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. IMPORTANCECHIKV 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 membranedistal 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.

Chikungunya virus pathogenesis: From bedside to bench

“…Human neutralizing monoclonal antibodies directed against E2 or E1 significantly delay lethality of CHIKV-infected mice, both in prophylactic or therapeutic settings [33,35]. Similarly, a human neutralizing monoclonal antibody directed against E2 protein is able to prophylactically protect adult C57BL/6 mice from viremia and foot swelling, and to therapeutically protect neonatal C57BL/6 mice from death [118].…”

Chikungunya virus vaccines: Current strategies and prospects for developing plant-made vaccines

“…In fact Kam et al [21,22] observed, using plasma samples from humans obtained during the early convalescent phase, that the naturally-acquired IgG response is dominated by neutralizing IgG3 antibodies which are mostly directed toward the single linear epitope E2EP3, a peptide located at the N-terminus of the E2 glycoprotein and exposed on the viral envelope. More recently, it was confirmed that epitopes on the exposed top-most and outer surfaces of the E2/E1 trimer structure may be useful for CHIKV neutralization by specific antibodies, whereas epitopes facing the interior of the trimer are not [64]. Overall, these findings pave the way for the development of CHIKV-specific multi-epitopic-or VLP-based vaccination strategies that can be efficiently produced in plant-based platforms.…”

DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops

“…VLP have the advantage that they have a much higher density of the target GPCR compared to cells and membrane fractions and can easily be coated onto an ELISA plate. 22 The molecular density of target molecules on VLPs is still much lower compared to a coating of recombinant protein.…”