We have developed technology that enables rapid, compI'ehensive and high-throughput analysis ofhumoral immune responses to infectious disease antigens that is being used fOf subunit vaccine antigendiscovery and to develop serodiagnostic tests. The method is based on printing microarray chips withproteins encoded by infectious microorganisms. Currently, we have expressed and printed more than 16,000individual proteins from 6 bacteria, 17 viruses, and 2 parasites. The chips are probed with serum frominfected patients and healthy controls to identify the dominant antibody responses, and identify a sel of 10-20serodiagnostic antigens for each infectious agent. By using a multiplicity of antigens for each infectiousagent, serodiagnostic tests with statistically improved sensitivity and specificity can be confIQured.Here we wiD fabricate whole proteome miaoarrays for Rickettsia prowazekH and R. rickettsii. The arrays'11 be probed with serum from well-<:haracterized infected humans and animals. and healthy controls 10identify the immunodominant and early reactive serodiagnostic antigen sets. The ability of this chip todistinguish welkharacterized sera collected from humans and animals infected with the different rlc:kettsiae'1 be determined and the results compared with established assays done on the same samples.
Specific Aims1. PCR and done 2246 ORFs from two rickettsiae.2. Express each ORF in Ihe cell free system and print each protein onto Microarray chips.3. Probe the microarrays with well characterized human and animal sera to identify the immunodominantantigen set for each agent.4. Transfer the immunodominant antigen sets to immunostrips and validate the assay perfonnance with wellcharacterized infected sera.The p1asmids, proteins. and protein mictoarrays generated from this project will be available to other RCEinvestigators ltv'ough the PSWRCE Protein Miaoarray Core. The validated immunostrips that result from thisproject win be transferred under a licensing agreement to a commercial business for GMP manufactUring.
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