AU710439B2 - Recombinant 170 KD subunit lectin of Entamoeba histolytica and methods of use - Google Patents

Description

RECOMBINANT 170 KD SUBUNIT LECTIN OF ENTAMOEBA HISTOLYTICA AND METHODS OF USE Field of the Invention The invention concerns the use of epitopebearing regions of the 170 kD subunit of Entamoeba histolytica Gal/GalNAc adherence lectin which are produced recombinantly in procaryotic systems in diagnosis and as vaccines. Thus, the inventicn relates to the determination of the presence, absence or amount of antibodies raised by a subject in response to infection by E. histolytica using these peptides and to vaccines incorporating them. This invention also particul-rly relates to reagents specific for a novel variant of the 170 kD subunit of E. histolytica Gal/GalNAc adherence lectin and to the gene (hgl3) which encodes this novel subunit form, which represents the third member of the multigene family encoding this 170 kD subunit.

Bakground Art Entamoeba histcoytica infection is extremely common and affects an estimated 480 milliro 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 WO 95/00849 PCT/US94/06890 2 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 conveniently distinguishable from the nonpathogenic counterparts using morphogenic criteria, but 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.

It is known that E. histolytica infection is mediated at least in part by the "Gal/GalNAc" adherence lectin which was isolated from a pathogenic strain and purified 500 fold by Petri, et al., J Biol Chem (1989) 264:3007-3012. The purified "Gal/GalNAc" lectin was shown to have a nonreduced molecular weight of 260 kD on SDS-PAGE; after reduction with beta-mercaptoethanol, the lectin separated into two subunits of 170 and 35 kD MW. Further studies showed that antibodies directed to the 170 kD subunit were capable of blocking surface adhesion to test cells (Petri, et al. J Biol Chem (1989) supra). Therefore, the 170 kD subunit is believed to be of primary importance in meditating adhesion.

In addition, the 170 kD subunit is described as constituting an effective vaccine to prevent E.

histolytica infection in U.S. Patent 5,004,608 issued 2 April 1991.

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 sera tested (Petri, Jr., et al., Am J Med Sci (1989) 296:163-165). The lectin heavy subunit is almost universally recognized by immune sera and T-cells from patients with invasive amebiasis (Petri, WO 95/00849 PCT/US94/06890 3 et al., Infect Immun (1987) 55:2327-2331; Schain, et al., Infect Immun (1992) 60:2143-2146).

DNA encoding both the heavy (170 kD) and light kD) subunits have been cloned. The heavy and light subunits are encoded by distinct mRNAs (Mann, et al., Proc Natl Acad Sci USA (1991) 88:3248-3252) and these subunits have different amino acid compositions and amino terminal sequences. The sequence of the cDNA encoding the 170 kD subunit suggests 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 USA (1991) supra; Tannich, et al., Proc Natl Acad Sci USA (1991) 88:1849-1853). The derived amino acid sequence of the 170 kD lectin shows that the extracellular domain can be divided into three regions on the basis of amino acid composition. The amino terminal amino acids 1-187 are relatively rich in cysteine and tryptophan Amino acid sequence at positions 188-378 does not contain cysteine, and the amino acid sequence at positions 379-1209 contains 10.8% cysteine residues. The obtention of clones encoding the heavy chain subunit is further described in U.S. Patent 5,260,429 issued 9 November 1993, the disclosure of which is incorporated herein by reference. In that patent, diagnostic methods for the presence of E. histolytica based on the polymerase chain reaction and the use of DNA probes is described.

The heavy subunit is considered to be encoded by a multigene family (Mann, et al., Parasit Today (1991) 1:173-176). Two different heavy subunit genes, hgll and hgl2, have been sequenced by separate laboratories. While hgl2 was isolated from an HM-1:IMSS cDNA library in its entirety (Tannich, E. et al. Proc Natl Acad Sci USA (1991) 88:1849-1853), hgll was isolated in part from an H-302:NIH cDNA library and in part by PCR amplification of the gene from the HM-1:IMSS genome WO 95/00849 PCT/US94/06890 4 (Mann, B.J. et al. Proc Natl Acad Sci USA (1991) 88:3248- 3252). As the amino acid sequence of these two genes is 87.6% identical (Mann, B.J. et al. Parasit Today (1991) 7:173-176), the differences could be explained by strain variation alone. The presence of multiple bands hybridizing to an hgl probe on Southern blots, however, in consistent with the existence of a 170 kDa subunit gene family (Tannich, E. et al. Proc Natl Acad Sci USA (1991) 88:1849-1853).

Monoclonal antibodies specifically immunoreactive with various epitope-bearing regions of the 170 kD heavy chain subunit have also been disclosed in U.S. Patent 5,272,058 issued 21 December 1993, the disclosure of which is incorporated herein by reference in its entirety. This application also describes use of these antibodies to detect the 170 kD heavy chain and the use of the 170 kD subunit to detect antibodies in serum or other biological samples. The experimental work described utilizes the native protein. Further characterization of these antibodies is described in a publication by Mann, et al., Infect Immun (1993) 61:1772-1778 also incorporated herein by reference.

Various immunoassay techniques have been used to diagnose E. histolytica infection. ELISA techniques have been used to detect the presence or absence of E. histolytica antigens both in stool specimens and in sera, though these tests do not seem to distinguish between the pathogenic and nonpathogenic strains. In a seminal article, Root, et al., Arch Invest Med (Mex) (1978) 9: Supplement 1:203, described the use of ELISA techniques for the detection of amoebic antigen in stool specimens using rabbit polyclonal antiserum, and various forms of this procedure have been used, some in conjunction with microscopic studies. Palacios et al., Arch Invest Med (Mex) (1978) 9: Supplement 1:203; Randall et al., Trans Roy Soc Trop Med Hyq (1984) 78:593; Grundy, WO 95/00849 PCT/US94/06890 5 Trans Roy Soc Trop Med Hyq (1982) 76:396; Ungar, Am J Trop Med Hyq (1985) 34:465. These studies on stool specimens and on other biological fluids are summarized in Amebiasis: Human Infection by Entamoeba Histolytica, J. Ravdin, ed. (1988) Wiley Medical Publishing, pp. 646- 648.

Conversely, amebic serology is also a critical component in the diagnosis of invasive amebiasis. One approach utilizes conventional serologic tests, such as the indirect hemagglutinin test. These tests are very sensitive but seropositivity is persistent for years (Krupp, Am J Trop Med Hyg (1970) 19:57-62; Lobel, H.O. et al., Ann Rev Microbiol (1978) 32:379-347). Thus, healthy subjects may give positive responses to the assay, creating an undesirable high background. Similar problems with false positives are found in using immunoassay tests involving a monoclonal antibody and purified native 170 kD protein (Ravdin, et al., J Infect Dis (1990) 162:768-772.) Recombinant E. histolytica proteins other than the 170 kD subunit have been used as the basis for serological tests. Western blotting using a recombinant form of the "52 kD serine-rich protein" was highly specific for invasive disease and had a higher predictive value (92 vs. 65%) than an agar gel diffusion test for diagnosis of acute amebiasis (Stanley, Jr., et al., Proc Natl Acad Sci U.S.A. (1990) 87:4976-4980; Stanley, Jr., et al., JAMA (1991) 266:1984-1986). However, the overall sensitivity was lower than for the conventional agar gel test (82% vs. 90-100%).

Thus, there remains a need for serological tests which will provide optimum sensitivity while minimizing the number of false positives retained. The present invention provides such a test by utilizing, as antigen, epitope-bearing portions of the 170 kD subunit WO 95/00849 PCTIS94/06890 6 of the adherence lectin produced recombinantly in procaryotic systems.

It is particularly advantageous to use recombinantly produced, nonglycosylated peptides or proteins in this assay since these peptides are easily and efficiently obtained and are easily standardized.

Furthermore, since selected portions of the lectin heavy chain subunit can be produced, epitopes characteristic of the pathogenic or nonpathogenic forms of E. histolytica can be produced and used to distinguish these forms in the assays. Subsequent to the invention herein, a report of immunoreactivity of recombinant 170 kd lectin with immune sera was published by Zhang, Y, et al. J. Clin Micro-immunol (1992) 2788-2792. Applicants incorporate by reference their own publication: Mann, B.J et al.

Infect and Immun (1993) 61: 1772-1778.

Similarly, although it is known that the 170 kD subunit may be used as a vaccine as described in the above-referenced U.S. Patent 5,004,608, recombinantly produced forms of the 170 kD subunit, specifically those obtained from procaryotic cells that lack glycosylation may offer advantages in reproducibility of product and in ease of preparation of subunit vaccines. The present invention is directed to this desirable result.

Disclosure of the Invention The invention provides diagnostic tests which permit the assessment of patients for invasive E.

histolytica infection and vaccines for prevention of infection. The invention also provides a novel third variant of the 170 kD subunit of the Gal/GalNAc adherence lectin and a gene (hgl3) which encodes this novel protein. Accordingly, the diagnostic tests of the invention are based on the genetic sequences of all three variants of the 170 kD subunit of the Gal/GalNAc WO 95/00849 PCTIUS94/06890 7 adherence lectin which are encoded by three different genes in a multigene family.

Pathogenic and nonpathogenic strains can be distinguished by use of the invention diagnostic method, if desired. The tests use, as antigen, an epitopebearing portion of the 170 kD subunit of the Gal/GalNAc adherence lectin recombinantly produced in procaryotic systems. Despite the absence of glycosylation from such portions and despite the lack of post-translational modifications characteristic of the native protein or peptide, the recombinantly produced proteins are effective antigens in these assays.

Thus, in one aspect, the invention is directed to a method to detect the presence or absence of antibodies immunoreactive with pathogenic and/or nonpathogenic E. histolytica in a biological sample which method comprises contacting the fluid with an epitopebearing portion of the 170 kD heavy chain of the Gal/GalNAc adherence lectin wherein the lectin is nonglycosylated and in a form obtainable from procaryotic cells. If distinction between antibodies to the pathogenic and nonpathogenic forms is desired, the portion may be chosen so as to be characteristic of the pathogenic or nonpathogenic form. Alternatively, the assay may be conducted as a competition assay using MAbs with such characteristics. The contacting is conducted under conditions where the epitope-bearing portion forms complexes with any antibodies present in the biological fluid which are immunoreactive with an epitope on the portion. The presence, absence or amount of such complexes is then assessed, either directly or in a competition format, as a measure of the antibody contained in the biological sample. The invention is also directed to materials and kits suitable for performing the methods of the invention.

WO 95/00849 PCT/US94/06890 8 In a second aspect, the invention is directed to methods to prevent E. histolytica infection using vaccines containing, as active ingredient, epitopebearing portions of the 170 kD subunit produced recombinantly in procaryotic systems, as described above.

The invention is also directed to vaccines containing this active ingredient.

In other aspects, the invention is directed to epitope-bearing portions of the 170 kD subunit produced recombinantly in procaryotic systems and thus in a form characteristic of such production. One characteristic is lack of glycosylation; in addition, secondary structure of proteins produced by procaryotic hosts differs from that of proteins produced by the natural source.

In yet another aspect, the invention is directed to a DNA in purified and isolated form which consists essentially of a DNA encoding the 170 kd heavy chain subunit of pathogenic E. histolytica Gal/GalNAc adherence lectin, which subunit is encoded by the hgl3 gene for which the nucleotide sequence and deduced amino acid sequence are shown in Figure 4. In further aspects, the invention is directed to both nucleic acid and immunological reagents which are enabled by the discovery of the hgl3 gene, reagents which are specific for each of the hgll, hgl2 or hgl3 genes, as well as reagents which detect common regions of all three hgl genes or their nucleic acid or protein products. For example, oligonucleotide probes specific for any one of these three genes or for a sequence common to all three genes may be identified by one of ordinary skill in the art, using conventional nucleic acid probe design principles, by comparisons of the three DNA sequences for these genes. See Example 6.

In still further aspects, the invention is directed to a method to detect the presence, absence, or amount of a pathogenic or nonpathogenic form of Entamoeba WO 95/00849 PCT/US94/06890 9 histolytica, where E. histolytica has both pathogenic and nonpathogenic forms, in a biological sample, which method comprises contacting the sample with a monoclonal antibody immunospecific for an epitope of the 170 kd subunit of Gal/GalNAc lectin unique to the pathogenic or to the nonpathogenic form, or shared by the pathogenic and nonpathogenic forms of E. histolytica, to form an immunocomplex when the pathogenic and/or nonpathogenic form is present, and detecting the presence, absence or amount of the immunocomplex. In this method, the epitope is selected to be specific for one of 170 kD subunits encoded by the hgll, hgl2 or hgl3 genes, or for a common region of the subunits from all three hgl genes.

In another aspect, the invention is directed to a method to determine the presence, absence or amount of antibodies specifically immunoreactive with the Gal/GalNAc lectin derived from E. histolytica, which method comprises contacting a biological sample with the Gal/GalNAc lectin or the 170 kd subunit thereof in purified and isolated form, under conditions wherein antibodies immunospecific for said lectin or subunit will forma complex, and detecting the presence, absence or amount of the complex, wherein the purified and isolated Gal/GalNAc lectin or subunit is derived from either a pathogenic or nonpathogenic form of E. histolytica, and is a 170 kD subunit encoded by one of the hgll, hgl2 or hgl3 genes. Detailed descriptions of these and related methods for detecting pathogenic or nonpathogenic forms of E. histolytica and antibodies specifically immunoreactive with the Gal/GalNAc lectin derived from E.

histolytica, as well as reagent kits suitable for the conduct of such methods, are disclosed in U.S. Patent 5,272,058, the entire disclosure of which is incorporated herein by reference.

WO 95/00849 PCT/US94/06890 10 Brief Description of the Drawings Figure 1A shows the DNA and amino acid sequence deduced from the nucleotide sequence corresponding to the 170 kD heavy chain of the adherence lectin from pathogenic strain HM1:IMSS, designated hgll.

Figure 1B shows the deduced amino acid sequence of hgll with the amino-terminal amino acid of the mature protein designated as amino acid number 1.

Figure 2A is a diagram of the construction of expression vectors for recombinant production of specified portions of the 170 kD subunit; Figure 2B shows the pattern of deletion mutants.

Figure 3 is a diagram of the location of human B cell epitopes and pathogenic-specific epitopes on the 170 kD heavy chain.

Figure 4A shows the DNA and amino acid sequence deduced from the nucleotide sequence corresponding to the 170 kD heavy chain of the adherence lectin from pathogenic strain HM1:IMSS, designated hgl3.

Figure 4B shows the deduced amino acid sequence of hgl3 with the amino-terminal amino acid of the mature protein designated as amino acid number 1. The putative signal sequence and transmembrane domains are overlined and underlined respectively. Conserved cysteine residues and potential sites of glycosylation are indicated.

Figure 5 shows in schematic form a comparison of amino acid sequences of three heavy subunit genes.

The top diagram represents a schematic representation of a heavy subunit gene. Starting at the amino terminus, regions include the cysteine/tryptophan rich domain, the cysteine-free (C-free) domain, the cysteinerich (C-rich) domain, and the putative transmembrane (TM) sequence and cytosolic domains (Mann, B.J. et al. Parasit Today (1991) 2:173-176). Amino acid sequence comparisons of hgll, hgl2 and hgl3 are shown. Upright lines indicate WO 95/00849 PCTIS94/06890 11 nonconservative amino acid substitutions in the amino acid sequence of the second gene as compared to the first gene listed to the right. Downward arrowheads indicate a deletion while upright arrowheads indicate an insertion.

The number of residues inserted or deleted are listed below the arrowheads and the total percent amino acid sequence identity is listed at right.

Modes of Carrving Out the Invention The invention provides methods and materials which are useful in assays to detect antibodies directed to pathogenic and/or nonpathogenic forms of E.

histolytica and in vaccines. The diagnostic assays can be conducted on biological samples derived from subjects at risk for infection or suspected of being infected.

The assays can be designed to distinguish pathogenic from nonpathogenic forms of the amoeba if desired. The vaccines are administered to subjects at risk for amebic infections.

The assays of the invention rely on the ability of an epitope-bearing portion of the 170 kD subunit produced recombinantly in procaryotic cultures to immunoreact with antibodies contained in biological samples obtained from individuals who have been infected with E. histolytica. Even though the relevant peptide or protein is produced in a procaryotic system, and is thus not glycosylated or processed after translation in a manner corresponding to the native protein, the epitopebearing portions thus prepared are useful antigens in immunoassays performed on samples prepared from biological fluids, cells, tissues or organs, or their diluted or fractionated forms. Similarly, these peptides are also immunogenic.

The use of recombinant forms of the antigen or offers advantages of cost-effective, reliable production of pure antigen, thus assuring the uniformity of the WO 95/00849 PCT/US94/06890 12 assay materials. Recombinant production in bacteria is a particularly efficient and useful method. It is surprising that such procaryotic systems can produce successful antigens and immunogens, since the peptides produced are not processed in a manner analogous to the reactive native forms.

Furthermore, recombinant production facilitates the preparation of specific epitopes, thus providing a means for detecting antibodies specifically immunoreactive with pathogenic or nonpathogenic forms of the amoeba, as well as offering the opportunity to provide subunit vaccines.

Thus, the invention is directed to methods to detect antibodies in biological samples and to immunize subjects at risk using these recombinantly produced epitope-bearing portions as antigens or immunogens as well as to the recombinantly produced peptides themselves and to materials useful in performing the assays and in administering the vaccines.

Definitions The diagnostic assays may be designed to distinguish antibodies raised against nonpathogenic or pathogenic forms of the amoeba. "Pathogenic forms" of E.

histolytica refers to those forms which are invasive and which result in symptomology to infected subjects.

"Nonpathogenic forms" refers to those forms which may be harbored asymptomatically by carriers.

The assays and vaccines of the invention utilize an epitope-bearing portion of the 170 kD subunit of the Gal/GalNAc lectin. "Gal/GalNAc lectin" refers to 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 Nacetylgalactosamine. The Gal/GalNAc lectin refers specifically to the lectin reported and isolated by WO 95/00849 PCTIUS94/06890 13 Petri, et al. (supra) from the pathogenic strain HMI- IMSS, and to the corresponding lectin found in other strains of E. histolytica. The "170 kD subunit" refers to the large subunit, upon reduction of the Gal/GalNAc lectin, such as that obtained by Petri, et al. and shown in Figure 1 as well as to its corresponding counterparts in other strains.

Diagnostic Assays With respect to the diagnostic assays of the invention, the complete 170 kD antigen or an epitopebearing portion thereof can be used in the assays. Such epitope-bearing portions can be selected as characteristic of pathogens or nonpathogens or common to both.

As shown hereinbelow, the portion of the 170 kD protein which contains epitopes for all monoclonal antibodies prepared against the lectin is found at amino acid positions 596-1138. There appears to be an epitope characteristic of pathogens between each of amino acid positions 596-818, 1082-1138, and 1033-1082. Positions 895-998 contain epitopes which are shared by pathogens and nonpathogens as well as epitopes characteristic of pathogenic strains. Thus, to utilize fragments of the recombinantly produced protein for detection of antibodies, a peptide representing positions 596-818, 1033-1082 or 1082-1138 may be used to detect antibodies raised against pathogens by hosts in general; however, the epitope at positions 596-816 is not recognized by human antisera. Mixtures of these peptides could also be used. Alternatively, longer forms of the antigen can be used by selecting the appropriate positions depending on whether pathogenic and nonpathogenic amoebae are to be distinguished.

As shown in Example 4, below, epitope-bearing portions relevant for human testing include portions 2- WO 95/00849 PCT/US94/06890 14 482, 1082-1138, 1032-1082 and 894-998. Only the portions represented by 1082-1138 and 1032-1082 appears specific for antibodies against pathogenic ameba. These epitopebearing portions may be used as single peptides, as uniquely lectin-derived portions of chimeric proteins, as mixtures of peptides or of such proteins, or as portions of a single, multiple-epitope-bearing protein.

Procedures for preparing recombinant peptide proteins containing only a single epitope-bearing portion identified above, or multiples of such portions (including tandem repeats) are well understood in the art.

The assays are designed to detect antibodies in biological samples which are "immunospecific" or "immunoreactive" with respect to the epitope-bearing portion i.e. with respect to at least one epitope contained in this portion. As used herein, "immunospecific" or "immunoreactive" 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 haptens. 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 or magnitude greater than with which it binds alternate haptens.

The assays can be performed in a wide variety of protocols depending on the nature of the sample, the circumstances of performing the assays, and the particular design chosen by the clinician. The biological sample is prepared in a manner standard for the conduct of immunoassays; such preparation may involve dilution if the sample is a biological fluid, fractionation if the sample is derived from a tissue or organ, or other standard preparation procedures which are WO 95/00849 PCTIUS94/06890 15 known in the art. Thus, "biological sample" refers to the sample actually used in the assay which is derived from a fluid, cell, tissue or organ of a subject and prepared for use in the assay using the standard techniques. Normally, plasma or serum is the source of biological sample in these assays.

The assays may be conducted in a competition format employing a specific binding partner for the epitope-bearing portion. As used herein, "specific binding partner" refers to a substance which is capable of specific binding to a targeted substance, such as the epitope-bearing portion of the 170 kD subunit. In general, such a specific binding partner will be an antibody, but any alterative substance capable of such specific binding, such as a receptor, enzyme or arbitrarily designed chemical compound might also be used. In such contexts, "antibody" refers not only to immunoglobulin per se, but also to fragments of immunoglobulin 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') 2 fragments. The term "antibody" also includes not only native forms of immunoglobulin, but forms of the immunoglobulin 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.

A particularly useful form of specific binding reagents useful in the assay methods of the invention is as monoclonal antibodies. Three categories of monoclonal antibodies have been prepared to the 170 kD subunit. One category of antibody is immunospecific for epitopes WO 95/00849 PCT/US94/06890 16 "unique" to pathogenic forms. These antibodies are capable, therefore, of immunoreaction to a significant extent only with the pathogenic forms of the amoeba or to the 170 kD subunit of lectin isolated from pathogenic forms. A second set of monoclonal antibodies is immunoreactive with epitopes which are "unique" to nonpathogenic forms. Thus, these antibodies are 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 immunoreactive with epitopes common to pathogenic and nonpathogenic forms and these antibodies are capable of immunoreaction with the subunit or with the amoeba regardless of pathogenicity.

With respect to the monoclonal antibodies described herein, those immunoreactive with epitopes 1 and 2 of the 170 kD subunit isolated from the pathogenicstrain exemplified are capable of reacting, also, with the corresponding epitopes on nonpathogens. On the other hand, those immunoreactive with epitopes 3-6 are capable of immunoreaction only with the 170 kD subunit of pathogenic strains. By applying the techniques for isolation of the pathogenic 170 kD subunit to amoeba which are nonpathogenic, a 170 kD subunit can be obtained for immunization protocols which permit the analogous preparation of MAbs immunoreactive with counterpart epitopes 3-6 in the nonpathogenic forms.

Of course, with respect to antibodies found in the biological sample, in general, these will be found in the form of immunoglobulins. However, pretreatment of the sample with an enzyme, for example, to remove the F c portions of the antibodies contained therein, does not debilitate the sample with respect to its ability to respond to the assay.

WO 95/00849 PCTIUS94/06890 17 Assay Procedure For the conduct of the assays of the invention, in general, the biological sample is contacted with the epitope-bearing portion used as an antigen in the immunoassay. The presence, absence or amount of the resulting complex formed between any antibody present in the sample and the epitope-bearing portion is measured directly or competitively.

As is well understood in the art, once the biological sample is prepared, there is a multiplicity of alternative protocols for conduct of the actual assay.

In one rather straightforward protocol, the epitopebearing portion provided as antigen may be coupled to a solid support, either by adsorption or by covalent linkage, and treated with the biological sample. The ability of any antibodies in the sample to bind to coupled antigen is then determined.

This ability may be determined in a "direct" form of the assay in which the level of complex formation by the antibody is measured directly. In one particularly convenient format of this approach, the antigen may be supplied as a band on a polyvinylidene difluoride (PVDF) and contacted with the biological sample; any resulting complexes formed with antibody on the PVDF membrane are then detected as described above for Western blot procedure. This protocol is substantially a Western Blot procedure. Alternatively, microtiter plates or other suitable solid supports may be used. The binding of antibody to the antigen coupled to support can then be detected as described above for Western blot procedure using conventional techniques generally involving secondary labeling using, for example, antibodies to the species from which the biological sample is derived. Such labels may include radioisotopes, fluorescent tags, enzyme labels and the like, as is conventionally understood.

WO 95/00849 PCT/US94/06890 18 The assay may also be formatted as a competition assay wherein the antigen coupled to solid support is treated not only with the biological sample but also with competing specific binding partner immunospecific for at least one epitope contained in the antigen. The competing binding partner is preferably an antibody. The competing antibody may be polyclonal or monoclonal and may itself be labeled or may be capable of being labeled in a secondary reaction. In a typical conduct of such a competitive test, a competitive specific binding partner for the antigen is generally supplied in labeled form and the success of the competition from the biological sample is measured as a reduction in the amount of label bound in the resulting complex or increased levels of label remaining in the supernatant. If monoclonal antibodies are used, the assay can readily be made specific for pathogenic or nonpathogenic reacting antibodies, if desired, by choosing antibodies of the appropriate specificity.

Thus, if the assay is to be made specific for antibodies raised against pathogenic forms of E. histolytica, the competition will be provided by a monoclonal antibody specific for an epitope characteristic of pathogenic strains.

Another manner in which the assay may be made specific for pathogenic or nonpathogenic forms is in the choice of the epitope-bearing portion. If antibodies specific to the pathogens are to be detected, an epitopebearing portion is chosen which bears only epitopes characteristic of pathogenic strains. Conversely, antibodies immunospecific for nonpathogens can be conducted by utilizing as antigen only portions of the subunit which contain epitopes characteristic of nonpathogens. Where characterization as pathogen or nonpathogen-specific antibodies is unnecessary, antigen WO 95/00849 PCT/US94/06890 19 containing both such epitopes or epitopes shared by both forms may be used.

Additional ways to distinguish between antibodies immunospecific for pathogens and for nonpathogens employ competition assays with monoclonal antibodies of such specificities, as described above.

Alternatively, the biological sample can be coupled to solid support and the desired epitope-bearing portion added under conditions where a complex can be formed to the epitope-bearing portion, which is then used to treat the support. Subsequent treatment of the support with antibodies known to immunoreact with the antigen can then be used to detect whether antigen has been bound.

Thus, the biological sample to be tested is contacted with the epitope-bearing portion, which is derived either from a pathogenic or nonpathogenic from one both of E. histolytica so that a complex is formed.

The complex is then detected by suitable labeling, either by supplying the antigen in labeled from, or by a secondary labeling process which forms a ternary complex.

The reaction is preferably conducted using a solid phase to detect the formation of the complex attached to solid support, or the complex can be precipitated using conventional precipitating agents such as polyethylene glycol.

In a more complex form of the assay, competitive assays, can be used wherein the biological sample, preferably serum or plasma, provides the cold antibody to compete with a specific binding partner, such as a labeled monoclonal antibody preparation known to bind specifically to an epitope unique to the Gal/GalNAc lectin or its 170 kD subunit of a pathogenic or nonpathogenic from. In this embodiment, the binding to labeled specific monoclonal antibody is conducted in the presence and absence of biological sample, and the WO 95/00849 PCT/US94/06890 20 diminution of labeling of the resulting complex in the presence of sample is used as an index to determine the level of competing antibody.

Kits suitable for the conduct of these methods include the appropriate labeled antigen or antibody reagents and instructions for conducting the test. The kit may include the antigen coupled to solid support as well as additional reagents.

Methods of Protection and Vaccines The recombinant 170 kD subunit or an epitopebearing portion thereof may be used as active ingredient.

Preferred regions include positions 482-1138, 596-1138, 885-998, 1033-1082 and 1082-1138.

The 170 kD subunit or its epitope-bearing regions may also be produced recombinantly in procaryotic cells for the formulation of vaccines. The recombinantly produced 170 kD protein or an epitope-bearing region thereof can be used as an active ingredient in vaccines for prevention of E. histolytica infection in subjects who are risk for such condition. Sufficiently large portions of the 170 kD protein can be used per se; if only small regions of the molecules for example containing 20 amino acids or less or to be used, it may advantageous to couple the peptide to a neutral carrier to enhance its immunogenicity. Such coupling techniques are well known in the art, and include standard chemical coupling techniques optionally effected through linker moieties such as those available from Pierce Chemical Company, Rockford, Illinois. Suitable carriers may include, for example, keyhole limpid hemocyanin (KLH) E.

coli pilin protein k99, BSA, or the VP6 protein of rotavirus. Another approach employs production of fusion proteins which include the epitope-bearing regions fused to additional amino acid sequence. In addition, because of the ease with which recombinant materials can be WO 95/00849 PCT/US94/06890 21 manipulated, the epitope-bearing region may be included in multiple copies in a single molecule, or several epitope-bearing regions can be "mixed and matched" in a single molecule.

The active ingredient, or mixture of active ingredients, in the vaccine is formulated using standard formulation for administration of proteins or peptides and the compositions may include an immunostimulant or adjuvant such as complete Freund's adjuvant, aluminum hydroxide, liposomes, ISCOMs, and the like. General methods to prepare vaccines are described in Remingtons's Pharmaceutical Science; Mack Publishing Company Easton, PA (latest edition). The compositions contain an effective amount of the active ingredient peptide or peptides together with a suitable amount of carrier vehicle, including, if desired, preservatives, buffers, and the like. Other descriptions of vaccine formulations are found in "New Trends and Developments in Vaccines", Voller, et al., University Park Press, Baltimore, Maryland (1978).

The vaccines are administered as is generally understood in the art. Ordinarily, administration is systemic through injection; however, other effective means of administration are included. With suitable formulation, for example, peptide vaccines may be administered across the mucus membrane using penetrants such as bile salts or fusidic acids in combination, usually, with a surfactant. Transcutaneous means for administering peptides are also known. Oral formulations can also be used. Dosage levels depend on the mode of administration, the nature of the subject, and the nature of carrier/adjuvant formulation. Typical amounts of protein are in the range of .01 Ag- mg/kg. However, this is an arbitrary range which is highly dependent on the factors cited above. In general, multiple WO 95/00849 PCTIUS94/06890 22 administrations in standard immunization protocols are preferred; such protocols are standard in the art.

A preferred epitope-bearing region of the 170 kD subunit is that represented by amino acids 482-1138 which includes the cysteine-rich domain. This region is encoded by nucleotides 1492-3460 shown in Figure 1 herein. Preferred regions include those bearing epitopes which are specific for antibodies against pathogenic amoeba regions 1082-1138 and 1032-1082.

However, the epitope-bearing region at positions 894-998 may also be used. For regions of this length, production of peptides with multiple copies of the epitope-bearing regions is particularly advantageous.

Production of Recombinant Epitope-bearing Portions The epitope-bearing portions of the 170 kD subunit can be conveniently prepared in a variety of procaryotic systems using control sequences and hosts ordinarily available in the art. The portions may be provided as fusion proteins or as mature proteins and may be produced intracellularly or secreted. Techniques for constructing expression systems to effect all of these outcomes is well understood in the art. If the epitopebearing portion is secreted, the medium can be used directly in the assay to provide the antigen, or the antigen can be recovered from the medium and further purified if desired. If the protein is produced intracellularly, lysates of cultured cells may be used directly or the protein may be recovered and further purified. In the Examples below, the epitope-bearing portion is provided as a fusion protein using the commercially available expression vector pGEX.

Alternative constructions and alternative hosts can also be used as is understood in the art.

Reagents and assays for a novel 170 kD lectin subunit WO 95/00849 PCT/US94/06890 23 To determine the existence and complexity of the 170 kDa subunit gene family, hgl, an amebic genomic library in lambda phage was hybridized with DNA fragments from the 5' or 3' ends of hgll. Termini from three distinct heavy subunit genes were identified including hgll, hgl2, and a third, unreported gene designated hgl3.

The open reading frame of hgl3 was sequenced in its entirety (Figure 4A). Nonstringent hybridization of a genomic Southern blot with heavy subunit specific DNA labeled only those bands predicted by hgll-3. The amino acid sequence of hgl3 (Figure 4B) was 95.2% identical to hgll and 89.4% identical to hgl2. All 97 cysteine residues present in the heavy subunit were conserved in hgll-3. Analysis of amebic RNA showed that all three heavy subunit genes were expressed in the amebae and that hgl message became less abundant as the amebae entered a stationary growth phase.

Accordingly, the present invention provides both nucleic acid and immunological reagents specific for 170 kDa subunits encoded by each of the hgll, hgl2 or hgl3 genes, as well as reagents which detect common regions of all three hgl genes and their nucleic acid or protein products. For example, oligonucleotide probes specific for any one of these three genes may be identified by one of ordinary skill in the art, using conventional nucleic acid probe design principles, by comparisons of the three DNA sequences for these genes, which sequences are disclosed in Figure 1A and Figure 4A for hgll and hgl3, respectively, and for hgl2, in Tannich, E. et al. Proc Natl Acad Sci USA (1991) 88:1849- 1853, the entire disclosure of which is hereby incorporated herein by reference. Example 6 illustrates the use of oligonucleotide probes specific for each of the three hgl genes, for determining the level of expression of RNA from each gene using Northern blot analyses. Other methods of using hgl-specific nucleic WO 95/00849 PCTIS94/06890 24 acids for diagnostic purposes, for pathogenic and/or nonpathogenic forms of E. histolytica, are described in U.S. Patent 5,260,429, the entire disclosure of which is incorporated herein by reference.

The following Examples are intended to illustrate but not to limit the invention.

Example 1 Construction of Expression Vectors The 170 kD subunit of the galactose lectin is encoded by at least two genes. The DNA used for all of the constructions described herein encodes the 170 kD lectin designated hgll (Fig. 1A). The nucleotide position designations refer to the numbering in Figure 1A.

The DNA sequence encoding hgll was expressed in three portions: fragment C (nucleotides 46-1833) included the cysteine- and tryptophan-rich region, the cysteine-free region, and 277 amino acids of the cysteine-rich domain, i.e. amino acid residues 2-596; fragment A (nucleotides 1492-3460) encoded the majority of the cysteine-rich domain, i.e. amino acid residues 482-1138; fragment B (3461-3892) included 70 amino acids of the cysteine-rich domain, the putative membranespanning region, and the cytoplasmic tail, i.e. amino acid residues 1139-1276.

See Fig. 2B.

Each of these three fragments was inserted in frame by ligation into pGEX2T or pGEX3X to obtain these proteins as GST fusions. A diagram of the vectors constructed is shown in Figure 2A.

Fragment C was produced by PCR amplification.

Primers were designed so that a BamHI site was added to the 5' end and an EcoRI site was added to the 3' end WO 95/00849 PCT/US94/06890 25 during the PCR process. The PCR product, fragment C, was then digested with restriction enzymes BamHI and EcoRI, purified, and ligated into similarly digested pGEX3X.

Fragments A and B were produced by digestion with EcoRI from plasmid clones (Mann, BJ et al. Proc Natl Acad Sci USA (1991) 88:3248-3252) and ligated into pGEX2T that had been digested with EcoRI. In the pGEX expression system a recombinant protein is expressed as a fusion protein with glutathione S-transferase (GST) from Schistosoma japonicum and is under the control of the tac promoter.

The tac promoter is inducible by IPTG. The construction of the vectors and subsequent expression is further described in Mann, BJ et al. Infec and Immun (1993) 61:1772-1778, referenced above, and incorporated herein by reference.

Expression in the correct reading frame was verified for all constructs by sequencing and Western immunoblot analysis by testing for reactivity with antiadhesion antisera (data not shown). Expression of the hgll fusion proteins was shown to be inducible by IPTG.

The GST protein produced from the original pGEX2T did not react with the anti-adhesion sera. The GST portion of the fusion protein has a molecular mass of 27.5 kD.

Example 2 Production of Recombinant Protein The four vectors described above, as well as the host vector were transfected into competent E. coli hosts and expression of the genes encoding the fusion proteins was effected by induction with IPTG. Production of the fusion proteins was determined by Western blot SDS-PAGE analysis of the lysates.

Example 3 Reactivity of Recombinant 170 kD Subunit Fusion Proteins with MAbs WO 95/00849 PCT/US94/06890 26 Induced cultures containing bacterial strains expressing hgll fragment A, B, or C were harvested, lysed in sample buffer, and applied to an SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to Immobilon and incubated with anti-170-kD MAbs, specific for seven different epitopes.

Characteristics of the individual MAbs are shown in Table 1. It will be noted that all the known epitopes are in the region of amino acids 596-1138.

TABLE 1. Characteristics of monoclonal antibodies directed against the galactose adhesion 170 kD subunit Epitope Designation Isotypel Adherencel Cytotoxicity 2 C5b9 Resistance 3 p4 NP4 Location 1 3F4 IgG 1 Increases Decreases No effect 895-998 2 8A3 IgG 1 Increases No effect Decreases 895-998 3 7F4 IgG2b No effect No effect Decreases 1082-1138 4 8C12 IgG 1 Inhibits Inhibits Decreases 895-998 5 1G7 IgG2b Inhibits Inhibits Decreases 596-818 6 6 H85 IgG2b Inhibits Inhibits Blocks 1033-1082 7 3D12 IgG 1 No effect Not tested Blocks 895-998 1 Adherence was assayed by thelbgding of Chinese hamster ovary (CHO) cells to E. histolytica trophozoites and by binding of I labeled purified colonic mucins to trophozoites. Petri, W.A.

Jr., et al., J Immunol (1990) 144:4803-4809.

2 The assay for cytotoxicity was CHO cell killing by E. histolytica trophozoites as measured by Cr release from labeled CHO cells. Saffer, et al. Infect Immun (1991) 59:4681-4683.

3 0 3C5b9 resistance was assayed by the addition of purified complement components to E. histolytica trophozoites. The percent of amebic lysis was determined microscopically. Braga, et al. J Clin Invest (1992) 90:1131-1137.

4p and NP refer to reactivity of the MAb with pathogenic and nonpathogenic (NP) species of E. histolytica as determined in an Elisa assay. Petri, W.A. Jr., et al. Infect Immun (1991) 58:1802-1806.

Location of antibody binding site by amino acid number. Results presented herein.

6Inhibits adherence to CHO cells but not human colonic mucin glycoproteins. Petri, W.A. Jr., et al., J Immunol (1990) 144:4803-4809.

WO 95/00849 PCT/US94/06890 27 Fusion proteins B and C failed to react with any of the seven MAbs (data not shown). Fusion protein A, representing positions 482-1132, reacted with all seven MAbs representing all 7 epitopes and not a negative control developed with an irrelevant MAb, MOPC21. The MAbs were used at 10 pg/ml and polyclonal antibodies at 1:1000 dilution. These results'indicated that these seven epitopes were contained within the 542 amino acids of the cysteine-rich extracellular domain of the 170 kD subunit.

The generation of 3' deletions by controlled ExoIII digestion of fragment A of the 170 kD subunit is outlined in Figure 2B. Al contains amino acid residues 482-1082; A2 contains amino acid residues 482-1032; A3 contains amino acid residues 482-998. The reactivities of the fusion proteins that include fragment A or either of two carboxy-terminal deletions (A3 and A4) with the seven distinct 170 kD-specific MAbs were determined.

Deletion 3 reacted with MAb against epitopes 1-2, and 7 but failed to react with MAbs recognizing epitopes 3 and 6; Deletion 4 which contains residues 498-894 reacted only with the MAb which recognizes epitope The five deletion derivatives of fusion protein A shown in Figure 2B, ranging in estimated size from to 68 kD, were tested for reactivity to each MAb, and the reactivities of the deletions with each MAb are summarized in Fig. 3. The endpoints of the various deletions were determined by DNA sequencing with primers specific for the remaining hgll sequence. MAbs recognizing epitopes 1 and 2, which increase amebic adherence to target cells, failed to react with recombinant lectin fusion proteins lacking amino acids 895 to 998. Similarly, MAbs recognizing epitope 4, an inhibitory epitope, and epitope 7, which has the effect of abrogating amebic lysis by complement, failed to react WO 95/00849 PCT/US94/06890 28 with deletion mutants lacking this region. The MAb specific for epitope 6, which has inhibitory effects on amebic adherence and abrogates amebic lysis by complement, did not react with a recombinant protein missing amino acids 1033 to 1082. Recombinant proteins lacking amino acids 1082 to 1138 did not react with a MAb which is specific for the neutral epitope 3. Finally, a construct containing amino acids 482 to 818 was recognized only by the adherence-inhibitory epitope MAb. The thus predicted locations of the MAb epitopes are listed in Table 1 above.

Example 4 Reactivity of 170 kD Fusion Proteins with Human Immune Sera Since the galactose adhesion is a major target of the humoral immune response in the majority of immune individuals, the mapping of human B-cell epitopes of the 170 kD subunit was undertaken. The recombinant fusion proteins and ExoIII-generated deletion constructs of the 170 kD subunit were tested for reactivity with pooled human immune sera in the same manner as described for MAb reactivity. Nonimmune sera was used as a control.

Fusion proteins A and C reacted with immune sera, whereas fusion protein B did not (data not shown). Human immune sera also reacted with deletion constructs Al, A2, and A3 but not with A4 or AI0. Reactivity of immune sera with the different deletions localized major human B-cell epitopes to be within the first 482 amino acids and between amino acids 895 and 1138 (Fig. This second region is the same area which contains six of the MAb epitopes. These results are consistent with a report by Zhang et al. supra, who found that sera from immune individuals reacted primarily with recombinant adhesion constructs containing amino acids 1 to 373 and 649 to 1202.

WO 95/00849 PCT/US94/06890 29 Thus, for use in assays to detect human antisera against E. histolytica, the useful epitopebearing portions are as shown in Table 2.

Table 2 Positions Epitope P/NP 2-482 1082-1138 3 P 1033-1082 6 P 895-998 1,2,4,7 both The epitope-bearing portions indicated can be used alone, as fragments or as portions of chimeric or fusion proteins, or any combination of these epitopebearing portions can be used.

Example Immunization Using Recombinant Subunit Protein A GST fusion protein with fragment A was prepared in E. coli as described in Example 1 above.

This peptide contains an upstream GST derived peptide sequence followed by and fused to amino acids 432-1138 encoded by nucleotides 1492-3460 in Figure 1 herein. The protein is produced intracellularly; the cells were harvested and lysed and the lysates subjected to standard purification techniques to obtain the purified fusion protein.

Gerbils were immunized by intraperitoneal injection with 30 Ag of purified fusion protein in complete Freund's adjuvant and then boosted at 2-4 weeks with 30 Ag of the fusion protein in incomplete Freund's adjuvant.

The gerbils were challenged at 6 weeks by intrahepatic injection of 5x10 5 amebic trophozoites and sacrificed 8 weeks later. The presence and size of amoebic liver abscesses was determined.

WO 95/00849 PCTIUS94/06890 30 The results of the two experiments described above are shown in the tables below. The administration of the fusion protein reduced the size of abscesses in a statistically significant manner.

In experiment 1, six animals were used as controls and nine were administered the fusion protein; in experiment 2, seven animals were used as controls and seven were provided the fusion protein.

Experiment 1 Experiment 2 Abscess with Abscess with Weight Abscess Weight Abscess Control 1.44±1.64 71% 4.76±1.78 100% GST (482-1138) 0.81±0.10' 100% 2.35±1.99 100% P<0.03 compared to control.

P<0.24 compared to control.

Example 6 Analysis of the Gene Family Encoding the 170 kD Subunit of E. histolytica Gal/GalNAc Adherence Lectin This Example shows that the adhesin 170 kDa subunit of HM-1:IMSS strain E. histolytica is encoded by a gene family that includes hgll, hgl2 and a previously undescribed third gene herein designated hgl3. Since hgll and hgl2 were originally sequenced, in part, from different cDNA libraries, it was possible that they represented strain differences of a single gene.

However, in this report both 5' and 3' termini of hgll, hgl2, and hgl3 were isolated and sequenced from the same lambda genomic library demonstrating unambiguously that hgl is a gene family.

Comparison of the amino acid sequences of the three heavy subunit genes showed that hgll and hgl2 are 89.2% identical, hgll and hgl3 are 95.2% identical, and hg12 and hgl3 are 89.4% identical. Sequence variation within the gene family, however, appears to be nonrandomly distributed within the coding sequence. The WO 95/00849 PCT/US94/06890 31 majority of the nonconservative amino acid substitutions as well as insertions and deletions occur in the amino third of the molecule. Comparison of the amino acid sequences of hgl2 and hgl3 reveal that 11 of the 19 nonconservative amino acid substitutions and 11 of the 13 residues inserted or deleted reside within the first 400 amino acid residues. A similar pattern of variation is present when hgll and hgl2 are compared. While hgll and hgl3 contain only two nonconservative substitutions, both are found within the first 400 residues although the 57 conservative substitutions appear to be more randomly distributed throughout the coding sequence. The high degree of sequence conservation between hgl3 and hgll suggest that they may have arisen from a recent gene duplication event.

All 97 cysteine residues were maintained in the three heavy subunit genes. The hgl2 gene was originally reported lacking a single cysteine present in both hgll and hgl3. However, this discrepancy has since been recognized as a sequencing error (Dr. E. Tannich, Bernhard Nocht Institute, Hamburg, Germany). The cysteine residues are nonrandomly distributed throughout the gene (Figure 4) with the highest concentration within the cysteine-rich domain between amino acid residues 379- 1210. All seven identified epitopes recognized by murine monoclonal antibodies map to this region (Mann, B.J. et al. Infect Immun (1993) 61:1772-1778). As these monoclonal antibodies can block target cell adhesion, target cell lysis (Saffer, L.D. et al. Infect Immun (1991) 59:4681-4683), and/or resistance to host complement-mediated lysis (Braga, L.L. et al. J Clin Invest (1992) 90:1131-1137), the conservation of cysteine residues may play an important role in maintaining the conformation of this important region of hgl.

A minimum of three genes are shown to make up the heavy subunit gene family. While it is not possible WO 95/00849 PCT/US94/06890 32 to rule out the existence of additional hgl genes, the Southern blot and library screen data can be explained by a gene family of three members. As the genomic library was screened separately with a 5' and a 3' hgl specific probe, additional heavy subunit genes would be isolated even if they contained only partial identity with the gene family at only one end or even if one termini of an additional gene had been lost during library amplification. The library screen looked at more than 3.2x10 8 bases of genomic DNA in an organism with an estimated genome size of i07 5 bases (Gelderman, A.H. et al. J Parasitol (1971) 57:906-911). Thus, a full genomic equivalent was screened at low stringency for genes containing identity at either end.

The Northern data indicated that all three genes were expressed in the amebae. As the messages of hgll-3 are predicted to comigrate at 4.0 kb, differential hybridization was required to ascertain expression of individual genes. Due to the high degree of identity between hgll-3, relatively short oligonucleotides (17-21 bases) were synthesized specific for regions where the three genes diverge. Each probe was compared by computer analysis to the other hgl genes to be certain that they were sufficiently divergent to prevent cross hybridization. Hybridization and wash conditions were highly stringent for such A/T rich probes and were done at temperatures 5C or less below the predicted Tm based upon nearest neighbor analysis. While it is impossible to rule out cross hybridization with other hgl gene members, these precautions make such an event less likely.

The Northern blot also indicates that abundance of mRNA for all three genes decreased as the amebae progressed from log to stationary growth. This finding correlates with data which indicate that late log and stationary phase amebae have a decreased ability to WO 95/00849 PCT/US94/06890 33 adhere to, lyse, and phagocytose target cells (Orozco, E.

et al. (1988) "The role of phagocytosis in the pathogenic mechanism of Entamoeba histolytica. In: Amebiasis: Human infection by Entamoeba histolytica (Ravdin J.I., ed), pp. 326-338. John Wiley Sons, Inc., New York.

Details of the experimental methods and results of the characterization of the hgl multigene family are presented below.

Library Screen. A lambda Zap® II library containing randomly sheared 4-5 kb fragments of genomic DNA from HM-1:IMSS strain E. histolytica was kindly provided by Dr. J. Samuelson at Harvard University (Kumar, A. et al.

Proc Natl Acad Sci USA (1992) 89:10188-10192). Over 80,000 plaques from the library were screened on a lawn of XL-1 Blue E. coli (Strategene, La Jolla, CA).

Duplicate plaque lifts, using Hybond-N membranes (Amersham, Arlington Heights, IL), were placed in a prehybridization solution consisting of 6x SSC (.89 M sodium chloride and 90 mM sodium citrate), 5x Denhardts solution, SDS, 50 mM NaPO 4 (pH and 100 Jg/ml salmon sperm DNA for a minimum of 4 hours at 55 0 C. A and 3' DNA fragment of hgll (nucleotides 106-1946 and 3522-3940 respectively) were [a- 32 p]dCTP (Amersham) labeled using the Random Primed DNA labeling Kit according to the manufacturer's instructions (Boehringer Mannheim, Mannheim, Germany) and hybridized separately to the membranes overnight at 55 0 C in prehybridization solution. Membranes were rinsed once and washed once for minutes at room temperature in 2x SSC, SDS, then washed once for 15 minutes at room temperature, and twice at 550C for 20 minutes in .Ix SSC, SDS. Plaques that hybridized with the 5' or the 3 radiolabeled probe on both duplicate filters were isolated and purified.

Northern blot and hybridization. Total RNA was harvested from amebae using the guanidinium isothiocyanate method (RNagen, Promega, Madison, WI).

WO 95/00849 PCT/US94/06890 34 Polyadenylated RNA was purified from total RNA using PolyATract System 1000 (Promega). RNA was electrophoresed through a formaldehyde gel and transferred to a nylon Zetabind membrane (Cuno) using mM phosphate buffer (pH 7.5) as described (Sambrook, J.

et al. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The membrane was incubated in prehybridization solution and incubated at 37 0 C for at least two hours. Oligonucleotides (18-22 nucleotides long) were end-labeled using polynucleotide kinase and [y-P 2 ]ATP (Sambrook, J. et al. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), added to the hybridization mixture and the membrane, and incubated at 37 0

C

overnight. The membrane was then washed once at room temperature for 10 minutes, once at 37 0 C for 10 minutes; and twice at 40-44 0 C for 15 minutes each in 2x SSC, .1% SDS. The radiolabeled probes used were: 5'-TTTGTCACTATTTTCTAC-3', hgll; 5'-TATCTCCATTTGGTTGA-3', hgl2; 5'-TTTGTCACTATTTTCTAC-3', hgl3; and 5'-CCCAAGCATATTTGAATG-3', EF-la (Plaimauer, B. et al. DNA Cell Biol (1993) 12:89-96).

Characterization of the hgl3 gene. The hgl3 open reading frame was 3876 bases and would result in a predicted translation product of 1292 amino acids (Figure The predicted translation products of hgll and hgl2 would be 1291 and 1285 amino acids respectively. A putative signal sequence and a transmembrane domain were identified in the amino acid sequence of hgl3 similar to hgll and hgl2. The amino-terminal amino acid sequence of the mature hgl3 protein, determined by Edman degradation (Mann, B.J. et al. Proc Natl Acad Sci USA (1991) 88:3248- 3252), was assigned residue number 1. Previous analysis of hgll and hgl2 identified a large, conserved, extracellular region which was 11% cysteine, designated WO 95/00849 PCTIUS94/06890 35 the cysteine-rich domain (Mann, B.J. et al. Parasit Today (1991) 1:173-176) (Fig. Sequence analysis of hgl3 revealed that all 97 cysteine residues present within this region were also conserved in both of the previously reported heavy subunit genes.

A schematic comparison (Figure 5) of heavy subunit gene sequences revealed a high degree of amino acid sequence identity. However, seven sites, ranging from 3-24 nucleotides, were found where an insertion or deletion had occurred in one subunit relative to another, all of which maintained the open reading frame. Both hgll and hgl3 contained a large number of nonconservative amino acid substitutions when compared to hgl2, making them 89.2% and 89.4% identical to hgl2 respectively.

While the comparison of hgll and hgl3 revealed only two nonconservative substitutions, 57 conservative amino acid substitutions and 3 single residue insertion/deletions making them 95.2% identical.

All 16 potential sites of glycosylation present in hgll were conserved in hgl3. A sequence analysis of hgl2 indicated that it contained only 9 such sites, although all 9 were present in hgll and hgl3.

Glycosylation appears to account for approximately 6% of the heavy subunits' apparent molecular mass (Mann, B.J.

et al. Proc Natl Acad Sci USA (1991) 88:3248-3252).

All three heavy subunits are expressed. Since hgl3 was isolated from a genomic library, it was unknown if this gene was transcribed. Polyadenylated RNA was harvested from amebae in both log and stationary phase growth. Probes specific for hgll, hgl2, or hgl3 were hybridized to a Northern blot and identified an RNA band of the predicted size of 4.0 kb.

As the messages of hgll-3 are predicted to comigrate at 4.0 kb, differential hybridization was required to ascertain expression of individual genes using Northern blots. Due to the high degree of identity WO 95/00849 PCTIUS94/06890 36 between hgll-3, relatively short oligonucleotides (17-21 bases) were synthesized specific for regions where the three genes diverge. Each probe was compared by computer analysis to the other hgl genes to be certain that they were sufficiently divergent to prevent cross hybridization. Hybridization and wash conditions were highly stringent for such A/T rich probes and were done at temperatures 5 0 C or less below the predicted Tm based upon nearest neighbor analysis. While it is impossible to rule out cross hybridization with other hgl gene members, these precautions make such an event less likely.

The message abundance decreased significantly as the amebic trophozoites passed from log phase growth (lane A) to stationary phase growth (lane B) while the control gene, EF-la, either remained constant or increased slightly. This finding correlates with data indicating that late log and stationary phase amebae have a decreased ability to adhere to, lyse, and phagocytose target cells (Orozco, E. et al. (1988) "The role of phagocytosis in the pathogenic mechanism of Entamoeba histolytica. In: Amebiasis: Human infection by Entamoeba histolytica (Ravdin ed), pp. 326-338.

John Wiley Sons, Inc., New York.

Estimation of the number of heavy subunit genes.

The observations herein confirm that the adhesin 170 kDa subunit of HM-1:IMSS strain E. histolytica is encoded by a gene family that includes hgll, hgl2 and a previously undescribed third gene which is designated hgl3. Since hgll and hgl2 were originally sequenced, in part, from different cDNA libraries, it was possible that they represented strain differences of a single gene.

However, in the present work both 5' and 3' termini of hgll, hgl2, and hgl3 were isolated and sequenced from the same lambda genomic library, demonstrating unambiguously that hgl is a gene family.

WO 95/00849 PCTIUS94/06890 37 Comparison of the amino acid sequences of the three heavy subunit genes found that hgll and hgl2 are 89.2% identical, hgll and hgl3 are 95.2% identical, and hgl2 and hgl3 are 89.4% identical. Sequence variation within the gene family, however, appears to be nonrandomly distributed within the coding sequence. The majority of the nonconservative amino acid substitutions as well as insertions and deletions occur in the amino third of the molecule. Comparison of the amino acid sequences of hgl2 and hgl3 reveal that 11 of the 19 nonconservative amino acid substitutions and 11 of the 13 residues inserted or deleted reside within the first 400 amino acid residues.

A similar pattern of variation is present when hgll and hgl2 are compared. While hgll and hgl3 contain only two nonconservative substitutions, both are found within the first 400 residues although the 57 conservative substitutions appear to be more randomly distributed throughout the coding sequence. The high degree of sequence conservation between hgl3 and hgll suggest that they may have arisen from a recent gene duplication event.

All 97 cysteine residues were maintained in the three heavy subunit genes. The hgl2 gene was originally reported lacking a single cysteine present in both hgll and hgl3. However, this discrepancy has since been recognized as a sequencing error (Dr. E. Tannich, Bernhard Nocht Institute, Hamburg, Germany, personalcommunication). The cysteine residues are nonrandomly distributed throughout the gene (Fig. 1) with the highest concentration within the cysteine-rich domain between amino acid residues 379-1210. All seven identified epitopes recognized by murine monoclonal antibodies map to this region (Mann, B.J. et al. Infect Immun (1993) 61:1772-1778). As these monoclonal antibodies can block target cell adhesion, target cell lysis (Saffer, L.D. et al. Infect Immun (1991) 59:4681-4683), and/or resistance WO 95/00849 PCTIUS94/06890 38 to host complement-mediated lysis (Braga, L.L. et al. J Clin Invest (1992) 90:1131-1137), the conservation of cysteine residues may play an important role in maintaining the conformation of this important region of hgl.

A minimum of three genes have been shown to make up the heavy subunit gene family, as described herein.

While it is not possible to rule out the existence of additional hgl genes, Southern blot analyses and library screen data can best be explained by a gene family of three members. For Southern blots, two restriction enzymes were identified, DdeI and HindIII, that cut genomic DNA to completion and resulted in analyzable restriction fragments. As the membrane was hybridized with a fragment of hgll corresponding to nucleotides 1556 to 3522, two bands of >976 and 1965 nucleotides should have been present from hgl3. This central hgll radioprobe would hybridize with three bands of 1158, 810 and >1080 nucleotides from hgll and would hyribidze with five bands of 819, 312, 55, 755, and >1080 nucleotides from hgl2. The Southern blot showed 7 bands for genomic DNA disgested with DdeI, at 4200, 3700, 2100, 1800, 1300, 840, and 760 nucleotides. As the 819 and 810 nucleotide bands would be expected to comigrate, all the bands observed with DdeI digestion are explained by the restriction maps of hgll-3.

HindIII has no restriction sites in hgll-3 within the coding region and would result in each gene being represented by a single band greater than 4.0 kb.

The Southern blot showed three bands at 17500, 5600, and 4200 nucleotides. Should an additional heavy subunit gene exist, its DdeI and HindIII fragments would need to comigrate with hgll-3 bands, be so divergent that they failed to hybridize with the hgll probe under very low stringency, or be too large to be resolved and transferred.

WO 95/00849 PCT/US94/06890 39 As to the genomic screening data, the genomic library was screened separately with a 5' and a 3' hgl specific probe, additional heavy subunit genes would be isolated even if they contained only partial identity with the gene family at only one end or even if one termini of an additional gene had been lost during library amplification. The library screen looked at more than 3.2x10 8 bases of genomic DNA in an organism with an estimated genome size of 10 75 bases (Gelderman, A.H. et al. J Parasitol (1971) 57:906-911). Thus, a full genomic equivalent was screened at low stringency for genes containing identity at either end. Of 7 clones identified with the 5' heavy subunit-specific probe, 4 contained inserts that matched the reported sequence for hgll, 2 matched the sequence of hgl2, and 1 clone represented hgl3. Of eight clones obtained using the 3' radiolabeled fragment, 1 matched the sequence for hgll, matched the sequence of hgl2, and 2 represented hgl3. No termini were found that did not match the sequence of hgll, hgl2 or hgl3.

Claims (4)

1. A vaccine composition for immunising a subject against Entamoeba histolvica infection, the vaccine composition comprising an effective amount of a recombinant, non-glycosylated epitope-bearing peptide of the 170 kDa subunit if E. histolytica Gal/GalNAc adherence lectin capable of eliciting an immune response to E. histolytica in a subject together with a pharmaceutically acceptable diluent or excipient, which peptide is selected from: the region of amino acids 596-1138 as shown in Figure 1B; or the corresponding amino acids in a naturally occurring variant of the 170 kD subunit; wherein the peptide being in a form produced in prokaryotic cells.

2. The vaccine according to claim 1 wherein the epitope-bearing portion of the peptide comprises amino acids of the 170 kD subunit selected from the group consisting of amino acids 596-818 and 895-998 as shown in Figure 1B, and the corresponding amino acids in a naturally occurring variant of the 170 kD subunit.

3. The vaccine of claim 2 wherein the epitope-bearing portion comprises amino acids 596-818 as shown in Figure lB.

4. The vaccine of claim 2 wherein the epitope-bearing portion comprises amino acids 895-998 as shown in Figure lB. A vaccine for immunising a subject against E. histolytica infection, the vaccine comprising an effective amount of a non-glycosylated epitope- bearing peptide capable of eliciting an immune response to E. histolytica in a subject consisting of amino acids selected from the group consisting of positions 596-1138, 895-998, 1033-1082, and 1082-1138 of the 170 kD subunit of E. histolytica Gal/GalNAc adherence lectin as shown in Figure 1B, and the corresponding amino acids in a naturally occurring variant thereof, wherein the peptide is in a form produced by recombinant prokaryotic cell culture. Dated this 23rd day of July 1999 UNIVERSITY OF VIRGINIA PATENT FOUNDATION Patent Attorneys for the Applicant: F B RICE CO