【作者】 李芸；

【导师】 曾毅；

【作者基本信息】 北京工业大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 艾滋病（AIDS），是当今对人类健康威胁最大的疾病之一，自从引起艾滋病的HIV病毒被发现以来，科学家们始终致力于寻找科学有效的预防和治疗手段，但由于HIV本身高度变异性及易于产生耐药性的特点，至今仍然没有研制出治愈艾滋病的有效药物。从疾病控制长远战略来说，有效的HIV疫苗才是战胜HIV流行的最佳希望。本课题组前期进行了多载体HIV疫苗序贯及重复免疫的研究，接受免疫的动物体内可长期维持高水平细胞免疫反应。本研究主要对表达gag基因的重组腺病毒疫苗（Ad5-HIVgag）进行临床前药效学研究，为该疫苗的临床应用提供依据。腺病毒载体一直是基因治疗和疫苗研究的热点，这主要是由于腺病毒载体具有安全性高、宿主范围广泛、感染滴度高、稳定性好，腺病毒双链DNA基因组易于重组基因操作等特点。核心蛋白Gag具有较高的保守性，富含CTL表位，是细胞免疫应答的主要靶标。Ad5-HIVgag疫苗是插入了HIV-1gag基因的、缺失病毒E1及E3区的非复制型5型重组腺病毒。疫苗中gag基因来自HIV-1B亚型gag共享序列。本研究首先对Ad5-HIVgag疫苗检定的PCR方法、酶切方法、Western blot方法及ELISpot方法进行了方法学验证并制定了相应的质量检定规程。对纯化的三批重组Ad5-HIVgag疫苗制品进行了相应的质量检定。用PCR方法可以扩增出gag特异性产物，酶切方法证明HIV-1gag基因插入位置正确，Western blot方法证实Gag蛋白可以有效表达，免疫小鼠后用ELISpot方法可检测到Gag特异性细胞免疫反应。本研究重点评价了小鼠及猴体内的细胞免疫反应效果。用不同剂量的Ad5-HIVgag疫苗(2×10⁶VP/只/次、2×10⁷VP/只/次、2×10⁸VP/只/次、2×10⁹VP/只/次、2×10¹⁰VP/只/次)局部肌肉注射小鼠两次或三次，最后一次免疫后1周用ELISpot方法检测Gag特异性细胞免疫反应。结果2×10⁶VP的剂量就可以在小鼠体内获得明显的细胞免疫应答。以2×10⁷VP的剂量注射产生的细胞免疫反应水平最高，当高于此剂量时，细胞免疫水平反而降低。为观察Ad5-HIVgag疫苗在食蟹猴体内所引起的特异性免疫反应，在猴长毒试验过程中同时进行了药效学观察。分别使用低（0.99×1011VP/只/次）、中（4.94×1011VP/只/次）、高（24.68×1011VP/只/次）不同的病毒剂量，肌肉注射食蟹猴，于0、3、6、9、12周各肌肉注射1次。于不同时间点用ELISpot方法检测疫苗诱导的Gag特异性细胞免疫反应，同时也检测了动物血清抗腺病毒的中和抗体。结果从初次免疫后5周开始，免疫的动物即检测到了Gag特异性细胞免疫反应，随着时间的延长，8周时Gag特异性细胞免疫反应水平有增加。12周时Gag特异性细胞免疫反应水平有所下降，但随后15周及18周的检测，Gag特异性细胞免疫反应水平又有升高。说明，食蟹猴在一定的剂量范围内用Ad5-HIVgag免疫后，可产生针对Gag的特异性免疫反应。三个实验组动物都检测到了较高水平的腺病毒中和抗体，但是对Ad5疫苗反复应用诱导的Gag特异性细胞免疫反应抑制作用并不明显。总之，Ad5-HIVgag疫苗可以诱导机体产生Gag特异性T细胞应答，有可能成为治疗艾滋病的有效疫苗。依据国家对生物制品注册的管理规定，对Ad5-HIVgag疫苗相关的质量研究及药效学研究资料进行了整理，并已向北京市药品监督管理局递交了Ⅰ期临床研究申请。更多还原

【Abstract】 AIDS is one of the most serious diseases threatening human health nowadays.Since HIV was identified as the pathogen of AIDS, scientists have been focused ondeveloping effective strategies for prevention and treatment of AIDS. However, dueto the features of high variability and drug resistance of HIV effective drugs thatcould cure AIDS is still no available. Towards a long-term strategy for preventionand control of AIDS, the discovery of effective HIV vaccines is the best hope toconquer AIDS.We developed sequential and repeated immunization with multi-vector based HIVvaccines, the vaccinated animals could maintain high level of cellular immuneresponses for long time. The main objective of this study is to evaluate thepre-clinical immunogenicity of recombinant adenovirus vaccine expressing gag gene（Ad5-HIVgag）, and provide evidence for the clinical application of this vaccine.Adenoviral vector is widely used in gene therapy and vaccines research.Adenoviral vector has many advantages. It is safe, it can infect a wide variety of celltypes and tissues including both dividing and non-dividing cells for gene transfer orexpression. And it can be produced to high titers and purified easily. The viruschromosome remains episomal in the transduced cell, thus avoiding the possibilityof insertional mutagenesis. Besides, the adenovirus double-stranded DNA is easy forgenetic recombination. The core protein （Gag） is highly conservative, rich in CTLepitopes and is the major target of cellular immune responses. Ad5-HIVgag isnon-replicate recombinant virus which lacks of E1and E3region, and is insertedwith HIV-1subtype B gag consensus sequence.Firstly the methodological verification of PCR, restriction enzyme digestions,Western blot and ELISpot assay was performed and the products inspectioninstruction was prepared. Then three lots of purified rAd5-HIVgag vaccines wereverified by these assays.The effects of cellular immune responses in mice and monkeys were evaluated.The mice were randomly divided into six groups of10. Different amount of thepurified rAd5-HIVgag (2×10⁶VP,2×10⁷VP,2×10⁸VP,2×10⁹VP,2×10¹⁰VP) orPBS were administered two or three times. Gag-specific cellular immune responseswere detected by ELISpot assay at one week post last immunization. The results showed that significant Gag-specific cellular immune responses were detected inimmunized mice. And the mice immunized with2×10⁷VP of rAd5-HIVgag elicitedhighest level of cellular immune responses. To observe the specific cellular immuneresponses in Macaca fascicular is immunized with different dosage of Ad5-HIVgagby repeated in tramuscular injection, the Macaca fascicularis were randomly dividedinto four groups of6. Different amount of the purified Ad5-HIVgag （0.99×1011VP,4.94×1011VP,24.68×1011VP） or PBS were administered in3weeks interval andfive times. Gag-specific cellular immune responses and the level of anti-adenovirusneutralizing antibodies at different time points were detected by ELISpot assay andneutralization assay respectively. The results showed that significant Gag-specificcellular immune responses were detected in all Ad5-HIVgag immunized groups at5weeks post first immunization and then increased at8weeks. The Gag-specificcellular immune responses declined at12weeks and then increased with time. Theseindicated that Gag-specific cellular immune responses could be induced in Macacafascicularis immunized with Ad5-HIVgag in a certain range of dosages. Thepresence of anti-adenovirus neutralizing antibodies induced by vaccination withadenovirus vectors in Ad5-na ve animals did not further reduce Gag-specific cellularimmune responses.In summary, Ad5-HIVgag vaccine can induce Gag-specific cellular immuneresponses. It may be an effective vaccine for the treatment of AIDS. According toChinese registration administration of biological product and technical requirements,we organized the data about the quality control and pharmacodynamic research ofAd5-HIVgag and submitted the application of clinical research to Beijing DrugAdministration.更多还原