PG and JC wrote the first draft of the paper
PG and JC wrote the first draft of the paper. proteomic sequencing approaches to assess the composition of the ebolavirus glycoprotein (GP)-reactive antibody repertoire in the plasma of an EVD survivor. We first identified 1,512 GP-specific mAb variable gene sequences from single cells in the memory B cell compartment. Using mass spectrometric analysis of the corresponding GP-specific plasma IgG, we found that only a portion of the large B cell antibody repertoire was represented in the plasma. Molecular and functional analysis of proteomics-identified mAbs revealed recognition of epitopes in three major antigenic sites – the GP head domain, the glycan cap, and the base region, with a high prevalence of neutralizing and protective mAb specificities that targeted the base and glycan cap regions on the GP. Polyclonal plasma antibodies from the survivor reacted broadly to EBOV, BDBV, and SUDV GP, while reactivity of the potently neutralizing mAbs we identified was limited mostly to the homologous EBOV GP. Together these results reveal a restricted diversity of neutralizing humoral response in which mAbs targeting two antigenic sites on GP glycan cap and base play a principal role in plasma-antibody-mediated protective immunity against EVD. Keywords:ebolavirus, ebolavirus infection, glycoprotein, proteo-genomics, convalescent plasma, viral antibodies, neutralizing antibodies, epitope mapping == Introduction == Ebolaviruses are responsible for severe disease and occasional deadly outbreaks in Africa posing a significant health threat. The Ebolavirus genus consists of six species, including Zaire ebolavirus [represented by Ebola virus (EBOV)], Sudan ebolavirus [Sudan virus (SUDV)], Bundibugyo ebolavirus [Bundibugyo virus (BDBV)], Ta Forest ebolavirus [Ta Forest virus (TAFV)], Reston ebolavirus [Reston virus (RESV)] (1), and Bombali ebolavirus [Bombali virus (BOMV)] (2). EBOV, BDBV, and SUDV are the medically important causative agents of symptomatic infections and ebolavirus disease (EVD) in humans. A total of 41 confirmed EVD outbreaks have been documented, and the largest EVD epidemic to date occurred in 2013-2016 in West Africa with a total of 28,610 disease cases and 11,308 deaths reported (3). The unpredictable nature of EVD outbreaks and public health challenges stemming from the severity of the disease underscores the need for development of medical countermeasures and systematic studies to elucidate correlates of immune response-mediated Benzocaine protection against EVD. The evidence to date suggests an indispensable role for antibody-mediated immunity in the protection against EVD. Several investigational treatments based on human monoclonal antibodies (mAb) showed therapeutic efficacy in animal models of EVD (48) and clinical trials in the Democratic Republic of Congo outbreak demonstrated high efficacy of antibody-based therapeutics for acute EVD treatment in patients (9). By 2020, two monoclonal antibody-based therapeutics ansuvimab-zykl (Ebanga) and atoltivimab + maftivimab + odesivimab-ebgn (Inmazeb) were developed and approved by the Food and Drug Administration (FDA) for clinical use (10,11). A landmark achievement was the development and FDA approval of a recombinant viral vector-based vaccine (Ervebo) for prevention of EVD (12,13), vaccination with which has been shown to induce long-lasting antibody responses in clinical trials (14). The key target for protective antibodies is the ebolavirus glycoprotein (GP), which is a single surface protein of the viral envelope. GP forms a trimer, in which each protomer consists MMP3 of two subunits, designated GP1 and GP2. The GP1 subunit contains a heavily glycosylated mucin-like domain (MLD) and a glycan cap, which shields the host receptor binding site (RBS). The RBS is exposed after proteolytical cleavage in the Benzocaine host endosome and binds to domain C of its endosomal receptor, the protein Niemann-Pick C1 (NPC1-C). The GP2 subunit contains the internal fusion loop (IFL) and stalk and is anchored into the viral membrane by a transmembrane domain (1517). Recent improvement of Benzocaine instruments and Benzocaine technologies for high-throughput single B cell analysis enabled isolation of thousands of ebolavirus GP-reactive mAbs from the circulating memory B cells of EVD survivors or vaccinees (1820). Hundreds of mAbs were characterized at the molecular level in studies that revealed a diverse landscape of epitope recognition in which.