【作者】 王敏；

【导师】 洪涛； 王健伟；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2008， 博士

【摘要】 A组轮状病毒(Group A Rotavirus,ARV)是引起全世界婴幼儿严重腹泻的最重要病原,在发展中国家,每年至少约600,000儿童死于轮状病毒感染。鉴于轮状病毒危害严重且缺乏有效治疗手段,世界卫生组织一直将发展轮状病毒疫苗列为最优先发展的疫苗项目之一。由于腺病毒能感染呼吸道和肠道,在诱导全身免疫的同时产生局部粘膜免疫,安全可靠,可以通过口服或滴鼻给药,适于婴幼儿使用,因此,腺病毒载体轮状病毒基因工程疫苗具有良好的前景。我们实验室前期利用腺病毒载体表达轮状病毒保护性抗原,对轮状病毒基因工程疫苗进行了系统研究。我们课题组前期研究表明:用Ad5表达的轮状病毒的VP6和VP7基因,采用滴鼻或灌胃的给药方式,可以在小鼠实验模型上取得良好的细胞免疫和体液免疫效果,并对轮状病毒攻击鼠有一定的保护作用。但是,由于腺病毒对轮状病毒的野生型基因表达量比较低,因此,增强轮状病毒抗原的表达,优化免疫效果,降低重组腺病毒的用量,提高疫苗的安全性等诸多问题就成了发展该疫苗的重要课题。本文通过密码子优化,人工合成了轮状病毒的VP6,VP7基因,通过对蛋白表达量及免疫效果的比较,鉴定了密码优化确实提高了VP6,VP7蛋白的表达量,从而减少了病毒的用量。本研究还检测了口服腺病毒免疫后,不同时间点病毒在小鼠体内的分布,为该疫苗的进一步研究提供了实验资料。1.利用密码子优化提高重组腺病毒中人轮状病毒基因的表达量ARV基因的密码子使用与人类密码子相差很远,其AT含量很高。大量研究证明,通过基因密码改造可以有效提高基因的表达量。我们对RV VP6、G1VP7、G2VP7和G3VP7 4个基因依据人的偏爱密码子进行了密码优化,人工合成了优化基因,利用腺病毒Ad5载体(AdEasy系统)在人胚肾细胞293中进行了表达。结果显示,经过密码子优化后,与野生型病毒基因相比,4个基因的蛋白表达量均有显著提高。同时,我们对这4种重组腺病毒进行了连续20代的传代培养,在连续传代过程中,插入的轮状病毒基因一直稳定存在和表达。重组腺病毒rvAdG2VP7(o)在连续传代15代后,重组腺病毒rvAdG1VP7(o)和rvAdG2VP7(o)在连续传代20代后检测到复制型腺病毒(Replication-Competent AdenovirusRCA)的存在。说明重组腺病毒在293细胞中连续传代具有良好的遗传稳定性,传代10代以内一般检测不到RCA,可望满足腺病毒载体疫苗的研发需要。2.密码子优化增强了轮状病毒VP6基因重组腺病毒的免疫效果在证实了通过密码子优化可以提高蛋白表达量后,我们以VP6基因为例,以表达野生型RVVP6基因的重组病毒rvAdVP6为对照,在小鼠模型上通过等量病毒(10~8TCID50/只/次)3次滴鼻免疫,观察了基因优化重组腺病毒rvAdVP6(o)的免疫效果。结果显示:(1)三次免疫rvAdVP6(o)产生的抗VP6血清IgG抗体水平均明显高于rvAdVP6,说明优化后的重组病毒产生了更强的体液免疫反应;(2)两种重组腺病毒均可诱导粘膜免疫,在肺灌洗液、肺匀浆液、肠匀浆液和粪便中均能检测出较高水平的特异性IgG和IgA,其中,rvAdVP6(o)肺灌洗液、肺匀浆液和粪便中的IgG和IgA水平均显著高于rvAdVP6;rvAdVP6(o)肠匀浆液IgG水平显著高于rvAdVP6,说明优化后的病毒产生了更强的粘膜免疫效果;(3)用ELISpot检测脾细胞培养上清中的γ干扰素(IFN-γ),结果显示,rvAdVP6(o)产生的斑点数多于rvAdVP6产生的斑点数,说明优化后的病毒产生了更强的细胞免疫效果;(4)RV攻击后检测小鼠的排毒量,发现rvAdVP6(o)免疫组的RV排毒减少率明显高于rvAdVP6,说明优化后的病毒对小鼠的保护性也增强了。综上所述,在等量重组腺病毒免疫的情况下,优化后的VP6重组病毒在体液免疫、粘膜免疫、细胞免疫和攻毒保护方面,均优于优化前,可望在以后的疫苗应用中,达到减少病毒用量的目的。3.重组腺病毒口服免疫后在小鼠体内的生物分布为了探讨重组腺病毒作为口服疫苗的可行性,研究了经灌胃免疫后重组腺病毒在各组织器官中的分布以及抗原表达情况。将小鼠分为两组:rvAdVP6(o)免疫组和PBS对照组,每组70只小鼠,免疫后在7个时间点(4h、12h、1d、4d、7d、14d和28d)分别取5只小鼠,采集14种组织标本,用免疫组织化学方法检测腺病毒载体和轮状病毒VP6抗原,用荧光定量PCR检测腺病毒载体和轮状病毒VP6基因的存在。结果显示:用重组腺病毒灌胃免疫小鼠后,在4h～28d内,在肝、肾、脾、心脏、肺、大肠、小肠、胃、食管、舌、脑、气管、派氏结和卵巢14种组织标本中,均无明显的病理变化;免疫组织化学检测结果显示,只在4h时在大肠和小肠中检测到腺病毒和轮状病毒VP6抗原;荧光定量PCR在4h时在大肠、小肠、胃、食管和派氏结中检测到腺病毒载体和轮状病毒VP6基因;12h时后在大肠、小肠、胃和食管中仍能检测到腺病毒载体和轮状病毒VP6基因,但其拷贝数明显降低;1d后只在大肠和食管中检测到少量的腺病毒载体和轮状病毒VP6基因;在4d、7d后,食管、胃和大肠仍能检测到腺病毒载体基因的存在,其他组织中均检测不到;至14d时,只有在食管中仍能检测到腺病毒载体基因的存在,在其他组织中均检测不到。说明灌胃免疫后,腺病毒载体和VP6基因在小鼠体内可以表达,并在多种组织器官中存在。综上所述,本研究构建了4株表达轮状病毒密码子优化基因的VP6和VP7的重组腺病毒,与野生型基因相比,其在蛋白表达水平和免疫效果上均明显得到提高,在此基础上检测了灌胃免疫后,其在小鼠体内的分布情况,这些研究均未见报道。这些结果的获得,为研制我国具有自主知识产权的新型轮状病毒基因工程疫苗进一步奠定了基础。更多还原

【Abstract】 Group A rotaviruses (ARV) are the single most significant cause of severe dehydrating diarrhea in infants and young children in both developed and developing countries worldwide. In the developing countries, the problem seems even more serious and urgent, an estimated 18 million cases of moderately severe diarrhea and over 600,000 children died of this illness yearly. Because of the high morbidity and mortality associated with rotavirus diarrhea, it is apparently urgent need to develop some more effective and safe rotavirus vaccines to prevent rotavirus infections.In the recent years, adenoviruses have been used to make recombinant genetic engineering vaccines, and the potential and perspective seem to be rather encouraging. In our previous work, we have found that mice immunized either intranasally or orally with the adeno-based rotavirus recombinants could expressing human ARV protein VP6 and VP7, initiating a high level of systemic immune response against rotavirus infection.For designing an acceptable vaccine, safety is always the paramount significance of any other criteria to be considered, it is particularly true when develop a vaccine for use in infants and young children. The present study was carried out based on our previous work, trying to optimize the codons of the recombinant virus aiming to appropriately elevate the expressing level of the encoded human rotavirus protein (VP6 and VP7), the designed titer of the expressed proteins should be the titer that could evoke a sufficient protective immunity, safe and acceptable to children. The optimized codons of the gene VP6 and VP7were artificially synthesized and inserted into the adenovirus backbone plasmid, the plasmid then transfected into 293 cells for generating mature viral particles. Four recombinants were successfully constructed and designated as: rvAdVP6(o), rvAdG1VP7(o), rvAdG2VP7(o) and rvAdG3VP7(o). The expression level of the optimized and wild type VP6 and VP7 gene, as well as the subsequently induced immune responses in mice were identified and evaluated. The main results of the experiments are summarized as follows:1. Improved expression of human rotavirus antigens in the recombinant adenoviruses by codon optimizationIt has been known that codon optimization can raise the expression level of genes of many viral proteins. In the present study, we have artificially synthesized four genes of group A human rotavirus that encode VP6, G1VP7, G2VP7 and G3VP7 according to the human biased codon. The modified genes were transfected into 293 cells using adenovirus vectors and the gene products, the respective proteins were produced. The expression level of each gene was detected by Western Blot. The results showed a remarkable increase of the expression level in comparison with the wild type control. For evaluating the genetic stability of the optimized genes, the four 293 cell lines with non-replicating adenoviruses bearing the genes of VP6, G1VP7, G2VP7 and G3VP7 were continuously propagated up to 20 passages. The results demonstrated a steady stability of the cells in the passage, namely, the recombinant adenoviruses remained genetically stable and kept a potent and high level of expression ability. What is more, the unwanted Replication-Competent Adenoviruses were not found at least within the first 10 passages for all the four recombinants. The above results indicate that our recombinant adenoviruses are potentially applicable for further development and improvement of the adenovirus-vectored vaccines.2. Optimization of the codon strengthened the immune responses in mice model6～8-week female BALB/c mice were randomly grouped and immunized intranasally with 10~8 TCID50 rvAdVP6(o) and rvAdVP6, respectively. After the first immunization, the mice were boosted twice with a 14-day interval. The immunization results were recorded as follows:(a) Both adenoviruses with optimized and wild type genes of RV could generate high-level of serum IgG against rotavirus. However, the serum antibody level induced by rvAdVP6(o) was much higher than that of rvAdVP6.(b) Murine mucosal immunity was induced after intranasal administration of the both optimized and wild type recombinant adenoviruses. whereas the higher-level of sIgG and sIgA was only detected from the lung lavage, lung and intestinal homogenates and feces in rvAdVP6(o) immunized mice.(c) Cell mediated immunity was observed in rvAdVP6(o) administered mice and in rvAdVP6 administered mice. But the rvAdVP6(o) could induce a stronger cell mediated immune response in comparison with the rvAdVP6 as seen in the ELISpot results in which much more spots appeared signifying the higher level of cell mediated immune response.(d) The immunized mice shed significantly lower amount of viral antigens in feces as compared with the control group inoculated with an empty vector rvAdpSC. Also, the viral antigen shed from mice immunized with rvAdVP6(o) was lower than that immunized with rvAdVP6.In summary, the recombinant adenoviruses which encode optimized human rotavirus VP6 proteins (rvAdVP6(o)) could induce stronger immune and protective responses against the challenge of the rotavirus than the wild type(rvAdVP6) at the same immunizing dosage.3. Bio-distribution and persistence of VP6 gene expressed recombinant adenovirus in mice modelFor safe application as the oral vaccine, it is necessary to investigate the bio-distribution of the recombinant adenovirus after oral administration. Seven groups of mice (ten adult female mice per time point) were orally inoculated with 109 TCID50 rvAdVP6(o). Control group was given phosphate buffered saline (PBS) instead. Fourteen kinds of tissues were harvested at each time point (4h, 12h, 1d, 4d, 7d, 14d, 28d), including brain, tongue, trachea, esophagus, lung, liver, spleen, stomach, kidney, large intestine, small intestine, heart, ovary and Peyer’s patchs. Immunohistochemistry was used to determine the histological localization of the recombinant adenoviruses and the expression of VP6 protein in the immunized mice. The results showed that the endothelial cells in large intestine were positively stained at 4 hours post-infection. Meanwhile, Real-time PCR was used to quantify recombinant adenoviruses and VP6 gene copies in the different organs of the immunized mice. The result showed that 4 hours after oral vaccination, the recombinant adenoviruses and VP6 gene could be detected in large intestine, stomach, small intestine, esophagus and Peyer’s patchs in most of the mice. After12 hours, copy numbers of the recombinant adenoviruses and VP6 gene were reduced significantly. while they could still be detected in large intestine, stomach, small intestine, esophagus, but could not be detected in Peyer’s patchs. By day 1, the recombinant adenoviruses and VP6 sequence could only be detected in large intestine and esophagus. And by day 4 and 7, the recombinant adenoviruses could still be detected in large intestine, esophagus and stomach. At day 14, the recombinant adenoviruses could only be detected in esophagus. This result indicated that the recombinant adenoviruses and VP6 gene had widely distributed and durably expressed in vivo.In conclusion, four recombinant adenoviruses encoding the optimized human rotavirus antigens were successfully constructed, the expression of optimized genes significantly increased in comparison with the wild type RV genes. Immune responses in mice induced by optimized recombinant adenovirus also showed a higher level than the wide type ones. The Bio-distribution and persistence were detected upon oral application in mice. The result, therefore, provide an important clue and experimental support for the development of rotavirus genetic engineering vaccine against rotavirus infection.更多还原