Staphylococcus aureus vaccine and its preparation method, and the use of PLGA-PEG copolymer in vaccine preparation.

Abstract

This invention relates to Staphylococcus aureus vaccines and their preparation methods, as well as the use of PLGA-PEG copolymers in vaccine preparation. According to embodiments of the invention, the vaccine contains: adjuvant nanoparticles containing PLGA or PLGA-PEG copolymers; and Staphylococcus aureus antigen, wherein the Staphylococcus aureus antigen is covalently linked to the adjuvant nanoparticles.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to Staphylococcus aureus vaccine and its preparation method, and the use of PLGA-PEG copolymer in vaccine preparation. Background Technology

[0002] Staphylococcus aureus (S. aureus), also known as "Staphylococcus aureus," is a common foodborne pathogen. Staphylococcus aureus commonly resides on the skin, nasal cavity, pharynx, gastrointestinal tract, boils, and abscesses of humans and animals. It is also ubiquitous in the environment, including air and sewage. Staphylococcus aureus often causes opportunistic infections, leading to varying degrees of suppurative inflammatory diseases such as boils, carbuncles, otitis media, sinusitis, osteomyelitis, and sepsis.

[0003] Staphylococci, especially methicillin-resistant Staphylococcus aureus (MRSA), exhibit multidrug resistance, posing a significant challenge to clinical treatment. The development of antibiotics is lagging far behind the rate of bacterial resistance growth, thus necessitating the search for safe and effective treatment methods.

[0004] Methicillin-resistant Staphylococcus aureus (MRSA) is a unique strain of Staphylococcus aureus that is resistant to almost all penicillin antibiotics, including methicillin and other β-lactamase-resistant penicillins. First discovered in the UK in 1961, MRSA is now widespread and is known as a "superbug."

[0005] Vaccines have become an effective means of preventing Staphylococcus aureus (especially MRSA) infection for the following reasons: 1. Vaccine use is not affected by existing bacterial resistance mechanisms in clinical practice; 2. Vaccine use can greatly reduce bacterial infection, thereby reducing antibiotic use and breaking the vicious cycle of "antibiotic use - resistance - antibiotic abuse - widespread resistance"; 3. Vaccines have very high specificity, targeting only specific pathogens and not affecting the normal human flora, overcoming the side effect of flora imbalance caused by antibiotic use.

[0006] However, there are currently no superbug vaccines developed and marketed internationally. The main reason is that traditional vaccine theory and technology have failed to screen out a sufficient amount of protective target components. At the same time, although traditional adjuvants can promote T-cell immune response, prolong the retention time of antigens in the body, and enhance humoral immunity, they also have disadvantages such as swelling, pain, and induration at the injection site; systemic reactions such as fever, dizziness, diarrhea, and vomiting; and easy aggregation to form molecular structures of different sizes.

[0007] Therefore, existing Staphylococcus aureus vaccines still need further improvement. Summary of the Invention

[0008] This invention is based on the following discoveries of the inventors:

[0009] During their in-depth research on Staphylococcus aureus vaccines, the inventors of this invention discovered that by using adjuvant nanoparticles to load antigens, the small size, large specific surface area, strong adsorption capacity, and high adjuvant activity of the adjuvant nanoparticles can enhance the body's immune response, reduce side effects, and achieve a relatively ideal immune-enhancing effect. Furthermore, using nanoparticles (NPs) as antigen carriers / adjuvants can simultaneously enhance humoral and cellular immunity while reducing the required antigen dosage. In their further research on adjuvant nanoparticles, the inventors unexpectedly discovered that the mechanical properties of adjuvant nanoparticles, such as Young's modulus, are important determinants of immune cell activation, and that these mechanical properties can regulate humoral and cellular immune responses. Therefore, through screening and activity verification of a series of adjuvant nanoparticles, the inventors proposed adjuvant nanoparticles suitable for different immunization routes, such as those suitable for intravenous or subcutaneous immunization. The resulting vaccines can produce more antibodies and effectively neutralize the toxicity of Staphylococcus aureus.

[0010] In view of this, the present invention proposes a Staphylococcus aureus vaccine that can effectively neutralize the toxicity of Staphylococcus aureus and a method for preparing the same.

[0011] In a first aspect, the present invention provides a Staphylococcus aureus vaccine, wherein, according to an embodiment of the present invention, the vaccine comprises: adjuvant nanoparticles containing PLGA or PLGA-PEG copolymer; and Staphylococcus aureus antigen, wherein the Staphylococcus aureus antigen is covalently linked to the adjuvant nanoparticles.

[0012] Through arduous research, the inventors of this invention unexpectedly discovered that selecting PLGA or PLGA-PEG copolymers as adjuvants can effectively obtain adjuvant nanoparticles with desired mechanical properties, thus adapting to different immunization scenarios, such as intravenous or subcutaneous immunization. Furthermore, by covalently linking the adjuvant nanoparticles to Staphylococcus aureus, the activity of the vaccine in activating immune cells can be further enhanced, generating more neutralizing antibodies to effectively neutralize the toxicity of Staphylococcus aureus. In addition, the inventors found that based on the novel adjuvant nanoparticles proposed in this invention, the loading capacity of vaccine antigens can be further increased, thereby improving the immunization efficiency of the vaccine. Furthermore, using the novel adjuvant nanoparticles, the uptake of vaccine antigens can be further increased, thereby improving the immunization efficiency per unit amount of vaccine antigen. Therefore, according to the embodiments of this application, compared to traditional aluminum-based vaccines, the vaccine of this application has at least one of the following advantages: low side effects, long antibody maintenance level, high antibody titer level, and neutralizing antibody activity. In addition, the adjuvant nanoparticles according to the embodiments of this application have good biocompatibility and self-degradable properties.

[0013] The adjuvant nanoparticles according to embodiments of this application can be degraded in vivo. For example, according to embodiments of this application, under body temperature (37 degrees Celsius) conditions in a body fluid environment, the degradation time of the adjuvant nanoparticles is 1 day to 1 month, for example, 1 day to 2 weeks. Therefore, the vaccine according to embodiments of this invention has high biocompatibility, higher safety, and can be used as an effective means of regulating immunization by adjusting the PEG content.

[0014] In a second aspect, this application proposes a method for preparing a Staphylococcus aureus vaccine. According to an embodiment of this application, the method includes: (1) dissolving PLGA or PLGA-PEG copolymer in an organic solvent, preferably a mixture of dichloromethane and acetone; (2) adding the mixture obtained in step (1) dropwise to a polyvinyl alcohol solution, followed by ultrasonic emulsification, room temperature reaction in water, and microporous membrane filtration to obtain adjuvant nanoparticles; and (3) covalently linking Staphylococcus aureus antigen to the adjuvant nanoparticles to obtain the Staphylococcus aureus vaccine.

[0015] Using this method, the Staphylococcus aureus vaccine described in the first aspect can be effectively obtained. Therefore, by selecting PLGA or PLGA-PEG copolymer as an adjuvant, adjuvant nanoparticles with the desired mechanical properties can be effectively obtained, thus adapting to different immunization scenarios, such as intravenous or subcutaneous immunization. Furthermore, by covalently linking the adjuvant nanoparticles to Staphylococcus aureus, the activity of the vaccine in activating immune cells can be further enhanced, generating more antibodies to effectively neutralize the toxicity of Staphylococcus aureus.

[0016] According to an embodiment of this application, step (3) further includes: (3-1) adding the adjuvant nanoparticles to morpholine ethanesulfonic acid buffer, and adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; (3-2) centrifuging the mixture obtained in step (3-1) and resuspending the precipitate with a solution containing the Staphylococcus aureus antigen to obtain a resuspension; (3-3) adjusting the pH of the resuspension to 8 and incubating it overnight at 4 degrees Celsius to obtain a crude vaccine; and (3-4) subjecting the crude vaccine to ultrasonic dispersion to obtain the Staphylococcus aureus vaccine. This further improves the efficiency of vaccine preparation and reduces production costs.

[0017] In a third aspect, this application also proposes the use of PLGA or PLGA-PEG copolymers as adjuvants in the preparation of vaccines for intravenous or subcutaneous immunization. As mentioned above, by selecting PLGA or PLGA-PEG copolymers as adjuvants, the inventors of this application can effectively obtain adjuvant nanoparticles with desired mechanical properties, thereby adapting to different immunization scenarios, such as intravenous or subcutaneous immunization. In fact, the PLGA or PLGA-PEG copolymer as an adjuvant can also be used in a variety of vaccines. In addition, the inventors have found that, based on the novel adjuvant nanoparticles proposed in this invention, the loading of vaccine antigens can be further increased, thereby improving the immunization efficiency of the vaccine. Furthermore, by using the novel adjuvant nanoparticles, the uptake of vaccine antigens can be further increased, thereby improving the immunization efficiency per unit amount of vaccine antigen. Thus, according to the embodiments of this application, compared with traditional aluminum-based vaccines, the vaccine of this application has at least one of the following advantages: low side effects, long antibody maintenance level, high antibody titer level, and neutralizing antibody activity.

[0018] The adjuvant nanoparticles according to embodiments of this application can be degraded in vivo. For example, according to embodiments of this application, under body temperature (37 degrees Celsius) conditions in a body fluid environment, the degradation time of the adjuvant nanoparticles is 1 day to 1 month, for example, 1 day to 2 weeks. Therefore, vaccines using the adjuvant nanoparticles of the embodiments of this invention have high biocompatibility and higher safety.

[0019] According to an embodiment of this application, the vaccine is a Staphylococcus aureus vaccine. According to an embodiment of this application, the vaccine is used to resist methicillin-resistant Staphylococcus aureus. Thus, a novel vaccine effectively resisting Staphylococcus aureus, especially drug-resistant Staphylococcus aureus, can be provided, offering a new and effective solution for the prevention or treatment of Staphylococcus aureus infections.

[0020] Therefore, according to the fourth aspect of this application, this application also proposes a method for treating or preventing Staphylococcus aureus-related diseases, comprising: administering the aforementioned Staphylococcus aureus vaccine to a subject.

[0021] According to an embodiment of this application, the Staphylococcus aureus-related disease is a methicillin-resistant Staphylococcus aureus-related disease. Attached Figure Description

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0023] Figure 1 The illustration shows schematic results of Young's modulus characterization of a series of PLGA-PEG X%NPs (X = 0, 14, 20, 25, 33, i.e., PLGA NPs, PLGA-PEG 14%NPs, PLGA-PEG 20%NPs, PLGA-PEG 25%NPs, PLGA-PEG 33%NPs) using AFM according to one embodiment of the present application.

[0024] Figure 2 The results of antibody titer detection according to one embodiment of this application are shown.

[0025] Figure 3 The results of antibody titer detection according to another embodiment of this application are shown.

[0026] Figure 4 The figure shows the secretion levels of IL-4 and IFN-γ in the mouse spleen according to one embodiment of this application. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be described below.

[0028] definition

[0029] In this article, unless otherwise specified, the term "Staphylococcus aureus vaccine" should be interpreted broadly to include both preventative and therapeutic vaccines.

[0030] Unless otherwise specified herein, the term "Staphylococcus aureus antigen" refers to a molecular entity capable of inducing a specific immune response against Staphylococcus aureus in a host. According to embodiments of this application, the antigen may primarily comprise at least a portion of the surface proteins of Staphylococcus aureus. According to embodiments of this application, Staphylococcus aureus antigen may be obtained through in vitro synthesis, for example, by expression and purification in a microbial or animal expression system based on the amino acid sequence of the corresponding protein antigen, for example, by using the IPTG induction method.

[0031] Unless otherwise specified, the term "PLGA" in this document refers to poly(lactide / glycolic acid).

[0032] Unless otherwise specified, the term "PEG" in this document refers to polyethylene glycol.

[0033] Unless otherwise specified herein, the term "PLGA-PEG copolymer" refers to a polymer containing both PEG and PLGA units in the copolymer, such as a PLGA-PEG block copolymer. In this application, PLGA-PEG copolymers with different PEG contents are used for different immune scenarios. Specifically, monomethoxy PEG can be used as a macromolecular initiator, and the desired PLGA-PEG copolymer can be obtained through the ring-opening polymerization reaction of D,L-lactide and glycolide. Furthermore, by controlling the proportions of the reactants, a series of PLGA-PEG copolymers meeting specific requirements can be obtained. Alternatively, those skilled in the art can also purchase PLGA-PEG copolymers with different PEG contents.

[0034] Unless otherwise specified, the term "nanoparticle" in this document refers to a particulate dispersion with a particle size in the range of 10 to 1000 nanometers.

[0035] Unless otherwise specified herein, the term "PEG content in PLGA-PEG copolymer" refers to the weight percentage of PEG in the copolymer, that is, the proportion of the molecular weight of PEG in the total molecular weight of the PLGA-PEG copolymer. According to embodiments of this application, by fixing the molecular weight of PEG within a predetermined molecular weight range, such as 5000 Da, a series of PLGA-PEG copolymers with different PEG contents can be obtained by adjusting the amount of PLGA.

[0036] In this document, unless otherwise specified, the term "at least a portion of at least one of Csa1A, EsxA, Hla, and EsxB" means that the full length or a portion thereof, such as a portion of the extracellular segment, of the proteins Csa1A, EsxA, Hla, and EsxB from Staphylococcus aureus can be used as an antigen. The term "at least a portion" here is not particularly limited in length, as long as it can induce an immune response in the host.

[0037] Unless otherwise specified herein, the term "Staphylococcus aureus antigen content" is used to characterize the antigen loading of a Staphylococcus aureus vaccine, and refers to the weight percentage of Staphylococcus aureus antigen based on the total weight of the adjuvant nanoparticles and the Staphylococcus aureus antigen. Of course, those skilled in the art will understand that other methods can be used to characterize the antigen loading of a Staphylococcus aureus vaccine, such as determining the crosslinking rate of the PLGA-PEG copolymer and EsxB using the BCA method.

[0038] This invention is based on the following discoveries of the inventors:

[0039] During their in-depth research on Staphylococcus aureus vaccines, the inventors of this invention discovered that by using adjuvant nanoparticles to load antigens, the small size, large specific surface area, strong adsorption capacity, and high adjuvant activity of the adjuvant nanoparticles can enhance the body's immune response, reduce side effects, and achieve a relatively ideal immune-enhancing effect. Furthermore, using nanoparticles (NPs) as antigen carriers / adjuvants can simultaneously enhance humoral and cellular immunity while reducing the required antigen dosage. In their further research on adjuvant nanoparticles, the inventors unexpectedly discovered that the mechanical properties of adjuvant nanoparticles, such as Young's modulus, are important determinants of immune cell activation, and that these mechanical properties can regulate humoral and cellular immune responses. Therefore, through screening and activity verification of a series of adjuvant nanoparticles, the inventors proposed adjuvant nanoparticles suitable for different immunization routes, such as those suitable for intravenous or subcutaneous immunization. The resulting vaccines can produce more antibodies and effectively neutralize the toxicity of Staphylococcus aureus.

[0040] In view of this, the present invention proposes a Staphylococcus aureus vaccine that can effectively neutralize the toxicity of Staphylococcus aureus and a method for preparing the same.

[0041] In a first aspect, the present invention provides a Staphylococcus aureus vaccine, wherein, according to an embodiment of the invention, the vaccine comprises: adjuvant nanoparticles containing PLGA or PLGA-PEG copolymer; and Staphylococcus aureus antigen, wherein the Staphylococcus aureus antigen is linked to the adjuvant nanoparticles.

[0042] Through arduous research, the inventors of this invention unexpectedly discovered that selecting PLGA or PLGA-PEG copolymers as adjuvants can effectively yield adjuvant nanoparticles with desired mechanical properties, thus adapting to different immunization scenarios, such as intravenous or subcutaneous immunization. Furthermore, by covalently linking the adjuvant nanoparticles to Staphylococcus aureus, the activity of the vaccine in activating immune cells can be further enhanced, generating more antibodies to effectively neutralize the toxicity of Staphylococcus aureus. In addition, the inventors found that based on the novel adjuvant nanoparticles proposed in this invention, the loading capacity of vaccine antigens can be further increased, thereby improving the immunization efficiency of the vaccine. Furthermore, using the novel adjuvant nanoparticles, the uptake of vaccine antigens can be further increased, thereby improving the immunization efficiency per unit amount of vaccine antigen. Therefore, according to the embodiments of this application, compared to traditional aluminum-based vaccines, the vaccine of this application has at least one of the following advantages: low side effects, long antibody maintenance level, high antibody titer level, and neutralizing antibody activity.

[0043] Furthermore, according to embodiments of this application, the adjuvant nanoparticles exhibit good biocompatibility and self-degradability. Specifically, according to embodiments of this application, under body temperature (approximately 37 degrees Celsius) conditions in a bodily fluid environment, the degradation time of the adjuvant nanoparticles is 1 day to 1 month, for example, 1 day to 2 weeks. Among these embodiments, the inventors of this invention have discovered that the degradation time of the adjuvant nanoparticles is related to the PEG content in the PLGA-PEG copolymer. Specifically, the higher the PEG content in the PLGA-PEG copolymer, the shorter the degradation time of the adjuvant nanoparticles, which can range from 1 day to 1 month, for example, 1 day to 2 weeks.

[0044] According to embodiments of this application, the inventors discovered that the self-degradation properties of adjuvant nanoparticles, in addition to improving the biocompatibility of materials, are also related to immunomodulatory properties. According to embodiments of this application, the inventors unexpectedly discovered that in the subcutaneous immunization route, antigen uptake rate is related to softness / hardness; nanovaccines with high PEG content (PEG content greater than 20%) are more easily phagocytosed by dendritic cells, eliciting a stronger immune response and producing more protective antibodies. The inventors also found that, unlike the subcutaneous immunization route, in the intravenous immunization route, the correlation between antigen uptake rate and softness / hardness is relatively low, but the antigen uptake rate is highly correlated with the self-degradation performance of the nanovaccines. Specifically, for nanovaccines with slow self-degradation rates and low PEG content (PEG content less than 20%), the binding time between the antigen and adjuvant nanoparticles is longer, resulting in a higher uptake rate by immune organs, thereby inducing a stronger immune response and producing more protective antibodies.

[0045] According to embodiments of the present invention, the inventors selected a PLGA-PEG copolymer to construct an adjuvant system for developing a Staphylococcus aureus nanovaccine system. Both PLGA and PEG are FDA-approved biocompatible biomaterials, and adjusting the copolymer's ratio and synthesis process allows for precise control of the physicochemical parameters of different nanoparticles, including different sizes, morphologies, degradation characteristics, and hardness / softness. According to embodiments of this application, the hardness / softness of the nanoparticles can be adjusted. Using the hardness / softness of dendritic cell membranes as a reference, nanoparticles with gradient hardness are screened as research objects to identify advantageous nano-adjuvants. The nanoparticles are covalently bound to the antigen, and the antigen-nanoparticle ratio is cross-validated. The composition of the nanovaccine is determined using the highest antigen binding rate as a reference indicator.

[0046] According to embodiments of this application, the linkage between Staphylococcus aureus antigen and adjuvant nanoparticles is not particularly limited; they can be linked through physical interactions or chemical bonds. According to embodiments of this application, Staphylococcus aureus antigen and adjuvant nanoparticles can be linked through at least one of electrostatic adsorption, covalent bonding, hydrophobic interaction, ligand exchange, amide bonds, disulfide bonds, linkers, and pyrophosphate diester bonds. According to embodiments of the present invention, the Staphylococcus aureus antigen and the adjuvant nanoparticles are linked by non-hydrolyzable covalent bonds. The inventors of this invention have discovered that, unlike the delivery mechanism of therapeutic drugs, linking Staphylococcus aureus antigen and adjuvant nanoparticles through non-hydrolyzable covalent bonds can enhance the activity of vaccines in activating immune cells. Furthermore, according to embodiments of the present invention, the covalent bond includes at least one of amide bonds and disulfide bonds. The inventors have discovered that, since the three-dimensional structure of antigen proteins is relatively easily affected, the reaction conditions for forming amide bonds or disulfide bonds do not damage or negatively affect the three-dimensional structure of the antigen protein, thereby maintaining the immunogenicity of the antigen protein.

[0047] According to embodiments of the present invention, the form of the Staphylococcus aureus vaccine is not particularly limited, and it can be in the form of lyophilized powder, aqueous solution, suspension, microemulsion, dispersion, or liposome, wherein lyophilized powder is preferred. The inventors of the present invention have found that the vaccine according to the embodiments of this application is suitable for preparation in the form of lyophilized powder, thereby having a longer shelf life, and subsequently only requiring mixing with a solution such as PBS to prepare an injectable solution, while the performance in activating immune cells is not affected.

[0048] According to embodiments of this application, the route of administration for the vaccine is not particularly limited, and it can be administered via at least one of the following methods: intravenous administration, subcutaneous administration, intramuscular administration, parenteral administration, rectal administration, spinal administration, epidermal administration, infusion administration, intraperitoneal administration, and intralymphatic injection. Furthermore, the inventors have analyzed the influence of PEG content on the immunization effect of various administration routes through a series of experiments. Therefore, according to embodiments of the present invention, the Staphylococcus aureus vaccine is used for intravenous immunization, and the adjuvant nanoparticles contain PLGA or a PLGA-PEG copolymer, wherein the PEG content in the PLGA-PEG copolymer does not exceed 15%. According to embodiments of the present invention, the Staphylococcus aureus vaccine is used for subcutaneous immunization, and the adjuvant nanoparticles contain a PLGA-PEG copolymer, wherein the PEG content in the PLGA-PEG copolymer is 20-35%, preferably 25-30%. The inventors have demonstrated through a series of experiments that, for different immunization scenarios, the optimal immunization effect can be achieved by adjusting the mechanical properties (e.g., Young's modulus) of the adjuvant nanoparticles by changing the PEG content. According to embodiments of this application, the adjuvant nanoparticles have a particle size in the range of 150 nm to 200 nm, and the particle size dispersibility (PDI) value of the adjuvant nanoparticles does not exceed 0.06. Optionally, the mechanical strength and Young's modulus of the adjuvant nanoparticles are between 800 Pa and 1 MPa. Optionally, the surface of the adjuvant nanoparticles carries surface modification groups, and the modification groups may include at least one of carboxyl, amino, thiol, methoxy, and aldehyde groups. This can further improve the immunogenicity of Staphylococcus aureus vaccines.

[0049] According to embodiments of the present invention, the Staphylococcus aureus antigen includes at least a portion of at least one selected from Csa1A, EsxA, Hla, and EsxB. This effectively stimulates the host's immune system to produce effective antigens to resist Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA). In a specific embodiment of the present invention, EsxB can be used as an MRSA-specific antigen, primarily based on two points: the EsxB protein is present in most Staphylococcus aureus substrains, and EsxB promotes IgG secretion; additionally, EsxB has a preventive effect against Staphylococcus aureus infection.

[0050] According to embodiments of the present invention, based on the total weight of the adjuvant nanoparticles and the Staphylococcus aureus antigen, the content of the Staphylococcus aureus antigen is 3-10%, preferably 4-6%. This effectively stimulates the host's immune system to produce effective antigens to resist Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA).

[0051] According to embodiments of the present invention, the molecular weight of PEG in the PLGA-PEG copolymer is 4000-6000 Daltons, preferably 5000 Daltons. This effectively stimulates the host's immune system to produce effective antigens to resist Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA).

[0052] According to embodiments of the present invention, compared with existing aluminum adjuvant vaccines, the survival rate of animals infected with Staphylococcus aureus is higher at the same antigen dose. This effectively avoids the current situation where superbug infections caused by antibiotic resistance are untreatable.

[0053] In a second aspect, this application proposes a method for preparing a Staphylococcus aureus vaccine. According to an embodiment of this application, the method includes: (1) dissolving PLGA or PLGA-PEG copolymer in an organic solvent, preferably a mixture of dichloromethane and acetone; (2) adding the mixture obtained in step (1) dropwise to a polyvinyl alcohol solution, followed by ultrasonic emulsification, room temperature reaction in water, and microporous membrane filtration to obtain adjuvant nanoparticles; and (3) covalently linking Staphylococcus aureus antigen to the adjuvant nanoparticles to obtain the Staphylococcus aureus vaccine.

[0054] Using this method, the Staphylococcus aureus vaccine described in the first aspect can be effectively obtained. Therefore, by selecting PLGA or PLGA-PEG copolymer as an adjuvant, adjuvant nanoparticles with the desired mechanical properties can be effectively obtained, thus adapting to different immunization scenarios, such as intravenous or subcutaneous immunization. Furthermore, by covalently linking the adjuvant nanoparticles to Staphylococcus aureus, the activity of the vaccine in activating immune cells can be further enhanced, generating more antibodies to effectively neutralize the toxicity of Staphylococcus aureus.

[0055] According to an embodiment of this application, step (3) further includes: (3-1) adding the adjuvant nanoparticles to morpholine ethanesulfonic acid buffer, and adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; (3-2) centrifuging the mixture obtained in step (3-1) and resuspending the precipitate with a solution containing the Staphylococcus aureus antigen to obtain a resuspension; (3-3) adjusting the pH of the resuspension to 8 and incubating it overnight at 4 degrees Celsius to obtain a crude vaccine; and (3-4) subjecting the crude vaccine to ultrasonic dispersion to obtain the Staphylococcus aureus vaccine. This further improves the efficiency of vaccine preparation and reduces production costs.

[0056] Thirdly, this application also proposes the use of PLGA or PLGA-PEG copolymers as adjuvants in the preparation of vaccines for intravenous or subcutaneous immunization. As mentioned above, by selecting PLGA or PLGA-PEG copolymers as adjuvants, the inventors of this application can effectively obtain adjuvant nanoparticles with desired mechanical properties, thereby adapting to different immunization scenarios, such as intravenous or subcutaneous immunization. In fact, the PLGA or PLGA-PEG copolymer as an adjuvant can also be used in a variety of vaccines.

[0057] According to an embodiment of this application, the vaccine is a Staphylococcus aureus vaccine. According to an embodiment of this application, the vaccine is used to resist methicillin-resistant Staphylococcus aureus. Therefore, a novel vaccine effectively resisting Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus, can be provided, offering a new and effective solution for the prevention or treatment of Staphylococcus aureus infection. It effectively prevents Staphylococcus aureus infection, especially methicillin-resistant Staphylococcus aureus, and the mortality rate from methicillin-resistant Staphylococcus aureus infection is significantly reduced after nano-vaccination.

[0058] Furthermore, the inventors discovered that, based on the novel adjuvant nanoparticles proposed in this invention, the loading capacity of vaccine antigens can be further increased, thereby improving the immunization efficiency of the vaccine. Additionally, by using the novel adjuvant nanoparticles, the uptake of vaccine antigens can be further increased, thereby improving the immunization efficiency per unit amount of vaccine antigen. Therefore, according to the embodiments of this application, compared to conventional aluminum-based vaccines, the vaccine of this application has at least one of the following advantages: low side effects, long-lasting antibody maintenance levels, high antibody titer levels, and antibodies with neutralizing activity.

[0059] The adjuvant nanoparticles according to embodiments of this application can be degraded in vivo. For example, according to embodiments of this application, under body temperature (37 degrees Celsius) conditions in a body fluid environment, the degradation time of the adjuvant nanoparticles is 1 day to 1 month, for example, 1 day to 2 weeks. Therefore, the vaccine according to embodiments of this invention has high biocompatibility, higher safety, and can be used as an effective means of regulating immunization by adjusting the PEG content.

[0060] Therefore, according to the fourth aspect of this application, this application also proposes a method for treating or preventing Staphylococcus aureus-related diseases, comprising: administering the aforementioned Staphylococcus aureus vaccine to a subject.

[0061] According to an embodiment of this application, the Staphylococcus aureus-related disease is a methicillin-resistant Staphylococcus aureus-related disease.

[0062] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention.

[0063] General methods

[0064] Unless otherwise specified, in the following examples, EsxB-loaded PLGA-PEG copolymer adjuvant nanoparticles were prepared using the following methods.

[0065] Raw materials (and intermediates): PLGA or PLGA-X% PEG copolymer (commercially available, where X represents the weight percentage of PEG in the copolymer), EsxB, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, morpholine ethanesulfonic acid buffer, polyvinyl alcohol, dichloromethane, acetone, sodium bicarbonate.

[0066] Adjuvant Construction: The chain-like PLGA-X%PEG copolymer was dissolved in a mixture of dichloromethane and acetone. The resulting mixture was added dropwise to a polyvinyl alcohol solution, and the mixture was ultrasonically emulsified (ultrasonic power 20W, pulsed ultrasonication, 2s intervals, ultrasonication time 4min). The emulsion was then added dropwise to deionized water and reacted at room temperature. The resulting emulsion was filtered through a microporous membrane and centrifuged to obtain the crude nano-adjuvant product. The crude product was centrifuged and washed with water to obtain the final nano-adjuvant product (also referred to as PLGA-PEG X% nanoparticles (NPs) in later examples).

[0067] Vaccine Development: The recombinant Staphylococcus aureus protein antigen EsxB was selected and bound to the surface of the nano-adjuvant via amide bonds. First, the previously obtained nano-adjuvant was dissolved in morpholine ethanesulfonic acid buffer, and N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added. After activation at room temperature and centrifugation, the precipitate was obtained, resuspended in EsxB solution, and the pH was adjusted to approximately 8 with sodium bicarbonate. The mixture was incubated overnight at 4°C, and centrifuged to obtain the crude nano-vaccine. After centrifugation and washing with water, the finished nano-vaccine was obtained.

[0068] Example 1: Construction and Physicochemical Characterization of a Nanoadjuvant System

[0069] In this embodiment, following general methods, a series of PLGA-PEG X% nanoparticles (NPs) (X = 0, 14, 20, 25, 33) with different PEG contents (i.e., different hardness) were prepared, and their Young's modulus was characterized by atomic force microscopy (AFM). Schematic results are shown below. Figure 1As shown. Among them, when X=33, its Young's modulus is comparable to that of normal human cells. Therefore, in the following embodiments, the inventors will use nanoparticles with different mechanical properties of PLGA-PEG X%NPs (X=0, 14, 20, 25, 33) as adjuvants to construct the nanovaccine system.

[0070] Furthermore, the inventors characterized the hydrated particle size and surface potential of the nanoparticles using a dynamic light scattering method. The hydrated particle size of the five nanoparticles used (PLGA-PEG X%NPs (X = 0, 14, 20, 25, 33)) was approximately 170 nm, and there was no statistically significant difference in the hydrated particle size among the five nanoparticles. With increasing PEG content, the surface potential of the nanoparticles changed from negative to slightly positive. Therefore, the particle size range of the obtained adjuvant nanoparticles allows the vaccine to easily enter the lymph nodes via lymphatic vessels, thereby effectively inducing the body's immune response.

[0071] Example 2 Expression and purification of EsxB and EsxA

[0072] In this embodiment, the inventors synthesized EsxB recombinant antigen (the amino acid sequence used is from Staphylococcus aureus strain ATCC25923 as shown in SEQ ID NO: 2) and EsxA recombinant antigen (the amino acid sequence used is from Staphylococcus aureus strain ATCC25923 as shown in SEQ ID NO: 1) by IPTG method, and purified them with nickel column. The purified products were verified by SDS-PAGE and Western Blot. The results showed that the synthesized EsxB and EsxA recombinant antigens were consistent with those reported in the literature.

[0073] in,

[0074] The full-length amino acid sequence of EsxA is as follows:

[0075] MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEEIKQ QLNSTADAVQEQDQQLSNNFGLQ(SEQ ID NO: 1)

[0076] The full-length amino acid sequence of EsxB is as follows:

[0077] MGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMA DHQKAIDNLSQNLAKYDTLSIKQGLDRVNP (SEQ ID NO: 2)

[0078] In short, the specific steps by which IPTG induces the expression of EsxB or EsxA are as follows:

[0079] After thawing competent E. coli cells in an ice bath, add 1 ng of plasmid carrying the EsxB-encoding nucleic acid sequence to the cell suspension, gently tap to mix, and incubate on ice for 30 min. Place the centrifuge tube in a 42°C water bath for 90 s, then quickly transfer it to an ice bath to cool for 2–3 min, without shaking the centrifuge tube. Add 3 mL of sterile LB medium to the centrifuge tube, mix well, and incubate at 37°C on a shaker for 45 min. Mix the contents of the centrifuge tube, and add 100 μL of the transformed competent cells to LB solid agar medium containing ampicillin sodium. Gently spread the cell suspension evenly with a sterile bent glass rod until all liquid is absorbed. Invert the plate and incubate at 37°C for 12–16 hrs. Select colonies with a bright and smooth surface from the solid agar medium, pick them up, and place them in LB medium containing ampicillin sodium. Incubate at 37°C on a shaker until OD reaches 1000 oz. 600 The concentration of the solution was 0.6–0.8; IPTG was added to induce the bacterial culture to a final concentration of 2 mM / mol, and the induction was carried out at 37°C for 4 hours; the solution was centrifuged at 3000 rpm for 10 minutes and the precipitate was discarded; the supernatant was sonicated and centrifuged at 3000 rpm for 10 minutes, and the above steps were repeated until no precipitate was produced after centrifugation; the protein solution was purified by condensation using a nickel column, dialyzed, and then stored at -20°C for later use.

[0080] Example 3: The effect of the binding mode of adjuvant nanoparticles to antigens on the immunization effect

[0081] In this embodiment, the inventors used EsxA as a model antigen, linked PLGA NPs (PEG-free PLGA nanoparticles) and EsxA in different cross-linking methods, and immunized mice. The antibody titers of IgG, IgG1, and IgG2a in the serum of different groups of immunized mice were measured, as well as the secretion levels of IFN-γ and IL-17 in the spleen of immunized mice. The schematic results show... Figure 2 ( Figure 2 In the diagram, FREE indicates free EsxA; PLGA indicates that EsxA and PLGA NPs are physically bound; AHG indicates that EsxA is adsorbed by aluminum adjuvant; SURF indicates that EsxA and PLGA NPs are bound by covalent bonds; and ENCAP indicates that EsxA is encapsulated in PLGA NPs. Figure 2As can be seen, when PLGA NPs and EsxA are covalently cross-linked, the serum antibody titer levels are higher than those of other cross-linking methods, and also higher than the positive control (aluminum adjuvant) group. When PLGA NPs and EsxA are covalently cross-linked, the secretion of IFN-γ is higher than that of other cross-linking methods, and also higher than the aluminum adjuvant control group. When PLGA NPs and EsxA are covalently cross-linked, the secretion of IL-17 is higher than that of other cross-linking methods, and comparable to the secretion level in the aluminum adjuvant group.

[0082] In addition, the inventors characterized the hydrated particle size and surface potential of the products covalently crosslinked with PLGA-PEG X%NPs (X = 0, 14, 20, 25, 33) and EsxA using DLS. They found that the hydrated particle size of all five nanovaccine systems was around 200 nm, an increase of approximately 30 nm compared to nanoparticles, and there was no statistically significant difference in the average particle size among the five nanovaccine systems; the surface potential of all five nanovaccine systems exhibited a weak negative charge.

[0083] In addition, the inventors characterized the formation of amide bonds in the products of covalently crosslinking PLGA NPs (X = 0, 14, 20, 25, 33) and Esx B using infrared spectroscopy. They found that in the infrared spectrum of the pure Esx B antigen, the amide bond formation was observed at ~3500 cm⁻¹. -1 The spectrum shows a double peak (primary amine) at ~3500 cm⁻¹, while in the infrared spectrum of PLGA-PEG X%NPs-EsxB, the peak is ~3500 cm⁻¹. -1 The single peak (secondary amine) indicates that the primary amine of the antigen is transformed into the secondary amine in the complex, meaning that PLGA-PEG X%NPs can covalently bind with EsxB through an amide bond.

[0084] Example 4: Determination of the crosslinking rate of EsxB

[0085] To further determine the dosage for mice in subsequent embodiments, the crosslinking rate of PLGA-PEG X%NPs and EsxB was measured in this embodiment using the BCA method. In short, the specific steps are as follows:

[0086] Take an appropriate amount of 5 mg / ml standard protein and dilute it to 0.5 mg / ml with PBS.

[0087] Prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly.

[0088] Diluting BSA standards: In microplates, dilute BSA standards directly with a diluent consistent with the protein sample to be tested, as follows.

[0089] Add 2 μL of the protein sample to be tested to each well of the microplate, and label it. Perform three parallel reactions for each sample.

[0090] Add 200 μL of BCA working solution to each well of the standard and the sample to be tested, incubate at 37°C for 30 min, and measure the absorbance at 562 nm.

[0091] Use Excel or other software to plot a standard curve and calculate the protein concentration in the sample.

[0092] The crosslinking rate is calculated using the following formula:

[0093] Crosslinking rate = (number of moles of EsxB on nanoparticles) / (number of moles of nanoparticles) * 100%

[0094] The results showed that the cross-linking rate between PLGA-PEG X%NPs and EsxB increased continuously with the increase of PEG content. Therefore, the subsequent drug dosage for mice was determined to be 25 micrograms of antigen per mouse.

[0095] Example 5: Safety assessment of the nano-vaccine system

[0096] In this embodiment, the inventors used the CCK8 method to verify the cytotoxicity of PLGA-PEG X% NPs-EsxB (a covalently linked product obtained by conventional methods) in vitro using the L929 cell line. In short, the specific steps are as follows:

[0097] The L929 cell density was adjusted to 5*102 4 Inoculate 100 μL per well in a 96-well plate and incubate at 37°C for 24 hours.

[0098] EsxB, PLGA NPs, PLGA-PEG 14% NPs, PLGA-PEG 20% NPs, PLGA-PEG 25% NPs, PLGA-PEG 33% NPs, PLGA NPs-EsxB, PLGA-PEG 14% NPs-EsxB, PLGA-PEG 20% NPs-EsxB, PLGA-PEG 25% NPs-EsxB, and PLGA-PEG 33% NPs-EsxB were added respectively to achieve final concentrations of 1 mg / mL, 500 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 100 ng / mL, and 10 ng / mL, respectively, and incubated at 37°C for 24 hours.

[0099] Add 10 μL of CCK8 reagent to each well and incubate at 37°C for an appropriate time, then measure the absorbance at 450 nm.

[0100] The results showed that the cell viability of all experimental groups (i.e., EsxB, PLGA NPs, PLGA-PEG 14% NPs, PLGA-PEG 20% NPs, PLGA-PEG 25% NPs, PLGA-PEG 33% NPs, PLGA NPs-EsxB, PLGA-PEG 14% NPs-EsxB, PLGA-PEG 20% NPs-EsxB, PLGA-PEG 25% NPs-EsxB, PLGA-PEG 33% NPs-EsxB) was greater than 95%.

[0101] In addition, the inventors investigated the biocompatibility of the nanovaccine system using BALB / c mice. No significant weight loss was observed in any of the immunized mouse groups (PLGA NPs-EsxB, PLGA-PEG 14% NPs-EsxB, PLGA-PEG 20% NPs-EsxB, PLGA-PEG 25% NPs-EsxB, PLGA-PEG 33% NPs-EsxB, PBS, EsxB, AHG-EsxB) (following the immunization procedure and dosage described in Example 6 below). Furthermore, after euthanizing the animals, the inventors performed H&E staining on the major organs of the mice. The results showed that no significant lesions were observed in any organ samples regardless of whether the nanovaccine system was administered subcutaneously or via tail vein.

[0102] Example 6: Evaluation of the efficacy of the nano-vaccine system

[0103] In this embodiment, BALB / c mice were immunized via subcutaneous and tail vein routes using EsxB and EsxA, respectively, at a dose of 25 micrograms per mouse, administered every 2 weeks for a total of 3 immunizations. The antibody titer levels in the serum of the immunized mice were measured using ELISA to evaluate the adjuvant performance of the species nanosystem. In short, the specific steps (taking EsxB as an example) are as follows:

[0104] Step 1: Dilute EsxB solution to 5 μg / mL with coating buffer, add 100 μL to each well to coat the ELISA plate, and incubate overnight at 4°C;

[0105] Step 2: After discarding the excess EsxB antigen in the ELISA plate, wash the ELISA plate three times with 1 / 1000 PBST and pat dry for later use;

[0106] Step 3: After serially diluting the test sample in a 96-well plate, add the diluted test sample sequentially to the ELISA plate at 100 μL / well and incubate at 37°C for 1 hour.

[0107] Step 4: Discard the liquid in the plate and wash three times with 1 / 1000 PBST;

[0108] Step 5: Dilute the secondary antibody and add it to the ELISA plate, 100 μL / well, and incubate at 37°C for 45 min;

[0109] Step 6: Discard the liquid in the plate and wash three times with 1 / 1000 PBST;

[0110] Step 7: 100 μL / well of developing solution, incubate at room temperature for 30 min, and measure absorbance at 405 nm. Results are shown in... Figure 3 middle.

[0111] Next, on day 7 after the third immunization, the IL-4 and IFN-γ secretion levels in the spleen of the immunized mice were measured using the ELISPOT method. In short, the specific steps are as follows:

[0112] Step 1: Add 100 μL of 70% ethanol to each well of the ELISPOT plate for 2 min to pre-wet, then wash the plate 5 times with 200 μL / well of sterile water.

[0113] Step 2: Dilute the coated antibody to 15 μg / mL with sterile PBS, add 100 μL / well to the diluted coated antibody in an ELISPOT plate, and incubate overnight at 4°C.

[0114] Step 3: Discard the liquid in the plate and wash 5 times with sterile PBS, 200 μL / well;

[0115] Step 4: Add cell culture medium containing 10% FCS to the ELISPOT plate, 200 μL / well, incubate at room temperature for 30 min, then discard.

[0116] Step 5: Adjust the density of the spleen cell suspension to 2*10 7 Incubate at 100 μL / well for 12-48 hours at 37°C;

[0117] Step 6: Discard the liquid in the plate and wash 5 times with sterile PBS, 200 μL / well;

[0118] Step 7: Dilute the detection antibody to 1 μL / mL with sterile PBS containing 0.5% FCS, and incubate at room temperature for 2 hours at 100 μL / well.

[0119] Step 8: Discard the liquid in the plate and wash 5 times with sterile PBS, 200 μL / well;

[0120] Step 9: Dilute Streptavidin-ALP 1:1000 with sterile PBS containing 0.5% FCS at a volume ratio of 100 μL / well and incubate at room temperature for 1 hour.

[0121] Step 10: Discard the liquid in the plate and wash 5 times with sterile PBS, 200 μL / well;

[0122] Step 11: Add 100 μL of substrate solution to each well until spots appear, then rinse the plate with plenty of tap water to prevent further color development;

[0123] Step 12: Dry the ELISPOT plate at room temperature and count it on the reader.

[0124] The results show Figure 4 In the middle. Fourteen days after the third immunization, lethal doses of the ATCC25923 substrain (6.8*10) were administered to each immunized mouse via tail vein. 9 CFU / ml (0.1ml / mouse), and clinical signs and mortality were monitored in mice daily. Survival rates in almost all nanovaccine systems were higher than those in the positive control (aluminum adjuvant) and negative control (PBS and EsxB).

[0125] like Figure 3 As shown, for both EsxB and EsxBA, in subcutaneous immunization, the antibody titer level in the nanovaccine group was positively correlated with the PEG content, with higher antibody titers than the aluminum adjuvant group, and a longer duration of antibody action, while the onset time was comparable to the aluminum adjuvant group. In tail vein immunization, the antibody titer level in the nanovaccine group was negatively correlated with the PEG content, with higher antibody titers than the aluminum adjuvant group, and a longer duration of antibody action, while the onset time was faster.

[0126] like Figure 4 As shown, in the subcutaneous immunization route, the secretion of IL-4 in the nanovaccine group was positively correlated with the PEG content. In the tail vein immunization route, the secretion of IL-4 in the nanovaccine group was negatively correlated with the PEG content. In both subcutaneous and tail vein immunization, the secretion of IFN-γ in the nanovaccine group was lower than that in the aluminum adjuvant control group. This phenomenon suggests that humoral immunity plays a dominant role in the nanovaccine group of this invention.

[0127] Example 7: Study on the mechanism of action of the nano-vaccine system of the present invention

[0128] Based on the positive correlation between serum antibody titers and PEG content in subcutaneous immunization, the applicant proposes a novel viewpoint: after subcutaneous immunization, the nanovaccine does not easily diffuse at the injection site, resulting in a high local concentration. Furthermore, the soft nanoadjuvant is more easily phagocytosed by dendritic cells (DCs), leading to higher antibody levels. Therefore, in this embodiment, the inventors verified the above viewpoint by examining the release rate of PLGA-PEG X%NPs and the rate at which DCs phagocytosed PLGA-PEG X%NPs.

[0129] In the tail vein immunization method, based on the negative correlation between serum antibody titers and PEG content in immunized mice, the applicant proposes a new viewpoint: After tail vein immunization, the nano-vaccine enters the bloodstream, and its concentration is rapidly diluted. The amount of each nano-vaccine phagocytosed by dendritic cells (DCs) is roughly equivalent, theoretically resulting in comparable antibody levels across the nano-vaccine groups. However, ELISA results show that the hard nano-vaccine produced higher antibody titers. Therefore, it is possible that the degradation properties of PLGA-PEG X%NPs themselves influence the higher antibody levels in the hard nano-vaccine group. Therefore, in this embodiment, the inventors verified the above viewpoint by examining the release rate of PLGA-PEG X%NPs, the rate at which DCs phagocytosed PLGA-PEG X%NPs, and the distribution of PLGA-PEG X%NPs in vivo.

[0130] Specifically, in this embodiment, FITC is cross-linked with PLGA-PEG X%NPs in the same manner. Therefore, the release rate of antigens in the nanovaccine system can be examined by measuring the release amount of FITC on PLGA-PEG X%NPs-FITC. In vitro, using 50% FCS to simulate the in vivo environment, the results showed that the higher the PEG content, the faster the FITC release rate. Furthermore, in this embodiment, the inventors simulated the local environment of subcutaneous immunization and the in vivo environment of tail vein immunization of the nanovaccines in vitro. Dendritic cells (DCs) were used to examine the phagocytosis of five nanovaccine systems by antigen-presenting cells. The results showed that in subcutaneous immunization, there was no difference in phagocytosis of the five nanovaccine systems by DCs at 0.5 hours, but the higher the PEG content over time, the higher the phagocytosis of the nanovaccines by DCs. In tail vein immunization, there was no difference in phagocytosis of the five nanovaccine systems by DCs. Furthermore, the inventors encapsulated ICG in nano-adjuvants and injected it into BALB / c mice via the tail vein, observing the in vivo distribution of ICG@PLGA-PEG X%NPs. The results showed that ICG@PLGA-PEG X%NPs were mainly distributed in the mouse intestines. Since the mesenteric lymph nodes are the largest lymphoid tissues in mice, tail vein immunization would produce a higher antibody titer level. This proves the inventors' aforementioned viewpoint.

[0131] Furthermore, based on the lethal challenge experiment, the survival rate of almost all mice in the nanovaccine group was higher than that in the aluminum adjuvant group, regardless of whether they were immunized subcutaneously or via tail vein. The inventors proposed a new perspective: although there were some differences in antibody titers in the serum of mice in the nanovaccine groups after subcutaneous and tail vein immunization, the neutralizing capacity of the antibodies in the serum was higher than that in the aluminum adjuvant group. Therefore, the inventors used a lysis assay to examine the neutralizing capacity of the serum antibodies in each group. The results showed that, regardless of whether immunized subcutaneously or via tail vein, the ability of all nanovaccine groups to lyse bacteria was indeed higher than that of the aluminum adjuvant group and the negative control group (EsxB and PBS).

[0132] The scope of this invention is not limited to the content specifically shown and described above. Variations, modifications, and other implementations of the content described herein will be apparent to those skilled in the art without departing from the spirit and scope of this invention.

Claims

1. A Staphylococcus aureus vaccine, characterized in that, Contains: adjuvant nanoparticles, wherein the adjuvant nanoparticles are PLGA or PLGA-PEG copolymers; and Staphylococcus aureus antigen, wherein the Staphylococcus aureus antigen is covalently linked to the adjuvant nanoparticles. The adjuvant nanoparticles have a particle size in the range of 150 nm to 200 nm, and the particle size dispersibility (PDI) value of the adjuvant nanoparticles does not exceed 0.

06. The Staphylococcus aureus antigen is EsxA or EsxB, the full-length amino acid sequence of EsxA is shown in SEQ ID NO: 1, and the full-length amino acid sequence of EsxB is shown in SEQ ID NO:

2. Based on the total weight of the adjuvant nanoparticles and the Staphylococcus aureus antigen, the content of the Staphylococcus aureus antigen is 3-10%. The molecular weight of PEG in the PLGA-PEG copolymer is 4000-6000 Daltons. The adjuvant nanoparticles have a mechanical strength and Young's modulus between 800 Pa and 1 MPa.

2. The Staphylococcus aureus vaccine according to claim 1, characterized in that, The Staphylococcus aureus antigen is linked to the adjuvant nanoparticles by non-hydrolyzable covalent bonds.

3. The Staphylococcus aureus vaccine according to claim 2, characterized in that, The covalent bond includes at least one of an amide bond and a disulfide bond.

4. The Staphylococcus aureus vaccine according to claim 1, characterized in that, The Staphylococcus aureus vaccine is in the form of freeze-dried powder, aqueous solution, suspension, microemulsion or liposome.

5. The Staphylococcus aureus vaccine according to claim 1, characterized in that, The Staphylococcus aureus vaccine is suitable for intravenous or subcutaneous administration.

6. The Staphylococcus aureus vaccine according to claim 1, characterized in that, The Staphylococcus aureus vaccine is used for intravenous immunization, and the adjuvant nanoparticles are PLGA-PEG copolymers, wherein the PEG content in the PLGA-PEG copolymer does not exceed 15%.

7. The Staphylococcus aureus vaccine according to claim 1, characterized in that, The Staphylococcus aureus vaccine is used for subcutaneous immunization, and the adjuvant nanoparticles are PLGA-PEG copolymers, wherein the PEG content in the PLGA-PEG copolymer is 20-35%.

8. The Staphylococcus aureus vaccine according to claim 7, characterized in that, The PEG content in the PLGA-PEG copolymer is 25-30%.

9. The Staphylococcus aureus vaccine according to claim 1, characterized in that, The adjuvant nanoparticles carry surface-modifying groups on their surface.

10. The Staphylococcus aureus vaccine according to claim 9, characterized in that, The modifying group includes at least one of carboxyl, amino, mercapto, methoxy, and aldehyde groups.

11. The Staphylococcus aureus vaccine according to claim 1, characterized in that, In a bodily fluid environment, the degradation time of the adjuvant nanoparticles is 1 day to 1 month.

12. The Staphylococcus aureus vaccine according to claim 11, characterized in that, In a bodily fluid environment, the degradation time of the adjuvant nanoparticles is 1 day to 2 weeks.

13. A method for preparing the Staphylococcus aureus vaccine according to any one of claims 1-5 and 9-12, characterized in that, include: (1) Dissolve PLGA or PLGA-PEG copolymer in an organic solvent; (2) The mixture obtained in step (1) is added dropwise to a polyvinyl alcohol solution, followed by ultrasonic emulsification, room temperature reaction in water, and microporous membrane filtration in sequence to obtain adjuvant nanoparticles. (3) Covalently link the Staphylococcus aureus antigen to the adjuvant nanoparticles to obtain the Staphylococcus aureus vaccine.

14. The method according to claim 13, characterized in that, The solvent is a mixture of dichloromethane and acetone.

15. The method according to claim 13 or 14, characterized in that, Step (3) further includes: (3-1) The adjuvant nanoparticles were added to morpholine ethanesulfonic acid buffer, and N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added; (3-2) After centrifuging the mixture obtained in step (3-1) to obtain a precipitate, the precipitate is resuspended in a solution containing the Staphylococcus aureus antigen to obtain a resuspension. (3-3) Adjust the pH of the resuspension to 8 and incubate it overnight at 4 degrees Celsius to obtain crude vaccine; (3-4) The crude vaccine is subjected to ultrasonic dispersion treatment in order to obtain the Staphylococcus aureus vaccine.

16. The use of PLGA or PLGA-PEG copolymer as an adjuvant in vaccine preparation, characterized in that, The vaccine is a Staphylococcus aureus vaccine, comprising: adjuvant nanoparticles, wherein the adjuvant nanoparticles are PLGA or PLGA-PEG copolymers; and Staphylococcus aureus antigen, wherein the Staphylococcus aureus antigen is covalently linked to the adjuvant nanoparticles. The adjuvant nanoparticles have a particle size in the range of 150 nm to 200 nm, and the particle size dispersibility (PDI) value of the adjuvant nanoparticles does not exceed 0.

06. The Staphylococcus aureus antigen is EsxA or EsxB, the full-length amino acid sequence of EsxA is shown in SEQ ID NO: 1, and the full-length amino acid sequence of EsxB is shown in SEQ ID NO:

2. Based on the total weight of the adjuvant nanoparticles and the Staphylococcus aureus antigen, the content of the Staphylococcus aureus antigen is 3-10%. The molecular weight of PEG in the PLGA-PEG copolymer is 4000-6000 Daltons. The adjuvant nanoparticles have a mechanical strength and Young's modulus between 800 Pa and 1 MPa.

17. The use according to claim 16, characterized in that, The vaccine is intended for intravenous or subcutaneous administration.

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