Mycobacterium tuberculosis antigen combination, mRNA encoding same, and vaccine

Abstract

The invention relates to a mycobacterium tuberculosis antigen combination, mRNA (messenger Ribonucleic Acid) for coding the same and a vaccine. The antigen combination comprises antigens in a group I and a group II or antigen fragments thereof: I: Rv3619, Rv3620 and Rv1468; and, II: at least one of Rv1886, Rv2029, Rv1813, Rv2234, Rv3310, Rv0125, and Rv1196, as well as a method for preparing the same. The mycobacterium tuberculosis mRNA vaccine is prepared by using the mycobacterium tuberculosis mRNA vaccine to help a mycobacterium tuberculosis infected person to remove and reduce pathogens in pulmonary infection focuses and macrophages, so that latent tuberculosis is inhibited from being converted into active tuberculosis, and / or an uninfected person is protected from mycobacterium tuberculosis infection or severe infection.

Description

Technical Field

[0001] The present application relates to the fields of genetic engineering and mRNA vaccine development, and specifically relates to Mycobacterium tuberculosis antigen combinations, mRNAs encoding the same, and vaccines. Background Art

[0002] Tuberculosis (TB) is a serious respiratory infectious disease caused by Mycobacterium tuberculosis (MTB). According to the World Health Organization's Global Tuberculosis Report 2022, there were approximately 10 million newly registered TB cases globally, and the number of TB deaths in the human immunodeficiency virus (HIV)-negative population reached 1.2 million, with an additional 251,000 deaths in the HIV-positive population. In addition, it is estimated that approximately one-third of the world's population has latent tuberculosis infection (LTBI).

[0003] Bacillus Calmette-Guérin (BCG) is currently the only available TB vaccine, which can effectively prevent miliary tuberculosis and tuberculous meningitis. However, its protective effect on adults is limited, and revaccination is ineffective. Moreover, BCG is a prophylactic vaccine and cannot effectively protect individuals with latent infection. There are some human tuberculosis vaccines still in the R & D or clinical stage, all of which are traditional vaccine technologies or DNA vaccine technologies. For example, the technical steps of inactivated vaccines are to culture a large number of Mycobacterium tuberculosis bacilli and then perform bacterial population disinfection while retaining the natural conformation of the bacterial proteins after disinfection of the bacterial strains, so as to use them as structural information to induce the body's immune system to recognize and remember them; attenuated vaccines are to perform single-round or multi-round point mutations through point mutations to cause the main toxic genes to lose their toxicity, thereby reducing their damage to the vaccinated host and achieving a safe immunization purpose. Recombinant protein subunits and DNA vaccines both recombine the antigen DNA sequence to be expressed into a plasmid vector through molecular cloning, and then express the recombinant protein through a prokaryotic or eukaryotic expression system or directly replicate a large amount of the plasmid to produce enough DNA for immunization. Traditional vaccines have the following defects:

[0004] 1. Poor cellular immunity: Traditional vaccine development technologies cannot activate a strong cellular immune response in the body to eliminate pathogens that have been latent in the body's cells for a long time. Appropriate adjuvants are needed for cooperation and the immunization dose needs to be increased to match the immunogenicity of mRNA vaccines.

[0005] 2. Difficult antigen screening: The R & D cycle is long. When it is difficult to determine the antigen, a large number of screenings need to be carried out. However, due to process and technical cycle characteristics and other reasons, traditional technologies cannot rapidly express, combine, and screen a large number of antigens. The cost of consumables and reagents used is too high.

[0006] 3. High trial-and-error costs and low flexibility: The time and economic costs are too high, and the steps are cumbersome. The feasibility of mRNA antigen combination screening can be known at the in vitro expression stage. If there is no expression or incorrect folding, it can be judged and the sequence design can be redone, saving time and effort. Only simple gene fragment combinations are needed.

[0007] 4. Inability to express some important antigens: Inactivated vaccines and attenuated vaccines cannot achieve the expression of some key proteins related to important immune escape mechanisms because some of them are stress gene proteins that are temporarily upregulated according to the surrounding microenvironment conditions and the pathogen genome rather than structural proteins. These proteins are likely to have low expression levels and cannot form strong enough immunogenicity, but are crucial for bacterial latency.

[0008] Since the outbreak of the COVID-19 pandemic in 2019, the vaccine industry has developed rapidly, especially mRNA vaccines. The emergence of mRNA vaccine technology has provided potential solutions and technical means for the development of multi-antigen bacterial pathogen vaccines. The strong self-adjuvant effect of mRNA vaccines themselves and their flexible design characteristics make it possible to screen potential Mycobacterium tuberculosis target antigens on a large scale in a short time. It is necessary to screen vaccine target antigens from more than 4,000 antigens to help the body's immune system clear the bacteria latent in cells and achieve intracellular clearance. And based on the target antigens, an mRNA tuberculosis vaccine that can comprehensively consider the antigen diversity and complexity of the pathogen has become an efficient, highly feasible, and technically reliable means for the prevention and treatment of Mycobacterium tuberculosis. Summary of the Invention

[0009] Based on this, an embodiment of the present application provides an antigen combination of Mycobacterium tuberculosis and uses it to prepare an mRNA vaccine for tuberculosis, helping tuberculosis-infected individuals clear and reduce the pathogen in the pulmonary infection foci and macrophages in the lungs, thereby inhibiting the transformation of latent tuberculosis into active tuberculosis, and / or protecting uninfected individuals from Mycobacterium tuberculosis infection or preventing severe infection.

[0010] The technical solution includes the following:

[0011] An antigen combination of Mycobacterium tuberculosis, the antigen combination including antigens or their antigen fragments in Group I and Group II:

[0012] I: Rv3619, Rv3620, and Rv1468; and

[0013] II: at least one of Rv1886, Rv2029, Rv1813, Rv2234, Rv3310, Rv0125, and Rv1196.

[0014] In one embodiment, the antigen combination includes antigens or their antigen fragments in at least one of the following groups:

[0015] (1) Rv3619, Rv3620, Rv2234, Rv3310 and Rv1468;

[0016] (2) Rv3619, Rv3620, Rv1886, Rv2029 and Rv1468;

[0017] (3) Rv3619, Rv3620, Rv1886, Rv1813 and Rv1468;

[0018] (4) Rv3619, Rv3620, Rv1468, Rv2234, Rv3310, Rv1886, Rv1813 and Rv2029.

[0019] In one embodiment, the antigen combination has a sequence shown in any one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 27 and SEQ ID NO: 28.

[0020] mRNA encoding the Mycobacterium tuberculosis antigen combination.

[0021] Optionally, the mRNA is transcribed from DNA having a sequence shown in any one of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 36 and SEQ ID NO: 37.

[0022] Optionally, the mRNA includes modified nucleotides.

[0023] Optionally, the modified nucleotides include one or more of N1-methylpseudouridine triphosphate, pseudouridine triphosphate, 5-methoxyuridine triphosphate and 5-methylcytidine triphosphate.

[0024] In one embodiment, it further includes one or more of the following elements: Kozak sequence, 5' cap structure, 5' UTR, signal peptide coding sequence, 2A peptide coding sequence, Ub coding sequence, 3' UTR and PolyA tail.

[0025] Optionally, the mRNA has the following structure: 5' cap structure, 5' UTR, open reading frame, 3' UTR and PolyA tail.

[0026] Optionally, the open reading frame includes the coding sequence of the Mycobacterium tuberculosis antigen combination.

[0027] Optionally, the open reading frame further includes one or more of Kozak sequence, signal peptide coding sequence, 2A peptide coding sequence, Ub coding sequence and linker sequence.

[0028] DNA that transcribes the aforementioned mRNA.

[0029] Recombinant expression vector that produces the aforementioned mRNA.

[0030] Optionally, the recombinant expression vector includes the aforementioned DNA.

[0031] mRNA vaccine, including the aforementioned mRNA and a delivery vector.

[0032] In one embodiment, the mRNA is encapsulated in the delivery vector.

[0033] Optionally, the delivery vector includes a lipid component.

[0034] Optionally, the mass ratio of the lipid component to the mRNA is (10 - 30):1.

[0035] Optionally, the lipid component includes one or more of cationic lipid, helper phospholipid, cholesterol, and PEG lipid.

[0036] Further optionally, in the lipid component, the molar ratio of cationic lipid, helper phospholipid, cholesterol, and PEG lipid is (45 - 55):(8 - 12):(35 - 40):(1 - 2).

[0037] In one embodiment, the preparation method includes: mixing an aqueous solution of the mRNA with the delivery vector.

[0038] Optionally, the preparation method includes:

[0039] Dissolving the mRNA in an aqueous solution to prepare phase A;

[0040] Dissolving the lipid component in an organic solvent to prepare phase B; and,

[0041] Mixing phase A and phase B to prepare the mRNA vaccine.

[0042] Optionally, the organic solvent is absolute ethanol.

[0043] Use of the aforementioned Mycobacterium tuberculosis antigen combination, the aforementioned mRNA, the aforementioned DNA, the aforementioned recombinant expression vector, or the aforementioned mRNA vaccine in the following:

[0044] (1) Use in the preparation of a drug for preventing or treating Mycobacterium tuberculosis infection;

[0045] (2) Use in the preparation of a vaccine for preventing or treating Mycobacterium tuberculosis infection; Optionally, the vaccine is an mRNA vaccine.

[0046] A medicament, including the mRNA or the mRNA vaccine described above.

[0047] Optionally, it further includes a pharmaceutically acceptable carrier.

[0048] Optionally, the medicament is suitable for sequential administration.

[0049] Compared with the traditional technology, the present application has the following beneficial effects:

[0050] In the present application, a large amount of bioinformatics is used for diversified analysis, and the skills and experience of designing agglutinated mRNA sequences are applied. Coupled with the screening of a high-throughput AI automated screening platform, the currently performance-optimal Mycobacterium tuberculosis antigen combination is finally designed. Its mRNA vaccine has been verified in animal models, P3 latency, and protective animal models to have excellent humoral and cellular immune responses, Mycobacterium tuberculosis clearance ability, etc. It has good protective and therapeutic effects. It has obvious advantages over the products of other current traditional Mycobacterium tuberculosis vaccine technologies in terms of the breadth, depth, efficacy, and safety of antigen screening, and it covers the optimal antigen combination strategy obtained after screening nearly a hundred antigens.

[0051] The present application provides a vaccine that supplements the performance of commercially available Bacillus Calmette-Guérin (BCG) and controls latent tuberculosis infection. The candidate vaccine can be used as: a booster vaccine after initial immunization with BCG; a therapeutic vaccine for controlling latent infection; a therapeutic vaccine for controlling tuberculosis in combination with a medicament. Description of the Drawings

[0052] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following-described drawings are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0053] Figure 1 In vitro expression of all tuberculosis mRNA candidates detected by the HiBiT kit; Figure A shows that all tuberculosis mRNA candidates except TB8 expressed a considerable amount of the target antigen protein; Figure B shows the detection of GAPDH and β-actin as experimental controls.

[0054] Figure 2 Schematic vaccination process in mouse models without infection (A), latent infection (B), and with protection (C).

[0055] Figure 3Schematic diagram of the mRNA vaccine structure; the above shows the molecular structure of all mRNA vaccines, which consists of a T7 promoter, a 5' cap structure, a 5' untranslated region, a Kozak sequence, a signal peptide, a tuberculosis antigen, a P2A element, a 3' untranslated region, and a poly-A tail; different colors are used to represent antigens from different tuberculosis functional categories (green represents cell wall and cell processes, light blue represents the PE-PPE family, yellow represents lipid metabolism, magenta represents regulatory proteins, gray represents intermediate metabolism and respiration, and red represents hypothetical proteins retained); all fusion polyproteins translated from each mRNA carry a HiBiT short peptide tag, which is not shown in this schematic diagram; each mRNA vaccine is designed to contain as many antigen categories as possible to achieve the best effect in the most mechanisms of action; all vaccine constructs are predicted by AlphaFold and RNA-fold to verify the minimum internal cross-interference caused by the interaction between each antigen RNA and protein structure.

[0056] Figure 4 Record of the body weights of mice in all tuberculosis mRNA candidate vaccine inoculation groups before and after the first vaccination.

[0057] Figure 5 Immunogenicity assessment of tuberculosis mRNA vaccines on splenocytes isolated from naïve C57BL / 6j female mice; splenocytes were isolated from naïve C57BL / 6j female mice in all groups, and the immunogenicity induced by 5 μg of tuberculosis mRNA vaccines was evaluated by three methods: enzyme-linked immunospot assay (ELISpot), cytokine bead array (CBA), and flow cytometry; the spot-forming units (SFU) of interleukin-2 (IL-2) and interferon-γ (IFN-γ) released by splenocytes stimulated with an antigen-specific peptide library were counted and plotted as color overlays representing various antigens ( Figure 5 A and B in); the supernatant of splenocytes plated and stimulated with the antigen peptide library was collected for cytokine bead array (CBA); the secretion of IL-2, IFN-γ, tumor necrosis factor-α (TNF-α), IL-4, IL-6, IL-10, and IL-17A in all groups was quantified and plotted as a heat map ( Figure 5 C in); the immune cell CD clusters and cytokine secretion of splenocytes in all groups were recorded by flow cytometry, indicating different functional cell populations that perform humoral and cell-mediated immune regulation ( Figure 5 D-K in); blank lipid nanoparticles (LNP) and phosphate-buffered saline (dPBS) immunization were used as negative controls.

[0058] Figure 6Antigen - specific IgG, IgG1, and IgG2a were detected in serum samples collected from all groups of naïve C57BL / 6j female mice immunized with tuberculosis mRNA vaccines; naïve C57BL / 6j mice had blood samples collected at three time points (day 14, 21, and 35) and were tested for IgG, IgG1, and IgG2a; considering different antigen combinations of tuberculosis mRNA candidate vaccines, each well of a 96 - well plate was individually coated with 100 ng of various relevant antigen proteins; antibody levels against different antigens were plotted in different colors and superimposed to present the total antibody level stimulated by all antigens contained in a specific tuberculosis candidate vaccine; all antigens including Rv3619 - 3620, Rv2234, Rv3310, Rv1468, Rv1886, Rv1738, Rv2029, Rv1985, Rv3425, Rv0125, Rv1196, Rv1813, Rv2608, Rv3874 - 3875, and Rv2660 were separately synthesized and purified in a eukaryotic expression system; an irrelevant protein was used as a control antigen; blank lipid nanoparticle (LNP) and phosphate - buffered saline (dPBS) immunized groups served as negative controls.

[0059] Figure 7 Long - term efficacy assessment was performed on splenocytes and sera of all vaccine groups of naïve C57BL / 6j female mice after 6 months and 12 months; the proportions of CD8+ T cells releasing interleukin - 2 (IL - 2), interferon - γ (IFN - γ), granzyme B, and perforin in all groups were plotted in different colors ( Figure 7 A in); the proportions of CD4+ T cells releasing IL - 2 and IFN - γ, as well as the proportions of macrophages and M1 subtypes were quantified ( Figure 7 B and C in); CD44 / CD62 antibodies were used to sort effector memory T cells and central memory T cells, indicating the positive T cell populations releasing cytokines ( Figure 7 D and E in) and the proportions of different populations of CD8+ and CD4+ T memory cells ( Figure 7 F and G in); CBA measurements of 6 - month and 12 - month long - term samples showed cytokine release in splenocytes of mice in all groups after peptide library stimulation in the form of a heatmap ( Figure 7 H in); the overall quantification of long - term efficacy of all vaccinations was summarized in the panel for comparison ( Figure 7 I, J for 6 months, Figure 7 K and L for 12 months).

[0060] Figure 8Transcriptomic and immunomic analyses were performed by RNA-seq and CDR3 sequencing of BCR and TCR on serum and splenocyte samples from uninfected C57BL / 6j mice; the top 30 significantly regulated genes were classified and listed in a heatmap, with unbalanced regulation represented by different colors compared to the blank lipid nanoparticle group ( Figure 8 in A); the functions and categories of all 30 genes were collected in Table 5; the regulation status (red dots, blue dots, and gray dots) and differentially expressed genes (DEGs) were presented in a volcano plot, showing significant differences in the immune response stimulated by the tuberculosis mRNA vaccine ( Figure 8 in B, with the abscissa log2(Fold Change) and the ordinate -log10(qvalue)); the Kyoto Encyclopedia of Genes and Genomes (KEGG) of B cell receptor (BCR) and T cell receptor (TCR) was analyzed, and 26 responsible signaling pathways related to immune response and regulation after vaccination or infection were selected and plotted; the number of upregulated genes was labeled under each pathway ( Figure 8 in C), and the abundances of BCR and TCR were plotted in different colors and sizes ( Figure 8 in D); the blank lipid nanoparticle and phosphate-buffered saline groups were used as negative controls.

[0061] Figure 9 To conduct a cellular immune detection experiment on a latent infection mouse model. Four indicators of CD8 T cell secretion of IL-2, TNF-α, granzyme, and perforin were detected by flow cytometry ( Figure 9 in A-D), and TB14 and TB18 showed outstanding comprehensive performance, stronger than other antigen combinations; in the heatmap of more immune evaluation indicators ( Figure 9 in E), the average performance of TB14 and TB18 was stronger than that of other combinations; in the detection of the bacterial load in the in vivo isolated lung tissue (G), the CFU count of TB18 was lower than that of the BCG control and lower than other antigen combinations, and in the pathological scoring ( Figure 9 in H), the pathological score of TB18 was lower than that of the BCG control group and other antigen combinations except higher than that of TB12; in the detection of H&E stained pathological sections ( Figure 9 in I), several representative examples of each antigen combination were shown.

[0062] Figure 10 To evaluate the effectiveness of all vaccine groups on splenocytes, serum, and organs collected from a C57BL / 6j female mouse protection model; the proportions of CD8+ T cells releasing IL-2, IFN-γ, granzyme B, and perforin and CD4+ T cells releasing IL-2 and IFN-γ were quantified by flow cytometry and plotted in a heatmap ( Figure 10in A); quantifying the ratio of CD8+ and CD4+ activated T cells by a bar chart ( Figure 10 in B and C); counting the colony-forming units (CFUs) of Mycobacterium tuberculosis in the harvested lung tissues of all groups of mice challenged with Mycobacterium tuberculosis (a mouse model with protective effects) Figure 10 in D); grading the pathological scores based on the H&E staining images of all groups Figure 10 in E); performing statistical analyses between the BCG booster group and the control group, and between the tuberculosis candidate vaccine group and the BCG booster group. Any p-value less than 0.3 is marked on the graphs; immunization with blank lipid nanoparticles (LNPs) and phosphate-buffered saline (dPBS) serves as negative controls. Representative pathological H&E staining images magnified 20-fold and 200-fold from all groups are selected and presented Figure 10 in F); respectively labeling granulocyte infiltration (yellow arrow), mild focal lymphocyte infiltration (red arrow), congestion (orange arrow), leukocytes visible in blood vessels (gray arrow), insoluble fibrin (purple arrow), mild hyperplasia and thickening of bronchial epithelium (brown arrow), focal cell necrosis (black arrow), eosinophilic tissue fluid (blue arrow).

[0063] Figure 11 This is a schematic flow chart for screening antigen combinations in this application.

[0064] Figure 12 It is the spatial structure of a single antigen among all the screened antigens and the spatial structure of a polyprotein generated from a single mRNA sequence in which multiple antigens predicted by AlphaFold V2.3.2 are concatenated and separated by a flexible linker or P2A. Detailed implementation manners

[0065] To make the above objects, features, and advantages of this application more obvious and understandable, the following provides a detailed description of the specific implementation manners of this application. Many specific details are set forth in the following description to facilitate a full understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of this application. Therefore, this application is not limited by the specific embodiments disclosed below.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0067] The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0068] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, in other cases where "one or more" or other expressions indicating "one or more" are used, the same understanding shall apply unless otherwise specified.

[0069] In this application, "optionally", "optional", and "option" mean optional, that is, it means any one of the two alternative options of "yes" or "no". If "optional" appears multiple times in a technical solution, unless otherwise specified and there are no contradictions or mutual restrictions, each "optional" is independent. Unless otherwise specified, descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or may not include".

[0070] In this application, when it comes to a numerical interval (that is, a numerical range), unless otherwise specified, the distribution of the optional numerical values within this numerical interval is considered continuous, and includes the two numerical endpoints of this numerical interval (that is, the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein shall be understood to include any and all sub-ranges subsumed therein. The "numerical value" in this numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" allows for a broad inclusion of numerical interval types such as a percentage interval, a ratio interval, a ratio interval, etc.

[0071] Since Mycobacterium tuberculosis is an intracellular parasitic bacillus with a large genome and complex immune evasion mechanisms, how to select vaccine target antigens from more than 4,000 antigens to help the body's immune system clear the bacteria latent in the cells has become a worldwide problem. In the early years, although antibiotics could inhibit the spread and recurrence of pulmonary tuberculosis to a certain extent, with the increase in drug-resistant strains of pulmonary tuberculosis, the use of vaccines that utilize the body's own immune system for prevention and treatment has become the only viable method to address drug-resistant strains. Currently, there are as many as more than one billion suspected or infected people worldwide. Therefore, with the advent of mRNA vaccine technology, inventing an mRNA tuberculosis vaccine that can comprehensively consider the antigen diversity and complexity of pathogens has become an efficient, highly feasible, and technically reliable preventive and treatment means against Mycobacterium tuberculosis. Traditional vaccine development methods, whether it is whole-cell vaccines, virus vector-expressed recombinant protein vaccines, recombinant protein subunit vaccines, attenuated vaccines, or inactivated vaccines, cannot solve problems such as a large number of antigen screenings, correct folding of fusion proteins, weak cellular immunity, and lack of good applicable adjuvants, resulting in slow R & D progress and poor effects. mRNA vaccine technology has inherent technical advantages and high adaptability for bacterial multi-antigen complex mechanism pathogens, and it can be said to be the only vaccine technology choice and the best way to overcome the worldwide problem of drug-resistant bacteria.

[0072] Through a large number of literature research, bioinformatics analysis, antigen combination test verification, etc., this application selected excellent immune effect vaccine backbone antigens and antigens in the effective vaccine selection pool from more than 4,000 antigens for combination. After combination, it has excellent humoral and cellular immune responses, Mycobacterium tuberculosis clearance ability, etc.

[0073] One embodiment of this application provides an antigen combination of Mycobacterium tuberculosis, which includes antigens or their antigen fragments in backbone antigen (I) and selection antigen (II); wherein, the backbone antigen includes Rv3619, Rv3620, and Rv1468, and the selection antigen includes at least one of Rv1886, Rv2029, Rv1813, Rv2234, Rv3310, Rv0125, and Rv1196.

[0074] This application realizes large-scale antigen screening and completes trial and error to find some better antigen combinations, which can be used as tuberculosis vaccine target antigens to help the body's immune system clear the bacteria latent in the cells.

[0075] In a specific example, the antigen combination includes antigens or their antigen fragments in backbone antigen (I) and selection antigen (II); wherein, the backbone antigen includes Rv3619, Rv3620, and Rv1468, and the selection antigen includes at least one of Rv2234, Rv3310, Rv1886, and Rv2029.

[0076] In a specific example, the antigen combination includes Rv3619, Rv3620, Rv2234, Rv3310, and Rv1468. This antigen combination has excellent immunogenicity and good protective effect in the protection model, and can be used as an antigen selection for preventive tuberculosis vaccine.

[0077] In a specific example, the antigen combination includes Rv3619, Rv3620, Rv1886, Rv2029, and Rv1468. This antigen combination has better immunogenicity and shows good therapeutic effect in the latent model, and can be used as an antigen selection for therapeutic tuberculosis vaccine.

[0078] In a specific example, the antigen combination includes Rv3619, Rv3620, Rv1886, Rv1813, and Rv1468.

[0079] In a specific example, the antigen combination includes Rv3619, Rv3620, Rv1468, Rv2234, Rv3310, Rv1886, Rv1813, and Rv2029. This antigen combination has better immunogenicity and has the effects of both therapeutic and preventive tuberculosis vaccines, and can be used as an antigen combination for tuberculosis vaccines with both therapeutic and preventive effects.

[0080] In a specific example, the antigen combination is a fusion antigen or a mixed antigen.

[0081] In a specific example, when the antigen combination is a fusion antigen, the connection order between antigens is not limited. For example (but not limited to), Rv3619-Rv3620-Rv2234-Rv3310-Rv1468, Rv3619-Rv3620-Rv1886-Rv2029-Rv1468, Rv3619-Rv3620-Rv1886-Rv1813-Rv1468. As long as the expressed antigens are independent of each other in structure and do not affect each other, retain their respective immunogenicity, and produce corresponding antibodies, they are all within the protection scope of this application. Optionally, the antigens are connected by at least one of a linker sequence, 2A peptide, ubiquitin (Ub), and HiBiT tag. Optionally, the linker is a flexible linker, and the flexible linker includes glycine and serine arranged in any combination according to a ratio of 4:1 or 3:1. For example, the sequence is (GGGGS) n , (GGSGG) n , (GSGGG) n or (GGGS) n , where n is any integer from 1 to 10. Optionally, the 2A peptide between antigens includes P2A. For example, the sequence of P2A includes ATNFSLLKQAGDVEEN PGP.

[0082] Among them, the 2A peptide causes ribosomes to "jump" during translation, enabling the independent expression of multiple proteins on the same transcript. In fields such as vaccine research and development, it is often necessary to simultaneously express multiple genes in the same cell or vector to achieve the synergistic effect of multiple functional proteins. Adding ubiquitin between antigens can accelerate the degradation of the synthesized antigen proteins into small fragments, facilitating MHC presentation on the cell surface and thus enhancing cellular immunity and the activation of corresponding TCR T cells. Protein stability, activity, localization, and interactions can be regulated through ubiquitination modification. The HiBiT tag between antigens, for example, the HiBiT tag sequence includes VSGWRLFKKIS, is used for later protein function studies such as protein detection and quantification, protein localization and tracking, etc.

[0083] In a specific example, the amino acid sequence of Rv3619 is as shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity.

[0084] In a specific example, the amino acid sequence of Rv3620 is as shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity.

[0085] In a specific example, the amino acid sequence of Rv1468 is as shown in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity.

[0086] In a specific example, the amino acid sequence of Rv1886 is as shown in SEQ ID NO: 10, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity.

[0087] In a specific example, the amino acid sequence of Rv2029 is as shown in SEQ ID NO: 17, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity.

[0088] In a specific example, the amino acid sequence of Rv1813 is as shown in SEQ ID NO: 13, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity as it.

[0089] In a specific example, the amino acid sequence of Rv2234 is as shown in SEQ ID NO: 15, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity as it.

[0090] In a specific example, the amino acid sequence of Rv3310 is as shown in SEQ ID NO: 5, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity as it.

[0091] In a specific example, the amino acid sequence of Rv0125 is as shown in SEQ ID NO: 18, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity as it.

[0092] In a specific example, the amino acid sequence of Rv1196 is as shown in SEQ ID NO: 6, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and having the same or substantially the same immunogenicity as it.

[0093] In a specific example, the antigen combination has a sequence shown in any one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 27 and SEQ ID NO: 28. In a specific example, the antigen combination has an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in any one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 27 and SEQ ID NO: 28 and having the same or substantially the same immunogenicity as it.

[0094] One embodiment of the present application also provides an mRNA encoding the above-mentioned Mycobacterium tuberculosis antigen combination. In a specific example, the mRNA sequence includes 1, 2, 3, 4, 5, 6, 7, or 8, and the above antigen combination is encoded by 1, 2, 3, 4, 5, 6, 7, or 8 mRNA sequences. For example, the antigen combination is Rv3619, Rv3620, Rv1468, Rv2234, Rv3310, Rv1886, Rv1813, and Rv2029. The mRNA encoding the antigen combination may include 8, each encoding an individual antigen, or may be 7. For example, the Rv3619 and Rv3620 antigens are linked and encoded as 1 mRNA, and the other antigens are independently encoded as 6 mRNAs.

[0095] In a specific example, the mRNA is transcribed from DNA shown by the sequence at positions 130-3591 in SEQ ID NO:30 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0096] In a specific example, the mRNA is transcribed from DNA shown by the sequence at positions 130-3843 in SEQ ID NO:31 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0097] In a specific example, the mRNA is transcribed from DNA shown by the sequence at positions 130-4221 in SEQ ID NO:36 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0098] In a specific example, the mRNA is transcribed from DNA shown by the sequence at positions 130-3633 in SEQ ID NO:37 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0099] In a specific example, the mRNA is transcribed from DNA with a sequence shown by at least one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, and SEQ ID NO:37. It should be noted that sequences encoding the same protein as the nucleotide sequences shown by SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, or SEQ ID NO:37 but different from the above nucleotide sequences due to the degeneracy of the genetic code are all within the scope of protection of this application. In a specific example, the mRNA is transcribed from DNA with a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown by at least one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36, and SEQ ID NO:37.

[0100] In a specific example, the nucleotide sequence of the mRNA is codon-optimized.

[0101] In a specific example, the mRNA includes modified nucleotides.

[0102] In a specific example, the modified nucleotides include, but are not limited to, one or more of nucleotides such as N1-methylpseudo-UTP, pseudo-UTP, 5-methoxy-UDP, and 5-methyl-CTP.

[0103] In a specific example, it further includes one or more elements selected from the following: Kozak sequence, 5' cap structure, 5' UTR, signal peptide coding sequence, 2A peptide coding sequence, Ub coding sequence, 3' UTR, and PolyA tail.

[0104] Optionally, the 5' UTR has the following sequence: cttgttctttttgcagaagctcagaataaacgctcaactttgg.

[0105] Optionally, the 3' UTR has the following sequence: AACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCAC.

[0106] Optionally, the 5' cap structure includes a first and a second structural unit. Among them, the chemical structural formulas of the first and second structural units are

[0107]

[0108] Optionally, the PolyA tail is 80 to 200 As, optionally 80 - 150 As.

[0109] Optionally, the signal peptide includes the following sequence: MFVFLVLLPLVSSQCV.

[0110] In a specific example, the mRNA includes the following structure: 5' cap structure, 5' UTR, open reading frame, 3' UTR, and PolyA tail.

[0111] In a specific example, the open reading frame includes the encoding of an antigen combination; optionally, the open reading frame further includes at least one of a Kozak sequence, a signal peptide encoding sequence, a 2A peptide encoding sequence, a Ub encoding sequence, a linker sequence, and a terminator.

[0112] In a specific example, in the structure of the mRNA, antigens are connected to each other through at least one of a 2A peptide encoding sequence, a Ub encoding sequence, and a linker sequence.

[0113] One embodiment of the present application also provides a DNA for transcribing the above mRNA or a DNA encoding the combination of Mycobacterium tuberculosis antigens.

[0114] In a specific example, the DNA sequence encoding the Rv3619 antigen is as shown in the sequence from position 130 to position 411 in SEQ ID NO:30.

[0115] In a specific example, the DNA sequence encoding the Rv3620 antigen is as shown in the sequence from position 490 to position 783 in SEQ ID NO:30.

[0116] In a specific example, the DNA sequence encoding the Rv1468 antigen is as shown in the sequence from position 2482 to position 3591 in SEQ ID NO:30.

[0117] In a specific example, the DNA sequence encoding the Rv1886 antigen is as shown in the sequence from position 904 to position 1878 in SEQ ID NO:36.

[0118] In a specific example, the DNA sequence encoding the Rv2029 antigen is as shown in the sequence from position 1957 to position 2973 in SEQ ID NO:36.

[0119] In a specific example, the DNA sequence encoding the Rv1813 antigen is as shown in the sequence at positions 1957 - 2385 of SEQ ID NO:37.

[0120] In a specific example, the DNA sequence encoding the Rv2234 antigen is as shown in the sequence at positions 973 - 1461 of SEQ ID NO:30.

[0121] In a specific example, the DNA sequence encoding the Rv3310 antigen is as shown in the sequence at positions 1540 - 2436 of SEQ ID NO:30.

[0122] In a specific example, the DNA sequence encoding the Rv0125 antigen is as shown in the sequence at positions 130 - 1194 of SEQ ID NO:32.

[0123] In a specific example, the DNA sequence encoding the Rv1196 antigen is as shown in the sequence at positions 1378 - 2550 of SEQ ID NO:32.

[0124] Those skilled in the art know that codons are degenerate, and nucleic acid molecules (such as mRNA and DNA) that can encode the above antigen combinations are all within the protection scope of this application.

[0125] One embodiment of this application also provides a recombinant expression vector for producing the above mRNA, including the above DNA sequence.

[0126] One embodiment of this application also provides an mRNA vaccine, including the above mRNA and a delivery vector. The vaccine screened and obtained in this application uses the mRNA sequence form as a vaccine for tuberculosis. The screened and constructed vaccine can effectively activate cellular immunity and humoral immunity, and strengthen the immune protection effect of BCG against Mycobacterium tuberculosis.

[0127] In a specific example, the mRNA is encapsulated in the delivery vector.

[0128] In a specific example, the delivery vector can be a lipid nanoparticle or other delivery vector.

[0129] In a specific example, the delivery vector includes a lipid component. In a specific example, the mass ratio of the lipid component to the mRNA is (10 - 30):1. Optionally, the mass ratio of the lipid component to the mRNA includes but is not limited to 10:1, 15:1, 20:1, 25:1, 30:1, or any range composed of any two of the above values.

[0130] In a specific example, the lipid component includes one or more of cationic lipids, helper phospholipids, cholesterol, and PEG lipids.

[0131] In a specific example, the cationic lipid may include, but is not limited to, one or more of ALC0315, SM102, 3-(dodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(docosylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-tricosyl-15,18,21,24-tetraazaoctaporous alkane (KL25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, Octyl-CLinDMA, Octyl-CLinDMA(2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Choi, DOSPA, DOGS, DODAP, DODMA, and DMRIE.

[0132] In a specific example, the phospholipid may include, but is not limited to, one or more of DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine.

[0133] In a specific example, in the lipid component, the molar ratio of the cationic lipid, co-phospholipid, cholesterol, and PEG lipid is (45 - 55):(8 - 12):(35 - 40):(1 - 2), and may optionally be 50:10:38.5:1.5.

[0134] In a specific example, the PEG lipid may include, but is not limited to, a polyethylene glycol chain with a length of up to 5 kDa covalently linked to a lipid with an alkyl chain having a length of C6 - C20. For example, PEG2000-DMG.

[0135] In a specific example, in the lipid component, the molar ratio of the cationic lipid, phosphatidylcholine, cholesterol, and PEG lipid is (45 - 55):(8 - 12):(35 - 40):(1 - 2), and may optionally be 50:10:38.5:1.5.

[0136] In a specific example, the mRNA vaccine is in the form of an injection, nasal spray, oral preparation, or parenteral preparation such as a suppository.

[0137] In a specific example, the nasal spray is selected from aerosols, sprays, and powder sprays.

[0138] In a specific example, the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film-coated tablets, pellets, sublingual tablets, and ointments;

[0139] In a specific example, the parenteral preparation is a transdermal agent, ointment, plaster, topical liquid, injectable or pushable preparation.

[0140] The vaccine of the present application achieves a strong cellular immune effect with the cooperation of an excellent antigen combination to eliminate intracellular pathogens.

[0141] One embodiment of the present application also provides a method for preparing the above-mentioned mRNA vaccine, including:

[0142] Dissolve the mRNA in an aqueous solution as the aqueous phase;

[0143] Dissolve the lipid component in an organic solvent as the organic phase; and,

[0144] Mix the aqueous phase and the organic phase to prepare the mRNA vaccine.

[0145] In a specific example, the organic solvent is absolute ethanol.

[0146] One embodiment of the present application also provides the use of the above-mentioned Mycobacterium tuberculosis antigen combination, the above-mentioned mRNA, the above-mentioned DNA, the above-mentioned recombinant expression vector or the above-mentioned mRNA vaccine in the preparation of a drug for preventing or treating Mycobacterium tuberculosis infection.

[0147] One embodiment of the present application also provides the use of the above-mentioned Mycobacterium tuberculosis antigen combination, the above-mentioned mRNA, the above-mentioned DNA, the above-mentioned recombinant expression vector or the above-mentioned mRNA vaccine in the preparation of a vaccine for preventing or treating Mycobacterium tuberculosis infection. Optionally, the vaccine is an mRNA vaccine.

[0148] One embodiment of the present application also provides a pharmaceutical composition, including the above-mentioned mRNA or the above-mentioned mRNA vaccine and / or a pharmaceutically acceptable carrier thereof.

[0149] In a specific example, the pharmaceutical composition is suitable for sequential administration.

[0150] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions in the following examples, the guidelines given in the present application are preferentially referred to, and it is also possible to follow the experimental manuals or conventional conditions in the art, or the conditions recommended by the manufacturer, or refer to the experimental methods known in the art.

[0151] In the following specific examples, for the measurement parameters of the raw material components, if there is no special explanation, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by the instrument test accuracy or operation accuracy are allowed.

[0152] The mRNA vaccine for tuberculosis in this application selects and designs a variety of antigens and combinations through literature research, database query, and bioinformatics analysis of a large number of antigens of Mycobacterium tuberculosis, aiming to achieve a Mycobacterium tuberculosis mRNA vaccine sequence with multiple mechanisms, multiple immune types, and strong cellular immune function, so as to enable the immunized person to prevent and treat the clearance of Mycobacterium tuberculosis in the body. The major technical background is based on mRNA vaccine technology. By reading small background technical literature and retrieving the VOILIN vaccine database, the mechanisms of intracellular latency and immune escape of Mycobacterium tuberculosis and antigen characteristics are understood. And the antigens used in traditional Mycobacterium tuberculosis vaccines in the research and development or clinical stage in the world are used for antigen collection. After combining the years of experience of the Zhang Wenhong team at Huashan Hospital, Fudan University in the screening and research and development of Mycobacterium tuberculosis antigens, all the collected antigens are classified according to function and immune type, which can be roughly divided into: Cell Wall and cell process, PE-PPE family, lipid metabolism, conserved hypotheticals, regulatory proteins, intermediary metabolism and respiration. Then, bioinformatics analysis is carried out on all antigens in each category, including transmembrane analysis by TMHMM2.0, glycosylation analysis by NetNGlyc1.0, 3D spatial structure analysis by Pymol / Uniprot, hydrophilicity and hydrophobicity analysis, CD4+ MHC-II immunogenicity scoring, MHC-I immunogenicity scoring, Mycobrowser antigen function classification, Uniprot structure analysis, alphafold structure prediction, mRNA sequence MFE, CAI analysis, etc., to obtain the basic and immunological characteristics and data of all antigens (see Table 1). Then, antigen diversification combinations are carried out according to the functions required by the vaccine candidates to achieve the best therapeutic and preventive purposes. In the design of the mRNA vaccine sequence, common technologies such as T7 promoter combined with Kozak sequence, 5'-Cap cap, 3'-polyA tail, P2A element, signal peptide of the new coronavirus, flexible linker such as GGGGS, UTRs region screening combined with the strongest immunogenic expression, pseudouridine modification, and human codon optimization are used to design 20 multi-antigen combination mRNA sequences that are most likely to express the target fusion protein.After multiple rounds of various evaluation methods using multiple animal models (natural mouse model, latent mouse model, challenge protection mouse model), a candidate vaccine antigen combination backbone and an effective antigen selection pool with good cellular immunity and humoral immunity were identified. Whether detected by ELISpot, flow cytometry, or CBA, the immune animal spleen cell samples or serum samples stimulated by the candidate antigen combination showed high levels of CD8+ activated T cells and the immune cytokines IL-2, IFN-γ, and TNF-α secreted by them, or high titers of specific antibodies. For the specific screening process, please refer to... Figure 11 。

[0153] Table 1 Classification and immunogenicity analysis of M.tb antigens

[0154]

[0155]

[0156] All the selected TB antigens of the Rv37H strain are listed in Table 1. According to the functions of the Mycobrowser database, the antigens are divided into 7 groups, including cell wall and cell processes, PE-PPE family, lipid metabolism, conserved hypothetical proteins, regulatory proteins, intermediary metabolism and respiration, and information pathways. Bioinformatics analysis was performed on each antigen, including glycosylation, transmembrane domains, immunogenicity scores (due to IEDB, MHC-I, and MHC-II immunogenicity predictions), and 3D structures.

[0157] The following three animal models were used in all in vivo experiments in the examples:[[]]

[0158] For the uninfected mouse model, 6-week-old uninfected C57BL / 6j female mice were vaccinated with 5 μg of the tuberculosis mRNA candidate vaccine (TB1-TB30) by the prime-boost immunization strategy, with a 3-week interval between the two immunizations. At 2 weeks after the boost immunization, i.e., on the 35th day, all the mice were sacrificed. Splenocytes were isolated from the spleens of the experimental group and control group mice, and then enzyme-linked immunospot (ELISpot) assay, flow cytometry, and CBA assay ( Figure 2 in A).

[0159] For the latent infection mouse model, 6-week-old C57BL / 6j female mice were inoculated with 1×10 6 cfu of BCG 11 weeks before vaccination with the tuberculosis mRNA candidate vaccine. Six weeks later, all the mice were nasally infected with 300 cfu of Mycobacterium tuberculosis Rv37H. Five weeks later, some of the mice were sacrificed to examine the Mycobacterium tuberculosis burden. The Mycobacterium tuberculosis burden was between 10 4.4 and 10 5It indicated that a latent infection animal model was successfully established. Mice in the latent infection state of Mycobacterium tuberculosis were inoculated with 10 μg of tuberculosis mRNA candidate vaccines (TB10, TB12, TB14, TB15, TB18, TB20) through the prime-boost immunization strategy, with a 3-week interval between the two immunizations. Two weeks later, spleens, lungs, and peripheral blood mononuclear cells were collected from all experimental group and control group mice, and then colony-forming unit counting, flow cytometry, and H&E staining were performed on the relevant samples ( Figure 2 in B).

[0160] For the protective mouse model, 6-week-old female C57BL / 6j mice were inoculated with 1×10 6 cfu of BCG 4 weeks before inoculation with the tuberculosis mRNA candidate vaccines. Then, the mice were inoculated with 10 μg of tuberculosis mRNA candidate vaccines (TB10, TB12, TB14, TB15, TB18, TB20) at 3-week intervals through the prime-boost immunization strategy. Three weeks later, the mice in all groups were challenged with 300 cfu of Mycobacterium tuberculosis Rv37H by nasal infection. Four weeks later, all the mice were sacrificed, and lungs, spleens, and peripheral blood mononuclear cells were collected for further immune evaluation ( Figure 2 in C). As Figure 2 shown, all blood samples were collected at the necessary time points.

[0161] Example 1

[0162] After the mRNA design and sequence confirmation, the company was entrusted to synthesize the full-length protein DNA gene fragments of tuberculosis antigens used in all mRNA candidate vaccine sequences, including the synthetic gene products of 5'UTR, open reading frame, 3'UTR, and poly(A) tail. Subsequently, the DNA template for in vitro transcription of RNA was cloned into the pUC57-mini vector using methods well-known to those skilled in the art. Then, it was transformed into competent Escherichia coli cells, and positive strains were obtained through resistance screening and expanded culture. The plasmid DNA template of Escherichia coli was extracted, purified, digested with plasmid linearization enzymes, and then purified. Using the linearized product as a template, in vitro transcription (IVT) of RNA was carried out. T7 RNA polymerase, CTP, GTP, ATP, m1Ψ (1-methyl-3'-pseudouridylyl)-modified UTP, i.e., N1-methylpseudouridine triphosphate (N1-Methylpseudo-UTP), a Cap analog, i.e., a compound containing three bases including the first and second structural units in the 5'-Cap structure ((3'-OMe-m7G)(5')ppp(5')(2'-OMeA)pG, 100 mM Ammonium Salt Solution, with the structural formula as described above, purchased from Hongene Biotech, Product Number ON-205), and other necessary components well-known to those skilled in the art were added and incubated at 37 °C for 1 - 5 hours. After the reaction was completed, DNase was used for digestion to remove DNA, and further purification was carried out to obtain the mRNA product. The concentration and integrity of the mRNA were detected using methods well-known to those skilled in the art. 30 tuberculosis mRNA antigen combination sequences were prepared. The obtained mRNA was encapsulated: Cationic lipid (DLin-MC3-DMA), DSPC, cholesterol, and PEG-lipid (DMG-PEG2000) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. In this process, the aqueous mRNA solution at acidic pH was rapidly mixed with the ethanol solution of the lipid. Then, ethanol in the crude product was removed by tangential flow ultrafiltration (TFF), and then buffer exchange was carried out using PBS solution (1×, pH 7.4). Next, the mRNA in the neutralized product was diluted to 0.5 mg / mL using a sucrose solution. Finally, the product was aseptically filtered through a 0.22 μm Sartopore PES membrane, and then the aliquots were stored at room temperature or frozen at -80 °C (1.0 mL filled).

[0163] Characterization of mRNA-LNP: The particle size, polydispersity (PDI), and Zeta potential of mRNA-LNP were measured by dynamic light scattering (Malvern Nano ZS Zetasizer). The diameter was characterized by Z average. The encapsulation efficiency (E.E.) of mRNA in LNP was defined as the mass ratio of the encapsulated mRNA to the total mRNA in the final mRNA-LNP product. A specific embedded fluorescent dye (Quant-iT TM RNA Reagent) was used to quantitatively detect nucleic acids such as mRNA. The mRNA concentration was calculated according to the calibration curve generated using the mRNA standard. The relative proportion of the encapsulated mRNA in mRNA-LNP was determined by the ratio of the fluorescence signals in the absence and presence of the surfactant in the dispersed LNP. The signal in the absence of the surfactant indicated the level of free mRNA, while the signal in the presence of the surfactant served as a measure of the total mRNA in the sample.

[0164] The particle size, homogeneity, integrity, surface potential, particle size distribution, etc. of the mRNA-LNP coated with mRNA at room temperature (RT) are shown in Table 3. All tuberculosis mRNA solutions have been encapsulated using the lipid nanoparticle formulation licensed by Acuitas. Quality tests were performed on all encapsulated tuberculosis mRNA-LNP samples. The encapsulation efficiency was calculated based on the encapsulated RNA concentration and the RNA concentration after demulsification. Integrity was measured by Agilent 5400 capillary electrophoresis. The particle size, Zeta potential, and polydispersity index (PDI) were detected by a Darwin analyzer. The encapsulation efficiency was calibrated using the Quant-iT TM RiboGreen RNA kit. The particle size range of mRNA-LNP was from 70 nanometers to 100 nanometers.

[0165] Table 2 Physicochemical properties of the encapsulated tuberculosis mRNA vaccine

[0166]

[0167] 0.5 μg of mRNA-LNP of all tuberculosis candidates was transfected into HEK293T cells. Cells were collected after overnight culture and subjected to Western Blot analysis. The expression level of mRNA was quantified by detecting the HiBiT tag using a commercial kit from Promega. For expression comparison, three separate blots were arranged. The cell lysate and the group treated with empty LNP served as internal negative controls. Except for TB8( Figure 1Except for A) in it, all tuberculosis mRNA candidates expressed a considerable amount of the target antigen protein. Multiple bands indicated the functional P2A device and the expression of separated fusion proteins with corresponding molecular weights, which were calculated and presented in Table 3. The full-size protein bands predicted the possibility of incomplete P2A cleavage. GAPDH and β-actin were detected as experimental controls ( Figure 1 in B). The full-length expression of the multi-antigen combination fusion protein of Mycobacterium tuberculosis was detected by Western Blot. Ctrl was the negative control group, and no expression of the multi-antigen combination fusion protein was observed; the mRNA-LNP group showed protein products with the same molecular weight of the multi-antigen combination fusion protein and different band combinations. Figure 1 indicating that the mRNA could be translated into the multi-antigen combination fusion protein of Mycobacterium tuberculosis.

[0168] Table 3

[0169]

[0170] Table 4 shows the antigen combinations of the tuberculosis mRNA vaccine.

[0171] Table 4. Antigen Combinations of the Tuberculosis mRNA Vaccine

[0172] Name Antigens combination Size(nt) Amino Acid TB1 Rv3874-3875-P2A-2234-3310 2641 801 TB2 Rv3874-3875-2608-P2A-2234-3310 4410 1381 TB3 Rv3874-3875-1705-P2A-2234-3310 3835 1186 TB4 Rv3874-3875-1485-P2A-2234-3310 3712 1145 TB5 Rv3874-3875-P2A-2234-3310-1468 3780 1171 TB6 Rv3619-3620-P2A-2234-3310 2632 800 TB7 Rv3619-3620-2608-P2A-2234-3310 4417 1380 TB8 Rv3619-3620-1705-P2A-2234-3310 3832 1185 TB9 Rv3619-3620-1485-P2A-2234-3310 3709 1144 TB10 Rv3619-3620-P2A-2234-3310-1468 3777 1170 TB11 Rv3619-3620-P2A-Ub-2234-3310-P2A-1468 4077 1254 TB12 Rv1886-P2A-1738-2029 2895 860 TB13 Rv1886-P2A-1985-3425 3198 961 TB14 Rv0125-P2A-1196 2847 844 TB15 Rv3619-3620-P2A-1813-P2A-2608 3537 1074 TB16 Rv3875-P2A-1886-P2A-2660 2229 638 TB17 Rv3874-3875-P2A-1886 2145 610 TB18 Rv3619-3620-P2A-1886-2029-P2A-1468 4431 1380 TB19 Rv3619-3620-P2A-1886-1813-P2A-1468 3843 1184 TB20 Rv1886-3425-P2A-1985 3174 961

[0173] The antigen combination of each tuberculosis mRNA vaccine has been shown in Table 4. The number of amino acids of each mRNA vaccine indicates the size of the fusion polyprotein translated before P2A self-cleavage. The number of nucleotides represents the length of the mRNA sequence, including all gene devices. All tuberculosis antigen genes start with four digits and are named "RV", "Ub" represents the ubiquitin sequence, and "P2A" is located at the self-cleavage site.

[0174] Figure 3 Shows the structures of 20 TB mRNA vaccines, including the RNA elements, antigen combinations, and arrangements contained in the sequences, etc.

[0175] The amino acid and nucleotide sequences related to the above antigens are as follows:

[0176] SEQ ID NO:1: Rv3619(esxV)(94aa)

[0177] MTINYQFGDVDAHGAMIRAQAGSLEAEHQAIISDVLTASDFWGGAGSAACQGFITQLGRNFQVIYEQAN

[0178] AHGQKVQAAGNNMAQTDSAVGSSWA

[0179] SEQ ID NO:2: Rv3620(esxW)(98aa)

[0180] MTSRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMTQMNQAFRN

[0181] IVNMLHGVRDGLVRDANNYEQQEQASQQILSS

[0182] SEQ ID NO:3: Rv3874(esxB)(100aa)

[0183] MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQ

[0184] ELDEISTNIRQAGVQYSRADEEQQQALSSQMGF

[0185] SEQ ID NO:4: Rv3875(esxA)(95aa)

[0186] MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNA

[0187] LQNLARTISEAGQAMASTEGNVTGMFA

[0188] SEQ ID NO:5: Rv3310(SapM)(299aa)

[0189] MLRGIQALSRPLTRVYRALAVIGVLAASLLASWVGAVPQVGLAASALPTFAHVVIVVEENRSQAAIIGNKS

[0190] APFINSLAANGAMMAQAFAETHPSEPNYLALFAGNTFGLTKNTCPVNGGALPNLGSELLSAGYTFMGFA

[0191] EDLPAVGSTVCSAGKYARKHVPWVNFSNVPTTLSVPFSAFPKPQNYPGLPTVSFVIPNADNDMHDGSIAQ

[0192] GDAWLNRHLSAYANWAKTNNSLLVVTWDEDDGSSRNQIPTVFYGAHVRPGTYNETISHYNVLSTLEQIY

[0193] GLPKTGYATNAPPITDIWGD

[0194] SEQ ID NO:6:Rv1196(MTB39A)(391aa)

[0195] MVDFGALPPEINSARMYAGPGSASLVAAAQMWDSVASDLFSAASAFQSVVWGLTVGSWIGSSAGLMVA

[0196] AASPYVAWMSVTAGQAELTAAQVRVAAAAYETAYGLTVPPPVIAENRAELMILIATNLLGQNTPAIAVNEA

[0197] EYGEMWAQDAAAMFGYAAATATATATLLPFEEAPEMTSAGGLLEQAAAVEEASDTAAANQLMNNVPQA

[0198] LQQLAQPTQGTTPSSKLGGLWKTVSPHRSPISNMVSMANNHMSMTNSGVSMTNTLSSMLKGFAPAAAA

[0199] QAVQTAAQNGVRAMSSLGSSLGSSGLGGGVAANLGRAASVGSLSVPQAWAAANQAVTPAARALPLTSLT

[0200] SAAERGPGQMLGGLPVGQMGARAGGGLSGVLRVPPRPYVMPHSPAAG

[0201] SEQ ID NO:7:Rv2608(580aa)

[0202] MNFAVLPPEVNSARIFAGAGLGPMLAAASAWDGLAEELHAAAGSFASVTTGLAGDAWHGPASLAMTRA

[0203] ASPYVGWLNTAAGQAAQAAGQARLAASAFEATLAATVSPAMVAANRTRLASLVAANLLGQNAPAIAAA

[0204] EAEYEQIWAQDVAAMFGYHSAASAVATQLAPIQEGLQQQLQNVLAQLASGNLGSGNVGVGNIGNDNIGN

[0205] ANIGFGNRGDANIGIGNIGDRNLGIGNTGNWNIGIGITGNGQIGFGKPANPDVLVVGNGGPGVTALVMGG

[0206] TDSLLPLPNIPLLEYAARFITPVHPGYTATFLETPSQFFPFTGLNSLTYDVSVAQGVTNLHTAIMAQLAAGN

[0207] EVVVFGTSQSATIATFEMRYLQSLPAHLRPGLDELSFTLTGNPNRPDGGILTRFGFSIPQLGFTLSGATPADA

[0208] YPTVDYAFQYDGVNDFPKYPLNVFATANAIAGILFLHSGLIALPPDLASGVVQPVSSPDVLTTYILLPSQDL

[0209] PLLVPLRAIPLLGNPLADLIQPDLRVLVELGYDRTAHQDVPSPFGLFPDVDWAEVAADLQQGAVQGVNDA

[0210] LSGLGLPPPWQPALPRLF

[0211] SEQ ID NO:8:Rv1705(PPE22)(385aa)

[0212] MDFGALPPEVNSGRMYCGPGSAPMVAAASAWNGLAAELSVAAVGYERVITTLQTEEWLGPASTLMVEA

[0213] VAPYVAWMRATAIQAEQAASQARAAAAAYETAFAAIVPPPLIAANRARLTSLVTHNVFGQNTASIAATEA

[0214] QYAEMWAQDAMAMYGYAGSSATATKVTPFAPPPNTTSPSAAATQLSAVAKAAGTSAGAAQSAIAELIAH

[0215] LPNTLLGLTSPLSSALTAAATPGWLEWFINWYLPISQLFYNTVGLPYFAIGIGNSLITSWRALGWIGPEAAE

[0216] AAAAAPAAVGAAVGGTGPVSAGLGNAATIGKLSLPPNWAGASPSLAPTVGSASAPLVSDIVEQPEAGAAG

[0217] NLLGGMPLAGSGTGTGGAGPRYGFRVTVMSRPPFAG

[0218] SEQ ID NO:9:Rv3425(176aa)

[0219] MHPMIPAEYISNIIYEGPGADSLFFASGQLRELAYSVETTAESLEDELDELDENWKGSSSDLLADAVERYLQ

[0220] WLSKHSSQLKHAAWVINGLANAYNDTRRKVVPPEEIAANREERRRLIASNVAGVNTPAIADLDAQYDQY

[0221] RARNVAVMNAYVSWTRSALSDLPRWREPPQIYRGG

[0222] SEQ ID NO:10:Rv1468c(370aa)

[0223] MSFVVANTEFVSGAAGNLARLGSMISAANSAAAAQTTAVAAAGADEVSAAVAALFGAHGQTYQVLSAQAAAFHSQFVQALSGGAQAYAAAEATNFGPLQPLFDVINAPTLALLNRPLIGNGADGTAANPNGQAGGLLIGNGGNGFSPAAGPGGNGGAAGLLGHGGNGGVGALGANGGAGGTGGWLFGNGGAGGNSGGGGGAGGIGGSAVLFGAGGAGGISPNGMGAGGSGGNGGLFFGNGGAGASSFLGGGGAGGRAFLFGDGGAGGAALSAGSAGRGGDAGFFYGNGGAGGSGAGGASSAHGGAGGQAGLFGNGGEGGDGGALGGNGGNGGNAQLIGNGGDGGDGGGAGAPGLGGRGGLLLGLPGANGT

[0224] SEQ ID NO:11: Rv1886 (Ag85B) (325aa)

[0225] MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG

[0226] SEQ ID NO:12: Rv3804 (Ag85A) (338aa)

[0227] MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA

[0228] SEQ ID NO:13: Rv1813 (Hypothetical protein) (143aa)

[0229] MITNLRRRTAMAAAGLGAALGLGILLVPTVDAHLANGSMSEVMMSEIAGLPIPPIIHYGAIAYAPSGASGKAWHQRTPARAEQVALEKCGDKTCKVVSRFTRCGAVAYNGSKYQGGTGLTRRAAEDDAVNRLEGGRIVNWACN

[0230] SEQ ID NO:14:Rv2660c(Hypothetical protein)(75aa)

[0231] MIAGVDQALAATGQASQRAAGASGGVTVGVGVGTEQRNLSVVAPSQFTFSSRSPDFVDETAGQSWCAILGLNQFH

[0232] SEQ ID NO:15:Rv2234(ptpA)(163aa)

[0233] MSDPLHVTFVCTGNICRSPMAEKMFAQQLRHRGLGDAVRVTSAGTGNWHVGSCADERAAGVLRAHGYPTDHRAAQVGTEHLAADLLVALDRNHARLLRQLGVEAARVRMLRSFDPRSGTHALDVEDPYYGDHSDFEEVFAVIESALPGLHDWVDERLARNGPS

[0234] SEQ ID NO:16:Rv1985c(303aa)

[0235] MVDPQLDGPQLAALAAVVELGSFDAAAERLHVTPSAVSQRIKSLEQQVGQVLVVREKPCRATTAGIPLLRLAAQTALLESEALAEMGGNASLKRTRITIAVNADSMATWFSAVFDGLGDVLLDVRIEDQDHSARLLREGVAMGAVTTERNPVPGCRVHPLGEMRYLPVASRPFVQRHLSDGFTAAAAAKAPSLAWNRDDGLQDMLVRKAFRRAITRPTHFVPTTEGFTAAARAGLGWGMFPEKLAASPLADGSFVRVCDIHLDVPLYWQCWKLDSPIIARITDTVRAAASGLYRGQQRRRRPG

[0236] SEQ ID NO:17: Rv2029c, PfkB (339aa)

[0237] MTEPAAWDEGKPRIITLTMNPALDITTSVDVVRPTEKMRCGAPRYDPGGGGINVARIVHVLGGCSTALFPAGGSTGSLLMALLGDAGVPFRVIPIAASTRESFTVNESRTAKQYRFVLPGPSLTVAEQEQCLDELRGAAASAAFVVASGSLPPGVAADYYQRVADICRRSSTPLILDTSGGGLQHISSGVFLLKASVRELRECVGSELLTEPEQLAAAHELIDRGRAEVVVVSLGSQGALLATRHASHRFSSIPMTAVSGVGAGDAMVAAITVGLSRGWSLIKSVRLGNAAGAAMLLTPGTAACNRDDVERFFELAAEPTEVGQDQYVWHPIVNPEASP

[0238] SEQ ID NO:18: Rv0125 (MTB32A) (355aa)

[0239] MSNSRRRSLRWSWLLSVLAAVGLGLATAPAQAAPPALSQDRFADFPALPLDPSAMVAQVGPQVVNINTKLGYNNAVGAGTGIVIDPNGVVLTNNHVIAGATDINAFSVGSGQTYGVDVVGYDRTQDVAVLQLRGAGGLPSAAIGGGVAVGEPVVAMGNSGGQGGTPRAVPGRVVALGQTVQASDSLTGAEETLNGLIQFDAAIQPGDSGGPVVNGLGQVVGMNTAASDNFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHIGPTAFLGLGVVDNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAPINSATAMADALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPA

[0240] SEQ ID NO:19: Rv1485 HemZ (344aa)

[0241] MQFDAVLLLSFGGPEGPEQVRPFLENVTRGRGVPAERLDAVAEHYLHFGGVSPINGINRTLIAELEAQQELPVYFGNRNWEPYVEDAVTAMRDNGVRRAAVFATSAWSGYSSCTQYVEDIARARRAAGRDAPELVKLRPYFDHPLFVEMFADAITAAAATVRGDARLVFTAHSIPTAADRRCGPNLYSRQVAYATRLVAAAAGYCDFDLAWQSRSGPPQVPWLEPDVTDQLTGLAGAGINAVIVCPIGFVADHIEVVWDLDHELRLQAEAAGIAYARASTPNADPRFARLARGLIDELRYGRIPARVSGPDPVPGCLSSINGQPCRPPHCVASVSPARPSAGSP

[0242] SEQ ID NO:20:Rv1738(Hypothetical protein)(94aa)

[0243] MCGDQSDHVLQHWTVDISIDEHEGLTRAKARLRWREKELVGVGLARLNPADRNVPEIGDELSVARALSDLGKRMLKVSTHDIEAVTHQPARLLY

[0244] SEQ ID NO:21:TB10,Rv3619-3620-2234-3310-1468(1170aa)

[0245] MFVFLVLLPLVSSQCV MTINYQFGDVDAHGAMIRAQAGSLEAEHQAIISDVLTASDFWGGAGSAACQG FITQLGRNFQVIYEQANAHGQKVQAAGNNMAQTDSAVGSSWAGGGGSGGGGSGGGGSVSGWRLFKKISMTSRFMTD PHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMTQMNQAFRNIVNMLHGVRDGLVRDANN YEQQEQASQQILSS ATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCVWLWYIKLFIMIVGGLVGLRIVFAVLSIV MSDPLHVTFVCTGNICRSPMAEKMFAQQLRHRGLGDAVRVTSAGTGNWHVGSCADERAAGVLRAHGYPTDHRAAQ VGTEHLAADLLVALDRNHARLLRQLGVEAARVRMLRSFDPRSGTHALDVEDPYYGDHSDFEEVFAVIESALPGLHD WVDERLARNGPS GGGGSGGGGSGGGGSVSGWRLFKKIS MLRGIQALSRPLTRVYRALAVIGVLAASLLASWVGAVP QVGLAASALPTFAHVVIVVEENRSQAAIIGNKSAPFINSLAANGAMMAQAFAETHPSEPNYLALFAGNTFGLTKNT CPVNGGALPNLGSELLSAGYTFMGFAEDLPAVGSTVCSAGKYARKHVPWVNFSNVPTTLSVPFSAFPKPQNYPGLP TVSFVIPNADNDMHDGSIAQGDAWLNRHLSAYANWAKTNNSLLVVTWDEDDGSSRNQIPTVFYGAHVRPGTYNETI SHYNVLSTLEQIYGLPKTGYATNAPPITDIWGD GGGGSGGGGSGGGGS MSFVVANTEFVSGAAGNLARLGSMISAA NSAAAAQTTAVAAAGADEVSAAVAALFGAHGQTYQVLSAQAAAFHSQFVQALSGGAQAYAAAEATNFGPLQPLFDV INAPTLALLNRPLIGNGADGTAANPNGQAGGLLIGNGGNGFSPAAGPGGNGGAAGLLGHGGNGGVGALGANGGAGG TGGWLFGNGGAGGNSGGGGGAGGIGGSAVLFGAGGAGGISPNGMGAGGSGGNGGLFFGNGGAGASSFLGGGGAGGR AFLFGDGGAGGAALSAGSAGRGGDAGFFYGNGGAGGSGAGGASSAHGGAGGQAGLFGNGGEGGDGGALGGNGGNGG NAQLIGNGGDGGDGGGAGAPGLGGRGGLLLGLPGANGT; Note: The underlined part is the antigen; Signal peptide: MFVFLVLLPLVSSQCV; Linker: GGGGSGGGGSGGGGS; HiBiT tag: VSGWRLFKKIS; P2A: ATNFSLLKQAGDVEENPGP.

[0246] SEQ ID NO:22:TB11,Rv3619 - 3620 - Ub - 2234 - 3310 - 1468(1254aa)

[0247] MFVFLVLLPLVSSQCV MTINYQFGDVDAHGAMIRAQAGSLEAEHQAIISDVLTASDFWGGAGSAACQG FITQLGRNFQVIYEQANAHGQKVQAAGNNMAQTDSAVGSSWA GGGGSGGGGSVSGWRLFKKISGGGGS MTSRFMTD PHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMTQMNQAFRNIVNMLHGVRDGLVRDANN YEQQEQASQQILSS GGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCVMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGA MSDPLHVTFVCTGNICRSPMAE KMFAQQLRHRGLGDAVRVTSAGTGNWHVGSCADERAAGVLRAHGYPTDHRAAQVGTEHLAADLLVALDRNHARLLR QLGVEAARVRMLRSFDPRSGTHALDVEDPYYGDHSDFEEVFAVIESALPGLHDWVDERLARNGPS GGGGSGGGGSVSGWRLFKKISGGGGS MLRGIQALSRPLTRVYRALAVIGVLAASLLASWVGAVPQVGLAASALPTFAHVVIVVEENR SQAAIIGNKSAPFINSLAANGAMMAQAFAETHPSEPNYLALFAGNTFGLTKNTCPVNGGALPNLGSELLSAGYTFM GFAEDLPAVGSTVCSAGKYARKHVPWVNFSNVPTTLSVPFSAFPKPQNYPGLPTVSFVIPNADNDMHDGSIAQGDA WLNRHLSAYANWAKTNNSLLVVTWDEDDGSSRNQIPTVFYGAHVRPGTYNETISHYNVLSTLEQIYGLPKTGYATN APPITDIWGD GGGSGGGSATNFSLLKQAGDVEENPGPVSGWRLFKKISGGGSGGGS MSFVVANTEFVSGAAGNLAR LGSMISAANSAAAAQTTAVAAAGADEVSAAVAALFGAHGQTYQVLSAQAAAFHSQFVQALSGGAQAYAAAEATNFG PLQPLFDVINAPTLALLNRPLIGNGADGTAANPNGQAGGLLIGNGGNGFSPAAGPGGNGGAAGLLGHGGNGGVGAL GANGGAGGTGGWLFGNGGAGGNSGGGGGAGGIGGSAVLFGAGGAGGISPNGMGAGGSGGNGGLFFGNGGAGASSFL GGGGAGGRAFLFGDGGAGGAALSAGSAGRGGDAGFFYGNGGAGGSGAGGASSAHGGAGGQAGLFGNGGEGGDGGAL GGNGGNGGNAQLIGNGGDGGDGGGAGAPGLGGRGGLLLGLPGANGT ; Note: The underlined part is the antigen; Ub: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGA; P2A: ATNFSLLKQAGDVEENPGP; HiBiT tag: VSGWRLFKKIS.

[0248] SEQ ID NO:23:TB14,Rv0125 - 1196(844aa)

[0249] MFVFLVLLPLVSSQCVMSNSRRRSLRWSWLLSVLAAVGLGLATAPAQAAPPALSQDRFADFPALPLDPSAMVAQVGPQVVNINTKLGYNNAVGAGTGIVIDPNGVVLTNNHVIAGATDINAFSVGSGQTYGVDVVGYDRTQDVAVLQLRGAGGLPSAAIGGGVAVGEPVVAMGNSGGQGGTPRAVPGRVVALGQTVQASDSLTGAEETLNGLIQFDAAIQPGDSGGPVVNGLGQVVGMNTAASDNFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHIGPTAFLGLGVVDNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAPINSATAMADALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPAGGGGSGGGGSVSGWRLFKKISGGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCVMVDFGALPPEINSARMYAGPGSASLVAAAQMWDSVASDLFSAASAFQSVVWGLTVGSWIGSSAGLMVAAASPYVAWMSVTAGQAELTAAQVRVAAAAYETAYGLTVPPPVIAENRAELMILIATNLLGQNTPAIAVNEAEYGEMWAQDAAAMFGYAAATATATATLLPFEEAPEMTSAGGLLEQAAAVEEASDTAAANQLMNNVPQALQQLAQPTQGTTPSSKLGGLWKTVSPHRSPISNMVSMANNHMSMTNSGVSMTNTLSSMLKGFAPAAAAQAVQTAAQNGVRAMSSLGSSLGSSGLGGGVAANLGRAASVGSLSVPQAWAAANQAVTPAARALPLTSLTSAAERGPGQMLGGLPVGQMGARAGGGLSGVLRVPPRPYVMPHSPAAGGGGGSGGGGSVSGWRLFKKIS。

[0250] SEQ ID NO:24:TB15,Rv3619-3620-1813-2608(1074aa)

[0251]

[0252] SEQ ID NO:25: TB16, Rv3875 - 1886 - 2660 (638aa)

[0253] MFVFLVLLPLVSSQCVMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAGGGGSVSGWRLFKKISGGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCVMTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGGGGSVSGWRLFKKISGGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCVMIAGVDQALAATGQASQRAAGASGGVTVGVGVGTEQRNLSVVAPSQFTFSSRSPDFVDETAGQSWCAILGLNQFHGGGGSVSGWRLFKKIS。

[0254] SEQ ID NO:26: TB17, Rv3874 - 3875 - 1886 (610aa)

[0255] MFVFLVLLPLVSSQCVTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFAGGGGSVSGWRLFKKISGGGGSAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGFGGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCVMTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGGGGSVSGWRLFKKIS。

[0256] SEQ ID NO:27:TB18,Rv3619-3620-1886-2029-1468(1380aa)

[0257] MFVFLVLLPLVSSQCV MTINYQFGDVDAHGAMIRAQAGSLEAEHQAIISDVLTASDFWGGAGSAACQG FITQLGRNFQVIYEQANAHGQKVQAAGNNMAQTDSAVGSSWA GGGGSGGGGSVSGWRLFKKISGGGGS MTSRFMTD PHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMTQMNQAFRNIVNMLHGVRDGLVRDANN YEQQEQASQQILSS GGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQ CVMTDVSRKIRAWGRRLMIGTAAA VVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAF EWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMI LAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVY CGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG GGGGSGGGGSVSGWRLFKKISGGGGS MTEPAAWDEGKPRIITLTMNPALDITTSVDVVRPTEKMRCGAPRYDPGGGG INVARIVHVLGGCSTALFPAGGSTGSLLMALLGDAGVPFRVIPIAASTRESFTVNESRTAKQYRFVLPGPSLTVAE QEQCLDELRGAAASAAFVVASGSLPPGVAADYYQRVADICRRSSTPLILDTSGGGLQHISSGVFLLKASVRELREC VGSELLTEPEQLAAAHELIDRGRAEVVVVSLGSQGALLATRHASHRFSSIPMTAVSGVGAGDAMVAAITVGLSRGWSLIKSVRLGNAAGAAMLLTPGTAACNRDDVERFFELAAEPTEVGQDQYVWHPIVNPEASP GGGSGGGSATNFSLLKQAGDVEENPGPVSGWRLFKKISGGGSGGGS MSFVVANTEFVSGAAGNLARLGSMISAANSAAAAQTTAVAAAGADE VSAAVAALFGAHGQTYQVLSAQAAAFHSQFVQALSGGAQAYAAAEATNFGPLQPLFDVINAPTLALLNRPLIGNGA DGTAANPNGQAGGLLIGNGGNGFSPAAGPGGNGGAAGLLGHGGNGGVGALGANGGAGGTGGWLFGNGGAGGNSGGG GGAGGIGGSAVLFGAGGAGGISPNGMGAGGSGGNGGLFFGNGGAGASSFLGGGGAGGRAFLFGDGGAGGAALSAGS AGRGGDAGFFYGNGGAGGSGAGGASSAHGGAGGQAGLFGNGGEGGDGGALGGNGGNGGNAQLIGNGGDGGDGGGAG APGLGGRGGLLLGLPGANGT ; The underlined part is the antigen.

[0258] SEQ ID NO:28:TB19,Rv3619 - 3620 - 1886 - 1813 - 1468(1184aa)

[0259] MFVFLVLLPLVSSQCV MTINYQFGDVDAHGAMIRAQAGSLEAEHQAIISDVLTASDFWGGAGSAACQG FITQLGRNFQVIYEQANAHGQKVQAAGNNMAQTDSAVGSSWA GGGGSGGGGSVSGWRLFKKISGGGGS MTSRFMTD PHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMTQMNQAFRNIVNMLHGVRDGLVRDANN YEQQEQASQQILSS GGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQ CVMTDVSRKIRAWGRRLMIGTAAA VVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAF EWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMI LAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVY CGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG GGGGSGGGGSVSGWRLFKKISGGGGS MITNLRRRTAMAAAGLGAALGLGILLVPTVDAHLANGSMSEVMMSEIAGLP IPPIIHYGAIAYAPSGASGKAWHQRTPARAEQVALEKCGDKTCKVVSRFTRCGAVAYNGSKYQGGTGLTRRAAEDD AVNRLEGGRIVNWACN GGGSGGGSATNFSLLKQAGDVEENPGPVSGWRLFKKISGGGSGGGS MSFVVANTEFVSGA AGNLARLGSMISAANSAAAAQTTAVAAAGADEVSAAVAALFGAHGQTYQVLSAQAAAFHSQFVQALSGGAQAYAAA EATNFGPLQPLFDVINAPTLALLNRPLIGNGADGTAANPNGQAGGLLIGNGGNGFSPAAGPGGNGGAAGLLGHGGN GGVGALGANGGAGGTGGWLFGNGGAGGNSGGGGGAGGIGGSAVLFGAGGAGGISPNGMGAGGSGGNGGLFFGNGGA GASSFLGGGGAGGRAFLFGDGGAGGAALSAGSAGRGGDAGFFYGNGGAGGSGAGGASSAHGGAGGQAGLFGNGGEG GDGGALGGNGGNGGNAQLIGNGGDGGDGGGAGAPGLGGRGGLLLGLPGANGT ; The underlined part is the antigen.

[0260] SEQ ID NO:29:TB20,Rv1886 - 3425 - 1985(961aa)

[0261] MFVFLVLLPLVSSQCVMTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGGGGSGGGGSVSGWRLFKKISGGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCVMHPMIPAEYISNIIYEGPGADSLFFASGQLRELAYSVETTAESLEDELDELDENWKGSSSDLLADAVERYLQWLSKHSSQLKHAAWVINGLANAYNDTRRKVVPPEEIAANREERRRLIASNVAGVNTPAIADLDAQYDQYRARNVAVMNAYVSWTRSALSDLPRWREPPQIYRGGGGGSGGGGSVSGWRLFKKISGGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCVMVDPQLDGPQLAALAAVVELGSFDAAAERLHVTPSAVSQRIKSLEQQVGQVLVVREKPCRATTAGIPLLRLAAQTALLESEALAEMGGNASLKRTRITIAVNADSMATWFSAVFDGLGDVLLDVRIEDQDHSARLLREGVAMGAVTTERNPVPGCRVHPLGEMRYLPVASRPFVQRHLSDGFTAAAAAKAPSLAWNRDDGLQDMLVRKAFRRAITRPTHFVPTTEGFTAAARAGLGWGMFPEKLAASPLADGSFVRVCDIHLDVPLYWQCWKLDSPIIARITDTVRAAASGLYRGQQRRRRPGGGGGSGGGGSVSGWRLFKKIS。

[0262] SEQ ID NO:30: TB10, pUC57-Mini-AmpR-RV3619-HiBiT-3620-2234-HiBiT-3310-1468c(5676bp)

[0263] CCAATGATGAATTCTAATACGACTCACTATAGcttgttctttttgcagaagctcagaataaacgctcaactttggGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG ATGACAATCAATTAC CAATTCGGCGATGTCGATGCACACGGGGCCATGATCCGCGCACAGGCAGGGAGTCTGGAGGCTGAGCACCAGGCCA TCATCAGCGATGTGCTGACCGCTAGTGATTTCTGGGGGGGCGCTGGATCCGCTGCTTGTCAGGGCTTCATTACCCA ACTCGGCAGAAATTTCCAGGTGATTTACGAGCAGGCTAATGCCCATGGGCAGAAGGTTCAAGCCGCCGGCAATAAC ATGGCGCAGACAGACTCCGCCGTAGGGTCCTCCTGGGCT GGAGGAGGCGGTTCTGGAGGCGGAGGAAGCggaggtggaggcagcGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC ATGACAAGTCGATTCATGACGGACCCACATGCAAT GCGAGACATGGCCGGTAGATTCGAAGTGCATGCCCAGACAGTGGAAGACGAGGCACGCAGGATGTGGGCCTCCGCC CAGAATATCAGCGGGGCAGGATGGAGTGGCATGGCAGAGGCCACTAGCCTGGACACTATGACTCAGATGAATCAGG CTTTTCGAAACATCGTGAATATGCTGCATGGAGTGAGAGATGGCCTGGTAAGAGACGCGAACAATTACGAGCAGCA GGAACAAGCTAGCCAGCAGATCTTGAGCTCT gccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGTGGCTGTGGTATATCAAGTTGTTCATTATGATCGTGGGCGGGCTGGTGGGCCTGAGAATTGTGTTCGCCGTCCTGAGCATTGTG ATGAGCGA TCCACTGCATGTGACTTTCGTGTGTACGGGGAACATTTGCCGGTCCCCTATGGCCGAGAAAATGTTCGCCCAGCAG CTGCGCCATAGAGGGCTCGGGGATGCCGTCAGAGTGACCAGCGCAGGTACTGGCAACTGGCACGTTGGCAGCTGTG CCGACGAAAGAGCGGCAGGTGTCCTGCGAGCCCACGGTTACCCCACAGACCACAGAGCCGCTCAGGTGGGAACTGA GCATCTGGCCGCCGACCTTCTGGTCGCCTTGGATAGAAACCATGCCAGACTGCTGAGACAGCTCGGCGTGGAGGCT GCCAGGGTCCGGATGCTGAGATCATTTGACCCACGGAGCGGCACACACGCTCTGGACGTGGAAGACCCTTACTACG GTGACCACTCTGACTTCGAGGAAGTCTTCGCAGTGATCGAATCCGCCCTTCCAGGTCTGCACGACTGGGTCGATGA AAGACTGGCTCGCAACGGCCCCTCA GGAGGAGGCGGTTCTGGAGGCGGAGGAAGCggaggtggaggcagcGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC ATGCTTAGAGGCATCCAAGCCCTGTCACGACCACTGACAAGAGTCTACC GAGCCCTGGCTGTGATTGGGGTATTGGCCGCATCACTGCTGGCCTCTTGGGTGGGCGCAGTCCCACAAGTTGGTTT GGCTGCCTCCGCCCTGCCTACATTTGCCCATGTGGTTATTGTGGTTGAGGAAAATAGGTCCCAGGCCGCCATCATA GGAAACAAATCAGCCCCCTTCATTAATAGCCTGGCCGCCAATGGAGCCATGATGGCACAGGCTTTCGCTGAGACTC ATCCGAGTGAGCCCAACTACCTGGCACTGTTTGCCGGAAATACATTTGGACTCACTAAAAACACATGTCCGGTTAA CGGAGGAGCACTTCCGAACCTGGGCAGCGAACTCCTTTCTGCCGGCTACACCTTTATGGGGTTTGCCGAGGATTTG CCCGCTGTAGGTTCCACGGTCTGTTCTGCTGGGAAGTACGCCCGGAAGCATGTGCCCTGGGTCAACTTCAGTAATG TGCCTACAACTCTGTCTGTGCCTTTCAGTGCTTTCCCAAAACCTCAGAATTATCCCGGCCTTCCTACTGTTAGTTT CGTCATCCCAAATGCTGATAACGACATGCACGATGGGTCTATTGCCCAGGGGGATGCATGGCTGAATAGGCATCTT TCCGCCTATGCCAATTGGGCAAAGACAAATAACAGTCTGCTTGTAGTGACATGGGATGAGGATGATGGATCTTCAC GAAATCAGATCCCTACCGTGTTTTACGGAGCCCATGTCAGGCCGGGCACATACAATGAGACTATCAGCCATTACAA CGTCCTGTCCACACTGGAGCAAATCTACGGGTTGCCCAAGACAGGGTATGCCACAAACGCTCCCCCTATAACCGAT ATCTGGGGGGATGGAGGAGGCGGTTCTGGAGGCGGAGGAAGCggaggtggaggcagc ATGTCCTTTGTGGTTGCCA ATACTGAATTTGTAAGTGGTGCCGCGGGTAATTTGGCACGCCTGGGCAGCATGATATCAGCTGCTAATTCCGCCGC CGCCGCCCAGACAACAGCTGTGGCAGCAGCGGGCGCCGATGAAGTGAGCGCTGCCGTCGCTGCTCTTTTCGGAGCT CATGGACAAACCTACCAAGTGCTGAGCGCCCAGGCTGCCGCTTTCCACAGTCAATTCGTGCAAGCTCTGTCAGGGG GAGCTCAAGCTTATGCCGCCGCAGAAGCAACCAACTTCGGACCACTCCAGCCCTTGTTCGATGTCATTAACGCCCC AACTCTCGCTCTGCTCAACCGGCCACTTATTGGCAACGGGGCCGATGGCACTGCCGCTAACCCTAACGGTCAAGCC GGTGGTCTGTTGATCGGCAATGGCGGAAACGGCTTCAGCCCAGCAGCGGGCCCCGGAGGAAACGGCGGCGCCGCCG GCCTTCTCGGTCATGGTGGAAACGGTGGGGTAGGGGCACTGGGGGCTAATGGCGGGGCTGGCGGAACAGGAGGCTG GCTGTTCGGCAACGGCGGCGCGGGCGGAAACAGCGGGGGAGGCGGGGGTGCAGGTGGAATTGGGGGTAGCGCCGTG CTGTTCGGGGCAGGCGGTGCCGGCGGGATCAGCCCAAATGGCATGGGTGCAGGAGGAAGCGGCGGGAATGGCGGTC TGTTCTTCGGGAATGGCGGAGCAGGAGCATCCTCCTTCCTGGGAGGTGGGGGAGCGGGAGGGCGGGCCTTCCTCTT CGGAGATGGAGGCGCGGGGGGGGCCGCCCTGAGCGCCGGCTCTGCAGGAAGGGGAGGAGATGCCGGTTTCTTCTAC GGGAATGGCGGCGCAGGGGGGAGCGGAGCCGGCGGCGCCTCAAGCGCCCACGGCGGGGCTGGGGGCCAGGCCGGTC TGTTCGGAAACGGCGGTGAGGGGGGCGATGGCGGTGCACTTGGGGGAAATGGCGGGAACGGGGGCAATGCACAGCT GATCGGAAATGGAGGTGACGGAGGGGACGGCGGCGGAGCAGGGGCCCCGGGTCTGGGAGGAAGAGGCGGGCTGCTG CTGGGGCTGCCTGGTGCCAACGGGACG

[0264] SEQ ID NO:31: TB11, pUC57-Mini-AmpR-RV3619-HiBiT-3620-P2A-Ub-2234-HiBiT-3310-P2A-HiBiT-1468c (5925bp)

[0265] CCAATGATGAATTCTAATACGACTCACTATAGcttgttctttttgcagaagctcagaataaacgctcaactttggGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG ATGACAATCAATTAC CAATTCGGCGATGTCGATGCACACGGGGCCATGATCCGCGCACAGGCAGGGAGTCTGGAGGCTGAGCACCAGGCCA TCATCAGCGATGTGCTGACCGCTAGTGATTTCTGGGGGGGCGCTGGATCCGCTGCTTGTCAGGGCTTCATTACCCA ACTCGGCAGAAATTTCCAGGTGATTTACGAGCAGGCTAATGCCCATGGGCAGAAGGTTCAAGCCGCCGGCAATAAC ATGGCGCAGACAGACTCCGCCGTAGGGTCCTCCTGGGCT GGAGGAGGCGGTTCTGGAGGCGGAGGAAGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAGGCGGTT CTATGACAAGTCGATTCATGACGGACCCACATGCAAT GCGAGACATGGCCGGTAGATTCGAAGTGCATGCCCAGACAGTGGAAGACGAGGCACGCAGGATGTGGGCCTCCGCC CAGAATATCAGCGGGGCAGGATGGAGTGGCATGGCAGAGGCCACTAGCCTGGACACTATGACTCAGATGAATCAGG CTTTTCGAAACATCGTGAATATGCTGCATGGAGTGAGAGATGGCCTGGTAAGAGACGCGAACAATTACGAGCAGCA GGAACAAGCTAGCCAGCAGATCTTGAGCTCT GGAGGAGGCGGTTCTgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGATGCAGATTTTCGTAAAGACCCTCACAGGCAAAACCATCACCCTCGAAGTGGAACCTAGTGATACCATCGAAAACGTGAAGGCTAAGATCCAGGACAAGGAAGGCATTCCTCCAGACCAGCAGCGGCTGATTTTCGCCGGCAAACAACTGGAAGATGGTCGGACACTGTCCGATTACAACATCCAGAAAGAGAGCACCCTCCACTTGGTGCTTCGATTGAGGGGAGCC A TGAGCGATCCACTGCATGTGACTTTCGTGTGTACGGGGAACATTTGCCGGTCCCCTATGGCCGAGAAAATGTTCGC CCAGCAGCTGCGCCATAGAGGGCTCGGGGATGCCGTCAGAGTGACCAGCGCAGGTACTGGCAACTGGCACGTTGGC AGCTGTGCCGACGAAAGAGCGGCAGGTGTCCTGCGAGCCCACGGTTACCCCACAGACCACAGAGCCGCTCAGGTGG GAACTGAGCATCTGGCCGCCGACCTTCTGGTCGCCTTGGATAGAAACCATGCCAGACTGCTGAGACAGCTCGGCGT GGAGGCTGCCAGGGTCCGGATGCTGAGATCATTTGACCCACGGAGCGGCACACACGCTCTGGACGTGGAAGACCCT TACTACGGTGACCACTCTGACTTCGAGGAAGTCTTCGCAGTGATCGAATCCGCCCTTCCAGGTCTGCACGACTGGG TCGATGAAAGACTGGCTCGCAACGGCCCCTCAGGAGGAGGCGGTTCTGGAGGCGGAGGAAGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAGGCGGTTCT ATGCTTAGAGGCATCCAAGCCCTGTCACGACCACTGACAAGA GTCTACCGAGCCCTGGCTGTGATTGGGGTATTGGCCGCATCACTGCTGGCCTCTTGGGTGGGCGCAGTCCCACAAG TTGGTTTGGCTGCCTCCGCCCTGCCTACATTTGCCCATGTGGTTATTGTGGTTGAGGAAAATAGGTCCCAGGCCGC CATCATAGGAAACAAATCAGCCCCCTTCATTAATAGCCTGGCCGCCAATGGAGCCATGATGGCACAGGCTTTCGCT GAGACTCATCCGAGTGAGCCCAACTACCTGGCACTGTTTGCCGGAAATACATTTGGACTCACTAAAAACACATGTC CGGTTAACGGAGGAGCACTTCCGAACCTGGGCAGCGAACTCCTTTCTGCCGGCTACACCTTTATGGGGTTTGCCGA GGATTTGCCCGCTGTAGGTTCCACGGTCTGTTCTGCTGGGAAGTACGCCCGGAAGCATGTGCCCTGGGTCAACTTC AGTAATGTGCCTACAACTCTGTCTGTGCCTTTCAGTGCTTTCCCAAAACCTCAGAATTATCCCGGCCTTCCTACTG TTAGTTTCGTCATCCCAAATGCTGATAACGACATGCACGATGGGTCTATTGCCCAGGGGGATGCATGGCTGAATAG GCATCTTTCCGCCTATGCCAATTGGGCAAAGACAAATAACAGTCTGCTTGTAGTGACATGGGATGAGGATGATGGA TCTTCACGAAATCAGATCCCTACCGTGTTTTACGGAGCCCATGTCAGGCCGGGCACATACAATGAGACTATCAGCC ATTACAACGTCCTGTCCACACTGGAGCAAATCTACGGGTTGCCCAAGACAGGGTATGCCACAAACGCTCCCCCTAT AACCGATATCTGGGGGGAT GGAGGAGGCTCTGGAGGCGGAAGCgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAGGCTCTGGAGGCGGAAGC ATGTCCTTTGTGGTTGCCAATACTGAATTTGTAAGTGGTGCCGCGGGTAATTTGGCACGCCTGGGCAGCAT GATATCAGCTGCTAATTCCGCCGCCGCCGCCCAGACAACAGCTGTGGCAGCAGCGGGCGCCGATGAAGTGAGCGCT GCCGTCGCTGCTCTTTTCGGAGCTCATGGACAAACCTACCAAGTGCTGAGCGCCCAGGCTGCCGCTTTCCACAGTC AATTCGTGCAAGCTCTGTCAGGGGGAGCTCAAGCTTATGCCGCCGCAGAAGCAACCAACTTCGGACCACTCCAGCC CTTGTTCGATGTCATTAACGCCCCAACTCTCGCTCTGCTCAACCGGCCACTTATTGGCAACGGGGCCGATGGCACT GCCGCTAACCCTAACGGTCAAGCCGGTGGTCTGTTGATCGGCAATGGCGGAAACGGCTTCAGCCCAGCAGCGGGCC CCGGAGGAAACGGCGGCGCCGCCGGCCTTCTCGGTCATGGTGGAAACGGTGGGGTAGGGGCACTGGGGGCTAATGG CGGGGCTGGCGGAACAGGAGGCTGGCTGTTCGGCAACGGCGGCGCGGGCGGAAACAGCGGGGGAGGCGGGGGTGCA GGTGGAATTGGGGGTAGCGCCGTGCTGTTCGGGGCAGGCGGTGCCGGCGGGATCAGCCCAAATGGCATGGGTGCAG GAGGAAGCGGCGGGAATGGCGGTCTGTTCTTCGGGAATGGCGGAGCAGGAGCATCCTCCTTCCTGGGAGGTGGGGG AGCGGGAGGGCGGGCCTTCCTCTTCGGAGATGGAGGCGCGGGGGGGGCCGCCCTGAGCGCCGGCTCTGCAGGAAGG GGAGGAGATGCCGGTTTCTTCTACGGGAATGGCGGCGCAGGGGGGAGCGGAGCCGGCGGCGCCTCAAGCGCCCACG GCGGGGCTGGGGGCCAGGCCGGTCTGTTCGGAAACGGCGGTGAGGGGGGCGATGGCGGTGCACTTGGGGGAAATGG CGGGAACGGGGGCAATGCACAGCTGATCGGAAATGGAGGTGACGGAGGGGACGGCGGCGGAGCAGGGGCCCCGGGT CTGGGAGGAAGAGGCGGGCTGCTGCTGGGGCTGCCTGGTGCCAACGGGACG

[0266] SEQ ID NO:32: TB14, pUC57-Mini-AmpR-RV0125-HiBiT-P2A-Rv1196-HiBIT (4695bp)

[0267] CCAATGATGAATTCTAATACGACTCACTATAGcttgttctttttgcagaagctcagaataaacgctcaactttggGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG ATGAGCAATTCACGA AGACGCAGTTTGCGCTGGTCATGGCTCCTTAGTGTGCTCGCGGCCGTCGGCCTGGGACTCGCTACTGCTCCAGCTC AGGCCGCCCCACCAGCTCTTTCTCAGGATCGGTTTGCTGACTTCCCTGCCTTGCCATTGGATCCATCCGCAATGGT TGCACAGGTAGGCCCGCAGGTCGTCAACATCAACACCAAGCTTGGGTACAATAACGCCGTGGGTGCCGGTACTGGC ATTGTGATTGACCCCAACGGAGTTGTGCTTACTAATAACCACGTGATCGCCGGTGCCACCGATATAAACGCCTTCA GCGTGGGGTCCGGACAGACATACGGAGTCGATGTGGTAGGCTACGATCGCACCCAAGATGTGGCCGTGCTCCAACT GCGGGGTGCAGGAGGCCTTCCCAGCGCTGCCATTGGAGGCGGCGTGGCCGTGGGAGAGCCGGTCGTAGCTATGGGC AATAGCGGCGGACAGGGGGGTACGCCGCGGGCCGTCCCTGGGAGAGTGGTGGCTCTGGGGCAGACAGTACAAGCCT CAGACAGCCTGACCGGGGCAGAGGAGACCCTGAACGGGCTGATTCAATTTGACGCTGCCATACAGCCCGGCGATTC AGGAGGACCAGTGGTGAACGGACTGGGCCAGGTAGTGGGAATGAACACCGCCGCCTCTGATAACTTCCAGCTGTCA CAGGGCGGACAGGGATTTGCCATTCCCATTGGTCAGGCTATGGCCATCGCCGGCCAGATAAGGTCTGGTGGCGGCT CACCTACAGTGCACATCGGCCCTACCGCATTCCTGGGATTGGGTGTAGTGGACAACAACGGTAATGGGGCCAGGGT CCAGCGAGTTGTCGGGTCCGCTCCTGCAGCTTCCCTGGGCATCAGTACTGGGGACGTCATCACCGCCGTAGATGGA GCTCCCATAAACAGCGCCACCGCCATGGCCGACGCATTGAACGGGCATCATCCTGGAGACGTCATCTCAGTCACCT GGCAGACTAAAAGCGGGGGAACACGGACTGGAAATGTTACACTGGCGGAAGGACCGCCGGCC GGAGGAGGCGGTTCTGGAGGCGGAGGAAGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAGGCGGTTCTgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG ATGGTCGACTTCGGCGCCCTTCCCCCAGAAATCAATTCCGCTAGAATGTACGCAGGCCC TGGAAGCGCCTCTCTTGTAGCAGCTGCGCAGATGTGGGACAGTGTGGCCTCAGACCTCTTTTCCGCCGCATCAGCT TTCCAAAGCGTCGTGTGGGGTCTGACGGTAGGCTCCTGGATCGGGTCCTCCGCAGGCCTCATGGTGGCCGCCGCCT CTCCATACGTGGCCTGGATGAGCGTTACCGCTGGACAGGCTGAGCTGACGGCTGCACAGGTCAGGGTCGCCGCGGC AGCCTATGAGACCGCCTATGGTCTGACCGTGCCACCTCCAGTTATTGCCGAGAACAGGGCGGAGCTTATGATCCTG ATCGCTACAAATCTGCTCGGCCAGAATACACCGGCGATTGCGGTGAACGAGGCTGAATACGGGGAGATGTGGGCGC AGGACGCAGCAGCCATGTTCGGCTACGCAGCTGCTACCGCAACTGCCACTGCAACTCTTCTGCCCTTCGAAGAAGC CCCCGAAATGACAAGCGCAGGCGGCCTGCTGGAGCAGGCCGCCGCAGTAGAAGAGGCATCCGATACCGCCGCCGCC AACCAACTCATGAACAATGTGCCTCAAGCTCTGCAGCAATTGGCACAGCCGACTCAGGGAACCACTCCTAGCTCCA AACTGGGCGGGCTGTGGAAAACTGTGAGCCCTCATAGAAGCCCTATCAGCAATATGGTCTCCATGGCCAACAACCA CATGAGCATGACTAACAGCGGGGTTAGCATGACTAATACCCTGAGCTCAATGCTGAAAGGATTTGCACCTGCCGCA GCGGCCCAGGCAGTCCAAACTGCGGCTCAGAACGGTGTTCGAGCCATGTCCTCCCTGGGGAGTTCTCTGGGGTCTT CTGGCCTGGGGGGCGGCGTGGCAGCCAACCTGGGCCGGGCCGCAAGTGTTGGAAGCCTTAGCGTCCCTCAAGCCTG GGCCGCCGCTAATCAAGCTGTGACTCCTGCCGCTCGGGCACTCCCTCTCACCTCATTGACAAGCGCAGCAGAGAGA GGGCCCGGTCAGATGCTGGGCGGCCTCCCTGTTGGACAGATGGGAGCAAGGGCTGGAGGCGGTCTGTCAGGTGTGC TGCGGGTTCCTCCTCGGCCCTACGTAATGCCCCACAGCCCAGCCGCCGGG

[0268] SEQ ID NO:33: TB15, pUC57-Mini-AmpR-RV3619-HiBiT-3620-P2A-Rv1813-HiBiT-P2A-Rv2608-HiBIT (5385bp)

[0269]

[0270] SEQ ID NO:34: TB16, pUC57 - Mini - AmpR - RV3875 - HiBiT - P2A - Rv1886 - HiBiT - P2A - Rv2660 - HiBIT (4080bp)

[0271]

[0272] SEQ ID NO:35: TB17, pUC57-Mini-AmpR-Rv3874-HiBiT-3875-P2A-Rv1886-HiBiT (3993bp)

[0273]

[0274] SEQ ID NO:36: TB18, pUC57-Mini-AmpR-RV3619-HiBiT-3620-P2A-1886-HiBiT-2029-P2A-HiBiT-1468c (6303bp)

[0275] CCAATGATGAATTCTAATACGACTCACTATAGcttgttctttttgcagaagctcagaataaacgctcaactttggGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG ATGACAATCAATTAC CAATTCGGCGATGTCGATGCACACGGGGCCATGATCCGCGCACAGGCAGGGAGTCTGGAGGCTGAGCACCAGGCCA TCATCAGCGATGTGCTGACCGCTAGTGATTTCTGGGGGGGCGCTGGATCCGCTGCTTGTCAGGGCTTCATTACCCA ACTCGGCAGAAATTTCCAGGTGATTTACGAGCAGGCTAATGCCCATGGGCAGAAGGTTCAAGCCGCCGGCAATAAC ATGGCGCAGACAGACTCCGCCGTAGGGTCCTCCTGGGCT GGAGGAGGCGGTTCTGGAGGCGGAGGAAGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAGGCGGTTCT ATGACAAGTCGATTCATGACGGACCCACATGCAAT GCGAGACATGGCCGGTAGATTCGAAGTGCATGCCCAGACAGTGGAAGACGAGGCACGCAGGATGTGGGCCTCCGCC CAGAATATCAGCGGGGCAGGATGGAGTGGCATGGCAGAGGCCACTAGCCTGGACACTATGACTCAGATGAATCAGG CTTTTCGAAACATCGTGAATATGCTGCATGGAGTGAGAGATGGCCTGGTAAGAGACGCGAACAATTACGAGCAGCA GGAACAAGCTAGCCAGCAGATCTTGAGCTCT GGAGGAGGCGGTTCTgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG A TGACCGACGTGAGCAGAAAGATACGGGCATGGGGTAGAAGACTGATGATCGGTACTGCGGCAGCTGTAGTGCTGCC TGGCTTGGTAGGGCTCGCGGGAGGTGCTGCTACGGCGGGAGCATTCTCACGCCCTGGACTCCCAGTGGAGTATCTT CAGGTCCCATCTCCTAGTATGGGCCGCGACATAAAAGTGCAGTTCCAGAGCGGTGGGAATAACTCTCCTGCCGTAT ATCTGTTGGACGGCCTGCGGGCCCAAGATGACTACAACGGCTGGGATATAAACACCCCCGCTTTCGAGTGGTATTA CCAGAGTGGCCTGAGCATCGTAATGCCCGTCGGGGGCCAATCTTCTTTCTACAGTGATTGGTACTCTCCCGCATGC GGTAAGGCAGGGTGTCAGACTTATAAGTGGGAAACCTTCCTGACATCCGAACTGCCCCAGTGGCTGAGCGCCAATA GGGCAGTAAAACCAACTGGGAGCGCCGCCATTGGACTGAGTATGGCCGGCAGTAGCGCGATGATATTGGCTGCCTA TCATCCCCAGCAGTTCATTTACGCTGGATCCTTGTCTGCGCTCCTGGACCCAAGTCAGGGGATGGGTCCTTCTCTT ATCGGGTTGGCTATGGGGGACGCAGGAGGTTACAAAGCCGCCGATATGTGGGGGCCATCATCTGACCCCGCATGGG AACGAAATGACCCCACGCAACAGATCCCCAAATTGGTTGCTAACAACACTAGGTTGTGGGTCTATTGCGGAAACGG AACCCCTAACGAGCTGGGAGGCGCGAACATACCCGCTGAGTTTCTGGAGAACTTTGTCCGATCCTCCAACCTGAAG TTCCAGGACGCTTACAATGCTGCTGGGGGACACAACGCGGTCTTCAACTTTCCTCCAAACGGCACGCACAGCTGGG AATACTGGGGAGCACAGCTGAACGCCATGAAGGGCGATCTGCAGTCCAGCCTGGGCGCTGGC GGAGGAGGCGGTTCTGGAGGCGGAGGAAGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAGGCGGTTCT ATGACAGAACCC GCCGCATGGGACGAAGGAAAGCCACGGATAATAACACTGACTATGAACCCAGCCTTGGATATTACAACGTCCGTGG ACGTTGTAAGGCCCACCGAAAAGATGCGATGTGGAGCTCCTCGATACGATCCTGGGGGCGGAGGAATCAATGTGGC GCGGATCGTGCATGTGCTTGGTGGGTGTTCCACCGCTCTCTTCCCTGCCGGGGGAAGTACTGGATCTCTCCTGATG GCTCTGCTCGGAGACGCGGGCGTCCCATTCCGGGTTATACCCATCGCCGCATCCACCAGAGAAAGTTTCACCGTGA ATGAGAGCAGGACTGCCAAGCAGTATCGGTTCGTGCTTCCAGGCCCCTCCCTGACTGTTGCAGAGCAAGAGCAGTG CCTTGACGAGCTGAGAGGGGCTGCAGCGAGCGCTGCCTTCGTGGTGGCCAGCGGCTCCCTTCCCCCTGGGGTGGCT GCTGACTACTATCAGCGGGTGGCCGATATATGCAGGAGGAGTTCAACCCCTTTGATTCTCGACACGAGCGGAGGTG GACTTCAGCACATTAGCTCCGGCGTATTCCTGCTCAAAGCCTCAGTTCGGGAGTTGAGAGAATGCGTGGGCAGTGA ACTTTTGACCGAACCTGAGCAGCTGGCCGCCGCCCATGAACTTATTGACAGAGGTAGAGCAGAGGTCGTGGTCGTATCACTCGGATCTCAGGGTGCTCTGCTCGCTACTCGACACGCCTCCCACCGATTTAGTAGTATCCCAATGACCGCGG TGAGCGGAGTTGGCGCTGGTGACGCTATGGTCGCTGCCATTACCGTGGGCCTCAGCAGGGGATGGAGCCTGATCAA GTCAGTCCGCTTGGGTAACGCGGCTGGTGCGGCCATGTTGCTGACCCCAGGAACAGCTGCCTGTAACAGGGACGAC GTCGAACGATTCTTCGAACTGGCGGCTGAGCCAACCGAAGTGGGGCAAGACCAGTACGTCTGGCACCCCATTGTGA ATCCTGAAGCCAGTCCT GGAGGAGGCTCTGGAGGCGGAAGCgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAGGCTCTGGAGGCGGAAGC ATGTCCTTTGTGGTTGCCAATACTGAATTTGTAAGTGGTGCCGCGGGTAATTTGGCACGCCTGGGCAGCATGA TATCAGCTGCTAATTCCGCCGCCGCCGCCCAGACAACAGCTGTGGCAGCAGCGGGCGCCGATGAAGTGAGCGCTGC CGTCGCTGCTCTTTTCGGAGCTCATGGACAAACCTACCAAGTGCTGAGCGCCCAGGCTGCCGCTTTCCACAGTCAA TTCGTGCAAGCTCTGTCAGGGGGAGCTCAAGCTTATGCCGCCGCAGAAGCAACCAACTTCGGACCACTCCAGCCCT TGTTCGATGTCATTAACGCCCCAACTCTCGCTCTGCTCAACCGGCCACTTATTGGCAACGGGGCCGATGGCACTGC CGCTAACCCTAACGGTCAAGCCGGTGGTCTGTTGATCGGCAATGGCGGAAACGGCTTCAGCCCAGCAGCGGGCCCC GGAGGAAACGGCGGCGCCGCCGGCCTTCTCGGTCATGGTGGAAACGGTGGGGTAGGGGCACTGGGGGCTAATGGCG GGGCTGGCGGAACAGGAGGCTGGCTGTTCGGCAACGGCGGCGCGGGCGGAAACAGCGGGGGAGGCGGGGGTGCAGG TGGAATTGGGGGTAGCGCCGTGCTGTTCGGGGCAGGCGGTGCCGGCGGGATCAGCCCAAATGGCATGGGTGCAGGA GGAAGCGGCGGGAATGGCGGTCTGTTCTTCGGGAATGGCGGAGCAGGAGCATCCTCCTTCCTGGGAGGTGGGGGAG CGGGAGGGCGGGCCTTCCTCTTCGGAGATGGAGGCGCGGGGGGGGCCGCCCTGAGCGCCGGCTCTGCAGGAAGGGG AGGAGATGCCGGTTTCTTCTACGGGAATGGCGGCGCAGGGGGGAGCGGAGCCGGCGGCGCCTCAAGCGCCCACGGC GGGGCTGGGGGCCAGGCCGGTCTGTTCGGAAACGGCGGTGAGGGGGGCGATGGCGGTGCACTTGGGGGAAATGGCG GGAACGGGGGCAATGCACAGCTGATCGGAAATGGAGGTGACGGAGGGGACGGCGGCGGAGCAGGGGCCCCGGGTCT GGGAGGAAGAGGCGGGCTGCTGCTGGGGCTGCCTGGTGCCAACGGGACG

[0276] SEQ ID NO:37: TB19, pUC57 - Mini - AmpR - RV3619 - HiBiT - 3620 - P2A - 1886 - HiBiT - 1813 - P2A - HiBiT - 1468c (5715bp)

[0277] CCAATGATGAATTCTAATACGACTCACTATAGcttgttctttttgcagaagctcagaataaacgctcaactttggGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG ATGACAATCAATTAC CAATTCGGCGATGTCGATGCACACGGGGCCATGATCCGCGCACAGGCAGGGAGTCTGGAGGCTGAGCACCAGGCCA TCATCAGCGATGTGCTGACCGCTAGTGATTTCTGGGGGGGCGCTGGATCCGCTGCTTGTCAGGGCTTCATTACCCA ACTCGGCAGAAATTTCCAGGTGATTTACGAGCAGGCTAATGCCCATGGGCAGAAGGTTCAAGCCGCCGGCAATAAC ATGGCGCAGACAGACTCCGCCGTAGGGTCCTCCTGGGCT GGAGGAGGCGGTTCTGGAGGCGGAGGAAGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAGGCGGTTCT ATGACAAGTCGATTCATGACGGACCCACATGCAAT GCGAGACATGGCCGGTAGATTCGAAGTGCATGCCCAGACAGTGGAAGACGAGGCACGCAGGATGTGGGCCTCCGCC CAGAATATCAGCGGGGCAGGATGGAGTGGCATGGCAGAGGCCACTAGCCTGGACACTATGACTCAGATGAATCAGG CTTTTCGAAACATCGTGAATATGCTGCATGGAGTGAGAGATGGCCTGGTAAGAGACGCGAACAATTACGAGCAGCA GGAACAAGCTAGCCAGCAGATCTTGAGCTCT GGAGGAGGCGGTTCTgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG A TGACCGACGTGAGCAGAAAGATACGGGCATGGGGTAGAAGACTGATGATCGGTACTGCGGCAGCTGTAGTGCTGCC TGGCTTGGTAGGGCTCGCGGGAGGTGCTGCTACGGCGGGAGCATTCTCACGCCCTGGACTCCCAGTGGAGTATCTT CAGGTCCCATCTCCTAGTATGGGCCGCGACATAAAAGTGCAGTTCCAGAGCGGTGGGAATAACTCTCCTGCCGTAT ATCTGTTGGACGGCCTGCGGGCCCAAGATGACTACAACGGCTGGGATATAAACACCCCCGCTTTCGAGTGGTATTA CCAGAGTGGCCTGAGCATCGTAATGCCCGTCGGGGGCCAATCTTCTTTCTACAGTGATTGGTACTCTCCCGCATGC GGTAAGGCAGGGTGTCAGACTTATAAGTGGGAAACCTTCCTGACATCCGAACTGCCCCAGTGGCTGAGCGCCAATA GGGCAGTAAAACCAACTGGGAGCGCCGCCATTGGACTGAGTATGGCCGGCAGTAGCGCGATGATATTGGCTGCCTA TCATCCCCAGCAGTTCATTTACGCTGGATCCTTGTCTGCGCTCCTGGACCCAAGTCAGGGGATGGGTCCTTCTCTT ATCGGGTTGGCTATGGGGGACGCAGGAGGTTACAAAGCCGCCGATATGTGGGGGCCATCATCTGACCCCGCATGGG AACGAAATGACCCCACGCAACAGATCCCCAAATTGGTTGCTAACAACACTAGGTTGTGGGTCTATTGCGGAAACGG AACCCCTAACGAGCTGGGAGGCGCGAACATACCCGCTGAGTTTCTGGAGAACTTTGTCCGATCCTCCAACCTGAAG TTCCAGGACGCTTACAATGCTGCTGGGGGACACAACGCGGTCTTCAACTTTCCTCCAAACGGCACGCACAGCTGGG AATACTGGGGAGCACAGCTGAACGCCATGAAGGGCGATCTGCAGTCCAGCCTGGGCGCTGGC GGAGGAGGCGGTTCTGGAGGCGGAGGAAGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAGGCGGTTCT ATGATCACTAAC TTGCGACGAAGAACCGCCATGGCTGCCGCCGGACTGGGGGCTGCACTGGGGTTGGGCATTCTGCTTGTTCCAACCG TGGATGCGCATCTCGCCAACGGGAGCATGAGTGAAGTTATGATGTCAGAGATCGCGGGACTCCCTATCCCCCCTAT CATACACTATGGCGCCATTGCCTACGCACCAAGTGGCGCTTCCGGCAAAGCTTGGCACCAGAGGACACCTGCTCGG GCCGAGCAAGTAGCACTGGAAAAGTGCGGGGATAAGACGTGCAAGGTCGTCTCTCGGTTTACACGATGCGGCGCCG TGGCGTATAACGGGTCAAAGTACCAGGGAGGGACCGGACTGACCAGGAGAGCTGCTGAGGATGATGCAGTGAATAG GCTCGAAGGGGGACGCATCGTGAACTGGGCCTGTAACGGAGGAGGCTCTGGAGGCGGAAGCgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAGGCTCTGGAGGCGGAAGC ATGTCCTTTGTGGTTGCCAATACTGAATTTGTAAGTGGTGCCGCGGGTAATTT GGCACGCCTGGGCAGCATGATATCAGCTGCTAATTCCGCCGCCGCCGCCCAGACAACAGCTGTGGCAGCAGCGGGC GCCGATGAAGTGAGCGCTGCCGTCGCTGCTCTTTTCGGAGCTCATGGACAAACCTACCAAGTGCTGAGCGCCCAGG CTGCCGCTTTCCACAGTCAATTCGTGCAAGCTCTGTCAGGGGGAGCTCAAGCTTATGCCGCCGCAGAAGCAACCAA CTTCGGACCACTCCAGCCCTTGTTCGATGTCATTAACGCCCCAACTCTCGCTCTGCTCAACCGGCCACTTATTGGC AACGGGGCCGATGGCACTGCCGCTAACCCTAACGGTCAAGCCGGTGGTCTGTTGATCGGCAATGGCGGAAACGGCT TCAGCCCAGCAGCGGGCCCCGGAGGAAACGGCGGCGCCGCCGGCCTTCTCGGTCATGGTGGAAACGGTGGGGTAGG GGCACTGGGGGCTAATGGCGGGGCTGGCGGAACAGGAGGCTGGCTGTTCGGCAACGGCGGCGCGGGCGGAAACAGC GGGGGAGGCGGGGGTGCAGGTGGAATTGGGGGTAGCGCCGTGCTGTTCGGGGCAGGCGGTGCCGGCGGGATCAGCC CAAATGGCATGGGTGCAGGAGGAAGCGGCGGGAATGGCGGTCTGTTCTTCGGGAATGGCGGAGCAGGAGCATCCTC CTTCCTGGGAGGTGGGGGAGCGGGAGGGCGGGCCTTCCTCTTCGGAGATGGAGGCGCGGGGGGGGCCGCCCTGAGC GCCGGCTCTGCAGGAAGGGGAGGAGATGCCGGTTTCTTCTACGGGAATGGCGGCGCAGGGGGGAGCGGAGCCGGCG GCGCCTCAAGCGCCCACGGCGGGGCTGGGGGCCAGGCCGGTCTGTTCGGAAACGGCGGTGAGGGGGGCGATGGCGG TGCACTTGGGGGAAATGGCGGGAACGGGGGCAATGCACAGCTGATCGGAAATGGAGGTGACGGAGGGGACGGCGGC GGAGCAGGGGCCCCGGGTCTGGGAGGAAGAGGCGGGCTGCTGCTGGGGCTGCCTGGTGCCAACGGGACG

[0278] SEQ ID NO: 38: TB20, pUC57-Mini-AmpR-RV1886c-HiBiT-P2A-Rv3425-HiBiT-P2A-Rv1985c-HiBIT (5046bp)

[0279]

[0280] Example 2

[0281] The percentage of weight loss from all immunized mice was collected and calculated. The inconsistent number of mice in each experimental group was due to the cumulative experiments conducted on any specific tuberculosis candidate vaccine. Weight loss indicates a potential adverse effect of vaccination. All weight changes caused by various immunizations were considered to be within an acceptable range of 0% to 10%. The weight loss of the blank lipid nanoparticle (LNP) group served as the baseline value of immunity caused by lipid nanoparticles. The phosphate-buffered saline (dPBS) group served as a negative control.

[0282] Figure 4 It was demonstrated that the immunotoxicity of all mRNA vaccine antigen combinations was not significant, and there was not much difference in the weight changes of animals in the blank control group and the empty LNP group. Within the acceptable range, that is, within 10% weight loss, it can be considered non-toxic or with low side reactions.

[0283] Example 3

[0284] After filtering the liposomal nanoparticle formulation of mRNA, female C57 mice at 6 - 8 weeks old (uninfected mouse model) were injected, and the control group was injected with boric acid buffer (dPBS). Fourteen days after the first immunization, mouse blood was collected to detect the titers of specific antibodies IgG, IgG1, and IgG2a against all tuberculosis antigens contained in the mRNA of this group in the serum. On the 21st day after the primary immunization, mouse blood was collected again to detect the titers of specific antibodies IgG, IgG1, and IgG2a against all tuberculosis antigens contained in the mRNA of this group in the serum, and the mice were injected again with the filtered liposomal nanoparticle formulation, which was the secondary immunization. Two weeks after the secondary immunization, mouse blood was collected to detect the titers of specific antibodies IgG, IgG1, and IgG2a against all tuberculosis antigens contained in the mRNA of this group in the serum, and the results were as Figure 6 . The experimental results showed that: the liposomal nanoparticles prepared from the mRNA and liposomal components provided in this application, when injected into mice, induced the production of specific antibodies IgG, IgG1, and IgG2a against tuberculosis antigens, and the antibody levels increased with the increase in the concentration of mRNA liposomal nanoparticles.

[0285] Subsequently, the T cell immune response of the collected mouse spleen cell samples was detected. The cytokines induced by re-stimulating with different antigenic protein peptide libraries were detected by ELISpot ( Figure 5 A - B in Figure 5 ), flow cytometry ( Figure 5In C), the specific cellular immune function of the multi-antigen combination fusion protein was evaluated. The vaccination protocol was the same as that for humoral antibody detection, i.e., the two-dose method. The second immunization was carried out three weeks after the first immunization (at the same dose as the first immunization), and spleen cell collection and analysis were carried out two weeks later. The results showed that the mRNA-LNP provided by the present application could induce a strong cellular immune response against Mycobacterium tuberculosis antigens, namely, the antigen-specific responses of CD8 and CD4 T cells ( Figures 5 to 6 ).

[0286] The splenocytes of mice stimulated by the 24 peptide libraries corresponding to all mRNAs showed a significant increase in IL2+, IFN-γ, and TNF-α lymphocytes in the vaccinated mice. At the same time, IL-4, IL-6, and IL-10 did not increase in all samples, and the ratio of specific antibody IgG2a to IgG1 against multiple Mycobacterium tuberculosis antigens was greater than 1, indicating that the candidate vaccine mainly induced a specific Th1 immune response rather than a Th2 immune response.

[0287] Example 4

[0288] Long-term efficacy assessment of all vaccine groups was carried out on splenocytes and sera from naïve C57BL / 6j female mice 6 months and 12 months later. The long-term cellular immune response was quantified by flow cytometry and cytokine bead array (CBA) methods. Six months and 12 months after the first vaccination with 10 μg of TB10, TB18, and TB19 mRNA candidate vaccines, splenocytes from all groups (N = 5) of mice were collected. The proportion of CD8+ T cells releasing interleukin-2 (IL-2), interferon-γ (IFN-γ), granzyme B, and perforin in all groups was plotted in different colors ( Figure 7 in A). The proportion of CD4+ T cells releasing IL-2 and IFN-γ and the proportion of macrophages and M1 subtypes were quantified ( Figure 7 in B and C). T effector memory cells and central memory cells were sorted using CD44 / CD62 antibodies to indicate the positive T cell population releasing cytokines ( Figure 7 in D and E) and the proportion of different populations of CD8+ and CD4+ T memory cells ( Figure 7 in F and G). CBA measurements of long-term samples at 6 months and 12 months showed cytokine release in splenocytes of mice in all groups after peptide library stimulation in the form of a heat map ( Figure 7 in H). The overall quantified long-term efficacy of all vaccinations was summarized in the panel for comparison ( Figure 7I and J are 6 months, and K and L are 12 months). TB10, TB18, and TB19 were used as the experimental groups, while blank lipid nanoparticles (LNP) and phosphate-buffered saline (dPBS) immunizations were used as negative controls. All quantified values in the data are relative quantified values relative to the control group dPBS, and the quantified value of dPBS is 1.

[0289] As described above, the two combinations of TB10 and TB18 have good potential for long-term prevention and treatment.

[0290] Example 5

[0291] Uninfected mice were inoculated with 10 μg of tuberculosis mRNA candidate vaccines (TB10, TB18, and TB20), as well as corresponding amounts of controls (blank lipid nanoparticles and phosphate-buffered saline), following Figure 2 the protocol of A in. Serum and splenocytes were collected from all groups on the 35th day after the first inoculation and subjected to RNA-seq and CDR3 sequencing. The top 30 significantly regulated genes were classified and listed in a heat map. Compared with the blank lipid nanoparticle group, the unbalanced regulation was represented by different colors ( Figure 8 A in). The functions and categories of all 30 genes were collected in Table 5. The regulation conditions (red dots, blue dots, and gray dots) and differentially expressed genes (DEGs) were presented in a volcano plot, showing significant differences in the immune responses stimulated by the tuberculosis mRNA vaccine ( Figure 8 B in). The Kyoto Encyclopedia of Genes and Genomes (KEGG) of B cell receptors (BCRs) and T cell receptors (TCRs) was analyzed, and 26 responsible signaling pathways related to immune responses and regulations after inoculation or infection were selected and plotted. The number of upregulated genes was marked under each pathway ( Figure 8 C in), and the abundances of BCR and TCR were plotted in different colors and sizes ( Figure 8 D in). The blank lipid nanoparticle and phosphate-buffered saline groups were used as negative controls.

[0292] Table 5 Functions of 30 regulated genes detected by RNA-Seq

[0293]

[0294]

[0295] Example 6

[0296] See Figure 2 B in, and 10 cfu of BCG was performed 11 weeks (-11 weeks) before vaccine immunization 5Pre-immunization, 6 weeks later (-5 weeks), intranasal infection with 300 cfu of Mycobacterium tuberculosis was performed; before vaccination (0 weeks), some mice were sacrificed to detect the bacterial load in the lungs. If the bacterial load cfu was less than 10 5， then it was considered that the latent infection model was successfully established and could be used for subsequent vaccination. Mice with successfully established latent models were vaccinated with 10 μg of tuberculosis mRNA vaccine in different groups (0 weeks). Three weeks later (3 weeks), a second dose of 10 μg mRNA vaccine was administered. Two weeks later (5 weeks), the animals were euthanized to collect blood samples, lung tissues, and spleen tissues for subsequent experimental detection. Four indicators of IL-2, IFN-γ, granzyme, and perforin secreted by CD8 T cells detected by flow cytometry ([ Figure 9 A-D in) TB14 and TB18 showed outstanding comprehensive performance and were stronger than other antigen combinations; in the heat maps of more immune evaluation indicators (CD8+activated, CD8+IL-2, CD8+IFN-γ, CD8+Granzyme B, CD8+perforin, CD4+activated, CD4+IL-2, and CD4+IFN-γ) ([ Figure 9 E in), the average performance of TB14 and TB18 was stronger than other combinations; in the detection of the bacterial load in the lung tissues isolated in vivo ([ Figure 9 G in), the CFU count of TB18 was lower than that of the BCG control and lower than other antigen combinations. In the pathological scoring ([ Figure 9 H in), the pathological score of TB18 was lower than that of the BCG control group and other antigen combinations except higher than that of TB12; in the detection of H&E stained pathological sections ([ Figure 9 I in), several representative examples of each antigen combination were shown; from a comprehensive view of multiple evaluation methods, the two antigen combinations of TB14 and TB18 showed good effects in inhibiting the growth of Mycobacterium tuberculosis, treating tuberculosis infection, and alleviating pathological symptoms in the latent animal model.

[0297] The pathological scoring criteria are shown in Table 6.

[0298] Table 6

[0299]

[0300] Figure 9 It was proved that TB18 had very good therapeutic effects; in addition, the two antigens Rv0125 and Rv1196 in TB14 showed good therapeutic effects.

[0301] Example 7

[0302] Spleen cells were collected from all groups of mice (protective mouse models) inoculated with 10 μg of the tuberculosis candidate vaccine, and the cellular immune response was evaluated. The proportions of CD8+ T cells releasing interleukin-2 (IL-2), interferon-γ (IFN-γ), granzyme B, and perforin, and CD4+ T cells releasing IL-2 and IFN-γ were quantified by flow cytometry and plotted as a heat map ( Figure 10 in A). The proportions of CD8+ and CD4+ activated T cells were quantified as bar graphs ( Figure 10 in B and C). Mycobacterium tuberculosis colony-forming units (CFUs) were counted in harvested lung tissues from all groups of mice (protective mouse models) challenged with Mycobacterium tuberculosis (M. tb) ( Figure 10 in D). Pathological scoring was graded based on H&E staining images of all groups ( Figure 10 in E). Statistical analysis was performed between the BCG boost group and the control group, and between the tuberculosis candidate vaccine group and the BCG boost group. Any p-value less than 0.3 was marked on the graphs. Blank lipid nanoparticles (LNPs) and phosphate-buffered saline (dPBS) immunization served as negative controls. Representative pathological H&E staining images at 20x and 200x magnifications from all groups were selected and presented ( Figure 10 in F).

[0303] Figure 10 It was demonstrated that both TB10 and TB18 had advantages in protective efficacy, especially TB10 had excellent advantages compared with other antigen combinations.

[0304] Example 8

[0305] The spatial structure of the polyprotein generated from a single mRNA sequence in which multiple antigens of the present application were concatenated and separated by a flexible linker or P2A predicted by AlphaFold V2.3.2 was used.

[0306] Figure 12 It was shown that each single antigen part of the polyprotein was well separated due to the presence of the linker and did not interact with each other, and this conclusion was also confirmed in the above in vitro expression and antibody detection experiments, and its spatial structure was basically consistent with the natural conformation of the single antigen.

[0307] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0308] The above-described embodiments merely represent several implementation manners of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A combination of Mycobacterium tuberculosis antigens, characterized in that: The antigen combination includes antigens or antigen fragments thereof in Group I and Group II: I: Rv3619, Rv3620, and Rv1468; and II: at least one of Rv1886, Rv2029, Rv1813, Rv2234, Rv3310, Rv0125 and Rv1196.

2. The Mycobacterium tuberculosis antigen combination according to claim 1, characterized in that: The antigen combination comprises at least one of the following antigens or antigen fragments thereof: (1) Rv3619, Rv3620, Rv2234, Rv3310 and Rv1468; (2) Rv3619, Rv3620, Rv1886, Rv2029 and Rv1468; (3) Rv3619, Rv3620, Rv1886, Rv1813 and Rv1468; (4) Rv3619, Rv3620, Rv1468, Rv2234, Rv3310, Rv1886, Rv1813 and Rv2029.

3. The Mycobacterium tuberculosis antigen combination according to claim 1 or 2, characterized in that: The antigen combination has a sequence shown in any one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 27 and SEQ ID NO:

28.

4. mRNA encoding the combination of Mycobacterium tuberculosis antigens according to any one of claims 1 to 3; Optionally, the mRNA comprises a DNA transcribed from the sequence shown in any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:36 and SEQ ID NO:37; Optionally, the mRNA comprises modified nucleotides; Optionally, the modified nucleotides include one or more nucleotides of N1-methylpseudouridine triphosphate, pseudouridine triphosphate, 5-methoxyuridine triphosphate and 5-methylcytidine triphosphate.

5. The mRNA according to claim 4, characterized in that It also includes one or more of the following elements: Kozak sequence, 5' cap structure, 5' UTR, signal peptide coding sequence, 2A peptide coding sequence, Ub coding sequence, 3' UTR and PolyA tail; Optionally, the mRNA includes the following structures: a 5' cap structure, a 5' UTR, an open reading frame, a 3' UTR and a PolyA tail; Optionally, the open reading frame includes a coding sequence for the combination of Mycobacterium tuberculosis antigens; Optionally, the open reading frame further comprises one or more of a Kozak sequence, a signal peptide coding sequence, a 2A peptide coding sequence, an Ub coding sequence and a linker sequence.

6. A DNA that transcribes the mRNA of claim 4 or 5, or a DNA that encodes the combination of Mycobacterium tuberculosis antigens of any one of claims 1 to 3.

7. A recombinant expression vector for producing the mRNA according to claim 4 or 5; Optionally, the recombinant expression vector comprises the DNA of claim 6.

8. mRNA vaccine, characterized in that Comprising the mRNA of claim 4 or 5, and a delivery vector.

9. The mRNA vaccine according to claim 8, characterized in that The mRNA is encapsulated in the delivery vector; Optionally, the delivery vehicle comprises a lipid component; Optionally, the mass ratio of the lipid component to the mRNA is (10-30):1; Optionally, the lipid component includes one or more of a cationic lipid, an auxiliary phospholipid, cholesterol and a PEG lipid; Further optionally, in the lipid component, the molar ratio of cationic lipid, auxiliary phospholipid, cholesterol and PEG lipid is (45-55):(8-12):(35-40):(1-2).

10. The method for preparing the mRNA vaccine according to claim 8 or 9, characterized in that: The preparation method comprises: mixing the aqueous solution of the mRNA with the delivery carrier; Optionally, the preparation method comprises: Dissolving the mRNA in an aqueous solution to prepare phase A; dissolving the lipid component in an organic solvent to prepare phase B; and, Mixing the phase A and the phase B to prepare the mRNA vaccine; Optionally, the organic solvent is anhydrous ethanol.

11. Use of the Mycobacterium tuberculosis antigen combination according to any one of claims 1 to 3, the mRNA according to claim 4 or 5, the DNA according to claim 6, the recombinant expression vector according to claim 7, or the mRNA vaccine according to claim 8 or 9 in the following: (1) Use in the preparation of drugs for preventing or treating Mycobacterium tuberculosis infection; (2) Use in the preparation of a vaccine for preventing or treating Mycobacterium tuberculosis infection; optionally, the vaccine is an mRNA vaccine.

12. A drug, characterized in that Comprising the mRNA of claim 4 or 5 or the mRNA vaccine of claim 8 or 9; Optionally, it further comprises a pharmaceutically acceptable carrier; Optionally, the medicaments are suitable for sequential administration.

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