CHAPTER 1: LITERATURE REVIEW
1.10 Antibody production using phage display
21 the action of OPB, is thought to contribute to disease progression (Morty et al., 2005a;
Munday et al., 2011).
22 The process of phage display is based on the linkage of foreign antibody gene fragments to the minor phage coat protein (pIII) of the M13 bacteriophage (Smith, 1985). As a result, the genes encoding the antibody (genotype) is directly linked to the displayed antibody on the phage particle (phenotype). This feature allows the coding sequence of the antibody to be exactly known and exactly reproduced. The antibody::pIII fusion protein are assembled in E. coli and secreted into the periplasmic space and are often soluble, stable and folded in their native form (Marston, 1986).
The process of phage display is illustrated in Fig. 1.8 which starts with the construction of a phagemid library whereby the immunoglobulin VH and VL genes are amplified and ligated into a phagemid plasmid (Step A). After the propagation of E. coli transfected with the phagemid cells (Step B), the scFv::pIII fusion proteins are displayed on the bacterial cell surface (Step C). The phagemids are then rescued by the M13KO7 helper phage which provides the essential proteins (dark grey outline) for the packaging of the recombinant phage DNA within the phage and the display of the fusion proteins on the bacterial cell surface (Webster, 1996) (Step D). As a result, a mosaic population where E. coli containing either phagemid or the helper phage exists whereby the WT pIII (dark gray) competes with the scFv::pIII fusion proteins for incorporation into the phage particle (Step E). In Step F, the mosaic population is panned against the immobilised antigen to isolate antigen specific scFv (light blue), whilst non-specific scFvs are discarded (pale yellow). The bound phagemids are eluted from the E. coli host, and under antibiotic selection, only the phagemids are transfected back into E. coli (Step G). It is at this step, that the WT phagemid is lost. The resulting phagemid population is rescued once again (Step H) and used to pan against the immobilised antigen once again. This process is repeated numerous times to select for scFv with a high affinity for the immobilised antigen.
23 Figure 1. 8: Schematic representation of the genotype:phenotype linkage of scFv and the process of phage display. The resultant myc-tagged scFv antibody fragment::pIII fusion is displayed by the bacterial host (light pink). The genes coding for the immunoglobulin VH and VL genes are coloured in light green and turquoise, respectively, and are joined by a linker region (GGGGS)3 which prevents dissociation. A myc sequence, coloured in bright pink, is included to facilitate affinity purification. The pIII phage coat protein is coloured in light grey and the WT pIII in dark grey. The essential packaging proteins, supplied by the helper phage, is shown as the grey outline of the E. coli. The process by which the scFv antibodies are selected from the phagemid library is detailed in steps A to H. The stepwise explanation of the process is given in the text. Adapted from McCafferty et al. (1990).
24
1.10.1 Antibody formats produced by phage display
The most common antibody fragments produced by phage display (Fig. 1.9) are the single chain variable fragment (scFv) and the antigen binding fragment (Fab) of all mammalian species as well as the variable heavy domain (VHH) of camel antibodies (Holt et al., 2003; Hoet et al., 2005; Hust and Dübel, 2005). The monovalent scFv (VL
and VH), Fab (VL, CL, VH, CH) and nanobody (VHH) fragments retain their respective specific antigen binding affinity compared to that of the VH1 and CH1, since the antigen binding surface is not altered (Bird et al., 1988; Huston et al., 1988).
Figure 1.9: Commonly produced antibody fragments using phage display.
(Hammarström and Marcotte, 2015).
The evolution of high affinity single V-like domains (VHH) is an integral part of the camelid immune system (Fig. 1.9, panel B) (De Genst et al., 2005). The VHH domain possesses extended surface loops which are able to penetrate the narrow cavities on various pathogens’ surfaces (Nuttall et al., 2004; Streltsov et al., 2004). In order to escape immunodetection, many pathogens have evolved narrow cavities in their surface antigens in order to make them inaccessible by host antibodies (Janeway Jnr and Medzhitov, 2002). A prime example is evident in the close packing of the VSG dimers on the parasite surface. A VHH antibody fragment was able to penetrate further between the VSGs on the surface of T. b. brucei compared to the Fab antibody fragment (Fig. 1.10) and the anti-VSG IgM antibody (Fig. 1.4) (Stijlemans et al., 2004).
The development of combinatorial libraries for human or mouse V genes is complex due to the requirement for multiple primer sets. In birds, the genes for the heavy (H)
25 and light (L) chains are subjected to VDJ and VJ rearrangement, respectively. As a result of the incorporation of pseudo V region genes, variability arises by resultant gene conversion (Reynaud et al., 1985; Reynaud et al., 1987; Thompson and Neiman, 1987;
Reynaud et al., 1989). This results in the V regions of the chicken immunoglobulins, having identical amino acid residues at both termini. This simplifies the development of a combinatorial library of the naïve chicken antibody repertoire as only one set of primers is required: one for the H and another for the L chain. This characteristic of the genes coding for chicken antibodies was first exploited by Davies et al. (1995) using chicken bursal lymphocyte RNA to produce a naïve scFv library, from which scFv antibodies were produced against three proteins. Since chickens are able to produce antibodies against a wide range of antigens (Conroy et al., 2012; Shih et al., 2012), together with the their phylogenetic distance from mammalian species, a combinatorial library using chicken antibody coding genes for the development of diagnostic antibodies would be advantageous (Conroy et al., 2014). In 2004, one such phage display library, using chicken immunoglobulin genes, was developed and named the Nkuku® library (van Wyngaardt et al., 2004). The Nkuku® phagemid library will be used in the present study.
Figure 1.10: Comparison of the penetration of VHH and Fab antibody fragments in between VSG dimers on trypanosome surfaces. The VSG dimer is coloured green and blue, the VH, and VL, CH and CL of the Fab fragment are coloured red and black, respectively. The structure of VHH is coloured in light blue and the complementary determining loops in yellow. (Stijlemans et al., 2004).
1.10.2 Applications of phage display
A study of protein-protein interactions (Hertveldt et al., 2009), identification of immunogenic proteins from pathogens (Stijlemans et al., 2004) and of agonists and antagonists to probe receptor site function (Koolpe et al., 2005) can all be achieved using phage display technology.
26 In addition, phage display is a cost effective and efficient method to map the epitopes of various antigens which are involved in antibody interaction, thus, providing vital information for the development of diagnostics, immunotherapies and vaccines (Böttger and Böttger, 2009; Wang and Yu, 2009). The Nkuku® phage display library was used to map the epitopes of the SAT2 foot-and-mouth disease virus in an attempt to neutralise the virus (Opperman et al., 2012).
Antibodies are used directly in diagnostics for pathogen antigen detection (Dussart et al., 2008) and for competition with serum antibodies for binding to the pathogen antigen (Dong et al., 2007). Single chain variable fragment antibodies can be used in many standard immunodiagnostic tests (Thompson and Neiman, 1987; Nissim et al., 1994;
van Wyngaardt and Du Plessis, 1998).
The Nkuku® phage display library has been used to generate antibody fragments for the use in ELISA immunodiagnostic tests against bluetongue virus (Fehrsen et al., 2005; Rakabe et al., 2011), African horse sickness virus (van Wyngaardt et al., 2004;
van Wyngaardt et al., 2013), a 16 kDa antigen of Mycobacterium tuberculosis (Sixholo et al., 2011), and the 65 kDa HSP of Mycobacterium bovis (Wemmer et al., 2010). In addition, the HSP65 antibody fragments were engineered to form bivalent constructs,
“gallibodies”, which were conjugated to gold particles and used in a sandwich RDT (Wemmer et al., 2010).