本文關鍵詞： 腺病毒載體 預存免疫 中和抗體 HIV疫苗 流行病學　出處：《吉林大學》2012年博士論文　論文類型：學位論文

【摘要】：重組腺病毒(recombinant adenovirus)廣泛應用于腫瘤基因治療和疫苗載體的研究。以人腺病毒制備的載體具有以下特點：①安全性好；②外源基因容量大；③可在懸浮細胞內獲得較高的滴度；④腺病毒無需整合進宿主細胞基因組中，整合突變致癌可能性小，基因毒性低；⑤宿主范圍廣，可感染分裂期及非分裂期細胞；⑥免疫原性強；⑦可通過聯(lián)合免疫策略增強免疫效果。正是由于具有以上這些優(yōu)點，腺病毒載體被極其廣泛地應用于體外基因轉導、體內接種疫苗和基因治療等各個領域。 目前應用最多的腺病毒載體是人血清5型腺病毒（Ad5），它也是當前腫瘤基因治療和人類免疫缺陷病毒(HIV)疫苗的臨床研究的主要病毒載體。另外，諸如乙肝病毒、瘧疾、結核等病原體的疫苗研制也都十分重視5型腺病毒載體的應用。然而，重組5型腺病毒(rAd5)的一個主要問題是人體針對載體本身存在十分普遍的預存免疫。臨床試驗表明，人體內較高滴度的Ad5中和抗體水平會顯著地降低載體的轉導效率和轉基因表達水平，并且可能引發(fā)機體的不良反應。最新的腺病毒流行病學研究顯示：西方發(fā)達國家Ad5的血清陽性率約60-70％，而非洲的撒哈拉和東南亞等一些發(fā)展中國家和地區(qū)的陽性率甚至高達98％。目前，我國關于腺病毒載體應用于腫瘤基因治療和疫苗的相關研究已經很多，但仍缺乏Ad5及一些有潛在應用價值的稀有血清型腺病毒的流行病學調查數據。除了預存免疫問題，rAd5可以說是一種近乎完美的基因治療和疫苗載體，因此研究Ad5預存免疫的機理，開發(fā)能避免預存免疫的重組腺病毒載體具有很強的科學意義和應用價值。 本論文通過分析機體內Ad5中和抗體的主要中和抗原位點，結合腺病毒流行病學調查和序列分析，對Ad5進行基因工程改造：即以稀有血清型腺病毒的中和抗原位點基因替換rAd5來構建嵌合載體，希望獲得一種易于包裝、免疫原性強且能有效避免機體預存免疫的新型rAd5載體。 論文首先通過在全國各地收集健康人群血清，應用國際標準化的病毒中和試驗檢測了超過1000份血清中Ad5的中和抗體滴度。結果表明，我國健康成人血清中72%為Ad5中和抗體陽性（滴度＞18），與歐美等發(fā)達國家的感染率接近。結果同時顯示，我國南方地區(qū)的Ad5血清陽性率較其他地區(qū)略高。綜合分析地域、年齡、型別和民族等因素發(fā)現，地域和氣候差異是影響腺病毒在成人中感染的主要因素。兒童是疫苗應用的重要群體，為進一步分析潛在的腺病毒載體應用人群，我們又對東北地區(qū)的兒童血清中Ad2和Ad5中和抗體水平進行了分組研究。結果表明，兒童的Ad5感染率較成人略低，其中7-12個月兒童血清中的Ad2和Ad5中和抗體水平最低，是較適合的腺病毒載體疫苗應用的候選群體。由于病毒中和試驗操作難度較大且價格昂貴，難以應用于更多的腺病毒血清型的流行病學研究。為了方便研究一些稀有血清型病毒在我國感染情況，我們通過分析人血清中腺病毒中和抗體的性質，原核表達了多種血清型腺病毒衣殼蛋白的功能結構域，建立了一種方便、快速的分型檢測人血清中腺病毒中和抗體的血清學方法。對比病毒中和試驗發(fā)現，我們建立的方法準確度較高，與中和試驗的吻合度在80%以上。應用該方法檢測東北地區(qū)兒童血清表明，Ad37和Ad43等國外稀有血清型在我國的感染率同樣較低。該部分的研究說明，解決Ad5預存免疫問題對于Ad5載體在我國的進一步研究和應用具有十分重要的意義。 隨后，在蛋白水平上對腺病毒的免疫學性質進行了研究，目的是尋找腺病毒衣殼蛋白最主要的中和抗原表位，為Ad5嵌合表位的選擇以及替換策略提供依據。我們首先利用原核表達的5型腺病毒六鄰體超變區(qū)（HVRs）和纖維蛋白頭節(jié)區(qū)（Knob）蛋白研究了腺病毒衣殼蛋白特異的體液免疫反應，證實了在高感染人群以及動物免疫后均會產生主要針對六鄰體的中和抗體。由于腺病毒載體的構建難度較大，我們利用構建的HVRs嵌合蛋白預先在蛋白水平上評價了六鄰體蛋白的中和抗原表位。結果表明，原核表達的HVRs嵌合蛋白可以體現其在病毒中的結構和部分功能。并且與結構分析不同，HVR5和HVR7具有部分六鄰體蛋白的中和抗原表位，而HVR1-7則完全體現了六鄰體的抗原特異性。該結果為接下來的腺病毒載體嵌合改造基因和策略的選擇提供了依據。 為了獲得既易于包裝又能逃避機體預存免疫的嵌合型rAd5載體，根據序列分析和以上實驗結果，我們選擇性的構建了來自三種稀有血清型（Ad37、Ad43、Ad26）的Loop1,2、HVRs和Loop4基因的共14個嵌合腺病毒骨架質粒。Loop4基因的選擇是希望其能夠提高替換了來源于同血清型病毒抗原基因的嵌合載體的包裝能力。將構建成功的嵌合骨架質粒與表達綠色熒光蛋白（GFP）的穿梭質粒共同轉染HEK293細胞，檢測嵌合載體的包裝能力。結果顯示，Loop1和Loop2以及全長HVRs的替換難以包裝或復制能力較低，而同時替換Loop4基因也未使載體的包裝能力有所提升。而基于Ad26、Ad37和Ad43，僅替換HVR5和HVR7的嵌合骨架質粒與穿梭質粒重組成功并獲得復制能力較強的嵌合型病毒。另外，我們發(fā)現替換Ad43HVR5,7的嵌合質粒的包裝效率顯著地增加。隨后，，我們對成功包裝的嵌合載體進行了包裝、復制等體外活性的評價。結果表明，成功包裝嵌合病毒載體保持了rAd5較強的轉基因表達能力和轉基因的穩(wěn)定性。 最后，以表達HIV1-Gag的嵌合型rAd5載體免疫小鼠發(fā)現，HVR5,7嵌合腺病毒載體針對Gag基因的免疫原性較強，其中rAd5HVR43（5,7）-Gag的免疫背景和免疫原性均與rAd5-Gag相當。然后我們按照人體感染腺病毒的程度差異，建立了具有不同Ad5中和抗體水平的預存免疫小鼠模型。以嵌合病毒載體通過不同的免疫策略免疫空白和預存模型小鼠，證實HVR5,7嵌合型rAd5載體在小鼠體內可以有效逃避中低程度的預存免疫。而通過Prime-Boost策略，嵌合載體可以一定程度的避免高水平的預存免疫效應。另外，結果同時表明一定程度的同亞群其它血清型的預存免疫并不影響rAd5載體疫苗的免疫原性。 本論文的研究表明：我國人群感染5型腺病毒的程度同樣較高，Ad5的預存免疫問題極大的限制了其進一步的研究和應用。然而，由于腺病毒在機體內免疫性質的復雜性，僅僅以現有的信息對其進行嵌合改造仍不足以完全避免人體內普遍存在的高水平的預存免疫，而過于復雜的基因工程修飾又會造成腺病毒載體的難以包裝。因此提示我們必須進一步深入地研究腺病毒特別是Ad5的免疫機制，才有可能構建出更為理想的基因治療和疫苗載體。盡管如此，本論文的相關研究仍然對于腺病毒的流行病學、免疫學性質、腺病毒載體的包裝和免疫原性具有一定意義，也為腺病毒載體在我國的進一步應用提供了一些依據。
[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.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392.1

【參考文獻】

相關期刊論文 前4條

1 蘇曉波;馬鑫;洪U嗚

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