title: Exposure of epitope residues on the outer face of the chikungunya virus envelope trimer determines antibody neutralizing efficacy.
abstract: Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer.
  
  
  OBJECTIVE
  CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally, and the spread of CHIKV to the Americas is now beginning, with over 100,000 cases occurring in the Caribbean within 6 months of its arrival. Our study reports on seven human MAbs against the CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane-distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV, and antibodies targeting the exposed topmost and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response.
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Rachel H
authors.initials: RH
authors.lastname: Fong
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Soma S R
authors.initials: SS
authors.lastname: Banik
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Mattia
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Trevor
authors.initials: T
authors.lastname: Barnes
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: David
authors.initials: D
authors.lastname: Tucker
authors.affiliation: Blood Systems Research Institute, San Francisco, California, USA.
authors.forename: Nathan
authors.initials: N
authors.lastname: Liss
authors.affiliation: Blood Systems Research Institute, San Francisco, California, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Lu
authors.affiliation: Blood Systems Research Institute, San Francisco, California, USA.
authors.forename: Suganya
authors.initials: S
authors.lastname: Selvarajah
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Surabhi
authors.initials: S
authors.lastname: Srinivasan
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Manu
authors.initials: M
authors.lastname: Mabila
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Miller
authors.affiliation: Blood Systems Research Institute, San Francisco, California, USA Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Marcus O
authors.initials: MO
authors.lastname: Muench
authors.affiliation: Centre Hospitalier, Groupe Hospitalier Sud-Reunion, La Réunion, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Michault
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Rucker
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Paes
authors.affiliation: Blood Systems Research Institute, San Francisco, California, USA Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Graham
authors.initials: G
authors.lastname: Simmons
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Kahle
authors.affiliation: Integral Molecular Inc., Philadelphia, Pennsylvania, USA bdoranz@integralmolecular.com.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Doranz
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D057134:Antibodies, Neutralizing / Q000276:immunology* / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000276:immunology* / Q000302:isolation & purification; D001665:Binding Sites; D061505:Cell Surface Display Techniques; D065632:Chikungunya Fever / Q000517:prevention & control; D002646:Chikungunya virus / Q000276:immunology*; D004195:Disease Models, Animal; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006801:Humans; D007116:Immunization, Passive; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D011487:Protein Conformation; D016019:Survival Analysis; D014759:Viral Envelope Proteins / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000939:Epitopes; D014759:Viral Envelope Proteins