Patent Application
20080226661
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).
Bacteriophages (or phages) 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 M13, fd and fl), double stranded DNA (e.g. the T series phages and λ phages), double stranded RNA (e.g. f6) or single stranded RNA (e.g. MS2 and Qβ).
Recently, whole bacteriophage λ particles have been described as efficient delivery vehicles for administration of genetic or ‘DNA’ vaccines (rather than purified DNA as a vaccine) (Clark & March, 2004). This is in contrast to the procedure of DNA vaccination, wherein a vaccine gene is cloned into a eukaryotic expression cassette, consisting of a promoter and an upstream processing region, in a plasmid. The whole plasmid (“naked DNA”) is injected and the host raises an immune response to vaccine protein synthesised in vivo. DNA vaccination is particularly effective at inducing cytotoxic T-lymphocyte (CTL) responses (Leitner et al, 1999). due to intracellular expression of protein. However, in the phage DNA vaccination system, the gene encoding the vaccine antigen, under the control of a suitable eukaryotic promoter, is cloned into the bacteriophage genome and subsequently, the whole bacteriophage particle is used to inoculate the host for the purposes of raising a specific immune response against said vaccine antigen. In general, DNA vaccines are efficient at stimulating the cellular arms of the immune system (in particular CD8+ cytolytic T-cell responses) and are useful at overcoming maternal antibody and for producing conformationally active epitopes. This procedure is referred to here as Phage DNA Vaccination, and the basic principle is shown in
There is, however, a constant need to identify and develop vaccines of defined antigenic profile for inoculation of subjects against known or less well-characterised/unknown pathogens, such as the causative pathogen of Severe Acute Respiratory Syndrome (SARS,) for example.
One such exemplary pathogen is Mycoplasma mycoides subsp. mycoides small colony type (MmmSC), the causative agent of contagious bovine pleuropneumonia (CBPP), an economically important disease of cattle currently affecting much of Africa and parts of southern Europe. 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 T144 or T1 SR) exhibit relatively poor efficacy, and repeated vaccination is necessary to maintain protection (Thiaucourt et al., 2000). 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). Although it has been suggested that 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). 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.
It is amongst the objects of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages.
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.
In a first aspect of the present invention there is provided a method for identifying a nucleic acid sequence or polypeptide for potential use in a vaccine, said method comprising:
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.
Typically, 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.
Typically 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.
Moreover, said phage may be engineered to express more than one polypeptide or antigen, as encoded by said nucleic acid. Typically, a known expression vector is used to express pathogen polypeptide(s) in said phage. For example, the cloning vehicles λ-gt11 or λ 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. Typically 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. Alternatively 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.
If there are any antibodies present in the antiserum, which specifically react with an expressed polypeptide, such 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 radio-immunoassay (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.
It is also a straight-forward task to isolate the phage clone capable of expressing a positively reactive polypeptide(s) and to sequence the nucleic acid of the phage, so as to identify the cloned nucleic acid from the disease causing agent or diseased cell and consequently the expressed polypeptide and associated nucleic acid to which the antiserum has reacted. This may thereafter be used to check available databases to ascertain the identity of the polypeptide/gene if required.
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. Alternatively, 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.
Optionally, 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. Typically the promoter may be a eukaryotic promoter such as CMV, SV40, thymidine kinase, RSV promoter or the like. Conveniently the promoter may be a constitutive promoter. However, controllable promoters known to those of skill in the art may also be used. For example 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. In this manner it may be possible to cause expression of the exogenous nucleic acid initially by way of the constitutive promoter and at a second time point by expression from the controllable promoter. This may result in a stronger immune response when following the techniques described in WO/0207/6498.
The term nucleic acid may refer to ribo- or deoxy ribo-nucleic acid (RNA or DNA, respectively). Conveniently, genomic DNA may be cloned into a vector of choice to be expressed in said phage. Preferably, 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.
In general, the term “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. Naturally the skilled addressee will appreciate that a modified polypeptide should retain physiological function i.e. be capable of eliciting an immune response.
The term phage according to the present invention includes single stranded DNA phages (such as the filamentous phages M13, fd and fl), double stranded DNA phages (e.g. the T series phages and λ phages), double stranded RNA phages (e.g. f6) or single stranded RNA phages (e.g. MS2 and Qβ). Preferably, double stranded DNA phage λ 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.
In a second aspect of tie present invention there is provided use of an polypeptide(s) identified by a method according to the present invention for manufacture of a vaccine for prophylactic or therapeutic administration.
It is to be appreciated that according to the second aspect, 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).
The term pathogen according to the present invention 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. and Collier L. H., Vol IV (Index), Edward Arnold, London 1990, The Zoonoses: Infections Transmitted from Animals to Man. Bell J. C. et al., Edward Arnold, London 1988 and Parasitology: The Biology of Animal Parasites 6th Ed. Noble E. R. et al., Lea & Febiger, Philadelphia, 1989. In addition, 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). Thus, in a yet further aspect of the present invention there is provided 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
In a yet further aspect of the present invention there is provided a modified phage(s) capable of expressing a polypeptide encoded by a nucleotide sequence B1, or functional fragment, homologue or derivative thereof for use in a vaccine for the prevention of disease caused by the pathogen MmmSC, wherein B1 is the nucleotide sequence as shown in
In a yet further aspect of the present invention there is provided 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
It should be understood that “functional fragment, homologue or derivative thereof” relates to 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).
Generally speaking, “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
The BLASTP program compares an amino acid query sequence against a protein sequence database.
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_sw/) [Smith-Waterman, (1981). J. Mol. Biol., 147, 195-197.] is a mathematically rigorous algorithm for sequence alignments.
FASTA (see [Pearson et al., (1988). Proc. Natl. Acad. Sci. USA, 85, 2444-2448.] is a heuristic approximation to the Smith-Waterman algorithm. For a general discussion of the procedure and benefits of the BLAST, 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 B1 of the present invention and/or lipoproteins as shown in
In a preferred presentation, 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. Examples of 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.
Further adjuvants of use in the invention include encapsulators comprising agents capable of forming microspheres (1-10 μm) 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:
Such cationic lipid vectors can be combined with further agents such as L-dioleoyl phosphatidyl ethanolamine (DOPE) to form multilamellar vesicles such as liposomes.
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. However, 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.
Generally, 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. 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. Optionally the formulation may further comprise a suitable carrier therefore.
The present invention will now be described by way of example and figures as follows:
pre-vaccination,
post-vaccination,
post-challenge.
Media (−ve control),
Live Mycoplasma,
Dead Mycoplasma.
Such cationic lipid vectors can be combined with further agents such as L-dioleoyl phosphatidyl ethanolamine (DOPE) to form multilamellar vesicles such as liposomes.
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. However, 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.
Generally, 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. 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. Optionally the formulation may further comprise a suitable carrier therefore.
The present invention will now be described by way of example and figures as follows:
pre-vaccination,
post-vaccination,
post-challenge. Group 1: Lambda A8; Group 2: Lambda B1; Group 3: Lambda gt11.
Media (−ve control),
Live Mycoplasma,
Dead Mycoplasma.
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 CO2 5%. For the challenge experiment, 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 1010 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.
Express recombinant proteins in E. coli and check sizes using immunoblotting and rabbit anti-serum (Makrina's blot and gel), about 30 and 65 Kb
Grow clones up in E. coli
A8 is IPTG inducible (expression controlled by the promoters β-gal and CMV)
The commonly used cloning vehicle k-gt11 was used throughout. A single colony of E. coli strain Y1090 was added to 100 ml NZCYM broth (Fluka, Biochemika, Switzerland) and grown overnight at 37° C., with vigorous agitation. The cell concentration was calculated, assuming 1OD600=8×108 cells/ml. Four aliquots of the E. coli suspension, each containing 1010 cells were centrifuged at 3300 g for 5 minutes at room temperature (19° C.). Each bacterial pellet was re-suspended in 3 ml phage (SM) buffer (5.8% Sodium Chloride (Fisher Scientific, UK Ltd.), 2% Magnesium sulphate (Fisher Scientific, UK Ltd.), 50 mM Tris.HCl (Sigma Ltd. UK), 2% Gelatine (Sigma Ltd. UK)). 1 μl of λ-gt11/λ-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 500 ml NZCYM broth and left at 37° C., with vigorous agitation overnight. 10 ml of chloroform (Fisher Scientific, UK Ltd.) was added to each of the flasks, which were then incubated for 10 minutes at 37° C., with shaking. The flasks were left to stand at an angle to allow the chloroform to pool to the bottom, before being removed with a pipette.
Purification of λgt11-Bacteriophage
Lysed cultures were cooled to room temperature. Deoxyribonuclease I (Sigma Ltd. UK) and Ribonuclease A (Sigma Ltd. UK) were added to each flask at a final concentration of 1 μg/ml in each. These were incubated for 30 minutes at room temperature before the addition of 1M Sodium Chloride, which was dissolved before the flasks were left to stand on ice for an hour. Cell debris was removed by centrifugation at 11,000 g for 10 minutes at 4° C. and the collected supernatants were pooled. Solid Polyethylene glycol (PEG, Sigma Ltd. UK) was added to a final concentration of 10% (w/v), dissolved slowly at room temperature, then the flasks incubated at 4° C. for at least one hour. The precipitated bacteriophage particles were recovered by centrifugation at 11,000 g for 10 minutes at 4° C. and the pellet re-suspended in SM buffer (8 ml for each 500 ml of supernatant). The PEG was removed by the addition of an equal volume of chloroform, before centrifugation at 300g for 15 minutes at 4° C. The aqueous phase was then recovered and ultra-centrifuged at 26,000 rpm for 2 hours at 4° C. The bacteriophage pellet was re-suspended in 1-2 ml of SM buffer and incubated at 4° C. overnight.
Titration of λgt11-Bacteriophage
E. coli was diluted 1/10 in Luria broth (Sigma Ltd. UK), (containing 10 mM 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 10 mM Magnesium sulphate and 0.2% Maltose) and incubated at 37° C., with vigorous shaking, for 3 hours. The culture was then centrifuged at 3300 g for 5 minutes at room temperature and the pellet re-suspended in 10 mM Magnesium sulphate to an OD600 of 0.6.
Appropriate serial dilutions of the purified λ-gt11 bacteriophage suspension were prepared in SM buffer. 5 μl of each dilution was diluted in 195 μl of the E. coli culture and incubated at 37° C. for 15 minutes.
Each bacteriophage/E. coli mixture was added to 2.5-3 ml 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 λ-gt11 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 μl SM saline buffer containing 5×108 bacteriophage particles. The 3 groups used were as follows: (1) λ-A8; (2) λ-B1 and (3) negative control, λgt11. 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.
At week 13, all three groups of BALB/c mice (8 mice per group) were challenged by the intraperitoneal injection of 0.5 ml (1010 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° C. for 7 days. Growth in liquid medium (observed as a colour change from red to yellow of the 1 in 10 dilutions) was confirmed with an MmmSC-specific latex agglutination test (March et al. 2000b; March and Cloughley 2001), or by plating on solid medium and observing mycoplasma colonies.
Antibody titres against bacteriophage λ coat proteins and whole MmmSC antigens in mouse sera (pre-vaccination, pre-challenge and final bleed post-challenge) were measured by indirect ELISA. Microtitre plates (96-well; Greiner Ltd, Brunel Way, Stonehouse, Gloucestershire) were coated overnight at 4° C. in 0.05M sodium carbonate buffer at pH 9.2 with either whole sonicated MmmSC(N6) at a concentration of 5 μg/ml or 109 bacteriophage (50 ng) per well. Plates were blocked for 30 minutes with PBS supplemented with Tween 20 (Sigma) 0.5% (PBST) and dry skimmed milk 5% w/v (blocking buffer) before incubation with a 1 in 400 dilution of primary mouse antiserum in blocking buffer (100 μl/well) overnight at 4° C., in triplicate. Plates were subsequently washed three times with PBST and incubated with goat anti-mouse horseradish peroxidase [HRP]-labelled secondary antibody (DAKO A/S, Glostrup, Denmark) diluted 1 in 2000 in blocking buffer for 2 hours at 37° C. Plates were then washed three times in PBST and “developed” with o-phenylenediamine dichloride (OPD) (Sigma), in the dark at room temperature for 10 minutes. The reaction was stopped by the addition of 3M H2SO4 and the optical density (OD) was measured at 492 nm with an IEMS Plate Reader (Labsystems, Life Sciences International, Edison Road, Basingstoke, Hampshire).
At week 13, day 4 following challenge, 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 2 ml 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 1500 rpm at 16° 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 1500 rpm 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% 1M Hepes (Sigma Ltd, UK)). This cell suspension was centrifuged again (as above), and the pellet re-suspended in 1 ml 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 1×106 cells/ml in complete RPMI. Using sterile 96-well tissue culture plates, 100 μl of the viable splenocytes were seeded onto 100 μl volumes of sterile diluted antigens in triplicate. Mycoplasma live and dead (heat killed) were diluted in complete RPMI, to give a concentration of 106 cells/ml for each. Also used was λgt11, diluted in complete RPMI to give concentrations of 5 and 2.5 μg/ml. As a positive control, cells were cultured with concavalin A (Sigma Ltd., UK) at a concentration of 2.5 μg/ml. Plates were incubated in a humid (5% CO2) environment at 37° C. for 96 hours. After 4 days incubation, the cell cultures were pulsed for 18 hours with 18.25 KBq (1 μCi) [3H] thymidine (Amersham Biosciences UK), per well. The cells were harvested in a Packard Harvester onto glass fibre filters (Packard, Netherlands), and activity counted in a direct beta counter (Packard, Netherlands). Results are expressed as average counts per minute (cpm) (±standard deviation), from which stimulation indices (SI) were calculated using the following equation: SI=average cpm of test/average cpm of media control (no antigenic stimulus).
The immune responses were analysed by applying the Mann-Whitney test to comparisons of the mean weekly increases in the ELISA titre. The results of challenge were analysed by applying the one-sided Fisher's Exact Probability test to comparisons of the numbers of mycoplasmaemic mice in different groups on days 2 and 3.
Clones A8 and B1 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% β-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. 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 0.1 mg/ml diaminobenzidine (Sigma) in PBST containing 0.1 ml of 30% H2O2 per 100 ml of substrate solution.
Examination of the amplified λ-ZAP express library with the specific labelled insertion sequences confirmed that library construction was successful and that a high proportion of the clones contained T1/44 DNA. A significant number of clones were positive, when tested with both rabbit and immune serum and bovine convalescent sera.
Table 1a, b and c show the results of challenge (week 13) with Mycoplasma. Group 1 mice, immunized with the λ-A8 vaccine, were almost completely protected from infection with live mycoplasma, as judged by the absence of mycoplasmaemia (only ⅛ still infected) on day 4 post-challenge. Less animals in group 2 (3 out of 8 mice), vaccinated with the λ-B1 vaccine had successfully cleared the infection from the blood by day 4 as compared with the control group 3 (immunised with λ-gt11), where 4 out of the 8 mice were protected.
The whole MmmSC immune responses before and after vaccination and following challenge, for the three different mouse groups are shown in
Immune Responses of Mice to Whole Live/Dead Mycoplasma and λgt11, as Measured by LSA
Following four days culture, cellular proliferation was measured by the incorporation of 3H-thymidine (counts per minute (cpm)), in response to stimuli from whole live/dead mycoplasma and phage (λ-gt11). From these values, the stimulation index for each group, in response to whole mycoplasma, was calculated (
Specific immune responses were detected by immuno-blotting mice anti-sera from groups 1, 2 and 3 final bleeds against A8 and B1 proteins. The two mice tested in group 1 (mouse 4 and 8, vaccinated with the λ-A8 construct), against E. coli expressing the A8 protein, each showed a positive band of <30 kD, compared to group 3 serum (mouse 4, vaccinated with λgt11), which showed no positive band of this size (
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 1010 viable organisms in growth medium. In some cases (group 1, A8 vaccinated mice), vaccination produced a protective immune response, demonstrated by a reduction in the mycoplasmaemia produced by challenge.
The factors that may affect the mouse immune response to MmmSC antigens have not been evaluated. It has been suggested that C57BL/6 mice are resistant to infection with the human pathogen Mycoplasma pneumoniae because of an innate immunity associated with alveolar macrophages and humoral immunity (Hickman-Davis et al., 1997).
A relation was observed between a high anti-MmmSC titre and protection against challenge, Smith (1971b) showed that mice vaccinated intravenously with heat-killed MmmSC were completely protected from mycoplasmaemia after challenge with live organisms. However, when the 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. In contrast, the same volume of a 1 in 50 dilution of serum taken from rabbits after recovery from infection with MmmSC protected mice from mycoplasmaemia. Thus, the rabbit antiserum was much more protective than the mouse antiserum. 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0415428.8 | Jul 2004 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2005/002732 | 7/11/2005 | WO | 00 | 11/15/2007 |
