In order to gain a better understanding of the targets and mechanisms of EBOV NAbs we studied a panel of known EBOV-specific antibodies; three mouse mAbs that conferred protection in mice against a mouse-adapted EBOV (Wilson et al., 2000
), one human mAb (Lee et al., 2008
) that protected guinea pigs (Parren et al., 2002
) but not monkeys (Oswald et al., 2007
), and one monkey survivor mAb specific for GP (Meissner et al., 2002
). The N-terminus of GP1 contains the putative RBD that is required for receptor binding and virus fusion (Brindley et al., 2007
; Kuhn et al., 2006
; Manicassamy et al., 2005
) and therefore may serve as a potent target for NAbs. The neutralizing mAbs examined herein had epitopes comprising N-terminal GP1 amino acids since deletion of the N-terminal GP1 region ablated Ab-binding. These NAbs could be further segregated based on their dependence on residues within GP2; only KZ52 and JP3K11 required the presence of GP2 for recognition. 6D3 and 13C6 were capable of binding free sGP, which does not contain any GP2 residues. Thus, it would be reasonable to hypothesize that binding by these N-terminal specific Abs may interfere with a number of events required for virus entry including cell attachment, receptor binding, and/or membrane fusion. It is noteworthy that the binding determinants for 6D3 and 13C6 are more accessible in sGP than virion GP (), suggesting that a weaker binding affinity for native GP may explain their reduced potency for neutralization when compared with KZ52. Additionally, it should be noted that the efficacy of NAbs that recognize free sGP, like 6D3 and 13C6, may be diminished in vivo
by the fact that their ligand of greatest affinity, sGP is present at higher frequencies than virus-associated GP during natural infection and may form complexes (and deplete) antibodies with this specificity. On the contrary, while recognition of GP by KZ52 and JP3K11 was also dependent on amino acids present in N-terminal GP1, binding was dependent on the presence of GP2 residues which are not present in sGP. Binding results for KZ52 are consistent with KZ52-GP contacts identified in co-crystals that reveal specific residues in GP2 that are situated at a distant location from the putative RBD in GP1 (Lee et al., 2008
). While KZ52 was previously the only known mAb to bridge both attachment (GP1) and fusion (GP2) subunits of any viral GP (Lee et al., 2008
), the present findings identify another antibody, JP3K11, with similar properties.
Neutralization studies herein revealed that all mAbs examined but one, 6D8, were capable of neutralizing an EBOV GP-bearing pseudovirus in vitro
. The lack of neutralization by 6D8 was not predicted since studies in mice show protection by passive transfer (Wilson et al., 2000
). However, it is quite possible that the protection observed in this model may be influenced by the use of a serially-passaged virus adapted for infection of non-natural host species (adult mice are naturally resistant to EBOV infection), in which important biological differences in viral pathology and GP amino acid sequences between the mouse-adapted and the naïve viruses may affect the recapitulation of clinical severity (Bray et al., 1998
). Also, such protection could be mediated by an alternative mechanism distinct from interference with virus entry, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The lack of neutralization by MUC-directed 6D8 in this report is consistent with a model whereby the EBOV MUC expresses a protective role against antibody binding not unlike the “glycan shield” of HIV which protects the receptor binding domain from NAbs (Wei et al., 2003
), since the putative RBD of EBOV is recessed beneath a glycan cap (Lee et al., 2008
). This domain is completely dispensable for infection (Simmons et al., 2002
; Takada et al., 2004
) and is immunodominant for humoral responses during non-lethal EBOV infection since the majority of EBOV-specific Abs reported to date recognize continuous epitopes in this region (Wilson et al., 2000
), likely the result of MUC comprising the most exposed elements of virus-bound GP (Vanderzanden et al., 1998
). Thus, the value of such a domain that protects critical functional regions from humoral immunity seems evident.
Although KZ52 and JP3K11 both bound epitopes dependent on amino acids in GP1 and GP2, only JP3K11 was able to bind and neutralize cathepsin-processed GP-pseudoviruses. These results demonstrate that JP3K11 recognizes a critical neutralizing determinant that is differentially accessible in native and CatL-cleaved GP. The mechanism of JP3K11 neutralization is consistent with a model whereby JP3K11 binding, occurring either before or after endosomal compartmentalization, blocks triggering of a final, activated form of GP, which may require the participation of a cellular reductase (Schornberg et al., 2006
) or another cofactor(s), the fusion event itself, or downstream events required for virus entry. Moreover, data demonstrate that J3PK11 may also exhibit a capacity for CatL inhibition by virtue of its proximity to the GPCatL1-2
cleavage site, in addition to its primary mechanism of neutralization as described herein. However, since the mechanism of EBOV entry has yet to be fully characterized it is difficult to predict at what point during virus entry the JP3K11 neutralizing determinant is accessible to this antibody. To date, JP3K11 is the first mAb described to preferentially bind the Cat-L cleaved form of EBOV GP. Importantly, this characteristic was associated with a greater potency for neutralization, albeit at higher mAb concentrations, than other antibodies studied. Since KZ52 was not as potent as JP3K11 at higher concentrations in neutralizing virus in vitro
, antibody-mediated interference of CatL cleavage alone may not be as effective in neutralizing virus as a blocking mechanism acting on later events required for fusion, such as that observed by JP3K11. Additionally, CatL cleavage eventually progresses, despite KZ52 binding, following longer incubation periods, raising the possibility that “leakiness” by KZ52 could explain the observation of virus escape after passive transfer into macaques. While these data do not directly explain why passively transferred KZ52 alone failed to protect monkeys from lethal virus challenge (Oswald et al., 2007
), they do suggest a mechanism for KZ52 that may be suboptimal for potent in vivo
neutralization when compared to the multiple neutralization activities exhibited by JP3K11.
Since KZ52 did not bind CatL-processed GP nor inhibit CatL-cleaved viral pseudotypes, we hypothesize that proteolytic cleavage, which is critically required for viral entry (Chandran et al., 2005
), may act to disrupt the binding epitope. Ablation of KZ52 binding may possibly occur by the removal of contact residues or by disrupting GP conformation. Indeed, the presence of KZ52 was sufficient to inhibit formation of the critical 22-kD fragment required for viral entry, and a larger intermediary fragment containing the RBD was observed (56-kD fragment or intermediate GPCatL1
). Intermediates of cathepsin proteolysis of GP1 have been observed (Kaletsky, Simmons, and Bates, 2007
; Schornberg et al., 2006
), and data herein show that EBOV GP contains at least two separate CatL cleavage sites; the GPCatL1
cleavage site occurs before the MUC yielding the 22-kD fragment containing the RBD and the second CatL cleavage site, GPCatL0-1
, likely occurs somewhere in the MUC. Furthermore, due to the proximity of the KZ52 binding site to the presumptive RBD, the contribution of direct receptor antagonism by the Ab should not be discounted entirely. Thus, these data are consistent with a model of Ab-mediated neutralization whereby KZ52 interferes with cathepsin cleavage of GP by blocking enzyme attachment or activity, and hinders cathepsin-activation of the fusogenic form of GP containing the RBD or receptor binding directly.
In conclusion, we show here for the first time that two neutralizing Abs, KZ52 and JP3K11, inhibit viral transduction by two fundamentally different mechanisms. Neutralization by KZ52 exploits the EBOV requirement for endosomal proteolysis required for virus entry. On the contrary, JP3K11 expresses a classic mechanism of Ab-mediated neutralization by inhibiting triggering of fusogenic GP, fusion events, and/or receptor binding. In addition, this NAb also exhibits a moderate capacity for inhibiting CatL proteolysis of EBOV GP. Since there is currently no vaccine or treatment for EBOV disease, future vaccine strategies may be more effective in the provision of antibodies like JP3K11 that recognize post-cleavage GP. While it remains to be determined whether JP3K11 can provide protection in vivo, its evaluation in future studies will be informative.