Kousoulas K G, Pellett P E, Pereira L, Roizman B
Virology. 1984 Jun;135(2):379-94. doi: 10.1016/0042-6822(84)90194-6.
Three classes of HSV-1(F) mutants expressing a resistance phenotype to two highly potent-type common monoclonal antibodies, H126-5 and H233, to glycoprotein B (gB) were selected. Class 1 mutants, selected for resistance to neutralization from nonmutagenized virus stocks, expressed a gB which reacted in biotin-avidin-enhanced surface immunoassays and in immune precipitation tests with the selecting antibodies. Class 2 and 3 mutants were selected for nonreactivity in the biotin-avidin-enhanced surface immunoassay from BUdR-mutagenized, preneutralized virus stocks, but differ in that the selecting antibodies immune precipitated the gB of Class 2 but not that of Class 3. Mutants expressing a resistance phenotype to one monoclonal antibody (H126-5 or H233) invariably retained reactivity in all tests with the heterologous antibody, and recombinants resistant to both antibodies were produced by cotransfection of intact DNA of one mutant with a cloned DNA fragment from another mutant. Class 1 mutations were mapped by marker transfer to a 1734-bp DNA fragment. Class 2 and 3 mutations were mapped to a region defined by a maximum of 377 bp and a minimum of 46 bp, in a biotin-avidin-enhanced surface immunoassay with a panel of DNA fragments of HSV-1(F) BamHI G carrying staggered deletions across the region encoding gB. This region does not overlap the neutralizing antibody determinant site mapped by T.C. Holland, R.M. Sandri-Goldin, L.E. Holland, S.D. Marlin, M. Levine, and J. Glorioso (1983, J. Virol. 46, 649-652) and is located 3' to the ts lesion of HSV-1(HFEM)tsB5 and 5' to the syn3 locus of that virus. It was concluded that (i) inasmuch as the biotin-avidin-enhanced surface immunoassay does not destroy the virus contained in the plaque, it is a rapid and convenient method for both identification and selection of mutants reactive and nonreactive to specific monoclonal antibodies. (ii) gB may contain multiple domains carrying epitopic sites of neutralizing monoclonal antibodies. (iii) The resistance phenotype may arise from mutations which alter the conformation or the amino acid sequence of the epitope. These mutations might be differentiable on the basis of reactivity of mutated gB with selecting monoclonal antibody in nondenaturing and denaturing environments, respectively.
我们筛选出了三类对两种高效通用型单克隆抗体H126 - 5和H233具有抗性表型的单纯疱疹病毒1型（HSV - 1(F)）突变体，这两种抗体针对糖蛋白B（gB）。第1类突变体是从未经诱变的病毒株中筛选出的具有中和抗性的突变体，其表达的gB在生物素 - 抗生物素蛋白增强的表面免疫测定以及与筛选抗体的免疫沉淀试验中均有反应。第2类和第3类突变体是从经5 - 溴脱氧尿苷（BUdR）诱变且预先中和的病毒株中筛选出的，在生物素 - 抗生物素蛋白增强的表面免疫测定中无反应，但不同之处在于筛选抗体能免疫沉淀第2类突变体的gB，而不能沉淀第3类突变体的gB。对一种单克隆抗体（H126 - 5或H233）具有抗性表型的突变体在所有与异源抗体的试验中始终保持反应性，并且通过将一个突变体的完整DNA与另一个突变体的克隆DNA片段共转染产生了对两种抗体均有抗性的重组体。通过标记转移将第1类突变定位到一个1734 bp的DNA片段上。在使用一组携带编码gB区域交错缺失的HSV - 1(F) BamHI G DNA片段进行的生物素 - 抗生物素蛋白增强的表面免疫测定中，将第2类和第3类突变定位到一个最大为377 bp、最小为46 bp的区域。该区域不与T.C. Holland、R.M. Sandri - Goldin、L.E. Holland、S.D. Marlin、M. Levine和J. Glorioso（1983年，《病毒学杂志》46卷，649 - 652页）所定位的中和抗体决定簇位点重叠，并且位于HSV - 1(HFEM)tsB5的温度敏感（ts）损伤位点的3'端以及该病毒syn3位点的5'端。得出的结论是：（i）由于生物素 - 抗生物素蛋白增强的表面免疫测定不会破坏噬斑中所含的病毒，所以它是一种快速且便捷的方法，可用于鉴定和筛选对特定单克隆抗体有反应和无反应的突变体。（ii）gB可能包含多个携带中和单克隆抗体表位的结构域。（iii）抗性表型可能源于改变表位构象或氨基酸序列的突变。这些突变可能分别根据突变的gB在非变性和变性环境中与筛选单克隆抗体的反应性来区分。
