Lopez Job E, Beare Paul A, Heinzen Robert A, Norimine Junzo, Lahmers Kevin K, Palmer Guy H, Brown Wendy C
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, United States.
J Immunol Methods. 2008 Mar 20;332(1-2):129-41. doi: 10.1016/j.jim.2007.12.018. Epub 2008 Jan 28.
The ability to rapidly screen a complex pathogen proteome for proteins that elicit recall T-lymphocyte responses from immune individuals would accelerate vaccine development. An outer membrane fraction of the rickettsial pathogen Anaplasma marginale induces protective immunity against infection and disease in cattle. We have used this immunization model to evaluate high-throughput screening of proteins expressed by in vitro transcription and translation (IVTT) for recognition by memory CD4(+) T-lymphocytes. Fifty selected vaccine candidate antigens identified from the A. marginale genome were expressed from transcriptionally active PCR products using an Escherichia coli-based IVTT system, and bead-affinity purified using antibodies to His and FLAG epitope tags. IVTT-expressed bead-bound antigens were processed and presented by antigen presenting cells to T-lymphocytes from outer membrane immunized animals and evaluated for immunogenicity in proliferation assays. Antigens that consistently stimulated responses were known T-cell antigens major surface protein (MSP)2, MSP3, VirB9, and VirB10 and newly identified T-cell antigens outer membrane protein (OMP)4, OMP9, elongation factor-Tu, Ana29, and OMA87. Specific T-cell stimulation was achieved even at low antigen concentration, and was highly sensitive when compared with unbound IVTT reaction products. This method allows rapid expression and identification of T-lymphocyte antigens for any pathogen for which the genome sequence is available.
能够快速从复杂的病原体蛋白质组中筛选出能引发免疫个体回忆性T淋巴细胞反应的蛋白质，将加速疫苗的研发。边缘无形体这种立克次氏体病原体的外膜组分可诱导牛产生针对感染和疾病的保护性免疫。我们利用这种免疫模型来评估通过体外转录和翻译（IVTT）表达的蛋白质进行高通量筛选，以检测记忆性CD4(+) T淋巴细胞的识别情况。从边缘无形体基因组中鉴定出的50种选定疫苗候选抗原，使用基于大肠杆菌的IVTT系统从转录活性PCR产物中表达出来，并使用针对His和FLAG表位标签的抗体进行磁珠亲和纯化。IVTT表达的磁珠结合抗原由抗原呈递细胞处理并呈递给来自外膜免疫动物的T淋巴细胞，并在增殖试验中评估其免疫原性。持续刺激反应的抗原包括已知的T细胞抗原主要表面蛋白（MSP）2、MSP3、VirB9和VirB10，以及新鉴定的T细胞抗原外膜蛋白（OMP）4、OMP9、延伸因子-Tu、Ana29和OMA87。即使在低抗原浓度下也能实现特异性T细胞刺激，与未结合的IVTT反应产物相比，其敏感性很高。这种方法能够快速表达和鉴定任何具有可用基因组序列的病原体的T淋巴细胞抗原。
