【作者】 李凯；

【导师】 徐志凯； 张芳琳；

【作者基本信息】 第四军医大学 ， 微生物学， 2013， 博士

【摘要】 【背景】汉坦病毒感染可引起两类不同的严重感染性疾病，即肾综合征出血热（Hemorrhagic fever with renal syndrome，HFRS）和汉坦病毒肺综合征（Hantaviruspulmonary syndrome，HPS）。其中HFRS多见于亚洲及东北欧部分地区，主要由汉滩病毒（Hantaan virus，HTNV）、首尔病毒（Seoul virus，SEOV）和普马拉病毒（Puumalavirus，PUUV）等引起。我国是世界上HFRS疫情最严重的国家，流行范围广，发病人数多，病死率较高。HFRS是我国危害最严重的急性传染病之一。目前我国已研制成功HFRS灭活疫苗，其推广使用对预防HFRS的发生和流行起到了积极作用。但该类疫苗仍存在一些问题，主要是不能有效刺激细胞免疫应答，诱导机体产生中和抗体的能力也较弱，抗体滴度不高；此外，目前对于灭活疫苗在制备与使用中的安全性尚存在一些争议。因此，研发新型的HFRS疫苗将有助于进一步提高我国对HFRS的防控水平。汉坦病毒基因组包括L、M、S等3个片段，分别编码病毒的RNA聚合酶、包膜糖蛋白（glycoprotein，GP）Gn、Gc以及核衣壳蛋白（nucleoprotein，NP）。GP和NP都是诱导机体免疫应答的主要结构蛋白，其中的Gn及Gc可刺激机体产生中和抗体，在机体的保护性体液免疫应答中起主要作用，但其免疫原性较弱，特别是刺激机体细胞免疫应答的能力较弱；NP的免疫原性很强，特别是在NP上存在多个CTL表位，在诱导机体细胞免疫应答中起主要作用，可有效保护汉坦病毒对实验动物的攻击。因此，同时应用汉坦病毒GP与NP作为重组疫苗的组分有可能达到优势互补，提高免疫应答效果的目的。本实验室前期构建了含HTNV S基因0.7kb片段与M基因嵌合基因的一系列原核与真核表达载体，并进行了融合表达以及基因免疫。结果表明，嵌合基因表达的融合蛋白能有效刺激机体的体液免疫应答以及部分细胞免疫应答；在不同的表达系统中，以重组腺病毒免疫的效果为好，但仍存在融合蛋白的表达量偏低以及诱导机体免疫应答水平尚不理想的问题。针对这些问题，本实验室正试图通过对表达载体进行改建，改善目的基因的构成，采用不同的表达方式，联合使用不同的分子佐剂等多种策略，来提高目的抗原的表达量及其诱导机体免疫应答的能力，本研究的目的亦在于此。【方法】本研究在采用CAG启动子及HSP70C分子佐剂的基础上，采取两条不同的技术路线，构建多株含HTNV不同结构蛋白及其CTL表位、不同组合方式、不同表达方式的重组腺病毒，并对构建的重组腺病毒诱导小鼠体液免疫应答和细胞免疫应答的情况进行观察和比较。（1）考虑到HTNV GP的表位为空间构像依赖，将GP与NP融合表达可能会影响其空间构型，从而影响到免疫原性，本研究使用脑心肌炎病毒内核糖体进入位点（ECMV IRES）将GP、NP在同一腺病毒中分别表达，更有利于保持其免疫学活性。（2）加入有效的HTNV CTL表位基因（NP127～141aa、NP331～345aa及NP421～429aa，以加间隔序列AAY的方式串联），以提高融合蛋白刺激机体免疫应答的能力，特别是提高细胞免疫应答的能力。【结果】第一部分含HTNV结构基因双顺反子及HSP70C的重组腺病毒的制备及免疫学特性研究1．构建了可分别表达HTNV GP、NP及HSP70C的重组腺病毒载体，转染HEK293A细胞后，获得了8株重组腺病毒，分别命名为rAd-GnH-IRES-S0.7H、rAd-GcH-IRES-S0.7H、 rAd-GnH-IRES-S0.7、 rAd-GcH-IRES-S0.7、 rAd-Gn-IRES-S0.7H、rAd-Gc-IRES-S0.7H、rAd-Gn-IRES-S0.7、rAd-Gc-IRES-S0.7。重组腺病毒经纯化并测定滴度后，以100pfu/cell的病毒量感染HEK293A细胞，免疫荧光检测结果显示，所有重组腺病毒均可表达目的蛋白。2．将上述各重组腺病毒通过腹腔途径免疫C57BL/6小鼠，以腺病毒空载体、PBS及HFRS灭活疫苗为对照。通过ELISA及微量细胞培养中和试验对各组小鼠体液免疫应答的情况进行了检测。结果显示，各重组腺病毒均可刺激小鼠产生针对HTNVGP、NP的特异性抗体及中和抗体，且添加HSP70C的各重组腺病毒免疫组抗体的几何平均滴度（GMT）均高于对应的未添加组；在含Gn的各重组腺病毒组中，rAd-GnH-IRES-S0.7H免疫组小鼠血清的抗GP、NP抗体及中和抗体的GMT均为最高，分别为95.1、320.0和30.3；在含Gc的各重组腺病毒组中，rAd-GcH-IRES-S0.7H免疫组小鼠血清的抗GP、NP抗体及中和抗体的GMT均为最高，分别为80.0、380.5和40.0；该两种重组腺病毒诱导的中和抗体均高于灭活疫苗组（p<0.05）。3．对上述各重组腺病毒免疫小鼠的细胞免疫应答情况进行了检测。CTL杀伤实验的结果表明，融合HSP70C的两种重组腺病毒（rAd-GnH-IRES-S0.7H和rAd-GcH-IRES-S0.7H）免疫组小鼠的脾细胞在各效靶比下杀伤效应均为最好，且均高于灭活疫苗组（p<0.05）。ELISPOT检测的结果表明，在各肽池刺激下rAd-GnH-IRES-S0.7H及rAd-GcH-IRES-S0.7H免疫组小鼠脾细胞的IFN-分泌水平均为最高，且均高于灭活疫苗组（p<0.05）。表明融合HSP70C的重组腺病毒能更好的诱导机体的细胞免疫应答。在使用重叠肽段作为刺激物进行的ELISPOT实验中，NP3肽池显示出了最好的刺激效果，表明HTNV的NP在诱导机体细胞免疫应答中的作用比GP更为重要。4．用HTNV76-118株攻击各重组腺病毒免疫的小鼠，采用ELISA及RT-PCR对感染小鼠的部分脏器进行检测，结果显示，各重组腺病毒免疫组小鼠脏器中的病毒抗原及病毒核酸量均低于PBS对照组及空载免疫组，其中rAd-GnH-IRES-S0.7H免疫组及rAd-GcH-IRES-S0.7H免疫组小鼠的HTNV抗原及核酸含量均为最低，且与灭活疫苗组无显著差别（p>0.05），表明其保护效果与灭活疫苗组相当。第二部分含HTNV嵌合基因、CTL串联表位及HSP70C的重组腺病毒的制备及免疫学特性研究1．构建了含HTNV S基因0.7kb片段与M基因的嵌合基因、CTL串联表位及HSP70C的重组腺病毒载体，转染HEK293A细胞后，获得了4株重组腺病毒，分别命名为rAd-GnS0.7-CTLH、rAd-GnS0.7-CTL、rAd-GcS0.7-CTLH、rAd-GcS0.7-CTL。重组腺病毒经纯化并测定滴度后，以100pfu/cell的病毒量感染HEK293A细胞，免疫荧光检测结果显示，所有重组腺病毒均可表达目的蛋白。2．将上述各重组腺病毒通过腹腔途径免疫C57BL/6小鼠，以腺病毒空载体、PBS及HFRS灭活疫苗为对照。通过ELISA、微量细胞培养中和试验对各组小鼠体液免疫应答的情况进行了检测。结果显示，各重组腺病毒均可刺激小鼠产生针对HTNVGP、NP的特异性抗体及中和抗体，且添加HSP70C的两个重组腺病毒免疫组抗体的GMT均高于对应的未添加组；其中rAd-GnS0.7-CTLH免疫组小鼠血清的抗GP、NP抗体及中和抗体的GMT分别为80.0、320.0和26.4，均高于rAd-GnS0.7-CTL免疫组；rAd-GcS0.7-CTLH免疫组小鼠血清的抗GP、NP抗体及中和抗体的GMT分别为113.1、452.5和30.3，该两种重组腺病毒诱导的中和抗体均高于灭活疫苗组（p<0.05）。3．对上述各重组腺病毒免疫小鼠的细胞免疫应答情况进行了检测。CTL杀伤实验的结果表明，rAd-GnS0.7-CTLH免疫组在各效靶比下，杀伤效应均强于rAd-GnS0.7-CTL组，且在效靶比为100:1时也强于灭活疫苗组（p<0.05）；rAd-GcS0.7-CTLH免疫组在各效靶比下，杀伤效应均强于rAd-GcS0.7-CTL组，且在效靶比为10:1及100:1时也强于灭活疫苗组（p<0.05）。ELISPOT检测的结果表明，在各肽池刺激下rAd-GnS0.7-CTLH及rAd-GcS0.7-CTLH免疫组小鼠脾细胞的IFN-分泌水平均为最高（p<0.05）；rAd-GcS0.7-CTLH免疫组IFN-分泌水平高于灭活疫苗组（p<0.05），在除Gn2肽池外的各肽池刺激下rAd-GnS0.7-CTLH组IFN-γ的分泌水平也高于灭活疫苗组（p<0.05）。同样表明融合HSP70C的重组腺病毒能更好的诱导机体的细胞免疫应答。以本研究所采用的CTL表位序列的三个单独的15aa的肽段分别作为刺激物进行ELISOPT实验，结果显示NP48及NP60肽段的刺激效果最好，表明其包含的CTL表位在诱导细胞免疫应答中的作用更为突出。4．用HTNV76-118株攻击各重组腺病毒免疫后的小鼠，采用ELISA及RT-PCR对感染小鼠的部分脏器进行检测，结果显示，各重组腺病毒免疫组小鼠脏器中的病毒抗原及病毒核酸量均低于PBS对照及空载免疫组，其中rAd-GnS0.7-CTLH免疫组及rAd-GcS0.7-CTLH免疫组小鼠的HTNV抗原及核酸含量均为最低，且与灭活疫苗组无显著差别（p>0.05），表明其保护效果与灭活疫苗组相当。【结论】本研究在采用CAG启动子及HSP70C分子佐剂的基础上，采取两条不同的技术路线进一步优化了HTNV结构蛋白在重组腺病毒中的表达，并藉此提高其诱导机体免疫应答的效果。一系列体内外实验的结果表明，采用将HTNV GP和NP分别与HSP70C融合，并用IRES将两种融合蛋白分别表达的策略构建的重组腺病毒，其诱导体液免疫应答和细胞免疫应答的能力均得到了提升；采用同时添加CTL串联表位及HSP70C的策略构建重组腺病毒，也可有效提升其诱导体液免疫应答及细胞免疫应答的能力。上述两种策略构建的重组腺病毒诱导的免疫应答在多数指标上均优于灭活疫苗或与之相同；相比较而言，前一种策略构建的重组腺病毒在诱导体液免疫应答方面更具优势，而后一种策略在诱导细胞免疫应答方面更具优势。未来我们将综合运用上述两种策略，以期获得更好的免疫保护效果，为HFRS新型疫苗的研制提供理论、实验和物质基础。更多还原

【Abstract】 【Background】Hantavirus infection is manifested as two different forms of severe febrile diseases,hemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome(HPS). Most HFRS infections occur in East Asia and Eastern Europe coursed by Hantaanvirus (HTNV), Seoul virus (SEOV) and Puumala virus (PUUV).In China, Hantaviruses result in most severe morbidity and mortality. Inactivatedvaccines have been produced by using rodent brain and cell cultures in China. Althoughthe protective efficacy of several inactivated vaccine have been confirmed, there remains cellular immunity. Moreover, there still some problems concerning their production andhuman use. There is an urgent need to develop new vaccines to prevent the epidemic ofHFRS in China.Hantaviruses are enveloped viruses with a genome that consists of threesingle-stranded, negative sense RNA segments designated S (small), M (medium), and L(large). The S RNA encodes the nucleocapsid (N) protein. The M RNA encodes apolyprotein that is co-translationally cleaved to yield the envelope glycoproteins Gn andGc. Both GP and NP play important role in elicit protective immune response duringHantavirus infection. The Gn and Gc are presumed to be the major elements involved ininduction of neutralizing antibodies during Hantavirus infection. Nevertheless, theimmunogenicity of GP is feeble, the antibody elicited by GP appeared later, the antibodytiter is low. Cellular immunity is believed to be associated with the Hantavirus NP. It issuggested that NP contains antigenic sites involved in CTL responses, which have a grateinfluence on elicit cellular immune response. Experiments show that immunization withNP induces a protective immune response which can increase the survival of animalsfollowing challenge with a lethal dose of Hantavirus. Therefore, utilization of both theglycoprotein and NP components for vaccine development could be a very promisingapproach.Our lab has constructed a series of prokaryotic and eukaryotic expression vectorcontaining HTNV chimeric genes (GnS0.7and GnS0.7) in previous studies. Data showsthat the fusion protein expressed by chimeric genes could elicit humoral immunity andpart of cellular immunity. During our research the adenovirus expression system shows thebest result, but there are still problems in fusion proteins express level and eliciting betterimmune response. Aiming at these problems, we try to optimize the expression vector andimprove the form of interest genes, joint application of molecular adjuvant to elevateantigen expression level and immune response level. The purpose of this study lies here.【Methods】Based on the CAG promoter and HSP70C molecular adjuvant, we take two strategiesto increase the humoral and cellular immune response. Adenovirus vectors containing HTNV structural protein and CTL were constructed through different combinations waysand different expression methods. The humoral and cellular immune response wereassessed by detect mice immunized with recombinant adenoviruses.(1) In consideration of the spatial conformation of GP, fusion expression coulddestruction immunity of GP; we take advantage of ECMV IRES to independently expressGP and NP.(2) Joint application of CTL epitopes (NP127-141aa, NP331-345aa and NP421-429aa)and HSP70C to increase cellular immunity.【Results】Part1Construction and identification of the immunological properties of adenovirusvector containing HTNV GP, IRES, NP and HSP70C1. Adenovirus vector containing HTNV GP, IRES, NP and HSP70C was constructed.Recombinant adenoviruses were obtained by infected HEK293A cells and namedrAd-GnH-IRES-S0.7H, rAd-GcH-IRES-S0.7H, rAd-GnH-IRES-S0.7, rAd-GcH-IRES-S0.7, rAd-Gn-IRES-S0.7H, rAd-Gc-IRES-S0.7H, rAd-Gn-IRES-S0.7and rAd-Gc-IRES-S0.7. The interest proteins of different recombinant adenoviruses were identified by IFA.Specific fluorescence was observed in cells after infection.2. C57BL/6mice were immunized with recombinant adenoviruses. Adeno-lacZ, PBSand inactivated vaccine immunized mice were set as controls. Humoral immunity levelwere assessed by ELISA and cell micro-neutralization test. Results showed that alladenovirus could elicit humoral immune responses incorporation of neutralizingantibodies and GP and NP specific antibodies. Adenovirus incorporation of HSP70C elicitshigher geometric mean titer (GMT)(glycoprotein and NP-specific) than others. Amongadenovirus containing Gn, rAd-GnH-IRES-S0.7H elicit highest GMT (glycoprotein andNP-specific), which were95.1and320.0. Among adenovirus containing Gc, miceimmunized with rAd-GcH-IRES-S0.7H elicit highest GMT (glycoprotein and NP-specific),which were80.0and380.5. Adenovirus incorporation of HSP70C also elicits higherneutralizing antibodies GMT than others. The neutralizing antibodies GMT of miceimmunized with rAd-GnH-IRES-S0.7H (30.3) and rAd-GcH-IRES-S0.7H (40.0) were higher than that mice immunized with inactivated vaccine (p<0.05).3. During CTL assay, the spleno6cytes of mice immunized withrAd-GnH-IRES-S0.7H and rAd-GcH-IRES-S0.7H showed highest specific Tcell-mediated cytotoxicity and the cytotoxicity were higher than inactivated vaccine atdifferent E/T ratio (p<0.05). ELISPOT results showed that mice were immunized withrecombinant adenoviruses which containing HSP70C exhibited higher frequency ofsplenic CD8+T cells secreting IFN-(p<0.05). rAd-GnH-IRES-S0.7H andrAd-GcH-IRES-S0.7H elicited higher frequency of splenic CD8+T cells secreting IFN-than vaccine during stimulated with different peptides（p<0.05）.4. Immunized mice were challenged by HTNV76-118, virus antigens and nucleicacid was detected by ELISA and RT-PCR. The amount of HTNV antigens and nucleicacids in organs of mice immunized with recombinant adenoviruses were lower thanimmunized with Adeno-lacZ and PBS. It is suggested that all recombinant adenovirusesimmunized groups show protectiveness against HTNV infection. Mice immunized withrAd-GnH-IRES-S0.7H and rAd-GcH-IRES-S0.7H exhibited the lowest HTNV antigensand nucleic acids capacity equal to the vaccine (p>0.05).Part2Construction and identification of the immunological properties of adenovirusvector containing HTNV chimeric genes and CTL1. Adenovirus vector containing HTNV chimeric genes (GnS0.7and GnS0.7) andCTL was constructed. After infected HEK293A cells, recombinant adenoviruses wereobtained and named rAd-GnS0.7-CTLH, rAd-GnS0.7-CTL, rAd-GcS0.7-CTLH andrAd-GcS0.7-CTL. The interest proteins of different recombinant adenoviruses wereidentified by IFA. Specific fluorescence was observed in cells after infection.2. C57BL/6mice were immunized with recombinant adenoviruses. Adeno-lacZ, PBSand inactivated vaccine immunized mice were set as controls. Humoral immunity levelwere assessed by ELISA and cell micro-neutralization test. Results showed that alladenovirus could elicit humoral immune responses incorporation of neutralizingantibodies and GP and NP specific antibodies. Adenovirus incorporation of HSP70C elicitshigher geometric mean titer (GMT)(glycoprotein and NP-specific) than others. Among adenovirus containing Gn, rAd-GnS0.7-CTLH elicit highest GMT (glycoprotein andNP-specific), which were80.0and320.0. Among adenovirus containing Gc, miceimmunized with rAd-GcS0.7-CTLH elicit highest GMT (glycoprotein and NP-specific),which were113.1and452.5. Adenovirus containing HSP70C also elicits higherneutralizing antibodies GMT than others. The neutralizing antibodies GMT of miceimmunized with rAd-GnS0.7-CTLH (26.4) and rAd-GcS0.7-CTLH (30.3) were higherthan that mice immunized with inactivated vaccine (p<0.05).3. During CTL assay, the splenocytes of mice immunized with rAd-GnS0.7-CTLHand rAd-GcS0.7-CTLH showed higher specific T cell-mediated cytotoxicity thanrAd-GnS0.7-CTLH and rAd-GcS0.7-CTLH. The cytotoxicity of rAd-GnS0.7-CTLH washigher than inactivated vaccine at E/T ratio of100:1(p<0.05). The cytotoxicity ofrAd-GcS0.7-CTLH was higher than inactivated vaccine at E/T ratio of10:1and100:1(p<0.05). ELISPOT results showed that mice were immunized with recombinantadenoviruses which containing HSP70C exhibited higher frequency of splenic CD8+Tcells secreting IFN-(p<0.05). rAd-GcS0.7-CTLH elicited higher frequency of splenicCD8+T cells secreting IFN-than vaccine during stimulated with different peptides(p<0.05). Besides stimulated with Gn2peptides pool rAd-GnS0.7-CTLH elicited higherfrequency of splenic CD8+T cells secreting IFN-than vaccine (p<0.05). Set15aaoverlapping peptide containing CTL epitopes as stimulator, data shows that NP48andNP60exhibit the best effects. It is suggested that the amino acid sequence in NP48andNP60play an important role in elicit CTL response.4. Immunized mice were challenged by HTNV76-118, virus antigens and nucleicacid was detected by ELISA and RT-PCR. The amount of HTNV antigens and nucleicacids in organs of mice immunized with recombinant adenoviruses were lower than whichimmunized with Adeno-lacZ and PBS. Suggested that all recombinant adenovirusesimmunized groups show protectiveness against HTNV infection. Mice immunized withrAd-GnS0.7-CTLH and rAd-GcS0.7-CTLH exhibited the lowest HTNV antigens andnucleic acids capacity equal to the vaccine (p>0.05).【Conclusion】 During this study, based on the CAG promoter and HSP70C molecular adjuvant, we take twostrategies to promote humoral and cellular immune response. We optimize the expression ofinterest genes in adenovirus expression system. A series of experiments in vivo and in vitroshows that take advantage of ECMV IRES to independently express GP and NP(fused toHSP70C respectively) could elevate humoral and cellular immune response. Joint applicationof CTL epitopes and HSP70C also could increase humoral and cellular immunity. Data showsthat both strategies acquired success, in most respects (humoral and cellular immune response)even exceed vaccine. By contrast, the former strategies elicit better humoral immunity and thelatter strategies elicit better cellular immune response. In future studies, we will integrateapplication of both strategies to acquire better immune response, which will provide theory,experiment and material basis for HTNV vaccine research.更多还原