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EP0081575A1 - Antigene pour anaplasma - Google Patents

Antigene pour anaplasma

Info

Publication number
EP0081575A1
EP0081575A1 EP82902304A EP82902304A EP0081575A1 EP 0081575 A1 EP0081575 A1 EP 0081575A1 EP 82902304 A EP82902304 A EP 82902304A EP 82902304 A EP82902304 A EP 82902304A EP 0081575 A1 EP0081575 A1 EP 0081575A1
Authority
EP
European Patent Office
Prior art keywords
antigen
anaplasma
marginale
anablock
virulent
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP82902304A
Other languages
German (de)
English (en)
Other versions
EP0081575A4 (fr
Inventor
Miodrag Ristic
Michael G. Levy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Illinois at Urbana Champaign
University of Illinois Foundation
Original Assignee
University of Illinois at Urbana Champaign
University of Illinois Foundation
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 University of Illinois at Urbana Champaign, University of Illinois Foundation filed Critical University of Illinois at Urbana Champaign
Publication of EP0081575A1 publication Critical patent/EP0081575A1/fr
Publication of EP0081575A4 publication Critical patent/EP0081575A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/0233—Rickettsiales, e.g. Anaplasma
    • 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 materials and methods useful in immunization of animals against infection by parasites of the genus Anaplasma (Order Rickettsiales, family Anaplasmatacae). More specifically, the present invention provides novel, in vitro produced and isolated, water soluble antigen materials specific for Anaplasma __., which, when administered as an active component of a vaccine, provoke develop ⁇ ment of an in vivo protective immune response in the vaccinates as well as a characteristic serologic profile in in vitro tests.
  • Anaplasmosis is a tick-borne hemotropic disease of cattle and certain other ruminants. Its causative agents include the three recognized species of Anaplasma, i.e., A. marginale, A. eentrale, and A. ovis. Occa ⁇ sionally, anaplasmosis has been noted to involve a "mixed" infection of A. marginale and related Par anaplasma species, __ caudatum and/or P. dis- coides. Bovine anaplasmosis is manifested by progressive severe anemia associated with the presence of parasite "inclusion bodies" in erythrocytes. The disease is quite frequently fatal, especially among cattle initially infected as adults.
  • A. eentrale also has cattle as its principal host but is rela ⁇ tively less virulent than A. marginale.
  • Anaplasmosis involving A. eentrale is frequently distinguished from the disease state involving A. marginale by the location of the parasite within infected erythrocytes.
  • A. ovis has sheep as its principal host but also infects goats and deer, causing mild to severe anaplasmosis symptoms.
  • Anaplasma sp. parasites are sensitive to treatment with broad-spectrum antibiotics such as the tetraeyclines. Due to a characteristic long delay between initial infection and onset of clinical symptoms of anaplasmosis, however, tetraeycline treatment is seldom initiated until late in the acute disease phase when it is generally least effective. Because of the lack of effectiveness of therapeutic measures, significant effort has been directed to immunoprophylactic measures for control of the disease.
  • premunition a "premunition" procedure involving purposeful infection of an animal (with, e.g., whole blood of an A. marginale infected animal) followed by treatment with antibiotics in the hope of lessening clinical signs of the disease.
  • premunition has proven to be too hazardous, costly and time- consuming to warrant widespread application.
  • a first proposed vaccination technique involves administering less virulent A. eentrale parasites for the purpose of developing a cross-protective immune response to A. marginale.
  • a second proposed technique involves use of an "inactivated” or “killed” immunogen (consisting of marginal bodies and erythrocyte ghosts) derived from the blood of acutely infected cattle. See, e.g., U-S. Letters Patent No. 3,511,908.
  • the vaccine comprises a live, attenuated form of A. marginale selected by gradual adaptation of the organism to growth in an atypical host, the sheep.
  • Anaplasma Compounding the search ' for non-virulent Anaplasma immuno- gens suitable for use as vaccine components is the antigenic complexity of anaplasmal species.
  • the known antigenic profile of Anaplasma includes both intraerythrocytic and serum "antigens" of widely varying constitution and properties.
  • CF complement fixation test
  • the active component of the well-known complement fixation test is an intraerythrocytic lipoprotein. See, Gates, et aL, Proc. A.V.M.A., 91st Ann. Mtg. (1954).
  • the particulate "antigen" of the capillary tube agglutination (CA) test has been shown to consist of struc ⁇ tures closely resembling Anaplasma initial bodies. See, Ristic, J.A.Y.M.A., Ml, p. 588 (1962). None of the intraerythrocytic "antigens" has been useful in eliciting a protective response when administered as a vaccine compo ⁇ nent.
  • Soluble isolates obtained from serum of infected animals have displayed lipoprotein or glycoprotein electrophoretic mobility.
  • Vaccines containing certain of these soluble serum "antigens" are capable of eliciting in vivo production of precipitating antibodies. None, however, is capable of
  • MASP macroaerophilous stationary phase
  • a suitable anaplasmal immunogen should have the following characteristics which are not presently possessed by any single immunogen available to the art. It should be susceptible to production in vitro, preferably by means of generally continuous cultivation of parasites. It should have relatively distinct physical properties so that it can be isolated with- ease. It should be water soluble, thereby allowing for uniform formulation in a vaccine and for admixture with soluble drugs and other antigens used in vaccination against other diseases. Both the immunogen and vaccines prepared therefrom should be storage stable for long periods of time without the need for costly or difficult maintenance procedures.
  • the immunogen should be non-virulent and should not induce iso-blood group immime responses, yet it should be capable of provoking a long-term, anemnestic, protective response to challenge of virulent anaplasmal infection.
  • the protective response developed should preferably be of a character permitting distinction of vaccinated animals from diseased or recovered animals.
  • the present invention comprehends novel Anaplasma antigen preparations, vaccines including i munologically effective amounts of the same as an active immunogen, and methods for immunoprophylactic treat ⁇ ment of animals susceptible to anaplasmal infection with such vaccines. More specifically, the invention provides new, in vitro produced and isolated, water soluble antigen substances specific for Anaplasma sp., especially A_ marginale.
  • Presently preferred methods for in vitro preparation of a specific A. marginale antigen (designated "Anablock”) involve parasite propagation in erythrocyte host cells according to hereinafter specified modifications of published cultivation techniques, followed by isolation of the soluble antigen from the medium supporting culture growth.
  • Anablock antigen so prepared and isolated is characterized by: solubility in water and normal saline solution; insolubility in from about 40 to about 75% ammonium sulfate solution; the presence of carbohydrate and protein components (as well as the absence of any functionally essential lipid component); a molecular weight oi the order of about 53,000; an isoelectrie point of about 6.0; heat stability at 56° C for at least 30 minutes; and immunodiffusion test reactivity with antibodies from animals recovered from virulent A. mar ⁇ ginale infection.
  • Vaccine preparations comprising immunologically effective quantities of Anablock antigen of the invention combined with a suitable diluent, adjuvant and/or carrier substance are storage stable at ambient conditions over long periods of time, are non-inflammatory upon parenteral administration, and are uniquely characterized by in vivo immunological effects including the absence of virulence, the absence of manifest induc ⁇ tion of iso immunity and, most significantly, the provocation of a long-term anemnestic protective response to virulent infection by A. marginale.
  • Immunoprophylactic methods of the invention include, e.g., parenterally administering Anablock antigen-containing vaccine to develop protective immunity in vaccinates which is characterized by a serological property profile directing positive reaction in fluorescent antibody (I A) and serum latex agglutination (LAT) test procedures but negative response in complement fixation (CF) and capillary tube agglutination (CA) tests.
  • I A fluorescent antibody
  • LAT serum latex agglutination
  • CF complement fixation
  • CA capillary tube agglutination
  • the following illustrative examples relate to presently pre- ferred methods of practice of the invention. More specifically, the examples describe: a method for in vitro propagation of A. marginale which gives rise to a specific Anablock antigen-enriched growth medium; an alternate in vitro propagative method; methods for isolation of Anablock from culture growth medium; physical and biological characteristics of Anablock antigen; methods for formulating vaceine preparations; and, in vivo tests of the efficacy of vaccine preparations including Anablock antigen.
  • An A. marginale enriched growth medium from which soluble Anablock antigen of the invention can be isolated is obtained by practice of the Anaplasma propagative techniques described by Kessler, et al. Am. Jour. Vet. Res., 40, pp.1767-73 and 1777-80 (1979). Blood from cattle infected with virulent A ⁇ marginale is collected in acid-eitrate-dextrose solution during the early stages of parasitemia. Erythrocytes are separated by centrifugation at 600 x g for 15 minutes at room temperature.
  • the erythrocytes are resuspended and washed twice in a medium consisting of RPMI 1640 supplemented with 25 mM HEPES buffer and 10% bovine serum (?H 7.4). Cells are resuspended in a ratio of 1:8 (v/v) with culture medium, resulting in a final packed cell volume (PCV) of 8% to 9%.
  • PCV packed cell volume
  • OMPI ⁇ WIPO resuspending these cells with sufficient fresh medium and normal, freshly collected bovine erythrocytes to obtain the desired dilution.
  • An antigen enriched growth medium from which a soluble antigen of the invention can be isolated is also obtained through modifica ⁇ tion of the macroaerophilous stationary phase propagation techniques of the co-inventors as set out in Science, 207, p. 1218 (1980). More specifically, blood is collected from a splenectomized Bos tauras calf infected with virulent A. marginale parasites and immediately defibrinated by shaking with glass beads. The defibrinated blood is sedimented at 2,000 x g for 15 minutes and the serum and buffy coat cells are removed.
  • the remaining erythrocytes are washed two times in phosphate buffered saline (pH 6.8) and added to a medium consisting of 90 percent Medium 199 (containing 25 mm HEPES) and 10 percent heat inactivated (56° C/30min) normal bovine serum. The final content of the mixture is about 9%. This mixture (pH 7.2) is transferred to plastic culture flasks so that the final depth is approximately
  • any in vitro propagative technique which promotes growth stage development of Ana ⁇ plasma initial bodies should provide for enrichment of the growth culture medium with soluble antigens of the invention.
  • Availability of the soluble specific antigen by the procedure of Examples 1 and 2 will make possible further purification by straightforward means and the eventual determina ⁇ tion of whether lower molecular weight fragments of the relatively crude antigen isolates are or can be responsible for in vivo protective activity.
  • Chemical synthesis or recombinant DNA techniques may thereafter be employed to generate in large quantities the antigen or any lower molecular weight, immunologically active fragment or fragment analog.
  • the supernatant containing the soluble antigen is centrifuged at 12,000 x g for 30 minutes and passed through a 0.22 micron millipore filter.
  • the antigen-containing filtrate may be maintained in a frozen state for long periods of time or lyophilized and stored at 4° C. under vaceum.
  • the antigen was found to be soluble in water and normal saline but insoluble in 40 to 75% solutions of ammonium sulfate.
  • a vaccine is prepared in the following manner. Cultures were prepared in flasks according to Example 2 utilizing cells with an initial parasitema of 8.0 percent and a total culture volume of about 110 ml per flask containing about 9% erythrocytes. Supernatants were collected at 2 day intervals and processed according to Example 3. For each vaccine dose, twenty ml of supernatant collected at 96 hours a ter initiation of cultures is lyophilized and stored under vacuum at 4° C. This quantity of the lyophilized material (about 400 mg) was reconstituted in 1 mL of 1 • gram/liter aqueous solution of Saponin adjuvant (Quil-A of Superfos Export Co., Copenhagen, Denmark) to yield 1 ml of vaccine.
  • the two test animals were vaccinated on day 0 of the test procedure and again on day 21 with 1 ml of Anablock antigen vaccine preparation obtained according to Example 5.
  • the four cows were chal ⁇ lenged with virulent A. marginale on test day 4L
  • the challenge consisted of intramuscular administration of 1 ml of bovine blood containing virulent A. marginale organisms in 73 percent of the erythrocytes.
  • the latex particles in suspension were thereafter sensitized for 15 minutes at 56° C with an equal volume of an aqueous solution of lyophilized antigen according to Example 3 which had been restored to a volume equal to 0.625 to L25 times the original culture supernatant volume with distilled water.
  • the agglutination test was performed on glass slides marked into 2.5 cm squares. One drop of sensitized latex particles was applied to each square and one drop of undiluted serum was added, mixed, hand-rotated, and the suspension exam- ined over indirect lighting for evidence of agglutination.
  • Both of the control animals developed characteristic symp ⁇ toms of anaplasmosis, i.e., anemia, anorexia and high temperature, while the vaccinated animals remained clinically normal throughout the experiment.
  • one vaccinate and one control animal were pregnant at the time of the experiment.
  • the vaccinated animal delivered a healthy calf at 45 days after challenge.
  • the calf was allowed to obtain colostrum and milk from its immunized mother and exhibited a passive IFA titer of 1:20,400 for more than one month.
  • Both the cow and calf remained free of hemolytic disease indicating lack of isoimmune response to vaccination.
  • patent A. marginale infection however, the pregnant control animal aborted on day 34 after challenge.
  • Example 3 A second study was conducted using the soluble Anablock antigen of Example 3. Four test and four control Angus heifers were employed and vaccination was according to the procedures of Example 6. Two of the test and three of the control animals had been used as vaccinate test animals in a Babesia bovis vaccine trial and entered this experiment about four months after _ bovis challenge. No variations in serologic profile in this test were attributable to the prior B. bovis vaccination history of the heifers. The test animals were vaccinated at day 0 of the test (1 mL vaccine subcutaneously at the right side of the neck), and again on day 21 (1 ml. vaccine subcutaneously at the left side of the neck).
  • results for IFA, CA and LAT tests are set out in Table IL
  • Table II the following symbols are used to represent LAT test data: "-" for a negative response characterized by uniform turbity without floceulation; "+” for a weak response characterized by fine clumps, granular appearance; "++” for a moderate response characterized by dis ⁇ tinctly larger clumps; and "+++” for a strong response characterized by large definitive clumps with complete aggregation.
  • "-" indicates a negative response and "+” indicates a positive response and "+” indicates aft equivocal test result.
  • IFA and CF titers of less than 1:20 and CF titers of less than 1:5 were not considered to be significant and are reported as "0".
  • Example 6 While the experimental data do not illustrate as dramatic a protective response to anaplasmosis as was noted in Example 6, all measured parameters of severity of infection indicated significant advantages in response to infectious challenge for the vaccinated test animals over the controls. Because none of the animals died as a result of challenge, one of the most significant distinctions between test and control animals was the difference in average lowest packed cell volume. The vaccinates displayed lowest PCV's of 22 percent while that of the controls was 15 percent.
  • vaccine dosages employed in Examples 6 and 7 provided a total dose of about 40 mg. of the relatively crude Anablock antigen of Example 3 per 100 pounds of body weight, it will be understood that immunoprophylaxis may be provoked by vaccines providing from about 0.1 to about 1000 mg. of antigen per 100 pounds of body weight.
  • subcutaneous administration of vaccines is presently preferred other parenteral modes of administration (intravenous, intramuscular, etc.) are within the contemplation of the invention and may involve use of adjuvants other than saponin.
  • Oral administration is also contemplated, provided that the antigen is capable of surviving digestive conditions (with or without enteric coating) and is absorbable in immunologically active form through digestive tract tissue.
  • vaccines may be prepared which contain the antigen in combination with other therapeutically and immunologically active water soluble substances * such as drugs and antigens protective against other diseases.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Des antigènes solubles dans l'eau et spécifiques de l'Anaplasma sp. sont produits et isolés in vitro. Par exemple, un antigène d'Anaplasma marginal soluble, stable et non vivant (désigné "Anablock") se caractérise, inter alia, en tant que glycoprotéine possédant un poids moléculaire d'environ 53.000 et un poids isoélectrique d'environ 6. Des compositions de vaccins contenant des quantités immunologiquement efficaces d'un antigène spécifique soluble et d'un adjuvant approprié peuvent provoquer une réponse immunitaire protectrice contre l'Anaplasma virulent, par exemple, en bloquant le développement de l'infection provoquée par l'A. marginal virulent. La réponse immunitaire protectrice dans le sujet vacciné se caractérise sérologiquement par des résultats positifs dans le test d'agglutination de latex (LAT) et d'anticorps fluorescents (ISA), mais par des résultats négatifs dans les tests de fixation de compléments (CS) et d'agglutination dans des tubes capillaires (CA).
EP19820902304 1981-06-22 1982-06-17 Antigene pour anaplasma. Withdrawn EP0081575A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US27544681A 1981-06-22 1981-06-22
US275446 1981-06-22

Publications (2)

Publication Number Publication Date
EP0081575A1 true EP0081575A1 (fr) 1983-06-22
EP0081575A4 EP0081575A4 (fr) 1983-10-26

Family

ID=23052321

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19820902304 Withdrawn EP0081575A4 (fr) 1981-06-22 1982-06-17 Antigene pour anaplasma.

Country Status (2)

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EP (1) EP0081575A4 (fr)
WO (1) WO1983000017A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GR860772B (en) * 1985-03-25 1986-07-21 Univ Washington Anaplasma marginale subunit antigen for vaccination and diagnosis
US4956278A (en) * 1988-02-23 1990-09-11 Louisiana State University Anaplasma marginale antigen, antigen compositions, vaccine and process for the production of said antigen, antigen compositions and vaccine
US5869335A (en) * 1995-08-25 1999-02-09 Regents Of The University Of Minnesota Method of growing rickettsiae in Ixodes scapularis tick cell culture and preparing antigens and vaccines of rickettsiae
GB9726009D0 (en) * 1997-12-10 1998-02-04 Metal Box Plc Can base reforming
RU2503461C2 (ru) * 2012-03-05 2014-01-10 Государственное научное учреждение Всероссийский научно-исследовательский институт экспериментальной ветеринарии имени Я.Р. Коваленко Способ получения антигена для серологической диагностики анаплазмоза мелкого рогатого скота

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3511908A (en) * 1965-06-23 1970-05-12 Univ Oklahoma State Method of manufacturing anaplasmosis vaccine
US3674860A (en) * 1969-07-14 1972-07-04 Diamond Lab Inc Anaplasmosis vaccines
US3616202A (en) * 1970-06-15 1971-10-26 Research Corp Culture of anaplasma marginale

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8300017A1 *

Also Published As

Publication number Publication date
WO1983000017A1 (fr) 1983-01-06
EP0081575A4 (fr) 1983-10-26

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