NCBI and against the E. histolytica ORFs generated from the TIGR sequenced genome database. Search results were analyzed using minimum cutoffs (Xcorr>1.5 for +1, >2.0 for +2 and >2.5 for +3). Any proteins of interest were confirmed by manual validation of the spectra. Production of anti-EEAKl rabbit serum

The peptide EIQKQNPISTSLKISKISSD (SEQ ID NO:7), (amino acids 130-150 of EEAKl) was synthesized, conjugated to KLH and used to immunize New Zealand White Rabbits (Covance, Princeton, NJ). Resultant serum was protein G purified using packed protein G columns (Pierce, Rockford, IL) and affinity purified against bound peptide. The resultant serum was dialyzed against PBS and stored at -20⁰C until use. Fluorescent labeling and antibody pre-incubation.

Prior to calcium treatment, erythrocytes were fluorescently labeled by incubation at 37°C for 20 to 25 minutes in phosphate -buffered saline (PBS) containing 5 μM 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR). Unbound dye was quenched by incubation with an excess of fetal bovine serum at 37°C for 20 min, and the cells were washed twice more with M199s medium before use. Where indicated, erythrocytes were washed once in M199S and re-suspended at 10⁶ cells/ml and incubated with (antibody concentration) per 25 minutes at 4°C. The cells were then washed twice in M199S before they were added to amebae [30]. Immunoprecipitation and Western Blotting

Soluble proteins were extracted from ameba by harvesting 5 x 10⁷ trophozoites expressing FLAG epitope-tagged EEAKl by incubation on ice for 10 minutes, followed by centrifugation (200 x g at 4°C for 5 minutes). The amebae were lysed in Lysis Buffer (150 mM NaCl, 50 mM Tris-HCl, and 1% NP-40, complemented with protease inhibitor cocktail I (Sigma, St. Louis, MO) per the manufacturer's directions). The amebic lysate was incubated with anti-FLAG-M2 affinity gel (Sigma, St. Louis, MO) for 30 minutes at 4°C. Resin was washed once in lysis buffer and twice in PBS pH 7.4, then boiled in SDS-PAGE loading buffer. All samples were then separated on 8-10% polyacrylamide gels and subjected to immunob lotting by standard techniques [31] with either 5 μg/ml anti-EEAKl rabbit serum or pre -immune serum. Confocal microscopy

E. histolytica trophozoites (1 x 10⁶) were bound to glass coverslips in a 24-well plate for 30 minutes at 37°C in TYI-S-33 medium. Adherent amebae were washed twice in phosphate buffered saline (PBS) and fixed in 3% paraformaldehyde for 30 minutes at room temperature. Where indicated 10⁷ CFSE-labeled erythrocytes were incubated for 10 minutes with washed ameba in M199S at 37°C, prior to formaldehyde fixation. Next, amebae were solubilized in 0.2% Triton X 100 in PBS for 1 minute. Nonspecific binding was blocked by incubation with 20% goat serum and 5% bovine serum albumin (Sigma, St. Louis, MO) in PBS for 1 h at 37°C. Detection of kinases was performed by incubation with protein A purified anti-EEAKl rabbit polyclonal antibody (anti-EEAKl) diluted to 200 μg/ml and incubated with fixed cells for 1 hour at 37°C. Detection of the Gal/GalNAc adherence lectin was detected by addition of anti-lectin rabbit polyclonal antibody (shiro) diluted to 6.0 μg/ml. Two quick washes were performed before Cy3- conjugated goat anti-rabbit secondary antibodies (Jackson Laboratories, Bar Harbor, ME) were added at a 1 : 160 dilution for 1 hour at 37°C. The coverslips were washed twice more and mounted with Gel/Mount (Biomeda, Foster City, CA) and sealed to slides. Confocal images were visualized using a Zeiss LSM 510 laser scanning microscope. Construct assembly

Cloning of EEAKl cterm, for expression of 6XHIS tagged protein: the kinase containing region of EEAKl was PCR amplified with the primers: CAATTTAGAGAAGGAATTCCT (5' primer) (SEQ ID NO:8) and TCACATTAATTGAAGATGTTTTAAAACAACA (3' primer) (SEQ ID NO:9). This 1000 bp fragment was then cloned into TOPO NT/T7 (Invitrogen, Carlsbad, California), in frame with an amino-terminal 6X HIS tag.

Cloning of U6 driven RNAi hairpin constructs for EEAKl knockdown (Figure 2- 1): short hairpin RNAs were expressed from by the E. histolytica RNA polymerase III, U6 promoter (GenBank accession number U43841) [32]. This approach utilized a 29- base base pair stem, with a 9 bp loop. Four sequences were targeted and construction of the vector was done in 2 parts. Three experimental constructs were made (325, 2273, and 3552) each corresponding to the corresponding beginning nucleotide sequence as well as a control with the same nucleotide content as 3552, but in random order (scrambled). These constructs were made using two rounds of PCR, each using the same 5' oligo for each round, (CTACTGAAGCTTGTTTTTATGAAAAAGTGT ATTTGC) (SEQ ID

NO: 10) but different 3' oligos as listed: 325 first round

TCTCTTGAAAGAATCTTATCAACTGATAAGTCTCCAGGGCCCAATTTTATTTTT CTTTTTATCC (SEQ ID NO : 11 ), second round

GAATGCGGCCGCAAAAAATGGAGACTTATCAGTTGATAAGATTCTTCTCTTGA

A (SEQ ID NO: 12); 2273 first round

TCTCTTGAAACACTCATATTGTTCTAAATAATACCCTTGGGCCCAATTTTATTT

TTCTTTTTATCC (SEQ ID NO: 13), second round GAATGCGGCCGCAAAAAAGGGTATTATTTAGAACAATATGAGTGTTCTCTTG

AA (SEQ ID NO: 14); and 3552 first round

TCTCTTGAAGCCATATAAATTGGACTTCCTATTCCCTTGGGCCCAATTTTATTT

TTCTTTTTATCC (SEQ ID NO: 15), second round

GAATGCGGCCGCAAAAAAGGGAATAGGAAGTCCAATTTATATGGCTCTCTTG AA (SEQ ID NO: 16). PCR products were cloned into pBluescript II, sequence-verified, then subcloned into the amebic expression vector pGIR310.

Cloning of carboxy-FLAG epitope tagged EEAKl (EEAKl 1279) and truncated

EEAKl at residue 932 (EEAKIΛ932), was performed by PCR of genomic DNA with the same forward oligo, which added a Bglϊl site to the 5' region of the gene (AAAGATCTTCAATGAGCATTATTCCATTTCAATGGTGCTAT) (SEQ ID NO: 17).

Two different 3' oligos terminating at nucleotide 3834 to create EEAKl 1279 (sequence:

AACTCGAGTTAGCCCTTGTCGTCGTCGTCCTTGTAGTCCATTAATTGAAGATG

TTTTAAAACAACATCAATGGGTAT) (SEQ ID NO: 18) or nucleotide 2796 to create

EEAK1Λ932 (sequence: AACTCGAGTTAGCCCTTGTCGTCGTCGTCCTTGTAGTCAATTTTTAATGGATTT

TTCCTTATGCTTGCTAT) (SEQ ID NO: 19). Each 3' oligo also contained the FLAG epitope tag and an Xhol site for detection and cloning respectively. PCR products were cloned into vector pEhEX (a gift from T. Nozaki) at B gill and Xhol sites (Figure 2-2).

Vector pEhEx without an insert was used for the empty vector controls. Phagocytosis assays.

Phagocytosis was assayed by microscopy as previously described [12].

Phagocytosis positive amebae were defined by microscopy as ameba containing one or more ingested erythrocytes. Both the numbers of positive amebae, as well as the numbers of intact, engulfed, erythrocytes were counted. These results were expressed as a phagocytic index, which was the percentage of amebic trophozoites that had engulfed erythrocytes multiplied by the average number of erythrocytes ingested per ameba [33]. Anti-EEAKl blocking experiments were performed using this same approach with the addition of a 20 minute incubation on ice with the antibody, control antibody, or antibody and the peptide-antigen. Excess antibody and peptide were washed away in two rinses in M 199s, prior to incubation with erythrocytes.

Flow Cytometry

Ameba cell surface changes were assessed by anti-lectin staining (10 μg/ml anti- lectin, Rabbit IgG) of paraformaldehyde fixed trophozoites for 1 hour at RT and analyzed by using a FACScan cytometer and CellQuest 3.3 software (Becton Dickinson, Franklin Lakes, N.J.).

Animal Models

CBA mice were challenged with 2 x 10⁶ trophozoites by cecal inoculation, by previously described methods [34]. Mice were sacrificed 72-96 hours following challenge and the cecca removed for culture in TYI-S-33 medium and paraffin embedding for histological scoring as previously described [35]. Gerbils were challenged with 5 x 10⁵ trophozoites by direct hepatic inoculation by previously described methods [36]. Gerbils were sacrificed 5-8 d following challenge and liver abscess weights were determined and cultures started in TYI-S-33.

Results

Sequencing of phagosomal preparations revealed proteins with conserved functions in endocvtosis. Ingested carboxylated magnetic beads within intact phagosomes were separated from amebic lysate and subjected to delipidation and mass spectrometry sequencing. Phagosome preparations were performed at 0, 5, 10, and >30 minutes following centrifugation of the beads into contact with trophozoites. To eliminate proteins from the medium and lysed ameba, which interacted with the carboxylated beads, beads were also centrifuged through amebic lysate and, delipidated and sequenced. Resultant proteins from this negative control were subtracted from the entire dataset. Using this approach, we identified 217 proteins with known homologues, and 100 novel amebic proteins. The proteome assembled revealed dynamic changes in the phagosome over time (Figure 1). Many conserved proteins consistent with the endocytic process were found. These included small GTPases, (Rab7, Rab9, Rabl 1, Racl and Rho), surface lectins (IgI), and endoplasmic reticulum proteins (calreticulin, and Seel), which appeared throughout maturation of the phagosomes (Figure 1).

Analysis of this data was done first by subtraction of all proteins which were found in the lysed ameba preparation. This was a stringent analysis to remove charge interacting proteins, but was necessary because of the amount of DNA/RNA binding proteins that contaminated all preparations. Following subtraction of the control the dataset was sorted for membrane proteins and a putative receptor kinase was discovered at early time points (5 and 10 minutes) but this protein did not appear at 0, or >30 minutes (EEAKl, highlighted in red in Figure 1). EEAKl was predicted by bioinformatics to be 146 kDa, with a 21 amino acid signal peptide sequence, an ectodomain containing 25 CXXC repeats, a 22 amino acid membrane spanning domain and an intracellular domain with the key catalytic residues of a kinase (Figure 2a). Rabbit anti-serum against a peptide specific for the ectodomain of EEAKl (EIQKQNPISTSLKISKISSD; SEQ ID NO:7) revealed a single band at -140 kDa. This band disappeared when antibody was pre-absorbed against the antigen peptide in amounts ranging from 10 μM to 50OnM, and was not evident in rabbit matched pre- immune serum (Figure 2b). Anti-EEAKl sera stained the surface of both permeabilized and non-permeabilized cells, whereas pre-immune serum left no staining (Figure 2c). Anti-EEAKl pre-incubation of ameba blocks ingestion of human erythrocytes. Amebae were pre-incubated for 20 minutes on ice in medium containing 50 μg/ml of anti-EEAKl serum. Pre-incubation led to a reduction in ingestion of healthy (5.3% ± 2.5% vs. 22.0% ± 3.0%, p< 0.0018) and calcium-treated erythrocytes (15.0% ± 4.0% vs. 30.7 ± 1.5%, p< 0.0032) compared with pre-immune incubation in M199S medium. Inhibition was also observed for the ingestion of calcium treated erythrocytes in the presence of 55 mM galactose, by ameba pre-incubated with 50 μg/ml of anti-EEAKl serum compared with pre-immune serum (2.7% ± 2.1% vs. 12.7 ± 5.7, p< 0.0459). Anti- IgI serum had no affect on ingestion of either healthy or calcium treated erythrocytes. The effect of anti-EEAKl serum could be reversed by pre-absorbing the serum against the antigen peptide at a concentration of 25 μM (Figure 3 a). EEAKl transiently localizes to the interface of ameba with erythrocytes. In order to further understand the role EEAKl played in ingestion, E. histolytica trophozoites were stained in the presence of fluorescently labeled (CFSE) erythrocytes (Figure 3b). EEAKl staining on non-permeabilized trophozoites appeared to enrich at the site of contact with CFSE labeled erythrocytes (arrows, Figure 3b). This staining could explain the ability of this anti-serum to interfere with ingestion, by interfering with the binding of the ameba to the erythrocyte either by specific competition or creating steric hindrance.

EEAKl protein levels were decreased following expression of interfering RNA which led to a reduction in ervthrophagocvtosis. In order to further address EEAKl 's role in phagocytosis, RNA interference (RNAi) was used to knock down its protein expression. Short interfering RNAs were expressed using the U6 promoter as hairpin loops in E. histolytica with identical sequence to three regions of EEAKl (Figure 4a). Knockdown of EEAKl was seen upon expression of hairpin loops identical to two regions of EEAKl, nucleotides 2273-2302, and 3552-3581 (Figure 4b). The RNAi sequences were SEQ ID NOs :5 and 6, respectively. Concomitant with knockdown of EEAKl, amebae were demonstrated to have a reduction in erythrophagocytosis. In the absence of galactose, ingestion of calcium-treated erythrocytes was statistically significantly reduced by 48.3% by the 3552-3581 corresponding hairpin (RNAi SEQ ID NO:6) (58.0% ± 9.8% vs. 30.0% ± 2.6%, p< 0.0176) and 35.6% by the 2273-2302 hairpin (RNAi SEQ ID NO:5) (58.0% ± 9.8% vs. 37.3% ± 6.4%, p< 0.0383) compared with the scrambled control. In the presence of 55 mM galactose, both constructs (2273 and 3552) reduced ingestion of calcium treated erythrocytes by more than 65% (22.0% ± 6.6% (scrambled control) vs. 5.7% ± 2.5% (3552) or 7.0% ± 2.6% (2273), p< 0.02). The amino most construct (325-354; RNAi SEQ ID NO:4) neither reduced EEAKl protein levels nor had a significant effect on ingestion of calcium treated erythrocytes.

Expression of a truncated form of EEAKl at residue 932 (EEAKl ΛQ^?; SEQ ID NO:2), caused a reduction in host cell ingestion. In order to produce a stable, but growth competent ameba, three constructs, EEAKl 1279 (full length carboxy-FLAG epitope- tagged EEAKl ; encoding SEQ ID NO: 1), EEAKl Λ932 (a truncated, carboxy-FLAG epitope-tagged EEAKl encoding SEQ ID NO:2), and empty vector control (figure 5) were transfected into HM-I :IMSS trophozoites. Immunoprecipitations were performed using anti-FLAG binding resin, and Western blots revealed that both EEAKl 1279, and EEAK1Λ932 were being expressed (Figure 5a). The truncated form of EEAKIΛ932 was co- immunoprecipitated with native EEAKl , as indicated by western blots of the immunoprecipitate indicating interaction of the truncated and wild type proteins. These amebic transfectants were assayed for phagocytosis of calcium-treated erythrocytes, and truncation of EEAKl caused impairment in ingestion (Figure 5b). The expression of a truncated form of EEAKl reduced ingestion by 67% in M199S medium (11.33 ± 2.52 vs. 34.33 ± 4.93, p < 0.002) and by 81% in the presence of 55 mM D-galactose (3.33 ± 2.08 vs. 17.67 ± 3.06, p < 0.003) compared with the empty vector control. Expression of the EEAKl 1279 had no statistical impact on erythrophagocytosis. Expression of EEAKIΛ932 did not amebic surface lectin, as demonstrated by staining with anti-Gal/GalNAc lectin heavy subunit antibodies (Figure 5c). Hence, no change was seen in surface expression of the principle adherence molecule of E. histolytica.

the intestinal model of amebiasis, but has no affect on formation of liver abscesses. The infection rate of HM- 1 :IMSS strain ameba transfected with EEAKl^₂ vs. empty vector control (Table 1) was significantly decreased in the murine model of amebiasis (2/24 EEAK1_Λ932 VS. 9/24 empty vector, p < 0.0157). However, no reduction was seen in the ability of these cells to cause liver abscesses in gerbils (Table 2). Both the percentage of liver that comprises abscess, as well as the number of gerbils infected by EEAKl^₂ was statistically similar to empty vector controls.

Table 1. Expression of an EEAKl truncation led to a reduction in E. histolytica infection in the intestinal model of amebiasis. EEAKl Λ932 (protein having the sequence of SEQ ID NO:2) and empty vector control ameba were introduced via cecal inoculation. Mice were sacrificed 72 to 96 hours later, and infection was assayed by culture, cecal antigen ELISA, and the cecum was sectioned for histological slides. Data are reported as means. P values were determined by a two-tailed t test (* indicates p < 0.0157).

Infection Cecal Average

Rate Culture Antigen Histology Score

(Gross) Positive Positive Amoeba Inflammation

EEAKl Δ932 2/24* 2/24* 2/24 1/24* 1.125

Control 9/24 9/24 8/24 8/24 2.125

Table 2. Expression of an EEAKl truncation was not sufficient to prevent liver abscess formation in gerbils. EEAKl Λ932 (protein having the sequence of SEQ ID NO:2) and empty vector control ameba were introduced into gerbils through direct hepatic inoculation. Animals were sacrificed 5 to 7 days later and both the liver weight and the weight of the abscesses were measured.

Culture Average Average % Positive Abscess Size of Liver

EEAK1_A932 7/12 0.07 g 1.51% Control 3/6 0.03 g 2.68%

Discussion

This work identified EEAKl as a phagosome associated protein. Furthermore, manipulation of EEAKl protein levels or oligomerization interfered with erythrophagocytosis by Entamoeba histolytica. Expression of a dominant negative truncation of EEAKl (EEAKIΛ932; protein having the sequence of SEQ ID NO:2) reduces virulence in the intestinal mouse model of amebiasis, revealing some necessity for EEAKl function in disease.

Although multiple research groups have performed proteomic analysis such as this, each group has identified new molecules of interest. The first analysis of this type in E. histolytica, published by Okada et al. , revealed many of the GTP binding proteins that are important for maturation of phagosomes [22]. However, this screen did not reveal any endoplasmic reticulum resident proteins which the presence of these proteins have be controversially identified in metazoan systems [18, 21]. More recent work by Marion et al. identified ER resident proteins in E. histolytica phagosomes such as calreticulin as well as significant involvement by myosin IB, actin and actin accessory proteins [23]. Later this group identified a small number of surface proteins which were suggested as possible receptors [37]. The screen reported here was focused on early time points following ingestion with the goal of identifying new surface molecules with roles in this process. In addition proteins were identified such as amoebapore A and B, which were known to be involved in endocytosis but had remained absent in other screens. The most obvious conclusion from this collection of proteomics is that not one effort has taken its screen to saturation. Incompleteness of these screens may explain the differences that have been published between clinical isolates of Entamoeba histolytica [38].

One major flaw with this screen was the dramatic amount of contaminating proteins. Greater than 25% of all our protein sequence was made up of histones and ribosomal subunit proteins. One explanation for this is that the strong negative charge on the beads attracted these positively charged proteins. Unfortunately, beads concealed in uncompromised phagosomes should not have come in contact with these proteins. This made it necessary to perform the lysed ameba control. Although subtraction of this control from the data set reduced the amount of noise in the screen, it clearly reduced the number of true positives as well. One case in point is that LgI was found in the final data (following subtraction) but not HgI, which is both covalently attached to IgI, as well as already confirmed to be a phagosomal constituent. This issue could best have been addressed by more repetition of each time point, as well as the control and zero, such that there was better coverage of all proteins. Using percent coverage instead of the binary present/absent would provide a much better analysis of this data.

Manipulation of EEAKl by binding the ectodomain with antibody (75% reduction of ingestion of calcium treated erythrocytes in M199S with 55 mM D-galactose), reducing the protein levels with interfering RNA (65% reduction in M199S with 55 mM D-galactose), or by expressing a truncated protein (81%) all produced a similar reduction of erythrophagocytosis in vitro. The mechanism of this interference is not entirely clear. Given that anti-EEAKl serum co-localizes with erythrocytes in contact with ameba, we assume that antibody against this protein blocks uptake of erythrocytes by interfering with receptor function, but it is very possible that this could be blocking interaction with a different molecule, by steric hindrance. Given that native EEAKl was seen by western of a FLAG immunoprecipitation of EEAKl Λ932, it is reasonable to think that this disruption is because of formation of incompetent dimers. Clearly all three approaches support a role for this protein in host cell ingestion by Entamoeba histolytica.

Although no kinase activity has been demonstrated, we theorize that EEAKl may act in a model similar to the Mer family of tyrosine kinases in metazoans [39-42]. Mer tyrosine kinase interacts with the bridging molecule GAS6 to recognize PS on apoptotic cells. This causes MER signaling, leading to activation of FAK, which then associates with α_vβ3 integrins [42]. α_vβ3 integrins provide the platform for pl30cas, CrkII, and dockl80 to activate Racl, which leads to actin polymerization and ingestion of the particle [40]. Our model proposes that the Gal/GalNAc lectin may provide the platform for which actin polymerization is signaled. FAK has been shown to be activated in E. histolytica during contact with fibronectin [43, 44], although no interaction has been demonstrated between FAK and the Gal/GalNAc lectin. All three subunits of the galactose inhibitable lectin have been found within endosomes, and there is evidence that expression of IgI truncation mutants cause deficiencies in host cell ingestion [15].

Virulence of E. histolytica has been long associated with the parasite's ability to ingest host cells. This work suggests that this may be more important in intestinal disease than in liver abscesses. Ameba expressing EEAKIΛ932 had reduced ability to infect the intestine of susceptible CBA mice. However, this strain was not impaired at causing gerbil liver abscesses. This disconnect has been seen with other mutant strains of E. histolytica. Cysteine protease 2 over-expression, was found to reduce in vitro monolayer destruction but had no effect on liver abscess formation [45], and amoebapore A silencing led to inability to cause liver abscesses although these parasites still caused tissue damage in a colonic xenograft model of amebiasis [46]. These results suggest that expression of EEAKl Λ932 does not simply produce an impaired ameba, but that its failure to infect mice is more likely due to its deficiency in host cell phagocytosis. Little is known why this parasite ingests host cells. However, erythrophagocytosis is a hallmark of E. histolytica infection. One could envision that this behavior provides an advantage during infection by clearing dying and dead cells and thereby reducing the infiltration of inflammatory cells and release of toxic cellular content. EEAKl may have a pivotal role in allowing this parasite to persist longer in the host. Interfering with this pathway may produce a more robust immune response to E. histolytica and clearance of infection.

Other methods which were used but not described herein are well known and within the competence of one of ordinary skill in the art of chemistry, cell biology and molecular biology, microbiology, and clinical medicine.

The invention should not be construed to be limited solely to the assays and methods described herein, but should be construed to include other methods and assays as well. One of skill in the art will know that other assays and methods are available to perform the procedures described herein. Headings are included herein for reference and to aid in locating certain sections.

These headings are not intended to limit the scope of the concepts described therein under, and these concepts may have applicability in other sections throughout the entire specification.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by the previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Accordingly, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

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Claims

CLAIMSWhat is claimed is:

1. A method of inhibiting or preventing amebiasis, said method comprising administering to a subject a pharmaceutical composition comprising an effective amount of at least one inhibitor of EEAKl function or EEAKl levels, a pharmaceutically-acceptable carrier, and optionally an antibiotic.

2. The method of claim 1, wherein said method inhibits or prevents amebic colitis.

3. The method of claim 1, wherein said method inhibits or prevents host cell phagocytosis by E. histolytica.

4. The method of claim 3, wherein said phagocytosis is erythrophagocytosis.

5. The method of claim 1, wherein said method inhibits virulence of E. histolytica.

6. The method of claim 1 , wherein said method inhibits or prevents infection by E. histolytica.

7. The method of claim 1, wherein said inhibitor of EEAKl function or levels is selected from the group consisting of an EEAKl protein comprising SEQ ID NO:1 or a fragment thereof, an antibody directed against an EEAKl protein comprising SEQ ID NO:1 or a fragment of SEQ ID NO:1, an RNAi complementary to an EEAKl nucleic acid sequence, an expression vector encoding an RNAi complementary to an EEAKl nucleic acid sequence, and an isolated nucleic acid comprising a nucleic acid sequence encoding a protein having SEQ ID NO: 1 or encoding a fragment of SEQ ID NO: 1.

8. The method of claim 7, wherein said RNAi reduces EEAKl protein levels.

9. The method of claim 7, wherein said RNAi has a sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6.

10. The method of claim 7, wherein said nucleic acid sequence encoding the protein having SEQ ID NO: 1 or a fragment thereof, has the sequence of SEQ ID NO:20 or a fragment of SEQ ID NO:20.

11. The method of claim 7, wherein said fragment of SEQ ID NO: 1 has the sequence of SEQ ID NO:2 or SEQ ID NO:7.

12. The method of claim 7, wherein said antibody is directed against SEQ ID NO:1 or a fragment thereof.

13. The method of claim 12, wherein said antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, and a humanized antibody.

14. The method of claim 12, wherein said antibody is directed against SEQ ID NO:7.

15. The method of claim 7, wherein said nucleic acid sequence has the sequence of SEQ ID NO:20 or a fragment thereof.

16. A method of inducing an immune response against E. histolytica, said method comprising administering to a subject a pharmaceutical composition comprising a protein having the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1, or an isolated nucleic acid comprising a nucleic acid sequence encoding SEQ ID NO:1 or a fragment of SEQ ID NO:1, a pharmaceutically-acceptable carrier, and optionally an adjuvant.

17. The method of claim 16, wherein said fragment has the sequence of SEQ ID NO:2 or SEQ ID NO:7.

18. The method of claim 16, wherein said nucleic sequence encoding SEQ ID NO:1 or a fragment of SEQ ID NO: 1 is SEQ ID NO:20 or a fragment of SEQ ID NO:20.

19. A method of inhibiting or reducing phagocytosis by E. histolytica, said method comprising contacting E. histolytica with a composition comprising at least one of an EEAKl protein comprising SEQ ID NO:1 or a fragment thereof, an antibody directed against an EEAKl protein comprising SEQ ID NO:1 or a fragment of SEQ ID NO:1, an RNAi complementary to an EEAKl nucleic acid sequence, an expression vector encoding an RNAi complementary to an EEAKl nucleic acid sequence, and an isolated nucleic acid comprising a nucleic acid sequence encoding a protein having SEQ ID NO: 1 or encoding a fragment of SEQ ID NO: 1.

20. The method of claim 19, wherein said inhibitor is an antibody directed against EEAKl.

21. The method of claim 20, wherein said antibody is directed against a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:7.

22. A method of reducing EEAKl protein levels in E. histolytica, said method comprising contacting E. histolytica with a composition comprising an RNAi complementary to an EEAKl nucleic acid sequence or an expression vector encoding an RNAi complementary to an EEAKl nucleic acid sequence.

23. The method of claim 22, wherein said EEAKl nucleic acid sequence comprises the sequence of SEQ ID NO:20 or a fragment thereof.

24. The method of claim 22, wherein said RNAi is a short hairpin RNA

25. The method of claim 22, wherein said RNAi comprises a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO:6.