Castilla J, Sola I, Pintado B, Sánchez-Morgado J M, Enjuanes L
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Madrid, Spain.
Adv Exp Med Biol. 1998;440:675-86. doi: 10.1007/978-1-4615-5331-1_87.
Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection against neonatal infections of the enteric tract.
口服病毒中和抗体可提供针对冠状病毒感染的保护。为了提供乳汁源性免疫，制备了18株分泌重组IgG1单克隆抗体（rIgG1）的转基因小鼠品系和10株分泌重组IgA单克隆抗体（rIgA）的转基因小鼠品系，这些抗体可在乳汁中中和传染性胃肠炎冠状病毒（TGEV）。编码单克隆抗体（MAb）6A.C3轻链和重链的基因在源自编码乳清酸性蛋白（WAP）和β-乳球蛋白（BLG）的小鼠基因组DNA的调控序列控制下表达，WAP和BLG是乳汁中含量非常丰富的蛋白质。MAb 6A.C3与几种冠状病毒中存在的一个高度保守表位结合。该单克隆抗体不允许选择逃避中和的病毒突变体。通过放射免疫分析测定，该抗体在转基因小鼠乳汁中的表达滴度为100万，并且以每毫升5至6毫克的免疫球蛋白浓度将TGEV感染性中和了100万倍。基质附着区（MAR）序列对于rIgG1转基因表达不是必需的，但MAR与抗体基因共显微注射导致在50%的所产生的rIgG1转基因动物中抗体滴度提高了20至10000倍。基因组BLG基因与rIgA轻链和重链基因共显微注射导致产生携带这三个转基因的转基因小鼠。整合了抗体和BLG基因的转基因小鼠产生的抗体滴度最高，尽管所产生的转基因动物数量不允许就BLG共整合的增强作用得出明确结论。抗体表达水平与转基因拷贝数无关，而与整合位点有关。产生在乳汁中具有病毒中和抗体的转基因动物可能是一种提供针对肠道新生儿感染保护的通用方法。
