柯萨奇病毒B3腺病毒载体疫苗Ad/sVP1的构建及免疫效果的研究
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摘要
目的:柯萨奇病毒B组3型(Coxsackievirus group B type 3,CVB3)感染所致的病毒性心肌炎(viral myocarditis,VMC)严重危害人类健康,也是新生儿猝死的重要原因,目前尚无有效的预防方法。核酸疫苗的问世,为建立有效的预防方法提供了机会。本室前期的大量研究证明,以pcDNA3质粒为载体构建的多种核酸疫苗免疫小鼠后,仅可诱导产生低效价的中和抗体,不能有效阻止致死量病毒感染。因此,提高免疫原性、增强免疫效果是制备CVB3疫苗亟需解决的问题。
目前提高免疫效果的关键因素之一是开发具有较高基因转移率和靶向特异性的转基因载体。在众多载体体系中,复制缺陷型腺病毒载体系统(Replication-Defective Adenovirus Vector System)以其广泛的宿主范围、极高的转染效率、外源基因高表达以及病毒滴度高等特点,在疫苗载体选择中表现出优势,成为极具应用前景的基因转移载体。
VP1是CVB3的主要中和抗原,能诱导机体产生体液和细胞免疫,但用VP1基因构建的核酸疫苗免疫小鼠后产生的中和抗体效价偏低。其主要原因是VP1蛋白在转染细胞中可以表达,但却不能从转染细胞中自主释放,因而不能有效激发抗体应答。
人白细胞介素2(Human interleukin-2, hIL-2)信号肽是由20个氨基酸组成的高度疏水性序列,它可以将合成的蛋白质移向细胞膜并与细胞膜结合,然后把合成的蛋白质分泌到胞外,且可以在不同种属蛋白质间相互使用。本室前期曾将CVB3 VP1基因拼接到人白介素2(hIL-2)信号肽5’端构建了分泌性重组真核表达质粒pcDNA3/sVP1,免疫小鼠可以增强中和抗体应答。
本研究利用AdEasy腺病毒载体系统构建、包装重组腺病毒Ad/sVP1,并通过研究该疫苗免疫BALB/c小鼠后诱生的特异性免疫应答和病毒攻击后的免疫保护作用探讨这种疫苗的应用效果。
方法: (1)目的基因的扩增与鉴定以重组质粒pcDNA3/sVP1为模板,扩增带有信号肽的VP1基因。将扩增产物与pGEM-T载体连接,构建质粒pGEM-T/sVP1。转化DH5α大肠杆菌,提取质粒进行酶切鉴定和测序,筛选重组子。(2)重组穿梭质粒的构建将质粒pGEM-T/sVP1以合适的限制性内切酶进行双酶切,回收带粘末端的目的片段,将目的片断分别与经合适的限制性内切酶双酶切的腺病毒穿梭载体pAdTrack-CMV连接。经转化、筛选和酶切鉴定,构建重组穿梭质粒pAdTrack-CMV/sVP1。(3)重组腺病毒质粒的构建将质粒用内切酶PmeⅠ线性化。利用AdEasy系统,经两步法分别在大肠杆菌BJ-5183中与骨架载体pAdEasy-1同源重组,构建重组腺病毒质粒pAd/sVP1。用PacⅠ酶切鉴定。将阳性质粒转化入感受态E.coli DH5α,挑取阳性克隆。以碱裂解法大量提取,并用聚乙二醇(polyethylene glycol, PEG)沉淀法进行纯化。(4)重组腺病毒Ad/sVP1的包装与扩增将重组腺病毒质粒pAd/sVP1用内切酶PacⅠ线性化,以阳离子脂质体法转染HEK293(humanembryo kidney 293 derived cell line)细胞,并观察绿色荧光蛋白(green fluorescent protein,GFP)的表达。冻融法收集初代病毒液。取初代病毒液感染293细胞,逐日观察细胞病变(cytopathic effect,CPE),及有否彗星样斑块(FOCI)形成。并经第三轮大量扩增,以提高病毒滴度。(5)病毒感染293细胞后48小时,提取细胞总蛋白及细胞培养液,通过SDS-PAGE和Western blot检测VP1蛋白的表达。(6)大量扩增重组腺病毒取Ad/sVP1以及本室保存Ad/VP1第三代病毒液感染293细胞,大量扩增重组腺病毒,并以冻融法收集病毒液。将收集到的病毒液利用大量腺病毒纯化试剂盒纯化病毒。(7)重组腺病毒滴度测定待293细胞在96孔板中生长至70-80%汇合时,以不同稀释倍数的上述病毒液感染细胞,感染后48小时GFP阳性细胞计数法测定病毒滴度。病毒滴度=荧光细胞数×病毒液的稀释倍数/病毒液量。(8)动物实验:将6～8周龄雄性BALB/c小鼠随机分为3组,分别为PBS对照组、Ad/VP1组、Ad/sVP1组,每组18只,病毒液以PBS稀释后,股四头肌注射免疫小鼠,每次每只接种1×10~7pfu,第0、16天分别免疫,共免疫2次;每次免疫后第14天,内眦静脉取血,分离血清,用微量中和试验(固定病毒-稀释血清法)及ELISA法检测血清CVB3特异性中和抗体和IgG抗体水平;第2次免疫后3周,每组3只小鼠取脾脏制备淋巴细胞悬液,采用细胞计数试剂盒(cell counting kit-8, CCK-8)法进行特异性淋巴细胞增殖活性和特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)杀伤活性的检测;另每组12只小鼠用4LD_(50)CVB3进行腹腔注射,观察并记录小鼠存活情况至感染后第21天;每组剩余的3只小鼠用3LD_(50)CVB3攻击,注射病毒后第7天取血,用于血中病毒滴度的测定。
结果:(1)成功构建了pGEM-T/sVP1,测序和酶切鉴定证明符合预期结果。(2)成功构建穿梭质粒pAdTrack-CMV/sVP1,酶切分析证明符合预期设计。(3)成功构建重组腺病毒质粒pAd/sVP1,用PacⅠ酶切得到约30kb的片段和一个3.0或4.5kb的片段,PCR鉴定目的基因长度与预期值相符。(4)转染293细胞48小时后,荧光显微镜下观察到报告基因GFP的表达,重组腺病毒Ad/sVP1包装成功。初代病毒再次感染293细胞,在荧光显微镜下观察到逐日明显的细胞病变和荧光聚集,各代病毒感染细胞后均有彗星样荧光斑块形成。(5)重组腺病毒Ad/sVP1感染293细胞48小时后,经Western Blot检测,在细胞培养液中检测到VP1蛋白。(6)重组腺病毒Ad/vp1、Ad/sVP1大量扩增后的病毒滴度依次为: 3.0×10~9 pfu/ml、5.0×10~8 pfu/ml。(7)各次免疫后血清CVB3 VP1特异性IgG水平分别为:Ad/VP1组: 1:56.10,1:168.27;Ad/sVP1组: 1:66.68,1:503.50;而PBS组未检测到。单因素方差分析表明,Ad/VP1和Ad/sVP1组VP1特异性IgG逐次增加(P<0.01);末次免疫后二组比较,Ad/sVP1组抗体滴度高于Ad/VP1组(P<0.01)。(8)各次免疫后血清CVB3中和抗体水平分别为:Ad/VP1组:1:8.41,1:31.77;Ad/sVP1组:1:8.91,1:44.87;PBS组未检测到。单因素方差分析表明Ad/VP1和Ad/sVP1组中和抗体水平逐次增加(P<0.01);第2次免疫后Ad/sVP1组抗体滴度高于Ad/VP1组(P<0.05)。(9)小鼠脾淋巴细胞增殖增殖活性经单因素方差分析表明,以CVB3刺激时,各组间比较无统计学意义;以ConA刺激时, Ad/sVP1组高于其他二组(P<0.05)。(10)小鼠脾脏特异性CTL杀伤活性经单因素方差分析表明,Ad/sVP1组与Ad/VP1组杀伤率无统计学意义,但二者均高于PBS对照组(P<0.05)。(11)用3LD_(50) CVB3攻击小鼠后第7d,血清病毒滴度测定结果表明,Ad/sVP1和Ad/VP1组明显低于PBS组(P<0.05),且Ad/sVP1组较Ad/VP1组显著降低(P<0.05)。(12)4LD_(50) CVB3感染小鼠后21d,Ad/sVP1组和Ad/VP1组生存率分别为:58.33% ,41.67%,PBS组无存活。经过χ2检验分析,尚不能确定三组之间有统计学差异。用Kaplan-Meier法进行生存分析,生存率曲线分布有差异,Ad/sVP1组生存状况好于PBS组(p<0.01),但与Ad/VP1组比较无统计学意义。
结论:(1)本研究利用AdEasy腺病毒载体系统成功包装了重组腺病毒载体疫苗Ad/sVP1,在293细胞内可以表达并分泌至细胞外。(2)Ad/VP1和Ad/sVP1均能诱导小鼠产生中和抗体和特异性IgG抗体,增强脾淋巴细胞增殖活性,降低病毒攻击后血清病毒滴度,提高小鼠的生存率。(3)与Ad/VP1比较,分泌型重组腺病毒Ad/sVP1能更有效的提高体液和脾淋巴细胞增殖活性,降低病毒攻击后的小鼠血中病毒滴度,免疫效果优于Ad/VP1。
Objective: Coxsackievirus Group B Type 3(CVB3) is a major pathogen of human viral myocarditis, which is the leading cause of neonate sudden death as well. There is no virus-specific preventive procedure against CVB3 infection available today.
Gene vaccine affords an opportunity to pave a new way to prevent CVB3 infection. Although different vaccines constructed with pcDNA3 vector could induce the production of antibody, the titers of antibody induced were usually too low to protect the host from lethal CVB3 challenge. Therefore, it appears to be an important strategy to improve immunogencity of the DNA vaccine so as to induce stronger CVB3-specific immune responses and enhance the protective efficacy.
One of the key points of gene therapy is the exploitation of a transfer vector with high efficiency of gene transfer and target specificity. In the past years, a recombinant, replication-defective adenovirus system has been used as a transfer vector for experimental models of gene therapy. The fact that adenovirus could infect most types of cells with no requirement for cell division, transfer and express gene in a higher efficiency, and therefore robust the protective effect of the recombinant adenovirus-based vaccine, has made it a promising system for human gene therapy in vivo.
VP1 is the major capsid protein of CVB3, which can induce both humoral and cellular immune responses. It had been reported that DNA vaccine expressing VP1 alone could induce the production of antibody, but the titers of antibody were usually low. The probable reason might be that VP1 protein could be expressed by the transfected cells but released from these cells and thus can not elicit antibody response effectively.
Signal peptides may help proteins transport out of cells, so adding signal peptide gene to VP1 cDNA can probably promote the secretion of VP1 protein by the transfected cells. Human IL-2(hIL2) signal peptide is a quite hydrophobic sequence consisted of 20 amino acids. In the early study, we have constructed a secretable VP1 eukaryotic expressing plasmid pcDNA3/sVP1, which was constructed by fusing DNA sequence of the 11 N-terminal amino acid residues of mature hIL-2 with CVB3 VP1 and could express VP1 secretively. It has been shown that the gene vaccine could protect mice against CVB3 challenge.
In this study, we constructed an adenovirus-based CVB3 vaccine Ad/sVP1, which could express sVP1 in 293 cells and evaluated the immune protective effect of the vaccine in mice.
Methods: (1) The DNA fragment of sVP1 was amplified by PCR from the plasmid pcDNA3 encoding sVP1 protein. Gene fragments were inserted into pGEM-T vector respectively. Competent E.coli DH5αwere transformed and selected by Ampicillin, the recombinants were verified by electrophoresis analyses and gene sequencing. (2) The gene fragments were purified from agarose gel, and cloned into the transfer vector pAdTrack-CMV cut by the same endonucleases to construct the transfer plasmids AdTrack-CMV/sVP1. The transformed bacteria were selected by Kanamycin, the recombined plasmids extracted were verified by endonuclease analyses. (3) The recombinant plasmids were linearized with Pme I and co-transformed into E. coli strain BJ5183 respectively together with pAdEasy-1, the viral DNA plasmid. Recombinants were selected with kanamycin. Once achieved and verified, the recombinant adenoviral plasmids were transformed to competent E. coli DH5αfor greater yields and then purified by PEG. (4) Screening and amplification of recombinant adenoviral particles Linearized by PacI digestion, the recombinant adenoviral plasmids were transferred into HEK 293 cells (human embryo kidney 293 derived cell line) using LipofectamineTM2000 according to the manufacturer's guidelines, to produce viral particles, followed by screening the expression of green fluorescent protein(GFP). The recombinant viruses further amplified for 2 to 3 passages to get higher titers of the virus. (5) Cell lysate and supernatant of the cell culture were analyzed by Western blotting for the expression of the protein 48h post infection. (6) Viral particle titration Cell-free virus stocks were diluted serially in serum-free medium at ten times, and were titrated by blue forming units (BFU) methods according to the manual of Ad-Easy vector system application. (7)BALB/c mice aged 6-8 weeks were inoculated intramuscularly (i.m.) twice on 0、16 day with 1×10~7 pfu/ml of Ad/VP1、Ad/sVP1 and PBS respectively. Fourteen days after every injection, sera of each group were collected and the titers of CVB3-specific neutralizing antibodies and specific VP1 IgG were measured. Three weeks after the second immunization, splenocytes from three immunized mice of each group were stimulated by inactivated CVB3 and harvested to analyze the lymphocytic proliferative activity and specific CTL cytotoxic activity by CCK-8 assay. At the same time, the other eight mice were challenged with 4LD_(50) of CVB3 and the number of surviving animals was monitored up to three weeks post infection. Furthermore, the rest mice of each group were challenged with 3LD_(50) CVB3 and sacrificed seven days later to evaluate the titers of blood viruses.
Result: (1)The clone vector pGEM-T/sVP1 was constructed successfully; endonuclease analyses and sequencing result showed that the inserted genes were the same as reported.(2)Recombinant adenoviral transfer plasmid AdTrack-CMV/sVP1 was constructed successfully.(3) Recombinant adenoviral plasmid pAd/sVP1 was generated successfully. Restriction digest with Pac I yielded a large fragment of approximately 30 kb, and a smaller fragment of either 3.0 kb or 4.5 kb. (4) Recombinant adenoviruses Ad/sVP1 was generated successfully. The infected cells were lysed by freeze/thaw cycles, and supernatant was used to reinfect fresh HEK 293 cells.(5) The cell culture medium was screened for VP1 expression by Western blotting.(6)The titers of recombinant adenoviruses Ad/VP1 and Ad/sVP1 after passage 4 were as follows: 3.0×10~9 pfu/ml、5.0×10~8 pfu/ml。(7)When mice were immunized with the vaccines, the antibody titers increased time-dependently with the time of inoculation. The mean titers of neutralizing antibody after every immunization in Ad/VP1 group were 1:8.41,1:31.77, respectively, and that in Ad/sVP1 group were 1:8.91,1:44.87, while it was always lower than 1:5 in PBS group. More specifically, mice with Ad/sVP1 elicited the highest level of neutralizing antibody after the second immunization. When compared with that of Ad/VP1, the difference was significant (P<0.05).(8)The titers of specific VP1 IgG after each inoculation were 1:56.10,1:168.27 in Ad/VP1 group, and 1:66.68,1:503.50 in Ad/sVP1 group. The mice immunized with Ad/sVP1 elicited the strongest specific VP1 IgG after the second immunization. When compared with that of Ad/VP1, the difference was significant (P<0.01).(9) Splenocytes from vaccinated mice showed different proliferative activity to different stimulants of CVB3 or ConA. When stimulated by CVB3, the difference of the three groups was not significant. While, when stimulated by ConA, the proliferative activity of mice of Ad/sVP1 group was higher than those of the other two groups (p<0.05).(10) The adenovirus-based CVB3 vaccines Ad/VP1 and Ad/sVP1 enhanced the specific lymphocytic CTL cytotoxic activity, which was remarkably stronger than that that of the mice immunized with PBS (p<0.05).(11) After 3LD_(50) of CVB3 challenge, the virus titers of blood in Ad/sVP1 group were only 2.58±0.25, significantly lower than that of Ad/VP1 group (3.41±0.21). (12) Protection of mice from death after lethal CVB3 (4LD_(50)) challenge was augmented to 58.33% with Ad/sVP1, while mice with Ad/VP1 was only 41.67% and no one survived in PBS group. Chi-square test indicated that differences between any two groups were not significant by the multiple comparisons.
Conclusion: (1)Adenovirus-based CVB3 vaccine Ad/sVP1 was constructed successfully. And the expression of VP1 was verified by Western blotting.(2)After administration of Recombinant adenovirus vaccine Ad/sVP1 to BALB/c mice, the neutralizing antibody increased along with the inoculation times, the virus titers of blood were much lower, and protection of mice from death after lethal CVB3 (4LD_(50)) challenge was augmented to 58.33%.
目前提高免疫效果的关键因素之一是开发具有较高基因转移率和靶向特异性的转基因载体。在众多载体体系中,复制缺陷型腺病毒载体系统(Replication-Defective Adenovirus Vector System)以其广泛的宿主范围、极高的转染效率、外源基因高表达以及病毒滴度高等特点,在疫苗载体选择中表现出优势,成为极具应用前景的基因转移载体。
VP1是CVB3的主要中和抗原,能诱导机体产生体液和细胞免疫,但用VP1基因构建的核酸疫苗免疫小鼠后产生的中和抗体效价偏低。其主要原因是VP1蛋白在转染细胞中可以表达,但却不能从转染细胞中自主释放,因而不能有效激发抗体应答。
人白细胞介素2(Human interleukin-2, hIL-2)信号肽是由20个氨基酸组成的高度疏水性序列,它可以将合成的蛋白质移向细胞膜并与细胞膜结合,然后把合成的蛋白质分泌到胞外,且可以在不同种属蛋白质间相互使用。本室前期曾将CVB3 VP1基因拼接到人白介素2(hIL-2)信号肽5’端构建了分泌性重组真核表达质粒pcDNA3/sVP1,免疫小鼠可以增强中和抗体应答。
本研究利用AdEasy腺病毒载体系统构建、包装重组腺病毒Ad/sVP1,并通过研究该疫苗免疫BALB/c小鼠后诱生的特异性免疫应答和病毒攻击后的免疫保护作用探讨这种疫苗的应用效果。
方法: (1)目的基因的扩增与鉴定以重组质粒pcDNA3/sVP1为模板,扩增带有信号肽的VP1基因。将扩增产物与pGEM-T载体连接,构建质粒pGEM-T/sVP1。转化DH5α大肠杆菌,提取质粒进行酶切鉴定和测序,筛选重组子。(2)重组穿梭质粒的构建将质粒pGEM-T/sVP1以合适的限制性内切酶进行双酶切,回收带粘末端的目的片段,将目的片断分别与经合适的限制性内切酶双酶切的腺病毒穿梭载体pAdTrack-CMV连接。经转化、筛选和酶切鉴定,构建重组穿梭质粒pAdTrack-CMV/sVP1。(3)重组腺病毒质粒的构建将质粒用内切酶PmeⅠ线性化。利用AdEasy系统,经两步法分别在大肠杆菌BJ-5183中与骨架载体pAdEasy-1同源重组,构建重组腺病毒质粒pAd/sVP1。用PacⅠ酶切鉴定。将阳性质粒转化入感受态E.coli DH5α,挑取阳性克隆。以碱裂解法大量提取,并用聚乙二醇(polyethylene glycol, PEG)沉淀法进行纯化。(4)重组腺病毒Ad/sVP1的包装与扩增将重组腺病毒质粒pAd/sVP1用内切酶PacⅠ线性化,以阳离子脂质体法转染HEK293(humanembryo kidney 293 derived cell line)细胞,并观察绿色荧光蛋白(green fluorescent protein,GFP)的表达。冻融法收集初代病毒液。取初代病毒液感染293细胞,逐日观察细胞病变(cytopathic effect,CPE),及有否彗星样斑块(FOCI)形成。并经第三轮大量扩增,以提高病毒滴度。(5)病毒感染293细胞后48小时,提取细胞总蛋白及细胞培养液,通过SDS-PAGE和Western blot检测VP1蛋白的表达。(6)大量扩增重组腺病毒取Ad/sVP1以及本室保存Ad/VP1第三代病毒液感染293细胞,大量扩增重组腺病毒,并以冻融法收集病毒液。将收集到的病毒液利用大量腺病毒纯化试剂盒纯化病毒。(7)重组腺病毒滴度测定待293细胞在96孔板中生长至70-80%汇合时,以不同稀释倍数的上述病毒液感染细胞,感染后48小时GFP阳性细胞计数法测定病毒滴度。病毒滴度=荧光细胞数×病毒液的稀释倍数/病毒液量。(8)动物实验:将6～8周龄雄性BALB/c小鼠随机分为3组,分别为PBS对照组、Ad/VP1组、Ad/sVP1组,每组18只,病毒液以PBS稀释后,股四头肌注射免疫小鼠,每次每只接种1×10~7pfu,第0、16天分别免疫,共免疫2次;每次免疫后第14天,内眦静脉取血,分离血清,用微量中和试验(固定病毒-稀释血清法)及ELISA法检测血清CVB3特异性中和抗体和IgG抗体水平;第2次免疫后3周,每组3只小鼠取脾脏制备淋巴细胞悬液,采用细胞计数试剂盒(cell counting kit-8, CCK-8)法进行特异性淋巴细胞增殖活性和特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)杀伤活性的检测;另每组12只小鼠用4LD_(50)CVB3进行腹腔注射,观察并记录小鼠存活情况至感染后第21天;每组剩余的3只小鼠用3LD_(50)CVB3攻击,注射病毒后第7天取血,用于血中病毒滴度的测定。
结果:(1)成功构建了pGEM-T/sVP1,测序和酶切鉴定证明符合预期结果。(2)成功构建穿梭质粒pAdTrack-CMV/sVP1,酶切分析证明符合预期设计。(3)成功构建重组腺病毒质粒pAd/sVP1,用PacⅠ酶切得到约30kb的片段和一个3.0或4.5kb的片段,PCR鉴定目的基因长度与预期值相符。(4)转染293细胞48小时后,荧光显微镜下观察到报告基因GFP的表达,重组腺病毒Ad/sVP1包装成功。初代病毒再次感染293细胞,在荧光显微镜下观察到逐日明显的细胞病变和荧光聚集,各代病毒感染细胞后均有彗星样荧光斑块形成。(5)重组腺病毒Ad/sVP1感染293细胞48小时后,经Western Blot检测,在细胞培养液中检测到VP1蛋白。(6)重组腺病毒Ad/vp1、Ad/sVP1大量扩增后的病毒滴度依次为: 3.0×10~9 pfu/ml、5.0×10~8 pfu/ml。(7)各次免疫后血清CVB3 VP1特异性IgG水平分别为:Ad/VP1组: 1:56.10,1:168.27;Ad/sVP1组: 1:66.68,1:503.50;而PBS组未检测到。单因素方差分析表明,Ad/VP1和Ad/sVP1组VP1特异性IgG逐次增加(P<0.01);末次免疫后二组比较,Ad/sVP1组抗体滴度高于Ad/VP1组(P<0.01)。(8)各次免疫后血清CVB3中和抗体水平分别为:Ad/VP1组:1:8.41,1:31.77;Ad/sVP1组:1:8.91,1:44.87;PBS组未检测到。单因素方差分析表明Ad/VP1和Ad/sVP1组中和抗体水平逐次增加(P<0.01);第2次免疫后Ad/sVP1组抗体滴度高于Ad/VP1组(P<0.05)。(9)小鼠脾淋巴细胞增殖增殖活性经单因素方差分析表明,以CVB3刺激时,各组间比较无统计学意义;以ConA刺激时, Ad/sVP1组高于其他二组(P<0.05)。(10)小鼠脾脏特异性CTL杀伤活性经单因素方差分析表明,Ad/sVP1组与Ad/VP1组杀伤率无统计学意义,但二者均高于PBS对照组(P<0.05)。(11)用3LD_(50) CVB3攻击小鼠后第7d,血清病毒滴度测定结果表明,Ad/sVP1和Ad/VP1组明显低于PBS组(P<0.05),且Ad/sVP1组较Ad/VP1组显著降低(P<0.05)。(12)4LD_(50) CVB3感染小鼠后21d,Ad/sVP1组和Ad/VP1组生存率分别为:58.33% ,41.67%,PBS组无存活。经过χ2检验分析,尚不能确定三组之间有统计学差异。用Kaplan-Meier法进行生存分析,生存率曲线分布有差异,Ad/sVP1组生存状况好于PBS组(p<0.01),但与Ad/VP1组比较无统计学意义。
结论:(1)本研究利用AdEasy腺病毒载体系统成功包装了重组腺病毒载体疫苗Ad/sVP1,在293细胞内可以表达并分泌至细胞外。(2)Ad/VP1和Ad/sVP1均能诱导小鼠产生中和抗体和特异性IgG抗体,增强脾淋巴细胞增殖活性,降低病毒攻击后血清病毒滴度,提高小鼠的生存率。(3)与Ad/VP1比较,分泌型重组腺病毒Ad/sVP1能更有效的提高体液和脾淋巴细胞增殖活性,降低病毒攻击后的小鼠血中病毒滴度,免疫效果优于Ad/VP1。
Objective: Coxsackievirus Group B Type 3(CVB3) is a major pathogen of human viral myocarditis, which is the leading cause of neonate sudden death as well. There is no virus-specific preventive procedure against CVB3 infection available today.
Gene vaccine affords an opportunity to pave a new way to prevent CVB3 infection. Although different vaccines constructed with pcDNA3 vector could induce the production of antibody, the titers of antibody induced were usually too low to protect the host from lethal CVB3 challenge. Therefore, it appears to be an important strategy to improve immunogencity of the DNA vaccine so as to induce stronger CVB3-specific immune responses and enhance the protective efficacy.
One of the key points of gene therapy is the exploitation of a transfer vector with high efficiency of gene transfer and target specificity. In the past years, a recombinant, replication-defective adenovirus system has been used as a transfer vector for experimental models of gene therapy. The fact that adenovirus could infect most types of cells with no requirement for cell division, transfer and express gene in a higher efficiency, and therefore robust the protective effect of the recombinant adenovirus-based vaccine, has made it a promising system for human gene therapy in vivo.
VP1 is the major capsid protein of CVB3, which can induce both humoral and cellular immune responses. It had been reported that DNA vaccine expressing VP1 alone could induce the production of antibody, but the titers of antibody were usually low. The probable reason might be that VP1 protein could be expressed by the transfected cells but released from these cells and thus can not elicit antibody response effectively.
Signal peptides may help proteins transport out of cells, so adding signal peptide gene to VP1 cDNA can probably promote the secretion of VP1 protein by the transfected cells. Human IL-2(hIL2) signal peptide is a quite hydrophobic sequence consisted of 20 amino acids. In the early study, we have constructed a secretable VP1 eukaryotic expressing plasmid pcDNA3/sVP1, which was constructed by fusing DNA sequence of the 11 N-terminal amino acid residues of mature hIL-2 with CVB3 VP1 and could express VP1 secretively. It has been shown that the gene vaccine could protect mice against CVB3 challenge.
In this study, we constructed an adenovirus-based CVB3 vaccine Ad/sVP1, which could express sVP1 in 293 cells and evaluated the immune protective effect of the vaccine in mice.
Methods: (1) The DNA fragment of sVP1 was amplified by PCR from the plasmid pcDNA3 encoding sVP1 protein. Gene fragments were inserted into pGEM-T vector respectively. Competent E.coli DH5αwere transformed and selected by Ampicillin, the recombinants were verified by electrophoresis analyses and gene sequencing. (2) The gene fragments were purified from agarose gel, and cloned into the transfer vector pAdTrack-CMV cut by the same endonucleases to construct the transfer plasmids AdTrack-CMV/sVP1. The transformed bacteria were selected by Kanamycin, the recombined plasmids extracted were verified by endonuclease analyses. (3) The recombinant plasmids were linearized with Pme I and co-transformed into E. coli strain BJ5183 respectively together with pAdEasy-1, the viral DNA plasmid. Recombinants were selected with kanamycin. Once achieved and verified, the recombinant adenoviral plasmids were transformed to competent E. coli DH5αfor greater yields and then purified by PEG. (4) Screening and amplification of recombinant adenoviral particles Linearized by PacI digestion, the recombinant adenoviral plasmids were transferred into HEK 293 cells (human embryo kidney 293 derived cell line) using LipofectamineTM2000 according to the manufacturer's guidelines, to produce viral particles, followed by screening the expression of green fluorescent protein(GFP). The recombinant viruses further amplified for 2 to 3 passages to get higher titers of the virus. (5) Cell lysate and supernatant of the cell culture were analyzed by Western blotting for the expression of the protein 48h post infection. (6) Viral particle titration Cell-free virus stocks were diluted serially in serum-free medium at ten times, and were titrated by blue forming units (BFU) methods according to the manual of Ad-Easy vector system application. (7)BALB/c mice aged 6-8 weeks were inoculated intramuscularly (i.m.) twice on 0、16 day with 1×10~7 pfu/ml of Ad/VP1、Ad/sVP1 and PBS respectively. Fourteen days after every injection, sera of each group were collected and the titers of CVB3-specific neutralizing antibodies and specific VP1 IgG were measured. Three weeks after the second immunization, splenocytes from three immunized mice of each group were stimulated by inactivated CVB3 and harvested to analyze the lymphocytic proliferative activity and specific CTL cytotoxic activity by CCK-8 assay. At the same time, the other eight mice were challenged with 4LD_(50) of CVB3 and the number of surviving animals was monitored up to three weeks post infection. Furthermore, the rest mice of each group were challenged with 3LD_(50) CVB3 and sacrificed seven days later to evaluate the titers of blood viruses.
Result: (1)The clone vector pGEM-T/sVP1 was constructed successfully; endonuclease analyses and sequencing result showed that the inserted genes were the same as reported.(2)Recombinant adenoviral transfer plasmid AdTrack-CMV/sVP1 was constructed successfully.(3) Recombinant adenoviral plasmid pAd/sVP1 was generated successfully. Restriction digest with Pac I yielded a large fragment of approximately 30 kb, and a smaller fragment of either 3.0 kb or 4.5 kb. (4) Recombinant adenoviruses Ad/sVP1 was generated successfully. The infected cells were lysed by freeze/thaw cycles, and supernatant was used to reinfect fresh HEK 293 cells.(5) The cell culture medium was screened for VP1 expression by Western blotting.(6)The titers of recombinant adenoviruses Ad/VP1 and Ad/sVP1 after passage 4 were as follows: 3.0×10~9 pfu/ml、5.0×10~8 pfu/ml。(7)When mice were immunized with the vaccines, the antibody titers increased time-dependently with the time of inoculation. The mean titers of neutralizing antibody after every immunization in Ad/VP1 group were 1:8.41,1:31.77, respectively, and that in Ad/sVP1 group were 1:8.91,1:44.87, while it was always lower than 1:5 in PBS group. More specifically, mice with Ad/sVP1 elicited the highest level of neutralizing antibody after the second immunization. When compared with that of Ad/VP1, the difference was significant (P<0.05).(8)The titers of specific VP1 IgG after each inoculation were 1:56.10,1:168.27 in Ad/VP1 group, and 1:66.68,1:503.50 in Ad/sVP1 group. The mice immunized with Ad/sVP1 elicited the strongest specific VP1 IgG after the second immunization. When compared with that of Ad/VP1, the difference was significant (P<0.01).(9) Splenocytes from vaccinated mice showed different proliferative activity to different stimulants of CVB3 or ConA. When stimulated by CVB3, the difference of the three groups was not significant. While, when stimulated by ConA, the proliferative activity of mice of Ad/sVP1 group was higher than those of the other two groups (p<0.05).(10) The adenovirus-based CVB3 vaccines Ad/VP1 and Ad/sVP1 enhanced the specific lymphocytic CTL cytotoxic activity, which was remarkably stronger than that that of the mice immunized with PBS (p<0.05).(11) After 3LD_(50) of CVB3 challenge, the virus titers of blood in Ad/sVP1 group were only 2.58±0.25, significantly lower than that of Ad/VP1 group (3.41±0.21). (12) Protection of mice from death after lethal CVB3 (4LD_(50)) challenge was augmented to 58.33% with Ad/sVP1, while mice with Ad/VP1 was only 41.67% and no one survived in PBS group. Chi-square test indicated that differences between any two groups were not significant by the multiple comparisons.
Conclusion: (1)Adenovirus-based CVB3 vaccine Ad/sVP1 was constructed successfully. And the expression of VP1 was verified by Western blotting.(2)After administration of Recombinant adenovirus vaccine Ad/sVP1 to BALB/c mice, the neutralizing antibody increased along with the inoculation times, the virus titers of blood were much lower, and protection of mice from death after lethal CVB3 (4LD_(50)) challenge was augmented to 58.33%.
引文
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12 Smith TA, Mehaffey MG, Kayda DB, et al. Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice. Nat Genet , 1993, 5(4):397-402
13 Orin JE, Lubeck MD, Barton JE, et al. Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters. Proc Natl Acad Sci U S A, 1987, 84(13):4626-30
14 Shiver JW, Fu T-M, Chen L, et al. Replication incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature, 2002, 415:331-335
15 Chen W Z, Liu M Q, Jiao Y, et a1. Adenovirus-mediated RNA interference against Foot - and - mouth disease virusinfection both in vitro and in vivo . Virolgy , 2006, 80(7) 3559-3566
16 Ponnazhagan S, Erik son D, Kearns W G, et al. Lack of site-specific integration of the recombinant adeno-associated virus-genomes in human cells. Hum Gene Ther, 1997, 8: 275-284
17 Flotte TR. Gene therapy progress and prospects: recombinant adeno-associated virus ( rAAV ) vectors. Gene Ther, 2004, 11: 805-810
18 Davidoff AM, Nathwani AC, Spurbeck WW, et al. rAAV-mediated long-term liver-generated expression of an angiogenesis inhibitor can restrict renal tumor growth in mice. Cancer Res, 2002, 62(11): 307-323
19鲁晓春,李小鹰,马鑫等.腺相关病毒载体对心肌细胞感染和外源基因表达的作用.中国临床康复, 2003, 7: 4056-4057
20 Ho shijima M , Ikeda Y, Iwanaga Y, et al. Chronic suppression of heart-failure progression by a pseudo phosphorrylated mutant of phospho lamban via in vivo cardiac rAAV gene delivery. Nat Med, 2002, 8: 864-871
21 Bartlett JS,Wilcher R, Samulsi RJ. Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors. J Viro l, 2000, 74: 2777-2785
22黄志军,袁洪,曾钧发等.重组腺相关病毒载体介导人血管内皮生长因子165基因对缺血心肌血管新生的影响.中国动脉硬化杂志, 2004, 12: 627- 631
23 Sugiyama A , Ho tto ri S, Tanaka S, et al. Defective adeno-associated viral-mediated transfection of insulin gene by direct injection into liver parenchyma decreases blood glucose of diabetic mice. Horm Metab Res, 1997, 29: 599-603
24 Yang Z, Chen M , Wu R, et al. Suppression of auto immune diabetes by viral IL-10 gene transfer. J Immuno l, 2002, 168:6479-6485
25 Shklyaev S, Aslanidi G, Tennant M , et al. Sustained peripheral exp ression of transgene adiponectin offsets the development of diet-induced obesity in rats. Proc Natl Acad Sci USA , 2003, 25: 14217-14222
26张明满,李德华,严律南等.携反义mrp和mdr1双耐药基因的重组腺相关病毒载体的构建与鉴定.四川大学学报(医学版) , 2005, 36: 765-769
27赵春霞,赵荫涛,汪培华等.重组腺相关病毒介导核心蛋白聚糖基因转染对SiHa细胞周期和凋亡的影响.癌症, 2005, 24: 28-32
28 DavisN I, Willis LW, Smith JF, et al. In vitro synthesis of infectious Venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of aviable deletion mutant. Virology, 1989, 171 (1) : 189-204
29 Hang CS, Hang YS, Braciale TJ, et al. Infectious sindbis virus transient expression vectors for studying antigen processing and presentation. PNAS, 1992, 89 (4) : 2679-2683
30 DiCiommo DP, Bremner R. Rapid, high level protein production using DNA-based Semliki Forest virus vectors. J Biol Chem, 1998, 273 (29): 18060-18066
31 The Pipeline Project: HVTN vaccines in development. http://chi.ucsf.edu/vaccines updated May 2007
32 Lundstrom K. Biology and application of alphaviruses in gene therapy. Gene Ther 2005, 12:92-97
33 Thornburg NJ, Ray CA, Collier ML, et al. Vaccination with Venezuelan equine encephalitis replicons encoding cowpox virus structural proteins protects mice from intranasal cowpox virus challenge. Virology, 2007, 362:441-452
34 Greer CE, Zhou F, Leeg HS, et al. A chimeric alphavirus RNA replicon gene-based vaccine for human parainfluenza virus type 3 induces protective immunity against intranasal virus challenge. Vaccine , 2007, 25:481 -489
35 Watanabe D, Brockman MA, Ndung T, et al. Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector. Virology, 2007, 357:186-198
36 Argnani R, Lufino M, Manservigi M, et al. Replication competent herpes simplex vectors: design and applications. Gene Ther, 2005, 12:170-177
37 Berto E, Bozac A, Marconi P. Development and application of replication-incompetent HSV-1-based vectors. Gene Ther, 2005, 12:98-102
38 Murphy CG, Lucas WT, Means RE, et al. Vaccine protectionagainst simian immunodeficiency virus by recombinant strains of herpes simplex virus. J Virol 2000, 74:7745-7754
39 Zuniga A, Wang Z, Liniger M, et al. Attenuated measles virus as a vaccine vector. Vaccine, 2007, 25:2974-2983
40 Hoffman SJ, Polack FP, Hauer DA, et al. Vaccination of rhesus macaques with a recombinant measles virus expressing interleukin-12 alters humoral and cellular immune responses . J Infect Dis.2003,188(10):1553-1561
41 Parrino J, Graham BS. Smallpox vaccines: past, present, and future. J Allergy Clin Immunol, 2006, 118:1320-1326
42殷震,刘景华.动物病毒学.第2版北京:科学出版社,1997,939-987
43 The Pipeline Project: HVTN vaccines in development. http://chi.ucsf.edu/vaccines updated May 2007.
44 Aspden K, Passmore J, Tiedt F, et al. Evaluation of lumpy skin disease virus, a capripoxvirus, as a replication deficient vaccine vector. J General Virol, 2003, 84:1985-1996
45 Fischer T, Planz O, Stitz L, et al. Novel recombinant parapoxvirus vectors induce protective humoral and cellular immunity against Lethal herpesvirus challenge infection in mice. J Virol, 2003, 77(17):9312-9323
46 Jones SM, Feldmann H, Stroher U, et al.. Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses. Nat Med, 2005, 11:786-790.
47 Majid AM, Ezelle H, Shah S, et al. Evaluating replication defective vesicular stomatitis virus as a vaccine vehicle. JVirol, 2006, 80:6993-7008
48 Johnson JE, Nasar F, Coleman JW, et al. Neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates. Virology, 2007, 360:36-49
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1 Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998, 392:245-252
2 Ballay A ,Levrero M ,Buendia MA ,et al. In vitro and in vivo synthesis of the hepatitis B virus surface antigen and of the receptor for polymerized human serum albumin from recombinant human adenoviruses. EMBO J ,1985, 4 :3861-3865
3 Robert-Guroff M, Nabel GJ, Shiver JW, et al. Replication- defective and competent adenovirus recombinants as vaccine vectors. In New Generation Vaccines, 2007, 18:127-134
4 Zoltick PW, Chirmule N, Schnell MA, et al. Biology of E1-deleted adenovirus vectors in nonhuman primate muscle. J Virol, 2001, 75: 5222- 5229
5 Lieber A, He CY, Kirillova I, et al. Recombinant adenoviruses with large deletions generated by remediated ecision exhibit different biological properties compared with first generation vectors in vitro and in vivo. J Virol, 1996, 70: 8944- 8960
6 Gorziglia MI, Lapcevich C, Roy S, et al. Generation of an adenovirus vector lacking E1, e2a, E3, and all of E4 except open reading frame 3. J Virol, 1999, 73 (7): 6048- 6055
7 Raper SE, Haskal ZJ, Ye X, et al. Selective gene transfer into the liver of non-human primates with E1-deleted, E2Adefective, or E1-E4 deleted recombinant adenoviruses. Hum Gene Ther, 1998, 9 (5): 671- 679
8 Alba R, Bosch A, Chillon M, et al .Gutless adenovirus: last-generation adenovirus for gene therapy. Gene Therapy, 2005,12:18-20
9 He TC, Zhou S, Costa L T, et al. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA ,1998, 95 (5):509-2514
10 Anderson R D, Haskell RE, Xia H ,et a1 .A simple method for the rapid generation of recombinant adenovirus vectors. Gene therapy, 2000, 7:1034-1038
11普卢,曾军.在新腺病毒载体及其在口蹄疫疫苗研究中的应用.动物医学进展, 2007 , 28 (8): 59-64
12 Smith TA, Mehaffey MG, Kayda DB, et al. Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice. Nat Genet , 1993, 5(4):397-402
13 Orin JE, Lubeck MD, Barton JE, et al. Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters. Proc Natl Acad Sci U S A, 1987, 84(13):4626-30
14 Shiver JW, Fu T-M, Chen L, et al. Replication incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature, 2002, 415:331-335
15 Chen W Z, Liu M Q, Jiao Y, et a1. Adenovirus-mediated RNA interference against Foot - and - mouth disease virusinfection both in vitro and in vivo . Virolgy , 2006, 80(7) 3559-3566
16 Ponnazhagan S, Erik son D, Kearns W G, et al. Lack of site-specific integration of the recombinant adeno-associated virus-genomes in human cells. Hum Gene Ther, 1997, 8: 275-284
17 Flotte TR. Gene therapy progress and prospects: recombinant adeno-associated virus ( rAAV ) vectors. Gene Ther, 2004, 11: 805-810
18 Davidoff AM, Nathwani AC, Spurbeck WW, et al. rAAV-mediated long-term liver-generated expression of an angiogenesis inhibitor can restrict renal tumor growth in mice. Cancer Res, 2002, 62(11): 307-323
19鲁晓春,李小鹰,马鑫等.腺相关病毒载体对心肌细胞感染和外源基因表达的作用.中国临床康复, 2003, 7: 4056-4057
20 Ho shijima M , Ikeda Y, Iwanaga Y, et al. Chronic suppression of heart-failure progression by a pseudo phosphorrylated mutant of phospho lamban via in vivo cardiac rAAV gene delivery. Nat Med, 2002, 8: 864-871
21 Bartlett JS,Wilcher R, Samulsi RJ. Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors. J Viro l, 2000, 74: 2777-2785
22黄志军,袁洪,曾钧发等.重组腺相关病毒载体介导人血管内皮生长因子165基因对缺血心肌血管新生的影响.中国动脉硬化杂志, 2004, 12: 627- 631
23 Sugiyama A , Ho tto ri S, Tanaka S, et al. Defective adeno-associated viral-mediated transfection of insulin gene by direct injection into liver parenchyma decreases blood glucose of diabetic mice. Horm Metab Res, 1997, 29: 599-603
24 Yang Z, Chen M , Wu R, et al. Suppression of auto immune diabetes by viral IL-10 gene transfer. J Immuno l, 2002, 168:6479-6485
25 Shklyaev S, Aslanidi G, Tennant M , et al. Sustained peripheral exp ression of transgene adiponectin offsets the development of diet-induced obesity in rats. Proc Natl Acad Sci USA , 2003, 25: 14217-14222
26张明满,李德华,严律南等.携反义mrp和mdr1双耐药基因的重组腺相关病毒载体的构建与鉴定.四川大学学报(医学版) , 2005, 36: 765-769
27赵春霞,赵荫涛,汪培华等.重组腺相关病毒介导核心蛋白聚糖基因转染对SiHa细胞周期和凋亡的影响.癌症, 2005, 24: 28-32
28 DavisN I, Willis LW, Smith JF, et al. In vitro synthesis of infectious Venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of aviable deletion mutant. Virology, 1989, 171 (1) : 189-204
29 Hang CS, Hang YS, Braciale TJ, et al. Infectious sindbis virus transient expression vectors for studying antigen processing and presentation. PNAS, 1992, 89 (4) : 2679-2683
30 DiCiommo DP, Bremner R. Rapid, high level protein production using DNA-based Semliki Forest virus vectors. J Biol Chem, 1998, 273 (29): 18060-18066
31 The Pipeline Project: HVTN vaccines in development. http://chi.ucsf.edu/vaccines updated May 2007
32 Lundstrom K. Biology and application of alphaviruses in gene therapy. Gene Ther 2005, 12:92-97
33 Thornburg NJ, Ray CA, Collier ML, et al. Vaccination with Venezuelan equine encephalitis replicons encoding cowpox virus structural proteins protects mice from intranasal cowpox virus challenge. Virology, 2007, 362:441-452
34 Greer CE, Zhou F, Leeg HS, et al. A chimeric alphavirus RNA replicon gene-based vaccine for human parainfluenza virus type 3 induces protective immunity against intranasal virus challenge. Vaccine , 2007, 25:481 -489
35 Watanabe D, Brockman MA, Ndung T, et al. Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector. Virology, 2007, 357:186-198
36 Argnani R, Lufino M, Manservigi M, et al. Replication competent herpes simplex vectors: design and applications. Gene Ther, 2005, 12:170-177
37 Berto E, Bozac A, Marconi P. Development and application of replication-incompetent HSV-1-based vectors. Gene Ther, 2005, 12:98-102
38 Murphy CG, Lucas WT, Means RE, et al. Vaccine protectionagainst simian immunodeficiency virus by recombinant strains of herpes simplex virus. J Virol 2000, 74:7745-7754
39 Zuniga A, Wang Z, Liniger M, et al. Attenuated measles virus as a vaccine vector. Vaccine, 2007, 25:2974-2983
40 Hoffman SJ, Polack FP, Hauer DA, et al. Vaccination of rhesus macaques with a recombinant measles virus expressing interleukin-12 alters humoral and cellular immune responses . J Infect Dis.2003,188(10):1553-1561
41 Parrino J, Graham BS. Smallpox vaccines: past, present, and future. J Allergy Clin Immunol, 2006, 118:1320-1326
42殷震,刘景华.动物病毒学.第2版北京:科学出版社,1997,939-987
43 The Pipeline Project: HVTN vaccines in development. http://chi.ucsf.edu/vaccines updated May 2007.
44 Aspden K, Passmore J, Tiedt F, et al. Evaluation of lumpy skin disease virus, a capripoxvirus, as a replication deficient vaccine vector. J General Virol, 2003, 84:1985-1996
45 Fischer T, Planz O, Stitz L, et al. Novel recombinant parapoxvirus vectors induce protective humoral and cellular immunity against Lethal herpesvirus challenge infection in mice. J Virol, 2003, 77(17):9312-9323
46 Jones SM, Feldmann H, Stroher U, et al.. Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses. Nat Med, 2005, 11:786-790.
47 Majid AM, Ezelle H, Shah S, et al. Evaluating replication defective vesicular stomatitis virus as a vaccine vehicle. JVirol, 2006, 80:6993-7008
48 Johnson JE, Nasar F, Coleman JW, et al. Neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates. Virology, 2007, 360:36-49