AU6530394A - 35/31 kda subunit of the (entamoeba histolytica) adherence lectin - Google Patents

Description

35/31 KDA SUBUNIT OF THE ENTAMOEBA

HISTOLYTICA ADHERENCE LECTIN

This research was supported in part by U.S. government grants; the U.S. government has certain rights in this invention.

This application is a continuation-in-part of U.S. application Ser. No. 08/045,679, filed April 9, 1993.

Technical Field

The invention relates to diagnostics and therapeutics for Entamoeba histolytica infection. More specifically, the invention is directed to therapeutics which take advantage of the genes encoding the Gal/GalNAc lectin of this amoeba, and specifically cDNA encoding the 35/31 kDa light chain subunit, and antibodies directed to the protein product thereof.

Background Art

Entamoeba histolytica infection is extremely common and affects an estimated 480 million individuals annually. However, only about 10% of these persons develop symptoms such as colitis or liver abscess. The low incidence of symptom occurrence is putatively due to the existence of both pathogenic and nonpathogenic forms of the amoeba. As of 1988, it had been established that the subjects who eventually exhibit symptoms harbor pathogenic "zymodemes" which have been classified as such on the basis of their distinctive hexokinase and phosphoglucomutase isoenzymes. The pathogenic forms are not, however, conveniently distinguishable from the nonpathogenic counterpart zymodemes using morphogenic criteria.

The distinction between pathogenic and nonpathogenic strains in diagnosis is of great practical importance, because only persons infected with E. histolytica who will develop the disease should be treated. This is bad enough in developed countries where it would at least be possible economically to treat every carrier with a known effective drug (metronidazole) ; it is, of course, undesirable to administer such drugs unnecessarily. In less developed countries, the cost of these unnecessary administrations is significant enough to have a dramatic negative impact on the resources for overall health care.

There is an almost perfect correlation between infection with a pathogenic zymodeme and development of symptoms and between infection with a nonpathogenic zymodeme and failure to develop these symptoms. As a general proposition, only pathogenic strains can be grown axenically (i.e., in the absence of an associated micro- organism) and nonpathogenic strains have been made to grow in this manner only by "training" them to do so in a series of media alterations beginning with attenuated bacteria. The adaptation was accompanied by exhibition of the enzyme pattern characteristic of pathogenic strains (Mirelman, D., et al. , Infect Immun (1986)

5_4:827-832) . This work has not been repeatable in other laboratories, and more recent work on genomic differences (see below) indicates that the pathogenic and nonpathogenic forms are separate species. Despite the generally interesting and useful results cited above, the ability to diagnose the presence or absence of pathogenic strains of E. histolytica has proved difficult. Since both pathogens and nonpathogens are morphologically similar, microscopic tests are not particularly useful . ELISA techniques have been used to detect the presence or absence of E. histolytica antigen in both stool specimens and in sera, but these tests do not seem to distinguish between the pathogenic and nonpathogenic strains. Root et al . , Arch Invest Med (Mex) (1978) S_ : Supplement 1:203, pioneered the use of ELISA techniques for the detection of amoebic antigen in stool specimens using rabbit polyclonal antiserum. Various forms of this procedure have been used since, some in correlation with microscopic studies, and all using polyclonal antisera. None of these, apparently, pinpoints the instances of infection with the pathogenic as opposed to nonpathogenic form. See, for example, Palacios et al. , Arch Invest Med (Mex) (1978) 9_:

Supplement 1:203; Randall et al. , Trans Roy Soc Trop Med Hyg (1984) 78_.:593; Grundy, Trans Roy Soc Trop Med Hyg (1982) 16.:396; Ungar, Am J Trop Med Hvg (1985) 34:465. Studies on stool specimens are summarized in Amebiasis: Human Infection by Entamoeba Histolytica. J. Ravdin, ed. (1988) Wiley Medical Publishing, pp. 646-648. Similar methods to detect characteristic E. histolytica antigens in serum and in liver abscess fluid are equally unable to distinguish pathogens from non- pathogens (ibid., pp. 661-663) . As summarized in this article, as of 1988, the only known way to distinguish pathogenic from nonpathogenic forms of this amoeba was through characterizing the isoenzyme pattern using electrophoresis. Recently it has been shown by two different groups that differences between pathogenic and nonpathogenic strains can be demonstrated using comparisons of DNA isolates. Garfinkel, L.I., et al. , Infect Immun (1989) 57: 926-931 developed DNA probes which hybridize to DNA isolated from E. histolytica and four types of restriction fragment length patterns were obtained. These patterns correlated with pathogenic/nonpathogenic distinctions. Similarly, Tannich, E., et al. , Proc Natl Acad Sci (1989) .86.:5118-5122 probed cDNA libraries constructed from various strains and showed that pathogenic isolates were genetically distinct from nonpathogenic ones. However, these techniques require the culture of the organisms isolated from patients to obtain sufficient quantities for testing, and are thus time consuming and labor intensive.

Strachan, W.D., et al. , Lancet (1988) 561-562, report the production of two monoclonal antibodies designated 22.3 and 22.5 which were members of a large group prepared by standard procedures from mice immunized with axenic cultures of a pathogenic E. histolytica strain NIH200/ATCC 30458. These monoclonal antibodies were tested in an immunofluorescence assay with cultures obtained from both putatively invasive and noninvasive strains, and appeared to immunoreact only with culture samples of invasive strains. There is no indication in this publication as to the manner of screening for antibodies with this characteristic, it is not known to what target these antibodies bind, nor would it be possible, without these specific antibodies, to reproduce this result . The test described requires intact E. histolytica and therefore cannot be applied in serum, urine or liver abscess fluid and can only be applied to stool samples which are freshly collected. Adherence and subsequent destruction of host target cells by the protozoan parasite Entamoeba histolytica is mediated by galactose and N-acetyl-D-galactosamine- specific cell-surface lectin (Gal/GalNAc lectin) . Mann, et al. Proc Natl Acad Sci (1991) 3248-3252; Petri, et al., J Biol Chem (1989) 264=3007-3012; Petri, et al . Infect Immun (1991) 5_9_:97-101; Ravdin, et al . J Exp Med (1980) 152_.:377-390. This lectin has a light and heavy chain and a MW of 260 kDa; the light subunit as further described below has a MW of 31/35 kDa; the heavy chain has a MW of about 170 kDa.

Recently, preparation of monoclonal antibodies which are capable of distinguishing pathogenic and nonpathogenic forms of E. histolytica have been described (Petri, W.A. et al. Infect Immunol (1990) 5_8:1802-1806) . These antibodies bind to various epitopes on the 170 kDa heavy subunit of the lectin.

Inhibition of the normal adherence via the lectin prevents contact-dependent killing of mammalian cells by E. histolytica (Ravdin, et al. J Clin Invest (1981)

£8:1305-1313; Saffer, et al. Exp Parasit (1991) 72:106- 108) . The lectin heavy subunit is almost universally recognized by immune sera and T cells from patients with invasive amebiasis (Petri, et al . , Infect Immun (1987) 55_.:2327-2331; Schain, et al . Infect Immun (1992) 60:2143- 2146) . Immunization of gerbils with the purified lectin has been demonstrated to provide complete protection against amebic liver abscess in 67% of animals challenged intrahepatically with E. histolytica (Petri, et al. , Infect Immun (1991) 5_9:97-101) . The 170 kDa subunit is regarded as the active component of the vaccine (U.S. patent 5,004,608, issued 2 April 1991) .

In general, carbohydrate-binding proteins or lectins are ubiquitous in nature and are involved in a myriad of recognition processes including mammalian fertilization, adhesion of cells to the extracellular matrix, and cellular interactions of the immune system. Lectin- carbohydrate interactions play a crucial role in the specificity of cell adhesion processes. In contrast to peptides and oligonucleotides, whose information content is based only on the number of monomeric units and their sequence, carbohydrates contain the potential to encode vastly greater amounts of information as a result of the position and anomeric configuration of the glycosidic units and in the occurrence of branch points. The importance of carbohydrates in cell-cell adhesion has begun to receive increasing attention since the discovery of the role of the homing receptor selection in the binding of leukocytes to endothelium during lymphocyte recirculation. In addition to mammalian cells, bacteria and other microorganisms utilize protein-carbohydrate interactions to colonize and invade host tissue.

The above-discussed Gal/GalNAc adherence lectin of E. histolytica was isolated from a pathogenic strain and purified 500 fold by Petri, W.A. , et al. , J Biol Chem (1989) 264 :3007-3012. This successful isolation and purification was made possible by the production of mouse monoclonal antibodies which inhibit the jLn vitro adherence of the amoebic trophozoites; the antibodies were prepared from immortalized cells from spleens of mice immunized with sonicated trophozoites grown in axenic culture after having originally been isolated as a pathogenic strain from an affected subject. (Ravdin, J.I., et al., Infect Immun (1986) 53_:l-5.) The cells were screened by the ability of the supematants to inhibit adherence of the trophozoites to target tissue. All of these reported monoclonal antibodies, therefore, are presumably immunoreactive with the Gal/GalNAc surface adhesion region of the pathogen. The Gal/GalNAc lectin itself was prepared by galactose affinity chromatography and reported in 1987. (Petri, W.J., et al. , J Clin Invest (1987) 8^:1238-1244) . Studies of serological cross-reactivity among patients having symptomology characteristic of E. histolytica pathogenic infection, including liver abscess and colitis, showed that the adherence lectin was recognized by all patients' sera tested (Petri, Jr., W.A. , et al . , Am J Med Sci (1989) 296_:163-165) .

The purified 260 kDa galactose-binding lectin is stable in sodium dodecyl sulfate (SDS) , but dissociates in the presence of 2-mercaptoethanol into heavy (170 kDa) and light (35/31 kDa) glycoprotein subunits, suggesting intersubunit disulfide bonds. As used herein, the "light subunit" intends either or both of the 35 or 31 kDa isoforms of the light glycoprotein subunit . The heavy and light subunits are encoded by distinct mRNAs (Mann et al., Proc Natl Acad Sci (1991) j38_.:3248-3252) have different amino acid compositions and amino-terminal sequences (Petri, et al . , J Biol Chem (1989) 264 :3007- 3012) . Mouse polyclonal anti-lectin antisera, which recognizes only the heavy subunit on Western blots, completely inhibited amebic adherence, suggesting that the heavy subunit contains the galactose-binding domain (Petri, et al. , J Biol Chem (1989) 264:3007-3012) . Analysis of the sequence of a cDNA of the 170 kDa subunit revealed it to be an integral membrane protein with a large cysteine-rich extracellular domain and a short cytoplasmic tail (Mann, et al. , Proc Natl Acad Sci (1991) supra; Tannich, et al . Proc Natl Acad Sci (1991) 88 :1849- 1853) . Recent reports indicate that the heavy subunit is a member of a multi-gene family (Mann, et al . Parasit Today (1991) 1:173-176; Tannich, et al . , supra) . The amino-terminal-sequence of the 35/31 kDa subunit exhibited microheterogeneity, indicating the presence of multiple light subunit isoforms.

In a later paper by Tannich, E. et al . Molec Biochem Parasitol (1992) 5_5:225-228, cloning of the cDNA encoding what was described as a 35 kDa light subunit of the Gal/GalNAc lectin was reported and the deduced amino acid sequence disclosed. (This paper characterizes the total molecular weight of the lectin as 220 kDa.) No suggestion was made that the 35 kDa subunit for which the DNA was reported would be useful as a vaccine. Indeed, citing a communication from the present applicant, the article states that rabbits immunized with the isolated 35 kDa molecule failed to produce antibodies against either the heterodimer or its small subunit.

The prior art has shown an immune response only to the E. histolytica 170 kDa subunit; antibodies to the light subunit have not been detected in infected animals or in animals administered the Gal/GalNAc lectin. Thus, based on the prior art, one would not expect the light subunit to contribute any advantage to a vaccine formulated against E. histolytica, nor to be diagnostic of infection. Further, antibodies immunospecific for this submit have not been available.

Disclosure of the Invention

It has now been found that the 35/31 kDa subunit of E. histolytica Gal/GalNAc lectin is an effective active ingredient of an E. histolytica vaccine. The structure of the 35/31kDa subunit has been elucidated and recombinant materials for its production have been prepared. The present invention therefore provides an improved vaccine against E. histolytica which would not have been predicted to confer protection.

Until the present invention, the unavailability of light subunit-specific antibodies has made structural and functional analysis of the 35/31 kDa subunit difficult, hence its role in adherence and contact-dependent cytolysis has been unknown. Attempts at developing vaccines have focused solely on the 170 kDa subunit. In the present invention, the galactose-binding lectin light (35/31 kDa) subunit was characterized in order to determine its structure and mechanism of membrane anchoring. The deduced amino acid sequence of the 35/31 kDa subunit cDNA demonstrates a putative glycosyl- phosphatidylinositol (or "GPI") anchor addition signal, and biochemical and immunological analysis supports this discovery. One aspect of the present invention is a vaccine comprising the 35/31 kDa subunit of E. histolytica Gal/GalNAc adherence lectin or a functional portion thereof. The vaccine results in an immune response and is protective against infection by E. histolytica . The 170 kDa heavy subunit or functional portion thereof may also be included. The protein or portion thereof may itself be used as active ingredient, or recombinant expression vectors, especially those which take advantage of viral infection and expression systems can be used as vaccines.

Accordingly, in other aspects, the invention is directed to recombinant materials useful for the production of the 35/31 kDa subunit either in cell culture conditions or in situ in an immunized host. The invention is also directed to chimeric fusion peptides which include the 35/31kDa subunit. Still another aspect is directed to recombinant materials which produce the complement of DNA encoding the light chain subunit and the complementary oligonucleotides per se which are useful in interrupting the growth of E. histolytica in culture or .in situ.

Still other aspects of the present invention relate to methods of immunizing against Entamoeba histolytica using the vaccines of the invention and of producing antibodies immunospecific for the 35/31 kDa subunit, and to these antibodies per se.

Another aspect of the invention is a method to detect the presence or absence of a non-pathogenic or pathogenic E. histolytica in a biological sample, which method comprises subjecting the sample to polymerase chain reaction (PCR) using primers framing a region of the 35/31 kDa light chain of the Gal/GalNAc adherence lectin, and hybridizing the amplified DNA under stringent conditions with an oligomer corresponding to said region. By choosing regions of DNA characteristic of pathogenic or non-pathogenic E. histolytica or both, these zymodemes may be distinguished or determined collectively. Other methods employ the monoclonal antibodies of the invention.

Brief Description of the Drawings

Figures la and lb show the nucleotide sequence and the derived amino acid sequence of the 35/31 kDa subunit encoded by two members of the gene family. Figure 2 is a photocopy of two-dimensional SDS-PAGE of affinity-purified lectin.

Figure 3 is a diagram showing the Peptide Map of the 31 and 35 kDa subunits after CNBr digestion.

Figure 4 is a photocopy of an SDS-PAGE autoradiograph of galactose lectin metabolically labeled with palmitic and myristic acid.

Figure 5 is a photocopy of an SDS-PAGE autoradiograph of [³H] glucosamine-labeled lectin.

Figure 6a is a drawing showing the results of thin- layer chromatography of the lipid product from nitrous acid deamination of the 31 kDa band from total amebic proteins. Figure 6b is a drawing showing the results of thin- layer chromatography of the lipid product from nitrous acid deamination of the 31 kDa band from immunoprecipitated lectin. Figure 7 is a photocopy of Western blots of electroeluted 31 and 35 kDa subunits using anti-CRD antibodies.

Figure 8a is a diagram showing the results of an assay of E. histolytica proteins for GPI-PLC activity. Figure 8b is a drawing showing the results of TLC of lipid product from GPI-PLC assay.

Figure 9a is a photocopy of Southern blots of E. histolytica DNA probed with the 35/31 kDa subunit PCR (polymerase chain reaction) fragment. Figure 9b is a photocopy of Northern blot analysis of E. histolytica RNA with a 35/31 kDa subunit probe.

Figure 10 is a photocopy of a Western Blot of fusion protein with the subunit or isolated subunit with immune and pre-immune sera.

Modes of Carrying Out the Invention

The present invention provides materials which are useful in developing vaccines and diagnostic assays for both pathogenic and nonpathogenic forms of E. histolytica . The vaccines developed from the present invention enable the production of a vaccine directed against both subunits of the Gal/GalNac adherence lectin, in particular the light, or 35/31 kDa, subunit. The diagnostic assays can be conducted on biological samples derived from cell cultures or from subjects at risk for infection. The assays utilize hybridization probes or antibodies and by the design of the assay can distinguish pathogenic from nonpathogenic forms of the amoeba. In addition, the availability of the cDNA provides an opportunity for preventing the production of the adherence lectin using an "antisense" approach.

Knowledge of the DNA sequence of the light subunit also enables the production of synthetic peptides based upon the sequence. These peptides can be specifically engineered to contain, for example, the T-cell epitope(s) or the B-cell epitope(s) of the light subunit. See, for instance, Example 16, which describes a vaccine containing a synthetic peptide representing a predicted B-cell epitope of the light subunit. Methods to identify synthetic peptides based on the sequence of the 31/35 kDa subunit which are recognized by the T cells and antibodies of immune individuals are known in the art. The invention provides the primary amino acid sequence of the 35/31 kDa subunit of the galactose- binding lectin of E. histolytica and demonstrates that it contains a GPI anchor. Sequence analysis of the light subunit cDNA shows that the subunit contains a potential GPI-anchor addition signal; further, the light subunit reacts with anti-CRD antibodies after treatment with PI- PLC. ("CRD" represents cross reacting determinant, which is a determinant exposed by cleavage of diacylglycerol from the anchor by phosphatidylinositol-specific phospholipase C ("PI-PLC") . Nitrous acid deamination of the lipid-labeled 31 kDa subunit isoform releases lipid products which co-migrate with phosphatidylinositol, also consistent with the presence of a GPI anchor. The galactose lectin is a unique example of a GPI-anchored protein in that it is a heterodimer, while other GPI- anchored proteins described to date have been monomers or homodimers. The cDNA sequence of the 170 kDa subunit has been previously shown to contain a 26 amino acid hydrophobic domain followed by a 41 amino acid hydrophilic domain at its carboxy terminus, consistent with it being an integral-membrane protein. The two lectin subunits thus use different mechanisms of membrane anchoring.

The isoforms of the light subunit result in two lectin heterodimers i.e., 170/35 and 170/31. Although the light subunit is demonstrated by Southern blots to be encoded by a gene family, and direct sequencing of the 35 kDa subunit protein reveals microheterogeneity consistent with the existence of more than one gene, the 35 and 31 kDa isoforms are antigenically cross-reactive (Example 15) and have very similar amino acid compositions and CNBr-digested peptide patterns. They are demonstrated herein to contain the same primary amino acid sequence. The 31 kDa and 35 kDa subunits show differential labeling with glucosamine, and in the presence of labeled fatty acids. The accessibility of their amino termini to Edman degradation also differs. Thus the biochemical differences between the isoforms are complex.

Both the 31 kDa and 35 kDa subunits are encoded by the same genes. The nucleotide sequence and deduced amino acid sequence of these isoforms for one member of the 35/31 KDa subunit multigene family (designated "lgll") is shown in Figure la. Residues underlined with dashed lines indicate three residues of the putative amino-terminal leader sequence and the hydrophobic carboxy-terminal domain. Residues underlined with solid lines indicate the amino-terminal and CNBr sequences determined by Edman degradation. Potential N-linked glycosylation sites are starred. The putative GPI-anchor cleavage/addition site is marked with solid circles. Amino acids 79-90 of the mature subunit protein, for which a synthetic peptide has been shown to be active in a vaccine (see Example 16) , are enclosed in a box. Residue 1 (K) is the amino terminus of the mature protein. The amino acid sequences were determined by Edman degradation for the amino terminus (KTN/QDN/GR/KDQF/LSPNYPYG/DKMDN) and CNBr peptide (MSTSYAIPKSV/DISARAP) . Underlined residues indicate differences from the derived sequence, which were predominantly located at areas of microheterogeneity.

Figure lb shows the nucleotide sequence and deduced amino acid sequence of a clone encoding the 35/31 kDa subunit which is another member of the multigene family ("lgl2") . This clone was retrieved in a manner analogous to that set forth hereinbelow in Example 12. The amino acid sequence obtained is, of course, substantially homologous to that of the gene shown in Figure la. Sequences of other clones of these two 35/31 kDa subunit genes, obtained from different strains of E. histolytica, have been shown to be nearly identical to those in Figures la and lb with at most only a few amino acids differing from strain to strain.

E. histolytica was also demonstrated to have a GPI- PLC activity. Although the lectin exists in both soluble and membrane forms (Petri, et al. , J Clin Invest (1987) _,80.:1238-1244, the CRD has not been identified in amebic conditioned medium. The covalent association of the light subunit with the 170 kDa subunit, which appears to be an integral membrane protein, makes it unlikely that

GPI anchor hydrolysis would be an effective mechanism for lectin release in E. histolytica .

Since a heterodimeric protein of this nature has not been previously described, the question of why E. histolytica would link a GPI-anchored polypeptide with an integral membrane polypeptide must be raised. Possible answers to this question are based on biochemical and biological roles of GPI-anchored proteins. It has been shown that GPI-anchored proteins have lateral diffusion coefficients similar to those of membrane phospholipids which might allow GPI-anchored receptor-like proteins to migrate rapidly to regions of contact with other cells in cell-cell interactions. This property could be relevant to the lectin if the heterodimeric structure is dynamic, with the 35/31 kDa subunit at times free to diffuse in the membrane dissociated from the 170 kDa putative integral membrane subunit. Second, GPI-anchored proteins are believed to play a role in cell signalling. Evidence has shown that a number of GPI-anchored proteins on T lymphocytes (i.e. Thy-1, TAP, and Ly-6) mediate cell activation signals. Immunoprecipitation studies have shown an association between GPI-anchored cell-surface molecules and p56lck, a member of the src family of tyrosine kinases in a number of mammalian cells, linking these GPI-anchored proteins with known signal transduction molecules. Inhibition of the lectin with galactose prevents amebic contact-dependent killing of target cells even when the amebic and target cell membranes are brought into contact by centrifugation. This suggests that the lectin, and potentially its GPI anchor, could be involved in signalling initiation of the cytolytic process.

Definitions As used herein, a DNA or RNA "corresponding to" a referent refers to a DNA or RNA which encodes the same functional protein (e.g. the E. histolytica 31/35 kDa subunit) or portion thereof or is complementary thereto.

A protein "corresponding to" a referent refers to the same functional protein in the same or another strain of

E. histolytica .

By "substantially" homologous to is meant homology is sufficient to provide the requisite hybridization to target under the conditions the DNA is employed. Similarly, by "effective fragment" in this context is meant a fragment of sufficient size to provide the requisite hybridization to target under the conditions the DNA is employed.

A nucleotide sequence or epitope "characteristic of" a pathogenic or nonpathogenic strain refers to a nucleotide sequence or epitope which differentiates between these classes. Thus, a nucleotide sequence or epitope "characteristic of" a pathogenic E. histolytica refers to a nucleotide sequence or epitope wherein the corresponding sequence or epitope in the nonpathogenic strain is not identical to and is characteristically different from that of the sequence or epitope in the pathogenic strain.

A "composite" nucleotide sequence refers to a nucleotide sequence which in part corresponds to the sequence as it occurs in a pathogenic strain, and in part as it occurs in a nonpathogenic strain of E. histolytica . "Replicon" refers to a DNA vector which is capable of self-replication when transformed into a suitable host, the context most frequently used herein when transformed into E. coli .

Diagnostic tests involve hybridization under various levels of hybridization stringency, and these levels are defined as follows: low stringency corresponds to washing filters in 0.2 x SSC, 0.1% SDS at 37°C (1 x SSC is 0.15 M NaCl, 0.015 M Na citrate, SDS- Na lauryl sulfate) ; high stringency corresponds to washing in 0.2 x SSC,

0.1% SDS at 65°C; moderate stringency corresponds to washing in 0.2 x SSC, 0.1% SDS at 45°C. As used herein, "immunospecific" with respect to a specified target means that the antibody thus described binds that target with significantly higher affinity than that with which it binds to alternate epitope or antigenic determinant. The degree of specificity required may vary with circumstances, but typically an antibody immunospecific for a designated target will bind to that target with an affinity which is at least one or two, or preferably several orders of magnitude greater than that with which it binds alternate epitopes or antigenic determinants.

The term "antibody" refers not only to immunoglobulins per se, but also to fragments of immunoglobulins which retain the immunospecificity of the complete molecule. Examples of such fragments are well known in the art, and include, for example, Fab, Fab', and F(ab')_ fragments. The term "antibody" also includes not only native forms of immunoglobulins, but forms of the immunoglobulins which have been modified, as techniques become available in the art, to confer desired properties without altering the immunospecificity. For example, the formation of chimeric antibodies derived from two species is becoming more practical. In short, "antibodies" refers to any component of or derived form of an immunoglobulin which retains the immunospecificity of the immunoglobulin per se.

The term "pathogenic forms" of E. histolytica refers to those forms which are invasive and which result in symptomology in infected subjects. "Nonpathogenic forms" refer to those forms which may be harbored asympto- matically by carriers.

"Gal/GalNAc lectin" refers to the above described glycoprotein found on the surface of E. histolytica which mediates the adherence of the amoeba to target cells, and which mediation is inhibited by galactose or N-acetylgalactosamine. The Gal/GalNAc lectin refers specifically to the lectin reported and isolated by Petri et al. (supra) from the pathogenic strain HMI-IMSS, and to the corresponding lectin found in other species of E. histolytica . The "35/31 kDa subunit" or "light subunit" refers to the small subunit obtained by reduction of the Gal/GalNAc lectin and its corresponding counterparts in other species.

Preparation of Purified Gal/GalNAc Lectin and the 35/31 kDa Subunit

The preparation of a highly purified form of the Gal/GalNAc lectin derived from a pathogenic E. histolytica is described in detail in Example 2. The preparation comprises an affinity chromatography step wherein monoclonal antibodies prepared by immunization with the Gal/GalNAc lectin are used as an affinity ligand. The isolated lectin is then reduced with a sulfhydryl reducing agent, for example dithiothreitol or beta-mercaptoethanol, to obtain the 35/31 kDa subunit. The isolated 35/31 kDa light subunit or both the light and heavy subunits of the purified lectin may be used for immunization.

In a manner similar to that set forth in Example 2, 35/31 kDa subunit maybe isolated from nonpathogenic strains by utilizing those antibodies as affinity ligands which immunoreact with lectin epitopes characteristic of nonpathogenic forms or which are shared by pathogens and nonpathogens. As described hereinbelow, the purified forms of the

35 kDa and 31 kDa forms of the lectin were hydrolyzed by CNBr and sequenced. Based on the resulting sequence, primers were prepared to amplify cDNA for use as a probe. Using this probe, cDNA encoding the pathogenic form of the 35/31 kDa subunit was obtained; both isoforms are encoded by the same gene and share the same primary structure. The deduced amino acid sequence for these isoforms is shown in Figures la and lb, and described above.

Preparation of Monoclonal Antibodies

The monoclonal antibodies described below are prepared by immunization protocols using the isolated and purified Gal/GalNAc lectin light subunits of the invention. Use of these lectins in purified and isolated form as immunogens, as well as their availability for use in screening the monoclonal preparations obtained greatly facilitates the preparation and identification of suitable monoclonal antibodies.

For immunization, standard protocols are employed, and any suitable vertebrate, typically a mammal, such as rats, mice, rabbits, and the like, can be used as the subject. When sufficient titers are obtained, the sera are harvested. The sera prepared as above are useful polyclonal compositions which, it has been found, are required for recovery of recombinant clones which express the genes encoding the 35/31 kDa light subunit.

If monoclonals are desired, the antibody-producing cells of the subject, preferably spleen cells, are subjected to immortalization protocols, most conveniently those for the formation of hybridomas as set forth originally by Kohler and Millstein. However, additional techniques for immortalization such as viral infection may also be used.

The immortalized cells are then screened for the production of the desired mAbs. Generally, the super- natants of the cultured immortalized cells are tested in standard immunoassays, such as ELISA or RIA, which employ as antigen the purified lectin or subunit used as an immunogen. Positively reacting supernatants are then further tested. It is convenient to verify immunoreactivity with the 35/31 kDa subunit by using, as antigen in the assay or in Western blots, the reduced form of the isolated lectin.

The supernatants are then tested for cross-reactivity with the alternate forms of the lectin or subunit. For example, supernatants of antibody- secreting cells prepared from subjects immunized by pathogenic E. histolytica are tested by immunoassay against the purified lectin, or other lectin-containing antigen composition of nonpathogenic amoeba. Conversely, supernatants of antibody-secreting cells of subjects immunized with the lectin from nonpathogenic forms are checked for cross-reactivity with the lectin or other antigen-containing composition derived from the pathogenic alternatives. Thus, monoclonal antibody preparations are obtained which are either immunoreactive with epitopes shared by both pathogens and nonpathogens, or with epitopes which are unique to the form from which they are derived. Thus, three categories of monoclonal antibodies may be prepared, with respect to the 35/31 kDa subunit. One category of antibody is immunospecific for epitopes which are found on the 35/31 kDa subunit are characteristic of pathogenic forms. These antibodies are capable, there¬ fore, of immunoreaction to a significant extent only with the pathogenic forms of the amoeba or to the 35/31 kDa subunit of lectin thereof . A second set of monoclonal antibodies is immuno specific for epitopes which are characteristic of the 35/31 kDa subunit of nonpathogenic forms; thus, immunoreactive to a substantial degree only with the nonpathogenic amoeba or their lectins and not to the pathogenic forms. A third category of monoclonal antibodies is immunospecific for epitopes of the 35/31 kDa subunit common to pathogenic and nonpathogenic forms. These antibodies are capable of immunoreaction with the subunit or with the amoeba regardless of pathogenicity. The monoclonal antibodies reported in the art, as set forth in the Background section above, were prepared using a screening procedure which screens for inhibition of an adherence of the amoeba to target cells.

Therefore, the prior art antibodies are distinct from those of the present invention since the art heretofore has been unable to prepare antibodies against the 35/31 kDa subunit. The monoclonal antibodies of the invention are prepared by culturing immortalized cell lines which are capable of secreting them. The culturing of these lines is, as is known in the art, generally done in two ways--through in vitro culture methods with nutrients as generally understood, or by injection into suitable hosts, such as mice, in order to permit proliferation in vivo, with subsequent recovery of the mAbs from ascites fluid. As used herein, "culturing" an immortalized cell line and "recovering the mAbs from the culture" includes the procedures using both of these approaches.

Vaccines

Similarly, vaccines can be prepared with an immunoresponse to epitopes shared by both pathogens and nonpathogens, or with epitopes which are unique to the form from which they are derived. Alternatively, vaccines can be prepared to both the light and heavy subunits of the Gal/GalNAc lectin. Methods for formulating vaccines are known in the art, and the vaccine may contain carriers and/or adjuvants. Protocols for optimal administration (namely the minimum amount of antigen required to produce a protective response) can be determined using techniques known in the art. The present invention provides the DNA encoding the

35/31 kDa light subunit thus permitting recombinant production of this protein, including fusion proteins that include it. Presence of these proteins in vaccines enhances the immune response in an animal to be vaccinated against E. histolytica . Suitable fusion proteins include but are not limited to the maltose binding protein of E. coli or glutathione transferase. Alternatively, the DNA encoding the 31/35 kDa light subunit could be directly used as a vaccine, by the method of Wolff, et al. , Science (1990) 247:1465.

Protein can be produced alone (i.e., not as a fusion protein) in either eucaryotic or procaryotic systems which are well known in the art. These systems include, but are not limited to mammalian cells, yeast cells, insect cells, etc. Alternatively, the DNA could be used for expression in live, attenuated vaccine vectors, such as vaccinia virus, Bacillus Calmette Guerin, attenuated Salmonella, etc.

The DNA-Based Assay For the conduct of the assay of the invention, samples are prepared and any amoebae contained therein solubilized according to standard procedures for the type of sample provided. Stool samples are treated as described, for example, by Ungar, et al. , Am J Trop Med Hyg (1985) 3_4:465. Serum or plasma samples are diluted serially in phosphate buffered saline. Stool, serum or plasma samples are preferred, although other biological fluids or biopsy materials can also be used. The DNA is extracted from the sample by the method of Kawasaki, E.S., in "PCR Protocols," Innis, M.A. , et al., Eds., (1990) Academic Press, Ch. 18, p. 153 et seq. Briefly, in this protocol, the sample is lyophilized and resuspended in 100 μl of 50 mM Tris, pH 8.3, 150 mM NaCl, 0.5% NP40. Proteinase K is added to 100 μg/ml . The sample is incubated at 55°C for one hour, boiled for 3 minutes, and then placed on ice. After cooling, the sample is pelleted for 5 minutes in a microfuge and the supernatant is saved and either stored at 4°C or used immediately. About 5 μl of the supernatant is used as a DNA template for further amplification per 50 μl reaction mixture in this procedure.

For preparation of standards, identified trophozoites are used in the initial preparation.

Approximately 2 x 10° trophozoites are washed once with PBS, pelleted, lyophilized and stored at 20°C for future use. About 10 mg of lyophilized amoebae are then used in the extraction. The presence or absence of DNA associated with pathogenic or nonpathogenic strains can then be assessed using a protocol which takes advantage of nucleic acid sequence differences between these types of strains. All protocols involve amplification of the DNA using the polymerase chain reaction.

The PCR reaction is conducted using standard procedures, with primers designed to match specific regions of the target. In one approach, the primers are designed to hybridize to a region of the DNA which is characteristic of the pathogenic or nonpathogenic form as desired. Thus, only a characteristic region will be amplified for detection with probe. The probe is then selected to correspond to that region specifically. Pathogenic strains are then distinguishable from nonpathogenic strains by their ability to hybridize to a probe corresponding to the region as it appears in the pathogenic strains; similarly, nonpathogens are detectable by their ability to hybridize with probes which correspond to sequences characteristic of nonpathogenic strains.

In an alternate approach, the choice of primer is used to distinguish between the pathogens and nonpathogens. In this approach, at least one of the primers used in the PCR reaction will correspond to the sequence which is characteristic of one or the other type of strain. Thus only the desired sequences will be amplified and detected by probe. In this instance, the probe used can be from either pathogenic or nonpathogenic strains or a composite thereof, and the stringency of the hybridization conditions can be adjusted to accommodate the selection of the probe; the stringency will be high, low or moderate depending on the degree of probe homology. Where probes corresponding to the region to be detected are used, high stringency is generally employed. In the first method set forth above, high stringency is needed in order to distinguish between pathogenic and nonpathogenic strains; in the second approach which relies on characterization through selection of PCR primers, low stringency can be used since the distinction between pathogen/nonpathogen has already taken place. The hybridization to probe is detected using conventional techniques and under the stringency conditions set forth hereinabove.

Kits suitable for the above-described methods of diagnosis are also provided by the invention. These kits include, at a minimum, the appropriate primers, probes, additional reagents if desired, and instructions for conduct of the assay.

Antisense Methods

The DNAs of the invention can also be used to control the production of the 35/31 kDa Gal/GalNAc binding lectin subunit using antisense technology. The complement to the DNA of the 35/31 kDa subunit, or a significant portion thereof, is supplied to an amoeba culture or to host organism harboring E. histolytica infection using standard methods of administration, such as those set forth in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, latest edition. Preferably, for in vivo treatment of a subject, the complement is provided by injection, and formulated using conventional excipients therefor, such as Ringer's solution, Hank's solution, and the like. Oral administration with proper formulation can also be effected. While most administration is systemic, in the case of localized conditions such as solid tumor growth, administration may be topical or otherwise local. Slow release mechanisms for drug delivery may also be used.

Alternatively, the complementary nucleotide sequence may be generated in situ by providing an expression system which contains the cDNA of the 35/31 kDa subunit or its analogous DNA from other E. histolytica strains or an effective fragment thereof in a "reverse orientation" expression system. The expression system may either be designed to be operable in the host subject, such as a mammalian subject wherein the reverse oriented E. histolytica encoding sequence is under the control of, for example, an SV-40 promoter, an adenovirus promoter, and the like so that the complement is transcribed in situ. When used in a cell culture of E. histolytica, the expression system will be provided on a replicon compatible with the E. histolytica strain.

The following examples are intended to illustrate but not to limit the invention:

Example 1

Cultivation and Harvesting of E. histolytica Axenic E. histolytica, strain HMI1-IMSS, was grown in medium TYI-S-33 (trypticase and yeast extract, iron and serum) with 100 units/ml penicillin and 100 μg/ml streptomycin sulfate (Pfizer) at 37°C in 15 ml glass culture tubes. Amebic trophozoites were harvested after 72 h of growth by centrifugation at 150 x g for 5 min at 4°C and washed in 75 mM Tris (Sigma) , 65 mM NaCl, pH 7.2 (Ravdin, et al. , 1981) . Metabolic labeling with [³H]palmitate and [³H]myristate was accomplished by adding 50 μCi/ml [³H]palmitic or myristic acid (New England Nuclear) to TYI-S-33 medium and growing amebae for 24 h at 37°C.

Example 2 Purification of the Galactose Lectin by

Monoclonal Antibody Affinity Chromatography Amebic trophozoites harvested from a 72-h culture were washed as described and solubilized in 150 mM NaCl, 50 mM Tris, pH 8.3, 0.5% Nonidet P-40 (NP-40) (Sigma) , 5 mM ethylenediaminetetraacetic acid (EDTA) (Sigma) , and 2 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma) . The solubilized amebae were centrifuged at 15,000 x g for 10 min and the supernatant applied at 4°C to a monoclonal antibody affinity column consisting of 2 mg each of protein A-purified anti-lectin monoclonal antibodies H8- 5, 7F-4, 5B-8, 3F-4, and 6D-2 immobilized on 1-2 ml of Affi-Gel 10 (Bio-Rad) . The supernatant was recirculated through the column with a peristaltic pump overnight, and the column was then extensively washed with solubilization buffer. The bound amebic lectin was eluted with 0.2 N acetic acid, pH 2.5, immediately frozen and lyophilized. In some preparations, 5 mM p- chloromercuriphenyl-sulfonic acid (PCMS) (Sigma) was included in the solubilization buffer to inhibit amebic GPI-PLC. Example 3

SDS-PAGE and Autoradiography SDS-PAGE was performed using standard procedures previously described by Laemmli (1970) , using 6-15% acrylamide running gels. Molecular weight determinations were made using Rainbow Markers (14,300-200,000) from

Amersham. All proteins for electrophoresis were boiled in 4% SDS and 10% /3-mercaptoethanol unless otherwise indicated. For autoradiography, SDS-PAGE gels were soaked in Entensify A and B (DuPont) prior to drying and exposed to Kodak XAR-5 film at -70°C for one week or more.

One dimensional SDS-PAGE of both immunoprecipitated and affinity-purified lectin identified bands of 170, 35, and 31 kDa (not shown) . Since the 31 kDa band has not been previously resolved in purified lectin preparations (Petri, et al. , J Biol Chem (1989) 64_.:3007-3012, we wished to characterize both it and the 35 kDa subunit. Neither the 31 nor the 35 kDa protein migrated free of the heterodimer on non-reducing gels, indicating that both are disulfide-linked to the 170 kDa subunit (Petri, et al. , 1989 supra) .

To determine whether the 31 kDa band was a previously unidentified subunit of a heterotrimeric protein, the lectin was analyzed by two-dimensional gel analysis. The purified lectin was electrophoresed in a 6% tube gel under non-reducing conditions in the horizontal dimension and in a 10% slab gel reducing conditions in the vertical dimension. Staining was performed with Coomassie blue. The results are shown in Figure 2, arrowheads indicate the 170, 35, and 31 kDa subunits . Molecular weight markers in kDa are indicated on the left for the second dimension. The 170 kDa subunit appeared as a major broad band in the horizontal (nonreduced) dimension with minor bands of 160-170 kDa. As shown, the 35 and 31 kDa subunits appeared below the 170 kDa band in the vertical dimension but migrated separately in the horizontal dimension, suggesting that they are not members of a heterotrimeric protein, but rather represent light subunits of two different heterodimers; one containing the 170 and 35 kDa subunits, and the other containing the 170 and 31 kDa subunits.

Example 4 Amino Acid Composition of the Lectin Light Subunits The amino acid compositions of the two subunits were nearly identical, and compared closely with the amino acid composition derived from the 35/31 kDa cDNA (see below) and the previously published 35/31 kDa amino acid composition.

Table 1

Amino Acid composition of the galactose lectin subunits

Residue

Residues/100 amino acids

Cys ND^a ND 1.8 ND

Ala 4.1 4.5 5.8 6.8

He 3.6 3.7 4.4 4.4

Leu 5.6 5.4 4.4 5.8

Asp 9.6 10.8 6.2 7.0

Asn ND ND 7.3 ND

Gly 12.8 10.0 5.1 8.5

Glu 12.1 12.1 4.0 9.2

Gin ND ND 5.5 ND

Ser 8.0 6.9 5.1 7.3

Val 5.0 4.9 4.7 5.1

Tyr 5.0 4.6 8.0 6.6

Phe 3.9 4.1 5.1 5.1

His 1.4 1.3 1.1 1.9

Lys 11.6 12.0 8.4 9.4

Met 0.4 0.4 1.4 1.5

Pro 4.3 4.4 5.8 5.9

Arg 4.5 5.4 6.5 6.9

Thr 7.9 9.4 8.0 7.7

Trp ND ND 1.5 ND ^aND, not determined.

Column 1, electroeluted 31 kDa subunit

Column 2, electroeluted 35/31 kDa subunit

Column 3, deduced from cDNA

Column 4, from [Petri, 1989]

The slight differences observed may be due to the various E. histolytica strains used (HM-1:IMSS for amino acid analyses, H-302:NIH for cDNA sequencing) , or due to discrete members of the light subunit gene family. Example 5 Cyanogen Bromide Digestion of the Lectin Light Subunits To further characterize the 31 and 35 kDa subunits, each subunit was digested with CNBr and the resultant peptides were analyzed by tricine-SDS-PAGE (Figure 3) . The electroeluted 31 and 35 kDa subunits were dried and digested overnight with CNBr. The digested proteins were analyzed by tricine-SDS-PAGE, electrophoretically transferred onto a PVDF filter, and stained with Coomassie blue. Left lane, 35 kDa subunit; right lane, 31 kDa subunit. Molecular weight markers in kDa are shown on the right.

The peptide fragment patterns from CNBr digestion were almost identical for the two subunits, indicating that they are closely related, if not identical, proteins. Attempts to determine amino acid sequences from the CNBr fragments of the 31 kDa subunit by Edman degradation were unsuccessful. In addition, the amino terminus of the 31 kDa, but not the 35 kDa subunit, was blocked to Edman degradation, suggesting that one difference between these two proteins is a post- translational modification which blocks the 31 kDa amino terminus.

Example 6 Metabolic Labeling

A. Fatty acids: To examine the light subunit for the presence of a GPI anchor, the lectin was purified from amebae metabolically labeled with [³H]palmitate and [³H]myristate. Amebic trophozoites were metabolically labeled with 50 μCi/ml of [³H]palmitic acid or

[³H]myristic acid for 24 h in TYI-S-33 media. Following metabolic labeling amebae were pelleted, washed 2 x in 75 mM Tris, 65mM NaCl pH 7.2, and solubilized in 150 mM NaCl, 50mM tris, pH 8.3, 0.5% NP-40 (Nonidet P-40) , 5mM EDTA, 2mM PMSF, and 5mM PCMS. The results are shown in Figure 4. Lanes 1 and 4 , total amebic proteins; lanes 2 and 5, immunoprecipitation with mouse preimmune serum; lanes 3 and 6, immunoprecipitation with mouse anti-lectin antiserum. A number of amebic proteins were also labeled with both fatty acids as shown in Lanes 1 and 4.

B. [³H] Glucosamine and Incubation with PI-PLC: The 35 kDa subunit was more intensely labeled by [³H] glucosamine than the 31 kDa subunit, indicating that the two polypeptides are differentially glycosylated.

This variation in labeling intensity is not merely due to a quantitative difference as the two subunits appear to be about equally abundant as shown by Coomassie blue- stained gels of the lectin (Figure 2) . SDS-PAGE has shown that T. Jbrucei VSGs in detergent lysate have apparent molecular weights 800-2,000 daltons lower than soluble VSGs. To determine whether migration of the lectin on SDS-PAGE was affected by PI-PLC, [³H] glucosamine-labeled lectin was incubated with B. cereus PI-PLC or PCMS, a PI-PLC inhibitor, and analyzed by SDS-PAGE and autoradiography. As shown in Figure 5, E. histolytica trophozoites were metabolically labeled for 1 h at 37°C in glucose-free TYI-S-33 media with 50 μCi/ml [³H] glucosamine in the presence of 5mM PCMS (lane A) , or 1 unit/ml B. cereus PI-PLC (lane B) , immunoprecipitated with anti-lectin antisera, and analyzed by SDS-PAGE and autoradiography. Molecular weight markers in kDa are on the left. PI-PLC digestion of [³H] glucosamine-labeled lectin had no apparent effect on the migration of either the 35 or 31 kDa subunits, suggesting that the GPI-anchor on the 31 kDa subunit is resistant to B . cereus PI-PLC. Differential resistance to PI-PLC from various sources has been shown for other GPI-anchored proteins. Example 7 Immunoprecipitation of Galactose Lectin from Solubilized Amebae Amebae were metabolically labeled with [³H] palmitate and solubilized as described, then centrifuged at 4°C for 10 min to remove insoluble matter. In some experiments, solubilized amebic proteins were incubated with 0.5 units Bacillus cereus PI-PLC (Sigma) for 1 h at 37°C prior to addition of the antibodies. The solubilized galactose lectin was incubated overnight with mouse anti-heavy subunit mAb or mouse anti-lectin antisera, using mouse IgG_j or mouse pre-immune serum as controls. The immune complexes were precipitated overnight at 4°C with 50 μl of 50% protein-A agarose beads (Sigma) and washed 5 x with solubilization buffer prior to SDS-PAGE.

Immunoprecipitation with mouse anti-lectin antisera demonstrated that the 170 and 31 kDa, but not the 35 kDa, subunits were metabolically labeled by [³H] palmitate. (Figure 4, Lanes 3 and 6.) The lipid modification on the heavy subunit has not been investigated, however cDNA sequence analysis indicates that it is an integral- membrane protein. The [³H]myristate-labeled band at 21 kDa does not appear to be associated with the lectin as it was non-specifically immunoprecipitated with control antisera. Figure 4, Lanes 5 and 6.

Example 8 Nitrous Acid Deamination of Immunoprecipitated Lectin Nitrous acid deamination of GPI-anchored proteins cleaves the glucosamine- (1-6) -myoinositol glycosidic linkage, releasing phosphatidylinositol or acyl- phosphatidylinositol, and is diagnostic for the presence of a GPI anchor. In order to identify the nature of the lipid modification, the [³H]palmitate-labeled, 31 kDa band from total amebic proteins was deaminated with nitrous acid and the resulting lipid product was extracted into n-butanol. The released lipid product exactly comigrated with phosphatidylinositol when analyzed by TLC. Amebic trophozoites were metabolically labeled for

24 h with 50 μCi/ml of [³H]palmitate in TYI-S-33 media. [³H] Palmitate-labeled amebic proteins were immunoprecipitated with anti-lectin antisera, separated by SDS-PAGE, dried, and autoradiographed to identify the lipid-labeled 31 kDa subunit. The 31 kDa band was cut from the gel, rehydrated in deionized water, and electroeluted. The electroeluted protein was dialyzed against water and evaporated. The dried protein was resuspended in 400 μl of freshly prepared deamination buffer (0.1 M CH₃COONa, pH 3.7, 0.1% NP-40, 0.25 M NaN0₂) , incubated for 12^' h at room temperature, and acidified with 6 μl of 5 M HC1. The reaction products were partitioned between 400 μl of water-saturated n-butanol :water (1:1) and the aqueous phase was extracted again with 400 μl of water-saturated n-butanol to remove residual salt. The butanol phases were pooled and evaporated, and TLC conducted.

Briefly, TLC was performed as follows. Lipids were resuspended in 9:1 chloroform:methanol and chromatography was performed on Kieselgel 60 plastic-backed silica gel plates (EM Science) that had been heated at 80°C for 30 min prior to use. The solvent system used was 4:4:1 chloroform:methanol :water. Lanes were cut into 1 cm strips and analyzed by scintillation counting. Lipid standards were chromatographed simultaneously with the labeled lipids and visualized in iodine vapor. Standards were (a) phosphatidylcholine; (b) phosphatidylinositol; (c) phosphatidylethanolamine; (d) dimyristoylglycerol . The results are shown in Figure 6a. To verify that the 31 kDa band from SDS-PAGE represented the 31 kDa lectin light subunit, the lectin was immunoprecipitated with mouse anti-lectin antiserum and the experiment was repeated. Amebic proteins were metabolically labelled with [³H]palmitate and detergent- solubilized. Following solubilization, immunoprecipitation was performed with mouse anti-lectin antiserum, and the lectin subunits were separated by SDS- PAGE. The 31 kDa band was identified by autoradiography, cut from the gel and analyzed for lipid composition as above. On this chromatograph, the solvent front was allowed to migrate further on the TLC plate, which caused both the phosphatidylinositol standard and the labeled lipid to appear as broad bands, however the radioactive label again comigrated with phosphatidylinositol, verifying the identity of the lipid on the 31 kDa subunit (Figure 6b) . The release of phosphatidylinositol, a GPI anchor component, confirmed the presence of a GPI anchor on the 31 kDa subunit.

Example 9

Western Blots with Anti-CRD Antibodies GPI anchors from several species have been shown to share a carbohydrate epitope known as the cross-reacting determinant (CRD) , which is exposed by cleavage of diacylglycerol from the anchor by phosphatidylinositol- specific phospholipase C (PI-PLC) . To determine whether the lectin contained the CRD, the 31 and 35 kDa subunits prepared as in Example 3 were electroeluted, incubated with B . cereus PI-PLC, and immunoblotted with anti-CRD antibodies. Ten μg of each subunit was incubated with

0.1 unit B . cereus PI-PLC (phosphatidylinositol-specific phospholipase C) for 30 min at 37°C, electrophoresed by SDS-PAGE, and transferred to a PVDF filter for Coomassie blue staining or immunoblotting with anti-CRD antibodies diluted 1:500 (courtesy of Dr. Tamara Doering, the Johns Hopkins University) . Prior to immunoblotting the filter was incubated overnight at 4°C in 5% nonfat dry milk (Carnation) to block excess protein-binding capacity. Incubation times with the antibodies were 1 h at room temperature followed by washings of 3 x 10 min in 50 mM Tris-HCl, 200 mM NaCl, 0.1% Tween 20, pH 7.35/31. The first wash following incubation with primary antibody was at 50°C. The secondary antibody was an anti-rabbit IgG alkaline phosphatase conjugate (Promega) . Blots were developed in Western Blue (Promega) .

The results are shown in Figure 7, Lane 1, affinity purified galactose-binding lectin; Lanes 2 and 4, electroeluted 35/31 kDa subunit; Lanes 3 and 5, electroeluted 31 kDa subunit. Lanes 1, 2, and 3, Coomassie blue-stained proteins. Lanes 4 and 5, immunoblots with anti-CRD antibodies. Both subunits were bound by anti-CRD antibodies (Lanes 4 and 5) . In the absence of PI-PLC, the antibodies bound equally well, based on blot intensity (not shown) .

Weak reactivity of membrane form VSG with anti-CRD antibodies has been reported and is most likely due to a CRD epitope which is exposed on membrane form GPI anchors. The anti-CRD antibodies did not react with the lectin 170 kDa subunit, nor did the secondary antibody alone bind the 31 or 35 kDa subunits (not shown) .

Example 10 Cellular Fractionation of E. histolytica and Assay of Amebic GPI-PLC Activity

The discovery of a GPI-anchored protein suggests that E. histolytica contains an endogenous GPI-PLC activity. To assay for amebic GPI-PLC, [³H]myristate- labeled VSG was purified and used as the substrate. The release of diacylglycerol from the VSG is diagnostic for GPI-PLC activity.

Amebic cytosolic and membrane fractions were prepared as previously described (Fox, et al . J Biol Chem (1986) 261:15767-15771) , with minor alterations. Briefly, 5 x IO⁷ amebic trophozoites were pelleted and washed in 5 ml of 75 mM Tris 65 mM NaCl, pH 7.2, then incubated on ice for 30 min in the same buffer with 1:1000 diisopropylfluorophosphate (Sigma) . Trophozoites were then osmotically lysed in 5 ml of 10 mM sodium phosphate buffer, pH 8.0 containing 2 mM PMSF. Following a 5 min incubation at 37°C, the preparation was freeze- thawed and centrifuged at 50,000 x g for 1 h at 4°C. The supernatant was assayed as the cytosolic fraction. The resulting pellet was resuspended in fresh lysis buffer and centrifuged at 100,000 x g for 1 h at 4°C. This pellet was homogenized on ice in a dounce homogenizer in 5 ml of 1% n-octyl glucoside, 25 mM Tris HCl, pH 8.0 with 2 M PMSF. The homogenate was then centrifuged at

100,000 x g for 1 h at 4°C. This supernatant was assayed as the membrane fraction.

Amebic GPI-PLC activity was assayed using 2μg/15 ml [³H]myristate-labeled VSG (variant surface glycoprotein) as the substrate in 1% NP-40, 50mM Tris-HCl, 5mM EDTA, pH 8.0 for 30 min at 37°C as described (Hereld, et al . , J Biol Chem (1986) 13813-13819) . Each fractions contained 1 x IO⁷ amebic equivalents. One unit of purified B . cereus PI-PLC was used as a positive control, and the assay buffer (1% NP-40, 50 mM Tris HCl pH 8.0, 5 mM EDTA) was used as a negative control. For the assays with dithiothreitol (DTT) and EDTA, 25 mM DTT and 5 mM EDTA were added to the cytosolic fractions. The DTT and EDTA assays were done in duplicate and results were expressed as the mean of two assays. Following the PI-PLC assays the n-butanol phase was evaporated on a Speed Vac, resuspended in 10 μl of chloroform/methanol (9:1) , and chromatographed on a thin layer of plastic-backed Silica Gel 60 (heated at 80°C for 30 min prior to- use) using petroleum ether/diethyl ether/acetic acid (80:20:1) as the solvent . The lanes were cut into 0.5 cm strips and analyzed by scintillation counting.

Results are expressed as cpm in butanol phase/total cpm in both phases. Column 1, 1 unit purified B . cereus PI-PLC; column 2, buffer alone; column 3, E. histolytica membrane fraction; column 4, E. histolytica cytosolic fraction.

As shown in Figure 8a, E. histolytica contained a GPI-PLC in the cytosol in contrast to T. Brucei , where the endogenous GPI-PLC activity was identified in membrane fractions. Addition of 5 mM EDTA or 25 mM DTT increased cleavage of [³H]membrane form VSG by 23% and 41% respectively (not shown) , which indicated that like the T. brucei GPI-PLC, the amebic enzyme is somewhat inhibited by Ca²⁺, and may contain an active site thiol .

To confirm that the lipid released from the VSG was due to an amebic GPI-PLC, TLC was performed on the organic-phase product cleaved from [³H]myristate-labeled VSG by the amebic cytosolic fraction. The butanol phase from the GPI-PLC assay was analyzed by TLC along with the product of a purified B . cereus PI-PLC digestion. The butanol phase was evaporated, lipids were resuspended in 9:1 chloroform:methanol, spotted onto plastic backed silica gel 60 TLC plates (heated at 80°C for 30 min prior to use) , and analyzed using petroleum ether:diethyl ether:acetic acid (80:20:1) as the solvent. Lanes were cut into 0.5 cm strips and analyzed by scintillation counting. Lipid standards were developed in iodine vapor. Standards were (a) dimyristoylglycerol; (b) myristic acid; (c) dimyristoyl phosphatidic acid. A single radioactive band appeared on TLC that comigrated with purified dimyristoylglycerol and the lipid product of purified [³H]VSG digested with B . cereus PI-PLC (Figure 8b) . An identical experiment using the amebic membrane fraction failed to release radioactive label into the organic phase (not shown) .

Example 11 Amino Acid Analysis and Cyanogen Bromide Digestion Affinity-purified lectin was electrophoresed as described above. The 31 and 35 kDa subunit bands were identified by staining at room temperature for 20 min using 0.5% Coomassie blue in acetic acid:isopropanol :water (1:3:6) and destained at 4°C in acetic acid:methanol :water (50:165:785) . The bands of interest were cut from the gel and electroeluted. The electroeluted proteins were analyzed for amino acid composition at the Protein and Nucleic Acid Sequencing Center of the University of Virginia. For peptide mapping, 50 μg of dried protein was digested overnight at room temperature in the dark using 50 μl of a 70 mg/ml solution of CNBr in 70% formic acid. The digested proteins were dried and boiled in 2X reducing sample buffer for tricine-SDS-PAGE analysis (Schagger, & von

Jagow, 1987) . Following tricine-SDS-PAGE, proteins were electrophoretically transferred to PVDF filters and stained with Coomassie blue. The amino terminus of the lectin light subunit and one CNBr fragment were sequenced by Edman degradation as described in Mann, et al. , Proc Natl Acad Sci (1991) 85: 2444-2448. Example 12 PCR and Se uencing Reactions Using the E. histolytica codon bias, two degenerate oligonucleotides were designed from the sequence results obtained in Example 11 sequencing and used as primers in a polymerase chain reaction (PCR) to amplify a 400 bp fragment from genomic E. histolytica DNA. The primers used were: 5'GCGCAAGCTTCCAAA(C,T)TA(C,T)CCATA(C,T)GG3' (sense) and 5'GCGCCTCGAG(T,A)AC(T,A)GA(CT)TTTGGAAT(T,A)G (antisense) . PCR reactions were carried out using Pfu polymerase (Stratagent) . Reaction times were 1 min at 93°C, 1 min at 37°C, and 3 min at 72°C for 30 cycles. The 400 bp PCR product was labeled with α[³²P]dCTP (Amersham) using the random primed DNA labeling kit (Boehringer-Mannheim) . The [³²P] -labeled PCR fragment was used to screen an E. histolytica λgtll cDNA library from strain H-302:NIH (courtesy of Dr. Bruce Torian, Louisiana State University (Torian, et al. Proc Natl Acad Sci (1990) 7:6358-6362)). A single plaque was identified which hybridized with the labeled PCR fragment. The plaque was isolated and purified, and additional PCR products were generated using the purified plaque as a source of template DNA with primers designed from the 400 bp fragment and λgtll flanking sequences. PCR products were prepared for sequencing by polyethylene glycol precipitation as described (Mann, et al. , Proc Natl Acad Sci (1991) 88_.=3248-3252. The 400bp fragment hybridized to a single plaque which was subsequently purified and sequenced and were sequenced by Sanger di-deoxy sequencing using the TAQuenase system (USB) .

Gel readings were assembled using the Sequence Assembly Manager. Comparisons of the derived protein sequence with the National Biomedical Research Foundation, Swiss-Prot, and Genbank data bases were done using the FASTA program (Pearson, et al. Proc Natl Acad Sci (1988) 5:2444-2448) . The cDNA insert from the purified plaque (designated lgll) contained an open reading frame (ORF) encoding a polypeptide of 278 amino acids with a predicted molecular weight of 32.1 kDa

(Figure la) . Analysis of the deduced amino acid sequence identified the amino-terminal and CNBr sequences determined by Edman degradation, as well as three amino acids amino-terminal to the mature polypeptide which most likely are components of the leader sequence common to membrane and secreted proteins. (The presence of alanine and glycine at the -3 and -1 positions respective to the amino terminus of the mature protein is consistent with the requirement for small amino acids at these positions in signal peptides) . The remainder of the leader sequence, including the expected amino-terminal translation initiation codon ATG, was not present in this clone.

The amino-terminal sequence and the total amino acid composition were nearly identical to that previously described for the light subunit (Petri, et al. , 1989), verifying the identity of this cDNA (Figure 1 and Table 1) . The amino acid sequence contained two potential N- linked glycosylation sites, supporting metabolic labeling studies which showed incorporation of [³H] glucosamine by the 35/31 kDa subunit. No sequence similarity was found between the 35/31 kDa subunit and the classical carbohydrate-binding domains of C- and S-type lectins. FASTA searches of the complete light subunit amino acid sequence found no significant similarity to other proteins in the National Biomedical Research Foundation data bank (Pearson, et al . , 1988) .

A hydropathy plot showed that the polypeptide is relatively hydrophilic and lacks a membrane-spanning domain. Instead, a hydrophobic sequence of 7 amino acids was identified at the carboxy terminus. A hydrophobic carboxy-terminal domain is unique to GPI-anchored proteins, although the hydrophobic carboxy-terminal domain of 7 residues is relatively short compared with most other GPI-anchored proteins. However, T. Brucei VSGs anchored through serine have short precursor tails and terminate in a stretch of only 8 hydrophobic amino acids. It is believed that GPI anchor addition is an early post-translational event in which 15-30 carboxy- terminal residues are cleaved from the nascent protein and the GPI anchor is subsequently attached. Fifteen residues amino-terminal to the hydrophobic carboxy- terminus the cDNA encodes the tripeptide C-S-A. This tripeptide is consistent at all three positions with previously sequenced GPI anchor cleavage/addition sites, but is three amino acids closer to the carboxy terminus than the shortest signal identified to date.

Only about 20 of the approximately 100 known GPI- anchored proteins have been characterized for the site of GPI attachment and considerable heterogeneity has been found for the GPI addition signal in other organisms. Therefore, the exact addition site for the anchor cannot yet be determined based on sequence information alone. The identification of amino- and carboxy-terminal signal peptides together with a potential cleavage/addition site, plus the lack of putative transmembrane and cytoplasmic domains, suggested that this cDNA encodes a GPI-anchored protein. An additional clone, retrieved using the above protocol, represents an additional member of the gene family (lgl2) and is shown in Figure lb. The sequence of lgl2 includes a single open reading frame of 864 nucleotides encoding a polypeptide of 288 amino acids with a calculated molecular weight of 32.4 kDa for the mature protein.

Example 12 Southern and Northern Analysis A. Southern Blot: To analyze E. histolytica for the presence of multiple lectin light subunit genes, genomic DNA from strain HM-1:IMSS was isolated, digested to completion with the restriction enzymes Bglll and EcoRI , and transferred to Zetabind filters (CUNO) according to published procedures (Maniatis, et al. ,

Molecular Cloning: A Laboratory Manual (1982) . The 400 bp PCR fragment was random prime-labeled (Boehringer Mannheim) with α [³²P] dCTP (Amersham) and denatured by boiling for 10 min prior to use. Hybridization was performed at 37°C. Washes were done at 50°C in 0.IX SSC, 0.1% SDS. Figure 9a shows the results for Bglll (lane A) or EcoRI (lane B digestion after) , separation by agarose gel electrophoresis and transfer onto a nylon filter. Molecular size markers are shown in kilobase pairs at the left. The labeled PCR product bound to fragments of 8.3 and 4.8 kb from Bglll digested DNA, and to fragments of 10.5 and 6.8 kb from EcoRI digested DNA. The hybridization of the probe to two DNA fragments after restriction enzyme digestion suggested the presence of a light subunit gene family, since neither restriction site was found in the 35/31 kDa subunit cDNA. Previous work had identified microheterogeneity in the amino acid sequence of the 35/31 kDa subunit, consistent with it being encoded by more than one gene '(Petri, et al . , 1989) .

B. Northern Blot Analysis: For Northern blot analysis, total E. histolytica RNA was purified and analyzed as previously described (Mann, et al . , Proc Natl Acad Sci (1991) £8:3248-3252. Total E. histolytica RNA was purified and separated by formaldelhyde gel electrophoresis and transferred to a nylon filter. The filter was hybridized with the [³P] -labeled 35/31 kDa subunit 400 bp PCR fragment as paragraph A of this example. Molecular size markers are shown in kilobase pairs at the left of Figure 9b which shows the results. A single mRNA of approximately 1.0 kb encoding the 35/31 kDa subunit was identified. This corresponded well with the cDNA size of 837 bp and an earlier estimation of 1.2 kb (Mann, et al . , Natl Acad Sci (1991) 3428-3252. The presence of a single band on the Northern blot indicated that the multiple light subunit mRNAs must be similar in size.

Example 14 Recognition of the Adherence Lectin Light Subunits Both the 35 kDa and 31 kDa isoforms were recognized by antisera produced against a recombinantly-expressed glutathione-S-transferase (GST) -light subunit fusion protein. The cDNA sequence encoding the entire amino acid sequence of the 35/31 kDa lectin light subunit was expressed as a GST fusion protein in E. coli using the pGEX expression plasmid. The fusion protein thus consists of the GST as amino terminus and light subunit as carboxy terminus. The fusion protein was purified by French press disruption and electrophoresis and electroelution and used to immunize mice. The immune sera from the mice were reacted with the purified fusion protein and the adherence lectin on Western blots. The results are shown in Figure 10. Lane 1 is the fusion protein reacted with pre-immune sera; lane 2 is the fusion protein reacted with the immune sera of mice immunized with the GST-35 kDa subunit fusion protein; lane 3 is purified lectin reacted with pre-immune sera; lane 4 is the 35/31 kDa isoforms reacted with immune sera to GST-35 kDa. Recognition of the 35 kDa and 31 kDa subunit isoforms by the antisera is confirmed by the results in lanes 2 and 4.

Example 15

Immunization of the Gerbils with the

Adherence Lectin 35/31 kDa subunit Gerbils were immunized intraperitoneally with 50μg of purified 35/31 kDa subunit-GST fusion protein in complete Freund's adjuvant. After two weeks the animals were given a second intraperitoneal injection of 50 μg in complete Freund's adjuvant. Four weeks after the second injection the animals were challenged by direct injection of amebic trophozoites in to the liver. Two weeks later the animals were sacrificed and examined for the presence of amebic liver abscess. As a control, one group of animals was immunized with the GST protein alone. Immunization with 35/31 kDa fusion protein resulted in a decrease in the size of liver abscess when compared to animals immunized with GST alone.

IMMUNIZATION ABSCESS WEIGHT (g)

Animal #1 GST 6.14

#2 GST .68

#3 GST 13.87

#4 GST 1 .66

#5 GST 4.39

#6 GST ,33

#7 GST ,27

#8 GST-35/31 kDa ,16

#9 GST " ,33

#10 GST " ,81

#11 GST " ,44

#12 GST " ,12

#13 GST " ,34

#14 GST " ,75

#15 GST ,85

MEAN of ABSCESS WEIGHT

GST 35/31kDa

MEAN 4.76 1.73

SD (+/- 4.71 1.73S

SE 1.78 61

Example 16 Immunization of Gerbils with a Synthetic Peptide Consisting of Amino Acids 79-90 of the Adherence Lectin 35/31 kPa Subunit

A light subunit peptide, consisting of amino acids 79-80 of the mature lgll 35/31 kPa subunit of the adherence lectin (amino acids boxed in Figure la) , was identified as a potential B-cell epitope from analysis of a hydropathicity profile of the 35/31 kPa subunit amino acid sequence. See, for instance, T. P. Hopp et al . , "Prediction of Protein antigenic determinants from amino acid sequences," Proc. Natl. Acad. Sci., USA (1981) 7£:3824-3828. The peptide was synthesized and coupled to keyhole limpet hemocyanin (KLH) carrier by routine peptide chemistry methods known in the art. See, for instance, U.S. Patent No. 4,708,871, the entire disclosure of which is hereby incorporated herein by reference. Gerbils were immunized with 50 μg of the light subunit expressed as a glutathione-S-transferase (GST) fusion protein ("light subunit"), for comparison, or with the light subunit peptide or GST alone ("controls") . Gerbils were immunized intraperitoneally (ip) using Freund's adjuvant and were boosted at 2 weeks with an additional 50 μg of immunogen ip in incomplete Freund's adjuvant. At 6 weeks the animals were challenged by the direct injection of amebic trophozoites into the liver. Two weeks later the animals were sacrificed and examined for the presence of amebic liver abscess. The results in the following table show that the light subunit peptide vaccine was about as effective as the 35 kPa subunit fusion product vaccine.

Animals with

Immunization Abscess Weight (g) Abscesses

Controls 1.19±0.53 (n=6) 83%

35 KPa subunit 0.31±0.09 (n=7)^* 100%

35 KPa peptide 0.40±0.34 (n=6)^# 50%

(amino acids 79- -90)

^*(p<0.06 compared to control) ^#(p<0.20 compared to control)

Claims (26)

1. A vaccine comprising the 35/31 kDa subunit of the Gal/GalNAc adherence lectin of Entamoeba histolytica, or a functional portion thereof, in admixture with at least one pharmaceutically acceptable excipient.

2. The vaccine of claim 1 wherein said lectin is derived from a pathogenic strain.

3. The vaccine of claim 1 which further includes the 170 kDa subunit of E. histolytica Gal/GalNAc lectin or a functional portion thereof.

4. The vaccine of claim 1 wherein the 35/31 kDa subunit is in the form of a fusion protein, fused to additional amino acid sequence.

5. The vaccine of claim 4 wherein said additional amino acid sequence is that of glutathione-S-transferase or a substantial portion thereof.

6. The vaccine of claim 1 comprising as an active ingredient a peptide consisting essentially of amino acids 79-90 of said 35/31 kDa subunit.

7. A process of immunizing a host against

Entamoeba histolytica comprising: administering a vaccine containing, as active ingredient, a 35/31 kDa subunit of the Gal/GalNAc adherence lectin of E. histolytica or a functional portion thereof, whereby said vaccine elicits an immune response in the host against invasive amebiasis by E. histolytica trophozoites.

8. The process of claim 10 wherein said active ingredient is said functional portion and wherein said functional portion elicits antibodies which block adherence of the lectin.

9. A DNA in purified and isolated form which consists essentially of a DNA encoding the 35/31 kDa subunit of E. histolytica Gal/GalNAc adherence lectin, or a functional portion thereof.

10. A recombinant DNA which comprises a DNA encoding the 35/31 kDa subunit of E. histolytica Gal/GalNAc adherence lectin, or a functional portion thereof, ligated to a heterologous DNA.

11. An expression system capable of producing the 35/31 kDa subunit of E. histolytica Gal/GalNAc adherence lectin, or a functional portion thereof, which comprises DNA encoding said subunit or portion operably linked to control sequences capable of effecting its expression.

12. The expression system of claim 11 wherein said control sequences are viral control sequences.

13. The expression system of claim 12 contained in a live attenuated virus.

14. The expression system claim 13 wherein said virus is a vaccinia virus.

15. A recombinantly produced fusion protein which comprises the amino acid sequence of the 35/31 kDa subunit of the Gal/GalNAc adherence lectin of E. histolytica or a functional portion thereof fused to additional heterologouε amino acid sequence.

16. A method to inhibit the production of the 35/31 kDa subunit of Gal/GalNAc adherence lectin in a culture of E. histolytica which method comprises contacting said culture with an oligonucleotide which is the complement to the nucleotide sequence of Figure la or lb, or with a corresponding oligonucleotide or an effective fragment thereof, wherein said oligonucleotide is of a pathogenic strain, and wherein said contacting is effected by providing an expression system for said oligonucleotide under conditions wherein expression is effected.

17. A polynucleotide in recombinant form ligated to a heterologous polynucleotide, or in purified and isolated form, which comprises the complement to the nucleotide sequence of Figure la or lb or a corresponding oligonucleotide^'or an effective fragment thereof.

18. An expression system which comprises the nucleotide sequence of Figure la or Figure lb or a corresponding nucleotide sequence of an E. histolytica strain, or an effective fragment thereof, in antisense orientation under control of a compatible control sequence.

19. A method to detect the presence or absence of E. histolytica in a biological sample which has been treated so as to contain single-stranded polynucleotides, which method comprises subjecting the treated sample to polymerase chain reaction (PCR) amplification using primers framing a region of the 35/31 kDa subunit of the Gal/GalNAc adherence lectin of E. histolytica, and probing the amplified DNA under stringent conditions with an oligomer corresponding to said region.

20. Antibodies immunospecific for the 35/31 kDa subunit of E. histolytica Gal/GalNAc lectin.

21. The antibodies of claim 20 which are monoclonal antibodies.

22. The antibodies of claim 21 which are immunospecific for an epitope characteristic of a pathogenic strain.

23. The antibodies of claim 21 which are immunospecific for an epitope characteristic of a nonpathogenic strain.

24. The antibodies of claim 21 which are immunospecific for an epitope characteristic of both pathogenic and nonpathogenic strains.

25. An immortalized cell line capable of secreting the antibodies of claim 21.

26. A method to detect the presence or absence of E. histolytica in a biological sample which method comprises contacting said sample with the antibodies of any of claims 22 to 24 under conditions wherein any E. histolytica present forms a complex with any of said antibodies; and detecting the presence or absence of said complex.