WO2007020633A2 - Avirulent trophozoites of entamoeba histolytica for vaccination against amoebiasis - Google Patents

Abstract

The present invention relates to a novel pharmaceutical composition and methods of production thereof for the prevention of amoebiasis and, more particularly, to a live vaccine in which more than one Entamoeba histolytica virulence factor gene has been silenced.

Description

AVIRULENT TROPHOZOITES OF ENTAMOEBA HISTOLYTICA FOR VACCINATION AGAINST AMOEBIASIS

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to a novel pharmaceutical composition and methods of production thereof for the prevention of amoebiasis and, more particularly, to a live vaccine in which more than one Entamoeba histolytica virulence factor gene has been silenced.

Amoebiasis is the third most common cause of death (after Schistosomiasis and Malaria) from parasitic infections. According to WHO estimates there are over

50 million cases of infection per year accompanied by approximately 100,000 deaths.

The disease is endemic in communities with poor sanitary conditions where drinking water is polluted with sewage.

Entamoeba histolytica (E. histolytica) is the protozoan parasite which is the causative agent of amoebiasis. E. histolytica exhibits a typical fecal-oral life cycle consisting of infectious cysts passed in the feces and trophozoites which replicate within the large intestine.

The infection is acquired through the ingestion of cysts which pass through the stomach and excyst in the lower portion of the small intestine. Excystation involves a disruption of the cyst wall and the quadranucleated amoebas emerge through the opening. The amoebas undergo another round of nuclear division followed by three successive rounds of cytokinesis (i.e. cell division) to produce eight small uninucleated trophozoites. These trophozoites colonize the large intestine, especially the cecal and sigmoidorectal regions, where they feed on bacteria and cellular debris and undergo repeated rounds of binary fission. As an alternative to asexual replication trophozoites can also encyst. Cyst maturation involves two rounds of nuclear replication without cell division and cysts with 1-4 nuclei are found in feces.

Cysts are immediately infective upon excretion with the feces and are viable for weeks-to-months depending on environmental conditions. E. histolytica is a facultative pathogen that exhibits a wide range of virulence.

It frequently lives commensally within the large intestine with no overt clinical manifestations. However, trophozoites can invade the colonic epithelium and produce ulcers and dysentery. This invasive disease can become progressively worse and may ultimately lead to extraintestinal amoebiasis. The non-invasive disease is most often asymptomatic, but may cause diarrhea or other gastro-intestinal symptoms such as abdominal pain or cramps. The noninvasive infection can persist or progress to an invasive disease in which trophozoites penetrate the intestinal mucosa and kill the epithelial cells. The early lesion is a small area of necrosis, or ulcer, characterized by raised edges and virtually no inflammation between lesions. Trophozoites may then spread laterally in the submucosa (beneath the epithelium) killing host cells as they progress. Trophozoites are most numerous at the boundary between the healthy tissue and the necrotic tissue. Hematophagous trophozoites are sometimes found in the dysenteric feces. Cyst production decreases during the invasive stage of the infection and are never found in the tissue lesions.

The ulcerative process may continue to expand laterally or downward. If large numbers of ulcers are present, they may coalesce, which could lead to a localized sloughing off of the intestinal wall. Ulcer expansion can also penetrate the serous layer and lead to perforation of the intestinal wall. This perforation can lead to a local abscess or a generalized peritonitis.

Amoebiasis can also progress to a systemic or extraintestinal infection. Dissemination from the primary intestinal lesion is predominantly via the blood stream, but can also occur by direct extension of the lesion. The liver is the most commonly affected organ and this is probably due to the direct transport of trophozoites from the large intestine to the liver via the hepatic portal vein. Hematogenous spread of trophozoites to other sites, such as the lungs or brain, is uncommon, but does occur. Pulmonary infections generally result from a direct extension of the hepatic lesion across the diaphragm and into the pleura and lungs. Cutaneous lesions formed as a result of hepatic or intestinal fistula can also occur, although they are extremely rare.

Invasion of intestinal mucosa by E. histolytica is an active process mediated by the parasite. Initially, trophozoites adhere to the mucus layer. Depletion of the mucus barrier allows the trophozoite to come in contact with epithelial cells. Epithelial cells are then killed in a contact dependent manner leading to a disruption of the intestinal mucosa. The trophozoites continue to kill host cells in the submucosa and further disrupt the tissue as they advance. Disruption of the intestinal wall or metastasis via the circulatory system is also possible. Adherence, cytotoxicity, and disruption of the tissues are important factors in the pathogenesis of E. histolytica. Parasite proteins (also termed virulence factors) which play a role in these processes include galactose-specific lectin molecules that mediate the adherence of the parasite to carbohydrate containing surface receptors on the target cell; cysteine proteinases which degrade mammalian matrix proteins and mucosal tissues including mucus and antibodies (slgA); and the small polypeptide family of amoebapores (AP). There are three amoebapore isoforms of which AP-A comprises approximately 70 %. Following their initial adherence, the amoeba trophozoites inject the amoebapores into mammalian target cell membranes, where they form pores or ion channels which depolarize the membrane causing cell death. Several drugs are available for the treatment of amoebiasis and the choice of drug(s) depends on the clinical stage of the infection. However, in many endemic areas, where the rates of re-infection are high and treatment is expensive, the standard practice is to only treat symptomatic cases. Metronidazole or tinidazole is recommended for all symptomatic infections. This treatment is usually followed by or combined with luminal antiamoebic drugs.

Total eradication of amoebiasis is believed to be feasible. As the disease is not vector-borne and the parasite naturally infects only humans, control of amoebiasis could be achieved solely by implementing adequate water management systems in the endemic areas and respecting good hygienic practices. However, as developing countries generally cannot economically afford sewage treatment and water purification facilities, and their inhabitants are exposed every day to food and water contaminated with E. histolytica cysts, there is a widely recognized need for, and it would be highly advantageous to have, an affordable method of prevention.

Vaccination against amoebiasis may be a viable alternative. Acquired immunity to invasive amoebiasis has been achieved repeatedly in rodent models of the disease. Protection was obtained by immunization with total amoebic antigen or some of its chromatographic fractions [Krupp, I.M., Am J Trop Med Hyg 1974; 23: 255-260; Vinayak V.K et a!., Trans R Soc Trop Med Hyg 1980; 74: 483-^187; Ghadirian E et a!., Am J Trop Med Hygl980; 29: 779-784] and laboratory animals experimentally infected with E. histolytica developed resistance to a challenge infection upon cure with metronidazole [Campbell D, Chadee K₅ J Infect Dis., 1997, 175: 1176-1183]. Recent epidemiological data obtained with a cohort of children in Bangladesh shows a correlation between presence of anti-amoebic antibodies present in the lumen of the gastro-intestinal tract and acquired resistance to infection [Haque R, Duggal P, AIi IM et al, J Infect Dis 2002; 186: 547-552]. This suggests that protective immunity can also occur in humans and that an amoebiasis vaccine is possible. These studies also indicate that a mucosal immune response against E. histolytica is protective against intestinal amoebic invasion. In recent years attempts have been made to prepare vaccines based on presentation of recombinant antigens. For example, a number of Gal-lectin-based DNA vaccines have been developed [Gaucher D. and Chadee K., Parasite Immunology, 2003, 25, 55-58]. Some advances have been made in animal models with recombinant bacteria which expresses antigens of Entamoeba histolytica [Lotter H. et al, Infection and Immunity, December 2004, p. 7318-7321, Vol. 72, No. 12]. Specifically, protection against invasive amoebiasis was achieved in the gerbil model for amebic liver abscess by oral immunization with live attenuated Yersinia enterocolitica expressing the Entamoeba histolytica galactose-inhibitable lectin that has been fused to the Yersinia outer protein E (YopE). It was shown that protection was dependent on the presence of the YopE translocation domain but independent of the antibody response to the ameba lectin. However, the length of time that these vaccines were capable of protection varied from pathogen to pathogen.

Additionally, the present authors irreversibly silenced the transcription of the amoebapore gene [Bracha, R., Y. Nuchamowitz, et al. (2003) Eukaryot. Cell 2: 295- 305] producing a strain of E. histolytica that is non-virulent in some animal models, although retained some virulent properties in other animal models such as the Hu- mice intestinal model [Zhang X., Zhang Z., (2004) Infect Immun 72, 678-83]. Preliminary studies also demonstrated that vaccination of hamsters with live silenced amoeba afforded considerable, although not total immuno-protection [Bujanover S. et al, 2003, Int J Parasitol. 33(14): 1655-63]. The partial virulence of this strain of E. histolytica does not favor its use for immunization purposes.

Since none of the above mentioned vaccines conferred complete protection against challenge or undergone clinical trials in humans, there still remains a need for a safe and effective vaccine for protection against amoebiasis in humans. SUMMARY OF THE INVENTION

It is an object of the present invention to provide viable avirulent amoeba of E. histolytica.

It is another object of the present invention to provide pharmaceutical compositions containing these agents.

It is another object of the present invention to provide therapeutic methods using the agents and/or pharmaceutical compositions.

According to one aspect of the present invention there is provided a viable, avirulent amoeba of E. histolytica. According to further features in preferred embodiments of the invention described below, the viable, avirulent amoeba of E. histolytica are genetically modified.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising the genetically modified viable, avirulent amoeba of E. histolytica.

According to yet a further aspect of the present invention there is provided a method of generating an anti-amoebiasis vaccine, the method comprising down- regulating gene expression of at least two genes of a genome of E. histolytica, each being down regulated by independent exogenous genetic means, thereby generating the anti-amoebiasis vaccine.

According to still a further aspect of the present invention there is provided a method of preventing amoebiasis, comprising administering to a subject in need thereof an immunogenically effective amount of the pharmaceutical composition of claim 2, thereby preventing amoebiasis. According to still a further aspect of the present invention there is provided a method of down-regulating gene expression in a genome of an amoeba of E. histolytica, comprising introducing into the amoeba of E. histolytica a first nucleic acid construct comprising a regulatory sequence as set forth in SEQ ID NO: 1 or a functional equivalent thereof; and subsequently introducing into the amoeba of E. histolytica a second nucleic acid construct comprising the regulatory sequence and a polynucleotide encoding a polypeptide of E. histolytica, operably linked to the regulatory sequence, thereby down-regulating gene expression in a genome of an amoeba of E. histolytica. According to still a further aspect of the present invention there is provided a nucleic acid construct system comprising a first nucleic acid construct comprising a regulatory sequence as set forth in SEQ ID NO: 1 or a functional equivalent thereof; and a second nucleic acid construct comprising the regulatory sequence and a polynucleotide encoding a polypeptide of E. histolytica other than a amoebapore polypeptide, operably linked to the regulatory sequence.

According to still further features in the described preferred embodiments the genetic modification comprises down-regulation of at least two genes, each being down regulated by independent exogenous genetic means. According to still further features in the described preferred embodiments the genetic modification comprises down-regulation of at least three genes, each being down regulated by independent exogenous genetic means.

According to still further features in the described preferred embodiments at least one of the two down-regulated genes is epigenetically down-regulated. According to still further features in the described preferred embodiments the at least two down-regulated genes are virulent factor genes.

According to still further features in the described preferred embodiments the virulent factor genes are selected from the group consisting of an amoebapore gene, a galactose specific lectin gene and a cysteine proteinase gene. According to still further features in the described preferred embodiments the at least one virulent factor genes is ap-a.

According to still further features in the described preferred embodiments the ap-a is epigenetically silenced.

According to still further features in the described preferred embodiments the amoebapore gene is selected from the group consisting of ap-a, ap-b, ap-c and saposin-like.

According to still further features in the described prefeiτed embodiments the amoebapore gene is ap-a.

According to still further features in the described preferred embodiments the galactose specific lectin gene is selected from the group consisting of IgI- 1, lgl-2, IgI- 3, hgl-1, hgI-2, hgI-3, hgl-4 and hgl-5.

According to still further features in the described preferred embodiments the galactose specific lectin is IgI- 1. According to still further features in the described preferred embodiments the cysteine proteinase is selected from the group consisting of cysteine proteinase 1, cysteine proteinase 2 cysteine proteinase 3 cysteine proteinase 4 and cysteine proteinase 5, cysteine proteinase 6, cysteine proteinase 7, cysteine proteinase 8, cysteine proteinase 9, cysteine proteinase 10, cysteine proteinase 1 1, cysteine proteinase 12, cysteine proteinase 13, cysteine proteinase 14, cysteine proteinase 15, cysteine proteinase 16, cysteine proteinase 17, cysteine proteinase 18, cysteine proteinase 19 and cysteine proteinase 112.

According to still further features in the described preferred embodiments the cysteine proteinase is cysteine proteinase 5.

According to still further features in the described preferred embodiments the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

According to still further features in the described preferred embodiments the avirulent amoeba of E. histolytica comprises an epigenetic nucleic acid construct comprising a regulatory sequence, as set forth in SEQ ID NO: 1 or a functional equivalent thereof.

According to still further features in the described preferred embodiments the strain of the viable, avirulent amoeba of E. histolytica is selected from the group consisting of HM-UMSS, HU-21 :AMC, Rahman, HU1 :MUSC, HU-1:CDC, DKB, SAW 755CR, SAW 89 IR, SD 157, SD 184, HK-9, HB-301 :NIH, 200:NIH, HU- 2:MUSC, HU-I :CDC, H-302:NIH, H-303:NIH, H-458:CDC, HM-3:IMSS, HU22:AMC, HI- 1295: AIIMS and HB-301 :NIH.

According to still further features in the described preferred embodiments the strain of viable, avirulent amoeba of E. histolytica is HM-I.¹IMSS. According to still further features in the described preferred embodiments the viable, avirulent amoeba of E. histolytica is stably avirulent.

According to still further features in the described preferred embodiments the down-regulating gene expression of the at least two genes is effected by an agent selected from the group consisting of a regulatory sequence, a dsRNA, an antisense RNA and a ribozyme.

According to still further features in the described preferred embodiments the down-regulating gene expression of the at least two genes is effected sequentially. According to still further features in the described preferred embodiments the sequential down-regulation of the at least two genes is effected by introducing the epigenetic nucleic acid construct into a viable amoeba of E. histolytica.

According to still further features in the described preferred embodiments the method of down-regulating gene-expression further comprises removing the nucleic acid construct from the viable amoeba of E. histolytica.

According to still further features in the described preferred embodiments the polynucleotide encodes at least a portion of an ORF of said polypeptide.

The present invention successfully addresses the shortcomings of the presently known configurations by providing a method and pharmaceutical composition for protection against amoebiasis in humans.

Unless otherwise defined, 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 methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. AU publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the ait how the several forms of the invention may be embodied in practice. In the drawings: FIG. IA is a photograph depicting changes in the transcription of amoebapore genes in G3 trophozoites as demonstrated by RT-PCR analyses. Total RNA was prepared from HMl-IMSS and G3 freshly harvested trophozoites. The RNA was treated with RNase Free DNase, and reverse transcribed using oligo dT-adaptor primer (Table 1, SEQ ID NO: 2). PCR was then performed using a sense primer from the gene of interest (Table 1) and the adaptor primer (Table 1, SEQ ID NO: 3) as antisense for all the genes. Lanes: 1- gene EhRPLIl; 2- Ehap-a (ace. no X70851); 3- Ehap-b (GenBank Ace. No X76904.1); 4- Ehap-c (GenBank Ace. No. X76903.1); and the saposin-like protein genes: lane: 5 - SAPLIP 1 (GenBank Ace. No. AZ529784); 6- SAPLIP 5 (GenBank Ace. No. AZ530711 ); 7- SAPLIP 14 (GenBank Acc. No. AZ690015).

FIGs. IB-C are sequence comparisons between the three silenced genes: Ehap-a (GenBank Ace. No. X70851), Ehap-b (GenBank Ace. No. X76904.1) and SAPLIP 1 (GenBank Ace. No. AZ529784). Figure IB is a sequence comparison between Ehap-a and Ehap-b. Figure 1 C is a sequence comparison between Ehap-a and SAPLIP 1.

FIG. ID is a schematic representation illustrating the generation of a single gene silenced trophozoite mutant. Initially, the amoebapore gene in trophozoites of virulent strain HM-1 :IMSS is silenced using plasmid psAP-2 (described herein) which contained 473 bp (SEQ ID NO.l) of the 5' upstream flanking region of the amoebapore gene. After transfection of the plasmid to parent HM-1 :IMSS virulent strain and silencing of the ap-a gene, the plasmid is removed by omission of the selection antibiotic G418, and a single amoeba is cloned, tested for plasmid absence and cultured to generate the G3 isolate. FIGs. IE-F are schematic representations illustrating the construct systems used to produce double gene silenced trophozoites.

FIGs. 2A-C are photographs and diagrams illustrating silencing of the Ehlgll (GenBank Ace. No. M96024) gene. Figure 2 A is a diagram of the Ehlgll - containing plasmids pB33 and pAY. Regions of the 473 bp Ehap-a promoter, SINE and T- rich (Tr) are marked. Plasmid pAY includes 44 bp of the signal peptide (sp) of the Ehap-a gene. Both plasmids contain the ORF of Ehlgll gene and the 3 'regulatory sequence from the Ehactin gene. Figure 2B is a photograph depicting changes in the transcription of Ehlgll genes in amoebic RNA extracts as demonstrated by Northern blot analysis. Lanes: 1- Parent strain- HM1 :IMSS; 2- HM- 1:IMSS transfected with plasmid pB33; 3- HM-1:IMSS transfected with pAY; 4- Ehap-a silenced G3 trophozoites; 5- G3 transfected with pB33; and 6- G3 transfected with pAY. Blots were probed as indicated. Figure 2C is a photograph of a Western blot analysis of protein lysates separated on SDS-PAGE (12 %) reacted with polyclonal antibodies against the LgIl protein. Samples in lanes 1-6 are as in Figure 2B. Lane 7 are lysates from the plasmid-less RBV trophozoites.

FIGs. 3A-C are diagrams and photographs identifying the source of Ehlgll (GenBank Ace. No. M96024) transcripts. Figures 3A-B are diagrams of the location of the two different sense primers used to differentiate between the two types of Ehlgll transcripts (genomic and plasmid derived). Figure 3C are photographs of an RT-PCR analysis of different transfectants. Isolated RNA was subjected to reverse transcription and PCR for several types of transcripts. Genomic Ehlgll (table 3 primers SEQ ID NOs: 23 and 27); Ehlgll derived from plasmid (primers SEQ ID NOs: 22 and 27); Ehap-a (primers SEQ ID NOs: 3 and 4) and: ribosomal protein L21 (primers SEQ ID NOs: 3 and 5). Samples: Lane 1, HM-I . -IMSS- transfected with pB33; lane 2, HM-UMSS- pAY transfected; lane 3, Silenced G3- pB33 transfected; lane 4, G3 -pAY transfected; lane 5, HM-I :IMSS; lane 6, G3 control trophozoites.

FIGs. 4A-F are photomicrographs of confocal microscopy illustrating the induction of capping of the Gal/GalNAc-lectin to the uroid region of the trophozoites. Figures 4A, 4C and 4E illustrate trophozoites incubated at 4 ⁰C and Figures 4B, 4D and 4F illustrate trophozoites which were incubated at 37 ⁰C for 20 minutes to observe the induction of capping using two monoclonal antibodies against the heavy subunit of the Gal-lectin. Figures 4 A-F show the fluorescent Gal-lectin superimposed on a Nomarsky section. Samples: HM-1 :IMSS, G3 silenced culture and RBV (the plasmid-less, two gene (Ehlgll and Ehap-ά) silenced culture).

FIGs. 5A-B are schematic diagrams and photographs illustrating silencing of the CP-5 gene (GenBank Ace No. X91644.2). Figure 5 A is a schematic diagram of the plasmid pAP-CP5. Figure 5B is a photograph of a Northern blot analysis of RNA extracts from: Lanel- Parent strain HM1:IMSS; lane 2- G3; lane 3- G3- pAP-CP5 transfected (probes used are as indicated).

FIGs. 6A-B are photographs illustrating in vivo labeling of cysteine proteinsases. Figure 6A: The different trophozoite cultures were grown for 18 hours with the radiolabeled cysteine proteinase inhibitor Fmoc-[I^125]Tyr-Ala- diazomethylketone (10 μg/ml, 10 μCi/ml), harvested, washed and lysed. 25 μg protein of each cell lysate was separated on 12 % acrylamide reducing gel and exposed to X-ray film to reveal the different cysteine proteinase bands. Samples are: Lane 1- parent HM-I: IMSS; lane 2- G3; lane 3- G3-transfected with pAP-CP5 and grown with 50ug/ml of G418; lane 4- RB8 trophozoites after removal of the plasmid; lane 5- HM-L¹IMSS grown with a lower specific activity inhibitor (0.05 μCi/μg). Figure 6B demonstrates the location of the CP-5 band in HM-I: IMSS and its absence from the E.dispar culture.

FIG. 7 is a photograph illustrating an RT PCR analysis for the different Ehlgl transcripts (GenBank Ace. No. M96024). Total RNA was prepared from G3 and RBV freshly harvested trophozoites. PCR was performed using antisense conserved primer for Ehlgll, Ehlgl2 and EhIgU (Table 3, primer SEQ ID NO:24), and a specific sense primer for each of the genes as indicated: Lane 1- primer specific for Ehlgll (Table 3, primer SEQ ID NO: 23), Lane 2- for EhIgU (Table 2, primer SEQ ID NO: 25), Lane 3 - for EhIgB (Table 2, primer SEQ ID NO: 26), Lane 4- primers for EhRPUl (tables 1 and 2 primers SEQ ID NOs: 3 + 10).

FIGs. 8A-C are schematic diagrams of various plasmids. Figure 8A is a diagram of plasmid pSG in which the truncated SINE 1 and the T-rich sequences were ligated upstream to the Ehlgll promoter and ORF regions. Figure 8B is a diagram of Plasmid pPIO in which the SINEl sequences were removed from the Ehap-a promoter region. Figure 8C is a diagram of Plasmid pTL containing a truncated segment of the Ehlgll gene without the 3' regulatory region.

FIG. SD is a photograph of Northern blots of RNA extracts of trophozoite transfectants. Probes used as indicated. Lane 1- HM-:IMSS; lane 2-HM-l .TMSS transfected with plasmid pSG; lane 3- HM-LIMSS transfected with pPIO; lane 4- G3; lane 5- G3 transfected with pSG; lane 6- G3 transfected with pPIO.

FIG. 9 is a dot blot of RNA extracts from G3 trophozoites transfected with plasmid pTL and probed with either EhRP -L21 or Ehlgll.

FIG. 10 is a schematic diagram of the plasmid used to generate triple silenced trophozoites of the present invention. FIG. 11 is a photograph of Northern blots of RNA extracts of triple silenced trophozoites (RB9a and RJB9b - lanes 1 and 2), double silenced trophozoites (RB8 and RBV - lanes 3 and 4) and a single silenced trophozoite (G3 - lane 5). Probes used as indicated. DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a viable avirulent amoeba of E. histolytica, pharmaceutical compositions containing same and methods of producing same, which can be used for protecting against amoebiasis. The principles and operation of the vaccine according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Entamoeba histolytica (E. histolytica) is the aetiological agent of invasive amoebiasis, the third leading parasitic cause of mortality in the world. With the discovery of a number of E. histolytica virulence factors in recent years (e.g., amoebapores, galactose-specific lectins and cysteine proteinases) vaccination against amoebiasis may be considered a viable method of prevention of the disease.

The present authors have previously demonstrated that down-regulating expression of any one of three virulence factors individually, either by down- regulation of expression of their genes using antisense RNA, with small molecular weight inhibitors (e.g. GaINAc) or irreversible inhibitors, (e.g. E-64) reduced some aspects of pathogenicity of the parasite [Bracha R. et al, MoI. Microbiol. 34, 463- 472, 1999; Ankri S et al, 28, 777-785, 1998; Ankri, S. et al, MoI. Microbiol. 33:327- 337, 1999].

Additionally, the present authors irreversibly silenced the transcription of the amoebapore gene [Bracha, R., Y. Nuchamowitz, et al. (2003) Eukaryot. Cell 2: 295- 305] producing a strain of E. histolytica that is non-virulent in some animal models, although remained virulent in other animal models such as the Hu-mice intestinal model [Zhang X., Zhang Z., (2004) Infect Immun 72, 678-83]. Preliminary studies also demonstrated that vaccination of hamsters with live silenced amoeba afforded considerable, although not total immuno-protection [Bujanover S. et al, 2003, Int J Parasitol. 33(14): 1655-63]. This irreversible silencing was achieved by transfection with homologous sequences of the amoebapore gene including the 5 'and 3' regulatory regions. The partial virulence of the above described mutant strains of E. histolytica together with the fact that only one gene in these initial experiments was down- regulated and therefore there remains a chance of reversion does not favor their use for immunization purposes. While searching for the role of different virulence factors in the pathogenicity of E. histolytica amoeba, the present inventors unexpectedly found a novel approach for irreversible down-regulating gene expression in amoeba. The present invention exploits this finding to provide a novel phaπnaceutical composition for use as a vaccine for the prevention of amoebiasis. As is illustrated herein below and in the Examples section which follows, down-regulation of amoebapore gene expression by amoeba transfection with homologous sequences of the amoebapore gene, is a first step in the irreversible down-regulation of genes such as virulent factor genes. For example, down-regulation of a second virulent factor gene (the galactose-specific lectin gene, IgI 1 or the cysteine proteinase gene, cp-5) following down-regulation of an amoebapore gene was achieved, as verified by Northern and Western blot analyses.

Thus, according to one aspect of the present invention there is provided a viable avirulent amoeba of E. histolytica.

As used herein the phrase "amoeba of E. histolytica" refers to the parasitic unicellular amoeba protozoa responsible for the disease amoebiasis either in a cyst or an active trophozoite state. Preferably the E. histolytica amoeba is in the trophozoite state. Any strain of E. histolytica amoeba may be used for the present invention. Examples of E. histolytica amoeba strains include HM-I rIMSS (also known as HM- 1), HU-21 :AMC, Rahman, HUI rMUSC, HU-I :CDC, DKB, SAW 755CR, SAW 89 IR, SD157, SDl 84, HK-9, HB-301:NIH, 200:NIH, HU-2:MUSC, HU-I :CDC, H- 302:NIH, H-303:NIH, H-458:CDC, HM-3:IMSS, HU22:AMC, HI-1295:AIIMS and HB-301:NIH.

According to a presently known embodiment of this aspect of the present invention the strain of E. histolytica amoeba is HM-I. As used herein the phrase "viable avirulent amoeba of E. histolytica" refers to an intact form of the above described E. histolytica pathogen, which is capable of replication whilst it's cytopathic and/or cytotoxic activities are reduced (such as can be demonstrated in in vitro systems) and it is no longer capable of inducing the clinical manifestations of the invasive disease in vivo such as ulcer formation, dysentery, liver abscess and colitis in humans (further described in the preceding Background section) and in animal models described hereinbelow.

Preferably, the avirulent amoebas of the present invention comprise less than 5 % and even more preferably less than 1 % of the cytopathic or cytotoxic activity of an identical concentration of a virulent amoeba of E. histolytica (e.g., naturally occurring amoeba of E. histolytica).

Preferably, the amoeba of this aspect of the present invention is a stably avirulent amoeba.

As used herein, the phrase "stably avirulent amoeba" refers to amoebas which remain avirulent for at least 2000 generations, preferably at least 10,000 generations and more preferably at least 100,000 generations.

According to one embodiment of this aspect of the present invention, the viable avirulent amoeba of E. histolytica is genetically modified.

As used herein the phrase "genetically modified amoeba" refers to directed or non-directed introduction of exogenous genetic information (e.g., encoding genetic information and/or regulatory genetic information) into the amoeba.

Preferably, E. histolytica amoebas of this aspect of the present invention are genetically modified to down-regulate (i.e., reduce or totally inhibit the mRNA level prior to synthesis of the complete transcript or following synthesis of the complete transcript) at least two genes such that the residual activities of the down-regulated genes are limited to less than 20 %, more preferably less than 10 %, more preferably less than 5 %, more preferably less than 1 % and more preferably to less than 0.1 % of their original protein expressing capability. Preferably, at least one of the two genes is a virulent factor gene and more preferably both of the at least two genes are virulent factor genes.

As used herein the phrase "each being down regulated by independent exogenous genetic means" refers to the process of down-regulating a particular gene by exogenous means (genetic manipulation), whereby each gene which is down- regulated involves an exogenous step. According to one embodiment the individual down-regulation of at least two genes is performed sequentially. Thus, it should be evident that the phrase does not include down-regulation of genes (e.g. genes which comprise homogenous sequences) as a natural byproduct of the active down- regulation of a particular gene - see Example 1 of the Examples section herein below. E. histolytica virulent factor genes include genes encoding polypeptides belonging to three virulent factor families - the amoebapores, the galactose specific lectins and the cysteine proteinases. Examples of amoebapore genes include ap-a, (GenBank Accession No. X70851), ap-b (GenBank Accession No. X76904), ap-c (GenBank Accession No. X76903) and saposin-like proteins 1, 5 and 14 (GenBank Accession Nos. AZ529784, AZ530711 and AZ690015 respectively ) [Bruhn H and Leippe M. Biochim Biophys Acta. 2001 Sep 3;1514(l):14-20]. Examples of galactose-specific lectin genes include, but are not limited to IgU (Accession No. M96024), lgl-2, lgl-3, HgI-I₁ hgl-2, hgl-3, hgl-4 and hgl-5 [Ramakrishnan et al., 1996, MoI. Microbiol. 19, 91-100]. Example of cysteine proteinase genes include but are not limited to cp-1 (GenBank Accession No. Q01957); cp-2 (GenBank Accession No. Q01958); cp-3 (GenBank Accession No. CAA60673); cp-4 (GenBank Accession No. CAA62833); cp-5 [Alon, R., Bracha R., MoI. Biochem. Parasitol, 90: 193-201, 1997] (GenBank Accession No. CAA62835); cp-6 (GenBank Accession No. CAA62835); cp-1 (GenBank Accession No. CAC34069); cp-8 (GenBank Accession No. AY156066); cp-9 (GenBank Accession No. AY156067); cp-10 (GenBank Accession No. AYl 56068); cp-11 (GenBank Accession No. AYl 56096); cp-12 (GenBank Accession No. AY156070); cp-13 (GenBank Accession No. AYl 56071); cp-14 (GenBank Accession No. AYl 56072); cp-15 (GenBank Accession No. AYl 56073); cp-16 (GenBank Accession No. AyI 56074); cp-17 (GenBank Accession No. AY156075); cp-18 (GenBank Accession No. AY156076; cp-19 (GenBank Accession No. AY156077 and cp-112 (GenBank Accession No. AAF04255) [Bruchhaus I, et al, Eukaryot Cell. 200, (3):501-9].

Preferably, the at least two down-regulated virulent factor genes belong to different families of virulent factors since each family of virulent factors is responsible for a different aspect of virulence as described in the Background section hereinabove. For example, genetically modified E. histolytica amoeba may comprise a down-regulated apa-a gene and a down-regulated cp-5 gene or alternatively genetically modified E. histolytica amoeba may comprise a down-regulated apa-a gene and a down-regulated IgI-I gene (see Examples section below). Preferably, the genetically modified E. histolytica amoebas comprise down-regulated virulent factor genes from all three families of virulent factors. For example, genetically modified E. histolytica amoeba may comprise a down-regulated apa-a gene, a down-regulated cp- 5 gene and a down-regulated lgl-1 gene. The genetically modified E. histolytica amoebas may comprise at least two down-regulated genes which have been epigenetically modified. As used herein the phrase "epigenetically modified gene" refers to an alteration in the chromatin structure of a gene domain leading to a stable and inheritable change. Examples of epigenetic modifications include but are not limited to DNA methylation, histone deacetylation, histone methylation, histone demethylation thereby effecting chromatin structure. An epigenetic modification typically does not effect the sequence of the endogeonous gene itself. Without being bound to theory, it is believed that the down- regulation of genes using the ap-a gene regulatory sequence in constructs described herein above is effected by a demethylation of lysine 4 in Histone H3, thereby affecting chromatin structure.

The epigenetic down-regulation of at least two genes in the E. histolytica genome may be effected by the epigenetic down-regulation of the ap-a gene followed by the down-regulation of the second gene. A preferred method of epigenetically down-regulating gene expression in E. histolytica comprises (a) introducing into the amoeba of E. histolytica a nucleic acid construct comprising: a regulatory sequence as set forth in SEQ ID NO: 1 or a functional equivalent thereof; and subsequently (b) introducing into the amoeba of E. histolytica a second nucleic acid construct comprising: (i) the regulatory sequence as set forth in SEQ ID NO: 1 ; and (ii) a polynucleotide encoding a polypeptide of E. histolytica, operably linked to said regulatory sequence.

The plirase "regulatory sequence" refers to a deoxyribonucleic acid sequence composed of naturally-occurring bases, sugars and covalent internucleoside linkages (e.g., backbone) as set forth in SEQ ID NO: 1 as well as oligonucleotides having non- naturally-occurring portions which function similarly to respective naturally- occurring portions.

As used herein, the phrase "functional equivalent thereof refers to a nucleic acid sequence which exhibits functional properties similar to SEQ ID NO: 1 (down- regulation of gene expression preferably in a non-reverting fashion). The phrase "a polynucleotide encoding a polypeptide of E. histolytica'¹ refers to any polynucleotide encoding a polypeptide of E. histolytica except the polynucleotide encoding AP-A. For example, the additional pol^ynucleotide may encode for a membrane protein to generate amoebas with a modified membrane composition for use as carriers as discussed herein below. Preferably it refers to a polynucleotide encoding a virulent factor polypeptide as discussed herein above.

According to one embodiment the polynucleotide encodes at least a portion of an open reading frame of a polypeptide of E. histolytica. Determination of the maximum number of base-pairs which may be deleted for each particular gene is well within the scope of one skilled in the art. Preferably, the polynucleotide comprises at least 10 % of the first base pairs of the open reading frame of the polypeptide of E. histolytica.

Thus, as described in Example 7, a plasmid was generated (pTL) which only comprised 421 base pairs of the 894 bp open reading frame of EhIgIl. Silencing of the chromosomal Ehlgll gene was successful in the pTL transfectants as shown by the complete absence of transcripts of Ehlgll in RNA dot blots (Figure 9).

According to another embodiment the polynucleotide comprises a fragment of a 3' regulatory region of the polypeptide of E. histolytica. According to this embodiment the 3' regulatory region may be partially or fully removed. Thus, as described in Example 7, the plasmid pTL was devoid of the 3' regulatory sequence of Ehlgll, yet sill was able to silence the chromosomal Ehlgll gene.

As used herein, the phrase "operably linked" refers to a functional positioning of the regulatory sequence so as to allow down-regulation of the selected nucleic acid sequence. For example, the regulatory sequence of this aspect of the present invention may be located upstream of the polynucleotide sequence in terms of the direction of transcription and translation.

According to a particular embodiment, the 473 bp regulatory sequence is placed directly adjacent to the 5' starting codon of the polynucleotide sequence of the gene to be down-regulated. As described in Example 2 of the Examples section herein below, a plasmid was generated (pAY) in which the 44 bp sequences of the Ehap-a signal peptide were placed before the Ehlgll gene. No down-regulation of Ehlgll in the G3 pAY transfectants was observed as opposed to the marked down- regulation of Ehlgll in the pB33 transfectants of G3 (Figure 2B). As used herein the term "polynucleotide" refers to a double stranded nucleic acid sequence including for example, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above). IS

As used herein the phrase "complementary polynucleotide sequence" refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.

As used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

As used herein the phrase "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.

In addition to the above described nucleic acid sequences, nucleic acid constructs of the present invention may also comprise other elements such as, for example, an antibiotic resistance gene. Examples of antibiotic resistance genes include, but are not limited to, genes encoding neomycin phosphotransferase, which provides resistance to G418; hygromycin phosphotransferase, which provides resistance to hygromycin; bleomycin resistance protein, which provides resistance to bleomycin; and puromycin acetyltransferase, which provides resistance to puromycin.

Methods of introducing the nucleic acid construct of this aspect of the present invention into E. histolytica amoeba include, but are not limited to electroporation or other methods known to one with ordinary skill in the art such as lipofection.

Following each introduction of the first and second nucleic acid construct, the transfected amoeba are grown under conditions facilitating isolation of the specific transfectants, either under conditions under which such transfectants have a selective advantage over parental amoeba or under conditions allowing their easy recognition from unaltered amoeba or amoeba of other types. In each case, the nucleic acid constructs of this aspect of the present invention may then be removed by omission of the selection antibiotic, and single amoebas may be cloned and cultured as described in the Examples section herein below. ^

It will be appreciated that although down-regulation of the first and second gene is preferably effected sequentially, as described hereinabove, a third gene does not necessarily have to be down-regulated sequentially. Thus, as described in Example 8 herein below, triple silenced trophozoites of E. histolytica were generated by down-regulation of apa-1, followed by the simultaneous down-regulation of a second and third gene. According to this method, the number of genes that ma}' be down-regulated is limited by the number of the repeat sequences of the ap-a promoter that can be introduced into the same plasmid.

It will be appreciated that down-regulating gene expression of at least two genes in the genome of the E. histolytica may also be effected by other genetic down- regulatory agents. According to presently known embodiments of this aspect of the present invention, the introduced plasmids should be self-replicating and stable. One example of an agent capable of down-regulating an E. histolytica gene is an oligonucleotide agent. For example, oligonucleotides directed against endogenous nucleic acid sequences expressing at least two virulent factor proteins may be used.

Small interfering RNA (siRNA) molecules are one type of oligonucleotide agent capable of down-regulating at least two virulent factor genes.

RNA interference is a two-step process. During the first step, which is termed the initiation step, input dsRNA is digested into 21-23 nucleotides (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which cleaves dsRNA (introduced directly or via an expressing vector, cassette or virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each strand with 2-nucleotide 3" overhangs [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature 409:363-366 (2001)].

In the effector step, the siRNA duplexes bind to a nuclease complex to form the RNA-induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); Hammond et al, (2001) Nat. Rev. Gen. 2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although the mechanism of cleavage is still to be elucidated, research indicates that each RISC contains a single siRNA and an RNase [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].

Because of the remarkable potency of RNAi, an amplification step within the RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs, which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC [Hammond et al., Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev. 15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)]. For more information on RNAi see the following reviews Tuschl CheniBiochem. 2:239-245 (2001); Cullen Nat. Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575: 15-25 (2002).

Synthesis of RNAi molecules suitable for use with the present invention can be affected as follows. First, the mRNA sequence target is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl CheniBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 3' UTR mediated about 90 % decrease in cellular GAPDH mRNA and significantly reduced protein level (http://www.ambion.com/techlib/tn/93/935.html).

Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/). Putative target sites that exhibit significant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.

U.S. Pat. App. No. 20020182223 teaches methods of reducing virulence of protozoan parasites using dsRNA.

Another oligonucleotide agent capable of down-regulating at least two genes in the genome of the E. histolytica is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or a DNA sequence of the target. DNAzymes are single- stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 1997;94:4262). A general model (the "10-23" model) for the DNAzyme has been proposed. "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM [Curr Opin MoI Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes have been used to successfully down-regulate microorganism genes. For instance, a variety of therapeutically relevant viral genes have been down-regulated by DNAzymes in cell culture experiments. For example a 10-23 DNAzyme has been employed as an antiviral agent either directly targeting HIV-I RNA [Zhang, X.; Xu, Y.; Ling, H. and Hattori, T. (1999) FEBS Lett. ,458(2), 151-156] or preventing virus entry by downregulation of the CCR5 co-receptor [Goila, R. and Banerjea, A.C, (1998) FEBS Lett., 436(2), 233-238].

Down-regulation of at least two genes in the genome of the E. histolytica can also be affected by using antisense polynucleotides capable of specifically hybridizing with mRNA transcripts endogenous to the E. histolytica genome (e.g., an antisense oligonucleotide directed at virulent factor genes).

Design of antisense molecules, which can be used to efficiently down-regulate at least two genes in the genome of the E. histolytica, must be effected while considering which oligonucleotides specifically bind to the designated niRNA within the amoebas in a way that inhibits translation thereof.

Algorithms for identifying those sequences with the highest predicted binding affinity for their target niRNA based on a thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotide are also available [see, for example, Walton et ah, Biotechnol Bioeng 65: 1-9 (1999)].

Such algorithms have been successfully used to implement an antisense approach in cells. For example, the algorithm developed by Walton et al., enabled scientists to successfully design antisense oligonucleotides for rabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNF alpha) transcripts. The same research group has more recently reported that the antisense activity of rationally selected oligonucleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat gpl30) in cell culture as evaluated by a kinetic PCR technique proved to be effective in almost all cases, including tests against three different targets in two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries.

In addition, several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system were also published (Matveeva et al., Nature Biotechnology 16: 1374 - 1375 (1998)]. Antisense-mediated suppression of single virulent factor genes in E. histolytica has been successfully performed for the inhibition of the ap-a gene [Bracha, R., Y. Nuchamowitz, M. Leippe, and D. Mirelman (1999) MoI. Microbiol. 34:463-472]; the cp-5 gene [Ankri S et al., 28, 777-785, 1998] and the lgl-1 gene [Ankri, S. et al., MoI. Microbiol. 33:327-337, 1999]. In all these cases, a reduction in amoeba pathogenicity was clearly demonstrated, however avirulence was not achieved since total inhibition of the associated genes were never attained. By down- regulating at least two virulent factor genes in the same amoeba, avirulence may be accomplished.

The current consensus is that recent developments in the field of antisense technology which, as described above, have led to the generation of highly accurate antisense design algorithms, enable an ordinarily skilled artisan to design and implement antisense approaches suitable for down-regulating expression of known sequences without having to resort to undue trial and error experimentation. Another agent capable of down-regulating E. histolytica virulent factor genes are ribozyme molecules. Endogenous ribozymes were first discovered in the protozoa Tetrahymena thermophilia. Ribozymes are being increasingly used for the sequence- specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest rendering them valuable tools in both basic research and therapeutic applications [Welch et aL, Curr Opin Biotechnol. 9:486-96 (1998)]. Thus it is possible to design ribozymes capable of specifically cleaving virulent factors of E. histolytica. Ribozymes have been developed to inhibit microorganisms including viruses (e.g. HIV-I). Specifically, two ribozymes targeted against different sites on the RNA of HIV-I were shown to inhibit viral replication in cell culture experiments [Ojwang, J.; et aL, (1992) Proc. Natl. Acad. Sci. USA, 89(22), 10802-10806].

An additional method of down-regulating the expression of virulence factor genes in E. histolytica amoeba is via triplex forming oligonucleotides (TFOs). In the last decade, studies have shown that TFOs can be designed which can recognize and bind to polypurine/polypirimidine regions in double-stranded helical DNA in a sequence-specific manner. These recognition rules are outlined by Maher III, L. J., et aL, Science (1989) 245:725-730; Moser, H. E., et aL, Science (1987)238:645-630; Beal, P. A., et aL, Science (1991) 251 :1360-1363; Cooney, M., et aL, Science(1988)241:456-459; and Hogan, M. E., et aL, EP Publication 375408. Modification of the oligonucleotides, such as the introduction of intercalators and backbone substitutions, and optimization of binding conditions (pH and cation concentration) have aided in overcoming inherent obstacles to TFO activity such as charge repulsion and instability, and it was recently shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review see Seidman and Glazer (2003) J Clin Invest; 112:487-94).

In general, the triplex-forming oligonucleotide has the sequence correspondence: oligo 3'-A G G T duplex 5'-A G C T duplex 3'-T C G A

However, it has been shown that the A-AT and G-GC triplets have the greatest triple helical stability (Reither and Jeltsch (2002), BMC Biochem, , Septl2, Epub). The same authors have demonstrated that TFOs designed according to the A-AT and G- GC rule do not form non-specific triplexes, indicating that the triplex formation is indeed sequence specific.

Thus for any given sequence in the regulatory region a triplex forming sequence may be devised. Triplex-forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.

Transfection of E. histolytica amoeba with TFOs (e.g. by electroporation), and formation of the triple helical structure with the target DNA induces steric and functional changes thereby blocking transcription initiation and elongation resulting in the specific downregulation of gene expression. Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal siφFGl and endogenous HPRT genes in mammalian cells (Vasquez et al, Nucl Acids Res. (1999)

27:1 176-81, and Puri, et a/., J Biol Chem, (2001) 276:28991-98). TFOs were also shown to cause conformational changes in the protozoan parasite Leishmania amazonensis gene [Brossalina et al.. Nucleic acids research, 1996, 3392-3398].

Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn. It will be appreciated that oligonucleotides may further include base and/or backbone modifications to reduce cytotoxicity such that the genetically modified E histolytica amoebas of the present invention remain viable. Such modifications are described in Younes (2002) Current Pharmaceutical Design 8:1451-1466.

Another mode of down-regulation may be the use of non-functional isoforms of virulent genes which may exert dominant negative effect. For example mutated or truncated forms of a virulent factor gene may be introduced into the amoeba, and through a dominant negative effect, down-regulate the endogenous virulent factor.

This technique was successfully employed for the down-regulation of the lgl-1 gene

[Katz U., et al, MoI. Biol. Cell. 13, 4256-4265, 2002; Vines R.R., et al, MoI Biol Cell. 1998 Aug;9(8):2069-79J.

Oligonucleotide agents or mutated genes of the present invention can be introduced as part of a nucleic acid construct into the E. histolytica amoeba using any method known in the art (for example electroporation). When two or more E. histolytica genes are to be down-regulated, the oligonucleotides or mutated genes may be introduced into the amoeba in a sequential manner - i.e. first one virulent factor gene is inhibited using a first nucleic acid construct, positive clones are selected and subsequently a second virulent factor gene is inhibited using a second nucleic acid construct. Alternatively, two or more virulent factor genes may be down-regulated using a single nucleic acid construct comprising at least two promoters and two operably linked sequences, each sequence encoding an oligonucleotide or mutated virulent factor and each being responsible for the down-regulation of a different virulent factor gene. Alternatively, the two virulent factor genes can be co-transcribed as a polycistronic message from a single promoter sequence of the nucleic acid construct.

To enable co-translation of both virulent factor genes from a single polycistronic message, the first and second polynucleotide segments can be transcriptionally fused via a linker sequence including an internal ribosome entry site (IRES) sequence which enables the translation of the polynucleotide segment downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule including the coding sequences of both the first and the second growth factors will be translated from both the capped 5' end and the internal IRES sequence of the polycistronic RNA molecule to thereby produce both the first and the second virulent factor. In addition, the oligonucleotide or mutated (i.e. non-functional isofomi) gene constructs can be adapted in such a way so that their synthesis can be regulated (stimulated or inhibited) by the addition of a compound. This enables the concentration of the inhibitory molecules to be tightly controlled. Concentration increases may be obtained in a step-like manner by increasing the concentration of the inducer compound without the need for further transfection. It will be appreciated that for vaccination use, no induction is necessary.

For example, a construct might comprise tetracycline-inducible promoters. Immediately downstream of each of the two promoters, a tetracycline operator region could be inserted. In the absence of tetracycline the tetracycline repressor protein binds to these operator sites, preventing transcription. When tetracycline is present, it binds to the repressor and the repressor-tetracycline complex is unable to interact with the operator region. A tetracycline-inducible gene expression system in Entamoeba histolytica has been described by Ramakrishnan G. et al, in MoI Biochem Parasitol. 1997 Jan;84(l):93-100 and Hamman, L, et al, MoI Biochem Parasitol. 1997 Jan;84(l):83-91.

Other inducible promoters, and their corresponding inducers could be used in the present invention. For example, the bacterial lactose operator could be used such that the adjacent promoter would be induced by the addition of isopropylthiogalactoside (IPTG). Another example utilizing a different molecular mechanism is the placement of a transcription termination sequence immediately downstream of the promoter. The termination sequence is flanked by short regions that are recognized by excision- or "flipase" enzymes. Transcription would be terminated until the termination sequence is deleted or inverted via induction of the expression of an excision- or "flipase" enzyme that recognizes the flanking regions and excises or inverts ("flips") the termination sequence. The Cre-loxP system is one example of this excision/inversion system in which Cre is the enzyme and loxP is the recognition sequence of Cre that would flank the transcription termination sequence. An exemplary plasmid that may be used in the present invention is the pEhActNeo shuttle vector (as described in the Example section below).

Whichever method is used, following the generation of genetically modified E. histolytica amoebas, their virulence is preferably assayed.

Methods of quantifying cytopathic activity include analyzing the destruction rate of cell monolayers (e.g. baby hamster kidney cells) as described by Bracha and Mirelman, 1984, J. Exp. Med. 160:353-368.

Cytotoxic activity may be measured using the vital dye exclusion technique [Leippe, M., J. et al, 1994, Proc. Natl. Acad. Sci. USA 91:2602-2606]. Essentially, freshly harvested baby hamster kidney cells are incubated with the avirulent amoeba of the present invention. Samples are examined microscopically in a hemocytometer chamber at different time points during the incubation. Viability is indicated by exclusion of trypan blue (0.1 %). The cytotoxic activity is expressed as the percentage of stained cells in each sample minus the percentage of stained cells in the sample with BHK cells alone for the same time point. Preferably, the number of cells that incorporate the dye is less than 10 %, more preferably less than 4 % and even more preferably less than 1 % during the period tested.

Other assays which may be used to assay the virulence of the modified E. histolytica amoeba include an erythrophagocytosis assay [Mora-Galindo et al, 1997, Arch. Med. Res. 28, 200-201] and the rosette formation assay [Ravdin and Guerrant,

1981, J. Clin. Invest. 68, 1305-1313].

The capability of inducing clinical manifestations of the invasive disease in vivo may be examined in animal models such as SCID mouse model of amebic liver or tissue models such as a mouse human colonic xenograft, a model of amebic colitis. The viable avirulent E. histolytica amoeba of the present invention may be used as a vaccine to prevent an amoebiasis disease in a subject in need thereof. As used herein, the term "preventing" refer to the protection, amelioration or incidence reduction of E. histolytica colonization in the subject. As used herein, the phrase "subject in need thereof refers to a human or primate which is at the risk of an E. histolytica infection.

The viable avirulent E. histolytica amoeba of the present invention may alternatively be used to treat other diseases such as inflammatory gut disorders. For example, the amoebas of the present invention may be further modified so that they are capable of secreting other polypeptides such as cytokines. The production and secretion of biologically active mammalian polypeptides by a genetically modified gut commensal amoeba could lead to the development of new long-term immunotherapies for inflammatory gut diseases. Farrar et al. teach the use of a gut commensal bacterium, Bacteroidesovatus for the treatment of inflammatory gut disorders [Farrar M., et al, (2005), J. Applied microbiology, vol. 98, no. 5, pp. 1191-

1197(7)].

As used herein the term "treat" means alleviation of some or all of the symptoms associated with a disease, prolongation of life expectancy of patients having a disease, as well as complete recovery from a disease. The viable avirulent amoeba of E. histolytica of the present invention may be administered to a human subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to the active ingredient described herein with other chemical components such as physiologically suitable carriers. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the preparation of viable avirulent amoeba of E. histolytica accountable for the biological effect. Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

To prepare an anti-amoebiasis vaccine, the genetically modified amoebas of the present invention are isolated. The amount of these cells may then be adjusted to an appropriate concentration, optionally combined with a suitable vaccine adjuvant, and packaged for use. The formulation and packaging of the anti-amoebiasis vaccine for the present invention must endure that the genetically modified E. histolytica amoeba of the present invention remain viable. Suitable adjuvants include but are not limited to surfactants, e.g., hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N'-N-bis(2-hydroxyethyl- propane di-amine), methoxyhexadecyl-glycerol, and pluronic polyols; polanions, e.g., pyran, dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g., muramyl dipeptide, MPL, aimethylglycine, tuftsin, oil emulsions, alum, and mixtures thereof. Other potential adjuvants include the B peptide subunits of E. coli heat labile toxin or of the cholera toxin. McGhee, J. R., et ah, "On vaccine development," Sem. Hematol., 30:3-15 (1993).

Preferable routes of administration of the viable amoeba of the present invention include oral, rectal, nasal or enteral administration. More preferably, the viable amoeba of the present invention are administered orally.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes as long as the amoeba of the present invention remain viable. Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, an immunogenically effective amount means an amount of active ingredients (avirulent genetically modified E. histolytica amoebas) effective to elicit an immune reaction which protects the subject from an amoebiasis disease.

Determination of an immunogenically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the immunogenically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in primates to achieve a desired concentration or titer. Such information can. be used to more accurately determine useful doses in humans.

It will be appreciated that treatment of a disease (i.e. amoebiasis) would require additional studies for an estimation of a therapeutically effective amount. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or in primates. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et oL, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Dosage amount and interval may be adjusted individually to provide plasma levels of the active ingredient sufficient to have a biological effect (e.g. protect against amoebiasis). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the rapidity with which protection is required and the amount of spread of amoebiasis in the vicinity, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. The quantity of the anti-amoebiasis vaccine of the present invention also depends upon the capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034.074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1

Simultaneous down-regulation of other genes during the epigenetic silencing of the

Ehap-a gene

In order to ascertain whether down-regulation of Ehap-a was accompanied by down-regulation of other genes comprising similar sequence homologies, the transcription levels of such genes was examined.

MATERIALS AND METHODS

Total RNA was prepared from the plasmid-less, ap-a gene silenced, G3 amoeba culture [Bracha, R., Y. Nuchamowitz, et al. (2003). Eukaryot. Cell 2: 295- 305] using the RNA isolation kit TRI Reagent according to the manufacturers protocol (Sigma, Co). RNA samples were treated with RNase free DNase (Promega) and after phenolation and sedimentation, 2ug RNA were reacted with AMV reverse transcriptase (Promega) according to the manufacturer's protocol using an oligo dT- adaptor as primer (Table 1 herein below). The cDNA product was diluted 5 times and used for the subsequent PCR reactions.

Table 1

RESULTS

As shown in Figure IA, the transcripts of Ehap-a, Ehap-b and SAPLIP 1 (ace. no. AZ529784) were down-regulated in the silenced G3 trophozoites while others like Ehap-c, and SAPLIP 5, (ace. number AZ53071 1) and SAPLIP 14, (ace. number AZ690015) showed similar transcription levels as in the parent strain HM-1:IMSS. Similar levels of transcription were also detected for the ribosomal protein gene- EhRP-LIl, and Ehactin gene (not shown). Considerable sequence homology was found between Ehap-a, Ehap-b and SAPLIP 1 [Bruhn H Leippe M, ( 2001) Biochim Biophys Acta 1514: 14-20; Winkelmann J, et al., (2006). MoI Biochem Parasitol. Feb 14; [Epub ahead of print] - (Figures IB-C). According to the phylogenetic tree of SAPLIP's and amoebapore proteins, Ehap-b and SAPLIP 1. are on the same branch as the Ehap-a gene. This homology was restricted to the ORF region of those genes. No homology was found in the regulatory sequences and SINEl sequences are not present upstream to those genes. Furthermore, the two non-silenced SAPLIP genes tested (SAPLIP's 5 and 14), as well as Ehap-c (Figure IA) have no significant sequence homology to Ehap-a . CONCLUSION

Down-regulation of Ehap-a according to the method of the present invention is accompanied by the simultaneous down-regulation of other E. histolytica genes which have significant sequence homology in their ORFs.

EXAMPLE 2

Generation of double gene silenced trophozoites by sequential down-regulation.

The objective of this study was to ascertain whether more than one virulence gene in amoeba could be silenced, thus decreasing residual virulence and diminishing the chance of reversion to virulence.

MATERIALS AND METHODS

Strain and culture conditions: Trophozoites of E. histolytica strain HM- 1:IMSS and of the plasmid-less gene silenced substrain G3 were grown at 37 ⁰C in TYI-S-33 medium. The substrain G3 was generated as previously described [Bracha, R., Y. Nuchamowitz, et al. (2003). Eukaryot. Cell 2: 295-305] and as illustrated in Figure ID using a construct containing the 5' upstream 473 bp. Transfection of trophozoites was performed as previously described [Bracha, R., Y. Nuchamowitz, et al. (2003). Eukaryot. Cell 2: 295-305] and cultures were then grown in the presence of the neomycin derivative G418. The removal of plasmids was performed by growing transfectants without G418 for 3 weeks followed by cloning of trophozoites and testing for cultures that were devoid of plasmid by PCR amplification of the Neo resistance gene in genomic DNA. Constructs: The pEhActNeo shuttle vector served as the basic construct. It contains the Neo gene that confers resistance to G418 flanked by the 5' and 3' regulatory sequences of the amoeba actiii 1 gene [Alon, R., Bracha R., MoI. Biochem. Parasitol, 90: 193-201, 1997] and the E. histolytica autonomous replication sequence, both cloned in pBluescript II SK (-). Primers used for the construction of the different cassettes are listed in Table 2 hereinbelow. A schematic representation of the constructs is provided in Figures IE-F.

Table 2

a. PlasmidB33 (Figure 2A)

Production of the cassette: The 5' upstream segment (473 bp) of the Ehap-a gene was amplified by PCR from psAP-1 [Bracha R, et al., (2003) Eukaryot Cell 2: 295-305] using primers SEQ ID NOs: 11 and 12 (Table 2). The ORF region of the Ehlgll gene and the 3' Ehactin flanking region were prepared by digestion of the intermediate plasmid construct pSA21 with NcoI/BamHI [Katz U et al, 2002, MoI Biol Cell 13: 4256-4265]. This plasmid is based on pBluescript HKS and includes a cassette containing the 5' flanking region of the Ehactin gene, the ORF of the Ehlgll (867bp) gene and the 3' flanking region of Ehactin. b. Plasmid pΛY (Figure 2A)

Production of the cassette: A 521 bp fragment was generated by PCR, using plasmid psAP-1 as a template and primers SEQ ID NOs: 11 and 13 (Table 2) which included the 473 bp of the 5' upstream region of the Ehap-a gene and 44 bp of the signal peptide of the Ehap-a gene. This fragment was then ligated to the segment containing the ORF of EhIgIl with the 3' Ehactin flanking region as above. c. Plasmid pAP-CP5 (Figure 5A)

Production of the cassette: The construction of this plasmid was similar to pB33. The 473 bp 5 'upstream of the Ehap-a gene was generated by PCR using primers SEQ ID NOs: 11 and 12 (Table 2) on plasmid psAP-1 [Bracha R₅ et al.,

(2003) Eukaryot Cell 2: 295-305]. The ORF of EhCP-5 [Bruchhaus I et al., (2003)

Eukaryotic Cell 2: 501-509] was generated by PCR using primers SEQ ID NOs: 14 and 15 (Table 2) on genomic DNA of strain HM-I : IMS S and the 3' flanking region of Ehactin was generated by digestion of plasmid pSA21 [Ankri S. et al (1999) MoI

Microbiol 33: 2096-2102] with Sall/BamHl. d. Plasmid pSG (Figure 8A)

Production of the cassette: A DNA fragment containing 188 bp of the 5' upstream region of the ap-a gene consisting of the truncated SINEl sequence and the T rich region was amplified by PCR from plasmid psAP-1 using primers SEQ ID NOs: 11 and 16 (Table 2). This 188 bp fragment was ligated to the 5' end of another segment (1203 bp) consisting of the Ehlgll gene ORF and 338 bp of the EhIgIl 5' upstream region. This latter fragment was generated by PCR amplification of genomic DNA of strain HM-1:IMSS using primers SEQ ID NOs: 17 and 18 (Table 2) followed by ligation to the 3^' 'Ehactin regulatory segment. e. Plasmid pPIO (Figure 8B)

Production of the cassette: A DNA fragment of 275 bp of the 5'upstram region of the ap-a gene was generated by PCR from plasmid psAP-1 using primers SEQ ID NOs: 19 and 12 (Table 2) and ligated to the ORF of Ehlgll with the 3'actin flanking region as in pB33.

/ Plasmid pTL (Figure 8C)

Production of the cassette: The 473 bp fragment of the 5' upstream region of Ehap-a and a fragment of 421 bp starting from the 5' end of the ORF of Ehlgll (truncated Ehlgll) (894 bp) were generated by PCR from plasmid pB33 using primers SEQ ID NOs: 1 1 and 21 (Table 2). EXAMPLE 3

Analysis of transcriptionally silenced genes in B33 and pAY transfectants MATERIALS AND METHODS

Preparation of RNA extracts: RNA extracts were prepared from parent strain HM1 :IMSS, silenced strain G3; silenced strain G3 transfected with plasniid B33 following G418 selection and silenced strain G3 transfected with plasmid pAY. Total RNA was prepared using the RNA isolation kit TRI Reagent (Sigma).

Preparation of Protein extracts; Protein extracts were prepared from parent strain HM1:IMSS, silenced strain G3, silenced strain G3 transfected with plasmid B33 and silenced strain G3 transfected with plasmid pAY. Extracts were also prepared from the plasmid-less double silenced trophozoites RBV (see below).

Transcriptionally silenced genes were analyzed using Northern and Western blot analysis.

Northern blot analysis: RNA (5 μg) was size fractionated on a 4 % polyacrylamide denaturing gel containing 8 M urea and subsequently blotted to a nylon membrane. Using stringent conditions, hybridization was carried out with different probes (0.1 % sodium dodecyl sulfate [SDS], O.lx SSC [Ix SSC is 0.15 M NaCl plus 0.015 M sodium citrate]). Probes were randomly labeled using the Rediprime II kit (Amersham Life Science). Probes for Northern blots were made by PCR from the genomic sequences of the genes AP-A (Genebank accession No. X- 70851) and LgIl (GenBank accession No. M96024).

Western blot analysis: Protein extracts were subjected to separation on a 20 % polyacrylamide gel under non-reducing conditions for the AP- proteins and on a 12 % polyacrylamide gel under reducing conditions for the lectin proteins (LgIl). Gels were blotted on a nitrocellulose membrane and subjected to immunoreactions with polyclonal antibodies prepared against high-pressure liquid chromatography-purified AP-A kindly supplied by M. Leippe, U. of Kiel, Kiel, Germany. The rabbit polyclonal antibody against the LgIl protein was produced at the Weizmann Institute using as an antigen the recombinant LgIl protein produced in bacteria from the cloned gene. The blots were washed and incubated with horseradish peroxidase conjugated to donkey anti-rabbit immunoglobulin whole antibody (Amersham Pharmacia Biotech) and developed by an enhanced chemiluminescence kit (ECL) (Amersham, Little Chalfont, Buckinghamshire, United Kingdom). The procedure was carefully optimized using various protein and antibody concentrations. Gal/GalNAc-lectin Capping Assay: Capping of the Gal-Lectin surface molecules was performed essentially as previously described [Katz U et al., 2002, MoI Biol Cell 13: 4256-4265]. Freshly harvested trophozoites were washed in PBS and divided into two tubes each (~2xlO /tube). To each tube two monoclonal antibodies ( 3F4 and 7F4; a gift from Dr. Richard Vines, TechLab, Blacksburg, VA) against the heavy (170 kDa) subunit of the Gal-lectin [34] were added at 1 :30 dilution. One of the tubes was kept at 4 ^°C and the other incubated at 37 ^°C to induce capping for 20 minutes. Fixation of trophozoites was performed with the addition of paraformaldehyde to a final concentration of 3.7 % for 15 minutes followed by a wash with 50 mM NFI₄Cl in order to block free aldehydes. Final blocking was with 2 % fetal calf serum. After washing the fixed trophozoites were incubated with FITC- labeled goat anti-mouse antibodies ( Jackson Immuno Research ) at 1: 200 dilution. Finally, samples were observed under confocal microscope (Fluoview FV500; Olympus, Tokyo, Japan). RESULTS

Analysis of transcriptionally silenced genes:

The two plasmids were transfected into trophozoites of both HM-1 :IMSS and G3. RNA isolated from the four transfectants was analyzed by northern blot (Figure 2B). The northern blot revealed that a marked down-regulation occurred in the expression of Ehlgll in the pB33 transfectants of G3. No such effect was seen in the pAY transfectants of G3 in which the 44 bp sequences of the Ehap-a signal peptide were placed before the Ehlgll gene. Transfections of parent strain HM-I : IMS S showed an increase in the level of Ehlgll transcript in both pB33 and pAY transfectants (Figure 2B) which indicates that the plasmid expressed the downstream gene. The level of ribosomal protein EhRP-LIl transcript served as loading control and was similar in all cultures. The Ehap-a gene transcript was totally absent in G3 derived samples but was equally expressed in all the HM-1:IMSS transfected cultures. Western blots of the above samples revealed a similar picture (Figure 2C). EhLgIl protein (35 KDa ) was not observed in G3 transfected with pB33 plasmid as well as in the plasmid-less RBV culture (see below) but was present in all other cultures. An unidentified lower band which cross-reacted with the anti-lgll antibodies appears in all the samples. The EIiAP-A protein, as expected, was absent from cultures derived from G3 but was present in all the HM-I. 'IMSS transfected cultures (not shown). 2

39

The source of the Ehlgll transcript in the different transfectants:

Since the Ehlgll chromosomal gene copy contains the sequences of the Ehlgll UTR while the transgene Ehlgll copy contains sequences of the UTR of Ehap-a, mRNA from the above (B33 and AY) transfectants may be transcribed from two sources. RT PCR reactions were performed on the respective mRNA samples using primers designed for the different UTR' s as set forth in Table 3 herein below and as illustrated in Figures 3A-B.

Table 3

As illustrated in Figure 3C, the G3 cultures traiisfected with pB33 plasmid had no Ehlgll transcripts, neither from the chromosomal gene nor from the plasmid copy. Trophozoites of parent strain HM-I: IMS S transfected with pB33, showed high levels of transcription of Ehlgll from the transgene copy of the gene and similar levels from the chromosomal copy as in the non-transfected control. G3 trophozoites transfected with plasmid pAY revealed that the transcript of Ehlgll, which can be seen in Figure 2B, originated from the chromosomal gene copy while the transgene copy was not transcribed and remained silenced. Transfectants of parent strain HM-1:IMSS with plasmid pAY also showed transcription of Ehlgll both from the transgene and the chromosomal copy. A similar level of ribosomal protein L21 (EhRP-L21) RT-PCR product was observed in all cultures and the Ehap-a product was absent from all G3 derived RNA as seen on Northern blots (Figure 2B). The difference between the results obtained with the two plasmids (pB33 and pAY) in G3 transfectants indicate that the ability to spread the silencing to an additional chromosomal gene, in trans, from the already silenced promoter of the Ehap-a gene, required the direct ligation of the ORF of EhIgIl to the 473 bp 5¹ upstream region of Ehap-a gene, as was the case with plasmid pB33. In plasmid pAY where there was an interruption due to the introduction of a 44 bp sequence of the Ehap-a gene ORF, only the EhIgU from the episomal gene was silenced while the chromosomal gene remained active.

Analysis of the double gene silenced cultures following removal of the plasmids

The double silenced trophozoites were cultured for several weeks (> 30 generations) without the addition of the selective drug G418 followed by cloning of single selected trophozoites. Cultures were grown from the clones which were found to be devoid of the neomycin resistance gene (Neo), as determined by PCR amplification of their genomic DNA's. The plasmid-less cultures were silenced in two genes each, the Ehap-a and the Ehlgll genes in the pB33 derived trophozoites (termed RBV) (Figure 2C).

In addition, it was observed that in RBV trophozoites, there was also no transcription of the two additional Gal/GalNAcl lectin light subunit genes Ehlgll and EhIgB (Figure 7).

G3 silenced trophozoites are required for silencing of a second gene

As described above, the successful silencing of Ehlgll was achieved by transfection of plasmid pB33 into the plasmid-less G3 trophozoites which were originally derived from the silencing with plasmid psAP-2 that only contained 473 bp of the 5' upstream region of the Ehap-a gene [Bracha R, et al., (2003) Eukaryot Cell 2: 295-305]. Attempts to silence the Ehlgll gene by transfection of plasmid pB33 into the plasmid-less trophozoites (termed Fl), derived from the silencing event which was induced with plasmid psAP-1, which also included the ORF and 3' region of the Ehap-a gene [Bracha R, et at., (2003) Eukaryot Cell 2: 295-305], failed. Transfectants of Fl trophozoites with plasmid pB33 remained silenced in the Ehap-a gene but were not silenced in the Ehlgll gene (not shown) in contrast to transfectants of G3 shown above (Figures 2B-C). Phenotype of the trophozoite cultures silenced in Ehap-a and Eltlgll genes

In order to find a specific effect of silencing of the Ehlgll gene, the ability to perform cell capping of a surface antigen of the plasmid-less RBV trophozoites silenced in the two genes was examined. As shown in Figures 4A-F₅ the capacity of the RBV trophozoites to cap the Gal/GalNAc lectin molecules following their interaction with monoclonal antibodies against the heavy subunit of the lectin (hgl) molecules was drastically reduced whereas a high degree of capping was observed in G3 or HM-l:IMSS trophozoites.

EXAMPLE 4

Analysis of transcriptionally silenced genes in pAP~CP5 transfectants MATERIALS AND METHODS

Preparation of RNA extracts: RNA extracts were prepared from parent strain HM1:IMSS, silenced strain G3; and silenced strain G3 transfected with plasmid pAP- CP5 following G418 selection. Total RNA was prepared using the RNA isolation kit TRI Reagent (Sigma).

Detection of cysteine proteinases: The cysteine proteinase inhibitor, Fmoc-

Tyr-Ala-diazomethylketone (Bachem, Switzerland) which covalently binds to cysteine proteinases, was labeled at its tyrosine residue with I¹²⁵ using the iodogen protocol (Pierce Co., USA). The [Iⁱ²⁵]-labeled inhibitor (10 μCi, 10 μg/ml) was added to logarithmically growing cultures of trophozoites (5xl0⁴/ml) for 18 hours.

The trophozoites were then harvested, washed and lysed in the presence of protease inhibitors. Bands containing the covalently bound [I¹²⁵]-labeled inhibitor were detected after SDS-PAGE separation on reducing gels and overnight exposure of the dried gel to X-ray film.

Cysteine proteinase activity: Proteinase activity of trophozoite lysates was determined spectrophotometrically by the rate of degradation of the the cysteine proteinase fluorogenic substrate Z-Arg-Arg-pNA (Bachem) in the presence or absence of DTT (20 niM) as described previously [Hellberg A, et al., (2002) Protein Expr Purif 24: 131-137]. RESULTS

Transfection of plasmid pAP-CP5 into the G3 culture resulted in total silencing of the transcription of the EhCP-5 gene as revealed by Northern blot (Figure 5B). No transcript was observed indicating that both the endogenous gene and the transgene were silenced. The lack of expression of the CP-5 enzyme was also demonstrated by labeling the cysteine proteinases of the trophozoites, in vivo, with Fmoc-[I^I25]-Tyr-Ala-diazomethylketone_; a specific inhibitor of cysteine proteinases that binds covalently to such enzymes. The labeling results (Figures 6A-B), demonstrate the disappearance of the CP-5 band in the pAP-CP-5 transfected culture as well as from the plasmid-less RB8 culture derived from it (see below). In the parent HM-1 :IMSS strain and the G3 culture the bands corresponding to CP-5 were detected (Figure 6A). The result seen with the CP-5 deficient culture is similar to the result obtained from trophozoites of the avirulent strain, E. dispar which is known to be devoid of CP-5 (Figure 6B). The total cysteine proteinase activity detected in trophozoite lysates of the AP-A and CP-5-deficient, plasmid-less RB8 trophozoites (see below), was approximately 30 % less than that present in lysates of G3 or HM- 1 :IMSS trophozoites when determined spectrophotometrically (in units per mg protein) from the degradation of the cysteine proteinase specific substrate Z-Arg- Arg-pNA (not shown). This result correlates well with the similar level of CP active bands in the in vivo labeling experiment (Figure 6A). Analysis of the double gene silenced cultures following removal of the plasmids

The double silenced trophozoites were cultured for several weeks (> 30 generations) without the addition of the selective drug G418 followed by cloning of single selected trophozoites. Cultures were grown from the clones which were found to be devoid of the neomycin resistance gene (Neo), as determined by PCR amplification of their genomic DNA's. The plasmid-less cultures were silenced in two genes each, the Ehap-a and the EhCP -5 gene in the pAP-CP5 derived trophozoites (termed RB8) (Figure 6A).

EXAMPLE 5

Virulence of double gene silenced cultures

As previously reported, [Bracha R, et al., (2003) Eukaryot Cell 2: 295-305], the G3 culture was avirulent in many of the assays used to evaluate virulence. Since the double gene silenced plasmid-less trophozoites RBV and RB8 are derived from the plasmid-less Ehap-a' silenced culture G3, it was reasonable to assume that RBV or RB8 trophozoites would also possess low virulence. MATERIALS AND METHODS

Liver lesions in hamsters: Amoebic liver abscesses in Golden Syrian hamsters were performed following laparatomy by injecting freshly harvested trophozoites (5x10⁵) directly into the frontal liver lobe as previously described [Katz U, et al., (2002) MoI Biol Cell 13: 4256-4265]. Hamsters were sacrificed after one week and the hepatic lesions determined. RESULTS

In contrast to trophozoites of strain HM-1:IMSS which gave very large liver abscesses in Syrian Golden hamsters (4 animals), the three types of silenced trophozoites (G3, RBV and RB8) were incapable of inducing hepatic lesions following injection of 5x10⁵ trophozoites/liver in none of the four animals tested per group.

EXAMPLE 6

Sequences required for the spreading of gene silencing to a second gene It was previously shown that in order to silence the Ehap-a gene, the plasmid has to contain in addition to the 5' upstream region of the Ehap-a gene, a truncated segment (> 80 bp ) of a SINEl repetitive element that is situated at the 5'end of the Ehap-a regulatory region. Omission of the truncated sequences of the SINEl element resulted in over-expression of the Ehap-a gene. The following experiment was conducted in order to ascertain which are the sequences required for the silencing of the second gene.

MATERIALS AND METHODS

Northern blot and RT-PCR: Northern blot and RT-PCR were performed as described in Example 1 and 2 hereinabove. Primers used for RT-PCR are set forth in Table 4 hereinbelow.

Table 4

Plasmid construction: pSG and pPIO were constructed as described in Example 2 hereinabove. The two additional plasmids each carried different regions ^

of the 473 bp 5 'upstream flanking region of the Ehap-a gene. In the first plasmid, pSG (Figure 8A), 195 bp of the truncated SINEl and the T-rich region that preceeds it were ligated to 300 bp of the 5 'upstream region of the Ehlgll gene followed by the EhIgU ORF and the 3 ' regulatory sequence of the Ehactin gene. The second plasmid pPIO (Figure 8B) lacked the sequences of the SINEl element and contained only 275 bp of the 5' upstream region of Ehap-a fused to the Ehlgll gene as above. Both plasmids, pSG and pPIO, were transfected into the parent strain HM-1:IMSS as well as into the G3 trophozoites. RESULTS The results from Northern blots (Figure 8D) and from RT-PCR experiments on total RNA extracted from the transfectants indicate that there was no silencing of the EhIgJl gene in either of the two transfected cultures. RNA extracts from transfectants of both HM-I. TMSS and G3 with plasmid pPIO, revealed that Ehlgll transcripts were produced from both the plasmid encoded Ehlgll gene, as revealed by RT-PCR using primers SEQ ID NOs: 22 and 27 as well as from the chromosomal copy which remained at a similar level as the control (data not shown). CONCLUSION

The conclusion from these different transfectants as well as from those shown in Figures 2 and 5 indicates (i) that the presence of both, the SINE truncated sequences and the proximal sequences of the 5' upstream region of the Ehap-a gene are essential for the silencing of the second gene and (ii) the first ATG start codon of the second gene has to be placed directly at the 3' end of the 473 bp of the 5' upstream fragment.

EXAMPLE 7 Is the entire ORF of the second gene needed to induce silencing?

The transcription initiation site of a gene usually contains sequences from the 5' UTR as well as sequences from the 5' region of the ORF. This region is known to be crucial for induction or inhibition of transcription. The following experiment was carried out in order to ascertain whether it is sufficient to introduce a truncated ORF of the second gene (starting at its start codon and without any 3' regulatory sequences) in order to maintain the silencing the chromosomal copy of that second gene. MATERIALS AND METHODS

Plasmid construction: pTL (Figure 8C) was constructed as described in Example 2 herein above, in which the 473 bp fragment of the 5' upstream region of the Ehap-a gene ligated to a truncated EhIgH fragment of 421 bp (+1-+ 421) and without 3 ' regulatory sequences, was transfected into G3 trophozoites.

RESULTS

Silencing of the chromosomal Ehlgll gene was successful in the pTL transfectants as shown by the complete absence of transcripts of Ehlgll in RNA dot blots (Figure 9). CONCLUSION

Silencing of a second gene can be accomplished with a truncated second gene and in the absence of a 3' regulatory region.

EXAMPLE 8 Triple gene silenced trophozoites

The following experiment was carried out in order to ascertain whether it is possible to generate trophozoites with three silenced virulence genes.

MATERIALS AND METHODS

Production of the cassette (Figure 10): The 473 bp fragment of the 5' upstream region of Ehap-a and a fragment of 421 bp starting from the 5' end of the

ORF of Ehlgll (truncated Ehlgll) were generated by PCR from plasmid pB33 using primers SEQ ID NOs: 11 and 21 (Table 5, hereinbelow). In parallel, the 473 bp fragment of the 5' upstream region of Ehap-a and a fragment of 396 bp starting from the 5' end of the ORF of EhC P -5 (truncated EhCP -5) were generated by PCR from plasmid pAP-CP5 SEQ ID Nos 14 and 28 (Table 5, hereinbelow). Both fragments were BamHl digested and ligated tail to tail. The resulting cassette, contained the two truncated open reading frames, each preceded by the 473 bp fragment of the 5' upstream region of Ehap-a.

Table 5

Transfection of plasmid into G3 trophozoites: G3 trophozoites of E. histolytica strain HM-1 :IMSS (as described herein above) were grown at 37 ⁰C in TYI-S-33 medium. Transfection of trophozoites was performed as previously described [Bracha, R., Y. Nuchamowitz, et al. (2003). Eukaryot. Cell 2: 295-305] and cultures were then grown in the presence of the neomycin derivative G418. The removal of plasmids was performed by growing transfectants without G418 for 3 weeks followed by cloning of trophozoites and testing for cultures that were devoid of plasmid by PCR amplification of the Neo resistance gene in genomic DNA. Northern blot analysis: RNA (5 μg) was size fractionated on a 4 % polyacrylamide denaturing gel containing 8 M urea and subsequently blotted to a nylon membrane. Using stringent conditions, hybridization was carried out with different probes (0.1 % sodium dodecyl sulfate [SDS], O.lx SSC [Ix SSC is 0.15 M NaCl plus 0.015 M sodium citrate]). Probes were randomly labeled using the Rediprime II kit (Amersham Life Science). Probes for Northern blots were made by PCR from the genomic sequences of the genes AP-A (Genbank accession No. X- 70851), LgIl (GenBank accession No. M96024) and CP-5 (GenBank Ace No. X91644.2).

RESULTS As illustrated in Figure 1 1, the generated trophozoites comprised three down- regulated genes. The AP-A gene (not shown) was absent in all the samples. The LGL gene and the CP-5 gene had no transcripts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the ait. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

WHAT IS CLAIMED IS:

1. A viable, avirulent amoeba of E. histolytica.

2. The viable, avirulent amoeba of E. histolytica of claim 1, being genetically modified.

3. A pharmaceutical composition comprising the viable, avirulent amoeba of E. histolytica of claim 2.

4. A method of generating an anti-amoebiasis vaccine, the method comprising down-regulating gene expression of at least two genes of a genome of E. histolytica, each being down regulated by independent exogenous genetic means, thereby generating the anti-amoebiasis vaccine.

5. A method of preventing amoebiasis, comprising administering to a subject in need thereof an immunogenically effective amount of the pharmaceutical composition of claim 3, thereby preventing amoebiasis.

6. A method of down-regulating gene expression in a genome of an amoeba of E. histolytica, comprising

(a) introducing into the amoeba of E, histolytica a first nucleic acid construct comprising a regulatory sequence as set forth in SEQ ID NO: 1 or a functional equivalent thereof; and subsequently

(b) introducing into the amoeba of E. histolytica a second nucleic acid construct comprising:

(i) said regulatory sequence; and

(ii) a polynucleotide encoding a polypeptide of E. histolytica, operably linked to said regulatory sequence, thereby down-regulating gene expression in a genome of an amoeba of E. histolytica.

7. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claims 2 or 3, wherein said genetic modification comprises down- regulation of at least two genes, each being down regulated by independent exogenous genetic means.

8. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claims 2 or 3, wherein said genetic modification comprises down- regulation of at least three genes, each being down regulated by independent exogenous genetic means.

9. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claims 7 or 8, wherein at least one of said down-regulated genes is epigenetically down-regulated.

10. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claims 7 or 8, wherein said down-regulated genes are virulent factor genes.

11. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 10, wherein said virulent factor genes are selected from the group consisting of an amoebapore gene, a galactose specific lectin gene and a cysteine proteinase gene.

12. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 10, wherein at least one of said virulent factor genes is ap-a.

13. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 12, wherein said ap-a is epigenetically silenced.

14. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 11 , wherein said amoebapore gene is selected from the group consisting of ap-a, ap-b, ap-c and saposin-like.

15. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 14, wherein said amoebapore gene is ap-a.

16. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 11, wherein said galactose specific lectin gene is selected from the group consisting of IgI- 1 , lgl-2, lgl-3, hgl-1, hgl-2, hgl-3, hgl-4 and hgl-5.

17. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 16, wherein said galactose specific lectin is IgI- 1.

18. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 11, wherein said cysteine proteinase is selected from the group consisting of cysteine proteinase 1, cysteine proteinase 2 cysteine proteinase 3 cysteine proteinase 4 and cysteine proteinase 5, cysteine proteinase 6, cysteine proteinase 7, cysteine proteinase 8, cysteine proteinase 9, cysteine proteinase 10, C3'steine proteinase 11, cysteine proteinase 12, cysteine proteinase 13, cysteine proteinase 14, cysteine proteinase 15, cysteine proteinase 16, cysteine proteinase 17, cysteine proteinase 18, cysteine proteinase 19 and cysteine proteinase 1 12.

19. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 11, wherein said cysteine proteinase is cysteine proteinase 5.

20. The pharmaceutical composition of claim 3, further comprising a pharmaceutically acceptable carrier.

21. The viable avirulent amoeba of E. histolytica or phaπnaceutical composition of claims 7 or 8, wherein said viable, avirulent amoeba of E. histolytica comprises an epigenetic nucleic acid construct comprising a regulatory sequence, as set forth in SEQ ID NO: 1 or a functional equivalent thereof.

22. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 2 or 3, wherein a strain of said viable, avirulent amoeba of E. histolytica is selected from the group consisting of HM-1:IMSS, HU -21: AMC, Rahman, KUlMUSC, HU-1:CDC, DKB, SAW 755CR, SAW 891R, SD157, SD184, HK-9, HB-301:NIH, 200:NIH, HU-2:MUSC, HU-I :CDC, H-302:NIH, H-303:NIH, H-458:CDC, HM-3:IMSS, HU22.-AMC, HI- 1295. -AIIMS and HB-301 :NIH.

23. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 22, wherein said strain of viable, avirulent amoeba of E. histolytica is HM-l :IMSS.

24. The viable avirulent amoeba of E. histolytica or pharmaceutical composition of claim 2 or 3, wherein said viable, avirulent amoeba of E. histolytica is stably avirulent.

25. The method of claim 4, wherein said down-regulating gene expression of said at least two genes is effected by an agent selected from the group consisting of a regulatory sequence, a dsRNA, an antisense RNA and a ribozyme.

26. The method of claim 4, wherein said down-regulating gene expression of said at least two genes is effected sequentially.

27. The method of claim 26, wherein said sequential down-regulation of said at least two genes is effected by introducing the epigenetic nucleic acid construct of claim 20 into a viable amoeba of E. histolytica.

28. The method of any of claims 6 or 27 further comprising removing said nucleic acid construct from said viable amoeba of E. histolytica.

29. A nucleic acid construct system comprising: (a) a first nucleic acid construct comprising:

(i) a regulatory sequence as set forth in SEQ ID NO: 1 or a functional equivalent thereof; and (b) a second nucleic acid construct comprising:

(i) said regulatory sequence; and

(ii) a polynucleotide encoding a polypeptide of E. histolytica other than an amoebapore polypeptide, operably linked to said regulatory sequence.

30. The method or nucleic acid construct of claims 6 or 29, wherein said polynucleotide encodes at least a portion of an ORF of said polypeptide.