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

重组糖蛋白疫苗目前正在开发中，有几种 疱疹病毒，包括单纯疱疹病毒和EB病毒，以及艾滋病毒。EB病毒膜抗原 (EBV-MA)gp350是一种含有病毒中和表位的糖蛋白 目前正在作为可能的EBV候选疫苗进行测试 英国。制备了14株抗EBV-MA的单抗。 从Louis Qualtiere和Gary Pearson那里获得。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。最初十个重叠克隆表达不同的 未糖基化的gp350的一部分在大肠杆菌中构建。14次中的4次 被测试的单抗与重组的大肠杆菌抗原发生反应，三个 抗原表位(Bam H中的核苷酸1980-2307、3186-3528和3528-3576 L)已确认。克隆表达的蛋白质被用来生产 兔抗血清。当检测组织中EBV的中和性时 培养、兔抗血清以及识别这三种病毒的单抗 表位未能中和，这表明这些表位不是 参与中和的。因为剩余的单抗可能只识别 全长糖基化形式的gp350，完整的gp350编码序列 也在杆状病毒表达系统中表达，该系统产生 糖基化蛋白质。所有的单抗都与全长的 杆状病毒表达的蛋白质。Sf9细胞的免疫荧光研究 表达gp350表明该蛋白是膜相关的。五 表达gp350不同部分的其他亚克隆 杆状病毒表达系统已被制备并用于另外的 表位图谱研究。就像这些大肠杆菌表达的片段一样 亚基因组蛋白只与识别该蛋白的14株单抗中的4株发生反应。 杆状病毒表达的完整gp350。脱糖基糖基化gp350保留复活剂 当变性的gp350失去与大多数单抗的反应性时， 提示需要正确的构象而不是糖基化 为了得到认可。一份描述这些数据的手稿正在《J.Gen. 维罗尔。比较天然和非天然免疫原性的更多研究 变性杆状病毒在兔体内表达的gp350正在进行中。

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.