RNA for preventing or treating tuberculosis

Abstract

The present disclosure provides agents and methods for preventing or treating tuberculosis using RNA. RNA encoding an antigen, immunogenic variant, or fragment thereof of Mycobacterium tuberculosis is formulated and administered such that the antigen, variant, or fragment is produced by cells of the subject, particularly after intramuscular or intravenous administration of the RNA.

Description

[Technical Field]

[0001] The present disclosure provides agents and methods for preventing or treating tuberculosis using RNA. RNA encoding an antigen, immunogenic variant, or fragment thereof of Mycobacterium tuberculosis is formulated and administered such that the antigen, variant, or fragment is produced by cells of the subject, particularly after intramuscular or intravenous administration of the RNA. [Background technology]

[0002] The use of RNA to deliver foreign genetic information to target cells offers an attractive alternative to DNA. The advantages of RNA include its transient expression and non-transforming properties. RNA does not require nuclear penetration for expression and cannot be integrated into the host genome, eliminating the risk of oncogenicity.

[0003] Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), is a leading cause of death from a single infectious agent. Mtb is a Gram-positive, rod-shaped bacterium of the Mycobacterium family. More than 4,000 genes encoded within its approximately 4 million base pair genome make Mtb a complex pathogenic organism. This is further accentuated by the atypical composition of its cell wall, which has a high lipid content.

[0004] Despite a declining trend in TB cases and TB-related deaths over the past two decades, 1.42 million people died from TB alone in 2019. In addition to active TB, challenges arise from latent TB infection (LTBI), when infected patients do not show clinical symptoms. An estimated 2 billion latently infected individuals worldwide represent a huge and unpredictable reservoir of Mtb (WORLD HEALTH ORGANIZATION. Global tuberculosis report 2019. Geneva, WORLD HEALTH ORGANIZATION; 2019. ISBN: 978-92-4-156571-4). The high prevalence of HIV-1 infection further increases the risk of TB disease acquisition, activation of latent TB infection, and death from HIV-TB coinfection. In 2009, 200,000 deaths were associated with HIV-TB comorbidity. The complexity of the Mtb cell wall renders the bacterium resistant to environmental influences and treatment with certain antibiotics. The latter further complicates anti-TB treatment, especially in low- and middle-income countries (WORLD HEALTH ORGANIZATION. Global tuberculosis report 2020. Geneva, WORLD HEALTH ORGANIZATION; 2020. ISBN: 978-92-4-001313-1).

[0005] Bacillus Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis, is the only licensed TB vaccine introduced in 1921. The use of live BCG is not recommended for immunocompromised individuals, and the protection against pulmonary TB conferred by immunization with BCG varies widely, ranging from 50 to 80%. Furthermore, decades of BCG passage have further attenuated currently used BCG strains, reducing their protective efficacy (Brosch R, et al. Proc. Natl. Acad. Sci. USA, 2007;104(13):5596-5601). Therefore, there is an unmet medical need for safer and more effective vaccines to prevent TB, particularly those that can be administered to immunocompromised individuals.

[0006] The clinical trial pipeline for TB vaccine candidates includes the use of live, live-attenuated, and inactivated mycobacteria, as well as Mtb antigens as recombinant proteins (subunit vaccines) (TuBerculosis Vaccine Initiative (TBVI). Available from https: / / www.tbvi.eu / what-we-do / pipeline-of-vaccines / ). The challenges with these vaccine platforms are: i) their low safety profile due to the replicative live vaccines, which are still infectious; ii) the low immunogenicity of inactivated vaccines; and iii) the need to add adjuvants to subunit vaccines to enhance immunogenicity. To date, most vaccine candidates have failed to demonstrate TB or better protection against TB development compared to placebo in clinical trials.

[0007] For all these reasons, new drugs are needed to prevent or treat tuberculosis. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] WORLD HEALTH ORGANIZATION.Global tuberculosis report 2019.Geneva,WORLD HEALTH ORGANIZATION;2019.ISBN:978-92-4-156571-4 [Non-patent document 2] WORLD HEALTH ORGANIZATION.Global tuberculosis report 2020.Geneva,WORLD HEALTH ORGANIZATION;2020.ISBN:978-92-4-001313-1 [Non-patent document 3] Brosch R,et al.Proc.Natl.Acad.Sci.USA,2007;104(13):5596-5601 [Non-patent document 4] TuBerculosis Vaccine Initiative(TBVI).https: / / www.tbvi.eu / what-we-do / pipeline-of-vaccines / Summary of the Invention

[0009] The present disclosure provides compositions useful as TB vaccines. The compositions provided herein include RNA for delivering Mtb antigens to a subject. The findings described herein demonstrate that the RNA described herein, e.g., unmodified uridine-containing mRNA (uRNA) or nucleoside-modified mRNA (modRNA), expressing an antigen of Mycobacterium tuberculosis, its immunogenic variant, or fragment, is useful for preventing or treating tuberculosis. The RNA encoding the antigen of Mycobacterium tuberculosis, its immunogenic variant, or fragment, is formulated and administered such that the antigen, variant, or fragment can be produced, and preferably secreted, by the patient's cells to prevent or combat tuberculosis.

[0010] In particular, the present disclosure describes, in some embodiments, RNA components encoding antigen 85A (Ag85A), antigen Mtb 72F (M72; a recombinant protein derived from two Mtb proteins, Mtb32A and Mtb39A; hereafter referred to as M72 only), 6-kilodalton early secretory antigenic target (ESAT6), resuscitation-promoting factor A (RpfA), resuscitation-promoting factor D (RpfD), hypoxia-response protein 1 (Hrp1), virulence-associated protein B47 (VapB47), and heparin-binding hemagglutinin A (HbhA). The present disclosure observed that immunization of mice with these RNA components induced strong antigen-specific T cell and B cell responses, such that the immune response elicited by two intramuscular administrations administered 21 days apart was higher than the immune response elicited by a single subcutaneous immunization with BCG.

[0011] Mtb exhibits differential gene expression patterns during its active and dormant (non-dividing) stages (Andersen P, et al. Cold Spring Harb Perspect Med, 2014;4(6):a018523). To prevent the development of TB, immunity to antigens specific to each of the various stages of Mtb infection should exist. The TB vaccine candidate developed herein, containing the above-described RNA components, is designed to induce protective immunity against antigens specific to different stages of Mtb infection.

[0012] Unlike the attenuated vaccine BCG, this TB vaccine candidate does not carry the risks associated with infection and can therefore be administered to people who cannot receive live organisms (such as pregnant women and immunocompromised individuals).

[0013] In one aspect, the present disclosure provides a composition or pharmaceutical formulation comprising at least one RNA molecule, wherein the at least one RNA molecule encodes a set of antigenic amino acid sequences, wherein the set of antigenic amino acid sequences comprises (i) at least one Mtb antigen, immunogenic variant thereof, or immunogenic fragment of the Mtb antigen or its immunogenic variant from the acute phase of the Mtb life cycle, (ii) at least one Mtb antigen, immunogenic variant thereof, or immunogenic fragment of the Mtb antigen or its immunogenic variant from the latent phase of the Mtb life cycle, and (iii) at least one Mtb antigen, immunogenic variant thereof, or immunogenic fragment of the Mtb antigen or its immunogenic variant from the resuscitation phase of the Mtb life cycle.

[0014] In some embodiments, at least one Mtb antigen from the acute phase of the Mtb life cycle, an immunogenic variant thereof, or an immunogenic fragment of an Mtb antigen or an immunogenic variant thereof is (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof; and / or (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof; Includes:

[0015] PepA (a component of the M72 fusion protein) has been reported to be expressed during the acute phase of Mtb infection. Thus, at least one Mtb antigen from the acute phase of the Mtb life cycle, an immunogenic variant thereof, or an immunogenic fragment of an Mtb antigen or its immunogenic variant comprises (optionally in addition to one or more of the above) an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or its immunogenic variant.

[0016] In some embodiments, at least one Mtb antigen from the latent stage of the Mtb life cycle, an immunogenic variant thereof, or an immunogenic fragment of an Mtb antigen or an immunogenic variant thereof is (i) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof; and / or (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof; Includes:

[0017] In some embodiments, at least one Mtb antigen from the resuscitation phase of the Mtb life cycle, an immunogenic variant thereof, or an immunogenic fragment of an Mtb antigen or an immunogenic variant thereof is (i) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof; and / or (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof; Includes:

[0018] In one aspect, the present disclosure provides a composition or pharmaceutical formulation comprising at least one RNA molecule, wherein the at least one RNA molecule encodes a set of antigenic amino acid sequences, each antigenic amino acid sequence comprising an Mtb antigen, an immunogenic variant thereof, or an immunogenic fragment of an Mtb antigen or an immunogenic variant thereof, and each RNA molecule encodes at least two of the antigenic amino acid sequences as a fusion molecule.

[0019] In some embodiments, each RNA molecule encodes two of the antigen amino acid sequences as a fusion molecule.

[0020] In some embodiments, the Mtb antigens, immunogenic variants, or immunogenic fragments in the fusion molecule are not linked by a linker comprising a sequence heterologous to the Mtb antigen, immunogenic variant, or immunogenic fragment.

[0021] In some embodiments, the set of antigenic amino acid sequences includes two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more, or all of the following: (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof; (vii) an amino acid sequence comprising Mtb32a, an immunogenic variant thereof, or an immunogenic fragment of Mtb32a or an immunogenic variant thereof; (viii) an amino acid sequence comprising Mtb39a, an immunogenic variant thereof, or an immunogenic fragment of Mtb39a or an immunogenic variant thereof; and (ix) An amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof.

[0022] In one aspect, the present disclosure provides a composition or pharmaceutical formulation comprising at least one RNA molecule, wherein the at least one RNA molecule encodes at least one antigen amino acid sequence comprising an Mtb antigen, an immunogenic variant thereof, or an immunogenic fragment of an Mtb antigen or an immunogenic variant thereof, wherein the RNA encodes an amino acid sequence comprising a secretory signal peptide at the N-terminus of the encoded amino acid sequence, wherein the secretory signal peptide is not endogenous to the Mtb antigen. In some embodiments, the secretory signal peptide is of human origin. In some embodiments, the secretory signal peptide is not of human origin.

[0023] In some embodiments, (i) the secretory signal peptide comprises the amino acid sequence of positions 1 to 26 of SEQ ID NO: 44, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 26 of SEQ ID NO: 44, or a functional fragment of the amino acid sequence of positions 1 to 26 of SEQ ID NO: 44 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 26 of SEQ ID NO: 44; and / or (ii) The RNA molecule encoding the secretory signal peptide comprises the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43, or a fragment of the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43.

[0024] In some embodiments, (i) the secretory signal peptide comprises the amino acid sequence of positions 1 to 25 of SEQ ID NO: 63, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 25 of SEQ ID NO: 63, or a functional fragment of the amino acid sequence of positions 1 to 25 of SEQ ID NO: 63 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 25 of SEQ ID NO: 63; and / or (ii) The RNA molecule encoding the secretory signal peptide comprises the nucleotide sequence of positions 1 to 75 of SEQ ID NO:62, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1 to 75 of SEQ ID NO:62, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1 to 75 of SEQ ID NO:62 or the nucleotide sequence of positions 1 to 75 of SEQ ID NO:62.

[0025] In some embodiments, (i) the secretory signal peptide comprises the amino acid sequence of positions 1 to 13 of SEQ ID NO: 71, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 13 of SEQ ID NO: 71, or a functional fragment of the amino acid sequence of positions 1 to 13 of SEQ ID NO: 71 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 13 of SEQ ID NO: 71; and / or (ii) The RNA molecule encoding the secretory signal peptide comprises the nucleotide sequence of positions 1 to 39 of SEQ ID NO:70, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1 to 39 of SEQ ID NO:70, or a fragment of the nucleotide sequence of positions 1 to 39 of SEQ ID NO:70 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1 to 39 of SEQ ID NO:70.

[0026] In one aspect, the present disclosure provides a composition or pharmaceutical formulation comprising at least one RNA molecule, wherein the at least one RNA molecule encodes at least one antigenic amino acid sequence comprising an Mtb antigen, an immunogenic variant thereof, or an immunogenic fragment of an Mtb antigen or an immunogenic variant thereof, wherein the RNA comprises modified uridines and / or is formulated in a lipid nanoparticle.

[0027] In some embodiments, at least one RNA molecule encodes two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more of the following: (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof; (vii) an amino acid sequence comprising Mtb32a, an immunogenic variant thereof, or an immunogenic fragment of Mtb32a or an immunogenic variant thereof; (viii) an amino acid sequence comprising Mtb39a, an immunogenic variant thereof, or an immunogenic fragment of Mtb39a or an immunogenic variant thereof; and (ix) An amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof.

[0028] In some embodiments, at least one RNA molecule encodes the following amino acid sequence: (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof; (vii) an amino acid sequence comprising Mtb32a, an immunogenic variant thereof, or an immunogenic fragment of Mtb32a or an immunogenic variant thereof; (viii) an amino acid sequence comprising Mtb39a, an immunogenic variant thereof, or an immunogenic fragment of Mtb39a or an immunogenic variant thereof; and (ix) An amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof.

[0029] In one aspect, the present disclosure provides a composition or pharmaceutical formulation comprising at least one RNA molecule, wherein the at least one RNA molecule encodes two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the following amino acid sequences: (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof; (vii) an amino acid sequence comprising Mtb32a, an immunogenic variant thereof, or an immunogenic fragment of Mtb32a or an immunogenic variant thereof; (viii) an amino acid sequence comprising Mtb39a, an immunogenic variant thereof, or an immunogenic fragment of Mtb39a or an immunogenic variant thereof; and (ix) An amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof.

[0030] In some embodiments, the amino acid sequence comprising Mtb32a, an immunogenic variant thereof, or an immunogenic fragment of Mtb32a or an immunogenic variant thereof and the amino acid sequence comprising Mtb39a, an immunogenic variant thereof, or an immunogenic fragment of Mtb39a or an immunogenic variant thereof exist as a fusion protein.

[0031] In some embodiments, the fusion protein comprises an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof.

[0032] In some embodiments, at least one RNA molecule encodes two or more, three or more, four or more, five or more, six or more, seven or more, or all of the following amino acid sequences: (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of said Hrp1 or its immunogenic variant; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or an immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or its immunogenic variant; (vii) an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof; and (viii) an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof.

[0033] In one aspect, the present disclosure provides a composition or pharmaceutical formulation comprising at least one RNA molecule, wherein the at least one RNA molecule encodes two or more, three or more, four or more, five or more, six or more, seven or more, or all of the following amino acid sequences: (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of said Hrp1 or its immunogenic variant; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or an immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or its immunogenic variant; (vii) an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof; and (viii) an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof.

[0034] In some embodiments, (i) the amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof comprises the amino acid sequence of positions 2 to 298 of SEQ ID NO: 20, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 298 of SEQ ID NO: 20, or an immunogenic fragment of the amino acid sequence of positions 2 to 298 of SEQ ID NO: 20 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 298 of SEQ ID NO: 20; and / or (ii) An RNA sequence encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof comprises the nucleotide sequence of positions 4 to 894 of SEQ ID NO: 19, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 894 of SEQ ID NO: 19, or a fragment of the nucleotide sequence of positions 4 to 894 of SEQ ID NO: 19 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 894 of SEQ ID NO: 19.

[0035] In some embodiments, (i) the amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof comprises the amino acid sequence of positions 27 to 323 of SEQ ID NO: 44, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 323 of SEQ ID NO: 44, or an immunogenic fragment of the amino acid sequence of positions 27 to 323 of SEQ ID NO: 44 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 323 of SEQ ID NO: 44; and / or (ii) An RNA sequence encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 1022 of SEQ ID NO:43, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1022 of SEQ ID NO:43, or a fragment of the nucleotide sequence of positions 132 to 1022 of SEQ ID NO:43 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1022 of SEQ ID NO:43.

[0036] In some embodiments, (i) the amino acid sequence comprising ESAT6, its immunogenic variant, or an immunogenic fragment of ESAT6 or its immunogenic variant comprises the amino acid sequence of positions 2 to 95 of SEQ ID NO: 4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 95 of SEQ ID NO: 4, or an immunogenic fragment of the amino acid sequence of positions 2 to 95 of SEQ ID NO: 4 or the amino acid sequence of positions 2 to 95 of SEQ ID NO: 4; and / or (ii) An RNA sequence encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof comprises the nucleotide sequence of positions 4 to 285 of SEQ ID NO:21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 285 of SEQ ID NO:21, or a fragment of the nucleotide sequence of positions 4 to 285 of SEQ ID NO:21 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 285 of SEQ ID NO:21.

[0037] In some embodiments, (i) the amino acid sequence comprising ESAT6, its immunogenic variant, or an immunogenic fragment of ESAT6 or its immunogenic variant comprises the amino acid sequence of positions 27 to 120 of SEQ ID NO: 46, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 120 of SEQ ID NO: 46, or an immunogenic fragment of the amino acid sequence of positions 27 to 120 of SEQ ID NO: 46 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 120 of SEQ ID NO: 46; and / or (ii) An RNA sequence encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 413 of SEQ ID NO: 45, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 413 of SEQ ID NO: 45, or a fragment of the nucleotide sequence of positions 132 to 413 of SEQ ID NO: 45 or the nucleotide sequence of positions 132 to 413 of SEQ ID NO: 45.

[0038] In some embodiments, (i) the amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the amino acid sequence of positions 2 to 99 of SEQ ID NO: 6, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 99 of SEQ ID NO: 6, or an immunogenic fragment of the amino acid sequence of positions 2 to 99 of SEQ ID NO: 6 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 99 of SEQ ID NO: 6; and / or (ii) An RNA sequence encoding an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the nucleotide sequence of positions 4 to 297 of SEQ ID NO:22, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 297 of SEQ ID NO:22, or a fragment of the nucleotide sequence of positions 4 to 297 of SEQ ID NO:22 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 297 of SEQ ID NO:22.

[0039] In some embodiments, (i) the amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the amino acid sequence of positions 749 to 846 of SEQ ID NO: 52, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 749 to 846 of SEQ ID NO: 52, or an immunogenic fragment of the amino acid sequence of positions 749 to 846 of SEQ ID NO: 52 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 749 to 846 of SEQ ID NO: 52; and / or (ii) An RNA sequence encoding an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the nucleotide sequence of positions 2298 to 2591 of SEQ ID NO:51, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 2298 to 2591 of SEQ ID NO:51, or a fragment of the nucleotide sequence of positions 2298 to 2591 of SEQ ID NO:51 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 2298 to 2591 of SEQ ID NO:51.

[0040] In some embodiments, (i) the amino acid sequence comprising Hrp1, its immunogenic variant, or an immunogenic fragment of Hrp1 or its immunogenic variant comprises the amino acid sequence of positions 2 to 143 of SEQ ID NO: 8, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 143 of SEQ ID NO: 8, or an immunogenic fragment of the amino acid sequence of positions 2 to 143 of SEQ ID NO: 8 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 143 of SEQ ID NO: 8; and / or (ii) An RNA sequence encoding an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the nucleotide sequence of positions 4 to 429 of SEQ ID NO:23, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 429 of SEQ ID NO:23, or a fragment of the nucleotide sequence of positions 4 to 429 of SEQ ID NO:23 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 429 of SEQ ID NO:23.

[0041] In some embodiments, (i) the amino acid sequence comprising Hrp1, its immunogenic variant, or an immunogenic fragment of Hrp1 or its immunogenic variant comprises the amino acid sequence of positions 324 to 465 of SEQ ID NO: 44, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 324 to 465 of SEQ ID NO: 44, or an immunogenic fragment of the amino acid sequence of positions 324 to 465 of SEQ ID NO: 44 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 324 to 465 of SEQ ID NO: 44; and / or (ii) An RNA sequence encoding an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the nucleotide sequence of positions 1023 to 1448 of SEQ ID NO: 43, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1023 to 1448 of SEQ ID NO: 43, or a fragment of the nucleotide sequence of positions 1023 to 1448 of SEQ ID NO: 43 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1023 to 1448 of SEQ ID NO: 43.

[0042] In some embodiments, (i) the amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof comprises the amino acid sequence of positions 2 to 407 of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 2 to 407 of SEQ ID NO: 10, or an immunogenic fragment of the amino acid sequence of positions 2 to 407 of SEQ ID NO: 10 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 2 to 407 of SEQ ID NO: 10; and / or (ii) An RNA sequence encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof comprises the nucleotide sequence of positions 4 to 1221 of SEQ ID NO:24, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 1221 of SEQ ID NO:24, or a fragment of the nucleotide sequence of positions 4 to 1221 of SEQ ID NO:24 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 1221 of SEQ ID NO:24.

[0043] In some embodiments, (i) the amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof comprises the amino acid sequence of positions 27 to 432 of SEQ ID NO: 48, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 432 of SEQ ID NO: 48, or an immunogenic fragment of the amino acid sequence of positions 27 to 432 of SEQ ID NO: 48 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 432 of SEQ ID NO: 48; and / or (ii) An RNA sequence encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 1349 of SEQ ID NO: 47, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1349 of SEQ ID NO: 47, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1349 of SEQ ID NO: 47 or the nucleotide sequence of positions 132 to 1349 of SEQ ID NO: 47.

[0044] In some embodiments, (i) the amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the amino acid sequence of positions 2 to 154 of SEQ ID NO: 12, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 154 of SEQ ID NO: 12, or an immunogenic fragment of the amino acid sequence of positions 2 to 154 of SEQ ID NO: 12 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 154 of SEQ ID NO: 12; and / or (ii) An RNA sequence encoding an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the nucleotide sequence of positions 4 to 462 of SEQ ID NO:25, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 462 of SEQ ID NO:25, or a fragment of the nucleotide sequence of positions 4 to 462 of SEQ ID NO:25 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 462 of SEQ ID NO:25.

[0045] In some embodiments, (i) the amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the amino acid sequence of positions 121 to 273 of SEQ ID NO: 46, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 121 to 273 of SEQ ID NO: 46, or an immunogenic fragment of the amino acid sequence of positions 121 to 273 of SEQ ID NO: 46 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 121 to 273 of SEQ ID NO: 46; and / or (ii) An RNA sequence encoding an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the nucleotide sequence of positions 414 to 872 of SEQ ID NO: 45, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 414 to 872 of SEQ ID NO: 45, or a fragment of the nucleotide sequence of positions 414 to 872 of SEQ ID NO: 45 or the nucleotide sequence of positions 414 to 872 of SEQ ID NO: 45.

[0046] In some embodiments, (i) the amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof comprises the amino acid sequence of positions 2 to 723 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 723 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of positions 2 to 723 of SEQ ID NO:29 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 723 of SEQ ID NO:29; and / or (ii) An RNA sequence encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof comprises the nucleotide sequence of positions 4 to 2169 of SEQ ID NO:28, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 2169 of SEQ ID NO:28, or a fragment of the nucleotide sequence of positions 4 to 2169 of SEQ ID NO:28 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 2169 of SEQ ID NO:28.

[0047] In some embodiments, (i) the amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof comprises the amino acid sequence of positions 27 to 748 of SEQ ID NO: 52, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 748 of SEQ ID NO: 52, or an immunogenic fragment of the amino acid sequence of positions 27 to 748 of SEQ ID NO: 52 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 748 of SEQ ID NO: 52; and / or (ii) An RNA sequence encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 2297 of SEQ ID NO:51, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 2297 of SEQ ID NO:51, or a fragment of the nucleotide sequence of positions 132 to 2297 of SEQ ID NO:51 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 2297 of SEQ ID NO:51.

[0048] In some embodiments, (i) the amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the amino acid sequence of positions 2 to 199 of SEQ ID NO: 18, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 199 of SEQ ID NO: 18, or an immunogenic fragment of the amino acid sequence of positions 2 to 199 of SEQ ID NO: 18 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 2 to 199 of SEQ ID NO: 18; and / or (ii) An RNA sequence encoding an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the nucleotide sequence of positions 4 to 597 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 597 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of positions 4 to 597 of SEQ ID NO: 30 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 597 of SEQ ID NO: 30.

[0049] In some embodiments, (i) the amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the amino acid sequence of positions 433 to 630 of SEQ ID NO: 48, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 433 to 630 of SEQ ID NO: 48, or an immunogenic fragment of the amino acid sequence of positions 433 to 630 of SEQ ID NO: 48 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 433 to 630 of SEQ ID NO: 48; and / or (ii) An RNA sequence encoding an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the nucleotide sequence of positions 1350 to 1943 of SEQ ID NO: 47, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1350 to 1943 of SEQ ID NO: 47, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1350 to 1943 of SEQ ID NO: 47 or the nucleotide sequence of positions 1350 to 1943 of SEQ ID NO: 47.

[0050] In some embodiments, the composition or pharmaceutical formulation comprises: (i) an RNA molecule encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or its immunogenic variant, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or its immunogenic variant; (ii) an RNA molecule encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or its immunogenic variant, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or its immunogenic variant; (iii) an RNA molecule encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof; and (iv) an RNA molecule encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof. Includes:

[0051] In some embodiments, (i) the amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the amino acid sequence of positions 27 to 465 of SEQ ID NO: 44, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 465 of SEQ ID NO: 44; and / or (ii) An RNA sequence encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof, comprises the nucleotide sequence of positions 132 to 1448 of SEQ ID NO: 43, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1448 of SEQ ID NO: 43.

[0052] In some embodiments, (i) the amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the amino acid sequence of positions 27 to 273 of SEQ ID NO: 46, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 273 of SEQ ID NO: 46; and / or (ii) An RNA sequence encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 872 of SEQ ID NO: 45, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 872 of SEQ ID NO: 45.

[0053] In some embodiments, (i) the amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the amino acid sequence of positions 27 to 630 of SEQ ID NO: 48, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 27 to 630 of SEQ ID NO: 48; and / or (ii) RNA sequences encoding amino acid sequences comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof, comprise the nucleotide sequence of positions 132 to 1943 of SEQ ID NO: 47, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1943 of SEQ ID NO: 47.

[0054] In some embodiments, (i) the amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the amino acid sequence of positions 27 to 846 of SEQ ID NO: 52, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 846 of SEQ ID NO: 52; and / or (ii) An RNA sequence encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, comprises the nucleotide sequence of positions 132 to 2591 of SEQ ID NO:51, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 2591 of SEQ ID NO:51.

[0055] In some embodiments, (i) the amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 44; and / or (ii)(a) the RNA sequence encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof, comprises the nucleotide sequence of positions 54 to 1448 of SEQ ID NO: 43, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 1448 of SEQ ID NO: 43; or (b) An RNA sequence encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:43 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:43.

[0056] In some embodiments, (i) the amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 46; and / or (ii)(a) the RNA sequence encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the nucleotide sequence of positions 54 to 872 of SEQ ID NO: 45, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 872 of SEQ ID NO: 45; or (b) The RNA sequence encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:45 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:45.

[0057] In some embodiments, (i) the amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 48; and / or (ii)(a) the RNA sequence encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the nucleotide sequence of positions 54 to 1943 of SEQ ID NO: 47, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 1943 of SEQ ID NO: 47; or (b) The RNA sequence encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:47 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:47.

[0058] In some embodiments, (i) the amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:52 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO:52; and / or (ii)(a) an RNA sequence encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, comprises the nucleotide sequence of positions 54 to 2591 of SEQ ID NO: 51, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 2591 of SEQ ID NO: 51; or (b) RNA sequences encoding amino acid sequences comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, comprise the nucleotide sequence of SEQ ID NO:51 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:51.

[0059] In some embodiments, (i) the amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 63; and / or (ii) The RNA sequence encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:62 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:62.

[0060] In some embodiments, (i) the amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 65; and / or (ii) The RNA sequence encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:64 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:64.

[0061] In some embodiments, (i) the amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 67; and / or (ii) The RNA sequence encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:66 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:66.

[0062] In some embodiments, (i) the amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 69; and / or (ii) The RNA sequence encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:68 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:68.

[0063] In some embodiments, (i) the amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 71; and / or (ii) The RNA sequence encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:70 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:70.

[0064] In some embodiments, (i) the amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 73; and / or (ii) The RNA sequence encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO: 72 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 72.

[0065] In some embodiments, (i) the amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 75 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 75; and / or (ii) The RNA sequence encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:74 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:74.

[0066] In some embodiments, (i) the amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 77 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 77; and / or (ii) RNA sequences encoding amino acid sequences comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, comprise the nucleotide sequence of SEQ ID NO:76 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:76.

[0067] In some embodiments, the RNA encodes an amino acid sequence that includes a secretory signal peptide.

[0068] In some embodiments, the secretory signal peptide is fused to the amino acid sequence, preferably to the N-terminus.

[0069] In some embodiments, the secretory signal peptide is not endogenous to the Mtb antigen. In some embodiments, the secretory signal peptide is of human origin. In some embodiments, the secretory signal peptide is not of human origin.

[0070] In some embodiments, (i) the secretory signal peptide comprises the amino acid sequence of positions 1 to 26 of SEQ ID NO: 44, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 26 of SEQ ID NO: 44, or a functional fragment of the amino acid sequence of positions 1 to 26 of SEQ ID NO: 44 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 26 of SEQ ID NO: 44; and / or (ii) The RNA molecule encoding the secretory signal peptide comprises the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43, or a fragment of the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43.

[0071] In some embodiments, (i) the secretory signal peptide comprises the amino acid sequence of positions 1 to 25 of SEQ ID NO: 63, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 25 of SEQ ID NO: 63, or a functional fragment of the amino acid sequence of positions 1 to 25 of SEQ ID NO: 63 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 25 of SEQ ID NO: 63; and / or (ii) The RNA molecule encoding the secretory signal peptide comprises the nucleotide sequence of positions 1 to 75 of SEQ ID NO:62, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1 to 75 of SEQ ID NO:62, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1 to 75 of SEQ ID NO:62 or the nucleotide sequence of positions 1 to 75 of SEQ ID NO:62.

[0072] In some embodiments, (i) the secretory signal peptide comprises the amino acid sequence of positions 1 to 13 of SEQ ID NO: 71, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 13 of SEQ ID NO: 71, or a functional fragment of the amino acid sequence of positions 1 to 13 of SEQ ID NO: 71 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 1 to 13 of SEQ ID NO: 71; and / or (ii) The RNA molecule encoding the secretory signal peptide comprises the nucleotide sequence of positions 1 to 39 of SEQ ID NO:70, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1 to 39 of SEQ ID NO:70, or a fragment of the nucleotide sequence of positions 1 to 39 of SEQ ID NO:70 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1 to 39 of SEQ ID NO:70.

[0073] In some embodiments, the composition or pharmaceutical formulation comprises: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 43, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 43; (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 45, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 45; (iii) an RNA comprising the nucleotide sequence of SEQ ID NO: 47, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 47; and (iv) RNA comprising the nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 51. Includes:

[0074] In some embodiments, the composition or pharmaceutical formulation comprises: (i) RNA comprising the nucleotide sequence of SEQ ID NO: 43; (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 45; (iii) RNA comprising the nucleotide sequence of SEQ ID NO: 47; and (iv) RNA comprising the nucleotide sequence of SEQ ID NO: 51 Includes:

[0075] In some embodiments, the composition or pharmaceutical formulation comprises: (i) an RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 44; (ii) an RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 46; (iii) an RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 48; and (iv) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 52 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 52. Includes.

[0076] In some embodiments, the composition or pharmaceutical formulation comprises: (i) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 44; (ii) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 46; (iii) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 48; and (iv) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 52 Includes.

[0077] In some embodiments, the composition or pharmaceutical formulation comprises: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 62, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 62; (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 64, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 64; (iii) an RNA comprising the nucleotide sequence of SEQ ID NO: 66, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 66; and (iv) RNA comprising the nucleotide sequence of SEQ ID NO: 68 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 68. Includes.

[0078] In some embodiments, the composition or pharmaceutical formulation comprises: (i) RNA comprising the nucleotide sequence of SEQ ID NO: 62; (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 64; (iii) RNA comprising the nucleotide sequence of SEQ ID NO: 66; and (iv) RNA comprising the nucleotide sequence of SEQ ID NO: 68 Includes.

[0079] In some embodiments, the composition or pharmaceutical formulation comprises: (i) an RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 63; (ii) an RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 65; (iii) an RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 67; and (iv) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 69. Includes.

[0080] In some embodiments, the composition or pharmaceutical formulation comprises: (i) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 63; (ii) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 65; (iii) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 67; and (iv) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 69 Includes:

[0081] In some embodiments, the composition or pharmaceutical formulation comprises: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 70, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 70; (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 72, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 72; (iii) an RNA comprising the nucleotide sequence of SEQ ID NO: 74, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 74; and (iv) RNA comprising the nucleotide sequence of SEQ ID NO: 76 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 76. Includes:

[0082] In some embodiments, the composition or pharmaceutical formulation comprises: (i) RNA comprising the nucleotide sequence of SEQ ID NO: 70; (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 72; (iii) RNA comprising the nucleotide sequence of SEQ ID NO: 74; and (iv) RNA comprising the nucleotide sequence of SEQ ID NO: 76 Includes.

[0083] In some embodiments, the composition or pharmaceutical formulation comprises: (i) an RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 71; (ii) an RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 73; (iii) an RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 75 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 75; and (iv) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 77 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 77. Includes.

[0084] In some embodiments, the composition or pharmaceutical formulation comprises: (i) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 71; (ii) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 73; (iii) an RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 75; and (iv) RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 77 Includes.

[0085] In some embodiments, the RNA is formulated in lipid nanoparticles.

[0086] In some embodiments, the lipid nanoparticles comprise: (i) cationic ionizable lipids; (ii) steroids; (iii) neutral lipids; and (iv) Polymer-conjugated lipids Each of the following is included.

[0087] In some embodiments, the cationic ionizable lipid is present at a concentration ranging from about 40 to about 60 mol % of the total lipid.

[0088] In some embodiments, the steroid is present at a concentration ranging from about 30 to about 50 mol % of the total lipid.

[0089] In some embodiments, the neutral lipid is present at a concentration ranging from about 5 to about 15 mol % of the total lipid.

[0090] In some embodiments, the polymer-conjugated lipid is present at a concentration ranging from about 1 to about 10 mol % of the total lipid.

[0091] In some embodiments, the cationic ionizable lipid is in the range of about 40 to about 60 mol%, the steroid is in the range of about 30 to about 50 mol%, the neutral lipid is in the range of about 5 to about 15 mol%, and the polymer-conjugated lipid is in the range of about 1 to about 10 mol%.

[0092] In some embodiments, the cationic ionizable lipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0093] In some embodiments, the steroid is or comprises cholesterol.

[0094] In some embodiments, the neutral lipid is or comprises a phospholipid.

[0095] In some embodiments, the phospholipid is or comprises distearoylphosphatidylcholine (DSPC).

[0096] In some embodiments, the polymer-conjugated lipid is or comprises a polyethylene glycol (PEG) lipid.

[0097] In some embodiments, the PEG lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

[0098] In some embodiments, the lipid nanoparticles comprise: (a) ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); (b) cholesterol; (c) distearoylphosphatidylcholine (DSPC); and (d) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide Includes:

[0099] In some embodiments, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) is in the range of about 40 to about 60 mol %, cholesterol is in the range of about 30 to about 50 mol %, distearoylphosphatidylcholine (DSPC) is in the range of about 5 to about 15 mol %, and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide is in the range of about 1 to about 10 mol %.

[0100] In some embodiments, the RNA comprises a 5' cap.

[0101] In some embodiments, the 5' cap is or comprises a Cap1 structure.

[0102] In some embodiments, the 5' cap is m2 7,3’ -OGppp(m1 2’-O ) ApG or containing it.

[0103] In some embodiments, the RNA comprises a 5'-UTR.

[0104] In some embodiments, the 5'-UTR is or comprises a 5'-UTR of modified human alpha-globin.

[0105] In some embodiments, the 5'-UTR is or comprises the nucleotide sequence of SEQ ID NO:56, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:56.

[0106] In some embodiments, the 5'-UTR is or comprises the nucleotide sequence of SEQ ID NO:57 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:57.

[0107] In some embodiments, the RNA comprises a 3'-UTR.

[0108] In some embodiments, the 3'-UTR is or comprises a first sequence derived from a split amino-terminal enhancer (AES) messenger RNA and a second sequence derived from a mitochondrially encoded 12S ribosomal RNA.

[0109] In some embodiments, the 3'-UTR is or comprises the nucleotide sequence of SEQ ID NO:58 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:58.

[0110] In some embodiments, the RNA comprises a polyA sequence.

[0111] In some embodiments, the polyA sequence is an interrupted sequence of A nucleotides.

[0112] In some embodiments, the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence of 10 nucleotides.

[0113] In some embodiments, the polyA sequence is or comprises the nucleotide sequence of SEQ ID NO:59 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:59.

[0114] In some embodiments, the sequence downstream of the open reading frame, i.e., the 3'-UTR and polyA sequence, is or comprises the nucleotide sequence of SEQ ID NO:60 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:60.

[0115] In some embodiments, the RNA comprises a 5' cap, a 5'-UTR, a 3'-UTR, and a polyA sequence.

[0116] In some embodiments, the RNA comprises a modified uridine.

[0117] In some embodiments, the RNA comprises modified uridines in place of every uridine.

[0118] In some embodiments, the modified uridine is N1-methyl-pseudouridine.

[0119] In some embodiments, the coding sequence of the RNA is codon-optimized and / or characterized by its increased G / C content compared to the parent sequence.

[0120] In some embodiments, the RNA is present in a liquid formulation.

[0121] In some embodiments, the RNA is present in a frozen formulation.

[0122] In some embodiments, the RNA is present in a lyophilized formulation.

[0123] In some embodiments, the RNA is formulated for injection.

[0124] In some embodiments, the RNA is formulated for intramuscular administration.

[0125] In some embodiments, the composition or pharmaceutical formulation is a pharmaceutical composition.

[0126] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0127] In some embodiments, the composition or pharmaceutical preparation is a vaccine.

[0128] In some embodiments, the composition or pharmaceutical preparation is a kit.

[0129] In some embodiments, the different RNA molecules are present in separate vials.

[0130] In some embodiments, the composition or pharmaceutical preparation further comprises instructions for using the composition or pharmaceutical preparation to treat or prevent tuberculosis.

[0131] In some embodiments, the composition or pharmaceutical formulation is for pharmaceutical use.

[0132] In some embodiments, the medical use includes the therapeutic or prophylactic treatment of a disease or disorder.

[0133] In some embodiments, therapeutic or prophylactic treatment of a disease or disorder includes treating or preventing tuberculosis.

[0134] In some embodiments, the composition or pharmaceutical preparation is for administration to a human.

[0135] In one aspect, the present disclosure provides a method of vaccinating a subject, comprising administering to the subject a composition described herein.

[0136] In some embodiments, the vaccination is to prevent tuberculosis.

[0137] In some embodiments, administration is by intramuscular administration.

[0138] In some embodiments, the method comprises administering at least one dose of the composition to the subject.

[0139] In some embodiments, the method comprises administering at least two doses of the composition to the subject.

[0140] In some embodiments, an amount of RNA of at least 10 μg / dose is administered.

[0141] In some embodiments, the subject is a human.

[0142] In a further aspect, the present disclosure provides: A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and comprising the nucleotide sequence of positions 4 to 894 of SEQ ID NO: 19, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 894 of SEQ ID NO: 19, or a fragment of the nucleotide sequence of positions 4 to 894 of SEQ ID NO: 19 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 894 of SEQ ID NO: 19.

[0143] A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and comprising the nucleotide sequence of positions 4 to 285 of SEQ ID NO: 21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 285 of SEQ ID NO: 21, or a fragment of the nucleotide sequence of positions 4 to 285 of SEQ ID NO: 21 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 285 of SEQ ID NO: 21.

[0144] A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, the amino acid sequence comprising the nucleotide sequence of positions 4 to 297 of SEQ ID NO:22, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 297 of SEQ ID NO:22, or a fragment of the nucleotide sequence of positions 4 to 297 of SEQ ID NO:22 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 297 of SEQ ID NO:22.

[0145] A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof, the amino acid sequence comprising the nucleotide sequence of positions 4 to 429 of SEQ ID NO:23, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 429 of SEQ ID NO:23, or a fragment of the nucleotide sequence of positions 4 to 429 of SEQ ID NO:23 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 429 of SEQ ID NO:23.

[0146] A nucleic acid, such as RNA, encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and comprising the nucleotide sequence of positions 4 to 1221 of SEQ ID NO:24, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 1221 of SEQ ID NO:24, or a fragment of the nucleotide sequence of positions 4 to 1221 of SEQ ID NO:24 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 1221 of SEQ ID NO:24.

[0147] A nucleic acid, such as RNA, encoding an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof, and comprising the nucleotide sequence of positions 4 to 462 of SEQ ID NO: 25, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 462 of SEQ ID NO: 25, or a fragment of the nucleotide sequence of positions 4 to 462 of SEQ ID NO: 25 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 462 of SEQ ID NO: 25.

[0148] A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, the amino acid sequence comprising the nucleotide sequence of positions 4 to 2169 of SEQ ID NO:28, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 2169 of SEQ ID NO:28, or a fragment of the nucleotide sequence of positions 4 to 2169 of SEQ ID NO:28 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 2169 of SEQ ID NO:28.

[0149] A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof, and comprising the nucleotide sequence of positions 4 to 597 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 597 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of positions 4 to 597 of SEQ ID NO: 30 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 4 to 597 of SEQ ID NO: 30.

[0150] A polypeptide comprising an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof.

[0151] A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or its immunogenic variant, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or its immunogenic variant.

[0152] A polypeptide comprising an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof.

[0153] A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof.

[0154] A polypeptide comprising an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof.

[0155] Nucleic acids, e.g., RNA, encoding amino acid sequences comprising RpfA, its immunogenic variants, or immunogenic fragments of RpfA or its immunogenic variants, and HbhA, its immunogenic variants, or immunogenic fragments of HbhA or its immunogenic variants.

[0156] A polypeptide comprising an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof.

[0157] Nucleic acids, e.g., RNA, encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof.

[0158] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the amino acid sequence of positions 27 to 465 of SEQ ID NO: 44, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 465 of SEQ ID NO: 44; and / or (ii) A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof, comprises the nucleotide sequence of positions 132 to 1448 of SEQ ID NO: 43, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1448 of SEQ ID NO: 43.

[0159] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the amino acid sequence of positions 27 to 273 of SEQ ID NO: 46, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 273 of SEQ ID NO: 46; and / or (ii) A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof, comprises the nucleotide sequence of positions 132 to 872 of SEQ ID NO: 45, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 872 of SEQ ID NO: 45.

[0160] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the amino acid sequence of positions 27 to 630 of SEQ ID NO: 48, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 27 to 630 of SEQ ID NO: 48; and / or (ii) Nucleic acids, e.g., RNAs, encoding amino acid sequences comprising RpfA, its immunogenic variants, or immunogenic fragments of RpfA or its immunogenic variants, and HbhA, its immunogenic variants, or immunogenic fragments of HbhA or its immunogenic variants, comprise the nucleotide sequence of positions 132 to 1943 of SEQ ID NO: 47, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1943 of SEQ ID NO: 47.

[0161] In some embodiments, (i) a polypeptide comprising an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, comprising the amino acid sequence of positions 27 to 846 of SEQ ID NO: 52, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 846 of SEQ ID NO: 52; and / or (ii) A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, comprises the nucleotide sequence of positions 132 to 2591 of SEQ ID NO:51, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 2591 of SEQ ID NO:51.

[0162] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:44 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:44; and / or (ii)(a) a nucleic acid, e.g., RNA, encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof, comprises the nucleotide sequence of positions 54 to 1448 of SEQ ID NO: 43, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 1448 of SEQ ID NO: 43; or (b) A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:43 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:43.

[0163] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 46; and / or (ii) (a) the nucleic acid, e.g., RNA, encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof, comprises the nucleotide sequence of positions 54 to 872 of SEQ ID NO: 45, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 872 of SEQ ID NO: 45; or (b) A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:45 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:45.

[0164] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 48; and / or (ii)(a) a nucleic acid, e.g., RNA, encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof, comprises the nucleotide sequence of positions 54 to 1943 of SEQ ID NO: 47, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 1943 of SEQ ID NO: 47; or (b) The nucleic acid, e.g., RNA, encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:47 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:47.

[0165] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:52 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO:52; and / or (ii)(a) a nucleic acid, e.g., RNA, encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, comprises the nucleotide sequence of positions 54 to 2591 of SEQ ID NO: 51, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 2591 of SEQ ID NO: 51; or (b) A nucleic acid, e.g., RNA, encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:51 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:51.

[0166] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 63; and / or (ii) The nucleic acid, e.g., RNA, encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:62 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:62.

[0167] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 65; and / or (ii) The nucleic acid, e.g., RNA, encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:64 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:64.

[0168] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 67; and / or (ii) The nucleic acid, e.g., RNA, encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:66 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:66.

[0169] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:69 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO:69; and / or (ii) The nucleic acid, e.g., RNA, encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:68 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:68.

[0170] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 71; and / or (ii) The nucleic acid, e.g., RNA, encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or an immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:70 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:70.

[0171] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 73; and / or (ii) The nucleic acid, e.g., RNA, encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or an immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO: 72 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 72.

[0172] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 75 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 75; and / or (ii) The nucleic acid, e.g., RNA, encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or an immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:74 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:74.

[0173] In some embodiments, (i) the polypeptide comprising an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 77 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 77; and / or (ii) The nucleic acid, e.g., RNA, encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or an immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or an immunogenic variant thereof, comprises the nucleotide sequence of SEQ ID NO:76 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:76. [Brief explanation of the drawings]

[0174] [Figure 1]RNA mixes of four modRNAs encoding four (RNA mix 1), six (RNA mix 2), or eight (RNA mix 3) Mtb antigens. Top: General mRNA construct structure with 5' cap, 5' and 3' untranslated regions (UTRs), open reading frame (ORF), and polyadenosine tail. Bottom: RNA mixes 1–3 containing the four to eight Mtb antigen sequences tested. All antigen-coding sequences were fused at the N-terminus to a major histocompatibility complex (MHC) class I signal peptide fragment (sec), which mediates translocation to the endoplasmic reticulum. A) The modRNA constructs in RNA mix 1 separately encode the Mycobacterium tuberculosis (Mtb) antigens Ag85A(Δ1-41), M72, ESAT6, and HbhA. B) The modRNA constructs in RNA mix 2 encode antigens Ag85A(Δ1-41) and M72 separately, as well as two fusion antigens: Hrp1-ESAT6 and RpfD-HbhA. C) The modRNA constructs in RNA mix 3 encode four fusion antigens: ESAT6-RpfD, Ag85A(Δ1-41)-Hrp1, RpfA-HbhA, and M72-vapB47. The modRNA mixture was formulated in lipid nanoparticles 315 (LNP-315). UTR: untranslated region; poly(A): polyadenosine tail. [Figure 2] Immunization schedule for in vivo mouse immunogenicity studies. A) Mice received two intramuscular (im) injections (days 0 and 21). B) Mice received three intravenous (iv) injections (days 0, 7, and 21). Arrowheads indicate the days of injection. Blood samples (indicated by arrows) were collected for serum analysis of antigen-specific IgG antibodies 14, 28, and 42 days after the first injection. On the final day of the experiment (day 42), mouse spleens were dissected to isolate splenocytes for subsequent analysis of T cell responses to antigen-specific peptides. [Figure 3]Scheme of the mRNA constructs used to determine the most immunogenic mRNA platform and Mtb antigen. Top: General mRNA construct structure with 5' cap, 5' and 3' untranslated regions (UTRs), open reading frame (ORF), and polyadenosine tail. Bottom: A-E) Antigen-encoding mRNA constructs contained within the ORF. All antigen-encoding sequences were fused to an N-terminal major histocompatibility complex (MHC) class I signal peptide fragment (sec), which mediates translocation to the endoplasmic reticulum. One construct (B) contained a C-terminal MHC class I transmembrane and cytoplasmic domain (MITD), a cellular transport signal for cell membrane anchoring. M. tuberculosis antigens tested: Ag85A, ESAT6, HbhA, Hrp1, M72, RpfA, RpfD, and vapB47. [Figure 4]PPD stimulation of splenocytes demonstrated better immune response induction in mice immunized with pseudouridine-modified mRNA compared with unmodified and self-replicating mRNA. C57BL / 6 mice (5 animals per group) were immunized with the indicated mix of mRNA / mRNA constructs (6-antigen cassette, 6-antigen cassette with MITD, 6-antigen mix, 2-antigen mix, 6-antigen cassette + 2 antigens) as unmodified mRNA (uRNA), pseudouridine-modified mRNA (modRNA), or self-amplifying mRNA (saRNA). uRNA was formulated in lipoplexes, while modRNA and saRNA were formulated in C12 lipid nanoparticles (LNP-C12). Mice were injected intravenously with 20 μg (100 μL dose volume) of uRNA three times (days 0, 7, and 21) or intramuscularly with 4 μg (20 μL dose volume) of modRNA or saRNA twice (days 0 and 21). Mice in the reference group were injected subcutaneously (sc) once on day 0 with 10 colony-forming units (CFU; 100 μL dose volume) of bacillus Calmette-Guérin (BCG). Mice in the control group were injected intramuscularly with 20 μL of phosphate-buffered saline (buffer). On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 hours) with 10 μg / mL purified protein derivative (PPD), and interferon gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate, and bars represent group mean spot-forming units (SFU) ± SD. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test at α = 0.05. *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001, MITD: MHC class I transmembrane and cytoplasmic domain, modRNA: nucleoside-modified mRNA, saRNA: self-amplifying mRNA, uRNA: unmodified mRNA, SD: standard deviation, SFU: spot-forming unit. [Figure 5A]Immunogenicity induced by a six-antigen cassette using three mRNA platforms. C57BL / 6 mice (five animals per group) were immunized with a six-antigen cassette mRNA construct (encoding the Mycobacterium tuberculosis antigens Ag85A, ESAT6, vapB47, Hrp1, RpfA, and RpfD) as unmodified mRNA (uRNA), pseudouridine-modified mRNA (modRNA), or self-amplifying mRNA (saRNA). uRNA was formulated in lipoplexes, while modRNA and saRNA were formulated in C12 lipid nanoparticles (LNP-C12). Mice were injected intravenously with 20 μg of uRNA (100 μL dose volume) three times (days 0, 7, and 21) or intramuscularly with 4 μg of modRNA or saRNA (20 μL dose volume) twice (days 0 and 21). Control mice were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the entire length of each antigen encoded in the construct or the nonspecific peptide TRP1, and interferon gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled samples were measured in triplicate; bars represent group mean spot-forming units (SFU) ± SD. [Figure 5C]C) Blood samples were collected from mice on days 14, 28, and 42 after the first immunization to obtain serum. Antigen-specific IgG was measured in serum samples (1:100 dilution) by ELISA. Results are shown as ΔOD. Samples were measured in duplicate; bars represent group means ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm; MITD: MHC class I transmembrane and cytoplasmic domain; modRNA: nucleoside-modified mRNA; saRNA: self-amplifying mRNA; uRNA: unmodified mRNA; SD: standard deviation; SFU: spot-forming unit. [Figure 6A]Immunogenicity induced by a six-antigen cassette with MITD using three mRNA platforms. C57BL / 6 mice (five animals per group) were immunized with a six-antigen cassette mRNA construct (encoding Mycobacterium tuberculosis antigens Ag85A, ESAT6, vapB47, Hrp1, RpfA, and RpfD) containing the C-terminal MHC class I transmembrane and cytoplasmic domain (MITD) as unmodified mRNA (uRNA), pseudouridine-modified mRNA (modRNA), or self-amplifying mRNA (saRNA). uRNA was formulated in lipoplexes, while modRNA and saRNA were formulated in C12 lipid nanoparticles (LNP-C12). Mice were injected intravenously with 20 μg of uRNA (100 μL dose volume) three times (days 0, 7, and 21) or intramuscularly with 4 μg of modRNA or saRNA (20 μL dose volume) twice (days 0 and 21). Control mice were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the entire length of each antigen encoded in the construct or the nonspecific peptide TRP1, and interferon gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent group mean spot-forming units (SFU) ± SD. [Figure 6C]C) Blood samples were collected from mice on days 14, 28, and 42 after the first immunization to obtain serum. Antigen-specific IgG was measured in serum samples (1:100 dilution) by ELISA. Results are shown as ΔOD. Samples were measured in duplicate; bars represent group means ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm; MITD: MHC class I transmembrane and cytoplasmic domain; modRNA: nucleoside-modified mRNA; saRNA: self-amplifying mRNA; uRNA: unmodified mRNA; SD: standard deviation; SFU: spot-forming unit. [Figure 7A]Immunogenicity induced by a six-antigen mix using three mRNA platforms. C57BL / 6 mice (five animals per group) were immunized with a six-antigen mix mRNA construct (separately encoded Mycobacterium tuberculosis antigens Ag85A, ESAT6, vapB47, Hrp1, RpfA, and RpfD) as unmodified mRNA (uRNA), pseudouridine-modified mRNA (modRNA), or self-amplifying mRNA (saRNA). uRNA was formulated in lipoplexes, while modRNA and saRNA were formulated in C12 lipid nanoparticles (LNP-C12). Mice were injected intravenously with 20 μg of uRNA (100 μL dose volume) three times (days 0, 7, and 21) or intramuscularly with 4 μg of modRNA or saRNA (20 μL dose volume) twice (days 0 and 21). Control mice were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the entire length of each antigen encoded in the construct or the nonspecific peptide TRP1, and interferon gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent group mean spot-forming units (SFU) ± SD. [Figure 7C]C) Blood samples were collected from mice on days 14, 28, and 42 after the first immunization to obtain serum. Antigen-specific IgG was measured by ELISA in serum samples (1:300 dilution for Ag85A and Hrp1, and 1:100 dilution for other antigens). Results are shown as ΔOD. Samples were measured in duplicate; bars represent group means ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm; MITD: MHC class I transmembrane and cytoplasmic domain; modRNA: nucleoside-modified mRNA; saRNA: self-amplifying mRNA; uRNA: unmodified mRNA; SD: standard deviation; SFU: spot-forming unit. [Figure 8]Immunogenicity induced by a two-antigen mix using three mRNA platforms. C57BL / 6 mice (five animals per group) were immunized with a two-antigen mix mRNA construct (separately encoded Mycobacterium tuberculosis antigens HbhA and M72) as unmodified mRNA (uRNA), pseudouridine-modified mRNA (modRNA), or self-amplifying mRNA (saRNA). uRNA was formulated in lipoplexes, while modRNA and saRNA were formulated in C12 lipid nanoparticles (LNP-C12). Mice were injected intravenously with 20 μg of uRNA (100 μL dose volume) three times (days 0, 7, and 21) or intramuscularly with 4 μg of modRNA or saRNA (20 μL dose volume) twice (days 0 and 21). Control mice received intramuscular injections of 20 μL of phosphate-buffered saline (buffer). A) Mice were sacrificed 42 days after the first immunization, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 hours) with 2 μg / mL of overlapping peptide pools covering the full length of each of the antigens encoded in the constructs or the nonspecific peptide TRP1. Interferon-gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate, and bars represent the group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent the group mean spot-forming units (SFU) ± SD. C) Blood samples were collected from mice on days 14, 28, and 42 after the first immunization to obtain serum. Antigen-specific IgG was measured in serum samples (1:100 dilution) by ELISA. Results are shown as ΔOD. Samples were measured in duplicate; bars represent group means ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test at α = 0.05. **** = P < 0.0001. ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm. MITD: MHC class I transmembrane and cytoplasmic domains. modRNA: nucleoside-modified mRNA. saRNA: self-amplifying mRNA. uRNA: unmodified mRNA. SD: standard deviation. SFU: spot-forming units. [Figure 9] Immunogenicity induced by the M72 antigen using the modRNA platform. C57BL / 6 mice (5 animals per group) were immunized with modRNA encoding M72 formulated in C12 lipid nanoparticles (LNP-C12). Mice were intramuscularly injected with 4 μg of modRNA or saRNA (20 μL dose volume) twice (days 0 and 21). Control mice were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the full length of each of the antigens encoded in the constructs or the nonspecific peptide TRP1. Interferon gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate, and the bars represent the group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (105 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent the mean spot-forming units (SFU) ± SD of group replicates. C) Blood samples were collected from mice on days 14, 28, and 42 after the first immunization to obtain serum. Antigen-specific IgG was measured in serum samples (1:25 dilution) by ELISA. Results are shown as ΔOD. Samples were measured in duplicate; bars represent the group mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test at α = 0.05. *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001, ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm, MITD: MHC class I transmembrane and cytoplasmic domain, modRNA: nucleoside-modified mRNA, saRNA: self-amplifying mRNA, uRNA: unmodified mRNA, SD: standard deviation, SFU: spot-forming unit. [Figure 10A]Immunogenicity induced by a six-antigen cassette + two antigens using three mRNA platforms. C57BL / 6 mice (five animals per group) were immunized with a six-antigen cassette + two antigen mixture of mRNA constructs (encoding fusion proteins of separately encoded Mycobacterium tuberculosis antigens M72 and HbhA, and antigens Ag85A, ESAT6, vapB47, Hrp1, RpfA, and RpfD) as unmodified mRNA (uRNA), pseudouridine-modified mRNA (modRNA), or self-amplifying mRNA (saRNA). uRNA was formulated in lipoplexes, and modRNA and saRNA were formulated in C12 lipid nanoparticles (LNP-C12). Mice were injected intravenously with 20 μg of uRNA (100 μL dose volume) three times (days 0, 7, and 21) or intramuscularly with 4 μg of modRNA or saRNA (20 μL dose volume) twice (days 0 and 21). Control mice were injected intramuscularly with 20 μL of phosphate-buffered saline (PBS). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the full length of each of the antigens encoded by the constructs or the nonspecific peptide TRP1. Interferon gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate, and the bars represent the group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (105 cells) were selected from mouse splenocytes pooled by treatment group and processed as in A. Pooled T cell samples were measured in triplicate; bars represent the mean spot-forming units (SFU) ± SD of group replicates. [Figure 10C]C) Blood samples were collected from mice on days 14, 28, and 42 after the first immunization to obtain serum. Antigen-specific IgG was measured in serum samples (1:100 dilution) by ELISA. Results are shown as ΔOD. Samples were measured in duplicate; bars represent group means ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test at α = 0.05. ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm; MITD: MHC class I transmembrane and cytoplasmic domain; modRNA: nucleoside-modified mRNA; saRNA: self-amplifying mRNA; uRNA: unmodified mRNA; SD: standard deviation; SFU: spot-forming unit. [Figure 11] Scheme of mRNA constructs used to test fusions of signal peptides alone or in combination with transmembrane domains using two different codon optimizations. Top: General mRNA construct structure with 5' cap, 5' and 3' untranslated regions (UTRs), open reading frame (ORF), and polyadenosine (A) tail. Bottom: Antigen-encoding mRNA constructs contained within the ORF. All antigen-encoding sequences were codon-optimized (opt1 or opt10). Antigen-encoding sequences were either included alone in the mRNA backbone or fused at their N-terminus to a secretory signal peptide (sec, SP1, or SP2) with or without a transmembrane domain (TMD1, TMD2, or TMD3) fused at their C-terminus. M. tuberculosis antigens tested: Ag85A (Δ1-41), RpfA, and vapB47. [Figure 12]Immunogenicity induced by Ag85A with or without a signal peptide. As depicted in Figure 11, C57BL / 6 mice (five animals per group) were immunized with codon-optimized (opt1 or opt10) pseudouridine-modified mRNA (modRNA) constructs encoding the Mycobacterium tuberculosis antigen Ag85A (Δ1-41) alone or with a secretory signal peptide sequence (sec, SP1, or SP2) fused to its N-terminus. The modRNA was formulated in C12 lipid nanoparticles (LNP-C12). Mice were intramuscularly injected twice (days 0 and 21) with 4 μg of modRNA (20 μL dose volume). Mice in the control group were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the Ag85A (Δ1-41) antigen or the nonspecific peptide TRP1, and interferon-gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent group mean spot-forming units (SFU) ± SD. C) On day 42 after the first immunization, blood samples were collected from mice to obtain serum. Ag85A-specific IgG was measured by ELISA in serum samples (1:2700 dilution). Results are shown as ΔOD. Samples were measured in duplicate; bars represent group mean ± SD. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm, SD: standard deviation, SFU: spot-forming unit, SP: secretory signal peptide. [Figure 13A]Immunogenicity induced by signal peptide-fused Ag85A with or without a transmembrane domain sequence. As depicted in Figure 11, C57BL / 6 mice (five animals per group) were immunized with codon-optimized (opt1 or opt10) pseudouridine-modified mRNA (modRNA) constructs encoding the Mycobacterium tuberculosis antigen Ag85A (Δ1-41) with a secretory signal peptide sequence (sec, SP1, or SP2) fused to its N-terminus and a transmembrane domain sequence fused to its C-terminus. The modRNA was formulated in C12 lipid nanoparticles (LNP-C12). Mice were intramuscularly injected twice (days 0 and 21) with 4 μg of modRNA (20 μL dose volume). Control mice were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the Ag85A (Δ1-41) antigen or the nonspecific peptide TRP1, and interferon-gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent group mean spot-forming units (SFU) ± SD. [Figure 13C] C) Blood samples were collected from mice 42 days after the first immunization to obtain serum. Ag85A-specific IgG was measured in serum samples (1:300 dilution) by ELISA. Results are shown as ΔOD. Samples were measured in duplicate; bars represent group mean ± SD. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm; SD: standard deviation; SFU: spot-forming unit; SP: secretory signal peptide; TMD: transmembrane domain. [Figure 14]Immunogenicity induced by RpfA with or without a signal peptide. As depicted in Figure 11, C57BL / 6 mice (five animals per group) were immunized with codon-optimized (opt1 or opt10) pseudouridine-modified mRNA (modRNA) constructs encoding the Mycobacterium tuberculosis antigen RpfA alone or with a secretory signal peptide sequence (sec, SP1, or SP2) fused to its N-terminus. The modRNA was formulated in C12 lipid nanoparticles (LNP-C12). Mice were intramuscularly injected twice (days 0 and 21) with 4 μg of modRNA (20 μL dose volume). Mice in the control group were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the RpfA antigen or the nonspecific peptide TRP1, and interferon-gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent group mean spot-forming units (SFU) ± SD. C) On day 42 after the first immunization, blood samples were collected from mice to obtain serum. RpfA-specific IgG was measured by ELISA in serum samples (1:300 dilution). Results are shown as ΔOD. Samples were measured in duplicate; bars represent group mean ± SD. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm, SD: standard deviation, SFU: spot-forming unit, SP: secretory signal peptide. [Figure 15]Immunogenicity induced by signal peptide-fused RpfA with or without a transmembrane domain sequence. As described in Figure 11, C57BL / 6 mice (five animals per group) were immunized with codon-optimized (opt1 or opt10) pseudouridine-modified mRNA (modRNA) constructs encoding the Mycobacterium tuberculosis antigen RpfA with a secretory signal peptide sequence (sec, SP1, or SP2) fused to its N-terminus and a transmembrane domain sequence fused to its C-terminus. The modRNA was formulated in C12 lipid nanoparticles (LNP-C12). Mice were intramuscularly injected twice (days 0 and 21) with 4 μg of modRNA (20 μL dose volume). Control mice were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the RpfA antigen or the nonspecific peptide TRP1, and interferon-gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent group mean spot-forming units (SFU) ± SD. C) On day 42 after the first immunization, blood samples were collected from mice to obtain serum. RpfA-specific IgG was measured by ELISA in serum samples (1:300 dilution). Results are shown as ΔOD. Samples were measured in duplicate; bars represent group mean ± SD. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm, SD: standard deviation, SFU: spot-forming unit, SP: secretory signal peptide, TMD: transmembrane domain. [Figure 16]Immunogenicity induced by VapB47 with or without a signal peptide. As depicted in Figure 11, C57BL / 6 mice (five animals per group) were immunized with codon-optimized (opt1 or opt10) pseudouridine-modified mRNA (modRNA) constructs encoding the Mycobacterium tuberculosis antigen VapB47 alone or with a secretory signal peptide sequence (sec, SP1, or SP2) fused to its N-terminus. ModRNA was formulated in C12 lipid nanoparticles (LNP-C12). Mice were intramuscularly injected twice (days 0 and 21) with 4 μg of modRNA (20 μL dose volume). Control mice were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the VapB47 antigen or the nonspecific peptide TRP1, and interferon-gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent group mean spot-forming units (SFU) ± SD. C) On day 42 after the first immunization, blood samples were collected from mice to obtain serum. VapB47-specific IgG was measured by ELISA in serum samples (1:300 dilution). Results are shown as ΔOD. Samples were measured in duplicate; bars represent group mean ± SD. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm, SD: standard deviation, SFU: spot-forming unit, SP: secretory signal peptide. [Figure 17]Immunogenicity induced by signal peptide-fused VapB47 with or without a transmembrane domain sequence. As depicted in Figure 11, C57BL / 6 mice (five animals per group) were immunized with codon-optimized (opt1 or opt10) pseudouridine-modified mRNA (modRNA) constructs encoding the Mycobacterium tuberculosis antigen VapB47, carrying a secretory signal peptide sequence (sec, SP1, or SP2) fused to its N-terminus and a transmembrane domain sequence fused to its C-terminus. The modRNA was formulated in C12 lipid nanoparticles (LNP-C12). Mice were intramuscularly injected twice (days 0 and 21) with 4 μg of modRNA (20 μL dose volume). Control mice were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (5 × 10 cells) in culture were treated overnight (12–16 h) with 2 μg / mL of overlapping peptide pools covering the VapB47 antigen or the nonspecific peptide TRP1, and interferon-gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent group mean spot-forming units (SFU) ± SD. C) On day 42 after the first immunization, blood samples were collected from mice to obtain serum. VapB47-specific IgG was measured by ELISA in serum samples (1:300 dilution). Results are shown as ΔOD. Samples were measured in duplicate; bars represent group mean ± SD. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, ΔOD: absorbance at 620 nm subtracted from absorbance at 450 nm, SD: standard deviation, SFU: spot-forming unit, SP: secretory signal peptide, TMD: transmembrane domain. [Figure 18]PPD-induced cellular immune responses in mice immunized with three different modRNA mixes. C57BL / 6 mice (five animals per group) were immunized with a mixture of codon-optimized (opt10) pseudouridine-modified mRNA (modRNA) constructs described in Figure 1. The modRNA mixture was formulated in Acuitas ALC-315 lipid nanoparticles (LNP-315). Mice were intramuscularly injected twice (days 0 and 21) with 4 μg of modRNA (20 μL dose volume). Mice in the reference group were subcutaneously (sc) injected once on day 0 with 10 colony-forming units (CFU; 100 μL dose volume) of Bacillus Calmette-Guérin (BCG). Mice in the control group were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). A) On day 42 after the first immunization, mice were sacrificed, and spleens were dissected to isolate splenocytes. Splenocytes (1.25 × 10 cells) in culture were treated overnight (12–16 h) with 10 μg / mL purified protein derivative (PPD), and interferon-gamma (IFNγ) secretion was assessed by ELISpot assay. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. B) CD4+ and CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent group mean spot-forming units (SFU) ± SD. Samples were measured in duplicate; bars represent group mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. SD = standard deviation. [Figure 19]Antigen-specific cellular immune responses in mice immunized with three different modRNA mixes. C57BL / 6 mice (five animals per group) were immunized with a mixture of codon-optimized (opt1) pseudouridine-modified mRNA (modRNA) constructs as described in Figure 1. The modRNA mixture was formulated in Acuitas ALC-315 lipid nanoparticles (LNP-315). Mice were intramuscularly injected twice (days 0 and 21) with 4 μg of modRNA (20 μL dose volume). Control mice were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). Mice were sacrificed 42 days after the first immunization, and spleens were dissected to isolate splenocytes. A) Splenocytes (5 x 10 cells) in culture were treated overnight (12-16 hours) with 2 µg / mL of overlapping peptide pools covering the entire length of each of the antigens encoded in the constructs or the nonspecific peptide TRP1, and interferon gamma (IFNγ) secretion was assessed by ELISpot assay. B) Because the spot counts from treatment of splenocytes with Ag85A- and M72-specific peptide pools were too high when 5 x 10 splenocytes were used, responses to these antigens were analyzed using 1.25 x 10 splenocytes treated as in A. Samples were measured in duplicate; bars represent group mean spot-forming units (SFU) ± SD. C) CD4+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. D) CD8+ T cells (10 cells) were selected from mouse splenocytes pooled by treatment group and treated as in A. Pooled T cell samples were measured in triplicate; bars represent the mean spot-forming units (SFU) ± SD of group replicates. Samples were measured in duplicate; bars represent the group mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test at α = 0.05. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, SD: standard deviation. [Figure 20]Antigen-specific humoral immune responses in mice immunized with three different modRNA mixes. C57BL / 6 mice (five animals per group) were immunized with a mixture of codon-optimized (opt1) pseudouridine-modified mRNA (modRNA) constructs as described in Figure 1. The modRNA mixture was formulated in Acuitas ALC-315 lipid nanoparticles (LNP-315). Mice were intramuscularly injected with 4 μg of modRNA (20 μL dose volume) twice (days 0 and 21). Mice in the control group were intramuscularly injected with 20 μL of phosphate-buffered saline (buffer). Blood samples were collected from mice on day 42 after the first immunization to obtain serum. Antigen-specific IgG was measured in serum samples (serum dilutions indicated in each graph title) by ELISA. Results are shown as ΔOD. Samples were measured in duplicate; bars represent group means ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test at α = 0.05. *=P<0.05, **=P<0.01, ***=P<0.001, ****P<0.0001, SD: standard deviation. [Figure 21A] In vitro expression of uRNA Mix 3 and modRNA Mix 3. HEK293T / 17 cells were transfected with equal amounts of unmodified or nucleoside-modified mRNAs as single mRNAs (0.25 μg of mRNA) contained within a mixture of each of the four fusion mRNAs (Mix 1; 0.25 μg of each mRNA, 1 μg total), or with the drug substance containing the four fusion mRNAs (Mix 2; 1 μg total mRNA). Untransfected HEK293T / 17 cells were used as a control (NT). Whole cell lysates of untreated and transfected cells were generated and separated by SDS-PAGE. Proteins were blotted onto nitrocellulose membranes, and expression of the fusion proteins was assessed using antigen-specific antibodies. Expected molecular weights: A: Ag85A-Hrp1, 50 kDa; B: ESAT-6-RpfD, 28 kDa. [Figure 21C] Expression of the fusion proteins was evaluated as in Figure 21A. Predicted molecular weights: C: RpfA-HbhA, 120 kDa; D: M72-VapB47, 86 kDa. [Figure 22]T cell responses induced by immunization with uRNA Mix 3 and modRNA Mix 3. Splenocytes were isolated from C57BL / 6 mice injected with uRNA Mix 3, modRNA Mix 3, or control (saline) on study day 42. Cells were stimulated with an antigen-specific overlapping peptide pool at 2 μg / mL per peptide for 18 hours, and responses were assessed by IFN-γ ELISpot assay. Bars represent the mean of the treatment groups. One-way analysis of variance (ANOVA), α=0.05. ***=p<0.001, ****=p<0.0001. Significance between treatment and control groups is not shown. [Figure 23-1] Antibody responses induced by immunization with uRNA mix 3 and modRNA mix 3. Antigen-specific immunoglobulin G (IgG) antibodies were assessed by ELISA in serum from C57BL / 6 mice immunized with uRNA mix 3, modRNA mix 3, or saline control. Data shown are from day 42 after the first immunization. No IgG was detected in serum from mice in the control group. Bars represent treatment group means; symbols represent values for individual mouse samples. One-way analysis of variance (ANOVA), α = 0.05. ** = p < 0.01. Significant differences between treatment and control groups are not shown. [Figure 23-2] Antibody responses induced by immunization with uRNA mix 3 and modRNA mix 3 evaluated as in Figure 23-1 [Figure 24]CD4+ and CD8+ T cell-specific responses assessed by intracellular cytokine staining of cells from mice injected with uRNA Mix 3 and modRNA Mix 3. Splenocytes were isolated from C57BL / 6 mice injected with uRNA Mix 3, modRNA Mix 3, or control (saline) on study day 42. (A) Cells were stimulated for 5–6 hours with a mix of overlapping peptide pools covering all antigens at 1 μg / mL per peptide in the presence of costimulatory antibodies (CD28 and CD49d), Golgi Stop, and Golgi Plug (protein transport inhibitors). (B) Cells were stimulated for 5–6 hours with a mix of overlapping peptide pools covering all antigens at 1 μg / mL per peptide in the presence of costimulatory antibodies (CD28 and CD49d), Golgi Stop, and Golgi Plug (protein transport inhibitors). Cells were stained for intracellular and extracellular markers, including viability, CD3, CD4, CD8, IFN-γ, IL-2, and TNFα. Cells were analyzed using a BD-Celesta flow cytometer to identify specific cell types. (A) CD4+ and (B) CD8+ cells. (C) Splenocytes were stained with a different panel of surface markers in the presence of Mtb32A tetramer (PepA, a component of M72), and cells were acquired and analyzed on a BD-Celesta. Data represent cells positive for single cytokines and polyfunctional T cells (IFN-γ, IL-2, and TNFα secreting cells). Bars represent treatment group means; symbols represent values for individual mouse samples. One-way analysis of variance (ANOVA), α = 0.05. Significance between treatment and control groups is not shown. [Figure 25] T cell responses induced by immunization with uRNA Mix 3 and modRNA Mix 3. Splenocytes were isolated from BALB / c mice injected with uRNA Mix 3, modRNA Mix 3, or control (saline) on study day 42. Whole splenocytes were stimulated with an antigen-specific overlapping peptide pool at 2 μg / mL per peptide for 18 hours, and responses were assessed by IFN-γ ELISpot assay. Bars represent treatment group means (± standard deviation). One-way analysis of variance (ANOVA), α = 0.05. Significance between treatment and control groups is not shown. [Figure 26-1] Antibody responses induced by immunization with uRNA mix 3 and modRNA mix 3. Antigen-specific immunoglobulin G (IgG) antibodies were assessed by ELISA in serum from BALB / c mice immunized with uRNA mix 3, modRNA mix 3, or saline control. Data shown are from day 42 after the first immunization. No IgG was detected in serum from mice in the control group. Bars represent treatment group means; symbols represent values for each mouse sample. One-way analysis of variance (ANOVA), α = 0.05. * = p < 0.05. Significant differences between treatment and control groups are not shown. [Figure 26-2] Antibody responses induced by immunization with uRNA mix 3 and modRNA mix 3 evaluated as in Figure 26-1 [Figure 27] Cellular immune responses induced by immunization with uRNA Mix 3 or modRNA Mix 3 in a humanized mouse model. Splenocytes were isolated on day 42 from humanized mice (transgenic for HLA allele A2.1 / DR1) injected with 4 μg of uRNA Mix 3, 4 μg of modRNA Mix 3, or saline control (buffer). Splenic CD4+ and CD8+ T cells were magnetically isolated and stimulated with an antigen-specific peptide pool in the presence of autologous bone marrow-derived dendritic cells. Cellular responses were assessed by IFN-γ ELISpot assay after approximately 18 hours of incubation. The bars (± standard deviation) indicate the mean number of spots (measured in triplicate wells) per group using T cells. Counts above 1,500 SFU were considered too high to count (TNTC). [Figure 28]Cellular responses induced by immunization with modRNA Mix 3 or individual RNAs containing modRNA Mix 3. Splenocytes were isolated from C57BL / 6 mice injected with 4 μg of modRNA Mix 3 or 1 μg of RNA-LNPs (Ag85A-Hrp1, ESAT6-RpfD, RpfA-HbhA, or M72-VapB47) on day 42 postprime. Control groups received saline (buffer). Cells were stimulated with antigen-specific peptide pools, and responses were assessed by IFN-γ ELISpot assay after approximately 18 hours of incubation. Bars indicate group mean spot counts (± standard deviation). One-way analysis of variance (ANOVA) with Tukey's multiple comparison test was performed; P values: * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Not shown: IFN-γ secretion from both test groups was significant compared to the saline group, except for HbhA and VapB47. Because the SFU of 5 × 10 cells per well was above the detectable range of the assay for Ag85A and M72, lower cell numbers were used for stimulation. [Figure 29] Humoral responses induced by immunization with modRNA Mix 3 or individual RNAs containing modRNA Mix 3. Antigen-specific IgG antibodies were assessed by ELISA in serum from C57BL / 6 mice immunized with 4 μg of modRNA Mix 3 or 1 μg of RNA-LNPs (Ag85A-Hrp1, ESAT6-RpfD, RpfA-HbhA, or M72-VapB47). Control groups received saline (buffer). Data shown are from day 42 postprime. Group means are indicated by horizontal bars (± standard deviation), and means from individual mice (measured in duplicate) are shown as circles. One-way analysis of variance (ANOVA) with Tukey's multiple comparison test was performed; P values: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Not shown: significant values for test groups compared with the saline group. Numbers in parentheses above the graph indicate serum dilutions. [Figure 30]Cellular and humoral responses to Ag85A and Hrp1 induced by immunization with modRNA Mix 3, individual RNAs contained in modRNA Mix 3, or single RNAs encoding single antigens. Splenocytes and blood were isolated 42 days postprime from C57BL / 6 mice injected with 4 μg of modRNA Mix 3 or 1 μg of Ag85A-Hrp1 or RNA-LNP encoding Ag85A or Hrp1. Control groups received saline (buffer). (A) Splenocytes were stimulated with antigen-specific peptide pools, and responses were assessed by IFN-γ ELISpot assay. Group means are shown as bars (± standard deviation). (B) Antigen-specific immunoglobulin G (IgG) antibodies in serum were assessed by ELISA. Group means are shown as horizontal bars (± standard deviation), and means from individual mice (measured in duplicate) are shown as circles. One-way analysis of variance (ANOVA) with Tukey's multiple comparison test was performed, P values: *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. Not shown: significance values for test groups compared to the saline group. Numbers in parentheses above the graph in B indicate serum dilutions. [Figure 31A] Humoral responses induced by immunization with uRNA mix 3 and modRNA mix 3 in Wistar Han rats. (A) Immunization schedule. [Figure 31B] (B) Antigen-specific IgG antibodies in serum from Wistar Han rats immunized with 30 μg of uRNA Mix 3, 30 μg of modRNA Mix 3, or saline control (buffer) were assessed by ELISA 28 days after the first immunization. Group means are shown as horizontal bars (± standard deviation), and means from individual mice (measured in duplicate) are shown as circles. One-way analysis of variance (ANOVA) with Tukey's multiple comparison test was performed; P values: * = p < 0.05, ** = p < 0.01. Not shown: significant values for test groups compared to the saline group. Numbers in parentheses above the graph indicate serum dilutions. [Figure 32]Cellular responses induced by injection of uRNA mix 3 and modRNA mix 3 in Wistar Han rats. Wistar Han rats were administered 30 μg of uRNA mix 3 or modRNA mix 3 intramuscularly on days 0, 7, 14, and 21. Control groups received saline. IFN-γ ELISpot assays were performed using splenocytes isolated on day 28 and stimulated for approximately 36 hours with each of eight Mtb antigen-specific overlapping peptide pools or respective recombinant Mtb proteins. Group means are indicated by bars (± standard deviation). One-way analysis of variance (ANOVA) with Tukey's multiple comparison test was performed; P values: * = p < 0.05, ** = p < 0.01. [Figure 33] Figure 1. Study design and readout of the in vivo Mtb challenge study in C57BL / 6 mice. The image shows the immunization schedule for the different groups and the two endpoints. The arrows below the blood sampling and endpoints indicate the respective assays performed. [Figure 34-1] Humoral responses induced by immunization with uRNA mix 3 and modRNA mix 3 in C57BL / 6 mice after boosting and challenge with M. tuberculosis H37Rv. Antigen-specific IgG antibodies in serum from C57BL / 6 mice were assessed by ELISA for the indicated groups at various time points. Data shown are from days 43, 87, and 117 (days). "Prime" refers to a single dose, and "prime and boost" refers to two doses. Group means are indicated by horizontal bars (± standard deviation), and means from individual mice (measured in duplicate) are shown as circles. One-way analysis of variance (ANOVA) with Tukey's multiple comparison test was performed; P values: ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Not shown: significant values for test groups compared with saline or NTL groups. Numbers in parentheses above the graph indicate serum dilutions. [Figure 34-2] Humoral responses induced by immunization with uRNA mix 3 and modRNA mix 3 in C57BL / 6 mice after boosting and challenge with M. tuberculosis H37Rv, assessed as in Figure 34-1. [Figure 35] Enumeration of Mtb H37Rv from the lungs and spleens of infected C57BL / 6 mice after immunization. Saline-injected or vaccinated C57BL / 6 mice were aerosol-infected with approximately 100 colony-forming units (CFU) of Mtb H37Rv strain. "Priming" refers to one dose, whereas "priming and boosting" refers to two doses. (A) Bacterial burdens in the right lung lobe and whole spleen were determined on day 87 (n = 10), 30 days after infection. (B) Bacterial burdens in the right lung lobe and whole spleen were determined on day 117 (n = 5), 60 days after infection. Group means are indicated by horizontal bars (± standard deviation), and means from individual mice (duplicate determinations) are shown as circles. One-way analysis of variance (ANOVA) with Dunnett's multiple comparison test was performed between the saline-treated group and all other groups, P values: *=p<0.05; **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 36A] Effect of the third immunization with uRNA mix 3 and modRNA mix 3 on humoral responses. (A) Immunization schedule and time points for serum collection. [Figure 36B-1] (B) Antigen-specific IgG antibodies in serum from C57BL / 6 mice immunized with uRNA mix 3 or modRNA mix 3 were assessed by ELISA at the indicated time points. Data shown are from day 42 to day 155 (d). Group means are indicated by symbols (± standard deviation). Numbers in parentheses above the graph indicate serum dilutions. The dotted line indicates the third immunization on day 134. [Figure 36B-2] Effect of the third immunization with uRNA mix 3 and modRNA mix 3 on humoral responses assessed as in Figure 36B-1. [Figure 37]N-terminal fusion of alternative signal peptides to uRNA Mix 3 and modRNA Mix 3. Top: General mRNA construct structure with a 5' cap, 5' and 3' untranslated regions (UTRs), open reading frames (ORFs), and polyadenosine tails. Bottom: RNA Mix 3 containing Mtb antigen sequences. All antigen-coding sequences were fused at the N-terminus to a major histocompatibility complex (MHC) class I signal peptide fragment (sec) or an alternative signal peptide fragment that mediates translocation to the endoplasmic reticulum. The RNA mixture was formulated in lipid nanoparticles 315 (LNP-315). UTR: untranslated region; poly(A): polyadenosine tail. [Figure 38-1] In vitro expression of uRNA Mix 3 fused to alternative signal peptides. HEK293T cells were transfected with 1 µg of uRNA Mix 3, which contains four fusion antigens with either SP1, SP2, or sec. 18 h after transfection, cells were stained with specific antibodies for viability and Mtb antigens included in uRNA Mix 3 (Ag85A, Hrp1, ESAT-6, RpfD, RpfA, HbhA, M72, and vapB47). Data show the mean fluorescence intensity of antigen-specific staining within the live cell population. Bars represent mean values, and symbols represent technical replicates of transfection and staining. Circles (first bar) indicate untransfected (negative control), upward-pointing triangles (second bar) indicate sec, squares (third bar) indicate SP1, and diamonds (fourth bar) indicate SP2. One-way analysis of variance (ANOVA) with Dunnett's multiple comparison test, *=p<0.05, **=p<0.01, ***=p<0.001. Not shown: significance values of test items compared to non-transfected controls. [Figure 38-2] In vitro expression of uRNA mix 3 fused to alternative signal peptides evaluated as in Figure 38-1 [Figure 39-1]In vitro expression of modRNA Mix 3 fused to alternative signal peptides. HEK293T cells were transfected with 1 µg of modRNA Mix 3, which contains four fusion antigens with either SP1, SP2, or sec. 18 h after transfection, cells were stained with specific antibodies for viability and Mtb antigens included in modRNA Mix 3 (Ag85A, Hrp1, ESAT-6, RpfD, RpfA, HbhA, M72, and vapB47). Data show the mean fluorescence intensity of antigen-specific staining within the live cell population. Bars represent mean values, and symbols represent technical replicates of transfection and staining. Circles (first bar) indicate untransfected (negative control), upward-pointing triangles (second bar) indicate sec, squares (third bar) indicate SP1, and diamonds (fourth bar) indicate SP2. One-way analysis of variance (ANOVA) with Dunnett's multiple comparison test, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. Not shown: significance values of test items compared to non-transfected controls. [Figure 39-2] In vitro expression of modRNA mix 3 fused to alternative signal peptides evaluated as in Figure 39-1 [Figure 40-1] Humoral responses induced by immunization with uRNA mix 3 or modRNA mix 3 fused to alternative signal peptides. Antigen-specific IgG antibodies in serum from C57BL / 6 mice were assessed by ELISA for the indicated groups on day 42 after the first immunization. Group means are indicated by horizontal bars (± standard deviation), and means from individual mice (measured in duplicate) are shown as symbols. Circles indicate buffer (negative control), upward-pointing triangles indicate sec, squares indicate SP1, and diamonds indicate SP2. Black symbols represent uRNA mix 3, and white symbols represent modRNA mix 3. One-way analysis of variance (ANOVA) with Tukey's multiple comparison test was performed; P values: * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Not shown: significant values for test groups compared to the buffer group. Numbers in parentheses above the graph indicate serum dilutions. [Figure 40-2]Humoral responses induced by immunization with uRNA mix 3 or modRNA mix 3 fused to alternative signal peptides evaluated as in Figure 40-1 [Figure 41-1] Cellular responses from whole splenocytes induced by immunization with uRNA Mix 3 or modRNA Mix 3 fused to alternative signal peptides. Splenocytes were isolated from C57BL / 6 mice injected with 4 μg of uRNA Mix 3 or modRNA Mix 3 (containing the fusion antigens Ag85A-Hrp1, ESAT6-RpfD, RpfA-HbhA, or M72-VapB47 with SP1, SP2, or sec) on day 42 after the first immunization. Control groups received saline (buffer). Cells were stimulated with antigen-specific peptide pools, and responses were assessed by IFN-γ ELISpot assay after approximately 18 hours of incubation. Bars represent the group mean spot counts (± standard deviation). Symbols indicate the average responses from individual mice measured in duplicate. Circles indicate buffer (negative control), upward-pointing triangles indicate sec, squares indicate SP1, and diamonds indicate SP2. Black symbols represent uRNA Mix 3, and white symbols represent modRNA Mix 3. The y-axis indicates IFN-γ-secreting cells / 5 × 10 splenocytes for Trp1, Hrp1, ESAT-6, RpfA, HbhA, and VapB47, and IFN-γ-secreting cells / 1.25 × 10 splenocytes for Ag85A, RpfD, and M72. One-way analysis of variance (ANOVA) with Tukey's multiple comparison test was performed between test groups immunized with uRNA-encoding mix or modRNA-encoding mix, respectively. P values: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Not shown: significant IFN-γ secretion in test groups compared to buffer. Because a spot number of 5 × 10 cells per well exceeded the detectable range of the assay for Ag85A, RpfD, and M72, lower cell numbers were used for stimulation. [Figure 41-2] Cellular responses from whole splenocytes induced by immunization with uRNA mix 3 or modRNA mix 3 fused to alternative signal peptides evaluated as in Figure 41-1 [Figure 42-1] Cellular responses from CD4+ T cells induced by immunization with uRNA mix 3 or modRNA mix 3 fused to alternative signal peptides. CD4+ T cells were isolated from mouse splenocytes pooled by treatment group and stimulated with an antigen-specific peptide pool in the presence of autologous bone marrow-derived dendritic cells. Pooled T cell samples were measured in duplicate or triplicate, where possible; bars represent group replicate mean spot-forming units (SFU) ± standard deviation. Bars represent group mean values. From left to right, bars represent buffer, sec uRNA mix 3, SP1 uRNA mix 3, SP2 uRNA mix 3, sec modRNA mix 3, SP1 modRNA mix 3, and SP2 modRNA mix 3. The Y-axis indicates IFN-γ-secreting cells / 1 × 10 CD4+ cells for all panels. [Figure 42-2] Cellular responses from CD4+ T cells induced by immunization with uRNA mix 3 or modRNA mix 3 fused to alternative signal peptides evaluated as in Figure 42-1 [Figure 42-3] Cellular responses from CD4+ T cells induced by immunization with uRNA mix 3 or modRNA mix 3 fused to alternative signal peptides evaluated as in Figure 42-1 [Figure 43-1] Cellular responses from CD8+ T cells induced by immunization with uRNA mix 3 or modRNA mix 3 fused to alternative signal peptides. CD8+ T cells were isolated from mouse splenocytes pooled by treatment group and stimulated with an antigen-specific peptide pool in the presence of autologous bone marrow-derived dendritic cells. Pooled T cell samples were measured in duplicate or triplicate, where possible; bars represent group replicate mean spot-forming units (SFU) ± standard deviation. Bars represent group mean values. From left to right, bars represent buffer, sec uRNA mix 3, SP1 uRNA mix 3, SP2 uRNA mix 3, sec modRNA mix 3, SP1 modRNA mix 3, and SP2 modRNA mix 3. The Y-axis indicates IFN-γ-secreting cells / 1 × 10 CD8+ cells for all panels. [Figure 43-2]Cellular responses from CD8+ T cells induced by immunization with uRNA mix 3 or modRNA mix 3 fused to alternative signal peptides evaluated as in Figure 43-1 [Figure 43-3] Cellular responses from CD8+ T cells induced by immunization with uRNA mix 3 or modRNA mix 3 fused to alternative signal peptides evaluated as in Figure 43-1 DETAILED DESCRIPTION OF THE INVENTION

[0175] Although the present disclosure will be further described in more detail below, it should be understood that the disclosure is not limited to the specific methods, protocols and reagents described herein, which may vary. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0176] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the present disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise.

[0177] For example, the present disclosure describes combinations of sequence molecules that may have different levels of sequence identity to a particular sequence, such as (i) sequence molecule A comprising the sequence of SEQ ID NO: a, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the sequence of SEQ ID NO: a, (ii) sequence molecule B comprising the sequence of SEQ ID NO: b, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the sequence of SEQ ID NO: b, etc. It should be understood that sequence molecules can be combined at any of the specified identity levels. In some embodiments, the sequence molecules are combined so that the identity levels are the same, for example, (i) sequence molecule A comprising the sequence of SEQ ID NO: a, (ii) sequence molecule B comprising the sequence of SEQ ID NO: b, etc., or (i) sequence molecule A comprising a sequence having at least 90% identity with the sequence of SEQ ID NO: a, (ii) sequence molecule B comprising a sequence having at least 90% identity with the sequence of SEQ ID NO: b, etc. In some embodiments, the identity levels are independently selected and partially or completely different from each other, i.e., the sequence molecules are combined so that the identity levels are not the same, for example, (i) sequence molecule A comprising the sequence of SEQ ID NO: a, (ii) sequence molecule B comprising a sequence having at least 90% identity with the sequence of SEQ ID NO: b, etc., or (i) sequence molecule A comprising a sequence having at least 90% identity with the sequence of SEQ ID NO: a, (ii) sequence molecule B comprising a sequence having at least 85% identity with the sequence of SEQ ID NO: b, etc.

[0178] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemical, biochemical, cell biology, immunological, and recombinant DNA techniques described in the art.

[0179] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprises" and variations such as "comprising" are understood to imply the inclusion of a stated feature, element, member, integer, or step or group of features, elements, members, integers, or steps, but not the exclusion of any other feature, element, member, integer, or step or group of features, elements, members, integers, or steps. The term "consisting essentially of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps and to those that do not materially affect the basic and novel characteristics of the claim or disclosure. The term "consisting of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, in each occurrence in this application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of."

[0180] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0181] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0182] The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better describe the disclosure and does not pose a limitation on the scope of the claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0183] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes cases where said event, circumstance, or condition occurs and cases where it does not occur.

[0184] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" should be interpreted as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0185] In the context of the present disclosure, the term "about" indicates an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation from the indicated numerical value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, e.g., ±0.01%. In some embodiments, "about" indicates a deviation of ±10% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±4% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±3% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±2% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±1% from the indicated numerical value. In some embodiments, "about" indicates a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.2% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.01% deviation from the indicated numerical value. As will be understood by one of ordinary skill in the art, such specific deviations from the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than artificial or engineered technical effects.

[0186] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually listed herein.

[0187] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0188] For clarity, it should be noted that whenever a sequence is referred to as being the sequence between a nucleotide at position x and a nucleotide at position y, the resulting sequence includes both the nucleotide at position x and the nucleotide at position y. Similarly, whenever a sequence is referred to as being the sequence between an amino acid at position x and an amino acid at position y, the resulting sequence includes both the amino acid at position x and the amino acid at position y. Furthermore, although the sequences set forth herein, and particularly the Sequence Listing, refer to DNA molecules, when it is stated in the description or claims herein that an RNA comprises a nucleotide sequence set forth herein, and particularly the Sequence Listing, it is clear that the nucleotide sequence referred to is actually identical to the base sequence of a DNA molecule set forth herein, and particularly the Sequence Listing, e.g., as represented by the referenced SEQ ID NO:, except that thymine is replaced by uracil.

[0189] The following provides definitions and embodiments that apply to all aspects of this disclosure. Terms defined below have the defined meanings unless otherwise indicated. Terms not defined have their art-wide accepted meanings.

[0190] Mycobacterium tuberculosis (M. tuberculosis) is a non-motile, slow-growing, rod-shaped bacterium (2-4 μm long and 0.2-0.5 μm wide). M. tuberculosis is a Gram-positive, obligate aerobic bacterium that requires a host for growth and reproduction and does not form spores.

[0191] The terms "tuberculosis" or "TB" are used to describe the infection caused by the infectious agent Mycobacterium tuberculosis (M. tuberculosis). TB is a potentially fatal contagious disease that can affect almost any part of the body, but is most frequently a pulmonary infection. M. tuberculosis, the causative agent of tuberculosis, is transmitted by airborne droplet nuclei produced when individuals with active disease cough, speak, or sneeze. When inhaled, the droplet nuclei reach the alveoli of the lungs. In susceptible individuals, the organism then multiplies and can spread via lymphatic vessels to lymph nodes and via the bloodstream to other sites, such as the lung apex, bone marrow, kidneys, and meninges. Once acquired immunity develops within 2–10 weeks, bacterial growth ceases. Lesions heal, and the individual remains asymptomatic. M. tuberculosis can remain dormant in the body (latent TB) for years after infection, hiding in phagocytosed cells and preventing disease development. Such individuals are said to have asymptomatic tuberculosis infection and exhibit a positive tuberculin skin test. The clinical state of latent TB is traditionally associated with the transition of M. tuberculosis to a dormant state in response to suboptimal growth conditions in vivo resulting from activation of the host immune response. Dormancy is a specific physiological state characterized by a significant cessation of metabolic activity and proliferation, while resuscitation from dormancy is a process that restores cellular activity and subsequent bacterial growth, which, in the case of M. tuberculosis, can lead to disease progression. The risk of developing active disease with clinical symptoms decreases over time and may never occur, but it is a lifelong risk. Approximately 5% of individuals with tuberculosis infection progress to active disease.

[0192] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0193] As used herein, terms such as "enhance" refer to the ability to cause an overall increase or enhancement in a level, for example, by at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more.

[0194] As used herein, "physiological pH" refers to a pH of about 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0195] As used in this disclosure, "% w / v" refers to weight-to-volume percent, a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of a solution in milliliters (mL).

[0196] As used in this disclosure, "wt. %" refers to weight percent, a unit of concentration that measures the amount of a substance in grams (g) expressed as a percentage of the total weight of the entire composition in grams (g).

[0197] As used in this disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100.

[0198] As used in this disclosure, "mol % of total lipid" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like substances.

[0199] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Thus, ionic strength, I, is calculated by the formula:

number

[0200] According to the present disclosure, the term "ionic strength" in some embodiments refers to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is, in some embodiments, sufficiently low to prevent degradation of nucleic acids. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of phosphodiester bonds between nucleotides, such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 μM or less. In some embodiments, free divalent ions are absent or essentially absent.

[0201] "Osmolality" refers to the concentration of a particular solute expressed as osmoles of solute per kilogram of solvent.

[0202] The term "lyophilize" or "freeze-drying" refers to the freeze-drying of a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or 1 Pa or less) to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilize" and "freeze-dry" are used interchangeably herein.

[0203] The term "spray drying" refers to spray drying a substance by mixing a fluid and a (heated) gas that is atomized (atomized) in a vessel (spray dryer), where the solvent from the droplets formed evaporates, resulting in a dry powder.

[0204] The term "reconstitute" relates to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

[0205] The term "recombinant" in the context of this disclosure means "produced through genetic engineering." In some embodiments, "recombinant" in the context of this disclosure is not naturally occurring.

[0206] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in living organisms (including viruses), can be isolated from natural sources, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature", and includes known objects and objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0207] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0208] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given in terms of EDTA disodium salt.

[0209] The term "cryoprotectant" relates to a substance added to a formulation to protect the active ingredient during the freezing step.

[0210] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying step.

[0211] According to the present disclosure, the term "peptide" refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together by peptide bonds. The term "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids. Although both "peptide" and "polypeptide" are protein molecules, the terms "protein" and "polypeptide" are generally used synonymously herein.

[0212] The term "biological activity" refers to a response of a biological system to a molecule. Such a biological system can be, for example, a cell or an organism. In some embodiments, such a response is therapeutically or pharmaceutically useful.

[0213] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" may refer to a contiguous or discontinuous fraction of said structure.

[0214] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or protein, refers to a continuous element of said structure. When used in reference to a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0215] With respect to an amino acid sequence (peptide or polypeptide), the term "fragment" refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence comprises, for example, at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. Fragments of an amino acid sequence include, for example, sequences of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the amino acid sequence.

[0216] As used herein, "variant" with respect to an amino acid sequence (peptide or polypeptide) means an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence can be a native or wild-type (WT) amino acid sequence, or can be a modified version of the wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences compared to the parent, e.g., 1 to about 10 or 1 to about 5 amino acid differences.

[0217] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or polypeptide has an amino acid sequence that has not been intentionally modified by man.

[0218] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide or polypeptide) can include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformational variants, isoform variants, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0219] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion can be at any position in the protein. Amino acid deletion variants containing deletions at the N- and / or C-termini of a protein are also called N- and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions within the amino acid sequence that are not conserved between homologous peptides or peptides and / or replacement of amino acids with other amino acids with similar properties are preferred. In some embodiments, amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions of one member of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: -Glycine, alanine; -valine, isoleucine, leucine; -Aspartic acid, glutamic acid; -Asparagine, glutamine; -Serine, threonine; -Lysine, arginine; and -Phenylalanine, tyrosine.

[0220] In some embodiments, the degree of similarity, such as identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given for, for example, at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, such as sequence identity, can be performed using tools known in the art, for example, using best sequence alignment, for example, Align, using standard settings, preferably EMBOSS::Needle, matrix:Blosum62, gap open 10.0, gap extension 0.5.

[0221] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0222] The terms "% identical" and "% identity" or similar terms are intended to refer to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences being compared.The percentage is purely statistical, and the differences between two sequences may be, but are not necessarily, randomly distributed over the entire length of the sequences being compared.The comparison of two sequences is usually carried out by comparing the sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, or the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, or the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use the above algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 28; (iii) a maximum match within the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter for low-complexity regions being used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 3; (iii) a maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to presence: 11, extension: 1; and (vi) a conditional composition score matrix adjustment.

[0223] The percent identity is obtained by determining the number of identical positions where the compared sequences match, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0224] In some embodiments, the degree of similarity or identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, consecutive nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0225] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, for example at least 95%, at least 98 or at least 99% of the amino acid residues.

[0226] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or polypeptides with substitutions, additions, insertions, or deletions is described, for example, in Molecular Cloning: A Laboratory Manual, 4 th Edition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, the peptides, polypeptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, such as, for example, solid phase synthesis and similar methods.

[0227] In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" specifically refers to a variant molecule or sequence that contains an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of performing one or more functions of the parent molecule or sequence, e.g., capable of inducing an immune response. In some embodiments, alterations to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, for example, the function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments, the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0228] An amino acid sequence (peptide or polypeptide) "derived from" a specified amino acid sequence (peptide or polypeptide) refers to the origin of the initial amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be modified to differ in sequence from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0229] In some embodiments, "isolated" means removed (e.g., purified) from a natural state or from an artificial composition, such as a composition from a manufacturing process. For example, a nucleic acid, peptide, or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid, peptide, or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid, peptide, or polypeptide can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0230] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into or the uptake of nucleic acids by such cells, and the cells may be present in a subject, e.g., a patient, or the cells may be present in vitro, e.g., outside the patient. Thus, according to this disclosure, cells for transfection of nucleic acids described herein can be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue, and / or body of a patient. According to this disclosure, transfection can be transient or stable. In some applications of transfection, it is sufficient for the transfected genetic material to be expressed only transiently. RNA can be transfected into cells to transiently express its encoded protein. Nucleic acids introduced during the transfection process are typically not integrated into the nuclear genome, and the foreign nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must occur.Such stable transfection can be achieved, for example, by using a virus-based system or a transposon-based system for transfection.Generally, the nucleic acid encoding the antigen is transiently transfected into the cell.RNA can be transfected into the cell to transiently express the encoded protein.

[0231] The present disclosure includes analogs of peptides or polypeptides. According to the present disclosure, a peptide or polypeptide analog is a modified form of the peptide or polypeptide from which it is derived, and retains at least one functional property of the peptide or polypeptide. For example, a pharmacologically active analog of a peptide or polypeptide retains at least one pharmacological activity of the peptide or polypeptide from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecule associated with the peptide or polypeptide, such as carbohydrates, lipids, and / or peptides or polypeptides. In some embodiments, a "peptide or polypeptide analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term "analog" also covers all functional chemical equivalents of the peptides and polypeptides.

[0232] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements or components or domains.

[0233] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue, or system.

[0234] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0235] According to various embodiments of the present disclosure, a nucleic acid, such as an RNA, encoding a peptide or polypeptide is taken up or introduced, i.e., transfected or transduced, into a cell, which may be present in vitro or in a subject, resulting in expression of the peptide or polypeptide. The cell may, for example, express the encoded peptide or polypeptide intracellularly (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptide or polypeptide, and / or express it on its surface. In some embodiments, the cell secretes the encoded peptide or polypeptide.

[0236] In accordance with the present disclosure, terms such as "expressing nucleic acid" and "encoding nucleic acid" or similar terms are used interchangeably herein and mean that, with respect to a particular peptide or polypeptide, the nucleic acid is capable of being expressed to produce said peptide or polypeptide when present in an appropriate environment, e.g., a cell.

[0237] In particular, the term "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or RNA (especially mRNA), to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene encodes a protein when transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0238] In this context, an "open reading frame" or "ORF" is a contiguous stretch of codons beginning with a start codon and ending with a stop codon.

[0239] As used herein, the term "expression" includes the transcription and / or translation of a particular nucleotide sequence.

[0240] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA (particularly mRNA), which can then be translated into peptides or polypeptides.

[0241] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0242] The pharmaceutical preparations, particularly kits, described herein may include instruction materials or instructions. As used herein, "instruction materials" or "instructions" include publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present disclosure. The instruction materials of the kits of the present disclosure may, for example, be attached to a container containing the composition / formulation of the present disclosure or may be shipped together with a container containing the composition / formulation. Alternatively, the instruction materials may be shipped separately from the container, with the intention that the instruction materials and the composition will be used in conjunction with each other by the recipient.

[0243] The term "set," as used herein, e.g., in the context of "a set of antigen amino acid sequences," means more than one, e.g., two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more.

[0244] The term "at least one," as used herein in the context of "at least one RNA molecule," means one or more, e.g., two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more. In some embodiments, the term "at least one" refers to one, two, three, four, five, six, seven, or eight. In some embodiments, "at least one RNA molecule" refers to a set of RNA molecules, e.g., a set of four RNA molecules, each RNA molecule encoding an amino acid sequence comprising at least two different Mtb antigens, immunogenic variants, or fragments thereof, e.g., an amino acid sequence comprising two different Mtb antigens, immunogenic variants, or fragments thereof. In some embodiments, such at least one RNA molecule or set of RNA molecules comprises RNA molecules in a mixture, which mixture can be obtained by transcribing, in a common reaction, a mixture of DNA templates encoding said RNA molecules.

[0245] A prodrug of a particular compound described herein is a compound that undergoes chemical conversion under physiological conditions to provide the particular compound upon administration to an individual. Furthermore, a prodrug can be converted to the particular compound by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be slowly converted to the particular compound when placed in a transdermal patch reservoir with an appropriate enzyme or chemical reagent. Exemplary prodrugs are in vivo hydrolyzable esters (using alcohol or carboxy groups contained in the particular compound) or amides (using amino or carboxy groups contained in the particular compound). Specifically, any amino group contained in the particular compound that has at least one hydrogen atom can be converted to a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[0246] In this specification, the structural formula of a compound may represent a specific isomer of the compound. However, it should be understood that the present disclosure includes all isomers and isomer mixtures, such as structurally occurring geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and is not limited to the description of the formula. Also, in this specification, the structural formula of a compound may represent a specific salt and / or solvate of the compound. However, it should be understood that the present disclosure includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates), and is not limited to the description of a specific salt and / or solvate.

[0247] "Isomers" are compounds that have the same molecular formula but differ in structure ("structural isomers") or in the geometric (spatial) arrangement of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains equal amounts of a pair of enantiomers and is designated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and are not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that, even when pure, spontaneously and reversibly interconvert due to the migration of individual atoms or groups of atoms; i.e., tautomers are in dynamic chemical equilibrium with each other. One example of a tautomer is keto-enol tautomeric isomers. "Conformers" are stereoisomers that can formally be interconverted by rotation about a single bond alone, and include in particular those resulting in different three-dimensional forms of a (hetero)cyclic ring, such as the chair, half-chair, boat, and twist-boat forms of cyclohexane.

[0248] The term "solvate," as used herein, refers to an addition complex of a substance dissolved in a solvent (e.g., an organic solvent (e.g., an aliphatic alcohol (e.g., methanol, ethanol, n-propanol, isopropanol, etc.), acetone, acetonitrile, ether, etc.), water, or a mixture of two or more of these liquids), where the addition complex exists in crystalline or mixed crystal form. The amount of solvent in the addition complex can be stoichiometric or non-stoichiometric. A "hydrate" is a solvate in which the solvent is water.

[0249] In an isotopically labeled compound, one or more atoms are replaced by a corresponding atom having the same number of protons but a different number of neutrons. For example, a hydrogen atom can be replaced with a deuterium or tritium atom. Exemplary isotopes that can be used in the present disclosure include deuterium, tritium, 11 C. 13 C. 14 C. 15 N, 18 F, 32 P, 32 S, 35 S, 36 Cl, and 125 Contains I.

[0250] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which results in a so-called Z dimension with a length dimension. 平均 , and the dimensionless polydispersity index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle is Z 平均 Used synonymously with this value.

[0251] In some embodiments, the "polydispersity index," as mentioned in the definition of "mean diameter," is calculated based on dynamic light scattering measurements by so-called cumulant analysis. Under certain prerequisites, it can be considered as a measure of the size distribution of an ensemble of nanoparticles.

[0252] The "radius of gyration" of the particle around the axis of rotation (R g ) is the radial distance from the axis of rotation of the point at which the moment of inertia of a particle about a given axis is the same as its actual mass distribution, if the entire mass of the particle were assumed to be concentrated. Mathematically, R g is the root mean square distance of a particle's components from either its center of mass or a given axis. For example, if the particle is at a fixed distance s from the center of mass, i Mass m located at i For a polymer consisting of n mass elements (i=1, 2, 3, ..., n), R g is the s over all mass elements i 2 is the mass-averaged square root of and can be calculated as follows:

number

[0253] The radius of gyration can be determined experimentally or calculated, for example, by using light scattering. In particular, for small scattering vectors

number

number

[0254] The "hydrodynamic radius" (sometimes called the "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere diffusing at the same rate as the particle. The hydrodynamic radius is related to the particle's mobility, taking into account not only size but also solvent effects. For example, a smaller charged particle with stronger hydration may have a larger hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags more water molecules along as it moves through the solution. Since the actual dimensions of a particle in a solvent cannot be measured directly, the hydrodynamic radius may be defined by the Stokes-Einstein equation:

number

[0255] As used herein, the expression "light scattering" refers to the physical process by which light is caused to deviate from a straight line trajectory by one or more paths due to local inhomogeneities in the medium through which the light passes.

[0256] The term "UV" means ultraviolet and refers to the band of the electromagnetic spectrum having wavelengths between 10 nm and 400 nm, i.e., shorter than those of visible light but longer than X-rays.

[0257] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring light scattered at multiple angles by a sample. "Multi-angle" in this context means that the scattered light can be detected at different discrete angles, as measured, for example, by a single detector moving over a range that includes a selected specific angle, or by an array of detectors fixed at specific angular positions. In certain embodiments, the light source used in MALS is a laser source (MALLS: Multi-Angle Laser Light Scattering). Based on the MALS signal of a composition containing particles, the radius of gyration (R) can be calculated by using an appropriate format (e.g., Zimm plot, Berry plot, or Debye plot). g ), and thus it is possible to determine the size of the particles. Preferably, the Zimm plot is a graphical representation using the following formula:

number

number

number

[0258] As used herein, the term "dynamic light scattering" or "DLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of particles. A monochromatic light source, usually a laser, is incident on a sample through a polarizer. The scattered light then passes through a second polarizer, where it is detected, and the resulting image is projected onto a screen. Particles in solution strike the light and diffract it in all directions. The diffracted light from the particles can interfere constructively (bright areas) or destructively (dark areas). This process is repeated over short time intervals, and the resulting set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity at each spot over time.

[0259] As used herein, the term "static light scattering" or "SLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the particle's radius of gyration and / or molar mass. A high-intensity monochromatic light, usually a laser, is emitted into a solution containing the particles. One or more detectors are used to measure the scattered intensity at one or more angles. The angular dependence is necessary to obtain accurate measurements of both the molar mass and size of all macromolecules in the radius. Therefore, simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), are generally considered the standard implementation of static light scattering.

[0260] nucleic acid The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. This term includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is a mixture of DNA and RNA. The nucleic acid can exist as a single-stranded or double-stranded molecule, and as a linear or covalently closed circular molecule. A nucleic acid can be isolated. The term "isolated nucleic acid," according to the present disclosure, means that the nucleic acid has been (i) amplified in vitro, e.g., by polymerase chain reaction (PCR) of DNA or in vitro transcription of RNA (e.g., using RNA polymerase); (ii) recombinantly produced by cloning; (iii) purified, e.g., by cleavage and separation by gel electrophoresis; or (iv) synthesized, e.g., by chemical synthesis.

[0261] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be thought of as a nucleotide without the phosphate group. A nucleoside is a nucleic acid base linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0262] The five standard nucleosides that commonly make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are commonly abbreviated by their single-letter codes: U, A, T, C, and G, respectively. However, thymidine is more commonly designated "dT" (the "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) but not ribonucleic acid (RNA). Conversely, uridine is found in RNA but not DNA. The remaining three nucleosides can be found in both RNA and DNA. In RNA, they are designated as A, C, and G, while in DNA, they are designated as dA, dC, and dG.

[0263] The modified purine (A or G) or pyrimidine (C, T, or U) base moiety may, in some embodiments, be one or more alkyl groups, e.g., one or more C 1-4 Modified by alkyl groups, e.g., one or more methyl groups. Specific examples of modified purine or pyrimidine base moieties include N 7 -Alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(1)-alkyl-uracil, e.g., N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C 1-4 Alkyl-uracil, and N(1)-C1-4 Alkyl-uracil, preferably N 7 -methyl-guanine, N 6 -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(1)-methyl-uracil.

[0264] DNA As used herein, the term "DNA" refers to a nucleic acid molecule consisting entirely or at least substantially of deoxyribonucleotide residues. In preferred embodiments, DNA contains all or most of the deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal DNA nucleotides or to either or both ends of the DNA. It is also contemplated herein that the nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. In the present disclosure, these modified DNAs are considered analogs of naturally occurring DNA. A molecule contains a "majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0265] The DNA may be recombinant DNA and may be obtained by cloning a nucleic acid, in particular cDNA, which may be obtained by reverse transcription of RNA.

[0266] RNA The term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends of the RNA (either or both). It is also contemplated herein that the nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA containing such modified / modified nucleotides (i.e., modified / modified RNA) may be referred to as analogs of naturally occurring RNA. A molecule contains a "majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0267] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (e.g., antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activator RNA (e.g., small activator RNA), and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0268] As used herein, the term "in vitro transcription" or "IVT" means that transcription (i.e., production of RNA) is performed acellularly. That is, IVT does not use live / cultured cells, but rather uses transcription machinery extracted from cells (e.g., cell lysates or isolated components thereof, including RNA polymerase (preferably T7, T3, or SP6 polymerase)).

[0269] According to the present disclosure, the term "RNA" includes "mRNA." According to the present disclosure, the term "mRNA" means "messenger RNA" and includes "transcripts" that can be produced by using a DNA template. Generally, mRNA encodes a peptide or polypeptide.

[0270] Although mRNA is single-stranded, it may contain self-complementary sequences that allow part of the mRNA to fold back on itself and pair with itself to form a double helix.

[0271] According to the present disclosure, "dsRNA" means double-stranded RNA, which is RNA having two partially or completely complementary strands.

[0272] In a preferred embodiment of the present disclosure, mRNA relates to an RNA transcript that encodes a peptide or polypeptide.

[0273] In some embodiments, preferably the mRNA encoding the peptide or polypeptide has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0274] As is well-established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide / polypeptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, mRNA is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art; for example, Molecular Cloning:4 thEdition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Additionally, various in vitro transcription kits are commercially available from, for example, Thermo Fisher Scientific (TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®, etc.), New England BioLabs Inc. (HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit, etc.), Promega (RiboMAX™, HeLaScribe®, Riboprobe® system, etc.), Jena Bioscience (SP6 or T7 transcription kit, etc.), and Epicentre (AmpliScribe™, etc.). To provide a modified mRNA, correspondingly modified nucleotides, e.g., modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be made in and / or added to the mRNA after transcription.

[0275] In some embodiments, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0276] In some embodiments of the present disclosure, the RNA is a "replicon RNA" or simply a "replicon," particularly a "self-replicating RNA" or "self-amplifying RNA." In certain embodiments, the replicon or self-replicating RNA is derived from or contains elements derived from a ssRNA virus, particularly a positive-strand ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication and protein modification) and structural proteins (forming viral particles). Typically, two open reading frames (ORFs) are present in the genome. The four nonstructural proteins (nsP1-nsP4) are typically encoded together by a first ORF that begins near the 5' end of the genome, while the alphavirus structural proteins are encoded together by a second ORF that is found downstream of the first ORF and extends toward the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins is translatable from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87, pp. 111-124). After infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts directly like a messenger RNA to translate the open reading frame encoding the nonstructural polyprotein (nsP1-nsP4).

[0277] Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication (trans-amplification) systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase, and the other nucleic acid molecule can be replicated in trans by the replicase (hence the name trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules that can be replicated in trans by the replicase must contain specific alphavirus sequence elements to enable recognition and RNA synthesis by the alphavirus replicase.

[0278] In some embodiments of the present disclosure, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations of the present disclosure and / or used in the methods of the present disclosure) contains one or more modifications, for example, to increase its stability, and / or translation efficiency, and / or reduce immunogenicity and / or cytotoxicity. For example, to increase the expression of the RNA (particularly mRNA), modifications can be made within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without changing the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include: 5' cap structure; extension or truncation of a naturally occurring poly(A) tail; modification of the 5' and / or 3' untranslated region (UTR), for example, introduction of a UTR not associated with the coding region of the RNA; substitution of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to change, preferably increase, the GC content of the RNA). Combinations of the above-mentioned modifications, i.e., incorporation of a 5' cap structure, incorporation of a polyA sequence, unmasking of a polyA sequence, alteration of the 5'-UTR and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine in the case of cytidine, and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U) in the case of uridine), and codon optimization have a synergistic effect on increasing RNA (preferably mRNA) stability and translation efficiency.Thus, in some embodiments, RNA (particularly mRNA) described in the present disclosure includes a combination of at least two, at least three, at least four, or all five of the above-mentioned modifications, namely: (i) incorporation of a 5' cap structure; (ii) incorporation of a polyA sequence, unmasking of a polyA sequence; (iii) modification of the 5'-UTR and / or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine in the case of cytidine, and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U) in the case of uridine); and (v) codon optimization.

[0279] 5' Cap In some embodiments, the RNA (particularly mRNA) described herein comprises a 5' cap structure. In some embodiments, the RNA does not have an uncapped 5'-triphosphate. In some embodiments, the RNA (particularly mRNA) may comprise a conventional 5' cap and / or a 5' cap analog. The term "conventional 5' cap" refers to a cap structure found at the 5' end of an RNA molecule, generally comprising guanosine 5'-triphosphate (Gppp) connected via its triphosphate moiety to the 5' end of the next nucleotide of the RNA (i.e., the guanosine is connected to the remainder of the RNA via a 5'-5' triphosphate bond). The guanosine is N 7 can be methylated at the cap structure m 7 The term "5' cap analog" is based on the traditional 5' cap, but uses a 5' cap analog with a reverse orientation to avoid incorporation of the 5' cap analog. 7These include 5' caps modified at either the 2' or 3' position of the guanosine structure (such 5' cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5' cap analogs are those with one or more substitutions at the bridging and non-bridging oxygens in the phosphate bridge, such as phosphorothioate-modified 5' cap analogs at the β-phosphate (e.g., m2), as described in PCT / EP2019 / 056502. 7,2’O G(5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA). Providing RNA (particularly mRNA) having a 5' cap structure as described herein can be achieved by in vitro transcription of a DNA template in the presence of the corresponding 5' cap compound, with the 5' cap structure being co-transcriptionally incorporated into the generated RNA (particularly mRNA) strand, or RNA (particularly mRNA) can be generated, for example, by in vitro transcription, and the 5' cap structure can be attached to the RNA post-transcriptionally using a capping enzyme, for example, vaccinia virus capping enzyme.

[0280] In some embodiments, the RNA (particularly mRNA) is m2 7,2’O G(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3’O G(5')ppp(5')G, and m2 7,3’-O Gppp(m1 2’-O In some embodiments, the RNA comprises a 5' cap structure selected from the group consisting of m2 7,2’O G(5')ppSp(5')G (particularly its D1 diastereomer). In some embodiments, the RNA comprises m2 as the 5' cap structure. 7,3’-O Gppp(m1 2’-O )Contains ApG.

[0281] In some embodiments, RNA (particularly mRNA) comprises cap 0, cap 1, or cap 2, preferably cap 1 or cap 2. According to the present disclosure, the term "cap 0" refers to the structure "m 7GpppN" where N is any nucleoside having an OH moiety at the 2' position. According to the present disclosure, the term "Cap 1" refers to the structure "m 7 GpppNm, where Nm is any nucleoside having an OCH3 moiety at the 2' position. According to the present disclosure, the term "cap 2" refers to the structure "m 7 GpppNmNm" where each Nm is independently any nucleoside having an OCH3 moiety at the 2' position.

[0282] The 5' cap analog beta-S-ARCA (β-S-ARCA) has the following structure: [ka] The "D1 diastereomer of beta-S-ARCA" or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)). The HPLC is preferably analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column, preferably in a 5 μm, 4.6 x 250 mm format, is used for the separation, allowing a flow rate of 1.3 ml / min to be applied. In some embodiments, a gradient of methanol in ammonium acetate is used, e.g., a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH 5.9, within 15 minutes. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0283] 5' cap analog m2, a building block of cap 1 7,3’-O Gppp(m1 2’-O )ApG(m2 7,3’O G(5')ppp(5')m 2’-O ApG (also called ApG) has the following structure: [ka] An exemplary cap 0 mRNA, including β-S-ARCA, has the following structure: [ka] m2 7,3’O An exemplary cap 0 mRNA containing G(5')ppp(5')G and mRNA has the following structure: [ka] m2 7,3’-O Gppp(m1 2’-O ) An exemplary cap 1 mRNA containing ApG and mRNA has the following structure: [ka]

[0284] Poly A tail As used herein, the term "poly A tail" or "poly A sequence" refers to a continuous or intermittent sequence of adenylate residues typically located at the 3' end of an RNA (particularly an mRNA) molecule. Poly A tails or poly A sequences are known to those skilled in the art and may follow the 3'-UTR of an RNA (particularly an mRNA) described herein. A continuous poly A tail is characterized by consecutive adenylate residues. In nature, continuous poly A tails are typical. The RNA (particularly an mRNA) disclosed herein may have a poly A tail attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription, or a poly A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0285] Poly(A) tails of approximately 120 A nucleotides have been demonstrated to have beneficial effects on the levels of RNA in transfected eukaryotic cells and on the levels of proteins translated from open reading frames located 5' upstream of the poly(A) tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0286] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "consisting essentially of" means that most of the nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly-A tail, are A nucleotides, while allowing for the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides of the poly A tail, i.e., 100% of the number of nucleotides in the poly A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0287] In some embodiments, the poly(A) tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template containing repeated dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is called a poly(A) cassette.

[0288] In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of the four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length. Such cassettes are disclosed in International Publication No. 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication No. 2016 / 005324 A1 may be used in the present disclosure. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences with an equal distribution of the four nucleotides (dA, dC, dG, dT) and a length of, for example, 5-50 nucleotides, exhibit sustained propagation of plasmid DNA in Escherichia coli (E. coli) at the DNA level and are still associated with beneficial properties for supporting RNA stability and translation efficiency at the RNA level. As a result, in some embodiments, the poly-A tails included in the RNA molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0289] In some embodiments, the poly(A) tail comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a 10 nucleotide linker sequence.

[0290] In some embodiments, no nucleotides other than A nucleotides are adjacent to the polyA tail at its 3' end, i.e., the polyA tail is not masked or followed by a nucleotide other than A at its 3' end.

[0291] In some embodiments, the poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises the poly-A tail set forth in SEQ ID NO:59. In some embodiments, the polyA tail comprises at least 100 nucleotides. In some embodiments, the polyA tail comprises about 150 nucleotides. In some embodiments, the polyA tail comprises about 120 nucleotides.

[0292] Untranslated Regions (UTRs) In some embodiments, the RNA (particularly mRNA) described in the present disclosure includes a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of the protein-coding region. If present, the 5'-UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon of the protein-coding region, although the term "3'-UTR" generally does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence. The incorporation of a 3'-UTR into the 3' untranslated region of an RNA (preferably mRNA) molecule can result in improved translation efficiency. By incorporating two or more such 3'-UTRs (preferably arranged in a head-to-tail orientation; see, for example, Holtkamp et al., Blood 108, 4009-4017 (2006)), a synergistic effect can be achieved. 3'-UTRs can be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced. In certain embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as α2-globin, α1-globin, or β-globin, e.g., β-globin, e.g., human β-globin gene or mRNA. For example, an RNA (e.g., an mRNA) can be modified by replacing or inserting an existing 3'-UTR with one or more, e.g., two, copies of a 3'-UTR from a globin gene, e.g., α2-globin, α1-globin, β-globin, e.g., β-globin, e.g., human β-globin.

[0293] In some embodiments, the 5'-UTR is or comprises a modified human alpha-globin 5'-UTR. A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 56. In some embodiments, the 3'-UTR comprises a first sequence derived from a split amino-terminal enhancer (AES) messenger RNA and a second sequence derived from a mitochondrially encoded 12S ribosomal RNA. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 58.

[0294] In some embodiments, the RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO:56, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:56.

[0295] In some embodiments, the RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO:58, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:58.

[0296] chemical modification The RNA (particularly mRNA) described herein can have modified ribonucleotides to increase its stability, and / or reduce its immunogenicity, and / or reduce its cytotoxicity.For example, in some embodiments, the uridine in the RNA (particularly mRNA) described herein is replaced by modified nucleoside (partially or completely, preferably completely).In some embodiments, the modified nucleoside is modified uridine.

[0297] In some embodiments, the modified uridine substituting for uridine is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U), and combinations thereof.

[0298] In some embodiments, modified nucleosides that replace (partially or completely, preferably completely) uridine in RNA are 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 4-thiouridine (s4U), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine). , uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyluridine (cm5U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-methyl 5-methylaminomethyluridine (mnm5U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyluridine (ncm5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine (τm5U), 1-taurinomethyluridine Methylpseudouridine, 5-taurinomethyl-2-thiouridine (m5s2U), 1-taurinomethyl-4-thiopseudouridine), 5-methyl-2-thiouridine (m5s2U), 1-methyl-4-thiopseudouridine (m1s4Ψ), 4-thio-1-methylpseudouridine, 3-methylpseudouridine (m3Ψ), 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m5D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m5Um), 2'-O-methylpseudouridine The uridine may be any one or more of uridine (Ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-aruridine, 2'-F-uridine, 2'-OH-aruridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine]uridine, or any other modified uridine known in the art.

[0299] RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as "Ψ-modified." The term "mΨ-modified" means that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (partially or completely, preferably completely replacing uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (partially or completely, preferably completely replacing uridine). Such Ψ-modified or mΨ-modified or m5U-modified RNAs typically exhibit reduced immunogenicity compared to their unmodified forms and are therefore preferred in applications where induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(1)-methylpseudouridine, completely replacing uridine.

[0300] Codon optimization and GC enrichment The codons of the RNA (particularly mRNA) described herein can be further optimized, for example, to increase the GC content of the RNA and / or to replace codons that are rare in a cell (or subject) in which a peptide or polypeptide of interest is to be expressed with codons that are synonymous and frequently occurring in the cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA (particularly mRNA) described herein is encoded by a codon-optimized coding sequence and / or a coding sequence whose G / C content is increased compared to a wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of a wild-type coding sequence. In some embodiments, the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0301] The term "codon-optimized" refers to the modification of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, preferably without modifying the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, the coding region can be codon-optimized for optimal expression in a subject treated with the RNA (especially mRNA) described herein. Codon optimization is based on the observation that translation efficiency is also determined by the different frequencies of occurrence of tRNA in cells. Therefore, the sequence of RNA (especially mRNA) can be modified so that codons for which frequently occurring tRNAs are available are inserted instead of "rare codons".

[0302] In some embodiments, the guanosine / cytosine (G / C) content of the coding region of an RNA (especially an mRNA) described herein is increased compared to the G / C content of the corresponding coding sequence of a wild-type RNA, and the amino acid sequence encoded by the RNA is preferably unaltered compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased A (adenosine) / U (uracil) content. Given the fact that several codons encode identical amino acids (the so-called degeneracy of the genetic code), the most favorable codons for stability can be determined (the so-called alternative codon usage). Depending on the amino acids encoded by the RNA, there are various possibilities for modifying the RNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acids but do not contain A and / or U nucleotides or contain a lower content of A and / or U nucleotides.

[0303] In various embodiments, the G / C content of the coding region of the RNA (particularly mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the coding region of the wild-type RNA.

[0304] non-immunogenic RNA As used herein, the term "non-immunogenic RNA" (e.g., "non-immunogenic mRNA") refers to RNA that, for example, when administered to a mammal, does not induce a response by the immune system or that induces a weaker response than that induced by the same RNA that differs only in that it has not been subjected to modifications and processing that render the non-immunogenic RNA non-immunogenic, i.e., a weaker response than that induced by standard RNA (stdRNA). In certain embodiments, the non-immunogenic RNA is made non-immunogenic by incorporating modified nucleosides into the RNA that suppress RNA-mediated activation of innate immune receptors and / or by limiting the amount of double-stranded RNA (dsRNA), for example, by limiting the formation of double-stranded RNA (dsRNA) during in vitro transcription, and / or by removing double-stranded RNA (dsRNA), for example, after in vitro transcription. In certain embodiments, the non-immunogenic RNA is made non-immunogenic by incorporating modified nucleosides into the RNA that suppress RNA-mediated activation of innate immune receptors and / or by removing double-stranded RNA (dsRNA), for example, after in vitro transcription.

[0305] To render non-immunogenic RNA (especially mRNA) non-immunogenic by incorporating modified nucleosides, any modified nucleoside may be used as long as it reduces or suppresses the immunogenicity of the RNA. Modified nucleosides that suppress RNA-mediated activation of innate immune receptors are particularly preferred. In some embodiments, the modified nucleoside comprises the substitution of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is 3-methyl-uridine (m3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 In certain embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (mψ), or 5-methyl-uridine (m5U), particularly N1-methyl-pseudouridine.

[0306] In some embodiments, the substitution of one or more uridines with nucleosides comprising modified nucleobases comprises substitution of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.

[0307] During the synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase, significant amounts of aberrant products, including double-stranded RNA (dsRNA), are produced due to the enzyme's unconventional activity. dsRNA induces inflammatory cytokines and activates effector enzymes, leading to the inhibition of protein synthesis. The formation of dsRNA can be limited during the synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP can be added once or several times during mRNA synthesis. Also, dsRNA can be removed from RNA, such as IVT RNA, by ion-pair reverse-phase HPLC using, for example, a nonporous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method using Escherichia coli RNase III can be used, which specifically hydrolyzes dsRNA but not ssRNA, thereby removing dsRNA contaminants from IVT RNA preparations. Furthermore, dsRNA can be separated from ssRNA by using cellulose materials. In some embodiments, RNA preparation is contacted with cellulose material, and ssRNA is separated from the cellulose material under conditions that allow dsRNA to bind to the cellulose material and do not allow ssRNA to bind to the cellulose material.Suitable methods for providing ssRNA are disclosed in, for example, WO2017 / 182524.

[0308] "Removing" or "removal," as used herein, refers to the characteristic of a population of a first substance, such as non-immunogenic RNA, that is separated from the vicinity of a population of a second substance, such as dsRNA, where the population of the first substance is not necessarily devoid of the second substance, and the population of the second substance is not necessarily devoid of the first substance. However, the population of the first substance that is characterized by the removal of the population of the second substance has a measurably lower content of the second substance compared to an unseparated mixture of the first substance and the second substance.

[0309] In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, and dsRNA (particularly dsmRNA) is removed from non-immunogenic RNA, for example, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in non-immunogenic RNA composition is dsRNA.In some embodiments, non-immunogenic RNA (particularly mRNA) does not contain or essentially does not contain dsRNA.In some embodiments, non-immunogenic RNA (particularly mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA. In some embodiments, the non-immunogenic RNA (particularly mRNA) composition comprises single-stranded nucleoside-modified RNA (particularly mRNA) and is substantially free of double-stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA (particularly mRNA) composition comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, at least 99.993%, at least 99.994%, at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single-stranded nucleoside-modified RNA compared to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0310] Various methods can be used to determine the amount of dsRNA.For example, sample can be contacted with dsRNA specific antibody, and the amount of antibody that binds to RNA can be considered as the measure of the amount of dsRNA in sample.The sample that contains known amount of dsRNA can be used as reference.

[0311] For example, RNA can be spotted on a membrane, for example, a nylon blotting membrane. For example, the membrane can be blocked in TBS-T buffer solution (20 mM TRIS pH 7.4, 137 mM NaCl, 0.1% (v / v) TWEEN®-20) containing 5% (w / v) skim milk powder. For detecting dsRNA, the membrane can be incubated with a dsRNA-specific antibody, for example, a dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). For example, after washing with TBS-T, the membrane can be incubated with a secondary antibody, for example, HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, catalog number 715-035-150), and the signal provided by the secondary antibody can be detected.

[0312] In some embodiments, non-immunogenic RNA (especially mRNA) is translated in cells more efficiently than standard RNA having the same sequence. In some embodiments, translation is enhanced 2-fold compared to its unmodified counterpart. In some embodiments, translation is enhanced 3-fold. In some embodiments, translation is enhanced 4-fold. In some embodiments, translation is enhanced 5-fold. In some embodiments, translation is enhanced 6-fold. In some embodiments, translation is enhanced 7-fold. In some embodiments, translation is enhanced 8-fold. In some embodiments, translation is enhanced 9-fold. In some embodiments, translation is enhanced 10-fold. In some embodiments, translation is enhanced 15-fold. In some embodiments, translation is enhanced 20-fold. In some embodiments, translation is enhanced 50-fold. In some embodiments, translation is enhanced 100-fold. In some embodiments, translation is enhanced 200-fold. In some embodiments, translation is enhanced 500-fold. In some embodiments, translation is enhanced 1000-fold. In some embodiments, translation is enhanced 2000-fold. In some embodiments, translation is enhanced 1000-fold. In some embodiments, translation is enhanced 2000-fold. In some embodiments, the factor is between 10-1000 fold. In some embodiments, the factor is 10-100 fold. In some embodiments, the factor is 10-200 fold. In some embodiments, the factor is 10-300 fold. In some embodiments, the factor is 10-500 fold. In some embodiments, the factor is 20-1000 fold. In some embodiments, the factor is 30-1000 fold. In some embodiments, the factor is 50-1000 fold. In some embodiments, the factor is 100-1000 fold. In some embodiments, the factor is 200-1000 fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0313] In some embodiments, non-immunogenic RNA (especially mRNA) exhibits significantly lower natural immunogenicity than standard RNA having the same sequence. In some embodiments, non-immunogenic RNA (especially mRNA) exhibits a 2-fold lower natural immune response than its unmodified counterpart. In some embodiments, the natural immunogenicity is reduced 3-fold. In some embodiments, the natural immunogenicity is reduced 4-fold. In some embodiments, the natural immunogenicity is reduced 5-fold. In some embodiments, the natural immunogenicity is reduced 6-fold. In some embodiments, the natural immunogenicity is reduced 7-fold. In some embodiments, the natural immunogenicity is reduced 8-fold. In some embodiments, the natural immunogenicity is reduced 9-fold. In some embodiments, the natural immunogenicity is reduced 10-fold. In some embodiments, the natural immunogenicity is reduced 15-fold. In some embodiments, the natural immunogenicity is reduced 20-fold. In some embodiments, the natural immunogenicity is reduced 50-fold. In some embodiments, the natural immunogenicity is reduced 100-fold. In some embodiments, the natural immunogenicity is reduced 200-fold. In some embodiments, the natural immunogenicity is reduced by 500-fold, in some embodiments, the natural immunogenicity is reduced by 1000-fold, in some embodiments, the natural immunogenicity is reduced by 2000-fold.

[0314] The term "exhibiting significantly reduced natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In some embodiments, this term refers to a reduction such that an effective amount of non-immunogenic RNA (especially mRNA) can be administered without eliciting a detectable natural immune response. In some embodiments, this term refers to a reduction such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting a natural immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting a natural immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

[0315] "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in humans or other animals. The innate immune system is a relatively nonspecific, immediate component of the immune system. It is one of the two main components of the vertebrate immune system, along with the adaptive immune system.

[0316] Antigen-encoding RNA and its use for inducing an immune response Generally, the RNA (particularly mRNA) described in this disclosure comprises a nucleic acid sequence that encodes a peptide or polypeptide comprising one or more Mycobacterium tuberculosis antigens, immunogenic variants or fragments thereof, for inducing an immune response against Mycobacterium tuberculosis in a subject. The peptides or polypeptides for inducing an immune response are also referred to herein as "vaccine antigens" or simply "antigens."

[0317] In some embodiments, the RNA (particularly mRNA) is translated into the respective protein once it enters the cells of the subject to which it is administered, such as muscle cells or antigen-presenting cells (APCs).

[0318] In some embodiments, RNA encoding the vaccine antigen is expressed in the subject's cells to provide the vaccine antigen. In some embodiments, RNA encoding the vaccine antigen is transiently expressed in the subject's cells. In some embodiments, the vaccine antigen is presented in the context of MHC. In some embodiments, the vaccine antigen is secreted by the subject's cells.

[0319] In some embodiments, RNA encoding a vaccine antigen is administered intramuscularly.

[0320] In some embodiments, the RNA encoding the vaccine antigen is administered systemically, for example, intravenously. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen occurs in the spleen. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen occurs in antigen-presenting cells, preferably professional antigen-presenting cells. In some embodiments, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, no or essentially no expression of the RNA encoding the vaccine antigen occurs in the lung and / or liver. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen in the spleen is at least five times higher than the expression level in the lung.

[0321] Vaccine antigens comprise epitopes for inducing immune responses to disease-related antigens, such as proteins of infectious agents (e.g., Mtb antigens) in subjects.Therefore, vaccine antigens comprise antigen sequences for inducing immune responses to disease-related antigens in subjects.Such antigen sequences can correspond to target antigens or disease-related antigens, their immunogenic variants, or immunogenic fragments of target antigens or disease-related antigens or their immunogenic variants.Therefore, antigen sequences can comprise at least one epitope of target antigens or disease-related antigens or their immunogenic variants.

[0322] Antigen sequences, e.g., epitopes, suitable for use in accordance with the present disclosure may typically be derived from target antigens, i.e., antigens against which an immune response is elicited. For example, the antigen sequence included within a vaccine antigen may be the target antigen or a fragment or variant of the target antigen.

[0323] The antigen sequence or its processing product, e.g., a fragment thereof, can bind to an antigen receptor, such as a TCR, carried by an immune effector cell. In some embodiments, the antigen sequence is selected from the group consisting of an antigen or fragment thereof expressed by a target cell targeted by the immune effector cell, or a variant of the antigen sequence or fragment.

[0324] The vaccine antigens that can be provided to a subject according to the present disclosure by administering RNA encoding the vaccine antigen preferably result in the induction of an immune response in the subject to which the vaccine antigen is provided, e.g., stimulation, priming, and / or expansion of immune effector cells. The immune response, e.g., stimulated, primed, and / or expanded immune effector cells, is preferably directed against a target antigen, particularly a target antigen expressed in diseased cells, tissues, and / or organs, i.e., a disease-associated antigen. Thus, the vaccine antigen can include a disease-associated antigen, or a fragment or variant thereof. In some embodiments, such a fragment or variant is immunologically equivalent to the disease-associated antigen.

[0325] The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to a subject's immune system, the amino acid sequence induces an immune response with specificity that reacts with the reference amino acid sequence. Thus, in some embodiments, a molecule immunologically equivalent to an antigen exhibits the same or essentially the same properties as the antigen targeted by T cells and / or exerts the same or essentially the same effect with respect to stimulating, priming, and / or expanding T cells.

[0326] In the context of the present disclosure, the term "antigen fragment" or "antigen variant" refers to an agent that induces an immune response, e.g., stimulation, priming, and / or expansion of immune effector cells, where the immune response, e.g., stimulated, primed, and / or expanded immune effector cells, target an antigen, i.e., a disease-associated antigen, particularly when presented by diseased cells, tissues, and / or organs. Thus, a vaccine antigen may correspond to or comprise a disease-associated antigen, a fragment of a disease-associated antigen, or an antigen homologous to a disease-associated antigen or its fragment. When a vaccine antigen comprises a fragment of a disease-associated antigen or an amino acid sequence homologous to a fragment of a disease-associated antigen, the fragment or amino acid sequence may comprise an epitope of the disease-associated antigen or a sequence homologous to an epitope of the disease-associated antigen targeted by an antigen receptor of an immune effector cell. Thus, according to the present disclosure, a vaccine antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that can induce, e.g., stimulate, prime, and / or expand, an immune response against immune effector cells bearing an antigen receptor that binds to the antigen or cells expressing the antigen. Preferably, the vaccine antigen (analogous to a disease-associated antigen) provides a relevant epitope for binding by an antigen receptor present on immune effector cells. In some embodiments, the vaccine antigen or a fragment thereof (analogous to a disease-associated antigen) is expressed (optionally in the context of an MHC) on the surface of a cell, such as an antigen-presenting cell, to provide a relevant epitope for binding by the immune effector cell. The vaccine antigen may be a recombinant antigen.

[0327] In some embodiments of all aspects described herein, RNA encoding the vaccine antigen is expressed in cells of a subject to provide the antigen or its processing products for binding by antigen receptors expressed by immune effector cells, which binding results in stimulation, priming, and / or expansion of the immune effector cells.

[0328] An "antigen" according to the present disclosure encompasses any substance that elicits an immune response and / or any substance against which an immune response or mechanism, such as a cellular and / or humoral response, is directed. This also includes situations in which an immune response or mechanism is directed against one or more antigenic peptides, particularly when the antigen is processed into antigenic peptides and presented in the context of MHC molecules. In particular, "antigen" relates to any substance, such as a peptide or polypeptide, that specifically reacts with antibodies or T lymphocytes (T cells). The term "antigen" can include molecules that contain at least one epitope, such as a T cell epitope. In some embodiments, an antigen is a molecule that, optionally after processing, induces an immune response that may be specific to the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-associated antigen, such as an Mtb antigen.

[0329] In some embodiments, the antigen is presented or present on the surface of a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or macrophage. In some embodiments, the antigen or its processing product, such as a T cell epitope, is bound by an antigen receptor. Thus, the antigen or its processing product can specifically react with immune effector cells, such as T lymphocytes (T cells).

[0330] According to the present disclosure, the antigens or combinations of antigens described herein can induce an immune response, which may include a humoral or cellular immune response, or both. In some embodiments of the present disclosure, the antigens are presented by cells, such as antigen-presenting cells, in association with MHC molecules, resulting in an immune response against the antigen. The antigens can correspond to naturally occurring antigens or products derived from naturally occurring antigens. According to the present disclosure, the antigens can correspond to naturally occurring products.

[0331] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. In some embodiments, a disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms, typically microbial antigens (bacterial or viral antigens, such as Mtb antigens), or antigens associated with cancer, typically tumors, e.g., tumor antigens.

[0332] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane. The term "bacterial antigen" includes Mtb antigens, such as the Mtb antigens described herein.

[0333] The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, particularly when presented in the context of an MHC molecule. An epitope of a protein can include a continuous or discontinuous portion of the protein and can be, e.g., about 5 to about 100, about 5 to about 50, about 8 to about 30, or about 10 to about 25 amino acids in length; for example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope in the context of the present disclosure is a T cell epitope.

[0334] Terms such as "epitope," "fragment of an antigen," "immunogenic peptide," and "antigenic peptide" are used interchangeably herein and may refer, for example, to an incompletely displayed form of an antigen that can elicit an immune response against the antigen or a cell that expresses or contains and presents the antigen. In some embodiments, these terms relate to an immunogenic portion of an antigen. In some embodiments, this is the portion of the antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly when presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100 amino acids in length, e.g., about 5 to about 50, about 8 to about 30, or about 8 to about 25 amino acids in length; for example, an epitope can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T-cell epitopes.

[0335] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or disease cells during the immune response; they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. For class I MHC / peptide complexes, the bound peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides can be effective. For class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer and shorter peptides may be effective.

[0336] Peptide and polypeptide antigens can be 2 to 100 amino acids in length, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0337] A peptide or polypeptide antigen can be any peptide or polypeptide that is capable of inducing or increasing the ability of the immune system to generate antibody and T cell responses against the peptide or polypeptide.

[0338] In some embodiments, vaccine antigens, i.e., antigens whose inoculation into a subject induces an immune response, are recognized by immune effector cells. In some embodiments, when recognized by immune effector cells, vaccine antigens can, in the presence of appropriate costimulatory signals, induce the stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the vaccine antigen. In connection with embodiments of the present disclosure, vaccine antigens can be presented or present on the surface of cells, such as, for example, antigen-presenting cells.

[0339] In some embodiments, the antigen is expressed in diseased cells (such as infected cells).

[0340] In some embodiments, the antigen is presented by a diseased cell (such as an infected cell). In some embodiments, the antigen receptor is a TCR that binds to an epitope of the antigen presented in the context of MHC. In some embodiments, binding of a TCR, when expressed by and / or present on a T cell, to an antigen presented by a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or expansion of the T cell. In some embodiments, binding of a TCR, when expressed by and / or present on a T cell, to an antigen presented on a diseased cell results in cytolysis and / or apoptosis of the diseased cell, and the T cell releases cytotoxic factors, such as perforin and granzymes.

[0341] In some embodiments, the antigen receptor is an antibody or B cell receptor that binds to an epitope in the antigen. In some embodiments, the antibody or B cell receptor binds to a natural epitope of the antigen.

[0342] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells), including cytolytic T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes the antigen it targets, particularly when presented on the surface of an antigen-presenting cell or diseased cell in association with an MHC molecule, and preferably exerts T cell effector function. A T cell is considered specific for an antigen if it kills a target cell expressing the antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, in a chromium release assay or proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured.

[0343] In some embodiments, the term "target" refers to an agent, such as a cell or tissue, that is the target of an immune response, such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen. In some embodiments, target cells are cells that express an antigen and present the antigen in association with class I MHC.

[0344] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a polypeptide into peptides), and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by cells such as antigen-presenting cells. Antigen-presenting cells can be distinguished into professional and non-professional antigen-presenting cells.

[0345] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules, which are necessary for interaction with naive T cells. When T cells interact with the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0346] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do so upon stimulation with certain cytokines, such as interferon gamma. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0347] The term "dendritic cell" (DC) refers to a subtype of phagocyte belonging to the class of antigen-presenting cells. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitors. These progenitor cells first transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Upon contact with presentable antigens, they are activated to become mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, degrade their proteins into small fragments, and upon maturation, present these fragments on their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that function as coreceptors in T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that directs dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to lymph nodes. Here, they act as antigen-presenting cells, activating helper T cells, killer T cells, and B cells by presenting antigens together with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce immune responses associated with T cells or B cells. In some embodiments, the dendritic cells are splenic dendritic cells.

[0348] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf and kill foreign pathogens within the macrophage through hydrolytic and oxidative attack, resulting in the degradation of the pathogen. Peptides derived from degraded proteins are presented on the macrophage cell surface, where they can be recognized by T cells and directly interact with antibodies on the surface of B cells, leading to the activation of T cells and B cells and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In some embodiments, the macrophages are splenic macrophages.

[0349] "Antigen-responsive CTL" refers to a CD8 CTL that is responsive to an antigen or a peptide derived from the antigen, which is presented together with class I MHC on the surface of an antigen-presenting cell. + It means T cells.

[0350] According to the present disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing tumor antigens. CTL responsiveness can also be determined using artificial reporters that accurately represent CTL responsiveness.

[0351] As used herein, "activation" or "stimulation" refers to the state of cells, such as immune effector cells, such as T cells, that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cells" refers, among other things, to immune effector cells undergoing cell division.

[0352] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, resulting in differentiation into an effector cell, such as an effector T cell.

[0353] The term "expansion" refers to the process by which a particular entity multiplies. In some embodiments, the term is used in the context of an immunological response in which immune effector cells are stimulated by an antigen, causing proliferation and amplification of specific immune effector cells that recognize the antigen. In some embodiments, expansion results in differentiation of immune effector cells.

[0354] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense to refer to the integrated body's response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the terms "immune response to" or "immune response against," in reference to an agent such as an antigen, cell, or tissue, refer to an immune response, such as a cellular response, to the agent. An immune response is characterized by the development of antibodies against one or more antigens, as well as antigen-specific T lymphocytes, e.g., CD4, which can be detected in various in vitro proliferation or cytokine production tests. + and CD8 + T lymphocytes, e.g., CD8 + The reaction may include one or more responses selected from the group consisting of: T lymphocyte expansion.

[0355] The terms "inducing an immune response" and "eliciting an immune response" and similar terms in the context of the present disclosure refer to the induction of an immune response, for example, the induction of a cellular immune response, a humoral immune response, or both. The immune response can be protective / protective / prophylactic and / or therapeutic. The immune response can be directed against any immunogen or antigen or antigenic peptide, such as a pathogen-associated antigen (e.g., an antigen of Mtb). "Inducing" in this context can mean that there was no immune response against a particular antigen or pathogen before induction, but it can also mean that there was a certain level of immune response against a particular antigen or pathogen before induction, and that the immune response is enhanced after induction. Thus, "inducing an immune response" in this context also includes "enhancing an immune response." In some embodiments, after inducing an immune response in an individual, the individual is protected from developing a disease, such as an infectious disease, or the disease state is ameliorated by inducing an immune response.

[0356] The terms "cellular immune response," "cellular response," "cellular immunity," or similar terms are intended to include a cellular response to cells characterized by expression of an antigen and / or presentation of the antigen by class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as either "helpers" or "killers." Helper T cells (CD4 + T cells (also called T cells) play a central role by regulating the immune response and are responsible for the production of killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells, including diseased cells.

[0357] The term "humoral immune response" refers to the process in living organisms by which antibodies are produced in response to agents and organisms, ultimately neutralizing and / or eliminating them. The specificity of the antibody response is mediated by T cells and / or B cells through membrane-bound receptors that bind to a single specific antigen. After binding the appropriate antigen and receiving various other activation signals, B lymphocytes divide, producing memory B cells and antibody-secreting plasma cell clones, each of which produces antibodies that recognize the same antigen epitope recognized by its antigen receptor. Memory B lymphocytes remain dormant until subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and the amplified antibody response that occurs upon re-exposure to a specific antigen.

[0358] The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to an epitope on an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2, and HCDR3, and the CDRs of VL are called LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), and the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from amino terminus to carboxy terminus: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or immunologically active portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0359] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, e.g., antibodies or antigen receptors such as B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains, with a characteristic immunoglobulin (Ig) fold. The term encompas...

Claims

1. 1. A composition or pharmaceutical formulation comprising at least one RNA molecule, wherein said at least one RNA molecule encodes a set of antigenic amino acid sequences, wherein said set of antigenic amino acid sequences comprises (i) at least one Mtb antigen from the acute phase of the Mtb life cycle, an immunogenic variant thereof, or an immunogenic fragment of said Mtb antigen or said immunogenic variant thereof, (ii) at least one Mtb antigen from the latent phase of the Mtb life cycle, an immunogenic variant thereof, or an immunogenic fragment of said Mtb antigen or said immunogenic variant thereof, and (iii) at least one Mtb antigen from the resuscitation phase of the Mtb life cycle, an immunogenic variant thereof, or an immunogenic fragment of said Mtb antigen or said immunogenic variant thereof.

2. the at least one Mtb antigen from the acute phase of the Mtb life cycle, an immunogenic variant thereof, or an immunogenic fragment of the Mtb antigen or the immunogenic variant thereof, (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of said Ag85A or said immunogenic variant thereof; and / or (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of said ESAT6 or said immunogenic variant thereof; 2. The composition or pharmaceutical formulation of claim 1, comprising:

3. the at least one Mtb antigen from the latent stage of the Mtb life cycle, an immunogenic variant thereof, or an immunogenic fragment of the Mtb antigen or the immunogenic variant thereof, (i) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of said VapB47 or said immunogenic variant thereof; and / or (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of said Hrp1 or said immunogenic variant thereof; 3. The composition or pharmaceutical formulation of claim 1 or 2, comprising:

4. the at least one Mtb antigen from the resuscitation stage of the Mtb life cycle, an immunogenic variant thereof, or an immunogenic fragment of the Mtb antigen or the immunogenic variant thereof, (i) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or said immunogenic variant thereof; and / or (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or said immunogenic variant thereof The composition or pharmaceutical formulation according to any one of claims 1 to 3, comprising:

5. 1. A composition or pharmaceutical formulation comprising at least one RNA molecule, wherein said at least one RNA molecule encodes a set of antigenic amino acid sequences, each antigenic amino acid sequence comprising an Mtb antigen, an immunogenic variant thereof, or an immunogenic fragment of said Mtb antigen or said immunogenic variant thereof, and wherein each RNA molecule encodes at least two of said antigenic amino acid sequences as a fusion molecule.

6. 6. The composition or pharmaceutical preparation of claim 5, wherein each RNA molecule encodes two of the antigenic amino acid sequences as a fusion molecule.

7. 7. The composition or pharmaceutical formulation of claim 5 or 6, wherein the Mtb antigen, immunogenic variant, or immunogenic fragment in the fusion molecule is not linked by a linker comprising a sequence heterologous to the Mtb antigen, immunogenic variant, or immunogenic fragment.

8. The set of antigen amino acid sequences is (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of said Ag85A or said immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of said ESAT6 or said immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of said VapB47 or said immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of said Hrp1 or said immunogenic variant thereof; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or said immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or said immunogenic variant thereof; (vii) an amino acid sequence comprising Mtb32a, an immunogenic variant thereof, or an immunogenic fragment of said Mtb32a or said immunogenic variant thereof; (viii) an amino acid sequence comprising Mtb39a, an immunogenic variant thereof, or an immunogenic fragment of said Mtb39a or said immunogenic variant thereof; and (ix) an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of said HbhA or said immunogenic variant thereof; 8. The composition or pharmaceutical formulation of claim 1, comprising two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more of the following:

9. A composition or pharmaceutical formulation comprising at least one RNA molecule, wherein said at least one RNA molecule encodes at least one antigenic amino acid sequence comprising an Mtb antigen, an immunogenic variant thereof, or an immunogenic fragment of said Mtb antigen or said immunogenic variant thereof, said RNA encoding an amino acid sequence comprising a secretory signal peptide at the N-terminus of said encoded amino acid sequence; (i) the secretory signal peptide comprises the amino acid sequence of positions 1 to 26 of SEQ ID NO:44, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 1 to 26 of SEQ ID NO:44, or a functional fragment of the amino acid sequence of positions 1 to 26 of SEQ ID NO:44 or the amino acid sequence of positions 1 to 26 of SEQ ID NO:44 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 1 to 26 of SEQ ID NO:44; and / or (ii) the RNA sequence encoding the secretory signal peptide comprises the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43 or the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43; Composition or pharmaceutical preparation.

10. 1. A composition or pharmaceutical formulation comprising at least one RNA molecule, wherein said at least one RNA molecule encodes at least one antigenic amino acid sequence comprising an Mtb antigen, an immunogenic variant thereof, or an immunogenic fragment of said Mtb antigen or said immunogenic variant thereof, wherein said RNA comprises modified uridines and / or is formulated in a lipid nanoparticle.

11. the at least one RNA molecule (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of said Ag85A or said immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of said ESAT6 or said immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of said VapB47 or said immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of said Hrp1 or said immunogenic variant thereof; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or said immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or said immunogenic variant thereof; (vii) an amino acid sequence comprising Mtb32a, an immunogenic variant thereof, or an immunogenic fragment of said Mtb32a or said immunogenic variant thereof; (viii) an amino acid sequence comprising Mtb39a, an immunogenic variant thereof, or an immunogenic fragment of said Mtb39a or said immunogenic variant thereof; and (ix) an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of said HbhA or said immunogenic variant thereof; 11. The composition or pharmaceutical formulation of claim 1, comprising two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more of the following:

12. The at least one RNA molecule has the following amino acid sequence: (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of said Ag85A or said immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of said ESAT6 or said immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of said VapB47 or said immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of said Hrp1 or said immunogenic variant thereof; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or said immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or said immunogenic variant thereof; (vii) an amino acid sequence comprising Mtb32a, an immunogenic variant thereof, or an immunogenic fragment of said Mtb32a or said immunogenic variant thereof; (viii) an amino acid sequence comprising Mtb39a, an immunogenic variant thereof, or an immunogenic fragment of said Mtb39a or said immunogenic variant thereof; and (ix) an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of said HbhA or said immunogenic variant thereof; The composition or pharmaceutical preparation according to any one of claims 1 to 11, which encodes

13. 1. A composition or pharmaceutical formulation comprising at least one RNA molecule, wherein said at least one RNA molecule has the following amino acid sequence: (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of said Ag85A or said immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of said ESAT6 or said immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of said VapB47 or said immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of said Hrp1 or said immunogenic variant thereof; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or said immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or said immunogenic variant thereof; (vii) an amino acid sequence comprising Mtb32a, an immunogenic variant thereof, or an immunogenic fragment of said Mtb32a or said immunogenic variant thereof; (viii) an amino acid sequence comprising Mtb39a, an immunogenic variant thereof, or an immunogenic fragment of said Mtb39a or said immunogenic variant thereof; and (ix) an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of said HbhA or said immunogenic variant thereof; A composition or pharmaceutical formulation encoding the

14. 14. The composition or pharmaceutical preparation of any one of claims 8 and 11 to 13, wherein the amino acid sequence comprising Mtb32a, an immunogenic variant thereof, or an immunogenic fragment of said Mtb32a or said immunogenic variant thereof, and the amino acid sequence comprising Mtb39a, an immunogenic variant thereof, or an immunogenic fragment of said Mtb39a or said immunogenic variant thereof, are present as a fusion protein.

15. 15. The composition or pharmaceutical preparation of claim 14, wherein the fusion protein comprises an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or the immunogenic variant thereof.

16. The at least one RNA molecule has the following amino acid sequence: (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of said Ag85A or said immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of said ESAT6 or said immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of said VapB47 or said immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of said Hrp1 or said immunogenic variant thereof; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or said immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or said immunogenic variant thereof; (vii) an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of said M72 or said immunogenic variant thereof; and (viii) an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of said HbhA or said immunogenic variant thereof; The composition or pharmaceutical preparation according to any one of claims 1 to 15, which encodes

17. 1. A composition or pharmaceutical formulation comprising at least one RNA molecule, wherein said at least one RNA molecule has the following amino acid sequence: (i) an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of said Ag85A or said immunogenic variant thereof; (ii) an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of said ESAT6 or said immunogenic variant thereof; (iii) an amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of said VapB47 or said immunogenic variant thereof; (iv) an amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of said Hrp1 or said immunogenic variant thereof; (v) an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or said immunogenic variant thereof; (vi) an amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or said immunogenic variant thereof; (vii) an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of said M72 or said immunogenic variant thereof; and (viii) an amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of said HbhA or said immunogenic variant thereof; A composition or pharmaceutical formulation encoding the

18. (i) the amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or of said immunogenic variant thereof comprises the amino acid sequence of positions 27 to 323 of SEQ ID NO:44, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 27 to 323 of SEQ ID NO:44, or an immunogenic fragment of the amino acid sequence of positions 27 to 323 of SEQ ID NO:44 or the amino acid sequence of positions 27 to 323 of SEQ ID NO:44 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 27 to 323 of SEQ ID NO:44; and / or (ii) the RNA sequence encoding the amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or the immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 1022 of SEQ ID NO:43, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1022 of SEQ ID NO:43, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1022 of SEQ ID NO:43 or the nucleotide sequence of positions 132 to 1022 of SEQ ID NO:43; A composition or pharmaceutical formulation according to any one of claims 8 and 11 to 17.

19. (i) the amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or said immunogenic variant thereof comprises the amino acid sequence of positions 27 to 120 of SEQ ID NO:46, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 120 of SEQ ID NO:46, or an immunogenic fragment of the amino acid sequence of positions 27 to 120 of SEQ ID NO:46 or the amino acid sequence of positions 27 to 120 of SEQ ID NO:46 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 120 of SEQ ID NO:46; and / or (ii) the RNA sequence encoding the amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of ESAT6 or the immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 413 of SEQ ID NO:45, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 413 of SEQ ID NO:45, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 413 of SEQ ID NO:45 or the nucleotide sequence of positions 132 to 413 of SEQ ID NO:45; A composition or pharmaceutical formulation according to any one of claims 8 and 11 to 18.

20. (i) the amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or of said immunogenic variant thereof comprises the amino acid sequence of positions 749 to 846 of SEQ ID NO: 52, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said amino acid sequence of positions 749 to 846 of SEQ ID NO: 52, or an immunogenic fragment of said amino acid sequence of positions 749 to 846 of SEQ ID NO: 52 or said amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said amino acid sequence of positions 749 to 846 of SEQ ID NO: 52; and / or (ii) the RNA sequence encoding the amino acid sequence comprising VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or the immunogenic variant thereof comprises the nucleotide sequence of positions 2298 to 2591 of SEQ ID NO:51, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 2298 to 2591 of SEQ ID NO:51, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 2298 to 2591 of SEQ ID NO:51 or the nucleotide sequence of positions 2298 to 2591 of SEQ ID NO:51; A composition or pharmaceutical formulation according to any one of claims 8 and 11 to 19.

21. (i) the amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or the immunogenic variant thereof comprises the amino acid sequence of positions 324 to 465 of SEQ ID NO: 44, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 324 to 465 of SEQ ID NO: 44, or an immunogenic fragment of the amino acid sequence of positions 324 to 465 of SEQ ID NO: 44 or the amino acid sequence of positions 324 to 465 of SEQ ID NO: 44 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 324 to 465 of SEQ ID NO: 44; and / or (ii) the RNA sequence encoding the amino acid sequence comprising Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or the immunogenic variant thereof comprises the nucleotide sequence of positions 1023 to 1448 of SEQ ID NO:43, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1023 to 1448 of SEQ ID NO:43, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1023 to 1448 of SEQ ID NO:43 or the nucleotide sequence of positions 1023 to 1448 of SEQ ID NO:43; A composition or pharmaceutical formulation according to any one of claims 8 and 11 to 20.

22. (i) the amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or of said immunogenic variant thereof comprises the amino acid sequence of positions 27 to 432 of SEQ ID NO: 48, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said amino acid sequence of positions 27 to 432 of SEQ ID NO: 48, or an immunogenic fragment of said amino acid sequence of positions 27 to 432 of SEQ ID NO: 48 or said amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said amino acid sequence of positions 27 to 432 of SEQ ID NO: 48; and / or (ii) the RNA sequence encoding the amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or the immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 1349 of SEQ ID NO: 47, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of positions 132 to 1349 of SEQ ID NO: 47, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of positions 132 to 1349 of SEQ ID NO: 47 or the nucleotide sequence of positions 132 to 1349 of SEQ ID NO: 47; A composition or pharmaceutical formulation according to any one of claims 8 and 11 to 21.

23. (i) the amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or of said immunogenic variant thereof comprises the amino acid sequence of positions 121 to 273 of SEQ ID NO: 46, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said amino acid sequence of positions 121 to 273 of SEQ ID NO: 46, or an immunogenic fragment of said amino acid sequence of positions 121 to 273 of SEQ ID NO: 46 or said amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said amino acid sequence of positions 121 to 273 of SEQ ID NO: 46; and / or (ii) the RNA sequence encoding the amino acid sequence comprising RpfD, an immunogenic variant thereof, or an immunogenic fragment of RpfD or the immunogenic variant thereof comprises the nucleotide sequence of positions 414 to 872 of SEQ ID NO: 45, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of positions 414 to 872 of SEQ ID NO: 45, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of positions 414 to 872 of SEQ ID NO: 45 or the nucleotide sequence of positions 414 to 872 of SEQ ID NO: 45; A composition or pharmaceutical formulation according to any one of claims 8 and 11 to 22.

24. (i) the amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or of said immunogenic variant thereof comprises the amino acid sequence of positions 27 to 748 of SEQ ID NO: 52, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 27 to 748 of SEQ ID NO: 52, or an immunogenic fragment of the amino acid sequence of positions 27 to 748 of SEQ ID NO: 52 or the amino acid sequence of positions 27 to 748 of SEQ ID NO: 52 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 27 to 748 of SEQ ID NO: 52; and / or (ii) the RNA sequence encoding the amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or the immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 2297 of SEQ ID NO:51, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 2297 of SEQ ID NO:51, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 2297 of SEQ ID NO:51 or the nucleotide sequence of positions 132 to 2297 of SEQ ID NO:51; A composition or pharmaceutical formulation according to any one of claims 8 and 15 to 23.

25. (i) the amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or of said immunogenic variant thereof comprises the amino acid sequence of positions 433 to 630 of SEQ ID NO: 48, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 433 to 630 of SEQ ID NO: 48, or an immunogenic fragment of the amino acid sequence of positions 433 to 630 of SEQ ID NO: 48 or the amino acid sequence of positions 433 to 630 of SEQ ID NO: 48 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 433 to 630 of SEQ ID NO: 48; and / or (ii) the RNA sequence encoding the amino acid sequence comprising HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or the immunogenic variant thereof comprises the nucleotide sequence of positions 1350 to 1943 of SEQ ID NO: 47, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1350 to 1943 of SEQ ID NO: 47, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 1350 to 1943 of SEQ ID NO: 47 or the nucleotide sequence of positions 1350 to 1943 of SEQ ID NO: 47; A composition or pharmaceutical formulation according to any one of claims 8 and 11 to 24.

26. (i) an RNA molecule encoding an amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of said Ag85A or said immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of said Hrp1 or said immunogenic variant thereof; (ii) an RNA molecule encoding an amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of said ESAT6 or said immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or said immunogenic variant thereof; (iii) an RNA molecule encoding an amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or said immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of said HbhA or said immunogenic variant thereof; and (iv) an RNA molecule encoding an amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of said M72 or said immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of said VapB47 or said immunogenic variant thereof; 26. The composition or pharmaceutical formulation of any one of claims 1 to 25, comprising:

27. (i) the amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or the immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or the immunogenic variant thereof comprises the amino acid sequence of positions 27 to 465 of SEQ ID NO: 44, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 465 of SEQ ID NO: 44; and / or (ii) the RNA sequence encoding the amino acid sequence comprising Ag85A, an immunogenic variant thereof, or an immunogenic fragment of Ag85A or the immunogenic variant thereof, and Hrp1, an immunogenic variant thereof, or an immunogenic fragment of Hrp1 or the immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 1448 of SEQ ID NO: 43, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 1448 of SEQ ID NO: 43; 27. A composition or pharmaceutical formulation according to claim 26.

28. (i) the amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of said ESAT6 or said immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or said immunogenic variant thereof comprises the amino acid sequence of positions 27 to 273 of SEQ ID NO: 46, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of positions 27 to 273 of SEQ ID NO: 46; and / or (ii) the RNA sequence encoding the amino acid sequence comprising ESAT6, an immunogenic variant thereof, or an immunogenic fragment of said ESAT6 or said immunogenic variant thereof, and RpfD, an immunogenic variant thereof, or an immunogenic fragment of said RpfD or said immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 872 of SEQ ID NO: 45, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 132 to 872 of SEQ ID NO: 45; 28. A composition or pharmaceutical formulation according to claim 26 or 27.

29. (i) the amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of said RpfA or said immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of said HbhA or said immunogenic variant thereof comprises the amino acid sequence of positions 27 to 630 of SEQ ID NO: 48, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said amino acid sequence of positions 27 to 630 of SEQ ID NO: 48; and / or (ii) the RNA sequence encoding the amino acid sequence comprising RpfA, an immunogenic variant thereof, or an immunogenic fragment of RpfA or the immunogenic variant thereof, and HbhA, an immunogenic variant thereof, or an immunogenic fragment of HbhA or the immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 1943 of SEQ ID NO: 47, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of positions 132 to 1943 of SEQ ID NO: 47; A composition or pharmaceutical formulation according to any one of claims 26 to 28.

30. (i) the amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of said M72 or said immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of said VapB47 or said immunogenic variant thereof comprises the amino acid sequence of positions 27 to 846 of SEQ ID NO: 52, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said amino acid sequence of positions 27 to 846 of SEQ ID NO: 52; and / or (ii) the RNA sequence encoding the amino acid sequence comprising M72, an immunogenic variant thereof, or an immunogenic fragment of M72 or the immunogenic variant thereof, and VapB47, an immunogenic variant thereof, or an immunogenic fragment of VapB47 or the immunogenic variant thereof comprises the nucleotide sequence of positions 132 to 2591 of SEQ ID NO:51, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of positions 132 to 2591 of SEQ ID NO:51; A composition or pharmaceutical formulation according to any one of claims 26 to 29.

31. The composition or pharmaceutical preparation of any one of claims 1 to 8 and 10 to 30, wherein the RNA encodes an amino acid sequence including a secretory signal peptide.

32. 32. The composition or pharmaceutical preparation of claim 31 , wherein the secretory signal peptide is fused to the amino acid sequence, preferably at the N-terminus.

33. (i) the secretory signal peptide comprises the amino acid sequence of positions 1 to 26 of SEQ ID NO:44, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 1 to 26 of SEQ ID NO:44, or a functional fragment of the amino acid sequence of positions 1 to 26 of SEQ ID NO:44 or the amino acid sequence of positions 1 to 26 of SEQ ID NO:44 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of positions 1 to 26 of SEQ ID NO:44; and / or (ii) the RNA sequence encoding the secretory signal peptide comprises the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43 or the nucleotide sequence of positions 54 to 131 of SEQ ID NO: 43; 33. A composition or pharmaceutical formulation according to claim 31 or 32.

34. (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 43, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said nucleotide sequence of SEQ ID NO: 43; (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 45, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to said nucleotide sequence of SEQ ID NO: 45; (iii) an RNA comprising the nucleotide sequence of SEQ ID NO: 47, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 47; and (iv) RNA comprising the nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:

51.

34. The composition or pharmaceutical formulation of any one of claims 1 to 33, comprising:

35. (i) RNA comprising the nucleotide sequence of SEQ ID NO: 43; (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 45; (iii) RNA comprising the nucleotide sequence of SEQ ID NO: 47; and (iv) RNA comprising the nucleotide sequence of SEQ ID NO: 51 35. The composition or pharmaceutical formulation of any one of claims 1 to 34, comprising:

36. 36. The composition or pharmaceutical formulation of any one of claims 1 to 35, wherein the RNA is formulated in lipid nanoparticles.

37. The lipid nanoparticles are (i) a cationic ionizable lipid; (ii) steroids; (iii) neutral lipids; and (iv) polymer-conjugated lipids 37. The composition or pharmaceutical formulation of claim 36, comprising each of:

38. 38. The composition or pharmaceutical formulation of claim 37, wherein the cationic ionizable lipid is present at a concentration ranging from about 40 to about 60 mol % of total lipid.

39. 39. The composition or pharmaceutical formulation of claim 37 or 38, wherein the steroid is present at a concentration ranging from about 30 to about 50 mol% of total lipids.

40. 40. The composition or pharmaceutical formulation of any one of claims 37 to 39, wherein the neutral lipid is present at a concentration ranging from about 5 to about 15 mol% of total lipid.

41. 41. The composition or pharmaceutical formulation of any one of claims 37 to 40, wherein the polymer-conjugated lipid is present at a concentration ranging from about 1 to about 10 mol % of total lipid.

42. 42. The composition or pharmaceutical formulation of any one of claims 37 to 41, wherein the cationic ionizable lipid is in the range of about 40 to about 60 mol%, the steroid is in the range of about 30 to about 50 mol%, the neutral lipid is in the range of about 5 to about 15 mol%, and the polymer-conjugated lipid is in the range of about 1 to about 10 mol%.

43. 43. The composition or pharmaceutical formulation of any one of claims 37 to 42, wherein the cationic ionizable lipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

44. 44. A composition or pharmaceutical formulation according to any one of claims 37 to 43, wherein the steroid is or comprises cholesterol.

45. 45. A composition or pharmaceutical formulation according to any one of claims 37 to 44, wherein the neutral lipid is or comprises a phospholipid.

46. 46. The composition or pharmaceutical formulation of claim 45, wherein the phospholipid is or comprises distearoylphosphatidylcholine (DSPC).

47. 47. The composition or pharmaceutical formulation of any one of claims 37 to 46, wherein the polymer-conjugated lipid is or comprises a polyethylene glycol (PEG) lipid.

48. 48. The composition or pharmaceutical formulation of claim 47, wherein the PEG lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

49. The lipid nanoparticles are (a) ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); (b) cholesterol; (c) distearoylphosphatidylcholine (DSPC); and (d) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide 49. The composition or pharmaceutical formulation of any one of claims 36 to 48, comprising:

50. 50. The composition or pharmaceutical formulation of claim 49, wherein ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) is in the range of about 40 to about 60 mol %, cholesterol is in the range of about 30 to about 50 mol %, distearoylphosphatidylcholine (DSPC) is in the range of about 5 to about 15 mol %, and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide is in the range of about 1 to about 10 mol %.

51. 51. The composition or pharmaceutical preparation of any one of claims 1 to 50, wherein the RNA comprises a 5' cap.

52. 52. The composition or pharmaceutical formulation of claim 51, wherein the 5' cap is or comprises a Cap 1 structure.

53. The 5' cap is ２ ７，３’ -OGppp(m １ ２’－Ｏ 53. A composition or pharmaceutical preparation according to claim 51 or 52, which is or comprises ApG.

54. 54. The composition or pharmaceutical preparation of any one of claims 1 to 53, wherein the RNA comprises a 5'-UTR.

55. 55. The composition or pharmaceutical preparation of claim 54, wherein the 5'-UTR is or comprises a modified human alpha-globin 5'-UTR.

56. 56. The composition or pharmaceutical formulation of claim 54 or 55, wherein the 5'-UTR is or comprises the nucleotide sequence of SEQ ID NO: 56 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:

56.

57. 57. The composition or pharmaceutical preparation of any one of claims 1 to 56, wherein the RNA comprises a 3'-UTR.

58. 58. The composition or pharmaceutical preparation of claim 57, wherein the 3'-UTR is or comprises a first sequence derived from a split amino-terminal enhancer (AES) messenger RNA and a second sequence derived from a mitochondrially encoded 12S ribosomal RNA.

59. 58. The composition or pharmaceutical formulation of claim 56 or 57, wherein the 3'-UTR is or comprises the nucleotide sequence of SEQ ID NO: 58 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:

58.

60. 60. The composition or pharmaceutical preparation of any one of claims 1 to 59, wherein the RNA comprises a polyA sequence.

61. 61. The composition or pharmaceutical preparation of claim 60, wherein the polyA sequence is an interrupted sequence of A nucleotides.

62. 62. The composition or pharmaceutical preparation of claim 60 or 61, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence of 10 nucleotides.

63. 63. The composition or pharmaceutical formulation of any one of claims 60 to 62, wherein the polyA sequence is or comprises the nucleotide sequence of SEQ ID NO: 59 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:

59.

64. 64. The composition or pharmaceutical preparation of any one of claims 1 to 63, wherein the RNA comprises a 5' cap, a 5'-UTR, a 3'-UTR and a polyA sequence.

65. 65. The composition or pharmaceutical preparation of any one of claims 1 to 64, wherein the RNA comprises a modified uridine.

66. 66. The composition or pharmaceutical preparation of any one of claims 1 to 65, wherein the RNA comprises modified uridines in place of all uridines.

67. 67. The composition or pharmaceutical preparation of claim 65 or 66, wherein the modified uridine is N1-methyl-pseudouridine.

68. 68. The composition or pharmaceutical formulation of any one of claims 1 to 67, wherein the coding sequence of said RNA is codon-optimized and / or has an increased G / C content compared to the parent sequence.

69. 69. The composition or pharmaceutical formulation of any one of claims 1 to 68, wherein the RNA is in a liquid formulation.

70. 69. The composition or pharmaceutical preparation of any one of claims 1 to 68, wherein the RNA is in a frozen preparation.

71. 69. The composition or pharmaceutical formulation of any one of claims 1 to 68, wherein the RNA is a lyophilized formulation.

72. 72. The composition or pharmaceutical preparation of any one of claims 1 to 71, wherein the RNA is formulated for injection.

73. 73. The composition or pharmaceutical preparation of any one of claims 1 to 72, wherein the RNA is formulated for intramuscular administration.

74. 74. The composition or pharmaceutical formulation of any one of claims 1 to 73, which is a pharmaceutical composition.

75. 75. The composition or pharmaceutical formulation of claim 74, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

76. 76. A composition or pharmaceutical preparation according to any one of claims 1 to 75 which is a vaccine.

77. 74. The composition or pharmaceutical preparation of any one of claims 1 to 73, which is a kit.

78. 78. The composition or pharmaceutical formulation of claim 77, wherein the different RNA molecules are in separate vials.

79. 79. The composition or pharmaceutical preparation of claim 77 or 78, further comprising instructions for using the composition or pharmaceutical preparation to treat or prevent tuberculosis.

80. A composition or pharmaceutical formulation according to any one of claims 1 to 79 for pharmaceutical use.

81. 81. The composition or pharmaceutical formulation of claim 80, wherein the pharmaceutical use comprises the therapeutic or prophylactic treatment of a disease or disorder.

82. 82. The composition or pharmaceutical preparation of claim 81, wherein said therapeutic or prophylactic treatment of a disease or disorder comprises treating or preventing tuberculosis.

83. 83. A composition or pharmaceutical formulation according to any one of claims 1 to 82, for administration to a human.

84. 84. A method of vaccinating a subject, the method comprising administering to said subject a composition according to any one of claims 1 to 83.

85. 85. The method of claim 84, wherein the vaccination is for preventing tuberculosis.

86. 86. The method of claim 84 or 85, wherein the administration is by intramuscular administration.

87. 87. The method of any one of claims 84 to 86, comprising administering at least one dose of the composition to the subject.

88. 88. The method of any one of claims 84 to 87, comprising administering at least two doses of the composition to the subject.

89. 89. The method of any one of claims 84 to 88, wherein an amount of said RNA of at least 10 μg / dose is administered.

90. 90. The method of any one of claims 84 to 89, wherein the subject is a human.

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