Identifying serological markers for the diagnosis of acute infectious diseases with standard immunological approaches is laborious and requires, at a minimum, prior knowledge of the etiologic agents and the ability either to culture them as sources of antigens or to clone the appropriate antigens and produce recombinant proteins. The advent of phage-displayed peptide technology, in which large, complex libraries of filamentous bacteriophage bearing random peptide sequences on their coat proteins can be generated and screened with antibodies, has provided a new approach called “epitope discovery” that can circumvent these problems. In this approach, phage-displayed peptide libraries are screened with sera from patients who have suffered a particular disease or pathological condition to discover peptide epitopes that are specifically recognized by sera from patients with the same disease or condition (1
). The sequence of peptides from phage clones bearing diagnostic epitopes can be readily determined, and the peptide epitopes can be synthesized relatively cheaply to provide inexpensive diagnostic tests (18
Perhaps of equal importance, “epitope discovery” has the potential to identify the antigen(s) responsible for eliciting antibody responses in patients experiencing a particular infection or condition. In theory, “mimotopes” identified by antibody screening are likely to have some sequence similarity to epitopes on the original antigen that elicited the antibody response. Thus, by performing similarity searches of protein databases, one may be able to identify the original antigenic stimulus to the patient's immune system. This reverse-discovery approach has the potential to identify previously unknown antigens and/or new epitopes on already known antigens. Moreover, epitope discovery has the potential to identify antigens in diseases of unknown etiology that stimulate an antibody response. However, one obvious limitation of applying “epitope discovery” to the identification of antigens with diagnostic relevance is that databases of microbial protein sequences are by no means complete, and hence, similarity searches will miss any antigens that are not represented in the available databases. A second problem arises in that phage libraries displaying peptides with the potential for forming “constrained epitopes” due to sulfhydryl bonds between cystine residues in the peptide sequence may mimic the conformation but not the sequence of an eliciting antigenic epitope. This problem can be minimized by screening libraries displaying linear, unconstrained peptides but carries the risk of missing conformational epitopes that might be valuable as diagnostic reagents.
The value of “epitope discovery” in developing diagnostics for infectious diseases is strongly supported by the report of Kouzmitcheva et al. (10
), who identified a set of diagnostic peptides in patients with Lyme disease. They devised a protocol in which 12 different phage-displayed peptide libraries were first absorbed with immunoglobulin G (IgG) from pooled sera of healthy donors to remove epitopes recognized by normal human sera. The depleted libraries were then panned over all possible pairwise combinations of IgG purified from the sera of eight different Lyme disease patients. From the 336 sublibraries that were created by this panning procedure, they identified a set of 12 peptides comprising five different sequence motifs that could specifically identify Lyme disease patients. While the diagnostic sensitivity of any individual peptide was not more than 50%, the combined set of peptides was 100% sensitive and highly specific for the small cohorts (10 patients each) of Lyme disease and control patients that were tested.
We wished to evaluate the diagnostic utility of these peptides and to determine whether similarities existed between the peptide motifs and proteins in sequence databases that might reveal the bacterial antigens responsible for eliciting antibodies in patients with Lyme disease. The 12 peptide libraries employed by Kouzmitcheva et al. (10
) included random 8-mer and 15-mer linear peptides and 15-mer peptides containing two cystine residues spaced between 0 and 6 residues apart that could potentially introduce conformational constraints on the epitopes. Although searches conducted in the original report did not reveal any convincing sequence similarities with Borrelia burgdorferi
proteins or other bacterial proteins in the TIGR protein database, we found similarities between several of their peptide motifs (designated A, B, C, F, and H) and bacterial antigens in the NCBI database by utilizing the phi-BLAST search algorithm. The most notable similarity was between the motif A sequence and a nine-peptide C-terminal sequence in the VlsE antigen of B. burgdorferi
. We present evidence which indicates that this VlsE sequence represents a previously undiscovered epitope recognized by antibodies in Lyme disease patient sera.