A diphtheria-pertussis-tetanus combined adjuvant vaccine

Abstract

The application discloses a composite adjuvant combined vaccine, which comprises an immunogenic composition, a composite adjuvant and / or a pharmaceutically acceptable carrier, the immunogenic composition comprises a pertussis antigen, a diphtheria antigen and a tetanus antigen, and the composite adjuvant is composed of an aluminum adjuvant and a TLR9 receptor agonist. The application also discloses use of the composite adjuvant in preparation of the aforementioned composite adjuvant combined vaccine for preventing pertussis, diphtheria and tetanus in a subject. The composite adjuvant combined vaccine of the application can induce a higher immune response intensity and achieve a better immune effect compared with a vaccine using a single adjuvant.

Description

Technical Field

[0001] This invention relates to the field of biological products, and in particular to a combined adjuvant vaccine containing pertussis, diphtheria and tetanus antigens. Background Technology

[0002] Whooping cough is a severe acute respiratory infectious disease caused by Bordetella pertussis. People of any age can be infected, but infants under five years old are more susceptible. The disease is transmitted through droplets and is highly contagious. Typical clinical symptoms of whooping cough include persistent paroxysmal coughing, accompanied by inspiratory codas or vomiting. It is prone to complications such as pneumonia and encephalitis, which are leading causes of death.

[0003] Diphtheria is an acute upper respiratory tract infection caused by Corynebacterium diphtheriae, a Gram-positive bacterium. Clinically, it manifests as inflammation of the upper respiratory tract, usually in the pharynx, sometimes in the posterior nasal cavity, larynx, and trachea, and can even damage other organs, primarily the myocardium and peripheral nerves. The toxin-producing strains of Corynebacterium diphtheriae produce a highly pathogenic exotoxin that can form local membranes or cause systemic organ damage. Its clinical features include pseudomembrane formation in the pharynx, larynx, and nose, and systemic poisoning symptoms such as fever, fatigue, nausea, vomiting, and headache. In severe cases, complications such as myocarditis and nerve paralysis can occur.

[0004] Tetanus is a serious disease that endangers people's lives and health. It is an acute, specific infection caused by Clostridium tetani invading a wound, multiplying, and producing toxins. Clostridium tetani and its toxins cannot penetrate normal skin and mucous membranes; therefore, tetanus always occurs after injury. All open wounds can potentially lead to tetanus. Tetanus is an acute, specific infection caused by Clostridium tetani invading a wound, multiplying, and secreting toxins. Its main symptoms are persistent muscle contractions and paroxysmal spasms, either systemically or locally. The condition is more severe in impoverished and underdeveloped countries and regions.

[0005] Vaccination is an effective means of preventing the aforementioned diseases. Whole-cell pertussis vaccine has played a significant role in the prevention and control of pertussis, making it a vaccine-preventable infectious disease and one of the infectious diseases to be controlled and eradicated under the World Health Organization's Expanded Programme for Immunization (EPI). Diphtheria toxoid is an effective vaccine against diphtheria, controlling its spread and ensuring children's health and safety. Currently, the WHO and the Chinese government have included diphtheria as one of the six key infectious diseases controlled by EPI. Epidemiological studies have proven that diphtheria toxoid is a safe and effective vaccine for preventing diphtheria outbreaks. Tetanus toxoid is an effective vaccine against tetanus; immunization with tetanus toxoid can induce an immune response in 74-90% of recipients.

[0006] my country's "National Immunization Program Vaccine Immunization Schedule and Instructions (2021 Edition)" stipulates that children should receive one dose of adsorbed acellular DPT combined vaccine at 3, 4, 5, and 18 months of age, and one dose of adsorbed diphtheria-tetanus combined vaccine at 6 years of age. The DPT vaccine currently marketed in my country uses ammonium sulfate precipitation to purify the pertussis antigen, producing a co-purified vaccine containing two main antigens: pertussis toxoid (PT) and filamentous hemagglutinin (FHA). In Europe and the United States, the DPT vaccine primarily uses column chromatography to purify the pertussis antigen, obtaining a component vaccine containing three components with clearly defined dosages: pertussis toxoid, filamentous hemagglutinin, and pertussis pertactin (PRN). The advantages of component vaccines are their clearly defined components, higher antigen purity, and fewer side effects. Currently, vaccine research in my country containing PT, FHA, PRN, diphtheria, and tetanus antigen components has entered the clinical research stage.

[0007] With the widespread use of vaccines, the incidence of pertussis, diphtheria, and tetanus has decreased significantly. However, in recent years, the incidence of pertussis has increased among preschool children and adolescents.

[0008] Currently available DPT (diphtheria, pertussis, tetanus) combined vaccines all use aluminum adjuvants to enhance the immune response. Aluminum adjuvants can effectively induce a strong humoral immune response, i.e., a Th2-type immune response; however, they are weaker in inducing cellular immunity (Th1-type immune response). CpG ODN is a synthetically produced ODN containing an unmethylated CpG motif, which has a strong immunostimulatory effect. When used in combination with other adjuvants, it can significantly enhance specific antigen-mediated humoral and cellular immune responses. Summary of the Invention

[0009] Therefore, the present invention aims to provide a combined adjuvant vaccine comprising pertussis toxoid (PT), filamentous hemagglutinin (FHA), diphtheria toxoid (DT), and tetanus toxoid (TT) antigens, and further comprising a combination adjuvant, such as an aluminum adjuvant and a TLR9 receptor agonist. The combined adjuvant vaccine can be used to prevent pertussis, diphtheria, and tetanus in infants, adolescents, and adults.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides an immunogenic composition comprising pertussis antigen, diphtheria antigen, and tetanus antigen. Preferably, it comprises pertussis PT antigen, pertussis FHA antigen, diphtheria DT antigen, and tetanus TT antigen.

[0011] Secondly, the present invention provides a composite adjuvant composed of an aluminum adjuvant and a TLR9 receptor agonist. Preferably, the aluminum adjuvant is aluminum hydroxide, and the TLR9 receptor agonist is CpG ODN.

[0012] Thirdly, the present invention provides a combined adjuvant vaccine comprising the immunogenic composition of the first aspect and the combined adjuvant and / or pharmaceutically acceptable carrier of the second aspect. Preferably, the combined adjuvant is a composition of aluminum hydroxide and CpGODN.

[0013] Preferably, each dose of the combined adjuvant vaccine contains: 1-25 μg of pertussis PT antigen, 1-25 μg of pertussis FHA antigen, 1-20 Lf of diphtheria DT antigen, and 1-5 Lf of tetanus TT antigen; more preferably, the pertussis PT antigen is 4, 8, 16, and 25 μg, the pertussis FHA antigen is 4, 8, 16, and 25 μg, the diphtheria DT antigen is 1, 2, 4, and 20 Lf, and the tetanus TT antigen is 1.5, 2.5, and 5 Lf; most preferably, the pertussis PT antigen is 8 μg, the pertussis FHA antigen is 8 μg, the diphtheria DT antigen is 2 Lf, and the tetanus TT antigen is 2.5 Lf.

[0014] Preferably, each dose of the combined adjuvant vaccine contains: 0.01~1mg of aluminum and 0.1~100μg of CpG ODN; more preferably, each dose of the combined adjuvant vaccine contains: 0.1~0.5mg of aluminum and 25~100μg of CpG ODN; most preferably, each dose of the combined adjuvant vaccine contains: 0.24mg of aluminum and 50 μg or 100μg of CpG ODN.

[0015] Fourthly, the present invention provides the use of the compound adjuvant of the second aspect in the preparation of a compound adjuvant combined vaccine of the third aspect for the prevention of pertussis, diphtheria and tetanus in subjects.

[0016] Preferably, the combined adjuvant vaccine comprises an immunogenic composition and a combined adjuvant.

[0017] Preferably, the subject is a mammal; more preferably, the subject is a human.

[0018] The combined adjuvant vaccine prepared by this invention has the following advantages: using aluminum adjuvant and TLR9 receptor agonist as the combined adjuvant, it can achieve high immunogenicity with a lower antigen dosage, and the potency of each component meets the relevant requirements of the 2020 edition of the Chinese Pharmacopoeia. This invention maintains strong immunogenicity and protective effect even without pertussis adhesin antigen, and can be applied to the prevention of pertussis, diphtheria, and tetanus in infants, adolescents, and adults. Detailed Implementation

[0019] This invention provides a combined adjuvant vaccine comprising pertussis PT antigen, pertussis FHA antigen, diphtheria DT antigen, and tetanus TT antigen, and further comprising a combined adjuvant composed of an aluminum adjuvant and a TLR9 receptor agonist. The combined adjuvant vaccine can prevent pertussis, diphtheria, and tetanus in infants, adolescents, and adults.

[0020] The present invention will be further illustrated below with reference to the embodiments, but the present invention is not limited to these embodiments: Example 1: Exploration of adjuvant dosage in combined adjuvant vaccines 1) The stock solution of pertussis (PT antigen and FHA antigen) (prepared according to Examples 1, 2, 4 and 5 of patent publication number CN103242434B) and the stock solution of purified diphtheria toxoid (fermentation reference: Cox, J C. New method for the large-scale preparation of diphtheria toxoid: purification of toxin[J]. Applied Microbiology, 1975, 29(4):464-468. Preparation and purification reference: Robb LA, Stainer DW, Scholte MJ, Preparation and properties of diphtheria toxoids in submerged culture. IV. A revised method for the purification of diphtheria toxin.[J]. Can J Microbiol, 1970, 16: 639. Preparation and detoxification reference: Xiao Xiling, Shi Cuifeng, Ma Xueyan, et al. Complete toxoidization of purified diphtheria toxin[J]. Chinese Journal of Biological Products, 1990, 1(3):26-29.), and refined tetanus toxoid stock solution (fermentation and detoxification reference: Lu Jinhan. Research on tetanus toxin and toxoid[J]. Acta Microbiologica Sinica, 1953, 1(1):113-126. preparation and purification reference: Stojićević Ivana, Dimitrijević Ljiljana, Dovezenski Nebojša et al. Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation.[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2011, 879: 2213-2219. preparation) were mixed with aluminum hydroxide in proportion, adsorbed, and the pH value was adjusted to 6.5. Then, CpG ODN 2006 adjuvant was added and mixed evenly; 2) Mix the adsorbed components so that each 0.5ml dose contains 8μg of pertussis PT antigen, 8μg of pertussis FHA antigen, 2Lf of diphtheria DT antigen, 2.5Lf of tetanus TT antigen, 0.24mg of aluminum, and 25, 50, and 100μg of CpG ODN 2006; the specific groups are: CpG ODN 2006 25μg group (group 1), CpG ODN 2006 50μg group (group 2), and CpG ODN 2006 100μg group (group 3).

[0021] 3) Simultaneously, DPT vaccine group with single Al adjuvant (Group 4), DPT vaccine group with single CpG ODN 2006 adjuvant (Group 5), aluminum hydroxide control group (Group 6), CpG ODN 2006 control group (Group 7), and antigen control group (Group 8) were prepared. The composition and dosage of antigen and adjuvant for each component in this embodiment are shown in Table 1.

[0022] Table 1. Composition and dosage of antigens and adjuvants Example 2: Study on the effect of adjuvant dosage on protective potency in combined adjuvant vaccines 1) Pertussis potency: Referencing the 2020 edition of the Chinese Pharmacopoeia. National potency standards (provided by the China National Institutes for Food and Drug Control) were diluted to 1, 0.2, and 0.04 IU / ml, and then combined with the compound adjuvant vaccine prepared in Table 1 to immunize experimental animals. The experimental animals were clean-grade NIH mice, administered 0.5 ml intraperitoneally per mouse. Twenty-one days after immunization, the mice were challenged. Fourteen days post-challenge, animal survival was observed, and potency was calculated.

[0023] 2) Diphtheria potency: Referencing the 2020 edition of the Chinese Pharmacopoeia. National potency standards (provided by the China National Institutes for Food and Drug Control) were diluted to 5.54, 2.77, 1.38, and 0.69 IU / ml, and then combined with the compound adjuvant vaccine prepared in Table 1 to immunize experimental animals. The experimental animals were clean-grade NIH mice, with 0.5 ml injected subcutaneously per mouse. Blood was collected 5 weeks post-immunization, serum was separated, and a toxin neutralization test was performed using Vero cells. Diphtheria potency was calculated based on cell viability.

[0024] 3) Tetanus potency: Referencing the 2020 edition of the Chinese Pharmacopoeia. National potency standards (provided by the China National Institutes for Food and Drug Control) were diluted to 9.64, 4.82, 2.41, and 1.205 IU / ml, and then combined with the compound adjuvant vaccine prepared in Table 1 to immunize experimental animals. The experimental animals were clean-grade NIH mice, administered 0.5 ml subcutaneously per mouse. Four weeks after immunization, the mice were challenged. Five days post-challenge, animal survival was observed, and potency was calculated.

[0025] 4) The protective potency of each component is shown in Table 2.

[0026] Table 2. Results of valence testing for each group Example 3: Exploration of antigen dosage in combined adjuvant vaccines 1) The stock solution of pertussis (PT antigen and FHA antigen) (prepared according to Examples 1, 2, 4 and 5 of patent publication number CN103242434B) and the stock solution of purified diphtheria toxoid (fermentation reference: Cox, J C. New method for the large-scale preparation of diphtheria toxoid: purification of toxin[J]. Applied Microbiology, 1975, 29(4):464-468. Preparation and purification reference: Robb LA, Stainer DW, Scholte MJ, Preparation and properties of diphtheria toxoids in submerged culture. IV. A revised method for the purification of diphtheria toxin.[J]. Can J Microbiol, 1970, 16: 639. Preparation and detoxification reference: Xiao Xiling, Shi Cuifeng, Ma Xueyan, et al. Complete toxoidization of purified diphtheria toxin[J]. Chinese Journal of Biological Products, 1990, 1(3):26-29.), and refined tetanus toxoid stock solution (fermentation and detoxification reference: Lu Jinhan. On the study of tetanus toxin and toxoid[J]. Acta Microbiologica Sinica, 1953, 1(1):113-126. preparation and purification reference: Stojićević Ivana, Dimitrijević Ljiljana, Dovezenski Nebojša et al. Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation.[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2011, 879: 2213-2219. preparation) were mixed with aluminum hydroxide in proportion, adsorbed, and the pH value was adjusted to 6.5. Then, CpG ODN2006 adjuvant was added and mixed evenly; 2) Mix the adsorbed components so that each dose contains 4, 8, 16 and 25 μg of pertussis PT antigen, 4, 8, 16 and 25 μg of pertussis FHA antigen, 1, 2, 4 and 20 Lf of diphtheria DT antigen, 1.5, 2.5 and 5 Lf of tetanus TT antigen, 0.24 mg of aluminum, and 50 and 0 μg of CpGODN 2006; group them into groups 1, 2, 3, 4 and 5, and detailed information on each component is shown in Table 3.

[0027] The composition and dosage of each component antigen and adjuvant in this embodiment are shown in Table 3.

[0028] Table 3 Composition and dosage of antigens and adjuvants Example 4: Study on the effect of antigen dosage on protective potency in combined adjuvant vaccines 1) Pertussis potency: Referencing the 2020 edition of the Chinese Pharmacopoeia. National potency standards (provided by the China National Institutes for Food and Drug Control) were diluted to 1, 0.2, and 0.04 IU / ml, and used to immunize experimental animals with the combined adjuvant vaccine prepared in Table 3 and a commercially available vaccine (adsorbed acellular pertussis-diphtheria-tetanus-inactivated poliomyelitis and Haemophilus influenzae type b combined vaccine, purchased from Sanofi Pasteur S.A.). The experimental animals were clean-grade NIH mice, administered 0.5 ml intraperitoneally per mouse. Twenty-one days after immunization, the mice were challenged. Fourteen days post-challenge, animal survival was observed, and potency was calculated.

[0029] 2) Diphtheria potency: Referencing the 2020 edition of the Chinese Pharmacopoeia. National potency standards (provided by the China National Institutes for Food and Drug Control) were diluted to 5.54, 2.77, 1.38, and 0.69 IU / ml, and used to immunize experimental animals with the combined adjuvant vaccine prepared in Table 3 and a commercially available vaccine (adsorbed acellular pertussis-diphtheria-tetanus-inactivated polio vaccine and Haemophilus influenzae type b combined vaccine, purchased from Sanofi Pasteur SA). The experimental animals were clean-grade NIH mice, with 0.5 ml injected subcutaneously per mouse. Blood was collected 5 weeks post-immunization, serum was separated, and toxin neutralization was performed using Vero cells. Diphtheria potency was calculated based on cell viability.

[0030] 3) Tetanus potency: Referencing the 2020 edition of the Chinese Pharmacopoeia. National potency standards (provided by the China National Institutes for Food and Drug Control) were diluted to 9.64, 4.82, 2.41, and 1.205 IU / ml, and used to immunize experimental animals with the combined adjuvant vaccine prepared in Table 3 and a commercially available vaccine (adsorbed acellular pertussis-diphtheria-tetanus-inactivated poliomyelitis and Haemophilus influenzae type b combined vaccine, purchased from Sanofi Pasteur SA). The experimental animals were clean-grade NIH mice, administered 0.5 ml subcutaneously per mouse. Four weeks after immunization, the mice were challenged. Five days post-challenge, animal survival was observed, and potency was calculated.

[0031] 4) The potency results of each component are shown in Table 4.

[0032] Table 4. Protective efficacy results for each group In summary, the experimental results show that: As shown in Examples 1 and 2, under the same antigen and aluminum adjuvant content, the potency of each antigen (except diphtheria) exhibits a dose-response effect with the increase of CpG ODN2006 content. That is, the potency of each antigen (except diphtheria) increases with the increase of CpG ODN2006 content, and the compound adjuvants have a synergistic effect, that is, the potency of the groups using compound adjuvants (groups 1, 2, and 3) is higher than the sum of the potencies of the single adjuvant groups (groups 4 and 5). The potency of each compound adjuvant group meets the standards of the 2020 edition of the Chinese Pharmacopoeia, that is, each human dose of 0.5 mL should contain a cell-free pertussis vaccine with a potency of not less than 4.0 IU, a diphtheria vaccine with a potency of not less than 30 IU, and a tetanus vaccine with a potency of not less than 40 IU.

[0033] As shown in Examples 3 and 4, the use of compound adjuvant DPT vaccine can effectively reduce the amount of antigen used. In Group 2, PT and FHA are each 8 μg, while in the commercially available vaccine group, PT and FHA are both 25 μg, and their pertussis potency is almost the same. When PT and FHA are each 16 μg in Group 3, the pertussis potency is significantly higher than that of the commercially available vaccine group. Since the diphtheria and tetanus antigen content in the instructions of the commercially available vaccine is different from the domestic definition, no further comparison is made here.

[0034] As shown in Examples 1, 2, 3, and 4, when the concentration of CpG ODN 2006 in the compound adjuvant is increased to 50-100 μg / dose, its pertussis potency is equivalent to or significantly higher than that of commercially available vaccines.

[0035] Therefore, by using an aluminum hydroxide-CpG ODN 2006 composite adjuvant, this invention increases the potency of pertussis, diphtheria, and tetanus with increasing dosage, without the presence of pertussis adhesin antigen. Compared with using aluminum adjuvant or CpG ODN 2006 adjuvant alone, the antigen dosage can be reduced while maintaining the same potency. This invention can be applied to prevent pertussis, diphtheria, and tetanus in infants, adolescents, and adults.

Claims

1. A combined adjuvant vaccine, wherein the active ingredient of the combined adjuvant vaccine comprises an immunogenic composition and a combined adjuvant, wherein the antigens in the immunogenic composition comprise pertussis PT antigen, pertussis FHA antigen, diphtheria DT antigen, and tetanus TT antigen, wherein the combined adjuvant comprises an aluminum adjuvant and a TLR9 receptor agonist; wherein the aluminum adjuvant is aluminum hydroxide, wherein the aluminum content is 0.1~0.5 mg per dose of the combined adjuvant vaccine, and the TLR9 receptor agonist is CpG ODN 2006, the content of which is 25~100 μg per dose of the combined adjuvant vaccine; In each dose of the combined adjuvant vaccine, the pertussis PT antigen is 4-25 μg, the pertussis FHA antigen is 4-25 μg, the diphtheria DT antigen is 1-20 Lf, and the tetanus TT antigen is 1.5-5 Lf. The dosage of each dose of the combined adjuvant vaccine is 0.5 ml.

2. The combined adjuvant vaccine according to claim 1, wherein each dose of the combined adjuvant vaccine contains 4, 8, 16 or 25 μg of pertussis PT antigen, 4, 8, 16 or 25 μg of pertussis FHA antigen, 1, 2, 4 or 20 Lf of diphtheria DT antigen, and 1.5, 2.5 or 5 Lf of tetanus TT antigen.

3. The combined adjuvant vaccine according to claim 2, wherein each dose of the combined adjuvant vaccine contains 8 μg of pertussis PT antigen, 8 μg of pertussis FHA antigen, 2 Lf of diphtheria DT antigen, and 2.5 Lf of tetanus TT antigen.

4. The combined adjuvant vaccine according to claim 1, wherein each dose of the combined adjuvant vaccine contains 0.24 mg of aluminum and 50 μg or 100 μg of CpG ODN 2006.

5. The use of the compound adjuvant combined vaccine according to any one of claims 1-4 in the preparation of a vaccine for the prevention of pertussis, diphtheria and tetanus in subjects.

6. The use according to claim 5, wherein the subject is a mammal.

7. The use according to claim 5, wherein the subject is a human.

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