Aqueous immunologic adjuvant composition and application thereof

Abstract

The invention belongs to the field of veterinary biological products, and particularly relates to a water-based immunologic adjuvant composition and application thereof. The water-soluble adjuvant prepared from carbomer and synanthrin is a safe, efficient, easy-to-absorb and stress-free water-soluble adjuvant, and in addition, the combination of the carbomer and the synanthrin has an immunological enhancement effect.

Description

Technical Field

[0001] This application belongs to the field of veterinary biological products, and particularly relates to an aqueous immunoadjuvant composition and its application. Background Art

[0002] An immunologic adjuvant is an important component for preparing inactivated vaccines. It can significantly enhance the humoral or cellular immune response of antigens. With the least amount of antigen and the fewest number of inoculations, it can stimulate the body to produce sufficient immune responses and high-titer antibodies, maintain for a long time, and exert a lasting effect.

[0003] Since adjuvants containing white oil for injection in animals have strong side effects, aqueous adjuvants have been developed, such as polyacrylic acid adjuvants represented by Carbopol with the trade name and model number 934, including the commercial Montanide GEL01 adjuvant of Seppic. However, such aqueous adjuvants can cause side effects such as stress and pustules in pets, such as cats. Therefore, there is an urgent need to develop highly efficient, safe, easily absorbable, and stress-free aqueous adjuvants.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] One of the purposes of the present invention is to provide an aqueous immunoadjuvant composition and its application to alleviate the problem of relatively large side effects of existing aqueous adjuvants, especially for pets.

[0006] To achieve the above purpose, the present invention proposes the following technical solutions.

[0007] An aqueous immunoadjuvant composition, the aqueous immunoadjuvant composition contains Carbopol and inulin.

[0008] Further, the mass ratio of the Carbopol to the inulin is 1:(0.2 - 2).

[0009] Further, the content of the Carbopol is 0.2% w / v - 0.5% w / v.

[0010] Further, the content of the inulin is 0.1% w / v - 0.4% w / v.

[0011] The application of the above aqueous immunoadjuvant composition in preparing a vaccine composition.

[0012] A vaccine composition, the vaccine composition contains an immunizing amount of antigen and the aqueous immunoadjuvant composition of the present invention.

[0013] Further, the concentration of the aqueous immunocomposition in the vaccine composition is 5% V / V to 50% V / V.

[0014] Furthermore, the concentration of the aqueous immune composition is 10% V / V.

[0015] Furthermore, the antigen includes inactivated antigen or subunit antigen.

[0016] Furthermore, the antigen includes porcine circovirus type 2 antigen, Mycoplasma hyopneumoniae antigen, feline calicivirus antigen, feline herpesvirus type 1 antigen or feline panleukopenia virus antigen.

[0017] Compared with the prior art, the technical effect of the present invention is as follows:

[0018] The water-soluble adjuvant prepared from carbomer and inulin in the present invention is a safe, highly efficient, easily absorbable and stress-free aqueous adjuvant, which creatively solves the problems of side effects such as stress and pustules caused by the polyacrylic acid aqueous adjuvant represented by carbomer 934 in pets; moreover, it is unexpectedly found that the combination of these two components has an immune-enhancing effect. Detailed Embodiments

[0019] Hereinafter, the embodiments of the present application will be described in detail.

[0020] The present invention provides an aqueous immune adjuvant composition, wherein the aqueous immune adjuvant composition contains carbomer and inulin. The synergistic use of carbomer and inulin can alleviate the problems of side effects such as stress and pustules caused by the aqueous adjuvant in pets (such as cats), and at the same time, there is a synergistic effect between the two, which can enhance the immune effect.

[0021] In some embodiments, the mass ratio of carbomer to inulin is 1:(0.2 - 2). The two components can achieve a better synergistic effect within this range, and the mass ratio can be but is not limited to 1:0.2, 1:0.5, 1:1 or 1:2.

[0022] In some embodiments, the content of carbomer is 0.2% w / v - 0.5% w / v. The content of carbomer can be but is not limited to 0.2% w / v, 0.25% w / v, 0.3% w / v, 0.35% w / v, 0.4% w / v, 0.45% w / v, 0.5% w / v. The content of inulin is 0.1% w / v - 0.4% w / v. The content range of inulin can be selected from 0.1% w / v, 0.15% w / v, 0.2% w / v, 0.25% w / v, 0.3% w / v, 0.35% w / v, 0.4% w / v.

[0023] The present invention also provides the application of the aqueous immune adjuvant composition in the preparation of a vaccine composition. The aqueous immune adjuvant composition of the present invention can be used as a common aqueous adjuvant in the art for the preparation of various common vaccines, and the antigen can be inactivated antigen, subunit antigen, attenuated antigen, etc.

[0024] The present invention also provides a vaccine composition, comprising the aqueous immunoadjuvant composition and an immunologically effective amount of an antigen.

[0025] As an embodiment of the present invention, in the vaccine composition of the present invention, the concentration range of the adjuvant is from 5% V / V to 50% V / V, preferably 10% V / V.

[0026] In the vaccine composition of the present invention, the content of the adjuvant composition can be selected from 5% V / V, 10% V / V, 15% V / V, 20% V / V, 25% V / V, 30% V / V, 35% V / V, 40% V / V, 45% V / V, 50% V / V.

[0027] As an embodiment of the present invention, in the vaccine composition of the present invention, the antigen comprises an inactivated antigen or a subunit antigen. The antigen comprises a porcine circovirus type 2 antigen, a Mycoplasma hyopneumoniae antigen, a feline calicivirus antigen, a feline herpesvirus type 1 antigen or a feline panleukopenia virus antigen.

[0028] The aqueous immunoadjuvant composition of the present invention can be used in combination with a variety of inactivated antigens or subunit antigens, and can effectively enhance the immune effect of these antigens. These antigens can be porcine circovirus antigen, Mycoplasma hyopneumoniae antigen, pseudorabies virus antigen, Haemophilus parasuis antigen, porcine reproductive and respiratory syndrome virus antigen, porcine parvovirus antigen, Pasteurella multocida antigen, Streptococcus suis antigen, Staphylococcus aureus antigen, Bordetella bronchiseptica antigen, Salmonella choleraesuis antigen, Salmonella enteritidis antigen, porcine respiratory coronavirus antigen, porcine epidemic diarrhea virus antigen, porcine rotavirus antigen, transmissible gastroenteritis virus antigen, porcine circovirus antigen, porcine cytomegalovirus antigen, encephalomyocarditis virus antigen, porcine influenza virus antigen, classical swine fever virus antigen or a combination thereof.

[0029] Explanation of relevant terms in the present invention:

[0030] The term "adjuvant" refers to a compound that, when administered together with an antigen, enhances the immune response of the body to that antigen. The adjuvant-mediated enhancement of the immune response can be evaluated by any method known in the art, including (but not limited to) one or more of the following methods: (i) an increase in the number of antibodies produced in response to immunization with the adjuvant / antigen combination compared to the number of antibodies produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells that recognize the antigen or the adjuvant; (iii) an increase in the levels of one or more type I cytokines; and (iv) protection in vivo after challenge. An enhanced immune response is considered when any measurable parameter of antigen-specific immunoreactivity (e.g., antibody titer or T cell production) is increased by at least 10% when the body is challenged with the antigen and the adjuvant compared to the body challenged with the antigen alone. In certain embodiments, an enhanced immune response is considered when any measurable parameter of antigen-specific immunoreactivity is increased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, at least 250%, at least 275%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, or at least 1000%.

[0031] "Acrylic polymer" refers to any polymer or copolymer containing an acrylic moiety. Exemplary acrylic polymers include, for example, polyacrylic acid, methacrylic acid, methacrylates, acrylamides, acrylates, acrylonitrile, and alkyl esters of polyacrylic acid. Examples of acrylic copolymers include, for example, polyacrylamide-co-butyl methacrylate, acrylic acid-methacrylic acid, acrylic acid-acrylamide, and polymethacrylates. Examples of commercially available acrylic polymers include carbomer, which is also commonly referred to and known in the art as a water-soluble polymer of acrylic acid crosslinked with polyallylsucrose. The amount used is generally from about 0.0001% volume / volume (v / v) to about 75% v / v. In other embodiments, the amount used is selected from about 0.001% v / v to about 50% v / v, about 0.005% v / v to about 25% v / v, about 0.01% v / v to about 10% v / v, about 0.05% v / v to about 2% v / v, and about 0.1% v / v to about 0.75% v / v.

[0032] The term "inulin" is also known as artichoke mannan and inulin, and is widely present in plant tissues. In particular, Jerusalem artichoke tubers and chicory roots are rich in inulin. Jerusalem artichoke, also known as sunchoke, is a perennial herbaceous plant that is widely planted in China. It has wide adaptability, is tolerant to barrenness, has high yields, and is easy to grow. In addition to water, Jerusalem artichoke tubers contain 15% - 20% inulin, making them good raw materials for the processing and production of inulin and its products. The amount of inulin used is usually selected from about 0.001% v / v to about 50% v / v, about 0.005% v / v to about 25% v / v, about 0.01% v / v to about 10% v / v, about 0.05% v / v to about 2% v / v, and about 0.1% v / v to about 0.75% v / v.

[0033] The term "vaccine composition" refers to a composition that can be used to induce protective immunity in an organism. Therefore, after the organism has been inoculated with an antigen, the vaccine can prevent, delay, or reduce the severity of disease development in the organism exposed to the same or related antigen (compared to an unvaccinated organism). The protective immunity provided by the vaccine can be humoral (antibody-mediated) immunity, cellular immunity, or both.

[0034] The term "antigen" refers to a substance that can induce an immune response in an organism, that is, a substance that can be specifically recognized and bound by the antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activate T / B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro.

[0035] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0036] The chemical reagents used in the embodiments of the present invention are all of analytical grade and are purchased from the Sinopharm Group. The experimental methods described in the present invention are all conventional methods unless otherwise specified; the biological materials, unless otherwise specified, can be obtained from commercial sources.

[0037] Example 1 Preparation of Carbomer Solution

[0038] 1 g of Carbomer 934 was added to 50 ml of water, stirred to dissolve it, then 1.5 g of sodium chloride solution was added, and the pH was adjusted to 7.0 with sodium hydroxide solution. After autoclaving, it was reserved for use, with a content of 2% w / v.

[0039] Example 2 Preparation of Inulin Solution

[0040] 5 g of inulin (GR type, Beneo-orafti brand from Belgium) was added to 100 ml of water, incubated in a water bath at 55 °C for 1 hour, then heated to 85 °C for complete dissolution, filtered through a 0.22-μm filter membrane and reserved for use, with a content of 5% w / v.

[0041] Preparation of the adjuvant composition in Example 3

[0042] The carbomer solution prepared in Example 1 and the inulin solution prepared in Example 2 were mixed evenly according to the ratio in Table 1 and made up to 100 ml with PBS solution at pH 7.0.

[0043] Table 1 Ratio of the adjuvant composition

[0044] Safety test of adjuvant 1 in Example 4

[0045] The Mycoplasma hyopneumoniae antigen solution was stirred evenly with adjuvant 1 in Example 3 to prepare Vaccine 1 and Vaccine 2. Among them, 2 ml of the finished vaccine contained 3×10 9 CCU of pre-inactivated bacteria. Vaccine 1 contained 10% adjuvant, and Vaccine 2 contained 20% adjuvant. Twenty-five 14-day-old piglets were selected, divided into 5 groups with 5 piglets in each group. The first group and the second group were immunized with 2 ml of Vaccine 1 and Vaccine 2 respectively, the third group and the fourth group were immunized with 4 ml of Vaccine 1 and Vaccine 2 respectively, and the fifth group was immunized with 4 ml of sterile normal saline as the control group. All piglets were continuously observed for 14 days after immunization, including whether there were any abnormalities in spirit, food intake, water intake, behavior status, etc. At the same time, whether there were any abnormalities such as swelling and redness at the vaccine injection site was observed. The results showed that no adverse reactions were observed in all piglets immunized with the vaccine and the control piglets during the 14-day observation. The mental state was good, the food intake and water intake were normal, and there was no death. No abnormal changes were found in all organs after dissection, and there was no abnormal situation such as swelling and redness at the injection site.

[0046] The feline panleukopenia virus antigen solution was stirred evenly with adjuvant 1 in Example 3 to prepare Vaccine 3 and Vaccine 4. Among them, 1 ml of the finished vaccine contained 10 6 FAID 50, Vaccine 3 contains 10% adjuvant, and Vaccine 4 contains 20% adjuvant. Twenty-five healthy and susceptible antigen-antibody negative cats at 2 months of age were randomly divided into 5 groups, with 5 cats in each group. Cats in Group 6 and Group 7 were immunized with 1 ml of Vaccine 3 and Vaccine 4 respectively, cats in Group 8 and Group 9 were immunized with 2 ml of Vaccine 3 and Vaccine 4 respectively, and cats in Group 10 were immunized with 2 ml of sterile normal saline as the control group. All cats were continuously observed for 14 days after immunization, including whether there were any abnormalities in spirit, food intake, feces, body temperature, etc., and at the same time, whether there were any abnormalities such as redness and swelling at the vaccine injection site. The results showed that all cats in the vaccine immunization groups showed listlessness, loss of appetite, diarrhea, and elevated body temperature. At the same time, redness, induration and other phenomena were observed at the vaccine injection site; the side effects in Group 9 were the most obvious; the cats in the control group were in good spirit, with no abnormal behavior, normal feces, normal food intake, normal body temperature, and no local redness, induration and other phenomena at the injection site.

[0047] It shows that although the aqueous adjuvant represented by carbomer solves the safety problem of livestock vaccines, it still has strong side effects on pets and cannot be actually applied.

[0048] Example 5 Preparation of Feline Panleukopenia Virus Vaccine Composition

[0049] The feline panleukopenia virus antigen solution was uniformly stirred with Adjuvant 2, Adjuvant 3, Adjuvant 4, and Adjuvant 5 in Example 3 to prepare Vaccine 5, Vaccine 6, Vaccine 7, Vaccine 8, and Vaccine 9. The specific formula of the vaccine is shown in Table 2.

[0050] Table 2 Ratio of Feline Panleukopenia Virus Vaccine

[0051] Example 6 Evaluation Test of Feline Panleukopenia Virus Vaccine Composition

[0052] Thirty healthy and susceptible antigen-antibody negative cats at 2 months of age were randomly divided into 6 groups, with 5 cats in each group. Cats in Group 11 to Group 15 were immunized with 1 ml of Vaccine 5, Vaccine 6, Vaccine 7, Vaccine 8, and Vaccine 9 respectively, and cats in Group 16 were immunized with 1 ml of sterile normal saline as the control group. All cats were continuously observed for 14 days after immunization, including whether there were any abnormalities in spirit, food intake, feces, body temperature, etc., and at the same time, whether there were any abnormalities such as redness and swelling at the vaccine injection site. The results showed that no adverse reactions occurred in all cats in the vaccine immunization groups and the control group during the 14-day observation. The mental state was good, the feces were normal, the food intake and drinking water were normal, the body temperature was normal, and no local redness, induration and other phenomena were observed at the injection site.

[0053] Twenty-one days after the above cats were immunized, they were challenged with feline panleukopenia virus, and each cat was inoculated with 4 ml (10 5.5 FAID 50( / ml). After challenging, the clinical manifestations such as body temperature, appetite, and feces of the cats were observed and recorded daily, and the peripheral blood white blood cell count was measured regularly. The specific results are shown in Table 3.

[0054] Table 3 Results of the challenge test of the feline panleukopenia virus vaccine composition

[0055] The results showed that all the cats vaccinated with the feline panleukopenia virus vaccine composition prepared with the adjuvant containing carbomer and inulin were protected. Only partial protection was obtained in the cats vaccinated with the feline panleukopenia virus vaccine composition prepared with inulin alone as the adjuvant, while all the cats in the control group developed the disease and died. The safety test also showed that the adjuvant containing carbomer and inulin solved the problem of relatively large side effects of the carbomer adjuvant on pets. In addition, both Group 13 and Group 14 corresponding to Vaccines 7 and 8 solved the safety problem and had good immune efficacy. The components of carbomer and inulin in Adjuvants 3 and 4 used by the two were relatively low, and it could be considered that there was a synergistic effect between the two, which could enhance the immune effect.

[0056] Example 7 Preparation of the feline herpesvirus type 1 vaccine composition

[0057] The feline herpesvirus type 1 antigen solution was uniformly stirred with Adjuvant 3, Adjuvant 4, and Adjuvant 5 of Example 3 to prepare Vaccines 10, 11, and 12. The specific formula of the vaccine is shown in Table 4.

[0058] Table 4 Ratio of the feline herpesvirus type 1 vaccine

[0059] Example 8 Evaluation test of the feline herpesvirus type 1 vaccine composition

[0060] Twenty 2-month-old healthy susceptible antigen-antibody negative cats were randomly divided into 4 groups, with 5 cats in each group. Groups 17 to 19 were immunized with 1 ml of Vaccines 10, 11, and 12 respectively, and Group 20 was immunized with 1 ml of sterile normal saline as the control group. Immunization was carried out twice at an interval of 21 days. All the cats were continuously observed for 14 days after immunization, including whether there were abnormalities in spirit, feeding, body temperature, etc., and whether there were abnormalities such as redness and swelling at the vaccine injection site. The results showed that no adverse reactions occurred in the cats in the vaccine immunization group and the control group during the 14-day observation. The mental state was good, the feeding and drinking were normal, the body temperature was normal, and no local redness, induration, etc. were observed at the injection site.

[0061] Fourteen days after the second immunization, the above cats were challenged with the feline herpesvirus type 1. Each cat was instilled nasally with 0.5 ml (10 4.5 TCID 50( / ml). After virus challenge, the body temperature of the cats, as well as the clinical manifestations such as whether conjunctivitis occurred, eye and nasal secretions, and sneezing, were observed and recorded daily. According to the duration of the clinical symptoms, the clinical symptoms were scored daily according to the scoring criteria in Table 5, and the total clinical score of each cat within 14 days after virus challenge was calculated. The differences in the clinical scores between the immunized group and the control group were compared. The specific results are shown in Table 6.

[0062] Table 5 Scoring criteria for clinical symptoms after challenge with feline herpesvirus type 1

[0063] Table 6 Results of the challenge test of the feline herpesvirus type 1 vaccine composition

[0064] Note: Different superscript letters indicate significant differences (P < 0.05). The above clinical scores were daily scores and were calculated based on the total score.

[0065] The results showed that all cats in the control group developed the disease, and the clinical scores of the immunized group with the feline herpesvirus type 1 vaccine composition prepared with the adjuvant containing carbomer and inulin were significantly lower than those of the control group after virus challenge. The safety test further demonstrated that the adjuvant containing carbomer and inulin solved the problem of relatively large side effects of the carbomer adjuvant on pets.

[0066] Example 9 Preparation of a feline calicivirus vaccine composition

[0067] The feline calicivirus antigen solution was uniformly mixed with Adjuvant 3, Adjuvant 4, and Adjuvant 5 in Example 3 to prepare Vaccine 13, Vaccine 14, and Vaccine 15, respectively. The specific formula of the vaccine is shown in Table 7.

[0068] Table 7 Formulation ratio of the feline calicivirus vaccine

[0069] Example 10 Evaluation test of the feline calicivirus vaccine composition

[0070] Twenty 2-month-old healthy susceptible antigen-antibody negative cats were randomly divided into 4 groups, with 5 cats in each group. Cats in groups 21 to 23 were immunized with 1 ml of Vaccine 13, Vaccine 14, and Vaccine 15, respectively, and cats in group 24 were immunized with 1 ml of sterile normal saline as the control group. Immunization was carried out twice at an interval of 21 days. All cats were continuously observed for 14 days after immunization, including whether there were abnormalities in spirit, food intake, body temperature, etc., and at the same time, whether there were abnormalities such as redness and swelling at the vaccine injection site was observed. The results showed that no adverse reactions occurred in the cats in the vaccine immunized group and the control group during the 14-day observation. The mental state was good, the food intake and drinking water were normal, the body temperature was normal, and no local redness, induration, etc. were observed at the injection site.

[0071] After 14 days of the second immunization, the cats were challenged with feline calicivirus. Each cat was instilled with 1 ml (10 7.0 TCID 50 / ml) by nasal drip. After the challenge, the body temperature, appetite, eye and nasal secretions, oral cavity, foot pads and other clinical manifestations of the cats were observed and recorded daily. According to the duration of the clinical symptoms, the clinical symptoms were scored daily according to the scoring criteria in Table 8, and the total clinical score within 14 days after the challenge for each cat was calculated. The differences in the clinical scores between the immunized group and the control group were compared. The specific results are shown in Table 9.

[0072] Table 8 Clinical symptom scoring criteria after feline calicivirus challenge

[0073] Table 9 Results of the feline calicivirus vaccine composition challenge test

[0074] Note: Different superscript letters indicate significant differences (P < 0.05). The above clinical scores were daily scores and were calculated based on the total score.

[0075] The results showed that all the cats in the control group developed the disease, and the clinical scores of the immunized group with the feline calicivirus vaccine composition prepared with the adjuvant containing carbomer and inulin were significantly lower than those of the control group after the challenge. The safety test further demonstrated that the adjuvant containing carbomer and inulin solved the problem of relatively large side effects of the carbomer adjuvant on pets.

[0076] Example 11 Preparation of a Mycoplasma hyopneumoniae vaccine composition for pigs

[0077] The Mycoplasma hyopneumoniae antigen solution was respectively and uniformly stirred with Adjuvant 3, Adjuvant 4, and Adjuvant 5 of Example 3 to prepare Vaccines 16, 17, and 18. The specific formulations of the vaccines are shown in Table 10. Table 10 Ratio of the Mycoplasma hyopneumoniae vaccine for pigs

[0078] Example 12 Evaluation test of the Mycoplasma hyopneumoniae vaccine composition for pigs

[0079] Twenty healthy piglets at 21 days old and negative for Mycoplasma hyopneumoniae were selected and randomly divided into 4 groups of 5 piglets each. Piglets in groups 25, 26, and 27 were each intramuscularly injected with 1 ml of vaccine 16, vaccine 17, and vaccine 18 respectively in the neck. Piglets in group 28 were immunized with 1 ml of sterile saline as the control group. All piglets were continuously observed for 14 days after immunization, including for any abnormalities in mental state, food intake, water intake, and behavior. At the same time, the injection sites of the vaccines were observed for any swelling or other abnormalities. The results showed that no adverse reactions were observed in the piglets immunized with the vaccines for 14 days. The mental state was good, food intake and water intake were normal, there were no deaths, and no local swelling or induration was observed at the injection sites.

[0080] On the 35th day after immunization, all pigs were intratracheally injected with the highly virulent tissue strain CVCC354 (purchased from the China Institute of Veterinary Drug Control, and this strain is the strain for potency test of Mycoplasma hyopneumoniae vaccine preserved by the China Institute of Veterinary Drug Control) at 5 ml per pig (100 MID). They were observed for 28 days, then sacrificed and their lungs were taken. The mycoplasmal pneumonia lesions of the test pigs were scored according to the 28-point method, and the reduction rate of pneumonia lesions was calculated according to the following formula.

[0081] Reduction rate of pneumonia lesions = (average score of pneumonia lesions in challenged control pigs - average score of pneumonia lesions in immunized pigs) / average score of pneumonia lesions in challenged control pigs × 100%

[0082] The results of the challenge protection are shown in Table 11. The results indicate that there were extremely significant differences in the average lung lesions between the immunized groups with vaccine 16, vaccine 17, and vaccine 18 and the challenged control group. This shows that the Mycoplasma hyopneumoniae vaccine composition prepared with the adjuvant containing carbomer and inulin has good immunogenicity.

[0083] Table 11 Results of the challenge test of the Mycoplasma hyopneumoniae vaccine composition

[0084] Note: Different superscript letters indicate significant differences (P < 0.05).

[0085] Example 13 Preparation of a porcine circovirus type 2 vaccine composition

[0086] The porcine circovirus type 2 subunit antigen solution was uniformly mixed with adjuvant 3, adjuvant 4, and adjuvant 5 of Example 3 to prepare vaccine 19, vaccine 20, and vaccine 21 respectively. The specific formulations of the vaccines are shown in Table 12.

[0087] Table 12 Formulation ratio of the porcine circovirus type 2 vaccine Component Vaccine 19 Vaccine 20 Vaccine 21 Antigen (μg / ml) 20 20 20 Adjuvant 3 (V / V%) 10％ - - Adjuvant 4 (V / V%) - 10％ - Adjuvant 5 (V / V%) - - 10％

[0088] Example 14 Evaluation test of the porcine circovirus type 2 vaccine composition

[0089] Twenty healthy piglets at 21 days of age that were negative for porcine circovirus type 2 were selected and randomly divided into 4 groups, with 5 piglets in each group. Piglets in groups 29, 30, and 31 were each intramuscularly injected with 1 ml of vaccine 19, vaccine 20, and vaccine 21 respectively in the neck muscle, and piglets in group 32 were immunized with 1 ml of sterile normal saline as the control group. All piglets were continuously observed for 14 days after immunization, including whether there were any abnormalities in spirit, food intake, water intake, and behavior status, etc., and at the same time, whether there were any abnormalities such as swelling at the vaccine injection site. The results showed that no adverse reactions were observed in the vaccinated piglets during the 14-day observation period. Their mental state was good, food intake and water intake were normal, there were no deaths, and no local swelling or induration was observed at the injection site.

[0090] Thirty-five days after the above pigs were immunized, all pigs were challenged with porcine circovirus type 2 (containing 10 6.0 TCID 50 / ml). Each pig was intranasally inoculated with 1 ml / head and intramuscularly injected with 2 ml / head. On the 4th and 7th days after challenge, keyhole limpet hemocyanin emulsified with Freund's incomplete adjuvant (KLH / ICFA, 0.5 mg / ml) was inoculated into all pigs at a total of 4 points on both sides of the axilla and both sides of the buttocks of each challenged pig, with 1 ml inoculated at each point (4 ml / head). At the same time, thioglycolate medium was intraperitoneally inoculated, 10 ml / head; on the 11th and 19th days after challenge, thioglycolate medium was intraperitoneally inoculated again, 10 ml / head. The pigs were continuously observed for 25 days after challenge, weighed and euthanized on the 25th day after challenge, and necropsied. The determination was made based on the body temperature, relative daily weight gain, and virus antigen detection results.

[0091] After challenge with porcine circovirus type 2, the changes in body temperature are shown in Table 13. Only individual piglets in the immunized groups of vaccine 19, vaccine 20, and vaccine 21 showed transient body temperature elevation, and the body temperature returned to normal quickly after one day of elevation, without other clinical symptoms; after challenge, the body temperature of all pigs in the control group rose above 40.5 °C, lasted for 3 - 5 days, with reduced appetite, listlessness, rough hair, weight loss, and slowed growth rate.

[0092] Table 13 Changes in body temperature after challenge with porcine circovirus type 2 (number of consecutive days)

[0093] The morbidity and protection status after virus challenge are shown in Tables 14 and 15. After virus challenge, the piglets immunized with Vaccine 19, Vaccine 20, and Vaccine 21 showed no obvious clinical symptoms, no specific pathological changes were observed, and all antigen PCR tests were negative, with a protection effect of 5 / 5; while all the piglets in the control group developed the disease, with obvious clinical symptoms and pathological changes, and all pathogen PCR tests were positive. By the end of the virus challenge observation, there was no significant difference in the average daily weight gain of the piglets immunized with Vaccine 19, Vaccine 20, and Vaccine 21. Compared with the control group, the average daily weight gain of the piglets immunized with Vaccine 19, Vaccine 20, and Vaccine 21 was extremely significantly higher than that of the control group. This indicates that the porcine circovirus type 2 vaccine composition prepared with the adjuvant containing carbomer and inulin has a good protective effect against PCV2 challenge after immunization.

[0094] Table 14 Results of morbidity determination after porcine circovirus type 2 challenge

[0095] After PCV2 challenge, if any 2 of the following 3 items are met, it can be judged as morbidity.

[0096] A: Clinical symptoms: The body temperature of the piglets rises (≥40°C), which should last for at least 3 days, showing obvious loss of appetite, listlessness, rough hair, weight loss, and slow growth rate;

[0097] B: Pathological changes: Edema of the inguinal and tracheal lymph nodes, mild pulmonary edema, and the kidneys are yellowish or have punctate necrosis. Histological lesions show obvious lymphocyte infiltration in the lymph nodes or multinucleated giant cells;

[0098] C: Virus detection: PCV2 is detected by PCR in lymph node tissues.

[0099] Table 15 Protection status after porcine circovirus type 2 challenge Group Number of piglets Challenge protection rate Average daily gain (kg) 29 5 5 / 5 <![CDATA[0.0253 a > 30 5 5 / 5 <![CDATA[0.0255 a > 31 5 5 / 5 <![CDATA[0.0252 a > 32 5 0 / 5 <![CDATA[0.0136 b >

[0100] Note: Different superscript letters indicate significant differences (P < 0.05).

[0101] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the above-disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change, and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

[0102] Unless otherwise defined, all technical and scientific terms used throughout this application shall have the same meaning as commonly understood by those skilled in the art to which this application pertains. In case of any inconsistency, the meaning set forth in this application or the meaning derived from the content recorded in this application shall prevail. Additionally, the terms used in this specification are for the purpose of describing the embodiments of this application only and are not intended to limit this application.

Claims

1. An aqueous immunoadjuvant composition, characterized in that, The aqueous immune adjuvant composition contains carbomer and inulin.

2. The aqueous immunoadjuvant composition according to claim 1, wherein The mass ratio of the carbomer to the inulin is 1:(0.2 - 2).

3. The aqueous immunoadjuvant composition according to claim 1 or 2, characterized in that, The content of the carbomer is 0.2% w / v - 0.5% w / v.

4. The aqueous immunoadjuvant composition according to claim 3, characterized in that, The content of the inulin is 0.1% w / v - 0.4% w / v.

5. Use of the aqueous immune adjuvant composition according to any one of claims 1 to 4 in the preparation of a vaccine composition.

6. A vaccine composition, characterized in that, The vaccine composition contains an immunogenic amount of an antigen and the aqueous immune adjuvant composition according to any one of claims 1 to 4.

7. The vaccine composition according to claim 6, wherein, The concentration of the aqueous immune composition in the vaccine composition is 5% V / V to 50% V / V.

8. The vaccine composition according to claim 7, wherein, The concentration of the aqueous immune composition is 10% V / V.

9. The vaccine composition according to any one of claims 6 to 8, characterized in that, The antigen includes an inactivated antigen or a subunit antigen.

10. The vaccine composition according to any one of claims 6 to 8, characterized in that, The antigen includes a porcine circovirus type 2 antigen, a Mycoplasma hyopneumoniae antigen, a feline calicivirus antigen, a feline herpesvirus type 1 antigen, or a feline panleukopenia virus antigen.

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