EP1766008A1 - Dosage de criblage phage - Google Patents

Dosage de criblage phage

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Publication number
EP1766008A1
EP1766008A1 EP05759229A EP05759229A EP1766008A1 EP 1766008 A1 EP1766008 A1 EP 1766008A1 EP 05759229 A EP05759229 A EP 05759229A EP 05759229 A EP05759229 A EP 05759229A EP 1766008 A1 EP1766008 A1 EP 1766008A1
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European Patent Office
Prior art keywords
polypeptide
phage
vaccine
nucleic acid
mycoplasma
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EP05759229A
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German (de)
English (en)
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John Bernard Moredun Research Institute MARCH
Jason Clark
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Moredun Research Institute
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Moredun Research Institute
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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
    • 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
    • C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/00041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/00043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a method for identifying an agent for potential use in a vaccine.
  • the present invention also provides agents identified by this method for use in a vaccine.
  • the present further describes peptides of the pathogen Mycoplasma for use as a vaccine to treat or prevent diseases caused by Mycoplasma species, such as, Contagious bovine pleuropneumonia (CBPP).
  • CBPP Contagious bovine pleuropneumonia
  • Bacteriophages are viruses of bacteria, which bind to specific receptors on their host cell and inject their DNA, which then either incorporates into the host genome (lysogeny) or is used to synthesise new phage particles which either extrude from the membrane without disrupting the cell (as with filamentous phage) or lyse the cell and release all the phage produced in the cell in a single burst (lytic growth).
  • Phages can be single stranded DNA (such as the filamentous phages Ml 3, fd and fl), double stranded DNA (e.g. the T series phages and ⁇ phages), double stranded RNA (e.g. f6) or single stranded RNA (e.g. MS2 and Q ⁇ ).
  • DNA vaccines are efficient at stimulating the cellular arms of the immune system (in particular CD 8+ cytolytic T-cell responses) and are useful at overcoming maternal antibody and for producing conformational ⁇ active epitopes. This procedure is referred to here as Phage DNA Vaccination, and the basic principle is shown in Figure 5.
  • MmmSC Mycoplasma mycoides subsp. mycoides small colony type
  • CBPP contagious bovine pleuropneumonia
  • Recent vaccination programmes based on freeze-dried cultures of the causative organism (MmmSC) have been unable to halt the re-emergence of the disease seen in recent years.
  • Current OIE-recommended (O.I.E., 1996) vaccines freeze-dried broth cultures of live attenuated MmmSC strains Ti44 or T 1 SR
  • T 1 SR live attenuated MmmSC strains Ti44 or T 1 SR
  • Vaccination can cause severe, sometimes fatal, reactions, leading to evasion by farmers.
  • Other problems include the requirement for refrigeration in the field and the possibility of reversion to virulence (Rweyemamu et al., 1995, Hubschle 2003).
  • current live vaccines can be made considerably more stable and efficacious by following modifications to culture media and reconstitution procedures, it is apparent that a stable inexpensive and efficacious CBPP vaccine would offer many advantages, particularly if it offered a defined and recognisable antigenic profile, since this would allow the opportunity to differentiate between vaccinated and infected cattle, and would therefore be useful for control in Europe should the need arise.
  • Inactivated CBPP vaccines have been field tested, but results have generally been inconclusive, and detailed efficacy studies have not been performed (Garba et al., 1986). Successful immunization with an inactivated CBPP vaccine has been reported (Gray et al., 1986), but only when an extreme form of vaccination was used (two large doses of MmmSC in Freund's complete adjuvant). More recently, immunostimulating complex (ISCOM) protein sub-unit vaccines have been developed. Encouraging antibody responses were observed in vaccinated mice and cattle (including the induction of growth inhibiting antibodies); protection against CBPP field infection, however, has not yet been demonstrated (Abusugra et al., 1997; Abusugra and Morein, 1999).
  • ISCOM immunostimulating complex
  • a major problem in designing an effective CBPP vaccine is the lack of understanding of the basic mechanism of immunity in cattle, although the apparent lack of correlation between antibody response and protection suggests that protection may be cell mediated, and the poor results with inactivated vaccines suggests that conformational epitopes may be important for protective efficacy.
  • the present invention is based on the inventor's discovery of a technique using bacteriophage to screen and identify immunogenic polypeptides which may be suitable vaccine candidates.
  • the present inventors have also identified vaccine candidates using the assay of the present invention for the prevention of contagious bovine pleuropneumonia (CBPP), a disease of cattle.
  • CBPP contagious bovine pleuropneumonia
  • a method for identifying a nucleic acid sequence or polypeptide for potential use in a vaccine comprising: a) providing a genetically modified phage comprising nucleic acid from a disease causing agent or diseased cell; b) expressing a polypeptide(s) encoded by said nucleic acid sequence; c) contacting said expressed polypeptide(s) with antiserum from an animal which has previously been infected with said disease causing agent and/or has said diseased cell(s); and d) identifying said polypeptide(s) which specifically reacts with said antiserum. It is understood that the invention may also be used to identify a phage comprising said nucleic acid and/or nucleic acid capable of expressing said polypeptide.
  • the phage is modified using standard molecular biology techniques so as to allow expression of said pathogen polypeptide(s) encoded by said pathogen nucleic acid sequence(s) by a phage infected host cell (see for example Sambrook et al, 2001).
  • the phage may be designed using techniques known to those skilled in the art, to be capable of expressing the polypeptide(s) in a cell to be infected by said phage and/or to express the polypeptide(s) on the surface of the phage particle, by a technique known as phage display.
  • nucleic acid such as genomic nucleic acid genome fragments may be cloned into the phage such that it is capable of being expressed by the phage once the phage transfects a target host cell which in the case of lambda phage is Escherichia coli.
  • said phage may be engineered to express more than one polypeptide or antigen, as encoded by said nucleic acid.
  • a known expression vector is used to express pathogen polypeptide(s) in said phage.
  • the cloning vehicles ⁇ -gtl 1 or ⁇ ZAP express may be used.
  • the polypeptide(s) encoded by said nucleic acid sequence are generally expressed by transfecting said phage into a host cell appropriate for each particular phage, so that the host cell's "machinery" will cause expression of the protein.
  • the phage will comprise appropriate transcription/translation signals for the given host cell, so that the cloned nucleic acid may be transcribed and the polypeptide thereafter translated and expressed.
  • Antiserum may be obtained from an animal which has previously been infected with the disease causing agent (live or dead), or comprises diseased cells, such as cancer cells, which may express polypeptides which a host would raise a humoral (antibody) immune response thereto.
  • the antiserum will therefore comprise antibodies specifically reactive against antigens from said disease causing agent or diseased cells. Nevertheless, prior to carrying out the method according to the present invention, it would not be known to which antigens such antibodies have been raised against.
  • the animal may for example be a mammal such as a human, cow, sheep, pig, goat, rabbit, mouse or rat.
  • the animal may be a bird, such as a chicken, duck or turkey, or a fish, such as salmon, sea bass, trout or the like.
  • the antiserum is brought into contact with said expressed polypeptides.
  • This may be achieved for example by first immobilising the phage to a substrate, such as nitrocellulose and lysing, if appropriate, the phage, in order to allow the polypeptide(s) to be exposed to the antiserum.
  • the polypeptide may then be exposed or contacted with the antiserum, by for example washing the immobilised polypeptide(s) with a solution of neat or diluted antiserum.
  • antibodies will bind to the polypeptide and this may be detected by, for example Western blotting techniques known in the art (see Sambrook et al, 2001) or, for example by way of a further labelled antibody, using techniques well known to those skilled in the art, such as ELISA,or radioimmunoassay (Sambrook et al, 2001).
  • a phage clone from which a positively reacting polypeptide has been identified may be subjected to a further round of screening to ensure/confirm the positive result.
  • a phage identified by the method according to the present invention containing nucleic acid and encoding an immunogenic polypeptide may be used directly to vaccinate a host animal (e.g. mammals, birds, fish, reptiles or amphibians) as described for example in WO/02076498.
  • the polypeptide may be expressed by said phage, or nucleic acid encoding said polypeptide excised and cloned into another suitable vector and expressed thereby, such that the polypeptide may be purified and subsequently used in the provision of a so-called sub-unit vaccine.
  • the phage of the present invention preferably further comprises appropriate transcription/translation regulators such as promoters, enhancers, terminators and/or the like for controlling expression of polypeptides in eukaryotic host cells.
  • the promoter may be a eukaryotic promoter such as CMV, SV40, thymidine kinase, RSV promoter or the like.
  • the promoter may be a constitutive promoter.
  • controllable promoters known to those of skill in the art may also be used.
  • constructs may be designed which comprise the exogenous (pathogen) nucleic acid under control of a constitutive promoter and a controllable promoter by way of cloning into an expression vector of choice.
  • nucleic acid may refer to ribo- or deoxy ribo- nucleic acid (RNA or DNA, respectively).
  • genomic DNA may be cloned into a vector of choice to be expressed in said phage.
  • a random or semi-random genome library of the genome of a disease causing agent or diseased cell of choice may be cloned into a phage vector under a promoter of choice. In this manner the nucleic acid of many, if not a majority of the polypeptides expressed by said disease causing agent or diseased cell may be cloned in order to allow the screening of suitable immunogenic polypeptides.
  • polypeptide refers to a chain or sequence of amino acids displaying an antigenic activity and does not refer to a specific length of the product as such.
  • the polypeptide if required, can be modified in vivo and/or in vitro, for example by glycosylation, amidation, carboxylation, phosphorylation and/or post translational cleavage, thus inter alia, peptides, oligo -peptides, proteins and fusion proteins are encompassed thereby.
  • a modified polypeptide should retain physiological function i.e. be capable of eliciting an immune response.
  • phage according to the present invention includes single stranded DNA phages (such as the filamentous phages Ml 3, fd and fl), double stranded DNA phages (e.g. the T series phages and ⁇ phages), double stranded RNA phages (e.g. f6) or single stranded RNA phages (e.g. MS2 and Q ⁇ ).
  • double stranded DNA phage ⁇ is used, as such phage have the additional advantages of stability under high ambient conditions, ease and cheapness of production, together with providing a highly immunological signal (against the phage coat) which provides an easily assayed marker with which to check for vaccination.
  • an polypeptide(s) identified by a method according to the present invention for manufacture of a vaccine for prophylactic or thereapeutic administration.
  • the present invention is applicable to the preparation of a vaccine for practically any disease, such as those caused by a pathogen, providing that a suitable immuno-protective response can be raised to a protein or proteins of an infectious agent (pathogen).
  • pathogen encompasses virus, bacteria, fungi, yeast, protozoa, helminths, insecta, and transmissible spongiform encephalopathies, for example.
  • the pathogens for which the agents of the present invention would be applicable to, include pathogen-causing infectious diseases of both humans and animals. Lists of suitable diseases are well known to those versed in the art and examples are to be found in the O.I.E. Manual of Standards and Diagnostic Tests 3rd Ed., OIE, Paris 1996, Topley & Wilson's Principles of Bacteriology, Virology and Immunity 8th Ed., Eds. Parker M.T.
  • agents identified by the present invention for use as a vaccine could be used to elicit an immune response against cancer cells by means of the expression of a cancer cell specific antigen as the immunogenic polypeptide.
  • the present invention further provides novel modified phage expressing peptides of the pathogen Mycoplasma, eg., Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) for use as a vaccine to treat or prevent Contagious bovine pleuropneumonia (CBPP).
  • MmmSC Mycoplasma mycoides subsp. mycoides small colony type
  • CBPP Contagious bovine pleuropneumonia
  • a modified phage(s) capable of expressing a polypeptide encoded by a nucleotide sequence A8, or functional fragment, homologue or derivative thereof for use in a vaccine for the prevention of disease caused by the pathogen MmmSC, wherein A8 comprises the nucleotide sequence as shown in Figure 6c.
  • a modified phage(s) capable of expressing a polypeptide encoded by a nucleotide sequence B 1 , or functional fragment, homologue or derivative thereof for use in a vaccine for the prevention of disease caused by the pathogen MmmSC, wherein Bl is the nucleotide sequence as shown in Figure 7a.
  • a modified phage(s) capable of expressing a polypeptide or functional fragment, homologue or derivative thereof for use in a vaccine for the prevention of disease caused by the pathogen MmmSC, wherein said polypeptide is at least one prolipoprotein as shown in Figures 6 (d) or 7 (d) or functional fragment, homologue or derivative therof.
  • nucleic acid or polypeptide sequences with a similar function. That is, nucleic acid sequences or polypeptides capable of effecting a suitable immuno-protective response to an infectious agent (pathogen).
  • homologue relates to nucleic acid or polypeptide sequences sharing at least 25%, 50%, particularly 60, 70 and 80%, and especially 90 and 95% identity to the nucleic acid sequences as shown in Figures 6(c) or 7(a) or prolipoprotein amino acid sequences as shown in Figures 6(d) or 7(d).
  • % sequence identity may be determined when the alignment or comparison is conducted by a computer homology program or search algorithm known in the art.
  • useful computer homology programs include the following: Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov) [Altschul et al., (1990). The BLAST Algorithm. J. MoI.
  • the BLASTN program compares a nucleotide query sequence against a nucleotide sequence database.
  • the BLASTX program compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.
  • the TBLASTN program compares a protein query sequence against a nucleotide sequence database translated in all six reading frames (both strands).
  • the TBLASTX program compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • Smith- Waterman database: European Bioinformatics Institute www, ebi. ac. uk/bic_ s w/
  • FASTA (see [Pearson et al., (1988). Proc. Natl. Acad. Sci. USA, 85, 2444-2448.] is a heuristic approximation to the Smith-Waterman algorithm.
  • Smith-Waterman and FASTA algorithms see [Nicholas et al., (1998). A tutorial on searching sequence databases and sequence scoring methods. www.psc.edu.] and references cited therein.
  • the present invention further describes use of the phage expressing polypeptides encoding nucleotide sequences A8 and/or Bl of the present invention and/or lipoproteins as shown in Figure 6d) and/or 7d) as described herein for the manufacture of vaccines for the prevention or treatment of Contagious bovine pleuropneumonia (CBPP) caused by the pathogen Mycoplasma mycoides subsp. mycoides small colony type (MmmSC, as well as a method of prophylactically treating CBPP using a nucleic acid and/or polypeptide(s) encoding a prolipoprotein or lipoprotein as shown in Fugures 6d and/or 7d or functional fragment, homologue or derivative thereof.
  • CBPP Contagious bovine pleuropneumonia
  • MmmSC mycoides small colony type
  • the vaccine potential of such phage ⁇ constructs carrying MmmSC nucleic acid sequences A8 and Bl are assessed herein, showing antibody responses and cellular proliferation in vaccinated mice following infection with MmmSC, and additionally measuring the length of the mycoplasmaemia in infected animals by means of a mouse infection technique (Smith 1965, 1969, 1971a,b).
  • the vaccine can also comprise an adjuvant.
  • Adjuvants in general comprise substances that boost the immune response of the host in a non-specific manner. A number of different adjuvants are known in the art.
  • adjuvants may include Freund's Complete adjuvant, Freund's Incomplete adjuvant, liposomes, and niosomes as described in WO90/11092, mineral and non-mineral oil-based water-in-oil emulsion adjuvants, cytokines, short immunostimulatory polynucleotide sequences, for example in plasmid DNA containing CpG dinucleotides such as those described by Sato Y.et al. (1996); and Krieg A.M. (1996).
  • Such adjuvants may in fact be expressed by the phage which is capable of expressing a polypeptide(s) identified by the method of the present invention.
  • encapsulators comprising agents capable of forming microspheres (1-1 O ⁇ m) such as poly(lactide-coglycolide), facilitating agents which are capable of interacting with polynucleotides such that the said polynucleotide is protected from degradation and which agents facilitate entry of polynucleotides such as DNA into cells.
  • Suitable facilitating agents include cationic lipid vectors such as: 1 ,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER), N-[l-(2,3-dioleoyloxy) ⁇ ro ⁇ yl]-N,N,N-trimethylammoniummethylsulfate (DOTAP), N-[l-(2,3-dioleoyloxy)propyl)]-N,N,N-trimethylammonium chloride (DOTMA), (N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxylethyl)-2,3-dioleoyloxy-l, 4-butanediammonium iodide, bupivacaine-HCl, non-ionic polyoxypropylene/polyoxyethylene block copolymers, polyvinyl polymers and the like.
  • Such cationic lipid vectors can be combined with further agents
  • the mode of administration of the vaccine of the invention may be by any suitable route that delivers a suitable amount of the nucleic acid construct, or vector of the invention to the subject.
  • the vaccine is preferably administered parenterally via the intramuscular or deep subcutaneous routes.
  • Other modes of administration may also be employed, where desired, such as oral administration or via other parenteral routes, i.e., intradermally, intranasally, or intravenously.
  • the vaccine will usually be presented as a pharmaceutical formulation including a carrier or excipient, for example an injectable carrier such as saline or a pyro genie water.
  • a carrier or excipient for example an injectable carrier such as saline or a pyro genie water.
  • the formulation may be prepared by conventional means. It will be understood, however, that the specific dose level for any ' particular recipient animal will depend upon a variety of factors including age, general health, and sex; the time of administration; the route of administration; synergistic effects with any other drugs being administered; and the degree of protection being sought. Of course, the administration can be repeated at suitable intervals if necessary.
  • the present invention further provides a vaccine formulation comprising nucleic acid and/or polypeptide sequences as described herein or identified by the methods described herein.
  • the formulation may further comprise a suitable carrier therefore.
  • Fig.l Number of mice in each vaccinated group exhibiting mycoplasmaemia after intraperitoneal challenge
  • Fig. 2 Anti-Mycoplasma immune responses (measured by ELISA) in BALB/c mice before and after vaccination and pre and post challenge. Error bars are standard deviations of the mean OD 492 nm for each mouse on each separate bleed. Key:
  • DOTAP N- [ 1 -(2,3 -dioleoyloxy)propyl] -N,N,N-trimethylammoniummethylsulfate
  • DOTMA N-[l-(2,3-dioleoyloxy)
  • Such cationic lipid vectors can be combined with further agents such as L- dioleoyl phosphatidyl ethanolamine (DOPE) to form multilamellar vesicles such as liposomes.
  • DOPE L- dioleoyl phosphatidyl ethanolamine
  • the mode of administration of the vaccine of the invention may be by any suitable route that delivers a suitable amount of the nucleic acid construct, ,or vector of the invention to the subject.
  • the vaccine is preferably administered parenterally via the intramuscular or deep subcutaneous routes.
  • Other modes of administration may also be employed, where desired, such as oral administration or via other parenteral routes, i.e., intradermally, intranasally, or intravenously.
  • the vaccine will usually be presented as a pharmaceutical formulation including a carrier or excipient, for example an injectable carrier such as saline or a pyrogenic water.
  • a carrier or excipient for example an injectable carrier such as saline or a pyrogenic water.
  • the formulation may be prepared by conventional means. It will be understood, however, that the specific dose level for any particular recipient animal will depend upon a variety of factors including age, general health, and sex; the time of administration; the route of administration; synergistic effects with any other drugs being administered; and the degree of protection being sought. Of course, the administration can be repeated at suitable intervals if necessary.
  • the present invention further provides a vaccine formulation comprising nucleic acid and/or polypeptide sequences as described herein or identified by the methods described herein.
  • the formulation may further comprise a suitable carrier therefore.
  • Fig.l Number of mice in each vaccinated group exhibiting mycoplasmaemia after intraperitoneal challenge
  • mice before and after vaccination and pre and post challenge. Error bars are standard deviations of the mean OD 492 nm f° r each mouse on each separate bleed. Key: QJ pre-vaccination, [J post-vaccination, ⁇ post-challenge. Group 1 : Lambda A8; Group 2: Lambda B 1 ; Group 3 : Lambda gtl 1.
  • Fig. 4 a) Immunoblot of mice 4 and 8 from group 1 ( ⁇ A8) and 3 ( ⁇ gtl 1) against A8 proteins. Rabbit anti-serum used for positive control, b) Immunoblot of mice 4 and 8 from group 2 ( ⁇ Bl) and 3 ( ⁇ gtl 1) against Bl proteins. Rabbit anti ⁇ serum used for positive control.
  • FIG. 5 Schematic diagram outlining basic principle of phage DNA vaccination. A strong immune response to the "vaccine” proteins is seen circa 4 weeks post vaccination.
  • MmmSC challenge strain N6 (March et al., 2000a) was obtained from Willie Amanfu, National Veterinary Laboratory, Gaborone, Botswana.
  • Mycoplasmas were grown in Gourlay's broth or agar (1%) medium (modified Newings tryptose broth; Gourlay, 1964), containing thallous acetate 0.04% and ampicillin 0.4 mg/ml unless otherwise stated, at 37°C, in an atmosphere containing CO 2 5%.
  • mycoplasmas were grown to mid-logarithmic phase, concentrated 10-fold by centrifugation and re-suspended in fresh medium immediately before challenge to give a titre of 10 10 organisms/ml.
  • Rabbit hyperimmune serum (R55) against MmmSC strain N6 was produced by two subcutaneous injections of mycoplasma (gluteraldehyde inactivated, followed by quenching with glycine). Sterility was confirmed by the absence of growth after streaking on Gourlay's agar plates.
  • the final vaccine constitution was 5.6 mg /ml protein in phosphate-buffered saline (PBS) containing gluteraldehyde 0.01%, 0.01M glycine, thimerosal 0.01% in an equal volume of oil adjuvant (Montanide ISA50; Seppic, 75, quai d'Orsay, 75321 Paris, France), followed by one intravenous injection of an aqueous suspension. Injections were given every three weeks.
  • the phagemids excised from two strongly immunodominant clones have been named ⁇ -A8 and ⁇ -Bl . Sequence using internal priming sites
  • the commonly used cloning vehicle ⁇ -gtl 1 was used throughout.
  • a single colony of E. coli strain Y 1090 was added to 100ml NZCYM broth (Fluka, Biochemika, Switzerland) and grown overnight at 37°C, with vigorous agitation. The cell concentration was calculated, assuming 1 OD 6O0 — 8x10 cells/ml.
  • Four aliquots of the E. coli suspension, each containing 10 10 cells were centrifuged at 3300g for 5 minutes at room temperature (19 0 C).
  • SM phage
  • SM phage
  • l ⁇ l of ⁇ -gtl 1/ ⁇ -HBsAg bacteriophage was added to each bacterial suspension and incubated at 37°C, with intermittent shaking for 20 minutes.
  • Each infected aliquot was then added to pre- warmed (37°) flasks of 500ml NZCYM broth and left at 37°C, with vigorous agitation overnight.
  • E. coli was diluted 1/10 in Luria broth (Sigma Ltd. UK), (containing 1OmM Magnesium sulphate and 0.2% Maltose (Sigma Ltd. UK)) and incubated overnight at 37°C, with vigorous shaking. This overnight culture was sub-cultured 1/10 in Luria broth (supplemented with 1OmM Magnesium sulphate and 0.2% Maltose) and incubated at 37 0 C, with vigorous shaking, for 3 hours. The culture was then centrifuged at 3300g- for 5 minutes at room temperature and the pellet re-suspended in 1OmM Magnesium sulphate to an OD 600 of 0.6.
  • Appropriate serial dilutions of the purified ⁇ -gtll bacteriophage suspension were prepared in SM buffer. 5 ⁇ l of each dilution was diluted in 195 ⁇ l of the E. coli culture and incubated at 37°C for 15 minutes. Each bacteriophage/E. coli mixture was added to 2.5-3ml NZCYM top agar (0.6% purified agar (Oxoid Ltd. England), mixed gently and poured onto Luria broth agar (1% purified agar) plates. The plates were left to set at room temperature for 5 minutes, then incubated at 37°C overnight. The plaques present on each plate were counted and the ⁇ -gtl 1 bacteriophage concentration per ml calculated.
  • mice were strain BALB/c, female and 10 weeks of age, eight per group. All mice were injected intramuscularly on week 0, 4 and 8 with 50 ⁇ l SM saline buffer containing 5x10 8 bacteriophage particles. The 3 groups used were as follows: (1) ⁇ - A8; (2) ⁇ -Bl and (3) negative control, ⁇ gtl 1. Mice were tail bled pre-vaccination at week 0, and post vaccination at weeks 4 and 8, then again on days 0, 2 and 3 post- challenge at week 13. Finally, all mice were humanely killed and bled out on day 4 post-challenge and spleens obtained for stimulation assays.
  • mice All three groups of BALB/c mice (8 mice per group) were challenged by the intraperitoneal injection of 0.5ml (10 10 organisms) of MmmSC strain N6, grown in Gourlay's broth without thallous acetate or ampicillin. (Previous research had indicated that this strain produced a particularly high degree of mycoplasmaemia in mice [March and Brodlie 2000]. All mice were held in negative pressure isolators (one group per isolator) to satisfy current disease security requirements. A drop of blood from tail-tip bleeds taken on days 2, 3 and 4 after challenge, was placed in 3 ml of liquid growth medium before being diluted 1 in 10 in the same medium and incubated at 37 0 C for 7 days.
  • Antibody titres against bacteriophage ⁇ coat proteins and whole MmmSC antigens in mouse sera were measured by indirect ELISA.
  • Microtitre plates (96-well;Greiner Ltd, Brunei Way, Stonehouse, Gloucestershire) were coated overnight at 4 0 C in 0.05M sodium carbonate buffer at pH 9.2 with either whole sonicated MmmSC (N6) at a concentration of 5 ⁇ g/ml or 10 9 bacteriophage (50ng) per well.
  • the spleens were harvested from each mouse and combined for each of the four groups (8 spleens per group).
  • the splenocytes were recovered from the spleens via injection with 2ml mouse wash medium (MWM, containing Hanks Balanced salt Solution, 2% Foetal Bovine Serum (FBS), 2% Penicillin/Streptomycin, 0.25% Nystatin and 0.2% gentamycin (Lloyds Chemist PLC, UK)) and light bashing with sterile microscope slides.
  • This cell suspension was then filtered through lens tissue (Whatman International Ltd, England), before being centrifuged for 5 minutes at 1500rpm at 16 0 C.
  • the pellet was re-suspended in lysis buffer (nine parts 0.16M ammonium chloride (Sigma Ltd., UK) and one part 0.17M Tris (Sigma Ltd., UK) at pH 7.65). After 10 minutes at 4°C, lysis was stopped by the addition of MWM. Following centrifugation at 1500rpm for 5 minutes at 16°C, the pellet was re-suspended in complete RPMI (RPMI 1640, 10% FBS, 2% glutamine, 1% penicillin/streptomycin, 1% gentamycin, 0.5% 2- mercaptoethanol, 2.5% Sodium Bicarbonate (8%) and 1.2% IM Hepes (Sigma Ltd, UK)).
  • lysis buffer no parts 0.16M ammonium chloride (Sigma Ltd., UK) and one part 0.17M Tris (Sigma Ltd., UK) at pH 7.65. After 10 minutes at 4°C, lysis was stopped by the addition of MWM. Following centrifugation at 1500rpm for 5 minutes at 16°C, the
  • This cell suspension was centrifuged again (as above), and the pellet re- suspended in ImI complete RPMI.
  • a 1 :10 dilution of cell suspension in 0.1% nigrosin (Sigma Ltd., UK) in phosphate buffered saline was prepared for a viability count, using a modified Neubauer counting chamber. Cell concentrations were adjusted to lxl ⁇ 6 cells/ml in complete RPMI. Using sterile 96-well tissue culture plates, lOO ⁇ l of the viable splenocytes were seeded onto lOO ⁇ l volumes of sterile diluted antigens in triplicate.
  • Mycoplasma live and dead (heat killed) were diluted in complete RPMI, to give a concentration of 10 cells/ml for each. Also used was ⁇ gtl 1, diluted in complete RPMI to give concentrations of 5 and 2.5 ⁇ g/ml.
  • As a positive control cells were cultured with concavalin A (Sigma Ltd., UK) at a concentration of 2.5 ⁇ g/ml. Plates were incubated in a humid (5% CO2) environment at 37 0 C for 96 hours. After 4 days incubation, the cell cultures were pulsed for 18 hours with 18.25KBq (l ⁇ Ci) [3H] thymidine (Amersham Biosciences UK), per well.
  • Clones A8 and Bl were prepared in SDS-PAGE sample buffer (0.1M Tris- HCl pH 6.8, 40% (v/v) glycerol, 4% (w/v) SDS, 0.25% bromophenol blue, 2% ⁇ - mercaptoethanol). The samples were boiled for 5 minutes and separated by SDS- PAGE using a 12% homogeneous polyacrylamide gel followed by electrophoresis transfer to nitrocellulose membranes (Hybond C-pure, Amersham, UK) using standard techniques. Rainbow markers (Amersham, UK) were run alongside the samples.
  • SDS-PAGE sample buffer 0.1M Tris- HCl pH 6.8, 40% (v/v) glycerol, 4% (w/v) SDS, 0.25% bromophenol blue, 2% ⁇ - mercaptoethanol.
  • the samples were boiled for 5 minutes and separated by SDS- PAGE using a 12% homogeneous polyacrylamide gel followed by electrophoresis transfer to nitrocellulose
  • Efficiency of transfer was estimated by staining the membranes with 0.1% Ponceau Red (Sigma) in 1% acetic acid solution, followed by destain in 1% acetic acid. The membrane was then rinsed twice in PBST (PBS + 0.5% Tween 20), and blocked in 5% (w/v) dry skimmed milk in PBST for 30 minutes before primary specific mouse polyclonal antibody was added at a dilution of 1:100. Biorad Mini- Protean II Multi-Screen blotting apparatus was used to allow multiple primary serum samples to be tested against the same protein.
  • the membrane was subsequently incubated for 1 hour at room temperature, rinsed five times in PBST, then anti -mouse horse radish peroxidase-labelled secondary antibody (DAKO) was added diluted in 5% (w/v) dry skimmed milk in PBST as per manufacturers instructions, and the membrane incubated for a further hour at room temperature. Three 5 minute washes in PBST were performed, before incubation of the membrane in O.lmg/ml diaminobenzidine (Sigma) in PBST containing 0.1ml of 30% H 2 O 2 per 100ml of substrate solution.
  • DAKO anti -mouse horse radish peroxidase-labelled secondary antibody
  • Table Ia, b and c show the results of challenge (week 13) with Mycoplasma.
  • mice The whole MmmSC immune responses before and after vaccination and following challenge, for the three different mouse groups are shown in Fig. 2.
  • Five out of eight mice in group 1 show a higher response (OD value) against whole Mycoplasma in the. post vaccination sera compared to that of the pre-vaccination sera. This was most apparent in mice 2 and 5, where an OD value of > 1.5 is seen.
  • mice In group 2 mice (vaccinated with ⁇ -Bl construct) only two out of the eight mice gave a higher response in the post- vaccination bleed compared to pre-vaccination bleed.
  • mice anti-sera from groups I 5 2 and 3 final bleeds against A8 and Bl proteins.
  • group 1 mouse 4 and 8, vaccinated with the ⁇ -A8 construct
  • group 3 serum mouse 4, vaccinated with ⁇ gtl 1
  • mice sera from group 2 mouse 4 and 8, vaccinated with the ⁇ -Bl construct
  • mice Sera from both mice showed two distinct positive bands at ⁇ 30kD and ⁇ 60kD, compared with that of group 3 mouse (mouse 4, vaccinated with ⁇ gtl I) 5 which had no visible bands of these sizes (figure 3b)).
  • group 3 mouse mouse 4, vaccinated with ⁇ gtl I
  • a mouse infection technique (Smith, 1965, 1969, 1971a,b) was used to test the vaccines.
  • a protracted mycoplasmaemia could be produced in laboratory mice by intraperitoneal inoculation with approximately 10 10 viable organisms in growth medium.
  • vaccination produced a protective immune response, demonstrated by a reduction in the mycoplasmaemia produced by challenge.
  • mice vaccinated intravenously with heat-killed MmmSC were completely protected from mycoplasmaemia after challenge with live organisms.
  • serum from these vaccinated mice (0.25 ml) was transferred to unvaccinated mice, they were not protected upon challenge with live organisms; however, a dose of 1 ml of undiluted serum per mouse was slightly protective.
  • the same volume of a 1 in 50 dilution of serum taken from rabbits after recovery from infection with MmmSC protected mice from mycoplasmaemia.
  • the rabbit antiserum was much more protective than the mouse antiserum.
  • mice When cattle, rabbits and mice were immunized subcutaneously with heat-killed MmmSC in adjuvant, the mouse-protective effective of bovine and rabbit antisera was high; in contrast, however, the protection of mice against challenge was slight or nil (Hooker et al., 1980). These observations are consistent with findings that rabbit serum R54 inhibited MmmSC growth but mouse serum did not.
  • ISCOM is an efficient mucosal delivery system for Mycoplasma mycoides subsp. mycoides (MmmSC) antigens inducing high mucosal and systemic antibody responses. FEMS Immunology and Medical Microbiology, 23, 5-12.

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Abstract

L'invention concerne un procédé destiné à identifier un agent pour une utilisation potentielle dans un vaccin. Cette invention concerne également des agents identifiés par ce procédé en vue d'une utilisation dans un vaccin. Elle décrit des peptides de Mycoplasma pathogène pour une utilisation en tant que vaccin dans le traitement ou la prévention de maladies entraînées par des espèces de Mycoplasma, notamment, la pleuropneumonie contagieuse bovine (CBPP).
EP05759229A 2004-07-09 2005-07-11 Dosage de criblage phage Withdrawn EP1766008A1 (fr)

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CN109970844A (zh) * 2019-03-14 2019-07-05 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 丝状支原体丝状亚种p35脂蛋白及其在诊断牛传染性胸膜肺炎中的应用

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CA2993076A1 (fr) * 2015-07-22 2017-01-26 University Of Saskatchewan Vaccins contre mycoplasma et leurs utilisations
CN110607284A (zh) * 2019-10-23 2019-12-24 青岛农业大学 大肠杆菌噬菌体vB_EcoM_swi3及其应用

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CN109970844A (zh) * 2019-03-14 2019-07-05 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 丝状支原体丝状亚种p35脂蛋白及其在诊断牛传染性胸膜肺炎中的应用
CN109970844B (zh) * 2019-03-14 2022-08-23 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 丝状支原体丝状亚种p35脂蛋白及其在诊断牛传染性胸膜肺炎中的应用

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