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Isolation and characterization of protective anti-LPS nanobody against V. cholerae O1 recognizing Inaba and Ogawa serotypes

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Abstract

Vibrio cholerae is considered one of the major health threats in developing countries. Lack of efficient vaccine, short incubating time of the disease, and bacterium ability to survive in aquatic environment have made cholera one of the most epidemic diseases yet known. The lipopolysaccharide is one of the bacterium key antigens used to classify V. cholerae into 206 serogroups. V. cholerae serogroup O1 is a causative agent of all cholera pandemics. Research has shown that anti-lipopolysaccharide (LPS) antibodies could provide protective immunity in cholera cases. In this research, we used N-terminal fragments of the camel's heavy-chain antibodies called VHH or nanobodies and produced a phagemid library. The obtained library was panned against V. cholerae O1 LPS, and four monoclonal nanobodies were isolated. Isolated nanobodies were tested in LPS ELISA and bacterial ELISA. The nanobody with the highest affinity toward the bacterium was used in an in vivo challenge and successfully neutralized the bacterium infection. The isolated nanobody showed high thermostability and proteolytic resistance in characterization tests.

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References

  • Alam M, Hasan NA, Sadique A, Bhuiyan N, Ahmed KU, Nusrin S, Nair GB, Siddique A, Sack RB, Sack DA (2006) Seasonal cholera caused by Vibrio cholerae serogroups O1 and O139 in the coastal aquatic environment of Bangladesh. Appl Environ Microbiol 72(6):4096–4104

    Article  CAS  Google Scholar 

  • Alam M, Sultana M, Nair GB, Siddique A, Hasan NA, Sack RB, Sack DA, Ahmed K, Sadique A, Watanabe H (2007) Viable but nonculturable Vibrio cholerae O1 in biofilms in the aquatic environment and their role in cholera transmission. Proc Natl Acad Sci 104(45):17801

    Article  CAS  Google Scholar 

  • Ashbolt NJ (2004) Microbial contamination of drinking water and disease outcomes in developing regions. Toxicology 198(1):229–238

    Article  CAS  Google Scholar 

  • Beatty JD, Beatty BG, Vlahos WG (1987) Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods 100(1–2):173–179

    Article  CAS  Google Scholar 

  • Behdani M, Zeinali S, Khanahmad H, Karimipour M, Asadzadeh N, Azadmanesh K, Khabiri A, Schoonooghe S, Habibi Anbouhi M, Hassanzadeh-Ghassabeh G (2011) Generation and characterization of a functional nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor. Mol Immunol 50:35–41

    Article  Google Scholar 

  • Bhattacharya K, Kanungo S, Sur D, Sarkar BL, Manna B, Lopez AL, Bhattacharya M, Nandy S, Niyogi SK (2011) Tetracycline-resistant Vibrio cholerae O1, Kolkata, India. Emerg Infect Dis 17(3):568

    Article  Google Scholar 

  • Bhunia R, Ramakrishnan R, Hutin Y, Gupte MD (2009) Cholera outbreak secondary to contaminated pipe water in an urban area, West Bengal, India, 2006. Indian J Gastroenterol 28(2):62–64

    Article  Google Scholar 

  • Bishop AL, Schild S, Patimalla B, Klein B, Camilli A (2010) Mucosal immunization with Vibrio cholerae outer membrane vesicles provides maternal protection mediated by antilipopolysaccharide antibodies that inhibit bacterial motility. Infect Immun 78(10):4402–4420

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254

    Article  CAS  Google Scholar 

  • Bronze MS, Greenfield RA (eds) (2005) Biodefense: principles and pathogens. Horizon bioscience, Greenfield, 838 pp

    Google Scholar 

  • Brooks GF, Butel JS, Morse SA (2004) Jawetz, Melnick, & Adelberg’s medical microbiology. McGraw-Hill, USA, 880 pp

    Google Scholar 

  • Dharmasena MN, Krebs SJ, Taylor RK (2009) Characterization of a novel protective monoclonal antibody that recognizes an epitope common to Vibrio cholerae Ogawa and Inaba serotypes. Microbiology 155(7):2353–2364

    Article  CAS  Google Scholar 

  • Dubois M, Gilles KA, Hamilton JK, Rebers P, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28(3):350–356

    Article  CAS  Google Scholar 

  • Faruque SM, Sack DA, Sack RB, Colwell RR, Takeda Y, Nair GB (2003) Emergence and evolution of Vibrio cholerae O139. Proc Natl Acad Sci U S A 100(3):1304

    Article  CAS  Google Scholar 

  • Ghassabeh GH, Muyldermans S, Saerens D (2010) Nanobodies, single-domain antigen-binding fragments of camelid heavy-chain antibodies. In: Shire SJ, Gombotz W, Bechtold-Peters K, Andya J (eds) Current trends in monoclonal antibody development and manufacturing. Springer, New York, pp 29–48

    Chapter  Google Scholar 

  • Goodchild S, Hopkins N, Love T, Patel V, Houghton B, Mayers C (2004) Engineering antibodies to detect biological warfare agents. DTIC Document

  • Haley BJ, Grim CJ, Hasan NA, Taviani E, Chun J, Brettin TS, Bruce DC, Challacombe JF, Detter JC, Han CS (2010) The pre-seventh pandemic Vibrio cholerae BX 330286 El Tor genome: evidence for the environment as a genome reservoir. Environ Microbiol Rep 2(1):208–216

    Article  CAS  Google Scholar 

  • Harmsen M, De Haard H (2007) Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 77(1):13–22

    Article  CAS  Google Scholar 

  • Harris AM, Bhuiyan MS, Chowdhury F, Khan AI, Hossain A, Kendall EA, Rahman A, LaRocque RC, Wrammert J, Ryan ET (2009) Antigen-specific memory B-cell responses to Vibrio cholerae O1 infection in Bangladesh. Infect Immun 77(9):3850–3856

    Article  CAS  Google Scholar 

  • Huang L, Muyldermans S, Saerens D (2010) Nanobodies: proficient tools in diagnostics. Expert Rev Mol Diagn 10(6):777–785

    Article  Google Scholar 

  • Igbinosa EO, Okoh AI (2008) Emerging Vibrio species: an unending threat to public health in developing countries. Res Microbiol 159(7):495–506

    Article  Google Scholar 

  • Igbinosa EO, Okoh AI (2009) Toxigenic Vibrio cholerae strains and their associated malaises. Afr J Microbiol Res 3(5):200–211

    CAS  Google Scholar 

  • Kolkman JA, Law DA (2010) Nanobodies—from llamas to therapeutic proteins. Drug Discov Today: Technol 7(2):e139–e146

    Article  CAS  Google Scholar 

  • Krickeberg K, Pham V, Pham T (2012) Diarrhoea and Cholera. In: Epidemiology. Statistics for Biology and Health. Springer, New York, pp 47–52. doi:10.1007/978-1-4614-1205-2_6

  • Laeremans T, Van Bergen PPMP (2010) Camelidae single domain antibodies vhh directed against epidermal growth factor receptor and uses thereof. EP Patent 1:687,338

    Google Scholar 

  • Liu JL, Anderson GP, Delehanty JB, Baumann R, Hayhurst A, Goldman ER (2007) Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library. Mol Immunol 44(7):1775–1783

    Article  CAS  Google Scholar 

  • Malekshahi ZV, Gargari SLM, Rasooli I, Zadeh WE (2011) Treatment of Helicobacter pylori infection in mice with oral administration of egg yolk-driven anti-UreC immunoglobulin. Microb Pathog 51:366–372

    Article  CAS  Google Scholar 

  • Mandal S (2011) Cholera epidemic in and around Kolkata, India: endemicity and management. Oman Med J 26(4):288

    Article  Google Scholar 

  • Mishra M, Mohammed F, Akulwar S, Katkar V, Tankhiwale N, Powar R (2004) Re-emergence of El Tor Vibrio in outbreak of cholera in and around Nagpur. Indian J Med Res 120:478–480

    Google Scholar 

  • Murad F, Alam K, Mazumdar R, Islam S, Nipa M, Iqbal A, Bhuiyan H (2010) Anti-bacterial activity of the extract of Terminalia arjuna against multi antibiotic resistant Vibrio cholerae. J Sci Res 3(1):129

    Article  Google Scholar 

  • Musekiwa A, Volmink J (2011) Oral rehydration salt solution for treating cholera:≤ 270 mOsm/L solutions vs ≥310 mOsm/L solutions. Cochrane Database Syst Rev (12):CD003754

  • Muyldermans S, Baral T, Retamozzo VC, De Baetselier P, De Genst E, Kinne J, Leonhardt H, Magez S, Nguyen V, Revets H (2009) Camelid immunoglobulins and nanobody technology. Vet Immunol Immunopathol 128(1–3):178–183

    Article  CAS  Google Scholar 

  • Mwansa J, Mwaba J, Lukwesa C, Bhuiyan N, Ansaruzzaman M, Ramamurthy T, Alam M, Balakrish Nair G (2007) Multiply antibiotic-resistant Vibrio cholerae O1 biotype El Tor strains emerge during cholera outbreaks in Zambia. Epidemiol Infect 135(5):847–853

    Article  CAS  Google Scholar 

  • Nakano T, Matsui M, Inoue I, Awata T, Katayama S, Murakoshi T (2011) Free immunoglobulin light chain: its biology and implications in diseases. Clin Chim Acta 412:843–849

    Article  CAS  Google Scholar 

  • Nguyen VK, Muyldermans S, Hamers R (1998) The specific variable domain of camel heavy-chain antibodies is encoded in the germline. J Mol Biol 275(3):413–418

    Article  CAS  Google Scholar 

  • Ogunbanjo PGA, Durrheim DN (2011) Cholera—are we containing the crisis? SA Fam Prac 23(2):2078–6204

    Google Scholar 

  • Qadri F, Svennerholm AM, Faruque A, Sack RB (2005) Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev 18(3):465–483

    Article  Google Scholar 

  • Roovers RC, van der Linden E, Zijlema H, de Bruïne A, Arends JW, Hoogenboom HR (2001) Evidence for a bias toward intracellular antigens in the local humoral anti-tumor immune response of a colorectal cancer patient revealed by phage display. Int J Cancer 93(6):832–840

    Article  CAS  Google Scholar 

  • Saerens D, Huang L, Bonroy K, Muyldermans S (2008) Antibody fragments as probe in biosensor development. Sensors 8(8):4669–4686

    Article  CAS  Google Scholar 

  • Safa A, Bhuiyan NA, Alam M, Sack DA, Nair GB (2005) Genomic relatedness of the new Matlab variants of Vibrio cholerae O1 to the classical and El Tor biotypes as determined by pulsed-field gel electrophoresis. J Clin Microbiol 43(3):1401–1404

    Article  CAS  Google Scholar 

  • Sánchez J, Holmgren J (2011) Cholera toxin—a foe & a friend. Indian J Med Res 133(2):153

    Google Scholar 

  • Schägger H (2006) Tricine–SDS-PAGE. Nat Protoc 1(1):16–22

    Article  Google Scholar 

  • Shale M, Seow C, Coffin C, Kaplan G, Panaccione R, Ghosh S (2010) Review article: chronic viral infection in the anti-tumour necrosis factor therapy era in inflammatory bowel disease. Aliment Pharmacol Ther 31(1):20–34

    Article  CAS  Google Scholar 

  • Sharma D, Malik A (2012) Incidence and prevalence of antimicrobial resistant Vibrio cholerae from dairy farms. Afr J Microbiol Res 6(25):5331–5334

    Google Scholar 

  • Shrestha B, Brien JD, Sukupolvi-Petty S, Austin SK, Edeling MA, Kim T, O'Brien KM, Nelson CA, Johnson S, Fremont DH (2010) The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1. PLoS Pathog 6(4):e1000823

    Article  Google Scholar 

  • Siddique A, Nair G, Alam M, Sack D, Huq A, Nizam A, Longini I, Qadri F, Faruque S, Colwell R (2010) El Tor cholera with severe disease: a new threat to Asia and beyond. Epidemiol Infect 138(3):347

    Article  CAS  Google Scholar 

  • Svennerholm A, Holmgren J (1976) Synergistic protective effect in rabbits of immunization with Vibrio cholerae lipopolysaccharide and toxin/toxoid. Infect Immun 13(3):735–740

    CAS  Google Scholar 

  • Taneja N, Kaur J, Sharma K, Singh M, Kalra J, Sharma N, Sharma M (2003) A recent outbreak of cholera due to Vibrio cholerae O1 Ogawa in & around Chandigarh, North India. Indian J Med Res 117:243–246

    Google Scholar 

  • Thompson CC, Freitas FS, Marin MA, Fonseca EL, Okeke IN, Vicente ACP (2011) Vibrio cholerae O1 lineages driving cholera outbreaks during seventh cholera pandemic in Ghana. Infect Genet Evol 11:1951–1956

    Article  Google Scholar 

  • Weil AA, Arifuzzaman M, Bhuiyan TR, LaRocque RC, Harris AM, Kendall EA, Hossain A, Tarique AA, Sheikh A, Chowdhury F (2009) Memory T-cell responses to Vibrio cholerae O1 infection. Infect Immun 77(11):5090–5096

    Article  CAS  Google Scholar 

  • Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, Cauerhff A, Danquah W, Rissiek B, Scheuplein F (2009) Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol 198(3):157–174

    Article  CAS  Google Scholar 

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Acknowledgments

The authors like to thank the Biotechnology Development Council of I. R. Iran and Shahed University, Tehran, Iran, for supporting this study.

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The authors declare no conflict of interests.

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Correspondence to Seyedlatif Mousavi Gargari.

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Ebrahimizadeh, W., Mousavi Gargari, S., Rajabibazl, M. et al. Isolation and characterization of protective anti-LPS nanobody against V. cholerae O1 recognizing Inaba and Ogawa serotypes. Appl Microbiol Biotechnol 97, 4457–4466 (2013). https://doi.org/10.1007/s00253-012-4518-x

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