[go: up one dir, main page]

WO1997019107A1 - Identification d'antigenes a partir de nematodes post-infectieux pour le developpement de nouveaux anthelmintiques et vaccins - Google Patents

Identification d'antigenes a partir de nematodes post-infectieux pour le developpement de nouveaux anthelmintiques et vaccins Download PDF

Info

Publication number
WO1997019107A1
WO1997019107A1 PCT/EP1996/004918 EP9604918W WO9719107A1 WO 1997019107 A1 WO1997019107 A1 WO 1997019107A1 EP 9604918 W EP9604918 W EP 9604918W WO 9719107 A1 WO9719107 A1 WO 9719107A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigens
larvae
stage
nematodes
nematode
Prior art date
Application number
PCT/EP1996/004918
Other languages
German (de)
English (en)
Inventor
Achim Harder
Michael Londershausen
Wilhelm Friedrich Eisenbeiss
Original Assignee
Bayer Aktiengesellschaft
Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Aktiengesellschaft, Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin filed Critical Bayer Aktiengesellschaft
Priority to JP9519341A priority Critical patent/JP2000500474A/ja
Priority to EP96938168A priority patent/EP0868433A1/fr
Priority to AU75693/96A priority patent/AU7569396A/en
Publication of WO1997019107A1 publication Critical patent/WO1997019107A1/fr

Links

Classifications

    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • 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
    • 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
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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

Definitions

  • the invention relates to the isolation and characterization of antigens from nematodes in a substantially pure form, which react immunologically with protective antisera, and a method for obtaining these antigens. Furthermore, the present invention relates to the use of these antigens for the development of new anthelmintics and vaccines
  • Parasitic nematodes which can affect human and animal lymphatic vessels and tissues, cause chronic diseases.
  • Human pathogenic nematode species occur in tropical regions, where the WHO estimates that there are more than 100 million infected people.Nematode diseases in farm animals and pets occur throughout World and cause high economic damage and costs
  • a protective antiserum can be isolated from immunized host animals which is directed against antigenic substances which are secreted between the L3 and L4 stages during molting. However, this secretion takes place only for a very short period and in very small amounts, so that isolation of the antigens in a pure form is not possible.
  • the present invention thus relates to isolated antigens from nematodes in a substantially pure form, which react immunologically with a protective antiserum which is directed against nematode larvae at the molting stage from L3 to L4, the antigens being converted by in vitro
  • protective antiserum in the sense of the present application means that the serum is obtained from animals whose immune system can completely eliminate a new infection due to a corresponding pre-immunization.
  • antigens which react immunologically with protective antisera can be isolated from a large number of nematode species, in particular from filaria species, for example from free-living nematode species, such as Caenorhabditis elegans, or parasitic nematodes, such as, for example, Acanthocheilonema viteae, Monanemaia martini and Dirof ⁇ lar immitis,
  • Protective antisera are available from host animals which are exposed to a primary infection with live nematode larvae before the L3 / L4 skin stage, and which are at least partially inhibited in their development by a second exposure infection with nematode larvae of the same species or a different species.
  • Protective antisera from the Gerbils gerbil M unguiculatus, the African striped mouse Lemniscomys striatus or other natural hosts from nematodes are preferably used for primary infection.
  • the interval between the administration of the larvae and the number of larvae can vary depending on the nematode species Immunization of M unguiculatus can, for example, live infectious L3 larvae of the nematode species A viteae 3 times at 3-day intervals and in one
  • protective antisera can also be obtained by immunization with living L4 A vitaea larvae, with living in vitro cultivated L3 / L4 or L4 A vitaea larvae, with living in vitro cultivated "exsheathed" L3 H contortus
  • Larvae are obtained with living in vitro cultivated L3 (80% Lethargus) C elegans larvae
  • a protective antiserum obtained from the natural host of the Fila ⁇ e A viteae, the Meriones unguiculatus shows an immunological cross-reactivity against antigens from the nematode species M martini, L. sigmodontis, D. immitis, H. contortus and even the free-living nematode species C. elegans.
  • the antigens according to the invention are preferably polypeptides, which can optionally be glycosylated. Different antigens are secreted, which can be separated according to their molecular weight and obtained in isolated form. The amount of antigens obtained is easily sufficient for a partial determination of the amino acid sequence. Based on this information, recombinant DNA techniques can then also be used to isolate DNA molecules that code for the respective antigens. After isolation of the coding DNA sequences, the antigens according to the invention can also be obtained by recombinant
  • Suitable host cells e.g. Bacteria, yeast or insect cell culture lines, or with transgenic C. elegans.
  • antigens are antigens which are obtainable from A. viteae and have molecular weights of approximately 4, 14, 18, 25, 29-30, 40, 44, 52, 56, 60, 66 or 68 kD after sieve gel chromatography, or are thus immunologically cross-reactive Antigens. Also preferred are antigens which are obtainable from A viteae and have molecular weights of about 17, 40, 52, 56, 60, 63, 67, 90, 94, 1 16, 180, 212, 400 or 800 kD according to SDS-PAGE, or thus immunologically cross-reacting antigens. Immunologically cross-reacting antigens, for example, have already been found in D. immitis, M. martini, L. sigmodontis,
  • H. contortus or C. elegans can be detected
  • antigens which are obtainable from C elegans and have molecular weights of about 14, 16, 26, 28, 30, 46, 49, 58 and 66 kD according to sieve gel chromatography, or antigens which are obtainable from C. elegans and molecular weights of 46, 60, 97, 109, 212 or 400 kD according to SDS
  • PAGE exhibit, or thus immunologically cross-reactive antigens. Immunologically cross-reacting antigens have already been detected in A. vitaea, for example.
  • Antigens which have a molecular weight of approximately 60 kD and are particularly preferred
  • (b) have an N-terminal amino acid sequence which is at least 80% and in particular at least 90% homologous to the sequence from (a).
  • antigens can have serine / threonine protein kinase activity.
  • Another object of the present invention is a method for obtaining antigens from nematodes which react immunologically with a protective antiserum which is directed against nematode larvae at the molting stage from L3 to L4, larvae of nematodes at the L4 stage cultured in vitro, the culture supernatant separated and the antigens isolated from the culture supernatant.
  • the isolation preferably comprises a sieve gel chromatography step.
  • the use of A. viteae larvae at the stage 13 days after infection is preferred.
  • suitable larval stages in which the production of the antigens according to the invention takes place can be identified in a simple manner by determining the immunological activity
  • Yet another object of the present invention is a pharmaceutical composition which comprises one or more of the aforementioned antigens as an active ingredient and, optionally, pharmaceutically customary additives, carriers and auxiliaries.
  • Composition can be used for diagnosis, prevention and therapy of nematode infections.
  • the present invention thus also relates to a method for immunizing against a nematode infection, in which one or more of the aforementioned antigens are administered in pharmaceutically acceptable form.
  • the administration is preferably in a parenteral or oral formulation in a dose between 0.5 and 250 mg / kg, particularly preferably between 10 and 100 mg / kg on 1 to 4 consecutive days. Single administration is particularly preferred.
  • the invention thus also relates to a pharmaceutical composition which contains attenuated larvae of parasitic nematodes in the L4 stage as an active ingredient and, if appropriate, pharmaceutically customary additives, auxiliaries and carriers.
  • the attenuated larvae and a pharmaceutical composition containing them can be used for the prevention and therapy of nematode infections, optionally in combination with one or more of the aforementioned antigens.
  • Gerbils (M unguiculatus) were subjected to a primary infection by subcutaneous administration of 10 - 20 live infectious larvae of A viteae at intervals of three days. Serum was taken 25 days after the initial infection
  • the antisera obtained in this way recognize nematode antigens which are secreted from L3 to L4 during the skin stage Example 2
  • viteae larvae were isolated from M unguiculatus as described in Eisenbeiss, J Parasitol 77 (1991) 580-586. For this purpose, the bodies of infected and then killed gerbils in
  • RPMI1640 medium added and chopped. The larvae were isolated from the tissue fractions and stored in RPMI
  • Larvae at different stages of maturity were isolated, namely larvae in the L3 stage (0-6 days p i), larvae in the skin stage from L3 to L4 (6-7 days p i) and larvae in the L4 stage (more than 8 days p I)
  • the larvae were cultivated in vitro at a concentration of 500-1000 larvae / ml culture medium RPMI 1640 at 37 ° C. and 5% CO 2
  • antigens could be detected in the supernatant of the culture medium from 13-day-old larvae (L4 stage) in a high yield, namely with a cultivation period of 0-2, 2-4, 4-6 and 6-8 days
  • the antigens were detected by an ELISA as follows. 96-well microtiter plates were incubated with 100 ⁇ l of crude culture medium from 1000 larvae / ml overnight at 4 ° C. Three different dilutions of the crude culture medium, namely 1 10, 1 100 and 1 1000, were used in
  • Elution buffer (0.1 M K-phosphate buffer pH 7.0), used All wells were blocked with 150 ⁇ l 3% bovine serum albumin (RSA), 10% milk powder and 0.05% Tween 20 in elution buffer.
  • RSA bovine serum albumin
  • Rabbit anti-M was used as the second antibody.
  • a total of 12 fractions could be separated from the collected culture supernatants from larvae cultured in vitro using an FPLC gel filtration method. Of these, seven fractions were of particular interest because they either appeared to be dominant and / or were recognized as antigens by antibodies from the serum of 100% immune-protected animals. The separation was carried out using a Superdex 75 HR 10/30 gel filtration column in 0.5 ml fractions. Elution was carried out with a 0.1 M potassium phosphate buffer pH 7.0. The immunologically relevant fractions were determined by means of ELISA with sera from protected animals.
  • Example 5 A culture medium isolated as a control for the FPLC column was free of protein components.
  • Proteins in the FPLC fractions obtained in Example 4 were concentrated in bands via SDS-PAGE and then transferred electrophoretically to a support membrane
  • Protein-containing FPLC fractions were then selected for the protein sequence analysis.
  • the individual fractions contained approx. 7 pmol protein in 400 ⁇ l elution buffer
  • the selected fractions were transferred in a concentrated form to PVDF membranes using a suction device with which large application volumes can be filtered through small membrane areas.
  • the filters were washed in water and stored in an argon atmosphere until protein sequencing
  • Xaa means any amino acid
  • the identified sequence region does not start with a methionine, although the native protein has been sequenced N-terminally. As a result, the protein probably originally had a leader sequence.
  • Gerbils (unguiculatus) were immunized with 15-20 mL3 larvae of the nematode species Monanema martini and Litomosoides sigmodontis. This immunization resulted in significant protection against subsequent stress infections with A. viteae.
  • Cross immunization against A viteae can be achieved in the gerbil M unguiculatus.
  • C elegans larvae in the L3 / L4 stage (L3 / S0% lethargus larvae), which had preferably been incubated in vitro beforehand for 1.5 to 3 hours, were used to immunize M. unguiculatus used against a subsequent infection of A viteae
  • the 60 kD antigen is expressed by adult C. elegans worms and molting C. elegans L3 larvae.
  • the other antigens are also expressed by adult C. elegans worms and by mL3 larvae and are immunologically cross-reactive with corresponding antigens which are expressed by 13-day-old L4-A. viteae larvae.
  • antigens with molecular weights of approx. 14, 16, 26, 28, 30, 46, 49, 58 and 66 kD could be identified by sieve gel chromatographic analysis of the culture supernatants.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Communicable Diseases (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne des antigènes isolés à partir de nématodes sous une forme essentiellement pure. Ces antigènes réagissent immunologiquement avec un antisérum protecteur, lequel est dirigé contre les larves de nématodes au stade de la mue de L3 à L4. Les antigènes sont obtenus par culture in vitro de larves de nématodes au stade L4 et par extraction à partir de l'excès de culture. L'invention concerne également un procédé de production de ces antigènes et leur utilisation pour développer de nouveaux anthelmintiques et vaccins.
PCT/EP1996/004918 1995-11-22 1996-11-11 Identification d'antigenes a partir de nematodes post-infectieux pour le developpement de nouveaux anthelmintiques et vaccins WO1997019107A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP9519341A JP2000500474A (ja) 1995-11-22 1996-11-11 新規な駆虫剤およびワクチンを開発する目的のための感染後の線虫からの抗原の同定
EP96938168A EP0868433A1 (fr) 1995-11-22 1996-11-11 Identification d'antigenes a partir de nematodes post-infectieux pour le developpement de nouveaux anthelmintiques et vaccins
AU75693/96A AU7569396A (en) 1995-11-22 1996-11-11 Identification of antigenes from post-infection nematodes for the purpose of developing new anthelmintics and vaccines

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19543554A DE19543554A1 (de) 1995-11-22 1995-11-22 Identifizierung von Antigenen aus post-infektiösen Nematoden zur Entwicklung neuer Anthelmintika und Vakzinen
DE19543554.0 1995-11-22

Publications (1)

Publication Number Publication Date
WO1997019107A1 true WO1997019107A1 (fr) 1997-05-29

Family

ID=7778135

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1996/004918 WO1997019107A1 (fr) 1995-11-22 1996-11-11 Identification d'antigenes a partir de nematodes post-infectieux pour le developpement de nouveaux anthelmintiques et vaccins

Country Status (5)

Country Link
EP (1) EP0868433A1 (fr)
JP (1) JP2000500474A (fr)
AU (1) AU7569396A (fr)
DE (1) DE19543554A1 (fr)
WO (1) WO1997019107A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013560A1 (fr) * 1991-02-12 1992-08-20 Colorado State University Research Foundation Reactifs et procedes d'identification de vaccins
WO1993010225A1 (fr) * 1991-11-12 1993-05-27 The Colorado State University Research Foundation Vaccin a base de protease anti-dirofilaria immitis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013560A1 (fr) * 1991-02-12 1992-08-20 Colorado State University Research Foundation Reactifs et procedes d'identification de vaccins
WO1993010225A1 (fr) * 1991-11-12 1993-05-27 The Colorado State University Research Foundation Vaccin a base de protease anti-dirofilaria immitis

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
EISENBEISS ET AL: "PROTECTIVE IMMUNITY LINKED WITH A DISTINCT DEVLOPMENTAL STAGE OF A FILARIAL PARASITE", JOURNAL OF IMMUNOLOGY, vol. 152, 1994, pages 735 - 742, XP002026644 *
FRANK ET AL: "PURIFICATION AND CHARACTERIZATION OF THREE LARVAL EXCRETORY-SECRETORY PROTEINS OF DIROFILARIA IMMITIS", MOLECULAR AND BIOCHEMICAL PARASITOLOGY, vol. 75, January 1996 (1996-01-01), pages 221 - 229, XP000618683 *
GANGADHARA ET AL: "LITOMOSOIDES CARINII: EFFECT OF IRRADIATION ON THE DEVELOPMENT AND IMMUNOGENICITY OF THE LARVAL FORMS", EXPERIMENTAL PARASITOLOGY, vol. 43, 1977, pages 39 - 44, XP000644679 *
WONG ET AL: "DIROFILARIA IMMITIS: FATE AND IMMUNOGENICITY OF IRRADIATED INFECTIVE STAGE LARVAE IN BEAGLES", EXPERIMENTAL PARASITOLOGY, vol. 35, 1974, pages 465 - 474, XP000644663 *

Also Published As

Publication number Publication date
AU7569396A (en) 1997-06-11
DE19543554A1 (de) 1997-05-28
EP0868433A1 (fr) 1998-10-07
JP2000500474A (ja) 2000-01-18

Similar Documents

Publication Publication Date Title
DE69123012T2 (de) Rekombinantes Milben-Allergen
SCHALLIG et al. Protective immunity induced by vaccination with two Haemonchus contortus excretory secretory proteins in sheep
DE69030383T2 (de) Impfstoff-Konjugate zur Behandlung von Vogelkrankheiten
DE69432658T2 (de) Rib-protein, ein oberflächenprotein welches immunität gegen viele streptococcus-stämme der gruppe b verleiht, reinigungsverfahren für das rib-protein, testsatz und pharmazeutische zusammenstellung (arznei)
AT503530A1 (de) Polypeptid mit allergenen eigenschaften
Adams et al. Cross-immunity between Haemonchus contortus and Trichostrongylus colubriformis in sheep
DE3751670T2 (de) Coccidiosis-Impfstoff
DE69535378T2 (de) Allergieauslösendes Protein Hev b III aus natürlichem Gummi-Latex, seine Herstellung und Anwendung in Nachweismethoden
DE3750225T2 (de) Herstellung und verwendung von anti-helminthischen wirkstoffen und schutzantigenen.
DE3751777T2 (de) Impfstoff, der zecken-antigene enthält
Selkirk et al. Immunity and the prospects for vaccination against filariasis
DE69732630T2 (de) Vasoaktive, aminbindende moleküle
DE69332977T2 (de) Thiolprotease enthaltender impfstoff
DE69931518T2 (de) Chimäres Gen, das für die antigenischen Determinaten von vier Proteinen aus L. Infantum kodiert
DE69032323T2 (de) Produktion und verwendung von antihelmintischen substanzen und schützenden immunogenen
DE69532964T2 (de) Neue proteine aus ektoparasitenspeichel und vorrichtung zur sammlung davon
EP1363937B1 (fr) Immunomodulants provenant de vers parasites et procede pour leur separation
DE68926712T2 (de) Gen zum kodieren eines antigens gegen menschlichen malaria impfstoff
MEJIA et al. An analysis of the humoral immune response of dogs following vaccination with irradiated infective larvae of Dirofilaria immitis
DE60220988T2 (de) Herstellung und verwendung eines plasmodiumfusionsantigen
DD283331A5 (de) Verfahren zur herstellung eines impfstoffes zum schutz von gefluegel vor escherichia coli-septikaemie
DE69734761T2 (de) Aus hemophilus paragallinarum abstammendes polypeptid und verfahren zu dessen herstellung
DE602005006096T2 (de) Apikomplex-vakzine-stämme einer familie von sarcocystidae
DE69025414T2 (de) Vakzin-Zusammensetzung
DE4419264B4 (de) Antigen, um Menschen und Tieren eine schützende Immunität gegen Wurminfektionen zu verleihen

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR BY CA CN CZ HU IL JP KR KZ LK MX NO NZ PL RO RU SK TR UA US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1996938168

Country of ref document: EP

ENP Entry into the national phase

Ref country code: JP

Ref document number: 1997 519341

Kind code of ref document: A

Format of ref document f/p: F

WWP Wipo information: published in national office

Ref document number: 1996938168

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1996938168

Country of ref document: EP