Intradermal immune water-based adjuvant porcine circovirus subunit vaccine as well as preparation method and application thereof

Abstract

The invention discloses a porcine circovirus subunit vaccine suitable for needle-free intradermal immunization and a preparation method of the porcine circovirus subunit vaccine. The vaccine comprises a porcine circovirus type 2 (PCV2) Cap protein, an M101 adjuvant and a PBS buffer solution, and the adding proportion of the M101 adjuvant is 5% (volume ratio) and is obviously higher than 0.5% of a conventional vaccine. Compared with the prior art, by optimizing an adjuvant formula and an immunization mode, the limitation of a traditional water adjuvant in needle-free injection is overcome. Meanwhile, the proportion of the M101 adjuvant is increased to 5%, so that the antibody level after secondary immunization is equivalent to that of a commercial vaccine intramuscular injection group, and the antibody level is slightly high in the first two weeks after immunization. In addition, the vaccine is a clear transparent liquid, is free of precipitation or flocculation and is suitable for high-pressure jet delivery, and the oil-in-water vaccine easily causes the safety problems of skin rupture, absorption disorder and the like in needle-free intradermal immunization. The vaccine is simple in preparation process and good in stability, and an efficient and safe new choice is provided for prevention and control of the porcine circovirus disease.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an aqueous adjuvant porcine circovirus subunit vaccine for needle-free intradermal immunization, and a preparation method and application thereof. Background Art

[0002] Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus of the family Circoviridae, and is an unenveloped single-stranded, positive-sense, circular DNA virus. PCV2 is the main pathogen of porcine circovirus-associated diseases (PCVAD) such as post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), and porcine respiratory disease complex (PRDC), and is one of the most widely spread porcine viruses globally. After PCV2 infects pigs, it will weaken the protective effects of vaccines against viruses such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), and classical swine fever virus (CSFV), thus leading to mixed infections or secondary infections of pigs with other pathogens.

[0003] Since 2006, the widespread use of vaccines in major pig-producing countries has significantly reduced the losses caused by PCV2 infection. However, the use of vaccines can only prevent the clinical syndromes induced by PCV2 infection, but cannot prevent the transmission of the virus in vaccinated herds. Therefore, it is still necessary to develop new vaccines for PCV2. Most of the existing PCV2 vaccines on the market use inactivated PCV1-2 chimeric strains, inactivated whole viruses, or recombinant envelope proteins encoded by ORF2, and the adjuvants are mostly aqueous polymers (such as carbomer), Dl-α-tocopheryl acetate plus liquid paraffin and light paraffin oil. The immunization method is mostly intramuscular injection (IM), but intramuscular injection of pigs has a greater stress response, and the needles are not changed during the immunization process, increasing the risk of cross-infection. Different from intramuscular immunization, intradermal vaccination (ID) has better safety and protective effects after immunizing pigs than intramuscular injection.

[0004] Needle-free intradermal immunization is a technique that can deliver vaccines into the body without traditional injection needles, and can stimulate the host's protective immune response through antigen-presenting cells in the dermis near the skin lymph nodes. Such techniques aim to improve vaccination safety, convenience and acceptance. Technologies such as human patch vaccines and spray vaccines have been widely studied in the prevention and control of influenza and COVID-19 vaccines. Currently, the needle-free immunization technology for swine vaccines in China is mainly needle-free syringe-type vaccines, and its mechanism of action is to use the spring power in high-pressure air flow to penetrate the skin with extremely fine jets of liquid vaccine. This immunization method is simple and convenient to operate, and can significantly reduce the pain and stress response of pigs. Since there is no need to change the needle, the risk of cross-infection is reduced, the possibility of injection site infection is lowered, and biosafety is ensured. Moreover, the dosage of needle-free injection vaccine is only 0.2 - 0.5 mL, which greatly reduces the vaccine cost. The common dosage forms of needle-free injection vaccines on the market are mostly emulsions or freeze-dried products combined with diluents (for live vaccines), the adjuvants are mostly Dl-α-tocopheryl acetate and light liquid paraffin, and excipients mostly contain polysorbate 80 and dimethicone. Currently, needle-free injector-type vaccines are widely used clinically in China, and the clinical use effect is good, but there is no safe and efficient needle-free vaccine adjuvant available yet.

[0005] In order to save costs for pig farm immunization, enhance animal welfare, reduce the risk of cross-infection during pig injection, and avoid accidental injury to personnel during immunization, a series of needle-free injection vaccine products have emerged on the market. Currently, there is only one intradermal immunization vaccine for pigs - PCV ID approved in China. The adjuvant used in this vaccine is a mineral oil-based oil-in-water emulsion, which appears as a uniform white to nearly white emulsion after being shaken well. When using these vaccines for intradermal immunization, not only do they need to be used in combination with the needle-free injection equipment developed by the same company, but also the safety is not good (there will be large bumps at the injection site and they cannot subside within 2 weeks). Aqueous adjuvants may be more suitable for intradermal immunization due to their low viscosity and easy injection, but currently there is no intradermal immunization porcine circovirus subunit vaccine based on aqueous adjuvants approved. However, existing research shows that traditional aqueous adjuvants such as aluminum gel not only have significantly lower immunization effects in needle-free injection than in intramuscular injection, but also lower than those of oil-in-water adjuvants in needle-free injection. However, oil-in-water vaccines are prone to serious safety problems such as skin rupture and vaccine non-absorption in needle-free intradermal immunization. Therefore, there is an urgent need to develop an aqueous adjuvant specifically for intradermal injection immunization. Summary of the Invention

[0006] Vaccine adjuvants play a crucial role in the development and improvement of new vaccines, and thus have received increasing attention in the 21st century. However, to date, all these adjuvants have been approved for intramuscular immunization, and few adjuvants have been approved for intradermal immunization. Some studies have predicted the immunogenicity and local reactogenicity of approved adjuvants in current human vaccines for needle-free immunization based on their immunogenicity and local reactogenicity after intramuscular immunization. Among them, the AS01 adjuvant containing monophosphoryl lipid A (MPL) and QS21 in liposomes has high immunogenicity for intradermal immunization. However, liposomes may rupture during needle-free high-pressure or microneedle processing, requiring optimization of the formulation, which increases the complexity of vaccine research. The AS04 adjuvant is composed of MPL adsorbed on alum (aluminum salt), and may cause induration and a relatively high risk of inflammation after immunization. AS03 is an oil-in-water nanoemulsion adjuvant containing squalene and Dl-α-tocopheryl acetate, which is widely used in human influenza vaccines and malaria vaccines, but its intradermal immunogenic response is low.

[0007] Vaccines applied to needle-free injection need to have characteristics such as low viscosity and small particle size (less than 100 μm), and will not clog the injection device. Live vaccines also need to ensure their stability, which limits the application of oil adjuvants in needle-free immunization. Currently, all the approved needle-free injection vaccines for porcine circovirus in the world are oil emulsion adjuvants, and there is no approved aqueous adjuvant for intradermal immunization of porcine circovirus subunit vaccines.

[0008] The aqueous adjuvant YSK M101 (abbreviated as M101) is a commercial animal vaccine adjuvant developed by our company. The main component of this adjuvant is a compound saponin adjuvant, and it has been approved by the China Veterinary Drug Association group standard (《Animal Vaccine Compound Saponin Adjuvant YSK M101》, T / CVDA 24-2024). The M101 adjuvant is a light yellow clear and transparent liquid, which is applicable to various veterinary vaccines, including inactivated vaccines, genetic engineering vaccines, etc. It has the advantages of being safe, non-toxic and metabolizable, without stress reaction; high standard and stable quality; significantly improved immune effect, rapid immune response, and can stimulate humoral immunity and cellular immunity; easy to use, directly mixed, and simple to operate. Due to its characteristics of low viscosity and small particle size, in this invention, a vaccine is prepared by using the M101 adjuvant and PCV2 Cap protein to verify the feasibility of this vaccine for needle-free injection immunization of piglets. Therefore, the purpose of this invention is to provide an aqueous adjuvant intradermal immunization porcine circovirus subunit vaccine for needle-free intradermal immunization of pigs.

[0009] Therefore, on the one hand, this invention discloses a porcine circovirus subunit vaccine, which contains porcine circovirus type 2 Cap protein, M101 adjuvant and PBS buffer solution, wherein the addition amount of the M101 adjuvant according to the volume ratio is 5%.

[0010] Preferably, the vaccine of the present invention is inoculated by needle-free intradermal injection, with a dose of 0.2 mL per dose, containing 20 μg of PCV2 Cap protein.

[0011] Preferably, the M101 adjuvant of the present invention is a high-purity aqueous molecular adjuvant, mainly composed of immunologically active substances, with a pH value of 4.0 - 6.5, and enhances the immune response by enhancing immune cell differentiation and antigen presentation ability.

[0012] In one aspect, the present invention also discloses a method for preparing the vaccine, which is: mixing PCV2 Cap protein with PBS to form an aqueous phase, adding M101 adjuvant at a volume ratio of 5%, shaking and mixing evenly, then aliquoting and storing at 4°C for later use.

[0013] In one aspect, the present invention also discloses the application of the M101 adjuvant in the preparation of a needle-free intradermal immunization vaccine against porcine circovirus type 2.

[0014] It should be noted that although the present invention does not provide experimental data on the application of the M101 adjuvant in the needle-free intradermal immunization of other porcine diseases, the researchers of this study have applied this adjuvant to the needle-free intradermal immunization of other porcine diseases (such as PRV / PEDV / PRRSV / CSFV, etc.), and have also achieved the same immune effect as that against porcine circovirus type 2, that is, it has good safety and effectiveness. Therefore, the M101 adjuvant used in the present invention is also equally applicable to the preparation of needle-free intradermal immunization vaccines for other porcine diseases.

[0015] Therefore, the beneficial effects of the present invention are summarized as follows:

[0016] 1. Adjuvant optimization breaks through traditional limitations: The present invention increases the proportion of the M101 adjuvant from the conventional 0.5% to 5%, significantly enhancing the antigen presentation efficiency (the researchers of this study prepared the vaccine using a proportion of 0.5%, and the effect after needle-free injection was far inferior to that of the vaccine prepared with the existing 5% proportion). Comparative tests show that the antibody level of traditional aqueous adjuvants (such as aluminum hydroxide gel) in needle-free injection is significantly lower than that of oil-in-water adjuvants, while the antibody level after the second immunization of the vaccine of the present invention is comparable to that of the commercial vaccine intramuscular injection group, and the antibody level is slightly higher at 6 - 7 weeks after immunization. In addition, the M101 adjuvant promotes immune cell differentiation and antigen presentation, making the immune response more persistent, and the antibody persistence period is the same as that of the intramuscular injection group.

[0017] 2. Safety is significantly better than traditional formulations: Traditional oil-in-water vaccines are prone to problems such as skin rupture and vaccine absorption failure in needle-free intradermal immunization, while the body temperature fluctuation of piglets after immunization with the vaccine of the present invention is <1°C, and the dermal papules naturally subside in about 1 week, with significantly improved safety. The M101 adjuvant is controlled by the quality standards of human vaccines, without irritating components, avoiding the risk of local granuloma of traditional oil adjuvants.

[0018] 3. Physical characteristics adapted to needle-free injection technology: The vaccine of the present invention is a colorless, clear and transparent liquid without precipitation or flocculation, and is suitable for high-pressure jet delivery (injection pressure 60 - 75 psi). In contrast, the oil-in-water vaccine is prone to clogging the needles of needle-free syringes due to its high viscosity and poor stability, and has absorption obstacles after immunization. In addition, the dose of the vaccine of the present invention is only 0.2 mL / dose, which is 80% less than the traditional intramuscular injection dose (1 mL / dose), significantly reducing the cost and enhancing the biosafety. At the same time, while achieving basically the same or better immune effect, the immune dose of the antigen of the present invention is only 20 μg / head / time, while the immune dose of the control vaccine by intramuscular injection of antigen is 60 μg / head / time. Therefore, the immune dose of the present invention is lower, further reducing the production cost of the antigen.

[0019] 4. Process innovation and industrialization advantages: In the prior art, water adjuvant vaccines often require complex emulsification processes (such as double-phase adjuvants), while the present invention is prepared by a direct mixing method without the need for emulsification equipment, and the production cost is reduced by more than 30%. Yisikang Company has established a production line with an annual output of more than 100 tons of water adjuvant, which can meet the demand for 2 billion doses of vaccines, and the industrialization prospect is clear.

[0020] In summary, the porcine circovirus vaccine for needle-free immunization prepared by the present invention reduces the stress response in piglets and has better safety (see the specific results in Example 2). In terms of effectiveness, the antibody level of piglets in the needle-free group of the 1# porcine circovirus subunit vaccine was higher than that of the intramuscular injection group of the 2# commercial circovirus vaccine in the first two weeks after the second immunization, indicating that reducing the immunization dose by needle-free injection can achieve the same effect as intramuscular injection, and has good effectiveness (see the specific results in Example 2). Needle-free immunization simplifies the immunization operation on pig farms, providing a new, fast, safe, cost-reducing and efficiency-enhancing immunization route for the prevention and control of infectious diseases on pig farms, and has broad application prospects and use value. Description of the Drawings

[0021] Figure 1 Purification results of the Cap protein of porcine circovirus type 2.

[0022] Figure 2 Appearance of the porcine circovirus subunit vaccine.

[0023] Figure 3 Condition of the skin bumps in the needle-free group of the 1# porcine circovirus subunit vaccine after the first immunization.

[0024] Figure 4 Body temperature condition of piglets after the first immunization.

[0025] Figure 5 Condition of the skin bumps in the needle-free group of the 1# porcine circovirus subunit vaccine after the second immunization.

[0026] Figure 6Body temperature of piglets after secondary immunization.

[0027] Figure 7 Antibody level of piglets after immunization. Specific implementation manners

[0028] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0029] Example 1: Preparation of porcine circovirus vaccine

[0030] 1.1 Test materials

[0031] Porcine circovirus type 2 Cap protein was prepared by Zhejiang Hongsheng Biotechnology Co., Ltd. through a baculovirus expression system, and the protein purity > 90% ( Figure 1 ); M101 adjuvant was produced and inspected by Chengdu Yisikang Biotechnology Co., Ltd. and was a commercial adjuvant; the commercial vaccine was a porcine circovirus type 2 baculovirus vector inactivated vaccine (aqueous adjuvant).

[0032] 1.2 Test methods

[0033] 1.2.1 Vaccine preparation

[0034] A vaccine was prepared from porcine circovirus type 2 Cap protein and M101 adjuvant. The normal immunization dose was 0.2 mL / dose, and each 0.2 mL contained 20 μg of porcine circovirus type 2 Cap protein. The recommended ratio of M101 was 1:199 (the addition amount was 0.5%). Since the immunization amount for needle-free immunization was only 0.2 mL, in order to ensure the immunization effect, the present invention increased the addition ratio of M101 when preparing the porcine circovirus subunit vaccine. M101 was added to the aqueous phase (mixture of Cap protein and PBS) at a ratio of 5% (the addition amount of M101 in each 0.2 mL of vaccine was 1.0%), shaken and mixed evenly, then dispensed into vaccine bottles and stored at 4 °C for standby. The formulation details of each group are shown in Table 1.

[0035] Table 1 Vaccine preparation information

[0036]

[0037] 1.2.2 Detection of physical properties of the vaccine

[0038] The porcine circovirus subunit vaccine prepared from aqueous adjuvant M101 and PCV2 Cap protein was a colorless, clear and transparent liquid, and there was no turbidity, flocculation and precipitation visually. A certain commercial circovirus vaccine was a light yellow, clear and transparent liquid, see Figure 2 .

[0039] Example 2: Evaluation of Porcine Circovirus Vaccine

[0040] 2.1 Experimental Materials

[0041] The needleless syringe (Model: DERMU-G1-03) was purchased from Jiangsu Leju Medical Technology Co., Ltd.; 3- to 5-week-old piglets negative for porcine circovirus (negative for antigen and antibody) were purchased from Hunan Zhong'an Biopharmaceutical Co., Ltd.; The porcine circovirus 2-dCap-ELISA antibody detection kit (Lot No.: 20240802) was purchased from Beijing Jinnuobaitai Biotechnology Co., Ltd.

[0042] 2.2 Experimental Procedures and Results

[0043] 2.2.1 Safety Verification

[0044] Select 3- to 5-week-old piglets negative for porcine circovirus (negative for antigen and antibody), and randomly divide them into 2 groups with 5 piglets in each group. The needleless group of 1# porcine circovirus subunit vaccine was injected intradermally behind the ear and neck at a dose of 0.2 mL per head (the injection pressure for the first immunization was 60 psi, and the injection pressure for the second immunization was 75 psi). The intramuscular injection group of 2# a commercial porcine circovirus vaccine was injected intramuscularly in the neck at a dose of 1 mL per head. The second immunization was carried out 5 weeks after the first immunization, and the body temperature of the pigs was monitored continuously for 7 days after each immunization.

[0045] When using a needleless syringe for intradermal immunization, the pressure needs to be adjusted according to the size of the pig first. After pressure testing, observe the shape of the wheal and whether there is leakage or bleeding. If there is leakage, it means the pressure is too low and the pressure needs to be increased by 3-5 psi. If the wheal is small or there is bleeding, it means the pressure is too high and the pressure needs to be increased by 3-5 psi. Until the wheal is normal without leakage or bleeding, the immunization can be carried out at this pressure. When 0.2 mL is injected intradermally into normal 3- to 5-week-old piglets, the diameter of the wheal ranges from 1.0 to 2.0 cm. The appearance of the wheal indicates that the vaccine has been injected into the dermis. According to the situation of the piglets, the wheal will last for about 1 week and then disappear, returning to normal. This phenomenon is a normal phenomenon after needleless injection.

[0046] After the first immunization, the needleless group of 1# porcine circovirus subunit vaccine had a large wheal after intradermal immunization. The wheal gradually decreased day by day in the 3 days before immunization and basically returned to normal on the 4th day, as shown in Figure 3 . There was no wheal in the intramuscular injection group of 2# a commercial porcine circovirus vaccine.

[0047] On the 3rd day after intradermal immunization by needleless injection, the body temperature of the piglets rose to 39.6 °C, and the increase in body temperature did not exceed 1 °C. After the 4th day, the body temperature decreased, and the safety was good, as shown in Figure 4 .

[0048] After the second immunization, the wheals produced by the needle-free group of the porcine circovirus subunit vaccine in Pig No. 1 were larger than those produced during the first immunization. On the second day after immunization, the immunization site of one piglet became inflamed and damaged, and returned to normal on the ninth day after immunization; the rest of the piglets basically returned to normal on the fourth day after immunization, see Figure 5 . There were no wheals in the intramuscular injection group of a certain commercial circovirus vaccine (No. 2).

[0049] After the second immunization, the body temperature of the piglets was generally between 38.0 - 39.6 °C, and the increase in body temperature did not exceed 1 °C, and the safety was relatively good, see Figure 6 .

[0050] The above results all indicate that the needle-free immunization with the porcine circovirus vaccine prepared by combining M101 with PCV2 Cap protein has very good safety for pigs.

[0051] In the subsequent R & D process of our company, an oil-in-water vaccine was used for needle-free intradermal immunization. After immunization, phenomena such as skin rupture, non-disappearance of wheals, and non-absorption of the vaccine occurred at the immunization site. This proves that the oil-in-water vaccine has poor safety in needle-free intradermal immunization, which limits the application of this type of vaccine in needle-free intradermal immunization.

[0052] 2.2.2 Efficacy test

[0053] Select 3 - 5-week-old circovirus-negative piglets (antigen-negative, antibody-negative), and randomly divide them into 2 groups, with 5 pigs in each group. The needle-free group of the porcine circovirus subunit vaccine (No. 1) was given a needle-free injection of 0.2 mL / dose behind the ear and on the back of the neck (the injection pressure for the first immunization was 60 psi, and the injection pressure for the second immunization was 75 psi). The intramuscular injection group of a certain commercial circovirus vaccine (No. 2) was given an intramuscular injection of 1 mL / dose in the neck. The second immunization was carried out 5 weeks after the first immunization, and blood was collected weekly after immunization for ELISA antibody titer detection.

[0054] The results are as Figure 7 shown. In the second, third, and fourth weeks after the first immunization, the PCV2 antibody levels of the two groups of piglets were comparable, with no significant difference. In the first two weeks after the second immunization, that is, the sixth and seventh weeks, the PCV2 antibody level of the needle-free group of the porcine circovirus subunit vaccine (No. 1) was slightly higher than that of the intramuscular injection group of a certain commercial circovirus vaccine (No. 2). In the eighth and ninth weeks, the PCV2 antibody levels of the two groups of piglets were comparable, with no significant difference.

[0055] The above results all show that the needle-free immunization with the porcine circovirus vaccine prepared by combining M101 with PCV2 Cap protein has good efficacy.

[0056] 2.3 Conclusion

[0057] After the porcine circovirus subunit vaccine of the present invention is immunized intradermally without needles, it has good safety, which is superior to the oil-in-water vaccine. In terms of effectiveness, the porcine circovirus subunit vaccine has the same effect as the commercial vaccine by intramuscular injection after needle-free immunization, and the immune effect is good. Therefore, the porcine circovirus subunit vaccine is a new type of vaccine that is simple to prepare, safe, effective, and can be used for needle-free intradermal immunization.

[0058] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A porcine circovirus subunit vaccine, characterized in that, The vaccine contains the Cap protein of porcine circovirus type 2, M101 adjuvant and PBS buffer, wherein the addition amount of the M101 adjuvant by volume ratio is 5%.

2. The vaccine according to claim 1, characterized in that, The vaccine is inoculated by needle-free intradermal injection, and the dose per dose is 0.2 mL, containing 20 μg of PCV2 Cap protein.

3. The vaccine according to claim 1, characterized in that, The M101 adjuvant is a high-purity aqueous molecular adjuvant, the main component of which is an immunologically active substance, with a pH value of 4.0 - 6.5, and it enhances the immune response by enhancing the differentiation of immune cells and the ability of antigen presentation.

4. A method for preparing the vaccine according to claim 1, characterized in that, The method is as follows: Mix the PCV2 Cap protein with PBS to form an aqueous phase, add the M101 adjuvant according to a volume ratio of 5%, shake and mix evenly, then subpackage and store at 4°C for standby.

5. Use of the M101 adjuvant as described in claim 1 in the preparation of a needle-free intradermal immunization vaccine against porcine circovirus type 2.

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