甲型流感病毒樣顆粒疫苗的初步研究
發(fā)布時間：2018-08-14 10:49
【摘要】：流感是世界范圍內廣泛流行的呼吸系統(tǒng)傳染病,每年都造成大量的住院及死亡病例。截至2010年7月18日,爆發(fā)于墨西哥的甲型H1N1流感已在全球214個國家造成18,336人死亡;自2003年以來,H5N1高致病性禽流感病毒已在世界范圍內造成310人死亡,且死亡率高達59%。疫苗是防控流感最為有效的手段。流感疫苗的發(fā)展經歷了滅活疫苗、裂解疫苗、減毒活疫苗、亞單位疫苗等階段,季節(jié)性流感裂解疫苗雖然能在人群中產生有效保護作用,但是依然存在許多問題。首先,傳統(tǒng)流感疫苗的生產工藝依賴于雞胚,難以應對流感大規(guī)模爆發(fā)對疫苗的需求,且雞胚卵清蛋白易引起過敏,至今還沒有針對老年人、兒童及特殊人群理想的流感疫苗,因此現(xiàn)有流感疫苗的生產工藝亟待更新。其次,現(xiàn)有流感疫苗的有效成分是流感病毒HA和NA抗原。流感病毒具有易錯傾向的RNA依賴性RNA聚合酶,使得HA和NA抗原不斷發(fā)生抗原漂移;流感病毒的宿主極為廣泛,可以感染鳥類、馬、豬等多種動物并在這些動物體內發(fā)生基因節(jié)段的重組。因此,現(xiàn)有的季節(jié)性流感疫苗每年都需要更新。這不但需要對每年的病毒流行株進行預測,而且,每年接種流感疫苗,除了加重國家公共衛(wèi)生經費的負擔造成大量不必要的浪費之外,還不可避免的在人體中引入大量外源核酸,對人體健康造成潛在的威脅。因此,更新現(xiàn)有流感疫苗生產工藝,研發(fā)新類型的流感疫苗成為流感疫苗研究領域的發(fā)展方向。 病毒樣顆粒(Virus-like particle, VLP)作為一種候選疫苗具有免疫原性好,沒有感染性,穩(wěn)定性好,不易失活等特點,因此在病毒感染性疾病的疫苗研發(fā)中極具研究價值。國內外已有大量關于病毒樣顆粒的研究報道,包括人類乳頭瘤病毒樣顆粒、人類免疫缺陷病毒樣顆粒、輪狀病毒樣顆粒等。其中,HPV-VLP疫苗已經在歐洲和美國上市,展現(xiàn)出了VLP疫苗良好的應用前景。由昆蟲細胞衍生的流感VLP是最有希望替代現(xiàn)有流感疫苗的候選疫苗。目前已經成功重組的流感VLP包括H9N2、H3N2、H1N1、H5N1等多種亞型,這些VLP都具有免疫原性強,保護效果好,易于構建表達等優(yōu)點。因此,研制流感病毒樣顆粒疫苗具有極為重要的科學價值和社會效益。但是國內關于流感病毒樣顆粒的文獻還比較少,國外也未見關于將流感病毒樣顆粒與現(xiàn)行裂解疫苗進行比較的報道。 現(xiàn)有的大部分通用疫苗研究以人來源的流感病毒M2蛋白外功能區(qū)(M2e)序列作為靶點。然而,M2e免疫原性低,M2e氨基酸序列在人來源和禽來源的A型流感病毒之間存在5-6個氨基酸殘基的差異,且M2e誘導機體產生細胞免疫的能力較差。NP418-426表位是NP蛋白的免疫優(yōu)勢表位,與IFN-γ的產生高度相關并且能夠有效的誘導特異的CTL發(fā)生免疫應答;禽流感病毒來源的NP418-426表位可以被人流感病毒特異的CTL交叉識別。因此,NP418-426表位有希望成為基于人來源M2e序列的流感病毒通用疫苗的重要組成成分,用以提高流感通用疫苗的種間交叉免疫保護效果,進一步提高通用疫苗的廣譜性。乙肝核心蛋白(HBc)是使用最為廣泛的外源表位載體。HBc可在體內或體外自組裝成病毒樣顆粒(VLP),將外源表位與HBc構建融合蛋白可以有效的誘導T細胞和B細胞免疫反應。基于以上研究背景,本研究分為兩部分: 第一部分甲型H1N1病毒樣顆粒疫苗的初步研究 1.通過昆蟲-桿狀病毒表達系統(tǒng)表達A/California/07/2009(H1N1)毒株的VLP。使用DEAE陰離子交換介質、Sepharose 4FF凝膠介質純化甲型H1N1 VLP。電鏡觀察可知,純化的甲型H1N1 VLP結構完整,直徑在100nm左右。通過Western blot、血凝、單向免疫擴散等實驗進一步證實,獲得了重組甲型H1N1 VLP蛋白抗原并具有良好的免疫活性。 2.以10ug HA為免疫劑量,腹腔免疫4-6周齡雌性Balb/c小鼠,ELISA結果證實,研制的甲型H1N1 VLP能夠誘導機體產生高滴度的特異性抗體,且甲型H1N1 VLP較甲型H1N1裂解疫苗能夠誘導機體產生更高滴度的IgG抗體。 3.選用A/Beijing/501/2009 (H1N1)甲型H1N1流行株進行攻毒,結果顯示,甲型H1N1 VLP候選疫苗能夠保護小鼠抵抗50LD_(50)劑量的致死性攻擊,保護率達到100%。 結論:研制的甲型H1N1 VLP候選疫苗具有良好的免疫原性,能夠刺激機體產生體液免疫應答和細胞免疫應答,且生產較為便利。昆蟲細胞生產的流感病毒VLP是值得深入研究,以期有希望替代現(xiàn)有流感疫苗的候選疫苗之一。 第二部分甲型流感通用病毒樣顆粒疫苗的初步研究 1.使用大腸桿菌表達融合蛋白3M2e-NP-HBc、3M2e-HBc以及HBc N端149個氨基酸HBc-N149。采用His-trap純化介質對重組蛋白進行純化。電鏡觀察可知,純化的重組蛋白可形成大小均一,結構完整的病毒樣顆粒。通過M2e單克隆抗體進行Western blot實驗可知,3M2e-NP-HBc及3M2e-HBc VLP抗原具有良好的M2e特異的免疫活性。 2.以20ug重組蛋白為免疫劑量,選取4-6周齡雌性Balb/c小鼠進行動物實驗可知,重組3M2e-NP-HBc和3M2e-HBc VLP抗原均能夠誘導機體產生高滴度的M2e特異性抗體。3M2e-NP-HBc較3M2e-HBc能夠誘導機體產生更高滴度的IgG2a抗體且3M2e-NP-HBc較3M2e-HBc能夠刺激機體產生更多的IFN-γ分泌型淋巴細胞。 3.分別使用A/Beijing/501/2009 (H1N1)甲型H1N1毒株、A/PR/8/34(H1N1)及A/Ostrich/SuZhou/097/2003 (H5N1)高致病性禽流感毒株進行攻毒實驗。結果顯示,3M2e-NP-HBc和3 2e HBc均能夠保護小鼠抵抗10LD_(50)的A/Beiji g/501/2 09 (H1N1)、A/PR/8/34(H1N1)毒株的致死性攻擊,保護率達到100%。3M2e-NP-HBc較3M2e-HBc能夠更好的保護小鼠抵抗10LD_(50)的A/Ostrich/SuZhou/097/2003 (H5N1)毒株的致死性攻擊。 結論:NP418-426表位的嵌入能夠有效的誘導機體產生細胞免疫應答,有效的提高基于M2e的甲型流感通用候選疫苗的種間交叉保護效果。流感病毒內部蛋白作為潛在的通用疫苗候選抗原應當進行深入的研究。
[Abstract]:As of July 18, 2010, influenza A (H1N1) outbreak in Mexico has killed 18,336 people in 214 countries worldwide; since 2003, the highly pathogenic H5N1 avian influenza virus has caused 310 deaths worldwide. Vaccines are the most effective means to prevent and control influenza. The development of influenza vaccines has gone through inactivated vaccines, lysed vaccines, live attenuated vaccines, subunit vaccines and so on. Although seasonal split influenza vaccines can produce effective protection in the population, there are still many problems. First, the traditional influenza vaccines The production process is dependent on chicken embryos, which is difficult to meet the demand for vaccines in large-scale influenza outbreaks, and egg albumin is susceptible to allergies. So far, there is no ideal influenza vaccine for the elderly, children and special populations. Therefore, the existing influenza vaccine production process needs to be updated urgently. Secondly, the effective ingredient of the existing influenza vaccine is influenza virus H. A and NA antigens. Influenza viruses have a RNA dependent RNA polymerase which is prone to be misaligned, which makes the antigen shift of HA and NA antigens. The host of influenza virus is very extensive, * it can infect many kinds of animals such as birds, horses, pigs and so on, and reorganize gene segments in these animals. New. This requires not only the annual prediction of the virus epidemic strains, but also the annual vaccination of influenza vaccines, in addition to increasing the burden of national public health expenditure caused by a large number of unnecessary waste, but also the inevitable introduction of a large number of exogenous nucleic acids in the human body, a potential threat to human health. The development of new types of influenza vaccine has become the development direction of influenza vaccine research.
Virus-like particles (VLP), as a candidate vaccine, have good immunogenicity, no infectivity, good stability, and are not easy to inactivate. Therefore, it is of great value in the research and development of vaccine for viral infectious diseases. Among them, HPV-VLP vaccines have been marketed in Europe and the United States, showing a promising future for VLP vaccines. Influenza VLP derived from insect cells is the most promising alternative to existing influenza vaccines. H5N1 and other subtypes, these VLPs have the advantages of strong immunogenicity, good protective effect, easy construction and expression. Therefore, the development of influenza-like granule vaccine has extremely important scientific value and social benefits. Comparison of split vaccine was reported.
However, M2e immunogenicity is low. There are 5-6 amino acid residues in the M2e amino acid sequence between human and avian influenza A viruses, and the ability of M2e to induce cellular immunity is poor. NP epitope is an immunodominant epitope of NP protein, which is highly correlated with the production of IFN-gamma and can effectively induce specific CTL immune response. NP418-426 epitope derived from avian influenza virus can be cross-identified by human influenza virus-specific CTL. Therefore, NP418-426 epitope is hopeful to become a universal influenza virus based on human M2e sequence. Hepatitis B core protein (HBc) is the most widely used exogenous epitope vector. HBc can self-assemble into virus-like particles (VLP) in vivo or in vitro, and construct fusion proteins between exogenous epitopes and HBc. In order to effectively induce T cell and B cell immune response, based on the above research background, the study is divided into two parts:
The first part is a preliminary study of a H1N1 virus like particle vaccine.
1. Expressing the VLP of A/California/07/2009 (H1N1) strain by insect-baculovirus expression system. Using DEAE anion exchange medium and Sepharose 4FF gel medium to purify A/H1N1 VLP. The structure of purified A/H1N1 VLP was intact and its diameter was about 100 nm. It was confirmed that the recombinant H1N1 VLP protein antigen was obtained and had good immunological activity.
2. Female Balb/c mice aged 4-6 weeks were immunized by intraperitoneal injection of 10ug HA. ELISA results showed that the developed A H1N1 VLP could induce high titer of specific antibodies, and A H1N1 VLP could induce higher titer of IgG antibodies than A H1N1 lysis vaccine.
3. A/Beijing/501/2009 (H1N1) influenza A H1N1 strain was selected to attack the virus. The results showed that the candidate vaccine of A H1N1 VLP could protect mice against lethal attack of 50 LD_ (50) dose, and the protection rate reached 100%.
CONCLUSION: The candidate vaccine for influenza A H1N1 VLP has good immunogenicity, can stimulate humoral and cellular immune responses, and is convenient for production.
The second part is a preliminary study of influenza A virus like particle vaccine.
1. The fusion protein 3M2e-NP-HBc, 3M2e-HBc and 149 amino acids HBc-N149 were expressed in E. coli. The recombinant protein was purified by his-trap purification medium. The purified recombinant protein could form virus-like particles with uniform size and complete structure. The Western blot test with M2e monoclonal antibody showed that the recombinant protein was 3. M2e-NP-HBc and 3M2e-HBc VLP antigen have good M2e specific immune activity.
2. Using 20ug recombinant protein as the immune dose, female Balb/c mice aged 4-6 weeks were selected for animal experiment. The results showed that recombinant 3M2e-NP-HBc and 3M2e-HBc VLP antigens could induce high titer of M2e-specific antibody. 3M2e-NP-HBc could induce higher titer of IgG2a antibody than 3M2e-HBc and 3M2e-NP-HBc could prick higher titer of IgG2a antibody than 3M2e-HBc. The stimulated organism produces more IFN- - ray secreting lymphocytes.
3. Attack experiments were carried out using A/Beijing/501/2009 (H1N1) A/H1N1 strain, A/PR/8/34 (H1N1) and A/Ostrich/SuZhou/097/2003 (H5N1) highly pathogenic avian influenza strains. The results showed that 3M2e-NP-HBc and 32e-HBc could protect mice against lethal attack by 10LD_ (50) A/Beiji g/501/209 (H1N1), A/PR/8/34 (H1N1) strains. Compared with 3M2e-HBc, 3M2e-NP-HBc could protect mice against lethal attack of 10LD_ (50) A/Ostrich/SuZhou/097/2003 (H5N1).
CONCLUSION: NP418-426 epitope insertion can effectively induce cellular immune response and improve the cross-protection effect of M2e-based universal vaccine candidates for influenza A. As a potential candidate antigen for universal vaccine, influenza virus internal protein should be further studied.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392
本文編號：2182618
[Abstract]:As of July 18, 2010, influenza A (H1N1) outbreak in Mexico has killed 18,336 people in 214 countries worldwide; since 2003, the highly pathogenic H5N1 avian influenza virus has caused 310 deaths worldwide. Vaccines are the most effective means to prevent and control influenza. The development of influenza vaccines has gone through inactivated vaccines, lysed vaccines, live attenuated vaccines, subunit vaccines and so on. Although seasonal split influenza vaccines can produce effective protection in the population, there are still many problems. First, the traditional influenza vaccines The production process is dependent on chicken embryos, which is difficult to meet the demand for vaccines in large-scale influenza outbreaks, and egg albumin is susceptible to allergies. So far, there is no ideal influenza vaccine for the elderly, children and special populations. Therefore, the existing influenza vaccine production process needs to be updated urgently. Secondly, the effective ingredient of the existing influenza vaccine is influenza virus H. A and NA antigens. Influenza viruses have a RNA dependent RNA polymerase which is prone to be misaligned, which makes the antigen shift of HA and NA antigens. The host of influenza virus is very extensive, * it can infect many kinds of animals such as birds, horses, pigs and so on, and reorganize gene segments in these animals. New. This requires not only the annual prediction of the virus epidemic strains, but also the annual vaccination of influenza vaccines, in addition to increasing the burden of national public health expenditure caused by a large number of unnecessary waste, but also the inevitable introduction of a large number of exogenous nucleic acids in the human body, a potential threat to human health. The development of new types of influenza vaccine has become the development direction of influenza vaccine research.
Virus-like particles (VLP), as a candidate vaccine, have good immunogenicity, no infectivity, good stability, and are not easy to inactivate. Therefore, it is of great value in the research and development of vaccine for viral infectious diseases. Among them, HPV-VLP vaccines have been marketed in Europe and the United States, showing a promising future for VLP vaccines. Influenza VLP derived from insect cells is the most promising alternative to existing influenza vaccines. H5N1 and other subtypes, these VLPs have the advantages of strong immunogenicity, good protective effect, easy construction and expression. Therefore, the development of influenza-like granule vaccine has extremely important scientific value and social benefits. Comparison of split vaccine was reported.
However, M2e immunogenicity is low. There are 5-6 amino acid residues in the M2e amino acid sequence between human and avian influenza A viruses, and the ability of M2e to induce cellular immunity is poor. NP epitope is an immunodominant epitope of NP protein, which is highly correlated with the production of IFN-gamma and can effectively induce specific CTL immune response. NP418-426 epitope derived from avian influenza virus can be cross-identified by human influenza virus-specific CTL. Therefore, NP418-426 epitope is hopeful to become a universal influenza virus based on human M2e sequence. Hepatitis B core protein (HBc) is the most widely used exogenous epitope vector. HBc can self-assemble into virus-like particles (VLP) in vivo or in vitro, and construct fusion proteins between exogenous epitopes and HBc. In order to effectively induce T cell and B cell immune response, based on the above research background, the study is divided into two parts:
The first part is a preliminary study of a H1N1 virus like particle vaccine.
1. Expressing the VLP of A/California/07/2009 (H1N1) strain by insect-baculovirus expression system. Using DEAE anion exchange medium and Sepharose 4FF gel medium to purify A/H1N1 VLP. The structure of purified A/H1N1 VLP was intact and its diameter was about 100 nm. It was confirmed that the recombinant H1N1 VLP protein antigen was obtained and had good immunological activity.
2. Female Balb/c mice aged 4-6 weeks were immunized by intraperitoneal injection of 10ug HA. ELISA results showed that the developed A H1N1 VLP could induce high titer of specific antibodies, and A H1N1 VLP could induce higher titer of IgG antibodies than A H1N1 lysis vaccine.
3. A/Beijing/501/2009 (H1N1) influenza A H1N1 strain was selected to attack the virus. The results showed that the candidate vaccine of A H1N1 VLP could protect mice against lethal attack of 50 LD_ (50) dose, and the protection rate reached 100%.
CONCLUSION: The candidate vaccine for influenza A H1N1 VLP has good immunogenicity, can stimulate humoral and cellular immune responses, and is convenient for production.
The second part is a preliminary study of influenza A virus like particle vaccine.
1. The fusion protein 3M2e-NP-HBc, 3M2e-HBc and 149 amino acids HBc-N149 were expressed in E. coli. The recombinant protein was purified by his-trap purification medium. The purified recombinant protein could form virus-like particles with uniform size and complete structure. The Western blot test with M2e monoclonal antibody showed that the recombinant protein was 3. M2e-NP-HBc and 3M2e-HBc VLP antigen have good M2e specific immune activity.
2. Using 20ug recombinant protein as the immune dose, female Balb/c mice aged 4-6 weeks were selected for animal experiment. The results showed that recombinant 3M2e-NP-HBc and 3M2e-HBc VLP antigens could induce high titer of M2e-specific antibody. 3M2e-NP-HBc could induce higher titer of IgG2a antibody than 3M2e-HBc and 3M2e-NP-HBc could prick higher titer of IgG2a antibody than 3M2e-HBc. The stimulated organism produces more IFN- - ray secreting lymphocytes.
3. Attack experiments were carried out using A/Beijing/501/2009 (H1N1) A/H1N1 strain, A/PR/8/34 (H1N1) and A/Ostrich/SuZhou/097/2003 (H5N1) highly pathogenic avian influenza strains. The results showed that 3M2e-NP-HBc and 32e-HBc could protect mice against lethal attack by 10LD_ (50) A/Beiji g/501/209 (H1N1), A/PR/8/34 (H1N1) strains. Compared with 3M2e-HBc, 3M2e-NP-HBc could protect mice against lethal attack of 10LD_ (50) A/Ostrich/SuZhou/097/2003 (H5N1).
CONCLUSION: NP418-426 epitope insertion can effectively induce cellular immune response and improve the cross-protection effect of M2e-based universal vaccine candidates for influenza A. As a potential candidate antigen for universal vaccine, influenza virus internal protein should be further studied.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392
【參考文獻】
相關期刊論文 前3條
1 吳小鋒;岳萬福;劉劍梅;李廣立;繆云根;;Bac-to-Bac系統(tǒng)的研究進展及在家蠶中的應用[J];蠶業(yè)科學;2007年01期
2 王惠明;吳玉章;;以病毒樣顆粒為平臺的分子載體和疫苗設計[J];免疫學雜志;2006年01期
3 姚志東;張良艷;劉坤;邢麗;王希良;;甲型H1N1流感病毒疫苗株高適應性MDCK細胞的篩選及鑒定[J];免疫學雜志;2011年03期
，本文編號：2182618
本文鏈接：http://sikaile.net/xiyixuelunwen/2182618.html
最近更新
教材專著
