腺病毒/甲病毒复制子嵌合载体猪瘟疫苗的构建及评价
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摘要
猪瘟（classical swine fever，CSF）是由猪瘟病毒（classical swine fever virus，CSFV）引起的一种烈性传染病，对养猪业危害极大。目前，免疫接种仍然是防制猪瘟的重要措施。国内外常用的猪瘟疫苗基本上是兔化弱毒疫苗，尽管弱毒疫苗对猪瘟的防控起到了关键性作用，但这类疫苗存在明显不足之处。首先，现行猪瘟弱毒疫苗的生产工艺存在较多问题，用于生产疫苗的犊牛睾丸细胞和犊牛血清经常污染牛病毒性腹泻病毒（BVDV）或其抗体，严重影响猪瘟疫苗的效价和质量；其次，弱毒疫苗受猪瘟母源抗体及其它疫苗的干扰，接种时如果免疫程序不当，容易造成免疫失败；此外，弱毒疫苗缺乏适当的标记，对免疫猪与野毒感染猪在血清学上难以区分，从而干扰猪瘟流行病学调查和净化。因此，各国学者不断探索研制和开发新型猪瘟疫苗的新策略。
本研究首先利用人5型复制缺陷性腺病毒载体系统构建了表达猪瘟病毒E2基因的重组腺病毒rAdV-E2，并对该重组腺病毒进行了鉴定和免疫原性分析。结果表明，E2基因可在重组腺病毒中稳定遗传，并可在rAdV-E2感染的HEK293细胞中正常表达。家兔免疫攻毒试验结果显示，rAdV-E2以108TCID50剂量一次免疫家兔后2w可诱导猪瘟特异性抗体的产生，并且能够完全抵抗猪瘟病毒C株的攻击。猪只试验结果显示，rAdV-E2以2×108TCID50免疫2次，于加强免疫后2w诱导了较高水平的猪瘟特异性抗体。攻毒后仅个别猪出现短暂发热和轻微病理变化；而野生型腺病毒免疫猪在攻毒前一直没有检测到猪瘟特异性抗体，攻毒后所有猪均出现了典型的猪瘟临床症状和严重病理变化。
随后将甲病毒复制子载体DNA疫苗pSFV1CS-E2和重组腺病毒疫苗rAdV-E2以异源prime-boost策略进行了联合免疫，即用甲病毒复制子载体DNA疫苗pSFV1CS-E2对猪只进行初免，3w后用重组腺病毒疫苗rAdV-E2进行加强免疫。结果显示，所有异源prime-boost免疫猪均产生了高水平的猪瘟特异性的抗体和相当水平的CD4、CD8T细胞增殖反应，用猪瘟强毒攻击后，pSFV1CS-E2/rAdV-E2异源prime-boost免疫组所有猪（n=5）均没有出现任何猪瘟临床症状和病理变化，攻毒后在血液中也没有检测到猪瘟病毒RNA，免疫效果与C株相当，而rAdV-E2/rAdV-E2免疫组（n=5）有1头猪出现短期发热和轻微的病毒血症和病理变化，而所有阴性对照猪均出现了典型的猪瘟临床症状和病理变化。
受此启发，我们考虑能不能将甲病毒复制子载体和腺病毒载体嵌合到一起，充分发挥甲病毒复制子载体高效表达外源基因的优势和腺病毒载体高效递送的优势，因此本研究构建了一株表达猪瘟病毒E2蛋白的腺病毒/甲病毒复制子嵌合病毒。首先，将表达猪瘟病毒E2基因的甲病毒复制子载体克隆到腺病毒穿梭载体中，然后，通过细菌内同源重组构建重组腺粒，最后，将线性化的重组腺粒经脂质体转染HEK293细胞，从而包装成腺病毒/甲病毒复制子载体嵌合病毒，并对获得的重组嵌合病毒进行了系列的鉴定。PCR证实外源基因（甲病毒复制子基因NCR及猪瘟病毒E2基因）在重组嵌合病毒中稳定存在；间接免疫荧光试验和Western blot证实，猪瘟病毒E2蛋白能够在嵌合载体病毒rAdV-SFV-E2感染的HEK293细胞中正常地表达。随后，将此重组嵌合病毒rAdV-SFV-E2以107TCID50剂量接种猪只2次，间隔3w，同时与先前构建的表达猪瘟病毒E2基因的重组腺病毒rAdV-E2、表达猪瘟病毒E2基因的甲病毒复制子载体猪瘟疫苗pSFV1CS-E2和猪瘟兔化弱毒疫苗C株进行了免疫效力比较，同时以不表达外源基因的嵌合病毒rAdV-SFV-empty及甲病毒复制空载体pSFV1CS作为阴性对照。结果表明，rAdV-SFV-E2、C株和rAdV-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，并于加强免疫后5w达到峰值，攻毒后rAdV-SFV-E2和C株免疫组所有猪均未出现任何其它临床症状，也没有出现病毒血症，剖检后没有发现任何猪瘟病变及组织病理学上的变化；而rAdV-E2和pSFV1CS-E2免疫组个别猪出现了短暂的发热、轻微的病毒血症及病理学变化。rAdV-SFV-empty及pSFV1CS免疫组（n=5）猪只在攻毒前一直没有检出特异性抗体，攻毒后全部出现典型的猪瘟临床症状和严重的病毒血症，剖检时可见典型猪瘟病理变化。猪只免疫试验结果初步证实，构建的腺病毒/甲病毒复制子嵌合载体病毒的免疫效果与猪瘟兔化弱毒疫苗C株相当，这种全新的疫苗策略结合了腺病毒载体与甲病毒复制子载体的优点，为新型疫苗研制开拓了新的思路和途径。
为了进一步评价该嵌合载体病毒的免疫效果及作为候选猪瘟疫苗的可能性，我们从对靶动物和非靶动物的安全性、一次免疫、不同免疫剂量、抗腺病毒载体抗体干扰、猪瘟母源抗体干扰及与伪狂犬病疫苗联合免疫等方面，对该腺病毒/甲病毒复制子嵌合病毒rAdV-SFV-E2进行了综合评价。研究结果显示，该嵌合载体病毒对靶动物猪及非靶动物家兔和小鼠非常安全，免疫后没有任何副反应和组织病理学变化。rAdV-SFV-E2以6.25×105TCID50免疫后能够完全抵抗猪瘟病毒强毒的攻击；当猪体内存在猪瘟母源抗体，且抗体阻断率在60%以上时，嵌合载体猪瘟疫苗rAdV-SFV-E2正常免疫后，抗体水平逐渐上升，并于二免后4w达到峰值，随后趋于稳定，攻毒后所有免疫猪均获得了完全保护，而猪瘟母源抗体阻断率在60%左右的非免疫猪，3~4w抗体转阴（抗体阻断率30%以下），攻毒后所有猪均未得到保护，并于攻毒后10d左右全部死亡；rAdV-SFV-E2一次免疫后，于攻毒前一直没有检测到猪瘟特异性抗体，但是攻毒后所有猪均没有出现猪瘟临床症状，攻毒后6d抗体迅速上升到80%左右，对猪瘟病毒的攻击提供了完全的保护；rAdV-SFV-E2与伪狂犬病疫苗同时免疫后相互不影响各自的免疫应答，所有免疫猪均产生了高水平的猪瘟抗体和伪狂犬病抗体，并对猪瘟病毒的攻击提供了完全的保护；rAdV-SFV-E2免疫后不影响腺病毒载体疫苗的再次免疫。
总之，从目前的结果来看，嵌合载体疫苗rAdV-SFV-E2能诱导猪只产生高水平的免疫反应，对猪瘟病毒强毒的攻击得到完全的保护，免疫效果与猪瘟兔化弱毒疫苗C株相当。这种策略给新型猪瘟基因工程疫苗带来了希望，也给其它疫苗的研究开创了新的途径。
Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is one of themost devastating epizootic diseases of pigs worldwide. At present, vaccination is still an importantmeasure for prevention and control of CSF. Efficacious and safe attenuated vaccines have played a keyrole in CSF control, but this kind of vaccine has some disadvantages. First of all, the currently used CSFvaccine production process usually meet with some problems, for example, the production of vaccinesby the use of calf testicle cells and calf sera is often contaminated with bovine viral diarrhea virus(BVDV) or its antibody, which seriously influences the CSF vaccine potency and quality. Secondly,improper immune inoculation procedure may cause immune failure. In addition, antibodies against liveattenuated vaccines do not allow differentiation of infected from vaccinated animals (DIVA principle).Therefore, researchers make efforts to develop new-type CSF marker vaccine.
In this study, we first constructed a recombinant human adenovirus type5expressing the CSFV E2gene (rAdV-E2) and evaluated its efficacy in rabbits and pigs. The results showed E2protein wasexpressed in rAdV-E2-infected HEK293cells. The rabbits and the pigs immunized with the rAdV-E2developed high-level CSFV-specific neutralizing antibodies. The rAdV-E2-immunized rabbits werecompletely protected from fever induced by infection with C-strain, and the rAdV-E2-immunized pigswere protected from lethal challenge with highly virulent Shimen strain, however, a few pigs showedshort-term fever and occasional pathological changes following virulent challenge.
We further evaluated the efficacy of the heterologous prime-boost immunization approach (primewith pSFV1CS-E2and boost with rAdV-E2, at three-week intervals) in pigs. The results showed thatthe pigs (n=5) receiving pSFV1CS-E2/rAdV-E2heterologous prime-boost immunization developedsignificantly higher titers of CSFV-specific neutralizing antibodies and comparable CD4and CD8Tcell proliferation. When challenged with virulent CSFV Shimen strain, the pigs of the heterologousprime-boost group did not show clinical symptoms or viremia, which were observed in one of the5pigsimmunized with rAdV-E2alone and in all the5control pigs immunized with wild-type adenovirus. Theresults demonstrate that the heterologous DNA prime and recombinant adenovirus boost strategy caninduce full protective immunity.
Later, we used the safe, efficient adenoviral vector to deliver the alphavirus replicon vector andconstructed an adenovirus/alphavirus replicon chimeric virus expressing the E2protein of CSFV. First,the alphavirus replicon vector expressing the E2gene was cloned into the adenovirus shuttle vector. Arecombinant adenovirus plasmid was constructed through homologous recombination in bacteria, andthe linearzied plasmid was transfected into HEK293cells by liposome to package adenovirus/alphavirusreplicon vector chimeric virus rAdV-SFV-E2. The recombinant chimeric virus was identified by PCR,IFA and Western blot and evaluated in pigs. PCR results confirmed that the exogenous gene can existstably in the recombinant chimeric virus. IFA and Western blot confirmed that the exogenous gene wasexpressed in rAdV-SFV-E2infected cells. Pigs immunized with rAdV-SFV-E2(n=5) developed robust humoral and cell-mediated responses to CSFV and were completely protected from subsequent lethalCSFV infection clinically and virologically. The level of immunity and protection induced byrAdV-SFV-E2was comparable to that provided by the currently used live attenuated vaccine, C-strain.By contrast, both the conventional alphavirus replicon-vectored vaccine pSFV1CS-E2and conventionaladenovirus-vectored vaccine rAdV-E2provided incomplete protection.
In order to further verify whether rAdV-SFV-E2can be used as an ideal CSFV marker vaccine, wecomprehensively evaluated the safety and efficacy of the chimeric virus with respect to differentimmunization times and doses, pre-existing antibodies to the adenovirus vector, maternal antibodyinterference and combination immunization with PRV vaccine. The results showed that the minimalimmunization dose is6.25×105TCID50in pigs. A single immunization provided complete protectionagainst lethal CSFV challenge, though specific neutralizing antibodies were undetectable beforechallenge. The chimeric virus can overcome CSF maternal antibody interference and immunization withthe chimeric virus did not affect re-immunization of adenovirus-vectored vaccines. When chimeric viruswas co-administered with pseudorabies vaccine, all immunized pigs developed high-level specificantibodies against CSFV and PRV compared to either vaccine administered alone.
The chimeric vector-based vaccine represents the first gene-based vaccine that is able to confersterile immunity and complete protection against CSFV. The novel vaccination strategy may also bevaluable in vaccine development against other pathogens.
本研究首先利用人5型复制缺陷性腺病毒载体系统构建了表达猪瘟病毒E2基因的重组腺病毒rAdV-E2，并对该重组腺病毒进行了鉴定和免疫原性分析。结果表明，E2基因可在重组腺病毒中稳定遗传，并可在rAdV-E2感染的HEK293细胞中正常表达。家兔免疫攻毒试验结果显示，rAdV-E2以108TCID50剂量一次免疫家兔后2w可诱导猪瘟特异性抗体的产生，并且能够完全抵抗猪瘟病毒C株的攻击。猪只试验结果显示，rAdV-E2以2×108TCID50免疫2次，于加强免疫后2w诱导了较高水平的猪瘟特异性抗体。攻毒后仅个别猪出现短暂发热和轻微病理变化；而野生型腺病毒免疫猪在攻毒前一直没有检测到猪瘟特异性抗体，攻毒后所有猪均出现了典型的猪瘟临床症状和严重病理变化。
随后将甲病毒复制子载体DNA疫苗pSFV1CS-E2和重组腺病毒疫苗rAdV-E2以异源prime-boost策略进行了联合免疫，即用甲病毒复制子载体DNA疫苗pSFV1CS-E2对猪只进行初免，3w后用重组腺病毒疫苗rAdV-E2进行加强免疫。结果显示，所有异源prime-boost免疫猪均产生了高水平的猪瘟特异性的抗体和相当水平的CD4、CD8T细胞增殖反应，用猪瘟强毒攻击后，pSFV1CS-E2/rAdV-E2异源prime-boost免疫组所有猪（n=5）均没有出现任何猪瘟临床症状和病理变化，攻毒后在血液中也没有检测到猪瘟病毒RNA，免疫效果与C株相当，而rAdV-E2/rAdV-E2免疫组（n=5）有1头猪出现短期发热和轻微的病毒血症和病理变化，而所有阴性对照猪均出现了典型的猪瘟临床症状和病理变化。
受此启发，我们考虑能不能将甲病毒复制子载体和腺病毒载体嵌合到一起，充分发挥甲病毒复制子载体高效表达外源基因的优势和腺病毒载体高效递送的优势，因此本研究构建了一株表达猪瘟病毒E2蛋白的腺病毒/甲病毒复制子嵌合病毒。首先，将表达猪瘟病毒E2基因的甲病毒复制子载体克隆到腺病毒穿梭载体中，然后，通过细菌内同源重组构建重组腺粒，最后，将线性化的重组腺粒经脂质体转染HEK293细胞，从而包装成腺病毒/甲病毒复制子载体嵌合病毒，并对获得的重组嵌合病毒进行了系列的鉴定。PCR证实外源基因（甲病毒复制子基因NCR及猪瘟病毒E2基因）在重组嵌合病毒中稳定存在；间接免疫荧光试验和Western blot证实，猪瘟病毒E2蛋白能够在嵌合载体病毒rAdV-SFV-E2感染的HEK293细胞中正常地表达。随后，将此重组嵌合病毒rAdV-SFV-E2以107TCID50剂量接种猪只2次，间隔3w，同时与先前构建的表达猪瘟病毒E2基因的重组腺病毒rAdV-E2、表达猪瘟病毒E2基因的甲病毒复制子载体猪瘟疫苗pSFV1CS-E2和猪瘟兔化弱毒疫苗C株进行了免疫效力比较，同时以不表达外源基因的嵌合病毒rAdV-SFV-empty及甲病毒复制空载体pSFV1CS作为阴性对照。结果表明，rAdV-SFV-E2、C株和rAdV-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，并于加强免疫后5w达到峰值，攻毒后rAdV-SFV-E2和C株免疫组所有猪均未出现任何其它临床症状，也没有出现病毒血症，剖检后没有发现任何猪瘟病变及组织病理学上的变化；而rAdV-E2和pSFV1CS-E2免疫组个别猪出现了短暂的发热、轻微的病毒血症及病理学变化。rAdV-SFV-empty及pSFV1CS免疫组（n=5）猪只在攻毒前一直没有检出特异性抗体，攻毒后全部出现典型的猪瘟临床症状和严重的病毒血症，剖检时可见典型猪瘟病理变化。猪只免疫试验结果初步证实，构建的腺病毒/甲病毒复制子嵌合载体病毒的免疫效果与猪瘟兔化弱毒疫苗C株相当，这种全新的疫苗策略结合了腺病毒载体与甲病毒复制子载体的优点，为新型疫苗研制开拓了新的思路和途径。
为了进一步评价该嵌合载体病毒的免疫效果及作为候选猪瘟疫苗的可能性，我们从对靶动物和非靶动物的安全性、一次免疫、不同免疫剂量、抗腺病毒载体抗体干扰、猪瘟母源抗体干扰及与伪狂犬病疫苗联合免疫等方面，对该腺病毒/甲病毒复制子嵌合病毒rAdV-SFV-E2进行了综合评价。研究结果显示，该嵌合载体病毒对靶动物猪及非靶动物家兔和小鼠非常安全，免疫后没有任何副反应和组织病理学变化。rAdV-SFV-E2以6.25×105TCID50免疫后能够完全抵抗猪瘟病毒强毒的攻击；当猪体内存在猪瘟母源抗体，且抗体阻断率在60%以上时，嵌合载体猪瘟疫苗rAdV-SFV-E2正常免疫后，抗体水平逐渐上升，并于二免后4w达到峰值，随后趋于稳定，攻毒后所有免疫猪均获得了完全保护，而猪瘟母源抗体阻断率在60%左右的非免疫猪，3~4w抗体转阴（抗体阻断率30%以下），攻毒后所有猪均未得到保护，并于攻毒后10d左右全部死亡；rAdV-SFV-E2一次免疫后，于攻毒前一直没有检测到猪瘟特异性抗体，但是攻毒后所有猪均没有出现猪瘟临床症状，攻毒后6d抗体迅速上升到80%左右，对猪瘟病毒的攻击提供了完全的保护；rAdV-SFV-E2与伪狂犬病疫苗同时免疫后相互不影响各自的免疫应答，所有免疫猪均产生了高水平的猪瘟抗体和伪狂犬病抗体，并对猪瘟病毒的攻击提供了完全的保护；rAdV-SFV-E2免疫后不影响腺病毒载体疫苗的再次免疫。
总之，从目前的结果来看，嵌合载体疫苗rAdV-SFV-E2能诱导猪只产生高水平的免疫反应，对猪瘟病毒强毒的攻击得到完全的保护，免疫效果与猪瘟兔化弱毒疫苗C株相当。这种策略给新型猪瘟基因工程疫苗带来了希望，也给其它疫苗的研究开创了新的途径。
Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is one of themost devastating epizootic diseases of pigs worldwide. At present, vaccination is still an importantmeasure for prevention and control of CSF. Efficacious and safe attenuated vaccines have played a keyrole in CSF control, but this kind of vaccine has some disadvantages. First of all, the currently used CSFvaccine production process usually meet with some problems, for example, the production of vaccinesby the use of calf testicle cells and calf sera is often contaminated with bovine viral diarrhea virus(BVDV) or its antibody, which seriously influences the CSF vaccine potency and quality. Secondly,improper immune inoculation procedure may cause immune failure. In addition, antibodies against liveattenuated vaccines do not allow differentiation of infected from vaccinated animals (DIVA principle).Therefore, researchers make efforts to develop new-type CSF marker vaccine.
In this study, we first constructed a recombinant human adenovirus type5expressing the CSFV E2gene (rAdV-E2) and evaluated its efficacy in rabbits and pigs. The results showed E2protein wasexpressed in rAdV-E2-infected HEK293cells. The rabbits and the pigs immunized with the rAdV-E2developed high-level CSFV-specific neutralizing antibodies. The rAdV-E2-immunized rabbits werecompletely protected from fever induced by infection with C-strain, and the rAdV-E2-immunized pigswere protected from lethal challenge with highly virulent Shimen strain, however, a few pigs showedshort-term fever and occasional pathological changes following virulent challenge.
We further evaluated the efficacy of the heterologous prime-boost immunization approach (primewith pSFV1CS-E2and boost with rAdV-E2, at three-week intervals) in pigs. The results showed thatthe pigs (n=5) receiving pSFV1CS-E2/rAdV-E2heterologous prime-boost immunization developedsignificantly higher titers of CSFV-specific neutralizing antibodies and comparable CD4and CD8Tcell proliferation. When challenged with virulent CSFV Shimen strain, the pigs of the heterologousprime-boost group did not show clinical symptoms or viremia, which were observed in one of the5pigsimmunized with rAdV-E2alone and in all the5control pigs immunized with wild-type adenovirus. Theresults demonstrate that the heterologous DNA prime and recombinant adenovirus boost strategy caninduce full protective immunity.
Later, we used the safe, efficient adenoviral vector to deliver the alphavirus replicon vector andconstructed an adenovirus/alphavirus replicon chimeric virus expressing the E2protein of CSFV. First,the alphavirus replicon vector expressing the E2gene was cloned into the adenovirus shuttle vector. Arecombinant adenovirus plasmid was constructed through homologous recombination in bacteria, andthe linearzied plasmid was transfected into HEK293cells by liposome to package adenovirus/alphavirusreplicon vector chimeric virus rAdV-SFV-E2. The recombinant chimeric virus was identified by PCR,IFA and Western blot and evaluated in pigs. PCR results confirmed that the exogenous gene can existstably in the recombinant chimeric virus. IFA and Western blot confirmed that the exogenous gene wasexpressed in rAdV-SFV-E2infected cells. Pigs immunized with rAdV-SFV-E2(n=5) developed robust humoral and cell-mediated responses to CSFV and were completely protected from subsequent lethalCSFV infection clinically and virologically. The level of immunity and protection induced byrAdV-SFV-E2was comparable to that provided by the currently used live attenuated vaccine, C-strain.By contrast, both the conventional alphavirus replicon-vectored vaccine pSFV1CS-E2and conventionaladenovirus-vectored vaccine rAdV-E2provided incomplete protection.
In order to further verify whether rAdV-SFV-E2can be used as an ideal CSFV marker vaccine, wecomprehensively evaluated the safety and efficacy of the chimeric virus with respect to differentimmunization times and doses, pre-existing antibodies to the adenovirus vector, maternal antibodyinterference and combination immunization with PRV vaccine. The results showed that the minimalimmunization dose is6.25×105TCID50in pigs. A single immunization provided complete protectionagainst lethal CSFV challenge, though specific neutralizing antibodies were undetectable beforechallenge. The chimeric virus can overcome CSF maternal antibody interference and immunization withthe chimeric virus did not affect re-immunization of adenovirus-vectored vaccines. When chimeric viruswas co-administered with pseudorabies vaccine, all immunized pigs developed high-level specificantibodies against CSFV and PRV compared to either vaccine administered alone.
The chimeric vector-based vaccine represents the first gene-based vaccine that is able to confersterile immunity and complete protection against CSFV. The novel vaccination strategy may also bevaluable in vaccine development against other pathogens.
引文
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