WO2009021291A1 - Formulation de vaccin contre des mycobactéries - Google Patents

Formulation de vaccin contre des mycobactéries Download PDF

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Publication number
WO2009021291A1
WO2009021291A1 PCT/AU2008/001186 AU2008001186W WO2009021291A1 WO 2009021291 A1 WO2009021291 A1 WO 2009021291A1 AU 2008001186 W AU2008001186 W AU 2008001186W WO 2009021291 A1 WO2009021291 A1 WO 2009021291A1
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mycobacterium
protein
vaccine
immune response
formulation
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PCT/AU2008/001186
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English (en)
Inventor
Richard Whittington
Deborah Taylor
Douglas Begg
Sanjeev Gumber
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The University Of Sydney
Meat And Livestock Australia
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Priority claimed from AU2007904380A external-priority patent/AU2007904380A0/en
Application filed by The University Of Sydney, Meat And Livestock Australia filed Critical The University Of Sydney
Publication of WO2009021291A1 publication Critical patent/WO2009021291A1/fr

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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
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis

Definitions

  • the invention relates to formulations and vaccines for inducing immune responses to mycobacterium infections and related pathologies, especially Johne's disease.
  • Johne's disease is a chronic, insidious wasting disease of livestock caused by infection with Mycobacterium avium subsp. paratuberculosis which is transmitted in faeces to young animals by infected adults.
  • the disease is characterized by intestinal lesions of granulomatous enteritis, lymphadenitis and progressive emaciation of the animal.
  • the disease has been reported in cattle in Australia since 1925 but only recently recognized in sheep.
  • the first report of JD in sheep in Australia was in 1980 and the first outbreak reported in New South Wales in 1981. Since then, JD has been recognised as being widespread in both domestic and wild ruminants in Australia.
  • JD typically progresses through three distinct stages of disease, the division of each stage being based on the detection of host immune responses to paratuberculosis antigen, detection of faecal shedding of the organism and the existence of clinical signs.
  • animals are infected although asymptomatic, and can remain so for many years without developing signs of clinical disease.
  • Subclinically infected animals can transmit the infection via faecal shedding of the causative organism into the environment, although in the very early stages of infection it cannot be detected by culture. Shedding has also been found to be intermittent.
  • a vaccine, GudairTM, in the form of killed M.ptb has been found to stimulate cell mediated and humoral responses, to reduce mortality due to Johne's disease and to delay fecal shedding for the first year post-vaccination.
  • this vaccine is that sub clinical infection may persist in vaccinated individuals, hence leading to a risk that unvaccinated individuals may acquire disease from vaccinated individuals. Further, some of the vaccinated individuals develop multibacillary lesions leading to a higher risk of transmission of disease amongst a population.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including an immunogen in the form of a stressed mycobacterium.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including an immunogen in the form of a mycobacterium that has been transformed with one or more genes for providing the transformed mycobacterium with a phenotype that resembles the phenotype of a stressed mycobacterium.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including an immunogen in the form of a bacterium that has been transformed with one or more genes for providing the transformed bacterium with a phenotype that resembles the phenotype of a stressed mycobacterium.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including an immunogen in the form of a molecule that is expressed by a stressed mycobacterium.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including an immunogen in the form of a eukaryotic cell having a molecule that is expressed by a stressed mycobacterium.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including a nucleic acid for encoding an immunogen in the form of a molecule that is expressed by a stressed mycobacterium.
  • a method for inducing an immune response against a mycobacterium including providing a formulation or vaccine described above to an individual.
  • kits for use in inducing an immune response against a mycobacterium including an immunogen in a form as described above.
  • the kit may further include an adjuvant.
  • the kit may further include written instructions for use of the immunogen to produce a formulation or vaccine as described above.
  • the kit may further include written instructions for use of the immunogen for inducing an immune response against a mycobacterium.
  • mycobacteria such as M.ptb existing in asymptomatic and subclinical hosts may be stressed and in some circumstances, dormant.
  • stressed or dormant mycobacteria have a phenotype that can be distinguished from the phenotype of mycobacteria existing in non stressed environments, such as in circumstances of symptomatic disease.
  • the inventors provide new formulations and vaccines for inducing, generating or raising an immune response against a mycobacterium, in particular a stressed mycobacterium such as a mycobacterium occurring in an individual having asymptomatic or sub-clinical disease.
  • the formulations and vaccines are effective for inducing cellular and/or humoral immune response.
  • the immune response induced by the formulations and vaccines is effective for preventing a mycobacterium from replicating in an individual infected with the mycobacterium. In other embodiments, the immune response is effective for preventing a mycobacterium from establishing a microbial load that is commensurate with asymptomatic or sub clinical disease in an individual infected with the mycobacterium that is caused or mediated by, or associated with the infection.
  • the immune response is effective for minimising the likelihood of an individual infected with a mycobacterium from developing, obtaining or progressing to an asymptomatic or sub clinical disease caused by the infection or progressing to a clinical disease.
  • the immune response is effective for providing a protective immune response to a mycobacterium.
  • Individuals in which a protective immune response has been generated may be considered as "carriers" in the sense that they do not develop the clinical symptoms of disease associated with infection and yet they are not free of pathogen.
  • the immune response is effective for treating an individual for symptomatic disease that is caused by a mycobacterium infection.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including an immunogen in the form of a stressed mycobacterium.
  • Stressed bacteria are generally defined as cells that have been exposed to environmental conditions that impact on the bacteria in a way which impinges on one or more physiological processes, culminating in a bacterium having a particular phenotype that is distinguished from that of bacteria which have not been exposed to said conditions.
  • these conditions include those related to temperature, atmosphere, pH and like conditions that are provided in an environment analogous to that found in living tissue, in particular living mammalian tissue.
  • Dormancy is one example of a particular phenotype that may be displayed by stressed bacteria. However, it is not the only phenotype.
  • Others include increased or decreased sensitivities to temperature, atmosphere, pH, nutrient levels, oxidative conditions or fluxes in any of these parameters. Bacteria may display a new phenotype in order to withstand these and other stresses.
  • Dormant bacteria are generally defined as cells having a reversible state of low metabolic activity. These cells can persist for extended periods without division.
  • Dormant cells are distinguished from dead cells on the basis of reversibility i.e. dormant cells can regain their growth under suitable conditions, a process referred to as "resuscitation".
  • Dormant cells are distinguished from "growing cells". Growing cells are generally characterised as cells existing in a lag phase (i.e. a phase in which the cells adapt ) themselves to growth conditions and mature to a point at which they become able to divide) or an exponential or log phase (i.e a phase in which mature cells undergo cell division so that the number of new bacteria appearing per unit time is proportional to the present population) which is followed by a stationery phase and then a decline phase as resources for sustained growth are exhausted.
  • a lag phase i.e. a phase in which the cells adapt
  • an exponential or log phase i.e a phase in which mature cells undergo cell division so that the number of new bacteria appearing per unit time is proportional to the present population
  • Dormant cells are particularly distinguished from “growing cells” observed in a lag phase, in that when dormant cells are placed in conditions conducive for the cells to enter log phase, there is generally required a period of 4 to 6 weeks, or longer for mycobacteria, before the cells are resuscitated from dormancy and are able to enter log phase.
  • “growing cells” that are in what is conventionally known as a "lag
  • phase tend to enter a log phase within hours or, in the case of mycobacteria, days, of being provided with conditions conducive for growth.
  • Dormant cells are sometimes known as “latent cells” or cells that exist in a “latent phase”.
  • the stressed cells of the formulation or vaccine are dormant cells.
  • Some cells become dormant as a function of environmental conditions that apply stress to the cells. Examples of these conditions include hypoxia, nutrient starvation, temperature flux and elevated temperature. Many other conditions are known in the art to induce dormancy in a growing bacterium.
  • stress conditions include temperature flux, hypoxia and nutrient starvation.
  • the stress condition is temperature flux.
  • the temperature to which the mycobacterium is exposed is raised and lowered.
  • the temperature may be lowered to about 10 0 C and raised to about 60 0 C.
  • the temperature change may be cyclical.
  • a cycle in which the temperature is raised from about 10 0 C to 50 0 C lasts about 3 to 4 minutes.
  • the number of cycles that are required to induce stress or dormancy are a function of temperature range and the length of each cycle. In one embodiment the number of cycles is from about 3 to 150. The appropriate number of cycles varies with the initial load of bacteria.
  • the stress condition is hypoxia.
  • the mycobacterium is partially or completely deprived of oxygen. Hypoxia may be achieved by any number of methods known to the skilled worker, including nitrogen flushing and the methods exemplified in the Examples below.
  • the stress condition is nutrient starvation.
  • the mycobacterium is partially or completely deprived of elements that are necessary for the metabolism that is generally observed in growing mycobacterium.
  • Nutrient starvation may be achieved by any number of methods know to the skilled worker. It may involve the complete or partial deprivation of all or a selected few nutrients. Examples of key nutrients to be removed from the mycobacterium include those that are a carbon or nitrogen source. Exemplary methods are described in the Examples below. Dormancy can be assessed by a number of methods including those exemplified in the Examples below. Some key characteristics of dormant bacteria are discussed above. Other features that especially define the dormant mycobacterium are discussed in Examples 1 and 2 below.
  • the stressed mycobacterium may be Mycobacterium avium subsp. paratuberculosis (M.ptb).
  • M.ptb Mycobacterium avium subsp. paratuberculosis
  • S M.ptb sheep
  • C M.ptb cattle
  • the stressed mycobacterium has a protein described in any one of Tables 8 to 13 herein, or peptide or fragment thereof.
  • the bacteria may be another species or sub species of Mycobacterium.
  • Mycobacterium examples include M. tuberculosis, M.avium, M.bovis, M. avium-intracellulare-scofulaccum complex, M.ulcerans, M. leprae. M.kansasii, M.gordonae, M.celatum. M.abscessus, M.africarum, M.asiaticum, M.avum, M.chelorae, M.flavescers, M.fortiutum, M.gastri, M.haemophilum, M.intracellulare, M.interjectum, M.
  • a stressed mycobacterium as an immunogen in the manufacture of a formulation or vaccine for inducing an immune response against a mycobacterium.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including an immunogen in the form of a mycobacterium that has been transformed with one or more genes for providing the transformed mycobacterium with a phenotype that resembles the phenotype of a stressed mycobacterium.
  • This phenotype may be referred to as a "stress phenotype”.
  • the mycobacterium may be provided for use in the formulation or vaccine by the steps of:
  • a molecule that is expressed by a mycobacterium in a host having an asymptomatic or sub clinical infection is a molecule that is expressed by a stressed mycobacterium.
  • stressed mycobacterium examples include the protein and peptides (herein “stress proteins” and “stress peptides” shown in Tables 8 to 13).
  • a molecule that is expressed by a mycobacterium in a host having an asymptomatic or sub clinical mycobacterium infection can be identified by one of the following steps:
  • Examples 1 and 2 demonstrate detecting differential expression of proteins in dormant and growing cells using 2D gel electrophoresis and tandem mass spectrometry. Any molecule that is expressed by dormant or stressed mycobacteria may be potentially useful in the formulation or vaccine of these embodiments of the invention. Examples of molecules include proteins, carbohydrates, lipids and like molecules.
  • the molecule is a protein, peptide or fragment thereof. Proteins are particularly useful as when presented in the context of MHC class II, they can provoke a specific immune response.
  • the stress protein or stress peptide is a protein described in any one of Tables 8 to 13 herein, or peptide or fragment thereof.
  • the mycobacterium that is selected for transformation is the same species, sub-species or strain of mycobacterium as the mycobacterium that causes the asymptomatic or sub-clinical disease.
  • mycobacterium for use in these embodiments of the invention are as described above.
  • a transformed mycobacterium having a stress phenotype as an immunogen in the manufacture of a formulation or vaccine for inducing an immune response against a mycobacterium.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including an immunogen in the form of a bacterium that has been transformed with one or more genes for providing the transformed bacterium with a phenotype that resembles the phenotype of a stressed mycobacterium.
  • bacterium examples include but are not limited to E.coli, Pseudomonas, Bacillus. Also, baculovirus or yeast expression or plant/plant cell expression systems could be used.
  • the bacterium may be provided for use in the formulation or vaccine by the steps of:
  • a molecule that is expressed by a mycobacterium in a host having an asymptomatic or sub clinical mycobacterium infection such as a stress protein or stress peptide can be identified as described above.
  • the stress protein or stress peptide is a protein described in any one of Tables 8 to 13 herein, or peptide or fragment thereof.
  • a transformed bacterium having a stress phenotype as an immunogen in the manufacture of a formulation or vaccine for inducing an immune response against a mycobacterium.
  • the bacteria (mycobacteria or non-mycobacteria) for use in the above described formulations has been killed or attenuated.
  • the bacteria can be killed by heating.
  • mycobacteria may be killed at 60 0 C for 20 minutes.
  • Bacteria may also be killed by exposure to formalin for 1 hour.
  • the formulation or vaccine of the above described embodiments contains a portion or fragment of one or more of a stressed mycobacterium or a transformed or recombinant bacterium having the phenotype of a stressed mycobacterium.
  • the formulation or vaccine may be substantially free of whole bacteria.
  • fragments include one or more of the proteins described in any one of Tables 8 to 13 herein, or peptide or fragment thereof.
  • the formulation or vaccine includes more than one immunogen, i.e. more than one species of molecule that is expressed by a mycobacterium in a host having an asymptomatic or sub clinical mycobacterium infection.
  • the formulation or vaccine may include more than one stress protein or stress peptide shown in any one of Tables 8 to 13 herein.
  • the immunogen is provided in the range of from 1 to 1000 ⁇ g, preferably 10 to 100 ⁇ g, in some embodiments about 25, 50 or 75 ⁇ g.
  • the immunogen is a pure or substantially pure molecule that is expressed by a mycobacterium in a host having an asymptomatic or sub clinical mycobacterium infection.
  • the formulation or vaccine does not include other immunogens in the form of whole bacteria or fragments of bacteria.
  • the formulation or vaccine may further include adjuvants in the form of fragments of bacteria, such as muramyl dipeptide.
  • the immunogen may be produced by recombinant expression of a stress protein or stress peptide or other molecule that is expressed by a mycobacterium in a host having an asymptomatic or sub clinical mycobacterium infection. Alternatively, these molecules may be isolated from a stressed mycobacterium.
  • a molecule that is expressed by a mycobacterium in a host having an asymptomatic or sub clinical mycobacterium infection as an immunogen in the manufacture of a formulation or vaccine for inducing an immune response against a mycobacterium.
  • the formulation or vaccine described above further includes an adjuvant.
  • An adjuvant is generally a compound or composition that enhances the effectiveness of the formulation or vaccine for inducing an immune response, including for example by potentiating the immunogenicity of the immunogen or by skewing a response to become predominantly humoral or cell-mediated.
  • Adjuvants that are particularly useful are those that induce cell-mediated immunity. These adjuvants may provide an immune response that essentially consists of cell mediated immunity, for example a response where little or no humoral responses can be detected.
  • Examples include adjuvants that primarily induce a Th1 response, such as LPS, Lipid A, Muramyl dipeptide lipophil, CpG ODN, ISCOMS. However adjuvants may also stimulate a lesser Th2 response.
  • adjuvants are those that induce humoral immunity and little or no cell mediated responses.
  • Examples include: adjuvants that primarily induce a Th2 response, such as Aluminium salts, Muramyl dipeptide hydrophil, Vit D3, CTA-DD and Cholera toxin. However adjuvants may also stimulate a lesser Th1 response.
  • adjuvants that primarily induce a Th2 response, such as Aluminium salts, Muramyl dipeptide hydrophil, Vit D3, CTA-DD and Cholera toxin. However adjuvants may also stimulate a lesser Th1 response.
  • the adjuvant may provide for an immune response that consists of both cell mediated immunity and humoral immunity.
  • the adjuvant is selected according to the route of administration of the formulation or vaccine that is desired.
  • a commercial oil adjuvant manufactured from plant sources may be used.
  • Other adjuvants that could be used include CpG motifs, lipid formulations, liposomes, Quil-A, immuno-stimulating complexes (ISCOMS), virus like particles or other microemulsions and nanoparticles.
  • receptor specific mucosal adjuvants such as ganglioside receptor binding toxins, surface immunoglobulin binding CTA1-DD, pattern recognition receptor binding adjuvants and cytokine or chemokine receptor binding adjuvants may be used.
  • Particular mucosal adjuvants such as Quil A and ISCOMS may also be used.
  • Systemic immunisation including vitD3 or cholera toxin as adjuvant can be used to modulate a mucosal response that is beneficial in preventing mycobacterial establishment in the gut or lung.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including an immunogen in the form of a eukaryotic cell having a molecule that is expressed by a mycobacterium in a host having an asymptomatic or sub clinical mycobacterium infection, the molecule being located on the surface of the cell.
  • transgenic plants such as Arabidopsdis thalaria which have been engineered to express the mycobacterial antigen. Plants which are palatable to livestock may also be used, in which case oral delivery of vaccine in feed is used to stimulate a protective immune response.
  • a formulation or vaccine for inducing an immune response against a mycobacterium including an immunogen in the form of a nucleic acid for encoding a molecule that is expressed by a stressed mycobacterium.
  • Examples include a DNA vaccine where the gene for the mycobacterial antigen is inserted into a bacterial plasmid vector which can be amplified within the host bacterium, purified from the host bacterium. Single or multiple plasmids or multipromoter plasmids containing several mycobacterial genes, with adjuvant are injected into a skeletal muscle of the target animal. This is a priming dose. A booster vaccine comprising the mycobacterial antigen, or mycobacterial cell or other host expressing the antigen with adjuvant is injected.
  • a method for inducing an immune response against a mycobacterium including providing a formulation or vaccine described above to an individual.
  • the individual receiving the formulation or vaccine may be uninfected or known to have a mycobacteria infection.
  • the individual may have an asymptomatic or sub clinical infection.
  • an individual having an asymptomatic infection is one which does not show obvious signs or symptoms of disease caused by mycobacterium infection.
  • an asymptomatic individual having Johne's disease generally does not display any symptoms of the disease that are apparent in animals exhibiting clinical manifestations.
  • an individual having an early sub clinical infection is one without clinical manifestations of the infection or pathology accompanying it.
  • an early sub clinical individual having Johne's disease generally does not display intestinal lesions of granulomatous enteritis, lymphadenitis or progressive emaciation.
  • the individual may display a very mild symptom of an infection, such as an elevated interferon level in response to challenge with M.ptb or extracts thereof.
  • Late sub clinical infection may be accompanied by pathology such as intestinal lesions of granulomatous enteritis, lymphadenitis, prior to clinical signs of progressive emaciation becoming apparent.
  • individuals having an asymptomatic or subclinical infection contain proteins, peptides or fragments thereof that are expressed by stressed or dormant mycobacteria at a higher level than in growing mycobacteria.
  • the detection of these molecules is a hallmark of asymptomatic or sub-clinical infection.
  • a host contains a protein, peptide or fragment thereof that is expressed in a stressed mycobacterium and not expressed in a growing mycobacterium or;
  • the presence of a given protein, or level of expression of a given protein in a host can be detected by any number of assays. Examples include immunoassays, chromatography and mass spectrometry.
  • Immunoassays i.e. assays involving an element of the immune system are particularly preferred. These assays may generally be classified into one of:
  • purified antigen for example, an antigen that is expressed in dormant mycobacteria and not growing mycobacteria
  • purified antigen is bound to solid phase by adsorption or indirectly through another molecule and host serum is applied followed by another antibody for detecting presence or absence of host antibody;
  • purified antigen for example, an antigen that is expressed in dormant mycobacteria and not growing mycobacteria
  • immune cells such as T and B lymphocytes.
  • peripheral white cells are purified from a host and cultured with purified antigen. The presence or absence of one or more factors indicating immunity are then detected.
  • the ELISPOT and Cell ELISA methods discussed in Examples 4 and 5 are examples.
  • Other examples include assays that measure cell proliferation (lymphocyte proliferation or transformation assays) following exposure to purified antigen, and assays that measure cell death (including apoptosis) following exposure to purified antigen;
  • purified antibody specific for antigen for example, an antigen that is expressed in dormant or stressed mycobacteria and not growing mycobacteria
  • purified antibody is bound to solid phase, host tissue is then applied followed by another antibody specific for the antigen to be detected.
  • this approach including ELISA, RIA;
  • the immunoassays can be applied in vitro or in vivo.
  • An example of an in vivo diagnostic test is a DTH assay using purified mycobacterial antigen.
  • the infection status of the individual may be unknown.
  • the individual may or may not have been infected with mycobacterium.
  • the individual or host is an ovine, such as a sheep. Particular sheep breeds include: Merino, Rambouillet, Romney, Lincoln, Drysdale, Herdwick, Suffolk, Hampshire, Dorset, Columbia, Texel, Montadale, Coopworth,
  • the host may be another mammal, such as a bovine, especially cattle and other or a human, goats, deer, antelope, ruminants and carnivores such as foxes, ferrets, and some non-ruminant herbivores such as rabbits.
  • a bovine especially cattle and other or a human
  • goats deer
  • antelope ruminants
  • carnivores such as foxes, ferrets
  • some non-ruminant herbivores such as rabbits.
  • the mycobacterium is Mycobacterium tuberculosis and the host is a human being, such as an individual having an acute or chronic, asymptomatic or sub clinical infection of Mycobacterium tuberculosis.
  • the individual receiving a formulation or vaccine of the invention is at least 1 month old, preferably 4 to 6 months old, in some embodiments more than 1 year old.
  • the individual is provided with the formulation or vaccine at about the time that the symptoms of disease are detected.
  • the formulation or vaccine is first provided when the individual is from 1 to 3 months old, but could be older.
  • the formulation or vaccine of the invention is typically provided in more than one dose, and in certain embodiments, up to 2 doses of vaccine are provide in an interval of one month between doses.
  • One or more doses may be required depending on whether an adjuvant is used and the measure of the immune response provided.
  • a number of approaches may be used for assessing whether an immune response has been induced in an individual who has received a formulation or vaccine of the invention.
  • antibody responses can be measured using ELISA to determine the amount of antibody being produced in various fluid samples including blood, faecal samples, saliva and mucus.
  • Various isotypes of antibody such as IgM, IgE, IgG, IgGI , lgG2 or IgA may be measured as well as total antibody.
  • Cytokines such as IFN-gamma and IL-10 may be measured using a number of different techniques including ELISPOT, CeI-ELISA, IFN-gamma ELISA, IL-10 ELISA.
  • Proliferation of lymphocytes isolated from blood can also be measured using flow cytometry with the stain CFSE and simulation with identified antigens.
  • Individual populations of lymphocytes such as CD4 and CD8 cells can also be measured using surface markers and flow cytometry.
  • Changes in the amount of apoptosis in blood samples can be measured by several techniques including caspase assays.
  • kits for use in inducing an immune response against a mycobacterium including an immunogen in a form as described above.
  • kit may further include an adjuvant.
  • the kit may further include written instructions for use of the immunogen to produce a formulation or vaccine as described above.
  • the kit may further include written instructions for use of the immunogen for inducing an immune response against a mycobacterium.
  • Example 1 Inducing dormancy in Mycobacterium avium subsp. paratuberculosis by temperature flux.
  • Suspensions of M.pth S and C strains with a concentration of 4.3x10 7 viable cells/ml were used.
  • the S strain isolate used Telford 9.2, has an S1 ⁇ S900 RFLP pattern and an IS 7377 S pattern.
  • the C strain isolate used was a field isolate (CMOO/416), has a C3 IS900 RFLP pattern and IS 7377 C pattern, and appeared identical to strain K-10 in whole genome microarray analysis.
  • Suspensions were stored at -80 0 C. At the start of the experiment, the stock was thawed at room temperature followed by thorough mixing by vortexing.
  • Hourly temperature records from shaded and unshaded locations in a previous experiment were analysed to determine the pattern of temperature change each day at Camden (elevation 70 m above sea level, latitude 34°S) in southeast New South Wales, Australia, over a 12 month period. Peak daily temperatures were reached each day at about 1 :30 pm but a broad peak (defined as maximum temperature minus 10%) occurred from 11 :30 am to 4:00 pm. This was defined as the peak period. The daily minimum temperature occurred at about 7:00 am each day and a broad trough (defined as minimum temperature plus 10%) occurred from 12:00 am to 8:30 am. This was defined as the trough period. The periods between the peak and the trough were referred to as the ascending and descending periods.
  • the daily ascending period occurred from 8:30 am to 11 :30 am each day while the descending period occurred from 4:00 pm to 12:00 am each day.
  • each day could be broken down into 4 phases of relative length of 8.5 (trough period), 3 (ascending period), 4.5 (peak period) and 8 (descending period) units duration (1 cycle).
  • This pattern was programmed into the computer that controlled a thermocycler (Corbett Research, Sydney, Australia). However, in some experiments this ideal pattern was not possible due to programming limitations and a pattern of 4 (trough period), 8 (ascending period), 4 (peak period) and 8 (descending period) unit duration (1 cycle) was used. In the diurnal cycle of nature 1 unit is equal to 1 h, but in these experiments 1 unit was made equal to 1 min.
  • BACTEC vials were incubated for 12 weeks at 37°C. The growth in BACTEC vials was measured weekly with an automated BACTEC 460 ion chamber machine.
  • Peak temperature conditions were simulated in a thermocycler in thin walled 200 ⁇ l PCR tubes. 100 ⁇ l of S strain suspension (4.3x10 5 viable cells/ml) per PCR tube was used. A positive and a negative control were used in every experiment. The timing of sample collection in all experiments was based on data from earlier studies with various species of mycobacterium in which organisms were shown not to be viable after prolonged heating. However, due to lack of information for the S strain of M.ptb, the time period was extended in all the experiments beyond the expected duration of survival for that temperature.
  • each tube was removed from the thermocycler at intervals as summarized in Table 4, held at room temperature until all the samples had been collected, and then inoculated into the culture media.
  • M.ptb was confirmed by ⁇ S900 PCR in serially diluted template DNA samples in these experiments.
  • M.ptb suspensions with and without growth in BACTEC vials were also inoculated onto modified Middlebrook 7H10 slopes with and without mycobactin J.
  • 2c 70 2 h 4.3x10 5 15 s, 30 s, 45 s, 60 s, 75 S, 90 s, 2 m, 3 m, 4 m, 5 m, 6 m, 7 m, 8 m, 9 m, 10 m, 20 m, 30 m, 40 m, 50 m, 1 h, 2 h
  • 2d 80 1 h 4.3x10 5 15 s, 30 s, 45 s, 60 s, 75 s, 90 s, 2 m, 3 m, 4 m, 5 m, 6 m, 7 m, 8 m, 9 m, 10 m, 20 m, 30 m, 40 m, 50 m, 1 h
  • M.ptb suspensions were heated in 200 ⁇ l PCR tubes in thermocycler machine
  • 4b 70 55 s 4.3*10° 15 s, 20 s, 25 s, 30 s, 35 s, 40 S 1 45 S, 50 s
  • 4c 80 50 s 4.3x10° 15 s, 20 s, 25 s, 30 s, 35 s, 40 s, 45 s, 50 s
  • CGIs cumulative growth indices
  • lag phase of the bacterial growth cycle there is no increase in cell number.
  • the length of lag phase can vary considerably depending on inoculum size.
  • An inoculum size of 1 viable M.ptb in a BACTEC culture medium will result in peak growth index within 6 weeks.
  • time required to reach peak Gl following commencement of growth is about 1-2 weeks, an inoculum with a lag phase of >4 weeks was deemed to be dormant.
  • Protein samples were prepared with the 160 ⁇ l of the cell lysis buffer (8 M Urea, 100 mM Dithiothreitol, 4% w/v CHAPS, 0.8% v/v carrier ampholytes, 40 mM Tris). The lysed cell suspension was centrifuged at 16,060 x g for 1 h at 4°C to pellet the cellular debris and the supernatant was collected. The aliquots of supernatant were stored at -80 0 C for 2-D PAGE. A Bradford assay for the estimation of protein concentration was performed. 60 ⁇ g of protein sample was processed with 2D clean up kit (Amersham Biosciences, catalogue number 80648451 ) as described by the manufacturer.
  • IPG strips were washed for 10 min in equilibration buffer 1 (6 M Urea, 375 mM Tris-HCI pH 8.8, 2% w/v SDS, 20% v/v glycerol and 2% w/v DTT) followed by a second 10 min wash in equilibration buffer 2 (6 M Urea, 375 mM Tris-HCI pH 8.8, 2% w/v SDS, 20% v/v glycerol and 2.5% w/v iodoacetimide).
  • IPG strips were rinsed in SDS-PAGE tank buffer and run in the 2 nd dimension on 12% vertical SDS-PAGE gels. Gels were rinsed in MQW and stained with silver stain using one of the published methods. Duplicate gels were run for each sample. The 2D gels were scanned with ImageScannerTM Il via LabScanTM software (Amersham Biosciences).
  • gel spots were incubated with DTT (20 mM) in NH 4 HCO 3 (30 ⁇ l, 25 mM) for 30 min at 37°C; after removal of the liquid the spot was incubated with iodoacetamide (30 ⁇ l, 40 mM) in NH 4 HCO 3 (30 ⁇ l, 25 mM) for 30 min at 37°C.
  • the spot was washed with CH 3 CN (2 x 50 ⁇ l, 10 min). Trypsin (-100 ng) in NH 4 HCO 3 (10 mM, 25 ⁇ l) was added and the solution was left at 37°C for 14 h.
  • the gel pieces were washed with H 2 O (0.1% formic acid, 50 ⁇ l) and H 2 OiCH 3 CN (1 :1) (0.1% formic acid, 50 ⁇ l) for 15 min and the combined extracts were dried and peptides dissolved in H 2 O with 0.05% heptafluorobutyric acid and 0.1% formic acid, 10 ⁇ l.
  • Digested peptides were separated by nano-Liquid Chromatography (LC) using a Cap- LC autosampler system (Waters, Milford MA). Samples (5 ⁇ l) were concentrated and desalted onto a micro C18 precolumn (500 ⁇ m x 2 mm, Michrom Bioresources, Auburn, CA) with H 2 OiCH 3 CN (98:2, 0.05 % HFBA) at 15 ⁇ l/min. After a 4 min wash the precolumn was automatically switched (Valco 10 port valve, Houston, TX) into line with a fritless nano column as described previously.
  • LC Liquid Chromatography
  • Peptides were eluted using a linear gradient of H 2 0:CH 3 CN (98:2, 0.1 % formic acid) to H 2 0:CH 3 CN (50:50, 0.1 % formic acid) at ⁇ 200 nl/min over 30 min.
  • the precolumn was connected via a fused silica capillary (10 cm, 25 ⁇ ) to a low volume tee (Upchurch Scientific), where high voltage (2600 V) was applied and the column tip positioned ⁇ 1 cm from the Z-spray inlet of an Ultima API hybrid Quadrupole Time-of-Flight (Q-TOF) tandem mass spectrometer (Micromass, Manchester, UK).
  • Q-TOF Quadrupole Time-of-Flight
  • the matches with Mascot probability scores greater than 50 were used to query the NCBI nr database using the BLASTp algorithm.
  • the matches were further subjected to searches of the Mtb H37Rv genome sequence database (Tuberculist, Institute Pasteur, Paris, http://genolist.pasteur.fr/Tuberculist/).
  • the complete amino acid sequence for each of the M.ptb proteins identified was obtained from the M.ptb K10 genome (GenBank accession, AE016958).
  • the theoretical pi of proteins was calculated using Biomanager, Australian National Genomic Information Service (ANGIS) (http://www.angis.org.au/).
  • the expressed protein sequences were further analysed to identify the pattern or profiles of proteins using the lnterpro Scan algorithm of the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB) (http://us.expasy.org/tools/).
  • the ratios or magnitude of differences in protein expression level between control and temperature flux (320 cycles) 2D gels were calculated by ImageMaster 2D Platinum v5.0 software (Amersham Biosciences). Briefly, after automated spot detection, spots were checked manually to eliminate any possible background streaks. The patterns of each sample were overlapped and matched by selecting four landmarks in both the images.
  • %Vol relative volumes
  • the resulting bacterial pellet was washed twice in 200 ⁇ of sterile phosphate-buffered saline, resuspended in 50 ⁇ of sterile distilled water, and lysed at 100 0 C for 20 min. The lysate was then stored at - 2O 0 C.
  • a crude suspension of DNA was obtained by suspending a colony in distilled water, washing the cells three times in water, suspending the cells in 100 ⁇ of water, and boiling the washed cells for 20 min.
  • a DNA dilution series was prepared in a class Il biological safety cabinet. Concentrated DNA prepared above was serially diluted in TE (10 mM Tris HcI, 0.1 mM EDTA, pH 8.8) from 10° to lO "8 .
  • the delayed then rapid growth pattern was also observed at this temperature range; there was no growth or lag phase up to 6 weeks after 140 cycles of heat treatment for C strain. After 3 cycles of heat treatment (12-60 0 C), the C strain achieved peak growth within 7 weeks of incubation, but S strain was unable to survive after 2 cycles and achieved peak growth after 5 weeks of incubation. A lag phase of 3-4 weeks was observed for S and C strains after 2-3 cycles, respectively.
  • S and C strain were inactivated after 20 and 30 min of heating, respectively. There were intermediate points where no growth was recorded (16 min, 18 min for S strain; 26 min and 28 min for C strain).
  • the last time point with viable M.ptb was 30 s for S strain and 50 s for C strain. Again, there were intermediate time points where growth was negative (40 s and 45 s). A lag phase of 4 weeks was observed for C strain after 35 s of heating. The last time point with viable M.ptb was 25 s for S strain and 30 s for C strain at 8O 0 C. A lag phase of 2 weeks was observed for C strain after 25 s. DNA was extracted from samples in Experiment 3, which had shown growth in culture vials, and also from some samples without growth in culture vials. These samples were also inoculated on modified Middlebrook 7H10 slopes and showed colonies with morphology typical of S and C strains of M.ptb.
  • M.ptb was 30 s for S strain and 35 s for C strain at 70 0 C. Heating at 70 0 C resulted in a lag phase of 2-3 weeks for S and C strains after 30-35 s, respectively. At 8O 0 C, the last time point with viable M.ptb was 25 s and 30 s for S and C strains, respectively. A lag phase of 3 weeks was seen after 25-30 s of heating at 80 0 C.
  • Table 6 summarizes data for the S and C strains from the experiments (3.2a, 4a, 4b, 4c, 1d, 3.1a, 3.1b and 3.1c) described above.
  • MAP2698c desaturase dodecameric ferritin homologue that binds and protects DMA
  • MAP0540 Hypothetical protein MAP0540 Tranferases composed of 17.6 6.13 Rv3525c hexapeptide, LpxA-like
  • MAP3268 Respond to heat shock or other environmental stress and act as chaperons
  • Ratios represent relative protein abundance or the ratio of protein expression of cells grown exponentially to protein expression of cells after temperature flux.
  • Asterisks (*) represent proteins that were not identified in control samples
  • Ratios represent relative protein abundance or the ratio of protein expression of cells grown exponentially to protein expression of cells after temperature flux.
  • Asterisks ( * ) represent proteins that were not identified in control samples, t indicates downregulation and was not identified in the S strain after 322 cycles
  • Example 2 Inducing dormancy in Mycobacterium avium subsp. paratuberculosis bv hypoxia and nutrient starvation.
  • BACTEC culture was as per Example 1 except egg yolk was not included in culture vials in hypoxia experiments. Protein extraction, 2-D gel chromatography and in gel digestion were as per Example 1.
  • the bacteria were initially exposed to aerobic conditions for entry into exponential growth phase which was followed by sudden exposure to anaerobic conditions.
  • the bacteria were provided only an anaerobic environment. The purpose of these experiments was to identify the differences in the proteome of M.ptb during hypoxic shock under two different conditions.
  • 1 ml thawed M.ptb suspension having 4.3x10 7 viable cells/ml (S and C strains) was pelleted by centrifugation (21 , 910 x g) and resuspended into 100 ⁇ l of MQW for inoculation into BACTEC12B culture vials.
  • the atmosphere was changed to anaerobic as the growth index (Gl) in culture vials was measured every day using a BACTEC 460 ion chamber machine.
  • the removed gases were exchanged with anaerobic gases (10% CO 2 , 90% N 2 ) for 4 weeks of incubation at 37 0 C.
  • the air valve of the BACTEC 460 ion chamber machine was connected to an anaerobic gas cylinder (BOC, Australia) with a gas pressure of 800 psi.
  • a positive control culture vial i.e. under aerobic conditions was included for each time point for both the strains, and the Gl was also recorded every day. Samples were collected at weekly intervals for 4 weeks for protein extraction.
  • culture media from one culture vial for each strain was pelleted by centrifugation (21 , 910 x g) and the pellet was stored at -8O 0 C until protein extraction.
  • the second culture vial for each time point was shifted to aerobic conditions for 8 weeks at 37°C to investigate the resuscitation capacity of M.ptb following hypoxic shock and the Gl was recorded after every week.
  • Proteome analysis was conducted for the samples collected after 1 week and 4 weeks of hypoxic stress to estimate the temporal differences in proteome expression.
  • the experimental conditions were similar to Experiment 1 except the culture vials were preequilibrated once with anaerobic gases for 5 min before and immediately after inoculation of M.ptb and so the bacteria were not exposed to an initial period of aerobic conditions, although there may have been residual dissolved oxygen in the broth.
  • a total of 16 plastic tubes (Becton Dickinson, 15 ml polypropylene tube), with each tube containing 1 ml of freshly thawed M.ptb suspension (C or S strain) having 4.3x10 7 viable, cells/ml in 9 ml of deionised water (MiIIiQ, Millipore) were incubated at 37°C for 16 weeks. Samples (one tube for each strain) were collected after every 2 weeks to analyze the growth pattern of M.ptb after different periods of starvation. 100 ⁇ l of starved mycobacterial suspension was inoculated into radiometric BACTEC culture media with egg yolk after every 2 weeks. A total of 8 samples were inoculated into the culture media for each strain.
  • the Gl of the anaerobic treated cultures of S strain did not increase beyond 400-600.
  • the C strain did not reach a Gl of >300 during the anaerobic phase in this experiment.
  • the Gl of C strain under anaerobic conditions appeared consistently to be less than that for S strain.
  • the culture appears to remain viable throughout the period of anaerobic incubation up to 28 days, and the inferred lag phase upon restoration of aerobic conditions was less than 2 weeks.
  • M.ptb cells achieved peak Gl after 4-5 weeks of incubation at 37°C following 12-16 weeks of starvation. After starvation for 14 weeks, the viability of M.ptb cells was determined by an MPN method. No growth was recorded for the S strain after 14 weeks of starvation and MPN counts revealed ⁇ 0.3 x 10° viable cells or -100% loss in viability. In contrast, 9.3 x 10 1 viable cells or >99% loss in viability was observed for the C strain. A lag phase of 2-3 weeks was observed after starvation for 12-16 weeks.
  • MAP2450C involved in ATP synthesis coupled proton transport
  • Ratios represent relative protein abundance or the ratio of protein expression of cells grown exponentially to protein expression of cells after hypoxia.
  • Asterisks ( * ) represent proteins that were not identified in control samples, i indicates downregulation and was not identified in the S strain after hypoxia
  • MAP0593C Hypothetical protein Histidine triad protein, diade ⁇ osine 14.8 5.25 RvO759c 0.088 3.328 MAP0593c polyphospahte hydrolases and function as tumor suppressors in human and mice
  • UspA gene of E coli occurs during growth arrest conditions.
  • a Spot numbers correspond to those in the relevant 2D gel image (i.e. a figure)
  • c Homologues in M.ptb were identified when significant hits belong to other Mycobacterium spp.
  • Theoretical isoelectric point (pi) of matching protein was calculated by Biomanager (http://www.angis.org.au/) e Homologues in M.
  • tuberculosis were identified by H37Rv genome sequence database (http://qenolist.pasteur.fr/Tuberculist/) f Mean squared deviation was calculated by lmageMaster 2D Platinum software 9 Ratios represent relative protein abundance or the ratio of protein expression of cells grown exponentially to protein expression of cells after hypoxia. Asterisks (*) represent proteins that were not identified in control samples.
  • a Spot numbers correspond to those in the relevant 2D gel image (i.e. a figure)
  • Theoretical isoelectric point (pi) of matching protein was calculated by Biomanager (http.7/www.angis.org.au/) e Homologues in M. tuberculosis were identified by H37Rv genome sequence database (http://genolist.pasteur.fr/Tuberculist/) f Mean squared deviation was calculated by ImageMaster 2D Platinum software
  • Ratios represent relative protein abundance or the ratio of protein expression of cells grown exponentially to protein expression of cells after starvation.
  • Asterisks (*) represent proteins that were not identified in control samples, I indicates downregulation and was not identified in the S strain after starvation
  • MAP0593c polyphospahte hydrolases and function as tumor suppressors in human and mice
  • UspA gene of E coli occurs during growth arrest conditions.
  • a Spot numbers correspond to those in the relevant 2D gel image (i.e. a figure)
  • Ratios represent relative protein abundance or the ratio of protein expression of cells grown exponentially to protein expression of cells after starvation.
  • Example 3 Antigenicity of proteins having increased expression in dormant or stressed Mycobacterium avium subsp. paratuberculosis as compared with growing cells.
  • E.coli TOP 10 cells Genotype: F r mcrA, A ⁇ mrr-hsdRMS-mcr&C), ⁇ 80/acZ ⁇ M15, ⁇ /acX74, recA1 , araD139, ⁇ (araleu) 7697, galU, galK, rpsl (StrR), enc/A1 , nupG
  • E.coli BL21(DE3)pLysS cells (Novagen): Genotype: F' ompT, hsdSB (r B “ m B “ ), gal dcm (DE3)pLysS (CamR)
  • the mycobacterial pellet was resuspended in 1ml TE pH 8.0 in a sterile 1.5 ml centrifuge tube.
  • the pellet was vigorously mixed with a sterile inoculation loop followed by vortexing (1-2 min) to break up any clumps of aggregated M.ptb.
  • Mycobacteria were then killed by incubation at 80 0 C for 30 min in a hybridization oven after which they were allowed to cool at room temperature for 10 min.
  • DNA was extracted with the addition of 120 ⁇ i of lysozyme solution (200 mg/ml) and 200 units of mutanolysin (20 ⁇ l of a 10,000 units per ml stock) to the tube.
  • the cells were gently mixed and incubated overnight at 37 0 C with very gentle end over end mixing on a suspension mixer (Ratek Instruments, Australia).
  • the cells were transferred to a 10 ml centrifuge tube followed by addition of 120 ⁇ l proteinase K solution (10 mg/ml), 210 ⁇ l of 10% (wt/vol) SDS and incubated at 65°C (hybridisation oven) for 20 min with gentle mixing every 5 min.
  • 195 ⁇ l 5 M NaCI and 165 ⁇ l CTAB/ 5 M NaCI both prewarmed to 65°C were added and gently mixed until "milky” followed by incubation at 65°C (hybridisation oven) for 10 min.
  • the DNA was extracted and purified using a modified chloroform/isoamyl alcohol technique. An equal volume of 24:1 chloroform/isoamyl alcohol was added and mixed gently end over end, for 10 s or until an emulsion formed.
  • the aqueous phase containing DNA was separated using a phase lock gel system (Eppendorf Cat No. 0032.005.250) according to manufacturer's instructions. Following this, the aqueous phase was poured off into a fresh 10 ml centrifuge tube and 0.6 volumes of isopropanol was added. This was mixed by inversion and incubated at -20 0 C for 2 h. The precipitated DNA strands were transferred to a new 10 m!
  • DNA amplification was carried out in a 50 ⁇ l reaction containing 10 ng M.ptb DNA (S strain, Telford 9.2), 200 ⁇ M dNTPs, 0.5 ⁇ M each primer and 2U Taq polymerase (Expand High Fidelity PCR System, Roche).
  • reaction was subjected to the following conditions: 1 cycle of denaturation at 95°C for 3 min followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 1 min and extension at 72°C for 30 s. However, atpC was annealed at 52°C for 30 s. After 30 cycles of amplification, final extension was carried at 72°C for 10 min. PCR products were visualized by 2% agarose gel electrophoresis using ethidium bromide.
  • a single bacterial colony was allowed to grow overnight in 10 ml of Luria Bertani (LB) broth at 37°C with shaking. 100 ⁇ l of overnight culture was inoculated into prewarmed 100 ml of LB broth and grown with shaking to an OD ⁇ oo 0.6. The cells were decanted into ice-cold sterile centrifuge tubes and incubated on ice for 7 min before harvesting by centrifugation (2400 rpm, 10 min, 4°C).
  • the pellet was resuspended in 40 ml ice cold TFB-1 (3OmM KAc, 50 mM MnCI 2 , 10 mM CaCI 2 100 mM KCI, 15% glycerol), re- pelleted and resuspended in 4 ml ice cold TFB-2 (10 mM NaMOPS, 75 mM CaCI2, 10 mM KCI, 15% glycerol).
  • the cell suspension was rapidly aliquoted into chilled eppendorf tubes (100 ⁇ l), snap frozen on liquid nitrogen and stored at -80°C.
  • a tube of TOP 10 competent cells was removed from -80 0 C and placed on ice for 10 min to thaw. 50 ⁇ l was dispensed into a tube containing ligated product and incubated on ice for 30 min. The cells were then heat shocked at 37°C for 1 min, placed on ice for 2 min and 500 ⁇ l of LB broth was added. The tubes were further incubated at 37°C for 45 min.
  • the cells were pelleted by centrifugation at 16,100 x g for 30 s and small amount of supernatant was decanted and resuspended in the residual liquid and plated on LB agar plates containing appropriate selection 100 ⁇ g/ml ampicillin, 1 mM IPTG and 20 ⁇ g/ml X-gal. The plates were incubated for 16-18 h at 37°C. White colonies were selected and grown overnight in LB broth with 100 ⁇ g/ml ampicillin and inserts were confirmed by restriction digestion. Plasmids were recovered with a QIAprep® Miniprep (Qiagen) kit.
  • Amplified PCR products were purified using the Min Elute PCR purification kit (Qiagen) according to the manufacturer's instructions and were cloned into the pCR2.1 vector (TA cloning kit, Invitrogen) according to the manufacturer's instructions. Briefly, 1 ⁇ l of fresh PCR product was ligated with 2 ⁇ l of pCR2.1 vector (25 ng/ ⁇ l) with 1 ⁇ l of 10X ligation buffer and 1 ⁇ l of T4 DNA ligase, the reaction volume was made up to 10 ⁇ l and incubated at room temperature overnight. The cloning reaction was transformed into chemically competent TOP10 E.coli cells.
  • the isolated plasmids were digested with restriction endonucleases Nde ⁇ and Sa/nHI (NEB).
  • the reaction was carried out as a double digest. Briefly, 1 ⁇ g of DNA, 1 ⁇ l of enzyme (A/de ⁇ , BamHl), 2 ⁇ l of reaction buffer (NEB 2) and 0.2 ⁇ l of BSA (100X) were made up to 20 ⁇ l with nuclease free water. The reaction was mixed gently and incubated at 37 0 C for 2 h. The digest was verified by DNA electrophoresis on a 0.8% agarose gel.
  • Plasmids containing an insert were sent for DNA sequencing using M13 Forward and M13 Reverse primers. DNA sequencing was performed at the Australian Genome
  • the expression vector pET-15b was prepared by digesting with Nde ⁇ and BamHI. Briefly, 500 ng of pET-15b DNA, 1.5 ⁇ l of each enzyme ( ⁇ /cfel, BamHI) and 10 ⁇ l of reaction buffer (NEB 2) were made up to 100 ⁇ l with nuclease free water. The reaction was mixed gently and incubated at 37°C for 2 h. The digest was verified by DNA electrophoresis on a 0.8% agarose gel and stored at -20 0 C.
  • the DNA inserts were excised from the agarose gel and purified using a Qiagen Gel Purification kit.
  • the pET-15b vector was prepared using a double digest reaction with
  • Properly oriented pET-15b constructs were transformed into a chemically competent BL21(DE3)pLysS strain of E.coli.
  • a single colony was inoculated into 20 ml of LB broth containing ampicillin (100 ⁇ g/ml) and chloramphenicol (34 ⁇ g/ml) and grown at 37°C on an orbital shaker incubator overnight.
  • the following day 15 ml of cell culture was diluted into 150 ml of LB broth having ampicillin (100 ⁇ g/ml) and chloramphenicol (34 ⁇ g/ml).
  • the culture was grown to a cell density of OD value 0.5-0.6 and recombinant protein expression was induced by addition of 1 mM IPTG.
  • the pET-15b vector encodes an N-terminal polyhistidine tag which is fused to the proteins expressed in this study. Therefore, the expressed proteins were purified using TALON metal affinity resin (Clontech) or Ni-NTA agarose resin (Invitrogen). TALON resin was used for purification of proteins MAP2411 , Ppa, GreA under native conditions.
  • the cell pellets were resusp ' ended in 1 ml of binding buffer pH 7.8 (50 mM sodium phosphate, 500 mM NaCI, 0.1% Triton-X-100, 20 mM PMSF, 1 ⁇ l of Protease inhibitor cocktail, Sigma) and 0.5 g of 0.1 mm zirconium beads were added to the respective cell pellets.
  • the pellets were lysed using a cell homogenizer (FastPrep ® FP120, Thermo Electron Corporation) using 7 x 15 s pulses at maximum speed (6.5) with 2 min rest on ice between pulses.
  • each Fastprep tube with a tuberculin syringe and each tube was secured in another microfuge tube.
  • These secured tubes were placed in 50 ml plastic tubes and centrifuged at 360 x g for 5 min (Beckman coulter, AllegraTM-X12R centrifuge) to obtain the whole lysed cell suspension without zirconium beads.
  • Cell debris (insoluble fraction) was pelleted at 16,100 x g for 10 min at 4°C, the supernatant (soluble fraction) was collected and both were stored at -20 0 C for SDS-PAGE analysis.
  • the lysate prepared above was passed through a 0.45 ⁇ m filter to prevent clogging of the column.
  • Protein was eluted with 8 ml of elution buffer pH 6.0 (50 mM sodium phosphate, 500 mM NaCI, 150 mM imidazole). Protein concentrations of fractions were measured using the Bio-Rad Protein assay. The fractions were initially analysed by SDS-PAGE and subsequently pooled and dialyzed through 3kDa dialysis membrane with PBS using a microdialyzer (Pierce) and stored at -80 0 C.
  • elution buffer pH 6.0 50 mM sodium phosphate, 500 mM NaCI, 150 mM imidazole.
  • Nickel based resin was used for purification of CIpP and MAP0593c proteins under denaturing conditions. Denaturing purification was performed at room temperature. Briefly, the lysate was prepared in denaturing binding buffer pH 7.8 (8M Urea, 20 mM sodium phosphate, 500 mM NaCI) and was allowed to bind to nickel resin for 1.5 h at room temperature on suspension mixture as described above. The resin was then washed once with denaturing binding buffer pH 7.8 followed by two washes with denaturing wash buffer pH 6.0 (8M Urea, 20 mM sodium phosphate, 500 mM NaCI) and finally two washes with denaturing wash buffer pH 5.3 (8M Urea, 20 mM sodium phosphate, 500 mM NaCI).
  • the bound protein was eluted with 6 ml of denaturing elution buffer pH 4.0 (8M Urea, 20 mM sodium phosphate, 500 mM NaCI). The protein concentration of eluted fractions was determined using the Bio-Rad Protein assay according to manufacturer's instructions. Following SDS-PAGE analysis, the pooled fractions were dialyzed with PBS and 0.05% Tween20 using a SnakeSkin pleated dialysis tubing, 3.5kDa MWCO (Pierce) to refold the protein for use in ELISA and stored at -8O 0 C.
  • denaturing elution buffer pH 4.0 8M Urea, 20 mM sodium phosphate, 500 mM NaCI.
  • the protein concentration of eluted fractions was determined using the Bio-Rad Protein assay according to manufacturer's instructions. Following SDS-PAGE analysis, the pooled fractions were dialyzed with PBS and 0.05% Tween20 using a SnakeS
  • the recombinant proteins were diluted in carbonate buffer pH 9.6 to a final concentration of 5 ⁇ g/ml (GreA), 10 ⁇ g/ml (Ppa), 30 ⁇ g/ml (MAP 2411), 10 ⁇ g/ml (MAP0593c), 5 ⁇ g/ml (CIpP), a cocktail of 2.5 ⁇ g/ml GreA and 5 ⁇ g/ml Ppa, a cocktail of 2.5 ⁇ g/ml MAP0593c and 5 ⁇ g/ml CIpP and another cocktail of 2.5 ⁇ g/ml GreA, 2.5 ⁇ g/ml Ppa, 2.5 ⁇ g/ml CIpP and 2.5 ⁇ g/ml MAP0593c.
  • Protein expression was induced with 1 mM IPTG in BL21(DE3)pLysS competent expression cells having T7 RNA polymerase for the pET-15b expression.
  • SDS-PAGE analysis of recombinant clones indicated high levels of production of recombinant proteins. It was found that four recombinant proteins, MAP2411 , Ppa, GreA and CIpP, were soluble while MAP4107, sMAP0834, MAP0593c and Hsp were insoluble in nature. The findings were also confirmed by Western blot analysis. However, BL21(DE3)pLysS cells failed to produce overexpressed recombinant proteins for atpC. No difference was observed on SDS-PAGE and Western blot analysis of uninduced BL21 and cells induced with 1 mM IPTG.
  • MAP2411 soluble and one insoluble recombinant proteins expressed in E.coli were purified using nickel and cobalt based resins. The results show that after purification His-tag proteins with the appropriate predicted molecular masses became the major protein band in the eluent. Dialysis of the samples resulted in reduction of multiple bands. In each case the recombinant protein was the predominant protein in the sample. The total yield of purified proteins was estimated to be 1.2-1.4 mg/150 ml of induced E. coli culture.
  • n a number of animals tested
  • Example 4 ELlSPOT method for diagnosis of Mycobacterium avium subsp. paratuberculosis infection.
  • ELISPOT plates For ruminants the ELISPOT plates (Millipore) are coated with 50 ⁇ L of IFN6.19 or MCA2112 (Serotec) and incubated overnight at 4°C. Other antibodies are used for tests for other species. The plates are then washed 6 times with phosphate buffered saline (PBS) to remove excess antibody. Purified white blood cells at a concentration of 2.5 x 10 6 cells per mL are also diluted to concentrations of 1.25 x 10 6 cells per mL and 6.25 x 10 5 cells per mL with 100 ⁇ L of the three dilutions being placed into each well as required into a nitrocelluose plate (Millipore).
  • PBS phosphate buffered saline
  • the cells were incubated with 5OmL of either culture media (unstimulated), M.ptb 316v antigen 30ug/mL (EMAI Australia), purified protein derivative from Mycobacterium avium 30ug/mL (PPDA, CSL) and Pokeweed Mitogen 10ug/mL (Sigma) and one or more of the antigens identified in Example 3.
  • culture media unstimulated
  • M.ptb 316v antigen 30ug/mL EHA, CSL
  • PPDA Mycobacterium avium 30ug/mL
  • Pokeweed Mitogen 10ug/mL Sigma
  • Other antigens including those in Tables 8 to 13 can be used.
  • the plates are washed 6 times again in using PBS to remove the cells and 50 ⁇ L of the secondary antibody is added MCA1783b (serotec) at a concentration of 0.5ug/mL diluted in PBS. The plate is then incubated at 37°C for 1 hour. After this incubation the plates are washed again in PBS and 50 ⁇ L of a 1ug/mL concentration of alkaline phosphatase streptavidin (Vector Labs) is added to the wells. The plate is then incubated at 37°C for 1 hour.
  • the spots are differentiated and enumerated visually or by using an automated image analysis system, typically a low magnification microscope interfaced to a computer. A positive result is presented as number of spots greater than a value determined from a standard included in the assay.
  • Example 5 Cell ELISA method for diagnosis of Mycobacterium avium subsp. paratuberculosis infection.
  • the ELISA plates (Nunc, Maxisorb) are coated with 50 ⁇ L of MCA2112 (Serotec) and incubated overnight at 4°C. Other antibodies are used for tests for other species. The plates are then washed 6 times with PBS to remove excess antibody. 100 ⁇ L of purified white blood cells at a concentration of 2.5 x 10 6 cells per mL each well as required into plate ELISA plate.
  • the cells were incubated with 5OmL of either culture media (unstimulated), M.ptb 316v antigen 30ug/mL (EMAI Australia) Purified protein derivative from Mycobacterium avium 30ug/mL (PPDA 1 CSL) and Pokeweed Mitogen 10ug/mL (Sigma), and one or more of the antigens identified in Example 3.
  • culture media unstimulated
  • M.ptb 316v antigen 30ug/mL EHA 1 CSL
  • Pokeweed Mitogen 10ug/mL Sigma
  • the plates are washed 6 times again in using PBS + 0.05% tween 20 to remove the cells and 50 ⁇ L of the secondary antibody is added MCA1783b (serotec) at a concentration of 0.5 ⁇ g/mL diluted in PBS. The plate is then incubated at 37 0 C for 1 hour. After this incubation the plates are washed again in PBS + 0.05% tween 20 and 50 ⁇ L of a 0.01 ⁇ g/mL concentration of Horse-radish peroxidase streptavidin (Vector Labs) is added to the wells. The plate is then incubated at 37°C for 1 hour.
  • the plate is washed 5 times in PBS + 0.05% tween 20 and 100 ⁇ L TMB substrate (Pierce) is added for 30 minutes hour at room temperature. After the incubation the substrate is stopped by 100 ⁇ L 2M sulfuric acid and read in an ELISA plate reader at 450nm.
  • a positive result is presented as optical density greater than a value determined from a standard included in the assay.
  • Lambs are separated into groups of 40 to allow for variation in responses between individuals and a separate antigen or combination of antigens is administered to each group in the vaccinated groups, while the control lambs are sham vaccinated with saline.
  • Vaccine or saline is administered by subcutaneous injection high on the lamb's neck behind the ear using a 6 mm needle.
  • a DNA vaccine administered intramuscularly followed for example one month later by a protein vaccine as a booster as above would be given.
  • an oral vaccine comprising recombinant antigen expressed in plant cells would be given in feed over one month.
  • Trial sheep are subjected to the usual farm management practices, along with the rest of the sheep on each farm. To increase the M.ptb challenge to the trial sheep, they are depastured in paddocks previously grazed by older sheep in which clinical cases of OJD are occurring. In addition, suspected clinical cases from the remainder of the flock are regularly grazed with the trial sheep. Alternatively, an artificial oral infection can be used, with sheep grazed on uninfected pasture to ensure a uniform challenge dose. In this case sheep would be challenged orally with a pure culture derived from a seed stock of M.paratuberculosis strain Telford 9.2 using a dose of about 10 7'8 cells as a suspension in 10 ml of PBS. The dose is repeated four times at weekly intervals.
  • Moribund sheep are euthanased and post mortem examinations are conducted on-farm during scheduled visits, and where possible at other times when moribund sheep are observed.
  • the prevalence of subclinical lesions is assessed by histopathology after abattoir slaughter in a sample of clinically normal sheep on each farm at about 2 years of age (hogget cull), and again at the end of the trial, 54,48 and 42 months pv on farms 1 , 2 and 3, respectively.
  • These sheep are also routinely examined for gross lesions of OJD (abattoir surveillance).
  • Hogget cull animals are selected by the cooperating farmers based on fleece characteristics, as part of their normal management practice.
  • Adult sheep slaughtered at the end of the trial are randomly selected.
  • Samples are collected into 10% neutral buffered formalin from the ileocaecal valve (ICV), two sites in terminal ileum (Tl) and three mesenteric lymph nodes (MLN).
  • Fixed tissues are processed routinely for histopathology, then stained with hematoxylin and eosin (H&E) and a Ziehl-Neelsen (ZN) method.
  • Paratuberculous lesions are graded, based on the classification of Perez, as focal (grade I or 2), multifocal (3a), diffuse paucibacillary (3c) or diffuse multibacillary (3b).
  • the minimum criterion for a positive result is the finding of at least two clumps of macrophages with typical epithelioid morphology in a usual predilection site, with or without the presence of acid-fast bacilli.
  • Tissue samples from sheep slaughtered at the end of the trial are held at 4 0 C for less than 24 h, then frozen at -80 0 C for up to 6 months prior to processing.
  • a single pooled sample from the ileocaecal valve (ICV) 1 two sites in terminal ileum (Tl) and three mesenteric lymph nodes (MLN) from each sheep is prepared using previously described techniques including a centrifugation step.
  • Radiometric culture is done in modified BACTEC medium, with confirmation of M.ptb growth by PCR and REA analysis.
  • the prevalence of individual sheep excreting M.ptb in each group at each sampling time is estimated from the PFC results using the pooled prevalence calculator (http://www.ausvet.com.au/pprev/).
  • Method 2 for fixed pool size, with exact binomial confidence limits, is used. The method assumes 100% sensitivity and specificity for PFC. This method is chosen, acknowledging that sensitivity is not 100%, thus the prevalences derived are underestimates of the true prevalence. However, this method gives meaningful results across the spectrum of PFC results, allowing meaningful comparison between the vaccinated and control groups at each sampling time on each farm.
  • Immunological responses to M. a. paratuberculosis are measured using a gamma interferon (IFN-y) assay (BovigamTM). Stimulation of whole blood is done within 8 h of blood collection, using 100 ⁇ of Johnin PPD (300 pg/ml), avian PPD (300 ⁇ g/mL), or any of the antigens or combinations of antigens in Tables 8 to 13, PBS (negative control) and pokeweed mitogen (150 ( ⁇ g/mL, positive control). Stimulated plasma is held overnight at 4 0 C prior to enzyme immunoassay.
  • IFN-y gamma interferon assay
  • a sample is considered positive only if OD (Johnin) is greater than OD (PBS), and OD (Johnin) exceeds OD (avian) by at least 0.05.
  • immune responses would be evaluated using ELISPOT, CELLELISA and lymphocyte proliferation assays.
  • Humoral immune responses are measured using a commercial ELISA test (ParachekTM). The test is performed on the plasma from blood samples stimulated with PBS in the IFN-y test. A sample is considered positive if sample OD exceeds negative control OD by at least 0.2.
  • the vaccination site of all sheep is inspected visually and palpated. The presence of lesions, diameter of the lesions, and any discharging sinuses are recorded. At the first pv visit, the draining prescapular lymph nodes are also palpated.
  • a sheep is classified as a shedder of M.ptb if at any sampling time it has a positive IFC.
  • a sheep is classified as infected with M.ptb if it is a shedder, or if at necropsy it has histological lesions consistent with M.ptb infection, or is positive by culture of tissues at slaughter.
  • Moribund sheep that are shown to have severe diffuse lesions of OJD at subsequent necropsy, are classified as having died of OJD (OJD-mortality).
  • Infected sheep that do not die of OJD are classified as subclinical ⁇ infected.
  • the x ⁇ test on 2 x 2 contingency tables, or Fisher exact test (if an expected cell value in the ⁇ -test was less than 5) is used to test the significance of association of each of the following with vaccination on each farm, and using stratified analysis, across all farms: OJD-mortality, infection status, presence of histological lesions, presence of multibacillary lesions, shedding of M.ptb, immunological responses.
  • the same tests, using stratified analysis across vaccinates and controls where appropriate, are used to test the significance of association of each of the following with OJD-mortality, with shedding, and with infection status: pv IFN-y and ELISA responses, maternal antibodies (P2 only), vaccine-site lesions.
  • the effect of maternal antibodies on pv IFN-y and ELISA responses on P2 may also be tested.
  • An example is a whole mycobacterial cell preparation in which the mycobacteria have been inactivated by heating and then suspended in PBS. This is homogenised typically with an equal volume of an oil adjuvant to produce a stable emulsion. A preservative such as thiomersal is included at a concentration of about 0.013% VA/ in the final volume. The dose size is 1 to 2 ml.
  • An example is a whole bacterial cell preparation in which the bacteria have been inactivated by heating and then suspended in PBS. This is homogenised typically with an equal volume of an oil adjuvant to produce a stable emulsion. A preservative such as thiomersal would typically be included at a concentration of about 0.013% VA/ in the final volume.
  • the dose size is 1 to 2 ml.
  • Vaccine containing an immunogen in the form of a molecule that is expressed by a stressed mycobacterium.
  • An example is a purified protein preparation in which the recombinant antigen has been purified using metal affinity chromatography, quantified and then suspended in PBS. This is homogenised typically with an equal volume of an oil adjuvant to produce a stable emulsion. A preservative such as thiomersal would typically be included at a concentration of about 0.013% VA/ in the final volume. The dose size is 1 to 2 ml.
  • An example is a plant expressing the mycobacterial antigen. This is dried, chopped and fed to animals as an ingredient in a feed ration.
  • E. Vaccine containing a nucleic acid for encoding a molecule that is expressed by a stressed mycobacterium.
  • An example is a purified bacterial plasmid in which the gene for the mycobacterial antigen has been inserted suspended in PBS. This is mixed with an adjuvant such as cationic lipid vaxfectin. A plasmid encoding GM-CSF or IL-2 may also be included to enhance immunogenicity. A preservative such as thiomersal would typically be included at a concentration of about 0.013% VA/ in the final volume. The dose size is 1 to 2 ml.
  • the vaccine may be prepared for injection and/or electroporation, and typically would be packaged in a kit in which the booster vaccine consists of examples A 1 B or C. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

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Abstract

L'invention porte sur des formulations et sur des vaccins visant à induire une réponse immunitaire contre une mycobactérie. L'immunogène de la formulation ou du vaccin peut avoir la forme d'une mycobactérie stressée, d'une mycobactérie transformée par un de plusieurs gènes pour fournir la mycobactérie transformée dont le phénotype ressemble au phénotype d'une mycobactérie stressée, ou d'une bactérie ou autre hôte transformé par un ou plusieurs gènes pour fournir la bactérie transformée dont le phénotype ressemble au phénotype d'une mycobactérie stressée. L'immunogène de la formulation ou du vaccin peut également avoir la forme d'une molécule exprimée par une mycobactérie stressée, un acide nucléique pour coder une molécule exprimée par une mycobactérie stressée, ou une cellule eucaryote dont la molécule est exprimée par une mycobactérie stressée située sur la surface de la cellule.
PCT/AU2008/001186 2007-08-16 2008-08-15 Formulation de vaccin contre des mycobactéries WO2009021291A1 (fr)

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AU2007904380A AU2007904380A0 (en) 2007-08-16 Mycobacterium vaccine formulation
AU2007904380 2007-08-16

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001013944A2 (fr) * 1999-08-19 2001-03-01 Immunobiology Limited Vaccins contre des agents infectieux
WO2002020045A2 (fr) * 2000-09-04 2002-03-14 Immunobiology Limited Vaccin contre des microbes pathogenes
WO2002067982A2 (fr) * 2001-02-20 2002-09-06 Imperial College Innovations Ltd. Methodes et compositions d'intervention therapeutique dans le cadre d'une maladie infectieuse

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001013944A2 (fr) * 1999-08-19 2001-03-01 Immunobiology Limited Vaccins contre des agents infectieux
WO2002020045A2 (fr) * 2000-09-04 2002-03-14 Immunobiology Limited Vaccin contre des microbes pathogenes
WO2002067982A2 (fr) * 2001-02-20 2002-09-06 Imperial College Innovations Ltd. Methodes et compositions d'intervention therapeutique dans le cadre d'une maladie infectieuse

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