Human mycoplasma pneumoniae inactivated vaccine preparation and application thereof

Abstract

The invention provides a human mycoplasma pneumoniae inactivated vaccine preparation and application thereof. The human mycoplasma pneumoniae inactivated vaccine preparation comprises inactivated mycoplasma pneumoniae thallus antigens and mucous membrane adjuvants. Wherein the mass ratio of the inactivated mycoplasma pneumoniae thallus antigen to the mucous membrane adjuvant is (2-5): 1. The inactivated vaccine preparation disclosed by the invention can induce an organism to generate mycoplasma pneumoniae specific IgG and IgA antibody titers after immunization in a nasal drop way, can protect the organism after mycoplasma pneumoniae infection, avoids weight loss and severe lung injury, fills the blank in the field of mycoplasma pneumoniae vaccines, and meets clinical requirements.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a human Mycoplasma pneumoniae inactivated vaccine preparation and application thereof. Background Art

[0002] Mycoplasma pneumoniae (Mp) is a small prokaryotic microorganism lacking a cell wall, exhibiting high polymorphism, and capable of growth in inanimate culture media. Studies have shown that Mp is the leading cause of community-acquired pneumonia (CAP) in both children and adults, accounting for 10% to 40% of upper respiratory tract infections, respectively. While Mp infections are generally self-limited, they can also cause severe lung infections, encephalitis, and other neurological complications.

[0003] Macrolide antibiotics are commonly used as first-line antimicrobials in clinical practice and are also used to treat M. pylori infection. Unfortunately, with the increasing prevalence of antibiotic overuse, the global emergence of macrolide resistance in M. pylori is becoming increasingly serious. Therefore, the most cost-effective means of preventing and controlling M. pylori infection and epidemics is vaccination of susceptible populations. However, no vaccine is currently available for general use, necessitating the urgent need to research and develop a M. pylori vaccine with consistent safety and efficacy.

[0004] Although the development of a variety of human MP vaccines is underway, no human MP vaccine has been successfully developed. MP vaccine research mainly includes inactivated vaccines, live attenuated vaccines, protein vaccines and nucleic acid vaccines. MP inactivated vaccines are usually inactivated using chemical methods (paraformaldehyde, β-propiolactone) and prepared with adjuvants. Commonly used adjuvants include aluminum, CpG, MF59, etc. Previous studies on MP inactivated vaccines have found that although they can induce host immune responses and inhibit the load of MP in the body, their protective effects are limited, and the vaccine is prone to cause severe histopathological reactions after injection. Therefore, it is necessary to consider improving the current MP inactivated vaccine formula in order to avoid the occurrence of pathological damage while inducing immune responses. Summary of the Invention

[0005] In response to the gap in the existing Mycoplasma pneumoniae vaccine field, the present invention aims to provide a human Mycoplasma pneumoniae inactivated vaccine preparation and its application. This vaccine preparation can induce the body to produce Mycoplasma pneumoniae-specific IgG and IgA antibody titers after immunization via nasal drops, and can protect the body after Mycoplasma pneumoniae infection, preventing weight loss and severe lung damage. This fills the gap in the field of Mycoplasma pneumoniae vaccines and meets clinical needs.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a human Mycoplasma pneumoniae inactivated vaccine preparation, wherein the vaccine preparation comprises an inactivated Mycoplasma pneumoniae bacterial antigen and a mucosal adjuvant;

[0008] The mass ratio of the inactivated Mycoplasma pneumoniae bacterial antigen and the mucosal adjuvant in the vaccine preparation is 2 to 5:1; preferably, the mass ratio of the two is 4:1.

[0009] Furthermore, the mucosal adjuvant includes bacterial flagellin FLA-ST (flagellin from Salmonella typhimurium) and other mucosal adjuvants with similar functions, including Escherichia coli heat-sensitive toxin LT and cholera toxin CT.

[0010] Furthermore, the Mycoplasma pneumoniae cells are inactivated chemically or physically to obtain inactivated Mycoplasma pneumoniae cell antigens. In some embodiments, the Mycoplasma pneumoniae cells are inactivated by adding propiolactone.

[0011] Preferably, each dose of the vaccine preparation contains 20 μg of inactivated Mycoplasma pneumoniae antigen and 5 μg of mucosal adjuvant.

[0012] Furthermore, the inactivated vaccine preparation is in the form of mucosal immunization, and the administration methods include nasal drops, oral inhalation, and vaginal suppositories.

[0013] The second aspect of the present invention provides use of the inactivated vaccine preparation described in the first aspect in preparing a vaccine for inducing an antibody response and a mucosal system response against Mycoplasma pneumoniae in a subject.

[0014] The third aspect of the present invention provides use of the inactivated vaccine preparation described in the first aspect in preparing a vaccine for preventing Mycoplasma pneumoniae infection.

[0015] Compared with the prior art, the present invention has the following significant beneficial effects:

[0016] 1. A breakthrough solution to the problem of low antigen-specific antibody production induced by inactivated Mycoplasma pneumoniae bacteria and poor protection against pathogen infection, significantly improving the effectiveness of vaccine protection:

[0017] Traditional unadjuvanted inactivated MP vaccines have significant limitations: on the one hand, the levels of antigen-specific antibodies (including IgG and IgA) induced by them are extremely low (as shown in the experimental data of the present invention, the serum IgG titer of the unadjuvanted group is significantly lower than that of the aluminum adjuvanted group, while the IgA titer of the mucosal sample is significantly lower than that of the mucosal adjuvanted group); on the other hand, due to insufficient antibody levels, the protective effect against pathogen infection is almost negligible - the lung pathogen load remains high and cannot effectively resist MP infection ( Figure 1 A-lung disease load detection); The weight of mice continued to decrease after the challenge ( Figure 1 (B-weight monitoring) Even the weight of mice in the non-adjuvant group was significantly lower than that of the model group starting from the first day after challenge (1 dpi);

[0018] The present invention significantly enhances the intensity of vaccine-induced immune response by adding mucosal adjuvants: not only the specific IgA titer in mucosal samples is greatly increased (the IgA level in alveolar lavage fluid, saliva and vaginal swabs is significantly better than that in the adjuvant-free group), but also the weight of mice is stable and the MP load in the lungs is significantly reduced after infection, proving that the addition of adjuvants can effectively solve the problem of "low antibody level and poor protection effect" of inactivated vaccines without adjuvants, giving practical application value to MP inactivated vaccines.

[0019] 2. A breakthrough solution to the Enhanced Respiratory Disease (ERD) problem caused by the application of traditional adjuvants in the development of inactivated Mycoplasma pneumoniae vaccines, significantly improving vaccine safety and protective effectiveness

[0020] In the research of inactivated Mp vaccines, the choice of adjuvant has long faced a contradiction: without an adjuvant, the vaccine has no protective effect; if a traditional aluminum adjuvant is added, although it may increase some antibody levels, it will induce enhanced respiratory disease (ERD) - that is, the lung pathological damage is aggravated after vaccination, and the degree of damage is significantly higher than that of the non-adjuvanted group (the lung pathology scoring of Example 7 of the present invention confirms that the alveolar inflammation, alveolar wall thickening and fibrosis in the aluminum adjuvanted group are much higher than those in the non-adjuvanted group). This phenomenon of "vaccine exacerbating pathological damage" is a key bottleneck restricting the development of Mp vaccines.

[0021] This invention's innovative use of a mucosal adjuvant completely resolves this contradiction: experimental data showed that the mucosal adjuvant group not only did not experience ERD, but also had lung pathology scores close to normal levels (mild inflammatory cell infiltration, no significant bleeding or fibrosis), and the protective effect after challenge was significantly better than the aluminum adjuvant and non-adjuvant groups. This breakthrough proves that the addition of an adjuvant is necessary for inactivated Mp vaccines, but traditional aluminum adjuvants must be abandoned. Mucosal adjuvants can simultaneously achieve "no pathology risk" and "highly effective protection," providing a new solution for balancing vaccine safety and efficacy.

[0022] 3. Subvert the traditional antibody evaluation system, identify mucosal-specific IgA as the key protection indicator, and accurately activate the mucosal immune barrier

[0023] Traditionally, it is believed that high titers of IgG antibodies in the serum induced by vaccines are the core protection against pathogen infection. However, experimental data from this study confirms that the key to defense against Mycoplasma pneumoniae infection lies in the mucosal immune response, rather than simply serum IgG levels.

[0024] The experimental results showed that although the serum IgG antibody titer of the mucosal adjuvant group was lower than that of the aluminum adjuvant group, the specific IgA antibody titer in mucosal samples such as alveolar lavage fluid, saliva swabs, and vaginal swabs was significantly higher than that of the non-adjuvant group and the aluminum adjuvant group. At the same time, this high titer of IgA was directly associated with the stable weight of mice after challenge, reduced lung load, and reduced pathological damage, proving that the specific IgA produced by mucosal adjuvants through activation of the mucosal immune pathway is the core protective factor against Mycoplasma pneumoniae infection. This discovery redefines the effectiveness evaluation criteria for Mycoplasma pneumoniae vaccines and provides a precise scientific basis for vaccine development.

[0025] 4. The mucosal immune pathway is efficient and convenient, and is suitable for the mucosal targeted defense needs of Mycoplasma pneumoniae infection.

[0026] This invention uses mucosal immunization methods, such as nasal drops and oral inhalation, to directly target the respiratory mucosa (the primary site of infection for Mycoplasma pneumoniae). This rapidly activates the local mucosal immune response, establishing the first line of defense at the infection portal. Compared to traditional routes such as intramuscular injection, mucosal immunization is not only more convenient and easily accepted by recipients (especially children), but also more precisely targets and prevents Mycoplasma pneumoniae from adhering to and invading the respiratory mucosa, significantly enhancing the immediacy and specificity of immune protection.

[0027] 5. Fill the gap in the field of Mycoplasma pneumoniae vaccines and meet urgent clinical needs

[0028] Currently, there is no approved vaccine for Mycoplasma pneumoniae, posing significant challenges to clinical prevention and treatment. The inactivated vaccine formulation provided by the present invention, through its innovative "whole-inactivated bacteria + mucosal adjuvant" formulation and mucosal immunization pathway, can induce a strong and sustained mucosal immune response (specific IgA) while avoiding the pathological risks of traditional adjuvants. It can effectively prevent Mycoplasma pneumoniae infection and related complications (such as severe lung infection and nervous system damage). It is particularly suitable for children and adults, a high-risk population for community-acquired pneumonia. It also provides a new approach for the prevention and treatment of Mycoplasma pneumoniae infection and has significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below with reference to the accompanying drawings and examples:

[0030] Figure 1 The figures show the MP load in the lungs of animals 7 days after infection (A) and the changes in body weight monitored for 7 consecutive days (B); in Figure A, the data of the four groups, namely, no adjuvant, aluminum adjuvant, mucosal adjuvant and model group, were significantly different by one-way analysis of variance, with the model group as the control for the analysis data, and the mark *** represents the difference value p < 0.001.

[0031] Figure 2Figure 2 shows antigen-specific antibody titers (IgG, IgG2a, and IgG1) measured in serum samples obtained from animals immunized with different vaccine groups before challenge. (A) Specific IgG antibody titer; (B) Specific IgG2a antibody titer; (C) Specific IgG1 antibody titer. Data from the unadjuvanted, aluminum-adjuvanted, and mucosal adjuvanted groups showed significant differences using one-way ANOVA. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. (D) Calculation of the IgG2a to IgG1 ratio; the dashed line represents a ratio of 1.

[0032] Figure 3 Figure 2 shows the determination of antigen-specific antibody titers in mucosal samples taken 7 days after animals were immunized with different groups of vaccines and challenged with pathogens, including (A) alveolar lavage fluid, (B) vaginal swabs, and (C) saliva swabs; the data of the three groups of no adjuvant, aluminum adjuvant, and mucosal adjuvant were significantly different by one-way analysis of variance, and the * in the figure represents the difference value p < 0.05.

[0033] Figure 4 Figure 2 shows the lung pathological changes measured 7 days after animals were immunized with different groups of vaccines and challenged with pathogens; (A) is a representative image of each group of slices, with the standard line representing 200 μm; (B) is the statistical summary of the values ​​of each group after randomly selecting a visual field for each mouse; the data of the five groups of no adjuvant, aluminum adjuvant, mucosal adjuvant, model group and blank control group were significantly different by one-way analysis of variance, with the model group as the control for the analyzed data. *** represents a difference value of p < 0.001, and **** represents a difference value of p < 0.0001. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be further described below in conjunction with the embodiments and drawings, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely illustrative and do not limit the scope of the present invention.

[0035] It should be noted that the experimental methods used in the following examples are conventional methods in the art unless otherwise specified. Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention relates.

[0036] Example 1: Culture and counting of Mp

[0037] Take one tube (1 mL) of Mp stock solution and place it in a 37°C water bath to quickly melt. Add 9 mL of preheated PPLO culture medium and 1 mL of Mp stock solution to a 15 mL centrifuge tube. Perform the entire process aseptically, mix well in a vortex shaker after addition, and place the mixed centrifuge tube in a constant temperature incubator at 37°C containing 5% carbon dioxide for incubation for 3-5 days, observing the color change of the culture medium every day. According to the physical and chemical properties of Mp, during the metabolic process, the sugar substances in the PPLO liquid culture medium can be converted into acid substances, thereby reducing the pH of the PPLO culture medium from 7.8 to 6.8. At this time, the color of the PPLO liquid culture medium will also change from red to orange. Generally speaking, the color change occurs on the third day after recovery, that is, when the Mp growth enters the logarithmic phase.

[0038] Dilute the MP culture solution 10x, 100x, and 1000x, and evenly spread 100 μL of each dilution onto a PPLO solid culture medium plate. Incubate the solid culture medium in a 37°C incubator with 5% carbon dioxide for 5-7 days, observing colony growth daily. After seven days of incubation, place the culture plate under an inverted microscope, count the colonies, and calculate the CFU value using the following formula:

[0039] CFU / mL=(number of colonies in 10 fields of view / 10)*(area of ​​a single field of view / area of ​​the plate)*dilution factor*10.

[0040] Example 2: Preparation of Mp inactivated whole bacteria

[0041] Mp was cultured in PPLO medium to the logarithmic phase. Take 1000 mL of Mp culture medium cultured to the logarithmic phase, add 1 mL of β-propiolactone (0.1% by volume) to inactivate Mp, and inactivate for 10 hours at room temperature. Then place it in a 37°C water bath for 2 hours and 30 minutes to terminate the inactivation. Collect the bacteria by centrifugation (12000 rpm, 1 hour), resuspend twice with PBS and wash the bacteria by centrifugation. In the last step, resuspend the Mycoplasma pneumoniae bacteria with 10 mL of PBS.

[0042] Example 3: Preparation, Immunization and Related Detection of Mp Inactivated Vaccine

[0043] The antigen concentration in the inactivated whole-cell Mp prepared in Example 2 was quantified using a BCA quantitative kit. Vaccine preparation and immunization were performed according to the grouping in Table 1.

[0044] Experimental animals: Pathogen-free female BALB / c mice aged 6-8 weeks provided by the Experimental Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences were used. The mice were randomly divided into 5 groups, with 6 mice in each group. They were housed under SPF conditions and had free access to food and water.

[0045] Immunization doses: According to the settings in Table 1, mice were immunized by intramuscular injection or intranasal drops, 50 μL per mouse. The blank group was injected with an equal amount of PBS. 4 weeks later, a second dose of vaccine was administered in the same manner.

[0046] Table 1

[0047] Group antigen adjuvant Immunization method Fighting the virus No adjuvant group 20μg / injection whole bacterial antigen PBS muscle immunity Nasal drops Mp Aluminum adjuvant group 20μg / injection whole bacterial antigen 50μg / injection aluminum adjuvant muscle immunity Nasal drops Mp Mucosal adjuvant group 20μg / injection whole bacterial antigen 5μg / injection FLA-ST Nasal drop immunization Nasal drops Mp challenge control group PBS PBS muscle immunity Nasal drops Mp Blank control group PBS PBS muscle immunity PBS nasal drops

[0048] Two weeks after the second immunization, mice in each group were infected with MP or PBS. Mice were anesthetized with isoflurane gas at a concentration of 5×10 8 Each mouse was challenged with 50 μL of Mp bacterial solution containing 100 CFU / mL. A blank control group received 50 μL of PBS intranasally. This was designated as day 0 of challenge (dpi). Following challenge, all mice were weighed daily, and weight changes were recorded for 5 days (0, 1, 2, 3, and 4 dpi).

[0049] like Figure 1 Middle A shows that Mp challenge (model group) can significantly increase the lung pathogen load of animals to 1.85×10 6 CFU / mL, the mucosal adjuvant group was able to reduce the lung load to the lowest level, which was 4.7×10 5 CFU / mL, while the lung load in the non-adjuvant group and the aluminum adjuvant group was reduced to 9.6×10 5 CFU / mL, 9.5×10 5 CFU / mL. Figure 1 Middle B shows that the body weight of animals after Mp challenge (model group) dropped to the lowest value on the second day after challenge (2dpi), which was 83.426% ± 1.324% of the initial body weight. The mucosal adjuvant group had the best protection effect, with the lowest body weight occurring at 2dpi, which was 92.067% ± 4.176% of the initial body weight. The protective effect of the no adjuvant group and the aluminum adjuvant group on weight loss was similar, and neither group reduced the degree of weight loss after Mp challenge. Compared with the model group, which saw weight recovery at 3dpi, the weight of the no adjuvant group and the aluminum adjuvant group continued to decrease at 3dpi, reaching the lowest value, which was 79.346% ± 4.64% and 80% ± 1.902%, respectively.

[0050] Acquisition and testing of immune serum:

[0051] Two weeks after the last immunization and before challenge, venous blood was collected from mice. The blood was incubated at 4°C overnight and centrifuged at 3000 rpm for 20 minutes to obtain serum. Mp-specific antibody titers were determined using an indirect enzyme-linked immunosorbent assay (ELISA).

[0052] The specific implementation method is as follows: Mp whole bacterial protein is plated at a concentration of 10 μg / mL, with a volume of 50 μL per well. After incubation at 4°C overnight, the plates are washed twice with PBST. 50 μL of 5% skim milk is added to each well and blocked at 37°C for 1 hour. After blocking, the plates are washed three times, and the serially diluted serum samples are added to each well. The plates are incubated at 37°C for another 1 hour. After the incubation period, the plates are washed four times, and HRP-labeled IgG antibody (10,000-fold diluted in 1% skim milk) is added to each well. After incubation for 30 minutes, the plates are washed five times, the liquid in the plates is patted dry, and 50 μL of TMB colorimetric solution is added to each well. After 5 minutes, 50 μL of sulfuric acid (2 M) is added to each well to stop the color development. The absorbance value is read at 450 nm. The readings of the blank control group are averaged and multiplied by 2.1 to serve as the cutoff value. That is, the serum dilution multiple with readings higher than the cutoff value in other groups was defined as the IgG antibody titer value of the serum in that group.

[0053] like Figure 2 As shown, immunization with inactivated Mycoplasma pneumoniae alone (no adjuvant group) increased the IgG antibody titer to 10,240 ( Figure 2 A), IgG2a antibody titer increased to 1,024 ( Figure 2 Middle B), IgG1 antibody titer increased to 5,376 ( Figure 2 After the whole bacteria were supplemented with aluminum adjuvant, the titers of IgG, IgG2a and IgG1 antibodies were significantly increased to 40,960 ( Figure 2 A), 9,728 ( Figure 2 B) and 16,384 ( Figure 2 In comparison, the mucosal adjuvant group induced the lowest IgG, IgG2a, and IgG1 antibody titers, which were 880 ( Figure 2 Middle A), 192( Figure 2 Middle B) and 112( Figure 2 However, it is worth noting that the mucosal adjuvant group induced the highest IgG2a / IgG1 ratio ( Figure 2 The average value was 3.45. The IgG2a / IgG1 ratio was higher than 1, indicating that the induced immunity had a Th1 bias. The IgG2a / IgG1 ratios of the no adjuvant group and the aluminum adjuvant group were both less than 1 ( Figure 2 Middle D), indicating that the immune responses generated by these two groups were Th2-biased.

[0054] Obtaining and examining mucosal samples:

[0055] Four days after the challenge (4 dpi), mice were euthanized by an overdose of tribromoethanol (100 mg / kg). Saliva swabs and vaginal swabs were taken from the mice and stored in 300 μL PBS. The skin of the mouse neck was opened to expose the trachea, a tracheal tube was inserted, 1 mL of PBS was injected to rinse the lungs, and the liquid was aspirated and stored as bronchoalveolar lavage fluid (BALF). Saliva swab samples, vaginal swab samples, and BALF of the mice were centrifuged at 1000 rpm for 10 min, and the samples were stored for IgA antibody titer detection.

[0056] The specific implementation method is as follows: Mp whole bacterial protein is plated at a concentration of 10 μg / mL, with a volume of 50 μL per well. After being placed at 4°C overnight, the plate is washed twice with PBST. 50 μL of 5% skim milk is added to each well and blocked at 37°C for 1 hour. After blocking, the plate is washed three times, and the diluted serum sample is added to each well. The plate is continued to be incubated at 37°C for 1 hour. After the incubation is completed, the plate is washed four times, and HRP-labeled IgA antibody (200-fold diluted with 1% skim milk) is added to each well. After incubation for 30 minutes, the plate is washed five times, the liquid in the plate is patted dry, and 50 μL of TMB color development solution is added to each well. After 5 minutes, 50 μL of sulfuric acid (2M) is added to each well to stop the color development, and the absorbance value is read at 450 nm. The readings of the blank control group are averaged and multiplied by 2.1 as the cutoff value. That is, the serum dilution multiple with readings of other groups higher than the cutoff value was defined as the IgA antibody titer of the serum in that group.

[0057] like Figure 3 As shown in Figure 2, in the mucosal samples of BALF, vaginal swabs, and saliva swabs, the IgA antibody titer induced by the mucosal adjuvant group was the highest, with an average value of 168 ( Figure 3 Medium A), 7.1( Figure 3 Medium B) and 2.8( Figure 3 (C) was significantly higher than that in the other groups, including the no adjuvant group, the aluminum adjuvant group, the model group, and the blank control group. The IgA levels in the mucosal samples induced by the other groups were almost zero.

[0058] Detection of lung pathology:

[0059] After euthanasia, the lung tissues of the mice were aseptically separated and promptly placed in 4% paraformaldehyde solution for fixation. The tissues were then sent to Wuhan Sevier Company for subsequent embedding, sectioning, and hematoxylin-eosin (H&E) staining. Finally, histopathological analysis was performed and each section was scored for histopathology using a double-blind scheme. Among them, a four-level score (0-4 points) was given based on basic lung tissue lesions, including the degree of alveolar and bronchial inflammatory cell infiltration, the presence of congestion and hemorrhage between alveoli, the presence of thickening of the alveolar wall, and the presence of hyperplasia and fibrosis of connective tissue.

[0060] 0 points: represents within the normal range;

[0061] 1 point: very mild, the lesion just exceeds the normal range;

[0062] 2 points: Mild, lesions can be observed but not too serious;

[0063] 3 points: moderate, obvious lesions, and may continue to worsen;

[0064] 4 points: Severe, the lesion is very severe and occupies the entire lung tissue.

[0065] like Figure 4 As shown, the model group showed severe lung necrosis, inflammatory cell infiltration, and alveolar wall thickening, indicating that the MP challenge caused lung lesions, indicating that the model was successfully established. Correspondingly, the no-adjuvant and mucosal adjuvant groups showed significant reductions in lung lesions, while the aluminum adjuvant group did not show any significant reduction in lesions.

[0066] In summary, the present invention has achieved breakthrough innovations in vaccine safety, understanding of immune mechanisms, and clinical applicability, providing an efficient, safe, and convenient solution for the prevention of Mycoplasma pneumoniae infection, and has significant scientific significance and application prospects.

[0067] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A human Mycoplasma pneumoniae inactivated vaccine preparation, characterized in that: The vaccine preparation includes inactivated Mycoplasma pneumoniae bacterial antigen and mucosal adjuvant; The mass ratio of inactivated Mycoplasma pneumoniae bacterial antigen to mucosal adjuvant in the vaccine preparation is 2 to 5:

1.

2. The vaccine preparation according to claim 1, characterized in that The mucosal adjuvants include bacterial flagellin FLA-ST, Escherichia coli heat-sensitive toxin LT, and cholera toxin CT.

3. The vaccine preparation according to claim 1, characterized in that The Mycoplasma pneumoniae bacteria are inactivated by chemical or physical means to obtain inactivated Mycoplasma pneumoniae bacteria antigens.

4. The vaccine preparation according to claim 3, characterized in that The inactivation of Mycoplasma pneumoniae bacteria is carried out by adding propiolactone.

5. The vaccine preparation according to any one of claims 1 to 4, characterized in that Each dose of the vaccine preparation contains 20 μg of inactivated Mycoplasma pneumoniae antigen and 5 μg of mucosal adjuvant.

6. The vaccine preparation according to any one of claims 1 to 4, characterized in that The inactivated vaccine preparation is in the form of mucosal immunization, and the administration methods include nasal drops, oral inhalation, and vaginal suppositories.

7. Use of the inactivated vaccine preparation according to any one of claims 1 to 6 in the preparation of a vaccine for inducing an antibody response or mucosal system response against Mycoplasma pneumoniae in a subject.

8. Use of the inactivated vaccine preparation according to any one of claims 1 to 6 in the preparation of a vaccine for preventing Mycoplasma pneumoniae infection.

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