甲型H1N1流感病毒特异性抗原捕获ELISA方法的建立及通用型猪流感疫苗基础研究
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摘要
流感是人类面临的主要公共健康问题之一。流感病毒在进化过程中发生导致抗原性改变的突变(抗原漂移)或不同流感病毒之间发生基因重配而产生一种新的流感病毒(抗原转变)是其逃避宿主免疫系统、得以在感染群体之间传播的主要策略。2009年开始流行的猪源新型H1N1流感病毒(以下简称甲流病毒)导致了人类历史的第四次流感大流行,该病毒比普通季节流感病毒具备更强的传播能力,在流行过程中病毒又由人回传给了猪。虽然该病毒已不再呈大流行态势,但其与其他流感病毒发生基因重配可能会产生毒力更强的病毒,因此有必要建立准确、特异的病原学检测方法以及可以评价不同宿主群体感染状态的血清学诊断方法,以监测病毒的流行和传播规律,掌握其在不同宿主群体中的感染状况,继而帮助为下一次流感爆发做出预警。
本研究从病原学和血清学两个方面分别建立了病毒及血清中和抗体的检测方法。首先利用构建的杆状病毒表面展示系统筛选到一株甲流病毒特异性的单抗,对单抗的表位进行鉴定后发现60KLRG63为表位的最小活性单元,同时该表位不能被全病毒感染的阳性血清识别,提示表位不是HA分子的优势表位,其所受的免疫压力要小于HA分子上的优势表位,故在病毒流行过程中不易发生变异,从而可保证单抗对病毒的有效识别；同时对抗原表位的保守性分析发现该表位是甲流病毒特有的,表明可以利用单抗特异地检测甲流病毒。结合兔抗HA蛋白多抗建立了抗原捕获ELISA方法,并对各个抗体的工作浓度进行了优化；敏感性试验表明所建立的方法比常规血凝试验至少敏感8倍；特异性试验表明用建立的方法检测不到人的季节性H1N1流感病毒及H1N1、H3N2猪流感病毒,对实验室感染样品的检测发现该方法与鸡胚病毒分离法具有较好的符合率,但不如病毒分离敏感。以上结果表明利用甲流病毒特异性的单抗建立的抗原捕获ELISA方法可以作为甲流病毒特异性的病原学检测的工具。
将人工合成的甲流病毒A/Califorina/04/2009株HA基因连接至真核表达载体pcDNA3.1,该重组质粒与表达逆转录病毒相关元件的骨架质粒pHIT111及pHIT60共转染人胚胎肾细胞293T,构建了以鼠白血病病毒为核心、包装有HA蛋白的伪型病毒。通过对伪型病毒感染细胞中报告基因表达产物的检测,证明伪型病毒可成功感染MDCK及293T细胞；同时其感染过程可被重组甲流病毒免疫的小鼠的阳性血清所阻断,表明该伪型病毒可模拟野生型病毒完成对宿主细胞的感染过程,提示该伪型病毒系统可作为一个特异性检测甲流病毒中和抗体的应用平台,从而为准确的评价机体感染状态或抗病毒药物筛选、疫苗评价等提供辅助工具。同时,本研究在缺少野生型病毒的条件下,利用公共数据库所登载的目的序列构建了与天然病毒具有相似感染特性的伪型病毒。因此理论上,其他相似的病毒表面膜蛋白也可根据本研究报道的方法构建伪病毒系统,而不依赖于某种特定病原的分离、鉴定。该方法特别对于高度危险性病原研究具有一定的意义。
猪在流感病毒生态中扮演着重要的角色。由于猪的呼吸道上皮细胞同时具有人流感病毒和禽流感病毒的受体,使其极有可能成为新流感病毒的产生源头。控制猪流感(Swine influenza, SI)的发生和发展有助于减少流感病毒对人类的健康威胁。疫苗免疫是预防SI的有效手段。当前商品化的SI疫苗为包括Hl和/或H3亚型病毒的灭活疫苗。与人流感疫苗不同,SI疫苗并不会按照当年流行的优势毒株更换疫苗种子毒,由于流行毒株的抗原性变异,经常导致现有商品化疫苗无法对其提供有效免疫保护的情况。鉴于此,本研究尝试利用保守的SIV抗原组分构建具有广谱交叉保护效力的DNA疫苗。第一组DNA疫苗将H3亚型SIV共有的M2e及HA蛋白序列的编码基因连接至表达载体(MHa);第二组在此基础上添加一个保守的T细胞表位(MNHa),以增强疫苗诱导T细胞免疫应答的能力。通过基因枪免疫小鼠后发现,两组DNA疫苗能有效刺激机体产生体液免疫应答,同时MNHa比MHa诱导了更高水平的T细胞免疫应答。攻毒保护实验表明两组疫苗都能够对同源H3亚型处在不同进化谱系的病毒攻击提供完全免疫保护,提示增强的T细胞免疫应答对于同亚型病毒的交叉保护来讲可能不是必需的；而与MHa相比,MNHa提供了更显著的对于异源H1亚型病毒攻击的保护效力,用H1亚型病毒攻击小鼠后,MNHa免疫组小鼠肺脏内病毒滴度及组织病理学损伤显著低于MHa免疫组,表明T细胞免疫应答在提供异源毒株的交叉保护方面起到重要作用。本研究在通用型SI疫苗研究方面进行了有益探索,并为未来SI疫苗的研制提供了可借鉴的思路。
Influenza is one of the major threats to global public health. Influenza viruses escape from host immune system and transmit among host species through the accumulation of changes on antigenic sites (antigenic drift) and genetic reassortment of different strains (antigenic shift). The2009swine-origin influenza A H1N1virus (hereafter referred to as pH1N1) resulted in the4th influenza global pandemic in human history. pH1N1transmit more efficiently in humans than that of seasonal influenza viruses, and transmission of pH1N1from human to pigs has also been reported. Although the pH1N1virus is now considered to be post-pandemic, there are still possibilities that the virus recombines with other influenza viruses in pigs then yielding a novel potential epidemic or pandemic strain. It is therefore necessary to establish accurate and specific virus detection and serological diagnostic methods to monitor the spread of virus and the infection state of different hosts, which may provide useful information in predicting the next influenza pandemic.
In this study, we established virus-and neutralization antibody-detection methods from the perspective of etiology and serology, respectively. A pH IN1-specific monoclonal antibody (mAb) was first developed based on a baculoviurs surface disply system, the epitope of the mAb was then identified. Results indicated that60KLRG63motif on hemagglutinin (HA) protein is the minimal requirement for the reactivity of the epitope recognized by mAb. Whole virus-positive mouse serum did not reacted with this epitope, suggesting that the epitope is not the immunodominant epitope of HA protein, thus the immune pressure posed by the hosts against the epitope is much less than that against immunodominant epitope, and thereby the identified epitope do not tend to shift. Further, conservation analysis of epitope showed that this epitope was pH1N1-specific, suggesting that the mAb is suitable material for virus-detection. An antigen-capture ELISA (AC-ELISA) method was developed in combinnation with rabbit HA-specific polyclonal antibody. Working dilution of each antibody were optimiezed. Sensitivity assay showed that AC-ELISA was at least8times more sensitive than hemagglutination test. Besides, specificity assay demonstrated that human seasonal H1NI, swine influenza virus (SIV) H1N1and H3N2can not be detected by AC-ELISA. The AC-ELISA was then employed to test the experimental infection samples, it was found that the results of AC-ELISA showed high agreement rate with virus isolation (VI), but was less sensitive than VI. It is conclued that the pH IN1-specific AC-ELISA represents a valuable tool for virus-detection.
A recombinant plasmid expressing HA gene of pH1N1A/Califorina/04/2009was co-transfected into human embryonic kidney293T cells with retroviral vector pHIT111and pHIT60to construct Murine leukemia virus (MuLV) pseudotyped virus. The pseudotyped virus infected Madin-Darby canine kidney (MDCK) and293T cells as indicated by the detection of the P-galactosidase as reporter gene. Furthermore, the infections were neutralized by the recombinant pHlN1virus-positive serum, suggesting that the HA-pseudotyped particles can mimic the surface of influenza virions. The pseudotyped virus reported here represents a powerful surrogate for pH1N1-specific neutralization antibody detection, which may be useful in assessing hosts'infection state, screening the antiviral regents, as well as developing vaccines. In addition, we constructed pseudotyped virus with similar infection property with wild type viruses by using the synthetized HA gene which is available in public database, thus, theoretically, other pseudotyped virus can also be constructed based on the method reported here, without the need of viral isolation and identification, especially for highly pathogenic and contagious ones.
Pigs play important roles in the ecology of influenza viruses, and could be the source of new strains since pigs express sialic acid receptors for both mammalian and avian strains of influenza viruses on their tracheal epithelial cells. The control of swine influeza (SI) is helpful to reduce the significant health risk for the human population by reducing virus shed and thereby transmission. Vaccination is considered to be the most effective method to control SI. Currently, commercially available swine influenza vaccines are adjuvanted, whole-virus killed vaccines containing of both HI and H3subtypes. Unlike human influenza vaccine, strains used for SI vaccine preparation are not regularly replaced, making the vaccines do not provide consistent protection from infection, because the viruses continuously evolve by antigenic drift and shift. Cross-protective SI vaccine designed based on the conerved viral proteins would be of great help to solve this problem. To this end, we constructed two forms of DNA vaccines, one was made by fusing M2e to consensus H3HA (MHa), which represents the majority of the HA sequences of H3N2viruses, another was made by fusing M2e and a conserved CTL epitope (NP147-155) to consensus H3HA (MNHa). BALB/c mice were immunized twice with the two DNA vaccines via gene gun. It was shown that the two vaccines elicited substantial antibody responses, and MNHa induced more significant T cell-mediated immune response than MHa did. Then two H3N2strains representative of different evolutional clusters were used to challenge the vaccine-immunized mice. Results indicated that both of the two DNA vaccines prevented homosubtypic virus infections completely, suggesting the ehanced T cell-mediated immune response may be non-essential for homologous protection. The vaccines'heterologous protective efficacies were further tested by challenging with a H1N1SI strain and a reassortant pH1N1strain. It was found that MNHa, but not MHa, induces immune responses partially protect against heterologous influenza infection as indicted by the reduced lung viral titer and less lung histopathological damage, suggesting that CTL responses play important roles for heterologous protection. Reults of the current study indicate that strengthening CTL responses are promising ways for universal influenza vaccines development.
本研究从病原学和血清学两个方面分别建立了病毒及血清中和抗体的检测方法。首先利用构建的杆状病毒表面展示系统筛选到一株甲流病毒特异性的单抗,对单抗的表位进行鉴定后发现60KLRG63为表位的最小活性单元,同时该表位不能被全病毒感染的阳性血清识别,提示表位不是HA分子的优势表位,其所受的免疫压力要小于HA分子上的优势表位,故在病毒流行过程中不易发生变异,从而可保证单抗对病毒的有效识别；同时对抗原表位的保守性分析发现该表位是甲流病毒特有的,表明可以利用单抗特异地检测甲流病毒。结合兔抗HA蛋白多抗建立了抗原捕获ELISA方法,并对各个抗体的工作浓度进行了优化；敏感性试验表明所建立的方法比常规血凝试验至少敏感8倍；特异性试验表明用建立的方法检测不到人的季节性H1N1流感病毒及H1N1、H3N2猪流感病毒,对实验室感染样品的检测发现该方法与鸡胚病毒分离法具有较好的符合率,但不如病毒分离敏感。以上结果表明利用甲流病毒特异性的单抗建立的抗原捕获ELISA方法可以作为甲流病毒特异性的病原学检测的工具。
将人工合成的甲流病毒A/Califorina/04/2009株HA基因连接至真核表达载体pcDNA3.1,该重组质粒与表达逆转录病毒相关元件的骨架质粒pHIT111及pHIT60共转染人胚胎肾细胞293T,构建了以鼠白血病病毒为核心、包装有HA蛋白的伪型病毒。通过对伪型病毒感染细胞中报告基因表达产物的检测,证明伪型病毒可成功感染MDCK及293T细胞；同时其感染过程可被重组甲流病毒免疫的小鼠的阳性血清所阻断,表明该伪型病毒可模拟野生型病毒完成对宿主细胞的感染过程,提示该伪型病毒系统可作为一个特异性检测甲流病毒中和抗体的应用平台,从而为准确的评价机体感染状态或抗病毒药物筛选、疫苗评价等提供辅助工具。同时,本研究在缺少野生型病毒的条件下,利用公共数据库所登载的目的序列构建了与天然病毒具有相似感染特性的伪型病毒。因此理论上,其他相似的病毒表面膜蛋白也可根据本研究报道的方法构建伪病毒系统,而不依赖于某种特定病原的分离、鉴定。该方法特别对于高度危险性病原研究具有一定的意义。
猪在流感病毒生态中扮演着重要的角色。由于猪的呼吸道上皮细胞同时具有人流感病毒和禽流感病毒的受体,使其极有可能成为新流感病毒的产生源头。控制猪流感(Swine influenza, SI)的发生和发展有助于减少流感病毒对人类的健康威胁。疫苗免疫是预防SI的有效手段。当前商品化的SI疫苗为包括Hl和/或H3亚型病毒的灭活疫苗。与人流感疫苗不同,SI疫苗并不会按照当年流行的优势毒株更换疫苗种子毒,由于流行毒株的抗原性变异,经常导致现有商品化疫苗无法对其提供有效免疫保护的情况。鉴于此,本研究尝试利用保守的SIV抗原组分构建具有广谱交叉保护效力的DNA疫苗。第一组DNA疫苗将H3亚型SIV共有的M2e及HA蛋白序列的编码基因连接至表达载体(MHa);第二组在此基础上添加一个保守的T细胞表位(MNHa),以增强疫苗诱导T细胞免疫应答的能力。通过基因枪免疫小鼠后发现,两组DNA疫苗能有效刺激机体产生体液免疫应答,同时MNHa比MHa诱导了更高水平的T细胞免疫应答。攻毒保护实验表明两组疫苗都能够对同源H3亚型处在不同进化谱系的病毒攻击提供完全免疫保护,提示增强的T细胞免疫应答对于同亚型病毒的交叉保护来讲可能不是必需的；而与MHa相比,MNHa提供了更显著的对于异源H1亚型病毒攻击的保护效力,用H1亚型病毒攻击小鼠后,MNHa免疫组小鼠肺脏内病毒滴度及组织病理学损伤显著低于MHa免疫组,表明T细胞免疫应答在提供异源毒株的交叉保护方面起到重要作用。本研究在通用型SI疫苗研究方面进行了有益探索,并为未来SI疫苗的研制提供了可借鉴的思路。
Influenza is one of the major threats to global public health. Influenza viruses escape from host immune system and transmit among host species through the accumulation of changes on antigenic sites (antigenic drift) and genetic reassortment of different strains (antigenic shift). The2009swine-origin influenza A H1N1virus (hereafter referred to as pH1N1) resulted in the4th influenza global pandemic in human history. pH1N1transmit more efficiently in humans than that of seasonal influenza viruses, and transmission of pH1N1from human to pigs has also been reported. Although the pH1N1virus is now considered to be post-pandemic, there are still possibilities that the virus recombines with other influenza viruses in pigs then yielding a novel potential epidemic or pandemic strain. It is therefore necessary to establish accurate and specific virus detection and serological diagnostic methods to monitor the spread of virus and the infection state of different hosts, which may provide useful information in predicting the next influenza pandemic.
In this study, we established virus-and neutralization antibody-detection methods from the perspective of etiology and serology, respectively. A pH IN1-specific monoclonal antibody (mAb) was first developed based on a baculoviurs surface disply system, the epitope of the mAb was then identified. Results indicated that60KLRG63motif on hemagglutinin (HA) protein is the minimal requirement for the reactivity of the epitope recognized by mAb. Whole virus-positive mouse serum did not reacted with this epitope, suggesting that the epitope is not the immunodominant epitope of HA protein, thus the immune pressure posed by the hosts against the epitope is much less than that against immunodominant epitope, and thereby the identified epitope do not tend to shift. Further, conservation analysis of epitope showed that this epitope was pH1N1-specific, suggesting that the mAb is suitable material for virus-detection. An antigen-capture ELISA (AC-ELISA) method was developed in combinnation with rabbit HA-specific polyclonal antibody. Working dilution of each antibody were optimiezed. Sensitivity assay showed that AC-ELISA was at least8times more sensitive than hemagglutination test. Besides, specificity assay demonstrated that human seasonal H1NI, swine influenza virus (SIV) H1N1and H3N2can not be detected by AC-ELISA. The AC-ELISA was then employed to test the experimental infection samples, it was found that the results of AC-ELISA showed high agreement rate with virus isolation (VI), but was less sensitive than VI. It is conclued that the pH IN1-specific AC-ELISA represents a valuable tool for virus-detection.
A recombinant plasmid expressing HA gene of pH1N1A/Califorina/04/2009was co-transfected into human embryonic kidney293T cells with retroviral vector pHIT111and pHIT60to construct Murine leukemia virus (MuLV) pseudotyped virus. The pseudotyped virus infected Madin-Darby canine kidney (MDCK) and293T cells as indicated by the detection of the P-galactosidase as reporter gene. Furthermore, the infections were neutralized by the recombinant pHlN1virus-positive serum, suggesting that the HA-pseudotyped particles can mimic the surface of influenza virions. The pseudotyped virus reported here represents a powerful surrogate for pH1N1-specific neutralization antibody detection, which may be useful in assessing hosts'infection state, screening the antiviral regents, as well as developing vaccines. In addition, we constructed pseudotyped virus with similar infection property with wild type viruses by using the synthetized HA gene which is available in public database, thus, theoretically, other pseudotyped virus can also be constructed based on the method reported here, without the need of viral isolation and identification, especially for highly pathogenic and contagious ones.
Pigs play important roles in the ecology of influenza viruses, and could be the source of new strains since pigs express sialic acid receptors for both mammalian and avian strains of influenza viruses on their tracheal epithelial cells. The control of swine influeza (SI) is helpful to reduce the significant health risk for the human population by reducing virus shed and thereby transmission. Vaccination is considered to be the most effective method to control SI. Currently, commercially available swine influenza vaccines are adjuvanted, whole-virus killed vaccines containing of both HI and H3subtypes. Unlike human influenza vaccine, strains used for SI vaccine preparation are not regularly replaced, making the vaccines do not provide consistent protection from infection, because the viruses continuously evolve by antigenic drift and shift. Cross-protective SI vaccine designed based on the conerved viral proteins would be of great help to solve this problem. To this end, we constructed two forms of DNA vaccines, one was made by fusing M2e to consensus H3HA (MHa), which represents the majority of the HA sequences of H3N2viruses, another was made by fusing M2e and a conserved CTL epitope (NP147-155) to consensus H3HA (MNHa). BALB/c mice were immunized twice with the two DNA vaccines via gene gun. It was shown that the two vaccines elicited substantial antibody responses, and MNHa induced more significant T cell-mediated immune response than MHa did. Then two H3N2strains representative of different evolutional clusters were used to challenge the vaccine-immunized mice. Results indicated that both of the two DNA vaccines prevented homosubtypic virus infections completely, suggesting the ehanced T cell-mediated immune response may be non-essential for homologous protection. The vaccines'heterologous protective efficacies were further tested by challenging with a H1N1SI strain and a reassortant pH1N1strain. It was found that MNHa, but not MHa, induces immune responses partially protect against heterologous influenza infection as indicted by the reduced lung viral titer and less lung histopathological damage, suggesting that CTL responses play important roles for heterologous protection. Reults of the current study indicate that strengthening CTL responses are promising ways for universal influenza vaccines development.
引文
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