Good initial 2D classes were chosen for template picking of particles. Africa, yet effective therapies or vaccines are lacking. The isolation of potent and protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccines candidates have generally been unsuccessful in doing so and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron-microscopy based epitope mapping pipeline that enables high-resolution structural characterization of polyclonal antibodies to GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization which involve epitopes of the GPC-C, GPC-A, and GP1-A competition clusters. Furthermore, by identifying previously undescribed immunogenic off-target epitopes, we expose challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design. Introduction Lassa fever, a viral hemorrhagic fever which is caused by the Lassa virus (LASV), is a persistent public health and socioeconomic burden to affected countries in West Africa. LASV is predominantly transmitted to humans from its natural reservoir or the GPC undergoes antibody-facilitated disassembly. This latter phenomenon has been observed extensively for influenza and HIV-1 where specific antibody responses have been shown to facilitate hemagglutinin and envelope protein dissolution, respectively.69C71 Our observations that interior-targeting responses were already elicited after the first immunization suggest that the GPCysR4-I53-50A readily disassemble before the emergence of other p53 and MDM2 proteins-interaction-inhibitor racemic responses to the exterior of the trimer. As a result, GPC exposes its largest non-glycosylated surface. According to our analyses, this interior surface contains epitopes that are the dominant target during early immunizations and likely compete with B cells in germinal centers that recognize functional epitopes. Our monomer EMPEM data also correspond to previous reports of non-NAbs targeting the GP1-B and GP2-B epitopes.15 We offer structural definition to these antibody clusters with data that strongly suggests these epitopes are present only on disassembled, non-trimeric GPCs. The fact that these mAbs were isolated from Lassa fever survivors, suggests that natural infection presents GPC interiors, though the mechanism by which this happens is still unknown. Regardless, our results imply that fusing GPC to a trimerization domain is insufficient and further stabilization of the trimeric interface should dictate the design of the next generation of recombinant GPC vaccines. Our EMPEM work also revealed vaccine-induced pAb responses remarkably similar to previously characterized NAbs isolated from Lassa fever survivors, supporting the use of rabbits as vaccination models. GPCysR4-I53-50A induced a response in two out of six rabbits that p53 and MDM2 proteins-interaction-inhibitor racemic correlated strongly with neutralization and which we demonstrated parallels the canonical human GPC-A NAbs. With its high similarity to GPC-A-targeting NAbs 36.1F and 25.10C, GPC-A-1 likely neutralizes LASV by blocking LAMP-1. 40 While the FP-1 pAb response did not fully mirror the defined GPC-B epitope, it engages with some of the same residues that are targeted by potent GPC-B NAbs.27,28 Focusing heavily on the fusion peptide, we speculate that the FP-1 response may have the potential to mature towards neutralization, perhaps by increasing its affinity or making it resilient to acidic pH. Interestingly, the GPC-derived VLP, which induced markedly more potent neutralizing responses than GPCysR4-I53-50A, elicited very different on-target responses. This vaccine induced responses very similar to the GPC-C and GP1-A NAbs, 8.9F and 12.1F, respectively; two of the three NAbs that make up Arevirumab-3.29,43 The p53 and MDM2 proteins-interaction-inhibitor racemic engagement of GPC-C-1 with the native-cleavage site reinforces earlier suggestions that these residues are necessary for the elicitation of apex-targeting antibodies and provides one explanation for the differences in NAb responses between the vaccine modalities.29 Another explanation may be differences in epitope presentation (i.e. valency of immunogen display and accessibility of epitopes) between the two vaccines. It is tempting to perceive the remarkable neutralization titers this rabbit generated in the context of the VLPs inability to induce off-target responses to the base and interior. It should be considered, however, that this immunogen required an Mouse Monoclonal to S tag extensive prime-boost regimen to p53 and MDM2 proteins-interaction-inhibitor racemic achieve its potent neutralization. We do note that membrane-embedded GPCs are generally poorly cleaved (the GPC-derived.