The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity

Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2(c,d,e,f,g,h)) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE. We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants. Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 A resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope. The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.