Vaccine constructs comprising tuberculosis antigens

Abstract

The present invention relates to a polygene nucleic acid construct comprising a nucleotide sequence encoding a Mycobacterium tuberculosis antigen, and an mRNA vaccine construct transcribed or obtained therefrom. Also provided are lipid nanoparticles comprising the mRNA vaccine constructs and vaccine compositions comprising the constructs. The constructs, lipid nanoparticles containing them and the vaccine compositions can be used in methods for eliciting a protective immune response against Mycobacterium tuberculosis in a subject.

Description

Technical Field Background of the Invention

[0002] The present invention relates to the identification of Mycobacterium tuberculosis (M.tb) antigens recognized by antigen-specific T cells associated with the control of tuberculosis (TB). The present invention further relates to the use of antigens identified as targets of these antigen-specific T cells in the development of mRNA vaccine constructs that induce an immune response to TB. The present invention also relates to a vaccine composition comprising at least one mRNA encoding at least one or more identified antigens for use in eliciting an immune response against TB. Background Art

[0003] TB is a disease that causes significant morbidity and mortality worldwide, particularly affecting low- and middle-income countries. Until 2019, and again since the end of the Covid-19 pandemic, TB was and is the single largest infectious killer of humans by a pathogen. TB is also a significant burden on healthcare systems worldwide, and an effective vaccine would help society by reducing the burden of the disease.

[0004] Antigen-specific CD4 T cells are required for protective immunity against M.tb, the causative agent of tuberculosis, but the ability to extensively study these responses is limited. Experimental and clinical evidence suggests that the major T-cell mediators of this protection are IFN-γ-expressing Th1 cells, although recent evidence from non-human primates suggests that Th1 / Th17 cells may be correlates of protection. A comprehensive characterization of αβ T-cell responses in M.tb-infected individuals is hampered by the complexity and heterogeneity of the TB clinical phenotype, the high inter-individual diversity of the major histocompatibility complex (MHC), which limits antigen presentation to T cells, and the remarkable diversity of the T-cell receptor (TCR), even within a single host.

[0005] There are various TB vaccine candidates that contain M.tb proteins and are targeted against TB. Some of the candidate vaccines are in the form of subunit vaccines that contain certain M.tb proteins, others are live attenuated M.tb, and others are genetically modified versions of the Bacillus Calmette-Guérin (BCG) vaccine.

[0006] The inventors of the present invention have developed single and multigene vaccine constructs containing one or four antigens for inducing an immune response against M.tb. Summary of the invention

[0007] The present invention relates to single gene and multigene nucleic acid constructs comprising nucleotide sequences encoding Mycobacterium tuberculosis (M.tb) antigens, and to mRNA vaccine constructs transcribed or obtained therefrom. The present invention further relates to lipid nanoparticles comprising mRNA vaccine constructs, and to vaccine compositions comprising said constructs. The constructs, lipid nanoparticles containing them, and vaccine compositions can be used in methods for eliciting an immune response against M.tb in a subject.

[0008] According to a first aspect of the present invention, a single gene nucleic acid construct is provided, which comprises at least one nucleotide sequence selected from: a) a nucleotide sequence encoding a WbbL1 antigen having an amino acid sequence of SEQ ID NO: 1; b) a nucleotide sequence encoding a CFP-10 antigen having an amino acid sequence of SEQ ID NO: 2; c) a nucleotide sequence encoding a PPE18 antigen having an amino acid sequence of SEQ ID NO: 3; and d) a nucleotide sequence encoding a PE13 antigen having an amino acid sequence of SEQ ID NO: 4; wherein two or more single gene nucleic acid constructs can be provided to a subject in combination; and / or a multi-gene nucleic acid construct, which comprises at least two nucleotide sequences selected from: a) a nucleotide sequence encoding a WbbL1 antigen having an amino acid sequence of SEQ ID NO: 1; b) a nucleotide sequence encoding a CFP-10 antigen having an amino acid sequence of SEQ ID NO: 2; c) a nucleotide sequence encoding a PPE18 antigen having an amino acid sequence of SEQ ID NO: 3; and d) a nucleotide sequence encoding a PE13 antigen having an amino acid sequence of SEQ ID NO: 4.

[0009] According to the first embodiment of the single-gene and multi-gene nucleic acid constructs of the present invention, (a) the nucleotide sequence encoding the WbbL1 antigen has a nucleic acid sequence substantially identical to SEQ ID NO: 5, (b) the nucleotide sequence encoding the CFP-10 antigen has a nucleic acid sequence substantially identical to SEQ ID NO: 6, (c) the nucleotide sequence encoding the PPE18 antigen has a nucleic acid sequence substantially identical to SEQ ID NO: 7, and (d) the nucleotide sequence encoding the PE13 antigen has a nucleic acid sequence substantially identical to SEQ ID NO: 8.

[0010] In a second embodiment of the polygenic nucleic acid construct of the present invention, each of the at least two nucleotide sequences may be separated by a nucleotide sequence encoding a linker. A variety of linkers are known in the art, and the linker may be selected from a glycine-serine flexible linker, a glycine flexible linker, and a 2A-derived peptide. In particular, each linker may independently have a sequence selected from the linker sequence shown in any one of SEQ ID NOs: 9-19.

[0011] According to the third embodiment of the single gene and multi-gene nucleic acid constructs of the present invention, the nucleic acid construct may further comprise a leader nucleotide sequence encoding a secretory peptide signal. In some embodiments of the multi-gene nucleic acid constructs of the present invention, each of the at least two nucleotide sequences comprises a leader nucleotide sequence encoding a secretory peptide signal. The leader sequence may be derived from a M.tb secretory peptide signal or from another source. In particular, each secretory peptide signal may independently have an amino acid sequence selected from SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 28. In addition, the nucleotide sequence encoding each secretory peptide signal may be encoded by a nucleotide sequence substantially identical to the sequence shown in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29. It will be appreciated by those skilled in the art that the leader sequence is generally located upstream of the antigenic polypeptide, preferably in the reading frame. However, in some suitable embodiments of the present invention, the leader sequence may also be located downstream of the antigenic polypeptide.

[0012] In the fourth embodiment of the polygenic nucleic acid construct of the present invention, the polygenic nucleic acid construct may comprise all of the nucleotide sequences in (a) to (d). An exemplary polygenic nucleic acid construct of the present invention comprises a polygenic nucleic acid construct encoding a polyprotein having an amino acid sequence of any one of SEQ ID NOs: 33-36. For example, the polygenic nucleic acid construct may have a nucleic acid sequence substantially identical to any one of SEQ ID NOs: 37-40.

[0013] Those skilled in the art will further recognize that the first (N-terminal) antigen sequence in a single / multi-gene construct will contain a start codon (ATG). However, when a single / multi-gene construct contains a leader sequence, the first antigen sequence will not contain the first codon (start codon) because the leader sequence contains a start codon (ATG). A single antigen sequence may also contain a start codon. Similarly, the last (C-terminal) antigen sequence in the construct may contain one or more stop codons, including two, three or four stop codons, each stop codon having a TAA, TAG or TGA sequence. In particular, the last sequence contains a stop codon having a design consisting of four consecutive stop sequences in a row, and its nucleotide sequence is TGATAATGATAG (SEQ ID NO: 32). A single antigen sequence may also contain the stop sequence of SED ID NO: 32.

[0014] According to a second aspect of the present invention, there is provided an mRNA construct transcribed from the single / multi-gene nucleic acid construct described herein.

[0015] In a first embodiment of the mRNA construct of the present invention, the mRNA construct may be capped at the 5' end.

[0016] According to the second embodiment of mRNA construct of the present invention, mRNA construct can include one or more modified nucleotides.For example, mRNA construct can include one or more modified nucleotides selected from N1-methyl-pseudouridine and pseudouridine.

[0017] According to a third aspect of the present invention, there is provided a lipid nanoparticle comprising the mRNA construct described herein.

[0018] According to a fourth aspect of the present invention, a vaccine composition is provided, comprising a single / multi-gene nucleic acid construct as described herein, or an mRNA construct as described herein, or a lipid nanoparticle as described herein, and one or more pharmaceutically acceptable diluents or excipients, wherein the vaccine composition can induce an immune response against M.tb. In some embodiments, a combination of two or more single gene nucleic acid constructs or mRNA constructs is provided in a single vaccine composition.

[0019] In one aspect of the present invention, a vaccine composition is provided, comprising at least two nucleic acids selected from: a) a nucleic acid encoding a WbbL antigen having an amino acid sequence of SEQ ID NO: 1; b) a nucleic acid encoding a CFP-10 antigen having an amino acid sequence of SEQ ID NO: 2; c) a nucleic acid encoding a PPE18 antigen having an amino acid sequence of SEQ ID NO: 3; and d) a nucleic acid encoding a PE13 antigen having an amino acid sequence of SEQ ID NO: 4.

[0020] In the first embodiment of the vaccine composition, the vaccine composition may comprise all of the nucleic acids in (a) to (d).

[0021] According to a second embodiment of the vaccine composition, (a) has a nucleotide sequence substantially identical to SEQ ID NO:5, (b) has a nucleotide sequence substantially identical to SEQ ID NO:6, (c) has a nucleotide sequence substantially identical to SEQ ID NO:7, and (d) has a nucleotide sequence substantially identical to SEQ ID NO:8.

[0022] In a third embodiment of the vaccine composition, at least one nucleic acid may further comprise a leader nucleotide sequence encoding a secretory peptide signal. In some embodiments, each nucleic acid may comprise a leader nucleotide sequence encoding a secretory peptide signal. The leader sequence may be derived from a M.tb secretory peptide signal or from another source. In particular, each secretory peptide signal may independently have an amino acid sequence selected from SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28. In addition, the nucleotide sequence encoding each secretory peptide signal may be encoded by a nucleotide sequence substantially identical to the sequence shown in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29. It will be appreciated by those skilled in the art that the leader sequence is generally located upstream of the sequence encoding the antigenic polypeptide, preferably in the reading frame. However, in some suitable embodiments of the present invention, the leader sequence may also be located downstream of the antigenic polypeptide.

[0023] According to a fourth embodiment of the vaccine composition of the present invention, at least two nucleic acid sequences may be provided on a single nucleic acid construct.

[0024] In another embodiment of the vaccine composition, each nucleic acid provided on the nucleic acid construct may be separated by a nucleotide sequence encoding a linker. A variety of linkers are known in the art, and the linker may be selected from a glycine-serine flexible linker, a glycine flexible linker, and a 2A-derived peptide. In particular, each linker may independently have a sequence selected from the linker sequences shown in any one of SEQ ID NOs: 9-19.

[0025] In another embodiment of the vaccine composition of the present invention, the nucleic acid construct may encode a polyprotein having an amino acid sequence of any one of SEQ ID NOs: 33-36. For example, the polygenic nucleic acid construct may have a nucleic acid sequence substantially identical to any one of SEQ ID NOs: 37-40. It will be further understood by those skilled in the art that the first (N-terminal) antigen sequence in the polygenic nucleic acid construct will contain a start codon (ATG). However, when the polygenic construct contains a leader sequence, the first antigen sequence will not contain the first codon (start codon) because the leader sequence contains a start codon (ATG). Similarly, the last (C-terminal) antigen sequence in the polygenic nucleic acid construct may contain one or more stop codons, including two, three or four stop codons, each stop codon having the sequence TAA, TAG or TGA. In particular, the last sequence contains a stop codon having a design consisting of four consecutive stop sequences in a row, and its nucleotide sequence is TGATAATGATAG (SEQ ID NO: 32).

[0026] In another embodiment of the vaccine composition, the nucleic acid may be mRNA, or the nucleic acid construct may be an mRNA construct.

[0027] In some embodiments of the vaccine compositions of the invention, the mRNA or mRNA construct may be capped at the 5' end.

[0028] According to another embodiment of the vaccine composition of the present invention, mRNA or mRNA construct can include one or more modified nucleotides. For example, mRNA or mRNA construct can include one or more modified nucleotides selected from N1-methyl-pseudouridine and pseudouridine.

[0029] In another embodiment of the vaccine composition, the mRNA may be contained in lipid nanoparticles.

[0030] In another embodiment of the vaccine composition, the vaccine composition is capable of eliciting a protective immune response against Mycobacterium tuberculosis.

[0031] According to another aspect, an mRNA vaccine composition is provided, comprising at least two mRNAs selected from: a) mRNA encoding a WbbL antigen having an amino acid sequence of SEQ ID NO: 1; b) mRNA encoding a CFP-10 antigen having an amino acid sequence of SEQ ID NO: 2; c) mRNA encoding a PPE18 antigen having an amino acid sequence of SEQ ID NO: 3; and d) mRNA encoding a PE13 antigen having an amino acid sequence of SEQ ID NO: 4.

[0032] In the first embodiment of the mRNA vaccine composition, the mRNA vaccine composition may comprise all of the mRNAs in (a) to (d).

[0033] According to a second embodiment of the mRNA vaccine composition, (a) is transcribed from a nucleotide sequence substantially identical to SEQ ID NO:5, (b) is transcribed from a nucleotide sequence substantially identical to SEQ ID NO:6, (c) is transcribed from a nucleotide sequence substantially identical to SEQ ID NO:7, and (d) is transcribed from a nucleotide sequence substantially identical to SEQ ID NO:8.

[0034] In the third embodiment of the mRNA vaccine composition, the nucleic acid that transcribes at least one mRNA may further comprise a leader nucleotide sequence encoding a secretory peptide signal. In some embodiments, each nucleic acid may comprise a leader nucleotide sequence encoding a secretory peptide signal. The leader sequence may be derived from an M.tb secretory peptide signal or from another source. In particular, each secretory peptide signal may independently have an amino acid sequence selected from SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28. In addition, the nucleotide sequence encoding each secretory peptide signal may be encoded by a nucleotide sequence substantially identical to the sequence described in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29. It will be understood by those skilled in the art that the leader sequence is generally located upstream of the sequence encoding the antigenic polypeptide, preferably in the reading frame. However, in some suitable embodiments of the present invention, the leader sequence may also be located downstream of the antigenic polypeptide.

[0035] According to a fourth embodiment of the mRNA vaccine composition of the present invention, at least two mRNAs can be transcribed from a single nucleic acid construct.

[0036] In another embodiment of the mRNA vaccine composition, each nucleic acid provided on the nucleic acid construct can be separated by a nucleotide sequence encoding a linker. A variety of linkers are known in the art, and the linker can be selected from a glycine-serine flexible linker, a glycine flexible linker, and a 2A-derived peptide. In particular, each linker can independently have a sequence selected from the linker sequence shown in any one of SEQ ID NOs: 9-19.

[0037] In another embodiment of the mRNA vaccine composition of the present invention, mRNA can be transcribed from a nucleic acid construct encoding a polyprotein having an amino acid sequence of any one of SEQ ID NOs: 33-36. For example, mRNA can be transcribed from a polygene nucleic acid construct having a nucleic acid sequence substantially identical to any one of SEQ ID NOs: 37-40. It will be further understood by those skilled in the art that the first (N-terminal) antigen sequence in the polygene nucleic acid construct will contain a start codon (ATG). However, when the polygene construct contains a leader sequence, the first antigen sequence will not contain the first codon (start codon) because the leader sequence contains a start codon (ATG). Similarly, the last (C-terminal) antigen sequence in the polygene nucleic acid construct may contain one or more stop codons, including two, three or four stop codons, each of which has the sequence TAA, TAG or TGA. In particular, the last sequence contains a stop codon having a design consisting of four consecutive stop sequences in a row, and its nucleotide sequence is TGATAATGATAG (SEQ ID NO: 32).

[0038] According to one embodiment of the mRNA vaccine composition of the present invention, the mRNA may be capped at the 5' end.

[0039] According to another embodiment of the mRNA vaccine composition of the present invention, the mRNA may include one or more modified nucleotides. For example, the mRNA may include one or more modified nucleotides selected from N1-methyl-pseudouridine and pseudouridine.

[0040] In another embodiment of the mRNA vaccine composition, the mRNA may be contained in lipid nanoparticles.

[0041] In another embodiment of the mRNA vaccine composition, the vaccine composition is capable of eliciting a protective immune response against Mycobacterium tuberculosis.

[0042] In another aspect of the present invention, a single gene and / or multi-gene nucleic acid construct, mRNA construct, lipid nanoparticle, vaccine composition or mRNA vaccine composition as described herein is provided, for use in a method for eliciting an immune response against M.tb, wherein the method comprises administering the vaccine composition to a subject.

[0043] In another aspect of the present invention, a method for eliciting an immune response against M.tb in a subject is also provided, the method comprising administering to the subject a single gene and / or multi-gene nucleic acid construct, mRNA construct, lipid nanoparticle, vaccine composition or mRNA vaccine composition as described herein.

[0044] According to another aspect of the present invention, there is provided use of the single / multi-gene nucleic acid construct, mRNA construct, lipid nanoparticle, vaccine composition or mRNA vaccine composition described herein in the manufacture of a vaccine in a method for eliciting an immune response against M.tb, the method comprising administering the vaccine to a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Non-limiting embodiments of the present invention will now be described, by way of example only, and with reference to the following drawings:

[0046] Figure 1 : Identification of TCR sequences expressed by antigen-specific T cells in controls and progressors and antigens recognized by M.tb lysate-reactive T cells: The figure depicts the longitudinal study time points (points) of PBMC sample analysis for each control (blue) or progressor (red, synchronized with TB diagnosis) in the Adolescent Cohort Study (ACS) (a) or GC6-74 cohort (b). Each horizontal line or symbol represents an individual.

[0047] Figure 2:Identification of TCR sequences expressed by antigen-specific T cells in controls and progressors and antigens recognized by M.tb lysate-reactive T cells: Experimental workflow and analytical methods for identifying mycobacterium-reactive CDR3αβ sequences and determining their frequency in blood. First, single-cell TCR-seq (scTCR-seq) was performed on sorted mycobacterium-reactive T cells expressing activation markers CD69 and CD154 or CD137 after in vitro M.tb lysate stimulation. GLIPH2 analysis clustered the TCR sequences expressed by mycobacterium-reactive T cells into TCR similarity groups. At the same time, batch TCR-seq was performed on PBMCs (unstimulated) to analyze the library and determine the frequency of occurrence of CDR3β sequences in each sample. The total frequency of occurrence of CDR3β sequences within the GLIPH2 TCR similarity group for each control and progressor sample was determined using batch TCR-seq data. For controls and progressors with samples collected at multiple study time points, the total frequency of CDR3β sequences within the TCR similarity group was determined at each time point. The total frequency of CDR3β sequences within the TCR similarity group was compared between controls and progressors. To identify the antigens recognized by these antigen-specific T cells, transduced NFAT reporter gene-stable J76-NFATRE-luc T cell line cells expressing representative TCRαβ chains from the TCR similarity group that were found to be differentially abundant in controls and progressors were co-incubated with artificial antigen presenting cells (aAPCs) to screen for antigens representing the M.tb proteome.

[0048] Figure 3 : Analysis workflow for measuring the frequency of occurrence of GLIPH2 TCR similarity groupings of mycobacterium-reactive (M.tb) or CMV, EBV, or influenza A virus (Infl.A)-specific CDR3b sequences in controls and progressors. CDR3b sequences of mycobacterium-reactive T cells were sorted from TCR sequences expressed by mycobacterium-reactive cells in controls, progressors, and healthy M.tb-infected adolescents. CMV, EBV, and Infl.A-specific CDR3b sequences were from VDJdb. GLIPH2 analysis was performed and the resulting GLIPH2 similarity groups were initially filtered using the criteria listed under Filter 1. TCR similarity groups with significant HLA allele associations in the progressor / control cohort were selected (Filter 2). Finally, similarity groups with differences in abundance in controls and progressors carrying the relevant HLA alleles were determined (Filter 3).

[0049] Figure 4: CMV-, EBV-, influenza A-, or M.tb-specific TCR specificity group: The frequency of HLA combinations associated with clinical outcome (significantly more abundant in controls or progressors), expressed as the percentage of all TCR specificity groups: HLA combinations for that pathogen (i.e., in Figure 3 before applying filter 3).

[0050] Figure 5 : Relative frequency of occurrence of the number of TCR-specific group:HLA combinations found to be significantly different between the two groups. Permutation analysis was performed with 200 iterations of randomized disease outcome labels. The vertical lines indicate the actual number of M.tb-specific TCR-specific group:HLA combinations found to be significantly different between controls and progressors when having the correct disease outcome labels.

[0051] Figure 6 : mRNA construct design: The linkers at position 1, positions 1 and 2 and / or positions 1, 2 and 3 can be selected from a nucleic acid having a nucleotide sequence of any one of SEQ ID NOs: 9-19, and the antigen sequence is selected from a codon-optimized sequence encoding PE13, CFP-10, WbbL1 and PPE18 proteins in any order within the polyprotein expression sequence.

[0052] Figure 7 : Exemplary constructs for obtaining mRNA vaccines of the present invention: A) A construct comprising a codon-optimized nucleotide sequence encoding each of the four antigens PE13, CFP-10, WbbL1 and PPE18, separated by a linker sequence and without a leader sequence. B) A construct comprising a codon-optimized nucleotide sequence encoding each of the four antigens PE13, CFP-10, WbbL1 and PPE18, separated by a linker sequence and comprising a codon-optimized nucleotide sequence encoding an N-terminal leader sequence.

[0053] Figure 8 : Figure 7 The nucleotide sequence of the nucleic acid construct provided in A (SEQ ID NO: 30) comprises a codon-optimized nucleotide sequence encoding PE13 (Rv1195) (SEQ ID NO: 8), a linker of SEQ ID NO: 11, a codon-optimized nucleotide sequence encoding PPE18 (Rv1196) (SEQ ID NO: 7), a linker of SEQ ID NO: 16, a codon-optimized nucleotide sequence encoding Wbbl1 (Rv3265c) (SEQ ID NO: 5), a linker of SEQ ID NO: 14, and a codon-optimized nucleotide sequence encoding CFP-10 (Rv3874) (SEQ ID NO: 6).

[0054] Fig. 9 : Figure 7 The nucleotide sequence of the nucleic acid construct provided in B (SEQ ID NO: 31) includes a leader sequence of SEQ ID NO: 25, followed by a codon-optimized nucleotide sequence encoding PE13 (Rv1195) (SEQ ID NO: 8), a linker of SEQ ID NO: 11, a codon-optimized nucleotide sequence encoding PPE18 (Rv1196) (SEQ ID NO: 7), a linker of SEQ ID NO: 16, a codon-optimized nucleotide sequence encoding Wbbl1 (Rv3265c) (SEQ ID NO: 5), a linker of SEQ ID NO: 14, and a codon-optimized nucleotide sequence encoding CFP-10 (Rv3874) (SEQ ID NO: 6).

[0055] Fig.10 : Size and integrity analysis of in vitro transcribed TB antigen encoding mRNA (single gene) using capillary gel electrophoresis.

[0056] Fig.11 : In vitro potency assay in cultured cells. Immunofluorescence staining of HepG2 cells transfected with CFP-10 mRNA confirmed the in situ expression of the transcript and the intracellular detection of CFP-10 protein.

[0057] Fig.12 : Particle size analysis of formulated mRNA encoding TB antigens showing size distribution by intensity. mRNA encoding TB antigens were formulated into lipid nanoparticles (LNPs) and LNPs coated with CFP-10 (A), WbbL1 (B), PE-13 (C), and PPE-18 (D) mRNA were measured by size.

[0058] Fig.13 : IFN-γ, TNF-α and IL-2 cytokine secretion profiles of in vitro stimulated splenocytes of immunized BALB / c mice. Increased concentrations of IFN-γ, TNF-α and IL-2 were observed in BALB / c mice immunized with WbbL1, PPE-18 and mixed (CWPP) mRNA. Increased IL-2 was detected in mice immunized with PE-13 mRNA.

[0059] Fig.14 : IFN-γ, TNF-α and IL-2 cytokine secretion profiles of immunized C57Bl / 6 mice. Increased IFN-γ, TNF and IL-2 concentrations were observed in C57BL / 6 mice immunized with CFP-10, WbbL1, PE-13, PPE-18 and mixed (CWPP) mRNA.

[0060] Fig.15: IFN-γ, TNF-α and IL-2 cytokine secretion profiles of immunized C3HeB / FeJ mice. Increased concentrations of IFN-γ, TNF and IL-2 were observed in C3HeB / FeJ mice immunized with CFP-10 and mixed (CWPP) mRNA.

[0061] Fig.16 : IL-17A and IL-22 cytokine secretion profiles of immunized C3HeB / FeJ mice. Increased IL-17A and IL-22 concentrations were observed in C3HeB / FeJ mice immunized with CFP-10 and mixed (CWPP) mRNA.

[0062] Fig.17 : Detection of IFN-γ positive CD4 T cells based on flow cytometry. CD4 T cells from immunized C57BL / 6 mice were stimulated ex vivo with peptide pools covering the M.tb antigens CFP-10, WbbL1, PE-13, and PPE-18, and their activation was assessed by IFN-γ staining. An increase in the percentage of IFN-γ+CD4+ T cells was observed after PE-13, PPE-18, and mixed (CWPP) vaccination.

[0063] Fig.18 : Detection of IFN-γ positive CD4 T cells (A) and CD8 T cells (B) based on flow cytometry. CD4 and CD8 T cells from immunized C3HeB / FeJ mice were stimulated ex vivo with peptide pools covering M.tb antigens CFP-10, WbbL1, PE-13, PPE-18 or mixed antigen peptide pools (Peptide pool: CWPP), and activation was assessed by IFN-γ staining. An increase in the percentage of IFN-γ+CD4+ T cells was observed after vaccination with CFP-10, PE-13, and mixed (CWPP) mRNA vaccines. IFN-γ+CD8+ T cells were detected after CFP-10 mRNA vaccination.

[0064] Fig.19 : M.tb derived leader sequences L1 (SEQ ID NO: 21), L2 (SEQ ID NO: 23), L3 (SEQ ID NO: 25) and L4 (SEQ ID NO: 27) were included in the mRNA vaccine encoding CFP-10. When leader sequences 2, 3 and 4 were added, an antigen-specific increase in the percentage of IFN-γ+CD4+ T cells was observed in immunized C3HeB / FeJ mice, while the CFP-10 mRNA vaccine without leader sequence (NL) and L1-CFP-10 was not observed.

[0065] Fig. 20: A diagram depicting an exemplary construct for obtaining the polyprotein mRNA vaccine of the present invention.

[0066] Fig.21 : Electropherograms and capillary gel images depicting the size and integrity of two multigene mRNA transcripts, TB-M3 and TB-M4.

[0067] Fig. 22 : Capillary gel images depicting the size and integrity of TB-M3 and TB-M4 multi-gene mRNA transcripts with and without leader sequences (L1, L2, L3, L4, L5) and CFP-10 single-gene mRNA transcripts with and without leader sequences (L1, L2, L3, L4).

[0068] Fig.23 : TB-M7 polyprotein mRNA vaccination. Cytokine profiles secreted in ex vivo stimulated splenocytes of mice immunized with intramuscular injection of saline or control mRNA encoding a bioluminescent reporter gene or 10 μg LNP formulated with multigene TB-M7 mRNA. Exemplary cytokine profiles of IFN-γ and IL-2 of BALB / c, C57BL6 and C3HeB / FeJ mice are shown. Unstimulated splenocytes of mice vaccinated with TB-M7 mRNA served as controls. These results confirm individual antigen-specific T cell activation of all four TB antigens contained in the polyprotein. A) Stimulation with the CFP-10 peptide pool shows increased IFN-γ and IL-2 concentrations in C3HeB / FeJ mice. B) Stimulation with the Wbbl1 peptide pool shows increased IFN-γ and IL-2 concentrations in BALB / c, C57BL6 and C3HeB / FeJ mice. C) Stimulation with PE-13 peptide pool shows increased IFN-γ and IL-2 concentrations in C57BL6 and C3HeB / FeJ mice. D) Stimulation with PPE-18 peptide pool shows increased IFN-γ and IL-2 concentrations in BALB / c, C57BL6 and C3HeB / FeJ mice.

[0069] Fig.24 : TB-M7 polyprotein mRNA vaccination. Exemplary secreted cytokine profiles in in vitro stimulated splenocytes of BALB / c, C57BL6 and C3HeB / FeJ mice stimulated with mixed antigenic peptide pool (Peptide pool: CWPP). IFN-γ, IL-2, TNF-α and IL-17A cytokine concentrations are shown. BALB / c, C57BL6 and C3HeB / FeJ mice showed an increase in IFN-γ and IL-2 after vaccination with TB-M7 mRNA vaccine. In addition, BALB / c mice showed an increase in TNF-α and IL-17A, and C57BL6 mice showed an increase in TNF-α.

[0070] Fig.25 : IL-6 concentrations secreted by BALB / c, C57BL6 and C3HeB / FeJ mice vaccinated with TB-M7 polyprotein mRNA. Splenocytes were stimulated with CFP-10 (A), PE-13 (B) and PPE-18 (C) peptide pools.

[0071] Fig.26 : IL-22 concentrations secreted by BALB / c, C57BL6 and C3HeB / FeJ mice vaccinated with TB-M7 polyprotein mRNA. Splenocytes were stimulated with WBBL1 (A), PE-13 (B) and PPE-18 (C) peptide pools.

[0072] Fig. 27 : Flow cytometry-based detection of IFN-γ-positive CD3 (A) and CD4 (B) T cells in BALB / c mice vaccinated with TB-M7 polyprotein mRNA. The percentage of IFN-γ+CD3+ T cells increased after stimulation of cells with WbbL1, PPE-18, and CWPP peptide pools. PPE-18-specific IFN-γ+CD4+ T cells are shown.

[0073] Fig.28 : Flow cytometry-based detection of IFN-γ-positive CD4 T cells in C57BL / 6 mice vaccinated with TB-M7 polyprotein mRNA. The percentage of IFN-γ+CD4+ T cells increased after cell stimulation with WbbL1, PE-13, PPE-18, and CWPP peptide pools in C57BL6 mice.

[0074] Fig.29 : Based on flow cytometry detection of IFN-γ positive CD4 T cells in BALB / c, C57BL / 6 and C3HeB / FeJ mice vaccinated with TB-M7 polyprotein mRNA. Splenocytes of vaccinated mice were stimulated ex vivo with TB antigen (TB10.4) not included in the polyprotein. Based on flow cytometry detection of IFN-γ positive CD4 T cells, no IFN-γ activation was shown, confirming that the results describe antigen-specific T cell stimulation.

[0075] Fig.30: TB-M3 and TB-M4 polyprotein mRNA vaccination. Cytokine profiles secreted in ex vivo stimulated splenocytes of mice injected intramuscularly with 10 μg LNP-formulated control mRNA encoding a bioluminescent reporter gene (control) or 10 μg LNP-formulated multigene TB-M3 or TB-M4 mRNA. Exemplary cytokine profiles of IFN-γ and IL-2 for BALB / c, C57BL6, and C3HeB / FeJ mice are shown. These results confirm individual antigen-specific T cell activation for all four TB antigens contained in the polyprotein. A) Stimulation with the CFP-10 peptide pool showed increased IFN-γ and IL-2 concentrations in C3HeB / FeJ mice. B) Stimulation with the Wbbl1 peptide pool showed increased IFN-γ and IL-2 concentrations in BALB / c, C57BL6, and C3HeB / FeJ mice. C) Stimulation with the PE-13 peptide pool showed increased IFN-γ and IL-2 concentrations in C57BL6 and C3HeB / FeJ mice. D) Stimulation with PPE-18 peptide pools shows increased IFN-γ concentrations in BALB / c and C57BL6 mice, and increased IL-2 concentrations in BALB / c, C57BL6, and C3HeB / FeJ mice. DETAILED DESCRIPTION

[0076] Sequence Listing

[0077] The nucleic acid and amino acid sequences listed in the attached sequence listing use standard letter abbreviations to represent nucleotide bases and standard three-letter abbreviations to represent amino acids. It will be understood by those skilled in the art that only one strand of each nucleic acid sequence is shown, but any reference to a displayed strand includes the complementary strand. In the attached sequence listing:

[0078] SEQ ID NO: 1 - Amino acid sequence of Wbbl1 (Rv3265c)

[0079] SEQ ID NO: 2 - Amino acid sequence of CFP-10 (Rv3874)

[0080] SEQ ID NO: 3—Amino acid sequence of PPE18 (Rv1196)

[0081] SEQ ID NO:4—Amino acid sequence of PE13 (Rv1195)

[0082] SEQ ID NO: 5—Codon-optimized nucleotide sequence of Wbbl1 (Rv3265c)

[0083] SEQ ID NO: Codon-optimized nucleotide sequence of 6-CFP-10 (Rv3874)

[0084] SEQ ID NO: 7—Codon-optimized nucleotide sequence of PPE18 (Rv1196)

[0085] SEQ ID NO: 8—Codon-optimized nucleotide sequence of PE13 (Rv1195)

[0086] SEQ ID NO: 9—Nucleotide sequence of linker 1

[0087] SEQ ID NO: 10—Nucleotide sequence of linker 2

[0088] SEQ ID NO: 11—Nucleotide sequence of linker 3

[0089] SEQ ID NO: 12—Nucleotide sequence of linker 4

[0090] SEQ ID NO: 13—Nucleotide sequence of linker 5

[0091] SEQ ID NO: 14—Nucleotide sequence of linker 6

[0092] SEQ ID NO: 15—Nucleotide sequence of linker 7

[0093] SEQ ID NO: 16—Nucleotide sequence of linker 8

[0094] SEQ ID NO: 17—Nucleotide sequence of linker 9

[0095] SEQ ID NO: 18—Nucleotide sequence of linker 10

[0096] SEQ ID NO: 19—Nucleotide sequence of linker 11

[0097] SEQ ID NO: 20 - Amino acid sequence of M.tb Rv2878c derived leader sequence

[0098] SEQ ID NO: 21 - Codon-optimized nucleotide sequence of M.tb Rv2878c derived leader sequence

[0099] SEQ ID NO: 22 - Amino acid sequence of M.tb MT18B_2507 derived leader sequence

[0100] SEQ ID NO: 23 - Codon-optimized nucleotide sequence of M.tb MT18B_2507 derived leader sequence

[0101] SEQ ID NO: 24 - Amino acid sequence of M.tb Rv3803c derived leader sequence

[0102] SEQ ID NO: 25 - Codon-optimized nucleotide sequence of M.tb Rv3803c derived leader sequence

[0103] SEQ ID NO: 26 - Amino acid sequence of M.tb Rv1860 derived leader sequence

[0104] SEQ ID NO: 27 - Codon-optimized nucleotide sequence of M.tb Rv1860 derived leader sequence

[0105] SEQ ID NO: 28 - Amino acid sequence of human interleukin 2 derived leader sequence

[0106] SEQ ID NO: 29 - Codon-optimized nucleotide sequence of human interleukin 2 derived leader sequence

[0107] SEQ ID NO:30—Nucleotide sequence of an exemplary construct without a leader sequence

[0108] SEQ ID NO:31 - Nucleotide sequence of an exemplary construct with a leader sequence

[0109] SEQ ID NO: 32—Stop codon nucleotide sequence

[0110] SEQ ID NO: 33 - Amino acid sequence of TB-M3 polyprotein

[0111] SEQ ID NO: 34 - Amino acid sequence of TB-M4 polyprotein

[0112] SEQ ID NO: 35 - Amino acid sequence of TB-M7 polyprotein

[0113] SEQ ID NO: 36 - Amino acid sequence of TB-M8 polyprotein

[0114] SEQ ID NO: 37 - Codon-optimized nucleotide sequence of TB-M3 multigene construct

[0115] SEQ ID NO: 38 - Codon-optimized nucleotide sequence of TB-M4 multigene construct

[0116] SEQ ID NO: 39 - Codon-optimized nucleotide sequence of TB-M7 multigene construct

[0117] SEQ ID NO:40—Codon-optimized nucleotide sequence of TB-M8 multigene construct. DETAILED DESCRIPTION OF THE INVENTION

[0119] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0120] The described invention is not to be limited to the specific embodiments disclosed and modifications and other embodiments are also intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0121] As used in the specification and the following claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0122] The terms and expressions used herein are for descriptive purposes only and should not be considered as limiting. The terms "comprising", "containing", "having" and "including" and variations thereof used herein are intended to encompass the items listed thereafter and their equivalents as well as additional items. However, as a specific embodiment of the present disclosure, the term "comprising" encompasses the possibility that no other members are present, that is, in some embodiments, "comprising" should be understood to have the meaning of "consisting of..."

[0123] The inventors of the present invention have developed an mRNA vaccine construct containing a unique combination of M.tb proteins that have been found to be associated with human control of M.tb infection. The present invention is a messenger RNA (mRNA)-based vaccine that induces an immune response to tuberculosis (TB). The vaccine is an mRNA expression cassette that encodes single / multimeric proteins of M.tb proteins Rv3265c (WbbL1), Rv1195 (PE13), Rv1196 (PPE18) and Rv3874 (CFP-10), and produces an adaptive immune response to these proteins. To the best of the inventors' knowledge, the antigen selection of all other TB vaccine candidates is not based on an understanding of human control of M.tb infection, as such knowledge is lacking to date. The present mRNA vaccine construct comprises a modified bacterial RNA sequence encoding the amino acid sequences of WbbL1, PE13, PPE18 and CFP-10, which is codon-optimized for mammalian expression and immune recognition. The nanoparticle formulation of the vaccine construct also uses a novel renewable lipid formulation to integrate the RNA sequence.

[0124] Specifically, the selection of proteins contained in the vaccine construct was based on the results of determining T cell responses and their protein targets associated with human control of M.tb infection. The inventors of the present invention used single-cell and bulk TCR sequencing and the GLIPH2 algorithm to analyze millions of M.tb-specific sequences in two longitudinal cohorts, which included 184 M.tb-infected individuals who either progressed to tuberculosis (TB) disease (progressors) or whose infection was controlled (controls). They found dozens of T cell groups with similar TCRβ sequences, which GLIPH2 predicted to have common antigenic specificities, some of which were associated with infection control and others with disease progression. Using whole-genome M.tb antigen screening, they identified antigens enriched in controls or progressors and peptides targeted by similar groups of T cells.

[0125] This enabled the inventors to rationally select four antigens for vaccine development among the approximately 4,000 proteins expressed by M.tb. In addition, the inventors modified the RNA sequences encoding the selected WbbL1, PE13, PPE18, and CFP-10 proteins to optimize the expression and uptake of the vaccine antigens in human cells.

[0126] Other researchers have identified vaccine antigens by observing which M.tb proteins are recognized by infected humans or which M.tb proteins induce protective responses in animal models of TB. To the inventors’ knowledge, this is the first time that samples from human controls and progressors have been used to measure T cell responses associated with controlling infection and to identify specific M.tb-expressed proteins that these T cells target.

[0127] Traditionally, antigen discovery for vaccine development begins with the most immunogenic antigens in a particular pathogen. Many of the M.tb antigens used in candidate TB vaccines have been identified in this way. However, M.tb expresses approximately 4000 gene products, and it remains assumed that the most immunogenic antigens in natural infection are the most critical immune targets for disease control, particularly because an important resistance strategy of the pathogen is to avoid shedding prior to transmission. By combining the power of new sequencing and TCR profiling methods with clinically relevant cohorts, the inventors employed a novel strategy to profile the repertoire of αβ TCR responses to M.tb in controls and progressors without specifying the antigen involved and focusing on TCR specificities associated with clinical outcomes.

[0128] Immunogenicity analysis of mRNA vaccine constructs has been evaluated in three different mouse strains (BALB / c, C57BL / 6, and C3HeB / FeJ mice). The results demonstrated that delivery of mRNA vaccines by intramuscular administration induced T cell responses to all four antigenic components of the mRNA vaccine constructs in genetically distinct mouse strains. These immune responses primarily included CD4 T cells expressing Th1 cytokines (IFNγ, TNF, and IL-2) as well as some IL-17 and IL-22, although CD8 T cell responses expressing IFN-γ were also induced. Since Th1 CD4 T cell responses are necessary for protective immunity against TB, these results suggest that these mRNA vaccine constructs may be able to protect against challenge with M. tuberculosis.

[0129] A "protein," "peptide," or "polypeptide" is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogs, regardless of post-translational modification (eg, glycosylation or phosphorylation).

[0130] An "antigen" is a compound, composition or substance that stimulates an animal or human to produce antibodies and / or a CD4+ or CD8+ T cell response, including compositions that are injected or absorbed into an animal or human. Antigens react 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 a chemical group or peptide sequence on a molecule that is antigenic, i.e., a chemical group or peptide sequence that elicits a specific immune response. Antibodies specifically bind to specific epitopes on polypeptides.

[0131] The terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and encompass ribonucleotides (RNA) and deoxyribonucleotides (DNA), including cDNA, genomic DNA and synthetic DNA. Nucleic acids can be double-stranded or single-stranded. When the nucleic acid is single-stranded, the nucleic acid can be a sense strand or an antisense strand. A nucleic acid molecule can be any chain of two or more covalently bound nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. "RNA" refers to a sequence of two or more covalently bound, naturally occurring or modified ribonucleotides. The term "DNA" refers to a sequence of two or more covalently bound, naturally occurring or modified deoxyribonucleotides. "cDNA" refers to a complementary or replicating DNA produced from an RNA template by the action of an RNA-dependent DNA polymerase (reverse transcriptase). Therefore, a "cDNA clone" refers to a double-stranded DNA sequence that is complementary to a target RNA molecule and is carried in a cloning vector.

[0132] The term "complementary" refers to two nucleic acid molecules, such as DNA or RNA, that are capable of forming Watson-Crick base pairs, thereby producing a double-stranded region between the two nucleic acid molecules. It will be appreciated by those skilled in the art that each nucleotide in a nucleic acid molecule does not need to form a matching Watson-Crick base pair with a nucleotide in an opposite complementary strand to form a double strand. Therefore, if a nucleic acid molecule hybridizes with a second nucleic acid molecule under high stringency conditions, the nucleic acid molecule is "complementary" to the second nucleic acid molecule. Nucleic acid molecules according to the present invention comprise two complementary molecules.

[0133] It is generally understood by those skilled in the art that DNA can be transcribed into mRNA by in vitro or in vivo transcription processes. Alternatively, RNA can be synthesized based on the corresponding DNA sequence by methods known in the art. The DNA sequence provided herein can be interchangeable with the RNA sequence, wherein the thymine residues in the DNA sequence are replaced by the uracil residues in the corresponding RNA sequence.

[0134] In some embodiments, nucleic acid constructs of the present invention may include but are not limited to nucleotide sequences encoding antigenic peptides, the antigenic peptides comprising amino acid sequences substantially identical to the amino acid sequences of M.tb proteins Rv3265c (WbbL1), Rv1195 (PE13), Rv1196 (PPE18) and Rv3874 (CFP-10). Another embodiment of the present invention includes but is not limited to nucleic acid molecules encoding the above-mentioned antigenic peptides substantially identical to the nucleotide sequences described herein.

[0135] As used herein, "substantially identical" sequences refer to amino acid or nucleotide sequences that differ from a reference sequence by only one or more conservative substitutions, or one or more non-conservative substitutions, deletions or insertions, which are located at sequence positions that do not destroy or substantially reduce the antigenicity of one or more expressed polypeptides or polypeptides encoded by nucleic acid molecules. The comparisons performed to determine the percentage of sequence identity can be achieved in various ways known to those skilled in the art. These methods include the use of computer software, such as ALIGN, Megalign (DNASTAR), CLUSTALW or BLAST software. Those skilled in the art can easily determine the appropriate parameters for measuring the comparison, including any algorithm required for achieving maximum comparison over the full length of the compared sequences. In one embodiment of the invention, a polypeptide or polynucleotide sequence is provided, which has at least about 80% sequence identity, at least about 90% sequence identity or even higher sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the sequences described herein.

[0136] Alternatively, or in addition, two nucleic acid sequences can be "substantially identical" if they hybridize under high stringency conditions. The "stringency" of a hybridization reaction is readily determined by one skilled in the art and is typically calculated empirically and depends on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures to anneal properly, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. A typical example of such "stringent" hybridization conditions is hybridization at 65°C with gentle shaking for 18 hours, a first wash at 65°C in wash buffer A (0.5% SDS; 2XSSC) for 12 minutes, and a second wash at 65°C in wash buffer B (0.1% SDS; 0.5% SSC) for 10 minutes.

[0137] It will be appreciated by those skilled in the art that polypeptides, peptides or peptide analogs can be synthesized using standard chemical techniques, for example, by automatic synthesis using solutions or solid phase synthesis methods. Automatic peptide synthesizers are commercially available and use techniques known in the art. Recombinant DNA technology can also be used to prepare polypeptides, peptides and peptide analogs from corresponding nucleic acid molecules.

[0138] As used herein, the term "gene" refers to a nucleic acid encoding a functional product (eg, RNA, polypeptide, or protein). A gene may include a regulatory sequence upstream or downstream of the sequence encoding the functional product.

[0139] As used herein, the term "coding sequence" refers to a nucleic acid sequence that encodes a specific amino acid sequence. On the other hand, a "regulatory sequence" refers to a nucleotide sequence located upstream, downstream or within a coding sequence. In general, a regulatory sequence affects the transcription, RNA processing or stability or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, effector binding sites, enhancers, introns, polyadenylation recognition sequences, promoters, RNA processing sites, stem-loop structures, translation leader sequences.

[0140] In some embodiments, the gene used in the inventive method can be operably connected to other sequences." operably connected " refers to that the nucleic acid molecule encoding the recombinant polypeptide of the present invention and the regulating sequence are connected in such a way that, when the appropriate molecule is combined with the regulating sequence, protein expression is allowed. This operably connected sequence can be included in a vector or an expression construct, and the vector or expression construct can be transformed or transfected into a host cell for expression. It is to be understood that any vector or a plurality of vectors can be used to express recombinant antigen polypeptides of the present invention.

[0141] The term "promoter" refers to a DNA sequence that can control the expression of a nucleic acid coding sequence or a functional RNA. A promoter can be completely based on a natural gene, or it can contain different elements of different promoters present in nature. Different promoters can direct the expression of genes in different cell types, different developmental stages, or in response to different environmental or physiological conditions. A "constitutive promoter" refers to a promoter that directs the expression of a target gene at most times in most host cell types.

[0142] The term "recombinant" means that something has been recombined. When used to refer to a nucleic acid construct, the term refers to a molecule comprising a nucleic acid sequence that is connected together or produced by molecular biological techniques. The term "recombinant" when used to refer to a protein or polypeptide refers to a protein or polypeptide molecule expressed by a recombinant nucleic acid construct produced by molecular biological techniques. A recombinant nucleic acid construct can include a nucleotide sequence that is connected to a nucleic acid sequence that is not connected to it in nature or to a nucleic acid sequence that is connected to a different position in nature, or a nucleotide sequence that is manipulated to be connected to the nucleic acid sequence. Therefore, a recombinant nucleic acid construct means that a nucleic acid molecule has been manipulated using genetic engineering (i.e., by human intervention). A recombinant nucleic acid construct can be introduced into a host cell by transformation. Such recombinant nucleic acid constructs can include sequences from the same host cell species or different host cell species.

[0143] The term "vector" refers to a means by which a polynucleotide or gene sequence can be introduced into a cell. Various types of vectors are known in the art, including plasmids, viruses, bacteriophages, and cosmids. Typically, a polynucleotide or gene sequence is introduced into a vector via a cassette vector. The term "cassette vector" refers to one or more gene sequences inserted into a vector, which in some embodiments provides regulatory sequences for expressing a polynucleotide or gene sequence. In other embodiments, the vector provides regulatory sequences for expressing a polypeptide of the present invention. In additional embodiments, the vector provides some regulatory sequences, and the nucleotide or gene sequence provides other regulatory sequences.

[0144] The construct of the present invention can be provided alone or in combination with other compounds (e.g., nucleic acid molecules, small molecules, peptides or peptide analogs), in the presence of liposomes, adjuvants or any carrier (e.g., pharmaceutically acceptable carrier), in a form suitable for administration to mammals (e.g., humans, cattle, sheep, etc.).

[0145] As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any and all antibacterial and antifungal agents, coatings, dispersion media, solvents, isotonic agents and absorption delaying agents, etc. that are physiologically compatible. "Pharmaceutically acceptable carrier" may include solid or liquid fillers, diluents or encapsulating materials that can be safely used to administer the recombinant vaccine construct to a subject. Pharmaceutically acceptable carriers may be suitable for intramuscular, intradermal, intravenous, intraperitoneal, subcutaneous, oral or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, dispersions and sterile powders for preparing sterile solutions. The use of media and medicaments to prepare pharmaceutically active substances is well known in the art. When any conventional media or medicaments are incompatible with the active compound, they are not considered for use in the pharmaceutical composition of the present invention. Supplementary active compounds may also be added to the composition.

[0146] Suitable formulations or compositions for administering the vaccine constructs of the present invention to a subject also fall within the scope of the present invention. Any suitable route of administration may be used, such as parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, topical or oral administration.

[0147] Typically, an effective amount of a vaccine construct, or a formulation or composition comprising a vaccine construct, will be administered to a subject.As used herein, the term "subject" includes all mammals, particularly human subjects.

[0148] The present invention also relates in part to a method of eliciting an immune response against M.tb or treating M.tb infection or TB disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount, an immunologically effective amount, or a prophylactically effective amount of a construct or composition or formulation of the present invention to prevent or treat TB in the subject.

[0149] Vaccine formulations and compositions useful in the present invention include the multigene or single gene nucleic acid constructs described herein, preferably mRNA constructs, and / or lipid nanoparticles including nucleic acid constructs that prime and / or enhance an immune response to M.tb.

[0150] In one embodiment, the vaccine formulation is capable of "priming" an immune response to M.tb. In another embodiment, the vaccine formulation is capable of "enhancing" an immune response to M.tb, for example, when a subject has been vaccinated with a priming vaccine. It should be further recognized that the "enhancing" composition can include a nucleic acid construct or vaccine formulation that is administered to a subject in two or more doses after the initial priming vaccination.

[0151] The "effective amount" of the vaccine construct and / or composition of the present invention includes a therapeutically effective amount, an immunologically effective amount, or a prophylactic effective amount. "Therapeutically effective amount" refers to an amount that effectively achieves a desired therapeutic outcome within the necessary dosage and time period, such as treating an M.tb infection or a condition associated with the infection. The therapeutically effective amount of a compound can vary according to factors such as individual disease state, age, sex, and body weight, and the ability of the composition to cause a desired response in an individual. The dosage regimen can be adjusted to provide the best therapeutic response. The therapeutically effective amount is also the amount in which any toxic or deleterious effects of the composition are offset by the therapeutically beneficial effects.

[0152] The dosage of the vaccine construct or pharmaceutical composition of the present invention will vary according to the symptoms, age and weight of the subject, the nature and severity of the disease to be treated or prevented, the route of administration and the form of the composition. Any composition of the present invention can be administered in a single dose or multiple doses. The dosage of the composition of the present invention can be easily determined by techniques known to those skilled in the art or the techniques taught herein.

[0153] An "immunologically effective amount" refers to an amount effective to achieve a desired immune response, at dosages and for periods of time necessary. A desired immune response may include stimulating or inducing a protective immune response, such as a T cell response.

[0154] "Prophylactically effective amount" refers to an amount that is effective to achieve the desired prophylactic effect (e.g., inducing protective immunity against M.tb infection) at the necessary dosage and time period. Typically, a prophylactic dose is used for a subject before or in the early stages of infection, so the prophylactic effective amount may be lower than the therapeutic effective amount.

[0155] The amount of mRNA construct in the composition can vary according to factors such as individual infection status, age, sex and body weight. The dosage regimen can be adjusted to provide the best therapeutic response. For example, a single dose can be used, or multiple doses can be used over time. It may be advantageous to formulate the composition into a dosage unit form, so that it is easy to use and the dosage is uniform.

[0156] The term "prevention" as it applies to infectious diseases (e.g., TB) is well known in the art and includes the administration of a composition that can reduce the frequency of occurrence of an infectious organism or delay infection with an infectious organism (infection vaccine prevention), or, in a population already infected with an organism, reduce the frequency of disease onset or delay the onset of disease progression, pathology, or symptoms of a condition in a subject relative to a subject that has not received the composition (disease prevention). Disease prevention includes, for example, reducing the severity of pathology or the number of disease diagnoses in a treated population compared to an untreated control population, and / or delaying the onset of disease progression, disease symptoms in a treated population compared to an untreated control population.

[0157] The term "treating" or "treatment" includes administering a therapeutically effective amount of a construct or composition of the invention to a subject in need thereof, and includes prophylaxis and / or treatment with a construct or composition. Treatment may be combined with another agent, including one or more antibiotics, to mitigate, ameliorate or accelerate recovery from TB disease or prevent recurrence of TB disease.

[0158] The term "prophylactic and / or therapeutic" treatment is well known to those skilled in the art and includes administering one or more compositions of the invention to a subject. If the composition is administered prior to infection with an organism or prior to clinical manifestation of an adverse condition (e.g., a disease or other adverse condition in a subject), the treatment is prophylactic, i.e., it protects the host from developing an adverse condition, whereas if the composition is administered after manifestation of an adverse condition, the treatment is therapeutic (i.e., it is intended to alleviate, ameliorate, or stabilize an existing adverse condition or its side effects).

[0159] As defined herein, the vaccination scheme for inducing an immune response against mycobacterium tuberculosis in a subject generally includes a series of single doses of nucleic acid constructs or compositions as described herein. Single doses (dose) or dosages (dosage) used herein refer to a starting dose (i.e., an initial first dose or second dose with the same antigen) and any subsequent doses, respectively, and they are preferably administered to "enhance" immune responses. In this case, each single dose includes administering one of the antigens or compositions of the present invention, wherein the interval between two single doses can be at least one week, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks apart. Most preferably, the antigens or compositions of the present invention are administered at intervals of 4 or 8 weeks. It should be understood that the intervals between single doses can be constant, or change during the immunization scheme, for example, the interval can be shorter (e.g., 4 weeks apart) at the beginning, and can be longer (e.g., 8 weeks apart) at the end of the scheme. In addition, depending on the total number of single doses and the intervals between single doses, the immunization regimen can be extended for a period of time, preferably for at least one week, more preferably for several weeks, and even more preferably for several months (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months). Each single dose includes administration of one of the antigens or mRNA vaccine constructs described herein.

[0160] Toxicity and therapeutic efficacy of the compositions of the invention can be determined by standard pharmaceutical procedures in cell cultures or using experimental animals, for example by determining the LD 50 and ED 50 The data obtained from cell culture and / or animal studies can be used in formulating a range of dosage for use in subjects. The dosage of any composition of the invention is preferably in the range of ED 50However, the toxicity is small or no concentration range. The dosage can vary within this range, depending on the dosage form used and the administration route used. For the composition of the present invention, the therapeutically effective dose can be estimated first from cell culture assays.

[0161] In some embodiments, the vaccine construct or composition according to the present invention may be provided in the form of a kit, optionally together with a carrier and / or an adjuvant, and accompanied by instructions for use.

[0162] The following examples are for illustrative purposes only and are not intended to be limiting.

[0163] Example 1

[0164] Identification of antigens involved in the αβ T cell response to Mycobacterium tuberculosis infection

[0165] Defining M.tb-specific T cells and their repertoire

[0166] The inventors first determined the TCRαβ sequences expressed by mycobacterium-reactive T cells in adolescents with controlled M.tb infection ("controls") and adolescents who progressed to TB disease ("progressors"). These adolescents were selected from adolescents with evidence of M.tb infection who participated in the Adolescent Cohort Study (ACS), a large TB epidemiological study. Progressors (n=44) developed microbiologically confirmed intrathoracic TB during 2 years of follow-up. Controls (n=44) also had evidence of M.tb infection but did not develop TB during follow-up. Mycobacterium-reactive T cells ( Figure 1 and Figure 2). Activated T cells were identified by their elevated expression levels of CD69, as well as CD154 or CD137, for single-cell TCR sequencing as described by Huang et al. (2020). Using this single-cell TCR sequencing approach, the TCRαβ repertoire of M.tb lysate-reactive T cells was successfully captured from PBMC samples collected from 35 controls and 35 progressors. Among 37,674 sorted T cells from progressors and controls, 22,276 (59.1%) CDR3α and 21,404 (56.8%) CDR3β sequences were detected, of which 15,272 and 16,517 were unique, respectively. A higher frequency of activated T cells was observed after stimulation with M.tb lysate compared with PBS, but there was no difference in the frequency of activated T cells between controls and progressors, nor was there a difference in the number of CDR3β sequences detected. In addition, the frequency of activated T cells remained unchanged during the two-year follow-up period. Clonal expansion (≥2 clones) was observed in the single-cell TCR data for all but four samples. More than 90% of sorted M.tb lysate-reactive T cells were CD4 T cells, 2.2% were CD8 T cells, and 6.5% expressed typical mucosal-associated invariant T (MAIT) cell CDR3α sequences, which were independent of CD4 and CD8 expression. Cells expressing known typical MAIT CDR3α sequences expressed significantly higher levels of CD26 (a MAIT cell-associated marker) compared to CD4 and CD8 T cells, indicating that the phenotype of the single-cell sorted cells was fully consistent with TCR identity. The expected levels of mRNA expression of known functional markers by sorted CD4, CD8, and MAIT cells further validated the experimental TCR sequencing pipeline used. For example, a higher proportion of M.tb lysate-reactive MAIT cells expressed IFNG mRNA compared with CD4 and CD8 T cells, while a higher proportion of CD4 T cells expressed TNF, IL2, IL17A, and IL13 than CD8 and MAIT cells, and a higher proportion of CD8 T cells and MAIT cells expressed EOMES and PERF than CD4 T cells. However, the proportion of T cells expressing any of the 20 functional transcripts measured previously to assess T cell responses to M.tb lysates (Han et al., 2014) was not significantly different between controls and progressors. Since T cells expressing a Th1 / Th17 phenotype are associated with the control of M.tb, the inventors compared the proportion of Th1 / Th17 T cells (defined as cells expressing at least two Th1 genes (i.e., IFNG, TNF, IL2, or TBX21) plus IL17A or RORC) in controls and progressors; the proportion of these cells in controls was also not different.

[0167] Comparison of M.tb TCR similarity groups in single-cell libraries

[0168] The inventors then combined CDR3β sequences obtained from mycobacterium-reactive CD4 T cells from controls, progressors, and previously published TCR datasets (Huang et al., 2020 and Glanville et al., 2017), totaling 25,256 CDR3β sequences. To determine whether M.tb-specific T cells are preferentially enriched at sites of recent or ongoing TB disease, a large set of TCR data generated from blood and resected lung tissue samples collected from an independent TB patient cohort was compared. M.tb lysate-reactive CD4 TCR sequences were significantly enriched in lung tissue compared to corresponding peripheral blood samples. In contrast, the frequency of CMV-, EBV-, and influenza A-specific CDR3β sequences did not differ between blood and lung resection samples, consistent with the expansion of M.tb-specific TCRs at sites of recent or ongoing disease.

[0169] The striking diversity and privacy of CDR3β sequences necessitated the development of clustering methods that group CDR3β sequences that are likely to share epitope specificity. This clustering approach enables inter-individual comparisons of CDR3β sequences that are likely to share antigen specificity. The inventors sought to determine whether such TCR clusters were differentially associated with controls or progressors. They used GLIPH2 (Huang et al., 2020) to cluster TCRb sequences expressed by mycobacterium-reactive T cells and identified 3,417 M.tb TCR similarity groups. 54% of the TCR similarity groups contained CDR3b sequences observed in classification experiments performed in at least two independent studies (Huang et al., 2020 and Glanville et al., 2017). This observation strongly suggests that most TCR similarity groups contain TCRs that target antigens in M.tb lysates.

[0170] Huang et al. (2020) reported that applying filters to GLIPH2 output parameters narrowed the number of TCR similarity groups and enriched groups that were more likely to be correctly clustered. Therefore, the inventors selected TCR similarity groups shared by ≥3 participants, which were consistent with ≥3 unique CDR3b sequences, had enriched common V genes (vb_score<0.05), had a limited distribution of CDR3 lengths (length_score<0.05), and had statistically significant motifs from the reference CDR3b sequence set (Fisher_score<0.05). This filtering produced 290 TCR similarity groups. They then investigated whether any of the selected TCR similarity groups were significantly enriched in sorted M.tb lysate-reactive CD4 T cells from controls or progressors. Most TCR similarity groups were shared between controls and progressors, indicating a high degree of overlap in T cell specificity between the two groups. However, some TCR similarity groups appeared to be enriched in one group. The degree of TCR sequence diversity may be related to the control of M.tb, or it may be related to progression. The large size of the M.tb TCR sequence dataset enables assessment of TCR similarity group diversity within individuals.

[0171] M.tb TCR similarity groups associated with controls and progressors in the bulk TCR repertoire

[0172] Single-cell TCR sequencing of M.tb-specific cells is necessary to identify TCR-like groups that may target M.tb antigens and to identify TCRα and TCRβ pairs that allow the establishment of peptide-MHC specificity. However, single-cell TCR sequencing cannot accurately quantify clonotypes in the entire TCR repertoire in peripheral blood. To estimate the relative frequency of occurrence of individual TCR sequences expressed by mycobacterium-reactive T cells, bulk TCRβ repertoire analysis was performed on unstimulated PBMC samples from a subset of ACS study participants (n=30) who had remaining PBMC samples after single-cell sorting, as well as in a second longitudinal cohort of adult progressors and controls enrolled in Grand Challenges 6-74 (GC6-74). The GC6-74 cohort included household contacts of South African TB patients who either developed microbiologically confirmed pulmonary TB (progressors, n=12) or remained healthy (controls, n=25) ( Figure 1 From the combined ACS and GC6-74 bulk TCR sequencing data, only CDR3b sequences associated with mycobacterium-reactive T cells (i.e., CDR3b expressed in sorted mycobacterium-reactive T cells) were selected.

[0173] From the 290 mycobacterium-reactive TCR similarity groups initially screened based on GLIPH2 output parameters, additional TCR similarity groups with significant HLA associations were selected using Fisher's exact test p-value threshold < 0.05 (HLA alleles defined by two-digit typing). Among the 148 TCR similarity group:HLA allele combinations that met this criterion, the frequency of occurrence of TCRs belonging to each similarity group in unstimulated PBMC samples from controls and progressors carrying the relevant HLA alleles was compared. A total of 40 TCR similarity group:HLA allele combinations (including 25 GLIPH2 TCR similarity groups) were differentially abundant in controls and progressors with a p-value threshold below 0.05 after controlling the false discovery rate using the Benjamini Hochberg method (q < 0.2). Thirty TCR similarity group:HLA allele combinations were more frequent in controls than in progressors, while 10 TCR similarity group:HLA allele combinations were more abundant in progressors. Among them, the DRB1*15-related TCR similarity group "SVAL" was highly enriched in controls, which is consistent with the finding that TCR diversity is higher in controls expressing DRB1*15. To investigate the specificity of TCR similarity groups associated with disease outcomes, the inventors compared the frequencies of CMV, EBV, and influenza A TCR similarity groups identified using the GLIPH2-based pipeline in controls and progressors ( Figure 3 ). Two CMV, one EBV, and five influenza A-specific TCR similarity groups showed significant differences in abundance between controls and progressors ( Figure 3 and Figure 4 To test whether the outcome-associated M.tb-reactive TCR-like groups:HLA allele combinations were nonrandom, a permutation analysis was performed with random disease outcome labels and the number of significantly associated clusters was determined for 200 iterations. The number of 40 M.tb-specific GLIPH2-specific groups associated with clinical outcome significantly exceeded the number obtained from 200 iterations with random disease outcome ( Figure 5 ). Furthermore, the number of identified CMV-, EBV-, or influenza-specific TCR sets fully matched the distribution obtained from analyses with random outcome labels. These results validate the specificity of the outcome-related M.tb-reactive TCR set discovery approach and suggest that the identified TCR sets are non-random.

[0174] The inventors also sought to investigate the longitudinal dynamics of similar groups of differentially abundant TCRs in samples collected at different time points before TB diagnosis in progressors or in controls throughout the study follow-up, modeled by fitting nonlinear spline curves. Overall, these analyses produced large 95% confidence intervals, highlighting the high inter-sample and inter-individual heterogeneity of M.tb-specific TCR data. However, the results showed that for many clusters that were more frequently identified in controls, TCRs in controls were elevated throughout the study period. Similarly, TCR clusters that were more frequently identified in progressors were also generally elevated in progressors throughout the study period.

[0175] To determine whether these results were robust to the TCR clustering algorithm, the TCR similarity group discovery analysis associated with outcomes was repeated using another clustering algorithm, TCRdist3. The TCRdist3 pipeline identified 246 unique mycobacterium-reactive metaclonal clusters with significant HLA allele associations. Of these, 46 metaclonal clusters consisting of 33 unique metaclonal clusters: HLA allele combinations were differentially abundant in controls and progressors. Overall, 64% of GLIPH2-identified clusters associated with clinical outcomes were also identified by TCRdist3, and 44.4% of all clinical outcome-associated clusters identified by either GLIPH2 or TCRdist3 were identified by both. There was substantial overlap between clusters with differential abundance identified using either the GLIPH2 or TCRdist3 analysis pipelines, suggesting that these results were largely independent of the TCR clustering algorithm.

[0176] Example 2

[0177] Identification of epitopes targeted by similar groups of control- and progressor-associated TCRs

[0178] Next, the inventors sought to identify antigens and epitopes targeted by TCRs belonging to GLIPH2 TCR-like groups that differ in abundance (ie, either a control-associated or a progressor-associated similarity group).

[0179] The selection of the 4 M.tb proteins that make up the mRNA vaccine construct was guided by the data generated in Example 1, where the inventors measured T cell responses and their antigenic targets in controls and progressors. It is noteworthy that peripheral blood mononuclear cells (PBMCs) were collected from progressors before TB disease was diagnosed. Therefore, the inventors were able to compare the frequency of antigen-specific T cell responses in controls and progressors up to 24 months before clinical disease onset. Figure 1 and Figure 2A summary of the study design and experimental methods used to identify M.tb proteins preferentially recognized by controls is depicted.The mRNA vaccine constructs of the present invention comprise modified bacterial RNA sequences encoding the amino acid sequences of WbbL1, PE13, PPE18, and CFP-10 that are codon-optimized for mammalian expression and immune recognition.

[0180] As detailed in Example 1, T cell clones that responded to M.tb in controls and progressors were first identified. This was accomplished by stimulating PBMCs with M.tb lysate (an antigen preparation containing M.tb proteins, lipids, and carbohydrates) for 12 hours. Using flow cytometry, the inventors identified single TCRαβ+ T cells that co-expressed activation markers CD69+CD137+ and / or CD69+CD154+ and sorted them into 96-well plates. Plates containing one-step RT-PCR buffer (Qiagen) and a set of primers were then subjected to TCRαβ sequence-specific amplification in a nested PCR, and the TCRs were then sequenced on a MiSeq (Illumina) instrument as described in Han et al. (2014), Glanville et al. (2017), and Huang et al. (2020). A clustering algorithm called GLIPH2 (Huang et al., 2020) was used to group T cell clones that were predicted to recognize the same M.tb protein based on their TCR CDR3β amino acid motifs. These TCR groups are called GLIPH2-specific clusters. Next, a complementary sequencing method called bulk TCR sequencing (Adaptive Biotechnologies immmunoSEQ) was used to measure the frequency of occurrence of these individual TCRs in PBMC samples collected from controls and progressors, thereby estimating the abundance of antigen-specific T cells in peripheral blood. The inventors then evaluated whether certain GLIPH2-specific clusters were more abundant in controls than in progressors, and identified multiple clusters or T cell responses that were significantly more abundant in controls (protective T cells), as well as some clusters that were significantly more abundant in progressors (pathogenic T cells).

[0181] For a subset of these GLIPH2-specific clusters, the inventors identified the M.tb proteins recognized by T cells belonging to these GLIPH2-specific clusters. This was achieved by cloning representative TCR α and β chains from a particular GLIPH2-specific cluster into the NFAT reporter stable J76-NFATRE-luc T cell line, which lacks TCR α and TCR β chains, as described by Huang et al. (2020) to allow TCR expression, thereby conferring antigen specificity to the recombinant T cell line. Then, CD80 and HLA-DM molecules were transduced into K562 cells using lentivirus to construct artificial antigen presenting cells (aAPCs), as well as candidate HLA alleles corresponding to the HLA alleles of the donor possessing the GLIPH2-specific cluster. This enabled the identification of GLIPH2-specific clusters or protective T cells that recognized PE13, CFP-10, WbbL1, and PPE18, which were more abundant in controls than in progressors. These proteins were subsequently incorporated into the mRNA vaccine construct.

[0182] Example 3

[0183] Designing mRNA vaccine constructs

[0184] Codon modifications in mRNA sequences optimize antigen expression, humoral and T-cell immune responses in mammalian cells and impair the induction of strong type I IFN responses

[0185] Codon-optimized and uridine-depleted sequences encoding bacterial PE13, CFP-10, WbbL1, and PPE18 proteins have been designed, synthesized, and cloned into plasmid templates for in vitro transcription (IVT). Codon optimization and uridine depletion can be used to enhance protein expression and attenuate innate immune stimulation (type I IFN response), respectively.

[0186] Table 1 provides the amino acid sequences of PE13, CFP-10, WbbL1 and PPE18 proteins. Table 2 provides the codon-optimized and uridine-depleted nucleic acid sequences encoding PE13, CFP-10, WbbL1 and PPE18 proteins designed for mRNA vaccines.

[0187] Table 1. Amino acid sequences of PE13, CFP-10, WbbL1 and PPE18 proteins.

[0188]

[0189] Table 2. Codon-optimized and uridine-depleted nucleic acid sequences encoding PE13, CFP-10, WbbL1 and PPE18 proteins.

[0190]

[0191]

[0192] *Any sequence in Table 1 may contain a methionine residue at the beginning, particularly when the sequence is the first sequence in a construct.

[0193] **Any sequence in Table 2 may include a start codon (ATG) at the 5' end, particularly when the sequence is the first sequence in the construct. In addition, any sequence may include a stop codon at the 3' end selected from TAA, TAG or TGA, particularly when the sequence is the last sequence in the construct. In particular, the inventors of the present invention have designed a specific stop codon sequence comprising four consecutive stop codons, the nucleotide sequence of which is TGATAATGATAG (SEQ ID NO: 32). A single antigen sequence may also include the stop sequence of SED ID NO: 32.

[0194] Various construct designs have been employed, including wherein the polyprotein expression sequence comprises two or more codon optimized sequences encoding PE13, CFP-10, WbbL1 and PPE18 proteins in the polyprotein expression sequence in any order, and is connected by glycine-serine and / or glycine flexible linkers. In other designs, the polyprotein expression sequence comprises two or more codon optimized sequences encoding PE13, CFP-10, WbbL1 and PPE18 proteins in the polyprotein expression sequence in any order, and is connected by 2A derived peptides. Alternatively, the polyprotein expression sequence comprises two or more codon optimized sequences encoding PE13, CFP-10, WbbL1 and PPE18 proteins in the polyprotein expression sequence in any order, and is connected by glycine-serine and / or glycine flexible linkers and 2A derived peptides. Table 3 below provides glycine-serine, glycine and 2A derived sequences studied for incorporation into vaccine constructs. Figure 6 Various polyprotein constructs designed are shown. The linkers in position 1, position 1 and 2 and / or position 1, 2 and 3 are derived from the sequences provided in Table 3, and the antigen sequences are selected from the codon optimized sequences encoding PE13, CFP-10, WbbL1 and PPE18 proteins in any order in the polyprotein expression sequence. The first four and / or the last four codons of each codon optimized sequence encoding PE13, CFP-10, WbbL1 and PPE18 proteins can be modified or removed to facilitate the insertion of the linker.

[0195] Table 3. Glycine-serine, glycine and 2A derived linker sequences investigated in multiprotein construct design.

[0196]

[0197] Analysis of native sequences encoding CFP-10, WbbL1, and PPE18 did not reveal a mammalian secretory peptide signal (SP), so these proteins would remain in the cytosol. Therefore, to enhance antigen secretion, sequences encoding SPs with defined efficiencies were incorporated into the mRNA template plasmid. These are typically located upstream and in-frame of the M.tb antigen ORF (i.e., at the N-terminus of the protein). The immunogenicity of constructs with the following leader sequences was studied:

[0198] · Leader sequence derived from the Mycobacterium tuberculosis Rv2878c sequence

[0199] · Leader sequence derived from the Mycobacterium tuberculosis MT18B_2507 sequence

[0200] · Leader sequence derived from the Mycobacterium tuberculosis Rv3803c sequence

[0201] · Leader sequence derived from the Mycobacterium tuberculosis Rv1860 sequence

[0202] · Leader sequence derived from human interleukin-2 sequence

[0203] · Leader sequence derived from Gaussia luciferase

[0204] Table 4 below provides the leader sequences studied for incorporation into vaccine constructs, and the codon optimized sequences encoding the leader peptides. The leader sequence is included at the N-terminus of a single antigen sequence or the N-terminus of a multimeric protein comprising two or more antigen sequences, wherein the antigen sequence is selected from the codon optimized sequences encoding PE13, CFP-10, WbbL1 and PPE18 proteins in any order in the multimeric protein expression sequence. The first four and / or the last four codons of each codon optimized sequence encoding PE13, CFP-10, WbbL1 and PPE18 proteins can be modified or removed to facilitate the inclusion of the leader sequence.

[0205] Table 4. Leader sequences and codon-optimized sequences encoding leader sequences investigated in the design of multiprotein constructs.

[0206]

[0207]

[0208] Cloning and amplification of plasmids encoding M.tb proteins

[0209] DNA sequences encoding individual M.tb antigens (i.e., WbbL1, PE13, PPE18, and CFP-10) and polyprotein combinations were generated using gene synthesis as described above. Antigen coding sequences were subcloned into a T7 RNA polymerase-based vector to facilitate in vitro transcription of these sequences into eukaryotic mRNA-like transcripts. Leader sequences were generated by oligonucleotide extension and polymerase chain reaction (PCR) or by gene synthesis and cloned upstream of individual M.tb antigens or polyproteins. Figure 7 Two exemplary constructs are provided. The sequences of these two constructs correspond to SEQ ID NO: 30 and SEQ ID NO: 31, respectively. Figure 7 The construct provided in A does not include a leader sequence, and Figure 7 The construct provided in B includes a leader sequence of SEQ ID NO: 24 encoded by a codon-optimized sequence of SEQ ID NO: 25. In both constructs, "Linker 1" is a linker of SEQ ID NO: 11, "Linker 2" is a linker of SEQ ID NO: 16, and "Linker 3" is a linker of SEQ ID NO: 14. Figure 8 and Fig. 9 Further provided are the nucleic acid sequences of these exemplary constructs, wherein Figure 8 The sequence in corresponds to Figure 7 The construct without the leader sequence shown in A, Fig. 9 The sequence in corresponds to Figure 7 The construct including the leader sequence shown in B. The plasmid was propagated in bacteria and purified using an anion exchange resin column.

[0210] Example 4

[0211] Preparation of single-gene M.tb mRNA vaccine

[0212] Synthesis of mRNA

[0213] In vitro transcription (IVT) is performed according to established methods that employ restriction linearized plasmid DNA followed by IVT. Capping is performed co-transcriptionally (e.g., using CleanCap substrate from TriLink) or post-transcriptionally using vaccinia capping enzyme. Modified nucleotides (e.g., N1-methyl-pseudouridine and pseudouridine) are included in the IVT reaction. This is intended to reduce the innate immune response to the synthetic mRNA candidates. In this example, linearized plasmids encoding four monogenic M.tb antigens CFP-10, WbbL1, PE-13, and PPE-18 (with or without a leader sequence) were used as templates for chemical synthesis of mRNA. IVT reactions were performed using bacteriophage T7 polymerase in the presence of Reagent AG (TriLink BioTechnologies) and pseudouridine.

[0214] Purification and analysis of in vitro transcribed mRNA

[0215] mRNA for evaluation in cultured cells and in vivo was purified according to established methods, including lithium chloride precipitation and oligo(dT) column chromatography. The size and integrity of in vitro transcribed mRNA was assessed using capillary gel electrophoresis on a 5200 Fragment Analyzer using the DNF-471 RNA Kit (15 nt) (Agilent). Fig.10 As shown, in vitro transcribed mRNAs of all four M.tb antigens CFP-10, WbbL1, PE-13, and PPE-18 were successfully obtained.

[0216] In vitro characterization of mRNA

[0217] Immunofluorescence staining of M.tb antigens expressed from mRNA in cultured mammalian cells was assessed using established methods. Briefly, HepG2-hNTCP cells were seeded in 96-well plates at a density of approximately 15,000 cells / well (DMEM / F12 supplemented with GlutaMax TM , 10% FBS, 10 mM HEPES, 50 μM hydrocortisone, 200 units / mL penicillin, 200 μg / mL streptomycin, 5 μg / mL insulin, and 400 μg / mL G418). The next day, Lipofectamine TM MessengerMAX TM Cells were transfected with 100 ng of mRNA encoding CFP-10 (Thermo Fisher Scientific) and incubated at 37°C and 5% CO2. 24 hours after transfection, the growth medium was removed and the cells were fixed and permeabilized with 4% PFA. Rabbit polyclonal anti-CFP-10 primary antibody (Thermo Fisher Scientific, PA5-122324) and Alexa-fluor TM Cells were stained with 488-conjugated goat anti-rabbit secondary antibody (Thermo Fisher Scientific, A-11008). Cells were counterstained with DAPI and imaged using EVOS FL (Thermo Fisher Scientific) under DAPI and GFP filters. Fig.11 Confirmation that CFP-10 protein is expressed in situ from CFP-10 mRNA is provided.

[0218] Formulation of lipid nanoparticles (LNP)

[0219] mRNA was incorporated into LNPs using validated microfluidics and diafiltration or tangential flow filtration methods. The component lipids of the formulations included previously described ionizable lipids as well as novel renewable lipids. In this example, microfluidics-based assembly ( Ignite TM , Precision Nanosystems), mRNA encoding TB antigens was formulated into lipid nanoparticles (LNPs) containing a helper lipid (DSPC), a pegylated lipid (PEG2000-DMG), cholesterol, and an ionizable lipid SM102. Surface charge analysis, particle size determination, and polydispersity index were used for characterization using a Zetasizer Pro (Malvern) ( Fig.12 ). The size of mRNA antigen LNP of single gene mRNA ranges from 58nm to 75.5nm.

[0220] Example 5

[0221] Induction of cellular immune responses to single-gene M.tb antigen vaccines

[0222] Immunization of mice and preparation of spleen cells

[0223] All animal work was performed in accordance with protocols approved by the University of the Witwatersrand Animal Ethics Screening Committee. On day zero (D0), female BALB / c, C57BL6, and C3HeB / FeJ mice were injected intramuscularly with lipid nanoparticle-formulated mRNA vaccines expressing TB antigens, including mRNA encoding four M.tb antigens CFP-10, WbbL1, PE-13, and PPE-18 (10 μg / mouse per inoculation, in a total volume of 50 μl), and an equal proportion of vaccine mixture containing all four single gene mRNAs mixed before inoculation of mice (2.5 μg of each single gene mRNA / mouse per inoculation, in a total volume of 50 μl). Control groups were injected intramuscularly with saline (50 μl / mouse per inoculation) or lipid nanoparticle-formulated control mRNA encoding firefly luciferase (10 μg / mouse per inoculation, in a total volume of 50 μl). Mice received booster vaccinations three weeks later. Serum was collected directly before vaccination (day 0 and week 3) and at week 5. Spleens were collected at week 5 to analyze antigen-specific T cells. Table 5 below shows the animal groups and vaccine doses administered for the single gene mRNA vaccine.

[0224] Table 5. Animal groups and doses of single gene TB mRNA vaccines.

[0225]

[0226] Splenocytes were isolated from mice according to standard protocols. Briefly, spleens were mashed through a 70 μm cell strainer and washed in PBS pH 7.2 supplemented with 1% FBS. Red blood cells were lysed using potassium ammonium chloride buffer. Cells were washed and resuspended in complete RPMI 1640 medium supplemented with GlutaMax (Gibco), 10% FBS and antibiotics. Cells were seeded in 96-well U-bottom plates at a density of 1x10 6 cells per well for ex vivo stimulation.

[0227] Quantification of T cell stimulation and secreted cytokines

[0228] Peptide pools covering the M.tb mRNA vaccine antigens CFP-10, WbbL1, PE-13, and PPE-18 and the M.tb non-vaccine antigen TB10.4 were synthesized by GenScript. Cells isolated from mouse spleens were stimulated with a cell stimulation cocktail (PMA and ionomycin) or 2 μg / ml of the peptide pool in the presence of 1 μg / ml anti-CD28 (clone 37.51) and incubated at 37°C and 5% CO2 for 24 h. TM MU Th1 / Th2 Panel V03 (8-plex) (BioLegend) quantifies the concentration of secreted cytokines in the cell supernatant. The 8-plex panel detects IL-5, IL-13, IL-2, IL-6, IL-10, IFN-γ, TNF-α, and IL-4, and the 12-plex panel detects IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IFN-γ, and TNF-α. Figures 13 to 15 Provided are the IFN-γ, TNF-α, and IL-2 cytokine secretion profiles in ex vivo stimulated splenocytes of immunized BALB / c, C57Bl / 6, and C3HeB / FeJ mice, respectively. Fig.16 IL-17A and IL-22 cytokine secretion profiles of C3HeB / FeJ mice are shown.

[0229] from Figures 13 to 16 As can be seen, due to the different haplotypes, the antigen-specific responses of different mouse strains are also different, which is expected. Fig.13As can be seen in the Figure 2, the concentrations of IFN-γ, TNF-α, and IL-2 increased in BALB / c mice immunized with WbbL1, PPE-18, and mixed (CWPP) mRNA. In addition, an increase in IL-2 was detected in mice immunized with PE-13 mRNA. Fig.14 As can be seen in Figure 2, the concentrations of IFN-γ, TNF-α, and IL-2 increased in C57BL / 6 mice immunized with CFP-10, WbbL1, PE-13, PPE-18, and mixed (CWPP) mRNA. Fig.15 Increased concentrations of IFN-γ, TNF-α, and IL-2 were detected in C3HeB / FeJ mice immunized with CFP-10 and mixed (CWPP) mRNA. Fig.16 The concentrations of IL-17A and IL-22 were shown to be increased, indicating a Th17 response in vaccinated C3HeB / FeJ mice.

[0230] T cell stimulation and intracellular cytokine staining

[0231] IFN-γ positive CD3, CD4 and CD8 T cells were further detected using flow cytometry. Cell stimulation and intracellular cytokine staining were performed according to standard protocols. Briefly, cells were stimulated with cell stimulation cocktail (PMA and ionomycin) or 2 μg / ml peptide pool in the presence of 1 μg / ml anti-CD28 (clone 37.51) at 37°C and 5% CO2 for 1 hour, followed by the addition of brefeldin A and incubation overnight (15 hours). The cells were washed, stained with a fixable viability dye, fixed and permeabilized using BioLegend Fixation Buffer and Intracellular Staining Permeabilization Wash Buffer Set. The cells were stained for intracellular IFN-γ-APC (clone XMG1.2), CD3-PerCP-eFluor TM 710 (clone 17A2) and CD4-FITC (clone GK1.5) or CD8-FITC (clones 53-67). TM Cellular events were captured on a C6 plus flow cytometer (Beckton Dickinson) and analyzed using FlowJo TM The percentage of intracellular IFN-γ staining was determined using ELISA software (BD Life Sciences). Antigen-specific activation of CD4+ T cells was confirmed in vaccinated C57BL / 6 mice ( Fig.17 In C3HeB / FeJ mice, CD4 and CD8 IFN-γ positive T cells were detected in the 10 μg dose of CFP-10 mRNA vaccine and the 2.5 μg dose of CFP-10 mRNA vaccine contained in the mixed (CWPP) mRNA vaccine ( Fig.18 ). The secreted cytokine profiles indicated that vaccination induced enhanced M.tb antigen-specific T helper cell type 1 (Th1) immune responses (increased concentrations of IFN-γ, TNF-α, and IL-2) in all three vaccinated mouse strains. Multi-antigen Th1 responses were also detected in C57Bl / 6 and C3HeB / FeJ mice immunized with a vaccine mixture containing all four M.tb antigens. Increased concentrations of IL-17A and IL-22 in C3HeB / FeJ-vaccinated mice indicated the presence of a Th17 response, which contributes to the control of initial TB infection. Intracellular cytokine staining showed that IFN-γ-positive CD4 T cells (CD3 + CD4 + IFN-γ + Intracellular cytokine staining showed that IFN-γ-positive CD4 and CD8 T cells (CD3 + CD8 + IFN-γ + In addition, the percentage of IFN-γ-positive CD4 T cells showed a multi-antigen increase in C3HeB / FeJ mice vaccinated with the mixed (CWPP) mRNA vaccine.

[0232] M.tb derived leader sequences L1 (SEQ ID NO: 21), L2 (SEQ ID NO: 23), L3 (SEQ ID NO: 25) and L4 (SEQ ID NO: 27) were included in the mRNA vaccine encoding CFP-10. When leader sequences 2, 3 and 4 were added, an increase in the percentage of IFN-γ+CD4+ T cells was observed in immunized C3HeB / FeJ mice compared to CFP-10 mRNA vaccines without leader sequence (NL) and L1-CFP-10 ( Fig.19 ).

[0233] Thus, these results provide evidence that a single gene mRNA construct is able to elicit antigen-specific T cell responses to four M.tb antigens and that the mRNA vaccine is immunogenic in mice.

[0234] Example 6

[0235] Preparation of multigene M.tb mRNA vaccine

[0236] Synthesis of mRNA

[0237] The same methods as above were used to develop, synthesize and purify various polyprotein mRNA vaccine constructs. The polygene constructs included the M.tb antigens CFP-10, WbbL1, PE-13 and PPE-18 at different locations, with and without leader sequences (L1, L2, L3, L4, L5), such as Fig. 20 The complete amino acid sequences of the four polyproteins (referred to as TB-M3, TB-M4, TB-M7 and TB-M8) are provided as SEQ ID NOs: 33 to 36, as shown in Table 6 below. The complete nucleic acid sequences of the multigene constructs are provided as SEQ ID NOs: 37 to 40, as shown in Table 7 below. In addition, any of the sequences in Table 6 may include one or more leader sequences selected from the group consisting of the leader sequences shown in SEQ ID NOs: 20, 22, 24, 26 and 28, and any of the sequences in Table 7 may include one or more leader sequences selected from the group consisting of the leader sequences shown in SEQ ID NOs: 21, 23, 25, 27 and 29, as described in Table 4 above. The plasmids were propagated in bacteria and purified using an anion exchange resin column.

[0238] Table 6. Amino acid sequences of TB-M3, TB-M4, TB-M7, and TB-M8 proteins.

[0239]

[0240]

[0241] Table 7. Codon-optimized and uridine-depleted nucleic acid sequences encoding TB-M3, TB-M4, TB-M7, TB-M8 proteins.

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] *Any of the sequences in Table 6 may contain a methionine residue at the beginning.

[0248] **Any sequence in Table 7 may include a start codon (ATG) at the 5' end of the construct. In addition, any sequence may include a stop codon selected from TAA, TAG or TGA at the 3' end of the construct. In particular, the inventors of the present invention designed a specific stop codon sequence comprising four consecutive stop codons, the nucleotide sequence of which is TGATAATGATAG (SEQ ID NO: 32).

[0249] Purification and analysis of in vitro transcribed mRNA

[0250] The size and integrity of exemplary in vitro transcribed CFP-10 single gene mRNAs encoding TB antigens, multigene mRNAs and multigene mRNAs with leader sequences were analyzed using capillary gel electrophoresis on a 5200 fragment analyzer using the DNF-471 RNA kit (15 nt) (Agilent). Fig.21 As shown, in vitro transcribed mRNAs of the longer polyprotein transcripts TB-M3 and TB-M4 as well as transcripts containing leader sequences were successfully obtained. Fig. 22 Capillary gel images are shown depicting the size and integrity of TB-M3 and TB-M4 multi-gene mRNA transcripts with and without leader sequences (L1, L2, L3, L4, L5) and CFP-10 single-gene mRNA transcripts with and without leader sequences (L1, L2, L3, L4).

[0251] Formulation of lipid nanoparticles (LNP)

[0252] As described above, the multi-gene mRNA was incorporated into LNPs. The size range of the LNPs of the multi-gene mRNA was 68.12 nm-100.7 nm.

[0253] Example 7

[0254] Induction of cellular immune response to multigene M.tb antigen vaccine

[0255] Immunization of mice and preparation of splenocytes

[0256] All animal experiments were performed in accordance with protocols approved by the Animal Ethics Review Committee of the University of Witwatersrand. On day 0 (D0), female BALB / c, C57BL6 and C3HeB / FeJ mice were intramuscularly injected with lipid nanoparticle-formulated mRNA vaccines TB-M3, TB-M4 and TB-M7 expressing multiple TB antigens. The control group was intramuscularly injected with saline (50 μl / mouse per vaccination) or lipid nanoparticle-formulated control mRNA encoding firefly luciferase (10 μg / mouse, total volume of 50 μl per vaccination). Mice received booster vaccinations three weeks later. Serum was collected directly before vaccination (day 0 and week 3) and at week 5. Spleens were collected at week 5 for analysis of antigen-specific T cells. Table 8 below shows the animal groups and vaccine doses administered for multi-gene mRNA vaccines.

[0257] Table 8. Animal groups and doses of polyprotein TB mRNA vaccines.

[0258]

[0259]

[0260] Splenocytes were isolated from mice following the standard protocol described in Example 5.

[0261] T cell stimulation, quantification of secreted cytokines, and intracellular cytokine staining.

[0262] A peptide pool covering the M.tb mRNA vaccine antigens CFP-10, WbbL1, PE-13 and PPE-18, a mixture of all four antigens (peptide mixture CWPP) and the M.tb non-vaccine antigen TB10.4 was used for T cell stimulation. Samples from vaccinated mice were stimulated and processed for quantification of secreted cytokines and intracellular cytokine staining according to the standard protocol described in Example 5.

[0263] Fig.23Cytokine profiles are shown in ex vivo stimulated splenocytes of mice immunized with saline or control mRNA encoding a bioluminescent reporter gene or 10 μg LNP formulated multigene TB-M7 mRNA. Exemplary cytokine profiles of IFN-γ and IL-2 are shown for BALB / c, C57BL6, and C3HeB / FeJ mice. Unstimulated splenocytes from mice vaccinated with TB-M7 mRNA served as controls. These results confirm individual antigen-specific T cell activation for all four TB antigens contained in the polyprotein. Specifically, the concentrations of IFN-γ and IL-2 increased when C3HeB / FeJ mice were stimulated with the CFP-10 peptide pool, the concentrations of IFN-γ and IL-2 increased when BALB / c, C57BL6, and C3HeB / FeJ mice were stimulated with the Wbbl1 peptide pool, the concentrations of IFN-γ and IL-2 increased when C57BL6 and C3HeB / FeJ mice were stimulated with the PE-13 peptide pool, and the concentrations of IFN-γ and IL-2 increased when BALB / c, C57BL6, and C3HeB / FeJ mice were stimulated with the PPE-18 peptide pool.

[0264] Fig.24 The exemplary IFN-γ, IL-2, TNF-α and IL-17A cytokine profiles secreted in ex vivo stimulated splenocytes of BALB / c, C57BL6 and C3HeB / FeJ mice stimulated with mixed antigenic peptide pools (peptide pool: CWPP) after inoculation with TB-M7 polyprotein mRNA are shown. IFN-γ and IL-2 increased in BALB / c, C57BL6 and C3HeB / FeJ mice after inoculation with TB-M7 mRNA. In addition, TNF-α and IL-17A increased in BALB / c mice and TNF-α increased in C57BL6 mice.

[0265] For splenocytes stimulated with CFP-10, PE-13, and PPE-18 peptide pools, the concentration of IL-6 secreted by C57BL6 mice vaccinated with TB-M7 polyprotein mRNA increased in all three peptide pools ( Fig.25 ). In addition, BALB / c mice showed an increase in IL-6 after stimulation with the PPE-18 peptide pool.

[0266] In BALB / c, C57BL6, and C3HeB / FeJ mice vaccinated with TB-M7 polyprotein mRNA, splenocytes stimulated with WBBL1, PE-13, and PPE-18 peptide pools secreted increased IL-22 concentrations ( Fig.26 ).

[0267] from Fig. 27It can be seen that in BALB / c mice vaccinated with TB-M7 polyprotein mRNA, the percentage of IFN-γ+CD3+ T cells increased after stimulation of cells with WbbL1, PPE-18, and CWPP peptide pools. PPE-18-specific IFN-γ+CD4+ T cells were observed. In addition, after stimulation of cells from C57BL6 mice with WbbL1, PE-13, PPE-18, and CWPP peptide pools, the percentage of IFN-γ+CD4+ T cells increased in mice vaccinated with TB-M7 polyprotein mRNA ( Fig.28 ). In addition, flow cytometry-based detection of IFN-γ-positive CD4 T cells showed no activation of IFN-γ in ex vivo stimulation of splenocytes from vaccinated BALB / c, C57BL6, and C3HeB / FeJ mice in which the TB antigen (TB10.4) was not contained in the polyprotein ( Fig.29 ). This confirms the results described above for antigen-specific T cell stimulation.

[0268] mRNA vaccines encoding TB-M3 and TB-M4 polyproteins were also investigated, as described above in Table 8. The cytokine profiles secreted in ex vivo stimulated splenocytes of mice injected intramuscularly with 10 μg LNP formulated control mRNA encoding a bioluminescent reporter gene (control) or 10 μg LNP formulated multigenic TB-M3 or TB-M4 mRNA were analyzed. Fig.30 The exemplary cytokine profiles of IFN-γ and IL-2 of BALB / c, C57BL6 and C3HeB / FeJ mice are shown. The concentration of IFN-γ and IL-2 of C3HeB / FeJ mice is increased by stimulation with the CFP-10 peptide pool. The concentration of IFN-γ and IL-2 of BALB / c, C57BL6 and C3HeB / FeJ mice is increased by stimulation with the Wbbl1 peptide pool. The concentration of IFN-γ and IL-2 of C57BL6 and C3HeB / FeJ mice is increased by stimulation with the PE-13 peptide pool. Finally, the concentration of IFN-γ and IL-2 of BALB / c and C57BL6 mice is increased by stimulation with the PPE-18 peptide pool.

[0269] These results confirm single antigen-specific T cell activation for all four TB antigens contained in the polyprotein and that the polyprotein mRNA vaccine is immunogenic in mice.

[0270] References

[0271] Han, A., Glanville, J., Hansmann, L. & Davis, M. M. Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nat. Biotechnol. 32, 684–692 (2014).

[0272] Glanville, J. et al. Identifying specificity groups in the T cell receptor repertoire. Nature 547. (2017).

[0273] Huang, H., Wang, C., Rubelt, F., Scriba, T. J. & Davis, M. M. Analyzing the Mycobacterium tuberculosis immune response by T-cell receptor clustering with GLIPH2 and genome-wide antigen screening. Nat. Biotechnol. (2020).

Claims

1. A vaccine composition comprising at least two nucleic acids selected from the group consisting of: a. a nucleic acid encoding the WbbL antigen having the amino acid sequence of SEQ ID NO: 1; b. a nucleic acid encoding a CFP-10 antigen having an amino acid sequence of SEQ ID NO: 2; c. a nucleic acid encoding a PPE18 antigen having an amino acid sequence of SEQ ID NO: 3; and d. A nucleic acid encoding the PE13 antigen having the amino acid sequence of SEQ ID NO:

4.

2. The vaccine composition according to claim 1, wherein the vaccine composition comprises all nucleic acids in (a) to (d).

3. A vaccine composition according to claim 1 or 2, wherein (a) has a nucleotide sequence substantially identical to SEQ ID NO: 5, (b) has a nucleotide sequence substantially identical to SEQ ID NO: 6, (c) has a nucleotide sequence substantially identical to SEQ ID NO: 7, and (d) has a nucleotide sequence substantially identical to SEQ ID NO:

8.

4. The vaccine composition according to any one of claims 1 to 3, wherein at least one nucleic acid further comprises a leader nucleotide sequence encoding a secretory peptide signal.

5. The vaccine composition of claim 4, wherein each nucleic acid comprises a leader nucleotide sequence encoding a secretory peptide signal.

6. The vaccine composition according to claim 4 or 5, wherein each secretory peptide signal is independently selected from SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO:

28.

7. The vaccine composition according to any one of claims 4 to 6, wherein each secretory peptide signal is encoded by a nucleotide sequence substantially identical to the sequence shown in the group consisting of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO:

29.

8. The vaccine composition according to any one of claims 1 to 7, wherein at least two nucleic acid sequences are present on a single nucleic acid construct.

9. The vaccine composition of claim 8, wherein each nucleic acid is separated by a nucleotide sequence encoding a linker.

10. The vaccine composition of claim 9, wherein the linker is selected from the group consisting of a glycine-serine flexible linker, a glycine flexible linker and a 2A-derived peptide.

11. The vaccine composition according to claim 9 or 10, wherein each linker is independently selected from the linker sequence of any one of SEQ ID NOs: 9-19.

12. The vaccine composition according to any one of claims 8 to 11, wherein the nucleic acid construct encodes a polyprotein having an amino acid sequence of any one of SEQ ID NOs: 33-36.

13. The vaccine composition according to any one of claims 8 to 12, wherein the nucleic acid construct has a nucleotide sequence substantially identical to any one of SEQ ID NOs: 37-40.

14. The vaccine composition of any one of claims 1 to 14, wherein the nucleic acid is mRNA.

15. The vaccine composition of claim 14, wherein the mRNA is capped at the 5' end.

16. The vaccine composition of claim 14 or 15, wherein the mRNA comprises one or more modified nucleotides.

17. The vaccine composition of claim 16, wherein the one or more modified nucleotides are selected from N1-methyl-pseudouridine and pseudouridine.

18. The vaccine composition of any one of claims 14 to 17, wherein the mRNA is contained in a lipid nanoparticle.

19. The vaccine composition according to any one of claims 1 to 18, wherein the vaccine composition is capable of eliciting a protective immune response against Mycobacterium tuberculosis.

20. Use of the vaccine composition of any one of claims 1 to 19 for use in a method of eliciting a protective immune response against Mycobacterium tuberculosis, the method comprising administering the vaccine composition to a subject.

21. A method of eliciting a protective immune response against Mycobacterium tuberculosis in a subject, the method comprising administering to the subject a vaccine composition according to any one of claims 1 to 19.

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