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Construction of Antibody Phage Libraries and Their Application in Veterinary Immunovirology

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Construction of Antibody Phage Libraries and Their Application in Veterinary Immunovirology antibodies Review Construction of Antibody Phage Libraries and Their Application in Veterinary Immunovirology Shahbaz Bashir and Jan Paeshuyse * Department of Biosystems, Division of Animal and Human Health Engineering, Laboratory of Host Pathogen Interaction in Livestock, KU Leuven University, 3000 Leuven, Belgium; [email protected] * Correspondence: [email protected]; Tel.: +32-16-37-4654 Received: 23 March 2020; Accepted: 28 April 2020; Published: 3 June 2020 Abstract: Antibody phage display (APD) technology has revolutionized the field of immunovirology with its application in viral disease diagnostics and antiviral therapy. This robust and versatile technology allows the expression of an antibody fused to a phage coat protein on the surface of a filamentous phage. The DNA sequence coding for the antibody is packaged within the phage, linking the phenotype to genotype. Antibody phage display inherits the ability to rapidly generate and modify or improve high-affinity monoclonal antibodies, rendering it indispensable in immunology. In the last two decades, phage-display-derived antibodies have been extensively used in human medicine as diagnostic and therapeutic modalities. Recently, they are also gaining significant ground in veterinary medicine. Even though these advancements are mainly biased towards economically important animals such as chicken, cattle, and pigs, they are laying the foundation of fulfilling the unmet needs of veterinary medicine as antibody-based biologics in viral diagnostics, therapeutics, and immunoprophylaxis. This review provides a brief overview of the construction of antibody phage libraries and their application in diagnosis, prevention, and control of infectious viral diseases in veterinary medicine in detail. Keywords: phage display; immunovirology; monoclonal antibody; veterinary medicine 1. Introduction Phage display has emerged as a very powerful, robust, and effective molecular technique to generate vast libraries containing millions or even billions of different peptides or proteins. It includes batch cloning of DNA encoding for millions of variants of certain ligands (e.g., peptides and proteins or fragments thereof) into the phage genome as part of one of the phage coat proteins (p III, p VI, or p VIII). Exposition of proteins or polypeptides on the surface of phage is achieved by fusion of the coding sequence of one of the coat proteins to the gene of interest, which helps in isolating the specific binding ligands by a series of recursive cycles of selection on antigen or ligand with each cycle comprising of binding, washing, elution, and amplification. Generally, proteins are displayed on phage particle tethered to either minor coat proteins like p III or the major coat proteins like p VIII, yet retaining its structure/function. Based on the direct linkage between phenotype and genotype, phage display technology (PDT) provides an expedient approach to study the genetics and functionality of the interacting proteins and peptides [1]. Following the initial demonstration of peptide display, antibodies were the first functional proteins to be successfully displayed on the phage surface. In a humoral immune response, antibodies serve as antigen-targeting effector molecules. Their unique tetrameric structure consists of two identical light and two identical heavy chains joined together by disulfide bridges and non-covalent interactions. Each chain is comprised of a series of discreet repeats, forming a compactly folded regions of proteins called “antibody domains” (Figure1). Antibodies are modular protein defense systems possessing a paratope (variable domain), an antigen-binding site located at Antibodies 2020, 9, 21; doi:10.3390/antib9020021 www.mdpi.com/journal/antibodies Antibodies 2020, 9, x FOR PEER REVIEW 2 of 13 Antibodies 2020, 9, 21 2 of 13 Antibodies are modular protein defense systems possessing a paratope (variable domain), an antigen-binding site located at the upper tips of the “Y”-shaped structure. These paratopes identify thethe upperspecific tips epitope of the “Y”-shapeddisplayed by structure. the antigen These and paratopes tag the microbe identify theas well specific as the epitope infected displayed cell to bybe therecognized antigen andby the tag immune the microbe system as well for asneutralizatio the infectedn. cellTherefore, to be recognized these variable by the domains immune serve system as forparadigmatic neutralization. proteinaceous Therefore, scaffolds these variable to be expresse domainsd serveon the as phage paradigmatic surface for proteinaceous the isolation sca of ffnovelolds tobinders be expressed against a on myriad the phage of antigens surface. Phage for the display isolation of ofcombinatorial novel binders an againsttibody libraries a myriad is of an antigens. efficient Phagetechnique display by which of combinatorial monoclonal antibodyantibodies libraries (mAbs) isof an a desired efficient specificity technique can by be which selected monoclonal without antibodiesthe use of (mAbs)conventional of a desired hybridoma specificity technology can be selected[2]. Their without uniquethe maturation use of conventional process helps hybridoma them to technologyevolve to be [2 highly]. Their specific unique to maturation viral antigens, process opening helps new them horizons to evolve in to viral be highlydisease specific diagnosis to viral and antigens,therapeutics. opening In the new last horizons few decades, in viral the disease prime diagnosis focus of andthe therapeutics.developed recombinant In the last few antibodies decades, was the primetargeted focus towards of the developedhuman medicine, recombinant but now antibodies the shift was in targetedtrends towards towards veterinary human medicine, medicine but seems now thepromising. shift in trendsIn spite towards of skewed veterinary research medicine trends towa seemsrds promising.economically In important spite of skewed livestock research species trends such towardsas cows, economicallypoultry, sheep, important goats, and livestock pigs, there species is sucha vast as field cows, of poultry,veterinary sheep, medicine goats, andwaiting pigs, for there the isapplication a vast field of of mAbs veterinary developed medicine through waiting antibody for the applicationphage display. of mAbs Furthermore, developed efficient through techniques antibody phagehave been display. established Furthermore, and optimize efficientd to techniques design, build, have and been manipula establishedte the and vast optimized antibody tofragment- design, build,based andlibraries manipulate in order the to vast derive antibody the antibodies fragment-based of desired libraries characteristics in order to deriveand affinity. the antibodies Here, we of desiredencompass characteristics the progress and in affi thisnity. rapidly Here, wegrowing encompass field theand progress discuss in its this application rapidly growing in finding field new and discussdiagnostics its application and therapeutic in finding viral new diagnosticstargets in andveterinary therapeutic medicine viral targets in association in veterinary with medicine other incomplementary association with technologies. other complementary technologies. (A) (B) FigureFigure 1.1. ((AA)) StructureStructure ofof aa conventionalconventional antibodyantibody andand itsits derivatives.derivatives. Abbreviations:Abbreviations: Fv,Fv, fragmentfragment variable;variable; Fc,Fc, fragmentfragment crystallizable;crystallizable; Fab, fragmentfragment antigenantigen binding;binding; scFv,scFv, single-chainsingle-chain variablevariable fragmentfragment with linker linker (yellow); (yellow); scFab, scFab, single-chain single-chain Fa Fab;b; VH, VH, variable variable domain domain heavy heavy chain; chain; F(ab)2, F(ab)2, two twoFab Fabfragments fragments joined joined together; together; scFv–Fc, scFv–Fc, scFv scFv linked linked to to Fc Fc (inter- (inter- and and intra-disulfide intra-disulfide linkages linkages are notnot shownshown herehere inin thethe allall formats).formats). (B) Camelid heavy chain antibody (HCAb);(HCAb); VHH, nanobody (modified(modified fromfrom [[3]).3]). 2. Antibody Phage Display Library Construction and Biopanning 2. Antibody Phage Display Library Construction and Biopanning An antibody phage library is the collection of antibody variable domains displaying phages An antibody phage library is the collection of antibody variable domains displaying phages with with library size 106–1011, depending on the library type, fused to their coat proteins as a result library size 106–1011, depending on the library type, fused to their coat proteins as a result of antibody- of antibody-variable fragment (gene) cloning. Here, we shall briefly describe the construction of variable fragment (gene) cloning. Here, we shall briefly describe the construction of a phagemid- a phagemid-based antibody (scFv and Fab) phage display library from immunized or naïve sources. based antibody (scFv and Fab) phage display library from immunized or naïve sources. The basic The basic methodology to build APD library from the V-gene repertoire (Figure2) is almost the same, methodology to build APD library from the V-gene repertoire (Figure 2) is almost the same, starting starting with the extraction of mRNA from B-cells isolated from blood, spleen, tonsils, and tumor tissue with the extraction of mRNA from B-cells isolated from blood, spleen, tonsils, and tumor tissue samples. Using oligo (dt) primers or random primers, cDNA is synthesized from mRNA through samples. Using oligo
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