阅读说明：本技术 一种带状疱疹疫苗组合物及其制备方法和应用 (Herpes zoster vaccine composition, and preparation method and application thereof ) 是由 刘存宝 曹晗 齐家龙 王云飞 于 2020-05-14 设计创作，主要内容包括：本发明公开了一种带状疱疹疫苗组合物及其制备方法和应用,属于疫苗领域。所述疫苗组合物中包含水痘-带状疱疹糖蛋白E、聚乳酸-聚羟基乙酸共聚物(PLGA)、双链聚胞嘧啶核苷酸片段(Poly I:C)和富含GC的单链寡聚脱氧核苷酸片段(CpG ODN),通过微流控设备或双乳化媒蒸发法制备为直径20-400纳米的颗粒。所述疫苗组合物能特异性增强针对水痘-带状疱疹糖蛋白E的体液免疫应答和细胞免疫应答,可用作带状疱疹疫苗；并且所述疫苗组合物中各成分廉价易得,有效降低了疫苗成本、提高疫苗产量。(The invention discloses a herpes zoster vaccine composition, a preparation method and an application thereof, and belongs to the field of vaccines. The vaccine composition comprises varicella-zoster glycoprotein E, polylactic acid-polyglycolic acid copolymer (PLGA), double-chain Poly cytosine nucleotide fragment (Poly I: C) and single-chain oligodeoxynucleotide fragment (CpG ODN) rich in GC, and is prepared into particles with the diameter of 20-400 nanometers by a micro-fluidic device or a double-emulsion medium evaporation method. The vaccine composition can specifically enhance the humoral immune response and the cellular immune response aiming at the varicella-zoster glycoprotein E, and can be used as a herpes zoster vaccine; and the components in the vaccine composition are cheap and easy to obtain, so that the vaccine cost is effectively reduced, and the vaccine yield is improved.)

1. A herpes zoster vaccine composition comprising varicella-zoster glycoprotein E (gE), polylactic-co-glycolic acid (PLGA), double-stranded Poly-cytosine nucleotide fragments (Poly I: C) and GC-rich single-stranded oligodeoxynucleotide fragments (CpG ODN).

2. The vaccine composition according to claim 1, wherein the content of each raw material in the single injection vaccine composition is:

3. the vaccine composition of claim 1, wherein the gE is a hydrophilic extracellular region of viral glycoprotein E prepared using Chinese Hamster Ovary (CHO) secretory expression.

4. The vaccine composition of claim 1, wherein the mole percent of lactic acid and glycolic acid of the PLGA is in the range of 45: 55 to 85:15, the intrinsic viscosity is in the range of 5-100ml/g, the weight average molecular weight is 7000 and 170000, and the molecular weight distribution coefficient is not more than 2.5.

5. The vaccine composition of claim 1, wherein the Poly I: C has an average molecular weight of between 0.2 and 8 kb.

6. The vaccine composition of claim 1, wherein the CpG ODN comprises three A, B, C classes, including native structures and thio-oxidized forms.

7. The vaccine composition according to any one of claims 1 to 6, wherein the vaccine composition is a particulate composition having a diameter of between 20 and 400 nm.

8. The vaccine composition according to any one of claims 1 to 6, wherein the vaccine composition is used for preventing varicella or herpes zoster by subcutaneous or intramuscular injection.

9. A method of preparing a vaccine composition according to any one of claims 1 to 8, wherein the vaccine composition is prepared using a microfluidic device or a double emulsion medium evaporation method.

10. Use of a vaccine composition according to any one of claims 1 to 8 for the manufacture of a medicament for the prevention or amelioration of varicella and/or herpes zoster and/or post herpetic neuralgia.

Technical Field

The invention belongs to the field of vaccines, and particularly relates to a herpes zoster vaccine composition, and a preparation method and application thereof.

Background

Almost all children are infected with Varicella-zoster virus (VZV) before the adult, the primary infection produces Varicella, the virus remains in the ganglia after the Varicella heals, and impaired cellular immune response (such as HIV infection or immunosuppression) due to aging or other causes induces reactivation of the virus in the body, resulting in the development of shingles.

The protective rate of the Merk attenuated vaccine Zostavax to the population 50-59, 60-69 and over 70 years old is 70%, 64% and 38% respectively. This reduction in protection rate with age is primarily due to the impaired cellular immune response that occurs with aging of the immune system. In addition, compared with the preparation of varicella vaccine with low titer, the technical difficulty of preparing and storing the herpes zoster attenuated live vaccine with high virus titer in technical aspect is higher, and no related product is available on the market in domestic vaccine enterprises.

The herpes zoster genetic engineering subunit vaccine Shingrix of GSK uses conserved virus glycoprotein E (gE) expressed by Chinese hamster ovary Cells (CHO) AS an antigen, uses an adjuvant AS01B to effectively enhance a cell immune response specific to VZV-gE, ensures that the protection rate of the vaccine in healthy people over 50 years old is up to 97.2 percent (the protection rates of the vaccine in healthy people over 50-59, 60-69 and 70 years old are 96.6 percent, 97.3 percent and 91.3 percent respectively) and shows good safety and effectiveness in immunodeficiency people including HIV carriers. The key component QS21 of AS01B is a polysaccharide mixture which can only be extracted from saponin of Quillaja saponaria of south American honeylocust tree by reverse high performance liquid chromatography, cannot be artificially synthesized at present, and has serious limitations of limited sources (the annual yield in the world is only 600 million (the annual yield is consistent with the selling amount of the saponin in 2018), and the intellectual property of the producing country starts to limit the felling of related honeylocust trees), large difficulty in quality control in the preparation process (the purification components are non-monomers with complex components, the active components are sensitive to temperature), and the need of adding a detoxifying agent due to hemolytic activity, and the like. In addition, according to the instruction of Shingrix, the adjuvant component AS01B needs to be mixed with antigen (Bedsidemix) before use, which indirectly suggests that the stability of the liposome adjuvant system needs to be further improved when used in vaccine.

The above background resulted in the shingles subunit vaccine being expensive ($ 150-.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a herpes zoster vaccine composition, a preparation method and an application thereof, which can effectively enhance the specific cellular immune response to VZV-gE, make the vaccine be used as a subunit vaccine of herpes zoster, and simultaneously effectively reduce the vaccine cost and improve the vaccine yield.

The purpose of the invention is realized by the following technical scheme:

a herpes zoster vaccine composition comprises varicella-zoster glycoprotein E (gE), polylactic-co-glycolic acid (PLGA), double-stranded Poly-cytosine nucleotide fragment (Poly I: C) and GC-rich single-stranded oligodeoxynucleotide fragment (CpG ODN).

Further, the content of each raw material in the single injection vaccine composition is as follows:

further, gE is a hydrophilic extracellular region of the virus glycoprotein E prepared using Chinese Hamster Ovary (CHO) secretory expression.

Further, the mole percentage of lactic acid and glycolic acid of the PLGA ranges from 45: 55 to 85:15, the intrinsic viscosity is in the range of 5-100ml/g, the weight average molecular weight is 7000 and 170000, and the molecular weight distribution coefficient is not more than 2.5.

Further, the average molecular weight of the Poly I: C is between 0.2 kb and 8 kb.

Further, the CpG ODN includes A, B, C three classes, and the specific form includes a natural structure and a sulfo-oxidized form.

Further, the vaccine composition is a particle composition with the diameter of 20-400 nanometers.

Further, the vaccine composition is used for preventing varicella or herpes zoster by subcutaneous or intramuscular injection.

Another aspect of the invention:

the preparation method of the vaccine composition adopts a micro-fluidic device or a double-emulsifying medium evaporation method to prepare the vaccine composition.

The application of the vaccine composition can be used for preparing a medicament for preventing or improving varicella and/or herpes zoster and/or postherpetic neuralgia.

Compared with the prior art, the invention has the beneficial effects that:

1. in the herpes zoster vaccine composition, PLGA is used for preparing nanoparticles: the vaccine can effectively promote the phagocytosis of antigen presenting cells and efficiently deliver antigens, and realizes the slow release and continuous stimulation of an organism to generate specific cellular immune response aiming at VZV-gE. ② the introduction of the strictly controlled antibiotic in the vaccine can be avoided by improving the stability of Poly I: C by adding kanamycin sulfate and calcium chloride. ③ the non-sulfo-oxidation form CpG ODN wrapped by the nano-particles can effectively avoid the degradation of nuclease, and can achieve the similar effect of the sulfo-oxidation form CpG ODN with higher cost (the half-life period of the two exposed forms in vivo is 5-10min and 30-60min respectively); on the other hand, the non-sulfo oxidized form CpG ODN escaping before phagocytosis of the presented cells can be rapidly degraded by nuclease in vivo, thereby effectively avoiding systemic inflammation side effects possibly caused by nonspecific diffusion of the CpG ODN from the vaccine injection site, and conforming to the characteristics of 'locality' and 'transient' of a safety adjuvant;

2. according to the herpes zoster vaccine composition, the used Poly I: C can be taken up by TLR3 distributed in endocytosis, so that the maturation of corresponding dendritic cells and the initiation of downstream cellular immune response are promoted. C may respectively activate a Retinoic acid-induced gene 1(RIG-I) and a melanoma differentiation-associated protein 5(MDA5) antiviral natural immune response pathway in cytoplasm to trigger corresponding acquired immune response;

3. CpG ODN can be taken up by TLR9 distributed within the endocytotic body, resulting in massive secretion of interferon, and efficient activation of antigen-specific CD8+ T cells by promoting cross-presentation of antigen. The A class of the CpG ODN can stimulate dendritic cells to generate I-type interferon and activate natural killer cells, the B class can be rapidly transferred from early endosome to late endosome, stimulate B cell proliferation, stimulate plasmacytoid dendritic cell maturation and the generation of TNF-alpha, IL-6 and IL-12, and the C class has the action characteristics of the A class and the B class and can balance and promote humoral immunity and cellular immune response. CpGODN type C, which may form a local stem-loop structure, escaping into the cytoplasm may then induce a relevant adaptive immune response by activating the cyclic GMP-AMP synthsase (cGAS) and subsequently the stimulator of IFN genes (STING) innate immune pathway;

4. the invention simultaneously utilizes Poly I, C and CpG ODN which have good synergistic effect on the aspect of inducing antigen specific cellular immune response;

5. the CpG ODN in the herpes zoster vaccine composition can be in a sulfo-oxidation form and a natural structural form; the cost of the non-sulfo-oxidized CpG ODN is extremely low compared with the sulfo-oxidized CpG ODN, and the non-sulfo-oxidized CpG ODN is wrapped in PLGA to avoid nuclease hydrolysis, so that the similar immune stimulation effect of the expensive sulfo-oxidized CpG ODN which resists the enzyme hydrolysis by introducing sulfo-oxidized groups through chemical modification can be obtained;

6. animal experiments prove that the herpes zoster vaccine composition can specifically enhance humoral immune response and cellular immune response aiming at varicella-zoster glycoprotein E, and can be used as a herpes zoster vaccine; according to the invention, PLGA is used for coating Poly I: C and CpG ODN to avoid degradation of nuclease, so that the dosage and cost of the nucleic acid adjuvant are further reduced, and the rash vaccine composition comprising the antigen, the vector and the nucleic acid adjuvant can be lyophilized together, so that the vaccine production and packaging cost is further reduced.

Drawings

FIG. 1 shows TEM photographs of nanoparticle vaccines prepared by examples 5-9 using double emulsification medium evaporation;

FIG. 2 shows the production of IgG antibodies specific for the gE antigen, as determined by example 10 and example 11, for vaccines prepared in examples 1-9;

FIGS. 3-8 show the vaccines prepared in examples 1-9, respectively, and the numbers of IFN-. gamma.and IL-2 secreting cells specific for the gE antigen as determined by examples 10, 12 and 13;

FIGS. 9-12 show vaccines prepared in examples 1-9, respectively, and the ratios of IFN-. gamma.and IL-2 producing cells specific for the gE antigen as determined by examples 10, 12 and 14.

Detailed Description

The non-nanoparticle non-nucleic acid adjuvant vaccine (examples 1-4) was prepared by directly mixing 100% of the formulation of the single injection vaccine in table 1 below, and the PLGA nanoparticle vaccine (examples 5-9) was administered 200% (i.e., encapsulation efficiency 50%) to calculate 50 injections of the vaccine.

TABLE 1 Single agent vaccine composition

Note: wherein, the first to the fourth is a mixed preparation control, and the fifth to the fifth is a nano-particle vaccine prepared by a double-emulsifying medium evaporation method.