EP1100920A2 - Acides nucleiques et proteines de streptococcus groupe b - Google Patents

Acides nucleiques et proteines de streptococcus groupe b

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Publication number
EP1100920A2
EP1100920A2 EP99934984A EP99934984A EP1100920A2 EP 1100920 A2 EP1100920 A2 EP 1100920A2 EP 99934984 A EP99934984 A EP 99934984A EP 99934984 A EP99934984 A EP 99934984A EP 1100920 A2 EP1100920 A2 EP 1100920A2
Authority
EP
European Patent Office
Prior art keywords
group
dna
streptococcus
proteins
protein
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Withdrawn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
EP99934984A
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German (de)
English (en)
Inventor
Richard William Falla Le Page
Jeremy Mark Institute of Food Research WELLS
Sean Bosco University of Cambridge HANNIFFY
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.)
Microbial Technics Ltd
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Microbial Technics Ltd
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Publication date
Priority claimed from GBGB9816335.5A external-priority patent/GB9816335D0/en
Application filed by Microbial Technics Ltd filed Critical Microbial Technics Ltd
Publication of EP1100920A2 publication Critical patent/EP1100920A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus
    • 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
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci

Definitions

  • the present invention relates to proteins derived from Streptococcus agalactiae, nucleic acid molecules encoding such proteins, and the use of the proteins as antigens and/or immunogens and in detection/diagnosis. It also relates to a method for the rapid screening of bacterial genomes to isolate and characterise bacterial cell envelope associated or secreted proteins.
  • the Group B Streptococcus (GBS) (Streptococcus agalactiae) is an encapsulated bacterium which emerged in the 1970s as a major pathogen of humans causing sepsis and meningitis in neonates as well as adults.
  • GBS Group B Streptococcus
  • the incidence of early onset neonatal infection during the first 5 days of life varies from 0.7 to 3.7 per 1000 live births and causes mortality in about 20% of cases. Between 25-50% of neonates surviving early onset infections frequently suffer neurological sequalae. Late onset neonatal infections occur from 6 days to three months of age at a rate of about 0.5 - 1.0 per 1000 live births.
  • GBS colonisation of the maternal genetic tract by GBS at the time of birth and the risk of neonatal sepsis.
  • the rectum may act as a reservoir for GBS.
  • Susceptibility in the neonate is correlated with the a low concentration or absence of IgG antibodies to the capsular polysaccharides found on GBS causing human disease.
  • Type VIII GBS is the major cause of neonatal sepsis in Japan.
  • a possible means of prevention involves intra or postpartum administration of antibiotics to the mother but there are concerns that this might lead to the emergence of resistant organisms and in some cases allergic reactions.
  • Vaccination of the adolescent females to induce long lasting maternally derived immunity is one of the most promising approaches to prevent GBS infections in neonates.
  • the capsular polysaccharide antigens of these organisms have attracted most attention as with regard to vaccine development. Studies in healthy adult volunteers have shown that serotype la, II and III polysaccharides are non-toxic and immunogenic in approximately 65%, 95% and 70% of non-immune adults respectively.
  • capsule antigens as vaccines.
  • the response rates vary according to pre-immunisation status and the polysaccharide antigen and not all vaccinees produce adequate levels of IgG antibody as indicated in vaccination studies with GBS polysaccharides in human volunteers.
  • Rib A protein which is found on most serotype III strains but rarely on serotypes la, lb or II confers immunity to challenge with Rib expressing GBS in animal models (Stalhammar-Carlemalm et al, Journal of Experimental Medicine 177:1593-1603 (1993)).
  • Another surface protein of interest as a component of a vaccine is the alpha antigen of the C proteins which protected vaccinated mice against lethal infection with strains expressing alpha protein. The amount of antigen expressed by GBS strains varies markedly.
  • This invention seeks to overcome the problem of vaccination against GBS by using a novel screening method specifically designed to identify those Group B Streptococcus genes encoding bacterial cell surface associated or secreted proteins (antigens).
  • the proteins expressed by these genes may be immunogenic, and therefore may be useful in the prevention and treatment of Group B Streptococcus infection.
  • immunogenic means that these proteins will elicit a protective immune response within a subject.
  • the present invention provides a Group B Streptococcus protein, having a sequence selected from those shown in figure 1 , or fragments or derivatives thereof.
  • proteins and polypeptides included within this group may be cell surface receptors, adhesion molecules, transport proteins, membrane structural proteins, and/or signalling molecules.
  • Alterations in the amino acid sequence of a protein can occur which do not affect the function of a protein. These include amino acid deletions, insertions and substitutions and can result from alternative splicing and/or the presence of multiple translation start sites and stop sites. Polymorphisms may arise as a result of the infidelity of the translation process. Thus changes in amino acid sequence may be tolerated which do not affect the proteins function.
  • the present invention includes derivatives or variants of the proteins, polypeptides, and peptides of the present invention which show at least 50%> identity to the proteins, polypeptides and peptides described herein.
  • the degree of sequence identity is at least 60%> and preferably it is above 75%o. More preferably still is it above 80%, 90% or even 95%.
  • identity can be used to describe the similarity between two polypeptide sequences.
  • a software package well known in the art for carrying out this procedure is the CLUSTAL program. It compares the amino acid sequences of two polypeptides and finds the optimal alignment by inserting spaces in either sequence as appropriate.
  • amino acid identity or similarity identity plus conservation of amino acid type
  • BLASTx a software package such as BLASTx. This program aligns the largest stretch of similar sequence and assigns a value to the fit. For any one pattern comparison several regions of similarity may be found, each having a different score.
  • two polypeptides of different lengths may be compared over the entire length of the longer fragment. Alternatively small regions may be compared. Normally sequences of the same length are compared for a useful comparison to be made.
  • Manipulation of the DNA encoding the protein is a particularly powerful technique for both modifying proteins and for generating large quantities of protein for purification purposes. This may involve the use of PCR techniques to amplify a desired nucleic acid sequence.
  • sequence data provided herein can be used to design primers for use in PCR so that a desired sequence can be targeted and then amplified to a high degree.
  • primers will be at least five nucleotides long and will generally be at least ten nucleotides long (e.g. fifteen to twenty-five nucleotides long). In some cases primers of at least thirty or at least thirty-five nucleotides in length may be used.
  • chemical synthesis may be used. This may be automated. Relatively short sequences may be chemically synthesised and ligated together to provide a longer sequence.
  • the present invention provides , a nucleic acid molecule comprising or consisting of a sequence which is: (i) any of the DNA sequences set out in figure 1 herein or their RNA equivalents; (ii) a sequence which is complementary to any of the sequences of (i); (iii) a sequence which codes for the same protein or polypeptide, as those sequences of (i) or (ii);
  • identity can also be used to describe the similarity between two individual
  • the 'bestfit' program (Smith and Waterman, Advances in applied Mathematics, 482-489 (1981)) is one example of a type of computer software used to find the best segment of similarity between two nucleic acid sequences, whilst the GAP program enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate.
  • RNA equivalent' when used above indicates that a given RNA molecule has a sequence which is complementary to that of a given DNA molecule, allowing for the fact that in RNA 'U' replaces 'T' in the genetic code.
  • the nucleic acid molecule may be in isolated or recombinant form.
  • the nucleic acid molecule may be in an isolated or recombinant form.
  • DNA constructs can readily be generated using methods well known in the art. These techniques are disclosed, for example in J. Sambrook et al, Molecular Cloning 2 nd Edition, Cold Spring Harbour Laboratory Press (1989). Modifications of DNA constructs and the proteins expressed such as the addition of promoters, enhancers, signal sequences, leader sequences, translation start and stop signals and DNA stability controlling regions, or the addition of fusion partners may then be facilitated.
  • the DNA construct will be inserted into a vector which may be of phage or plasmid origin. Expression of the protein is achieved by the transformation or transfection of the vector into a host cell which may be of eukaryotic or prokaryotic origin.
  • Such vectors and suitable host cells form yet further aspects of the present invention.
  • the Group B Streptococcus proteins (antigens) described herein can additionally be used to raise antibodies, or to generate affibodies. These can be used to detect Group B Streptococcus.
  • the present invention provides, an antibody, affibody, or a derivative thereof which binds to any one or more of the proteins, polypeptides, peptides, fragments or derivatives thereof, as described herein.
  • Antibodies within the scope of the present invention may be monoclonal or polyclonal.
  • Polyclonal antibodies can be raised by stimulating their production in a suitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, goat or monkey) when a protein as described herein, or a homologue, derivative or fragment thereof, is injected into the animal.
  • a suitable animal host e.g. a mouse, rat, guinea pig, rabbit, sheep, goat or monkey
  • an adjuvant may be administered together with the protein.
  • Well- known adjuvants include Freund 's adjuvant (complete and incomplete) and aluminium hydroxide.
  • the antibodies can then be purified by virtue of their binding to a protein as described herein.
  • Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing myeloma cells and spleen cells which produce the desired antibody in order to form an immortal cell line. Thus the well-known Kohler & Milstein technique (Nature 256 (1975)) or subsequent variations upon this technique can be used.
  • the present invention includes derivatives thereof which are capable of binding to proteins etc as described herein.
  • the present invention includes antibody fragments and synthetic constructs. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtech 12 372-379 (September 1994).
  • Antibody fragments include, for example, Fab, F(ab') 2 and Fv fragments.
  • Fab fragments include, for example, Fab, F(ab') 2 and Fv fragments.
  • Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining V n and Vi regions, which contributes to the stability of the molecule.
  • Other synthetic constructs that can be used include CDR peptides. These are synthetic peptides comprising antigen-binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings that mimic the structure of a CDR loop and that include antigen-interactive side chains.
  • Synthetic constructs include chimaeric molecules.
  • humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention.
  • a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions. Ways of producing chimaeric antibodies are discussed for example by Morrison et al in PNAS, 81, 6851-6855 (1984) and by Takeda et al in Nature. 314, 452-454 (1985).
  • Synthetic constructs also include molecules comprising an additional moiety that provides the molecule with some desirable property in addition to antigen binding.
  • the moiety may be a label (e.g. a fluorescent or radioactive label).
  • it may be a pharmaceutically active agent.
  • Affibodies are proteins which are found to bind to target proteins with a low dissociation constant. They are selected from phage display libraries expressing a segment of the target protein of interest (Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA, Department of Biochemistry and Biotechology, Royal Institute of Technology (KTH), Sweden).
  • the invention provides an immunogenic composition comprising one or more proteins, polypeptides, peptides, fragments or derivatives thereof, or nucleotide sequences described herein.
  • a composition of this sort may be useful in the treatment or prevention of Group B Streptococcus infection in subject.
  • the immunogenic composition is a vaccine.
  • the invention provides:
  • an immunogenic composition as described herein in the preparation of a medicament for the treatment or prophylaxis of Group B Streptococcus infection.
  • the medicament is a vaccine.
  • a method of detection of Group B Streptococcus which comprises the step of bringing into contact a sample to be tested with at least one antibody, affibody, or a derivative thereof, as described herein.
  • a method of detection of Group B Streptococcus which comprises the step of bringing into contact a sample to be tested with at least one protein, polypeptide, peptide, fragments or derivatives as described herein.
  • a method of detection of Group B Streptococcus which comprises the step of bringing into contact a sample to be tested with at least one nucleic acid molecule as described herein.
  • a kit for the detection of Group B Streptococcus comprising at least one antibody, affibody, or derivatives thereof, described herein.
  • kits for the detection of Group B Streptococcus comprising at least one Group B Streptococcus protein, polypeptide, peptide, fragment or derivative thereof, as described herein.
  • kits for the detection of Group B Streptococcus comprising at least one nucleic acid of the invention.
  • novel proteins described herein are identified and isolated using a novel screening method which specifically identifies those Group B Streptococcus genes encoding bacterial cell envelope associated or secreted proteins.
  • Staphylococcal nuclease is a naturally secreted heat-stable, monomeric enzyme which has been efficiently expressed and secreted in a range of Gram positive bacteria (Shortle., Gene 22:181-189 (1983), Kovacevic et al, J. Bacteriol. 162:521-528 (1985), Miller et al, J. Bacteriol. 169:3508-3514 (1987), Liebl et al, J. Bacteriol. 174:1854-1861(1992), Le Loir et al, J. Bacteriol. 176:5135-5139 (1994), Poquet et al, 1998 [supra]).
  • the screening vector contains the pAM ⁇ l replicon which functions in a broad host range of Gram-positive bacteria in addition to the ColEl replicon that promotes replication in Escherichia coli and certain other Gram negative bacteria.
  • Unique cloning sites present in the vector can be used to generate transcriptional and translational fusions between cloned genomic DNA fragments and the open reading frame of the truncated nuc gene devoid of its own signal secretion leader.
  • the nuc gene makes an ideal reporter gene because the secretion of nuclease can readily be detected using a simple and sensitive plate test: Recombinant colonies secreting the nuclease develop a pink halo whereas control colonies remain white
  • a direct screen to identify and isolate DNA encoding bacterial cell envelope associated or secreted proteins (antigens). in pathogenic bacteria has been developed by the present inventors which utilises a vector-system (pTREPl expression vector) in
  • Lactococcus lactis that specifically detects DNA sequences which are adjacent to, and associated with DNA encoding surface proteins from Group B Streptococcus.
  • the screening vector also incorporates the nuc gene encoding the Staphylococcal nuclease as a reporter gene.
  • nuc gene encoding the mature nuclease protein (minus its signal peptide sequence) is cloned into the pTREPl expression vector in L. lactis. In this form, the nuc-encoded nuclease cannot be secreted even when expressed intracellularly.
  • the reporter vector is then randomly combined with appropriately digested genomic DNA from Group B Streptococcus, cloned into L. lactis and used as a screening system for sequences permitting the export of nuclease. In this way gene/partial gene sequences encoding exported proteins from Group B Streptococcus are isolated. Once a partial gene sequence is obtained, full length sequences encoding exported proteins can readily be obtained using techniques well known in the art.
  • the pTREPl -nuc vectors differ from the pFUN vector described by Poquet et al. (1998) [supra], which was used to identify L. lactis exported proteins by screening directly for Nuc activity directly in L. lactis.
  • the pFUN vector does not contain a promoter upstream of the nuc open reading frame the cloned genomic DNA fragment must also provide the signals for transcription in addition to those elements required for translation initiation and secretion of Nuc. This limitation may prevent the isolation of genes that are distant from a promoter for example genes which are within polycistronic operons. Additionally there can be no guarantee that promoters derived from other species of bacteria will be recognised and functional in L. lactis.
  • promoters may be under stringent regulation in the natural host but not in L. lactis.
  • the presence of the PI promoter in the pTREPl -nuc series of vectors ensures that promoterless DNA fragments (or DNA fragments containing promoter sequences not active in L. lactis) may still be transcribed.
  • promoterless DNA fragments or DNA fragments containing promoter sequences not active in L. lactis
  • genes missed in other screening methods may be identified.
  • the present invention provides a method of screening for DNA encoding bacterial cell wall associated or surface antigens in gram positive bacteria comprising the steps of:
  • the reporter vector is one of the pTREPl -nuc vectors shown in figure 4.
  • the present invention provides a vector as shown in figure 4 for use in screening for DNA encoding exported or surface antigens in gram positive bacteria.
  • gram positive bacteria which may be screened include Group B Streptococcus, Streptococcus pneumoniae, Staphylcoccus aureus or pathogenic Group A Streptococci.
  • the present invention also provides a method of determining whether a protein or polypeptide as described herein represents a potential anti-microbial target which comprises inactivating said protein and determining whether Group B Streptococcus is still viable.
  • a suitable method for inactivating the protein is to effect selected gene knockouts, ie prevent expression of the protein and determine whether this results in a lethal change. Suitable methods for carrying out such gene knockouts are described in Li et al , P.N.A.S., 94: 13251-13256 (1997) and Kolkman et al
  • the present invention provides the use of an agent capable of antagonising, inhibiting or otherwise interfering with the function or expression of a protein or polypeptide of the invention in the manufacture of a medicament for use in the treatment or prophylaxis of Group B Streptococcus infection.
  • Fig 1 Shows a number of full length nucleotide sequences encoding antigenic Group B Streptococcus proteins.
  • B Shows the corresponding amino acid sequences.
  • Fig 2 Shows a number of oligonucleotide primers used in the screening process
  • nucSl primer designed to amplify a mature form of the nuc A gene
  • nucS2- primer designed to amplify a mature form of the nuc A gene
  • nucS3 primer designed to amplify a mature form of the nuc A gene
  • nucR primer designed to amplify a mature form of the nuc A gene
  • nucseq primer designed to sequence DNA cloned into the pTREP-Nuc vector pTREPF nucleic acid sequence containing recognition site for ECORV. Used for cloning fragments into pTREX7.
  • pTREPR nucleic acid sequence containing recognition site for BAMH1. Used for cloning fragments into pTREX7.
  • PUCF forward sequencing primer enables direct sequencing of cloned DNA fragments.
  • VR example of gene specific primer used to obtain further antigen DNA sequence by the method of DNA walking VI example of gene specific primer used to obtain further antigen DNA sequence by the method of DNA walking.
  • Fig 3 (i) Schematic presentation of the nucleotide sequence of the unique gene cloning site immediately upstream of the mature nuc gene in pTREPl-nwcl, pTREPl -nuc2 and pTREPl -nuc3.
  • Each of the pTREP- ⁇ uc vectors contain an EcoRV (a Smal site in pTREPl -nuc2) cleavage site which allows cloning of genomic DNA fragments in 3 different frames with respect to the mature nuc gene.
  • Fig 4 Shows the results of various DNA vaccine trials
  • Fig 5 Shows the results of a second group of DNA vaccine trials
  • Figs 6-11 Show various Southern Blot analyses of different Group B streptococcus strains.
  • All putative surface proteins are analysed for leader/signal peptide sequences.
  • Bacterial signal peptide sequences share a common design. They are characterised by a short positively charged N-terminus (N region) immediately preceding a stretch of hydrophobic residues (central portion-h region) followed by a more polar C-terminal portion which contains the cleavage site (c-region).
  • Computer software is used to perform hydropathy profiling of putative proteins (Marcks, Nuc. Acid. Res., 16:1829-1836 (1988)) which is used to identify the distinctive hydrophobic portion (h-region) typical of leader peptide sequences.
  • the presence/absence of a potential ribosomal binding site is also noted.
  • Putative S. agalactiae surface proteins are also be assessed for their novelty. Some of the identified proteins may or may not possess a typical leader peptide sequence and may not show homology with any DNA/protein sequences in the database. Indeed these proteins may indicate the primary advantage of our screening method, i.e. isolating atypical surface-related proteins, which would have been missed in all previously described screening protocols.
  • the pTREPl plasmid is a high-copy number (40-80 per cell) theta-replicating gram positive plasmid, which is a derivative of the pTREX plasmid which is itself a derivative of the the previously published pIL253 plasmid.
  • pIL253 incorporates the broad Gram-positive host range replicon of pAM ⁇ l (Simon and Chopin, 1988) and is non-mobilisable by the L lactis sex-factor.
  • pIL253 also lacks the tra function which is necessary for transfer or efficient mobilisation by conjugative parent plasmids exemplified by pIL501.
  • the Enterococcal pAM ⁇ l replicon has previously been transferred to various species including Streptococcus, Lactobacillus and Bacillus species as well as Clostridium acetobutylicum, (LeBlanc et al, Proceedings of the National Academy of Science USA 75:3484-3487 (1978)) indicating the potential broad host range utility.
  • the pTREPl plasmid represents a constitutive transcription vector.
  • the pTREX vector was constructed as follows. An artificial DNA fragment containing a putative RNA stabilising sequence, a translation initiation region (TIR), a multiple cloning site for insertion of the target genes and a transcription terminator was created by annealing 2 complementary oligonucleotides and extending with Tfl DNA polymerase. The sense and anti-sense oligonucleotides contained the recognition sites for Nhel and BamHI at their 5' ends respectively to facilitate cloning. This fragment was cloned between the Xbal and BamHI sites in pUC19NT7, a derivative of pUC19 which contains the T7 expression cassette from pLETl (Wells et al, J. Appl. Bacteriol.
  • pUCLEX The complete expression cassette of pUCLEX was then removed by cutting with Hindlll and blunting followed by cutting with EcoRI before cloning into EcoRI and Sad (blunted) sites of pIL253 to generate the vector pTREX (Wells and Schofield, In Current advances in metabolism, genetics and applications-NATO ASI Series. H 98:37-62. (1996)).
  • RNA stabilising sequence and TIR are derived from the Escherichia coli T7 bacteriophage sequence and modified at one nucleotide position to enhance the complementarity of the Shine Dalgarno (SD) motif to the ribosomal 16s RNA of Lactococcus lactis (Schofield et al. pers. corns. University of Cambridge Dept. Pathology.).
  • a Lactococcus lactis MG1363 chromosomal DNA fragment exhibiting promoter activity which was subsequently designated P7 was cloned between the EcoRI and Bglll sites present in the expression cassette, creating pTREX7. This active promoter region had been previously isolated using the promoter probe vector pSB292 (Waterfield et al, Gene 165:9-15 (1995)). The promoter fragment was amplified by
  • the pTREPl vector was then constructed as follows. An artificial DNA fragment which included a transcription terminator, the forward pUC sequencing primer, a promoter multiple cloning site region and a universal translation stop sequence was created by annealing two overlapping partially complementary synthetic oligonucleotides together and extending with sequenase according to manufacturers instructions.
  • the sense and anti-sense (pTREPF and pTREP ) oligonucleotides contained the recognition sites for EcoRV and BamHI at their 5' ends respectively to facilitate cloning into pTREX7.
  • the transcription terminator was that of the Bacillus penicillinase gene, which has been shown to be effective in Lactococcus (Jos et al, Applied and Environmental Microbiology 50:540-542 (1985)). This was considered necessary as expression of target genes in the pTREX vectors was observed to be leaky and is thought to be the result of cryptic promoter activity in the origin region (Schofield et al. pers. corns. University of Cambridge Dept. Pathology.).
  • the forward pUC primer sequencing was included to enable direct sequencing of cloned DNA fragments.
  • the translation stop sequence which encodes a stop codon in 3 different frames was included to prevent translational fusions between vector genes and cloned DNA fragments.
  • the pTREX7 vector was first digested with EcoRI and blunted using the 5' - 3' polymerase activity of T4 DNA polymerase (NEB) according to manufacturer's instructions.
  • the EcoRI digested and blunt ended pTREX7 vector was then digested with Bgl II thus removing the P7 promoter.
  • the artificial DNA fragment derived from the annealed synthetic oligonucleotides was then digested with EcoRV and Bam HI and cloned into the EcoRI(blunted)-Bgl II digested pTREX7 vector to generate pTREP.
  • a Lactococcus lactis MG1363 chromosomal promoter designated PI was then cloned between the EcoRI and Bglll sites present in the pTREP expression cassette forming pTREPl.
  • This promoter was also isolated using the promoter probe vector pSB292 and characterised by Waterfield et al, (1995) [supra].
  • the PI promoter fragment was originally amplified by PCR using vent DNA polymerase according to manufacturers instructions and cloned into the pTREX as an EcoRI-Bglll DNA fragment.
  • the EcoRI-Bglll PI promoter containing fragment was removed from pTREXl by restriction enzyme digestion and used for cloning into pTREP (Schofield et al pers. corns. University of Cambridge, Dept. Pathology.).
  • the nucleotide sequence of the S. aureus nuc gene (EMBL database accession number V01281) was used to design synthetic oligonucleotide primers for PCR amplification.
  • the primers were designed to amplify the mature form of the nuc gene designated nuc A which is generated by proteolytic cleavage of the N-terminal 19 to 21 amino acids of the secreted propeptide designated Snase B (Shortle, 1983 [supra]).
  • Three sense primers «wcSl, nucS2 and nucS3, shown in figure 3
  • Bglll and BamHI were incorporated at the 5' ends of the sense and anti-sense primers respectively to facilitate cloning into BamHI and Bglll cut pTREPl.
  • the sequences of all the primers are given in figure 3.
  • Three nuc gene DNA fragments encoding the mature form of the nuclease gene (NucA) were amplified by PCR using each of the sense primers combined with the anti-sense primer.
  • the nuc gene fragments were amplified by PCR using S. aureus genomic DNA template, Vent DNA Polymerase (NEB) and the conditions recommended by the manufacturer.
  • the purified nuc gene fragments described in section b were digested with Bgl II and BamHI using standard conditions and li gated to BamHI and Bglll cut and dephosphorylated pTREPl to generate the pTREPl -nucl, pTREPl -nuc2 and pTREPl -nuc3 series of reporter vectors. These vectors are described in figure 4.
  • General molecular biology techniques were carried out using the reagents and buffers supplied by the manufacturer or using standard techniques (Sambrook and Maniatis, Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbour (1989)).
  • the expression cassette comprises a transcription terminator, lactococcal promoter PI, unique cloning sites (Bglll, EcoRV or Smal) followed by the mature form of the nuc gene and a second transcription terminator.
  • a transcription terminator lactococcal promoter PI
  • unique cloning sites Bglll, EcoRV or Smal
  • sequences required for translation and secretion of the nuc gene were deliberately excluded in this construction.
  • Such elements can only be provided by appropriately digested foreign DNA fragments (representing the target bacterium) which can be cloned into the unique restriction sites present immediately upstream of the nuc gene.
  • Genomic DNA isolated from and Group B Streptococcus (S. agalactiae) was digested with the restriction enzyme Tru9I.
  • This enzyme which recognises the sequence 5'- TTAA -3' was used because it cuts A/T rich genomes efficiently and can generate random genomic DNA fragments within the preferred size range (usually averaging 0.5 - 1.0 kb).
  • This size range was preferred because there is an increased probability that the PI promoter can be utilised to transcribe a novel gene sequence.
  • the PI promoter may not be necessary in all cases as it is possible that many Streptococcal promoters are recognised in L. lactis.
  • DNA fragments of different size ranges were purified from partial Tru9I digests of and S.
  • Tru9I digested DNA was dissolved in a solution (usually between 10-20 ⁇ l in total) supplemented with T4 DNA ligase buffer (New England Biolabs; NEB) (IX) and 33 ⁇ M of each of the required dNTPs, in this case dATP and dTTP.
  • Klenow enzyme was added (1 unit Klenow enzyme (NEB) per ⁇ g of DNA) and the reaction incubated at 25 °C for 15 minutes. The reaction was stoped by incubating the mix at 75 °C for 20 minutes. EcoRV or Smal digested pTREP- ⁇ wc plasmid DNA was then added (usually between 200-400 ng). The mix was then supplemented with 400 units of T4 DNA ligase (NEB) and T4 DNA ligase buffer (IX) and incubated overnight at 16°C. The ligation mix was precipiated directly in 100%) Ethanol and 1/10 volume of 3M sodium acetate (pH 5.2) and used to transform L. lactis MG1363 (Gasson, J.
  • the gene cloning site of the pTREP- «uc vectors also contains a Bglll site which can be used to clone for example Sau3AI digested genomic DNA fragments.
  • pcDNA3.1 The commercially available pcDNA3.1+ plasmid (Invitrogen), referred to as pcDNA3.1 henceforth, was used as a vector in all DNA immunisation experiments involving gene targets derived using the LEEP system.
  • pcDNA3.1 is designed for high-level stable and transient expression in mammalian cells and has been used widely and successfully as a host vector to test candidate genes from a variety of pathogens in DNA vaccination experiments (Zhang et al, 1997; Kurar and Splitter, 1997; Anderson et al, 1996).
  • the vector possesses a multiple cloning site which facilitates the cloning of multiple gene targets downstream of the human cytomegalovirus (CMV) immediate-early promoter/enhancer which permits efficient, high-level expression of the target gene in a wide variety of mammalian cells and cell types including both muscle and immune cells. This is important for optimal immune response as it remains unknown as to which cells types are most important in generating a protective response in vivo.
  • the plasmid also contains the ColEl origin of replication which allows convenient high- copy number replication and growth in E. coli and the ampicillin resistance gene (B- lactamase) for selection in E. coli.
  • pcDNA 3.1 possesses a T7 promoter/priming site upstream of the MCS which allows for in vitro transcription of a cloned gene in the sense orientation.
  • Oligonucleotide primers were designed for each individual gene of interest derived using the LEEP system. Each gene was examined thoroughly, and where possible, primers were designed such that they targeted that portion of the gene thought to encode only the mature portion of the protein (APPENDIX I). It was hoped that expressing those sequences that encode only the mature portion of a target gene protein, would facilitate its correct folding when expressed in mammalian cells. For example, in the majority of cases primers were designed such that putative N-terminal signal peptide sequences would not be included in the final amplification product to be cloned into the pcDNA3.1 expression vector.
  • the signal peptide directs the polypeptide precursor to the cell membrane via the protein export pathway where it is normally cleaved off by signal peptidase I (or signal peptidase II if a lipoprotein). Hence the signal peptide does not make up any part of the mature protein whether it be displayed on the bacterium's surface or secreted. Where a N-terminal leader peptide sequence was not immediately obvious, primers were designed to target the whole of the gene sequence for cloning and ultimately, expression in pcDNA3.1.
  • All forward and reverse oligonucleotide primers incorporated appropriate restriction enzyme sites to facilitate cloning into the pcDNA3.1 MCS region. All forward primers were also designed to include the conserved Kozak nucleotide sequence 5'-gccacc-3' immediately upstream of an 'atg' translation initiation codon in frame with the target gene insert. The Kozak sequence facilitates the recognition of initiator sequences by eukaryotic ribosomes. Typically, a forward primer incorporating a BamHI restriction enzyme site the primer would begin with the sequence 5'-cgggatccgccaccatg-3', followed by a sequence homologous to the 5' end of that part of a gene being amplified. All reverse primers incorporated a Not I restriction enzyme site sequence 5' -ttgcggccgc-3'. All gene-specific forward and reverse primers were designed with compatible melting temperatures to facilitate their amplification.
  • All gene targets were amplified by PCR from S. agalactiae genomic DNA template using Vent DNA polymerase (NEB) or x th DNA polymerase (PE Applied Biosystems) using conditions recommended by the manufacturer.
  • a typical amplification reaction involved an initial denaturation step at 95 °C for 2 minutes followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at the appropriate melting temperature for 30 seconds, and extension at 72 °C for 1 minute (1 minute per kilobase of DNA being amplified). This was followed by a final extension period at 72°C for 10 minutes. All PCR amplified products were extracted once with phenol chloroform (2:1:1) and once with chloroform (1 :1) and ethanol precipitated.
  • agalactiae serotype III (strain 97/0099) does contain the rib gene, hence the rib gene as part of a DNA vaccine would represent a potential positive control for all DNA immunisation experiments.
  • Oligonucleotide primers were designed (Appendix I) that targeted only the mature portion of the rib gene and which included appropriate restriction enzyme sites for cloning into pcDNA3.1.
  • rib was amplified using rTtA DNA polymerase (PE Applied Biosystems) using conditions recommended by the manufacturer. Conditions for cloning were similar to that described previously.
  • S. agalactiae serotype III (strain 97/0099) is a recent clinical isolate derived from the cerebral spinal fluid of a new born baby suffering from meningitis.
  • This haemolytic strain of Group B Streptococcus was epidemiologically tested and validated at the Respiratory and Systemic Infection Laboratory, PHLS Central Public health laboratory, 61 Collindale Avenue, London NW9 5HT. The strain was subcultured only twice prior to its arrival in the laboratory. Upon its arrival on a agar slope, a sweep of 4-5 colonies was immediately used to inoculate a Todd Hewitt/5%> horse blood broth which was incubated overnight statically at 37 C .
  • mice were challenged with various concentrations of the pathogen ranging from 10 ⁇ to 10 ⁇ colony forming units (cfu). Mice that developed symptoms were terminally anaesthetized and cardiac punctures were performed (Only mice that had been challenged with the highest doses, i.e. 1 X 10 ⁇ cfu, developed symptoms).
  • the retrieved unclotted blood was used to inoculate directly a 50ml serum broth (Todd Hewitt/20%) inactivated foetal calf serum).
  • the culture was constantly monitored and allowed to grow to late logarithmic phase. The presence of blood in the medium interfered with OD600 readings as it was being increasingly lysed with increasing growth of the bacterium, hence the requirement to constantly monitor the culture.
  • the culture was transferred to a fresh 50 ml tube in order to exclude dead bacterial cells and remaining blood cells which would have sedimented at the bottom of the tube.
  • 0.5 ml aliquots were then transferred to sterile cryovials, frozen in liquid nitrogen and stored at -70 C .
  • a viable count was carried out on a single standard inoculum aliquot in order to determine bacterial numbers. This was determined to be approximately 5 X10 ⁇ cfu per ml.
  • the optimal dose was estimated to be approximately 2.5 X10 ⁇ cfu. This represented a
  • mice were accomplished by the administration of DNA to 6 week old CBA/ca mice (Harlan, UK). Mice to be vaccinated were divided into groups of six and each group were immunised with recombinant pcDNA3.1 plasmid DNA containing a specific target-gene sequence derived using the LEEP system. A total of 100 ⁇ g of
  • mice groups were included in all vaccine trials. These control groups were either not DNA-vaccinated or were immunised with non-recombinant pcDNA3.1 plasmid DNA only, using the same time course described above. Four weeks after the second immunisation, all mice groups were challenged intra-peritoneally with a lethal dose of S. agalactiae serotype III (strain 97/0099).
  • mice were killed 3 or 4 days after infection.
  • challenged mice were monitored for the development of symptoms associated with the onset of S. agalactiae induced-disease.
  • Typical symptoms in an appropriate order included piloerection, an increasingly hunched posture, discharge from eyes, increased lethargy and reluctance to move which was often the result of apparent paralysis in the lower body/hind leg region. The latter symptoms usually coincided with the development of a moribund state at which stage the mice were culled to prevent further suffering.
  • mice were deemed to be very close to death, and the time of culling was used to determine a survival time for statistical analysis. Where mice were found dead, a survival time was calculated by averaging the time when a particular mouse was last observed alive and the time when found dead, in order to determine a more accurate time of death.
  • a positive result was taken as any DNA sequence that was cloned and used in challenge experiments as described above and gave protection against that challenge.
  • DNA sequences were determined to be protective
  • mice may survive for significantly longer time periods when compared with control mice.
  • the time to first death may also be prolonged when compared to counterpart mice in control groups.
  • p value 1 refers to statistical significance when compared to pcDNA3.1 controls.
  • p value 2 refers to statistical significance when compared to rib positive control.
  • mice immunised with the '17 (ID-8)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there are two outlying mice one of which survived the term of the experiment despite developing symptoms. The group also exhibited a much wider range of survival times reflected by a mean survival value which is approximately 14 hours higher than that demonstrated by the unvaccinated control group.
  • mice immunised with the '20 (ID-25)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there was one outlying mouse which survived the term of the experiment despite developing symptoms.
  • p value 1 refers to statistical significance when compared to pcDNA3.1 controls.
  • p value 2 refers to statistical significance when compared to unvaccinated controls.
  • mice immunised with the '22 (ID-10)' DNA vaccine exhibited significantly longer survival times when compared with the unvaccinated control group but not when compared with the pcDNA3.1 control group.
  • the time to first death in this group was prolonged by approximately 12 hours when compared to the pcDNA3.1 and unvaccinated control groups.
  • the mean survival time of 43.691 hours is also considerbly higher than that determined for both control groups.
  • mice immunised with the '28 (ID-13)' DNA vaccine did not show significantly longer survival times when compared with the pcDNA3.1 and unvaccinated control groups. However there are three outlying mice, two of which survived the term of the experiment despite showing symptoms. In addition, the time to first death in this group was prolonged by approximately 9 hours when compared to the pcDNA3.1 and unvaccinated control groups. The mean survival time of 52.449 hours is also considerbly higher than that determined for both control groups, as well demonstrating a wider range of survival times.
  • p value refers to statistical significance when compared to unvaccinated controls.
  • mice immunised with the '70 (ID-42)' DNA vaccine marginally did not show significantly longer survival times when compared with the unvaccinated control group.
  • the first death in this group is prolonged (by approximately 3 hours ) when compared with the unvaccinated group.
  • the group has a mean survival time which is approximately 8 hours longer than the unvaccinated group.
  • mice immunised with the '94 (ID-48)' DNA vaccine exhibited significantly longer survival times when compared with the unvaccinated control group.
  • mice immunised with the '51 (ID-37)' DNA vaccine exhibited significantly longer survival times when compared with the unvaccinated control group.
  • p value 2 refers to statistical significance when compared to unvaccinated controls.
  • mice immunised with the '32 (ID-15)' DNA vaccine did not show significantly longer survival times when compared with the pcDNA3.1 and unvaccinated control groups.
  • the '32 (ID-15)' group has two outlying mice one of which survived the term of the experiment despite showing symptoms. This group also exhibits a wide range of survival times.
  • mice immunised with the '39 (ID-17)' DNA vaccine exhibited significantly longer survival times when compared with the unvaccinated control group but was not significant when compared with the pcDNA3.1 control group.
  • the group has a considerbly higher mean survival time of 44.016 hours than that determined for either of the control groups.
  • mice immunised with the '32 (ID-15)' DNA vaccine did not show significantly longer survival times when compared with the pcDNA3.1 and unvaccinated control groups.
  • the '32 (ID-15)' group has one outlying mouse which survived the term of the experiment despite showing symptoms.
  • p value 1 refers to statistical significance when compared to pcDNA3.1 controls.
  • p value 2 refers to statistical significance when compared to unvaccinated controls.
  • mice immunised with the '13 (ID-72)' DNA vaccine did not show significantly longer survival times when compared with the pcDNA3.1 and unvaccinated control groups. However, there is one outlying mouse which survived approximately 24 hours longer than the longest surviving mice in the pcDNA3.1 and unvaccinated control groups respectively. In addition, the time to first death in this group was prolonged when compared to the pcDNA3.1 and unvaccinated control groups. The mean survival time of 43.582 hours is considerbly higher than that determined for both control groups.
  • p value refers to statistical significance when compared to unvaccinated controls.
  • mice immunised with the '3-5 (ID-66)' DNA vaccine exhibited significantly longer survival times when compared with the unvaccinated control group.
  • p value refers to statistical significance when compared to unvaccinated controls
  • mice immunised with the '3-40 (ID-67)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there is one outlying mouse which survived approximately 43 hours longer than the longest surviving mice in the unvaccinated control group.
  • mice immunised with the '2-19 (ID-73)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there was one outlying mouse which survived approximately 32 hours longer than the longest surviving mice in the unvaccinated control group. In addition, the time to first death was prolonged (by approximately 8 hours) when compared to the unvaccinated controls.
  • p value - refers to statistical significance when compared to unvaccinated controls.
  • mice immunised with the '3-20 (ID-71)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there is one outlying mouse which survived approximately 10 hours longer than the longest surviving mice in the unvaccinated control group.
  • mice immunised with the '2-19 (ID-73)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there are three outlying mouse which survived approximately 4, 10 and 23 hours longer than the longest surviving mice in the unvaccinated control group. This is reflected in the higher mean survival time of 48.749 hours and a much wider range of survival times.
  • mice immunised with the '3-6 (ID-74)' DNA vaccine did not show significantly longer survival times when compared with the unvaccinated control group. However, there are three outlying mouse which survived approximately 4, 10 and 23 hours longer than the longest surviving mice in the unvaccinated control group. This is reflected in the higher mean survival time of 49.599 hours and a much wider range of survival times.
  • p value 1 refers to statistical significance when compared to pcDNA3.1 controls
  • p value 2 refers to statistical significance when compared to unvaccinated controls.
  • mice immunised with the '3-51 (ID-75)' DNA vaccine did not show significantly longer survival times when compared with the pcDNA3.1 control group but was relatively close to significant when compared with the unvaccinated control group.
  • the '3-51' group has two outlying mouse one of which survived the term of the experiment despite developing symptoms.
  • the mean survival time of 44.499 hours is considerbly higher than that determined for both control groups and the group also demonstrates as a much wider range of survival times.
  • mice immunised with the '3-56 (ID-76)' DNA vaccine exhibited significantly longer survival times when compared with the pcDNA3.1 control group but were marginally not significant when compared with unvaccinated control group.
  • Example 3 Conservation and varability of candidate vaccine antigen genes among different isolates of Group B Strepococci
  • Oligonucleotide primers were designed for each individual gene of interest derived using the LEEP system. Primers were designed to target the whole of the gene being investigated (All primers are listed in APPENDIX III). Specific gene targets were amplified by PCR using Vent DNA polymerase (NEB) according to the manufacturers instructions. Typical reactions were carried out in a 100 ⁇ l volume containing 50 ng of GBS template DNA, a one tenth volume of enzyme reaction buffer, 1 ⁇ M of each primer, 250 ⁇ M of each dNTP and 2 units of Vent DNA polymerase.
  • NEB Vent DNA polymerase
  • a typical reaction contained an initial 2 minute denaturation at 95°C, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at the appropriate melting temperature for 30 seconds, and extension at 72°C for 1 minute (1 minute per kilobase of DNA being amplified). The annealing temperature was determined by the lower melting temperature of the two oligonucleotide primers. The reaction was concluded with a final extension period of 10 minutes at 72°C. All PCR amplified products were extracted once with phenol chloroform (2:1 :1) and once with chloroform (1 :1) and ethanol precipitated. Specific DNA fragments were isolated from agarose gels using the QIAquick Gel Extraction Kit (Qiagen). For use as DNA probes, purified amplified gene DNA fragments were labelled with digoxygenin using the DIG Nucleic Acid Labelling Kit (Boehringer Mannheim) according to the manufacturer's instructions.
  • DNA concentrations were digested using either Hin Dili, Eco RI or Bgl Ilrestriction enzymes (NEB) according to manufacturer instructions and analysed by agarose gel electrophoresis. Following agarose gel electrophoresis of DNA samples, the gel was denatured in 0.25M HCl for 20 minutes and DNA was transferred onto HybondTM N + membrane (Amersham) by overnight capillary blotting. The method is essentially as described in Sambrook et al. (1989) using Whatman 3MM wicks on a platform over a reservoir of 0.4M NaOH. After transfer, the filter was washed briefly in 2x SSC and stored at 4 C in Saran wrap (Dow chemical company).
  • Filters were prehybridised, hybridised with the digoxygenin labelled DNA probes and washed using conditions recommended by Boehringer Mannheim when using their DIG Nucleic Acid Detection Kit. Filters were prehybridised at 68°C for one hour in hybridisation buffer (1% w/v supplied blocking reagent, 5x SSC, 0.1% v/v N-lauryl sarcosine, 0.02%) v/v sodium dodecyl sulphate[SDS]). The digoxygenin labelled DNA probe was denatured at 99.9°C for 10 minutes before being added to the hybridisation buffer.
  • Hybridisation was allowed to proceed overnight in a rotating Hybaid tube in a Hybaid Mini-hybridisation oven. Unbound probe was removed by washing the filter twice with 2x SSC- 0.1 % SDS for 5 minutes at room temperature. For increased stringency filters were then washed twice with O.lx SSC-0.1%o SDS for 15 minutes at 68°C.
  • the DIG Nucleic Acid Detection Kit (Boehringer Mannheim) was used to immunologically detect specifically bound digoxygenin labelled DNA probes. Results of Southern blot analysis
  • Genomic DNA from each strain was digested completely with Hin Dili (NEB) and elecfrophoresed at 40 Volts for 6 hours in 0.8% o agarose, transferred onto Hybond ⁇ NT (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled rib gene probe. Specifically bound DNA probe was identified using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
  • rib appears to be absent from all serotype la and lb strains (lanes 2 to 5) and from strains 118/158 and 97/0057 of serotype II (lanes 8 and 9). However, rib would appear to present in strains 18RS21 and 1954/92 of serotype II (lanes 6 and 7) and in all strains of serotype III (lanes 10 to 13). This is in agreement with previously published data (Stalhammar-Carlemalm et al, 1993). rib would also appear to be present in strains representing serotypes VII and VII (lanes 17 and 18) but was absent from strains representing serotypes IV, V and V (lanes 14 to
  • the rib. gene probe did hybridise with lower intensity to genomic DNA fragments from strains representing serotypes la, lb, IV, VI, VII and serotype II strains 118/158 and 97/0057. This may indicate the presence of a gene in these strains with a lower level of homology to rib.
  • These hybridising DNA fragments may contain a homologue of the GBS bca gene encoding the Ca protein antigen which has been shown to be closely homologous to the Rib protein (Wastfelt et al, 1996).
  • Genomic DNA from each strain was digested completely with Hin Dili (NEB) and elecfrophoresed at 40 Volts for 6 hours in 0.8% agarose, transfe ⁇ ed onto Hybond N + (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled 4 (ID-1) gene probe. Specifically bound DNA probe was identified using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
  • Genomic DNA from each strain was digested completely with Eco RI (NEB) and elecfrophoresed at 40 Volts for 6 hours in 0.8%> agarose, transferred onto Hybond N " (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled
  • ID-2 gene probe. Specifically bound DNA probe was identified using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
  • Genomic DNA from each strain was digested completely with Eco RI (N ⁇ B) and elecfrophoresed at 40 Volts for 6 hours in 0.8%> agarose, transferred onto Hybond N + (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled 15 (ID-7) gene probe. Specifically bound DNA probe was identified using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
  • the gene probe hybridised specifically with Eco RI -digested DNA fragments ranging from approximately 3.5 kb to 5.2 kb in size.
  • Genomic DNA from each strain was digested completely with Hin Dili (N ⁇ B) and elecfrophoresed at 40 Volts for 6 hours in 0.8%> agarose, transferred onto Hybond NT 1" (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled 17 (ID-8) gene probe. Specifically bound DNA probe was identified using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim).
  • Genomic DNA from each strain was digested completely with Bgl II (NEB) and elecfrophoresed at 40 Volts for 6 hours in 0.8%> agarose, transferred onto Hybond N 1" (Amersham) membrane by Southern blot and hybridised with the digoxigenin-labelled 22 (ID-10) gene probe. Specifically bound DNA probe was identified using the DIG
  • Il-digested genomic DNA fragment of approximately 3.1 kb in DNA digests from all GBS representatives except serotype lb strain H36B, where the gene probe hybridised specifically to a a Bgl Il-digested genomic DNA fragment.
  • Gene 22 (ID-10) was absent from both the control strains (lanes 19 and 20).
  • a group A Streptococcal strain (serotype Ml, strain NCTC8198) and Streptococcus pneumoniae (serotype 14) were also included in the analysis for control pu ⁇ oses.
  • Protein Rib a novel Group B Streptococcal protein that confers protective immunity and is expressed by most strains causing invasive infections: J. Exp. Med. Ill: 1593-1603
  • DNA vaccination with the major outer-membrane protein genes induces acquired immunity to Chlamydia trachomatis (mouse pneumonitis) infection. Infection and Immunity, 176, 1035-40.

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Abstract

L'invention concerne de nouveaux antigènes protéiques de Streptococcus groupe B. ainsi que les séquences d'acides nucléiques codant pour eux. Elle concerne également leur utilisation dans des vaccins et des procédés de criblage.
EP99934984A 1998-07-27 1999-07-27 Acides nucleiques et proteines de streptococcus groupe b Withdrawn EP1100920A2 (fr)

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PCT/GB1999/002444 WO2000006736A2 (fr) 1998-07-27 1999-07-27 Acides nucleiques et proteines de streptococcus groupe b

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