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• W2022202320 abstract "Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two high molecular weight exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease and are targets for C. difficile-associated disease therapy. Here, recombinant single-domain antibody fragments (VHHs), which specifically target the cell receptor binding domains of TcdA or TcdB, were isolated from an immune llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8), all shown to recognize conformational epitopes, were potent neutralizers of the cytopathic effects of toxin A on fibroblast cells in an in vitro assay. The neutralizing potency was further enhanced when VHHs were administered in paired or triplet combinations at the same overall VHH concentration, suggesting recognition of nonoverlapping TcdA epitopes. Biacore epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Further binding assays revealed TcdA-specific VHHs neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin. With favorable characteristics such as high production yield, potent toxin neutralization, and intrinsic stability, these VHHs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal tract. Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two high molecular weight exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease and are targets for C. difficile-associated disease therapy. Here, recombinant single-domain antibody fragments (VHHs), which specifically target the cell receptor binding domains of TcdA or TcdB, were isolated from an immune llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8), all shown to recognize conformational epitopes, were potent neutralizers of the cytopathic effects of toxin A on fibroblast cells in an in vitro assay. The neutralizing potency was further enhanced when VHHs were administered in paired or triplet combinations at the same overall VHH concentration, suggesting recognition of nonoverlapping TcdA epitopes. Biacore epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Further binding assays revealed TcdA-specific VHHs neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin. With favorable characteristics such as high production yield, potent toxin neutralization, and intrinsic stability, these VHHs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal tract. Clostridium difficile is a Gram-positive, anaerobic, endospore-forming gastrointestinal pathogen responsible for C. difficile-associated disease (CDAD) 2The abbreviations used are: CDAD, C. difficile-associated disease; CD, CD-grease trisaccharide; HCAb, heavy-chain immunoglobulin; HLF, human lung fibroblast; LeX, LeX-AmHex trisaccharide; RBD, receptor binding domain; SPR, surface plasmon resonance; TcdA, toxin A; TcdA-RBD-f1, recombinant fragment of TcdA receptor binding domain; TcdB, toxin B; TcdB-RBD-f1, recombinant fragment of TcdB receptor binding domain; VHH, variable domain of heavy-chain immunoglobulin; RU, resonance unit; AP, alkaline phosphatase. in humans and animals with symptoms ranging in severity from mild cases of antibiotic-associated diarrhea to fatal pseudomembranous colitis (1Rupnik M. Wilcox M.H. Gerding D.N. Nat. Rev. Microbiol. 2009; 7: 526-536Crossref PubMed Scopus (1100) Google Scholar, 2Leffler D.A. Lamont J.T. Gastroenterology. 2009; 136: 1899-1912Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 3Songer J.G. Anim. Health Res. Rev. 2004; 5: 321-326Crossref PubMed Scopus (92) Google Scholar, 4Kelly C.P. Pothoulakis C. LaMont J.T. N. Engl. J. Med. 1994; 330: 257-262Crossref PubMed Scopus (1046) Google Scholar). Each year in North America, 1–3% of hospitalized patients receiving antibiotics become infected with C. difficile, leading to thousands of deaths and over $1 billion in associated costs to the health-care system (4Kelly C.P. Pothoulakis C. LaMont J.T. N. Engl. J. Med. 1994; 330: 257-262Crossref PubMed Scopus (1046) Google Scholar, 5Wilkins T.D. Lyerly D.M. J. Clin. Microbiol. 2003; 41: 531-534Crossref PubMed Scopus (139) Google Scholar, 6Kyne L. Hamel M.B. Polavaram R. Kelly C.P. Clin. Infect. Dis. 2002; 34: 346-353Crossref PubMed Scopus (614) Google Scholar). C. difficile produces two primary virulence factors, toxin A (TcdA) and toxin B (TcdB), which are large (308 and 269 kDa, respectively) single-subunit exotoxins composed of a catalytic, a translocation, and a cell receptor binding domain (RBD) (7Jank T. Aktories K. Trends Microbiol. 2008; 16: 222-229Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 8Jank T. Giesemann T. Aktories K. Glycobiology. 2007; 17: 15R-22RCrossref PubMed Scopus (141) Google Scholar). It has been suggested TcdB is solely responsible for C. difficile virulence (9Lyras D. O'Connor J.R. Howarth P.M. Sambol S.P. Carter G.P. Phumoonna T. Poon R. Adams V. Vedantam G. Johnson S. Gerding D.N. Rood J.I. Nature. 2009; 458: 1176-1179Crossref PubMed Scopus (573) Google Scholar), although a recent study found both TcdA and TcdB knock-out strains are capable of causing mortality in hamsters (10Kuehne S.A. Cartman S.T. Heap J.T. Kelly M.L. Cockayne A. Minton N.P. Nature. 2010; 467: 711-713Crossref PubMed Scopus (615) Google Scholar). This latter finding is in agreement with earlier work that showed both anti-TcdA and anti-TcdB mAbs were required for full protection of hamsters from CDAD (11Babcock G.J. Broering T.J. Hernandez H.J. Mandell R.B. Donahue K. Boatright N. Stack A.M. Lowy I. Graziano R. Molrine D. Ambrosino D.M. Thomas Jr., W.D. Infect. Immun. 2006; 74: 6339-6347Crossref PubMed Scopus (205) Google Scholar, 12Kink J.A. Williams J.A. Infect. Immun. 1998; 66: 2018-2025Crossref PubMed Google Scholar), and anti-TcdA mAbs were required for protection in mice (13Corthier G. Muller M.C. Wilkins T.D. Lyerly D. L'Haridon R. Infect. Immun. 1991; 59: 1192-1195Crossref PubMed Google Scholar). Patients suffering from CDAD are most commonly treated with metronidazole or vancomycin (2Leffler D.A. Lamont J.T. Gastroenterology. 2009; 136: 1899-1912Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). However, there are several emerging challenges warranting the development of novel therapeutics. First, there is no acute CDAD treatment targeting TcdA/B. These toxins are responsible for loss of epithelial barrier function in the colon by disrupting tight junctions and increasing membrane permeability, causing diarrhea and promoting severe inflammation (1Rupnik M. Wilcox M.H. Gerding D.N. Nat. Rev. Microbiol. 2009; 7: 526-536Crossref PubMed Scopus (1100) Google Scholar, 7Jank T. Aktories K. Trends Microbiol. 2008; 16: 222-229Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Second, hypervirulent strains of C. difficile, such as the NAP1/027 isolate, overexpress TcdA and TcdB (14Warny M. Pepin J. Fang A. Killgore G. Thompson A. Brazier J. Frost E. McDonald L.C. Lancet. 2005; 366: 1079-1084Abstract Full Text Full Text PDF PubMed Scopus (1198) Google Scholar) and have been associated with increased mortality rates and disease severity (15O'Connor J.R. Johnson S. Gerding D.N. Gastroenterology. 2009; 136: 1913-1924Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 16Pépin J. Valiquette L. Cossette B. CMAJ. 2005; 173: 1037-1042Crossref PubMed Scopus (506) Google Scholar). Third, an estimated 20–25% of patients suffering from CDAD experience symptomatic relapse after the initial infection is cleared, with 45% of these patients prone to subsequent relapses (17Johnson S. J. Infect. 2009; 58: 403-410Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Taken together, there is a need for nonantibiotic-based reagents that target and inhibit TcdA and TcdB for CDAD therapy. Individuals who are asymptomatic C. difficile carriers and patients who experience mild cases of CDAD tend to possess high anti-TcdA titers (18Kyne L. Warny M. Qamar A. Kelly C.P. Lancet. 2001; 357: 189-193Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 19Kyne L. Warny M. Qamar A. Kelly C.P. N. Engl. J. Med. 2000; 342: 390-397Crossref PubMed Scopus (779) Google Scholar, 20Warny M. Vaerman J.P. Avesani V. Delmée M. Infect. Immun. 1994; 62: 384-389Crossref PubMed Google Scholar, 21Viscidi R. Laughon B.E. Yolken R. Bo-Linn P. Moench T. Ryder R.W. Bartlett J.G. J. Infect. Dis. 1983; 148: 93-100Crossref PubMed Scopus (149) Google Scholar). Conversely, patients susceptible to relapsing C. difficile infection have low anti-TcdA immunoglobulin titers, specifically IgM, IgG2, and IgG3 isotypes (18Kyne L. Warny M. Qamar A. Kelly C.P. Lancet. 2001; 357: 189-193Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 22Katchar K. Taylor C.P. Tummala S. Chen X. Sheikh J. Kelly C.P. Clin. Gastroenterol. Hepatol. 2007; 5: 707-713Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). TcdA-neutralizing secretory IgA antibodies are also thought to play a role in regulating CDAD severity (23Johal S.S. Lambert C.P. Hammond J. James P.D. Borriello S.P. Mahida Y.R. J. Clin. Pathol. 2004; 57: 973-979Crossref PubMed Scopus (45) Google Scholar, 24Kelly C.P. Pothoulakis C. Orellana J. LaMont J.T. Gastroenterology. 1992; 102: 35-40Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Therefore, the introduction of antitoxin antibodies to patients suffering from severe C. difficile infection may be a therapeutically useful approach. A limited number of animal and human studies have illustrated the effectiveness of antitoxin Abs for treatment of CDAD (25Hussack G. Tanha J. Toxins. 2010; 2: 998-1018Crossref PubMed Scopus (31) Google Scholar). Babcock et al. (11Babcock G.J. Broering T.J. Hernandez H.J. Mandell R.B. Donahue K. Boatright N. Stack A.M. Lowy I. Graziano R. Molrine D. Ambrosino D.M. Thomas Jr., W.D. Infect. Immun. 2006; 74: 6339-6347Crossref PubMed Scopus (205) Google Scholar) intraperitoneally administered anti-TcdA and anti-TcdB mAbs to hamsters and found a significant reduction in hamster mortality in prophylactic, primary disease and relapse models when both antitoxin mAbs were administered. A recently completed human trial involving these two mAbs appears promising for treating CDAD relapse (26Lowy I. Molrine D.C. Leav B.A. Blair B.M. Baxter R. Gerding D.N. Nichol G. Thomas Jr., W.D. Leney M. Sloan S. Hay C.A. Ambrosino D.M. N. Engl. J. Med. 2010; 362: 197-205Crossref PubMed Scopus (627) Google Scholar). In another study, intravenous administration of anti-TcdA mAbs raised against the RBD followed by oral challenge with C. difficile resulted in protection of mice (13Corthier G. Muller M.C. Wilkins T.D. Lyerly D. L'Haridon R. Infect. Immun. 1991; 59: 1192-1195Crossref PubMed Google Scholar). Elsewhere, a toxoid vaccine given by the intraperitoneal route to hamsters conferred protection against oral C. difficile challenge (27Giannasca P.J. Zhang Z.X. Lei W.D. Boden J.A. Giel M.A. Monath T.P. Thomas Jr., W.D. Infect. Immun. 1999; 67: 527-538Crossref PubMed Google Scholar), and mice vaccinated with DNA encoding the TcdA RBD resulted in full protection from oral TcdA challenge (28Gardiner D.F. Rosenberg T. Zaharatos J. Franco D. Ho D.D. Vaccine. 2009; 27: 3598-3604Crossref PubMed Scopus (59) Google Scholar). In humans, a number of studies have reported intravenous immunoglobulin therapy to be successful for the treatment of severe CDAD (29Juang P. Skledar S.J. Zgheib N.K. Paterson D.L. Vergis E.N. Shannon W.D. Ansani N.T. Branch R.A. Am. J. Infect. Control. 2007; 35: 131-137Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 30Hassoun A. Ibrahim F. Am. J. Geriatr. Pharmacother. 2007; 5: 48-51Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 31McPherson S. Rees C.J. Ellis R. Soo S. Panter S.J. Dis. Colon Rectum. 2006; 49: 640-645Crossref PubMed Scopus (178) Google Scholar, 32Wilcox M.H. J. Antimicrob. Chemother. 2004; 53: 882-884Crossref PubMed Scopus (169) Google Scholar, 33Salcedo J. Keates S. Pothoulakis C. Warny M. Castagliuolo I. LaMont J.T. Kelly C.P. Gut. 1997; 41: 366-370Crossref PubMed Scopus (223) Google Scholar, 34Leung D.Y. Kelly C.P. Boguniewicz M. Pothoulakis C. LaMont J.T. Flores A. J. Pediatr. 1991; 118: 633-637Abstract Full Text PDF PubMed Scopus (247) Google Scholar), although most lacked proper control groups. Intravenous immunoglobulin involves administration of high concentrations (150–400 mg/kg) of human immunoglobulins from healthy donors that are thought to contain neutralizing antitoxin antibodies, because an estimated 60% of healthy adults have detectable TcdA- and TcdB-specific serum IgG antibodies (21Viscidi R. Laughon B.E. Yolken R. Bo-Linn P. Moench T. Ryder R.W. Bartlett J.G. J. Infect. Dis. 1983; 148: 93-100Crossref PubMed Scopus (149) Google Scholar). Given that C. difficile toxins rely on attachment to epithelial cells for entry (7Jank T. Aktories K. Trends Microbiol. 2008; 16: 222-229Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 8Jank T. Giesemann T. Aktories K. Glycobiology. 2007; 17: 15R-22RCrossref PubMed Scopus (141) Google Scholar), the toxins could conceivably be neutralized within the lower gastrointestinal tract, thereby preventing the critical first step in CDAD pathogenesis. In animals, orally administered bovine immunoglobulin concentrate containing TcdA- and TcdB-neutralizing IgGs were able to prevent hamster mortality when used as a prophylactic (35Lyerly D.M. Bostwick E.F. Binion S.B. Wilkins T.D. Infect. Immun. 1991; 59: 2215-2218Crossref PubMed Google Scholar) and protected rats from the enterotoxic effects of TcdA in vivo (36Kelly C.P. Pothoulakis C. Vavva F. Castagliuolo I. Bostwick E.F. O'Keane J.C. Keates S. LaMont J.T. Antimicrob. Agents Chemother. 1996; 40: 373-379Crossref PubMed Google Scholar). Chicken IgY antibodies specific for toxin RBDs were shown to reduce hamster mortality when administered orally to infected animals (12Kink J.A. Williams J.A. Infect. Immun. 1998; 66: 2018-2025Crossref PubMed Google Scholar). In humans, there have been limited reports on CDAD therapy with orally delivered Abs. Tjellström et al. (37Tjellström B. Stenhammar L. Eriksson S. Magnusson K.E. Lancet. 1993; 341: 701-702Abstract PubMed Scopus (57) Google Scholar) reported the successful treatment of a 3½-year-old boy suffering from severe CDAD with IgA antibody orally. Warny et al. (38Warny M. Fatimi A. Bostwick E.F. Laine D.C. Lebel F. LaMont J.T. Pothoulakis C. Kelly C.P. Gut. 1999; 44: 212-217Crossref PubMed Scopus (97) Google Scholar) and Kelly et al. (39Kelly C.P. Chetham S. Keates S. Bostwick E.F. Roush A.M. Castagliuolo I. LaMont J.T. Pothoulakis C. Antimicrob. Agents Chemother. 1997; 41: 236-241Crossref PubMed Google Scholar) examined the passage of antitoxin bovine IgG through the human gastrointestinal tract and found a significant reduction in IgG activity, likely due to proteolytic degradation within the upper gastrointestinal tract. The limited success of both oral and systemic antitoxin immunotherapy in clinical settings has likely been hampered by the high immunoglobulin dose requirements (150–400 mg/kg), the associated costs of these doses, and a lack of published clinical data showing the effectiveness of these treatments. Alternatives to mAbs for antitoxin immunotherapy are variable heavy-chain single-domain antibodies (VHHs), which are isolated from the heavy-chain IgG (HCAb) of Camelidae species (40Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Nature. 1993; 363: 446-448Crossref PubMed Scopus (2177) Google Scholar, 41Arbabi Ghahroudi M. Desmyter A. Wyns L. Hamers R. Muyldermans S. FEBS Lett. 1997; 414: 521-526Crossref PubMed Scopus (583) Google Scholar). These domains maintain many characteristics of conventional mAbs, including high target affinity (42Koide A. Tereshko V. Uysal S. Margalef K. Kossiakoff A.A. Koide S. J. Mol. Biol. 2007; 373: 941-953Crossref PubMed Scopus (66) Google Scholar) and specificity, with the added advantages of small size (∼15 kDa), easy genetic manipulation, amenability to library screening and selection (43Arbabi-Ghahroudi M. Tanha J. MacKenzie R. Methods Mol. Biol. 2009; 502: 341-364Crossref PubMed Scopus (25) Google Scholar), inherent thermal and proteolytic stability (44Harmsen M.M. van Solt C.B. van Zijderveld-van Bemmel A.M. Niewold T.A. van Zijderveld F.G. Appl. Microbiol. Biotechnol. 2006; 72: 544-551Crossref PubMed Scopus (101) Google Scholar, 45Dumoulin M. Conrath K. Van Meirhaeghe A. Meersman F. Heremans K. Frenken L.G. Muyldermans S. Wyns L. Matagne A. Protein Sci. 2002; 11: 500-515Crossref PubMed Scopus (479) Google Scholar, 46van der Linden R.H. Frenken L.G. de Geus B. Harmsen M.M. Ruuls R.C. Stok W. de Ron L. Wilson S. Davis P. Verrips C.T. Biochim. Biophys. Acta. 1999; 1431: 37-46Crossref PubMed Scopus (353) Google Scholar), and high yield, low cost recombinant production in expression hosts such as bacteria (47Arbabi-Ghahroudi M. Tanha J. MacKenzie R. Cancer Metastasis Rev. 2005; 24: 501-519Crossref PubMed Scopus (139) Google Scholar), yeasts (48Thomassen Y.E. Verkleij A.J. Boonstra J. Verrips C.T. J. Biotechnol. 2005; 118: 270-277Crossref PubMed Scopus (21) Google Scholar), plants (49Teh Y.H. Kavanagh T.A. Transgenic Res. 2010; 19: 575-586Crossref PubMed Scopus (31) Google Scholar), and mammalian cells (50Zhang J. MacKenzie R. Durocher Y. Methods Mol. Biol. 2009; 525: 323-336Crossref PubMed Scopus (20) Google Scholar) (reviewed in Refs. 51Harmsen M.M. De Haard H.J. Appl. Microbiol. Biotechnol. 2007; 77: 13-22Crossref PubMed Scopus (593) Google Scholar, 52Holliger P. Hudson P.J. Nat. Biotechnol. 2005; 23: 1126-1136Crossref PubMed Scopus (1501) Google Scholar, 53Holt L.J. Herring C. Jespers L.S. Woolven B.P. Tomlinson I.M. Trends Biotechnol. 2003; 21: 484-490Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). An increasing number of VHHs have been isolated against targets relevant to infection and immunity (reviewed in Ref. 54Wesolowski J. Alzogaray V. Reyelt J. Unger M. Juarez K. Urrutia M. Cauerhff A. Danquah W. Rissiek B. Scheuplein F. Schwarz N. Adriouch S. Boyer O. Seman M. Licea A. Serreze D.V. Goldbaum F.A. Haag F. Koch-Nolte F. Med. Microbiol. Immunol. 2009; 198: 157-174Crossref PubMed Scopus (382) Google Scholar). Several of these VHHs were effective neutralizers of toxins, viruses, and enzymes as follows: scorpion toxin AahI′ (55Hmila I. Saerens D. Ben Abderrazek R. Vincke C. Abidi N. Benlasfar Z. Govaert J. El Ayeb M. Bouhaouala-Zahar B. Muyldermans S. FASEB J. 2010; 24: 3479-3489Crossref PubMed Scopus (90) Google Scholar, 56Hmila I. Abdallah R.B.A. Saerens D. Benlasfar Z. Conrath K. Ayeb M.E. Muyldermans S. Bouhaouala-Zahar B. Mol. Immunol. 2008; 45: 3847-3856Crossref PubMed Scopus (117) Google Scholar); Escherichia coli heat-labile toxin (57Harmsen M.M. van Solt C.B. Fijten H.P. Appl. Microbiol. Biotechnol. 2009; 84: 1087-1094Crossref PubMed Scopus (28) Google Scholar); foot and mouth disease virus (58Harmsen M.M. Fijten H.P. Engel B. Dekker A. Eblé P.L. Vaccine. 2009; 27: 1904-1911Crossref PubMed Scopus (28) Google Scholar); ART2.2, an ecto-enzyme related to ADP-ribosylating bacterial toxins (59Koch-Nolte F. Reyelt J. Schössow B. Schwarz N. Scheuplein F. Rothenburg S. Haag F. Alzogaray V. Cauerhff A. Goldbaum F.A. FASEB J. 2007; 21: 3490-3498Crossref PubMed Scopus (89) Google Scholar); verotoxin 1 (60Stone E. Hirama T. Chen W. Soltyk A.L. Brunton J. MacKenzie C.R. Zhang J. Mol. Immunol. 2007; 44: 2487-2491Crossref PubMed Scopus (25) Google Scholar); HIV-1 envelope protein gp120 (61Forsman A. Beirnaert E. Aasa-Chapman M.M. Hoorelbeke B. Hijazi K. Koh W. Tack V. Szynol A. Kelly C. McKnight A. Verrips T. de Haard H. Weiss R.A. J. Virol. 2008; 82: 12069-12081Crossref PubMed Scopus (103) Google Scholar); rotovirus (62Garaicoechea L. Olichon A. Marcoppido G. Wigdorovitz A. Mozgovoj M. Saif L. Surrey T. Parreño V. J. Virol. 2008; 82: 9753-9764Crossref PubMed Scopus (95) Google Scholar, 63van der Vaart J.M. Pant N. Wolvers D. Bezemer S. Hermans P.W. Bellamy K. Sarker S.A. van der Logt C.P. Svensson L. Verrips C.T. Hammarstrom L. van Klinken B.J. Vaccine. 2006; 24: 4130-4137Crossref PubMed Scopus (89) Google Scholar); and p2 phage (64Ledeboer A.M. Bezemer S. de Hiaard J.J. Schaffers I.M. Verrips C.T. van Vliet C. Düsterhöft E.M. Zoon P. Moineau S. Frenken L.G. J. Dairy Sci. 2002; 85: 1376-1382Abstract Full Text PDF PubMed Scopus (43) Google Scholar). These VHHs neutralized or inhibited the function of their targets by blocking enzyme-active sites (59Koch-Nolte F. Reyelt J. Schössow B. Schwarz N. Scheuplein F. Rothenburg S. Haag F. Alzogaray V. Cauerhff A. Goldbaum F.A. FASEB J. 2007; 21: 3490-3498Crossref PubMed Scopus (89) Google Scholar), preventing protein-receptor binding through site-specific binding or steric hindrance (56Hmila I. Abdallah R.B.A. Saerens D. Benlasfar Z. Conrath K. Ayeb M.E. Muyldermans S. Bouhaouala-Zahar B. Mol. Immunol. 2008; 45: 3847-3856Crossref PubMed Scopus (117) Google Scholar, 57Harmsen M.M. van Solt C.B. Fijten H.P. Appl. Microbiol. Biotechnol. 2009; 84: 1087-1094Crossref PubMed Scopus (28) Google Scholar), and by possibly inducing conformational changes in the target protein (62Garaicoechea L. Olichon A. Marcoppido G. Wigdorovitz A. Mozgovoj M. Saif L. Surrey T. Parreño V. J. Virol. 2008; 82: 9753-9764Crossref PubMed Scopus (95) Google Scholar). Here, we set out to isolate VHHs capable of binding and neutralizing C. difficile TcdA and TcdB. We hypothesized that VHHs targeting the RBD region of toxin will block the toxin-receptor interaction, thereby preventing toxin entry into the host cell, which is a critical initial step in the TcdA/B mechanism of action (7Jank T. Aktories K. Trends Microbiol. 2008; 16: 222-229Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). To do so, an immune llama VHH phage display library was constructed and panned with recombinant RBD fragments. The isolated VHHs were then characterized for their ability to bind native toxins and recombinant RBD fragments and the nature and relative positioning of epitopes. In addition, the ability of VHHs to neutralize toxins in an in vitro cell cytotoxicity assay was assessed. These findings and the potential applications of VHHs in treating infectious disease are discussed. TcdA and TcdB were isolated from C. difficile strain 10463 (ATCC, Manassas, VA) as described previously (65Keel M.K. Songer J.G. Vet. Pathol. 2007; 44: 814-822Crossref PubMed Scopus (44) Google Scholar) and were stored in 50 mm Tris-HCl buffer, pH 7.5, at 4 °C. Recombinant fragments of TcdA (amino acid residues 2304–2710) and TcdB (amino acid residues 2286–2366), which are fragments of the RBD, were cloned (as a BamHI-HindIII fragment for tcdA and a BamHI-EcoRI fragment for tcdB) into pTrcHisB (Invitrogen), transforming E. coli DH5αMCR. Expression was induced by isopropyl 1-thio-β-d-galactopyranoside, and cells were harvested and lysed in a French pressure cell, and proteins TcdA-RBD-f1 and TcdB-RBD-f1 were purified by immobilized metal-affinity chromatography. Recombinant RBD fragments were dialyzed into PBS, pH 7.3, and stored at 4 °C. One male llama (Lama glama) was immunized by subcutaneous lower back injection of TcdA-RBD-f1 and TcdB-RBD-f1 antigens. On day 1, 200 μg of each antigen diluted in PBS to 1 ml was injected with 1 ml of Freund's complete adjuvant (Sigma). Three more injections of 100 μg of each antigen + Freund's incomplete adjuvant (Sigma) were performed on days 22, 36, and 50. A final injection of 100 μg of each antigen with no adjuvant was performed on day 77. Preimmune blood was drawn before the first injection on day 1 and served as a negative control. Blood (10–15 ml) was collected on days 29, 43, 57, and 84. Preimmune and postimmune total serum was analyzed for a specific response to TcdA-RBD-f1 and TcdB-RBD-f1 by ELISA on day 57 (see below). Llama serum from day 84 was fractionated as before (66Doyle P.J. Arbabi-Ghahroudi M. Gaudette N. Furzer G. Savard M.E. Gleddie S. McLean M.D. Mackenzie C.R. Hall J.C. Mol. Immunol. 2008; 45: 3703-3713Crossref PubMed Scopus (72) Google Scholar). The resulting fractions, A1 (HCAb), A2 (HCAb), G1 (HCAb), and G2 (conventional IgG), were analyzed for specific binding to TcdA-RBD-f1 and TcdB-RBD-f1 by ELISA. Briefly, 5 μg of TcdA-RBD-f1 or TcdB-RBD-f1 diluted in PBS was coated overnight (100 μl/well, 18 h, 4 °C) in 96-well MaxisorpTM plates (Nalge Nunc International, Rochester, NY). Plates were blocked with bovine BSA and washed with PBS-T (PBS + 0.05% (v/v) Tween 20), and serial dilutions of preimmune total serum, postimmune total serum (day 57), and fractionated serum (day 84; 100 μg/ml starting concentration) were applied. After incubation at room temperature for 1.5 h and washing with PBS-T, goat anti-llama IgG (1:1,000 in PBS) was added for 1 h at 37 °C. After washing with PBS-T, pig anti-goat IgG-HRP conjugate (1:3,000 in PBS) was added for 1 h at 37 °C. A final PBS-T wash precluded the addition of 100 μl/well TMB substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 10 min. The reaction was stopped with 100 μl/well 1 m H3PO4 and read on a Bio-Rad plate reader at 450 nm. Library construction and panning were performed essentially as described previously (67Arbabi-Ghahroudi M. MacKenzie R. Tanha J. Methods Mol. Biol. 2009; 525: 187-216Crossref PubMed Scopus (25) Google Scholar, 68Arbabi-Ghahroudi M. To R. Gaudette N. Hirama T. Ding W. MacKenzie R. Tanha J. Protein Eng. Des. Sel. 2009; 22: 59-66Crossref PubMed Scopus (69) Google Scholar, 69Tanha J. Muruganandam A. Stanimirovic D. Methods Mol. Med. 2003; 89: 435-449PubMed Google Scholar). Total RNA was isolated from ∼5 × 106 lymphocytes collected on day 84 post-immunization using the QIAamp RNA blood mini kit (Qiagen, Mississauga, Ontario, Canada). About 5 μg of total RNA was used as template for first strand cDNA synthesis with oligo(dT) primers using the first-Strand cDNA synthesis kit (GE Healthcare). The cDNA was PCR-amplified by an equimolar mix of three variable region-specific sense primers (MJ1, 5′-GCCCAGCCGGCCATGGCCSMKGTGCAGCTGGTGGAKTCTGGGGGA-3′; MJ2, 5′-CAGCCGGCCATGGCCCAGGTAAAGCTGGAGGAGTCTGGGGGA-3′; and MJ3, 5′-GCCCAGCCGGCCATGGCCCAGGCTCAGGTACAGCTGGTGGAGTCT-3′) and two antisense CH2-specific primers (CH2, 5′-CGCCATCAAGGTACCAGTTGA-3′ and CH2b3, 5′-GGTACCTGTCATCCACGGACCAGCTGA-3′). Briefly, the PCR mixture was set up in a total volume of 50 μl with the following components: 1–3 μl of cDNA, 5 pmol of MJ1–3 primer mixture, 5 pmol of either CH2 or CH2b3 primers, 5 μl of 10× reaction buffer, 1 μl of 10 mm dNTP, and 2.5 units of TaqDNA polymerase (Hoffmann-La Roche). The PCR protocol consisted of an initial step at 94 °C for 3 min, followed by 30 cycles of 94 °C for 1 min, 55 °C for 30 s, 72 °C for 30 s, and a final extension step at 72 °C for 7 min. The amplified PCR products were run in a 1% agarose gel, and two major bands were observed as follows: a band of about 850 bp, corresponding to conventional IgG, and a second band of around 600 bp, corresponding to HCAbs. The smaller bands were cut and purified using the QIAquick gel extraction kit (Qiagen) and re-amplified in a second PCR in a total volume of 50 μl using 1 μl of DNA template, 5 pmol of each of MJ7 primer (5′-CATGTGTAGACTCGCGGCCCAGCCGGCCATGGCC-3′) and MJ8 primer (5′-CATGTGTAGATTCCTGGCCGGCCTGGCCTGAGGAGACGGTGACCTGG-3′), 5 μl of 10× reaction buffer, 1 μl of 10 mm dNTP, and 2.5 units of TaqDNA polymerase. The PCR protocol consisted of an initial step at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min and a final extension step at 72 °C for 7 min. The amplified PCR products, ranging between 340 and 420 bp and corresponding to VHH fragments of HCAbs, were purified using the QIAquick PCR purification kit (Qiagen), digested with SfiI restriction enzyme (New England Biolabs, Pickering, Ontario, Canada), and re-purified using the same kit. Eighty micrograms of pMED1 phagemid (67Arbabi-Ghahroudi M. MacKenzie R. Tanha J. Methods Mol. Biol. 2009; 525: 187-216Crossref PubMed Scopus (25) Google Scholar) was digested with SfiI overnight at 50 °C. To minimize self-ligation, 20 units of XhoI and PstI restriction enzymes were added, and the digestion reaction was incubated for an additional 2 h at 37 °C. Sixty micrograms of digested phagemid DNA was ligated with 6 μg of digested VHH fragments for 3 h at room temperature using LigaFast Rapid DNA ligation system (Promega, Madison, WI) and its protocol. The ligated materials were purified using the QIAquick PCR purification kit in a final volume of 100 μl and electroporated in 5-μl portions into commercial electrocompetent TG1 E. coli cells (Stratagene, La Jolla, CA) as described previously (67Arbabi-Ghahroudi M. MacKenzie R. Tanha J. Methods Mol. Biol. 2009; 525: 187-216Crossref PubMed Scopus (25) Google" @default.
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• W2022202320 date "2011-03-01" @default.
• W2022202320 modified "2023-10-03" @default.
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