Industrial production method of pseudomonas aeruginosa vaccine

Abstract

The invention relates to an industrial production method of a pseudomonas aeruginosa vaccine, which adopts a series of standardized, programmed and digital settings to ensure that a vaccine which is stable in quality and contains pseudomonas aeruginosa whole thallus and various immunogenic components in the pseudomonas aeruginosa whole thallus is produced. The obtained vaccine has good immunogenicity, can prevent infectious diseases caused by various pseudomonas aeruginosa, and is small in side effect and high in safety.

Description

Industrial production method of Pseudomonas aeruginosa vaccine Technical Field

[0001] The invention belongs to the field of bioengineering, and particularly relates to an industrial production method of Pseudomonas aeruginosa vaccine. Background Art

[0002] Pseudomonas aeruginosa (P. aeruginosa), also known as Pseudomonas aeruginosa, is a common opportunistic pathogen that is highly resistant to various environmental factors and inactivation measures, and can survive for long periods in humid environments. It is found in soil, water, air, and the skin, mucous membranes, intestines, and upper respiratory tract of humans and animals. Its pathogenicity is characterized by secondary infections, which often occur in patients with compromised immune systems, such as those with extensive burns, long-term immunosuppressant use, tumors, and hypoproteinemia. Infections caused by P. aeruginosa can occur anywhere in the body, with common clinical manifestations including skin and subcutaneous tissue infections, including otitis media, meningitis, respiratory tract infections, urinary tract infections, and sepsis. Recent surveys have shown that over 30% of hospital-acquired infections are caused by P. aeruginosa. Currently, infections caused by P. aeruginosa rely primarily on medication. However, the recent overuse of antibiotics has led to the emergence of numerous drug-resistant strains, exacerbating the threat posed by P. aeruginosa to patients. The currently well-researched mechanisms of antibiotic resistance in Pseudomonas aeruginosa include the following: ① Reduced permeability of the bacterial outer membrane, hindering antibiotic access to the bacterial inner membrane target; ② Active efflux by Pseudomonas aeruginosa cells, which can excrete a wide range of structurally unrelated antibiotics or other toxic substances; ③ Alterations in the number or structure of penicillin-binding proteins (PBPs), the antibiotic's target, to reduce the affinity of the drug for PBPs, thereby generating intrinsic resistance; and ④ Plasmid- or chromosome-mediated β-lactamases inactivate antibiotics. Therefore, Pseudomonas aeruginosa infections have now reached a stage where no antibiotics are available, and prevention and control of Pseudomonas aeruginosa infections through immunization is the prevailing trend.

[0003] A vaccine is a substance that stimulates the immune system, activating T and B lymphocytes to produce antibodies or immune cells specific for target antigens (viruses, bacteria, etc.). Currently, research worldwide on Pseudomonas aeruginosa vaccine preparation methods covers multiple technical aspects, including heat- or formaldehyde-treated killed bacteria vaccines, attenuated vaccines, subunit vaccines, and single-component vaccines (such as pili, flagella, and outer membrane proteins OprI / OprF). In basic experiments, single-component vaccines have achieved good preventive effects, but due to their single antigenic component, they can only target infections caused by a single disease (such as pneumonia). Whole-bacterium vaccines that are inactivated (heat, formaldehyde, etc.) or attenuated (genetic mutation) have a relatively broad spectrum of protection against diseases. However, due to the inactivation of certain unknown antigenic components during the preparation process, bacterial metabolic activity is reduced, resulting in poor immunogenicity and preventive efficacy. Incomplete inactivation also results in high residual toxicity in the vaccine. Research and development of whole-cell vaccines has been conducted for numerous bacterial species (such as Shigella, Neisseria gonorrhoeae, and Neisseria meningitidis), but the vast majority of these vaccines have failed, making the development of effective whole-cell vaccines extremely difficult. Furthermore, current whole-cell Pseudomonas aeruginosa vaccine preparations still rely primarily on traditional formaldehyde inactivation, a substance classified by the World Health Organization as a Class 1 carcinogen and not suitable for use by those allergic to formaldehyde. Therefore, new inactivation methods are urgently needed.

[0004] The methods disclosed in the prior art for producing whole-cell Pseudomonas aeruginosa vaccines are only suitable for small-batch production and are not suitable for large-scale, industrialized production. Therefore, the present invention provides a method for industrially producing multi-component and whole-cell Pseudomonas aeruginosa vaccines, which can alleviate one or more of the existing problems.

[0005] Summary of the Invention

[0006] The present invention provides an industrial production method of a Pseudomonas aeruginosa vaccine, and a Pseudomonas aeruginosa vaccine prepared using the method. The specific technical scheme is as follows.

[0007] An industrial production method for a Pseudomonas aeruginosa vaccine comprises the following steps:

[0008] S1: Cultivating the Pseudomonas aeruginosa production strain with a suitable culture medium to prepare a seed solution; the culture medium should preferably be one that does not contain animal-derived components, and is not limited to the culture medium involved in the embodiments of the present invention.

[0009] S2: inoculating the seed liquid into a fermentation tank at 2% to 10% of the fermentation volume for fermentation, wherein the fermentation temperature is 30 to 40°C, the pH value is 5 to 9, the rotation speed is 100 to 400 rpm, the ventilation volume is 2 to 5 L / min, the dissolved oxygen is 10% to 30%, and the fermentation time is 3 to 8 hours;

[0010] S3 monitors the bacterial cell density in the fermentation tank. When the bacterial cell density in the fermentation tank reaches the standard, the bacterial liquid in the fermentation tank is directly centrifuged at a centrifugal force of 3000 to 8000×g, and the bacterial cells are collected after 10 to 30 minutes.

[0011] S4: resuspending the bacterial cells with isotonic injection solution and adjusting the concentration, and then irradiating the bacterial cells with radiation to make them lose their proliferation activity. The radiation irradiation includes X-rays, gamma rays and isotope radiation sources Co 60 One or more of the rays produced, the isotonic injection solution includes a solution such as physiological saline;

[0012] S5: Check the irradiated bacterial solution. The bacterial solution is qualified if the proportion of whole bacterial cells and immunogenic components in the bacteria is greater than 80%.

[0013] S6: resuspend the bacterial solution that has passed the inspection in step S5 with the isotonic injection solution and adjust the concentration to 1.0×10 7 ~3.0×10 7 / ml, and a Pseudomonas aeruginosa vaccine was prepared.

[0014] Furthermore, the absorbance value of the bacterial cell density in the fermentation tank reaches 1 to 3 OD, which is considered to be up to standard.

[0015] Furthermore, the intrabacterial immunogenic components include membrane vesicles, nucleic acids and bacterial fragments.

[0016] Furthermore, step S4 further includes the step of determining the composition and content of the bacterial liquid after the radiation irradiation, and the method for determining the composition and content of the bacterial liquid after the radiation irradiation includes one or more of a spectrophotometer method, a density gradient centrifugation method, a scanning electron microscope, and a transmission electron microscope.

[0017] Furthermore, the basic inactivation dose of the radiation irradiation shall not be less than 1000 Gy, and the total dose of the radiation irradiation shall be greater than the basic inactivation dose of the radiation irradiation.

[0018] Furthermore, the radiation irradiation is carried out in a low-dose-rate, long-term, continuous manner; the dose rate is preferably 5 to 15 Gy / min, and the continuous irradiation time is preferably greater than 2 hours.

[0019] Furthermore, the step of determining the inactivation time or inactivation dose of the radiation includes:

[0020] a) determining the material and shape of the container for loading the irradiated sample, wherein the material is a medical-grade plastic material, including but not limited to polypropylene (PP), polystyrene (PS), etc., and the shape is preferably cylindrical, barreled, or bagged;

[0021] b) Determine the maximum loading volume for inactivation;

[0022] c) Determine the concentration of the bacterial solution: Determine the total amount of inactivated bacteria in each batch based on the production design, and then select the appropriate bacterial solution concentration based on the loading requirements. The calculation formula for the bacterial solution concentration is: Where N represents the concentration of bacterial solution, A represents the yield per person, B represents the bacterial dosage per person, V represents the total volume of bacterial solution, and V≤maximum loading volume;

[0023] d) Selecting a dose rate: Designing different dose rate groups, performing the inactivation of the maximum load according to steps a) to c), and setting the continuous irradiation time to be no less than 2 hours, specifically, it can be set to 2 hours or any time greater than or equal to 2 hours;

[0024] e) determination of a sterilization curve: performing multiple batches of irradiation according to the dose rate and the irradiation time determined in step d), and counting viable bacteria at intermediate time points or extended time points, or counting viable bacteria at intermediate irradiation dose points or extended irradiation dose points, and determining the sterilization curve;

[0025] 6) Determine the inactivation time or inactivation dose: According to the sterilization curve, take the adjacent first time point, second time point, and third time point, or take the adjacent first irradiation dose point, second irradiation dose point, and third irradiation dose point for live bacteria counting. The time point interval is not less than 20 minutes, and the irradiation dose point interval is not less than 200 Gy. When the counting results are less than 100 CFU / ml, 0 CFU / ml, and 0 CFU / ml, respectively, the second time point is the inactivation time, and the second irradiation dose point is the inactivation dose.

[0026] After further optimization and preliminary determination of the inactivation time or inactivation dose, the key parameters for further optimization include dose rate, irradiation time, loading volume, bacterial concentration, etc., and may also include inactivation dose; the above parameters can be adjusted within an appropriate range to adapt to industrial production.

[0027] Furthermore, after determining the inactivation time or the inactivation dose, inactivation verification is further included, and the inactivation verification includes sterility inspection and stability investigation after inactivation; the stability investigation includes accelerated investigation at 25°C or 37°C and real-time investigation at 2-8°C. The purpose of this step is to ensure that the vaccine will not proliferate during long-term storage.

[0028] Furthermore, industrial production verification should be carried out. After selecting appropriate industrial production parameters, multiple batches of production verification should be carried out to ensure that each batch can achieve complete bacterial inactivation.

[0029] The Pseudomonas aeruginosa vaccine produced by the industrial production method provided by the present invention is preferably in the form of an injection and / or a lyophilized powder, and can be administered by one or more of subcutaneous injection, intramuscular injection, intradermal scarification, nasal administration, and oral administration. The vaccine is a whole-bacterial vaccine or a multicomponent vaccine containing both whole bacteria and intrabacterial immunogenic components such as membrane vesicles, nucleic acids, and bacterial fragments.

[0030] The Pseudomonas aeruginosa vaccine prepared by the present invention is used in the preparation of drugs for preventing infectious diseases caused by Pseudomonas aeruginosa. The infectious diseases generally include Pseudomonas aeruginosa pneumonia, chronic obstructive pulmonary disease combined with Pseudomonas aeruginosa infection, burns combined with Pseudomonas aeruginosa infection, Pseudomonas aeruginosa keratitis, etc.

[0031] Glossary

[0032] The "basic inactivation dose" mentioned in the present invention refers to the dose that can basically inactivate bacteria, and the lower limit of this dose is 1000Gy.

[0033] The “total dose” mentioned in the present invention refers to the total radiation dose accumulated over time during the irradiation process.

[0034] The "inactivation dose" mentioned in the present invention refers to the irradiation dose (also understood as the "optimal inactivation dose") that just inactivates all bacteria in the inactivated bacterial solution (also understood as the "inactivated stock solution"). "Just inactivated" means that all bacteria in the inactivated bacterial solution just reach irreversible death. In some embodiments of the present invention, according to the drawn sterilization curve, when three adjacent irradiation dose points are taken for live bacteria counting and the counting results are less than 100 CFU / ml, 0 CFU / ml, and 0 CFU / ml respectively, the irradiation dose point in the middle of the three adjacent irradiation dose points is the inactivation dose, and the intervals between the irradiation metering points are the same and not less than 200 Gy; further, for example, when the dose is in the "inactivation dose-200 Gy" condition, the inactivated stock solution contains less than 100 CFU / ml of live bacteria; when the dose is the inactivation dose, the inactivated stock solution contains 0 CFU / ml of live bacteria; when the dose is in the "inactivation dose + 200 Gy" condition, the inactivated stock solution contains 0 CFU / ml of live bacteria. Similarly, in some embodiments of the present invention, according to the drawn sterilization curve, when live bacteria are counted at three adjacent time points and the counting results are less than 100 CFU / ml, 0 CFU / ml, and 0 CFU / ml respectively, the middle time point among the three adjacent time points is the inactivation time, and the intervals between the time points are the same and not less than 20 min; further, for example, when the time is in the "inactivation time - 20 min" condition, the number of live bacteria contained in the inactivated stock solution is less than 100 CFU / ml; when the time is the inactivation time, the number of live bacteria contained in the inactivated stock solution is 0 CFU / ml; when the time is in the "inactivation time + 20 min" condition, the number of live bacteria contained in the inactivated stock solution is 0 CFU / ml.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The present invention provides a method for industrial production of Pseudomonas aeruginosa vaccine, that is, using a series of standardized, programmed, and digital settings to industrially produce a vaccine with stable quality containing whole Pseudomonas aeruginosa bacteria and multiple immunogenic components within the bacteria. Specifically, the present invention directly centrifuges the bacterial solution with a cell density that meets the standard, eliminating the purification step of repeated centrifugation and washing, thereby improving the industrial production efficiency and avoiding the cell rupture caused by repeated centrifugation and washing in large-scale, industrial-grade production processes. After rupture, the cells will release more harmful substances such as endotoxins. Therefore, in the industrial-grade production process, repeated centrifugation and washing will increase the safety risk of the prepared vaccine; the large number of cell ruptures caused by repeated centrifugation and washing will reduce the number of whole cells and produce more cell fragments, which will be removed by centrifugation and washing. In addition, the purification step of repeated centrifugation and washing will also remove the membrane vesicles and nucleic acids in the immunogenic components of the vaccine prepared by the present invention, ultimately resulting in unstable quality of the industrially produced vaccine and reduced types and quantities of immunogenic components. Based on this, the direct centrifugation in the industrial production method provided by the present invention avoids the above-mentioned risks caused by the purification step and improves the effectiveness and safety of the vaccine.

[0037] (2) The present invention optimizes the irradiation method of radiation, namely, adopting a low dose rate (5-15Gy / min), long-term, continuous irradiation (greater than 2h), reducing the total dose of radiation (≤2000Gy), thereby avoiding the damage to bacteria by large doses of radiation and improving the immune efficacy and safety of the vaccine.

[0038] (3) The vaccine prepared by the present invention has good immunogenicity. Not only is the actual vaccination amount low, but it can also prevent a variety of infectious diseases caused by Pseudomonas aeruginosa, such as Pseudomonas aeruginosa pneumonia, chronic obstructive pulmonary disease combined with Pseudomonas aeruginosa infection, burns combined with Pseudomonas aeruginosa infection, Pseudomonas aeruginosa keratitis, etc. The vaccine prepared by the present invention can usually be adjuvant-free (i.e., no adjuvant is needed to enhance the body's immune response). Of course, in certain scenarios (for example, it is necessary to prepare a Pseudomonas aeruginosa vaccine with extremely strong immunogenicity), an adjuvant can also be used. The realization of the above-mentioned good immunogenicity (protective efficacy of the vaccine) is not only due to the optimization in the preparation method as described above, but also lies in the optimization of the actual immunization program of the vaccine: while reducing the total number of vaccinations, the immunization interval is extended. The present invention found that this optimized immunization program is conducive to improving the actual efficacy of the vaccine, and the extended immunization interval is more in line with the body's immune response to the vaccine.

[0039] (4) The technical solution of the present invention adopts physical methods (i.e., radiation) for inactivation. Compared with traditional chemical methods (for example, using formaldehyde as a chemical inactivator), no chemical substances (chemical inactivators) remain. To a certain extent, it avoids allergic reactions and carcinogenic risks caused by chemical inactivators, improves the safety of vaccines and reduces side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0041] FIG1 shows the protective effect of the vaccine prepared by the method provided by the present invention in a mouse Pseudomonas aeruginosa pneumonia model;

[0042] FIG2 shows the protective effect of the vaccine prepared by the method provided by the present invention in a COPD combined with Pseudomonas aeruginosa infection model;

[0043] FIG3 shows the protective effect of the vaccine prepared by the method provided by the present invention in a mouse burn model;

[0044] FIG4 shows the protective effect of the vaccine prepared by the method provided by the present invention in a mouse corneal infection model;

[0045] FIG5 shows a schematic diagram of a sterilization curve. DETAILED DESCRIPTION

[0046] To further clarify the objectives, technical solutions, and advantages of the embodiments of the present invention, the following will provide a clear and complete description of the technical solutions in the embodiments of the present invention, in conjunction with the accompanying drawings. It should be understood that the described embodiments are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0047] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0048] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0049] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that such descriptions of "being within a certain range" are merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of a range of 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0050] Example 1: Preparation method of Pseudomonas aeruginosa vaccine

[0051] S1: Cultivate the Pseudomonas aeruginosa production strain in a suitable culture medium to prepare a seed solution. The culture medium should preferably be one that does not contain animal-derived components, such as Tryptone Soy Broth (TSB).

[0052] S2: inoculate the seed liquid into a fermentation tank according to 2% to 10% of the fermentation volume for fermentation, wherein the fermentation temperature is 30 to 40°C, the pH value is 5 to 9 (preferably 7), the rotation speed is 100 to 400 rpm, the ventilation volume is 2 to 5 L / min, the dissolved oxygen is 10% to 30%, and the fermentation time is 3 to 8 hours.

[0053] S3 monitors the bacterial density in the fermentation tank. After the bacterial density in the fermentation tank reaches the standard (i.e., the bacterial density absorbance value reaches 1-3 OD), the fermented bacterial liquid is directly centrifuged at a centrifugal force of 3000-8000×g, and the bacteria are collected after 10-30 minutes.

[0054] S4: resuspend the bacteria in isotonic injection solution and adjust the concentration, and then irradiate the bacteria to lose their proliferation activity. The irradiation rays include X-rays, gamma rays and isotope radiation sources Co 60 One or more of the radiations produced, the isotonic injection solution includes a solution such as physiological saline. After irradiation, a step of testing the composition and content of the bacterial solution can be performed using one or more of the following methods: spectrophotometry, density gradient centrifugation, scanning electron microscopy, and transmission electron microscopy.

[0055] S5: The irradiated bacterial solution is inspected for the presence of whole bacteria and immunogenic components. A percentage of whole bacteria exceeding 80% is considered acceptable. These immunogenic components include membrane vesicles, nucleic acids, and bacterial fragments. Typically, the percentage of whole bacteria in the bacterial solution obtained using the industrialized production method provided herein should be maintained above 80%. The purpose of inspecting the bacterial solution is to ensure that the amounts of components (whole bacteria and immunogenic components) in the solution remain stable while adjusting other parameters.

[0056] S6: resuspend the qualified bacterial solution in step S5 with isotonic injection solution and adjust the concentration to 1.0×10 7 ~3.0×10 7 / ml, and Pseudomonas aeruginosa vaccine was prepared.

[0057] Furthermore, the basic inactivation dose of the radiation exposure should not be less than 1000 Gy, and the total radiation exposure dose should be greater than the basic inactivation dose of the radiation exposure. The radiation exposure method is low-dose-rate, long-term, continuous exposure. The radiation exposure dose rate is 5 to 15 Gy / min, and the continuous exposure time is greater than 2 hours. In some embodiments, the total radiation exposure dose of the present invention can be controlled within the range of ≤2000 Gy.

[0058] The steps of determining the inactivation time or inactivation dose of the radiation are:

[0059] a. Determine the material and shape of the container for the irradiated sample. The material should be medical-grade plastic, including but not limited to polypropylene (PP) and polystyrene (PS). The shape should preferably be one or more of a column, barrel, and bag.

[0060] b. Determine the maximum loading volume for inactivation. The loading volume is determined by the shape of the container and the liquid level. The difference between the dose rate when the radiation passes through the highest (near) point of the liquid level and the dose rate when the radiation passes through the lowest (far) point of the liquid level shall not exceed 20% of the dose rate at the highest (near) point of the liquid level. The volume of the bacterial liquid corresponding to the highest (near) point of the liquid level is the maximum loading volume for inactivation. Use a radiation dose detector to place the detection probe at the highest (near) and lowest (far) point of the liquid level from the radiation source, respectively. Start the irradiator and the radiation dose detector will read the irradiation dose rate. 20% is based on quality control requirements to ensure the uniformity of the radiation dose received by the irradiated samples.

[0061] c. Determine the concentration of the bacterial solution. Determine the total amount of bacteria inactivated in each batch based on the production design, and then select the appropriate bacterial solution concentration based on the loading requirements. The calculation formula is: Where N represents the concentration of the bacterial solution, A represents the output per person, B represents the bacterial dosage per person, V represents the total volume of the bacterial solution, and V≤maximum loading volume.

[0062] d. Select a dose rate. Design different dose rate groups and perform the maximum inactivation according to steps a through c. Set the continuous irradiation time to be no less than 2 hours. Specifically, it can be set to 2 hours or any time greater than or equal to 2 hours.

[0063] e. Determination of sterilization curve. According to the dose rate and irradiation time determined in step d, multiple batches of irradiation are carried out, and live bacteria are counted at intermediate time points or extended time points, or live bacteria are counted at intermediate irradiation dose points or extended irradiation dose points to determine the sterilization curve. It should be noted that the inactivation process can be controlled by controlling the irradiation time or the irradiation dose, and then the inactivation parameters can be examined from the perspective of controlling the time or the dose. For example, after determining the inactivation time, samples can be taken at the time point before the inactivation time (e.g., the "intermediate time point") and the time point after the inactivation time (e.g., the "extended time point") to verify whether the inactivation time can meet the requirements for complete inactivation; similarly, after determining the inactivation dose, samples can be taken at the irradiation dose point before the inactivation dose (e.g., the "intermediate irradiation dose point") and the irradiation dose point after the inactivation dose (e.g., the "extended irradiation dose point") to verify whether the inactivation dose can meet the requirements for complete inactivation.

[0064] f. Determine the inactivation dose or inactivation time. Based on the sterilization curve, the inactivation time or inactivation dose is determined by counting viable bacteria at three adjacent time points (the first time point, the second time point, and the third time point) or three adjacent irradiation dose points (the first irradiation dose point, the second irradiation dose point, and the third irradiation dose point), with an interval of no less than 20 minutes between time points and an interval of no less than 200 Gy between irradiation dose points. If the count results are less than 100 CFU / ml, 0 CFU / ml, and 0 CFU / ml, respectively, the middle time point (i.e., the second time point) is the inactivation time, and the middle irradiation dose point (i.e., the second irradiation dose point) is the inactivation dose. The schematic diagram of the sterilization curve is shown in Figure 5. According to the sterilization curve, when the three adjacent irradiation dose points of "1200Gy", "1400Gy" and "1600Gy" are taken for live bacteria counting and the counting results are less than 100CFU / ml, 0CFU / ml and 0CFU / ml respectively, the irradiation dose point "1400Gy" in the middle of the three adjacent irradiation dose points is the inactivation dose; similarly, according to the drawn sterilization curve, when the three adjacent time points of "120min", "140min" and "160min" are taken for live bacteria counting and the counting results are less than 100CFU / ml, 0CFU / ml and 0CFU / ml respectively, the time point "140min" in the middle of the three adjacent time points is the inactivation time.

[0065] After the inactivation time or inactivation dose is preliminarily determined, the key parameters for further optimization include dose rate, irradiation time, loading volume, bacterial liquid concentration, etc., and may also include inactivation dose; the above parameters can be adjusted within an appropriate range to adapt to industrial production.

[0066] After determining the inactivation time or inactivation dose, inactivation verification is carried out, including sterility inspection and stability study after inactivation; the stability study includes accelerated study at 25°C or 37°C and real-time study at 2-8°C. The purpose of this step is to ensure that the vaccine will not proliferate during long-term storage.

[0067] Conduct industrial production verification. After selecting appropriate industrial production parameters, multiple batches of production verification should be carried out to ensure that each batch can achieve complete bacterial inactivation.

[0068] The Pseudomonas aeruginosa vaccine produced by the industrial production method provided by the present invention is a whole-bacterial vaccine or a multi-component vaccine containing both whole bacteria and intrabacterial immunogenic components such as membrane vesicles, nucleic acids, and bacterial fragments. The vaccine is in the form of an injection and / or a lyophilized powder. The vaccine can be administered by one or more of subcutaneous injection, intramuscular injection, skin scarification, nasal administration, and oral administration.

[0069] In some embodiments of the present invention, the mass ratio of whole bacteria, membrane vesicles, nucleic acids, and bacterial fragments is preferably 98:1:0.1:1. Under this ratio, the vaccine has better immunogenicity and less side effects.

[0070] Example 2: Protective effect of vaccine in a mouse Pseudomonas aeruginosa pneumonia model

[0071] There are 20 serotypes of Pseudomonas aeruginosa. The main serotypes of Pseudomonas aeruginosa isolated clinically are O6, O11, and O4, accounting for approximately 20%, 15%, and 10%, respectively. In this example, a vaccine was prepared using Pseudomonas aeruginosa PA1 (serotype O5) according to the above process. The vaccine was administered subcutaneously to mice at 0, 14, and 28 days. 7Seven days after the last immunization, mice were infected with Pseudomonas aeruginosa PA1(O5), the clinically isolated carbapenem-resistant Pseudomonas aeruginosa C58(O6), the standard strains ATCC33358(O11), ATCC33351(O4), and the laboratory strain PA14(O14) through their airways. Lung tissue was collected 24 hours after infection for bacterial load enumeration, as shown in Figure 1. The vaccine effectively reduced the bacterial loads of PA1(O5) (P<0.001), C58(O6) (P<0.05), ATCC33358(O11) (P<0.01), ATCC33351(O4) (P>0.05), and PA14 (P<0.01). These results demonstrate that the vaccine protects against infections caused by different serotypes of Pseudomonas aeruginosa and can prevent infections caused by a variety of Pseudomonas aeruginosa strains.

[0072] Example 3: Protective effect of the vaccine in a mouse model of chronic obstructive pulmonary disease (COPD) combined with Pseudomonas aeruginosa infection

[0073] In this example, a vaccine was prepared using Pseudomonas aeruginosa PA1 (serotype O5) according to the above process. The vaccine was administered subcutaneously to mice with 1.0×10 7 The COPD model was established by intratracheal infusion of elastase 10 days before infection. Seven days after the last immunization, the mice were infected with the bacteria through the airways and lungs. 24 hours after infection, the mice were sacrificed, and the lung tissues were collected for bacterial load counts. The results are shown in Figure 2. Both immunization schedules showed a significant in vivo protective effect against COPD combined with Pseudomonas aeruginosa lung infection (P < 0.01), reducing the bacterial load in lung tissue by approximately 2 log. 10 The results showed that the vaccine has a significant protective effect on COPD combined with Pseudomonas aeruginosa lung infection.

[0074] Example 4: Protective effect of the vaccine in a mouse burn model

[0075] In this example, Pseudomonas aeruginosa PA1 (serotype O5) was used to prepare the vaccine according to the above process. The vaccine was administered subcutaneously to mice at 0, 14, and 28 days. 7 Seven days after the last immunization, mice were scalded on the dorsal skin with a hot air blower to establish a burn model. Two hours after the burn, the wound was subcutaneously infected with Pseudomonas aeruginosa PA1. Bacterial loads were counted 24 hours later. The results are shown in Figure 3. Vaccination significantly reduced the bacterial load in burn wounds complicated by Pseudomonas aeruginosa infection (P < 0.001). These results demonstrate that this vaccine has a significant protective effect against burns complicated by Pseudomonas aeruginosa infection.

[0076] Example 5: Protective effect of the vaccine in a mouse corneal infection model

[0077] In this example, Pseudomonas aeruginosa PA1 (serotype O5) was used to prepare the vaccine according to the above process. The vaccine was administered to mice via nasal drops (containing 1.0×10 7 Seven days after the last immunization, the mice's corneas were scratched with a needle tip and then infected with the bacteria. Bacterial loads were counted 24 hours later. The results are shown in Figure 4. Both immunization schedules showed significant protection against corneal infection with Pseudomonas aeruginosa (P < 0.01). The results demonstrate that the vaccine has a significant protective effect against corneal infection with Pseudomonas aeruginosa.

[0078] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. An industrial production method for Pseudomonas aeruginosa vaccine, characterized in that: The steps include: S1: culturing the production strain of Pseudomonas aeruginosa in a suitable culture medium to prepare a seed solution; S2: inoculating the seed liquid into a fermentation tank at 2% to 10% of the fermentation volume for fermentation, wherein the fermentation temperature is 30 to 40°C, the pH value is 5 to 9, the rotation speed is 100 to 400 rpm, the ventilation volume is 2 to 5 L / min, the dissolved oxygen is 10% to 30%, and the fermentation time is 3 to 8 hours; S3 monitors the bacterial cell density in the fermentation tank. When the bacterial cell density in the fermentation tank reaches the standard, the bacterial liquid in the fermentation tank is directly centrifuged at a centrifugal force of 3000 to 8000×g, and the bacterial cells are collected after 10 to 30 minutes. S4: resuspending the bacterial cells with isotonic injection solution and adjusting the concentration, and then irradiating the bacterial cells with radiation to make them lose their proliferation activity. The radiation irradiation includes X-rays, gamma rays and isotope radiation sources Co 60 One or more of the rays produced; S5: Check the irradiated bacterial solution. The bacterial solution is qualified if the proportion of whole bacterial cells and immunogenic components in the bacteria is greater than 80%. S6: resuspend the bacterial solution that has passed the inspection in step S5 with the isotonic injection solution and adjust the concentration to 1.0×10 7 ~3.0×10 7 / ml, to prepare the Pseudomonas aeruginosa vaccine.

2. The method according to claim 1, wherein The bacterial cell density in the fermentation tank is considered to be up to standard when the absorbance value reaches 1 to 3 OD.

3. The method according to claim 1, wherein The intrabacterial immunogenic components include membrane vesicles, nucleic acids and bacterial fragments.

4. The method according to claim 1, wherein After step S4, the step of determining the composition and content of the bacterial solution after the radiation irradiation is further included. The method of determining the composition and content of the bacterial solution after the radiation irradiation includes one or more of a spectrophotometer, a density gradient centrifugation method, a scanning electron microscope, and a transmission electron microscope.

5. The method according to claim 1, wherein The basic inactivation dose of the radiation shall not be less than 1000 Gy, and the total dose of the radiation shall be greater than the basic inactivation dose of the radiation.

6. The method according to claim 5, wherein The radiation irradiation method is low dose rate, long time, continuous irradiation; the dose rate is preferably 5 to 15 Gy / min, and the continuous irradiation time is preferably greater than 2 hours.

7. The method according to claim 1, wherein The step of determining the inactivation time or inactivation dose of the radiation comprises: a) Determine the material and shape of the container for the irradiated sample; b) Determine the maximum loading volume for inactivation; c) Determine the concentration of the bacterial solution: Determine the total amount of inactivated bacteria in each batch based on the production design, and then select the appropriate bacterial solution concentration based on the loading requirements. The calculation formula for the bacterial solution concentration is: Where N represents the concentration of bacterial solution, A represents the yield per person, B represents the bacterial dosage per person, V represents the total volume of bacterial solution, and V≤maximum loading volume; d) selecting a dose rate: designing different dose rate groups, performing the inactivation of the maximum load according to steps a) to c), and setting the continuous irradiation time to be no less than 2 hours; e) determining a sterilization curve: performing multiple batches of irradiation according to the dose rate and the irradiation time determined in step d), and counting viable bacteria at intermediate time points or extended time points, or counting viable bacteria at intermediate irradiation dose points or extended irradiation dose points, and determining the sterilization curve; f) Determine the inactivation time or inactivation dose: According to the sterilization curve, take the adjacent first time point, second time point, and third time point, or take the adjacent first irradiation dose point, second irradiation dose point, and third irradiation dose point for viable bacteria counting, wherein the time point interval is not less than 20 minutes, and the irradiation dose point interval is not less than 200 Gy. When the counting results are less than 100 CFU / ml, 0 CFU / ml, and 0 CFU / ml, respectively, the second time point is the inactivation time, and the second irradiation dose point is the inactivation dose.

8. The method according to claim 7, wherein After determining the inactivation time or the inactivation dose, inactivation verification is further performed, and the inactivation verification includes sterility inspection and stability investigation after inactivation; the stability investigation includes accelerated investigation at 25°C or 37°C and real-time investigation at 2-8°C.

9. The Pseudomonas aeruginosa vaccine produced by the industrial production method according to any one of claims 1 to 8, wherein the dosage form of the vaccine is preferably an injection and / or a lyophilized powder, and the vaccination method of the vaccine optionally includes one or more of subcutaneous injection, intramuscular injection, skin scarification, nasal administration and oral administration.

10. Use of the vaccine according to claim 9 in the preparation of a medicament for preventing infectious diseases caused by Pseudomonas aeruginosa.

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