【作者】 江云波；

【导师】 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2007， 博士

【摘要】 猪繁殖与呼吸综合征（PRRS）是由猪繁殖与呼吸综合征病毒（PRRSV）引起的一种以妊娠母猪严重繁殖障碍以及仔猪的呼吸道症状和高死亡率为特征的传染病。PRRS自1987年首次报道发现于美国以来，现已遍及世界各主要养猪国家与地区，并已成为危害养猪业最严重的传染病之一。同其它病毒病的防制一样，免疫接种仍是预防与控制PRRS的主要措施。目前用于预防PRRS的主要商用疫苗是传统的常规弱毒疫苗和灭活疫苗，但由于存在弱毒疫苗散毒和灭活疫苗免疫效果不理想等原因，使得这两种疫苗的使用都存在着一定的争议。因此，研制更安全、高效、廉价的新型疫苗成为越来越多研究者关注的焦点，但目前的试验性疫苗普遍存在诱发保护性免疫不高的问题，使得对于PRRSV的新型疫苗的研究进展一直比较缓慢。鉴于此，本研究对PRRSV新型疫苗设计进行了新的探索，构建了以西门利克森林病毒（SFV）为复制子的“自杀性”DNA疫苗和伪狂犬病毒（PRV）为载体的重组PRV-PRRSV新型基因工程疫苗，并对构建的候选疫苗进行了免疫效力和免疫保护研究，主要研究内容如下：1、天然ORF5基因的修饰ORF5基因编码的囊膜糖蛋白GP5是PRRSV的主要免疫原性蛋白，也是目前研制PRRSV新型疫苗的主要靶蛋白。但单独基于天然ORF5基因构建的一些试验性疫苗普遍存在诱发中和抗体缓慢和不高的问题。为了获得用于研制PRRSV新型疫苗的良好靶基因，将通用型辅助性T淋巴细胞表位（PADRE）插入ORF5基因的中和表位和覆盖表位间，获得修饰后的ORF5基因ORF5m，以期达到充分暴露中和抗体表位，诱发更强的免疫应答的目的。进一步以DNA疫苗的形式进行了ORF5m的体外表达和免疫原性研究。Wsetern blot证实ORF5m能够表达25～26 kDa的GP5m蛋白，并能够较好地与PRRSV的高免猪血清发生特异性结合，间接免疫荧光试验（IFA）表明GP5m蛋白与天然的GP5蛋白同样定位于细胞质中。小鼠免疫试验结果表明：修饰后的DNA疫苗pCI-ORF5m诱导的中和抗体、抗GP5蛋白ELISA抗体和淋巴细胞增殖反应均明显优于未经修饰的DNA疫苗pCI-ORF5，表明修饰型ORF5m基因具有更好的免疫原性，可作为PRRSV新型疫苗开发的候选靶基因。2、GP5／M异源二聚体的形成及其对GP5蛋白亚细胞定位和诱发免疫反应的影响由ORF5基因编码的GP5蛋白与ORF6基因编码的M蛋白在成熟的病毒粒子和感染的细胞内可以形成异源二聚体GP5／M。为了探讨GP5蛋白和M蛋白体外共表达特性及对免疫反应的影响，分别构建了PRRSV ORF5、ORF6单基因真核表达质粒pCI-ORF5、pCI-ORF6和双基因共表达的真核表达质粒pCI-ORF5／ORF6。转染BHK-21细胞，Westem blot检测证实共表达的GP5和M蛋白能够形成异源二聚体，并且这种异源二聚体的形成能促进GP5蛋白从内质网向高尔基体转运。将构建的真核表达质粒免疫小鼠后，发现共表达GP5和M蛋白的DNA疫苗pCI-ORF5／ORF6免疫小鼠于首免后4周开始产生可检测特异性中和抗体，到首免后8周达到最高，而pCI-ORF5和pCI-ORF6免疫组只有部分小鼠产生了可检测特异性中和抗体。并且pCI-ORF5／ORF6免疫组产生的特异性淋巴细胞增殖反应也最高。而且，GP5和M蛋白共表达免疫小鼠所产生的特异性抗GP5蛋白的ELISA抗体水平也显著增强。进一步进行仔猪免疫试验证实，pCI-ORF5／ORF6免疫仔猪于首免后10周中和抗体全部阳转（≥1∶8），而单独表达GP5蛋白的pCI-ORF5免疫仔猪至试验结束时未检测到特异性中和抗体的产生。研究表明GP5／M异源二聚体的形成可能与GP5蛋白的翻译后修饰、转运、定位相关，并可促进免疫动物产生特异性的免疫应答。3、PRRSV DNA疫苗的构建和免疫效果以小鼠为模型动物，探讨了ORF5m和ORF6双基因共表达的常规DNA疫苗pCI-ORF5m／ORF6和“自杀性”DNA疫苗pSFV-ORF5m／ORF6的免疫效力。结果，所有DNA疫苗免疫小鼠均于首免后4周产生了可检测的特异性中和抗体，于首免后8周达到最高。共表达修饰型ORF5m和ORF6基因较共表达天然ORF5和ORF6基因的DNA疫苗能够诱发更高的中和抗体，而“自杀性”DNA疫苗又较常规DNA疫苗诱发更强的淋巴细胞增殖水平。结果表明ORF5m和ORF6双基因共表达的“自杀性”DNA疫苗pSFV-ORF5m／ORF6能够诱发最高的中和抗体和细胞免疫应答，是一种具有良好发展前景的PRRSV新型候选疫苗。4、伪狂犬病毒-猪繁殖与呼吸综合征病毒（rPRV-PRRSV）重组基因工程疫苗构建了以致弱的伪狂犬病毒（PRV）为载体表达PRRSV两种主要膜相关蛋白（GP5和M）的四种重组病毒，分别为rPRV-GP5（单独表达GP5）、rPRV-GP5m（单独表达修饰型GP5蛋白（GP5m））、rPRV-GP5-M（共表达GP5和M）和rPRV-GP5m-M（共表达GP5m和M）。体外表达试验表明，构建的重组病毒均能较好的表达外源目的基因，并且表达的GP5（或GP5m）和M蛋白能够形成GP5／M（或GP5m／M）异源二聚体。将这四种重组病毒免疫小鼠证实，四种PRV重组体均能诱发免疫动物产生与PRV亲本株相当的特异性抗PRV抗体和抵抗PRV致死剂量的攻击，并观察到诱发不同程度的特异性抗PRRSV的免疫应答。其中共表达GP5和M蛋白的重组病毒rPRV-GP5-M较单独表达GP5的重组病毒rPRV-GP5能够诱发更高的中和抗体。相比较而言，以ORF5m为候选基因构建的重组病毒rPRV-GP5m和rPRV-GP5m-M展示了更强的诱发中和抗体和淋巴细胞增殖能力。共表达GP5m和M蛋白的重组体rPRV-GP5m-M表现出最佳的免疫效果，于首免后10周6只免疫小鼠有两只的中和抗体达到了1∶32，并且淋巴细胞增殖能力也显著高于其他PRV重组体免疫组，表明rPRV-GP5m-M是一种新型的PRV-PRRSV二价基因工程候选疫苗。5、PRRSV新型候选疫苗对猪的免疫保护研究为了充分评价构建的两种PRRSV新型候选疫苗（“自杀性”DNA疫苗pSFV-ORF5m／ORF6和重组伪狂犬病毒rPRV-GP5m-M）的免疫效力和免疫保护，进一步以PRRSV感染自然宿主仔猪为动物模型，分析其诱发特异性体液免疫和细胞免疫应答以及保护免疫动物免受强毒攻击的能力，并与PRRSV灭活苗进行比较。结果，rPRV-GP5m-M能够诱发与PRV亲本株相当的针对PRV的特异性中和抗体和ELISA抗体。pSFV-ORF5m／ORF6和rPRV-GP5m-M免疫仔猪于首免后42d产生可检测的特异性抗PRRSV的中和抗体（1∶4），随后逐渐上升，而PRRSV灭活苗免疫仔猪在攻毒前PRRSV中和抗体一直为阴性。所有免疫仔猪于首免后63d用5×10^5.0TCID₅₀（5mL）PRRSV YAl强毒株进行攻击后，pSFV-ORF5m／ORF6和rPRV-GP5m-M免疫仔猪的中和抗体水平进一步快速上升，于攻毒后17-21d时达到1∶128～1∶256。并且，pSFV-ORF5m／ORF6和rPRV-GP5m-M免疫仔猪在攻毒前也产生了较强的淋巴细胞增殖，并于攻毒后进一步增强，而灭活苗免疫仔猪对照组在攻毒后仅产生了较微弱的淋巴细胞增殖反应。在攻毒后的临床保护方面，与灭活苗相比，pSFV-ORF5m／ORF6和rPRV-GP5m-M免疫仔猪显示了更平稳的体温波动、更短时间的病毒血症、更低的鼻拭子、扁桃体拭子和组织病毒检出率、更温和的肺和淋巴结损伤，展示了更为有效的免疫保护。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a serious infectious disease of swine. It is characterized by severe reproductive failure in sows, and respiratory distress in piglets and growing pigs. Since it was first reported in the United States in 1987, PRRS has already been one of the most economically important diseases of the global swine industry. Vaccine inoculation is the main method to prevent and control this disease. Currently, the commercial vaccines to prevent PRRS are mainly attenuated and inactivated vaccines. A discrepancy of inadequate efficacy and safety concerns surrounds the modified live or killed vaccines. It has become a focus to develop safer and more effective and cheap vaccines to prevent and control PRRS. However, due to limited immunity protection in several experimental vaccines, the progress of new vaccines against PRRSV maintains slowly.Therefbre, in the present study, we explored the new vaccine design against PRRSV, and constructed suicidal DNA vaccines based on Semiliki Forest virus (SFV)-replicon and the recombinant viruses based on pseudorabies virus (PRV)-vector. Furthermore, the immune efficacy and protection of the candidate vaccines were investigated. The most research works were as following:1.2.1 Functional modification of native ORF5 geneThe ORF5-encoded major envelope glycoprotein (GP5) is one of the key immunogenic proteins of PRRSV and is the leading target for the development of the new generation of vaccines against PRRSV. However, weak and tardy neutralizing antibodies have been elicited in several developed experimental vaccines expressing native GP5 protein. In order to obtain the new perfected target genes for developing new vaccines against PRRSV, we modified ORF5 gene by inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the decoy epitoe to minimize or eliminate the decoy effect of the non-neutralizing epitope. The invitro expression and immunogenicity of the modified ORF5 (ORF5m) was further evaluated using DNA vaccination. Western blot showed that ORF5m gene can express GP5m protein with about 25～26 kDa molecular mass, which reacted well with porcine polyclonal antiserum against PRRSV. IFA assay showed that the intracellular location of GP5m was equal to that of native GP5. The results of the mice immunization suggested that significantly enhanced neutralizing antibodies were elicited in mice immunized with the DNA construct expressing ORF5m gene compared with the native ORF5. Significantly increased levels of GP5-specific ELISA antibodies and T-cell proliferative activities were also observed. These results indicated that the high immunogenicity of ORF5m gene might facilitate the development of improved PRRS vaccines in the future.2.2.1 Formation of GP5/M heterodimer and influence on the intracellular localization and induced immune responsesThe two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In the present study, three different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed to investigate the biological characterization and immunological responses to co-expressing GP5 and M proteins. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells, and heterodimerization facilitated the transport of GP5 from endocytoplasmic reticulum (ER) to Golgi’s body. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 4 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibodies could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies (≥1:8) at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. These results indicate that the formation of GP5/M heterodimers may be involved in posttranslational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.3.2.1 Construction and immune effect of new DNA vaccines against PRRSVThe immunogenicity of general DNA vaccine pCI-ORF5m/ORF6 and suicidal DNA vaccine pSFV-ORF5m/ORF6 co-expressing ORF5m and ORF6 genes was investigated in mouse model. The results showed that all immunized mice developed neutralizing antibodies (≥1:8) at 4 weeks after primary immunization and achieved the peak at 8 weeks after primary immunization. The neutralizing antibodies level induced by co-expression of ORF5m and ORF6 genes was higher than that induced by co-expression of native ORF5 and ORF6 genes. And suicidal DNA vaccine also displayed the enhanced lymphocyte proliferative compared with general DNA vaccine. These results indicated that a suicidal DNA vaccine pSFV-ORF5m/ORF6 co-expressing ORF5m and ORF6 genes elicited the immunized animals to produce highest neutralizing antibodies and cellular immune responses, and is a new promising vaccine against PRRSV.4.2.1 The recombinant genetically-engineering vaccines against PRRSV based on PRV-vector (rPRV-PRRSV)A live attenuated pseudorabies virus (PRV) was used as vaccine vector to express the two major membrane-associated proteins (GP5 or M) of PRRSV in various forms. Four PRV recombinants, rPRV-GP5 (expressing native GP5), rPRV-GP5m (expressing GP5m, a modified GP5), rPRV-GP5-M (co-expressing GP5 and M proteins), rPRV-GP5m-M (co-expressing GP5m and M proteins) were generated. The results of Western blot using anti-PRRSV hyperimmune sera also demonstrated correct expression of PRRSV genes in the four identified recombinant viruses. Furthermore, under non-reducing condition, GP5 or GP5m and M could form heterodimers in cells infected by rPRV-GP5-M or rPRV-GP5m-M. Mice immunized with all these recombinants developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. However, immune responses to PRRSV induced by these PRV recombinants showed distinctness, rPRV-GP5-M co-expressing GP5 and M proteins elicited higher neutralizing antibody levels than rPRV-GP5 expressing GP5 alone. In contrast, the recombinant viruses using ORF5m as the target protein including rPRV-GP5m and rPRV-GP5m-M displayed a stronger capacity eliciting neutralizing antibodies and lymphocyte proliferative responses. The highest level of PRRSV-specific immune responses was observed in mice immunized with rPRV-GP5m-M, in which 2 out of 6 mice produce 1:32 neutralizing antibodies level at 10 weeks after primary immunization and induced lymphocyte proliferative responses were significantly higher than the other PRV recombinants. These results indicated that the recombinant rPRV-GP5m-M is a promising candidate bivalent vaccine against both PRV and PRRSV infection.5.2.1 Immunological protection induced by the new candidates vaccines against PRRSVTo evaluate sufficiently the immune efficacy and protection provided by two new candidates vaccines (suicidal DNA vaccine pSFV-ORF5rn/ORF6 and the recombinant rPRV-GP5m-M), specific humoral and cellular immunity and the capacity prevented the immunized animals from the virulent infection were investigated and compared with PRRSV killed vaccine in pig model. The recombinant rPRV-GP5m-M elicited comparative neutralizing antibodies and ELISA antibodies against PRV with the PRV parental virus. The piglets immunized with pSFV-ORF5m/ORF6 and rPRV-GP5m-M produced detectable PRRSV-specific neutralizing antibodies at 42d after primary immtmization. Subsequently, the immune responses were further enhanced gradually. But no neutralizing antibodies were detected in the group vaccinated with PRRSV killed vaccine before PRRSV challenge (63 days after primary immunization). After PRRSV challenge, piglets vaccinated with rPRV-GP5m-M and pSFV-ORF5rm/ORF6 developed rapidly increased neutralizing antibodies and reached 1: 128-1: 256 neutralizing antibodies at 17 and 21 dpc. In additional, vaccination with PRRSV killed vaccine produced very low specific lymphocyte proliferative responses before challenge, and it was not significantly increased by the subsequent infectious challenge. In contrast, rPRV-GP5m-M vaccination resulted in significantly proliferative responses at days 42 and 63 after primary immunization, and virus challenge resulted in a faster and higher improvement. Compared with the protection provided by killed vaccine, more efficient protection against a PRRSV challenge was obtained in piglets immunized with rPRV-GP5m-M and pSFV-ORF5m/ORF6, as showed by the balanced body-temperature fluctuation, shorter-term viremia, lower proportion of virus load in nasal and oropharyngeal scrapings and tissues, and milder lung lesions.更多还原