Mycobacterium vaccine formulation

Field of the invention

The invention relates to formulations and vaccines for inducing immune responses to mycobacterium infections and related pathologies, especially Johne's disease.

Background of the invention

Johne's disease (JD) 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. In the early subclinical stages, 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. As animals progress through the late subclinical phase and into clinical disease, they begin to shed high numbers of bacteria into the environment, which can be detected by culture and antibody response to Mycobacterium avium subsp. paratuberculosis (M.ptb) antigen.

A vaccine, Gudair™, 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. However, one problem with 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.

There is a need for new formulations and vaccines for inducing an immune response against mycobacteria.

Summary of the invention

In one embodiment there is provided a formulation or vaccine for inducing an immune response against a mycobacterium, the formulation or vaccine including an immunogen in the form of a stressed mycobacterium.

In another embodiment there is provided 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.

In further embodiments there is provided 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.

In further embodiments there is provided 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.

In other embodiments there is provided 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.

In other embodiments there is provided 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.

In other embodiments there is provided a method for inducing an immune response against a mycobacterium, the method including providing a formulation or vaccine described above to an individual.

In other embodiments there is provided a kit for use in inducing an immune response against a mycobacterium, the kit including an immunogen in a form as described above. In certain embodiments 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.

Detailed description of the embodiments

The inventors recognise that mycobacteria, such as M.ptb existing in asymptomatic and subclinical hosts may be stressed and in some circumstances, dormant. The inventors also recognise that 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.

In certain embodiments the formulations and vaccines are effective for inducing cellular and/or humoral immune response.

In certain embodiments 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.

In certain embodiments 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.

In certain embodiments, 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.

In certain embodiments the immune response is effective for treating an individual for symptomatic disease that is caused by a mycobacterium infection.

In one embodiment there is provided a formulation or vaccine for inducing an immune response against a mycobacterium, the formulation or vaccine 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. Examples of 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.

5 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.

> 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. In contrast "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".

In one embodiment, the stressed cells of the formulation or vaccine are dormant cells.

5 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.

In one embodiment a stressed mycobacterium may be provided for use in the formulation or vaccine by the steps of:

- providing a growing mycobacterium;

- providing stress conditions to the growing mycobacterium to form a stressed mycobacterium, such as a dormant mycobacterium. Examples of stress conditions include temperature flux, hypoxia and nutrient starvation.

In one embodiment the stress condition is temperature flux. In this embodiment the temperature to which the mycobacterium is exposed is raised and lowered. For example the temperature may be lowered to about 10 ⁰ C and raised to about 60 ⁰ C. The temperature change may be cyclical. In one embodiment 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.

In another embodiment, the stress condition is hypoxia. In this embodiment 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.

In another embodiment, the stress condition is nutrient starvation. In this embodiment, 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.

In the above described embodiment, the stressed mycobacterium may be Mycobacterium avium subsp. paratuberculosis (M.ptb). Examples include the M.ptb sheep (S) strains and the M.ptb cattle (C) strains. The former are distinguished from the latter by a point mutation in IS1311 and a major genomic deletion including the deletion of mmpL8.

In one embodiment the stressed mycobacterium has a protein described in any one of Tables 8 to 13 herein, or peptide or fragment thereof.

It will be understood that in certain embodiments, the bacteria may be another species or sub species of 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. intermedium, M.karsasii, M.malmoense, M.marirum, M.non-chromogenicum, M.phlei, M.shimodei, M.simiae, M.smegmatis, M.szulgai, M. terrae, M.trivale, M.ulcerars and M.xerzopi.

Thus in certain embodiments there is provided a use of a stressed mycobacterium as an immunogen in the manufacture of a formulation or vaccine for inducing an immune response against a mycobacterium.

In one embodiment there is provided 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".

In one embodiment the mycobacterium may be provided for use in the formulation or vaccine by the steps of:

- providing a growing mycobacterium;

- introducing a nucleic acid for encoding a molecule that is expressed by a mycobacterium in a host having an asymptomatic or sub clinical infection into the mycobacterium; or

- introducing a nucleic acid for causing the expression of said molecule into the mycobacterium.

One example 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. 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, such as a stress protein or stress peptide can be identified by one of the following steps:

- identifying a molecule that is expressed by a stressed mycobacterium but not by a growing mycobacterium, or

- identifying a molecule that is expressed at a higher level in a stressed mycobacterium than in a growing mycobacterium.

Methods known for detecting differential expression of molecules (i.e. expression between a test sample and control) are known in the art. With respect to proteins, examples include immunoassays, chromatography and mass spectrometry. 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.

Typically 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.

In one embodiment the stress protein or stress peptide is a protein described in any one of Tables 8 to 13 herein, or peptide or fragment thereof.

Methods for the introduction of a nucleic acid into a mycobacterium, or in other words, for transformation of mycobacterium are well known by the skilled worker.

Typically 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. Examples of mycobacterium for use in these embodiments of the invention are as described above.

Thus in certain embodiments there is provided a use of 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.

In addition to using mycobacteria in the formulation or vaccine, other non- mycobacterium bacteria may be transformed such that their phenotype resembles the phenotype of a stressed mycobacterium. Accordingly, in further embodiments, there is provided 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.

Examples of bacterium that may be transformed in accordance with this embodiment of the invention include but are not limited to E.coli, Pseudomonas, Bacillus.

Also, baculovirus or yeast expression or plant/plant cell expression systems could be used.

In these embodiments, the bacterium may be provided for use in the formulation or vaccine by the steps of:

- providing a growing bacterium;

- introducing a nucleic acid for encoding a molecule that is expressed by a mycobacterium in a host having an asymptomatic or sub clinical infection into the bacterium; or

- introducing a nucleic acid for causing expression of said molecule into the bacterium.

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.

In one embodiment the stress protein or stress peptide is a protein described in any one of Tables 8 to 13 herein, or peptide or fragment thereof.

In certain embodiments there is provided a use of 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.

Typically 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. For example mycobacteria may be killed at 60 ⁰ C for 20 minutes. Bacteria may also be killed by exposure to formalin for 1 hour.

In certain embodiments 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. In these embodiments, the formulation or vaccine may be substantially free of whole bacteria.

Typically the fragments include one or more of the proteins described in any one of Tables 8 to 13 herein, or peptide or fragment thereof.

In certain embodiments 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. For example the formulation or vaccine may include more than one stress protein or stress peptide shown in any one of Tables 8 to 13 herein.

Typically the immunogen is provided in the range of from 1 to 1000μg, preferably 10 to 100μg, in some embodiments about 25, 50 or 75μg.

In these embodiments, 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. Typically, the formulation or vaccine does not include other immunogens in the form of whole bacteria or fragments of bacteria. However, 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.

Thus in certain embodiments there is provided a use of 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.

In certain embodiments 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.

Other 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.

In certain embodiments, the adjuvant may provide for an immune response that consists of both cell mediated immunity and humoral immunity.

In certain embodiments, the adjuvant is selected according to the route of administration of the formulation or vaccine that is desired. For example, where systemic administration is intended, 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.

For mucosal immunisation, 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.

In other embodiments there is provided 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.

Examples include 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.

In other embodiments there is provided 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.

In other embodiments there is provided a method for inducing an immune response against a mycobacterium, the method 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. For example, the individual may have an asymptomatic or sub clinical infection.

As generally understood, an individual having an asymptomatic infection is one which does not show obvious signs or symptoms of disease caused by mycobacterium infection. For example, an asymptomatic individual having Johne's disease generally does not display any symptoms of the disease that are apparent in animals exhibiting clinical manifestations.

As generally understood, an individual having an early sub clinical infection is one without clinical manifestations of the infection or pathology accompanying it. For example, an early sub clinical individual having Johne's disease generally does not display intestinal lesions of granulomatous enteritis, lymphadenitis or progressive emaciation. However, 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.

As demonstrated in Examples 1 and 2, 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.

Accordingly, one can determine whether a host has an asymptomatic or sub clinical mycobacterium infection by a process including:

- determining whether a host contains a protein, peptide or fragment thereof that is expressed in a stressed mycobacterium and not expressed in a growing mycobacterium or;

- determining whether a host contains a protein, peptide or fragment thereof that is expressed in a stressed mycobacterium at a higher level than a growing mycobacterium; or

- determining whether a host contains a protein, peptide or fragment thereof described in any one of Tables 8 to 13 herein.

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:

(i) assays in which purified antigen (for example, an antigen that is expressed in dormant mycobacteria and not growing mycobacteria) is used to detect an antibody in host serum. For example, 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;

(ii) assays in which purified antigen (for example, an antigen that is expressed in dormant mycobacteria and not growing mycobacteria) is used to detect immune cells, such as T and B lymphocytes. For example, 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;

(iii) assays in which purified antibody specific for antigen (for example, an antigen that is expressed in dormant or stressed mycobacteria and not growing mycobacteria) is used to detect antigen in the host. For example, purified antibody is bound to solid phase, host tissue is then applied followed by another antibody specific for the antigen to be detected. There are many examples of this approach including ELISA, RIA;

(iv) assays in which a purified anti-idiotypic antibody is used to detect host antibody. For example, anti-idiotypic antibody is adsorbed to solid phase, host serum is added and

anti-Fc antibody is added to bind to any host antibodies having been bound by the anti- idiotypic antibody.

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.

In other embodiments, the infection status of the individual may be unknown. In these embodiments, the individual may or may not have been infected with mycobacterium. Typically, 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,

Corriedale, St. Croix, Barbados Blackbelly, Mouflon, Santa Inez and Royal White and genetic crosses between these breeds.

It will be understood that in certain embodiments, including where the mycobacteria is other than M.ptb, 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.

In one particular embodiment, 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.

Typically 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.

Where the purpose of intervention is to treat an individual having clinical symptoms of disease, the individual is provided with the formulation or vaccine at about the time that the symptoms of disease are detected.

In certain embodiments where the mycobacterium is M.ptb and the individual to be provided with the formulation or vaccine is ovine or bovine, 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.

As discussed in more detail in the Examples, 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.

For example, 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.

In other embodiments there is provided a kit for use in inducing an immune response against a mycobacterium, the kit including an immunogen in a form as described above.

In certain embodiments 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.

Examples

Example 1 : Inducing dormancy in Mycobacterium avium subsp. paratuberculosis by temperature flux.

Materials and methods

1.1 Preparation of bacterial suspensions

Suspensions of M.pth S and C strains with a concentration of 4.3x10 ⁷ viable cells/ml (stock suspensions) 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. Some was diluted in PBS with Tween 80 (0.1%v/v), to prepare the required viable cells/ml for different experiments (see below). Suspensions were then dispensed into 100 μl aliquots in 200 μl thin walled PCR Eppendorf tubes (Scientific Specialties Inc., CA, USA) for use in thermocycler experiments.

1.2 Design of temperature and time patterns

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. Conceptually, 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.

1.3 BACTEC culture

After inoculation with 100 μl suspension, 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.

1.4 Experiment 1 -Temperature flux experiments

Experimental parameters are summarized in Table 1. In each experiment, 100 μl of S strain suspension was placed in each PCR tube. A positive and a negative control were included in every experiment. The positive control consisted of 100 μl bacterial suspension in culture media and a negative control consisted of culture media without bacterial suspension. Two tubes were removed from the thermocycler after every nth cycle as shown in Table 1 and inoculated immediately into radiometric BACTEC media.

1.5 Experiment 2-Effect of constant heating on survival of M.ptb

Peak temperature conditions were simulated in a thermocycler in thin walled 200 μl PCR tubes. 100 μl 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.

The experimental conditions are summarized in Table 2. One tube was removed from the thermocycler at each time point and immediately inoculated into the culture media, except in experiments 2c and 2d, where tubes were held at room temperature until all the samples had been collected, and then all were inoculated into the culture media.

1.6 Experiment 3-Evaluation of thermal tolerance of the C and S strains due to temperature flux and constant heating

Experimental parameters for temperature flux experiments (Experiment 3.1 a-c) are summarized in Table 3. For each strain, a tube was removed from thermocycler after intervals as shown in Table 3 and immediately inoculated into the culture media.

In an experiment on constant heating (Experiment 3.2), 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.

1.7 Experiment 4-Further evaluation of thermal tolerance and comparison of C and S strains

This experiment was carried out to assess the repeatability of Experiment 3.2 and to test viability at intermediate time points from the previous experiment. M.ptb suspensions were passed through a 26 g tuberculin syringe to remove the clumps before heating in the thermocycler. Experimental parameters are summarized in Table 5. Each tube was removed from the thermocycler at intervals as summarized in Table 5 and inoculated into culture media as described previously. Experiments 3.2 and 4a-c were designed to provide exposure to peak temperatures matched to the total duration of peak periods stated for Experiment 3.1b and 3.1c, to determine whether temperature

flux or time at peak was the most detrimental to the organism. Cycle equivalents were calculated to compare the detrimental effect of temperature flux and time at peak (constant temperature). One cycle equivalent was equal to 4.5 min (i.e. peak time period in one cycle) in Experiment 3.1b and 3.1c.

Table 1 Thermocycler parameters for Experiment 1 to evaluate the effect of temperature flux on the viability of the S strain of M.ptb

Experiment Number Temperature Temperature Concentration Sample Total duration of cycles range ( ⁰ C) pattern for of M.ptb collection of each cycle (viable every nth thermocycling ⁰

(mins) ^c cells/ml) cycle

N ^a

1a 28 4 14-30 4-8-4 -8 4.3x10' 4 11 h 12m

8 18-34

8 18-42

8 18-50

1b 42 18-50 4-8-4 -8 4.3*10 ⁷ 14 16 h 48 m

4.3x10 ⁵

1c 84 18-50 4-8-4 -8 4.3x10 ⁵ 14 33 h 36 m

1d 42 20-60 4-8-4 -8 4.3x10 ⁷ 14 16 h 48 m

4.3x10 ⁵

1e 42 12-60 8.5 - 3 - 4.5 - 4.3x10 ⁵ 3 16 h 48 m 8

^a Total number of cycles b Subcycles in Experiment 1 a

Temperature pattern as trough-ascending-peak-descending phases d h = hour; m = minute

Table 2 Thermocycler parameters for Experiment 2 to evaluate the effect of constant heating on the survival of the S strain of M.ptb. M.ptb suspensions were heated in 200 μl PCR tubes in a thermocycler machine and sampled at the times shown

Experiment Temperature Total Concentration of Sampling times ' duration M.ptb (viable ( ⁰ C) cells/ml)

2a 50 48 h 4.3x10° 2 h, 16 h, 32 h, 48 h 2b 60 6 h 4.3x10 ⁵ 10 m, 20 m, 30 m, 40 m, 50 m, 1h, 2 h, 3 h, 4 h, 5 h, 6 h

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

a u h - = hour; m = minute; s = seconds

Table 3 Thermocycler parameters for Experiment 3.1 to evaluate the effect of temperature flux on the viability of the S and C strains M.ptb

Experiment Number of Temperature Temperature pattern Concentration of Sample collection Total duration of cycles range ( ⁰ C) for each cycle (mins) ^a M.ptb C and S strains every nth cycle thermocycling ^b

(viable cells/ml)

3.1a 168 18-50 4-8-4-8 4.3x10° 7 67 h 12 m 3.1b 168 10-50 7.5-4-4.5-8 4.3x10 ⁶ 7 67 h 48 m 3.1c 10 12-60 8.5-3-4.5-8 4.3^10 ^β 1 4h

^a Temperature pattern as trough-ascending-peak-descending phases b h = hour; m = minute

Table 4 Thermocycler parameters for Experiment 3.2 to evaluate the effect of constant heating on the survival of the C and S strains of M.ptb

Experiment Temperature Total Concentration of M.ptb Sampling times ^a duration C and 5 strains (viable ( ⁰ C) cells/ml)

3.2a 50 48 h 4.3x10° 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, 42 h, 44 h, 46 h, 48 h

3.2b 60 40 m 4.3x10 ^d 0.5 m, 1 , 1.5 m, 2 m, 2.5 m, 3 m, 3.5 m, 4 m, 4.5 m, 5 m, 5.5 m, 6 m, 6.5 m, 7 m, 7.5 m, 8 m, 8.5 m, 9 m, 9.5 m, 10 m, 12 m, 14 m, 16 m, 18 m, 20 m, 22 m, 24 m, 26 m, 28 m, 30 m, 35 m, 40 m to

3.2c 70 3 m 4.3x10 ⁶ 5 s, 10 s, 15 s, 20 s, 25 s, 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, 60 s, 65 s, 70 s, 75 s, 80 s, 85 s, 90 s, 95 s, 100 s, 105 s, 110 s, 115 s, 120 s

3.2d 80 3 m 4.3x10 ⁶ 5 s, 10 s, 15 s, 20 s, 25 s, 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, 60 s, 65 s, 70 s, 75 s, 80 s, 85 s, 90 s, 95 s, 100 s, 105 s, 110 s, 115 s, 120 s

^a h = hour; m = minute; s = seconds

M.ptb suspensions were heated in 200 μl PCR tubes in thermocycler machine

Table 5 Thermocycler parameters for Experiment 4 to evaluate the reproducibility of Experiment 3.2

Experiment Temperature Total Concentration of Sampling times ^a duration M.ptb C and S ( ⁰ C) strains (viable cells/ml)

4a 60 45 m 4.3x10° 8 m, 10 m, 12 m, 14 m, 16 m, 18 m, 20 m, 22 m, 24 m, 26 m, 28 m, 30 m, 35 m, 40 m

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

^a m = minute; s = seconds M.ptb suspensions were heated in 200 μl PCR tubes in thermocycler machine

1.8 Dormancy based on M.ptb inoculum size and lag phase

To determine the inoculum size for M.ptb, cumulative growth indices (CGIs) were calculated and plotted against days of incubation.

Log<ιo inoculum size = 8.59-0.185 days to CGH 000

During the lag phase of the bacterial growth cycle there is no increase in cell number. However, 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. By inference, noting that 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.

1.9 Experiment 5-ldentification of proteins expressed by C and S strains of M.ptb after temperature flux treatment

Fresh suspensions of C and S strain of M.ptb (4.3x10 ⁷ cells/ml) were subjected to temperature flux treatment to induce stress proteins. Experimental parameters were 10- 50 ⁰ C except that the duration of thermocycling was extended to 420 cycles (168 h) and samples were collected at 322 and 420 cycles. PCR tubes (n = 10 per sample) each with 100 μl of mycobacterial suspension were used. At each time point, the whole suspension (1 ml) after thermocycling was pooled into 2 ml screw cap polypropylene tubes (Scientific Specialties Inc, catalogue number 233000) separately for S and C strain. 0.9 ml of the treated suspension was centrifuged at 21 ,910 x g and the pellet was stored at -80 ⁰ C until processing for protein extraction and 100 μl was inoculated into BACTEC culture media to determine the growth pattern. Two samples were collected for each strain at each time point for protein analysis.

1.10 Protein sample preparation Protein samples were prepared with the 160 μl 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 μg of protein sample was processed with 2D clean up kit (Amersham Biosciences, catalogue number 80648451 ) as described by the manufacturer.

1.11 2-D PAGE

For each strain (C and S) of M.ptb, 60 μl (~ 60 μg) of the cleaned protein sample was diluted in 90 μl of protein sample extraction buffer and used to re-hydrate a 7 cm IPG strip (pH 4-7) passively, according to the BioRad ReadyStrip™ IPG strip instruction manual. Iso-electric focusing (IEF) was undertaken in a BioRad Protean IEF unit using the conditions described in the instruction manual and IPG strips were focused at 15000 Vh. IPG strips were equilibrated in a two-step equilibration procedure as described in the BioRad ReadyStrip™ IPG strip instruction manual. Briefly, 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 ImageScanner™ Il via LabScan™ software (Amersham Biosciences).

1.12 In gel digestion

The protein expression patterns of control M.ptb cells and cells that had been subjected to temperature flux were compared. Proteins spots showing distinctly different expression patterns between control and temperature flux samples were identified from 2-D gels. Gels from which spots were excised for mass spectrometric analysis were prepared by washing the gels in MQW for 5 min. The spots of interest were excised using a fresh scalpel blade with the excision being made as close as possible to the boundary of the stain. The gel pieces were then placed in clean-labelled 2 ml screwcap plastic tubes and a small volume of liquid was left to keep the gel slice hydrated. Silver stained spots were digested as follows. Briefly, gel spots were incubated with DTT (20

mM) in NH ₄ HCO ₃ (30 μl, 25 mM) for 30 min at 37°C; after removal of the liquid the spot was incubated with iodoacetamide (30 μl, 40 mM) in NH ₄ HCO ₃ (30 μl, 25 mM) for 30 min at 37°C. The spot was washed with CH ₃ CN (2 x 50 μl, 10 min). Trypsin (-100 ng) in NH ₄ HCO ₃ (10 mM, 25 μl) 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 μl) and H ₂ OiCH ₃ CN (1 :1) (0.1% formic acid, 50 μl) 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 μl.

1.13 Mass spectrometry

Digested peptides were separated by nano-Liquid Chromatography (LC) using a Cap- LC autosampler system (Waters, Milford MA). Samples (5 μl) were concentrated and desalted onto a micro C18 precolumn (500 μm x 2 mm, Michrom Bioresources, Auburn, CA) with H ₂ OiCH ₃ CN (98:2, 0.05 % HFBA) at 15 μl/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. 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 μ) 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). Positive ions were generated by electrospray and the Q- TOF operated in data dependent acquisition mode (DDA). A Time of Flight Mass Spectrometer (TOF-MS) survey scan was acquired (m/z 350-1700, 1 sec) and the 2 largest multiply charged ions (counts > 20) were sequentially selected by Q1 for tandem mass spectrometry or MS-MS analysis. Argon was used as collision gas and an optimum collision energy chosen (based on charge state and mass). Tandem mass spectra were accumulated for up to 8 sec (m/z 50-2000). Peak lists were generated by MassLynx (Micromass) using the Mass Measure program and submitted to the database search program Mascot (version 2.1 , Matrix Science, London, England). Search parameters were: Precursor and product ion tolerances ± 0.25 and 0.2 Da respectively; Met (O) and Cys-carboxyamidomethylation specified as variable modification, enzyme specificity was trypsin, 1 missed cleavage was possible. Mass

spectrometric analysis for this work was carried out at the Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Sydney, Australia.

The matches with Mascot probability scores greater than 50 (p<0.05) 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. Spot normalization was made using relative volumes (%Vol) to quantify and compare the gel spots; %Vol corresponds to the volume of each spot divided by the total volume of all the spots in the gels. The normalized volume of an identified protein spot in control and temperature flux gels was compared to calculate the ratio value. The analysis was conducted only on spots which were identified by mass spectrometry.

1.14 Preparation of samples prior to PCR and dilution series of DNA The preparation of BACTEC samples and colonies for PCR was conducted as described previously. Briefly, the rubber stopper-lid of the radiometric culture vial was disinfected with 70% ethanol, the contents were mixed by inverting the tube, and 200 μ\ of medium was removed and transferred to a microcentrifuge tube. Absolute ethanol (500 μ\) was added, and the tube was left to stand for 2 min and then was vortexed for 5 s and centrifuged at 8 x g for 10 min at 22 ⁰ C. The supernatant was transferred to a

clean tube and then centrifuged at 18,000 x g for 5 min. The resulting bacterial pellet was washed twice in 200 μ\ of sterile phosphate-buffered saline, resuspended in 50 μ\ of sterile distilled water, and lysed at 100 ⁰ C for 20 min. The lysate was then stored at - 2O ⁰ C. For solid media, 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 μ\ 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 .

Results

1.15 Experiment 1 -Temperature flux experiments

The controls achieved peak Growth Index (Gl) after 2-3 weeks of incubation in each experiment. In Experiment 1a, there was no effect on growth pattern of various temperature cycle ranges. However, growth was delayed progressively with increasing number of cycles at temperature cycle range of 18-50 ⁰ C. At higher temperature flux and greater peak temperature (20-60 ⁰ C and 12-60 ⁰ C), no growth was recorded from either cell concentration after 3-14 cycles, but there were no intermediate time points to assess survival.

1.16 Experiment 2-Constant heating in a PCR thermocycler

The controls achieved peak Gl after 2-3 weeks of incubation in each experiment. At 50 ⁰ C, the last time point with viable S strain of M.ptb was 2 h (Table 2). However, M.ptb did not survive heating for 10 min at 60°C. M.ptb survived at 7O ⁰ C and 80 ⁰ C for 30 s, where peak Gl was observed after 4-5 weeks of incubation, and complete inactivation was achieved within 45 s.

1.17 Experiment 3-Evaluation of thermal tolerance of C and S strains The controls achieved peak Gl after 2-3 weeks of incubation in each experiment. Using a temperature cycle with a range of 18-50 ⁰ C ₁ both S and C strains survived even after 168 cycles, but the time to reach peak Gl was delayed as the number of cycles increased. After 161 cycles of temperature flux treatment, a unique growth pattern was

observed; there was a lag phase or no growth in BACTEC cultures up to 5 weeks, but afterwards growth occurred quickly and peak Gl was attained within 6-7 weeks. In contrast, a greater temperature flux of 10-50 ⁰ C resulted in loss of viability of S and C strains after only 147 cycles. 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.

1.18 Effect of constant heating

The controls achieved peak Gl after 2-3 weeks of incubation in each experiment. There was a progressive increase in time to peak Gl with increasing heating times. The C strain was unable to survive after 18 h (240 cycle equivalents) of heating at 50 ⁰ C; however, the S strain did not survive constant heating at 5O ⁰ C for more than 16 h (213 cycle equivalents). A lag phase of 4 weeks was observed for C strain after 18 h of heating. At 60 ⁰ C, 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). A lag phase of 4 weeks was seen for S and C strains after 20 and 20, 24 and 30 min of heating, respectively. At 70 ⁰ C, 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. DNA extracts were serially diluted (10 ^'1 to 10 ^"8 ) and confirmed by PCR. Colonies from the modified Middlebrook 7H10 slopes were also tested. The samples without growth had either negative or trace results in the PCR, suggestive of the residual inoculum. Samples with evidence of growth (GMO) were positive in PCR at a dilution of 10 ^"3 .

1.19 Experiment 4-Further evaluation of thermal tolerance and comparison of C and S strains

The controls achieved peak Gl after 2-3 weeks of incubation in each experiment. At

60 ⁰ C, S and C strains were inactivated after 28 min (6 cycle equivalents) and 30 min (6.5 cycle equivalents) of heating, respectively. A lag phase of 3 weeks was recorded for S and C strains after 28-30 min of heating at 60 ⁰ C. The last time point with viable

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.

Table 6 Summarized dataset of constant heating and temperature flux experiments for M.ptb. Dormancy was defined as growth in BACTEC medium after a lag phase > 4 weeks

Temperature Viable Maximum duration of Dormancy after nth cycle cells/ml survival at peak temperature (°C) a

S strain C strain S strain C strain

Constant temperature

50 4.3x10 ⁶ 16 n or 18 n or - -

213 CE ^b 240 CE

60 4.3x10 ⁶ 28 m or 30 m or - -

6 CE 6.5 CE

70 4.3x10 ⁶ 30 s 35 s - -

80 4.3*10 ⁶ 25 s 30 s - -

Temperature flux

10-50 4.3x10 ⁶ 11 h ^c 11 h ^c 140 140

18-50 4.3x10 ⁶ 11 h 12 m ^c 11 h 12 m ^c 161 161

12-60 4.3x10 ⁶ 9 m 13.5 m 20-60 4.3x10 ,5-7 nd nd

^a h = hour; m = minute; s = seconds b Cycle equivalents

⁰ Total duration for which the organism was exposed to peak period in temperature flux experiments nd = not detected

1.20 Lag phase and inoculum size of M.ptb after constant heating and temperature flux

The inoculum size of M.ptb was calculated for all the samples in Experiments 3, 4 and 5 to assess the evidence for dormancy (Table 7). Data for lag phase after constant heating and temperature flux have been described in Table 7. It would appear that a consistent pattern of lag phase ^ weeks was observed for temperature flux experiments except a hypothetical temperature range of 12-60°C resulted in a lag phase of 3 weeks. Moreover, <10 viable cells were found throughout the experiment conducted at different times (Table 7). At 60°C and 7O ⁰ C, albeit a lag phase of 4 weeks or inoculum size of <10 viable cells was seen but the results were not reproduced in another experiment (Experiments 4a and 4b).

Table 7 Lag phase and inoculum size of M.ptb in constant heating and temperature flux experiments

Experiment Strain Temperature Viable Lag Log-ioinoculum 'Viable cells ("C) cells/ml ^a phase size (Weeks)

Constant temperature

3.2a S 50 4.3x10 ⁶ 3 1.75 55

C 50 4.3x10 ⁶ 4 1.19 16

3.2b S 60 4.3x10 ⁶ 4 0.82 7

C 60 4.3x10 ⁶ 4 0.82 7

3.2c S 70 4.3x10 ⁶ 3 4.33 21627

C 70 4.3x10 ⁶ 4 1.00 10

3.2d S 80 4.3x10 ⁶ 2 4.33 21627

C 80 4.3x10 ⁶ 1 4.33 21627

4a S 60 4.3x10 ⁶ 3 2.11 130

C 60 4.3x10 ⁶ 3 2.3 200

4b S 70 4.3x10 ⁶ 2 3.04 1097

C 70 4.3x10 ⁶ 3 2.67 468

4c S 80 4.3x10 ⁶ 3 2.48 306

C 80 4.3x10 ⁶ 3 2.11 130

Temperature flux

3.1a S 18-50 4.3x10 ⁶ 5 0.08 1

C 18-50 4.3x10 ⁶ 5 0.08 1

3.1b S 10-50 4.3x10 ⁶ 5 0.45 3

C 10-50 4.3x10 ⁶ 6 -0.66 < 1

3.1c S 12-60 4.3x10 ⁶ 3 2.11 130

C 12-60 4.3x10 ⁶ 4 1.00 10

5 S 10-50 4.3x10 ⁷ 4 0.26 2

C 10-50 4.3x10 ⁷ 5 0.08 1 ^a viable cells at he start of experiment b Log ₁₀ inoculum size of M.ptb was calculated after temperature flux and constant heating as described previously ⁴⁵⁹

1.21 Experiment 5-Protein expression by C and S strains of M.ptb after temperature flux treatment

The controls achieved peak Gl after 2 weeks of incubation. At a temperature range of 10-50 ⁰ C, both S and C strains survived even after 420 cycles of temperature flux. The S and C strain achieved peak Gl in 7 weeks after 420 cycles, with a lag phase of 4-5 weeks. Protein expression was investigated for both strains at 322 and 420 cycles. ImageMaster 2D Platinum generated 598 match pairs between control and temperature flux gels for the C strain. In C strain, a total of 27 differentially expressed proteins after temperature flux were selected for identification by mass spectrometry. Similarly, ImageMaster 2D platinum generated 493 match pairs between control and temperature flux gels for the S strain. Eleven protein spots showed alteration in protein expression for the S strain and these were selected for identification by mass spectrometry. The protein expression for both strains was similar at lower (322 cycles) and higher levels (420 cycles) of exposure to temperature flux. Protein identification data are provided in Tables 8 and 9.

Table 8 Differentially expressed protein profiles of C strain using a temperature flux cycle of 10-50 ⁰ C for 322-420 cycles

Spot Accession Gene/Locus Protein description lnterpro Scan Family/Function M.ptb Mass pr Tuberculist M.S.D ^f Ratio ⁹ No." No. * tag ⁶ homologue ^c

(kDa) Synonym ^e

41408796 desA2, Acyl-Acyl Carrier Protein Fatty acid metabolism, 31.4 4.7 Rv1094

MAP2698c desaturase dodecameric ferritin homologue that binds and protects DMA

41410223 rplJ, 50s ribosomal protein L10 protein synthesis 20.1 5.08 Rv0651 0.054 2.351

MAP4125

41409675 fabG3_2, FabG3 2 Oxidoreductase activity 25.9 5.62 Rv2002 0.075 3.368 ω

MAP3577 OO

41409675 fabG3_2, FabG3 2 Oxidoreductase activity 25.9 5.62 Rv2002 0.022 1.595

MAP3577

41407266 MAP1168c Hypothetical protein MAP1168c Oxidation of long chain aldehydes 31.9 5.92 0.063 2.907 and releases energy in the form of visible light

6 41409665 MAP3567 Hypothetical protein MAP3567 Oxidoreductase activity 30.1 5.98 RvO148 0.151 1.700

7 41408970 fabG, 3-ketoacyl- reductase Oxidoreductase activity 26.7 5.94 Rv2766c 0.075 1.982

MAP2872c

8 41406614 ecM20, Enoyl-CoA hydratase Fatty acid metabolism 26.8 5.91 Rv3550 0.056 1.930

MAP0516c echA20

41406606 MAP0508 Short chain dehydrogenase Oxidoreductase activity 27.5 5.82 Rv3559c 0.008 1.184

10 41407115 echA8_1, Enoyl-CoA hydratase Fatty acid metabolism 27.8 5.51 Rv1070c 0.068 1.962

MAPI 017c

11 41409665 MAP3567 Hypothetical protein MAP3567 Oxidoreductase activity 30.1 5.98 RvO148 0.120 3.500

12 41407693 bfrA, Bacterioferritin subunit Iron ion transport and storage 18.4 4.41 bfr, Rv1876 0.034 2.479

MAP1595

13 41407751 tpx Putative thiol peroxidase Antioxidant enzymes and defense 16.4 4.24 tpx, cfp20, - *

MAPI 653 against sulphur containing radicals Rv1932

14 41409491 purE, Phosphoribosyi aminoimidazole 'de novo' IMP biosynthesis 17.5 5.68 Rv3275c

MAP3393C carboxylase catalytic subunit purE

15 41408585 MAP2487C Hypothetical protein MAP2487c Carbonic anhydrase, Carbon 17.8 5.61 Rv1284 0.032 1.519 utilization and prevents depletion CD of cellular bicarbonate

16 41406638 MAP0540 Hypothetical protein MAP0540 Tranferases composed of 17.6 6.13 Rv3525c hexapeptide, LpxA-like

17 41409366 hsp18_3, Hsp18_3 Alpha crystallin family protein. 16.4 4.88 - - *

MAP3268 Respond to heat shock or other environmental stress and act as chaperons

18 41407796 hsp18_2, Hsp18_2 Ditto 16.3 5.06 0.093 6.009

MAPI698c

19 41407796 hsp18_2, Hsp18_2 Ditto 16.3 5.06 -

MAPI698c

20 41406691 MAP0593c Hypothetical protein MAP0593c diadenosine polyphospahte 14.8 5.25 -

hydrolases and function as tumor suppressors in human and mice

21 41409799 hsp, Hsp Alpha crystallin family protein 16.2 5.32 RvO251c, 0.045 55.067

MAP3701c acr2

22 13879620 41409636 Hypothetical protein MAP3538 Oxidoreductase activity, synthesis 15.9 6.1 Rv0130 - * of monoamine oxidase

23 41410196 MAP4098 Cyanate hydratase Bacteria can overcome the toxicity 18.7 5.8 of environmental cyanate by hydrolysis of cyanate

24 41408021 MAP1923C Hypothetical protein MAPI 923c Antioxidant enzymes and defense 16.4 5.15 Rv2185c, against sulphur containing TB16.3

4^ radicals, σ

25,26 136429 Trypsin precursor a Spot numbers correspond to those in the relevant 2D gel image (i.e. a figure) b Accession number and locus tags are from NCBInr database (http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?db=Protein&itool=toolbar) c Homologues in M.ptb were identified when significant hits belong to other Mycobacterium spp. d Theoretical isoelectric point (pi) of matching protein was calculated by Biomanager (http://www.angis.org.au/) 5 ^e Homologues in M. tuberculosis were identified by H37Rv genome sequence database (http://genolist.pasteur.fr/Tuberculist/) 1 Mean squared deviation was calculated by lmageMaster2D Platinum

⁹ 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

Table 9 Differentially expressed protein profiles of S strain using a temperature flux cycle of 10-50 ⁰ C for 322-420 cycles

Spot Accession Gene/Locus tag Protein description lnterpro Scan Family/Function Homologue Mass pi Tuberculist M.S.D Ratio ^g No. ^a No. " to M.ptb" (kDa) Synonym , ' e

41409288 fadB4, MAP3190 FadB4 Zinc ion binding, Oxidoreductase - 33.3 5.27 Rv3141 activity. Similar to GroES family which helps in protein folding and intercellular signaling.

41408379 clpP, M AP2281c ATP dependant CIp Proteolysis 21.6 4.62 Rv2460c protease proteolytic subunit 4^

41408379 clpP, MAP2281c ditto Proteolysis 24.5 6.36 dp,

Rv2461c

4,5 13883596 ppa, MT3730 Inorganic Phosphate metabolism, ppa, 18.3 4.58 pyrophosphatase (M. magnesium ion binding MAP0435c tuberculosis CDC1551)

41409653 MAP3555 Hypothetical protein Unknown 18.8 4.76 RvO138

41410205 MAP4107 Hypothetical protein Unknown 17.7 4.56

41408548 afpC,MAP2450c ATP synthase subunit membrane bound enzyme 13.1 4.23 Rv1311 0.039 1.691 epsilon complexes involved in ATP synthesis coupled proton transport

62616 GAPDH Glyceraldehydephosphate Glycolysis and gluconeogenesis gap, 35.6 8.81 Rv1436, dehydrogenase (Coturnix MAP1164 coturnix) gap

10 7331218 Keratin

£

11 136429 Trypsin precursor

Spot numbers correspond to those in the relevant 2D gel image (i.e. a figure)

⁶ Accession number and locus tags are from NCBInr database (http://www.ncbi.nIm.nih.gov/entrez/query.fcgi?db=Protein&am p;itool=toolbar) 0 Homologues to M.ptb were identified when significant hits belong to other Mycobacterium spp. d Theoretical isoelectric point (pi) of matching protein was calculated by Biomanager (http://www.angis.org.au/) 8 Homologues to M. tuberculosis were identified by H37Rv genome sequence database (http://genolist.pasteur.fr/Tuberculist/) f Mean squared deviation was calculated by ImageMaster 2D Platinum

³ 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.

Materials and methods

Bacteria! suspensions were prepared as per Example 1. 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.

2.1 Experiment 1 - Experimental design for hypoxia induced dormancy model

The study was conducted in two different ways to examine the responses of hypoxic shock on M.ptb. In the first experiment, the bacteria were initially exposed to aerobic conditions for entry into exponential growth phase which was followed by sudden exposure to anaerobic conditions. In contrast, in the second experiment 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.

2.2 Experiment 2 - Hypoxic shock after a shift from aerobic to anaerobic conditions

In the first experiment, the culture vials (in duplicate) for each time point (n = 8) were initially preequilibrated once with aerobic gases by exchanging the gas phase of the culture vials with a BACTEC 460 ion chamber machine and were incubated at 37°C for 1 week. 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 μl of MQW for inoculation into BACTEC12B culture vials. After 1 week of aerobic conditions, 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. After each week, 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.

2.3 Experiment 3 - Hypoxic shock with anaerobic conditions only

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.

2.4 Experiment 4 - Experimental design for nutrient starvation induced dormancy model

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 μl 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. Following 8 and 14 weeks of nutrient starvation, samples were pelleted by centrifugation (21 , 910 x g) and stored at - 80 ⁰ C for proteome analysis of M.ptb. To assess the loss in viability of M.ptb subsequent to starvation, viable cells/ml were enumerated from 14 week starved samples by the most probable number (MPN) method.

2.5 Mass spectrometry

The protein expression patterns of control M.ptb cells and cells under the effects of starvation and hypoxia were compared. Proteins spots differing between control and starvation or hypoxia samples were excised from 2-D gels and identified by mass spectrometry as described above. 2-D gel analyses were conducted using ImageMaster 2D Platinum (Amersham Biosciences).

Results

2.6 Growth pattern of M.ptb during hypoxia

In Experiment 1 , culture vials with M.ptb were initially preequilibrated with aerobic conditions for a week before exposure to anaerobic gases. No substantial difference in Gl was observed between controls and the culture vials under anaerobic conditions for both strains of M.ptb after 7-28 days of hypoxic stress. There was a period of rapid increase and plateauing of Gl followed by a gradual decline after about 4 days. There was a slight increase in Gl after restoration of aerobic conditions. In contrast, in Experiment 2, when not exposed to an initial anaerobic period, the growth of both strains was restricted compared to control cultures, but rapid growth ensured upon provision of aerobic atmosphere with Gl of 999 reached in 2-4 weeks. 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.

2.7 Growth pattern of M.ptb during starvation 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.

2.8 Proteome of M.ptb during hypoxia

Sequential protein expression was investigated for both the strains after 1 and 4 weeks of hypoxic stress. ImageMaster 2D Platinum (Amersham Biosciences) generated 568 match pairs between controls and cells for the hypoxia treatment for the S strain. However, a total of 15 spots were differentially expressed after hypoxic stress in the S strain relative to the control. Likewise 559 match pairs were generated by ImageMaster 2D Platinum for the control and hypoxic cells of the C strain.10 protein spots showed differential expression for the C strain. The protein expression pattern for both the strains was similar at lower (1 week) and higher duration (4 weeks) of exposure to hypoxic stress. Further, the protein expression was also identical in both the experiments (described above) under hypoxia irrespective of the pattern of anaerobic condition. Protein identification data are provided in Tables 10 and 11.

Table 10 Differentially expressed protein profiles of the S strain after 1- 4 weeks of hypoxic stress

Spot Accession Gene/Locus Protein description lnterpro Scan M.ptb Mass pi Tuberculist M.S.D Ratio ³

No. ^a No. ' tag* Family/Function homologue ^c (kθa) Synonym ^e

41407395 hisA 1-(5-phosphoribosyl)-5-[(5- Enzymes that use - 25.3 4.53 hisA phosphoribosylamino) FMN as a cof actor,

MAP1297 Histidine biosynthesis Rv1603 methylideneamino] imidazole- 4-carboxamide isomerase

41406932 MAP0834c Hypothetical protein Two component 24.9 4.81 Rv0903c MAP0834c response regulator activity and respond to prrA environmental changes

41406741 MAP0643C Hypothetical protein Unknown 25.3 5.21 MAP0643c

4,5 41408379 clpP, ATP dependant CIp protease Proteolysis 21.6 4.62 Rv2460c proteolytic subunit

MAP2281C

6,7 41406533 ppa, inorganic pyrophosphatase Phosphate 18.6 4.59 Rv3628 metabolism,

MAP0435C magnesium ion binding

41408548 afpC, ATP synthase subunit epsilon membrane bound 13.1 4.23 Rv1311 enzyme complexes

MAP2450C involved in ATP synthesis coupled proton transport

33327135 h hbbhhaa Heparin binding Signal recognistion 20.7 7.11 RvO475 haemagglutiniπ adhesiπ like particle protein, RNA

Oo

MAP3968 protein binding and GTP hbhA binding

10 48928144 greA Probable transcription Necessary for efficient greA 17.8 4.66 Rv1080c elongation factor G (M avium) RNA polymerase transcription past MAPI 027c greA template encoded arresting sites

11 41410205 MAP4107 Hypothetical protein MAP4107 Unintegrated 17.7 4.56

12 41408803 MAP2705c Hypothetical protein Nuclear taπsport of 13.9 4.59 0.050 2.89 MAP2705c cargo protein, Steroid delata isomerase catlyzes isomerisatioπ of unsaturated ketosteroids

13 41407174 MAP1076 Hypothetical protein MAPI 076 Unknown 14.4 5.17

14 41407684 MAPI 586 Hypothetical protein MAP1586 Unknown 16.9 7.1

CO

15 136429 - Trypsin precursor a Spot numbers correspond to those in the relevant 2D gel image (i.e. a figure) b Accession number and locus tags are from NCBInr database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein&am p;itool=toolbar)

⁰ Homologues in M.ptb were identified when significant hits belong to other Mycobacterium spp. d 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 ImageMaster 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, i indicates downregulation and was not identified in the S strain after hypoxia

Table 11 Differentially expressed protein profiles of the C strain after 1- 4 weeks of hypoxic stress

Spot Accession Gene/ Protein description lnterpro Scan Family/Function M.ptb Mass pi " Tuberculist M.S.D ' Ratio ⁹ No. ³ No. * Locus tag* homologue ⁰ (kDa) Synonym ^e

41406533 ppa, Inorganic Phosphate metabolism, magnesium - 18.6 4.59 Rv3628 0.018 1.762 pyrophosphatase ion binding

MAP0435C

41406533 ppa, Inorganic Phosphate metabolism, magnesium 18.6 4.59 Rv3628 0.028 2.388 pyrophosphatase ion binding

MAP0435C

O

73698151 tpx, MAPI 653 Putative thiol Antioxidant enzymes and defense 16.4 4.24 Tpx, cfp20, - peroxidase against sulphur containing radicals Rv1932

41407686 ahpD, AhpD aromatic compound metabolism, 18.8 4.79 Rv2429 AhpD for peroxidase activity

MAP1588c

41406166 ssb, Single strand DNA DNA replication, recombination and 17.5 4.94 RvOO54 0.028 2.388 binding protein repair

MAP0068

41409491 purE, Phosphoribosyl 'de novo' IMP biosynthesis 17.5 5.68 Rv3275c 0.029 3.488 aminoimidazoie

MAP3393c carboxylase catalytic subunit

41407658 MAPI 560 Hypothetical protein Catalytic activity with thioesterases 15.2 4.94 MAPI 560

41409298 MAP3200 Hypothetical protein Two component response regulator 14.7 5.67 Rv3143

MAP3200 activity and respond to environmental changes. These have been detected during host Cλ invasion and drug resistance

41406691 MAP0593C Hypothetical protein Histidine triad protein, diadeπosine 14.8 5.25 RvO759c 0.088 3.328 MAP0593c polyphospahte hydrolases and function as tumor suppressors in human and mice

10 41407437 MAP1339 Hypothetical protein Universal stress protein, Response 15.4 6.12 MAPI 339 to stress, Trancriptional induction of

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) b Accession number and locus tags are from NCBInr database (http://www.ncbi.nlm.nih.gov/entrez/query .fcgi?db=Protein&itool=toolbar) c Homologues in M.ptb were identified when significant hits belong to other Mycobacterium spp. d 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.

2.9 Proteome of M.ptb during starvation

2D gel analyses revealed synthesis of starvation induced proteins and repression of many proteins. ImageMaster 2D platinum generated 635 match pairs between controls and gels produced under starvation for the S strain and a total of 25 spots were detected by mass spectrometry that were differentially expressed after 8 weeks of starvation. However, 389 match pairs were detected by 2D gel analyses for the C strain and 16 spots with differential protein expression were identified by mass spectrometry. Protein identification data are provided in Tables 12 and 13.

Table 12 Differentially expressed protein profiles of the S strain after 8 weeks of starvation

Spot Accession Gene/Locus tag Protein description lnterpro Scan M.ptb Mass pi Tuberculist M.S.D Ratio ⁹

No. ^a No. ' Family/Function homologue ⁰ (kDa) Synonym '

41409105 MAP3007 Hypothetical protein MAP3007 Oxidoreductase activity 29.9 4.37 Rv2971

41407987 wag31, MAP1889c Wag31 DivlVA 28 4.45 Rv2145c, 0.190 ^" 4.579 ag84

Ol

41407932 prcA, MAPI 834c PrcA Proteasome subunits, 27.9 5.16 Rv2109c, 0.050 30.986 protein degradation prcA

41407944 hisG, MAPI 846c ATP phosphoribosyl transferase Histidine biosynthesis 30.5 4.67 Rv2121c 0.038 3.807 M.ptb hisG

41407459 argC, MAPI 361 N-acetyl gamma glutamyl Arginine metabolism 35.2 6.28 Rv1652 phosphate reductase argC

41408962 cfapA,MAP2864c Dihydrodipicolinate synthase Key enzyme in lysine 30.9 5.62 Rv2753c biosynthesis dapA

41406809 MAP0711c hypothetical protein MAP0711 Oxidoreductase activity

41410223 rpU, MAP4125 50s ribosomal protein L10 Protein synthesis 20.1 5.08 RvO651

rpU en

41408379 cIpP, MAP2281c ATP dependant CIp protease Proteolysis 21.6 4.62 Rv2460c proteolytic subunit

10 13883596 ppa, MT3730 inorganic pyrophosphatase (Mtb Phosphate metabolism, ppa, 18.3 4.58 0.005 8.272

CDC1551) magnesium ion binding MAP0435c

11 13883596 ppa, MT3730 inorganic pyrophosphatase {Mtb Phosphate metabolism, ppa, 18.3 4.58 0.067 18.441

CDC1551) magnesium ion binding MAP0435c

12 41407983 MAPI 885c Hypothetical protein MAPI 885c Regulation of protein 18.4 4.73 Rv2140c phosphorylation by kinases, lipid binding TB18.6

13 41410205 MAP4107 Hypothetical protein MAP4107 Unintegrated 17.7 4.56

14 41408548 atpC, ATP synthase subunit Membrane bound enzyme 13.1 4.23 Rv1311 0.068 3.652 epsilon complexes involved in ATP

MAP2450C synthesis coupled proton transport

15 41408509 MAP2411 Hypothetical protein Electron transfer pathway at low 15.5 4.63 0.021 5.727 MAP2411 redox potential

16 41406691 MAP0593C Hypothetical protein Histidine triad protein, diadenosine 14.8 5.25 RvO759c MAP0593c polyphospahte hydrolases and function as tumor suppressors in human and mice

17 13881271 Ws/, MT1641.1 Phosphoribosyl AMP Histidine biosynthetic pathway hisl2, 12.4 5.15 Rv1606 cyclohydrolase Mtb MAPI 300

18 41407684 MAPI 586 Hypothetical protein Unknown - 16.9 7.1 - i

MAPI 586

19- 136429 - Trypsin precursor - . . . . . .

25

a Spot numbers correspond to those in the relevant 2D gel image (i.e. a figure) b Accession number and locus tags are from NCBInr database (http://www.ncbi.nlm.nih.gov/entrez/query .fcgi?db=Protein&itool=toolbar)

⁰ Homologues in M.ptb were identified when significant hits belong to other Mycobacterium spp. - _^ l

"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

⁹ 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

Table 13 Differentially expressed protein profiles of C strain after 8 weeks of starvation

Spot Accession Gene/Locus Protein description lnterpro Scan Family/Function Homologue Mass pi Tuberculist M.S.D Ratio ⁹

No. ^a No. ' tag' to M.ptb"

(kDa) Synonym

41408156 cysQ_2, CysQ_2 Enhance the synthesis or degradation - 25.4 4.64 Rv2131c

MAP2058C of phosphorylated messenger molecules cysQ

41408970 fabG, 3-ketoacyl- reductase Oxidoreductase activity 26.7 5.94 Rv2766c 0.018 - 1.289

MAP2872C

03

41406614 echA20, Enoyl-CoA hydratase Fatty acid metabolism 26.8 5.91 Rv3550 0.038 - 2.482

MAP0516c

41406606 MAP0508 Short chain dehydrogenase Oxidoreductase activity 27.5 5.82 Rv3559c

41407687 ahpC, AhpC Antioxidant enzymes and defense 21.6 4.32 Rv2428

MAPI 589c against sulphur containing radicals ahpC

41408378 clpP2, ATP dependant CIp protease Proteolysis 23,2 4.84 Rv2460c

MAP2280C proteolytic subunit clpP2

41409962 MAP3864 hypothetical protein Unknown 16.4 4.47 Rv0360c MAP3864

73698151 tpx, Putative thiol peroxidase Antioxidant enzymes and defense 16.4 4.24

MAPI 653 against sulphur containing radicals

Ol

41410438 trxC, TrxC Electron carrier activity and protein 12.4 4.56 mpt46, 0.179 4.353 CD

MAP4340 disulphide oxidoreductase activity Rv3914, trx, trxA

10 41410196 MAP4098 Cyanate hydratase Bacteria can overcome the toxicity of 18.7 5.8 0.147 3.589 environmental cyanate by hydrolysis of cyanate

11 41406691 MAP0593C Hypothetical protein Histidine triad protein, diadenosine 14.8 5.25 RvO759c

MAP0593c polyphospahte hydrolases and function as tumor suppressors in human and mice

12,13 41407437 MAP1339 Hypothetical protein Universal stress protein, Response to - 15.4 6,12 - - *

MAPI 339 stress, Trancriptional induction of

UspA gene of E coli occurs during growth arrest conditions.

14-16 136429 - Trypsin precursor - . . . . . .

a Spot numbers correspond to those in the relevant 2D gel image (i.e. a figure)

"Accession number and locus tags are from NCBInr database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein&am p;itool=toolbar) c Homologues in M.ptb were identified when significant hits belong to other Mycobacterium spp. d Theoretical isoelectric point (pi) of matching protein was calculated by Biomanager (http://www.angis.org.au/) . ._ o 5 ^e Homologues in M. tuberculosis were identified by H37Rv genome sequence database (http://qenolist.pasteur.fr/Tuberculist/) f Mean squared deviation was calculated by ImageMaster 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 starvation. Asterisks ( ^* ) represent proteins that were not identified in control samples i indicates downregulation and was not identified in the C strain after starvation, -ve ratio value = dowπregulation

Example 3: Antigenicity of proteins having increased expression in dormant or stressed Mycobacterium avium subsp. paratuberculosis as compared with growing cells.

Materials and Methods

3.1 Bacterial strains used during this study E.coli TOP 10 cells (Invitrogen): Genotype: F ^r mcrA, A {mrr-hsdRMS-mcr&C), ^80/acZδM15, δ/acX74, recA1 , araD139, δ (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)

3.2 Extraction of M.ptb DNA

Briefly, 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 μi of lysozyme solution (200 mg/ml) and 200 units of mutanolysin (20 μl 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 μl proteinase K solution (10 mg/ml), 210 μl of 10% (wt/vol) SDS and incubated at 65°C (hybridisation oven) for 20 min with gentle mixing every 5 min. 195 μl 5 M NaCI and 165 μl 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! centrifuge tube containing ice cold 70% (vol/vol) ethanol, using a sterile plastic inoculation loop. The DNA was centrifuged at 16,100 x g for 5 min. The supernatant was removed and the DNA was allowed to sit at room temperature with the lid slightly off until dry (1-2 h). The purified DNA was resuspended in sterile TE pH 8.0 by gentle end over mixing at 37°C for 1 h. If the DNA was not fully dissolved this was continued overnight at 4°C. The concentration of DNA samples was determined by spectrophotometry at 260 nm and stored at -2O ⁰ C.

3.3 PCR amplification

To amplify 9 genes which were expressed during various stressful conditions in Examples 1 and 2, primers containing Ncfel and BamHI restriction endonuclease sites were designed on the basis of DNA sequence available for the M.ptb K-10 genome. DNA amplification was carried out in a 50 μl reaction containing 10 ng M.ptb DNA (S strain, Telford 9.2), 200 μM dNTPs, 0.5 μM each primer and 2U Taq polymerase (Expand High Fidelity PCR System, Roche). Each 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.

3.4 Preparation of chemically competent E.coli

A single bacterial colony was allowed to grow overnight in 10 ml of Luria Bertani (LB) broth at 37°C with shaking. 100 μl of overnight culture was inoculated into prewarmed 100 ml of LB broth and grown with shaking to an ODβoo 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 μl), snap frozen on liquid nitrogen and stored at -80°C.

3.5 Transformation of chemically competent E.coli

Briefly, a tube of TOP 10 competent cells was removed from -80 ⁰ C and placed on ice for 10 min to thaw. 50 μl 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 μl 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 μg/ml ampicillin, 1 mM IPTG and 20 μg/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 μg/ml ampicillin and inserts were confirmed by restriction digestion. Plasmids were recovered with a QIAprep® Miniprep (Qiagen) kit.

3.6 DNA ligations

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 μl of fresh PCR product was ligated with 2 μl of pCR2.1 vector (25 ng/μl) with 1 μl of 10X ligation buffer and 1 μl of T4 DNA ligase, the reaction volume was made up to 10 μl and incubated at room temperature overnight. The cloning reaction was transformed into chemically competent TOP10 E.coli cells.

3.7 Restriction Digestion of DNA plasmids

The isolated plasmids were digested with restriction endonucleases Nde\ and Sa/nHI (NEB). The reaction was carried out as a double digest. Briefly, 1 μg of DNA, 1 μl of enzyme (A/de\, BamHl), 2 μl of reaction buffer (NEB 2) and 0.2 μl of BSA (100X) were made up to 20 μl 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.

3.8 DNA Sequencing

Plasmids containing an insert were sent for DNA sequencing using M13 Forward and M13 Reverse primers. DNA sequencing was performed at the Australian Genome

Research Facility Ltd (AGRF Ltd., Australia). Nucleotide sequences were translated to amino acid sequences using the Emboss Transeq database (European Bioinformatics

Institute, http://www.ebi.ac.uk/emboss/transeq/) and were aligned with the nucleotide and amino acid sequences of the K-10 strain of M.ptb using the CLUSTALW alignment tool of the Genome Net database (Bioinformatics Center Institute for Chemical

Research Kyoto University, http://align.genome.jp/).

3.9 Preparation of expression vector pET-15b

The expression vector pET-15b was prepared by digesting with Nde\ and BamHI. Briefly, 500 ng of pET-15b DNA, 1.5 μl of each enzyme (λ/cfel, BamHI) and 10 μl of reaction buffer (NEB 2) were made up to 100 μl 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.

3.10 Subcloning into expression vector pET-15b

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

Nde\ and BamH\ in a total volume of 100 μl as described above. Purified insert DNA was ligated to the digested pET-15b vector. An insert to vector ratio of 3:1 was determined by comparing the gel intensity. Ligation was performed using 1 μl of digested pET-15b vector, 3 μl insert DNA, 1 μl 10X ligation buffer, 1 μl T4 DNA ligase (Roche) brought to a total volume of 10 μl with nuclease free water. The reaction was carried out overnight at 4°C. Transformations were performed into TOP10 E.coli cells and plated on to LB plates containing the appropriate antibiotic selection. The plasmids

were isolated and verified by restriction digestion and sequencing using the T7 promoter primer.

3.11 Expression of recombinant protein

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 μg/ml) and chloramphenicol (34 μg/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 μg/ml) and chloramphenicol (34 μg/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. After three hours of induction, cultures were harvested by centrifugation (1 min, 16,100 x g) in 2 ml screw cap polypropylene tubes (Scientific Specialties Inc.). The supernatant was discarded and the cell pellet was stored at -2O ⁰ C for analysis.

3.12 SDS-PAGE and Western blot analysis Unless stated otherwise, 12% SDS-PAGE gels and Coomassie blue staining were used for all analyses. For Western blot, monoclonal antipolyhistidine (mouse, Sigma H1029) was used as primary antibody and antimouse IgG peroxidase (Sigma A9044) was used as a secondary antibody.

3.13 Protein purification 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 μl 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. The beads were allowed to settle for 2 min, small outlets were created at the bottom of 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, Allegra™-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 0.45μm filter to prevent clogging of the column. Native purification of proteins was performed at 4 ⁰ C. Briefly, the cleared lysate was mixed with 2 ml pre-washed TALON resin in a column and suspended on a suspension mixer (Ratek) for 40 min and then resin was allowed to settle for 30 min. The settled resin in the column was washed with 6 bed volumes of binding buffer (pH 7.8) which was followed by 4 washes with wash buffer pH 6.0 (50 mM sodium phosphate, 500 mM NaCI, 10 mM imidazole, 20 mM PMSF). The flow through of each wash was collected and stored for SDS-PAGE analysis. 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.

3.14 Ni-NTA agarose resin

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.

3.15 Preparation of E.coli proteins for serum preabsorption

Overnight grown cultures of E.coli BL21(DE3)pLysS in LB broth (150 ml) were centrifuged at 3000 x g for 30 min to obtain bacterial pellets. The bacterial pellets were heat killed at 90 ⁰ C for 10 min, followed by resuspension in lysis buffer (PBS, 1% Tween20) and were sonicated for 7 x 30 s. Cell debris was pelleted at 16,100 x g for 5 min, the supernatant was collected and stored at -20 ⁰ C for sera absorption. Protein concentration was determined using the Bio-Rad Protein assay.

3.16 ELISA

The recombinant proteins were diluted in carbonate buffer pH 9.6 to a final concentration of 5 μg/ml (GreA), 10 μg/ml (Ppa), 30 μg/ml (MAP 2411), 10 μg/ml (MAP0593c), 5 μg/ml (CIpP), a cocktail of 2.5 μg/ml GreA and 5 μg/ml Ppa, a cocktail of 2.5 μg/ml MAP0593c and 5 μg/ml CIpP and another cocktail of 2.5 μg/ml GreA, 2.5 μg/ml Ppa, 2.5 μg/ml CIpP and 2.5 μg/ml MAP0593c. 100 μl of each antigen was coated onto a 96 well plate (Maxisorp, Nunc International) at 4°C overnight. After being washed in PBS and 0.05% v/v Tween20 wash buffer, the wells were blocked with 3% skim milk powder in PBS and 0.05% Tween20 at room temperature for 1.5 h. The serum samples were absorbed with 500 μg/ml proteins extracted from E.coli BL21(DE3)pLysS cells (described above) in M. phlei buffer (Pourquier reagent, Batch number 12-371) for 30 min and ELISA was performed as described previously. However, for the CIpP ELISA sera was diluted 1 :10 in M. phlei buffer with E.coli soluble proteins. To assess the immunogenicity of recombinant proteins, serum samples collected from the animals at different stages of disease were tested. A total of 41 known negative sheep sera from Western Australia and 41 positive sera from NSW were included in this study. The infection status of positive animals was confirmed by histopathology and both positive and negative sera were previously tested using

Pourquier ELISA. An arbitrary cut off value was selected for each recombinant protein ELISA to differentiate between positive and negative serum samples.

Results

3.17 Expression of dormancy-associated genes in E.colii BL21(DE3)pLysS cells

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.

3.18 Recombinant protein purification

Four soluble and one insoluble recombinant proteins (MAP2411 , GreA, Ppa, CIpP, MAP0593c) 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.

3.19 lmmunogenicity of recombinant proteins

The relationship between humoral immune responses and the different stages of paratuberculosis was investigated by measuring serum antibody to the five recombinant antigens that were suitable for ELISA. The serum samples used in this study were previously screened by the Pourquier ELISA test and comparative data were collected (Table 14). An arbitrary mean OD value was selected for each recombinant protein to

differentiate the disease positive and negative sera. It can be seen in Table 14 that the recombinant antigens detected 9-19 paucibacillary cases. In contrast, the Pourquier ELISA detected 15 animals from the same group of samples. Interestingly, all the recombinant antigens detected animals in early stages of disease and more multibacillary cases were identified by the MAP0593c, CIpP and Ppa antigens than by the Pourquier ELISA.

Table 14 Comparison of immunoreactivity of dormancy-associated recombinant proteins and the Pourquier ELISA kit. Data are the number of animals

Histopathology Immunoreactivity of different ELISA tests

Category n ^a GreA Ppa CIpP MAP0593c GreA+Ppa CIpP +MAP0593C GreA+Ppa+ CIpP Pourquier +MAP0593C

Paucibacillary

Type 1

O

Type 2 1 0 0

Type 3a 20 12 11

Type 3c 1 1

Multibacillary (Type 3b)

Total 41 10 14 19 24 12 14 17 18

n ^a = number of animals tested

Example 4: ELlSPOT method for diagnosis of Mycobacterium avium subsp. paratuberculosis infection.

For ruminants the ELISPOT plates (Millipore) are coated with 50μL 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 μL of the three dilutions being placed into each well as required into a nitrocelluose plate (Millipore). 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. The plates were incubated for 18-24 hours at 37°C with 5% CO ₂ . 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 50μL 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 50μL 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.

After this incubation the plate is washed 5 times in PBS and 10OuL BCIP substrate (Vector Labs) is added for 1 hour at room temperature. After the incubation the BCIP substrate is removed and the plate is washed 6 times in water.

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.

For ruminants the ELISA plates (Nunc, Maxisorb) are coated with 50μL 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. 100μL 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. The plates were incubated for 18-24 hours at 37°C with 5% CO2. Other antigens including those in Tables 8 to 13 can be used.

The plates are washed 6 times again in using PBS + 0.05% tween 20 to remove the cells and 50μL of the secondary antibody is added MCA1783b (serotec) at a concentration of 0.5μg/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 50μL of a 0.01μg/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.

After this incubation the plate is washed 5 times in PBS + 0.05% tween 20 and 100μL TMB substrate (Pierce) is added for 30 minutes hour at room temperature. After the incubation the substrate is stopped by 100μL 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.

Example 6: Immunisation against M.ptb infection.

Lamb selection

400 lambs are selected to enter a trial, and are double ear tagged for individual identification. Lambs are randomly allocated to treatment groups (vaccinated, n = 200 and unvaccinated controls, n = 200).This number was chosen to allow for normal farm culling and deaths unrelated to OJD, so that about 300 sheep on each farm remain in to the fourth year of their life. The vaccinated and control lambs in each flock are mainly female, and are grazed together for the whole of the trial.

Lamb treatment

Each farm is visited once for lamb selection and again for each vaccination when lambs are 1-3 months old. 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. Alternatively a DNA vaccine administered intramuscularly followed for example one month later by a protein vaccine as a booster as above would be given. Alternatively 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.

Sampling schedule

Measurements are taken, and samples collected, at the time of vaccination, approximately 2 and 6 months post-vaccination (pv), then six monthly thereafter.

Necropsy examinations

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.

Histόpathological examination

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.

Culture from tissues

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.

Faecal sampling and culture

Samples for pooled faecal culture (PFC) are collected at each scheduled visit. Initially pools of 40 sheep are used. This is reduced over time to 20 sheep per pool and finally to 10 sheep per pool, to provide greater discrimination between group excretion levels as prevalence increased, and to facilitate the identification of individual culture-positive sheep from positive pools. These samples are held at 4 ⁰ C, and processed within 72 h of collection. Samples for individual faecal culture (IFC) are collected at each scheduled visit, and held at -80 ⁰ C for up to 12 months. Individual samples from positive pools and from all sheep at the final sampling, are cultured, PFC and IFC samples processed by a double incubation and centrifugation method as previously described with subsequent radiometric culture as above.

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.

The numbers of M.ptb organisms per gram in each culture-positive pooled faecal sample are estimated based on the days taken for the cumulative growth index in BACTEC culture to reach 1000 and allowing for the effect of decontamination procedures. Then an estimate of the total daily excretion by the sheep contributing to each positive pool is calculated: excretion by sheep in pool = excretion per gram x total grams of faeces (allowing for I kg of faeces per sheep per day). Finally, the contributions from each positive pool are added to yield an approximation of the numbers of organisms excreted daily by each group of sheep.

Immunological responses to M. a. paratuberculosis

Cell-medicated immune responses are measured using a gamma interferon (IFN-y) assay (Bovigam™). Stimulation of whole blood is done within 8 h of blood collection, using 100 μ\ of Johnin PPD (300 pg/ml), avian PPD (300 μg/mL), or any of the antigens or combinations of antigens in Tables 8 to 13, PBS (negative control) and pokeweed mitogen (150 (μg/mL, positive control). Stimulated plasma is held overnight at 4 ⁰ C prior to enzyme immunoassay. 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. Alternatively or in addition, immune responses would be evaluated using ELISPOT, CELLELISA and lymphocyte proliferation assays.

Humoral immune responses are measured using a commercial ELISA test (Parachek™). 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.

Assessment of local reactions to vaccination

At each scheduled farm visit, 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.

Collection of production data

Liveweight is measured for each sheep at each visit. Greasy fleece weights and fibre diameter measurements are collected from most sheep when shorn for the first time as adults.

Statistical methods

For the purposes of analysis, 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.

The agreement between markers of vaccine take (vaccine-site lesions, IFN-y response, ELISA response) are assessed using Kappa coefficient.

General linear models (Minitab statistical software) are used to compare production data across the three farms for surviving sheep at each sampling time. The models included farm as a random factor, vaccination status, infection status and their interactions. Weight at the time of vaccination is included as a covariate in the analyses of liveweight for young sheep up 10 to 12 months of age.

Example 7. Vaccine formulations

A. Vaccines containing a stressed mycobacterium

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.

B. Vaccines containing a transformed bacterium having a stress phenotype

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.

C. 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.

D. Vaccine containing a eukaryotic cell having a molecule that is expressed by a stressed mycobacterium located on the surface of the cell.

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.

It will also be understood that the term "comprises" (or its grammatical variants) as used in this specification is equivalent to the term "includes" and should not be taken as excluding the presence of other elements or features.