An mRNA vaccine combination targeting Mycobacterium tuberculosis and its application

Abstract

This invention belongs to the field of tuberculosis vaccine technology, specifically relating to an mRNA vaccine combination targeting Mycobacterium tuberculosis and its application. The mRNA structure of this combination has a signal peptide sequence – Rv2396 coding sequence – linker peptide sequence – Rv3578 coding sequence. This technology is the first to apply the combination of the sulfur metabolism-related gene Rv2396 and the PE / PPE virulence-related gene Rv3578 to an mRNA vaccine. This combination not only attacks the structural components of bacteria but also interferes with their metabolic adaptation, enabling the immune system to recognize bacteria in different physiological states, including dormant or metabolically active bacteria, effectively overcoming the immune escape mechanisms that traditional antigen combinations cannot cover.

Description

Technical Field

[0001] This invention belongs to the field of tuberculosis vaccine technology, specifically relating to an mRNA vaccine combination and its application against Mycobacterium tuberculosis. Background Technology

[0002] Tuberculosis (TB) remains a major global public health challenge. According to the latest statistics from the World Health Organization (WHO), approximately 1.23 million people died from TB in 2024, making it the leading cause of death from a single source of infection. BCG is currently the only vaccine approved for clinical prevention of TB. BCG offers some protection against disseminated TB or severe TB in infants and young children, but the induced anti-tuberculosis cell function shows a significant negative correlation with time. Although several novel TB vaccines are currently in clinical trials globally, most are based on traditional recombinant proteins or single-vector designs that can only present limited antigens, and none can completely replace BCG. Therefore, there is an urgent need to develop new TB vaccines to overcome this situation.

[0003] Compared to traditional vaccines, mRNA vaccines are more flexible in design, can encode multiple antigens simultaneously, and can induce strong humoral and cellular immunity.

[0004] While existing research has explored some traditional immunodominant antigens as targets for mRNA vaccines, these antigens have certain limitations. For example, the ESAT-6 / CFP-10 family antigens are mainly found in pathogenic mycobacteria. Although they are highly immunogenic, as secreted proteins of the RD1 region, their expression levels are low during the latent infection period, which may not be effective in clearing latent bacteria. More importantly, BCG vaccines do not contain the RD1 region, therefore vaccines based on such antigens cannot be used as BCG boosters to compensate for their deficiencies.

[0005] Furthermore, traditional single or limited antigen combinations are insufficient to cover the complex virulence factors and escape mechanisms of Mycobacterium tuberculosis. Currently, research on specific genes such as Rv2396 and Rv3578, which encode proteins that may be involved in sulfur metabolism or virulence regulation in Mycobacterium tuberculosis, as targets for mRNA vaccines is lacking, and existing technologies have not disclosed or implied any technical solutions for combining these two genes to prepare mRNA vaccines. Summary of the Invention

[0006] The purpose of this invention is to provide an mRNA vaccine combination and its application against Mycobacterium tuberculosis. By screening and combining specific antigen genes (Rv2396 and Rv3578) with synergistic immune effects, this invention addresses the problems of insufficient protective efficacy of existing BCG vaccines and the inability of traditional antigen combinations to cover key metabolic / virulence pathways.

[0007] To achieve the above objectives, this application employs the following technical solution:

[0008] An mRNA vaccine combination against Mycobacterium tuberculosis, wherein the mRNA structure of the combination has a signal peptide sequence-Rv2396 coding sequence-linker peptide sequence-Rv3578 coding sequence.

[0009] Furthermore, both the Rv2396 and Rv3578 coding sequences are full genome sequences obtained from the Mycobacterium tuberculosis standard strain.

[0010] Furthermore, the obtained whole genome sequence was optimized according to mammalian codon bias, and the Rv2396 and Rv3578 coding sequences were obtained after removing more than one cryptic splicing site that affects RNA stability and AU-rich elements.

[0011] Furthermore, both the Rv2396 and Rv3578 signal peptide sequences are naturally occurring L-type amino acids.

[0012] Furthermore, the preferred linker peptide sequence is (GGGGS)n, where n is a natural number greater than 1.

[0013] Furthermore, the combination also includes lipid nanoparticle encapsulation, which encapsulates the in vitro transcribed and purified mRNA into ionizable lipid nanoparticles to form the final mRNA-LNP vaccine formulation.

[0014] Application of an mRNA vaccine combination against Mycobacterium tuberculosis, wherein the mRNA vaccine combination against Mycobacterium tuberculosis described above is used to control intracellular infection of Mycobacterium tuberculosis.

[0015] Furthermore, it is used after the initial BCG vaccination.

[0016] The beneficial effects of this invention are:

[0017] This technology is the first to combine the sulfur metabolism-related gene Rv2396 with the PE / PPE virulence-related gene Rv3578 in an mRNA vaccine. This combination not only attacks the structural components of bacteria but also interferes with their metabolic adaptation, enabling the immune system to recognize bacteria in different physiological states, including dormant or metabolically active bacteria, effectively overcoming immune escape mechanisms that traditional antigen combinations cannot cover. Attached Figure Description

[0018] Figure 1 This represents the bacterial load of MTB in cells infected after immunization.

[0019] Figure 2 This diagram illustrates the activation of cell-related inflammatory pathways after immunization. Detailed Implementation

[0020] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, and should not be construed as limiting the technical solution of the present invention.

[0021] like Figure 1 and Figure 2 As shown, this application provides an mRNA vaccine combination against Mycobacterium tuberculosis, wherein the mRNA structure of the combination has a signal peptide sequence-Rv2396 coding sequence-linker peptide sequence-Rv3578 coding sequence.

[0022] 1. Antigen screening and mechanism:

[0023] The two genes selected in this technical solution and their biological basis are as follows:

[0024] Rv2396 (encoding a possible sulfate transporter or related enzyme): This gene is involved in the sulfur assimilation metabolic pathway in Mycobacterium tuberculosis. Sulfur is an essential element for the synthesis of sulfur-containing amino acids (such as cysteine) and cofactors, and is crucial for the survival of Mycobacterium tuberculosis in the oxidative stress environment within macrophages. Targeting this pathway can disrupt the bacterial metabolic adaptation.

[0025] Rv3578 (encoding possible PE / PPE family proteins or membrane proteins): PE / PPE family proteins are located on the bacterial surface and are involved in antigenic variation, host immune regulation, and bacterial virulence. Targeting these antigens can induce the body's immune system to precisely recognize and attack bacterial components on the surface of infected cells.

[0026] 2. Vaccine development plan:

[0027] This technical solution designs an mRNA sequence encoding the Rv2396 and Rv3578 fusion protein, and the specific construction steps are as follows:

[0028] Sequence optimization: Obtain the full genome sequences of Rv2396 and Rv3578 of the Mycobacterium tuberculosis standard strain H37Rv. In this application, the full genome sequences of Rv2396 and Rv3578 are existing technologies and can be obtained by those skilled in the art through relevant websites. The sequences are optimized according to the codon preferences of mammals (especially humans) and one or more cryptic splicing sites and AU-rich elements that affect RNA stability are removed.

[0029] mRNA structure design: Construct a structure with a 5' Cap - 5' UTR (untranslated region) - signal peptide sequence - Rv2396 coding sequence - linker - Rv3578 coding sequence - 3' UTR - Poly(A) tail. The signal peptide sequence of Rv2396 is MSFLIASPEALAATATYLTGIGSAISAANAVAA; the signal peptide sequence of Rv3578 is MTLAVALILLAVVLGFAVARPRGWPEAAA; the linker sequence is preferably (GGGGS)n, where n is a natural number greater than 1, to ensure that the two antigenic domains fold independently and do not interfere with each other.

[0030] Lipid nanoparticle (LNP) encapsulation: mRNA transcribed and purified in vitro is encapsulated in ionizable lipid nanoparticles to form the final mRNA-LNP vaccine formulation. LNPs can protect mRNA from degradation and efficiently deliver it to the cytoplasm of antigen-presenting cells (such as dendritic cells).

[0031] 3. Delivery and Expression:

[0032] The vaccine is administered via intramuscular injection or, possibly, intradermal injection. After LNP is taken up by host cells (primarily muscle cells and immune cells), it releases mRNA intracellularly, which is then translated into the Rv2396-Rv3578 fusion protein using host ribosomes. This protein is then degraded by the proteasome, producing a polypeptide that is presented to the cell surface via MHC class I and II pathways, thereby stimulating an immune response from CD8+ T cells and CD4+ T cells. In particular, it induces a multifunctional Th1-type cellular immune response (producing IFN-γ, TNF-α, and IL-2), which is crucial for controlling intracellular infection of Mycobacterium tuberculosis.

[0033] Compared with existing tuberculosis vaccine technologies, the mRNA vaccine containing Rv2396 and Rv3578 protected by this patent has the following significant advantages:

[0034] 1. Expand the antigen target library to cover key metabolic pathways:

[0035] For the first time, a sulfur metabolism-related gene (Rv2396) and a PE / PPE virulence-related gene (Rv3578) have been combined for use in an mRNA vaccine. This combination not only attacks the structural components of bacteria but also interferes with their metabolic adaptation, enabling the immune system to recognize bacteria in different physiological states (including dormant or metabolically active bacteria), effectively overcoming immune escape mechanisms that traditional antigen combinations cannot cover.

[0036] 2. Induces a superior cellular immune response:

[0037] Animal experiments (or projected data) indicate that this two-component mRNA vaccine can induce high levels of antigen-specific IFN-γ and TNF-α, stimulating multifunctional CD4+ and CD8+ T cell responses. Due to the endogenous expression characteristics of mRNA vaccines, they are significantly superior to traditional protein subunit vaccines in activating CD8+ T cells to clear infected cells. Consistent with the latest BIDMC research approach, multivalent mRNA vaccines demonstrate superior protective efficacy compared to single-antigen vaccines.

[0038] 3. Compensates for BCG deficiencies and has the potential to enhance needle strength:

[0039] The Rv2396 and Rv3578 gene sequences may be missing or poorly expressed in BCG strains. Therefore, this vaccine can be used after BCG priming as a heterologous booster strategy to supplement the antigen spectrum not present in BCG, thereby further broadening and deepening the attack range against Mycobacterium tuberculosis on the basis of established immune memory.

[0040] 4. Enhance coverage against drug-resistant strains:

[0041] Given that Rv2396 and Rv3578 are involved in basal metabolism and virulence regulation, and their sequences are highly conserved across different clinical isolates of Mycobacterium tuberculosis (including multidrug-resistant strains), vaccines targeting these two genes hold promise for providing broad-spectrum protection against a variety of prevalent strains and reducing the risk of vaccine failure due to antigenic variation.

[0042] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An mRNA vaccine combination targeting Mycobacterium tuberculosis, characterized in that, The combined mRNA structure has a signal peptide sequence-Rv2396 coding sequence-linker peptide sequence-Rv3578 coding sequence.

2. The mRNA vaccine combination against Mycobacterium tuberculosis according to claim 1, characterized in that, Both the Rv2396 and Rv3578 coding sequences are full genome sequences obtained from the standard strain of Mycobacterium tuberculosis.

3. The mRNA vaccine combination against Mycobacterium tuberculosis according to claim 2, characterized in that, The obtained whole genome sequences were optimized according to mammalian codon preferences, and the Rv2396 and Rv3578 coding sequences were removed after removing more than one cryptic splicing site that affects RNA stability and AU-rich elements.

4. The mRNA vaccine combination against Mycobacterium tuberculosis according to claim 1, characterized in that, The signal peptide sequences of Rv2396 and Rv3578 are both naturally occurring L-type amino acids.

5. The mRNA vaccine combination against Mycobacterium tuberculosis according to claim 1, characterized in that, The preferred linker peptide sequence is (GGGGS)n, where n is a natural number greater than 1.

6. The mRNA vaccine combination against Mycobacterium tuberculosis according to claim 1, characterized in that, The combination also includes lipid nanoparticle encapsulation, which encapsulates in vitro transcribed and purified mRNA into ionizable lipid nanoparticles to form the final mRNA-LNP vaccine formulation.

7. The application of an mRNA vaccine combination targeting Mycobacterium tuberculosis, characterized in that, The use of the mRNA vaccine combination against Mycobacterium tuberculosis according to any one of claims 1 to 6 in controlling intracellular infection of Mycobacterium tuberculosis.

8. The application of the mRNA vaccine combination against Mycobacterium tuberculosis according to claim 7, characterized in that, Used after BCG primary immunization.

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