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Human monoclonal antibodies against West Nile virus induced by natural infection neutralize at a postattachment step.

ABSTRACT: West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies against the DIII-lr epitope comprise, at best, a minor component of the human anti-WNV antibody response. Herein, we characterize in detail two WNV-specific human MAbs, CR4348 and CR4354, that were isolated from B-cell populations of convalescent patients. These MAbs strongly neutralize WNV infection of cultured cells, protect mice against lethal infection in vivo, and yet poorly recognize recombinant forms of the E protein. Instead, CR4348 and CR4354 bind determinants on intact WNV virions and subviral particles in a pH-sensitive manner, and neutralization is altered by mutations at the dimer interface in domain II and the hinge between domains I and II, respectively. CR4348 and CR4354 human MAbs neutralize infection at a postattachment step in the viral life cycle, likely by inhibiting acid-induced fusion within the endosome.

SUBMITTER: Vogt MR

PROVIDER: S-EPMC2698525 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Human monoclonal antibodies against West Nile virus induced by natural infection neutralize at a postattachment step.

Vogt Matthew R MR Moesker Bastiaan B Goudsmit Jaap J Jongeneelen Mandy M Austin S Kyle SK Oliphant Theodore T Nelson Steevenson S Pierson Theodore C TC Wilschut Jan J Throsby Mark M Diamond Michael S MS

Journal of virology 20090422 13

West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies aga ...[more]

PMID: 19386704

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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.

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.

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Human monoclonal antibodies against West Nile virus induced by natural infection neutralize at a postattachment step.

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Human monoclonal antibodies against West Nile virus induced by natural infection neutralize at a postattachment step.

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