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WO1995000849A1 - Lectine recombinee a sous-unite de 170 kd d'entomoeba histolytica et ses procedes d'utilisation - Google Patents

Lectine recombinee a sous-unite de 170 kd d'entomoeba histolytica et ses procedes d'utilisation Download PDF

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Publication number
WO1995000849A1
WO1995000849A1 PCT/US1994/006890 US9406890W WO9500849A1 WO 1995000849 A1 WO1995000849 A1 WO 1995000849A1 US 9406890 W US9406890 W US 9406890W WO 9500849 A1 WO9500849 A1 WO 9500849A1
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WIPO (PCT)
Prior art keywords
epitope
subunit
bearing portion
histolytica
amino acids
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PCT/US1994/006890
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English (en)
Inventor
Barbara J. Mann
William A. Petri
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University Of Virginia Patent Foundation
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Publication date
Application filed by University Of Virginia Patent Foundation filed Critical University Of Virginia Patent Foundation
Priority to AU71123/94A priority Critical patent/AU710439B2/en
Priority to US08/569,214 priority patent/US6165469A/en
Priority to EP94920262A priority patent/EP0708923A4/fr
Priority to JP7502991A priority patent/JPH09502165A/ja
Publication of WO1995000849A1 publication Critical patent/WO1995000849A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention concerns the use of epitope- bearing regions of the 170 kD subunit of Entamoeba histolytica Gal/GalNAc adherence lectin which are produced recombinantly in procaryotic systems in diagnosis and as vaccines.
  • the invention relates to the determination of the presence, absence or amount of antibodies raised by a subject in response to infection by E. histolytica using these peptides and to vaccines incorporating them.
  • This invention also particularly relates to reagents specific for a novel variant of the 170 kD subunit of E. histolytica Gal/GalNAc adherence lectin and to the gene (hgl3 ) which encodes this novel subunit form, which represents the third member of the multigene family encoding this 170 kD subunit.
  • Entamoeba histolytica infection is extremely common and affects an estimated 480 million individuals annually. However, only about 10% of these persons develop symptoms such as colitis or liver abscess. The low incidence of symptom occurrence is putatively due to the existence of both pathogenic and nonpathogenic forms of the amoeba. As of 1988, it had been established that the subjects who eventually exhibit symptoms harbor pathogenic "zymodemes" which have been classified as such on the basis of their distinctive hexokinase and phosphoglucomutase isoenzy es.
  • the pathogenic forms are not conveniently distinguishable from the nonpathogenic counterparts using morphogenic criteria, but there is an almost perfect correlation between infection with a pathogenic zymodeme and development of symptoms and between infection with a nonpathogenic zymodeme and failure to develop these symptoms.
  • E. histolytica infection is mediated at least in part by the "Gal/GalNAc" adherence lectin which was isolated from a pathogenic strain and purified 500 fold by Petri, .A. , et al. , J Biol Chem (1989) 264:3007-3012.
  • the purified "Gal/GalNAc” lectin was shown to have a nonreduced molecular weight of 260 kD on SDS-PAGE; after reduction with beta-mercaptoethanol, the lectin separated into two subunits of 170 and 35 kD MW.
  • Further studies showed that antibodies directed to the 170 kD subunit were capable of blocking surface adhesion to test cells (Petri, et al. J Biol Chem (1989) supra) . Therefore, the 170 kD subunit is believed to be of primary importance in meditating adhesion.
  • the 170 kD subunit is described as constituting an effective vaccine to prevent E. histolytica infection in U.S. Patent 5,004,608 issued 2 April 1991.
  • DNA encoding both the heavy (170 kD) and light (35 kD) subunits have been cloned.
  • the heavy and light subunits are encoded by distinct mRNAs (Mann, B., et al. , Proc Natl Acad Sci USA (1991) . 38 :3248-3252) and these subunits have different amino acid compositions and amino terminal sequences.
  • the sequence of the cDNA encoding the 170 kD subunit suggests it to be an integral membrane protein with a large cysteine-rich extracellular domain and a short cytoplasmic tail (Mann, B., et al. , Proc Natl Acad Sci USA (1991) supra; Tannich, et al.
  • the derived amino acid sequence of the 170 kD lectin shows that the extracellular domain can be divided into three regions on the basis of amino acid composition.
  • the amino terminal amino acids 1-187 are relatively rich in cysteine (3.2%) and tryptophan (2.1%) .
  • Amino acid sequence at positions 188-378 does not contain cysteine, and the amino acid sequence at positions 379-1209 contains 10.8% cysteine residues.
  • the obtention of clones encoding the heavy chain subunit is further described in U.S. Patent 5,260,429 issued 9 November 1993, the disclosure of which is incorporated herein by reference. In that patent, diagnostic methods for the presence of E. histolytica based on the polymerase chain reaction and the use of DNA probes is described.
  • the heavy subunit is considered to be encoded by a multigene family (Mann, B., et al. , Parasit Today (1991) 1:173-176) .
  • Two different heavy subunit genes, hgll and hgl2 have been sequenced by separate laboratories. While hgll was isolated from an HM-1:IMSS cDNA library in its entirety (Tannich, E. et al. Proc Natl Acad Sci USA (1991) £8:1849-1853), hgll was isolated in part from an H-302:NIH cDNA library and in part by PCR amplification of the gene from the HM-1:IMSS genome (Mann, B.J. et al .
  • Patent 5,272,058 issued 21 December 1993, the disclosure of which is incorporated herein by reference in its entirety.
  • This application also describes use of these antibodies to detect the 170 kD heavy chain and the use of the 170 kD subunit to detect antibodies in serum or other biological samples.
  • the experimental work described utilizes the native protein. Further characterization of these antibodies is described in a publication by Mann, B.J., et al. , Infect Immun (1993) .61:1772-1778 also incorporated herein by reference.
  • amebic serology is also a critical component in the diagnosis of invasive amebiasis.
  • One approach utilizes conventional serologic tests, such as the indirect hemagglutinin test. These tests are very sensitive but seropositivity is persistent for years (Krupp, I.M., Am J Trop Med Hv ⁇ (1970) l£:57-62; Lobel, H.O. et al., Ann Rev Microbiol (1978) 32 . :379-347) .
  • healthy subjects may give positive responses to the assay, creating an undesirable high background.
  • the present invention provides such a test by utilizing, as antigen, epitope-bearing portions of the 170 kD subunit of the adherence lectin produced recombinantly in procaryotic systems.
  • the 170 kD subunit may be used as a vaccine as described in the above-referenced U.S. Patent 5,004,608, recombinantly produced forms of the 170 kD subunit, specifically those obtained from procaryotic cells that lack glycosylation may offer advantages in reproducibility of product and in ease of preparation of subunit vaccines.
  • the present invention is directed to this desirable result.
  • the invention provides diagnostic tests which permit the assessment of patients for invasive E. histolytica infection and vaccines for prevention of infection.
  • the invention also provides a novel third variant of the 170 kD subunit of the Gal/GalNAc adherence lectin and a gene (hgrl3) which encodes this novel protein. Accordingly, the diagnostic tests of the invention are based on the genetic sequences of all three variants of the 170 kD subunit of the Gal/GalNAc adherence lectin which are encoded by three different genes in a multigene family.
  • Pathogenic and nonpathogenic strains can be distinguished by use of the invention diagnostic method, if desired.
  • the tests use, as antigen, an epitope- bearing portion of the 170 kD subunit of the Gal/GalNAc adherence lectin recombinantly produced in procaryotic systems. Despite the absence of glycosylation from such portions and despite the lack of post-translational modifications characteristic of the native protein or peptide, the recombinantly produced proteins are effective antigens in these assays.
  • the invention is directed to a method to detect the presence or absence of antibodies immunoreactive with pathogenic and/or nonpathogenic E. histolytica in a biological sample which method comprises contacting the fluid with an epitope- bearing portion of the 170 kD heavy chain of the Gal/GalNAc adherence lectin wherein the lectin is nonglycosylated and in a form obtainable from procaryotic cells.
  • the portion may be chosen so as to be characteristic of the pathogenic or nonpathogenic form.
  • the assay may be conducted as a competition assay using MAbs with such characteristics.
  • the contacting is conducted under conditions where the epitope-bearing portion forms complexes with any antibodies present in the biological fluid which are immunoreactive with an epitope on the portion.
  • the presence, absence or amount of such complexes is then assessed, either directly or in a competition format, as a measure of the antibody contained in the biological sample.
  • the invention is also directed to materials and kits suitable for performing the methods of the invention.
  • the invention is directed to methods to prevent E. histolytica infection using vaccines containing, as active ingredient, epitope- bearing portions of the 170 kD subunit produced recombinantly in procaryotic systems, as described above.
  • the invention is also directed to vaccines containing this active ingredient.
  • the invention is directed to epitope-bearing portions of the 170 kD subunit produced recombinantly in procaryotic systems and thus in a form characteristic of such production.
  • One characteristic is lack of glycosylation; in addition, secondary structure of proteins produced by procaryotic hosts differs from that of proteins produced by the natural source.
  • the invention is directed to a DNA in purified and isolated form which consists essentially of a DNA encoding the 170 kd heavy chain subunit of pathogenic E. histolytica Gal/GalNAc adherence lectin, which subunit is encoded by the hg!3 gene for which the nucleotide sequence and deduced amino acid sequence are shown in Figure 4.
  • the invention is directed to both nucleic acid and immunological reagents which are enabled by the discovery of the hgl3 gene, reagents which are specific for each of the hgll , hgl2 or hgl3 genes, as well as reagents which detect common regions of all three hgl genes or their nucleic acid or protein products.
  • oligonucleotide probes specific for any one of these three genes or for a sequence common to all three genes may be identified by one of ordinary skill in the art, using conventional nucleic acid probe design principles, by comparisons of the three DNA sequences for these genes. See Example 6.
  • the invention is directed to a method to detect the presence, absence, or amount of a pathogenic or nonpathogenic form of Entamoeba histolytica, where E. histolytica has both pathogenic and nonpathogenic forms, in a biological sample, which method comprises contacting the sample with a monoclonal antibody immunospecific for an epitope of the 170 kd subunit of Gal/GalNAc lectin unique to the pathogenic or to the nonpathogenic form, or shared by the pathogenic and nonpathogenic forms of E. histolytica, to form an immunocomplex when the pathogenic and/or nonpathogenic form is present, and detecting the presence, absence or amount of the immunocomplex.
  • the epitope is selected to be specific for one of 170 kD subunits encoded by the hgll , hgl2 or hgl3 genes, or for a common region of the subunits from all three hgl genes.
  • the invention is directed to a method to determine the presence, absence or amount of antibodies specifically immunoreactive with the Gal/GalNAc lectin derived from E.
  • the histolytica which method comprises contacting a biological sample with the Gal/GalNAc lectin or the 170 kd subunit thereof in purified and isolated form, under conditions wherein antibodies immunospecific for said lectin or subunit will forma complex, and detecting the presence, absence or amount of the complex, wherein the purified and isolated Gal/GalNAc lectin or subunit is derived from either a pathogenic or nonpathogenic form of E. histolytica, and is a 170 kD subunit encoded by one of the hgll , hgl2 or gl3 genes. Detailed descriptions of these and related methods for detecting pathogenic or nonpathogenic forms of E.
  • Figure 1A shows the DNA and amino acid sequence deduced from the nucleotide sequence corresponding to the 170 kD heavy chain of the adherence lectin from pathogenic strain HM1:IMSS, designated hgll .
  • Figure IB shows the deduced amino acid sequence of hgll with the amino-terminal amino acid of the mature protein designated as amino acid number 1.
  • Figure 2A is a diagram of the construction of expression vectors for recombinant production of specified portions of the 170 kD subunit;
  • Figure 2B shows the pattern of deletion mutants.
  • Figure 3 is a diagram of the location of human B cell epitopes and pathogenic-specific epitopes on the 170 kD heavy chain.
  • Figure 4A shows the DNA and amino acid sequence deduced from the nucleotide sequence corresponding to the 170 kD heavy chain of the adherence lectin from pathogenic strain HM1:IMSS, designated hgl3 .
  • Figure 4B shows the deduced amino acid sequence of hgl3 with the amino-terminal amino acid of the mature protein designated as amino acid number 1.
  • the putative signal sequence and transmembrane domains are overlined and underlined respectively. conserveed cysteine residues (•) and potential sites of glycosylation (*) are indicated.
  • Figure 5 shows in schematic form a comparison of amino acid sequences of three heavy subunit genes.
  • the top diagram represents a schematic representation of a heavy subunit gene. Starting at the amino terminus, regions include the cysteine/tryptophan (C-W) rich domain, the cysteine-free (C-free) domain, the cysteine- rich (C-rich) domain, and the putative transmembrane (TM) sequence and cytosolic domains (Mann, B.J. et al . Parasit Today (1991) 2:!73-176) . Amino acid sequence comparisons of hgll , hgl2 and hgl3 are shown.
  • Upright lines indicate nonconservative amino acid substitutions in the amino acid sequence of the second gene as compared to the first gene listed to the right.
  • Downward arrowheads indicate a deletion while upright arrowheads indicate an insertion.
  • the number of residues inserted or deleted are listed below the arrowheads and the total percent amino acid sequence identity is listed at right.
  • the invention provides methods and materials which are useful in assays to detect antibodies directed to pathogenic and/or nonpathogenic forms of E. histolytica and in vaccines.
  • the diagnostic assays can be conducted on biological samples derived from subjects at risk for infection or suspected of being infected.
  • the assays can be designed to distinguish pathogenic from nonpathogenic forms of the amoeba if desired.
  • the vaccines are administered to subjects at risk for amebic infections.
  • the assays of the invention rely on the ability of an epitope-bearing portion of the 170 kD subunit produced recombinantly in procaryotic cultures to immunoreact with antibodies contained in biological samples obtained from individuals who have been infected with E. histolytica .
  • the relevant peptide or protein is produced in a procaryotic system, and is thus not glycosylated or processed after f :nslation in a manner corresponding to the native protein, the epitope- bearing portions thus prepared are useful antigens in immunoassays performed on samples prepared from biological fluids, cells, tissues or organs, or their diluted or fractionated forms. Similarly, these peptides are also immunogenic.
  • recombinant production facilitates the preparation of specific epitopes, thus providing a means for detecting antibodies specifically immunoreactive with pathogenic or nonpathogenic forms of the amoeba, as well as offering the opportunity to provide subunit vaccines.
  • the invention is directed to methods to detect antibodies in biological samples and to immunize subjects at risk using these recombinantly produced epitope-bearing portions as antigens or immunogens as well as to the recombinantly produced peptides themselves and to materials useful in performing the assays and in administering the vaccines.
  • the diagnostic assays may be designed to distinguish antibodies raised against nonpathogenic or pathogenic forms of the amoeba.
  • “Pathogenic forms” of E. histolytica refers to those forms which are invasive and which result in symptomology to infected subjects.
  • “Nonpathogenic forms” refers to those forms which may be harbored asymptomatically by carriers.
  • the assays and vaccines of the invention utilize an epitope-bearing portion of the 170 kD subunit of the Gal/GalNAc lectin.
  • Gal/GalNAc lectin refers to glycoprotein found on the surface of E. histolytica which mediates the adherence of the amoeba to target cells, and which mediation is inhibited by galactose or N- acetylgalactosamine.
  • the Gal/GalNAc lectin refers specifically to the lectin reported and isolated by Petri, et al. (supra) from the pathogenic strain HMI- IMSS, and to the corresponding lectin found in other strains of E. histolytica .
  • the "170 kD subunit” refers to the large subunit, upon reduction of the Gal/GalNAc lectin, such as that obtained by Petri, et al. and shown in Figure 1 as well as to its corresponding counterparts in other strains.
  • the complete 170 kD antigen or an epitope- bearing portion thereof can be used in the assays.
  • Such epitope-bearing portions can be selected as characteristic of pathogens or nonpathogens or common to both.
  • the portion of the 170 kD protein which contains epitopes for all monoclonal antibodies prepared against the lectin is found at amino acid positions 596-1138. There appears to be an epitope characteristic of pathogens between each of amino acid positions 596-818, 1082-1138, and 1033-1082. Positions 895-998 contain epitopes which are shared by pathogens and nonpathogens as well as epitopes characteristic of pathogenic strains.
  • a peptide representing positions 596-818, 1033-1082 or 1082-1138 may be used to detect antibodies raised against pathogens by hosts in general; however, the epitope at positions 596-816 is not recognized by human antisera. Mixtures of these peptides could also be used. Alternatively, longer forms of the antigen can be used by selecting the appropriate positions depending on whether pathogenic and nonpathogenic amoebae are to be distinguished. As shown in Example 4, below, epitope-bearing portions relevant for human testing include portions 2- 482, 1082-1138, 1032-1082 and 894-998.
  • epitope- bearing portions may be used as single peptides, as uniquely lectin-derived portions of chimeric proteins, as mixtures of peptides or of such proteins, or as portions of a single, multiple-epitope-bearing protein. Procedures for preparing recombinant peptide proteins containing only a single epitope-bearing portion identified above, or multiples of such portions
  • the assays are designed to detect antibodies in biological samples which are "immunospecific” or “immunoreactive” with respect to the epitope-bearing portion -- i.e. with respect to at least one epitope contained in this portion.
  • immunospecific or “immunoreactive” with respect to a specified target means that the antibody thus described binds that target with significantly higher affinity than that with which it binds to alternate haptens.
  • the degree of specificity required may vary with circumstances, but typically an antibody immunospecific for a designated target will bind to that target with an affinity which is at least one or two, or preferably several orders or magnitude greater than with which it binds alternate haptens.
  • the assays can be performed in a wide variety of protocols depending on the nature of the sample, the circumstances of performing the assays, and the particular design chosen by the clinician.
  • the biological sample is prepared in a manner standard for the conduct of immunoassays; such preparation may involve dilution if the sample is a biological fluid, fractionation if the sample is derived from a tissue or organ, or other standard preparation procedures which are known in the art.
  • biological sample refers to the sample actually used in the assay which is derived from a fluid, cell, tissue or organ of a subject and prepared for use in the assay using the standard techniques. Normally, plasma or serum is the source of biological sample in these assays.
  • the assays may be conducted in a competition format employing a specific binding partner for the epitope-bearing portion.
  • specific binding partner refers to a substance which is capable of specific binding to a targeted substance, such as the epitope-bearing portion of the 170 kD subunit.
  • a specific binding partner will be an antibody, but any alterative substance capable of such specific binding, such as a receptor, enzyme or arbitrarily designed chemical compound might also be used.
  • antibody refers not only to immunoglobulin per se, but also to fragments of immunoglobulin which retain the immunospecificity of the complete molecule.
  • antibody also includes not only native forms of immunoglobulin, but forms of the immunoglobulin which have been modified, as techniques become available in the art, to confer desired properties without altering the immunospecificity. For example, the formation of chimeric antibodies derived from two species is becoming more practical. In short, “antibodies” refers to any component of or derived form of an immunoglobulin which retains the immunospecificity of the immunoglobulin per se.
  • a particularly useful form of specific binding reagents useful in the assay methods of the invention is as monoclonal antibodies.
  • Three categories of monoclonal antibodies have been prepared to the 170 kD subunit.
  • One category of antibody is immunospecific for epitopes "unique" to pathogenic forms. These antibodies are capable, therefore, of immunoreaction to a significant extent only with the pathogenic forms of the amoeba or to the 170 kD subunit of lectin isolated from pathogenic forms.
  • a second set of monoclonal antibodies is immunoreactive with epitopes which are "unique" to nonpathogenic forms. Thus, these antibodies are immunoreactive to a substantial degree only with the nonpathogenic amoeba or their lectins and not to the pathogenic forms.
  • a third category of monoclonal antibodies is immunoreactive with epitopes common to pathogenic and nonpathogenic forms and these antibodies are capable of immunoreaction with the subunit or with the amoeba regardless of pathogenicity.
  • those immunoreactive with epitopes 1 and 2 of the 170 kD subunit isolated from the pathogenic- strain exemplified are capable of reacting, also, with the corresponding epitopes on nonpathogens.
  • those immunoreactive with epitopes 3-6 are capable of immunoreaction only with the 170 kD subunit of pathogenic strains.
  • a 170 kD subunit By applying the techniques for isolation of the pathogenic 170 kD subunit to amoeba which are nonpathogenic, a 170 kD subunit can be obtained for immunization protocols which permit the analogous preparation of MAbs immunoreactive with counterpart epitopes 3-6 in the nonpathogenic forms.
  • the biological sample is contacted with the epitope-bearing portion used as an antigen in the immunoassay.
  • the presence, absence or amount of the resulting complex formed between any antibody present in the sample and the epitope-bearing portion is measured directly or competitively.
  • the epitope- bearing portion provided as antigen may be coupled to a solid support, either by adsorption or by covalent linkage, and treated with the biological sample. The ability of any antibodies in the sample to bind to coupled antigen is then determined.
  • the antigen may be supplied as a band on a polyvinylidene difluoride (PVDF) and contacted with the biological sample; any resulting complexes formed with antibody on the PVDF membrane are then detected as described above for Western blot procedure.
  • PVDF polyvinylidene difluoride
  • This protocol is substantially a Western Blot procedure.
  • microtiter plates or other suitable solid supports may be used.
  • the binding of antibody to the antigen coupled to support can then be detected as described above for Western blot procedure using conventional techniques generally involving secondary labeling using, for example, antibodies to the species from which the biological sample is derived.
  • Such labels may include radioisotopes, fluorescent tags, enzyme labels and the like, as is conventionally understood.
  • the assay may also be formatted as a competition assay wherein the antigen coupled to solid support is treated not only with the biological sample but also with competing specific binding partner immunospecific for at least one epitope contained in the antigen.
  • the competing binding partner is preferably an antibody.
  • the competing antibody may be polyclonal or monoclonal and may itself be labeled or may be capable of being labeled in a secondary reaction.
  • a competitive specific binding partner for the antigen is generally supplied in labeled form and the success of the competition from the biological sample is measured as a reduction in the amount of label bound in the resulting complex or increased levels of label remaining in the supernatant.
  • the assay can readily be made specific for pathogenic or nonpathogenic reacting antibodies, if desired, by choosing antibodies of the appropriate specificity.
  • the competition will be provided by a monoclonal antibody specific for an epitope characteristic of pathogenic strains.
  • the assay may be made specific for pathogenic or nonpathogenic forms is in the choice of the epitope-bearing portion. If antibodies specific to the pathogens are to be detected, an epitope- bearing portion is chosen which bears only epitopes characteristic of pathogenic strains. Conversely, antibodies immunospecific for nonpathogens can be conducted by utilizing as antigen only portions of the subunit which contain epitopes characteristic of nonpathogens. Where characterization as pathogen or nonpathogen-specific antibodies is unnecessary, antigen
  • Kits suitable for the conduct of these methods include the appropriate labeled antigen or antibody reagents and instructions for conducting the test.
  • the kit may include the antigen coupled to solid support as well as additional reagents.
  • the recombinant 170 kD subunit or an epitope- bearing portion thereof may be used as active ingredient.
  • Preferred regions include positions 482-1138, 596-1138, 885-998, 1033-1082 and 1082-1138.
  • the 170 kD subunit or its epitope-bearing regions may also be produced recombinantly in procaryotic cells for the formulation of vaccines.
  • the recombinantly produced 170 kD protein or an epitope-bearing region thereof can be used as an active ingredient in vaccines for prevention of E. histolytica infection in subjects who are risk for such condition.
  • Suitable carriers may include, for example, keyhole limpid hemocyanin (KLH) E. coli pilin protein k99, BSA, or the VP6 protein of rotavirus.
  • KLH keyhole limpid hemocyanin
  • Another approach employs production of fusion proteins which include the epitope-bearing regions fused to additional amino acid sequence.
  • the epitope-bearing region may be included in multiple copies in a single molecule, or several epitope-bearing regions can be "mixed and matched" in a single molecule.
  • the active ingredient, or mixture of active ingredients, in the vaccine is formulated using standard formulation for administration of proteins or peptides and the compositions may include an immunostimulant or adjuvant such as complete Freund's adjuvant, aluminum hydroxide, liposomes, ISCOMs, and the like. General methods to prepare vaccines are described in Remingtons ' s Pharmaceutical Science; Mack Publishing Company Easton, PA (latest edition) .
  • compositions contain an effective amount of the active ingredient peptide or peptides together with a suitable amount of carrier vehicle, including, if desired, preservatives, buffers, and the like.
  • suitable amount of carrier vehicle including, if desired, preservatives, buffers, and the like.
  • the vaccines are administered as is generally understood in the art. Ordinarily, administration is systemic through injection; however, other effective means of administration are included. With suitable formulation, for example, peptide vaccines may be administered across the mucus membrane using penetrants such as bile salts or fusidic acids in combination, usually, with a surfactant. Transcutaneous means for administering peptides are also known. Oral formulations can also be used. Dosage levels depend on the mode of administration, the nature of the subject, and the nature of carrier/adjuvant formulation. Typical amounts of protein are in the range of .01 ⁇ g-1 mg/kg. However, this is an arbitrary range which is highly dependent on the factors cited above. In general, multiple administrations in standard immunization protocols are preferred; such protocols are standard in the art.
  • a preferred epitope-bearing region of the 170 kD subunit is that represented by amino acids 482-1138 which includes the cysteine-rich domain. This region is encoded by nucleotides 1492-3460 shown in Figure 1 herein. Preferred regions include those bearing epitopes which are specific for antibodies against pathogenic amoeba -- i.e., regions 1082-1138 and 1032-1082. However, the epitope-bearing region at positions 894-998 may also be used. For regions of this length, production of peptides with multiple copies of the epitope-bearing regions is particularly advantageous.
  • the epitope-bearing portions of the 170 kD subunit can be conveniently prepared in a variety of procaryotic systems using control sequences and hosts ordinarily available in the art.
  • the portions may be provided as fusion proteins or as mature proteins and may be produced intracellularly or secreted. Techniques for constructing expression systems to effect all of these outcomes is well understood in the art.
  • the medium can be used directly in the assay to provide the antigen, or the antigen can be recovered from the medium and further purified if desired.
  • the protein is produced intracellularly, lysates of cultured cells may be used directly or the protein may be recovered and further purified.
  • the epitope-bearing portion is provided as a fusion protein using the commercially available expression vector pGEX. Alternative constructions and alternative hosts can also be used as is understood in the art.
  • the present invention provides both nucleic acid and immunological reagents specific for 170 kDa subunits encoded by each of the hgll, hgl2 or hgl3 genes, as well as reagents which detect common regions of all three hgl genes and their nucleic acid or protein products.
  • oligonucleotide probes specific for any one of these three genes may be identified by one of ordinary skill in the art, using conventional nucleic acid probe design principles, by comparisons of the three DNA sequences for these genes, which sequences are disclosed in Figure 1A and Figure 4A for hgll and hgl3 , respectively, and for hgl2 , in Tannich, E. et al .
  • Example 6 illustrates the use of oligonucleotide probes specific for each of the three hgl genes, for determining the level of expression of RNA from each gene using Northern blot analyses.
  • Other methods of using hgl-specific nucleic acids for diagnostic purposes, for pathogenic and/or nonpathogenic forms of E. histolytica are described in U.S. Patent 5,260,429, the entire disclosure of which is incorporated herein by reference. The following Examples are intended to illustrate but not to limit the invention.
  • Example 1 Construction of Expression Vectors The 170 kD subunit of the galactose lectin is encoded by at least two genes.
  • the DNA used for all of the constructions described herein encodes the 170 kD lectin designated hgll (Fig. 1A) .
  • the nucleotide position designations refer to the numbering in Figure 1A.
  • fragment C included the cysteine- and tryptophan-rich region, the cysteine-free region, and 277 amino acids of the cysteine-rich domain, i.e. amino acid residues 2-596
  • fragment A encoded the majority of the cysteine-rich domain, i.e. amino acid residues 482-1138
  • fragment B (3461-3892) included 70 amino acids of the cysteine-rich domain, the putative membrane- spanning region, and the cytoplasmic tail, i.e. amino acid residues 1139-1276. See Fig. 2B.
  • Each of these three fragments was inserted in frame by ligation into pGEX2T or pGEX3X to obtain these proteins as GST fusions.
  • Figure 2A A diagram of the vectors constructed is shown in Figure 2A.
  • Fragment C was produced by PCR amplification. Primers were designed so that a BairiHI site was added to the 5' end and an BcoRI 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 Ec ⁇ RI from plasmid clones (Mann, BJ et al . Proc Natl Acad Sci USA (1991) 81:3248-3252) and ligated into pGEX2T that had been digested with EcoRI .
  • a recombinant protein is expressed as a fusion protein with glutathione S-transferase (GST) from Schistosoma japonicum and is under the control of the tac promoter.
  • GST glutathione S-transferase
  • the tac promoter is inducible by IPTG.
  • Fusion Proteins with MAbs Induced cultures containing bacterial strains expressing hgll fragment A, B, or C were harvested, lysed in sample buffer, and applied to an SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to Immobilon and incubated with anti-170-kD MAbs, specific for seven different epitopes. Characteristics of the individual MAbs are shown in Table 1. It will be noted that all the known epitopes are in the region of amino acids 596-1138.
  • Adherence was assayed by the.b ⁇ iding of Chinese hamster ovary (CHO) cells to E. histolytica trophozoites and by binding of I labeled purified colonic mucins to trophozoites.
  • CHO Chinese hamster ovary
  • the assay for cytotoxicity was CHO cell killing by E. histolytica trophozoites as measured by
  • P and NP refer to reactivity of the MAb with pathogenic (P) and nonpathogenic (NP) species of E. histolytica as determined in an Elisa assay.
  • P pathogenic
  • NP nonpathogenic
  • epitope-bearing portions indicated can be used alone, as fragments or as portions of chimeric or fusion proteins, or any combination of these epitope- bearing portions can be used.
  • a GST fusion protein with fragment A was prepared in E. coli as described in Example 1 above.
  • This peptide contains an upstream GST derived peptide sequence followed by and fused to amino acids 432-1138 encoded by nucleotides 1492-3460 in Figure 1 herein.
  • the protein is produced intracellularly; the cells were harvested and lysed and the lysates subjected to standard purification techniques to obtain the purified fusion protein.
  • Gerbils were immunized by intraperitoneal injection with 30 ⁇ g of purified fusion protein in complete Freund's adjuvant and then boosted at 2-4 weeks with 30 ⁇ g of the fusion protein in incomplete Freund's adjuvant.
  • E. histolytica is encoded by a gene family that includes hgll, hgl2 and a previously undescribed third gene herein designated hgl3. Since hgll and hgl2 were originally sequenced, in part, from different cPNA libraries, it was possible that they represented strain differences of a single gene. However, in this report both 5' and 3' termini of hgll, hgl2 , and hg!3 were isolated and sequenced from the same lambda genomic library demonstrating unambiguously that hgl is a gene family.
  • genomic library was screened separately with a 5' and a 3' hgl specific probe, additional heavy subunit genes would be isolated even if they contained only partial identity with the gene family at only one end or even if one termini of an additional gene had been lost during library amplification.
  • the library screen looked at more than 3.2xl0 8 bases of genomic DNA in an organism with an estimated genome size of 10 7*5 bases (Gelderman, A.H. et al . J Parasitol (1971) 57_:906-911) . Thus, a full genomic equivalent was screened at low stringency for genes containing identity at either end.
  • the Northern data indicated that all three genes were expressed in the amebae.
  • the Northern blot also indicates that abundance of mRNA for all three genes decreased as the amebae progressed from log to stationary growth. This finding correlates with data which indicate that late log and stationary phase amebae have a decreased ability to adhere to, lyse, and phagocytose target cells (Orozco, E. et al . (1988) "The role of phagocytosis in the pathogenic mechanism of En amoeJba histolytica . In: Amebiasis: Human infection by Entamoeba histolytica (Ravdin J.I., ed) , pp. 326-338. John Wiley & Sons, Inc., New York.
  • Duplicate plague lifts using Hybond-N membranes (Amersham, Arlington Heights, IL) , were placed in a prehybridization solution consisting of 6x SSC (.89 M sodium chloride and 90 mM sodium citrate) , 5x Denhardts solution, .5% SDS, 50 mM NaP0 4 (pH 6.7), and 100 ⁇ g/ml salmon sperm DNA for a minimum of 4 hours at 55°C.
  • 6x SSC .89 M sodium chloride and 90 mM sodium citrate
  • 5x Denhardts solution 5x Denhardts solution
  • 50 mM NaP0 4 pH 6.7
  • 100 ⁇ g/ml salmon sperm DNA for a minimum of 4 hours at 55°C.
  • a 5' and 3' DNA fragment of hgll (nucleotides 106-1946 and 3522-3940 respectively) were [ ⁇ - 32 P]dCTP (Amersham) labeled using the Random Primed DNA labeling Kit according to the manufacturer's instructions (Boehringer Mannheim, Mannheim, Germany) and hybridized separately to the membranes overnight at 55°C in prehybridization solution. Membranes were rinsed once and washed once for 15 minutes at room temperature in 2x SSC, .1% SDS, then washed once for 15 minutes at room temperature, and twice at 55°C for 20 minutes in .lx SSC, .1% SDS. Plaques that hybridized with the 5' or the 3 radiolabeled probe on both duplicate filters were isolated and purified.
  • 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 _7-P 32 ]ATP (Sambrook, J. et al . (1989) Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) , added to the hybridization mixture and the membrane, and incubated at 37°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 2x SSC, .1% SDS.
  • radiolabeled probes used were: 5' -TTTGTCACTATTTTCTAC-3' , hgll; 5' -TATCTCCATTTGGTTGA-3' , hgl2; 5' -TTTGTCACTATTTTCTAC-3' , hgl3; and
  • the hg 3 open reading frame was 3876 bases and would result in a predicted translation product of 1292 amino acids ( Figure 4) .
  • the predicted translation products of hgll and hgl2 would be 1291 and 1285 amino acids respectively.
  • a putative signal sequence and a transmembrane domain were identified in the amino acid sequence of hg!3 similar to hgll and hgl2 .
  • the amino-terminal amino acid sequence of the mature hg!3 protein determined by Edman degradation (Mann, B.J. et al. Proc Natl Acad Sci USA (1991) 88:3248- 3252), was assigned residue number 1.
  • FIG. 5 A schematic comparison ( Figure 5) of heavy subunit gene sequences revealed a high degree of amino acid sequence identity. However, seven sites, ranging from 3-24 nucleotides, were found where an insertion or deletion had occurred in one subunit relative to another, all of which maintained the open reading frame. Both hgll and hgl3 contained a large number of nonconservative amino acid substitutions when compared to hgl2, making them 89.2% and 89.4% identical to hg!2 respectively. While the comparison of hgll and hgl3 revealed only two nonconservative substitutions, 57 conservative amino acid substitutions and 3 single residue insertion/deletions making them 95.2% identical.
  • hgl3 was isolated from a genomic library, it was unknown if this gene was transcribed. Polyadenylated RNA was harvested from amebae in both log and stationary phase growth. Probes specific for hgll, hgl2, or hgl3 were hybridized to a Northern blot and identified an RNA band of the predicted size of 4.0 kb.
  • This central hgll radioprobe would hybridize with three bands of 1158, 810 and > 1080 nucleotides from hgll and would hyribidze with five bands of 819, 312, 55, 755, and >1080 nucleotides from hg!2.
  • the Southern blot showed 7 bands for genomic DNA disgested with Ddel, at 4200, 3700, 2100, 1800, 1300, 840, and 760 nucleotides. As the 819 and 810 nucleotide bands would be expected to comigrate, all the bands observed with Ddel digestion are explained by the restriction maps of hgll-3.
  • Hindlll has no restriction sites in hgll-3 within the coding region and would result in each gene being represented by a single band greater than 4.0 kb.
  • the Southern blot showed three bands at 17500, 5600, and 4200 nucleotides. Should an additional heavy subunit gene exist, its Ddel and Hindlll fragments would need to comigrate with hgll-3 bands, be so divergent that they failed to hybridize with the hgll probe under very low stringency, or be too large to be resolved and transferred.
  • genomic screening data the genomic library was screened separately with a 5' and a 3' hgl specific probe, additional heavy subunit genes would be isolated even if they contained only partial identity with the gene family at only one end or even if one termini of an additional gene had been lost during library amplification.
  • the library screen looked at more than 3.2xl0 8 bases of genomic DNA in an organism with an estimated genome size of 10 7S bases (Gelderman, A.H. et al . J Parasitol (1971) 57:906-911) . Thus, a full genomic equivalent was screened at low stringency for genes containing identity at either end.

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Abstract

Une sous-unité lourde de 170 kDa produite par recombinaison de lectine d'adhésion Gal/GalNAc d'Entamoeba histolytica ou une partie porteuse d'épitopes de celle-ci peut être utilisée en tant qu'antigène dans l'analyse sérologique de l'infection par E.histolytica ou en tant que vaccin d'immunisation contre l'infection. La production par recombinaison s'effectue dans un système procaryote afin de produire des antigènes ou des immunogènes non glycosylés immunologiquement réactifs. Trois gènes codant les parties porteuses d'épitopes de la lectine sont également décrits.
PCT/US1994/006890 1988-01-13 1994-06-17 Lectine recombinee a sous-unite de 170 kd d'entomoeba histolytica et ses procedes d'utilisation WO1995000849A1 (fr)

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AU71123/94A AU710439B2 (en) 1993-06-17 1994-06-17 Recombinant 170 KD subunit lectin of Entamoeba histolytica and methods of use
US08/569,214 US6165469A (en) 1988-01-13 1994-06-17 Recombinant Entamoeba histolytica lectin subunit peptides and reagents specific for members of the 170 kD subunit multigene family
EP94920262A EP0708923A4 (fr) 1993-06-17 1994-06-17 Lectine recombinee a sous-unite de 170 kd d'entomoeba histolytica et ses procedes d'utilisation
JP7502991A JPH09502165A (ja) 1993-06-17 1994-06-17 赤痢アメーバの組換型１７０ｋｄサブユニットレクチン及びその使用方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997011669A3 (fr) * 1995-09-26 1997-04-24 Us Gov Health & Human Serv Antigenes limites du melanome, appartenant a la classe ii du mhc, et leur utilisation dans des methodes therapeutiques
WO2002032936A3 (fr) * 2000-10-20 2003-03-13 Kevin C Kain Expression de lectine gal/galnac de recombinaison biologiquement active de entamoeba histolytica et analyse diagnostique d'une infection de e. histolytica dans des echantillons fecaux conserves
US7501501B2 (en) 1995-09-26 2009-03-10 The United States Of America As Represented By The Secretary Department Of Health And Human Services MHC-Class II restricted melanoma antigens and their use in therapeutic methods

Families Citing this family (1)

* Cited by examiner, † Cited by third party
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JP6799927B2 (ja) * 2016-02-23 2020-12-16 古河電気工業株式会社 生体分子検出用試験キット、及びこれを用いた生体分子の検出方法、並びにこれらに用いられる生体分子検出用標識試薬

Citations (2)

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Publication number Priority date Publication date Assignee Title
US5004608A (en) * 1988-01-13 1991-04-02 The University Of Virginia Alumni Patents Foundation Amebiasis vaccine
WO1991012529A1 (fr) * 1990-02-13 1991-08-22 The University Of Virginia Alumni Patents Foundation LECTINE Gal/GalNAc DE L'ENTAMOEBA HISTOLYTICA

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US5004608A (en) * 1988-01-13 1991-04-02 The University Of Virginia Alumni Patents Foundation Amebiasis vaccine
WO1991012529A1 (fr) * 1990-02-13 1991-08-22 The University Of Virginia Alumni Patents Foundation LECTINE Gal/GalNAc DE L'ENTAMOEBA HISTOLYTICA

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Title
J. CLIN. MICROBIOL., Volume 30, No. 11, issued November 1992, ZHANG et al.: "Use of a Recombinant 170-Kilodalton Surface Antigen of Entamoeba histolytica for Serodiagnosis of Amebiasis and Indentification of Immunodominant Domains of the Native Molecule", pages 2780-2792, especially see page 2788, col. 2, lines *
JAMA, Volume 266, No. 14, issued 09 October 1991, STANLEY et al.: "Serodiagnosis of Invasive Amebiasis Using a Recombinant Entamoeba histolytica Protein", pages 1984-1986, especially see Abstract on page 1984. *
MOL. BIOCHEM. PARASITOL., Volume 62, No. 1, issued November 1993, PURDY et al.: "Analysis of the gene family encoding the Entamoeba histolytica galactose-specific adhesin 170-kDa subunit", pages 53-60, especially see Abstract. *
PLOTKIN et al., "VACCINES", published 1988 by W.B. SAUNDERS COMPANY (Philadelphia), pages 568-575, especially see page 572, first full paragraph to page 573, line 4 and Table 29-6. *
PROC. NATL. ACAD. SCI. USA, Volume 88, issued April 1991, MANN et al.: "Sequence of a cysteine-rich galactose-specific lectin of Entamoeba histolytica", pages 3248-3252, especially see Abstract on page 3248 and Fig. 1. *
PROC. NATL. ACAD. SCI. USA, Volume 88, issued March 1991, TANNICH et al., "Primary structure of the 170-kDa surface lectin of pathogenic Entamoeba histolytica", pages 1849-1853, especially Fig. 3 on page 1851. *
See also references of EP0708923A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997011669A3 (fr) * 1995-09-26 1997-04-24 Us Gov Health & Human Serv Antigenes limites du melanome, appartenant a la classe ii du mhc, et leur utilisation dans des methodes therapeutiques
US6951917B1 (en) 1995-09-26 2005-10-04 The United States Of America As Represented By The Department Of Health And Human Services MHC-class II restricted melanoma antigens and their use in therapeutic methods
US7501501B2 (en) 1995-09-26 2009-03-10 The United States Of America As Represented By The Secretary Department Of Health And Human Services MHC-Class II restricted melanoma antigens and their use in therapeutic methods
WO2002032936A3 (fr) * 2000-10-20 2003-03-13 Kevin C Kain Expression de lectine gal/galnac de recombinaison biologiquement active de entamoeba histolytica et analyse diagnostique d'une infection de e. histolytica dans des echantillons fecaux conserves

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