Screening and identification of human antibodies against dengue virus (DENV) envelope protein
GAO Bo1, 2, ZHENG Hui2, LI Shu2, XIE Lian-hui3
1.School of Life Science, Fujian Agriculture and Forestry University,Fuzhou 350001 China; 2.Fujian International Travel Health Center, Fuzhou 350001 China; 3.Institute of Plant Virology,Fujian Agriculture and Forestry University, Fuzhou 350001,China
Abstract：We amied to establish a method for rapid screening of human antibodies against dengue virus(DENV) capsids from peripheral blood, and to screen specific antibodies from peripheral blood memory B cells of virus-infected patients for qualitative analysis. By flow cytometry, DENV envelope protein-specific memory B cells in peripheral blood were single-cell sorted. The gene sequence of antibody was amplified using efficient single-cell PCR. We determined the binding activity and neutralization potency of recombinant full-length antibodies. Six human monoclonal antibodies recognizing DENV-1 envelope protein were successfully screened, and four of them had strong binding activity and neutralizing activity. DENV-specific memory B cells were successfully sorted by flow cytometry, and DENV-specific human monoclonal antibodies were efficiently obtained using single-cell PCR. The results revealed that it is hopeful to obtain DENV-specific antibody with broadly neutralizing activity for the prevention and treatment of dengue fever.
[1] Kuhn RJ, Zhang W, Rossmann MG,et al. Structure of dengue virus: implications for flavivirus organization,maturation, fusion[J].Cell, 2002, 108(5):717-725. PMID:11893341 [2] Zhang X, Ge P, Yu X, et al.Cryo-EM structure of the mature dengue virus at 3.5-A resolution[J]. Nat Struct Mol Biol, 2013, 20(1): 105-110. DOI:10.1038/nsmb.2463 [3] ModisY, Ogata S, Clements D, et al. A ligand-binding pocket in the dengue virus envelope glycoprotein[J]. Proc Natl Acad Sci U S A, 2003, 100(12): 6986-6991. DOI:10.1073/pnas.0832193100 [4] Pierson TC,Diamond MS.Molecular mechanisms of antibody-mediated neutralisation of flavivirus infection[J]. Expert Rev Mol Med, 2008, 10: e12. DOI:10.1017/S1462399408000665 [5] Halstead SB.Dengue[J]. The Lancet, 2007, 370(9599): 1644-1652. DOI:10.1016/S0140-6736(07)61687-0 [6] Simmons CP,Farrar JJ,van Vinh Chau N,et al.Dengue[J]. New Engl J Med, 2012, 366(15): 1423-1432. [7] Morens DM, Rigau-Pérez JG,López-Correa R H,et al.Dengue in puerto rico, 1977: public health response to characterize and control an epidemic of multiple serotypes[J]. The American Journal of Tropical Medicine and Hygiene, 1986, 35(1): 197-211. DOI:10.4269/ajtmh.1986.35.197 [8] Wu W, Bai Z, Zhou H,et al.Molecular epidemiology of dengue viruses in southern China from 1978 to 2006[J]. Virol J, 2011, 8: 322. DOI:10.1186/1743-422X-8-322 [9] Jiang T, Yu XD, Hong WX, et al.Co-circulation of two genotypes of dengue virus serotype 3 in Guangzhou, China, 2009[J]. Virol J, 2012, 9: 125. DOI:10.1186/1743-422X-9-125 [10] Zhao H, Deng YQ, Hong WX,et al.Complete genome sequence of dengue virus serotype 2 Cosmopolitan genotype strain in Guangdong, China[J]. J Virol, 2012, 86(24): 13808-13809. DOI:10.1128/JVI.02562-12 [11] Bhatt S, Gething PW, Brady OJ,et al.The global distribution and burden of dengue[J]. Nature, 2013, 496(7446): 504-507. DOI:10.1038/nature12060 [12] Shepard DS, Undurraga EA, Halasa YA,et al.The global economic burden of dengue: a systematic analysis[J]. The Lancet Infectious Diseases, 2016, 16(8): 935-941. DOI:10.1016/s1473-3099(16)00146-8 [13] Hadinegoro SR, Arredondo-Garcia JL, Capeding MR,et al.Efficacy and long-term safety of a Dengue vaccine in regions of endemic disease[J]. N Engl J Med, 2015, 373(13): 1195-206. DOI:10.1056/NEJMoa1506223 [14] Utidjian LH, Hogan A, Michel J, Localio AR,et al.Clinical decision support and palivizumab: a means to protect from respiratory syncytial virus[J]. Appl Clin Inform, 2015, 6(4): 769-784. DOI:10.4338/ACI-2015-08-RA-0096 [15] Goodwin E, Gilman MSA, Wrapp D,et al.Infants infected with respiratory syncytial virus generate potent neutralizing antibodies that lack somatic hypermutation[J]. Immunity, 2018, 48(2): 339-349. DOI:10.1016/j.immuni.2018.01.005 [16] Tiller T, Meffre E, Yurasov S,et al.Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning[J]. J Immunol Methods, 2008, 329(1/2): 112-124. DOI:10.1016/j.jim.2007.09.017 [17] Xiao X, Chen Y, et al.Varkey R.A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells[J]. MAbs, 2016, 8(5): 916-927. DOI:10.1080/19420862.2016.1170263 [18] Lu J, Wang R, Xia B,et al.Potent neutralization ability of a human monoclonal antibody against serotype 1 Dengue virus[J]. Front Microbiol, 2018, 9: 1214. DOI:10.3389/fmicb.2018.01214 [19] Xu M, Zuest R, Velumani S,et al.A potent neutralizing antibody with therapeutic potential against all four serotypes of dengue virus[J]. NPJ Vaccines, 2017, 2: 2. DOI:10.1038/s41541-016-0003-3 [20] Koelsch K, Zheng NY, Zhang Q, et al.Mature B cells class switched to IgD are autoreactive in healthy individuals[J]. J Clin Invest, 2007, 117(6): 1558-1565. DOI:10.1172/JCI27628 [21] Desai NR, Giugliano RP, Zhou J,et al.AMG 145, a monoclonal antibody against PCSK9, facilitates achievement of national cholesterol education program-adult treatment panel III low-density lipoprotein cholesterol goals among high-risk patients: an analysis from the LAPLACE-TIMI 57 trial (LDL-C assessment with PCSK9 monoclonal antibody inhibition combined with statin thErapy-thrombolysis in myocardial infarction 57)[J]. J Am Coll Cardiol, 2014, 63(5): 430-433. DOI:10.1016/j.jacc.2013.09.048 [22] Mizutani N, Nabe T, Yoshino S.IgE/antigen-mediated enhancement of IgE production is a mechanism underlying the exacerbation of airway inflammation and remodelling in mice[J]. Immunology, 2015, 144(1): 107-115. DOI:10.1111/imm.12355 [23] Savic S, Diebold J, Zimmermann AK,et al.Screening for ALK in non-small cell lung carcinomas: 5A4 and D5F3 antibodies perform equally well, but combined use with FISH is recommended[J]. Lung Cancer, 2015, 89(2): 104-109. DOI:10.1016/j.lungcan.2015.05.012 [24] Beck A, Wurch T, Bailly C, et al.Strategies and challenges for the next generation of therapeutic antibodies[J]. Nat Rev Immunol, 2010, 10(5): 345-352. DOI:10.1038/nri2747 [25] Hussman JM, Li A, Paes B, et al.A review of cost-effectiveness of palivizumab for respiratory syncytial virus[J]. Expert Rev Pharmacoecon Outcomes Res, 2012, 12(5): 553-567. DOI:10.1586/erp.12.45 [26] Zhang TY, Yuan Q, Zhao JH, et al.Prolonged suppression of HBV in mice by a novel antibody that targets a unique epitope on hepatitis B surface antigen[J]. Gut, 2016, 65(4): 658-671. DOI:10.1136/gutjnl-2014-308964 [27] Tian M, Cheng C, Chen X, et al.Induction of HIV neutralizing antibody lineages in mice with diverse precursor repertoires[J]. Cell, 2016, 166(6): 1471-1484. DOI:10.1016/j.cell.2016.07.029
