以HBc颗粒为载体甲型流感通用疫苗的初步研究
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摘要
【研究背景】
流感病毒(Influenza virus )是一种能够引起严重性呼吸道传播疾病的正粘科属病毒。根据其内部核蛋白(NP)和基质蛋白(M)的抗原性不同,可分为甲(A)、乙(B)、丙(C)三种类型。其中,甲型流感病毒曾引起多次大范围的流行,上世纪共出现四次大流行,最为严重的是1918年的大流感,造成了全世界5千多万人死亡。2009年世界范围内暴发的猪源性甲型流感病毒流行,传染性强,波及范围广,再次突显了甲型流感病毒感染的严重性及其对人类社会造成的巨大经济负担。
接种疫苗是预防流感发生与传播的最有效方式。目前市场应用的流感疫苗主要有三种:全病毒灭活疫苗、裂解疫苗、亚单位疫苗。这些疫苗对同亚型流感病毒感染预防有效,但在不同亚型病毒之间的保护效果较弱。此外,流感病毒的变异速度非常快,流感疫苗每年都要更换,给疫苗的生产制备造成了诸多不便。世界卫生组织是依据当年全世界范围流感病毒变化情况来预测并推荐下一年流感疫苗生产用组分的,预测的准确度将会直接影响疫苗的保护效率,如预测失败将造成流感爆发流行的潜在威胁。因此,研制出一种具有广泛保护作用的通用型疫苗对于应对流感病毒突变后的突然爆发,迅速建立人群免疫屏障,阻断流感大流行蔓延,降低其危害性方面具有极其重要的实际意义。
甲型流感通用疫苗的研发主要基于流感病毒基质蛋白胞外功能区M2e (Ectodomain of matrix protein 2),其在各种甲型流感病毒中具有极高的保守性,且可诱导特异性保护抗体产生。此外还有高度保守的流感病毒核蛋白NP(Nucleoprotein),其表面具有多个细胞毒性T淋巴细胞(CTL)识别表位,可诱导细胞免疫。但M2e和NP表位的分子量小、免疫原性低、易降解,在体内难以产生足够的免疫反应。所以,使用结构复杂病毒蛋白作为免疫原性放大载体成为表位疫苗研发的热点。其中,乙肝病毒核心蛋白HBc(Hepatitis B virus core protein , HBc)蛋白具有天然的颗粒组装能力,能特异性激发针对外源表位的体液、细胞免疫。近年来,HBc颗粒的应用方面已经有了较成熟研究结果。疫苗研究中以HBc作为载体的疫苗抗原有很多,如乙肝病毒表面抗原、人乳头瘤病毒E7蛋白、B群链球菌CPS糖蛋白等,在临床前实验研究中已观察到肯定的疗效。因此,发展以HBc颗粒为载体的流感病毒表位疫苗研究具有很好的前景和应用价值。
【目的】
本研究的目的是构建以乙肝病毒核心蛋白(HBc)为载体的基于M2e蛋白的甲型流感通用疫苗抗原,利用杆状病毒表达系统及大肠杆菌表达系统表达、纯化和鉴定,并进行疫苗免疫效果初步评价,为发展安全、有效、具有广泛保护作用的甲型流感通用疫苗奠定基础。
【方法与结果】
第一部分sf9昆虫细胞表达甲型流感M2e通用疫苗抗原的构建、表达、纯化及鉴定通过基因工程方法,构建含M2e重复片段的3M2e-HBc-pFastBacHTA重组质粒。将该重组质粒与DH10Bac中的穿梭载体Bacmid同源重组,获得3M2e-HBc-Bacmid重组杆粒。提取杆粒DNA转染sf9昆虫细胞,得到含有重组杆状病毒。继续扩增病毒,感染细胞后,免疫荧光检测成功表达出3M2e-HBc目的蛋白。Western blot检测该蛋白具有免疫活性。利用pFastBacHTA载体上带有的组氨酸标签,进行亲和层析,纯化获得3M2e-HBc重组蛋白。电镜结果显示,3M2e-HBc目的基因可以通过杆状病毒sf9昆虫细胞表达系统表达并正确装配出病毒样颗粒。
第二部分大肠杆菌表达甲型流感M2e-NP通用疫苗抗原的构建、表达、纯化及鉴定通过基因工程方法,构建并鉴定获得3M2e-NP-HBc-pET 21a重组表达质粒,并转化大肠杆菌细胞,IPTG诱导表达首先获得了包涵体形式表达的重组蛋白。经变性及复性纯化处理后,包涵体蛋白形成的重组病毒颗粒的状态不理想。经进一步优化诱导表达条件获得了部分可溶性表达的重组蛋白,Western blot检测证明该蛋白具有免疫活性。采用层析方法获得纯化的3M2e-NP-HBc重组蛋白。电镜观察结果显示,大肠杆菌表达的可溶性重组蛋白能够自动装配成大小约为30nm的病毒样颗粒。
第三部分甲型流感通用疫苗免疫效果初步评价
通过动物免疫实验,初步评价甲型流感通用疫苗的免疫应答效果及交叉保护作用。采用间接ELISA法、流式细胞术、ELISOPT技术,对疫苗在Balb/C小鼠体内的免疫应答效果进行了评估。在对大肠杆菌表达的流感通用疫苗评价的结果中显示,疫苗以滴鼻和腹腔注射两种途径免疫小鼠,均可以诱导产生针对不同亚型流感病毒的特异性体液及细胞免疫应答,并且滴鼻免疫能够激发更高的黏膜局部免疫应答,说明采用滴鼻免疫这种更接近于流感病毒自然感染途径的方式免疫对提高流感疫苗的保护效果具有重要意义。同时,攻毒试验结果显示,以A/Beijing/501和A/PR/8/34两株代表性的甲型流感病毒攻毒后,疫苗免疫组相比对照组明显降低小鼠的死亡率,表明大肠杆菌表达的以HBc为载体的甲型流感通用疫苗具有良好的免疫原性交叉保护作用。
【结论】
本研究利用Bac-to-Bac杆状病毒sf9昆虫细胞表达系统和大肠杆菌原核表达系统,分别成功制备了两种的以HBc为载体的基于流感病毒M2e蛋白的流感通用疫苗,并主要对大肠杆菌表达的流感通用疫苗进行了动物免疫效果评价。实验结果显示,该疫苗具有很好的免疫原性,可以刺激机体产生系统免疫应答和黏膜局部免疫应答,并且能产生有效的交叉保护作用,为甲型流感通用疫苗的深入研究提供了理论依据。
【Backgrounds】
Influenza is a severely contagious respiratory disease caused by influenza virus which belongs to the family of Orthomyxoviridae. Influenza virus is classified into three types based on the antigenicity of their nucleoprotein (NP) and matrix protein (M). Influenza A viruses have caused four pandemics during the last century. The pandemic of 1918 killed as many as 50 million people worldwide. The pandemic of influenza A (H1N1)2009 virus, characterized by its rapid transmission and large scale epidemics, has a potential to cause fatal infection and exert high economic burden to humans.
Inoculation remains the most effective approach for influenza prophylaxis and control. The inactivated vaccine, split vaccine and subunit vaccines are currently used in humans. These vaccines are effective to the homosubtypes of influenza virus, but have little cross protection to the different subtype of influenza virus. In addition, the genome of influenza virus is vulnerable to mutate. This phenomenon makes it more difficult to produce vaccines to counter the newly mutated influenza virus annually. WHO will recommend the component of influenza virus annually based on the changing of influenza virus.However, there will be less efficient if there is any inaccuracy in predicting the influenza virus strains. It brings a latent threat of abrupt pandemic of influenza. Therefore, it is greatly significant to develop one universal influenza vaccines which could provide broad-spectrum protection for controlling the abrupt pandemic caused by mutated influenza virus, building the immunologic barrier, block the epidemics of influenza and abase the perniciousness of influenza pandemics.
Matrix protein (M)2 is an Influenza A membrane protein with an extracellular domain of 24 amino acid residues, which is strongly conserved among influenza A virus strains, and its use as a vaccine antigen can induce special protective antibody. NP is one of influenza nucleoproteins, and its sequence is highly conserved. On the surface of NP, there are many CTL epitopes which can induce cellular immunity. Accordingly, the highly conserved M2e and CTL epitopes on NP are thought to be excellent universal influenza vaccine antigen targest. However, the small molecules of M2e and NP epitopes are and weak antigens and vulnerable to degradation, which only induce poor immunity response in body and hardly induce enough immunoprotection. Therefore, using adjuvant or virus protein constructs as vector to elevate immunogenicity has been a hot topic in development of universal influenza vaccine. Hepatitis B virus core protein (HBc) is capable of constructing into VLP and inducing specific humoral and cellular immunity targeting to the exogenous epitopes. Recently, more successful results gained in the application of HBc particles. The satisfied effectiveness was observed in clinical with HBc as the vector in vaccination with the hepatitis B surface antigen, E7 of human papilloma virus (HPV), and CPS of Streptococcus B antigens. Therefore, it is promising and practical to develop universal influenza epitop vaccine with Hepatitis B virus core protein as vector.
In animal experiments, the immune response and cross-protection effect of candidate universal influenza vaccine were preliminary evaluated. By ELISA, FACS, ELISPOT, the vaccine immune response effect in Balb/C mice were evaluated. Results showed, special humoral immunity and cellular immunity against different subtypes influenza virus were induced through both intranasal and intraperitoneal vaccination. And intranasal vaccination could induce higher level mucosa local immune response. This demonstrated that intranasal vaccination which more like to natural immunity could significantly enhance protection effect of influenza vaccine. Meanwhile, challenge experiments results showed, mice mortality in vaccine group was distinguishably lower than control group. It indicated that our universal influenza virus vaccine with hepatitis B virus core (HBc) particle as a vector could induce fine cross-protection immunity.
【Objective】
Our objective is to develop a universal A influenza vaccine antigen based on M2e with Hepatitis B virus core protein as vector. The vaccine antigen will be generated in Baculovirus and Escherichia coli expression system. After purification and identification of antigen, we will preliminarily evaluate the efficiency of vaccine by animal experiments. This research lays a foundation for developing a safe and effective universal A influenza vaccine.
【Methods and Results】
1. Construction, expression, purification and identification of universal influenza vaccine by Bac-to-Bac baculovirus insect cell expression system 3M2e-HBc-pFastBacHTA recombinant plasmid was constructed by genetic engineering methods. Then 3M2e-HBc-pFastBacHTA plasmid was transformed into DH10Bac cells. Through homologous recombination, we got the 3M2e-HBc-Bacmid recombinant bacmid. Then, the isolated Bacmic DNA was transfected into sf9 nsect cells. By immunofluorescence, the recombinant protein was detected in insect cells infected by recombinant baculovirus. Immunological activity was detected by WB assay. 3M2e-HBc recombinant protein was purified by affinity chromatograph. Electron microscope images displayed that 3M2e-HBc gene correctly expressed and assembled to form VLP in Bac-to-Bac baculovirus expression system.
2. Construction, expression, purification and identification of universal influenza vaccine by Escherichia coli expression system
3M2e-NP-HBc-pET21a recombinant expression plasmid was constructed by genetic engineering methods and was transform into Escherichia coli cells. At the beginning, inclusion body protein was got after inducing with IPTG. However, undertaking denaturation and renaturation procedure, the state of VLP formed by inclusion body protein was bad. Through the optimization of induction condition, partly dissoluble protein was expressed and gained. Then, 3M2e-NP-HBc recombinant protein was purified by affinity chromatograph and gel chromatograph. Western blot result indicated the purified protein had immunogenicity. Electron microscope images displayed that, the dissoluble 3M2e-NP-HBc recombinant protein could correctly assembled to form VLP in Escherichia coli cells. The size of VLP was approximately 30nm.It was shown that we successfully construct universal influenza vaccine expressed by Escherichia coli expression system.
3. Evaluation of protective efficiency of universal influenza vaccines on Balb/C mice. In animal experiments, the immune response and cross-protection effect of candidate universal influenza vaccine were preliminary evaluated. By ELISA, FCM, ELISPOT, the vaccine immune response effect in Balb/C mice were evaluated. Results showed, special humoral immunity and cellular immunity against different subtypes influenza virus were induced through both intranasal and intraperitoneal vaccination. And intranasal vaccination could induce higher level mucosa local immune response. This demonstrated that intranasal vaccination which more like to natural immunity could significantly enhance protection effect of influenza vaccine. Meanwhile, challenge experiments results showed, mice mortality in vaccine group was distinguishably lower than control group. It indicated that our universal influenza virus vaccine with hepatitis B virus core (HBc) particle as a vector could induce fine cross-protection immunity.
【Conclusion】
In this study, we constructed two universal influenza virus vaccines with hepatitis B virus core (HBc) particle as a vector by Bac-to-Bac baculovirus insect cell expression system and Escherichia coli procaryotic expression system. By animal experiments, we evaluated the vaccine which was expressed in Escherichia coli. It showed that our influenza vaccine had fine immunegenicity. This vaccine induced system immune response and mucosa local immune response, and provided effective cross-protection. This research provided a base theory for the further development of universal influenza vaccine.
流感病毒(Influenza virus )是一种能够引起严重性呼吸道传播疾病的正粘科属病毒。根据其内部核蛋白(NP)和基质蛋白(M)的抗原性不同,可分为甲(A)、乙(B)、丙(C)三种类型。其中,甲型流感病毒曾引起多次大范围的流行,上世纪共出现四次大流行,最为严重的是1918年的大流感,造成了全世界5千多万人死亡。2009年世界范围内暴发的猪源性甲型流感病毒流行,传染性强,波及范围广,再次突显了甲型流感病毒感染的严重性及其对人类社会造成的巨大经济负担。
接种疫苗是预防流感发生与传播的最有效方式。目前市场应用的流感疫苗主要有三种:全病毒灭活疫苗、裂解疫苗、亚单位疫苗。这些疫苗对同亚型流感病毒感染预防有效,但在不同亚型病毒之间的保护效果较弱。此外,流感病毒的变异速度非常快,流感疫苗每年都要更换,给疫苗的生产制备造成了诸多不便。世界卫生组织是依据当年全世界范围流感病毒变化情况来预测并推荐下一年流感疫苗生产用组分的,预测的准确度将会直接影响疫苗的保护效率,如预测失败将造成流感爆发流行的潜在威胁。因此,研制出一种具有广泛保护作用的通用型疫苗对于应对流感病毒突变后的突然爆发,迅速建立人群免疫屏障,阻断流感大流行蔓延,降低其危害性方面具有极其重要的实际意义。
甲型流感通用疫苗的研发主要基于流感病毒基质蛋白胞外功能区M2e (Ectodomain of matrix protein 2),其在各种甲型流感病毒中具有极高的保守性,且可诱导特异性保护抗体产生。此外还有高度保守的流感病毒核蛋白NP(Nucleoprotein),其表面具有多个细胞毒性T淋巴细胞(CTL)识别表位,可诱导细胞免疫。但M2e和NP表位的分子量小、免疫原性低、易降解,在体内难以产生足够的免疫反应。所以,使用结构复杂病毒蛋白作为免疫原性放大载体成为表位疫苗研发的热点。其中,乙肝病毒核心蛋白HBc(Hepatitis B virus core protein , HBc)蛋白具有天然的颗粒组装能力,能特异性激发针对外源表位的体液、细胞免疫。近年来,HBc颗粒的应用方面已经有了较成熟研究结果。疫苗研究中以HBc作为载体的疫苗抗原有很多,如乙肝病毒表面抗原、人乳头瘤病毒E7蛋白、B群链球菌CPS糖蛋白等,在临床前实验研究中已观察到肯定的疗效。因此,发展以HBc颗粒为载体的流感病毒表位疫苗研究具有很好的前景和应用价值。
【目的】
本研究的目的是构建以乙肝病毒核心蛋白(HBc)为载体的基于M2e蛋白的甲型流感通用疫苗抗原,利用杆状病毒表达系统及大肠杆菌表达系统表达、纯化和鉴定,并进行疫苗免疫效果初步评价,为发展安全、有效、具有广泛保护作用的甲型流感通用疫苗奠定基础。
【方法与结果】
第一部分sf9昆虫细胞表达甲型流感M2e通用疫苗抗原的构建、表达、纯化及鉴定通过基因工程方法,构建含M2e重复片段的3M2e-HBc-pFastBacHTA重组质粒。将该重组质粒与DH10Bac中的穿梭载体Bacmid同源重组,获得3M2e-HBc-Bacmid重组杆粒。提取杆粒DNA转染sf9昆虫细胞,得到含有重组杆状病毒。继续扩增病毒,感染细胞后,免疫荧光检测成功表达出3M2e-HBc目的蛋白。Western blot检测该蛋白具有免疫活性。利用pFastBacHTA载体上带有的组氨酸标签,进行亲和层析,纯化获得3M2e-HBc重组蛋白。电镜结果显示,3M2e-HBc目的基因可以通过杆状病毒sf9昆虫细胞表达系统表达并正确装配出病毒样颗粒。
第二部分大肠杆菌表达甲型流感M2e-NP通用疫苗抗原的构建、表达、纯化及鉴定通过基因工程方法,构建并鉴定获得3M2e-NP-HBc-pET 21a重组表达质粒,并转化大肠杆菌细胞,IPTG诱导表达首先获得了包涵体形式表达的重组蛋白。经变性及复性纯化处理后,包涵体蛋白形成的重组病毒颗粒的状态不理想。经进一步优化诱导表达条件获得了部分可溶性表达的重组蛋白,Western blot检测证明该蛋白具有免疫活性。采用层析方法获得纯化的3M2e-NP-HBc重组蛋白。电镜观察结果显示,大肠杆菌表达的可溶性重组蛋白能够自动装配成大小约为30nm的病毒样颗粒。
第三部分甲型流感通用疫苗免疫效果初步评价
通过动物免疫实验,初步评价甲型流感通用疫苗的免疫应答效果及交叉保护作用。采用间接ELISA法、流式细胞术、ELISOPT技术,对疫苗在Balb/C小鼠体内的免疫应答效果进行了评估。在对大肠杆菌表达的流感通用疫苗评价的结果中显示,疫苗以滴鼻和腹腔注射两种途径免疫小鼠,均可以诱导产生针对不同亚型流感病毒的特异性体液及细胞免疫应答,并且滴鼻免疫能够激发更高的黏膜局部免疫应答,说明采用滴鼻免疫这种更接近于流感病毒自然感染途径的方式免疫对提高流感疫苗的保护效果具有重要意义。同时,攻毒试验结果显示,以A/Beijing/501和A/PR/8/34两株代表性的甲型流感病毒攻毒后,疫苗免疫组相比对照组明显降低小鼠的死亡率,表明大肠杆菌表达的以HBc为载体的甲型流感通用疫苗具有良好的免疫原性交叉保护作用。
【结论】
本研究利用Bac-to-Bac杆状病毒sf9昆虫细胞表达系统和大肠杆菌原核表达系统,分别成功制备了两种的以HBc为载体的基于流感病毒M2e蛋白的流感通用疫苗,并主要对大肠杆菌表达的流感通用疫苗进行了动物免疫效果评价。实验结果显示,该疫苗具有很好的免疫原性,可以刺激机体产生系统免疫应答和黏膜局部免疫应答,并且能产生有效的交叉保护作用,为甲型流感通用疫苗的深入研究提供了理论依据。
【Backgrounds】
Influenza is a severely contagious respiratory disease caused by influenza virus which belongs to the family of Orthomyxoviridae. Influenza virus is classified into three types based on the antigenicity of their nucleoprotein (NP) and matrix protein (M). Influenza A viruses have caused four pandemics during the last century. The pandemic of 1918 killed as many as 50 million people worldwide. The pandemic of influenza A (H1N1)2009 virus, characterized by its rapid transmission and large scale epidemics, has a potential to cause fatal infection and exert high economic burden to humans.
Inoculation remains the most effective approach for influenza prophylaxis and control. The inactivated vaccine, split vaccine and subunit vaccines are currently used in humans. These vaccines are effective to the homosubtypes of influenza virus, but have little cross protection to the different subtype of influenza virus. In addition, the genome of influenza virus is vulnerable to mutate. This phenomenon makes it more difficult to produce vaccines to counter the newly mutated influenza virus annually. WHO will recommend the component of influenza virus annually based on the changing of influenza virus.However, there will be less efficient if there is any inaccuracy in predicting the influenza virus strains. It brings a latent threat of abrupt pandemic of influenza. Therefore, it is greatly significant to develop one universal influenza vaccines which could provide broad-spectrum protection for controlling the abrupt pandemic caused by mutated influenza virus, building the immunologic barrier, block the epidemics of influenza and abase the perniciousness of influenza pandemics.
Matrix protein (M)2 is an Influenza A membrane protein with an extracellular domain of 24 amino acid residues, which is strongly conserved among influenza A virus strains, and its use as a vaccine antigen can induce special protective antibody. NP is one of influenza nucleoproteins, and its sequence is highly conserved. On the surface of NP, there are many CTL epitopes which can induce cellular immunity. Accordingly, the highly conserved M2e and CTL epitopes on NP are thought to be excellent universal influenza vaccine antigen targest. However, the small molecules of M2e and NP epitopes are and weak antigens and vulnerable to degradation, which only induce poor immunity response in body and hardly induce enough immunoprotection. Therefore, using adjuvant or virus protein constructs as vector to elevate immunogenicity has been a hot topic in development of universal influenza vaccine. Hepatitis B virus core protein (HBc) is capable of constructing into VLP and inducing specific humoral and cellular immunity targeting to the exogenous epitopes. Recently, more successful results gained in the application of HBc particles. The satisfied effectiveness was observed in clinical with HBc as the vector in vaccination with the hepatitis B surface antigen, E7 of human papilloma virus (HPV), and CPS of Streptococcus B antigens. Therefore, it is promising and practical to develop universal influenza epitop vaccine with Hepatitis B virus core protein as vector.
In animal experiments, the immune response and cross-protection effect of candidate universal influenza vaccine were preliminary evaluated. By ELISA, FACS, ELISPOT, the vaccine immune response effect in Balb/C mice were evaluated. Results showed, special humoral immunity and cellular immunity against different subtypes influenza virus were induced through both intranasal and intraperitoneal vaccination. And intranasal vaccination could induce higher level mucosa local immune response. This demonstrated that intranasal vaccination which more like to natural immunity could significantly enhance protection effect of influenza vaccine. Meanwhile, challenge experiments results showed, mice mortality in vaccine group was distinguishably lower than control group. It indicated that our universal influenza virus vaccine with hepatitis B virus core (HBc) particle as a vector could induce fine cross-protection immunity.
【Objective】
Our objective is to develop a universal A influenza vaccine antigen based on M2e with Hepatitis B virus core protein as vector. The vaccine antigen will be generated in Baculovirus and Escherichia coli expression system. After purification and identification of antigen, we will preliminarily evaluate the efficiency of vaccine by animal experiments. This research lays a foundation for developing a safe and effective universal A influenza vaccine.
【Methods and Results】
1. Construction, expression, purification and identification of universal influenza vaccine by Bac-to-Bac baculovirus insect cell expression system 3M2e-HBc-pFastBacHTA recombinant plasmid was constructed by genetic engineering methods. Then 3M2e-HBc-pFastBacHTA plasmid was transformed into DH10Bac cells. Through homologous recombination, we got the 3M2e-HBc-Bacmid recombinant bacmid. Then, the isolated Bacmic DNA was transfected into sf9 nsect cells. By immunofluorescence, the recombinant protein was detected in insect cells infected by recombinant baculovirus. Immunological activity was detected by WB assay. 3M2e-HBc recombinant protein was purified by affinity chromatograph. Electron microscope images displayed that 3M2e-HBc gene correctly expressed and assembled to form VLP in Bac-to-Bac baculovirus expression system.
2. Construction, expression, purification and identification of universal influenza vaccine by Escherichia coli expression system
3M2e-NP-HBc-pET21a recombinant expression plasmid was constructed by genetic engineering methods and was transform into Escherichia coli cells. At the beginning, inclusion body protein was got after inducing with IPTG. However, undertaking denaturation and renaturation procedure, the state of VLP formed by inclusion body protein was bad. Through the optimization of induction condition, partly dissoluble protein was expressed and gained. Then, 3M2e-NP-HBc recombinant protein was purified by affinity chromatograph and gel chromatograph. Western blot result indicated the purified protein had immunogenicity. Electron microscope images displayed that, the dissoluble 3M2e-NP-HBc recombinant protein could correctly assembled to form VLP in Escherichia coli cells. The size of VLP was approximately 30nm.It was shown that we successfully construct universal influenza vaccine expressed by Escherichia coli expression system.
3. Evaluation of protective efficiency of universal influenza vaccines on Balb/C mice. In animal experiments, the immune response and cross-protection effect of candidate universal influenza vaccine were preliminary evaluated. By ELISA, FCM, ELISPOT, the vaccine immune response effect in Balb/C mice were evaluated. Results showed, special humoral immunity and cellular immunity against different subtypes influenza virus were induced through both intranasal and intraperitoneal vaccination. And intranasal vaccination could induce higher level mucosa local immune response. This demonstrated that intranasal vaccination which more like to natural immunity could significantly enhance protection effect of influenza vaccine. Meanwhile, challenge experiments results showed, mice mortality in vaccine group was distinguishably lower than control group. It indicated that our universal influenza virus vaccine with hepatitis B virus core (HBc) particle as a vector could induce fine cross-protection immunity.
【Conclusion】
In this study, we constructed two universal influenza virus vaccines with hepatitis B virus core (HBc) particle as a vector by Bac-to-Bac baculovirus insect cell expression system and Escherichia coli procaryotic expression system. By animal experiments, we evaluated the vaccine which was expressed in Escherichia coli. It showed that our influenza vaccine had fine immunegenicity. This vaccine induced system immune response and mucosa local immune response, and provided effective cross-protection. This research provided a base theory for the further development of universal influenza vaccine.
引文
[1]、Kobasa D, Kawaoka Y. Emerging influenza viruses, Past and present. [J] Curr Mol Med, 2005, 5: 791-803.
[2]、Webster RG, Bean WJ, Gorman OT et al. Evolution and ecology of influenza A viruses. [J] Microbiol Rev, 1992, 56(1):152-79.
[3]、Russell CJ, Webster RG. The genesis of a pandemic influenza virus. [J] Cell, 2005, 123(3):368–371.
[4]、Fedson DS. Vaccine development for an imminent pandemic: why we should worry, what we must do. [J] Hum Vaccine, 2006, 2: 38-42.
[5]、Alan W Hampson. Vaccines for Pandemic Influenza. The History of our Current Vaccines, their Limitations and the Requirements to Deal with a Pandemic Threat. [J] Ann Acad Med Singapore, 2008, 37(6): 510-517.
[6]、Gerhard W, Mozdzanowska K, Zharikova D. Prospects for universal influenza virus vaccine. [J] Infect. Dis. Emerg, 2006, 12(4): 569–574.
[7]、Czabotar PE, Martin SR, Hay AJ. Studies of structural changes in the M2 proton channel of influenza A virus by tryptophan fluorescence. [J] Virus Res, 2004, 99(1):57-61.
[8]、Wu F, Huang JH, Yuan XY, Huang WS, Chen YH. Characterization of immunity induced by M2e of influenza virus. [J] Vaccine, 2007, 25(52):8868-73.
[9]、Fan J, Liang X, Horton MS, et al. Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. [J] Vaccine, 2004, 22(23–24): 2993–3003.
[10]、Sch?del F, Peterson D, Milich D. Hepatitis B virus core and e antigen: immune recognition and use as a vaccine carrier moiety. [J] Intervirology, 1996, 39(1-2):104-10
[11]、Guus F. Rimmelzwaan, Joost H.C.M.Kreijtz, Rogier Bodewes, et al. Influenza virus CTL epitopes, remarkably conserved and remarkably variable. [J] Vaccine, 2009, 27(45): 6363-6365.
[12]、Greory A. Stoloff, Wilson Caparros-Wanderley. Synthetic multi-epitope peptides identified in silico induce protective immunity against multiplt influenza serotypes. [J] Eur. J. Immunol, 2007, 37: 2441-2449.
[13]、Xavier Saelens. The influenza matric protein 2 as a vaccine target. [J] Future Virol, 2008, 3(2): 167-178.
[14]、Bui HH, Peters B, Assarsson E, et al. Ab and T cell epitopes of Influenza A virus, knowledge and opportunities. [J] Proc. Natl Acad. Sci. USA, 2007, 104(1): 246–251
[15]、Pumpers P, Grens E. HBV Core Particles as a Carrier for B Cell/T Cell Epitopes. [J] Intervirology, 2001, 44: 98-114.
[16]、Kratz PA, Bottcher B, Nassal M. Nature display of complete foreign protein domains on the surface of hepatitis B virus capsids. [J] Proc Natl Acad Sci USA,1999, 96: 1915-1920.
[17]、Mayer L. Mucosal immunity and gastrointestinal antigen processing. [J]. Pediatr Gastroenterol Nutr, 2000, 30(5):4-12.
[18]、Ogra PL, Fishaut M, Gallagher MR. Viral vaccination via the mucosal routes. [J] Rev Infect Dis.1980, 2(3):352-69.
[19]、Ogra PL, Faden H, Welliver RC. Vaccination strategies for mucosal immune responses. [J] Clin Microbiol Rev, 2001,14(2):430-45.
[20]、Anjuère F, Czerkinsky C. Mucosal immunity and vaccine development. [J] Med Sci (Paris), 2007, 23(4):371-8.
[21]、Michael Schotsaert, Marina De Filette, Walter Fiers, Xavier Saelens. Universal M2 etodomain-based influenza A vaccines: preclinical and clinical developments. [J] Expert Rev.Vaccines, 2009, 8(4): 499-508.
[22]、Marina De Filette, Walter Fiers, Wouter Martens, et al. Improved design and intranasal delivery of an M2e-based human influenza A vaccine. [J] Vaccine, 2006, 6(24): 6597-6601.
[23]、Harley VR, Mather KA, Power BE, McKimm-Breschkin JL, Hudson PJ. Characterization of an avian influenza virus nucleoprotein expressed in E. coli and in insect cells. [J] Arch Viral, 1990; 113(3-4):267-77.
[24]、Grgacic EV, Anderson DA, [J] Methods, Virus-like particles: passport to immune recognition, 2006, 40: 60-65.
[25]、Huleatt JW, Nakaar V, Desai P, et al. Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin. [J] Vaccine, 2008, 26(2):201-14.
[26]、Fiers W, De Filette M, Birkett A, Neirynck S, Min Jou W. A "universal" human influenza A vaccine. [J] Virus Res, 2004, 103(1-2):173-6.
[27]、Jennings GT, Bachmann MF. The coming of age of virus-like particle vaccines. [J] Biol Chem,2008, 389(5):521-36.
[28]、Banks L, Matlashewski G, Pim D, Churcher M, Roberts C, Crawford L. Expression of human papillomavirus type 6 and type 16 capsid proteins in bacteria and their antigenic characterization. [J] Gen Virol, 1987, 68(12):3081-9.
[29]、Chen XS, Casini G, Harrison SC, Garcea RL. Papillomavirus capsid protein expression in Escherichia coli: purification and assembly of HPV11 and HPV16 L1. [J] Mol Biol, 2001, 307(1):173-82.
[30]、.Zhang YL, Guo YJ, Wang KY, et al. Enhanced immunogenicity of modified hepatitis B virus core particle fused with multiepitopes of foot-and-mouth disease virus. [J] Scand J Immunol, 2007, 65(4):320-8.
[31]、Paul Pumpens Elmars Grens. HBV Core Particles as a Carrier for B Cell/T Cell Epitopes. [J] Intervirology, 2001, 44:98–114.
[32]、Marina De Filette, Walter Fiers, Wouter Martens, et al. Improved design and intranasal delivery of an M2e-based human influenza A vaccine. [J] Vaccine, 2006, 24:6597-6601.
[33]、Mozdzanowska K, Feng J, Eid M et al. Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2. [J] Vaccine, 2003, 21(19–20): 2616–2626.
[34]、Brandtzaeg P, Farstad IN, Haraldsen G, Jahnsen FL. Cellular and molecular mechanisms for induction of mucosal immunity. [J] Dev Biol Stand, 1998, 92:93-108.
[1]、Russell CJ, Webster RG. The genesis of a pandemic influenza virus. Cell, 2005, 123(3):368–371.
[2]、Fouchier RA, Munster V, Wallensten A, et al. Characterization of a novel Influenza A virus hemagglutinin subtype (H16) obtained from black-headedgulls. J. Virol, 2005, 79(5): 2814–2822.
[3]、Taubenberger JK, Reid AH, Lourens RM et al. Characterization of the 1918 influenza virus polymerase genes. Nature, 2005, 437(7060):889–893.
[4]、Leroux-Roels I, Borkowski A, Vanwolleghem T, et al. Antigen sparing and cross-reactive immunity with an adjuvanted rH5N1 prototype pandemic influenza vaccine: a randomised controlled trial. Lancet, 2007, 370(9587): 580–589.
[5]、Venkataraman P, Lamb RA, Pinto LH .Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of Influenza A virus. J. Biol. Chem, 2005, 280(22): 21463–21472.
[6]、Feng J, Zhang M, Mozdzanowska K, et al. Influenza A virus infection engenders a poor antibody response against the ectodomain of matrix protein 2. Virol. J, 2006, 102(3).
[7]、Kitikoon P, Strait EL, Thacker EL. The antibody responses to swine influenza virus (SIV)recombinant matrix 1 (rM1), matrix 2 (M2), and hemagglutinin (HA) proteins in pigs with different SIV exposure. Vet. Microbiol, 2007, 126(1-3):51–62.
[8]、Bui HH, Peters B, Assarsson E, et al. Ab and T cell epitopes of Influenza A virus, knowledge and opportunities. Proc. Natl Acad. Sci. USA, 2007, 104(1): 246–251.
[9]、Treanor JJ, Tierney EL, Zebedee SL, et al. Passively transferred monoclonal antibody to the M2 protein inhibits Influenza A virus replication in mice. J. Virol, 1990, 64(3):1375–1377.
[10]、Slepushkin VA, Katz JM, Black RA, et al. Protection of mice against Influenza A virus challenge by vaccination with baculovirus expressed M2 protein. Vaccine, 1995, 13(15):1399–1402.
[11]、Neirynck S, Deroo T, Saelens X, et al. A universal Influenza A vaccine based on the extracellular domain of the M2 protein. Nat. Med, 1999,5(10):1157–1163.
[12]、De Filette M, Min Jou W, Birkett A, et al. Universal Influenza A vaccine: optimization of M2-based constructs. Virology, 2005, 337(1):149–161.
[13]、Eriksson AM, Schon KM, Lycke NY.The cholera toxin-derived CTA1-DD vaccine adjuvant administered intranasally does not cause inflammation or accumulate in the nervous tissues. J. Immunol, 2004, 173(5):3310–3319.
[14]、De Filette M, Fiers W, Martens W, et al. Improved design and intranasal delivery of an M2e-based human Influenza A vaccine. Vaccine, 2006, 24(44–46):6597–6601.
[15]、Frace AM, Klimov AI, Rowe T, et al. Modified M2 proteins produce heterotypic immunity against Influenza A virus. Vaccine, 1999, 17(18):2237–2244.
[16]、Jegerlehner A, Tissot A, Lechner F, et al. A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses. Vaccine, 2002, 20(25–26):3104–3112.
[17]、Fan J, Liang X, Horton MS, et al. Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. Vaccine, 2004, 22(23–24): 2993–3003.
[18]、Jegerlehner A, Schmitz N, Storni T,et al. Influenza A vaccine based on the extracellular domain of M2: weak protection mediated via antibody-dependent NK cell activity. J. Immunol, 2004, 172(9):5598–5605
[19]、Tompkins SM, Zhao ZS, Lo CY, et al. Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1. Emerg. Infect. Dis, 2007, 13(3):426–435.
[1]、Kobasa D, Kawaoka Y. Emerging influenza viruses: Past and present. Curr Mol Med, 2005, 5: 791-803.
[2]、Fedson DS. Vaccine development for an imminent pandemic: why we should worry, what we must do. Hum Vaccine, 2006, 2: 38-42.
[3]、Alan W Hampson. Vaccines for Pandemic Influenza. The History of our Current Vaccines, their Limitations and the Requirements to Deal with a Pandemic Threat. Ann Acad Med Singapore, 2008, 37(6): 510-517.
[4]、Gerhard W, Mozdzanowska K, Zharikova D. Prospects for universal influenza virus vaccine. Emerg. Infect. Dis, 2006, 12(4): 569–574.
[5]、Greory A. Stoloff, Wilson Caparros-Wanderley. Synthetic multi-epitope peptides identified in silico induce protective immunity against multiplt influenza serotypes. Eur. J. Immunol, 2007, 37: 2441-2449.
[6]、Bui HH, Peters B, Assarsson E, et al. Ab and T cell epitopes of Influenza A virus, knowledge and opportunities. Proc. Natl Acad. Sci. USA, 2007, 104(1): 246–251.
[7]、Fan J, Liang X, Horton MS, et al. Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. Vaccine, 2004, 22(23–24): 2993–3003.
[8]、Michael Schotsaert, Marina De Filette, Walter Fiers, Xavier Saelens. Universal M2 etodomain-based influenza A vaccines: preclinical and clinical developments. 2009, 8(4): 499-508.
[9]、Guus F. Rimmelzwaan, Joost H.C.M.Kreijtz, Rogier Bodewes, et al. Influenza virus CTL epitopes, remarkably conserved and remarkably variable. Vaccine, 2009, 27(45): 6363-6365.
[10]、Marina De Filette, Walter Fiers, Wouter Martens, et al. Improved design and intranasal delivery of an M2e-based human influenza A vaccine. Vaccine, 2006, 6(24): 6597-6601.
[11]、Pumpers P, Grens E.HBV Core Particles as a Carrier for B Cell/T Cell Epitopes. Intervirology, 2001, 44: 98-114.
[12]、Kratz PA, Bottcher B, Nassal M. Nature display of complete foreign protein domains on the surface of hepatitis B virus capsids. Proc Natl Acad Sci USA, 1999, 96: 1915-1920.
[13]、Grgacic EV, Anderson DA. Virus-like particles: passport to immune recognition. Methods, 2006, 40: 60-65.
[14]、Neirynck S, Deroo T, Saelens X, et al. A universal Influenza A vaccine based on the extracellular domain of the M2 protein. Nat. Med, 1999, 5(10): 1157-1163.
[15]、Xavier Saelens. The influenza matric protein 2 as a vaccine target. Future Virol, 2008, 3(2): 167-178.
[2]、Webster RG, Bean WJ, Gorman OT et al. Evolution and ecology of influenza A viruses. [J] Microbiol Rev, 1992, 56(1):152-79.
[3]、Russell CJ, Webster RG. The genesis of a pandemic influenza virus. [J] Cell, 2005, 123(3):368–371.
[4]、Fedson DS. Vaccine development for an imminent pandemic: why we should worry, what we must do. [J] Hum Vaccine, 2006, 2: 38-42.
[5]、Alan W Hampson. Vaccines for Pandemic Influenza. The History of our Current Vaccines, their Limitations and the Requirements to Deal with a Pandemic Threat. [J] Ann Acad Med Singapore, 2008, 37(6): 510-517.
[6]、Gerhard W, Mozdzanowska K, Zharikova D. Prospects for universal influenza virus vaccine. [J] Infect. Dis. Emerg, 2006, 12(4): 569–574.
[7]、Czabotar PE, Martin SR, Hay AJ. Studies of structural changes in the M2 proton channel of influenza A virus by tryptophan fluorescence. [J] Virus Res, 2004, 99(1):57-61.
[8]、Wu F, Huang JH, Yuan XY, Huang WS, Chen YH. Characterization of immunity induced by M2e of influenza virus. [J] Vaccine, 2007, 25(52):8868-73.
[9]、Fan J, Liang X, Horton MS, et al. Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. [J] Vaccine, 2004, 22(23–24): 2993–3003.
[10]、Sch?del F, Peterson D, Milich D. Hepatitis B virus core and e antigen: immune recognition and use as a vaccine carrier moiety. [J] Intervirology, 1996, 39(1-2):104-10
[11]、Guus F. Rimmelzwaan, Joost H.C.M.Kreijtz, Rogier Bodewes, et al. Influenza virus CTL epitopes, remarkably conserved and remarkably variable. [J] Vaccine, 2009, 27(45): 6363-6365.
[12]、Greory A. Stoloff, Wilson Caparros-Wanderley. Synthetic multi-epitope peptides identified in silico induce protective immunity against multiplt influenza serotypes. [J] Eur. J. Immunol, 2007, 37: 2441-2449.
[13]、Xavier Saelens. The influenza matric protein 2 as a vaccine target. [J] Future Virol, 2008, 3(2): 167-178.
[14]、Bui HH, Peters B, Assarsson E, et al. Ab and T cell epitopes of Influenza A virus, knowledge and opportunities. [J] Proc. Natl Acad. Sci. USA, 2007, 104(1): 246–251
[15]、Pumpers P, Grens E. HBV Core Particles as a Carrier for B Cell/T Cell Epitopes. [J] Intervirology, 2001, 44: 98-114.
[16]、Kratz PA, Bottcher B, Nassal M. Nature display of complete foreign protein domains on the surface of hepatitis B virus capsids. [J] Proc Natl Acad Sci USA,1999, 96: 1915-1920.
[17]、Mayer L. Mucosal immunity and gastrointestinal antigen processing. [J]. Pediatr Gastroenterol Nutr, 2000, 30(5):4-12.
[18]、Ogra PL, Fishaut M, Gallagher MR. Viral vaccination via the mucosal routes. [J] Rev Infect Dis.1980, 2(3):352-69.
[19]、Ogra PL, Faden H, Welliver RC. Vaccination strategies for mucosal immune responses. [J] Clin Microbiol Rev, 2001,14(2):430-45.
[20]、Anjuère F, Czerkinsky C. Mucosal immunity and vaccine development. [J] Med Sci (Paris), 2007, 23(4):371-8.
[21]、Michael Schotsaert, Marina De Filette, Walter Fiers, Xavier Saelens. Universal M2 etodomain-based influenza A vaccines: preclinical and clinical developments. [J] Expert Rev.Vaccines, 2009, 8(4): 499-508.
[22]、Marina De Filette, Walter Fiers, Wouter Martens, et al. Improved design and intranasal delivery of an M2e-based human influenza A vaccine. [J] Vaccine, 2006, 6(24): 6597-6601.
[23]、Harley VR, Mather KA, Power BE, McKimm-Breschkin JL, Hudson PJ. Characterization of an avian influenza virus nucleoprotein expressed in E. coli and in insect cells. [J] Arch Viral, 1990; 113(3-4):267-77.
[24]、Grgacic EV, Anderson DA, [J] Methods, Virus-like particles: passport to immune recognition, 2006, 40: 60-65.
[25]、Huleatt JW, Nakaar V, Desai P, et al. Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin. [J] Vaccine, 2008, 26(2):201-14.
[26]、Fiers W, De Filette M, Birkett A, Neirynck S, Min Jou W. A "universal" human influenza A vaccine. [J] Virus Res, 2004, 103(1-2):173-6.
[27]、Jennings GT, Bachmann MF. The coming of age of virus-like particle vaccines. [J] Biol Chem,2008, 389(5):521-36.
[28]、Banks L, Matlashewski G, Pim D, Churcher M, Roberts C, Crawford L. Expression of human papillomavirus type 6 and type 16 capsid proteins in bacteria and their antigenic characterization. [J] Gen Virol, 1987, 68(12):3081-9.
[29]、Chen XS, Casini G, Harrison SC, Garcea RL. Papillomavirus capsid protein expression in Escherichia coli: purification and assembly of HPV11 and HPV16 L1. [J] Mol Biol, 2001, 307(1):173-82.
[30]、.Zhang YL, Guo YJ, Wang KY, et al. Enhanced immunogenicity of modified hepatitis B virus core particle fused with multiepitopes of foot-and-mouth disease virus. [J] Scand J Immunol, 2007, 65(4):320-8.
[31]、Paul Pumpens Elmars Grens. HBV Core Particles as a Carrier for B Cell/T Cell Epitopes. [J] Intervirology, 2001, 44:98–114.
[32]、Marina De Filette, Walter Fiers, Wouter Martens, et al. Improved design and intranasal delivery of an M2e-based human influenza A vaccine. [J] Vaccine, 2006, 24:6597-6601.
[33]、Mozdzanowska K, Feng J, Eid M et al. Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2. [J] Vaccine, 2003, 21(19–20): 2616–2626.
[34]、Brandtzaeg P, Farstad IN, Haraldsen G, Jahnsen FL. Cellular and molecular mechanisms for induction of mucosal immunity. [J] Dev Biol Stand, 1998, 92:93-108.
[1]、Russell CJ, Webster RG. The genesis of a pandemic influenza virus. Cell, 2005, 123(3):368–371.
[2]、Fouchier RA, Munster V, Wallensten A, et al. Characterization of a novel Influenza A virus hemagglutinin subtype (H16) obtained from black-headedgulls. J. Virol, 2005, 79(5): 2814–2822.
[3]、Taubenberger JK, Reid AH, Lourens RM et al. Characterization of the 1918 influenza virus polymerase genes. Nature, 2005, 437(7060):889–893.
[4]、Leroux-Roels I, Borkowski A, Vanwolleghem T, et al. Antigen sparing and cross-reactive immunity with an adjuvanted rH5N1 prototype pandemic influenza vaccine: a randomised controlled trial. Lancet, 2007, 370(9587): 580–589.
[5]、Venkataraman P, Lamb RA, Pinto LH .Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of Influenza A virus. J. Biol. Chem, 2005, 280(22): 21463–21472.
[6]、Feng J, Zhang M, Mozdzanowska K, et al. Influenza A virus infection engenders a poor antibody response against the ectodomain of matrix protein 2. Virol. J, 2006, 102(3).
[7]、Kitikoon P, Strait EL, Thacker EL. The antibody responses to swine influenza virus (SIV)recombinant matrix 1 (rM1), matrix 2 (M2), and hemagglutinin (HA) proteins in pigs with different SIV exposure. Vet. Microbiol, 2007, 126(1-3):51–62.
[8]、Bui HH, Peters B, Assarsson E, et al. Ab and T cell epitopes of Influenza A virus, knowledge and opportunities. Proc. Natl Acad. Sci. USA, 2007, 104(1): 246–251.
[9]、Treanor JJ, Tierney EL, Zebedee SL, et al. Passively transferred monoclonal antibody to the M2 protein inhibits Influenza A virus replication in mice. J. Virol, 1990, 64(3):1375–1377.
[10]、Slepushkin VA, Katz JM, Black RA, et al. Protection of mice against Influenza A virus challenge by vaccination with baculovirus expressed M2 protein. Vaccine, 1995, 13(15):1399–1402.
[11]、Neirynck S, Deroo T, Saelens X, et al. A universal Influenza A vaccine based on the extracellular domain of the M2 protein. Nat. Med, 1999,5(10):1157–1163.
[12]、De Filette M, Min Jou W, Birkett A, et al. Universal Influenza A vaccine: optimization of M2-based constructs. Virology, 2005, 337(1):149–161.
[13]、Eriksson AM, Schon KM, Lycke NY.The cholera toxin-derived CTA1-DD vaccine adjuvant administered intranasally does not cause inflammation or accumulate in the nervous tissues. J. Immunol, 2004, 173(5):3310–3319.
[14]、De Filette M, Fiers W, Martens W, et al. Improved design and intranasal delivery of an M2e-based human Influenza A vaccine. Vaccine, 2006, 24(44–46):6597–6601.
[15]、Frace AM, Klimov AI, Rowe T, et al. Modified M2 proteins produce heterotypic immunity against Influenza A virus. Vaccine, 1999, 17(18):2237–2244.
[16]、Jegerlehner A, Tissot A, Lechner F, et al. A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses. Vaccine, 2002, 20(25–26):3104–3112.
[17]、Fan J, Liang X, Horton MS, et al. Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys. Vaccine, 2004, 22(23–24): 2993–3003.
[18]、Jegerlehner A, Schmitz N, Storni T,et al. Influenza A vaccine based on the extracellular domain of M2: weak protection mediated via antibody-dependent NK cell activity. J. Immunol, 2004, 172(9):5598–5605
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