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• W2051298245 abstract "Antibodies protect against homologous Dengue virus (DENV) infection but can precipitate severe dengue by promoting heterotypic virus entry via Fcγ receptors (FcγR). We immortalized memory B cells from individuals after primary or secondary infection and analyzed anti-DENV monoclonal antibodies (mAbs) thus generated. MAbs to envelope (E) protein domain III (DIII) were either serotype specific or cross-reactive and potently neutralized DENV infection. DI/DII- or viral membrane protein prM-reactive mAbs neutralized poorly and showed broad cross-reactivity with the four DENV serotypes. All mAbs enhanced infection at subneutralizing concentrations. Three mAbs targeting distinct epitopes on the four DENV serotypes and engineered to prevent FcγR binding did not enhance infection and neutralized DENV in vitro and in vivo as postexposure therapy in a mouse model of lethal DENV infection. Our findings reveal an unexpected degree of cross-reactivity in human antibodies against DENV and illustrate the potential for an antibody-based therapy to control severe dengue. Antibodies protect against homologous Dengue virus (DENV) infection but can precipitate severe dengue by promoting heterotypic virus entry via Fcγ receptors (FcγR). We immortalized memory B cells from individuals after primary or secondary infection and analyzed anti-DENV monoclonal antibodies (mAbs) thus generated. MAbs to envelope (E) protein domain III (DIII) were either serotype specific or cross-reactive and potently neutralized DENV infection. DI/DII- or viral membrane protein prM-reactive mAbs neutralized poorly and showed broad cross-reactivity with the four DENV serotypes. All mAbs enhanced infection at subneutralizing concentrations. Three mAbs targeting distinct epitopes on the four DENV serotypes and engineered to prevent FcγR binding did not enhance infection and neutralized DENV in vitro and in vivo as postexposure therapy in a mouse model of lethal DENV infection. Our findings reveal an unexpected degree of cross-reactivity in human antibodies against DENV and illustrate the potential for an antibody-based therapy to control severe dengue. A large panel of human mAbs against DENV was isolated from immune donors DIII-mAbs neutralized infection, whereas DI/DII-mAbs neutralized poorly and enhanced infection DI/DII-specific mAbs from secondary infection cross-reacted with the four DENV serotypes mAbs engineered to prevent binding to FcRs protected mice from lethal DENV infection Dengue virus (DENV) is a mosquito-borne Flavivirus responsible for tens of millions of human cases of dengue annually, including 500,000 hospitalizations and 20,000 deaths (Gibbons and Vaughn, 2002Gibbons R.V. Vaughn D.W. Dengue: an escalating problem.BMJ. 2002; 324: 1563-1566Crossref PubMed Scopus (547) Google Scholar), with an economic burden rivaling that of malaria. A primary infection is believed to provide effective, durable, and possibly life-long protection against reinfection with the same serotype but only short-term protection against other serotypes (Rothman, 2004Rothman A.L. Dengue: defining protective versus pathologic immunity.J. Clin. Invest. 2004; 113: 946-951Crossref PubMed Scopus (272) Google Scholar). Classical epidemiologic studies suggested that immunity to one of the four DENV serotypes can increase disease severity upon subsequent challenge with a different serotype, leading, in some cases, to severe dengue, a disease characterized by plasma leakage and hemorrhagic manifestations (Halstead, 1970Halstead S.B. Observations related to pathogensis of dengue hemorrhagic fever. VI. Hypotheses and discussion.Yale J. Biol. Med. 1970; 42: 350-362PubMed Google Scholar). Poorly neutralizing cross-reactive antibodies raised in response to a previous serotype are believed to contribute to pathogenesis of severe dengue by promoting virus entry via Fcγ receptors (FcγR) and infection of myeloid cells (Halstead, 2003Halstead S.B. Neutralization and antibody-dependent enhancement of dengue viruses.Adv. Virus Res. 2003; 60: 421-467Crossref PubMed Scopus (549) Google Scholar), leading to antibody-dependent enhancement (ADE) of infection. The role of antibodies in severe dengue is supported by epidemiological studies showing that infants with waning levels of maternal antibodies (age 6–9 months) are most vulnerable to severe DENV disease (Halstead et al., 2002Halstead S.B. Lan N.T. Myint T.T. Shwe T.N. Nisalak A. Kalyanarooj S. Nimmannitya S. Soegijanto S. Vaughn D.W. Endy T.P. Dengue hemorrhagic fever in infants: research opportunities ignored.Emerg. Infect. Dis. 2002; 8: 1474-1479Crossref PubMed Scopus (179) Google Scholar, Nguyen et al., 2004Nguyen T.H. Lei H.Y. Nguyen T.L. Lin Y.S. Huang K.J. Le B.L. Lin C.F. Yeh T.M. Do Q.H. Vu T.Q. et al.Dengue hemorrhagic fever in infants: a study of clinical and cytokine profiles.J. Infect. Dis. 2004; 189: 221-232Crossref PubMed Scopus (219) Google Scholar) and that serum from these infants enhances DENV infection in vitro (Chau et al., 2008Chau T.N. Quyen N.T. Thuy T.T. Tuan N.M. Hoang D.M. Dung N.T. Lien le B. Quy N.T. Hieu N.T. Hieu L.T. et al.Dengue in Vietnamese infants—results of infection-enhancement assays correlate with age-related disease epidemiology, and cellular immune responses correlate with disease severity.J. Infect. Dis. 2008; 198: 516-524Crossref PubMed Scopus (132) Google Scholar, Kliks et al., 1988Kliks S.C. Nimmanitya S. Nisalak A. Burke D.S. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants.Am. J. Trop. Med. Hyg. 1988; 38: 411-419Crossref PubMed Scopus (460) Google Scholar). The difficulty of balancing immunity to the four serotypes and minimizing incomplete response and the risk of ADE are major hurdles in the development of a tetravalent vaccine against DENV (Whitehead et al., 2007Whitehead S.S. Blaney J.E. Durbin A.P. Murphy B.R. Prospects for a dengue virus vaccine.Nat. Rev. Microbiol. 2007; 5: 518-528Crossref PubMed Scopus (431) Google Scholar). The 10.7 Kb RNA genome of DENV encodes three structural proteins, the capsid protein (C), a membrane-associated protein (prM), and an envelope protein (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The E protein is structurally conserved among flaviviruses and consists of three distinct domains. Domain I (DI) participates in the conformational changes required for viral entry and nucleocapsid escape from the endosomal compartment, domain II (DII) contains the fusion loop, and domain III (DIII) has been suggested to bind cellular receptors (Bhardwaj et al., 2001Bhardwaj S. Holbrook M. Shope R.E. Barrett A.D. Watowich S.J. Biophysical characterization and vector-specific antagonist activity of domain III of the tick-borne flavivirus envelope protein.J. Virol. 2001; 75: 4002-4007Crossref PubMed Scopus (116) Google Scholar, Chin et al., 2007Chin J.F. Chu J.J. Ng M.L. The envelope glycoprotein domain III of dengue virus serotypes 1 and 2 inhibit virus entry.Microbes Infect. 2007; 9: 1-6Crossref PubMed Scopus (107) Google Scholar, Chu et al., 2005Chu J.J. Rajamanonmani R. Li J. Bhuvanakantham R. Lescar J. Ng M.L. Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein.J. Gen. Virol. 2005; 86: 405-412Crossref PubMed Scopus (138) Google Scholar, Rey et al., 1995Rey F.A. Heinz F.X. Mandl C. Kunz C. Harrison S.C. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution.Nature. 1995; 375: 291-298Crossref PubMed Scopus (1197) Google Scholar). Partially mature virions also express varying levels of prM protein on their surface, which is normally cleaved by a furin-like cellular protease to generate the mature virion (Stadler et al., 1997Stadler K. Allison S.L. Schalich J. Heinz F.X. Proteolytic activation of tick-borne encephalitis virus by furin.J. Virol. 1997; 71: 8475-8481Crossref PubMed Google Scholar). The most potent neutralizing antibodies against DENV, or other flaviviruses such as West Nile Virus (WNV), bind to DIII and have been shown in some cases to be effective as passive prophylaxis or therapy in rodents (Beasley and Barrett, 2002Beasley D.W. Barrett A.D. Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope protein.J. Virol. 2002; 76: 13097-13100Crossref PubMed Scopus (216) Google Scholar, Goncalvez et al., 2008Goncalvez A.P. Chien C.H. Tubthong K. Gorshkova I. Roll C. Donau O. Schuck P. Yoksan S. Wang S.D. Purcell R.H. Lai C.J. Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo.J. Virol. 2008; 82: 7009-7021Crossref PubMed Scopus (60) Google Scholar, Gromowski et al., 2008Gromowski G.D. Barrett N.D. Barrett A.D. Characterization of dengue virus complex-specific neutralizing epitopes on envelope protein domain III of dengue 2 virus.J. Virol. 2008; 82: 8828-8837Crossref PubMed Scopus (130) Google Scholar, Kaufman et al., 1987Kaufman B.M. Summers P.L. Dubois D.R. Eckels K.H. Monoclonal antibodies against dengue 2 virus E-glycoprotein protect mice against lethal dengue infection.Am. J. Trop. Med. Hyg. 1987; 36: 427-434PubMed Google Scholar, Oliphant et al., 2005Oliphant T. Engle M. Nybakken G.E. Doane C. Johnson S. Huang L. Gorlatov S. Mehlhop E. Marri A. Chung K.M. et al.Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus.Nat. Med. 2005; 11: 522-530Crossref PubMed Scopus (421) Google Scholar, Sánchez et al., 2005Sánchez M.D. Pierson T.C. McAllister D. Hanna S.L. Puffer B.A. Valentine L.E. Murtadha M.M. Hoxie J.A. Doms R.W. Characterization of neutralizing antibodies to West Nile virus.Virology. 2005; 336: 70-82Crossref PubMed Scopus (111) Google Scholar, Shrestha et al., 2010Shrestha B. Brien J.D. Sukupolvi-Petty S. Austin S.K. Edeling M.A. Kim T. O'Brien K.M. Nelson C.A. Johnson S. Fremont D.H. Diamond M.S. The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1.PLoS Pathog. 2010; 6: e1000823Crossref PubMed Scopus (177) Google Scholar, Sukupolvi-Petty et al., 2007Sukupolvi-Petty S. Austin S.K. Purtha W.E. Oliphant T. Nybakken G.E. Schlesinger J.J. Roehrig J.T. Gromowski G.D. Barrett A.D. Fremont D.H. Diamond M.S. Type- and subcomplex-specific neutralizing antibodies against domain III of dengue virus type 2 envelope protein recognize adjacent epitopes.J. Virol. 2007; 81: 12816-12826Crossref PubMed Scopus (211) Google Scholar). DIII-reactive antibodies produced by mice immunized with virus and boosted with recombinant E protein are largely serotype-specific and do not neutralize all of the genotypes within a given serotype (Shrestha et al., 2010Shrestha B. Brien J.D. Sukupolvi-Petty S. Austin S.K. Edeling M.A. Kim T. O'Brien K.M. Nelson C.A. Johnson S. Fremont D.H. Diamond M.S. The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1.PLoS Pathog. 2010; 6: e1000823Crossref PubMed Scopus (177) Google Scholar). The role of antibodies to DI/DII is less clear, as they tend to be more cross-reactive and less potent in neutralization (Crill and Chang, 2004Crill W.D. Chang G.J. Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes.J. Virol. 2004; 78: 13975-13986Crossref PubMed Scopus (211) Google Scholar, Goncalvez et al., 2004Goncalvez A.P. Men R. Wernly C. Purcell R.H. Lai C.J. Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses.J. Virol. 2004; 78: 12910-12918Crossref PubMed Scopus (39) Google Scholar, Oliphant et al., 2006Oliphant T. Nybakken G.E. Engle M. Xu Q. Nelson C.A. Sukupolvi-Petty S. Marri A. Lachmi B.E. Olshevsky U. Fremont D.H. et al.Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein.J. Virol. 2006; 80: 12149-12159Crossref PubMed Scopus (236) Google Scholar). Antibodies to prM generally have poor neutralizing and enhancing activity (Falconar, 1999Falconar A.K. Identification of an epitope on the dengue virus membrane (M) protein defined by cross-protective monoclonal antibodies: design of an improved epitope sequence based on common determinants present in both envelope (E and M) proteins.Arch. Virol. 1999; 144: 2313-2330Crossref PubMed Scopus (98) Google Scholar, Huang et al., 2006Huang K.J. Yang Y.C. Lin Y.S. Huang J.H. Liu H.S. Yeh T.M. Chen S.H. Liu C.C. Lei H.Y. The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection.J. Immunol. 2006; 176: 2825-2832Crossref PubMed Scopus (143) Google Scholar), although recent studies suggest that some anti-prM mAbs can augment infectivity of poorly infectious immature virions (Rodenhuis-Zybert et al., 2010Rodenhuis-Zybert I.A. van der Schaar H.M. da Silva Voorham J.M. van der Ende-Metselaar H. Lei H.Y. Wilschut J. Smit J.M. Immature dengue virus: a veiled pathogen?.PLoS Pathog. 2010; 6: e1000718Crossref PubMed Scopus (189) Google Scholar). Antibodies against NS1, a secreted nonstructural glycoprotein that is absent from the virion but expressed on the cell surface, can also protect against infection in vivo through FcγR-dependent and -independent mechanisms (Chung et al., 2006Chung K.M. Nybakken G.E. Thompson B.S. Engle M.J. Marri A. Fremont D.H. Diamond M.S. Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through Fc gamma receptor-dependent and -independent mechanisms.J. Virol. 2006; 80: 1340-1351Crossref PubMed Scopus (200) Google Scholar) or can possibly contribute to pathogenesis (Falconar, 2007Falconar A.K. Antibody responses are generated to immunodominant ELK/KLE-type motifs on the nonstructural-1 glycoprotein during live dengue virus infections in mice and humans: implications for diagnosis, pathogenesis, and vaccine design.Clin. Vaccine Immunol. 2007; 14: 493-504Crossref PubMed Scopus (54) Google Scholar, Lin et al., 2008Lin C.F. Wan S.W. Chen M.C. Lin S.C. Cheng C.C. Chiu S.C. Hsiao Y.L. Lei H.Y. Liu H.S. Yeh T.M. Lin Y.S. Liver injury caused by antibodies against dengue virus nonstructural protein 1 in a murine model.Lab. Invest. 2008; 88: 1079-1089Crossref PubMed Scopus (60) Google Scholar). Our current knowledge of the human antibody response to DENV is mostly based on serological studies. In this study, we used an improved method of memory B cell immortalization (Traggiai et al., 2004Traggiai E. Becker S. Subbarao K. Kolesnikova L. Uematsu Y. Gismondo M.R. Murphy B.R. Rappuoli R. Lanzavecchia A. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.Nat. Med. 2004; 10: 871-875Crossref PubMed Scopus (532) Google Scholar) combined with a broad screening approach to isolate a large panel of DENV-reactive mAbs from human donors. MAbs that bound to DIII of the E protein potently neutralized DENV infection, although they also showed enhancing activity at lower concentrations. In contrast, mAbs that bound to DI/DII of E protein or prM neutralized poorly yet potently enhanced infection of FcγR-bearing cells. Surprisingly, some of the human DIII-specific mAbs and all DI/DII-specific mAbs isolated from secondary infection showed a broad pattern of cross-reactivity with the four DENV serotypes. Based on an improved understanding of the functional humoral response against DENV, we produced a cocktail of three variant recombinant mAbs that neutralized all four DENV serotypes without causing immune enhancement in vitro and in vivo. Blood samples were collected from donors at different time points after diagnosis of either primary (donor 7, donor 13, and donor 76) or secondary (donor 12 and donor 92) DENV infection. IgG+ memory B cells isolated from frozen PBMC were immortalized with EBV and CpG in multiple replicate wells as previously described (Traggiai et al., 2004Traggiai E. Becker S. Subbarao K. Kolesnikova L. Uematsu Y. Gismondo M.R. Murphy B.R. Rappuoli R. Lanzavecchia A. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.Nat. Med. 2004; 10: 871-875Crossref PubMed Scopus (532) Google Scholar). Culture supernatants were collected after 2 weeks and analyzed by staining of fixed and permeabilized C6/36 mosquito cells infected with DENV or by ELISA using recombinant E protein from DENV-1, DENV-2, and DENV-4 or lysates of DENV-infected cells (Table 1). The frequency of IgG+ memory B cells reactive against DENV-infected cells, calculated from the number of positive cultures and the number of input cells after corrections for the efficiency of B cell immortalization, was high in all donors, ranging from 5.4% to 16% of IgG+ memory B cells. In the three donors that were screened using E protein, the frequency of E-reactive IgG+ memory B cells ranged from 1.5% to 3.2%. These findings indicate that DENV-immune donors maintain a very large pool of memory B cells reactive against DENV proteins, even several years after infection.Table 1DENV-Immune Donors and B Cell Repertoire AnalysisInfecting SerotypeTime p.i.Immortalization EfficiencyPrimary ScreeningPositive Cultures/TotalDENV- or E-Reactive B Cells (%)aFrequency of DENV- or E-reactive IgG+ B cells was calculated from the number of positive cultures and the number of input cells per well after correction for the efficiency of B cell immortalization.Donor 7primary DENV-4200 days9.1%ELISA E4 protein81/28441.5%Donor 13bSupernatants were tested using two different screening assays.primary DENV-2> 8 years6.5%staining of infected C6/36 cellscC6/36 cells were infected with DENV-2, WHO reference strain 516803.567/201610.7%ELISA E2 protein152/20162.9%Donor 76primary DENV-3241 days14%staining of infected C6/36 cellsdC6/36 cells were infected with DENV isolated from the same donor.434/96016%Donor 12eTwo independent immortalizations were performed.secondary DENV-1510 days13.8%staining of infected C6/36 cellsdC6/36 cells were infected with DENV isolated from the same donor.245/16325.4%12.7%ELISA E1 protein313/138243.2%Donor 92secondary DENV-2212 days9%ELISA DENV lysates344/22088.6%PBMC were obtained from four donors after a primary or secondary DENV infection and frozen until the day of use. Shown is the infecting serotype and the days postinfection (p.i.) when blood was collected. IgG+ memory B cells were immortalized with EVB and CpG. Also shown is the efficiency of immortalization and the number and fraction of cultures containing antibodies against DENV identified using different screening methods.a Frequency of DENV- or E-reactive IgG+ B cells was calculated from the number of positive cultures and the number of input cells per well after correction for the efficiency of B cell immortalization.b Supernatants were tested using two different screening assays.c C6/36 cells were infected with DENV-2, WHO reference strain 516803.d C6/36 cells were infected with DENV isolated from the same donor.e Two independent immortalizations were performed. Open table in a new tab PBMC were obtained from four donors after a primary or secondary DENV infection and frozen until the day of use. Shown is the infecting serotype and the days postinfection (p.i.) when blood was collected. IgG+ memory B cells were immortalized with EVB and CpG. Also shown is the efficiency of immortalization and the number and fraction of cultures containing antibodies against DENV identified using different screening methods. Because the E protein, and in particular DIII, is the main target of neutralizing anti-WNV antibodies in mice (Oliphant et al., 2007Oliphant T. Nybakken G.E. Austin S.K. Xu Q. Bramson J. Loeb M. Throsby M. Fremont D.H. Pierson T.C. Diamond M.S. Induction of epitope-specific neutralizing antibodies against West Nile virus.J. Virol. 2007; 81: 11828-11839Crossref PubMed Scopus (144) Google Scholar), we performed a large screen to gain insights into the domain specificity and cross-reactivity of E-specific antibodies isolated from donor 13 (primary DENV-2) and donor 12 (secondary DENV-1). Eighteen out of 152 antibodies from donor 13 bound to DIII of DENV-2. Of these, 13 were serotype specific, 1 cross-reacted with DENV-2, -3, and -4, and 4 cross-reacted with all four DENV serotypes. In the case of donor 12, a large fraction of E-reactive antibodies bound to DIII (138 out of 313). Of these, 44 were specific for DIII of DENV-1, whereas the remaining 94 were cross-reactive with two, three, or all four DENV serotypes (16, 15, and 63, respectively). Taken together, these findings indicate that the human antibody response to DIII of DENV E protein elicited by natural infection comprises serotype-specific as well as broadly cross-reactive antibodies. In order to better characterize the human antibody response to DENV, we isolated B cell clones from several independent cultures and characterized 70 DENV-reactive monoclonal antibodies (mAbs) (68 IgG1, either κ or λ, 1 IgG3, λ, and 1 IgG4, λ). All DENV-reactive mAbs bound to one or more DENV serotypes, as shown by staining of Vero cells infected with DENV-1, DENV-2, DENV-3, or DENV-4 (see examples in Figure 1A ). Specificity and cross-reactivity were assessed using binding assays (ELISA with recombinant E, NS1, and NS3 proteins, staining of yeast displaying DI/DII or DIII, or western blot of infected cell lysates) (see examples in Figures 1B–1D). Mabs were also characterized using functional assays (neutralization or enhancement of infection by DENV vaccine strains using flow cytometry assays with Vero and K562 cells, respectively; Kraus et al., 2007Kraus A.A. Messer W. Haymore L.B. de Silva A.M. Comparison of plaque- and flow cytometry-based methods for measuring dengue virus neutralization.J. Clin. Microbiol. 2007; 45: 3777-3780Crossref PubMed Scopus (120) Google Scholar, Littaua et al., 1990Littaua R. Kurane I. Ennis F.A. Human IgG Fc receptor II mediates antibody-dependent enhancement of dengue virus infection.J. Immunol. 1990; 144: 3183-3186PubMed Google Scholar) (see examples in Figures 1E and 1F). Of the 70 DENV-reactive mAbs isolated, 13 mapped to DIII of E protein, and of these, 5 were serotype specific, whereas 8 were cross-reactive with two, three, or even all four DENV serotypes (Table 2). Of the five serotype-specific mAbs, three (DV3.7, DV25.5, and DV470.12) potently neutralized infection of Vero cells by either DENV-1 or DENV-2 vaccine strains, with EC50 values in the low ng/ml range, whereas two mAbs (DV1.6 and DV14.5) showed decreased potency against DENV-2, suggesting that they may represent low-affinity antibodies or may target private epitopes within DENV-2. Of the eight cross-reactive mAbs, two (DV63.1 and DV55.1) potently neutralized DENV-1 and DENV-3, whereas mAb DV87.1 potently neutralized DENV-1, DENV-2, and DENV-3. Strikingly, the cross-reactive mAbs DV63.1 and DV87.1 neutralized infection in the same range of concentrations as observed for serotype-specific mAbs (Table 2). Finally, four mAbs bound all serotypes by ELISA, and of these, two neutralized, albeit with low potency, all serotypes (DV21.5 and DV257.13), whereas the remaining mAbs showed a more restricted pattern of neutralization.Table 2Characterization of DIII-E-Reactive Human mAbsELISA E ProteinYeasts DV2Neutralization (EC50 μg/ml)Range of Enhancement (log10 μg/ml)mAbsDonorIsotypeSpecificityDV 1DV 2DV 3DV 4DIII DV 3DI/DIIDIIIDV 1DV 2DV 3DV 4DV 1DV 2DV 3DV 4DV 1.6Don 13γ1, κE, DIII—+———n.d.+—3.5——————DV 3.7Don 13γ1, λE, DIII—+———n.d.+—0.003———−3, 1——DV 14.5Don 13γ1, λE, DIII—+———n.d.+—0.043———−1, 1——DV 25.5Don 13γ1, κE, DIII—+———n.d.+—0.005———−3, 1——DV 470.12Don 12γ1, λE, DIII+————n.d.—0.002———−3, 1———DV 63.1Don 76γ1, κE, DIII+—+—+n.d.—0.006—0.006—−4, −1—−3, 0—DV 55.1Don 76γ1, λE, DIII+++—+n.d.+0.0130.5770.014—−3, 0−1, 1−3, 1—DV 87.1Don 12γ1, κE, DIII+++—+n.d.+0.0040.0040.008—−3, 1−2, 1−2, 0—DV 291.7Don 12γ1, κE, DIII+++—+n.d.+0.1470.0560.820—−4, 0−2, 0−2, 0—DV 10.16Don 13γ1, λE, DIII+++++n.d.+—0.083—0.4110, 3−2, 1−1, 1−2, 1DV 21.5Don 13γ1, λE, DIII+++++n.d.+0.4101.0110.7264.037−1, 2−1, 3−1, 30, 3DV 257.13Don 12γ1, κE, DIII+++++n.d.+0.1070.1540.1930.223n.d.n.d.n.d.n.d.DV 415.8Don 12γ1, κE, DIII+++++n.d.+0.1340.1630.127—n.d.n.d.n.d.n.d.The table reports the phenotype of DIII-E-specific mAbs. The target specificity of the mAbs was determined by ELISA. The donor from which the mAbs were isolated and the mAb isotype and light-chain usage are indicated. Viral neutralization was performed using Vero cells and DENV-1-4 vaccine strains. Shown are EC50 values. Enhancement of infectivity was performed using K562 cells. Shown is the range of mAb concentrations for which infection enhancement was observed (see example in Figure 1F). The data shown are representative of two independent experiments. n.d., not done. (+) OD values > 0.5. (–) OD values < 0.5 or no neutralization at the highest concentration tested (5 or 10 μg/ml). Open table in a new tab The table reports the phenotype of DIII-E-specific mAbs. The target specificity of the mAbs was determined by ELISA. The donor from which the mAbs were isolated and the mAb isotype and light-chain usage are indicated. Viral neutralization was performed using Vero cells and DENV-1-4 vaccine strains. Shown are EC50 values. Enhancement of infectivity was performed using K562 cells. Shown is the range of mAb concentrations for which infection enhancement was observed (see example in Figure 1F). The data shown are representative of two independent experiments. n.d., not done. (+) OD values > 0.5. (–) OD values < 0.5 or no neutralization at the highest concentration tested (5 or 10 μg/ml). Consistent with what is known about the stoichiometric relationship between neutralization and enhancement (Pierson et al., 2007Pierson T.C. Xu Q. Nelson S. Oliphant T. Nybakken G.E. Fremont D.H. Diamond M.S. The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection.Cell Host Microbe. 2007; 1: 135-145Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar), DIII-specific mAbs also enhanced infection of K562 cells over a given range of concentrations with the same serotype specificity as shown in the neutralization assay (Table 2). These results indicate that it is possible to isolate DIII-reactive human mAbs that potently neutralize infection of two or more DENV serotypes. Of the 70 DENV-reactive mAbs, 34 mapped to DI/DII of E protein (Table 3). Of these, 8 were serotype-specific, 1 cross-reacted with DENV-1 and DENV-2, and 25 cross-reacted with all four DENV serotypes. When compared to DIII-specific mAbs, the DI/DII-specific mAbs showed in general a 10- to 50-fold lower potency of neutralization and enhanced infection over a wide range of concentrations (Table 3). Of interest, whereas DI/DII-reactive mAbs isolated from primary infections (donors 7, 13, and 76) were mostly serotype specific, all DI/DII-reactive mAbs isolated from secondary infection (donors 12 and 92) neutralized and enhanced infection by all four DENV serotypes. These findings suggest that broadly cross-reactive DI/DII-specific memory B cells may be selected during the course of secondary DENV infection.Table 3Characterization of DI/DII E-Reactive Human mAbsELISA E ProteinYeasts DV2Neutralization (EC50 μg/ml)Range of Enhancement (log10 μg/ml)mAbsDonorIsotypeSpecificityDV 1DV 2DV 3DV 4DIII DV 3/4DI/DIIDIIIDV 1DV 2DV 3DV 4DV 1DV 2DV 3DV 4DV 13.8Don 13γ1, λE, DI/DII—+———n.d.——0.241———−2, 1——DV 16.8Don 7γ1, κE, DI/DII———+—n.d.————0.039———−2, 0DV 18.21Don 13γ1, κE, DI/DII—+———n.d.——0.003———−3, 0——DV 22.3Don 7γ1, λE, DI/DII———+—n.d.————0.006———−2, 1DV 35.3Don 13γ1, κE, DI/DII—+———n.d.——0.918———−1, 1——DV 74.4Don 76γ1, λE, DI/DII——+——n.d.———0.020———−2,0—DV 79.3Don 76γ1, κE, DI/DII——+——n.d.———0.023———−2, 0—DV 76.5Don 76γ1, κE, DI/DII——+——n.d.—————−1, 2−2, 1−2, 1−2, 1DV 90.3Don 76γ1, λE, DI/DII+—+——n.d.—0.342—0.454—−2, 1—−1, 1—DV 1.1Don 92γ1, λE, DI/DII++++—+—0.2090.0660.2620.183−2, 1−2, 1−2, 1−2, 1DV 4.4Don 92γ1, λE, DI/DII++++—+—0.2480.2140.1670.760−2, 1−2, 1−1, 1−2, 1DV 5.1Don 92γ1, κE, DI/DII++++—+—0.1560.1030.0920.069−2, 1−2, 1−2, 1−3, 1DV 6.1Don 92γ1, λE, DI/DII++++—+—0.3820.2960.3510.170−1, 1−2, 1−1, 1−2, 1DV 7.5Don 92γ1, κE, DI/DII++++—+—0.0840.0750.1300.031−2, 1−2, 1−2, 1−2, 1DV 8.1Don 92γ1, λE, DI/DII++++—+—0.1030.0220.0310.024−3, 2−2, 1−3, 1−3, 0DV 13.4Don 92γ1, λE, DI/DII++++—+—0.0630.0230.0160.045−2, 1−3, 0−3, 0−3, 0DV 14.5Don 92γ1, λE, DI/DII++++—+—0.0530.0490.0600.040−2, 0−2, 0−2, 0−3, 0DV 15.7Don 92γ1, λE, DI/DII++++—+—0.1230.0550.1730.070−2, 1−3, 1−2, 1−3, 1DV 16.5Don 92γ1, κE, DI/DII++++—+—0.2480.0890.2190.101−2, 1−2, 1−2, 1−2, 1DV 17.6Don 92γ1, λE, DI/DII++++—+—0.2180.1030.2560.087−2, 1−2, 1−2, 1−2, 2DV 18.4Don 92γ1, κE, DI/DII++++—+—0.6700.2410.7990.699−2, 1−2, 1−2, 1−2, 1DV 19.3Don 92γ1, κE, DI/DII++++—+—0.6310.1890.3680.114−2, 1−2, 1−2, 1−2, 1DV 20.1Don 92γ1, λE, DI/DII++++—+—0.2440.1280.7770.226−2, 1−2, 1−1, 1−2, 1DV 21.1Don 92γ1, κE, DI/DII++++—+—0.1610.0550.0690.101−2, 1−2, 1−3, 1−3, 1DV 23.13Don 13γ1, λE, DI/DII++++—n.d.—n.d.0.013n.d.n.d.n.d.−3, 1n.d.n.d.DV 28.1Don 12γ1, λE, DI/DII++++—n.d.—0.0930.0270.0840.027−3, 1−3, 1−3, 1−3, 1DV 28.8Don 92γ1, κE, DI/DII++++—+—0.1060.0480.2330.092−2, 0−2, 0−2, 0−2, 1DV 38.1Don 92γ1, λE, DI/DII++++—+—0.1110.0360.2390.106−2, 1−3, 1−2, 1−3, 1DV 78.6Don 76γ1, κE, DI/DII++++—n.d.—0.5910.2510.8090.367−2, 1−2, 1−2, 1−2, 1DV 82.11Don 76γ1, λE, DI/DII++++—n.d.—0.0430.0240.0900.117−2, 1−2, 1−2, 1−2, 1DV 143.6Don 12γ1,κE, DI/DII++++—n.d.—0.2530.0830.2550.070−2, 1−2, 1−2, 1n.d.DV 163.3Don 12γ1,κE, DI/DII++++—n.d.—0.1700.1000.1870.542−2, 1−2, 0−1, 0n.d.DV 378.12Don 12γ1,κE, DI/DII++++—n.d.—0.1490.0720.2510.498−2, 1−2, 1−3, 1n.d.DV" @default.
• W2051298245 created "2016-06-24" @default.
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• W2051298245 date "2010-09-01" @default.
• W2051298245 modified "2023-10-11" @default.
• W2051298245 title "The Human Immune Response to Dengue Virus Is Dominated by Highly Cross-Reactive Antibodies Endowed with Neutralizing and Enhancing Activity" @default.
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• W2051298245 doi "https://doi.org/10.1016/j.chom.2010.08.007" @default.
• W2051298245 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3884547" @default.
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• W2051298245 hasPublicationYear "2010" @default.
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