H5N1亞型禽流感病毒樣顆粒構(gòu)建、優(yōu)化與實(shí)驗(yàn)免疫研究
發(fā)布時(shí)間：2019-06-16 19:26
【摘要】：自2003年起,高致病性H5N1亞型流感病毒感染人的事件不斷發(fā)生,并且衍生出新的基因譜系,其中,2.3.2.1和2.3.4譜系流行最為廣泛。高致病性H5N1病毒存在大規(guī)模流行的可能性是被大家所公認(rèn)的,需要對存在流行風(fēng)險(xiǎn)的多個(gè)譜系禽流感病毒進(jìn)行疫苗的研究。目前廣泛應(yīng)用的滅活疫苗只能針對同譜系病毒感染提供免疫保護(hù),利用桿狀病毒表達(dá)系統(tǒng)生產(chǎn)的無感染性的病毒樣顆粒疫苗,因生產(chǎn)成本低、無生物安全風(fēng)險(xiǎn)等優(yōu)勢受到廣泛關(guān)注,特別是因其具有誘導(dǎo)細(xì)胞免疫能力可能針對同型的異源病毒提供免疫保護(hù),而可能用于變異頻繁的流感病毒疫苗研究。本研究的目的是應(yīng)用昆蟲桿狀病毒表達(dá)系統(tǒng),通過表達(dá)模式篩選、佐劑效應(yīng)蛋白偶聯(lián)等系列優(yōu)化研究,研制H5N1病毒樣顆粒,并評估對異源病毒感染的免疫保護(hù)作用。首先,利用昆蟲桿狀病毒表達(dá)系統(tǒng)研制了包含H5N1 A/meerkat/Shanghai/SH-1 2012 (clade 2.3.2.1) HA、NA和M1蛋白的病毒樣顆粒。通過系列試驗(yàn)鑒定證明,包含HA、NA和M1蛋白的H5N1病毒樣顆粒與天然流感病毒具有相似的形態(tài)結(jié)構(gòu)和大小,并且具有血凝活性。與全病毒滅活疫苗相比,小鼠通過免疫病毒樣顆粒疫苗能夠誘導(dǎo)產(chǎn)生更強(qiáng)的體液免疫和細(xì)胞免疫,在加強(qiáng)免疫后,可以產(chǎn)生針對A/meerkat/Shanghai/SH-1/2012特異性IgG,其滴度為全病毒滅活組的20倍。接下來的攻毒結(jié)果表明,全病毒滅活組表現(xiàn)出對同源病毒80%的保護(hù)率,對異源病毒(A/duck/Jilin/JL-SIV/2013, clade 2.3.4)的保護(hù)率只有40%,而病毒樣顆粒免疫組小鼠則完全存活,保護(hù)率均達(dá)到100%。結(jié)果顯示,流感病毒樣顆粒疫苗可以作為一種具有交叉保護(hù)活性的流感疫苗能夠控制高致病H5N1流感病毒的爆發(fā)。為進(jìn)一步提高病毒樣顆粒的免疫原性,我們在疫苗中增加具有免疫佐劑活性的蛋白以期增強(qiáng)疫苗誘導(dǎo)的適應(yīng)性免疫反應(yīng)水平。以膜錨定形式構(gòu)建了包含大腸桿菌不耐熱腸毒素B亞基(LTB),鞭毛蛋白(Toll樣受體5的配體,Flic)和粒細(xì)胞-巨噬細(xì)胞集落刺激因子(GM-CSF)的嵌合病毒樣顆粒。鑒定表明,LTB-、Flic-和GM-CSF-VLPs病毒樣顆粒包含所特有的蛋白,與天然流感病毒具有相似的形態(tài)結(jié)構(gòu)和大小,并且具有血凝活性。與未偶聯(lián)免疫刺激蛋白病毒樣顆粒疫苗相比,LTB-、 Flic-和GM-CSF-VLPs病毒樣顆粒疫苗能夠誘導(dǎo)產(chǎn)生更強(qiáng)的體液免疫和細(xì)胞免疫。所有肌肉注射和滴鼻免疫組小鼠均能夠保護(hù)同源和異源H5N1流感病毒的攻擊,然而在口服免疫組中LTB-VLPs、Flic-VLPs或GM-CSF-VLPs能夠?qū)ν春彤愒碒5N1流感病毒的攻擊提供部分的保護(hù)活性,而單獨(dú)VLPs組則不能保護(hù)流感病毒的攻擊。LTB-或Flic-VLPs口服免疫組誘導(dǎo)產(chǎn)生比單獨(dú)VLPs組高10倍的病毒特異性IgG抗體。說明具有免疫刺激活性的LTB-、Flic-和GM-CSF嵌合病毒樣顆粒能夠誘導(dǎo)產(chǎn)生更強(qiáng)的免疫反應(yīng)。我們的研究結(jié)果表明,基于昆蟲桿狀病毒表達(dá)系統(tǒng)的流感病毒樣顆粒可為同源甚至是抗原性存在差異的流感病毒感染提供免疫保護(hù),偶聯(lián)了具有免疫刺激活性的LTB-VLPs、Flic-VLPs或GM-CSF-VLPs可進(jìn)一步提高病毒樣顆粒的免疫原性。本研究為研發(fā)能夠控制高致病H5N1亞型禽流感流感病毒流行具有交叉保護(hù)活性的疫苗奠定了基礎(chǔ),同時(shí)也為新型疫苗研究提供了可借鑒的數(shù)據(jù)與經(jīng)驗(yàn)。
[Abstract]:Since 2003, the events of highly pathogenic H5N1 subtype influenza virus infections have continued and new genetic lineages have been derived, where 2.3. 2.1 and 2.3.4 are among the most popular. The possibility of a large-scale epidemic of highly pathogenic H5N1 viruses is recognized by all, and there is a need for a study of the vaccine of a number of lineages of avian influenza that have an epidemic risk. the inactivated vaccine which is widely used at present can only provide the immune protection against the virus infection of the same lineage, and the non-infectious virus-like particle vaccine produced by the baculovirus expression system is widely concerned by the advantages of low production cost, no biological safety risk and the like, In particular, because of its ability to induce cellular immunity, it is possible to provide an immune protection against a homotype of heterologous virus, which may be used in that study of influenza virus vaccine with frequent variation. The purpose of this study is to use the insect baculovirus expression system to study and develop the H5N1 virus-like particles by means of a series of optimization studies such as the expression pattern selection and the coupling of adjuvant effect protein, and to evaluate the immune protection effect on the heterologous virus infection. First, the virus-like particles containing the HA, NA and M1 proteins of the HA, NA and M1 proteins of the H5N1 A/ meerkat/ Shanhai/ SH-1 2012 (clade 2.3. 2.1) were developed using the insect baculovirus expression system. The identification of the series of tests demonstrated that the H5N1 virus-like particles containing the HA, NA and M1 proteins have similar morphological structure and size to the natural influenza virus and have a blood-clotting activity. Compared with the full-virus inactivated vaccine, the mice can induce stronger humoral and cellular immunity through the immune virus-like particle vaccine, and after the booster immunization, the specific IgG of the A/ meerkat/ Shanghai/ SH-1/2012 can be generated, and the titer is 20 times of that of the whole virus inactivation group. The next challenge results showed that the whole-virus inactivated group exhibited a protection rate of 80% against the homologous virus, with a protection rate of only 40% for the heterologous virus (A/ duck/ Jilin/ JL-SIV/2013, clade 2.3.4), while the virus-like particle-like mice were fully alive and the protection rate was 100%. The results show that influenza virus-like particle vaccine can be used as a kind of influenza vaccine with cross-protection activity to control the outbreak of highly pathogenic H5N1 influenza virus. To further improve the immunogenicity of the virus-like particles, we increase the immune-adjuvant-active protein in the vaccine with a view to enhancing the adaptive immune response level induced by the vaccine. The chimeric virus-like particles of the non-heat-labile enterotoxin B subunit (LTB), the flagellin (Toll-like receptor 5, and the granulocyte-macrophage colony-stimulating factor (GM-CSF) were constructed in the form of membrane-anchoring. The identification shows that LTB-, Flic-and GM-CSF-VLPs virus-like particles contain the specific proteins and have a similar morphological structure and size to the natural influenza virus and have a blood-clotting activity. LTB-, public-and GM-CSF-VLPs viral-like particle vaccines can induce stronger humoral and cellular immunity as compared to unconjugated immunostimulatory protein-like particle vaccines. All of the intramuscular and nasal immune group mice are capable of protecting both homologous and heterologous H5N1 influenza viruses, whereas LTB-VLPs, public-VLPs, or GM-CSF-VLPs in the oral immunization group can provide a partial protection activity against the attack of homologous and heterologous H5N1 influenza viruses, And the individual VLPs group cannot protect the attack of the influenza virus. The LTB-or the Flic-VLPs oral immune group induced a 10-fold higher virus-specific IgG antibody than the individual VLPs group. The LTB-, Flic-and GM-CSF chimeric virus-like particles with immunostimulatory activity can induce a stronger immune response. Our research results show that the influenza virus-like particles based on the insect baculovirus expression system can provide the immune protection for influenza virus infection which is homologous or even antigenic, and the LTB-VLPs with immunostimulating activity are coupled, The public-VLPs or GM-CSF-VLPs can further improve the immunogenicity of the virus-like particles. The research has laid a foundation for the development of a vaccine which can control the epidemic of highly pathogenic H5N1 subtype avian influenza virus and has cross-protection activity, and also provides useful data and experience for new vaccine research.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R392
[Abstract]:Since 2003, the events of highly pathogenic H5N1 subtype influenza virus infections have continued and new genetic lineages have been derived, where 2.3. 2.1 and 2.3.4 are among the most popular. The possibility of a large-scale epidemic of highly pathogenic H5N1 viruses is recognized by all, and there is a need for a study of the vaccine of a number of lineages of avian influenza that have an epidemic risk. the inactivated vaccine which is widely used at present can only provide the immune protection against the virus infection of the same lineage, and the non-infectious virus-like particle vaccine produced by the baculovirus expression system is widely concerned by the advantages of low production cost, no biological safety risk and the like, In particular, because of its ability to induce cellular immunity, it is possible to provide an immune protection against a homotype of heterologous virus, which may be used in that study of influenza virus vaccine with frequent variation. The purpose of this study is to use the insect baculovirus expression system to study and develop the H5N1 virus-like particles by means of a series of optimization studies such as the expression pattern selection and the coupling of adjuvant effect protein, and to evaluate the immune protection effect on the heterologous virus infection. First, the virus-like particles containing the HA, NA and M1 proteins of the HA, NA and M1 proteins of the H5N1 A/ meerkat/ Shanhai/ SH-1 2012 (clade 2.3. 2.1) were developed using the insect baculovirus expression system. The identification of the series of tests demonstrated that the H5N1 virus-like particles containing the HA, NA and M1 proteins have similar morphological structure and size to the natural influenza virus and have a blood-clotting activity. Compared with the full-virus inactivated vaccine, the mice can induce stronger humoral and cellular immunity through the immune virus-like particle vaccine, and after the booster immunization, the specific IgG of the A/ meerkat/ Shanghai/ SH-1/2012 can be generated, and the titer is 20 times of that of the whole virus inactivation group. The next challenge results showed that the whole-virus inactivated group exhibited a protection rate of 80% against the homologous virus, with a protection rate of only 40% for the heterologous virus (A/ duck/ Jilin/ JL-SIV/2013, clade 2.3.4), while the virus-like particle-like mice were fully alive and the protection rate was 100%. The results show that influenza virus-like particle vaccine can be used as a kind of influenza vaccine with cross-protection activity to control the outbreak of highly pathogenic H5N1 influenza virus. To further improve the immunogenicity of the virus-like particles, we increase the immune-adjuvant-active protein in the vaccine with a view to enhancing the adaptive immune response level induced by the vaccine. The chimeric virus-like particles of the non-heat-labile enterotoxin B subunit (LTB), the flagellin (Toll-like receptor 5, and the granulocyte-macrophage colony-stimulating factor (GM-CSF) were constructed in the form of membrane-anchoring. The identification shows that LTB-, Flic-and GM-CSF-VLPs virus-like particles contain the specific proteins and have a similar morphological structure and size to the natural influenza virus and have a blood-clotting activity. LTB-, public-and GM-CSF-VLPs viral-like particle vaccines can induce stronger humoral and cellular immunity as compared to unconjugated immunostimulatory protein-like particle vaccines. All of the intramuscular and nasal immune group mice are capable of protecting both homologous and heterologous H5N1 influenza viruses, whereas LTB-VLPs, public-VLPs, or GM-CSF-VLPs in the oral immunization group can provide a partial protection activity against the attack of homologous and heterologous H5N1 influenza viruses, And the individual VLPs group cannot protect the attack of the influenza virus. The LTB-or the Flic-VLPs oral immune group induced a 10-fold higher virus-specific IgG antibody than the individual VLPs group. The LTB-, Flic-and GM-CSF chimeric virus-like particles with immunostimulatory activity can induce a stronger immune response. Our research results show that the influenza virus-like particles based on the insect baculovirus expression system can provide the immune protection for influenza virus infection which is homologous or even antigenic, and the LTB-VLPs with immunostimulating activity are coupled, The public-VLPs or GM-CSF-VLPs can further improve the immunogenicity of the virus-like particles. The research has laid a foundation for the development of a vaccine which can control the epidemic of highly pathogenic H5N1 subtype avian influenza virus and has cross-protection activity, and also provides useful data and experience for new vaccine research.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R392
【共引文獻(xiàn)】
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