Nucleic acid vaccine against tuberculosis

Abstract

The present invention is directed to a vaccine composition against a disease caused by Mycobacterium tuberculosis, said composition comprising at least four nucleic acids selected from the following groups: a. a nucleic acid encoding an Ag85A, Ag85B, or Ag85C antigen; b. a nucleic acid encoding a resuscitation promoting factor (Rpf) selected from the group consisting of: RpfA, RpfB, RpfC, RpfD and RpfE; c. a nucleic acid encoding a PE / PPE antigen selected from the group consisting of: PE5, PE13, PE15, PE29, PE31, PPE1, PPE2, PPE18, PPE20, and PPE68; d. a nucleic acid encoding a disease-reactivation-related antigen selected from the group consisting of: Rv1234 / MMAR_4207, and Rv0359 / MMAR_0678, wherein said vaccine composition comprises at least one nucleic acid from each of group a, b, and c; and wherein the nucleic acids are provided on one or more nucleic acids constructs.

Description

Nucleic acid vaccine against tuberculosisFIELD

[0001] The present invention relates to the field of vaccines. Particularly, the present invention provides a vaccine comprising nucleic acids encoding tuberculosis antigens.BACKGROUND

[0002] Tuberculosis is a chronic infection by Mycobacterium tuberculosis that results in over 1.5 million deaths worldwide each year. Currently, there is only one licensed vaccine against tuberculosis, the Bacillus Calmette-Guerin (BCG) vaccine comprising live attenuated vaccine form of Mycobacterium bovis. Despite widespread vaccination programs, over 10 million new M. tuberculosis infections are diagnosed yearly, with almost half a million cases caused by antibiotic-resistant strains. Efforts to produce new vaccines against an M. tuberculosis infection have encountered several challenges, including the complexity of M. tuberculosis pathogenesis and limited knowledge of the protective immune responses. The preclinical evaluation of novel tuberculosis vaccine candidates is also hampered by the lack of an appropriate animal model that could accurately predict the protective effect of vaccines in humans.

[0003] Despite the widespread use of the BCG vaccine, tuberculosis is still a major cause of morbidity and mortality worldwide. Vaccination with BCG does not prevent a M. tuberculosis infection, nor does it inhibit the reactivation of latent tuberculosis.

[0004] Roughly one third of the human population carries a latent M. tuberculosis infection, with a 5-10% lifetime risk of reactivation to active tuberculosis and further spreading the disease. The mechanisms leading to the reactivation of a latenttuberculosis infection are insufficiently understood.

[0005] Accordingly, the development of novel, improved tuberculosis vaccines is important for reducing the disease burden caused by tuberculosis. An improved vaccine should i) protect against primary infection, ii) enhance immunity generated by BCG vaccination, and / or iii) protect against reactivation of latent tuberculosis infection.

[0006] W02024023790 discloses a AL tuberculosis vaccine composition comprising at least two nucleic acids selected from: a nucleic acid encoding a Wbbl 1 antigen; a nucleic acidencoding a CFP-10 antigen; a nucleic acid encoding a PPE18; and a nucleic acid encoding a PEI 3 antigen.

[0007] WO2024138121 discloses a lipid nanoparticle (LNP) composition consisting of: a messenger ribonucleic acid (mRNA) encoding one or more Mycobacterium tuberculosis (Mtb) proteins selected from the group consisting of CFP10 / Rv3874, ESAT-6 / Rv3875, Mtb32A / RvO125, Mtb39A / Rvl 196, Ag85B / Rvl886c, EsxW / Rv3620c, EsxV / Rv3619c, PE13 / Rvll95, PPE30 / Rvl802, PPE40 / Rv2356c and TB10.4 / Rv0288.

[0008] WO2023201199 discloses a nucleic acid vaccine composition comprising a synthetic polynucleotide encoding a Mycobacterium tuberculosis (Mtb) RelA-SpoT homolog (RSH) protein, RelMtb, conjugated to a macrophage inflammatory protein-3 alpha (MIP-3a) or other chemokine that binds to a chemokine receptor 6 (CCR6).SUMMARY

[0009] The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims.

[0010] The present invention provides a novel nucleic-acid-based M. tuberculosis vaccine composition with a unique selection of antigens providing improved and enhanced immunization protecting from tuberculosis (TB) including protection against reactivation of latent TB.

[0011] According to a first aspect of the present invention, there is provided a vaccine composition against a disease caused by Mycobacterium tuberculosis, said composition comprising at least four nucleic acids selected from the following groups:a. a nucleic acid encoding an Ag85A, Ag85B, or Ag85C antigen;b. a nucleic acid encoding a resuscitation promoting factor (Rpf) selected from the group consisting of: RpfA, RpfB, RpfC, RpfD and RpfE;c. a nucleic acid encoding a PE / PPE antigen selected from the group consisting of: PE5, PE13, PE15, PE29, PE31, PPE1, PPE2, PPE18, PPE20, and PPE68;d. a nucleic acid encoding a disease-reactivation-related antigen selected from the group consisting of: Rvl234 / MMAR_4207, and Rv0359 / MMAR_0678,wherein said vaccine composition comprises at least one nucleic acid from each of group a, b, and c; andwherein the nucleic acids are provided on one or more nucleic acid constructs.

[0012] According to a second aspect of the present invention, there is provided a vaccine composition against a disease caused by Mycobacterium tuberculosis, said composition comprising a nucleic acid encoding a disease-reactivation-related antigen selected from the group consisting of: Rvl234 / MMAR_4207, and Rv0359 / MMAR_0678.

[0013] According to a third aspect of the present invention, there is provided a recombinant nucleic acid vector encoding the nucleic acids as defined in the vaccine composition of the present invention.

[0014] According to a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising the recombinant nucleic acid vector of the present invention and a pharmaceutically acceptable carrier, preservative, and / or buffer.

[0015] According to a fifth aspect of the present invention, there is provided a method of preventing a disease caused by Mycobacterium tuberculosis comprising administering a prophylactically or therapeutically effective amount of the vaccine composition according to the present invention to a mammal, preferably to a human.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGURE 1. The structural elements of the mRNA vaccine against M. tuberculosis. In a preferred vaccine, each mRNA construct consists of the coding sequence for a single M. tuberculosis (Mtb) antigen, 3'- and 5' untranslated regions (UTR) from Xenobus P-globin mRNA and post-transcriptionally synthetized 5' Cap and 3' PolyA tail. The range of length for both the coding sequences and the polyA tails in mRNAs coding for different antigens are shown as subscripts. To obtain nucleoside-modified mRNA, Nl-methylpseudouridine-5'- triphosphate instead of regular uridine-5'-triphosphate is used in the synthesis. The mRNA is encapsulated into lipid nanoparticles consisting of 1,2-distearoyl-sn-glycero-3-PC (1,2-DSPC), SM-102, cholesterol and DMG-PEG(2000). The mRNAs encoding different antigens can be encapsulated separately after which they are combined to obtain multivalent vaccines.

[0017] FIGURE 2. Test efficacy of mRNA vaccines to protect mice from M. tuberculosis aerosol infection. Mice were injected intramuscularly with 50pl of PBS as a control or a vaccine in a total amount of 2 pg of mRNA / dose, according to the dosage regime shown. BCG vaccine with 5 x 105CFU was given subcutaneously once only on Day 0 (DO). There were 6 mice / group.

[0018] FIGURE 3. Bacterial counts in the lung. On Day 28 (D28 / D91) after aerosol TB infection, all mice were harvested and bacterial counts were performed on lung tissue. Bar graphs represent group means ± SEM; ns, not significant; ****, P < 0.0001.

[0019] FIGURE 4. The nucleoside modified mRNA is efficiently translated and well-tolerated in zebrafish Danio rerio) embryos. 450 pg of pcDNA3.1 plasmid coding for egfp (enhanced green fluorescent protein, eGFP) or 370 pg of nucleoside modified egfp-mRNA (synthetized using l-methylpseudouridine-5’-triphosphate=m1'PU) was microinjected into the cytosol of 1-cell stage zebrafish embryos and the GFP expression in live larvae was visualized with Lumar Vl.l fluorescence stereomicroscope (Carl Zeiss MicroImaging GmbH, Gottingen, Germany) 24 hours post injection. Uninjected embryos were used as negative controls in the assay. The images indicate no gfp expression in uninjected or plasmid-injected embryos but clear, universal expression in embryos injected with nucleoside-modified mRNA. Noteworthy, modified mRNA was well-tolerated and did not affect the early development of the embryos.

[0020] FIGURE 5. mRNA vaccine induces antigen specific Interferon Gamma (IFNy) response in mice. Eight C57BL / 6 female mice were intramuscularly immunized three times (at three-week intervals) with 2 pg of mRNA vaccine coding for six M. tuberculosis antigens (mRNA vaccine), 2 pg of control mRNA vaccine coding for GFP (GFP vaccine), PBS or subcutaneously once only with 5x105cfu of BCG. Mouse splenocytes were isolated three days after the last dose and subjected to analysis by ImmunoSpot Murine IFNy Single Color Enzymatic ELISPOT Assay (Cellular Technology Limited, Ohio, USA). In the assay, 300000 splenocytes were stimulated for 16 h with 1 pg / ml GFP, Rvl886 or Rvl234 peptide library consisting of 15-mer synthetic peptides overlapping by 11-mer and covering the full protein. IFN / detection was carried out according to the manufacturer’s instructions after which the wells were scanned with ImmunoSpot Analyzer for spots representing the frequency of IFN producing T cells. Each assay was carried out as duplicates or triplicates and the results were normalized to the number of spots in the wells mock-stimulated with cell culture medium. The statistical analysis was performed with Kruskal -Wallis test with Dunn’s multiple comparisontest. The comparisons were made within samples stimulated with the same peptides and so that each group was compared with PBS group, p-value of <0.05 was considered significant. ***= <0.001, ****= <0.0001.EMBODIMENTS

[0021] In the present context, the term “antigen” is a compound, composition, or substance that can stimulate the production of antibodies and / or a CD4+ or CD8+ T cell response in an animal or human, including compositions that are injected or absorbed into an animal or human. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term “antigen” includes all related antigenic epitopes. An “epitope” refers to a site on an antigen, including chemical groups or peptide sequences on a molecule that are antigenic, i.e., that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide antigen.

[0022] A number of M. tuberculosis antigens for use in candidate TB vaccines have already been identified (see, e.g., W02024023790 and WO2024138121). However, M. tuberculosis expresses over 4000 gene products so uncovering novel antigens and selection of antigens for a vaccine are not trivial undertakings. In the present invention, the inventors have rationally selected at least four antigens from the groups a. to d. for vaccine development:a. Ag85A, Ag85B, and Ag85C;b. RpfA, RpfB, RpfC, RpfD and RpfE;c. PE5, PE13, PE15, PE29, PE31, PPE1, PPE2, PPE18, PPE20, and PPE68; andd. Rvl234 / MMAR_4207, and Rv0359 / MMAR_0678.

[0023] In an embodiment, the present invention provides a vaccine composition which improves safety of TB vaccinations without compromising efficacy. It is well-known that the BCG vaccine may cause serious side effects, such as osteomyelitis, particularly in infants, which is why comprehensive BCG vaccinations of infants have been abandoned in many countries. In contrast to the BCG vaccine, the vaccine composition of the present invention potentially does not have toxic side effects (Figure 4).

[0024] The terms “nucleic acid” and “nucleic acid construct” encompass both ribonucleotides (RNA) and deoxyribonucleotides (DNA), including synthetic DNA and RNA. The nucleic acid may be double-stranded or single-stranded. Where the nucleic acid is single-stranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. By “RNA” is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. The term “DNA” refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides. In an embodiment, said RNA is messenger RNA (mRNA). As used herein, the term "mRNA” means "messenger RNA” and refers to a "transcript” which can be generated by using a DNA template and encodes an antigen peptide or antigen polypeptide. Typically, an mRNA comprises a 5'-UTR, a protein coding region, and a 3'-UTR. In the context of aspects of the present disclosure, mRNA may be generated by in vitro transcription from a DNA template.

[0025] In an embodiment, an mRNA construct may be capped at the 5’ end. According to another embodiment, the mRNA construct may include one or more modified nucleotides. For example, the mRNA construct may include one or more modified nucleotides selected from the group consisting of Nl-methyl-pseudouridine and pseudouridine.

[0026] In some embodiments, nucleic acid constructs of the invention may include, without limitation, nucleotide sequences encoding antigenic peptides including amino acid sequences substantially identical to the amino acid sequences of the antigens selected from the following groups:a. Ag85A, Ag85B, and Ag85C;b. RpfA, RpfB, RpfC, RpfD and RpfE;c. PE5, PE13, PE15, PE29, PE31, PPE1, PPE2, PPE18, PPE20, and PPE68; andd. Rvl234 / MMAR_4207, and Rv0359 / MMAR_0678.

[0027] Another embodiment of the invention includes, without limitation, nucleic acid molecules encoding the aforementioned antigenic peptides that are substantially identical to the nucleotide sequences described herein.

[0028] As used herein a “substantially identical” sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the antigenicity of one or more ofthe expressed polypeptides or of the polypeptides encoded by the nucleic acid molecules. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as ALIGN, CLUSTALW or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment of the invention there is provided for a polypeptide or polynucleotide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences of SEQ ID NOS: 1-40 described herein.

[0029] Accordingly, the present invention provides a vaccine composition against a disease caused by Mycobacterium tuberculosis, said composition comprising at least four nucleic acids selected from the following groups:a. a nucleic acid encoding an Ag85A, Ag85B, or Ag85C antigen;b. a nucleic acid encoding a resuscitation promoting factor (Rpf) selected from the group consisting of: RpfA, RpfB, RpfC, RpfD and RpfE;c. a nucleic acid encoding a PE / PPE antigen selected from the group consisting of: PE5, PE13, PE15, PE29, PE31, PPE1, PPE2, PPE18, PPE20, and PPE68;d. a nucleic acid encoding a disease-reactivation-related antigen selected from the group consisting of: Rvl234 / MMAR_4207, and Rv0359 / MMAR_0678,wherein said vaccine composition comprises at least one nucleic acid from each of group a, b, and c; andwherein the nucleic acids are provided on one or more nucleic acid constructs.

[0030] In a preferred embodiment, the present invention provides said vaccine composition comprises a lipid nanoparticle enclosing said one or more nucleic acids constructs.

[0031] In another preferred embodiment, said lipid nanoparticle comprises i) a first phospholipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), and dipalmitoylphosphatidylcholine (DPPC);ii) a second phospholipid selected from the group consisting of sphingomyelins (SM), iii) PEG(2000)-dimyristoylglycerol (PEG-DMG); and iv) cholesterol.

[0032] In another preferred embodiment, said lipid nanoparticle comprises 1,2-distearoyl-sn-glycero-3-PC (1,2-DSPC), SM-102, cholesterol, and PEG-DMG.

[0033] In another preferred embodiment, said at least four nucleic acids encode Ag85B, RpfE, PE13, and PE15, or at least the nucleic acids encoding Ag85B, RpfE, PE13, and PE15.

[0034] In another preferred embodiment, said nucleic acids are mRNAs comprising one or more nucleic acid sequences selected from the group consisting of: SEQ ID NOS: 1-4.

[0035] In another preferred embodiment, said the nucleic acids are mRNAs encoding one or more amino acid sequences selected from SEQ ID NOs:21-24 or amino acid sequences which have at least 80% sequence identity to SEQ ID NOs:21-24.

[0036] In another preferred embodiment, said vaccine composition comprises at least one nucleic acid from each of group a, b, c, and d.

[0037] In another preferred embodiment, said at least four nucleic acids encode at least a nucleic acid encoding Rv0359 / MMAR_0678.

[0038] In another preferred embodiment, said vaccine composition further comprises a nucleic acid encoding Rvl234 / MMAR_4207.

[0039] In another preferred embodiment, said composition comprises nucleic acids encoding Ag85B, RpfE, PE13, PE15, Rv0359 / MMAR_0678, and Rvl234 / MMAR_4207 or at least the nucleic acids encoding Ag85B, RpfE, PE13, PE15, Rv0359 / MMAR_0678, and Rvl234 / MMAR_4207.

[0040] In another preferred embodiment, said at least four nucleic acids are messenger ribonucleic acids (mRNAs). More preferably, said messenger ribonucleic acids (mRNAs) encode antigens Ag85B, RpfE, PE13, PE15, Rv0359 / MMAR_0678, and Rvl234 / MMAR_4207.

[0041] In another preferred embodiment, the mRNAs comprise one or more nucleic acid sequences selected from the group consisting of: SEQ ID NOS: 1-6.

[0042] In another preferred embodiment, the nucleic acids are mRNAs encoding one or more amino acid sequences selected from SEQ ID NOs:21-26 or amino acid sequences which have at least 80% sequence identity to SEQ ID NOs:21-26.

[0043] In another preferred embodiment, said vaccine composition comprises mRNAs comprising one or more nucleic acid sequences selected from the group consisting of: SEQ ID NOS: 1-20.

[0044] In another preferred embodiment, the nucleic acids are mRNAs encoding one or more amino acid sequences selected from SEQ ID NOs:21-40 or amino acid sequences which have at least 80% sequence identity to SEQ ID NOs:21-40.

[0045] In an alternative embodiment, the present invention provides a vaccine composition against a disease caused by Mycobacterium tuberculosis, said composition comprising a nucleic acid, preferably an mRNA construct, encoding a disease-reactivation-related antigen selected from the group consisting of: Rvl234 / MMAR_4207, and Rv0359 / MMAR_0678.

[0046] In another embodiment, the present invention is directed to a recombinant nucleic acid vector encoding the nucleic acids as defined in the vaccine composition of the present invention.

[0047] In another embodiment, the present invention is directed to a pharmaceutical composition comprising the recombinant nucleic acid vector of the present invention and a pharmaceutically acceptable carrier, preservative, and / or buffer.

[0048] In another embodiment, the present invention is directed to a method of preventing a disease caused by Mycobacterium tuberculosis comprising administering a prophylactically or therapeutically effective amount of the vaccine composition according to the present invention to a mammal, preferably to a human. Preferably, said disease is lung tuberculosis.

[0049] In another embodiment, the present invention is directed to a use of nucleic acids, preferably mRNAs, encoding at least Ag85B, RpfE, PE13, and PE15 in the manufacturing of a vaccine, preferably an mRNA vaccine, for inducing an immune response in a mammal, preferably a human.

[0050] The vaccination protocol for eliciting an immune response against Mycobacterium tuberculosis in a subject as defined herein typically comprises a series of single doses of the nucleic acid constructs or compositions described herein. A single dose or dosage, as used herein, refers to the priming dose (i.e., initial first or second dose with the same antigens), and any subsequent dose, respectively, which are preferably administered in order to “boost” the immune reaction. In a specific embodiment, the vaccine composition of the present invention can be administered to boost a previous BCG administration to a subject.

[0051] In an embodiment, each single dosage comprises the administration of the vaccine composition according to the invention, wherein the interval between the administration of two single dosages can vary from at least one week, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 26 or 52 weeks apart. Most preferably, the composition of the invention is administered at intervals of either 4 or 8 weeks apart. It will be appreciated that the intervals between single dosages may be constant or vary over the course of the immunization protocol, e.g., the intervals may be shorter in the beginning (such as 4 weeks apart) and longer towards the end of the protocol (such as 8 weeks apart). Additionally, depending on the total number of single dosages and the interval between single dosages, the immunization protocol may extend over a period of time, which preferably lasts at least one week, more preferably several weeks, even more preferably several months (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months). Each single dosage encompasses the administration of the vaccine composition described herein.

[0052] It is to be understood that the embodiments of the invention disclosed are not limited to the particular structures, process steps, or materials disclosed herein, but are extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0053] Reference throughout this specification to “an embodiment” or “a preferred embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in an embodiment” or “a preferred embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.

[0054] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separateand unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. In addition, various embodiments and example of the present invention may be referred to herein along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives are not to be construed as de facto equivalents of one another, but are to be considered as separate and autonomous representations of the present invention.

[0055] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as examples of lengths, widths, shapes, etc., to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

[0056] While the forgoing examples are illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below.

[0057] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. a singular form, throughout this document does not exclude a plurality.EXPERIMENTAL SECTIONMaterials and methodsPreparation of mRNA constructs. pT7TS-rich plasmids encoding the desired Mycobacterium tuberculosis (Mtb) gene sequences flanked by 3’ - and 5’ UTR sequences were used to synthetize antigen encoding mRNAs by in vitro transcription. Each plasmid encoding a singleantigen was linearized with Xbal FastDigest restriction enzyme (Thermo Scientific) and the mRNA synthesis was done with MEGAScript T7 Transcription Kit (Invitrogen) according to the manufacturer’s instructions. To generate nucleoside modified mRNA, 1-methylpseudouridine-5’ -triphosphate (mlvPTP) (Trilink) was used instead of uridine-5’-triphosphate (UTP). After transcription, 5’ capl structure (m7G(5’)pppNi-) was synthetized using the ScriptCap Capl Capping system (Cell Script) and a poly A tail was synthetized using the A-Plus Poly(A) Polymerase Tailing Kit (CellScript). Finally, the capped and tailed mRNAs were purified with Lithium Chloride precipitation and analyzed with capillary electrophoresis (5200 Fragment Analyzer system, Agilent, California, USA) for concentration, integrity and polyA tail length. mRNA was stored in -80°C until further use.Testing mRNA translatability and tolerability in vivo. The translatability and the tolerability of mRNA in living organism was verified in zebrafish embryos by injecting 370 pg of egfp encoding mRNA into the cytosol of 1-cell stage zebrafish embryo. Embryos injected with 450 pg of plasmid encoding egfp (pcDNA3.1-egfp) as well as uninjected embryos were used as negative controls. 24 hours post injection, embryos were visualized and imaged under Lumar VI. 1 fluorescence stereomicroscope (Carl Zeiss MicroImaging GmbH).Preparation of lipid nanoparticles. Synthetized mRNA was encapsulated into lipid nanoparticles (LNP) using the LipidLaunch™ LNP-102 Exploration Kit (Cayman Chemical). mRNAs encoding different antigens were encapsulated separately to ensure desired concentration of each antigen encoding mRNA in the final vaccine. To prepare the lipid solution for LNP synthesis, SM-102, l,2-distearoyl-sn-glycero-3-PC, cholesterol andDMG-PEG(2000) were mixed in ethanol in molar ratios of 50:10:38.5:1.5. The lipid solution was mixed with aqueous mRNA-solution (pH adjusted to 4.0 with Sodium acetate) with ethanol: aqueous ratio 1:3 and lipid:oligonucleotide ratio 10:1. To ensure the self-assembly of mRNA-LNPs, the lipid solution was rapidly pipetted into the mRNA solution under continuous stirring and the particles were let to assemble for 30 min. The LNPs were dialyzed against lOOOx volume of PBS at +4°C overnight after which the LNPs were concentrated using the Amicon ultra-4 centrifugal lOkDa filters (Millipore). mRNA concentration in and outside the particles was evaluated with Qubit BR RNA Kit (Invitrogen). To release mRNA from the particles, the LNPs were treated with 2% Triton-X at 37°C for 10 min. The difference of RNA in Triton-X treated and untreated LNPs was used to evaluate the encapsulation efficiency. Finally, the LNPs were stored at +4°C until further use.Table 1. Mtb antigens included in vaccine formulations A and B. The tables summarize the antigens used in the tested multivalent vaccines, their protein products, if known, the amount of mRNA used in one immunization per mice, the targeted use (prevention of primary infection / reactivation), and citation to the original data from zebrafish / Mycobacterium marinum studies.amount ofVaccine A gene (product) encapsulated mRNA protection against referenceRvli95 (PE13) 0. S ug primary infection Myllymaki et ai., 2017, Myliymaki et at, 2018 RV1388 (REIS) 0,5 ug primary infection Myllymaki et ai., 2017, Myllymaki et at, 2018 Rv2450c (RpfE) 0,5 ug primary infection Myllymaki et ai., 2017, Myllymaki et al., 2018 Rvl886c • Ag85b) 0,5 ug primary infectiontatai rr. RNA / dose 2 ugamount ofVaccine 8 gene {product) encapsulated mRNARV1195 (PE13) 0.333 ug primary infection MyBymaki et ai., 2017, Myliymaki et at, 2018 Rvl388(PE15) 0.333 ng primary infection Myllymaki et a!., 2017, Mykymaki et at, 2018 Rv2450c (RpfE) 0.333 ng primary infection Myliymaki et at, 2017, Myiiymaki et at, 2018 RvlB80c (Ag85b) 0.333 ug primary infectionRV0359 0.333 ug reactivation unpublishedRV1234 0.333 ug reactivation Myiiymakt et st, 2017, MyHymak; et at, 2018 totai mRNA / dose 2 ugTesting efficacy of mRNA vaccines. Mice were injected intramuscularly with 50.l of PBS as a control or a vaccine in a total amount of 2 pg of mRNA / dose, according to the dosage regime as shown in Figure 2. BCG vaccine with 5 x 105CFU was given subcutaneously once only on Day 0 (DO). There were 6 mice / group. Mice were aerosolized with -100 M. tuberculosis on Day 63 (D63). On Day 28 after the infection (D91) all mice were harvested and lung samples were prepared for bacterial counts and histology.Testing the immunogenicity of mRNA vaccines. Eight C57BL / 6 female mice were intramuscularly immunized three times (at three-week intervals) with 2 pg of mRNA vaccine coding for six M. tuberculosis antigens (mRNA vaccine B), 2 pg of control mRNA vaccine coding for GFP (GFP vaccine), PBS or subcutaneously once only with 5xl05cfu of BCG.Mouse splenocytes were isolated three days after the last dose and subjected to analysis by ImmunoSpot Murine IFN / Single Color Enzymatic ELISPOT Assay (Cellular Technology Limited, Ohio, USA). In the assay, 300000 splenocytes were stimulated for 16 h with 1 pg / ml GFP, Rvl886 or Rvl234 peptide library consisting of 15-mer synthetic peptides overlapping by 11-mer and covering the full protein. IFN detection was carried outaccording to the manufacturer’s instructions after which the wells were scanned with ImmunoSpot Analyzer for spots representing the frequency of IFN producing T cells. Each assay was carried out as duplicates or triplicates and the results were normalized to the number of spots in the wells mock-stimulated with cell culture medium.Results- During the vaccinations according to the dose regime of Figure 2, no loss of condition was noted for any of the mice indicating acceptable tolerability of the vaccines.- Day 64 (a day after the aerosolization) harvest of 3 mice showed -300 bacilli per lung sample demonstrating the TB infection.- BCG gave over 1 log of protection against Mtb in the lung compared to the PBS control (see Figure 3).Vaccines A and B also provided significant protection in the lung, i.e. over 1 log of protection compared to the PBS control (see Figure 3).Control mRNA (“Control vac” comprising mRNA encoding egfp) did not provide protection (Figure 3).- Protection provided by Vaccines A and B was equivalent to the protection provided by BCG (Figure 3).mRNA vaccine induces antigen specific Interferon Gamma (lENy) response in mice (Figure 5).Table 2. Examples of the Mtb antigen coding sequences in the mRNA constructs of the present invention, each containing a 5’ UTR, Kozak sequence, restriction sites for template cloning, the protein coding sequence, a 3’ UTR and extra nucleotides from the DNA template.Rvl886c / Ag85B RNA (1271 nt) GAGACAAGCUUGCUUGUUCUUUUUGCAGAAGCUCAGAAUAAACGCUCAACUUUGGCAGAUCUCCACCA UGACAGACGUGAGCCGAAAGAUUCGAGCUUGGGGACGCCGAUUGAUGAUCGGCACGGCAGCGGCUGUA GUCCUUCCGGGCCUGGUGGGGCUUGCCGGCGGAGCGGCAACCGCGGGCGCGUUCUCCCGGCCGGGGCU GCCGGUCGAGUACCUGCAGGUGCCGUCGCCGUCGAUGGGCCGCGACAUCAAGGUUCAGUUCCAGAGCG GUGGGAACAACUCACCUGCGGUUUAUCUGCUCGACGGCCUGCGCGCCCAAGACGACUACAACGGCUGG GAUAUCAACACCCCGGCGUUCGAGUGGUACUACCAGUCGGGACUGUCGAUAGUCAUGCCGGUCGGCGG GCAGUCCAGCUUCUACAGCGACUGGUACAGCCCGGCCUGCGGUAAGGCUGGCUGCCAGACUUACAAGU GGGAAACCUUCCUGACCAGCGAGCUGCCGCAAUGGUUGUCCGCCAACAGGGCCGUGAAGCCCACCGGC AGCGCUGCAAUCGGCUUGUCGAUGGCCGGCUCGUCGGCAAUGAUCUUGGCCGCCUACCACCCCCAGCA GUUCAUCUACGCCGGCUCGCUGUCGGCCCUGCUGGACCCCUCUCAGGGGAUGGGGCCUAGCCUGAUCG GCCUCGCGAUGGGUGACGCCGGCGGUUACAAGGCCGCAGACAUGUGGGGUCCCUCGAGUGACCCGGCA UGGGAGCGCAACGACCCUACGCAGCAGAUCCCCAAGCUGGUCGCAAACAACACCCGGCUAUGGGUUUA UUGCGGGAACGGCACCCCGAACGAGUUGGGCGGUGCCAACAUACCCGCCGAGUUCUUGGAGAACUUCG UUCGUAGCAGCAACCUGAAGUUCCAGGAUGCGUACAACGCCGCGGGCGGGCACAACGCCGUGUUCAAC UUCCCGCCCAACGGCACGCACAGCUGGGAGUACUGGGGCGCUCAGCUCAACGCCAUGAAGGGUGACCU GCAGAGUUCGUUAGGCGCCGGCUGAACUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCUCA AGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUUACAAAAUGUUGUCCCCCAA AAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCUGCAGGUCGACU ( SEQ ID NO: 1 ) Rv2450c / RpfE RNA (812 nt) GAGACAAGCUUGCUUGUUCUUUUUGCAGAAGCUCAGAAUAAACGCUCAACUUUGGCAGAUCUCCACCA UGAAGAACGCCCGUACGACGCUCAUCGCCGCCGCGAUUGCCGGGACGUUGGUGACCACGUCACCAGCC GGUAUCGCCAAUGCCGACGACGCGGGCUUGGACCCAAACGCCGCAGCCGGCCCGGAUGCCGUGGGCUU UGACCCGAACCUGCCGCCGGCCCCGGACGCUGCACCCGUCGAUACUCCGCCGGCUCCGGAGGACGCGG GCUUUGAUCCCAACCUCCCCCCGCCGCUGGCCCCGGACUUCCUGUCCCCGCCUGCGGAGGAAGCGCCU CCCGUGCCCGUGGCCUACAGCGUGAACUGGGACGCGAUCGCGCAGUGCGAGUCCGGUGGAAACUGGUC GAUCAACACCGGUAACGGUUACUACGGCGGCCUGCGGUUCACCGCCGGCACCUGGCGUGCCAACGGUG GCUCGGGGUCCGCGGCCAACGCGAGCCGGGAGGAGCAGAUCCGGGUGGCUGAGAACGUGCUGCGUUCG CAGGGUAUCCGCGCCUGGCCGGUCUGCGGCCGCCGCGGCUGAACUAGUGACUGACUAGGAUCUGGUUA CCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUUAC AAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUC UAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCUGCAGGUCGACU( SEQ ID NO: 2 )Rvll95 / PE13 RNA (593 nt) GAGACAAGCUUGCUUGUUCUUUUUGCAGAAGCUCAGAAUAAACGCUCAACUUUGGCAGAUCUCCACCA UGUCUUUCGUGAUGGCAUACCCAGAGAUGUUGGCGGCGGCGGCUGACACCCUGCAGAGCAUCGGUGCU ACCACUGUGGCUAGCAAUGCCGCUGCGGCGGCCCCGACGACUGGGGUGGUGCCCCCCGCUGCCGAUGA GGUGUCGGCGCUGACUGCGGCGCACUUCGCCGCACAUGCGGCGAUGUAUCAGUCCGUGAGCGCUCGGG CUGCUGCGAUUCAUGACCAGUUCGUGGCCACCCUUGCCAGCAGCGCCAGCUCGUAUGCGGCCACUGAA GUCGCCAAUGCGGCGGCGGCCAGCUAAACUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCU CAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCUGCAGGUCGACU ( SEQ ID NO: 3 ) Rvl386 / PE15 RNA (602 nt) GAGACAAGCUUGCUUGUUCUUUUUGCAGAAGCUCAGAAUAAACGCUCAACUUUGGCAGAUCUCCACCA UGACGUUGCGAGUCGUUCCCGAAAGCCUGGCAGGCGCCAGCGCUGCCAUCGAAGCAGUGACCGCUCGC CUGGCCGCCGCGCACGCCGCGGCGGCCCCGUUUAUCGCGGCGGUCAUCCCGCCUGGGUCCGACUCGGU UUCGGUGUGCAACGCCGUUGAGUUCAGCGUUCACGGUAGUCAGCAUGUGGCAAUGGCCGCUCAGGGGG UUGAGGAGCUCGGCCGCUCGGGGGUCGGGGUGGCCGAAUCGGGUGCCAGUUAUGCCGCUAGGGAUGCG CUGGCGGCGGCGUCGUAUCUCAGCGGUGGGCUAUGAACUAGUGACUGACUAGGAUCUGGUUACCACUA AACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUUACAAAAUG UUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCUGCAGGUCGACU ( SEQ ID NO: 4 )Rvl234 / MMAR 4207 RNA (821 nt) GAGACAAGCUUGCUUGUUCUUUUUGCAGAAGCUCAGAAUAAACGCUCAACUUUGGCAGAUCUCCACCA UGACUAGCCCAUUCCAGCCCAGACAGGUUCCCGGUUCAACACCCGCCGCCGCAGGUGCGGGUCGACGU GGUGUGCCCGCAUUGCCCACCCCGCCGAAAGGUUGGCCAGUCGGGUCGUAUCCCACCUAUGCCGAGGC GCAACGUGCGGUCGACUAUCUAUCCGAACAGCAGUUCCCGGUCCAGCAGGUGACCAUCGUUGGCGUGG ACCUCAUGCAGGUUGAACGGGUCACAGGCCGGCUGACCUGGCCCAAAGUGCUUGGUGGCGGCGUGCUG AGUGGCGCCUGGCUGGGCCUGUUCAUCGGGUUGGUGCUCGGGUUCUUCAGUCCCAAUCCAUGGUCCGC GCUGGUUACCGGCCUGGUGGCCGGGGUGUUCUUCGGGCUGAUCACCUCUGCAGUGCCGUACGCAAUGG CUCGCGGCACAAGGGAUUUCAGCUCGACCAUGCAACUGGUUGCCGGUCGCUACGACGUACUUUGUGAU CCGCAAAAUGCGGAAAAGGCACGGGAUCUGCUGGCGCGUCUGGCGAUCUGAACUAGUGACUGACUAGG AUCUGGUUACCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUU ACACUUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUC UUCACAUUCUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCUGCAGGU CGACU ( SEQ ID NO: 5 )RvO359 / MMAR 0678 RNA (1073 nt) GAGACAAGCUUGCUUGUUCUUUUUGCAGAAGCUCAGAAUAAACGCUCAACUUUGGCAGAUCUCCACCA UGAGCGAGACGGGCCAGCGCGAGUCGGUGCGACCCAGCCCGAUCUUUCUGGGCCUGCUCGGAUUGACG GCCGUCGGGGGCGCGCUGGCCUGGCUGGCCGGGGAGACGGUGCAGCCGCUGGCCUACGCCGGGGUGUU CGUCAUGGUGAUCGCCGGCUGGCUGGUGUCGCUGUGCCUGCACGAGUUCGGUCACGCGUUCACCGCUU GGCGUUUCGGUGACCACGACGUCGCAGUGCGCGGCUACCUGACGCUGGAUCCCCGCCGCUACAGCCAU CCCAUGCUCUCGCUCGGUCUGCCGAUGCUGUUCAUCGCCCUGGGCGGGAUCGGUCUGCCGGGUGCCGC GGUGUAUGUGCACACCUGGUUCAUGACGACGGCGCGCCGCACCCUGGUCAGUUUGGCGGGGCCGACGG UCAACCUGGCGCUGGCCAUGUUGCUGCUGGCGGCGACCCGGUUGUUGUUCGACCCGAUCCACGCGGUG UUAUGGGCCGGGGUGGCGUUCCUAGCAUUCCUUCAGCUCACCGCGCUGGUGUUAAACCUGCUACCCAU CCCGGGUCUGGACGGCUAUGCGGCCCUGGAGCCGCACCUGAGACCCGAGACGCAGCGCGCCCUGGCGC CGGCCAAGCAGUUCGCUUUGGUGUUUCUGCUGGUCCUGUUCCUGGCGCCGACGCUGAACGGGUGGUUU UUCGGGGUGGUGUACUGGCUCUUCGACCUGUCUGGCGUGUCGCACCGGCUGGCCGCCGCGGGCAGCGU GCUGGCCCGUUUCUGGAGUAUCUGGUUCUGAACUAGUGACUGACUAGGAUCUGGUUACCACUAAACCA GCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUUACAAAAUGUUGUC CCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCUGCAGGUCGACU ( SEQ ID NO: 6 )Table 3. Examples of alternative Mtb antigen coding sequences for the mRNA constructs of the present invention.Rv3804c / Ag85A RNA (1017 nt) AUGCAGCUUGUUGACAGGGUUCGUGGCGCCGUCACGGGUAUGUCGCGUCGACUCGUGGUCGGGGCCGU CGGCGCGGCCCUAGUGUCGGGUCUGGUCGGCGCCGUCGGUGGCACGGCGACCGCGGGGGCAUUUUCCC GGCCGGGCUUGCCGGUGGAGUACCUGCAGGUGCCGUCGCCGUCGAUGGGCCGUGACAUCAAGGUCCAA UUCCAAAGUGGUGGUGCCAACUCGCCCGCCCUGUACCUGCUCGACGGCCUGCGCGCGCAGGACGACUU CAGCGGCUGGGACAUCAACACCCCGGCGUUCGAGUGGUACGACCAGUCGGGCCUGUCGGUGGUCAUGC CGGUGGGUGGCCAGUCAAGCUUCUACUCCGACUGGUACCAGCCCGCCUGCGGCAAGGCCGGUUGCCAG ACUUACAAGUGGGAGACCUUCCUGACCAGCGAGCUGCCGGGGUGGCUGCAGGCCAACAGGCACGUCAA GCCCACCGGAAGCGCCGUCGUCGGUCUUUCGAUGGCUGCUUCUUCGGCGCUGACGCUGGCGAUCUAUC ACCCCCAGCAGUUCGUCUACGCGGGAGCGAUGUCGGGCCUGUUGGACCCCUCCCAGGCGAUGGGUCCC ACCCUGAUCGGCCUGGCGAUGGGUGACGCUGGCGGCUACAAGGCCUCCGACAUGUGGGGCCCGAAGGA GGACCCGGCGUGGCAGCGCAACGACCCGCUGUUGAACGUCGGGAAGCUGAUCGCCAACAACACCCGCG UCUGGGUGUACUGCGGCAACGGCAAGCCGUCGGAUCUGGGUGGCAACAACCUGCCGGCCAAGUUCCUC GAGGGCUUCGUGCGGACCAGCAACAUCAAGUUCCAAGACGCCUACAACGCCGGUGGCGGCCACAACGG CGUGUUCGACUUCCCGGACAGCGGUACGCACAGCUGGGAGUACUGGGGCGCGCAGCUCAACGCUAUGA AGCCCGACCUGCAACGGGCACUGGGUGCCACGCCCAACACCGGGCCCGCGCCCCAGGGCGCCUAG( SEQ ID NO: 7 )RvO129c / Ag85C RNA (1023 nt) AUGACGUUCUUCGAACAGGUGCGAAGGUUGCGGAGCGCAGCGACAACCCUGCCGCGCCGGCUGGCUAU CGCGGCUAUGGGGGCUGUCCUGGUUUACGGUCUGGUCGGUACCUUCGGCGGGCCGGCCACCGCGGGCG CAUUCUCUAGGCCCGGUCUUCCAGUGGAAUAUCUGCAGGUGCCAUCCGCGUCGAUGGGCCGCGACAUC AAGGUCCAGUUCCAGGGCGGCGGACCGCACGCGGUCUACCUGCUCGACGGUCUGCGGGCCCAGGAUGA CUACAACGGCUGGGACAUCAACACCCCGGCCUUCGAGGAGUACUACCAGUCAGGGUUGUCGGUGAUCA UGCCCGUGGGCGGCCAAUCCAGUUUCUACACCGACUGGUAUCAGCCCUCGCAGAGCAACGGCCAGAAC UACACCUACAAGUGGGAGACCUUCCUUACCAGAGAGAUGCCCGCCUGGCUACAGGCCAACAAGGGCGU GUCCCCGACAGGCAACGCGGCGGUGGGUCUUUCGAUGUCGGGCGGUUCCGCGCUGAUCCUGGCCGCGU ACUACCCGCAGCAGUUCCCGUACGCCGCGUCGUUGUCGGGCUUCCUCAACCCGUCCGAGGGCUGGUGG CCGACGCUGAUCGGCCUGGCGAUGAACGACUCGGGCGGUUACAACGCCAACAGCAUGUGGGGUCCGUC CAGCGACCCGGCCUGGAAGCGCAACGACCCAAUGGUUCAGAUUCCCCGCCUGGUCGCCAACAACACCC GGAUCUGGGUGUACUGCGGUAACGGCACACCCAGCGACCUCGGCGGCGACAACAUACCGGCGAAGUUC CUGGAAGGCCUCACCCUGCGCACCAACCAGACCUUCCGGGACACCUACGCGGCCGACGGUGGACGCAA CGGGGUGUUUAACUUCCCGCCCAACGGAACACACUCGUGGCCCUACUGGAACGAGCAGCUGGUCGCCA UGAAGGCCGAUAUCCAGCAUGUGCUCAACGGCGCGACACCCCCGGCCGCCCCUGCUGCGCCGGCCGCC UGA ( SEQ ID NO: 8 )RvO867c / RpfA RNA (1224 nt) AUGAGUGGACGCCACCGUAAGCCCACCACAUCCAACGUCAGCGUCGCCAAGAUCGCCUUUACCGGCGC AGUACUCGGUGGCGGCGGCAUCGCCAUGGCCGCUCAGGCGACCGCGGCCACCGACGGGGAAUGGGAUC AGGUGGCCCGCUGCGAGUCGGGCGGCAACUGGUCGAUCAACACCGGCAACGGUUACCUCGGUGGCUUG CAGUUCACUCAAAGCACCUGGGCCGCACAUGGUGGCGGCGAGUUCGCCCCGUCGGCUCAGCUGGCCAG CCGGGAGCAGCAGAUUGCCGUCGGUGAGCGGGUGCUGGCCACCCAGGGUCGCGGCGCCUGGCCGGUGU GCGGCCGCGGGUUAUCGAACGCAACACCCCGCGAAGUGCUUCCCGCUUCGGCAGCGAUGGACGCUCCG UUGGACGCGGCCGCGGUCAACGGCGAACCAGCACCGCUGGCCCCGCCGCCCGCCGACCCGGCGCCACC CGUGGAACUUGCCGCUAACGACCUGCCCGCACCGCUGGGUGAACCCCUCCCGGCAGCUCCCGCCGACC CGGCACCACCCGCCGACCUGGCACCACCCGCGCCCGCCGACGUCGCGCCACCCGUGGAACUUGCCGUA AACGACCUGCCCGCACCGCUGGGUGAACCCCUCCCGGCAGCUCCCGCCGACCCGGCACCACCCGCCGA CCUGGCACCACCCGCGCCCGCCGACCUGGCGCCACCCGCGCCCGCCGACCUGGCGCCACCCGCGCCCGCCGACCUGGCACCACCCGUGGAACUUGCCGUAAACGACCUGCCCGCGCCGCUGGGUGAACCCCUCCCG GCAGCUCCCGCCGAACUGGCGCCACCCGCCGAUCUGGCACCCGCGUCCGCCGACCUGGCGCCACCCGC GCCCGCCGACCUGGCGCCACCCGCGCCCGCCGAACUGGCGCCACCCGCGCCCGCCGACCUGGCACCAC CCGCUGCGGUGAACGAGCAAACCGCGCCGGGCGAUCAGCCCGCCACAGCUCCAGGCGGCCCGGUUGGC CUUGCCACCGAUUUGGAACUCCCCGAGCCCGACCCCCAACCAGCUGACGCACCGCCGCCCGGCGACGU CACCGAGGCGCCCGCCGAAACGCCCCAAGUCUCGAACAUCGCCUAUACGAAGAAGCUGUGGCAGGCGA UUCGGGCCCAGGACGUCUGCGGCAACGAUGCGCUGGACUCGCUCGCACAGCCGUACGUCAUCGGCUGA( SEQ ID NO: 9 )Rvl009 / RpfB RNA (1089 nt) AUGUUGCGCCUGGUAGUCGGUGCGCUGCUGCUGGUGUUGGCGUUCGCCGGUGGCUAUGCGGUCGCCGC AUGCAAAACGGUGACGUUGACCGUCGACGGAACCGCGAUGCGGGUGACCACGAUGAAAUCGCGGGUGA UCGACAUCGUCGAAGAGAACGGGUUCUCAGUCGACGACCGCGACGACCUGUAUCCCGCGGCCGGCGUG CAGGUCCAUGACGCCGACACCAUCGUGCUGCGGCGUAGCCGUCCGCUGCAGAUCUCGCUGGAUGGUCA CGACGCUAAGCAGGUGUGGACGACCGCGUCGACGGUGGACGAGGCGCUGGCCCAACUCGCGAUGACCG ACACGGCGCCGGCCGCGGCUUCUCGCGCCAGCCGCGUCCCGCUGUCCGGGAUGGCGCUACCGGUCGUC AGCGCCAAGACGGUGCAGCUCAACGACGGCGGGUUGGUGCGCACGGUGCACUUGCCGGCCCCCAAUGU CGCGGGGCUGCUGAGUGCGGCCGGCGUGCCGCUGUUGCAAAGCGACCACGUGGUGCCCGCCGCGACGG CCCCGAUCGUCGAAGGCAUGCAGAUCCAGGUGACCCGCAAUCGGAUCAAGAAGGUCACCGAGCGGCUG CCGCUGCCGCCGAACGCGCGUCGUGUCGAGGACCCGGAGAUGAACAUGAGCCGGGAGGUCGUCGAAGA CCCGGGGGUUCCGGGGACCCAGGAUGUGACGUUCGCGGUAGCUGAGGUCAACGGCGUCGAGACCGGCC GUUUGCCCGUCGCCAACGUCGUGGUGACCCCGGCCCACGAAGCCGUGGUGCGGGUGGGCACCAAGCCC GGUACCGAGGUGCCCCCGGUGAUCGACGGAAGCAUCUGGGACGCGAUCGCCGGCUGUGAGGCCGGUGG CAACUGGGCGAUCAACACCGGCAACGGGUAUUACGGUGGUGUGCAGUUUGACCAGGGCACCUGGGAGG CCAACGGCGGGCUGCGGUAUGCACCCCGCGCUGACCUCGCCACCCGCGAAGAGCAGAUCGCCGUUGCC GAGGUGACCCGACUGCGUCAAGGUUGGGGCGCCUGGCCGGUAUGUGCUGCACGAGCGGGUGCGCGCUGA ( SEQ ID NO: 10 )Rvl884c / RpfC RNA (531 nt) GUGCAUCCUUUGCCGGCCGACCACGGCCGGUCGCGGUGCAAUAGACACCCGAUCUCACCACUCUCUCU AAUCGGUAACGCUUCGGCCACUUCCGGCGAUAUGUCGAGCAUGACAAGAAUCGCCAAGCCGCUCAUCA AGUCCGCCAUGGCCGCAGGACUCGUCACGGCAUCCAUGUCGCUCUCCACCGCCGUUGCCCACGCCGGU CCCAGCCCGAACUGGGACGCCGUCGCGCAGUGCGAAUCCGGGGGCAACUGGGCGGCCAACACCGGAAA CGGCAAAUACGGCGGACUGCAGUUCAAGCCGGCCACCUGGGCCGCAUUCGGCGGUGUCGGCAACCCAG CAGCUGCCUCUCGGGAACAACAAAUCGCAGUUGCCAAUCGGGUUCUCGCCGAACAGGGAUUGGACGCG UGGCCGACGUGCGGCGCCGCCUCUGGCCUUCCGAUCGCACUGUGGUCGAAACCCGCGCAGGGCAUCAA GCAAAUCAUCAACGAGAUCAUUUGGGCAGGCAUUCAGGCAAGUAUUCCGCGCUGA ( SEQ ID NO: 11 )Rv2389c / RpfD RNA (465 nt) AUGACACCGGGUUUGCUUACUACUGCGGGUGCUGGCCGACCACGUGACAGGUGCGCCAGGAUCGUAUG CACGGUGUUCAUCGAAACCGCCGUUGUCGCGACCAUGUUUGUCGCGUUGUUGGGUCUGUCCACCAUCA GCUCGAAAGCCGACGACAUCGAUUGGGACGCCAUCGCGCAAUGCGAAUCCGGCGGCAAUUGGGCGGCC AACACCGGUAACGGGUUAUACGGUGGUCUGCAGAUCAGCCAGGCGACGUGGGAUUCCAACGGUGGUGU CGGGUCGCCGGCGGCCGCGAGUCCCCAGCAACAGAUCGAGGUCGCAGACAACAUUAUGAAAACCCAAG GCCCGGGUGCGUGGCCGAAAUGUAGUUCUUGUAGUCAGGGAGACGCACCGCUGGGCUCGCUCACCCAC AUCCUGACGUUCCUCGCGGCCGAGACUGGAGGUUGUUCGGGGAGCAGGGACGAUUGA ( SEQ ID NO: 12 )RvO285 / PE5 RNA (309 nt) AUGACGUUGCGAGUGGUUCCGGAGGGGCUGGCCGCAGCCAGCGCUGCGGUGGAAGCGCUGACGGCGCG GUUGGCCGCCGCGCAUGCGAGCGCAGCGCCGGUGAUUACCGCGGUAGUGCCGCCGGCGGCGGAUCCGG UGUCGCUGCAGACCGCGGCCGGGUUCAGUGCACAGGGCGUCGAGCACGCGGUCGUCACCGCCGAAGGU GUCGAAGAGCUGGGACGCGCCGGCGUUGGUGUGGGCGAAUCCGGCGCCAGCUACCUGGCCGGUGAUGC GGCCGCCGCCGCUACGUACGGGGUCGUGGGCGGCUGA ( SEQ ID NO: 13 )Rv3022A / PE29 RNA (315 nt) GUGACGCUGAGAGUGGUUCCUGAGGGUUUGGCGGCCGCCAGUGCGGCGGUGGAGGCGUUGACCGCACG GCUGGCCGCCGCACACGCUGGCGCGGCGCCGGCGAUUACGGCGGUGGUGGCGCCGGCGGCGGAUCCGG UGUCGUUGCAGAGUGCGGUGGGGUUUAGCGCCUUAGGUAGCGAGCAUGCGGCGAUCGCGGGCGAAGGG GUCGAGGAGCUGGGUCGUUCCGGGGUCGCUGUGGGUGAGUCUGGGAUCGGUUAUGCCGCCGGUGAUGC GGUGGCGGCGGCGACGUAUCUGGUUUCGGGUGGGUCGUUGUGA ( SEQ ID NO: 14 ) Rv3477 / PE31 RNA (297 nt) GUGUCUUUCACUGCGCAACCGGAGAUGUUGGCGGCCGCGGCUGGCGAACUUCGUUCCCUGGGGGCAAC GCUGAAGGCUAGCAAUGCCGCCGCAGCCGUGCCGACGACUGGGGUGGUGCCCCCGGCUGCCGACGAGG UGUCGCUGCUGCUUGCCACACAAUUCCGUACGCAUGCGGCGACGUAUCAGACGGCCAGCGCCAAGGCC GCGGUGAUCCAUGAGCAGUUUGUGACCACGCUGGCCACCAGCGCUAGUUCAUAUGCGGACACCGAGGC CGCCAACGCUGUGGUCACCGGCUAG ( SEQ ID NO: 15 )Rv0096 / PPEl RNA (1392 nt) AUGGCUAUACCACCGGAGGUGCACUCGGGCCUGUUGAGCGCCGGGUGCGGUCCGGGAUCAUUGCUUGU UGCCGCGCAGCAGUGGCAAGAACUUAGUGAUCAGUACGCACUCGCAUGCGCCGAGUUGGGCCAAUUGU UGGGCGAGGUUCAGGCCAGCAGCUGGCAGGGAACCGCCGCCACCCAGUACGUGGCUGCCCAUGGCCCC UAUCUGGCCUGGCUUGAGCAAACCGCGAUCAACAGCGCCGUCACCGCCGCACAGCACGUAGCGGCUGC CGCUGCCUACUGCAGCGCCCUGGCCGCGAUGCCCACCCCAGCAGAGCUGGCCGCCAACCACGCCAUUC AUGGCGUUCUGAUCGCCACCAACUUCUUCGGGAUCAACACCGUUCCGAUCGCGCUCAACGAAGCCGAU UAUGUCCGCAUGUGGCUGCAAGCCGCCGACACCAUGGCCGCCUACCAGGCCGUCGCCGAUGCGGCCAC GGUGGCCGUACCGUCCACCCAACCGGCGCCACCGAUCCGCGCGCCCGGCGGCGAUGCCGCAGAUACCC GGCUAGACGUAUUGAGUUCAAUUGGUCAGCUCAUCCGGGAUAUCUUGGAUUUCAUUGCCAACCCGUAC AAGUAUUUUCUGGAGUUUUUCGAGCAAUUCGGCUUCAGCCCGGCCGUAACGGUCGUCCUUGCCCUUGU UGCCCUGCAGCUGUACGACUUUCUUUGGUAUCCCUAUUACGCCUCGUACGGCCUGCUCCUGCUUCCGU UCUUCACUCCCACCUUGAGCGCGUUGACCGCCCUAAGCGCGCUGAUCCAUUUGCUGAACCUGCCCCCG GCUGGACUGCUUCCUAUCGCCGCAGCGCUCGGUCCCGGCGACCAAUGGGGCGCAAACUUGGCUGUGGC UGUCACGCCGGCCACGGCGGCCGUGCCCGGCGGAAGCCCGCCCACCAGCAACCCCGCGCCCGCCGCUC CCAGCUCGAACUCGGUUGGCAGCGCUUCGGCUGCACCCGGCAUCAGCUAUGCCGUGCCCGGCCUGGCG CCACCCGGGGUUAGCUCUGGCCCUAAAGCCGGCACCAAAUCACCUGACACCGCCGCCGACACCCUUGC AACCGCGGGCGCAGCACGACCGGGCCUCGCCCGAGCCCACCGAAGAAAGCGCAGCGAAAGCGGCGUCG GGAUACGCGGUUACCGCGACGAAUUUUUGGACGCGACCGCCACGGUGGACGCCGCUACGGAUGUGCCC GCUCCCGCCAACGCGGCUGGCAGUCAAGGUGCCGGCACUCUCGGCUUUGCCGGUACCGCACCGACAAC CAGCGGCGCCGCGGCCGGAAUGGUUCAACUGUCGUCGCACAGCACAAGCACUACAGUCCCGUUGCUGC CCACUACCUGGACAACCGACGCCGAACAAUGA ( SEQ ID NO: 16 )RvO256c / PPE2 RNA (1671 nt) AUGACCGCCCCGAUCUGGAUGGCUUCGCCCCCAGAGGUGCACUCGGCGCUGCUAAGCAGCGGGCCUGG GCCCGGUCCGCUGCUGGUGUCGGCCGAGGGGUGGCACUCGUUGAGCAUCGCCUACGCGGAGACGGCCG ACGAGCUGGCCGCGCUGUUGGCCGCCGUACAGGCCGGCACCUGGGACGGCCCGACCGCCGCGGUUUAC GUGGCCGCCCAUACCCCUUAUCUGGCGUGGCUGGUGCAGGCCAGCGCUAACAGCGCGGCCAUGGCCAC CCGGCAAGAAACCGCGGCCACCGCCUACGGCACCGCCUUGGCCGCGAUGCCAACGUUGGCCGAGCUGGGCGCCAACCACGCCCUCCACGGCGUGCUGAUGGCGACGAACUUCUUCGGCAUCAACACCAUCCCGAUC GCGCUCAACGAGUCCGACUACGCGCGGAUGUGGAUCCAGGCCGCCACCACGAUGGCCAGCUAUCAAGC GGUCUCGACCGCGGCGGUGGCCGCCGCACCGCAGACCACCCCAGCCCCCCAGAUCGUGAAAGCCAACG CGCCGACAGCGGCUUCCGACGAGCCGAACCAAGUCCAGGAAUGGCUGCAAUGGUUGCAGAAGAUCGGG UAUACCGACUUCUACAACAACGUUAUACAACCGUUCAUCAACUGGCUGACCAACCUCCCCUUUUUGCA AGCGAUGUUUUCCGGAUUUGAUCCGUGGCUGCCCUCGCUGGGUAAUCCGCUAACCUUCCUAAGCCCGG CCAACAUUGCGUUCGCCCUUGGCUACCCCAUGGACAUCGGAUCUUAUGUCGCCUUCCUGUCGCAAACC UUCGCAUUCAUCGGGGCGGAUCUGGCGGCGGCGUUCGCGUCGGGCAAUCCCGCAACUAUCGCUUUUAC CCUGAUGUUCACCACGGUCGAAGCCAUUGGCACGAUCAUCACCGAUACCAUCGCGCUGGUUAAAACGC UGCUCGAGCAAACUCUUGCGUUGCUCCCGGCGGCGCUGCCCCUGCUAGCCGCCCCGCUGGCGCCGUUG ACCCUUGCCCCUGCGAGUGCAGCAGGCGGCUUCGCGGGCUUGUCUGGGCUGGCGGGCCUGGUUGGCAU CCCGCCAUCCGCGCCGCCCGUCAUCCCGCCGGUCGCGGCGAUUGCCCCGAGUAUCCCGACCCCCACCC CAACUCCAGCCCCAGCCCCGGCCCCCACGGCCGUGACCGCGCCGACGCCCCCGCCCGGGCCGCCACCG CCGCCGGUGACCGCCCCGCCGCCGGUGACCGGAGCCGGCAUACAAAGCUUCGGGUACCUGGUGGGUGA CCUGAACUCGGCGGCGCAGGCCAGGAAGGCCGUCGGCACCGGCGUUCGAAAGAAGACGCCGGAACCCG ACAGCGCCGAGGCCCCAGCGGCCGCGGCGGCGCCCGAGGAACAGGUUCAACCGCAGCGGCGUCGGCGG CCAAAGAUCAAACAGCUCGGCCGCGGUUACGAAUACCUGGAUUUAGACCCCGAAACCGGCCACGACCC GACGGGUUCGCCUCAAGGAGCGGGAACCCUGGGUUUCGCCGGAACAACCCACAAGGCCAGUCCCGGAC AAGUCGCAGGACUGAUCACGUUACCCAACGACGCGUUCGGCGGCAGCCCACGCACGCCAAUGAUGCCC GGAACCUGGGACACCGACUCAGCGACCCGGGUGGAGUGA ( SEQ ID NO: 17 ) Rvll96 / PPE18 RNA (1176 nt) AUGGUGGAUUUCGGGGCGUUACCACCGGAGAUCAACUCCGCGAGGAUGUACGCCGGCCCGGGUUCGGC CUCGCUGGUGGCCGCGGCUCAGAUGUGGGACAGCGUGGCGAGUGACCUGUUUUCGGCCGCGUCGGCGU UUCAGUCGGUGGUCUGGGGUCUGACGGUGGGGUCGUGGAUAGGUUCGUCGGCGGGUCUGAUGGUGGCG GCGGCCUCGCCGUAUGUGGCGUGGAUGAGCGUCACCGCGGGGCAGGCCGAGCUGACCGCCGCCCAGGU CCGGGUUGCUGCGGCGGCCUACGAGACGGCGUAUGGGCUGACGGUGCCCCCGCCGGUGAUCGCCGAGA ACCGUGCUGAACUGAUGAUUCUGAUAGCGACCAACCUCUUGGGGCAAAACACCCCGGCGAUCGCGGUC AACGAGGCCGAAUACGGCGAGAUGUGGGCCCAAGACGCCGCCGCGAUGUUUGGCUACGCCGCGGCGAC GGCGACGGCGACGGCGACGUUGCUGCCGUUCGAGGAGGCGCCGGAGAUGACCAGCGCGGGUGGGCUCC UCGAGCAGGCCGCCGCGGUCGAGGAGGCCUCCGACACCGCCGCGGCGAACCAGUUGAUGAACAAUGUG CCCCAGGCGCUGCAACAGCUGGCCCAGCCCACGCAGGGCACCACGCCUUCUUCCAAGCUGGGUGGCCU GUGGAAGACGGUCUCGCCGCAUCGGUCGCCGAUCAGCAACAUGGUGUCGAUGGCCAACAACCACAUGU CGAUGACCAACUCGGGUGUGUCGAUGACCAACACCUUGAGCUCGAUGUUGAAGGGCUUUGCUCCGGCG GCGGCCGCCCAGGCCGUGCAAACCGCGGCGCAAAACGGGGUCCGGGCGAUGAGCUCGCUGGGCAGCUC GCUGGGUUCUUCGGGUCUGGGCGGUGGGGUGGCCGCCAACUUGGGUCGGGCGGCCUCGGUCGGUUCGU UGUCGGUGCCGCAGGCCUGGGCCGCGGCCAACCAGGCAGUCACCCCGGCGGCGCGGGCGCUGCCGCUG ACCAGCCUGACCAGCGCCGCGGAAAGAGGGCCCGGGCAGAUGCUGGGCGGGCUGCCGGUGGGGCAGAU GGGCGCCAGGGCCGGUGGUGGGCUCAGUGGUGUGCUGCGUGUUCCGCCGCGACCCUAUGUGAUGCCGC AUUCUCCGGCGGCCGGCUAG ( SEQ ID NO: 18 )Rvl387 / PPE20 RNA (1620 nt) AUGACCGAGCCGUGGAUAGCCUUCCCUCCCGAGGUGCACUCGGCGAUGCUGAACUACGGUGCGGGCGU UGGGCCGAUGUUGAUCUCCGCCACGCAGAAUGGGGAGCUCAGCGCCCAAUACGCAGAAGCGGCAUCAG AGGUCGAGGAAUUGUUGGGGGUGGUGGCCUCCGAGGGAUGGCAGGGGCAAGCCGCCGAGGCGUUUGUC GCCGCGUACAUGCCGUUUCUGGCGUGGCUGAUCCAAGCCAGCGCCGACUGCGUGGAAAUGGCCGCCCA GCAACACGUCGUCAUCGAGGCCUACACUGCCGCGGUAGAGCUGAUGCCUACUCAGGUCGAACUGGCCG CCAACCAAAUCAAGCUCGCGGUGUUGGUAGCGACCAAUUUCUUUGGCAUCAACACCAUUCCCAUUGCG AUCAAUGAGGCCGAGUACGUGGAGAUGUGGGUUCGGGCCGCCACCACGAUGGCGACCUAUUCAACAGU CUCCAGAUCGGCGCUCUCCGCGAUGCCGCACACCAGCCCCCCGCCGCUGAUCCUGAAAUCCGAUGAAC UGCUCCCCGACACCGGGGAGGACUCCGAUGAAGACGGCCACAACCAUGGCGGUCACAGUCAUGGCGGUCACGCCAGGAUGAUCGAUAACUUCUUUGCCGAAAUCCUGCGUGGCGUCAGCGCGGGCCGCAUUGUUUG GGACCCCGUCAACGGCACCCUCAACGGACUCGACUACGACGAUUACGUCUACCCCGGUCACGCGAUCU GGUGGCUGGCUCGAGGCCUCGAGUUUUUUCAGGAUGGUGAACAAUUUGGCGAACUGUUGUUCACCAAU CCGACUGGGGCUUUUCAGUUCCUCCUCUACGUCGUUGUGGUGGAUUUGCCGACGCACAUAGCCCAGAU CGCUACCUGGCUGGGCCAGUACCCGCAGUUGCUGUCGGCUGCCCUCACUGGCGUCAUCGCCCACCUGG GAGCAAUAACUGGUUUGGCGGGCCUAUCCGGCCUGAGCGCCAUUCCGUCUGCUGCGAUACCCGCCGUU GUACCGGAGCUGACACCCGUCGCGGCCGCGCCGCCUAUGUUGGCGGUCGCCGGGGUGGGCCCUGCAGU CGCCGCGCCGGGCAUGCUCCCCGCCUCAGCACCCGCACCGGCGGCAGCGGCCGGCGCCACCGCAGCCG GCCCGACGCCGCCGGCGACUGGUUUCGGAGGCUUCCCGCCCUACCUGGUCGGCGGUGGCGGCCCAGGA AUAGGGUUCGGCUCGGGACAGUCGGCCCACGCCAAGGCCGCGGCGUCCGAUUCCGCUGCAGCCGAGUC GGCGGCCCAGGCCUCGGCGCGUGCGCAGGCGCGUGCUGCACGGCGGGGCCGCUCGGCGGCGAAGGCAC GUGGCCAUCGUGACGAAUUCGUCACGAUGGACAUGGGUUUCGACGCGGCAGCUCCGGCCCCAGAGCAC CAGCCGGGUGCCCGGGCGUCCGACUGUGGUGCGGGACCUAUCGGAUUUGCUGGCACGGUGCGCAAAGA GGCGGUCGUGAAAGCGGCGGGGUUGACCACGCUGGCCGGUGACGACUUCGGCGGCGGCCCAACGAUGC CGAUGAUGCCCGGCACCUGGACCCAUGAUCAGGGCGUGUUCGACGAGCAUCGCUGA ( SEQ ID NO: 19 )Rv3873 / PPE68 RNA (1107 nt) AUGCUGUGGCACGCAAUGCCACCGGAGCUAAAUACCGCACGGCUGAUGGCCGGCGCGGGUCCGGCUCC AAUGCUUGCGGCGGCCGCGGGAUGGCAGACGCUUUCGGCGGCUCUGGACGCUCAGGCCGUCGAGUUGA CCGCGCGCCUGAACUCUCUGGGAGAAGCCUGGACUGGAGGUGGCAGCGACAAGGCGCUUGCGGCUGCA ACGCCGAUGGUGGUCUGGCUACAAACCGCGUCAACACAGGCCAAGACCCGUGCGAUGCAGGCGACGGC GCAAGCCGCGGCAUACACCCAGGCCAUGGCCACGACGCCGUCGCUGCCGGAGAUCGCCGCCAACCACA UCACCCAGGCCGUCCUUACGGCCACCAACUUCUUCGGUAUCAACACGAUCCCGAUCGCGUUGACCGAG AUGGAUUAUUUCAUCCGUAUGUGGAACCAGGCAGCCCUGGCAAUGGAGGUCUACCAGGCCGAGACCGC GGUUAACACGCUUUUCGAGAAGCUCGAGCCGAUGGCGUCGAUCCUUGAUCCCGGCGCGAGCCAGAGCA CGACGAACCCGAUCUUCGGAAUGCCCUCCCCUGGCAGCUCAACACCGGUUGGCCAGUUGCCGCCGGCG GCUACCCAGACCCUCGGCCAACUGGGUGAGAUGAGCGGCCCGAUGCAGCAGCUGACCCAGCCGCUGCA GCAGGUGACGUCGUUGUUCAGCCAGGUGGGCGGCACCGGCGGCGGCAACCCAGCCGACGAGGAAGCCG CGCAGAUGGGCCUGCUCGGCACCAGUCCGCUGUCGAACCAUCCGCUGGCUGGUGGAUCAGGCCCCAGC GCGGGCGCGGGCCUGCUGCGCGCGGAGUCGCUACCUGGCGCAGGUGGGUCGUUGACCCGCACGCCGCU GAUGUCUCAGCUGAUCGAAAAGCCGGUUGCCCCCUCGGUGAUGCCGGCGGCUGCUGCCGGAUCGUCGG CGACGGGUGGCGCCGCUCCGGUGGGUGCGGGAGCGAUGGGCCAGGGUGCGCAAUCCGGCGGCUCCACC AGGCCGGGUCUGGUCGCGCCGGCACCGCUCGCGCAGGAGCGUGAAGAAGACGACGAGGACGACUGGGA CGAAGAGGACGACUGGUGA ( SEQ ID NO: 20 )Table 4. Antigen amino acid sequences expressed by the mRNA constructs in candidate vaccines A and B of the present disclosure.Rvl886c / Ag85B peptide (325 aa) MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQS GGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYK WETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLI GLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENF VRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG ( SEQ ID NO: 21 ) Rv2450c / RpfE peptide (172 aa) LKNARTTLIAAAIAGTLVTTSPAGIANADDAGLDPNAAAGPDAVGFDPNLPPAPDAAPVDTPPAPEDA GFDPNLPPPLAPDFLSPPAEEAPPVPVAYSVNWDAIAQCESGGNWSINTGNGYYGGLRFTAGTWRANG GSGSAANASREEQIRVAENVLRSQGIRAWPVCGRRG ( SEQ ID NO: 22 )Rvll95 / PE13 peptide (99 aa) VSFVMAYPEMLAAAADTLQSIGATTVASNAAAAAPTTGVVPPAADEVSALTAAHFAAHAAMYQSVSAR AAAIHDQFVATLASSASSYAATEVANAAAAS ( SEQ ID NO: 23 )Rvl386 / PE15 peptide (102 aa) VTLRVVPESLAGASAAIEAVTARLAAAHAAAAPFIAAVIPPGSDSVSVCNAVEFSVHGSQHVAMAAQG VEELGRSGVGVAESGASYAARDALAAASYLSGGL ( SEQ ID NO: 24 )Rvl234 / MMAR 4207 peptide (175 aa) MTSPFQPRQVPGSTPAAAGAGRRGVPALPTPPKGWPVGSYPTYAEAQRAVDYLSEQQFPVQQVTIVGV DLMQVERVTGRLTWPKVLGGGVLSGAWLGLFIGLVLGFFSPNPWSALVTGLVAGVFFGLITSAVPYAM ARGTRDFSSTMQLVAGRYDVLCDPQNAEKARDLLARLAI ( SEQ ID NO: 25 ) RvO359 / MMAR 0678 peptide (259 aa) VSETGQRESVRPSPIFLGLLGLTAVGGALAWLAGETVQPLAYAGVFVMVIAGWLVSLCLHEFGHAFTA WRFGDHDVAVRGYLTLDPRRYSHPMLSLGLPMLFIALGGIGLPGAAVYVHTWFMTTARRTLVSLAGPT VNLALAMLLLAATRLLFDPIHAVLWAGVAFLAFLQLTALVLNLLPIPGLDGYAALEPHLRPETQRALA PAKQFALVFLLVLFLAPTLNGWFFGVVYWLFDLSGVSHRLAAAGSVLARFWSIWF ( SEQ ID NO: 26 )Table 5. Antigen amino acid sequences expressed by the alternative Mtb antigen coding sequences.Rv3804c / Ag85A (338aa) MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQ FQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQ TYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGP TLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFL EGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA(SEQ ID NO: 27 )RvO129c / Ag85C (340aa) MTFFEQVRRLRSAATTLPRRLAIAAMGAVLVYGLVGTFGGPATAGAFSRPGLPVEYLQVPSASMGRDI KVQFQGGGPHAVYLLDGLRAQDDYNGWDINTPAFEEYYQSGLSVIMPVGGQSSFYTDWYQPSQSNGQN YTYKWETFLTREMPAWLQANKGVSPTGNAAVGLSMSGGSALILAAYYPQQFPYAASLSGFLNPSEGWW PTLIGLAMNDSGGYNANSMWGPSSDPAWKRNDPMVQIPRLVANNTRIWVYCGNGTPSDLGGDNIPAKF LEGLTLRTNQTFRDTYAADGGRNGVFNFPPNGTHSWPYWNEQLVAMKADIQHVLNGATPPAAPAAPAA SEQ ID NO: 28 )RvO867c / RpfA (407 aa) MSGRHRKPTTSNVSVAKIAFTGAVLGGGGIAMAAQATAATDGEWDQVARCESGGNWSINTGNGYLGGL QFTQSTWAAHGGGEFAPSAQLASREQQIAVGERVLATQGRGAWPVCGRGLSNATPREVLPASAAMDAP LDAAAVNGEPAPLAPPPADPAPPVELAANDLPAPLGEPLPAAPADPAPPADLAPPAPADVAPPVELAV NDLPAPLGEPLPAAPADPAPPADLAPPAPADLAPPAPADLAPPAPADLAPPVELAVNDLPAPLGEPLP AAPAELAP PADLAP AS AD LAP PAPADLAP PAPAELAP PAPADLAP PAAVNEQT APGDQPAT APGGPVG LATDLELPEPDPQPADAPPPGDVTEAPAETPQVSNIAYTKKLWQAIRAQDVCGNDALDSLAQPYVIG SEQ ID NO: 29 )Rvl009 / RpfB (362 aa) MLRLVVGALLLVLAFAGGYAVAACKTVTLTVDGTAMRVTTMKSRVIDIVEENGFSVDDRDDLYPAAGV QVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGMALPVV SAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPIVEGMQIQVTRNRIKKVTERL PLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKP GTEVPPVIDGSI DAIAGCEAGGNWAINTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREEQIAVA EVTRLRQGWGAWPVCAARAGAR (SEQ ID NO: 30 )Rvl884c / RpfC (176 aa) VHPLPADHGRSRCNRHPISPLSLIGNASATSGDMSSMTRIAKPLIKSAMAAGLVTASMSLSTAVAHAG PSPNWDAVAQCESGGNWAANTGNGKYGGLQFKPATWAAFGGVGNPAAASREQQIAVANRVLAEQGLDA WPTCGAASGLPIALWSKPAQGIKQIINEIIWAGIQASIPR ( SEQ ID NO: 31 ) Rv2389c / RpfD (154aa) MTPGLLTTAGAGRPRDRCARIVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQCESGGNWAA NTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTH ILTFLAAETGGCSGSRDD (SEQ ID NO: 32 )RvO285 / PE5 (102aa) MTLRVVPEGLAAASAAVEALTARLAAAHASAAPVITAVVPPAADPVSLQTAAGFSAQGVEHAVVTAEG VEELGRAGVGVGESGASYLAGDAAAAATYGVVGG ( SEQ ID NO: 33 )Rv3022A / PE29 (104aa) VTLRVVPEGLAAASAAVEALTARLAAAHAGAAPAITAVVAPAADPVSLQSAVGFSALGSEHAAIAGEG VEELGRSGVAVGESGIGYAAGDAVAAATYLVSGGSL ( SEQ ID NO: 34 )Rv3477 / PE31 (98aa) VSFTAQPEMLAAAAGELRSLGATLKASNAAAAVPTTGVVPPAADEVSLLLATQFRTHAATYQTASAKA AVIHEQFVTTLATSASSYADTEAANAVVTG ( SEQ ID NO: 35 )Rv0096 / PPEl (463aa) MAIPPEVHSGLLSAGCGPGSLLVAAQQWQELSDQYALACAELGQLLGEVQASSWQGTAATQYVAAHGP YLAWLEQTAINSAVTAAQHVAAAAAYCSALAAMPTPAELAANHAIHGVLIATNFFGINTVPIALNEAD YVRMWLQAADTMAAYQAVADAATVAVPSTQPAPPIRAPGGDAADTRLDVLSSIGQLIRDILDFIANPY KYFLEFFEQFGFSPAVTVVLALVALQLYDFLWYPYYASYGLLLLPFFTPTLSALTALSALIHLLNLPP AGLLPIAAALGPGDQWGANLAVAVTPATAAVPGGSPPTSNPAPAAPSSNSVGSASAAPGISYAVPGLA PPGVSSGPKAGTKSPDTAADTLATAGAARPGLARAHRRKRSESGVGIRGYRDEFLDATATVDAATDVP APANAAGSQGAGTLGFAGTAPTTSGAAAGMVQLSSHSTSTTVPLLPTTWTTDAEQ ( SEQ ID NO: 36 )RvO256c / PPE2 (556aa) MTAPIWMASPPEVHSALLSSGPGPGPLLVSAEGWHSLSIAYAETADELAALLAAVQAGTWDGPTAAVY VAAHTPYLAWLVQASANSAAMATRQETAATAYGTALAAMPTLAELGANHALHGVLMATNFFGINTIPI ALNESDYARMWIQAATTMASYQAVSTAAVAAAPQTTPAPQIVKANAPTAASDEPNQVQEWLQWLQKIG YTDFYNNVIQPFINWLTNLPFLQAMFSGFDPWLPSLGNPLTFLSPANIAFALGYPMDIGSYVAFLSQT FAFIGADLAAAFASGNPATIAFTLMFTTVEAIGTIITDTIALVKTLLEQTLALLPAALPLLAAPLAPL TLAPASAAGGFAGLSGLAGLVGIPPSAPPVIPPVAAIAPSIPTPTPTPAPAPAPTAVTAPTPPPGPPP PPVTAPPPVTGAGIQSFGYLVGDLNSAAQARKAVGTGVRKKTPEPDSAEAPAAAAAPEEQVQPQRRRR PKIKQLGRGYEYLDLDPETGHDPTGSPQGAGTLGFAGTTHKASPGQVAGLITLPNDAFGGSPRTPMMP GTWDTDSATRVE (SEQ ID NO: 37 )Rvll96 / PPE18 (391aa) MVDFGALPPEINSARMYAGPGSASLVAAAQMWDSVASDLFSAASAFQSVVWGLTVGSWIGSSAGLMVA AASPYVAWMSVTAGQAELTAAQVRVAAAAYETAYGLTVPPPVIAENRAELMILIATNLLGQNTPAIAV NEAEYGEMWAQDAAAMFGYAAATATATATLLPFEEAPEMTSAGGLLEQAAAVEEASDTAAANQLMNNV PQALQQLAQPTQGTTPSSKLGGLWKTVSPHRSPISNMVSMANNHMSMTNSGVSMTNTLSSMLKGFAPA AAAQAVQTAAQNGVRAMSSLGSSLGSSGLGGGVAANLGRAASVGSLSVPQAWAAANQAVTPAARALPL TSLTSAAERGPGQMLGGLPVGQMGARAGGGLSGVLRVPPRPYVMPHSPAAG ( SEQ ID NO: 38 ) Rvl387 / PPE20 (539aa) MTEPWIAFPPEVHSAMLNYGAGVGPMLISATQNGELSAQYAEAASEVEELLGVVASEGWQGQAAEAFV AAYMPFLAWLIQASADCVEMAAQQHVVIEAYTAAVELMPTQVELAANQIKLAVLVATNFFGINTIPIA INEAEYVEMWVRAATTMATYSTVSRSALSAMPHTSPPPLILKSDELLPDTGEDSDEDGHNHGGHSHGG HARMIDNFFAEILRGVSAGRIVWDPVNGTLNGLDYDDYVYPGHAIWWLARGLEFFQDGEQFGELLFTN PTGAFQFLLYVVVVDLPTHIAQIATWLGQYPQLLSAALTGVIAHLGAITGLAGLSGLSAIPSAAIPAV VPELTPVAAAPPMLAVAGVGPAVAAPGMLPASAPAPAAAAGATAAGPTPPATGFGGFPPYLVGGGGPG IGFGSGQSAHAKAAASDSAAAESAAQASARAQARAARRGRSAAKARGHRDEFVTMDMGFDAAAPAPEH QPGARASDCGAGPIGFAGTVRKEAVVKAAGLTTLAGDDFGGGPTMPMMPGTWTHDQGVFDEHR ( SEQ ID NO: 39 )Rv3873 / PPE68 (368aa) MLWHAMPPELNTARLMAGAGPAPMLAAAAGWQTLSAALDAQAVELTARLNSLGEAWTGGGSDKALAAA TPMVVWLQTASTQAKTRAMQATAQAAAYTQAMATTPSLPEIAANHITQAVLTATNFFGINTIPIALTE MDYFIRMWNQAALAMEVYQAETAVNTLFEKLEPMASILDPGASQSTTNPIFGMPSPGSSTPVGQLPPA ATQTLGQLGEMSGPMQQLTQPLQQVTSLFSQVGGTGGGNPADEEAAQMGLLGTSPLSNHPLAGGSGPS AGAGLLRAESLPGAGGSLTRTPLMSQLIEKPVAPSVMPAAAAGSSATGGAAPVGAGAMGQGAQSGGST RPGLVAPAPLAQEREEDDEDDWDEEDDW ( SEQ ID NO: 40 )CITATION LISTPatent LiteratureW02024023790WO2024138121WO2023201199Non-patent LiteratureMyllymaki, H., Niskanen, M., Oksanen, K. E., Sherwood, E., Ahava, M., Parikka, M. and Ramet, M. (2017). Identification of novel antigen candidates for a tuberculosis vaccine in the adult zebrafish (Danio rerio). PLoS ONE 12, e0181942.Myllymaki, H., Niskanen, M., Luukinen, H., Parikka, M. and Ramet, M. (2018). Identification of protective postexposure mycobacterial vaccine antigens using an immunosuppression-based reactivation model in the zebrafish. Dis. Model. Mech. 11, dmm033175. doi:10.1242 / dmm.033175.

Claims

CLAIMS1. A vaccine composition against a disease caused by Mycobacterium tuberculosis, said composition comprising at least four nucleic acids selected from the following groups: a. a nucleic acid encoding an Ag85A, Ag85B, or Ag85C antigen;b. a nucleic acid encoding a resuscitation promoting factor (Rpf) selected from the group consisting of: RpfA, RpfB, RpfC, RpfD and RpfE;c. a nucleic acid encoding a PE / PPE antigen selected from the group consisting of: PE5, PE13, PE15, PE29, PE31, PPE1, PPE2, PPE18, PPE20, and PPE68;d. a nucleic acid encoding a disease-reactivation-related antigen selected from the group consisting of: Rvl234 / MMAR_4207, and Rv0359 / MMAR_0678,wherein said vaccine composition comprises at least one nucleic acid from each of group a, b, and c; andwherein the nucleic acids are provided on one or more nucleic acid constructs.

2. The vaccine composition according to claim 1, wherein said composition comprises a lipid nanoparticle enclosing said one or more nucleic acids constructs.

3. The vaccine composition according to claim 2, wherein said lipid nanoparticle comprises i) a first phospholipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), and dipalmitoylphosphatidylcholine (DPPC); ii) a second phospholipid selected from the group consisting of sphingomyelins (SM), iii) PEG(2000)-dimyristoylglycerol (PEG-DMG); and iv) cholesterol.

4. The vaccine composition according to claim 3, wherein said lipid nanoparticle comprises l,2-distearoyl-sn-glycero-3-PC (1,2-DSPC), SM-102, cholesterol, and PEG-DMG.

5. The vaccine composition according to any one of claims 1-4, wherein said at least four nucleic acids encode at least antigens Ag85B, RpfE, PE13, and PE15.

6. The vaccine composition according to any one of claims 1-4, wherein said vaccine composition comprises at least one nucleic acid from each of group a, b, c, and d.

7. The vaccine composition according to any one of claims 1-6, wherein said at least four nucleic acids encode at least a nucleic acid encoding antigen Rv0359 / MMAR_0678.

8. The vaccine composition according to claim 6 or 7 further comprising a nucleic acid encoding antigen Rvl234 / MMAR_4207.

9. The vaccine composition according to any one of claims 1-8, wherein said composition comprises at least six nucleic acids encoding antigens Ag85B, RpfE, PE13, PE15, Rv0359 / MMAR_0678, and Rvl234 / MMAR_4207.

10. The vaccine composition according to any one of claims 1-9, wherein said at least four nucleic acids are messenger ribonucleic acids (mRNAs).

11. The vaccine composition according to claim 10, wherein said messenger ribonucleic acids (mRNAs) encode antigens Ag85B, RpfE, PE13, and PE15.

12. The vaccine composition according to claim 11, wherein the mRNAs comprise one or more nucleic acid sequences selected from the group consisting of: SEQ ID NOS: 1-4.

13. The vaccine composition according to claim 5 or 11, wherein the nucleic acids are mRNAs encoding one or more amino acid sequences selected from SEQ ID NOs:21-24 or amino acid sequences which have at least 80% sequence identity to SEQ ID NOs:21-24.

14. The vaccine composition according to claim 10 or 11, wherein said messenger ribonucleic acids (mRNAs) encode antigens Ag85B, RpfE, PE13, PE15, Rv0359 / MMAR_0678, and Rvl234 / MMAR_4207.

15. The vaccine composition according to claim 14, wherein the mRNAs comprise one or more nucleic acid sequences selected from the group consisting of: SEQ ID NOS: 1-6.

16. The vaccine composition according to claim 9 or 14, wherein the nucleic acids are mRNAs encoding one or more amino acid sequences selected from SEQ ID NOs:21-26 or amino acid sequences which have at least 80% sequence identity to SEQ ID NOs:21-26.

17. The vaccine composition according to claim 1, wherein said vaccine composition comprises mRNAs comprising one or more nucleic acid sequences selected from the group consisting of: SEQ ID NOS: 1-20.

18. The vaccine composition according to claim 1 or 17, wherein the nucleic acids are mRNAs encoding one or more amino acid sequences selected from SEQ ID NOs:21-40 or amino acid sequences which have at least 80% sequence identity to SEQ ID NOs:21-40.

19. A vaccine composition against a disease caused by Mycobacterium tuberculosis, said composition comprising a nucleic acid encoding a disease-reactivation-related antigen selected from the group consisting of: Rvl234 / MMAR_4207, and Rv0359 / MMAR_0678.

20. A recombinant nucleic acid vector encoding the nucleic acids in the vaccine composition of any of claims 1-18.

21. A pharmaceutical composition comprising the recombinant nucleic acid vector of claim 20 and a pharmaceutically acceptable carrier, preservative, and / or buffer.

22. A method of preventing a disease caused by Mycobacterium tuberculosis comprising administering a prophylactically or therapeutically effective amount of the vaccine composition according to any one of claims 1-18 to a mammal, preferably to a human.

23. The method according to claim 22, wherein said disease is lung tuberculosis.

24. Use of nucleic acids encoding at least antigens Ag85B, RpfE, PE13, and PE15 in the manufacturing of a vaccine for inducing an immune response in a mammal, preferably a human, or of a pharmaceutical composition for the treatment or prevention of a disease caused by Mycobacterium tuberculosis.

25. The use according to claim 24, wherein nucleic acids encoding at least antigens Ag85B, RpfE, PE13, PE15, Rv0359 / MMAR_0678, and Rvl234 / MMAR_4207 are used in the manufacturing of said vaccine.

26. The use according to claim 24 or 25, wherein said nucleic acids are mRNAs and said vaccine is a mRNA vaccine.

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