Technical field

The invention belongs to molecular biosciences field of immunology.It is being prepared more particularly, to Artesunate as immunologic adjuvant Application in rabies vacciness.

Background technique

Hydrophobin (Rabies virus, RABV) is the strong people beast caused with the characteristics of infecting central nervous system Suffer from the rabic cause of disease of infectious disease altogether, once morbidity, lethality are up to 100%.China is rabic district occurred frequently, number of the infected It is only second to India, is in second place of the world.With the increase of domestic pets, the rabic control in China human world and prevention and treatment are still appointed Weight road is remote.However rabies pathogenesis is also imperfectly understood so far, also without treating rabic specific drug, so prevention is mad The most effective measure of dog disease is timely vaccine inoculation.

Currently, be both at home and abroad the protein formulation purified for the rabies vacciness of the mankind, due to a lack of corresponding adjuvant, first It is secondary it is immune need to inject 3-5 needle, take one month.As for animal rabies vaccine, import inactivated vaccine have using it is safe, be easy to The advantages such as preservation, pollution-free danger.Although China's approved several moneys inactivated vaccines with independent intellectual property rights at present, because The factors such as higher of production-scale limitation and production cost, these inactivated vaccines hair more backward for Asia and African economy Price is too high for area in exhibition, limits them in the use of developing country, and most of economically developed city is still Preferred import inactivated vaccine.Domestic inactivated vaccine needs for animals further reduce the cost.

Summary of the invention

The defects of higher the technical problem to be solved by the present invention is to overcome existing vaccine cost and deficiency, provide Artesunate As application of the immunologic adjuvant in preparation rabies vacciness.

The object of the present invention is to provide Artesunate (referred to as (ART)) as active constituent in preparation rabies vacciness adjuvant In application.

By ART and hydrophobia strain mixed immunity mouse, immune in mouse of enhancing inactivation hydrophobin is answered It answers.And the recruitment evaluation of vaccine after identifying and mixing with vaccine, experimental result hair are further carried out as vaccine adjuvant to ART Existing, Artesunate can significantly increase the immunogenicity of rabies vacciness, can be used as new semple type rabies vaccine candidate adjuvant.

In one embodiment of this invention, the Artesunate is as sole active agent.

The present invention also provides a kind of rabies vacciness adjuvant, the rabies vacciness adjuvant includes Artesunate.

In one embodiment of this invention, the Artesunate is as sole active agent.

The present invention also provides application of the Artesunate in preparation rabies vacciness, and the Artesunate is as adjuvant.

The present invention also provides a kind of vaccine compositions, and it includes the hydrophobin strains and Artesunate of inactivation.

In one embodiment of this invention, the hydrophobin strain of the inactivation is selected from CVS-11 strain, rHEP-dG poison Strain, SAD plants, CTN-1 plants, PV plants, at least one of FluryLEP plants.Certainly, above-mentioned strain, all rabies diseases are not limited to Poison strain is suitable for the present invention.

In one embodiment of this invention, in the vaccine composition hydrophobin strain and Artesunate amount ratio It is 0.9 × 10⁵~1.1 × 10⁵The μ of FFU:120~130 g.

In one embodiment of this invention, the content of hydrophobin strain is 1.0 × 10 in the vaccine composition⁵~ 1.1×10⁵The μ of FFU:120~130 g.

In one embodiment of this invention, the dosage of Artesunate is 5 μ g/g the weight of animals in the vaccine composition.It is green The dosage of artemisic succinate is determined according to the weight of animals such as mouse, and by taking mouse as an example, actual use concentration is 5 μ g/g mouse weights, If every mouse weight is about 25g, the dosage of Artesunate is about 125 μ g.

In one embodiment of this invention, further include dimethyl sulfoxide (DMSO), other solvents can also be used, DMSO exists When dissolving the reagent of some good water solubilities, solute effect is more preferable.

In one embodiment of this invention, Artesunate (g) in the vaccine composition: dimethyl sulfoxide (mL)=1:(8 ~12), preferably 1:10.

It in one embodiment of this invention, further include PBS buffer solution.PBS buffer solution has effects that physiological saline, in stabilization More advantageous, pH 7.20-7.40 in terms of albumen.

In one embodiment of this invention, by volume, Artesunate in the vaccine composition: PBS buffer solution=1: (90~110), preferably 1:100.

The present invention also provides application of the Artesunate in preparation rabies vacciness.The rabies vacciness is for preventing or controlling Treat people or the rabies of other animals.

Optionally, the Artesunate is as adjuvant.

Term " adjuvant " is the pharmacy or immunological reagent or composition for changing other agent efficacies, refer in a broad sense from Body does not provide wide spectrum substance that is immune, but can increasing the immunogenicity of the antigen of co-administration.Adjuvant can be added to vaccine In, increase the quantity and lasting protection of antibody by increasing immune response, to reduce the external of injection to the maximum extent Substance.Adjuvant can also be used for improving the effect of vaccine, be changed by helping to the immune anti-of certain types of immune system cell It answers, for example, according to the purpose of vaccine, by activating T cell rather than the B cell of secretory antibody.Therefore, adjuvant can be advantageously Adjust cytokine-expressing/secretion, antigen presentation, type of immune response etc..In the present invention, the situation of the term refers to work For the compound or composition of the carrier or auxiliary substance of immunogene and/or other drugs reactive compound.

Wherein above-mentioned " antigen ", which is typically meant that, can be identified by immune system and can for example be adapted to by being formed to be used as Property immune response a part antibody or antigen specific T-cell come trigger antigen specific immune reaction substance.Antigen It is divided into two classes: comlete antigen and incomplete antigen according to property.Comlete antigen (complete antigen) abbreviation antigen, is one The existing immunogenicity of class, and have immunoreactive substance, such as most protein, bacterium, virus, bacterial exotoxin and intestines poison Element etc..Incomplete antigen, that is, haptens (hapten) is that only have immunoreactivity, and the substance of non-immunogenicity, such as absolutely mostly The hydrolysate and all lipoids of number polysaccharide (capsular polysaccharide of such as pneumococcus), capsular polysaccharide.

The invention has the following advantages:

Present invention firstly provides artemisinin derivative Artesunate (ART) is used as rabies vacciness adjuvant.

The present invention passes through experimental studies have found that artemisinin derivative Artesunate (ART) can be shown as rabies vacciness adjuvant The immunogenicity for writing enhancing rabies vacciness, can be used as new semple type rabies vaccine candidate adjuvant.

The Artesunate (ART) of vaccine effect studies have shown that relatively low-dose of the invention can promote exempting from for rabies vacciness Epidemic disease effect can reduce the cost of production vaccine.

Vaccine effect studies have shown that artemisinin derivative Artesunate (ART) of the invention is used as rabies vacciness adjuvant Better protection can be generated.

Detailed description of the invention

Fig. 1 is shown as a kind of chemical formula of artemisinin derivative Artesunate (ART).

Fig. 2, which is shown as ART, influences statistical chart to KM mouse weight, while setting up PBS control group.

Peripheral blood anti-rabies virus neutralizing antibody water after mouse is immunized in the CVS-11 that Fig. 3 is shown as combining ART inactivation Flat statistical chart, while setting up PBS control group (P < 0.05 *).

Peripheral blood anti-rabies virus neutralizing antibody water after mouse is immunized in the rHEP-dG that Fig. 4 is shown as combining ART inactivation Flat statistical chart, while setting up PBS control group (P < 0.05 *).

Fig. 5 is shown as the survival rate statistical chart of CVS-11 attack mouse after ART is immunized mouse 14 days as vaccine adjuvant.

Specific embodiment

The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art Agent, method and apparatus.

Unless stated otherwise, following embodiment agents useful for same and material are commercially available.

Currently, the World Health Organization is recommended to use inactivation rabies vacciness according to its safety, in order to develop it is more effective and Cheaper inactivated vaccine, we, which can take, improves the means such as virus titer or the better adjuvant of searching.

Fig. 1 is shown as the chemical formula of artemisinin derivative Artesunate (ART) used by following embodiment, Artesunate (ART) No. CAS is 88495-63-0, molecular formula: C₁₉H₂₈O₈, molecular weight: 384.42, No. EINECS: 618-170-5.

Artesunate used in following embodiment is purchased from TCI AmericaPortland company (Tokyo chemical conversion industry Co., Ltd.).

Artemisinin derivative Artesunate (ART) adjuvant in following embodiment, is the CVS-11 or rHEP-dG with inactivation Strain is mixed with ART, forms rabies virus vaccine.

Specifically: ART is initially dissolved in DMSO, and the CVS-11 in PBS with inactivation is dissolved in when then using in vivo Or rHEP-dG hydrophobin mixing.According to Artesunate (g): DMSO (mL)=1:10 ratio, the i.e. Artesunate of 1g are molten In 10mL DMSO, the Artesunate being then dissolved in DMSO is redissolved in PBS buffer solution solution, Artesunate and PBS The volume ratio of buffer is 1:100, and PBS buffer solution is phosphate buffer, pH 7.2-7.4.

Use the ART as adjuvant, rabies vacciness induction can be promoted to generate in higher anti-rabies virus and anti- Body, and better protecting effect is generated, reduce the cost of Canine vaccine.

Specifically, in the immunologic adjuvant, Artesunate is the dosage and 1.0 × 10 by 5 μ g/g mouse weights⁵FFU inactivation CVS-11 or rHEP-dG mixing, by right leg gastrocnemius intramuscular injection be immunized mouse after collect peripheral blood, utilize neutralize experiment Study anti-rabies virus neutralize antibody titers.

In addition, artemisinin derivative Artesunate (ART) also exists in the application as vaccine adjuvant or in terms of preparing vaccine Within protection scope of the present invention.

Artemisinin derivative Artesunate (ART) is initially dissolved in DMSO, is dissolved in PBS when then using in vivo In, it is used after being mixed with CVS-11 the or rHEP-dG hydrophobin of inactivation.The immunologic adjuvant Artesunate is by 5 μ g/g Dosage is mixed with the CVS-11 of inactivation or rHEP-dG, collects serum after mouse is immunized by right leg gastrocnemius intramuscular injection, is used Neutralize experiment detection anti-rabies virus neutralize antibody titers.The present invention is also to ART side effect, ART Adjuvanted vaccines immunogenicity It is studied with the biological characteristics such as malicious protection are attacked, to assess the safety and effect of its as vaccine adjuvant.The results show that ART will not generate significant side effect as new vaccine adjuvant in Mice Body, and ART can significantly increase vaccine as adjuvant Immunogenicity promotes vaccine-induced body to generate the anti-rabies virus neutralizing antibody with Vaccine effectiveness, can preferably protect Protect the attack that body resists lethal rabies virus.

In terms of specific research method, the present invention utilizes immunology the relevant technologies, has studied in Mice Body ART as novel The recruitment evaluation of vaccine adjuvant.

Embodiment 1

The research that novel rabies virus vaccine adjuvant ART influences mouse weight

SPF grade Kunming (KM) mouse of 3-4 week old is divided into 2 groups, i.e. ART group and PBS control group, every group comprising 6 small Mouse.ART is injected with the dosage of 5 μ g/g by the right leg gastrocnemius muscle of mouse, and control group injects equivalent PBS.It is every after administration The weight of its detection mouse, monitors 15 days altogether.By the weight before respectively injecting on the weight ratio of all mouse, weight ratio is studied Example situation of change.As shown in Figure 2, the results showed that, ART not will lead to treatment mouse weight and mitigate, this is similar to PBS treatment.More Importantly, there is not abnormal symptom in treatment mouse.Therefore, the ART treatment that dosage is 5 μ g/g is safe to mouse.

Embodiment 2

Artesunate (ART) in the intracorporal immunogenicity of KM mouse and attacks malicious Protective strategy to rabies vacciness

1, experimental method

CVS-11 and rHEP-dG are inactivated with UV respectively.Then ART is mixed with the CVS-11 of inactivation or rHEP-dG, PBS is used as vehicle control.The SPF grade kunming mouse of 6~7 week old is divided into 4 groups, i.e., ART+CVS-11 group, PBS+CVS-11 group, ART+rHEP-dG group and PBS+rHEP-dG control group, every group includes 6 mouse.The right leg gastrocnemius injection of every mouse of experimental group 1.0×10⁵The hydrophobin of FFU inactivation and the mixture of ART, the volume of mixture are 200 μ L, and ART's contains in mixture Measuring is 5 μ g ART/g mouse weights, Artesunate (g): dimethyl sulfoxide (mL)=1:10, by volume, Artesunate: PBS Buffer=1:100, control group intramuscular injection penetrate 1.0 × 10⁵The hydrophobin of FFU inactivation and PBS mixture.7th after immune It and the 14th day acquisition peripheral blood, and by fluorescence antibody virus neutralization (FAVN) testing inspection serum anti-rabies virus And antibody level, rabies standard serum are purchased from the World Health Organization (WHO), detection method is in strict accordance with world animal health Organize the standard practice instructions of (World Organisation for Animal Health, OIE).

2, experimental result

As shown in Figure 3, the results showed that, the hydrophobin that mouse generates is immunized with the CVS-11 joint ART of inactivation and neutralizes Antibody level is significantly higher than PBS control group.As shown in figure 4, the mad dog that mouse generates is immunized with the rHEP-dG joint ART of inactivation Sick virucidin's level is significantly higher than PBS control group.

Fig. 3 and Fig. 4's the result shows that, when ART is applied to rabies vacciness as adjuvant, it is not limited to a certain specific Inactivation hydrophobin strain, but can be adapted for the hydrophobin strain of various inactivations.

3, further, in order to verify after mouse obtains high level immunity whether there is protection to strong virus attack, In CVS-11 strain living is passed through intramuscular injection infecting mouse by 14d after immune, while being arranged without immune rabies vacciness Control group, the morbidity and death condition of continuous 3 weeks observation mouse, records survival rate daily.As shown in Figure 5, the results showed that, it uses ART as adjuvant mouse than there is higher survival rate with the mouse of PBS control.

The above results show that artemisinin derivative Artesunate (ART) enhances inactivation rabies epidemic disease when being used as adjuvant The immune response of seedling.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

技术领域

本发明属于分子生物免疫学领域。更具体地，涉及青蒿琥酯作为免疫佐剂在制备狂犬病疫苗中的应用。

背景技术

狂犬病病毒(Rabies virus，RABV)是引起以侵染中枢神经系统为特点的烈性人兽共患传染病狂犬病的病原，一旦发病，致死率高达100％。我国是狂犬病的高发区，发病人数仅次于印度，高居世界第二位。随着家养宠物的增加，我国人间狂犬病的控制和防治依然任重道远。然而狂犬病致病机理至今还不完全清楚，也没有治疗狂犬病的特效药，所以预防狂犬病最有效的措施是及时接种疫苗。

目前，国内外用于人类的狂犬病疫苗为纯化的蛋白制剂，因缺乏相应的佐剂，第一次免疫需注射3-5针，需时一个月。至于动物狂犬病疫苗，进口灭活疫苗具有使用安全、易于保存、无污染危险等优势。我国目前虽然已批准了几款具有自主知识产权的灭活疫苗，但因生产规模的限制和生产成本的偏高等因素，这些灭活疫苗对于亚洲和非洲经济较落后的发展中地区来说价格太高，限制了它们在发展中国家的使用，而大部分经济发达的城市仍然首选进口灭活疫苗。国产兽用灭活疫苗需要进一步降低成本。

发明内容

本发明要解决的技术问题是克服现有疫苗成本较高等缺陷和不足，提供青蒿琥酯作为免疫佐剂在制备狂犬病疫苗中的应用。

本发明的目的是提供青蒿琥酯(简称(ART))作为活性成分在制备狂犬病疫苗佐剂中的应用。

将ART与狂犬病毒疫苗株混合免疫小鼠，增强灭活狂犬病病毒在小鼠中的免疫应答。并进一步对ART作为疫苗佐剂进行鉴定以及与疫苗混合后疫苗的效果评估，实验结果发现，青蒿琥酯可显著增强狂犬病疫苗的免疫原性，可以作为新型狂犬病疫苗候选佐剂。

在本发明的一实施例中，所述青蒿琥酯作为唯一活性成分。

本发明还提供一种狂犬病疫苗佐剂，所述狂犬病疫苗佐剂包含青蒿琥酯。

在本发明的一实施例中，所述青蒿琥酯作为唯一活性成分。

本发明还提供青蒿琥酯在制备狂犬病疫苗中的应用，所述青蒿琥酯作为佐剂。

本发明还提供一种疫苗组合物，其包含灭活的狂犬病病毒毒株和青蒿琥酯。

在本发明的一实施例中，所述灭活的狂犬病病毒毒株选自CVS-11毒株、rHEP-dG毒株、SAD株、CTN-1株、PV株、FluryLEP株中的至少一种。当然，不限于上述毒株，所有狂犬病病毒毒株均适用于本发明。

在本发明的一实施例中，所述疫苗组合物中狂犬病病毒毒株与青蒿琥酯的用量比为0.9×10⁵～1.1×10⁵FFU：120～130μg。

在本发明的一实施例中，所述疫苗组合物中狂犬病病毒毒株的含量为1.0×10⁵～1.1×10⁵FFU：120～130μg。

在本发明的一实施例中，所述疫苗组合物中青蒿琥酯的用量为5μg/g动物体重。青蒿琥酯的用量是根据小鼠等动物体重确定，以小鼠为例，实际使用浓度为5μg/g小鼠体重，如果每只小鼠体重约为25g，那么，青蒿琥酯的用量约为125μg。

在本发明的一实施例中，还包括二甲基亚砜(DMSO)，也可以采用其他溶剂，DMSO在溶解一些水溶性好的试剂时，溶解效果更好。

在本发明的一实施例中，所述疫苗组合物中青蒿琥酯(g)：二甲基亚砜(mL)＝1：(8～12)，优选为1:10。

在本发明的一实施例中，还包括PBS缓冲液。PBS缓冲液具有生理盐水功效，在稳定蛋白方面更具有优势，pH为7.20-7.40。

在本发明的一实施例中，按体积计，所述疫苗组合物中青蒿琥酯：PBS缓冲液＝1：(90～110)，优选为1：100。

本发明还提供青蒿琥酯在制备狂犬病疫苗中的应用。该狂犬病疫苗用于预防或治疗人或其他动物的狂犬病。

可选地，所述青蒿琥酯作为佐剂。

术语“佐剂”是改变其他药剂功效的药学或免疫学试剂或组合物，在广义上是指自身不提供免疫，但能够增加共同施用的抗原的免疫原性的广谱物质。佐剂可以添加到疫苗中，通过增加免疫应答来增加抗体的数量和持久的保护，从而最大限度地减少注射的外来物质。佐剂也可用于提高疫苗的效力，通过帮助改变对特定类型的免疫系统细胞的免疫反应，例如，根据疫苗的目的，通过激活T细胞而不是分泌抗体的B细胞。因此，佐剂可以有利地调节细胞因子表达/分泌、抗原呈递、免疫应答的类型等。在本发明中，该术语的情形是指作为免疫原和/或其他药物活性化合物的载体或辅助物质的化合物或组合物。

其中上述“抗原”典型地是指可以由免疫系统识别并能够例如通过形成作为适应性免疫应答的一部分的抗体或抗原特异性T-细胞来触发抗原特异性免疫反应的物质。抗原根据性质分为两类：完全抗原和不完全抗原。完全抗原(complete antigen)简称抗原，是一类既有免疫原性，又有免疫反应性的物质，如大多数蛋白质、细菌、病毒、细菌外毒素和肠毒素等。不完全抗原即半抗原(hapten)是只具有免疫反应性，而无免疫原性的物质，如绝大多数多糖(如肺炎球菌的荚膜多糖)、荚膜多糖的水解产物和所有的类脂等。

本发明具有以下有益效果：

本发明首次提出将青蒿素衍生物青蒿琥酯(ART)作为狂犬病疫苗佐剂。

本发明通过实验研究发现青蒿素衍生物青蒿琥酯(ART)作为狂犬病疫苗佐剂能显著增强狂犬病疫苗的免疫原性，可以作为新型狂犬病疫苗候选佐剂。

本发明的疫苗效果研究显示，较低剂量的青蒿琥酯(ART)能促进狂犬病疫苗的免疫效果，可以降低制作疫苗的成本。

本发明的疫苗效果研究显示，青蒿素衍生物青蒿琥酯(ART)作为狂犬病疫苗佐剂能产生更好的保护力。

附图说明

图1显示为一种青蒿素衍生物青蒿琥酯(ART)的化学式。

图2显示为ART对KM小鼠体重影响统计图，同时设立PBS对照组。

图3显示为将ART联合灭活的CVS-11免疫小鼠后外周血抗狂犬病病毒中和抗体水平统计图，同时设立PBS对照组(*P<0.05)。

图4显示为将ART联合灭活的rHEP-dG免疫小鼠后外周血抗狂犬病病毒中和抗体水平统计图，同时设立PBS对照组(*P<0.05)。

图5显示为ART作为疫苗佐剂免疫小鼠14天后CVS-11攻击小鼠的存活率统计图。

具体实施方式

以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。

除非特别说明，以下实施例所用试剂和材料均为市购。

目前，世界卫生组织根据其安全性推荐使用灭活狂犬病疫苗，为了开发更有效和更便宜的灭活疫苗，我们可以采取提高病毒滴度或寻找更好的佐剂等手段。

图1显示为以下实施例所采用的青蒿素衍生物青蒿琥酯(ART)的化学式，青蒿琥酯(ART)的CAS号为88495-63-0，分子式：C₁₉H₂₈O₈，分子量：384.42，EINECS号：618-170-5。

以下实施例中所使用的青蒿琥酯购于TCI AmericaPortland公司(东京化成工业株式会社)。

以下实施例中的青蒿素衍生物青蒿琥酯(ART)佐剂，是以灭活的CVS-11或rHEP-dG毒株与ART混合，形成狂犬病病毒疫苗。

具体地：ART首先溶解在DMSO中，然后在体内使用时溶解在PBS中与灭活的CVS-11或rHEP-dG狂犬病病毒混合。按照青蒿琥酯(g)：DMSO(mL)＝1:10的比例，即1g的青蒿琥酯溶解在10mL DMSO中，然后将溶解在DMSO中的青蒿琥酯再溶解到PBS缓冲液中，青蒿琥酯与PBS缓冲液的体积比为1:100，PBS缓冲液即为磷酸盐缓冲液，pH为7.2-7.4。

使用该ART作为佐剂，能够促进狂犬病疫苗诱导产生更高的抗狂犬病病毒中和抗体，且产生更好的保护效果，降低了犬用疫苗的成本。

具体地，该免疫佐剂中，青蒿琥酯是按5μg/g小鼠体重的剂量与1.0×10⁵FFU灭活的CVS-11或rHEP-dG混合，通过右腿腓肠肌肌内注射免疫小鼠后收集外周血，利用中和实验研究抗狂犬病病毒中和抗体效价。

另外，青蒿素衍生物青蒿琥酯(ART)在作为疫苗佐剂或制备疫苗方面的应用，也在本发明的保护范围之内。

青蒿素衍生物青蒿琥酯(ART)首先溶解在DMSO中，然后在体内使用时溶解在PBS中，与灭活的CVS-11或rHEP-dG狂犬病病毒混合后使用。该免疫佐剂青蒿琥酯是按5μg/g的剂量与灭活的CVS-11或rHEP-dG混合，通过右腿腓肠肌肌内注射免疫小鼠后收集血清，采用中和实验检测抗狂犬病病毒中和抗体效价。本发明还对ART副作用、ART佐剂疫苗免疫原性和攻毒保护力等生物学特性进行研究，以评估其作为疫苗佐剂的安全性和效果。结果显示，ART作为新型疫苗佐剂在小鼠体内不会产生显著的副作用，ART作为佐剂能显著增强疫苗的免疫原性，促进疫苗诱导机体产生具有保护效力的抗狂犬病病毒中和抗体，能够更好的保护机体抵抗致死性狂犬病病毒的攻击。

具体研究方法方面，本发明利用免疫学相关技术，研究了在小鼠体内ART作为新型疫苗佐剂的效果评估。

实施例1

新型狂犬病病毒疫苗佐剂ART对小鼠体重影响的研究

将3-4周龄的SPF级昆明(KM)小鼠分为2组，即ART组和PBS对照组，每组包含6只小鼠。ART以5μg/g的剂量通过小鼠右腿腓肠肌肌肉进行注射，对照组注射等量PBS。给药后每天检测小鼠的体重，一共监测15天。将所有小鼠的体重比上各自注射前的体重，研究体重比例变化情况。如图2所示，结果表明，ART不会导致治疗小鼠体重减轻，这与PBS治疗相似。更重要的是，治疗小鼠没有出现异常症状。因此，剂量为5μg/g的ART治疗对小鼠是安全的。

实施例2

青蒿琥酯(ART)对狂犬病疫苗在KM小鼠体内的免疫原性及攻毒保护研究

1、实验方法

分别将CVS-11和rHEP-dG用UV灭活。然后将ART与灭活的CVS-11或rHEP-dG混合，PBS用作佐剂对照。6～7周龄的SPF级昆明系小鼠分为4组，即ART+CVS-11组、PBS+CVS-11组、ART+rHEP-dG组和PBS+rHEP-dG对照组，每组包含6只小鼠。实验组每只小鼠右腿腓肠肌注射1.0×10⁵FFU灭活的狂犬病病毒和ART的混合物，混合物的体积为200μL，混合物中ART的含量为5μg ART/g小鼠体重，青蒿琥酯(g)：二甲基亚砜(mL)＝1：10，按体积计，青蒿琥酯：PBS缓冲液＝1：100，对照组肌注射1.0×10⁵FFU灭活的狂犬病病毒和PBS混合物。在免疫后第7天和第14天采集外周血，并且通过荧光抗体病毒中和(FAVN)试验检测血清抗狂犬病病毒中和抗体水平，抗狂犬病标准血清购自世界卫生组织(WHO)，检测方法严格按照世界动物卫生组织(World Organisation for Animal Health,OIE)的标准操作规程。

2、实验结果

如图3所示，结果表明，用灭活的CVS-11联合ART免疫小鼠产生的狂犬病病毒中和抗体水平显著高于PBS对照组。如图4所示，用灭活的rHEP-dG联合ART免疫小鼠产生的狂犬病病毒中和抗体水平显著高于PBS对照组。

图3和图4的结果表明，ART作为佐剂应用于狂犬病疫苗时，并不局限于某一种特定的灭活狂犬病病毒毒株，而是可以适用于各种灭活的狂犬病病毒毒株。

3、进一步地，为了验证小鼠获得高水平免疫力后是否具有对强毒攻击的保护，在免疫后第14d将活的CVS-11毒株通过肌肉注射感染小鼠，同时设置没有免疫狂犬病疫苗的对照组，连续3周观察小鼠的发病及死亡情况，每天记录存活率。如图5所示，结果表明，用ART作为佐剂的小鼠比用PBS对照的小鼠具有更高的存活率。

上述结果表明，青蒿素衍生物青蒿琥酯(ART)在用作佐剂时增强了灭活狂犬病疫苗的免疫应答。

上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。