CN109701010B - Vaccine composite adjuvant system and application thereof in antigen - Google Patents
Vaccine composite adjuvant system and application thereof in antigen Download PDFInfo
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Abstract
The invention provides a vaccine composite adjuvant system and application thereof in antigen. The composition consists of an aluminum adjuvant, a double-stranded polynucleotide-epsilon-polylysine compound and an aqueous solvent, wherein the clinical use concentration of the aluminum adjuvant is 0.1-10.0mg/ml, and the clinical use concentration of the double-stranded polynucleotide-epsilon-polylysine compound is 10000 mu g/ml in terms of the double-stranded polynucleotide. The formed composite adjuvant system has good RNase hydrolysis resistance, stability, safety and immunostimulation activity, and can be combined with hepatitis B vaccines, inactivated rabies vaccines and other vaccines in different forms to obviously improve the immunogenicity of the vaccines.
Description
Technical Field
The invention relates to the technical field of immunology, in particular to a compound adjuvant system and application thereof in antigen, which is used for immunotherapy and prevention.
Background
The vaccine Adjuvant referred to herein is an Adjuvant (Adjuvant), also known as an immunomodulator or immunopotentiator (immunopotentiator), derived from latin, "Adjuvant", in the sense of either Adjuvant or booster. An adjuvant is an additive to a vaccine, which, when injected into the body prior to or mixed with an antigen, can enhance the immune response of the body to the antigen or change the type of immune response, and is a non-specific immunopotentiator, which is not antigenic by itself. The ideal adjuvant can not only enhance the immune response, but also can enable the body to obtain the optimal protective immunity.
The application of the vaccine adjuvant aims to consider that the adjuvant mainly comprises a plurality of action modes such as immunoregulation, cytotoxic T lymphocyte induction, antigen presentation, antigen targeting and storage and the like. Through the above several modes, the use purpose that reaches has:
(1) enhancing the immunogenicity, immune response speed and tolerance of purified or recombinant antigens;
(2) reducing the amount of antigen or the amount of vaccination required to achieve immune protection;
(3) increasing the immune potency of the vaccine in infants, elderly, or people with compromised immune systems;
(4) as an antigen delivery system for uptake of antigen through the mucosa, absorption of the vaccine by the gastrointestinal mucosa can be promoted. The concept of adjuvant is derived from ulcers that form at the site of vaccination and promote the production of high levels of specific antibodies, even ulcers produced by substances unrelated to vaccination are able to induce the production of high specific antibodies;
(5) adjuvants can increase infiltration into cells, prevent antigen degradation, transport antigen to specific antigen presenting cells, enhance antigen presentation or induce cytokine release.
The types of vaccine adjuvants are currently, no unified standard exists for the classification of adjuvants internationally, and can be classified into aluminum salt adjuvants, protein adjuvants, nucleic acid adjuvants, lipid-containing adjuvants, mixed adjuvants and the like according to the difference of chemical components.
1. Aluminium salt adjuvant
Aluminum salts have been used clinically for over 80 years and are the first classic adjuvants approved by the FDA in the united states for use in humans. Many vaccines contain aluminium salts as components, such as diphtheria, tetanus and haemophilus influenzae vaccines. According to different preparation processes, the vaccine taking the aluminum salt as the adjuvant can be divided into two types, namely an aluminum adsorption vaccine and an aluminum precipitation vaccine. The aluminum adsorption vaccine is prepared by adding antigen into aluminum hydroxide or aluminum phosphate solution; the aluminum precipitation vaccine is prepared by adding aluminum agent suspension into antigen solution. Aluminum hydroxide or aluminum phosphate is an aluminum adjuvant that is often used. Researches show that the aluminum adjuvant vaccine can reduce the using amount of antigen and enhance the strength and the durability of the immune response of the organism. The action mechanism of aluminum salt is not completely clear up to now, and it is widely believed that antigen adsorbs aluminum salt particles to form a gel state, and after the gel state is injected into an animal body, an antigen storage library is formed, and the insoluble particles can adsorb antigen substances, so that the antigen surface area is increased. In addition, the adjuvant can form granuloma rich in macrophage at an injection site, delay the absorption of antigen, further prolong the stimulation time of the antigen, retain the antigen existing for several days under normal conditions for several weeks, and enhance the antigen intake capability of the injection site. The research shows that aluminum hydroxide used as an adjuvant can also activate Th2 cells to secrete IL-4, induce the expression of CD83, CD86 and MHC-II molecules and further generate Th2 type humoral immune response. Aluminum salts have many advantages as vaccine adjuvants, but also have disadvantages. It is effective in inducing humoral immune responses, but does not contribute to cellular immunity and does not induce cellular immune responses.
2. Protein adjuvant
The adjuvant mostly belongs to small molecular polypeptide or glycoprotein, is a bioactive substance synthesized and secreted by immune cells and certain non-immune cells through stimulation, is generally cytokine, plays an important role in the differentiation of Th cells, can also enhance the functions of NK cells and T lymphocytes, and plays a role in widely up-regulating the immune response of organisms. The protein adjuvant is used together with antigen to enhance the immune efficacy of the vaccine, and can also be assembled into plasmid and then mixed with antigen for injection. IL-12 is produced by monocyte and B cell, has various biological activities, can obviously reduce the invasion number of bacteria, improves the expression level of IgG2a and IgA in mucous membrane and immune system, and is a cytokine adjuvant with wider application prospect. It induces Th1 type immune response, and the treatment of tumors and AIDS is in clinical trial.
3. Nucleic acid adjuvant
In the process of research on vaccines, some nucleic acids are also found to have adjuvant properties, most typically CPGDNA, an oligomer of which sequence takes unmethylated cytosine deoxynucleotide and guanine deoxynucleotide as units, and an agonist of TLR9 are hot spots of current adjuvant research. It plays an important role in enhancing specific immune response, inducing non-specific immune response of organisms, regulating immune response types and the like. The characteristic sequence of the CPG-ODN can activate various immune effector cells, such as T cells, B cells, NK and other immune active cells, so that the CPG-ODN is applied to more experimental researches. Bacterial DNA is the source of CPG-ODN and its role includes both humoral and cellular immunity enhancement. The possibility of its application in the fields of tumors and infectious diseases will be high. There is no report that CPG-ODN has serious side effects on human, but CPG-ODN has been found to induce autoimmune diseases in animal models. A double-stranded polynucleotide: the double-stranded polynucleotide or its derivative has various pharmacological activities, and is obtained by natural extraction or artificial synthesis. Artificially synthesized double-stranded polynucleotide such as polyinosinic acid (polyI: C) can induce in vivo production of interferon, protect cells from virus infection, and has therapeutic effects of preventing and treating viral diseases; the naturally extracted double-stranded polynucleotide such as virus double-stranded RNA extracted from reoviridae has antiviral and antitumor activities. Polyinosinic acid (PolyIC, PIC) is a double-chain product formed by pairing artificially synthesized poly-hypoxanthine nucleotide (PolyI) and poly-cytosine nucleotide (PolyC), and is a high-efficiency interferon inducer. However, PIC is susceptible to hydrolysis by nucleases in human and primate serum, limiting clinical use. The polyinosinic cell is mainly used for preventing and treating viral infection, such as influenza, viral hepatitis and herpetic conjunctivitis. Also used for the adjuvant treatment of tumor. A few patients have transient low fever, and are occasionally lack of strength, dry mouth, nausea and the like.
4. Lipid-containing adjuvant
Lipid-containing adjuvants include both Lipopolysaccharide (LPS) and liposomes. LPS is a gram-negative outer membrane lipopolysaccharide, and lipid a is the major component of LPS acting as an adjuvant. Researchers use LPS as adjuvant of pertussis vaccine to immunize mice together, and experimental results show that LPS not only improves the immune efficiency of the vaccine, but also reduces the occurrence of type I hypersensitivity. Liposomes are similar to biological membranes, and typically comprise a bilayer of phospholipids and cholesterol encapsulating an antigen to form a microspheroidal formulation that is capable of both transporting the antigen and serving as an adjuvant for a vaccine. The liposome adjuvant has no toxicity and can reduce the toxicity of the antigen, and can be degraded in a host body by self. Research shows that the liposome can enhance the humoral immune response and the cellular immune response of the organism, and in addition, the structure of the liposome is beneficial to presenting antigen to antigen processing cells. Studies have also shown that liposomes used in combination with freund's reagent or aluminum hydroxide have a much more than half the effort. But it also has disadvantages such as poor stability, easy oxidation, high production cost, etc. Thus, current research pauses for liposome applications remain in medical research.
5. Mixed adjuvant
MF59 is an oil-in-water emulsion, which is a uniform droplet emulsion formed by mixing Tween 80, sorbitol trioleate and squalene and then microfluidizing under high pressure. The mixed adjuvant can induce local immunostimulation environment at injection site, increase contents of chemotactic factor and cytokine, and gather MHC + cells in muscle. Furthermore, MF59 can also enhance the ability of dendritic cells to take up antigen. MF59 was approved as an adjuvant for influenza vaccines in the 90 s of the 20 th century because it can enhance the immunogenicity of influenza in immunocompromised people. A large amount of data shows that MF59 is safer for use in influenza vaccines.
6. Aggregate structure adjuvant
The investigators compared the immunopotentiation of 3 novel molecular aggregate formulation adjuvants, tomato glycoside (RAM1), glycosyl amide lipid (RAM2) and generation 5 dendrimer (RAM3), and evaluated the ability of these adjuvants to enhance Th1 or Th2 responses when used with soluble protein antigens. The study used Ovalbumin (OVA) as antigen, the above 3 novel aggregates as adjuvants, and tuberculin, Al (OH)3 and Freund's incomplete adjuvant as controls. Results of 3 adjuvants, RAM1 was superior to the other adjuvants, and its induced cytokine was predominantly Th1 type and induced Th2 type response in the late phase of vaccination.
Early adjuvant research was largely empirical, and adjuvant development was relatively delayed from vaccine development due to lack of support for technical and immunological mechanism studies. Since aluminum adjuvant was first applied to humans for nearly 70 years, it was the only human adjuvant (O' Hagan DT et al Expert Rev Vaccines 2013 Jan; 12(1): 13-30). Although the aluminum adjuvant has low cost and good safety, the adjuvant mainly activates Th2 immune response, has no strengthening effect on cellular immunity, and has no obvious effect of strengthening immune response in many human vaccines (particularly recombinant protein vaccines and polypeptide vaccines). Thus, with the emergence of more and more candidate vaccines and the emergence of vaccine economics, the need for new adjuvants that are clinically useful is rapidly increasing.
Molecules with immunostimulatory activity are one of the development directions for novel adjuvants. In particular, TLR stimulators such as Pam3Cys, poly IC, MPL, Imiquimod (Imiquimod), CpG ODN, and the like.
The invention patent of Suwenquan 'invention-double-stranded polynucleotide-epsilon-polylysine compound with immunoregulation function and the preparation and use method thereof' 201610056797.6 describes that epsilon-polylysine is used as a poly IC stabilizer, and the stability and immunoregulation activity in a poly IC organism are better improved.
Because the mechanism of the activated immune reaction of the currently known aluminum adjuvant and the double-chain polynucleotide-epsilon-polylysine compound is obviously different, the types of the activated immune reaction are different, and the aluminum adjuvant and the double-chain polynucleotide-epsilon-polylysine are combined for use, the immune reaction can be expected to be more comprehensively activated through synergistic effect. The aluminum adjuvant with good safety is mixed with the double-chain polynucleotide-epsilon-polylysine compound with strong immunostimulation activity for use, the dosage of the antigen can also be expected to be reduced, the strength of immunoreaction is controlled within a certain range, and both safety and effectiveness are considered. Therefore, the inventor mixes the aluminum adjuvant and the double-chain polynucleotide-epsilon-polylysine compound according to a certain proportion and then jointly immunizes with different types of vaccines.
Disclosure of Invention
The invention aims to provide a novel vaccine composite adjuvant system with strong RNase hydrolysis resistance, good stability, good immunostimulation activity and human application prospect, application of the vaccine composite adjuvant system in antigens and research of application of the vaccine composite adjuvant system and an immune composition in the fields of immunotherapy and prevention.
The inventor finds that the isoelectric point of epsilon-polylysine is similar to that of an aluminum adjuvant, the two components in an adjuvant system have good compatibility, the existence of the aluminum adjuvant improves the anti-RNase hydrolysis capability of a double-chain polynucleotide-epsilon-polylysine compound, the double-chain polynucleotide-epsilon-polylysine compound and the aluminum adjuvant are combined by adopting a proper compatibility mode, and the formed composite adjuvant system has good anti-RNase hydrolysis capability, stability, safety and immunostimulation activity, and can obviously improve the immunogenicity of vaccines when being combined with hepatitis B vaccines, inactivated rabies vaccines and other vaccines in different forms. The present invention is based on the above findings, which have now been completed, and data is provided in the examples herein.
The invention provides a composite adjuvant system consisting of a double-stranded polynucleotide-epsilon-polylysine compound and an aluminum adjuvant, which comprises the aluminum adjuvant and the double-stranded polynucleotide-epsilon-polylysine compound, wherein the clinical use concentration of the aluminum adjuvant is 0.1mg/ml-10.0mg/ml, and the clinical use concentration of the double-stranded polynucleotide-epsilon-polylysine compound is 100 mu g/ml-10000 mu g/ml.
Specifically, in the composite adjuvant system provided by the first aspect of the invention, the double-stranded polynucleotide-epsilon-polylysine compound is characterized by containing a compound formed by mutually compounding double-stranded polynucleotide, cation epsilon-polylysine and metal cation,
the double-stranded polynucleotide refers to anionic double-stranded polynucleotide or derivatives thereof, and the source can be selected from but not limited to natural extraction or artificial synthesis; the naturally extracted double-stranded polynucleotide is derived from a nucleic acid extract of reoviridae virus, and comprises chicken infectious bursal disease virus RNA, rotavirus RNA, bluetongue virus RNA and African horse sickness virus RNA; the artificially synthesized double-stranded polynucleotide comprises polyinosinic cells; preferably, the double-stranded polynucleotide is polyinosinic acid-polycytidylic acid poly IC;
the cation epsilon-polylysine is a monomer polymer with epsilon-amino homotype of cation; the preferable ratio of the double-stranded polynucleotide to the epsilon-polylysine is such that the ratio of the anionic charge number of the phosphate group of the double-stranded polynucleotide to the cationic charge number of the amino group of the epsilon-polylysine is 1: 1;
the metal cation can be selected from but not limited to Ca2+、Zn2+、Mg2+、Mn2+One or more than one of them, and its metal ion is in the form of soluble metal salt, the clinical use concentration is 0.01 mmol-100 mmol/L; preferably, the metal cation is Ca2+。
The aqueous solvent used comprises a human-tolerable pH buffer which may be selected from, but not limited to, citrate or phosphate, and an ionic strength modifier comprising sodium chloride and/or potassium chloride, the pH of the aqueous phase components being in the range of 6.0-9.0,
preferably, the aqueous phase component is PBS buffer.
In another aspect, the present invention provides the use of an immunogenic composition, i.e. a vaccine adjuvant complex system, in an antigen, the components of which consist of the adjuvant composition according to the first aspect of the present invention and a different form of antigen. Wherein the antigen may be selected from, but not limited to, antigens derived from bacteria, viruses, parasites, fungi, tumors, antigens prepared by genetic engineering and the like, human self-antigens and/or allergens, and the like, and the number of antigens in the immunological composition may be one or more.
Preferably, the antigen of the immunological composition is derived from an antigen of a pathogenic microorganism, which may be selected from, but not limited to, antigens of the poxviridae, herpesviridae, adenoviridae, polyomaviridae, papilloma viridae, parvoviridae, circoviridae, hepadnaviridae, retroviridae, reoviridae, paramyxoviridae, rhabdoviridae, filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae, picornaviridae, caliciviridae, astroviridae, pentaviridae, flaviviridae, coronaviridae, togaviridae, actinomycetes, chlamydia, firmicutes, proteobacteria, spirochetes, ascomycetes, basidiomycetes, serotrichuris, acrophylum, ciliate, phylum platyphylla, phylum nematoda, and/or arthropoda.
More preferably, the antigen is derived from human papilloma virus, rabies virus, encephalitis B virus, hepatitis B virus, influenza virus, poliovirus, hepatitis A virus, varicella virus, and renal syndrome hemorrhagic fever virus.
Particularly preferably, the antigen is an inactivated virus, such as but not limited to: rabies virus, influenza virus, poliovirus, hepatitis A virus, encephalitis B virus, varicella virus and hemorrhagic fever virus with renal syndrome.
Particularly preferably, the antigen of the immunological composition is a recombinant protein antigen and/or a polypeptide or epitope antigen of viral origin, such as, but not limited to: human papilloma virus or hepatitis b virus-like particle VLP, chimeric virus-like particle VLP, human papilloma virus major coat protein L1 and/or minor coat protein L2 derived recombinant protein antigen or polypeptide or epitope antigen.
The invention also relates to the use of the composite adjuvant system and the immune composition, which can be applied to the preparation of preparations with various uses such as immunotherapy, prevention and the like, including but not limited to: (1) preventing pathogenic microorganism infection and various diseases related to infection, such as infection and infection related diseases of influenza virus, rabies virus, HIV, HPV and the like; (2) prevention and treatment of tumors, and (3) treatment of immune-related diseases such as allergic reactions.
According to the present invention, the compound adjuvant system and the immunological composition can be inoculated in a form acceptable to human body, and the inoculation position includes but is not limited to parenteral injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, respiratory inhalation, rectal administration, vaginal administration, nasal administration, oral administration, ocular administration, local administration, transdermal or intradermal administration and the like.
Description and explanation of related terms
According to the present invention, the term "antigen" refers to various substances recognized by the immune system under appropriate circumstances, and may be derived from pathogens, the human body itself, tumors, substances prepared by genetic engineering and the like, and the like.
Detailed Description
The invention will now be further illustrated by the following non-limiting examples, and it will be apparent to those skilled in the art that many modifications can be made without departing from the spirit of the invention, such modifications also falling within the scope of the invention. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, as the embodiments are necessarily diverse. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be defined in the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. The following experimental procedures are all the ones described in the conventional methods or product specifications unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and/or materials in the publications.
Example 1: stability experiment of composite adjuvant system consisting of double-chain polynucleotide-epsilon-polylysine compound and aluminum adjuvant
The 21-day-old chicks are randomly divided into eight groups, each group comprises 10 chickens, one group is injected with a composite adjuvant system consisting of double-stranded polynucleotide-epsilon-polylysine compounds (each ml contains poly IC 2mg, epsilon-polylysine 1.0mg and calcium chloride 0.4mmol/L), the second group is injected with double-stranded polynucleotide-epsilon-polylysine compounds and aluminum adjuvants (each ml contains poly IC 2mg, epsilon-polylysine 1.0mg, calcium chloride 0.4mmol/L and aluminum hydroxide adjuvant 1.6mg), the third group is injected with a composite adjuvant system consisting of double-stranded polynucleotide-epsilon-polylysine compounds and aluminum adjuvants (each ml contains poly IC 2mg, epsilon-polylysine 1.0mg, calcium chloride 0.4mmol/L and aluminum phosphate adjuvant 1.0mg), and the fourth group is injected with a composite adjuvant system consisting of double-stranded polynucleotide-epsilon-polylysine and aluminum adjuvants (each ml contains poly IC 2mg, epsilon-polylysine 1.0mg, calcium chloride 0mmol/L and aluminum phosphate adjuvant 1.0mg), 1.0mg of epsilon-polylysine, 0.4mmol/L of calcium chloride and 2.0mg of aluminum sulfate adjuvant). Injecting each chicken according to 0.01ml/Kg body weight, injecting once more every three days, collecting blood 12 hours and 36 hours after the last injection, and measuring the content of alpha interferon in blood by an ELISA method; secondly, placing a composite adjuvant system consisting of the double-stranded polynucleotide-epsilon-polylysine compound, the double-stranded polynucleotide-epsilon-polylysine compound and the aluminum adjuvant at 37 ℃ for 2 weeks, and detecting the content of alpha interferon by the same method; and thirdly, adding RNase with the final concentration of 50ug/ml into a composite adjuvant system consisting of the double-stranded polynucleotide-epsilon-polylysine compound, the double-stranded polynucleotide-epsilon-polylysine compound and the aluminum adjuvant, standing at room temperature for 15min, and detecting the content of the alpha interferon for 12 hours by the same method. The detection results are as follows:
test results show that the composite adjuvant system consisting of the double-stranded polynucleotide-epsilon-polylysine compound, the double-stranded polynucleotide-epsilon-polylysine compound and the aluminum adjuvant has good interferon induction effect. After being placed at 37 ℃ for 2 weeks, the stability of the two groups of samples is obviously different, and the stability of the double-stranded polynucleotide-epsilon-polylysine compound is improved by adding the aluminum adjuvant.
Example 2: abnormal toxicity detection of composite adjuvant system consisting of double-chain polynucleotide-epsilon-polylysine composite and aluminum adjuvant
The composite adjuvant system consisting of the double-stranded polynucleotide-epsilon-polylysine composite prepared in the example 1 and the aluminum adjuvant is subjected to abnormal toxicity detection according to a method of Chinese pharmacopoeia, and the detection results are as follows:
test results show that the detection result of the abnormal toxicity of the mouse in the compound adjuvant system consisting of the double-stranded polynucleotide-epsilon-polylysine compound prepared in the example 1 and the aluminum adjuvant is qualified.
Example 3: titer detection of double-chain polynucleotide-epsilon-polylysine compound and aluminum adjuvant combined inactivated rabies vaccine by using compound adjuvant system
The composite adjuvant system consisting of the double-chain polynucleotide-epsilon-polylysine compound prepared in the example 1 and the aluminum phosphate adjuvant (the content of the aluminum phosphate adjuvant is 1.0mg/ml) is fully mixed with the inactivated rabies virus antigen according to the proportion of 1:1 to be used as a sample vaccine group, and the inactivated rabies virus antigen is used as a control vaccine group. Then carrying out titer detection according to a rabies vaccine titer detection method (NIH method), wherein the detection result is as follows:
| group of | Vaccine potency (IU/ml) |
| Sample vaccine group | 6.6 |
| Control vaccine group | 4.0 |
The test result shows that the titer of the sample vaccine group is higher than that of the control vaccine group.
Example 4: neutralization index T value detection of composite adjuvant system combined inactivated Japanese encephalitis vaccine consisting of double-chain polynucleotide-epsilon-polylysine compound and aluminum adjuvant
A composite adjuvant system consisting of the double-stranded polynucleotide-epsilon-polylysine compound prepared in example 1 and aluminum sulfate adjuvant (aluminum sulfate adjuvant content is 2.0mg/ml) was mixed with the inactivated Japanese encephalitis virus antigen in a ratio of 1:1, and the mixture was used as a sample vaccine group, and PBS and the inactivated Japanese encephalitis virus antigen in a ratio of 1:1 were mixed together, and the sample vaccine group was used as a control vaccine group. Then, the neutralization index T value is detected according to a method of Chinese pharmacopoeia, and the detection result is as follows:
| group of | Vaccine neutralization index (T value) |
| Sample vaccine group | 1.62 |
| Control vaccine group | 1.36 |
The test results show that the neutralization index of the sample vaccine group is higher than that of the control vaccine group.
Example 5: titer detection of hepatitis B virus surface antigen (HBsAg) by combining composite adjuvant system consisting of double-stranded polynucleotide-epsilon-polylysine compound and aluminum adjuvant with recombinant expression
A composite adjuvant system composed of the double-stranded polynucleotide-. epsilon. -polylysine complex prepared in example 1 and aluminum hydroxide adjuvant (aluminum hydroxide adjuvant content 1.6mg/ml) was mixed well with the recombinantly expressed hepatitis B virus surface antigen (HBsAg) in a ratio of 1:2 to prepare a sample vaccine group, and PBS containing 1.6mg/ml aluminum hydroxide was mixed well with the recombinantly expressed hepatitis B virus surface antigen (HBsAg) in a ratio of 1:2 to prepare a control vaccine group. Then, the titer is measured according to the method of Chinese pharmacopoeia, and the detection result is as follows:
| group of | Potency assay |
| Sample vaccine group | 2.2 |
| Control vaccine group | 1.4 |
The test results show that the titer of the sample vaccine group is higher than that of the control vaccine group.
Example 6: potency detection of combined adjuvant system formed by double-chain polynucleotide-epsilon-polylysine compound and aluminum adjuvant for inactivating I and II type renal syndrome hemorrhagic fever virus antigens
A composite adjuvant system consisting of the double-stranded polynucleotide-epsilon-polylysine compound prepared in example 1 and an aluminum hydroxide adjuvant (the content of the aluminum hydroxide adjuvant is 1.6mg/ml) is fully mixed with the inactivated hemorrhagic fever virus antigens of the type I and the type II in a ratio of 1:2 to serve as a sample vaccine group, and PBS containing 1.6mg/ml of aluminum hydroxide is fully mixed with the inactivated hemorrhagic fever virus antigens of the type I and the type II in a ratio of 1:2 to serve as a control vaccine group. Then, the rabbit serum titer detection is carried out according to a Plaque Reduction Neutralization Test (PRNT) method, and the detection result is as follows:
the test results show that the titer of the sample vaccine group is higher than that of the control vaccine group.
Claims (5)
1. A vaccine composite adjuvant system, which is characterized in that: the composition comprises an aluminum adjuvant, a double-stranded polynucleotide-epsilon-polylysine compound and an aqueous solvent, wherein the clinical use concentration of the aluminum adjuvant is 0.1-10.0mg/ml, and the clinical use concentration of the double-stranded polynucleotide-epsilon-polylysine compound is 10000 mu g/ml calculated by the double-stranded polynucleotide;
the aluminum adjuvant refers to aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum sulfate adjuvant, ammonium alum adjuvant or potassium alum adjuvant;
the double-stranded polynucleotide-epsilon-polylysine compound is a compound formed by mutually compounding double-stranded polynucleotide, cation epsilon-polylysine and metal cation, wherein the double-stranded polynucleotide is polyinosinic acid-polycytidylic acid poly IC;
the cation epsilon-polylysine is a monomer polymer with epsilon-amino homotype of cation; the ratio of the double-stranded polynucleotide to the epsilon-polylysine is that the ratio of the anionic charge number of the phosphate radical of the double-stranded polynucleotide to the cationic charge number of the amino group of the epsilon-polylysine is 1: 1;
the metal cation is Ca2+、Zn2+、Mg2+、Mn2+And the metal ion is in the form of soluble metal salt, and the clinical use concentration is 0.01-100 mmol/L.
2. The vaccine composite adjuvant system according to claim 1, wherein the aqueous solvent used is a human body-tolerable pH buffer comprising citrate or phosphate and an ionic strength modifier comprising one or both of sodium chloride and potassium chloride, the pH value of the aqueous solvent is in the range of 6.0-9.0, and the aqueous solvent is PBS buffer.
3. Use of the vaccine composite adjuvant system according to claim 1 or 2 for the preparation of an immunological composition characterized in that: the antigen in the immune composition is derived from one or more of bacteria, viruses, parasites, fungi, tumors, antigens prepared by genetic engineering and other methods, human autoantigens and antigens of allergens.
4. Use of the vaccine composite adjuvant system according to claim 3 for the preparation of an immunological composition, characterized in that: the antigen is inactivated virus, and the inactivated virus is one or more selected from inactivated rabies virus, influenza virus, poliovirus, hepatitis A virus, encephalitis B virus, varicella virus and renal syndrome hemorrhagic fever virus.
5. Use of the vaccine composite adjuvant system according to claim 3 for the preparation of an immunological composition, characterized in that: the antigen is one or more of recombinant protein antigen, polypeptide and epitope antigen derived from virus, and is selected from the following group: one or more of human papilloma virus or hepatitis B virus-like particle VLP, chimeric virus-like particle cVLP, human papilloma virus major coat protein L1, minor coat protein L2-derived recombinant protein antigen, polypeptide and epitope antigen.
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| Poly(I:C)/Alum Mixed Adjuvant Priming Enhances HBV Subunit Vaccine-Induced Immunity in Mice When Combined with Recombinant Adenoviral-Based HBV Vaccine Boosting.;Chuai X等;《PLoS ONE》;20130115;第1-9页 * |
| 佐剂铝和PolyIC 在结核亚单位疫苗中的佐剂效应;付丽丽等;《2013年中国药学大会暨第十三届中国药师周论文集》;20131102;第539页 * |
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