WO2024078170A1 - 一种百白破复合佐剂联合疫苗 - Google Patents

一种百白破复合佐剂联合疫苗 Download PDF

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WO2024078170A1
WO2024078170A1 PCT/CN2023/115711 CN2023115711W WO2024078170A1 WO 2024078170 A1 WO2024078170 A1 WO 2024078170A1 CN 2023115711 W CN2023115711 W CN 2023115711W WO 2024078170 A1 WO2024078170 A1 WO 2024078170A1
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antigen
pertussis
diphtheria
tetanus
combined vaccine
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French (fr)
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王梦舒
杨军
姜涛
武艳春
闫昆明
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长春百克生物科技股份公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0016—Combination vaccines based on diphtheria-tetanus-pertussis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biological products, in particular to a composite adjuvant combined vaccine containing pertussis, diphtheria and tetanus antigens.
  • Whooping cough is a serious 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 spread through droplets and is highly contagious. The typical clinical symptoms of whooping cough are persistent paroxysmal coughs with inspiratory tails or vomiting, which are easily complicated by pneumonia and encephalitis and are the main causes of death. In developed countries, the mortality rate of whooping cough is 0.1%, while in developing countries, the average mortality rates for infants and children aged 1-4 are 3.9% and 1% respectively. According to the World Health Organization (WHO), there are about 60 million new cases of whooping cough each year in the world, of which 500,000 to 1 million deaths, seriously endangering the lives and health of children.
  • WHO World Health Organization
  • Diphtheria is an acute upper respiratory tract infectious disease caused by Gram-positive Corynebacterium diphtheriae.
  • Clinical manifestations include inflammation of the upper respiratory tract, usually in the pharynx, sometimes in the posterior nasal cavity, larynx and trachea, and even damage to other organs, mainly the myocardium and peripheral nerves.
  • the highly pathogenic exotoxins produced by toxin-producing strains of Corynebacterium diphtheriae can produce membranes locally and cause systemic organ damage. Its clinical characteristics are the formation of pseudomembranes in the pharynx, larynx, nose, etc. and systemic poisoning symptoms such as fever, fatigue, nausea, vomiting, headache, etc. In severe cases, myocarditis and nerve paralysis may occur.
  • diphtheria was the leading cause of death among children in the UK. Since the diphtheria toxoid vaccination, the incidence has dropped significantly, but it continues to occur in some other areas. It is estimated that before the implementation of WHO's EPI, there were nearly 1 million cases of diphtheria in third world countries each year, with 50,000 to 60,000 deaths.
  • Tetanus is a disease that seriously endangers people's lives and health. It is an acute specific infection caused by Clostridium tetani invading human wounds, growing and multiplying, and producing toxins. Clostridium tetani and its toxins cannot invade normal skin and mucous membranes. Therefore, tetanus occurs after injury. All open injuries are likely to cause tetanus. Tetanus is an acute specific infection caused by Clostridium tetani invading wounds, multiplying, and secreting toxins. The main manifestations are persistent contractions and paroxysmal spasms of systemic or local muscles. The situation is more serious in poor and backward countries and regions. It is estimated that there are about 1 million cases of tetanus in the world every year, with a mortality rate of about 50%. In developing countries, the mortality rate of neonatal tetanus is as high as 90%.
  • Vaccine immunization is an effective means to prevent the above-mentioned diseases. Since the whole cell pertussis vaccine was developed in the 1940s, it has been used in countries around the world for nearly 60 years. The whole cell pertussis vaccine has played a huge role in the prevention and control of pertussis, making pertussis an infectious disease that can be prevented by vaccines. It has been listed as one of the infectious diseases to be controlled and eliminated in the Expanded Program of Immunization (EPI) promoted by the World Health Organization. Diphtheria toxoid can effectively prevent diphtheria, control the prevalence of diphtheria, and ensure the health and life safety of children.
  • EPI Expanded Program of Immunization
  • diphtheria toxoid is a safe and effective vaccine to prevent diphtheria epidemics.
  • Tetanus toxoid can effectively prevent tetanus.
  • Immunization with tetanus toxoid can induce immune responses in 74-90% of vaccine recipients.
  • Tetanus vaccine was launched in the United States in 1938 and was used on a large scale in the military in 1941.
  • Epidemiological studies have shown that diphtheria toxoid is a safe and effective vaccine to prevent tetanus epidemics. In 1948, the whole-cell DTP vaccine was launched in the United States. At the end of 1996, the acellular DTP vaccine was approved for marketing.
  • diphtheria-tetanus-pertussis vaccine currently on the market in my country uses the ammonium sulfate precipitation method to purify pertussis antigens, producing a co-purified vaccine containing two main antigens: pertussis toxoid (PT) and filamentous hemagglutinin (FHA).
  • PT pertussis toxoid
  • FHA filamentous hemagglutinin
  • the diphtheria-tetanus-pertussis vaccine currently used in Europe and the United States mainly uses the column-column method to purify pertussis antigens, obtaining a component vaccine containing three components and a clear dose of pertussis toxoid, filamentous hemagglutinin, and pertussis adhesin (PRN).
  • the advantages of component vaccines are that they have clear components, high antigen purity, and fewer side effects.
  • my country's vaccine research containing PT, FHA, PRN, diphtheria and tetanus antigen components has also entered the clinical research stage.
  • CpG ODN is an artificially synthesized ODN containing unmethylated CpG motifs, which has a strong immunostimulatory effect. When used in combination with other adjuvants, it can significantly enhance specific antigen humoral immunity and cellular immune responses.
  • the object of the present invention is to provide a composite adjuvant combined vaccine, comprising pertussis toxoid (PT), filamentous hemagglutinin (FHA), diphtheria toxoid (DT) and tetanus toxoid (TT) antigens, and further comprising a composite adjuvant, such as a composition of an aluminum adjuvant and a TLR9 receptor agonist.
  • PT pertussis toxoid
  • FHA filamentous hemagglutinin
  • DT diphtheria toxoid
  • TT tetanus toxoid
  • the composite adjuvant combined vaccine can be used to prevent pertussis, diphtheria and tetanus in infants, adolescents and adults.
  • the present invention provides an immunogenic composition comprising a pertussis antigen, a diphtheria antigen and a tetanus antigen, preferably, a pertussis PT antigen, a pertussis FHA antigen, a diphtheria DT antigen and a tetanus TT antigen.
  • the present invention provides a composite adjuvant, which is composed of an aluminum adjuvant and a TLR9 receptor agonist.
  • the aluminum adjuvant is aluminum hydroxide
  • the TLR9 receptor agonist is CpG ODN.
  • the present invention provides a composite adjuvant combined vaccine, which comprises the immunogenic composition of the first aspect and the composite adjuvant and/or pharmaceutically acceptable carrier of the second aspect.
  • the composite adjuvant is a composite of aluminum hydroxide and CpG ODN.
  • each dose of the composite adjuvant combination vaccine contains: 1-25 ⁇ g of pertussis PT antigen, 1-25 ⁇ g 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 ⁇ g, the pertussis FHA antigen is 4, 8, 16 and 25 ⁇ g, 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 ⁇ g, the pertussis FHA antigen is 8 ⁇ g, the diphtheria DT antigen is 2 Lf, and the tetanus TT antigen is 2.5 Lf.
  • each dose of the composite adjuvant combination vaccine contains: 0.01-1 mg aluminum, 0.1-100 ⁇ g CpG ODN; more preferably, each dose of the composite adjuvant combination vaccine contains: 0.1-0.5 mg aluminum, 25-100 ⁇ g CpG ODN; most preferably, each dose of the composite adjuvant combination vaccine contains: 0.24 mg aluminum, 50 ⁇ g or 100 ⁇ g CpG ODN.
  • the present invention provides use of the composite adjuvant of the second aspect in preparing the composite adjuvant combination vaccine of the third aspect for preventing pertussis, diphtheria and tetanus in a subject.
  • the composite adjuvant combination vaccine comprises an immunogenic composition and a composite adjuvant.
  • the subject is a mammal; more preferably, the subject is a human.
  • the composite adjuvant combined vaccine prepared by the present invention has the following advantages: aluminum adjuvant and TLR9 receptor agonist are used as composite adjuvants, and a higher immunogenicity can be achieved with a lower antigen dosage, and the titer of each component meets the relevant requirements of the 2020 edition of the Chinese Pharmacopoeia.
  • the present invention still has strong immunogenicity and protective effect without the presence of pertussis antigen, and can be used to prevent pertussis, diphtheria and tetanus in infants, adolescents and adults.
  • the present invention provides a composite adjuvant combined vaccine, comprising pertussis PT antigen, pertussis FHA antigen, diphtheria DT antigen and tetanus TT antigen, and further comprising a composite adjuvant consisting of an aluminum adjuvant and a TLR9 receptor agonist.
  • the composite adjuvant combined vaccine can prevent pertussis, diphtheria and tetanus in infants, adolescents and adults.
  • Preparation and detoxification reference Xiao Xiling, Shi Cuifeng, Ma Xueyan, et al. Complete toxinization of purified diphtheria toxin [J]. Chinese Journal of Biological Products, 1990, 1 (3): 26-29.), purified 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.
  • each 0.5 ml dose contains 8 ⁇ g of pertussis PT antigen, 8 ⁇ g of pertussis FHA antigen, 2 Lf of diphtheria DT antigen, 2.5 Lf of tetanus TT antigen, 0.24 mg of aluminum content, and 25, 50, and 100 ⁇ g of CpG ODN 2006; specifically divided into groups: CpG ODN 2006 25 ⁇ g group (Group 1), CpG ODN 2006 50 ⁇ g group (Group 2), and CpG ODN 2006 100 ⁇ g group (Group 3).
  • Example 2 Study on the effect of adjuvant dose on protective titer in composite adjuvant combined vaccine
  • Pertussis titer refer to the 2020 edition of the Chinese Pharmacopoeia.
  • the national titer standard (provided by the China Food and Drug Administration) was diluted to 1, 0.2, and 0.04 IU/ml, and the composite adjuvant combined vaccine prepared in Table 1 was used to immunize the experimental animals.
  • the experimental animals were clean-grade NIH mice, and 0.5 ml/mouse was injected intraperitoneally. After 21 days of immunization, the virus was challenged. 14 days after the virus was challenged, the animals were observed. The survival of the animals was observed and the titer was calculated.
  • Diphtheria titer refer to the 2020 edition of the Chinese Pharmacopoeia.
  • the national titer standard (provided by the China Food and Drug Inspection Institute) was diluted to 5.54, 2.77, 1.38, and 0.69 IU/ml, and the test animals were immunized with the composite adjuvant combined vaccine prepared in Table 1.
  • the test animals were clean-grade NIH mice, subcutaneously injected with 0.5 ml/mouse, and blood was collected 5 weeks after immunization to separate serum. Toxin neutralization test was performed with Vero cells, and diphtheria titer was calculated by cell survival.
  • Tetanus titer refer to the 2020 edition of the Chinese Pharmacopoeia.
  • the national titer standard (provided by the China Food and Drug Inspection Institute) was diluted to 9.64, 4.82, 2.41, and 1.205 IU/ml, and the test animals were immunized with the composite adjuvant combined vaccine prepared in Table 1.
  • the test animals were clean-grade NIH mice, subcutaneously injected with 0.5 ml/mouse, and challenged 4 weeks after immunization. Five days after challenge, the survival of the animals was observed and the titer was calculated.
  • Preparation and detoxification reference Xiao Xiling, Shi Cuifeng, Ma Xueyan, et al. Complete toxinization of purified diphtheria toxin [J]. Chinese Journal of Biological Products, 1990, 1(3):26-29.), purified 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.
  • each dose contained 4, 8, 16 and 25 ⁇ g pertussis PT antigen, 4, 8, 16 and 25 ⁇ g pertussis FHA antigen, 1, 2, 4 and 20 Lf diphtheria DT antigen, 1.5, 2.5 and 5 Lf tetanus TT antigen, 0.24 mg aluminum content, 50 and 0 ⁇ g CpG ODN 2006; the doses were divided into Group 1, Group 2, Group 3, Group 4 and Group 5. The detailed information of each component is shown in Table 3.
  • composition and dosage of each component antigen and adjuvant of this example are shown in Table 3.
  • Example 4 Study on the effect of antigen dose on protective titer in composite adjuvant combined vaccine
  • Pertussis titer refer to the 2020 edition of the Chinese Pharmacopoeia.
  • the national potency standard (provided by the China Food and Drug Inspection Institute) was diluted to 1, 0.2, and 0.04 IU/ml, and the test animals were immunized with the composite adjuvant combined vaccine prepared in Table 3 and the commercially available vaccine (adsorbed acellular diphtheria, pertussis, inactivated poliomyelitis and type b Haemophilus influenzae combined vaccine, purchased from Sanofi Pasteur S.A).
  • the test animals were clean-grade NIH mice, which were injected intraperitoneally with 0.5 ml/mouse. After 21 days of immunization, the virus was challenged. 14 days after the virus challenge, the survival of the animals was observed and the titer was calculated.
  • Diphtheria potency refer to the 2020 edition of the Chinese Pharmacopoeia. Dilute the national potency standard (provided by the China Food and Drug Inspection Institute) to 5.54, 2.77, 1.38, and 0.69 IU/ml.
  • the experimental animals were immunized with the composite adjuvant combination vaccine prepared in Table 3 and the commercial vaccine (adsorbed acellular diphtheria, pertussis, inactivated poliomyelitis and Haemophilus influenzae type b combined vaccine, purchased from Sanofi Pasteur SA). The experimental animals were clean NIH mice, and 0.5 ml/mouse was injected subcutaneously. Blood was collected 5 weeks after immunization, serum was separated, toxin neutralization test was performed with Vero cells, and diphtheria titer was calculated by cell survival.
  • Tetanus titer refer to the 2020 edition of the Chinese Pharmacopoeia.
  • the national potency standard (provided by the China Food and Drug Inspection Institute) was diluted to 9.64, 4.82, 2.41, and 1.205 IU/ml, and the experimental animals were immunized with the composite adjuvant combined vaccine prepared in Table 3 and the commercially available vaccine (adsorbed acellular diphtheria, pertussis, inactivated poliomyelitis and type b Haemophilus influenzae combined vaccine, purchased from Sanofi Pasteur S.A).
  • the experimental animals were clean-grade NIH mice, subcutaneously injected with 0.5 ml/mouse, and challenged 4 weeks after immunization. Five days after challenge, the survival of the animals was observed and the titer was calculated.
  • the titer of each antigen showed a dose effect with the increase of CpG ODN 2006 content, that is, the titer of each antigen (except diphtheria) increased with the increase of CpG ODN 2006 content, and the composite adjuvant had a synergistic effect. That is, the titers of the groups using composite adjuvants (Groups 1, 2, and 3) are all higher than the sum of the titers of the single adjuvant groups (Groups 4 + Group 5).
  • each composite adjuvant group meets the standards of the 2020 edition of the "Chinese Pharmacopoeia", that is, each human dose of 0.5mL, the titer of acellular pertussis vaccine should not be less than 4.0IU, the titer of diphtheria vaccine should not be less than 30IU, and the titer of tetanus vaccine should not be less than 40IU.
  • the present invention uses aluminum hydroxide-CpG ODN 2006 composite adjuvant, in the absence of pertussis antigen, to increase the titers of pertussis, diphtheria and tetanus with increasing doses.
  • the antigen dose can be reduced under the condition of equivalent titers, and the present invention can be used to prevent pertussis, diphtheria and tetanus in infants, adolescents and adults.

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Abstract

本发明公开了一种复合佐剂联合疫苗，其包含一种免疫原性组合物及复合佐剂。所述免疫原性组合物包含百日咳抗原、白喉抗原和破伤风抗原，所述复合佐剂由铝佐剂和TLR9受体激动剂组成。本发明还公开了所述复合佐剂在制备用于在受试者中预防百日咳、白喉以及破伤风的前述复合佐剂联合疫苗中的用途。

Description

一种百白破复合佐剂联合疫苗 技术领域
本发明涉及生物制品领域，特别涉及含百日咳、白喉及破伤风抗原的复合佐剂联合疫苗。
背景技术
百日咳是由百日咳杆菌引起的一种严重急性呼吸道传染病，任何年龄的人都可感染，但对五岁以下婴幼儿更为敏感。本病通过飞沫传播，传染性极强。百日咳典型的临床症状为持续性阵发性咳嗽，带有吸气性尾声或呕吐，易合并肺炎和脑炎，是导致死亡的主要原因。在发达国家，百日咳的致死率为0.1％，而在发展中国家，婴儿和1-4岁儿童的平均致死率分别为3.9％和1％。据世界卫生组织(WHO)估计，全世界每年大约有6000万百日咳新病例，其中有50-100万例死亡，严重地危害着儿童的生命和健康。
白喉是由革兰氏阳性白喉棒状杆菌引起一种急性上呼吸道传染病。临床表现为上呼吸道炎症，通常在咽部，有时在后鼻腔、喉部和气管，甚至损伤到其它器官，主要是心肌及周围神经。白喉杆菌产毒菌株产生的致病性很强的外毒素，可在局部产生膜也可导致全身器官损害。其临床特征是咽、喉、鼻等处假膜形成和全身中毒症状如发热、乏力、恶心呕吐、头痛等，严重者可并发心肌炎和神经瘫痪。20世纪初，白喉是引起英国儿童死亡的主要原因。自白喉类毒素疫苗接种以来，发病率显著降低，但在其他一些地区仍继续发生。据估计，在WHO的EPI实施之前，第三世界国家每年白喉发病接近100万例，5万～6万人死亡。
破伤风是一种严重危害人民生命健康的疾病，是由于破伤风杆菌(Clostridium tetani)侵入人体伤口、生长繁殖、产生毒素可引起的一种急性特异性感染。破伤风杆菌及其毒素不能侵入正常的皮肤和粘膜，因此，破伤风都发生在受伤之后。一切开放性损伤，均有发生破伤风的可能。破伤风是破伤风杆菌侵入伤口内繁殖、分泌毒素引起的急性特异性感染，主要表现为全身或局部肌肉的持续性收缩和阵发性痉挛。在广 大贫困落后的国家和地区更为严重。据估计，世界上每年约100万病例发生，死亡率在50％左右。在发展中国家，新生儿破伤风死亡率高达90％。
疫苗免疫接种是预防上述几种疾病的有效手段。自20世纪40年代制成全细胞百日咳疫苗以来，已在世界各国使用近60年。全细胞百日咳疫苗在预防和控制百日咳中发挥了巨大的作用，使百日咳成为可以用疫苗预防的传染病，已列入世界卫生组织推行的扩大免疫计划(Expend Programmed Immunization,EPI)中要控制的和消灭的传染病之一。白喉类毒素可以有效的预防白喉，控制了白喉的流行,确保了儿童的身体健康和生命安全。当前WHO和我国政府已把白喉列入了EPI重点控制的6种传染病之一。流行病学研究证明白喉类毒素是一种安全有效预防白喉流行的疫苗。破伤风类毒素可以有效的预防破伤风，用破伤风类毒素进行免疫，可以在74～90％接种者体内诱导免疫应答。破伤风疫苗1938年在美国上市，1941年在军队大规模使用。流行病学研究证明白喉类毒素是一种安全有效预破伤风流行的疫苗。1948年全细胞百白破联合疫苗在美国上市应用。1996年底，无细胞组分百白破疫苗获批上市。
在我国《国家免疫规划疫苗儿童免疫程序及说明(2021年版)》中规定，3月龄、4月龄、5月龄、18月龄各接种1剂吸附无细胞百白破联合疫苗，6周岁接种1剂吸附白喉破伤风联合疫苗。我国目前上市应用的百白破疫苗是采用硫酸铵盐析的方法进行百日咳抗原纯化，生产出主要含百日咳类毒素(Pertussis toxoid，PT)和丝状血细胞凝集素(Filamentous hemagglutinin，FHA)两种抗原的共纯化疫苗。欧美国家目前应用的百白破疫苗主要是采用柱层柱的方法进行百日咳抗原纯化，获得含有百日咳类毒素、丝状血细胞凝集素及百日咳粘附素(Pertussis pertactin,PRN)三个组分及剂量明确的组分疫苗。组分疫苗的优势在于其组分明确、抗原纯度较高、副作用较小。目前，我国含PT、FHA、PRN、白喉、破伤风抗原组分的疫苗研究也已进入临床研究阶段。
随着疫苗的广泛使用，百日咳、白喉及破伤风疾病的发病率明显降低。但近年来在学龄前儿童及青少年人群中百日咳发病率有所增高。在1922-2017年间，欧美国家百日咳继续感染所有年龄段人群且病例逐年增加。2005年，免疫实践咨询委员会(ACIP)建议为青少年和小于65岁 的成人一次性接种无细胞百日咳、(白喉和Tdap)破伤风类毒素疫苗。2012年，ACIP将该建议扩大到包括所有成年人，建议每隔10年对10岁以上的人群进行一次百白破联合疫苗的接种。欧美等国家现已开始在青少年及成人人群中采用加强免疫的方式控制感染。但是我国目前尚没有自主研发的百白破三组分青少年及成人用疫苗，因此青少年及成人百白破疫苗在我国的研发具有重大意义。
目前上市应用的百白破联合疫苗均采用铝佐剂增强免疫应答，铝佐剂可以有效地诱导机体产生较强的体液免疫反应，即Th2型免疫反应；但在诱导细胞免疫能力上(Th1型免疫反应)较弱。CpG ODN是人工合成的含非甲基化CpG基序的ODN，具有较强的免疫刺激作用。与其他佐剂联合应用时可显著增强特异性的抗原体液免疫和细胞免疫应答。
发明内容
鉴于此，本发明的目的在于提供一种复合佐剂联合疫苗，包括百日咳类毒素(Pertussis toxoid，PT)、丝状血细胞凝集素(Filamentous hemagglutinin，FHA)、白喉类毒素(Diphtheria toxoid，DT)和破伤风类毒素(Tetanus toxoid，TT)抗原，另外还包含一种复合佐剂，如铝佐剂及TLR9受体激动剂的组合物。所述复合佐剂联合疫苗可应用于预防婴幼儿、青少年及成人的百日咳、白喉及破伤风。
为了实现上述发明目的，本发明提供以下技术方案：
第一方面，本发明提供了一种免疫原性组合物，包含百日咳抗原、白喉抗原及破伤风抗原。优选地，其包括百日咳PT抗原、百日咳FHA抗原、白喉DT抗原及破伤风TT抗原。
第二方面，本发明提供了一种复合佐剂，其由铝佐剂及TLR9受体激动剂组成。优选地，所述铝佐剂为氢氧化铝，所述TLR9受体激动剂为CpG ODN。
第三方面，本发明提供了一种复合佐剂联合疫苗，其包括第一方面的免疫原性组合物以及第二方面的复合佐剂和/或可药用载体。优选地，所述复合佐剂为氢氧化铝和CpG ODN组合物。
优选地，每剂复合佐剂联合疫苗含：百日咳PT抗原1～25μg，百日咳FHA抗原1～25μg，白喉DT抗原1～20Lf，破伤风TT抗原1～5Lf；更优选地，百日咳PT抗原为4、8、16和25μg，百日咳FHA抗原4、8、16和25μg，白喉DT抗原1、2、4和20Lf，破伤风TT抗原1.5、2.5和5Lf；最优选地，百日咳PT抗原8μg，百日咳FHA抗原8μg，白喉DT抗原2Lf，破伤风TT抗原2.5Lf。
优选地，每剂复合佐剂联合疫苗含：铝含量为0.01～1mg，CpG ODN0.1～100μg；更优选地，每剂复合佐剂联合疫苗含：铝含量0.1～0.5mg，CpG ODN 25～100μg；最优选地，每剂复合佐剂联合疫苗含：铝含量为0.24mg，CpG ODN 50μg或100μg。
第四方面，本发明提供了第二方面的复合佐剂在制备用于在受试者中预防百日咳、白喉以及破伤风的第三方面的复合佐剂联合疫苗中的用途。
优选地，所述复合佐剂联合疫苗包括免疫原性组合物和复合佐剂。
优选地，所述受试者是哺乳动物；更优选地，所述受试者是人。
本发明制备的复合佐剂联合疫苗具有以下优点：以铝佐剂及TLR9受体激动剂作为复合佐剂，能够用较低的抗原用量达到较高的免疫原性，且各组分效价均符合《中国药典》2020版相关要求。本发明在不含百日咳粘附素抗原下仍有较强的免疫原性和保护效果，可应用于预防婴幼儿、青少年及成人的百日咳、白喉及破伤风。
具体实施方式
本发明提供了一种复合佐剂联合疫苗，包含百日咳PT抗原、百日咳FHA抗原、白喉DT抗原及破伤风TT抗原，另外还包含一种复合佐剂，由铝佐剂及TLR9受体激动剂组成。所述复合佐剂联合疫苗能预防婴幼儿、青少年及成人百日咳、白喉及破伤风。
下面结合实施例，进一步阐述本发明，但本发明并不限于这些实施例：
实施例1：复合佐剂联合疫苗中佐剂剂量的摸索
1)将百日咳(PT抗原和FHA抗原)原液(按照公开号CN103242434B专利实施例1、2、4和5制备)和精制白喉类毒素原液(发酵参考：Cox,J C.New method for the large-scale preparation of diphtheria toxoid:purification of toxin[J].Applied Microbiology,1975,29(4):464-468.制备、纯化参考：Robb L A,Stainer D W,Scholte M J,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.制备、脱毒参考：肖锡岭,史翠凤,马学严,等.精制白喉毒素的完全类毒化[J].中国生物制品学杂志,1990,1(3):26-29.)、精制破伤风类毒素原液(发酵及脱毒参考：卢锦漢.关于破伤风毒素及类毒素的研究[J]. 微生物学报,1953,1(1):113-126.制备、纯化参考：Ivana,Ljiljana,Dovezenskiet 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.制备)按比例分别与氢氧化铝混合、吸附，调节pH值至6.5，再加入CpG ODN 2006佐剂，混合均匀；
2)将吸附后的各组分混合，使每0.5ml剂量含百日咳PT抗原8μg，百日咳FHA抗原8μg，白喉DT抗原2Lf，破伤风TT抗原2.5Lf，铝含量0.24mg，CpG ODN 2006 25、50、100μg；具体分组：CpG ODN 200625μg组(组1)、CpG ODN 2006 50μg组(组2)、CpG ODN 2006 100μg组(组3)。
3)同时制备单Al佐剂百白破疫苗组(组4)、单CpG ODN 2006佐剂百白破疫苗组(组5)、氢氧化铝对照组(组6)、CpG ODN 2006对照组(组7)及抗原对照组(组8)。本实施例各组分抗原及佐剂的组成和剂量见表1。
表1抗原及佐剂的组成及剂量
实施例2：复合佐剂联合疫苗中佐剂剂量对保护性效价的影响研究1)百日咳效价：参照《中国药典》2020年版进行。将国家效价标准品(中国食品药品检定研究院提供)稀释至1、0.2、0.04IU/ml，与表1中制备的复合佐剂联合疫苗分别免疫试验动物。试验动物为清洁级NIH小鼠，腹腔注射0.5ml/只，免疫21天后，进行攻毒，攻毒后14天，观 察动物存活情况，并计算效价。
2)白喉效价：参照《中国药典》2020年版进行。将国家效价标准品(中国食品药品检定研究院提供)稀释至5.54、2.77、1.38、0.69IU/ml，与表1中制备的复合佐剂联合疫苗分别免疫试验动物。试验动物为清洁级NIH小鼠，皮下注射0.5ml/只，免疫后5周采血，分离血清，用Vero细胞进行毒素中和试验，并通过细胞存活情况计算白喉效价。
3)破伤风效价：参照《中国药典》2020年版进行。将国家效价标准品(中国食品药品检定研究院提供)稀释至9.64、4.82、2.41、1.205IU/ml，与表1中制备的复合佐剂联合疫苗分别免疫试验动物。试验动物为清洁级NIH小鼠，皮下注射0.5ml/只，免疫4周后进行攻毒，攻毒后5天，观察动物存活情况，并计算效价。
4)各组分保护效价见表2所示。
表2各组效价检测结果
实施例3：复合佐剂联合疫苗中抗原剂量的摸索
1)将百日咳(PT抗原和FHA抗原)原液(按照公开号CN103242434B专利实施例1、2、4和5制备)和精制白喉类毒素原液(发酵参考：Cox,J C.New method for the large-scale preparation of diphtheria toxoid:purification of toxin[J].Applied Microbiology,1975,29(4):464-468.制备、纯化参考：Robb L A,Stainer D W,Scholte M J,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.制备、脱毒参考：肖锡岭,史翠凤,马学严,等.精制白喉毒素的完全类毒化[J].中国生物制品学杂志,1990,1(3):26-29.)、精制破伤风类毒素原液(发酵及脱毒参考：卢锦漢.关于破伤风毒素及类毒素的研究[J].微生物学报,1953,1(1):113-126.制备、纯化参考：Ivana,Ljiljana,Dovezenskiet 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.制备)按比例分别与氢氧化铝混合、吸附，调节pH值至6.5，再加入CpG ODN2006佐剂，混合均匀；
2)将吸附后的各组分混合，使每剂含百日咳PT抗原4、8、16和25μg，百日咳FHA抗原4、8、16和25μg，白喉DT抗原1、2、4和20Lf，破伤风TT抗原1.5、2.5、和5Lf，铝含量0.24mg，CpG ODN 2006 50和0μg；分组为组1、组2、组3、组4和组5，各组分详细信息见表3。
本实施例各组分抗原及佐剂的组成和剂量见表3。
表3抗原及佐剂的组成及剂量
实施例4：复合佐剂联合疫苗中抗原剂量对保护性效价的影响研究
1)百日咳效价：参照《中国药典》2020年版进行。将国家效价标准品(中国食品药品检定研究院提供)稀释至1、0.2、0.04IU/ml，与表3中制备的复合佐剂联合疫苗及市售疫苗(吸附无细胞百白破灭活脊髓灰质炎和b型流感嗜血杆菌联合疫苗，购自Sanofi Pasteur S.A)分别免疫试验动物。试验动物为清洁级NIH小鼠，腹腔注射0.5ml/只，免疫21天后，进行攻毒，攻毒后14天，观察动物存活情况，并计算效价。
2)白喉效价：参照《中国药典》2020年版进行。将国家效价标准品(中国食品药品检定研究院提供)稀释至5.54、2.77、1.38、0.69IU/ml， 与表3中制备的复合佐剂联合疫苗及市售疫苗(吸附无细胞百白破灭活脊髓灰质炎和b型流感嗜血杆菌联合疫苗，购自Sanofi Pasteur S.A)分别免疫试验动物。试验动物为清洁级NIH小鼠，皮下注射0.5ml/只，免疫后5周采血，分离血清，用Vero细胞进行毒素中和试验，并通过细胞存活情况计算白喉效价。
3)破伤风效价：参照《中国药典》2020年版进行。将国家效价标准品(中国食品药品检定研究院提供)稀释至9.64、4.82、2.41、1.205IU/ml，与表3中制备的复合佐剂联合疫苗及市售疫苗(吸附无细胞百白破灭活脊髓灰质炎和b型流感嗜血杆菌联合疫苗，购自Sanofi Pasteur S.A)分别免疫试验动物。试验动物为清洁级NIH小鼠，皮下注射0.5ml/只，免疫4周后进行攻毒，攻毒后5天，观察动物存活情况，并计算效价。
4)各组分效价结果见表4。
表4各组保护效价结果
综上试验结果表明：
由实施例1、2可知，相同抗原及铝佐剂含量下，各抗原(除白喉)效价随CpG ODN 2006含量的增加呈现剂量效应，即各抗原(除白喉)效价随CpG ODN 2006含量的增加而升高，且复合佐剂存在协同效应， 即采用复合佐剂的组别(组1、2、3)效价均高于单一佐剂组别效价的加和(组4+组5)。各复合佐剂组别效价均满足2020版《中国药典》标准，即每一次人用剂量0.5mL，含无细胞百日咳疫苗效价应不低于4.0IU，白喉疫苗效价应不低于30IU，破伤风疫苗效价应不低于40IU。
由实施例3、4可知，采用复合佐剂百白破疫苗可以有效降低抗原使用量，组2中PT、FHA各为8μg，市售疫苗组PT、FHA均为25μg，其百日咳效价几乎相当，当组3中PT、FHA各为16μg时，百日咳效价显著高于市售疫苗组，由于市售疫苗说明书对白喉及破伤风抗原含量与国内定义不同，在此不做进一步比较。
由实施例1、2、3、4可知，当提高复合佐剂中CpG ODN 2006浓度在50～100μg/剂时，其百日咳效价等同或显著高于市售疫苗组。
因此，本发明通过使用氢氧化铝-CpG ODN 2006复合佐剂，在不含百日咳粘附素抗原下，使百日咳、白喉及破伤风效价随剂量增加而升高，与单独使用铝佐剂或CpG ODN 2006佐剂相比，效价相当的情况下，可以减少抗原剂量，可应用于预防婴幼儿、青少年及成人的百日咳、白喉及破伤风。

Claims (10)

  1. 一种复合佐剂联合疫苗，其中所述复合佐剂联合疫苗包含一种免疫原性组合物和一种复合佐剂和/或可药用载体，其中所述免疫原性组合物包括百日咳PT抗原、百日咳FHA抗原、白喉DT抗原及破伤风TT抗原，其中所述复合佐剂由铝佐剂及TLR9受体激动剂组成。
  2. 根据权利要求1所述的复合佐剂联合疫苗，其中每剂所述复合佐剂联合疫苗含：所述百日咳PT抗原1～25μg，所述百日咳FHA抗原1～25μg，所述白喉DT抗原1～20Lf，所述破伤风TT抗原1～5Lf。
  3. 根据权利要求2所述的复合佐剂联合疫苗，其中每剂所述复合佐剂联合疫苗含：所述百日咳PT抗原为4、8、16和25μg，所述百日咳FHA抗原4、8、16和25μg，所述白喉DT抗原1、2、4和20Lf，所述破伤风TT抗原1.5、2.5和5Lf。
  4. 根据权利要求3所述的复合佐剂联合疫苗，其中每剂所述复合佐剂联合疫苗含：所述百日咳PT抗原8μg，所述百日咳FHA抗原8μg，所述白喉DT抗原2Lf，所述破伤风TT抗原2.5Lf。
  5. 根据权利要求1-4中任一项所述的复合佐剂联合疫苗，其中所述铝佐剂为氢氧化铝，所述TLR9受体激动剂为CpG ODN。
  6. 根据权利要求5中所述的复合佐剂联合疫苗，其中所述CpG ODN为CpG ODN 2006。
  7. 根据权利要求5所述的复合佐剂联合疫苗，其中每剂所述复合佐剂联合疫苗含：铝含量0.01～1mg，CpG ODN 0.1～100μg。
  8. 根据权利要求7所述的复合佐剂联合疫苗，其中每剂所述复合佐剂联合疫苗含：铝含量0.1～0.5mg，CpG ODN 25～100μg；优选地，每剂复合佐剂联合疫苗含：铝含量为0.24mg，CpG ODN 50μg或100μg。
  9. 一种复合佐剂在制备用于在受试者中预防百日咳、白喉以及破伤风的权利要求1-8中任一项所述的复合佐剂联合疫苗中的用途，所述复合佐剂由铝佐剂及TLR9受体激动剂组成。
  10. 根据权利要求9所述的用途，其中所述受试者为哺乳动物，更优选地，所述受试者是人。
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