CN107496914B - Adjuvant composition and preparation method and application thereof - Google Patents

Abstract

The invention relates to an adjuvant composition and a preparation method and application thereof in the technical field of biological products for livestock. The adjuvant composition comprises acrylic polymer-polylysine nanoparticles; specifically, 100 parts by weight of the adjuvant composition comprises 0.001-5 parts by weight of polylysine nanoparticles, which is an acrylic polymer. The acrylic polymer-polylysine nanoparticles contained in the adjuvant composition are prepared by simple electrostatic interaction, do not have cross-linking between proteins harmful to organisms, have high safety and effectiveness, are suitable for being used as adjuvants, and overcome the problems existing in the use of polyacrylic acid as adjuvants. In addition, the vaccine composition formed from the adjuvant composition and antigen has good stability for long-term storage and can be administered safely and effectively to a wide variety of subjects.

Description

Adjuvant composition and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological products for livestock, and particularly relates to an adjuvant composition and a preparation method and application thereof.

Background

Adjuvants are materials used to produce vaccines with increased antigenicity or to achieve therapy and prevention by increasing the non-specific immune response to an antigen. At low antigen levels, adjuvants are used to maintain a strong and rapid immune response to the antigen for a longer period of time, and these adjuvants can be used in the preparation of vaccines. Adjuvants can use a particular antigen or can alter the level of antigen, thereby modulating the immune response to the antigen or controlling the type and subclass of antibody to the antigen. In addition, adjuvants may also be used to enhance the immune response, particularly in the induction of mucosal immunity in immunocompromised or senescent humans.

Most adjuvants are found in natural materials by repeated experiments. In the first report of adjuvants worldwide in 1925, Ramon (france) reported that tapioca starch (Casaba) used in food mixed with diphtheria and tetanus toxoids would effectively increase antigen-specific antibody production. Next, the immunopotentiating effect of the aluminum adjuvant was found, and an effective emulsion-type adjuvant comprising inactivated mycobacteria was developed as an immunomodulator. When polyacrylic acid is used as an adjuvant in veterinary vaccines, polyacrylic acid itself has negative charges in a physiological environment and is difficult to adsorb negatively charged antigens. In addition, polyacrylic acid precipitates in sodium chloride solution, which also affects its use as an adjuvant. The technical scheme commonly adopted in the prior art is as follows: the polyacrylic acid is added with other adjuvants and immunopotentiators to form a compound adjuvant for use.

The prior art lacks an adjuvant composition which can enhance the adjuvant immune effect and applicable antigen range of polyacrylic acid, can improve the stability of polyacrylic acid solution, has simple preparation and does not generate side effect.

Polylysine, which is formed by the linkage of the amino group of the essential amino acid L-lysine to the alpha-carboxyl group of another L-lysine in an amide bond. The prior art has no report that the prior art has adjuvant effect.

Disclosure of Invention

The technical problem to be solved by the present invention is to provide an adjuvant composition which has high safety and effectiveness, and a vaccine composition formed from the adjuvant composition and an antigen has good stability for long-term storage, and can be safely and effectively administered to a wide variety of subjects, in view of the shortcomings of the prior art.

To this end, a first aspect of the invention provides an adjuvant composition comprising an acrylic polymer, polylysine nanoparticles.

In some embodiments of the invention, the acrylic polymer, polylysine nanoparticles, are included in an amount of 0.001 to 5 parts by weight (dry weight) per 100 parts by weight of the adjuvant composition. Preferably, the polylysine nanoparticles, which are acrylic polymers, are included in an amount of 0.01 to 3 parts by weight (dry weight) per 100 parts by weight of the adjuvant composition. Further preferably, the polylysine nanoparticles, an acrylic polymer, are included in an amount of 0.45 to 1 part by weight (dry weight) per 100 parts by weight of the adjuvant composition. Still more preferably, the adjuvant composition comprises 0.55 to 1 part by weight (dry weight) of polylysine nanoparticles, an acrylic polymer, per 100 parts by weight of the adjuvant composition. Most preferably, the polylysine nanoparticles, an acrylic polymer, are included in an amount of 0.6 to 0.75 parts by weight (dry weight) per 100 parts by weight of the adjuvant composition. When 100 parts by weight of the acrylic polymer-polylysine nanoparticles included in the adjuvant composition is less than 0.001 parts by weight, the adjuvant composition will not have the ability to produce antibodies; when the polylysine nanoparticle, which is an acrylic polymer, is included in an amount of more than 5 parts by weight, the viscosity of the adjuvant composition is greatly increased.

In the present invention, the weight parts of the acrylic polymer, i.e., polylysine nanoparticles, in the adjuvant composition are the dry weight of the acrylic polymer, i.e., polylysine nanoparticles, in the adjuvant composition. The nanoparticles of the adjuvant composition may be dispersed in water or an aqueous electrolyte solution.

In the invention, the acrylic polymer-polylysine nanoparticles are prepared by forming ionic bonds through electrostatic interaction between negative charges of carboxyl reaction groups of the acrylic polymer and positive charges of protonation of amino reaction groups of polylysine. In some embodiments of the invention, the mass ratio of acrylic polymer to polylysine is (1-10): 1. In other embodiments of the present invention, the acrylic polymer, polylysine nanoparticles, have a particle size of 200nm to 1500 nm.

In some embodiments of the invention, the acrylic polymer is selected from one or more of polyacrylic acid, polymethacrylic acid, poly (acrylic acid-co-methacrylic acid), and poly (acrylic acid-co-acrylamide). However, the acrylic polymer according to the present invention is not limited thereto, and may be polymethacrylate (preferably, alkyl ester of polyacrylic acid), polyacrylamide, polyacrylate, polyacrylonitrile, or poly (acrylamide-co-butyl methacrylate), which are all anionic compounds, in which carboxyl groups are only partially substituted and carboxyl groups still exist.

Acrylic polymers can swell in water up to 1000 times their original volume and 10 times their original diameter to form a gel when exposed to a pH environment above the pKa of the carboxylate group. At higher pH than the pKa of the carboxylate group, the carboxylate group ionizes, resulting in repulsion between negatively charged groups, increasing the volume expansion of the polymer.

In some preferred embodiments of the invention, the acrylic polymer is polyacrylic acid having an average equivalent weight of 76 and is prepared from starting polymer particles having an average diameter of about 0.2 to 6.0 μm. Further preferably, the polyacrylic acid is a commercially available carbomer (trade name), which in the art also refers generally to a water soluble polymer of acrylic acid cross-linked with polyallyl sucrose. In some embodiments of the invention, the polyacrylic acid is commercially available carbomer 934P (trade name).

In the invention, the relative molecular mass of the polylysine is 1000kDa-15000 kDa. If the relative molecular mass of polylysine is less than 1000kDa, its immunopotentiating effect will be low; if the relative molecular mass of polylysine is greater than 15,000kDa, the viscosity of the adjuvant composition formed therefrom will increase substantially.

In a second aspect, the present invention provides a process for the preparation of an adjuvant composition according to the first aspect of the invention, which comprises: the acrylic polymer solution and the polylysine solution were mixed under stirring to obtain an adjuvant composition.

In some embodiments of the invention, the acrylic polymer solution and the polylysine solution each have a pH of 6.9 to 7.5; preferably, the pH of the acrylic polymer solution and the polylysine solution is 7.2. The pH value of the solution is too low, so that the particle size of the precipitated acrylic polymer, namely polylysine nano-particles is too large; too high a pH will result in less acrylic polymer, polylysine nanoparticles, being precipitated. In the present invention, the solution for dissolving the acrylic polymer and polylysine is preferably a NaCl solution; specifically, the concentration of the NaCl solution is 0.85%.

In other embodiments of the present invention, the mass ratio of the acrylic polymer solution to the polylysine solution is (1-10): 1.

In a third aspect, the invention provides a vaccine composition comprising an adjuvant composition according to the first aspect of the invention or prepared by a method according to the second aspect of the invention and an immunologically effective amount of an antigen.

The term "immunologically effective amount" also referred to as an immunoprotective amount or an amount effective to produce an immune response, is an amount effective to induce an immunogenic response in a recipient. The immune response may be sufficient for diagnostic purposes or other testing, or may be suitable for use in preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.

The term "antigen", also called immunogen, refers to any substance that stimulates an immune response, is a molecule that can be bound by antibodies and is also capable of inducing a humoral and/or cellular immune response that produces B-and/or T-cells, and also has one or more epitopes (B-and T-epitopes). The antigens are viruses (inactivated viruses, attenuated and modified live viruses), bacteria, parasites, nucleotides, polynucleotides, peptides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, carbohydrates (e.g., polysaccharides), fatty acids, teichoic acids (teichoc acids), peptidoglycans, lipids and glycolipids, individually or in any combination thereof.

Viruses that can be used as antigens include, but are not limited to: avian herpes virus, bovine herpes virus, canine herpes virus, equine herpes virus, ovine herpes virus, porcine herpes virus, feline viral rhinotracheitis virus, marek's Disease virus, pseudorabies virus, avian paramyxovirus, bovine respiratory syncytial virus, canine distemper virus, canine parainfluenza virus, canine adenovirus, canine parvovirus, bovine parainfluenza virus 3, ovine parainfluenza virus 3, bove Disease virus, border Disease virus, Bovine Viral Diarrhea Virus (BVDV), BVDV type I, BVDV type II, Classical swine fever virus (classic swine fever virus), avian leukemia virus, bovine immunodeficiency virus, bovine leukemia virus, bovine tuberculosis virus, porcine infectious anemia virus, feline immunodeficiency virus, feline leukemia virus (FeLV), Newcastle Disease virus (Newcastle Disease virus), progressive pneumonia virus of ovine, pneumoconiosis virus, Canine Coronavirus (CCV), Pantropic CCV (pantopicCCV), canine respiratory coronavirus, bovine coronavirus, feline calicivirus, feline enteric coronavirus, feline infectious peritonitis virus, porcine epidemic diarrhea virus, porcine thromboencephalomyelitis virus, porcine parvovirus, porcine circovirus type I (PCV), PCV type II, Porcine Reproductive and Respiratory Syndrome (PRRS) virus, transmissible gastroenteritis virus, turkey coronavirus, bovine epidemic fever virus, rabies rotavirus, vesicular stomatitis virus, one or more of lentivirus, avian influenza virus, rhinovirus, equine influenza virus, swine influenza virus, canine influenza virus, feline influenza virus, human influenza virus, eastern equine encephalitis virus (EEE), Venezuelan equine encephalitis virus, West Nile virus, Western equine encephalitis virus, human immunodeficiency virus, human papilloma virus, varicella zoster virus, hepatitis B virus, rhinovirus, and measles virus.

The peptide antigens include one or more of Bordetella bronchiseptica p68, GnRH, IgE peptide, Fel d1, and cancer antigens.

Parasites that may be used as antigens include, but are not limited to: one or more members of the genera Anamorpha, Fasciola hepatica, Coccidia, Eimeria, Neosporon Canitis, Toxoplasma gondii, Giardia, Dirofilaria, Trypanosoma, Leishmania, Trichomonas, Cryptosporidium parvum, Babesia, Schistosoma, Taenia, Toxoid, ascaris, Trichostrongylus, Sarcocystis, Hammond and Isospora.

Bacteria that can be used as antigens include, but are not limited to: acinetobacter calcoaceticus (Acinetobacter calcoaceticus), Acinetobacter pasteurianus (Acetobacter pasania), Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Aeromonas hydrophila (Aeromonas hydrophyllum), Alicyclobacillus acidocaldarius (Alicyclobacillus acidocaldarius), Archaeoglobus fulgidus (Arhaplobus fulgidus), Bacillus pumilus (Bacillus pumilus), Bacillus stearothermophilus (Bacillus stearothermophilus), Bacillus subtilis (Bacillus subtilis), Bacillus thermocatenulatus (Bacillus thermocatenulatus), Bordetella bronchiseptica (Bordetella bronchus), Chlamydia cepacis (Campylobacter), Escherichia coli (Campylobacter), Campylobacter coli (Campylobacter), Escherichia coli (Campylobacter coli), Campylobacter coli (Campylobacter coli), Campylobacter coli (Campylobacter coli), Campylobacter coli (Campylobacter coli), salmonella erythraea (Erysipelothrix rhusiopathiae), Listeria monocytogenes (Listeria monocytogenes), Escherichia canis (Ehrlichia canis), Escherichia coli (Escherichia coli), Haemophilus influenzae (Haemophilus influenzae), Haemophilus somnus, Helicobacter suis (Helicobacter suis), Lawsonia intracellularis (Lawsonia intracellularis), Legionella pneumophila (Legiomonas pneumophila), Morobacterium morchelli (Moraxella sp.), Mycobacterium bovis (Mycobacterium triborum), Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), Mycoplasma subspecies (Mycoplasma hyopneumoniae), Mycoplasma hyopneumoniae (Mycoplasma), Mycoplasma hyopneumoniae (Porphyromonas), Mycoplasma urens (Porphyromonas), Mycoplasma urena (Porphyromonas), Mycoplasma urena, Mycoplasma uremia, Mycosphaea, Mycosphaedomonas, Mycoplasma ure, Propionibacterium acnes (Propionibacterium acnes), Proteus vulgaris (Proteus vulgaris), Pseudomonas wisoniensis (Pseudomonas wisconsiensis), Pseudomonas aeruginosa, Pseudomonas fluorescens C9(Pseudomonas fluorescens C9), Pseudomonas fluorescens SIKW1(Pseudomonas fluorescens SIKW1), Pseudomonas rubus practical (Pseudomonas fragi), Pseudomonas flava (Pseudomonas fragilis), Pseudomonas aeruginosa (Pseudomonas lutea), Pseudomonas oleovora (Pseudomonas oleovora), Pseudomonas pseudomonads B11-1(Pseudomonas sp B11-1), Alcaligenes eutrophus (Alcaligenes eutrophus), Pseudomonas nonmotile (Pseudomonas momordialis), Salmonella typhimurium (Salmonella typhimurium), Salmonella typhimurium (Salmonella typhimurium), Salmonella typhi (Salmonella typhi), Salmonella typhi, and Salmonella typhi, Salmonella, Serratia marcescens (Serratia marcescens), Spirulina platensis (Spirulina platensis), Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus suis (Staphylococcus hyicus), Streptomyces albus (Streptomyces albus), Streptomyces cinnamomi (Streptomyces cinnnanenus), Streptococcus suis (Streptococcus suis), Streptomyces exfoliates (Streptomyces exfoliates), Streptomyces scabies (Streptomyces scabies), Sulfolobus acidocaldarius (Sulfobacus), Synechocystis sp), Vibrio cholerae (Vibrio cholerae), Spirospira Borrelia (Borrelia burgdorferi), Spirothrix denticola (Pennelida), Spirothrix parvulus (Spirulina parvulus), Spirothrix lepta (Spirulina platensis), Treponensis (Treponema), Treponema pallidum (Treponema), Leponema pallidum (Leponema pallidum), Leponema pallida), Leponema pallidum (Leponema pallida), Leponema pallida (Leponema pallida), and the strain (Leponema pallida), and the like, Leptospira hazorum (Leptospira hardjo), Leptospira borgripponica (Leptospira borgpetersenii hardjo-bovis), Leptospira paludi Leptospira gordonii hardjo-prajitno, Leptospira interrogans (Leptospira interrogans), Leptospira icterohaemorrhagiae (Leptospira icherohaemomorrhagiae), Leptospira pomonella (Leptospira pomona), and Leptospira bratislava (Leptospira bratislava).

The preparation method of the vaccine composition comprises the following steps: mixing the antigen solution containing effective immunization amount with the adjuvant composition solution, and stirring uniformly to obtain the vaccine composition.

The term "vaccine" refers to a composition comprising an antigen as defined above. Administration of a vaccine to a subject can generate an immune response that is substantially directed against one or more specific diseases. The amount of vaccine that is therapeutically effective may vary depending on the particular antigen used and the condition of the subject, and may be determined by one skilled in the art.

One of the requirements for commercial vaccine adjuvant formulations is to establish the stability of the adjuvant solution for long term storage. Adjuvant compositions are provided herein that are easy to manufacture and can remain stable for at least 18 months, and vaccine compositions containing the adjuvant compositions can also remain stable for at least 18 months. In one embodiment, the vaccine composition may remain stable for about 18 months. In another embodiment, the vaccine composition is stable for about 18 to about 24 months. In another embodiment, the vaccine composition is stable for about 24 months. Accelerated testing procedures also indicate that the vaccine compositions described herein are stable.

Vaccine compositions formulated from the adjuvant compositions of the present invention can be safely and effectively administered to a variety of subjects. It is expected in the art that combinations of adjuvants will exhibit greater reactogenicity than the individual components. However, the adjuvant compositions described herein exhibit reduced reactogenicity when compared to either or both of the components of the composition, while still maintaining the adjuvant effect. It has also been surprisingly found that the safety of the adjuvant compositions described herein is also improved when compared to other adjuvant compositions.

Vaccine compositions formulated with the adjuvant compositions of the present invention can be used to generate a desired immune response in a subject that is effective in a variety of species. Any animal to which administration of the vaccine composition is desired is a suitable subject. It includes mammals and non-mammals, including primates, domestic animals, companion animals, laboratory test animals, captive wild animals, birds (including eggs), reptiles, and fish. Specifically, subjects include, but are not limited to: monkey, human, pig, cow, sheep, goat, horse, mouse, rat, dutch pig, hamster, rabbit, cat, dog, chicken, turkey, duck, other avian species, frog, and lizard.

In a fourth aspect, the invention provides the use of a vaccine composition according to the third aspect of the invention in the manufacture of a prophylactic and/or therapeutic medicament.

The invention has the beneficial effects that: the acrylic polymer, namely polylysine nanoparticles, contained in the adjuvant composition are prepared by simple electrostatic interaction, and do not have cross-linking between proteins harmful to organisms, so that the adjuvant composition has high safety and effectiveness, is suitable for being used as an adjuvant, and overcomes the problems existing in the use of polyacrylic acid as the adjuvant. In addition, vaccine compositions formed from the adjuvant compositions of the present invention and antigens have good stability for long-term storage and can be administered safely and effectively to a wide variety of subjects.

Detailed Description

In order that the present invention may be more readily understood, the invention will be further described in detail with reference to the following examples, which are intended to be illustrative only and are not to be construed as limiting the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.

Examples

Example 1: preparation of adjuvant composition containing polyacrylic acid-polylysine nanoparticles

Polyacrylic acid (commercially available carbomer 934P) and polylysine were dissolved in 0.85% NaCl solution and adjusted to pH 7.2 to obtain polyacrylic acid solution and polylysine solution. And mixing the polyacrylic acid solution and the polylysine solution under the stirring condition to obtain the adjuvant composition containing the polyacrylic acid-polylysine nano-particles with negative charges on the surface.

The particle size of the formed nanoparticles was measured using DLS (dynamic light scattering), showing that the particle size of the formed nanoparticles was 200 and 1500 nm. The formulation of the adjuvant composition is shown in table 1.

Table 1: formulation of adjuvant compositions

Group of	Carbomer 934P	Polylysine	Sodium chloride	Immunopotentiating agent	Water (W)
						1 (adjuvant 1)	0.5g	0.5g	0.85g	—	Adding to 100ml
2 (adjuvant 2)	0.5g	0.25g	0.85g	—	Adding to 100ml
						3 (adjuvant 3)	0.5g	0.05g	0.85g	—	Adding to 100ml
4 (adjuvant 4)	0.5g	0.25g	0.85g	QuilA 0.01g	Adding to 100ml
						5 (adjuvant 5)	0.3g	0.3g	0.85g	—	Adding to 100ml
6 (adjuvant 6)	0.3g	0.15g	0.85g	—	Adding to 100ml
						Comparative adjuvant	0.5g	—	0.85g	—	Adding to 100ml

Example 2: the adjuvant composition of the invention has the promotion effect on the generation of neutralizing antibodies of porcine circovirus

2.1 preparation of vaccine compositions

To 50g of the adjuvant composition prepared in example 1, 20 μ g/head of PCV2 ORF2 protein was added, and the volume was adjusted with phosphate buffer at pH 7.4 to obtain a vaccine composition. The stability of the obtained vaccine composition was observed at 4 ℃ and the results are shown in Table 2.

Table 2: formulation and stability of vaccine compositions

As can be seen from Table 2, the vaccine composition prepared by using the adjuvant composition of the present invention has high stability, and can be kept stable for at least 18 months at 4 ℃.

2.2 neutralizing antibody titer assay

30 PCV2 negative healthy piglets, which were 30 days old or so, were randomly divided into 6 groups of 5 piglets each. Injecting the 5 vaccine compositions into piglets of groups 1-5 with 1 mL/head respectively; group 6 was injected with an equal amount of physiological saline as a blank control group. Neutralizing antibody titer of piglets was measured 4 weeks after immunization, and the measurement results are shown in table 3.

The detection of neutralizing antibodies was as follows: serum with different dilutions was mixed with PCV2 virus, subjected to 37 ℃ water bath for 1h, taken out and added with PK-15 cells which had been confluent with a monolayer of cells, 100. mu.L per well, 4 wells were inoculated per serum dilution, and a positive control well, a normal cell negative control well and a negative serum control well were set to which only PCV2 had been inoculated. PCV2 Virus final concentration of 1000TCID₅₀and/mL. After 24h, the cells were treated with 300mmol/L D-glucosamine and then cultured for 48h for indirect immunofluorescence assay. The neutralizing antibody titer was given as the reciprocal of the highest dilution of the fluorescent serum reduced by 70% or more.

Table 3: neutralizing antibody titer of piglets in each test group

Group of

A

B

C

D

Comparison vaccine

Control

Neutralizing antibody titer

1:252

1:263

1:286

1:216

1:75

Negative of

As can be seen from Table 3, the adjuvant composition of the present invention has an antibody-promoting effect superior to that of the comparative adjuvant using carbomer 934P alone, and the neutralizing antibody titer was improved by 3-4 times.

3.3 piglet challenge protection test

30 PCV2 negative healthy piglets, which were about 35 days old, were randomly divided into 6 groups of 5 piglets each. Injecting the vaccine composition prepared by 2.1 into the piglets of the 1 st to 5 th groups respectively, wherein each group is 1 mL; group 6 was injected with an equal amount of physiological saline as a control group. After 35d of immunization, the piglets of each test group and the control group use PCV2 SH strain (containing 106.0 TCID)₅₀(ml) nasal drip 1 ml/head, intramuscular injection 2 ml/head, 4 spots on the axilla and hip of each pig 4 th and 7 th days after challenge, respectively, inoculating keyhole limpet hemocyanin (KLH/ICFA, 0.5mg/ml) emulsified with Freund's incomplete adjuvant to all pigs, and inoculating 1ml (4 m) per spotl/head), simultaneously inoculating thioglycollic acid culture medium into the abdominal cavity, 10 ml/head; the thioglycollic acid culture medium was re-inoculated into the peritoneal cavity at 10 ml/head on days 11 and 19 after challenge. Continuously observing for 25 days after challenge, weighing for killing on 25 th day after challenge, and performing autopsy. The body temperature, the relative daily gain and the virus antigen detection result are used for judgment, and the change condition of the body temperature is shown in a table 4.

Table 4: days for which the body temperature of each group of piglets exceeds 40.5 ℃ after PCV2 challenge

As can be seen from table 4, the piglets in the vaccine composition a, the vaccine composition B, the vaccine composition C and the vaccine composition D immunized groups hardly showed the body temperature rise phenomenon, while the comparative vaccine group had 2 pigs showing the body temperature rise phenomenon once, and the piglets quickly recovered to be normal after the body temperature rise for one day without other clinical symptoms; after the control group piglets challenge, all the piglets rise to the temperature of more than 40.5 ℃ for 3-5 days, and have anorexia, mental depression, rough and disordered hair, emaciation and slow growth speed.

The onset and protection of the various groups of piglets after challenge are shown in tables 5 and 6. As can be seen from Table 5, no obvious clinical symptoms appear after the vaccine composition A, B, C, D is used for immunizing piglets after challenge, no specific pathological change is observed, the antigen PCR detection is negative, and the protection effect reaches 5/5. After the control vaccine is used for immunizing piglets, 1 pig suffers from the disease, and the protection effect reaches 4/5. The piglets in the control group are all attacked, the clinical symptoms and the pathological changes are obvious, and the pathogen PCR detection is positive. As can be seen from table 6, by the end of the challenge observation, there was no significant difference in the average daily gain of the piglets immunized with vaccine composition A, B, C, D and the control vaccine, and the average daily gain of the piglets in each vaccine group was significantly higher than that in the control group.

Table 5: results of disease determination of piglets in each group after PCV (viral infection attack)

After PCV attacks toxic materials, the PCV accords with any 2 items in the following 3 items, and then the PCV can be judged to be the onset of disease.

A. Clinical symptoms: the piglet body temperature is increased (more than or equal to 40 ℃) and lasts for at least 3 days, and obvious anorexia, mental depression, rough and disorderly fur, emaciation and slow growth speed appear.

B. Pathological changes are as follows: inguinal and tracheal lymph node edema, mild edema of the lung, yellow or somewhat necrotic kidneys. The histological lesion is the lymph node with obvious lymphocyte invasion or multinucleated giant cells.

C. And (3) virus detection: lymph node tissue was detected by PCR and PCV2 was detected.

TABLE 6 post-challenge protection of immunized piglets by PCV

Example 3: protection test of adjuvant composition of the invention against Mycoplasma hyopneumoniae

3.1 preparation of vaccine compositions

To 50g of the adjuvant composition prepared in example 1, 2X 10 of the adjuvant composition before inactivation of swine pneumonia was added⁸50g of mycoplasma antigen per head, obtaining the vaccine composition. The stability of the obtained vaccine composition was observed at 4 ℃ and the results are shown in Table 7.

Table 7: formulation and stability of vaccine compositions

3.2 immunization of piglets with vaccine for testing

30 piglets of 14-21 days old are selected and randomly divided into 6 groups and 5 piglets per group. Respectively injecting the vaccine compositions I, II, III and IV prepared in the step 3.1 and the comparison vaccine into piglets of groups 1-5, and respectively injecting the corresponding inactivated vaccine 1ml into neck muscles of each piglet; group 6 was injected with an equal amount of physiological saline as a control group.

3.3 post-immunization challenge of piglets

And (3) toxin attacking of mycoplasma hyopneumoniae: after 70 days of primary immunization, a virus challenge test is carried out by adopting mycoplasma hyopneumoniae, piglets of each immunization group and a control group are injected with 5 ml/head (100MID) of CVCC354 strain (purchased from China institute of veterinary drugs) through air pipes, observed for 30 days after virus challenge, and killed by dissecting. According to the mycoplasma hyopneumoniae pulmonary lesion index scoring standard, the pulmonary lesions of the test piglets are scored, and the pulmonary lesion index difference analysis is performed on each immune group and the control group, and the results are shown in table 8.

Table 8: lung injury score condition of piglets of each test group after mycoplasma hyopneumoniae challenge

Group of	Number of piglets	Mean lung lesion index
			Vaccine composition I	5	3.61±0.52Bd
Vaccine composition II	5	3.10±0.33Bd
			Vaccine composition III	5	3.12±0.31Bd
Vaccine composition IV	5	3.06±0.51Bd
			Comparison vaccine	5	6.30±0.53Bb
Control group	5	15.90±0.63Aa

Note that in statistical analysis of differences, when compared among groups, the ones with the same letter mean no significant difference, the ones with different capital letters mean very significant difference (P < 0.01), and the ones with different lower case letters mean significant difference (P < 0.05).

As can be seen from Table 8, each immunization group had a significant effect in preventing pulmonary disease changes caused by Mycoplasma hyopneumoniae, indicating that the adjuvant compositions of the invention have a significant synergistic effect in combination. The technical personnel in the field know that the mycoplasma hyopneumoniae immunity needs the combined action of cellular immunity and humoral immunity to achieve the ideal effect, and the test result shows that the adjuvant composition can promote the dual functions of the humoral immunity and the cellular immunity.

Example 4: preparation of pseudorabies vaccine composition and detection of neutralizing antibody titer

4.1 formulation of vaccine compositions

To a phosphate buffer solution at pH 7.4 was added a mixture of porcine pseudorabies gB and gD antigens expressed with baculovirus (equimolar ratio of gB and gD) 200 micrograms per head as antigen phase. 50g of the antigen phase was mixed with 50g of the adjuvant composition prepared in example 1 to obtain a vaccine composition.

Table 9: formulation and stability of vaccine compositions

Group of	Adjuvant	Stability at 4 deg.C
			Vaccine composition a	Adjuvant 1	Is stable for 18 months
Vaccine composition b	Adjuvant 3	Is stable for 18 months
			Vaccine composition c	Adjuvant 4	Is stable for 18 months
Vaccine composition d	Adjuvant 6	Is stable for 18 months
			Comparison vaccine	Comparative adjuvant	After 2 months, the layers appeared and the upper layer was clear

2.2 neutralizing antibody titer assay

30 PRV antibody-negative piglets at 21 days of age were randomly divided into 6 groups, 5 groups/group. Respectively injecting the vaccine compositions a, b, c and d prepared by 4.1 and the comparative vaccine into piglets of groups 1-5 with 2 ml/head; group 6 was injected with an equal amount of physiological saline as a control group. After immunization, neutralizing antibody titers were determined weekly in each group of piglets with reference to the GB/T18641-2002 serum neutralization assay, and the results are shown in Table 10.

Table 10: antibody titer conditions of immunized piglets at different times

The above test results show that the adjuvant composition of the present invention not only allows the antibody to be produced more prematurely, but also allows the antibody to be maintained for a longer period of time.

It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Claims (7)

1. An adjuvant composition comprising poly (acrylic acid) -polylysine nanoparticles;

the polyacrylic acid-polylysine nano-particles are prepared by ionic bonding of polyacrylic acid and polylysine, and the relative molecular mass of the polylysine is 1000Da-15000 Da;

the particle size of the polyacrylic acid-polylysine nano-particles is 200-1500 nm;

the mass ratio of the polyacrylic acid to the polylysine is (1-10) to 1.

2. The adjuvant composition according to claim 1, wherein the polyacrylic acid-polylysine nanoparticles are included in an amount of 0.001 to 5 parts by weight based on 100 parts by weight of the adjuvant composition.

3. The adjuvant composition according to claim 2, wherein the polyacrylic acid-polylysine nanoparticles are included in an amount of 0.01 to 3 parts by weight based on 100 parts by weight of the adjuvant composition.

4. The adjuvant composition of claim 1, wherein the polyacrylic acid is a commercially available carbomer.

5. A process for preparing an adjuvant composition according to any one of claims 1 to 4, comprising: mixing a polyacrylic acid solution and a polylysine solution under stirring to obtain an adjuvant composition; wherein the pH values of the polyacrylic acid solution and the polylysine solution are both 7.2; the mass ratio of the polyacrylic acid solution to the polylysine solution is (1-10) to 1.

6. A vaccine composition comprising the adjuvant composition of any one of claims 1-4 or the adjuvant composition prepared by the method of claim 5 and an immunologically effective amount of an antigen; wherein the antigen is selected from the group consisting of a mixture of porcine circovirus, mycoplasma hyopneumoniae, porcine pseudorabies gB and gD antigens.

7. Use of a vaccine composition according to claim 6 in the manufacture of a prophylactic and/or therapeutic medicament.