US 6,187,310 B1Grant
Recombinant Entamoeba histolytica lectin subunit peptides and reagents specific for members of the 170 kDa subunit multigene family
Issue Date:2001-02-13
•22 Claims
•19 Drawing Sheets
Abstract
The 170 kDa adhesin subunit of the Entamoeba histolyticaGal/GalNAc adherence lectin is encoded by members of a gene family that includes hgl1, hgl2 and a newly discovered gene, hgl3. The DNA and encoded protein sequences of the hgl genes are disclosed. A number of proteins and peptide fragments of the adhesin as well as other functional derivatives, preferably produced by recombinant methods in prokaryotic cells are disclosed. A preferred peptide for a vaccine composition corresponds to amino acids 896-998 of the mature 170 kDa lectin and contains the galactose- and N-acetylgalactosamine-binding activity of the native lectin. These compositions are useful as immunogenic vaccine components and as diagnostic reagents. Methods are provided for a vaccine comprising one or more peptides of the lectin to immunize subjects at risk for infection by E. histolytica. Additionally, immunoassay methods are disclosed for measuring antibodies specific for an epitope of the lectin. These methods detect E. histolytica-specific antibodies, some of which are specific for epitopes characteristic of pathogenic strains, nonpathogenic strains, or both.
Metadata
Assignee
- University of Virginia Patent Foundation
Inventors
- Barbara J. Mann
- James M. Dodson
- William A. Petri, Jr.
Application Information
Application Number:US 08/937,236
Filing Date:1997-09-16
Priority Date:1988-01-13
Art Unit:7
Classifications
IPC:
A61K 39002C07K 1444
Field of Search:
5303005303504241301424185142426514242691
Patent Drawings (19 sheets)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application is a continuation-in-part of U.S. Ser. No. 08/569,214, filed Apr. 29, 1996 which was a National Stage filing of PCT/US94/06890, filed Jun. 17, 1994, which is a continuation of U.S. applications: U.S. Ser. No. 08/078,476, filed Jun. 17, 1993 (now abandoned), which is a continuation-in-part, U.S. Ser. No. 08/130,735, filed Oct. 1, 1993 (now abandoned).
[0003] U.S. Ser. No. 08/078,476 was a continuation-in-part of two applications: U.S. Ser. No. 07/615,719, filed Nov. 21, 1990 (issued as U.S. Pat. No. 5,260,429) and U.S. Ser. No. 08/075,226, filed Jun. 10, 1993 (issued as U.S. Pat. No. 5,401,831). U.S. Ser. No. 08/075,226 and U.S. Ser. No. 07/615,719 both claimed priority (as a division and as a continuation-in-part, respectively) from U.S. Ser. No. 07/479,691, filed Feb. 13, 1990 (issued as U.S. Pat. No. 5,272,058), which was a continuation-in-part of U.S. Ser. No. 07/456,579, filed Dec. 29, 1989 (issued as U.S. Pat. No. 5,004,608), which was a continuation of U.S. Ser. No. 07/143,626, filed Jan. 13, 1988 (abandoned).
[0004] All the applications cited above are hereby incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
[0006] 1. Field of the Invention
[0007] The invention in the field of infectious diseases and immunology concerns vaccine and diagnostic compositions and methods. The compositions comprise peptides and proteins which include epitope-bearing regions of the 170 kDa subunit (or adhesin) of the Entamoeba histolyticaGal/GalNAc adherence lectin. The compositions are produced recombinantly in prokaryotic hosts. These peptides are used to measure subunit-specific antibodies in a subject infected by E. histolyticaor responding to these vaccines. This invention includes the discovery of a novel variant of the 170 kDa subunit and the gene (hgl3) which encodes it. Hgl3 is the third member of a multigene family each member of which encodes a 170 kDa subunit of the lectin.
[0008] 2. Description of the Background Art
[0009] Entamoeba histolyticainfection 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 symptoms is thought to be due to the existence of nonpathogenic as well as pathogenic strains of this ameba. As of 1988, it had been established that the subjects who eventually exhibit symptoms harbor pathogenic “zymodemes” classified 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 pathogenicity of the infecting zymodeme and development of symptoms.
[0010] It is known that E. histolyticainfection 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, W. A. et al., J Biol Chem(1989) 264:3007-3012. This nomenclature derives from the fact that adherence of the organism to target cells via this lectin is inhibited by the saccharides galactose and N-acetylgalactosamine. The purified lectin was shown to have a nonreduced molecular weight of 260 kDa on SDS-PAGE; reduction with β-mercaptoethanol yielded two subunits having molecular masses of 170 kDa and 35 kDa. The 170 kDa subunit is also referred to herein as the 170 kDa adhesin or the 170 kDa protein. Further studies showed that antibodies directed to the 170 kDa subunit were capable of blocking surface adhesion to test cells (Petri, et al., supra). Therefore, the 170 kDa subunit is believed to be of primary importance in mediating adhesion, hence is designation as the 170 kDa adhesin.
[0011] U.S. Pat. No. 5,004,608 (Apr. 2, 1991), describes the entire lectin as an effective vaccine to prevent E. histolyticainfection.
[0012] Studies of serological cross-reactivity of sera from patients having symptoms, characteristic of pathogenic E. histolyticainfection, including liver abscess and colitis, showed that the adherence lectin was recognized by all sera tested Petri, Jr., W. A. et al., Am J Med Sci(1989) 296:163-165). The 170 kDa subunit is recognized almost universally 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).
[0013] DNA molecules encoding both the heavy (170 kDa) and light (35 kDa) subunits have been cloned. The heavy and light subunits are encoded by distinct mRNAs (Mann, B. et al., Proc Natl Acad Sci USA(1991) 88:3248-3252), and these subunits have different amino acid compositions and N-terminal sequences. The sequence of cDNA encoding the 170 kDa subunit suggests that it is an integral membrane protein with a large cysteine-rich extracellular domain and a short cytoplasmic tail (Mann, B. et al., supra; Tannich et al., Proc Natl Acad Sci USA(1991) 88:1849-1853). The deduced amino acid sequence of the 170 kDa adhesin shows that the extracellular domain can be divided into three regions based on amino acid composition. The N-terminal amino acids 1-187 are relatively rich in cysteine (3.2%) and tryptophan (2.1%). The convention for amino acid numbering of the 170 kDa subunit is to start with the N-terminus of the mature (processed) protein as #1. The sequence from positions 188-378 lacks cysteine. In the stretch of residues from 379-1209, 10.8% are cysteine. Clones encoding the 170 kDa subunit are further described in U.S. Pat. No. 5,260,429 (Nov. 9, 1993), the disclosure of which is incorporated herein by reference. This patent describes methods for diagnosing the presence of E. histolyticausing the polymerase chain reaction (PCR) and DNA probes.
[0014] The 170 kDa subunit is thought to be encoded by a multigene family (Mann, B. et al., Parasit Today(1991) 1:173-176). Two different 170 kDa subunit genes, hgl1 and hgl2, have been sequenced by separate laboratories. While hgl2 was isolated in its entirety from an HM-1:IMSS cDNA library (Tannich, E. et al. Proc Natl Acad Sci USA(1991) 88:1849-1853), hgl1 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 (Mann et al., supra). As the amino acid sequences of these two gene products have 87.6% identity (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, is consistent with the existence of a gene family (Tannich, E. et al. Proc Natl Acad Sci USA(1991) 88:1849-1853).
[0015] U.S. Pat. No. 5,272,058 (Dec. 21, 1993; incorporated herein by reference in its entirety) discloses monoclonal antibodies (mAbs) immunoreactive with various epitopes of the 170 kDa subunit. This document also describes use of these antibodies to detect the 170 kDa protein and use of the protein to detect antibodies in serum or other biological samples. It is noteworthy that all the experimental work described in this document was limited to the native protein. These mAbs were further characterized by the present inventors' group (Mann, B. J. et al., Infect Immun(1993) 61:1772-1778; also incorporated by reference).
[0016] Various immunoassay techniques have been used to diagnose E. histolyticainfection. E. histolyticaantigens have been detected by ELISA of stool specimens and sera, though these tests do not seem to distinguish between the pathogenic and nonpathogenic strains. Root et al., Arch Invest Med(Mex) (1978) 9: Supplement1:203, described ELISAs with rabbit polyclonal antiserum to detect amebic antigens in stool specimens, and various forms of this procedure have been adapted by others (Palacios et al., Arch Invest Med(Mex) (1978) 9 Supplement1:203; Randall et al., Trans Roy Soc Trop Med Hyg(1984) 78:593; Grundy, Trans Roy Soc Trop Med Hyg(1982) 76:396; Ungar, Am J Trop Med Hyg(1985) 34:465). These studies on stool specimens and other biological fluids are summarized in Amebiasis: Human Infection by Entamoeba histolyticaJ. Ravdin, ed. (1988) Wiley Medical Publishing, pp. 646-648.
[0017] Serological analysis is also a critical tool in the diagnosis of invasive amebiasis. One approach utilizes conventional serologic tests, such as indirect hemagglutination. These tests are very sensitive, but seropositivity persists for years (Krupp, I. M., Am J Trop Med Hyg(1970) 19:5762; Lobel, H. O. et al., Ann Rev Microbiol(1978) 32:379-347). Thus, healthy subjects may give positive assay responses, creating an undesirably high background. Similar problems with false positives have been observed in immunoassays using a mAb and purified native 170 kDa protein (Ravdin, J. I. et al., J Infect Dis(1990) 162:768-772.)
[0018] Recombinant E. histolyticaproteins other than the 170 kDa subunit have been used as the basis for serological tests. Western blotting using a recombinant form of the “52 kDa 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., S. L. et al., Proc Natl Acad Sci USA(1990) 87:4976-4980; Stanley, Jr., S. L. et al., JAMA(1991) 266:1984-1986). However, the overall sensitivity was lower than for the conventional agar gel test (82% vs. 90-100%).
[0019] Thus, there remains a need for serological tests which will provide optimal sensitivity while minimizing the number of false positives. The present invention provides such a test by utilizing, as antigen, epitope-bearing portions of the 170 kDa subunit of the adherence lectin produced recombinantly in prokaryotic systems.
[0020] It is particularly advantageous to use recombinantly produced, nonglycosylated peptides or proteins in this assay because of the ease of their preparation and standardization. Furthermore, since selected portions of the 170 kDa subunit can be generated, epitopes characteristic of the pathogenic or nonpathogenic forms of E. histolyticacan be produced and used to distinguish these parasite forms. Subsequent to the making of this invention, Zhang, Y et al. (J Clin Micro-immunol(1992) 2788-2792) reported on immunoreactivity of immune sera with recombinant 170 kDa protein.
[0021] Although it was known that the 170 kDa subunit may be used in a vaccine (U.S. Pat. No. 5,004,608, supra), the present invention, directed to recombinantly produced 170 kDa subunit fragments made in prokaryotic cells and lacking in glycosylation, offers significant advantages in (a) reproducibility of the product, (b) ease of preparation of potent “subunit” vaccines and (c) the biologic (carbohydrate-binding) activity is contained in recombinant product.
[0022] Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
SUMMARY OF THE INVENTION
[0023] The invention provides (1) vaccine compositions for prevention of E. histolyticainfection and (2) diagnostic compositions and assays that permit assessment of subjects for the presence of invasive E. histolyticainfection. The invention also provides a novel third variant protein of the 170 kDa subunit and a gene (designated hgl3) which encodes this novel protein. Accordingly, the vaccines and diagnostics of the invention are based on the sequences of all three variants of the 170 kDa subunit which are encoded by three different genes of this multigene family.
[0024] Pathogenic and nonpathogenic strains of E. histolyticacan be distinguished by the present diagnostic methods. The tests use as antigen an epitope-bearing protein or peptide of 170 kDa subunit that is preferably recombinantly produced in a prokaryotic system. Despite the absence of glycosylation from such peptides, and despite the lack of post-translational modifications characteristic of the native protein or peptide, such recombinantly produced proteins or peptides are effective antigens in these assays.
[0025] Thus, the present invention is directed to a protein or peptide which comprises a 170 kDa subunit of E. histolyticaadherence lectin or a functional derivative thereof, which protein, peptide or functional derivative:
[0026] (a) is nonglycosylated and
[0027] (b) bears at least one epitope that reacts with antibodies made in a subject infected with E. histolyticainfection or immunized with the adherence lectin or a portion thereof.
[0028] The protein, or peptide fragment is preferably encoded by one, two or all of the hgl1 gene, the hgl2 gene and the hgl3 gene. The above protein, peptide or functional derivative is preferably produced recombinantly in prokaryotic cells.
[0029] The protein may be the full length 170 kDa subunit. Preferably, however, a peptide is a fragment of SEQ ID NO:3 or SEQ ID NO:6 selected from the group consisting of 482-1138, 596-1138, 895-998, 946-970, 976-1000, 991-1015, 1006-1030, 1036-1060, 1033-1082 and 1082-1138 or a functional derivative of any of the foregoing. The functional derivative may be a naturally occurring variant of the peptide fragment. Most preferred is a peptide which has the carbohydrate binding domain of the lectin, and preferably has the sequence of 895-998.
[0030] In another embodiment, this invention is directed to a vaccine composition for immunizing a subject against E. histolyticainfection. The vaccine comprises one or more of the proteins, peptides or functional derivatives described above and a pharmaceutically acceptable vehicle or carrier. A preferred vaccine comprises a fusion protein which includes a peptide fragment as above. The vaccine composition may further comprise an adjuvant or other immune stimulating agent.
[0031] Also provided is a method for immunizing a subject against Entamoeba histolyticainfection which method comprises administering to the subject an effective amount of a vaccine as described above, in particular fragment 895-998 which contains the carbohydrate-binding domain. Vaccination with 895-998 (also designated here as Δ3′) will result in an immune response which blocks the carbohydrate-binding activity of the organism which is needed for both colonization and host cell killing.
[0032] In yet another embodiment, the invention is directed to an immunoassay method for detecting or quantitating Entamoeba histolytica-specific antibodies in a biological sample comprising
[0033] (a) contacting the sample with an epitope-bearing protein, peptide or functional derivative as described above, under conditions wherein the protein, peptide or functional derivative binds to the specific antibodies immunoreactive to form a complex; and
[0034] (b) determining the presence or amount of the complex,
[0035] thereby detecting or quantitating the antibodies.
[0036] In the immunoassay method, the protein, peptide or functional derivative is preferably immobilized to a solid support.
[0037] A competitive immunoassay format further comprises, during step (a), adding a competing specific binding partner for the epitope, preferably an antibody or antigen binding fragment thereof, more preferably, a detectably labeled antibody. The binding partner competes with antibody in the sample for binding to the epitope.
[0038] In the above immunoassay the epitope of the protein, peptide or functional derivative is one which is characteristic of a pathogenic form of E. histolytica. Alternatively, the epitope is characteristic of a nonpathogenic form of E. histolyticaIn another embodiment, the epitope is characteristic of both a pathogenic and a nonpathogenic form of E. histolytica.
[0039] The invention also provides an article of manufacture useful in the above immunoassay method, comprising a solid support or matrix to which is immobilized an epitope-bearing protein, peptide or functional derivative as described above. Also provided is a kit for carrying out the immunoassay, the kit being adapted to receive therein one or more containers, the kit comprising:
[0040] (a) a first container containing an epitope-bearing protein, peptide or functional derivative as described above in soluble form, or preferably immobilized to a solid support;
[0041] (b) a second container or plurality of containers containing a reagent or reagents capable of detecting or quantitating the binding of the sample antibodies to the epitope-bearing protein, peptide or functional derivative.
[0042] In yet another aspect, the invention is directed to a purified and isolated DNA molecule consisting essentially of a DNA encoding the 170 kDa subunit of pathogenic E. histolyticaadherence lectin. A preferred DNA molecule has the sequence SEQ ID NO:4, the sequence determined for the hgl3 gene. In further aspects, the invention is directed to both nucleic acid and immunological reagents which can be produced in view of the discovery of the hgl3 gene. These reagents are specific for each of the hgl1, hgl2 or hgl3 genes, as well as their RNA 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 VI.
[0043] Detailed descriptions of methods for detecting pathogenic or nonpathogenic forms of E. histolyticaand 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. Pat. No. 5,272,058, the entire disclosure of which is incorporated herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1A (sheets FIG. 1A-1 through FIG. 1A-6) shows the nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO:2) from the DNA encoding the 170 kDa subunit of the adherence lectin from pathogenic strain HM1:IMSS, designated hgl1.
[0045] FIG. 1B shows the deduced amino acid sequence (SEQ ID NO:2) of the hgl1 encoded protein (1291 residues) with the N-terminal amino acid of the mature protein numbered as residue 1. The mature protein has the sequence SEQ ID NO:3.
[0046] FIG. 2 is a diagram of the construction of expression vectors for recombinant production of specified portions of the 170 kDa subunit
[0047] FIG. 3 is a diagram of the location of various epitopes on the 170 kDa subunit. In particular, the location of epitopes recognized by mAbs specific for the 170 kDa subunit are shown below the rectangular box which is the domain diagram. The epitopes were mapped by measuring reactivity of the mAbs with deletion constructs of the subunit. The domains of the 170 kDa subunit are labeled left to right as follows: “C-W” is the N-terminal region rich in cysteine and tryptophan; “CF” is the cysteine-free domain; “C-Rich” is the cysteine-rich domain; the transmembrane (TM) domain is shown as a dark box; CD is the cytosolic domain. (See: Mann, B. J. et al. Parasit Today(1991) 7:173-176). The amino acids included in the recombinant deletion variants of the subunit are shown in parentheses beside the deletion designation and are indicated by black bars below the domain diagram. The mAbs which recognized each deletion derivative are listed by number to the right of the horizontal bars (see also Table 1). Also shown above the domain diagram are maps of epitopes in terms of their reactivity human B cells, pathogen specificity and the effects of the antibodies on biological activity (adherence, complement resistance).
[0048] FIGS. 4A-4F shows the nucleotide sequence (SEQ ID NO:4) of DNA encoding the 170 kDa subunit of the adherence lectin from pathogenic strain HM1:IMSS, designated hgl3 and the deduced amino acid sequence (SEQ ID NO:5) encoded thereby.
[0049] FIG. 5 shows the deduced amino acid sequence (SEQ ID NO:5) encoded by hgl3 with the N--terminal amino acid of the mature protein numbered as residue 1. The putative signal sequence is overlined, and the putative transmembrane domain is underlined. Conserved cysteine residues (●) and potential sites of glycosylation (★) are indicated. The mature protein has the sequence SEQ ID NO:6.
[0050] FIG. 6 is a schematic representation of the protein product of the 170 kDa subunit gene (top panel) and a pairwise comparison of the amino acid sequences of the products of three known members of this gene family, hgl1, hgl2 and hgl3. The protein structure is as in FIG. 3, above. Amino acid sequence comparisons of the protein products of hgl1, hgl2 and hgl3 are shown in the lower three panels. Upright lines indicate nonconservative amino acid substitutions in the second protein of the comparison (listed at right). Downward arrows indicate a deletion while downward lines ending in upward-pointing arrowheads indicate insertions. The number of inserted or deleted residues are given below the arrowheads. The % amino acid sequence identity is indicated at right.
[0051] FIG. 7 is a dot blot showing the specific binding of GalNAc by purified 170 kDa lectin and by two concentrations of the fragment Δ3′. Purified lectin or fragment was spotted onto a PVDF membrane using a dot blot apparatus. Inhibition of binding to 125I-GalNAc20BSA is shown by either glucose (strip A) or galactose, is shown.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0052] This invention provides vaccine and diagnostic compositions and methods. Diagnostic methods detect antibodies specific for pathogenic and/or nonpathogenic forms of E. histolyticaand are conducted on biological samples derived from subjects at risk for infection or suspected of being infected. The assays are also designed to distinguish pathogenic from nonpathogenic forms of the ameba. The vaccines compositions are administered to subjects at risk for E. histolyticainfection.
[0053] The assays of the invention rely on the ability of an epitope-bearing fragment of the 170 kDa subunit, produced recombinantly in prokaryotic cells, to react immunologically with antibodies in biological samples from infected individuals. Even though the preferred peptide or protein is produced in a prokaryotic system, and is thus not glycosylated or processed after translation in the same way as the native protein, the epitope-bearing fragments are nevertheless useful as antigens in these immunoassays. “Biological samples” include (a) biological fluids such as blood, serum, urine or cerebrospinal fluid, (b) diluted or fractionated forms of such fluids, (c) cells, tissues or organs, or extracts or lysates thereof. These epitope-bearing peptides are also immunogenic or can be made immunogenic by coupling to an appropriate carrier, and are therefore used in vaccine preparation as well.
[0054] The advantages of the recombinant forms of the 170 kDa fragments as antigens include cost-effectiveness and reliable production of pure material, thereby assuring uniformity of vaccine or diagnostic assay compositions. Importantly, the Δ3′ fragment (aa-895-998) has the GalNAc-binding activity of specificity identical to that of the native lectin. Recombinant production in bacteria is particularly efficient and preferred. It is surprising that successful antigens and immunogens can be made in such prokaryotic hosts, since the peptides are not processed in a manner analogous to the native protein. Recombinant methods also facilitate the preparation of single epitopes or selected combinations of epitopes, thereby providing a means for detecting antibodies specific for pathogenic or nonpathogenic forms of the organism. Such recombinant production allows preparation of subunit vaccines with the desired epitope or combination of epitopes.
[0055] Thus, the invention is directed chiefly to (1) recombinantly produced peptides and materials useful as vaccine compositions and as diagnostic reagents, (2) methods to immunize subjects at risk of infection with such vaccine compositions, and (3) methods to evaluate biological samples for the presence of antibodies or 170 kDa subunit antigens using these recombinantly produced diagnostic reagents.
[0056] Definitions
[0057] The diagnostic assays may be designed to distinguish antibodies raised against nonpathogenic or pathogenic forms of the ameba. “Pathogenic form” of E. histolyticarefers to those forms which are invasive and result in symptoms in infected subjects. “Nonpathogenic form” refers to those forms which may be harbored asymptomatically by carriers of the infection.
[0058] The assays and vaccines of the invention utilize an epitope-bearing “portion,” “fragment” or “peptide” of the 170 kDa subunit of the Gal/GalNAc lectin, a glycoprotein found on the surface of E. histolyticawhich mediates the adherence of the amebas to target cells, and which adherence is inhibited by the saccharides Gal or GalNAc. The “lectin,” as the term is used herein, is the molecule isolated by Petri et al. (supra) from the pathogenic strain HMI-IMSS of E. histolytica, or is the corresponding or homologous glycoprotein found in other strains of E. histolytica.
[0059] The “170 kDa subunit” refers to the large subunit obtained upon reduction of the Gal/GalNAc lectin, such as that obtained by Petri et al. (supra), the amino acid sequence of which may be SEQ ID NO:3 or SEQ ID NO:6, as well as to its counterparts in other strains. See, also, FIGS. 1A (sheets 1A-1-1A-6), FIG. 1B, and FIGS. 4A-F and FIG. 5.
[0060] Diagnostic Assays
[0061] The complete 170 kDa subunit (antigen) or an epitope-bearing fragment thereof can be used in the present assays. The peptide, and therefore, the epitope(s) may be selected to configure an assay that detects pathogenic strains, nonpathogenic strains or both. This is accomplished by selecting strain-specific epitopes. To detect infection by either or both types of amebae in a single assay, either a combination of epitopes, or an epitope(s) common to both pathogenic and nonpathogenic strains, are used.
[0062] As shown below, the segments of the 170 kDa protein which contain epitopes reactive with all mAbs prepared thus far against the complete lectin subunit are found between amino acid positions 596 and 1138 (SEQ ID NO:3). An epitope characteristic of pathogens is found in peptide fragments 596-818, 1082-1138 and 1033-1082. Epitopes which are shared by pathogens and nonpathogens as well as epitopes characteristic of pathogens are found in peptide 896-998. Thus, an assay to detect antibodies against pathogens using a fragment of recombinantly produced 170 kDa protein preferably employs a peptide representing positions 596-818, 895-998, 1033-1082 or 1082-1138. Mixtures of the above peptides may also be used. Alternatively, longer forms of antigen can be used by selecting and combining the appropriate peptides depending on whether infections with pathogenic or nonpathogenic amebae are to be diagnosed.
[0063] As shown in Examples IV, V, VII and VIII, below, useful epitope-bearing fragments for serodiagnosis include peptides 2-482, 1082-1138, 1032-1082 and 895-998. Peptides 1082-1138 and 1032-1082 appear to be recognized by antibodies against pathogenic amebae. These epitope-bearing peptides may be used as single peptides, basically as they appear in the lectin, or as portions of chimeric or fusion proteins, as mixtures of peptides or of chimeric proteins, or as portions of a distinct protein construct bearing multiple epitopes not necessarily ordered relative to one another in the same order in which they exist in the native protein. Procedures for preparing recombinant peptides and proteins containing only a single epitopic fragment, as identified above, or containing multiples of such epitopic fragments (including tandem repeats) are routine in the art.
[0064] Fusion proteins between GST and epitopes of the present invention have been exemplified in several of the Examples herein. Other fusion proteins can be used to enhance antigenicity. Thus, heterologous oligopeptide epitopes of immunological interest have been inserted in-frame into bacterial flagellin (Newton, S. et al, (1989) Science244:70-72; Jennings et al., (1989) Protein Eng.2:365), influenza virus nucleoprotein (Chimini, G. et al. (1989) J. Exp. Med.169:97-302), hepatitis B surface antigen (Rutgers et al., (1988) Bio/Technology6:1065) and in the complementarity determining regions (CDR) of immunoglobulin (Ig) molecules (Billetta, R. et al., (1991) Proc. Natl. Acad. Sci. USA88:4713-4717; Zanetti et al. (1992) Nature,355:476; Zanetti et al. WO90/090804); Zaghouani, H. et al. (1993) Science259:224-227; Zaghouani, H. et al., (1993) Int. Rev. Immunol.10:265-278; Zaghouani, H. et al. (1995) Proc. Natl. Acad. Sci. USA92: 631-635). Such a recombinant protein can induce an enhanced immune response to the heterologous oligopeptide. (See, also, Zauderer, M., Nature Biotechnol. (1996) 12:703-704, and the references cited therein)
[0065] Thus in the present invention, a peptide-immunoglobulin fusion protein is used to induce immunity following the approach described by Billetta et al., supra. There, a fusion Ig was made which expressed in the CDR3 of its VHregion the repetitive tetrapeptide Asn-Ala-Asn-Pro (designated (NANP)n) of the circumsporozoite protein of Plasmodium falciparum, an etiologic agent of malaria. Immunization of rabbits and mice with the engineered fusion Ig in adjuvant elicited antibodies to the (NANP)3synthetic peptide and to P. falciparumparasite. Such antibodies efficiently inhibited the invasion of cultured liver cells by P. falciparum. Thus, immunity to malaria was induced in the absence of the parasite using antibody V regions engineered to mimic the parasite's molecular structure. C. Bona et al., (1994) Cell Mol. Biol.40 Suppl1:21-30 expressed viral epitopes on Ig molecules by replacing the D segment of a VHgene with (a) a B cell epitope from the V3-loop of HIV-1 gpl20, (b) a cytotoxic T lymphocyte-epitope from influenza virus nucleoprotein or (c) a T helper epitope from influenza hemagglutinin. The T cell-targeted fusion Ig molecules (produced by cells transfected with chimeric genes) activated specific T cells. The authors suggested practical applications for Ig molecules bearing foreign epitopes for the development of prophylactic and immunotherapeutic reagents. In summary, it is known that recombinant fusion proteins, including fusion Ig proteins, may be useful immunogens to induce immune responses to a heterologous oligopeptide. Hence, an immunogenic fusion Ig is made with a desired epitope-bearing fragment of the 170 kDa subunit by conventional recombinant techniques. This protein can serve as a vaccine composition in accordance with this invention.
[0066] The present assays are designed to detect in biological samples antibodies which are “immunospecific for” or “immunoreactive with” the epitope-bearing fragment—i.e., having at least one epitope contained in this fragment. As used herein, an “immunospecific” or “immunoreactive” antibody binds to its target with significantly higher affinity or avidity compared to its binding to any other antigen, hapten or epitope. 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, preferably two, or more preferably greater than 2 orders or magnitude higher than its affinity for alternate targets for which is not “specific.”
[0067] The present assays can be performed using 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 practitioner. The biological sample is prepared in a conventional manner for immunoassays; such preparation may involve dilution or fractionation of the biological fluid or initial cell, tissue or organ extract. A “biological sample” refers to the sample actually used in the assay whether it is derived from a fluid, cells, tissue or an organ of a subject and prepared for use in the assay using any known technique. Normally, plasma or serum is the preferred source of the biological sample. For a detailed discussion of immunoassays, including ELISA, use of immobilized antigens and antibodies, conventional solid supports, and the like, see, for example Hartlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988); Monroe, D., Anal. Chem.56: 920A-931A (1984); Voller, A et al. (eds)., Immunoassays for the1980's, University Park Press, Baltimore, Md., 1981; Butt, W. R. (ed.) Practical Immunoassay: The State of the Art, Dekker, New York, 1984; Bizollon, C., ed., Monoclonal Antibodies and New Trends in Immunoassays, Elsevier, New York, 1984; Butler, J. E., The Behavior of Antigens and Antibodies Immobilized on a Solid Phase (Chapter 11) In: Structure of Antigens, Vol. 1 (Van Regenmortel, M., CRC Press, Boca Raton 1992, pp. 209-259; Butler, J. E., ELISA (Chapter 29), In: van Oss, C. J. et al., (eds), Immunochemistry, Marcel Dekker, Inc., New York, 1994, pp. 759-803; Butler, J. E. (ed.), Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton, 1991; Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, 1980; Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986; Work, T. S. et al., Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, NY, (1978.)
[0068] 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 binding specifically to a targeted substance, such as an epitope-bearing peptide of the 170 kDa subunit. Commonly, such a specific binding partner is an antibody, but any alternative substance capable of such specific binding, such as a receptor, enzyme or any other chemical compound with the requisite property may be used. In this context, “antibody” refers not only to an intact immunoglobulin (Ig) molecule with two heavy and two light chains, but also to Ig fragments which retain the antigen-binding specificity of the intact antibody molecule. Examples of such fragments are well known in the art, and include, for example, Fab, Fab′, F(ab′)2and Fv fragments. The term “antibody” includes not only native forms of Ig, but forms which have been modified, as techniques become available in the art, to confer desired properties without altering the specificity. For example, the chimeric antibodies derived from two different species, such as mouse and man, are becoming more practical (e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP173494; Neuberger et al., WO8601533; Robinson et al., WO 8702671). Single chain antibodies are known (U.S. Pat. No. 4,946,778). In short, “antibody” refers to any component of, or derivative of an Ig molecule which retains the antigen binding specificity of the Ig.
[0069] A particularly useful type of antibody in the assay methods of the invention is a mAb. Three groups of mAbs have been prepared to the 170 kDa subunit. One group is immunospecific for epitopes “unique” to pathogenic forms. These mAbs therefore bind to a significant extent only to the pathogenic forms of the ameba or the 170 kDa subunit isolated from pathogenic forms. A second group is immunoreactive with epitopes “unique” to nonpathogenic forms. Thus, these mAbs bind to a substantial degree only to the nonpathogenic ameba or their lectins but not to the pathogenic forms. A third group of mAbs binds to epitopes shared among pathogenic and nonpathogenic strains and are thus capable of binding to an ameba, a subunit or a fragment, regardless of the organism's pathogenicity.
[0070] As shown in Table 1, below, seven different epitopes (designated by the numbers 1-7) of the 170 kDa subunit have been defined based on the reactivity patterns of seven different mAbs. The mAbs reactive with epitopes 1 and 2 of the subunit isolated from the pathogenic-strain react also with the corresponding epitopes on nonpathogens. On the other hand, those mAbs immunoreactive with epitopes 3-6 react only with the 170 kDa subunit of pathogenic strains. Techniques for isolating the subunit of pathogenic amebae is therefore used to isolate the 170 kDa subunit from nonpathogenic strains. This isolated material is then used to immunize animals and generate new mAbs immunoreactive with “counterpart” epitopes 3-6 of the 170 kDa subunit of nonpathogenic organisms.
[0071] Of course, antibodies found in a biological sample, in general, will be intact Ig molecules. However, pretreatment of the sample with an enzyme, for example, an enzyme which removes the Fc portion of the antibodies contained therein, does not destroy the sample's ability to react in the assay.
[0072] Assay Procedure
[0073] For the conduct of the present immunoassays, in general, the biological sample is contacted with an epitope-bearing peptide of the invention. The presence, absence or amount of the resulting immune complex formed between an antibody in the sample and the peptide is measured directly or in a competitive format.
[0074] As is well understood in the art, once the biological sample is prepared, a multiplicity of alternative protocols may be used to conduct the assay. In a preferred protocol, the epitope-bearing peptide is immobilized to a solid support, either by adsorption or by covalent linkage, and incubated with the biological sample. Any specific antibodies in the sample will bind to the peptide and become immobilized. Their presence on the solid support is then determined. This binding may be determined in by “direct” assay of the sample antibody bound to the support. In one particularly convenient format, the antigen is immobilized as a band on polyvinylidene difluoride (PVDF) (or other) membrane and contacted with the biological sample. Any antibody binding to the PVDF membrane is detected as described herein for Western blot procedures. This protocol is, in fact, a modified Western blot procedure without the electrophoretic step. Alternatively, microtiter plates or other suitable solid supports may be used.
[0075] The binding of antibody to the immobilized antigen is detected using conventional techniques. These generally involve a secondary labeling agent, for example, labeled a reagent comprising antibodies specific for the Ig species (e.g., human Ig for a human sample) or the Ig isotype or an Ig allotype. Detectable labels include radioisotopes, fluorescent tags, enzyme labels and the like, as is conventionally understood.
[0076] The assay may also be formatted as a competitive assay wherein the immobilized antigen (epitope-bearing fragment) is allowed to react with the biological sample in the presence of a competing specific binding partner 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 competitive assay, the competing binding partner is detectably labeled. Successful competition with antibodies in the biological sample is measured as a reduction in the amount of label bound in the resulting complex (or, alternatively as an increase in the amount of label remaining unbound in the supernatant. If mAbs are used, the assay can readily be adapted for antibodies reactive to pathogenic or nonpathogenic amebic strains by choosing competing antibodies of the appropriate specificity. Thus, if the assay is to be specific for antibodies against pathogenic E. histolytica, the competitor is a mAb specific for an epitope characteristic of pathogenic strains.
[0077] The assay may also be made specific for pathogenic vs. nonpathogenic strains by the choice of the epitope-bearing fragment. If antibodies specific to the pathogens are to be detected, the epitope-bearing peptide(s) chosen bears only an epitope(s) characteristic of pathogenic strains. Conversely, detection of antibodies specific for nonpathogens requires that the peptide(s) comprise an epitope(s) characteristic of nonpathogens. Where such distinction among antibodies is unnecessary, the antigen used in the assay may contain either or both types of epitopes.
[0078] In another embodiment, the antibody-containing biological sample is immobilized to a solid support, and the desired epitope-bearing peptide or protein, preferably having at least two epitopes, is added under conditions wherein the epitope) binds to the immobilized sample antibody. Addition of a detectably labeled second antibody specific for an epitope of the now bound antigen different from the epitope bound by the sample antibody) will result in binding of the label to the solid phase, thereby detecting the presence of antigen bound to the immobilized sample antibody.
[0079] In summary, the biological sample to be tested is contacted with the epitope-bearing fragment from a pathogenic or nonpathogenic strain of E. histolyticaso that a complex is formed. The complex is then detected by suitable labeling, either of the E. histolyticaantigen or of a secondary detection molecule which forms a ternary complex. The reaction is preferably conducted using a solid support to detect the formation of the complex attached to the support. Alternatively, the complex can be precipitated using conventional precipitating agents such as polyethylene glycol.
[0080] In a more complex, competitive assay, the biological sample, preferably serum or plasma, provides the unlabeled antibody which competes with a specific binding partner, preferably a labeled mAb specific for an epitope unique to the Gal/GalNAc lectin or its 170 kDa subunit. In this embodiment, the binding of the labeled specific mAb is conducted in the presence and absence of the biological sample, and the diminution of labeling of the resulting complex by the sample is a measure of the level of competing antibodies present in the sample.
[0081] Kits suitable for the conduct of the foregoing assays 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.
[0082] Vaccine Compositions
[0083] The recombinant 170 kDa subunit or an epitope-bearing peptide thereof is used as the active ingredient of a vaccine composition. A preferred region of the protein is from 482-1138 (of SEQ ID NO:2) which includes the cysteine-rich domain. This region is encoded by nucleotides 1492-3460 of SEQ ID NO: 1. More preferred “subregions” of the 482-1138 fragment are the following regions: 596-1138, 885-998, 1033-1082 and 1082-1138. One particularly preferred peptide is 895-998, which is advantageously prepared as a larger construct comprising multiple repeats of one or more of these epitopic regions.
[0084] Additional short peptide epitopes useful in vaccine compositions have been found (Lotter, H. et al., J. Exp. Med. (1997) 185:1793-1801) to react with antibodies from gerbils vaccinated with recombinant fragments corresponding to domains of the 170 kDa subunit which have been discovered by the present inventors to be efficacious vaccine preparations. Such peptides, corresponding to regions in the “C-Rich” domain (FIG. 3) include:
[0085] For use in vaccines, the full 170 kDa subunit or an epitope-bearing peptide thereof is produced recombinantly, preferably in prokaryotic cells. The recombinant product is administered as a protein or peptide vaccine. In another embodiment, the vaccine is in the form of a strain of bacteria (preferably a known “vaccine strain”) which has been genetically transformed to express the epitope-bearing peptide. Some known vaccine strains of Salmonella are described in Example VIII and in references cited therein. It may be practical to prepare the peptide by chemical synthesis if is sufficiently short, using methods described below.
[0086] Any functional derivative of the 170 kDa adhesin is intended within the scope of the invention. “Functional derivative” as used herein means a “fragment,” a “variant,” a “homologue,” an “analogue,” or a “chemical derivative” of the 170 kDa adhesin, which terms are defined below. A functional derivative retains at least a portion of the function of the 170 kDa adhesin which permits its utility in accordance with the present invention. Generally, this means that at least that portion of the sequence which is needed to retain the requisite biochemical and biological activity, in particular the antigenicity and immunogenicity.
[0087] A “fragment” of the 170 kDa adhesin refers to any subset of the molecule, that is, a shorter peptide, and is the preferred functional derivative.
[0088] A “variant” of the 170 kDa adhesin refers to a molecule substantially similar to either the entire protein or to a peptide fragment thereof. Variant peptides may be conveniently prepared by recombinant methods or by direct chemical synthesis on solid phase supports and their subsequent separation from the support. Such methods well-known in the art, and are described in the following references, hereby incorporated by reference: Merrifield, B., J. Amer. Chem. Soc.85:2149-2154 (1963); Merrifield, B., Science232:341-347 (1986); Wade, J. D. et al., Biopolymers25:S21-S37 (1986); Fields, G. B., Int. J. Peptide Prot. Res.35:161 (1990); MilliGen Report Nos. 2 and 2a, Millipore Corporation, Bedford, Mass., 1987). For example, the more classical method, “tBoc method,” or the more recent improved “F-moc” technique may be used (Atherton, E. et al., J. Chem. Soc. Perkin Trans.1:538-546 (1981)).
[0089] A preferred group of variants are peptides in which at least one amino acid residue and preferably, only one, has been removed and a different residue inserted in its place. For a detailed description of protein chemistry and structure, see Schulz, G. E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1979, and Creighton, T. E., Proteins: Structure and Molecular Principles, W. H. Freeman & Co., San Francisco, 1984, which are hereby incorporated by reference. The types of substitutions which may be made in the peptide molecule of the present invention are conservative substitutions and are defined herein as exchanges within one of the following groups:
[0090] 1. Small aliphatic, nonpolar or slightly polar residues: e.g., Ala, Ser, Thr, Gly;
[0091] 2. Polar, negatively charged residues and their amides: e.g., Asp, Asn, Glu, Gln;
[0092] 3. Polar, positively charged residues: e.g., His, Arg, Lys;
[0093] 4. Large aliphatic, nonpolar residues: Met, Leu, Ble, Val (Cys); and
[0094] 5. Large aromatic residues: Phe, Tyr, Trp.
[0095] Pro, because of its unusual geometry, tightly constrains the chain. Substantial changes in functional properties are made by selecting substitutions that are less conservative, such as between, rather than within, the above groups (or two other amino acid groups not shown above), which will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Most substitutions according to the present invention are those which do not produce radical changes in the characteristics of the peptide molecule. Even when it is difficult to predict the exact effect of a substitution in advance of doing so, one skilled in the art will appreciate that the effect can be evaluated by routine screening assays, preferably the immunoassays described herein. Modifications of peptide properties including redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan.
[0096] The term “homologue” as used here in is well-defined in the art, and can be described (at the protein level) as the product any member of a set of genes or DNA sequences from different organisms whose nucleotide sequences show a high degree of one-to-one correspondence with the DNA encoding the 170 kDa subunit. (See, for example, Micklos, D. A. et al., DNA SCIENCE, Cold Spring Harbor Press, 1990, p. 468). In particular, the 170 kDa subunit protein (or DNA coding therefore) from phylogenetically related amebic species is considered to be a homologue of the protein (or DNA) may be used.
[0097] An “analogue” of the 170 kDa subunit refers to a non-natural molecule substantially similar to either the entire molecule or to a fragment thereof.
[0098] A “chemical derivative” of the 170 kDa subunit contains additional chemical moieties not normally a part of the peptide. Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
[0099] Production of Recombinant Epitope-Bearing Compositions
[0100] The epitope-bearing portions of the 170 kDa subunit can be conveniently prepared in a variety of prokaryotic systems using control sequences and hosts well known and readily available in the art. The epitope-containing peptides may be fusion proteins or mature proteins which are produced intracellularly or are secreted. Techniques for constructing appropriate expression systems are well known in the art. If the epitope-bearing peptide is secreted, the culture medium can be used directly as a source of antigen in the assays described above. The antigen can also 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 after further purification. In the Examples below, the epitope-bearing fragment is provided as a fusion protein using the commercially available expression vector, pGEX. Alternative constructs and alternative hosts can also be used as is understood in the art.
[0101] Methods of Protecting a Subject from Infection
[0102] The present vaccine compositions are used primarily for prevention of E. histolyticainfection in subjects at risk for such an infection. Full length subunit or large fragments of the 170 kDa protein can be used as immunogens. If a shorter epitope-bearing fragment, for example containing 20 amino acids or less, is the active ingredient of the vaccine, it is advantageous to couple the peptide to an immunogenic carrier to enhance its immunogenicity. Such coupling techniques are well known in the art, and include standard chemical coupling techniques using linker moieties such as those available from Pierce Chemical Company, Rockford, Ill. Suitable carriers are proteins such as keyhole limpet hemocyanin (KLH), E. colipilin protein k99, BSA, or rotavirus VP6 protein.
[0103] Another embodiment is a fusion protein which comprise the epitope-bearing peptide region fused linearly to an additional amino acid sequence. Because of the ease with which recombinant materials can be manipulated, multiple copies a selected epitope-bearing region may be included in a single fusion protein molecule. Alternatively, several different epitope-bearing regions can be “mixed and matched” in a single fusion protein.
[0104] The active ingredient, or mixture of active ingredients, in the vaccine composition is formulated conventionally using methods well-known for formulation of protein or peptide vaccines. Vaccine compositions may include an immunostimulant or adjuvant such as complete or incomplete Freund's adjuvant, aluminum hydroxide, liposomes, beads such as latex or gold beads, ISCOMs, and the like. General methods to prepare vaccines are described in Remington's Pharmaceutical Science; Mack Publishing Company Easton, Pa. (latest edition). Liposomes are pharmaceutical compositions in which the active protein is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The active protein is preferably present in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non-homogeneous system generally known as a liposomic suspension. The hydrophobic layer, or lipidic layer, generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and/or other materials of a hydrophobic nature. Adjuvants, including liposomes, are discussed in the following references, incorporated herein by reference: Gregoriades, G. et al., Immunological Adjuvants and Vaccines, Plenum Press, New York, 1989 Michalek, S. M. et al., “Liposomes as Oral Adjuvants,” Curr. Top. Microbiol. Immunol.146:51-58 (1989).
[0105] The vaccine compositions preferably contain (1) an effective amount of the active ingredient, that is, the peptide or peptides, together with (2) a suitable amount of a carrier vehicle, and, if desired, (3) preservatives, buffers, and the like. Descriptions of vaccine formulations are found in Voller, A. et al., New Trends and Developments in Vaccines, University Park Press, Baltimore, Md. (1978).
[0106] The vaccines are administered as is generally understood in the art. Ordinarily, systemic administration is by injection; however, other effective means of administration are known. With suitable formulation, 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 administration of peptides is 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. Preferably, an effective amount of the protein or peptide is between about 0.01 μg/kg-1 mg/kg body weight. In general, multiple administrations of the vaccine in a standard immunization protocol are used, as is standard in the art.
[0107] Induction of Immunological Tolerance to “Enhancing” Epitopes
[0108] Antibodies specific for certain epitopes in certain regions of the 170 kDa subunit, in particular the C-W domain (in the vicinity of residues 1-436) act to enhance amebic adherence, thereby promoting the infectivity and pathology. The present invention provides an additional approach to preventing infection by selectively inhibiting the generation of antibodies with such specificity. Thus, in conjunction with the vaccines are provided tolerogenic compositions capable of inducing and maintaining epitope-specific tolerance in a subject.
[0109] Immunological tolerance is known to be inducible by attaching an epitope to be tolerated to isologous or heterologous Ig molecules, primarily of the IgG isotype (Borel, Y. (1980) Immunol. Rev.50:71; Scott, D. W. (1976) Cell Immunol.22:311). Such molecules are termed “tolerogenic carriers” or “tolerogens” (Scott, D. W. (1979) Immunol. Rev.43:241). These carriers owe their superior tolerogenicity to their persistence in vivo and the ability of an epitope chemically coupled to the IgG molecule to crosslink membrane IgM on the surface of B lymphocytes with surface Fc receptors. However, chemical coupling of epitopes to IgG carriers can be limited by the availability of free reactive amino groups, structural change of the epitope as a result of the coupling reaction, and the uncontrolled targeting of the added epitope to different (disadvantageous ) sites of the IgG molecule. Protein engineering strategies are used to create molecules containing heterologous epitopes. This has been discussed above for the amplification of specific immune responses. Recently, however, strategies for fusing DNA encoding a desired epitope to an Ig H chain gene to create tolerogenic fusion Ig's have been described (Scott and Zambidis, WO95/21926; Zambidis, E. T. et al., (1996) Proc. Natl. Acad. Sci. USA93:5019-5024; Zambidis, E. T. et al., J. Immunol. (1997) 158:2174-2182; Zambidis, E. T. et al., Mol. Med. (1997) 3:212-24)). By fusing an otherwise immunodominant epitope (termed 12-26) of the phage λcI repressor protein to the N-terminus of an Ig H chain, Scott, Zambidis and coauthors induced epitope-specific tolerance. Hematopoietic progenitor cells or B cells transfected using a retroviral vector expressed the fusion protein which could be used to induce tolerance by administration to a subject. More importantly, long lasting tolerance could be induced and maintained in an immunocompetent recipient by transfer of such hematopoietic progenitor cells or resting or activated B cells which had been transfected with the engineered fusion Ig to express this tolerogenic construct. Certain of these compositions could even overcome a state of pre-existing immunity.
[0110] Thus, according to the present invention, it is desired to induce tolerance to one or more peptides from the C-W domain of the 170 kDa subunit (or any other epitopes in other regions which are found to be associated with undesired “enhancing” antibodies that promote pathogenesis). A fusion Ig is made with the selected single epitope or combination of epitopes. The DNA encoding these epitopes is fused in-frame in the variable (V) region of a gene encoding an Ig chain (H or L), preferably the VHgene. An effective amount of this tolerogenic fusion protein, or of appropriate cells, preferably hematopoietic progenitors or B cells, expressing the fusion protein, are administered to a subject susceptible to infection with E. histolytica. This treatment can be given alone in conjunction with a vaccine directed to epitopes which are associated with protective immunity, such as the vaccine epitopes discussed herein.
[0111] An example of a preferred tolerogenic peptide is one corresponding to residues which are associated with an adherence-enhancing antibody response such as residues 436-624 of hgl2 (corresponding to 443-461 of hgl1) or the peptides recognized by the enhancing mAbs indicated in Table 1, below.
[0112] Treatment of a subject using the tolerogenic fusion Ig comprises parenterally administering a single or multiple doses of the fusion Ig to the subject, preferably a human. The fusion Ig is preferably an isologous Ig, that is, of the same species as the subject, most preferably human. Most preferred is a fusion IgG molecule. An effective tolerogenic dose is a function of the size and number of particular epitopes included in a particular fusion construct, the subject and his clinical status, and can vary from about 0.01 mg/kg body weight to about 1 g/kg body weight. A subject can be given this amount in a single dose or in multiple repeated doses. Doses of hematopoietic cells or B cells expressing the fusion Ig are preferably administered at a dose of between about 106and about 1010cells on one or several occasions. The route of administration may include intravenous (iv), subcutaneous (sc), intramuscular, intrapulmonary, intraperitoneal or other known routes. The preferred route for administration of such proteins or cells for tolerogenesis is by iv injection.
[0113] Reagents and Assays for a Novel 170 kDa Lectin Subunit
[0114] To discover the existence and determine the 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 hgl1. Termini from three distinct 170 kDa subunit genes were identified including hgl1, hgl2, and a third, previously unreported gene designated hgl3. The open reading frame of hgl3 was sequenced in its entirety (FIGS. 4A-F; SEQ ID NO:4). Nonstringent hybridization of a genomic Southern blot with 170 kDa subunit-specific DNA labeled only those bands predicted by hgl1-3. The amino acid sequence encoded by hgl3 FIGS. 4A-F; SEQ ID NO:5) was 95.2% identical to that encoded by hgl1 and 89.4% identical to that encoded by hgl2. All 97 cysteine residues in the 170 kDa subunit were conserved in hgl1-3. Nucleotide sequences for two additional members of this gene family, hgl4 and hgl5 are disclosed in a paper by the present inventors' laboratory (Ramakrishnan, G. et al., Mol. Microbiol.,1996, 19:91-100; hereby incorporated by reference). Analysis of amebic RNA showed that all three 170 kDa subunit genes were expressed in the amebae and that the abundance of the hgl message dropped as the amebae entered a stationary growth phase.
[0115] Accordingly, the present invention provides both nucleic acid and immunological reagents specific for 170 kDa subunits encoded by each of the hgl1, hgl2 or hgl3 genes. Also included are reagents which detect regions common to all three hgl genes and the nucleic acid and protein products of these regions. For example, oligonucleotide probes specific for any one of these three genes are readily identified by one of ordinary skill in the art, using conventional nucleic acid probe design principles, by comparisons of the three DNA sequences which are disclosed in FIG. 1A as the hgl1 gene (SEQ ID NO: 1), in FIGS. 4A-4F as the hgl3gene (SEQ ID NO:4), and, in Tannich, E. et al., (supra) (the hgl2 gene). Example VI illustrates the use of oligonucleotide probes specific for each of the three hgl genes, for determining the level of RNA expression from each gene by Northern analysis. Other methods of using hgl-specific nucleic acids diagnostically, for pathogenic and/or nonpathogenic forms of E. histolytica, are described in U.S. Pat. No. 5,260,429.
[0116] Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.
EXAMPLE I
Construction of Expression Vectors
[0117] The 170 kDa subunit of the galactose lectin is encoded by at least five genes. The DNA used for all of the constructions described herein encodes the 170 kDa lectin designated hgl1 (FIG. 1A, sheets 1-6)). The nucleotide position designations refer to the numbering in FIG. 1A. The DNA sequence encoding hgl1 was expressed in three portions (see FIG. 2B):
[0118] (1) fragment C (nucleotides 46-1833 of SEQ ID NO: 1) included (a) the cysteine- and tryptophan-rich region, (b) the cysteine-free region, and (c) 277 amino acids of the cysteine-rich domain, (residues 2-596 of SEQ ID NO:3);
[0119] (2) fragment A (nucleotides 1492-3460 of SEQ ID NO:1) encoded the majority of the cysteine-rich domain, (residues 482-1138 of SEQ ID NO:3);
[0120] (3) fragment B (nucleotides 3461-3892 of SEQ ID NO:1) included 70 amino acids of the cysteine-rich domain, the putative membrane-spanning region, and the cytoplasmic tail (residues 1139-1276 of SEQ ID NO:3).
[0121] Each of these three fragments was inserted in-frame by ligation into pGEX2T or pGEX3X to obtain these proteins as GST fusions. A schematic diagram of the vectors is shown in FIG. 2.
[0122] 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 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, B J et al. Proc Natl Acad Sci USA(1991) supra) 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 japonicumand 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, B J et al. Infec. Immun(1993) supra.
[0123] Expression of all constructs in the correct reading frame was verified by sequencing and by Western immunoblot analysis using anti-adhesion antisera. Expression of the hgl1 fusion proteins was inducible by IPTG. The GST protein produced from the original pGEX2T (molecular mass of 27.5 kDa) did not react with the anti-adhesion sera.
EXAMPLE II
Production of Recombinant Protein
[0124] The four vectors described above, as well as the host vector were transfected into competent E. colihosts. IPTG was used to induce expression of the genes encoding the fusion proteins. Production of the fusion proteins was determined by Western blot SDS-PAGE analysis of the lysates.
EXAMPLE III
Reactivity of Recombinant 170 kDa Subunit Fusion Proteins with mAbs
[0125] Induced cultures of bacterial strains expressing hgl1 fragment A, B, or C were harvested, lysed in sample buffer, and the lysate was applied to an SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to Immobilon and incubated with seven different anti-170-kDa mAbs, each specific for a different epitope. 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 of SEQ ID NO:3).
[0126] Fusion proteins B and C failed to react with any of the seven mAbs. Fusion protein A, representing positions 482-1132, reacted with all seven mAbs but not with a negative control mAb, MOPC21. The mAbs were used at 10 μg/ml and polyclonal antisera at 1:1000 dilution. These results indicated that all seven epitopes were contained within the 542 amino acids of the cysteine-rich extracellular domain of the 170 kDa subunit.
[0127] The generation of 3′ deletions by controlled ExoIII digestion of fragment A of the 170 kDa subunit is outlined in FIG. 3. Deletion Δ1 contains amino acid residues 482-1082; Deletion Δ2 contains amino acid residues 482-1032; deletion Δ3 contains amino acid residues 482-998. The positions refer to SEQ ID NO:3 but may be replaced by corresponding stretches from SEQ ID NO:5 or from the gene products of hgl2, hgl4 or hgl5. The reactivities of the fusion proteins that include fragment A or either of two C-terminal deletions (Δ3 and Δ4) with the seven mAbs (Table 1) were determined. Deletion Δ3 reacted with mAbs against epitopes 1, 2, 4, 5 and 7 but failed to react with mAbs recognizing epitopes 3 and 6; Deletion Δ4 (residues 498-894) reacted only with the mAb specific for epitope 5.
[0128] The five deletion derivatives of fusion protein A shown in FIG. 3, ranging in estimated size from 35 to 68 kDa, were tested for reactivity with each mAb as is summary in FIG. 3. The endpoints of the various deletions were determined by DNA sequencing with primers specific for the remaining hgl1 sequence. mAbs recognizing epitopes 1 and 2, which increase amebic adherence to target cells (“enhancing antibodies”), failed to react with recombinant 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 with deletion mutants lacking 895-998. The mAb specific for epitope 6, which has inhibitory effects on amebic adherence and abrogates lysis of amebae 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 the mAb specific for the neutral epitope 3. Finally, a construct containing amino acids 482 to 818 was recognized only by the mAb to epitope 5 (an adherence-inhibitory epitope). The predicted locations of the epitopes recognized by the 7 mAbs are listed in the right-most column of Table 1 above and are shown in FIG. 3.
EXAMPLE IV
Reactivity of 170 kDa Fusion Proteins with Human Immune Sera
[0129] Since the galactose adhesin 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 kDa subunit was undertaken. The recombinant fusion proteins and ExoIII-generated deletion constructs were tested for reactivity with pooled human immune sera in the same manner as described above 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. Human immune sera also reacted with deletion constructs Δ1, Δ2, and Δ3 but not with Δ4 or Δ10. Reactivity of immune sera with the different deletions localized major human B-cell epitopes to two primary sites: (a) within the first 482 amino acids and (b) between amino acids 895 and 1138 (FIG. 3). This second region is the site of 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.
[0130] Thus, for assays to detect human antibodies against E. histolytica, the useful epitope-bearing portions are those shown in Table 2.
[0131] The epitope-bearing fragments indicated can be used alone as fragments or as parts of a chimeric or a fusion protein, or any combination of the above peptide fragments can be used.
EXAMPLE V
Immunization Using Recombinant Subunit Protein
[0132] A GST fusion protein with fragment A was prepared in E. colias described in Example I. This peptide contains an upstream GST-derived peptide sequence followed by, and fused to, amino acids 432-1138 encoded by nucleotides 1492-3460 of SEQ ID NO: 1. The protein was produced intracellularly. The cells were harvested and lysed and the lysates subjected to standard purification techniques to obtain the purified fusion protein.
[0133] Gerbils were immunized by an intraperitoneal injection of 30 μg of purified fusion protein in complete Freund's adjuvant and then boosted at 2-4 weeks with 30 μg of the fusion protein in incomplete Freund's adjuvant. The gerbils were challenged at 6 weeks by intrahepatic injection of 5×105amebic trophozoites and were sacrificed 8 weeks later. The presence and size of amebic liver abscesses was determined.
[0134] The results of these two experiments are shown in Table 3, below. 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 immunized with the fusion protein. The administration of the fusion protein resulted in a statistically significant reduction in the size of abscesses.
EXAMPLE VI
Analysis of the Gene Family Encoding the 170 kDa Subunit of
[0135] This Example shows that the adhesin 170 kDa subunit of E. histolyticaHM-1 :IMSS strain is encoded by a gene family that includes hgl1, hgl2 and a previously undescribed third gene, herein designated hgl3. Since hgl1 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 hgl1, hgl2, and hgl3 were isolated and sequenced from the same lambda genomic library demonstrating unambiguously that hgl is a gene family.
[0136] Comparison of the amino acid sequences of the three 170 kDa subunit genes showed that the products of hgl1 and hgl2 are 89.2% identical, hgl1 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 C-terminal one-third of the molecule. Comparison of the amino acid sequences of the products of hgl2 and hgl3 revealed that 11 of the 19 nonconservative amino acid substitutions and 11 of the 13 residues inserted or deleted reside within the N-terminal 400 residues. A similar pattern of variation is present when hgl1- and hgl2-encoded proteins are compared. While the proteins of hgl1 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 hgl1 suggest that they may have arisen from a recent gene duplication event.
[0137] All 97 cysteine residues were conserved in the three genes. The hgl2 gene was originally reported to lack a single cysteine codon present in both hgl1 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 protein (FIG. 4) with the highest concentration in the cysteine-rich domain between residues 379-1210. All seven mAb-identified epitopes map to this region (Mann, B. J. et al. Infect Immun(1993) 61:1772-1778). As these mAbs 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.
[0138] The Northern hybridization results indicated that all three genes were expressed in the amebae. As the messages of hgl1-3 are predicted to comigrate at 4.0 kb, differential hybridization was required to ascertain expression of individual genes. Due to the high degree of sequence similarity between hgl1-3, relatively short oligonucleotides (17-21 bases) specific for regions where the three genes diverge were synthesized. 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 50° C. or less below the predicted Tm based upon nearest neighbor analysis. These precautions make unlikely the possibility of cross hybridization with other hgl gene members.
[0139] The Northern blots also indicated that mRNA from all three genes fell in abundance as the amebae progressed from logarithmic to stationary growth. This finding correlates with the observation that late logarithmic and stationary-phase amebae have decreased ability to adhere to, lyse, and phagocytose target cells (Orozco, E. et al. (1988) In: Amebiasis: Human infection by Entamoeba histolytica(Ravdin J. I., ed), pp. 326-338. John Wiley & Sons, Inc., New York.
[0140] Details of the experimental methods and results of the characterization of the hgl multigene family are presented below.
[0141] Library Screen. A lambda Zap® II library containing randomly sheared 4-5 kb fragments of genomic DNA from HM-1:IMSS strain E. histolyticawas 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. histolytica(Stratagene, La Jolla, Calif.). Duplicate plaque lifts, using Hybond-N membranes (Amersham, Arlington Heights, Ill.), were placed in a prehybridization solution consisting of 6× SSC (0.89 M sodium chloride and 90 mM sodium citrate), 5× Denhardt's solution, 0.5% SDS, 50 mM NaPO4(pH 6.7), and 100 μg/ml salmon sperm DNA for a minimum of 4 hours at 55° C. A 5′- and 3′-DNA fragment of hgl1 (nucleotides 106-1946 and 3522-3940 respectively) were labeled with [32P]dCTP (Amersham) using the Random Primed® DNA labeling kit according to the manufacturer's instructions (Boehringer Mannheim, Mannheim, Germany) and were hybridized separately to the membranes overnight at 55° C. in prehybridization solution. Membranes were rinsed once and washed once for 15 minutes at room temperature in 2× SSC, 0.1% SDS, then washed once for 15 minutes at room temperature, and twice at 55° C. for 20 minutes in 0.1× SSC, 0.1% SDS. Plaques that hybridized with the 5′- or the 3′-radiolabeled probe on both duplicate filters were isolated and purified.
[0142] Northern blots and hybridization. Total RNA was harvested from amebae using the guanidinium isothiocyanate method (RNagen®, Promega, Madison, Wis.). 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 25 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, N.Y.). The membrane was incubated in prehybridization solution and incubated at 37° C. for at least two hours. Oligonucleotides (18-22 nucleotides long) were end-labeled using polynucleotide kinase and [γ-32P]ATP (Sambrook, J. et al., supra), were added to the hybridization mixture and the membrane, and were incubated at 37° C. overnight. The membrane was then washed once at room temperature for 10 minutes, once at 37° C. for 10 minutes; and twice at 40-44° C. for 15 minutes each in 2× SSC, 0.1% SDS. The following radiolabeled probes were used (Plaimauer, B. et al. DNA Cell Biol(1993) 12:89-96):
[0143] Characterization of the hgl3 gene. The hgl3 open reading frame was 3876 bases (SEQ ID NO:4) with a predicted translation product of 1292 amino acids (SEQ ID NO:5) as shown in FIGS. 4A-4F. The predicted translation products of hgl1 and hgl2 are 1291 and 1285 amino acids long, respectively. A putative signal sequence and a transmembrane domain were identified in the amino acid sequence of hgl3 similar to hgl1 and hgl2. The N-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 hgl1 and hgl2 identified a large, conserved, extracellular region which was 11% cysteine, designated the “cysteine-rich domain” (Mann, B. J. et al. Parasit Today(1991) 7:173-176) (See FIGS. 3 an 6). Sequence analysis of hgl3 revealed that all 97 cysteine residues present within this region were also conserved in both of the previously reported 170 kDa subunit genes.
[0144] A schematic comparison (FIG. 6) of 170 kDa subunit gene sequences at the protein level revealed a high degree of sequence identity. However, seven sites, ranging from 3-24 nucleotides, were identified as resulting from an insertion or deletion, and which maintained the same open reading frame, in one subunit relative to another. Both hgl1 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. The comparison of hgl1 and hgl3 revealed two nonconservative substitutions, 57 conservative amino acid substitutions and 3 single residue insertion/deletions, making them 95.2% identical.
[0145] All 16 potential sites of glycosylation present in hgl1 were conserved in hgl3. A sequence analysis of hgl2 indicated that it shared only 9 of these sites. Glycosylation appears to account for approximately 6% of the apparent molecular mass of the 170 kDa subunit Mann, B. J. et al. Proc Natl Acad Sci USA(1991) 88:3248-3252).
[0146] All three 170 kDa 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 hgl1, hgl2, or hgl3 were hybridized to a Northern blot and identified an RNA band of the predicted size of 4.0 kb.
[0147] As the mRNAs of hgl1, hgl2 and hgl3 were predicted to comigrate at 4.0 kb, differential hybridization was required to ascertain expression of individual genes by Northern analysis. Due to the high degree of identity between the three hgl1 genes, 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 it was sufficiently divergent to prevent cross hybridization. Hybridization and washing conditions were highly stringent for such A/T rich probes, and hybridization was done at temperatures ≦5° C. below the predicted Tm based upon nearest neighbor analysis. While it is impossible to rule out cross hybridization with other hgl gene family members, these precautions significantly lowered the likelihood of such an event.
[0148] The abundance of message decreased significantly as the amebic trophozoites passed from logarithmic phase growth (lane A) to stationary growth (lane B) while expression of the control gene, EF-II, remained constant or increased slightly. This finding correlates with results indicating that late logarithmic and stationary phase amebae have a decreased ability to adhere to, lyse and phagocytose target cells (Orozco et al., supra).
[0149] Estimation of the number of 170 kDa subunit genes. The observations herein confirm that the adhesin 170 kDa subunit of HM-1 :IMSS strain E. histolyticais encoded by a gene family that includes hgl1, hgl2 and a previously undescribed third gene which is designated hgl3. Since hgl1 and hgl2 were originally sequenced, in part, from different cDNA libraries, it was possible that they represented polymorphs of a single gene. However, in the present work both 5′ and 3′ termini of hgl1, hgl2, and hgl3 were isolated and sequenced from the same lambda genomic library, demonstrating unambiguously that hgl is a gene family.
[0150] As stated above, Southern blot analyses and library screening data best support the conclusion of a gene family of three members. For Southern blots, two restriction enzymes, DdeI and HindIII, cut genomic DNA to completion and yielded analyzable restriction fragments. As the membrane was hybridized with a fragment of hgl1 corresponding to nucleotides 1556 to 3522, two bands of>976 and 1965 nucleotides should have been present from hgl3. This central hgl1 radioprobe would hybridize with three bands of 1158, 810 and >1080 nucleotides from hgl1 (SEQ ID NO: 1) and would hybridize with five bands of 819, 312, 55, 755, and >1080 nucleotides from hgl2. The Southern blot showed 7 bands for genomic DNA digested with DdeI, at sizes of 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 hgl1-3.
[0151] Because no HindIII restriction sites are within the coding region in hgl1, hgl2 and hgl3, each gene should be represented by a single band greater than 4.0 kb. The Southern blot showed three bands of 17500, 5600, and 4200 nucleotides. Should an additional 170 kDa subunit gene exist, its DdeI and HindIII fragments would need to comigrate with hgl1, hgl2 and hgl3 bands, and be so divergent that they would fail to hybridize with the hgl1 probe under very low stringency, or be too large to be resolved and transferred.
[0152] The genomic library was screened separately with a 5′ and a 3′ hgl specific probe, such that additional 170 kDa 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 3.2×108bases of genomic DNA in an organism with an estimated genome size of 107.5bases (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 seven clones identified with the 5′ 170 kDa subunit-specific probe, four contained inserts that matched the reported sequence for hgl1, two matched the sequence of hgl2, and one clone represented hgl3. Of eight clones obtained using the 3′ radiolabeled fragment, one matched the sequence for hgl1, five matched the sequence of hgl2, and two represented hgl3. These studies did not reveal any termini that did not match the sequence of hgl1, hgl2 or hgl3.
EXAMPLE VII
Oral Immunization with Salmonella Expressing the Gal/GalNAc Inhibitable Lectin of
[0153] An effective vaccine must be able to protect against both mucosal and systemic disease. In this study, an attenuated Salmonella strain that expressed a portion of the E. histolyticaGal/GalNAc lectin 170 kDa subunit was used to orally immunize gerbils. Animals were challenged by intrahepatic injection of amebic trophozoites. A significant decrease in size of amebic liver abscesses was observed in orally immunized animals. Oral immunization with a Salmonella-based vaccine was as effective as systemic immunization for protection against systemic challenge.
MATERIALS AND METHODS
Bacterial Strains, Plasmids, and Media
[0154] Salmonella dublinlive vaccine strain SL5928 aroA148 fliC(i)::Tn10 and S. typhimuriumLB5000 hsdSB121 leu-3121, were donated by B. Stocker, Stanford University (Newton S. M. et al., Science1989, 244: 70. The host strain for pGEX vectors (Pharmacia Biotech, Uppsala, Sweden) was Escherichia coliMC1061. JM109 was the host strain used to express proteins generated from pRSET vectors (Invitrogen, San Diego, Calif.). All bacterial strains were grown in Luria Broth (LB). The concentration of antibiotic, when added, was 50 μg/ml for ampicillin, or 40 μg/ml for spectinomycin. Plasmid pADE171, which encodes spectinomycin resistance (Hone D. et al., Microbial. Path.1988, 5, 407 kindly supplied by Eric Elsinghorst, Naval Medical Research Institute.
Expression and Purification of Recombinant Proteins
[0155] Recombinant lectin proteins encoded by pGEX plasmids were induced and expressed according to the procedure described by D. B. Smith and K. S. Johnson (Gene1988, 67, 31). Induction and expression recombinant proteins encoded by pRSET plasmids was carried out according to manufacturers instructions.
[0156] Recombinant proteins from induced bacterial cultures were separated by electrophoresis on 10% SDS polyacrylamide gel electrophoresis (PAGE) overnight at 40° C. Gels were briefly stained with 0.5% Coomassie Blue in acetic acid:isopropanol:water (1:3:6) and the recombinant protein band excised. The protein was eluted and concentrated from the gel using an Amicon centrilutor (Beverly, Mass.). Protein concentration was determined using BCA Protein Assay system (Pierce, Rockford, Ill.). The eluted protein was incubated with anti-lectin antibodies on immunoblots to confirm identity.
Immunoblotting
[0157] Proteins, separated by electrophoresis on 10% SDS-PAGE, were electrophoretically transferred to a PVDF Immobilon-P® membrane (Millipore Corp., Bedford, Mass.) at 40° C. for 2 h at 0.5 mA in 20% methanol-25 mM Tris base-192 mM glycine. After transfer, the membranes were blocked in 3% bovine serum albumin in blot wash buffer (BWB) (50 mM Tris base, 200 mM NaCl, 0.1% Tween 20) at 40° C. overnight. Membranes were incubated with pooled protein A-purified gerbil sera (50 μg/ml) or rabbit anti-lectin polyclonal antibody for 1 h at room temperature, washed, then incubated with protein A conjugated with horse radish peroxidase (HRP) for 1 h at room temperature. Bound protein A-HRP was visualized using chemiluminescent detection as recommended by the manufacturer (LumiGLO®, Kirkegaard & Perry Laboratories, Inc, Gaithersburg, Md.).
Construction of a Salmonella Strain Expressing Fragment A
[0158] A pADE171-based plasmid encoding fragment A, pADE-A, was constructed as follows: A DNA fragment containing the ptac promoter and fragment A of the hgl1 gene was first amplified by PCR using pGEX-A as a template (Mann B. J. et al., Infec. Immun.,1993, supra). The sequence of the two primers used corresponded to pGEX2T sequences located upstream of the ptac promoter or downstream of the 3′ end of fragment A, and included extra sequences that specified a Pst 1 site. The sequence of the sense primer was
[0159] and the sequence of the anti-sense primer was
[0160] PCR was performed using the conditions recommended by the manufacturer Perkin Elmer Cetus, Norwalk Conn.) and incubated for 30 cycles at 94° C. for 1 min, 48° C. for 1 min, 72° C. for 3 min. The reaction was preceded by a 5 min incubation at 94° C. and terminated with an incubation at 72° C. for 15 min. The PCR fragment was digested with Pst1 and ligated to Pst-digested pADE171. The recombinant plasmid was first selected in E. colistrain MC1061. The purified plasmid was then introduced into S. typhimuriumstrain LB5000 so that the plasmid DNA would be properly modified for introduction into Salmonella strains. Plasmid DNA isolated from LB5000 was introduced into S. dublinstrain SL5928 by electroporation. Expression of the lectin fragment encoded by pADE-A in SL5928 was verified by Western blots of bacterial lysates and anti-170 kDa lectin subunit antibody.
Immunization and Challenge Protocol
[0161] For i.p. immunizations animals were injected with 30 μg of electro-eluted fragments of the 170 kDa subunit or glutathione S-transferase (GST) in complete Freund's adjuvant. Four weeks later animals received booster immunizations with 30 μg of antigen in incomplete Freund's.
[0162] For oral immunization animals were fed 108-1010bacteria on days 1, 5 and 7 by oral gavage. Six weeks after the first immunization the animals were challenged by direct intrahepatic injection with 5×105amebic trophozoites (Chadee, K. et al., Am. J. Pathol.,1984, 117:71; Chadee, K., et al., Am. J. Trop. Med. Hyg.,1985, 34:283. Fourteen days post-challenge the gerbils were sacrificed and any liver abscess was dissected away from the remaining liver tissue and weighed. Each abscess was cultured for E. histolyticain TYI medium25. Results were analyzed using Student's t test.
RESULTS
Intraperitoneal Immunization with Purified 170 kDa Polypeptides
[0163] To test the ability of different regions of the 170 kDa subunit to elicit protection against amebic liver abscess formation portions of the 170 kDa subunit, fragment A (containing amino acids 482-1138 of the cysteine-rich region) and fragment C (amino acid s 2-596) were expressed in E. coliand purified as described herein. Gerbils were immunized by i.p. injection with the purified recombinant lectin polypeptides, then challenged by intrahepatic injection of amebic trophozoites and analyzed for the presence and size of an amebic liver abscess 14 days later. The results of three independent trials are presented in Table 4, below.
[0164] In Experiment 3, animals were immunized with a polyhistidine fusion version of fragment A that was encoded by an pRSET vector containing fragment A. In Experiment 2, animals were immunized with a combination of fragments A and C; however there was no significant additional decrease in liver abscess size compared to animals immunized with fragment A alone. Since the addition of fragment C did not enhance protection, further studies with Salmonella expression were confined to fragment A.
Construction of Salmonella Expressing the Gal/GalNAc Lectin
[0165] The first step in testing the effectiveness of an oral anti-amebic Salmonella vaccine was to create a stable recombinant strain expressing fragment A of the 170 kDa subunit. Initially a pGEX plasmid containing DNA encoding fragment A was introduced into Salmonella strains, however the stability of this plasmid was very low. To try to improve stability of expression, the DNA encoding fragment A was inserted into plasmid vector pADE171 (Hone et al., supra), creating pADE-A. This plasmid was introduced into S. dublinstrain SL5928 creating strain SL5928-A. The expression of fragment A in SL5928-A was verified by Western blots of bacterial lysates using anti-170 kDa subunit antibody. A strain carrying the parent plasmid, pADE171, was also created and designated SL5928-171.
[0166] To test the stability of the fragment A expression in SL5928-A in vitro, an overnight culture grown in the presence of antibiotic was diluted 1:500 in LB without antibiotic and grown for an additional 24 hours. This dilution procedure was repeated for 5 consecutive days using the previous day's culture. The percentage of spectinomycin-resistant bacteria was determined by plating the bacteria on LB and LB plus spectinomycin plates each day. The percentage of spectinomycin resistant bacteria dropped to 60% on day two and by day six it had dropped to 23%. Fragment A protein was detectable by immunoblotting in cultures grown up to four days without antibiotic.
[0167] In vivo stability of the expression of fragment A in 5928-A was tested by examining the bacteria isolated from the livers and spleens of gerbils that had been fed bacteria. Gerbils were fed 1010bacteria on days 1 and 5. On day 8, the gerbils were sacrificed and the spleens and livers harvested. The organs were homogenized in LB and spun at low speed to pellet cellular debris. The supernatants were plated on LB plus spectinomycin. Six out of six spectinomycin resistant bacteria tested expressed fragment A as measured on Westerns blots of total bacterial protein. The estimated titer of spectinomycin resistant bacteria isolated from the livers and spleens of immunized animals in this experiment was 3×102/ml.
Oral Immunization with Recombinant Salmonella Expressing the A Fragment
[0168] The ability of recombinant Salmonella expressing fragment A of the 170 kDa subunit to protection against amebic liver abscess formation by oral immunization was examined. Gerbils were immunized by oral gavage with the various strains of attenuated Salmonella. Animals were given 3 doses of bacteria on days 1, 5 and 7. Six weeks after the final dose animals were challenged with amebic trophozoites by intrahepatic injection and sacrificed 2 weeks later. In two independent experiments animals orally immunized with Salmonella expressing fragment A had a decrease in the size of liver abscess compared to animals immunized with the bacteria carrying the plasmid vector (Table 5).
Presence of Antibody in Immunized Animals
[0169] To determine if the oral immunization protocols used above induced anti-GalNAc lectin antibodies, serum samples were collected both before and after intrahepatic challenge with amebic trophozoites. Pooled protein A purified antisera from Experiment 5 were incubated with purified denatured Gal/GalNAc lectin immobilized to a PVDF membrane. Bound antibody was visualized with protein A labeled with horseradish peroxidase and a chemiluminescent substrate. No anti-lectin antibody could be detected in pre-challenge sera from animals orally immunized with SL5928-A. In addition no anti-lectin antibody could be detected in pre-challenge sera from SL5928-A immunized animals by ELISA. Anti-lectin antibody reactivity was observed in post-challenge sera from both SL5928-171 and SL5928-A immunized animals. It should be noted that pre-challenge sera from SL5928-A immunized animals did recognize total Salmonella proteins on Western blots whereas pre-immune gerbil sera did not.
DISCUSSION
[0170] This study demonstrated that oral administration of attenuated Salmonella, expressing a portion of the GalNAc lectin, can provide protection against amebic liver abscess formation in the gerbil model of amebiasis. A S. dublinstrain of bacteria was created that expressed a portion of the cysteine-rich region of the Gal/GalNAc lectin, designated fragment A. Protection against amebic liver abscess formation was observed in animals that had been orally immunized with fragment A-expressing bacteria. An oral vaccine for amebiasis has the potential to be more effective than a systemic vaccine since the primary site of protection against infection with E. histolyticais at the intestinal mucosal surface. Even though the mucosal immune response was not measured in this study, vaccination via the mucosal route was effective in preventing systemic disease.
[0171] Previous vaccine trials demonstrated that systemic immunization with this lectin conferred protection in the gerbil model of amebiasis. Petri and Ravdin first identified the Gal/GalNAc lectin as a protective antigen by i.p. immunization with native heterodimeric protein that had been purified using monoclonal antibody affinity chromatography (Petri W. A. Jr. and Ravdin J. I., Infect. Immun.1991, 59:97). Protection was observed in 67% of immunized animals. Zhang et al., Infect. Immun.1994, 62:2605, reported that i.p. immunization with a portion of the cysteine-rich region of the 170 kDa subunit conferred protection against liver abscess formation in gerbils with a vaccine efficacy of 81%. In a similar study Soong et al. (J. Inf. Dis.1995, 171:645) observed a significant number of gerbils protected against liver abscess formation with i.p. immunization with the cysteine-rich region. Their vaccine efficacy was 71% but there were no significant difference in abscess size in immunized unprotected animals when compared to controls. The cysteine-rich region used by Zhang et al. corresponds to amino acids 649-1201. Soong et al. used amino acids 767-1138, while amino acids 483-1138 of the cysteine-rich region were used in the present example. The lack of complete protection observed in these experiments and the inability to detect antibody may be due to the low level of infectivity of the bacteria in the spleen and liver and/or instability of the plasmid. The plasmid appeared to be somewhat unstable in culture and the number of spectinomycin resistant bacteria recovered from the liver and spleen was low. However, all of the spectinomycin resistant bacteria isolated from the spleen and liver that were tested still expressed fragment A, indicating that the plasmid was not substantially rearranged.
[0172] The cysteine-rich region of the 170 kDa subunit has been shown to be the immunologically reactive portion of the protein. Six different mAbs that recognize functionally distinct epitopes located within a portion of the cysteine-rich region containing amino acids 597-1138 were shown to neutralize t he virulence traits of adherence, contact-dependent cytolysis, and amebic resistance to lysis by complement components C5b-9 (see above and Mann B. J. et al., 1993, supra. This region has also been implicated in stimulating cell mediated immune responses. The lectin has been shown to stimulate TNF-α in naive macrophages and to stimulate nitric oxide production and amebicidal activity by activated macrophages in vitro (Seguin R. et al., Proc. Natl. Acad. Sci. USA1996, 92:12175). This stimulatory activity was blocked by some of the mAbs that recognize amino acids 597-1138 of the cysteine-rich region. The cysteine-rich region and peptides derived from this region have also been shown to cause proliferation of lymphocytes from patients recovered from amebic disease (C. Velasquez et al., J. Euk. Microbiol.,1995, 42:636).
[0173] Anti-lectin antibodies were not detected by Western blots of reduced lectin in pre-challenge immune sera from gerbils orally immunized with Salmonella expressing fragment A. The lack of an antibody response to antigens introduced by oral immunization with recombinant strains of Salmonella has been observed for several other antigens (Sadoff, J. C. et al., Science,1988, 240:336; Fouts, R. R. et al., Vaccine,1995, 17:1697. These results suggest that a humoral response was not primarily responsible for protection in this model system. Previous vaccine trials have found no correlation between pre-challenge antibody titer and protection (Petri & Ravdin, supra; Soong et al., supra). However the inability to detect anti-lectin antibody could have been due to the protein A purification of the gerbil sera. Protein A purification was found to be necessary in order to reduce the background and visualize a specific signal. Protein A preferentially binds to specific antibody isotypes and isotype binding can also vary depending upon the animal species. If the response was primarily of antibody isotype that did not bind well to protein A these antibodies would have been lost during purification and therefore go undetected. Improved methods for detecting gerbil antibodies should alleviate this problem. The nature of the protective immune response in the gerbil model still requires further resolution.
[0174] The foregoing study therefore demonstrated that oral immunization with Salmonella expressing an immunogenic fragment of the Gal/GalNAc lectin of E. histolyticamay be the basis of a protective vaccine strategy.
EXAMPLE VIII
Identification of an Adherence and Cytotoxicity Inhibitory Epitope of the
[0175] In order to identify a region of the lectin which binds only adherence inhibitory antibodies, the present inventors expressed a 312 bp region of the 170 kDa subunit DNA encoding amino acids 896-998 as a recombinant GST fusion protein in E. coli. This region has been designated Δ3′. The 895-998 domain was tested with anti-adhesin mAbs. On Western blots, only mAb 8C12 which is directed against amino acids 895-998 recognized Δ3′. This mAb was previously shown to inhibit both adherence and cytotoxicity.
[0176] Serodiagnostic studies were conducted using sera from patients with current or past amebic infection and compared to control sera. Using ELISA with immobilized Δ3′ protein, 11 of 13 patients with current amebiasis had increased levels of anti-Δ3′ antibodies. On Western blots, similar results were seen with 7 of 10 sera from patients with current infection reacting and with Δ3′, whereas sera from past infections and negative control sera did not react with Δ3′.
[0177] The specificity of sugar binding of purified lectin (intact 170 kDa) and the Δ3′ fragment were tested by allowing these substances to bind to 125I-GalNAc20BSA in the presence of competing monosaccharides. In this experiment, PVDF membranes were spotted with purified lectin or fragment. The strips were blocked with 10 mg/ml BSA in PBS for 1 hr and then incubated in 1 mg/ml BSA/PBS containing 106cpm of the radioiodinated GalNAc-BSA and either glucose or galactose as inhibitor. After 1 hr, strips were washed three times for 5 minutes each in 1 mg/ml BSA/PBS. All incubations and washes were at room temperature. Air dried strips were exposed to a phosphorimager screen for 48 hrs.
[0178] The results in FIG. 7 show that both the fill length lectin and the specific fragment share Gal/GalNAc-binding activity which was specific (not inhibited by glucose). Thus, the binding specificity of the lectin is possessed by this short fragment.
[0179] In summary, then the Δ3′ domain of the E. histolyticalectin contains an adherence and cytotoxicity inhibitory epitope as well as the sugar-binding specificity of the lectin. Furthermore, Δ3′ is a marker for current invasive amebiasis. This peptide would therefore be particularly useful in a vaccine composition as described herein.
[0180] The references cited above are all incorporated by reference herein, whether specifically incorporated or not.
[0181] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
[0182] While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.
Claims
What is claimed is:
1. A recombinant, nonglycosylated, epitope-bearing peptide of the 170 kDa subunit of E. histolyticaGal/GalNac adherence lectin, which peptide bears at least one epitope that reacts with antibodies made in a subject infected with E. histolyticainfection or immunized with said adherence lectin or an epitope-bearing portion thereof, with the proviso that said peptide is not
(i) the full length 170 kDa subunit, or
(ii) amino acid sequence residues 480-1138 of SEQ ID NO:3.
2. A peptide according to claim 1 produced in prokaryotic cells.
3. A peptide according to claim 1 which binds to Gal or GalNAc.
4. A peptide according to claim 1 encoded by one, two or all of the hgl1 gene, the hgl2 gene and the hgl3 gene.
5. A vaccine composition for immunizing a subject against E. histolyticainfection comprising one or more peptides according to claim 1 and a pharmaceutically acceptable vehicle or carrier.
6. A vaccine composition for immunizing a subject against E. histolyticainfection comprising one or more peptides according to claim 2 and a pharmaceutically acceptable vehicle or carrier.
7. A vaccine composition for immunizing a subject against E. histolyticainfection comprising a pharmaceutically acceptable vehicle or carrier and one or more peptides having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5, said peptide being selected from the group consisting of:
(a) residues 895-998 or a corresponding peptide of a naturally occurring variant of the 170 kD subunit of E. histolyticaGal/GalNac adherence lectin encoded by an hgl gene of any strain of E. histolytica;
(b) residues 946-970, or a corresponding peptide of a naturally occurring variant of the 170 kD subunit of E. histolyticaGal/GalNac adherence lectin encoded by an hgl gene of any strain of E. histolytica;
(c) residues 976-1000, or a corresponding peptide of a naturally occurring variant of the 170 kD subunit of E. histolyticaGal/GalNac adherence lectin encoded by an hgl gene of any strain of E. histolytica;
(d) residues 991-1015, or a corresponding peptide of a naturally occurring variant of the 170 kD subunit of E. histolyticaGal/GalNac adherence lectin encoded by an hgl gene of any strain of E. histolytica;
(e) residues 1006-1030, or a corresponding peptide of a naturally occurring variant of the 170 kD subunit of E. histolyticaGal/GalNac adherence lectin encoded by an hgl gene of any strain of E. histolytica; and
(f) residues 1036-1060, or a corresponding peptide of a naturally occurring variant of the 170 kD subunit of E. histolyticaGal/GalNac adherence lectin encoded by an hgl gene of any strain of E. histolytica.
8. A vaccine composition according to claim 7, wherein said peptide binds to Gal or GalNAc.
9. A vaccine composition according to claim 7, wherein said peptide has the amino acid sequence of residues 895-998 or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica.
10. A vaccine composition for immunizing a subject against E. histolyticainfection comprising one or more peptides according to claim 4 and a pharmaceutically acceptable vehicle or carrier.
11. A vaccine composition according to claim 5, 6, 7, 8, 9 or 10, further comprising an adjuvant or other immune stimulating agent.
12. A method for immunizing a subject against Entamoeba histolyticainfection which method comprises administering to said subject an effective amount of a vaccine composition according to claim 5.
13. A method for immunizing a subject against Entamoeba histolyticainfection which method comprises administering to said subject an effective amount of a vaccine composition according to claim 6.
14. A method for immunizing a subject against Entamoeba histolyticainfection which method comprises administering to said subject an effective amount of a vaccine composition according to claim 7.
15. A method for immunizing a subject against Entamoeba histolyticainfection which method comprises administering to said subject an effective amount of a vaccine composition according to claim 8.
16. A method for immunizing a subject against Entamoeba histolyticainfection which method comprises administering to said subject an effective amount of a vaccine composition according to claim 9.
17. A method for immunizing a subject against Entamoeba histolyticainfection which method comprises administering to said subject an effective amount of a vaccine composition according to claim 10.
18. A method for immunizing a subject against Entamoeba histolyticainfection which method comprises administering to said subject an effective amount of a vaccine composition according to claim 11.
19. A peptide according to claim 1, which is useful in a diagnostic, a vaccine or a tolerogenic composition, which peptide has an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5 selected from the group consisting of:
(a) residues 596-1138, or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica;
(b) residues 895-998 or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica, and
(c) residues 946-970, or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica;
(d) residues 976-1000, or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica;
(e) residues 991-1015, or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica;
(f) residues 1006-1030, or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica;
(g) residues 1036-1060, or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica;
(b) residues 1033-1082, or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica;
(i) residues 1082-1138, or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica; and
(j) residues 443-461 of hgl 1 (SEQ ID NO:2), or a corresponding peptide of a naturally occurring variant of said 170 kD subunit encoded by an hgl gene of any strain of E. histolytica.
20. A vaccine composition for immunizing a subject against E. histolyticainfection comprising a fusion protein that includes the peptide of claim 1.
21. A vaccine composition for immunizing a subject against E. histolyticainfection comprising a fusion protein that includes a peptide according to claim 19.
22. The peptide of claim 19 which comprises residues 895-998 of SEQ ID NO:2 or SEQ ID NO:5.
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