Protein Microarray for Profiling Antibody Responses to Yersinia pestis Live Vaccine

Abstract

A protein microarray representing 149 Yersinia pestis proteins was developed to profile antibody responses in EV76-immunized rabbits. Antibodies to 50 proteins were detected. There are 11 proteins besides F1 and V antigens to which the predominant antibody response occurred, and these proteins show promise for further evaluation as candidates for subunit vaccines and/or diagnostic antigens.Plague, one of the most dangerous diseases, is caused by Yersinia pestis. The increasing possibility of antibiotic-resistant Y. pestis strains means that a vaccine effective against bubonic and pneumonic plague is urgently needed (12,14,18). The current interest is in developing plague vaccines that consist of purified protein subunits, with improved protection and reduced side effects (25,31,34). The F1 or V single-subunit vaccine and the F1 plus V combination vaccine have been shown to provide effective protection against bubonic and pneumonic plague in animal models (1,31,34). There may still exist other Y. pestis antigens that provide protection. These novel vaccine candidates in conjunction with F1 and V can be developed as multicomponent subunit vaccines (21). It may improve protection against F1-and/or V-antigen mutant but virulent strains. Therefore, evaluation of Y. pestis proteins beside F1 and V for their efficacy in inducing specific antibody in the infected animals or human patients is urgently needed for plague vaccine development.Genomic sequences of Y. pestis CO92 (27), KIM (9), and 91001 (30) have been released in the past 3 years. Decoding of whole-genome sequence provides unprecedented opportunities for vaccine design. The microarray immobilized with multiple antigens, using a simple fluorescence analysis, allows high-throughput parallel detection and quantification of multiple specific antibodies in a miniaturized, low-sample-consumption format. In this study, a microarray representing 149 Y. pestis proteins was developed to profile the serum antibodies of rabbits that were immunized with live plague vaccine, providing an overall picture of the immunogenicity of the proteins tested.Construction of an antigen microarray representing 149 Y. pestis proteins. In our present work, 202 genes were selected for cloning and expression (additional information is available at http://bioinflab.org/journals/ruifu/supplementary_TableS1 .pdf/). The digested PCR products of specific genes were cloned into expression plasmid vector pET-32a (Novagen), and recombinant plasmids were transformed into BL21(DE3) cells. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, 172 genes were successfully expressed in Escherichia coli. The expressed proteins were subjected to purification in a 96-well format using Ni-NAT agarose (QIA-GEN) according to Brauns' method with a few modifications (5). For quality control, the purified proteins were printed onto silylated glass slides (CEL) and incubated with Cy5-labeled antibody specific for the six-His tag. Only the proteins giving a signal-to-background ratio of ...

Current Trends in Plague Research: From Genomics to Virulence

“…Indeed, a recent protein microarray study showed that the Y. pestis-specific genes YPO0388, YO2090 and YPO2093 encoded proteins that induced significant antibody response in the immunized rabbit. 77 These results yield clues about the role of these genes in pathogenicity and immunity, and beg for further investigation into these possibilities and for the potential use of these immunogens as vaccine candidates.…”

Seromic profiling of ovarian and pancreatic cancer

“…Defined protein collections assembled from phage expression systems or purified from recombinant sources were used to detect antibody responses in the serum of ovarian (11,12), breast (13), colorectal (2), pancreatic (14), or lung (15,16) cancer patients, and some of these responses appear to have diagnostic significance. In addition, protein array tools are also useful to measure changes in antibody responses to vaccination or immunotherapies, although it has yet to be applied to the monitoring of cancer treatments (17,18). So far, studies have often concentrated either on small numbers of defined antigenic targets tested across large serum datasets, or on large numbers of proteins probed by a limited number of patient sera, likely because of prohibitive cost constraints and arduous handling of large data samples.…”

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

“…They produced antigen microarrays with 430 different proteins and overlapping peptides spanning the whole SHIV proteome and identified eight immunodominant epitopes. Antibody responses of immunized rabbits to Yersinia pestis vaccine was performed by Li and colleagues [168]. They applied protein microarrays harboring 149 Yersinia pestis proteins and were able to detect 13 imunogenic proteins.…”