摘要
目的 探讨体外筛选的呼吸道合胞病毒抗体逃逸株生长特性、F蛋白免疫反应性及在棉鼠体内对抗体免疫预防的抵抗 ,旨在证实用单克隆抗体免疫预防的负面效应 ,为今后临床可能出现的抗体逃逸株提供线索。方法 呼吸道合胞病毒 (RSV)A2株在逐渐升高的Palivizumab(PZ)浓度环境中连续传代 5次并经单噬斑纯化后获得PZ逃逸株MP4。用RT PCR法扩增A2及MP4全长F基因并自动测序 ;用微量中和试验鉴定MP4在体外对PZ中和反应的抗性 ;用Westernblot鉴定其F蛋白免疫反应性的变化 ;最后在棉鼠模型内观察PZ预防A2和MP4感染的效果。结果 与A2母株比较 ,MP4于F基因 82 8位核苷酸发生突变 (A→T) ,导致 2 72位推导氨基酸由赖氨酸变为甲硫氨酸。MP4在体外及在棉鼠肺内的生长能力无明显改变。MP4F蛋白虽仍可表达于受感染细胞膜上 ,但不再被PZ识别。动物实验证实 ,即使大剂量PZ亦不能预防MP4感染。结论 体外筛选的PZ逃逸株在棉鼠模型体内对同一抗体的免疫预防具有极强的抗性 ,类似情况是否会在人体内发生及是否会导致严重后果尚有待进一步研究。
Objective Palivizumab (PZ), a humanized monoclonal antibody against RSV fusion protein, is prophylactically given in certain high risk children. Expanding use in children and in individuals with ineffective cell mediated viral clearing will provide opportunities for the emergence of antibody resistant viruses. The ability of PZ resistant virus to evade PZ prophylaxis was evaluated in cotton rats. Methods A2 RSV was passed in culture in increasing PZ concentrations (0.4 to 40 μg/ml) and the resulting virus was characterized from a single plaque (MP4). Antigenecity, resistance to PZ in vitro and in vivo were assessed by use of Western blot, microneutralization assay and prophylactic experiment in cotton rats, respectively. Results MP4 was 100 fold less susceptible to PZ neutralization as compared to A2. The F protein gene of MP4 had a nucleotide substitution (base 828 A to T) that resulted in a coding change (amino acid 272 Lys to Met). Cotton rats were given prophylactic PZ and challenged with virus intranasally. The MP4 challenged rats had no reduction in lung viral titers as compared to rats that did not receive PZ. The A2 challenged rats revealed the expected PZ protection against RSV replication. Conclusion A virus resistant to PZ in culture was also resistant to the prophylactic effect of PZ in cotton rats. Whether PZ resistant viruses will occur in humans and result in clinical consequences is unknown.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2005年第3期222-226,共5页
Chinese Journal of Microbiology and Immunology
