BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to compositions and methods of use of immunodominant surface antigen polypeptides from Entamoeba protozoa and, more particularly, to the preparation of reagents useful for diagnosing, treating, or inhibiting Entamoeba protozoa infections.

Description of the Background Art

Entamoeba histolytica is a common human pathogenic protozoa. moebias-'^, or infection by E. histolytica, causes a spectrum c. disease ranging from a commensal state in asymptomatic carriers to fulminant diarrhea or extra-intestinal abscess formation in invasive infections. See, e.g., Merck Manual. (15th Ed.), chapter 13. Entamoeba histolytica exists in two forms, a motile trophozoite or a dormant cyst. Virulent amoebae infections can cause ulceration of the intestinal epithelium and may penetrate the bowel wall to form extra-intestinal abscesses, primarily in the liver. The infection rate in the United States is about 1%, but the carrier rate may exceed 50% in certain areas of the world. With the availability of effective treatment regimens, early diagnosis is crucial for the prevention of disease and transmission. However, much controversy has centered on benefits and drawbacks of initiating therapy in asymptomatic in actions. Thus recent investigations have focussed on a molecular genetic analysis of virulence and the definition of marker molecules which have high predictive value and can be applied in a clinically feasible fashion.

Several molecular activities thought to correlate with the virulent phenotype have been partially characterized, but the role of each of these in pathogenesis is not understood. Until recently, it was unclear whether invasiveness is a stable or a variable genotypic characteristic of a particular strain. Polymorphisms in the electrophoretic mobility of the glycolytic enzymes phosphoglucomutase (PGM) , hexokinase (HK) , and phosphoglucoisomerase (PGI) have been used to identify pathogenic and non-pathogenic strains.

More recently several other diagnostic reagents have been suggested for distinguishing pathogenic strains, based either on DNA sequences, or the detection of specific antigens. See Garfinkel, et al. (1989) Infect. Immun. 57:926-931; Samuelson, et al. (1989) J. Clinic. Microbiol. 27:671-676; Tannich, et al. (1989) Proc. Nat'l. Acad. Sci. USA 86:5118-5122; and Strachnan, et al. (1988) Lancet i:561-562. However, these tests typically require axenic cultivation and cloning of amoebae directly from fresh stool samples prior to assay. These cultivation steps are difficult and, in most cases, have not been achieved. None of these probes have been validated by large scale screening of clinically defined strain isolates in comparison with extant criteria, such as accepted zymodeme identifying characteristics.

Moreover, axenization and cloning of amoebae from patient isolates often favors outgrowth of the less fragile pathogenic strains and has been known to reversibly attenuate virulence. Thus, it is important to develop direct, accurate, and quick tests which can identify pathogenic amoebae in fresh isolates. Diagnostic reagents are needed which allow clinical characterization of pathogenic infections caused by a mixture of strains or infections which may have interconverted phenotypically from pathogenic to non-pathogenic strains. In addition, new reagents for treatment and prevention of infection by amoeba are always valuable. The present invention

provides these and other important reagents and methods for their effective use.

SUMMARY OF THE INVENTION Various alleles of an immunodominant surface antigen from Entamoeba histolytica have been identified and characterized. Genes encoding the antigens have been isolated and sequenced, thus providing detailed information on their structure. Both polypeptides and nucleic acids and fragments thereof are provided. Highly specific antibody preparations, both polyclonal and monoclonal, against epitopes on the immunodominant surface antigen have been produced. Treatment and vaccination methods for amoebiasis are provided.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Western blot of whole Entamoeba extract fractionated by 5-15% SDS-PAGE; lanes 2, 7, and 12, polyxenic pathogenic E. histolytica isolate SD-4, lanes 3, 8, and 13, polyxenic non-pathogenic E. histolytica isolate SD116, lanes 4, 9, and 14, E. histolytica-like Laredo, lanes 5, 10, and 15, E. histolytica HK-9, lanes 6, 11, and 16, E. histolytica HM1:IMSS probed with anti-membrane fraction serum (lanes 2 through 6) , pooled human immune sera (lanes 7 through 11) , and monoclonal antibody FA7 (lanes 12-16) ; molecular weights are given in kilo-Dalton (molecular weight standards lane 1: 200, 97, 68, 43, 28 kDa) .

Figure 2: Photographs (800-fold magnification) of

E. histolytica HM1:IMSS trophozoites labeled in vivo with primary antibodies. A: pool of human anti-E. histolytica immune sera at 1:500 dilution, B: pool of human anti-E. histolytica immune sera purified by binding to λcM17 phage lysates, C: monoclonal FA7 harvest fluid at 1:1000 dilution, D: monoclonal anti-E. histolytica actin antibody at 1:1000 dilution; secondary

antibodies, fluorescein isothiocyanate goat-anti-human and fluorescein isothiocyanate goat-anti-mouse.

Figure 3: Transfer blot of E. histolytica HM1:IMSS RNA probed with cDNA clone λcM17 indicates a single hybridizing band migrating at ~3 kb. Hybridization conditions were 50% formamide, 0. 2 X SSC, 42*C. Autoradiography shown required a 72 hr exposure.

Figure 4: Restriction endonuclease (Eco RV lower case letters, Ssp I capital letters) digests of PCR products generated by amplification of genomic DNA from E. histolytica isolates/strains using oligonucleotide primers SR018 + SR021 and SR019 + SR022. a/A = #43, b/B = #44, c/C = SD116, d/D = REF291, e/E = E. histolytica-like Laredo, f/F = #46, g/G = HK9, h/H = HM1:IMSS.

DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention provides novel compositions and methods for diagnosing, treating and vaccinating Entamoeba parasitic infections. The present invention is based, in part, on the discovery of a class of highly immunogenic, or immunodominant, 125 kDa proteins, designated M17 proteins, which are localized on the membrane of the trophozoite form of E. histolytica. Particular allelic forms of this 125 kDa antigen have been isolated and found to be characteristic of pathogenic types of amoeba, and such identification and sequencing of the antigens provides a basis for preparation of novel compositions including vaccines, polypeptides, and polypeptide fragments. Other compositions according to the present invention include nucleic acids encoding the various surface antigens and homologous polypeptides, nucleic acids homologous to those encoding peptides, as well as antibodies raised against the proteins, fragments, and homologous polypeptides. Methods for the use of these compositions are provided in view of the discoveries related to biological function.

The genus Entamoeba is defined by a number of cellular and biological markers. These markers define a genus Entamoeba, which exhibit common characteristics, but which may vary as better detection methods and functional tests are developed. However, the term Entamoeba, as used herein, is intended to include organisms which now are classified therein or are sufficiently similar as to share significant epitopes or biological properties characteristic of organisms assigned to this genus.

These E tamoeba strains often invade the body causing a oebiasis. An invasiv infection includes infection within the intestines, or into the body through the intestinal wall. The reagents provided herein are useful for both types of infections.

An immunodominant 125 kDa surface antigen has been isolated from various strains of Entamoeba. Table I sets forth the genomic sequences and corresponding amino acid sequences of two alleles of the 125 kDa immunodominant surface antigen, from a pathogenic strain designated HM1:IMSS and a non-pathogenic strain designated REF 291 strains. It will be appreciated that other alleles will exist in nature and that the compositions may be derived from such other alleles or the nucleic acid or amino acid sequences of such alleles, as described in more detail herein after.

Table I: Inferred amino acid sequence and nucleotide sequence of coding region and flanking region obtained from genomic clone pBSgM17-l. The sequence of the internal EcoRI fragment was identical in both genomic clones (pBSgM17-l/2) and the cDNA clone λcM17. Shown aligned below is the partial nucleotide sequence of PCR amplification products derived from non-pathogenic isolate REF291. Nucleotide substitutions are underlined and amino acid substitutions are indicated below the partial sequence derived from REF291. Two boundaries are marked between Glyl86 and Ilel87 (corresponding to A558 and A559) and between Phe825 and Gln826 (corresponding to C2475 and C2476) . These boundaries separate region I (amino or 5' proximal of the former boundary) from region II (between the boundaries) and region II from region III (carboxy or 3 ' proximal of the latter boundary) .

The amino acid sequence inferred from the nucleotide sequence of the coding region of the 125 kDa antigen is unusual with respect to its high Asn (90 =8.2%), Tyr (70=6.3%) and hydroxyl amino acid residue (Ser, 85=7.6%; Thr, 90=8.1%) content. While a total of 17 N-linked glycosylation sites suggests that the 125 kDa antigen may be glycosylated, Western blot analysis shows that this antigen migrates as a compact band on SDS-PAGE. A distinctly hydrophobic amino terminal region of 35 amino acids may serve as anchor or signal sequence. Compared to known prokaryotic and eucaryotic signal sequences this region contains an unusually long (20 amino acids) N-terminal (n) region with a single positively charged residue, an 8 amino acid long hydrophobic core (h) region and a 7 amino acid long polar C-terminal (c) region with an amino acid composition similar to those seen in other signal sequences. By extrapolation this would imply that the antigen is either a peripheral membrane protein or it may be anchored in the membrane by other means such as a glycophospholipid anchor. Alternatively, the hydrophobic amino terminal itself may serve to anchor the antigen in the membrane with the C-terminal externally exposed as no additional trans membrane domains could be discern _* d.

Table I, panel 1 gaagctataaataagttatagaaatataaaagaatg ttaaaaatgaaaacaaacataaaaaataagtgtatttaaagtgtttttaaaaaaactaat t*ATTCATAAATTAAAGTT

20

61

121

181

241

301

361

421

481

541

601

661

721

781

841

901

961

Table I, panel 2

1021

1081

1141

1201

1261

1321

480

His Leu Arg Ser Tyr Val Asn Met Ala His Ala Phe Gly Thr Asp Thr Leu He Ala Leu

1381 CAT TTA AGG TCT TAT GTT AAT ATG GCA CAT GCA TTT GGA ACA GAC ACT TTA ATT GCT TTA CAT TTA AGG TCT TAT GTT AAT ATT GCA CAT GCA TTT GGA ACA GAT ACT TTA ATT GCT TTA

He

500

Val Lys Ser Tyr Tyr Gly Leu Trp Tyr Glu Asn Asn Phe Glu Ser Lys Tyr Ser He Lys

1441 GTT AAA TCT TAT TAT GGA TTA TGG TAT GAA AAT AAT TTT GAA AGT AAA TAT TCA ATT AAA GTT AAA TCT TAT TAT GGG CTA TGG TAT GAA AAT AAT TAT GAA GGT GAG TAT TCA ATT AAG

Tyr Gly Glu

520

Arg Asp Ser Thr Ser Ala Phe Cys Leu Leu Ala Ala Leu Val Thr Lys Arg Asp Thr Arg

1501 AGA GAT TCT ACC TCT GCT TTC TGT TTG TTA GCT GCA TTA GTT ACA AAA AGA GAT ACT AGA AGA GAT TCA ACT TCA GCT TTC TGT TTG TTA GCT GCA ATI ~~~ ACA AAA AGA GAT ACT AGA

He AΪa

540

Tyr Leu Cys Ser Leu Phe Lys Tyr Asp He Gin Ser Asn Val Ser Glu Ala He Lys Asn

1561 TAC TTA TGT TCT CTA TTT AAA TAT GAT ATA CAA TCA AAT GTT TCA GAA GCA ATT AAA AAT TAT TTA TGT TCT CTT TTT AAA TAC GAT ATA CAA £AA AAT GTT TCA GAA GCA ATT AAA AAC

Gin

560

Met Asn Tyr Pro Thr Tyr Tyr Pro Phe Phe Asn Leu Tyr Ala Met Ser Tyr Asn Gly Asn

1621 ATG AAT TAT CCA ACT TAT TAT CCA TTC TTC AAC CTC TAT GCC ATG AGT TAT AAT GGA AAT ATG AAT TAT CCA ACT TAT TAT CCA TTC TTC AAT jGTT TAT GCT ATG AGT TAC AAT GGA AAT

Val

Table I, panel 3

580

Tyr Tyr Gly Arg Pro Tyr Lys He Pro Tyr Gly Arg Thr Arg Leu Asn Phe Thr Ala Thr

1681 TAC TAT GGA AGA CCC TAT AAA ATT CCA TAT GGA AGA ACT AGA TTG AAT TTC ACT GCA ACT

TAT TAT GGA AGA A.CA TAT AAA ATT CCA TAT GGT ACA ACT AGA TTG AAT TTT ACA GCA ACC

Thr Thr

600

1741

1801

1861

1921

1981

2041

2101

2161

2221

2281

Table I, panel 4

Phe He Arg He Gly Tyr Cys Tyr 2341 TTT ATT AGA ATA GGA TAT TGT TAT TTT ATT AGA ATA GGG TAT TGT TAT

Cys Ser Val Ser Asp He Gly Ser

2401 TGC AGT GTA TCA GAT ATT GGA AGC TGT AGT G£A TTA GAT ATT GGA AGC

Gly Leu

840

Lys Glu Pro Glu Phe|Gin He Pro Pro He Lys Tyr Ser Arg Pro Thr Arg Phe Leu Thr

2461 AAA GAA CCA GAA TTC|CAA ATT CCA CCA ATT AAA TAC AGC AGA CCA ACA CGT TTC TTA ACT 860

2521

2581

2641

2701

2761

2821

2881

2941

3001

3061

3121

3181

3241

3301

3444 3365 cattttaaataatgtagtgttattttaattttattgagaaaattttgagtctattteatt acatattgaatcatgattg

The M17 protein has been determined to be membrane associated. When these proteins are cross- linked with multivalent antibody molecules, live trophozoites cap the complexes in a manner characteristic of membrane capping. See Figure 2. This result is achieved with pooled patient sera, with the FA7 monoclonal antibody, or with immunoselected antibody preparations. Cells also appear to round upon antibody binding, are likely disrupted biologically, and perhaps even unable to divide. Thus, the immunodominant antigen is determined to be a surface antigen and attachment by antibodies is likely to cause significant disruption of the infective cycle of the trophozoites.

The present invention, in one embodiment, provides polypeptides which are related to the 125 kDa surface antigen and which will usually be either haptenic or antigenic, typically including at least about 6 amino acids, usually at least about 9 amino acids, and more usually about 12 or more amino acids found contiguously within a natural form of the 125 kDa immunodominant surface antigen protein. Longer polypeptides will also find use, up to and including substantially full length of the natural protein and larger. The contiguous amino acids may be located within any region of the polypeptide, but preferably in regions I or III of Table I, and will correspond to at least one epitope site which is characteristic of the particular immunodominant surface antigen protein. By characteristic, it is meant that the epitope site will allow immunologic detection of the exposed polypeptide segment in a cell sample with reasonable assurance, in most cases allowing immunodominant surface antigen of a pathogenic form to be immunologically distinguished from other related proteins, such as an immunodominant surface antigen from non-pathogenic strains. The polypeptides will also be capable of inducing an immune response in a host when

used in vaccines, in therapeutic compositions, and for preparing polyclonal and monoclonal antibodies.

The compositions and methods of the present invention are particularly useful for identifying and treating pathogenic amoebic infection, but it will also be possible to identifying epitopes of the surface antigen which are conserved among pathogenic and non- pathogenic amoeba. Use of polypeptides and nucleic acid probes based on the sequences of such conserved epitopes allows detection and treatment of both pathogenic and non-pathogenic amoeba. In contrast compositions based on epitopes found in pathogenic forms only allows specific detection and treatment of pathogenic amoeba. Conversely, epitopes found in non-pathogenic forms only allow specific detection and treatment of non-pathogenic amoeba. Regions of particular importance for distinguishing pathogenic strains from non-pathogenic strains will be those located at the sites of difference between the amino acid or nucleotide differences, as indicated in Table III.

In a preferred aspect, the polypeptide compositions will be either identical or equivalent to a sequence set forth in Table I. By equivalent, it is meant for the purposes of defining polypeptides that a substantial identity of amino acid sequences exists over a stretch of at least about 10 residues, where each position of the corresponding sequences is either identical to or has a conservative substitution in at least about 60% of the residues, preferably at least about 70% of the residues and more preferably at least about 80% of the residues. The compared sequence segments may, however, be modified by occasional deletions, additions, or replacements in accordance with known methods for comparison. See, e.g.. Sequence Analysis Software Package. Univ. Wisconsin Biotechnology Center, Madison, Wisconsin. Conservative substitutions are replacements within the groups gly, ala; val, ile,

leu; asp, glu; asn, gin; ser, thr; lys , arg; and phe, tyr.

Synthetic polypeptides which are immunologically cross-reactive with a natural immunodominant surface antigen protein (i.e., immunological analogs) may be produced by either of at least two general approaches. First, polypeptides having fewer than about 50 amino acids, more usually fewer than about 20 amino acids, can be synthesized by the Merrifield solid-phase synthesis method where amino acids are sequentially added to a growing chain. See Merrifield (1963) J. Am. Chem. Soc. 85:2149-2156. The amino acid sequences of such synthetic polypeptides will usually be based on the sequences described in Table I, preferably regions I or III.

A second and preferred method for synthesizing the polypeptides of the present invention involves the expression in cultured cells of recombinant DNA molecules encoding a desired portion of an immunodominant surface antigen gene. The gene may itself be natural or synthetic as described below. The natural gene is obtainable from cDNA or genomic libraries, as described herein. Using such segments or genes, additional homologous gene sequences might be isolated from other related strains or sequences which encode peptides equivalent to those described above. Such genes represent, among other possibilities, other alleles of or phantom genes of related polypeptides. By using sequences from such genes, segments may be found which have similar biological functions which might be equivalent to these described and would be substituted for the listed segments. In particular, epitopic similarity can serve as a means for selecting equivalent peptides. To be useful in the detection methods of the present invention, the polypeptides are usually obtained in substantially pure form, that is, typically about 50%

w/w or more purity, substantially free of interfering proteins and contaminants. Preferably, the immunodominant surface antigen polypeptides are isolated or synthesized in a purity of at least about 80% w/w and, more preferably, in at least about 95% w/w purity. Using conventional protein purification techniques, homogeneous polypeptides of at least 99% w/w can be obtained. For example, the proteins may be purified by use of the antibodies described hereinafter using immunoadsorbent affinity chromatography. Such affinity chromatography is performed by first linking antibodies or appropriate affinity reagents to the solid support and then contacting the linked antibodies or affinity reagents with the source of the immunodominant surface antigen proteins, e.g., lysates of protozoa which naturally produce immunodominant surface antigen or which produce immunodominant surface antigen as a result of introduction of a recombinant immunodominant surface antigen DNA molecule. Useful production cultures include the insect

Bacculovirus system, see Luckow and Summers (1988) Biotechnology 6: 47-55, or the T7 polymerase gene with a φlO promoter, see Rosenberg et al. (1987) Gene 56: 125- 135, and Studier and Moffatt (1986) J. Mol. Biol. 189: 113-130, each jf which is hereby incorporated herein by reference. Various other high capacity systems expressing recombinant nucleic acids, described below, are provided herein. See, e.g., Sambrook, et al. (1989) Molecular Cloning: A Laboratory Method. Cold Spring Harbor Press.

In accordance with the present invention, nucleic acid seσ snces encoding portions of the polypeptide sequence of distinct forms of immunodominant surface antigens have been isolated and characterized. Isolated DNA segments encoding the immunodominant surface antigens can be expressed to provide isolatable quantities of polypeptides displaying biological (e.g.,

immunological) properties of naturally-occurring immunodominant surface antigens. Other useful nucleic acids are substantially homologous to encoding sequences, either natural or artificial. These homologous polynucleotides will find use as probes or primers for locating or characterizing natural or artificial nucleic acids encoding the surface antigen peptides.

Substantial homology or substantial identity of a nucleic acid sequence indicates either that: a) there is greater than about 65%, typically greater than about 75%, more typically greater than about 85%, preferably greater than about 95%, and more preferably greater than about 98% homology with a disclosed segment of at least about 10 contiguous nucleotides; or b) the homologous nucleic acid sequence will hybridize to the consensus sequence or its complementary strand under stringent conditions of temperature and salt concentration. These stringent conditions will generally be at temperatures greater than about 22"C, usually greater than about 30"C and more usually greater than about 45"C. Salt concentrations are generally less than about 1 M, usually less than about 500 mM, and preferably less than about 200 mM. The combined conditions will be more important than either the salt concentration or the temperature alone. Other parameters which are used to define stringency include GC content of the sequence, extent of complementarity of the sequences and length of segments involved in the hybridization, besides composition of buffer solutions used in the hybridization mixture. Nucleic acids can be synthesized based directly on the DNA sequences reported in Table I. Polynucleotides may be synthesized by known techniques, for example, short single-stranded DNA fragments may be prepared by the phosphoramidite method described by Beaucage and Carruthers (1981) Tett. Letters 22:1859- 1862. A double-stranded fragment may then be obtained either by synthesizing the complementary strand and

annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Polymerase chain reaction techniques may be used for production of probes or amplification of polynucleotides for synthetic purposes. See Innis et al. (Ed.) (1990) PCR Protocols. Academic Press, N.Y., which is hereby incorporated herein by reference.

The natural or synthetic DNA fragments coding for a desired immunodominant surface antigen fragment will often be incorporated in DNA constructs capable of introduction to and expression in an in vitro cell culture. Usually, the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction and integration within the genome of cultured mammalian, protozoa, or other eucaryotic cell lines. DNA constructs prepared for introduction into bacteria or yeast will usually include a replication system recognized by the host, the appropriate immunodominant surface antigen DNA fragment encoding the desired polypeptide product, transcriptional and translational initiation regulatory sequences joined to the 5'-end of the immunodominant surface antigen DNA sequence, and transcriptional ε_._d translational termination regnlatory sequences joined to the 3*-end of the immunodomin..nt surface antigen sequence. The transcriptional regulatory sequences will typically include a heterologous promoter which is recognized by the host. Conveniently, available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with an insertion site for the immunodominant surface antigen DNA sequence may be employed. Synthesis and production of the immunodominant surface antigen polypeptides, fragments, fusion proteins, and variants thereof have been described above.

Similarly, the nucleic acids and fragments which encode or are homologous to sequences which encode these epitopes have been described. Expression of characteristic forms of the immunodominant surface antigens have been associated with amoebiasis, and even distinction between pathogenic or non-pathogenic iiinfection may be achieved. As further demonstrated herein, pathogenic infection is characterized by detectable expression of the polypeptide epitopes of pathogenic-specific immunodominant surface antigen proteins. Non-pathogenic infection is likewise detectable using non-pathogenic-specific epitopes.

Production of antibodies to characteristic epitopes will allow for distinguishing between pathogenic and non-pathogenic strains. General detection of infection may use epitopes common to both pathogenic and non-pathogenic strains, but distinguishing will usually be directed to epitopes involving amino acids which differ and are indicated in Table I. Once a sufficient quantity of intestinal immunodominant surface antigen polypeptide has been obtained, polyclonal antibodies specific for the immunodominant surface antigen protein can be produced by in vitro or i__ vivo techniques. Jin vitro techniques involve .in vitro exposure of lymphocytes to the antigenic polypeptides or fragments, while in vivo techniques require the injection of the polypeptides or fragments into any of a wide variety of target immune systems, such as vertebrates. Suitable vertebrates are typically non- human, including mice, rats, rabbits, sheep, goats, and the like. Polypeptides having more than about 5 to 30 amino acids, particularly more than about 50 to 100 amino acids, may serve directly as immunogens. if the polypeptide is smaller than about 10 kD, particularly less than about 6 kD, it may be necessary to join the polypeptide to a larger molecule to elicit the desired immune response. The immunogens are then injected into

the animal according to a predetermined schedule, and the animals are bled periodically with successive bleeds generally having improved titer and specificity. The injections may be made intramuscularly, intraperitoneally, subcutaneously, or the like, and usually an adjuvant, such as incomplete Freund's adjuvant, will be employed.

While the invenntion embraces antibodies made against polypeptides provided, it also embraces proteins which are specifically recognized by antibodies made against epitopes provided herein. Thus, the FA7 monoclonal antibody also defines a class of proteins which are targets for specific binding.

If desired, monoclonal antibodies can be obtained by preparing immortalized cell lines capable of producing antibodies having the desired specificity. The FA7 monoclonal antibody is produced by such a cell line. Such immortalized cell lines may be produced in a variety of ways depending upon the target immune system. Conveniently, a small vertebrate, such as a mouse, is hyperimmunized with the desired antigen by the method just described. The vertebrate is then killed, usually several days after the final immunization, the spleen removed, and the spleen cells immortalized. The manner of immortalization is not critical. Presently, the most common technique is fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519. Other techniques include EBV transformation, transformation with bare DNA, e.g., oncogenes, retroviruses, etc., or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies. Common techniques are described, e.g., in Lane and Harlow, (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Press, N.Y.; and Goding (1986) Monoclonal Antibodies:

Principles and Practice. (Second Edition) Academic Press, N.Y., each of which is hereby incorporated herein by

reference. New techniques for in vitro production of antibodies may also be applied. See, e.g., Huse et al. (1989) Science 246: 1275-1281, Ward et al. (1989) Nature 341: 544-546, each of which is hereby incorporated herein by reference.

When employing fusion with a fusion partner, the manner of fusion is usually not critical and various techniques may be employed. Conveniently, the spleen cells and myeloma cells are combined in the presence of a nonionic detergent, usually polyethylene glycol, and other additives such as Dulbecco's Modified Eagle's Medium, for a few minutes. At the end of the fusion, the nonionic detergent is rapidly removed by washing the cells. The fused cells are promptly dispensed in small culture wells (usually in a microtiter plate) at relatively low density, ranging from about 1-5x10 per well, in a selective medium chosen to support growth of the hybrid cells while being lethal to the myeloma cells. Usually, the myeloma cell line has been mutated to be sensitive to a lethal agent, typically being HAT sensitive. After sufficient time, usually from one to two weeks, colonies of hybrids are observed and plates containing hybrid positive wells are identified. The plates and wells having only one colony per well are selected, and supernatants from these wells are tested for binding activity against the desired intestinal immunodominant surface antigen protein or the isolated antigen. Once positive hybridomas are identified, the cell line can be maintained as viable cultures and/or by lyophilization or frozen storage.

Depending on the desired use for the antibodies, further screening of the hybridomas may be desirable. Hybridomas providing high titers are desirable. Furthermore, cytotoxic antibodies, e.g., ^I 9 ^G ₂ a' ^IgG 2b' ^IσG 3 ^{and IσM} ' ^{may be selec} ted for use in therapeutic treatment of pathogenic infections. For use

in immunodiagnostic assays, antibodies having very high specificity fsr the antigenic site are desirable.

Once the desired hybridomas have been selected, monoclonal antibodies may be isolated from the supernatants of the growing colonies. The yield of antibodies obtained, however, is usually low. The yield may be enhanced by various techniques, such as injection of the hybridoma cell line into the peritoneal cavity of a vertebrate host which will accept the cells. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Proteinaceous and other contaminants will usually be removed from the monoclonal antibodies prior to use by conventional technique, e.g., chromatography, gel filtration, precipitation, extraction, or the like.

By properly selecting polypeptides used as the immunogen, antibodies having high specificity and affinity for the desired immunodominant surface antigen epitope can be obtained. The polypeptide selected should represent one or more epitopic sites which are unique to the desired immunodominant surface antigen protein and which can distinguish immunodominant surface antigen from closely related proteins. Such unique epitopes are found on polypeptides expressed by cells containing sequences disclosed in Table I. One particular example of such is the FA7 monoclonal antibody.

The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labelled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibi¬ tors, fluorescers, chemiluminescers, magnetic particles, and the like. Patents teaching the use of such labels

include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, which are incorporated herein by reference.

Antibodies and polypeptides prepared as described above can be used in various immunological techniques for detecting immunodominant surface antigen proteins in biological specimens, particularly cell samples such as biopsy tissue samples and body fluid samples, including blood, plasma, serum, urine, stool, and the like. As amoebiasis infections typically originate in the intestinal flora, stool samples will routinely be the samples of choice. Depending on the nature of the sample, both liquid phase assays and solid- phase immunohistochemical staining techniques will find use. Conveniently, immunohistochemical staining techniques may be used with cell samples including tissue samples, sputum, and lung lavage samples. For example, a tissue sample may be fixed in formalin, B-5, or other standard histological preservative, dehydrated and embedded in paraffin as is routine in any hospital pathology laboratory. Sections may then be cut from the paraffinized tissue block and mounted on glass slides. The immunodominant surface antigen proteins, if present, may then be detected in the cytoplasm or extracellular space by exposure with labeled immunodominant surface antigen antibody or exposure to unlabelled anti- immunodominant surface antigen antibody and a labeled secondary antibody. Because the antigen is a surface antigen and exposed extracellularly, it is unnecessary to denature a sample, allowing assay of non-denatured target sample. This may also allow further live characterization of the same tested sample. Sputum and lavage samples are typically prepared in a similar manner where the sample is first dehydrated by exposure to a dehydrating agent, typically a low molecular weight alcohols

Liquid phase immunoassays or Western blot analysis will also find use in the detection of the immunodominant surface antigen proteins particularly in body fluids when the proteins are shed into such fluids, e.g., blood or stool. Solid tissue and sputum samples may also be assayed in liquid phase systems by lysing the cellular sample in order to release the protein. Once the protein is released, the sample will be placed in a suitable buffer, the sample buffer subjected to a suitable im unoassay. Numerous competitive and non- competitive immunoassays are available and described in the scientific and patent literature. Having described how to make various diagnostic reagents, particularly antibodies polyclonal and monoclonal, and fragments thereof with binding sites, diagnostic kits for using them are also provided. Similar kits may be prepared using particular nucleic acid probes. The kits will typically have at least one compartment comprising the detection reagent to be applied to an appropriate sample. The reagent may be attached to a dipstick or similar physical entity. Alternatively, the reagent may be contained in a liquid solution to which a sample is added. A compartment containing the active ingredient may be a sealed envelope, a plastic bag, a vial, a bottle, a jar, an ampule, a well, or any other protective package.

The antibodies of the present invention may also find use in therapy and other medical applications. For example, immunodominant surface antigen antibodies, may be coupled to toxins, such as diphtheria toxin and the ricin A chain, and administered to patients, or hosts, suffering from pathogenic Entamoeba infections. The use of antibody conjugated toxins in cancer therapy is described generally in U.S. Patent Nos. 4,093,607; 4,340,535; 4,379,145; and 4,450,154. Antibodies alone may also find use in treatment, particularly by blocking or interrupting some functional activity of

immunodominant surface antigen protein which contributes to the pathogenic phenotype.

In a particular embodiment of the present invention, the binding fragments will be joined to active substances, such as proteolytic enzymes or glycosidases, in order to enhance interference with amoeba infection. The binding fragments will specifically bind to the trophozoites and the active substances will act to destroy the protozoa. Vaccines against Entamoeba infections are producible using the compositions of the present invention. These vaccines may be passive, consisting of Ig supplementation which interferes with amoeba growth or toxicity. Alternatively, vaccines may be active producing a cellular response providing protective immunity by cytotoxic or other active suppression of amoeba infections.

A vaccine prepared utilizing the immunodominant surface antigen or immunogenic equivalents thereof can consist of: (a) fixed cells either recombinantly altered to produce these antigen proteins or cells from the Entamoeba itself; (b) a crude cell extract; (c) a partially or completely purified immunodominant surface antigen preparation. Fusion proteins combining a segment of the immunodominant surface antigen will be readily prepared. In some embodiments, a multiple vaccination may be achieved by fusing these antigens with other target antigens on a single protein, inducing protection against multiple infectant vectors. Alternatively, a "cocktail' ¹ of different immunogens may be simultaneously administered or inoculated. These immunogens can be prepared in vaccine dose form by well-known procedures. These vaccines can be in the form of an injectable dose and may be administered intramuscularly, intravenously, or subcutaneously. These vaccines can also be administered intranasally by aspiration, or orally by mixing the active components with food or water,

providing a tablet form, and the like. Means for administering, or more typically inoculating, these vaccines should be apparent to those skilled in the art from the teachings herein; accordingly, the scope of the invention is not limited to any particular delivery form.

For parenteral administration, such as subcutaneous injection, these immunogens can be combined with a suitable physiologically acceptable carrier, for example, it can be administered in water, saline, alcohol, fats, waxes, or buffered vehicles with or without various adjuvants or immunomodulating agents. Suitable immunological adjuvants or agents include, but are not limited to, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum) , beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Proplonobacterium acnes) , Bordetolla pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan) . Other suitable adjuvants are Amphigen (oil-in-water) , Alhydrogel (aluminum hydroxide) , or a mixture of Amphigen and Alhydrogel. The Rotavirus VP6 carrier system developed by

VIDO (Veterinary Infectious Disease Organization, Saskatoon, Canada) , although not an adjuvant, is also suitable.

The proportion of xmmunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts. On a per-dose basis, the amount of the im unogen can range broadly from about l.o pg to

about 100 mg per kg of host, usually at least about 10 pg, typically at least about 100 pg, and preferably at least about l ng per kg of host weight, and usually less than about 1 mg, typically less than about 10 μg, and more typically less than about 1 μg, and preferably less than about 100 ng per kg of host. A preferable range is from about 10 pg to about 100 ng per dose. A suitable dose size will usually be between about .01 and 5 ml, preferably about 0.5 ml for a 20-59 kg organism. Comparable dose forms can also be prepared for parenteral administration to smaller or larger animals, but the amount of immunogen per dose will usually be smaller, for a smaller animal.

For the initial vaccination of immunologically naive animals, a regiment of between 1 and 4 doses can be used with the injections spaced out over a 2 to 6-week period. Typically, a two-dose regimen is used. The second dose of the vaccine then should be administered some weeks after the first dose, for example, about 2 to 4 weeks later. Animals that have been previously exposed to Entamoeba or have received colostral antibodies from the mother may require booster injections. The booster injection is preferably timed to coincide with the vulnerable point in the life cycle of the Entamoeba. Periodic revaccination is advisable under certain conditions.

The vaccine may also be combined with other vaccines for other diseases to produce multivalent vaccines. It may also be combined with other medicaments, for example, antibiotics. A pharmaceutically effective amount of the vaccine can be employed with a pharmaceutically acceptable carrier such as viral capsid protein complex or diluent understood to be useful for the vaccination of animals. Other vaccines may be prepared according to methods well-known to those skilled in the art as set forth, for example, in Tizard, L. , An Introduction to

Veterinary Immunology, 2nd Ed (1982) , which is incorporated herein by reference.

The following experiments are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

I. Entamoeba Isolates and Cell Culture

II. Human Immune Sera

III. Anti-membrane Fraction Serum IV. Monoclonal Antibody FA7

V. Western Blot Analysis

VI. Antibody Capping by Live Trophozoites

VII. Preparation and Screening of Libraries

VIII. Sequence Analysis IX. Primer Extension Sequence Analysis

X. Gene Copy Number

XI. Detection of Sequences Related to M17 in Non- Pathogenic E. histolytica

XII. Restriction Fragment Length Polymorphisms

Using pooled human immune sera a cDNA clone (cM17) encoding this antigen (M17) was isolated from a λgtll expression library of the virulent strain E. histolytica HMI:IMSS. Monospecific antibodies, purified by binding to phage lysate of cM17, and monoclonal antibody FA7 reacted exclusively with the 125 kDa antigen by Western blot analysis. Surface binding and cap formation was observed with patient sera, purified monospecific antiseruro and monoclonal antibody FA7. Corresponding genomic clones (pBSgM17-l/2/3) were isolated by hybridization with the cDNA clone. These contained an open-reading frame of 3345 bp which is in good agreement with the mRNA size of approximately 3.0 kb as revealed by Northern hybridization with λcM17. The inferred amino acid sequence predicted a 125,513 Da protein which contains 17 potential N-linked glycosylation sites and is unusually rich in tyrosine and asparagine residues. A distinctly hydrophobic amino terminal region may serve as membrane anchor or signal sequence. Some restriction enzymes were found which allowed PCR diagnosis of non-pathogenic and pathogenic

isolates with the exclusion of E. histolytica-like Laredo.

I. Entamoeba Isolates and Cell Culture Trophozoites of the axenized E. histolytica strains (HM1:IMSS, NIH:HK9) and E. histolytica-like Laredo were grown in TYI-S-33 media as described by Diamond, et al. (1978) "A New Medium for the Axenic Cultivation of Entamoeba histolytica and Other Entamoeba," Trans. R. Soc. Trop. Med. Hvg. 72: 431-432.

Polyxenic isolates were grown in liquid Robinson's medium supplemented with 10% bovine serum and containing 5 μg per ml of medium of each of the following antibiotics: kanamycin, erythromycin, and ampicillin. Amoebae were pelleted by centrifugation at 900 rpm and washed twice with phosphate buffered saline, pH 7.5 (PBS). Polyxenic amoebae were further purified by centrifugation through a Percoll/PBS cushion at 3000 rpm in a refrigerated Accuspin centrifuge. Isolates SD4 (pathogenic, zymodemes II) and REF 291 and SD116 (non-pathogenic, zymodemes III and I) , were a generous gift of Dr. Sharon Reed from the University of California, San Diego. Non-pathogenic isolates #43 and #44 and pathogenic isolate #46, classified by zymodeme analysis using gradient PAGE were isolated in Mexico City. See Meza, et al. (1986)

"Isoenzyme Patterns of Entamoeba Histolytica Isolates from Asymptomatic Carriers: Use Of Gradient Aσrylamide Gels." Am. J. Trop. Med. Hvg. 35: 1134-1139. They correspond to Sargeaunt zymodemes I, I, and II, respectively.

II. Human Immune Sera

Sera from 108 patients with amoebic liver abscesses were obtained from Drs. A. Isibasi and R. Landa at the Instituto Nacional de la Nutricion and La

Raza-IMSS Hospitals, Mexico City. Diagnosis of hepatic abscess in patients was established by clinical symptoms,

countercurrent immunoelectrophoresis, ELISA and rectosigmoidoscopy. Human sera from donors without history of amoebiasis and negative for anti-amoebic antibodies as tested by immunoblot served as controls. Western blots of whole trophozoites were prepared by suspending washed cells in PBS containing 10 mM p-hydroxymercuribenzoate and Laemli sample buffer, boiling for 5 min, fractionation by 10% or 5-15% gradient SDS-PAGE and electrophoretic transfer to nitrocellulose filters. All sera were evaluated by Western blot analysis on extracts of whole amoebae. Twenty-nine sera with the highest titer were selected from the 108 samples and were pooled.

III. Anti-membrane Fraction Serum

A membrane fraction was prepared as described in Aley, et al. (1980) J. EXP. Med. 152: 391-404, and diluted 1:1 with PBS and complete Freund's adjuvant.

Mice were immunized intraperitoneally with 300 μg every two weeks until titers reached 1:5000 as assayed by

Western blot.

IV. Monoclonal Antibody FA7

Whole amoebic extract from 2 x 10 amoebae was fractionated by preparative 5-15% gradient SDS-PAGE.

After electrophoretic transfer to nitrocellulose the 125 kDa region was excised from the blot, ground to a powder and suspended in PBS. One hundred μl of the suspension was diluted 1:9 with PBS and injected three times intraperitoneally into mice at two week intervals with a final boost before the fusion. Hybridomas were selected by positive reaction with the 125 kDa band in Western transfers of E. histolytica extracts. Harvest fluid from clone FA7 was used at a 1:1000 dilution in Western blot analysis.

V. Western Blot Analysis

Western blot analyses were performed using standard procedures. See, e.g., Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York, which is hereby incorporated herein by reference. E. histolytica whole cell extracts were reacted with individual serum samples from each of the 108 infected patients. Seven antigens (220, 190, 160, 125-129, 96, 75, 46 kDa) were detected by more than 62% of the sera. Amongst these seven, a 125 kDa antigen was immunodominant, reacting strongly and being recognized by more than 70% of the serum samples. We assume, based on their molecular weight and serological reactivity, that the 220 kDa antigens represents an N- acetyl-gluσosamine adherence lectins, the 160 kDa antigen represents an N-acetyl-D-galactosamine adherence lectin, and the 96 kDa antigen represents an integral membrane protein. A Western blot of whole cell extracts of axenically or polyxenically propagated pathogenic and polyxenically propagated non-pathogenic E. histolytica isolates was assayed with a pooled subset of 29 human immune sera. The sera reacted strongly with a 125 kDa antigen in all isolates regardless of source. See Figure 1. Polyspecific antiserum prepared against amoebic plasma membrane also reacted strongly with the 125 kDa antigen. The monoclonal antibody FA7, prepared against partially purified 125 kDa antigen, reacted specifically with an epitope of the 125 kDa antigen; by Western analysis with FA7 this epitope was detected in different strains and species of Entamoeba. In the Western blot probed with monoclonal antibody FA7, additional bands of lower molecular weight and varying intensity are apparent in most of the isolates. Potent proteases are present in whole amoebic extracts, so we assume that these are degradation products of the 125 kDa antigen, though processing intermediates of unknown origin cannot be ruled out.

VI. Antibody Capping by Live Trophozoites

Human immune serum, hybπdoma harvest fluid from clone FA7, and purified monospecific antiseru was added to live trophozoites at 1:500, 1:2000, and undiluted, respectively. After formation of caps (10 min at 37*C), cells were fixed with 3.7% formaldehyde, washed with PBS and stained with fluorescein-isothiocyanate labeled anti-human or anti-mouse IgG. Undiluted harvest fluid from an anti-actin producing clone was used as control for a non-surface antigen.

Live trophozoites cap antibody-antigen complexes bound to their surface. Antibody-antigen caps were induced in HM1:IMSS trophozoites by incubation with the pooled patient serum, monoclonal antibody FA7, or monospecific antibody recovered after specific binding and elution of pooled patient sera to phage lysates of cDNA clone λcM17. See Fig. 2. A negative control antibody (monoclonal anti-actin antibody) neither bound to trophozoite surfaces nor induced cap formation. These results indicate that the 125 kDa antigen is localized to the surface of the amoebae. Besides capping the cross-linked antigens, it appeared that the cells also rounded up. This is indicative of an interruption of normal biological function and probably indicates that attachment to antibodies would disrupt cellular functions, including cell division and infective cycle functions.

VII. Preparation and Screening of Libraries

Genomic DNA and poly(A) ⁺ RNA isolation and construction of the λgtll cDNA library from strain E. histolytica HM1:IMSS has been described previously. See, e.g. Edman, et al. (1987) Proc. Natl. Acad. Sci. USA. 84: 3024-3028. A genomic library from E. histolytica

HM1:IMSS was made by adding 600 μl of Nal (GeneClean kit;

BiolOl) to 200 μl (approximately 20 μg DNA) of agarose

embedded nuclei in an Eppendorf tube and melted by incubation at 60"C for 5 min. Twenty μl of glassmilk was added, and suspended well and the mixture incubated at room temperature for 5 min. The sample was vortexed for 1 min to shear the DNA and spun in a microfuge for 5 sec. After removal of the supernatant the pellet was suspended in 1 ml wash buffer by vortexing for 30 sec. The glassmilk was pelleted by a 5 sec spin in the microfuge and the supernatant was removed. The wash was repeated twice and the sheared and purified DNA eluted into 100 μl TE (10 mM Tris-HCl (pH 8) , 1 mM EDTA) by incubation at 37 ^# C for 5 min. Recovery and degree of shearing were assessed by agarose gel electrophoresis. All subsequent steps including addition of Eco RI linkers, ethylation, ligation into the vector ZAPII and packaging reaction were performed by standard procedures. See, e.g., Sambrook et al. (1989) ; Gubler, U. and B. J. Hoffman (1983) Gene. 25: 263-269; and Morgan, et al. (1987), Nature. 329: 301-307. The λgtll cDNA library (3 x 10 ⁵ phage) was screened with the pool of 29 patient sera at a 1:200 dilution. The genomic library was screened with the ³² P-α-dCTP labeled EcoRI fragment of λcM17.

Forty-six reactive clones were each plaque purified and tested for recognition by each of the 29 patient sera included in the serum pool and by the anti-membrane antibody. Clone λcM17 strongly reacted with 26 out of 29 patient sera as well as with the anti-membrane serum. Monospecific antibody was selected from the pooled human sera by elution from filter-bound phage lysate of λcM17. This eluate reacted with a single polypeptide of 125 kDa by Western blot analysis of whole amoebic extracts. Phage lysate of λgtll, serving as negative control, did not bind antibodies reacting with amoebic antigens. Plasmids were rescued from genomic λZAPII clones. Phage DNA and plasmid DNA were purified by standard methods.

VIII. Sequence Analysis

With the exception of the first 207 bp, the entire sequence of gene M17 was determined on both strands in genomic clone pBSgM17-l and on one strand in genomic clone pBSgM17-2. See Table I. The internal Eco RI fragment representing the cDNA insert was also sequenced on both strands using nested deletion templates created with the Promega system. Double-stranded sequence was also determined for two PCR fragments obtained by amplification of genomic DNA from isolate REF 291, Zymodeme III, derived from an asymptomatic Costa Rican refugee and kindly provided by Dr. S.L. Reed. Several oligonucleotides were used as primers in single-stranded DNA (M13mpl8/19) and double-stranded DNA (pBSKS(+)) sequencing reactions with the Sequenase system (US Biochemicals) or ABI Sequencer (Applied Biosyste s Inc.) .

The 1.9 kB insert of λcM17 revealed an ORF (open reading frame) spanning the entire insert. See Table I. The lack of a 5* initiating methionine, the absence of a poly(A)-tail and hybridization to a -3 kb mRNA by Northern blot analysis indicated that amino and carboxy terminal sequences were lacking in λcM17. See Fig. 3. To isolate a genomic clone and obtain the amino and carboxy terminal sequences, a λZAPII library from E. histolytica HM1:IMSS was screened using the 1.9 kb insert of ΛcM17 as a probe. Three genomic clones were identified, and two of these were sequenced using oligonucleotide primers derived from the cDNA sequence.

The nucleotide sequence of the cDNA was identical in both genomic clones. An additional 556 bp of 5' and 870 bp of 3' sequence yielded an ORF of 3345 bp which was also identical in both genomic clones. The size of this ORF (gene M17) is in reasonable agreement with the mRNA size of * 3000 bp determined by Northern blot analysis. The

inferred amino acid sequence predicts a 125 kDa protein.

The 5 ^» flanking sequence of gene M17 shares striking similarities with the 5' flanking region of both actin and ferredoxin genes, the only other genes of E. histolytica where sequence has been determined. See Table II. The transcriptional start site of M17 was mapped to an adenine residue 17 bp 5' of the start codon by primer extension sequence analysis using an oligonucleotide SR09. 5' untranslated regions of actin (11 bp) and ferredoxin (9 bp) genes were likewise very short as compared with other eukaryotic gene transcripts. A common sequence motif, 5ΑTTCA3', is present at the transcriptional start site of both M17 and actin genes, the initiating nucleotide being an adenine residue as is most frequently found in other eukaryotes. While the same motif is also present in the flanking sequence of the ferredoxin gene, its cap site was mapped to the 3' thymidine rather than the 5' adenine residue. An additional sequence motif shared amongst these genes is YATTTAAA present at -29, -31, -32, and -32 for the M17, actin*, actin§, and ferredoxin gene flanking sequences, respectively. This sequence motif does not conform with the Goldberg-Hogness promoter consensus sequence TATAAATA, which in eukaryotic genes is located 25-30 bp upstream of the transcriptional start site.

Nevertheless, this sequence is similar in the three E. histolytica genes, both in sequence and relative position, suggesting a consensus which in E. histolytica serves as the entry point for RNA polymerase.

Table II: Alignment of the 5* flanking sequences from genes M17, actin* (one reported sequence), actin§ (a second reported sequence) and ferredoxin § ; a likely Goldberg-Hogness consensus sequence at -29, -31, -32, and -32, respectively, is bracketed. The 5' end of the mRNA is in bold face and follows the { symbol, and the initial ATG methionine codon is indicated. Sequence similarities around the cap site are ATTCA or ATTAA.

TABLE II Ml 7* T Λ G A A A T A T A A A A G A A T G T T A A A A A T G A A A Λ C A A A C A T A A A A A A T A A Q T β

ACTIN* A A A A T A A C G T A A G G A A C T A T G A A β T T C A C C T T C A G T A A A A A A Q A A Q A A A Q

ACTIN* G T T A A C T C C A A A C A A A

FERREDOXIN* T A T T A C A C T A A C T A A A T T T C T T A T T A T T C T A T T T A A A T C A C A A A C A A A C T

> > > > > > > > > > > > > <

M17* [T A T T T A A A]G T G T T T T T A A A A A A A C T A A T TJA T T C A T A A A T T A A A fl T T A T α i

ACTIN* A C A C(T A T T T A A AJG A C T G A C A A A A A C T Q A A T T G A A CJA T T C A A T A &Λ T Λ T Q g

ACTIN* A A G[T A T T T A A A)G A T C A T A A T G A A C T G A A T T A A A T CJA T T A A T T A A T T A T ^Q 2

FERREDOXIN* T G T[C Λ T T T Λ Λ A]T Λ C Λ Λ C Λ A Λ T T Λ A T C T T T T T Λ T C|Λ T T C Λ Λ Λ T £ T Λ A T Q g < < < < < < < < < < < < M e t

IX. Primer Extension Sequence Analysis

Primer extension sequence analysis was performed by reverse transcriptase mediated extension of oligonucleotide primer SR09 [5ΑACTACTCCTGTGACTATTGCAGAAG3'] annealed to 10 μg poly(A) ⁺ enriched RNA in the presence of deoxyadenosine 5•-[a-[ ³⁵ S]thio]triphosphate.

X. Gene Copy Number Southern blot and sequence analysis of the M17 gene and limited flanking regions indicate that this surface antigen is encoded by a single copy gene. A Southern blot of genomic DNA from E. histolytica, restricted with Bgl II and EcoR V in single and double digests, probed with Bam Hl-Bgl II, Bgl II-Eco RV, and Eco RV fragments of λcM17, only unique restriction fragments hybridized with each probe. Furthermore, the nucleotide sequence of both genomic clones and the cDNA clone is identical.

XI. Detection of Sequences Related to M17 in Non- Pathogenic E. histolytica

Western blot analysis suggested that the 125 kDa antigen or a closely related antigen which shared the epitope recognized by poly- and monoclonal antisera was found in both pathogenic and non-pathogenic E. histolytica isolates as well as E. histolytica-like Laredo. By Southern blot analysis, even under low stringency hybridization and wash conditions (25% formamide, 2 X SSC, 37*C) sequences related to M17 were difficult to detect in non-pathogenic E. histolytica isolates and E. histolytica-like Laredo.

PCR was performed using a Cetus/Perkin-Elmer DNA thermocycler. Reaction mixtures (50 μl) contained 25 pmol of each of the two oligonucleotide primer pairs SR018 [5-GCAACTAGTGTTAGTTATAC3'] with SR021 [5-GGTGGAATTTGGAATTCTGG3'] and SR019

[5-GTATAACTAACACTAGT3-] with SR022

[5•GCTGTTACACTTGAAAATAT3•] , approximately 500 ng of genomic DNA, all four dNTPs each at 1.5 mM, 60 mM KCl, 25 mM Tris-HCl (pH 8), 0 to 20 mM MgCl ₂ , 0.1% bovine serum albumin, and 10% DMSO. The reaction mixture was overlaid with a drop of paraffin oil and denatured at 94"C for 10 min, and amplification was initiated by addition of 2.5 units of Thermus aquaticus DNA polymerase (Cetus) . PCR parameters were 35 thermal cycles consisting of a 1 min denaturation at 94 ^• c followed by a 3 min annealing period at 42*C, a 3 min ramp and a 4-min extension period at 72 *C The amplification products were restricted with Eco RI and Spe I endonucleases and purified for subcloning into M13 by 1% low-melting-point agarose gel electrophoresis.

Two fragments spanning most of the sequence contained within the cDNA clone λcM17 were amplified in a PCR using oligonucleotide pairs SR019/SR022, and SR018/SR021 as primers on genomic template DNA derived from non-pathogenic isolate REF 291. By nucleotide sequence analysis of the two subcloned PCR amplification products, REF 291 had 145 nucleotide substitutions over 1410 residues (10.3%) as compared to the sequence of λcM17 (HM1:IMSS) See Table I. These substitutions result in 57 amino acid differences per 470 residues (12.1%). A computer search of published protein sequences with the entire 3345 bp M17 gene sequence revealed that the internal gene fragment represented by the λcM17 insert encoded a protein sequence similar to that deduced for a DNA fragment isolated from non-pathogenic and pathogenic strains, of E___ histolytica by Tannich et al. (1989) Proc. Nat'l Acad. Sci.. U.S.A.. 86:5118-5122, and proposed by these authors as a potential diagnostic probe for strain differentiation. Specifically, when we compare the amino acid sequences of Tannich et al. with that of λcM17, we detected five substitutions between pathogenic HM1:IMSS isolates (1%) . See Table III. The nucleotide sequence

of the DNA fragment was not published by Tannich et al., so we infer from the amino acid sequence that at least three of these five differences between the E. histolytica HM1:IMSS laboratory strains must have arisen from more than one nucleotide substitution and are therefore unlikely to represent cDNA synthesis or sequencing artifacts. When the 470 amino acid sequence derived from the PCR product of non-pathogenic isolate REF291 was compared to isolate SAW 1734, six amino acid substitutions (1.3%) were detected. Over the same 470 amino acids, 61 amino acid residues (12.9%) differ among the pathogenic HM1:IMSS and non-pathogenic SAW 1734 strains. Overall there are 65 variable residues over a stretch of 470 amino acids (13.8%) when these four isolates were compared. See Table III.

Table III: Alignment of the amino acid sequences inferred from nucleotide sequences of cDNA and genomic clones (HM1:IMSS*) and of PCR amplification products (REF291*) with those published by Tannich et al.

(HM1:IMSS#, SAW 1734#) . Conserved amino acids are in plain type, positions of variable residues differentiating pathogenic from non-pathogenic isolates are indicated below by an *, and positions of additional variable residues are indicated below by a ^A .

ill

TABLE HI

HM1:IMSS* PITLNFDORVDAGAAVAYVGRWFTONPSDWAAACVGKDGLINYGN GPLHEMN

HM1:IMSS* PITI_NFDORVDAGAAVAYVDR FTONPSDWAAACVGKDGLINYGN GPLHEMN

REF 291* PITLNFDORVDAGAAVAFVGRWFTOHPSDWASGCVNKDRLINSGNWGPLHEMN

SAW 1734* PITLNFDQRVDAGAAVAFVGRWFTOHPSDWASGCVNKEGLINSGNWGPLHEMN

HM1:IMSS* lJHMQGTYLKGGNWGISNPGEETNNVMTSINYILYTNIAGHRNQGLSGWNYVSD

HM1:IMSS* HHMQGPYLKGGNWGISNPGEETN VMTSINYILYTNIAGHRNOGLSGW YVSD

REF 291* HHMOGTYI_RGGNGGIKEPGEET_TOVMTSI YILYTNIAGHRKOGLSGWNYVSD

SAW 1734* HHMOGTYLRGG WGIKEPGEETNNVMTSINYILYTNIAGHR OGLSGWNYVSD

HM1:IMSS* GYSTIYKILKGE DQPHLRSYVNMAHAFGTDTLIALVKSYYGLWYEN FESKY

HM1:IMSS* GYSTIYKILKGE DOPHLRSYVNMAHAFGTDTLIALVKSYYGLWYENNFESKY

REF 291* GYSTIYKILKGENDQPHLRSYVNIΛHAFGTDTLIALVKSYYGLWYENNYEGEY

SAW 1734* GYSTIYKILNGENDQPHLRSYVNIAHAFGTDTLIALVKSYYGLWYENNYEGEY

HM1:IMSS* SIKRDSTSAFCLLAALVTKRDTRYLCSLFKYDIOSNVSEAIKNMNYPTYYPFF

HM1:IMSS* SIKRDSTSAFCLLAALVTKRDTRYLCSLFKYDIQSNVSEAIKNM YPTYYPFF

REF 291* SIKRDSTSAFCLLAAIATKRDTRYLCSLFKYDIOONVSEAIKNMNYPTYYPFF

SAW 1734* SIKRDSTSAFCLLAAIATKRDTRYLCSLFKYDIOONVSEAIKNMNYPTYYPFF

HM1:IMSS* NLYAMSYNGNYYGRPYKIPYGRTRL FTATTAIDPKATSVSYTIKSGLTKG L

HM1:IMSS* NLYAMSYNGNYYGRPYKIPYGRTRLNFTATCSIDPKATSVSYTIKSGLTKGKL

REF 291* NVYAMSYNGNYYGRTYKIPYGTTRLNFTATTAIDPSATSVSHTIKSGLTKGKL

SAW 1734* VYAMSYNGNYYGRTYKIPYGTTRLNFTATTAIDPSATSVSYTIKSGLTKGKL

HM1:IMSS* ERVEDNVYDYTPFFGIEENDTFVLNIDCWNGEKVHIEOEGGTFELDPHOVEY

HM1:IMSS* ERVEDNVYDYTPFFGIEENDTFVLNIDCW GEKVHIEOEGGTFELDPHOVEY

REF 291* EOVEENVYDYTPNFGADENDTFVLNIDCIVNGEKVHIEODGGTFELDPHOVEY

SAW 1734* EOVEENVYDYTPNFGADENDTFVLNIDCIVNGEICVHIEODGGTFELDPHOVEY

HM1:IMSS* EVY DVOTRDMAOAINIIQNKTRNDTGRASFFGIGTYNDGSMOSMLVEKGKLI

HM1:IMSS* EVYKDVQTRDMAQAINIIQNKTRNDTGRASFFDIGTYNDGSMQSMLVEKGKLI

REF 291* EVYKDVKTKDMEOALNTIONKTSNYTGTSTFFGIGNYDDGTMOSMLVEKGKLI

SAW 1734* EVYKDVKTKDMEOALNTIONKTSNYTGTSTFFGIGNYDDGTMOSMLVEKGKLI

HM1:IMSS* VPKSGYYTLFMKADDLGRLLLNITGEYEOLLDVKTYLGGYSKTLNGSYATVKL

HM1:IMSS* VPKSGYYTLF KADDLGRLLLNITGEYEQLLDVKTYLGGYSKTL GSYATVKL

REF 291* VPTSGYYTLFMKADDLGRLLL VNGEYEOLLNVKTYLGGYSKTINGTYATVKL

SAW 1734* VPTSGYYTLI^OCADDIΛRU NVNGEYEOLLNV TYLGGYSKTINGTYATVKL

HM1:IMSS* EKDVGYPFILYNLNTGGOGFIRIGYCYHGTEESSVDVSKCSVSDIGS

HM1:IMSS* EKDVGYPFILYNLNTGGOGFIRIGYCYHGTEESSVDVSKCSVSDIGS

REF 291* EKDTEYPFILYNLNTGGOGFIRIGYCYQGTEOSSV VSKCSGLDIGS

SAW-1734* - EKDTEYPFILYNL TGGQGFIRIGYCYQGTDQSSVNVSKCSGLDIGS

XII. Restriction Fragment Length Polymorphisms

The partial M17 amino acid sequences of non-pathogenic strains SAW 1734 and REF 291 were significantly more similar to one another than to their pathogenic counterparts, so PCR amplification of the same gene fragments from six additional strains was undertaken to examine the possibility of defining restriction length polymorphisms that could reliably differentiate pathogenic from non-pathogenic amoeba isolates. Using oligonucleotide primers SR019, SR022, SR018 and SR021,

PCR products of the same size were amplified from genomic DNA of strains SD116, SD4, #43, #44, #46 and HK9. Based on our nucleotide sequence of these fragments from HM1:IMSS and REF291, we predicted that restriction endonucleases Eco RV, Ssp I, Pvu II, Ace I and Hinc II among others would cleave the PCR products into restriction fragments which might be expected to correlate with the pathogenic or non-pathogenic phenotype of the isolate. An example of such an analysis with Eco RV and Ssp I is presented. See Fig. 4. A restriction site for Eco RV is absent in non-pathogenic #43, #44 and REF291 but present in non-pathogenic SD116 and Laredo as well as pathogenic HM1:IMSS, HK9, SD4, and #46. Digestion with restriction endonuclease Ssp I shows a distinct pattern for pathogenic (HM1:IMSS, HK9, SD4, 46) versus non-pathogenic (#43, #44, SD116, REF291) strains with the exception of E. histolytica-like Laredo, which would appear pathogenic by this criterion. See, e.g., Fig. 4. Similarly, restriction with Ace I distinguishes pathogenic from non-pathogenic isolates with the exception of Laredo which appears to have an additional restriction site for the enzyme. Hinc II digestion shows the same restriction fragments in non-pathogenic isolates #43, #44 and REF291 and pathogenic isolate SD4 but no restriction sites in commensal Laredo and pathogenic isolates #46, HM1:IMSS and HK9.

All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the claims.