DNA molecular combination, antigen expression vector combination, mRNA molecular combination and staphylococcus aureus mRNA vaccine

Abstract

The invention relates to the technical field of immunity, and particularly discloses a DNA molecular combination, an antigen expression vector combination, an mRNA molecular combination and a staphylococcus aureus mRNA vaccine. Wherein the DNA molecule combination comprises at least two of a first DNA molecule, a second DNA molecule, a third DNA molecule, a fourth DNA molecule and a fifth DNA molecule; the first DNA molecule comprises a first coding region, and the first coding region comprises a nucleotide sequence for coding MnTc protein with a sequence as shown in SEQ ID No.1; the second DNA molecule comprises a second coding region, and the second coding region comprises a nucleotide sequence for coding SEB protein with a sequence shown as SEQ ID No.2; the third DNA molecule comprises a third coding region, and the third coding region comprises a nucleotide sequence for coding the Hla protein with the sequence as shown in SEQ ID No.3; the fourth DNA molecule comprises a fourth coding region, and the fourth coding region comprises a nucleotide sequence for coding the FnBPA protein with the sequence as shown in SEQ ID No.4; the fifth DNA molecule comprises a fifth coding region, and the fifth coding region comprises a nucleotide sequence for coding IsdB protein with the sequence shown as SEQ ID No.5. The mRNA vaccine finally synthesized by using the DNA molecule combination can prevent and / or treat staphylococcus aureus infection.

Description

Technical Field

[0001] The present application relates to the field of immunotechnology, and in particular to a DNA molecule combination, an antigen expression vector combination, an mRNA molecule combination, and a Staphylococcus aureus mRNA vaccine. Background Art

[0002] Staphylococcus aureus belongs to the Micrococcaceae family and appears as a gram-positive coccus, 1 micron in diameter, in grape-like clusters. S. aureus is a commensal bacterium with a nasal carriage rate of approximately 30% in the human population. As a ubiquitous human pathogen, S. aureus is the most common cause of skin or soft tissue infections and endocarditis. S. aureus is also a major cause of various nosocomial infections, including ventilator-associated pneumonia, intravenous catheter-related infections, postoperative infections, infections in immunosuppressed patients, and invasive infections. In healthcare settings, immunocompetent patients or those undergoing procedures such as surgery or intravenous catheterization are at higher risk for contracting S. aureus infections.

[0003] Methicillin-resistant Staphylococcus aureus (MRSA) strains are resistant to many antibiotics, even those approved to treat S. aureus infections such as linezolid, daptomycin, vancomycin, and clindamycin. The development of this antibiotic resistance poses a global challenge to the treatment of S. aureus infections. The growth of bacterial resistance outpaces the development of new antibiotics, so safe and effective vaccines are urgently needed.

[0004] In recent years, breakthroughs have been made in the field of RNA molecules, and mRNA vaccines have achieved certain research results in a variety of infectious diseases. mRNA vaccines deliver mRNA to cells to express and produce proteins, thereby providing immune protection to the body. Therefore, it is necessary to develop an mRNA vaccine that can respond to MRSA intracellular infection and new immune escape mechanisms for latent transmission, to prevent MRSA infection and latent intracellular transmission. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a Staphylococcus aureus mRNA vaccine, aiming to solve the problem that the existing Staphylococcus aureus vaccine has poor immune effect against its new immune escape mechanism of intracellular infection and latent transmission.

[0006] The technical solution of this application is as follows:

[0007] In a first aspect of the present application, a DNA molecule combination is provided, the DNA molecule combination comprising at least one of a first DNA molecule, a second DNA molecule, a third DNA molecule, a fourth DNA molecule, and a fifth DNA molecule; wherein the first DNA molecule comprises a first coding region, the first coding region comprising a nucleic acid sequence encoding an MnTc protein having a sequence as shown in SEQ ID No.1; the second DNA molecule comprises a second coding region, the second coding region comprising a nucleic acid sequence encoding an SEB protein having a sequence as shown in SEQ ID No.2; the third DNA molecule comprises a third coding region, the third coding region comprising a nucleic acid sequence encoding an Hla protein having a sequence as shown in SEQ ID No.3; the fourth DNA molecule comprises a fourth coding region, the fourth coding region comprising a nucleic acid sequence encoding an FnBPA protein having a sequence as shown in SEQ ID No.4; the fifth DNA molecule comprises a fifth coding region, the fifth coding region comprising a nucleic acid sequence encoding an IsdB protein having a sequence as shown in SEQ ID No.5.

[0008] Optionally, the first coding region, the second coding region, the third coding region, the fourth coding region or the fifth coding region comprises a nucleic acid sequence encoding a signal peptide of the sequence shown in SEQ ID No. 7 and / or a nucleic acid sequence encoding a tag protein.

[0009] Optionally, the upstream and downstream of the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region further include a 5' untranslated region and a 3' untranslated region, respectively; the 5' untranslated region is as shown in SEQ ID No.8; and the 3' untranslated region is as shown in SEQ ID No.9.

[0010] Optionally, the downstream of the first coding region, the second coding region, the third coding region, the fourth coding region or the fifth coding region includes polyadenylic acid, which is located downstream of the 3' non-translated region, and the sequence of the polyadenylic acid is shown in SEQ ID No.10; the upstream of the first coding region, the second coding region, the third coding region, the fourth coding region or the fifth coding region includes a KOZAK sequence, which is located downstream of the 5' non-translated region, and the KOZAK sequence is shown in SEQ ID No.6.

[0011] Optionally, the upstream of the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region further includes a 5' cap structure, and the 5' cap structure is located upstream of the 5' untranslated region.

[0012] In a second aspect of the present application, an antigen expression vector combination is provided, wherein the antigen expression vector combination comprises the above-mentioned DNA molecule combination.

[0013] The third aspect of the present application provides an mRNA molecule combination, wherein the mRNA molecule combination is prepared by the above-mentioned antigen expression vector combination.

[0014] In a fourth aspect of the present application, a Staphylococcus aureus mRNA vaccine is provided, wherein the Staphylococcus aureus mRNA vaccine comprises the above-mentioned mRNA molecule combination.

[0015] Optionally, the Staphylococcus aureus mRNA vaccine includes a pharmaceutically acceptable carrier; the carrier is a liposome nanoparticle.

[0016] The fifth aspect of the present application provides a preparation, which includes the above-mentioned Staphylococcus aureus mRNA vaccine.

[0017] Beneficial effects of this application:

[0018] It can be seen from the in vivo bactericidal activity tests in the examples and the related immune experiments in mice that the DNA molecule combination, antigen expression vector combination, isolated mRNA molecule combination, vaccine, and preparation in the present application can be applied to the preparation of products for preventing and / or treating Staphylococcus aureus, thereby efficiently and simultaneously activating relatively independent humoral immune responses and cellular immune responses to respond to Staphylococcus aureus infection and its hidden spread within cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.

[0020] Figure 1 A schematic diagram of the components of the mRNA vaccine provided in the embodiments of the present application;

[0021] Figure 2 The pVAX1-MnTc plasmid map provided in the examples of this application;

[0022] Figure 3 The pVAX1-SEB plasmid map provided in the examples of this application;

[0023] Figure 4 The pVAX1-Hla plasmid map provided in the examples of this application;

[0024] Figure 5 The pVAX1-FnBPA plasmid map provided in the examples of this application;

[0025] Figure 6The pVAX1-IsdB plasmid map provided in the examples of this application;

[0026] Figure 7 A diagram showing the nucleic acid analysis results of mRNA provided in the examples of the present application;

[0027] Figure 8 This is a test chart of the uniformity of mRNA-LNP particles provided in the examples of the present application;

[0028] Fig. 9 This is a test graph of mRNA-LNP encapsulation efficiency provided in the examples of the present application;

[0029] Fig.10 Western Blot detection diagram of mRNA provided in the examples of this application;

[0030] Fig.11 Western Blot detection diagram of mRNA provided in the examples of this application;

[0031] Fig.12 This is a SDS-PAGE identification scan of the MnTc, SEB, Hla, FnBPA, and IsdB proteins provided in the examples of the present application;

[0032] Fig.13 This is a graph showing the survival rate of mice immunized with the single-component vaccine provided in the examples of the present application and infected with Staphylococcus aureus;

[0033] Fig.14 Antibody titer diagram of the single-component vaccine provided in the examples of this application:

[0034] a is the antibody titer of the single-component vaccine at 14 days; b is the antibody titer of the single-component vaccine at 21 days; c is the antibody titer of the single-component vaccine at 28 days;

[0035] Fig.15 Antibody titer analysis chart of the single-component vaccine provided in the examples of this application:

[0036] a is the IgM antibody titer of the single-component vaccine at 7 days; b is the IgG antibody titer of the single-component vaccine at 28 days

[0037] 1 is the antibody titer; c is the IgG2a antibody titer at 28 days for the single-component vaccine;

[0038] Fig.16 This is an analysis chart of the antibody titer of the vaccine provided in the examples of this application:

[0039] a is the antibody titer at 14 days after vaccination; b is the antibody titer at 21 days after vaccination; c is the antibody titer at 28 days after vaccination;

[0040] Fig.17This is an analysis chart of the antibody titer of the vaccine provided in the examples of this application:

[0041] a is a comparison of antibody titers between the mixed component mRNA vaccine and the MnTc protein vaccine; b is a comparison of antibody titers between the mixed component mRNA vaccine and the SEB protein vaccine; c is a comparison of antibody titers between the mixed component mRNA vaccine and the Hla protein vaccine; d is a comparison of antibody titers between the mixed component mRNA vaccine and the FnBPA protein vaccine; e is a comparison of antibody titers between the mixed component mRNA vaccine and the IsdB protein vaccine;

[0042] Fig.18 Mouse spleen cell ELISpot count diagram provided in the examples of this application:

[0043] a is the secretion of IL-4 cytokine; b is the secretion of IL-2 cytokine; c is the secretion of IFN-γ cytokine;

[0044] Fig.19 This is a HE staining image of mouse lung tissue provided in the examples of the present application. DETAILED DESCRIPTION

[0045] In order to make it easier to understand this application, some terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art.

[0046] KOZAK sequence: a nucleic acid sequence located behind the cap structure at the 5' end of eukaryotic mRNA, which can bind to the translation initiation factor and mediate the translation initiation of mRNA containing the 5' cap structure. It corresponds to the SD sequence of prokaryotes. It is a sequence present in eukaryotic mRNA and plays an important role in the initiation of translation.

[0047] Capillary electrophoresis (CE): also known as high-performance capillary electrophoresis (HPCE) or capillary separation method (CESM), is a type of liquid phase separation technology that uses capillaries as separation channels and high-voltage DC electric fields as driving forces to achieve separation based on the differences in migration speed and distribution behavior between components in the sample. Capillary gel electrophoresis is an electrophoresis that moves the gel on the plate into the capillary as a support. The gel is porous and acts like a molecular sieve, separating solutes one by one according to molecular size.

[0048] Liposome nanoparticles (LNPs) are lipid vesicles with a uniform lipid core. They are widely used in the delivery of small molecules and nucleic acid drugs because of their high delivery efficiency and good safety.

[0049] ELISPOT (Enzyme-linked Immunospot Assay): The full name is enzyme-linked immunospot assay, which combines cell culture technology with enzyme-linked immunosorbent assay technology and can detect the cytokines secreted by single cells.

[0050] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0051] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0052] At present, 10 Staphylococcus aureus vaccines from 8 companies, including Merck, Nabi, Vaccine Research International Plc, Pfizer, Novartis, GSK, NIAID and Olin Bio, have entered the clinical research stage. However, no vaccine has been successfully launched. The two vaccines that have entered the third phase of clinical trials, Merck's V710 and Nabi's Staphvax, have both failed. The reason why V710, which targets IsdB, was considered to have failed in subsequent studies was mainly attributed to the lack of protection caused by a single target, and non-protective antibodies would be produced in the body of people who had been previously infected with Staphylococcus aureus, blocking the effect of protective antibodies. Staphvax vaccine is a bivalent polysaccharide vaccine that mainly relies on the recognition of specific surface capsular polysaccharides to induce immunity. However, the main infectious strain USA300 prevalent in the United States lacks capsular polysaccharide components, and the clinical indication selected by Nabi is patients with advanced chronic kidney disease, who will undergo repeated hemodialysis, resulting in serious loss of antibodies that mainly play a bactericidal role.

[0053] Humoral immunity will produce specific antibodies to neutralize the function of independent factors or regulate pathogens to optimize phagocytosis and clearance. Inducing high humoral immunity is a traditional strategy for vaccines. However, it is traditionally believed that Staphylococcus aureus infects the human body as an extracellular bacterium, and the body's immunity relies on neutrophils and macrophages to kill bacteria. However, some studies have shown that some MRSA survives secretly in monocytes and macrophages through changes in the cell wall and colonizes in macrophages. These infected cells act as "Trojan horses", prompting the killing of intracellular bacterial immune cells by extracellular antibiotics and antibacterial substances in the body, promoting MRSA transmission and immune escape. In view of the new immune escape mechanism of MRSA intracellular infection and latent transmission, the development of new vaccines that activate specific and killer cellular immune responses and therapeutic vaccines that can produce strong T cell immune responses against MRSA provide new ideas for preventing MRSA infection and intracellular latent transmission.

[0054] In recent years, the relevant technologies in the field of RNA molecules have made breakthrough progress. mRNA vaccines have achieved certain research results in a variety of infectious diseases. mRNA vaccines deliver mRNA to cells to express and produce proteins, thereby providing immune protection to the body. Compared with traditional vaccines (including inactivated viruses, attenuated vaccines, subunit vaccines, and viral vector vaccines), mRNA vaccines have the following outstanding advantages: (1) mRNA vaccines can not only induce host cells to produce strong humoral immunity, but also activate cellular immunity including cytotoxic T cells, and activate innate immunity through the specificity of pattern recognition receptors; (2) mRNA vaccines have strong design, simple preparation process, rapid standardized production, and short R&D cycle; (3) mRNA vaccine R&D costs are highly controllable and relatively low; (4) mRNA vaccines are highly safe. After entering the body, they will undergo a natural degradation process, and side effects can be more accurately controlled. There is no risk of integration, induced gene mutation, and exogenous viral infection. In addition, with the development of mRNA structural engineering, such as 5'-end cap structure, nucleotide modification, mRNA sequence optimization, etc., the shortcomings of mRNA, such as poor stability, low translation efficiency, and easy degradation, have been greatly improved. mRNA has been widely used in cell programming and vaccine research, and shows great application potential.

[0055] Based on this, one aspect of an embodiment of the present application provides a DNA molecule combination, including at least one of a first DNA molecule, a second DNA molecule, a third DNA molecule, a fourth DNA molecule, and a fifth DNA molecule.

[0056] Among them, the first DNA molecule includes a first coding region, the first coding region includes a nucleic acid sequence encoding an MnTc protein with a sequence as shown in SEQ ID No.1; the second DNA molecule includes a second coding region, the second coding region includes a nucleic acid sequence encoding an SEB protein with a sequence as shown in SEQ ID No.2; the third DNA molecule includes a third coding region, the third coding region includes a nucleic acid sequence encoding an Hla protein with a sequence as shown in SEQ ID No.3; the fourth DNA molecule includes a fourth coding region, the fourth coding region includes a nucleic acid sequence encoding an FnBPA protein with a sequence as shown in SEQ ID No.4; the fifth DNA molecule includes a fifth coding region, the fifth coding region includes a nucleic acid sequence encoding an IsdB protein with a sequence as shown in SEQ ID No.5.

[0057] Staphylococcus aureus (MRSA) has a variety of protective antigens, including capsular polysaccharides, adhesion factors, enterotoxins (SE), toxic shock toxin-1 (TSST-1), Panton-Valentine leukocidin (PVL), α-hemolysin, coagulase, and extracellular regulatory proteins (RNAⅢ activating protein, RAP), which can be used for vaccine research. Among them, the expression of PVL, α-hemolysin, and coagulase in MRSA strains is quite different. Previous studies have also proved that it is difficult to obtain comprehensive and effective protection by selecting a single antigen. The failure of the Phase III clinical trial of the capsular polysaccharide vaccine has further increased the controversy over whether it can become a suitable protective antigen. MRSA pathogenic factors are numerous and complex, and a single antigen is difficult to stimulate a comprehensive and effective protective immune response. In order to improve the protectiveness of the vaccine, it is necessary to first make reasonable choices and combinations at the antigen level.

[0058] Toxin proteins are extremely lethal and incapacitating and are the focus of biosafety defense. Some successful vaccines have been developed for various microbial diseases. For example, diphtheria and tetanus can be prevented by toxoid vaccines, and rubella, measles, mumps, polio and smallpox vaccines can also prevent viral infections. The success of these vaccines has led researchers to believe that effective vaccines can also be produced against virulence factors in organisms such as Staphylococcus aureus.

[0059] Iron is a critical and essential nutrient for bacterial growth and reproduction, and iron deficiency inhibits the growth of invading bacteria, allowing the host's immune system to eliminate the infection. Staphylococcus aureus produces a high-affinity iron uptake system called siderophores, which are secreted small molecules that have an extremely high affinity for iron and outcompete host iron-binding proteins. S. aureus obtains iron during infection through the first binding of hemoglobin to the bacterial surface (IsdB). Heme is obtained from hemoglobin and transferred to the cell wall and cell membrane by translocation factors (IsdD, IsdE, and IsdF). The Isd system is essential for the growth of S. aureus, and heme is the only source of iron and is required for full virulence in several models of pathogenesis. Therefore, the Isd system is highly expressed in bacteria, and the human body produces a considerable humoral immune response to the Isd components, making it suitable as a target.

[0060] Fibronectin binding protein A (FnBPA) participates in the pathogenesis of Staphylococcus aureus infection by promoting bacterial attachment to host cells. The FnBPA protein has 10 fibronectin binding repeats, which have a high affinity for fibronectin. The SS sorting signal can help anchor peptidoglycan, help Staphylococcus aureus adhere to the bacterial extracellular matrix, and aggregate to form biofilms by binding to various extracellular matrix proteins such as elastin and collagen, resulting in a variety of clinical indications, such as internalization into osteoblasts. This internalization causes the pathogen to evade the immune system, making antibiotic treatment ineffective, leading to recurrent infections. Staphylococcus aureus is the leading cause of bloodstream infections, and therapeutic targets targeting the organism's ability to adhere, clump, and coagulate can reduce the severity of these infections.

[0061] α-Hemolysin (Hla) is expressed in 95% of Staphylococcus aureus strains as a water-soluble protein with pore-forming and pro-inflammatory properties. When bacteria invade epithelial cells and aggregate to a certain density, the bacteria produce invasin, and Hla binds to membrane receptors and forms heptamer pores, destroying a wide range of host cells, including epithelial cells, erythrocytes, fibroblasts, monocytes, macrophages, and lymphocytes, but not neutrophils. Experiments have found that histidine at position 35 plays an important role in the formation of the perforating complex. The histidine was mutated to leucine to remove the hemolytic activity of Hla. Immunization with the mutant form can produce antigen-specific immunoglobulin G responses and provide protection against staphylococci.

[0062] Enterotoxin B (SEB) is a diverse protein exotoxin that acts as a superantigen and belongs to the most potent T-cell mitogens. Through cross-linking between MHC-II and the β-chain of the T-cell receptor (TCR), they induce activation of antigen-presenting cells (APCs) and T lymphocytes, leading to the release of large amounts of proinflammatory cytokines. Amino acid mutations in the polar region of SEB (Y89A and Y94A) and a single amino acid mutation in the hydrophobic domain (L45R) can inactivate its ability to form bridges between major histocompatibility complex class II molecules and maximally restore the conformation of SEB.

[0063] Manganese transporter C (MnTc) is a highly conserved protein in MRSA and VRSA strains. It is a cofactor of superoxide dismutase and can neutralize superoxide free radicals produced during the oxidative burst of macrophage and neutrophil phagosomes. MnTc has been shown to significantly increase serum IgG levels and induce cellular immunity. It can be used as a candidate vaccine to confer protective immunity in animal model systems of Staphylococcus aureus infection.

[0064] Therefore, the following five immunodominant antigens were finally screened out from the whole genome of MRSA: α-hemolysin (Hla), iron surface determinant protein B (IsdB), fibronectin binding protein A (FnBPA), enterotoxin B (SEB), and manganese ion transporter C (MnTc). They target key pathogenic links such as MRSA adhesion and colonization, important metabolic pathways, toxin secretion, and immune escape pathways.

[0065] In some embodiments, the DNA molecule combination includes a first DNA molecule, a second DNA molecule, a third DNA molecule, a fourth DNA molecule, and a fifth DNA molecule. The mRNA vaccine made by using a DNA molecule combination containing five DNA molecules can work together from the perspectives of MRSA adhesion colonization, toxin secretion pathways, immune escape pathways, and important metabolic pathways to achieve effective regulation of MRSA growth and spread, so as to respond to the infection of Staphylococcus aureus and its hidden spread in cells.

[0066] Furthermore, in order to obtain an mRNA vaccine that can efficiently express exogenous target genes, it is necessary to consider many aspects and conduct continuous screening and optimization.

[0067] In some embodiments, the first coding region, the second coding region, the third coding region, the fourth coding region or the fifth coding region includes a nucleic acid sequence encoding a signal peptide of the sequence shown in SEQ ID No.7 and / or a nucleic acid sequence encoding a tag protein. The signal peptide is a secretion signal peptide (Tissue Plasminogen Activator, TPA), which can promote the secretion of the expressed protein outside the cell. By secreting the protein, the effect of the vaccine prepared subsequently can be effectively improved. The tag protein is conducive to the detection and purification of the expressed target protein. In some embodiments, the tag protein is selected from 6×His tag protein, 3×FLAG tag protein, HA tag protein, MBP tag protein, Myc tag protein, GST tag protein or Strep tag protein.

[0068] In some embodiments, the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region further include a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR) upstream and downstream, respectively. The 5' untranslated region is shown in SEQ ID No. 8; the 3' untranslated region is shown in SEQ ID No. 9.

[0069] In some embodiments, the downstream of the first coding region, the second coding region, the third coding region, the fourth coding region or the fifth coding region includes polyadenylic acid (Poly A tail), and the polyadenylic acid is located downstream of the 3' untranslated region. Polyadenylic acid can protect mRNA from degradation and increase the stability of mRNA.

[0070] In some embodiments, the length of the poly(A) is 80 to 150 bp, for example, the length of the poly(A) is 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp or 150 bp, etc.

[0071] In some embodiments, the upstream of the first coding region, the second coding region, the third coding region, the fourth coding region or the fifth coding region includes a KOZAK sequence, the KOZAK sequence is located downstream of the 5' untranslated region, and the KOZAK sequence is shown in SEQ ID No. 6. The KOZAK sequence can bind to a translation initiation factor to mediate the translation initiation of mRNA containing a 5' cap structure.

[0072] In some embodiments, the upstream of the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region further comprises a 5' cap structure, and the 5' cap structure is located upstream of the 5' untranslated region. In addition to recognizing the start of protein synthesis, the 5' cap structure also serves as a protective group for exonuclease cleavage from 5' to 3', and is also an identifier for recruiting protein factors for pre-mRNA splicing, polyadenylation, and nuclear export, and it also serves as an anchor point for recruiting initiation factors.

[0073] In some embodiments, the 5' cap structure is CAP type I.

[0074] In some embodiments, taking the first DNA molecule as an example, the first DNA molecule is Figure 1 The structure shown. From 5' to 3', it includes a 5' cap structure, a 5'UTR, a KOZAK sequence, a nucleic acid sequence encoding the MnTc protein as shown in SEQ ID No.1, a 3'UTR and polyadenylation. It can be understood by those skilled in the art that the nucleic acid sequence encoding the MnTc protein as shown in SEQ ID No.1 is replaced by a nucleic acid sequence encoding the SEB protein as shown in SEQ ID No.2, a nucleic acid sequence encoding the Hla protein as shown in SEQ ID No.3, a nucleic acid sequence encoding the FnBPA protein as shown in SEQ ID No.4, and a nucleic acid sequence encoding the IsdB protein as shown in SEQ ID No.5 in the second DNA molecule, the third DNA molecule, the fourth DNA molecule and the fifth DNA molecule, respectively.

[0075] In a second aspect of the embodiments of the present application, there is provided an antigen expression vector combination, which includes the DNA molecule combination provided in the first aspect of the embodiments of the present application. The antigen expression vector can use pVAX1, pcDNA3.1, etc. as a tool vector, and the first DNA molecule, the second DNA molecule, the third DNA molecule, the fourth DNA molecule, and the fifth DNA molecule can be obtained by direct synthesis, or by connecting the DNA molecule with the tool vector using the restriction site on the vector.

[0076] According to a third aspect of the embodiments of the present application, an mRNA molecule combination is provided, wherein the mRNA molecule combination is prepared by the antigen expression vector combination provided by the second aspect of the embodiments of the present application.

[0077] In some embodiments, the method for preparing the mRNA molecule combination includes: linearizing and purifying the antigen expression vector, and then preparing it through in vitro transcription of the linearized plasmid.

[0078] The fourth aspect of the embodiments of the present application provides a Staphylococcus aureus mRNA vaccine, which includes the mRNA molecule combination provided in the third aspect of the embodiments of the present application.

[0079] In some embodiments, the Staphylococcus aureus mRNA vaccine also includes a pharmaceutically acceptable carrier. The carrier can be a liposome nanoparticle (LNP). Since the mRNA chain is unstable and carries a negative charge, and the cell membrane surface also carries a negative charge, electrostatic repulsion makes it difficult for the mRNA molecule to pass through the cell membrane and enter the cell, so a special modification or package delivery system is required to achieve the intracellular expression of mRNA. Liposome nanoparticles (LNP) can be used as a delivery tool for mRNA because of their high delivery efficiency and good safety, and can transport the mRNA in the Staphylococcus aureus mRNA vaccine into cells.

[0080] The fifth aspect of the embodiments of the present application provides a preparation, which includes the Staphylococcus aureus mRNA vaccine provided in the fourth aspect of the embodiments of the present application.

[0081] The following is further explained through specific implementation.

[0082] Example 1 Construction of template plasmid

[0083] The amino acid sequence of the secretory signal peptide nucleic acid sequence shown in SEQ ID No.7 was added to the N-terminus of the protein nucleic acid sequence of MnTc, SEB, Hla, FnBPA, and IsdB, and a 6×His tag was added to the C-terminus. Under the premise of not changing the amino acid sequence, the codons were optimized to obtain the ORF (open reading frame) nucleic acid sequence of mRNA (shown in SEQ ID Nos.11 to 15). A 5′UTR (untranslated region) sequence (shown in SEQ ID No.8) and a KOZAK sequence (shown in SEQ ID No.6) were added to the 5′ end of the ORF nucleic acid sequence, and a 3′UTR sequence (shown in SEQ ID No.9) and a polyadenylic acid (PolyA) sequence (shown in SEQ ID No.10) were added to the 3′ end of the ORF nucleic acid sequence. No. 10), and obtained the constituent elements of 5 mRNA vaccines. The gene fragments of the constituent elements of the above 5 mRNA vaccines were synthesized by GENEWIZ and cloned into the pVAX1 vector to obtain the template plasmids pVAX1-MnTc, pVAX1-SEB, pVAX1-Hla, pVAX1-FnBPA and pVAX1-IsdB. The plasmid spectra of the 5 template plasmids are shown in Figure 2 to Figure 6 shown.

[0084] Example 2 In vitro co-transcription synthesis of mRNA

[0085] (1) Linearization and purification of template plasmid

[0086] The five template plasmids prepared in Example 1 were transformed into DH5α competent cells, respectively, and coated on solid LB (Luria-Bertani solid medium) plates containing Kan+ (kanamycin) resistance, and cultured at 37°C for about 12 to 15 hours (observe whether a single colony grows on the LB plate), and a single colony was picked and added to a liquid LB medium containing Kan+ resistance. After culturing at 37°C and 200rpm for 15 hours, the cells were collected by centrifugation, and the amplified template plasmid was extracted using a commercial plasmid extraction kit. The linearization restriction site XhoI adjacent to the PolyA tail was selected as the linearization restriction site, and the template plasmid was linearized and detected by DNA agarose gel electrophoresis. After the detection size was consistent, it was recovered by a PCR product recovery kit, and the concentration and quality of the recovered DNA were measured by using a NanoDrop ultra-micro spectrophotometer by measuring the OD of the DNA 260 and OD 260 / OD 280 Ratio judgment.

[0087] (2) Synthesizing mRNA using linearized plasmid

[0088] The correctly linearized plasmid determined in (1) was used as a DNA template. Nuclease-free water, NTPs (ATP, GTP, N1-pUTP, CTP), Reagent AG (Cap1) was mixed and centrifuged to collect the liquid; 10×HY T7 Buffer, DNA template (Template DNA), RNase Inhibitor and Pyrophosphatase Inorganic were added, and finally T7 RNA Polymerase was added. The proportions of each component are shown in Table 1. After mixing the components, the liquid was collected by centrifugation and reacted at 37°C for 3h. After the reaction, DNasel was added to remove the DNA template, and the transcription product was purified using Monarch RNAcleanup kit (NEB). The concentration of the obtained mRNA was calculated using the reading of the NanoDrop ultra-micro spectrophotometer.

[0089] Table 1. Ratio of components in mRNA in vitro transcription reaction

[0090]

[0091] (3) Using capillary electrophoresis to detect mRNA quality

[0092] After diluting the Lower marker, add mineral oil to seal it; dilute the mRNA to be tested to a concentration of 20-50 ng / μL using dilution buffer, then perform denaturation treatment, place the diluted mRNA to be tested in a 70°C metal bath for 2 minutes, immediately place it on ice for 5 minutes, and then use the Qsep series fully automatic nucleic acid analyzer for detection. The test results are as follows: Figure 7 As shown. Figure 7 It can be seen that the mRNA sample to be tested contains only mRNA and a small amount of water, indicating that the quality of the in vitro transcribed mRNA meets the standards for subsequent use.

[0093] Example 3 mRNA-LNP Preparation

[0094] (1) mRNA-LNP coating

[0095] The LNP (liposome nanoparticle) was dissolved in an ethanol solution containing an ionizable lipid (SM-102), 1,2-distearoylglycerol-3-phosphocholine (DSPC), cholesterol (cholesterol) and polyethylene glycol lipid (MPEG2000-DMG) to obtain a lipid mixture, wherein the molar ratio of SM-102: DSPC: Cholesterol: mPEG2000-DMG was 50:10:38.5:1.5); the lipid mixture was uniformly mixed with 20mM citrate buffer (pH4.0) containing the mRNA prepared in Example 2 at a ratio of 1:3 and a flow rate of 12ml / min using a microfluidic device through a microfluidic chip to obtain an mRNA-LNP solution; the mRNA-LNP solution was then concentrated into a PBS buffer at pH7.4 by ultrafiltration displacement, concentrated to the desired concentration, filtered through a filter head with a membrane pore size of 0.22μm and stored at 4°C for later use.

[0096] (2) Determination of mRNA-LNP particle size, uniformity, and encapsulation efficiency

[0097] All preparations obtained in (1) were subjected to particle size and distribution, RNA concentration, zeta potential and encapsulation efficiency determination. The particle size and uniformity of LNP were determined using a dynamic light scattering (DLS) instrument. The specific steps are as follows: dilute the preparation to be tested in sterile PBS at a ratio of 1:100, and use a HORIBA-SZ100 device to measure at dispersion angles of 25° and 90°, repeat three times, and obtain the particle size distribution and PDI value (Polymer dispersity index) of mRNA-LNP. Particle size distribution is as follows Figure 8 As shown. Figure 8It can be seen that the particle size uniformity of the five mRNA-LNPs is high. The particle size results are given as the ratio of particle size to intensity, and the stability of the mRNA-LNP dispersion system is predicted at the same time. The encapsulation efficiency is determined by destroying the liposome structure with a certain amount of TritonX-100 to obtain total mRNA, and the same amount of mRNA-LNP preparation without adding TritonX-100 to obtain unencapsulated mRNA. The mRNA concentration is determined using a fluorescence (RiboGreen) quantitative kit. The encapsulation efficiency calculation formula is as follows:

[0098]

[0099] The calculated encapsulation efficiency of the five mRNA-LNPs is shown in Table 2 and Fig. 9 As shown in Table 2 and Fig. 9 It can be seen that all five mRNA-LNPs have good encapsulation efficiency.

[0100] Table 2. mRNA-LNP particle size, PDI value, and encapsulation efficiency

[0101]

[0102] Example 3 mRNA-LNP cell transfection

[0103] In order to verify the expression and activity of mRNA at the cellular level, mRNA was transfected into 293T cells (human kidney epithelial cells) grown to 80% density in the form of LNP preparation, the culture supernatant and cells were collected, and Western Blot was used to verify the expression and secretion of the protein.

[0104] (1) Five mRNA-LNP preparations were transfected into 293T cells with a growth density of 80% and cultured to obtain cell culture supernatant and cell lysate. The cell culture supernatant and cell lysate were added to 5× loading buffer respectively, and subjected to 99°C metal bath for 10 min, followed by polyacrylamide gel electrophoresis. After the electrophoresis, the proteins on the gel were transferred to a PVDF membrane and blocked with 5% skim milk at room temperature. After 60 min, the membrane was washed three times with TBST (Tris-HCl buffer + Tween20). The membrane was incubated with anti-His-HRP antibody at 4°C overnight and washed three times with TBST. The enhanced chemiluminescence kit (ECL) was used to develop the color and scan the plate. The results are shown in Figure 2. Fig.10 shown. Fig.10 NC represents negative control, WCL represents whole cell lysate group, SUP represents cell culture supernatant group, and TUBULIN (β-tubulin) is the internal reference. Fig. 9It can be seen that the five mRNAs, MnTc, SEB, Hla, FnBPA and IsdB, were all expressed in the cell.

[0105] (2) The five mRNA-LNP preparations were transfected into J774A.1 cells with a growth density of 80% and the transfected cells were cultured to obtain cell culture supernatant and cell lysate. The same operation was performed as above, and the enhanced chemiluminescence kit (ECL) was used for color development and scanning of the plate. The results are as follows: Fig.11 shown. Fig.11 NC represents negative control, WCL represents whole cell lysate group, SUP represents cell culture supernatant group. Fig.11 It can be seen that the five mRNAs, MnTc, SEB, Hla, FnBPA and IsdB, are all expressed in macrophages.

[0106] Example 4 Immunogenicity

[0107] (1) Protein purification

[0108] The gene fragments of the constituent elements of the five mRNA vaccines obtained in Example 1 were expressed by means of E. coli expression vectors pET30a and pGEX-6P-1. Five expression plasmids, pGEX-6P-1-MnTc, pET30a-SEB, pGEX-6P-1-Hla, pET30a-FnBPA and pET30a-IsdB, were synthesized by GENEWISE. The five expression plasmids were transformed into E. coli competent cells BL21 (DE3), and single colonies were picked for amplification and culture. When the OD of the bacterial solution reached 600When the value was 0.8, 0.5 mM IPTG (isopropylthiogalactoside) was added, and the culture was carried out at 16°C and 200 rpm for 12 h to induce protein expression; the bacteria were collected after the culture was completed, and for mSEB, FnBPA, and IsdB proteins, they were resuspended in Ni-NTA buffer (50 mM Tris-HCl pH 7.8 + 300 mM NaCl) with 1 mM phenylmethylsulfonyl fluoride (PMSF), and high-pressure crushed for 30 min. After crushing, the cells were centrifuged at 4°C and 12000g for 30 min, and the clarified cell lysate was collected; the supernatant was purified by Ni NTA Beads 6FF loaded on a protein purifier. Before loading, 5 column volumes of Ni-NTA buffer were used to balance the nickel column, and the loading was repeated twice. The nickel column was balanced with 5 column volumes of Ni-NTA buffer again, and then the protein was gradient eluted with Ni-NTA washing buffer (50mM Tris-HCl+300mM NaCl+500mM imidazole). For MnTc and Hla proteins, the cells were resuspended in GST Binding Buffer (140mM NaCl+2.7mM KCl+10Mm Na2HPO4+1.8mM KH2PO4+10mM DTT, pH 7.4) and crushed under high pressure. The supernatant was collected by centrifugation and purified by Glutathione Beads 4FF loaded on the protein purifier. Before loading, 5 column volumes of GST Binding Buffer were used to balance the column, and the loading was repeated twice. Then, 5 column volumes of GST Binding Buffer were used to balance again, and then the protein was gradient eluted with GST Elution Buffer (140mM NaCl+2.7mM KCl+10Mm Na2HPO4+1.8mM KH2PO4+10mMDTT+10mM reduced glutathione, pH 7.4). PRESCISSION protease was added to react overnight at 4°C to remove the N-terminal GST tag of the protein. The protein purification instrument was used again. The flow-through that was not bound to the GST column was the target protein. The GST-tagged protein bound to the GST column was eluted and discarded with GST Elution Buffer. The protein concentration was determined by measuring the A280 of the purified protein using NanoDrop One ultra-micro spectrophotometer, and then the protein was concentrated in phosphate buffer using a 10kDa molecular weight cutoff (MWCO) dialysis tube to obtain the purified protein. The purified protein was identified by SDS-PAGE. Fig.12 As shown. Fig.12 It can be seen that the protein sizes of MnTc, SEB, Hla, FnBPA, and IsdB are correct, indicating that the protein purification was successful.

[0109] (2) Staphylococcus aureus culture

[0110] The standard strain of Staphylococcus aureus (MRSA252) was obtained from ATCC and streaked on tryptic soy agar (TSA) and grown overnight at 37° C. A single colony from a TSA plate was grown overnight in 3 mL tryptic soy broth (TSB) and transferred to fresh TSB at a ratio of 1:100 and grown for at least 6 h.

[0111] Example 5 Immunity Test

[0112] (1) Immunization regimen

[0113] The experimental animals used were 6-8 week old Balb / C female mice. In the active immunization experiment, five antigen Staphylococcus aureus protein vaccine, five mRNA mixed vaccines and five mRNA single component vaccines (Hla, FnBPA, isdB, MnTc and SEB) were injected intramuscularly on days 0 and 14, respectively. AlPO4 was added as an adjuvant during protein vaccine immunization, and phosphate buffer alone was used as a control.

[0114] (2) Systemic infection model

[0115] The systemic infection model was established by tail vein injection. Unimmunized female mice of the same age were used to test different doses of the virus in advance to find the appropriate lethal dose. The lethal dose of bacterial liquid was resuspended in phosphate buffer and used to infect immunized mice. The mice were given sufficient water and food, and observed every 12 hours for 7 days, and the survival rate of the mice was recorded. The results are shown in Fig.13 As shown. Fig.13 It can be seen that the mice immunized with the five mRNA mixed vaccines and the five mRNA single component vaccines all had a certain survival rate under Staphylococcus aureus infection, among which the mice immunized with the five mRNA mixed vaccines had the highest survival rate, which was higher than the group immunized with the five-antigen Staphylococcus aureus protein vaccine.

[0116] (3) Humoral immunity

[0117] Mouse serum samples were collected for enzyme-linked immunosorbent assay (ELISA). Hla, FnBPA, IsdB, MnTc and SEB (200 ng per well) proteins were diluted with 0.05 M carbonate buffer and added to the ELISA plate overnight at 4°C; Blocking solution was added to block at 37 degrees for two hours; the primary antibody was the diluted serum sample, incubated at 37°C for 1 hour; the secondary antibody was enzyme-labeled goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a or anti-mouse IgGM, incubated at 37°C for 1 hour, 100 μl of HRP colorimetric substrate TMB was added, and the reaction was carried out at room temperature for 15 minutes in the dark; 100 μl of 2% sulfuric acid stop solution was added, and the plate was washed with PBST at least 5 times after each step, each time for 5 minutes. The absorbance value was measured at 450 nm, and the titer was defined as the highest dilution. The absorbance value produced was more than twice the pre-immune serum value, which was positive. The results are as follows Figures 14 to 17 shown.

[0118] from Figures 14 to 17 It can be seen that the antibody titer increased rapidly after vaccination and reached a peak 14 days after the second vaccination ( Fig.14 ), the vaccine will also induce the production of IGM. Analysis of the isotype spectrum of antigen-specific IgG (IgG1, IgG2a) showed that the experimental group vaccines induced high levels of antigen-specific IgG1 and IgG2a subtypes. In addition, the level of antigen-specific IgG1 was higher than that of IgG2a. IgG1 and IgG2a are markers of Th2 and Th1 responses, respectively. These results show that the mRNA vaccine of the present application induces a Th2-biased response ( Fig.15 ). In addition, the mixed vaccine group was able to induce high antibody titers against each antigen ( Fig.16 ). Except for glycosylation targets (Hla, FnBPA), the antibody titers induced by the mixed mRNA vaccine group were higher than those induced by the mixed protein group ( Fig.17 ).

[0119] (4) Cellular immunity

[0120] Enzyme linked immunospot assay (ELISpot): Mouse ACK lysate-treated lymphocytes were plated at 2×10 6The cells were added to the ELISA plate coated with IL-2, IL-4, and IFN-γ antibodies at a density of 10 μg / mL. The cells were stimulated with 10 μg / ml antigen for 24 hours, and the liquid in the wells was poured out. Ice-cold deionized water was added to lyse the cells on the plate, and the plate was washed 6 times; Biotinylated antibody was added and incubated at 37°C for 1 hour, and the plate was washed 6 times; Streptavidin-HRP was added and incubated at 37°C for 1 hour, and the plate was washed 6 times; the freshly prepared AEC color development solution was added to each experimental well (100 μL / well), and allowed to stand at room temperature and away from light for 30 minutes; the liquid in the wells was poured out, the base of the plate was uncovered, and the front and back sides and the base were washed with deionized water for 3-5 times to stop the color development; the plate was placed in a cool place at room temperature, and the base was closed after it was naturally dried; the ELISPOT plate spots were counted, and various parameters of the spots were recorded for statistical analysis. The results are as follows Fig.18 As shown. Fig.18 It can be seen that compared with spleen cells from control mice, spleen cells from immunized mice produced significantly more IFN-γ, IL-2, and IL-4 under the action of stimulants, indicating that the immunized group can induce a good cellular immune response.

[0121] (5) Histological analysis

[0122] 48 hours after the infection, the lung tissue of the mice was completely removed, fixed with 4% paraformaldehyde solution, embedded in paraffin, and cut into 5μm slices. Dewax the paraffin sections to water: put the sections into environmentally friendly dewaxing solution I for 20min-environmentally friendly dewaxing solution II for 20min-anhydrous ethanol I for 5min-anhydrous ethanol II for 5min-75% alcohol for 5min, and wash with tap water. The sections were treated in high-definition constant staining pretreatment solution for 1min. The sections were dyed in hematoxylin stain solution for 3-5min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue reversion solution, and rinsed with running water. The sections were dehydrated in 95% alcohol for 1min and stained in eosin stain solution for 15s. The sections were placed in anhydrous ethanol I for 2min-anhydrous ethanol II for 2min-anhydrous ethanol III for 2min-n-butanol I for 2min-n-butanol II for 2min-xylene I for 2min-xylene II for 2min, and transparent, and sealed with neutral gum. Image analysis was performed, and the results are as follows Fig.19 As shown. Fig.19 In the experiment, enhanced neutrophil infiltration, alveolar hemorrhage, and alveolar structural destruction were observed in the sections of the control mice. In contrast, the immunized group significantly reduced inflammatory changes and tissue damage, indicating that pre-vaccination with S. aureus vaccine can significantly reduce lung damage caused by S. aureus infection.

[0123] In summary, any of the five mRNA vaccines can induce humoral and cellular immune responses, and the mouse experiment shows that the vaccine with a mixture of five mRNAs has a better immune effect. The DNA molecule combination, antigen expression vector combination, isolated mRNA molecule combination, vaccine, and preparation in this application can be applied to the preparation of products for the prevention and / or treatment of Staphylococcus aureus, thereby efficiently activating relatively independent humoral and cellular immune responses at the same time to respond to Staphylococcus aureus infection and hidden intracellular transmission.

[0124] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A DNA molecule combination, characterized in that: The DNA molecule combination includes at least two of a first DNA molecule, a second DNA molecule, a third DNA molecule, a fourth DNA molecule, and a fifth DNA molecule; Wherein, the first DNA molecule comprises a first coding region, and the first coding region comprises a nucleic acid sequence encoding the MnTc protein of the sequence shown in SEQ ID No.1; The second DNA molecule includes a second coding region, and the second coding region includes a nucleic acid sequence encoding a SEB protein as shown in SEQ ID No. 2; The third DNA molecule includes a third coding region, and the third coding region includes a nucleic acid sequence encoding the Hla protein as shown in SEQ ID No.3; The fourth DNA molecule includes a fourth coding region, and the fourth coding region includes a nucleic acid sequence encoding the FnBPA protein as shown in SEQ ID No. 4; The fifth DNA molecule includes a fifth coding region, and the fifth coding region includes a nucleic acid sequence encoding the IsdB protein as shown in SEQ ID No.

5.

2. According to the DNA molecule combination of claim 1, the first coding region, the second coding region, the third coding region, the fourth coding region or the fifth coding region comprises a nucleic acid sequence encoding a signal peptide of the sequence shown in SEQ ID No. 7 and / or a nucleic acid sequence encoding a tag protein.

3. The DNA molecule combination according to claim 1 or 2, characterized in that: The upstream and downstream of the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region further include a 5' untranslated region and a 3' untranslated region respectively; The 5' untranslated region is shown in SEQ ID No.8; The 3' untranslated region is shown as SEQ ID No.

9.

4. The DNA molecule combination according to claim 3, characterized in that: The downstream of the first coding region, the second coding region, the third coding region, the fourth coding region or the fifth coding region comprises polyadenylic acid, the polyadenylic acid is located downstream of the 3'-end untranslated region, and the sequence of the polyadenylic acid is shown in SEQ ID No.10; The upstream of the first coding region, the second coding region, the third coding region, the fourth coding region or the fifth coding region includes a KOZAK sequence, and the KOZAK sequence is located downstream of the 5' untranslated region. The KOZAK sequence is shown in SEQ ID No.

6.

5. The DNA molecule combination according to claim 3, characterized in that: The upstream of the first coding region, the second coding region, the third coding region, the fourth coding region and the fifth coding region also includes a 5' cap structure, and the 5' cap structure is located upstream of the 5' untranslated region.

6. An antigen expression vector combination, characterized in that: The antigen expression vector combination comprises the DNA molecule combination according to any one of claims 1 to 5.

7. An mRNA molecule combination, characterized in that: The mRNA molecule combination is prepared by the antigen expression vector combination according to claim 6.

8. A Staphylococcus aureus mRNA vaccine, characterized in that: The Staphylococcus aureus mRNA vaccine comprises the mRNA molecule combination of claim 7.

9. The Staphylococcus aureus mRNA vaccine according to claim 8, characterized in that The Staphylococcus aureus mRNA vaccine includes a pharmaceutically acceptable carrier; The carrier is liposome nanoparticles.

10. A preparation, characterized in that The preparation comprises the Staphylococcus aureus mRNA vaccine according to claim 8 or 9.

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