Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus - PubMed (original) (raw)

. 2006 Jul;80(14):6982-92.

doi: 10.1128/JVI.00551-06.

Cecile Geuijen, Jaap Goudsmit, Arjen Q Bakker, Jehanara Korimbocus, R Arjen Kramer, Marieke Clijsters-van der Horst, Maureen de Jong, Mandy Jongeneelen, Sandra Thijsse, Renate Smit, Therese J Visser, Nora Bijl, Wilfred E Marissen, Mark Loeb, David J Kelvin, Wolfgang Preiser, Jan ter Meulen, John de Kruif

Affiliations

Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus

Mark Throsby et al. J Virol. 2006 Jul.

Abstract

Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 mug/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Anti-WNV monoclonal antibody panel binding and functional characteristics. (a) ELISA immunoreactivities of selected monoclonal scFv phages for purified inactivated WNV along the x axis and purified VLP along the y axis and (b) purified inactivated WNV along the x axis and purified soluble E protein along the y axis. OD, optical density. (c) PRNT for the determination of in vitro neutralizing activities of monoclonal antibodies. Data from two independent experiments carried out in duplicate are plotted as percentage mean ± standard error of the mean of the test IgG1 plaque number compared to negative control IgG1. A nonlinear-regression line was fitted using the ordinal-regression model probit. CR4374, •; CR4353, ○; 7H2, dotted line; 6B6C-1, solid line. (d) The CF activity of anti-WNV IgG1 was measured by the degree of SRBC lysis by free complement after preincubation of complement with WNV and IgG1. The results of two independent experiments were calculated as the mean percentage of SRBC lysis compared to negative control IgG1 wells. Nonlinear-regression lines fitted to the data are shown, and data were plotted for CR4268 (•) and CR4381 (○).

FIG. 2.

FIG. 2.

West Nile virus-neutralizing MAb sequences. VH sequences and VL sequences of scFv were aligned with germ line sequences. Framework (FR), CDR, sequence identities (.) and deletions (-) outside the CDR3 sequence, and VL germ line usage are indicated.

FIG. 3.

FIG. 3.

Binding activities of functional anti-WNV antibodies. (a) Purified IgG1s were titrated by ELISA on directly coated WNV antigen, and the antibody concentration required for 50% saturated binding was calculated by nonlinear regression. The antibody names are ranked from lowest to highest concentration required for 50% binding activity. OD, optical density. (b) To correlate CF activity with binding activity, the MAb concentration required for 50% CF activity was plotted against the concentration required for 50% binding activity. Antibodies were grouped based on the VH CDR3H sequence, and linear-regression lines were plotted to determine if the binding target specificity influenced the interaction between MAb affinity and CF activity. (c) Representative sensorgram of MAb CR4283 analyzed by SPR, showing raw data and curves fitted using a bivalent analyte model.

FIG. 4.

FIG. 4.

Identification of anti-WNV IgG1 binding targets that are nonreactive in the E protein ELISA. (a and b) Purified IgG1s were incubated with membranes blotted with inactivated WNV separated by 4 to 12% SDS-PAGE under nonreducing conditions. The anti-E protein murine MAb 7H2 and rabbit polyclonal anti-M serum were included as positive controls. The migration of a molecular weight marker is indicated. (c) Immunoprecipitation of biotinylated VLP-transfected mammalian cell lysates with anti-WNV IgG1 expressed in serum-free medium was analyzed under nonreducing conditions by SDS-PAGE separation, membrane transfer, and probing with streptavidin-HRP. The migration of a molecular weight marker is indicated.

FIG. 5.

FIG. 5.

West Nile virus (WNV) domain mapping by competition binding ELISA. (a) E protein was captured in microtiter plates either by immobilized 6B6C-1 MAb (filled bars) or 7H2 (open bars). The wells were then incubated with a panel of anti-WNV IgG1 at saturating concentrations. Binding was detected after anti-human IgG-HRP incubation and O-phenylenediamine (OPD) deposition. A representative experiment is shown (* denotes neutralizing activity). (b) WNV antigen was immobilized overnight in microtiter plates and then incubated with saturating concentrations of IgG1 (columns), followed by biotinylated IgG1 (rows) at subsaturating concentrations for 5 min. Binding was detected after streptavidin-HRP incubation and OPD deposition. The tabulated data represent the mean percentage of binding compared to negative control IgG1 for three independent experiments. For clarity, percentage binding lower than or equal to 25 is shaded gray, and percentage binding of less than 50 is in boldface.

FIG. 6.

FIG. 6.

Demonstration of the protective activity of anti-West Nile virus (WNV) IgG1 in a lethal WNV challenge model. BALB/c mice (n = 5) were injected i.p. with (a) 15 mg/kg or (b and c) the indicated dose of purified IgG1 in PBS and 24 h later infected i.p. with WNV strain USA99b. The animals were monitored twice daily and euthanized when clinical signs of infection appeared. Kaplan-Meier survival curves are shown for (a) a combined neutralizing MAb panel, (b) CR4374, and (c) CR4353.

References

    1. Beasley, D. W., and A. D. Barrett. 2002. Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope protein. J. Virol. 76:13097-13100. - PMC - PubMed
    1. Ben-Nathan, D., S. Lustig, G. Tam, S. Robinzon, S. Segal, B. Rager-Zisman, J. T. Roehrig, L. A. Staudinger, A. R. Hunt, J. H. Mathews, and C. D. Blair. 2003. Prophylactic and therapeutic efficacy of human intravenous immunoglobulin in treating West Nile virus infection in mice. Antibody prophylaxis and therapy for flavivirus encephalitis infections. J. Infect. Dis. 188:5-12. - PubMed
    1. Boel, E., S. Verlaan, M. J. Poppelier, N. A. Westerdaal, J. A. Van Strijp, and T. Logtenberg. 2000. Functional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody fragments. J. Immunol. Methods 239:153-166. - PubMed
    1. Cardosa, M. J., S. Gordon, S. Hirsch, T. A. Springer, and J. S. Porterfield. 1986. Interaction of West Nile virus with primary murine macrophages: role of cell activation and receptors for antibody and complement. J. Virol. 57:952-959. - PMC - PubMed
    1. Daffis, S., R. E. Kontermann, J. Korimbocus, H. Zeller, H. D. Klenk, and J. Ter Meulen. 2005. Antibody responses against wild-type yellow fever virus and the 17D vaccine strain: characterization with human monoclonal antibody fragments and neutralization escape variants. Virology 337:262-272. - PubMed

MeSH terms

Substances

LinkOut - more resources