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WO1992017587A1 - ANTIGENE DE MEMBRANE EXTERIEURE DE $i(BORDETELLA BRONCHISEPTICA) - Google Patents

ANTIGENE DE MEMBRANE EXTERIEURE DE $i(BORDETELLA BRONCHISEPTICA) Download PDF

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Publication number
WO1992017587A1
WO1992017587A1 PCT/GB1992/000561 GB9200561W WO9217587A1 WO 1992017587 A1 WO1992017587 A1 WO 1992017587A1 GB 9200561 W GB9200561 W GB 9200561W WO 9217587 A1 WO9217587 A1 WO 9217587A1
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Prior art keywords
protein
nucleotide
sequence
dna sequence
amino acid
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Application number
PCT/GB1992/000561
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English (en)
Inventor
Ian George Charles
Gordon Dougan
Li Jing LI
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The Wellcome Foundation Limited
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Publication date
Application filed by The Wellcome Foundation Limited filed Critical The Wellcome Foundation Limited
Priority to JP4506936A priority Critical patent/JPH06506111A/ja
Priority to EP92907237A priority patent/EP0594631A1/fr
Publication of WO1992017587A1 publication Critical patent/WO1992017587A1/fr

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Classifications

    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • BORDETELLA BRONCHISEPTICA OUTER MEMBRANE ANTIGEN This invention relates to proteins suitable for use in vaccination against Bordetella bronchiseptica.
  • Bordetalla bronchiseptica is a bacterial pathogen associated with respiratory diseases in animals, particularly atrophic rhinitis in pigs. The disease state is recognised by severe changes in the nasal architecture of developing piglets, pneumonia and growth retardation.
  • protection against B.bronchiseptica mediated atrophic rhinitis correlates with the presence of an outer membrane protein with a molecular weight of 68kDa (P.68) as determined by SDS polyacrylamide gel electrophoresi ⁇ (Novotny et al. , 1985b) .
  • Antibodies to this 68kDa protein can be detected in high titre in protected piglets, while the titre is low or absent from non-protected animals (Novotny et al. , 1985b) . Furthermore, a naturally occurring mutant B ⁇ bronchiseptica lacking the 68kDa protein is unable to protect piglets when used as a vaccine and is unable to induce pathological changes in infected piglets (Novotny et al. , 1985b) .
  • a vaccine consisting of a purified preparation of the 68kDa protein to pregnant sows results in significant protection of the resulting piglets when challenged with virulent B.bronchiseptica (Kobisch & Novotny, 1990) .
  • passively administered monoclonal antibody BB05 Novotny et al. , 1985a specific for the 68kDa protein is able to prevent death from pneumonia and development of atrophic rhinitis in mice that had been aerosol infected with a virulent strain of B.bronchiseptica.
  • Native P.68 antigen can only be purified at low levels from cultures of B.bronchiseptica.
  • the gene from which P.68 is expressed in fact encodes a protein with a Mr of 93996 (P.94). It may be surmised that P.94 is processed to form P.68 on the surface of B.bronchiseptica.
  • heterologous expression of the full-length gene encoding P.94 in E.coli results in similar processing, with the P.68 antigen targeted to the bacterial outer membrane. This allows P.68 to be produced in large amounts uncontaminated by other B.bronchiseptica components.
  • the present invention provides a protein which is uncontaminated by components from B. bronchiseptica, which is capable of binding to antibody which also binds the native P.68 antigen of B___ bronchiseptica and which has (a) the amino acid sequence:
  • Amino acid sequence (a) is the amino acid sequence P.68.
  • Amino acid sequence (b) may contain one or more amino acid insertions, deletions or substitutions. Amino acid sequences (a) and (b) may therefore differ from each other at no more than twelve positions, for example at no more than eight positions or at no more than four positions. The homology between the sequences may therefore by 98% or more, for example 99% or more.
  • amino acid residues of amino acid sequence (a) may therefore be deleted or substituted or one or more additional amino acid residues may be inserted, provided the physicochemical character of the original sequence is retained for example in terms of charge density, hydrophobicity/hydrophilicity, size and configuration.
  • a protein composed of the modified amino acid sequence must also be capable of binding to antibody which is capable of binding the native P.68 antigen.
  • Candidate substitutions are: A for G and vice versa; V by A, L or G; K by R;
  • a protein of the invention is obtained by recombinant DNA technology. The preparation of the protein therefore depends upon the provision of a DNA sequence encoding protein. However, we have found that the protein of the invention is a processed form of a larger precursor protein designated P.94. A DNA sequence encoding P.94 may also be used to express a protein of the invention. In further aspects, therefore, the invention provides: a DNA sequence which encodes a protein of the invention composed of amino acid sequence (a) or (b) ; - a DNA sequence which consists essentially of the nucleotide sequence shown in Figure 1 from nucleotide
  • - a protein which as the amino acid sequence shown in Figure 1 or an amino acid sequence which has a homology of more than 98% with the amino acid sequence shown in Figure 1;
  • - a DNA sequence which encodes a protein as defined immediately above; a DNA sequence which consists essentially of the nucleotide sequence shown in Figure 1 from nucleotide 145 to nucleotide 2877; a DNA sequence which differs from the nucleotide sequence shown in Figure 1 from nucleotide 145 to 2877 at no more than twelve positions;
  • - a DNA sequence which consists essentially of the sequence shown in Figure 1 or a sequence which has a homology of more than 98% with the sequence shown in Figure 1.
  • nucleotide sequences shown in Figure 1 from nucleotide 247 to 2040 and from nucleotide 145 to 2877 encode P.68 and P.94 respectively.
  • DNA sequences which contain differences from these two sequences may have differences at up to twelve nucleotide positions, for example at no more than eight positions or at no more than four positions. These differences may or may not result in coding changes. Codons may be inserted or omitted, and nucleotides may be substituted. There may be a degree of homology of 98% or more, for example 99% or more, between the sequences shown in Figure 1 which encode P.68 and P.94 and modified versions of these DNA sequences.
  • a DNA sequence may be purified and isolated.
  • a DNA sequence may be synthesised de novo, for example by synthesising and annealing oligonucleotides of appropriate sequences.
  • a DNA sequence may be a cloned sequence.
  • the cloning of the DNA sequence may be carried out using standard procedures known in the art. However, it is particularly advantageous in such procedures to employ the sequence data disclosed herein so as to facilitate the identification and isolation of the desired cloned DNA sequences.
  • the DNA is isolated by the method described in Hull et aJL. (1981) as modified by Maskell et al., J. Bacteriol. 170: 2467-2471 (1988).
  • the DNA is then digested with a restriction enzyme to generate short fragments which are then inserted into a cloning vector, such as the cosmid pHC79 or a derivative thereof and the resulting recombinant DNA molecules used to transform E.coli and thus generate the desired library.
  • the library may be screened using a standard screening strategy. For example one may employ as hybridisation probes one or more labelled oligonucleotides synthesised using the DNA sequence information disclosed herein. One or more additional rounds of screening of one kind or another may be carried out to characterise and identify positive clones.
  • the library may be rescreened for additional positive clones using the first clone as a hybridisation probe.
  • further libraries may be prepared and these may be screened using hybridisation probes. In this way, further DNA sequences may be obtained.
  • the desired DNA sequences may be inserted into an expression vector using known and standard techniques.
  • the expression vector is normally cut using restriction enzymes and the DNA sequence inserted using blunt-end or staggered-end ligation.
  • the cut is usually made at a restriction site in a convenient position in the expression vector such that, once inserted, the DNA sequence is under the control of the elements of DNA that effects its expression.
  • Expression vectors of the present invention encompass both extrachromosomal vectors and vectors that are integrated into the host cell's chromosome.
  • the invention therefore also provides - an expression vector which contains a DNA sequence as herein defined, and which, when provided in a suitable host, is capable of expressing encoded protein of the invention; and - a host transformed with such an expression vector.
  • host cells of use with the invention include prokaryotic and eukaryotic cells, such as bacterial, yeast, mammalian and insect cells. Particular examples of such cells are E.coli. S.cerevisiae. P. pastoris, Chinese hamster ovary and mouse cells, and Spodoptera fru ⁇ iperda and Tricoplusia ni.
  • the choice of host cell may depend on a number of factors but, is post- translational modification of the antigen is important, then a prokaryotic host would be preferred.
  • Transformation of a host cell may be carried out using standard techniques.
  • a phenotypic marker is usually employed to distinguish between the transformants that have successfully taken up the expression vector and those that have not.
  • Culturing of the transformed host cell and isolation of the antigen may also be carried out using standard techniques.
  • the invention therefore provides a process for the preparation of a protein of the invention, which process comprises maintaining a host transformed with an expression vector according to the invention under such conditions that the said protein is expressed.
  • the process may comprise: cloning or synthesising a DNA sequence encoding the protein as herein defined; - inserting the DNA sequence into an expression vector such that it is capable in an appropriate host of being expressed; transforming a host cell with the expression vector; culturing the transformed host cell; and isolating the protein.
  • heterologous expression of P.68 or a modified version thereof according to the invention, or of P.94 or a modified version thereof according to the invention may be achieved.
  • These proteins may be expressed therefore in a host which is not B.bronchiseptica.
  • the proteins can thus be obtained free of any other components of B.bronchiseptica.
  • the P.94 or modified version thereof may be processed to P.68 or a modified version thereof.
  • P.68 or a modified version thereof can therefore be obtained from a host cell transformed with an expression vector containing a DNA sequence encoding P.68 or a modified version thereof or a DNA sequence encoding the precursor molecule P.94 or a modified version thereof.
  • the protein obtained in this way may be insoluble and thus may need to be refolded following the use of guanidinium hydrochloride as denaturant in conventional manner and in any event is preferably purified.
  • the invention additionally provides a veterinary composition comprising a protein of the invention and a veterinarily acceptable carrier or diluent.
  • This composition may be used as a vaccine.
  • the vaccine of the invention may optionally contain additional antigens of B.bronchiseptica.
  • the vaccine of the invention is normally associated with a veterinarily acceptable vehicle which allows the protein to be administered to an animal. Administration is usually carried out via the oral, intranasal, or preferably parenteral route. In the case of the parenteral route, the vehicle is generally liquid and the antigen is generally dissolved or suspended in it. An example of -a liquid vehicle is physiological saline solution.
  • the vaccine may also contain an adjuvant for stimulating the immune response and thereby enhancing the potency of the antigen.
  • Convenient adjuvants for use in the present invention include, for example aluminium hydroxide and aluminium phosphate.
  • the vaccine contains a final concentration of protein in the range of from 0.01 to 5mg/ml, preferably 0.03 to 2mg/ml, most preferably 0.3mg/ml.
  • the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4°C, or is freeze dried.
  • the invention also provides a method for inducing immunity to B.bronchiseptica in animals, comprising the administration to an animal of an effective amount of a protein of the present invention.
  • the antigen of the invention may therefore be used to induce immunity to B.bronchiseptica
  • each dose of the vaccine may be from 1ml to 5ml, preferably 2 to 4ml.
  • Figure 1 shows the DNA sequence of the prn gene encoding the P.68 pertactin from B.bronchiseptica. Downward arrows indicate the sites of two possible protein cleavage regions that result in the production of the mature polypeptide. Arrows over protein sequence denote two protein repeat motifs: (i) (GGXXP) 3 encoded by nucleotides 939-983 and (ii) (PQP) 7 encoded by nucleotides 1852 and 1947. Two RGD tripeptide sequences encoded by nucleotides 922-930 and 2245-2253 are shown overlined although only the first sequence occurs in the mature protein.
  • FIG. 1 An inverted repeat that is followed by a run of T-residues is shown between residues 2898-2909 and resembles a rho-independent transcription terminator.
  • Figure 2 is a line drawing representing the strategy used to generate the P.68 expression plasmid pBD881. Plasmids pBD875 and pBD856 contain the 5' and 3' sections of the gene encoding B.bronchiseptica prn respectively and were used to generate the full-length gene encoding P.94.
  • This full-length (non-expressing) gene harboured by pBD876 was excised by digestion with Aflll and Hindlll and ligated with Ncol-Hindlll digested pKK233-2 so as to generate the P.68 expressing plas id pBD881.
  • B. bronchiseptica strain CN7531 Bacterial strains, plasmids and phaqe.
  • B. bronchiseptica strain CN7531 was from the Wellcome culture collection (Wellcome Biotech, UK) .
  • E. coli K12 strains TGI and HB101 were as previously described (Carter et a . , 1985; Boyer & Roulland Dussoix, 1969) .
  • Cosmid pHC79 Hohn & Collins, 1980 was from Amersham International (Little Chalfont, Buckinghamshire, UK) .
  • Plasmid pKK233-2 (Amann & Brosius, 1985) and M13 mpl ⁇ and M13 mpl9 (Yanisch-Perron et al.
  • B. bronchiseptica was grown in Stainer-Scholte (Stainer & Scholte, 1962) broth or medium as previously described (Novotny et a_l. , 1985a) . DNA isolation and manipulations. B. bronchiseptica
  • CN7531 chromosomal DNA was prepared as described previously (Hull jet al. , 1981) . Plasmid and bacteriophage DNA were isolated and purified by standard methods (Maniatis et al. , 1982) . All DNA-modifying enzymes were from Gibco BRL (Paisley, Scotland) .
  • Reco binant phage DNA was sequenced after ligation of specific restriction endonuclease fragments of cosmid into the vector M13 mpl8 and M13 mpl9. Sequencing was carried out using universal primer [ 35 S] dATP and both gradient and wedge gels (Biggin et al. , 1983; Sanger et al. , 1977). Some clones were sequenced with modified T7 DNA poly erase (Tabor & Richardson, 1987) and 7-deaza-2-dGTP (Mizusawa et al.. 1986) using a kit supplied by Pharmacia. Gaps in the sequence were filled in using synthetic oligonucleotides made on a MilliGen 7500 DNA synthesizer (Millipore, UK) as specific primers (Charles et al. , 1986) .
  • the B.bronchiseptica genomic library was screened by hybridisation with a radioactively labelled 1.8kb Clal fragment of the prn gene encoding the P.70 antigen from B.parapertussis. Of the two cos ids returning a positive signal one, designated pBD844, was selected for further analysis. Restriction mapping and Southern blotting experiments carried out on pBD844 demonstrated that the Clal and Sail hybridization pattern of the prn gene from B.bronchiseptica was identical to that described for both B.pertussis (Charles et a_L. , 1989) and B.parapertussis (International Application No. PCT/GB91/02302) .
  • Nucleotide sequence of the prn gene encoding P.68 DNA sequence was obtained initially from the Clal and Sail fragments cloned into M13 and using universal primer; overlapping sequence was comp yield._.ed using a set of synthetic oligonucleotides as specific sequence primers.
  • Computer analysis of the DNA sequence (Fig. 1) demonstrates that the open reading frame for the prn gene of P.68 protein is capable of encoding a protein with a Mr of 93,996 (P.94).
  • the molecular weight of the mature protein found on the surface of B.bronchiseptica is however 68,000 as judged by SDS-PAGE, suggesting that this molecule is the processed form of the larger precursor.
  • N-terminal protein sequencing of purified preparations of P.68 from B.bronchiseptica gave the sequence Asp-Trp/Gln-Asn-Asn-Gln-Gln/Ser-Ile-Xaa-Lys-Ala confirming that a signal peptide is cleaved. Cleavage of this signal peptide, of between 32 or 34 amino acid residues depending on which ATG initiation codon is used, occurs after the sequence AYA which is in agreement with the Ala-Xaa-Ala motif reported to be recognized by E. coli signal peptidases (Perlman & Halvorson, 1983) .
  • Towards the C-terminus of the P.68 protein at residues 600-602 is a dibasic pair of amino acids, Lys-Arg, that occur in the same position, and are flanked by identical residues.
  • Colonies returning a positive signal were isolated and plasmid minipreps carried out to verify that a full- length insert has been cloned.
  • One such plasmid, pBD881 was selected for further study.
  • a Western blot of an SDS- PAGE gel of E. coli TGI harbouring pBD881 surprisingly did not produce a detectable 94kDa higher molecular weight band, but instead produces a pair of stronger bands at around 69kDa and 68kDa.
  • B. parapertussis chromosomal DNA (prepared by the method of Hull et a_l, 1981 as modified by Maskell et al. 1988) was partially digested with Sau3A. and fragments in the 40-50kb size range were ligated into the BamHI site (Maniatis et a_l, 1982) of cosmid pHC79 (Hohn & Collins, 1980) .
  • Recombinant cosmids in E. coli HB101 were plated out and transferred to microtiter plates following the method described by Charles et al, 1990 and transferred to Gene Screen Plus hybridized membranes (Du Pont, Stevenage, Hertfordshire) .
  • the cosmids were then screened for the presence of the prn gene encoding P.70 by DNA : DNA hybridization using a radioactively labelled 1.8kb Clal restriction fragment isolated from the related prn gene from B. pertussis.
  • the Clal fragment was gel purified (Tautz & Renz, 1983) following digestion of cosmid pI69 (Charles et aJL, 1989) .
  • the fragment was nick translated with a kit supplied by BCL, Mannheim and [ ⁇ 32 P]-ATP (Amersham) , and hybridized with the B. parapertussis gene bank filters as previously described (Charles et al. (1990)). Three positive colonies were identified and one, harbouring cosmid pBD ⁇ ll was selected for further analysis.
  • Oligonucleotides for use as specific sequencing primers were made on a SAM1 oligonucleotide synthesizer (Biolabs) .
  • Computer analysis of the DNA sequence revealed an open reading frame capable of encoding a protein of 922 amino acids with a calculated molecular weight of 95,177.
  • CARTER P., BEDOUELL, H. & WINTER, G. (1985). Improved oligonucleotide site directed utagenesis using M13 vectors. Nucleic Acids Research 13, 443-444.
  • CHARLES I. G., KEYTE, J., BRAMMAR, W. J. , SMITH, M. & HAWKINS, A. R. (1986) .
  • M13 phage vectors and host strains nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103- 119.

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Abstract

On décrit une protéine non contaminée par des constituants de B. bronchiseptica, capable de se lier à un anticorps qui se lie également à l'antigène P.68 natif de B. bronchiseptica et qui présente (a) la séquence d'acides aminés (I) ou (b) une séquence d'acides aminés qui présente une homologie supérieure à 98 % avec ladite séquence d'acides aminés (a).
PCT/GB1992/000561 1991-03-27 1992-03-27 ANTIGENE DE MEMBRANE EXTERIEURE DE $i(BORDETELLA BRONCHISEPTICA) WO1992017587A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP4506936A JPH06506111A (ja) 1991-03-27 1992-03-27 ボルデテラブロンキセプティカ外膜抗原
EP92907237A EP0594631A1 (fr) 1991-03-27 1992-03-27 ANTIGENE DE MEMBRANE EXTERIEURE DE $i(BORDETELLA BRONCHISEPTICA)

Applications Claiming Priority (2)

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GB919106568A GB9106568D0 (en) 1991-03-27 1991-03-27 Recombinant antigen
GB9106568.0 1991-03-27

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067031A1 (fr) * 2003-01-29 2004-08-12 Pfizer Products Inc. Vaccins canins contre la bordetella bronchiseptica

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015571A1 (fr) * 1990-04-02 1991-10-17 The Wellcome Foundation Limited Expression d'une proteine heterologue dans de la levure

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015571A1 (fr) * 1990-04-02 1991-10-17 The Wellcome Foundation Limited Expression d'une proteine heterologue dans de la levure

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
INFECTION AND IMMUNITY. vol. 47, no. 3, March 1985, WASHINGTON US pages 744 - 751; J. MONTAREZ ET AL.: 'Identification of a 68-kilodalton protective protein antigen from Bordetella bronchiseptica' *
MOLECULAR MICROBIOLOGY vol. 5, no. 2, 22 February 1991, pages 409 - 418; L. LI ET AL.: 'P.70 pertactin, an outer-membrane protein from Bordetella parapertussis: Cloning, nucleotide sequence and surface expression in E. coli' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 86, 1989, WASHINGTON US pages 3554 - 3558; I. CHARLES ET AL.: 'Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis' *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067031A1 (fr) * 2003-01-29 2004-08-12 Pfizer Products Inc. Vaccins canins contre la bordetella bronchiseptica
AU2004208556B2 (en) * 2003-01-29 2009-06-04 Zoetis Services Llc Canine vaccines against bordetella bronchiseptica
US7736658B2 (en) 2003-01-29 2010-06-15 Pfizer Inc. Canine combination vaccines

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GB9106568D0 (en) 1991-05-15
EP0594631A1 (fr) 1994-05-04
JPH06506111A (ja) 1994-07-14

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