WO1999026969A1 - Facteur d'immunite contre la zoocine a - Google Patents

Facteur d'immunite contre la zoocine a Download PDF

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Publication number
WO1999026969A1
WO1999026969A1 PCT/NZ1998/000171 NZ9800171W WO9926969A1 WO 1999026969 A1 WO1999026969 A1 WO 1999026969A1 NZ 9800171 W NZ9800171 W NZ 9800171W WO 9926969 A1 WO9926969 A1 WO 9926969A1
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WIPO (PCT)
Prior art keywords
organism
zoocin
activity
protein
dna molecule
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PCT/NZ1998/000171
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English (en)
Inventor
Robin Stuart Simmonds
Scott Alexander Beatson
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University Of Otago
New Zealand Pastoral Agriculture Research Institute Limited
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Publication date
Application filed by University Of Otago, New Zealand Pastoral Agriculture Research Institute Limited filed Critical University Of Otago
Priority to AU18926/99A priority Critical patent/AU748950B2/en
Priority to NZ505282A priority patent/NZ505282A/xx
Publication of WO1999026969A1 publication Critical patent/WO1999026969A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/34635—Antibiotics
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3571—Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the invention relates to a factor which has activity in protecting a cell producing zoocin A, to the gene encoding that factor, to vectors and organisms containing the gene and the use of such organisms as anti- bacterial agents.
  • Zoocin A is a unique domain-structured bacteriolytic enzyme produced by Streptococcus equi subsp. zooepidemicus 4881, which specifically attacks the cell walls of some closely related streptococcal species including the principal causative agents of group A streptococcal sore throat and dental caries respectively (Simmonds et al (1995); Simmonds et al (1996)). It was shown that zoocin A could suppress the growth of S. mutans in a triple species plaque model and that the initiation of the killing sequence occured very quickly.
  • the N- terminal catalytic domain of zoocin A has a high degree of homology with the N-terminal catalytic domain of a similar bacteriolytic enzyme lysostaphin, produced by Staphylococcus simulans biovar staphylolyticus, which specifically attacks the cell walls of other staphylococcal species.
  • the C- terminal substrate-binding domain of lysostaphin is known to have a high degree of homology to at least one other staphylococcal cell wall binding enzyme, a Staph. aureus amidase.
  • the substrate-binding domain of zoocin A has homology to no other known sequence.
  • Both enzymes appear to lyse cell walls by cleaving the peptide cross-links within the peptidoglycan (Simmonds et al (1996)).
  • the bacteriocidal nature of their mode of action and the high degree of species and strain specificity exhibited by these enzymes are characteristics of that group of proteinaceous inhibitory agents referred to as bacteriocin-like inhibitory substances (BLIS).
  • Zoocin A targets only a very limited range of bacteria, restricted to some species of Streptococcus only. This species-specific anti-bacterial action is useful. For example, it is active against two groups of medically significant human pathogens and at least one significant animal pathogen.
  • S. mutans and S. sobrinus are two of twenty or more species of bacteria present in dental plaque. Although not numerically dominant, these two species are considered to be the major aetiological agents of dental caries and their suppression in the oral cavity has been shown to reduce caries incidence (Loesche (1976); Loesche et al (1989)).
  • Group A streptococci (GAS) infect via the upper respiratory tract where the tonsillar region in particular is believed to be the primary site of colonization. GAS carriage in humans is relatively common and GAS pharyngitis left untreated can progress to more serious disease including rheumatic fever and nephritis (Bronze and Dale (1996)).
  • Vaccines are not available to prevent these infections and although it has been shown that these groups of microorganisms can be suppressed in the oral cavity by administration of antibacterial agents such as chlorhexidine (Loesche (1976)), polyvalent cations (Jones et al (1988)) and classical antibiotics (Loesche et al (1989)), the broad spectrum nature of these agents means that many commensal organisms are also suppressed, a condition which is known to pre-dispose the patient to superinfection by resistant microoganisms including gram-negative bacteria and yeasts. In each case the prolonged and widespread use of these agents has not been considered acceptable (Marsh (1991)).
  • antibacterial agents such as chlorhexidine (Loesche (1976)), polyvalent cations (Jones et al (1988)) and classical antibiotics (Loesche et al (1989)
  • the broad spectrum nature of these agents means that many commensal organisms are also suppressed, a condition which is known
  • zoocin A while having significant bacteriocidal activity against these groups of microorganisms has little or no activity against many other plaque species such as S. or ⁇ lis (Simmonds et al (1996)), S. sanguis or non-streptococcal species (Simmonds et al (1995)), or against the major groups colonizing the mucosal surfaces of the oral cavity such as S. salivarius (Simmonds et al (1995)). Therefore, administration of zoocin A to the oral cavity is unlikely to result in the complications seen with the previously mentioned broad spectrum anti-microbial agents, yet should lead to a decrease in the incidence of dental caries and carriage of GAS.
  • zoocin A Before zoocin A can be used for its desirable anti-bacterial properties, there is a need for it to be provided in a form that can be administered to a human or an animal safely. For many antibiotics this is achieved by batch fermentation of the organism producing the antibiotic and purifying the antibiotic molecule and adding it to a suitable carrier. This method would be very expensive for zoocin A which has a molecular weight of 28,000. For that reason, the more commercially attractive option is to produce the zoocin A in situ in a naturally fermented food such as yoghurt.
  • zoocin A is produced by S. equi subsp. zooepidemicus, a recognized animal and occasional human pathogen. Serious human disease has been shown to result from the ingestion of S. equi subsp. zooepidemicus contaminated unpasteurized milk (Francis et al (1993)). Therefore, use of the natural producer organism to incorporate zoocin A in a food product as part of a food fermentation process is unlikely to be acceptable, but one solution would be to move the genes required for zoocin A production from the natural host to an organism suitable for use in food fermentation processes. However, this approach presents some difficulties when zoocin A is lethal to the genetically transformed organism.
  • the invention provides zoocin A immunity factor, which is a protein which is capable of protecting a host cell expressing zoocin A against the potentially damaging activity of zoocin A.
  • the invention provides an isolated DNA molecule which has a nucleotide sequence which encodes zoocin A immunity factor (zij).
  • the DNA molecule is selected from the group comprising molecules having one or more of: the zif sequence shown in Figure 3, a sequence comprising that sequence, a sequence comprising a part of that sequence active in protecting an organism from zoocin A, a sequence encoding the same protein as the zif sequence of Figure 3 but differing in nucleic acid sequence by virtue of degeneracy of the genetic code and a sequence which is a functionally equivalent variant of the zif sequence shown in Figure 3.
  • a vector comprising the zif encoding molecule defined above, optionally together with a gene encoding the zoocin A active protein or variant defined above.
  • the invention provides a non-pathogenic organism containing the zif encoding molecule defined above, optionally together with a gene encoding a polypeptide sequence selected from the sequence for zoocin A or a functionally equivalent variant of that sequence.
  • the organism is a food-grade organism.
  • an antibacterial composition comprising a non-pathogenic organism as defined above.
  • the composition is suitable for ingestion, particularly human ingestion, and is a foodstuff, nutriceutical or confectionery.
  • the invention provides a method of preventing or inhibiting the growth of undesirable organisms susceptible to zoocin A which comprises the step of contacting said organisms or the environment thereof with a composition as defined above.
  • the organisms inhibited are S. mutans, S. sobrinus or S. pyogenes and the composition is administered to the oral cavity of a patient.
  • Figure 1 shows a map of pBluescript® II SK(+) phagemid vector and pVA838.
  • Figure 2 is a restriction map of PDN488L showing ORFs and s bclones.
  • the nucleotides are numbered from the first nucleotide of the EcoR I restriction site located proximal to the Sac I restriction site in the pBluescript® II SK(+) phagemid vector Sac I - Kpn I MCS of pDN488L.
  • the translation is in the direction indicated by the bold arrows.
  • Figure 3 shows the DNA sequence of 6.8 kb base .EcoR I fragment showing the nucleotide and amino acid sequences for both zooA and zif. It will be appreciated that the strand of nucleic acid coding for zif is complementary to the non-coding strand shown expressly in Figure 3. DESCRIPTION OF THE INVENTION
  • the focus of the invention is on the applicants identification of the gene encoding zoocin A immunity factor (zif) .
  • This gene is capable of protecting cells which express zoocin A against the effects of that enzyme.
  • the zif gene has been identified from S. equi subsp. zooepidemicus 4881 and has the sequence given in Figure 3. This sequence is of the non-coding strand, with the coding strand being complementary. The sequence of the coding strand is recited as SEQ ID NO. 2.
  • sequence need not always be that shown in Figure 3 but can instead be a functionally-equivalent variant of that sequence.
  • Such variants are in no way intended to be excluded and the resultant molecules are referred to herein as "zif-l ⁇ ke genes".
  • phase "functionally equivalent variants” recognises that it is possible to vary the amino acid/ nucleotide sequence of a protein while retaining substantially equivalent functionality.
  • a protein can be considered a functional equivalent of another protein for a specific function if the equivalent protein is immunologically cross-reactive with and has at least substantially the same function as, the original protein.
  • the equivalent can be, for example, a fragment of the protein, a fusion of the protein with another protein or carrier, or a fusion of a fragment which additional amino acids.
  • it is possible to substitute amino acids in a sequence with equivalent amino acids using conventional techniques. Groups of amino acids normally held to be equivalent are:
  • DNA sequences encoding a particular produce can vary significantly simply due to the degeneracy of the nucleic acid code.
  • the probability of one sequence being functionally equivalent to another can be measured by the computer algorithms BLASTP (Altschul, S. F. et al (1990)) and FASTA (Pearson, W. R. et al (1988)) for proteins and DNA respectively.
  • the zif gene or zi -like gene of the invention can be inserted into organisms which are to be transformed with the zooA gene (which encodes zoocin A) so that a recipient organism which is zoocin A sensitive is protected by expression of the zif gene.
  • the action of zif in protecting a zoocin A producer cell from the otherwise lethal action of its own product is believed to involve the modification of the cells peptidoglycan cross-links to a chemical form non-hydrolysed by zoocin A.
  • Organisms which may be usefully transformed with the zif gene include any food- acceptable or pharmaceutically acceptable non-pathogenic organism. When the gene is inserted into zoocin A susceptible organisms, these organisms can be subsequently or simultaneously transformed with zoo A in a manner which allows production of zoocin A. The zif gene protects the transformed organism from the lethal effects of zoocin A produced.
  • the vector pSBl 131 is a preferred vector for this purpose.
  • non-pathogenic organisms for use in the invention include yeasts and bacteria.
  • organisms having a genus selected from non-pathogenic strains of streptococcus are particularly useful.
  • non-pathogenic strains of Streptococcus gordonii are particularly preferred.
  • Organisms transformed with the gene of the invention may be used as preservatives in processed cheese, various pasteurised dairy products, canned vegetables, hot baked flour products and pasteurised liquid egg. They may also be used in preservation of naturally fermented foods such as beer, wine, yoghurt and cheeses.
  • the transformed organisms and/ or extracts of the organisms may also be used to prepare pharmaceutical compositions for use topically to prevent establishment of infectious diseases of humans and animals.
  • Such topical compositions are useful in treatment of skin conditions, such as ulcers, in which streptococci are significant pathogens and where poor blood supply limits the effectiveness of systemically administered antibiotics.
  • Group C streptococci are serious animal pathogens, particularly of horses and are responsible for considerable economic loss to the bloodstock industry.
  • the primary route of infection for these organisms is believed to be the respiratory tract and it is contemplated that the incorporation of organisms according to the invention which express zoocin A with animal feeds may reduce colonization rates in these animals, and hence the rate of serious disease.
  • the transformed organisms and/ or their zoocin A-containing culture fluid be included in a composition intended for human ingestion (such as a foodstuff, nutriceutical or confectionery). This is particularly the case where the intention is to treat or prevent problems associated with the organisms S. mutans and/ or S. sobrinus.
  • These organisms inhabit the oral cavity and, as stated previously, are considered to be the major aetiological agents of dental caries. Their suppression in the oral cavity reduces the incidence of dental caries.
  • Foodstuffs such as processed cheeses and yoghurts are particularly appropriate for such applications.
  • Confectioneries such as wine gums and chewing gums are also contemplated.
  • the transformed organism of the invention may be admixed with food products, confectioneries and pharmaceutical carriers by conventional means.
  • conventional methods may also be used including the step of adding the transformed microorganism at the time of culturing the product.
  • the transformed microorganism is of the same species as conventionally used for the preparation of the fermented product thus allowing the preparation of the zoocin A and the fermented product to occur simultaneously.
  • E. coli DH5 ⁇ F' Woodcock et al (1989), Raleigh et al (1989) was grown routinely at 37°C in air and S. equi subsp. zooepidemicus 4881 (Schofield and Tagg (1983)) and S.gordonii DL1 (Macrina et al (1982)) in 5% C0 2 in air atmosphere at 37°C.
  • E. coZi DH5 ⁇ F' was routinely cultured in 2xYT medium (16 g bacto-tryptone (Difco Laboratories, Detroit, MI, USA), 10 g bacto-yeast extract (Difco), and 5 g NaCl (Riedel-de Haen AG, Seeize, Germany) to one litre of distilled water, purified with a Milli-Q system (Millipore Inc., France) (MQ water), Luria-Bertani (LB) medium (10 g bacto-tryptone (Difco), 5 g bacto-yeast extract (Difco), and 10 g NaCl (Riedel-de Haen AG) to one litre of MQ water) or on LB agar (LBA) plates.
  • 2xYT medium (16 g bacto-tryptone (Difco Laboratories, Detroit, MI, USA), 10 g bacto-yeast extract (Difco), and 5 g NaCl (Ri
  • LBA was prepared by supplementing LB medium with 1.5% bacto-agar (Difco). Plates containing antibiotics were prepared by supplementing LBA with either 100 mg/ml ampicillin (LBA+Ap), 250 mg/ml erythromycin (LBA+Em250), 500 mg/ml erythromycin (LBA+Em500) or 25 mg/ml chloramphenicol (LBA+Cm). All antibiotics were manufactured by Sigma (Sigma Chemical Co., St. Louis, MO, USA). LBA containing antibiotics was stored at 4°C for periods of up to two weeks.
  • Streptococcus gordonii DL1 strains were routinely cultured in Todd Hewitt broth (THB) (Difco), on Columbia Agar Base (CAB) (GIBCO BRL, Life Tec. Ltd., Paisly UK) plates or on blood agar (BA) (CAB supplemented with 5% whole human blood (Dunedin Public Hospital, Dunedin, NZ)).
  • TLB Todd Hewitt broth
  • CAB Columbia Agar Base
  • BA blood agar
  • Antibiotic containing agar plates were prepared by supplementing CAB with 10 mg/ml erythromycin (CAB+Em). Prior to transformation S.
  • gordonii DL1 were grown in Brain Heart Infusion (BHI) (Difco) supplemented with 0.5% bacto-yeast extract (Difco), 1% membrane filtered horse serum (GIBCO BRL) and 0.1% glucose (Serva Feinbiochemica GmbH & Co. KG, Heidelberg, Germany) (BHS broth).
  • BHI Brain Heart Infusion
  • bacto-yeast extract Difco
  • GIBCO BRL 1% membrane filtered horse serum
  • glucose Serva Feinbiochemica GmbH & Co. KG, Heidelberg, Germany
  • Maps of pBluescript® II SK(+) phagemid vector (Stratagene, La Jolla, CA, USA) and pVA838 (Macrina et al (1982)) are given in Figure 1.
  • Ligations were performed at temperatures between 12°C and 15°C overnight using T4 DNA ligase (Boehringer Mannheim Gmbh) as per the manufacturers instructions. Prior to use in transformations, ligation mixtures were ethanol precipitated with 1 ⁇ l glycogen (Boehringer Mannheim Gmbh) and resuspended in 10 ⁇ l Milli-Q water.
  • gel electrophoresis was performed using 1% agarose (Sigma) gels prepared and run with Tris-acetate EDTA (TAE) buffer (per litre: 4.84 g Tris base (Serva), 1.142 ml glacial acetic acid (Rh ⁇ ne-Poulenc Chemicals Ltd., Bristol, UK), and 0.8 ml 0.5 M ethylenediaminetetra-acetate (BDH Laboratory Supplies, Poole, UK) (EDTA) at 75 - 100 V.
  • TAE Tris-acetate EDTA
  • Electrophoresis was performed using a Pharmacia Electrophoresis Constant Power Supply ECPS 2000/300 (Pharmacia Fine Chemicals AB, Uppsala, Sweden), and gel electrophoresis apparatus including a range of submarine gel tanks: 20 cm x 24 cm Model H4 (Betheseda Research Laboratories, Gaithersburg, MD, USA), 11 cm x 14 cm HORIZON 11*14 (GIBCO BRL), 8 cm x 6 cm minigel tank (Bio-rad).
  • Pharmacia Electrophoresis Constant Power Supply ECPS 2000/300 Pharmacia Fine Chemicals AB, Uppsala, Sweden
  • gel electrophoresis apparatus including a range of submarine gel tanks: 20 cm x 24 cm Model H4 (Betheseda Research Laboratories, Gaithersburg, MD, USA), 11 cm x 14 cm HORIZON 11*14 (GIBCO BRL), 8 cm x 6 cm minigel tank (Bio-rad).
  • E. coli DH5 ⁇ F' electro-transformations were performed with a Biotechnologies and Experimental Research Inc. (BTX) BTX® E. coli TransPoratorTM (BTX, SanDiego, CA, USA), a Pharmacia LKB 2197 Power Supply (Pharmacia LKB, Broma, Sweden), and 0. 1 cm electrode gap Gene PulserTM Cuvettes (Bio-rad Laboratories, Hercules, CA, USA). 40 ⁇ l aliquots of E.
  • coZi DH5 ⁇ F' electro- competent cells were maintained at -70°C until required. Following electro- poration, 1 ml of 2xYT broth was immediately added to the transformation mixture and the cells resuspended and transferred to a glass vial. Resuspended cells were incubated at 37°C with shaking at 200 rpm for 1 hour to enable the plasmid encoded antibiotic resistance genes to be expressed. Dilutions of the mixture were spread plated on appropriate antibiotic- containing media and incubated at 37°C overnight.
  • TAE buffer was then added to cover the gel and electrophoresis continued at 75 - 100 V until completion.
  • DNA bands were visualized by staining the gel for 10 minutes in 0.5 ⁇ g/ml ethidium bromide (Sigma) solution.
  • Supercoiled plasmids were clearly visible after ethidium bromide staining.
  • Recombinants were initially characterized by comparing their plasmid size with the plasmid size of supercoiled pBluescript® II SK(+) phagemid vector carrying no insert.
  • coZi DH5 ⁇ F' transformants yielding appropriately sized plasmids were used to inoculate 2.5 ml 2xYT broth supplemented with 100 ⁇ g/ml ampicillin.
  • plasmid DNA was extracted from 1.5 ml of each culture using the Quantum prepTM plasmid miniprep kit (miniprep) (Bio-rad) and the plasmid DNA eluted from the miniprep matrix in 100 ml of MQ water according to the manufacturers instructions. The eluted DNA was stored at -20°C. The remaining culture was centrifuged and the pellet resuspended in 10% skim milk and stored at -70°C.
  • Those transformants carrying pBluescript® II SK(+) phagemid vector with an insert were characterized by restriction digestion of miniprep plasmid DNA. Plasmid DNA was digested with restriction enzymes chosen to linearise the plasmid. EcoR I was used to linearise plasmid DNA from pSB1006, pSB 1291, pSB1205, and pSB1014 transformants. Sac I was used to linearise plasmid DNA from pSB 10313 and pSB 1047 transformants, Hind III to linearise plasmid DNA from pSB 1083 transformants, and Pst I to linearise plasmid DNA from pSB961 and pSB981 transformants.
  • the digested plasmid DNA was electrophoresed and the size of the plasmid determined relative to known DNA sizing standards (either Pst I or Hind III digested 1 DNA (New England Biolabs)). DNA bands were visualized by staining the gel for 10 minutes in 0.5 ⁇ g/ml ethidium bromide (Sigma) solution. The size estimate obtained for each plasmid was compared with the predicted size determined from the previously published restriction map of pDN488L (Simmonds et al (1997)).
  • E. coli DH5 F' transformants carrying recombinant pBluescript® II SK(+) phagemid vectors were initially characterized as previously described (Characterisation of E. coli DH5 F' transformants carrying recombinant pBluescript® II SK(+) phagemid vectors) and the size of their supercoiled plasmids compared with the size of supercoiled pVA838 (Macrina et al (1982)).
  • E. coli DH5 ⁇ F' isolates identified as carrying plasmids of the appropriate size were grown overnight at 37°C in 5 ml 2xYT broth supplemented with 500 ⁇ g/ml Em.
  • Plasmid DNA was extracted from 3 ml of each culture using the Quantum prepTM plasmid miniprep kit (Bio-rad) and the plasmid DNA eluted from the miniprep matrix in 100 ml of MQ water according to the manufacturers instructions. The eluted DNA was stored at -20°C. The remaining culture was centrifuged and the pellet resuspended in 10% skim milk and stored at -70°C.
  • Transformants carrying ⁇ VA838 vector with an insert were characterized by restriction digestion of miniprep plasmid DNA essentially as described previously
  • Plasmids were constructed using a subcloning strategy based on the previously published restriction map of pDN488L (Simmonds et al (1997)). The cloning of pDN488L, pDN2.2, and pDNO.8 has been previously described. Unless otherwise stated the following method was used to construct all pBluescript® II SK(+) phagemid vector subclones.
  • At least 1 ⁇ g pBluescript® II SK(+) phagemid vector miniprep DNA was digested with the appropriate restriction enzyme (s), treated with CIP and electrophoresed. Unless otherwise stated, at least 1 ⁇ g of the appropriate parent plasmid miniprep DNA was digested with the appropriate restriction enzyme(s), treated with CIP and electrophoresed. Bands corresponding to the 2.9 kb linearised pBluescript® II SK(+) phagemid vector, and the desired insert fragment (Table 1) were extracted from the gel using a Prep-A-GeneTM DNA purification kit (Bio-rad), eluted with 30 ⁇ l MQ water according to the manufacturers instructions and ligated.
  • a Prep-A-GeneTM DNA purification kit Bio-rad
  • E. coZi electro-transformation E. coZi electro-transformation
  • transformants isolated and characterized as previously described E. coZ transformants carrying recombinant pBluescript® II SK(+) phagemid vectors
  • An alternative method was used to construct pSB 1006, pSB1014, and pSB 1025.
  • a restriction enzyme was chosen that cut once within the 6.8 kb insert of pDN488L and once within the pDN488L multi-cloning site (MCS).
  • pSB 1083 was constructed similarly, differing in that the parent plasmid was pSB1014.
  • pSB 1047 was constructed similarly, differing in that the parent plasmid was pSB1006 and that two enzymes with unique but compatible restriction sites were used to digest pSB1006.
  • pSB961 was pBluescript® II SK(+) phagemid vector incorporating the 0.7 kb Eco RV
  • pSB981 was pBluescript® II SK(+) phagemid vector incorporating the 1.5 kb Eco RV
  • pSB 1006 was pBluescript® II SK(+) phagemid vector incorporating the 3.7 kb Cla I - EcoR I fragment of pDN488L. A CZ ⁇ I digestion of pDN488L was electrophoresed and the 6.6 kb band was extracted from the gel and self-ligated as described previously
  • pSB1014 was pBluescript® II SK(+) phagemid vector incorporating the 3.1 kb Hind III - EcoR I fragment of pDN488L.
  • a Hind III digestion of pDN488L was electrophoresed and the 6.0 kb band extracted from the gel and self-ligated as described previously (Construction of clones using pBluescript® II SK(+) phagemid vectors).
  • pSB1025 was pBluescript® II SK(+) phagemid vector incorporating the 3.4 kb Eco RV - EcoR I fragment of pDN488L.
  • An Eco RV digestion of pDN488L was electrophoresed and the 6.3 kb band extracted from the gel and self-ligated as described previously (Construction of clones using pBluescript® II SK(+) phagemid vectors) .
  • pSB1083 was pBluescript® II SK(+) phagemid vector incorporating the 2.3 kb Hind III - Xba I fragment of pSB 1014.
  • a Xba I digestion of pSB1014 was electrophoresed and the 5.2 kb band extracted from the gel and self-ligated as described previously
  • pSB 10313 was pBluescript® II SK(+) phagemid vector incorporating the 0.8 kb Xba I - EcoR I fragment of pSB1014.
  • pSB1047 was pBluescript® II SK(+) phagemid vector incorporating the 0.2 kb CZ ⁇ I -
  • pSB1097 was pBluescript® II SK(+) phagemid vector incorporating the 0.3 kb Hind III - EcoR I fragment of pSB 1025.
  • pSB1291 was pBluescript® II SK(+) phagemid vector incorporating the 4.0 kb Pst I - EcoR I fragment of pDN488L.
  • pSB 131 1 in £. coZi DH5 ⁇ F' The following procedure was used to construct pSB 131 1 in £. coZi DH5 ⁇ F'.
  • pVA838 miniprep DNA (at least 1 ⁇ g) was digested with EcoR I, treated with CIP and electrophoresed.
  • pDN488L miniprep DNA (at least 1 ⁇ g) was digested with EcoR I, treated with CIP and electrophoresed.
  • Bands corresponding to the 9.2 kb EcoR I digested pVA838 vector and the 6.8 kb EcoR I digested pDN488L insert were extracted from the gel using the Prep-A-GeneTM DNA purification kit (Bio-rad) and eluted with 30 ⁇ l MQ water according to the manufacturers instructions.
  • E. coZz electro-transformation E. coZz electro-transformation
  • transformants isolated and characterized as previously described E. coZz transformants carrying recombinant pVA838 vectors.
  • pSB1847 in E. coZi DH5 ⁇ F' The following procedure was used to construct pSB1847 in E. coZi DH5 ⁇ F'.
  • pVA838 miniprep DNA (at least 1 ⁇ g) was digested with EcoR I and P ⁇ u II, treated with CIP and electrophoresed.
  • pSB1291 miniprep DNA (at least 1 ⁇ g) was digested with EcoR I and Sma I and electrophoresed. Bands corresponding to the 8.9 kb EcoR I /P ⁇ u II digested pVA838 vector and the 4 kb EcoRI/ Sma I pSB 1291 insert were extracted using the Bio-rad Gel Extraction Kit (Bio-rad) and eluted with 30 ⁇ l MQ water according to the manufacturers instructions.
  • E. coZt electro-transformation E. coZt electro-transformation
  • transformants isolated and characterized as previously described E. coZt electro-transformation
  • S. gordonii DL1 was freshly subcultured on CAB prior to each transformation. 50 ⁇ l of an overnight culture of S. gordonii DL 1 in BHS broth was used to inoculate 5 ml of pre-warmed BHS broth and the culture incubated (with a loosened cap) at 37°C in 5% CO2 in air for 3 hours. 50 ⁇ l of this was used to inoculate 5 ml of pre-warmed BHS broth and the culture incubated (with a loosened cap) at 37°C in 5% C ⁇ 2 in air for a further one hour.
  • the culture was dispensed in 0.8 ml volumes into glass vials and mixed with 10 - 50 ⁇ l (containing a minimum of 1 ⁇ g of DNA) of pSB1311 and pSB1847 miniprep DNA obtained from E. coZi DH5 ⁇ F' (pSB1311) and (pSB 1847).
  • Vials containing S. gordonii DL1 cells and pVA838 with no insert or S.gordonii DL1 cells and no DNA were included in each experiment as positive and negative controls respectively.
  • Transformation mixtures were incubated for 3 - 4 hours at 37°C in 5% CO2 in air before dilutions of each mixture were spread plated on CAB+Em and the plates incubated for 24 hours at 37°C in 5% CO2 in air.
  • E. coZi DH5 ⁇ F' plasmid DNA used for comparison with the S. gordonii DLl plasmid DNA originated from the same miniprep sample used in the respective S. gordonii DLl transformation. Transformants were stored in 10% skim milk at -70°C.
  • BLIS production was assessed using the deferred antagonism procedure (Tagg & Bannister (1979)). Briefly, a 1-cm wide streak of the test strain was inoculated diametrically across the surface of CAB plates using a cotton swab heavily charged with cells from a freshly grown THB culture. The inoculated plates were incubated at 37°C for 18 hour in air plus 5% CO2 after which the visible growth was removed by scraping with the edge of a glass slide. The surface of the medium was sterilized by exposure to chloroform vapour for 30 minutes, aired for 30 minutes and the nine standard indicator strains (I I, Micrococcus luteus; 12, S. pyogenes; 13, S. anginosus; 14, S.
  • I I Micrococcus luteus
  • S. pyogenes S. anginosus
  • BLIS activity in liquid samples was quantitated using the surface spot method (SSM) described by Jack (1991). Briefly, a 20 ⁇ l droplet of the sample to be tested was spotted out on the surface of a CAB plate and left to soak into the agar plate. The plate surface was then sterilized by exposure to choloroform vapour for 30 minutes, aired for 30 minutes and standard indicator 12 (overnight culture in THB broth) swabbed evenly onto the surface of the plate. Following overnight incubation at 37°C for 18 hours in air plus 5% CO2, the presence of inhibitory activity was visualized as a circular zone of inhibition in the 12 lawn at the site of droplet deposition.
  • SSM surface spot method
  • the titre of inhibitory activity in the samples were determined by making doubling dilutions of the test samples and plating out 20 ml drops of each dilution. The reciprocal of the highest doubling dilution at which inhibitory action was observed is given as the titre.
  • S. gordonii DLl, S. gordonii DLl (pVA838) and S. gordonii DLl (pSB1311) and ( ⁇ SB 1847) were tested for zoocin A production by the deferred antagonism method.
  • S. gordonii DLl, S. gordonii DLl (pVA838) and S. gordonii DLl (pSB131 1) and (pSB 1847) were tested for sensitivity to zoocin A by both a modification of the deferred antagonism method, and a modification of the SSM.
  • the zoocin A producer strain, S. equi subsp. zooepidemicus 4881 was used as the test strain and S. gordonii DLl, S. gordonii DLl (pVA838) and S.
  • gordonii OLl (pSB 1311) and (pSB1847) , standard indicators II and 12 and S. equi subsp. zooepidemicus 4881 used as the indicator strains.
  • modified SSM a partially purified preparation of zoocin A was diluted two-fold and 20 ml drops spotted onto the surface of CAB plates. The presence of inhibitory activity was visualized by swabbing onto the surface of each plate a lawn of either S. gordonii DLl, S. gordonii OLl (pVA838) and S. gordonii DLl (pSB 1311) or (pSB 1847), standard indicator II or 12 or S. equi subsp. zooepidemicus 4881.
  • E. coli DH5 ⁇ F' and XL1 blue pBluescript® II SK(+) phagemid vector subclones have been previously described (See Figure 2 and section; Construction of subclones using pBluescript® II SK(+) phagemid vectors) .
  • Table 2 contains a description of the primers used in this study.
  • Universal M13 forward and reverse primers were synthesized by the Oligonucleotide Unit (Department of Biochemistry, University of Otago, Dunedin, NZ) and all other primers were synthesized by GIBCO BRL Custom Primers (GIBCO BRL). Universal M13 forward and reverse primers were used in sequencing reactions with pDN0.8, pSB961, pSB981, pSB1006, pSB 1025, pSB 10313, pSB1047, pSB 1083 and pSB 1291 plasmid DNA.
  • SB108.3F2 and SB 108.3R2 primers were designed from the sequence data obtained from sequencing pSB 1083 using universal M13 forward and reverse primers respectively.
  • Primers SB108.3F2 and SB 108.3R2 were used in sequencing reactions with pSB1083 plasmid DNA.
  • 6.8kbcontigl to 6.8kbcontigl2 primers were designed from contiguous sequence data obtained from sequencing pDN0.8, pSB961, pSB981, pSB1006, pSB1025, pSB10313, pSB1047, pSB 1083 and pSB1291 using universal M13 forward, universal M13 reverse, SB 108.3F2 and SB108.3R2 primers.
  • 6.8kbcontigl - 6.8kbcontigl2 primers were used in sequencing reactions with pDN488L plasmid DNA.
  • ZooA SBD primer 1 was designed from the previously reported zooA sequence (Simmonds et al (1997)).
  • ZooA SBD primer 1 was used in sequencing reactions with pSB981 plasmid DNA. Sequencing reactions were performed by the Centre for Gene Research (University of Otago, Dunedin, NZ) using an Applied Biosystems (ABI) 373 Version 3.0 DNA sequencer and the manufacturers' procedures and specifications.
  • DNA sequence analysis was performed using an series 6100/66 Power Macintosh Apple computer. The sequence chromatographs were viewed and trimmed using the SeqEd (ABI) application. DNA sequences were compiled and a contiguous sequence was constructed using the DNAstar Seqman application. Open reading frames and putative amino acid sequences were determined using the DNAstar EditSeq application and visualized using either the DNAstar MapDraw or GeneJockey (Biosoft, Cambridge, England) applications. DNA and amino acid sequence homology searches were performed using the non-redundant protein and nucleotide databases and the gapped basic local alignment search tool (BLAST) program of the National Centre for Biotechnology Information (NCBI) (NCBI, Bethesda, MD, USA). Sequence alignments and sequence similarity calculations were performed using the DNAstar Megalign application.
  • BLAST basic local alignment search tool
  • E. coli DH5 ⁇ F' were transformed by electro-poration with Bluescript® II SK(+) phagemid vector with a transformation efficiency of approximately 10" transformants per ⁇ g plasmid DNA. Transformation efficiency for the electro- transformations of pSB1006, pSB1014, pSB1025, pSB10313, pSB1083, and pSB1097 were less than 20 transformants per ⁇ g plasmid DNA. All other recombinant Bluescript® II SK(+) phagemid vectors gave transformation efficiencies of between 10 3 - 10 4 transformants per ⁇ g plasmid DNA. 2 - 50% of E.
  • coZi DH5 ⁇ F' pBluescript® II SK(+) phagemid vector transformants screened on LBA+Ap containing IPTG and X-gal produced white colonies. 5 - 100% of white transformants were initially characterized as containing the predicted recombinant pBluescript® II SK(+) phagemid vector. All pBluescript® II SK(+) phagemid vectors characterized by restriction analysis yielded banding patterns consistent with those predicted by the cloning strategy. The discrepancies observed between E.
  • pBluescript® II SK(+) phagemid vector subclones that involved self-ligation were the simplest to characterize. Although all arose from low efficiency transformations almost 100% of white colonies were shown to carry plasmids with an appropriate insert. In contrast, many of the isolates obtained from higher efficiency transformations were difficult to characterize because of the high background of blue colonies, and the lower proportion (as few as 5%) of white colonies that were subsequently shown to possess plasmids with an appropriate insert. The high background of blue colonies most likely arose as vectors cleaved with a single restriction enzyme recircularised due to incomplete phosphatase treatment. The high proportion of white colonies that did not harbour inserts was probably related to the use of LBA+Ap containing IPTG and X-gal plates unevenly spread with IPTG or X-gal, or the use of plates not prepared on the day of transformation.
  • coZi DH5 ⁇ F' were naturally partially resistant to erythromycin and very high concentrations were required to enable selection of pVA838 transformants expressing erythromycin resistance genes. It was noted that colonies that grew rapidly (within 12 - 16 hours) on LBA+250Em transformation plates were far more likely to contain pVA838 or recombinant pVA838 than those that grew after 16 hours. Only pVA838 or recombinant pVA838 transformants were subsequently able to grow on LBA+500Em overnight.
  • SK(+) phagemid vector subclones Presumably due to the low copy of pVA838, plasmid miniprep yields were only 25% of those obtained from minipreps of pBluescript® II SK(+) phagemid vector subclones. Doubling the amount of culture used to 3 ml increased yields, but increasing the volume of culture beyond 3 ml did not significantly enhance yield.
  • Quantum prepTM uses an adaptation of the standard alkaline lysis miniprep method (Sambrook et al (1989)) so there is a limit to the amount of cells that can effectively be lysed without increasing the volume of lysis buffer that is added at the same time. It is most likely that inefficient ligation due to their larger size caused the low transformation efficiencies observed with pSB131 1 and pSB 1847. Construction of E. coHDHSa ' subclones.
  • pVA838 has two restriction sites within the chloramphenicol resistance determinant that are suitable for shuttle cloning between E. coli DH5 ⁇ F' and S. gordonii DLl ie. EcoR I and Pirn II. Use of the EcoR I site enabled pSB1311 to be constructed without difficulty. In contrast it was more difficult to decide the best strategy to use in constructing pSB 1847. Although it was possible to use the Pvu II restriction sites flanking the pSB1291 MCS to directly transfer the 4.0 kb insert into pVA838 cleaved with P ⁇ u II, this strategy was not favoured for a number of reasons.
  • gordonii DLl (pVA838), S. gordonii DLl (pSB1847), S. gordonii DLl (pSB 131 1), standard indicator I I and S. equi subsp. zooepidemicus 4881 respectively.
  • ORF 1 encodes a 142 amino acid sequence with homology to the 5' region of rgg which regulates expression of glucosyltransf erase in S. gordonii CHI.
  • ORF 2 encodes a 244 amino acid sequence with homology to insertion sequence IS200 found in a range of bacteria including Clostridium perfringens, E. coli, and Yersinia pestis. However, ORF 2 is most closely related to an IS200 sequence identified in S. pneumoniae.
  • ORF 3 encodes a 394 amino acid sequence with homology to a transposase/insertion sequence also identified in S. pneumoniae.
  • Primer sequence is presented 5' to 3'.
  • b Primer position is given as the first nucleotide of the primer relative to the sequence of the pBluescript® II SK (+) phagemid vector as previously described (Short et al, 1988; Alting-Mees et al, 1989).
  • c Primer position is given as the first nucleotide of the primer relative to the sequence of the 6.8 kb fragment of pDN488L as designated in Figure 3.
  • Table 3 Production and sensitivity to BLIS of strains tested by deferred antagonism.
  • zoo A +/- denotes the prescence or absence of the gene encoding zoocin A
  • zif +/- denotes the prescence or absence of the gene encoding zoocin A immunity
  • Em R denotes the presence of the erythromycin resistance gene located on pVA838 and Em° indicates no erythromycin resistance gene.
  • Lambda ZAP a bacteriophage lambda expression vector with in ⁇ i ⁇ o excision properties. Nucleic Acids Res. 16: 7583-7600.
  • streptococcal bacteriocin-like inhibitory substance zoocin A
  • the streptococcal bacteriocin-like inhibitory substance, zoocin A reduces the proportion of Streptococcus mutans in an artificial plaque.
  • MOLECULE TYPE protein
  • Glu Glu lie Lys Val Val Ala Leu Thr Tyr Thr Gin Lys
  • Gly Asn lie Leu Asp Gly Arg His Arg Leu Glu Asn Lys
  • MOLECULE TYPE protein
  • CCTAACTGGC AAAATGGTTT TTCTGGAAGA ATAGATCCAA CCGGATACAT 470

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Abstract

Cette invention concerne un facteur dont l'activité protège la cellule contre une enzyme bactériologique, la zoocine A. L'acide nucléique qui code pour ce facteur convient pour transformer des organismes GRAS (inocuité généralement reconnue) de telle sorte qu'ils sont capables de produire la zoocine A sans devenir vulnérables à l'activité de l'enzyme elle-même. Les organismes qui en résultent peuvent être utilisés dans des compositions antibactériennes (en particulier dans des denrées alimentaires) permettant de combattre diverses bactéries, dont S.mutans, S.sobrinus et $i(S.pyogenes.)
PCT/NZ1998/000171 1997-11-21 1998-11-23 Facteur d'immunite contre la zoocine a WO1999026969A1 (fr)

Priority Applications (2)

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AU18926/99A AU748950B2 (en) 1997-11-21 1998-11-23 Zoocin A immunity factor
NZ505282A NZ505282A (en) 1997-11-21 1998-11-23 Gene encoding a zif amino acid sequence: a bacteriolytic enzyme (zoocin A) immunity factor capable of protecting a host cell expressing zoocin A against its potentially damaging activity

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NZ329227 1997-11-21
NZ32922797 1997-11-21

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US7709009B2 (en) 2003-07-31 2010-05-04 Novartis Vaccines And Diagnostics, Srl Immunogenic compositions for streptococcus pyogenes
US7731978B2 (en) 2007-12-21 2010-06-08 Novartis Ag Mutant forms of streptolysin O
US7838010B2 (en) 2004-10-08 2010-11-23 Novartis Vaccines And Diagnostics S.R.L. Immunogenic and therapeutic compositions for Streptococcus pyogenes
US7939087B2 (en) 2000-10-27 2011-05-10 Novartis Vaccines And Diagnostics, Inc. Nucleic acids and proteins from Streptococcus groups A & B
US8287885B2 (en) 2007-09-12 2012-10-16 Novartis Ag GAS57 mutant antigens and GAS57 antibodies
US8778358B2 (en) 2004-07-29 2014-07-15 Novartis Vaccines And Diagnostics, Inc. Immunogenic compositions for gram positive bacteria such as Streptococcus agalactiae
US8945589B2 (en) 2003-09-15 2015-02-03 Novartis Vaccines And Diagnostics, Srl Immunogenic compositions for Streptococcus agalactiae

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Volume 62, 1996, R.S. SIMMONDS et al., "Mode of Action of a Lysostaphin-Like Bacteriolytic Agent Produced by Streptococcus Zooepidemicus 4881", pages 4536-4541. *
FEMS MICROBIOLOGY LETTERS, Volume 163, 1998, S.A. BEATSON et al., "Zoocin A Immunity Factor: A Fem A-Like Gene Found in a Group C Streptococcus", pages 73-77. *
GENE, Volume 189, Genbank Accession Number U50357, April 1997, R.S. SIMMONDS et al., "Cloning and Sequence Analysis of zooA, a Streptococcus Zooepidemicus Gene Encoding a Bacteriocin-Like Inhibitory Substance Having a Domain Structure Similar to that of Lysostaphin", pages 255-261. *

Cited By (24)

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US7955604B2 (en) 2000-10-27 2011-06-07 Novartis Vaccines And Diagnostics, Inc. Nucleic acids and proteins from streptococcus groups A and B
US8431139B2 (en) 2000-10-27 2013-04-30 Novartis Vaccines And Diagnostics, Inc. Nucleic acids and proteins from Streptococcus groups A and B
US9738693B2 (en) 2000-10-27 2017-08-22 Novartis Ag Nucleic acids and proteins from streptococcus groups A and B
US10428121B2 (en) 2000-10-27 2019-10-01 Novartis Ag Nucleic acids and proteins from streptococcus groups A and B
US9840538B2 (en) 2000-10-27 2017-12-12 Novartis Ag Nucleic acids and proteins from Streptococcus groups A and B
US7939087B2 (en) 2000-10-27 2011-05-10 Novartis Vaccines And Diagnostics, Inc. Nucleic acids and proteins from Streptococcus groups A & B
US8137673B2 (en) 2000-10-27 2012-03-20 Novartis Vaccines And Diagnostics, Inc. Nucleic acids and proteins from Streptococcus groups A & B
US8025890B2 (en) 2000-10-27 2011-09-27 Novartis Vaccines And Diagnostics, Inc. Nucleic acids and proteins from streptococcus groups A and B
WO2003093306A3 (fr) * 2002-05-02 2004-02-12 Chir0N Srl Acides nucleiques et proteines tires des groupes de streptocoques a et b
WO2003093306A2 (fr) * 2002-05-02 2003-11-13 Chir0N Srl Acides nucleiques et proteines tires des groupes de streptocoques a et b
US8128936B2 (en) 2003-07-31 2012-03-06 Novartis Vaccines And Diagnostics, S.R.L. Immunogenic compositions for Streptococcus pyogenes
US7709009B2 (en) 2003-07-31 2010-05-04 Novartis Vaccines And Diagnostics, Srl Immunogenic compositions for streptococcus pyogenes
US9056912B2 (en) 2003-07-31 2015-06-16 Novartis Vaccines And Diagnostics, Srl Immunogenic compositions for Streptococcus pyogenes
US8529913B2 (en) 2003-07-31 2013-09-10 Novartis Vaccines And Diagnostics, Srl Immunogenic compositions for Streptococcus pyogenes
US8945589B2 (en) 2003-09-15 2015-02-03 Novartis Vaccines And Diagnostics, Srl Immunogenic compositions for Streptococcus agalactiae
US8778358B2 (en) 2004-07-29 2014-07-15 Novartis Vaccines And Diagnostics, Inc. Immunogenic compositions for gram positive bacteria such as Streptococcus agalactiae
US7838010B2 (en) 2004-10-08 2010-11-23 Novartis Vaccines And Diagnostics S.R.L. Immunogenic and therapeutic compositions for Streptococcus pyogenes
US8858957B2 (en) 2007-09-12 2014-10-14 Novartis Ag GAS57 mutant antigens and GAS57 antibodies
US8399651B2 (en) 2007-09-12 2013-03-19 Novartis Ag Nucleic acids encoding GAS57 mutant antigens
US8287885B2 (en) 2007-09-12 2012-10-16 Novartis Ag GAS57 mutant antigens and GAS57 antibodies
US9102741B2 (en) 2007-09-12 2015-08-11 Novartis Ag GAS57 mutant antigens and GAS57 antibodies
US8409589B2 (en) 2007-12-21 2013-04-02 Novartis Ag Mutant forms of streptolysin O
US8039005B2 (en) 2007-12-21 2011-10-18 Novartis Ag Mutant forms of streptolysin O
US7731978B2 (en) 2007-12-21 2010-06-08 Novartis Ag Mutant forms of streptolysin O

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