带有Tuftsin的分支状流感病毒M2e多肽疫苗的研究
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摘要
甲型流感病毒因其基因变异频繁、传播速度快、人群普遍易感、控制难度大等原因易发生世界范围的大流行,严重危害人类健康并影响社会经济发展。目前预防流感的主要措施仍是接种疫苗。目前应用的流感疫苗都是针对流感病毒膜蛋白HA和NA,通过诱导机体产生中和抗体发挥保护作用。但HA和NA频繁发生变异,每年都需要根据流行株的情况决定制备疫苗所用的毒株。因此,研发对不同亚型的甲型流感病毒株有交叉保护作用的通用疫苗是当前的研究热点之一。
M2蛋白是流感病毒表面的一种膜蛋白,由97个氨基酸组成。M2蛋白胞外区(M2e)的氨基酸序列高度保守,并且其抗血清有抑制流感病毒复制的功能,因而成为“通用流感疫苗”的重要候选抗原。但M2e仅含23个氨基酸,分子量小、免疫原性较差。分支状多抗原肽系统是利用赖氨酸(Lys)的两个氨基分别能与另一个氨基酸的羧基形成肽键这一特征,以数个Lys (n)作为核心,通过化学合成方法制备的大分子分支状多肽,可以使多肽分子的稳定性和免疫原性大大提高。
在分支状多肽分子的羧基端加上一段具有免疫活性的短肽,可以增强其免疫效果。Tuftsin就是这样一种免疫活性肽。它是IgG分子Fc段中的一个四肽片段。多种参与抗原呈递的细胞表面都有其受体,它自身的免疫原性极弱但能增强机体对与其连接的抗原肽的免疫反应。
本研究旨在结合并充分发挥分支状多抗原肽技术和Tuftsin的优势,研制针对流感病毒M2e蛋白的具有交叉保护效果的通用流感疫苗。本研究共包括以下四方面工作：
1.分支状多肽的理化性质及生物学活性鉴定
我们应用化学合成法得到分支状(M2e)4-Tuftsin的纯品,作为对照,我们又合成了用四个甘氨酸取代Tuftsin的分支状(M2e)4-G4以及单体M2e。得到纯品后,我们首先通过反相高效液相法检测合成肽的纯度,结果三种合成肽的纯度分别达到93-99%,可用于后续实验。用质谱法检测三种合成肽的分子量,M2e单体的理论值为2624.87,测得值为2625.77; (M2e)4-Tuftsin的理论值为11312.53,测得值为11558.00；(M2e)4-G4的理论值为11058.15测得值为11114.00,实测结果均与理论值相近似。表明合成的多肽与我们的设计相符。
应用SDS-PAGE进一步检测了分支状多肽的纯度与分子量,考马斯兰染色可在11kDa处看到明显条带,表明分支肽的分子量与理论值相似,说明其结构完整并与设计一致。用抗M2单克隆抗体做Western blot,结果显示上述条带可被抗M2抗体识别。将上述三种合成肽分别包被96孔板,ELISA检测结果表明三种多肽均能与抗M2单克隆抗体特异性结合。以上结果说明三种合成肽均具有甲型流感病毒M2蛋白的抗原性。
我们进而通过巨噬细胞结合实验检测与分支状多肽连接的Tuftsin能否促进其与巨噬细胞的结合。用荧光素FITC分别标记带有Tuftsin的分支肽(M2e)4-Tuftsin和对照肽(M2e)4-G4,然后将二者分别与小鼠腹腔巨噬细胞在4℃共同孵育。用1640培养液洗细胞两次后,在荧光显微镜下观察它们与巨噬细胞结合情况。FITC-(M2e)4-Tuftsin与小鼠腹腔巨噬细胞孵育后可见细胞表面有绿色荧光,说明前者能够与细胞表面的受体结合；而FITC-(M2e)4-G4孵育的细胞未见绿色荧光,说明对照分支肽不能与巨噬细胞结合。这一结果证实分支肽能借助Tuftsin与巨噬细胞表面的受体结合,有助于其被抗原呈递细胞处理。
2.分支状多肽疫苗肌肉注射免疫效果的研究
本研究进行了分支肽刺激体内免疫反应的研究。将6周龄雌性BALB/c小鼠随机分为四组：PBS组、M2e单体组、(M2e)4-Tuftsin组和(M2e)4-G4组。将PBS和三种合成肽分别与等体积氢氧化铝佐剂混合,肌肉注射接种小鼠。注射的合成肽总量为10μg/只。各组小鼠均免疫接种两次,每次免疫间隔两周。于每次免疫前1天和末次免疫后两周分别从小鼠眼眶后静脉采血,收集血清用于抗体检测。末次免疫后两周,用10×LD50流感病毒鼠肺适应株PR8株病毒攻击小鼠,每日观察各组小鼠的存活情况及体重变化,评价各种多肽的免疫保护效果。
(1)体液免疫水平评价用合成的M2e单体包被96孔ELISA板,采用间接ELISA方法检测不同免疫组小鼠免疫后血清中抗M2e IgG抗体效价。结果显示两次免疫后M2e单体组诱导的抗体滴度为10,而(M2e)4-Tuftsin和(M2e)4-G4组诱导产生的抗M2e抗体滴度分别为89144和29407,均显著比M2e单体组高,说明这两种分支状多肽都能诱导小鼠产生高水平抗M2e抗体,而其中(M2e)4-Tuftsin组诱导产生的抗体水平最高,说明与四分支M2e连接的Tuftsin确实有免疫增强作用。
(2)细胞免疫水平评价采用ELISPOT方法检测免疫后小鼠脾细胞中特异性产生γ干扰素的细胞数,间接反映Thl细胞的特异性反应功能。于末次免疫后第12天,取小鼠脾脏分离淋巴细胞。ELISPOT试验按照BD公司ELISPOT试剂盒说明书操作,用合成的M2e单体刺激各组小鼠脾细胞。结果显示,在M2e刺激下,PBS组平均每1×106细胞产生6个斑点,M2e单体组产生46个斑点,(M2e)4-Tuftsin组产生254个斑点,(M2e)4-G4组产生156个斑点,说明三种合成肽均能诱导小鼠产生针对M2e的特异性细胞免疫,其中分支肽免疫组明显高于M2e单体组,而(M2e)4-Tuftsin组诱导细胞免疫的能力最强。
(3)保护性评价流感病毒攻击实验结果显示,至攻毒后14天,PBS组存活率为10%,M2e单体组存活率为30%,分支状(M2e)4-G4组存活率为44%,而接种分支状(M2e)4-Tuftsin组小鼠的存活率达到80%。说明两种分支状多肽均能在一定程度上缓解小鼠由于病毒感染引发的症状,并显著降低小鼠死亡率。与体液免疫和细胞免疫结果相一致,其中(M2e)4-Tuftsin的免疫保护效果最好。
3.分支状多肽疫苗滴鼻接种免疫效果的研究
由于流感病毒是通过呼吸道感染,呼吸道的粘膜免疫会有重要的保护作用。肌肉注射免疫主要刺激产生血清抗体,不产生分泌型IgA。故而我们尝试通过鼻腔粘膜进行免疫。由于单独用合成肽免疫的效果不好,所以需要加上合适的佐剂。JY佐剂是将带有正电荷的壳聚糖与带有负电荷的具有免疫调节活性的白细胞介素-2(IL-2)所组成的复方。我们将JY佐剂与合成的几种多肽分别混合,滴鼻接种小鼠,观察其粘膜免疫效果。
将6周龄雌性BALB/c小鼠随机分为四组：PBS组、M2e单体组、(M2e)4-Tuftsin组和(M2e)4-G4组。将PBS和三种合成肽分别与等体积2×JY佐剂混合。先用戊巴比妥钠腹腔注射麻醉小鼠,待小鼠明显无力后,向小鼠鼻腔滴加10u 1候选疫苗(或PBS),接种多肽总量为10μg/只。各组小鼠均免疫三次,每次免疫间隔两周。于每次免疫前1天和末次免疫后两周分别从小鼠眼眶后静脉采血,用于血清抗体检测。末次免疫后两周,用10×LD50的PR8株流感病毒攻击小鼠,每日观察各组小鼠的存活情况及体重变化,以评价各组的粘膜免疫保护效果。
(1)体液免疫水平评价用合成的M2e单体包被96孔ELISA板,采用间接ELISA方法检测不同免疫组小鼠免疫后血清中抗M2e IgG抗体效价。ELISA检测结果显示,末次免疫后两周M2e单体、(M2e)4-Tuftsin和(M2e)4-G4诱导产生的抗M2e抗体滴度分别为8、4526和3592,两种分支状多肽诱导小鼠产生的抗体滴度均明显高于M2e单体,其中分支状(M2e)4-Tuftsin诱导产生的抗体水平最高,与肌肉注射途径免疫的结果一致。但滴鼻免疫小鼠的血清IgG抗体滴度远低于肌肉注射途径诱导的抗体滴度,分析原因可能是由于鼻腔免疫的作用机理与肌肉注射不同,它主要是诱导局部粘膜免疫而不是全身的体液免疫。
(2)细胞免疫水平评价采用ELISPOT方法检测免疫后小鼠脾细胞中特异性产生γ干扰素的细胞数,间接反映Thl细胞的特异性反应功能。于末次免疫后第12天,取小鼠脾脏分离淋巴细胞。ELISPOT试验按照BD公司ELISPOT试剂盒说明书操作,用合成的M2e单体刺激各组小鼠脾细胞。ELISPOT结果显示,在M2e单体刺激下,PBS组平均每1×106细胞产生22个斑点,M2e单体产生49个斑点,分支肽(M2e)4-Tuftsin组产生371个斑点,对照分支肽(M2e)4-G4组产生112个斑点,说明分支状M2e鼻腔免疫后诱导小鼠产生的细胞免疫水平显著高于M2e单体组,而(M2e)4-Tuftsin诱导细胞免疫的能力最强。
(3)保护性评价流感病毒攻击小鼠14天后PBS组小鼠全部死亡,而M2e单体组、(M2e)4-Tuftsin组和(M2e)4-G4组存活率均为40%,说明三种多肽鼻腔免疫均有一定免疫保护作用,但保护效果较肌肉注射途径弱。
上述结果证实,分支状多肽技术大大增强了短肽抗原的免疫原性,而带有Tuftsin的分支状多肽免疫效果最好,是一种值得开发和推广的新型疫苗制备技术。
4.可调控表达甲型流感病毒M2蛋白的哺乳动物细胞系的建立与鉴定
鉴于M2蛋白表达系统在研发与M2蛋白相关的流感通用疫苗中的重要作用,本研究构建了可调控表达M2蛋白的哺乳动物细胞系。首先应用PCR方法从含有流感病毒PR8株第七节段全长基因的质粒中扩增得到M2基因。将该片段亚克隆到真核表达载体pcDNA5/FRT/TO上,用BamHⅠ和NotⅠ双酶切鉴定正确后将重组质粒与表达Flp重组酶的pOG44质粒共转染Flp-In T-REx-293细胞,使目的基因整合到宿主细胞染色体。筛选具有Hygromycin B抗性的细胞株。在该细胞的培养基中加入四环素以诱导目的基因表达,48 h后通过间接免疫荧光方法检测到M2蛋白的表达。共得到16株高表达M2蛋白的重组细胞株,这些细胞株在传十代后仍能稳定表达目的蛋白。未加四环素诱导的细胞没有检测到M2蛋白,说明四环素调控系统严格控制着目的基因的表达。今后,该细胞系可用于流感病毒M2蛋白的功能研究、流感候选疫苗的免疫学评价以及流感病毒减毒活疫苗的研制。综上所述,本研究结果表明分支状多肽比单体短肽的免疫原性大大增强,而带有免疫活性肽Tuftsin的分支肽刺激免疫反应的效果最好。本文提供了一种非常有希望的新型疫苗制备技术,为进一步研发基于M2蛋白的广谱流感疫苗奠定了基础。
Influenza has become a serious global health threat. Vaccination is the most effective means for preventing influenza-associated morbidity and mortality. Since the HA, NA genes of influenza virus mutate frequently.The virus strains for new vaccine production should be changed according to predicted epidemic strains. Recent research focused on the conserved epitopes of the influenza virus proteins, which may protect against a much wider range of virus strains when used as vaccines.
Matrix protein 2 (M2) is a 97 aa-long transmembrane protein of IAV. The extracellular domain of M2 (M2e) is 23 amino acids long. The M2e is highly conserved and M2e-specific Abs has shown to display significant protective activity in animal models. There has been growing interest in M2 as a "universal" vaccine that may protect against a much wider range of IAVs than current vaccines.
In view of the relatively small size of M2e (23aa), most efforts to produce an M2e vaccine have made use of chemical or genetic M2e fusion constructs to a variety of carriers. While one drawback of the fusion protein is that owing to adding irrelevant protein, the composition and structure of them are ambiguous.
In 1988 James Tam first reported the multiple antigen peptide system, which is based on a small immunologically inert core molecule of radially branching lysine dendrites onto which a number of peptide antigens are anchored. The MAP system does not require a carrier protein for conjugation. The high molar ratio and dense packing of multiple copies of the antigenic epitope in a MAP has shown to produce strong immunogenic response.
Tuftsin is a tetrapeptide (Thr-Lys-Pro-Arg) produced by enzymatic cleavage of the Fc-domain of the heavy chain of IgG. It can be recognized by specific receptors on macrophages, and is capable of targeting protein and peptides to these sites. Tuftsin has been one of the promising carrier due to its immunomodulatory role in the immune system. Several studies indicated that conjugates with tuftsin increase the epitope specific antibody production.
In this study we formulated a novel construction of influenza vaccine based on M2e protein. The research is composed of four parts:
1. Appraisal of physicochemical and biological properties of synthetic peptides
The M2e peptide, (M2e)4-Tuftsin and (M2e)4-G4 peptides were synthesized using the standard solid-phase synthesizing methods. These peptides were analysed by-RP-HPLC, showing their purity were 93%,99% and 98% respectively. MALDI-TOF-MS spectrum of M2e peptide revealed the component with estimated masses of 2625.77(single M2e),11,558 Da for (M2e)4-Tuftsin and 11,114 Da for (M2e)4-G4.These results strongly suggest that the synthetic peptides are in accordance with the expected composition. Western blot and ELISA results showed that the synthetic peptides could recognize by M2 mAb specifically.
The branched peptides were labelled with FITC, the FITC-(M2e)4-Tuftsin was incubated with mouse peritoneal macrophage (MPM) at 4℃.The green fluorescent could be seen on the surface of MPM. However, this phenomenon was not seen when FITC-(M2e)4-G4 was incubated with MPM. This indicated that the Tuftsin attached to M2e MAP contributes to the binding and capturing of peptide by macrophage.
2. Immunostimulation and protection of mice by intramuscular vaccination with synthetic peptides
Six week old female BALB/c mice were devided into four groups, each group immunized intramuscularly (i.m.) with 10μg of one of synthetic peptides or PBS plus aluminum adjuvant, boosted at 2 week intervals. Two weeks post final immunization, anesthetized mice were challenged intranasally (i.n.) with 10 X LD50 of influenza virus PR8 strain. The mice were monitored for body weight and death for 14 days.
M2e-specific IgG antibodies were determined at 2 weeks after boost immunization by ELISA using the M2e peptide as a coating antigen. The M2e-specific IgG titers in (M2e)4-Tuftsin and (M2e)4-G4 groups were 89144 and 29407 respectively, which were significantly higher than that of M2e peptide group(10), and the antibody levels between the two M2e MAP groups were also significantly different from each other.These results indicate that the (M2e)4-Tuftsin induce highest level of M2 specific antibody.
The enzyme-linked immunospot (ELISPOT) assays were performed using commercial ELISPOT set (BD Biosciences). IFN-γproducing cell spots were detected in all groups, and the spot numbers were 46,254 and 156 respectively. As control, PBS plus adjuvant could not induce T cell response. The spot numbers of two M2e MAP groups were both more than M2e group. Furthermore, spot numbers of IFN-γsecreting cells were higher in (M2e)4-Tuftsin group than in (M2e)4-G4 group. These results provide evidence that (M2e)4-Tuftsin was the most effective in stimulating T cell responses.
Forteen days after challenge with influenza virus, mice in PBS group were not protected (10% survival). Survival rates of M2e group and (M2e)4-G4 group were 30% and 44% respectively, which were significantly lower than (M2e)4-Tuftsin group (80% survival).These results clearly demonstrate that the (M2e)4-Tuftsin could provide the most efficient protective immunity among synthetic peptides.
3. Immunostimulation and protection of mice by intranasal vaccination with synthetic peptides
Six week old female BALB/c mice were devided into four groups, each group were immunized intranasally (i.m.) with 10μg of one of synthetic peptides plus JY adjuvant, boosted twice at 2 week intervals. JY adjuvant is the compound of positively charged chitosan and negatively charged IL-2.Two weeks post final immunization, anesthetized mice were challenged intranasally (i.n.) with 10×LD50 of influenza virus PR8 strain.The mice were monitored for body weight and death for 14 days.
M2e-specific IgG antibodies were determined by ELISA using the M2e peptide as a coating antigen. After final immunization, the M2e-specific IgG titers in (M2e)4-Tuftsin and (M2e)4-G4 groups were 4526 and 3592 respectively, which were significantly higher than that in the M2e peptide group(8). These results indicate that the (M2e)4-Tuftsin induce highest level of M2 specific antibody. Because intranasal immunization mainly induced local mucosal immunity, the IgG titers of mice immunized by intramuscular injection were much lower than that of mice immunized by intramuscular injection.
Measurement of T cell responses by ELISPOT.IFN-Y producing cell spots were detected in all PBS, M2e, (M2e)4-Tuftsin and (M2e)4-G4 groups, and the spot numbers were 22,49,371 and 112 respectively. The spot numbers of two M2e MAP group were both more than M2e peptide. Furthermore, spot numbers of IFN-γsecreting cells were higher in (M2e)4-Tuftsin group than in (M2e)4-G4 group. These results provide evidence that (M2e)4-Tuftsin was the most effective in stimulating T cell responses.
Four day after challenging with influenza virus, mice in PBS group were not protected (0% survival).Survival rates of M2e group, (M2e)4-Tuftsin group and (M2e)4-G4 group were all 40%. The results revealed that all these synthetic peptides had protective effect, but the protective effect of intranasal immunization was much lower than intramuscular immunization.
4. Establishment of a stable and inducible mammalian cell line expressing influenza virus A M2 protein
By insertion of M2 gene into mammalian cell expression vectors and transfed into a mammalian cell line, we establish a stable 293 cell line that express M2 protein under the control of the tetracycline operator. The expression of M2 protein from hygromycin -resistant cell was induced by addition of tetracycline into the cell culture media, and then tested by indirect immunofluorescence assay.16 strains with high expression of M2 were selected. After subculturing for more than ten passages, the cell lines still stably expressed M2 protein. No M2 protein could be detected without tetracycline induction, suggesting that the expression was strictly controlled by tetracycline operator.
The cell lines expressing M2 will be useful for further functional studies of M2 protein, detection of immune response against natural structure M2 protein and development of live attenuated influenza virus vaccine with reverse genetics technique.
In summary, the three kinds peptides including M2e,(M2e)4-Tuftsin and (M2e)4-G4 were synthesized using the standard solid-phase methods. The purity and integrity of synthetic peptides was confirmed by HPLC and MS. The accuracy and antigenicity of synthetic peptides were analysed by SDS-PAGE, Western blot and ELISA. The rusults showed that the structure of synthetic peptides were inconsistent with design. The peptides mixed with aluminum adjuvant or JY adjuvant respectively, and were administered to BALB/c mice by intramuscular or intranasal route.The ability of induction of humoral and cellular immune response in (M2e)4-Tuftsin and (M2e)4-G4 group were greater than that in M2e group, and this ability in (M2e)4-Tuftsin group were greater than (M2e)4-G4 group. Coincidently, the protective effect in (M2e)4-Tuftsin and (M2e)4-G4 group were superior to that in the M2e group, and the protective effect in (M2e)4-Tuftsin group were greater than (M2e)4-G4 group. When comparing the two immunization approach, the protective effects of intramuscular immunization were superior to intranasal immunization.
This work has built a solid foundation for development of branched M2e peptide as a novel influenza vaccine.
M2蛋白是流感病毒表面的一种膜蛋白,由97个氨基酸组成。M2蛋白胞外区(M2e)的氨基酸序列高度保守,并且其抗血清有抑制流感病毒复制的功能,因而成为“通用流感疫苗”的重要候选抗原。但M2e仅含23个氨基酸,分子量小、免疫原性较差。分支状多抗原肽系统是利用赖氨酸(Lys)的两个氨基分别能与另一个氨基酸的羧基形成肽键这一特征,以数个Lys (n)作为核心,通过化学合成方法制备的大分子分支状多肽,可以使多肽分子的稳定性和免疫原性大大提高。
在分支状多肽分子的羧基端加上一段具有免疫活性的短肽,可以增强其免疫效果。Tuftsin就是这样一种免疫活性肽。它是IgG分子Fc段中的一个四肽片段。多种参与抗原呈递的细胞表面都有其受体,它自身的免疫原性极弱但能增强机体对与其连接的抗原肽的免疫反应。
本研究旨在结合并充分发挥分支状多抗原肽技术和Tuftsin的优势,研制针对流感病毒M2e蛋白的具有交叉保护效果的通用流感疫苗。本研究共包括以下四方面工作：
1.分支状多肽的理化性质及生物学活性鉴定
我们应用化学合成法得到分支状(M2e)4-Tuftsin的纯品,作为对照,我们又合成了用四个甘氨酸取代Tuftsin的分支状(M2e)4-G4以及单体M2e。得到纯品后,我们首先通过反相高效液相法检测合成肽的纯度,结果三种合成肽的纯度分别达到93-99%,可用于后续实验。用质谱法检测三种合成肽的分子量,M2e单体的理论值为2624.87,测得值为2625.77; (M2e)4-Tuftsin的理论值为11312.53,测得值为11558.00；(M2e)4-G4的理论值为11058.15测得值为11114.00,实测结果均与理论值相近似。表明合成的多肽与我们的设计相符。
应用SDS-PAGE进一步检测了分支状多肽的纯度与分子量,考马斯兰染色可在11kDa处看到明显条带,表明分支肽的分子量与理论值相似,说明其结构完整并与设计一致。用抗M2单克隆抗体做Western blot,结果显示上述条带可被抗M2抗体识别。将上述三种合成肽分别包被96孔板,ELISA检测结果表明三种多肽均能与抗M2单克隆抗体特异性结合。以上结果说明三种合成肽均具有甲型流感病毒M2蛋白的抗原性。
我们进而通过巨噬细胞结合实验检测与分支状多肽连接的Tuftsin能否促进其与巨噬细胞的结合。用荧光素FITC分别标记带有Tuftsin的分支肽(M2e)4-Tuftsin和对照肽(M2e)4-G4,然后将二者分别与小鼠腹腔巨噬细胞在4℃共同孵育。用1640培养液洗细胞两次后,在荧光显微镜下观察它们与巨噬细胞结合情况。FITC-(M2e)4-Tuftsin与小鼠腹腔巨噬细胞孵育后可见细胞表面有绿色荧光,说明前者能够与细胞表面的受体结合；而FITC-(M2e)4-G4孵育的细胞未见绿色荧光,说明对照分支肽不能与巨噬细胞结合。这一结果证实分支肽能借助Tuftsin与巨噬细胞表面的受体结合,有助于其被抗原呈递细胞处理。
2.分支状多肽疫苗肌肉注射免疫效果的研究
本研究进行了分支肽刺激体内免疫反应的研究。将6周龄雌性BALB/c小鼠随机分为四组：PBS组、M2e单体组、(M2e)4-Tuftsin组和(M2e)4-G4组。将PBS和三种合成肽分别与等体积氢氧化铝佐剂混合,肌肉注射接种小鼠。注射的合成肽总量为10μg/只。各组小鼠均免疫接种两次,每次免疫间隔两周。于每次免疫前1天和末次免疫后两周分别从小鼠眼眶后静脉采血,收集血清用于抗体检测。末次免疫后两周,用10×LD50流感病毒鼠肺适应株PR8株病毒攻击小鼠,每日观察各组小鼠的存活情况及体重变化,评价各种多肽的免疫保护效果。
(1)体液免疫水平评价用合成的M2e单体包被96孔ELISA板,采用间接ELISA方法检测不同免疫组小鼠免疫后血清中抗M2e IgG抗体效价。结果显示两次免疫后M2e单体组诱导的抗体滴度为10,而(M2e)4-Tuftsin和(M2e)4-G4组诱导产生的抗M2e抗体滴度分别为89144和29407,均显著比M2e单体组高,说明这两种分支状多肽都能诱导小鼠产生高水平抗M2e抗体,而其中(M2e)4-Tuftsin组诱导产生的抗体水平最高,说明与四分支M2e连接的Tuftsin确实有免疫增强作用。
(2)细胞免疫水平评价采用ELISPOT方法检测免疫后小鼠脾细胞中特异性产生γ干扰素的细胞数,间接反映Thl细胞的特异性反应功能。于末次免疫后第12天,取小鼠脾脏分离淋巴细胞。ELISPOT试验按照BD公司ELISPOT试剂盒说明书操作,用合成的M2e单体刺激各组小鼠脾细胞。结果显示,在M2e刺激下,PBS组平均每1×106细胞产生6个斑点,M2e单体组产生46个斑点,(M2e)4-Tuftsin组产生254个斑点,(M2e)4-G4组产生156个斑点,说明三种合成肽均能诱导小鼠产生针对M2e的特异性细胞免疫,其中分支肽免疫组明显高于M2e单体组,而(M2e)4-Tuftsin组诱导细胞免疫的能力最强。
(3)保护性评价流感病毒攻击实验结果显示,至攻毒后14天,PBS组存活率为10%,M2e单体组存活率为30%,分支状(M2e)4-G4组存活率为44%,而接种分支状(M2e)4-Tuftsin组小鼠的存活率达到80%。说明两种分支状多肽均能在一定程度上缓解小鼠由于病毒感染引发的症状,并显著降低小鼠死亡率。与体液免疫和细胞免疫结果相一致,其中(M2e)4-Tuftsin的免疫保护效果最好。
3.分支状多肽疫苗滴鼻接种免疫效果的研究
由于流感病毒是通过呼吸道感染,呼吸道的粘膜免疫会有重要的保护作用。肌肉注射免疫主要刺激产生血清抗体,不产生分泌型IgA。故而我们尝试通过鼻腔粘膜进行免疫。由于单独用合成肽免疫的效果不好,所以需要加上合适的佐剂。JY佐剂是将带有正电荷的壳聚糖与带有负电荷的具有免疫调节活性的白细胞介素-2(IL-2)所组成的复方。我们将JY佐剂与合成的几种多肽分别混合,滴鼻接种小鼠,观察其粘膜免疫效果。
将6周龄雌性BALB/c小鼠随机分为四组：PBS组、M2e单体组、(M2e)4-Tuftsin组和(M2e)4-G4组。将PBS和三种合成肽分别与等体积2×JY佐剂混合。先用戊巴比妥钠腹腔注射麻醉小鼠,待小鼠明显无力后,向小鼠鼻腔滴加10u 1候选疫苗(或PBS),接种多肽总量为10μg/只。各组小鼠均免疫三次,每次免疫间隔两周。于每次免疫前1天和末次免疫后两周分别从小鼠眼眶后静脉采血,用于血清抗体检测。末次免疫后两周,用10×LD50的PR8株流感病毒攻击小鼠,每日观察各组小鼠的存活情况及体重变化,以评价各组的粘膜免疫保护效果。
(1)体液免疫水平评价用合成的M2e单体包被96孔ELISA板,采用间接ELISA方法检测不同免疫组小鼠免疫后血清中抗M2e IgG抗体效价。ELISA检测结果显示,末次免疫后两周M2e单体、(M2e)4-Tuftsin和(M2e)4-G4诱导产生的抗M2e抗体滴度分别为8、4526和3592,两种分支状多肽诱导小鼠产生的抗体滴度均明显高于M2e单体,其中分支状(M2e)4-Tuftsin诱导产生的抗体水平最高,与肌肉注射途径免疫的结果一致。但滴鼻免疫小鼠的血清IgG抗体滴度远低于肌肉注射途径诱导的抗体滴度,分析原因可能是由于鼻腔免疫的作用机理与肌肉注射不同,它主要是诱导局部粘膜免疫而不是全身的体液免疫。
(2)细胞免疫水平评价采用ELISPOT方法检测免疫后小鼠脾细胞中特异性产生γ干扰素的细胞数,间接反映Thl细胞的特异性反应功能。于末次免疫后第12天,取小鼠脾脏分离淋巴细胞。ELISPOT试验按照BD公司ELISPOT试剂盒说明书操作,用合成的M2e单体刺激各组小鼠脾细胞。ELISPOT结果显示,在M2e单体刺激下,PBS组平均每1×106细胞产生22个斑点,M2e单体产生49个斑点,分支肽(M2e)4-Tuftsin组产生371个斑点,对照分支肽(M2e)4-G4组产生112个斑点,说明分支状M2e鼻腔免疫后诱导小鼠产生的细胞免疫水平显著高于M2e单体组,而(M2e)4-Tuftsin诱导细胞免疫的能力最强。
(3)保护性评价流感病毒攻击小鼠14天后PBS组小鼠全部死亡,而M2e单体组、(M2e)4-Tuftsin组和(M2e)4-G4组存活率均为40%,说明三种多肽鼻腔免疫均有一定免疫保护作用,但保护效果较肌肉注射途径弱。
上述结果证实,分支状多肽技术大大增强了短肽抗原的免疫原性,而带有Tuftsin的分支状多肽免疫效果最好,是一种值得开发和推广的新型疫苗制备技术。
4.可调控表达甲型流感病毒M2蛋白的哺乳动物细胞系的建立与鉴定
鉴于M2蛋白表达系统在研发与M2蛋白相关的流感通用疫苗中的重要作用,本研究构建了可调控表达M2蛋白的哺乳动物细胞系。首先应用PCR方法从含有流感病毒PR8株第七节段全长基因的质粒中扩增得到M2基因。将该片段亚克隆到真核表达载体pcDNA5/FRT/TO上,用BamHⅠ和NotⅠ双酶切鉴定正确后将重组质粒与表达Flp重组酶的pOG44质粒共转染Flp-In T-REx-293细胞,使目的基因整合到宿主细胞染色体。筛选具有Hygromycin B抗性的细胞株。在该细胞的培养基中加入四环素以诱导目的基因表达,48 h后通过间接免疫荧光方法检测到M2蛋白的表达。共得到16株高表达M2蛋白的重组细胞株,这些细胞株在传十代后仍能稳定表达目的蛋白。未加四环素诱导的细胞没有检测到M2蛋白,说明四环素调控系统严格控制着目的基因的表达。今后,该细胞系可用于流感病毒M2蛋白的功能研究、流感候选疫苗的免疫学评价以及流感病毒减毒活疫苗的研制。综上所述,本研究结果表明分支状多肽比单体短肽的免疫原性大大增强,而带有免疫活性肽Tuftsin的分支肽刺激免疫反应的效果最好。本文提供了一种非常有希望的新型疫苗制备技术,为进一步研发基于M2蛋白的广谱流感疫苗奠定了基础。
Influenza has become a serious global health threat. Vaccination is the most effective means for preventing influenza-associated morbidity and mortality. Since the HA, NA genes of influenza virus mutate frequently.The virus strains for new vaccine production should be changed according to predicted epidemic strains. Recent research focused on the conserved epitopes of the influenza virus proteins, which may protect against a much wider range of virus strains when used as vaccines.
Matrix protein 2 (M2) is a 97 aa-long transmembrane protein of IAV. The extracellular domain of M2 (M2e) is 23 amino acids long. The M2e is highly conserved and M2e-specific Abs has shown to display significant protective activity in animal models. There has been growing interest in M2 as a "universal" vaccine that may protect against a much wider range of IAVs than current vaccines.
In view of the relatively small size of M2e (23aa), most efforts to produce an M2e vaccine have made use of chemical or genetic M2e fusion constructs to a variety of carriers. While one drawback of the fusion protein is that owing to adding irrelevant protein, the composition and structure of them are ambiguous.
In 1988 James Tam first reported the multiple antigen peptide system, which is based on a small immunologically inert core molecule of radially branching lysine dendrites onto which a number of peptide antigens are anchored. The MAP system does not require a carrier protein for conjugation. The high molar ratio and dense packing of multiple copies of the antigenic epitope in a MAP has shown to produce strong immunogenic response.
Tuftsin is a tetrapeptide (Thr-Lys-Pro-Arg) produced by enzymatic cleavage of the Fc-domain of the heavy chain of IgG. It can be recognized by specific receptors on macrophages, and is capable of targeting protein and peptides to these sites. Tuftsin has been one of the promising carrier due to its immunomodulatory role in the immune system. Several studies indicated that conjugates with tuftsin increase the epitope specific antibody production.
In this study we formulated a novel construction of influenza vaccine based on M2e protein. The research is composed of four parts:
1. Appraisal of physicochemical and biological properties of synthetic peptides
The M2e peptide, (M2e)4-Tuftsin and (M2e)4-G4 peptides were synthesized using the standard solid-phase synthesizing methods. These peptides were analysed by-RP-HPLC, showing their purity were 93%,99% and 98% respectively. MALDI-TOF-MS spectrum of M2e peptide revealed the component with estimated masses of 2625.77(single M2e),11,558 Da for (M2e)4-Tuftsin and 11,114 Da for (M2e)4-G4.These results strongly suggest that the synthetic peptides are in accordance with the expected composition. Western blot and ELISA results showed that the synthetic peptides could recognize by M2 mAb specifically.
The branched peptides were labelled with FITC, the FITC-(M2e)4-Tuftsin was incubated with mouse peritoneal macrophage (MPM) at 4℃.The green fluorescent could be seen on the surface of MPM. However, this phenomenon was not seen when FITC-(M2e)4-G4 was incubated with MPM. This indicated that the Tuftsin attached to M2e MAP contributes to the binding and capturing of peptide by macrophage.
2. Immunostimulation and protection of mice by intramuscular vaccination with synthetic peptides
Six week old female BALB/c mice were devided into four groups, each group immunized intramuscularly (i.m.) with 10μg of one of synthetic peptides or PBS plus aluminum adjuvant, boosted at 2 week intervals. Two weeks post final immunization, anesthetized mice were challenged intranasally (i.n.) with 10 X LD50 of influenza virus PR8 strain. The mice were monitored for body weight and death for 14 days.
M2e-specific IgG antibodies were determined at 2 weeks after boost immunization by ELISA using the M2e peptide as a coating antigen. The M2e-specific IgG titers in (M2e)4-Tuftsin and (M2e)4-G4 groups were 89144 and 29407 respectively, which were significantly higher than that of M2e peptide group(10), and the antibody levels between the two M2e MAP groups were also significantly different from each other.These results indicate that the (M2e)4-Tuftsin induce highest level of M2 specific antibody.
The enzyme-linked immunospot (ELISPOT) assays were performed using commercial ELISPOT set (BD Biosciences). IFN-γproducing cell spots were detected in all groups, and the spot numbers were 46,254 and 156 respectively. As control, PBS plus adjuvant could not induce T cell response. The spot numbers of two M2e MAP groups were both more than M2e group. Furthermore, spot numbers of IFN-γsecreting cells were higher in (M2e)4-Tuftsin group than in (M2e)4-G4 group. These results provide evidence that (M2e)4-Tuftsin was the most effective in stimulating T cell responses.
Forteen days after challenge with influenza virus, mice in PBS group were not protected (10% survival). Survival rates of M2e group and (M2e)4-G4 group were 30% and 44% respectively, which were significantly lower than (M2e)4-Tuftsin group (80% survival).These results clearly demonstrate that the (M2e)4-Tuftsin could provide the most efficient protective immunity among synthetic peptides.
3. Immunostimulation and protection of mice by intranasal vaccination with synthetic peptides
Six week old female BALB/c mice were devided into four groups, each group were immunized intranasally (i.m.) with 10μg of one of synthetic peptides plus JY adjuvant, boosted twice at 2 week intervals. JY adjuvant is the compound of positively charged chitosan and negatively charged IL-2.Two weeks post final immunization, anesthetized mice were challenged intranasally (i.n.) with 10×LD50 of influenza virus PR8 strain.The mice were monitored for body weight and death for 14 days.
M2e-specific IgG antibodies were determined by ELISA using the M2e peptide as a coating antigen. After final immunization, the M2e-specific IgG titers in (M2e)4-Tuftsin and (M2e)4-G4 groups were 4526 and 3592 respectively, which were significantly higher than that in the M2e peptide group(8). These results indicate that the (M2e)4-Tuftsin induce highest level of M2 specific antibody. Because intranasal immunization mainly induced local mucosal immunity, the IgG titers of mice immunized by intramuscular injection were much lower than that of mice immunized by intramuscular injection.
Measurement of T cell responses by ELISPOT.IFN-Y producing cell spots were detected in all PBS, M2e, (M2e)4-Tuftsin and (M2e)4-G4 groups, and the spot numbers were 22,49,371 and 112 respectively. The spot numbers of two M2e MAP group were both more than M2e peptide. Furthermore, spot numbers of IFN-γsecreting cells were higher in (M2e)4-Tuftsin group than in (M2e)4-G4 group. These results provide evidence that (M2e)4-Tuftsin was the most effective in stimulating T cell responses.
Four day after challenging with influenza virus, mice in PBS group were not protected (0% survival).Survival rates of M2e group, (M2e)4-Tuftsin group and (M2e)4-G4 group were all 40%. The results revealed that all these synthetic peptides had protective effect, but the protective effect of intranasal immunization was much lower than intramuscular immunization.
4. Establishment of a stable and inducible mammalian cell line expressing influenza virus A M2 protein
By insertion of M2 gene into mammalian cell expression vectors and transfed into a mammalian cell line, we establish a stable 293 cell line that express M2 protein under the control of the tetracycline operator. The expression of M2 protein from hygromycin -resistant cell was induced by addition of tetracycline into the cell culture media, and then tested by indirect immunofluorescence assay.16 strains with high expression of M2 were selected. After subculturing for more than ten passages, the cell lines still stably expressed M2 protein. No M2 protein could be detected without tetracycline induction, suggesting that the expression was strictly controlled by tetracycline operator.
The cell lines expressing M2 will be useful for further functional studies of M2 protein, detection of immune response against natural structure M2 protein and development of live attenuated influenza virus vaccine with reverse genetics technique.
In summary, the three kinds peptides including M2e,(M2e)4-Tuftsin and (M2e)4-G4 were synthesized using the standard solid-phase methods. The purity and integrity of synthetic peptides was confirmed by HPLC and MS. The accuracy and antigenicity of synthetic peptides were analysed by SDS-PAGE, Western blot and ELISA. The rusults showed that the structure of synthetic peptides were inconsistent with design. The peptides mixed with aluminum adjuvant or JY adjuvant respectively, and were administered to BALB/c mice by intramuscular or intranasal route.The ability of induction of humoral and cellular immune response in (M2e)4-Tuftsin and (M2e)4-G4 group were greater than that in M2e group, and this ability in (M2e)4-Tuftsin group were greater than (M2e)4-G4 group. Coincidently, the protective effect in (M2e)4-Tuftsin and (M2e)4-G4 group were superior to that in the M2e group, and the protective effect in (M2e)4-Tuftsin group were greater than (M2e)4-G4 group. When comparing the two immunization approach, the protective effects of intramuscular immunization were superior to intranasal immunization.
This work has built a solid foundation for development of branched M2e peptide as a novel influenza vaccine.
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