Preparation method of aluminum MOF nano adjuvant and application thereof in polyvalent vaccine

Abstract

This invention discloses a method for preparing aluminum MOF nano-adjuvants and their application in multivalent vaccines, belonging to the field of biotechnology. The aluminum MOF nano-adjuvants are prepared with an Al content of 2-4:4-6. 3+ This invention develops a novel aluminum MOF nanoadjuvant, prepared via a solvothermal method using ligands rich in carboxylic acids as raw materials. Based on this adjuvant, a multivalent vaccine was prepared. By binding to inactivated antigens of various diseases, it exerts a broad delivery effect and can induce a superior long-lasting immune response. This demonstrates that the nanoadjuvant can elicit an effective immune defense system in the body, providing a new material for the preparation of multivalent vaccines and strong support for the prevention and control of swine diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing an aluminum MOF nanoadjuvant and its application in multivalent vaccines. Background Technology

[0002] Swine diseases pose a severe challenge to the healthy development of the pig farming industry. They not only reduce the growth performance of pigs and affect feed conversion rates, but also cause high mortality rates, resulting in huge economic losses for farms. Zoonotic diseases such as streptococcal disease in pigs also pose a potentially serious threat to human health. Therefore, strengthening the prevention and control of swine diseases is crucial for promoting high-quality development of animal husbandry and ensuring human safety.

[0003] Vaccination plays a crucial role in the prevention and control of swine diseases. Adjuvants, as an important component of vaccines, help the vaccine better stimulate the body's immune response, providing more durable and effective immune protection and enhancing the vaccine's preventative and resistant effects against pathogens. However, traditional adjuvants have a number of problems. For example, oil adjuvants can cause local swelling at the injection site, affecting vaccine absorption and immunogenicity; aluminum gel adjuvants have a relatively short duration of immunity. Given these shortcomings, the development of novel adjuvants is particularly important.

[0004] Vaccines, as one of the most effective means of preventing infectious diseases, play a crucial role in maintaining global public health security. Traditional vaccines typically target a single pathogen or a single serotype of the same pathogen, often requiring multiple doses of different vaccines. This not only causes inconvenience for recipients but can also lead to difficulties in completing the full vaccination course due to the cumbersome procedures, thus affecting immunization efficacy. Multivalent vaccines have emerged to address this issue, simultaneously preventing infection by multiple pathogens or multiple serotypes of the same pathogen. This significantly reduces the number of doses, improves prevention efficiency, and greatly facilitates vaccination efforts. However, multivalent vaccines are not simply a mixture of multiple vaccine components; they require careful design and development. The development process involves various technologies, such as antigen screening, antigen preparation, adjuvant optimization, and the determination of vaccine formulation and manufacturing processes. Different pathogens have different antigenic characteristics, and in terms of antigen compatibility, different antigens may interfere with each other, affecting the vaccine's immunization effect. For example, some antigens may undergo physical or chemical changes after mixing, leading to a decrease in immunogenicity. The complex manufacturing processes and high costs of some multivalent vaccines limit their large-scale production and widespread application to some extent. Furthermore, regarding immunization efficacy, although multivalent vaccines can prevent multiple diseases simultaneously, the efficacy of some multivalent vaccines still needs further improvement. After vaccination, the immune response generated by some multivalent vaccines may not be as strong as that of single vaccines, failing to provide sufficient protection for the recipient. In the face of complex situations involving multiple concurrent or secondary infectious diseases, existing multivalent vaccines may not meet the needs for comprehensive prevention. With the increasing global awareness of public health and the widespread implementation of vaccination programs, the market demand for multivalent vaccines continues to grow, placing higher demands on vaccine safety, efficacy, and convenience. Against this backdrop, developing a new adjuvant to enhance the immunization efficacy of multivalent vaccines, overcome the shortcomings of existing technologies, and meet market demands has significant practical implications and broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing an aluminum MOF nanoadjuvant and its application in multivalent vaccines, thereby addressing the problems existing in the prior art. This invention develops a novel aluminum MOF nanoadjuvant and prepares multivalent vaccines based on it. By binding to inactivated antigens of various diseases, it exerts a broad delivery effect and can induce a superior long-lasting immune response. This nanoadjuvant demonstrates that it can induce an effective immune defense system in the body, providing a new material for the preparation of multivalent vaccines and strong support for the prevention and control of swine diseases.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for preparing aluminum MOF nanoadjuvants, wherein the aluminum MOF nanoadjuvants are prepared in a molar ratio of 2-4:4-6 of Al. 3+ It is prepared by solvothermal method using ligands rich in carboxylic acids as raw materials.

[0008] Furthermore, it includes the following steps:

[0009] Al 3+ The aluminum salt was dissolved in N,N-dimethylformamide; the carboxylic acid-rich ligand was dissolved in N,N-dimethylformamide; the two solutions were mixed and subjected to a solvothermal reaction, followed by washing and drying to obtain the aluminum MOF nano-adjuvant.

[0010] Optionally, the aluminum salt includes Al(NO3)3·9H2O.

[0011] Optionally, the carboxylic acid-rich ligand includes biphenyl dicarboxylic acid.

[0012] Optionally, the temperature of the solvothermal reaction is 50-150℃ and the time is 6-72h.

[0013] The present invention also provides aluminum MOF nanoadjuvants prepared by the above preparation method.

[0014] This invention also provides the application of the above-mentioned aluminum MOF nanoadjuvant in the preparation of multivalent vaccines.

[0015] The present invention also provides a multivalent vaccine, which is prepared by mixing the antigen with the above-mentioned aluminum MOF nano-adjuvant at a mass ratio of 1-5:1.

[0016] Optionally, the antigen may include inactivated Streptococcus suis type 2, inactivated Gracilaria parasuis, and inactivated Mycoplasma hyopneumoniae.

[0017] Optionally, the multivalent vaccine contains 3.0 × 10⁻⁶ inactivated Streptococcus suis type 2. 9 CFU / mL, inactivated Gracilaria parasuis 5.0 × 10⁻⁶ 9 CFU / mL and inactivated Mycoplasma hyopneumoniae 100 μg / mL.

[0018] The present invention discloses the following technical effects:

[0019] This invention employs a solvothermal method to successfully convert Al under high-temperature conditions. 3+ By binding with ligands rich in carboxylic acids, the efficient preparation of Al-based MOFs (Al-MOFs) nanoadjuvants was achieved. This invention uses Al-MOFs nanoadjuvants as the core to prepare vaccines that effectively promote the body's immune response and achieve better protective effects without affecting pig growth.

[0020] This invention develops a method for preparing a multivalent vaccine based on Al-MOF nano-adjuvant materials. By binding these materials to inactivated antigens of various diseases, the vaccine exhibits broad delivery capabilities and can induce superior long-lasting immune responses. This demonstrates that the nano-adjuvant can elicit an effective immune defense system in the body, providing a new material for the preparation of multivalent vaccines and offering strong support for the prevention and control of swine diseases. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 XRD powder diffraction patterns of Al-MOFs;

[0023] Figure 2 TGA spectra of Al-MOFs;

[0024] Figure 3 SEM images of Al-MOFs;

[0025] Figure 4 DLS particle size distribution of Al-MOFs;

[0026] Figure 5 Changes in body temperature of piglets in different groups after challenge with Streptococcus suis type 2;

[0027] Figure 6 The level of Streptococcus suis type 2 antibody in the serum of piglets in each group after challenge with Streptococcus suis type 2;

[0028] Figure 7 The changes in body temperature of piglets in different groups after challenge with Grasserius parasuis type 5;

[0029] Figure 8 The levels of Graciela suis antibodies in the serum of piglets in each group after challenge with Graciela suis type 5.

[0030] Figure 9 The levels of Mycoplasma hyopneumoniae antibodies in the serum of piglets in each group after challenge with Mycoplasma hyopneumoniae were shown. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Example 1 Preparation of nano-adjuvants Al-MOFs

[0037] 0.45 g of Al(NO3)3·9H2O (1.2 mmol) was dissolved in 10 mL of DMF and sonicated to ensure complete dissolution. Similarly, 0.16 g of 4,4'-biphenyl dicarboxylic acid (0.72 mmol) was dissolved in another 10 mL of DMF and sonicated until completely dissolved. The two solutions were mixed (molar ratio of biphenyl dicarboxylic acid precursor to aluminum ions = 3:5) and transferred to a reaction vessel. The reaction was carried out at 120 °C for 24 h. After the reaction was completed, the product was washed three times with DMF, followed by one wash with anhydrous ethanol. Finally, it was vacuum dried at room temperature to obtain a white Al-MOF powder.

[0038] 0.525 g of Al(NO3)3·9H2O (1.4 mmol) was dissolved in 10 mL of DMF and sonicated to ensure complete dissolution. Similarly, 0.27 g of 4,4'-biphenyl dicarboxylic acid (1.2 mmol) was dissolved in another 10 mL of DMF and sonicated until completely dissolved. The two solutions were mixed (biphenyl dicarboxylic acid precursor to aluminum ions molar ratio = 6:7) and transferred to a reaction vessel. The reaction was carried out at 120 °C for 24 h. After the reaction, the product was washed three times with DMF, followed by one wash with anhydrous ethanol. Finally, it was vacuum dried at room temperature to obtain a white Al-MOF powder.

[0039] XRD powder diffraction confirmed that Al-MOFs prepared with a biphenyl phthalic acid precursor to aluminum ions molar ratio of 3:5 had higher crystallinity, while Al-MOFs prepared with a biphenyl phthalic acid precursor to aluminum ions molar ratio of 6:7 had poorer crystallinity. Figure 1 Therefore, a molar ratio of 3:5 between biphenyl dicarboxylic acid precursor and aluminum ions was chosen to prepare Al-MOF nano-adjuvants.

[0040] Furthermore, TGA spectra indicate that Al-MOFs prepared with a biphenyl dicarboxylic acid precursor to aluminum ions molar ratio of 3:5 exhibit good thermal stability. Figure 2 SEM images and DLS particle size distribution maps show that Al-MOFs are rod-like nanomaterials with an average particle size of 200 nm. Figure 3 and Figure 4 ).

[0041] Example 2: Vaccine Preparation

[0042] Based on an antigen-to-adjuvant ratio of 5:1 (w / w), the required amounts of PBS and three inactivated antigens (Streptococcus suis type 2 QH strain, Gracilaria parasuis LZ strain, and Mycoplasma hyopneumoniae) and adjuvant were calculated, ensuring that each milliliter of vaccine contained 3.0 × 10⁻⁶ inactivated Streptococcus suis QH strain. 9 CFU containing 5.0 × 10⁻⁶ inactivated Haemophilus parasuis strain LZ 9 CFU containing 100 μg of Mycoplasma hyopneumoniae.

[0043] First, fill the vaccine preparation tank with PBS, then add the tested and qualified concentrated bacterial solutions of Streptococcus suis type 2 (QH strain), Gracilaria parasuis (LZ strain), and Mycoplasma hyopneumoniae antigen solution. Stir at a low speed of 120-140 rpm, slowly add Al-MOFs, and simultaneously add 0.005% of the total vaccine volume of thimerosal. Stir thoroughly for 15-20 minutes to ensure complete mixing. Following the above method, prepare vaccines using Chemtrade aluminum gel adjuvant (Vaccine A); vaccines prepared with Al-MOFs (3:5) are designated Vaccine B; and vaccines prepared with Al-MOFs (6:7) are designated Vaccine C.

[0044] Example 3: Evaluation test of immunization effect against Streptococcus suis type 2 (SS2)

[0045] Twenty healthy, susceptible piglets aged 20 days were randomly divided into four groups of five each. Immunization was administered via intramuscular injection into the neck. Group A received 1 mL / pig of vaccine A, Group B received 1 mL / pig of vaccine B, Group C received 1 mL / pig of vaccine C, and Group D (Infected group) received 1 mL / pig of PBS. Twenty-one days post-immunization, groups A, B, C, and D underwent challenge experiments via intravenous injection of 2.0 mL of Streptococcus suis type 2 QH strain (9.68 × 10⁶ viable cells). 5 (CFU). Clinical symptoms and mortality were closely monitored in each group of piglets for 14 days post-infection. Blood samples were collected from each experimental group of piglets via the anterior vena cava on days 7, 14, 21, 28, and 35 post-immunization, and serum was separated. The level of Streptococcus suis type 2 antibody in the serum was detected using an ELISA antibody detection method.

[0046] The results showed that the body temperature of piglets in groups A and B increased to varying degrees within 3 days, and returned to normal after 3 days; the body temperature of piglets in group C increased significantly for the first 4 days, and returned to normal on the 5th day; the body temperature of piglets in group D increased significantly (…). Figure 5 Within one week after challenge, no pigs died in groups A and B, one pig died in group C, and all pigs died in group D (Table 1). Serum antibody levels of Streptococcus suis type 2 showed that 35 days after immunization, the antibody level in group B was significantly higher than that in groups A and C. Figure 6 This indicates that the adjuvant in group B has a better immunostimulatory effect.

[0047] Table 1. Results of vaccine immunization challenge (SS2)

[0048]

[0049] Example 4: Evaluation test of the immunization effect of Gracie parasuis (GPS)

[0050] Twenty healthy, susceptible piglets aged 25 days were randomly divided into four groups of five each. Immunization was administered via intramuscular injection into the neck. Group A received 1 mL / pig of vaccine A, Group B received 1 mL / pig of vaccine B, Group C received 1 mL / pig of vaccine C, and Group D (Infected group) received 1 mL / pig of PBS. A booster immunization was administered 21 days after the initial immunization via the same route and at the same dose. 21 days after the second immunization, groups A, B, C, and D were challenged via intraperitoneal injection with 2.0 ml / pig of *Glasgiella parasuis* strain 5, LZ (containing 6.34 × 10⁻⁶ live bacteria). 9 (CFU). Clinical symptoms, morbidity, and mortality of piglets in each group were closely observed and recorded within 14 days after challenge. Blood samples were collected from piglets in each experimental group via the anterior vena cava on days 7, 14, 21, 28, 35, 42, and 49 after the initial immunization. The collected blood samples were used to separate serum, and the level of *Glassiella parasuis* antibodies in the serum was detected using an ELISA antibody detection method.

[0051] The results showed that the body temperature of piglets in group D was significantly elevated; in group A, the body temperature was elevated 4-7 days after challenge and then returned to normal; in group B, the body temperature was transiently elevated 3-4 days after challenge and then returned to normal; and in group C, the body temperature was elevated 3-7 days after challenge and then returned to normal. Figure 7 In Group D, piglets exhibited lameness, recumbency, and difficulty breathing after challenge, with four pigs dying within 14 days. In Group A, one piglet became lame after challenge and subsequently died. No clinical symptoms or deaths were observed in Group B. In Group C, one piglet became lame on day 7 after challenge, and another died on day 10, with necropsy revealing a typical "villous heart" (Table 2). Serum antibody levels in *Glassella parasuis* showed that antibody levels in Groups A and B were significantly higher than in Group C after immunization. From 14-35 days post-immunization, there was no significant difference in antibody levels between Groups A and B. Starting at day 42, antibody levels in Group B gradually increased, and at day 49, antibody levels in Group B were significantly higher than in Groups A and C. Figure 8 This indicates that the adjuvant in group B has a better protective effect against Glasgow parasuis.

[0052] Table 2. Results of vaccine immunization challenge (GPS)

[0053]

[0054] Example 5: Evaluation Test of Immunization Efficacy Against Mycoplasma hyopneumoniae in Swine

[0055] Twenty-five healthy, susceptible piglets aged 25 days were randomly divided into five groups of five each. All piglets were immunized via neck muscle injection. Group A received 1 mL / pig of vaccine A, Group B received 1 mL / pig of vaccine B, Group C received 1 mL / pig of vaccine C, Group D (Infected group) received 1 mL / pig of PBS, and Group E (Control group) received no immunization or challenge treatment. Each piglet in groups A, B, and C received a booster immunization via the same route and dose 21 days after the initial immunization. Twenty-one days after the second immunization, piglets were inoculated intratracheally with a virulent, freeze-dried strain of *Mycoplasma hyopneumoniae* lung tissue. The challenge dose was 10.0 mL / pig (containing 0.016 g of lung tissue virus). Piglets were observed for 28 days after the second immunization, and the lung lesions in each group were recorded in detail. Blood samples were collected from piglets in each group via the anterior vena cava on days 7, 14, 21, 28, 35, 42, 49, 56, and 63 following the initial vaccination. The collected blood samples were used to separate serum, and the levels of Mycoplasma hyopneumoniae antibodies in the serum were subsequently determined using an ELISA antibody detection method.

[0056] The results showed that piglets in group D exhibited typical symmetrical lesions, while the lung lesions in groups A, B, and C were significantly less severe than those in group D. After challenge, one piglet in each of groups A and B developed coughing symptoms, two piglets in group C developed coughing symptoms, all piglets in group D became ill, and the control group remained normal (Table 3). Serum Mycoplasma hyopneumoniae antibody levels showed that the antibody levels in group B were higher than those in groups A and C from 14 to 63 days post-immunization. Figure 9 This indicates that group B can produce a stronger immune stimulus, protecting pigs against Mycoplasma pneumoniae infection.

[0057] Table 3 Results of vaccine challenge (Mycoplasma hyopneumoniae)

[0058]

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of an aluminum MOF nanoadjuvant in the preparation of a multivalent vaccine, characterized in that, The aluminum MOF nanoadjuvant is prepared in a molar ratio of 5:3 with Al. 3+ It is prepared by a solvothermal method using ligands rich in carboxylic acids as raw materials; The preparation method of the aluminum MOF nanoadjuvant includes the following steps: Al 3+ The aluminum salt was dissolved in N,N-dimethylformamide; the carboxylic acid-rich ligand was dissolved in N,N-dimethylformamide; the two solutions were mixed and subjected to a solvothermal reaction, followed by washing and drying to obtain the aluminum MOF nano-adjuvant; The aluminum salt is Al(NO3)3·9H2O; the carboxylic acid-rich ligand is biphenyl dicarboxylic acid; The solvothermal reaction was carried out at a temperature of 120°C for 24 hours.

2. A multivalent vaccine, characterized in that, The multivalent vaccine is prepared by mixing the antigen with the aluminum MOF nano-adjuvant described in claim 1 at a mass ratio of 1-5:

1.

3. The multivalent vaccine according to claim 2, characterized in that, The antigens include inactivated Streptococcus suis type 2, inactivated Gracilaria parasuis, and inactivated Mycoplasma hyopneumoniae.

4. The multivalent vaccine according to claim 3, characterized in that, The multivalent vaccine contains 3.0 × 10⁻⁶ inactivated Streptococcus suis type 2. 9 CFU / mL, inactivated Gracilaria parasuis 5.0 × 10⁻⁶ 9 CFU / mL and inactivated Mycoplasma hyopneumoniae 100 μg / mL.

Images