A Genome-Wide Proteome Array Reveals a Limited Set of Immunogens in Natural Infections of Humans and White-Footed Mice withBorrelia burgdorferi

Abstract

Humans and other animals with Lyme borreliosis produce antibodies to a number of components of the agent Borrelia burgdorferi, but a full accounting of the immunogens during natural infections has not been achieved. Employing a protein array produced in vitro from 1,292 DNA fragments representing ϳ80% of the genome, we compared the antibody reactivities of sera from patients with early or later Lyme borreliosis to the antibody reactivities of sera from controls. Overall, ϳ15% of the open reading frame (ORF) products ( Infectious disease research has advanced rapidly with the accumulation of whole-genome sequences of pathogens and the subsequent use of genome-wide DNA microarrays to study gene expression. Equipped with arrays in different formats, investigators have identified different genes in a variety of pathogens that are more highly expressed in host animals or under in vitro conditions mimicking the in vivo environment. With few exceptions (27), these array studies have been performed with experimental animals, usually rodents, and in laboratory settings. Less is known about the proteins that are expressed during natural infections of humans or other host animals. Detection of a specific antibody during the course of infection is indirect evidence of in vivo expression by the pathogen. But the use of this approach to study large numbers of proteins has been largely limited to one-dimensional and two-dimensional gel electrophoresis of whole cells having an in vitro origin, followed by identification of the more abundant antigens by partial amino acid sequencing of reactive bands or spots and then searches of the databases (22,23,38,44,52,60,66).An alternative to using the pathogen itself as the source of the proteins is to produce recombinant polypeptides based on the deduced open reading frames (ORFs) of the pathogen's genome and to determine whether these polypeptides are antibody targets (11,61). A potential shortcoming of using this approach with cells, such as Escherichia coli or yeast (Saccharomyces cerevisiae) cells, is that some foreign proteins may not be expressed or the quantity may be insufficient. Felgner and coinvestigators increased the success rate for expression and decreased the cost with a high-throughput, cell-free, coupled transcription-translation system (26,29,30). Hundreds to thousands of individual recombinant proteins are printed on chips, which are then used to capture antibodies present in serum from infected individuals and other animals. The amount of captured antibody is quantified using a labeled secondary antibody. Whole-proteome microarrays have been employed in studies of experimental Francisella tularensis infections in laboratory mice (35,57) and of immune responses of humans to immunization with live vaccinia virus (26,29,31). McKevitt et al. (61) and Brinkmann et al. (11) used the enzyme-linked immunosorbent assay (ELISA) format to study the binding of antibodies of experimentally infected rabbits and people with syphilis to a nearly complete representation of the OR...

Identification of Potential Serodiagnostic and Subunit Vaccine Antigens by Antibody Profiling of Toxoplasmosis Cases in Turkey

“…Microarray Fabrication and Probing-Proteome microarrays were fabricated essentially as described previously (45)(46)(47) by PCR amplification of coding sequences in genomic DNA, followed by insertion of amplicons into a T7 expression vector by homologous recombination, and expression in coupled transcription-translations in vitro before printing onto microarrays. Rather than use cDNA as the PCR template, which may under represent genes expressed at low levels in vivo, we used genomic DNA and amplified exons separately, a strategy we have used previously with the malaria parasite (46).…”

“…Meanwhile, the array employed here represents only a fraction of the nearly 8000 genes encoded in the T. gondii genome. Although the protein microarray strategy has been used extensively in bacteria (45,47,53,63,64,67,68), its application to antigen discovery in eukaryotic genomes is relatively novel, having been applied previously only to Plasmodium falciparum (46,69). Of the 15 known serodiagnostic antigens listed above, the smallest exons of four were represented on the current chip, namely GRA1 (070250_1 and _2), GRA2 (027620_2), GRA5 (086450_1), and MIC5 (077080_1).…”

Immunodominant “Asymptomatic” Herpes Simplex Virus 1 and 2 Protein Antigens Identified by Probing Whole-ORFome Microarrays with Serum Antibodies from Seropositive Asymptomatic versus Symptomatic Individuals

“…On the other hand, standard criteria for array production and data normalization with noise models, variance esti-mation, and differential expression analysis techniques have become powerful tools for the interpretation of results. Based on this progress, successful attempts have been made using protein microarrays for profiling the antibody responses to numerous infectious pathogens (3,21,40,54,66).…”

A Burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens

“…We have previously developed array technology that allows protein microarrays to be constructed from the predicted proteome of a microorganism (3)(4)(5)(6)(7). These arrays can be used to address basic questions about the interactions of a given pathogen with the host immune system (8)(9)(10), and allows the identification of proteins which can be used as diagnostic reagents or for inclusion in vaccines (3).…”