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標題: | 動物α-冠狀病毒之病毒馴化與三聚體棘狀醣蛋白應用 Viral Attenuation and Applications of Trimeric Spike Glycoproteins of Animal Alphacoronaviruses |
作者: | Yen-Chen Chang 張晏禎 |
指導教授: | 張惠雯 |
關鍵字: | 動物冠狀病毒,病毒馴化,三聚體棘狀醣蛋白, viral attenuation,trimeric spike,glycoproteins,animal Alphacoronavirus, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 在過去數十年裡,冠狀病毒引發數種新興疾病,不僅重創民生和經濟,對於人及動物皆造成重大影響。其中,同樣隸屬於α-冠狀病毒之豬流行性下痢病毒(porcine epidemic diarrhea virus; PEDV)及貓傳染性腹膜炎病毒(feline infectious peritonitis virus; FIPV),為目前豬隻及貓重要之動物冠狀病毒。基因型2b(Genogroup 2b,G2b)豬流行性下痢病毒為2010年之後出現的新興病原,引發豬隻嚴重下痢疫情並且於世界多國快速蔓延,因此,研發新一代針對G2b PEDV的疫苗是當務之急。本實驗首先成功由台灣病豬分離出G2b型PEDV屏東52病毒株(PEDVPT-52),將其於綠猴腎臟細胞株(Vero cell)進行連續繼代,並以口服接種離乳豬隻,結果發現,相較於接種第五代PEDVPT-52病毒株(PEDVPT-P5)的豬隻會出現典型下痢症狀,接種第96代病毒株PEDVPT-P96只會引發零星輕微泥便,甚至在大部分豬隻無臨床症狀。此外也證實接種PEDVPT-P96豬隻產生的中和抗體、免疫球蛋白G(immunoglobulin G; IgG)和IgA的力價雖較PEDVPT-P5接種豬隻低,但是都能保護豬隻免於後續以PEDVPT-P5攻毒之病害。當比較PEDVPT-P5和PEDVPT-P96的全長核酸序列之後,發現PEDVPT-P96雖未出現任何缺失或插入,但仍可見23處核苷酸序列改變所導致相關之19處的胺基酸置換,這些變異主要位於非結構蛋白2、3、4、9、14、15、棘狀蛋白(spike; S)、閱讀框(open reading frame; ORF)3及膜蛋白(membrane; M)(第二章)。
此外,根據以往文獻可知,冠狀病毒S蛋白功能為與細胞上受體結合並決定與組織及標的細胞之親和性,也為刺激免疫系統產生中和抗體之重要結構蛋白。因此,本研究中第二部分為建立以人體胚胎腎臟細胞293(human embryonic kidney 293; HEK 293)蛋白質表現系統以產生PEDV和FIPV之膜外全長S醣蛋白三聚體,以作為發展PEDV次單位疫苗與研究此二動物冠狀病毒S蛋白結構、功能及其與標的細胞之親和性之使用。為了模擬自然界病毒S蛋白及增加蛋白質表現量,此研究在核酸序列C端插入T4噬菌體fibritin三聚體的折疊序列,及將N端的訊號胜肽序列替換成組織胞漿素原活化訊號胜肽序列,而成功建立能夠穩定表現PEDVPT-52株及FIPV UU4株的膜外全長棘狀醣蛋白三聚體之HEK 293細胞株。本研究接著進行表現之S蛋白定性試驗,包含確認其三聚體結構、生物活性及其天冬醯胺醣化修飾情形(第3章和第4章),即利用西方墨點法或粒徑篩析層析-多角光散射檢測法(size exclusion chromatography coupled with multi-angle static light scattering; SEC-MALS)確認三聚體結構;以質譜儀確認FIPV UU4 S蛋白天冬醯胺醣化的位置;並使用西方墨點法、免疫組織化學染色法 (immunohistochemistry; IHC)和免疫細胞化學染色法(immunocytochemistry; ICC)驗證合成S蛋白的生物活性。質譜儀分析的結果顯示,在覆蓋率80%的情況下,FIPV-UU4 S蛋白單聚體有至少29處的天冬醯胺醣化,而且大多數的醣化修飾分布於S1蛋白的B子域、S1/S2分裂位和七肽重複區2(heptad repeat 2; HR 2)(第4章)。 在PEDVPT重組S蛋白應用方面,為了研發能夠誘發黏膜免疫的PEDV疫苗及藉由肌肉注射引發有效的全身及黏膜免疫反應,本研究利用研發之PEDVPT重組S蛋白結合強效的黏膜佐劑-大腸桿菌忌熱性腸毒素B次單元(B subunit of Escherichia coli heat-labile enterotoxin; LTB)作為佐劑,於五週齡田間離乳豬進行肌肉注射免疫以評估疫苗效力(第3章)。結果發現相對於控制組,肌肉注射S蛋白的豬隻能夠刺激PEDV S特異性IgG和中和抗體的產生,但糞便IgA力價則是低於檢測極限,故無法提供有效保護力,無法有效抵抗PEDV-PT株的攻毒。 在FIPV重組S蛋白應用方面,由於第一型貓冠狀病毒(feline coronavirus serotype I; serotype I FCoV)的細胞受器仍然未知,因此本論文應用合成的第一型FIPV UU4和貓腸道冠狀病毒(feline enteric coronavirus; FECV)UU7病毒株之S蛋白為探針,經由親和性結合測試與FCoV的宿主細胞受器結合特性,於石蠟包埋組織定位標的細胞(第5章)。免疫組織化學染色結果顯示,FECV-UU7 S蛋白無法與任何組織或細胞鍵結,但FIPV-UU4 S蛋白能夠辨識FIPV抗原陽性細胞和FIP病變區域,且偵測到之陽性細胞主要以巨噬細胞為主。此外,在以神經胺酸酶(neuraminidase; NA)去除細胞表面唾液酸(sialic acid)之後,亦不會影響FIPV-UU4 S蛋白對於巨噬細胞的親和性,因此,可排除唾液酸對於巨噬細胞與FIPV-UU4 S蛋白親和性的影響,進而了解病毒與宿主細胞的交互作用與尚未辨識的第一型FCoV細胞受器之生物特性。 綜合上述,本研究建立PEDVPT於田間離乳豬的攻毒模式,亦證實PEDVPT-P96具有發展為減毒疫苗的潛力。此外,亦成功表現PEDVPT與FIPV UU4的重組S蛋白,並證實此二重組蛋白質與其病毒表面之S蛋白有相當程度的相似性,故能夠被運用於次單位疫苗發展及作為研究病原致病機制的工具。接著,本研究進一步證實PEDVPT S醣蛋白三聚體為具免疫原性並能誘導中和抗體之抗原,但以LTB為佐劑進行肌肉注射並無法有效刺激黏膜免疫以達到完全保護豬隻免於PEDV之病害,未來應尋求及開發更好免疫佐劑以達到更好保護效果。本研究提供α-冠狀病毒中之PEDV減毒疫苗、PEDV S及FIPV S蛋白表現、結構分析及PEDV次單位疫苗研究模式及應用,對於未來豬隻及貓冠狀病毒疫苗研發及病毒學研究提供重要資訊及幫助。 In the past decades, coronaviruses have caused several emerging diseases and resulted in dramatic impacts on the economy and livelihood of human and animals. Porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV) belong to the Alphacoronavirus and are one of the important pathogens in pigs and cats, respectively. Genogroup 2b (G2b) PEDVs are emerging pathogens, which caused devastating outbreaks since 2010 and rapidly spread worldwide in late 2013. A new generation of vaccine against the PEDV is urgently needed. In the present study, firstly, a G2b Taiwan Pintung 52 (PEDVPT-52) strain was isolated and serially passaged in the Vero cells. While oral inoculation of postweaning piglets with the low-passage PEDVPT-52 strain (passage 5; PEDVPT-P5) showed typical diarrhea, the high-passage PEDVPT-52 strain (passage 96; PEDVPT-P96) only caused intermittent, mild to no diarrhea in piglets. Although animals inoculated with PEDVPT-P96 elicited slightly lower neutralizing antibody and PEDV-specific immunoglobulin G (IgG) and immunoglobulin A (IgA) titers than those inoculated with PEDVPT-P5, a reduction in the severity of disease after subsequent challenge with PEDVPT-P5 was noted. Alignment and comparison of the full-length sequences of PEDVPT-P5 and PEDVPT-P96 revealed 23 nucleotide changes and resultant 19 amino acid substitutions in the 2, 3, 4, 9, 14, 15 non-structure proteins, spike (S), open reading frame 3 (ORF3), and membrane (M) proteins with no detectable deletion or insertion in the genome (chapter II). Several studies have suggested that the S protein of coronaviruses is not only responsible for binding to the host cell receptor and determining the tissue or cell tropism and host ranges but also acts as an important structural protein that could induce immune response and neutralizing antibody. Therefore, the second part of the thesis is to establish stable HEK 293 cell lines producing the trimeric ectodomain of S glycoproteins of the PEDVPT-52 strain and the serotype I FIPV UU4 strain in order to develop a PEDV subunit vaccine and provide the assess to study the structure, function and cell tropism of the S protein of both animal coronaviruses. To mimic the native viral S glycoprotein and enhance the expressing efficiency, a T4 bacteriophage foldon sequence of fibritin trimerization domains was incorporated at the C-terminal end and the signal peptide of S protein were replaced by a tissue plasminogen activator signal peptide sequence at the N terminal end. The human embryonic kidney (HEK) 293 cell lines constitutively expressing the full-length trimeric S ectodomain glycoproteins of the PEDVPT-52 and FIPV UU4 strains were successfully established. In chapter III and IV, the protein properties were characterized, including the trimeric structure, bio-reactivity to specific antibodies, and the N-glycosylation modifications. The trimeric structures of both S proteins were confirmed by Western blotting with different levels of denaturing methods or size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS). The N-linked glycosylated modifications of both S proteins were also confirmed and further localized by using the general mass spectrometry-based proteomics strategy that uses PNGase F to introduce mass signatures and distinguish peptide glycosites that are unoccupied or occupied by glycans. Additionally, the bio-activity of both S proteins were proven by Western blotting, immunohistochemistry (IHC) or immunocytochemistry (ICC). The result of mass spectrometry demonstrated that, with 80% sequence coverage of the FIPV UU4 S protein, each S protein monomer had at least 29 N-linked glycosylation sites. We also found that most glycosites in FIPV UU4 S proteins were fully occupied by glycans, especially in the B subdomain regions of S1 protein, S1/S2 cleavage site, and heptad repeat (HR) 2 region (Chapter IV). As for the application of the recombinant PEDVPT S protein, we aim to develop a subunit vaccine capable of inducing sufficient systemic and mucosal immunity via intramuscular (IM) injection. In the current study, the trimeric S glycoprotein adjuvanted with the B subunit of Escherichia coli heat-labile enterotoxin (LTB), which was reported as a potent mucosal adjuvant, was intramuscularly administered in conventional 5-week-old pigs. The protective efficacy of the vaccine regimen was evaluated by PEDVPT challenge. Comparing with the control group, all piglets in the S protein-LTB immunized group generated systemic PEDV S-specific IgG and neutralizing antibody in the blood with undetectable levels of fecal PEDV-specific IgA and a limited protection against the PEDVPT challenge. Due to that the cellular receptor for serotype I FCoV is still unknown, the recombinant S protein of FIPV UU4 and feline enteric coronavirus (FECV) UU7 strains were used as probes to assess the host cell receptor of FCoV by affinity binding assay in paraffin-embedded tissues (Chapter V). While no tissue or enteric binding of FECV UU7 S protein was detected, specific binding of FIPV UU4 S protein on the host cells, predominantly macrophages, is correlated with FIPV antigen-positive cells identified by IHC and lesions associated with FIP. In addition, the affinity binding of FIPV UU4 S protein on macrophages was not affected by enzymatic removal of the host cell-surface sialic acid with neuraminidase suggesting that factors other than sialic acid may contribute to the macrophage tropism of the FIPV UU4. This approach allowed obtaining more information about both virus–host cell interactions and the biological characteristics of the remained to be identified cellular receptor of serotype I FCoVs. In conclusion, we have not only established a conventional pig PEDV challenge model but also confirmed the potential of PEDVPT-P96 as an attenuated vaccine candidate. Furthermore, the HEK 293 cell expressing systems producing the PEDVPT and FIPV-UU4 S proteins have also been successfully developed. The recombinant S proteins were proven harboring certain identity with the natural viral protein that could serve as immunogens for subunit vaccine development and an alternative platform to study the viral pathogenesis. Furthermore, our results have demonstrated that the trimeric PEDVPT S glycoprotein is immunogenic and capable of inducing neutralizing antibody. However, pigs intramuscularly inoculated with PEDVPT S protein adjuvanted with LTB could not elicit sufficient mucosal immunity and protection against PEDV challenge. The development of novel vaccine regimens as well as effective mucosal adjuvants will be an important task for the development of protect PEDV vaccine. In the present study, we have provided the rationales for an attenuated and a subunit PEDV vaccine development and the information of the establishment of expression system and structural analyses of PEDV S and FIPV S trimeric proteins, and the applications for the trimeric S proteins. The results would contribute to future vaccine development and virological research of coronaviruses. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69227 |
DOI: | 10.6342/NTU201801590 |
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顯示於系所單位: | 獸醫學系 |
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