基于甲病毒复制子载体的猪瘟DNA疫苗的免疫效力评价

此前已构建了基于Semliki Forest病毒（Semliki Forest virus,SFV）复制子载体的表达猪瘟病毒（classical swine fever virus,CSFV）E2基因的新型猪瘟DNA疫苗pFV1CS-E2,通过动物试验证实,该疫苗以600μg/头的剂量免疫3次,免疫猪能抵抗致死剂量猪瘟强毒的攻击。为进一步评价该疫苗在较低的免疫剂量和较少的免疫次数情况下的免疫效力,将DNA疫苗pSFV1CS-E2和空载体pSFV1CS按100μg/头的剂量,接种猪只2次,然后用致死剂量的猪瘟强毒石门株进行攻击。结果表明,pSFV1CS-E2免疫组（n=5）所有免疫猪在加强免疫后均产生了猪瘟特异性中和抗体,攻毒后所有猪只抗体迅速升高,除了短期体温升高外,未出现任何其它临床症状,部分猪出现短期轻微病毒血症,个别猪的部分脏器出现轻微病变;而空载体免疫组（n=3）猪只在攻毒前一直没有检出特异性抗体,攻毒后全都出现典型的猪瘟临床症状和严重的病毒血症,有2头猪分别于攻毒后第10和11d死亡,剖检时可见典型猪瘟病理变化。结果表明,基于甲病毒复制子载体的猪瘟DNA疫苗有望成为具有开发价值的猪瘟标记疫苗。     

猪瘟甲病毒复制子载体DNA疫苗与重组腺病毒活载体疫苗联合免疫效果评价

本实验室先前分别将构建的猪瘟病毒E2基因重组腺病毒疫苗(rAdV-E2)和猪瘟甲病毒复制子载体DNA疫苗(pSFV1CS-E2)在猪体上进行了免疫效力评价,结果显示,rAdV-E2免疫组所有猪虽然在加强免疫后产生了比较高的猪瘟特异性中和抗体,但攻毒后个别猪表现短期体温升高和轻微病变;而pSFV1CS-E2免疫组猪只虽然在攻毒后得到了保护,但产生的抗体水平较低。为了增强猪瘟甲病毒复制子载体疫苗和猪瘟重组腺病毒活载体疫苗的免疫效果,本研究应用了复制子载体DNA疫苗初免和重组腺病毒疫苗加强免疫的初免-加强(Prime-boost)免疫策略,并在猪体上进行了评价。结果显示,所有免疫猪均产生了高水平的猪瘟特异性的中和抗体,用猪瘟强毒攻击后,pSFV1CS-E2初免组所有猪(n=5)均没有出现任何猪瘟的临床症状和病理变化,攻毒后在猪血液中也没有检测到猪瘟病毒RNA,而重组腺病毒初免组(n=5)有一头猪出现短期发热和病毒血症及轻微病理变化。这表明初免-加强免疫策略能显著提高重组疫苗的免疫效力。     

基于Semliki Forest病毒RNA复制子的猪瘟RNA疫苗初步研究

将猪瘟病毒E2基因克隆于我们此前构建的衍生于Semliki Forest病毒(semliki forest virus,SFV)RNA复制子的新型真核表达载体pSFV1CS中,获得重组质粒pSFV1CS-E2。用纯化的pSFV1CS-E2分别转染BHK-21细胞和293T细胞,经间接免疫荧光试验检测显示,CSFV E2基因在转染细胞中得到表达。小鼠接种试验结果表明,10μg或100μg pSFV1CS-E2可诱导小鼠产生猪瘟特异性抗体。     

共表达PRRSV GP5蛋白和CSFV E2蛋白的“自杀性”DNA疫苗在小鼠体内诱导的免疫应答

为了获得新型双价"自杀性"DNA疫苗,将猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratorysyndrome virus,PRRSV)GP5基因克隆于此前构建的表达猪瘟病毒(Classical swine fever virus,CSFV)E2基因的甲病毒复制子载体疫苗pSFV1CS-E2中.为了增强免疫效果,在密码子优化的GP5基因中插入了泛DR表位(PADRE),在CSFV E2基因后融合伪狂犬病病毒(PrV)UL49基因,获得了6种重组质粒.间接免疫荧光试验显示,PRRSV GP5和CSFV E2基因在瞬时转染的293T细胞中得到同时表达,将6种重组质粒和空载体pSFV1CS分别免疫BALB/c小鼠,用间接ELISA方法检测血清抗体水平,通过基于CSFE/WST-8的淋巴细胞增殖试验和细胞因子ELISA评价疫苗诱导的细胞免疫.结果显示,除pSFV1CS组外,从各疫苗组小鼠血清中均可检测到低水平的针对GP5和E2蛋白的抗体;各疫苗组小鼠脾细胞经CSFV和PRRSV刺激后均能诱导特异性的淋巴细胞增殖:部分疫苗组小鼠脾细胞经CSFV和PRRSV刺激后可分泌较高水平的IFN-γ和IL-4;引入UL49的疫苗组细胞免疫应答显著高于其它疫苗组.结果表明,这些共表达GP5和E2蛋白的自杀性DNA疫苗可以诱导体液免疫和细胞免疫,PrV UL49可以增强其细胞免疫应答.     

乙肝病毒RNA复制子疫苗与DNA疫苗对小鼠免疫效率的比较

为建立并获取更有效的乙肝疫苗,本实验通过将所构建的HBV RNA复制子疫苗和DNA疫苗分别免疫小鼠,检测细胞免疫与体液免疫的效果.结果表明,以pSFV为基础构建的疫苗载体免疫小鼠后采集的血清中抗体效价不随免疫剂量的增加而提高,在较低剂量免疫的时候,RNA复制子疫苗所产生的抗体效价优于DNA疫苗.并且RNA复制子疫苗在以较低剂量免疫后脾细胞CTL活性高于DNA疫苗.本研究证明HBV RNA疫苗比DNA疫苗表达效果更好,安全性更高,更具有应用前景.     

猪瘟病毒E2基因真核表达质粒的构建及基因疫苗的研究

构建了猪瘟病毒(classical swine fever virus, CSFV)主要保护性抗原E2基因4种不同的真核表达质粒.小鼠免疫试验表明,E2基因上不同的功能区对基因疫苗的免疫应答有很大影响,有信号肽序列的E2基因可诱导产生特异性免疫反应,且无跨膜区序列的E2基因所诱导的免疫应答反应比有跨膜区序列的强,而无信号肽序列的E2基因则不能诱导产生CSFV特异性的免疫反应.攻毒保护试验表明,免疫家兔最少可抵抗10个最小感染剂量(MID)的猪瘟兔化弱毒苗(Hog cholera lap-inized virus, HCLV)的攻击;免疫猪可抵抗致死剂量的CSFV石门株强毒的攻击.     

表达猪瘟病毒E2蛋白的重组腺病毒的构建及其在兔体内的免疫原性分析

为了构建猪瘟重组腺病毒载体疫苗,通过细菌内同源重组法构建了含有猪瘟病毒E2基因的重组腺病毒rAdV-E2.测定其一步生长曲线,同时用间接免疫荧光试验和Western blotting检测外源基因表达,然后用rAdV-E2免疫家兔,免疫后6周用猪瘟兔化弱毒疫苗株(c株)进行攻击,攻毒后3 d取其脾脏,用实时荧光定量RT-PCR检测C株病毒RNA.结果表明,该重组腺病毒传至第10代时,毒价可达1.0×1010TCID<,50/mL;外源基因可在其中得到稳定表达;rAdV-E2接种兔免疫后2周产生猪瘟特异性抗体,免疫后5 W抗体达到峰值,攻毒后rAdV-E2接种兔和C株接种兔均未出现定型热反应,从其脾脏也未检测到C株病毒RNA,而野生型腺病毒接种兔均出现了定型热反应,并且从其脾脏检测大量C株病毒RNA,其含量达到了103拷贝/μL以上.由此表明,rAdV-E2可望开发为猪瘟候选疫苗.     

一种基于塞姆利基森林病毒复制子的新型复制子载体

RNA复制子是能自主复制的病毒RNA。基于RNA复制子的表达载体和基因疫苗比常规真核表达载体和DNA疫苗具有更大的优越性。以塞姆利基森林病毒RNA复制子衍生的真核表达载体pSFV1为骨架 ,插入CMV立即早期启动子和SV40晚期Poly(A)信号 ,构建了一种完全基于DNA的复制型表达载体pSFV1CS ,将增强型绿色荧光蛋白基因EGFP插入其中 ,构建了重组质粒pSFV1CS EGFP ,通过转染 2 93T细胞 ,证实外源基因能在其中高效表达。该载体可用于表达真核蛋白和构建复制子载体疫苗。     

表达猪圆环病毒Ⅱ型ORF2基因的重组猪伪狂犬病病毒的免疫原性

【目的】为研制预防猪圆环病毒II型(PCV2)感染的重组伪狂犬病病毒(PRV)活载体疫苗。【方法】将PCV2 ORF2基因插入到PRV通用载体pG中,利用脂质体LipofectamineTM2000试剂盒将重组转移质粒pGO与猪PRV弱毒HB98株DNA共转染猪睾丸(ST)细胞,通过3轮蚀斑纯化重组病毒。将重组病毒、商品化PCV2灭活苗及DMEM培养液分别免疫6周龄雌性昆明小鼠,4周后加强免疫1次,首免后第8周用PCV2强毒NY株对小鼠进行攻毒。【结果】成功获得表达ORF2基因的重组病毒PGO,首免重组病毒后小鼠体内抗PCV2的ELISA抗体水平很低,二免后小鼠PCV2特异的ELISA抗体水平明显升高,并且重组病毒组能够激发PCV2特异的淋巴细胞增殖效应。攻毒试验表明重组病毒组和PCV2灭活疫苗组均能有效抵抗PCV2强毒攻击。【结论】表明表达ORF2基因的重组病毒PGO具有良好免疫原性。     

马传染性贫血病毒弱毒疫苗株与亲本强毒株的体内病毒载量与诱导保护性免疫关系的研究

慢病毒免疫应答的载量阈值学说认为病毒载量决定了机体对病毒反应的类型。为了探讨马传染性贫血病毒(EIAV)血浆病毒载量与马体免疫保护的相关性,本研究利用Real-time RT-PCR方法对EIAV弱毒疫苗株(EIA-VDLV125)免疫马和EIAV强毒株(EIAVLN40)非致死剂量接种马血浆中病毒载量进行了动态比较。结果显示两组马在监测过程中皆可检测到相似水平的病毒载量(103～105copies/mL),且两者之间差异不显著(P0.05)。以上毒株接种23周后,对马匹进行了强毒株(EIAVLN40)的致死剂量攻毒,根据攻毒后典型马传贫急性发病与否确定接种保护率。结果显示,疫苗组的保护率为67%而非致死剂量强毒组的保护率为0。以上结果提示,病毒血浆载量不是决定EIAV弱毒疫苗诱导免疫保护能力的主要或单一因素。     

Generation and Efficacy Evaluation of Recombinant Classical Swine Fever Virus E2 Glycoprotein Expressed in Stable Transgenic Mammalian Cell Line

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which is a highly contagious swine disease that causes significant economic loses to the pig industry worldwide. The envelope E2 glycoprotein of CSFV is the most important viral antigen in inducing protective immune response against CSF. In this study, we generated a mammalian cell clone (BCSFV-E2) that could stably produce a secreted form of CSFV E2 protein (mE2). The mE2 protein was shown to be N-linked glycosylated and formed a homodimer. The vaccine efficacy of mE2 was evaluated by immunizing pigs. Twenty-five 6-week-old Landrace piglets were randomly divided into five groups. Four groups were intramuscularly immunized with mE2 emulsified in different adjuvants twice at four-week intervals. One group was used as the control group. All mE2-vaccinated pigs developed CSFV-neutralizing antibodies two weeks after the first vaccination with neutralizing antibody titers ranging from 1∶40 to 1∶320. Two weeks after the booster vaccination, the neutralizing antibody titers increased greatly and ranged from 1∶10,240 to 1∶81,920. At 28 weeks after the booster vaccine was administered, the neutralizing antibody titers ranged from 1∶80 to 1∶10240. At 32 weeks after the first vaccination, pigs in all the groups were challenged with a virulent CSFV strain at a dose of 1×10⁵ TCID₅₀. At two weeks after the challenge, all the mE2-immunized pigs survived and exhibited no obvious symptoms of CSF. The neutralizing antibody titer at this time was 20,480. Unvaccinated pigs in the control group exhibited symptoms of CSF 3–4 days after challenge and were euthanized from 7–9 days after challenge when the pigs became moribund. These results indicate that the mE2 is a good candidate for the development of a safe and effective CSFV subunit vaccine.     

A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs

Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas PCV1 is nonpathogenic. We previously demonstrated that a chimeric PCV1-2 virus (with the immunogenic capsid gene of PCV2 cloned into the backbone of PCV1) induces an antibody response to the PCV2 capsid protein and is attenuated in pigs. Here, we report that the attenuated chimeric PCV1-2 induces protective immunity to wild-type PCV2 challenge in pigs. A total of 48 specific-pathogen-free piglets were randomly and equally assigned to four groups of 12 pigs each. Pigs in group 1 were vaccinated by intramuscular injection with 200 microg of the chimeric PCV1-2 infectious DNA clone. Pigs in group 2 were vaccinated by intralymphoid injection with 200 microg of a chimeric PCV1-2 infectious DNA clone. Pigs in group 3 were vaccinated by intramuscular injection with 10(3.5) 50% tissue culture infective doses (TCID(50)) of the chimeric PCV1-2 live virus. Pigs in group 4 were not vaccinated and served as controls. By 42 days postvaccination (DPV), the majority of pigs had seroconverted to PCV2 capsid antibody. At 42 DPV, all pigs were challenged intranasally and intramuscularly with 2 x 10(4.5) TCID(50) of a wild-type pathogenic PCV2 virus. By 21 days postchallenge (DPC), 9 out of the 12 group 4 pigs were viremic for PCV2. Vaccinated animals in groups 1 to 3 had no detectable PCV2 viremia after challenge. At 21 DPC the lymph nodes in the nonvaccinated pigs were larger (P < 0.05) than those of vaccinated pigs. The PCV2 genomic copy loads in lymph nodes were reduced (P < 0.0001) in vaccinated pigs. Moderate amounts of PCV2 antigen were detected in most lymphoid tissues of nonvaccinated pigs but in only 1 of 36 vaccinated pigs. Mild-to-severe lymphoid depletion and histiocytic replacement were detected in lymphoid tissues in the majority of nonvaccinated group 4 pigs but in only a few vaccinated group 1 to 3 pigs. The data from this study indicated that when given intramuscularly in pigs, the attenuated chimeric PCV1-2 live virus, as well as the chimeric PCV1-2 infectious DNA clone, induces protective immunity against PCV2 infection and could potentially serve as an effective vaccine.     

A heterologous DNA prime-Venezuelan equine encephalitis virus replicon particle boost dengue vaccine regimen affords complete protection from virus challenge in cynomolgus macaques

A candidate vaccine (D1ME-VRP) expressing dengue virus type 1 premembrane and envelope proteins in a Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) system was constructed and tested in conjunction with a plasmid DNA vaccine (D1ME-DNA) expressing identical dengue virus sequences. Cynomolgus macaques were vaccinated with three doses of DNA (DDD), three doses of VRP (VVV group), or a heterologous DNA prime-VRP boost regimen (DDV) using two doses of DNA vaccine and a third dose of VRP vaccine. Four weeks after the final immunization, the DDV group produced the highest dengue virus type 1-specific immunoglobulin G antibody responses and virus-neutralizing antibody titers. Moderate T-cell responses were demonstrated only in DDD- and DDV-vaccinated animals. When vaccinated animals were challenged with live virus, all vaccination regimens showed significant protection from viremia. DDV-immunized animals were completely protected from viremia (mean time of viremia = 0 days), whereas DDD- and VVV-vaccinated animals had mean times of viremia of 0.66 and 0.75 day, respectively, compared to 6.33 days for the control group of animals.     

乙型脑炎减毒活疫苗(SA_(14)-14-2)在豚鼠体内保护作用的免疫机制的研究

本实验将乙脑减毒活疫苗ＳＡ_（１４）－１４－２株以不同疫苗病毒量（３．８７ＰＦＵ／ｍｌ和５．８７ＰＦＵ／ｍｌ）分别一次免疫豚鼠,观察其对强毒攻击后抑制毒血症和抗体形成的能力。结果显示疫苗（５．８７ＰＦＵ／ｍｌ）免疫组豚鼠攻击前虽然中和抗体阴性或很低,但经攻击感染后不同时间内均未出现病毒血症,对照组豚鼠则于第２,３,４天全部出现病毒血症。表明一次活疫苗免疫后能有效地抑制病毒血症的产生。免疫后３０天虽然免疫组的豚鼠中和抗体很低,但攻击感染后抗体迅速增长。第四天的抗体滴度为１：８～３２,第５天达１：１２８～２５６,第１４天抗体高达１：５１２～１０２４;而对照组抗体则上升很慢,第７天才出现低水平抗体（１：４）。血凝抑制抗体增长的动态与中和抗体近似。表明活疫苗免疫后虽然中和抗体水平不高,但一经感染可迅速产生高滴度抗体达到保护作用。     

Immunization of pigs with the attenuated S- strain of Japanese encephalitis virus.

The attenuated S- strain of Japanese encephalitis virus was produced from a wild strain of this virus by serial cultivation in primary bovine kidney cell cultures at 30 degrees C. Pigs were inoculated with it and examined for ability to produce antibody and protect themselves from infection with a wild strain used for challenge. In pigs inoculated with a single dose of 10(6.5) approximately 10(7.5) TCID50 of the S- strain, the neutralizing antibody titer or hemagglutination-inhibiting antibody (HI) titer increased to 10 approximately 320. An antibody titer exceeding 10 was maintained for 2 approximately 9 weeks. In pigs inoculated twice with 10(6.5) approximately 10(7.0) TCID50 of the S- strain, HI titer increased to 80 approximately 640. In many of these pigs, HI titers of 80 approximately 160 persisted for more than 6 weeks. Pigs inoculated once or twice with 10(7.0) approximately 10(7.5) TCID50 of the S- strain were challenged by inoculation with 10(4.5) approximately 10(5.5) TCID50 of a wild strain and examined for the occurrence of viremia. As a result, an ability to protect from infection was demonstrated in pigs which showed an antibody titer surpassing 10 at the time of challenge. Pregnant sows inoculated with 10(7.0) TCID50 of the S- strain were challenged by inoculation with 10(7.0) TCID50 of a wild strain. Neither death nor infection occurred to any fetus harbored by them. From these results, it is concluded that the S- strain can be used as live virus vaccine for porcine practice.     

猪霍乱沙门菌载体介导猪瘟病毒DNA免疫研究

构建了猪瘟病毒(CSFV)主要保护性抗原E2基因的真核表达质粒pVAXE2。将其电转化猪霍乱沙门氏菌C500疫苗株,得到了携带pVAXE2质粒的猪霍乱沙门氏菌工程菌株S.C500/pVAXE2,对该菌株的特征、培养特性和生化特性进行了鉴定。分别用1×10⁸CFU、2×10⁹CFU S.C500/pVAXE2经口服或肌肉注射免疫小鼠和家兔,间接ELISA检测免疫动物的特异性抗体,在第三次免疫后2周用20ID50猪瘟兔化弱毒和致死量猪霍乱沙门氏菌强毒株对免疫兔进行攻击。结果表明,S.C500/pVAXE2保持了猪霍乱沙门氏菌原有形态特征、培养特性和生化特性,免疫鼠和兔都产生了抗CSFV和猪霍乱沙门菌的ELISA抗体,免疫家兔能抵抗猪瘟兔化弱毒株和猪霍乱沙门氏菌强毒株的攻击。显示了以S.C500为DNA运输载体构建二联或多联猪用疫苗的可行性。