六邻体嵌合型重组5型腺病毒载体逃避预存免疫的研究

发布时间：2018-01-23 21:50

本文关键词： 腺病毒载体预存免疫中和抗体 HIV疫苗流行病学　出处：《吉林大学》2012年博士论文　论文类型：学位论文

【摘要】：重组腺病毒(recombinant adenovirus)广泛应用于肿瘤基因治疗和疫苗载体的研究。以人腺病毒制备的载体具有以下特点：①安全性好；②外源基因容量大；③可在悬浮细胞内获得较高的滴度；④腺病毒无需整合进宿主细胞基因组中，整合突变致癌可能性小，基因毒性低；⑤宿主范围广，可感染分裂期及非分裂期细胞；⑥免疫原性强；⑦可通过联合免疫策略增强免疫效果。正是由于具有以上这些优点，腺病毒载体被极其广泛地应用于体外基因转导、体内接种疫苗和基因治疗等各个领域。目前应用最多的腺病毒载体是人血清5型腺病毒（Ad5），它也是当前肿瘤基因治疗和人类免疫缺陷病毒(HIV)疫苗的临床研究的主要病毒载体。另外，诸如乙肝病毒、疟疾、结核等病原体的疫苗研制也都十分重视5型腺病毒载体的应用。然而，重组5型腺病毒(rAd5)的一个主要问题是人体针对载体本身存在十分普遍的预存免疫。临床试验表明，人体内较高滴度的Ad5中和抗体水平会显著地降低载体的转导效率和转基因表达水平，并且可能引发机体的不良反应。最新的腺病毒流行病学研究显示：西方发达国家Ad5的血清阳性率约60-70％，而非洲的撒哈拉和东南亚等一些发展中国家和地区的阳性率甚至高达98％。目前，我国关于腺病毒载体应用于肿瘤基因治疗和疫苗的相关研究已经很多，但仍缺乏Ad5及一些有潜在应用价值的稀有血清型腺病毒的流行病学调查数据。除了预存免疫问题，rAd5可以说是一种近乎完美的基因治疗和疫苗载体，因此研究Ad5预存免疫的机理，开发能避免预存免疫的重组腺病毒载体具有很强的科学意义和应用价值。本论文通过分析机体内Ad5中和抗体的主要中和抗原位点，结合腺病毒流行病学调查和序列分析，对Ad5进行基因工程改造：即以稀有血清型腺病毒的中和抗原位点基因替换rAd5来构建嵌合载体，希望获得一种易于包装、免疫原性强且能有效避免机体预存免疫的新型rAd5载体。论文首先通过在全国各地收集健康人群血清，应用国际标准化的病毒中和试验检测了超过1000份血清中Ad5的中和抗体滴度。结果表明，我国健康成人血清中72%为Ad5中和抗体阳性（滴度＞18），与欧美等发达国家的感染率接近。结果同时显示，我国南方地区的Ad5血清阳性率较其他地区略高。综合分析地域、年龄、型别和民族等因素发现，地域和气候差异是影响腺病毒在成人中感染的主要因素。儿童是疫苗应用的重要群体，为进一步分析潜在的腺病毒载体应用人群，我们又对东北地区的儿童血清中Ad2和Ad5中和抗体水平进行了分组研究。结果表明，儿童的Ad5感染率较成人略低，其中7-12个月儿童血清中的Ad2和Ad5中和抗体水平最低，是较适合的腺病毒载体疫苗应用的候选群体。由于病毒中和试验操作难度较大且价格昂贵，难以应用于更多的腺病毒血清型的流行病学研究。为了方便研究一些稀有血清型病毒在我国感染情况，我们通过分析人血清中腺病毒中和抗体的性质，原核表达了多种血清型腺病毒衣壳蛋白的功能结构域，建立了一种方便、快速的分型检测人血清中腺病毒中和抗体的血清学方法。对比病毒中和试验发现，我们建立的方法准确度较高，与中和试验的吻合度在80%以上。应用该方法检测东北地区儿童血清表明，Ad37和Ad43等国外稀有血清型在我国的感染率同样较低。该部分的研究说明，解决Ad5预存免疫问题对于Ad5载体在我国的进一步研究和应用具有十分重要的意义。随后，在蛋白水平上对腺病毒的免疫学性质进行了研究，目的是寻找腺病毒衣壳蛋白最主要的中和抗原表位，为Ad5嵌合表位的选择以及替换策略提供依据。我们首先利用原核表达的5型腺病毒六邻体超变区（HVRs）和纤维蛋白头节区（Knob）蛋白研究了腺病毒衣壳蛋白特异的体液免疫反应，证实了在高感染人群以及动物免疫后均会产生主要针对六邻体的中和抗体。由于腺病毒载体的构建难度较大，我们利用构建的HVRs嵌合蛋白预先在蛋白水平上评价了六邻体蛋白的中和抗原表位。结果表明，原核表达的HVRs嵌合蛋白可以体现其在病毒中的结构和部分功能。并且与结构分析不同，HVR5和HVR7具有部分六邻体蛋白的中和抗原表位，而HVR1-7则完全体现了六邻体的抗原特异性。该结果为接下来的腺病毒载体嵌合改造基因和策略的选择提供了依据。为了获得既易于包装又能逃避机体预存免疫的嵌合型rAd5载体，根据序列分析和以上实验结果，我们选择性的构建了来自三种稀有血清型（Ad37、Ad43、Ad26）的Loop1,2、HVRs和Loop4基因的共14个嵌合腺病毒骨架质粒。Loop4基因的选择是希望其能够提高替换了来源于同血清型病毒抗原基因的嵌合载体的包装能力。将构建成功的嵌合骨架质粒与表达绿色荧光蛋白（GFP）的穿梭质粒共同转染HEK293细胞，检测嵌合载体的包装能力。结果显示，Loop1和Loop2以及全长HVRs的替换难以包装或复制能力较低，而同时替换Loop4基因也未使载体的包装能力有所提升。而基于Ad26、Ad37和Ad43，仅替换HVR5和HVR7的嵌合骨架质粒与穿梭质粒重组成功并获得复制能力较强的嵌合型病毒。另外，我们发现替换Ad43HVR5,7的嵌合质粒的包装效率显著地增加。随后，，我们对成功包装的嵌合载体进行了包装、复制等体外活性的评价。结果表明，成功包装嵌合病毒载体保持了rAd5较强的转基因表达能力和转基因的稳定性。最后，以表达HIV1-Gag的嵌合型rAd5载体免疫小鼠发现，HVR5,7嵌合腺病毒载体针对Gag基因的免疫原性较强，其中rAd5HVR43（5,7）-Gag的免疫背景和免疫原性均与rAd5-Gag相当。然后我们按照人体感染腺病毒的程度差异，建立了具有不同Ad5中和抗体水平的预存免疫小鼠模型。以嵌合病毒载体通过不同的免疫策略免疫空白和预存模型小鼠，证实HVR5,7嵌合型rAd5载体在小鼠体内可以有效逃避中低程度的预存免疫。而通过Prime-Boost策略，嵌合载体可以一定程度的避免高水平的预存免疫效应。另外，结果同时表明一定程度的同亚群其它血清型的预存免疫并不影响rAd5载体疫苗的免疫原性。本论文的研究表明：我国人群感染5型腺病毒的程度同样较高，Ad5的预存免疫问题极大的限制了其进一步的研究和应用。然而，由于腺病毒在机体内免疫性质的复杂性，仅仅以现有的信息对其进行嵌合改造仍不足以完全避免人体内普遍存在的高水平的预存免疫，而过于复杂的基因工程修饰又会造成腺病毒载体的难以包装。因此提示我们必须进一步深入地研究腺病毒特别是Ad5的免疫机制，才有可能构建出更为理想的基因治疗和疫苗载体。尽管如此，本论文的相关研究仍然对于腺病毒的流行病学、免疫学性质、腺病毒载体的包装和免疫原性具有一定意义，也为腺病毒载体在我国的进一步应用提供了一些依据。
[Abstract]:The recombinant adenovirus (recombinant adenovirus) is widely used in tumor gene therapy and vaccine vector with human adenovirus vector. The preparation has the following characteristics: good safety; the exogenous gene capacity; the titer was higher in suspension cells; the adenovirus without integration into the host cell genome integration, gene mutation of the possibility of a small, low toxicity gene; the broad host range, infection division and non mitotic cells; the immunogenicity is strong; and can enhance the immune effect by combined immunization strategies. It is because of these advantages, adenovirus vector is widely used in gene transduction in vitro, various in the field of vaccination and gene therapy.
The adenovirus vector is the most widely used human adenovirus serotype 5 (Ad5), it is also the current tumor gene therapy and human immunodeficiency virus (HIV) virus vector vaccine mainly clinical research. In addition, such as hepatitis B virus, malaria, tuberculosis and other pathogen vaccine development also attaches great importance to the application of adenovirus type 5 carrier. However, recombinant adenovirus type 5 (rAd5) is one of the main issues for the human body itself is very common carrier of pre-existing immunity. Clinical trials show that the human body high titer of neutralizing antibody to Ad5 level will significantly reduce the vector transduction efficiency and transgene expression levels, adverse reactions and may cause the body adenovirus. Recent epidemiological studies showed that the positive rate of serum Ad5 in western developed countries is about 60-70%, while the positive rate of sub Saharan Africa and Southeast Asia and other countries and regions have some Up to 98%. at present, there have been a lot of research on the application of adenovirus vector in tumor gene therapy and vaccine in China, but still lack the epidemiological data of Ad5 and some of the potential value of the rare adenovirus serotypes. In addition to pre-existing immune problems, rAd5 can be said to be a near perfect gene therapy and so to study the mechanism of Ad5 vaccine vector, pre-existing immunity, can prevent the development of recombinant adenovirus vector of pre-existing immunity has great scientific significance and application value.
This paper through the analysis of main antigenic sites of Ad5 neutralizing antibody in the body, according to the investigation and sequence analysis of adenovirus epidemiology, genetic engineering reconstruction of Ad5: the antigenic sites of gene with rare adenovirus serotypes replace rAd5 to construct chimeric vector, hope to obtain an easy packaging, strong immunogenicity and can to avoid the new rAd5 carrier body pre-existing immunity.
Firstly, through collecting the serum of healthy individuals in the country, virus neutralization test application of international standard detection of more than 1000 copies of Ad5 in serum neutralizing antibody titers. The results showed that in healthy adults in the serum of 72% Ad5 antibody positive (titer > 18), close to the developed countries such as Europe and the United States the infection rate. The results also showed that the positive rate of serum Ad5 in southern China than other regions is slightly higher. The comprehensive analysis of regional, ethnic and age, found type and other factors, and regional differences in climate are the main factors influencing adenovirus infection in adults. Children are an important group of vaccines, for further analysis of adenovirus vector application groups potentially, we went on the Northeast children's serum Ad2 and Ad5 levels of neutralizing antibodies were grouped. The results showed that the infection rate of Ad5 in children than in adults is slightly lower, 7-12 of which the children's blood In the Ad2 and Ad5 neutralizing antibody level is the lowest, the candidate group of adenovirus vector vaccine is more suitable for the application. Because the virus neutralization test is difficult and expensive, difficult to be applied to epidemiological study on adenovirus serotype more. In order to facilitate the study of some rare serotype virus infection in China, we through the analysis of the nature of adenovirus neutralizing antibody in human serum, prokaryotic expression and functional domains of various serotypes of adenovirus capsid protein, establish a convenient, serological method for detection of human type fast adenovirus neutralizing antibody in serum. Compared the virus neutralization test, we establish a method of high accuracy with the neutralization test fit in more than 80%. This method was applied to detect the Northeast children's serum showed that Ad37 and Ad43 and other foreign rare serotype in China. The infection rate was low Some studies show that the solution of the immune problem of Ad5 is of great significance for further research and application of Ad5 in China.
Then, at the protein level on immunological properties of adenovirus were studied. The aim is to find the main capsid protein of the adenovirus neutralizing epitope, Ad5 chimeric epitope selection and provide the basis for the replacement strategy. We first use the prokaryotic expression of type 5 adenovirus six neighbor hypervariable region (HVRs) fiber and protein (Knob protein) of the scolex area humoral immune response to adenovirus capsid protein, confirmed the high infection and animal immunization will produce major neutralizing antibodies against six neighbor. Due to difficulty in construction of recombinant adenovirus vector, we use the HVRs chimeric protein constructed at the protein level in advance evaluation of the six neighbor protein neutralizing epitope. The results showed that the prokaryotic expression of HVRs chimeric protein can reflect the virus in the structure and function and structure analysis. HVR5 and HVR7 have different. The neutralization epitope of some six adjacent proteins, while HVR1-7 fully reflects the antigen specificity of the six neighbour. The results provide a basis for the next selection of recombinant adenovirus vectors and strategies.
In order to obtain chimeric rAd5 vector and packaging both easy to evade preexisting immunity, according to the sequence analysis and the experimental results, we constructed from selective three rare serotypes (Ad37, Ad43, Ad26) Loop1,2, a total of 14 chimeric adenovirus backbone plasmid.Loop4 of HVRs gene and Loop4 gene selection hope it can improve the ability to replace the packaging from the same serotype chimeric virus antigen gene. The chimeric plasmid successfully and the expression of green fluorescent protein (GFP) of the shuttle plasmid was co transfected into HEK293 cells, packaging the ability to detect the chimeric vector. The results showed that the replacement of Loop1 and Loop2 as well as the full length of HVRs is not the packaging or copy ability is low, while the replacement of Loop4 gene did not make the package carrier improved. Based on Ad26, Ad37 and Ad43, and only replace the HVR5 chimeric skeleton plasmid and HVR7 The recombinant shuttle plasmid success and a strong ability to replicate the chimeric virus. In addition, we found that the chimeric plasmid Ad43HVR5,7 to replace the packing efficiency increased markedly. Subsequently, we successfully packaged chimeric vector for the packaging, evaluation of replication activity in vitro. The results showed that the chimeric virus vector successfully packaged to keep the rAd5 strong GM transgenic expression ability and stability.
Finally, in order to find expression of chimeric rAd5 vector HIV1-Gag immunized mice, immune HVR5,7 chimeric adenovirus vector for Gag gene with high rAd5HVR43 (5,7), the immune background and the immunogenicity of the -Gag and rAd5-Gag. Then we according to the differences in the degree of human infection of adenovirus, of Ad5 neutralizing antibody the level of pre-existing immunity in mice model. The chimeric virus carrier through different immunization strategy of blank immunization and stored mice confirmed that HVR5,7 chimeric rAd5 vector can effectively avoid the low degree of pre-existing immunity in mice. The Prime-Boost chimeric vector strategy, can to a certain extent to avoid the high level of pre-existing immunity effect. In addition, the results also showed that certain subsets of other serotypes with pre-existing immunity does not affect the immunogenicity of the rAd5 vaccine.
This study showed that adenovirus type 5 infection in Chinese population the same high degree of Ad5, pre-existing immunity problem limits its further research and application. However, because of the complexity of adenovirus immunity in nature, only to the existing information of the chimeric transformation is still not enough to completely avoid common body of the high level of pre-existing immunity, but genetic engineering modification is too complex and will cause the adenovirus vector to packaging. Therefore we must further study on adenovirus especially the immune mechanism of Ad5, will it be possible to construct a more ideal vector for gene therapy and vaccine. However, this thesis research is still in epidemiology, adenovirus immunological properties, has certain significance of packaging and immunogenicity of adenoviral vectors for adenovirus vector in China into a The step application provides some basis.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.1

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