title: Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery.
abstract: Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immune-response profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naive humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naive mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.
authors.affiliation: Center for Virus Research, University of California, Irvine, CA 92697, USA.
authors.forename: D Huw
authors.initials: DH
authors.lastname: Davies
authors.forename: Xiaowu
authors.initials: X
authors.lastname: Liang
authors.forename: Jenny E
authors.initials: JE
authors.lastname: Hernandez
authors.forename: Arlo
authors.initials: A
authors.lastname: Randall
authors.forename: Siddiqua
authors.initials: S
authors.lastname: Hirst
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Mu
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Romero
authors.forename: Toai T
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Mina
authors.initials: M
authors.lastname: Kalantari-Dehaghi
authors.forename: Shane
authors.initials: S
authors.lastname: Crotty
authors.forename: Pierre
authors.initials: P
authors.lastname: Baldi
authors.forename: Luis P
authors.initials: LP
authors.lastname: Villarreal
authors.forename: Philip L
authors.initials: PL
authors.lastname: Felgner
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000917:Antibody Formation / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D006801:Humans; D007239:Infections / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D011323:Primates; D040081:Protein Array Analysis / Q000379:methods*; D020543:Proteome / Q000276:immunology; D012698:Serologic Tests / Q000379:methods; D014612:Vaccines / Q000276:immunology; D014616:Vaccinia virus / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D020543:Proteome; D014612:Vaccines