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WO1992019757A1 - Oligomere b de recombinaison de la toxine de coqueluche - Google Patents

Oligomere b de recombinaison de la toxine de coqueluche Download PDF

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Publication number
WO1992019757A1
WO1992019757A1 PCT/US1992/003485 US9203485W WO9219757A1 WO 1992019757 A1 WO1992019757 A1 WO 1992019757A1 US 9203485 W US9203485 W US 9203485W WO 9219757 A1 WO9219757 A1 WO 9219757A1
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WO
WIPO (PCT)
Prior art keywords
subunits
oligomer
recombinant
multimeric
chaotrope
Prior art date
Application number
PCT/US1992/003485
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English (en)
Inventor
W. Neal Burnette
Vernon L. Mar
Original Assignee
Amgen Inc.
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 Amgen Inc. filed Critical Amgen Inc.
Publication of WO1992019757A1 publication Critical patent/WO1992019757A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/235—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bordetella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the recombinant expression of the subunits of the B oligomer of pertussis toxin and association of the subunits in vitro to form a non-reactogenic multimeric composite capable of use in a vaccine for eliciting an immunoprotective response against infection, and the effects of infection, by Bordetella sp .
  • toxin-based pertussis vaccines a major concern in producing toxin-based pertussis vaccines is the elimination of the enzymatic activity of the SI subunit.
  • deactivation of SI is accomplished by treatment of vaccine materials with heat or toxoiding agents such as formalin and glutaraldehyde.
  • toxoiding agents such as formalin and glutaraldehyde.
  • the disadvantages of these methods of physical inactivation are that, if too stringent, they can substantially reduce the protective immunogenicity of the toxin; and if not stringent enough, the toxicity-related enzyme activity of the molecule can reappear upon shelf storage (5) .
  • B oligomer - is believed to be the delivery platform for the SI subunit, possessing cell receptor recognition domains (1,10-13); it can be isolated as a pentameric macromolecule without the SI subunit, comprised of subunits S2, S3, S4, and S5 in an approximate molar ratio of 1:1:2:1, respectively (1) . It has recently been shown that natural (native) B oligomer, which intrinsically possesses none of the ADP- ribosyltransferase activity associated with the toxicity of pertussis toxin, is by itself sufficient to elicit immunoprotective responses (14-16) ; further, in studies with recombinant proteins conducted in collaboration with Dr. Drusilla L. Burns and Dr. Juan L.
  • B oligomer Arciniega of the U.S. Food and Drug Administration, the SI subunit did not appear to contribute significantly to the protective response of the B oligomer. Thus, B oligomer clearly has the potential as a component of acellular pertussis vaccines. However, the natural B oligomer used in these studies was isolated from the holotoxin molecule, and while largely free of the SI subunit (>95%), B oligomer derived in such a manner has measurable amounts of Sl-related activity which may contribute significantly to its reactogenicity.
  • FIG. 1 This figure depicts the SDS-PAGE of recombinant B oligomer-like multimer in accordance with this invention (hereinafter referred to simply as recombinant B oligomer) .
  • B oligomer was assembled in vitro from recombinant E. coli-produced subunits S2, S3, S4, and S5, as described in the following text, and purified by affinity chromatography on fetuin-Sepharose (17) .
  • TCA trichloroacetic acid
  • the gel was subsequently stained with Coomassie Blue.
  • FIG. 2 This figure is a graphical representation of the mitogenic activity of B oligomer preparations, including recombinant B oligomer according to this invention.
  • Two-fold serial dilutions of the indicated B oligomer preparations were made in RPMI 1640 medium.
  • Mouse splenic lymphocytes were then added to the B oligomer preparations to yield concentrations of B oligomer indicated on the abscissa.
  • [ 3 H]thymidine incorporation was measured by conventional means (19) .
  • the recombinant B oligomer preparation was stored in high concentrations of urea (2 M) , and because of its relatively low initial protein concentration, the lower dilutions of the recombinant B oligomer preparations contained significant amounts of residual urea that resulted in cell death.
  • the preparations were: natural B oligomer (•) ; natural B oligomer which at the highest concentration shown contained 0.125 M urea ( ⁇ ) ; and recombinant B oligomer which at the highest concentration shown contained 0.125 M urea (o) .
  • FIG. 3 This figure illustrates the effect of immunization of mice with B oligomer preparations on the leukocytosis-promoting activity of PT.
  • Five mice were each injected intraperitoneally (i.p.) with preparations of recombinant B oligomer (rB) according to this invention, or natural B oligomer (B) , at doses of 8 ⁇ g (rB H /B H ) , 1 ⁇ g (rB M /B M ) , or 0.1 ⁇ g (rBi/Bi.) in a total volume of 0.5 ml of PBS-gel with alum (see Table 1) .
  • One group of 5 mice (marked "PT") were mock-immunized with PBS-gel.
  • the present invention provides a genetically-engineered B oligomer-like multimeric protein (recombinant B oligomer) of pertussis toxin derived from recombinant materials, as well as a method of preparation. Briefly described, the cistronic elements ("genes") for the S2, S3, S4, and S5 subunits of the toxin, and any analogs of such subunits produced by genetic manipulation (e . g.
  • site-specific mutagenesis and derivatives thereof, are individually expressed in separate transformed heterologous (“foreign") hosts, harvested, and then associated in vitro to form a multimeric composite which, like natural B oligomer, has the ability to elicit a mitogenic response in lymphocytes and, more important, to induce an immunoprotective response against pertussis toxin.
  • mice with the recombinant B oligomer of this invention, or with native B oligomer isolated from PT produced by B. pertussis resulted in antibodies reactive with PT in ELISA and which were able to neutralize the cytopathic effect of PT on cultured Chinese hamster ovary (CHO) cells; antibody titers evoked by each preparation were similar.
  • mice immunized with either natural or recombinant B oligomer were protected against the leukocytosis-promoting activity of PT.
  • each of the subunit polypeptides is synthesized with an initiating methionine residue substituting for its native signal peptide sequence; the heterologous amino-terminal methionine residue is substantially cleaved from each of the recombinant proteins, with the exception of subunit S4, by endogenous methionyl aminopeptidase.
  • the S4 subunit is thus a "methionyl-mature" polypeptide, i.e. it is an analog of the natural mature S4 subunit bearing a methionine residue at its amino terminus .
  • Each of the recombinant subunit proteins were synthesized in individual E. coli transformants in individual fermentations.
  • Cell pastes were recovered from the fermentation broths and were subsequently stored at -60°C to -80°C.
  • Cells in the individual cell pastes were disrupted by two passages each through a French press at approximately 10 psig in the presence of 1 mM dithiothreitol (DTT) at a temperature of approximately 4°C.
  • DTT dithiothreitol
  • inclusion bodies were recovered from the cell lysates, and washed at least twice, by differential centrifugation; the first wash was in 1% deoxycholate (DOC) and 25 mM Tris-HCl (pH 7.9), and the second wash was in 4 M urea and 25 mM Tris-HCl (pH 7.5) .
  • DOC deoxycholate
  • Tris-HCl pH 7.9
  • the individual inclusion body preparations were weighed and then mixed in a stoichiometric amount, based upon their relative percentage of subunit protein, approximating their molar ratio in natural B oligomer (S2:S3:S4:S5 at 1:1:2:1, respectively) .
  • the inclusion- body mixture was solubilized by adding a solution of 6 M guanidinium-HCl (GuHCl) and 10 mM DTT in a buffer of 25 mM Tris-HCl (pH 8.5); the mixture was then stirred gently overnight at room temperature. The mixture was centrifuged (18,000 rpm in a JA20 rotor for 30 minutes at 2°C-ro m temperature) to remove insoluble materials.
  • the soluble supernatant was recovered and excess DTT removed by desalting in a chromat ⁇ graphic column of GH25 with the same GuHCl-Tris buffer free of DTT.
  • the protein peak eluting at, and near, the void volume of the GH25 column was recovered.
  • the sample could be diluted with 6 M GuHCl anytime after this step.
  • CU2SO 4 was added to a final concentration of 50 ⁇ M; the sample was gently stirred overnight in an air atmosphere at room temperature. Following this reoxidation step, the sample was dialyzed against two changes (5 liters per change) of 100 mM potassium phosphate buffer (pH 7.4) containing 2 M urea; dialysis tubing should have the tolerance of 3,500-MW cut-off.
  • Fetuin-Sepharose affinity resins were prepared from commercial fetuin Sepharose 4B, purified by the Spiro method (available from Gibco) and covalently linked under published conditions.
  • the dialyzed sample was applied twice to a chromatographic column of fetuin- Sepharose at temperatures of 2°C-room temperature; bulk (non-column) methods of application, wash, and elution may also be utilized.
  • the loaded affinity resin was washed sequentially with PBS and with buffer A (0.05 M Tris-HCl, pH 7.5) containing 1 M NaCl. The sample binding to the resin under these conditions was eluted with buffer A containing 4 M MgCl 2 .
  • the individual recombinant subunits were isolated as insoluble inclusion bodies following lysis of the bacteria.
  • Individual inclusion- body preparations were solubilized in 6 M guanidinium hydrochloride (GuHCl) in the presence of 1 mM dithiothreitol (DTT) and combined to give a molar ratio approximately equivalent to that estimated to be found in natural B oligomer (i.e., S2:S3:S4:S5 at 1:1:2:1, respectively) .
  • Reductant is removed by size-exclusion chromatography, reoxidation enabled by stirring in an air atmosphere in the presence of 50 ⁇ M CU 2 SO 4 , and spontaneous association of the subunits facilitated by equilibrium dialysis against 2 M urea.
  • the recombinant B oligomer was purified to near homogeneity (as assessed by the purity of the individual subunits in the multimer) by affinity chromatography on fetuin-Sepharose (Fig. 1); in our experience, very little if any of the individual subunits bind specifically to the fetuin resin, indicating that the bulk of material eluting in gCl2 is in a multimeric form. The eluant was tested, and found to be positive, for agglutination of goose erythrocytes.
  • the isolated multimer contained a significant amount of the pentameric B oligomer species, however, is clearly demonstrated by the mitogenicity experiments (Fig. 2) : whereas B oligomer and certain of the individual subunits and dimers comprising it are capable of erythrocyte agglutination, only intact B oligomer is mitogenic (10,21) .
  • the recombinant B oligomer of this invention differs from the natural form in that it always possesses a methionyl-mature S4 subunit.
  • mice were inoculated intraperitoneally (i.p.) with graded doses of either natural or recombinant B oligomer. Two weeks after immunization, mice were bled to measure titers of antitoxin antibodies by ELISA and then challenged with lethal doses of PT; animals were observed for toxin-induced death and all were evaluated four days after challenge for toxin-mediated leukocytosis.
  • mice responded to each immunizing agent with significant antitoxin and toxin-neutralizing titers, the latter measured by reduction of PT-mediated CHO cell clustering; moreover, the neutralizing antibody titers induced by the recombinant B oligomer were similar to those observed when natural B oligomer was used as the immunogen.
  • the potency of the recombinant material at the lowest dose may be slightly lower than an equivalent amount of native B oligomer, this difference may more likely be ascribed to accuracy in determining the protein concentration of the recombinant B oligomer by integrative densitometry of stained SDS-polyacrylamide gels .
  • the results presented herein demonstrate the feasibility of creating complex heteromeric proteins from recombinant DNA-derived subunits. In the case of PT, it is now possible to produce a highly immunogenic subspecies of the toxin molecule. Although B oligomer can be produced in B .
  • pertus ⁇ ' is by inactivating the ability of the SI subunit to associate with B oligomer by mutations in its encoding cistronic element, little if any of the B oligomer is subsequently secreted by this organism (24) .
  • B oligomer can be purified from natural toxin, but this is an intensive process which still results in measurable contamination by Sl- containing holotoxin (9,19,25) .
  • the recombinant B oligomer of this invention lack intrinsic toxicity by virtue of deletion of the SI subunit, but removal of the pathogenic organism from the manufacturing process eliminates the potential for contamination by other B .
  • pertussis toxic components 26,27 which may also contribute to the adverse reactions of pertussis vaccines.
  • the genetically-engineered B oligomer-like multimer of this invention can be formulated in a conventional manner into a vaccine.
  • a toxin that has been "genetically" inactivated such as pertussis toxin in the present invention
  • further inactivating steps should not normally be required since these materials are produced in non-pathogenic organisms and are inherently free of the Sl-related enzyme activity that is generally accepted to elicit the adverse reactions to whole-cell pertussis vaccines and to untreated (or not stringently inactivated) pertussis holotoxin vaccines.
  • recombinant B subunit macromolecules described in the present disclosure as potential vaccinating antigens would be purified to >90% homogeneity.
  • the nature and estimated quantity of contaminants, if any, would be evaluated to ensure that the extent of endotoxin contamination meets the standards of the individual regulatory agencies.
  • the vaccine materials would normally be adsorbed onto aluminum adjuvants. This can be accomplished by at least two means: precipitation with preformed alum and precipitation with aluminum salts. The adsorbed precipitates are then resuspended in an excipient to yield a dosage concentration of vaccine antigen generally in the range of 5-100 ⁇ g per dose and an alum amount usually not exceeding 1.5 mg/dose; volume per dose is generally in the range of 0.1-1.0 ml.
  • the suspending excipient is commonly a buffered solution (e.g., phosphate-buffered saline, pH 7.0), may have added stabilizers (e.g., glycerol) , and will likely contain a preservative (e.g., 0.01% Thimerosal) to prevent microbial contamination and to extend shelf life.
  • a buffered solution e.g., phosphate-buffered saline, pH 7.0
  • stabilizers e.g., glycerol
  • a preservative e.g., 0.01% Thimerosal
  • the vaccine materials could be formulated in enteric-coated capsules or other delivery vehicle to prevent degradation of the vaccine product until it reaches lymphoid tissues of the gut (e.g., Peyer' s patches) .
  • Elicitation of a secretory immune response in the gut can be transmitted globally ( via slgA and/or slgA-secreting lymphocytes) to the lamina intestinal of other mucosal-lined organs, and particularly to the respiratory tract for protection against pertussis.
  • the vaccine product could be formulated as an aerosol or liquid for respiratory, buccal, or nasal delivery in an appropriate excipient and/or delivery vehicle (e.g., liposomes) , such that it stimulates a secretory immune response directly in the respiratory tree or is transmitted (as above) from the buccal or nasal lymphoid tissue to the lamina limbaloid tissue to the lamina limbal, and the like.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
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Abstract

Une protéine multimère de la toxine de coqueluche analogue à l'oligomère B, obtenue par technique de recombinaison et utile comme constituant non réactogène dans un vaccin anticoquelucheux est produite lorsqu'on exprime les éléments cistroniques individuels des sous-unités S2, S3, S4 et S5 séparément dans un hôte hétérologue, lorsqu'on récupère les polypeptides de sous-unités produits, et qu'on les réunit par association in vitro de façon à former un composite multimère.
PCT/US1992/003485 1991-05-03 1992-04-28 Oligomere b de recombinaison de la toxine de coqueluche WO1992019757A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US69535991A 1991-05-03 1991-05-03
US695,359 1991-05-03
US70543391A 1991-05-24 1991-05-24
US705,433 1991-05-24

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WO1992019757A1 true WO1992019757A1 (fr) 1992-11-12

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CN (1) CN1067678A (fr)
AU (1) AU1924292A (fr)
IE (1) IE921407A1 (fr)
IL (1) IL101692A0 (fr)
WO (1) WO1992019757A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0646599A3 (fr) * 1993-08-24 1996-05-15 Connaught Lab Modification de la toxine pertussis.
US6261560B1 (en) 1995-02-13 2001-07-17 Chugai Seiyaku Kabushiki Kaisha Method for inhibiting muscle protein proteolysis with antibodies to interleukin-6 receptor
EP1011718A4 (fr) * 1997-08-15 2003-01-08 Picower Inst Med Res Traitement antiviral utilisant le b-oligomere de la toxine coquelucheuse

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SI9720050B (en) * 1996-07-02 2001-12-31 Connaught Lab Multivalent dtp-polio vaccines

Citations (2)

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Publication number Priority date Publication date Assignee Title
US4511502A (en) * 1982-12-22 1985-04-16 Genentech, Inc. Purification and activity assurance of precipitated heterologous proteins
US5085862A (en) * 1987-11-24 1992-02-04 Connaught Laboratories Limited Genetic detoxification of pertussis toxin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4511502A (en) * 1982-12-22 1985-04-16 Genentech, Inc. Purification and activity assurance of precipitated heterologous proteins
US5085862A (en) * 1987-11-24 1992-02-04 Connaught Laboratories Limited Genetic detoxification of pertussis toxin

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIO/TECHNOLOGY, Volume 6, issued June 1988, W.N. BURNETTE et al., "Direct Expression of Bordetella Pertussis Toxin Subunits to High Levels in Escherichia Coli". *
BIO/TECHNOLOGY, Volume 8, issued November 1990, W.N. BURNETTE, "The Advent of Recombinant Pertussis Vaccines", pages 1002-1005. *
BIOCHEMISTRY, Volume 21, No. 22, issued 1982, M. TAMURA et al., "Subunit Structure of Islet-Activating Protein, Pertussis Toxin, in Conformity with the A-B Model", pages 5516-5522. *
INFECTION AND IMMUNITY, Volume 55, No. 5, issued May 1987, J.L. ARCIENEGA et al., "Immune Response to the B Oligomer of Pertussis Toxin". *
INFECTION AND IMMUNITY, Volume 58, No. 12, issued December 1990, R.S. SHAHIN et al., "Mechanism of Pertussis Toxin B Oligomer-Mediated Protection against Bordetella Pertussis Respiratory Infection", pages 4063-4068. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0646599A3 (fr) * 1993-08-24 1996-05-15 Connaught Lab Modification de la toxine pertussis.
US5856122A (en) * 1993-08-24 1999-01-05 University Of Alberta Modification of pertussis toxin
US5977304A (en) * 1993-08-24 1999-11-02 Connaught Laboratories Limited Modification of pertussis toxin
US6018022A (en) * 1993-08-24 2000-01-25 Connaught Laboratories Limited Modification of pertussis toxin
US6168928B1 (en) 1993-08-24 2001-01-02 Connaught Laboratories Limited Modification of pertussis toxin
US6261560B1 (en) 1995-02-13 2001-07-17 Chugai Seiyaku Kabushiki Kaisha Method for inhibiting muscle protein proteolysis with antibodies to interleukin-6 receptor
EP1011718A4 (fr) * 1997-08-15 2003-01-08 Picower Inst Med Res Traitement antiviral utilisant le b-oligomere de la toxine coquelucheuse

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IL101692A0 (en) 1992-12-30
IE921407A1 (en) 1992-11-04
CN1067678A (zh) 1993-01-06
AU1924292A (en) 1992-12-21

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