利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展
Summary
在这里，我们提出了一个协议，以产生证明性的原则，二价5型腺病毒（Ad5的）载体的Ad5 / H5-HVR1-KWAS-HVR5-他6通过利用衣壳抗原掺入策略。此载体被证明表现出定性的健身，能力逃脱的Ad5阳性血清在体外 ，和抗原性以及免疫原性的结合抗原。
Abstract
腺病毒血清型5（Ad5的）已被广泛地修改传统的转基因方法，所述疫苗开发。在临床试验中，这些传统改性Ad5载体的疗效降低可能主要与相关Ad5的预存免疫性（PEI）的大多数人口中。要通过解决的Ad5 PEI的担忧推动的Ad5载体疫苗的开发，创新的衣壳抗原掺入战略已经就业。通过这一战略的优点，的Ad5向量，我们首先构建了六邻体穿梭质粒HVR1-KWAS-HVR5-他6 / pH5S通过亚克隆高变区（HVR）1六邻体成以前构建穿梭质粒HVR5-他6 / pH5S，其中有他的6标签纳入HVR5。合成这种含有HIV抗原决定簇ELDKWAS HVR1 DNA片段。 HVR1-KWAS-HVR5-他6 / pH5S然后线性化和共转化用线性骨架质粒PAD5 /ΔH5（GL），用于同源重组。此重组质粒PAD5 / H5-HVR1-KWAS-HVR5-他6转染到细胞中，以产生病毒载体的Ad5 / H5-HVR1-KWAS-HVR5-他6。这个载体被验证，以具有通过病毒物理滴度（VP / ml）的指示的定性健身，感染滴度（IP / ml）和相应的VP / IP的比率。两者的HIV抗原决定簇和His 6标签进行表面暴露在Ad5的衣壳，并保留其自己的表位特异性的抗原性。一中和试验表明该向量价由Ad5的阳性血清体外规避中和能力。免疫的小鼠表现出强劲的体液免疫特定的生成艾滋病毒抗原表位和他的6。证明型原理这项研究表明，与衣壳抗原掺入战略相关联的协议，可以切实通过改变不同的衣壳蛋白用于中的Ad5载体疫苗的产生。这个协议甚至可以为further修改为稀土血清型腺病毒载体疫苗的产生。
Introduction
人腺病毒（AD）是一种中型，无包膜病毒含有双链DNA基因组的二十面体衣壳。广告属于腺病毒家族，与分类分为七组（A至G）。每个组都包含不同的血清型病毒。其中，腺病毒血清型5（Ad5的）从C组一直是最广泛研究和应用最广泛的用于矢量化方法，如基因治疗和疫苗接种。
传统的转基因策略已经被开发并应用于Ad5的修改，它的特点是由该病毒早期基因与基因的感兴趣的位移，并在宿主中的感兴趣基因的聚焦表达。例子是pENV9 /Ad5hrΔE3通过与猴免疫缺陷病毒1转基因替换早期基因3（E3）的建设，AdCMVGag通过与人类免疫缺陷的gag基因替换早期基因1（E1）的建设病毒（HIV）2，和广告LAC的Z用紫胶 ž基因3 E1更换施工。对Ad5的传统的转基因战略的广泛应用取决于以下优点：广泛用于Ad5的，对病毒和病毒传播的可行的基因工程，外源基因插入大量的住宿和4,5的Ad5的安全主机。然而，的Ad5向量临床治疗通过使用这种策略的功效降低一直是主要瓶颈，已经映射到主要与Ad5的PEI有关，因为Ad5的是广大儿童和成人4,6中如此普遍。
为了克服的Ad5的主要瓶颈，首要目标是开发一种替代策略规避的Ad5 PEI。先天免疫7，适应性免疫诸如中和抗体（NAb的）8-10和CD8 + T细胞应答10对Ad5的已示出的共同ntribute与Ad5 PEI，与Ad5的出现的NAb发挥贡献的主导作用与Ad5 PEI 10,11。此外，Ad5的NABS位于衣壳蛋白靶表位，包括主要的蛋白质六邻体，纤维和五邻体基底。其中，六邻体是的Ad5的NAbs 8,11-13的主要对象。基于这些发现，一个创新的衣壳抗原掺入战略已经出台。这种新颖的策略突出蛋白的感兴趣上的Ad5衣壳蛋白，其移位或屏蔽的Ad5中和表位的置换或掺入，导致降低识别由所述的NAb高效Ad5载体给药。值得注意的是，这一战略是有竞争力的，因为它也可以帮助主机引起强大的体液免疫和有效的细胞免疫功能通过直接抗原呈递的感兴趣的免疫系统4,14,15。基于这一战略，分子克隆和重组广告病毒载体抢救可分为结构分为四个主要步骤：（a）由任一聚合酶链式反应（PCR）或合成制备的基因的感兴趣的片段; （b）基因片段的连接到包含基因的感兴趣的片段和同源臂到腺病毒主链的穿梭质粒; （c）该同源重组通过共转化含基因的感兴趣的片段与线性化骨架质粒PAD5 /ΔH5（GL）16穿梭质粒; （四）线性化重组腺病毒质粒的转染营救与抗原的感兴趣并入重组Ad载体。
我们的团队和其他一些人已经扩展这种替代广告纳入战略针对不同传染病病原体广告载体疫苗的开发。我们报道了重组腺病毒载体的新一代广告-HVR1-LGS-他6 -V3通过将他标记的HIV-1抗原V3成的Ad5六邻体（hexon5）的HVR1区域。这个生成的载体引发STR具体到V3表位4翁体液免疫应答。我们还报道的Ad5 / HVR2-MPER-L15ΔE1的发展通过将HIV-1的近膜胞外区（MPER）到hexon5 2的HVR2区域。此外，周博士的研究小组已经使用这种广告策略结合的优势，开发广告3型（Ad3的）载体疫苗， 即 ，病毒载体R1SP70A3的通过将肠道病毒71型的中和表位SP70到Ad3的的HVR1的一代六邻体（hexon3）。 R1SP70A3产生了浓厚的NAbs和IFN-γ生产特定的抗原表位SP70，从而导致对肠病毒71型的挑战15率高保护。
对于技术参考的目的，我们的研究采取了定性衣壳抗原掺入战略的优势，专注于二价Ad5载体的Ad5 / H5-HVR1-KWAS-HVR5-他6的产生通过将一种HIV-1抗原进入HVR1一个DA他的标签为hexon5的HVR5。生成的病毒载体也被免疫评价。该衣壳抗原掺入策略可以被用来实现对不同传染病的Ad5疫苗向量方法的制定。
Protocol
Representative Results
Discussion
上的Ad5修改为疫苗的发展传统的转基因策略的应用已经被降低主要是由于与Ad5的PEI 4,6相关联的瓶颈。这个瓶颈可以通过应用替代抗原衣壳-团策略（ 图1A）被部分地减小，因为这种策略可以通过的Ad5的NAb逃避中和通过与抗原的感兴趣取代的Ad5的中和表位，以及促进健壮免疫力的产生到结合抗原的感兴趣。在这项研究中，修改后的二价病毒载体的产生的Ad5 / H5-HVR1-KWAS-HVR5-他6<…
Divulgations
The authors have nothing to disclose.
Acknowledgements
卫生部授予5T32AI7493-20和5R01AI089337-03部分这项工作是支持国家机构。该资助者在研究设计，数据收集和分析，发布决定，或准备的手稿没有作用。
Materials
| 1x DPBS | Thermo Scientific | SH30256.01 | for cell spliting |
| 10x DPBS | Thermo Scientific | SH30378.02 | for dialysis buffer preparation |
| glycerol | SIGMA | G5516-1L | for dialysis buffer preparation |
| SDS | BIO-RAD | 161-0301 | for virus lysis buffer preparation |
| Fetal Bovine Serum (FBS) | Thermo Scientific | SH30910.03 | component of culture medium |
| 100X Non-Essential Amino Acids | Thermo Scientific | SH30238.01 | component of culture medium |
| 200mM/L L-glutamine | Cellgro | 25-005-CI | component of culture medium |
| penicillin/streptomycin solution | Cellgro | 30-002-CI | component of culture medium |
| DMEM with high glucose | Thermo Scientific | SH30081.01 | for HEK293 cell culture |
| Minimum Essential Medium Eagle | SIGMA | M5650 | for HeLa cell culture |
| phenol:chloroform:isoamyl alcohol | SIGMA | P3803-100ML | for large size of DNA purification |
| cesium chloride | Research Products International Corp. | C68050 | for virus purificiation |
| HEPES | Cellgro | 25-060-CI | for CsCl solution preparation |
| HEK293 | ATCC | 51-0036 | for virus rescue and upscale |
| HeLa | ATCC | CCL-2 | for neutralization assay |
| T-25 flask | Thermo Scientific | 156367 | for cell culture |
| T-75 flask | CORNING | 430641 | for cell culture |
| T-175 flask | Thermo Scientific | 159910 | for cell culture |
| Ultracentrifuge | BECKMAN | NA | for virus purification |
| Ultracentrifuge tube | BECKMAN | 344059 | for virus purification |
| dialysis cassette | Thermo Scientific | 66380 | for virus dialysis |
| ELISA plate | Thermo Scientific | 442404 | for ELISA |
| human anti-gp41 (2F5) mAb | NIH AIDS Reagent Program | 1475 | for immunological assays |
| mouse anti-His tag mAb | GenScript | A00186 | for immunological assays |
| SOC medium | CORNING | 46-003-CR | for transformation |
| PCR master mix solution | QIAGEN | 201445 | for PCR |
| Animal lancet (point length at 5mm) | MEDIpoint | for mice bleeding | |
| Biophotometer | Eppendorf | for virus physical titer titration |
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