IMMUNOLOGIC ANALYSIS OF A HERPES VIRUS GLYCOPROTEIN VACCINE CANDIDATE--EBV GP350

疱疹病毒糖蛋白疫苗候选物--EBV GP350的免疫学分析

• 批准号: 3770329
• 负责人: C J MARCUS-SEKURA
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3770329
• 关键词: Baculoviridae; Epstein Barr virus; Escherichia coli; Herpesviridae vaccine; antiserum; antiviral antibody; chemical binding; conformation; epitope mapping; gene expression; glycoproteins; glycosylation; immunofluorescence technique; laboratory rabbit; microorganism immunology; molecular cloning; molecular shape; monoclonal antibody; neutralizing antibody; nucleic acid sequence; protein denaturation; recombinant proteins; virus antigen; virus protein

Recombinant glycoprotein vaccines are currently in development for several herpesviruses, including HSV and EBV, and for HIV. The EBV membrane antige (EBV-MA) gp350 is a glycoprotein which contains virus neutralizing epitopes and is currently being tested as a possible EBV vaccine candidate in England. A series of 14 monoclonal antibodies (mAbs) to EBV-MA were obtained from Louis Qualtiere and Gary Pearson. These have been used to screen antigen expressing clones for reactivity to different epitopes in order to identify the nucleic acid sequences important for virus neutralization. Initially ten overlapping clones which express different portions of unglycosylated gp350 were constructed in E. coli. Four of 14 mAbs tested reacted with the recombinant E. coli antigens, and three antigenic epitopes (nucleotides 1980-2307, 3186-3528 and 3528-3576 in Bam H L) identified. Proteins expressed by the clones were used to produce antisera in rabbits. When tested for neutralization of EBV in tissue culture, the rabbit antisera as well as the mAbs which recognized the three epitopes failed to neutralize, suggesting that these epitopes are not involved in neutralization. Since the remaining mAbs may only recognize the full-length glycosylated form of gp350, the entire gp350 coding sequenc has also been expressed in a Baculovirus expression system which produces glycosylated protein. All of the MAbs react with the full-length baculovirus-expressed protein. Immunofluorescence studies of Sf9 cells expressing gp350 suggest the protein is membrane associated. Five additional subclones which express different portions of gp350 in the Baculovirus expression system have been prepared and used for additional epitope mapping studies. Like the E. coli-expressed fragments these subgenomic proteins only react with 4 of the 14 mAbs which recognize the entire Baculovirus-expressed gp350. Deglycosylated gp350 retained reactivit with the mAbs, while denatured gp350 lost reactivity with most of the mAbs, suggesting that correct conformation rather than glycosylation is required for recognition. A manuscript describing these data is in press in J. Gen. Virol. Additional studies comparing the immunogenicity of native versus denatured baculovirus-expressed gp350 in rabbits are in progress.

重组糖蛋白疫苗目前正在开发中， 疱疹病毒，包括HSV和EBV，以及HIV。 EB病毒膜抗原 （EBV-MA）gp 350是一种含有病毒中和表位的糖蛋白 目前正在测试作为一种可能的EBV疫苗候选人， 英格兰 本研究制备了14株抗EBV-MA的单克隆抗体， 从路易斯·夸尔蒂尔和加里·皮尔森那里得到的。 这些已经被用来 筛选抗原表达克隆对不同表位的反应性， 为了鉴定对病毒重要的核酸序列， 中和 最初有10个重叠克隆， 在E.杆菌14个中的4个 检测的mAb与重组E.大肠杆菌抗原，和三个 抗原表位（BamH中的核苷酸1980-2307、3186-3528和3528-3576 （二）识别。 由克隆表达的蛋白质用于产生 兔抗血清。 当检测组织中EBV的中和作用时 培养，兔抗血清以及识别这三种抗体的mAb 表位未能中和，表明这些表位不是 参与中和。 因为剩下的单克隆抗体只能识别 gp 350的全长糖基化形式，整个gp 350编码序列， 也在杆状病毒表达系统中表达， 糖基化蛋白 所有的单克隆抗体都与全长 杆状病毒表达蛋白。Sf 9细胞的免疫荧光研究 表达gp 350表明该蛋白是膜结合的。 五 另外的亚克隆表达不同部分的gp 350， 已经制备了杆状病毒表达系统并用于另外的研究。 表位作图研究。 就像E大肠杆菌表达的片段， 亚基因组蛋白仅与14种mAb中的4种反应，所述mAb识别亚基因组蛋白。 完整杆状病毒表达的gp 350。去糖基化gp 350保留反应性 与单克隆抗体，而变性的gp 350失去了与大多数单克隆抗体的反应性， 表明需要正确的构象而不是糖基化 为了得到认可 描述这些数据的手稿在J. Gen. Virol. 比较天然与 在兔中变性杆状病毒表达的gp 350正在进行中。