EP1366068A1 - Polynucleotide et polypeptide de streptocoque du groupe b et leur utilisation dans la preparation d'un vaccin - Google Patents

Polynucleotide et polypeptide de streptocoque du groupe b et leur utilisation dans la preparation d'un vaccin

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Publication number
EP1366068A1
EP1366068A1 EP02704946A EP02704946A EP1366068A1 EP 1366068 A1 EP1366068 A1 EP 1366068A1 EP 02704946 A EP02704946 A EP 02704946A EP 02704946 A EP02704946 A EP 02704946A EP 1366068 A1 EP1366068 A1 EP 1366068A1
Authority
EP
European Patent Office
Prior art keywords
infection
peptide
gene
gbs
group
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Withdrawn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
EP02704946A
Other languages
German (de)
English (en)
Inventor
Joseph David Microscience Ltd. SANTANGELO
Robert Microscience Ltd FELDMAN
Jonathan Douglas Microscience Ltd LANE
Joanne Christine Microscience Ltd MOORE
Richard James ;Microscience Ltd DOBSON
Martin John Glenton Microscience Ltd HUGHES
Rebecca Kerry Wilson
Paul Everest
Gordon Depart.of Biochemistry ECSTM DOUGAN
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.)
Emergent Product Development UK Ltd
Original Assignee
Microscience Ltd
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 Microscience Ltd filed Critical Microscience Ltd
Publication of EP1366068A1 publication Critical patent/EP1366068A1/fr
Withdrawn legal-status Critical Current

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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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • 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
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • This invention relates to the identification of a bacterial gene and protein, and its use. More particularly, it relates to its use in therapy, for immunisation and in screening for drugs .
  • Background to the Invention
  • GBS Group B Streptococcus
  • Streptococcus agalactiae is the causative agent of various conditions.
  • GBS causes: Early onset neonatal infection .
  • This infection usually begins in utero and causes severe septicaemia and pneumonia in infants, which is lethal if untreated and even with treatment is associated with a 10-20% mortality rate. Late onset neonatal infection .
  • This infection occurs in the period shortly after birth until about 3 months of age. It causes a septicaemia, which is complicated by meningitis in 90% of cases. Other focal infections also occur including osteomyelitis, septic arthritis, abscesses and endopthalmitis . Adul t infections .
  • Urinary tract infections GBS is a cause of urinary tract infections and in pregnancy accounts for about 10% of all infections.
  • Veterinary infections .
  • GBS causes chronic mastitis in cows. This, in turn, leads to reduced milk production and is therefore of considerable economic importance.
  • GBS infections can be treated with antibiotics.
  • immunisation is preferable. It is therefore desirable to develop an immunogen that could be used in a therapeutically-effective vaccine .
  • the present invention is based on the identification of a gene in GBS, and also related organisms, the product of which may be localised on the outer surface of the organism and therefore may be used as a target for immuno- therapy.
  • a peptide is encoded by a gene identified herein as pho2-2, obtainable from Group B Streptococcus, or a homologue or functional fragment thereof. Such a peptide is suitable for therapeutic use, e.g. when isolated.
  • a functional fragment is used herein to define a part of the gene or peptide which retains the activity of the whole gene or peptide.
  • a functional fragment of the peptide may be used as an antigenic determinant, useful in a vaccine or in the production of antibodies .
  • a gene fragment may be used to encode the active peptide.
  • the gene fragment may have utility in gene therapy, targetting the -wild-type gene in vivo to exert a therapeutic effect.
  • a peptide according to the present invention may comprise the amino acid sequence identified herein as SEQ ID NO. 2, or a functional fragment thereof.
  • the peptide of the present invention may be a suitable candidate for the production of therapeutically-effective vaccines against GBS.
  • the term "therapeutically-effective" is intended to include the prophylactic effect of vaccines.
  • a vaccine may comprise a peptide according to the invention, or the means for its expression, for the treatment of infection.
  • the vaccine may be administered to females prior to or during pregnancy to protect mother and neonate against infection by GBS.
  • the peptide or gene may be used for screening potential antimicrobial drugs or for the detection of virulence.
  • a further aspect of this invention is the use of any of the products identified herein, for the treatment or prevention of a condition associated with infection by a Group B Streptococcal strain.
  • the protein has been described for use in the treatment of patients, veterinary uses of the products of the invention are also considered to be within the scope of the present invention.
  • the peptide or the vaccines may be used in the treatment of chronic mastitis, especially in cows. Description of the Drawings The invention is described with reference to the accompanying drawing where :
  • Figure 1 is a graphic illustration of the average level of protection offered by anti-pho2-2 anti-sera against a challenge dose of GBS. Description of the Invention
  • the present invention is described with reference to Group B Streptococcal strain M732.
  • GBS strains and many other bacterial strains are likely to include related peptides or proteins having amino acid sequence homology with the peptide of M732.
  • Organisms likely to contain the peptide include, but are not limited to, S. pneumoniae, S. pyogenes, S. suis, S. miller i , Group C and Group G Streptococci and Enterococci . Vaccines to each of these may be developed in the same way as described for GBS.
  • the peptides that may be useful for the production of vaccines have greater than 40% sequence similarity with the peptide identified herein. More preferably, the peptides have greater than 60% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity, e.g. 95% similarity.
  • related polynucleotides that may be useful in the various aspects of the invention may have greater than 40% identity with the sequence identified herein. More preferably, the polynucleotide sequences have greater than 60% sequence identity. Most preferably, the polynucleotide sequences have greater than 80% sequence identity, e.g. 95% identity.
  • similarity refers to a sequence comparison based on identical matches between correspondingly identical positions in the sequences being compared.
  • similarity refers to a comparison between amino acid sequences, and takes into account not only identical amino acids in corresponding positions, but also functionally similar amino acids in corresponding positions. Thus similarity between polypeptide sequences indicates functional similarity, in addition to sequence similarity.
  • Levels of identity between gene sequences and levels of identity or similarity between amino acid sequences can be calculated using known methods.
  • publicly available computer based methods for determining identity and similarity include the BLASTP, BLASTN and FASTA (Atschul et al . , J. Molec. Biol . , 1990; 215:403-410), the BLASTX program available from NCBI , and the Gap program from Genetics Computer Group, Madison WI .
  • sequence homologies may be established by searching in existing databases, e.g. EMBL or Genbank.
  • a fragment of the gene sequence disclosed herein may be used to prepare a vaccine product, or as a probe in a diagnostic method, or to identify homologues in other microorganisms.
  • the fragment will be at least 20 nucleic acids, more preferably at least 30 nucleic acids, and most preferably at least 70 nucleic acids.
  • hybridisation under stringent conditions means that a positive hybridisation signal is still observed after washing for 1 hour with 1 x SSC buffer and 0.1% SDS at 55°C, preferably at 62°C and most preferably at 68°C.
  • Peptides or proteins according to the invention may be purified and isolated by methods known in the art. In particular, having identified the gene sequence, it will be possible to use recombinant techniques to express the genes in a suitable host. Active fragments and homologues can be identified and may be useful in therapy. For example, the peptide or its active fragments may be used as antigenic determinants in a vaccine, to elicit an immune response. They may also be used in the preparation of antibodies, for passive immunisation, or diagnostic applications. Peptide fragments may be used to identify those parts of the protein that have the most favourable antigenic epitopes. The fragments can be generated by methods known to those skilled in the art. For example, partial digests of the complete protein can be made and tested.
  • synthetic peptide fragments can be made and tested. The fragments may be tested to establish which fragments elicit the strongest immune response .
  • a therapeutic antibody of the invention will have affinity for the protein (or peptide) of the invention and preferably should not cross-react with unrelated proteins or proteins in the patient.
  • Suitable antibodies which may be active against the peptide of the invention include monoclonal antibodies, or fragments thereof, including single chain fv fragments.
  • Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, and used in therapy, to provide effective immunisation against Group B Streptococci or other microorganisms that contain related proteins.
  • suitable carriers or adjuvants e.g. alum
  • the preparation of vaccine formulations will be apparent to the skilled person.
  • a suitable amount of an active component of the invention can be selected, for therapeutic use, as can suitable carriers or excipients, and routes of administration. These factors will be chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type or health of the subject etc.
  • the products of the invention may also be used in assays to screen potential antimicrobial drugs.
  • the protein of the invention may be used as a target for antimicrobial therapy, to localise a drug to the infecting microbe.
  • a partial gene library of GBS (strain M732) chromosomal DNA was prepared using the plasmid vectors pFW-phoAl, pFW- phoA2 and pFW-phoA3 (Podbielski, A. et al . 1996. Gene 177:137-147). These plasmids possess a constitutive spectinomycin adenyltransferase antibiotic resistance marker, which confers a high level of spectinomycin resistance and is therefore easily selected. Furthermore, these vectors contain a truncated (leaderless) Escherichia coli phoA gene for alkaline phosphatase.
  • the three vectors differ only with respect to the reading frame in which the leaderless phoA gene exists, as compared to an upstream in- frame BamHI restriction enzyme site. Because this truncated E. coli phoA gene lacks the appropriate leader sequence for export of this enzyme across the bacterial membrane, extracellular alkaline phosphatase activity is absent when these plasmids are propagated in an E. coli phoA mutant (e.g. strain DH5 ) .
  • the chromogenic alkaline phosphatase substrate, XP (5-bromo-4-chloro-3-indolyl-phosphate) does not enter intact bacterial cells and therefore only exported or surface associated alkaline phosphatase activity can be detected. When exported or surface associated alkaline phosphatase activity is present, the chromogenic XP substrate is cleaved to yield a blue pigment and the corresponding bacterial colonies can be identified by their blue colour.
  • Plasmid DNA was digested to completion with BamHI and dephosphorylated using shrimp alkaline phosphatase.
  • GBS genomic DNA was partially digested with Sau3AI , size fractionated on a sucrose gradient and fragments ⁇ lkb in size were ligated into the prepared pFW-p oA vectors.
  • E. coli strain DH5 was chosen as the cloning host since it lacks a functional phoA gene.
  • Recombinant plasmids were selected on Luria agar containing 100 ⁇ g/ml of spectinomycin and 40 ⁇ g/ml of the chromogenic XP substrate.
  • a comparison of the amino acid sequence of pho2-2 was performed. Homologues to the GBS pho2-2 gene product can be identified in Enterococcus faecalis, Escherichia coli ( alK and afuC) , Bacillus subtilis (glnO) , Haemophilus influenzae (yebM and potA) , Streptococcus pyogenes, Streptococcus pneumoniae and Salmonella typhi urium (malK) . The E. faecalis , S . pyogenes and S . pneumoniae homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products.
  • homologues represented ATP-binding transport proteins that are part of ABC type transporters.
  • Many of the components of ABC type transporters are membrane or cell surface associated, as these systems are involved in the transport of macromolecules from the extracellular environment to the intracellular compartment.
  • oligonucleotide primers were designed for genomic DNA sequencing. These primers were designed so as to sequence in an "outward 1 direction from the obtained sequence. Once read, the sequence obtained was checked to see if the 5' and 3' termini of the gene had been reached. The presence of these features was identified by checking against homologous sequences, and for the 5' end the presence of an AUG start codon (or accepted equivalent) preceded by a Shine-Dalgarno consensus sequence, and for the 3 1 end, the presence of a translation termination (Stop) codon.
  • AUG start codon or accepted equivalent
  • primers were designed for amplification of full-length product.
  • Primers used included restriction enzyme recognition sites (Ncol at the 5 ' end and EcoO109I at the 3 1 end) to allow subsequent cloning of the product into the Lactococcal expression system used.
  • PCR was carried out using the primers, and the products cloned into a pCR 2.1 cloning vector (Invitrogen) .
  • the vector into which this fragment was inserted was a modified version of pNZ8048 (Kuipers, O. P. et al . (1998) J. Biotech 64: 15-21).
  • This vector harbouring a lactococcal origin of replication, a chloramphenicol resistance marker, an inducible nisin promoter and a multicloning site was altered by the replacement of the multicloning site with two 10X His tags, flanked on the 5- most end with an Ncol site, split in the middle with a multicloning site (including an EcoO109I site) , and a stop (termination) codon at the 3' end of the His tags.
  • the gene of interest was inserted so that a 10X His tag was in the 3' position relative to the coding region.
  • a 400 ml liquid culture was set up and translation of the protein was induced by the addition of nisin to the culture. After a 2 hour incubation, the cells were harvested and lysed by bead beating. The resultant lysate was cleared by centrifugation, then passed over a metal affinity (Talon, Clonetech) column. The column was washed repeatedly before bound proteins were eluted with Imidazole.
  • the recombinant protein obtained was then used to immunise New Zealand white rabbits, with pre-immune sera being harvested prior to immunisation. Following a boost, the rabbits were sacrificed and sera collected. This sera was used in Western blots, ELISA and animal protection models .
  • Anti-sera against the pho2-2 outer surface protein and a further protein (pho3-9) identified in the same screen were raised in rabbits and the IgG from each anti-sera was purified using Protein A column chromatography. Newborn pups from time-mated Sprague Dawley rats were " "immunised' with 50 ⁇ l of the purified IgG intraperitoneally and returned to different mothers for at least 5 hours before they were challenged with the GBS.
  • the A909 strain of GBS was used as the challenge. The strain was streaked on to a blood agar plate, and allowed to grow over the weekend at room temperature. A single colony of GBS was used to produce the inoculum in THB. Following overnight growth, the GBS bacteria were centrifuged, and an appropriate volume of PBS added to produce a final dilution of 1 x 10 5 CFU/ml GBS (5 x 10 4 GBS/ 50 ⁇ l) . The innoculum was kept on ice until use in the challenge assay in the rat pups.
  • the GBS was administered subcutaneously to each rat and the time of challenge recorded. All pups were monitored for approximately 63 hours after the GBS challenge.
  • Table A shows the percentage of pups that survived 63 hours after challenge with GBS. This data has been represented in a graph in figure 1.
  • the pho2-2 protein offered significant protection against GBS infection compared to the PBS control and to the other outer surface protein pho3-9.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Une protéine du streptocoque de groupe B (streptococcus GBS) s'avère être une protéine de surface extérieure et constitue une cible utile pour une thérapie antimicrobienne.
EP02704946A 2001-03-09 2002-03-11 Polynucleotide et polypeptide de streptocoque du groupe b et leur utilisation dans la preparation d'un vaccin Withdrawn EP1366068A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB0105922 2001-03-09
GBGB0105922.9A GB0105922D0 (en) 2001-03-09 2001-03-09 Genes and proteins, and their use
PCT/GB2002/001089 WO2002072623A1 (fr) 2001-03-09 2002-03-11 Polynucleotide et polypeptide de streptocoque du groupe b et leur utilisation dans la preparation d'un vaccin

Publications (1)

Publication Number Publication Date
EP1366068A1 true EP1366068A1 (fr) 2003-12-03

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EP (1) EP1366068A1 (fr)
GB (1) GB0105922D0 (fr)
WO (1) WO2002072623A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6890539B2 (en) 1998-12-22 2005-05-10 Microscience, Ltd. Genes and proteins, and their use
EP2104512A2 (fr) * 2006-12-21 2009-09-30 Emergent Product Development UK Limited Proteines de streptococcus et leur utilisation comme vaccins

Family Cites Families (4)

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Publication number Priority date Publication date Assignee Title
US6015889A (en) * 1993-03-19 2000-01-18 Gunnar Lindahl Protein rib, a cell surface protein that confers immunity to many strains of the group B streptococcus: process for purification of the protein, reagent kit and pharmaceutical composition
CA2321106C (fr) * 1998-02-20 2013-07-23 Biochem Pharma Inc. Antigenes du streptocoque du groupe b
WO2000006736A2 (fr) * 1998-07-27 2000-02-10 Microbial Technics Limited Acides nucleiques et proteines de streptococcus groupe b
DK1141308T3 (da) * 1998-12-22 2007-06-04 Microscience Ltd Gruppe B streptococcus proteiner og deres anvendelse

Non-Patent Citations (1)

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Title
See references of WO02072623A1 *

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WO2002072623A1 (fr) 2002-09-19
GB0105922D0 (en) 2001-04-25

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