InVivoMAb anti-Venezuelan equine encephalitis virus E2 protein

in vivo protection against VEEV, in vitro neutralization of VEEV, Focus reduction neutralization test (FRNT), Plasma membrane fusion-from-without (FFWO), Inhibition of viral attachment on cells, Inhibition of viral egress, ELISA

Kafai NM, Williamson LE, Binshtein E, Sukupolvi-Petty S, Gardner CL, Liu J, Mackin S, Kim AS, Kose N, Carnahan RH, Jung A, Droit L, Reed DS, Handley SA, Klimstra WB, Crowe JE, Diamond MS. (2022). "Neutralizing antibodies protect mice against Venezuelan equine encephalitis virus aerosol challenge" J Exp Med 10.1084/jem.20212532. PubMed

Venezuelan equine encephalitis virus (VEEV) remains a risk for epidemic emergence or use as an aerosolized bioweapon. To develop possible countermeasures, we isolated VEEV-specific neutralizing monoclonal antibodies (mAbs) from mice and a human immunized with attenuated VEEV strains. Functional assays and epitope mapping established that potently inhibitory anti-VEEV mAbs bind distinct antigenic sites in the A or B domains of the E2 glycoprotein and block multiple steps in the viral replication cycle including attachment, fusion, and egress. A 3.2-Å cryo-electron microscopy reconstruction of VEEV virus-like particles bound by a human Fab suggests that antibody engagement of the B domain may result in cross-linking of neighboring spikes to prevent conformational requirements for viral fusion. Prophylaxis or postexposure therapy with these mAbs protected mice against lethal aerosol challenge with VEEV. Our study defines functional and structural mechanisms of mAb protection and suggests that multiple antigenic determinants on VEEV can be targeted for vaccine or antibody-based therapeutic development.

Basore K, Ma H, Kafai NM, Mackin S, Kim AS, Nelson CA, Diamond MS, Fremont DH. (2021). "Structure of Venezuelan equine encephalitis virus in complex with the LDLRAD3 receptor" Nature 10.1038/s41586-021-03963-9. PubMed

LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV)¹, a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2-E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus-receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.

in vitro neutralization of VEEV

Ma H, Kim AS, Kafai NM, Earnest JT, Shah AP, Case JB, Basore K, Gilliland TC, Sun C, Nelson CA, Thackray LB, Klimstra WB, Fremont DH, Diamond MS. (2020). "LDLRAD3 is a receptor for Venezuelan equine encephalitis virus" Nature 10.1038/s41586-020-2915-3. PubMed

Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans¹. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR-Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)-a highly conserved yet poorly characterized member of the scavenger receptor superfamily-as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)-Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)-Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.