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Appendix 41 Foot-and-mouth disease immunoprophylaxis using single-domain antibody fragments. Michiel. M. Harmsen*1, Conny B. van Solt1, Helmi P.D. Fijten1, Lonneke van Keulen1, Rodney A. Rosalia1, Klaas Weerdmeester1, Lisette A.H.M. Cornelissen1, Tiny G.M. de Bruin1, Phaedra L. Eblé2, Aldo Dekker2 2 1 Animal Sciences Group of Wageningen UR, P.O. Box 65, 8200 AB Lelystad, The Netherlands. Central Institute for Animal Disease Control, P.O. Box 2004, 8203 AA Lelystad, The Netherlands. Abstract: We aim to develop a method for the rapid control of FMDV in outbreak situations by passive immunisation with recombinant llama single-domain antibody fragments (VHHs). VHHs have a number of advantages for biotechnological applications, such as a high physicochemical stability and a high production level in micro-organisms. We obtained 24 FMD binding VHHs by phage display selection from llamas immunized with inactivated FMDV vaccine. They recognized 4 functionally independent antigenic sites of FMDV, one of which is the GH-loop. A major disadvantage of VHHs is their small size, resulting in a low serum half-life, which prohibits their therapeutic application. To increase their serum half-life we therefore produced genetic fusions of three FMDV binding VHHs with a single porcine Ig binding VHH by large scale yeast (Saccharomyces cerevisiae) fermentation. Passive immunisation with such fusion proteins reduced the mean daily viremia significantly (P=0.0031) and appeared to reduce the mean daily viral shedding (P=0.068) but did not prevent FMD transmission. Introduction: EU legislation (2003/85/EC) now favours emergency vaccination to control FMD outbreaks in Europe. It takes at least four days until such vaccination protects pigs (Salt et al., 1998). Within this time, the disease can spread further. Furthermore, although pigs vaccinated four days prior to challenge are protected against clinical disease, they can transmit FMD virus to contact susceptible animals, which then develop clinical FMD (Salt et al., 1998). Therefore, there is a need for therapies that provide rapid protection against FMD. Such rapid protection can be achieved by passive transfer of hyperimmune serum (Blancou, 2002) or neutralising monoclonal antibodies (mAbs) (McCullough et al., 1986). Since passive immunoprotection wanes fast, due to antibody clearance from the body, it should be used in conjunction with conventional emergency vaccination to induce both rapid and long lasting protection. We aim to develop such passive immunotherapy using recombinant mAbs. Previously isolated conventional FMDV neutralizing mAbs bind to the external surface of FMDV which is composed of 60 copies each of the viral proteins (VP) 1, VP2 and VP3. Many strongly neutralizing mAbs bind to a continuous antigenic site present on an exceptionally long flexible loop of VP1, the GH loop, that extrudes from the viral surface and can be removed by trypsin treatment (Strohmaier et al., 1982). The effectiveness of passive immunisation with mAbs may be complicated by the existence of at least 7 FMDV serotypes: A, O, C, Asia1, SAT1, SAT2 and SAT3. As a source of antibodies we use recombinant single-domain antibody fragments (VHHs) derived from heavy-chain antibodies as are found in camelids (Hamers-Casterman et al., 1993). Heavychain antibodies are naturally devoid of the immunoglobulin light chain. As compared to conventional antibodies VHHs have several advantages for biotechnological applications, such as their use as biopharmaceutical (Holliger and Hudson, 2005). Important advantages are the high production level of VHHs in micro organisms (Frenken et al., 2000), their high physicochemical stability (Van der Linden et al., 1999) and the facile construction of bispecific VHH dimers by genetic fusion of two VHH domains (Harmsen et al., 2005). A major disadvantage of the use of small antibody fragments is their rapid removal from the circulation of mammals with an elimination half-life of several hours (Batra et al., 2002). However, this half-life can be significantly extended by making genetic fusions of the VHH of interest to another VHH that binds to porcine immunoglobulin (Holliger et al., 1997; Harmsen et al., 2005). Here we describe the isolation and characterization of 24 FMDV binding VHHs. We next produced three bispecific VHH2s that consist of a VHH binding to FMDV fused to a VHH binding to porcine Ig and analysed their potential use in immunotherapy against experimental FMDV infection of pigs. 280 Materials and Methods: FMDV antigens: FMDV was cultured using BHK-21 cells grown in suspension. Virus released into the clarified culture supernatant was inactivated with 0.01 M binary ethyleneimine, concentrated using polyethylene glycol and sterile filtered. Trypsin-treated virus was prepared as described before (Strohmaier et al., 1982). Peptide PAT49 (acetyl-YGDGTVANVRGDLQVLAQKAARALPC-amide), corresponding to amino acid residues 136-160 of VP1 of the FMDV O1 Manisa strain and containing an additional C-terminal cysteine residue was coupled to maleimide-activated bovine serum albumin (Pierce, Rockford, IL). VHH selection : Llamas (Lama glama) were immunized or infected with live O1 Manisa (1 llama) or O1 BFS (1 (Frenken et al., 2000; Van der Linden et al., 2000). llama-derived immune libraries by phage display characterized by ELISA and sequencing. (2 llamas) with FMDV serotype O1 Manisa llama) for induction of antibody responses Antigen specific VHHs were isolated from (McCafferty and Johnson, 1996) and VHH production : Selected unique VHHs were subsequently expressed in bakers yeast under control of the GAL7 promotor and directed into the growth medium by fusion to the yeast invertase signal peptide using plasmid pRL188. Using this plasmid the resulting VHHs are fused to the llama long-hinge region containing a single C-terminal cysteine and a hexahistidine tag (EPKTPKPQPQPQPQPQPNPTTESKCPHHHHHH) for coupling and purification purposes, respectively. VHH encoding regions were inserted into the PstI and BstEII sites of plasmid pUR4585 (Harmsen et al., 2000) for yeast production of VHHs that instead of the long hinge region contain a C-terminal c-myc tag for specific detection of VHHs with the c-myc recognizing antibody 9E10. Fusion proteins consisting of two VHH domains separated by an artificial Gly-Gly-Ser linker were produced as described previously (Harmsen et al., 2005). Methods for VHH production in yeast and purification by immobilized-metal affinity chromatography from yeast culture supernatant have been desribed in detail previously (Van der Vaart, 2002). VHHs used in animal experiments were subsequently additionally purified by cation exchange chromatography. Characterization of antigen binding of VHHs: ELISAs used for screening of phage display libraries and yeast expressed VHHs were normally done by coating plates directly with 2 ug/ml of FMDV O1 Manisa/Turkey/69 146S particles. Plates were then incubated with serial twofold dilutions of 10 mg/L VHHs. Bound VHH was detected with peroxidase-conjugated polyclonal rabbit immunoglobulins anti-llama-immunoglobulin (Frenken et al., 2000) or peroxidase-conjugated antic-myc mAb 9E10 (Roche). Competition ELISAs were performed using VHHs without c-myc tag that were encoded by pRL188-derived plasmids and VHHs that contain a c-myc-tag that were encoded by pUR4585-derived plasmids. Affinity determinations were performed by competition ELISA in solution as described previously (Friguet et al., 1985). VHH concentrations required for neutralization of FMDV O1 Manisa were determined as described previously (De Leeuw et al., 1979) using 50 TCID50 in duplicate assays using monolayers of secondary swine kidney cells and serial twofold dilutions of 10 mg/ml VHHs. Animal experiments: Pigs weighing about 20 kg received 3 mg/kg of VHH2 intramuscularly. They were challenged 24 h later by inoculation of 104 TCID50 FMDV O1 Manisa into the left hind footpad and following the development of vesicular lesions typical of FMD on other feet the following days. The virus load in blood and oropharyngeal fluid (OPF) was determined daily by virus isolation 2 weeks following challenge. Results: We isolated 87 unique FMDV-binding VHHs by phage display selection from immunized llamas. The antigenic specificity of 24 clones was analyzed in more detail (Table 1). They recognize at least four functionally independent antigenic sites as determined by competition ELISA. Clones recognizing antigenic site I also bound to a peptide representing the GH-loop of VP1, which is a well-known neutralizing site involved in binding to the cellular receptor of FMDV (Sobrino et al., 2001). These clones, and an additional two clones, also failed to bind to trypsin-treated virus which is known to remove the GH-loop (Strohmaier et al., 1982). As expected, these clones are also the more potent in vitro virus neutralizing clones. Clones binding antigenic sites III and IV are more weakly neutralizing, whereas clones binding antigenic site II do not neutralize at all. Clones recognizing antigenic site III specifically recognize O serotype strains whereas clones recognizing the three other antigenic sites also cross react with A, Asia 1 and C serotype strains (SAT serotypes were not analysed). 281 We previously isolated several pIg binding VHHs that showed a hundredfold extended in vivo residence times in pigs as compared to a control VHH that does not bind pIg (Harmsen et al., 2005). We selected clone VI-4 for further work since it binds with high affinity (2 nM) and is specific for the Ig light chain. Thus we fused three FMDV binding VHHs with pIg binding clone VI-4. The resulting bispecific VHH2s were produced by 100-L yeast fermentations for analyzing their protective effect against FMD challenge infection of pigs. They were secreted at concentrations as high as 40-900 mg/L and had high affinity for FMDV (0.3-0.5 nM). The VHH2 M23ggsVI-4 reduced the mean daily viremia significantly (P=0.0031) and appeared to reduce the mean daily viral shedding (P=0.068) but failed to inhibit transmission. Discussion: We isolated a panel of 24 VHHs against FMDV O1 Manisa that recognize four functionally independent antigenic sites. The antigenic sites of FMDV serotype O strains were previously mapped into at least four antigenic sites, denominated sites 1-4 (Mateu and Verdaguer, 2004). A fifth site was identified later (Crowther et al., 1993), but this site appears to overlap with site 1 (Mateu and Verdaguer, 2004). Site 1 involves both the GH-loop and the C-terminus of VP1 (Kitson et al., 1990). It is destroyed by trypsin treatment of the virus, which excizes the GH-loop (Strohmaier et al., 1982). Many antibodies recognizing site 1 also bind to synthetic peptides representing the GH-loop (Parry et al., 1989; Verdaguer et al., 1995), which is therefore referred to as a continuous antigenic site, whereas sites 2-4 are discontinuous (Mateu and Verdaguer, 2004). Furthermore, immunization with synthetic peptides representing the GH-loop induces potent neutralizing antibody responses (Bittle et al., 1982). Neutralizing antibodies binding to the GH-loop recognize a highly conserved Arg-Gly-Asp (RGD) tripeptide motif that can bind to integrin receptors and facilitate the internalization of FMDV into target cells (Sobrino et al., 2001) suggesting that they prevent viral infectivity by abrogation of cellular attachment. We indicate the antigenic sites recognized by VHHs by roman numerals I – IV since we do not know for all sites how they correspond to the published antigenic sites 1-4 (arabic numerals) of FMDV O serotype. Only for antigenic site I could we show that it corresponds to antigenic site 1 because it is a trypsin-sensitive strongly neutralizing linear antigenic site present on a short synthetic peptide representing the VP1 GH-loop. VHHs binding the same antigenic site generally have the same serotype specificity. Thus the serotype specificity of VHHs further supports the antigenic site mapping by competition ELISA. Antigenic site III is specific for O-serotype strains whereas antigenic sites I, II and IV are also found on A, Asia1 and C serotype strains. Our primary aim was to develop passive FMDV immunotherapy using VHHs. Protection in vivo is primarily accomplished by phagocytosis of virus/antibody complexes by the reticuloendothelial system (McCullough et al., 1988). Blocking of viral cell entry, which is the predominant mechanism of in vitro FMDV neutralization, requires far higher antibody concentrations than are normally reached in vivo (McCullough et al., 1986). The importance of opsonophagocytosis for in vivo protection is also suggested by immunization with a five site escape virus that provides protection against challenge with wild type virus in spite of eliciting minimal neutralizing activity in vitro (Dunn et al., 1998). To enable passive FMDV immunotherapy with VHHs recruitment of opsonophagocytosis is therefore required. This can be accomplished indirectly using genetic fusions of two antibody fragments that are specific for the pathogen and for host immunoglobulin (Holliger et al., 1997). For this purpose we recently isolated VHHs binding to pig Ig. These VHHs have the additional advantage of an increased serum residence time (Harmsen et al., 2005). Therefore, we produced genetic fusions of three FMDV binding VHHs with a single pIg binding VHH. Passive immunisation studies with the resulting three VHH2s conferred partial protection against FMDV challenge infection of pigs, as exemplified by a reduced viremia and viral shedding, but failed to inhibit transmission. We are currently improving this strategy of passive immunoprophylaxis by producing improved VHHs by molecular engineering. Conclusions: • A well-characterized set of 24 recombinant single-domain antibody fragments binding to FMDV serotype O has been isolated. • A first generation of VHH2s confers partial protection of pigs against challenge infection, but does not inhibit transmission. 282 Recommendations: • The current VHHs should be further improved by molecular engineering to enable a more effective immunotherapy against FMDV. • It should be determined whether the bispecific VHH2s can indirectly recruit opsonophagocytosis in vitro. Acknowledgements: We thank A. Timmerman, A. Spithoven, S. Riemersma and M. Bleijenberg for expert biotechnical support. References: Batra, S.K., Jain, M., Wittel, U.A., Chauhan, S.C. & Colcher, D. 2002. Pharmacokinetics and biodistribution of genetically engineered antibodies. Curr. Opin. Biotechnol. 13(6): 603-608. Bittle, J.L., Houghten, R.A., Alexander, H., Shinnick, T.M., Sutcliffe, J.G., Lerner, R.A., Rowlands, D.J. & Brown, F. 1982. Protection against foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence. Nature 298: 30-33. Blancou, J. 2002. History of the control of foot and mouth disease. Comp. Immunol. Microbiol. Infect. Dis. 25(5-6): 283-296. Crowther, J.R., Farias, S., Carpenter, W.C. & Samuel, A.R. 1993. Identification of a fifth neutralizable site on type O foot-and-mouth disease virus following characterization of single and quintuple monoclonal antibody escape mutants. J. Gen. Virol. 74(8): 1547-1553. De Leeuw, P.W., Tiessink, J.W. & Frenkel, S. 1979. Vaccination of pigs with formaldehyde inactivated aluminium hydroxide foot-and-mouth disease vaccines, potentiated with diethylaminoethyldextran (DEAE-D). Zentralbl. Veterinarmed. B 26(2): 85-97. Dunn, C.S., Samuel, A.R., Pullen, L.A. & Anderson, J. 1998. The biological relevance of virus neutralisation sites for virulence and vaccine protection in the guinea pig model of foot-and-mouth disease. Virology 247(1): 51-61. Frenken, L.G.J., van der Linden, R.H., Hermans, P.W., Bos, J.W., Ruuls, R.C., de Geus, B. & Verrips, C.T. 2000. Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae. J. Biotechnol. 78(1): 11-21. Friguet, B., Chaffotte, A.F., Djavadi-Ohaniance, L. & Goldberg, M.E. 1985. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J. Immunol. Methods 77(2): 305-319. 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Biotechnol. 15(7): 632-636. 283 Kitson, J.D., McCahon, D. & Belsham, G.J. 1990. Sequence analysis of monoclonal antibody resistant mutants of type O foot and mouth disease virus: evidence for the involvement of the three surface exposed capsid proteins in four antigenic sites. Virology 179(1): 26-34. Mateu, M.G. & Verdaguer, N. 2004. Functional and structural aspects of the interaction of footand-mouth disease virus with antibodies, In: Sobrino, F., Domingo, E. (Eds.) Foot and mouth disease. Current perspectives. Horizon Bioscience, Norfolk, pp. 222-260. McCafferty, J. & Johnson, K.S. 1996. Construction and screening of antibody display libraries., In: Kay, B.K., Winter, J., McCafferty, J. (Eds.) Phage display of peptides and proteins. Academic Press, San Diego, pp. 79-111. McCullough, K.C., Crowther, J.R., Butcher, R.N., Carpenter, W.C., Brocchi, E., Capucci, L. & De Simone, F. 1986. 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Structure of the major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct involvement of the Arg-Gly-Asp motif in the interaction. EMBO J. 14(8): 1690-1696. 284 Table 1. Characteristics of selected FMDV binding VHHs. VHH Clone Eliciting a Antigen FMDV Anti- database neutrali- genic Coated trypsin- GH-loop accession zation site FMDV treated number (mg/ml) Selection EMBL b Antigen Titer in ELISA (mg/L) peptide c FMDV M3 C V AJ849380 >10 II 0.009 0.008 >10 M176 IV, O1 V AJ811555 >10 II 0.104 0.015 >10 M8 Man C SP, V AJ629049 0.15 I 0.007 4.4 0.10 M17 C V AJ811548 0.34 I 0.015 >10 0.40 M190 C SP, V AJ811560 0.20 I 0.011 >10 0.20 M191 C SP AJ811561 0.24 I 0.011 7.3 0.20 M200 C SP, V AJ811563 0.10 I 0.011 >10 0.23 M194 C SP AJ811562 >10 I 0.032 8.6 0.02 M170 IV, O1 V AJ811551 0.22 ? 0.057 9.1 >10 M210 BFS P V AJ811564 0.16 ? 0.059 >10 >10 M171 IV, O1 V AJ811552 0.30 ? 0.402 0.568 >10 M168 BFSO1 IV, V AJ811550 0.61 ? 0.031 0.027 >10 M172 BFS IV, O1 V AJ811553 0.30 ? 0.063 0.287 >10 M179 BFSO1 IV, V AJ811558 0.73 ? 0.013 0.046 >10 M211 BFS P V AJ811565 0.14 ? 0.005 0.004 >10 M178 IV, O1 V AJ811557 >10 ? 0.004 0.010 >10 M166 BFSO1 IV, V AJ811549 >10 ? 0.007 0.013 >10 M177 BFS IV, O1 V AJ811556 >10 ? 0.006 0.010 >10 M180 BFS IV, O1 V AJ811559 0.20 ? 0.010 0.009 >10 M31 BFS C V AJ629053 >10 ? 0.004 0.010 >10 M23 C V AJ629050 3.3 III 0.005 0.003 >10 M29 C V AJ629051 10 III 0.006 0.020 >10 M174 IV, O1 V AJ811554 >10 III 0.007 0.013 >10 M220 Man IV, O1 V AJ811566 1.4 IV 0.007 0.003 >10 a Man C, llama immunized with crude FMDV antigen; P, llama immunized with purified FMDV antigen; IV, llamas infected with FMDV O1 Manisa or O1 BFS, respectively. b V, O1 Manisa FMDV; SP, synthetic peptide PAT49 representing the VP1 GH-loop. Some clones were isolated several times, using both SP and V. c Underlined values are at least a hundred-fold increased compared to titers on directly coated intact FMDV, indicating binding to a trypsin-sensitive antigenic site. 285