The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.
References
[1]
Mackenzie JS, Gubler DJ, Petersen LR (2004) Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat Med 10: S98–S109. doi: 10.1038/nm1144
[2]
World Health Organization (2012) Global strategy for dengue prevention and control 2012–2020, WHO report, Geneva.
[3]
George R, Lum LCS (1997) Clinical spectrum of dengue infection. In: Gubler DJ, Kuno G, eds. Dengue and dengue hemorrhagic fever. CAB International, London, United Kingdom. 89–114.
[4]
Teles FR, Prazeres DM, Lima-Filho JL (2005) Trends in dengue diagnosis. Rev Med Virol 15: 287–302. doi: 10.1002/rmv.461
[5]
Whitehead SS, Blaney JE, Durbin AP, Murphy BR (2007) Prospects for a dengue virus vaccine. Nat Rev Microbiol 5: 518–528. doi: 10.1038/nrmicro1690
[6]
Flamand M, Megret F, Mathieu M, Lepault J, Rey FA, et al. (1999) Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion. J Virol 73: 6104–6110.
[7]
Mackenzie JM, Jones MK, Young PR (1996) Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology 220: 232–240. doi: 10.1006/viro.1996.0307
[8]
Alcon S, Talarmin A, Debruyne M, Falconar A, Deubel V, et al. (2002) Enzyme-linked immunosorbent assay specific to dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. J Clin Microbiol 40: 376–381. doi: 10.1128/jcm.40.02.376-381.2002
[9]
Xu H, Di B, Pan YX, Qiu LW, Wang YD, et al. (2006) Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1: Implications for early diagnosis and serotyping of dengue virus infections. J Clin Microbiol 44: 2872–2878. doi: 10.1128/jcm.00777-06
[10]
Yamada K, Takasaki T, Nawa M, Kurane I (2002) Virus isolation as one of the diagnostic methods for dengue virus infection. J ClinVirol 24(3): 203–209. doi: 10.1016/s1386-6532(01)00250-5
[11]
Guzmán MG, Kourí G (2004) Dengue diagnosis, advances and challenges. Int J Infect Dis 8(2): 69–80.
[12]
Ding X, Hu D, Chen Y, Di B, Jin J, et al. (2011) Full serotype- and group-specific NS1 capture enzyme-linked immunosorbent assay for rapid differential diagnosis of dengue virus infection. Clin Vaccine Immunol 18(3): 430–434. doi: 10.1128/cvi.00462-10
[13]
Qiu LW, Di B, Wen K, Wang XS, Liang WH, et al. (2009) Development of an antigen capture immunoassay based on monoclonal antibodies specific for dengue virus serotype 2 nonstructural protein 1 for early and rapid identification of dengue virus serotype 2 infections. Clin Vaccine Immunol 16(1): 88–95. doi: 10.1128/cvi.00212-08
[14]
Fry SR, Meyer M, Semple MG, Simmons CP, Sekaran SD, et al. (2011) The diagnostic sensitivity of dengue rapid test assays is significantly enhanced by using a combined antigen and antibody testing approach. PLoS Negl Trop Dis 5(6): e1199 doi:10.1371/journal.pntd.0001199.
[15]
Zhou P, Lu Y, Zhu J, Hong J, Li B, et al. (2004) Nanocolloidal gold-based immunoassay for the detection of N-Methylcarbamate pesticide carbofuran. J Agric Food Chem 52: 4355–4359. doi: 10.1021/jf0499121
[16]
Tanaka R, Yuhi T, Nagatani N, Endo T, Kerman K, et al. (2006) A novel enhancement assay for immunochromatographic test strips using gold nanoparticles. Anal Bioanal Chem 385(8): 1414–1420. doi: 10.1007/s00216-006-0549-4
Cortez-Retamozo V, Backmann N, Senter PD, Wernery U, De Baetselier P, et al. (2004) Efficient cancer therapy with a nanobody-based conjugate. Cancer Res 64: 2853–2857. doi: 10.1158/0008-5472.can-03-3935
[19]
Revets H, De Baetselier P, Muyldermans S (2005) Nanobodies as novel agents for cancer therapy. Expert Opin Biol Ther 5: 111–124. doi: 10.1517/14712598.5.1.111
[20]
McCoy LE, Quigley AF, Strokappe NM, Bulmer-Thomas B, Seaman MS, et al. (2012) Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization. J Exp Med 209(6): 1091–1103. doi: 10.1084/jem.20112655
[21]
Deffar K, Shi H, Li L, Wang X, Zhu X (2009) Nanobodies - the new concept in antibody engineering. Afr J Biotechnol 8 (12): 2645–2652.
[22]
Chen Y, Pan Y, Guo Y, Qiu L, Ding X, et al. (2010) Comprehensive mapping of immunodominant and conserved serotype and group specific B-cell epitopes on nonstructural protein 1 from dengue virus type 1. Virology 398: 290–298. doi: 10.1016/j.virol.2009.12.010
[23]
Jiang L, Zhou JM, Yin Y, Fang DY, Tang YX, et al. (2010) Selection and identification of B-cell epitope on NS1 protein of dengue virus type 2. Virus Res 150(1–2): 49–55. doi: 10.1016/j.virusres.2010.02.012
[24]
Ye W, Liu J, Wang H, Wang J, Wang X (2012) Cloning, expression, purification, and characterization of a glutamate-specific endopeptidase from Bacillus licheniformis. Protein Expr Purif 82(1): 138–143. doi: 10.1016/j.pep.2011.12.001
[25]
Allonso D, da Silva Rosa M, Coelho DR, da Costa SM, Nogueira RMR, et al. (2011) Polyclonal antibodies against properly refolded dengue virus NS1 protein expressed in E. coli enable sensitive and early dengue diagnosis. J Virol Methods 175: 109–116. doi: 10.1016/j.jviromet.2011.04.029
[26]
Che XY, Qiu LW, Pan YX, Wen K, Hao W, et al. (2004) Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome. J Clin Microbiol 42: 2629–2635. doi: 10.1128/jcm.42.6.2629-2635.2004
[27]
Doyle PJ, Arbabi-Ghahroudi M, Gaudette N, Furzer G, Savard ME, et al. (2008) Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acettyl-deoxynivalenol. Mol Immunol 45: 3703–3713. doi: 10.1016/j.molimm.2008.06.005
[28]
SAS Institute (2000) SAS user’s guide: statistics, SAS Institute, Cary, North Carolina.
[29]
Tjitra E, Suprianto S, Dyer M, Currie BJ, Anstey NM (1999) Field evaluation of the ICT malaria P.f/P.v immunochromatographic test for detection of Plasmodium falciparum and Plasmodium vivax in patients with a presumptive clinical diagnosis of malaria in eastern Indonesia. J Clin Microbiol 37: 2412–2417.
Davis MM (2004) The evolutionary and structural ‘logic’ of antigen receptor diversity. Semin Immunol 16: 239–243. doi: 10.1016/j.smim.2004.08.003
[32]
De Genst E, Saerens D, Muyldermans S, Conrath K (2006) Antibody repertoire development in camelids. Dev Comp Immunol 30(1–2): 187–198. doi: 10.1016/j.dci.2005.06.010
[33]
Alvarez-Rueda N, Behar G, Ferre V, Pugnière M, Roquet F, et al. (2007) Generation of llama single-domain antibodies against methotrexate, a prototypical hapten. Mol Immunol 44: 1680–1690. doi: 10.1016/j.molimm.2006.08.007
[34]
Monegal A, Ami D, Martinelli C, Huang H, Aliprandi M, et al. (2009) Immunological applications of single-domain llama recombinant antibodies isolated from a na？ve library. Protein Eng Des Sel 22(4): 273–280. doi: 10.1093/protein/gzp002
[35]
Zarebski LM, Urrutia M, Goldbaum FA (2005) Llama single domain antibodies as a tool for molecular Mimicry. J Mol Biol 349: 814–824. doi: 10.1016/j.jmb.2005.03.072
[36]
Moreland NJ, Tay MYF, Lim E, Paradkar PN, Doan DNP, et al. (2010) High affinity human antibody fragments to dengue virus non-structural protein 3. PLoS Negl Trop Dis 4(11): e881 doi: 10.1371/journal.pntd.0000881.
[37]
von Mehren M, Weiner LM (1996) Monoclonal antibody-based therapy. Curr Opin Oncol 8: 493–498. doi: 10.1097/00001622-199611000-00009
[38]
Verheesen P, Roussis A, de Hard HJ, Groot AJ, Stam JC, et al. (2006) Reliable and controllable antibody fragment selections from camelid non-immune libraries for target validation. Biochim Biophys Acta 1764: 1307–1319. doi: 10.1016/j.bbapap.2006.05.011
[39]
Klooster R, Rutgers KM, van der Maarel SM (2012) Selection of VHH antibody fregments that recognize different Aβ depositions using complex immune libraries. Methods Mol Biol 911(3): 241–253. doi: 10.1007/978-1-61779-968-6_15
[40]
Groot AJ, Verheesen P, Westerlaken EJ, Gort EH, van der Groep P, et al. (2006) Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha. Lab Invest 86(4): 345–356. doi: 10.1038/labinvest.3700395
[41]
Wei G, Meng W, Guo H, Pan W, Liu J, et al. (2011) Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein. PLoS One 6(12): e28309 doi:10.1371/journal.pone.0028309.
[42]
Strokappe N, Szynol A, Aasa-Chapman M, Gorlani A, Forsman Quigley A, et al. (2012) Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C. PLoS One. 7(3): e33298 doi:10.1371/journal.pone.0033298.
[43]
Andries A-C, Duong V, Ngan C, Ong S, Huy R, et al. (2012) Field evaluation and impact on clinical management of a rapid diagnostic kit that detects dengue NS1, IgM and IgG. PLoS Negl Trop Dis 6(12): e1993 doi:10.1371/journal.pntd.0001993.
[44]
Sekaran SD, Ew CL, Kantesh BM, Appana R, Subramaniam G (2007) Evaluation of a dengue NS1 capture ELISA assay for the rapid detection of dengue. J Infect Developing Countries 1: 182–188.
[45]
Blacksell SD, Bell D, Kelley J, Mammen MP Jr, Gibbons RV, et al. (2007) Prospective study to determine accuracy of rapid serological assays for diagnosis of acute dengue virus infection in Laos. Clin Vaccine Immunol 14: 1458–1464. doi: 10.1128/cvi.00482-06
[46]
Chuansumrit A, Chaiyaratana W, Pongthanapisith V, Tangnararatchakit K, Lertwongrath S, et al. (2008) The use of dengue nonstructural protein 1 antigen for the early diagnosis during the febrile stage in patients with dengue infection. Pediatr Infect Dis J 27: 43–48. doi: 10.1097/inf.0b013e318150666d
[47]
Phuong HL, Thai KT, Nga TT, Giao PT, Hung le Q, et al. (2009) Detection of dengue nonstructural 1 (NS1) protein in Vietnamese patients with fever. Diagn Microbiol Infect Dis 63: 372–378. doi: 10.1016/j.diagmicrobio.2008.12.009
[48]
Ramirez AH, Moros Z, Comach G, Zambrano J, Bravo L, et al. (2009) Evaluation of dengue NS1 antigen detection tests with acute sera from patients infected with dengue virus in Venezuela. Diagn Microbiol Infect Dis 65(3): 247–253. doi: 10.1016/j.diagmicrobio.2009.07.022
[49]
Malito E, Faleri A, Lo Surdo P, Veggi D, Maruggi G, et al. (2013) Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen. Proc Natl Acad Sci USA 110(9): 3304–3309. doi: 10.1073/pnas.1222845110
[50]
Wu HC, Huang YL, Chao TT, Jan JT, Huang JL, et al. (2001a) Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients. J Clin Microbiol 39: 977–982. doi: 10.1128/jcm.39.3.977-982.2001
[51]
Wu HC, Jung MY, Chiu CY, Chao TT, Lai SC, et al. (2003) Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen. J Gen Virol 84: 2771–2779. doi: 10.1099/vir.0.19228-0
