WO2024040363A1 - Staphylococcus aureus vaccine and preparation method therefor, use of plga-peg copolymer in vaccine preparation - Google Patents

Abstract

The present invention relates to a Staphylococcus aureus vaccine and a preparation method therefor, and a use of a PLGA-PEG copolymer in vaccine preparation. According to embodiments of the present invention, the vaccine contains: an adjuvant nanoparticle, the adjuvant nanoparticle comprising PLGA or a PLGA-PEG copolymer; and a Staphylococcus aureus antigen, the Staphylococcus aureus antigen being covalently linked to the adjuvant nanoparticle.

Description

Staphylococcus aureus vaccine and preparation method thereof, and use of PLGA-PEG copolymer in vaccine preparation Technical field

The present invention relates to the field of biomedicine, specifically to Staphylococcus aureus vaccine and its preparation method, and the use of PLGA-PEG copolymer in preparing the vaccine.

Background technique

Staphylococcus aureus (S.aureus), also known as "Staphylococcus aureus", is a common food-borne pathogenic microorganism. Staphylococcus aureus often parasitizes the skin, nasal cavity, throat, intestines, stomach, carbuncles, and purulent sores of humans and animals. It is also ubiquitous in the air, sewage and other environments. Staphylococcus aureus often causes opportunistic infections, leading to different diseases. Severe purulent inflammation spreads diseases, such as boils, carbuncles, otitis media, sinusitis, osteomyelitis and sepsis.

Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA), is multidrug-resistant and has become a difficulty in clinical treatment. The rate of antibiotic research and development cannot keep up with the development of bacterial resistance, so safe and effective treatments need to be found.

Methicillin-resistant Staphylococcus aureus (MRSA) is a unique strain of Staphylococcus aureus that is resistant to almost all penicillin antibiotics, including methicillin and other anti-β-lactams Enzyme of Penicillin. MRSA was first discovered in the United Kingdom in 1961. It has now spread widely and is known as a "superbug."

For the following reasons, vaccines have become an effective means to prevent Staphylococcus aureus (especially MRSA) infections: 1. The use of vaccines is not affected by the existing clinical bacterial resistance mechanisms; 2. The use of vaccines can greatly reduce bacterial infections. , thereby reducing the use of antibiotics and breaking the vicious cycle of "antibiotic use-drug resistance-antibiotic abuse-extensive drug resistance"; 3. The vaccine is very specific and only targets specific pathogenic bacteria and will not affect normal human bacteria. It has an impact on the flora and overcomes the side effects of antibiotic use leading to dysbiosis.

However, there is currently no superbug vaccine developed and marketed internationally. The main reason is that the use of traditional vaccine theory and technology cannot screen out a sufficient amount of protective target components. At the same time, although traditional adjuvants can promote T cell immune responses ability to prolong the residence time of antigens in the body and enhance humoral immunity; but at the same time, there are 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.

Therefore, existing S. aureus vaccines still need further improvement.

Contents of the invention

The present invention is completed based on the following findings of the inventor:

During the in-depth research on Staphylococcus aureus vaccine, the inventor of the present invention found that by using adjuvant nanoparticles to load antigens, the adjuvant nanoparticles are small, have a large specific surface area, strong adsorption capacity and adjuvant activity, so they can improve It can improve the body's immune response ability, reduce side effects, and has an ideal immune enhancement effect. In addition, by using nanoparticles (NP) as antigen carriers/adjuvants, humoral immunity and cellular immunity can be enhanced at the same time, and the antigen dosage can be reduced. During in-depth research on adjuvant nanoparticles, the inventor unexpectedly discovered that the mechanical properties of adjuvant nanoparticles, such as Young's modulus, are important determinants of immune cell activation, and found that the mechanical properties of adjuvant nanoparticles can be adjusted. Humoral and cellular immune responses. Therefore, the inventors screened and verified the activity of a series of adjuvant nanoparticles, and proposed adjuvant nanoparticles suitable for different immune pathways, such as intravenous immunization or subcutaneous immunization. , the resulting vaccine can produce more antibodies and effectively neutralize the toxicity of Staphylococcus aureus.

In view of this, the present invention proposes a Staphylococcus aureus vaccine that can effectively neutralize the toxicity of Staphylococcus aureus and a preparation method thereof.

In a first aspect, the present invention proposes a Staphylococcus aureus vaccine. According to an embodiment of the present invention, the vaccine contains: adjuvant nanoparticles, the adjuvant nanoparticles contain PLGA or PLGA-PEG copolymer; and gold A Staphylococcus aureus antigen covalently linked to the adjuvant nanoparticle.

The inventor of the present invention unexpectedly discovered through arduous research that choosing PLGA or PLGA-PEG copolymer as an adjuvant can effectively obtain adjuvant nanoparticles with desired mechanical properties, thereby being able to adapt to different immune scenarios, such as intravenous immunization. Or suitable for subcutaneous immunization. In addition, by covalently linking adjuvant nanoparticles to Staphylococcus aureus, the activity of the vaccine in activating immune cells can be further improved and more neutralizing antibodies can be produced to effectively neutralize the toxicity of Staphylococcus aureus. In addition, the inventor found that based on the new adjuvant nanoparticles proposed by the present invention, the loading capacity of vaccine antigens will be further improved, thereby improving the immune efficiency of the vaccine. In addition, using the new adjuvant nanoparticles, the uptake of vaccine antigens will be improved. The amount can be further increased, thereby improving the immune efficiency of the vaccine antigen per unit amount. Therefore, according to the embodiments of the present application, compared to traditional aluminum vaccines, the vaccine of the present application has at least one of the following advantages: low side effects, long-lasting antibody maintenance levels, high antibody titer levels, and the antibodies produced have neutralizing active. In addition, the adjuvant nanoparticles according to the embodiments of the present application have good biocompatibility and have self-degradation properties.

The adjuvant nanoparticles according to the embodiments of the present application can be degraded in the body. For example, according to the embodiments of the present application, under the conditions of body temperature (37 degrees Celsius) and in a body fluid environment, the degradation time of the adjuvant nanoparticles is 1 day to 1 day. Month, such as 1 day to 2 weeks. Therefore, vaccines according to embodiments of the present invention have higher biocompatibility and higher safety, and can be used as an effective means of regulating immune effects by adjusting the content of PEG.

In the second aspect, this application proposes a method for preparing 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 the solvent It is a mixture of methylene chloride and acetone; (2) add the mixture obtained in step (1) dropwise to the polyvinyl alcohol solution, and then perform ultrasonic emulsification, room temperature reaction in water, and microporous membrane filtration in sequence. to obtain adjuvant nanoparticles; and (3) covalently link Staphylococcus aureus antigen to the adjuvant nanoparticles to obtain the Staphylococcus aureus vaccine.

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 desired mechanical properties can be effectively obtained. This allows it to adapt to different immunization scenarios, such as intravenous immunization or subcutaneous immunization. In addition, by covalently linking the adjuvant nanoparticles to Staphylococcus aureus, the activity of the vaccine in activating immune cells can be further improved and more antibodies can be produced to effectively neutralize the toxicity of Staphylococcus aureus.

According to the embodiment of the present application, step (3) further includes: (3-1) adding the adjuvant nanoparticles to the morpholinoethanesulfonic acid buffer, and adding N-hydroxysuccinimide and 1- Ethyl-(3-dimethylaminopropyl)carbodiimide; (3-2) Centrifuge the mixture obtained in step (3-1), and use a solution containing the Staphylococcus aureus antigen to The pellet is resuspended to obtain a resuspension; (3-3) adjust the pH of the resuspension to 8 and incubate at 4 degrees Celsius overnight to obtain a crude vaccine product; and (3-4) The crude vaccine product is subjected to ultrasonic dispersion treatment to obtain the Staphylococcus aureus vaccine. Thus, the efficiency of preparing the above-mentioned vaccine can be further improved and the production cost can be reduced.

In the third aspect, this application also proposes the use of PLGA or PLGA-PEG copolymer as an adjuvant in preparing vaccines for intravenous immunization or subcutaneous immunization. As mentioned above, the inventor of the present application can effectively obtain adjuvant nanoparticles with desired mechanical properties by selecting PLGA or PLGA-PEG copolymer as an adjuvant, thereby being able to adapt to different immune scenarios, such as intravenous immunization or Suitable for subcutaneous immunization. In fact, the PLGA or PLGA-PEG copolymer can be used as an adjuvant in a variety of vaccines. In addition, the inventor found that based on the new adjuvant nanoparticles proposed by the present invention, the loading capacity of vaccine antigens will be further improved, thereby improving the immune efficiency of the vaccine. In addition, using the new adjuvant nanoparticles, the uptake of vaccine antigens will be improved. The amount can be further increased, thereby improving the immune efficiency of the vaccine antigen per unit amount. Therefore, according to the embodiments of the present application, compared to traditional aluminum vaccines, the vaccine of the present application has at least one of the following advantages: low side effects, long-lasting antibody maintenance levels, high antibody titer levels, and the antibodies produced have neutralizing active.

The adjuvant nanoparticles according to the embodiments of the present application can be degraded in the body. For example, according to the embodiments of the present application, under the conditions of body temperature (37 degrees Celsius) and in a body fluid environment, the degradation time of the adjuvant nanoparticles is 1 day to 1 day. Month, such as 1 day to 2 weeks. Therefore, vaccines using adjuvant nanoparticles according to embodiments of the present invention have higher biocompatibility and higher safety.

According to an embodiment of the present application, the vaccine is a Staphylococcus aureus vaccine. According to an embodiment of the present application, the vaccine is used against methicillin-resistant Staphylococcus aureus. Thus, new types of Staphylococcus aureus that can effectively resist Staphylococcus aureus, especially drug-resistant Staphylococcus aureus, can be provided, providing a new and effective solution for preventing or treating Staphylococcus aureus infection.

Therefore, according to the fourth aspect of the present application, the present application also proposes a method for treating or preventing Staphylococcus aureus-related diseases, which includes: administering the aforementioned Staphylococcus aureus vaccine to a subject.

According to an embodiment of the present application, the Staphylococcus aureus-related disease is resistance to methicillin-resistant Staphylococcus aureus-related disease.

Description of drawings

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:

Figure 1 shows the use of AFM to characterize a series of PLGA-PEG Schematic results of Young's modulus of %NPs, PLGA-PEG 25% NPs, PLGA-PEG 33% NPs).

Figure 2 shows the antibody titer detection results according to one embodiment of the present application.

Figure 3 shows the antibody titer detection results according to another embodiment of the present application.

Figure 4 shows the secretion amounts of IL-4 and IFN-γ in mouse spleen according to an embodiment of the present application.

Detailed ways

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

definition

In this article, unless otherwise stated, the term "Staphylococcus aureus vaccine" should be understood broadly to include both preventive and therapeutic vaccines.

In this article, unless otherwise specified, the term "Staphylococcus aureus antigen" refers to a molecular entity that is capable of inducing an immune response specifically against Staphylococcus aureus in the host body. According to the embodiments of the present application, The antigen used mainly includes at least part of the surface protein of Staphylococcus aureus. According to the embodiments of the present application, the Staphylococcus aureus antigen can be obtained through in vitro synthesis, for example, based on the amino acid sequence of the corresponding protein antigen, expressed in a microorganism or animal expression system and then purified, for example, by using the IPTG induction method. .

As used herein, the term "PLGA" refers to poly(lactide/glycolide) unless otherwise stated.

As used herein, the term "PEG" refers to polyethylene glycol unless otherwise stated.

In this article, unless otherwise specified, the term "PLGA-PEG copolymer" refers to a polymer containing PEG units 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 in different immune scenarios. Among them, monomethoxy PEG can be used as the macromolecule initiator, and through the development of D, L-lactide and glycolide The desired PLGA-PEG copolymer is obtained through the cyclopolymerization reaction, and the proportion of the corresponding reactants is controlled to obtain a series of PLGA-PEG copolymers that meet the needs. In addition, those skilled in the art can also purchase PLGA-PEG copolymers with different PEG contents.

In this article, unless otherwise stated, the term "nanoparticles" refers to a dispersion of particles with a particle size in the range of 10 to 1000 nanometers.

In this article, unless otherwise specified, the term "content of PEG in the PLGA-PEG copolymer" refers to the weight percentage of PEG in the copolymer, that is, the ratio of the molecular weight of PEG to the total molecular weight of the PLGA-PEG copolymer. According to the embodiments of the present application, the molecular weight of PEG is fixed within a predetermined molecular weight range, such as 5000 Da, and by adjusting the amount of PLGA, a series of PLGA-PEG copolymers with different PEG contents can be obtained.

In this article, unless otherwise specified, the term "at least a part of at least one of CsalA, EsxA, Hla and EsxB" refers to the full length or a part thereof of the proteins Csa1A, EsxA, Hla and EsxB from Staphylococcus aureus. , such as part of the extracellular segment as an antigen. The term "at least part" here is not particularly limited in length, as long as it can induce an immune response in the host.

In this article, unless otherwise specified, the term "Staphylococcus aureus antigen content" is used to characterize the antigen loading of the Staphylococcus aureus vaccine, which refers to the antigen loading based on the adjuvant nanoparticles and the Staphylococcus aureus The total weight of Kang antigen and the weight percentage of Staphylococcus aureus antigen. Of course, those skilled in the art can understand that other methods can also be used to characterize the antigen load of the Staphylococcus aureus vaccine, such as measuring the cross-linking rate of PLGA-PEG copolymer and EsxB through the BCA method.

The present invention is completed based on the following findings of the inventor:

During the in-depth research on Staphylococcus aureus vaccine, the inventor of the present invention found that by using adjuvant nanoparticles to load antigens, the adjuvant nanoparticles are small, have a large specific surface area, strong adsorption capacity and adjuvant activity, so they can improve It can improve the body's immune response ability, reduce side effects, and has an ideal immune enhancement effect. In addition, by using nanoparticles (NP) as antigen carriers/adjuvants, humoral immunity and cellular immunity can be enhanced at the same time, and the antigen dosage can be reduced. During in-depth research on adjuvant nanoparticles, the inventor unexpectedly discovered that the mechanical properties of adjuvant nanoparticles, such as Young's modulus, are important determinants of immune cell activation, and found that the mechanical properties of adjuvant nanoparticles can be adjusted. Humoral and cellular immune responses. Therefore, the inventors screened and verified the activity of a series of adjuvant nanoparticles, and proposed adjuvant nanoparticles suitable for different immune pathways, such as intravenous immunization or subcutaneous immunization. , the resulting vaccine can produce more antibodies and effectively neutralize the toxicity of Staphylococcus aureus.

In view of this, the present invention proposes a Staphylococcus aureus vaccine that can effectively neutralize the toxicity of Staphylococcus aureus and a preparation method thereof.

In a first aspect, the present invention proposes a Staphylococcus aureus vaccine. According to an embodiment of the present invention, the vaccine contains: adjuvant nanoparticles, the adjuvant nanoparticles contain PLGA or PLGA-PEG copolymer; and gold A Staphylococcus aureus antigen linked to the adjuvant nanoparticle.

The inventor of the present invention unexpectedly discovered through arduous research that choosing PLGA or PLGA-PEG copolymer as an adjuvant can effectively obtain adjuvant nanoparticles with desired mechanical properties, thereby being able to adapt to different immune scenarios, such as intravenous immunization. Or suitable for subcutaneous immunization. In addition, by covalently linking the adjuvant nanoparticles to Staphylococcus aureus, the activity of the vaccine in activating immune cells can be further improved and more antibodies can be produced to effectively neutralize the toxicity of Staphylococcus aureus. In addition, the inventor found that based on the new adjuvant nanoparticles proposed by the present invention, the loading capacity of vaccine antigens will be further improved, thereby improving the immune efficiency of the vaccine. In addition, using the new adjuvant nanoparticles, the uptake of vaccine antigens will be improved. The amount can be further increased, thereby improving the immune efficiency of the vaccine antigen per unit amount. Therefore, according to the embodiments of the present application, compared to traditional aluminum vaccines, the vaccine of the present application has at least one of the following advantages: low side effects, long-lasting antibody maintenance levels, high antibody titer levels, and the antibodies produced have neutralizing active.

In addition, according to embodiments of the present application, the adjuvant nanoparticles have good biocompatibility and have self-degradation characteristics. Specifically, according to the embodiments of the present application, under the conditions of body temperature (about 37 degrees Celsius) and in the body fluid environment, the degradation time of the adjuvant nanoparticles is 1 day to 1 month, for example, 1 day to 2 weeks. Among them, according to the embodiments of the present application, the inventor of the present invention found 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 higher the PEG content in the PLGA-PEG copolymer. Then the degradation time of the adjuvant nanoparticles is shorter, and the degradation time of the adjuvant nanoparticles can be 1 day to 1 month, for example, 1 day to 2 weeks.

According to the embodiments of the present application, the inventor found that the self-degradation characteristics of adjuvant nanoparticles, in addition to improving the biocompatibility of the material, are also related to immunomodulatory properties. According to the embodiments of this application, the inventor unexpectedly discovered that in the subcutaneous immunization route, the antigen uptake rate is related to the softness and hardness. Nano-vaccines with high PEG content (PEG content greater than 20%) are more likely to be phagocytosed by dendritic cells, causing stronger immune response, producing more protective antibodies. The inventors also found that, unlike the subcutaneous immunization route, in the intravenous immunization route, the correlation between the antigen uptake rate and the softness and hardness is relatively low, but the antigen uptake rate has a high correlation with the self-degradation performance of the nanovaccine. Specifically, for nanovaccines with slow self-degradation speed and low PEG content (PEG content less than 20%), the binding time of antigen and adjuvant nanoparticles is longer, and the uptake rate by immune organs is higher, thus inducing the body's immune response. Stronger, producing more protective antibodies.

According to the embodiment of the present invention, the inventor of the present invention uses PLGA-PEG copolymer to construct an adjuvant system to develop a Staphylococcus aureus nanovaccine system. Both PLGA and PEG are FDA-approved biomaterials with good biocompatibility, and adjusting the ratio and synthesis process of the copolymer can accurately control the physical and chemical parameters of different nanoparticles, including different sizes, morphologies, degradation characteristics, Softness and hardness, etc. According to the embodiments of the present application, the softness and hardness of nanoparticles can be adjusted. Using the softness and hardness of dendritic cell membranes as a reference, nanoparticles with gradient hardness are screened as research objects, and advantageous nanoadjuvants are screened out. The nanoparticles and antigens were combined in a covalent manner, the ratio of antigens to nanoparticles was cross-verified, and the composition of the nanovaccine was determined based on the maximum antigen binding rate as a reference indicator.

According to the embodiments of the present application, the way in which the Staphylococcus aureus antigen and the adjuvant nanoparticles are connected is not particularly limited, and they can be connected through physical effects or chemical bonds. According to the embodiments of the present application, Staphylococcus aureus antigen and adjuvant nanoparticles can be connected through electrostatic adsorption, covalent binding, hydrophobic interaction, ligand exchange, amide bond, disulfide bond, linker, and pyrophosphate diester bond. At least one of them is connected. According to an embodiment of the present invention, the Staphylococcus aureus antigen and the adjuvant nanoparticle are connected through a non-hydrolyzable covalent bond. The inventors of the present invention discovered that, unlike the delivery mechanism of therapeutic drugs, connecting Staphylococcus aureus antigens and adjuvant nanoparticles through non-hydrolyzable covalent bonds can improve the activity of the vaccine in activating immune cells. In addition, according to an embodiment of the present invention, the covalent bond includes at least one of an amide bond and a disulfide bond. The inventor found that since the three-dimensional structure of the antigen protein is relatively easily affected, the inventor found that the reaction conditions for forming amide bonds or disulfide bonds will not damage or negatively affect the three-dimensional structure of the antigen protein, thereby maintaining the antigen. Immunogenicity of proteins.

According to embodiments of the present invention, the form of the Staphylococcus aureus vaccine is not particularly limited. It can be in the form of freeze-dried powder, aqueous solution, suspension, microemulsion, dispersion, or liposome. Among them, frozen Dry powder. The inventor of the present invention found that the vaccine according to the embodiments of the present application is suitable for preparation into the form of a lyophilized powder, so that it has a longer storage period, and an injectable solution can be prepared by simply mixing with a solution such as PBS. At the same time, the performance of activating immune cells is not affected.

According to the embodiments of the present application, the route of administering the vaccine is not particularly limited, and it can be used for intravenous administration, subcutaneous administration, intramuscular administration, parenteral administration, rectal administration, spinal administration, epidermal administration. At least one of medicine, infusion administration, intraperitoneal administration, and intra-lymph node injection. In addition, the inventor also conducted a series of experiments to analyze the impact of PEG content on the immune effects of various administration routes. It is thus proposed that, according to an embodiment of the present invention, the Staphylococcus aureus vaccine is used for intravenous immunization, the adjuvant nanoparticles contain PLGA or PLGA-PEG copolymer, and the PEG content in the PLGA-PEG copolymer is No more than 15%. According to an embodiment of the present invention, the Staphylococcus aureus vaccine is used for subcutaneous immunization, the adjuvant nanoparticles contain PLGA-PEG copolymer, and the PEG content in the PLGA-PEG copolymer is 20 to 35%, preferably 25~30%. The inventors have demonstrated through a series of experiments that for different immune scenarios, the mechanical properties (mechanical properties) of the adjuvant nanoparticles (mechanical properties), such as Young's modulus, can be adjusted by changing the PEG content to achieve optimal immune effects. According to an embodiment of the present application, the particle size of the adjuvant nanoparticles is in the range of 150 nm to 200 nm, and the particle size dispersion PDI value of the adjuvant nanoparticles does not exceed 0.06. Optionally, the adjuvant nanoparticles The mechanical strength Young's modulus is between 800Pa and 1MPa. Optionally, the surface of the adjuvant nanoparticles carries surface modification groups. Optionally, the modification groups include carboxyl groups, amino groups, thiol groups, and methoxy groups. At least one of a group and an aldehyde group. Thus, the immune performance of Staphylococcus aureus vaccine can be further improved.

According to an embodiment of the present invention, the Staphylococcus aureus antigen includes at least a part of at least one selected from CsalA, EsxA, Hla and EsxB. Thus, the host's immune system can be effectively stimulated to produce effective antigens to resist Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus MRSA. According to specific embodiments of the present invention, EsxB can be used as the MRSA-specific antigen, mainly based on two points: EsxB protein exists in most Staphylococcus aureus substrains, and EsxB will promote the secretion of IgG; in addition, EsxB has no effect on Staphylococcus aureus. Coccal infection is preventive.

According to an embodiment 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 to 10%, preferably 4 to 6%. Thus, the host's immune system can be effectively stimulated to produce effective antigens to resist Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus MRSA.

According to an embodiment of the present invention, the molecular weight of PEG in the PLGA-PEG copolymer is 4000 to 6000 Daltons, preferably 5000 Daltons. Thus, the host's immune system can be effectively stimulated to produce effective antigens to resist Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus MRSA.

According to embodiments of the present invention, compared with existing aluminum adjuvant vaccines, at the same antigen dose, the survival rate of animals infected with Staphylococcus aureus is higher. It effectively avoids the current situation of untreatable super bacterial infections caused by antibiotic resistance.

In the second aspect, this application proposes a method for preparing 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 the solvent It is a mixture of methylene chloride and acetone; (2) add the mixture obtained in step (1) dropwise to the polyvinyl alcohol solution, and then perform ultrasonic emulsification, room temperature reaction in water, and microporous membrane filtration in sequence. to obtain adjuvant nanoparticles; and (3) covalently link Staphylococcus aureus antigen to the adjuvant nanoparticles to obtain the Staphylococcus aureus vaccine.

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 desired mechanical properties can be effectively obtained. This allows it to adapt to different immunization scenarios, such as intravenous immunization or subcutaneous immunization. In addition, by covalently linking the adjuvant nanoparticles to Staphylococcus aureus, the activity of the vaccine in activating immune cells can be further improved and more antibodies can be produced to effectively neutralize the toxicity of Staphylococcus aureus.

According to the embodiment of the present application, step (3) further includes: (3-1) adding the adjuvant nanoparticles to the morpholinoethanesulfonic acid buffer, and adding N-hydroxysuccinimide and 1- Ethyl-(3-dimethylaminopropyl)carbodiimide; (3-2) Centrifuge the mixture obtained in step (3-1), and use a solution containing the Staphylococcus aureus antigen to The pellet is resuspended to obtain a resuspension; (3-3) adjust the pH of the resuspension to 8 and incubate at 4 degrees Celsius overnight to obtain a crude vaccine product; and (3-4) The crude vaccine product is subjected to ultrasonic dispersion treatment to obtain the Staphylococcus aureus vaccine. Thus, the efficiency of preparing the above-mentioned vaccine can be further improved and the production cost can be reduced.

In a third aspect, this application also proposes the use of PLGA or PLGA-PEG copolymer as an adjuvant in the preparation of vaccines, which are used for intravenous immunization or subcutaneous immunization. As mentioned above, the inventor of the present application can effectively obtain adjuvant nanoparticles with desired mechanical properties by selecting PLGA or PLGA-PEG copolymer as an adjuvant, thereby being able to adapt to different immune scenarios, such as intravenous immunization or Suitable for subcutaneous immunization. In fact, the PLGA or PLGA-PEG copolymer can be used as an adjuvant in a variety of vaccines.

According to an embodiment of the present application, the vaccine is a Staphylococcus aureus vaccine. According to an embodiment of the present application, the vaccine is used against methicillin-resistant Staphylococcus aureus. This can provide new types of Staphylococcus aureus that can effectively resist Staphylococcus aureus, especially drug-resistant Staphylococcus aureus, and provide a new effective solution for preventing or treating Staphylococcus aureus infection, effectively preventing Staphylococcus aureus, especially drug-resistant Staphylococcus aureus. In the case of Oxicillin-resistant Staphylococcus aureus infection, the mortality rate of infection with drug-resistant Staphylococcus aureus is greatly reduced after nanovaccination.

In addition, the inventor found that based on the new adjuvant nanoparticles proposed by the present invention, the loading capacity of vaccine antigens will be further improved, thereby improving the immune efficiency of the vaccine. In addition, using the new adjuvant nanoparticles, the uptake of vaccine antigens will be improved. The amount can be further increased, thereby improving the immune efficiency of the vaccine antigen per unit amount. Therefore, according to the embodiments of the present application, compared to traditional aluminum vaccines, the vaccine of the present application has at least one of the following advantages: low side effects, long-lasting antibody maintenance levels, high antibody titer levels, and the antibodies produced have neutralizing active.

The adjuvant nanoparticles according to the embodiments of the present application can be degraded in the body. For example, according to the embodiments of the present application, under the conditions of body temperature (37 degrees Celsius) and in a body fluid environment, the degradation time of the adjuvant nanoparticles is 1 day to 1 day. Month, such as 1 day to 2 weeks. Therefore, vaccines according to embodiments of the present invention have higher biocompatibility and higher safety, and can be used as an effective means of regulating immune effects by adjusting the content of PEG.

Therefore, according to the fourth aspect of the present application, the present application also proposes a method for treating or preventing Staphylococcus aureus-related diseases, which includes: administering the aforementioned Staphylococcus aureus vaccine to a subject.

According to an embodiment of the present application, the Staphylococcus aureus-related disease is resistance to methicillin-resistant Staphylococcus aureus-related disease.

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present invention.

General method

Unless otherwise specified, in the following examples, EsxB-loaded PLGA-PEG copolymer adjuvant nanoparticles were prepared by using the following method.

Raw materials (and intermediates): PLGA or PLGA-X% PEG copolymer (commercially available, X represents the weight percentage of PEG in the copolymer), EsxB, N-hydroxysuccinimide, 1-ethyl- (3-Dimethylaminopropyl)carbodiimide, morpholinoethanesulfonic acid buffer, polyvinyl alcohol, methylene chloride, acetone, sodium bicarbonate.

Adjuvant construction: Dissolve chain PLGA-X% PEG copolymer in a mixture of methylene chloride and acetone, add the resulting mixture dropwise into the polyvinyl alcohol solution, and perform ultrasonic emulsification (ultrasonic power 20W, pulse type Ultrasonic, with an interval of 2s, ultrasonic time 4min), then drop into deionized water, and react at room temperature. The obtained emulsion is passed through a microporous filter membrane and centrifuged to obtain the crude nano-adjuvant product. The crude product was centrifuged and washed with water to obtain the finished product of the nanoadjuvant (also called PLGA-PEG X% nanoparticles (NPs) in the following examples).

Vaccine development: The recombinant protein antigen EsxB of Staphylococcus aureus is selected and bound to the surface of the nano-adjuvant through an amide bond. First, dissolve the finished nanoadjuvant obtained previously in morpholinoethanesulfonic acid buffer, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide , activate at room temperature and centrifuge to obtain the precipitate, resuspend with EsxB solution, adjust the pH value to about 8 with sodium bicarbonate, incubate at 4 degrees Celsius overnight, and centrifuge to obtain the crude nanovaccine. After centrifugation and water washing, the finished nano vaccine is obtained.

Example 1 Construction and physical and chemical characterization of nano-adjuvant system

In this example, according to the general method, a series of PLGA-PEG And its Young's modulus was characterized by atomic force microscopy (AFM). Schematic results are shown in Figure 1. Among them, when 25, 33) Nanoparticles with different mechanical properties are used as adjuvants to construct nanovaccine systems.

In addition, the inventor also characterized the hydrated particle size and surface potential of the nanoparticles through dynamic light scattering methods. The hydrated particle sizes of the five types of nanoparticles used (PLGA-PEG ; As the PEG content continues to increase, the surface potential of the nanoparticles changes from negative to slightly positive. As a result, the particle size range of the obtained adjuvant nanoparticles allows the vaccine to easily enter the lymph nodes through lymphatic vessels, thereby effectively inducing the body's immune response.

Example 2 Expression and purification of EsxB and EsxA

In this example, the inventor synthesized EsxB recombinant antigen through IPTG method (the amino acid sequence used is from Staphylococcus aureus strain ATCC25923 as shown in SEQ ID NO: 2) and EsxA (the amino acid sequence used is from Staphylococcus aureus strain ATCC25923) Strain ATCC25923 (shown as SEQ ID NO: 1) recombinant antigen and purified it with a nickel column. SDS-PAGE & Western Blot verified the purified product. The results showed that the synthesized EsxB recombinant antigen and EsxA recombinant antigen were consistent with reports in the literature.

in,

The full-length amino acid sequence of EsxA is:

The full-length amino acid sequence of EsxB is:

Briefly, the specific steps for IPTG to induce EsxB or EsxA expression are as follows:

Take the competent E. coli cells and place them in an ice bath until they thaw. Add 1ng of the plasmid carrying the EsxB encoding nucleic acid sequence to the competent cell suspension, gently blow evenly, and let stand in the ice bath for 30 minutes; place the centrifuge tube in Place it in a 42°C water bath for 90 seconds, then quickly transfer it to an ice bath to cool for 2 to 3 minutes. Do not shake the centrifuge tube during this process; add 3 mL of sterile LB culture medium to the centrifuge tube, mix and place it on a 37°C shaker for culture 45 minutes; mix the contents of the centrifuge tube, add 100 microliters of transformed competent cells to the LB solid agar medium containing ampicillin sodium, and use a sterile elbow glass rod to gently distribute the cell suspension evenly. Spread until all the liquid is absorbed, invert the plate, and incubate at 37°C for 12 to 16 hours; select colonies with bright and smooth surfaces on the solid medium and place them in LB medium containing ampicillin sodium, and culture on a shaking table at 37°C until OD ₆₀₀ is 0.6 ~ 0.8; add IPTG to induce the bacterial liquid, so that the final concentration of IPTG is 2mM/mol, induce for 4hrs at 37°C; centrifuge at 3000rpm for 10min, discard the precipitate; ultrasonically disrupt the supernatant, centrifuge at 3000rpm for 10min, repeat the above steps until centrifugation No precipitation occurred; the condensed protein solution was purified through a nickel column, dialyzed and stored at -20°C for later use.

Example 3 Effect of the combination of adjuvant nanoparticles and antigen on the immune effect

In this example, the inventor used EsxA as a model antigen, connected PLGA NPs (PEG-free PLGA nanoparticles) and EsxA in different cross-linking methods and immunized mice, and measured the IgG in the serum of different groups of immunized mice. , IgG1, IgG2a antibody titer levels, as well as the secretion of IFN-γ and IL-17 in the spleens of immunized mice. The schematic results are shown in Figure 2 (FREE in Figure 2 represents free EsxA; PLGA represents EsxA and PLGA NPs through Physically combined; AHG indicates that the aluminum adjuvant adsorbs EsxA; SURF indicates that EsxA and PLGA NPs are combined through covalent bonds; ENCAP indicates that EsxA is wrapped in PLGA NPs). As can be seen in Figure 2, when PLGA NPs and EsxA are cross-linked through covalent bonds, the levels of antibody titers in the serum are higher than those of several other cross-linking methods, and higher than the positive control (aluminum adjuvant) group. When PLGA NPs and EsxA were cross-linked by covalent bonds, the secretion amount of IFN-γ was higher than that of other cross-linking methods, and higher than that of the aluminum adjuvant control group. When PLGA NPs and EsxA were cross-linked through covalent bonds, the secretion amount of IL-17 was higher than that of other cross-linking methods, and was comparable to the secretion amount of the aluminum adjuvant group.

In addition, the inventor used DLS to characterize the hydrated particle size and surface potential of the product that covalently cross-linked PLGA-PEG X% NPs (X=0, 14, 20, 25, 33) and EsxA. It was found that the hydrated particle sizes of the five nanovaccine systems were all around 200nm, which was about 30nm larger than the nanoparticles, and there was no statistical difference in the average particle sizes of the five nanovaccine systems; the surface potentials of the five nanovaccine systems were all weak. of electronegative properties.

In addition, the inventors characterized the formation of amide bonds through infrared spectroscopy on products that covalently cross-linked PLGA NPs (X=0, 14, 20, 25, 33) and Esx B, and found that the pure EsxB antigen In the infrared spectrum ^, there is a double peak (primary amine) at ~3500cm ^-1 , while in the infrared spectrum of PLGA-PEG The primary amine becomes a secondary amine in the complex, that is, PLGA-PEG X%NPs can be covalently bound to EsxB through an amide bond.

Example 4 Determining the cross-linking rate of EsxB

In order to further determine the dosage of mice in the subsequent examples, in this example, the cross-linking rate of PLGA-PEG X% NPs and EsxB was measured by the BCA method. Briefly, the specific steps are as follows:

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

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

Dilute the BSA standard: In the microplate, directly dilute the BSA standard with a dilution consistent with the protein sample to be tested according to the table below.

In the sample wells of the microplate, add 2 μl of the protein sample to be tested, and label each sample to perform 3 parallel reactions.

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

Use Excel or other software to draw a standard curve and calculate the protein concentration in the sample.

Calculate the cross-linking ratio using the following formula:

Cross-linking rate = (moles of EsxB on nanoparticles)/(moles of nanoparticles)*100%

The results show that as the PEG content continues to increase, the cross-linking rate of PLGA-PEG X% NPs and EsxB continues to increase. Furthermore, it was determined that the dose for subsequent mice was 25 micrograms of antigen per mouse.

Example 5 Investigation of the safety of nano vaccine system

In this example, the inventor's CCK8 method verified the cytotoxicity of PLGA-PEG

Adjust the L929 cell density to 5*10 ⁴ cells/mL, inoculate 100 microliters into each well of a 96-well plate, and incubate at 37°C for 24hrs;

Add 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 respectively. -PEG 20% NPs-EsxB, PLGA-PEG 25% NPs-EsxB, PLGA-PEG 33% NPs-EsxB, so that the final concentrations are: 1 mg/mL, 500 μg/mL, 200 μg/mL, 100 μg/ mL, 50 μg/mL, 10 μg/mL, 5 μg/mL, 1 μg/mL, 100ng/mL, 10ng/mL, incubate at 37°C for 24hrs;

Add 10 μl of CCK8 reagent to each well and incubate at 37°C for an appropriate time and then measure the absorbance value at 450 nm.

The results show that 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) cell viability was greater than 95%.

In addition, the inventor also used BALB/c mice to examine the biocompatibility of the nanovaccine system. In all mouse groups (including subcutaneous immunization and tail vein immunization) (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), no significant weight loss was found. (Follow the immunization process and immunization dosage described in Example 6 below). Furthermore, after the animals were sacrificed, the inventors performed H&E staining on the main organs of the mice. The results showed that no obvious lesions were observed in all organ samples regardless of whether the nanovaccine system was immunized through subcutaneous immunization or tail vein immunization.

Example 6 Investigation of the efficacy of nano vaccine system

In this example, BALB/c mice were immunized with EsxB and EsxA through the subcutaneous and tail vein routes respectively, 25 μg/mouse, 2 weeks/time, for a total of 3 times, and the immunized mice were measured by ELISA. The antibody titer level in serum is used to evaluate the adjuvant performance of the species nanosystem. Briefly, the specific steps (taking EsxB as an example) are as follows:

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

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;

Step 3: After gradient dilution of the sample to be tested in a 96-well plate, add the diluted sample to be tested into the ELISA plate in sequence, 100 μl/well, and incubate at 37°C for 1HR;

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

Step 5: Dilute the secondary antibody and add it to the ELISA plate at 100 μl/well, and incubate at 37°C for 45 minutes;

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

Step 7: Add 100 μl/well of chromogenic solution, develop color at room temperature for 30 minutes, and measure the absorbance at 405nm. The results are shown in Figure 3.

Next, on the 7th day after the third immunization, the ELISPOT method was used to measure the secretion of IL-4 and IFN-γ in the spleens of the immunized mice. Briefly, the specific steps are as follows:

Step 1: Add 100 μl of 70% ethanol to each well of the ELISPOT plate and prewet it for 2 minutes, then wash the plate 5 times with 200 μl/well of sterile water;

Step 2: Dilute the coating antibody to 15 μg/mL with sterile PBS, add the diluted coating antibody to the ELISPOT plate, 100 μl/well, and incubate at 4°C overnight;

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

Step 4: Add cell culture medium containing 10% FCS to the ELISPOT plate, 200 μl/well, incubate at room temperature for 30 minutes and then discard;

Step 5: Adjust the splenocyte suspension density to 2*10 ⁷ cells/mL, and incubate 100 μl/well at 37°C for 12-48hrs;

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

Step 7: Dilute the detection antibody to 1 μl/mL with sterile PBS containing 0.5% FCS, and incubate 100 μl/well at room temperature for 2 hrs;

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

Step 9: Dilute Streptavidin-ALP 1:1000 by volume with sterile PBS containing 0.5% FCS, and incubate 100 μl/well at room temperature for 1 hrs;

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

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

Step 12: Dry the ELISPOT plate at room temperature and count on the reader.

The results are shown in Figure 4. Fourteen days after the third immunization, a lethal dose of ATCC25923 substrain (6.8*10 ⁹ CFU/ml, 0.1ml/mouse) was administered to each immunized mouse through the tail vein, and the clinical signs and mortality of the mice were monitored daily. The survival rates of almost all nanovaccine system groups were higher than those of the positive control (aluminum adjuvant) and negative control (PBS and EsxB).

As shown in Figure 3, whether it is EsxB or EsxBA, in subcutaneous immunization, the antibody titer level of the nanovaccine group is positively correlated with the PEG content. The antibody titer levels are higher than that of the aluminum adjuvant, and the antibody duration is longer than that of the aluminum adjuvant. In the adjuvant group, the onset time is comparable to that in the aluminum adjuvant group. In the tail vein immunization method, the antibody titer level of the nanovaccine group was negatively correlated with the PEG content. The antibody titer levels were higher than those of the aluminum adjuvant group, and the antibody duration was longer than that of the aluminum adjuvant group, and the onset of effect was faster than that of the aluminum adjuvant group. Aluminum adjuvant group.

As shown in Figure 4, in the subcutaneous immunization method, the secretion amount of IL-4 in the nanovaccine group was positively correlated with the PEG content. In the tail vein immunization method, the secretion amount of IL-4 in the nanovaccine group was negatively correlated with the PEG content. Whether in subcutaneous immunization or tail vein immunization, the secretion amount of IFN-γ in the nanovaccine group was lower than that in the aluminum adjuvant control group. This phenomenon indicates that humoral immunity in the nanovaccine group of the present invention plays a dominant role.

Example 7 Research on the mechanism of action of the nanovaccine system of the present invention

In the subcutaneous immunization method, based on the phenomenon that the serum antibody titer level of immunized mice is positively correlated with the PEG content, the applicant proposed a new point of view: after subcutaneous immunization, the nano vaccine is not easy to spread at the injection site, so the local concentration is high, and Soft nanoadjuvants are more easily phagocytosed by dendritic cells (DCs) and can produce higher antibody levels. Therefore, in this example, the inventor verified the above point by examining the release speed of PLGA-PEG X% NPs and the speed of DC phagocytosis of PLGA-PEG X% NPs.

In the tail vein immunization method, based on the negative correlation between the serum antibody titer level of immunized mice and the PEG content, the applicant proposed a new point of view: after tail vein immunization, the nano vaccine enters the blood circulation, and its concentration is rapidly diluted, DC The amount of phagocytosis of each nanovaccine is equivalent. Theoretically, the antibody levels of each nanovaccine group are equivalent. However, the ELISA experimental results show that the hard nanovaccine produced a higher level of antibody titer, so it may be affected by the degradation of PLGA-PEG X% NPs themselves. Performance implications, the stiff nanovaccine group produced higher antibody levels. Therefore, in this example, the inventor verified the above point by examining the release speed of PLGA-PEG X% NPs, the speed of DC engulfing PLGA-PEG X% NPs and the distribution of PLGA-PEG X% NPs in the body.

Specifically, in this example, FITC is cross-linked with PLGA-PEG speed. Using 50% FCS in vitro to simulate the in vivo environment, it was found that the higher the PEG content, the faster the release rate of FITC. In addition, in this example, the inventor simulated the local environment of subcutaneous immunity and the in vivo environment of tail vein immune nanovaccine in vitro, and examined the phagocytosis of five nanovaccine systems by antigen-presenting cells through dendritic cells (DC). , the results found that in subcutaneous immunization, there was no difference in the phagocytosis of the five nanovaccine systems by DCs at 0.5 hr, and as time went on, the higher the PEG content, the higher the phagocytosis of the nanovaccines by DCs; in tail vein immunization, There was no difference in the phagocytosis of the five nanovaccine systems by DC. In addition, the inventor wrapped ICG in nano-adjuvant and injected it into BALB/c mice through the tail vein, and observed the in vivo distribution of ICG@PLGA-PEG X% NPs. The results showed that the main distribution of ICG@PLGA-PEG X% NPs was In the intestine of mice, and mesenteric lymph nodes are larger lymphoid tissues in mice, tail vein immunization results in higher antibody titer levels. This proves the inventor's foregoing point of view.

In addition, based on the lethal challenge experiment, regardless of subcutaneous immunization or tail vein immunization, the survival rate of mice in almost all nanovaccine groups was higher than that of the aluminum adjuvant group. The inventor proposed a new point of view: Although there are certain differences in the antibody titers in the serum of mice in each nanovaccine group in subcutaneous immunization and tail vein immunization, the neutralizing ability of the antibodies in the serum is higher than that in the aluminum adjuvant group. Therefore, the inventors used bacteriolysis experiments to examine the neutralizing ability of serum antibodies in each group. The results showed that whether it was subcutaneous immunization or tail vein immunization, 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).

The scope of the invention is not limited by what is specifically shown and described above. Changes, modifications and other implementations of what is described herein will be apparent to those of ordinary skill in the art without departing from the spirit and scope of the invention.

Claims (25)

1. A Staphylococcus aureus vaccine, characterized in that it contains: adjuvant nanoparticles containing PLGA or PLGA-PEG copolymer; and Staphylococcus aureus antigen, which is identical to the Staphylococcus aureus antigen. The adjuvant nanoparticles are attached.
2. The Staphylococcus aureus vaccine according to claim 1, wherein the Staphylococcus aureus antigen and the adjuvant nanoparticles are connected through physical adsorption or chemical bonds.
3. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the Staphylococcus aureus antigen and the adjuvant nanoparticles are formed through electrostatic adsorption, covalent binding, hydrophobic interaction, and ligand exchange. At least one of an amide bond, a disulfide bond, a linker, and a pyrophosphate diester bond is connected.
4. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the Staphylococcus aureus antigen and the adjuvant nanoparticles are connected through a non-hydrolyzable covalent bond, preferably the covalent bond includes At least one of an amide bond and a disulfide bond.
5. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the Staphylococcus aureus vaccine is in the form of freeze-dried powder, aqueous solution, suspension, microemulsion, dispersion, or liposome.
6. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the Staphylococcus aureus vaccine is suitable for intravenous administration, subcutaneous administration, intramuscular administration, parenteral administration, and rectal administration. , at least one of spinal administration, epidermal administration, infusion administration, intraperitoneal administration, and intralymph node injection.
7. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the Staphylococcus aureus vaccine is used for intravenous immunization, the adjuvant nanoparticles contain PLGA or PLGA-PEG copolymer, and the PLGA - The PEG content in the PEG copolymer does not exceed 15%.
8. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the Staphylococcus aureus vaccine is used for subcutaneous immunization, the adjuvant nanoparticles contain PLGA-PEG copolymer, and the PLGA-PEG The content of PEG in the copolymer is 20 to 35%, preferably 25 to 30%.
9. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the particle size of the adjuvant nanoparticles is in the range of 150 nm to 200 nm, and the particle size dispersion PDI value of the adjuvant nanoparticles is not More than 0.06.
10. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the mechanical strength Young's modulus of the adjuvant nanoparticles is between 800 Pa and 1 MPa.
11. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the surface of the adjuvant nanoparticle carries a surface modification group, optionally, the modification group includes a carboxyl group, an amino group, a thiol group, At least one of methoxy group and aldehyde group.
12. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the degradation time of the adjuvant nanoparticles in a body fluid environment is 1 day to 1 month.
13. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the degradation time of the adjuvant nanoparticles in a body fluid environment is 1 day to 2 weeks.
14. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the Staphylococcus aureus antigen comprises at least a portion of at least one selected from the group consisting of CsalA, EsxA, Hla and EsxB.
15. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the content of the Staphylococcus aureus antigen is based on the total weight of the adjuvant nanoparticles and the Staphylococcus aureus antigen. 3 to 10%, preferably 4 to 6%.
16. The Staphylococcus aureus vaccine according to any one of the preceding claims, wherein the molecular weight of PEG in the PLGA-PEG copolymer is 4000-6000 Daltons, preferably 5000 Daltons.
17. A method for preparing the Staphylococcus aureus vaccine according to any one of claims 1 to 16, characterized by comprising:

    (1) Dissolve PLGA or PLGA-PEG copolymer in an organic solvent;

    (2) Add the mixture obtained in step (1) dropwise to the polyvinyl alcohol solution, and then sequentially perform ultrasonic emulsification, room temperature reaction in water, and microporous membrane filtration to obtain adjuvant nanoparticles;

    (3) Covalently link Staphylococcus aureus antigen to the adjuvant nanoparticles to obtain the Staphylococcus aureus vaccine.
18. The method according to claim 17, wherein the solvent is a mixture of dichloromethane and acetone.
19. The method according to claim 17 or 18, characterized in that step (3) further includes:

    (3-1) Add the adjuvant nanoparticles to the morpholinoethanesulfonic acid buffer, and add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbon Diimide;

    (3-2) Centrifuge the mixture obtained in step (3-1), and resuspend the precipitate with a solution containing the Staphylococcus aureus antigen to obtain a resuspension;

    (3-3) Adjust the pH of the resuspension to 8 and incubate it at 4 degrees Celsius overnight to obtain crude vaccine;

    (3-4) Perform ultrasonic dispersion treatment on the crude vaccine product to obtain the Staphylococcus aureus vaccine.
20. The use of PLGA or PLGA-PEG copolymer as an adjuvant in the preparation of vaccines.
21. The use according to claim 20, characterized in that the vaccine is used for intravenous administration, subcutaneous administration, intramuscular administration, parenteral administration, rectal administration, spinal administration, epidermal administration, and infusion. At least one of administration, intraperitoneal administration, and intralymph node injection, optionally, the vaccine is used for intravenous immunization or subcutaneous immunization.
22. The use according to claim 20, characterized in that the vaccine is Staphylococcus aureus vaccine.
23. The use according to claim 22, wherein the vaccine is used against methicillin-resistant Staphylococcus aureus.
24. A method for treating or preventing Staphylococcus aureus-related diseases, comprising: administering the Staphylococcus aureus vaccine according to any one of claims 1 to 16 to a subject.
25. The method of claim 24, wherein the Staphylococcus aureus-related disease is resistance to methicillin-resistant Staphylococcus aureus-related disease.

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