WO2022199317A1 - 一种金黄色葡萄球菌疫苗的工业化生产方法 - Google Patents

一种金黄色葡萄球菌疫苗的工业化生产方法 Download PDF

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WO2022199317A1
WO2022199317A1 PCT/CN2022/077827 CN2022077827W WO2022199317A1 WO 2022199317 A1 WO2022199317 A1 WO 2022199317A1 CN 2022077827 W CN2022077827 W CN 2022077827W WO 2022199317 A1 WO2022199317 A1 WO 2022199317A1
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staphylococcus aureus
bacterial
vaccine
production method
concentration
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PCT/CN2022/077827
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French (fr)
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王震玲
魏于全
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成都威斯克生物医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/44—Staphylococcus
    • C12R2001/445—Staphylococcus aureus

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to an industrialized production method of a Staphylococcus aureus vaccine.
  • Staphylococcus aureus is an important opportunistic pathogen. In the adult population, approximately 20% carry S. aureus persistently and another 30% carry it intermittently. Staphylococcus aureus can cause skin and soft tissue infections, life-threatening pneumonia, bacteremia, and serious complications including endocarditis, septic arthritis, and osteomyelitis. The exotoxin of Staphylococcus aureus can also cause food poisoning, epidermal relaxation syndrome and toxic shock syndrome.
  • MRSA Metal-resistant S. aureus
  • Staphylococcus aureus vaccines include whole-bacteria inactivated vaccines, genetically engineered vaccines, subunit vaccines, DNA vaccines, etc.
  • the existing preparation method of Staphylococcus aureus vaccine includes: 1) extracting one or more components of Staphylococcus aureus as an antigen to prepare, mainly using prokaryotic expression of one or more antigens of Staphylococcus aureus, and using an adjuvant 2) Extract and purify the capsular polysaccharide of Staphylococcus aureus, and add expressed one or more antigenic proteins of Staphylococcus aureus, or other exogenous carrier proteins to improve its immunogenicity; 3) Expression And purify one or more exotoxins secreted by Staphylococcus aureus as antigen, and combine with carrier protein to enhance its immunogenicity; 4) Insert one or more protein antigenic determinant coding sequences of Staphylococcus aureus into plasmid, construct Staphylococcus
  • the immunogenicity of the vaccine prepared by the above-mentioned methods is not as good as that of the whole cell vaccine, and does not cover most of the toxic proteins, conservative antigens, protective antigens, capsular polysaccharides, etc., so there are problems such as insufficient coverage and narrow application range.
  • the whole-cell inactivated vaccine can overcome these problems and stimulate the body to produce a large amount of immunoglobulin. Therefore, it is urgent to develop a Staphylococcus aureus vaccine with improved immunity and coverage and a wider range of applications, which can supplement and make up for the deficiencies of the existing technology.
  • the purpose of the present invention is to provide an industrialized production method of a Staphylococcus aureus vaccine, and the application of the vaccine.
  • An industrialized production method for a Staphylococcus aureus vaccine comprising the following steps:
  • S3 monitors the cell density in the fermenter, and when the cells in the fermenter reach the logarithmic growth phase, the bacterial liquid in the fermenter is directly centrifuged according to the centrifugal force of 2000-4000 ⁇ g for 10-30 min Then collect the bacteria;
  • S4 resuspend the thalline with isotonic injection and adjust the concentration, and then irradiate the thalline to lose the proliferation activity, and the rays irradiated by the rays include X-rays;
  • S5 The concentration of the irradiated bacterial solution is adjusted to 0.5-1 ⁇ 10 8 cells/ml with the isotonic injection to obtain the Staphylococcus aureus vaccine.
  • S1 includes the following steps:
  • b. Inoculate the first-class seeds into the TSB medium to expand the culture step by step, and the expansion times are not less than 2 times.
  • the bacterial concentration of the inoculation during each expansion is 0.01-0.1OD/ml, and the volume of the inoculation does not exceed the culture. At 10% of the volume, the final concentration of each grade of seed solution cultured was 0.8 ⁇ 0.2 OD/ml.
  • the specific operation steps of the S1 are: a. inoculating the Staphylococcus aureus strain on a TSA plate for cultivation to obtain first-class seeds; b. inoculating the first-class seeds into a TSB medium to adjust the bacterial concentration be 0.01-0.1OD/ml, then expand the culture to the logarithmic growth phase to obtain secondary seed liquid; c. Inoculate the secondary seed liquid into fresh TSB medium, adjust the bacterial concentration to 0.01-0.1OD/ml ml, continue to expand the culture to the logarithmic growth phase, and obtain the tertiary seed liquid.
  • the volume of each step of scale-up can be adjusted according to the final process scale-up requirements (for example, theoretically from 100ml to 1000ml, from 1000ml to 10L).
  • 2 additional amplifications ie, secondary seed liquid, tertiary seed liquid
  • the reason is that through 2 amplifications, The bacteria can be fully activated, the growth rate is faster, and the culture time is shortened; too many amplification times will increase the risk of contamination; and only one amplification will result in longer culture time, or insufficient bacterial production (extension). sex is not enough).
  • Staphylococcus aureus strains include one or more of ATCC25923, ATCC33591, SCPH-18 and SCPH-25.
  • the bacterial concentration of the seed solution inoculated in S2 is 0.01-0.1 OD/ml.
  • the fermenter can be a fermenter containing 1L-20L of fermentation broth; the actual fermentation volume is generally 1/3-1/2 of the volume of the fermenter. As a preference, a 10L fermenter is selected for fermentation.
  • the fermentation parameters are: aeration volume 3-5L/min, rotating speed 200-300rpm, temperature 35-39°C, online monitoring pH value and dissolved oxygen value, culturing cells to logarithmic growth phase (1.5 ⁇ 0.3OD/ml) .
  • the bacterial cells are resuspended with an isotonic injection and the concentration is adjusted to 0.5-1 ⁇ 10 10 CFU/ml, and the isotonic injection includes solutions such as physiological saline.
  • the dose rate of the radiation irradiation is about 5-20 Gy/min, and the total dose is about 2000-3000 Gy.
  • the bacterial liquid after irradiation with rays includes whole bacterial cells, nucleic acids, bacterial fragments and membrane vesicles.
  • the Staphylococcus aureus vaccine prepared by any one of the above-mentioned industrialized production methods.
  • the vaccine is a Staphylococcus aureus whole cell vaccine.
  • the vaccine also contains an adjuvant.
  • the adjuvant includes one or more of aluminum adjuvant, MF59, AS01, AS04, CpG and ISA51.
  • the Staphylococcus aureus vaccine of the present invention can be prepared in a type without adjuvant, or in a type with adjuvant added as required.
  • the dosage form of the vaccine is one or more of a subcutaneous injection preparation, an intramuscular injection preparation, an oral preparation and a nasal inhalation preparation.
  • the extracellular nucleic acid of the Staphylococcus aureus vaccine prepared by the present invention is increased by about 20%; , the Staphylococcus aureus vaccine prepared by the present invention after being stored at 2-8 DEG C for 4 weeks can increase the extracellular nucleic acid by 5-15 times.
  • Staphylococcus aureus vaccine described in any one of the above in the preparation of a medicine for preventing or treating bacteremia caused by Staphylococcus aureus.
  • the immunization procedure of the Staphylococcus aureus vaccine includes: subcutaneous inoculation, 3 injections of inoculation, and an interval of 2 weeks between each injection.
  • the whole Staphylococcus aureus cells contained in the Staphylococcus aureus vaccine are 1 ⁇ 10 7 cells/shot to 2 ⁇ 10 7 cells/shot.
  • Staphylococcus aureus vaccine described in any one of the above in the preparation of a medicine for preventing or treating pneumonia caused by Staphylococcus aureus.
  • the immunization procedure of the Staphylococcus aureus vaccine includes: subcutaneous inoculation, 3 injections of inoculation, and an interval of 2 weeks between each injection.
  • the whole Staphylococcus aureus cells contained in the Staphylococcus aureus vaccine are 1 ⁇ 10 7 cells/shot to 2 ⁇ 10 7 cells/shot.
  • the present invention has the following beneficial effects:
  • the present invention adopts X-rays to inactivate, and X-rays will not cause obvious damage to the S. aureus cell structure (that is, while X-rays inactivate S. aureus, the cell structure (antigen) is maintained. Integrity of Staphylococcus aureus), ensuring that the whole S. aureus cell becomes a more effective immune antigen; in addition, X-rays can also induce an increase in the release of S. aureus nucleic acid (ie, the level of S. aureus extracellular nucleic acid increases), improving the immunogen sex. In addition, the induction effect of X-rays on the release of extracellular nucleic acids from S.
  • the irradiated S. aureus can continue to release nucleic acids as time goes on.
  • X-rays also help to induce an increase in the release of S. aureus membrane vesicles, further improving the immunogenicity of the vaccine.
  • the irradiation method used in the present invention is intermittent irradiation, that is, a dose rate of 5-20 Gy/min, long-term, intermittent irradiation (preferably at an interval of 5-10 min) is used, which reduces the total dose of ray irradiation ( ⁇ 3000Gy ), thereby avoiding the destruction of bacteria by large doses of radiation, and improving the immune efficacy and safety of the vaccine.
  • the present invention provides a method for industrially producing a Staphylococcus aureus vaccine, so as to industrially produce a vaccine containing stable and controllable quality of Staphylococcus aureus whole cells and other immunogenic components.
  • the method of the present invention ensures the retention of immunogenic substances (such as bacterial cells, bacterial fragments, membrane vesicles and nucleic acids) and the removal of harmful substances (such as exotoxins), improving the vaccine efficacy and safety.
  • immunogenic substances such as bacterial cells, bacterial fragments, membrane vesicles and nucleic acids
  • harmful substances such as exotoxins
  • bacterial fragments are obtained by operations such as centrifugation.
  • the vaccine prepared by the present invention has good immunogenicity, not only the actual inoculation amount is low, but also can prevent a variety of infectious diseases caused by drug-resistant Staphylococcus aureus, such as Staphylococcus aureus bacteremia, gold Staphylococcal pneumonia, etc.
  • the vaccine prepared by the present invention can usually be free of adjuvant (that is, without adjuvant to enhance the immune response of the body), of course in some scenarios (for example, it is necessary to prepare a highly immunogenic Staphylococcus aureus vaccine) Adjuvants can also be used.
  • the above-mentioned good immunogenicity (protective efficacy of the vaccine) is achieved not only by the above-mentioned production method, but also by the actual immunization procedure of the vaccine: while reducing the total number of vaccinations, the immunization interval is extended.
  • the present invention finds that this immunization procedure is beneficial to improve the actual efficacy of the vaccine, and prolonging the immunization interval is more in line with the immune response law of the body to the vaccine.
  • the technical solution of the present invention uses X-rays for inactivation. Compared with traditional formaldehyde inactivation and thermal inactivation, the nucleic acid release level is increased and there is no chemical substance residue, which improves the immunogenicity of the vaccine. Allergic reactions and carcinogenic risks caused by chemical inactivators are avoided to a certain extent, the safety of vaccines is improved and side effects are reduced.
  • Figure 1 is a graph of irradiation dose and bacterial survival rate
  • Fig. 2 is the nucleic acid release situation graph under different inactivation modes
  • Figure 3 is a scanning electron microscope (SEM) image and a transmission electron microscope (TEM) image of the bacteria after inactivation;
  • Figure 4 is a graph showing the protective power of Staphylococcus aureus vaccine in a bacteremia model
  • Figure 5 is a graph of the protective power of Staphylococcus aureus vaccine in a pneumonia model.
  • the term "about” is typically expressed as +/- 5% of the stated value, more typically +/- 4% of the stated value, and more typically + /-3%, more typically +/-2% of said value, even more typically +/-1% of said value, even more typically +/-0.5% of said value.
  • TSA Tryptic Soy Agar
  • glycerol strains from -80°C ultra-low temperature refrigerator, streak inoculate on TSA plate, and cultivate at 37 ⁇ 1°C for 16 ⁇ 1h.
  • the strain used was ATCC25923.
  • the fermentation parameters were set as: aeration volume 3-5L/min, rotation speed 250 ⁇ 20rpm, temperature 37 ⁇ 1°C for cultivation, and online pH and dissolved oxygen monitoring were carried out to cultivate to 1.5 ⁇ 0.3OD/ml (logarithmic growth phase).
  • the bacterial solution was placed in a centrifuge bucket, centrifuged at 3000 ⁇ g for 20 min at room temperature, resuspended in 20 ml of sodium chloride injection (0.9%), centrifuged and washed once, and resuspended in 20 ml of sodium chloride injection (0.9%).
  • Dispense the bacterial liquid into a sealable container such as a 50ml centrifuge tube
  • the height of the liquid level should not exceed 1cm.
  • the bacterial liquid can be completely inactivated; from the perspective that the bacterial liquid is completely inactivated and the total dose should not be too large (too large will cause the whole cell to rupture and release cell toxins), the inactivated
  • the total dose was determined to be 2000-3000 Gy.
  • the dose rate is appropriate to set to 5-20Gy/min: when the dose rate is too low (ie ⁇ 5Gy/min), the total irradiation time (>400min) and the total production time will be prolonged (>8h), in addition to the impact of In addition to the production efficiency, the bacterial liquid at ambient temperature and in the production environment for a long time also has the risk of degradation and exposure to pollution; when the dose rate is too high (ie > 20 Gy/min), the total irradiation time ( ⁇ 100 min) is too short, and its Sufficient total irradiation time ( ⁇ 2h) cannot be guaranteed and will still negatively affect the degree of inactivation.
  • the detailed parameters of inactivation are affected by many factors such as the performance of the irradiation equipment, the shape of the bacterial solution container, the height of the liquid level, and the concentration of the bacterial solution. Adjust according to actual production needs.
  • the interval time of each irradiation is 5-10 min.
  • X-rays are used to inactivate Staphylococcus aureus.
  • X-rays have a unique bactericidal mechanism that induces DNA damage and inactivates bacteria.
  • the inactivation dose of Staphylococcus aureus ATCC25923 by X-ray is ⁇ 1950Gy, but considering that the bacterial solution needs to be absolutely completely inactivated in actual production, the inactivation dose can be set to ⁇ 2000Gy, Even the inactivation dose was delayed by one dose point to 2100Gy.
  • Half inactivation dose according to the X-ray inactivation procedure, the total dose is 1050Gy;
  • Inactivation dose according to the X-ray inactivation procedure, the total dose is 2100Gy;
  • Formaldehyde inactivation add formaldehyde solution to a final concentration of 1%, inactivate at 37 ⁇ 1°C for 24 hours, and wash with sodium chloride injection (0.9%) for 3 to 5 times after the inactivation is completed;
  • nucleic acid release assay After all samples are inactivated, immediately sample (0 week) and centrifuge, take the supernatant, and measure the nucleic acid concentration (A260) by UV spectrophotometer. Sampling and measurement were performed again after 2 weeks and 4 weeks.
  • Results as shown in Figure 2.
  • X-ray inactivation induced an increase in the level of extracellular nucleic acid in S. aureus, and this process of nucleic acid release continued over time.
  • Nucleic acid release is one of the important features that distinguish X-ray inactivation from formaldehyde inactivation and heat inactivation, which may bring more immunogenicity to vaccines and activate more immune signaling pathways, such as STING, TLR9, etc.
  • Activation of the STING pathway is conducive to promoting the recognition of bacterial infection by the immune system, promoting the production of type I interferon (enhancing cellular immunity), facilitating the presentation of vaccine antigen components and the recognition of the immune system in the immune stage, and is conducive to bacterial infection in the infection stage. of clearing.
  • the TLR9 pathway is the main receptor in the immune system that recognizes bacterial CpG DNA and induces the production of a series of pro-inflammatory cytokines and chemokines, ultimately causing a Th1-like inflammatory response.
  • Bacterial vaccines are mainly based on humoral immunity, and cellular immunity is weak. Both STING and TLR9 pathways activated by bacterial nucleic acid are beneficial to enhance Th1-type cellular immunity and improve the immune effect.
  • Sample preparation According to the preparation process of Staphylococcus aureus vaccine, after inactivating Staphylococcus aureus, take the original solution for scanning electron microscope sample preparation.
  • Fixation Take 200 ⁇ l of the inactivated vaccine stock solution, centrifuge at 3000 ⁇ g for 10 min, discard the supernatant, and add 1 ml of 2%-3% glutaraldehyde to fix at 4°C overnight.
  • Adhesion and coating paste the sample on the metal sample stage with a special double-sided tape, and coat the sample with a layer of gold film by ion sputtering.
  • Sample preparation According to the preparation process of Staphylococcus aureus vaccine, after inactivating Staphylococcus aureus, take the stock solution for transmission electron microscope sample preparation.
  • Pre-fixation Take 200 ⁇ l of the inactivated vaccine stock solution, centrifuge at 3000 ⁇ g for 10 min, discard the supernatant, and add 1 ml of 2%-3% glutaraldehyde to fix overnight at 4°C.
  • Negative staining drop 1 drop of 1% phosphotungstic acid for 1-2 minutes, filter paper to absorb the dyeing solution, drop 1 drop of pure water, absorb the filter paper, repeat two or three times, wash off excess phosphotungstic acid, and let stand dry.
  • Test grouping In this test, 4 strains of Staphylococcus aureus were selected to challenge the vaccine immune group respectively: including Methicillin Sensitive Staphylococcus aureus (MSSA) ATCC25923, Methicillin-resistant Staphylococcus aureus Coccus (Methicillin-resistant Staphylococcus aureus, MRSA) ATCC33591, and two clinically isolated multi-drug resistant (Multi-drug resistant, MDR) Staphylococcus aureus SCPH-18 and SCPH-25.
  • MSSA Methicillin Sensitive Staphylococcus aureus
  • MRSA Methicillin-resistant Staphylococcus aureus Coccus
  • MRSA Metal-resistant Staphylococcus aureus, MRSA
  • MDR multi-drug resistant Staphylococcus aureus SCPH-18 and SCPH-25.
  • the control group Unimmunized
  • the immune group Immunized
  • mice were injected with 0.1 ml/mice of bacterial solution (0.5-1 ⁇ 10 8 CFU/mice).
  • mice in the control group all died within 72-120 hours after each challenge strain was challenged, and the immune protection rates of the immunized group against each challenge strain challenged mice were 100% (ATCC25923) and 60%, respectively. (ATCC33591), 70% (SCPH-18), 80% (SCPH-25), that is, the protection rate of Staphylococcus aureus vaccine against Staphylococcus aureus bacteremia is 60% and above.
  • Test grouping In this test, 4 strains of Staphylococcus aureus were selected to challenge the vaccine immune group respectively: including Methicillin Sensitive Staphylococcus aureus (MSSA) ATCC25923, Methicillin-resistant Staphylococcus aureus Coccus (Methicillin-resistant Staphylococcus aureus, MRSA) ATCC33591, and two clinically isolated multi-drug resistant (Multi-drugresistant, MDR) Staphylococcus aureus SCPH-18 and SCPH-25.
  • MSSA Methicillin Sensitive Staphylococcus aureus
  • MRSA Methicillin-resistant Staphylococcus aureus Coccus
  • MDR Multi-drug resistant Staphylococcus aureus
  • mice were injected into the airway with 0.05ml/mice (1 ⁇ 2 ⁇ 10 7 CFU/mice).

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Abstract

本发明属于生物医药领域，具体涉及一种金黄色葡萄球菌疫苗的工业化生产方法。

Description

一种金黄色葡萄球菌疫苗的工业化生产方法 技术领域
本发明属于生物医药领域，具体涉及一种金黄色葡萄球菌疫苗的工业化生产方法。
背景技术
金黄色葡萄球菌(staphylococcus aureus)是一种重要的条件致病菌。在成年群体中，大约有20％的人持续携带金葡菌，另有30％的人间歇性携带。金葡菌可引起皮肤和软组织感染，还可导致威胁生命的肺炎、菌血症及包括心内膜炎、脓毒性关节炎、骨髓炎等在内的严重并发症。金葡菌的外毒素还可以引起食物中毒、表皮松弛征和中毒性休克综合征。
感染金黄色葡萄球菌，通常可选用红霉素、青霉素、庆大霉素、万古霉素或先锋霉素VI治疗，但由于抗生素的滥用，出现多种耐受抗生素的新菌株，特别是耐甲氧西林菌株(Methicillin-resistant S.aureus,MRSA)的迅速蔓延使得单纯依靠抗生素治疗金黄色葡萄球菌引发的相关疾病变得越来越不可靠。MRSA导致的感染用抗生素疗法很难治愈且致死率高。因此，针对金葡菌疫苗和免疫治疗的研究现已广泛开展。
金黄色葡萄球菌疫苗包括全菌灭活疫苗、基因工程疫苗、亚单位疫苗、DNA疫苗等。现有金黄色葡萄球菌疫苗的制备方法包括：1) 提取金黄色葡萄球菌的一个或多个组份作为抗原来制备，主要采用原核表达金葡菌的一种或多种抗原，并利用佐剂吸附后制成疫苗；2)提取并纯化金葡菌的荚膜多糖，并加入表达的一种或多种金葡菌抗原蛋白，或者其它外源载体蛋白以提高其免疫原性；3)表达并纯化金葡菌分泌的一种或多种外毒素作为抗原，与载体蛋白结合以增强其免疫原性；4)将金葡菌的一种或多种蛋白抗原决定簇编码序列插入质粒，构建金葡菌DNA疫苗。以上列举的方法制备得到的疫苗的免疫原性均不如全菌体疫苗，未涵盖大部分毒性蛋白、保守抗原、保护性抗原、荚膜多糖等，因此存在覆盖性不够，适用范围窄等问题。而全菌体灭活疫苗可以克服这些问题，并刺激机体产生大量的免疫球蛋白。因此亟待研究一种免疫性和覆盖性提高，适用范围更广的金黄色葡萄球菌疫苗，可以补充和弥补现有技术的不足。
发明内容
有鉴于此，本发明的目的在于提供一种金黄色葡萄球菌疫苗的工业化生产方法，以及所述疫苗的应用。
一种金黄色葡萄球菌疫苗的工业化生产方法，包括以下步骤：
S1用适宜的培养基培养金黄色葡萄球菌菌株，制成种子液；
S2将所述种子液接种于发酵罐中进行发酵；
S3监测所述发酵罐内的菌体密度，待所述发酵罐内的菌体达到对数生长期，取所述发酵罐内的菌液直接按照离心力2000-4000×g进行离心，10～30min后收集菌体；
S4将所述菌体用等渗注射液重悬并调整浓度，然后进行射线辐照使所述菌体失去增殖活性，所述射线辐照的射线包括X射线；
S5将射线辐照后的菌液再用所述等渗注射液调整浓度至0.5-1×10 8个/ml，得到所述金黄色葡萄球菌疫苗。
进一步，S1包括以下步骤：
a.将所述金黄色葡萄球菌菌株接种到TSA平板培养，得到一级种子；
b.将所述一级种子接种到TSB培养基中逐级扩大培养，扩大次数不少于2次，每次扩大培养时接种的菌浓度为0.01-0.1OD/ml，接种的体积不超过培养体积的10％，培养的每级种子液的终浓度为0.8±0.2OD/ml。
进一步，所述S1的具体操作步骤为：a.将所述金黄色葡萄球菌菌株接种到TSA平板培养，得到一级种子；b.将所述一级种子接种到TSB培养基中，调整菌浓度为0.01-0.1OD/ml，然后扩大培养至对数生长期，得到二级种子液；c.再将所述二级种子液接种到新鲜TSB培养基中，调整菌浓度为0.01-0.1OD/ml，继续扩大培养至对数生长期，得到三级种子液。在实际操作中，可根据最终工艺放大要求调整各步放大的体积(例如，理论上从100ml到1000ml，从1000ml到10L)。从菌种(工作种子)复苏(一级种子)，到接种至发酵罐前，还需要进行2次扩增(即，二级种子液、三级种子液)，原因在于通过2次扩增，可使细菌得到充分活化，生长速度更快，培养时间缩短；扩增次数过多则会增加污染风险；而仅扩增1次则会导致所需培养时 间更长，或菌体产量不足(延展性不够)。
进一步，所述金黄色葡萄球菌菌株包括ATCC25923、ATCC33591、SCPH-18和SCPH-25中的一种或多种。
进一步，S2中接种的所述种子液的菌浓度为0.01-0.1OD/ml。所述发酵罐可以为含有1L-20L发酵液的发酵罐；实际的发酵体积一般为发酵罐体积的1/3-1/2。作为一种优选，选择10L的发酵罐进行发酵。
进一步，发酵参数为：通气量3-5L/min，转速200-300rpm，温度35-39℃，在线监测pH值和溶氧值，培养菌体至对数生长期(1.5±0.3OD/ml)。
进一步，S3离心完成后弃掉上清液以收集菌体。
进一步，S4中将所述菌体用等渗注射液重悬并调整浓度为0.5-1×10 10CFU/ml，所述等渗注射液包括生理盐水等溶液。
进一步，所述射线辐照的剂量率约为5-20Gy/min，总剂量约为2000-3000Gy。
进一步，所述射线辐照后的菌液包括全菌体、核酸、菌体碎片和膜囊泡。
上述任一项所述工业化生产方法制备得到的金黄色葡萄球菌疫苗。
进一步，所述疫苗为金黄色葡萄球菌全菌体疫苗。
进一步，所述疫苗还含有佐剂。
进一步，所述佐剂包括铝佐剂、MF59、AS01、AS04、CpG和 ISA51中的一种或多种。本发明所述的金黄色葡萄球菌疫苗可以制备成不含佐剂的类型，也可以根据需要制备成添加佐剂的类型。
进一步，所述疫苗的剂型为皮下注射制剂、肌肉注射制剂、口服制剂和鼻腔吸入制剂中的一种或多种。
进一步，与未经射线辐照的金黄色葡萄球菌制得的金黄色葡萄球菌疫苗相比，本发明制得的金黄色葡萄球菌疫苗的胞外核酸提高约20％；与辐照完成时相比，于2-8℃存放4周后的本发明制得的金黄色葡萄球菌疫苗，胞外核酸可提高5-15倍。
上述任一项所述的金黄色葡萄球菌疫苗在制备预防或治疗金黄色葡萄球菌引起的菌血症药物中的应用。
进一步，所述金黄色葡萄球菌疫苗的免疫程序包括：皮下接种，接种3针，每针间隔2周。
进一步，所述金黄色葡萄球菌疫苗含有的金黄色葡萄球菌全菌体为1×10 7个/针-2×10 7个/针。
上述任一项所述的金黄色葡萄球菌疫苗在制备预防或治疗金黄色葡萄球菌引起的肺炎药物中的应用。
进一步，所述金黄色葡萄球菌疫苗的免疫程序包括：皮下接种，接种3针，每针间隔2周。
进一步，所述金黄色葡萄球菌疫苗含有的金黄色葡萄球菌全菌体为1×10 7个/针-2×10 7个/针。
与现有技术相比，本发明具有以下有益效果：
(1)本发明采用X射线来进行灭活，X射线不会对金葡菌菌 体结构造成明显的破坏(即X射线对金葡菌进行灭活的同时，维持了菌体结构(抗原)的完整)，保证金葡菌全菌体成为更有效的免疫抗原；此外，X射线还可以诱导金葡菌核酸的释放增多(即，金葡菌胞外核酸水平上升)，提高了疫苗的免疫原性。而且X射线对金葡菌释放胞外核酸的这种诱导作用，即使停止X射线的辐照，随着时间的延长，被辐照的金葡菌仍可以持续核酸释放。除此之外，X射线还有助于诱导金葡菌膜囊泡的释放增多，进一步提高了疫苗的免疫原性。
(2)本发明采用的辐照方式为间断照射，即采用5-20Gy/min的剂量率、长时间、间断照射(优选为间隔5～10min)，降低了射线辐照的总剂量(≤3000Gy)，从而避免了大剂量射线对菌体的破坏，提高了疫苗的免疫效力和安全性。
(3)本发明提供了一种工业化生产金黄色葡萄球菌疫苗的方法，以工业化生产出一种质量稳定可控的含有金黄色葡萄球菌全菌体等多种免疫原性成分的疫苗。具体而言，本发明方法提高了工业化生产效率的同时，保证了免疫原性物质(例如菌体、菌体碎片、膜囊泡和核酸)的保留和有害物质(例如外毒素)的去除，提高了疫苗的有效性和安全性。其中，菌体碎片是通过离心等操作获得。
(4)本发明制得的疫苗具备良好的免疫原性，不仅实际接种量低，而且可以预防多种由耐药性金黄色葡萄球菌引起的感染性疾病，例如金葡菌菌血症、金葡菌肺炎等。且本发明制得的疫苗通常可以不含佐剂(即无需佐剂来增强机体的免疫应答)，当然在某些场景 下(例如，需要制得免疫原性极强的金黄色葡萄球菌疫苗)也可以搭配佐剂。上述良好的免疫原性(疫苗的保护效力)的实现不仅得益于如上所述的生产方法，还在于对疫苗的实际免疫程序：降低接种总次数的同时，延长免疫间隔。本发明发现，这种免疫程序有利于提高疫苗的实际效力，且延长免疫间隔更符合机体对疫苗的免疫应答规律。
(5)本发明技术方案采用X射线来进行灭活，与传统的甲醛灭活、热灭活相比，核酸释放水平增加且无化学物质残留，提高了疫苗的免疫原性的同时，在一定程度上避免了因化学灭活剂造成的过敏反应和致癌风险，提高了疫苗的安全性并降低了副作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍。显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。
图1为辐照剂量与细菌存活率图；
图2为不同灭活方式下的核酸释放情况图；
图3为灭活后菌体的扫描电镜(SEM)图和透射电镜(TEM)图；
图4为金葡菌疫苗在菌血症模型中的保护力图；
图5为金葡菌疫苗在肺炎模型中的保护力图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。
如在本说明书中使用的，术语“大约”，典型地表示为所述值的+/-5％，更典型的是所述值的+/-4％，更典型的是所述值的+/-3％，更典型的是所述值的+/-2％，甚至更典型的是所述值的+/-1％，甚至更典型的是所述值的+/-0.5％。
在本说明书中，某些实施方式可能以一种处于某个范围的格式公开。应该理解，这种“处于某个范围”的描述仅仅是为了方便和简洁，且不应该被解释为对所公开范围的僵化限制。因此，范围的描述 应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如，范围
Figure PCTCN2022077827-appb-000001
的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。
实施例一
金黄色葡萄球菌疫苗制备方法
1.培养基及试剂
胰蛋白胨大豆肉汤(Tryptic Soy Broth,TSB)
胰蛋白胨大豆琼脂(Tryptic Soy Agar,TSA)
氯化钠注射液(0.9％)
2.疫苗制备过程
1)一级种子制备
从-80℃超低温冰箱中取出甘油菌种，划线接种于TSA平板，于37±1℃培养16±1h。在本实施例中，使用的菌种为ATCC25923。
2)二级种子液制备
刮取适量菌体于10ml TSB中，用分光光度计测定菌液浓度，接种适当体积菌液至100ml TSB中，终浓度约为0.05OD/ml，于37±1℃220rpm振荡培养至0.8±0.2OD/ml(对数生长期)。
3)三级种子液制备
取二级种子液，用分光光度计测定菌液浓度，接种适当体积菌液至1000ml TSB中，终浓度约为0.05OD/ml，于37±1℃220rpm振 荡培养至0.8±0.2OD/ml(对数生长期)。
4)发酵罐培养
取三级种子液，用分光光度计测定菌液浓度，接种适当体积菌液至4L TSB中，终浓度约为0.05OD/ml。发酵参数设置为：通气量3～5L/min，转速250±20rpm，温度37±1℃进行培养，并进行在线pH、溶氧监测，培养至1.5±0.3OD/ml(对数生长期)。
5)菌体收获
将菌液装入离心桶，3000×g室温离心20min，20ml氯化钠注射液(0.9％)重悬菌体，离心洗涤1次，20ml氯化钠注射液(0.9％)重悬。
6)调整浓度
调整菌液浓度至0.5～1×10 10CFU/ml。
7)X射线灭活
将菌液分装于可密封的容器中(如50ml离心管)，液面高度不超过1cm。当射线总剂量为≥2000Gy时，菌液可以被完全灭活；从菌液完全灭活以及总剂量不宜过大(过大会致使全菌体破裂，释放菌体毒素)的角度，将灭活的总剂量确定为2000-3000Gy。将剂量率设定为5-20Gy/min是合适的：剂量率过低(即＜5Gy/min)时，总辐照时间(＞400min)和总生产时间会被延长(＞8h)，除影响生产效率外，长时间处于环境温度下和生产环境中的菌液还存在降解和暴露污染风险；剂量率过高(即＞20Gy/min)时，总辐照时间(＜100min)过短，其无法保证足够的总辐照时间(≥2h)，仍会对灭活程度造成 负面影响。灭活的详尽参数受辐照仪器设备性能、菌液装载容器形状、液面高度、菌液浓度等多因素影响，因此，剂量率、总剂量、辐照次数以及每次辐照的间隔时间可根据实际生产需要进行调整。作为优选，每次辐照的间隔时间为5～10min。
8)原液
辐照完成后，取1/100体积菌液，涂布于TSA板，37±1℃培养48h，确定无菌生长。同时取1/100体积菌液按《中国药典》(通则1101)进行无菌检查。
9)疫苗成品
用氯化钠注射液(0.9％)调整疫苗菌体浓度至0.5～1×10 8个/ml，即疫苗成品。成品于2～8℃保存。需要说明的是，对于活菌菌体浓度，以每毫升细菌克隆形成单位“CFU/ml”表示；对于灭活菌菌体浓度，以每毫升细菌个数“个/ml”表示。如本文所使用，“1CFU/ml”被视为与“1个/ml”所表示的菌体浓度等同。
实施例二
灭活剂量考察
在本实施例中，采用X射线对金葡菌种进行灭活。
方法：辐照前对制备好的菌液进行稀释涂板计数，每次辐照完成后，取样稀释涂板计数，每次取样取3份分别进行计数，计算每次辐照后细菌存活率。
Figure PCTCN2022077827-appb-000002
结果：X射线具有独特的杀菌机制，即诱导DNA损伤，对细 菌进行灭活。如图1可知，金黄色葡萄球菌ATCC25923采用X射线的灭活剂量为≥1950Gy，但考虑到实际生产中需保证细菌菌液绝对地完全被灭活，可以将灭活剂量设定为≥2000Gy，甚至将灭活剂量往后延一个剂量点至2100Gy。
实施例三
不同灭活方式对疫苗核酸释放水平影响考察
方法：
1.同一批次制备一批菌液，均分为5份，按下列操作处理。
2.活菌对照：不作任何处理，室温放置；
3.半灭活剂量：按照X射线灭活程序进行处理，总剂量1050Gy；
4.灭活剂量：按照X射线灭活程序进行处理，总剂量2100Gy；
5.甲醛灭活：用加入甲醛溶液至终浓度为1％，37±1℃灭活处理24h，灭活完成后用氯化钠注射液(0.9％)进行换液洗涤3～5次；
6.热灭活：采用121℃高压蒸汽灭菌15min；
7.核酸释放测定：所有样品灭活处理完成后，立即取样(0week)离心，取上清，采用紫外分光光度计测定核酸浓度(A260)。2周、4周后再次进行取样测定。
结果：如图2所示。X射线灭活后诱导了金葡菌胞外核酸水平的上升，随着时间的延长，这种核酸释放的过程仍在持续。核酸释放是X射线灭活区别于甲醛灭活和热灭活方式的重要特征之一，可能为疫苗带来更多的免疫原性，激活更多免疫信号通路，如STING、 TLR9等。
STING通路的激活，有利于促进免疫系统对细菌感染的识别，促进I型干扰素的产生(增强细胞免疫)，在免疫阶段利于疫苗抗原成分的呈递和免疫系统识别，在感染阶段有利于对细菌的清除。TLR9通路是免疫系统中识别细菌CpG DNA的主要受体从而诱导产生一系列促炎细胞因子和趋化因子，最终引起Th1样炎症反应。细菌疫苗主要以体液免疫为主，细胞免疫较薄弱，通过细菌核酸激活的STING和TLR9通路均有利于增强Th1型细胞免疫，促进免疫效果提高。
实施例四
X射线灭活后的菌体电镜观察
扫描电镜样品制备方法：
1.样品制备：按照金黄色葡萄球菌疫苗制备流程，将金黄色葡萄球菌灭活后，取原液进行扫描电镜样品制备。
2.固定：取200μl灭活后的疫苗原液，3000×g离心10min弃上清，加入1ml 2％-3％戊二醛4℃固定过夜。
3.洗涤：0.1M PBS洗涤3次。
4.脱水：之后依次用30％、50％、70％、80％、90％的乙醇梯度脱水各一次，100％的无水乙醇脱水3次，每次脱水仅可能将菌体轻轻吹散，每次处理10min，3000×g离心5min。
5.干燥：CO 2临界点干燥法，35℃干燥1h。
6.粘托和镀膜：用特制的双面胶将样品粘贴到金属样品台上，采用离子溅射法将样品镀上一层金膜。
7.扫描成像。
结果：如图3扫描电镜结果所示。X射线灭活并未对金葡菌菌体结构造成明显的破坏，即X射线对金葡菌灭活的同时维持了菌体结构(抗原)的完整，可能使之成为更有效的免疫抗原。
透射电镜样品制备方法：
1.样品制备：按照金黄色葡萄球菌疫苗制备流程，将金黄色葡萄球菌灭活后，取原液进行透射电镜样品制备。
2.前固定：取200μl灭活后的疫苗原液，3000×g离心10min弃上清，加入1ml 2％-3％戊二醛4℃固定过夜。
3.洗涤：0.1M PBS洗涤3次。
4.后固定：1％锇酸固定液固定2h。
5.洗涤：0.1M PBS洗涤3次。
t6.脱水：之后依次用30％、50％、70％、80％、90％的丙酮梯度脱水各一次，100％的纯丙酮脱水3次，每次脱水仅可能将菌体轻轻吹散，每次处理30min，3000×g离心5min。
7.渗透：纯丙酮+包埋液(1:2)室温过夜。
8.包埋：将渗透好的样品挑到包埋板中，37℃过夜，45℃12h，60℃48h。
9.超薄切片。
10.负染色：滴1滴1％的磷钨酸染色1～2min，滤纸吸去染色液，滴1滴纯水，滤纸吸去，反复两三次，洗去多于的磷钨酸，静置干燥。
11.透射电镜成像。
结果：如图3透射电镜结果所示。X射线对金葡菌灭活的同时维持了菌体结构(抗原)的完整。
实施例五
金葡疫苗在金葡菌菌血症(血行感染)模型中的保护力考察
方法：
1.试验分组：本试验选用了4株金黄色葡萄球菌分别对疫苗免疫组进行了挑战试验：包括甲氧西林敏感金葡菌(Methicillin Sensitive Staphylococcus aureus,MSSA)ATCC25923，耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)ATCC33591，以及两株临床分离多药耐药(Multi-drug resistant,MDR)金黄色葡萄球菌SCPH-18和SCPH-25。其中对照组(Unimmunized)和免疫组(Immunized)，每组各10只。
2.免疫：取疫苗成品(0.5～1×10 8个/ml)，免疫6～8周龄的C57BL/6小鼠，腹股沟皮下接种0.2ml(1～2×10 7个/针)，免疫3针，间隔2周。末次免疫2周后进行攻毒挑战。
3.血行感染模型建立
3.1将攻毒挑战菌株(ATCC25923、ATCC33591、SCPH-18、SCPH-25)复苏于血平板，37±1℃培养16±1h。
3.2挑取单克隆接种于3ml TSB中，37±1℃培养16±1h。
3.3用分光光度计测定菌液浓度，接种适当体积菌液至20ml TSB中，终浓度约为0.05OD/ml，于37±1℃220rpm振荡培养至 0.8±0.2OD/ml(对数生长期)。
3.4 3000×g室温离心10min，2ml氯化钠注射液(0.9％)重悬菌体，调整菌液浓度至0.5～1×10 9CFU/ml。
3.5小鼠尾静脉注射菌液0.1ml/只(0.5～1×10 8CFU/只)。
3.6观察统计免疫组和对照组小鼠1周内的生存率。
结果：如图4所示，对照组小鼠在各挑战菌株攻毒后72～120h内全部死亡，免疫组对各挑战菌株攻毒小鼠的免疫保护率分别是100％(ATCC25923)、60％(ATCC33591)、70％(SCPH-18)、80％(SCPH-25)，即金葡菌疫苗对金葡菌菌血症的保护率在60％及以上。
实施例六
金葡疫苗在金葡菌肺炎(气道感染)模型中的保护力考察
方法：
1.试验分组：本试验选用了4株金黄色葡萄球菌分别对疫苗免疫组进行了挑战试验：包括甲氧西林敏感金葡菌(Methicillin Sensitive Staphylococcus aureus,MSSA)ATCC25923，耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)ATCC33591，以及两株临床分离多药耐药(Multi-drugresistant,MDR)金黄色葡萄球菌SCPH-18和SCPH-25。其中对照组(Unimmunized)和免疫组(Immunized)，每组各35只，每时间点3～5只。
2.免疫：取疫苗成品(0.5～1×10 8个/ml)，免疫6～8周龄的C57BL/6小鼠，腹股沟皮下接种0.2ml(1～2×10 7个/针)，免疫3针，间隔2周。末次免疫2周后进行攻毒挑战。
3.肺炎(气道感染)模型建立
3.1将攻毒挑战菌株(ATCC25923、ATCC33591、SCPH-18、SCPH-25)复苏于血平板，37±1℃培养16±1h。
3.2挑取单克隆接种于3ml TSB中，37±1℃培养16±1h。
3.3用分光光度计测定菌液浓度，接种适当体积菌液至20ml TSB中，终浓度约为0.05OD/ml，于37±1℃220rpm振荡培养至0.8±0.2OD/ml(对数生长期)。
3.4 3000×g室温离心10min，2ml氯化钠注射液(0.9％)重悬菌体，调整菌液浓度至2～4×10 8CFU/ml。
3.5小鼠气道注射菌液0.05ml/只(1～2×10 7CFU/只)。
3.6观察统计免疫组和对照组小鼠肺部1周内每天的细菌荷载量。
结果：如图5所示，各挑战菌株攻毒后，对照组小鼠肺部细菌荷载量呈明显的增长趋势，并且对照组小鼠72～120h内全部死亡；免疫组对挑战菌株呈现了明显的清除趋势，甚至是完全清除。
上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。

Claims (10)

  1. 一种金黄色葡萄球菌疫苗的工业化生产方法，其特征在于，包括以下步骤：
    S1用适宜的培养基培养金黄色葡萄球菌菌株，制成种子液；
    S2将所述种子液接种于发酵罐中进行发酵；
    S3监测所述发酵罐内的菌体密度，待所述发酵罐内的菌体达到对数生长期，取所述发酵罐内的菌液直接按照离心力2000-4000×g进行离心，10～30min后收集菌体；
    S4将所述菌体用等渗注射液重悬并调整浓度，然后进行射线辐照使所述菌体失去增殖活性，所述射线辐照的射线包括X射线；
    S5将射线辐照后的菌液再用所述等渗注射液调整浓度至0.5-1×10 8个/ml，得到所述金黄色葡萄球菌疫苗。
  2. 如权利要求1所述的工业化生产方法，其特征在于，S1包括以下步骤：
    a.将所述金黄色葡萄球菌菌株接种到TSA平板培养，得到一级种子；
    b.将所述一级种子接种到TSB培养基中逐级扩大培养，扩大次数不少于2次，每次扩大培养时接种的菌浓度为0.01-0.1OD/ml,接种体积不超过培养体积的10％，培养的每级种子液的终浓度为0.8±0.2OD/ml。
  3. 如权利要求1所述的工业化生产方法，其特征在于，所述金黄色葡萄球菌菌株包括ATCC25923、ATCC33591、SCPH-18和SCPH-25中的一种或多种。
  4. 如权利要求1所述的生产方法，其特征在于，S2中接种的所述种子液的菌浓度为0.01-0.1OD/ml。
  5. 如权利要求1所述的工业化生产方法，其特征在于，所述射线辐照后的菌液包括全菌体、核酸、菌体碎片和膜囊泡。
  6. 如权利要求1所述的工业化生产方法，其特征在于，所述射线辐照的剂量率为5-20Gy/min，总剂量≥2000Gy。
  7. 权利要求1-6任一项所述工业化生产方法制备得到的金黄色葡萄球菌疫苗。
  8. 权利要求7所述的金黄色葡萄球菌疫苗在制备预防或治疗金黄色葡萄球菌引起的菌血症药物中的应用。
  9. 权利要求7所述的金黄色葡萄球菌疫苗在制备预防或治疗金黄色葡萄球菌引起的肺炎药物中的应用。
  10. 如权利要求9所述的应用，其特征在于，所述金黄色葡萄球菌疫苗的免疫程序包括：皮下接种，接种3针，每针间隔2周。
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