WO2008140598A2

WO2008140598A2 - Novel immunotherapeutic mycobacteria, pharmaceutic formulations and uses thereof

Info

Publication number: WO2008140598A2
Application number: PCT/US2007/086335
Authority: WO
Inventors: David M. Hone
Original assignee: Bacilligen, Inc.
Priority date: 2006-12-04
Filing date: 2007-12-04
Publication date: 2008-11-20
Also published as: WO2008140598A3

Abstract

This invention provides genetically modified Mycobacterium strains that are useful as an anticancer immunotherapeutic. The genetically modified Mycobacterium strains preferably display at least one genetic modification resulting in increased tissue adherence, which can be combined with genetic modifications that increase immunopotency and result in adjuvant production. The invention also provides compositions and methods for the development of said Mycobacterium strains, and pharmaceutic compositions and methods for the use of said Mycobacterium strains as anticancer therapeutics.

Description

TITLE OF THE INVENTION

NOVEL IMMUNOTHERAPEUTIC MYCOBACTERIA, PHARMACEUTIC FORMULATIONS AND USES THEREOF

BACKGROUND OF THE INVENTION

Bacille Calmette-Guerin

[0001] The only licensed tuberculosis (TB) vaccine, bacille Calmette-Guerin (BCG), is a derivative of Mycobacterium bovis and was isolated empirically early last century following over 13 years of uninterrupted passage on a starch-based medium. Although the use of BCG was temporarily discontinued as a result of the Lubeck disaster, which was caused by a BCG vaccine lot contaminated with virulent M. tuberculosis (1), its use was reinitiated following a series of controlled studies conducted in Scandinavia during the 1930's, which demonstrated the safety of BCG when administered by the intradermal route (2). After World War II, BCG was administered to school-age children throughout Western Europe and is accredited with the decline of TB in that region {Reviewed in (3, 4)). The apparent effectiveness of BCG in Europe is exemplified by the results of a randomized- controlled clinical trial initiated in 1950 in the United Kingdom where BCG was shown to afford 84% protection over the first 5 years and 77% protection over the 20-year passive surveillance period (2, 5). BCG immunotherapy

[0002] In addition to its use as a TB vaccine, over the past 30 years, BCG has been utilized to prevent recurrences of superficial bladder cancer. Local immunotherapy with BCG is clinically established and efficacious against recurrences after transurethral resection of superficial bladder cancer (82, 83, 95). The immunologic mechanism of BCG immunotherapy is still not fully resolved (84, 85); however, when BCG is intravesically

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112275/F/l applied to treat bladder cancer, it induces local inflammation and an influx of various immune cells including CD4⁺ and CD8⁺ T cells, granulocytes and NK cells (86), which accumulate and form cellular infiltrates in the bladder wall. During this period of inflammation, BCG therapy also induces local secretion of cytokines, which can be detected in the urine of patients (87-92). Although both type 1 T helper (ThI) and type 2 T helper (Th2) cytokines are present in BCG-treated patient urine, animal studies have implicated ThI cytokines as being a critical factor for effective therapy (93).

[0003] Despite the success of BCG immunotherapy in superficial bladder cancer, a high proportion of non-responders and untoward side effects continue to be a major obstacle to broader application of BCG as an immunotherapeutic (94, 95). In this regard, the basis for the non-responsive state has not been defined and at present it is not clear whether it is possible to maintain the potency of BCG while ameliorating its toxicity.

[0004] Similar to the usefulness of BCG as a human cancer immunotherapy, there is an abundance of evidence showing the potential of BCG as a veterinary cancer immunotherapeutic (96-98). However, there has yet to be a BCG formulation developed and licensed for veterinary use. Factors influencing BCG potency

[0005] Despite the association of BCG vaccination with the decline of TB in Western Europe (1-3, 5), there is equivocal evidence as to whether this vaccine is effective against TB in the developing world (3, 6-9). Interestingly, there is convincing evidence that BCG induces long-lived immune responses in individuals living in poverty, and in controlled field trials, measurable protection against TB persisted for more than 20-50 years (5, 10). Nonetheless, the effectiveness of BCG as a public health measure against TB in the developing world remains unclear (3). Therefore, while immunity induced by BCG provides protection against severe forms of infant TB, field trials have shown that BCG-induced immunity does not

112275/F/l afford sufficient levels of protection against TB in adolescents and adults to serve as a reliable public health control measure {Reviewed in (3, 4)).

[0006] Although many causes have been proffered to explain the failure of BCG in developing countries (3, 6-9), two hypotheses based on the conserved core proteome of organisms within the Mycobacterium genus are supported by experimental data. A large number of species in this genus are non-pathogenic environmental mycobacterial species, which are ubiquitous in soil and water (11), and repeated exposure to these saprophytic organisms stimulates antigen-specific T cell responses, which have been quantified m vitro (12, 13) and in situ with skin tests (14, 15). Moreover, a large proportion of people that live in tropical regions display higher levels of responsiveness to antigens derived from environmental mycobacteria, than do individuals from developed countries (12, 13, 15). Although the basis for this difference in responsiveness has not been resolved, the greater level of responsiveness to mycobacterial antigens in individuals from developing countries is believed to influence the outcome of BCG vaccination trials in one of two ways: (i) The Masking Hypothesis proposes that responsiveness to mycobacterial antigens following exposure to environmental mycobacteria affords a quantifiable level of protection against TB and vaccination with BCG in this setting only marginally augments the protection against TB (16); and (ii) The Interference Hypothesis proposes that preexisting immunity to mycobacterial antigens reduces BCG replication and therefore interferes with vaccine take (17). In studies where interference has been reported as a possible mechanism underlying the failure of BCG, the study populations were school-aged children and young adults who had been exposed to environmental mycobacteria over their lifetimes (9, 18). Interference may also account for the failure of BCG to boost protection in BCG-primed individuals or in BCG-naϊve adolescents and adults who are responsive to mycobacterial antigens (17, 19).

112275/F/l [0007] Masking, on the other hand, is predicted to obscure the efficacy of vaccination with BCG in neonatal cohorts (3, 4, 16). Since vaccination with BCG clearly affords protection against severe forms of childhood TB (3, 4), exposure to environmental mycobacteria might play a favorable role in individuals who are naturally capable of controlling M. tuberculosis in childhood. Furthermore, immunity elicited by these saprophytic organisms is less potent against M. tuberculosis than immunity induced by BCG, probably due to the propensity of environmental mycobacteria to induce both ThI and Th2 responses (17). The fact that BCG induces some protection in low-economic groups where masking is expected to occur (10, 20), however, indicates that other factors may affect the efficacy of BCG. These observations notwithstanding, masking is not expected to play a role in influencing the efficacy of BCG in cancer immunotherapy and therefore is not a continuing topic of this disclosure. Recombinant BCG

[0008] In light of the failure of BCG to control TB in the developing world, a number of strategies have been developed to improve the effectiveness of BCG as a TB vaccine. In one study, inactivation of the leuD gene resulting in leucine dependence was shown to make BCG safer; however, this strain was significantly less effective as a vaccine (21). This observation suggests that replication by BCG post vaccination plays an important role in inducing an effective host immune response. On the other hand, two strategies have been shown to improve the potency of BCG in animal models: (i) recombinant BCG (herein referred to as "rBCG") strains that express expanded immunogen repertoires (22, 23) and (ii) rBCG strains that promote apoptosis (24).

[0009] The effectiveness of expanding the immunogen repertoire of BCG has been demonstrated by a handful of independent studies (22, 23, 25, 26). Moreover, although expression of M. tuberculosis proteins encoded in the RDl region, which are not present in

112275/F/l BCG, markedly improved the potency of BCG (23), expression of RDl proteins in BCG has practical limitations. However, rBCG-RDl⁺ isolates are more virulent in immunosuppressed mice (27) and proteins encoded in the RDl region, including ESAT-6 and CFPlO, are being used in diagnostic tests that differentiate between BCG vaccination and exposure to M. tuberculosis (28-31). There is little incentive to develop rBCG-RDl⁺ strains as TB vaccines and to date, the prior art does not delineate whether this strategy improves the anticancer therapeutic properties of BCG.

[0010] As mentioned, the second strategy that is believed to enhance the immunopotency of BCG involves genetic manipulations to BCG that result in rBCG strains that promote greater levels of apoptosis (99, 100). Increased apoptosis has been accomplished by reducing expression of anti-apoptotic factors in BCG (100) or by promoting degradation of the endosome (99). However, the precise basis through which apoptosis improves BCG potency has not been delineated. The link between apoptosis and the presentation of antigens by dendritic cells (DCs), termed cross-priming, is discussed elsewhere (32-41). Several lines of evidence implicate apoptosis as the initiating factor of afferent events that lead to the development of effector CD4⁺ and CD8⁺ T-cell responses that are capable of controlling intracellular pathogens (40). Macrophages, which harbor BCG bacilli after vaccination, are less effective than DCs at promoting the development of T cell responses (41). In this setting, therefore, BCG-induced apoptosis may provide a conduit through which BCG antigens are transferred to DCs leading to the induction of effector T cells (40, 41). Along these lines, macrophages undergoing apoptosis are more effective at killing BCG than macrophages undergoing necrosis (42), suggesting that apoptosis may enhance the processing of BCG antigens and facilitate the distribution of processed antigens to DCs. However, induction of apoptosis is a double-edged sword, which in certain circumstances enhances the pathogenesis of intracellular microbial pathogens (32, 33, 41, 43-

112275/F/l 48). Thus, the capacity of this strategy to improve the immunopotency of BCG while maintaining safety remains to be verified. Moreover, there is no information in the prior art as to whether BCG strains that promote apoptosis are improved immunotherapeutics against cancer.

Nonpathogenic Mycobacteria

[0011] The prior art also documents the genetic manipulation of nonpathogenic mycobacteria, such as M. vaccae and M. smegmatis, and the use of genetically modified nonpathogenic mycobacteria as TB vaccines (49, 101), as a vaccine vector carrying HIV antigens (50) and cancer immunotherapeutics (51). Recent evidence suggests that a genetically modified derivative of M. smegmatis that expressed tumor necrosis factor (herein referred to as "TNF") was effective as a cancer immunotherapeutic in the mouse bladder cancer model (51). Although the use of a human gene might be unacceptable to regulatory agencies due to the high risk of autoimmunity against TNF, this finding offers promise that nonpathogenic mycobacteria, which are less toxic than BCG, may have utility in the cancer immunotherapy setting. To date, the prior art does not provide nonpathogenic mycobacteria that are effective as anti-cancer therapies that do not carry significant regulatory concerns.

SUMMARY OF THE INVENTION

[0012] The invention relates, in part, to novel Mycobacteria with enhanced biological activities, such as, immunogenicity. A Mycobacteria of interest can serve as an improved adjuvant, resulting from modifications providing the bacteria with enhanced tissue attachment and adherence.

[0013] Additional features and advantages are described herein, and will be apparent from, the following Detailed Description and Figures.

112275/F/l BRIEF DESCRIPTION OF THE FIGURES

[0014] FIGURE 1 depicts a map of pB ACIL-101. [0015] FIGURE 2 depicts a cloning scheme. [0016] FIGURE 3 depicts a cloning scheme. [0017] FIGURE 4 depicts a cloning scheme.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0018] Mycobacterium is defined herein as an acid-fast bacterial genus that includes M. tuberculosis, M. bovis, M. smegmatis, M. microti, M. avium, M. vaccae and other species. This genus is divided into pathogenic organisms, such as M. tuberculosis and M. bovis, and nonpathogenic organisms, such as M. smegmatis and M. avium. Mycobacteria is a vernacular term that refers to organisms in the Mycobacterium genus, wherein mycobacterial is the adjectival form, thereof.

[0019] "Immunogen" and "antigen" are used interchangeably herein as a molecule that elicits a specific immune response containing an antibody that binds to that molecule. That molecule can contain one or more sites to which a specific antibody binds. As known in the art, such sites are known as epitopes. A vaccine is a form of immunogen or antigen. An antigen can be polypeptide, polynucleotide, polysaccharide, a lipid and so on, as well as a combination thereof. An immunogenic compound or product, and an antigenic compound or product is one which elicits a specific immune response, which can be a humoral, cellular or both.

[0020] A vaccine is an immunogen or antigen used to generate an immunoprotective response, that is, the antibody reduces the negative impact of the immunogen or antigen, or entity expressing same, in a host. The dosage is derived, extrapolated and/or determined

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112275/F/l from preclinical and clinical studies, as known in the art. Multiple doses can be administered as known in the art, and as needed to ensure a prolonged prophylactic state. The successful endpoint of the utility of a vaccine for the purpose of this invention is the resulting presence of an induced serum antibody, or antibody made by the host in any tissue or organ, that binds the antigen or immunogen of interest. In some embodiments, the induced antibody in some way, neutralizes and/or eliminates a pathogen, compound, molecule and the like carrying the cognate antigen or immunogen. Immunoprotection for the purposes of the instant invention is the presence of such circulating antibody. That can be determined using any known immunoassay, such as an ELISA. Alternatively, one an use a neutralizing assay to ascertain presence of circulating antibody. For the purposes of the instant invention, observing immunoprotection of at least thirty days is evidence of efficacy of a vaccine of interest. The time of immunoprotection can be at least 45 days, at least 60 days, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years or longer. Preferably the immunoprotection is observed in outbred populations, and to different forms, strains, variants, alleles and the like of a pathogen.

[0021] An "immunogenic factor" is one that supplements the immunogenicity of, antigenicity of or immune system reaction inducing ability of the host organism carrying or expressing same; or to an antigen associated or administered therewith. Thus, such a factor includes a TAP, a GGDEP containing peptide, a molecule with an adjuvant activity, RDl expression, a PAP and so on. Another factor is one which reduces expression of, for example, a mannosylated mannan, such as mannosylated lipoarabinomannan.

[0022] The phrases and terms, as well as combinations thereof, "functional fragment, portion, variant, derivative or analog" and the like, as well as forms thereof, of a bacterium or of a foreign antigen is a compound or molecule having qualitative biological activity in common with an intact bacterium or antigen of interest. For example, a functional portion,

112275/F/l fragment or analog of a bacterium, such as a cell wall/cell membrane preparation carrying an immunogenic and/or antigenic molecule, or of a foreign antigen is one which stimulates an immune response as does the native bacterium or portion thereof, or foreign antigen.

[0023] Thus, included within the scope of the invention are functional equivalents of a bacterium of interest or a foreign antigen of interest. The term "functional equivalents" includes the cell and portions thereof with the ability to stimulate an immune response, whether to the bacterium or to a foreign antigen.

[0024] Also, subcellular parts of a Mycobacterium of interest, such as a cell wall or cell membrane preparation can be obtained practicing methods known in the art. Recombinant expression of an adjuvant molecule or of a foreign antigen can be realized practicing methods known in the art.

[0025] A foreign antigen is a molecule that elicits an immune response in a host. The molecule is not of the Mycobacterium host species used as an adjuvant or expressing the foreign antigen.

[0026] Many techniques are available to one of ordinary skill in the art which permit manipulation of immunogenic structures. Typically, the techniques involve substitution of various amino acid residues at a site of interest, followed by a screening analysis of binding affinity of the mutant polypeptide for a binding partner thereof, such as an antibody.

[0027] One procedure for obtaining protein, adjuvant or foreign antigen mutants, variants, derivatives, muteins and the like is "alanine scanning mutagenesis" (Cunningham & Wells, Science 244: 1081-1085 (1989); and Cunningham & Wells, Proc Nat. Acad Sci USA 84:6434-6437 (1991)). One or more residues are replaced by alanine or polyalanine residue(s). Those residues demonstrating functional sensitivity to the substitutions then are refined by introducing further or other mutations at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature

112275/F/l of the mutation per se need not be predetermined. Similar substitutions can be attempted with other amino acids, depending on the desired property of the scanned residues.

[0028] A more systematic method for identifying amino acid residues to modify comprises identifying residues involved in immune system stimulation and those residues with little or no involvement with immune system stimulation. An alanine scan of the involved residues is performed, with each ala mutant tested for enhancing immune system stimulation. In another embodiment, those residues with little or no involvement in immune system stimulation are selected to be modified. Modification can involve deletion of a residue or insertion of one or more residues adjacent to a residue of interest. However, normally the modification involves substitution of the residue by another amino acid. A conservative substitution can be a first substitution. If such a substitution results in a change in immune system stimulation, then another conservative substitution can be made to determine if more substantial changes are obtained.

[0029] Even more substantial modification in the ability to stimulate the immune system can be accomplished by selecting an amino acid that differs more substantially in properties from that normally resident at a site. Thus, such a substitution can be made while maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0030] For example, the naturally occurring amino acids can be divided into groups based on common side chain properties:

[0031] (1) hydrophobic: methionine (M or met), alanine (A or ala), valine (V or val), leucine (L or leu) and isoleucine (I or ile);

[0032] (2) neutral, hydrophilic: cysteine (C or cys), serine (S or ser), threonine (T or thr), asparagine (N or asn) and glutamine (Q or gin);

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112275/F/l [0033] (3) acidic: aspartic acid (D or asp) and glutamic acid (E or glu);

[0034] (4) basic: histidine (H or his), lysine (K or lys) and arginine (R or arg);

[0035] (5) residues that influence chain orientation: glycine (G or gly) and proline (P or pro), and

[0036] (6) aromatic: tryptophan (W or trp), tyrosine (Y or tyr) and phenylalanine (F or phe).

[0037] Non-conservative substitutions can entail exchanging an amino acid with an amino acid from another group. Conservative substitutions can entail exchange of one amino acid for another within a group.

[0038] Preferred amino acid substitutions can include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter immune system stimulating activity and/or (4) confer or modify other physico-chemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally- occurring sequence (for example, in the portion of the polypeptide outside the functional domain(s)). A conservative amino acid substitution generally should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence) unless of a change in the bulk or conformation of the R group or side chain (Proteins, Structures and Molecular Principles (Creighton, ed., W. H. Freeman and Company, New York (1984); Introduction to Protein Structure, Branden & Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354: 105 (1991)).

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112275/F/l [0039] Ordinarily, the adjuvant or immunogen mutant with improved biological properties will have an amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of the parent molecule, at least 80%, at least 85%, at least 90% and often at least 95% identity. Identity or similarity with respect to parent antibody sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) or similar (i.e., amino acid residue from the same group based on common side-chain properties, supra) with the parent molecule residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0040] Covalent modifications of the molecules of interest are included within the scope of the invention. Such may be made by chemical synthesis or by enzymatic or chemical cleavage of the molecule, if applicable. Other types of covalent modifications of the molecule can be introduced into the molecule by reacting targeted amino acid residues of the molecule with an organic derivatizing agent that is capable of reacting with selected side chains or with the N-terminal or C-terminal residue.

[0041] Cysteinyl residues can be reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to yield carboxylmethyl or carboxyamidomethyl derivatives. Cysteinyl residues also can be derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercura-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-l,3- diazole, for example.

[0042] Histidyl residues can be derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0. p-bromophenacyl bromide also can be used, the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

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112275/F/l [0043] Lysinyl and α terminal residues can be reacted with succinic or other carboxylic acid anhydrides to reverse the charge of the residues. Other suitable reagents for derivatizing α-amino-containing residues include imidoesters, such as, methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea and 2,4-pentanedione, and the amino acid can be transaminase-catalyzed with glyoxylate.

[0044] Arginyl residues can be modified by reaction with one or several conventional reagents, such as, phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residues often requires alkaline reaction conditions. Furthermore, the reagents may react with lysine as well as the arginine ε-amino group.

[0045] The specific modification of tyrosyl residues can be made with aromatic diazonium compounds or tetranitromethane. For example, N-acetylimidizole and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues can be iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteins for use in a radioimmunoassay or with other radionuclides to serve as an imaging means.

[0046] Carboxyl side groups (aspartyl or glutamyl) can be modified by reaction with carbodiimides (R-N=C=C-R'), where R and R can be different alkyl groups, such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0047] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively, under neutral or basic conditions. The deamidated form of those residues falls within the scope of this invention.

[0048] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of serinyl or threonyl residues, methylation of the α-

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112275/F/l amino groups of lysine, arginine, and histidine side chains (Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)), and acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

[0049] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the molecules of interest. Depending on the coupling mode used, the sugar(s) may be attached to: (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine. Such methods are described in WO 87/05330 and in Aplin & Wriston, CRC Crit Rev Biochem, pp. 259-306 (1981).

[0050] Removal of any carbohydrate moieties present on the molecule of interest may be accomplished chemically or enzymatically. Chemical deglycosylation, for example, can require exposure of the molecule to the compound, trifluoromethanesulfonic acid, or an equivalent compound, resulting in cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the remainder of the molecule intact. Chemical deglycosylation is described, for example, in Hakimuddin et al. Arch Biochem Biophys 259:52 (1987) and in Edge et al., Anal Biochem 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on molecules can be achieved by any of a variety of endoglycosidases and exoglycosidases as described, for example, in Thotakura et al., Meth Enzymol 138:350(1987).

[0051] Another type of covalent modification of the molecule comprises linking the molecule to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol or polyoxylalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

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112275/F/l [0052] DNA encoding the adjuvant, immunogen, antigen and the like of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to the relevant genes, Innis et al. in PCR Protocols. A Guide to Methods and Applications, Academic (1990), and Sanger et al., Proc Natl Acad Sci 74:5463 (1977)). Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, NSO cells, COS cells, Chinese hamster ovary (CHO) cells or myeloma cells to obtain synthesis of the protein of interest in the recombinant host cells. The DNA also may be modified, for example, by substituting bases to optimize for codon usage in a particular host or by covalently joining to the coding sequence of a heterologous polypeptide. 2. Development of Mycobacterial strains with enhanced tissue adherence

[0053] Adherence to target tissue by means of the fibronectin attachment protein is a factor that can determine the success of cancer immunotherapy with Mycobacterium strains, such as BCG (52). Despite this knowledge, the prior art makes no mention of compositions with modified tissue adherence or whether such compositions have utility as cancer immunotherapeutics. As set forth below, Mycobacterium strains with modified tissue adherence were produced, and, surprisingly, although Mycobacterium strains such as BCG are capable of binding mammalian tissue, Mycobacterium strains with modified tissue adherence are substantially more effective cancer immunotherapeutics than are unmodified Mycobacterium strains. The precise mechanism through which Mycobacterium strains with modified tissue adherence induce an enhanced cancer immunotherapeutic effect has not been resolved. Although not wishing to be bound by theory, however, it is conceivable that Mycobacterium strains with modified tissue adherence are retained in the target tissue in greater numbers, therefore invoking a stronger inflammatory response to the target locale in which the tumor resides.

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112275/F/l [0054] An embodiment of the present invention provides mycobacterial strains engineered to express at least one recombinant DNA sequence (herein referred to as "RDS") comprised of DNA encoding a tissue attachment factor (herein referred to as "TAF"), which may be either derived from a Mycobacterium species, e.g. M. tuberculosis and M. bovis, or a TAF derived from an animal, plant, or fungal, viral, bacterial, protozoan or metazoan organism. The TAFs may be the full-length native protein, chimeric fusions between a TAF and an endogenous protein, heterologous protein or mimetic, or a fragment or fragments of a TAF or TAFs that originate from an animal, plant, or fungal, viral, bacterial, protozoan or metazoan organism. The RDS encoding the TAF is introduced into the chromosome or as part of an extrachromosomal element (i.e. plasmids) using compositions and methods well known in the art (102-108).

[0055] Examples of TAFs include, but are not restricted to, the fibronectin attachment protein of M. tuberculosis strain CDC 1551 (GenBank accession no. AAK46179), the fibronectin attachment protein of M. leprae (GenBank accession no. AAB34676), the fibronectin attachment protein of M. bovis (GenBank accession no. AAB71842), and fibronectin attachment protein of M. avium (GenBank accession no. AAG22111), for example. The fibronectin-binding activity of the aforementioned TAFs to yield a variant, derivative and the like can be enhanced by site-directed mutagenesis to either augment ligand-binding affinity, change the specificity of the ligand-binding activity to include additional targets on fibronectin or through a combination of both these approaches as discussed herein. Methods to make such modification are well-known in the art (e.g. Rauceo et al., Eukaryot Cell, 5(10): 1664-1673, (2006);Roche et al., J. Biol. Chem., 279(37):38433- 38440, (2004); and Terao et al., J. Biol. Chem., 277(49):47428-47435, (2002)).

Alternatively, the recombinant mycobacteria of the present invention can express other TAFs derived from an animal, plant, or fungal, viral, bacterial, protozoan or metazoan organism.

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112275/F/l Such heterologous TAFs include, but are not limited to, E-selectin (GenBank Accession no. AY367062), L-selectin (GenBank Accession no. AY367061), E. coli fimbrial adhesin subunit F 1845 antigen (GenBank Accession no. M27725), Escherichia coli adhesin (F17b-G) (GenBank Accession no. L14319), Klebsiella pneumoniae type 3 fimbrial adhesin (mrkD) (GenBank Accession no. M24536), B. parapertussis fimbrial adhesin FimD (GenBank Accession no. X75812), Streptococcus pneumoniae surface adhesin A (GenBank Accession no. U53509), Bordetella bronchiseptica strain GPl adhesin (fhaB) (GenBank Accession no. AFl 11796), Salmonella enterica subsp. enterica serovar Infantis fimbrial subunit (fimH) (GenBank Accession no. EF029034), Citrobacter freundii fimH gene fimbrial adhesin (FimH) (GenBank Accession no. AJ508060), Actinobacillus actinomycetemcomitans extracellular matrix protein adhesin A-like (emaA) protein (GenBank Accession no. DQ991438), Burkholderia pseudomallei aidA autotransporter diffuse adhesion protein (GenBank Accession no. NC 009075), lectins including, but not limited to, hemagglutinins, phytoagglutinins (Sharron and Lis, Glycobiol., 14(11):53R-62R, (2004)), S-type lectins (i.e. galectins; Hasan et al., Cancer Let., 2007)), endogenous glycan-binding proteins, such as, but not limited to, C-type lectins (collectins or selectins), mannose receptor, I-type lectins (siglecs and others), P-type lectins (phosphomannosyl receptors), pentraxins, tachylectins, etc.

[0056] In addition to the expression of a TAF, the Mycobacterium strains can be engineered to express at least one RDS comprised of DNA encoding an endogenous immunogen, such as, but not limited to, an autoimmune antigen or a tumor antigen. Examples of tumor specific antigens include prostate specific antigen (109); TAG-72 and CEA (110); MAGE-I; and tyrosinase (111). Recently, it has been shown in mice that immunization with non-malignant cells expressing a tumor antigen provides a vaccine effect, and also helps the animal mount an immune response to clear malignant tumor cells displaying the same antigen

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112275/F/l (112). Examples of transplant antigens include the CD3 molecule on T cells (113). Treatment with an antibody to CD3 receptor has been shown to rapidly clear circulating T cells and to reverse cell-mediated transplant rejection (113). An example of an autoimmune antigen includes IAS β chain (114). Vaccination of mice with an 18 amino acid peptide from IAS β chain has been demonstrated to provide protection and treatment in mice with experimental autoimmune encephalomyelitis (114).

[0057] The above description is not intended to restrict the present invention to the specific TAFs included by reference, and the skilled artisan will know of other TAFs, TAF domains and TAF mimetics, and can select them as appropriate depending on the application and the function desired. 3. BCG variants with enhanced immunopotency

[0058] This invention draws a novel distinction between the pro-inflammatory properties of BCG and the immunogenicity of BCG as a vaccine. As will be discussed below, the pro-inflammatory property of BCG results in an influx of host phagocytes, natural killer cells and lymphocytes to the site of BCG inoculation. Initially, these cells comprise the innate host response, which develops with time into an adaptive host response. The latter is central to the success of BCG as a vaccine and this invention shows that the former is central to the success of BCG as an anticancer therapy.

[0059] There are multiple cell wall and somatic factors in mycobacterial cells that activate host innate immune responses (53-60). However, in pathogenic and attenuated mycobacteria, the expression of these factors is precisely regulated, resulting in suboptimal activation of the host immune system (59). In addition, the mycobacterial cell wall component, mannosylated lipoarabinomannan, inhibits apoptosis, thereby restricting the delivery of antigens to dendritic cells via cross presentation pathways (61-64).

18

112275/F/l [0060] This invention, on the other hand, provides novel mycobacterial strains that undergo deregulated expression of immunostimulatory factors. Expression deregulation of an immunostimulatory factor in mycobacteria can be achieved by overexpressing a biosynthetic pathway that produces an immunostimulatory factor. In a preferred embodiment, mycobacteria can be engineered to overexpress a protein containing a GGDEF domain from a heterologous bacterium, such as AdrA from Salmonella enteriditis (GenBank Accession no. NP 806207), PIeD from unicellular cyanobacterium Synechocystis sp. strain PCC6803 (GenBank Accession no. NP 440351) or PIeD from Caulobacter crescentus (GenBank Accession no. AA87378; (65, 66). In a more preferred embodiment, mycobacteria are engineered to overexpress a protein containing a GGDEF domain, such as, but not limited to, the GGDEF domain contained within amino acids 1-360 in Mb 1389c (GenBank Accession no. NP 855043; SEQ ID NO: 1), the GGDEF domain contained within amino acids 1-360 in Rv 1354c of M tuberculosis (GenBank Accession no. NP_215870), etc.

[0061] Overexpression of proteins containing GGDEF (SEQ ID NO: 1) domains results in the over production of cyclic-di(3'-^5')-guanylic acid (herein referred to a "c-di- GMP"), which in and of itself, displays immunostimulatory and cancer immunotherapy properties (US Publication No. 20050203051; and US Publication No. 20060040887).

[0062] The above-described GGDEF (SEQ ID NO: 1) domains are included as a guide; however, given the ubiquitous presence of the GGDEF/cyclase superfamily, which forms a large diversified cluster of orthologous proteins present in bacteria, archaea and eukaryotes (67, 68), those skilled in the art will recognize that other proteins or fragments containing at least one GGDEF (SEQ ID NO: 1) domain exist or can be constructed, or possessing di-GMP cyclase activity, thereof, which are also suitable for use in the present invention.

19

112275/F/l [0063] The present invention also provides novel mycobacterial strains that undergo deregulated expression of immunostimulatory factors, which is achieved by diminishing the expression of a biosynthetic pathway that produces an immunosuppressive factor. In a preferred embodiment, mycobacteria can be engineered to underexpress or are rendered incapable of expressing mannosylated lipoarabinomannan (herein referred to as "ManLAM"). A preferred embodiment of the present invention provides a novel BCG carrying a defective embC gene (GenBank Accession no. CAB02472), which is incapable of expressing ManLAM. As discussed above, this molecule promotes suboptimal immune responses and blocks apoptosis (59, 61); however, despite there being other immunoregulatory factors in mycobacteria, the absence of ManLAM surprisingly enhances the cancer immunotherapeutic potential of mycobacteria.

[0064] The present invention also provides novel Mycobacterium strains that carry an RDS encoding an adjuvant, which is useful in eliciting augmented host immune responses, thereby improving the cancer immunotherapeutic efficacy of said mycobacteria. The specific adjuvant encoded by the RDS expressed by Mycobacterium is not critical to the present invention and may be, for example, the A subunit of cholera toxin (i.e. CtxA; GenBank accession no. X00171, AF175708, D30053, or D30052,) or parts, and/or mutant derivatives thereof (e.g. the Al domain of the A subunit of Ctx (i.e. CtxAl; GenBank accession no. K02679) from classical Vibrio cholerae (e.g. V. cholerae strain 395, ATCC # 39541) or from El Tor V. cholerae (e.g. V. cholerae strain 2125, ATCC # 39050) strain. Alternatively, the A subunit of heat-labile toxin (referred to herein as "EItA"; GenBank accession no. M35581) from enterotoxigenic Escherichia coli (ATCC# 35401) may be used in place of CtxA.

[0065] Secretion of CtxA and EItA by recombinant mycobacteria of the present invention is accomplished by generating a genetic fusion between DNA encoding a molecule which facilitates secretion, such as a leader sequence, a signal peptide, a targeting signal and

20

112275/F/l so on, for example, the Ag85A leader peptide (SEQ ID NO:2), herein referred to as "LPA_g85A") and DNA encoding the mature CtxA protein (amino acids 18-258) or mature EItA protein (amino acids 18-258). The sequences encoding QxA_18-258 and EltA_18-258 can be optimized for expression in mycobacteria by using the preferred codon bias of this genus (69, 70). Expression of LP_Ag85A::CtxA_18-258 and LP_Ag85A::EltA_18-258 (wherein "::" denotes the novel genetic junction) is accomplished by functionally linking synthetic DNA encoding said genetic fusions to, for example, the antigen 85A promoter (herein referred to as "P_Ag85A"; SEQ ID NO:3). Synthetic DNA encoding recombinant genes P_Ag8SA-LP_Ag8SA-QxA_18-258 and PA_g85A::LPA_g85A::EltA_18-258 can be purchased from commercial sources (i.e. Picoscript, Houston, Texas) and are introduced into mycobacterial strains as described here (see, for example, Example 1).

[0066] As outlined in the background above, immune interference can play an important role in limiting the effectiveness of mycobacterial vaccine BCG. Therefore, interference may limit the usefulness of BCG as a cancer immunotherapeutic, since the target population is adults who had been exposed to environmental mycobacteria over their lifetimes. To date, there is no guidance in the art as to whether a modified BCG that overcomes such interference displays improved rates of success when treating superficial bladder cancer or has therapeutic applications in other cancers. But that can be overcome, for example, by having a Mycobacterium of interest express RDl.

[0067] Genomic analysis of BCG compared to its parent M. bovis revealed that deletion in the so-called RDl region contributed to the hyper-attenuation of BCG (23). Thus, BCG-RDl⁺ strains, in which the RDl region has been functionally restored, display improved vaccinal properties in animals that have been pre-exposed to environmental mycobacteria (71). BCG-RDl⁺ strains also invoke a stronger influx of CD4⁺ and CD8⁺ T cells to the site of inoculation (27). However, BCG-RDl⁺ strains are only marginally more

21

112275/F/l effective than BCG at inducing protection against TB (23). Moreover, BCG-RDl⁺ strains induce immunity to ESAT-6 (Rv3875; GenBank Accession no. AAL 16895) and CFPlO (Rv3874; GenBank Accession no. CAAl 7966), which are important diagnostic antigens, since these two antigens distinguish between TB and BCG exposures (30). For this reason, there has been little impetus to move BCG-RDl⁺ strains forward as TB vaccines.

[0068] Notwithstanding the poor product profile of BCG-RDl⁺ strains as a TB vaccine, a surprising observation was that BCG and nonpathogenic mycobacterial strains when made RDl⁺ are more effective as cancer immunotherapeutics than are the parental strains. Strategies and methods for introducing RDl into target mycobacteria are well known (23, 72, 73). This region can be introduced in its entirety or as smaller components that are sufficient to complement deletions in the RDl region of the target strain (23, 72, 73). In a preferred embodiment, BCG strains are made RDl⁺ by introducing SEQ ID NO:4 or functional portions thereof. In a further preferred embodiment, BCG strains are made RDl⁺ by introducing SEQ ID NO:4, which carries a Q4L mutation in esxA (substituting Leu for GIn at position 4 of EsxA, Brodin et al., J Biol Chem 280(4):33953-33959, 2005) resulting in production of ESAT-6_Q4L that partially reduces the toxicity of the resulting recombinant strain BCG-RD I⁺-ES AT-6_Q4L. The mutation in esxA can be introduced by site-directed mutagenesis procedures using the QuikChange^® Site-Directed Mutagenesis Kit (Stratagene, La Jolla CA; Cat. No. 200518) according to the manufacturer's directions.

[0069] Alternatively, nonpathogenic mycobacteria can be made RDl⁺ by introducing SEQ ID NO:4. In a preferred embodiment, nonpathogenic mycobacteria strains can be made RDl⁺ by introducing SEQ ID NO:4, which carries a Q4L mutation in ESAT-6 that partially reduces the toxicity of the resulting BCG-RDl -ES AT-6_Q4L ⁺ strains.

[0070] The RDl sequences capable of complementing the RDl deletion in BCG can be obtained by PCR amplification of M. tuberculosis genomic DNA using SEQ ID NO: 5 as a

22

112275/F/l forward primer and SEQ ID NO: 6 as a reverse primer. An RDl sequence capable of complementing the RDl deletion in M. microti can be obtained by PCR amplification of M. tuberculosis genomic DNA using SEQ ID NO: 7 as a forward primer and SEQ ID NO: 6 as a reverse primer. Mutant RDl derivatives, such as RD1-ESAT-6_Q4L ⁺ can be made by site- directed mutagenesis of full-length or truncated RDl subclones using the QuikChange^® Site- Directed Mutagenesis Kit (Stratagene, La Jolla CA; Cat. No. 200518) according to the manufacturer's directions. Strategies and methods for introducing RDS encoding RDl, truncated RDl or mutant variants of RDl or truncated RDl, either into the chromosome or as part of a plasmid carried in the target mycobacterial strain, are well documented (115, 116).

[0071] In addition to encoding RDl, a truncated RDl or mutant variants of RDl, or mutant derivatives thereof, the RDS expressed by said mycobacteria strains for cancer immunotherapy can also encode any combination of TAFs, immunostimulatory factors, immunoregulatory factors and adjuvants described herein. In a preferred embodiment the RDS expressed by said mycobacteria strain for cancer immunotherapy can encode at least a TAF and can overexpress c-di-GMP. In another preferred embodiment, the RDS expressed by said mycobacteria strain for cancer immunotherapy can encode a TAF and the RDl region. In a further preferred embodiment, the RDS expressed by said mycobacteria strain for cancer immunotherapy can encode a TAF, the RDl region and overexpress c-di-GMP.

[0072] In a further alternative, this invention provides mycobacteria that encode a molecule that induces, stimulates, precipitates, causes etc. apoptosis, identified herein as a pro-apoptosis protein (herein referred to as "PAP"), and direct tumor antigens to cross-prime antigen presentation pathways and to elicit effector CD4⁺ and CD8⁺ T-cell responses. The link between apoptosis and the presentation of antigens by DCs, termed cross-priming (32- 41), has been implicated as a key initiating factor in the afferent events that lead to the development of effector CD4⁺ and CD8⁺ T-cell responses and results in the control and/or

23

112275/F/l clearance of tumor cells (74-78). Therefore, BCG-induced apoptosis provides a mechanism for the delivery of tumor-specific antigens to DCs, thereby leading to the induction of said T cells. It has also been observed that macrophages undergoing apoptosis are more effective at killing BCG than are macrophages undergoing necrosis (42), suggesting that apoptosis may improve the safety of cancer immunotherapeutics comprised of live attenuated or nonpathogenic mycobacteria, while facilitating the distribution of tumor antigens to DCs. To date, however, there is no guidance in the literature as to how mycobacterial strains can be engineered to promote apoptosis.

[0073] Accordingly, the present invention provides mycobacteria capable of expressing a PAP, such as, but not limited to, the mature activated form of caspase-8⁺ (GenBank Accession no. NP033942; i.e., amino acids 99-480). A preferred embodiment provides derivatives of mycobacteria capable of expressing a PAP from a microbial source, such as, but not limited to, the proteolytic domain of NS3 (spans amino acids 1-190; SEQ ID NO:8; herein designated "NS3_Pr") encoded by base pairs 6469-7039 of West Nile virus isolate Mex03 (GenBank Accession no. AY660002), the hepatitis C virus core protein (GenBank Accession no. AAXl 1912), the cytomegalovirus-encoded chemokine receptor (GenBank Accession no. AAQ24855), the human herpes virus chemokine receptor US28 (GenBank Accession no. AAN37944), the lyssavirus matrix protein (GenBank Accession no. AY540348), the IpaB protein of Shigella flexneri (GenBank Accession no. AAM89543), and the SipB protein of Salmonella enterica (GenBank Accession no. 2123407B).

[0074] The sequences encoding the PAP can be generated synthetically by a commercial source (e.g. Picoscript, Houston Texas) and can be optimized for expression in mycobacteria by using the preferred codon bias of this genus (69, 70). Secretion of the PAP by recombinant mycobacteria of the present invention can be accomplished by generating a genetic fusion between DNA encoding, for example, the Ag85A leader peptide (SEQ ID

24

112275/F/l NO:2; herein referred to as "LP_Ag85A") and DNA encoding the PAP. Transport of the PAP from the endosome to the cytoplasmic compartment of the host cell can be accomplished by creating a fusion between an intercellular trafficking protein (herein referred to as "ITP"; e.g. the human herpes virus tegument protein VP22 (GenBank Accession no. BAE87004), the human immunodeficiency virus Tat protein (GenBank Accession no. AAF35362), or parts thereof (e.g. VP22 amino acids 81-195 or Tat amino acids 40-65)), and the PAP. Expression of the PAP can be enhanced by placing a spacer between the ITP and the PAP, such as a flexible spacer (e.g. Serine-Glycine-Glycine-Glycine-Glycine-Serine; SEQ ID NO:9), an inflexible linker (e.g. Serine-Proline-Proline-Proline-Proline-Proline-Proline-Serine; SEQ ID NO: 10) or flexible linker with a furin degradation motif (e.g. Serine-Glycine-Glycine- Glycine-Glycine- Arginine-Threonine-Lysine-Arginine-Glycine-Glycine-Glycine-Gly cine- Serine; SEQ ID NO: 11), for example.

[0075] Expression of LP_Ag85A: ITP::Linker::PAP (wherein "::" denotes the novel genetic junction) is accomplished by functionally linking synthetic DNA encoding said genetic fusions to the antigen 85A promoter (herein referred to as "PA_g85A"; SEQ ID NO:3). Synthetic DNA encoding recombinant genes P_Ag85A::LP_Ag85A::ITP::Linker::PAP can be purchased from commercial sources (i.e. Picoscript, Houston, Texas) and can be introduced into mycobacterial strains as described (see, for example, Example 1).

[0076] In a preferred embodiment the RDS expressed by said mycobacteria strain for cancer immunotherapy can encode a TAF, the RDl region, overexpress c-di-GMP and a pro- apoptosis factor. 4. Useful attenuated and non-pathogenic Mycobacteria

[0077] In a preferred embodiment of the invention, the Mycobacterium strain that is genetically modified as set forth hereinabove is attenuated, as exemplified by BCG. Attenuated Mycobacterium strains can be derived from M. tuberculosis strain H37Rv

25

112275/F/l (ATCC#: 25618), M. tuberculosis strain Beijing (117), M. tuberculosis strain H37Ra (ATCC#: 25177), M. bovis (ATCC#: 19211), M. intracellular e (ATCC#: 35772), and M. gallinarum (ATCC#: 19711, andM marinarum (ATCC#: 11566). The strategies and methods to attenuate Mycobacterium are well known in the art and include inactivation of an essential metabolic pathway (e.g. (21, 79, 118); or inactivation of a specific virulence factor (e.g. (80, 81)) using well-documented compositions and methods thereof (21, 79-81, 118).

[0078] Examples of attenuated Mycobacterium strains include, but are not restricted to, M. tuberculosis pantothenate auxotroph strain (119), lysine and pantothenate auxotrophic strain M. tuberculosis AlysA, ApanCD (120), leucine auxotrophic strain M. tuberculosis AleuD (118), BCG Danish strain (ATCC # 35733), leucine and pantothenate auxotrophic strain M. tuberculosis AleuD, ApanCD (121), M. tuberculosis fadD26 mutant with impaired synthesis of phthiocerol dimycocerosates (122), Mycobacterium mce mutants with impaired synthesis of mammalian cell entry {mce) proteins (123), Mycobacterium sigC mutant strains (124), Mycobacterium leuD mutant strains (125), BCG Japanese strain (ATCC # 35737), BCG Copenhagen strain (ATCC #: 27290), BCG Pasteur strain (ATCC #: 35734), BCG Glaxo strain (ATCC #: 35741), and BCG Connaught strain (ATCC # 35745), for example.

[0079] Examples of non-pathogenic mycobacteria useful to the present invention include, but are not limited to, M. fortuitum (ATCC#: 15073), M. smegmatis (ATCC#: 12051 or 12549), M. intracellular e (ATCC#:35772 or 13209), M. kansasii (ATCC#:21982 or 35775), M avium (ATCC#: 19421 or 25291), and M microtti (ATCC#: 11152). Methods for genetic manipulation of nonpathogenic mycobacterial strains are extensively documented (21, 50, 79-81, 118). 5. Strategies to produce rBCG strains that meet regulatory standards

[0080] To streamline approval from regulatory agencies, such as the US Food and Drug Administration or European Medicines Agency for human products and the US

26

112275/F/l Department of Agriculture for veterinary products, biological pharmaceutics must meet purity, safety and potency standards defined by the pertinent regulatory agency. To produce an rBCG that meets these standards, the recombinant organisms should be maintained in culture media that is, for example, certified free of transmissible spongiform encephalopathies (herein referred to as "TSE"). Although it is possible to construct an rBCG strain harboring an RDS derived from a source organism (either by subcloning or polymerase chain reaction), preferably the RDS is derived from synthetic DNA. Synthetic DNA techniques are well known in the art and such DNA' s can be purchased from commercial sources, such as DNA 2.0 Inc. (Menlo Park CA, USA), Blue Heron Biotechnology (Bothell WA, USA), Geneart Inc. (Toronto, Ont, Canada), and Genscript Inc. (Piscataway NJ, USA). To synthesize expression cassettes, a series of short sequences 100-200 base pairs in length are generated and ligated together to form the full-length sequence using procedures well know in the art (126). The synthetic DNA can be produced using an Applied Biosystems International AB I™ 3900 High-Throughput DNA Synthesizer (Foster City, CA) and procedures provided by the manufacturer. Plasmids harboring an RDS of interest are introduced into mycobacteria by electroporation and selection of mycobacterial strains carrying such plasmids is achieved, for example, by antibiotic selection, such as hyg, encoding hygromycin resistance (GenBank accession no. AF025746; AF025747) and aph from Tn903, which confers kanamycin resistance (herein referred to as "Kan^R"; GenBank accession no. U75323).

[0081] Preferably, plasmids harboring the RDS carry a non-antibiotic selection marker, since it is not always ideal to use antibiotic resistance markers for selection and maintenance of plasmids in mycobacteria that are designed for use in humans and veterinary pharmaceutics. In a preferred embodiment, therefore, the present invention provides a novel selection strategy in which, for example, a catabolic enzyme is utilized as a selection marker

27

112275/F/l by enabling the growth of mycobacteria in medium containing a substrate of said catabolic enzyme as a carbon source. An example of such a catabolic enzyme includes, but is not restricted to, lacYZ encoding lactose uptake and β-galactosidase (Genbank accession no. J01636, J01637, K01483, or K01793). Other selection markers that provide a metabolic advantage in defined media include, but are not restricted to, galTK (GenBank Accession no. X02306) for galactose utilization, sacPA (GenBank Accession no. J03006) for sucrose utilization, trePAR (GenBank Accession no. Z54245) for trehalose utilization, xylAB (GenBank Accession no. CAB 13644 and AAB41094) for xylose utilization, etc. Alternatively, the selection can involve the use of antisense mRNA to inhibit a toxic allele, such as the sacB allele (GenBank Accession no. NP 391325), which renders Mycobacterium strains sensitive to sucrose.

[0082] Alternatively, a suicide plasmid harboring the RDS of interest can be introduced into mycobacteria by electroporation and selection of mycobacterial strains carrying such plasmids can be achieved by antibiotic selection, such as hyg encoding hygromycin resistance (GenBank accession no. AF025746; or AF025747) and Kan^R (GenBank accession no. U75323). The suicide plasmid can carry a sequence that is identical to a genomic homolog. The sequence allows recombination between the suicide plasmid and the genome resulting in integration of the suicide plasmid into the mycobacterial genome. Methods for allelic exchange are described in detail elsewhere (21, 79, 81).

[0083] Selective medium containing the metabolite as a carbon source can be a modified Sauton's medium (herein defined as "MSM") containing 0.5 g KH₂PO₄ (Sigma Cat. No. P9666), 0.5 g MgSO₄7H2O (Sigma Cat. No. M5921-500G), 0.1 ml of 1% (w/v) ZnSO₄ (Sigma Cat. No. 35392-1L) solution, 5 ml of a 5% (v/v) Triton WR1339 (Sigma Cat. No. T8761) solution, 2.0 g citric acid (Sigma Cat. No. 251275), 0.05 g ferric ammonium citrate (Sigma Cat. No. F5879), 4.0 g asparagine (Sigma Cat. No. A4159), and 0.6 ml oleic acid

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112275/F/l (Research Diagnostics Cat. No. 01257), and the substrate of interest at an appropriate concentration in place of glycerol (e.g. 5 g lactose (Sigma Cat. No. 17814) per liter of medium). Add water to 950 ml, and dissolve reagents. Check the pH and adjust to pH 7.4 with IM NaOH. Sterilize by autoclaving at 121⁰C for 15 min. 6. Additional utility for recombinant mycobacteria a) Utility as a TB vaccine and a vaccine vector

[0084] The recombinant mycobacteria of the present invention can be used to vaccinate against TB. Thus, mycobacteria that overexpress a fibronectin attachment protein, such as FapB encoded by the fapB gene (herein referred to as FapB, GenBank accession no. AAB71842) can be used as a TB vaccine using procedures described elsewhere (Horwitz and Harm, US Pat. No. 6,471,967; Bloom et al., US Pat. No. 5,504,005). In a preferred embodiment, mycobacteria that overexpress FapB and express a factor that enhances immunostimulating properties of said mycobacteria are used a TB vaccines. For example, an rBCG strain that overexpresses FapB and expresses a fusion protein comprised of the Ag85A leader peptide, VP22 and NS3_Pr has improved vaccinal properties due to the enhanced ability of this strain to adhere to tissue and to form a depot, and to promote apoptosis through delivery of NS3_Pr to the cytoplasm of host antigen-presenting cells triggering activation of caspase-8 and apoptosis.

[0085] Recombinant mycobacteria are useful as vaccine vectors, wherein the recombinant strains are engineered to express at least one passenger or foreign antigen, for example, from a second pathogen. The second pathogen can be a bacterium, virus, metazoa or a protozoa. Methods to produce vaccine vectors and antigen that are useful thereof are described in detail elsewhere (Bloom et al., US Pat. No. 5,504,005; and Sun et al., US Patent Publ. No. 20060121054).

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112275/F/l b) Utility as an adjuvant

[0086] Attenuated mycobacteria, such as BCG, have long been used as adjuvants, compounds or microbes (either live or inactivated) that quantitatively and/or qualitatively improve an immune response to an immunogen that is co-administered with the adjuvant. There is a long history of using mycobacteria as adjuvants. Inactivated mycobacteria are the immunostimulating component in Freund's Complete Adjuvant (e.g. Difco, Detroit, MI, Cat. No. 231131). More recently, mycobacteria have been used to increase the immunogenicity of sub unit vaccines and nucleic acid vaccines (129).

[0087] Accordingly, the recombinant mycobacteria of the present invention can be used as adjuvants. The particular recombinant mycobacterium strain of the present invention that is utilized as an adjuvant is not important and can be selected from, but not restricted to, recombinant mycobacteria that overexpress FapB. In a preferred embodiment, the adjuvant is selected from recombinant mycobacteria that overexpress FapB and a factor that enhances the immunostimulatory properties of said mycobacteria (e.g. an rBCG strain that overexpresses FapB and expresses a fusion protein comprised of the Ag85A leader peptide, VP22 and NS3p_r). Procedures for the administration of recombinant mycobacterial adjuvants are described elsewhere (Horwitz and Harth, US Pat. No. 6,471,967; Bloom et al., US Pat. No. 5,504,005; and Sun et al., US Pat. Publ. No. 20060121054).

[0088] The instant invention also includes kits, e.g., comprising a bacterium or functional portion thereof of interest, homolog, derivative thereof and so on, for use, such as a vaccine or an adjuvant, and instructions for the use of same and so on. The instructions may include directions for using the bacterium, derivative and so on. The bacterium can be in liquid form or presented as a solid form, generally, desiccated or lyophilized. The kit can contain suitable other reagents, such as a buffer, a reconstituting solution and other necessary ingredients for the intended use. A packaged combination of reagents in predetermined

30

112275/F/l amounts with instructions for use thereof, such as for a therapeutic use is contemplated. In addition, other additives may be included, such as, stabilizers, buffers and the like. The relative amounts of the various reagents may be varied to provide for concentrates of a solution of a reagent, which provides user flexibility, economy of space, economy of reagents and so on.

[0089] The bacterium of the present invention may be used to treat a mammal. In one embodiment, the bacterium of interest is administered to a nonhuman mammal for the purpose of obtaining preclinical data, for example. Exemplary nonhuman mammals include nonhuman primates, dogs, cats, rodents and other mammals. Such mammals may be established animal models for a disease to be treated with the formulation, or may be used to study toxicity of the bacterium of interest. In each of those embodiments, dose escalation studies may be performed in the mammal.

[0090] The specific method used to formulate the novel rdsRP vaccines and formulations described herein is not critical to the present invention and can be selected from a physiological buffer (Feigner et al., U.S. Pat. No. 5,589,466 (1996)); aluminum phosphate or aluminum hydroxyphosphate (e.g. Ulmer et al., Vaccine, 18: 18 (2000)), monophosphoryl- lipid A (also referred to as MPL or MPLA; Schneerson et al. J. Immunol., 147:2136-2140 (1991); e.g. Sasaki et al. Inf. Immunol., 65:3520-3528 (1997); Lodmell et al. Vaccine, 18: 1059-1066 (2000)), QS-21 saponin (e.g. Sasaki, et al., J. Virol., 72:4931 (1998); dexamethasone (e.g. Malone, et al., J. Biol. Chem. 269:29903 (1994)); CpG DNA sequences (Davis et al., J. Immunol., 15:870 (1998)); interferon-α (Mohanty et al., J. Chemother. 14(2): 194- 197, (2002)), or lipopolysaccharide (LPS) antagonist (Hone et al., J. Human Virol., 1 : 251-256 (1998)).

[0091] The formulation herein also may contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary

31

112275/F/l activities that do not adversely impact each other. For example, it may be desirable to further provide an immunogen. Such molecules suitably are present in combination in amounts that are effective for the purpose intended.

[0092] Thus, the bacterium can be used with a second component, such as a foreign antigen or a therapeutic moiety conjugated to or mixed with same, administered as a conjugate, separately in combination, mixed prior to use and so on as a therapeutic. When used as an adjuvant, the recombinant mycobacteria of the present invention are produced as live, inactivated or cell wall preparations as described above and can be admixed with an antigen, inactivated bacteria or live bacteria using methods well known in the art (e.g. Levine et al., Eds., New Generation Vaccines. 2^nd edition. Marcel Dekker, Inc., New York, N.Y. (1997)). The amount of antigen, inactive bacteria or live bacteria is not critical to the present invention but is typically an amount sufficient to induce the desired humoral and cell mediated immune response in the target host. The Mycobacteria of interest also can be configured to express a foreign antigen, or the adjuvant of interest can be administered sequentially, before of after antigen administration.

[0093] The adjuvant of interest can be used in any known manner where an enhancement of the immune response is desired or needed. Thus, an adjuvant of interest can be administered with a foreign antigen, a therapeutic agent and so on. The therapeutic agent can be any drug, vaccine and the like used for an intended purpose. Thus, the therapeutic agent can be a biological, a small molecule and so on.

[0094] The term "small molecule" as well as the "foreign antigen" and analogous terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogues, polynucleotides, polynucleotide analogues, carbohydrates, lipids, nucleotides, nucleotide analogues, organic or inorganic compounds (i.e., including heterorganic and/organometallic compounds) having a molecular weight less than about 10,000 grams per

32

112275/F/l mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, combinations thereof and other pharmaceutically acceptable forms of such compounds which stimulate an immune response or are immunogenic, or have a desired pharmacologic activity.

[0095] Thus, in the case of cancer, the bacterium of the invention may be administered alone or in combination with other types of cancer treatments, including conventional chemotherapeutic agents (paclitaxel, carboplatin, cisplatin, methotrexate and doxorubicin), anti-EGFR agents (gefitinib, erlotinib and cetuximab), anti-angiogenesis agents (bevacizumab and sunitinib), as well as immunomodulating agents, such as interferon-α and thalidomide. Alternatively, the bacterium of the invention can be administered with a cancer antigen, such as, CEA or TAG-72, or other isolated cancer-specific cell surface molecule.

[0096] In addition, the bacterium or product thereof of the instant invention may be conjugated to various effector molecules such as heterologous polypeptides, drugs, radionucleotides or toxins, see, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EPO 396,387. A bacterium or product thereof may be conjugated to a therapeutic moiety such as a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent or a radioactive metal ion (e.g., α emitters such as, for example, ²¹³Bi). A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-

33

112275/F/l mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil and decarbazine), alkylating agents (e.g., mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin, daunomycin and doxorubicin), antibiotics (e.g., dactinomycin, actinomycin, bleomycin, mithramycin and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0097] The present invention also is directed to Mycobacterium-based therapies which involve administering a bacterium or derivative of the invention to an animal, a mammal, or a human, for treating, for example, TB, HIV, an infectious disease, such as, malaria, a cancer, such as, bladder cancer, ocular squamous cell carcinoma, vulval papilloma and so on, or other disorder when used as an adjuvant. The animal or subject may be a mammal in need of a particular treatment, such as a mammal having been diagnosed with a particular disorder, e.g., TB or bladder cancer. For example, by administering a therapeutically acceptable dose of a bacterium of the instant invention, or a cocktail of a plurality of the instant bacterium or equivalents thereof, in combination with another therapeutic product or foreign antigen, disease symptoms may be ameliorated or prevented in the treated mammal, particularly humans.

[0098] Therapeutic compounds of the invention alleviate at least one symptom associated with Mycobacterium or any other disease, disorder, or condition amenable for treatment with an adjuvant of interest. The products of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein. The term "physiologically acceptable," "pharmacologically acceptable" and so on mean approved by a regulatory agency of the Federal or a state government or listed in the U.S.

34

112275/F/l Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.

[0099] The products of interest can be administered to a mammal in any acceptable manner. Methods of introduction include, but are not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation and oral routes, and if desired for immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intradermal, intravenous, intraarterial or intraperitoneal administration. The products or compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the therapeutic products or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. In addition, the product can be suitably administered by pulse infusion, particularly with declining doses of the products of interest. Preferably the dosing is given by injection, preferably intravenous or subcutaneous injections, depending, in part, on whether the administration is brief or chronic.

[00100] Various other delivery systems are known and can be used to administer a product of the present invention, including, e.g., encapsulation in liposomes, microparticles or microcapsules (see Langer, Science 249: 1527 (1990); Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein et al., eds., (1989)).

[00101] The active ingredients may be entrapped in a microcapsule prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule,

35

112275/F/l respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed. (1980).

[00102] Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. The composition of interest may also be administered into the lungs of a patient in the form of a dry powder composition, see e.g., U.S. Pat. No. 6,514,496.

[00103] It may be desirable to administer the therapeutic products or compositions of the invention locally to the area in need of treatment; that may be achieved by, for example, and not by way of limitation, local infusion, topical application, by injection, by means of a catheter, by means of a suppository or by means of an implant, said implant being of a porous, non-porous or gelatinous material, including hydrogels or membranes, such as sialastic membranes or fibers. Preferably, when administering a product of the invention, care is taken to use materials to which the protein does not absorb or adsorb.

[00104] In yet another embodiment, the product can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, Science 249: 1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N Engl J Med 321 :574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer et al., eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et al., eds., Wiley (1984); Ranger et al., J Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al., Science 228: 190 (1985); During et al., Ann Neurol 25:351 (1989); and Howard et al., J Neurosurg 71 : 105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target.

36

112275/F/l [00105] Therapeutic formulations of the product may be prepared for storage as lyophilized formulations or aqueous solutions by mixing the product having the desired degree of purity with optional pharmaceutically acceptable carriers, diluents, excipients or stabilizers typically employed in the art, i.e., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and other miscellaneous additives, see Remington's Pharmaceutical Sciences, 16th ed., Osol, ed. (1980). Such additives are generally nontoxic to the recipients at the dosages and concentrations employed, hence, the excipients, diluents, carriers and so on are pharmaceutically acceptable.

[00106] An "isolated" or "purified" bacterium is substantially free of contaminating proteins from the medium from which the cell is obtained, or substantially free of chemical precursors or other chemicals in the medium used which contains components that are chemically synthesized. The language "substantially free of subcellular material" includes preparations of a cell in which the cell is separated from subcellular components of the cells, such as dead cells, and portions of cells, such as cell membranes, ghosts and the like, from which same is isolated or recombinantly produced. Thus, a bacterium that is substantially free of subcellular material includes preparations of the cell having less than about 30%, 20%, 25%, 20%, 10%, 5%, 2.5% or 1%, (by dry weight) of non-bacterial, subcellular contaminants.

[00107] As used herein, the terms "stability" and "stable" in the context of a liquid formulation comprising a bacterium or product thereof refer to the resistance of the bacterium of product thereof in the formulation to thermal and chemical aggregation, degradation or fragmentation under given manufacture, preparation, transportation and storage conditions, such as, for one month, for two months, for three months, for four months, for five months, for six months or more. The "stable" formulations of the invention retain biological activity equal to or more than 80%, 85%, 90%, 95%, 98%, 99% or 99.5%

37

112275/F/l under given manufacture, preparation, transportation and storage conditions. The stability of said bacterium preparation can be assessed by degrees of aggregation, degradation or fragmentation by methods known to those skilled in the art, including, but not limited to, physical observation, such as, with a microscope, particle size and count determination and so on, compared to a reference.

[00108] The term, "carrier," refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered. Such physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a suitable carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[00109] The compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, depots and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate etc. Examples of suitable carriers are described in "Remington's Pharmaceutical Sciences," Martin. Such compositions will contain an effective amount of the bacterium or functional portion of variant thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration

38

112275/F/l to the patient. As known in the art, the formulation will be constructed to suit the mode of administration.

[00110] Buffering agents help to maintain the pH in the range which approximates physiological conditions. Buffers are preferably present at a concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the instant invention include both organic and inorganic acids, and salts thereof, such as citrate buffers (e.g., monosodium citrate-di sodium citrate mixture, citric acid-tri sodium citrate mixture, citric acid-monosodium citrate mixture etc.), succinate buffers (e.g., succinic acid- monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid- disodium succinate mixture etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-di sodium fumarate mixture etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture etc.). Phosphate buffers, carbonate buffers, histidine buffers, trimethylamine salts, such as Tris, HEPES and other such known buffers can be used.

[00111] Preservatives may be added to retard microbial growth, and may be added in amounts ranging from 0.2%-l% (w/v). Suitable preservatives for use with the present invention include phenol, benzyl alcohol, m-cresol, octadecyldimethylbenzyl ammonium chloride, benzyaconium halides (e.g., chloride, bromide and iodide),

39

112275/F/l hexamethonium chloride, alkyl parabens, such as, methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

[00112] Isotonicifϊers are present to ensure physiological isotonicity of liquid compositions of the instant invention and include polhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in an amount of between about 0.1% to about 25%, by weight, preferably 1% to 5% taking into account the relative amounts of the other ingredients.

[00113] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins, such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone, saccharides, monosaccharides, such as xylose, mannose, fructose or glucose; disaccharides, such as lactose, maltose and sucrose; trisaccharides, such as raffinose; polysaccharides, such as, dextran and so on. Stabilizers can be present in the range from 0.1 to 10,000 w/w per part of bacterium or product thereof.

40

112275/F/l [00114] Additional miscellaneous excipients include bulking agents, (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine or vitamin E) and cosolvents.

[00115] As used herein, the term "surfactant" refers to organic substances having amphipathic structures, namely, are composed of groups of opposing solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified, depending on the charge of the surface-active moiety, into anionic, cationic and nonionic surfactants. Surfactants often are used as wetting, emulsifying, solubilizing and dispersing agents for various pharmaceutical compositions and preparations of biological materials.

[00116] Non-ionic surfactants or detergents (also known as "wetting agents") may be added to help solubilize the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stresses without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80 etc.), polyoxamers (184, 188 etc.), Pluronic^® polyols and polyoxyethylene sorbitan monoethers (TWEEN-20^®, TWEEN-80^® etc.). Non-ionic surfactants may be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

[00117] As used herein, the term, "inorganic salt," refers to any compound, containing no carbon, that results from replacement of part or all of the acid hydrogen or an acid by a metal or a group acting like a metal, and often is used as a tonicity adjusting compound in pharmaceutical compositions and preparations of biological materials. The most common inorganic salts are NaCl, KCl, NaH₂PO₄ etc.

41

112275/F/l [00118] The present invention provides liquid formulations of a bacterium or product thereof, having a pH ranging from about 5.0 to about 7.0, or about 5.5 to about 6.5, or about 5.8 to about 6.2, or about 6.0, or about 6.0 to about 7.5, or about 6.5 to about 7.0.

[00119] The instant invention encompasses formulations, such as, liquid formulations having stability at temperatures found in a commercial refrigerator and freezer found in the office of a physician or laboratory, such as from about -20° C to about 5° C, said stability assessed, for example, by microscopic analysis, for storage purposes, such as for about 60 days, for about 120 days, for about 180 days, for about a year, for about 2 years or more. The liquid formulations of the present invention also exhibit stability, as assessed, for example, by particle analysis, at room temperatures, for at least a few hours, such as one hour, two hours or about three hours prior to use.

[00120] Examples of diluents include a phosphate buffered saline, buffer for buffering against gastric acid in the bladder, such as citrate buffer (pH 7.4) containing sucrose, bicarbonate buffer (pH 7.4) alone, or bicarbonate buffer (pH 7.4) containing ascorbic acid, lactose, or aspartame. Examples of carriers include proteins, e.g., as found in skim milk, sugars, e.g., sucrose, or polyvinylpyrrolidone. Typically these carriers would be used at a concentration of about 0.1-90% (w/v) but preferably at a range of 1-10% (w/v).

[00121] The formulations to be used for in vivo administration must be sterile.

That can be accomplished, for example, by filtration through sterile filtration membranes. For example, the subcellular formulations of the present invention may be sterilized by filtration.

[00122] Sustained-release preparations may be prepared for use with the products of interest. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the bacterium, or functional portion or variant thereof, and/or foreign antigen, which matrices are in the form of

42

112275/F/l shaped articles, e.g., films or matrices. Suitable examples of such sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate), poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as injectable microspheres composed of lactic acid-glycolic acid copolymer) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release cells, proteins and products for and during shorter time periods. Rational strategies can be devised for stabilization depending on the mechanism involved.

[00123] The bacterium or product thereof composition will be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of the bacterium or product thereof to be administered will be governed by such considerations, and can be the minimum amount necessary to prevent, ameliorate or treat a Mycobacterium based disease, condition or disorder.

[00124] The amount of the recombinant mycobacteria of the present invention to be administered as live bacteria, inactivated bacteria or as cell wall preparations will vary depending on the species of the subject, as well as the disease or condition that is being treated. Generally, the dosage employed will be about 10³ to 10¹¹ viable organisms, preferably about 10⁵ to 10⁹ viable organisms. Alternatively, when infecting individual cells, the dosage of viable organisms to administered will be at a multiplicity of infection ranging from about 0.1 to 10⁶, preferably about 10² to 10⁴. The number of inactivated bacteria may

43

112275/F/l vary and is adjusted based on a comparison of efficacy with live bacteria. The amount of a subcellular product or component of interest may vary and is adjusted based on a comparison of efficacy with live bacteria.

[00125] As used herein, the term "effective amount" refers to the amount of a therapy (e.g., a prophylactic or therapeutic agent), which is sufficient to reduce the severity and/or duration of a Mycobacterium-caused disease, ameliorate one or more symptoms thereof, prevent the advancement of a Mycobacterium-based disease or cause regression of a Mycobacterium-based disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of a Mycobacterium-based disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect(s) of another therapy (e.g., another therapeutic agent) useful for treating a disease where the bacterium or product thereof is used as an adjuvant. For example, a treatment of interest can increase survivability of the host, based on baseline or a normal level, by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In another embodiment, an effective amount of a therapeutic or a prophylactic agent reduces the symptoms of a Mycobacterium-based disease, such as a symptom of TB by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Also used herein as an equivalent is the term, "therapeutically effective amount."

[00126] Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine or other "caine" anesthetic to ease pain at the site of the injection.

44

112275/F/l [00127] Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided, for example, in a kit, so that the ingredients may be mixed prior to administration.

[00128] An article of manufacture containing materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for preventing or treating a mycobacterium-based condition or disease and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label on or associated with the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

[00129] While speculation is provided hereinabove on the potential mechanism through which genetically modified mycobacterial strains elicit enhanced anticancer immunotherapy, such speculation is not intended to limit the scope of the present invention. Furthermore, citation of any of the references discussed hereinabove shall not be construed as

45

112275/F/l an admission that any such reference is prior art to the present invention. All references cited herein are herein incorporated by reference in entirety.

[00130] The invention now will be exemplified by the following non-limiting examples.

Example 1

Procedures for the assembly of genetically modified mycobacteria

[00131] All media used in the production of recombinant mycobacterial strains for veterinary and human applications preferably should be certified TSE-free by the manufacturer. TSE-free liquid media for culturing mycobacterial strains include Middlebrook 7H9 (Difco) or Sauton's medium (Difco) or MSM (see above), which are normally maintained at 37⁰C but in certain circumstances these organisms can also be cultured at between 3O⁰C and 4O⁰C. The cultures are incubated with or without agitation at 150 oscillations per minute. The growth rate of mycobacteria can be enhanced by the addition of oleic acid (0.06% v/v; Research Diagnostics Cat. No. 01257) and a surfactant (such as Tyloxapol (0.05% v/v; Research Diagnostics Cat. No.70400)).

[00132] Liquid cultures of mycobacteria are stopped at an optical density (at

600 nm) of 0.2 - 4.0 relative to a sterile control, and the organisms are harvested by centrifugation at 8,000 x g for 5-10 min. The liquid supernatant is discarded and the mycobacterial bacilli are resuspended in PBS containing 10% (v/v) glycerol and 0.05% (v/v) tyloxapol (Research Diagnostics Cat. No.70400) at a density of 0.5 x 10⁷ to 5.0 x 10⁷ cfu/ml. Aliquots (1-5 ml) of these suspensions are dispensed into sterile appropriately sized sterile boron silicate freezer vials and stored at -8O⁰C.

[00133] TSE-free solid media for culturing mycobacterial strains includes

Middlebrook 7H10 (Difco) or Sauton's medium (Difco) and MSM (see above) containing 15 g/L agar (Difco), which is placed in 3.5 inch disposable plastic Petri dishes that are

46

112275/F/l maintained in gas-permeable bags to prevent desiccation of the plates and normally incubated at 37⁰C but in certain circumstances these organisms can also be incubated at between 3O⁰C and 4O⁰C.

[00134] The purity of mycobacterial cultures is evaluated by spreading small aliquots, typically 0.1 ml, of the culture in 10-fold serial dilutions from 10° - 10^"8 in phosphate buffered saline (herein referred to "PBS") on solid media, such as Middlebrook 7H10 at 37⁰C. The purity of the culture can be further assessed as described in US FDA document 21 CFR 610.12 using commercially available liquid media, such as Thioglycolate medium (Sciencelab, Cat #1891) and Soybean-Casein medium (Becton-Dickinson, Cat #: 211768).

[00135] All reagents used in the production of recombinant mycobacterial strains for veterinary and human applications preferably should be certified TSE-free by the manufacturer. TSE-free restriction endonucleases (New England Biolabs, Beverly, MA), T4 DNA ligase (New England Biolabs) and Taq polymerase (Invitrogen, Carlsbad, CA) are used according to the manufacturers' protocols. Plasmid DNA is prepared using small-scale (Qiagen Miniprep^R kit, Santa Clarita, CA) or large-scale (Qiagen Maxiprep^R kit, Santa Clarita, CA) plasmids DNA purification kits according to the manufacturer's protocols (Qiagen, Santa Clarita, CA). Nuclease-free, molecular biology grade milli-Q water, Tris-HCl (pH 7.5), EDTA pH 8.0, IM MgCl₂, 100% (v/v) ethanol, ultra-pure agarose, and agarose gel electrophoresis buffer are purchased from Invitrogen. Restriction endonuclease digestions, PCRs, DNA ligation reactions and agarose gel electrophoresis are conducted according to well-known procedures (127, 128). Nucleotide sequencing to verify the DNA sequence of each recombinant plasmid described in the following sections is accomplished by medium- high throughput automated DNA sequencing using an ABI 8-capillary 3730 DNA Analyzer (Applied Biosystems Inc., Foster City, CA) according to the manufacturer's directions.

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112275/F/l [00136] The PCR primers for the amplifications are designed using Clone

Manager Professional Suite version 8.0 (Scientific and Educational Software Inc., Durham, NC). This software enables the design of PCR primers and identifies compatible restriction endonuclease sites for the specific DNA fragments being amplified. PCR primers are purchased from commercial sources such as Sigma (St. Louis, MO) or are synthesized using an ABI model 3900 DNA synthesizer (Applied Biosystems Inc.) according to the manufacturer's directions. PCR primers are used at a concentration of 100-300 μM and annealing temperatures for the PCR reactions are determined using Clone Manager Professional Suite version 8.0 (Scientific and Educational Software Inc.). PCRs are run in a thermocycler device, such as the Stratagene Robocycler, model 400880 (Stratagene), and primer annealing, elongation and denaturation times in the PCRs are set according to standard procedures (128).

[00137] The DNA fragments produced by the restriction endonuclease digestions and PCRs are analyzed by agarose gel electrophoresis using standard procedures (127, 128). A positive clone is defined as one that displays the appropriate restriction endonuclease and/or PCR pattern. Plasmids identified through this procedure can be further evaluated by medium-high throughput automated DNA sequencing using an ABI 8-capillary 3730 DNA Analyzer (Applied Biosystems Inc., Foster City, CA) according to the manufacturer's directions.

[00138] Bacterial strains that serve as hosts and amplify recombinant plasmids, such as Escherichia coli strains DH5α and Stable2^R, are purchased from Invitrogen. Recombinant plasmids are introduced into E. coli strains by electroporation using an high- voltage electropulse device, such as the Gene Pulser (BioRad Laboratories, Hercules, CA), set at 100-200Ω, 15-25 μF and 1.0-2.5 kV, as described (126). Optimal electroporation

48

112275/F/l conditions are identified on a trial-by-error basis by determining settings that result in maximum transformation rates per meg DNA per bacterium.

[00139] Solid media for the growth of E. coli strains can be TSE-free tryptic soy agar (Difco, Detroit, MI) and liquid media for growth of the same can be TSE-free tryptic soy broth (Difco, Detroit, MI), which are made according to the manufacturer's directions. Unless stated otherwise, all E. coli are grown at 37⁰C with gentle agitation. When appropriate, the media are supplemented with antibiotics (Sigma, St. Louis, MO). Bacterial strains are stored at -8O⁰C suspended in tryptic soy broth (Difco) containing 30% (v/v) glycerol (v/v; Sigma, St. Louis, MO) at ca. 10⁹ colony-forming units (herein referred to as "cfu") per ml.

Example 2

Construction of mycobacteria with altered expression of a tissue adherence factor

[00140] This example describes procedures that are used to construct strains

Bacilligen-1010 (rBCG-FAP^c) and Bacilligen-1011 (rM. smegmati S-FAP⁺), which are recombinant mycobacterial strains that constitutively express fibronectin attachment protein (GenBank Accession no. AAB71842). To express FapB constitutively, a synthetic gene is generated that encodes the Ag85A promoter (herein referred to as "P_Ag85A"; SEQ ID NO:3) functionally linked to fapB (SEQ ID NO: 12). The synthetic gene, ^~P _A&_{5 A}-fa ' pB, flanked by Pad sites is purchased from Picoscript (Houston, Texas) and is ligated (Example 1) into the unique Pad site in plasmid pBACIL-101 (SEQ ID NO: 13). This latter plasmid is comprised of the lactose transporter LacY and β-galactosidase gene (herein referred to as "lacYZ"), which is under the control of the antigen-85B promoter (herein referred to as "P_Ag85B") and codon optimized for expression in mycobacteria, the Kan^R (GenBank accession no. U75323) and is flanked by Notl digestions sites, the E. coli plasmid origin of replication OriE (GenBank accession no. AY947541), the M. bovis genomic origin of replication (herein

49

112275/F/l referred to as "OriC"; Qin et al., J. Bacterid., 179(20):6311-6317, (1997); Salazar et al., Microbiol., 149:773-784, (2003)) and a unique Pad digestion site (Figure 1).

[00141] The ligated plasmid is introduced into E coli strain Stable2 as described in Example 1. An isolate harboring the recombinant plasmid designated pB ACIL- 102 (i.e. pBACIL-101 ::P_Ag85A-fapB), is amplified by culturing the resulting transformants in 100 ml liquid media at 37⁰C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1). The purified DNA is digested with Notl to remove Kan^R and the large fragment encoding the Ag85A promoter functionally linked to P_Agss_A-fapB, lacYZ, OriE and OriC is purified following fractionation by agarose gel electrophoresis (Example 1). The purified DNA fragment is ligated to form a closed circular DNA as described and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 1). Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 1) containing lactose in place of glycerol at 37⁰C. Resulting colonies are screened by PCR (Example 1), to demonstrate the presence of the plasmid, DNA sequencing (see recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for- service basis at ACE BioSciences (Odense, Denmark) to demonstrate that FabB is constitutively expressed in BCG.

[00142] The same procedure is used to construct a derivative of M. smegmatis

(ATCC#12051; American Type Culture Collection, Manassas, VA) that harbors pB ACIL- 102 [ΔKan^R]. Colonies that grow on the lactose-selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, automated DNA sequencing (Example 1) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that FapB is expressed in M. smegmatis.

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112275/F/l Example 3

Construction of mycobacteria that express an immunostimulatory factor

[00143] This example describes procedures that are used to construct strains

Bacilligen-1012 (rBCG-Mbl389c_1-36o^C) and Bacilligen-1014 (rM. smegmatis-Mbl3&9ci-₃6o^C), which are engineered to constitutively express GGDEF domain-containing amino acids 1-360 of Mbl389c (GenBank Accession no. NP855043). This truncated derivative is encoded by the complement of GenBank accession no. NC002945.3, from 1518040 to 1519911 and is shown in SEQ ID NO: 14. To enable constitutive expression the sequence encoding amino acids 1-360 of Mbl389c is functionally linked to P_Ag85A (SEQ ID NO:3).

[00144] To construct the recombinant gene, a synthetic gene is purchased from

Picoscript (Houston, Texas) encoding P_Ag85A-Mbl339c_1-36o and is ligated (Example 1) into the unique Pad site in plasmid pBACIL-101 (SEQ ID NO: 13). The ligated plasmid is introduced into E. coli strain Stable2 as described in Example 1. An isolate harboring the recombinant plasmid designated pBACIL-103 (i.e. pBACIL-lOl ::PAg85A::Mbl339c_1-36o), is amplified by culturing the resulting transformants in 100 ml liquid media at 37⁰C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1). The purified DNA is digested with Notl to remove Kan^R and the large fragment encoding PA_g85A::Mbl339c_1-36o, PAgssB-focFZ, OriE and OriC is purified following fractionation in agarose (Example 1). The purified DNA fragment is ligated to form a closed circular DNA as described and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 1). Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of glycerol at 37⁰C. Colonies that grow on this novel selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, DNA sequencing (see recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel

51

112275/F/l electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that Mb 1339C_1-36O is over-expressed in BCG.

[00145] The same procedure is used to construct a derivative of M. smegmatis

(ATCC#12051; American Type Culture Collection, Manassas, VA) that harbors pB ACIL- 101 ::P_Ag85A::Mbl339c_1-360 [ΔKan^R]). Colonies that grow on the lactose-selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, automated DNA sequencing (Example 1) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that Mbl339c_1-36o is over-expressed inM smegmatis.

Example 4

Construction of mycobacteria that express an adjuvant

[00146] This example describes procedures that are used to construct strains

Bacilligen-1015 (rBCG-CtxA⁺) and Bacilligen-1016 (rM. smegmatis-CtxA⁺\ which are engineered to constitutively express CtxA, using a similar approach as the two proceeding examples.

[00147] Secretion of CtxA by recombinant mycobacteria of the present invention is accomplished by generating a genetic fusion between DNA encoding LPA_g85A (SEQ ID NO:2) and DNA encoding the mature CtxA protein (amino acids 18-258). Expression of LPAg₈₅A-CtXA_18-258 (wherein "::" denotes the novel genetic junction) is accomplished by functionally linking synthetic DNA encoding said genetic fusion to PA_g85A (SEQ ID NO:3). Synthetic DNA encoding the recombinant gene, i.e. PAg8₅A::LPAg8₅A:

₂58, is purchased from Picoscript, Houston, Texas, and is ligated into plasmid pB ACIL-101 (SEQ ID NO: 13), as described (Example 1). The resulting recombinant plasmid designated pBACIL-104 (i.e. pBAdL-101 ::PA_g85A::LPAg8₅A::CtxA) is introduced into E. coli strain Stable2, as described in Example 1, and an isolate harboring the recombinant plasmid is

52

112275/F/l amplified by culturing the resulting transformants in 100 ml liquid media at 37⁰C with agitation (Example 1). Plasmid DNA is purified from this culture as described (Example 1). The purified DNA is digested with Notl to remove Kan^R and the large fragment encoding PA_g85A-CtxA, PA_g85B: .lacYZ, OriE and OriC is purified following fractionation in agarose (Example 1). The purified DNA fragment is ligated to form a closed circular DNA as described and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Examples 1 and 2). Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of glycerol at 37⁰C. Colonies that grow on this novel selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, DNA sequencing to demonstrate that the plasmid carries the correct sequence (Example 1) and 2D gel electrophoresis on a fee-for- service basis at ACE BioSciences (Odense, Denmark) to demonstrate that CtxA is expressed in BCG.

[00148] The same procedure is used to construct a derivative of M. smegmatis

(ATCC#12051; American Type Culture Collection, Manassas, VA) that harbors pB ACIL- 101 :: P_Ag85A-CtxA [ΔKan^R]). Colonies that grow on the lactose-selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, automated DNA sequencing (Example 1) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that CtxA is expressed in M. smegmatis.

Example 5

Construction of RDl -positive mycobacteria

[00149] This example describes procedures that are used to construct strains

Bacilligen-1017 (rBCG-RDl⁺) and Bacilligen-1018 (rM. smegmatis-RD\⁺\ which are

53

112275/F/l engineered to constitutively express a functional RDl region, using a similar approach as the proceeding examples.

[00150] The RDl deletion in BCG that results in an inability to secrete ESAT-6

(also know as Rv3875) and CFPlO (also known as Rv3874), which contributes to the virulence of Mtb, has been defined elsewhere (23, 27). The sequence to complement the RDl deletion in BCG (SEQ ID NO:4), which encodes the Mb3897 promoter region, Mb3901, Mb3902, Mb3903, Mb3904 (homologous to CFPlO), Mb3905 (homologous to ESAT6), Mb3906 and Mb3907, is made synthetically (Example 1) and is ligated (Example 1) into plasmid pBACIL-101 (SEQ ID NO: 13). The ligated plasmid is introduced into E. coli strain Stable2, also as described in Example 1. An isolate harboring the recombinant plasmid designated pBACIL-105 (i.e. pBACIL-101 ::SEQ ID NO:4), is amplified by culturing the resulting transformants in 100 ml liquid media at 37⁰C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1). The purified pBACIL-105 DNA is digested with Notl to remove Kan^R and the large fragment encoding SEQ ID NO:4, PA_g85B: :lacYZ, OriE and OriC is purified following fractionation in agarose (Example 1). The purified DNA fragment is ligated to form a closed circular DNA as described and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 1). Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 1) containing lactose in place of glycerol at 37⁰C. Colonies that grow on this novel selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, DNA sequencing (see recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-f or- service basis at ACE BioSciences (Odense, Denmark) to demonstrate that ESAT-6 and CFPlO are expressed by the rBCG strain, Bacilligen-1017 (rBCG-RDl⁺).

54

112275/F/l [00151] The same procedure is used to construct a derivative of M. smegmatis

(ATCC#12051; American Type Culture Collection, Manassas, VA) that harbors pB ACIL- 101 :: SEQ ID NO:4). Colonies that grow on the lactose-selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, automated DNA sequencing (Example 1) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that ESAT-6 and CFPlO are expressed in xM. Smegmatis strain, Bacilligen-1018 (rM. smegmatis-KDl⁺).

Example 6

Construction of pro-apoptosis mycobacteria

[00152] This example describes procedures that are used to construct strains

Bacilligen-1019 (rBCG-NS3⁺) and Bacilligen-1020 (rM. smegmatis-NS3⁺), which are engineered to constitutively express the proteolytic domain of NS3, using a similar approach as in the proceeding examples.

[00153] As discussed above, macrophages are less effective than DCs at promoting the development of CD4⁺ and CD8⁺ T cell responses (41). Therefore, induction of apoptosis in macrophages provides a delivery mechanism through which antigens from cells infected with mycobacteria are transferred to DCs leading to the induction of strong effector T cells (40, 41). Furthermore, macrophages undergoing apoptosis are more effective at killing mycobacteria than activated macrophages and macrophages undergoing necrosis (42), suggesting that mycobacteria that promote apoptosis will display an improved safety profile as anticancer immunotherapeutics.

[00154] To generate mycobacterial strains that promote apoptosis, it is necessary enable the transit of secreted mycobacterial proteins from the endosome into the cytoplasm of the infected host cell. The literature provides an example of a mycobacterial

55

112275/F/l strain that causes the endosome to become leaky (24); however, this example provides the materials and methods to construct novel rBCG and rM. smegmatis strains that harbor an RDS encoding a genetic fusion between the LP_Ag85A (SEQ ID NO:2), the intercellular trafficking domain of VP22 (SEQ ID NO: 15; herein referred to a "VP22i_TD"), which spans amino acids 81-195, and the proteolytic domain of NS3_Pr (SEQ ID NO:8); encoded by base pairs 6469-7039 of West Nile virus isolate Mex03 (GenBank Accession no. AY660002), which spans amino acids 1-190.

[00155] The sequence encoding the NS3p_r is generated synthetically by

Picoscript (Houston, Texas) and are optimized for expression in mycobacteria by using the preferred codon bias of this genus (SEQ ID NO:8; (69, 70)). Secretion of the NS3_Pr by the recombinant mycobacteria of the present invention is accomplished by generating a genetic fusion between DNA encoding LP_Ag85A (SEQ ID NO:2) and DNA encoding the PAP.

[00156] Transport of NS3_Pr from the endosome to the cytoplasmic compartment of the host cell is accomplished by inserting DNA encoding the ITP human herpes virus VP22 amino acids 81-195 (herein referred to as "VP22₈i-i₉5"; SEQ ID NO: 15) between DNA encoding LPA_g85A and NS3p_r. Expression of NS3p_r activity is enhanced by separating the fusion partners VP22₈i-i95 and NS3_Pr with DNA encoding a flexible linker, as known in the art, including, for example, one containing a furin degradation motif (herein referred to as "FLf_ur"; i.e Serine-Glycine-Glycine-Glycine-Glycine-Arginine-Threonine- Lysine-Arginine-Glycine-Glycine-Glycine-Glycine-Serine; SEQ ID NO: 11).

[00157] To instigate expression of LP_Ag85A::VP22iτ_D::FL_fur::NS3p_r, synthetic

DNA encoding the genetic fusion is functionally linked to PA_g85A (SEQ ID NO:3). Synthetic DNA encoding recombinant gene P_Ag85A-LP_Ag85A::VP22i_TD::FL_fur::NS3p_r is purchased from Picoscript (Houston, Texas) and is ligated (Example 1) into plasmid pB ACIL-101 (SEQ ID NO: 13). The ligated plasmid is introduced mto E. coli strain Stable2 as described in Example

56

112275/F/l 1. An isolate harboring the recombinant plasmid designated pBACIL-106 (i.e. pBACIL- 101 ::P_{Ag85 A}-LP _Ag85A::VP22iτ_D::FL_fur::NS3p_r), is amplified by culturing the resulting transformants in 100 ml liquid media at 37⁰C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1). The purified DNA is digested with restriction endonuclease Notl (New England Biolabs) to remove Kan^R and the large fragment encoding PA_g85A-LPAg85A::VP22iτD::FLf_ur::NS3p_r, PA_g85B: .lacYZ, OriE and OriC is purified following fractionation in agarose (Example 1). The purified DNA fragment is ligated to form closed circular DNA as described (Example 1) and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 2). Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of glycerol at 37⁰C. Colonies that grow on this novel selection medium are screened by PCR using forward primer: 5'-GGCGTGTTGTGGGACACTCCCTCA-S ' (SEQ ID NO: 16) and reverse primer 5'-GATCTGTTTTTTCCTCAGCATCTC-S ' (SEQ ID NO: 17) (Example 1) to demonstrate the presence of the plasmid, DNA sequencing (See recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that NS3_Pr is expressed in BCG.

[00158] The same procedure is used to construct a derivative of M. smegmatis

(ATCC#12051; American Type Culture Collection, Manassas, VA) that harbors pB ACIL- 106 encoding P_Ag85A-LP_Ag85A::VP22iτ_D::FL_fur::NS3p_r [ΔKan^R]). Colonies that grow on the lactose-selection medium are screened by PCR (Example 1), to demonstrate the presence of the plasmid, automated DNA sequencing (Example 1) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that NS3_Pr is expressed in M. smegmatis.

57

112275/F/l Example 7

Construction of mycobacteria carrying multiple modifications

[00159] This example describes procedures that are used to construct strains

Bacilligen-1021 (rBCG FAP^c-NS3_Pr ⁺-RDl⁺), Bacilligen-1022 (rBCG FAP^C- Mbl389c_1-360 ^C- RDl⁺) and Bacilligen-1023 (rM. smegmatis FAP^c-Mbl389c_1-36o^C), which are engineered to constitutively express the proteolytic domain of NS3, using a similar approach as the proceeding examples.

[00160] Strain Bacilligen-1021 is constructed by introducing a derivative of pBACIL-101 designated pBACIL-107, which carries synthetic sequences that express a FAP^C, NS3_Pr ⁺ and RDl⁺ phenotype in BCG. Plasmid pBACIL-107 is assembled using the cloning schematic shown in Figure 2. A small sequence encoding the Mycobacterial consensus ribosomal binding site (SEQ ID NO: 19 herein referred to as "RBS") is inserted upstream of the sequence encoding LP_Ag85A::VP22iτ_D::FL_fur::NS3p_r by PCR-directed insertional mutagenesis (Example 1). The sequence encoding

RBS::LP_Ag85A::VP22iτ_D::FL_fur::NS3p_r is then digested with Ascl (New England Biolabs, Cat. No. R0558S) and joined to Ascl-digested DNA encoding P_Ag85A-FAP (Figure 2). The resulting chimeric fragment is purified following agarose gel electrophoresis (Example 1) and digested with Pad (New England Biolabs, Cat. No. R0547S) and Fsel (New England Biolabs, Cat. No. R0588S). In parallel, DNA encoding RDl⁺ (SEQ ID NO:4) is amplified from plasmid pBACIL-105 (Example 5) by PCR so as to insert Fsel and Pad sites at the 5' and 3' ends, respectively. This PCR-generated fragment is digested with Pad and Fsel. Finally, pBACIL-101 is digested with Pad and equimolar amounts of the three digested DNA preparations are introduced into a ligation reaction, resulting in the generation of pBACIL-107 (Figure 2).

58

112275/F/l [00161] The ligated plasmid is introduced into E. coli strain Stable2 as described in Example 1. An isolate harboring the recombinant plasmid designated pB ACIL- 106 (i.e. pBACIL-101 ::P_Ag85A-NS3p_r), is amplified by culturing the resulting transformants in 100 ml liquid media at 37⁰C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1). The purified DNA is digested with restriction endonuclease Notl (New England Biolabs) to remove Kan^R and the large fragment encoding PA_g85A-FAP-RBS- NS3p_r-RDl⁺, PA_g85B: :lacYZ, OriE and OriC is purified following fractionation in agarose (Example 1). The purified DNA fragment is ligated to form closed circular DNA as described (Example 1) and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 2). Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of glycerol at 37⁰C. Colonies that grow on this novel selection medium are screened by PCR using forward primer: 5'- GGCGTGTTGTGGGAC ACTCCCTC A-3' (SEQ ID NO: 16) and reverse primer 5'-GATCTGTTTTTTCCTCAGCATCTC-S ' (SEQ ID NO: 17) (Example 1) to demonstrate the presence of plasmid pBACIL-107, DNA sequencing (see recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that FAP, NS3p_r, ESAT-6 and CFPlO are expressed in the resulting rBCG strains Bacilligen-1021.

[00162] Strain Bacilligen-1022 is constructed by introducing a derivative of pBACIL-101 designated pBACIL-108, which carries synthetic sequences that express a FAP^C (SEQ ID NO:9), Mbl389c_1-36o⁺ (SEQ ID NO: 14) and RDl⁺ (SEQ ID NO:4) phenotype in BCG. Plasmid pBACIL-108 is assembled using the cloning schematic shown in figure 3. A small sequence encoding the Mycobacterial consensus RBS (SEQ ID NO: 19) is inserted upstream of the sequence encoding Mbl389c_1-36o by PCR-directed insertional mutagenesis

59

112275/F/l (Example 1). The sequence encoding RB S-Mb 1389C_1-36O is then digested with Ascl and joined to Ascl-digested DNA encoding PA_g85A-FAP (Figure 3). The resulting chimeric fragment is purified following agarose gel electrophoresis (Example 1) and digested with Pad and Fsel. In parallel, DNA encoding RDl⁺ (SEQ ID NO:4) is amplified from plasmid pBACIL-105 (Example 5) by PCR so as to insert Fsel and Pad sites at the 5' and 3' ends, respectively. This PCR-generated fragment is digested with Pad and Fsel. Finally, pBACIL- 101 is digested with Pad and equimolar amounts of the three digested DNA preparations are introduced into a ligation reaction, resulting in the generation of pBACIL-108 (Figure 3).

[00163] The ligated plasmid is introduced into E. coli strain Stable2 as described in Example 1. An isolate harboring the recombinant plasmid designated pB ACIL- 108, is amplified by culturing the resulting transformants in 100 ml liquid media at 37⁰C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1). The purified DNA is digested with restriction endonuclease Notl (New England Biolabs) to remove Kan^R and the large fragment encoding P_Ag85A-FAP-RBS-Mbl389c_1-36o-RDl⁺, PA_g85B: :lacYZ, OriE and OriC is purified following fractionation in agarose (Example 1). The purified DNA fragment is ligated to form closed circular DNA as described (Example 1) and the resulting DNA is introduced into BCG strain Danish 1331 by electroporation as described (Example 2). Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of glycerol at 37⁰C. Colonies that grow on this novel selection medium are screened by PCR using forward primer: 5'- GGCGTGTTGTGGGAC ACTCCCTCA-3' (SEQ ID NO: 10) and reverse primer 5'- GATCTGTTTTTTCCTC AGC ATCTC-3' (SEQ ID NO: 17) (Example 1) to demonstrate the presence of plasmid pBACIL-108 [ΔKan^R], DNA sequencing (See recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to

60

112275/F/l demonstrate that FAP, Mb 1389C_1-36O, ESAT-6 and CFPlO are expressed in the resulting rBCG strains Bacilligen-1022.

[00164] Strain Bacilligen-1023 is constructed by introducing a derivative of pBACrL-101 designated pBACIL-109, which carries synthetic sequences that express a FAP^C (SEQ ID NO: 8) and Mbl389c_1-360 ⁺ (SEQ ID NO: 14) phenotype in M. smegmatis. Plasmid pBACIL-109 is assembled using the cloning schematic shown in Figure 4. A small sequence encoding the mycobacterial consensus RBS (SEQ ID NO: 19) is inserted upstream of the sequence encoding Mbl389c_1-36o by PCR-directed insertional mutagenesis (Example 1). The sequence encoding RBS-Mbl389c_1-36o is then digested with Ascl and joined to Ascl- digested DNA encoding PA_g85A-FAP (Figure 4). The resulting chimeric fragment is purified following agarose gel electrophoresis (Example 1) and digested with Pad. In parallel, pBACIL-101 is digested with Pad and equimolar amounts of the two digested DNA preparations are introduced into a ligation reaction, resulting in the generation of pBACIL- 109 (Figure 4).

[00165] The ligated plasmid is introduced into E. coli strain Stable2 as described in Example 1. An isolate harboring recombinant plasmid pBACIL-109 is amplified by culturing the resulting transformants in 100 ml liquid media at 37⁰C with agitation (Example 1) and the plasmid DNA is purified as described (Example 1). The purified DNA is digested with restriction endonuclease Notl (New England Biolabs) to remove Kan^R and the large fragment encoding PA_g85A-FAP-RBS-Mbl389c_1-36o, PA_g85B:J«c7Z, OriE and OriC is purified following fractionation in agarose (Example 1). The purified DNA fragment is ligated to form closed circular DNA as described (Example 1) and the resulting DNA is introduced into M. smegmatis (ATCC#12051; American Type Culture Collection, Manassas, VA) by electroporation as described (Example 2). Colonies harboring the recombinant plasmid are grown on Stauton's synthetic medium (Example 2) containing lactose in place of

61

112275/F/l glycerol at 37⁰C. Colonies that grow on this novel selection medium are screened by PCR using forward primer: 5'- GGCGTGTTGTGGGACACTCCCTCA-3' (SEQ ID NO: 16) and reverse primer 5'-GATCTGTTTTTTCCTCAGCATCTC-S ' (SEQ ID NO: 17) (Example 1) to demonstrate the presence of plasmid pBACIL-109 [ΔKan^R], DNA sequencing (See recombinant DNA methods above) to demonstrate that the plasmid carries the correct sequence and 2D gel electrophoresis on a fee-for-service basis at ACE BioSciences (Odense, Denmark) to demonstrate that FAP and Mb 1389C_1-36O are expressed in the resulting rM. smegmatis strains Bacilligen-1023.

Example 8

Safety, toxicity and potency of mycobacterial strains

[00166] The safety, toxicity and potency of recombinant mycobacterial strains are evaluated according to the guidelines in 21 CFR 610, which include: (i) general safety test; (ii) stringent safety test in immunocompetent mice; (iii) guinea pig safety test; and (iv) acute and chronic toxicity tests, as described below. (i) General safety test

[00167] Each strain is grown in 100 ml liquid cultures as described (Example

1); the bacteria are harvested by centrifugation (5000 x g for 10 min) and washed in 100 ml PBS. Inoculation suspensions are prepared by resuspending the washed bacteria in normal saline (0.85% w/v NaCl) containing 10% glycerol and 0.05% (v/v) tyloxapol to a density of 5 x 10⁷ cfu per ml. Groups of eight BALB/c mice are inoculated intraperitoneally with 100 μl of inoculation suspensions containing 5 x 10⁶ cfu of the recombinant mycobacterial strain of interest and the analogous parental strain, as shown in the table below.

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112275/F/l

[00168] Thee animals are monitored for general health and body weight for 14 days post infection. Similar to animals that receive BCG, animals that receive the recombinant mycobacterial strains remain healthy, and do not lose weight or display overt signs of disease during the observation period.

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112275/F/l (H) Stringent safety test in immunocompetent mice

[00169] Each strain is grown in 100 ml liquid cultures as described (Example

1); the bacteria are harvested by centrifugation (5000 x g for 10 min) and washed in 100 ml PBS. Inoculation suspensions are prepared by resuspending the washed bacteria in normal saline (0.85% w/v NaCl) containing 10% glycerol and 0.05% (v/v) tyloxapol to a density of 2 x 10⁷ cfu per ml. Groups of 15 healthy BALB/c mice are infected intravenously with 100 μl of the inoculation suspensions containing 2xlO⁶ viable recombinant mycobacteria and parental strains as shown in the table below.

64

112275/F/l [00170] One day after inoculation, 3 mice in each group are sacrificed and the cfu numbers in the spleen, lung and liver homogenates are analyzed to ensure each animal receives an equivalent infection dose. At week 4, 8, 12, and 16 post infection, 3 mice in each group are sacrificed and cfu numbers in spleen, live and lung homogenates are obtained to assess the in vivo growth of the recombinant mycobacterial strains as compared to the parental strains. Recombinant mycobacterial strains are expected to display similar or less growth to that of the parental strains, (iii) Guinea pig safety test

[00171] The safety of recombinant mycobacterial strains is also assessed in the guinea pig model in comparison to the licensed parenterally administered BCG vaccine, which has a well-established safety profile in humans. First, the effect of the recombinant strains on the general health status of the animals is examined, including weight gain.

[00172] Each strain is grown in 1.0 L liquid cultures as described (Example 1); the bacteria are harvested by centrifugation (5000 x g for 10 min) and washed in 1 L PBS. Inoculation suspensions are prepared by resuspending the washed bacteria in normal saline (0.85% w/v NaCl) containing 10% glycerol and 0.05% (v/v) tyloxapol to a density of 5 x 10⁹ cfu per ml. Groups of 8 guinea pigs are inoculated intramuscularly with 100 μl of the inoculation suspensions containing 5 x 10⁸ cfu (i.e. 100 x of human dose) of the recombinant mycobacterial and parental strain as shown in the table below.

65

112275/F/l

[00173] The general health and body weight of the animals are monitored for six weeks post inoculation. If any animals are sacrificed before the six-week period concludes due to serious adverse affects, each sacrificed animal will be subjected to a detailed postmortem examination. All animals are sacrificed at the end of six weeks post-inoculation and gross pathology is performed. The recombinant mycobacterial strains are deemed safe if no adverse health effects are observed and the animals gain weight at the normal rate compared to animals inoculated with BCG as an internal control.

[00174] At the same time, bacterial levels in animal organs are monitored.

Guinea pigs immunized with either the parental or recombinant strains are euthanized at various intervals after inoculation, after which cfu counts of the recombinant mycobacterial strains and parental strains are determined in lung, spleen, and regional (inguinal) lymph node homogenates.

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112275/F/l (iv) Acute and chronic toxicity test

[00175] Each strain is grown in 1.0 L liquid cultures as described (Example 1); the bacteria are harvested by centrifugation (5000 x g for 10 min) and washed in 1 L PBS. Inoculation suspensions are prepared by resuspending the washed bacteria in normal saline (0.85% w/v NaCl) containing 10% glycerol and 0.05% (v/v) tyloxapol to a density of 5 x 10⁷ (standard dose), 2 x 10⁸ (high dose) and 1.25 x 10⁷ (low dose) cfu per ml. To evaluate the acute and chronic toxicity of the recombinant mycobacterial strains, groups of 16 guinea pigs are inoculated intradermally with 100 μl of the inoculation suspensions containing 5 x 10⁶ cfu (i.e. 1 x human dose), 2 x 10⁷ cfu (i.e. 4 x human dose) and 1.25 x 10⁶ cfu (0.25 x human dose) of recombinant mycobacterial and parental strains or saline respectively as shown in the table below.

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112275/F/l

[00176] Three days post-inoculation, 8 animals in each group are sacrificed to access the acute effects of the recombinant strains on these animals. At 28-days post- inoculation, the remaining 8 animals in each group are sacrificed to evaluate any chronic effects on the animals. At both time points, the body weight of each animal is obtained. In addition, the gross pathology and appearance of the injection sites are examined. Blood is taken for blood chemistry, and the histopathology of the internal organs and injection sites are performed at each time point.

Example 9

Comparison of the anti-cancer potency of genetically modified mycobacteria

[0100] Currently, three are fundamental murine bladder tumor models: chemically induced bladder cancer (1, 2), the xenograft model (transplantation of human transitional cell

carcinoma into immunodeficient mice (3-5), and the syngeneic tumor model (transplantation of carcinogen-induced bladder cancer in syngeneic, immunocompetent mice (6-9). For the evaluation of immunotherapeutic approaches, the syngeneic murine bladder tumor model seems to be the most appropriate model because of the chance to study the local tumor in an immunocompetent host. Syngeneic tumor cells can be implanted either subcutaneously (heterotopic tumor) or intravesically (orthotopic).

[0101] To prepare the inocula, each strain is grown in 1.0 L liquid cultures as described (Example 1); the bacteria are harvested by centrifugation (5000 x g for 10 min) and washed in 1 L PBS. Inoculation suspensions are prepared by resuspending the washed bacteria in normal saline (0.85% w/v NaCl) containing 10% glycerol and 0.05% (v/v) tyloxapol to a density of 1 x 10⁷ cfu per ml.

68

112275/F/l [0102] Female C57B1/6 mice, 6-8 weeks old, each weighing 17 g, are purchased from Charles River (Maine) and maintained at an animal facility for 1 week prior to use. The mice are housed five per cage, in a limited access area at a room temperature of 20+1⁰C and a humidity of 50+10%, with food and water ad libitum.

[0103] Tumor cells used in this study are derived from the 7,12- dimethylbenzanthracene-induced murine bladder cancer MB49 (12). The cells are maintained in in vitro culture (DMEM, 10% FCS, and 1% w/v penicillin/streptomycin at 37°C and 5% CO₂). Tumor cells are harvested by trypsinization and suspended in DMEM without L- glutamine, FCS, and antibiotics. Viability is determined by trypan blue exclusion, and only tumor cell suspensions with 90% viable cells are used for tumor implantation. The concentrations of the tumor cell suspensions used for implantation are adjusted to 2 x 10⁶ cells/ml. Intravesical tumor implantation is performed as described by Soloway and Masters (8, 13) and Shapiro et al. (14) for the MBT-2 model and by Hudson et al. (7) for the MB49 model. Briefly, after a short ether inhalation anesthesia, the mice received an i.p. injection of diluted sodium pentobarbital (6 mg/ml) for general anesthesia of a single dose of 0.06 mg/g body weight. After shaving areas of 1 cm² on the backs of the mice, a 24-gauge Teflon i.v. catheter (Insyte-W; Becton Dickinson, Heidelberg, Germany) is inserted transurethrally into the bladder using a lubricant (Instilla Gel; Farco-Pharma, KoIn, Germany). Mice were placed with their backs on the ground plate of the cautery unit. To optimize contact, electrocardiogram electrode contact gel is used. The soft-tipped end of a spring-wire guide of a 24-gauge central venous catheter (Arrow, Erding, Germany) is inserted into the bladder via the Teflon catheter and gently pushed forward until it reaches the bladder wall. The guide wire is attached to the cautery unit (Elektrotom 500; Gebruder Martin, Tuttlingen, Germany), and a monopolar coagulation is applied for 5 s at the lowest setting (5 W). After removal of the guide wire, 0.05 ml of the tumor cell suspension is instilled. Unlike the conventional

69

112275/F/l procedure, in which catheters are removed after instillation, the catheters are pinched off with a clamp, kept locked with a Luer-Lock closing cone, and left in place until the mice awaken. Using this method, a dwell time of 3 h is ensured. DMEM was used as solvent for instilled tumor cells as a means for improving viability.

[0104] The animals are randomized into groups with 15 animals each to receive PBS or BCG/rBCG/rM smegmatis therapy as shown in the table below. Intravesical instillations of PBS, BCG, rBCG and rM. smegmatis are performed on days 1, 8, 15, and 22 after tumor implantation by the technique described above in a volume of 0.05 ml containing 5 x 10⁶ cfu.

70

112275/F/l

Example 10

Use of recombinant mycobacteria as an adjuvant

[0105] As mentioned above, recombinant mycobacteria are also useful as adjuvants. In this example, rBCG strain Bacilligen-1021, which expresses a FAP^C, NS3p_r ⁺ and RDl⁺ phenotype, is used to increase the immune response to influenza hemagglutinin. Accordingly, Bacilligen-1021 is grown as described in Example 1 in 100 ml 7H9 media supplemented with oleic acid (0.06% v/v; Research Diagnostics Cat. No. 01257) and Tyloxapol (0.05% v/v; Research Diagnostics Cat. No.70400) at 37⁰C to an OD₆₀₀ = 1.0. The bacteria are harvested by centrifugation and washed 3 times in 10 ml PBS containing 0.05% tyloxapol. The Bacilligen-1021 bacilli are resuspended in PBS containing 0.05% tyloxapol, 10% glycerol (Sigma, St Louis MO) at a density of 5 x 10⁶ CFU/ml and stored at -8O⁰C.

[0106] To utilize the preparation of Bacilligen-1021 as an adjuvant, 1.0 mg of purified endotoxin-free recombinant hemagglutinin purified from influenza A virus HlNl New Caledonia 20/99 (Prospec-Tany Technogen Ltd., Rehovot, Israel) in 1 ml PBS is admixed with 5 x 10⁶ Bacilligen-1021 cfu in 1 ml PBS containing 0.05% tyloxapol. To test the immunogenicity of this formulation, groups of 10 BALB/c mice (Jackson Laboratories, Bar Harbor, ME) are vaccinated as shown in the table below.

71

112275/F/l

[0107] The mice are given a total of 3 doses of vaccine at 0, 14 and 60 days and the immune response to hemagglutinin is measured by ELISA using sera collected from the tail vein of individual mice at 10 day intervals, as described (130). The neutralization of influenza virus is measured in the collected 80 days after the first vaccination, as described (131). The results of this study will show that Bacilligen-1021 has the capacity to substantially increase the magnitude and potency of the humoral response to hemagglutinin and therefore possesses useful adjuvant properties.

[0108] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be embraced by the appended claims. Recombinant DNA sequences

SEQ ID NO: 1 - GGDEF domain spanning amino acids 1-360 in Mbl389c ATGTGCAACGACACCGCGACGCCGCAGCTTGAGGAGCTCGTCACCACCGTAGCC AACCAGCTCATGACAGTCGACGCTGCCACGTCAGCCGAAGTCAGTCAGCGCGTTT TGGCCTATCTAGTGGAACAGCTGGGCGTAGATGTCAGCTTTTTGCGTCATAACGA TCGCGACAGGCGCGCGACGAGGCTGGTGGCCGAATGGCCACCTCGCCTCAACAT ACCGGACCCCGATCCGCTCAGGCTGATCTACTTCGCTGATGCCGACCCGGTGTTT GCGCTATGCGAACACGCCAAAGAGCCTCTCGTGTTCCGGCCCGAGCCGGCCACC GAGGACTATCAACGCCTCATCGAAGAAGCCCGCGGGGTTCCGGTAACGTCGGCT GCCGCCGTGCCGCTGGTATCTGGCGAGATCACCACTGGACTGCTGGGGTTCATCA AGTTCGGTGATCGGAAATGGCACGAGGCCGAGCTTAACGCCCTCATGACCATCG

72

112275/F/l VOVVDOVVOOOVOIVOOVOIVOVOIIODOIVO

OIVDOIVDVVVDDDODOIVOOVDIIVVIDDVOIDIOVDOIDDODOVDDDOOODD OIDVDOVODVDVIDVOIIOOIOIIOVVODDDODVOOIOOODOIVOIIDODOIOO DOODIIIOOOVVIVOODDODDVODVVOIODDOOIIVVODDDIOODIODODIODD OOVDIIDDVDDOIVVVOODOIDVDVOVDDDOIVDVDVODIOODOVIDVDOODDO VOIIVVDVDIVOVVDOVDVOIDVIVVDOOOOODODOVOIVVDOOOOVOIOVOO ODODDVDDODDDOODDDOOODVODDDIIOIVODIDOOOIOIOODDIOOIIVVOIV IDODODDIIODODDVODIVDODDVIIODDDVDIIOVODVIDDIIVDOIIOIDOVV

DDDDlDDOODDDDODDDlDHyDODOOYDODDyyDODDyDO

ODOODIOVDODOODIOOIDDOODDIDIOOIDVDOVDODOODIOVDODOODIODI

OOIDDODDODDDIOIVDOODDVDIOVDODOODODDIODODIVODIOOIDOVDOIV

VOIDVDVIDVVOIIVIVODVVDODOIDVVODOODOVDIOODIOIVD VOODODDVDIIIIVODODIODOVIVOVOODOODDODVDVVVDDOVDOOIDIDOD DOVVDDVODDOVODDODOIDDIDDVOODIODDVDVDVDOVDVOODDDIDVOOOV DIDDODIOIOODIVIOVDIODDVDODDDVDIDOIOOVOIOODOODIVIDODIODV DDVOOVVDIDOVDDVODODIIDIODVVODDODIDODDVVDIDODDOIVODDOIOV OIVVDDIDIVDODDDVIVDIDDIODIIVVODVODOODOODIDVODDDODIVOIDD OVOVODOOIIODIDVDODVOIOODIVOODVVDDDODIIOIOIVDDIVDIIOVDDV OIOODDODDODVDDOOOIDDVIDVODVVDIVDDOO VVDIDDODDVOOIIDVODI DIIIVIDODOODODIOODDDOOIDDVVDVOODDDDDOOIDVOVVVDDVOOIDDVD OVDOIDOIIODODODIODDVVIVDOIDVOODDVOIDIVODVOIVDDVODDOOIDI VIDODIIDOODODOOVODDODDODIODODIDOOVDOIOOVDDDODIIDIDVDVID

£CC980//.00ZSfl/13d 86S017Ϊ/800Z OΛV SEQ ID NO:4 - Sequence encoding complement to RDl deletion in BCG (8016 bp)

CGGCTGGGTGGAGATCGTTGCCAGCCAACGCCACCCCGGCGGCACCACGACGCA

GACCGACGCCGCCGCTGGCGTCCTGGACTCCAAGCTCGGTAGGCTGGTGTCGCTT

CCCCGCCGTGTTGGAGGCGACCTGTACGGAAGCTTCCTGCCCGGCACTCAGCAGA

ACTTGGAGCGTGCGCTGGACGGCTTGCTAGAGCTGCTCCCTGCGGGCGCTTGGCT

AGATCACACCTCAGATCACGCACAAGCCTCCTCCCGAGGCTGACCCCTCACATCT

CCGCTACGACTTCAGAAAGGGACGCCATGACTGCTGAACCGGAAGTACGGACGC

TGCGCGAGGTTGTGCTGGACCAGCTCGGCACTGCTGAATCGCGTGCGTACAAGAT

GTGGCTGCCGCCGTTGACCAATCCGGTCCCGCTCAACGAGCTCATCGCCCGTGAT

CGGCGACAACCCCTGCGATTTGCCCTGGGGATCATGGATGAACCGCGCCGCCATC

TACAGGATGTGTGGGGCGTAGACGTTTCCGGGGCCGGCGGCAACATCGGTATTG

GGGGCGCACCTCAAACCGGGAAGTCGACGCTACTGCAGACGATGGTGATGTCGG

CCGCCGCCACACACTCACCGCGCAACGTTCAGTTCTATTGCATCGACCTAGGTGG

CGGCGGGCTGATCTATCTCGAAAACCTTCCACACGTCGGTGGGGTAGCCAATCGG

TCCGAGCCCGACAAGGTCAACCGGGTGGTCGCAGAGATGCAAGCCGTCATGCGG

CAACGGGAAACCACCTTCAAGGAACACCGAGTGGGCTCGATCGGGATGTACCGG

CAGCTGCGTGACGATCCAAGTCAACCCGTTGCGTCCGATCCATACGGCGACGTCT

TTCTGATCATCGACGGATGGCCCGGTTTTGTCGGCGAGTTCCCCGACCTTGAGGG

GCAGGTTCAAGATCTGGCCGCCCAGGGGCTGGCGTTCGGCGTCCACGTCATCATC

TCCACGCCACGCTGGACAGAGCTGAAGTCGCGTGTTCGCGACTACCTCGGCACCA

AGATCGAGTTCCGGCTTGGTGACGTCAATGAAACCCAGATCGACCGGATTACCC

GCGAGATCCCGGCGAATCGTCCGGGTCGGGCAGTGTCGATGGAAAAGCACCATC

TGATGATCGGCGTGCCCAGGTTCGACGGCGTGCACAGCGCCGATAACCTGGTGG

AGGCGATCACCGCGGGGGTGACGCAGATCGCTTCCCAGCACACCGAACAGGCAC

CTCCGGTGCGGGTCCTGCCGGAGCGTATCCACCTGCACGAACTCGACCCGAACCC

74

112275/F/l GCCGGGACCAGAGTCCGACTACCGCACTCGCTGGGAGATTCCGATCGGCTTGCG

CGAGACGGACCTGACGCCGGCTCACTGCCACATGCACACGAACCCGCACCTACT

GATCTTCGGTGCGGCCAAATCGGGCAAGACGACCATTGCCCACGCGATCGCGCG

CGCCATTTGTGCCCGAAACAGTCCCCAGCAGGTGCGGTTCATGCTCGCGGACTAC

CGCTCGGGCCTGCTGGACGCGGTGCCGGACACCCATCTGCTGGGCGCCGGCGCG

ATCAACCGCAACAGCGCGTCGCTAGACGAGGCCGTTCAAGCACTGGCGGTCAAC

CTGAAGAAGCGGTTGCCGCCGACCGACCTGACGACGGCGCAGCTACGCTCGCGT

TCGTGGTGGAGCGGATTTGACGTCGTGCTTCTGGTCGACGATTGGCACATGATCG

TGGGTGCCGCCGGGGGGATGCCGCCGATGGCACCGCTGGCCCCGTTATTGCCGG

CGGCGGCAGATATCGGGTTGCACATCATTGTCACCTGTCAGATGAGCCAGGCTTA

CAAGGCAACCATGGACAAGTTCGTCGGCGCCGCATTCGGGTCGGGCGCTCCGAC

AATGTTCCTTTCGGGCGAGAAGCAGGAATTCCCATCCAGTGAGTTCAAGGTCAAG

CGGCGCCCCCCTGGCCAGGCATTTCTCGTCTCGCCAGACGGCAAAGAGGTCATCC

AGGCCCCCTACATCGAGCCTCCAGAAGAAGTGTTCGCAGCACCCCCAAGCGCCG

GTTAAGATTATTTCATTGCCGGTGTAGCAGGACCCGAGCTCAGCCCGGTAATCGA

GTTCGGGCAATGCTGACCATCGGGTTTGTTTCCGGCTATAACCGAACGGTTTGTG

TACGGGATACAAATACAGGGAGGGAAGAAGTAGGCAAATGGAAAAAATGTCAC

ATGATCCGATCGCTGCCGACATTGGCACGCAAGTGAGCGACAACGCTCTGCACG

GCGTGACGGCCGGCTCGACGGCGCTGACGTCGGTGACCGGGCTGGTTCCCGCGG

GGGCCGATGAGGTCTCCGCCCAAGCGGCGACGGCGTTCACATCGGAGGGCATCC

AATTGCTGGCTTCCAATGCATCGGCCCAAGACCAGCTCCACCGTGCGGGCGAAG

CGGTCCAGGACGTCGCCCGCACCTATTCGCAAATCGACGACGGCGCCGCCGGCG

TCTTCGCCTAATAGGCCCCCAACACATCGGAGGGAGTGATCACCATGCTGTGGCA

CGCAATGCCACCGGAGCTAAATACCGCACGGCTGATGGCCGGCGCGGGTCCGGC

TCCAATGCTTGCGGCGGCCGCGGGATGGCAGACGCTTTCGGCGGCTCTGGACGCT

75

112275/F/l CAGGCCGTCGAGTTGACCGCGCGCCTGAACTCTCTGGGAGAAGCCTGGACTGGA

GGTGGCAGCGACAAGGCGCTTGCGGCTGCAACGCCGATGGTGGTCTGGCTACAA

ACCGCGTCAACACAGGCCAAGACCCGTGCGATGCAGGCGACGGCGCAAGCCGCG

GCATACACCCAGGCCATGGCCACGACGCCGTCGCTGCCGGAGATCGCCGCCAAC

CACATCACCCAGGCCGTCCTTACGGCCACCAACTTCTTCGGTATCAACACGATCC

CGATCGCGTTGACCGAGATGGATTATTTCATCCGTATGTGGAACCAGGCAGCCCT

GGCAATGGAGGTCTACCAGGCCGAGACCGCGGTTAACACGCTTTTCGAGAAGCT

CGAGCCGATGGCGTCGATCCTTGATCCCGGCGCGAGCCAGAGCACGACGAACCC

GATCTTCGGAATGCCCTCCCCTGGCAGCTCAACACCGGTTGGCCAGTTGCCGCCG

GCGGCTACCCAGACCCTCGGCCAACTGGGTGAGATGAGCGGCCCGATGCAGCAG

CTGACCCAGCCGCTGCAGCAGGTGACGTCGTTGTTCAGCCAGGTGGGCGGCACC

GGCGGCGGCAACCCAGCCGACGAGGAAGCCGCGCAGATGGGCCTGCTCGGCACC

AGTCCGCTGTCGAACCATCCGCTGGCTGGTGGATCAGGCCCCAGCGCGGGCGCG

GGCCTGCTGCGCGCGGAGTCGCTACCTGGCGCAGGTGGGTCGTTGACCCGCACG

CCGCTGATGTCTCAGCTGATCGAAAAGCCGGTTGCCCCCTCGGTGATGCCGGCGG

CTGCTGCCGGATCGTCGGCGACGGGTGGCGCCGCTCCGGTGGGTGCGGGAGCGA

TGGGCCAGGGTGCGCAATCCGGCGGCTCCACCAGGCCGGGTCTGGTCGCGCCGG

CACCGCTCGCGCAGGAGCGTGAAGAAGACGACGAGGACGACTGGGACGAAGAG

GACGACTGGTGAGCTCCCGTAATGACAACAGACTTCCCGGCCACCCGGGCCGGA

AGACTTGCCAACATTTTGGCGAGGAAGGTAAAGAGAGAAAGTAGTCCAGCATGG

CAGAGATGAAGACCGATGCCGCTACCCTCGCGCAGGAGGCAGGTAATTTCGAGC

GGATCTCCGGCGACCTGAAAACCCAGATCGACCAGGTGGAGTCGACGGCAGGTT

CGTTGCAGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCAGGCCGCGGTGG

TGCGCTTCCAAGAAGCAGCCAATAAGCAGAAGCAGGAACTCGACGAGATCTCGA

CGAATATTCGTCAGGCCGGCGTCCAATACTCGAGGGCCGACGAGGAGCAGCAGC

76

112275/F/l AGGCGCTGTCCTCGCAAATGGGCTTCTGACCCGCTAATACGAAAAGAAACGGAG

CAAAAACATGACAGAGCAGCAGTGGAATTTCGCGGGTATCGAGGCCGCGGCAAG

CGCAATCCAGGGAAATGTCACGTCCATTCATTCCCTCCTTGACGAGGGGAAGCAG

TCCCTGACCAAGCTCGCAGCGGCCTGGGGCGGTAGCGGTTCGGAGGCGTACCAG

GGTGTCCAGCAAAAATGGGACGCCACGGCTACCGAGCTGAACAACGCGCTGCAG

AACCTGGCGCGGACGATCAGCGAAGCCGGTCAGGCAATGGCTTCGACCGAAGGC

AACGTCACTGGGATGTTCGCATAGGGCAACGCCGAGTTCGCGTAGAATAGCGAA

ACACGGGATCGGGCGAGTTCGACCTTCCGTCGGTCTCGCCCTTTCTCGTGTTTATA

CGTTTGAGCGCACTCTGAGAGGTTGTCATGGCGGCCGACTACGACAAGCTCTTCC

GGCCGCACGAAGGTATGGAAGCTCCGGACGATATGGCAGCGCAGCCGTTCTTCG

ACCCCAGTGCTTCGTTTCCGCCGGCGCCCGCATCGGCAAACCTACCGAAGCCCAA

CGGCCAGACTCCGCCCCCGACGTCCGACGACCTGTCGGAGCGGTTCGTGTCGGCC

CCGCCGCCGCCACCCCCACCCCCACCTCCGCCTCCGCCAACTCCGATGCCGATCG

CCGCAGGAGAGCCGCCCTCGCCGGAACCGGCCGCATCTAAACCACCCACACCCC

CCATGCCCATCGCCGGACCCGAACCGGCCCCACCCAAACCACCCACACCCCCCAT

GCCCATCGCCGGACCCGAACCGGCCCCACCCAAACCACCCACACCTCCGATGCC

CATCGCCGGACCTGCACCCACCCCAACCGAATCCCAGTTGGCGCCCCCCAGACCA

CCGACACCACAAACGCCAACCGGAGCGCCGCAGCAACCGGAATCACCGGCGCCC

CACGTACCCTCGCACGGGCCACATCAACCCCGGCGCACCGCACCAGCACCGCCC

TGGGCAAAGATGCCAATCGGCGAACCCCCGCCCGCTCCGTCCAGACCGTCTGCGT

CCCCGGCCGAACCACCGACCCGGCCTGCCCCCCAACACTCCCGACGTGCGCGCC

GGGGTCACCGCTATCGCACAGACACCGAACGAAACGTCGGGAAGGTAGCAACTG

GTCCATCCATCCAGGCGCGGCTGCGGGCAGAGGAAGCATCCGGCGCGCAGCTCG

CCCCCGGAACGGAGCCCTCGCCAGCGCCGTTGGGCCAACCGAGATCGTATCTGG

CTCCGCCCACCCGCCCCGCGCCGACAGAACCTCCCCCCAGCCCCTCGCCGCAGCG

77

112275/F/l CAACTCCGGTCGGCGTGCCGAGCGACGCGTCCACCCCGATTTAGCCGCCCAACAT

GCCGCGGCGCAACCTGATTCAATTACGGCCGCAACCACTGGCGGTCGTCGCCGC

AAGCGTGCAGCGCCGGATCTCGACGCGACACAGAAATCCTTAAGGCCGGCGGCC

AAGGGGCCGAAGGTGAAGAAGGTGAAGCCCCAGAAACCGAAGGCCACGAAGCC

GCCCAAAGTGGTGTCGCAGCGCGGCTGGCGACATTGGGTGCATGCGTTGACGCG

AATCAACCTGGGCCTGTCACCCGACGAGAAGTACGAGCTGGACCTGCACGCTCG

AGTCCGCCGCAATCCCCGCGGGTCGTATCAGATCGCCGTCGTCGGTCTCAAAGGT

GGGGCTGGCAAAACCACGCTGACAGCAGCGTTGGGGTCGACGTTGGCTCAGGTG

CGGGCCGACCGGATCCTGGCTCTAGACGCGGATCCAGGCGCCGGAAACCTCGCC

GATCGGGTAGGGCGACAATCGGGCGCGACCATCGCTGATGTGCTTGCAGAAAAA

GAGCTGTCGCACTACAACGACATCCGCGCACACACTAGCGTCAATGCGGTCAAT

CTGGAAGTGCTGCCGGCACCGGAATACAGCTCGGCGCAGCGCGCGCTCAGCGAC

GCCGACTGGCATTTCATCGCCGATCCTGCGTCGAGGTTTTACAACCTCGTCTTGG

CTGATTGTGGGGCCGGCTTCTTCGACCCGCTGACCCGCGGCGTGCTGTCCACGGT

GTCCGGTGTCGTGGTCGTGGCAAGTGTCTCAATCGACGGCGCACAACAGGCGTC

GGTCGCGTTGGACTGGTTGCGCAACAACGGTTACCAAGATTTGGCGAGCCGCGC

ATGCGTGGTCATCAATCACATCATGCCGGGAGAACCCAATGTCGCAGTTAAAGA

CCTGGTGCGGCATTTCGAACAGCAAGTTCAACCCGGCCGGGTCGTGGTCATGCCG

TGGGACAGGCACATTGCGGCCGGAACCGAGATTTCACTCGACTTGCTCGACCCTA

TCTACAAGCGCAAGGTCCTCGAATTGGCCGCAGCGCTATCCGACGATTTCGAGAG

GGCTGGACGTCGTTGAGCGCACCTGCTGTTGCTGCTGGTCCTACCGCCGCGGGGG

CAACCGCTGCGCGGCCTGCCACCACCCGGGTGACGATCCTGACCGGCAGACGGA

TGACCGATTTGGTACTGCCAGCGGCGGTGCCGATGGAAACTTATATTGACGACAC

CGTCGCGGTGCTTTCCGAGGTGTTGGAAGACACGCCGGCTGATGTACTCGGCGGC

TTCGACTTTACCGCGCAAGGCGTGTGGGCGTTCGCTCGTCCCGGATCGCCGCCGC

78

112275/F/l TGAAGCTCGACCAGTCACTCGATGACGCCGGGGTGGTCGACGGGTCACTGCTGA

CTCTGGTGTCAGTCAGTCGCACCGAGCGCTACCGACCGTTGGTCGAGGATGTCAT

CGACGCGATCGCCGTGCTTGACGAGTCACCTGAGTTCGACCGCACGGCATTGAAT

CGCTTTGTGGGGGCGGCGATCCCGCTTTTGACCGCGCCCGTCATCGGGATGGCGA

TGCGGGCGTGGTGGGAAACTGGGCGTAGCTTGTGGTGGCCGTTGGCGATTGGCA

TCCTGGGGATCGCTGTGCTGGTAGGCAGCTTCGTCGCGAACAGGTTCTACCAGAG

CGGCCACCTGGCCGAGTGCCTACTGGTCACGACGTATCTGCTGATCGCAACCGCC

GCAGCGCTGGCCGTGCCGTTGCCGCGCGGGGTCAACTCGTTGGGGGCGCCACAA

GTTGCCGGCGCCGCTACGGCCGTGCTGTTTTTGACCTTGATGACGCGGGGCGGCC

CTCGGAAGCGTCATGAGTTGGCGTCGTTTGCCGTGATCACCGCTATCGCGGTCAT

CGCGGCCGCCGCTGCCTTCGGCTATGGATACCAGGACTGGGTCCCCGCGGGGGG

GATCGCATTCGGGCTGTTCATTGTGACGAATGCGGCCAAGCTGACCGTCGCGGTC

GCGCGGATCGCGCTGCCGCCGATTCCGGTACCCGGCGAAACCGTGGACAACGAG

GAGTTGCTCGATCCCGTCGCGACCCCGGAGGCTACCAGCGAAGAAACCCCGACC

TGGCAGGCCATCATCGCGTCGGTGCCCGCGTCCGCGGTCCGGCTCACCGAGCGCA

GCAAACTGGCCAAGCAACTTCTCATCGGATACGTCACGTCGGGCACCCTGATTCT

GGCTGCCGGTGCCATCGCGGTCGTGGTGCGCGGGCACTTCTTTGTACACAGCCTG

GTGGTCGCGGGTTTGATCACGACCGTCTGCGGATTTCGCTCGCGGCTTTACGCCG

AGCGCTGGTGTGCGTGGGCGTTGCTGGCGGCGACGGTCGCGATTCCGACGGGTCT

GACGGCCAAACTCATCATCTGGTACCCGCACTATGCCTGGCTGTTGTTGAGCGTC

TACCTCACGGTAGCCCTGGTTGCGCTCGTGGTGGTCGGGTCGATGGCTCACGTCC

GGCGCGTTTCACCGGTCGTAAAACGAACTCTGGAATTGATCGACGGCGCCATGAT

CGCTGCCATCATTCCCATGCTGCTGTGGATCACCGGGGTGTACGACACGGTCCGC

AATATCCGGTTCTGA

79

112275/F/l 08

J₃S-A₁O-A₁O-A₁O-A₁O-J₃₈

VVIVOIDDVDIVVVDOVV

OVVOODDIDOIVVVOODDVVODIIDOOVDOODDDIVODDVVOIVOOIVOODVVO

DOOVVDOIODIVVDODDIDIVDVIDDIDOODVVODDOIVDIVOIODOODVVDOO

DVIDIDDOOIIVOIOIVODOODVVOVVIVOOIOIIVODDDDIDOODDIDDVDOOD

DVODDDIIIVODIDDDVIIOVDODOODIVVVODOOVVOODDDDVOVVDIIOIODO

OODDO VVDDVVVDIIODVVOVVIIODVVOVVDOOODDVVOIIOIIODIVOIVVV

DOIOVVOIVOVVDDOODVVOOIOVVDVDVVDDIDOVVOOIODDDOODOODVIDO

IDIDOODIVOVVOOVVIIODDIDOOOOIDVIDDDIVODIDOODDOOVVODOODDI

OIVDIDVDOVDODOOO VVODVODVDVDOOIDIDODVDVDDIIIIODOO VVOOIO

OIVIIODOOVDODOOVDO VVDDVIDDIDOODIDDIDDOOOODDDVOIVDIVOOD

DVIOIODOODDVDDVDDVIVODOOOVVOVVDVIVVOOVVDDDDDIDDDDDVIVO

OOIDIDOIODOODOODDDDIDDOODDDDODDDIDIIVDODOOVDODDVVDODDVD

OODOODIOVDODOODIODIDDOODDIDIODIDVDOVDODOODIOVDODOODIODI

ODIDDODDODDDIOIVDOODDVOIOVDODOODODOIODODIVODIOOIDOVDOIV uisiojd ^eSM pszxuiμdo uopo3 - SΌM αi 03S

« ε-DOIOVDOIOI VVDIDO VVIDOID- , ζ

IIOΛOIUI -JAl

Ui uoμapp aφ luauiajduioo ^~\vψ aouanbas ajmauas oj jauiud 3SJ₃Λ₃J ICTH ~ L OM QI 03S

. ε-ODDVOODOIIVI VOODDVVO VDI- , S

J3UIIJ^Q 3SJ3Λ3J ₊χQ^ " 9 OM CII OSS

«ε-DDOIIODIVOVOOIOOOIDOOD-,S

£CC980//.00ZSfl/13d 86S0H/800Z OΛV SEQ ID NO: 10 - Linker

Ser-Pro-Pro-Pro-Pro-Pro-Pro-Ser

SEQ ID NO: 11 - Linker

Ser-Gly-Gly-Gly-Gly-Arg-Thr-Lys-Arg-Gly-Gly-Gly-Gly-Ser

SEQ ID NO: 12 -M. bovis fibronectin attachment protein (lefapB; 978 bp)

ATGCATCAGGTGGACCCCAACTTGACACGTCGCAAGGGACGATTGGCGGCACTG

GCTATCGCGGCGATGGCCAGCGCCAGCCTGGTGACCGTTGCGGTGCCCGCGACC

GCCAACGCCGATCCGGAGCCAGCGCCCCCGGTACCCACAACGGCCGCCTCGCCG

CCGTCGACCGCTGCAGCGCCACCCGCACCGGCGACACCTGTTGCCCCCCCACCAC

CGGCCGCCGCCAACACGCCGAATGCCCAGCCGGGCGATCCCAACGCAGCACCTC

CGCCGGCCGACCCGAACGCACCGCCGCCACCTGTCATTGCCCCAAACGCACCCC

AACCTGTCCGGATCGACAACCCGGTTGGAGGATTCAGCTTCGCGCTGCCTGCTGG

CTGGGTGGAGTCTGACGCCGCCCACCTCGACTACGGTTCAGCACTCCTCAGCAAA

ACCACCGGGGACCCGCCATTTCCCGGACAGCCGCCGCCGGTGGCCAATGACACC

CGTATCGTGCTCGGCCGGCTAGACCAAAAGCTTTACGCCAGCGCCGAAGCCACC

GACTCCAAGGCCGCGGCCCGGTTGGGCTCGGACATGGGTGAGTTCTATATGCCCT

ACCCGGGCACCCGGATCAACCAGGAAACCGTCTCGCTCGACGCCAACGGGGTGT

CTGGAAGCGCGTCGTATTACGAAGTCAAGTTCAGCGATCCGAGTAAGCCGAACG

GCCAGATCTGGACGGGCGTAATCGGCTCGCCCGCGGCGAACGCACCGGACGCCG

GGCCCCCTCAGCGCTGGTTTGTGGTATGGCTCGGGACCGCCAACAACCCGGTGGA

CAAGGGCGCGGCCAAGGCGCTGGCCGAATCGATCCGGCCTTTGGTCGCCCCGCC

GCCGGCGCCGGCACCGGCTCCTGCAGAGCCCGCTCCGGCGCCGGCGCCGGCCGG

GGAAGTCGCTCCTACCCCGACGACACCGACACCGCAGCGGACCTTACCGGCCTG

A

81

112275/F/l 313VVODVDOIDDVDDIVIODOVOODDOIDDIOOODOIOODDIDDVDOOVDVVO

DDVDVDOVDDDIIDODIVOVDODVOIOOOOODODDVDIVODOOVOOIOOIDDVV

IVODDODOVDVDOIODOODVODIIOOVDDDOIODOODIVOIVOIDIVDDVDOVV

VVOOIVODIOIOVDOOODIOOODDIODIVVODOODDDIVOVODODDDVIIVOOD

DVODIVOVDDDVVVOIVVDIODVOIOOIIDOODDIIOVODIVOVVDDVDOODID

DVIDVODODIIOIODODIOVVOIDOVOVDVOOIDODVDDODVDDIDIVDIVDIOD

VDDIODOODIIODOOIDOOOOVDDDODDOOIDIVOVVDIIOOVDOOOOVOIIDD

VODDDDIIOVODOODIOIIIIOODDDOOIVOODVODIVDIVOIDIIIDIODVODO

ODVIVDDIVODDIODOIIODDDVVDIOVVDDIVODVOIODOIDOVDOODDVIOIV

OOODIVODIDOOOIOVODDVDVVOO VVDIIDDVDDVVVOOODVVDOODOIVDIO

DDOVVDOIVOVOVDODIOOIOOODDVVDIOOVVDVODDDOVODDIOODIVVDDO

VIOOOOIOODIODVDVDDIIDDVVVVODIDIVIDIVOIDOOODOODOOIOOVIDD

VODIVDOIIVIDIIOVDIIODVVDODODDVDIDVDVDVDDODDODDOODIOIVOI

OOIVODVOVDOIDVIDODVODIOVVOOODDVVVDIDDVDODOOOOOIIVIOODI

VDVVDOODOODDOOOODDIIIODVOVIODOOOOIOIOIVOOVDVIDIVDDODDO

DODDVVOIVOOIVDIVOOOOIDDDOIIIVODOIDDDDVVDVODOODIVOIODDDO

DIVDIDOVODVVDIDODDDIOODDIVVDDVOIIODDODDOIDOOIOIVOVVDVIO

DOIODODIVVOIDOIDVDOODIDOVDDVOOIDOIOIIOOVODODOIDODVOODVI

OVVOODDVVOIDOIDVOIVDDODVOOOVVVOVDIIDVODVIDODDIDIVDVDID

DDDVOIDOOVODDDIDDIDDOVVDVDODVDIVOVDIDDVDVDIVOVIDOOIIDOD

OOODOIDDDIDOIDOVOVIDOIIDOODVOOIDODOIODOVOOIIDVVOVDOVDID

VDOODDDOIDDIIDOVVOODVIOIDDVODOOVOOIIOIODDODDDDIIDODIOIO

OIDOOVIOODIDO VVDDIDVOOIDDIODOOIDODDODDODVODDVOVDODVODV

DDVDOODOODDDDVDDODVVDDOVDDOIIODIVOVOOIOOOIDOODVVIIVVII

(aq oirzj loi-iiDvaα pμusqd - εiON αi oas

£CC980//.00ZSfl/I3d 86S0H/800Z OΛV GACCCGAACCCGCCGGGACCAGAGTCCGACTACCGCACTCGCTGGGAGATTCCG

ATCGGCTTGCGCGAGACGGACCTGACGCCGGCTCACTGCCACATGCACACGAAC

CCGCACCTACTGATCTTCGGTGCGGCCAAATCGGGCAAGACGACCATTGCCCACG

CGATCGCGCGCGCCATTTGTGCCCGAAACAGTCCCCAGCAGGTGCGGTTCATGCT

CGCGGACTACCGCTCGGGCCTGCTGGACGCGGTGCCGGACACCCATCTGCTGGG

CGCCGGCGCGATCAACCGCAACAGCGCGTCGCTAGACGAGGCCGTTCAAGCACT

GGCGGTCAACCTGAAGAAGCGGTTGCCGCCGACCGACCTGACGACGGCGCAGCT

ACGCTCGCGTTCGTGGTGGAGCGGATTTGACGTCGTGCTTCTGGTCGACGATTGG

CACATGATCGTGGGTGCCGCCGGGGGGATGCCGCCGATGGCACCGCTGGCCCCG

TTATTGCCGGCGGCGGCAGATATCGGGTTGCACATCATTGTCACCTGTCAGATGA

GCCAGGCTTACAAGGCAACCATGGACAAGTTCGTCGGCGCCGCATTCGGGTCGG

GCGCTCCGACAATGTTCCTTTCGGGCGAGAAGCAGGAATTCCCATCCAGTGAGTT

CAAGGTCAAGCGGCGCCCCCCTGGCCAGGCATTTCTCGTCTCGCCAGACGGCAA

AGAGGTCATCCAGGCCCCCTACATCGAGCCTCCAGAAGAAGTGTTCGCAGCACC

CCCAAGCGCCGGTTAAGATTATTTCATTGCCGGTGTAGCAGGACCCGAGCTCAGC

CCGGTAATCGAGTTCGGGCAATGCTGACCATCGGGTTTGTTTCCGGCTATAACCG

AACGGTTTGTGTACGGGATACAAATACAGGGAGGGAAGAAGTAGGCAAATGGA

AAAAATGTCACATGATCCGATCGCTGCCGACATTGGCACGCAAGTGAGCGACAA

CGCTCTGCACGGCGTGACGGCCGGCTCGACGGCGCTGACGTCGGTGACCGGGCT

GGTTCCCGCGGGGGCCGATGAGGTCTCCGCCCAAGCGGCGACGGCGTTCACATC

GGAGGGCATCCAATTGCTGGCTTCCAATGCATCGGCCCAAGACCAGCTCCACCGT

GCGGGCGAAGCGGTCCAGGACGTCGCCCGCACCTATTCGCAAATCGACGACGGC

GCCGCCGGCGTCTTCGCCTAATAGGCCCCCAACACATCGGAGGGAGTGATCACC

ATGCTGTGGCACGCAATGCCACCGGAGCTAAATACCGCACGGCTGATGGCCGGC

GCGGGTCCGGCTCCAATGCTTGCGGCGGCCGCGGGATGGCAGACGCTTTCGGCG

83

112275/F/l GCTCTGGACGCTCAGGCCGTCGAGTTGACCGCGCGCCTGAACTCTCTGGGAGAA

GCCTGGACTGGAGGTGGCAGCGACAAGGCGCTTGCGGCTGCAACGCCGATGGTG

GTCTGGCTACAAACCGCGTCAACACAGGCCAAGACCCGTGCGATGCAGGCGACG

GCGCAAGCCGCGGCATACACCCAGGCCATGGCCACGACGCCGTCGCTGCCGGAG

ATCGCCGCCAACCACATCACCCAGGCCGTCCTTACGGCCACCAACTTCTTCGGTA

TCAACACGATCCCGATCGCGTTGACCGAGATGGATTATTTCATCCGTATGTGGAA

CCAGGCAGCCCTGGCAATGGAGGTCTACCAGGCCGAGACCGCGGTTAACACGCT

TTTCGAGAAGCTCGAGCCGATGGCGTCGATCCTTGATCCCGGCGCGAGCCAGAG

CACGACGAACCCGATCTTCGGAATGCCCTCCCCTGGCAGCTCAACACCGGTTGGC

CAGTTGCCGCCGGCGGCTACCCAGACCCTCGGCCAACTGGGTGAGATGAGCGGC

CCGATGCAGCAGCTGACCCAGCCGCTGCAGCAGGTGACGTCGTTGTTCAGCCAG

GTGGGCGGCACCGGCGGCGGCAACCCAGCCGACGAGGAAGCCGCGCAGATGGG

CCTGCTCGGCACCAGTCCGCTGTCGAACCATCCGCTGGCTGGTGGATCAGGCCCC

AGCGCGGGCGCGGGCCTGCTGCGCGCGGAGTCGCTACCTGGCGCAGGTGGGTCG

TTGACCCGCACGCCGCTGATGTCTCAGCTGATCGAAAAGCCGGTTGCCCCCTCGG

TGATGCCGGCGGCTGCTGCCGGATCGTCGGCGACGGGTGGCGCCGCTCCGGTGG

GTGCGGGAGCGATGGGCCAGGGTGCGCAATCCGGCGGCTCCACCAGGCCGGGTC

TGGTCGCGCCGGCACCGCTCGCGCAGGAGCGTGAAGAAGACGACGAGGACGACT

GGGACGAAGAGGACGACTGGTGAGCTCCCGTAATGACAACAGACTTCCCGGCCA

CCCGGGCCGGAAGACTTGCCAACATTTTGGCGAGGAAGGTAAAGAGAGAAAGTA

GTCCAGCATGGCAGAGATGAAGACCGATGCCGCTACCCTCGCGCAGGAGGCAGG

TAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCGACCAGGTGGAGTC

GACGGCAGGTTCGTTGCAGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCA

GGCCGCGGTGGTGCGCTTCCAAGAAGCAGCCAATAAGCAGAAGCAGGAACTCGA

CGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATACTCGAGGGCCGACGA

84

112275/F/l GGAGCAGCAGCAGGCGCTGTCCTCGCAAATGGGCTTCTGACCCGCTAATACGAA

AAGAAACGGAGCAAAAACATGACAGAGCAGCAGTGGAATTTCGCGGGTATCGA

GGCCGCGGCAAGCGCAATCCAGGGAAATGTCACGTCCATTCATTCCCTCCTTGAC

GAGGGGAAGCAGTCCCTGACCAAGCTCGCAGCGGCCTGGGGCGGTAGCGGTTCG

GAGGCGTACCAGGGTGTCCAGCAAAAATGGGACGCCACGGCTACCGAGCTGAAC

AACGCGCTGCAGAACCTGGCGCGGACGATCAGCGAAGCCGGTCAGGCAATGGCT

TCGACCGAAGGCAACGTCACTGGGATGTTCGCATAGGGCAACGCCGAGTTCGCG

TAGAATAGCGAAACACGGGATCGGGCGAGTTCGACCTTCCGTCGGTCTCGCCCTT

TCTCGTGTTTATACGTTTGAGCGCACTCTGAGAGGTTGTCATGGCGGCCGACTAC

GACAAGCTCTTCCGGCCGCACGAAGGTATGGAAGCTCCGGACGATATGGCAGCG

CAGCCGTTCTTCGACCCCAGTGCTTCGTTTCCGCCGGCGCCCGCATCGGCAAACC

TACCGAAGCCCAACGGCCAGACTCCGCCCCCGACGTCCGACGACCTGTCGGAGC

GGTTCGTGTCGGCCCCGCCGCCGCCACCCCCACCCCCACCTCCGCCTCCGCCAAC

TCCGATGCCGATCGCCGCAGGAGAGCCGCCCTCGCCGGAACCGGCCGCATCTAA

ACCACCCACACCCCCCATGCCCATCGCCGGACCCGAACCGGCCCCACCCAAACC

ACCCACACCCCCCATGCCCATCGCCGGACCCGAACCGGCCCCACCCAAACCACC

CACACCTCCGATGCCCATCGCCGGACCTGCACCCACCCCAACCGAATCCCAGTTG

GCGCCCCCCAGACCACCGACACCACAAACGCCAACCGGAGCGCCGCAGCAACCG

GAATCACCGGCGCCCCACGTACCCTCGCACGGGCCACATCAACCCCGGCGCACC

GCACCAGCACCGCCCTGGGCAAAGATGCCAATCGGCGAACCCCCGCCCGCTCCG

TCCAGACCGTCTGCGTCCCCGGCCGAACCACCGACCCGGCCTGCCCCCCAACACT

CCCGACGTGCGCGCCGGGGTCACCGCTATCGCACAGACACCGAACGAAACGTCG

GGAAGGTAGCAACTGGTCCATCCATCCAGGCGCGGCTGCGGGCAGAGGAAGCAT

CCGGCGCGCAGCTCGCCCCCGGAACGGAGCCCTCGCCAGCGCCGTTGGGCCAAC

CGAGATCGTATCTGGCTCCGCCCACCCGCCCCGCGCCGACAGAACCTCCCCCCAG

85

112275/F/l CCCCTCGCCGCAGCGCAACTCCGGTCGGCGTGCCGAGCGACGCGTCCACCCCGAT

TTAGCCGCCCAACATGCCGCGGCGCAACCTGATTCAATTACGGCCGCAACCACTG

GCGGTCGTCGCCGCAAGCGTGCAGCGCCGGATCTCGACGCGACACAGAAATCCT

TAAGGCCGGCGGCCAAGGGGCCGAAGGTGAAGAAGGTGAAGCCCCAGAAACCG

AAGGCCACGAAGCCGCCCAAAGTGGTGTCGCAGCGCGGCTGGCGACATTGGGTG

CATGCGTTGACGCGAATCAACCTGGGCCTGTCACCCGACGAGAAGTACGAGCTG

GACCTGCACGCTCGAGTCCGCCGCAATCCCCGCGGGTCGTATCAGATCGCCGTCG

TCGGTCTCAAAGGTGGGGCTGGCAAAACCACGCTGACAGCAGCGTTGGGGTCGA

CGTTGGCTCAGGTGCGGGCCGACCGGATCCTGGCTCTAGACGCGGATCCAGGCG

CCGGAAACCTCGCCGATCGGGTAGGGCGACAATCGGGCGCGACCATCGCTGATG

TGCTTGCAGAAAAAGAGCTGTCGCACTACAACGACATCCGCGCACACACTAGCG

TCAATGCGGTCAATCTGGAAGTGCTGCCGGCACCGGAATACAGCTCGGCGCAGC

GCGCGCTCAGCGACGCCGACTGGCATTTCATCGCCGATCCTGCGTCGAGGTTTTA

CAACCTCGTCTTGGCTGATTGTGGGGCCGGCTTCTTCGACCCGCTGACCCGCGGC

GTGCTGTCCACGGTGTCCGGTGTCGTGGTCGTGGCAAGTGTCTCAATCGACGGCG

CACAACAGGCGTCGGTCGCGTTGGACTGGTTGCGCAACAACGGTTACCAAGATTT

GGCGAGCCGCGCATGCGTGGTCATCAATCACATCATGCCGGGAGAACCCAATGT

CGCAGTTAAAGACCTGGTGCGGCATTTCGAACAGCAAGTTCAACCCGGCCGGGT

CGTGGTCATGCCGTGGGACAGGCACATTGCGGCCGGAACCGAGATTTCACTCGA

CTTGCTCGACCCTATCTACAAGCGCAAGGTCCTCGAATTGGCCGCAGCGCTATCC

GACGATTTCGAGAGGGCTGGACGTCGTTGAGCGCACCTGCTGTTGCTGCTGGTCC

TACCGCCGCGGGGGCAACCGCTGCGCGGCCTGCCACCACCCGGGTGACGATCCT

GACCGGCAGACGGATGACCGATTTGGTACTGCCAGCGGCGGTGCCGATGGAAAC

TTATATTGACGACACCGTCGCGGTGCTTTCCGAGGTGTTGGAAGACACGCCGGCT

GATGTACTCGGCGGCTTCGACTTTACCGCGCAAGGCGTGTGGGCGTTCGCTCGTC

86

112275/F/l CCGGATCGCCGCCGCTGAAGCTCGACCAGTCACTCGATGACGCCGGGGTGGTCG

ACGGGTCACTGCTGACTCTGGTGTCAGTCAGTCGCACCGAGCGCTACCGACCGTT

GGTCGAGGATGTCATCGACGCGATCGCCGTGCTTGACGAGTCACCTGAGTTCGAC

CGCACGGCATTGAATCGCTTTGTGGGGGCGGCGATCCCGCTTTTGACCGCGCCCG

TCATCGGGATGGCGATGCGGGCGTGGTGGGAAACTGGGCGTAGCTTGTGGTGGC

CGTTGGCGATTGGCATCCTGGGGATCGCTGTGCTGGTAGGCAGCTTCGTCGCGAA

CAGGTTCTACCAGAGCGGCCACCTGGCCGAGTGCCTACTGGTCACGACGTATCTG

CTGATCGCAACCGCCGCAGCGCTGGCCGTGCCGTTGCCGCGCGGGGTCAACTCGT

TGGGGGCGCCACAAGTTGCCGGCGCCGCTACGGCCGTGCTGTTTTTGACCTTGAT

GACGCGGGGCGGCCCTCGGAAGCGTCATGAGTTGGCGTCGTTTGCCGTGATCACC

GCTATCGCGGTCATCGCGGCCGCCGCTGCCTTCGGCTATGGATACCAGGACTGGG

TCCCCGCGGGGGGGATCGCATTCGGGCTGTTCATTGTGACGAATGCGGCCAAGCT

GACCGTCGCGGTCGCGCGGATCGCGCTGCCGCCGATTCCGGTACCCGGCGAAAC

CGTGGACAACGAGGAGTTGCTCGATCCCGTCGCGACCCCGGAGGCTACCAGCGA

AGAAACCCCGACCTGGCAGGCCATCATCGCGTCGGTGCCCGCGTCCGCGGTCCG

GCTCACCGAGCGCAGCAAACTGGCCAAGCAACTTCTCATCGGATACGTCACGTC

GGGCACCCTGATTCTGGCTGCCGGTGCCATCGCGGTCGTGGTGCGCGGGCACTTC

TTTGTACACAGCCTGGTGGTCGCGGGTTTGATCACGACCGTCTGCGGATTTCGCT

CGCGGCTTTACGCCGAGCGCTGGTGTGCGTGGGCGTTGCTGGCGGCGACGGTCGC

GATTCCGACGGGTCTGACGGCCAAACTCATCATCTGGTACCCGCACTATGCCTGG

CTGTTGTTGAGCGTCTACCTCACGGTAGCCCTGGTTGCGCTCGTGGTGGTCGGGT

CGATGGCTCACGTCCGGCGCGTTTCACCGGTCGTAAAACGAACTCTGGAATTGAT

CGACGGCGCCATGATCGCTGCCATCATTCCCATGCTGCTGTGGATCACCGGGGTG

TACGACACGGTCCGCAATATCCGGTTCTGAGGCCGGCCAAGCTGTTGCATTCCTA

CGAGTTCACCCGTTACCGCATCGACCGCGTTCCGCGCTATGAATTGGTCCGGTGT

87

112275/F/l GGGCTCGATGTTCCCGACGGGCCCGGCCCGCCACCGCGGGAGTGAGGGGCAATG

AGCGCGGGGGCAATACTGACAGCAAGATCACAATTGAGCCGGCACTAGCGGTCG

ACACATGCCCAGACACTGCGGAAATGCCACCTTCAGGCCGTCGCGTCGGTCCCG

AATTGGCCGTGAACGACCGCCGGATAAGGGTTTCGGCGGTGCGCTTGATGCGGG

TGGACGCCCGAAGTTGTGGTTGACTACACGAGCACTGCCGGGCCCAGCGCCTGC

AGTCTGACCTAATTCAGGATGCGCCCAAACATGCATGGATGCGTTGAGATGAGG

ATGAGGGAAGCAAGAATATATATGCATCAGGTGGACCCCAACTTGACACGTCGC

AAGGGACGATTGGCGGCACTGGCTATCGCGGCGATGGCCAGCGCCAGCCTGGTG

ACCGTTGCGGTGCCCGCGACCGCCAACGCCGATCCGGAGCCAGCGCCCCCGGTA

CCCACAACGGCCGCCTCGCCGCCGTCGACCGCTGCAGCGCCACCCGCACCGGCG

ACACCTGTTGCCCCCCCACCACCGGCCGCCGCCAACACGCCGAATGCCCAGCCG

GGCGATCCCAACGCAGCACCTCCGCCGGCCGACCCGAACGCACCGCCGCCACCT

GTCATTGCCCCAAACGCACCCCAACCTGTCCGGATCGACAACCCGGTTGGAGGAT

TCAGCTTCGCGCTGCCTGCTGGCTGGGTGGAGTCTGACGCCGCCCACCTCGACTA

CGGTTCAGCACTCCTCAGCAAAACCACCGGGGACCCGCCATTTCCCGGACAGCC

GCCGCCGGTGGCCAATGACACCCGTATCGTGCTCGGCCGGCTAGACCAAAAGCT

TTACGCCAGCGCCGAAGCCACCGACTCCAAGGCCGCGGCCCGGTTGGGCTCGGA

CATGGGTGAGTTCTATATGCCCTACCCGGGCACCCGGATCAACCAGGAAACCGTC

TCGCTCGACGCCAACGGGGTGTCTGGAAGCGCGTCGTATTACGAAGTCAAGTTCA

GCGATCCGAGTAAGCCGAACGGCCAGATCTGGACGGGCGTAATCGGCTCGCCCG

CGGCGAACGCACCGGACGCCGGGCCCCCTCAGCGCTGGTTTGTGGTATGGCTCG

GGACCGCCAACAACCCGGTGGACAAGGGCGCGGCCAAGGCGCTGGCCGAATCG

ATCCGGCCTTTGGTCGCCCCGCCGCCGGCGCCGGCACCGGCTCCTGCAGAGCCCG

CTCCGGCGCCGGCGCCGGCCGGGGAAGTCGCTCCTACCCCGACGACACCGACAC

CGCAGCGGACCTTACCGGCCTGATGAGGCGCGCCAGGAGACGCGTCCGACATGC

112275/F/l AGCTGGTCGATCGCGTCCGCGGCGCAGTCACCGGCATGTCCCGCCGCCTGGTCGT

CGGCGCAGTCGGCGCAGCACTGGTCTCCGGCCTGGTCGGCGCAGTCGGCGGCAC

CGCAACCGCAGGCGCATTCTCCCGCCCCGGCCTCCCCCGGCGGCGTGCTCTGGGA

TACCCCCTCCCCCAAGGAATACAAGAAGGGCGATACCACCACCGGCGTGTACCG

GATCATGACCCGGGGCCTCCTCGGCTCCTACCAAGCAGGCGCAGGCGTTATGGTG

GAAGGCGTTTTCCACACGCTCTGGCACACGACGAAGGGCGCAGCACTCATGTCC

GGCGAAGGCCGGCTCGATCCCTACTGGGGCTCCGTTAAGGAAGATCGGCTCTGCT

ACGGCGGCCCGTGGAAGCTCCAACACAAGTGGAACGGCCAAGATGAAGTGCAA

ATGATCGTTGTTGAACCGGGCAAGAACGTTAAGAACGTTCAAACCAAGCCGGGC

GTGTTCAAGACCCCGGAAGGCGAAATCGGCGCAGTTACCCTCGATTTCCCGACCG

GCACCTCCGGCTCCCCGATTGTGGATAAGAACGGCGATGTGATTGGCCTCTACGG

CAACGGCGTGATCATGCCGAACGGCTCCTACATCTCCGCAATCGTGCAAGGCGA

ACGGATGGATGAACCGATCCCGGCAGGCTTCGAACCGGAAATGCTCCGGAAGAA

GCAAATCACCTGATAATGATGAGCGGCCGCGCTAGCCAACAAAGCGACGTTGTG

TCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAA

TAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCA

ACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTA

TATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATC

GCTTGTATGGGAAGCCCCATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTA

GCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAAT

TTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTA

CTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTG

ATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTC

GATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGG

CGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCG

89

112275/F/l TAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAATCTTTTGCCATTC

TCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGA

CGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCG

ATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTAC

AGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCA

GTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAAC

ACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCG

AACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGT

TCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCT

CTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCT

GGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCACTAGTTCCACTGAGC

GTCAGACGCGGCCGCCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTT

TTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGT

TTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGC

AGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT

TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGT

GGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG

TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC

AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGA

GAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG

CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT

ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGA

TGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA

CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATCTGTCCGAGATGGCCGAGCGC

CGTGAGGTCTTTGATCACGTCAAAGAACTCACCACTCGCATCCGTCAGCGCTCCA

90

112275/F/l 16

311IVIDODOODODIOODDDOOIDDVVDVOODDDDDOOIDVOVVVDDVOOIDDVD

OVDOIDOIIODODODIODDVVIVDOIDVOODDVOIDIVODVOIVDDVODDOOIDI

VIDODIIDOODODOOVODDODDODIODODIDOOVDOIOOVDDDODIIDIDVDVID

ODIVDDVOIVDIDDDODVVIIDOVODDOOVODVDOOIVVVOODIVOIOODIIOV

VDIVDIIOOOOIDOIDVOOIDVDDVDIVOVODOOIDIVIOOIDODDOIODDODDO

IDOODIODVVIOODDIIOOOODODDDOVVOVVODIVDIDDODVVDIVIDVOOVO

DDVDDOODDOVODDDOODDIIOIODIDIDDOVOVVVDDODVDVVODOIVIDODO

IIIOIOODDDVODDOIVOIDODIIDVIDIVOIDOOVDIDODDIVODDDDVOODDV

IVDVVDIDDODIDDVDDOOIVVODDOOIOOIDOOVODVODODODOOVDVODODI

VODVVIVDIODOIIIIIDOVDIOIVOVIODOOOIDOVDVVOOIOVIDIVIDDOOI

IIIODODOVDIOVDIOVVODDOVDIODVDDOIDODVODIOVDVOIVDIDOVDDVV

DDOVIODDVDDVDIODIDOVOOVOIIDOVDODDODVODODDVDVODVVDOIOIV

(dq 980U uπmiop jaαOO suipooua 068εiqiΛ[ ^"3^"! - I7I OM αi 03S VVIIVVIIOIDIVVVVVIDOOIOOOIODIOIOODOIVODD

ODIIIDIOVOVODVIDOIIIODIIIODVOIIDVODDVDIDDOOIIOVOVVDVODV

IDOODODVOOIVVDODOOOVVVOIOOVOIODIVDIDVVODIVOOODVDDOVIDO

DOOOODOVIDODVDDDDIOVDOOIIODIODIIODOOOIVDIODODVDIOOODOO

IODIODDOODOIIOIDOIDVODIIOIIOIIOODVIDIDO VVOVOIODVOIIOVVD

IIIDVVOIVOIVVDIOOVOVDIOOOIOODODIODIDOOIDVVIVDODDDVDDVIV

OOIDODIIODIVOOOOIDODOVDVOVVVOOIIDIIOIIIODIODIIDIIIVOVOD

DVIIOIDVIVVIDIDDDVDVDOIDVVVDDDDIOIDVOVDDVOVVDDVDOIDDVOD

IODVODDVODDIDVVDVDDIVDIODDVDDODIVDIOVDVDDDDVDVDVDVVDDDV

ODDVDDDDIVDIDDODVDDDVVDVDIIODDIIOODOODDDIOVDVIVDIDVOVDV

DODODDVVVDVDOIOIDODIOIOVOIOIDOOIIVOVOVDVDIOVDDVDVDIOOV

DDDIDIDIDIIDVVVVVVVIIDIIODVVDVODDIIDIIOIODOODVDOVIDODOV

£CC980//.00ZSfl/13d 86S017Ϊ/800Z OΛV 16

VOIVOIOVODIODDDOODVOVDDDDODIDIIDODDIDODDDOVDDDDOVOD

IDDVODOVODDVDDDDODODIODOODOIDIIODOOOVODIDVODOODVDDIODO

DVODIODVOOVDOIOOIODVOVDDIVVOIOOIIOVODVVDDODODOVDIIDOID

DVVVVVDOOOVODOIDIOODVOIODODDIVDDVDDVDIVDOOIIDIIDVVODVV

DIDDVOVVODVOVDVDODODDIODODIOIVDVOOOIDIDOVDDIOODOODOOIV

OODDDOIVDOIVDDOODOOIDDODOOODIOODODDODOIDIIDIODODO VVDVVI

IIDOODDOOIOOODDDDDDVOOIVDDODODVODDDDVVVDDDDDDDODDVDOVII

IDVDOIDOVVVOVDDOOIDOOOOVDODODDDVDVOVVDDIVODDDVVDDODOOD

VODIODDDDDDODVOVDDDIDVDODDODDIVVOVDDOVDDDOODIVVVOOVDOD

ODDDVDDVOVOODOODOODDDDOODODDDDDOOVVIDVODOOIOOODOVDDVVO

DDDDDDOOODDDDDDODDVDDVDDDVDVDODVOODDOOOOVOODDIOOOIDODD

DODDODDIDDODOOODODOIDDOOOODDDOODDIOIIIIOODOOOODDIIIODDD

IODOODODVOODDDDDDDIDDOODDDDODDDIDIIVDODOOVDODDVVDODDVDO

ODOODIOVDODOODIOOIDDOODDIDIOOIDVDOVDODOODIOVDODOODIODI

OOIDDODDODDDIOIVDOODDVDIOVDODOODODDIODODIVODIOOIDOVDOIV

S61'18 spiOB oiπure Suipooua ureraop Suppijjmi .rejnjpojaiiπ ζζ_cLΛ_. - Sl OM QI 03S

OVIVVIDVDVIDVVOIIVIVODVVDODOIDVVODOODOVDIOODIOIVD

VOODODDVDIIIIVODODIODOVIVOVOODOODDODVDVVVDDOVDOOIDIDOD

DOVVDDVODDOVODDODOIDDIDDVOODIODDVDVDVDOVDVOODDDIDVOOOV

DIDDODIOIOODIVIOVDIODDVDODDDVDIDOIOOVOIOODOODIVIDODIODV

DDVOOVVDIDOVDDVODODIIDIODVVODDODIDODDVVDIDODDOIVODDOIOV

OIVVDDIDIVDODDDVIVDIDDIODIIVVODVODOODOODIDVODDDODIVOIDD

OVOVODOOIIODIDVDODVOIOODIVOODVVDDDODIIOIOIVDDIVDIIOVDDV

OIOODDODDODVDDOOOIDDVIDVODVVDIVDDOO VVDIDDODDVOOIIDVODI

£CC980//.00ZSfl/13d 86S017Ϊ/800Z OΛV SEQ ID NO: 16 - Forward Primer

GGCGTGTTGTGGGACACTCCCTCA

SEQ ID NO: 17 - Reverse Primer

GATCTGTTTTTTCCTCAGCATCTC

SEQ ID NO: 18 - Flexible linker containing furin degradation site

5 ' -AGCGGCGGCGGCGGCCGGACCAAGCGGGGCGGCGGCGGCAGC-S '

SEQ ID NO: 19 - Ribosomal binding site (RBS; 16 bp)

5 ' -AGGAGACGCGTCCGAC-3 '

93

112275/F/l References cited in text

1. Calmette, A. 1930. Au sujet des accidents de Lϋbeck chez des nourissons vaccines au BCG. Presse Med 44:748.

2. Evans, C. C. 1994. Historical background. Clinical Tuberculosis (Ed. Davies, P.D.O.), Chapman and Hall Medical, London. pp:l '-19.

3. Andersen, P., and T. M. Doherty. 2005. The success and failure of BCG - implications for a novel tuberculosis vaccine. Nat Rev Microbiol 3:656.

4. Lewinsohn, D. M., D. A. Lewinsohn, and J. E. Grotzke. 2003. TB vaccines at the turn of the century: insights into immunity to M. tuberculosis and modern approaches for prevention of an ancient disease. Semin Respir Infect 18:320.

5. Hart, P. D., and I. Sutherland. 1977. BCG and vole bacillus vaccines in the prevention of tuberculosis in adolescence and early adult life. Br Med J 2:293.

6. Colditz, G. A., T. F. Brewer, C. S. Berkey, M. E. Wilson, E. Burdick, H. V. Fineberg, and F. Mosteller. 1994. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. JAMA 271:698.

7. Colditz, G. A., C. S. Berkey, F. Mosteller, T. F. Brewer, M. E. Wilson, E. Burdick, and H. V. Fineberg. 1995. The efficacy of bacillus Calmette-Guerin vaccination of newborns and infants in the prevention of tuberculosis: meta-analyses of the published literature. Pediatrics 96:29.

8. Wilson, M. E., H. V. Fineberg, and G. A. Colditz. 1995. Geographic latitude and the efficacy of bacillus Calmette-Guerin vaccine. Clin Infect Dis 20:982.

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Claims

CLAIMSThe invention is claimed as follows:

1. A composition comprising a recombinant Mycobacterium comprising a nucleic acid encoding a polypeptide that enhances attachment of said Mycobacterium to a tissue.

2. The composition of claim 1, wherein said polypeptide comprises a tissue attachment factor (TAF), a functional variant thereof or a functional portion thereof.

3. The composition of claim 2, wherein said TAF comprises a fibronectin attachment protein, a functional variant thereof or a functional portion thereof.

4. The composition of claim 1, further comprising a nucleic acid encoding an immunogenic factor.

5. The composition of claim 4, wherein said immunogenic factor comprises a polypeptide comprising GGDEF.

6. The composition of claim 4, wherein said immunogenic factor comprises a polypeptide which is an adjuvant.

7. The composition of claim 4, wherein said immunogenic factor comprises RDl.

8. The composition of claim 4, wherein said immunogenic factor comprises a polypeptide that induces apoptosis.

9. The composition of claim 1, wherein said Mycobacterium underexpresses a carbohydrate associated with an immune response.

10. The composition of claim 9, wherein said carbohydrate comprises mannosylated lipoarabinomannan.

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11. The composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent.

12. The composition of claim 1 comprising a nucleic acid encoding an antigen foreign to said Mycobacterium.

13. The composition of claim 12, wherein said antigen is expressed on said Mycobacterium.

14. The composition of claim 1, further comprising an isolated antigen.

15. The composition of claim 1, wherein said Mycobacterium is attenuated.

16. The composition of claim 15, wherein said Mycobacterium comprises an inactivated essential metabolic pathway gene.

17. The composition of claim 15, wherein said Mycobacterium comprises an inactivated virulence factor.

18. The composition of claim 1, wherein said Mycobacterium comprises a non-antibiotic selection marker.

19. The composition of claim 18, wherein said marker is a catabolic enzyme.

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