WO2020259557A1 - 一种复合佐剂系统及制备该佐剂的方法 - Google Patents

一种复合佐剂系统及制备该佐剂的方法 Download PDF

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WO2020259557A1
WO2020259557A1 PCT/CN2020/098012 CN2020098012W WO2020259557A1 WO 2020259557 A1 WO2020259557 A1 WO 2020259557A1 CN 2020098012 W CN2020098012 W CN 2020098012W WO 2020259557 A1 WO2020259557 A1 WO 2020259557A1
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adjuvant
cpg
aluminum phosphate
aluminum
antigen
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PCT/CN2020/098012
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English (en)
French (fr)
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刁美君
魏健
史力
莫呈钧
张智
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怡道生物科技(苏州)有限公司
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Priority to US17/623,489 priority Critical patent/US20220331424A1/en
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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
    • A61K39/39—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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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

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  • the invention belongs to the technical field of medicine, and specifically relates to a new adjuvant system containing aluminum phosphate adjuvant and CpG.
  • Aluminum salt adjuvant is low cost and convenient to use. It is the most widely used adjuvant in the production of biological products. It is also the first adjuvant approved by the US Food and Drug Administration (FDA) for use in human vaccines. At present, the commonly used aluminum adjuvants are mainly aluminum hydroxide adjuvant and aluminum phosphate adjuvant. Aluminum salt has many advantages as a vaccine adjuvant. In addition to enhancing the immune response, it also has very solid safety data. But although it can effectively induce humoral immune response, it has no obvious effect on cellular immunity, and it is difficult to induce cellular immune response.
  • FDA US Food and Drug Administration
  • the isoelectric point and chemical physical structure of aluminum hydroxide adjuvant and aluminum phosphate adjuvant are different, the advantages and adsorption affinity of different vaccine antigens are also different. Some antigens are more suitable for aluminum hydroxide adjuvant, and some antigens are more suitable for aluminum phosphate adjuvant. In the aluminum phosphate adjuvant family, the content of phosphate is also different, and the corresponding isoelectric point is different.
  • CpG ODN adjuvant is one of the most interesting vaccine adjuvant technologies in the future.
  • Vaccines containing CpG ODN adjuvants from Coley and Dynavax in the United States have entered Phase III clinical trials and have been approved.
  • CpG OND is a short sequence of unmethylated short nucleotides (cytosine and guanine, CpG) as motifs of oligodeoxynucleotides (ODN), and is a strong immunostimulatory factor for humans and animals discovered so far.
  • CpG OND can not only enhance the humoral immune response of the vaccine, but also greatly enhance its cellular immune response, which is not achieved by aluminum adjuvants.
  • CpG is a new type of adjuvant that has been clinically proven to be safe and effective. It can enhance the immunogenicity of vaccines, increase the immune response rate, and stimulate the body to form more effective immune protection. It is one of the good adjuvant options for the development of new vaccines.
  • Aluminum hydroxide adjuvant exists in the form of aluminum hydroxyl, which is a fibrous particle with an isoelectric point of about 10-11. It exists in the form of positively charged micron particles in a solution of neutral pH, which can be adsorbed well Negatively charged CpG and negatively charged antigen; aluminum phosphate adjuvant is a complex of aluminum hydroxyphosphate, which is an aggregate of spherical particles.
  • the isoelectric point of commercial aluminum phosphate adjuvants is mostly around 4-5.
  • the neutral pH (pH6-7.4) solution exists in the form of negatively charged micron particles, which is not conducive to the adsorption of negatively charged CpG.
  • the prior art provides some solutions.
  • the prepared aluminum phosphate particle size can be effectively controlled between 7.5-15nm and the isoelectric point between 4.0-6.0, which can realize the surface charge of aluminum phosphate adjuvant And the particle size tends to be stable and the pH value is closer to the pH value of human body fluids.
  • Vaccine preparations containing adjuvants are all adjuvant-adsorbed antigenic vaccines (antigens in the vaccine are adsorbed on the adjuvant).
  • adjuvants due to the existence of adjuvants, the testing of some vaccine quality verification items will be interfered with or even impossible to carry out. Therefore, in actual operation, it is necessary to provide an adjuvant system with good desorption effect, and it is also necessary to free the antigen adsorbed on the adjuvant in a certain way for detection and analysis.
  • Chinese Patent 103471902A provides a method for desorption of vaccines containing aluminum phosphate adjuvants, stating that the vaccine containing adjuvants should be dissolved in an alkaline solution, and then neutralized by acidic chemical reagents containing metal complexing agents. . This method is very easy to cause the degradation of the antigen and lacks feasibility.
  • the patent CN1572324A improves the adsorption method of the adjuvant system, pointing out that for the adjuvant system containing aluminum adjuvant and CpG adjuvant, the antigen should only be adsorbed on the aluminum adjuvant, and it is not suitable to be adsorbed on the CpG adjuvant ( Immunostimulant), which is more conducive to achieving quality control requirements.
  • the antigen is hundreds of times the size of CpG, theoretically there is no possibility of antigen adsorption on CpG.
  • the present invention provides a new adjuvant system, including aluminum phosphate adjuvant and CpG adjuvant.
  • the new adjuvant system adjusts the isoelectric point of aluminum adjuvant and controls the adjuvant by carefully adjusting the ratio of phosphate to aluminum ion. Surface charge improves the adsorption rate of antigen and CpG.
  • the present invention also provides an optimized desorption method, which can give higher antigen desorption efficiency, provides a basis for subsequent antigen activity evaluation in vitro, and has good commercial value.
  • the present invention provides an improved adjuvant system containing aluminum phosphate adjuvant.
  • the inventor of the present invention unexpectedly discovered that, unlike traditional commercial options, if an aluminum phosphate adjuvant with an isoelectric point of 7.0-9.0 instead of 4.0-6.0 is selected, the antigen and CpG adjuvant can be adsorbed well.
  • Aluminum phosphate adjuvant if an aluminum phosphate adjuvant with an isoelectric point of 7.0-9.0 instead of 4.0-6.0 is selected, the antigen and CpG adjuvant can be adsorbed well.
  • Aluminum phosphate adjuvant isoelectric point of 7.0-9.0 instead of 4.0-6.0
  • the present invention finds that by matching the CpG adjuvant and the specially selected aluminum phosphate adjuvant, a corresponding compatible adjuvant system is obtained.
  • the adjuvant system has a better ability to adsorb antigen and CpG adjuvant, and also allows Antigen and CpG are effectively desorbed to obtain an antigen solution, which is beneficial for vaccine preparation, antigen content and activity evaluation.
  • the present invention found that when the ratio of aluminum element to phosphoric acid in aluminum phosphate increases, the adsorption strength of CpG increases linearly.
  • the present invention further focuses on when the mass ratio between the aluminum element content and the CpG content in the aluminum phosphate adjuvant is between 1:4-4:1, it is found that the CpG adjuvant can be better adsorbed on the aluminum phosphate adjuvant, and at the same time Antigen also has a good desorption effect. More preferably, when the mass ratio between aluminum element and CpG in the aluminum phosphate adjuvant is between 1:2-2:1, both CpG and the antigen achieve the best adsorption on the aluminum phosphate adjuvant.
  • the CpG adjuvant mentioned in the present invention refers to a DNA fragment containing an unmethylated cytosine-guanine dinucleotide (CpG) motif. It can activate B cells, NK, DC dendritic cells and induce the release of IL-12 and IFNr, thereby inducing a strong Th1-type response and cellular immunity.
  • an ideal CpG adjuvant can be CpG7909 (Coley) or CpG 1018ISS (Dynavax), etc.
  • the present invention obtains a series of aluminum phosphate adjuvants whose isoelectric point is greater than the isoelectric point of commercial aluminum phosphate adjuvant (isoelectric point is 5.0) by adjusting the ratio of soluble aluminum salt, soluble phosphate and alkaline solution. More preferably, the aluminum phosphate adjuvant prepared by the inventor of the present invention has an isoelectric point between 7.0 and 9.0. Preferably it is between 8.0-9.0. Most preferably, the aluminum phosphate adjuvant prepared by the inventor of the present invention has an isoelectric point between 8.5 and 9.0. On this basis, the inventor of the present invention provides a composite adjuvant system containing aluminum phosphate adjuvant.
  • the method for obtaining aluminum phosphate adjuvant with an isoelectric point between 7.0 and 9.0 provided by the present invention is:
  • the above-mentioned soluble aluminum salt is AlCl 3
  • the soluble phosphate is NaH 2 PO 4 , Na 2 HPO 4 or Na 3 PO 4
  • the alkaline solution is NaOH
  • the pH is 7-12.
  • the aluminum phosphate adjuvant adsorbs 85-100% vaccine antigen and 50-100% CpG adjuvant. More preferably, the aluminum phosphate adjuvant adsorbs 85-100% vaccine antigen in the adjuvant system. And 80-100% CpG adjuvant.
  • the present invention creates an optimized desorption method, which includes the following steps:
  • desorption solutions include sodium phosphate, sodium citrate, sodium chloride, and Tween 80.
  • the mass ratio between the desorption solution and the desorption sample is greater than 1:1.
  • the mass ratio between the desorption solution and the desorption sample is greater than 1:1.
  • the aforementioned desorption solution may contain 160mM-220mM sodium phosphate, 0.2-0.6M sodium citrate, 0.15-2M sodium chloride and 0.01-0.4% PS-80, and the pH is 6-7.
  • the novel adjuvant system provided by the present invention has better adsorption and desorption capabilities (detailed in the examples). Therefore, the adjuvant of the present invention has a wider application field and a better vaccine immunity effect.
  • Figure 1 shows the isoelectric points of aluminum phosphate adjuvants with different P/Al, where ⁇ represents the PI value of group A in Table 1; ⁇ represents the PI value of group B in Table 1; Indicates the PI value of group C in Table 1.
  • Al means aluminum
  • PI means isoelectric point
  • P/Al means the ratio of phosphorus content to aluminum content in aluminum phosphate.
  • Figure 2 shows the adsorption curves of Al adjuvants with different isoelectric points on CpG.
  • ⁇ represents the adsorption effect of AP (aluminum phosphate, isoelectric point of 9.0) prepared in the manner shown in Table 2 on CpG;
  • ⁇ represents the effect of AP (isoelectric point of 7.7) formulated in the manner shown in Table 2 on CpG
  • the adsorption effect of CpG It shows the adsorption effect of AP (isoelectric point 5.1) formulated in the manner shown in Table 2 on CpG.
  • Figure 3 shows the Western-blot detection of gE content in the supernatant after Al adsorbed gE antigen protein and CpG.
  • Figure 4 shows the adsorption rate of CpG after different concentrations of CpG are adsorbed by 100 ⁇ g gE/600 ⁇ g Al/mL sample.
  • Figure 5 shows the desorption recovery rates of gE and CpG adsorbed by the AH adjuvant under the action of different desorption solutions (desorption solutions A-E) after desorption for 2, 24 and 48 hours.
  • Figure 6 shows the effect of PS-80 on the desorption recovery rate of gE and CpG adsorbed by AH adjuvant after desorption for 3 hours.
  • Figure 7 shows the comparison of the desorption effects of different aluminum adjuvants (AP, AH) adsorbing gE samples.
  • the purpose of the present invention is to prepare aluminum phosphate (AP) adjuvant with an isoelectric point of 7.0 or higher by adjusting the ratio of the reaction solution, which is used to form a new adjuvant combination of aluminum phosphate and CpG with high adsorption rate, aiming to change the commercialization
  • Aluminum phosphate has a weak adsorption effect on CpG, which changes the current situation where only aluminum hydroxide can be used to adsorb CpG.
  • the inventor of the present application found that the adjuvant system of the present invention has a better ability to desorb antigen than the adjuvant system of aluminum hydroxide adjuvant for adsorbing CpG.
  • a new adjuvant system desorption method for aluminum phosphate and CpG with high adsorption rate is provided in the later research, which is more conducive to the preparation and evaluation in the field of vaccines, and has a broad application prospect.
  • reaction solution 1 a mixed solution of aluminum chloride and sodium dihydrogen phosphate
  • reaction solution 2 sodium hydroxide solution
  • the reactor is on a magnetic stirrer, stirring at a constant speed.
  • the suspension was collected, and after standing, the supernatant was discarded, and centrifuged at 8000 rpm for 15 min to obtain a precipitate. Disperse the precipitate in a certain volume of pure water to obtain an aluminum phosphate adjuvant solution with a certain concentration range.
  • the prepared aluminum phosphate suspension was sterilized by moist heat at 121°C for 30 min. Then store at 4°C. Determine the P/Al ratio and isoelectric point of the reaction product. Adjusting the composition ratio of PO 4 3- /Al 3+ in reaction solution 1 and the added volume of NaOH in reaction solution 2 to prepare a series of aluminum phosphate adjuvants with an isoelectric point (PI) value ranging from 4.5 to 9.2. The results are shown in Figure 1.
  • the adsorption strength of CpG is related to the isoelectric point of the aluminum adjuvant (AP) and the ratio of CpG to Al.
  • the common aluminum phosphate adjuvant (isoelectric point of about 5.0) in the prior art has an unsatisfactory adsorption effect on CpG.
  • the isoelectric point is above 7.0
  • the adsorption effect of aluminum phosphate adjuvant on CpG adjuvant has been greatly improved (several times), which has unexpected technical effects, and is compared with the commonly used aluminum hydroxide adjuvant. quite.
  • the aluminum phosphate adsorption rate of CpG in the improved adjuvant system provided by the present invention has a better and unexpected effect.
  • the adsorption of aluminum phosphate (AP) adjuvants with PI of 7.7, 8.5, 9.0 to CpG and gE antigens firstly adsorb the gE antigen stock solution onto the aluminum adjuvant (AP) to prepare gE antigen/Al(w/w) Adsorption sample with a ratio of 200 ⁇ g/1200 ⁇ g/ml; then add CpG samples of different concentrations to the gE antigen/Al adsorption sample: 1) 0 ⁇ g/ml, 2) 100 ⁇ g/ml, 3) 200 ⁇ g/ml, 4) 300 ⁇ g/ ml, 5) 500 ⁇ g/ml, 6) 600 ⁇ g/ml, 7) 900 ⁇ g/ml, 8) 1200 ⁇ g/ml, after adsorbing for 24h, centrifuge to take the supernatant, and calculate CpG and gE by westen-blot and UV second-order derivative respectively The adsorption effect.
  • AP aluminum phosphat
  • the aluminum phosphate adjuvant system provided by the present invention should have an adsorption rate of over 90% for gE antigen in the presence of CpG, and the increase in CpG content will not reduce the adsorption rate of aluminum adjuvant for gE antigen.
  • the amount of gE antigen protein in the supernatant of the sample after adsorption is less than 10 ⁇ g/ml, so the UV absorption value of the supernatant basically reflects the amount of CpG.
  • the adsorption curve of the CpG adjuvant with the gE antigen stock solution adsorbed by the UV second-order method is shown in Figure 4. It can be seen from Figure 4 that the aluminum phosphate (isoelectric point between 7.0-9.2) provided by the present invention and the CpG dual adjuvant system have good antigen adsorption capacity, and the increase in CpG content will not cause aluminum adjuvants The adsorption rate of gE antigen is reduced.
  • gE/Al Al element in aluminum adjuvant
  • w/w AH adjuvant
  • adsorption sample with a ratio of 200 ⁇ g/1200 ⁇ g/ml, under the desorption conditions as shown in Table 3, add desorption at a ratio of 1:3 liquid.
  • the supernatant was desorbed at 25 degrees Celsius for 2h, 24h, and 48h to detect the gE content of the second-order UV derivative and calculate the recovery rate. The results are shown in Figure 5.
  • the surfactant has a protective effect on the desorbed gE antigen.

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Abstract

本发明公开了一种包含磷酸铝佐剂及CpG佐剂的新佐剂系统及其制备方法。

Description

一种复合佐剂系统及制备该佐剂的方法 技术领域
本发明属于医药技术领域，具体涉及一种包含磷酸铝佐剂及CpG的新佐剂系统。
背景技术
近年来，随着免疫学研究的不断深入及基因工程技术的迅速发展，活载体疫苗、DNA疫苗和蛋白质疫苗等新型疫苗的研究取得了可喜的进步。这些新型疫苗纯度高、特异性强，但免疫原性弱，诱导机体产生的免疫应答不够强，因此，应用佐剂来增强其免疫原性或增强宿主对抗原的保护性应答就显得尤为重要。
铝盐佐剂成本低、使用方便，是生物制品生产中应用最广的一种佐剂，也是最早被美国食品药物监督管理局(FDA)批准用于人类疫苗的佐剂。目前常用的铝佐剂主要为氢氧化铝佐剂(aluminum hydroxide adjuvant)和磷酸铝佐剂(aluminum phosphate)两种。铝盐作为疫苗佐剂有很多优点，除了增强免疫应答，还具有非常坚实的安全性数据。但它虽然可以有效地诱导体液免疫应答，但是对细胞免疫无明显作用，难以诱导细胞免疫应答。由于氢氧化铝佐剂和磷酸铝佐剂的等电点和化学物理结构不一样，和不同疫苗抗原的复配优势和吸附亲和率也不一样。有些抗原更适合氢氧化铝佐剂，有些抗原更适合磷酸铝佐剂。就磷酸铝佐剂家族内也以磷酸根的含量不同，而有相应的等电点不同。
新型CpG ODN佐剂是未来最具看点的疫苗佐剂技术之一，美国的Coley公司和美国Dynavax公司的含CpG ODN佐剂的疫苗已分别进入三期临床和被批上是。CpG OND为非甲基化的短核苷酸(胞嘧啶和鸟嘌呤，CpG)为基序的寡脱氧核苷酸(ODN)短序列，是迄今发现的人和动物的强烈免疫刺激因子。CpG OND不仅可增强疫苗的体液免疫应答，尤其是可大大增强其细胞免疫应答，后者正是铝佐剂所达不到的。目前CpG是已被临床证明安全有效的新型佐剂，能够增强疫苗的免疫原性，提高免疫应答率，刺激机体形成更有效的免疫保护，是开发新型疫苗的良好佐剂选择之一。
调研目前相关科研文献发现CpG与铝佐剂联用，大多为氢氧化铝 佐剂而少有涉及磷酸铝佐剂。氢氧化铝佐剂以铝羟基形式存在的，是一种纤维状粒子，等电点一般在10～11左右，在中性pH的溶液中以带正电荷微米颗粒形式存在，可较好的吸附带负电荷的CpG以及带负电的抗原；磷酸铝佐剂是羟基磷酸铝复合物，为一种球形粒子的聚集体，商品化的磷酸铝佐剂的等电点多是4-5左右，在中性pH(pH6-7.4)的溶液中以带负电荷微米颗粒形式存在，不利于带负电荷的CpG的吸附。为了解决磷酸铝佐剂吸附率较差的问题，现有技术给出了一些解决方案，例如在中国专利CN106729702中，对现有磷酸铝佐剂配制工艺和后处理工艺进行了探索改良，以获得吸附能力和外观形态均良好的磷酸铝佐剂，制备得到的磷酸铝粒径可以有效的控制在7.5-15nm之间而等电点控制在4.0-6.0之间，可以实现磷酸铝佐剂表面电荷和粒径趋于稳定且pH值更加接近于人体体液pH值。
含有佐剂的疫苗制剂均为佐剂吸附抗原性型疫苗(疫苗中的抗原吸附于佐剂上)。而由于佐剂的存在，一些疫苗质量检定项目的检测会受到干扰，甚至无法开展。因此，实际操作中既需要提供一种具有较好解吸附效果的佐剂系统，又需要将吸附于佐剂上的抗原通过一定方式游离下来用于检测和分析。例如，中国专利103471902A中给出了对包含磷酸铝佐剂的疫苗的解吸附方式，指出应以碱溶液将含有佐剂的疫苗溶解，再采用含金属络合剂的酸性化学试剂中和的方法。这种方法极易造成抗原的降解，缺乏可行性。而专利CN1572324A则对佐剂系统的吸附方式给出了改进，指出对于包含铝佐剂及CpG佐剂的佐剂系统，抗原应仅吸附在铝佐剂上，而不宜吸附在CpG佐剂上(免疫刺激剂)，这样更有利于达到质量控制的要求。而事实上抗原是CpG的几百倍大小，理论上不存在抗原在CpG上吸附的任何可能。
本发明提供了一种新的佐剂系统，包括磷酸铝佐剂以及CpG佐剂，该新佐剂系统通过仔细调节磷酸根和铝离子的比例而实现调节铝佐剂等电点，控制佐剂表面电荷，提高抗原和CpG吸附率.本发明还提供了一种优化的解吸方法，可以给出较高的抗原解吸附效率，为后续抗原的体外活性评价提供基础，具有良好的商业价值。
发明内容
本发明给出了一种包含磷酸铝佐剂的改良型佐剂系统。本发明的 发明人出乎意料地发现，与传统商用选择不同，若选择等电点为7.0-9.0而非4.0-6.0左右的磷酸铝佐剂，将使得抗原和CpG佐剂能良好地吸附在磷酸铝佐剂上。
本发明发现，通过将CpG佐剂和特殊选出的磷酸铝佐剂符合使用，获得的相应的符合佐剂系统，该佐剂系统具有较好的吸附抗原和CpG佐剂的能力，也同时允许抗原和CpG被有效地解吸附，获得抗原溶液，有利疫苗的制备，抗原含量及活性等评价。其中，本发明发现，当磷酸铝中铝元素和磷酸的比值升高时，CpG的吸附强度会线性增加，这是因为在相同的中性pH环境下，随着磷酸铝中铝元素和磷酸的比值升高，磷酸铝的等电点也在线性升高，导致磷酸铝的表面电荷阳性增强。另一方面，CpG和抗原的吸附都将随着铝佐剂含量(以佐剂中铝元素含量计算)升高而加强。本发明进一步聚焦在磷酸铝佐剂中铝元素含量及CpG含量之间的质量比在1∶4-4∶1之间时，发现CpG佐剂可以较好地吸附在磷酸铝佐剂上，同时抗原也具有较好的解吸附效果。更优选地，磷酸铝佐剂中铝元素及CpG之间的质量比在1∶2-2∶1之间时，CpG和抗原在磷酸铝佐剂上都达到了最佳吸附。
本发明中所指出的CpG佐剂，是指含非甲基化胞嘧啶-鸟嘌呤二核苷酸(CpG)基序的DNA片段。它能激活B细胞、NK、DC树突状等细胞，诱导释放IL-12、IFNr，从而诱导强Th1型应答和细胞免疫。作为举例，理想的CpG佐剂可以是CpG7909(Coley公司)或CpG 1018ISS(Dynavax公司)等。
本发明通过调节可溶性铝盐、可溶性磷酸盐与碱性溶液三者的比例，获得了一系列等电点大于商品化磷酸铝佐剂等电点(等电点为5.0)的磷酸铝佐剂。更优选地，本发明的发明人制备得的磷酸铝佐剂，等电点在7.0-9.0之间。优选地为8.0-9.0之间。最优选地，本发明的发明人制备得的磷酸铝佐剂，等电点在8.5-9.0之间。在此基础上，本发明的发明人给出了一种包含磷酸铝佐剂的复合型佐剂系统，本发明所提供的获得等电点在7.0-9.0之间磷酸铝佐剂的方法为：
1)将可溶性铝盐(优选为AlCl 3)、可溶性磷酸盐(NaH 2PO 4，Na 2HPO 4或Na 3PO 4)混合，制备得到溶液1，其中PO 4 3-与Al 3+之间摩尔比在1∶3-1∶10之间；
2)将溶液1与碱溶液(优选为NaOH)混合，在预定pH条件下沉 淀来制备磷酸铝佐剂，其中PO 4 3-与Al 3+之间的摩尔比在1∶3-1∶10之间；为保证衡定pH，在使用NaH 2PO 4的情况下，PO 4 3-与OH-摩尔比在3∶5.56-10∶22.7之间.
3)吸附抗原和CpG佐剂，形成本发明中的复合型佐剂系统的疫苗产品；或者将直接与带负电荷的CpG佐剂合用，形成复合佐剂系统。关于上述方法，另一种表述方式为：
1.将可溶性铝盐、可溶性磷酸盐与不同浓度碱性溶液相混合，在；
2.通过调节反应溶液中可溶性铝盐和可溶性磷酸盐的配比，从而得到一系列不同等电点的磷酸铝佐剂；和
3.带负电荷的CpG佐剂合用，形成复合佐剂系统。
其中，上述可溶性铝盐为AlCl 3，可溶性磷酸盐为NaH 2PO 4，Na 2HPO 4或Na 3PO 4，所述碱性溶液为NaOH，所述pH值为7-12。
在本发明所提供的佐剂系统中，磷酸铝佐剂吸附85-100％疫苗抗原和50-100％CpG佐剂，更优选地，佐剂系统中磷酸铝佐剂吸附85-100％疫苗抗原和80-100％CpG佐剂。为了达到疫苗中抗原质量检定的目的，同时避免检测受到干扰，本发明创建了一种优化的解吸附方法，包括如下步骤：
1)将吸附了抗原的前述佐剂系统与解吸附液混合后温和地扰动或振荡1小时以上；
4)直接得清溶液即得解析附下来的抗原溶液。或适当离心后获得上清溶液以分离可能残存的铝佐剂颗粒。其中，优选的解吸附液包含磷酸钠、枸橼酸钠、氯化钠以及吐温80。解吸附液与解吸附样品(即吸附了抗原的佐剂系统)之间的质量比例大于1∶1。本领域技术人员可以理解的是，解吸附液体积越大，则解吸附效果将更好，但是考虑到陈本等因素，优选地为1∶1-1∶3之间。上述解吸附液可包含160mM-220mM的磷酸钠，0.2-0.6M的枸橼酸钠，0.15-2M的氯化钠以及0.01-0.4％PS-80，且pH为6-7。
本发明所提供的新型佐剂体系，与现有商用佐剂(尤其是CpG联合氢氧化铝佐剂)相比，具有更好的吸附及解吸附能力(实施例中将详述)。从而使得本发明的佐剂具有更广泛的运用领域及更优秀的疫苗免疫效果。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而 言是显而易见的。
附图说明
图1所示为不同P/Al的磷酸铝佐剂的等电点，其中，●表示表1中A组的PI值；■表示表1中B组的PI值；
Figure PCTCN2020098012-appb-000001
表示表1中C组的PI值。需要说明的是，Al表示铝；PI表示等电点；P/Al表示磷酸铝中磷含量与铝含量的比例。
图2所示为不同等电点的Al佐剂对CpG的吸附曲线。其中，■表示以表2中所示方式配制的AP(磷酸铝，等电点为9.0)对CpG的吸附效果；▲表示以表2中所示方式配制的AP(等电点为7.7)对CpG的吸附效果；
Figure PCTCN2020098012-appb-000002
表示以表2中所示方式配制的AP(等电点为5.1)对CpG的吸附效果。
图3所示为Western-blot检测Al吸附gE抗原蛋白、CpG后上清液中的gE含量。其中左图从左至右样品依次为：AP(PI＝9.0，1-5道，对应不同比例)、AH(氢氧化铝，6-10道，对应不同比例)、标记(Marker)、阳性对照。右图从左至右样品依次为：标记、AP(PI＝7.7，1-5道，对应不同比例)1、AH(氢氧化铝，6-10道，对应不同比例)、标记(Marker)、阳性对照。
图4所示为不同浓度的CpG被100μg的gE/600μg的Al/mL样品吸附后，CpG的吸附率。其中，▲表示AP(PI＝8.5)；■表示AP(PI＝7.7)。
图5所示为AH佐剂吸附的gE、CpG在不同解吸附液(解吸附液A-E)的作用下，在解吸附2小时、24小时以及48小时后的解吸附回收率。
图6所示为PS-80对AH佐剂吸附的gE、CpG在解吸附3小时后的解吸附回收率的影响。
图7所示为不同铝佐剂(AP、AH)吸附gE样品的解吸附效果对比。
具体实施方式
本发明的目的：通过调节反应液的比例，制备等电点为7.0以上的磷酸铝(AP)佐剂，应用于形成高吸附率的磷酸铝和CpG的新佐剂组合， 旨在改变商品化磷酸铝对CpG的弱吸附效果，从而改变目前只能使用氢氧化铝吸附CpG的现状。同时，本申请的发明人发现，相较氢氧化铝佐剂吸附CpG该佐剂系统，本发明的佐剂系统具有更优异的解吸附抗原能力。同时在后期研究中提供了一种针对高吸附率的磷酸铝和CpG的新佐剂系统的解吸附方法，更有利于，更有利疫苗领域中的制备及评价，具有广泛的应用前景。
实施例1高吸附率的磷酸铝和CpG的新佐剂组合的制备
1.不同等电点的磷酸铝佐剂的获得
分别采用蠕动泵将反应液1(氯化铝与磷酸二氢钠的混合溶液)和反应液2(氢氧化钠溶液)以一定的比例泵入容积为600mL的玻璃反应器中。调节PO 4 3-/Al 3+的组分比例以及NaOH的加入体积，具体信息见下表1。反应器至于磁力搅拌器上，以恒定的速度搅拌。收集混悬液，静置后，弃去上清夜，8000rpm离心15min，得沉淀。将沉淀分散于一定体积纯水中，即得某浓度范围的磷酸铝佐剂溶液。
表1不同等电点的磷酸铝佐剂反应信息汇总表
Figure PCTCN2020098012-appb-000003
将制备的磷酸铝悬浮液进行121℃、30min湿热灭菌。然后4℃条件下保存。测定反应产物的P/Al比以及等电点。调节反应液1中PO 4 3-/Al 3+的组分比例以及反应液2中NaOH的加入体积，制备得到一系列等电点(PI)值范围在4.5～9.2的磷酸铝佐剂。结果见图1。
从图1中可知，当反应液1中PO 4 3-与Al 3+之间摩尔比在1∶3-1∶10之间；而反应液1与反应液2中，PO 4 3-与Al 3+与NaOH溶液之间的摩尔比在1∶3∶5.56-1∶10∶22.7之间时，制备得到的磷酸铝佐剂等电点在7.0以上。且进一步以下实施例的进一步研究可知，通过上述等电点在7.0以上磷酸铝佐剂制备得到的磷酸铝及CpG佐剂系统，具有良好的吸附 效果及解吸附效果。
以下，将选择等电点(PI)分别为9.0、8.5、7.7、5.1的四种磷酸铝佐剂(AP)为代表，进行后续吸附、解吸附研究。需要说明的是，当等电点(PI)分别为9.0、8.5、7.7、5.1时，上述四种磷酸铝佐剂(AP)在制备时，P元素和Al元素之间的质量百分比分为：0.192、0.235、0.495以及0.526。本次研究所用商品化AH的PI(等电点)值为11.0。
2.等电点不同的磷酸铝(AP)佐剂与CpG的吸附作用
选择PI分别为5.1、7.7、8.5、9.0的磷酸铝(AP)佐剂，研究这些佐剂与CpG的吸附作用，具体方式如下：将CpG冻干粉使用缓冲液溶解稀释至200μg/ml；各铝佐剂自800μg/ml对倍梯度稀释，直至50μg/ml。各铝佐剂梯度稀释样品与CpG稀释液等体积对加，CpG佐剂与铝佐剂中铝元素的质量比如表2所示。随后，将上述溶液吸附4h，离心取上清，通过UV-MCA法计算CpG吸附率，结果见图2。
表2 CpG与铝佐剂(PI分别为5.1、7.7、8.5、9.0的AP)阶梯溶液配制比
Figure PCTCN2020098012-appb-000004
由图2可知，对CpG的吸附强弱与铝佐剂(AP)的等电点以及CpG与Al的比例有关。从图2可知，现有技术中常见的磷酸铝佐剂(等电点在5.0左右)对CpG的吸附效果不理想。而当等电点在7.0以上时，磷酸铝佐剂对CpG佐剂的吸附效果又来了大幅度(几十倍)的提升，具有出乎预料的技术效果，与常用的氢氧化铝佐剂相当。且更进一步地，当等电点在8.5-9.0之间时，本发明所提供的改良型佐剂系统中磷酸铝对CpG的吸附率具有更好的出乎意料的效果。
由此可知，通过调节反应中PO 4 3-/Al 3+的组分比例以及NaOH的加入体积，制备得到PI不同磷酸铝佐剂。当PI＞7.7时AP铝佐剂，对CpG的吸附效果较好，可替代氢氧化铝佐剂，形成新型磷酸铝-CpG佐剂组合。
实施例2磷酸铝(AP)与CpG佐剂应用于gE免疫疫苗
PI为7.7、8.5、9.0的磷酸铝(AP)佐剂与CpG、gE抗原的吸附作用：先将gE抗原原液吸附到铝佐剂(AP)上，制备成gE抗原/Al(w/w)比例为200μg/1200μg/ml的吸附样品；然后向该gE抗原/Al吸附样品中加入不同浓度的CpG样品：1)0μg/ml、2)100μg/ml、3)200μg/ml、4)300μg/ml、5)500μg/ml，6)600μg/ml，7)900μg/ml，8)1200μg/ml，吸附24h后，离心取上清，通过westen-blot、及UV二阶导分别计算CpG、gE的吸附效果。
由实验结果图3可知，PI大于7.7的磷酸铝佐剂系统吸附抗原后，制剂中吸附后游离gE抗原浓度＜10μg/ul，而gE抗原的总蛋白浓度为100μg/μl。由此可知，本发明所提供的磷酸铝佐剂系统在CpG存在条件下对gE抗原的吸附率应该超过90％，CpG含量的增加不会使铝佐剂对gE抗原吸附率降低。
根据WB结果，吸附后样品上清中gE抗原蛋白量小于10μg/ml，因此上清的UV吸收值基本反应CpG量。通过UV二阶导方法测得吸附有gE抗原原液对CpG佐剂的吸附曲线如图4所示。从图4中可以看出，本发明所提供的磷酸铝(等电点在7.0-9.2之间)与CpG双佐剂系统对抗原的吸附能力良好，且CpG含量的增加不会使铝佐剂对gE抗原吸附率降低。
实施例3的抗原解吸附方法
1)解吸附液种类的筛选。
gE/Al(铝佐剂中的Al元素)(w/w)(AH佐剂)比例为200μg/1200μg/ml的吸附样品，在解吸附条件如下表3，按照1∶3的比例加入解吸附液。25摄氏度解吸附2h、24h、48h离心上清检测UV二阶导gE含量，计算回收率，结果如图5所示。
CpG/Al(w/w)(AH佐剂)比例为600μg/1200μg/ml的吸附样品，按照1∶3的比例加入解吸附液。25摄氏度解吸附2h，24h，48h离心后上清检测UV二阶导CpG的含量，计算回收率，结果如图5所示。
通过图5的数据可知，解吸附液E对gE蛋白及CpG佐剂均具有良好的解吸附效果。
表3不同解吸附液组分的筛选
Figure PCTCN2020098012-appb-000005
2)表面活性剂对解吸附后的gE抗原有保护作用。
选择在解离液A和解离液E的组分中加入0.4％的PS-80，以及含有Triton X-100表面活性剂的解离液B，重复上述佐剂解吸附实验。37摄氏度解吸附3h离心后上清检测UV二阶导CpG的含量，计算回收率，结果如图6所示。由此可知，相较常规的Triton X-100表面活性剂，PS-80表明活性剂具有更良好的保护解吸附抗原的作用。
3)考察氢氧化铝(AH)、磷酸铝(AP)佐剂在使用相同解吸附条件时的gE抗原解吸附效果。
根据上述实验，最终将解吸附确定为解吸附液E，并添加PS-80表明活性剂。在此基础上，进一步研究氢氧化铝(AH)、磷酸铝(AP)佐剂在使用相同解吸附条件时的gE抗原解吸附效果。
gE疫苗，在解吸附条件如下表4所示条件下，经过24小时解吸的 样品，可观察到含有的AP佐剂样品离心后底部只有微量残余，而含有的氢氧化铝佐剂样品离心后底部仍有大量白色固体存在。通过对比图7相同解吸附条件下不同铝佐剂的gE的解吸附率，可知氢氧化铝佐剂吸附样品的gE解吸附回收率会低于磷酸铝佐剂吸附样品的gE解吸附回收率。在吸附样品/解吸附液的解吸附比例为1∶3时解吸附效果较好。由此可知，本发明所提供的新型佐剂，与现有商用CpG联合氢氧化铝佐剂相比，具有更好的解吸附能力。
表4氢氧化铝(AH)、磷酸铝(AP，PI＝7.0、8.5或9.0)佐剂吸附gE样品解吸附条件
Figure PCTCN2020098012-appb-000006
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种复合佐剂系统，其包含磷酸铝佐剂和CpG佐剂。
  2. 如权利要求1所述的佐剂系统，其特征在于，所述佐剂系统中磷酸铝佐剂吸附疫苗抗原和CpG佐剂。
  3. 如权利要求1所述的佐剂系统，其特征在于，所述佐剂系统中磷酸铝佐剂吸附85-100％疫苗抗原和50-100％CpG佐剂。
  4. 如权利要求3所述的佐剂系统，其特征在于，所述佐剂系统中磷酸铝佐剂吸附85-100％疫苗抗原和80-100％CpG佐剂。
  5. 如权利要求2所述的佐剂系统，其特征在于，所述磷酸铝佐剂与CpG之间的重量比为磷酸铝∶CpG＝1∶4-4∶1。
  6. 如权利要求1所述的佐剂系统，其特征在于，所述磷酸铝佐剂与CpG之间的重量比为1∶2-2∶1。
  7. 如权利要求1所述的佐剂系统，其特征在于，所述磷酸铝佐剂的等电点在7.0-9.0之间。
  8. 如权利要求1所述的佐剂系统，其特征在于，所述磷酸铝佐剂的等电点在8.0-9.0之间。
  9. 一种制备佐剂系统的方法，其包括以下步骤：
    1)将可溶性铝盐、可溶性磷酸盐与不同浓度碱性溶液相混合，在预定pH条件下沉淀来制备磷酸铝佐剂；
    2)通过调节反应溶液中可溶性铝盐和可溶性磷酸盐的配比，从而得到一系列不同等电点的磷酸铝佐剂；和
    3)带负电荷的CpG佐剂合用，形成复合佐剂系统。
  10. 如权利要求9所述的方法，其中，所述可溶性铝盐为AlCl 3，可溶性磷酸盐为NaH 2PO 4，Na 2HPO 4或Na 3PO 4所述碱性溶液为NaOH，所述pH值为7-12。
  11. 一种针对权利要求1-7所述的佐剂系统的抗原解吸附方法包括如下步骤：
    1)将吸附了抗原的权利要求1-7所述的佐剂系统与解吸附液混合后轻微振荡1小时以上；
    2)直接获得或经过离心后得到上清溶液获得解析附下来的抗原溶液。
  12. 如权利要求11所述的方法，其中，所述解吸附液包含160mM-220mM的磷酸钠，0.2-0.6M的枸橼酸钠，0.15-2M的氯化钠以及0.01-0.4％PS-80，且pH为6-7。
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