CN100479858C - A recombined smallpox vaccine - SARS vaccine and preparation method thereof - Google Patents

A recombined smallpox vaccine - SARS vaccine and preparation method thereof Download PDF

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CN100479858C
CN100479858C CN 200610003011 CN200610003011A CN100479858C CN 100479858 C CN100479858 C CN 100479858C CN 200610003011 CN200610003011 CN 200610003011 CN 200610003011 A CN200610003011 A CN 200610003011A CN 100479858 C CN100479858 C CN 100479858C
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sars
mva
vaccine
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ads
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CN1840188A (en
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何大一
张林琦
涂新明
川 秦
陈志伟
虹 高
强 魏
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中国医学科学院实验动物研究所;艾伦·戴蒙德艾滋病研究中心
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Abstract

本发明提供了一种用于预防或治疗SARS的重组痘苗SARS-CoV疫苗(ADS-MVA),以及在中国恒河猴感染模型体内进行预防性疫苗抗SARS-CoV感染的方法。 The present invention provides a recombinant vaccinia vaccine SARS-CoV (ADS-MVA) for the prevention or treatment of SARS, and rhesus monkey model of infection in vivo method of prophylactic vaccine against SARS-CoV infection in China. 本发明人用修饰后的减毒痘苗(MVA)为载体构建SARS疫苗,在恒河猴基础免疫和加强免疫后攻毒。 The present inventors have used an attenuated vaccinia (MVA) The modified build SARS vaccine as the carrier, in rhesus monkeys after basic immunization and booster challenge. 经疫苗免疫原性测定,疫苗安全性测定,疫苗有效性测定,以及抗体测定、病毒载量体内动态检测、SARS病理程度变化等指标确定,认为重组痘苗-SARS疫苗具有良好的免疫原性、安全性和有效性,为防治SARS筛选到理想的疫苗。 Determined by immunogenicity of the vaccine, vaccine safety measurement, determine the effectiveness of vaccines and antibody detection, motion detection in vivo viral load, degree of SARS pathological changes and other indicators to determine that the recombinant vaccinia -SARS vaccine has good immunogenicity, safety and effectiveness of screening for the prevention and treatment of SARS to the ideal vaccine.

Description

一种重组痘苗-SARS疫苗及其制备方法 Vaccinia -SARS one kind of a recombinant vaccine and its preparation method

技术领域 FIELD

.本发明涉及一种重组痘苗-SARS疫苗及其制备方法,尤其是涉及一种包含修饰后的减毒痘苗MVA和SARS冠状病毒S基因的SARS疫苗ADS-MVA 及其制备方法。 The present invention relates to a recombinant vaccinia virus vaccine and its preparation method -SARS, particularly, to a SARS ADS-MVA MVA Vaccinia vaccine and its preparation method and SARS S gene of coronavirus comprises an attenuated modified after. 背景技术 Background technique

SARS自2002年11月首次在广东省发现并报道以来,很快在全球范围内流行,WHO称其为重症急性呼吸综合征(Severe Acute Respiratory Syndrome, SARS)。 Since SARS was first discovered in November 2002 and reported in Guangdong Province since soon worldwide epidemic, WHO called severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS). 该病主要通过近距离空气飞沬和密切接触传播,具有普遍人群易感、传播快、发病急和一定病死率等特点。 The disease mainly through respiratory droplets and close air close contact, have generally susceptible populations, rapid spread, the incidence of acute mortality and some other characteristics. 目前,全球共有非典确诊病例和疑似病例5663例,截止到2004年5月15日死亡372例,中国截止到2004年5月26日已有5316例,死亡315例,疑似1573例,治愈2742例。 Currently, there are confirmed SARS cases and suspected cases of 5663 cases, as of May 15, 2004 death of 372 cases, China has as of May 26, 2004 5316 cases, 315 cases of death, suspected 1573 cases, 2742 cases were cured . SARS的广泛流行严重威胁着人类的健康,国内外研究表明,SARS病原是一种变异冠状病毒,长为29742bp的正单链RNA病毒。 Wide spread of SARS a serious threat to human health, research has shown that, SARS coronavirus pathogen is a variation of a length of 29742bp positive single-stranded RNA viruses.

我国对SARS病原、诊断、来源等方面的研究均已取得一定进展,在我们前期工作中已经建立了SARS感染动物模型。 Our research on the SARS pathogen, diagnosis, and other sources have made some progress in our previous work has established SARS animal model of infection. 2003年5月初,我们感染了恒河猴,系统地进行了病毒学、血清学以及病理学研究,重要的是在感染的猴体内检测到了高效价的中和抗体,这一结果证实了SARS病毒可以诱发机体产生保护性抗体,因而研制SARS病毒疫苗在理论上是可能的,感染的猴作为疫苗评价的模型动物是可靠的,这一工作解决了在相当一段时间内限制药物筛选、疫苗评价等研究的瓶颈问题,尤其为疫苗评价等方面的研究奠定了重要基础,该病的完全控制,依赖于有效疫苗的研制。 In early May 2003, we infected rhesus monkeys, were systematically virology, serology and pathology research, it is important in monkeys infected detected high titers of neutralizing antibodies, the results confirmed SARS virus can induce the body to produce protective antibodies, thus the development of the SARS virus vaccine is theoretically possible, as a monkey infected animal vaccine assessment model is reliable, this work addresses the limitations in drug screening for quite some time, vaccine evaluation research bottlenecks, especially laid an important foundation, complete control of the disease for the study of the vaccine evaluation, it depends on the development of an effective vaccine.

中国申请200410044291.0中公开了包含SARS相关冠状病毒S基因和真核表达质粒的SARS疫苗,以及中国申请200410044285.5中公开了包含腺病毒载体和SARS相关冠状病毒S基因的SARS疫苗等,但现有的SARS疫苗存在不同程度的安全性隐患,它们的有效性尚待进一步观察和验证。 Chinese application 200410044291.0 discloses a SARS-associated coronavirus vaccine containing S gene and eukaryotic expression plasmid of SARS, as well as Chinese application 200410044285.5 discloses a SARS vaccine comprising an adenovirus vector and SARS-associated coronavirus S gene, but the existing SARS vaccines exist to varying degrees of security risks, their effectiveness remains to be seen and verified. 发明内容 SUMMARY

现有的SARS疫苗本发明克服了现有诸多技术问题,成功的研制出具有良好的免疫原性、安全性和有效性的重组痘苗-SARS疫苗,并用本疫苗在动物感染模型中国恒河猴体内进行预防致病性SARS-CoV感染的实验,取得了满意的效果。 Existing SARS vaccine of the invention to overcome the existing number of technical problems, successfully developed a good immunogenicity, safety and efficacy of recombinant vaccinia -SARS vaccine and animals infected with this vaccine in rhesus monkey model of China experiments prevent pathogenic SARS-CoV infection, and achieved satisfactory results. 尤其与临床试验中的灭活SARS疫苗相比,本疫苗无需接触SARS-CoV,更为安全可靠。 Especially compared to the clinical trials of inactivated SARS vaccine, this vaccine without contact with SARS-CoV, more secure and reliable.

本发明的第一方面涉及一种在减毒痘苗病毒MVA基础上构建的SARS-CoV疫苗(ADS-MVA )。 The first aspect of the invention relates to an attenuated vaccinia virus of SARS-CoV vaccine constructs based on MVA (ADS-MVA).

上述SARS-CoV疫苗是将SARS相关冠状病毒S基因插入到MVA缺失III区而得到的。 SARS-CoV vaccine above is inserted into the SARS-associated coronavirus S gene into the deletion III region of the MVA obtained.

本发明还涉及一种SARS-CoV疫苗的制备方法,其包括: The present invention also relates to a method for preparing a SARS-CoV vaccine, comprising:

(1) 构建穿梭质粒,该质粒包含SARS-CoV抗原基因,以及减毒痘苗病毒 (1) Construction of shuttle plasmid, the plasmid comprising a SARS-CoV antigen gene, and attenuated vaccinia virus

MVA缺失lll区基因编码区序列或与编码区外两侧序列同源的侧翼序列; MVA deletion of the gene coding region sequence lll outer region or flanking sequences homologous to the flanking sequences of the coding region;

(2) 在痘苗病毒的允许细胞中使穿梭质粒与减毒痘苗病毒MVA株周源重 (2) and the shuttle plasmid attenuated vaccinia virus MVA strain of vaccinia virus in peripheral endogenous heavy manipulation allows cells

组; group;

(3) 通过筛选标记,对重组病毒进行单斑筛选,以分离获得纯的高度减毒痘苗ADS-MVA病毒。 (3), for screening recombinant viruses by a plaque single selection marker, separate to obtain pure ADS-MVA highly attenuated vaccinia virus.

本发明还涉及采用上述SARS-CoV疫苗的制备方法制得的SARS-CoV疫苗(ADS-MVA )。 The present invention also relates to a method for preparing the above SARS-CoV vaccine prepared SARS-CoV vaccine (ADS-MVA).

此外,本发明涉及前述减毒重组疫苗SARS-CoV疫苗ADS-MVA在治疗或预防SARS-CoV感染及疾病中的应用。 Further, the present invention relates to SARS-CoV infection and diseases of the application of recombinant attenuated SARS-CoV vaccine ADS-MVA vaccine in treatment or prevention.

本发明涉及前述构建减毒重组痘苗SARS疫苗ADS-MVA的方法在构建重组MVA表达外源基因中的应用。 The present invention relates to the recombinant vaccinia construct attenuated SARS vaccine ADS-MVA MVA method of application in the construction of a recombinant foreign gene expression.

本发明进一步涉及构建前述减毒重组痘苗SARS疫苗ADS-MVA的方法在构建重组MVA表达外源病原体抗原制备病原体检测和诊断试剂中的应用。 The present invention further relates to a recombinant vaccinia virus construct the attenuated SARS vaccine ADS-MVA method of constructing the recombinant MVA exogenously applied pathogen detection and diagnostic reagents prepared pathogen antigen expression.

此外,本发明涉及前述减毒重组痘苗SARS-CoV疫苗ADS-MVA在中国恒河猴体内预防致病性SARS-CoV感染的方法,其特征在于,在猴子攻毒试验中,恒河猴分别在第0和28天接种ADS-MVA两次,两次接种剂量都是5 X108TCID50,半数的疫苗通过肌肉接种,另外一半经过鼻腔给与,4周后每只动物用105TCID50通过鼻腔接种病毒SARS-CoVPUMCOl。 Further, the present invention relates to a method for attenuating vaccinia recombinant SARS-CoV vaccine ADS-MVA in Chinese rhesus monkey pathogenic prevention of SARS-CoV infection, characterized in monkeys challenge experiment, rhesus respectively seeded on day 0 and 28 ADS-MVA twice, inoculation dose is 5 X108TCID50, half of the vaccine is inoculated intramuscularly, the other half through the nasal cavity to give, after 4 weeks of each animal were inoculated intranasally with virus 105TCID50 SARS-CoVPUMCOl .

本发明还涉及一种假病毒作中和试验的方法,其特征在于,用分别携带SARS-CoV的S基因禾n HIV-1主要核心基因的pcDNASopt9和pNL4-3Luc+Env-Vpr-两个质粒共转染293T细胞,以制备假病毒;中和抗体活性用假病毒入侵试验检测,按一定比例稀释的血清样品和同等量的假病毒在37。 The present invention further relates to a process for virus neutralization test prosthesis, characterized in that, by carrying pcDNASopt9 of SARS-CoV and the S pNL4-3Luc Wo n HIV-1 gene main core gene + Env-Vpr- two plasmids co-transfected 293T cells, to prepare a false virus; neutralizing activity with fake test detecting viruses, diluted serum samples by a certain percentage, and the same amount of 37 sham virus. C下培养,血清/病毒混合物分别加入293-ACE2细胞中,感染细胞裂解检测荧光酶活性。 C under culture, the serum / virus mixture were added 293-ACE2 cells, infected cell lysate luciferase activity detected.

考虑到转入的S基因可能改变ADS-MVA的细胞趋向性,这可能影响疫苗的安全范围。 Taking into account the transfer of the S gene may alter ADS-MVA cell tropism, which may affect the safety of the vaccine range. 我们在哺乳动物中检测了ADS-MVA的生长特性,我们用ADS-MVA感染几种哺乳动物细胞系,293, Hela和Vero细胞。 We detected in mammals, the growth characteristics of ADS-MVA, we infect several mammalian cell lines with ADS-MVA, 293, Hela and Vero cells. 与在CEF细胞中观察到的典型的扩展性生长模式相比(图2A), ADS-MVA感染的哺乳细胞仅限于个别的细胞中。 Scalability compared to typical growth pattern observed in CEF cells (FIG. 2A), ADS-MVA infected mammalian cells is limited to individual cells. 因此,尽管S蛋白仍然表达,与野生型的MVA — 样,ADS-MVA没有在哺乳动物细胞中复制扩散。 Thus, while still expressing S protein, wild type MVA - like, ADS-MVA does not replicate in mammalian cells proliferation. 另外,ADS-MVA在CEF 细胞中传代9次,但是这些传代没有导致S蛋白表达的缺失,提示ADS-MVA 遗传稳定性很好 Further, in the ADS-MVA CEF cells were passaged 9 times, but did not result in deletion of these passaged S protein expression, suggesting good genetic stability ADS-MVA

在疫苗研制中,重组痘苗-SARS疫苗具有许多优点,有可能成为理想的一种疫苗,本发明利用修饰后的痘苗(MVA)为载体构建SARS疫苗。 In the vaccine, the recombinant vaccinia -SARS vaccine has many advantages, it may be desirable to a vaccine, vaccinia (MVA) The present invention utilizes the modified vector construct is a SARS vaccine. 不像传统的痘苗可以引起医院感染和死亡。 Unlike conventional vaccinia can cause hospital infections and deaths. MVA因为不含病毒基因组中的宿主基因,而不能在人体内和绝大多数哺乳动物细胞中复制,而且MVADNA基因表达没有受影响,在人体细胞内能合成早期和晚期基因。 Because the host genome MVA-free viral genome, and can not replicate in humans and in the majority of mammalian cells, and gene expression MVADNA not affected in human cells can synthesize early and late genes. 更重要的是,MVA 已经针对天花作了上亿次应用于大规模的疫苗试验和临床试验,在使用过程中没有发现副作用,即使是高危病人或实验免疫缺陷的猴子。 More importantly, MVA has been used in hundreds of millions of large-scale vaccine trials and clinical trials against smallpox, no known side effects during use, even for high-risk patients or experimental immunodeficiency monkeys. 在本发明中, 全长SARS-CoV包膜S糖蛋白基因转入了MVA缺失III区基因,我们选择S 蛋白是因为它作为细胞和体液免疫的主要靶蛋白介导了病毒的入侵,新产生的重组ADS-MVA能够在小鼠、兔子和猴子中经过两次免疫后引起高水平的中和抗体,在恒河猴中引起的中和抗体能够防止SARS病毒的入侵。 In the present invention, the full-length SARS-CoV S glycoprotein envelope gene into the MVA deletion III region of the gene, because we chose it as the S protein cellular and humoral immunity mediated primarily target the virus invasion, newly generated ADS-MVA can be recombinant after two immunization induced high levels of neutralizing antibodies in mice, rabbits and monkeys, neutralizing antibodies in rhesus monkeys caused by SARS virus invasion can be prevented. 经疫苗免疫原性测定,疫苗安全性测定,疫苗有效性测定,以及抗体测定,病毒载量体内动态检测,SARS病理程度变化等指标确定,重组痘苗SARS疫苗具有良好的免疫原性、安全性和有效性,可以作为一个潜在的安全的针对人类和动物SARS病毒感染的有用的预防性疫苗,为防治SARS筛选到理想的疫 Determined by vaccine immunogenicity and safety of the vaccine was measured, determine the effectiveness of vaccines and antibody assay, viral load body motion detection, and other indicators of the degree of pathological changes determined SARS, SARS recombinant vaccinia virus vaccine with good immunogenicity and safety effectiveness, can be used as a potential security useful prophylactic vaccines against human and animal SARS virus infection, screening for the prevention and treatment of SARS to the ideal of Phytophthora

苗是安全的,应尽早进行临床试验。 Vaccine is safe, clinical trials should be carried out as soon as possible.

附图说明 BRIEF DESCRIPTION

图1表示SARS疫苗ADS-MVA的构建方法的一例。 FIG 1 shows an example of the method for constructing the ADS-MVA SARS vaccine.

图2表示S蛋白和GFP (绿色荧光蛋白)共表达于ADS-MVA感染的CEF细胞中,其中A显示S蛋白,B显示GFP。 Figure 2 shows the S protein and GFP (green fluorescent protein) expressed in CEF cells were co-ADS-MVA infection, wherein A shows the S protein, B show GFP.

图3表示含有优化的S基因片段的DNA疫苗的构建和表达。 FIG 3 shows the construction and expression of the DNA vaccine containing S-optimized gene fragment.

图4表示ADS-MVA免疫BALB/c小鼠后的抗SARS-CoV特异性中和抗体反应的检测结果。 Figure 4 shows the result of the detection of anti-SARS-CoV specific for the ADS-MVA immunized BALB / c mice of antibody responses.

图5表示ADS-MVA免疫新西兰白兔后的抗SARS-CoV特异性中和抗体 Figure 5 shows the anti-SARS-CoV-specific post-ADS-MVA immunization of New Zealand White rabbits and antibodies

反应的检测结果。 A detection result of the reaction.

图6表示ADS-MVA免疫恒河猴后的抗SARS-CoV特异性中和抗体反应的检测结果。 6 shows the results of the detection of anti-SARS-CoV specific ADS-MVA after immunization and antibody response in rhesus monkeys.

图7表示DNA疫苗免疫新西兰白兔后的抗SARS-CoV特异性中和抗体反应的检测结果。 Figure 7 shows the detection result of specific anti-SARS-CoV vaccine after DNA in New Zealand White rabbits and antibody response.

图8显示融合有人类Fc的ACE2受体结合区域(RBR-Fc)对新西兰白兔(A) , BALB/c小鼠(B),恒河猴(C)的抗SARS-CoV特异性中和抗体的吸附和清除。 8 shows a fused human Fc receptor binding region of ACE2 (RBR-Fc) New Zealand White rabbits (A), BALB / c mice (B), rhesus (C) an anti-SARS-CoV specific neutralizing absorption and clearance of the antibodies. 具体实施方式 Detailed ways

实施例1 Example 1

构建pZC3d插入载体:把通过DNA合成的pH5启动子导入pLW7载体(NIH的Bernie Moss禾口Lynda Wyatt博士提供,参考Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model. Linda S. Wyatt. et. al. Vaccine. Vol. 14, No.l5, 1451-1456. 1996),构建新一代含双启动子的pZC3d载体。 Construction pZC3d inserted into a vector: the by DNA synthesis pH5 promoter introduced pLW7 vector (NIH's Bernie Dr. Moss Hekou Lynda Wyatt provided, reference Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model. linda S. Wyatt. et. al. Vaccine. Vol. 14, No.l5, 1451-1456. 1996), a new generation of vectors constructed with double pZC3d promoter. 在这个载体中,S基因在强合成启动子pSYN下,报告基因GFP基因在弱启动子pH5 (图l)下,两个基因转入相同的插入序列中,GFP作为代理标记用于携带S基因的重组MVA载体(NIH的Bernie Moss和Lynda Wyatt博士提供, 参考Vaccine Protocols, Second Edition, August 2003, pps. 51-68, ISBN1-59259-399-2 Series: Methods in Molecular Medicine; Volume #: 87; by Caroline Staib and Gerd Sutter)。 In this vector, S gene under a strong promoter pSYN synthesis, and GFP gene under a weak promoter pH 5 (Fig L), two gene into the same insertion sequence, for carrying GFP as a proxy marker genes S the recombinant MVA vector (NIH's Bernie Moss and Dr. Lynda Wyatt provided, reference Vaccine Protocols, Second Edition, August 2003, pps 51-68, ISBN1-59259-399-2 Series: Methods in Molecular Medicine; Volume #:. 87; by Caroline Staib and Gerd Sutter). pSYN和pH5这两个启动子都是痘苗特异性的启动子,新的载体允许多基因插入到单一重组MVA病毒。 pSYN and pH5 these two promoters are vaccinia-specific promoter, a new carrier allows multiple genes are inserted into a single recombinant MVA virus. 像pLW7—样, pZC3d的插入位点是MVA基因的缺失III区(参见图1)。 Like pLW7- like, pZC3d insertion sites of the MVA deletion III is the gene (see FIG. 1). 实施例2 Example 2

构建和纯化ADS-MVA:采用平端连接的方法,将GFP基因和S基因构建到pZC3d中。 Construction and purification of ADS-MVA: method of blunt end ligation, the construct GFP gene and the S gene into pZC3d. S基因来自SARS-CoVHKU39849病毒株的cDNA,该病毒分离自香港(S基因保藏于GenBank,保藏号AY27S491)。 S gene from cDNA SARS-CoVHKU39849 virus strain, the virus isolated from Hong Kong (deposited in GenBank Accession S gene, accession number AY27S491). 重组的ADS-MVA 通过同源重组的方法在鸡胚成纤维细胞(CEF)中产生:首先,用亲代MVA 感染CEF细胞(IMOI)。 Recombinant ADS-MVA by homologous recombination in chicken embryo fibroblast cells produced (CEF): first, by parental MVA CEF cells were infected (IMOI). 90分钟后,使用Effectene (Qiagen Cat: 301427), 用pZC3d转染同一群细胞;48小时后,阳性细胞群通过GFP在荧光显微镜下发出的荧光挑选出来;重组的ADS-MVA通过8次感染CEF细胞传代得以纯化。 After 90 minutes, using Effectene (Qiagen Cat: 301427), with pZC3d transfected with a population of cells; after 48 hours, the positive cell population chosen by GFP fluorescence emitted by fluorescence microscopy; recombinant ADS-MVA by 8 times in CEF infected cells were passaged to be purified. 由此,使用pZC3d,我们将野生型的SARS全长S基因和GFP重组到MVAIII区中,得到ADS-MVA (参见图1)。 Accordingly, the use of pZC3d, we recombinant wild type full-length SARS S gene and GFP into the region MVAIII afford ADS-MVA (see FIG. 1). 为了比较,采用同样的方法,我们将经过修饰HCV E1E2基因导入相同位点,构建了另一个重组病毒ADC-MVA。 For comparison, using the same method, we modified HCV E1E2 gene into the same site to construct a recombinant virus further ADC-MVA.

实施例3 Example 3

免疫荧光试验:为了检测细胞表面表达的S糖蛋白,我们做了免疫荧光 Immunofluorescence assay: In order to detect the S glycoprotein expressed on the cell surface, we did immunofluorescence

试验。 test. 简单步骤为第1天把CEF细胞铺到6孔板上(每个孔有2xl(^个细胞), 平板经ConA(100iig/ml)预处理30分钟,PBS清洗两次。第2天,用重组后的连续稀释的ADS-MVA (1:10)感染CEF细胞,两个小时后,细胞培养基用含2%的胎牛血清(FCS) DMEM替换,于37°C 5%(:02孵育48小时,感染后的细胞用热灭活的SARS病人血清(WH,1:500)孵育1小时,然后用Alexa Fluor594标记的羊抗人IgG (H+L) (Molecular Probes, USA)孵育30分钟, 细胞用PBS洗三遍,阳性群落用免疫荧光纤维镜鉴别,结果参见图2A。 CEF 细胞感染对照组ADC-MVA也在每个试验中作为对照染色。 实施例4 Simple steps Day 1 CEF cells plated onto 6-well plate (each well has a 2xl (^ cells) plates pretreated with ConA (100iig / ml) 30 minutes, washed twice with PBS. Day 2, with serially diluted ADS-MVA (1:10) CEF cells infected with the recombinant, two hours later, cell culture medium replaced with DMEM fetal calf serum (FCS) containing 2% is used, at 37 ° C 5% (: 02 of incubation 48 hours after the cells were infected with the SARS heat inactivated serum (WH, 1: 500) for 1 hour, and then labeled with Alexa Fluor594 goat anti-human IgG (H + L) (Molecular Probes, USA) for 30 min , the cells were washed with PBS three times, community positive identification by immunofluorescence microscopy fiber, see the results in FIG. 2A. CEF cells infected control ADC-MVA as a control in each test are also stained. Example 4

ADS-MVA病毒株的制备:该病毒株用CEF细胞经前面描述的方法纯化。 Preparation ADS-MVA strains: purified virus strain CEF cells by using the method previously described. 纯化的ADS-MVA在CEF细胞中用2000 ml转瓶培养技术扩增制备。 Purified ADS-MVA in CEF cells with 2000 ml spinner flasks culture techniques amplify the. 实施例5 Example 5

Western blot分析:为了确定ADS-MVA中全长S糖蛋白所含中和抗体决定表位的区域,我们制备了一批DNA疫苗,它们含不同长度的S基因。 Western blot analysis: To determine the full length S ADS-MVA glycoproteins contained in the region and the antibody epitope, we prepared a number of DNA vaccines, which contain the S gene of different lengths. 为了S蛋白更好的在哺乳细胞中表达,我们用基因工程的方法将S基因的密码子序列进行了优化。 In order to better the S protein expression in mammalian cells, we used genetic engineering to S sequence of a gene codon optimized. S基因的前导序列用组织纤维蛋白溶酶原激活剂基因(tissue plasminogen activator, Tpa)的前导序列替换,该前导序列能够通过促进蛋白质从内质网转移到高尔基氏体上而促进蛋白质的表达。 Leader sequence with a leader sequence of the S gene tissue plasminogen activator gene (tissue plasminogen activator, Tpa) is replaced by the preamble sequence is capable of promoting the transfer of protein from the endoplasmic reticulum to the Golgi apparatus to promote expression of the protein. 这些DNA疫苗在短暂表达试验中证明可以在哺乳细胞中表达。 These DNA vaccines proven to be expressed in mammalian cells in a transient expression assay. 主要方法是,用DNA疫苗哺乳类表达载体pcDNA-S200, pcDNA-S400, pcDNA-S600, pcDNA-S800, pcDNA-Sopt9,或pcDNA3.1转染293T细胞。 The main method is to use the expression vector pcDNA-S200 mammalian DNA vaccine, pcDNA-S400, pcDNA-S600, pcDNA-S800, pcDNA-Sopt9, pcDNA3.1 or 293T cells. 48小时后,转染后的细胞(lx106) 用PBS洗后放置干冰中用200(il细胞裂解液(50mM Tris-HCl (pH8.0), 137 mM NaCl, 2 mM EDTA, 0.5% NP-40, 10% glycerol, and lpg/ml of each of pepstatin, leupeptin and pefabloc)裂解30分钟。细胞碎片于4°C 、 14,000rpm离心10分钟,乘IJ下的细胞裂解上清液在10。/。SDS聚丙烯酰胺凝胶上电泳。将分离得到的蛋白转移到硝酸纤维素膜上,做抗体印记试验,结果参见图3 , S蛋白也结合N末端400个氨基酸的兔抗体,S蛋白在分子量为160和250Kd标记带中出现,这比实际预测值要大,这种改变可能是因为蛋白质翻译后加工所致。 阳性的血清样品来自pcDNA-S400经体内电转染技术免疫兔子得到的血清。 pcDNA-S400编码S基因N末端400个氨基酸,阴性血清来自使用安慰剂注射的健康的动物。为了S蛋白更好的在哺乳细胞中表达,我们用基因工程的方法将S基因的密码子序列进行了优化。S基 After 48 hours, cells (lx106) transfected with a dry ice is placed washed with PBS 200 (il cell lysate (50mM Tris-HCl (pH8.0), 137 mM NaCl, 2 mM EDTA, 0.5% NP-40 , 10% glycerol, and lpg / ml of each of pepstatin, leupeptin and pefabloc) lysed 30 min. cell debris at 4 ° C, centrifuged at 14,000rpm 10 minutes by the cell lysis supernatant in the IJ 10./.SDS polyacrylamide gel electrophoresis. the separated proteins were transferred to nitrocellulose, antibody do mark test, the results see Fig. 3, S protein also binds rabbit antibody N-terminal 400 amino acids, S protein of molecular weight 160 250Kd and occurring in the tag tape, which may be larger than the actual predicted values, because this change caused by protein post-translational processing. positive serum samples from serum by pcDNA-S400 vivo transfection electrical immunize rabbits obtained. pcDNA- S gene encoding the N-terminal 400 amino acids S400, negative serum from a healthy placebo injected animals. for better expression of protein S in mammalian cells, we used genetic engineering methods to S sequence of a gene codon optimized .S base 的前导序列用组织纤维蛋白溶酶原激活剂基因(tissue plasminogen activator, Tpa)的前导序列替换,该前导序列能够通过促进蛋白质从内质网转移到高尔基氏体上而促进蛋白质的表达,我们通过Western blot试验显示这些蛋白质能够在人293T细胞中表达, 为了确保产生抗体反应,我们通过体内电转染技术将DNA质粒转入兔子中。 The leader sequence leader sequence replaced the plasmin by tissue plasminogen activator gene (tissue plasminogen activator, Tpa) of the preamble sequence can be transferred by promoting protein from the endoplasmic reticulum to the Golgi apparatus to promote expression of the protein, by us Western blot test showed that these proteins can be expressed in human 293T cells, in order to ensure an antibody response, we have by in vivo electroporation of plasmid DNA transfection techniques into rabbits.

病毒接种方法SARS冠状病毒(SARS-CoV)分离自中国SARS病人, 患者为北京协和医院SARS病房患者,所分离的病毒经协和医院在Vero细胞上培养传代,经RT-PCR和电镜形态观察确定为SARS冠状病毒,病毒TCID50为106/ml。 Virus inoculation SARS coronavirus (SARS-CoV) isolated from Chinese SARS patient, patient for the Beijing Union Medical College Hospital in SARS patients, the isolated virus by Union Hospital cultured and propagated in Vero cells by RT-PCR and electronic form viewer identified as SARS coronavirus, the virus TCID50 to 106 / ml. 该病毒被我们命名为PUMC-01株,其序列与北京4株(BJ01-4) 有一定差异,而与香港SU10株,新加坡及加拿大的SARS-CoV株有更近的进化关系(Genbank Accession No., AY350750)。 The virus is named we PUMC-01 strain, whose sequence Beijing four (BJ01-4) have some differences, but the strain SU10 Hong Kong, Singapore and Canada's SARS-CoV strain have a closer evolutionary relationship (Genbank Accession No ., AY350750). SARS冠状病毒经鼻腔滴鼻接种,所有动物实验均在P3动物实验室中按有关实验动物所实验动物使用及实验规程进行。 SARS coronavirus intranasal inoculated intranasally All animal experiments were in P3 laboratory animals in experimental animals by about animals and experimental procedures experiments. 实施例7 Example 7

免疫动物:用ADS-MVA在第0, 3周分别经肌肉注射(im)免疫6〜8月龄的雌性BALB/c小鼠,按照免疫剂量的不同分为以下几组:ADS-MVA5x106 TCID50或5xl(fTCID50, ADC-MVA,生理盐水对照组,两周后作第二次注射,小鼠安乐死后采血液样品分析。在第0和28天,2只兔子用lx108 TCID50 ADS-MVA免疫,另外两只用1x1()8tCID50ADC-MVA免疫。8只恒河猴在第0和28天免疫两次,其中4只接种ADS-MVA,另外4只接种ADC-MVA。 最初免疫剂量是lxl08TCID50, 二次免疫剂量是3xl()STCID50。每次免疫后两周,采取血液样品分析。DNA疫苗接种,每组有2只兔子在第0和28天使用体内电穿孔技术,接种单纯的400pg DNA质粒(pcDNA-S200, -S400, -S600 and -S800)。而且,最后一次DNA免疫后2个月,这些兔子用lx108 TCID50ADS-MVA每4周加强一次。对照组兔子用表达HCV E1E2的相似的DNA质粒接种,对照组也用ADC-MVA以同样的方式加强,在每组中,都 Immunized animals: with ADS-MVA at 0 and 3 weeks, respectively, given intramuscularly (im) immunization of female BALB 8 ~ 6 months of age / c mice immunized according to different doses into the following groups: ADS-MVA5x106 TCID50 or 5xl (fTCID50, ADC-MVA, saline control group, made after two weeks a second injection, mice were euthanized after a blood sample analysis taken at 0 and 28 days, two rabbits with lx108 TCID50 ADS-MVA immunization, additional with two 1x1 () 8tCID50ADC-MVA immunization at 0 .8 macaques immunized twice, and 28 days, four vaccinated ADS-MVA, seeded four additional ADC-MVA. primary immunization dose lxl08TCID50, secondary immunizing dose is 3xl () STCID50. two weeks after each immunization, blood samples were taken to analyze .DNA vaccination, each with two rabbits using an in vivo electroporation at 0 and 28 days, inoculated 400pg DNA plasmid alone (the pcDNA -S200, -S400, -S600 and -S800). Further, after the last DNA immunization for 2 months, the rabbits with lx108 TCID50ADS-MVA reinforcing every 4 weeks the control group rabbit inoculated with a similar plasmid expressing HCV E1E2 DNA of the control group in the same manner also reinforcing ADC-MVA, in each group, are 取血清样品并在免疫2周后分析。 Serum samples were taken and analyzed 2 weeks after immunization.

在猴子攻毒试验中,共16只3岁恒河猴用于实验,分疫苗免疫和对照两组,每组各8只,疫苗免疫组8只猴编号为0411、 0412、 0413、 0414、 0415、 0416、 0417和0418;对照组8只编号为0419、 0420、 0421、 0422、 0423、 0424、 0425和0426。 Challenge test in monkeys, rhesus monkeys were 16 years old for 3 experiments, and the sub-control vaccine groups, n = 8, eight monkeys vaccinated group numbered 0411, 0412, 0413, 0414, 0415 , 0416, 0417 and 0418; 8 control group numbered 0419, 0420, 0421, 0422, 0423, 0424, 0425 and 0426. 恒河猴由中国医学科学院医学生物学研究所提供,接种前按实验用猴国家标准微生物SPF级进行检测,检查显示SARS病毒抗体为阴性。 Rhesus provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences, according to experiments with monkeys national standards for testing microbial SPF level before inoculation, examination showed that the SARS virus antibody negative. 16只恒河猴分别在第0和28天接种两次,其中8只接种ADS-MVA, 其他8只接种ADC-MVA。 16 rhesus monkeys were at 0 and 28 days vaccinated twice, eight vaccinated ADS-MVA, the other eight inoculated ADC-MVA. 两者接种的剂量都是5x108 TCID50。 Both doses were inoculated 5x108 TCID50. 半数的疫苗通过肌肉(im)接种的方式给予,另外一半经过鼻腔给予,4周后每只动物用105 TCID50 SARS-CoV PUMC01通过鼻腔接种。 Half way vaccine administered by intramuscular inoculation (IM), the other half through nasal administration, after 4 weeks of each animal with 105 TCID50 SARS-CoV PUMC01 inoculated through the nasal cavity. 需要注意的是,疫苗SARS-CoV HKU39849病毒株和SARS-CoV PUMC01的S基因序列(AY350750)完全一致,该实验在中国医学科学院实验动物研究所的P3实验室动物设施内完成。 It should be noted that the SARS-CoV HKU39849 vaccine virus strain and SARS-CoV PUMC01 the S gene sequence (AY350750) exactly the same, the completion of the experiment in P3 laboratory animal facility of the Institute of Chinese Academy of Medical Sciences experimental animals.

我们做了中和试验以检测疫苗产生的体液免疫的活性,本试验的优点在于消除使用活SARS-CoV在传统的中和试验中产生的不良影响,我们检测了ADS-MVA免疫动物后血清中产生的中和活性。 After we did the activity of humoral immune neutralization test to detect the vaccine, the advantage of this test is to eliminate the use of the adverse effects of live SARS-CoV produced in the traditional neutralization assay, we tested ADS-MVA immunized animal serum neutralizing activity generated. 热灭活动物血清中和抗体活性用假病毒入侵试验检测,该实验是根据文献中描述过的工作方法进行(参考Chen. Z., P. Zhou, et. al. Genetically divergent strains of simian immunodeficiency virus use CCR5 as a coreceptor for entry. J. Virol. 71: 2705-14, 1997.)。 Heat inactivated animal serum neutralizing activity with fake test detecting viruses, the experiment is carried out (according to Chen described with reference to the literature methods of work. Z., P. Zhou, et. Al. Genetically divergent strains of simian immunodeficiency virus . use CCR5 as a coreceptor for entry J. Virol 71:. 2705-14, 1997.). 假病毒用分别携带最优化的S基因和HIV-1主要基因的两个质粒pcDNASopt9和pNL4-3Luc+Env—Vpf共转染293T细胞得到,按一定比例稀释的血清样品和同等量的假病毒在37。 Pseudovirion optimized with, respectively, carry genes S and primary HIV-1 gene and two plasmids pcDNASopt9 pNL4-3Luc + Env-Vpf 293T cells were co-transfected to obtain, according to a certain percentage of serum samples diluted in the same amount and in pseudovirus 37. C下培养1小时,然后将血清/病毒混合物分别加入293-ACE2细胞中,56小时后,感染细胞裂解检测荧光酶活性。 Incubation at C 1 hour and then the serum / virus mixture was added to 293-ACE2 cells, respectively, after 56 hours, infected cells were lysed detecting luciferase activity.

首先,8只小鼠用ADS-MVA免疫。 First, the 8 mice immunized with ADS-MVA. 小鼠M260和M262用5xl06TCID50 的疫苗免疫,其它6只,M263, M264, MM1, MM2, MM3和MM4用5xl07 TCID50免疫,所有动物用肌肉注射方法接种,两次免疫间隔3周,在第2 次免疫后2周采取血液样品作中和抗体试验。 Mice M260 and M262 vaccine 5xl06TCID50 with the other six, M263, M264, MM1, MM2, MM3 and MM4 with 5xl07 TCID50 immunization, all animals by intramuscular inoculation, immunized twice 3 weeks apart, in the 2nd blood samples were taken two weeks for antibody test and after immunization. 在对照组,4只小鼠(M265,M266, M267 and M268)接种表达HCV E1E2蛋白的ADC-MVA,用于对照组的疫苗剂量是每只小鼠5xl(^TCID50,我们也用生理盐水接种了两只小鼠(Nl和N2) 作为对照,在用ADS-MVA免疫的8只小鼠中产生了高水平的中和抗体。在高剂量组,稀释IOOO倍以上的血清仍可抑制或中和50%的病毒(IC50)。 2个对照组中的小鼠没有产生中和抗体。另外,两个ADS-MVA剂量组有明显的剂量相关性(参见图4),因此,ADS-MVA在小鼠中诱导产生了SARS-CoV 特异性的中和抗体。 In the control group, 4 mice (M265, M266, M267 and M268) were inoculated HCV E1E2 protein expression ADC-MVA, the vaccine dose for each mouse of the control group is 5xl (^ TCID50, we inoculated with saline the two mice (Nl and N2) as a control, and produce high levels of neutralizing antibodies in the 8 mice immunized with ADS-MVA in the high dose group, more than IOOO-fold diluted serum can be suppressed or and 50% of the virus (IC50). 2 control groups of mice did not produce neutralizing antibodies. in addition, two ADS-MVA dose of clear dose related (see FIG. 4), thus, in the ADS-MVA induced in mice specific for the SARS-CoV neutralizing antibodies.

为了探讨在其它品系动物中,是否可以产生类似的中和抗体,我们用ADS-MVA免疫了两只兔子。 To explore other strains in animals, can produce similar neutralizing antibodies, we use the ADS-MVA immunization of two rabbits. 免疫程序在第2次免疫时有所不同,这次是间隔4周免疫,剂量的选择基于我们以前在小动物中检测MVAfflV-l疫苗试验的结果。 Immunization program has been in the second immunization, this time is the interval of 4 weeks immunization, dose selection is based on our previous detection result MVAfflV-l vaccine trials in small animals. 在接种ADS-MVA的两只兔子(R524禾D R525)中产生高水平的SARS-CoV特异性中和抗体。 Produce high levels of neutralizing SARS-CoV specific antibodies in the vaccinated ADS-MVA of two rabbits (R524 Wo D R525) in. 在血清稀释10000倍后仍可以检测到IC50。 After 10,000-fold serum dilution still can be detected IC50. 接种ADC-MVA的两只对照组的兔子(R520和R521)没有产生SARS-CoV中和抗体(参见图5),这些数据显示由ADS-MVA产生的高水平中和抗体并不具有品种特异性。 Two rabbits vaccinated control group of ADC-MVA (R520 and R521) did not produce neutralizing SARS-CoV strain-specific antibody (see FIG. 5), these data show that high levels of neutralizing antibodies produced by ADS-MVA does not have . 所有的测试的动物都能很好的耐受这种疫苗,实验中没有观察到明显的疾病或体重减轻。 All animals tested this vaccine can be well tolerated, the experiment did not observe significant disease or weight loss.

为了探讨ADS-MVA在人类中使用的可能性,我们进一步在恒河猴中检测了ADS-MVA。 In order to explore the possibility of ADS-MVA for use in humans, we further examined the ADS-MVA in rhesus monkeys. 本实验总共用了16只恒河猴,8只用ADS-MVA免疫,另外8只用ADC-MVA免疫,连续采取血清样品以检测中和抗体的出现。 This experiment used a total of 16 rhesus monkeys with 8 ADS-MVA immunization with eight additional ADC-MVA immunization, serum samples were taken continuously to detect the presence of neutralizing antibodies. 与对照组ADC-MVA相比,ADS-MVA免疫的恒河猴产生了高水平的SARS-CoV 中和抗体。 Compared with control ADC-MVA, ADS-MVA immunized rhesus produce high levels of neutralizing SARS-CoV antibodies. 因此,除了小动物之外,ADS-MVA在恒河猴中也可以产生中和抗体,考虑到SARS-CoV主要感染呼吸系统,我们也检测了肺脏和支气管中的中和抗体的滴度,肺和支气管组织样本和血清同时采取,但是在所有8只恒河猴中都没有检测到中和抗体,即使在最低的稀释度(<1:10)(参见图6)。 Therefore, in addition to small animals, ADS-MVA can produce neutralizing antibodies in rhesus monkeys, taking into account the major SARS-CoV infection in the respiratory system, we also examined the lungs and bronchi titers of neutralizing antibodies, lung serum and bronchial tissue samples taken at the same time, but was not detected in all eight antibodies in rhesus monkeys, even at the lowest dilution (<1:10) (see FIG. 6).

此外,采用体内电穿孔技术,分别用DNA疫苗S200 (R416, R417)、 S400 (R418, R419)、 S600 (R683, R684)以及表达HCVE1E2的DNA疫苗(R518, R519)对兔子进行了免疫,方法同上,均为2次免疫,间隔4周, 结果参见图7。 In addition, the use of in vivo electroporation techniques, respectively, with a DNA vaccine S200 (R416, R417), S400 (R418, R419), S600 (R683, R684) and expression of the DNA vaccine HCVE1E2 of (R518, R519) rabbits were immunized, method supra, were immunized twice, 4 weeks apart, the results of Figure 7.

临床观察和血液检测:16只动物实验前体温为(37.8〜38.rC),接种疫 Clinical observation and blood tests: first 16 animals of the experimental temperature (37.8~38.rC), inoculated with Phytophthora

苗和对照液后,未见体温升高。 After shoot and control solution, no temperature rise. 16只恒河猴在病毒接种的第1〜5天时有体温升高现象,为38.6〜39.2"C。部分对照动物有呼吸困难,摄食减少,体重减轻。未见咳嗽、流涕、呕吐、腹泻等临床表现。白细胞在感染前8只疫苗猴平均为(7.6士3.1)X103,疫苗免疫和病毒接种后未见明显改变;8只对照猴在感染前平均为(6.9土2.9)X103,病毒接种后的2, 5, 7和14天时明显降低, 平均范围(3.9土1.8〜5.4士1.9)X103。血液生化测定中AST在对照猴接种病毒后各吋间点有显著上升,但在疫苗猴病毒接种后7、 14天时有显著性上升, 其他指标如ALT, ALP, UREA, CRE, LDH, CK, TP和ALB在整个实验中未见明显改变。 实施例10 16 rhesus monkeys inoculated with the virus in the first 1 ~ 5 day phenomenon of elevated body temperature, as 38.6~39.2 "C. Part of the control animals had difficulty breathing, reduce food intake and weight loss. No cough, runny nose, vomiting, diarrhea . in front of white blood cells and other clinical manifestations of infection eight vaccine monkey average (7.6 persons 3.1) X103, the vaccine virus inoculation and no significant change; 8 prior to infection control monkeys average (6.9 earth 2.9) X103, virus inoculation after 2, 5, 7 and 14 days was significantly decreased, the average range (3.9 soil 1.8~5.4 disabilities 1.9) X103. biochemical assay AST in control monkeys after viral inoculation significantly increased point among inches, but the vaccine of the simian virus 7 after inoculation, 14 days significantly increased, other indicators such as ALT, ALP, UREA, CRE, LDH, CK, TP ALB and did not change significantly throughout the experiment. Example 10

抗体吸附试验:融合了人Fc的可溶性重组ACE2受体结合区域 Antibody assay: Fusion of human recombinant soluble Fc receptor binding region of ACE2

实施例9(S310-510) (RBR-Fc)用于吸附特异性结合受体结合区域的抗体。 Example 9 (S310-510) (RBR-Fc) specifically binds embodiment for adsorbing the receptor binding region of an antibody. 接着用特异性抗人IgG Fc-agarose (Sigma)去除RBR-Fc/抗体复合物。 Followed by removal of RBR-Fc / antibody complex with a specific anti-human IgG Fc-agarose (Sigma). 稀释的血清与可溶性的RBR-Fc在室温下孵育45分钟,然后在每个孔中加10^1预洗的抗人IgG Fc琼脂糖。 Diluted sera with soluble RBR-Fc incubated for 45 minutes at room temperature, each well was then added 10 ^ 1 anti-human IgG Fc Sepharose prewashed. 在室温下微平板震荡约2小时防止凝胶沉淀,进行吸附。 Microplate shaking at room temperature for about 2 hours to prevent gel precipitation, adsorption. 微平板在室温下3000转离心10分钟,剩余未结合的血清使用假病毒做中和试验。 Microplates at 3000 rpm for 10 min at RT, the remaining unbound serum using fake virus neutralization test done. 为了确保试验的特异性,在微平板中设置平行对照组,包括抗人IgG凝胶和RBR-Fc两组。 To ensure the specificity of the tests provided in the parallel control group microplate, a gel comprising anti-human IgG and RBR-Fc groups. 在另外一组实验中,由于含有抗体的猴子血清与包被在载体凝胶珠上的抗人Fc抗体交叉反应,,我们不得不采取不同的方法,我们使用产品使用手册中介绍的一种化学方法使Sepharose 4 Fast Flow beads (Amersham Biosciences)直接结合RBR-Fc。 In another set of experiments, since the packet monkey serum containing the antibody is cross-reactive anti-human Fc antibody on a carrier gel beads ,, we have to take a different approach, we use a chemical product described in the manuals The method of making Sepharose 4 Fast Flow beads (Amersham Biosciences) bind directly RBR-Fc. 没有用可溶性的RBR-Fc吸附抗体,而是使用RBR-Fc结合珠吸附和去除结合RBR部分的猴子抗体,其余的实验方法与前面相同。 With no adsorption of soluble RBR-Fc antibodies, but the use of RBR-Fc binding beads adsorb and remove RBR monkey antibody binding moiety, the remaining experimental procedure the same as before.

兔子和小鼠的血清根据中和抗体试验的结果稀释了1:300〜1:600倍,以便于消除过多的中和抗体对实验结果的影响,可溶性RBR-Fc能够吸附和去除兔子免疫血清中的大多数中和抗体,在RBR-Fc(-)组的琼脂糖珠对中和抗体没有作用。 Rabbits and mice sera diluted 1 antibodies tested and results: 300~1: 600 times, in order to eliminate the influence of excess neutralizing antibody of the experimental results, the soluble RBR-Fc rabbit serum can be adsorbed and removed most of neutralizing antibodies in RBR-Fc (-) group agarose beads no effect on neutralizing antibodies. 有意思的是,DNA疫苗免疫的动物(R681.D和R682.D)血清中的中和抗体活性几乎完全被消除。 Interestingly, animals immunized with DNA vaccine (R681.D and R682.D) serum neutralizing antibody activity was almost completely eliminated. 相对应的是,ADS-MVA疫苗免疫的动物, 其吸附后的血清保留部分中和活性,提示ADS-MVA可诱导广谱的中和抗体。 Correspondingly, ADS-MVA vaccine animal serum and the remaining portion of its activity after adsorption, suggesting ADS-MVA can induce broadly neutralizing antibodies. ADS-MVA比S600 DNA疫苗产生更广谱的中和抗体活性,即全长S蛋白可能存在多个中和抗体活性部位(参见图8A)。 ADS-MVA produce a broader spectrum of activity than the antibodies and S600 DNA vaccines, i.e., there may be a plurality of full-length S protein and the antibody active site in (see FIG. 8A).

ADS-MVA免疫小鼠,发现同样的结果。 ADS-MVA immunized mice, found similar results. 绝大多数的小鼠中和抗体被RBR-Fc重组蛋白吸附或去除,因此,我们的数据显示在ACE2结合区域含有一个主要的中和抗体决定位点(参见图8B)。 The vast majority of mice and antibodies are adsorbed or removed RBR-Fc recombinant protein, therefore, our data show contains a major neutralizing antibody sites determined (see FIG. 8B) in ACE2 binding region.

在检测猴子血清试验中,用RBR-Fc结合的琼脂糖4快流珠子(Sepharose 4 Fast Flow beads)去吸附和去除猴子血清中和RBR部分结合的中和抗体。 In the test monkey serum, with RBR-Fc binding sepharose 4 fast flow beads (Sepharose 4 Fast Flow beads) to adsorb and remove the monkey serum neutralizing antibodies bound and RBR portion. 在图8C所示,没有RBR-Fc的珠子(-)对中和抗体的活性没有作用;而RBR-Fc In FIG. 8C, no beads of RBR-Fc (-) activity neutralizing antibody had no effect; and RBR-Fc

珠子(+)能够去除大多数的中和抗体,这一结果类似于兔子和小鼠血清试验结果。 Beads (+) can be removed and most of the antibodies, the results were similar to the results of the test rabbit and mouse serum. 综合我们的实验结果,我们得出结论,ADS-MVA引起中和抗体,首先针对SARS-CoV受体ACE2的结合区域。 Comprehensive results of our experiments, we conclude, ADS-MVA induced neutralizing antibodies against first binding region of SARS-CoV receptor ACE2. 这个结论在3个物种中都有体现,同时提示由ADS-MVA产生的中和抗体没有种属特异性(参见图8C)。 This conclusion is reflected in the three species, and also suggest the antibodies produced by ADS-MVA did not species-specific (see FIG. 8C).

SARS-CoV分离试验:SARS-CoV病毒接种恒河猴后,在第2、 5、 7天收集鼻咽拭子标本,充分浸泡在lmlDMEM培养液中,经0.22pm滤膜过滤后, 接种Vero细胞,吸附1小时,病毒分离。 SARS-CoV separation test: After rhesus monkeys inoculated with SARS-CoV virus, nasopharyngeal swab specimens were collected at 2, 5, 7, lmlDMEM fully immersed in the culture broth, after 0.22pm membrane filter, Vero cells were seeded , 1 hour adsorption, the virus was separated. 在攻毒后7天和30天时安乐死, 每只动物的肺组织作匀浆后用于病毒分离。 7 days after challenge were euthanized 30 days, each animal after lung homogenate as for virus isolation. 将肺组织匀浆接种Vero细胞后进行病毒分离。 Virus isolation after lung homogenates inoculated Vero cells. 吸附1个小时后,洗脱后换新鲜的培养基,培养物观测细胞病变(CPE) 10天,如果没有发现CPE,用培养液盲接种第二批Vero细胞,再观察10天。 After 1 hour adsorption, the elution of fresh medium exchange, the culture was observed cytopathic effect (CPE) 10 days if no CPE was found, with a second batch of inoculated broth blind Vero cells, then observed for 10 days. 如果发现CPE,用免疫荧光试验(方法同上)检测Vero细胞中的SARS-CoV特异性抗原,以避免出现假阳性。 If the CPE found by immunofluorescence test (as above) Vero cells detect SARS-CoV-specific antigen, in order to avoid false positives. 分离检测结果参见表l Table l separation detection result

实施例12 Example 12

逆转录多聚酶链式反应(RT-PCR):在SARS-CoV病毒接种恒河猴的标本样品中检测病毒RNA,总RNA使用MagNA Pure LC总核酸分离试剂盒(Roche Diagnostics)从各种标本中分离,分离到的RNA再使用一种商业SARS-CoV RT-PCR kit (Roche Diagnostics)检测,结果参见表1 。 Reverse transcription polymerase chain reaction (RT-PCR): Detection of Viral RNA in the sample specimens of SARS-CoV virus vaccinated rhesus monkeys, total RNA using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics) isolated from various specimens , RNA was then isolated using a commercial SARS-CoV RT-PCR kit (Roche Diagnostics) test, summarized in Table 1.

表1.中国恒河猴接种SARS-CoV后病毒的检测结果。 Table detection result of the virus after vaccination SARS-CoV 1. Chinese rhesus monkeys. <table>table see original document page 13</column></row> <table>* *从肺组织匀浆中分离病毒***对鼻咽拭子标本进行RT-PCR <Table> table see original document page 13 </ column> </ row> <table> * * *** virus isolated from lung tissue of nasal swab specimens RT-PCR

实施例13 Example 13

病理组织切片制备:在病毒接种第7天和一个月后处死的动物中,取肺 Preparation of histopathology: after virus inoculation 7 days month animals were sacrificed, lung

等组织,10°/。 And other organizations, 10 ° /. 甲醛固定,石蜡包埋,常规病理组织切片处理后,HE染色, 镜检。 Formalin-fixed, paraffin-embedded, the processing routine histopathology, HE staining, microscopy. 结果显示,疫苗免疫组显示攻毒7天后2只动物出现肺间质性炎,攻毒35天后1只动物出现肺间质性炎,并出现肺胸膜水肿渗出,另3只动物呈局灶性肺间质性炎;对照组显示攻毒7天后4只动物中均存在肺间质性炎, 病变程度较重,各只动物病变范围大小略有差异。 The results showed that immunized group showed challenged 7 days after the 2 animals developed interstitial lung inflammation, a challenge 35 days after the animals developed interstitial lung inflammation, and pulmonary edema, pleural effusion, and the other three animals showed focal interstitial lung inflammation; control group showed four animals challenged 7 days in the presence of both interstitial lung inflammation, severe lesions, lesions in each animal slightly different size ranges. 攻毒35天后4只动物中均存在肺间质性炎,病变程度较攻毒7天后4只动物轻,各只动物病变范围大小略有差异。 Four animals challenged 35 days in both the presence of interstitial lung inflammation, lesions than the animals challenged four light 7 days, each animal lesion slightly different size ranges.

从以上病理比较结果看,与模型组相比,疫苗组动物的肺部病变较轻, 初步试验结果提示①此次受检的SARS相关疫苗可以减轻SARS病毒对受试动物恒河猴的攻击;②该疫苗对受检的其它脏器未见损伤性病理改变。 From the above comparison pathology results, compared with the model group, the vaccine group animals mild lung disease, preliminary test results suggest that ① the subject of SARS-associated SARS virus vaccine can reduce the tested animals rhesus monkeys attack; ② the vaccine of subjects no damage other organs pathological changes.

Claims (8)

1. 一种在减毒痘苗病毒MVA基础上构建的SARS-CoV疫苗ADS-MVA,所述的SARS-CoV疫苗ADS-MVA是将SARS-CoV的S基因插入到减毒痘苗病毒MVA缺失III区的MVA疫苗,其特征在于,所述SARS-CoV疫苗ADS-MVA包括SARS-CoV的S基因的启动子pSYN,且SARS-CoV的S基因位于该启动子pSYN的下游。 An attenuated vaccinia virus MVA constructed on the basis of the SARS-CoV vaccine ADS-MVA, the SARS-CoV vaccine ADS-MVA is a SARS-CoV S gene inserted into the attenuated vaccinia virus MVA deletion III the MVA vaccine, wherein said SARS-CoV vaccine ADS-MVA include promoters pSYN S gene of SARS-CoV, and the SARS-CoV S gene is located downstream of the promoter pSYN.
2. 根据权利要求1所述的SARS-CoV疫苗ADS-MVA,其中,所述MVA 缺失III区还插入了筛选标记。 The SARS-CoV vaccine as claimed in claim ADS-MVA claim 1, wherein the MVA deletion III region further inserted gene.
3. 根据权利要求2所述的SARS-CoV疫苗ADS-MVA,其中,所述筛选标记为GFP基因,所述GFP基因的启动子为pH5,且GFP基因位于该启动子pH5的下游。 According to claim SARS-CoV ADS-MVA vaccine of claim 2, wherein said selectable marker gene is GFP, the GFP gene promoter to pH5, and the GFP gene is located downstream of the promoter of pH5.
4. 权利要求l所述的SARS-CoV疫苗ADS-MVA的制备方法,其包括:a. 构建穿梭质粒,该质粒包含SARS-CoV的S基因,以及减毒痘苗病毒MVA缺失III区基因编码区序列或与编码区外两侧序列同源的侧翼序列,所述穿梭质粒的插入位点是减毒痘苗病毒MVA的缺失III区;b. 在痘苗病毒的允许细胞中使穿梭质粒与减毒痘苗病毒MVA株同源重组;c. 通过筛选标记,对重组病毒进行单斑筛选,以分离获得纯的高度减毒痘苗ADS-MVA病毒,其中,所述穿梭质粒是SARS-CoV的S基因位于启动子pSYN下游的穿梭质粒。 SARS-CoV vaccine prepared by the method of ADS-MVA according to claim l, comprising:. A shuttle plasmid, the plasmid containing the S gene of SARS-CoV, and the attenuated vaccinia virus MVA deletion III region coding region or sequences outside the coding region flanking sequences homologous to the flanking sequences of the insertion site of the plasmid shuttle attenuated vaccinia virus is the MVA deletion III region;. b vaccinia shuttle plasmid in the attenuated vaccinia virus and allow manipulation cells virus strains of MVA by homologous recombination;. c, recombinant virus plaques screened by a single selection marker, separate to obtain pure highly attenuated vaccinia virus ADS-MVA, wherein said shuttle plasmid is SARS-CoV S gene promoter located plasmids shuttle pSYN downstream.
5. 根据权利要求4所述的SARS-CoV疫苗ADS-MVA的制备方法,其中, 所述筛选标记是GFP, SARS-CoV的S基因和GFP基因转入穿梭质粒的相同的插入序列中,所述筛选标记位于启动子pH5下游。 The production method of claim 4 SARS-CoV vaccine ADS-MVA claim, wherein said selection marker is a GFP, the GFP gene and the S gene of SARS-CoV sequences inserted into the same shuttle plasmid, the said selectable marker is located downstream of the promoter pH5.
6. 根据权利要求4或5所述的SARS-CoV疫苗ADS-MVA的制备方法所制的SARS-CoV疫苗ADS-MVA。 Vaccine 6. SARS-CoV ADS-MVA SARS-CoV preparation process 4 or 5, wherein the ADS-MVA vaccine prepared according to claim.
7. 权利要求4或5所述的SARS-CoV疫苗ADS-MVA的制备方法在构建重组MVA表达外源基因中的应用。 SARS-CoV vaccine prepared ADS-MVA according to claim 4 or 5 in the construction of recombinant MVA applications foreign gene expression.
8. 根据权利要求7所述的应用,其中,所述应用为权利要求4或5所述的SARS-CoV疫苗ADS-MVA的制备方法在构建重组MVA表达外源病原体抗原制备病原体测验和诊断试剂中的应用。 8. The use according to claim 7, wherein the SARS-CoV application Preparation 4 or 5, wherein the ADS-MVA vaccine as claimed in claim in the construction of recombinant MVA expressing an exogenous pathogen antigen preparation pathogen tests and diagnostic reagents application.
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