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2). 3.2. virus, Neutralizing antibodies, Synthetic antibodies 1.?Introduction Members of the family cause severe hemorrhagic fever with a high percentage of fatal cases. Five different ebolaviruses have been isolated: Ebola virus (Zaire, EBOV), Bundibugyo virus (BDBV), Tai Forest virus (TAFV), Reston virus (RESTV), and Sudan virus (SUDV). Among these, EBOV and SUDV are responsible for most of the ebolavirus-related deaths [1]. The 2014C2016 EBOV epidemic in West Africa far exceeded the scale of any previous ebolavirus outbreak, with over 28,000 suspected cases of infection [2]. Prior to 2014, the largest ebolavirus outbreak was caused by SUDV in 2000, where at least 425 individuals were infected and the mortality rate was ~50% [3]. Monoclonal antibodies (mAbs) represent a promising therapeutic platform for the treatment of ebolavirus infections. For example, the mAb cocktail ZMapp? (Mapp Biopharmaceutical) has been shown to reverse the course of advanced Ebola virus disease (EVD) in non-human primates and recently completed clinical efficacy studies (PREVAIL II) [4]. However, although much effort has been made to isolate mAbs against EBOV, only a few mAbs have been shown to protect from lethal SUDV challenge [5-10]. The glycoproteins for EBOV and SUDV are ~45% divergent and thus mAbs that can cross-neutralize these viruses are rare [7,8,10]. We previously employed antibody engineering approaches to develop cross-protective ebolavirus antibodies [6,11]. The filovirus glycoprotein GP is the primary target of neutralizing antibodies [12-14]. The mature spike of the glycoprotein is a trimer comprised of three disulfide-linked GP1CGP2 heterodimers Complanatoside A that are generated by furin cleavage during virus assembly. The prefusion GP1-GP2 spike displays a chalice-and-bowl morphology. Three GP2 subunits form the chalice, while the bowl is represented by three GP1 subunits [12]. The head of GP1 contains a putative receptor-binding site (RBS). Complanatoside A The glycan cap and the mucin-like domain of the glycoprotein are extensively glycosylated and, in the prefusion form, likely sequester the critical RBS from the adaptive immune response [11,15,16]. Virus entry into cells is initiated by the interaction of GP1 with multiple cell-surface molecules and proceeds a macropinocytosis-like mechanism [17]. During the endolysosomal transport of the virus, major segments CD248 of GP1 (the glycan cap and mucin-like domain) are removed by host endosomal cysteine proteases (CathepsinL and Cathepsin B for EBOV). Next, the RBS engages the critical host receptor, Niemann Pick C1 (NPC1) its luminal C-loop [12,18]. Subsequent events lead ultimately to conformational changes in the GP2 subunit that promote viral membrane fusion and delivery of the viral contents into the cytosol [19-21]. A few studies to date have examined the intracellular fate of neutralizing ebolavirus mAbs, however these have all focused on EBOV. EBOV mAbs bind a number of epitopes on the viral glycoprotein and can potentially mediate different neutralizing effector functions [13]. The most potent antibodies bind epitopes at the GP1-GP2 interface, the glycan cap or the stem (C-heptad repeat region, CHR) of the glycoprotein [12,13]. Several studies suggest that protective antibodies directly inhibit the membrane fusion event between host and virus by either impeding necessary glycoprotein conformational changes or protection of the glycoprotein against the required proteolytic disassembly that reveals the RBS [12,22,23]. A number of neutralizing mAbs bind at the GP1-GP2 interface, and one Complanatoside A such EBOV-specific mAb, KZ52, seem to be able to protect GP from Cathepsin L-dependent activation, and thus may potentially block infection by preventing release of the GP2 fusion loop into the host endosomal membrane [23,24]. Here, we investigate the role of Fc for SUDV mAb F4 that was previously shown to provide potent post-exposure protection from viral challenge in mice [5]. The design of F4 is based on the humanized framework regions of Herceptin? and the complementarity determining regions (CDRs) of murine SUDV antibody.