Pseudoreceptors for Hepatitis B Virus Infection

B型肝炎病毒如何進入細胞至今仍具爭議。由於B型肝炎病毒只可感染人類以及某些靈長類，因此缺乏方便操作的實驗動物模型，B型肝炎病毒在動物體內的感染及致病基轉的研究也因此受限，另外，至今仍然沒有細胞株可提供B型肝炎病毒完成完整的生活史（從感染到複製）。在過去的研究， 關於B型肝炎病毒進入細胞之後的複製機轉研究的很清楚，但對於B型肝炎病毒藉由何種受體以及如何附著在肝細胞上，則所知甚少。B型肝炎病毒顆粒的外套膜蛋白，決定病毒與人類肝細胞結合的重要角色，很多研究已猜測多種細胞表面蛋白可能是B型肝炎病毒的受體，但是這些結果卻無法互相佐證，某些研究顯示，B型肝炎病毒顆粒上的preS1區域可能在與人類肝細胞結合上扮演重要的角色。此論文中，我們構築表現在細胞膜上的單鏈抗體以作為B型肝炎的偽受體，這個偽受體包含MA18/7（抗preS1區域的單株抗體）的單鏈抗體變異區（scFv），以及LDLR, EGFR, ICAM1的穿膜區域（transmembrane domain）和細胞質區域（cytoplasmic domain）。人類肝癌細胞（HepG2）在表現偽受體後確實可以與B型肝炎病毒結合，利用共軛焦顯微鏡﹙confocal﹚，可觀察到被吞噬的B型肝炎病毒顆粒與endosomal標記蛋白（運鐵蛋白）在細胞內位置是重疊的，但被吞噬的B型肝炎病毒顆粒無法成功地進入溶酶體（lysosome），因此無法確定B型肝炎病毒顆粒是否脫去外套膜成功地脫離endosome。如果成功，可利用此策略研究病毒與細胞之間的交互作用，將其轉殖在小鼠上也可以作為B型肝炎病毒感染的小動物模型，用以開發新疫苗及研究致病機轉的工具。 本論文根據已發表的核酸序列，設計寡聚核苷酸並利用疊合聚合酶連鎖反應得到MA18/7的基因，同時我們利用合成preS1胜肽鏈免疫老鼠，同時生產抗preS1區域的細胞融合瘤，並將這些單株抗體以酵素免疫分析法及西方點墨法作定性及測試其抗原決定位。Hepatitis B virus entry is controversial. Insight into the fundamentals of the HBV life cycle has been impeded by severe experimental obstacles posed by (a) the narrow host range of the virus (resulting in the absence of convenient animal models of infection and disease), and (b) the lack of cell lines that support productive HBV replication. Although much information about the molecular biology of HBV has been gained in the past decade, little is known about the mechanism of attachment and penetration of the HBV particle into human hepatocytes. The HBV envelope proteins are important for the interaction between HBV particle and the hepatocyte plasma membrane. A number of cellular protein have been suggested to be HBV receptors, but studies on the HBV receptor during the last decade produced controversial and confusing results. Some observations suggested that the preS1 domain is probably the most important attachment site of HBV to human hepatocytes. In this study, we proposed to engineer a membrane-anchored single chain antibody as an artificial receptor for HBV infection. We generated pseudoreceptors by fusing the single chain variable region (scFv) of MA18/7, a monoclonal antibody (mAb) against HBV preS1 domain, to transmembrane and cytoplasm domains of the low-density lipoprotein receptor (LDLR), epidermal growth factor receptor (EGFR), or intracellular adhesion molecule-1 (ICAM-1). HepG2 cells expressed the pseudoreceptors were shown to bind HBV particles. Using confocal microscope we visualized the colocalization of HBV particles with the endosomal marker transferrin. Internalized HBV was not efficiently moved to lysosomes. Therefore, it is still unknown internalized HBV can uncoat its envelope and escape the endosomes. This approach, if successful, may be useful for studying virus reception and for the development of safer vaccines against viral pathogens of animals and humans. According to published sequences, we designed oligonucleotide and employed overlapping PCR to generate variable region of MA18/7. At the same time, we produced anti-preS1 hybridoma by synthetic preS1 peptide. We characterize the monoclonal anti-preS1 antibodies by ELISA and immunoblotting, and briefly epitope mapping.Contents Chinese Abstract………………………………………………………………..1 English Abstract………………………………………………………………...2 1. Introduction…………………………………………………………………4 1.1 HBV overview……………………………………………………………...4 1.2 Hepadnaviruses……………………………………………………………..4 1.3 HBV structure and HBV surface protein…………………………………...6 1.4 HBV cellular binding protein………………………………………………7 1.4.1 Small HBsAg-binding domain………………………………………….8 1.4.2 PreS2 binding domain…………………………………………………..8 1.4.3 PreS1-binding domain…………………………………………………10 1.5 HBV infection models….............................................................................12 1.6 Pseudoreceptor applications………………………………………………13 1.7 Summary of the major routes of endocytosis used by viruses…………….13 1.8 Strategy……………………………………………………………………16 2. Material and methods……………………………………………………...18 2.1 Plasmids construction……………………………………………………..18 2.2 Transfection of transgenes………………………………………………...18 2.3 Immunoblotting…………………………………………………………...18 2.3.1 Immunoblotting of pseudoreceptors chimeric proteins………………..18 2.3.2 Immunoblotting of HBV particles……………………………………..19 2.4 Flow cytometer analysis…………………………………………………..20 2.5 Retrovirus-mediated expression…………………………………………..20 2.5.1 Transfection…………………………………………………………....20 2.5.2 Viral infection………………………………………………………….20 2.6 Cell sorting………………………………………………………………...21 2.7 Isolate HBV virions……………………………………………………….21 2.8 Electron microscopy (EM)………………………………………………..22 2.9 Confocal microscopy……………………………………………………...22 2.9.1 HBV binding ability…………………………………………………...22 2.9.2 Immmunofluorescence of HBV and human transferrin internalization………………………………………………………….23 2.9.3 HBV and lysotracker localization……………………………………...23 2.10 PreS1 peptide conjugated to BSA or OVA……………………………….24 2.11 Produce anti-preS1 hybridoma…………………………………………...25 2.11.1 Immunization of mice with OVA-preS1……………………………….25 2.11.2 Hybridoma fusion……………………………………………………...25 2.11.3 Screening primary hybridomas………………………………………...26 2.11.4 Limiting dilution……………………………………………………….26 2.12 Ascites generation………………………………………………………..26 2.13 Mouse IgG1 antibody purification by protein A…………………………27 2.14 Q-PCR……………………………………………………………………27 2.14.1 Non-hybridization probes……………………………………………...27 2.14.2 Hybridization probes…………………………………………………..28 2.15 Enzyme-linked immunosorbent assay(ELISA)…………………………..28 2.15.1 HBsAg detection……………………………………………………….28 2.15.2 Anti-preS1 hybridoma screening ELISA……………………………...29 2.16 HBV infection……………………………………………………………30 3. Results……………………………………………………………………..31 3.1 Generation of plasmids for surface expression of pseudoreceptors………31 3.2 Chemeric protein are expressed on the cell surface……………………….31 3.3 HBV purification………………………….………………………………32 3.4 HBV electron microscopy………………………………………………...33 3.5 Pseudoreceptors can bind HBV particles…………………………………34 3.6 HBV particles colocalized with endosomal marker………………………35 3.7 HBV particles little or no colocalized with lysosome…………………….36 3.8 Characteristics of anti-preS1 hybridoma………………………………….36 3.9 HBV replicated slowly in HepG2-MA18/7-LDLR……………………….38 4. Discussion…………………………………………………………………40 4.1 Comparison of HBV particles from HBV transgenic mice and chronic infected humans…………………………………………………………...40 4.2 Expression and binding ability of pseudorecepotors……………………...40 4.3 HBV entry into HepG2 cells through pseudoreceptors instead of transferring………………………………………………………………...41 4.4 HBV particles didn’t efficiently colocalize with lysosome……………….41 4.5 Applications of anti-preS1 hybridoma…………………………………….42 4.6 HBV infection……………………………………………………………..43 4.7 Workable applications of this study………………………………………44 5. Figures……………………………………………………………………..45 Figure 1. Strategy of the experiment………………………………………….45 Figure 2. Diagram of pseudoreceptor transgenes……………………………..46 Figure 3. Expression of pseudoreceptors in HepG2 human hepatoma cells…47 Figure 4. HBV purification-batch I…………………………………………...48 Figure 5. HBV purification batch II…………………………………………..49 Figure 6. Electron micrographs……………………………………………….50 Figure 7. Electron micrographs……………………………………………….51 Figure 8. MA18/7-eB7 pseudoreceptors bind to HBV particles…….………..52 Figure 9. MA18/7-LDLR pseudoreceptors bind to HBV particles…………...53 Figure 10. MA18/7-ICAM1 pseudoreceptors bind to HBV particles…….......54 Figure 11. MA18/7-EGFR pseudoreceptors bind to HBV particles…………..55 Figure 12. Anti-phOx-eB7 doesn’t bind to HBV at a MOI of 10000…………56 Figure 13. HBV didn’t entry into HepG2-MA18/7-LDLR at 4℃……………57 Figure 14. HBV colocalized with endosomal marker at 37℃ for 30min……..58 Figure 15. HBV colocalized with endosomal marker at 37℃ for 60 min…….59 Figure 16. HBV colocalized with endosomal marker at 37℃ for 120min……60 Figure 17. HBV didn’t entry into HepG2-MA18/7-eB7 at 4℃………………61 Figure 18. Few HBV particles entry into HepG2-MA18/7-eB7at 37℃ for 60 min...................................................................................................62 Figure 19. Few HBV particles colocalized with endosomal marker in HepG2-MA18/7-eB7 at 37℃ for 120min……………..………….63 Figure20. HBV particles don’t colocalize with LysoTracker after infection at 37℃...................................................................................................64 Figure 21. HBV particles don’t colocalize with LysoTracker after infection at 37℃, 60min…………………………………………………….......65 Figure 22. Few HBV particles colocalized with LysoTracker after infection at 37℃ for 60min……………………………………………………..66 Figure 23. Few HBV particles colocalized with LysoTracker after infection at 37℃, 2 hour………………………………………………………...67 Figure 24. Nucleotide sequence and derived amino acid sequence for the VH domain of the mAb MA18/7……………………………………......68 Figure 25. Nucleotide sequence and derived amino acid sequence for the VL domain of the mAb MA18/7………….…………………………….69 Figure 26. SMCC-conjugated BSA and OVA………………………………...70 Figure 27. Anti-preS1 polyclonal antibody titer………………………………71 Figure 28. 5 hours of post-fusion……………………………………………...72 Figure 29. Anti-preS1 antibodies recognize HBV virions…………………….73 Figure 30. Surface single chain antibody MA18/7 doesn’t compete with anti-preS1 antibody…………………………………………………74 Figure 31. 6F1E8D8 recognizes the first half of the preS1 peptides and 3C6B2 recognize the second half………………………………………..….75 Figure 32. Surface single chain antibody MA18/7 doesn’t compete with anti-preS1 antibody…………………………………………………76 Figure 33. The epitope of 3C6B2 clone of anti-preS1 antibody is different from MA18/7……………………………………………………………..77 Figure 34. 3C6 clone is considered to be usable for western blotting………...78 Figure 35. HBsAg in culture medium after HBV infection…………………...79 Figure 36. HBV viral DNA in culture medium after HBV infection…………80 6. Reference…………………………………………………………………..8