WO1992003457A1 - Entamoeba histolytica immunodominant surface antigens - Google Patents

Entamoeba histolytica immunodominant surface antigens Download PDF

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Publication number
WO1992003457A1
WO1992003457A1 PCT/US1991/005979 US9105979W WO9203457A1 WO 1992003457 A1 WO1992003457 A1 WO 1992003457A1 US 9105979 W US9105979 W US 9105979W WO 9203457 A1 WO9203457 A1 WO 9203457A1
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tat
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asn
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PCT/US1991/005979
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French (fr)
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Nina Martha Agabian
Ursula Edman
Isaura Meza Gomez-Palacio
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The Regents Of The University Of California
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Priority to BR919106968A priority Critical patent/BR9106968A/en
Publication of WO1992003457A1 publication Critical patent/WO1992003457A1/en

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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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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 present invention relates generally to
  • Entamoeba histolytica is a common human pathogenic protozoa. ⁇ moebiasis, or infection by E. histolytica, causes a spectrum c. disease ranging from a commensal state in asymptomatic carriers to fulminant diarrhea or extra-intestinal abscess formation in invasive
  • Entamoeba histolytica exists in two forms, a motile trophozoite or a dormant cyst. Virulent amoebae
  • the infection rate in the United States is about 1%, but the carrier rate may exceed 50% in certain areas of the world.
  • pathogenesis is not understood. Until recently, it was unclear whether invasiveness is a stable or a variable genotypic characteristic of a particular strain.
  • PGM phosphoglucomutase
  • HK hexokinase
  • PPI phosphoglucoisomerase
  • pathogenic infections caused by a mixture of strains or infections which may have interconverted phenotypically from pathogenic to non-pathogenic strains.
  • new reagents for treatment and prevention of infection by amoeba are always valuable.
  • the present invention provides these and other important reagents and methods for their effective use.
  • Figure 1 Western blot of whole Entamoeba extract fractionated by 5-15% SDS-PAGE; lanes 2, 7, and 12, polyxenic pathogenic E. histolytica isolate SD-4, lanes 3, 8, and 13, polyxenic non-pathogenic E. histolytica isolate SD116, lanes 4, 9, and 14, E. histolytica-like Laredo, lanes 5, 10, and 15, E. histolytica HK-9, lanes 6, 11, and 16, E.
  • E histolytica HM1:IMSS trophozoites labeled in vivo with primary antibodies.
  • histolytica immune sera at 1:500 dilution B: pool of human anti-E. histolytica immune sera purified by
  • C monoclonal FA7 harvest fluid at 1:1000 dilution
  • D monoclonal anti-E.
  • histolytica actin antibody at 1:1000 dilution secondary antibodies, fluorescein isothiocyanate goat-anti-human and fluorescein isothiocyanate goat-anti-mouse.
  • FIG. 3 Transfer blot of E. histolytica HM1:IMSS RNA probed with cDNA clone ⁇ cM17 indicates a single hybridizing band migrating at ⁇ 3 kb. Hybridization conditions were 50% formamide, 0. 2 X SSC, 42oC.
  • the present invention provides novel compositions and methods for diagnosing, treating and vaccinating Entamoeba parasitic infections.
  • the present invention is based, in part, on the discovery of a class of highly immunogenic, or immunodominant, 125 kDa
  • M17 proteins which are localized on the membrane of the trophozoite form of E. histolytica. Particular allelic forms of this 125 kDa antigen have been isolated and found to be characteristic of
  • compositions including vaccines, polypeptides, and polypeptide fragments.
  • compositions according to the present invention include nucleic acids encoding the various surface antigens and homologous polypeptides, nucleic acids homologous to those encoding peptides, as well as antibodies raised against the proteins, fragments, and homologous
  • polypeptides are provided. Methods for the use of these compositions are provided in view of the discoveries related to biological function.
  • the genus Entamoeba is defined by a number of cellular and biological markers. These markers define a genus Entamoeba, which exhibit common characteristics, but which may vary as better detection methods and functional tests are developed. However, the term
  • Entamoeba as used herein, is intended to include
  • E tamoeba strains often invade the body causing amoebiasis.
  • An invasiv infection includes infection within the intestines, or into the body through the intestinal wall.
  • the reagents provided herein are useful for both types of infections.
  • An immunodominant 125 kDa surface antigen has been isolated from various strains of Entamoeba. Table I sets forth the genomic sequences and corresponding amino acid sequences of two alleles of the 125 kDa
  • immunodominant surface antigen from a pathogenic strain designated HM1:IMSS and a non-pathogenic strain
  • compositions may be derived from such other alleles or the nucleic acid or amino acid sequences of such alleles, as described in more detail herein after.
  • Table I Inferred amino acid sequence and nucleotide sequence of coding region and flanking region obtained from genomic clone pBSgM17-1. The sequence of the internal EcoRI fragment was identical in both genomic clones (pBSgM17-1/2) and the cDNA clone ⁇ cM17. Shown aligned below is the partial nucleotide sequence of PCR amplification products derived from non-pathogenic isolate REF291. Nucleotide substitutions are underlined and amino acid substitutions are indicated below the partial sequence derived from REF291. Two boundaries are marked between Gly186 and Ile187 (corresponding to A558 and A559) and between Phe825 and Gln826 (corresponding to C2475 and C2476). These boundaries separate region I (amino or 5' proximal of the former boundary) from region II (between the boundaries) and region II from region III (carboxy or 3' proximal of the latter boundary).
  • N-terminal (n) region with a single positively charged residue an 8 amino acid long hydrophobic core (h) region and a 7 amino acid long polar C-terminal (c) region with an amino acid composition similar to those seen in other signal sequences.
  • the antigen is either a peripheral membrane protein or it may be anchored in the membrane by other means such as a glycophospholipid anchor.
  • the hydrophobic amino terminal itself may serve to anchor the antigen in the membrane with the C-terminal externally exposed as no additional trans membrane domains could be discerned.
  • Asp Thr Asn Ser lie Tyr Val Pro Asp Val Ile Thr Asn Asp Pro Gln Met Thr Asn Glu
  • M17 protein has been determined to be membrane associated. When these proteins are cross-linked with multivalent antibody molecules, live
  • the immunodominant antigen is determined to be a surface antigen and attachment by antibodies is likely to cause significant disruption of the infective cycle of the trophozoites.
  • the present invention in one embodiment, provides polypeptides which are related to the 125 kDa surface antigen and which will usually be either haptenic or antigenic, typically including at least about 6 amino acids, usually at least about 9 amino acids, and more usually about 12 or more amino acids found contiguously within a natural form of the 125 kDa immunodominant surface antigen protein.
  • Longer polypeptides will also find use, up to and including substantially full length of the natural protein and larger.
  • the contiguous amino acids may be located within any region of the
  • polypeptide but preferably in regions I or III of Table I, and will correspond to at least one epitope site which is characteristic of the particular immunodominant surface antigen protein.
  • epitope site it is meant that the epitope site will allow immunologic detection of the exposed polypeptide segment in a cell sample with reasonable assurance, in most cases allowing
  • polypeptides such as an immunodominant surface antigen from non-pathogenic strains.
  • the polypeptides will also be capable of inducing an immune response in a host when used in vaccines, in therapeutic compositions, and for preparing polyclonal and monoclonal antibodies.
  • compositions and methods of the present invention are particularly useful for identifying and treating pathogenic amoebic infection, but it will also be possible to identifying epitopes of the surface antigen which are conserved among pathogenic and non-pathogenic amoeba.
  • Use of polypeptides and nucleic acid probes based on the sequences of such conserved epitopes allows detection and treatment of both pathogenic and non-pathogenic amoeba.
  • compositions based on epitopes found in pathogenic forms only allows specific detection and treatment of pathogenic amoeba.
  • epitopes found in non-pathogenic forms only allow specific detection and treatment of non-pathogenic amoeba. Regions of particular importance for
  • pathogenic strains from non-pathogenic strains will be those located at the sites of difference between the amino acid or nucleotide differences, as indicated in Table III.
  • polypeptide In a preferred aspect, the polypeptide
  • compositions will be either identical or equivalent to a sequence set forth in Table I.
  • equivalent it is meant for the purposes of defining polypeptides that a substantial identity of amino acid sequences exists over a stretch of at least about 10 residues, where each position of the corresponding sequences is either
  • immunodominant surface antigen protein i.e., IL-12 immunodominant surface antigen protein
  • immunological analogs may be produced by either of at least two general approaches.
  • polypeptides having fewer than about 50 amino acids, more usually fewer than about 20 amino acids can be synthesized by the
  • amino acid sequences of such synthetic polypeptides will usually be based on the sequences described in Table I, preferably regions I or III.
  • a second and preferred method for synthesizing the polypeptides of the present invention involves the expression in cultured cells of recombinant DNA molecules encoding a desired portion of an immunodominant surface antigen gene.
  • the gene may itself be natural or
  • the natural gene is obtainable from cDNA or genomic libraries, as described herein. Using such segments or genes, additional
  • homologous gene sequences might be isolated from other related strains or sequences which encode peptides equivalent to those described above. Such genes
  • epitopic similarity can serve as a means for selecting equivalent peptides.
  • the polypeptides are usually obtained in substantially pure form, that is, typically about 50% w/w or more purity, substantially free of interfering proteins and contaminants.
  • the immunodominant surface antigen polypeptides are isolated or synthesized in a purity of at least about 80% w/w and, more
  • the proteins may be purified by use of the antibodies described hereinafter using immunoadsorbent affinity chromatography. Such affinity chromatography is performed by first linking antibodies or appropriate affinity reagents to the solid support and then
  • the linked antibodies or affinity reagents with the source of the immunodominant surface antigen proteins, e.g., lysates of protozoa which naturally produce immunodominant surface antigen or which produce immunodominant surface antigen as a result of
  • Useful production cultures include the insect
  • polypeptide sequence of distinct forms of immunodominant surface antigens have been isolated and characterized. Isolated DNA segments encoding the immunodominant surface antigens can be expressed to provide isolatable
  • nucleic acids are substantially homologous to encoding sequences, either natural or artificial. These homologous
  • polynucleotides will find use as probes or primers for locating or characterizing natural or artificial nucleic acids encoding the surface antigen peptides.
  • Substantial homology or substantial identity of a nucleic acid sequence indicates either that: a) there is greater than about 65%, typically greater than about 75%, more typically greater than about 85%, preferably greater than about 95%, and more preferably greater than about 98% homology with a disclosed segment of at least about 10 contiguous nucleotides; or b) the homologous nucleic acid sequence will hybridize to the consensus sequence or its complementary strand under stringent conditions of temperature and salt concentration. These stringent conditions will generally be at temperatures greater than about 22oC, usually greater than about 30oC and more usually greater than about 45oC. Salt
  • concentrations are generally less than about 1 M, usually less than about 500 mM, and preferably less than about 200 mM.
  • the combined conditions will be more important than either the salt concentration or the temperature alone.
  • stringency include GC content of the sequence, extent of complementarity of the sequences and length of segments involved in the hybridization, besides composition of buffer solutions used in the hybridization mixture.
  • Nucleic acids can be synthesized based directly on the DNA sequences reported in Table I.
  • Polynucleotides may be synthesized by known techniques, for example, short single-stranded DNA fragments may be prepared by the phosphoramidite method described by
  • a double-stranded fragment may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate
  • Polymerase chain reaction techniques may be used for production of probes or amplification of
  • DNA constructs capable of introduction to and expression in an in vitro cell culture will often be incorporated in DNA constructs capable of introduction to and expression in an in vitro cell culture.
  • the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction and integration within the genome of cultured mammalian, protozoa, or other eucaryotic cell lines.
  • DNA constructs prepared for introduction into bacteria or yeast will usually include a replication system recognized by the host, the appropriate immunodominant surface antigen DNA fragment encoding the desired polypeptide product, transcriptional and translational initiation regulatory sequences joined to the 5'-end of the immunodominant surface antigen DNA sequence, and transcriptional and translational termination regnlatory sequences joined to the 3'-end of the immunodominant surface antigen
  • the transcriptional regulatory sequences will typically include a heterologous promoter which is recognized by the host. Conveniently, available
  • expression vectors which include the replication system and transcriptional and translational regulatory
  • immunodominant surface antigen DNA sequence may be employed.
  • iiinfection may be achieved.
  • pathogenic infection is characterized by
  • Non-pathogenic infection is likewise
  • infection may use epitopes common to both pathogenic and non-pathogenic strains, but distinguishing will usually be directed to epitopes involving amino acids which differ and are indicated in Table I.
  • immunodominant surface antigen protein can be produced by in vitro or in vivo techniques.
  • In vitro techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or fragments, while in vivo techniques require the injection of the polypeptides or fragments into any of a wide variety of target immune systems, such as vertebrates. Suitable vertebrates are typically non- human, including mice, rats, rabbits, sheep, goats, and the like.
  • Polypeptides having more than about 5 to 30 amino acids, particularly more than about 50 to 100 amino acids, may serve directly as immunogens. if the
  • polypeptide is smaller than about 10 kD, particularly less than about 6 kD, it may be necessary to join the polypeptide to a larger molecule to elicit the desired immune response.
  • the immunogens are then injected into the animal according to a predetermined schedule, and the animals are bled periodically with successive bleeds generally having improved titer and specificity.
  • the injections may be made intramuscularly,
  • an adjuvant such as incomplete Freund's
  • monoclonal antibody also defines a class of proteins which are targets for specific binding.
  • monoclonal antibodies can be obtained by preparing immortalized cell lines capable of producing antibodies having the desired specificity.
  • the FA7 monoclonal antibody is produced by such a cell line.
  • Such immortalized cell lines may be produced in a variety of ways depending upon the target immune system.
  • a small vertebrate such as a mouse
  • the vertebrate is then killed, usually several days after the final immunization, the spleen removed, and the spleen cells immortalized.
  • the manner of immortalization is not critical.
  • the most common technique is fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519.
  • Other techniques include EBV transformation, transformation with bare DNA, e.g., oncogenes, retroviruses, etc., or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies. Common techniques are described, e.g., in Lane and Harlow, (1988)
  • the manner of fusion is usually not critical and various techniques may be employed.
  • the spleen cells and myeloma cells are combined in the presence of a nonionic detergent, usually polyethylene glycol, and other additives such as Dulbecco's Modified Eagle's
  • the nonionic detergent is rapidly removed by washing the cells.
  • the fused cells are promptly dispensed in small culture wells (usually in a microtiter plate) at
  • the myeloma cell line has been mutated to be sensitive to a lethal agent, typically being HAT
  • the cell line can be maintained as viable cultures and/or by lyophilization or frozen storage.
  • Hybridomas providing high titers are
  • cytotoxic antibodies e.g., IgG 2a , IgG 2b , IgG 3 and IgM, may be selected for use in therapeutic treatment of pathogenic infections.
  • antibodies having very high specificity fsr the antigenic site are desirable.
  • monoclonal antibodies may be isolated from the desired hybridomas.
  • the yield of antibodies obtained is usually low.
  • the yield may be enhanced by various techniques, such as injection of the hybridoma cell line into the peritoneal cavity of a vertebrate host which will accept the cells.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Proteinaceous and other contaminants will usually be removed from the monoclonal antibodies prior to use by conventional technique, e.g., chromatography, gel filtration, precipitation,
  • the polypeptide selected should represent one or more epitopic sites which are unique to the desired immunodominant surface antigen protein and which can distinguish immunodominant surface antigen from closely related proteins.
  • Such unique epitopes are found on polypeptides expressed by cells containing sequences disclosed in Table I.
  • One particular example of such is the FA7 monoclonal antibody.
  • polypeptides and antibodies of the present invention may be used with or without modification.
  • the polypeptides and antibodies will be labelled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include
  • tissue samples including tissue samples, sputum, and lung lavage samples.
  • a tissue sample may be fixed in formalin, B-5, or other standard histological preservative, dehydrated and embedded in paraffin as is routine in any hospital pathology laboratory. Sections may then be cut from the paraffinized tissue block and mounted on glass slides.
  • the immunodominant surface antigen proteins if present, may then be detected in the cytoplasm or extracellular space by exposure with labeled immunodominant surface antigen antibody or exposure to unlabelled anti- immunodominant surface antigen antibody and a labeled secondary antibody. Because the antigen is a surface antigen and exposed extracellularly, it is unnecessary to denature a sample, allowing assay of non-denatured target sample. This may also allow further live
  • Sputum and lavage samples are typically prepared in a similar manner where the sample is first dehydrated by exposure to a dehydrating agent, typically a low molecular weight alcohols
  • a dehydrating agent typically a low molecular weight alcohols
  • Liquid phase immunoassays or Western blot analysis will also find use in the detection of the immunodominant surface antigen proteins particularly in body fluids when the proteins are shed into such fluids, e.g., blood or stool.
  • Solid tissue and sputum samples may also be assayed in liquid phase systems by lysing the cellular sample in order to release the protein. Once the protein is released, the sample will be placed in a suitable buffer, the sample buffer subjected to a
  • kits for using them are also provided. Similar kits may be prepared using particular nucleic acid probes.
  • the kits will typically have at least one compartment comprising the detection reagent to be applied to an appropriate sample.
  • the reagent may be attached to a dipstick or similar physical entity.
  • the reagent may be contained in a liquid solution to which a sample is added.
  • a compartment containing the active ingredient may be a sealed envelope, a plastic bag, a vial, a bottle, a jar, an ampule, a well, or any other protective package.
  • immunodominant surface antigen antibodies may be coupled to toxins, such as diphtheria toxin and the ricin A chain, and administered to patients, or hosts, suffering from pathogenic Entamoeba infections.
  • toxins such as diphtheria toxin and the ricin A chain
  • the use of antibody conjugated toxins in cancer therapy is described generally in U.S. Patent Nos. 4,093,607;
  • Antibodies alone may also find use in treatment, particularly by blocking or interrupting some functional activity of immunodominant surface antigen protein which contributes to the pathogenic phenotype.
  • the binding fragments will be joined to active substances, such as proteolytic enzymes or glycosidases, in order to enhance interference with amoeba infection.
  • active substances such as proteolytic enzymes or glycosidases.
  • the binding fragments will specifically bind to the trophozoites and the active substances will act to destroy the protozoa.
  • Vaccines against Entamoeba infections are producible using the compositions of the present
  • vaccines may be passive, consisting of Ig supplementation which interferes with amoeba growth or toxicity.
  • vaccines may be active
  • a vaccine prepared utilizing the immunodominant surface antigen or immunogenic equivalents thereof can consist of: (a) fixed cells either recombinantly altered to produce these antigen proteins or cells from the
  • Entamoeba itself; (b) a crude cell extract; (c) a
  • these immunogens can be combined with a suitable physiologically acceptable carrier, for example, it can be administered in water, saline,
  • alcohol alcohol, fats, waxes, or buffered vehicles with or without various adjuvants or immunomodulating agents.
  • polyribonucleotides sodium alginate, lanolin,
  • lysolecithin lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
  • adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's
  • Comparable dose forms can also be prepared for parenteral administration to smaller or larger animals, but the amount of immunogen per dose will usually be smaller, for a smaller animal.
  • Periodic revaccination is advisable under certain conditions.
  • the vaccine may also be combined with other vaccines for other diseases to produce multivalent vaccines. It may also be combined with other agents.
  • medicaments for example, antibiotics.
  • vaccines may be prepared according to methods well-known to those skilled in the art as set forth, for example, in Tizard, L., An Introduction to Veterinary Immunology, 2nd Ed (1982), which is
  • Laredo were grown in TYI-S-33 media as described by
  • Polyxenic isolates were grown in liquid Robinson's medium supplemented with 10% bovine serum and containing 5 ⁇ g per ml of medium of each of the following antibiotics: kanamycin, erythromycin, and ampicillin. Amoebae were pelleted by centrifugation at 900 rpm and washed twice with phosphate buffered saline, pH 7.5 (PBS). Polyxenic amoebae were further purified by centrifugation through a Percoll/PBS cushion at 3000 rpm in a refrigerated
  • a membrane fraction was prepared as described in Aley, et al. (1980) J. EXP. Med. 152: 391-404, and diluted 1:1 with PBS and complete Freund's adjuvant.
  • mice were immunized intraperitoneally with 300 ⁇ g every two weeks until titers reached 1:5000 as assayed by
  • the 220 kDa antigens represents an N-acetyl-glu ⁇ osamine adherence lectins
  • the 160 kDa antigen represents an N-acetyl-D-galactosamine adherence lectin
  • the 96 kDa antigen represents an integral membrane protein.
  • Human immune serum, hyb ⁇ doma harvest fluid from clone FA7, and purified monospecific antiserum was added to live trophozoites at 1:500, 1:2000, and
  • Antibody-antigen caps were induced in HM1:IMSS trophozoites by incubation with the pooled patient serum, monoclonal antibody FA7, or monospecific antibody recovered after specific binding and elution of pooled patient sera to phage lysates of cDNA clone ⁇ cM17. See Fig. 2.
  • a negative control antibody monoclonal anti-actin antibody
  • histolytica HM1:IMSS has been described previously. See, e.g. Edman, et al. (1987) Proc. Natl. Acad. Sci. USA. 84: 3024-3028.
  • a genomic library from E. histolytica has been described previously. See, e.g. Edman, et al. (1987) Proc. Natl. Acad. Sci. USA. 84: 3024-3028.
  • BiolOl to 200 ⁇ l (approximately 20 ⁇ g DNA) of agarose embedded nuclei in an Eppendorf tube and melted by incubation at 60oC for 5 min. Twenty ⁇ l of glassmilk was added, and suspended well and the mixture incubated at room temperature for 5 min. The sample was vortexed for 1 min to shear the DNA and spun in a microfuge for 5 sec. After removal of the supernatant the pellet was suspended in 1 ml wash buffer by vortexing for 30 sec. The
  • the ⁇ gtll cDNA library (3 ⁇ 10 5 phage) was screened with the pool of 29 patient sera at a 1:200 dilution.
  • the genomic library was screened with the 32 P- ⁇ -dCTP labeled EcoRI fragment of ⁇ cM17.
  • oligonucleotide primers derived from the cDNA sequence.
  • the nucleotide sequence of the cDNA was identical in both genomic clones.
  • An additional 556 bp of 5' and 870 bp of 3' sequence yielded an ORF of 3345 bp which was also identical in both genomic clones.
  • the size of this ORF (gene M17) is in reasonable agreement with the mRNA size of ⁇ 3000 bp determined by Northern blot analysis.
  • the inferred amino acid sequence predicts a 125 kDa protein.
  • the 5' flanking sequence of gene M17 shares striking similarities with the 5' flanking region of both actin and ferredoxin genes, the only other genes of E. histolytica where sequence has been determined. See Table II.
  • the transcriptional start site of M17 was mapped to an adenine residue 17 bp 5' of the start codon by primer extension sequence analysis using an
  • sequence motif shared amongst these genes is YATTTAAA present at -29, -31, -32, and -32 for the M17, actin*, actin ⁇ , and ferredoxin gene flanking sequences, respectively. This sequence motif does not conform with the Goldberg-Hogness promoter consensus sequence
  • Table II Alignment of the 5' flanking sequences from genes M17, actin* (one reported sequence), actin ⁇ (a second reported sequence) and ferredoxin ⁇ ; a likely Goldberg-Hogness consensus sequence at -29, -31, -32, and -32, respectively, is bracketed.
  • the 5' end of the mRNA is in bold face and follows the ⁇ symbol, and the initial ATG methionine codon is indicated. Sequence similarities around the cap site are ATTCA or ATTAA.
  • Southern blot and sequence analysis of the M17 gene and limited flanking regions indicate that this surface antigen is encoded by a single copy gene.
  • the nucleotide sequence of both genomic clones and the cDNA clone is identical.
  • reaction mixtures 50 ⁇ l contained 25 pmol of each of the two oligonucleotide primer pairs SRO18 [5-GCAACTAGTGTTAGTTATAC3'] with SRO21
  • PCR parameters were 35 thermal cycles consisting of a 1 min denaturation at 94•c followed by a 3 min annealing period at 42oC, a 3 min ramp and a 4-min extension period at 72oC
  • the amplification products were restricted with Eco RI and Spe I endonucleases and purified for
  • SRO18/SRO21 as primers on genomic template DNA derived from non-pathogenic isolate REF 291.
  • REF 291 had 145 nucleotide substitutions over 1410 residues (10.3%) as compared to the sequence of ⁇ cM17 (HM1:IMSS) See Table I. These substitutions result in 57 amino acid differences per 470 residues (12.1%).
  • a computer search of published protein sequences with the entire 3345 bp M17 gene sequence revealed that the internal gene fragment represented by the ⁇ cM17 insert encoded a protein sequence similar to that deduced for a DNA fragment isolated from non-pathogenic and pathogenic strains, of E.
  • histolytica HM1:IMSS laboratory strains must have arisen from more than one nucleotide substitution and are therefore unlikely to represent cDNA synthesis or sequencing artifacts.
  • 470 amino acid sequence derived from the PCR product of non-pathogenic isolate REF291 was compared to isolate SAW 1734, six amino acid substitutions (1.3%) were detected. Over the same 470 amino acids, 61 amino acid residues (12.9%) differ among the pathogenic HM1:IMSS and non-pathogenic SAW 1734 strains. Overall there are 65 variable residues over a stretch of 470 amino acids (13.8%) when these four isolates were compared. See Table III.
  • Table III Alignment of the amino acid sequences inferred from nucleotide sequences of cDNA and genomic clones (HM1:IMSS*) and of PCR amplification products (REF291*) with those published by Tannich et al.
  • HM1 IMSS* PITLNFDORVDAGAAVAYVGRWFTONPSDWAAACVGKDGLINYGNWGPLHEMN
  • HM1 IMSS# PITINFDORVDAGAAVAYVDRWFTONPSDWAAACVGKDGLINYGNWGPLHEMN
  • HM1 IMSS* lJHMQGTYLKGGNWGISNPGEETNNVMTSINYILYTNIAGHRNQGLSGWNYVSD
  • HM1 IMSS* GYSTIYKILKGENDQPHLRSYVNMAHAFGTDTLIALVKSYYGLWYENNFESKY
  • HM1 IMSS# GYSTIYKILKGENDOPHLRSYVNMAHAFGTDTLIALVKSYYGLWYENNFESKY
  • HM1 IMSS* SIKRDSTSAFCLLAALVTKRDTRYLCSLFKYDIOSNVSEAIKNMNYPTYYPFF
  • HM1 IMSS# NLYAMSYNGNYYGRPYKIPYGRTRLNFTATCSIDPKATSVSYTIKSGLTKGKL
  • HM1 IMSS* ERVEDNVYDYTPFFGIEENDTFVLNIDCWNGEKVHIEOEGGTFELDPHOVEY
  • HM1 IMSS# ERVEDNVYDYTPFFGIEENDTFVLNIDCWNGEKVHIEOEGGTFELDPHOVEY
  • HM1 IMSS* EVYKDVOTRDMAOAINIIQNKTRNDTGRASFFGIGTYNDGSMOSMLVEKGKLI
  • HM1 IMSS# EVYKDVQTRDMAQAINIIQNKTRNDTGRASFFDIGTYNDGSMQSMLVEKGKLI
  • HM1 IMSS* VPKSGYYTLFMKADDLGRLLLNITGEYEOLLDVKTYLGGYSKTLNGSYATVKL
  • HM1 IMSS# VPKSGYYTLFMKADDLGRLLLNITGEYEQLLDVKTYLGGYSKTLNGSYATVKL
  • HM1 IMSS* EKDVGYPFILYNLNTGGOGFIRIGYCYHGTEESSVDVSKCSVSDIGS

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Abstract

A gene encoding a 125 kDa immunodominant surface antigen from Entamoeba histolytica is identified and characterized. The gene and protein provide treatment and vaccination reagents for amoebiasis.

Description

ENTAMOEBA HISTOLYTICA IMMUNODOMINANT SURFACE ANTIGENS
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates generally to
compositions and methods of use of immunodominant surface antigen polypeptides from Entamoeba protozoa and, more particularly, to the preparation of reagents useful for diagnosing, treating, or inhibiting Entamoeba protozoa infections. Description of the Background Art
Entamoeba histolytica is a common human pathogenic protozoa. \moebiasis, or infection by E. histolytica, causes a spectrum c. disease ranging from a commensal state in asymptomatic carriers to fulminant diarrhea or extra-intestinal abscess formation in invasive
infections. See, e.g., Merck Manual. (15th Ed.), chapter 13. Entamoeba histolytica exists in two forms, a motile trophozoite or a dormant cyst. Virulent amoebae
infections can cause ulceration of the intestinal
epithelium and may penetrate the bowel wall to form extra-intestinal abscesses, primarily in the liver. The infection rate in the United States is about 1%, but the carrier rate may exceed 50% in certain areas of the world.
With the availability of effective treatment regimens, early diagnosis is crucial for the prevention of disease and transmission. However, much controversy has centered on benefits and drawbacks of initiating therapy in asymptomatic in actions. Thus recent
investigations have focussed on a molecular genetic analysis of virulence and the definition of marker molecules which have high predictive value and can be applied in a clinically feasible fashion. Several molecular activities thought to
correlate with the virulent phenotype have been partially characterized, but the role of each of these in
pathogenesis is not understood. Until recently, it was unclear whether invasiveness is a stable or a variable genotypic characteristic of a particular strain.
Polymorphisms in the electrophoretic mobility of the glycolytic enzymes phosphoglucomutase (PGM), hexokinase (HK), and phosphoglucoisomerase (PGI) have been used to identify pathogenic and non-pathogenic strains.
More recently several other diagnostic reagents have been suggested for distinguishing pathogenic
strains, based either on DNA sequences, or the detection of specific antigens. See Garfinkel, et al. (1989)
Infect. Immun. 57:926-931; Samuelson, et al. (1989) J. Clinic. Microbiol. 27:671-676; Tannich, et al. (1989) Proc. Nat'l. Acad. Sci. USA 86:5118-5122; and Strachnan, et al. (1988) Lancet i:561-562. However, these tests typically require axenic cultivation and cloning of amoebae directly from fresh stool samples prior to assay. These cultivation steps are difficult and, in most cases, have not been achieved. None of these probes have been validated by large scale screening of clinically defined strain isolates in comparison with extant criteria, such as accepted zymodeme identifying characteristics.
Moreover, axenization and cloning of amoebae from patient isolates often favors outgrowth of the less fragile pathogenic strains and has been known to reversibly attenuate virulence.
Thus, it is important to develop direct, accurate, and quick tests which can identify pathogenic amoebae in fresh isolates. Diagnostic reagents are needed which allow clinical characterization of
pathogenic infections caused by a mixture of strains or infections which may have interconverted phenotypically from pathogenic to non-pathogenic strains. In addition, new reagents for treatment and prevention of infection by amoeba are always valuable. The present invention provides these and other important reagents and methods for their effective use.
SUMMARY OF THE INVENTION
Various alleles of an immunodominant surface antigen from Entamoeba histolytica have been identified and characterized. Genes encoding the antigens have been isolated and sequenced, thus providing detailed
information on their structure. Both polypeptides and nucleic acids and fragments thereof are provided. Highly specific antibody preparations, both polyclonal and monoclonal, against epitopes on the immunodominant surface antigen have been produced. Treatment and vaccination methods for amoebiasis are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Western blot of whole Entamoeba extract fractionated by 5-15% SDS-PAGE; lanes 2, 7, and 12, polyxenic pathogenic E. histolytica isolate SD-4, lanes 3, 8, and 13, polyxenic non-pathogenic E. histolytica isolate SD116, lanes 4, 9, and 14, E. histolytica-like Laredo, lanes 5, 10, and 15, E. histolytica HK-9, lanes 6, 11, and 16, E. histolytica HM1:IMSS probed with anti-membrane fraction serum (lanes 2 through 6) , pooled human immune sera (lanes 7 through 11) , and monoclonal antibody FA7 (lanes 12-16); molecular weights are given in kilo-Dalton (molecular weight standards lane 1: 200, 97, 68, 43, 28 kDa). Figure 2: Photographs (800-fold magnification) of
E. histolytica HM1:IMSS trophozoites labeled in vivo with primary antibodies. A: pool of human anti-E.
histolytica immune sera at 1:500 dilution, B: pool of human anti-E. histolytica immune sera purified by
binding to λcM17 phage lysates, C: monoclonal FA7 harvest fluid at 1:1000 dilution, D: monoclonal anti-E.
histolytica actin antibody at 1:1000 dilution; secondary antibodies, fluorescein isothiocyanate goat-anti-human and fluorescein isothiocyanate goat-anti-mouse.
Figure 3: Transfer blot of E. histolytica HM1:IMSS RNA probed with cDNA clone λcM17 indicates a single hybridizing band migrating at ~3 kb. Hybridization conditions were 50% formamide, 0. 2 X SSC, 42ºC.
Autoradiography shown required a 72 hr exposure. Figure 4: Restriction endonuclease (Eco RV lower case letters, Ssp I capital letters) digests of PCR products generated by amplification of genomic DNA from E. histolytica isolates/strains using oligonucleotide primers SRO18 + SRO21 and SRO19 + SRO22. a/A = #43, b/B = #44, c/C = SD116, d/D = REF291, e/E = E.
histolytica-like Laredo, f/F = #46, g/G = HK9, h/H = HM1:IMSS.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention provides novel compositions and methods for diagnosing, treating and vaccinating Entamoeba parasitic infections. The present invention is based, in part, on the discovery of a class of highly immunogenic, or immunodominant, 125 kDa
proteins, designated M17 proteins, which are localized on the membrane of the trophozoite form of E. histolytica. Particular allelic forms of this 125 kDa antigen have been isolated and found to be characteristic of
pathogenic types of amoeba, and such identification and sequencing of the antigens provides a basis for
preparation of novel compositions including vaccines, polypeptides, and polypeptide fragments. Other
compositions according to the present invention include nucleic acids encoding the various surface antigens and homologous polypeptides, nucleic acids homologous to those encoding peptides, as well as antibodies raised against the proteins, fragments, and homologous
polypeptides. Methods for the use of these compositions are provided in view of the discoveries related to biological function.
The genus Entamoeba is defined by a number of cellular and biological markers. These markers define a genus Entamoeba, which exhibit common characteristics, but which may vary as better detection methods and functional tests are developed. However, the term
Entamoeba, as used herein, is intended to include
organisms which now are classified therein or are
sufficiently similar as to share significant epitopes or biological properties characteristic of organisms
assigned to this genus.
These E tamoeba strains often invade the body causing amoebiasis. An invasiv infection includes infection within the intestines, or into the body through the intestinal wall. The reagents provided herein are useful for both types of infections. An immunodominant 125 kDa surface antigen has been isolated from various strains of Entamoeba. Table I sets forth the genomic sequences and corresponding amino acid sequences of two alleles of the 125 kDa
immunodominant surface antigen, from a pathogenic strain designated HM1:IMSS and a non-pathogenic strain
designated REF 291 strains. It will be appreciated that other alleles will exist in nature and that the
compositions may be derived from such other alleles or the nucleic acid or amino acid sequences of such alleles, as described in more detail herein after.
Table I: Inferred amino acid sequence and nucleotide sequence of coding region and flanking region obtained from genomic clone pBSgM17-1. The sequence of the internal EcoRI fragment was identical in both genomic clones (pBSgM17-1/2) and the cDNA clone λcM17. Shown aligned below is the partial nucleotide sequence of PCR amplification products derived from non-pathogenic isolate REF291. Nucleotide substitutions are underlined and amino acid substitutions are indicated below the partial sequence derived from REF291. Two boundaries are marked between Gly186 and Ile187 (corresponding to A558 and A559) and between Phe825 and Gln826 (corresponding to C2475 and C2476). These boundaries separate region I (amino or 5' proximal of the former boundary) from region II (between the boundaries) and region II from region III (carboxy or 3' proximal of the latter boundary).
The amino acid sequence inferred from the nucleotide sequence of the coding region of the 125 kDa antigen is unusual with respect to its high Asn (90 =8.2%), Tyr (70=6.3%) and hydroxyl amino acid residue (Ser, 85=7.6%; Thr, 90=8.1%) content. While a total of 17 N-linked glycosylation sites suggests that the 125 kDa antigen may be glycosylated, Western blot analysis shows that this antigen migrates as a compact band on SDS-PAGE. A distinctly hydrophobic amino terminal region of 35 amino acids may serve as anchor or signal sequence. Compared to known prokaryotic and eucaryotic signal sequences this region contains an unusually long (20 amino acids)
N-terminal (n) region with a single positively charged residue, an 8 amino acid long hydrophobic core (h) region and a 7 amino acid long polar C-terminal (c) region with an amino acid composition similar to those seen in other signal sequences. By extrapolation this would imply that the antigen is either a peripheral membrane protein or it may be anchored in the membrane by other means such as a glycophospholipid anchor. Alternatively, the hydrophobic amino terminal itself may serve to anchor the antigen in the membrane with the C-terminal externally exposed as no additional trans membrane domains could be discerned.
Table I, panel 1
gaagctataaataagttatagaaatataaaagaatg ttaaaaatgaaaacaaacataaaaaataagtgtatttaaagtgtttttaaaaaaactaatt*ATTCATAAATTAAAGTT
20
Met Leu Gly Ser Lys Ser Ile Ile Ala Val Val Ala Ile Ala Ser Ala Ile Val Thr Gly
ATG TTA GGT TCT AAA AGC ATT ATT GCT GTT GTG GCT ATA GCT TCT GCA ATA GTC ACA GGA
40
Val Val Val lle Val Val Val Val Thr Leu Ser Val Val Leu Thr Arg Ser Ser Val Lys
61 GTA GTT GTT ATA GTT GTT GTT GTT ACA CTT TCT GTG GTT TTA ACA AGA AGT AGT GTT AAA
60
Asp Thr Asn Ser lie Tyr Val Pro Asp Val Ile Thr Asn Asp Pro Gln Met Thr Asn Glu
121 GAC ACC AAC TCT ATT TAT GTT CCT GAT GTT ATT ACT AAC GAC CCA CAA ATG ACA AAT GAA
80
Met Asp Thr Leu Glu Val Ile Ser Ser Ser Lys Phe Ser Gly Thr Lye Pro Lys Glu Trp
181 ATG GAT ACA TTA GAG GTT ATT TCT TCT TCA AAA TTT AGT GGA ACA AAA CCA AAA GAA TGG
100
Thr Met Lys Tyr Thr Lys Tyr Pro Tyr Trp Thr Cys Gly Leu Thr Phe Thr Asn Glu Glu
241 ACT ATG AAA TAT ACA AAA TAT CCT TAT TGG ACA TGT GGA CTT ACA TTT ACT AAT GAG GAA
120
Lys Gln Asn Ile Val Asn Glu Asn Lys Glu Tyr Met Asn Ser Leu Leu Gln Leu Ile Asn
301 AAA CAA AAT ATT GTT AAT GAA AAT AAG GAA TAT ATG AAC TCA TTA TTA CAA CTT ATT AAT
140
Asn Gly Ser Leu Gly Arg Met Pro Glu Lys Tyr Gly Gly Asp Lys Gln Phe Glu Ala Asn
361 AAT GGA TCA TTA GGA AGA ATG CCT GAA AAA TAT GGT GGT GAT AAA CAA TTT GAA GCT AAT
Gly Val Asn Trp Glu Ala Asp Arg Leu Glu Val Arg Tyr Gly Leu Phe Gly Arg Val Phe
421 GGA GTT AAT TGG GAA GCT GAT AGA TTA GAA GTT AGA TAT GGT CTT TTC GGT AGA GTT TTT
180
Gly Gln Arg Ala Val Ala Trp Ala Phe Pro Gly Glu Ile Val Thr Ile Lys Phe Pro Lys
481 GGT CAA AGA GCT GTT GCA TGG GCT TTT CCA GGA GAA ATC GTT ACA ATT AAA TTC CCT AAA
200
Gly Met Ser Tyr Lys GlyiIle Gln Val Gly Ile Gly Lys Cys Asn His Asn Pro Ser Asp
541 GGA ATG AGT TAT AAG GGAiATT CAG GTT GGT ATT GGT AAG TGT AAC CAT AAT CCT TCT GAT
220
Gln Trp Leu Asn Val Asn Asn Trp Ser Asn Asp Arg Met Pro Ile Asp Ser Ile Gly Phe
601 CAA TGG TTA AAT GTT AAT AAC TGG TCA AAT GAT AGA ATG CCA ATC GAT TCA ATT GGA TTT
240
Asp Leu Gly Leu Asn Thr Thr Gln Pro Tyr Ile Ile Asn Asp Thr Phe Lys Ile Gly Ser
661 GAT TTA GGA CTT AAT ACA ACG CAA CCA TAC ATT ATT AAT GAT ACA TTT AAA ATA GGA TCA
260
Pro Phe Gly Gly Met Ile Tyr Leu Arg Ser Asp Thr Thr Phe Thr Asn Ser Phe Tyr Val 721 CCA TTT GGA GGT ATG ATT TAT TTA AGA TCT GAT ACA ACA TTT ACA AAT TCA TTT TAT GTC
280
Thr Phe Ser Asn Val Gly Arg Ala Pro Ile Ile Asn Tyr Asn Ile Thr Thr Asn Glu Glu
781 ACA TTC AGT AAT GTT GGA AGA GCT CCA ATT ATT AAT TAT AAT ATT ACA ACG AAT GAA GAA
300
Trp Asn Ser Val Leu Arg Asn Ala Pro Gly Asn Val Ala Glu Ile Arg Thr Pro Gly Asn
841 TGG AAT AGT GTT TTA AGA AAT GCA CCA GGA AAT GTT GCA GAA ATC AGA ACA CCA GGA AAT
320
Arg Leu Val Leu Thr Ser Arg Asn Ile Arg Ser Leu Glu Asp Ala Gln Tyr Ile Ser Asp
901 AGA CTT GTA CTT ACT TCA AGA AAT ATT AGA AGT TTG GAA GAT GCA CAA TAT ATT AGT GAT
340
Phe Trp Leu Lys Ala Ile Ser Ile Ser Asn Tyr Ala Val Thr Leu Glu Asn Ile Pro Ile
961 TTC TGG TTA AAA GCA ATT AGT ATT TCT AAT TAT GCT GTT ACA CTT GAA AAT ATT CCA ATT
CCA ATC Table I, panel 2
360
Thr Leu Asn Phe Asp Gln Arg Val Asp Ala Gly Ala Ala Val Ala Tyr Val Gly Arg Trp
1021 ACA TTA AAC TTT GAT CAA AGA GTT GAT GCA GGA GCT GCT GTT GCA TAT GTA GGA CGT TGG
ACA TTA AAC TTT GAT CAA AGA GTT GAT GCT GGA GCT GCT GTT GCA TTT GTA GGA AGA TGG
Phe
380
Phe Thr Gln Asn Pro Ser Asp Trp Ala Ala Ala Cys Val Gly Lys Asp Gly Leu Ile Asn
1081 TTT ACT CAA AAC CCA TCC GAT TGG GCA GCT GCA TGT GTT GGT AAA GAT GGA TTA ATA AAT
TTT ACT CAA CAC CCA TCT GAC TGG GCG TCT GGA TGC GTT AAC AAA GAA AGA TTA ATA AAT
His Ser Gly Asn Arg
400
Tyr Gly Asn Trp Gly Pro Leu His Glu Met Asn His His Met Gln Gly Thr Tyr Leu Lys
1141 TAT GGA AAT TGG GGA CCA TTA CAT GAA ATG AAT CAT CAT ATG CAA GGA ACT TAT TTA AAA
TC.T GGA AAT TGG GGA CCA TTA CAT GAA ATG AAT CAT CAC. ATG CAA GGA ACT TAT TTA AGA
Ser Arg
420
Gly Gly Asn Trp Gly Ile Ser Asn Pro Gly Glu Glu Thr Asn Asn Val Met Thr Ser Ile
1201 GGA GGA AAT TGG GGT ATT AGT AAT CCA GGA GAA GAA ACT AAT AAT GTT ATG ACA TCA ATT
GGA GGA AAT GGG GGT ATC. AAA G.AA CCA GGA GAA GAA ACT AAT AAT GTT ATG ACA TCA ATT
Gly Lys Glu
440
Asn Tyr Ile Leu Tyr Thr Asn Ile Ala Gly His Arg Asn Gln Gly Leu Ser Gly Trp Asn
1261 AAT TAT ATT TTG TAT ACA AAT ATT GCT GGA CAT AGA AAT CAA GGA CTT AGT GGT TGG AAT
AAT TAT ATT CTG TAT ACG AAT ATT GCA GGA CAT AGA AAA CAA GGG CTT AGT GGT TGG AAT
Lys
460
Tyr Val Ser Asp Gly Tyr Ser Thr Ile Tyr Lys Ile Leu Lys Gly Glu Asn Asp Gln Pro
1321 TAT GTT TCT GAT GGT TAT TCT ACA ATT TAT AAA ATT CTT AAA GGT GAA AAT GAT CAA CCT
TAT GTT TCT GAT GGC TAT TCT ACA ATA TAT AAA ATT CTT AAT GGT GAA AAT GAT CAA CCT
480
His Leu Arg Ser Tyr Val Asn Met Ala His Ala Phe Gly Thr Asp Thr Leu Ile Ala Leu
1381 CAT TTA AGG TCT TAT GTT AAT ATG GCA CAT GCA TTT GGA ACA GAC ACT TTA ATT GCT TTA CAT TTA AGG TCT TAT GTT AAT ATT GCA CAT GCA TTT GGA ACA GAT ACT TTA ATT GCT TTA
Ile
500
Val Lys Ser Tyr Tyr Gly Leu Trp Tyr Glu Asn Asn Phe Glu Ser Lys Tyr Ser Ile Lys
1441 GTT AAA TCT TAT TAT GGA TTA TGG TAT GAA AAT AAT TTT GAA AGT AAA TAT TCA ATT AAA GTT AAA TCT TAT TAT GGG CTA TGG TAT GAA AAT AAT TAT GAA GGT GAG TAT TCA ATT AAG
Tyr Gly Glu
520
Arg Asp Ser Thr Ser Ala Phe Cys Leu Leu Ala Ala Leu Val Thr Lys Arg Asp Thr Arg
1501 AGA GAT TCT ACC TCT GCT TTC TGT TTG TTA GCT GCA TTA GTT ACA AAA AGA GAT ACT AGA AGA GAT TCA ACT TCA GCT TTC TGT TTG TTA GCT GCA ATT GCT ACA AAA AGA GAT ACT AGA
Ile AΪa
540
Tyr Leu Cys Ser Leu Phe Lys Tyr Asp Ile Gln Ser Asn Val Ser Glu Ala Ile Lys Asn
1561 TAC TTA TGT TCT CTA TTT AAA TAT GAT ATA CAA TCA AAT GTT TCA GAA GCA ATT AAA AAT TAT TTA TGT TCT CTT TTT AAA TAC GAT ATA CAA CAA AAT GTT TCA GAA GCA ATT AAA AAC
Gln
560
Met Asn Tyr Pro Thr Tyr Tyr Pro Phe Phe Asn Leu Tyr Ala Met Ser Tyr Asn Gly Asn
1621 ATG AAT TAT CCA ACT TAT TAT CCA TTC TTC AAC CTC TAT GCC ATG AGT TAT AAT GGA AAT ATG AAT TAT CCA ACT TAT TAT CCA TTC TTC AAT jGTT TAT GCT ATG AGT TAC AAT GGA AAT
Val Table I, panel 3
580
Tyr Tyr Gly Arg Pro Tyr Lys Ile Pro Tyr Gly Arg Thr Arg Leu Asn Phe Thr Ala Thr
1681 TAC TAT GGA AGA CCC TAT AAA ATT CCA TAT GGA AGA ACT AGA TTG AAT TTC ACT GCA ACT
TAT TAT GGA AGA ACA TAT AAA ATT CCA TAT GGT ACA ACT AGA TTG AAT TTT ACA GCA ACC
Thr Thr
600
Thr Ala Ile Asp Pro Lys Ala Thr Ser Val Ser Tyr Thr Ile Lys Ser Gly Leu Thr Lys
1741 ACT GCT ATA GAT CCA AAA GCA ACT AGT GTT AGT TAT ACT ATT AAG TCT GGA TTA ACT AAA
ACT GCT ATA GAT CCA AGT GCA ACT AGT GTT AGT CAT AC£ ATT AAA TCT GGT TTA ACT AAA
Ser His
620
Gly Lys Leu Glu Arg Val Glu Asp Asn Val Tyr Asp Tyr Thr Pro Phe Phe Gly Ile Glu
1801 GGA AAA TTA GAA CGA GTT GAA GAC AAT GTT TAT GAC TAT ACA CCA TTC TTT GGA ATA GAA
GGA AAG TTA GAA CAA GTT GAA GAA AAT GTT TAT GAT TAT ACA CCA AAC TTT GGA GCA GAT
GTn Glu Asn Ala Asp
640
Glu Asn Asp Thr Phe Val Leu Asn Ile Asp Cys Val Val Asn Gly Glu Lys Val His Ile
1861 GAA AAT GAT ACA TTT GTT TTA AAT ATT GAT TGT GTT GTT AAT GGA GAA AAA GTA CAT ATC
GAA AAT GAT ACT TTT GTT TTG AAT ATT GAT TGT ATC GTT AAT GGA GAA AAG GTA CAT ATT
lle
660
Glu Gln Glu Gly Thr Phe Glu Leu Asp Pro His Gln Val Glu Tyr Glu Val Tyr Lys Asp
1921 GAA CAA GAA GGA ACA TTT GAA TTA GAT CCA CAT CAA GTA GAA TAT GAA GTT TAT AAA GAT
GAA CAA GAC GGA ACA TTT GAA CTA GAC CCA CAT CAA GTA GAG TAT GAA GTT TAT AAA GAT
Asp
680
Val Gln Thr Arg Asp Met Ala Gln Ala Ile Asn Ile Ile Gln Asn Lys Thr Arg Asn Asp
1981 GTT CAA ACA AGA GAT ATG GCA CAA GCT ATT AAT ATT ATT CAG AAT AAA ACT CGT AAT GAT
GTT AAA ACA AAA GAT ATG GAA CAA GCT CTT AAT ACT ATT CAG AAT AAA ACT JjCT AAT TAT
Lys Lys Glu Leu Thr Ser Tyr
700
Thr Gly Arg Ala Ser Phe Phe Gly Ile Gly Thr Tyr Asn Asp Gly Ser Met Gln Ser Met
2041 ACA GGA AGG GCT TCA TTC TTT GGA ATT GGA ACA TAC AAT GAT GGA TCA ATG CAA TCA ATG
ACA GGT ACG TCT ACA TTC TTT GGA ATT GGA AAT TAT gAT GAT GGA ACA ATG CAA TCA ATG
Thr Ser Thr Asn Asp Thr
720
Leu Val Glu Lys Gly Lys Leu Ile Val Pro Lys Ser Gly Tyr Tyr Thr Leu Phe Met Lys
2101 TTA GTA GAA AAA GGT AAA TTG ATA GTT CCA AAA TCT GGA TAT TAT ACA TTG TTT ATG AAA
TTA GTA GAA AAA GGT AAA CTG ATA GTT CCA ACA TCA GGA TAT TAT ACA TTG TTT ATG AAA
Leu Thr
740
Ala Asp Asp Leu Gly Arg Leu Leu Leu Asn Ile Thr Gly Glu Tyr Glu Gln Leu Leu Asp
2161 GCA GAT GAT TTA GGA AGA TTG TTA TTG AAT ATT ACT GGA GAG TAT GAA CAA TTA TTA GAT
GCA GAT GAT TTA GGA AGG TTG TTG TTA AAT GTT AAT GGA GAG TAT GAA CAA TTA TTA AAT
Val Asn Asn
760
Val Lys Thr Tyr Leu Gly Gly Tyr Ser Lys Thr Leu Asn Gly Ser Tyr Ala Thr Val Lys
2221 GTT AAA ACA TAT CTT GGA GGT TAT TCA AAA ACT CTT AAT GGA AGT TAT GCA ACT GTA AAA
GTT AAA ACA TAT CTT GGA GGT TAT TCA AAA ACT ATC AAT GGA ACT TAT GCA ACT GTA AAA
Ile Thr
780
Leu Glu Lys Asp Val Gly Tyr Pro Phe Ile Leu Tyr Asn Leu Asn Thr Gly Gly Gln Gly
2281 TTG GAA AAA GAT GTG GGA TAT CCA TTT ATT CTT TAT AAT TTG AAT ACT GGA GGA CAA GGA
TTA GAG AAA GAT ACT GAA TAT CCA TTT ATT CTT TAC AAC CTA AAT ACT GGA GGA CAA GGA Table I, panel 4
800
Phe Ile Arg Ile Gly Tyr Cys Tyr His Gly Thr Glu Glu Ser Ser Val Asp Val Ser Lys 2341 TTT ATT AGA ATA GGA TAT TGT TAT CAT GGA ACA GAA GAA TCA AGT GTT GAT GTT TCT AAA TTT ATT AGA ATA GGG TAT TGT TAT CAA GGA ACA GAA CAG TCT AGT GTT AAT GTT TCC AAA
Gln Gln Asn
820
Cys Ser Val Ser Asp Ile Gly Ser Ser Met Val Leu Asn Glu Lys Val Lys Thr Gly Ala
2401 TGC AGT GTA TCA GAT ATT GGA AGC TCT ATG GTT CTT AAT GAA AAA GTT AAA ACA GGA GCA TGT AGT GGA TTA GAT ATT GGA AGC
Gly Leu
840
Lys Glu Pro Glu Phe | Gln Ile Pro Pro Ile Lys Tyr Ser Arg Pro Thr Arg Phe Leu Thr
2461 AAA GAA CCA GAA TTC | CAA ATT CCA CCA ATT AAA TAC AGC AGA CCA ACA CGT TTC TTA ACT
860
Asn Ala Tyr Arg Thr Ile Pro Lys Cys Leu Asn Gly Asp Asp Ala Cys Ser Ile Lys Cys
2521 AAT GCA TAT AGA ACT ATT CCA AAA TGT TTG AAT GGT GAC GAT GCT TGT TCT ATT AAA TGT
880
Leu Ser Leu Leu Pro Leu Lys His Asp Asp Ser Ser Lys Cys Ser Asn Met Phe Asp Asp 2581 CTC TCC TTA TTA CCA CTT AAA CAT GAT GAT TCA AGT AAA TGT TCT AAT ATG TTT GAT GAT
900
Asn Tyr Ser Thr Met Tyr His Ser Arg Trp Thr Gly Gln Gly Thr Thr Phe Pro Val Asn
2641 AAT TAT TCT ACT ATG TAT CAT TCA AGA TGG ACT GGA CAA GGA ACT ACT TTC CCA GTT AAT
920
Tyr Thr Phe Glu Phe Ser Glu Asn Val Thr Phe Asn Asn Leu Tyr Val His His Arg Arg
2701 TAT ACA TTT GAA TTC TCA GAA AAT GTA ACA TTT AAT AAT CTT TAT GTT CAT CAT AGA AGA
940
Pro Glu Asp Ser Trp Gly Tyr Phe Glu Met Phe Val Lys Ser Pro Glu Thr Gly Glu Met 2761 CCT GAA GAT TCA TGG GGA TAC TTT GAA ATG TTT GTT AAA TCT CCA GAA ACA GGA GAA ATG
960
Glu Leu Leu Glu Lys Tyr Lys His Pro Lys Ser Thr Thr Thr Glu Leu Asn Phe Gln Lys
2821 GAG TTA TTA GAA AAA TAT AAG CAT CCA AAG TCT ACT ACA ACA GAA CTT AAT TTC CAA AAA
980
Leu Val Thr Thr Asp Arg Val Gln Phe Ile Val Tyr Asn Asn Ser Asn Gly Gly Asn Tyr 2881 TTA GTT ACA ACT GAT CGT GTC CAA TTT ATT GTC TAT AAT AAT TCA AAT GGT GGA AAT TAT
1000
Val Asn Val Val Glu Leu Ser Phe Asn Ile Lys Glu Thr Phe Lys Asn Tyr Thr Asn Ser
2941 GTC AAT GTT GTA GAA TTG TCT TTC AAT ATT AAG GAA ACC TTT AAG AAT TAT ACA AAT TCA
1020
Phe Gly Pro Lys Ile Lys Ser Thr Gly Phe Lys Lys Val Thr Thr Pro Gly Ala Ser Gly
3001 TTT GGA CCA AAG ATT AAA AGT ACT GGA TTT AAG AAA GTT ACT ACA CCA GGT GCT TCA GGA
1040
Gly Tyr Leu Ala Val Asn Glu Lys Glu Gly Glu Gly Ser Leu Cys Phe Lys Ala Lys Val
3061 GGA TAT CTT GCA GTA AAT GAA AAG GAA GGA GAA GGA TCA CTT TGT TTC AAA GCT AAA GTC
1060
Thr Lys Phe Gly Leu Tyr Gly Tyr Arg Lys Thr Thr Ser Gly Lys Phe Arg Val Thr Ile
3121 ACT AAA TTC GGT CTT TAT GGA TAT AGA AAA ACA ACA TCT GGA AAG TTT AGA GTT ACA ATT
1080
Asp Ser Gln Pro Gly Glu Val Thr Ser Gln Ser Tyr Phe Ser Asp Ser Glu Arg Thr Leu 3181 GAC TCA CAA CCA GGT GAA GTT ACT AGC CAA AGT TAT TTC TCT GAC TCT GAA CGA ACT TTG
1100
Phe Tyr Ala His Thr Phe Asp Glu Thr Glu Ala Asn Lys Val His Asn Ile Cys Met Glu 3241 TTC TAT GCT CAT ACT TTT GAT GAA ACT GAA GCA AAC AAA GTT CAT AAC ATT TGT ATG GAA
1114
Val Val Glu Gly Thr Val Asn Leu Asp He He Gly Ser Ser - - -
3301 GTT GTT GAA GGA ACA GTT AAT CTT GAT ATC ATT GGT TCT TCT TAA aegttaattgaagatattt
3444 3365 cattttaaataatgtagtgttattttaattttattgagaaaattttgagtctattteattacatattgaatcatgattg The M17 protein has been determined to be membrane associated. When these proteins are cross-linked with multivalent antibody molecules, live
trophozoites cap the complexes in a manner characteristic of membrane capping. See Figure 2. This result is achieved with pooled patient sera, with the FA7
monoclonal antibody, or with immunoselected antibody preparations. Cells also appear to round upon antibody binding, are likely disrupted biologically, and perhaps even unable to divide. Thus, the immunodominant antigen is determined to be a surface antigen and attachment by antibodies is likely to cause significant disruption of the infective cycle of the trophozoites.
The present invention, in one embodiment, provides polypeptides which are related to the 125 kDa surface antigen and which will usually be either haptenic or antigenic, typically including at least about 6 amino acids, usually at least about 9 amino acids, and more usually about 12 or more amino acids found contiguously within a natural form of the 125 kDa immunodominant surface antigen protein. Longer polypeptides will also find use, up to and including substantially full length of the natural protein and larger. The contiguous amino acids may be located within any region of the
polypeptide, but preferably in regions I or III of Table I, and will correspond to at least one epitope site which is characteristic of the particular immunodominant surface antigen protein. By characteristic, it is meant that the epitope site will allow immunologic detection of the exposed polypeptide segment in a cell sample with reasonable assurance, in most cases allowing
immunodominant surface antigen of a pathogenic form to be immunologically distinguished from other related
proteins, such as an immunodominant surface antigen from non-pathogenic strains. The polypeptides will also be capable of inducing an immune response in a host when used in vaccines, in therapeutic compositions, and for preparing polyclonal and monoclonal antibodies.
The compositions and methods of the present invention are particularly useful for identifying and treating pathogenic amoebic infection, but it will also be possible to identifying epitopes of the surface antigen which are conserved among pathogenic and non-pathogenic amoeba. Use of polypeptides and nucleic acid probes based on the sequences of such conserved epitopes allows detection and treatment of both pathogenic and non-pathogenic amoeba. In contrast compositions based on epitopes found in pathogenic forms only allows specific detection and treatment of pathogenic amoeba.
Conversely, epitopes found in non-pathogenic forms only allow specific detection and treatment of non-pathogenic amoeba. Regions of particular importance for
distinguishing pathogenic strains from non-pathogenic strains will be those located at the sites of difference between the amino acid or nucleotide differences, as indicated in Table III.
In a preferred aspect, the polypeptide
compositions will be either identical or equivalent to a sequence set forth in Table I. By equivalent, it is meant for the purposes of defining polypeptides that a substantial identity of amino acid sequences exists over a stretch of at least about 10 residues, where each position of the corresponding sequences is either
identical to or has a conservative substitution in at least about 60% of the residues, preferably at least about 70% of the residues and more preferably at least about 80% of the residues. The compared sequence
segments may, however, be modified by occasional
deletions, additions, or replacements in accordance with known methods for comparison. See, e.g., Sequence
Analysis Software Package. Univ. Wisconsin Biotechnology Center, Madison, Wisconsin. Conservative substitutions are replacements within the groups gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys , arg; and phe, tyr.
Synthetic polypeptides which are
immunologically cross-reactive with a natural
immunodominant surface antigen protein (i.e.,
immunological analogs) may be produced by either of at least two general approaches. First, polypeptides having fewer than about 50 amino acids, more usually fewer than about 20 amino acids, can be synthesized by the
Merrifield solid-phase synthesis method where amino acids are sequentially added to a growing chain. See
Merrifield (1963) J. Am. Chem. Soc. 85:2149-2156. The amino acid sequences of such synthetic polypeptides will usually be based on the sequences described in Table I, preferably regions I or III.
A second and preferred method for synthesizing the polypeptides of the present invention involves the expression in cultured cells of recombinant DNA molecules encoding a desired portion of an immunodominant surface antigen gene. The gene may itself be natural or
synthetic as described below. The natural gene is obtainable from cDNA or genomic libraries, as described herein. Using such segments or genes, additional
homologous gene sequences might be isolated from other related strains or sequences which encode peptides equivalent to those described above. Such genes
represent, among other possibilities, other alleles of or phantom genes of related polypeptides. By using
sequences from such genes, segments may be found which have similar biological functions which might be
equivalent to these described and would be substituted for the listed segments. In particular, epitopic similarity can serve as a means for selecting equivalent peptides.
To be useful in the detection methods of the present invention, the polypeptides are usually obtained in substantially pure form, that is, typically about 50% w/w or more purity, substantially free of interfering proteins and contaminants. Preferably, the immunodominant surface antigen polypeptides are isolated or synthesized in a purity of at least about 80% w/w and, more
preferably, in at least about 95% w/w purity. Using conventional protein purification techniques, homogeneous polypeptides of at least 99% w/w can be obtained. For example, the proteins may be purified by use of the antibodies described hereinafter using immunoadsorbent affinity chromatography. Such affinity chromatography is performed by first linking antibodies or appropriate affinity reagents to the solid support and then
contacting the linked antibodies or affinity reagents with the source of the immunodominant surface antigen proteins, e.g., lysates of protozoa which naturally produce immunodominant surface antigen or which produce immunodominant surface antigen as a result of
introduction of a recombinant immunodominant surface antigen DNA molecule.
Useful production cultures include the insect
Bacculovirus system, see Luckow and Summers (1988)
Biotechnology 6: 47-55, or the T7 polymerase gene with a ø10 promoter, see Rosenberg et al. (1987) Gene 56: 125-135, and Studier and Moffatt (1986) J. Mol. Biol. 189: 113-130, each of which is hereby incorporated herein by reference. Various other high capacity systems
expressing recombinant nucleic acids, described below, are provided herein. See, e.g., Sambrook, et al. (1989) Molecular Cloning: A Laboratory Method. Cold Spring Harbor Press.
In accordance with the present invention, nucleic acid seσ snces encoding portions of the
polypeptide sequence of distinct forms of immunodominant surface antigens have been isolated and characterized. Isolated DNA segments encoding the immunodominant surface antigens can be expressed to provide isolatable
quantities of polypeptides displaying biological (e.g., immunological) properties of naturally-occurring
immunodominant surface antigens. Other useful nucleic acids are substantially homologous to encoding sequences, either natural or artificial. These homologous
polynucleotides will find use as probes or primers for locating or characterizing natural or artificial nucleic acids encoding the surface antigen peptides.
Substantial homology or substantial identity of a nucleic acid sequence indicates either that: a) there is greater than about 65%, typically greater than about 75%, more typically greater than about 85%, preferably greater than about 95%, and more preferably greater than about 98% homology with a disclosed segment of at least about 10 contiguous nucleotides; or b) the homologous nucleic acid sequence will hybridize to the consensus sequence or its complementary strand under stringent conditions of temperature and salt concentration. These stringent conditions will generally be at temperatures greater than about 22ºC, usually greater than about 30ºC and more usually greater than about 45ºC. Salt
concentrations are generally less than about 1 M, usually less than about 500 mM, and preferably less than about 200 mM. The combined conditions will be more important than either the salt concentration or the temperature alone. Other parameters which are used to define
stringency include GC content of the sequence, extent of complementarity of the sequences and length of segments involved in the hybridization, besides composition of buffer solutions used in the hybridization mixture.
Nucleic acids can be synthesized based directly on the DNA sequences reported in Table I.
Polynucleotides may be synthesized by known techniques, for example, short single-stranded DNA fragments may be prepared by the phosphoramidite method described by
Beaucage and Carruthers (1981) Tett. Letters 22:1859- 1862. A double-stranded fragment may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate
conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
Polymerase chain reaction techniques may be used for production of probes or amplification of
polynucleotides for synthetic purposes. See Innis et al. (Ed.) (1990) PCR Protocols. Academic Press, N.Y., which is hereby incorporated herein by reference.
The natural or synthetic DNA fragments coding for a desired immunodominant surface antigen fragment will often be incorporated in DNA constructs capable of introduction to and expression in an in vitro cell culture. Usually, the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction and integration within the genome of cultured mammalian, protozoa, or other eucaryotic cell lines. DNA constructs prepared for introduction into bacteria or yeast will usually include a replication system recognized by the host, the appropriate immunodominant surface antigen DNA fragment encoding the desired polypeptide product, transcriptional and translational initiation regulatory sequences joined to the 5'-end of the immunodominant surface antigen DNA sequence, and transcriptional and translational termination regnlatory sequences joined to the 3'-end of the immunodominant surface antigen
sequence. The transcriptional regulatory sequences will typically include a heterologous promoter which is recognized by the host. Conveniently, available
expression vectors which include the replication system and transcriptional and translational regulatory
sequences together with an insertion site for the
immunodominant surface antigen DNA sequence may be employed.
Synthesis and production of the immunodominant surface antigen polypeptides, fragments, fusion proteins, and variants thereof have been described above. Similarly, the nucleic acids and fragments which encode or are homologous to sequences which encode these
epitopes have been described. Expression of
characteristic forms of the immunodominant surface antigens have been associated with amoebiasis, and even distinction between pathogenic or non-pathogenic
iiinfection may be achieved. As further demonstrated herein, pathogenic infection is characterized by
detectable expression of the polypeptide epitopes of pathogenic-specific immunodominant surface antigen proteins. Non-pathogenic infection is likewise
detectable using non-pathogenic-specific epitopes.
Production of antibodies to characteristic epitopes will allow for distinguishing between pathogenic and non-pathogenic strains. General detection of
infection may use epitopes common to both pathogenic and non-pathogenic strains, but distinguishing will usually be directed to epitopes involving amino acids which differ and are indicated in Table I.
Once a sufficient quantity of intestinal immunodominant surface antigen polypeptide has been obtained, polyclonal antibodies specific for the
immunodominant surface antigen protein can be produced by in vitro or in vivo techniques. In vitro techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or fragments, while in vivo techniques require the injection of the polypeptides or fragments into any of a wide variety of target immune systems, such as vertebrates. Suitable vertebrates are typically non- human, including mice, rats, rabbits, sheep, goats, and the like. Polypeptides having more than about 5 to 30 amino acids, particularly more than about 50 to 100 amino acids, may serve directly as immunogens. if the
polypeptide is smaller than about 10 kD, particularly less than about 6 kD, it may be necessary to join the polypeptide to a larger molecule to elicit the desired immune response. The immunogens are then injected into the animal according to a predetermined schedule, and the animals are bled periodically with successive bleeds generally having improved titer and specificity. The injections may be made intramuscularly,
intraperitoneally, subcutaneously, or the like, and usually an adjuvant, such as incomplete Freund's
adjuvant, will be employed.
While the invenntion embraces antibodies made against polypeptides provided, it also embraces proteins which are specifically recognized by antibodies made against epitopes provided herein. Thus, the FA7
monoclonal antibody also defines a class of proteins which are targets for specific binding.
If desired, monoclonal antibodies can be obtained by preparing immortalized cell lines capable of producing antibodies having the desired specificity. The FA7 monoclonal antibody is produced by such a cell line. Such immortalized cell lines may be produced in a variety of ways depending upon the target immune system.
Conveniently, a small vertebrate, such as a mouse, is hyperimmunized with the desired antigen by the method just described. The vertebrate is then killed, usually several days after the final immunization, the spleen removed, and the spleen cells immortalized. The manner of immortalization is not critical. Presently, the most common technique is fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519. Other techniques include EBV transformation, transformation with bare DNA, e.g., oncogenes, retroviruses, etc., or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies. Common techniques are described, e.g., in Lane and Harlow, (1988)
Antibodies: A Laboratory Manual. Cold Spring Harbor Press, N.Y.; and Goding (1986) Monoclonal Antibodies:
Principles and Practice. (Second Edition) Academic Press, N.Y., each of which is hereby incorporated herein by reference. New techniques for in vitro production of antibodies may also be applied. See, e.g., Huse et al. (1989) Science 246: 1275-1281, Ward et al. (1989) Nature 341: 544-546, each of which is hereby incorporated herein by reference.
When employing fusion with a fusion partner, the manner of fusion is usually not critical and various techniques may be employed. Conveniently, the spleen cells and myeloma cells are combined in the presence of a nonionic detergent, usually polyethylene glycol, and other additives such as Dulbecco's Modified Eagle's
Medium, for a few minutes. At the end of the fusion, the nonionic detergent is rapidly removed by washing the cells. The fused cells are promptly dispensed in small culture wells (usually in a microtiter plate) at
relatively low density, ranging from about 1-5×105 per well, in a selective medium chosen to support growth of the hybrid cells while being lethal to the myeloma cells. Usually, the myeloma cell line has been mutated to be sensitive to a lethal agent, typically being HAT
sensitive. After sufficient time, usually from one to two weeks, colonies of hybrids are observed and plates containing hybrid positive wells are identified. The plates and wells having only one colony per well are selected, and supernatants from these wells are tested for binding activity against the desired intestinal immunodominant surface antigen protein or the isolated antigen. Once positive hybridomas are identified, the cell line can be maintained as viable cultures and/or by lyophilization or frozen storage.
Depending on the desired use for the antibodies, further screening of the hybridomas may be desirable. Hybridomas providing high titers are
desirable. Furthermore, cytotoxic antibodies, e.g., IgG2a, IgG2b, IgG3 and IgM, may be selected for use in therapeutic treatment of pathogenic infections. For use in immunodiagnostic assays, antibodies having very high specificity fsr the antigenic site are desirable.
Once the desired hybridomas have been selected, monoclonal antibodies may be isolated from the
supernatants of the growing colonies. The yield of antibodies obtained, however, is usually low. The yield may be enhanced by various techniques, such as injection of the hybridoma cell line into the peritoneal cavity of a vertebrate host which will accept the cells.
Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Proteinaceous and other contaminants will usually be removed from the monoclonal antibodies prior to use by conventional technique, e.g., chromatography, gel filtration, precipitation,
extraction, or the like.
By properly selecting polypeptides used as the immunogen, antibodies having high specificity and
affinity for the desired immunodominant surface antigen epitope can be obtained. The polypeptide selected should represent one or more epitopic sites which are unique to the desired immunodominant surface antigen protein and which can distinguish immunodominant surface antigen from closely related proteins. Such unique epitopes are found on polypeptides expressed by cells containing sequences disclosed in Table I. One particular example of such is the FA7 monoclonal antibody.
The polypeptides and antibodies of the present invention may be used with or without modification.
Frequently, the polypeptides and antibodies will be labelled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, which are incorporated herein by reference.
Antibodies and polypeptides prepared as
described above can be used in various immunological techniques for detecting immunodominant surface antigen proteins in biological specimens, particularly cell samples such as biopsy tissue samples and body fluid samples, including blood, plasma, serum, urine, stool, and the like. As amoebiasis infections typically
originate in the intestinal flora, stool samples will routinely be the samples of choice. Depending on the nature of the sample, both liquid phase assays and solid-phase immunohistochemical staining techniques will find use. Conveniently, immunohistochemical staining
techniques may be used with cell samples including tissue samples, sputum, and lung lavage samples. For example, a tissue sample may be fixed in formalin, B-5, or other standard histological preservative, dehydrated and embedded in paraffin as is routine in any hospital pathology laboratory. Sections may then be cut from the paraffinized tissue block and mounted on glass slides. The immunodominant surface antigen proteins, if present, may then be detected in the cytoplasm or extracellular space by exposure with labeled immunodominant surface antigen antibody or exposure to unlabelled anti- immunodominant surface antigen antibody and a labeled secondary antibody. Because the antigen is a surface antigen and exposed extracellularly, it is unnecessary to denature a sample, allowing assay of non-denatured target sample. This may also allow further live
characterization of the same tested sample. Sputum and lavage samples are typically prepared in a similar manner where the sample is first dehydrated by exposure to a dehydrating agent, typically a low molecular weight alcohols Liquid phase immunoassays or Western blot analysis will also find use in the detection of the immunodominant surface antigen proteins particularly in body fluids when the proteins are shed into such fluids, e.g., blood or stool. Solid tissue and sputum samples may also be assayed in liquid phase systems by lysing the cellular sample in order to release the protein. Once the protein is released, the sample will be placed in a suitable buffer, the sample buffer subjected to a
suitable immunoassay. Numerous competitive and non-competitive immunoassays are available and described in the scientific and patent literature. Having described how to make various diagnostic reagents, particularly antibodies polyclonal and monoclonal, and fragments thereof with binding sites, diagnostic kits for using them are also provided. Similar kits may be prepared using particular nucleic acid probes. The kits will typically have at least one compartment comprising the detection reagent to be applied to an appropriate sample. The reagent may be attached to a dipstick or similar physical entity. Alternatively, the reagent may be contained in a liquid solution to which a sample is added. A compartment containing the active ingredient may be a sealed envelope, a plastic bag, a vial, a bottle, a jar, an ampule, a well, or any other protective package.
The antibodies of the present invention may also find use in therapy and other medical applications. For example, immunodominant surface antigen antibodies, may be coupled to toxins, such as diphtheria toxin and the ricin A chain, and administered to patients, or hosts, suffering from pathogenic Entamoeba infections. The use of antibody conjugated toxins in cancer therapy is described generally in U.S. Patent Nos. 4,093,607;
4,340,535; 4,379,145; and 4,450,154. Antibodies alone may also find use in treatment, particularly by blocking or interrupting some functional activity of immunodominant surface antigen protein which contributes to the pathogenic phenotype.
In a particular embodiment of the present invention, the binding fragments will be joined to active substances, such as proteolytic enzymes or glycosidases, in order to enhance interference with amoeba infection. The binding fragments will specifically bind to the trophozoites and the active substances will act to destroy the protozoa.
Vaccines against Entamoeba infections are producible using the compositions of the present
invention. These vaccines may be passive, consisting of Ig supplementation which interferes with amoeba growth or toxicity. Alternatively, vaccines may be active
producing a cellular response providing protective immunity by cytotoxic or other active suppression of amoeba infections.
A vaccine prepared utilizing the immunodominant surface antigen or immunogenic equivalents thereof can consist of: (a) fixed cells either recombinantly altered to produce these antigen proteins or cells from the
Entamoeba itself; (b) a crude cell extract; (c) a
partially or completely purified immunodominant surface antigen preparation. Fusion proteins combining a segment of the immunodominant surface antigen will be readily prepared. In some embodiments, a multiple vaccination may be achieved by fusing these antigens with other target antigens on a single protein, inducing protection against multiple infectant vectors. Alternatively, a "cocktail" of different immunogens may be simultaneously administered or inoculated. These immunogens can be prepared in vaccine dose form by well-known procedures. These vaccines can be in the form of an injectable dose and may be administered intramuscularly, intravenously, or subcutaneously. These vaccines can also be
administered intranasally by aspiration, or orally by mixing the active components with food or water, providing a tablet form, and the like. Means for
administering, or more typically inoculating, these vaccines should be apparent to those skilled in the art from the teachings herein; accordingly, the scope of the invention is not limited to any particular delivery form.
For parenteral administration, such as
subcutaneous injection, these immunogens can be combined with a suitable physiologically acceptable carrier, for example, it can be administered in water, saline,
alcohol, fats, waxes, or buffered vehicles with or without various adjuvants or immunomodulating agents.
Suitable immunological adjuvants or agents include, but are not limited to, aluminum hydroxide, aluminum
phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum
(Proplonobacterium acnes), Bordetolla pertussis,
polyribonucleotides, sodium alginate, lanolin,
lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's
Incomplete Adjuvant and Complete Adjuvant (Difco
Laboratories, Detroit, Michigan). Other suitable
adjuvants are Amphigen (oil-in-water), Alhydrogel
(aluminum hydroxide), or a mixture of Amphigen and
Alhydrogel.
The Rotavirus VP6 carrier system developed by
VIDO (Veterinary Infectious Disease Organization,
Saskatoon, Canada), although not an adjuvant, is also suitable.
The proportion of xmmunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts. On a per-dose basis, the amount of the immunogen can range broadly from about l.o pg to about 100 mg per kg of host, usually at least about 10 pg, typically at least about 100 pg, and preferably at least about l ng per kg of host weight, and usually less than about 1 mg, typically less than about 10 μg, and more typically less than about 1 μg, and preferably less than about 100 ng per kg of host. A preferable range is from about 10 pg to about 100 ng per dose. A suitable dose size will usually be between about .01 and 5 ml, preferably about 0.5 ml for a 20-59 kg organism.
Comparable dose forms can also be prepared for parenteral administration to smaller or larger animals, but the amount of immunogen per dose will usually be smaller, for a smaller animal.
For the initial vaccination of immunologically naive animals, a regiment of between 1 and 4 doses can be used with the injections spaced out over a 2 to 6-week period. Typically, a two-dose regimen is used. The second dose of the vaccine then should be administered some weeks after the first dose, for example, about 2 to 4 weeks later. Animals that have been previously exposed to Entamoeba or have received colostral antibodies from the mother may require booster injections. The booster injection is preferably timed to coincide with the vulnerable point in the life cycle of the Entamoeba.
Periodic revaccination is advisable under certain conditions.
The vaccine may also be combined with other vaccines for other diseases to produce multivalent vaccines. It may also be combined with other
medicaments, for example, antibiotics. A
pharmaceutically effective amount of the vaccine can be employed with a pharmaceutically acceptable carrier such as viral capsid protein complex or diluent understood to be useful for the vaccination of animals.
Other vaccines may be prepared according to methods well-known to those skilled in the art as set forth, for example, in Tizard, L., An Introduction to Veterinary Immunology, 2nd Ed (1982), which is
incorporated herein by reference.
The following experiments are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
I. Entamoeba Isolates and Cell Culture
II. Human Immune Sera
III. Anti-membrane Fraction Serum
IV. Monoclonal Antibody FA7
V. Western Blot Analysis
VI. Antibody Capping by Live Trophozoites
VII. Preparation and Screening of Libraries
VIII. Sequence Analysis
IX. Primer Extension Sequence Analysis
X. Gene Copy Number
XI. Detection of Sequences Related to M17 in Non- Pathogenic E. histolytica
XII. Restriction Fragment Length Polymorphisms
Using pooled human immune sera a cDNA clone (cM17) encoding this antigen (M17) was isolated from a λgtll expression library of the virulent strain E.
histolytica HMI:IMSS. Monospecific antibodies, purified by binding to phage lysate of cM17, and monoclonal
antibody FA7 reacted exclusively with the 125 kDa antigen by Western blot analysis. Surface binding and cap
formation was observed with patient sera, purified
monospecific antiseruro and monoclonal antibody FA7.
Corresponding genomic clones (pBSgM17-l/2/3) were
isolated by hybridization with the cDNA clone. These contained an open-reading frame of 3345 bp which is in good agreement with the mRNA size of approximately 3.0 kb as revealed by Northern hybridization with λcM17. The inferred amino acid sequence predicted a 125,513 Da protein which contains 17 potential N-linked
glycosylation sites and is unusually rich in tyrosine and asparagine residues. A distinctly hydrophobic amino terminal region may serve as membrane anchor or signal sequence. Some restriction enzymes were found which allowed PCR diagnosis of non-pathogenic and pathogenic isolates with the exclusion of E. histolytica-like
Laredo.
I. Entamoeba Isolates and Cell Culture
Trophozoites of the axenized E. histolytica strains (HM1:IMSS, NIH:HK9) and E. histolytica-like
Laredo were grown in TYI-S-33 media as described by
Diamond, et al. (1978) "A New Medium for the Axenic
Cultivation of Entamoeba histolytica and Other
Entamoeba," Trans. R. Soc. Trop. Med. Hvg. 72: 431-432.
Polyxenic isolates were grown in liquid Robinson's medium supplemented with 10% bovine serum and containing 5 μg per ml of medium of each of the following antibiotics: kanamycin, erythromycin, and ampicillin. Amoebae were pelleted by centrifugation at 900 rpm and washed twice with phosphate buffered saline, pH 7.5 (PBS). Polyxenic amoebae were further purified by centrifugation through a Percoll/PBS cushion at 3000 rpm in a refrigerated
Accuspin centrifuge. Isolates SD4 (pathogenic, zymodemes II) and REF 291 and SD116 (non-pathogenic, zymodemes III and I), were a generous gift of Dr. Sharon Reed from the University of California, San Diego. Non-pathogenic isolates #43 and #44 and pathogenic isolate #46,
classified by zymodeme analysis using gradient PAGE were isolated in Mexico City. See Meza, et al. (1986)
"Isoenzyme Patterns of Entamoeba Histolytica Isolates from Asymptomatic Carriers: Use Of Gradient Aσrylamide Gels." Am. J. Trop. Med. Hvg. 35: 1134-1139. They correspond to Sargeaunt zymodemes I, I, and II,
respectively.
II. Human Immune Sera
Sera from 108 patients with amoebic liver abscesses were obtained from Drs. A. Isibasi and R. Landa at the Instituto Nacional de la Nutricion and La
Raza-IMSS Hospitals, Mexico City. Diagnosis of hepatic abscess in patients was established by clinical symptoms, countercurrent immunoelectrophoresis, ELISA and
rectosigmoidoscopy. Human sera from donors without history of amoebiasis and negative for anti-amoebic antibodies as tested by immunoblot served as controls. Western blots of whole trophozoites were prepared by suspending washed cells in PBS containing 10 mM
p-hydroxymercuribenzoate and Laemli sample buffer, boiling for 5 min, fractionation by 10% or 5-15% gradient SDS-PAGE and electrophoretic transfer to nitrocellulose filters. All sera were evaluated by Western blot
analysis on extracts of whole amoebae. Twenty-nine sera with the highest titer were selected from the 108 samples and were pooled. III. Anti-membrane Fraction Serum
A membrane fraction was prepared as described in Aley, et al. (1980) J. EXP. Med. 152: 391-404, and diluted 1:1 with PBS and complete Freund's adjuvant.
Mice were immunized intraperitoneally with 300 μg every two weeks until titers reached 1:5000 as assayed by
Western blot.
IV. Monoclonal Antibody FA7
Whole amoebic extract from 2 × 106 amoebae was fractionated by preparative 5-15% gradient SDS-PAGE.
After electrophoretic transfer to nitrocellulose the 125 kDa region was excised from the blot, ground to a powder and suspended in PBS. One hundred μl of the suspension was diluted 1:9 with PBS and injected three times
intraperitoneally into mice at two week intervals with a final boost before the fusion. Hybridomas were selected by positive reaction with the 125 kDa band in Western transfers of E. histolytica extracts. Harvest fluid from clone FA7 was used at a 1:1000 dilution in Western blot analysis. V. Western Blot Analysis
Western blot analyses were performed using standard procedures. See, e.g., Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York, which is hereby incorporated herein by reference. E. histolytica whole cell extracts were reacted with individual serum samples from each of the 108 infected patients. Seven antigens (220, 190, 160, 125-129, 96, 75, 46 kDa) were detected by more than 62% of the sera. Amongst these seven, a 125 kDa antigen was immunodominant, reacting strongly and being
recognized by more than 70% of the serum samples. We assume, based on their molecular weight and serological reactivity, that the 220 kDa antigens represents an N-acetyl-gluσosamine adherence lectins, the 160 kDa antigen represents an N-acetyl-D-galactosamine adherence lectin, and the 96 kDa antigen represents an integral membrane protein. A Western blot of whole cell extracts of axenically or polyxenically propagated pathogenic and polyxenically propagated non-pathogenic E. histolytica isolates was assayed with a pooled subset of 29 human immune sera. The sera reacted strongly with a 125 kDa antigen in all isolates regardless of source. See Figure 1. Polyspecific antiserum prepared against amoebic plasma membrane also reacted strongly with the 125 kDa antigen. The monoclonal antibody FA7, prepared against partially purified 125 kDa antigen, reacted specifically with an epitope of the 125 kDa antigen; by Western analysis with FA7 this epitope was detected in different strains and species of Entamoeba. In the Western blot probed with monoclonal antibody FA7, additional bands of lower molecular weight and varying intensity are apparent in most of the isolates. Potent proteases are present in whole amoebic extracts, so we assume that these are degradation products of the 125 kDa antigen, though processing intermediates of unknown origin cannot be ruled out. VI. Antibody Capping by Live Trophozoites
Human immune serum, hybπdoma harvest fluid from clone FA7, and purified monospecific antiserum was added to live trophozoites at 1:500, 1:2000, and
undiluted, respectively. After formation of caps (10 min at 37ºC), cells were fixed with 3.7% formaldehyde, washed with PBS and stained with fluorescein-isothiocyanate labeled anti-human or anti-mouse IgG. Undiluted harvest fluid from an anti-actin producing clone was used as control for a non-surface antigen.
Live trophozoites cap antibody-antigen
complexes bound to their surface. Antibody-antigen caps were induced in HM1:IMSS trophozoites by incubation with the pooled patient serum, monoclonal antibody FA7, or monospecific antibody recovered after specific binding and elution of pooled patient sera to phage lysates of cDNA clone λcM17. See Fig. 2. A negative control antibody (monoclonal anti-actin antibody) neither bound to trophozoite surfaces nor induced cap formation. These results indicate that the 125 kDa antigen is localized to the surface of the amoebae. Besides capping the cross-linked antigens, it appeared that the cells also rounded up. This is indicative of an interruption of normal biological function and probably indicates that attachment to antibodies would disrupt cellular
functions, including cell division and infective cycle functions. VII. Preparation and Screening of Libraries
Genomic DNA and poly(A)+ RNA isolation and construction of the λgtll cDNA library from strain E.
histolytica HM1:IMSS has been described previously. See, e.g. Edman, et al. (1987) Proc. Natl. Acad. Sci. USA. 84: 3024-3028. A genomic library from E. histolytica
HM1:IMSS was made by adding 600 μl of Nal (GeneClean kit;
BiolOl) to 200 μl (approximately 20 μg DNA) of agarose embedded nuclei in an Eppendorf tube and melted by incubation at 60ºC for 5 min. Twenty μl of glassmilk was added, and suspended well and the mixture incubated at room temperature for 5 min. The sample was vortexed for 1 min to shear the DNA and spun in a microfuge for 5 sec. After removal of the supernatant the pellet was suspended in 1 ml wash buffer by vortexing for 30 sec. The
glassmilk was pelleted by a 5 sec spin in the microfuge and the supernatant was removed. The wash was repeated twice and the sheared and purified DNA eluted into 100 μl TE (10 mM Tris-HCl (pH 8), 1 mM EDTA) by incubation at 37ºC for 5 min. Recovery and degree of shearing were assessed by agarose gel electrophoresis. All subsequent steps including addition of Eco RI linkers, methylation, ligation into the vector ZAPII and packaging reaction were performed by standard procedures. See, e.g.,
Sambrook et al. (1989); Gubler, U. and B. J. Hoffman (1983) Gene. 25: 263-269; and Morgan, et al. (1987),
Nature. 329: 301-307. The λgtll cDNA library (3 × 105 phage) was screened with the pool of 29 patient sera at a 1:200 dilution. The genomic library was screened with the 32P-α-dCTP labeled EcoRI fragment of λcM17.
Forty-six reactive clones were each plaque purified and tested for recognition by each of the 29 patient sera included in the serum pool and by the anti-membrane antibody. Clone λcM17 strongly reacted with 26 out of 29 patient sera as well as with the anti-membrane serum. Monospecific antibody was selected from the pooled human sera by elution from filter-bound phage lysate of λcM17. This eluate reacted with a single polypeptide of 125 kDa by Western blot analysis of whole amoebic extracts. Phage lysate of λgtll, serving as negative control, did not bind antibodies reacting with amoebic antigens. Plasmids were rescued from genomic λZAPII clones. Phage DNA and plasmid DNA were purified by standard methods. VIII. Sequence Analysis
With the exception of the first 207 bp, the entire sequence of gene M17 was determined on both strands in genomic clone pBSgM17-l and on one strand in genomic clone pBSgM17-2. See Table I. The internal Eco RI fragment representing the cDNA insert was also
sequenced on both strands using nested deletion templates created with the Promega system. Double-stranded
sequence was also determined for two PCR fragments obtained by amplification of genomic DNA from isolate REF 291, Zymodeme III, derived from an asymptomatic Costa Rican refugee and kindly provided by Dr. S.L. Reed.
Several oligonucleotides were used as primers in
single-stranded DNA (M13mp18/19) and double-stranded DNA (pBSKS(+)) sequencing reactions with the Sequenase system (US Biochemicals) or ABI Sequencer (Applied Biosystems Inc.).
The 1.9 kB insert of λcM17 revealed an ORF (open reading frame) spanning the entire insert. See Table I. The lack of a 5' initiating methionine, the absence of a poly(A) -tail and hybridization to a -3 kb mRNA by Northern blot analysis indicated that amino and carboxy terminal sequences were lacking in λcM17. See Fig. 3.
To isolate a genomic clone and obtain the amino and carboxy terminal sequences, a λZAPII library from E. histolytica HM1:IMSS was screened using the 1.9 kb insert of λcM17 as a probe. Three genomic clones were
identified, and two of these were sequenced using
oligonucleotide primers derived from the cDNA sequence.
The nucleotide sequence of the cDNA was identical in both genomic clones. An additional 556 bp of 5' and 870 bp of 3' sequence yielded an ORF of 3345 bp which was also identical in both genomic clones. The size of this ORF (gene M17) is in reasonable agreement with the mRNA size of ≈ 3000 bp determined by Northern blot analysis. The inferred amino acid sequence predicts a 125 kDa protein.
The 5' flanking sequence of gene M17 shares striking similarities with the 5' flanking region of both actin and ferredoxin genes, the only other genes of E. histolytica where sequence has been determined. See Table II. The transcriptional start site of M17 was mapped to an adenine residue 17 bp 5' of the start codon by primer extension sequence analysis using an
oligonucleotide SRO9. 5' untranslated regions of actin (11 bp) and ferredoxin (9 bp) genes were likewise very short as compared with other eukaryotic gene transcripts. A common sequence motif, 5ΑTTCA3', is present at the transcriptional start site of both M17 and actin genes, the initiating nucleotide being an adenine residue as is most frequently found in other eukaryotes. While the same motif is also present in the flanking sequence of the ferredoxin gene, its cap site was mapped to the 3' thymidine rather than the 5' adenine residue. An
additional sequence motif shared amongst these genes is YATTTAAA present at -29, -31, -32, and -32 for the M17, actin*, actin§, and ferredoxin gene flanking sequences, respectively. This sequence motif does not conform with the Goldberg-Hogness promoter consensus sequence
TATAAATA, which in eukaryotic genes is located 25-30 bp upstream of the transcriptional start site.
Nevertheless, this sequence is similar in the three E. histolytica genes, both in sequence and relative
position, suggesting a consensus which in E. histolytica serves as the entry point for RNA polymerase.
Table II: Alignment of the 5' flanking sequences from genes M17, actin* (one reported sequence), actin§ (a second reported sequence) and ferredoxin § ; a likely Goldberg-Hogness consensus sequence at -29, -31, -32, and -32, respectively, is bracketed. The 5' end of the mRNA is in bold face and follows the { symbol, and the initial ATG methionine codon is indicated. Sequence similarities around the cap site are ATTCA or ATTAA.
Figure imgf000038_0001
IX. Primer Extension Sequence Analysis
Primer extension sequence analysis was
performed by reverse transcriptase mediated extension of oligonucleotide primer SRO9
[5ΑACTACTCCTGTGACTATTGCAGAAG3'] annealed to 10 μg poly(A)+ enriched RNA in the presence of deoxyadenosine 5'-[a-[35S]thio]triphosphate.
X. Gene Copy Number
Southern blot and sequence analysis of the M17 gene and limited flanking regions indicate that this surface antigen is encoded by a single copy gene. A Southern blot of genomic DNA from E. histolytica, restricted with Bgl II and EcoR V in single and double digests, probed with Bam Hl-Bgl II, Bgl II-Eco RV, and Eco RV fragments of λcM17, only unique restriction fragments hybridized with each probe. Furthermore, the nucleotide sequence of both genomic clones and the cDNA clone is identical.
XI. Detection of Sequences Related to M17 in Non- Pathogenic E. histolytica
Western blot analysis suggested that the 125 kDa antigen or a closely related antigen which shared the epitope recognized by poly- and monoclonal antisera was found in both pathogenic and non-pathogenic E.
histolytica isolates as well as E. histolytica-like
Laredo. By Southern blot analysis, even under low stringency hybridization and wash conditions (25%
formamide, 2 X SSC, 37ºC) sequences related to M17 were difficult to detect in non-pathogenic E. histolytica isolates and E. histolytica-like Laredo.
PCR was performed using a Cetus/Perkin-Elmer DNA thermocycler. Reaction mixtures (50 μl) contained 25 pmol of each of the two oligonucleotide primer pairs SRO18 [5-GCAACTAGTGTTAGTTATAC3'] with SRO21
[5-GGTGGAATTTGGAATTCTGG3'] and SRO19 [5'GTATAACTAACACTAGT3'] with SRO22
[5'GCTGTTACACTTGAAAATAT3'] , approximately 500 ng of genomic DNA, all four dNTPs each at 1.5 mM, 60 mM KCl, 25 mM Tris-HCl (pH 8), 0 to 20 mM MgCl2, 0.1% bovine serum albumin, and 10% DMSO. The reaction mixture was overlaid with a drop of paraffin oil and denatured at 94ºC for 10 min, and amplification was initiated by addition of 2.5 units of Thermus aquaticus DNA polymerase (Cetus). PCR parameters were 35 thermal cycles consisting of a 1 min denaturation at 94•c followed by a 3 min annealing period at 42ºC, a 3 min ramp and a 4-min extension period at 72ºC The amplification products were restricted with Eco RI and Spe I endonucleases and purified for
subcloning into M13 by 1% low-melting-point agarose gel electrophoresis.
Two fragments spanning most of the sequence contained within the cDNA clone λcM17 were amplified in a PCR using oligonucleotide pairs SRO19/SRO22, and
SRO18/SRO21 as primers on genomic template DNA derived from non-pathogenic isolate REF 291. By nucleotide sequence analysis of the two subcloned PCR amplification products, REF 291 had 145 nucleotide substitutions over 1410 residues (10.3%) as compared to the sequence of λcM17 (HM1:IMSS) See Table I. These substitutions result in 57 amino acid differences per 470 residues (12.1%). A computer search of published protein sequences with the entire 3345 bp M17 gene sequence revealed that the internal gene fragment represented by the λcM17 insert encoded a protein sequence similar to that deduced for a DNA fragment isolated from non-pathogenic and pathogenic strains, of E. histolytica by Tannich et al. (1989) Proc. Nat'l Acad. Sci., U.S.A.. 86:5118-5122, and proposed by these authors as a potential diagnostic probe for strain differentiation. Specifically, when we compare the amino acid sequences of Tannich et al. with that of λcM17, we detected five substitutions between pathogenic HM1:IMSS isolates (1%). See Table III. The nucleotide sequence of the DNA fragment was not published by Tannich et al., so we infer from the amino acid sequence that at least three of these five differences between the E.
histolytica HM1:IMSS laboratory strains must have arisen from more than one nucleotide substitution and are therefore unlikely to represent cDNA synthesis or sequencing artifacts. When the 470 amino acid sequence derived from the PCR product of non-pathogenic isolate REF291 was compared to isolate SAW 1734, six amino acid substitutions (1.3%) were detected. Over the same 470 amino acids, 61 amino acid residues (12.9%) differ among the pathogenic HM1:IMSS and non-pathogenic SAW 1734 strains. Overall there are 65 variable residues over a stretch of 470 amino acids (13.8%) when these four isolates were compared. See Table III.
Table III: Alignment of the amino acid sequences inferred from nucleotide sequences of cDNA and genomic clones (HM1:IMSS*) and of PCR amplification products (REF291*) with those published by Tannich et al.
(HM1:IMSS#, SAW 1734#). Conserved amino acids are in plain type, positions of variable residues
differentiating pathogenic from non-pathogenic isolates are indicated below by an *, and positions of additional variable residues are indicated below by a ^.
TABLE III
HM1 : IMSS* PITLNFDORVDAGAAVAYVGRWFTONPSDWAAACVGKDGLINYGNWGPLHEMN
HM1 : IMSS# PITINFDORVDAGAAVAYVDRWFTONPSDWAAACVGKDGLINYGNWGPLHEMN
REF 291* PITLNFDORVDAGAAVAFVGRWFTOHPSDWASGCVNKDRLINSGNWGPLHEMN
SAW 1734# PITLNFDQRVDAGAAVAFVGRWFTOHPSDWASGCVNKEGLINSGNWGPLHEMN
* ^ * ** * ^^ *
HM1 : IMSS* lJHMQGTYLKGGNWGISNPGEETNNVMTSINYILYTNIAGHRNQGLSGWNYVSD
HM1 :IMSS# HHMQGPYLKGGNWGISNPGEETNNVMTSINYILYTNIAGHRNOGLSGWNYVSD
REF 291* HHMOGTYLRGGNGGIKEPGEETNNVMTSINYILYTNIAGHRKOGLSGWNYVSD
SAW 1734# HHMOGTYLRGGNWGIKEPGEETNNVMTSINYILYTNIAGHRNOGLSGWNYVSD
^ * ** ^
HM1 : IMSS* GYSTIYKILKGENDQPHLRSYVNMAHAFGTDTLIALVKSYYGLWYENNFESKY
HM1 : IMSS# GYSTIYKILKGENDOPHLRSYVNMAHAFGTDTLIALVKSYYGLWYENNFESKY
REF 291* GYSTIYKILKGENDQPHLRSYVNIΛHAFGTDTLIALVKSYYGLWYENNYEGEY
SAW 1734# GYSTIYKILNGENDQPHLRSYVNIAHAFGTDTLIALVKSYYGLWYENNYEGEY
^ * * **
HM1 : IMSS* SIKRDSTSAFCLLAALVTKRDTRYLCSLFKYDIOSNVSEAIKNMNYPTYYPFF
HM1 : IMSS# SIKRDSTSAFCLLAALVTKRDTRYLCSLFKYDIQSNVSEAIKNMNYPTYYPFF
REF 291* SIKRDSTSAFCLLAAIATKRDTRYLCSLFKYDIOONVSEAIKNMNYPTYYPFF
SAW 1734# SIKRDSTSAFCLLAAIATKRDTRYLCSLFKYDIOONVSEAIKNMNYPTYYPFF
** *
HM1 :IMSS* NLYAMSYNGNYYGRPYKIPYGRTRLNFTATTAIDPKATSVSYTIKSGLTKGKL
HM1 : IMSS# NLYAMSYNGNYYGRPYKIPYGRTRLNFTATCSIDPKATSVSYTIKSGLTKGKL
REF 291* NVYAMSYNGNYYGRTYKIPYGTTRLNFTATTAIDPSATSVSHTIKSGLTKGKL
SAW 1734# NVYAMSYNGNYYGRTYKIPYGTTRLNFTATTAIDPSATSVSYTIKSGLTKGKL
* * * ^^ * ^
HM1 : IMSS* ERVEDNVYDYTPFFGIEENDTFVLNIDCWNGEKVHIEOEGGTFELDPHOVEY
HM1 : IMSS# ERVEDNVYDYTPFFGIEENDTFVLNIDCWNGEKVHIEOEGGTFELDPHOVEY
REF 291* EOVEENVYDYTPNFGADENDTFVLNIDCIVNGEKVHIEODGGTFELDPHOVEY
SAW 1734# EOVEENVYDYTPNFGADENDTFVLNIDCIVNGEICVHIEODGGTFELDPHOVEY
* * * ** * *
HM1 : IMSS* EVYKDVOTRDMAOAINIIQNKTRNDTGRASFFGIGTYNDGSMOSMLVEKGKLI
HM1 : IMSS# EVYKDVQTRDMAQAINIIQNKTRNDTGRASFFDIGTYNDGSMQSMLVEKGKLI
REF 291* EVYKDVKTKDMEOALNTIONKTSNYTGTSTFFGIGNYDDGTMOSMLVEKGKLI
SAW 1734# EVYKDVKTKDMEOALNTIONKTSNYTGTSTFFGIGNYDDGTMOSMLVEKGKLI
** * * * * * *** ^ * * *
HM1 : IMSS* VPKSGYYTLFMKADDLGRLLLNITGEYEOLLDVKTYLGGYSKTLNGSYATVKL
HM1 : IMSS# VPKSGYYTLFMKADDLGRLLLNITGEYEQLLDVKTYLGGYSKTLNGSYATVKL
REF 291* VPTSGYYTLFMKADDLGRLLLNVNGEYEOLLNVKTYLGGYSKTINGTYATVKL
SAW 1734# VPTSGYYTLI^OCADDIΛRUXNVNGEYEOLLNVKTYLGGYSKTINGTYATVKL
* ** * * *
HM1 : IMSS* EKDVGYPFILYNLNTGGOGFIRIGYCYHGTEESSVDVSKCSVSDIGS
HM1 : IMSS# EKDVGYPFILYNLNTGGOGFIRIGYCYHGTEESSVDVSKCSVSDIGS
REF 291* EKDTEYPFILYNLNTGGOGFIRIGYCYQGTEOSSVNVSKCSGLDIGS
SAW-1734# EKDTEYPFILYNLNTGGQGFIRIGYCYQGTDQSSVNVSKCSGLDIGS
** * ^* * ** XII. Restriction Fragment Length Polymorphisms
The partial M17 amino acid sequences of
non-pathogenic strains SAW 1734 and REF 291 were
significantly more similar to one another than to their pathogenic counterparts, so PCR amplification of the same gene fragments from six additional strains was undertaken to examine the possibility of defining restriction length polymorphisms that could reliably differentiate
pathogenic from non-pathogenic amoeba isolates. Using oligonucleotide primers SRO19, SRO22, SRO18 and SRO21,
PCR products of the same size were amplified from genomic DNA of strains SD116, SD4, #43, #44, #46 and HK9. Based on our nucleotide sequence of these fragments from
HM1:IMSS and REF291, we predicted that restriction endonucleases Eco RV,, Ssp I, Pvu II, Ace I and Hinc II among others would cleave the PCR products into
restriction fragments which might be expected to
correlate with the pathogenic or non-pathogenic phenotype of the isolate. An example of such an analysis with Eco RV and Ssp I is presented. See Fig. 4. A restriction site for Eco RV is absent in non-pathogenic #43, #44 and REF291 but present in non-pathogenic SD116 and Laredo as well as pathogenic HM1:IMSS, HK9, SD4, and #46.
Digestion with restriction endonuclease Ssp I shows a distinct pattern for pathogenic (HM1:IMSS, HK9, SD4, 46) versus non-pathogenic (#43, #44, SD116, REF291) strains with the exception of E. histolytica-like Laredo, which would appear pathogenic by this criterion. See, e.g., Fig. 4. Similarly, restriction with Ace I distinguishes pathogenic from non-pathogenic isolates with the
exception of Laredo which appears to have an additional restriction site for the enzyme. Hinc II digestion shows the same restriction fragments in non-pathogenic isolates #43, #44 and REF291 and pathogenic isolate SD4 but no restriction sites in commensal Laredo and pathogenic isolates #46, HM1:IMSS and HK9. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be
incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the claims.

Claims

WHAT IS CLAIMED IS:
1. A vaccine composition against invasive amoebiasis, said composition comprising a polypeptide having immunological cross-reactivity with an
immunodominant surface antigen of Entamoeba histolytica, said polypeptide being present in a physiologically acceptable carrier in an amount effective to elicit a protective immunity when administered to a susceptible host.
2. A vaccine composition as in claim 1, wherein the immunodominant surface antigen is
substantially homologous to the 125 kDa antigen whose amino acid sequence is set forth in Table I.
3. A vaccine composition as in claim l, wherein the polypeptide comprises at least six contiguous amino acids from the sequence set forth in Table I.
4. A recombinant nucleic acid composition comprising a sequence which is:
homologous to a sequence encoding a polypeptide encoded in region I or region III of Table I; or
capable of annealing under stringent hybridization conditions to said sequence.
5. A recombinant composition as in claim 4, wherein said sequence is at least about 15 nucleotides.
6. A recombinant composition as in claim 4, wherein said polypeptide comprises an extracellular domain.
7. A recombinant composition as in claim 4, wherein said nucleic acid comprises substantially the entire approximately 3342 nucleotide protein encoding sequence.
8. A cell comprising a recombinant
composition as in claim 4.
9. A diagnostic kit for detection of an Entamoeba histolytica surface antigen gene, said kit comprising a compartment containing a recombinant composition as in claim 4.
10. A substantially pure polypeptide
comprising a sequence homologous to a sequence of at least six contiguous amino acids disclosed in region I or region III of Table I.
11. A substantially pure polypeptide as in claim 10, wherein said polypeptide comprises an
extracellular domain.
12. A substantially pure polypeptide as in claim 10, wherein said polypeptide is a fusion protein.
13. A substantially pure polypeptide as in claim 10, wherein said polypeptide comprises
substantially the entire amino acid sequence of
approximately 1114 amino acids set forth in Table I.
14. An antibody capable of specifically binding an immunodominant surface antigen of Entamoeba histolytica.
15. An antibody as in claim 14, wherein the immunodominant surface antigen is substantially
homologous to the 125 kDa antigen whose amino acid sequence is set forth in Table I.
16. An antibody as in claim 14, wherein said antibody is the monoclonal antibody FA7.
17. A protein possessing an epitope specifically bound by an antibody of claim 15.
18. A diagnostic kit for detection of an
Entamoeba histolytica antigen, said kit comprising a compartment containing an antibody as in claim Dl.
19. A method for vaccinating a susceptible host to confer immunity against invasive amoebiasis, said method comprising administering the host with a
polypeptide having immunological activity cross-reactive with an immunodominant surface antigen of Entamoeba histolytica.
20. A method as in claim 19, wherein the host is administered prior to amoeba infection.
21. A method as in claim 19, wherein the host is administered after amoeba infection.
22. A method as in claim 19, wherein the immunodominant surface antigen is substantially
homologous to the 125 kDa antigen whose amino acid sequence is set forth in Table I.
23. A method as in claim 19, wherein the polypeptide comprises at least six contiguous amino acids from the sequence set forth in Table I.
24. A method for treatment of an animal infected with an amoeba of the genus Entamoeba comprising a step of injecting into said animal a recombinant protein containing a segment which is substantially homologous to a sequence of at least about 6 amino acids set forth in Table I.
25. A method as in claim 24, wherein said injecting induces an immune response.
26. A method as in claim 24, wherein said sequence is from region I or region III.
27. A method as in claim 24, wherein said sequence is the full length amino acid sequence set forth in Table I.
28. A method of immunizing an animal against an amoeba infection comprising introducing an immunogenic peptide having a sequence in common with a membrane bound protein found on said amoeba, said immunogenic peptide inducing an immune response affecting infectivity of said amoeba.
29. A method as in claim 28, wherein said infection is an extraintestinal infection.
30. A method as in claim 28, wherein said amoeba is from the genus Entamoeba.
31. A method as in claim 28, wherein said amoeba is Entamoeba histolytica.
32. A method as in claim 28, wherein said introducing is with a pharmaceutical carrier.
33. A method as in claim 28, wherein said introducing is with an immunological adjuvant.
34. A method as in claim 28, wherein said membrane bound protein is homologous to a protein
described in Table I.
35. A method as in claim 28, wherein said immunological response is production of IgA.
36. A method as in claim 28, wherein said affecting infectivity is an interference with amoeba reproduction.
37. A method for detecting pathogenic amoeba in a non-denatured target sample comprising a step of: detecting the presence in said sample of an epitope characteristic of pathogenic amoeba.
38. A method as in claim 37, wherein said test sample is a biopsy sample.
39. A method as in claim 37, wherein said detection is performed by binding an antibody to said epitope.
40. A method as in claim 39, wherein said antibody binds to an epitope from a sequence disclosed in Table I.
41. A method as in claim 39, wherein said epitope is located in regions I or III in Table I.
PCT/US1991/005979 1990-08-24 1991-08-21 Entamoeba histolytica immunodominant surface antigens WO1992003457A1 (en)

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US5004608A (en) * 1988-01-13 1991-04-02 The University Of Virginia Alumni Patents Foundation Amebiasis vaccine

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Title
Abstracts of Papers Presented at the Joint Meeting of American Society for Cell Biology and the American Society for Biochemistry and Molecular Biology, issued 29 January 1989, M.A. MERAZ et al., "Surface Molecules of Entamoeba Histolytica with Immunodominant Characteristics", page 746a, see the Abstract numbered 4231. *
Journal of Cellular Biochemistry, supplement 10A, issued March 1986, U. EDMAN et al., "Isolation of Entamoeba Surface Antigen Genes", page 129, see the Abstract numbered C39. *
Journal of Clinical Microbiology, Volume 27, No. 4, issued April 1989, J. SAMUELSON et al., "DNA Hybridization Probe for Clinical Diagnosis fo Entamoeba Histolytica", pages 671-676, see the Abstract and pages 672, left column. *
Proceedings of the National Academy of Sciences, Volume 84, issued May 1987, U. EDMAN et al., "Genomic and cDNA actin sequences from a virulent strain of Entamoeba Histolytica", pages 3024-3028, see the Abstract. *
Proceedings of the National Academy of Sciences, Volume 86, issued July 1981, E. TANNICH et al., "Genomic DNA Differences Between Pathogenic and Nonpathogenic Entamoeba Histolytica", pages 5118-5122, see the Abstract and page 5118, right column. *
Proceedings of the National Academy of Sciences, Volume 87, issued August 1990, B.E. TORIAN et al., "cDNA Sequence Analysis of a 29-KDa Cysteine-rich Surface Antigen of Pathogenic Entamoeba Histolytica", pages 6358-6362, see the Abstract. *
See also references of EP0550465A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
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WO2002012271A2 (en) * 2000-08-04 2002-02-14 Jeffrey Howard Treatment of microbial infections with bacterial proteins and peptides
WO2002012271A3 (en) * 2000-08-04 2003-03-13 Jeffrey Howard Treatment of microbial infections with bacterial proteins and peptides
US6727223B2 (en) 2000-08-04 2004-04-27 Urex Biotech, Inc. Treatment of microbial infections with bacterial proteins and peptides

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