Preparation method and application of sodium alginate-coated inactivated vaccine capable of retaining metabolic activity

Abstract

The invention belongs to the technical field of bacterial vaccines, particularly relates to a sodium alginate-coated inactivated vaccine capable of retaining metabolic activity, and further discloses a preparation method and application of the sodium alginate-coated inactivated vaccine. The sodium alginate-coated inactivated vaccine capable of retaining metabolic activity, disclosed by the invention, is prepared under a low-temperature condition, so that the obtained inactivated vaccine capable of retaining metabolic activity has higher metabolic activity. By wrapping sodium alginate (Alg) on the surface of the inactivated vaccine strain retaining metabolic activity, the stability of the vaccine in a gastric acid environment is further improved.

Description

Technical Field

[0001] This invention belongs to the field of bacterial vaccine technology, specifically relating to a novel inactivated vaccine based on sodium alginate coating that retains metabolic activity, and further discloses its preparation method and application. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus , S. aureus Staphylococcus aureus (S. aureus) is an important opportunistic pathogen with both symbiotic and pathogenic properties. Epidemiological studies show that approximately 20% of the global population are persistent carriers of S. aureus, 30% exhibit intermittent colonization, and the remaining 50% are non-carriers. The mortality rate of bacteremia caused by this bacterium is as high as 18% in developed countries, and is even more severe in developing countries with limited medical resources. Reported bacterial vaccines are mainly divided into three types: inactivated vaccines, live attenuated vaccines, and subunit vaccines. Each vaccine has its own advantages, but each also presents different problems during use. For example, while inactivated vaccines are safe, they induce a weaker immune response; while live attenuated vaccines can induce a strong immune response, they pose safety risks. To address these shortcomings, vaccines that kill the bacteria but retain their metabolic activity offer a new solution.

[0003] Killed but Metabolically Active (KBMA) vaccines combine the strong immunogenicity of live attenuated vaccines with the safety of inactivated vaccines. The molecular mechanism of this technology lies in the following: When strains deficient in the uvrA, uvrB, or uvrC genes (UV damage repair genes) are treated with a psoralen photosensitizer (S-59 / 8-MOP), psoralen molecules specifically insert into the double helix structure of bacterial DNA, forming adducts with single-stranded DNA. Subsequently, UVA activates these single-stranded adducts, causing them to form irreversible covalent cross-links with complementary DNA strands. Because the bacteria have lost a key component of the nucleotide excision repair (NER) system, they cannot repair the DNA damage induced by both UV light and psoralen, resulting in the loss of replication ability but preservation of metabolic activity.

[0004] Oral administration is widely recognized as the most ideal drug delivery method due to its excellent patient compliance and convenience. Sodium alginate (Alg), as a natural polysaccharide carrier, has demonstrated superior delivery performance. It can encapsulate drugs on the surface to form a protective barrier against gastric acid degradation, ensuring the bioactivity of antigens. Furthermore, its mucosal adhesion properties can significantly prolong the retention time of antigens in the intestine, promoting antigen uptake and immune response, thus making it an ideal carrier material for oral vaccine delivery systems.

[0005] Current research on Staphylococcus aureus largely focuses on candidate vaccines such as multi-component vaccines or recombinant protein vaccines. However, due to the diversity of Staphylococcus aureus virulence factors, most existing experiments have shown that these vaccines have failed to demonstrate effective protective efficacy. Therefore, the field anticipates the development of an inactivated vaccine targeting the complex virulence factors of Staphylococcus aureus, which would be of positive significance for advancing vaccine production and distribution. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a novel inactivated vaccine with preserved metabolic activity coated with sodium alginate, so as to solve the problem that the metabolic activity and stability of traditional inactivated vaccines in the prior art are not ideal. The second technical problem to be solved by the present invention is to provide a method for preparing and applying the novel inactivated vaccine with preserved metabolic activity coated with sodium alginate.

[0007] To solve the above-mentioned technical problems, the present invention provides a method for preparing a sodium alginate-coated inactivated vaccine that retains metabolic activity, comprising the following steps: (1) Selected gene-deleted strains were subjected to photochemical treatment to obtain inactivated vaccine strains that retain metabolic activity, denoted as KBMA; (2) The surface of the inactivated vaccine strain with preserved metabolic activity obtained in step (1) is coated with sodium alginate to obtain the desired sodium alginate-coated inactivated vaccine with preserved metabolic activity, denoted as KBMA / Alg.

[0008] Specifically, in the preparation method of the sodium alginate-coated inactivated vaccine with preserved metabolic activity, the photochemical treatment step (1) includes the steps of adding photosensitizer for co-culture and irradiation with UVA long-wave ultraviolet light.

[0009] Specifically, in the preparation method of the sodium alginate-coated inactivated vaccine with preserved metabolic activity, in step (1), the photosensitizer includes at least one of nucleic acid-targeting photosensitizers; Preferably, the amount of photosensitizer added is 80-90 ng / ml; Preferably, the photosensitizer includes psoralen; Preferably, the photosensitizer includes 8-methoxypsoralen and / or S-59 photosensitizer.

[0010] Specifically, in the preparation method of the sodium alginate-coated inactivated vaccine with preserved metabolic activity, step (1) includes: The wavelength of the UVA long-wave ultraviolet light is 360-370 nm, preferably 365 nm; and / or, The UVA long-wave ultraviolet light irradiation dose is 20-30 J / cm². 225 J / cm 2 ; and / or, The ambient temperature for UVA long-wave ultraviolet light irradiation is 3-5℃, preferably 4℃; and / or, The UVA long-wave ultraviolet light irradiation time is 160-240 s.

[0011] Specifically, the preparation method of the sodium alginate-coated inactivated vaccine with preserved metabolic activity includes step (2) of adding the inactivated vaccine strain to an Alg aqueous solution and mixing it, and step of incubation and culture.

[0012] Specifically, in the method for preparing the inactivated vaccine that retains metabolic activity, in step (2), the concentration of the Alg aqueous solution is 10-20 mg / ml.

[0013] Specifically, in the preparation method of the sodium alginate-coated inactivated vaccine with preserved metabolic activity, step (2) includes: The temperature for the incubation and culture step is 35-40℃; and / or, The rotation speed during the incubation and culture step is 180-250 rpm, preferably 220 rpm; and / or, The incubation and cultivation step takes 30-90 minutes, preferably 60 minutes.

[0014] Specifically, the method for preparing the sodium alginate-coated inactivated vaccine with preserved metabolic activity, wherein the vaccine strain includes methicillin-resistant Staphylococcus aureus.

[0015] The present invention also discloses an inactivated vaccine with preserved metabolic activity coated with sodium alginate prepared by the method.

[0016] The present invention also discloses the application of the sodium alginate-coated inactivated vaccine with preserved metabolic activity in the field of antibacterial infection.

[0017] The sodium alginate-coated inactivated vaccine of this invention, prepared under low-temperature conditions, exhibits higher metabolic activity. Furthermore, coating the surface of the inactivated vaccine strain with Alg further enhances its stability in acidic environments. This novel inactivated vaccine, capable of preserving metabolic activity, provides significant protection against bacterial infections in mice, achieving efficacy comparable to live vaccines but with greater safety, thus offering new insights for bacterial vaccine development and the treatment of bacterial infections.

[0018] The present invention describes a method for preparing a novel inactivated vaccine that retains metabolic activity. Under low-temperature conditions, photochemical treatment (including UVA long-wave ultraviolet light irradiation and photosensitizer treatment) is used to photorepair gene-deficient strains (deficient uvrA, uvrB, or uvrC), causing the strains to lose their reproductive ability while still maintaining high metabolic activity. Furthermore, sodium alginate (Alg) is coated onto the surface of the photochemically treated strains. This not only results in inactivated vaccines with higher metabolic activity prepared under low-temperature conditions but also effectively improves the stability of the vaccine in the acidic environment of the stomach by coating the surface with Alg, which helps to promote the practical production and application of vaccines. Attached Figure Description

[0019] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 (A) shows the results of incubation of bacteria with different concentrations of 8-MOP; (B) shows the photosensitivity comparison between uvrB gene knockout strains and wild-type strains; (C) shows the comparison of vaccine activity at room temperature and under environmental conditions; (D) shows the comparison of metabolic activity between live bacteria, vaccines and dead bacteria. Figure 2 (A) shows the observation results of bacterial cell membrane under transmission electron microscopy; (B) shows the observation results of vaccine Alg under transmission electron microscopy; (C) shows the results of KBMA / Alg metabolic activity before and after treatment with artificial gastric juice as detected by CCK-8 assay; (D) shows the results of KBMA / Alg metabolic activity before and after treatment with artificial gastric juice as detected by MTS assay. Figure 3 (A) shows the results of detecting vaccine metabolic activity in mannitol fermentation medium; (B) shows the results of detecting vaccine metabolic activity in freeze-dried rabbit plasma. Figure 4 (A) Results of weight change in USA300-infected mice (n=6) after immunization with the vaccine via gavage; (B) Results of bacterial colony counts in the liver of each group (n=6); (C) Results of bacterial colony counts in the spleen of each group (n=6); (D) Results of bacterial colony counts in the kidney of each group (n=6); (E) Results of bacterial colony counts in the lungs of each group (n=6). Figure 5 (A) shows the results of IL-1β level detection (n=6) in each group; (B) shows the results of IL-6 level detection (n=6) in each group; (C) shows the results of TNF-α level detection (n=6) in each group; and (D) shows the results of IL-17 level detection (n=6) in each group. Detailed Implementation

[0020] In the following embodiments of the present invention, the sodium alginate used can be a commercially available product. As an example, sodium alginate (M / G=1:2) purchased from McLean is selected.

[0021] Example 1 like Figure 1 The process shown in (A) and (B) of this embodiment describes the preparation method of the inactivated vaccine with preserved metabolic activity. The method involves co-incubating the bacterial strain with a photosensitizer followed by photochemical treatment to obtain the desired KBMA vaccine. Specifically, the method includes the following steps: Take the methicillin-resistant Staphylococcus aureus to be treated and culture it in the appropriate culture medium until OD200. 600 The concentration was set to 0.5, and 85 ng / ml of 8-methoxypsoralen was added for co-culturing for 1 h. Then, the bacterial culture was transferred to 6-well plates (2 ml per well) and incubated at 4°C with UVA (365 nm) at 25 J / cm². 2 Irradiation with a certain dose for 200 seconds yielded an inactivated vaccine strain that retained metabolic activity, denoted as KBMA vaccine.

[0022] Example 2 like Figure 2 The process shown in Figure (B) of this embodiment, the method for preparing the sodium alginate-coated inactivated vaccine with preserved metabolic activity, includes the following steps: (1) The methicillin-resistant Staphylococcus aureus to be treated was cultured in the appropriate medium until the OD600 reached 0.5, and then 85 ng / ml of 8-methoxypsoralen was added for co-culture for 1 h; then, the bacterial culture was transferred to a 6-well plate (2 ml per well) and incubated at 4℃ with UVA (365 nm) at 25 J / cm 2 Irradiation with a certain dose for 200 seconds yielded an inactivated vaccine strain that retained metabolic activity, denoted as KBMA vaccine; (2) The prepared KBMA vaccine was enriched (36 ml) and centrifuged at 5500 rpm for 5 min. A 14 mg / ml Alg (24 ml) aqueous solution was prepared, and the precipitate was resuspended in the Alg solution and mixed thoroughly. 40 ml of liquid paraffin, 120 μl of Span 80, and 40 μl of Tween 80 were mixed and stirred at 2000 r / min for 10 min. The resuspended KBMA sodium alginate solution was added to the mixture and stirred at 2000 r / min for 30 min to fully emulsify; this solution was named Solution A. 40 ml of CaCl2 (50 mg / ml) was then mixed with 40 ml of liquid paraffin and stirred for 5 min; this solution was named Solution B. Solution A was slowly added to Solution B, and the mixture was stirred at 2000 r / min to emulsify for 30 min. Add 2 ml of ethyl acetate as a demulsifier, place in a centrifuge at 4°C, centrifuge at 8000 rpm for 5 min, collect the precipitate, and wash twice with 0.01% (w / v) sodium acetate solution. The final precipitate is the KBMA / Alg vaccine.

[0023] Comparative Example 1 The preparation method of the inactivated vaccine that retains metabolic activity described in this comparative example is the same as that in Example 2, the only difference is that in step (1), light radiation is carried out at room temperature, and in step (2), sodium alginate is not coated, thus obtaining the KBMA vaccine.

[0024] Experimental Example 1. Vaccine characteristics This embodiment explores the toxicity of photosensitizers and the sensitivity of bacterial strains.

[0025] As attached Figure 1 The results of the effect of 8-MOP on bacterial viability are shown in Figure (A). After co-incubating different concentrations of 8-MOP with bacteria for 1 hour, the incubation products of different concentrations of 8-MOP and bacteria were diluted 10,000 times with physiological saline at a ratio of 100 μl bacterial culture to 900 μl physiological saline. 100 μl of each diluted solution was then spread onto solid culture medium and incubated overnight at 37°C for colony counting. The results show that 8-MOP does not affect bacterial viability.

[0026] As attached Figure 1 The UVA light energy shown in (B) is 25 J / cm². 2 The following figure shows the changes in the activity of USA300 and USA300ΔuvrB after treatment with different 8-MOP concentrations. As shown in the figure, the bacterial inactivation effect is more significant with increasing 8-MOP concentration, and the strain with the photorepair gene knocked out is more easily inactivated, indicating that it has better photosensitivity.

[0027] 2. Determine the metabolic activity and characterize the properties of the vaccine. As attached Figure 1 Figure (C) shows the metabolic capacity of inactivated but metabolically active vaccines prepared at room temperature and 4°C. The metabolic capacity of KBMA vaccines prepared at room temperature and 4°C was detected using an enhanced CCK-8 assay kit (Beyotime, C0042). The results showed that the metabolic capacity of the KBMA vaccine prepared at 4°C was stronger than that prepared at room temperature.

[0028] As attached Figure 1 Figure (D) shows the metabolic capacity of live bacteria, KBMA vaccine, and dead bacteria. The metabolic capacity of live bacteria, KBMA vaccine, and dead bacteria was detected using an enhanced CCK-8 assay kit (Beyotime, C0042). The results showed no significant difference in the metabolic capacity of KBMA vaccine compared to live bacteria.

[0029] As attached Figure 2 Image (B) shows the observation results of the KBMA / Alg vaccine under transmission electron microscopy. After Alg encapsulation, it is observed in conjunction with the attached... Figure 2Compared to the results of bacterial cell membrane under transmission electron microscopy in (A), the KBMA vaccine surface is clearly covered with a membrane-like substance.

[0030] As attached Figure 2 The metabolic activity of KBMA / Alg before and after treatment with artificial gastric fluid was detected using the CCK-8 assay. KBMA / Alg vaccine was immersed in 2 ml of artificial gastric fluid and incubated at 37°C for 1 min. The metabolic activity of KBMA / Alg was then detected using an enhanced CCK-8 assay kit (Beyotime, C0042). The results showed that KBMA / Alg maintained its metabolic activity after treatment with artificial gastric fluid.

[0031] As attached Figure 2 (D) shows the results of MTS assay for the metabolic activity of KBMA / Alg before and after treatment with artificial gastric fluid. KBMA / Alg vaccine was immersed in 2 ml of artificial gastric fluid and incubated at 37°C for 1 min. Bacterial viability (Promega, G3580) was measured using a cell proliferation assay kit to detect the metabolic activity of KBMA / Alg. The results showed that KBMA / Alg maintained good metabolic activity after treatment with artificial gastric fluid.

[0032] As attached Figure 3 (A) shows the results of detecting vaccine metabolic activity using mannitol fermentation medium. In this experimental example, the ability of the vaccine to metabolize was tested, and the ability of bacteria to produce acid through fermentation was detected using mannitol fermentation medium. Appendix Figure 3 (B) shows the results of detecting vaccine metabolic activity using freeze-dried rabbit plasma, and the ability of bacteria to secrete plasma coagulase using freeze-dried rabbit plasma. 3. Verify the protective effect of the vaccine against bacterial infection in mice. In this embodiment, the protective effect of the KBMA / Alg vaccine prepared in Example 2 on bacterial infection in mice was verified.

[0033] As attached Figure 4 The results (A) show the changes in body weight in mice (n=6). It can be seen that 7 days after infection with methicillin-resistant Staphylococcus aureus (MRSA), the body weight of the control group decreased significantly, while after KBMA / Alg vaccination, the weight loss was stopped and showed a recovery trend. This indicates that the vaccine can resist bacterial infection and play a protective role.

[0034] As attached Figure 4(B)-(E) represent the bacterial load in the liver, spleen, kidney, and lung of mice (n=6). It can be seen that the bacterial load in the lungs of mice in the KBMA / Alg group was 0.35±0.19 log, while that in the SA group was approximately 4.44±0.46 log. Mice immunized with KBMA / Alg showed a 4-log reduction in lung colonization. The bacterial load in the liver of mice immunized with KBMA / Alg was 1.26±1.09 log, while that in the SA group was 4.50±1.49 log. Mice immunized with KBMA / Alg showed a reduction of more than 3-log in liver colonization. In the kidneys, the colonization in mice immunized with KBMA / Alg was 2.32±2.13 log, while that in the SA group was 4.14±1.19 log. Mice immunized with KBMA / Alg showed a 2-log reduction in colonization. The bacterial load in the spleen of mice immunized with KBMA / Alg was 0.41±0.36 log, while that in the SA group was 3.59±1.63 log. Mice immunized with KBMA / Alg showed a reduction of more than 3 log in spleen colonization. These results demonstrate that KBMA / Alg can effectively protect critical organs from bacterial colonization.

[0035] As attached Figure 5 Tables (A)-(D) show the results of changes in inflammatory factors in mice (n=6). The IL-6 level in the KBMA / Alg group was only 5.96±1.60 pg / ml, while the IL-6 level in the infection group (SA group) was 38.6±1.74 pg / ml, the sodium alginate group was 50.6±5.98 pg / ml, and the KBMA group was 24.27±2.15 pg / ml. The KBMA / Alg immunization resulted in reduced levels of the inflammatory factors IL-1β, TNF-α, and IL-17 in mice, which may help alleviate inflammation.

[0036] In summary, the novel inactivated vaccine (KBMA / Alg vaccine) described in this invention, which retains metabolic activity, has a significant protective effect against bacterial infection in mice, achieving an effect comparable to that of live vaccines, but with greater safety, providing a new approach for bacterial vaccine development and the treatment of bacterial infectious diseases.

[0037] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a sodium alginate-coated inactivated vaccine that retains metabolic activity, characterized in that, Includes the following steps: (1) Selected gene-deleted bacteria were subjected to photochemical treatment to obtain inactivated vaccine strains that retain metabolic activity, denoted as KBMA; (2) The surface of the inactivated vaccine strain with preserved metabolic activity obtained in step (1) is coated with sodium alginate to obtain the desired sodium alginate-coated inactivated vaccine with preserved metabolic activity, denoted as KBMA / Alg.

2. The method for preparing the sodium alginate-coated inactivated vaccine with preserved metabolic activity according to claim 1, characterized in that, In step (1), the photochemical treatment step includes the step of adding a photosensitizer for co-culture and the step of irradiating with UVA long-wave ultraviolet light.

3. The method for preparing the sodium alginate-coated inactivated vaccine with preserved metabolic activity according to claim 2, characterized in that, In step (1), the photosensitizer includes at least one of nucleic acid-targeting photosensitizers; Preferably, the amount of photosensitizer added is 80-90 ng / ml; preferably, the photosensitizer includes 8-methoxypsoralen and / or S-59 photosensitizer.

4. The method for preparing the sodium alginate-coated inactivated vaccine with preserved metabolic activity according to claim 2 or 3, characterized in that, In step (1): The wavelength of the UVA long-wave ultraviolet light is 360-370 nm; and / or, The UVA long-wave ultraviolet light irradiation dose is 20-30 J / cm². 2 ; and / or, The ambient temperature for UVA long-wave ultraviolet light irradiation is 3-5℃; and / or, The UVA long-wave ultraviolet light irradiation time is 160-240 s.

5. The method for preparing the sodium alginate-coated inactivated vaccine with preserved metabolic activity according to any one of claims 1-4, characterized in that, Step (2) includes adding the inactivated vaccine strain with preserved metabolic activity to the Alg solution and mixing it, as well as incubating and culturing it.

6. The method for preparing the sodium alginate-coated inactivated vaccine with preserved metabolic activity according to claim 5, characterized in that, In step (2), the concentration of the Alg aqueous solution is 10-20 mg / ml.

7. The method for preparing the sodium alginate-coated inactivated vaccine with preserved metabolic activity according to claim 5 or 6, characterized in that, In step (2): The temperature for the incubation and culture step is 35-40℃; and / or, The rotation speed during the incubation and culture step is 120-200 rpm; and / or, The incubation and cultivation step takes 30-60 minutes.

8. The method for preparing a sodium alginate-coated inactivated vaccine with preserved metabolic activity according to any one of claims 1-7, characterized in that, The vaccine strains include methicillin-resistant Staphylococcus aureus.

9. The sodium alginate-coated inactivated vaccine with preserved metabolic activity prepared by the method according to any one of claims 1-8.

10. The application of the sodium alginate-coated inactivated vaccine with preserved metabolic activity as described in claim 9 in the field of antibacterial infection.

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