Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery.
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.
Davies
D Huw
DH
Center for Virus Research, University of California, Irvine, CA 92697, USA.
Liang
Xiaowu
X
Hernandez
Jenny E
JE
Randall
Arlo
A
Hirst
Siddiqua
S
Mu
Yunxiang
Y
Romero
Kimberly M
KM
Nguyen
Toai T
TT
Kalantari-Dehaghi
Mina
M
Crotty
Shane
S
Baldi
Pierre
P
Villarreal
Luis P
LP
Felgner
Philip L
PL
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
D000914:Antibodies, Viral; D000956:Antigens, Viral; D020543:Proteome; D014612:Vaccines