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WO1990011777A1 - Preparation de sous-unites de poils non assemblees et vaccins les contenant - Google Patents

Preparation de sous-unites de poils non assemblees et vaccins les contenant Download PDF

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Publication number
WO1990011777A1
WO1990011777A1 PCT/US1990/001592 US9001592W WO9011777A1 WO 1990011777 A1 WO1990011777 A1 WO 1990011777A1 US 9001592 W US9001592 W US 9001592W WO 9011777 A1 WO9011777 A1 WO 9011777A1
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WO
WIPO (PCT)
Prior art keywords
unassembled
pilin
vaccine
bacteria
solution
Prior art date
Application number
PCT/US1990/001592
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English (en)
Inventor
John C. Mcmichael
Original Assignee
Immunomed Corporation
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 Immunomed Corporation filed Critical Immunomed Corporation
Publication of WO1990011777A1 publication Critical patent/WO1990011777A1/fr

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    • 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
    • A61K39/104—Pseudomonadales, e.g. Pseudomonas
    • A61K39/1045—Moraxella

Definitions

  • the present invention relates to a unique method for obtaining unassembled bacterial pilus subunits suitable for use in preparing a vaccine, as well as vaccines containing unassembled pilin and which exhibit enhanced cross-reactivity with heterologous bacteria and protect animals from infection.
  • the primaty difficulties associated with producing a pili subunit vaccine still basically involve dete_rmining a commercially-acceptable procedure for deriving the pili subunits so that a subunit vaccine can be readily prepared in accord with accepted manufacturing processes.
  • those primary difficulties are substantially alleviated, for I have not only developed an improved method for obtaining unassembled pilus subunits, but also have developed a method utilizing unassembled pilin from bacteria capable of producing or modified to produce unassembled pilin, and utilizing those products have developed an improved vaccine having enhanced cross-reactivity with heterologous bacteria.
  • the present invention relates, first, to a unique method for obtaining unassembled bacterial pilus subunits directly from bacteria.
  • unassembled bacterial pilus subunits can be obtained from and vaccines can be developed against, virtually any piliated pathogenic bacteria, even those that are incapable of completing the assembly of the pilus.
  • unassembled pilin have been obtained from Moraxella bovis. and vaccines prepared from the M. bovis unassembled pilin have demonstrated enhanced efficacy.
  • Pili from M. bovis belong to the NMePhe pilus group, and this class of pili is found not only on M.
  • unassembled pilin may be obtained by first subjecting the pili to ultrasonication to dissociate the pili into pilin. This may normally be accomplished in a period of from 2 to 5 minutes using a sonication power of from about 35 to about 60 watts. The sonicated pilin are then dialysed against distilled water at a pH of from about 11.5 to about 12.5 for a period of from 24 to 72 hours. The dialysed material is then adjusted to substantial neutral pH.
  • unassembled pilin may be obtained directly from bacteria which synthesize pilin but are incapable of assembling it.
  • subjecting bacteria to subinhibitory concentrations of antibiotic penicillin will allow synthesis of the pilin to proceed, but will prevent its assembly into pili, also resulting in a source of unassembled pilin for use in preparing the vaccine of this inv ⁇ tion.
  • the unassembled pilin Once the unassembled pilin has been obtained, it may be preserved, stored or lyophylized for subsequent use in vaccine formulation, or one may proceed directly to vaccine formulation. In any event, of course, the concentration of the unassembled pilin would be adjusted as required-for vaccine preparation. Vaccines formulated with unassembled pilin would normally include an adjuvant, but the use of an adjuvant is not required within the scope of this invention. In similar fashion, vaccines utilizing the unassembled pilin of this invention might also include preservatives and stabilizers. Furthermore, it has been found desirable to include unassembled pilin from more than one bacterial strain in the vaccine.
  • the present invention relates to a method for obtaining unassembled pilus subunits as well as vaccines containing unassembled pilin which elicit antibodies with enhanced cross-reactivitywith heterologous bacteria, and protect animals from infection.
  • the method of this invention has been demonstrated to be suitable for use in obtaining unassembled pilin from a variety of pathogenic bacteria, and detailed studies have been conducted with regard to M. bovis including a particularly effective vaccine prepared using aggregates of unassembled pilin from different isolates of M. bovis.
  • the term "oligomer” is synonymous with "unassembled pilus subunits" and with "unassembled pilin.” The following examples, then, are set forth in order to describe more fully the method and vaccine of the present invention.
  • Moraxella bovis isolates FLA64(6), EPP63, MED72(23R), and NDL67(S-57R) were obtained from the National Animal Disease Center, AMES, Iowa.
  • the MISS isolate was supplied by the Mississippi Diagnostic Laboratory, Jackson, Mississippi, and the IM427 was isolated locally from a Holstein cow in Hillsborough County, Florida.
  • Pili were prepared from the M. bovis as follows. M. bovis, grown for 24 hours on Mueller-Hinton agar was scraped into lOmM Tris-HCl buffer adjusted to pH 7.0 and blended for 2 minutes to shear the pili from the bacteria. After centrifugation, the supernatant was retained and ammonium sulfate was added to 20% saturation.
  • Vaccines were prepared in three fashions. A first vaccine was prepared with intact pili in a Freund's adjuvant. A second vaccine was prepared utilizing intact pili in aluminum hydroxide adjuvant. A third vaccine was prepared utilizing the oligomers (unassembled pilin) in aluminum hydroxide adjuvant. Groups of two New Zealand white rabbits were immunized with a pilus preparation in a Freund's adjuvant or in aluminum hydroxide adjuvant, or with the unassembled pilin preparation in aluminum hydroxide adjuvant. The Immunization schedule was three injections 2 weeks apart followed by bleeding the rabbits one week after the final injection.
  • the rabbits receiving the pili in Frei ⁇ nd's adjuvant had the first injection formulated in complete Freund's adjuvant followed by 2 injections in incomplete Freund's adjuvant. These were prepared as an emulsion of a 1:1 mixture (v:v) of antigen to adjuvant, and administered subcutaneously.
  • the pili and unassembled pilin were absorbed with a final concentration of 1.2% aluminum hydroxide and were administered intramuscularly.
  • the amount of material injected was 50 micrograms protein per 0.45kg at each injection.
  • Sera were collected 1 week after the last injection and kept frozen at -40 C until needed.
  • the titers of the various antisera against purified intact pili were determined by an enzyme linked immunosorbent assay.
  • Moraxella bovis isolates FLA64(6), MED72(23R), and EPP63 were obtained from the National Animal Disease Center, AMES, Iowa.
  • M. bovis isolate IM427 was isolated locally from a Holstein cow in Hillsborough County, Florida.
  • the unassembled pilin form of the antigen was prepared from purified pili substantially as described in Example I, above. The pili were converted to unassembled pilin by dialysis for 48 hours against 0.15M phosphate buffer at pH 12.5 at 4 C followed by dialysis against a
  • vaccines were prepared having four different dosages (50 micrograms, 10 micrograms, 2 micrograms and 0 micrograms) of each antigen. All vaccine formulations included 1.2% aluminum hydroxide as adjuvant.
  • end point titers were determined by an enzyme linked immunosorbent assay.
  • the administered vaccines were prepared from the unassembled pilin of the FLA64 and MED72 isolates, mixed at various concentrations, adsorbed with an aluminum hydroxide adjuvant.
  • the titers were determined against the two pilus antigens from which the immunogens were prepared, and against pili from two heterologous isolates (EPP63 and IM427) .
  • adjuvants include alum, aluminum phosphate, emulsified paraffin, emulsified paraffin with lecithin, purified saponin, oils, liposomes, detergent-type micelles, ISCOM's and im unostimulants.
  • Preservatives such as those having USDA approval may be added to the vaccine formulation, and stabilizers such as boric acid, EDTA, bovine serum albumin, and saccharides may also be utilized.
  • the following test was conducted to investigate the efficacy of both the whole pilus and pilus oligomer isolated from Moraxella bovis as vaccines against IBK.
  • the vaccines were prepared from the same bacterial strain that was used as the challenge pathogen. This type of experiment is referred to as a homologous challenge experiment.
  • the first group of three were vaccinated with the whole pilus preparation.
  • the second group of three were vaccinated with the pilus oligomer preparation.
  • the third group of two received no vaccine and served as controls.
  • the vaccinated calves received two injections of the respective vaccines thirty days apart. Sera from all calves were collected periodically in order to follow anti-pilus antibody levels. Twenty days after the second vaccination, all calves were challenged after stressing their eyes with UV irradiation. Protection against IBK was monitored for 5 days after challenge by examination of the eyes for gross symptoms and by the recovery of beta hemolytic bacteria from the animals' eyes.
  • the vaccines were prepared from the EPP63 strain of M. bovis.
  • the intact pili were prepared by scraping the 24 hr. growth from Mueller-Hinton agar into 10 mM, pH 7.0 Tris-HCl buffer.
  • the pili were separated from the bacteria by blending 2 min. and then centrifuging for 20 min. at 20,000 RPM.
  • the supernatants were saved and ammonium sulfate was added to 20% saturation. After incubating 1 to 2 hr., this solution was centrifuged at 18,000 RPM for 2 hr.
  • the pellet was retained and resuspended in the Tris-HCl buffer. After resuspension, usually overnight, the solution was centrifuged at 18,000 RPM for 2 hr.
  • the oligomer was prepared from the purified pili by ultrasonication with a Branson W-350 apparatus using an 18 mm probe for 1 min. at a setting of 10. To complete the conversion to oligomers, the sonicated pili were dialyzed against distilled water adjusted to pH 11.5 with NaOH for 48 to 72 hr. The formation of oligomers was confirmed by "native" polyacrylamide gel electrophoresis and electron microscopy.
  • the vaccines were prepared by mixing the pili or oligomer for 1 hr. with Rehsorptar II (Armour Pharmaceuticals), an alum based adjuvant. The mixing ratio was 2 parts pili or oligomer to 3 parts Rehsorptar II. A total of 5 mg protein for the whole pilus and a total of 3.5 mg protein of the oligomer were administered intramuscularly (i.m.) per calf in a volume of 8 to 8.4 ml at each inoculation.
  • Rehsorptar II Armour Pharmaceuticals
  • the suspension medium was 10 ml of NIH thioglycollate medium containing 7% MgCl 2 .6H 2 0.
  • NIH thioglycollate medium containing 7% MgCl 2 .6H 2 0.
  • All eyes were swabbed the day prior to, just prior to, and each day for five days, after challenge.
  • Marion Scientific Culturettes were used for the swabbing and transport of the cultures to the laboratory. The swabs were then promptly streaked onto 5% blood agar plates and incubated overnight at 35 C. Small beta-hemolytic colonies were scored as being M. bovis.
  • Both vaccines elicited increased antibody titers as detected by the ELISA based on the whole pilus as the assay antigen. After the first injection, the titer rose to a plateau on about day 12. The second injection, on day 30, increased the titer even more with the maximum occurring on day 40. Both vaccines induced nearly equal titers after the first injection, but after the second injection, the whole pilus induced the highest titers. This difference in antibody induction, however, may be due to the use of only the whole pilus and not the oligomer as the antigen in the ELISA. The control animals exhibited no increase in antibody titers until after the bacterial challenge. Then a minimal increase in anti-pilus antibody was detected.
  • both the whole pilus and oligomer vaccines gave complete protection from the challenge of the homologous strain of M. bovis (EPP63) .
  • the oligomer vaccine gave antibody titers comparable to the intact pilus vaccine, as measured by ELISA against whole pili as the antigen.
  • the whole pilus vaccine gave the highest antibody titers.
  • One of the controls was challenged with the FLA 4 strain, and the remaining two control calves were challenged with the strain mixture. Protection against IBK was monitored for five days after challenge by examination of the eyes for gross clinical signs, and for four days by culturing the animals' eyes for M. bovis.
  • the oligomer vaccine was prepared from pili purified from strain EPP63 according to the procedure set forth in Example III, above. However, in this example, the pili were broken from the bacteria by blending for two minutes and then centrifuging for 20 minutes at 10,000 RPM. The final concentration of the vaccine preparation was 0.4 mg/ml. As described above, nine holstein calves received intramuscular injections of the vaccine. A total of '8.4 ml of the oligomer vaccine was injected into the left and right hip of each calf (4.2 ml per site) . The total dose per injection per calf was 3.4 mg protein. Three calves received no injections and served as controls. The first injection was given on day one and the second injection on day 30.
  • Example III In order to achieve infection, the calves' eyes were stressed using UV light as in Example III, but for ten minutes. The schedule for this treatment was 48 hours, 24 hours, and immediately prior to challenge, and 24 hours and 48 hours after challenge.
  • the bacterial challenge material was prepared as set forth in Example III, and the challenge dose was 1 ml of the bacterial suspension per eye instilled on day 45.
  • the eyes of each calf were examined for clinical signs of infection the day prior to challenge, the day of challenge, and for four days after challenge. On the fifth day after examination, all the calves were treated with antibiotic (Panalog) . All eyes were swabbed the day before, just prior to, and each day for four days after challenge.
  • Marion Scientific Culturettes were used for the swabbing and transport of the cultures to the laboratory.
  • the oligomer vaccine prepared from EPP63 gave protection against homologous (EPP63) challenge with the clinical signs resolving by day four. This confirmed the previous finding of protection against the homologous strains as set forth in Example III.
  • the vaccine showed protection against the heterologous FLA64 challenge, as well as the mixed homologous and heterologous challenge.
  • the clinical signs for three of six of the vaccinated calves were completely resolved by day five, and the remainder of these calves developed less severe signs than the unvaccinated calves.
  • the result of this test suggests a proper strategy for designing an M. bovis vaccine.
  • the oligomer has demonstrated a degree of cross-protection against a serologically different challenge strain and at a lower challenge dosage might have been completely protective.
  • an optimized vaccine should contain oligomers prepared from a few selected strains expressing the most divergent serotypes. The data from this test support this approach and further indicate that the vaccine should include oligomers prepared from both FLA64 and EPP63.

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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

On a mis au point un procédé d'obtention de sous-unités de poils bactériennes non assemblées à partir de bactéries spécifiées. La piline non assemblée ainsi obtenue est utile pour préparer des vaccins produisant des anticorps à réaction croisée améliorés à l'aide de bactéries hétérologues, et protégeant les animaux contre des infections.
PCT/US1990/001592 1989-03-31 1990-03-20 Preparation de sous-unites de poils non assemblees et vaccins les contenant WO1990011777A1 (fr)

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US331,177 1989-03-31

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5439808A (en) * 1993-07-23 1995-08-08 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US5747287A (en) * 1995-04-28 1998-05-05 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
WO1999065511A3 (fr) * 1998-06-12 2000-03-02 Univ Alberta COMPOSITION ET PROCEDE DE TRAITEMENT D'UNE INFECTION PAR $i(PSEUDOMONAS)
US6872398B2 (en) * 1995-12-22 2005-03-29 The United States Of America As Represented By The Secretary Of The Army Conjugate vaccine against gram-negative bacterial infections
US7135175B2 (en) 2002-02-27 2006-11-14 Duquesne University Of The Holy Ghost Compositions and methods for eliciting an immune response to gram-negative bacterial infections

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4461838A (en) * 1975-04-25 1984-07-24 Bactex, Inc. Gonococcal Pili processes for the preparation thereof and the use thereof for the detection of and prevention of infections caused by Neisseria gonorrhoeae
US4702911A (en) * 1985-09-27 1987-10-27 Immunomed Corporation Preparation of bacterium pili subunits and vaccines containing pili subunits
US4857318A (en) * 1983-11-07 1989-08-15 Syntex (U.S.A.) Inc. Bordetella bronchiseptica pilus subunit protein vaccine effective against bordetella pertussis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4461838A (en) * 1975-04-25 1984-07-24 Bactex, Inc. Gonococcal Pili processes for the preparation thereof and the use thereof for the detection of and prevention of infections caused by Neisseria gonorrhoeae
US4857318A (en) * 1983-11-07 1989-08-15 Syntex (U.S.A.) Inc. Bordetella bronchiseptica pilus subunit protein vaccine effective against bordetella pertussis
US4702911A (en) * 1985-09-27 1987-10-27 Immunomed Corporation Preparation of bacterium pili subunits and vaccines containing pili subunits

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CANADIAN JOURNAL OF MICROBIOLOGY, Volume 29, issued 1983, I.E. SALIT, "Effect of subinhibitory concentrations of antimicrobials on Meningococcal Adherence", See pages 369 and 374. *
JOURNAL OF BACTERIOLOGY, Volume 163, issued July 1985, C.F. MARRS et al.: "Cloning and sequencing of a Moraxella bovis pilin gene", See pages 132 and 137. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5439808A (en) * 1993-07-23 1995-08-08 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US5879686A (en) * 1993-07-23 1999-03-09 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US6013267A (en) * 1993-07-23 2000-01-11 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US5747287A (en) * 1995-04-28 1998-05-05 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US6872398B2 (en) * 1995-12-22 2005-03-29 The United States Of America As Represented By The Secretary Of The Army Conjugate vaccine against gram-negative bacterial infections
WO1999065511A3 (fr) * 1998-06-12 2000-03-02 Univ Alberta COMPOSITION ET PROCEDE DE TRAITEMENT D'UNE INFECTION PAR $i(PSEUDOMONAS)
US6767545B2 (en) 1998-06-12 2004-07-27 Governors Of The University Of Alberta Pseudomonas treatment composition and method
US7135175B2 (en) 2002-02-27 2006-11-14 Duquesne University Of The Holy Ghost Compositions and methods for eliciting an immune response to gram-negative bacterial infections

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