Persistent bacterial infections:
Identification of immunogenic structures of Borrelia burgdorferi sensu lato and
Chlamydophila pneumoniae by phage surface display
Dissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften
an der Universität Konstanz (Fachbereich Biologie) vorgelegt von
Markus Müller
Tag der mündlichen Prüfung: 13.Februar 2004 1. Referent: Prof. Dr. Dr. Thomas Hartung
2. Referent: PD. Dr. Klaus P. Schäfer
for Mom, Dad and my brother
2004 at the chair of Biochemical Pharmacology at the University of Konstanz under the instructions of Prof. Dr. Dr. Thomas Hartung and at the Swiss Institute of Allergy and Asthma Research (SIAF) in Davos under the instructions of Prof. Dr. Reto Crameri.
I would like to address my thanks to:
Prof. Dr. Dr. Thomas Hartung for the continuous support, providing the excellent working facilities, including the attendance at conferences that contribute substantially to the success of this PhD thesis. I like to thank him for the freedom in developing and implementing my own scientific ideas. His continuing mentorship and support throughout the last years is strongly appreciated.
Prof. Dr. Albrecht Wendel for giving me the opportunity to perform my PhD thesis in his group, his constant encouragement and interest in the work. I like to thank as well for his commitment to the Graduiertenkolleg “Biomedizinische Wirkstoff-Forschung”.
Prof. Dr. Reto Crameri for supporting me all along, his personal encouragement.
I would like to thank for the excellent working facilities offered to me in Davos and the inspiring atmosphere in- and outside of the lab.
Dr. Corinna Hermann for her continuous help and support, valuable scientific discussions, representing an inestimable contribution to this thesis.
Dr. Inge Mühldorfer, Dr. Stefan Postius and Jean-George Thimm from Altana Pharma for their contribution and support to the fruitful development of the animal model.
All the present and former members of the Graduiertenkolleg “Biomedizinische Wirkstoff-Forschung” for the help- and fruitful discussion. I am indebted for financial support and the organization of outstanding seminars and courses.
All the present and former members of the chair “Biochemical Pharmacology” and of the Swiss Institute of Allergy and As thma research (SIAF) for technical assistance, helpful discussions and critical comments. They all contributed to the exceptional working atmosphere. I am grateful to Margarete Kreuer-Ullmann and Anne Hildebrand, for excellent technical assistance. Furthermore the general support of Gregor Pinski, Annette Haas and Ina Seuffert was very helpful.
Thomas Meergans, Isabel Diterich, Sebastian Bunk, Stefan Michelfelder and Rebekka Munke for their assistance.
Carolin Rauter, Michael Weichel, Claudio Rhyner, Sabine Flückiger, Sven Klunker, Jan Wohlfahrt, Anja Mayer, Verena Tautorat and Astrid Leja for activities in- and outside the lab, which contribute to this productive and enjoyable time.
Dr. Derya Shimshek, Dr. Sonja v. Aulock and Laura Stamp for the critical reading of the manuscript, Wolf-Dieter Weissenbühler, Gerhard Hönig and Jürgen Müller for the assistance in problems with “Microsoft” and the hardware.
All my friends for going with me this long way. Sonja Lotz for being at my side.
My parents and my brother for supporting me all along
Major parts of this thesis are submitted for publications:
M. Mueller, S. Postius, J. G. Thimm, K. Gueinzius, I. Muehldorfer, T. Hartung, and C
Hermann. Toll-like receptor 2 and 4 do not contribute to clearance of Chlamydophila pneumoniae in mice, but are necessary for release of cytokines by macrophages (submitted to Infect Immun.)
M. Mueller, I. Diterich, M. Weichel, C. Rauter, Dieter Hassler, Reto Crameri and Thomas Hartung. Phage surface display as a tool to identify novel Borrelia antigens for serodiagnosis (revised to J. Clin. Microbiol.)
M. Mueller, S. Michelfelder, I. Muehldorfer, K. Maehnss, M. Weichel, Reto Crameri, T.
Hartung and C. Hermann. Identification of antigenic peptides from a genomic random phage surface display library of Chlamydophila pneumoniae (submitted to J. Clin.
Microbiol.)
M. Mueller, R. Crameri, T. Hartung. The use of phage display affinity selection in the post-genome era (submitted to trends Mol. Med.).
Table of Contents
1 Introduction ... 1
1.1 Persistent pathogens ... 1
1.1.1 Borrelia burgdorferi sensu lato... 1
1.1.2 Chlamydophila pneumoniae... 4
1.1.3 Starting point... 7
1.2 General aspects of display technologies... 7
1.2.1 In vitro display systems ... 8
1.2.2 Eukaryotic display systems ... 9
1.2.3 Prokaryotic display systems ... 10
1.3 Overview of phage display ... 10
1.4 Aim of the thesis... 12
2 The use of phage display affinity selection in the post-genome era ... 15
2.1 Abstract ... 15
2.2 General aspects of display technologies... 16
2.3 General aspects of phage display... 17
2.4 Components of phage display... 19
2.5 Principle and applications of phage libraries ... 22
2.5.1 Random peptide libraries ... 22
2.5.2 Antibody libraries ... 23
2.5.3 cDNA libraries ... 26
2.5.4 Whole genome libraries... 27
2.6 Affinity selection strategies ... 28
2.7 Summary of phage display... 32
3.1 Abstract ... 35
3.2 Introduction... 36
3.3 Materials and Methods ... 38
3.4 Results ... 43
3.5 Discussion... 48
3.6 Acknowledgments ... 48
4 Identification of antigenic peptides from a genomic random phage surface display library of Chlamydophila pneumoniae... 53
4.1 Abstract ... 53
4.2 Introduction... 54
4.3 Materials and Methods ... 56
4.4 Results ... 63
4.5 Discussion... 68
4.6 Acknowledgments ... 70
5 Toll-like receptor 2 and 4 do not contribute to clearance of Chlamydophila pneumoniae in mice, but are necessary for release of cytokines by macrophages ... 71
5.1 Abstract ... 71
5.2 Introduction... 72
5.3 Results ... 73
5.4 Discussion... 79
5.5 Materials and Methods ... 81
6.1 Phage surface display and antigen identification in persistent infection... 89
6.2 The murine C. pneumoniae infection mode l ... 92
7 Summary ... 95
8 Zusammenfassung ... 99
9 Abbreviations ... 103
10 References ... 105
1 Introduction
1.1 Persistent pathogens
The two bacteria Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) and Chlamydophila pneumoniae (C. pneumoniae) can cause persistent infections in human beings. These infections are correlated with chronic, clinical manifestations. The diagnosis of infection with these bacteria is made on the basis of serological tests. Nearly all these tests use as basis crude extract derived from bacterial cultures leading to many false-negative and false-positive results. Therefore, the serodiagnosis based on crude extracts lacks a standardization to improve both sensitivity and specificity. Recently, there were some reports where recombinant proteins were used for diagnostic applications indicating that recombinant antigens might have a great potential to improve the diagnosis of B.
burdorferi s.l. and C. pneumoniae infections. However, until to date none of these recombinant proteins has been used in routine diagnostics.
1.1.1 Borrelia burgdorferi sensu lato
B. burgdorferi s.l., the causative agent of Lyme Borreliosis, is a Gram-negative corkscrew shaped, microaerophilic bacterium of the family of Spirochaetaceae.
B. burgdorferi was first described in 1982 by W. Burgdorfer (31). In Europe, there are at least three species pathogenic for humans (B. burgdorferi sensu stricto (B. burgdorferi s.s.), B. afzelii and B. garinii). B. valaisiana might also be pathogenic for humans, as suggested by positive PCR results obtained from a skin biopsy (300). The pathogen is transmitted into man by hard ticks (Ixodidae). At the site of the tick bite the infection
starts primarily with a local skin infection (Erythema migrans (EM)), then the spirochetes disseminate into the whole body. They can persist for years if untreated and may result in a range of clinical symptoms such as arthritis, neurological disorders, skin manifestations and arrhythmia (333). There are studies which showed an indirect evidence for the association of B. garinii with neurological symptoms (79), while infections with B. burgdorferi s.s. and B. afzelii tend to lead to arthritic symptoms (356) and cutaneous manifestations, respectively (35).
According to the Centre of Disease Control and Prevention, Lyme Borreliosis (LB) accounts for 95% of all reported vector-borne diseases in the United States and incidence estimations in endemic areas range between 16 and 140 / 100,000 inhabitants per year (161, 377). Individuals with a high risk of tick exposure (forest workers) frequently have significantly increased antibody titers against the pathogen (262, 375).
At present, the diagnosis of LB is made on the basis of the clinical picture and serological tests (102). For serodiagnosis a combination of a screening enzyme-linked immunosorbent assay (ELISA) as first step and a Western blot for confirmation as second step are recommended (44). In Western blot analyses antibodies against individual Borrelia antigens, which have been separated by gel electrophoresis, can be detected. However, a positive serological result without any clinical symptoms is not sufficient for the diagnosis of LB. Other than serological methods for detection of the pathogen like cultivation of Borrelia from patients’ specimen is difficult, since the pathogen occurs at low number in infected tissue and is difficult to cultivate due to long doubling time and the need of complex media (12, 61, 242, 322). Only for patients presenting an EM the cultivation of Borrelia or the direct detection via PCR from skin biopsies is a suitable method (203, 295, 322). But all these methods are confined to their special indications.
The interpretation, especially of Western blot bands and their intensity, is difficult, labor-intensive and not standardized (160). Many tests use crude extracts from the cultured Borrelia (different strains, different antigen preparations), which leads to further problems. One major problem is cross reactions with antibodies against other bacteria like Treponema, Ehrlichia or Epstein-Barr-virus and furthermore Borrelia proteins which are expressed only under human immune pressure are missed in these tests. In summary, the tests available are not standardized and the results of different test are not comparable due to these problems.
Commercial serological tests or in-house produced ones can not distinguish between active and inactive infection, because the IgG and IgM antibodies remain detectable over years at high levels, even after a successful antibiotic treatment (333). There is a hope that recombinant antigens might overcome these limitations.
Recombinant antigens would probably contribute to improve specificity and sensitivity of the serodiagnosis of LB. They would offer the advantage of easier identification of the bands in the immunoblot, since specific antigens can be selected, antigens from different strains can be combined, the test would be standardizable and the missing antigens could be added (378). Furthermore, by the use of truncated proteins cross reactions can be avoided. In the last years, there were many efforts in the identification of recombinant antigens and many different antigens were reported (118, 119, 142, 144, 203, 217, 232, 233, 277, 278). The first test with promising results was reported by Gomez et al. (118). They used chimeric recombinant proteins of outer surface protein A (OspA), p93, OspB, OspC and p41. The recombinant VlsE seems to be another very promising and useful antigen (142, 217, 233). In EM patients a higher sensitivity than those obtained with commercial ELISA was reached with the recombinant antigen BBK32 (143, 203). Despite the advantages of recombinant antigens and identical specificity, the recombinant blot has not yet shown the equal sensitivity of the whole-cell lysate blot. At present, the diagnosis is still far away from
the standardized serological test with high sensitivity and specificity. Further recombinant antigens will be needed to succeed.
LB is treated with antibiotics, but results are poor for treatments at late stages of the disease (220, 269, 334, 337, 338, 384). The difficulties in treatment of late stages and the high sero-prevalence of LB (268) call for a vaccination against Borrelia.
Immunizations with several different recombinant B. burgdorferi s.l. proteins have been performed. Immunization with OspA was found to be safe and effective in a large clinical trial in the United States (335). The vaccine efficacy in preventing clinical LB was 49% after two doses of the vaccine and 76% after three injections (327). The vaccine was approved by the Food and Drug Administration in 1998, but in 2002 the vaccine was withdrawn from the market. During this time, the vaccine was not tested in Europe, due to the presence of three B. burgdorferi species (in the United States only B. burgdorferi s.s. is found) resulting in heterogeneity of the OspA protein. The OspA vaccine gives rise to the hope that a combination of different recombinant proteins can lead to the development of a safe and worldwide usable vaccine.
1.1.2 Chlamydophila pneumoniae
C. pneumoniae, a Gram-negative, obligate intracellular pathogen was first isolated 1965 in Taiwan (125) and was classified in 1989 as a new species of the genus Chlamydiaceae within the species Chlamydia, and was named Chlamydia pneumoniae (now Chlamydophila). The organism represents a common respiratory pathogen, leading to sinusitis, bronchitis and pneumonia (122) and is believed to be responsible for about 10% of community-acquired pneumonia (4, 85, 122, 180). The pathogen occurs worldwide with a high sero-prevalence (up to 70% in elder adults), with primary infection in teenage years and rising prevalence with age (123). The high prevalence of antibodies against C. pneumoniae suggests that re-infections often occur (4, 123), but
among the sero-postive individuals there is an assumed persistence up to 80% (136, 230). Persisting C. pneumoniae have been found in vivo in blood monocytes (18, 22, 230). The development of persistence in vitro is characterized by a reduction of intracellular growth and by the appearance of morphologically aberrant reticular bodies (non-infectious but metabolic active replicating stage of the pathogen) (198). C.
pneumoniae is still metabolic active in this stage (1, 36, 246), but nothing is known about the duration and the mechanism of persistence.
Recent attention has focused on the association of C. pneumoniae and several chronic and destructive diseases of the lung (asthma) (5, 58, 207), the nervous system (331, 349, 390) and atherosclerosis (121, 222, 312-314, 364). Especially in the field of atherosclerosis there is a controversial discussion. Evidences for a link come from sero-epidemiological studies (i) and direct identification of the pathogen in the atherosclerotic plaques (ii). The only causal evidence comes from animal models (iii).
(i) sero-epidemiological studies
The microimmunofluorecence (MIF) test is the best established serological test for determining C. peumoniae infections and is considered as the current gold standard (83). Primarily, the MIF was developed for C. trachomatis, but was later adopted for the serodiagnosis of C. pneumoniae (126, 372). The MIF uses formalin-fixed chlamydial elementary bodies (EB) (infectious but non-replicating stage of the pathogen) immobilized to glass slides as antigens. The MIF allows the quantitative determination of specific IgA and IgG antibodies of human sera, but the MIF itself shows several limitations like variability of antigen preparation, requiring experience of the performing person and interlaboratory variations (281). Additionally, the specificity of MIF has been questioned by several investigators (135, 180, 186, 274). In recent years, C.
pneumoniae specific ELISA and EIA have been developed, which are more objective, standardized and easier to perform. However, a comparison of 11 serological assays, including MIF and ELISA, showed high variations in sensitivity and specificity (146).
Proving the link between atherosclerosis and C. pneumoniae infection by serology is difficult, because the epidemiological studies so far did not use standardized serological methods. As a result there are opposing findings (47, 71, 313) which can be explained by the different accuracies of the diagnostic methods used in these studies, strongly influencing the composition of the different collectives. A real understanding of the role of C. pneumoniae in atherosclerosis needs standardization of the performance of the serological tests.
(ii) direct identification of the pathogen in the atherosclerotic plaque
The first direct identification was reported in 1992 by Shor et al., who identified C.
pneumoniae in atherosclerotic material from post mortem examination by electron microscopy and PCR (326). Until today, over 40 different studies reported the detection of the pathogen in atherosclerotic material, whereas the pathogen was never found in healthy tissue (120). Furthermore, viable C. pneumoniae have been isolated and successfully cultured from atheromatous plaques (229). Because C. pneumoniae are only found in atherosclerotic lesions a causal role of C. pneumoniae in the development of atherosclerosis is suggested.
(iii) animal models
The most reliable causal proof for a role of C. pneumoniae in atherosclerosis derives from animal models. Several studies have focused on the use of well-defined animal (mice and rabbit) models of atherosclerosis to address the putative role of C.
pneumoniae infection (37, 39, 157, 247, 255, 256). Campbell et al. showed for example the accelerated formation of atherosclerotic plaques in hypercholesterolemia mice and rabbit models after C. pneumoniae infection (41). All these models use hyperlipidemia or animals with deficiency in the lipid recycling (apolipoprotein-E deficient mice) to show the association between C. pneumoniae infection and atherosclerosis. Cortison treatment was shown to recover C. pneumoniae weeks after the last infection in mice (236), indicating a persistence of the pathogen. In summary, C. pneumoniae is, like in
humans, found in foam cells of atherosclerotic lesions or at the sites of inflammation in the aorta by PCR, immunohistology and by culture (41). C. pneumoniae infection can accelerate the progression of atherosclerosis in combination with hyperlipidemia and initiate changes in the aorta of mice and rabbits (41, 100, 256). There are also a few other reports, investigating the immunopathogenesis of C. pneumoniae in animals (88, 179, 387, 388).
1.1.3 Starting point
Both pathogens lack a standardized and reliable serodiagnostic method for the detection of infections. Several approaches, e. g. identification by Western blot, have shown that recombinant antigens have the potential to improve the serodiagnosis of B.
burgdorferi s.l. and C. pneumoniae, but none are yet used in routine diagnostics. Thus a major goal would be the identification of new and relevant antigens of the pathogens.
The availability of the genome information and the various display and screening technologies offer a great opportunity for identification of protein-protein interactions especially the identification such relevant antigens.
1.2 General aspects of display technologies
The basis for all biological screening approaches relies on the exploitation of molecular interaction through the link between the gene integrated into the host genome and the encoded gene product displayed on host surfaces. They are all based on molecular libraries, differing in complexity and size. Display technology of complex molecular libraries spans two major groups: those based on biological compounds (DNA, RNA, peptides and proteins) and the synthetic ones based on compounds generated by combinatorial chemistry. Regardless of the format, a display library consists of three
elementary components: the displayed entities, a common linker and the corresponding individualized codes, normally a gene or a part of it (figure1) (215). The linkage between the phenotype (displayed entity) and the genotype (code) allows a rapid identification of molecules of interest based on the power of affinity selection. This technological platform speeds up discovery in life sciences because complex and time- consuming experiments at the protein level can be bypassed.
The biological display systems are divided into three different groups of display systems: first in vitro display (see 1.2.1), second eukaryotic display (see 1.2.2) and third prokaryotic display (see 1.2.3).
displayed linker code entity
displayed linker code entity
Despite the effort and success made in the development and optimization of the different display systems, it is unlikely that one single method will replace all the other display technologies. The different requirements of each screening have to be considered in detail to find the optimal solution for the given scientific question. The best result will be achieved by matching the parameters to the desired characteristics of the target and its interaction with the binding partner.
1.2.1 In vitro display systems
These systems are characterized by the absence of a biological host, and are sub- classified into three main types: ribosome display (137), mRNA display (342) and in
figure 1: The elementary components of phage display:
The code, the linker and the displayed entity.
vitro compartmentalization (128). For ribosome and mRNA display the code is RNA, and they need an extra step of RNA synthesis in a pro- or eukaryotic system. In vitro compartmentalization uses an external source of transcription and translation, and therefore the size of the library is only limited by the amount of genetic information, that can be added to the cell-free protein synthesis system. Therefore, all the in vitro display systems enable the construction of libraries that are several orders of magnitude larger than using viruses or cell system (359). This is one of the major advantages of these systems, allowing to display extremely large libraries. For the identification of antigens from pathogens like Borrelia and Chlamydophila, with a relatively small amount of different genes (B. burgdorferi s.s. (strain B31) 1701 different genes, C. pneumoniae (strain TW-183) 1114 different genes), the amount of gene products which can be displayed on the surface, is not limiting the approach. Hence, it is not necessary to choose one of these display systems.
1.2.2 Eukaryotic display systems
The eurokaryotic display systems offer the advantage of high fidelity folding of eukaryotic proteins, and the advantage of eukaryotic post-translational modifications.
There are at least six different display systems, including yeast-two-hybrid (25), yeast- cell-surface-display (21), display on mammalian retroviruses (28), fusion to an integral membrane protein of mammalian cells (52), Baculovirus-display (253) and display on Baculovirus infected cells (89). However, eukaryotic display systems are strongly limited by the low complexity of the molecular libraries that can be displayed. The complexity of the library size is several orders of magnitude lower than the complexity reached in prokaryotic systems (21, 105, 178). They are also not as easy to adapt for screening against patient’s IgG. Furthermore, the different systems showed several specific limitations, the yeast two-hybrid for example is not applicable to secreted
proteins of the cell-surface (389) and hence not the method of choice to display all bacterial proteins where most of the known antigens are surface located. Due to the lack of post-translational modifications in bacteria and the limitations of eukaryotic display systems none of these systems were chosen.
1.2.3 Prokaryotic display systems
Phage display, peptides-on-plasmid display (70), bacterial cell wall display (51, 115) and bacterial periplasmic display (50) belong to the prokaryotic display systems. From these methods phage display technology, first described by Smith (329), is the most robust and widespread used technology. The phage expresses the entity as a fusion with the coat proteins of the bacteriophage on their surface. The method is easy to handle and allows a rapid identification of binding partners to a ligand out of a large library. Furthermore, patient antibodies are widespread used for the identification of antigens and allergens from pathogens (64). Therefore, it appeared to be the most reliable method for the identification of bacterial antigens through display of all proteins encoded in the bacterial genome, if a cDNA or genomic library was used.
1.3 Overview of phage display
As mentioned, phage display is a powerful in vitro selection technique to study protein ligand interactions and to isolate target-specific ligands. The peptide or protein is genetically fused to a coat protein of a bacteriophage, resulting in display of the fused protein on the surface of the phage, while the DNA encoding the fusion products is integrated within the phage genome. This physical linkage between the displayed protein and the DNA encoding allows a fast screening of enormous numbers of different proteins or peptides (phage library), by a simple in vitro selection procedure
called affinity selection. The affinity selection aims the enrichment and identification of phage displaying protein which bind to a target. The target will be incubated with the phage libraries, non-bound phage are washed away and specifically bound phage particles eluted afterwards with different methods, like pH change (176, 303) or oxidising agents (127). Eluted phage are amplified in an amplification step, which includes infection of E. coli with eluted phage and consecutive rounds of affinity selection can be performed. Phage are analyzed at the end of each affinity selection procedure, to identify the genetic code of the displayed entity (figure 2).
CH1
ori ampRCH1
ori ampRELUTION PHAGE BINDING
WASHING
(i) infection (ii) superinfection
AMPLIFICATION TARGET
BINDING
(iii) phage library
CH1
ori ampCH1
oriR ampRCH1
ori ampCH1
oriR ampRELUTION PHAGE BINDING
WASHING
(i) infection (ii) superinfection
AMPLIFICATION TARGET
BINDING
(iii) phage library
figure 2: Phage display affinity selection cycle in its simplest form (biopanning).
The selection is performed in several steps: Immobilization of the target, secondly incubation of the library with the target and phage binding, thirdly washing to remove non-bound phage.
Elution of bound phage is the next step followed by amplification of target-specific phage. The amplification is devided in three steps infection of E. coli (i), followed by an superinfection with helper phage for phage assembly (ii), which results again in a phage library (iii). This library is now more specific for the target and can used for a next cycle of affinity selection.
1.4 Aim of the thesis
New recombinant antigens might improve the serodiagnosis of B burgdorferi s.l. and C.
pneumoniae. The phage display technology allows a rapid identification of binding partners to a ligand out of a large library. The aim of the thesis was the identification of new antigens for diagnosis or vaccination purposes by the use of phage display.
In this approach a random genome library instead of a cDNA library was chosen, because not all antigens are expressed during bacterial culture. Hence, the phage display method, using the pJufo vector system (68), was adapted to display whole genomes of Borrelia and Chlamydophila resulting in libraries containing all bacterial proteins in overlapping fragments.
To identify the antigens, serum IgG-antibodies from sero-positive patients with clinical symptoms were used to screen the libraries by affinity selection. Using patient sera only antigenic structures relevant in humans will be enriched (figure 3A).
The identified proteins should be expressed and tested for their specificity and sensitivity with patient and control sera.
For the purpose of possible vaccination studies and to learn more about bacterial infection, animal models were necessary. Since a Borrelia infection model was available in-house, a further aim was to establish a murine C. pneumoniae infection model (figure 3B).
A
bacteria
patient
random phage library
infection
antibodies
anti B
anti D
affinity selection
D D B
B
antigens affinity
selection
mouse model
vaccination
B
serodiagnosis DNA
A B C D
figure 3: Aim of the PhD thesis.
A: The whole genome of the bacterium (Borrelia or Chlamydophila) was partially digested and ligated into a phage vector to construct a random phage library, presenting all proteins of the bacterium. However, patients produce antibodies against a few of these proteins only. In the affinity selection these antibodies were used for the identification and enrichment of the antigenic proteins.
B: From the affinity selection, antigenic structures were obtained. These antigens might be useful for an improvement of the serodiagnosis, as well as for the vaccination.
2 The use of phage display affinity selection in the post-genome era
Markus Mueller1, Reto Crameri2, Thomas Hartung1*
1Biochemical Pharmacology, University of Konstanz, Germany
2Swiss Institute of Allergy and Asthma Research, Davos, Switzerland
submitted to Trends Mol. Med.
2.1 Abstract
Phage display is a powerful screening method for the identification of predominantly protein-protein interactions. The peptide or protein is genetically fused to a coat protein of a bacteriophage, resulting in display of the fused protein on the surface of the phage, while the DNA encoding the fusion products is integrated within the phage genome.
This physical linkage between the displayed protein and the DNA encoding it, allows a fast screening of enormous numbers of different proteins or peptides (phage library) and the identification of the genetic code from peptides, proteins or antibodies with a high binding specificity for a desired target structure.
This review explains the basis of phage display and discusses the contribution of the different phage library technologies to the post-genome era.
2.2 General aspects of display technologies
The enormous amount of genetic information generated by genome projects world wide, led to the identification of thousands of uncharacterised open reading frames (ORFs). Consequently, turning sequence information into function is the major challenge of the post-genomic era and is well depicted by the fact that, for example, 44% of the 223 genes on human chromosome 21 represent ORFs encoding putative proteins with unknown function (138). This new dimension in life science demands methods for a rapid characterisation of gene products and protein-protein interaction networks (213). The post-genomic approach starts from the diversity of molecules encoded in the genomes and searches for biologically relevant networks of interacting molecules. Knowledge about these interactions facilitates rapid progress in life sciences and leads to the rational development of new drugs.
The basis for all biological screening approaches represents the exploitation of molecular interaction through the link between the gene integrated into the host genome and the encoded gene product displayed on the host surface. They are all based on molecular libraries, differing in complexity and size. Display technology of complex molecular libraries spans two major groups: those based on biological compounds (DNA, RNA, peptides and proteins) and the synthetic ones based on compounds generated by combinatorial chemistry. Regardless of the format, a display library consists of three elementary components: the displayed entities, a common linker and the corresponding individualized codes, normally a gene or a part of it (215).
The linkage between phenotype (displayed entity) and the genotype (code) allows a rapid identification of molecules of interest based on the power of affinity selection.
These technological platforms speed up discovery in life science because complex and time-consuming experiments at the protein level can be bypassed. The biological
display systems are divided into three different groups of display systems: in vitro, eukaryotic and prokaryotic display systems.
Despite the effort and success made in the development and optimization of each of these different display systems, it is unlikely that one single method will replace all the other display technologies. The different requirements of each screening have to be considered to find the optimal solution, granting all the methods their place in the field of library screening. The best result will be achieved by matching the characteristics of each technique to the characteristics of the target and its desired interaction with the binding partner. Nevertheless, phage display represent the most often used screening method at the moment.
2.3 General aspects of phage display
Phage display (329) is a versatile in vitro selection technique to study protein ligand interactions and to isolate target-specific ligands. The peptide or protein is genetically fused to a coat protein of a bacteriophage, resulting in display of the fused protein on the surface of the phage, while the DNA, coding for the fusion product, is integrated within the phage genome. This physical linkage between the displayed protein and the DNA encoding it, allows a fast screening of enormous numbers of different proteins or peptides (phage library), by a simple in vitro selection procedure called "affinity selection" (also "panning"). By the use of this technique, binding partners from a large phage library to a known structure (target) can be identified. During affinity selection, phage, displaying binding proteins, can be separated from those displaying proteins without affinity for the target molecule. Therefore, a desired target will be incubated with the phage library, non-bound phage are washed away and specifically bound phage particles are eluted afterwards with different methods, like pH change (303) or oxidizing agents (127). Eluted phage are amplified in an amplification step, which
includes infection of e.g. E. coli with the eluted phage. Consecutive rounds of affinity selection can be performed, phage are analyzed at the end of each affinity selection procedure, to identify the genetic code of the displayed entity (figure 1).
CH1 ori amp
RCH1 ori amp
RELUTION PHAGE BINDING
WASHING
(i) infection (ii) superinfection
AMPLIFICATION TARGET
BINDING
(iii) phage library
CH1 ori amp CH1 ori
Ramp
RCH1 ori amp CH1 ori
Ramp
RELUTION PHAGE BINDING
WASHING
(i) infection (ii) superinfection
AMPLIFICATION TARGET
BINDING
(iii) phage library
Figure 1: The phage display affinity selection cycle.
The cycle has several steps, i.e. immobilization of the target, incubation and binding of phage to the target. Washing steps follow, where non-bound phage are removed, and afterwards specifically bound phage are eluted by conditions that disrupt the protein-target interactions.
Eluted phage are amplified by infection of E. coli und superinfection with herlperphage. This new "second" library can be used for a next affinity selection cycle. After several rounds of affinity selection the phage displaying proteins with binding affinity are dominant and selected phage are analyzed individually.
Phage display technology has been used for a broad range of applications like the finding of antagonists or agonist of receptors (17, 82, 148, 317), epitope mapping (151, 261), identification of diagnostic proteins (64, 162, 260), engineering of the binding affinity of proteins (33) or simply for the identification of interacting proteins (45). The wide range of possible applications has established phage surface display technology as one of the most often used technologies in modern molecular biology.
2.4 Components of phage display
Three different components play an essential role in phage display :
(i) The target: Any structure can be used as target molecule in phage display. In most of the cases a component of a known protein/ protein complex or antibodies are used.
(ii) The phage: Different strains (strain M13, fd or f1) from the Ff filamentous phage family are commonly used in phage display. However, most vectors have been developed from the backbone of the fd and M13 filamentous phage.
(iii) The phage library: The quality (size and complexity, up to 1011) and source of a library is important for the success of an affinity selection. The libraries can be divided into different groups: antibody libraries, peptide libraries, cDNA libraries and whole genome libraries.
One of the most important prerequisites for success in phage display is an efficient, smart and discriminative design of the affinity selection strategy. The common rule is that "you get what you select for". Therefore, it is strongly recommended to link the selection criteria as closely as possible to the desired attribute. This can be achieved by different affinity selection strategies.
(i) The Target
As a target for phage display any structure of interest can be used, but most of the applications report to the use of naturally occurring molecules like peptides, proteins
and antibodies. Recently, phage display has been used to identify peptides that selectively bind to a target of inorganic material (376). The immobilization of the target e.g. to a solid phase is necessary to separate the target-phage-complex from the non- bound phage. Different immobilization methods are described in the literature, like immobilization on polysterol surfaces (66, 279) or magnetic immobilization (299, 371).
In general, it is better to have a defined target, whose characteristics and properties are known, than an ill-defined target. But nevertheless, there are reports of successful panning by affinity selection against whole cells (263), or in whole organisms (280), highlighting the versatility of the technology. One of the major goals is to fit the affinity selection procedure to the desired characteristics of the target.
(ii) The phage
Phage, or more correct bacteriophage were first described in 1915 by Frederick Twort (354). Because of their ability to lyse their bacterial host they were named bacteriophage, according to the Greek word "phagein" for "to eat". In the following years many phage with different characteristics were discovered.
The non-lytic filamentous phage M13 has been the platform of choice for most phage display applications so far. Other phage display systems have been developed as well, including the phage T4 (170, 238), T7 (72, 156, 216, 381), λ (55, 131, 393) and others (111, 141, 159, 358). Filamentous phage M13 are mainly composed of the major coat protein pVIII, (~2700 copies per phage), which is only 50 residues long, and each of the remaining (minor) coat proteins, pIII, pIV, pVIII and pIX, which occur in low copy number (3-5 per phage) (figure 2) (for review see (310)).
In phage display the recombinant proteins are displayed on the phage surface as a fusion (via a linker or direct) either to the coat protein pIII (11) or the coat protein pVIII (59, 86, 153, 177, 396). Few reports have used as fusion partners pIV, pIX and pX (107, 108, 158, 169). The fusion to the phage coat protein can be reached by cloning the respective code (DNA) into the phage genome or into a phage genome vector
(248). If this vector codes only parts of the whole phage (for example only the coat protein used for the fusion) it is named phagemid (13, 155, 250).
9 0 0 n m
6 n m
5 4 3 0 0 0 5 5
4 3 1 2 5 3 3 6 5 0 0 n t s n u m b e r
k D a
p I I I p V I p V I I I p V I I p I X g e n o m e
(s i n g l e s t r a n d ) c o a t
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Figure 2: Filamentous phage for displaying foreign peptide or protein.
Cartoon of wild-type Ff phage with the major (pVIII) and minor (pIII, PVI, pVII, PIX) coat proteins (adapted from Irving et al. (163)).
Phagemid vectors, commercial or non-commercial, contain a bacterial origin of DNA replication, a phage coat protein with a multiple cloning site for the code and a phage packing signal (13, 155, 250). This results in the possibility to replicate in the bacterial host as a plasmid, and the ability to be packed into a phagemid particle or recombinant phage upon infection with helper phage (superinfection). The helper phage provides the necessary genes for viral structure proteins, packing and phage assembly, which were not encoded on the phagemid vector. The phagemids offer several advantages compared to vectors with whole phage genome. They are easier to clone and amplify in bacteria, easier to manipulate by molecular biological standard procedures, easier to isolate from bacteria as double stranded DNA directly accessible for further
modifications and they are more stable than filamentous phage which are prone to mutational deletions.
Phagemids are transformed into a bacterial host e.g. E. coli with a F-conjugated plasmid for amplification and/or modification. After infection of phagemids carrying bacteria with natural helper phage, the phagemid DNA is packed into phage particles which display the product of the heterologous inserts as fusion to a phage coat protein on the phage surface. Each vector system can bee classified as type 3 or type 8 depending on wether the fusion links with protein pIII or pVIII (185).
2.5 Principle and applications of phage libraries
2.5.1 Random peptide libraries
Combinatorial peptide libraries obtained by random synthesis of short nucleotides is a powerful tool to study peptide-protein interactions. The pIII coat protein tolerates fusion peptides in the range from six (323) up to 38 (183) amino acids in length. The fusion to pVIII seems to be limited to six to eight amino acids (187). The formation of stable secondary structures is strongly limited by the shortness of the presented peptides.
Several approaches to introduce stable secondary structures have been reported;
O'Neil et al. introduced flanking cystein residues in the oligonucleotide synthesis design for the formation of intramolecular disulfide bridges to generate constrained peptides (270) whereas others cloned the peptides into the loop of structural scaffold proteins (244). These libraries have been largely used in finding binding partners to a wide range of biologically relevant targets as reviewed (46, 397). For example, they were used to screen for peptides that bind to pathogen specific antibodies (95, 195, 243, 254, 264, 345, 394) or peptide that bind to bacterial surface, which are of diagnostic
value (162). The selected sequence can together with the genome information of the organism be used for the identification of the natural protein by homology comparison (184). But in several approaches no correlating protein in the databases could be identified and thus the identified peptides represent immunogenic mimitopes.
Nevertheless, these peptides provide diagnostic tools and are putative vaccine candidates. Ide et al. identified a peptide that binds to H7 flagellin, showing the diagnostic potential of these peptides (162). Furthermore, a neutralizing effect of peptides in animal experiments was shown (80, 353). Together with the availability of genome information and the developing protein-modelling, it may be possible to identify the corresponding proteins and their nonlinear epitopes.
2.5.2 Antibody libraries
The isolation of antibody fragments raised against specific targets still represent the most powerful application of phage display. The first successful affinity selection was reported in 1990 from Mc Cafferty (248). The combinatorial antibody libraries were obtained by cloning the V-genes (genes of variable heavy (VH) and variable light (VL) chains, obtained from B-cells of different lymphoid sources like blood, spleen, tonsils or bone marrow. Two different types of antigen-binding fragments have been displayed on phage (i) the bigger FAB-fragments (110, 155), and (ii) the smaller single chain variable fragments (scFv) (248). Antibody libraries will reduce the time and money required to produce antibodies and to obtain the corresponding gene fragments (75). Many libraries, representing three different types of immune repertoires (immune, naïve, synthetic) have been cloned and displayed on phage. Their immune repertoire is illustrated in figure 3.
(1) Immune repertoires / immunized donor
Libraries with an immune antibody repertoire represent a mirror of the natural immune response. They have the advantage of presenting only antibodies produced against real antigenic structures seen by the immune system. Therefore, relatively small libraries can be used for a successful affinity selection. Furthermore, the antibodies are normally affinity matured, increasing the number of high affinity antibodies (34, 57). On the other hand, an individual has to be immunized with the target and libraries are dependent upon the individual immunresponse of the donor. Additional affinity selection fails for toxic targets, self-antigens and nonimmunogenic structures. The major drawback of immune libraries is that every new target needs a new library.
Libraries from many different donors are described in literature. Mice (6, 57) are most frequently used as donor, but rabbits (206), chicken (385), sheep (49) and primates (348) and even such exotic animals like dromedary (209) were used as sources for the construction of antibody libraries displayed on phage surface.
(2) Naive repertoires / naive donor
Antibody libraries are constructed with mRNA from B-cells of non-immunized donors.
Therefore, they are independent of the donor's immunological background. Compared to libraries constructed starting from mRNA of immunized donors, naïve libraries offer several advantages: These libraries are not biased to a given antigen-specific immune
Figure 3: Sources of antibody libraries.
The circles represent the immune repertoire of the libraries from different sources. The libraries are from an immunized donor (1), from a naïve donor (2), or represent a library with a synthetic repertoire (3) (adapted from (297)).
response since donors have not been immunised and can be used for selection against different targets. Furthermore, antibodies against self-antigens, toxic structures and non-immunogenic targets can be identified during affinity selection. On the other hand, the complexity of these libraries must be higher (>109) than those derived from immunized donors to obtain high-affinity antibodies (362). So far most of these libraries have been constructed starting from human V-genes. However, there is no technical limitation in constructing V-genes from other species such as mouse, rat or rabbit.
(3) Synthetic repertoire
The antigen bindingsite of an antibody is determined by six complementary determining regions (CDR). Only one (the CDR3 of the heavy chain) of these six CDRs shows high structural variations (53) and contributes up to 70% to the affinity of the antibody to its antigen. Therefore, libraries with a synthetic repertoire are artificially constructed in vitro by assembly of the V and D/J genes randomizing the VH-CDR3 region (9). Other approaches like randomization of light and heavy chain CDR3 (48) and diversification all three loops in one VH-segment (109) were reported. One striking advantage of semi-synthetic antibody repertoire libraries is their antigen-independency, allowing affinity selection of binding molecules raised against almost every target. Although in most cases the selected antibodies are of low affinity, the sequence information can be used as starting point for the development of high affinity binding partners by further randomization of the amino acid sequence of the CDR's followed by further selection against the antigen.
Applications:
Antibodies derived from one of these libraries can be used for a broad range of applications, like ELISA, Immunoprecipitation, Western blot and immunohistochemistry.
Microarray technology for the analysis of protein interactions will be most important in the future (192) and will benefit from antigens or antibodies derived from phage display.
There are calculations that several hundred thousand different antibodies are needed to screen for example the whole human proteome (78, 152). In vitro selection of antibodies seems to be the only rational way for this, promising a great future of antibody phage libraries in the post-genome era. Furthermore, antibodies directed against pathogens, have a great diagnostic and prophylactic impact. Tessmann et al.
for example identified high affinity antibody fragments against Hepatitis C virus, with potential diagnostic and therapeutic value (347).
2.5.3 cDNA libraries
The cDNA based phage libraries present the whole diversity of structures expressed by any organism or tissue at the moment of mRNA preparation. The affinity selection can be performed with any desired ligand or protein. An advantage is that cDNA libraries only code for natural ligands thus avoiding selection of ligands that only mimic biological structures with affinity to the target molecule. In many phage display systems, the fusion to the coat protein has to occur at the N-terminal end because the C-terminus is essential for the integration of the phage gene product into the phage coat. Full length cDNAs carry stop codons at the 3´-end hampering a direct fusion to the N-terminus. Crameri et al. solved this problem by introducing the fos/jun leucin zipper interaction to provide a covalent link between the coat protein and the expressed cDNA products (66, 68). Other approaches for cDNA libraries are described in the literature: Hufton et al. or Jespers et al. for example fused the cDNA to the carboxy- terminal end of the phage coat protein pVI (158, 169), and more recently different fusion approaches aimed to allow display of cDNA libraries on phage surface have been proposed (14, 150, 315). However, their usefulness in practice remains to be demonstrated.
Display of cDNA products on phage surface is limited by the feasibility of expression of the gene products by the bacterial host and the necessity to translocate them into the periplasmic space of the host (245).
One promising application is affinity selection against sera from patients. Nothing has to be known of the interacting partners in this case. Although allergens selected using the serum of sensitised patients has been the major field of this application of cDNA display as exemplified by the successful isolation of IgE-binding proteins from the allergenic sources Aspergillus fumigatus (65, 145), Malassezia furfur (221), Coprinus comatus (26), peanuts (188, 189) and mites (87), many other ligands have been used to screen cDNA expression libraries as recently reviewed (64).
The use of purified protein targets in affinity selection of cDNA libaries, has been employed in large approaches. In the field of pathogens for example interacting Plasmodium falciparum proteins with the erythrocyte membrane have been identified, leading to better understanding of this infection (208).
2.5.4 Whole genome libraries
cDNA libraries represent only the sequences of genes expressed during the mRNA preparation, missing those which are not expressed in culture, e.g. only under the pressure of the host immune response. For some bacteria, like Borrelia, the different expression of proteins is well investigated (42, 320, 386), leading to the absence of a unknown number of proteins. The use of whole genome libraries, obtained from randomly fragmented DNA, will overcome these limitations, because all genes are present in overlapping fragments, independent of their expression level in the donor.
Because phage of whole genome libraries express smaller fragments, they might also overcome some of the limitations the E. coli expression machinery. On the other hand, they contain many fragments in the wrong reading frame or orientation, which increase
the required library size for a successful affinity selection. For small genomes, like bacterial ones, this is not a major concern, but in case of large genomes one needs to overcome these limitation. Zacchi et al developed a method for selecting functional open reading frames, leading to phage display vectors with functional ORFs fused to coat protein gene gIII (391), which might represent a promising way for large genomes.
The application of whole genome libraries so far includes beside the identification of epitopes (94, 276, 302, 379), the identification of yeast interacting proteins (147), the identification of fibrinogen binding proteins (166) and fibronectin and IgG binding partners (165, 166, 223, 259, 260). We identified for example several proteins of Borrelia with a potential role in diagnosis and vaccination (260).
The field of using whole genome libraries is just at the beginning offering some of the greatest opportunities to phage display technology in the post genome era.
2.6 Affinity selection strategies
The general aim of the affinity selection is the separation of binding clones from the non-binding ones. Depending on the chosen strategy, ligands with different properties will be enriched. The affinity selection strategy determines what will be selected.
Beside the strategy of phage selection, the size of the library has a great impact on the kind of phage which will be affinity-enriched. Va ughan et al. showed an increasing affinity of selected phage antibodies with increased library size (224, 361). Increasing the size of a library from 3x107 up to 1x1010 independent clones enlarges the affinity of selected antibodies from 106-7 M- 1 up to 108-10 M-1.
However, the size of any library based on biological information is limited by the number of sequences which can be transformed into biological systems. In nature antibodies undergo "affinity maturation" through natural selection of antibodies with high affinity for the antigen. Several phage display approaches have been developed to
mimic this process. The diversity of a coding sequence can be introduced by several methods, like error-prone PCR (74, 139, 219), passing through mutator E. coli strains (164, 228), chain- or DNA shuffling (62, 172, 241, 291, 392) or site directed mutagenesis (117, 318, 365). These techniques primarily have been developed for antibody libraries, but analogous techniques have been used to increase the diversity of peptide libraries.
The second great impact on the affinity of selected clones originales from the kind of selection strategy which is chosen for the affinity selection of the library. Some of the currently most commonly used selection strategies are illustrated in figure 4 and figure 5. One can distinguish between different forms of the target and the way by which bound phage are eluted. Phage can be eluted either non-specific or specific. For non- specific elution, bound phage particles are eluted with non-specific methods like change of pH (acidic or alkaline, gradient or one step) (176, 303), chaotropic agents or by oxidising agents (127) (see figure 4 (I)). Because non-specific adsorption to the matrix or to the ligand itself occurs, a more specific method, is the competitive elution of bound phage either by an excess of the ligand or by displacement with target specific antibodies (57, 252) (see figure 4 (II)). In different experimental setups binding affinity might not be the major goal and as a consequence thereof, the selection strategy needs to be adapted e.g. to use the properties of the ligand in elution (101) (see figure 4 (III). All modifications of the elution strategy have the reduction non- specific "background" phage as final goal.
The simplest form of affinity selection is the screening of the library against a solid phase immobilized target, also called “biopanning”. For biopanning the target is immobilized and incubated with the phage library. Non-bound phage are washed away during different washing steps and bound phage particles eluted afterwards (see figure 5 (I)). Other affinity selection strategies to reduce non-specific phage and to raise affinity are the reduction of the target concentration during the different rounds of
affinity selection or increasing the numbers of washing steps or the enhancing of the washing stringency. A higher washing stringency can be achieved by performing the affinity selection on a column carrying the immobilized ligand (see figure 5 (III)) or by introducing an additional selection step via a labeled target (see figure 5 (II)). This approach reduces also conformational changes of the target occurring during direct coating on solid phases and enhance the chance of success. Sometimes the non- specific background phage can overgrow the selected phage population. Therefore, a second affinity selection step can be required without amplification of the phage between the selection rounds (239, 240).
(I) unspecific (II) specific (III) functional (I) unspecific (II) specific (III) functional
figure 4: Overview of currently most popular elution methods in affinity selection.
After several washing steps elution can be either performed non -specific (I), specific with antibodies or an excess of antigen or (III) by specifc catalysis.
In some cases, it is hard or still not possible to purify the target, and in such cases selection strategies other than biopanning or column selection must be applied.
Methods for affinity selection of cognate phage on tissue, cells or immunoblot have been described (see figure 5 (VI)) for example for the selection of receptor-specific phage using whole cells (see figure 5 (IV)). Cells carrying extremely abundant target molecules, or a biased library will be necessary for successful selections on these complex target systems, otherwise phage binding to irrelevant cellular structures will overgrow the specific ones. Because this is not always possible, a double selection strategy should be performed: substractive selection of the phage libraries on cells with
down regulated expression of the target of interest is the method of choice in such cases (see figure 5 (V)). By subtractive selection, phage specific for other cell surface proteins, are depleted from the library and pre-selected phage can the be used in further rounds of affinity selection of target cells expressing the target of interest. Cells overexpressing a given target can be separated from low expressing cells before elution of specific phage, e.g. via FACS (76), further increasing the chance to isolate target-specific phage. A second method is the change of the cell type during affinity selection, if both cell types express the target of interest. By this procedure, phage specific for other proteins do not find a binding-partner on the second type of cells which express different patterns of surface proteins. However, this method will only lead to success, if the cells differ in most surface proteins. By panning on whole cells, phage binding to yet unknown targets have been isolated (212, 357). Another method, that aims at co-selection of proteins and their associated targets is the selectively infective phage (SIP) (171, 197, 330) (see figure (VIII)). In this case, the infective region of pIII (the last two amino terminal domains) is deleted, so that the phage are no longer infective for E. coli. The infectivity is restored by the conjugation of the target with these two domains. By this way only phage bound to the desired target can infect the bacterial cells and will be amplified, whereas the "background" phage will not be amplified because they are not able to infect the host. However, further understanding of the infection process will probably be needed for further improvements of this method. Pasqualini et al. and others (280,Johns, 2000 #333, 350) performed affinity selections in vivo by injecting a library directly into living mice (see figure 5 (VII)). This approach enables for example the identification of peptides that selectively bind to markers that delineate the vasculature of different organs, or specifically bind to tumors (for review see (194)).
The best chance for a successful affinity selection of cognate phage is warranted by the use of a well defined and pure target, because selection against complex targets is
more difficult due to small amounts of the target present in a complex mixture leading to high backgrounds of non-specifically bound phage (214). The above described selection strategies represent useful methods to overcome these problems (6).
Cambridge antibody technology's "pathfinder" selection is a novel phage display affinity selection strategy (273) (see figure 5 (IX)). Either the target or a ligand neighboured to it is conjugated with a peroxidase enzyme, which catalyzes the release of biotin tyramine free radicals. Phage bound in the proximity are biotinylated and can be selected with streptavidin. This tequnique has been used for the identification of antibodies specific for receptors (273, 339).
2.7 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
infection E. coli
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bio HR P biotin tyramid
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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
