Dataset Information

Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein.

ABSTRACT: Recent epidemics of West Nile virus (WNV) around the world have been associated with significant rates of mortality and morbidity in humans. To develop standard WNV diagnostic tools that can differentiate WNV from Japanese encephalitis virus (JEV), four monoclonal antibodies (MAbs) specific to WNV envelope (E) protein were produced and characterized by isotyping, reactivity with denatured and native antigens, affinity assay, immunofluorescence assay (IFA), and epitope competition, as well as cross-reactivity with JEV. Two of the MAbs (6A11 and 4B3) showed stronger reactivity with E protein than the others (2F5 and 6H7) in Western blot analysis. 4B3 could bind with denatured antigen, as well as native antigens in indirect ELISA, flow cytometry analysis, and IFA; whereas 2F5 showed highest affinity with native antigen. 4B3 and 2F5 were therefore used to establish an antigen capture-ELISA (AC-ELISA) detection system. The sensitivity of this AC-ELISA was 3.95 TCID(50)/0.1 ml for WNV-infected cell culture supernatant. Notably, these MAbs showed no cross-reactivity with JEV, which suggests that they are useful for further development of highly sensitive, easy handling, and less time-consuming detection kits/tools in WNV surveillance in areas where JEV is epidemic.

SUBMITTER: Liu J

PROVIDER: S-EPMC7112808 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein.

Liu June J Liu Bohua B Cao Zhen Z Inoue Shingo S Morita Kouichi K Tian Kegong K Zhu Qingyu Q Gao George F GF

Journal of virological methods 20081105 1-2

Recent epidemics of West Nile virus (WNV) around the world have been associated with significant rates of mortality and morbidity in humans. To develop standard WNV diagnostic tools that can differentiate WNV from Japanese encephalitis virus (JEV), four monoclonal antibodies (MAbs) specific to WNV envelope (E) protein were produced and characterized by isotyping, reactivity with denatured and native antigens, affinity assay, immunofluorescence assay (IFA), and epitope competition, as well as cro ...[more]

PMID: 18948139

Similar Datasets

Project description:BackgroundDiagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs.MethodsSix MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity.ResultsAll MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys → Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser → Ile) or E278 (Thr → Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs.ConclusionsMAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization.

Project description:Envelope protein-targeted vaccines for flaviviruses are limited by concerns of antibody-dependent enhancement (ADE) of infections. Nonstructural protein 1 (NS1) provides an alternative vaccine target that avoids this risk since this protein is absent from the virion. Beyond its intracellular role in virus replication, extracellular forms of NS1 function in immune modulation and are recognized by host-derived antibodies. The rational design of NS1-based vaccines requires an extensive understanding of the antigenic sites on NS1, especially those targeted by protective antibodies. Here, we isolated human monoclonal antibodies (MAbs) from individuals previously naturally infected with WNV, mapped their epitopes using structure-guided mutagenesis, and evaluated their efficacy in vivo against lethal WNV challenge. The most protective epitopes clustered at three antigenic sites that are exposed on cell surface forms of NS1: (i) the wing flexible loop, (ii) the outer, electrostatic surface of the wing, and (iii) the spaghetti loop face of the β-ladder. One additional MAb mapped to the distal tip of the β-ladder and conferred a lower level of protection against WNV despite not binding to NS1 on the surface of infected cells. Our study defines the epitopes and modes of binding of protective anti-NS1 MAb antibodies following WNV infection, which may inform the development of NS1-based countermeasures against flaviviruses. IMPORTANCE Therapeutic antibodies against flaviviruses often promote neutralization by targeting the envelope protein of the virion. However, this approach is hindered by a possible concern for antibody-dependent enhancement of infection and paradoxical worsening of disease. As an alternative strategy, antibodies targeting flavivirus nonstructural protein 1 (NS1), which is absent from the virion, can protect against disease and do not cause enhanced infection. Here, we evaluate the structure-function relationships and protective activity of West Nile virus (WNV) NS1-specific monoclonal antibodies (MAbs) isolated from the memory B cells of a naturally infected human donor. We identify several anti-NS1 MAbs that protect mice against lethal WNV challenge and map their epitopes using charge reversal mutagenesis. Antibodies targeting specific regions in the NS1 structure could serve as the basis for countermeasures that control WNV infection in humans.

Dataset Information

Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein.

Publications

Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets