Industrial production method for Pseudomonas aeruginosa vaccine

Abstract

The industrial production method of Pseudomonas aeruginosa vaccine adopts a series of standardized, programmed and digitalized settings to ensure the production of a vaccine containing whole Pseudomonas aeruginosa cells and multiple intrabacterial immunogenic components with stable quality. The resulting vaccine has good immunogenicity and can prevent multiple infectious diseases caused by Pseudomonas aeruginosa, while also having few side effects and high safety.

Description

[Technical field]

[0001] [1] The present invention relates to the field of biotechnology, and more particularly to an industrial method for producing a Pseudomonas aeruginosa vaccine. [Background technology]

[0002] [2].Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is a common opportunistic pathogen that is highly resistant to various external environments and inactivation measures, and can survive for a long time, especially in humid environments. This bacterium is present in many places, such as soil, water, air, human and animal skin, mucous membranes, intestinal tract, and upper respiratory tract, and its pathogenic characteristic is to cause secondary infection. This secondary infection is often found in patients with low body resistance, such as patients with large-area burns, patients who have been using immunosuppressants for a long time, and patients with tumors or hypoproteinemia. Infections caused by Pseudomonas aeruginosa can occur in any part of the human body, but the most common clinical cases are skin and subcutaneous tissue infections such as otitis media, meningitis, respiratory infections, urinary tract infections, and sepsis. According to recent survey reports, more than 30% of hospital-acquired infections are caused by Pseudomonas aeruginosa. Currently, infections caused by P. aeruginosa are mainly treated with drugs, and the recent overuse of antibiotics has led to the development of a large number of drug-resistant strains, thereby exacerbating the threat of P. aeruginosa to patients. Currently, the mechanism of drug resistance of P. aeruginosa to antibiotics, which has already been well studied, has the following aspects: (1) The permeability of the bacterial outer membrane is reduced, inhibiting the entry of antibiotics into the bacterial inner membrane target site. (2) P. aeruginosa cells can excrete a wide range of structurally unrelated antibiotics and other toxic substances out of the cell through active excretion processes. (3) The number and structure of penicillin-binding proteins (PBPs), which are the action targets of antibiotics, are changed, reducing the affinity of drugs to PBPs, thereby generating intrinsic drug resistance. (4) Plasmid- or chromosomally mediated β-lactamase inactivates antibiotics. Therefore, at present, P. aeruginosa infection has reached a stage where there are no available antibiotics, and the prevention and control of P. aeruginosa infection by immunological methods is a general trend.

[0003] [3].A vaccine is a substance that can stimulate the body's immune system and activate T and B lymphocytes to produce antibodies or immune cells specific to a target antigen (virus, bacteria, etc.). Currently, the manufacturing methods of P. aeruginosa vaccines being researched worldwide involve multiple technical aspects, such as heat-treated or formaldehyde-treated killed vaccines, attenuated vaccines, subunit vaccines, and single-component (fimbria, flagella, outer membrane protein OprI / OprF, etc.) vaccines. In basic experiments, single-component vaccines have excellent preventive effects, but because they contain a single antigenic component, they can only respond to infections of a single disease (pneumonia, etc.). The disease resistance spectrum of inactivated (heat-treated, formaldehyde-treated, etc.) or attenuated (genetically mutated) whole-cell vaccines is relatively broad, but because certain unknown antigenic components are inactivated during the manufacturing process, the metabolic activity of the bacteria is reduced, resulting in poor immunogenicity and preventive effects. In addition, the inactivation is incomplete, which increases the residual toxicity of the vaccine. Research and development of whole cell vaccines has already been conducted for many bacterial species (Shiga bacteria, Neisseria gonorrhoeae, Neisseria meningitidis, etc.), but these vaccines have mostly ended in failure, making it very difficult to produce an effective whole cell vaccine. In addition, the current manufacturing process for whole cell Pseudomonas aeruginosa vaccine still mainly relies on traditional formaldehyde inactivation, but formaldehyde has already been classified as a Group 1 carcinogen by the WHO, and is not suitable for people with allergies to it, so there is an urgent need to adopt a new inactivation method.

[0004] [4] However, the methods for producing whole cell Pseudomonas aeruginosa vaccines disclosed in the prior art are only applicable to small-scale production, and not to large-scale industrial production. Based on this, the present invention provides an industrial method for producing a multi-component whole cell Pseudomonas aeruginosa vaccine, which can alleviate one or more of the problems of the prior art. Summary of the Invention

[0005] [5] The present invention provides an industrial production method for Pseudomonas aeruginosa vaccine and the Pseudomonas aeruginosa vaccine produced by the method. The specific technical solutions are as follows:

[0006] [6]. A method for industrially producing a Pseudomonas aeruginosa vaccine, comprising:

[0007] [7].S1 A process for culturing a Pseudomonas aeruginosa strain for production in an appropriate medium to produce a seed solution, wherein the medium is not limited to the medium according to the embodiment of the present invention, and it is preferable to select a medium that does not contain animal-derived components.

[0008] [8].S2 A step of inoculating the seed liquid into a fermenter according to 2% to 10% of the fermentation volume and fermenting the liquid, in which the fermentation temperature is 30 to 40°C, the pH value is 5 to 9, the rotation speed is 100 to 400 rpm, the aeration amount is 2 to 5 L / min, the dissolved oxygen is 10% to 30%, and the fermentation time is 3 to 8 h;

[0009] [9].S3 monitoring the density of the bacteria in the fermenter, and after the density of the bacteria in the fermenter reaches a standard, collecting the bacteria liquid in the fermenter and directly centrifuging it at a centrifugal force of 3,000 to 8,000 × g, and collecting the bacteria after 10 to 30 minutes;

[0010]

[10] .S4 A process of resuspending the bacterial cells in an isotonic injection solution, adjusting the concentration, and then irradiating the bacterial cells with radiation to eliminate their proliferation activity, wherein the radiation used for the irradiation is X-rays, gamma rays, or an isotope radiation source Co 60 and the isotonic injection solution comprises a solution such as saline;

[0011]

[11] .S5 A step of sampling the irradiated bacterial liquid and testing it, the bacterial liquid containing the whole bacterial cells and the bacterial immunogenic components, and the bacterial liquid being deemed to be passed if the ratio of the whole bacterial cells is more than 80%;

[0012]

[12] .S6 The bacterial solution that passed the inspection in step S5 is resuspended in the isotonic injection solution to a concentration of 1.0 x 10 7 ~3.0×10 7 and a step of adjusting the concentration to cells / ml to produce the Pseudomonas aeruginosa vaccine.

[0013]

[13] Furthermore, when the absorbance value, which is the density of the bacteria in the fermenter, reaches 1 to 3 OD, it is deemed that the standard has been reached.

[0014]

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

[0015]

[15] Furthermore, the method further includes a step of measuring the components and content of the bacterial liquid after the radiation exposure after step S4, and the method for measuring the components and content of the bacterial liquid after the radiation exposure includes one or more of spectrophotometry, density gradient centrifugation, scanning electron microscopy, and transmission electron microscopy.

[0016]

[16] Furthermore, the basic inactivating dose of the radiation exposure may not be less than 1,000 Gy, and the total dose of the radiation exposure may be greater than the basic inactivating dose of the radiation exposure.

[0017]

[17] . Furthermore, the radiation irradiation is performed in a manner of low dose rate, long time, and continuous irradiation, the dose rate is preferably 5-15 Gy / min, and the time of the continuous irradiation is preferably more than 2 hours.

[0018]

[18] Furthermore, the step of determining the inactivation time or inactivation dose of the radiation exposure comprises:

[0019]

[19] .a) determining the material and shape of a container for containing the sample to be irradiated, the material being a medical plastic material, the medical plastic material including but not limited to polypropylene (PP), polystyrene (PS) and the like, the shape being preferably one or more of a column shape, a barrel shape and a bag shape;

[0020]

[20] .b) determining the maximum capacity for inactivation;

[0021]

[21] .c) determining the concentration of the bacterial liquid, including determining the total amount of inactivated bacterial cells of each batch according to the yield design, and then selecting an appropriate concentration of the bacterial liquid according to the capacity requirement, wherein the concentration of the bacterial liquid satisfies the following formula:

number

[0022]

[22] .d) A step of selecting the dose rate, which includes designing different dose rate groups, inactivating the maximum capacity according to steps a) to c), and setting the time of the continuous irradiation to 2 hours or more, specifically, 2 hours or any time more than 2 hours;

[0023]

[23] .e) measuring a kill curve, which comprises irradiating multiple batches according to the dose rate and the duration of the irradiation determined in step d) and measuring the viable count at intermediate or extended time points, or measuring the viable count at intermediate or extended irradiation dose points to measure the kill curve;

[0024]

[24] .f) determining the inactivation time or the inactivation dose, which includes measuring the viable bacterial count at adjacent first, second and third time points or adjacent first, second and third exposure dose points according to the kill curve, wherein the interval between the time points is 20 min or more, the interval between the exposure dose points is 200 Gy or more, and the measurement results are less than 100 CFU / ml, 0 CFU / ml and 0 CFU / ml, respectively, and the second time point is the inactivation time and the second exposure dose point is the inactivation dose.

[0025]

[25] .Furthermore, after the inactivation time or inactivation dose is initially determined, the main parameters to be further optimized include dose rate, irradiation time, capacity, concentration of bacterial liquid, etc., and may also include inactivation dose. All the above parameters may be adjusted within appropriate ranges to accommodate industrial production.

[0026]

[26] .Furthermore, the method further includes a step of performing inactivation verification after the step of determining the inactivation time or the inactivation dose, the inactivation verification including a sterility test and a stability test after inactivation, and the stability test including an accelerated test under conditions of 25°C or 37°C and a real-time test under conditions of 2 to 8°C. The purpose of this step is to ensure that the bacterium (Pseudomonas aeruginosa) does not grow during long-term storage of the vaccine.

[0027]

[27] .Furthermore, industrial production validation should be performed. After selecting appropriate industrial production parameters, multiple batch validation should be performed to ensure that complete bacterial inactivation can be achieved in each batch.

[0028]

[28] . The present invention provides a Pseudomonas aeruginosa vaccine produced by the industrial production method. The vaccine is preferably in the form of an injection and / or a lyophilized powder. The vaccine is optionally administered by one or more of the following methods: subcutaneous injection, intramuscular injection, percutaneous scarification, nasal administration, and oral administration. The vaccine may be a whole cell vaccine or a multi-component vaccine. The multi-component vaccine contains both whole cells and intrabacterial immunogenic components such as membrane vesicles, nucleic acids, and cell fragments.

[0029]

[29] The present invention provides a use of the Pseudomonas aeruginosa vaccine produced by the present invention in the manufacture of a medicament for preventing an infectious disease caused by Pseudomonas aeruginosa, which may generally include Pseudomonas aeruginosa pneumonia, Pseudomonas aeruginosa infection associated with chronic obstructive pulmonary disease (COPD), Pseudomonas aeruginosa infection associated with burns, Pseudomonas aeruginosa keratitis, etc.

[0030]

[30] .Terminology

[0031]

[31] In the present invention, the "basic inactivation dose" refers to a dose that can basically inactivate the bacterial cells, and the lower limit of this dose is 1,000 Gy.

[0032]

[32] In the present invention, the term "total dose" refers to the total radiation dose accumulated over time during the irradiation process.

[0033]

[33] .The "inactivation dose" in the present invention refers to an irradiation dose (also understood as an "optimum inactivation dose") at which all bacteria in the inactivated bacteria liquid (also understood as an "inactivation stock solution") are just inactivated. "Just inactivated" refers to all bacteria in the inactivated bacteria liquid being just irreversibly killed. In some embodiments of the present invention, three adjacent irradiation dose points are taken according to the drawn sterilization curve, and the number of live bacteria is measured. If the measurement results are less than 100 CFU / ml, 0 CFU / ml, and 0 CFU / ml, respectively, the intermediate irradiation dose point among the three adjacent irradiation dose points is the inactivation dose, and the intervals between the irradiation dose points are the same and are 200 Gy or more. Furthermore, for example, when the irradiation dose is in the condition of "inactivation dose - 200 Gy", the number of live bacteria contained in the inactivation stock solution is less than 100 CFU / ml. When the irradiation dose is the inactivation dose, the number of viable bacteria contained in the inactivation stock solution is 0 CFU / ml. When the irradiation dose is in the condition of "inactivation dose + 200 Gy", the number of viable bacteria contained in the inactivation stock solution is 0 CFU / ml. Similarly, in some embodiments of the present invention, the number of viable bacteria is measured by taking three adjacent time points according to the drawn sterilization curve, and when the measurement results are less than 100 CFU / ml, 0 CFU / ml, and 0 CFU / ml, respectively, the intermediate time point between the three adjacent time points is the inactivation time, and the intervals between the time points are the same and are 20 min or more. Furthermore, for example, when the time point is in the condition of "inactivation time - 20 min", the number of viable bacteria contained in the inactivation stock solution is less than 100 CFU / ml. When the time point is the inactivation time, the number of viable bacteria contained in the inactivation stock solution is 0 CFU / ml. When the time point is in the condition of "inactivation time + 20 min", the number of viable bacteria contained in the inactivation stock solution is 0 CFU / ml. Effect of the Invention

[0034]

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

[0035]

[35] .(1) The present invention provides an industrial production method for Pseudomonas aeruginosa vaccine. That is, a series of standardized, programmed, and digitalized settings are adopted to realize the industrial production of a vaccine containing whole Pseudomonas aeruginosa cells and multiple intrabacterial immunogenic components with stable quality. Specifically, in the present invention, the bacterial liquid in which the density of the cells reaches a standard is directly centrifuged, eliminating the purification process of repeated centrifugation washing, improving the efficiency of industrial production and avoiding the bursting of the cells due to repeated centrifugation washing in large-scale industrial-grade production processes. Since the cells release more harmful substances such as endotoxins after bursting, repeated centrifugation washing in industrial-grade production processes actually increases the safety risk of the vaccine produced. The bursting of a large number of cells due to repeated centrifugation washing reduces the number of whole cells and generates more cell fragments, but these cell fragments are removed by centrifugation washing. In addition, the purification process of repeated centrifugation and washing also removes membrane vesicles and nucleic acids in the immunogenic components of the vaccine produced by the present invention, which ultimately leads to unstable quality of the industrially produced vaccine and a reduction in the types and number 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 process and improves the efficacy and safety of the vaccine.

[0036]

[36] . (2) The present invention optimizes the irradiation method of radiation, i.e., adopts a low dose rate (5-15 Gy / min) and long-term continuous irradiation (more than 2 h) method to reduce the total dose of radiation (≦2,000 Gy), thereby avoiding the destruction of bacterial cells caused by high doses of radiation and improving the immune efficacy and safety of the vaccine.

[0037]

[37] .(3) The vaccine produced by the present invention has good immunogenicity, so that not only the actual inoculation dose is small, but also it can prevent multiple infectious diseases caused by Pseudomonas aeruginosa, such as Pseudomonas aeruginosa pneumonia, Pseudomonas aeruginosa infection complicated with chronic obstructive pulmonary disease, Pseudomonas aeruginosa infection complicated with burn, Pseudomonas aeruginosa keratitis, etc. Furthermore, the vaccine produced by the present invention usually does not need to contain an adjuvant (i.e., does not need an adjuvant to enhance the body's immune response), but of course, in some scenarios (e.g., when it is necessary to produce a Pseudomonas aeruginosa vaccine with very good immunogenicity), an adjuvant may be used in combination. The above-mentioned good immunogenicity (protective efficacy of the vaccine) is achieved not only by the optimization in the above-mentioned production method, but also by optimizing the actual immunization procedure for the vaccine, i.e., by reducing the total number of inoculations and at the same time extending the immunization interval. The present invention has found that such an optimized immunization procedure contributes to improving the actual efficacy of the vaccine, and the extension of the immunization interval is more in line with the body's immune response rules to the vaccine.

[0038]

[38] .(4) The technical solution of the present invention adopts a physical method (i.e., radiation) to perform inactivation, which, compared with traditional chemical methods (e.g., using formaldehyde as a chemical inactivating agent), leaves no chemical residue (chemical inactivating agent), avoids to a certain extent the risks of allergic reactions and carcinogenesis caused by chemical inactivating agents, improves the safety of the vaccine, and reduces side effects.

[39] .In order to more clearly describe the technical solutions in the embodiments of the present invention or the prior art, the following briefly introduces the accompanying drawings used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings from these drawings without creative efforts. [Brief description of the drawings]

[0039] [Figure 1]

[40] FIG. 1 shows the protective effect of the vaccine produced by the method provided by the present invention in a mouse model of Pseudomonas aeruginosa pneumonia. [Diagram 2]

[41] FIG. 2 shows the protective effect of the vaccine produced by the method provided by the present invention in a model of Pseudomonas aeruginosa infection complicated with COPD. [Diagram 3]

[42] Figure 3 shows the protective effect of the vaccine produced by the method provided by the present invention in a mouse burn model. [Figure 4]

[43] Figure 4 shows the protective effect of the vaccine produced by the method provided by the present invention in a mouse model of corneal infection. [Diagram 5]

[44] . Figure 5 shows a schematic diagram of the killer kill curve. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040]

[45] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described below clearly and completely with the accompanying drawings. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and are not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive ideas are included in the protection scope of the present invention.

[0041]

[46] . It should be noted that, as used herein, the terms "comprise," "include," or any other variations thereof are intended to cover a non-exclusive inclusion. Thus, a process, method, article, or apparatus comprising a set of elements includes not only those elements but also other elements not expressly recited or inherent to such process, method, article, or apparatus. Absent further limitations, an element defined by the phrase "comprises a" does not preclude the presence of additional identical elements in the process, method, article, or apparatus that comprises that element.

[0042]

[47] For example, the term "about" as used herein typically refers to + / - 5% of the stated value, more typically + / - 4% of the value, more typically + / - 3% of the value, more typically + / - 2% of the value, even more typically + / - 1% of the value, and even more typically + / - 0.5% of the value.

[0043]

[48] . Certain embodiments may be disclosed herein in the form of a range. It should be understood that this "within a range" description is merely for convenience and brevity and should not be construed as an inflexible limitation on the disclosed range. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, a description of a range from 1 to 6 should be considered to have specifically disclosed 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within this range such as 1, 2, 3, 4, 5, and 6. The above rule applies regardless of the breadth of the range. EXAMPLES

[0044]

[49] . Example 1: Manufacturing method for Pseudomonas aeruginosa vaccine

[0045]

[50] .S1 The production P. aeruginosa strain is cultured in a suitable medium to produce a seed solution, preferably an animal-derived component-free medium such as tryptone soy broth (TSB).

[0046]

[51] .S2 The seed liquid is inoculated into a fermenter according to 2%-10% of the fermentation volume and fermented, where the fermentation temperature is 30-40°C, the pH value is 5-9 (preferably 7), the rotation speed is 100-400 rpm, the aeration amount is 2-5 L / min, the dissolved oxygen is 10%-30%, and the fermentation time is 3-8 h.

[0047]

[52] .S3 The density of the bacteria in the fermenter is monitored, and when the density of the bacteria in the fermenter reaches a standard (i.e., when the absorbance value, which is the density of the bacteria, reaches 1 to 3 OD), the fermented bacteria liquid is collected and directly centrifuged at a centrifugal force of 3,000 to 8,000 × g, and the bacteria are collected after 10 to 30 minutes.

[0048]

[53] .S4 The bacteria are resuspended in an isotonic injection solution, the concentration is adjusted, and then the bacteria are irradiated to eliminate the proliferation activity of the bacteria. The radiation used for the irradiation is X-rays, γ-rays, and isotope radiation source Co 60 The isotonic injection solution includes a solution such as physiological saline. After the irradiation, a step of measuring the components and content of the bacterial liquid may be performed. The components and content of the bacterial liquid may be measured by using one or more of spectrophotometry, density gradient centrifugation, scanning electron microscopy, and transmission electron microscopy.

[0049]

[54] .S5 The irradiated bacterial liquid is collected and tested. If the bacterial liquid contains whole bacterial cells and intrabacterial immunogenic components, and the proportion of the whole bacterial cells is greater than 80%, it is considered to be passed. The intrabacterial immunogenic components include membrane vesicles, nucleic acids, and bacterial cell fragments. In general, the proportion of the whole bacterial cells in the bacterial liquid obtained by the industrial production method provided by the present invention should be maintained at 80% or more. The purpose of the bacterial liquid test is to ensure that the number of components (whole bacterial cells and intrabacterial immunogenic components) in the bacterial liquid is stable when other parameters are adjusted.

[0050]

[55] .S6 The bacterial solution that passed the inspection in step S5 is resuspended in the isotonic injection solution to a concentration of 1.0 x 10 7 ~3.0×10 7 The concentration is adjusted to cells / ml to produce a Pseudomonas aeruginosa vaccine.

[0051]

[56] .Furthermore, the basic inactivation dose of the radiation exposure should not be less than 1,000 Gy, and the total dose of the radiation exposure is greater than the basic inactivation dose of the radiation exposure. The radiation exposure is performed in a manner of low dose rate, long time, and continuous exposure. The dose rate of the radiation exposure is 5-15 Gy / min, and the time of the continuous exposure is more than 2 hours. In some embodiments, the total dose of the radiation exposure in the present invention may be controlled in the range of ≦2000 Gy.

[0052]

[57] . The step of determining the inactivation time or inactivation dose of the radiation exposure comprises:

[0053]

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

[0054]

[59] .b. Determine the maximum capacity for inactivation. The capacity is determined by the shape of the container and the height of the liquid surface. The difference between the radiation dose rate passing through the highest (near) part of the liquid surface and the radiation dose rate passing through the lowest (farther) part of the liquid surface does not exceed 20% of the dose rate at the highest (near) part of the liquid surface, and the volume of the bacterial liquid corresponding to said highest (near) part of the liquid surface is the maximum capacity for inactivation. Using a radiation dose detector, the detection probes are placed at the highest (near) and lowest (farther) parts of the liquid surface from the radiation source, respectively, and the irradiator is started, so that the radiation dose detector can read the exposure dose rate. This 20% is based on the quality control requirement to ensure the uniformity of the exposure dose received by the irradiated sample.

[0055]

[60] .c. Determining the concentration of the bacteria liquid, which includes determining the total amount of inactivated bacteria of each batch according to the yield design, and then selecting an appropriate concentration of the bacteria liquid according to the capacity requirement, and the concentration of the bacteria liquid is determined according to the following formula:

number

[0056]

[61] .d. Select the dose rate. Design different dose rate groups and inactivate the maximum capacity according to steps a to c. Set the continuous irradiation time to 2 hours or more. Specifically, it may be set to any time of 2 hours or more.

[0057]

[62] .e. Measure the kill curve. According to the dose rate and the time of the irradiation determined in step d, multiple batches are irradiated, and the viable count is measured at the intermediate or extended time points, or the viable count is measured at the intermediate or extended irradiation dose points, to measure the kill curve. It should be noted that the inactivation process can be controlled by controlling the irradiation time or the irradiation dose, and thus the inactivation parameters can be considered from the control of the time or the control of the dose. For example, after the inactivation time is determined, samples may be taken at a time point before (e.g., "intermediate time point") and a time point after (e.g., "extended time point") the inactivation time to verify whether the inactivation time meets the complete inactivation requirement. Similarly, after the inactivation dose is determined, samples may be taken at an irradiation dose point before (e.g., "intermediate irradiation dose point") and an irradiation dose point after (e.g., "extended irradiation dose point") the inactivation dose to verify whether the inactivation dose meets the complete inactivation requirement.

[0058]

[63] .f. Determine the inactivation dose or the inactivation time. According to the sterilization curve, the inactivation time or the inactivation dose is obtained as follows: Three adjacent time points (first time point, second time point, third time point) or three adjacent irradiation dose points (first irradiation dose point, second irradiation dose point, third irradiation dose point) are taken to measure the viable bacteria count. The interval between the time points is 20 min or more, and the interval between the irradiation dose points is 200 Gy or more. If the measurement results are less than 100 CFU / ml, 0 CFU / ml, and 0 CFU / ml, respectively, the intermediate time point (i.e., the second time point) is the inactivation time, and the intermediate irradiation dose point (i.e., the second irradiation dose point) is the inactivation dose. A schematic diagram of the sterilization curve is shown in Figure 5. According to the sterilization curve, three adjacent irradiation dose points "1,200 Gy", "1,400 Gy", and "1,600 Gy" are taken to measure the viable bacteria count. If the measurement results are less than 100 CFU / ml, 0 CFU / ml, and 0 CFU / ml, respectively, the intermediate exposure dose point "1,400 Gy" among the three adjacent exposure dose points is the inactivation dose. Similarly, according to the drawn bactericidal curve, three adjacent time points "120 min", "140 min", and "160 min" are taken to measure the viable bacteria count. If the measurement results are less than 100 CFU / ml, 0 CFU / ml, and 0 CFU / ml, respectively, the intermediate time point "140 min" among the three adjacent time points is the inactivation time.

[0059]

[64] . After initially determining the inactivation time or inactivation dose, the main parameters to be further optimized include dose rate, irradiation time, capacity, concentration of bacterial liquid, etc. and may also include inactivation dose. All the above parameters may be adjusted within appropriate ranges to accommodate industrial production.

[0060]

[65] . After the inactivation time or inactivation dose is determined, inactivation verification is performed. The inactivation verification includes a sterility test and a stability test after inactivation. The stability test includes an accelerated test under conditions of 25°C or 37°C and a real-time test under conditions of 2-8°C. The purpose of this step is to ensure that the bacterium (Pseudomonas aeruginosa) does not grow during long-term storage of the vaccine.

[0061]

[66] . Validate industrial production. After selecting appropriate industrial production parameters, multiple batch validation should be performed to ensure that complete bacterial inactivation is achieved in each batch.

[0062]

[67] . The present invention provides a Pseudomonas aeruginosa vaccine produced by the industrial production method. The vaccine may be a whole cell vaccine or a multi-component vaccine. The multi-component vaccine contains both whole cells and intrabacterial immunogenic components such as membrane vesicles, nucleic acids, and cell fragments. The vaccine is in the form of an injection and / or a lyophilized powder. The vaccine is optionally administered by one or more of the following methods: subcutaneous injection, intramuscular injection, percutaneous scarification, nasal administration, and oral administration.

[0063]

[68] In some embodiments of the present invention, the mass ratio of whole cells, membrane vesicles, nucleic acids and cell fragments is preferably 98:1:0.1:1. Under this ratio, the vaccine has good immunogenicity and few side effects.

[0064]

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

[0065]

[70] . There are 20 serotypes of P. aeruginosa, and the major serotypes isolated in clinical trials are O6, O11, and O4, accounting for approximately 20%, 15%, and 10%, respectively. In this example, a vaccine was produced using P. aeruginosa PA1 (serotype O5) according to the process described above. Following the immunization procedure on days 0, 14, and 28, 1.0 × 10 7Mice were subcutaneously immunized with 0.5 mL of vaccine containing 1000 bacteria. Seven days after the last immunization, the mice were infected in the airways with P. aeruginosa PA1 (O5), carbapenem-resistant P. aeruginosa clinical isolate C58 (O6), standard strains ATCC33358 (O11), ATCC33351 (O4), and laboratory strain PA14 (O14), respectively. 24 h after infection, lung tissues were collected to measure bacterial load, and the detailed results are shown in Figure 1. The vaccine could effectively reduce the bacterial load 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). The results suggested that the vaccine had a protective effect against infection with different serotypes of Pseudomonas aeruginosa and could prevent multiple infectious diseases caused by Pseudomonas aeruginosa.

[0066]

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

[0067]

[72] . In this example, a vaccine was produced using Pseudomonas aeruginosa PA1 (serotype O5) according to the process described above. Two immunization procedures were performed on days 0, 3, and 7 and days 0, 7, and 14, and 1.0 × 10 7 Mice were subcutaneously immunized with 0.5 mL of vaccine containing the bacterium. Elastase was injected into the airways 10 days before infection to induce the establishment of a COPD model. Seven days after the last immunization, the lungs were infected with the bacterium (P. aeruginosa PA1) via the airways. 24 h after infection, the mice were killed and lung tissue was harvested to measure the bacterial load; detailed results are shown in Figure 2. The vaccine was immunized according to two procedures, and both had a significant internal protective effect against P. aeruginosa lung infection complicated by COPD (P<0.01), reducing the bacterial load in the lung tissue by approximately 2log 10 The results suggest that the vaccine has a significant protective effect against P. aeruginosa lung infection associated with COPD.

[0068]

[73] . Example 4: Protective effect of vaccine in a mouse model of thermal injury

[0069] In this example, a vaccine was produced using P. aeruginosa PA1 (serotype O5) according to the process described above. Following the immunization procedure on days 0, 14, and 28, 1.0 × 10 7 Mice were subcutaneously immunized with 0.5 mL of the vaccine containing the bacteria. Seven days after the last immunization, the back skin of the mice was burned with a heat gun to establish a burn model. Two hours after the burn, the wound was subcutaneously infected with P. aeruginosa PA1. 24 hours later, the skin including the wound was collected and the bacterial load was measured. For details of the results, see Figure 3. This vaccine significantly reduced the bacterial load of the P. aeruginosa-infected wound associated with burn (P<0.001). The results suggested that this vaccine has a significant protective effect against P. aeruginosa infection associated with burn.

[0070]

[75] . Example 5: Protective effect of vaccine in a mouse model of corneal infection

[0071]

[76] . In this example, a vaccine was produced using Pseudomonas aeruginosa PA1 (serotype O5) by the above-mentioned process. The vaccine (1.0 × 10 7 Mice were immunized with 20μL of vaccine containing 100 bacteria by nasal drop. Seven days after the last immunization, the corneas of the mice were scratched with a needle tip and infected with the bacteria (P. aeruginosa PA1). After 24h, the corneas were harvested and the bacterial load was measured. For details of the results, see Figure 4. The vaccine was immunized according to two procedures, and both had a significant protective effect against P. aeruginosa corneal infection (P<0.01). The results suggested that the vaccine has a significant protective effect against P. aeruginosa corneal infection.

[0072]

[77] Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is merely schematic and not limiting. There are many forms that a person skilled in the art may make by being inspired by the present invention without departing from the purpose of the present invention and the scope protected by the claims, and all of them fall within the scope of protection of the present invention.

Claims

1. A method for industrially producing a Pseudomonas aeruginosa vaccine, comprising: S1 Cultivating a production Pseudomonas aeruginosa strain in a suitable medium to produce a seed solution; S2: inoculating the seed liquid into a fermenter according to 2% to 10% of the fermentation volume, and fermenting the seed liquid in the fermenter, the fermentation temperature is 30 to 40° C., the pH value is 5 to 9, the rotation speed is 100 to 400 rpm, the aeration amount is 2 to 5 L / min, the dissolved oxygen is 10% to 30%, and the fermentation time is 3 to 8 h; S3: monitoring the density of the bacteria in the fermenter, and after the density of the bacteria in the fermenter reaches a standard, collecting the bacteria liquid in the fermenter and directly centrifuging it at a centrifugal force of 3,000 to 8,000 × g, and collecting the bacteria after 10 to 30 minutes; S4: A step of resuspending the bacterial cells in an isotonic injection solution, adjusting the concentration, and then irradiating the bacterial cells with radiation to eliminate their proliferation activity, the radiation used for the irradiation being X-rays, γ-rays, and isotope radiation source Co ６０ and S5: A step of sampling the bacterial liquid after the radiation irradiation and testing the bacterial liquid, the bacterial liquid containing all bacterial cells and intrabacterial immunogenic components, and a step of determining the bacterial liquid as passing when the ratio of the total bacterial cells is more than 80%; S6: The bacterial solution that passed the inspection in step S5 is resuspended in the isotonic injection solution to a concentration of 1.0 x 10 ７ ~3.0 x 10 ７ producing the Pseudomonas aeruginosa vaccine by adjusting the concentration to 0.1% by mass / ml; The industrial production method according to claim 1,

2. The method according to claim 1, wherein the standard is reached when an absorbance value, which is the density of the bacteria in the fermenter, reaches 1 to 3 OD.

3. 2. The method of claim 1, wherein the bacterial immunogenic components include membrane vesicles, nucleic acids, and bacterial somatic fragments.

4. 2. The method according to claim 1, further comprising a step of measuring components and contents of the bacterial liquid after the radiation exposure after step S4, wherein the method of measuring the components and contents of the bacterial liquid after the radiation exposure includes one or more of spectrophotometry, density gradient centrifugation, scanning electron microscopy, and transmission electron microscopy.

5. 2. The method of claim 1, wherein the basic inactivating dose of radiation exposure must not be less than 1,000 Gy and the total dose of radiation exposure is greater than the basic inactivating dose of radiation exposure.

6. The method according to claim 5, characterized in that the radiation exposure is carried out in a manner of low dose rate, long time, continuous exposure, the dose rate is preferably 5-15 Gy / min, and the time of the continuous exposure is preferably more than 2h.

7. The step of determining the inactivation time or inactivation dose of the radiation exposure comprises: determining the material and shape of a container for containing the sample to be irradiated; determining a maximum capacity for inactivation; determining the concentration of the bacterial liquid, comprising determining the total amount of inactivated bacterial cells of each batch according to yield design, and then selecting an appropriate concentration of the bacterial liquid according to capacity requirements, wherein the concentration of the bacterial liquid satisfies the following formula: [0010] In the formula, N is the concentration of the bacterial liquid, A is the number of batches to be produced, B is the amount of bacterial cells per batch, and V is the total volume of the bacterial liquid, and V is equal to or less than the maximum capacity. A step of selecting the dose rate, comprising designing different dose rate groups, inactivating the maximum capacity according to steps a) to c), and setting the time of the continuous irradiation to 2 hours or more; A step of measuring a kill curve, comprising: performing irradiation of multiple batches according to the dose rate and the irradiation time determined in step d) and measuring the viable count at intermediate or extended time points, or measuring the viable count at intermediate or extended irradiation dose points to measure the kill curve; determining the inactivation time or the inactivation dose, comprising measuring the number of viable bacteria at adjacent first, second and third time points or adjacent first, second and third exposure dose points according to the sterilization curve, wherein the interval between the time points is 20 min or more, the interval between the exposure dose points is 200 Gy or more, and when the measurement 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 exposure dose point is the inactivation dose; 2. The method of claim 1, comprising:

8. 8. The method according to claim 7, further comprising a step of performing an inactivation verification after the step of determining the inactivation time or the inactivation dose, the inactivation verification including a sterility test and a stability test after the inactivation, and the stability test including an accelerated test under conditions of 25°C or 37°C and a real-time test under conditions of 2-8°C.

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