About InVivoMAb anti-Eastern equine encephalitis virus E2 protein The EEEV-3 monoclonal antibody reacts with the B domain of the E2 glycoprotein on the eastern equine encephalitis virus (EEEV). In humans and horses, EEEV is transmitted primarily through the bite of the swamp mosquito Culiseta melanura (i.e., black-tailed mosquito). Infected mosquitoes transmit the EEEV to birds as well, and uninfected mosquitoes acquire the EEEV infection through feeding on infected birds. Human EEEV infection incidences are low, but the mortality rate can be up to 70%, and the survivors end up with significant brain damage. Structurally, the EEEV is an enveloped virus with single-stranded positive sense RNA, and the mature virion displays spikes that are made of E2-E1 heterodimers. The E2 glycoprotein binds to some host cell receptors, and it is suggested to be responsible for viral entry and endocytosis. In experimental studies, antibodies against epitopes on the E2 protein (e.g., EEEV-3) are used to elucidate the molecular mechanism of EEEV infections. The EEEV-3 monoclonal antibody is specific for EEEV’s E2 glycoprotein (B domain), and it does not bind other related viruses such as the Western equine encephalitis virus (WEEV) and the Venezuelan equine encephalitis virus (VEEV). Cryo-EM structure analysis and alanine-scanning mutagenesis studies demonstrated that the Fab fragments of the EEEV-3 monoclonal antibody interact exclusively with an epitope localized to residues 180–182 on the B domain of the EEEV E2 glycoprotein. In vitro experiments have shown that the EEEV-3 monoclonal antibody exhibits a modest inhibition of viral attachment to the plasma membrane of the cells. Post-attachment neutralization assays showed the in vitro inhibitory activity of the EEEV-3 monoclonal antibody against SINV-EEEV infection when the antibody was added after the virus was bound to cells. In the FFWO assay, the EEEV-3 monoclonal antibody was added after viral attachment to Vero cells, and it was found that the antibody significantly blocks the virus plasma membrane fusion. FRNT assays involving the infection of Vero cells with EEEV particles that were pre-incubated in vitro with the EEEV-3 monoclonal antibody or its Fab fragments demonstrated that the bivalent mouse IgG monoclonal antibodies efficiently inhibit infection while the Fab fragments does not. The in vivo efficacy of the EEEV-3 monoclonal antibody was evaluated in CD-1 mice, which were inoculated with the highly pathogenic EEEV strain FL93-939. A single 100-μg i.p. injection of the EEEV-3 monoclonal antibody one day before or after subcutaneous or aerosol inoculation of EEEV (lethal doses) provided significant protection against viral infection. InVivoMAb anti-Eastern equine encephalitis virus E2 protein Specifications IsotypeMouse IgG2c, κ Recommended Isotype Control(s)InVivoMAb mouse IgG2c isotype control, anti-dengue virus Recommended Dilution BufferInVivoPure pH 7.0 Dilution Buffer ImmunogenSINV-EEEV Reported Applicationsin vivo protection against EEEV in vitro neutralization of EEEV Flow cytometry ELISA Plasma membrane fusion-from-without (FFWO) Focus reduction neutralization tests (FRNT) Biolayer Interferometry (BLI) FormulationPBS, pH 7.0 Contains no stabilizers or preservatives Endotoxin<2EU/mg (<0.002EU/μg) Determined by LAL gel clotting assay Purity>95% Determined by SDS-PAGE Sterility0.2 μm filtration ProductionPurified from cell culture supernatant in an animal-free facility PurificationProtein A Molecular Weight150 kDa StorageThe antibody solution should be stored at the stock concentration at 4°C. Do not freeze. Application ReferencesInVivoMAb anti-Eastern equine encephalitis virus E2 protein (CLONE: EEEV-3)Ma H, Adams LJ, Raju S, Sariol A, Kafai NM, Janova H, Klimstra WB, Fremont DH, Diamond MS (2024). "The low-density lipoprotein receptor promotes infection of multiple encephalitic alphaviruses" Nat Commun 10.1038/s41467-023-44624-x. PubMedMembers of the low-density lipoprotein receptor (LDLR) family, including LDLRAD3, VLDLR, and ApoER2, were recently described as entry factors for different alphaviruses. However, based on studies with gene edited cells and knockout mice, blockade or abrogation of these receptors does not fully inhibit alphavirus infection, indicating the existence of additional uncharacterized entry factors. Here, we perform a CRISPR-Cas9 genome-wide loss-of-function screen in mouse neuronal cells with a chimeric alphavirus expressing the Eastern equine encephalitis virus (EEEV) structural proteins and identify LDLR as a candidate receptor. Expression of LDLR on the surface of neuronal or non-neuronal cells facilitates binding and infection of EEEV, Western equine encephalitis virus, and Semliki Forest virus. Domain mapping and binding studies reveal a low-affinity interaction with LA domain 3 (LA3) that can be enhanced by concatenation of LA3 repeats. Soluble decoy proteins with multiple LA3 repeats inhibit EEEV infection in cell culture and in mice. Our results establish LDLR as a low-affinity receptor for multiple alphaviruses and highlight a possible path for developing inhibitors that could mitigate infection and disease.Adams LJ, Raju S, Ma H, Gilliland T, Reed DS, Klimstra WB, Fremont DH, Diamond MS (2023). "Structural and functional basis of VLDLR receptor usage by Eastern equine encephalitis virus" bioRxiv 10.1101/2023.11.15.567188. PubMedThe very low-density lipoprotein receptor (VLDLR) is comprised of eight LDLR type A (LA) domains and supports entry of distantly related Eastern equine encephalitis (EEEV) and Semliki Forest (SFV) alphaviruses. Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage different LA domains simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection, highlighting complexity in domain usage. Whereas all EEEV strains show conservation of two VLDLR binding sites, the EEEV PE-6 strain and other EEE complex members feature a single amino acid substitution that mediates binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge.Williamson LE, Gilliland T, Yadav PK, Binshtein E, Bombardi R, Kose N, Nargi RS, Sutton RE, Durie CL, Armstrong E, Carnahan RH, Walker LM, Kim AS, Fox JM, Diamond MS, Ohi MD, Klimstra WB, Crowe JE (2020). "Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection" Cell 10.1016/j.cell.2020.11.011. PubMedEastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.Kim AS, Austin SK, Gardner CL, Zuiani A, Reed DS, Trobaugh DW, Sun C, Basore K, Williamson LE, Crowe JE, Slifka MK, Fremont DH, Klimstra WB, Diamond MS (2019). "Protective antibodies against Eastern equine encephalitis virus bind to epitopes in domains A and B of the E2 glycoprotein" Nat Microbiol 10.1038/s41564-018-0286-4. PubMedEastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus with a high case mortality rate in humans. EEEV is a biodefence concern because of its potential for aerosol spread and the lack of existing countermeasures. Here, we identify a panel of 18 neutralizing murine monoclonal antibodies (mAbs) against the EEEV E2 glycoprotein, several of which have 'elite' activity with 50 and 99% effective inhibitory concentrations (EC50 and EC99) of less than 10 and 100 ng ml-1, respectively. Alanine-scanning mutagenesis and neutralization escape mapping analysis revealed epitopes for these mAbs in domains A or B of the E2 glycoprotein. A majority of the neutralizing mAbs blocked infection at a post-attachment stage, with several inhibiting viral membrane fusion. Administration of one dose of anti-EEEV mAb protected mice from lethal subcutaneous or aerosol challenge. These experiments define the mechanistic basis for neutralization by protective anti-EEEV mAbs and suggest a path forward for treatment and vaccine design.Hasan SS, Sun C, Kim AS, Watanabe Y, Chen CL, Klose T, Buda G, Crispin M, Diamond MS, Klimstra WB, Rossmann MG (2018). "Cryo-EM Structures of Eastern Equine Encephalitis Virus Reveal Mechanisms of Virus Disassembly and Antibody Neutralization" Cell Rep 10.1016/j.celrep.2018.11.067. PubMedAlphaviruses are enveloped pathogens that cause arthritis and encephalitis. Here, we report a 4.4-Å cryoelectron microscopy (cryo-EM) structure of eastern equine encephalitis virus (EEEV), an alphavirus that causes fatal encephalitis in humans. Our analysis provides insights into viral entry into host cells. The envelope protein E2 showed a binding site for the cellular attachment factor heparan sulfate. The presence of a cryptic E2 glycan suggests how EEEV escapes surveillance by lectin-expressing myeloid lineage cells, which are sentinels of the immune system. A mechanism for nucleocapsid core release and disassembly upon viral entry was inferred based on pH changes and capsid dissociation from envelope proteins. The EEEV capsid structure showed a viral RNA genome binding site adjacent to a ribosome binding site for viral genome translation following genome release. Using five Fab-EEEV complexes derived from neutralizing antibodies, our investigation provides insights into EEEV host cell interactions and protective epitopes relevant to vaccine design.
