Mechanisms of Protective Humoral Immunity and Pathogenesis in Alphavirus Infection

Abstract

Alphaviruses are enveloped, mosquito-transmitted, positive-sense RNA viruses of the Togaviridae family classified into Old and New World groups based on endemic regions and human disease characteristics. Old World alphaviruses, including chikungunya virus preferentially cause arthritogenic disease, whereas New World viruses like Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and Western equine encephalitis virus (WEEV) can cause neurological disease. Since the 1920s, VEEV has been responsible for major periodic outbreaks affecting hundreds of thousands of humans and equines in South and Central America. Expansion of mosquito vectors raises concern for possible VEEV reemergence. In addition, VEEV is a bioterrorism threat and was weaponized in the 1960s and 1970s for possible aerosol dissemination. Thus, alphaviruses, including VEEV, have been responsible for numerous worldwide epidemic outbreaks in the past century and yet, approved vaccines or treatments still do not exist. Here we investigate mechanisms of antibody-mediated protection against alphaviruses and how understanding alphavirus-host interactions leading to pathogenesis is critical for mitigating disease through antiviral therapeutic design. We describe the molecular basis of neutralization by murine and human monoclonal antibodies (mAbs) targeting several envelope protein epitopes. These mAbs inhibited multiple steps in the viral replication cycle including viral attachment, fusion to host membranes, and egress. Epitope mapping identified antigenically distinct sites on the VEEV E2 protein targeted by mAbs. Cryo-electron microscopy (cryo-EM) analysis of a potently neutralizing human mAb bound to VEEV defined a critical binding site within domain B of the E2 protein. Anti-VEEV mAbs targeting each antigenic site conferred robust protection and post-exposure therapeutic efficacy in mice challenged with aerosolized VEEV, highlighting targets for potential therapeutic development and vaccine immunogen design. Our studies link epitopes recognized by inhibitory anti-VEEV mAbs elicited after immunization with mechanisms of neutralization and thus further define the structural and functional components that contribute to protective efficacy against aerosolized VEEV infection. While neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses had not been reported. We screened and identified several candidate anti-WEEV cross-reactive mAbs and then developed an immunization and B cell sorting strategy to generate pan-alphavirus cross-reactive mAbs. Biosafety level 2 mouse models of EEEV, WEEV, and VEEV were developed for use in lethality and virological endpoints as a tool for screening these mAbs. Ultimately, two pan-protective yet poorly neutralizing human mAbs were identified, which bind to viral antigen on the surface of alphavirus-infected cells. These mAbs engage a conserved epitope in the E1 protein. Treatment with these mAbs protected against arthritogenic and encephalitic alphaviruses through various mechanisms including inhibition of viral egress and monocyte-dependent Fc effector functions. To better understand mechanisms of protective humoral immunity that might inform anti-alphavirus therapeutic development, we also defined key alphavirus receptors critical for pathogenesis in vivo. A VEEV-specific entry receptor, low-density lipoprotein receptor class A domain containing 3 (LDLRAD3), was identified using a loss-of-infection-based CRISPR-Cas9 genome-wide screen. LDLRAD3 is a highly conserved type I membrane protein of the LDL receptor superfamily and has been reported to regulate amyloid processing and auto-ubiquitination in neurons, although its endogenous ligand(s) are unknown. The most membrane-distal domain 1 (D1) of the three extracellular domains of LDLRAD3 was shown to be necessary and sufficient for VEEV infection, and a cryo-EM structure showed that LDLRAD3 D1 binds in a cleft formed between adjacent VEEV E2 and E1 proteins on the virion surface. We investigated the role of the entry receptor LDLRAD3 in VEEV pathogenesis using newly generated LDLRAD3-deficient mice. We found consistently lower levels of VEEV infection in all target tissues of LDLRAD3-deficient mice after subcutaneous inoculation, as early as 6 hours post-infection and at every timepoint tested thereafter. While VEEV entry into the brain occurred in the absence of LDLRAD3 expression, spread was delayed, infection accumulated at substantially lower levels, and animals did not sustain weight loss or lethality. Bone marrow chimera studies established that VEEV pathogenesis was largely dependent on LDLRAD3 expression in radioresistant stromal cells. Direct inoculation of VEEV into the brain via intracranial or intranasal inoculation resulted in uniform lethality in wild-type mice, whereas in LDLRAD3-deficient mice, animals lost weight but survived infection. This phenotype was associated with reduced central nervous system (CNS) viral burdens in LDLRAD3-deficient mice. Using in situ hybridization and immunohistochemistry, the absence of LDLRAD3 was associated with marked decreases in infection of neurons in adult mouse brains and in mixed primary neuron cultures isolated from embryos. Overall, we establish a key role for LDLRAD3 in the infection, dissemination, and pathogenesis of VEEV in peripheral and CNS tissues