Summary of phage display

The phage display technology was introduced nearly 20 years ago, but is still at the starting point of its exploitation. Phage display offers a powerful platform technology for the rapid identification of interacting peptides, proteins or antibodies. The molecules can be selected and identified even in 2 to 4 weeks. The possible advantage of phage display technologies has been discussed in several fields. The diagnostic field will benefit from phage libraries, by identification of molecules that are unobtainable in such short time by classical methods. However, other areas like in the field of tumor research (395), in drug discovery (205, 227) or allergen identification (64) represent promising applications. Phage display will play a major role in the post-genome era, where the identification of function and characterisation of proteins are in the focus of research.

(I) biopanning (II) tagged target (III) antigen column

(VII) in vivoantigen (VIII) selective (IX) pathfinder infective phage

(IV) direct on cells (V) substractive (VI) on tissue/blots

binding

figure 5: Overview of currently most popular selection strategies.

(I) The simplest form is the affinity selection on target adsorbed onto a solid support. (II) To avoid conformational changes and crease specifity, selection of specific antibodies to biotinylated antigens is preferable. Bound and unbound phage are separated using streptavidin coated magnetic beads. (III) Immobilization on a column can be performed to allow to increase washing stringency. (IV) Selection on cells can be done directly by performing affinity selection on whole cells (V) or a substractive approach can be performed, e.g the cells of interest are separated via FACS. (VI) If the target is unknown or not purifiable, e.g. blots or even tissues or organs can be used for specific phage affinity selection. (VII) In vivo selection, (VIII) infection-mediated or (IX) the pathfinder method are possible procedures (adapted from Hoogenboom et al. (154)).

3 Phage surface display as a tool to identify novel Borrelia antigens for serodiagnosis

Markus Mueller¹, Michael Weichel², Isabel Diterich¹, Carolin Rauter¹, Dieter Hassler³, Reto Crameri², Thomas Hartung¹

1University of Konstanz, Biochemical Pharmacology, Germany,

2Swiss Institute of Allergy and Asthma Research, Switzerland and

3Private Practice, Kraichtal, Germany

revised to J. Clin. Microbiol.

3.1 Abstract

The serodiagnosis of Lyme Borreliosis (LB), the most common tick-borne disease, is still unsatisfactory. Serological tests using whole Borrelia lysate as antigen have several shortcomings including heterogeneity of antigen preparations and lack of standardization. The serodiagnosis could be substantially improved with recombinant and specific standardized antigens.

Here we present the application of a genomic phage surface display library as a tool to identify antigens with potential diagnostic value. This technique allows rapid isolation of even rare gene products from complex genomic or cDNA libraries by affinity selection using patient sera.

Random, genomic phage surface display libraries were generated from the three pathogenic European Borrelia strains. The libraries (>3x 10⁶ independent clones) were

enriched by affinity selection with a serum IgG pool (panning pool) derived from six patients suffering from different stages and manifestations of LB. Restriction analysis of the clones obtained after affinity enrichment of the phage library revealed nine different Borrelia proteins. One clone carried an open reading frame coding for the known antigenic protein BBK32 known to be immunogenic in Lyme Borreliosis. From the 9 identified proteins, two proteins (ctc, flgL) were produced sucessfully in E. coli and purified. Ctc showed higher reactivity with sera from 22 LB patients in ELISA than with sera from 13 seronegative controls. These data suggest that this identified protein represent a relevant antigenic structures of Borrelia.

Phage display technology represents a powerful platform technology to identify new antigens from the genome of pathogenic organisms by affinity selection using sera from affected patients.

3.2 Introduction

LB is increasingly recognized to cause chronic disease such as arthritis, neurological disorders, skin manifestations and arhythmia (333). LB is diagnosed based on clinical signs and patient history and confirmed by laboratory tests. Cultivation of Borrelia from patients' specimens is difficult: the pathogen is not found in blood, it occurs at low numbers in infected tissues, it grows slowly with a doubling time of around 20 h, and it requires expensive, complex culture media (61, 242, 322). Other direct methods to detect Borrelia like dark field microscopy or polymerase chain reaction (PCR) lack sensitivity, specificity and/or standardization (27, 118). Serodiagnosis still represents the method of choice despite several shortcomings such as the heterogeneity of the antigen preparations and lack of standardization. In Europe there are at least three species of Borrelia known to be pathogenic for humans, i.e. B. afzelii, B. garinii and B.

burgdorferi sensu stricto (s.s.), the latter being the only species found in the United

States (10). The high variability of antigen expressed by each strain, e.g. outer surface protein C (OspC) (202), further complicates the development of suitable diagnostic antigens.

It is well established that Borrelia express different surface components depending on temperature (320), pH (42) and cell density (386). Through differential gene expression the pathogen is able to adapt to different hosts and culture conditions (73, 97, 340).

Therefore the use of antigens derived from Borrelia cultures for serodiagnosis might not represent the antigens which are expressed in the human host. Although some Borrelia antigens are available as recombinant antigens, crude antigen extracts, i.e.

lysates of whole Borrelia cultures, are still used predominantly for serodiagnosis, despite considerable cross-reactivity in ELISA, mainly due to non-specific anti-flagellin antibodies (2, 234). Therefore, ELISA results need confirmation by immunoblot analysis in which individual antigens are separated according to their molecular mass to judge the specificity of the respective antibody response (332). A further limitation of these tests is the lack of discrimination between active and past LB (332). In addition, the variability of the antigen preparations in different serological tests and test kit lots, the lack of immunoblot standardization for both performance and interpretation, results in labor-intensive and subjective outcomes (160) and hampers long-term monitoring of therapeutic effects.

The relevance of LB becomes evident by its high seroprevalence in Europe and the United States (268, 272, 288), the high rate of infected ticks in endemic areas (up to 35%) (293), the putative link to diverse manifestations (334) and the poor results of antibiotic treatment at late stages of the disease (220, 337, 338, 384), calling for an improvement of diagnostic methods.

Here, a new strategy to identify specific and reliable antigens was developed. Random genomic libraries of three Borrelia species were generated and cloned into a phage surface display vector. The pJuFo phage surface display technology (68, 69) has

previously been successfully applied to identify allergens from cDNA libraries of Aspergillus fumigatus (65, 145), Malassezia furfur (221), Cladosporium herbarum (373, 374), Alternaria alternata (373), Coprinus comatus (26), peanuts (188, 189) as well as mites (87). Here, we extended this approach to identify phage displayed antigens from genomic Borrelia expression libraries by selecting with patients` IgG.

A pool of sera from patients suffering from LB was used to enrich phage encoding serologically relevant antigens. The respective full-length proteins were deduced from the nucleotide sequence after comparison with the published Borrelia genome (103).

Two proteins (ctc and flgL) were produced recombinantly and tested as to their suitability as antigens for serodiagnosis to show the potential of phage surface display as a rapid method for the identification of diagnostic targets from pathogenic organisms.