A recombinant bacterial strain as tuberculosis vaccine candidate, and methods of producing thereof

Abstract

The invention describes a recombinant Mycobacterium strain such as M. smegmatis that is recombinantly converted to express the M. tb complex ESX protein expression system, e.g. M. tb complex ESX-1 protein expression system. The recombinant strain exports virulence effector proteins (such as EspA, EspB, EspC, EsxA or EsxB) similar to M. tb, when administered to a subject, and is therefore useful as a potential tuberculosis vaccine candidate. The recombinant strain is non-pathogenic and the vaccinated subject does not give a false positive result when tested with any standard tuberculosis diagnostic test e.g. CCT, as the recombinant strain does not sensitize the vaccinated host to M. tb antigens or protein derivate of M. bovis.

Description

A RECOMBINANT BACTERIAL STRAIN AS TUBERCULOSIS VACCINE CANDIDATE, AND METHODS OF PRODUCING THEREOFFIELD OF INVENTION

[0001] The present invention relates to a recombinant Mycobacterium smegmatis strain expressing Mycobacterium tuberculosis Type VII protein secretion system for use as a tuberculosis vaccine, as well as methods of producing said vaccine.BACKGROUND OF THE INVENTION

[0002] Tuberculosis (TB) is a disease caused by the Mycobacterium tuberculosis (M. tb) complex. The bacteria usually attack the lungs, but TB bacteria can attack any part of the body such as the kidney, spine, and brain. If left unchecked, tuberculosis can lead to severe illness and even death.

[0003] The virulence of the M. tb complex is largely due to its export of a variety of effector proteins through five distinct forms of the Type VII protein secretion system (T7SS) - ESX-1 to ESX-5. The most critical for M. tb virulence is its T7SS ESX-1 , which based on structural studies is predicted to contain an oligomeric membrane-spanning translocon core of five conserved proteins (EccB1 , EccCal , EccCbl , EccD1 and EccE1). This translocon core works with EccA1 , PE35, PPE68, EspA, EspC, EspD, Espl, EspK, EspL and MycP1 proteins in an ATP-dependent and highly orchestrated manner to export EspA, EspB, EspC, EsxA and EsxB virulence effector proteins that are also highly immunogenic. Specifically, M. tb co-secretes EsxA, EsxB, EspA and EspC in an ESX-1 dependent manner while EspB which is not cosecreted with EsxA, EsxB, EspA or EspC, is still exported via the ESX-1 system. Finally, EspD is also exported by M. tb however its export does not require ESX-1.

[0004] The current state of the art suggests the minimal functioning unit of the M. tb ESX-1 system, which includes the translocon core proteins, associated chaperones and secreted protein substrates, is encoded by 19 genes arranged consecutively in the esx-1 locus [SEQ ID NO: 1] (espE / rv3864 to mycP1 / rv3883c as well as the distal co-transcribed espACD operon [SEQ ID NO: 52], (espA / rv3616c [SEQ ID. NO. 53 & 54], espC / rv3615c [SEQ ID. NO. 55 & 56] and espD / rv3614c [SEQ ID. NO. 57 & 58]) in the M. tb chromosome. These sequences are critical for the virulence of M. tb.

[0005] Tuberculosis is not only a major concern for human health, but also has significant repercussions for animal health, particularly in the cattle farming industry. Bovine tuberculosis (bTB) is caused by Mycobacterium bovis, but the acquired disease is zoonotic, and can be transmitted to humans and other wildlife. In addition to the toll on animal helath, the economic cost of bTB world-wide is estimated to be US$3 billion annually. The only strategy available to deal with bTB in many jurisdictions is to test and cull affected herds. This unsustainable approach often costs governments hundreds of millions of dollars in compensation to producers for culled animals, and months of lost productivity for producers. A vaccine, BCG (Bacille Calmette-Guerin), is currently in use to prevent TB in people and some wildlife species, however its use in livestock has been limited or not permitted in many jurisdictions. This is because the only authorized diagnostic test for bTB in cattle and livestock, the comparative cervical tuberculin (CCT) skin test, utilizes bovine purified protein derivative (PPD), which is a mixture of M. bovis cell surface and exported proteins. PPD is injected intradermally to trigger an immune cell, specifically a T-cell memory response characterized by the increased production of IFNy. Only animals infected with M. bovis and sensitized to PPD but not healthy animals show increased IFNy production when injected with PPD. Unfortunately, because M. bovis PPD is almost identical to that produced by BCG, animals given the vaccine also become sensitized to PPD of M. bovis origin. As such, the CCT skin test cannot be used to distinguish between animals given the BCG vaccine from those infected with M. bovis.SUMMARY OF THE INVENTION

[0006] It is an object of the invention to provide an improved tuberculosis vaccine candidate that provides preventative protection to a subject e.g. livestock like cattle, from the disease without triggering a false positive outcome on the standard CCT skin test.

[0007] The invention describes a recombinant bacterial strain comprising a Type VII Secretion System derived from M. tuberculosis (M. tb). The bacterial strain may be selected from any known Mycobacterium strain, and more specifically M. smegmatis, which is recombinantly converted to express the Type VII Secretion System from M. tb.

[0008] The Type VII secretion system could be one of M.tb complex ESX-1 , M.tb complex ESX-2, M.tb complex ESX-3 , M.tb complex ESX-4, or M.tb complex ESX-5, or a sequence that is at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that hasa sequence similarity of any integer in between 70%-100%, to the sequence of M.tb complex ESX-1 , M.tb complex ESX-2, M.tb complex ESX-3, M.tb complex ESX-4, or M.tb complex ESX-5, or repeats or concatemers of any of the above. Specifically, the application describes a recombinant M. smegmatis strain that is recombinantly converted to comprise the M. tb complex ESX-1 protein expression system.

[0009] The invention also describes an engineered or recombinant bacterial strain, or a Mycobacterium strain, or a M. smegmatis strain, that is recombinantly converted to express a sequence that is at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of the genetic material e.g. gene loci or operon system, responsible for expressing one of the Type VII protein expression systems from ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5.

[0010] For instance, Mycobacterium strain such as M. smegmatis can be recombinanty converted to express a M. tb complex esx-1 locus [SEQ ID NO: 1] or the M. tb espACD operon [SEQ ID NO: 52], or a combination of M. tb esx-1 locus [SEQ ID NO: 1] and the the M. tb espACD operon [SEQ ID NO: 52], that are responsible for expressing the M. tb ESX-1 protein expression system, or could recombinanty converted to express a sequence that is at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of M. tb esx-1 locus [SEQ ID NO: 1] or the M. tb espACD operon [SEQ ID NO: 52], or a combination of M. tb esx-1 locus [SEQ ID NO: 1] and the M. tb espACD operon [SEQ ID NO: 52],

[0011] The engineered or recombinant bacterial strain, such as M. smegmatis is non- pathogenic when administered to a subject in need thereof. The incorporation of M. tb genetic or protein material does not render the engineered strain pathogenic. The engineered or recombinant strain, such as the recombinant M. smegmatis strain, when administered to a subject does not sensitize the subject to antigens of M. tb or protein purified derivative of M. bovis. For instance, when the engineered strains is used as a tuberculosis vaccine, the vaccinated subject does not show sensitivity to antigens of M. tb or protein purified derivative of M. bovis, and thereby prevents a false positive outcome to a standard tuberculosis skin test or any tuberculosis diagnostic tests e.g. CCT.

[0012] Therefore, the recombinant strain can be used in preventative treatment of tuberculosis, for diagnostic testing of tuberculosis, as a vaccine candidate against tuberculosis, or for improving the immunity of patients susceptible to tuberculosis.

[0013] The application also describes vectors, translation cassettes, expression systems, compositions and kits comprising the above recombinant strains, or genetic material comprised in the recombinant strains.

[0014] The application also describes methods of production of the recombinant strains, and methods of vaccinating a subject, or methods of preventatively treating a subject against tuberculosis using the recombinant strains taught in the application.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0016] FIGURE 1 shows the expression and secretion of M. tb ESX-1 proteins by recombinant M. smegmatis strains. Immunoblots of cell filtrate (OF) proteins (A) and cell lysate (CL) proteins (B) of M. smegmatis::pYUB412+pMD31 , M. smegmatis::pYUB412+pMDespACD, M. smegmatis::2F9+pMD31 and M. smegmatis::2F9+pMDespACD (or MSX-1) after 10, 20, and 30 hours of growth in 7H9 media without Tween-80. Immunoblots shown are representative of 3 independent experiments.

[0017] FIGURE 2 shows the growth comparisons of recombinant M. smegmatis strains. M. smegmatis::pYUB412+pMD31 (blue), M. smegmatis::pYUB412+pMDespACD (green), M. smegmatis::2F9+pMD31 (orange) and M. smegmatis::2F9+pMDespACD (or MSX-1) (red) were cultured in 7H9 complete with Hyg and Kan, and OD600nm readings were taken at the indicated time-points. Data represent the means and error bars represent the standard deviation of the ODeoonm values from two independent experiments.

[0018] FIGURE 3 shows the viability of THP-1 cells infected with recombinant M. smegmatis strains. Human macrophage-like THP-1 cells were infected at an MOI of 1 with either M. smegmatis::pYUB412+pMD31 , M. smegmatis::pYUB412+pMDespACD, M. smegmatis::2F9+pMD31 , M. smegmatis::2F9+pMDespACD (or MSX-1) or left uninfected.Data shown here is the mean viability of THP-1 cells in multiple wells as measured using PrestoBlue reagent and is representative of at least three independent experiments.

[0019] FIGURE 4 shows Mycobacterium tuberculosis burden quantitation in vaccinated mice. Colony forming units (CFU) of challenge M. tb per organ in the lungs (A) and spleens (B) of mock vaccinated mice and mice vaccinated with either M. smegmaf / s::pYUB412+pMD31 , M. smegmaf / s::2F9+pMD31 , M. smegmaf / s::2F9+pMDespACD (or MSX-1) or BCG. Data points in each graph are the burden in CFU per organ of each mouse in a given treatment group. The bars represents the median CFU per organ (Significance in difference: * p<0.05; ** p<0.005 and ns: not significant).

[0020] FIGURE 5 shows Mycobacterium tuberculosis induced pathology in lungs of vaccinated mice. H&E staining of lung sections from M. to-challenged mice vaccinated with M. smegmaf / s::pYUB412+pMD31 (A), MSX-1 (B), BCG (C) and saline (D). Representative of 2 to 3 sections per mouse, with two mice per vaccination group.

[0021] FIGURE 6 shows the sensitization to PPD in vaccinated mice. Induction of IFNy by splenocytes isolated from mock vaccinated mice and mice vaccinated with either BCG, MSX- 1 , M. smegmaf / s::2F9+pMD31 or M. smegroaf / s::pYUB412+pMD31 upon stimulation with RPMI tissue culture medium, BCG CF, PPD and ConA. Data represent the means and error bars represent standard deviation of the concentration of IFNy in supernatants from duplicate wells of splenocytes from two mice per vaccination group; (Significance in difference: **** p<0.0005 and ns: not significant).

[0022] FIGURE 7 shows the sensitization status of splenocytes from vaccinated mice. Splenocytes were isolated from mice 26 days (A) and 47 days (B) after administration of different vaccines and saline and co-cultured in vitro with either media, ConA, EsxA peptide or tuberculin (PPD).

[0023] FIGURE 8 shows the survival of vaccinated mice after infectious challenge with virulent M. bovis. Mice administered the different vaccines and saline were challenged via the intranasal route with a lethal dose of 102CFU of virulent M. bovis, the causative agent of bovine TB and monitored for survival until humane intervention point.

[0024] FIGURE 9 shows the changes in median body weights of vaccinated mice after challenge with virulent M. bovis. Mice administered the different vaccines and saline were weighed and then challenged via the intranasal route with a lethal dose of 102CFU of virulent M. bovis. Mice were weighed again at HIP.

[0025] FIGURE 10 shows the pulmonary M. bovis burden in vaccinated mice. The median CFU of M. bovis per lung in different mice were enumerated. Each data point is the actual CFU / lung of M. bovis of each mouse. Statistical analysis using Kruskal-Wallis Dunn’s multiple comparisons test was performed. **** p < 0.0001 ; *** p = 0.0003; ** p = 0.0015; ns = not significant.DETAILED DESCRIPTION

[0026] The following description is of preferred embodiments by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

[0027] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0028] Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

[0029] The following definitions supplement those in the art and are directed to the current application. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0030] Definitions

[0031] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0032] In this application, the use of "or" means "and / or" unless stated otherwise. The terms "and / or" and "any combination thereof and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any and all combinations are specifically contemplated. The term "or" can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0033] Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[0034] Reference in the specification to "some embodiments," "an embodiment," "one embodiment" “alternate embodiment”, or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0035] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0036] The term "about" in relation to a reference numerical value and its grammatical equivalents as used herein can include the numerical value itself and a range of values plus or minus 10% from that numerical value. The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. , the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, upto 10%, up to 5%, or up to 1 % of a given value. In another example, the amount "about 10" includes 10 and any amounts from 9 to 11. In yet another example, the term "about" in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % from that value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0037] The term "isolated" and its grammatical equivalents as used herein refer to the removal of a nucleic acid from its natural environment. Whether the nucleic acid is removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and / or amplified under laboratory conditions, it is to be understood, however, that nucleic acids and proteins can be formulated with diluents or adjuvants and still for practical purposes be isolated. For example, nucleic acids typically are mixed with an acceptable carrier or diluent when used for introduction into cells.

[0038] As used herein, the term "gene" refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, siRNA, shRNA, miRNA, and the like. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements including promoters, enhancers, termination sequences and 5' and 3' untranslated regions). A gene may be "isolated" by which is meant a nucleic acid molecule that is substantially or essentially free from components normally found in association with the nucleic acid molecule in its natural state. Such components include other cellular material, culture medium from recombinant production, and / or various chemicals used in chemically synthesizing the nucleic acid molecule. Reference to a "gene" also includes within its scope reference to genes having a contiguous sequence, thus defining contiguous nucleic acid entities, as defined herein, or a non-contiguous sequence thus defining a non-contiguous nucleic acid entity as defined herein. In certain embodiments, the term "gene" includes within its scope the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression. In this regard, the gene may further comprise control sequences such as promoters, enhancers, termination and / or polyadenylation signals that are naturally associated with a given gene, or heterologous control sequences. The gene sequences maybe cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for introduction into a host.

[0039] "Genome" as used herein refers to the entirety of an organism's hereditary information, represented by genes and non-coding sequences of DNA, either chromosomal or non- chromosomal genetic elements such as, linear polynucleotides, e.g., including the gene(s) to be assembled and / or recombined. Thus, the term "genome" is intended to include the entire DNA of an organism, including the nuclear DNA component, chromosomal or extrachromosomal DNA, as well as the cytoplasmic domain (e.g., mitochondrial DNA).

[0040] The terms “Nucleic Acid(s)”, “Polynucleotide(s)", "oligonucleotide(s)", or "nucleotide(s)", or any grammatical equivalent as used herein refers to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double and single stranded DNA, triplex DNA, as well as double and single stranded RNA. It also includes modified, for example, by methylation and / or by capping, and unmodified forms of the polynucleotide. The term is also meant to include molecules that include non-naturally occurring or synthetic nucleotides as well as nucleotide analogs. The nucleic acid sequences and vectors disclosed or contemplated herein can be introduced into a cell by, for example, transfection, transformation, or transduction.

[0041] Nucleic acids and / or nucleic acid sequences are "homologous" when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and / or protein sequences are "homologous" when their encoding DNAs are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. The homologous molecules can be termed homologs. Homology is generally inferred from sequence identity between two or more nucleic acids (or sequences thereof). The precise percentage of identity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence identity is routinely used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology. Methods for determining sequence identity percentages (e.g., BI_ASTP and BI_ASTN using default parameters) are described herein and are generally available.

[0042] The terms "identical" and its grammatical equivalents as used herein or "sequence identity" in the context of two nucleic acid sequences or amino acid sequences of polypeptides refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.

[0043] The term "substantially identical" and its grammatical equivalents as applied to nucleic acid sequences mean that a nucleic acid or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, at least 95%, at least 98% and at least 99%), compared to a reference sequence using the programs described above, e.g., BLAST, using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11 , an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. In embodiments, the substantial identity exists over a region of the sequences that is at least about 25 bases in length, 50 bases in length, 100 bases in length, 125 bases in length, 150 bases in length and in embodiments, the sequences are substantially identical over at least about 180 bases. In embodiments, the sequences are substantially identical over the entire length of the coding regions.

[0044] An "expression vector" or "vector" is any genetic element, e.g., a plasmid, chromosome, virus, transposon, behaving either as an autonomous unit of polynucleotide replication within a cell. (i.e. capable of replication under its own control) or being rendered capable of replication by insertion into a host cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and / or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages and cosmids. Vectors can contain polynucleotide sequences which are necessary to effect ligation or insertion of the vector into a desired host cell and to effect the expression of the attached segment. Such sequences differ depending on the host organism;they include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosomal binding site sequences and transcription and translation termination sequences. Alternatively, expression vectors can be capable of directly expressing nucleic acid sequence products encoded therein without ligation or integration of the vector into host cell DNA sequences. In some embodiments, the vector is an episomal expression vector, which is able to replicate in a host cell, and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure. Vector also can comprise a selectable marker gene. The term "selectable marker gene" as used herein refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected for or against, in the presence of a corresponding selective agent.

[0045] The term "coding sequence" or “sequence encoding” as used herein refers to a segment of a polynucleotide that codes for protein. The region or sequence is bounded nearer the 5 ' end by a start codon and nearer the 3 ' end with a stop codon. Coding sequences can also be referred to as open reading frames. The present invention is further directed to a nucleotide construct comprising the nucleic acid as described above operatively linked to one or more regulatory elements or regulatory regions. By "regulatory element" or "regulatory region", it is meant a portion of nucleic acid typically, but not always, upstream of a gene, and may be comprised of either DNA or RNA, or both DNA and RNA. Regulatory elements may include those which are capable of mediating organ specificity, or controlling developmental or temporal gene activation. Furthermore, "regulatory element" includes promoter elements, core promoter elements, elements that are inducible in response to an external stimulus, elements that are activated constitutively, or elements that decrease or increase promoter activity such as negative regulatory elements or transcriptional enhancers, respectively. By a nucleotide sequence exhibiting regulatory element activity it is meant that the nucleotide sequence when operatively linked with a coding sequence of interest functions as a promoter, a core promoter, a constitutive regulatory element, a negative element or silencer (i.e. elements that decrease promoter activity), or a transcriptional or translational enhancer.

[0046] The present invention further includes vectors comprising the nucleic acids as described above. Suitable expression vectors for use with the nucleic acid sequences of the present invention include, but are not limited to, plasmids, phagemids, viral particles and vectors, phage and the like. For insect cells, baculovirus expression vectors are suitable. The entire expression vector, or a part thereof, can be integrated into the host cell genome.

[0047] Those skilled in the art will understand that a wide variety of expression systems can be used to produce the proteins or fragments thereof as defined herein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen by one skilled in the art to ensure the correct modification and processing of the expressed cardiac stem cell proliferation protein.

[0048] The term "operably linked" as used herein refers to the physical and / or functional linkage of a DNA segment to another DNA segment in such a way as to allow the segments to function in their intended manners. A DNA sequence encoding a gene product is operably linked to a regulatory sequence when it is linked to the regulatory sequence, such as, for example, promoters, enhancers and / or silencers, in a manner which allows modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter, in the correct reading frame with respect to the transcription initiation site and allows transcription elongation to proceed through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence coding for a gene product when it is ligated to the DNA sequence in such a manner as to increase or decrease, respectively, the transcription of the DNA sequence. Enhancers and silencers can be located upstream, downstream or embedded within the coding regions of the DNA sequence. A DNA for a signal sequence is operably linked to DNA coding for a polypeptide if the signal sequence is expressed as a pre-protein that participates in the secretion of the polypeptide. Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or via adapters or linkers inserted in the sequence using restriction endonucleases known to one of skill in the art.

[0049] The term "induce", "induction" and its grammatical equivalents as used herein refer to an increase in nucleic acid sequence transcription, promoter activity and / or expression brought about by a transcriptional regulator, relative to some basal level of transcription or control systems used. Increase in nucleic acid sequence transcription, promoter activity and / or expression can also be brought about by a translation regulator such as a translation enhancing UTR sequence.

[0050] Regulatory elements as used herein, also includes elements that are active following transcription initiation or transcription, for example, regulatory elements that modulate gene expression such as translational and transcriptional enhancers, translational and transcriptional repressors, and mRNA stability or instability determinants. In the context of this disclosure, the term "regulatory element" also refers to a sequence of DNA, usually, but not always, upstream (5') to the coding sequence of a structural gene, which includes sequences which control the expression of the coding region by providing the recognition for RNA polymerase and / or other factors required for transcription to start at a particular site. An example of a regulatory element that provides for the recognition for RNA polymerase or other transcriptional factors to ensure initiation at a particular site is a promoter element. A promoter element comprises a core promoter element, responsible for the initiation of transcription, as well as other regulatory elements that modify gene expression. It is to be understood that nucleotide sequences, located within introns, or 3' of the coding region sequence may also contribute to the regulation of expression of a coding region of interest. A regulatory element may also include those elements located downstream (3') to the site of transcription initiation, or within transcribed regions, or both. In the context of the present invention a post- transcriptional regulatory element may include elements that are active following transcription initiation, for example translational and transcriptional enhancers, translational and transcriptional repressors, and mRNA stability determinants.

[0051] The regulatory elements, or fragments thereof, may be operatively associated (operatively linked) with heterologous regulatory elements or promoters in order to modulate the activity of the heterologous regulatory element. Such modulation includes enhancing or repressing transcriptional activity of the heterologous regulatory element, modulating post- transcriptional events, or both enhancing / repressing transcriptional activity of the heterologous regulatory element and modulating post-transcriptional events. For example, one or more regulatory elements, or fragments thereof, may be operatively associated with constitutive, inducible, tissue specific promoters or fragment thereof, or fragments of regulatory elements, for example, but not limited to TATA or GC sequences may be operatively associated with the regulatory elements of the present invention, to modulate the activity of such promoters within plant, insect, fungi, bacterial, yeast, or animal cells.

[0052] There are several types of regulatory elements, including those that are developmentally regulated, inducible and constitutive. A regulatory element that isdevelopmentally regulated, or controls the differential expression of a gene under its control, is activated within certain organs or tissues of an organ at specific times during the development of that organ or tissue. However, some regulatory elements that are developmentally regulated may preferentially be active within certain organs or tissues at specific developmental stages, they may also be active in a developmentally regulated manner, or at a basal level in other organs or tissues within a plant as well.

[0053] The term "promoter" refers to a region of a polynucleotide that initiates transcription of a coding sequence. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5' region of the sense strand). Some promoters are constitutive as they are active in all circumstances in the cell, while others are regulated becoming active in response to specific stimuli, e.g., an inducible promoter. The term "promoter activity" and its grammatical equivalents as used herein refer to the extent of expression of nucleotide sequence that is operably linked to the promoter whose activity is being measured. Promoter activity can be measured directly by determining the amount of RNA transcript produced, for example by Northern blot analysis or indirectly by determining the amount of product coded for by the linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter. Some non-limiting examples are CMV, EF1a, CAG, PGK, TRE, U6, and UAS.

[0054] By "promoter" it is meant the nucleotide sequences at the 5' end of a coding region, or fragment thereof that contain all the signals essential for the initiation of transcription and for the regulation of the rate of transcription. There are generally two types of promoters, inducible and constitutive promoters.

[0055] A constitutive promoter directs the expression of a gene throughout the various parts of an organism and / or continuously throughout development of an organism. Any suitable constitutive promoter may be used to drive the expression of the proteins or fragments thereof as described herein.

[0056] The term "constitutive" as used herein does not necessarily indicate that a gene is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types, although some variation in abundance is often observed.

[0057] "Inducible promoter" as used herein refers to a promoter which is induced into activity by the presence or absence of transcriptional regulators, e.g., biotic or abiotic factors. Inducible promoters are useful because the expression of genes operably linked to them can be turned on or off with an inducer at certain stages of development of an organism or in a particular tissue. An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA sequences or genes will not be transcribed. Typically the protein factor that binds specifically to an inducible promoter to activate transcription is present in an inactive form which is then directly or indirectly converted to the active form by the inducer. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, or a physiological stress imposed directly by heat, cold, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus. Non-limiting examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, steroid- regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature-regulated promoters and light-regulated promoters, isopropyl-p- thiogalactopyranoside (IPTG) inducible promoter.

[0058] The nucleic acid and other constructs of the present invention can further comprise a 3' untranslated region. A 3' untranslated region refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by effecting the addition of polyadenylic acid tracks to the 3 prime end of the mRNA precursor. E.g. SV-40 polyadenylation site.

[0059] The gene construct of the present invention can also include further enhancers, either translation or transcription enhancers, as may be required. These enhancer regions are well known to persons skilled in the art, and can include the ATG initiation codon and adjacent sequences. The initiation codon must be in phase with the reading frame of the coding sequence to ensure translation of the entire sequence. The translation control signals and initiation codons can be from a variety of origins, both natural and synthetic. Translational initiation regions may be provided from the source of the transcriptional initiation region, or from the structural gene. The sequence can also be derived from the regulatory element selected to express the gene, and can be specifically modified so as to increase translation of the mRNA.

[0060] The term "transcriptional regulator" or “cis-acting regulatory elements” refers to a biochemical element that acts to prevent or inhibit the transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein), or to permit or stimulate the transcription of the promoter-driven DNA sequence under certain environmental conditions (e.g., an inducer or an enhancer).

[0061] The term "enhancer" or “translation enhancer” as used herein, refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoters (such as the commonly-used CMV promoter) also comprise enhancer sequences. Enhancers can be located upstream, within, or downstream of coding sequences.

[0062] "Patient" or "subject" as used herein refers to a mammalian subject diagnosed with or suspected of having or developing a medical condition, cellular defect or a disease or disorder e.g. tuberculosis.

[0063] "Administering" is referred to herein as providing one or more compositions described herein to a patient or a subject. By way of example and not limitation, composition administration, e.g., injection, can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by the oral route. Additionally, administration can also be by surgical deposition of a bolus or pellet of cells, or positioning of a medical device. In an embodiment, a composition of the present disclosure can comprise engineered cells or host cells expressing nucleic acid sequences described herein, or a vector comprising at least one nucleic acid sequence described herein, in an amount that is effective to treat or prevent proliferative disorders. A pharmaceutical composition can comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptablecarriers, diluents or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0064] As used herein, the term "treatment", "treating", “amelioration” or its grammatical equivalents refers to obtaining a desired pharmacologic and / or physiologic effect. In embodiments, the effect is therapeutic, i.e. , the effect partially or completely cures a disease and / or adverse symptom attributable to the disease.

[0065] Embodiments

[0066] Without wishing to be bound by theory or experimental results, the following paragraphs describe the nature of the invention by way of examples only. The experiments, or specific examples of materials, strains, promoters, enhancers, regulatory elements, vectors, cell lines and constructs described should not be construed as limiting the scope of the invention. A person skilled in the art would readily understand and appreciate that other materials, strains, promoters, enhancers, regulatory elements, vectors, cell lines and constructs not specifically described also form part of the invention.

[0067] In an embodiment of the invention, a recombinant bacterial strain comprising a Type VII Secretion System derived from M. tuberculosis (M. tb) is provided. The bacterial strain may be selected from any known bacterial family or genus which can be recombinantly converted to express the Type VII Secretion System from M. tb (also referred to herein as M. tb complex).

[0068] The Mycobacterium tuberculosis-complex (or M. tb-complex) are phylogenetically closely related organisms that cause TB disease. It includes M. tuberculosis, M. bovis, M. orygis, M. africanum, M. caprae, M. microti, M. canettii, M. pinnipedii, M. suricattae, M. mungi and M. dassie.

[0069] In some embodiments, the bacterial strain may be selected from Mycobacterium family, optionally wherein the bacterial strain is M. smegmatis. In a further embodiment, an M. smegmatis strain comprising or expressing a Type VII Secretion System derived from M. tuberculosis is provided.

[0070] In any of the above embodiments, the Type VII secretion system could be one of M.tb ESX-1 , M.tb ESX-2 , M.tb ESX-3 , M.tb ESX-4 , or M.tb ESX-5 , or a sequence that is at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of M.tb ESX-1 , M.tb ESX-2 , M.tb ESX-3 , M.tb ESX-4 , or M.tb ESX-5 . In a further embodiment, the Type VII secretion system could comprise two or more repeats or a concatemer of M.tb ESX-1 , M.tb ESX-2 , M.tb ESX-3 , M.tb ESX-4 , or M.tb ESX-5 , or a sequence that is at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of M.tb ESX-1 , M.tb ESX-2 , M.tb ESX-3 , M.tb ESX- 4 , or M.tb ESX-5 .

[0071] In some specific embodiments, the bacterial strain, or the Mcobacterium strain, or the M. smegmatis strain may be recombinantly converted to express M. tb complex ESX-1 protein expression system that’s derived from M. tuberculosis, or a sequence that is at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of M.tb ESX-1 protein expression system.

[0072] The recombinant bacterial strain, Mycobacterium strain or M. smegmatis strain as described above, with the M.tb ESX-1 protein expression system is capable of exporting one or more virulence effector proteins from EspA, EspB, EspC, EsxA or EsxB, when administered in a subject in need thereof.

[0073] In an embodiment of the invention, a recombinant bacterial strain expressing the genetic machinery for the Type VII protein secretion system is provided. For instance, the genome, of any bacterial strain could be recombinantly converted to express the genetic material e.g. gene loci or operon system, responsible for expressing one of the Type VII protein expression systems from ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5.

[0074] In some embodiments, the bacterial strain could be selected from Mycobacterium family, and in some specific embodiments the bacterial strain could be M. smegmatis.

[0075] In an further embodiment, the genome of any bacterial strain, or a Mycobacterium strain, or a M. smegmatis strain, could be engineered or recombinantly converted to express a sequence that is a sequence that is at least 70%, at least 80%, at least 90%, at least 95%similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of the genetic material e.g. gene loci or operon system, responsible for expressing one of the Type VII protein expression systems from ESX-1 , ESX-2, ESX-3, ESX- 4 or ESX-5.

[0076] In a further embodiment, the genome of the bacterial strain, Mycobacterium strain or M. smegmatis, could be engineered or recombinanty converted to express a M. tb complex esx-1 locus [SEQ ID NO: 1] or the M. tb espACD operon [SEQ ID NO: 52] , or a combination of M. tb esx-1 locus [SEQ ID NO: 1] and the the M. tb espACD operon [SEQ ID NO: 52], that are responsible for expressing the M. tb ESX-1 protein expression system. In another embodiment, the genome of the bacterial strain, Mycobacterium strain or M. smegmatis, could be engineered or recombinanty converted to express a sequence that is at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of M. tb locus or the M. tb espACD operon [SEQ ID NO: 52] , or a combination of M. tb esx-1 locus [SEQ ID NO: 1] and the the M. tb espACD operon [SEQ ID NO: 52],

[0077] In some specific embodiment, a recombinant or engineered M. smegmatis strain comprising a combination of the sequences of M. tb esx-1 locus [SEQ ID NO: 1] and M. tb espACD operon [SEQ ID NO: 52], a combination of the sequences that are at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequences of of M. tb esx-1 locus [SEQ ID NO: 1] and M. tb espACD operon [SEQ ID NO: 52] are provided.

[0078] In any of the above embodiments, the genome of the bacterial strain, Mycobacterium strain or M. smegmatis, could be engineered or recombinanty converted to express two or more copies or repeats of the sequences of M. tb esx-1 locus [SEQ ID NO: 1] or the M. tb espACD operon [SEQ ID NO: 52] , or a combination of M. tb esx-1 locus [SEQ ID NO: 1] and the the M. tb espACD operon [SEQ ID NO: 52], or two or more copies or repeats of a sequence that is 70-99% similar to the sequence of M. tb esx-1 locus [SEQ ID NO: 1] or the M. tb espACD operon [SEQ ID NO: 52], or a combination of both. In some embodiments, the genome of the bacterial strain, Mycobacterium strain or M. smegmatis, could be engineered or recombinanty converted to express concatemers of M. tb esx-1 locus [SEQ ID NO: 1] or the M. tb espACD operon [SEQ ID NO: 52],

[0079] The engineered or recombinant bacterial strain, or the engineered or recombinant Mycobaterium strain, or the engineered or recombinant M. smegmatis strain described in the above embodiments is non-pathogenic when administered to a subject in need thereof. The incorporation of M. tb genetic or protein material does not render the engineered or bacterial strains pathogenic.

[0080] The engineered or recombinant bacterial strain, or the engineered or recombinant Mycobaterium strain, or the engineered or recombinant M. smegmatis strain described in the above embodiments, when administered to a subject does not sensitize the subject to antigens of M. tb or protein purified derivative of M. bovis. The engineered strains described above when used as a tuberculosis vaccine candidate, the vaccinated subject does not show sensitivity to antigens of M. tb or protein purified derivative of M. bovis, thereby preventing false positive outcome to a standard tuberculosis skin test or any tuberculosis diagnostic tests e.g. CCT.

[0081] The engineered or recombinant bacterial strain, or the engineered or recombinant Mycobaterium strain, or the engineered or recombinant M. smegmatis strain described in the above embodiments could be used in preventative treatment of tuberculosis, for diagnostic testing of tuberculosis, as a vaccine candidate against tuberculosis, or for improving the immunity of patients susceptible to tuberculosis.

[0082] In an embodiment of the invention a vector, a translation cassette, a plasmid, a cosmid, or any known expression system is provided that comprises a DNA fragment of genetic material encoding ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5, or a DNA fragment with at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of genetic material encoding ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5, along with a promoter, one or more regulatory sequences, one or more restriction endonuclease or cloning sites, one or more polyadenylation sites or any combination of the above.

[0083] In an embodiment of the invention a vector, a translation cassette, a plasmid, a cosmid, or any known expression system is provided that comprises a DNA fragment of genetic material encoding ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5, or a DNA fragment with at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of genetic material encodingESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5, along with a promoter, one or more regulatory sequences, one or more restriction endonuclease or cloning sites, one or more polyadenylation sites or any combination thereof, wherein at least the promoter, the restriction endonuclease or cloning sites, the polyadenylation sites are heterologous to the genome of M. smegmatis.

[0084] In an embodiment of the invention a vector, a translation cassette, a plasmid, a cosmid, or any known expression system is provided that comprises a genome of any bacterial strain, Mycobacterium strain, or M. smegmatis strain, and a DNA fragment of genetic material encoding ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5, or a DNA fragment with at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of genetic material encoding ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5, along with a promoter, one or more regulatory sequences, one or more restriction endonuclease or cloning sites, one or more polyadenylation sites or any combination thereof, wherein at least the promoter, the restriction endonuclease or cloning sites, the polyadenylation sites are heterologous to the genome of M. smegmatis.

[0085] In a specific embodiment of the invention, the above vector, translation cassette, plasmid, cosmid, or any known expression systems, comprises a DNA fragment of M. tb complex esx-1 locus [SEQ ID NO: 1] , or a DNA fragment comprising M. tb espACD operon [SEQ ID NO: 52] ; or a DNA fragment comprising M. tb esx-1 locus [SEQ ID NO: 1] and M. tb espACD operon [SEQ ID NO: 52] , or a DNA fragment that is at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of M. tb complex esx-1 locus [SEQ ID NO: 1] , M. tb espACD operon [SEQ ID NO: 52] , or a combination of both. These specific vector, translation cassette, plasmid, cosmid, or expression systems, additionally comprise any known promoter, any known one or more regulatory sequences, any known one or more restriction endonuclease or cloning sites, any known one or more polyadenylation sites or any combination of the above. Alternatively, the promoter, the restriction endonuclease or cloning sites, the polyadenylation sites could be heterologous to the genome of M. smegmatis.

[0086] The transcription and translation machinery referred to above i.e. the promoter, one or more regulatory sequences, one or more restriction endonuclease or cloning sites, one or more polyadenylation sites may be selected from any known promoters, regulatory sequences, restriction endonucleases, cloning sites or polyadenylation sites.

[0087] The one or more regulatory sequences can be selected from any known promoters, enhancers, silencers, transcription factors, coactivators, or operators. The one or more restriction endonuclease or cloning sites are palindromic sequences that are recognizable by restriction endonucleases. The one or more polyadenylation sites could be a stretch of Adenine bases or SV40 site or any other site known in the art.

[0088] In a specific embodiment of the invention, a vector, a translation cassette, a plasmid, a cosmid, or any known expression system is provided that comprises a genome of M. smegmatis strain, and a DNA fragment of genetic material encoding ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5, or a DNA fragment with at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%- 100%, to the sequence of genetic material encoding ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5.

[0089] In another specific embodiment of the invention, a vector, a translation cassette, a plasmid, a cosmid, or any known expression system is provided that comprises a genome of M. smegmatis strain, and a DNA fragment of M. tb complex esx-1 locus [SEQ ID NO: 1] , or a DNA fragment comprising M. tb espACD operon [SEQ ID NO: 52] ; or a DNA fragment comprising M. tb complex esx-1 locus [SEQ ID NO: 1] and M. tb espACD operon [SEQ ID NO: 52] , or a DNA fragment that is at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of M. tb esx-1 locus [SEQ ID NO: 1] , M. tb espACD operon [SEQ ID NO: 52] , or a combination of both.

[0090] A few non-limiting examples of expression systems include gene delivery vehicles such as, a plasmid, a vector, a recombinant DNA or RNA construct, or an expression cassette or a nanoparticle, which could be a lipid nanoparticle. In some embodiments, the nucleotide machinery or DNA material described above may be inserted in a gene delivery vehicle such as a plasmid, a vector, a recombinant DNA or RNA construct, or an expression cassette. Some non-limiting examples of vector that can be employed included a viral vector, a live-viral vector, an oncolytic viral vector, an attenuated viral vector, a recombinant vector or an amplicon vector. In some alternate embodiments, the nucleic acid may be inserted in incorporated in a nanoparticle, or a lipid nanoparticle.

[0091] In another embodiment of the invention, a recombinant non-pathogenic bacterial strain, or a Mycobacterium strain or an M. smegmatis strain for preventative treatment of tuberculosis, wherein the bacterial strain, the Mycobacterium strain or the M. smegmatis strain comprises a DNA fragment of genetic material encoding ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5, or a DNA fragment with at least 70%, at least 80%, at least 90%, at least 95% similar to, or a sequence that has a sequence similarity of any integer in between 70%-100%, to the sequence of genetic material encoding ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5.

[0092] In another embodiment of the invention, a recombinant non-pathogenic bacterial strain, or a Mycobacterium strain or an M. smegmatis strain for preventative treatment of tuberculosis, wherein the bacterial strain, the Mycobacterium strain or the M. smegmatis strain comprises a DNA fragment of M. tb esx-1 locus [SEQ ID NO: 1] , a DNA fragment with M. tb espACD operon [SEQ ID NO: 52] , or a combination of both. In another embodiment, the recombinant non- pathogenic Mycobacterium strain may be a recombinant strain derived from the recombination or fusion of two or more Mycobacterium strains, optionally two or more M. smegmatis strains or a recombination of M. smegmatis with another Mycobacterium strain.

[0093] In an embodiment of the invention a tuberculosis vaccine, an injectable, a composition, a formulation, or a drug, is provide that comprises the recombinant or engineered bacterial strain, the Mycobacterium strain or the M. smegmatis strain, or the non-pathogenic bacterial strains described above.

[0094] In another embodiment of the invention a tuberculosis vaccine, an injectable, a composition, a formulation, or a drug, is provide that comprises the vector, translation cassette, plasmid, cosmid or any expression systems described above.

[0095] In some further embodiments of the invention, methods of engineering or recombinantly converting or constructing an engineered or recombinant bacterial strain. Mycobacterium strain or a M. smegmatis strain is provided. The method comprises electroporating the bacterial cell line, the Mycobacterium cell line or the M. smegmatis cell line with a cosmid, a vector, or a cassette comprising a M. tb chromosome that includes the genetic material for ESX-1 , ESX-2, ESX-3, ESX-4 or ESX-5. The method further comprises selecting the recombinant bacterial strain, the recombinant Mycobacterium strain or the recombinant M.smegmatis strain on a suitable culture medium. Any known growth culture mediums could be employed.

[0096] In some further embodiments of the invention, methods of engineering or recombinantly converting or constructing an engineered or recombinant bacterial strain. Mycobacterium strain or a M. smegmatis strain is provided. The method comprises electroporating the bacterial cell line, the Mycobacterium cell line or the M. smegmatis cell line with a cosmid, a vector, or a cassette comprising a M. tb chromosome that includes M.tb esx- 1 locus [SEQ ID NO: 1] [SEQ ID NO.6] or a DNA fragment thereof. The method further comprises selecting the recombinant bacterial strain, the recombinant Mycobacterium strain or the recombinant M. smegmatis strain comprising the M.tb esx-1 locus [SEQ ID NO: 1] [SEQ ID NO.6] or a DNA fragment thereof on a suitable culture medium. Any known growth culture mediums could be employed.

[0097] In a specific embodiment, the cosmid, vector, or cassette further comprises a Hyg- resistance gene. In a further specific embodiment, a culture medium comprising Hyg may be used to select the engineered or recombinantly converted bacterial, Mycobacterium, M. smegmatis strains described above.

[0098] In some further embodiments of the invention, methods of engineering or recombinantly converting or constructing an engineered or recombinant bacterial strain, Mycobacterium strain or a M. smegmatis strain is provided. The method comprises transforming the bacterial cell line, the Mycobacterium cell line or the M. smegmatis cell line with a cosmid, a vector, or a cassette comprising M. tb espACD operon [SEQ ID NO: 52] . The method further comprises selecting the recombinant bacterial strain, the recombinant Mycobacterium strain or the recombinant M. smegmatis strain comprising the M. tb espACD operon [SEQ ID NO: 52] on a suitable culture medium. Any known growth culture mediums could be employed.

[0099] In a specific embodiment, the cosmid, vector, or cassette further comprises a Hyg- resistance and a Kan-resistance gene. In a further specific embodiment, a culture medium comprising Hyg and / or Kan may be used to select the engineered or recombinantly converted bacterial, Mycobacterium, M. smegmatis strains described above.

[0100] In another embodiment, a method of vaccinating a subject against tuberculosis is provided. The method involves administering a dose of the recombinant bacterial strain, or the recombinant Mycobacterium strain or the recombinant M. smegmatis strain as described hereinbefore.

[0101] In another embodiment, a method of vaccinating a subject against tuberculosis is provided. The method involves administering a dose of the vector, translation cassette, plasmid, cosmid, or expression systems as described hereinbefore.

[0102] In another embodiment, a method of vaccinating a subject against tuberculosis is provided. The method involves administering a dose of the recombinant non-pathogenic bacterial strain, or the recombinant non-pathogenic Mycobacterium strain or the recombinant non-pathogenic M. smegmatis strain as described hereinbefore.

[0103] The method of vaccination as described above does not sensitize the vaccinated subject to M. tb antigens or purified protein derivative or M. bovis. Since, the vaccinated subject does not show sensitivity to antigens of M. tb or protein purified derivative of M. bovis, it prevents false positive outcome to a standard tuberculosis skin test or diagnostic tests e.g. CCT.

[0104] In another embodiment, a method of preventatively treating a subject against tuberculosis is provided. The method involves administering a dose of the recombinant bacterial strain, or the recombinant Mycobacterium strain or the recombinant M. smegmatis strain as described hereinbefore.

[0105] In another embodiment, a method of preventatively treating a subject against tuberculosis is provided. The method involves administering a dose of the vector, translation cassette, plasmid, cosmid, or expression systems as described hereinbefore.

[0106] In another embodiment, a method of preventatively treating a subject against tuberculosis is provided. The method involves administering a dose of the recombinant non- pathogenic bacterial strain, or the recombinant non-pathogenic Mycobacterium strain or the recombinant non-pathogenic M. smegmatis strain as described hereinbefore.

[0107] In another embodiment, various compositions are provided. The composition may comprise any of the recombinant bacterial strain, the recombinant Mycobacterium strain, the recombinant M. smegmatis strain, the recombinant non-pathogenic bacterial strain, the recombinant non-pathogenic Mycobacterium strain, the recombinant non-pathogenic M. smegmatis strain, the vector, the plasmid, the cosmid, the translation cassette or the various expression systems described herein before. The composition additionally comprises any known pharmaceutically acceptable excipient or carrier.

[0108] The composition may be in the form of a capsule, an injectable, a topical cream or a powder. In some embodiments, the composition may be in the form of an injectable.

[0109] In other embodiments, the composition may comprise the other recombinant strains, vectors, expression systems, and cell based formulations either alone or in combination, optionally comprising one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0110] In another embodiment, various kits are provided. The kit may comprise any of the recombinant bacterial strain, the recombinant Mycobacterium strain, the recombinant M. smegmatis strain, the recombinant non-pathogenic bacterial strain, the recombinant non- pathogenic Mycobacterium strain, the recombinant non-pathogenic M. smegmatis strain, the vector, the plasmid, the cosmid, the translation cassette or the various expression systems described herein before. The kit additionally comprises a syringe and / or instructions for using the any of the strains or expression systems listed above, or any pharmaceutically acceptable carriers, excipients, or diluents, or one or more buffers, wash or cell culture media, or one or more vessels for containing any of the above, or instructions for using any component of the kit, or any combination of the above components.

[0111] The kit may additionally comprise a container means which may include at least one vial, test tube, flask, bottle, syringe or other container means, into which the strains, expression systems, compositions and / or or other materials may be placed.

[0112] In some embodiments described above, the disclosure provides theory and speculation on the mechanism of biological processes. The present invention is not meant to be bound by theory or speculation as to the mechanisms involved in biological processes and the same should not be used to limit the invention in any way.

[0113] Experimental Discussion

[0114] Construction of recombinant M. smegmatis strains

[0115] Recombinant M. smegmatis strains were obtained by transformation of DNA constructs following standard electroporation procedures. Briefly, electrocompetent M. smegmatis mc2-155 cells were prepared and first electroporated with an integrative cosmid called 2F9 that harbours a DNA fragment consisting of 24 open reading frames (rv3861 to rv3885, as defined in SEQ ID Nos: 2 to ) of the M. tb chromosome which includes the esx-1 locus [SEQ ID NO: 1] to generate M. smeg matis ::2F9. M. smegmatis mc2-155 was also electroporated with the empty, integrative and Hyg-resistance cassette-containing cosmid vector pYUB41222 on which 2F9 is based to generate M. smegmatis::pYUB412. Both were selected for on 7H11 complete agar containing Hyg. Thereafter, M. smegmatis .2 9 and M. smegmatis::pYUB412 were transformed with an episomal Kan-resistance cassette-containing plasmid called pMDespACD which contains the M. tb espACD operon [SEQ ID NO: 52] and selected for on 7H11 complete agar containing Hyg and Kan. M. smegmatis .2 9 and M. smegmatis::pYUB412 were also transformed with the empty pMD31 plasmid on which pMDespACD is based, and selected for on 7H11 complete agar containing Hyg and Kan. In total, four different strains of recombinant M. smegmatis were created - 1) M. smegmatis::pYUB412+pMD31 (wild-type with empty vectors), 2) M. smegmatis::pYUB412+pMDespACD (with only the espACD operon [SEQ ID NO: 52]), 3) M. smegmatis::2F9+pMD31 (with only the esx-1 locus [SEQ ID NO: 1]) and 4) M. smegmatis::2F9+pMDespACD (with genes of the entire ESX-1 system) hereafter referred to as MSX-1.

[0116] M. smegmatis culture conditions and protein preparation for immunoblots

[0117] Recombinant M. smegmatis strains were grown in 7H9 complete with Kan and Hyg to mid-logarithmic phase of growth, centrifuged, cell pellet washed with PBS, and resuspended in modified 7H9 without ADC or Tween-80 at a starting ODeoonm of 0.2 and cultured at 37°C with agitation. Aliquots of cell cultures were then centrifuged at the indicated times to obtain culture supernatants and bacterial pellets. Culture supernatants were then concentrated in Vivaspin columns (Sartorius, Canada) with 5-kDa molecular weight cutoff membranes to obtain culture filtrates (CF). Cell lysates (CL) were prepared by resuspendingbacterial pellets in PBS containing EDTA-free protease inhibitor cocktail tablets (Roche, Canada) and bead-beating using 100 pm glass beads and clarified by centrifugation. Total protein concentrations in the CF and CL of the different mycobacterial strains were determined using the BCA assay (ThermoFisher, Canada) with bovine serum albumin as the standard.

[0118] Immunoblotting

[0119] Immunoblotting was conducted using 5 pg / well of CF proteins and 2.5 pg / well of CL proteins. These proteins were separated in NuPAGE 4-12% Bis-Tris gels (ThermoFisher, Canada) and transferred to nitrocellulose membranes using the iBlot2 system (ThermoFisher, Canada). Membranes were blocked with TBS-Milk (20 mM Tris-HCI, pH 7.5, 150 mM NaCI and 5% non-fat milk powder) and incubated overnight with primary antibody diluted in TNT-BSA (20 mM Tris-HCI, pH 7.5, 150 mM NaCI, 0.05% Tween-20 and 1 % BSA fraction V) at 4°C. Membranes were washed with TNT, incubated with the appropriate fluorescent secondary antibody in TNT-BSA, washed three times with TNT and scanned using the Odyssey CLx Imaging system (Li-Cor Biosciences, Canada). GroEL2 was used as a lysis control for CF and as a loading control for CL. ESX-1-independent secreted protein Ag85 was probed as a loading control for CF. Commercially produced rat polyclonal antibodies specific to M. tb EspB and EspD were used at dilutions of 1 :1000. Commercially obtained mouse monoclonal antibodies specific to M. tb EsxA and EsxB, mouse monoclonal antibody specific to mycobacterial GroEL2 (BEI Resources), and rabbit polyclonal antibody specific to mycobacterial Ag85 (BEI Resources) were used at dilutions of 1 :2000.

[0120] In vitro growth measurement of recombinant M. smegmatis strains

[0121] Each of the recombinant M. smegmatis strains were inoculated into flasks containing 7H9 complete media with Kan and Hyg at a starting ODeoonm of 0.2 and grown at 37°C with agitation. Aliguots were removed for ODeoonm measurements at the different indicated time points. At least two independent growth experiments were conducted.

[0122] TH P-1 cytotoxicity assays

[0123] Human THP-1 monocytic cells (TIB-202 - ATCC) were cultured, differentiated using phorbol ester (PMA) into macrophage-like cells, and seeded in 96-well tissue culture plates. Recombinant M. smegmatis strains for THP-1 infections were cultured in 7H9 completewith Kan and Hyg. After measuring ODeoonm and calculating the colony forming units per mL (CFU / rnL), aliquots of the bacteria were added to RPMI media supplemented with 10% fetal bovine serum to obtain the required CFU / rnL and then used to infect THP-1 cells in 96-well plates at a multiplicity of infection (MOI) of 1. Survival of THP-1 cells 12 hours post-infection was determined using the PrestoBlue reagent (ThermoFisher, Canada).

[0124] Protective efficacy trial in the mouse model of TB

[0125] For the mouse trial, six-week-old female C57BL / 6 mice were randomly assigned to 5 groups of 8 mice each and mice in each group were vaccinated subcutaneously once with 1x106CFU / animal of either M. smegmaf / s::pYUB412+pMD31 , M. smegmaf / s::2F9+pMD31 , MSX-1, M. bovis BCG Pasteur 1173P2 or saline. Three weeks postvaccination, two animals per vaccine group were sacrificed and spleens were collected for splenocyte assays to measure M. bovis antigen-specific T-cell mediated IFNy production (see procedure below). The remaining mice were then challenged via the intranasal route with 1x103CFU / animal of virulent M. tb Erdman, monitored daily and euthanized after 28 days. Spleens and lungs from all animals were collected, homogenized and plated out on 7H11 complete agar to enumerate M. tb burden in these organs (4 mice per vaccine group) and for histopathological analysis (2 mice per vaccine group).

[0126] Splenocyte assay

[0127] For the splenocyte assay, spleens collected from the different groups of vaccinated mice were macerated and the splenocytes isolated were cultured in 96-well plates (1 x 106cells / well). Duplicate wells of splenocytes from each mouse per vaccination group were incubated with either RPMI tissue culture media, 5pg / ml PPD (bovine tuberculin PPD 3000 - Phonics Lelystad B.V.), 5pg / ml CF of BCG (prepared in-house), and 1 pg / ml Concavalin A or ConA (Sigma-Aldrich, Canada) for 72 hours at 37°C and 5% CO2. The interferon-gamma (IFNy) produced and released by the splenocytes into the culture supernatant was quantified using the Invitrogen IFNy mouse ELISA kit (ThermoFisher, Canada).

[0128] Histopathology

[0129] Lung tissue samples from the different groups of vaccinated mice were held in 10% normal buffered formalin for about 1 week, embedded in paraffin, and sections were stained with hematoxylin and eosin (H&E) to assess inflammation.

[0130] Example 1 - Expression of M. tb ESX-1 system in M. smegmatis

[0131] In an embodiment of the present invention, the M. tb ESX-1 system was reconstituted in M. smegmatis transformed with the esx-1 locus [SEQ ID NO: 1] alone or both the esx-1 locus [SEQ ID NO: 1] and espACD operon [SEQ ID NO: 52]. Immunoblot analysis of culture filtrate (CF) and cell lysate (CL) proteins of the different recombinant M. smegmatis strains grown for 10, 20 and 30 hours was performed. M. tb EsxA and EsxB were not detected in the CF or CL of either M. smegmaf / s::pYUB412+pMD31 or M. smegmaf / s: :pYUB412+ pM DespA CD (Fig. 1A and B). However, M. tb EsxA and EsxB were detected in the CF and CL of both M. smegmaf / s::2F9+pMD31 and MSX-1 ; the signals for EsxA and EsxB were stronger in the latter strain (Fig. 1A and B). This suggests that the esx-1 locus [SEQ ID NO: 1] alone was capable of expressing EsxA and EsxB, as was the combination of both the esx-1 locus [SEQ ID NO: 1] (in 2F9) and the espACD operon [SEQ ID NO: 52] (in pMDespACD). The latter combination may be important for a more robust expression and export of M. tb EsxA and EsxB by M. smegmatis. Likewise, EspB was only detected in the CF and CL fractions of M. smegmaf / s::2F9+pMD31 and MSX-1 ; the signal for EspB was stronger in the latter strain (Fig. 1A and B). This indicates that the esx-1 locus [SEQ ID NO: 1] alone and the combination of the esx-1 locus [SEQ ID NO: 1] and the espACD operon [SEQ ID NO: 52] are able to express and export EspB from M. smegmatis.

[0132] Similarly, M. tb EspD was detected in the CL of M. smegmatis: :pYUB412+ pM DespA CD, with the amount of EspD in the CL of MSX-1 being higher (Fig. 1B). Additionally, secreted EspD was detected in the CF of MSX-1 after 20 and 30 hours of in vitro culture (Fig. 1A). This suggests that the esx-1 locus [SEQ ID NO: 1] alone or in combination with the espACD operon [SEQ ID NO: 52] can lead to the expression of M. tb EspD from M. smegmatis.

[0133] The differences in EspB, EspD, EsxA and EsxB levels in the CF noted above were likely not due to unequal total CF protein loading since the signal for Ag85, an ESX-1 independent secreted protein, was similar in the CF of all four strains at corresponding timepoints (Fig. 1A). Likewise the differences in levels of EspB, EspD, EsxA and EsxB in the CL noted above were likely not due to unequal loading of total CL proteins since the signal for GroEL2, a cell-associated chaperonin, and Ag85 were similar in the CL of all four strains at corresponding time points (Fig. 1 B). The overall absence of GroEL2 in the CF of all four strains also indicates that the noted differences in EsxA, EsxB, EspB and EspD levels were not due to cell lysis (Fig. 1A).

[0134] Example 2 - T ransformation of M. smegmatis with the different genetic elements encoding M. tb ESX-1 does not affect its growth in liquid culture media

[0135] To rule out differential growth rates as possible drivers of the variable ESX-1 protein expression and secretion noted above, the growth of M. smegmatis::pYUB412+pMD31 (shown in blus), M. smegmatis::pYUB412+pMDespACD (shown in green), M. smegmatis::2F9+pMD31 (shown in orange) and MSX-1 (shown in red) in 7H9 complete with Kan and Hyg was compared (Fig. 2). All four strains grew similarly and demonstrated similar lag, logarithmic and stationary phases of growth. This result clearly indicates that transformation of M. smegmatis with genes encoding the M. tb ESX-1 system does not impact their growth rates.

[0136] Example 3 - T ransformation of M. smegmatis with the different genetic elements that make up ESX-1 does not make it virulent

[0137] Given that the ESX-1 system is a critical virulence factor for M. tb and the secretion of EsxA, EsxB, EspA, EspB and EspC enables the TB bacillus to cause cell death in infected macrophages, the reconstitution of this system in MSX-1 was investigated to determine whether the genetic elements of the ESX-1 system might render it more cytopathic to macrophages. Accordingly, human THP-1 cells were infected with either M. smegmatis::pYUB412+pMD31 , M. smegmatis::pYUB412+pMDespACD, M. smegmatis::2F9+pMD31 or MSX-1 and their viability assessed. Compared to uninfected THP- 1 cells, none infected with recombinant M. smegmatis strains exhibited reduced viability (Fig. 3). This indicates that reconstitution of M. tb ESX-1 in M. smegmatis does not make it virulent.

[0138] Example 4 - M. smegmatis::2F9+pMDespACD (MSX-1) performs as well as the live attenuated M. bovis BCG vaccine in protecting mice against M. tb

[0139] Since EsxA, EsxB, EspA and EspC are potent T-cell antigens and the recombinant M. smegmatis strains were non-virulent, they were used in an attempt to protect against M. tb in the mouse model. This was then compared to the protection afforded by M. bovis BCG, the only licensed TB vaccine to date. To this end, groups of mice were vaccinated with either M. smegmaf / s::pYUB412+pMD31 , M. smegmaf / s::2F9+pMD31 , MSX-1 or BCG, subsequently challenged with virulent M. tb, and a month later, the burden of M. tb in the lungs and spleens of the mice was quantified. The burdens of M. tb in the lungs of mice vaccinated with MSX-1 or BCG were lower than the burdens in the lungs of mock-vaccinated mice (Fig. 4A). M. tb burdens in the spleens of mice vaccinated with either MSX-1 or BCG were qualitatively lower than the burdens in the spleens of mock-vaccinated mice (Fig. 4B).

[0140] Histopathological examination revealed multifocal neutrophilic infiltrations and thickening of the alveolar septa in the lungs of mice vaccinated with M. smegmaf / s::pYUB+pMD31 consistent with TB-mediated pathology (Fig. 5A). In mice given MSX-1 or BCG, the lungs presented with normal architecture and limited inflammation (Figs. 5B and C). In contrast, lungs of mock-vaccinated mice presented with necrotic regions surrounded by massive infiltration of neutrophils as well as significant loss of lung architecture consistent with severe TB-mediated pathology (Fig. 5D).

[0141] Collectively, these results indicate MSX-1 is protective against M. tb infection, and in some embodiments is as protective as BCG.

[0142] Example 5 - M. :2F9+pMDespACD (MSX-1) and host sensitizationto M. tb / M. bovis antigens in PPP

[0143] BCG vaccination causes sensitization to M. fb-specific antigens in the vaccinated host. This is manifested by the production of IFNy by a vaccinee’s primed T-cells upon stimulation with purified protein derivative (PPD), a complex mixture of secreted proteins that can be of M. tb or M. bovis origin. To determine if this sensitization occurred following vaccination with the recombinant M. smegmatis strains, splenocytes, which were expected to be enriched with primed T-cells were isolated from the spleens of mock-vaccinated mice as well as from mice vaccinated with M. smegmaf / s::pYUB412+pMD31 , M. smegmaf / s::2F9+pMD31 , MSX-1 or BCG. These splenocytes were stimulated with either RPMI media alone (negative control), PPD, BCG CF or ConA, and the amount of IFNy inducedquantified. RPMI media alone did not stimulate splenocytes from any of the mice while ConA, a non-specific stimulator of T-cells, stimulated high but equivalent levels of IFNy production by splenocytes from all mice (Fig. 6). Strikingly, PPD and BCG CF stimulated higher IFNy production by splenocytes from mice immunized with BCG compared to splenocytes from mice vaccinated with recombinant M. smegmatis strains.

[0144] These results indicate that unlike vaccination with BCG, vaccination with wildtype M. smegmatis (M. smegmaf / s::pYUB412+pMD31) and recombinant M. smegmatis strains, such as MSX-1 , do not sensitize the vaccinated host to PPD.

[0145] The inability of MSX-1 to sensitize a vaccinated host to M. tb and / or M. bovis- specific antigens, despite being protective (and in some embodiments, as protective as BCG) is notable and has significant implications for its use in the control of bovine TB (bTB). As bTB is a reportable zoonotic disease, MSX-1 could have an impact on human and wildlife health as well. Additionally, having a viable alternative to BCG may have substantial economic and resource management benefits.

[0146] In some embodiments of the present invention, it is envisioned that the surrogate M. smegmatis recombinant strain expressing a functional M. tb ESX-1 system could be used to study M. tb without the need for biocontainment level 3 facilities. This would substantially drop costs for conducting such research and greatly expand the number of researchers and companies that could work on tuberculosis.

[0147] Example 6 - MSX-1 as a vaccine candidate for bovine tuberculosis

[0148] Objectives: Bovine tuberculosis (bTB) is a contagious and reportable bacterial infectious disease that affects livestock and wildlife globally. Experiments were done to show that a new candidate vaccine called MSX-1 can protect against infection by Mycobacterium bovis, the causative agent of bTB. We also sought confirmation that MSX-1 will not sensitize animals to tuberculin, a mixture of M. bovis protein antigens, which is used in the diagnosis of bTB in cattle

[0149] Background: Although its livestock is officially designated bTB-free, outbreaks of bTB in Canada does occur. Indeed, outbreaks of bTB in Canada were reported in 2004, 2008, 2011 , 2016, 2018, 2023 and most recently in December of 2024. Testing andculling of affected animals are presently the only way bTB in cattle is dealt with in North America, including Canada. This strategy costs the Canadian federal government hundreds of millions of dollars to compensate producers for culled animals, months of lost productivity for cattle producers themselves and as such is unsustainable in the long-term. Importantly, these outbreaks which appear to be increasing in frequency, may well jeopardize the bTB- free status of Canadian livestock and block access of Canadian beef and dairy products to global markets.

[0150] A live attenuated vaccine called BCG which was discovered 100 years ago from M. bovis is used to prevent TB in people which is primarily caused by a close relative of M. bovis called M. tuberculosis. Its use in livestock however is prohibited in Canada. This is because the Canadian Food Inspection Agency’s approved diagnostic tests which include the comparative cervical tuberculin (CCT) skin test and in vitro blood-based Bovigam assay for bTB both utilize tuberculin. When tuberculin is injected into the skin of animals suspected of having bTB, only animals with bTB but not healthy animals are sensitized to tuberculin and as such give a positive CCT skin test result marked by increased production of a cytokine called interferon-gamma (I FNy) by the affected animal’s immune cells and inflammatory swelling at the tuberculin injection site. Likewise, when blood samples from suspect animals are mixed and incubated with tuberculin in vitro, only animals with bTB sensitized to tuberculin give a positive Bovigam test result which is also marked by increased I FNy production. Since BCG is derived from M. bovis and their protein antigens especially those that make up tuberculin are virtually identical, animals given this vaccine also become sensitized to tuberculin. As such, neither the CCT skin test nor Bovigam test can distinguish animals given BCG from those infected with M. bovis. Therefore, an alternative vaccine that can protect cattle from bTB but not sensitize them to tuberculin would be highly desirable.

[0151] Mycobacterium smegmatis is a non-pathogenic, fast-growing and genetically tractable mycobacterium that is used widely in TB research. We modified M. smegmatis by introducing genes that encode ESX-1 , a type-7 protein secretion system that is common to both M. tuberculosis and M. bovis. This recombinant M. smegmatis strain that we designated MSX-1 was shown to make and export key proteins of the ESX-1 system such as EsxA, EsxB, EspA and EspC that are known to be immunogenic T-cell antigens that drive IFNy production, an important way to provide immune protection against TB. Moreover, MSX-1 was found to be harmless in a macrophage model of infection and in mice. Notably, micevaccinated with MSX-1 was protected against infection by M. tuberculosis as well as mice vaccinated with BCG. Unlike BCG however, vaccination with MSX-1 did not sensitize mice to M. tuberculosis tuberculin which is identical to M. bovis tuberculin. With such features, we therefore hypothesized MSX-1 could be an ideal bTB DIVA (differentiate infected from vaccinated animals) vaccine.

[0152] Rationale, approach and methodology: It is well known that M. tuberculosis is human-adapted and as such is not as virulent or lethal as M. bovis in non-human animal models including mice and cattle. Therefore, to prove that MSX-1 is a good bTB vaccine, it is necessary to vaccinate an animal model with MSX-1 and then challenge it with the more virulent (in animals) M. bovis to assess its efficacy. Accordingly, we conducted two trials in mice to obtain proof-of-concept for MSX-1 as a viable bTB vaccine candidate before proceeding with far more costly MSX-1 vaccine trials in cattle.

[0153] In trial 1 , six groups of twenty lab mice (C57BL / 6) each were vaccinated once by subcutaneous injection with 1) saline (mock vaccine) or 106colony forming units (CFU) of 2) BCG::pYUB (wild-type BCG), 3) BCG::ESX-1 (a modified BCG that makes M. bovis ESX- 1), 4) M. smegmaf / s::pYUB+pMD31 (wild-type M. smegmatis and parental strain of MSX-1), 5) MSX-1 or 6) MSX-1 V2, a version of MSX-1 with minor genetic modifications that we have found produces and exports substantially lower amounts of immunogenic T-cell antigens EsxA, EsxB, EspA and EspC. To verify that MSX-1 exhibits DIVA potential, 4 mice from each vaccine treatment group were randomly selected and euthanized at 13-, 26- and 47-days post-vaccination. Splenocytes enriched with T-cells isolated from the spleens of these mice were incubated with 1) tissue culture media, 2) ConA (non-specific inducer of IFNy production by T-cells), 3) EsxA peptide or 4) tuberculin. The production of IFNy by spleen T- cells was measured by enzyme linked immunosorbent assay (ELISA). Weight loss upon infection with M. bovis and progression of active bTB is a hallmark of the disease. To determine if MSX-1 like BCG protects against bTB, at 47-days post-vacci nation the rest of the vaccinated mice (8 / group) were weighed and then challenged via the intranasal route with a lethal dose of 102CFU of virulent M. bovis per mouse and monitored for progression to humane intervention point (HIP) where the mice were euthanized once they started exhibiting well-defined clinical signs of terminal disease. All mice were weighed after being euthanized. In trial 2, six groups of eight mice (C57BL / 6) each were vaccinated once by subcutaneous injection with 1) saline or 106CFU of 2) BCG::pYUB, 3) BCG::ESX-1 , 4) M.smegmaf / s::pYUB+pMD31, 5) MSX-1 or 6) MSX-1 V2 as in trial 1. 45-days post-vaccination, all of the mice were challenged via the intranasal route with 10 CFU of virulent M. bovis per mouse. 30-days later, all mice were euthanized simultaneously, necropsied and the lungs collected. The lungs were homogenized and plated on agar to assess M. bovis burden.

[0154] Deliverables / outputs expected: The results of trial 1 is expected to confirm whether or not MSX-1 vaccination sensitizes the vaccinee to tuberculin and if the kinetics of sensitization is in MSX-1 vaccinated hosts is different from BCG vaccinated host. If MSX-1 has true DIVA potential, tuberculin will only trigger increased production of IFNy in mice vaccinated with BCG::pYUB and BCG::ESX-1 but not in mice vaccinated with MSX-1, M. smegmaf / s::pYUB+pMD31 or saline, regardless of the time since vaccination. We also expect trial 1 results to inform us if MSX-1 can protect against M. bovis infection-mediated lethality. We anticipate that after infection with M. bovis, mice vaccinated with MSX-1 , BCG::pYUB and BCG::ESX-1 will survive longer and show either actual weight gain or at least no weight loss than mice administered saline or M. smegmaf / s::pYUB+pMD31. The results of trial 2 is expected to inform us whether or not vaccination with MSX-1 can prime the immune system of a vaccinated host and control M. bovis growth in the lung and potentially mitigate bTB-driven tissue damage. We anticipate mice vaccinated with MSX-1, BCG::pYUB and BCG::ESX-1 will show reduced M. bovis burden in organs like the lungs and presumably present with less pathology compared to the lungs of mice administered saline or M. smegmaf / s::pYUB+pMD31.

[0155] Results

[0156] Sensitization to tuberculin and other antigens in vaccinated mice: While splenocytes from mice vaccinated with BCG::pYUB and BCG::ESX-1 became sensitized to tuberculin, only splenocytes from mice given BCG::ESX-1 which makes and exports EsxA became sensitized to EsxA peptide (Figure 7A and B). Regardless of the length of time postvaccination, neither M. smegmaf / s::pYUB+pMD31, MSX-1 nor MSX-1 V2 vaccinated mice became sensitized to tuberculin (Figure 7A and B). Surprisingly, neither MSX-1 nor MSX-1 V2 vaccinated mice became sensitized to EsxA (Figure 7A and B), even though these two strains have been shown to make and export EsxA. As expected splenocytes from all mice were equally induced to IFNy production by ConA (Figure 7A and B). Conversely, tissue culture media had no effect on splenocytes from all mice (Figure 7A and B).

[0157] Survival of vaccinated mice after challenge with lethal dose of M. bovis-.Mice vaccinated with BCG::pYUB and BCG::ESX-1 exhibited 100% survival while mice vaccinated with MSX-1 exhibited -82% survival upon infectious challenge with M. bovis (Figure 8). Mice vaccinated with saline, M. smegmat / s::pYUB+pMD31 and MSX-1 V2 exhibited -50%, 60% and 39% survival respectively.

[0158] Changes in body weight of vaccinated mice between time of infectious challenge and at humane intervention point (HIP): Only mice administered BCG::pYUB, BCG::ESX-1 and MSX-1 exhibited increases in median body weights from time of challenge with virulent M. bovis to time of HIP (Figure 9). In contrast, mice given saline, M. smegmaf / s::pYUB+pMD31 and MSX-1 V2 exhibited decreases in median body weights over the same timeframe (Figure 9).

[0159] M. bovis burden 30-days post-challenge in the lungs of vaccinated mice: Compared to mice administered saline, the median burden of M. bovis in the lungs was significantly lower only in mice administered BCG::pYUB, BCG::ESX-1 and MSX-1 (Figure 10). In contrast, the median burden of M. bovis was not significantly different in mice given M. smegmaf / s::pYUB+pMD31 and MSX-1 V2 (Figure 10).

[0160] Conclusion: The results of both trials 1 and 2 indicates that MSX-1 does protect the mouse model against infectious challenge with highly virulent M. bovis, the agent of bTB just as it protects against M. tuberculosis, the human-adapted agent of TB in people. As such, it is highly likely MSX-1 will also protect cattle against M. bovis without compromising the ability to diagnose the disease using the conventional tuberculin skin test.

[0161] The present invention has been described with regard to one or more embodiments, however, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined by the claims.

[0162] All citations and / or references recited herein are hereby incorporated by reference in their entirety.

[0163] References

[0164] Famelis, N.; Geibel, S.; van Tol, D., Mycobacterial type VII secretion systems. Biological chemistry 2023, 404 (7), 691-702. DOI: 10.1515 / hsz-2022-0350.

[0165] Bunduc, C. M.; Bitter, W.; Houben, E. N. G., Structure and Function of the Mycobacterial Type VII Secretion Systems. Annual review of microbiology 2020, 74, 315-335. DOI : 10.1146 / annurev-micro-012420-081657.

[0166] Drever, K.; Lim, Z. L.; Zriba, S.; Chen, J. M., Protein Synthesis and Degradation Inhibitors Potently Block Mycobacterium tuberculosis type-7 Secretion System ESX-1 Activity. ACS Infect Dis 2021, 7 (2), 273-280. DOI: 10.1021 / acsinfecdis.0c00741.

[0167] Brodin, P.; Majlessi, L.; Marsollier, L.; de Jonge, M. I.; Bottai, D.; Demangel, C.; Hinds, J.; Neyrolles, O.; Butcher, P. D.; Leclerc, C.; Cole, S. T.; Brosch, R., Dissection of ESAT- 6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence. Infection and immunity 2006, 74 (1), 88-98. DOI: 10.1128 / IAI.74.1.88-98.2006.

[0168] Chen, J. M.; Boy-Rottger, S.; Dhar, N.; Sweeney, N.; Buxton, R. S.; Pojer, F.; Rosenkrands, I.; Cole, S. T., EspD is critical for the virulence-mediating ESX-1 secretion system in Mycobacterium tuberculosis. Journal of bacteriology 2012, 194 (4), 884-93. DOI: 10.1128 / JB.06417-11.

[0169] Chen, J. M.; Zhang, M.; Rybniker, J.; Basterra, L.; Dhar, N.; Tischler, A. D.; Pojer, F.; Cole, S. T., Phenotypic profiling of Mycobacterium tuberculosis EspA point mutants reveals that blockage of ESAT-6 and CFP-10 secretion in vitro does not always correlate with attenuation of virulence. Journal of bacteriology 2013, 195 (24), 5421-30. DOI: 10.1128 / JB.00967-13.

[0170] Chen, J. M.; Zhang, M.; Rybniker, J.; Boy-Rottger, S.; Dhar, N.; Pojer, F.; Cole, S. T., Mycobacterium tuberculosis EspB binds phospholipids and mediates EsxA-independent virulence. Molecular microbiology 2013, 89 (6), 1154-66. DOI: 10.1111 / mmi.12336.

[0171] Fortune, S. M.; Jaeger, A.; Sarracino, D. A.; Chase, M. R.; Sassetti, C. M.; Sherman, D. R.; Bloom, B. R.; Rubin, E. J., Mutually dependent secretion of proteins required for mycobacterial virulence. Proceedings of the National Academy of Sciences of the United States of America 2005, 702 (30), 10676-81. DOI: 10.1073 / pnas.0504922102.

[0172] Lim, Z. L.; Drever, K.; Dhar, N.; Cole, S. T.; Chen, J. M., Mycobacterium tuberculosis EspK Has Active but Distinct Roles in the Secretion of EsxA and EspB. Journal of bacteriology 2022, 204 (4), e0006022. DOI: 10.1128 / jb.00060-22.

[0173] Lou, Y.; Rybniker, J.; Sala, C.; Cole, S. T., EspC forms a filamentous structure in the cell envelope of Mycobacterium tuberculosis and impacts ESX-1 secretion. Molecular microbiology 2017, 103 (1), 26-38. DOI: 10.1111 / mmi.13575.

[0174] Pym, A. S.; Brodin, P.; Brosch, R.; Huerre, M.; Cole, S. T., Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Molecular microbiology 2002, 46 (3), 709-17. DOI: 10.1046 / j.1365- 2958.2002.03237.x.

[0175] Zhang, M.; Chen, J. M.; Sala, C.; Rybniker, J.; Dhar, N.; Cole, S. T., Espl regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis. Molecular microbiology 2014, 93 (5), 1057-65. DOI: 10.1111 / mmi.12718.

[0176] Jia, Q.; Maslesa-Galic, S.; Nava, S.; Horwitz, M. A., Listeria-vectored multi- antigenic tuberculosis vaccine protects C57BL / 6 and BALB / c mice and guinea pigs against Mycobacterium tuberculosis challenge. Commun Biol 2022, 5 (1), 1388. DOI: 10.1038 / s42003- 022-04345-1.

[0177] Millington, K. A.; Fortune, S. M.; Low, J.; Garces, A.; Hingley-Wilson, S. M.; Wickremasinghe, M.; Kon, O. M.; Lalvani, A., Rv3615c is a highly immunodominant RD1 (Region of Difference Independent secreted antigen specific for Mycobacterium tuberculosis infection. Proceedings of the National Academy of Sciences of the United States of America 2011, 708 (14), 5730-5. DOI: 10.1073 / pnas.1015153108.

[0178] Pym, A. S.; Brodin, P.; Majlessi, L.; Brosch, R.; Demangel, C.; Williams, A.; Griffiths, K. E.; Marchal, G.; Leclerc, C.; Cole, S. T., Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nature medicine 2003, 9 (5), 533-9. DOI: 10.1038 / nm859.

[0179] Snapper, S. B.; Melton, R. E.; Mustafa, S.; Kieser, T.; Jacobs, W. R., Jr., Isolation and characterization of efficient plasmid transformation mutants of Mycobacteriumsmegmatis. Molecular microbiology 1990, 4 (11), 1911-9. DOI: 10.1111 / j.1365- 2958.1990. tb02040.x.

[0180] Converse, S. E.; Cox, J. S., A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis. Journal of bacteriology 2005, 787 (4), 1238-45. DOI: 10.1128 / jb.187.4.1238- 1245.2005.

[0181] Derbyshire, K. M.; Gray, T. A., Distributive Conjugal Transfer: New Insights into Horizontal Gene Transfer and Genetic Exchange in Mycobacteria. Microbiol Spectr 2014, 2 (1), Mgm2-0022-2013. DOI: 10.1128 / microbiolspec.MGM2-0022-2013.

[0182] Brosch, R.; Gordon, S. V.; Garnier, T.; Eiglmeier, K.; Frigui, W.; Valenti, P.; Dos Santos, S.; Duthoy, S.; Lacroix, C.; Garcia-Pelayo, C.; Inwald, J. K.; Golby, P.; Garcia, J. N.; Hewinson, R. G.; Behr, M. A.; Quail, M. A.; Churcher, C.; Barrell, B. G.; Parkhill, J.; Cole, S. T., Genome plasticity of BCG and impact on vaccine efficacy. Proceedings of the National Academy of Sciences of the United States of America 2007, 104 (13), 5596-601. DOI: 10.1073 / pnas.0700869104.

[0183] Goude, R.; Parish, T., Electroporation of mycobacteria. Methods in molecular biology 2009, 465, 203-15. DOI: 10.1007 / 978- 1-59745-207-6_13.

[0184] Wards, B. J.; Collins, D. M., Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria. FEMS microbiology letters 1996, 745 (1), 101-5. DOI: 10.1111 / j.1574-6968.1996.tb08563.x.

[0185] Bange, F. C.; Collins, F. M.; Jacobs, W. R., Survival of mice infected with Mycobacterium smegmatis containing large DNA fragments from Mycobacterium tuberculosis. Tubercle and Lung Disease 1999, 79 (3), 171-180. DOI: https: / / doi.Org / 10.1054 / tuld.1998.0201.

[0186] Garnier, T.; Eiglmeier, K.; Camus, J. C.; Medina, N.; Mansoor, H.; Pryor, M.; Duthoy, S.; Grondin, S.; Lacroix, C.; Monsempe, C.; Simon, S.; Harris, B.; Atkin, R.; Doggett, J.; Mayes, R.; Keating, L.; Wheeler, P. R.; Parkhill, J.; Barrell, B. G.; Cole, S. T.; Gordon, S. V.; Hewinson, R. G., The complete genome sequence of Mycobacterium bovis. Proceedingsof the National Academy of Sciences of the United States of America 2003, 100 (13), 7877- 82. DOI: 10.1073 / pnas.1130426100.

[0187] WorldOrganizationofAnimalHealth Controlling bovine tuberculosis: a One Health challenge. https: / / bulletin.woah. orq / ?panorama=3-05-tb-costs- en&edition=7591&pdf=panorama&article=8480.

[0188] CanadianFoodlnspectionAgency Bovine tuberculosis. https: / / inspection.canada.ca / animal-health / terrestrial-animals / diseases / reportable / bovine- tuberculosis / eng / 1330205978967 / 1330206128556.

[0189] CanadianCattlemen On the trail of bovine tuberculosis (bTB). https: / / www.canadiancattlemen.ca / features / on-the-trail-of-btb / .

[0190] SaskToday Bovine TB case in Sask, could have painful consequences. https: / / www.sasktoday.ca / hiqhliqhts / bovine-tb-case-in-sask-could-have-painful- consequences-7242648.

[0191] Balseiro, A.; Thomas, J.; Gortazar, C.; Risalde, M. A., Development and Challenges in Animal Tuberculosis Vaccination. Pathogens (Basel, Switzerland) 2020, 9 (6). DOI: 10.3390 / pathogens9060472.

[0192] Buddle, B. M.; Vordermeier, H. M.; Chambers, M. A.; de Klerk-Lorist, L. M., Efficacy and Safety of BCG Vaccine for Control of Tuberculosis in Domestic Livestock and Wildlife. Front Vet Sei 2018, 5, 259. DOI: 10.3389 / fvets.2018.00259.

[0193] Prasad, T. S. K.; Verma, R.; Kumar, S.; Nirujogi, R. S.; Sathe, G. J.; Madugundu, A. K.; Sharma, J.; Puttamallesh, V. N.; Ganjiwale, A.; Myneedu, V. P.; Chatterjee, A.; Pandey, A.; Harsha, H. C.; Narayana, J., Proteomic analysis of purified protein derivative of Mycobacterium tuberculosis. Clinical Proteomics 2013, 70 (1), 8. DOI: 10.1186 / 1559-0275- 10-8.

[0194] Yang, H.; Kruh-Garcia, N. A.; Dobos, K. M., Purified protein derivatives of tuberculin-past, present, and future. FEMS immunology and medical microbiology 2012, 66 (3), 273-80. DOI: 10.1111 / j.1574-695X.2O12.01002.x.SEQUENCES esx-1 (rv3861 to rv3885)DNA fragment (in 2F9) containing the Mtb esx-1 locus [SEQ ID NO: 1] gatcggatccgaccacaccagcccggcaccccgcggggatactcgccccgtccgccgtccggagatcgctgccccgcgccaccgc ctggccggcacgctgctgccgctacgccaccagggccgccgcgcctgccttcagctccactgcgtccattgccggacccggcttggc cacgccagccggaggccccgccaccgagcacctgggccgaccccgccctggcgccgatacgcagtcggacgcgacccggcgag cgtggttggcgacgcatggtgcggctggtcacctttggccttgtcggcctgggccggtcgggcatgcagcgccaggaggcccaattc gaagcaacgatacgaaccgtcctgcatggcaaccacaaggtcgccgtgctgggcaaaggaggtgtgggaaagacgtcggttgcgg cgtgcgtcggatcgatccttgccgaactgcgccagcaggaccgtatcgtcgggatcgacgccgacaccgccttcggcaggctgagc agccgaatcgatcctcgagcagctggttcgttctgggagctgaccaccgacacgaatctgcggtccttcaccgatatcaccgcgcgcc tgggccgaaattccgcgggactgtacgtcctggcaggccagccggcatccggtccgcgccgggtgctcgatccggccatctaccgc gaagccgccctaaggttggatcaccatttcgcaatctcggtgatcgactgcggttcctccatggaggcggcggtcacccaggaagtatt gcgcgatgtggatgctctgatcgtggtgtcctcgccctgggcggatggtgcctccgctgccgccaacaccatcgaatggctgtcggatt atggcctgacaggtttgttgcgacgcagcatcgtggtgctcaacgattcggacggacacgccgacaagcgcaccaagtcattgctggc ccaggaattcatcgaccacgggcagcctgtggtcgaggtgcccttcgatccccatttgcggcccgggggggtcatcgatatgagcca cgaaatggccccgacgacgcggctgaaaatcctgcaggtcgccgcgacggtgacggcgtacttcgcgtcgcgacccgccgacgca cacggcagcccgccccggtgacctggctggctgacccggtcggcaacagcaggatcgcccgagcgcaggcctgcaaaacgtcaat ctcggcgcccatcgtcgaatcctggcgggcgcaacgcggcgcgcaatgtggacagcgcgagaaatcttgtcgatgttctcgcgctgt ccacatccagggcatctcaccgccactgttccgcagacccctcgaaccagcggtccaggcggcggttgcgtcatgccgattgggcag acacccggtggtcgcgcaccgggtaaccgttgcgctcggccagggatcgcagctggcccaacgcgaatgcccgcgcccggcctga ttcgggaattacgacccctgcccacagcccttccgcacccgcggactcgacggcgtcgcgtgcacacagccaccggcgcgggcaa gcccggcacagggtcttggcctcgtcgtcgggagtcgtcgtccaacgatcgggatcttgcgtgcaaacgccgagcgggacctcatac agggcggttactgtcatgtctacgttcctccagaaagcgttgcaggttgtagcctctgccgcgaaagcgtatcgcattaaccatagcgat gcaacagtttcctcctctgcctgcctagcggtgctgcggctccggttcggcgagctccgagctctagtgcgcgcaccgccgagtacca gggcatagatcctgttaatcagctgtgtatctggcctcgccggcgcgtatccgaccccttcgggcagatcttccaggaaaagtgttctga catgcgacagttcaggtgtgaagtgaactgtagcggcagttcggtttggctaggaaactatttccatagcgggccgtcgcgtcgctagat ccaaaatgtagcgaagtcatagcagtagaagggtgcaacggttaggatggcgggcgagcggaaagtctgcccaccgtcccggctag tacccgcgaataagggatcaacgcagatgtctaaagcagggtcgactgtcggaccggcgccgctggtcgcgtgcagcggcggcacatcagacgtgattgagccccgtcgcggtgtcgcgatcattggccactcgtgccgagtcggcacccagatcgacgattctcgaatctctc agacacatctgcgagcggtatccgatgatggacggtggcggatcgtcggcaacatcccgagaggtatgttcgtcggcggacgacgc ggcagctcggtgaccgtcagcgataagaccctaatccgattcggcgatccccctggaggcaaggcgttgacgttcgaagtcgtcagg ccgtcggattccgctgcacagcacggccgcgtacaaccatcagcggacctgtcggacgacccggcgcacaacgctgcgccggtcg caccggaccccggcgtggttcgcgcaggggcggccgcggctgcgcgccgtcgtgaacttgacatcagccaacgcagcttggcggc cgacgggatcatcaacgcgggcgcgctcatcgcgttcgagaaaggccgtagttggccccgggaacggacccgggcaaaactcgaa gaagtgctgcagtggcccgctggaaccatcgcgcgaatccgtcggggcgagcccaccgagcccgcaacaaaccccgacgcgtcc cccggactccggcctgccgacggcccggcgtccttgatcgcgcaggctgtcaccgccgccgtagacggctgcagtctggctatcgc agcgttgccggcgaccgaggaccccgagttcaccgaacgtgccgcgccgatccttgctgatttgcgccagctcgaggcgattgccgt ccaagcaacccgcatcagccggattaccccggaattgatcaaggcgttgggcgcggtacgtcgccaccacgacgaattaatgaggct gggagcaaccgcccctggtgccacactggcgcagcgcttatatgccgcacggcggcgcgcgaacctttccaccctggagactgccc aagcggccggcgtcgcagaagaaatgatcgtcggcgccgaagccgaggaagagttgccagccgaggccaccgaagcgatcgaa gcactgatccgtcagatcaattgaggtcggctccgagcgtcccacaagtacaggcacgccgtaacgctcaagttcaacggtccgggg aacgcgcgcgttctccggcgtttgacggtgcgttccatcgtgccgcgaacttgaaaacgccagcgtcaccaaaaaattcgtgcaccaa cccccctccgagcgctgctaagctcaatgtgcagtgcaaaggtgcagataatgatggcgcaccggaacggcgagcgtaaggaaaca cataaatggcatcgggtagcggtctttgcaagacgacgagtaactttatttggggccagttactcttgcttggagagggaatccccgacc caggcgacattttcaacaccggttcgtcgctgttcaaacaaatcagcgacaaaatgggactcgccattccgggcaccaactggatcgg ccaagcggcggaagcttacctaaaccagaacatcgcgcaacaacttcgcgcacaggtgatgggcgatctcgacaaattaaccggca acatgatctcgaatcaggccaaatacgtctccgatacgcgcgacgtcctgcgggccatgaagaagatgattgacggtgtctacaaggtt tgtaagggcctcgaaaagattccgctgctcggccacttgtggtcgtgggagctcgcaatccctatgtccggcatcgcgatggccgttgt cggcggcgcattgctctatctaacgattatgacgctgatgaatgcgaccaacctgaggggaattctcggcaggctgatcgagatgttga cgaccttgccaaagttccccggcctgcccgggttgcccagcctgcccgacatcatcgacggcctctggccgccgaagttgcccgaca ttccgatccccggcctgcccgacatcccgggcctacccgacttcaaatggccgcccacccccggcagcccgttgttccccgacctccc gtcgttcccagggttccccgggttcccggagttccccgccatccccgggttccccgcactgcccgggttgcccagcattcccaacttgtt ccccggcttgccgggtctgggcgacctgctgcccggcgtaggcgatttgggcaagttacccacctggactgagctggccgctttgcct gacttcttgggcggcttcgccggcctgcccagcttgggttttggcaatctgctcagctttgccagtttgcccaccgtgggtcaggtgacc gccaccatgggtcagctgcaacagctcgtggcggccggcggtggccccagccaactggccagcatgggcagccaacaagcgcaa ctgatctcgtcgcaggcccagcaaggaggccagcagcacgccaccctcgtgagcgacaagaaggaagacgaggaaggcgtggcc gaggcggagcgtgcacccatcgacgctggcaccgcggccagccaacgggggcaggaggggaccgtcctttgatcggacaccga gtcgccagcaggtctgtgccatagcgagtcgaagccatagcgagtagaaagttaaacgtagaggagggttcaacccatgaccggatttctcggtgtcgtgccttcgttcctgaaggtgctggcgggcatgcacaacgagatcgtgggtgatatcaaaagggcgaccgatacggtcg ccgggattagcggacgagttcagcttacccatggttcgttcacgtcgaaattcaatgacacgctgcaagagtttgagaccacccgtagc agcacgggcacgggtttgcagggagtcaccagcggactggccaataatctgctcgcagccgccggcgcctacctcaaggccgacg atggcctagccggtgttatcgacaagattttcggttgatcatgacgggtccgtccgctgcaggccgcgcgggcaccgccgacaacgtg gtcggcgtcgaggtaaccatcgacggcatgttggtgatcgccgatcggttacacctggttgatttccctgtcacgcttgggattcggccg aatatcccgcaagaggatctgcgagacatcgtctgggaacaggtgcagcgtgacctcacagcgcaaggggtgctcgacctccacgg ggagccccaaccgacggtcgcggagatggtcgaaaccctgggcaggccagatcggaccttggagggtcgctggtggcggcgcga cattggcggcgtcatggtgcgcttcgtcgtgtgccgcaggggcgaccgccatgtgatcgcggcgcgcgacggcgacatgctggtgct gcagttggtggcgccgcaggtcggcttggcgggcatggtgacagcggtgctggggcccgccgaacccgccaacgtcgaacccctg acgggtgtggcaaccgagctagccgaatgcacaaccgcgtcccaattgacgcaatacggtatcgcaccggcctcggcccgcgtctat gccgagatcgtgggtaacccgaccggctgggtggagatcgttgccagccaacgccaccccggcggcaccacgacgcagaccgac gccgccgctggcgtcctggactccaagctcggtaggctggtgtcgcttccccgccgtgttggaggcgacctgtacggaagcttcctgc ccggcactcagcagaacttggagcgtgcgctggacggcttgctagagctgctccctgcgggcgcttggctagatcacacctcagatca cgcacaagcctcctcccgaggctgacccctcacatctccgctacgacttcagaaagggacgccatggtggacccgccgggcaacga cgacgaccacggtgatctcgacgccctcgatttctccgccgcccacaccaacgaggcgtcgccgctggacgccttagacgactatgc gccggtgcagaccgatgacgccgaaggcgacctggacgccctccatgcgctcaccgaacgcgacgaggagccggagctggagtt gttcacggtgaccaaccctcaagggtcggtgtcggtctcaaccctgatggacggcagaatccagcacgtcgagctgacggacaagg cgaccagcatgtccgaagcgcagctggccgacgagatcttcgttattgccgatctggcccgccaaaaggcgcgggcgtcgcagtac acgttcatggtggagaacatcggtgaactgaccgacgaagacgcagaaggcagcgccctgctgcgggaattcgtggggatgaccct gaatctgccgacgccggaagaggctgccgcagccgaagccgaagtgttcgccacccgctacgatgtcgactacacctcccggtaca aggccgatgactgatcgcttggccagtctgttcgaaagcgccgtcagcatgttgccgatgtcggaggcgcggtcgctagatctgttcac cgagatcaccaactacgacgaatccgcttgcgacgcatggatcggccggatccggtgtggggacaccgaccgggtgacgctgtttcg cgcctggtattcgcgccgcaatttcggacagttgtcgggatcggtccagatctcgatgagcacgttaaacgccaggattgccatcgggg ggctgtacggcgatatcacctacccggtcacctcgccgctagcgatcaccatgggctttgccgcatgcgaggcagcgcaaggcaatt acgccgacgccatggaggccttagaggccgccccggtcgcgggttccgagcacctggtggcgtggatgaaggcggttgtctacggc gcggccgaacgctggaccgacgtgatcgaccaggtcaagagtgctgggaaatggccggacaagtttttggccggcgcggccggtgt ggcgcacggggttgccgcggcaaacctggccttgttcaccgaagccgaacgccgactcaccgaggccaacgactcgcccgccggt gaggcgtgtgcgcgcgccatcgcctggtatctggcgatggcacggcgcagccagggcaacgaaagcgccgcggtggcgctgctg gaatggttacagaccactcaccccgagcccaaagtggctgcggcgctgaaggatccctcctaccggctgaagacgaccaccgccga acagatcgcatcccgcgccgatccctgggatccgggcagtgtcgtgaccgacaactccggccgggagcggctgctcgccgaggcccaagccgaactcgaccgccaaattgggctcacccgggttaaaaatcagattgaacgctaccgcgcggcgacgctgatggcccgggt ccgcgccgccaagggtatgaaggtcgcccagcccagcaagcacatgatcttcaccggaccgcccggtaccggcaagaccacgatc gcgcgggtggtggccaatatcctggccggcttaggcgtcattgccgaacccaaactcgtcgagacgtcgcgcaaggacttcgtcgcc gagtacgaggggcaatcggcggtcaagaccgctaagacgatcgatcaggcgctgggcggggtgcttttcatcgacgaggcttatgc gctggtgcaggaaagagacggccgcaccgatccgttcggtcaagaggcgctggacacgctgctggcgcggatggagaacgaccg ggaccggctggtggtgatcatcgccgggtacagctccgacatagatcggctgctggaaaccaacgagggtctgcggtcgcggttcgc cactcgcatcgagttcgacacctattcccccgaggaactcctcgagatcgccaacgtcattgccgctgctgatgattcggcgttgaccg cagaggcggccgagaactttcttcaggccgccaagcagttggagcagcgcatgttgcgcggccggcgcgccctggacgtcgccgg caacggtcggtatgcgcgccagctggtggaggccagcgagcaatgccgggacatgcgtctagcccaggtcctcgatatcgacaccc tcgacgaagaccggcttcgcgagatcaacggctcagatatggcggaggctatcgccgcggtgcacgcacacctcaacatgagagaa tgaactatggggcttcgcctcaccaccaaggttcaggttagcggctggcgttttctgctgcgccggctcgaacacgccatcgtgcgccg ggacacccggatgtttgacgacccgctgcagttctacagccgctcgatcgctcttggcatcgtcgtcgcggtcctgattctggcgggtg ccgcgctgctggcgtacttcaaaccacaaggcaaactcggcggcaccagcctgttcaccgaccgcgcgaccaaccagctttacgtgc tgctgtccggacagttgcatccggtctacaacctgacttcggcgcggctggtgctgggcaatccggccaacccggccaccgtgaagtc ctccgaactgagcaagctgccgatgggccagaccgttggaatccccggcgccccctacgccacgcctgtttcggcgggcagcacct cgatctggaccctatgcgacaccgtcgcccgagccgactccacttccccggtagtgcagaccgcggtcatcgcgatgccgttggaga tcgatgcttcgatcgatccgctccagtcacacgaagcggtgctggtgtcctaccagggcgaaacctggatcgtcacaactaagggacg ccacgccatagatctgaccgaccgcgccctcacctcgtcgatggggataccggtgacggccaggccaaccccgatctcggagggca tgttcaacgcgctgcctgatatggggccctggcagctgccgccgataccggcggcgggcgcgcccaattcgcttggcctacctgatg atctagtgatcggatcggtcttccagatccacaccgacaagggcccgcaatactatgtggtgctgcccgacggcatcgcgcaggtcaa cgcgacaaccgctgcggcgctgcgcgccacccaggcgcacgggctggtcgcgccaccggcaatggtgcccagtctggtcgtcaga atcgccgaacgggtatacccctcaccgctacccgatgaaccgctcaagatcgtgtcccggccgcaggatcccgcgctgtgctggtcat ggcaacgcagcgccggcgaccagtcgccgcagtcaacggtgctgtccggccggcatctgccgatatcgccctcagcgatgaacatg gggatcaagcagatccacgggacggcgaccgtttacctcgacggcggaaaattcgtggcactgcaatcccccgatcctcgatacacc gaatcgatgtactacatcgatccacagggcgtgcgttatggggtgcctaacgcggagacagccaagtcgctgggcctgagttcacccc aaaacgcgccctgggagatcgttcgtctcctggtcgacggtccggtgctgtcgaaagatgccgcactgctcgagcacgacacgctgc ccgctgaccctagcccccgaaaagttcccgccggagcctccggagccccctgatgacgaccaagaagttcactcccaccattacccg tggcccccggttgaccccgggcgagatcagcctcacgccgcccgatgacctgggcatcgacatcccaccgtcgggcgtccaaaaga tccttccctacgtgatgggtggcgccatgctcggcatgatcgccatcatggtggccggcggcaccaggcagctgtcgccgtacatgtt gatgatgccgctgatgatgatcgtgatgatggtcggcggtctggccggtagcaccggtggtggcggcaagaaggtgcccgaaatcaacgccgaccgcaaggagtacctgcggtatttggcaggactacgcacccgagtgacgtcctcggccacctctcaggtggcgttcttctcct accacgcaccgcatcccgaggatctgttgtcgatcgtcggcacccaacggcagtggtcccggccggccaacgccgacttctatgcgg ccacccgaatcggtatcggtgaccagccggcggtggatcgattattgaagccggccgtcggcggggagttggccgccgccagcgc agcacctcagccgttcctggagccggtcagtcatatgtgggtggtcaagtttctacgaacccatggattgatccatgactgcccgaaact gctgcaactccgtacctttccgactatcgcgatcggcggggacttggcgggggcagccggcctgatgacggcgatgatctgtcaccta gccgtgttccacccaccggacctgctgcagatccgggtgctcaccgaggaacccgacgaccccgactggtcctggctcaaatggctt ccgcacgtacagcaccagaccgaaaccgatgcggccgggtccacccggctgatcttcacgcgccaggaaggtctgtcggacctgg ccgcgcgcgggccacacgcacccgattcgcttcccggcggcccctacgtagtcgtcgtcgacctgaccggcggcaaggctggattc ccgcccgacggtagggccggtgtcacggtgatcacgttgggcaaccatcgcggctcggcctaccgcatcagggtgcacgaggatg ggacggctgatgaccggctccctaaccaatcgtttcgccaggtgacatcggtcaccgatcggatgtcgccgcagcaagccagccgta tcgcgcgaaagttggccggatggtccatcacgggcaccatcctcgacaagacgtcgcgggtccagaagaaggtggccaccgactg gcaccagctggtcggtgcgcaaagtgtcgaggagataacaccttcccgctggaggatgtacaccgacaccgaccgtgaccggctaa agatcccgtttggtcatgaactaaagaccggcaacgtcatgtacctggacatcaaagagggcgcggaattcggcgccggaccgcacg gcatgctcatcgggaccacggggtctgggaagtccgaattcctgcgcaccctgatcctgtcgctggtggcaatgactcatccagatca ggtgaatctcctgctcaccgacttcaaaggtggttcaaccttcctgggaatggaaaagcttccgcacactgccgctgtcgtcaccaacat ggccgaggaagccgagctcgtcagccggatgggcgaggtgttgaccggagaactcgatcggcgccagtcgatcctccgacaggcc gggatgaaagtcggcgcggccggagccctgtccggcgtggccgaatacgagaagtaccgcgaacgcggtgccgacctacccccg ctgccaacgcttttcgtcgtcgtcgacgagttcgccgagctgttgcagagtcacccggacttcatcgggctgttcgaccggatctgccgc gtcgggcggtcgctgagggtccatctgctgctggctacccagtcgctgcagaccggcggtgttcgcatcgacaaactggagccaaac ctgacatatcgaatcgcattgcgcaccaccagctctcatgaatccaaggcggtaatcggcacaccggaggcgcagtacatcaccaac aaggagagcggtgtcgggtttctccgggtcggcatggaagacccggtcaagttcagcaccttctacatcagtgggccatacatgccgc cggcggcaggcgtcgaaaccaatggtgaagccggagggcccggtcaacagaccactagacaagccgcgcgcattcacaggttcac cgcggcaccggttctcgaggaggcgccgacaccgtgacccgcgccggcgacgatgcaaagcgcagcgatgaggaggagcggcg ccaacggcccgcgccggcgacgatgcaaagcgcagcgatgaggaggagcggcgcgcatgactgctgaaccggaagtacggacg ctgcgcgaggttgtgctggaccagctcggcactgctgaatcgcgtgcgtacaagatgtggctgccgccgttgaccaatccggtcccgc tcaacgagctcatcgcccgtgatcggcgacaacccctgcgatttgccctggggatcatggatgaaccgcgccgccatctacaggatgt gtggggcgtagacgtttccggggccggcggcaacatcggtattgggggcgcacctcaaaccgggaagtcgacgctactgcagacg atggtgatgtcggccgccgccacacactcaccgcgcaacgttcagttctattgcatcgacctaggtggcggcgggctgatctatctcga aaaccttccacacgtcggtggggtagccaatcggtccgagcccgacaaggtcaaccgggtggtcgcagagatgcaagccgtcatgc ggcaacgggaaaccaccttcaaggaacaccgagtgggctcgatcgggatgtaccggcagctgcgtgacgatccaagtcaacccgttgcgtccgatccatacggcgacgtctttctgatcatcgacggatggcccggttttgtcggcgagttccccgaccttgaggggcaggttca agatctggccgcccaggggctggcgttcggcgtccacgtcatcatctccacgccacgctggacagagctgaagtcgcgtgttcgcga ctacctcggcaccaagatcgagttccggcttggtgacgtcaatgaaacccagatcgaccggattacccgcgagatcccggcgaatcgt ccgggtcgggcagtgtcgatggaaaagcaccatctgatgatcggcgtgcccaggttcgacggcgtgcacagcgccgataacctggt ggaggcgatcaccgcgggggtgacgcagatcgcttcccagcacaccgaacaggcacctccggtgcgggtcctgccggagcgtatc cacctgcacgaactcgacccgaacccgccgggaccagagtccgactaccgcactcgctgggagattccgatcggcttgcgcgagac ggacctgacgccggctcactgccacatgcacacgaacccgcacctactgatcttcggtgcggccaaatcgggcaagacgaccattgc ccacgcgatcgcgcgcgccatttgtgcccgaaacagtccccagcaggtgcggttcatgctcgcggactaccgctcgggcctgctgga cgcggtgccggacacccatctgctgggcgccggcgcgatcaaccgcaacagcgcgtcgctagacgaggccgttcaagcactggcg gtcaacctgaagaagcggttgccgccgaccgacctgacgacggcgcagctacgctcgcgttcgtggtggagcggatttgacgtcgtg cttctggtcgacgattggcacatgatcgtgggtgccgccggggggatgccgccgatggcaccgctggccccgttattgccggcggcg gcagatatcgggttgcacatcattgtcacctgtcagatgagccaggcttacaaggcaaccatggacaagttcgtcggcgccgcattcgg gtcgggcgctccgacaatgttcctttcgggcgagaagcaggaattcccatccagtgagttcaaggtcaagcggcgcccccctggcca ggcatttctcgtctcgccagacggcaaagaggtcatccaggccccctacatcgagcctccagaagaagtgttcgcagcacccccaag cgccggttaagattatttcattgccggtgtagcaggacccgagctcagcccggtaatcgagttcgggcaatgctgaccatcgggtttgttt ccggctataaccgaacggtttgtgtacgggatacaaatacagggagggaagaagtaggcaaatggaaaaaatgtcacatgatccgat cgctgccgacattggcacgcaagtgagcgacaacgctctgcacggcgtgacggccggctcgacggcgctgacgtcggtgaccggg ctggttcccgcgggggccgatgaggtctccgcccaagcggcgacggcgttcacatcggagggcatccaattgctggcttccaatgca tcggcccaagaccagctccaccgtgcgggcgaagcggtccaggacgtcgcccgcacctattcgcaaatcgacgacggcgccgccg gcgtcttcgccgaataggcccccaacacatcggagggagtgatcaccatgctgtggcacgcaatgccaccggagctaaataccgcac ggctgatggccggcgcgggtccggctccaatgcttgcggcggccgcgggatggcagacgctttcggcggctctggacgctcaggc cgtcgagttgaccgcgcgcctgaactctctgggagaagcctggactggaggtggcagcgacaaggcgcttgcggctgcaacgccg atggtggtctggctacaaaccgcgtcaacacaggccaagacccgtgcgatgcaggcgacggcgcaagccgcggcatacacccagg ccatggccacgacgccgtcgctgccggagatcgccgccaaccacatcacccaggccgtccttacggccaccaacttcttcggtatca acacgatcccgatcgcgttgaccgagatggattatttcatccgtatgtggaaccaggcagccctggcaatggaggtctaccaggccga gaccgcggttaacacgcttttcgagaagctcgagccgatggcgtcgatccttgatcccggcgcgagccagagcacgacgaacccgat cttcggaatgccctcccctggcagctcaacaccggttggccagttgccgccggcggctacccagaccctcggccaactgggtgagat gagcggcccgatgcagcagctgacccagccgctgcagcaggtgacgtcgttgttcagccaggtgggcggcaccggcggcggcaa cccagccgacgaggaagccgcgcagatgggcctgctcggcaccagtccgctgtcgaaccatccgctggctggtggatcaggcccc agcgcgggcgcgggcctgctgcgcgcggagtcgctacctggcgcaggtgggtcgttgacccgcacgccgctgatgtctcagctgatcgaaaagccggttgccccctcggtgatgccggcggctgctgccggatcgtcggcgacgggtggcgccgctccggtgggtgcggga gcgatgggccagggtgcgcaatccggcggctccaccaggccgggtctggtcgcgccggcaccgctcgcgcaggagcgtgaagaa gacgacgaggacgactgggacgaagaggacgactggtgagctcccgtaatgacaacagacttcccggccacccgggccggaaga cttgccaacattttggcgaggaaggtaaagagagaaagtagtccagcatggcagagatgaagaccgatgccgctaccctcgcgcag gaggcaggtaatttcgagcggatctccggcgacctgaaaacccagatcgaccaggtggagtcgacggcaggttcgttgcagggcca gtggcgcggcgcggcggggacggccgcccaggccgcggtggtgcgcttccaagaagcagccaataagcagaagcaggaactcg acgagatctcgacgaatattcgtcaggccggcgtccaatactcgagggccgacgaggagcagcagcaggcgctgtcctcgcaaatg ggcttctgacccgctaatacgaaaagaaacggagcaaaaacatgacagagcagcagtggaatttcgcgggtatcgaggccgcggca agcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggc ggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctgg cgcggacgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgttcgcatagggcaacgccgagttc gcgtagaatagcgaaacacgggatcgggcgagttcgaccttccgtcggtctcgccctttctcgtgtttatacgtttgagcgcactctgag aggttgtcatggcggccgactacgacaagctcttccggccgcacgaaggtatggaagctccggacgatatggcagcgcagccgttct tcgaccccagtgcttcgtttccgccggcgcccgcatcggcaaacctaccgaagcccaacggccagactccgcccccgacgtccgac gacctgtcggagcggttcgtgtcggccccgccgccgccacccccacccccacctccgcctccgccaactccgatgccgatcgccgc aggagagccgccctcgccggaaccggccgcatctaaaccacccacaccccccatgcccatcgccggacccgaaccggccccacc caaaccacccacaccccccatgcccatcgccggacccgaaccggccccacccaaaccacccacacctccgatgcccatcgccgga cctgcacccaccccaaccgaatcccagttggcgccccccagaccaccgacaccacaaacgccaaccggagcgccgcagcaaccg gaatcaccggcgccccacgtaccctcgcacgggccacatcaaccccggcgcaccgcaccagcaccgccctgggcaaagatgcca atcggcgaacccccgcccgctccgtccagaccgtctgcgtccccggccgaaccaccgacccggcctgccccccaacactcccgac gtgcgcgccggggtcaccgctatcgcacagacaccgaacgaaacgtcgggaaggtagcaactggtccatccatccaggcgcggct gcgggcagaggaagcatccggcgcgcagctcgcccccggaacggagccctcgccagcgccgttgggccaaccgagatcgtatct ggctccgcccacccgccccgcgccgacagaacctccccccagcccctcgccgcagcgcaactccggtcggcgtgccgagcgacg cgtccaccccgatttagccgcccaacatgccgcggcgcaacctgattcaattacggccgcaaccactggcggtcgtcgccgcaagcg tgcagcgccggatctcgacgcgacacagaaatccttaaggccggcggccaaggggccgaaggtgaagaaggtgaagccccagaa accgaaggccacgaagccgcccaaagtggtgtcgcagcgcggctggcgacattgggtgcatgcgttgacgcgaatcaacctgggc ctgtcacccgacgagaagtacgagctggacctgcacgctcgagtccgccgcaatccccgcgggtcgtatcagatcgccgtcgtcggt ctcaaaggtggggctggcaaaaccacgctgacagcagcgttggggtcgacgttggctcaggtgcgggccgaccggatcctggctct agacgcggatccaggcgccggaaacctcgccgatcgggtagggcgacaatcgggcgcgaccatcgctgatgtgcttgcagaaaaa gagctgtcgcactacaacgacatccgcgcacacactagcgtcaatgcggtcaatctggaagtgctgccggcaccggaatacagctcggcgcagcgcgcgctcagcgacgccgactggcatttcatcgccgatcctgcgtcgaggttttacaacctcgtcttggctgattgtggggc cggcttcttcgacccgctgacccgcggcgtgctgtccacggtgtccggtgtcgtggtcgtggcaagtgtctcaatcgacggcgcacaa caggcgtcggtcgcgttggactggttgcgcaacaacggttaccaagatttggcgagccgcgcatgcgtggtcatcaatcacatcatgc cgggagaacccaatgtcgcagttaaagacctggtgcggcatttcgaacagcaagttcaacccggccgggtcgtggtcatgccgtggg acaggcacattgcggccggaaccgagatttcactcgacttgctcgaccctatctacaagcgcaaggtcctcgaattggccgcagcgct atccgacgatttcgagagggctggacgtcgttgagcgcacctgctgttgctgctggtcctaccgccgcgggggcaaccgctgcgcgg cctgccaccacccgggtgacgatcctgaccggcagacggatgaccgatttggtactgccagcggcggtgccgatggaaacttatattg acgacaccgtcgcggtgctttccgaggtgttggaagacacgccggctgatgtactcggcggcttcgactttaccgcgcaaggcgtgtg ggcgttcgctcgtcccggatcgccgccgctgaagctcgaccagtcactcgatgacgccggggtggtcgacgggtcactgctgactct ggtgtcagtcagtcgcaccgagcgctaccgaccgttggtcgaggatgtcatcgacgcgatcgccgtgcttgacgagtcacctgagttc gaccgcacggcattgaatcgctttgtgggggcggcgatcccgcttttgaccgcgcccgtcatcgggatggcgatgcgggcgtggtgg gaaactgggcgtagcttgtggtggccgttggcgattggcatcctggggatcgctgtgctggtaggcagcttcgtcgcgaacaggttcta ccagagcggccacctggccgagtgcctactggtcacgacgtatctgctgatcgcaaccgccgcagcgctggccgtgccgttgccgc gcggggtcaactcgttgggggcgccacaagttgccggcgccgctacggccgtgctgtttttgaccttgatgacgcggggcggccctc ggaagcgtcatgagttggcgtcgtttgccgtgatcaccgctatcgcggtcatcgcggccgccgctgccttcggctatggataccagga ctgggtccccgcggggggatcgcattcgggctgttcattgtgacgaatgcggccaagctgaccgtcgcggtcgcgcggatcgcgctg ccgccgattccggtacccggcgaaaccgtggacaacgaggagttgctcgatcccgtcgcgaccccggaggctaccagcgaagaaa ccccgacctggcaggccatcatcgcgtcggtgcccgcgtccgcggtccggctcaccgagcgcagcaaactggccaagcaacttctg atcggatacgtcacgtcgggcaccctgattctggctgccggtgccatcgcggtcgtggtgcgcgggcacttctttgtacacagcctggt ggtcgcgggtttgatcacgaccgtctgcggatttcgctcgcggctttacgccgagcgctggtgtgcgtgggcgttgctggcggcgacg gtcgcgattccgacgggtctgacggccaaactcatcatctggtacccgcactatgcctggctgttgttgagcgtctacctcacggtagcc ctggttgcgctcgtggtggtcgggtcgatggctcacgtccggcgcgtttcaccggtcgtaaaacgaactctggaattgatcgacggcgc catgatcgctgccatcattcccatgctgctgtggatcaccggggtgtacgacacggtccgcaatatccggttctgagccggatcggctg attggcggttcctgacagaacatcgaggacacggcgcaggtttgcataccttcggcgcccgacaaattgctgcgattgagcgtgtggc gcgtccggtaaaatttgctcgatggggaacacgtataggagatccggcaatggctgaaccgttggccgtcgatcccaccggcttgagc gcagcggccgcgaaattggccggcctcgtttttccgcagcctccggcgccgatcgcggtcagcggaacggattcggtggtagcagc aatcaacgagaccatgccaagcatcgaatcgctggtcagtgacgggctgcccggcgtgaaagccgccctgactcgaacagcatcca acatgaacgcggcggcggacgtctatgcgaagaccgatcagtcactgggaaccagtttgagccagtatgcattcggctcgtcgggcg aaggcctggctggcgtcgcctcggtcggtggtcagccaagtcaggctacccagctgctgagcacacccgtgtcacaggtcacgacc cagctcggcgagacggccgctgagctggcaccccgtgttgttgcgacggtgccgcaactcgttcagctggctccgcacgccgttcagatgtcgcaaaacgcatcccccatcgctcagacgatcagtcaaaccgcccaacaggccgcccagagcgcgcagggcggcagcggc ccaatgcccgcacagcttgccagcgctgaaaaaccggccaccgagcaagcggagccggtccacgaagtgacaaacgacgatcag ggcgaccagggcgacgtgcagccggccgaggtcgttgccgcggcacgtgacgaaggcgccggcgcatcaccgggccagcagcc cggcgggggcgttcccgcgcaagccatggataccggagccggtgcccgcccagcggcgagtccgctggcggcccccgtcgatcc gtcgactccggcaccctcaacaaccacaacgttgtagaccgggcctgccagcggctccgtctcgcacgcagcgcctgttgctgtcctg gcctcgtcagcatgcggcggccagggcccggtcgagcaacccggtgacgtattgccagtacagccagtccgcgacggccacacgc tggacggccgcgtcagtcgcagtgtgcgcttggtgcagggcaatctcctgtgagtgggcagcgtaggcccggaacgcccgcagatg agcggcctcgcggccggtagcggtgctggtcatgggcttcatcagctcgaaccacagcatgtgccgctcatcgcccggtggattgac atccaccggcgccggcggcaacaagtcgagcaaacgctgatcggtagtgtcggccagctgagccgccgccgaggggtcgacgac ctccagccgcgaccggcccgtcattttgccgctctccggaatgtcatctggctccagcacaatcttggccacaccgggatccgaactgg ccaactgctccgcggtaccgatcaccgcccgcagcgtcatgtcgtggaaagccgcccaggcttgcacggccaaaaccgggtaggtg gcacagcgtgcaatttcgtcaaccgggattgcgtgatccgcgctggccaagtacaccttattcggcaattccatcccgtcgggtatgtag gccagcccatagctgttggccacgacgatggaaccgtcggtggtcaccgcggtgatccagaagaacccgtagtcgcccgcgttgttg tcggacgcgttgagcgccgccgcgatgcgtcgcgccaaccgcagcgcatcaccgcggccacgctggcgggcgctggcagctgca gtggcggcgtcgcgtgccgcccgagccgccgacaccgggatcatcgacaccggcgtaccgtcatctgcagactcgctgcgatcgg gtttgtcgatgtgatcggtcgacggcgggcgggcaggaggtgccgtccgcgccgaggccgcccgcgtgctcggtgccgccgccttg tccgaggtagccaccggcgcccgcccagtggcagcatgcgaccccgcgcccgaggccgcggccgtacccacgctcgaacgcgc gcccgctcccacggcggtaccgctcggcgcggcggccgccgcccgtgcgcccgggacaccggacgccgcagccggcgtcaccg acgcggcggattcgtccgcatgggcaggccccgactgcgtccccccgcccgcatgctggcccggcacaccaggttgctccgccaa cgccgcgggtttgacgtgcggcgccggctcgccccctggggtgcccggtgttgctggaccagacggaccgggagtggccggtgta accggctggggcccaggcgatggcgccggtgccggagccggctgcgggtgtggagcgggagctggggtaacgggcgtggccg gggttgccggtgtggccggggcgaccgggggggtgaccggcgtgatcggggttggctcgcctggtgtgcccggtttgaccggggt caccggggtgaccggcttgcccggggtcaccggcgtgacgggagtgccgggcgttggtgtgatcggagttaccggcgctcccggg atgggtgtgattggggttcccggggtgatcggggttcccggggtgatcggggttcccggtgtgcccggtgtgcccggggatggcacg accagggtaggcacgtctgggggtggcggcgacttctgctgaagcaaatcctcgagtgcgttcttcggaggtttccaattcttggattcc agcacccgctcagcggtctcggcgaccagactgacattggccccatgcgtcgccgtgaccaatgaattgatggcggtatggcgctcat cagcatccaggctagggtcattctccaggatatcgatctcccgttgagcgccatccacattattgccgatatcggatttagcttgctcaatc aacccggcaatatgcctgtgccaggtaatcaccgtggcgagataatcctgcagcgtcatcaattgattgatgtttgcacccagggcgcc gttggcagcattggcggcgccgccggaccataggccgccttcgaagacgtggcctttctgctggcggcaggtgtccaatacatcggt gaccctttgcaaaacctggctatattcctgggcccggtcatagaaagtgtcttcatcggcttccacccagccgcccggatccagcatctgtctggcatagctgcccgtcggcctggtaatactcatcccctactgccctccccaaaccgccagatcgcctcgcggatcaccgtccggtt ggcctccggcatttcacgccggctcggccgctggatccaccccgcgccggtattcgcagtaacccgttgaatccgcgcgcatgatgc accgcttgggcgatcagccgggtggtcacctcgcttgcgctggccgcgctgtcgcacggggcgctcggtggtaacggacgtcataat taaccagcgtaaccgaacctaagaccagctagctgcggcaatattggcgaccaggactatggcgccctccgaacccggccgatccat gtcaaaacattgacaatgcgtactcacgccgtgtcgggcgcgctgaatgaccgcattgcggcgctcattcggtgcgtagtcgctaccac cgcaacaatgggcttaggccattccttcgttcatcgcgcgggacatggccgataacgcagcggtcagctgctcgcccgccgcgtcgtt atacgcggacgccgcggcctgcgcattgtgcagcgcctcgttgacccgctgagccaccgcctcggcacccagcttcttcagcaaacc atcttcgatgcgcaggccggtgagccactggtgcccattgatcgtcacttcgacggtctcggcttcgtcggtggcgcggaaggatccgt tgttcatctgattgagcgtcccgtctagggccgactgaaaccgcgccgccagcgtcaacgcccgggcgacatgcgggtccaattcgtc catgctcacttcgactccttactgtcctggcgccgacggttaccaatgacggcctcggtccatgcccgatcctcggtgtagagcgcctcg tcttcctgctgagaacccttggacttggcgcccccttgtccctgatgcgcggcacccatcggcattcccatgccaccgccgcccagcgc ggcgccgccgccggcccttccctggcctaagccggcaatgtcaccagcgccagcgggccgcaccgattcggcgcccccgatcgcg gatcccaacggcgccgacggcaccccgccgcctccaccgccaccgagcgatgccgctttgaccgccacgtcgcccgacagcgctg cggcttcccgcccagccgacgtcagctgcgccgccgtgtcagccgggaggccaccacccggcgatccggtaggcggaaccatcg gtgcggctggcatcccggtaccgggagtcacaccggagccgtcagacggcggcatcaggaagccagggatcaatccctgctcttgc ggaggcgggggcgggtcgatcttgatggcggggggaggcttcggcgggtttaccggttccagggctgccttgttgttgtattcggtca gcaccttctccgacctctgctgatactccgcgtacaccgggagaatttggtcgcgggccgaagggttttccgcgtaaagccgttcgagc ccgactatgtcttcataagtcggatgttcccgcctagcccacacgtgcagctgcgcgacatattgagcctgcttggccatcgcagcgctc aatttggccatgtggagtatccattgccgttgttgatcgagcgaagcctcgcaagcggtagccgcatcgccttcccagttgtcaaacccc cggaaccgcttgacgtcgccttgcagcgtcaggttgaaagtgttccacccatccgcaaagtgcgcgagcgatgcgccttggtcgcccg tttcgagcttccttgccgcttctttgagatccatgaagttgggttcaccggccgtggccaccctcggcgtatcggttagttcggccgaact gtcccctccgacggccccggccgattctgcctgcacagttccttcgccgtcgttgtccagcgcggtcgcagcctcctcatcaacctcgc catacgccttggccgcgttgcgcagcgaggtcgccagacgctgccgctctttggcaccggccgccaggtattcccgcatgttgtcggc ggacaataccagctgttgggcggcgtttttagccgccgtgagttcgcacggtgtgatggggacatcagtcggtgggtccgccatcggg gcctccacctcgttggccctgttcaaaatctcttgctgatccaccgtcacggtctgcgactgcgtcatatcggatcatcctccttagtgctat agccattatcgtcgctaaactgaaaggttcctgcactaatttgatgccgcccgttcatgccggcatcgcgaacggatcgccctacttcgg cagcgccatctggtagcggctttcctcgggtggggaaacccggcgaatcggcagctgccgatgccgcggggtaccgatcacattgtg ccgcagaatcacccggtcaataccgggatgcgggccgagataggtcgtcgcattcggccacgccacctttacctcctgcccgatgtgt gcgccgatcaaccgggcaaattcctcgaactgtggcccgactgtgaccatcgcacctgccgccgccgcacgcaccacgaactgggt gaatgtctgagcgtcacccaggttgagggcgatgtcgacatcgtcgaagggcatgtagaccgggcatcggttcaccgtctcgccgaccagtaccccagctgacccgatcggcagctggcagtggcggttggccaccagatgctggccttgcagcgcgggccgctgcccgcca aataggcgggcgaagcccctgggtgtcttgggcttgtccgccgtggtcagcaacaccgtggactgcggggccatccccggcgcgac ccggactctggtgatggtgtggtccgcgcgcgccgaccaccatacatccggacctccgggcgccgcgtaggcggcagtgtaggcat cgcgccccttgatcatcgaccatttctcccgcacaaagccgatgtcggtggcgtggtcgtagtcatcgaagctgcggccacacaccgc gtcgacaccatggctagccagtcgatcggcaatgcgcgtcgcggacgccaccaaataccgggccagtcctgcgacgccttcatcgc ggcgctgcgccgatttgcgggtgcgttccgggtcggcgcgcagcacgatccaggtccggcggttcgccggcgccgggtctgtcccg atcacctgctgatacagactcaccacgtccggcgctgcggtattgccgacgcggtagccggctgagacgatatcggcctccaagtcg ggacagtgcaccgacaggagctcctccaccagtccggtgtccagcatgtcgtcggtgtgggcttgcccgtcgacgatgaccgtcggc gtgaatggtcggggaatgagctcgattacggcgaccagaaactcgccttgccagcgcaccgcaacgtgatctcctggcttcacggtg gccccgaccacaggttctgacgaggaatccgggggccgtcggcgccgccgcaaccacgcgtacaccgccgccacccagccggtg atccggcggccgtagaaagtgaccgtggccacgatgacgcccaacgaggccagcgcaatccccgcccaccagtagcgcgtctcca agaatgcgatgatgcatggcggggccaacgcggaggcaagcaaggcgtgcccggtgctgaaccgcagccctaaaggatttctcatc ggcggctcagcgcccgtctagccagcgcgcccaggcccagggccaacgtaaggccgacggccaccaacgccacagccgtaatcg ggcgacgatcgggacccggctccaccaccgggggtggaagtcgtctgacgttgtatggcgccgaagcagggccgggcggaatgtc ccacgtcagcgcggccaccgcatcgatgacgccggcgccgaccaggtcgtcgaccccgcccccggggtgtctcgcggtggcggt gatccggtggatgatctgcgccggcgtcaggtcggggaaccgctgccgaagcagggccgccagacccgacacatatgccgcggc aaacgaggtgccggcgatgggtaccggcccctcccggccttgcagcgcattcaccggttcaccggtgtcgccgagcgcgacgatgtt ttctgcgggcgcggccacgtccacccacggtccgtgcatcgagaacgagctgggcatcccggtctggccgataccgccgacgctta acaccagcggtgcgtaccacgccggggtgacaacggtctgcacattgttccagccgcgtgggtcgccgggtgtggacgggtccggc gccggattctgtacgcaatcgccaccggtgttgccggccgcgaccaccaccaccacgcctttgacgttgaccgcatagtcgatggatg cacccagtgaggtttcatcgatcggcctgctcaccttgtagcaggcggcttcactgatgttgatcacacccacgccgaggttggcggcg tgcaccacggcgcgggcaagactgcggatggaaccggcggccggggtggcgttggggtcattcgggttggcttgtgagccgaccg gttcgaaggcctcagacgtctgacgtagcgagagcagtcgagcgtcgggcgcgacgccgacgaacccgtcggtgggcgcgggcc ggcccgcgatgatggatgctgtgagagtcccatgggcatcacagtcagacaggccgttaccggcctggtcgacgaaatcgccgcca ggttccgccgggacccgtggcgaagcgtcgacaccggtgtcgatcaccgccaccgtcaccccggccccggtcgcgaacttgtggg catcggccacgcccagatacgtgttgctccacggcggatcgtggaacccggaccccggcagcgtggtgggcgacgcgcacaaaac gcgctgttcggtaggctgatccgggcccgtcacgtcgggcggcaacgcgcccggatcgatcggcggtggcgtgatggccgatgcg ggcgacgcggtgagcaacgccagcgccaccgtgatcagaaagatacggtgcactcccagaacactccattcgttgagattcattgcg attcattgagctgcgttgctaccttgggccacttgacggacctgtgtgcattttagacgtaacggctgggcaaacaacgctgtcacgcct gggctggtccgccgcgccgaccagggcgcgtaggcgctgtacctggaccacgccgggactcaacggttttgctaccgcactagccgatatgcggctgctaccaaacgatcgcggccatgtctcggttgtctgagcacacgctgcgtatcgcggcatcgatgtcggtggcggtgat gatctgcagatcctgaaccgataccggttggcccgcacgtttttgcgcaaccacccgggtgtcccggaacccttcggcgcgttcgatca cgttgcgggcgaaccgaccgttttgcatagcgtcgataccgtgctgcccactaggggtggtgtagttacggatggtggtgaccgcgtc gaggaatacctcccgtgcggcgtcatcgagctggctggcgcgcggtgtagcgtagcggtgtccaatctcgacgatctccaccggcga ataagactcgaaccgcagctttcggttgaaccggccagccaaacccgggttcacggtgaggaattcatccacctgatcctcatagccg gccccgatgaaacagaagtcgaatcggtgtgtttccaattgaaccaggagttgattgaccgcctccatgccgatcatgtccggtgttccg tcttgatgacgttcgatcagcgagtagaactcgtccatgaaaatgattcgcccgagtgacttttcgatcagctcgttcgtcttgggtcctga ctccccgatgtagtgcccacagaagtccgatcggcgaacttctcgaatttcggggtgacgcacgatccccatgccggcgtagatcttgc cgagcgcttcagcggtggttgtcttacctgtgcctggtggccccaccagcaacatgtggttggtctgcccctccaccggtaggccgtgc tctaggcgcatcatgcgcacctcgagttggtcttccagcgccgataccgcttgcttgaccgccgccaggcccacctgtttggccagcag ttcccggccctcggctagcagctcgccgcgccgctgcgctgcattgtcgtcatcgagctggtcgcggcttttcgccgtcgaagcatccc aacggtcggagcggctggcgatggttcgttcatcggtaacaatcaagcgcaggttcgggtccgccagggcttctttggcggcgtcggt gagcaccccgttgatggtggccttcgacagccagatctgggccttgtcctcctcatgcagttgccggtacaccatcccccgcacatacg ccaagtcggcgaccagcagcggaatatcggccggtccgatcgccgcggtgagcacgtcggcgccgaaccgctccgatgacctgct gtgtccgatcacgtccacccggtccagccagtccagggccactcgcccctgcccgagatgggcggcggcgtgggctgccagcgca caaatcgacgcggtcaccgccggcatgacgatcgcctgtggcggcagatcctcggcggccgtcgacaacacgtcgggccatcgctg cgtgacgtacatcaggaacgcccgagccagctgatgccactggtagttgcgccacgaatccaatagctcgcggtttgctaacagggc atcggccttcgcatactcccccgcgatcgtcaacgccgacgacagcgccagccccacctgagatgcgtcggtcaccgtgatcccgat ggatggtcccagctggacctcagcggccaacgtccggccgatccgcgtggtctcgcggtgcagccactcgctatgggcgttgagctg cttaagcgaggccagatcgcggtcaccgcaggcgatacgacccagccacgcgtcggccatcgacggatcggcctcggtggcagcc acaaactcaggcaacgccgccacgcatccctggccattcttgatcgtcatcgcccgatcgaaatgccggcgcgcagtgagtaaatcac ccatcgtgtccaccattctcgacatcgccgccgctgtcaccgcggttgcaacgtgtgtctgtcactctgtgcctcaaattccgttggcaac gttctaccggcctatcgacatcgtgaccggctcaaggctgacatagcggttctccgcacggaacatttccatctcaaccagccagttttgt cctgccgcaccgactttcaccgttgcccgatcgatttgttcgatggtcacctcgaagccatgccgatcgctctcggacagcgaggtacc gggtcgggcaatggtgatgacactggctggccgtggcgtgggcgaaatcgcgacatcgacaccgctgccttcagatttgccgtcatc gccgttcttgcgccgccgcacgtactccacgacgccgacagtggtgcgcggcgcgggccgtggtgtgccgacgatgctcaactgcg gcatgcgtacgctggcccaacgctcttggtcgcgagtgtgcacacacacccgctcaccggcaccgacgacgcgaatcacgatcctct tggcgatcgtgtcgtccgcggccacgaagacgcgcgacagctcaccggcgtcggtaacgggaatcatcagccggtccccgttgctc agcttgccaatcaacacccccgacggtccgatctcggtgactagctgcgccggcaacgggcagcgccgctgtccgcgtaggtgtgg acgtggcccgcacatgttggccgcagccgcggcggcttgctcaccattgagccgacgcaagatcacactgggcggggtaggcgccggcgtcggtgtgcgcacggtgatggtcgcggtgcacgtcgcgtccggatacaccgttacgttctggatgacctcatcggcacgcagc gtccaggcttgcgagagaacccgcgacgaaatcgcctcagccgggtacgcatacgtcgtcatccacccggcttcaccgcggatagct ttccagcgctgcgcactcccggctaccgcgtccgaccccagccggcgatcaagctcagccaagtctgttgcggtggccagtttggcg cgcaagccctgacagcgcagggagctggcaacgcgttgggcgaccgaaatggcagcggccccaacgctggtacgccagcgtaaa gcttgggtgttgccgatcaccggaagccgcatgatcagccacgtttcgcgccgcccggcatacggcggcgtaccgatctccgcgtcat acacccgcgggtaatcgccgacggtgccggttcgcgagccgaaggtgacgacgctgattgaatcgagttccaggtccagcgggtgg cgcagcaacggcgcgagctcaacgacgtcaatcacgttgtcgctttctacggtcaccgacccggtgaccgtagtcgcccggtgcgct cggccgagaagttgcaccgccaccaccgcgacaccgtcttgcacgcggacgccacccccggatcggttgttggccaaggtaattgg gtcattccatttgacgggacgccgaccccgcagccccagtaccgcccacgaccacgccggctgaccccaccactgtacgaacacca aggcgacgccgaccacgacagccatgaccgcacctagctggccgcccagcgcccagcccgccgacgcgagcacgaacactgtcc acaccccggcgacccgcctcgcactgcgcgggctgaacccggtcagcttggacgtcaacgcgccctccgtagccgagccccgattg ccattgccagcacaccggtggccactgcgccgacgaacccgatagcgatattgcgcgcccggtgatcrv3861 cds [SEQ ID NO: 2] gtgacctggctggctgacccggtcggcaacagcaggatcgcccgagcgcaggcctgcaaaacgtcaatctcggcgcccatcgtcga atcctggcgggcgcaacgcggcgcgcaatgtggacagcgcgagaaatcttgtcgatgttctcgcgctgtccacatccagggcatctca ccgccactgttccgcagacccctcgaaccagcggtccaggcggcggttgcgtcatgccgattgggcagacacccggtggtcgcgca ccgggtaaccgttgcgctcggccagggatcgcagctggcccaacgcgaatgcccgcgcccggcctgaRv3861 aa seq [SEQ ID NO: 3]VTWLADPVGNSRIARAQACKTSISAPIVESWRAQRGAQCGQREKSCRCSRAVHIQGIS PPLFRRPLEPAVQAAVASCRLGRHPVVAHRVTVALGQGSQLAQRECPRPA rv3862c (whiB6) cds [SEQ ID NO: 4] atgcgatacgctttcgcggcagaggctacaacctgcaacgctttctggaggaacgtagacatgacagtaaccgccctgtatgaggtcc cgctcggcgtttgcacgcaagatcccgatcgttggacgacgactcccgacgacgaggccaagaccctgtgccgggcttgcccgcgc cggtggctgtgtgcacgcgacgccgtcgagtccgcgggtgcggaagggctgtgggcaggggtcgtaattcccgaatcaggccggg cgcgggcattcgcgttgggccagctgcgatccctggccgagcgcaacggttacccggtgcgcgaccaccgggtgtctgcccaatcg gcatgaRv3862c (WhiB6) aa seq [SEQ ID NO: 5]MRYAFAAEATTCNAFWRNVDMTVTALYEVPLGVCTQDPDRWTTTPDDEAKTLCRA CPRRWLCARDAVESAGAEGLWAGVVIPESGRARAFALGQLRSLAERNGYPVRDHRV SAQSA rv3863 cds [SEQ ID NO: 6] atggcgggcgagcggaaagtctgcccaccgtcccggctagtacccgcgaataagggatcaacgcagatgtctaaagcagggtcgac tgtcggaccggcgccgctggtcgcgtgcagcggcggcacatcagacgtgattgagccccgtcgcggtgtcgcgatcattggccactc gtgccgagtcggcacccagatcgacgattctcgaatctctcagacacatctgcgagcggtatccgatgatggacggtggcggatcgtc ggcaacatcccgagaggtatgttcgtcggcggacgacgcggcagctcggtgaccgtcagcgataagaccctaatccgattcggcgat ccccctggaggcaaggcgttgacgttcgaagtcgtcaggccgtcggattccgctgcacagcacggccgcgtacaaccatcagcgga cctgtcggacgacccggcgcacaacgctgcgccggtcgcaccggaccccggcgtggttcgcgcaggggcggccgcggctgcgcgccgtcgtgaacttgacatcagccaacgcagcttggcggccgacgggatcatcaacgcgggcgcgctcatcgcgttcgagaaaggc cgtagttggccccgggaacggacccgggcaaaactcgaagaagtgctgcagtggcccgctggaaccatcgcgcgaatccgtcggg gcgagcccaccgagcccgcaacaaaccccgacgcgtcccccggactccggcctgccgacggcccggcgtccttgatcgcgcagg ctgtcaccgccgccgtagacggctgcagtctggctatcgcagcgttgccggcgaccgaggaccccgagttcaccgaacgtgccgcg ccgatccttgctgatttgcgccagctcgaggcgattgccgtccaagcaacccgcatcagccggattaccccggaattgatcaaggcgtt gggcgcggtacgtcgccaccacgacgaattaatgaggctgggagcaaccgcccctggtgccacactggcgcagcgcttatatgccg cacggcggcgcgcgaacctttccaccctggagactgcccaagcggccggcgtcgcagaagaaatgatcgtcggcgccgaagccg aggaagagttgccagccgaggccaccgaagcgatcgaagcactgatccgtcagatcaattgaRv3863 aa seq [SEQ ID NO: 7]MAGERKVCPPSRLVPANKGSTQMSKAGSTVGPAPLVACSGGTSDVIEPRRGVAIIGHS CRVGTQIDDSRISQTHLRAVSDDGRWRIVGNIPRGMFVGGRRGSSVTVSDKTLIRFGD PPGGKALTFEVVRPSDSAAQHGRVQPSADLSDDPAHNAAPVAPDPGVVRAGAAAAA RRRELDISQRSLAADGIINAGALIAFEKGRSWPRERTRAKLEEVLQWPAGTIARIRRGE PTEPATNPDASPGLRPADGPASLIAQAVTAAVDGCSLAIAALPATEDPEFTERAAPILA DLRQLEAIAVQATRISRITPELIKALGAVRRHHDELMRLGATAPGATLAQRLYAARRR ANLSTLETAQAAGVAEEMIVGAEAEEELPAEATEAIEALIRQIN rv3864 (espE) cds [SEQ ID NO: 8] atggcatcgggtagcggtctttgcaagacgacgagtaactttatttggggccagttactcttgcttggagagggaatccccgacccagg cgacattttcaacaccggttcgtcgctgttcaaacaaatcagcgacaaaatgggactcgccattccgggcaccaactggatcggccaa gcggcggaagcttacctaaaccagaacatcgcgcaacaacttcgcgcacaggtgatgggcgatctcgacaaattaaccggcaacatg atctcgaatcaggccaaatacgtctccgatacgcgcgacgtcctgcgggccatgaagaagatgattgacggtgtctacaaggtttgtaa gggcctcgaaaagattccgctgctcggccacttgtggtcgtgggagctcgcaatccctatgtccggcatcgcgatggccgttgtcggc ggcgcattgctctatctaacgattatgacgctgatgaatgcgaccaacctgaggggaattctcggcaggctgatcgagatgttgacgac cttgccaaagttccccggcctgcccgggttgcccagcctgcccgacatcatcgacggcctctggccgccgaagttgcccgacattccg atccccggcctgcccgacatcccgggcctacccgacttcaaatggccgcccacccccggcagcccgttgttccccgacctcccgtcg ttcccagggttccccgggttcccggagttccccgccatccccgggttccccgcactgcccgggttgcccagcattcccaacttgttccc cggcttgccgggtctgggcgacctgctgcccggcgtaggcgatttgggcaagttacccacctggactgagctggccgctttgcctgac ttcttgggcggcttcgccggcctgcccagcttgggttttggcaatctgctcagctttgccagtttgcccaccgtgggtcaggtgaccgccaccatgggtcagctgcaacagctcgtggcggccggcggtggccccagccaactggccagcatgggcagccaacaagcgcaactg atctcgtcgcaggcccagcaaggaggccagcagcacgccaccctcgtgagcgacaagaaggaagacgaggaaggcgtggccga ggcggagcgtgcacccatcgacgctggcaccgcggccagccaacgggggcaggaggggaccgtcctttgaRv3864 (EspE) aa seq [SEQ ID NO: 9]MASGSGLCKTTSNFIWGQLLLLGEGIPDPGDIFNTGSSLFKQISDKMGLAIPGTNWIGQ AAEAYLNQNIAQQLRAQVMGDLDKLTGNMISNQAKYVSDTRDVLRAMKKMIDGV YKVCKGLEKIPLLGHLWSWELAIPMSGIAMAVVGGALLYLTIMTLMNATNLRGILGRLIEMLTTLPKFPGLPGLPSLPDIIDGLWPPKLPDIPIPGLPDIPGLPDFKWPPTPGSPLFPD LPSFPGFPGFPEFPAIPGFPALPGLPSIPNLFPGLPGLGDLLPGVGDLGKLPTWTELAAL PDFLGGFAGLPSLGFGNLLSFASLPTVGQVTATMGQLQQLVAAGGGPSQLASMGSQ QAQLISSQAQQGGQQHATLVSDKKEDEEGVAEAERAPIDAGTAASQRGQEGTVL rv3865 (espF) cds [SEQ ID NO: 10] atgaccggatttctcggtgtcgtgccttcgttcctgaaggtgctggcgggcatgcacaacgagatcgtgggtgatatcaaaagggcgac cgatacggtcgccgggattagcggacgagttcagcttacccatggttcgttcacgtcgaaattcaatgacacgctgcaagagtttgaga ccacccgtagcagcacgggcacgggtttgcagggagtcaccagcggactggccaataatctgctcgcagccgccggcgcctacctc aaggccgacgatggcctagccggtgttatcgacaagattttcggttgaRv3865 (EspF) aa seq [SEQ ID NO: 11]MTGFLGVVPSFLKVLAGMHNEIVGDIKRATDTVAGISGRVQLTHGSFTSKFNDTLQE FETTRSSTGTGLQGVTSGLANNLLAAAGAYLKADDGLAGVIDKIFG rv3866 (espGl) cds [SEQ ID NO: 12] atgacgggtccgtccgctgcaggccgcgcgggcaccgccgacaacgtggtcggcgtcgaggtaaccatcgacggcatgttggtgat cgccgatcggttacacctggttgatttccctgtcacgcttgggattcggccgaatatcccgcaagaggatctgcgagacatcgtctggga acaggtgcagcgtgacctcacagcgcaaggggtgctcgacctccacggggagccccaaccgacggtcgcggagatggtcgaaac cctgggcaggccagatcggaccttggagggtcgctggtggcggcgcgacattggcggcgtcatggtgcgcttcgtcgtgtgccgca ggggcgaccgccatgtgatcgcggcgcgcgacggcgacatgctggtgctgcagttggtggcgccgcaggtcggcttggcgggcatggtgacagcggtgctggggcccgccgaacccgccaacgtcgaacccctgacgggtgtggcaaccgagctagccgaatgcacaac cgcgtcccaattgacgcaatacggtatcgcaccggcctcggcccgcgtctatgccgagatcgtgggtaacccgaccggctgggtgga gatcgttgccagccaacgccaccccggcggcaccacgacgcagaccgacgccgccgctggcgtcctggactccaagctcggtagg ctggtgtcgcttccccgccgtgttggaggcgacctgtacggaagcttcctgcccggcactcagcagaacttggagcgtgcgctggacg gcttgctagagctgctccctgcgggcgcttggctagatcacacctcagatcacgcacaagcctcctcccgaggctgaRv3866 (EspGl) aa seq [SEQ ID NO: 13]MTGPSAAGRAGTADNVVGVEVTIDGMLVIADRLHLVDFPVTLGIRPNIPQEDLRDIV WEQVQRDLTAQGVLDLHGEPQPTVAEMVETLGRPDRTLEGRWWRRDIGGVMVRFV VCRRGDRHVIAARDGDMLVLQLVAPQVGLAGMVTAVLGPAEPANVEPLTGVATEL AECTTASQLTQYGIAPASARVYAEIVGNPTGWVEIVASQRHPGGTTTQTDAAAGVLDSKLGRLVSLPRRVGGDLYGSFLPGTQQNLERALDGLLELLPAGAWLDHTSDHAQASS RG rv3867 (espH) cds [SEQ ID NO: 14] atggtggacccgccgggcaacgacgacgaccacggtgatctcgacgccctcgatttctccgccgcccacaccaacgaggcgtcgcc gctggacgccttagacgactatgcgccggtgcagaccgatgacgccgaaggcgacctggacgccctccatgcgctcaccgaacgc gacgaggagccggagctggagttgttcacggtgaccaaccctcaagggtcggtgtcggtctcaaccctgatggacggcagaatcca gcacgtcgagctgacggacaaggcgaccagcatgtccgaagcgcagctggccgacgagatcttcgttattgccgatctggcccgcc aaaaggcgcgggcgtcgcagtacacgttcatggtggagaacatcggtgaactgaccgacgaagacgcagaaggcagcgccctgct gcgggaattcgtggggatgaccctgaatctgccgacgccggaagaggctgccgcagccgaagccgaagtgttcgccacccgctac gatgtcgactacacctcccggtacaaggccgatgactgaRv3867 (EspH) aa seq [SEQ ID NO: 15]MVDPPGNDDDHGDLDALDFSAAHTNEASPLDALDDYAPVQTDDAEGDLDALHALT ERDEEPELELFTVTNPQGSVSVSTLMDGRIQHVELTDKATSMSEAQLADEIFVIADLA RQKARASQYTFMVENIGELTDEDAEGSALLREFVGMTLNLPTPEEAAAAEAEVFATR YDVDYTSRYKADD rv3868 (eccAl) cds [SEQ ID NO: 16]atgactgatcgcttggccagtctgttcgaaagcgccgtcagcatgttgccgatgtcggaggcgcggtcgctagatctgttcaccgagat caccaactacgacgaatccgcttgcgacgcatggatcggccggatccggtgtggggacaccgaccgggtgacgctgtttcgcgcctg gtattcgcgccgcaatttcggacagttgtcgggatcggtccagatctcgatgagcacgttaaacgccaggattgccatcggggggctgt acggcgatatcacctacccggtcacctcgccgctagcgatcaccatgggctttgccgcatgcgaggcagcgcaaggcaattacgccg acgccatggaggccttagaggccgccccggtcgcgggttccgagcacctggtggcgtggatgaaggcggttgtctacggcgcggc cgaacgctggaccgacgtgatcgaccaggtcaagagtgctgggaaatggccggacaagtttttggccggcgcggccggtgtggcgc acggggttgccgcggcaaacctggccttgttcaccgaagccgaacgccgactcaccgaggccaacgactcgcccgccggtgaggc gtgtgcgcgcgccatcgcctggtatctggcgatggcacggcgcagccagggcaacgaaagcgccgcggtggcgctgctggaatgg ttacagaccactcaccccgagcccaaagtggctgcggcgctgaaggatccctcctaccggctgaagacgaccaccgccgaacagat cgcatcccgcgccgatccctgggatccgggcagtgtcgtgaccgacaactccggccgggagcggctgctcgccgaggcccaagcc gaactcgaccgccaaattgggctcacccgggttaaaaatcagattgaacgctaccgcgcggcgacgctgatggcccgggtccgcgc cgccaagggtatgaaggtcgcccagcccagcaagcacatgatcttcaccggaccgcccggtaccggcaagaccacgatcgcgcgg gtggtggccaatatcctggccggcttaggcgtcattgccgaacccaaactcgtcgagacgtcgcgcaaggacttcgtcgccgagtacg aggggcaatcggcggtcaagaccgctaagacgatcgatcaggcgctgggcggggtgcttttcatcgacgaggcttatgcgctggtgc aggaaagagacggccgcaccgatccgttcggtcaagaggcgctggacacgctgctggcgcggatggagaacgaccgggaccgg ctggtggtgatcatcgccgggtacagctccgacatagatcggctgctggaaaccaacgagggtctgcggtcgcggttcgccactcgc atcgagttcgacacctattcccccgaggaactcctcgagatcgccaacgtcattgccgctgctgatgattcggcgttgaccgcagaggc ggccgagaactttcttcaggccgccaagcagttggagcagcgcatgttgcgcggccggcgcgccctggacgtcgccggcaacggtc ggtatgcgcgccagctggtggaggccagcgagcaatgccgggacatgcgtctagcccaggtcctcgatatcgacaccctcgacgaa gaccggcttcgcgagatcaacggctcagatatggcggaggctatcgccgcggtgcacgcacacctcaacatgagagaatgaRv3868 (EccAl) aa seq [SEQ ID NO: 17]MTDRLASLFESAVSMLPMSEARSLDLFTEITNYDESACDAWIGRIRCGDTDRVTLFRA WYSRRNFGQLSGSVQISMSTLNARIAIGGLYGDITYPVTSPLAITMGFAACEAAQGNY ADAMEALEAAPVAGSEHLVAWMKAVVYGAAERWTDVIDQVKSAGKWPDKFLAG AAGVAHGVAAANLALFTEAERRLTEANDSPAGEACARAIAWYLAMARRSQGNESA AVALLEWLQTTHPEPKVAAALKDPSYRLKTTTAEQIASRADPWDPGSVVTDNSGRE RLLAEAQAELDRQIGLTRVKNQIERYRAATLMARVRAAKGMKVAQPSKHMIFTGPP GTGKTTIARVVANILAGLGVIAEPKLVETSRKDFVAEYEGQSAVKTAKTIDQALGGV LFIDEAYALVQERDGRTDPFGQEALDTLLARMENDRDRLVVIIAGYSSDIDRLLETNEGLRSRFATRIEFDTYSPEELLEIANVIAAADDSALTAEAAENFLQAAKQLEQRMLRGR RALDVAGNGRYARQLVEASEQCRDMRLAQVLDIDTLDEDRLREINGSDMAEAIAAV HAHLNMRE rv3869 (eccBl) cds [SEQ ID NO: 18] atggggcttcgcctcaccaccaaggttcaggttagcggctggcgttttctgctgcgccggctcgaacacgccatcgtgcgccgggaca cccggatgtttgacgacccgctgcagttctacagccgctcgatcgctcttggcatcgtcgtcgcggtcctgattctggcgggtgccgcg ctgctggcgtacttcaaaccacaaggcaaactcggcggcaccagcctgttcaccgaccgcgcgaccaaccagctttacgtgctgctgt ccggacagttgcatccggtctacaacctgacttcggcgcggctggtgctgggcaatccggccaacccggccaccgtgaagtcctccg aactgagcaagctgccgatgggccagaccgttggaatccccggcgccccctacgccacgcctgtttcggcgggcagcacctcgatct ggaccctatgcgacaccgtcgcccgagccgactccacttccccggtagtgcagaccgcggtcatcgcgatgccgttggagatcgatg cttcgatcgatccgctccagtcacacgaagcggtgctggtgtcctaccagggcgaaacctggatcgtcacaactaagggacgccacg ccatagatctgaccgaccgcgccctcacctcgtcgatggggataccggtgacggccaggccaaccccgatctcggagggcatgttca acgcgctgcctgatatggggccctggcagctgccgccgataccggcggcgggcgcgcccaattcgcttggcctacctgatgatctag tgatcggatcggtcttccagatccacaccgacaagggcccgcaatactatgtggtgctgcccgacggcatcgcgcaggtcaacgcga caaccgctgcggcgctgcgcgccacccaggcgcacgggctggtcgcgccaccggcaatggtgcccagtctggtcgtcagaatcgc cgaacgggtatacccctcaccgctacccgatgaaccgctcaagatcgtgtcccggccgcaggatcccgcgctgtgctggtcatggca acgcagcgccggcgaccagtcgccgcagtcaacggtgctgtccggccggcatctgccgatatcgccctcagcgatgaacatgggga tcaagcagatccacgggacggcgaccgtttacctcgacggcggaaaattcgtggcactgcaatcccccgatcctcgatacaccgaatc gatgtactacatcgatccacagggcgtgcgttatggggtgcctaacgcggagacagccaagtcgctgggcctgagttcaccccaaaa cgcgccctgggagatcgttcgtctcctggtcgacggtccggtgctgtcgaaagatgccgcactgctcgagcacgacacgctgcccgc tgaccctagcccccgaaaagttcccgccggagcctccggagccccctgaRv3869 (EccBl) aa seq [SEQ ID NO: 19]MGLRLTTKVQVSGWRFLLRRLEHAIVRRDTRMFDDPLQFYSRSIALGIVVAVLILAG AALLAYFKPQGKLGGTSLFTDRATNQLYVLLSGQLHPVYNLTSARLVLGNPANPATV KSSELSKLPMGQTVGIPGAPYATPVSAGSTSIWTLCDTVARADSTSPVVQTAVIAMPL EIDASIDPLQSHEAVLVSYQGETWIVTTKGRHAIDLTDRALTSSMGIPVTARPTPISEG MFNALPDMGPWQLPPIPAAGAPNSLGLPDDLVIGSVFQIHTDKGPQYYVVLPDGIAQ VNATTAAALRATQAHGLVAPPAMVPSLVVRIAERVYPSPLPDEPLKIVSRPQDPALCWSWQRSAGDQSPQSTVLSGRHLPISPSAMNMGIKQIHGTATVYLDGGKFVALQSPDP RYTESMYYIDPQGVRYGVPNAETAKSLGLSSPQNAPWEIVRLLVDGPVLSKDAALLE HDTLPADPSPRKVPAGASGAP rv3870 (eccCal) cds [SEQ ID NO: 20] atgacgaccaagaagttcactcccaccattacccgtggcccccggttgaccccgggcgagatcagcctcacgccgcccgatgacctg ggcatcgacatcccaccgtcgggcgtccaaaagatccttccctacgtgatgggtggcgccatgctcggcatgatcgccatcatggtgg ccggcggcaccaggcagctgtcgccgtacatgttgatgatgccgctgatgatgatcgtgatgatggtcggcggtctggccggtagcac cggtggtggcggcaagaaggtgcccgaaatcaacgccgaccgcaaggagtacctgcggtatttggcaggactacgcacccgagtg acgtcctcggccacctctcaggtggcgttcttctcctaccacgcaccgcatcccgaggatctgttgtcgatcgtcggcacccaacggca gtggtcccggccggccaacgccgacttctatgcggccacccgaatcggtatcggtgaccagccggcggtggatcgattattgaagcc ggccgtcggcggggagttggccgccgccagcgcagcacctcagccgttcctggagccggtcagtcatatgtgggtggtcaagtttct acgaacccatggattgatccatgactgcccgaaactgctgcaactccgtacctttccgactatcgcgatcggcggggacttggcgggg gcagccggcctgatgacggcgatgatctgtcacctagccgtgttccacccaccggacctgctgcagatccgggtgctcaccgaggaa cccgacgaccccgactggtcctggctcaaatggcttccgcacgtacagcaccagaccgaaaccgatgcggccgggtccacccggct gatcttcacgcgccaggaaggtctgtcggacctggccgcgcgcgggccacacgcacccgattcgcttcccggcggcccctacgtag tcgtcgtcgacctgaccggcggcaaggctggattcccgcccgacggtagggccggtgtcacggtgatcacgttgggcaaccatcgc ggctcggcctaccgcatcagggtgcacgaggatgggacggctgatgaccggctccctaaccaatcgtttcgccaggtgacatcggtc accgatcggatgtcgccgcagcaagccagccgtatcgcgcgaaagttggccggatggtccatcacgggcaccatcctcgacaagac gtcgcgggtccagaagaaggtggccaccgactggcaccagctggtcggtgcgcaaagtgtcgaggagataacaccttcccgctgga ggatgtacaccgacaccgaccgtgaccggctaaagatcccgtttggtcatgaactaaagaccggcaacgtcatgtacctggacatcaa agagggcgcggaattcggcgccggaccgcacggcatgctcatcgggaccacggggtctgggaagtccgaattcctgcgcaccctg atcctgtcgctggtggcaatgactcatccagatcaggtgaatctcctgctcaccgacttcaaaggtggttcaaccttcctgggaatggaa aagcttccgcacactgccgctgtcgtcaccaacatggccgaggaagccgagctcgtcagccggatgggcgaggtgttgaccggaga actcgatcggcgccagtcgatcctccgacaggccgggatgaaagtcggcgcggccggagccctgtccggcgtggccgaatacgag aagtaccgcgaacgcggtgccgacctacccccgctgccaacgcttttcgtcgtcgtcgacgagttcgccgagctgttgcagagtcacc cggacttcatcgggctgttcgaccggatctgccgcgtcgggcggtcgctgagggtccatctgctgctggctacccagtcgctgcagac cggcggtgttcgcatcgacaaactggagccaaacctgacatatcgaatcgcattgcgcaccaccagctctcatgaatccaaggcggta atcggcacaccggaggcgcagtacatcaccaacaaggagagcggtgtcgggtttctccgggtcggcatggaagacccggtcaagttcagcaccttctacatcagtgggccatacatgccgccggcggcaggcgtcgaaaccaatggtgaagccggagggcccggtcaacag accactagacaagccgcgcgcattcacaggttcaccgcggcaccggttctcgaggaggcgccgacaccgtgaRv3870 (EccCal) aa seq [SEQ ID NO: 21]MTTKKFTPTITRGPRLTPGEISLTPPDDLGIDIPPSGVQKILPYVMGGAMLGMIAIMVA GGTRQLSPYMLMMPLMMIVMMVGGLAGSTGGGGKKVPEINADRKEYLRYLAGLRT RVTSSATSQVAFFSYHAPHPEDLLSIVGTQRQWSRPANADFYAATRIGIGDQPAVDRL LKPAVGGELAAASAAPQPFLEPVSHMWVVKFLRTHGLIHDCPKLLQLRTFPTIAIGGD LAGAAGLMTAMICHLAVFHPPDLLQIRVLTEEPDDPDWSWLKWLPHVQHQTETDAA GSTRLIFTRQEGLSDLAARGPHAPDSLPGGPYVVVVDLTGGKAGFPPDGRAGVTVITL GNHRGSAYRIRVHEDGTADDRLPNQSFRQVTSVTDRMSPQQASRIARKLAGWSITGT ILDKTSRVQKKVATDWHQLVGAQSVEEITPSRWRMYTDTDRDRLKIPFGHELKTGN VMYLDIKEGAEFGAGPHGMLIGTTGSGKSEFLRTLILSLVAMTHPDQVNLLLTDFKG GSTFLGMEKLPHTAAVVTNMAEEAELVSRMGEVLTGELDRRQSILRQAGMKVGAA GALSGVAEYEKYRERGADLPPLPTLFVVVDEFAELLQSHPDFIGLFDRICRVGRSLRV HLLLATQSLQTGGVRIDKLEPNLTYRIALRTTSSHESKAVIGTPEAQYITNKESGVGFL RVGMEDPVKFSTFYISGPYMPPAAGVETNGEAGGPGQQTTRQAARIHRFTAAPVLEE APTP rv3871 (eccCbl) cds [SEQ ID NO: 22] atgactgctgaaccggaagtacggacgctgcgcgaggttgtgctggaccagctcggcactgctgaatcgcgtgcgtacaagatgtgg ctgccgccgttgaccaatccggtcccgctcaacgagctcatcgcccgtgatcggcgacaacccctgcgatttgccctggggatcatgg atgaaccgcgccgccatctacaggatgtgtggggcgtagacgtttccggggccggcggcaacatcggtattgggggcgcacctcaa accgggaagtcgacgctactgcagacgatggtgatgtcggccgccgccacacactcaccgcgcaacgttcagttctattgcatcgacc taggtggcggcgggctgatctatctcgaaaaccttccacacgtcggtggggtagccaatcggtccgagcccgacaaggtcaaccggg tggtcgcagagatgcaagccgtcatgcggcaacgggaaaccaccttcaaggaacaccgagtgggctcgatcgggatgtaccggca gctgcgtgacgatccaagtcaacccgttgcgtccgatccatacggcgacgtctttctgatcatcgacggatggcccggttttgtcggcga gttccccgaccttgaggggcaggttcaagatctggccgcccaggggctggcgttcggcgtccacgtcatcatctccacgccacgctg gacagagctgaagtcgcgtgttcgcgactacctcggcaccaagatcgagttccggcttggtgacgtcaatgaaacccagatcgaccg gattacccgcgagatcccggcgaatcgtccgggtcgggcagtgtcgatggaaaagcaccatctgatgatcggcgtgcccaggttcgacggcgtgcacagcgccgataacctggtggaggcgatcaccgcgggggtgacgcagatcgcttcccagcacaccgaacaggcacct ccggtgcgggtcctgccggagcgtatccacctgcacgaactcgacccgaacccgccgggaccagagtccgactaccgcactcgctg ggagattccgatcggcttgcgcgagacggacctgacgccggctcactgccacatgcacacgaacccgcacctactgatcttcggtgc ggccaaatcgggcaagacgaccattgcccacgcgatcgcgcgcgccatttgtgcccgaaacagtccccagcaggtgcggttcatgct cgcggactaccgctcgggcctgctggacgcggtgccggacacccatctgctgggcgccggcgcgatcaaccgcaacagcgcgtcg ctagacgaggccgttcaagcactggcggtcaacctgaagaagcggttgccgccgaccgacctgacgacggcgcagctacgctcgc gttcgtggtggagcggatttgacgtcgtgcttctggtcgacgattggcacatgatcgtgggtgccgccggggggatgccgccgatggc accgctggccccgttattgccggcggcggcagatatcgggttgcacatcattgtcacctgtcagatgagccaggcttacaaggcaacc atggacaagttcgtcggcgccgcattcgggtcgggcgctccgacaatgttcctttcgggcgagaagcaggaattcccatccagtgagtt caaggtcaagcggcgcccccctggccaggcatttctcgtctcgccagacggcaaagaggtcatccaggccccctacatcgagcctcc agaagaagtgttcgcagcacccccaagcgccggttaaRv3871 (EccCbl) aa seq [SEQ ID NO: 23]MTAEPEVRTLREVVLDQLGTAESRAYKMWLPPLTNPVPLNELIARDRRQPLRFALGI MDEPRRHLQDVWGVDVSGAGGNIGIGGAPQTGKSTLLQTMVMSAAATHSPRNVQF YCIDLGGGGLIYLENLPHVGGVANRSEPDKVNRVVAEMQAVMRQRETTFKEHRVGS IGMYRQLRDDPSQPVASDPYGDVFLIIDGWPGFVGEFPDLEGQVQDLAAQGLAFGVH VIISTPRWTELKSRVRDYLGTKIEFRLGDVNETQIDRITREIPANRPGRAVSMEKHHLM IGVPRFDGVHSADNLVEAITAGVTQIASQHTEQAPPVRVLPERIHLHELDPNPPGPESD YRTRWEIPIGLRETDLTPAHCHMHTNPHLLIFGAAKSGKTTIAHAIARAICARNSPQQV RFMLADYRSGLLDAVPDTHLLGAGAINRNSASLDEAVQALAVNLKKRLPPTDLTTAQLRSRSWWSGFDVVLLVDDWHMIVGAAGGMPPMAPLAPLLPAAADIGLHIIVTCQM SQAYKATMDKFVGAAFGSGAPTMFLSGEKQEFPSSEFKVKRRPPGQAFLVSPDGKEV IQAPYIEPPEEVFAAPPSAG rv3872 (pe35) cds [SEQ ID NO: 24] atggaaaaaatgtcacatgatccgatcgctgccgacattggcacgcaagtgagcgacaacgctctgcacggcgtgacggccggctcg acggcgctgacgtcggtgaccgggctggttcccgcgggggccgatgaggtctccgcccaagcggcgacggcgttcacatcggagg gcatccaattgctggcttccaatgcatcggcccaagaccagctccaccgtgcgggcgaagcggtccaggacgtcgcccgcacctatt cgcaaatcgacgacggcgccgccggcgtcttcgccgaatagRv3872 (PE35) aa seq [SEQ ID NO: 25]MEKMSHDPIAADIGTQVSDNALHGVTAGSTALTSVTGLVPAGADEVSAQAATAFTSE GIQLLASNASAQDQLHRAGEAVQDVARTYSQIDDGAAGVFAE rv3873 (ppe68) cds [SEQ ID NO: 26] atgctgtggcacgcaatgccaccggagctaaataccgcacggctgatggccggcgcgggtccggctccaatgcttgcggcggccgc gggatggcagacgctttcggcggctctggacgctcaggccgtcgagttgaccgcgcgcctgaactctctgggagaagcctggactg gaggtggcagcgacaaggcgcttgcggctgcaacgccgatggtggtctggctacaaaccgcgtcaacacaggccaagacccgtgc gatgcaggcgacggcgcaagccgcggcatacacccaggccatggccacgacgccgtcgctgccggagatcgccgccaaccacat cacccaggccgtccttacggccaccaacttcttcggtatcaacacgatcccgatcgcgttgaccgagatggattatttcatccgtatgtgg aaccaggcagccctggcaatggaggtctaccaggccgagaccgcggttaacacgcttttcgagaagctcgagccgatggcgtcgat ccttgatcccggcgcgagccagagcacgacgaacccgatcttcggaatgccctcccctggcagctcaacaccggttggccagttgcc gccggcggctacccagaccctcggccaactgggtgagatgagcggcccgatgcagcagctgacccagccgctgcagcaggtgac gtcgttgttcagccaggtgggcggcaccggcggcggcaacccagccgacgaggaagccgcgcagatgggcctgctcggcaccag tccgctgtcgaaccatccgctggctggtggatcaggccccagcgcgggcgcgggcctgctgcgcgcggagtcgctacctggcgca ggtgggtcgttgacccgcacgccgctgatgtctcagctgatcgaaaagccggttgccccctcggtgatgccggcggctgctgccgga tcgtcggcgacgggtggcgccgctccggtgggtgcgggagcgatgggccagggtgcgcaatccggcggctccaccaggccgggt ctggtcgcgccggcaccgctcgcgcaggagcgtgaagaagacgacgaggacgactgggacgaagaggacgactggtgaRv3873 (PPE68) aa seq [SEQ ID NO: 27]MLWHAMPPELNTARLMAGAGPAPMLAAAAGWQTLSAALDAQAVELTARLNSLGE AWTGGGSDKALAAATPMVVWLQTASTQAKTRAMQATAQAAAYTQAMATTPSLPEI AANHITQAVLTATNFFGINTIPIALTEMDYFIRMWNQAALAMEVYQAETAVNTLFEK LEPMASILDPGASQSTTNPIFGMPSPGSSTPVGQLPPAATQTLGQLGEMSGPMQQLTQ PLQQVTSLFSQVGGTGGGNPADEEAAQMGLLGTSPLSNHPLAGGSGPSAGAGLLRAE SLPGAGGSLTRTPLMSQLIEKPVAPSVMPAAAAGSSATGGAAPVGAGAMGQGAQSG GSTRPGLVAPAPLAQEREEDDEDDWDEEDDW rv3874 (esxB) cds [SEQ ID NO: 28]atggcagagatgaagaccgatgccgctaccctcgcgcaggaggcaggtaatttcgagcggatctccggcgacctgaaaacccagat cgaccaggtggagtcgacggcaggttcgttgcagggccagtggcgcggcgcggcggggacggccgcccaggccgcggtggtgc gcttccaagaagcagccaataagcagaagcaggaactcgacgagatctcgacgaatattcgtcaggccggcgtccaatactcgagg gccgacgaggagcagcagcaggcgctgtcctcgcaaatgggcttctgaRv3874 (EsxB) aa seq [SEQ ID NO: 29]MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAV VRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF rv3875 (esxA) cds [SEQ ID NO: 30] atgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctccttg acgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatg ggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggcttcgac cgaaggcaacgtcactgggatgttcgcatagRv3875 (EsxA) aa seq [SEQ ID NO: 31]MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQ KWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA rv3876 (espl) cds [SEQ ID NO: 32] atggcggccgactacgacaagctcttccggccgcacgaaggtatggaagctccggacgatatggcagcgcagccgttcttcgacccc agtgcttcgtttccgccggcgcccgcatcggcaaacctaccgaagcccaacggccagactccgcccccgacgtccgacgacctgtc ggagcggttcgtgtcggccccgccgccgccacccccacccccacctccgcctccgccaactccgatgccgatcgccgcaggagag ccgccctcgccggaaccggccgcatctaaaccacccacaccccccatgcccatcgccggacccgaaccggccccacccaaaccac ccacaccccccatgcccatcgccggacccgaaccggccccacccaaaccacccacacctccgatgcccatcgccggacctgcacc caccccaaccgaatcccagttggcgccccccagaccaccgacaccacaaacgccaaccggagcgccgcagcaaccggaatcacc ggcgccccacgtaccctcgcacgggccacatcaaccccggcgcaccgcaccagcaccgccctgggcaaagatgccaatcggcga acccccgcccgctccgtccagaccgtctgcgtccccggccgaaccaccgacccggcctgccccccaacactcccgacgtgcgcgc cggggtcaccgctatcgcacagacaccgaacgaaacgtcgggaaggtagcaactggtccatccatccaggcgcggctgcgggcagaggaagcatccggcgcgcagctcgcccccggaacggagccctcgccagcgccgttgggccaaccgagatcgtatctggctccgcc cacccgccccgcgccgacagaacctccccccagcccctcgccgcagcgcaactccggtcggcgtgccgagcgacgcgtccaccc cgatttagccgcccaacatgccgcggcgcaacctgattcaattacggccgcaaccactggcggtcgtcgccgcaagcgtgcagcgc cggatctcgacgcgacacagaaatccttaaggccggcggccaaggggccgaaggtgaagaaggtgaagccccagaaaccgaagg ccacgaagccgcccaaagtggtgtcgcagcgcggctggcgacattgggtgcatgcgttgacgcgaatcaacctgggcctgtcaccc gacgagaagtacgagctggacctgcacgctcgagtccgccgcaatccccgcgggtcgtatcagatcgccgtcgtcggtctcaaaggt ggggctggcaaaaccacgctgacagcagcgttggggtcgacgttggctcaggtgcgggccgaccggatcctggctctagacgcgg atccaggcgccggaaacctcgccgatcgggtagggcgacaatcgggcgcgaccatcgctgatgtgcttgcagaaaaagagctgtcg cactacaacgacatccgcgcacacactagcgtcaatgcggtcaatctggaagtgctgccggcaccggaatacagctcggcgcagcg cgcgctcagcgacgccgactggcatttcatcgccgatcctgcgtcgaggttttacaacctcgtcttggctgattgtggggccggcttcttc gacccgctgacccgcggcgtgctgtccacggtgtccggtgtcgtggtcgtggcaagtgtctcaatcgacggcgcacaacaggcgtcg gtcgcgttggactggttgcgcaacaacggttaccaagatttggcgagccgcgcatgcgtggtcatcaatcacatcatgccgggagaac ccaatgtcgcagttaaagacctggtgcggcatttcgaacagcaagttcaacccggccgggtcgtggtcatgccgtgggacaggcacat tgcggccggaaccgagatttcactcgacttgctcgaccctatctacaagcgcaaggtcctcgaattggccgcagcgctatccgacgatt tcgagagggctggacgtcgttgaRv3876 (EspI) aa seq [SEQ ID NO: 33]MAADYDKLFRPHEGMEAPDDMAAQPFFDPSASFPPAPASANLPKPNGQTPPPTSDDL SERFVSAPPPPPPPPPPPPPTPMPIAAGEPPSPEPAASKPPTPPMPIAGPEPAPPKPPTPPM PIAGPEPAPPKPPTPPMPIAGPAPTPTESQLAPPRPPTPQTPTGAPQQPESPAPHVPSHGP HQPRRTAPAPPWAKMPIGEPPPAPSRPSASPAEPPTRPAPQHSRRARRGHRYRTDTER NVGKVATGPSIQARLRAEEASGAQLAPGTEPSPAPLGQPRSYLAPPTRPAPTEPPPSPS PQRNSGRRAERRVHPDLAAQHAAAQPDSITAATTGGRRRKRAAPDLDATQKSLRPA AKGPKVKKVKPQKPKATKPPKVVSQRGWRHWVHALTRINLGLSPDEKYELDLHAR VRRNPRGSYQIAVVGLKGGAGKTTLTAALGSTLAQVRADRILALDADPGAGNLADR VGRQSGATIADVLAEKELSHYNDIRAHTSVNAVNLEVLPAPEYSSAQRALSDADWHF IADPASRFYNLVLADCGAGFFDPLTRGVLSTVSGVVVVASVSIDGAQQASVALDWLR NNGYQDLASRACVVINHIMPGEPNVAVKDLVRHFEQQVQPGRVVVMPWDRHIAAG TEISLDLLDPIYKRKVLELAAALSDDFERAGRRrv3877 (eccDl) cds [SEQ ID NO: 34] ttgagcgcacctgctgttgctgctggtcctaccgccgcgggggcaaccgctgcgcggcctgccaccacccgggtgacgatcctgacc ggcagacggatgaccgatttggtactgccagcggcggtgccgatggaaacttatattgacgacaccgtcgcggtgctttccgaggtgtt ggaagacacgccggctgatgtactcggcggcttcgactttaccgcgcaaggcgtgtgggcgttcgctcgtcccggatcgccgccgct gaagctcgaccagtcactcgatgacgccggggtggtcgacgggtcactgctgactctggtgtcagtcagtcgcaccgagcgctaccg accgttggtcgaggatgtcatcgacgcgatcgccgtgcttgacgagtcacctgagttcgaccgcacggcattgaatcgctttgtggggg cggcgatcccgcttttgaccgcgcccgtcatcgggatggcgatgcgggcgtggtgggaaactgggcgtagcttgtggtggccgttgg cgattggcatcctggggatcgctgtgctggtaggcagcttcgtcgcgaacaggttctaccagagcggccacctggccgagtgcctact ggtcacgacgtatctgctgatcgcaaccgccgcagcgctggccgtgccgttgccgcgcggggtcaactcgttgggggcgccacaag ttgccggcgccgctacggccgtgctgtttttgaccttgatgacgcggggcggccctcggaagcgtcatgagttggcgtcgtttgccgtg atcaccgctatcgcggtcatcgcggccgccgctgccttcggctatggataccaggactgggtccccgcgggggggatcgcattcggg ctgttcattgtgacgaatgcggccaagctgaccgtcgcggtcgcgcggatcgcgctgccgccgattccggtacccggcgaaaccgtg gacaacgaggagttgctcgatcccgtcgcgaccccggaggctaccagcgaagaaaccccgacctggcaggccatcatcgcgtcgg tgcccgcgtccgcggtccggctcaccgagcgcagcaaactggccaagcaacttctgatcggatacgtcacgtcgggcaccctgattct ggctgccggtgccatcgcggtcgtggtgcgcgggcacttctttgtacacagcctggtggtcgcgggtttgatcacgaccgtctgcggat ttcgctcgcggctttacgccgagcgctggtgtgcgtgggcgttgctggcggcgacggtcgcgattccgacgggtctgacggccaaac tcatcatctggtacccgcactatgcctggctgttgttgagcgtctacctcacggtagccctggttgcgctcgtggtggtcgggtcgatggc tcacgtccggcgcgtttcaccggtcgtaaaacgaactctggaattgatcgacggcgccatgatcgctgccatcattcccatgctgctgtg gatcaccggggtgtacgacacggtccgcaatatccggttctgaRv3877 (EccDl) aa seq [SEQ ID NO: 35]LSAPAVAAGPTAAGATAARPATTRVTILTGRRMTDLVLPAAVPMETYIDDTVAVLSE VLEDTPADVLGGFDFTAQGVWAFARPGSPPLKLDQSLDDAGVVDGSLLTLVSVSRTE RYRPLVEDVIDAIAVLDESPEFDRTALNRFVGAAIPLLTAPVIGMAMRAWWETGRSL WWPLAIGILGIAVLVGSFVANRFYQSGHLAECLLVTTYLLIATAAALAVPLPRGVNSL GAPQVAGAATAVLFLTLMTRGGPRKRHELASFAVITAIAVIAAAAAFGYGYQDWVP AGGIAFGLFIVTNAAKLTVAVARIALPPIPVPGETVDNEELLDPVATPEATSEETPTWQ AIIASVPASAVRLTERSKLAKQLLIGYVTSGTLILAAGAIAVVVRGHFFVHSLVVAGLITTVCGFRSRLYAERWCAWALLAATVAIPTGLTAKLIIWYPHYAWLLLSVYLTVALV ALWVGSMAHVRRVSPVVKRTLELIDGAMIAAIIPMLLWITGVYDTVRNIRF rv3878 (espJ) cds [SEQ ID NO: 36] atggctgaaccgttggccgtcgatcccaccggcttgagcgcagcggccgcgaaattggccggcctcgtttttccgcagcctccggcg ccgatcgcggtcagcggaacggattcggtggtagcagcaatcaacgagaccatgccaagcatcgaatcgctggtcagtgacgggct gcccggcgtgaaagccgccctgactcgaacagcatccaacatgaacgcggcggcggacgtctatgcgaagaccgatcagtcactg ggaaccagtttgagccagtatgcattcggctcgtcgggcgaaggcctggctggcgtcgcctcggtcggtggtcagccaagtcaggct acccagctgctgagcacacccgtgtcacaggtcacgacccagctcggcgagacggccgctgagctggcaccccgtgttgttgcgac ggtgccgcaactcgttcagctggctccgcacgccgttcagatgtcgcaaaacgcatcccccatcgctcagacgatcagtcaaaccgcc caacaggccgcccagagcgcgcagggcggcagcggcccaatgcccgcacagcttgccagcgctgaaaaaccggccaccgagca agcggagccggtccacgaagtgacaaacgacgatcagggcgaccagggcgacgtgcagccggccgaggtcgttgccgcggcac gtgacgaaggcgccggcgcatcaccgggccagcagcccggcgggggcgttcccgcgcaagccatggataccggagccggtgcc cgcccagcggcgagtccgctggcggcccccgtcgatccgtcgactccggcaccctcaacaaccacaacgttgtagRv3878 (EspJ) aa seq [SEQ ID NO: 37]MAEPLAVDPTGLSAAAAKLAGLVFPQPPAPIAVSGTDSVVAAINETMPSIESLVSDGL PGVKAALTRTASNMNAAADVYAKTDQSLGTSLSQYAFGSSGEGLAGVASVGGQPSQ ATQLLSTPVSQVTTQLGETAAELAPRVVATVPQLVQLAPHAVQMSQNASPIAQTISQT AQQAAQSAQGGSGPMPAQLASAEKPATEQAEPVHEVTNDDQGDQGDVQPAEVVAA ARDEGAGASPGQQPGGGVPAQAMDTGAGARPAASPLAAPVDPSTPAPSTTTTL rv3879c (espK) cds [SEQ ID NO: 38] atgagtattaccaggccgacgggcagctatgccagacagatgctggatccgggcggctgggtggaagccgatgaagacactttctat gaccgggcccaggaatatagccaggttttgcaaagggtcaccgatgtattggacacctgccgccagcagaaaggccacgtcttcgaa ggcggcctatggtccggcggcgccgccaatgctgccaacggcgccctgggtgcaaacatcaatcaattgatgacgctgcaggattat ctcgccacggtgattacctggcacaggcatattgccgggttgattgagcaagctaaatccgatatcggcaataatgtggatggcgctca acgggagatcgatatcctggagaatgaccctagcctggatgctgatgagcgccataccgccatcaattcattggtcacggcgacgcat ggggccaatgtcagtctggtcgccgagaccgctgagcgggtgctggaatccaagaattggaaacctccgaagaacgcactcgagga tttgcttcagcagaagtcgccgccacccccagacgtgcctaccctggtcgtgccatccccgggcacaccgggcacaccgggaaccccgatcaccccgggaaccccgatcaccccgggaaccccaatcacacccatcccgggagcgccggtaactccgatcacaccaacgcc cggcactcccgtcacgccggtgaccccgggcaagccggtcaccccggtgaccccggtcaaaccgggcacaccaggcgagccaac cccgatcacgccggtcacccccccggtcgccccggccacaccggcaaccccggccacgcccgttaccccagctcccgctccacac ccgcagccggctccggcaccggcgccatcgcctgggccccagccggttacaccggccactcccggtccgtctggtccagcaacac cgggcaccccagggggcgagccggcgccgcacgtcaaacccgcggcgttggcggagcaacctggtgtgccgggccagcatgcg ggcggggggacgcagtcggggcctgcccatgcggacgaatccgccgcgtcggtgacgccggctgcggcgtccggtgtcccgggc gcacgggcggcggccgccgcgccgagcggtaccgccgtgggagcgggcgcgcgttcgagcgtgggtacggccgcggcctcgg gcgcggggtcgcatgctgccactgggcgggcgccggtggctacctcggacaaggcggcggcaccgagcacgcgggcggcctcg gcgcggacggcacctcctgcccgcccgccgtcgaccgatcacatcgacaaacccgatcgcagcgagtctgcagatgacggtacgc cggtgtcgatgatcccggtgtcggcggctcgggcggcacgcgacgccgccactgcagctgccagcgcccgccagcgtggccgcg gtgatgcgctgcggttggcgcgacgcatcgcggcggcgctcaacgcgtccgacaacaacgcgggcgactacgggttcttctggatc accgcggtgaccaccgacggttccatcgtcgtggccaacagctatgggctggcctacatacccgacgggatggaattgccgaataag gtgtacttggccagcgcggatcacgcaatcccggttgacgaaattgcacgctgtgccacctacccggttttggccgtgcaagcctggg cggctttccacgacatgacgctgcgggcggtgatcggtaccgcggagcagttggccagttcggatcccggtgtggccaagattgtgct ggagccagatgacattccggagagcggcaaaatgacgggccggtcgcggctggaggtcgtcgacccctcggcggcggctcagct ggccgacactaccgatcagcgtttgctcgacttgttgccgccggcgccggtggatgtcaatccaccgggcgatgagcggcacatgct gtggttcgagctgatgaagcccatgaccagcaccgctaccggccgcgaggccgctcatctgcgggcgttccgggcctacgctgccc actcacaggagattgccctgcaccaagcgcacactgcgactgacgcggccgtccagcgtgtggccgtcgcggactggctgtactgg caatacgtcaccgggttgctcgaccgggccctggccgccgcatgctgaRv3879c (EspK) aa seq [SEQ ID NO: 39]MSITRPTGSYARQMLDPGGWVEADEDTFYDRAQEYSQVLQRVTDVLDTCRQQKGH VFEGGLWSGGAANAANGALGANINQLMTLQDYLATVITWHRHIAGLIEQAKSDIGN NVDGAQREIDILENDPSLDADERHTAINSLVTATHGANVSLVAETAERVLESKNWKP PKNALEDLLQQKSPPPPDVPTLVVPSPGTPGTPGTPITPGTPITPGTPITPIPGAPVTPITP TPGTPVTPVTPGKPVTPVTPVKPGTPGEPTPITPVTPPVAPATPATPATPVTPAPAPHPQ PAPAPAPSPGPQPVTPATPGPSGPATPGTPGGEPAPHVKPAALAEQPGVPGQHAGGGT QSGPAHADESAASVTPAAASGVPGARAAAAAPSGTAVGAGARSSVGTAAASGAGSH AATGRAPVATSDKAAAPSTRAASARTAPPARPPSTDHIDKPDRSESADDGTPVSMIPV SAARAARDAATAAASARQRGRGDALRLARRIAAALNASDNNAGDYGFFWITAVTTDGSIVVANSYGLAYIPDGMELPNKVYLASADHAIPVDEIARCATYPVLAVQAWAAFH DMTLRAVIGTAEQLASSDPGVAKIVLEPDDIPESGKMTGRSRLEVVDPSAAAQLADT TDQRLLDLLPPAPVDVNPPGDERHMLWFELMKPMTSTATGREAAHLRAFRAYAAHS QEIALHQAHTATDAAVQRVAVADWLYWQYVTGLLDRALAAAC rv3880c (espL) cds [SEQ ID NO: 40] gtgagcatggacgaattggacccgcatgtcgcccgggcgttgacgctggcggcgcggtttcagtcggccctagacgggacgctcaat cagatgaacaacggatccttccgcgccaccgacgaagccgagaccgtcgaagtgacgatcaatgggcaccagtggctcaccggcct gcgcatcgaagatggtttgctgaagaagctgggtgccgaggcggtggctcagcgggtcaacgaggcgctgcacaatgcgcaggcc gcggcgtccgcgtataacgacgcggcgggcgagcagctgaccgctgcgttatcggccatgtcccgcgcgatgaacgaaggaatgg cctaaRv3880c (EspL) aa seq [SEQ ID NO: 41]VSMDELDPHVARALTLAARFQSALDGTLNQMNNGSFRATDEAETVEVTINGHQWLT GLRIEDGLLKKLGAEAVAQRVNEALHNAQAAASAYNDAAGEQLTAALSAMSRAMN EGMA rv3881c (espB) cds [SEQ ID NO: 42] atgacgcagtcgcagaccgtgacggtggatcagcaagagattttgaacagggccaacgaggtggaggccccgatggcggacccac cgactgatgtccccatcacaccgtgcgaactcacggcggctaaaaacgccgcccaacagctggtattgtccgccgacaacatgcggg aatacctggcggccggtgccaaagagcggcagcgtctggcgacctcgctgcgcaacgcggccaaggcgtatggcgaggttgatga ggaggctgcgaccgcgctggacaacgacggcgaaggaactgtgcaggcagaatcggccggggccgtcggaggggacagttcgg ccgaactaaccgatacgccgagggtggccacggccggtgaacccaacttcatggatctcaaagaagcggcaaggaagctcgaaac gggcgaccaaggcgcatcgctcgcgcactttgcggatgggtggaacactttcaacctgacgctgcaaggcgacgtcaagcggttccg ggggtttgacaactgggaaggcgatgcggctaccgcttgcgaggcttcgctcgatcaacaacggcaatggatactccacatggccaa attgagcgctgcgatggccaagcaggctcaatatgtcgcgcagctgcacgtgtgggctaggcgggaacatccgacttatgaagacat agtcgggctcgaacggctttacgcggaaaacccttcggcccgcgaccaaattctcccggtgtacgcggagtatcagcagaggtcgga gaaggtgctgaccgaatacaacaacaaggcagccctggaaccggtaaacccgccgaagcctccccccgccatcaagatcgacccg cccccgcctccgcaagagcagggattgatccctggcttcctgatgccgccgtctgacggctccggtgtgactcccggtaccgggatgc cagccgcaccgatggttccgcctaccggatcgccgggtggtggcctcccggctgacacggcggcgcagctgacgtcggctgggcgggaagccgcagcgctgtcgggcgacgtggcggtcaaagcggcatcgctcggtggcggtggaggcggcggggtgccgtcggcgc cgttgggatccgcgatcgggggcgccgaatcggtgcggcccgctggcgctggtgacattgccggcttaggccagggaagggccgg cggcggcgccgcgctgggcggcggtggcatgggaatgccgatgggtgccgcgcatcagggacaagggggcgccaagtccaagg gttctcagcaggaagacgaggcgctctacaccgaggatcgggcatggaccgaggccgtcattggtaaccgtcggcgccaggacagt aaggagtcgaagtgaRv3881c (EspB) aa seq [SEQ ID NO: 43]MTQSQTVTVDQQEILNRANEVEAPMADPPTDVPITPCELTAAKNAAQQLVLSADNM REYLAAGAKERQRLATSLRNAAKAYGEVDEEAATALDNDGEGTVQAESAGAVGGD SSAELTDTPRVATAGEPNFMDLKEAARKLETGDQGASLAHFADGWNTFNLTLQGDV KRFRGFDNWEGDAATACEASLDQQRQWILHMAKLSAAMAKQAQYVAQLHVWAR REHPTYEDIVGLERLYAENPSARDQILPVYAEYQQRSEKVLTEYNNKAALEPVNPPKP PPAIKIDPPPPPQEQGLIPGFLMPPSDGSGVTPGTGMPAAPMVPPTGSPGGGLPADTAA QLTSAGREAAALSGDVAVKAASLGGGGGGGVPSAPLGSAIGGAESVRPAGAGDIAG LGQGRAGGGAALGGGGMGMPMGAAHQGQGGAKSKGSQQEDEALYTEDRAWTEA VIGNRRRQDSKESK rv3882c (eccEl) cds [SEQ ID NO: 44] atgagaaatcctttagggctgcggttcagcaccgggcacgccttgcttgcctccgcgttggccccgccatgcatcatcgcattcttggag acgcgctactggtgggcggggattgcgctggcctcgttgggcgtcatcgtggccacggtcactttctacggccgccggatcaccggct gggtggcggcggtgtacgcgtggttgcggcggcgccgacggcccccggattcctcgtcagaacctgtggtcggggccaccgtgaa gccaggagatcacgttgcggtgcgctggcaaggcgagtttctggtcgccgtaatcgagctcattccccgaccattcacgccgacggtc atcgtcgacgggcaagcccacaccgacgacatgctggacaccggactggtggaggagctcctgtcggtgcactgtcccgacttgga ggccgatatcgtctcagccggctaccgcgtcggcaataccgcagcgccggacgtggtgagtctgtatcagcaggtgatcgggacag acccggcgccggcgaaccgccggacctggatcgtgctgcgcgccgacccggaacgcacccgcaaatcggcgcagcgccgcgat gaaggcgtcgcaggactggcccggtatttggtggcgtccgcgacgcgcattgccgatcgactggctagccatggtgtcgacgcggtg tgtggccgcagcttcgatgactacgaccacgccaccgacatcggctttgtgcgggagaaatggtcgatgatcaaggggcgcgatgcc tacactgccgcctacgcggcgcccggaggtccggatgtatggtggtcggcgcgcgcggaccacaccatcaccagagtccgggtcg cgccggggatggccccgcagtccacggtgttgctgaccacggcggacaagcccaagacacccaggggcttcgcccgcctatttggc gggcagcggcccgcgctgcaaggccagcatctggtggccaaccgccactgccagctgccgatcgggtcagctggggtactggtcggcgagacggtgaaccgatgcccggtctacatgcccttcgacgatgtcgacatcgccctcaacctgggtgacgctcagacattcaccca gttcgtggtgcgtgcggcggcggcaggtgcgatggtcacagtcgggccacagttcgaggaatttgcccggttgatcggcgcacacat cgggcaggaggtaaaggtggcgtggccgaatgcgacgacctatctcggcccgcatcccggtattgaccgggtgattctgcggcaca atgtgatcggtaccccgcggcatcggcagctgccgattcgccgggtttccccacccgaggaaagccgctaccagatggcgctgccg aagtagRv3882c (EccEl) aa seq [SEQ ID NO: 45]MRNPLGLRFSTGHALLASALAPPCIIAFLETRYWWAGIALASLGVIVATVTFYGRRIT GWVAAVYAWLRRRRRPPDSSSEPVVGATVKPGDHVAVRWQGEFLVAVIELIPRPFTP TVIVDGQAHTDDMLDTGLVEELLSVHCPDLEADIVSAGYRVGNTAAPDVVSLYQQVI GTDPAPANRRTWIVLRADPERTRKSAQRRDEGVAGLARYLVASATRIADRLASHGV DAVCGRSFDDYDHATDIGFVREKWSMIKGRDAYTAAYAAPGGPDVWWSARADHTI TRVRVAPGMAPQSTVLLTTADKPKTPRGFARLFGGQRPALQGQHLVANRHCQLPIGS AGVLVGETVNRCPVYMPFDDVDIALNLGDAQTFTQFVVRAAAAGAMVTVGPQFEEF ARLIGAHIGQEVKVAWPNATTYLGPHPGIDRVILRHNVIGTPRHRQLPIRRVSPPEESR YQMALPK rv3883c (mycPl) cds [SEQ ID NO: 46] gtgcaccgtatctttctgatcacggtggcgctggcgttgctcaccgcgtcgcccgcatcggccatcacgccaccgccgatcgatccgg gcgcgttgccgcccgacgtgacgggcccggatcagcctaccgaacagcgcgttttgtgcgcgtcgcccaccacgctgccggggtcc gggttccacgatccgccgtggagcaacacgtatctgggcgtggccgatgcccacaagttcgcgaccggggccggggtgacggtgg cggtgatcgacaccggtgtcgacgcttcgccacgggtcccggcggaacctggcggcgatttcgtcgaccaggccggtaacggcctg tctgactgtgatgcccatgggactctcacagcatccatcatcgcgggccggcccgcgcccaccgacgggttcgtcggcgtcgcgccc gacgctcgactgctctcgctacgtcagacgtctgaggccttcgaaccggtcggctcacaagccaacccgaatgaccccaacgccacc ccggccgccggttccatccgcagtcttgcccgcgccgtggtgcacgccgccaacctcggcgtgggtgtgatcaacatcagtgaagcc gcctgctacaaggtgagcaggccgatcgatgaaacctcactgggtgcatccatcgactatgcggtcaacgtcaaaggcgtggtggtg gtggtcgcggccggcaacaccggtggcgattgcgtacagaatccggcgccggacccgtccacacccggcgacccacgcggctgg aacaatgtgcagaccgttgtcaccccggcgtggtacgcaccgctggtgttaagcgtcggcggtatcggccagaccgggatgcccagc tcgttctcgatgcacggaccgtgggtggacgtggccgcgcccgcagaaaacatcgtcgcgctcggcgacaccggtgaaccggtgaa tgcgctgcaaggccgggaggggccggtacccatcgccggcacctcgtttgccgcggcatatgtgtcgggtctggcggccctgcttcggcagcggttccccgacctgacgccggcgcagatcatccaccggatcaccgccaccgcgagacaccccgggggcggggtcgacga cctggtcggcgccggcgtcatcgatgcggtggccgcgctgacgtgggacattccgcccggccctgcttcggcgccatacaacgtca gacgacttccacccccggtggtggagccgggtcccgatcgtcgcccgattacggctgtggcgttggtggccgtcggccttacgttggc cctgggcctgggcgcgctggctagacgggcgctgagccgccgatgaRv3883c (MycPl) aa seq [SEQ ID NO: 47]VHRIFLITVALALLTASPASAITPPPIDPGALPPDVTGPDQPTEQRVLCASPTTLPGSGF HDPPWSNTYLGVADAHKFATGAGVTVAVIDTGVDASPRVPAEPGGDFVDQAGNGLS DCDAHGTLTASIIAGRPAPTDGFVGVAPDARLLSLRQTSEAFEPVGSQANPNDPNATPAAGSIRSLARAVVHAANLGVGVINISEAACYKVSRPIDETSLGASIDYAVNVKGVVVVVAAGNTGGDCVQNPAPDPSTPGDPRGWNNVQTVVTPAWYAPLVLSVGGIGQTGM PSSFSMHGPWVDVAAPAENIVALGDTGEPVNALQGREGPVPIAGTSFAAAYVSGLAA LLRQRFPDLTPAQIIHRITATARHPGGGVDDLVGAGVIDAVAALTWDIPPGPASAPYN VRRLPPPVVEPGPDRRPITAVALVAVGLTLALGLGALARRALSRR rv3884c (eccA2) cds [SEQ ID NO: 48] atgtcgagaatggtggacacgatgggtgatttactcactgcgcgccggcatttcgatcgggcgatgacgatcaagaatggccagggat gcgtggcggcgttgcctgagtttgtggctgccaccgaggccgatccgtcgatggccgacgcgtggctgggtcgtatcgcctgcggtg accgcgatctggcctcgcttaagcagctcaacgcccatagcgagtggctgcaccgcgagaccacgcggatcggccggacgttggcc gctgaggtccagctgggaccatccatcgggatcacggtgaccgacgcatctcaggtggggctggcgctgtcgtcggcgttgacgatc gcgggggagtatgcgaaggccgatgccctgttagcaaaccgcgagctattggattcgtggcgcaactaccagtggcatcagctggct cgggcgttcctgatgtacgtcacgcagcgatggcccgacgtgttgtcgacggccgccgaggatctgccgccacaggcgatcgtcatg ccggcggtgaccgcgtcgatttgtgcgctggcagcccacgccgccgcccatctcgggcaggggcgagtggccctggactggctgg accgggtggacgtgatcggacacagcaggtcatcggagcggttcggcgccgacgtgctcaccgcggcgatcggaccggccgatat tccgctgctggtcgccgacttggcgtatgtgcgggggatggtgtaccggcaactgcatgaggaggacaaggcccagatctggctgtc gaaggccaccatcaacggggtgctcaccgacgccgccaaagaagccctggcggacccgaacctgcgcttgattgttaccgatgaac gaaccatcgccagccgctccgaccgttgggatgcttcgacggcgaaaagccgcgaccagctcgatgacgacaatgcagcgcagcg gcgcggcgagctgctagccgagggccgggaactgctggccaaacaggtgggcctggcggcggtcaagcaagcggtatcggcgct ggaagaccaactcgaggtgcgcatgatgcgcctagagcacggcctaccggtggaggggcagaccaaccacatgttgctggtgggg ccaccaggcacaggtaagacaaccaccgctgaagcgctcggcaagatctacgccggcatggggatcgtgcgtcaccccgaaattcgagaagttcgccgatcggacttctgtgggcactacatcggggagtcaggacccaagacgaacgagctgatcgaaaagtcactcgggc gaatcattttcatggacgagttctactcgctgatcgaacgtcatcaagacggaacaccggacatgatcggcatggaggcggtcaatcaa ctcctggttcaattggaaacacaccgattcgacttctgtttcatcggggccggctatgaggatcaggtggatgaattcctcaccgtgaacc cgggtttggctggccggttcaaccgaaagctgcggttcgagtcttattcgccggtggagatcgtcgagattggacaccgctacgctaca ccgcgcgccagccagctcgatgacgccgcacgggaggtattcctcgacgcggtcaccaccatccgtaactacaccacccctagtgg gcagcacggtatcgacgctatgcaaaacggtcggttcgcccgcaacgtgatcgaacgcgccgaagggttccgggacacccgggtg gttgcgcaaaaacgtgcgggccaaccggtatcggttcaggatctgcagatcatcaccgccaccgacatcgatgccgcgatacgcagc gtgtgctcagacaaccgagacatggccgcgatcgtttggtagRv3884c (EccA2) aa seq [SEQ ID NO: 49]MSRMVDTMGDLLTARRHFDRAMTIKNGQGCVAALPEFVAATEADPSMADAWLGRI ACGDRDLASLKQLNAHSEWLHRETTRIGRTLAAEVQLGPSIGITVTDASQVGLALSSA LTIAGEYAKADALLANRELLDSWRNYQWHQLARAFLMYVTQRWPDVLSTAAEDLP PQAIVMPAVTASICALAAHAAAHLGQGRVALDWLDRVDVIGHSRSSERFGADVLTA AIGPADIPLLVADLAYVRGMVYRQLHEEDKAQIWLSKATINGVLTDAAKEALADPNL RLIVTDERTIASRSDRWDASTAKSRDQLDDDNAAQRRGELLAEGRELLAKQVGLAA VKQAVSALEDQLEVRMMRLEHGLPVEGQTNHMLLVGPPGTGKTTTAEALGKIYAG MGIVRHPEIREVRRSDFCGHYIGESGPKTNELIEKSLGRIIFMDEFYSLIERHQDGTPDM IGMEAVNQLLVQLETHRFDFCFIGAGYEDQVDEFLTVNPGLAGRFNRKLRFESYSPVE IVEIGHRYATPRASQLDDAAREVFLDAVTTIRNYTTPSGQHGIDAMQNGRFARNVIER AEGFRDTRVVAQKRAGQPVSVQDLQIITATDIDAAIRSVCSDNRDMAAIVW rv3885c (eccE2) cds [SEQ ID NO: 50] ttgacgtccaagctgaccgggttcagcccgcgcagtgcgaggcgggtcgccggggtgtggacagtgttcgtgctcgcgtcggcggg ctgggcgctgggcggccagctaggtgcggtcatggctgtcgtggtcggcgtcgccttggtgttcgtacagtggtggggtcagccggc gtggtcgtgggcggtactggggctgcggggtcggcgtcccgtcaaatggaatgacccaattaccttggccaacaaccgatccgggg gtggcgtccgcgtgcaagacggtgtcgcggtggtggcggtgcaacttctcggccgagcgcaccgggcgactacggtcaccgggtc ggtgaccgtagaaagcgacaacgtgattgacgtcgttgagctcgcgccgttgctgcgccacccgctggacctggaactcgattcaatc agcgtcgtcaccttcggctcgcgaaccggcaccgtcggcgattacccgcgggtgtatgacgcggagatcggtacgccgccgtatgcc gggcggcgcgaaacgtggctgatcatgcggcttccggtgatcggcaacacccaagctttacgctggcgtaccagcgttggggccgctgccatttcggtcgcccaacgcgttgccagctccctgcgctgtcagggcttgcgcgccaaactggccaccgcaacagacttggctgag cttgatcgccggctggggtcggacgcggtagccgggagtgcgcagcgctggaaagctatccgcggtgaagccgggtggatgacga cgtatgcgtacccggctgaggcgatttcgtcgcgggttctctcgcaagcctggacgctgcgtgccgatgaggtcatccagaacgtaac ggtgtatccggacgcgacgtgcaccgcgaccatcaccgtgcgcacaccgacgccggcgcctaccccgcccagtgtgatcttgcgtc ggctcaatggtgagcaagccgccgcggctgcggccaacatgtgcgggccacgtccacacctacgcggacagcggcgctgcccgtt gccggcgcagctagtcaccgagatcggaccgtcgggggtgttgattggcaagctgagcaacggggaccggctgatgattcccgttac cgacgccggtgagctgtcgcgcgtcttcgtggccgcggacgacacgatcgccaagaggatcgtgattcgcgtcgtcggtgccggtg agcgggtgtgtgtgcacactcgcgaccaagagcgttgggccagcgtacgcatgccgcagttgagcatcgtcggcacaccacggccc gcgccgcgcaccactgtcggcgtcgtggagtacgtgcggcggcgcaagaacggcgatgacggcaaatctgaaggcagcggtgtc gatgtcgcgatttcgcccacgccacggccagccagtgtcatcaccattgcccgacccggtacctcgctgtccgagagcgatcggcatg gcttcgaggtgaccatcgaacaaatcgatcgggcaacggtgaaagtcggtgcggcaggacaaaactggctggttgagatggaaatgt tccgtgcggagaaccgctatgtcagccttgagccggtcacgatgtcgataggccggtagRv3885c (EccE2) aa seq [SEQ ID NO: 51]LTSKLTGFSPRSARRVAGVWTVFVLASAGWALGGQLGAVMAVVVGVALVFVQWW GQPAWSWAVLGLRGRRPVKWNDPITLANNRSGGGVRVQDGVAVVAVQLLGRAHR ATTVTGSVTVESDNVIDVVELAPLLRHPLDLELDSISVVTFGSRTGTVGDYPRVYDAE IGTPPYAGRRETWLIMRLPVIGNTQALRWRTSVGAAAISVAQRVASSLRCQGLRAKL ATATDLAELDRRLGSDAVAGSAQRWKAIRGEAGWMTTYAYPAEAISSRVLSQAWTL RADEVIQNVTVYPDATCTATITVRTPTPAPTPPSVILRRLNGEQAAAAAANMCGPRPH LRGQRRCPLPAQLVTEIGPSGVLIGKLSNGDRLMIPVTDAGELSRVFVAADDTIAKRIV IRVVGAGERVCVHTRDQERWASVRMPQLSIVGTPRPAPRTTVGVVEYVRRRKNGDD GKSEGSGVDVAISPTPRPASVITIARPGTSLSESDRHGFEVTIEQIDRATVKVGAAGQN WLVEMEMFRAENRYVSLEPVTMSIGR espACD (rv3614c to rv3616c)DNA fragment (in pMDespACD) containing the Mtb espACD operon [SEQ ID NO: 52] ccatcgtcggttttcgtcgccttatcaaacatgatcggagataatgacagatcggcctagctaggtgtttagcggacgcgatttaggacaa ccgagatttgctttgcctcgcaaccatgagagcgccccgcttcgacgccgaatcgggtgagtgatggtgggttagcacagccctgattgcgccaccggcgaggtgattgtgcccgccacgaggccgccgccggctagccccatgagcacactatatagactctcctgcaacagatc tcataccgatcgaaggcgaagcgcaggcatcgacgtcggagacactgccttgggatcgcgccgcctacacggcggttggcgcattgt cgcagcgcagttgcaggagggcaaatgtgcgcagacgatgtagtcgacaacaagtgaacatgccgtcttcacgaactcaaaactgac gatctgcttagcatgaaaaaaactgttgacatcggccaagcatgacagccagactgtaggccctacgcgtgcaatgcagaaccaagg ctatgcatggaatcgacgaccgttgagataggcggcaggcatgagcagagcgttcatcatcgatccaacgatcagtgccattgacggc ttgtacgaccttctggggattggaatacccaaccaagggggtatcctttactcctcactagagtacttcgaaaaagccctggaggagctg gcagcagcgtttccgggtgatggctggttaggttcggccgcggacaaatacgccggcaaaaaccgcaaccacgtgaattttttccagg aactggcagacctcgatcgtcagctcatcagcctgatccacgaccaggccaacgcggtccagacgacccgcgacatcctggagggc gccaagaaaggtctcgagttcgtgcgcccggtggctgtggacctgacctacatcccggtcgtcgggcacgccctatcggccgccttcc aggcgccgttttgcgcgggcgcgatggccgtagtgggcggcgcgcttgcctacttggtcgtgaaaacgctgatcaacgcgactcaac tcctcaaattgcttgccaaattggcggagttggtcgcggccgccattgcggacatcatttcggatgtggcggacatcatcaagggcatcc tcggagaagtgtgggagttcatcacaaacgcgctcaacggcctgaaagagctttgggacaagctcacggggtgggtgaccggactgt tctctcgagggtggtcgaacctggagtccttctttgcgggcgtccccggcttgaccggcgcgaccagcggcttgtcgcaagtgactgg cttgttcggtgcggccggtctgtccgcatcgtcgggcttggctcacgcggatagcctggcgagctcagccagcttgcccgccctggcc ggcattgggggcgggtccggttttgggggcttgccgagcctggctcaggtccatgccgcctcaactcggcaggcgctacggccccg agctgatggcccggtcggcgccgctgccgagcaggtcggcgggcagtcgcagctggtctccgcgcagggttcccaaggtatgggc ggacccgtaggcatgggcggcatgcacccctcttcgggggcgtcgaaagggacgacgacgaagaagtactcggaaggcgcggcg gcgggcactgaagacgccgagcgcgcgccagtcgaagctgacgcgggcggtgggcaaaaggtgctggtacgaaacgtcgtctaa cggcatggcgagccaaatccattgctagccagcgcctaacaacgcgcaatgctaaacggaagggacacgatcaatgacggaaaact tgaccgtccagcccgagcgtctcggtgtactggcgtcgcaccatgacaacgcggcggtcgatgcctcctcgggcgtcgaagctgccg ctggcctaggcgaatctgtggcgatcactcacggtccgtactgctcacagttcaacgacacgttaaatgtgtacttgactgcccacaatg ccctgggctcgtccttgcatacggccggtgtcgatctcgccaaaagtcttcgaattgcggcgaagatatatagcgaggccgacgaagc gtggcgcaaggctatcgacgggttgtttacctgaccacgtttgctgcccgcagtgcaggccacagcgtcttcccaacgacctgttcgga ctgaccacgccagctgcccaggccgacccttcccgggtggcaatgaattccgaagggacggtggacttgcccggaaatgactttgac agcaacgatttcgacgccgtggatctctggggtgccgacggcgcggagggctggactgcggatccgattattggcgtcgggtcggc ggcgaccccggacaccggacccgacctggacaatgcccacggtcaggcggagacggacaccgaacaagagatcgcgctttttacc gtgacgaatcccccacgcacggtgtcggtatcgacgctgatggacggccggattgaccatgtcgagctgtcggccagggtggcctgg atgagtgagtcgcagctcgcttctgagatcctggtgattgccgacctggcgcggcagaaggcgcagtcggcccagtacgccttcatcc ttgacaggatgagtcaacaggtcgatgcagatgaacaccgcgtcgcactgctacgtaagaccgtgggcgaaacctgggggttaccat cgccggaagaagccgcggcagcagaagctgaggtgttcgcgacgcgctacagcgacgattgtccagcaccagacgacgagagcgatccatggtgagttgaaccgccccggccgctgcggtgactctggtgctccgcgtgtttcccgactgggctcacaattcgcggctcagca ttaat rv3616c (espA) cds [SEQ ID NO: 53] atgagcagagcgttcatcatcgatccaacgatcagtgccattgacggcttgtacgaccttctggggattggaatacccaaccaaggggg tatcctttactcctcactagagtacttcgaaaaagccctggaggagctggcagcagcgtttccgggtgatggctggttaggttcggccgc ggacaaatacgccggcaaaaaccgcaaccacgtgaattttttccaggaactggcagacctcgatcgtcagctcatcagcctgatccac gaccaggccaacgcggtccagacgacccgcgacatcctggagggcgccaagaaaggtctcgagttcgtgcgcccggtggctgtgg acctgacctacatcccggtcgtcgggcacgccctatcggccgccttccaggcgccgttttgcgcgggcgcgatggccgtagtgggcg gcgcgcttgcctacttggtcgtgaaaacgctgatcaacgcgactcaactcctcaaattgcttgccaaattggcggagttggtcgcggcc gccattgcggacatcatttcggatgtggcggacatcatcaagggcaccctcggagaagtgtgggagttcatcacaaacgcgctcaacg gcctgaaagagctttgggacaagctcacggggtgggtgaccggactgttctctcgagggtggtcgaacctggagtccttctttgcggg cgtccccggcttgaccggcgcgaccagcggcttgtcgcaagtgactggcttgttcggtgcggccggtctgtccgcatcgtcgggcttg gctcacgcggatagcctggcgagctcagccagcttgcccgccctggccggcattgggggcgggtccggttttgggggcttgccgag cctggctcaggtccatgccgcctcaactcggcaggcgctacggccccgagctgatggcccggtcggcgccgctgccgagcaggtc ggcgggcagtcgcagctggtctccgcgcagggttcccaaggtatgggcggacccgtaggcatgggcggcatgcacccctcttcggg ggcgtcgaaagggacgacgacgaagaagtactcggaaggcgcggcggcgggcactgaagacgccgagcgcgcgccagtcgaa gctgacgcgggcggtgggcaaaaggtgctggtacgaaacgtcgtctaaRv3616c (EspA) aa seq [SEQ ID NO: 54]MSRAFIIDPTISAIDGLYDLLGIGIPNQGGILYSSLEYFEKALEELAAAFPGDGWLGSAA DKYAGKNRNHVNFFQELADLDRQLISLIHDQANAVQTTRDILEGAKKGLEFVRPVAV DLTYIPVVGHALSAAFQAPFCAGAMAVVGGALAYLVVKTLINATQLLKLLAKLAEL VAAAIADIISDVADIIKGTLGEVWEFITNALNGLKELWDKLTGWVTGLFSRGWSNLES FFAGVPGLTGATSGLSQVTGLFGAAGLSASSGLAHADSLASSASLPALAGIGGGSGFG GLPSLAQVHAASTRQALRPRADGPVGAAAEQVGGQSQLVSAQGSQGMGGPVGMGG MHPSSGASKGTTTKKYSEGAAAGTEDAERAPVEADAGGGQKVLVRNVV rv3615c (espC) cds [SEQ ID NO: 55]atgacggaaaacttgaccgtccagcccgagcgtctcggtgtactggcgtcgcaccatgacaacgcggcggtcgatgcctcctcgggc gtcgaagctgccgctggcctaggcgaatctgtggcgatcactcacggtccgtactgctcacagttcaacgacacgttaaatgtgtacttg actgcccacaatgccctgggctcgtccttgcatacggccggtgtcgatctcgccaaaagtcttcgaattgcggcgaagatatatagcga ggccgacgaagcgtggcgcaaggctatcgacgggttgtttacctgaRv3615c (EspC) aa seq [SEQ ID NO: 56]MTENLTVQPERLGVLASHHDNAAVDASSGVEAAAGLGESVAITHGPYCSQFNDTLN VYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLFT rv3614c (espD) cds [SEQ ID NO: 57] gtggacttgcccggaaatgactttgacagcaacgatttcgacgccgtggatctctggggtgccgacggcgcggagggctggactgcg gatccgattattggcgtcgggtcggcggcgaccccggacaccggacccgacctggacaatgcccacggtcaggcggagacggaca ccgaacaagagatcgcgctttttaccgtgacgaatcccccacgcacggtgtcggtatcgacgctgatggacggccggattgaccatgt cgagctgtcggccagggtggcctggatgagtgagtcgcagctcgcttctgagatcctggtgattgccgacctggcgcggcagaaggc gcagtcggcccagtacgccttcatccttgacaggatgagtcaacaggtcgatgcagatgaacaccgcgtcgcactgctacgtaagacc gtgggcgaaacctgggggttaccatcgccggaagaagccgcggcagcagaagctgaggtgttcgcgacgcgctacagcgacgatt gtccagcaccagacgacgagagcgatccatggtgaRv3614c (EspD) aa seq [SEQ ID NO: 58]VDLPGNDFDSNDFDAVDLWGADGAEGWTADPIIGVGSAATPDTGPDLDNAHGQAET DTEQEIALFTVTNPPRTVSVSTLMDGRIDHVELSARVAWMSESQLASEILVIADLARQ KAQSAQYAFILDRMSQQVDADEHRVALLRKTVGETWGLPSPEEAAAAEAEVFATRY SDDCPAPDDESDPW

Claims

WHAT IS CLAIMED IS:

1. A recombinant M. smegmatis strain comprising a Type VII Secretion System derived from the M. tuberculosis [M. tb] - complex.

2. The recombinant M. smegmatis strain of claim 1, wherein the Type VII Secretion System is / W.fb-complex ESX-1.

3. The recombinant M. smegmatis strain of claim 1 or 2, wherein the Type VII Secretion System comprises a combination of two or more repeats of / W.fb-complex ESX-1.

4. The recombinant M. smegmatis strain of claim 1, wherein the Type VII Secretion System is a concatamer of / W.tb-complex ESX-1.

5. The recombinant M. smegmatis strain of any one of claims 1-4, wherein / W.fb-complex ESX- 1 allows the strain to export one or more virulence effector proteins from EspA, EspB, EspC, EsxA or EsxB.

6. A recombinant M. smegmatis strain comprising: a) M. fb-complex esx-1 locus [SEQ ID NO: 1]; b) M. fb-complex espACD operon [SEQ ID NO: 52]; or c) a combination thereof.

7. The recombinant M. smegmatis strain of claim 6, comprising M. fb-complex esx-1 locus [SEQ ID NO: 1] and M. fb-complex espACD operon [SEQ ID NO: 52],8. The recombinant M. smegmatis strain of claim 7, comprising two or more copies of M. tb- complex esx-1 locus [SEQ ID NO: 1] or M. fb-complex espACD operon [SEQ ID NO: 52],9. The recombinant M. smegmatis strain of any one of claims 1-8, wherein the strain is non- pathogenic when administered to a subject.

10. The recombinant M. smegmatis strain of any one of claims 1-9, wherein the strain when administered does not sensitize the subject to antigens of M. tb or protein purified derivative of M. bovis.

11. The recombinant M. smegmatis strain of any one of claims 1-10 for use in prevention or treatment of tuberculosis.

12. The recombinant M. smegmatis strain of any one of claims 1-10 for use in diagnostic testing of tuberculosis.

13. The recombinant M. smegmatis strain of any one of claims 1-10 for use as a vaccine against tuberculosis.

14. A vector, a translation cassette or a plasmid comprising: a) a DNA fragment comprising M. tb-complex esx-1 locus [SEQ ID NO: 1]; b) a DNA fragment comprising M. tb-complex espACD operon [SEQ ID NO: 52]; or c) a DNA fragment comprising M. tb-complex esx-1 locus [SEQ ID NO: 1] and M. tb- complex espACD operon [SEQ ID NO: 52]; and a promoter, one or more regulatory sequences, one or more restriction endonuclease or cloning sites, one or more polyadenylation sites or any combination thereof.

15. A vector, a translation cassette or a plasmid comprising: a) a DNA fragment comprising M. tb-complex esx-1 locus [SEQ ID NO: 1]; b) a DNA fragment comprising M. tb-complex espACD operon [SEQ ID NO: 52]; or c) a DNA fragment comprising M. tb-complex esx-1 locus [SEQ ID NO: 1] and M. tb- complex espACD operon [SEQ ID NO: 52]; and a promoter, one or more regulatory sequences, one or more restriction endonuclease or cloning sites, one or more polyadenylation sites or any combination thereof, wherein at least the promoter, the restriction endonuclease or cloning sites, the polyadenylation sites are heterologous to the genome of M. smegmatis.

16. A vector, a translation cassette or a plasmid comprising: a) a genome of M. smegmatis or any part thereof; andb) a DNA fragment comprising M. tb-complex esx-1 locus [SEQ ID NO: 1]; a DNA fragment comprising M. tb-complex espACD operon [SEQ ID NO: 52]; or a DNA fragment comprising M. tb-complex esx-1 locus [SEQ ID NO: 1] and M. tb-complex espACD operon [SEQ ID NO: 52],17. A recombinant non-pathogenic Mycobacterium strain for prevention or treatment of tuberculosis, wherein the Mycobacterium strain comprises a DNA fragment with M. tb-complex esx-1 locus [SEQ ID NO: 1], a DNA fragment with M. tb-complex espACD operon [SEQ ID NO: 52], or a combination of both.

18. The recombinant non-pathogenic Mycobacterium strain of claim 17, wherein the Mycobacterium strain is engineered from one or more members of the Mycobacterium family.

19. The recombinant non-pathogenic bacterial strain of claim 17, wherein the Mycobacterium strain is Mycobacterium smegmatis.

20. A tuberculosis vaccine comprising the recombinant M. smegmatis strain of any one of claims 1-13, the vector, translation cassette or plasmid of claims 14-16, or the recombinant non-pathogenic strain of claims 17-19.

21. A method of constructing a recombinant M. smegmatis strain with M. tb-complex esx-1 locus [SEQ ID NO: 1]: a) electroporating a M. smegmatis cell line with a cosmid, a vector, or a cassette comprising a M. tb-complex chromosome including M. tb-complex esx-1 locus [SEQ ID NO: 1]; b) selecting the recombinant M. smegmatis strain with M. tb-complex esx-1 locus [SEQ ID NO: 1] on a culture medium.

22. A method of constructing a recombinant M. smegmatis strain with M. tb-complex espACD operon [SEQ ID NO: 52]: a) transforming a M. smegmatis cell line with a cosmid, a vector, or a cassette comprising M. tb-complex espACD operon [SEQ ID NO: 52];b) selecting the recombinant M. smegmatis strain with M. tb-complex espACD operon [SEQ ID NO: 52] on a culture medium.

23. A method of vaccinating a subject against tuberculosis comprising: administering a dose of the recombinant M. smegmatis strain of any one of claims 1-13, the vector, translation cassette or plasmid of claims 14-16, or the recombinant non-pathogenic strain of claims 17- 19, to the subject in need thereof.

24. The method of claim 23, wherein the vaccination does not sensitize the subject to specific antigens or purified protein derivative of the M. fb-complex.

25. The method of claim 23 or 24, wherein the vaccinated subject does not show sensitivity to a tuberculosis diagnostic test when not infected with M. fb-complex.

26. A method of preventating or treating a subject against tuberculosis comprising: administering a dose of the recombinant M. smegmatis strain of any one of claims 1-13, the vector, translation cassette or plasmid of claims 14-16, or the recombinant non-pathogenic strain of claims 17-19, to the subject.

27. A composition comprising: a) the recombinant M. smegmatis strain of any one of claims 1- 13, the vector, translation cassette or plasmid of claims 14-16, or the recombinant non- pathogenic strain of claims 17-19; and b) a pharmaceutically acceptable excipient, carrier, adjuvant or combination thereof28. A kit comprising: a) the recombinant M. smegmatis strain of any one of claims 1-13, the vector, translation cassette or plasmid of claims 14-16, or the recombinant non-pathogenic strain of claims 17-19; b) a syringe and / or instructions for using the strain, the vector, the cassette, the plasmid, or the recombinant non-pathogenic strain.

Images