Expression Libraries as Tools for the
Development of Subunit Vaccines and Novel
Detection Molecules for Orthopoxviruses
vorgelegt von
Diplom‐Ingenieurin
L
ilija Mille
r
aus Berlin
Von d
er Fakultät III – Prozesswissenscha
ften
der Technischen Universität Berlin
z
ur Erlangung des akademischen Grades
Doktorin der In
genieurw
issenschaften
‐Dr.‐Ing.‐
genehmigte Dissertation
romotionsausschuss:
P
er:
Vorsitzend
Prof. Dr. L.‐A. Garbe
r
Berichter:
Prof. Dr. R. Lauste
Berichter:
PD Dr. A. Nitsche
erichter:
Prof. Dr. J. Kurreck
B
ag der wissenschaftlichen Aussprache: 02. Dezember 2011
T
Berlin 2011
D83
“You can accurately judge the research of others only after you’ve done your own and can understand the messy reality behind what is so smoothly and confidently presented in your textbooks or by experts on TV.” [1]
To my parents, my sister and my other half
Acknowledgments
As with all projects in life, this one wouldn’t have been possible to complete without the help of many persons, including my dear colleagues but also friends and family. It is thus highly likely that I won’t be able to acknowledge every single contribution by name. Therefore, in order to do justice, I would first like to thank everyone who helped me through this demanding but interesting time, independent of the kind of support! I am grateful to Andreas Nitsche for giving me the opportunity to do my PhD project and for continuously providing motivation and support. Sincere thanks to my colleague, my best friend, and my fellow in misery Daniel Stern! Thank you for the innumerable, valuable scientific discussions, for frequent encouragement and for always having a sympathetic ear. Without you the time would have been much harder! Further, I’d like to thank all members of the ZBS1 group for the friendly atmosphere, company during lunch, and the badminton tournaments.A great contribution to this thesis was done by the students I supervised during my PhD project. Lisa Schlicher, Isabel Choschzick, and Christoph Hapke contributed to the “Expression library project” during their internships. Marco Richter and Christoph Hapke further contributed to this project by completing their master’s thesis or bachelor’s thesis, respectively. Johannes von Recum contributed to the “Phage Display project” within the scope of his master’s thesis. I am thankful to Janine Michel, who helped me with phage display selections and subsequent peptide characterization. Wojtek P. Dabrowski contributed to the same project by programming the “Library Insert Finder” software and thus considerably simplified the search for the random peptide sequences.
I am also grateful to the staff of the photo laboratory for taking great pictures of various agar plates and nitrocellulose filters and for printing my posters.
Jung‐Won Sim‐Brandenburg and Delia Barz gave me excellent technical assistance, whereas the staff of the sequencing lab, namely Julia Tesch, Silvia Muschter, Julia Hinzmann, and Marlies Panzer provided me with numerous DNA sequences. I am grateful to all of you for becoming more than colleagues but also valuable friends.
Sincere thanks to Ursula Erikli for copy‐editing and continuous suggestions for improving my English language skills.
This work was funded within the BMBF/VDI‐financed BiGRUDI network of the Ro of bert Koch Institute (RKI; Berlin). In this context I am also thankful to the members the “Aptamer work group” for the valuable scientific discussions. Most grateful I am to my parents who shaped me to be the person I am today. They always give me the feeling of being the best person in the entire world and thus help me to overcome moments of uncertainty. Last but not least, I am grateful to my partner for sharing the cheery moments with me as well as for supporting me in times of despair. Thank you so much for always having the right word at the right moment and for all the cooking.
Declaration of Authorship
I certify that the work presented here is, to the best of my knowledge and belief, original and the result of my own investigations, except as acknowledged. The present work has not been submitted, either in part or whole, for a degree at this or any other University. Parts of this work have been published under the following title:
Miller, L., Richter, M., Hapke, C., Stern, D., & Nitsche, A. (2011) Genomic Expression Libraries for the Identification of Cross‐Reactive Orthopoxvirus Antigens. PLoS ONE 6(7): e21950. Epub 2011 Jul 14. erlin, B Lilija Miller
nservation among orthopoxviruses.
Additionally, combinatorial phage display methodology was utilized to identify poxvirus‐specific peptide ligands as novel detection molecules. Affinity selections of random peptide phage display libraries against infectious virus particles yielded 17 recurring peptide sequences indicating an enrichment of poxvirus‐binding phage clones. After characterization of these 17 binding clones, three peptide sequences were synthesized and characterized further. Thereby, one phage‐derived synthetic peptide was shown to bind selectively and specifically to vaccinia virus particles. This provides important insights into applicability of synthetic molecules for detection of biothreat agents.
Abstract
The global eradication of smallpox led to the cessation of routine smallpox vaccination due to rare but severe adverse reactions. Today, more than 30 years later, the majority of the world’s population has no protective immunity against poxviruses. Concurrently, the frequency of zoonotic poxvirus infections with monkeypox and cowpox virus is increasing, accompanied by the fear of bioterrorist attacks with smallpox. These developments emphasize the need for bio‐preparedness programs to enable rapid and effective prevention and control of poxvirus‐associated disease spread. Bio‐preparedness includes the availability of rapid detection methods as well as the existence of safe vaccines and therapeutics that can be administered to the majority of the population. Various existing poxvirus detection methods are well‐ established and highly sensitive. However, they are usually not suitable for rapid on‐ site detection of biothreat agents. Conventional smallpox vaccines, on the other hand, show excellent immunogenic properties. Yet today, they can not be administered to a growing proportion of individuals with impaired immunity, urging the development of safer vaccines. Thereby, recombinant subunit vaccines are considered to be a safer alternative to conventional smallpox vaccines.
To speed up the development of subunit vaccines and to evaluate the applicability of synthetic peptide aptamers for poxvirus detection, screenings of bacteriophage‐based libraries were utilized in the present study. First, a low‐cost approach for antigen discovery was established and evaluated. This approach is based on serological screenings of bacteriophage‐based genomic expression libraries. These screenings resulted in the identification of 21 antigenic proteins. Sixteen of these 21 antigens were also found to be cross‐reactive among cowpox and vaccinia virus. In addition, seven of identified antigenic cross‐reactive proteins A3, A4, D13, E2, E3, E9, and H6 are proposed to be included in subunit vaccines due to their antigenicity and
it für die Verwendung in Subunit‐Impfstoffen vorgeschlagen.
Außerdem wird die kombinatorische Phagen‐Display‐Methode und ihre Verwen‐ dung zur Identifizierung pockenspezifischer Peptidliganden als neuartige Detektions‐ moleküle vorgestellt. Affinitätsselektionen von Phagen‐Display‐Bibliotheken gegen infektiöse Vaccinia‐Virus‐Partikel führten zur erfolgreichen Anreicherung von 17 wiederkehrenden Peptidsequenzen, was auf eine Anreicherung von Pockenvirus‐ bindenden Phagenklonen hindeutet. Nach einer Charakterisierung dieser 17 bindenden Klone, wurden drei Peptidsequenzen ausgewählt, synthetisiert und weiter charakterisiert. Dabei konnte für eins dieser drei synthetischen Peptide eine spezifi‐ sche Bindung an Vaccinia‐Virus‐Partikel gezeigt werden. Dies demonstriert die Eignung synthetischer Peptide für die Detektion von Bioterror‐Agenzien.
Zusammenfassung
Nach der erfolgreichen globalen Ausrottung der Pockenkrankheit wurde die Pockenimpfung aufgrund seltener aber schwerer Komplikationen eingestellt. Heute, nach mehr als 30 Jahren, weist die Mehrheit der Welt‐Bevölkerung keinen Immun‐ schutz mehr auf. Gleichzeitig steigt neben der Häufigkeit zoonotischer Pockenvirus‐ infektionen mit Affen‐ oder Kuhpockenviren auch die Angst vor Bioterror‐Anschlägen mit Variolaviren. Diese Entwicklungen unterstreichen die Notwendigkeit von Projekten, die vorbereitende Maßnahmen für den Fall eines Bioterroranschlags treffen, um die Ausbreitung von Krankheitserregern zu verhindern. Hierzu zählt neben der Entwicklung von schnellen und einfach zu bedienenden Diagnostikplattformen auch die Verfügbarkeit von Impfstoffen und Therapeutika, die der Mehrheit der Bevölkerung verabreicht werden können. Obwohl bereits zahlreiche, gut etablierte Methoden für den Nachweis von Pockenviren existieren, sind diese für eine schnelle Vor‐Ort‐Diagnostik häufig nicht geeignet. Vorhandene konventionelle Pockenimpf‐ stoffe besitzen gute immunogene Eigenschaften, können jedoch einer steigenden Anzahl immunsupprimierter Menschen nicht verabreicht werden. Somit ist eine Entwicklung sicherer Pockenimpfstoffe erforderlich. Hierbei, stellen Subunit‐Impf‐ stoffe eine potentiell sicherere Alternative zu konventionellen Impfstoffen dar.
Um die Entwicklung von Subunit‐Impfstoffen voranzubringen und die Anwendbar‐ keit synthetischer Peptid‐Aptamere für Pockenvirusdetektion zu beurteilen, wurden in der vorliegenden Arbeit Bakteriophagen‐basierte Bibliotheken gescreent. Zuerst wurde eine kostengünstige Methode zur Antigen‐Identifizierung etabliert und evaluiert. Bei dieser Methode werden Bakteriophagen‐basierte Expressionsbibliothe‐ ken mit Pocken‐Antiseren gescreent. Dank dieser serologischen Screenings konnten 21 immunogene Pockenproteine identifiziert werden. Sechzehn dieser 21 Proteine wurden auch als kreuzreaktiv zwischen Vaccinia‐Virus und Kuhpockenvirus identifiziert. Nach einer Auswertung, werden sieben der identifizierten kreuzreaktiven Proteine A3, A4, D13, E2, E3, E9 und H6 aufgrund ihrer Antigenität und Konserviert‐ he
3.4.2. Purification of OPV particles ...30 3.4.3. Purification of viral genomic DNA ...30 3.4.4. Quantification of extracted DNA with real‐time PCR ...31
Table of contents
Acknowledgments...III Declaration of Authorship... IV Abstract...V Zusammenfassung ... VI 1. Introduction...1 1.1. Orthopoxviruses (OPVs)...1 1.1.1. Human‐pathogenic OPVs... 1 1.1.2. Virion structure ... 4 1.1.3. OPV genome ... 5 1.1.4. OPV proteome... 8 1.2. OPV vaccines and vaccine candidates...9 1.2.1. First generation vaccines... 9 1.2.2. Second‐generation vaccines...10 1.2.3. Third‐ and fourth‐generation vaccines...11 1.3. Immune response to an OPV infection ...12 1.3.1. Humoral immune response ...12 1.3.2. Cellular immune response ...13 1.4. Immunogenic OPV proteins ...14 1.5. Detection of OPVs...14 1.5.1. BiGRUDI network... 1.5.2. Non‐antibody‐based detection of pathogens...15 1.6 de ...14 . Library screenings for the advancement of OPV vaccine and detection molecule velopment ...15 1.6.1. Bacteriophage λ‐based expression libraries (ELs)...16 1.6.2. Phage display of random peptide libraries...18 2. Aims of Study... 22 3. Materials and Methods ... 23 3.1. Materials ...23 3.2. Cell culture ...28 3.2.1. Maintenance and subculture routine ...28 3.2.2. Cell preservation and recovery...28 3.3. Virus propagation...29 3.3.1. Virus stock production ...29 3.3.2. Plaque assay ...29 3.4. Preparation of genomic viral DNA for cloning ...30 3.4.1. OPV propagation ...30Table of contents 3.5. Construction of genomic OPV ELs ...32 3.5.1. Partial digestion of genomic DNA ...32 3.5.2. Size fractionation of partially digested DNA ...33 3.5.3. Ligation reaction ...33 3.5.4. Packaging reaction ...34 3.5.5. Amplification of the primary EL ...34 3.6. Library validation...34 3.6.1. Mathematical validation...34 3.6.2. Blue‐white screening...35 3.7. Characterization of recombinant clones...35 3.7.1. Excision of the pBK‐CMV vector ...35 3.7.2. Plasmid DNA isolation ...36 3.7.3. Restriction analysis...36 3.7.4. PCR amplification of insert DNA...37 3.7.5. Sequencing of DNA inserts...38 3.7.6. Data processing...38 3.8. Anti‐rA27 enzyme‐linked immunosorbent assay (ELISA)...39 3.9. Serological screening of ELs ...39 3.9.1. Immunofluorescence assay (IFA) ...40 3.9.2. Ethics statement ...40 3.9.3. Primary antibody screening...40 3.9.4. Secondary screening procedure ...42 3.10. Analysis of immunopositive plaques...43 3.11. Random peptide phage display libraries ...43 3.12. Bacterial strain maintenance and culture ...43 3.13. Phage titering ...44 3.14. Identification of peptide ligands to infectious VACV particles...44 3.14.1. Propagation and purification of VACV particles ...44 3.14.2. Biopanning against VACV particles ...44 3.14.3. Amplification of binding phage clones...45 3.14.4. DNA sequencing of selected phage clones...46 3.14.5. Identification of consensus peptide sequences...46 3.14.6. Phage ELISA...46 3.15. Peptide synthesis...46 3.16. Characterization of synthetic anti‐OPV peptide ligands by peptide ELISA ...47 4. Results ... 48 4.1. Construction of primary genomic ELs ...48 4.2. Amplified ELs ...50 4.3. Validation of the constructed ELs...50 4.3.1. Restriction analysis...51 4.3.2. Sequencing of recombinant clones...51 4.3.3. Control screenings...55 4.4. Identification of immunoreactive proteins of VACV...57 4.5. Identification of immunoreactive proteins of CPXV ...58 4.6. DNA sequences contained in immunoreactive clones ...60 4.7. Genes encoding immunoreactive proteins are distributed genome‐wide ...61
Table of contents 4.8. Cross‐reactive proteins of CPXV and VACV ...61 4.8.1. Cross‐reactive proteins of VACV and CPXV show wide functional diversity ...62 4.9. Identification of peptide ligands to infectious VACV particles ...64 4.9.1. Identification of consensus peptide sequences...64 4.9.2. Preliminary characterization of enriched phage clones with phage ELISA...66 4.10. Synthetic peptide ligands ...67 4.11. Peptide ELISA...68 5. Discussion... 71 5.1. Recombinant vs. non‐recombinant titer of the constructed genomic ELs ...71 5.2. ELs with different insert size enable complete representation of viral sequences ...72 5.3. DNA sequences found in recombinant phage clones...73 5.4. Antigenicity of EL‐derived proteins...74 5.5. Comparison of the bacteriophage‐based screening system to alternative methods for antigen discovery...75 5.6. Bacteriophage‐based ELs allow identification of antigenic, cross‐reactive proteins ...77 5.6.1. Primary vs. boosted immune response sera ...77 5.6.2. Screening ELs with sera from different species...78 5.7. Large parts of the OPV genome encode immunoreactive proteins ...78 5.8. Limitations for the identification of antigenic, cross‐reactive proteins by screening bacteriophage‐based ELs...79 5.9. Relevance of identified cross‐reactive OPV proteins for subunit vaccine design...79 5.9.1. Highly conserved proteins are best suited for subunit vaccine design ...80 5.9.2. Potential role of non‐membrane proteins in subunit vaccine development...81 5.10. Techniques for improving binding properties of peptide ligands ...83 5.11. Synthetic peptides as surrogate antibodies for pathogen detection?...83 5.12. Implementing synthetic peptides for the detection of biothreat agents ...85 5.13. Conclusions and perspectives ...86 Abbreviations ... 88 Figures... 90 Tables ... 91 Formulas... 91 References ... 92 List of Publications ...102 Conference and workshop participation...103
1. Introduction
This chapter summarizes relevant knowledge in the field of poxvirology with special emphasis on two aspects: (1) prevention of viral infections through vaccination and (2) development of novel detection molecules. To highlight the relevance of both aspects, human‐pathogenic poxviruses and their biology are described first. Subsequently, different generations of poxvirus vaccines as well as the requirement for the improvement of existing vaccines are outlined. This is followed by a description of subunit‐based vaccines and their components. Thereby, special emphasis is laid on immunogenic poxvirus proteins and methods for their identification. Following this, currently available detection methods as well as the requirement for the development of non‐antibody‐based detection methods are outlined. In this context, the phage display of random peptides methodology and its potential use in poxvirus detection are described.
1.1. Orthopo
(OPVs)
The family Poxviridae comprises a large number of complex viruses that infect vertebrates, birds, and insects [2;3]. Poxviruses are classified into two subfamilies Chordopoxvirinae and Entomopoxvirinae, based on vertebrate and insect host range [3]. The subfamily Chordopoxvirinae contains eight genera, with one of them being Orthopoxvirus (OPV). The genus OPV is further divided into different virus species, including camelpox, cowpox, ectromelia, monkeypox, vaccinia, and variola virus [2;3]. Al
xviruses
l OPV species show serological cross‐reactivity and cross‐protection [4]. OPV infections can be classified as systemic or localized (at the site of virus entry) illnesses [5]. Generalized disease usually manifests with rash [5]. The type of infection depends on OPV species, the route of entry, and the genus/species of the susceptible animal and its immune status [5]. Four OPV species are known to infect humans. 1.1.1. um npathogenic OPVs HThe OPV species vaccinia virus (VACV), cowpox virus (CPXV), monkeypox virus (MPXV), and variola virus (VARV) can infect humans [5]. While VARV is an exclusively human pathogen, the other species are transmitted from animals to humans (zoonoses) [6‐8]. The properties of human‐pathogenic OPVs are listed in
a
Table 1 and described below in more detail.
1. Introduction
Table 1. Properties of humanpathogenic OPVs
Property Vaccinia Cowpox Monkeypox Variola
Host range Broad Broad Broad Narrow
Genome DN
(kbpb)) A
a) size
192 220 191 186
Geographic
distribution Brazilc) Western Eurasia Western and central Africa Eradicated (formerly worldwide) Direct contact, respiratory droplets Mod
transmission e of Direct contact Direct contact Direct contact, respiratory droplets a) DNA – Deoxyribonucleic acid
b) kbp – kilo base pairs
c) Naturally occurring infections Vaccinia virus
VACV is the agent that has been most widely used for vaccination to successfully eradicate smallpox [4]. The exact origin of VACV remains unknown but different explanations have been proposed. These include such possibilities as: (1) VACV is an extinct, once zoonotic virus, (2) VACV was artificially derived from another OPV during vaccine production, and (3) VACV may be a recombinant between VARV and the original species of OPV once used for vaccination [9]. There are many VACV strains with different biological properties. However, all of these have a broad host range and some of them have been used for decades as vaccines or in laboratory research [4;9]. Due to this long‐lasting utilization, VACV has become the most investigated prototype OPV virus. Despite the cessation of the worldwide vaccination campaign, VACV infections still occur worldwide. Today three different infection sources exist: (1) natural human infections (zoonoses) in Brazil transmitted by cattle [10;11], (2) accidental laboratory infections (Figure 1A) [9;12‐16], and (3) infections after contact to a recent vaccinee [17‐20].
Cowpox virus
CPXV infections first became known through an infection of milkers by contact with ulcers on the teats of cattle [4]. Human cowpox is a zoonotic disease which usually causes a self‐limiting local infection [21]. In recent years, more diverse sources of a CPXV infection have been reported. Human cowpox has been transmitted by cats [22‐ 25], by rats [26‐29], and by elephants [30;31]. Cases of infected dogs [32] and exotic zoo animals [33] as well as associated transmissions to humans [34;35] have also been reported. CPXV thus has an extremely broad host range and is prevalent in Western Eurasia [21]. Human cowpox is a self‐limiting disease, not highly infective in immunocompetent humans. Immunocompetent patients present with localized lesions or crusted ulcerated nodules or blisters on the fingers, arms, legs or face (Figure 1B). The virus infects through skin abrasions and the resulting lesions can leave scars after healing [21]. Beside self‐limiting, local infections, severe clinical courses resulting in prolonged treatment have been described [28;34]. Furthermore, a case of generalized, fatal CPXV infection in an immunocompromised patient with a life‐long history of
1. Introduction
atopic dermatitis has been reported [22]. Human cowpox particularly affects young people [24], indicating that the lack of vaccination against smallpox may render today’s population more susceptible to OPV infections including cowpox [21;36;37].
A
B
C
D
Figure 1. Clinical presentation of humanpathogenic OPV infections (A) Accidential needlestick inoculation of a laboratory worker with VACV. Shown are the lesions on the fourth and fifth fingers on day 12 after accidential inoculation [15]. (B) A fully developed ulcerated cowpox lesion on the forehead of a 15 year old patient [28]. (C) A VARV infection on the upper arm of a 9‐month‐old unvaccinated child on the ninth day of rash onset. The pustules have reached their maximum size and are becoming flattened [4]. (D) A MPXV infection in a six‐year‐old girl on about the eighth day of rash [38]. Variola virusVARV is the most prominent member of the OPV species since it once caused the highly lethal smallpox disease [4;39‐41]. This disease was characterized by a distinctive pustular rash (Figure 1C) [41]. Infection occurred by close face‐to‐face contact, commonly between members of the same household [41].
In 1980 the World Health Organization declared the world free of smallpox [4]. This eradication was achieved through a worldwide vaccination campaign [42‐44]. Smallpox was thus the first human disease that was deliberately eradicated globally [45]. The global eradication of smallpox was possible due to two essential factors: (1) cross‐reactivity and cross‐protection among members of OPV, and (2) the absence of a natural reservoir of VARV outside of humans [4]. Since its eradication, the only legally held (declared) VARV stocks are stored at the Centers for Disease Control and Prevention (CDC) in United States of America (USA) and at the State Research Center for Virology and Biotechnology (VECTOR) in Russia [46]. Originally, these stocks have been retained to gain possible new insights into the biology of VARV [47].
1. Introduction
Nevertheless, the controversy about retention or destruction of these stocks began shortly after the declaration of smallpox eradication in 1980 and continues to this day [45;47‐49].
Generally, there is a growing public concern of bioterrorist attacks with VARV [50‐ 56]. The CDC in the USA included VARV in a list of critical biologic agents that have potential for use in bioterrorism. This list includes different biologic agents, prioritized into three categories A, B, or C. Thereby, VARV was classified into the Category A, which includes biologic agents with the highest public risk in the case of a bioterrorist attack [57]. Monkeypox virus The first case of human monkeypox was found in the Democratic Republic of Congo in 1970 in a 9‐month‐old boy [58], 9 months after the eradication of smallpox in this country [59]. Clinically, human monkeypox closely resembles discrete, ordinary‐type smallpox; lesions evolve in the same stage on any part of the body (Figure 1D) [4;5]. In humans, clinical disease is believed to result from either respiratory, percutaneous, or permucosal exposures [5]. Unvaccinated persons are more likely to become infected than those who received the vaccine against smallpox [4]. Human monkeypox occurs mostly in central and western Africa [60‐64]. However, in 2003 a monkeypox outbreak occurred in the USA for the first time, marking the first documented human infections in the Western Hemisphere [65‐69]. These cases had similar clinical features to previously described African cases, but they were generally less severe [59].
1.1.2. Virion structure
Electron microscopic investigations suggest that OPV virions are brick‐ or barrel‐ shaped particles (Figure 2A and 2B) [70] with dimensions of about 360×270×250 nm [3;71]. VACV is the prototype OPV and has been studied most extensively. There are two major infectious forms of VACV, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV)1 [73‐75]. Basically, these distinct forms of virions
are surrounded by different numbers of lipid membranes which have various proteins on their surface (Figure 2C). IMVs are surrounded by a single membrane, are more abundant, and can be released by cell lysis [74;76]. EEVs differ from IMVs by the presence of an additional membrane that contains at least six proteins unique to the EEV envelope, namely the proteins A33, A34, A36, A56, B5, and F13 [75]. The outer membrane of EEV is extremely fragile and is easily destroyed by the process of virus purification [77]. However, after rupture of the outer EEV membrane the particle presumably retains full infectivity as an IMV [78;79]. IMVs are thought to be important for long‐term stability and transmission of the virus between hosts in the environment
1 An alternative nomenclature designates the IMV as mature virions (MV) and EEV as extracellular virions (EV)
1. Introduction
[71]. In contrast, EEV is responsible for fast cell‐to‐cell spread and long‐range dissemination [80;81].
The internal structure of IMV particles has been primarily gained through the use of thin sections (see Figure 2B) [71]. IMVs generally contain two distinct boundaries, a lipid membrane bilayer that surrounds the entire particle and the core wall surrounding an internal core (Figure 2C) [71]. The biconcave core contains the linear genome which is associated with viral proteins [2]. The function and origin of the lateral bodies is not exactly known but may be due to electron microscopy preparation artifacts [2].
A
B
C
Figure 2. Virion structure of OPV particles(A) Negative stain electron microscopic appearance of VACV (100,000×), (B) Ultrathin sectioning of VARV (51,000×), (C) Schematic diagram of an OPV particle. (A and B – kindly provided by Hans R. Gelderblom, RKI, Berlin; C – Swiss Institute of Bioinformatics [82]) 1.1.3. OPV genome OPVs have a linear double‐stranded DNA genome of about 200 kbp which encodes approximately 200 genes (Figure 3). The genome size and its coding capacity depend on the OPV species and strain (see Table 1) [71]. The genes are compactly organized along the genome and do not contain introns [71;83]. The ends of the OPV genomes contain inverted terminal repetitions (ITRs) which consist of identical but oppositely oriented sequences at the two ends of the genome [83]. A comparison of 19 members
1. Introduction
of the Chordopoxvirinae subfamily (vertebrate poxviruses) showed that 90 genes are completely conserved [84]. Generally, genes that are highly conserved and concerned with essential replication functions are located in the central region of the OPV genome. Variable genes concerned with host interactions are usually located in the end regions of the genome (Figure 3) [3]. Figure 3. A schematic representation of the OPV genome The genome can be divided into a central region which mainly encodes conserved genes that are essential for virus replication. The terminal regions are more variable and encode proteins that are non‐essential for virus replication in cell culture. ITR – Inverted Terminal Repetitions (adapted from [47]).
The first complete OPV genome sequence published was that of the VACV strain Copenhagen (VACV‐Cop) [83]. The publication of this sequence resulted in establishing a convention for naming VACV genes or open reading frames (ORFs). This convention consists of using the HindIII restriction endonuclease DNA fragment letter A – P, where A is the longest and P is the shortest fragment. The fragment letter is followed by the ORF number (from left to right) within the fragment, and L or R, depending on the direction of the ORF (Figure 4) [3;71;84]. An exception was made for the HindIII C fragment, where the ORFs were numbered from the right to avoid starting at the highly variable left end of the genome [3]. Thus, over the years the VACV‐Cop nomenclature has become the most familiar one among poxvirologists [71], and gene names of other OPV strains are provided along with VACV‐Cop genes for easier reference.
1. Introduction Figure 4. HindIII restriction map of a VACVCop genome with restriction fragments A to P Genes are named according to their position relative to the left end of individual HindIII restriction fragments (A ‐ P) and to their transcriptional orientation. Colored bars indicate ORFs and the number of viruses in which an ortholog is conserved. Dark blue bars indicate the 49 genes conserved in all 21 poxviruses analyzed, and lighter blue bars indicate genes conserved in at least 19 of the 21 genomes. ORFs transcribed rightward are shown above the line, and ORFs transcribed leftward are shown below the line. Only larger ORFs are labeled for easier reference (adapted from [84]).
1. Introduction
1.1.4. OPV proteome
Consistent with the size of their genome, OPVs encode numerous viral proteins [3]. Considerable knowledge about OPV proteomics was gained through investigations of the protein composition of VACV IMVs by mass spectrometry analysis [85‐87]. A number of 63 ‐ 80 different proteins could be identified as IMV components using this method [85‐87]. The convention of naming OPV polypeptides is similar to the convention of naming genes, except for dropping the letter indicating the ORF directionality (L or R) [3;85]. A possible way to classify the IMV proteins identified according to their function is shown in Figure 5 [85]. The most abundant protein group was found to consist of four core proteins involved in IMV structure and morphogenesis, namely A3, A4, A10, and F17 [85;87]. Transcription 20 (25%) Other 11 (14%) Host defense 3 (4%) Disulfide 3 (4%) Unknown 3 (4%) Fusion/Entry membrane 6 (7%) Other membrane 10 (12%) Structural/ Morphogenesis 24 (30%) Figure 5. Functional characterization of IMV proteins IMV components can be divided into non‐membrane proteins involved in IMV structure and morphogenesis (A3, A4, A10, A11, A12, A15, A30, D2, D3, D13, E8, E11, F10, F17, G1, G7, H5, I1, I3, I6, I7, J1, L4, VACV‐WR148), proteins involved in transcription (A5, A7, A18, A24, A29, D1, D6, D11, D12, E1, E4, G5.5, H1, H4, H6, I8, J3, J4, J6, L3), membrane proteins (A9, A13, A14, A17, A26, A27, D8, F9, H3, L1), membrane proteins involved in fusion/entry (A21, A28, G9, H2, J5, L5), components of the disulfide bond formation pathway (A2.5, E10, G4), host defense proteins (C22, E3, N1), proteins with other functions (A42, A45, A50, B1, E9, F4, F8, F13, K4, M1, O2), and proteins with unknown function (A6, A19, E6) (modified from [85]).
Beside these IMV proteins, there are at least six proteins unique to the EEV envelope, namely the proteins A33, A34, A36, A56, B5, and F13 [75].
1. Introduction
1.
2. OPV vaccines nd vaccine candidates
a
Currently, there is no U.S. Food and Drug Administration‐approved drug for the prevention or treatment of poxvirus infections [88;89]. However, ST‐246®
(Tecovirimat), an oral therapeutic agent developed by SIGA Technologies was shown to prevent disease symptoms in animal models for smallpox [88]. Nevertheless, the best way to prevent a viral infection still remains a protective vaccination [90].
Because of the exciting history of smallpox and its global eradication, the production and improvement of smallpox vaccines received special attention [91‐95]. Today, smallpox vaccines or vaccine candidates are grouped into four generations, depending on their properties and the wealth of experience in applying them (Table 2) [95‐98]. In contrast to other viral pathogens (e.g. Hepatitis B virus [99]), all efforts of producing an inactivated virus‐based vaccine remained unsuccessful [100].
Table 2. Smallpox vaccines and vaccine candidates
Generation Description VACV strain Advantage Disadvantage
1st Conventional vaccines, produced in animals NYCBH;
Lister/Elstree Historical experience in smallpox eradication Rare but severe adverse reactions
2nd Replication‐ competent, ed tissue‐cultur NYCBH, Lister/Elstree Historical experience; improved manufacturing process; replication‐competent – ce easier to produ Rare but severe adverse reactions 3rd Replication‐ competent or –deficient, highly attenuated MVA, Lister, Copenhagen Clinically safe (theoretically) and immunogenic Unproven efficacy 4th Subunit vaccines Protein‐/DNA‐ based Safe (theoretically) Unproven efficacy, most efficient antigen position nown com unk NYCBH – New York City Board of Health; MVA – Modified Vaccinia virus Ankara 1.2.1. First generation vaccines
The production of first‐generation smallpox vaccines involved superficial scarification of the abdomen skin of calves and sheep and subsequent inoculation of seed virus. For this purpose, the seed virus was rubbed into the scarified skin. Generally, the VACV strain New York City Board of Health (NYCBH) was used as seed virus [98]. After incubation, the skin was rinsed and the pulp was scraped from the skin with a curette to harvest the virus. The harvested virus was further processed to remove impurities like blood and animal hair and to reduce the amount of contaminating bacteria. VACV strain Lister‐based vaccines were produced on chick chorioallantoic membranes [98]. The resulting liquid vaccine was either directly used for immunization or, later on, freeze‐dried (e. g. Dryvax vaccine) for longer storage [4;93;94].
1. Introduction
During the global eradication campaign, the bifurcated needle (Figure 6A) was universally used for multiple‐puncture vaccination applied to the upper arm of the vaccinee (Figure 6B) [91]. When VACV is inoculated into the superficial layers of the skin, the virus grows and induces an immune response that protects against smallpox [91]. The reaction that follows is termed “a take”, or “primary reaction” from first vaccination (Figure 6C and 6D), and a “major reaction” from additional vaccinations [91]. Figure 6. Conventional smallpox vaccination (A) Bifurcated needle for smallpox vaccination shown both empty and containing vaccine in the bifurcation. (B) Proper position for a multipuncture vaccination (3 – 15 insertions) by a bifurcated needle. Progression of primary vaccination showing a typical lesion at (C) and day 5 (D) day 14 after vaccination (all adapted from [91]).
After smallpox was declared eradicated, the routine vaccination was discontinued [4]. This discontinuation was due to the occurrence of some rare but severe adverse reactions (Figure 7) [4;92;101]. Among known contraindications to conventional smallpox vaccination are: (1) immunodeficiency (Figure 7A and 7B), (2) certain skin disorders, especially true atopic dermatitis, (3) ocular disease, (4) immunosuppressive therapy, (5) HIV infection and acquired immunodeficiency syndrome (AIDS), as well as (6) pregnancy [91]. Moreover, certain genetic factors have been proposed to be associated with the development of adverse events [102;103]. Today, the number of individuals with contraindications is estimated to be increasing [104].
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1.2.2. econdgeneration accines S v
The historical methods of smallpox vaccine production could not meet the requirements for the production of modern human vaccines [90]. This led to the development of second‐generation vaccines. These vaccines utilize the same historical vaccine strains Lister and NYCBH that are amplified in tissue culture in an improved
1. Introduction
manufacturing process [96;98]. After the development, licensing, and mass production of second‐generation vaccine stocks (ACAM2000TM [105;106]) remaining stocks of
first‐generation vaccines were destroyed [107]. Despite their improved quality, second‐generation vaccines are likewise contraindicated for application to a significant part of today’s population [104;108]. Figure 7. Examples of adverse events occurring after smallpox vaccination (A) True generalized vaccinia, usually developing in individuals with abnormalities in T‐ cell function [109]. (B) Progressive vaccinia in a child with intact antibody synthesis but pted from [92]). lacking T cell function (both ada
Nowadays, the conventional smallpox vaccination can be recommended to certain individuals, including health care and laboratory workers, military personnel, and some first responders as one aspect of bio‐preparedness [92]. In the case of a bioterrorist attack a resumption of the mass smallpox vaccination could be necessary. Different scientific groups have theoretically addressed the consequences of such an event, including the frequency of expected adverse effects [104;108;110;111]. A statistical analysis of historical vaccination data extracted from literature demonstrated vaccine virus strain differences regarding the severity of expected adverse effects. It was estimated that vaccination with the NYCBH strain would result on average in 1.4 deaths per million vaccinations, whereas a vaccination with Lister vaccine would lead to an average 8.4 deaths per million vaccinations [108]. Thus, conventional smallpox vaccines have a higher complication rate than any other vaccine currently being used [104]. To decrease the number of direct and indirect adverse events, high‐risk individuals and their contacts would need to be excluded from vaccination. However, this would also mean that some proportion of the population would remain susceptible to smallpox [104].
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1.2.3. Third and fourthgeneration vaccines
Because of numerous potential contraindications to second‐generation vaccines, considerable effort has been made to develop safer third‐ and fourth‐generation vaccines [98;112]. The evaluation and licensing of these new vaccines is complicated by the fact that smallpox no longer exists in nature [98]. Thus, the evaluation of the efficacy of new vaccines and drugs against smallpox has to be carried out and assessed in various animal models [113‐116].
1. Introduction
Third‐generation vaccines are based on live but attenuated VACV with established safety and immunogenicity records from clinical testing in humans (e.g. strains Modified Vaccinia virus Ankara [MVA] or LC16m8) [98;117‐119].
The fourth‐generation smallpox vaccines include subunit‐based vaccines [96]. Subunit vaccines are defined as those containing one or more pure or semipurified antigens [90]. Much research remains to be done to explore the full potential of subunit vaccines [120]. However, in addition to some drawbacks, these vaccines also have important advantages. The drawbacks include difficulty in mimicking the conformation of antigen polymers found with many viruses [121], the fact that B‐cell epitopes recognized by neutralizing antibodies are sometimes discontinuous sequences, and the susceptibility of peptides to proteolysis [120]. The advantages of subunit vaccines include the fact that the product is chemically defined, stable and safe, and contains only important B‐cell and T‐cell epitopes [120].
1.3. Immune response to an OPV infection
Smallpox was eradicated before the onset of the era of molecular biology and the current advances of immunology. Consequently, the mechanisms of protection triggered by a VARV infection could not be exactly dissected [5]. Therefore, most knowledge about immunity to OPV infections was obtained from observed immune response patterns generated by first‐ and second‐generation vaccines (Figure 8) [122].
Early responses to virus infection include the production of interferon, nitric oxide, and elicitation of natural killer and macrophage cellular functions [5]. These non‐ specific innate responses activated by pattern recognition receptors serve to inhibit initial viral replication and to activate antigen‐presenting cells for the proper activation of the ensuing adaptive immunity (Figure 8) [122]. Innate inflammatory cytokines and chemokines then attract effector lymphocytes into infected tissues [122].
1.3.1. Humoral immune response
Smallpox vaccines induce strong humoral responses. B cells produce antibodies that agglutinate, opsonize, and neutralize viral particles, fix complement, and allow for antibody‐dependent cell cytotoxicity (ADCC) [122]. Antibody responses are detectable by immunoassay techniques from around day 10 post immunization (p.im.), reaching a peak at around one month p.im. [123]. Multiple studies have shown that antibody responses are long lived [124;125] and antibodies are detectable up to 75 years p.im. [126;127].
1. Introduction
Figure 8. Immune response pathways activated by conventional smallpox vaccines
Immunization with a conventional smallpox vaccine elicits a cascading network of integrated immune pathways. The combination of innate and adaptive responses halt viral replication, lyse infected cells, and remove viral particles from the host. Virus‐ specific lymphocyte numbers then contract to a small, long‐lived memory population capable of rapidly responding to subsequent OPV infection. See main text for more details. Abs – Antibodies, ADCC – Antibody‐dependent cell cytotoxicity, TLR – Toll‐like receptor, RLH – RIG‐like helicase, HLA – Human Leukocyte Antigen, CTL – Cytotoxic T lymphocyte, NK cell – Natural killer cell, Th cell – Helper T cell (adapted from [122]).
1.3.2. Cellular immune response
Smallpox vaccines induce strong CD4+ and CD8+ T cell responses that peak at two to
four weeks p.im. and then contract to form a stable memory population of T cells that remain detectable for several decades after vaccination [123;125;128]. Activated T helper (Th) cells supply necessary cytokines (IL‐4, IL‐5) and costimulatory signals (CD40L) for the B cell maturation, replication, and isotype switching [122]. T cell help (IL‐2, IFNγ) also promotes cytotoxic T lymphocyte (CTL) activation, clonal expansion,
1. Introduction
and effector function [122]. VACV‐specific Th cells can have direct lytic activity, and their secreted cytokines (IFNγ, TNFα) can have direct antiviral activity [122].
A recent study suggests that every antibody response needs to be accompanied by a matched CD4+ T cell response to the same protein [129]. This indicates that cognate
Th cell–B cell interactions may be required to generate robust anti‐VACV antibody responses [122]. It is assumed today that T cell immunity is most important during primary acute viral infections for directly controlling viral replication as well as for helping to establish the humoral immune response [130]. Protection against re‐ infection is primarily antibody‐mediated for OPV infections, and T cells play a secondary role in vaccine‐induced protective immunity [123;130;131].
1.4. Immunogenic OPV proteins
The foundation for the development of subunit vaccines is the understanding of the viral pathogenesis and the proteins, glycoproteins, or carbohydrates involved in inducing protective immunity [90]. Thus, it is crucial to identify these individual viral components [90]. Consistent with their size and the large number of proteins, humoral and cellular responses elicited by an OPV infection target a large number of antigens and epitopes [100;132]. The most comprehensive studies for the identification of VACV‐specific antigens and epitopes can be achieved through genome‐ and/or proteome‐based approaches. For verification of results obtained, the potential immunogenicity of individual proteins or protein classes can be confirmed in small‐ scale projects using molecular techniques [133].
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5. Detection o OPVs
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The availability of sensitive and rapid detection methods is a prerequisite for bioterrorism preparedness and control [134]. Thus, the development of rapid and sensitive detection methods remains an essential part of pathogen research. OPVs can be reliably detected by various methods including real‐time polymerase chain reaction (PCR) [135‐137] and electron microscopy [138]. While these methods are reliable, they require a high technical expenditure and generally cannot be used in the field for the rapid detection of biothreat agents [139]. Thus, there is a critical need to develop more rapid, accurate methods for the detection and identification of biothreat agents including OPVs [139].
1.5.1. BiGRUDI network
Following the bioterrorist attacks in October 2001 in the USA with Bacillus anthracis‐contaminated mail [140], the public awareness of the bioterrorism threat increased [141]. As a result, the bioterrorist potential of different infectious agents was evaluated and classified by the CDC into different categories according to the risks associated [57]. Soon after, European public health institutes also started to evaluate
1. Introduction
tection molecule development
To advance the prophylaxis of OPV infections and to improve diagnostic approaches for OPV detection, screening of bacteriophage‐based libraries was utilized to identify subunit vaccine‐suitable antigens and novel detection molecules. Thereby, two different types of libraries were used: (1) genomic λ‐based expression libraries (ELs) containing a complete OPV genome (see
their preparedness for potential bioterrorist attacks, discovering the demand for the development of bio‐ reparedness programs [142;143]. p
Consequently, in 2008 Robert Koch Institute (RKI), the German central federal institution responsible for disease control and prevention, initiated a network project named “Biologische Gefahrenlagen: Risikobewertung, ultraschnelle Detektion und Identifizierung von bioterroristisch relevanten Agenzien (BiGRUDI)”. The main goal of this project is the development of an easy‐to‐use, mobile diagnostic platform for the rapid on‐site detection of bioterrorist agents. This diagnostic platform should ideally enable multiplex detection of viruses, bacteria, and toxins and thus allow for a rapid response to bioterrorist attacks. Beside well‐established detection molecules like antibodies, the project aims at researching and developing innovative, synthetic molecules for the detection of pathogens such as poxviruses.
1.5.2. Nonantibodybased detection of pathogens
The development of diagnostic platforms for rapid on‐site detection requires the availability of highly specific, chemically stable detection molecules. Antibodies still remain the most versatile and widely used protein‐binding agents [144]. A good antibody can bind its target protein with an equilibrium dissociation constant (KD) of
10‐9 M, which can further be optimized to pico‐ or even femtomolar range [144].
Antibodies can further have extremely high specificities, recognizing only the target protein in a crude cellular extract containing different other proteins [144]. Despite all these advantages, antibodies have considerable limitations including tedious and expensive production, limited shelf‐life, and the requirement for animal use [144]. Thus, there is an increasing interest in employing alternative recognition species to detect pathogens [134].
Aptamers are specific binders for targets selected from huge libraries of molecules containing randomly created sequences [134]. Aptamers can be divided into those created from polymers of nucleic acids (DNA or RNA aptamers) [145‐151] or amino acids (peptide aptamers) [134;144;152]. For the selection of peptide aptamers, a variety of methods of directed evolution have been developed [134]. One of these methods is phage display.
1.6. Library screenings for the advancement of OPV vaccine and
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1.6.1), and (2) random peptide libraries displayed on filamentous phage (see 1.6.2). For the construction of λ‐based ELs, fragments of the viral genome are cloned into an expression vector. The resulting
1. Introduction
genomic λ‐based EL can be screened with various antisera to identify target proteins of the naturally produced OPV binders, the antibodies. The knowledge about the identified antigenic OPV proteins is most important for vaccine development but can also be used for the development of diagnostic assays. To improve these diagnostic tools in terms of availability, a second type of library, a combinatorial peptide library displayed on filamentous phage was utilized. Screening against replication competent virus particles can lead to the identification of short synthetic OPV binders called peptide aptamers that can be implemented on various detection platforms either alone or in combination with antibodies.
1.6.1. Bacteriophage λbased expression libraries (ELs)
A genomic expression library (EL) is a collection of genomic DNA fragments that together ideally represent the entire genome of the host organism. These fragments are contained within a self‐replicating expression vector that enables them to be maintained and propagated within the cells of microorganisms, such as Escherichia coli (E. coli) [153]. Additionally, promoters present in the expression vector allow the inserted DNA fragments to be translated into the encoded protein. The libraries obtained can be used for serological screenings to identify targets of the humoral immune response. Prior to construction of a genomic EL, an appropriate cloning vector has to be chosen.
Bacteriophage λ as a cloning vector
Bacteriophages (also called phages) are viruses that infect bacteria. Bacteriophage λ is one of the most popular virus‐based cloning vectors used in the construction of libraries due to its high infectivity, clone‐style propagation, and specific features of the genome organization [153]. The genome of wild‐type bacteriophage λ is a double‐ stranded DNA molecule of 48,502 base pairs (bp) in length [154]. The DNA is carried in phage particles as a linear double‐stranded molecule [154]. The genes of λ-phage are organized into functionally related clusters. The left‐hand region includes genes whose products are used to package viral DNA into bacteriophage heads and to assemble infectious virions from filled heads and preformed tails. The right‐hand region contains essential genes used in replication of phages and lysis of infected bacteria. The central third of the genome is not essential for lytic growth and can be replaced by segments of foreign DNA [154].
Many λ-phage‐based vectors that differ in their own special features have been constructed. There is no universal λ vector and the choice of a vector depends on the experimental conditions [154;155]. Some considerations influencing this choice include: (1) the restriction enzyme to be utilized, (2) the size of the foreign DNA fragment to be inserted, (3) whether the vector is to be used to express cloned DNA sequences in E. coli, and (4) whether the foreign DNA is to be rescued from the phage vector in the form of a plasmid [154].
1. Introduction Construction of viral genomic ELs
The construction of viral genomic bacteriophage λ‐based ELs is a complex process consisting of different methodical steps. Simplified, this process can be subdivided into multiple steps shown in Figure 9. To obtain genomic viral DNA, viruses have to be propagated in cell culture (1). After propagation, the virus particles have to be separated from the remaining cell debris prior to viral DNA purification (2). The purified viral genomic DNA has then to be fractionated to provide fragments of suitable size [156]. For this purpose, the DNA usually is partially digested with a frequently cutting restriction enzyme (3). After digestion, the DNA size range required can be selected and cloned into the phage‐based expression vector (4). The ligated DNA can then be packaged in vitro which results in a primary EL (5). This primary EL is amplified by infecting the bacterial host (6). The amplified EL can be stored in aliquots and used for screening experiments [154].
Figure 9. Construction procedure of viral, genomic λ-phagebased ELs
Host cells are cultured and infected with the virus strain needed. After virus propagation (1) and purification of viral particles from the host cells, the genomic, viral DNA is extracted (2). This DNA is then partially digested (3) with a frequently cutting restriction enzyme and the appropriate DNA fragment range selected on an agarose gel. The DNA fragments selected are ligated (4) to the λ-vector arms and packaged in vitro (5) using phage protein extract, yielding the unamplified primary EL. The primary EL is amplified (6) by infecting E. coli cells to result in the ready‐to‐use viral EL.
Screening of viral genomic ELs
Once constructed, genomic ELs can be used for serological screenings to identify targets of adaptive immunity to pathogens (Figure 10). Simplified, the screening
1. Introduction
procedure starts with an infection of E. coli cells with EL‐derived phages (1). Infected bacteria are then plated onto an agar plate (2) and incubated until plaques appear (3). The agar plates are overlaid with nitrocellulose membranes for a transfer of recombinant proteins (4). The recombinants that express a desired gene can be picked up by their selective reaction with the specific antibodies (5). The immunoreactive plaques are then selected (6) and the fragment of OPV DNA contained can be identified through DNA sequencing and alignment to a reference OPV genome. Figure 10. Serological screening procedure for a λ-phagebased EL For serological screening of phage‐based EL, E. coli cells are infected (1) and plated (2) onto agar plates. After appearance of plaques (3), the agar plates are overlaid with nitrocellulose filters for transfer of recombinant proteins (4). The filters are subsequently incubated with anti‐virus serum (5). Stained filters are aligned with the master agar plate to identify immunoreactive plaques (6) which are then selected and characterized.
1.6.2. Phage display of random peptide libraries
Phage display methodology was first introduced by Smith in 1985 [157] and since then has undergone a rapid application expansion. Phage display is a molecular methodology by which foreign proteins or peptides are expressed at the surface of phage particles [158]. Most phage display work has used filamentous phage strains M13, fd, and f1 as vectors [159]. The E. coli‐specific bacteriophage M13 is about 1 µm long and only 5–7 nm in diameter (Figure 11) [160]. The particle consists of a single‐ stranded DNA core surrounded by a proteinaceous coat. The coat contains five different proteins, but the vast majority consists of several thousand copies of the major coat protein VIII (pVIII) which covers the length of the particle [161]. The four
1. Introduction minor coat proteins are present at about five copies per particle; pVII and pIX cap the one end of the particle while pIII and pVI are at the opposite end of the particle [161]. Figure 11. Schematic representation of the M13 phage particle M13 bacteriophages are widely used for phage display. The pIII coat protein can be used as a fusion partner for a limited number (maximum of five) of protein copies, while numerous numbers of protein copies can be expressed at the phage surface if pVIII is used as a fusion partner. The approximate number of copies of each M13 coat protein is indicated in parenthesis.
There are numerous applications of the phage display methodology [162]. One important application is the construction and screening of combinatorial peptide libraries [162]. These libraries are constructed by cloning synthetic oligonucleotides, fixed in length but with randomized codons, as fusions to genes III or VIII of M13 where they are multiply expressed as peptide–capsid fusion proteins (Figure 12) [162].
Figure 12. Random peptide phage display libraries
Using phage display methodology, a library of variant nucleotide sequences can be converted into a library of variant peptides. In such a library every phage clone displays its own individual amino acid sequence. Because of this amino acid variability, random peptide libraries can be screened against a plethora of targets to isolate phages displaying peptides with properties required (modified from [158]).
The resulting libraries, often referred to as random peptide libraries, contain a relatively large variety of amino acid sequences (107 – 109) [163] and can be tested for
binding to target molecules of interest. This is most often done using a form of affinity selection known as “biopanning” [162]. A plethora of targets has been applied in biopanning experiments performed in vitro and in vivo [160;163‐165]. Possible targets include but are not limited to antibodies [166], enzymes [167], receptors, virus
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particles [168;169], and even whole cells [159;160;170;171]. Biopanning in its simplest form consists of multiple steps illustrated in Figure 13.
Figure 13. General biopanning procedure
In its simplest form biopanning is carried out by immobilizing the target molecule of interest by passive adsorption to a microtiter plate. Unbound target is washed off and the remaining sites in the well are blocked with unspecific proteins. The random peptide phage display library is added to the target‐coated well to allow phage binding. The unbound phages are repeatedly washed away. Target‐bound phages are eluted and titered. Subsequently, the eluted phages are amplified, titered again, and applied to the next selection round. Individual phage clones from the unamplified eluates are analyzed after every selection round by DNA sequencing.
Here, the target is immobilized onto a surface (surface panning procedure) and subsequently incubated with the phage display library. The unbound phages are washed away, whereas the bound phage clones are eluted. The specific elution can be carried out in a solution containing either free target or a competing ligand. Due to stability of filamentous phages, extremes of pH, denaturants, or ionic strength can be used for non‐specific elution of bound phages. The phage eluates are titered on agar plates. A part of the resulting phage plaques from these plates are randomly selected and analyzed by DNA sequencing. The eluted, titered phages are still infectious and are amplified by infecting bacterial host cells. The resulting “amplified” eluate can serve as input to another round of affinity selection. The biopanning process is repeated three to six times, depending on the complexity of the target. After every selection round,
1. Introduction
binding phage clones are randomly selected, propagated, and analyzed individually by DNA sequencing to identify the target‐binding peptide sequence [159;17 ]. 2
A successful biopanning experiment results in the identification of consensus peptide sequences or amino acid motives. The binding phage clones selected can be tested for their binding specificity to the target used during selection. However, for more detailed binding studies, the sequences selected can be synthesized as free, soluble peptides. Without the phage attached the peptide can be used at a defined concentration. Moreover, the peptide can be modified during synthesis and can thus be analyzed using various assays. Phage‐borne synthetic peptides could be used as probes for the detection of biological threat agents [152], either alone or in combination with antibodies.
