Epitopes Involved in Antibody-Mediated Protection from Ebola Virus

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Science  03 Mar 2000:
Vol. 287, Issue 5458, pp. 1664-1666
DOI: 10.1126/science.287.5458.1664


To determine the ability of antibodies to provide protection from Ebola viruses, monoclonal antibodies (mAbs) to the Ebola glycoprotein were generated and evaluated for efficacy. We identified several protective mAbs directed toward five unique epitopes on Ebola glycoprotein. One of the epitopes is conserved among all Ebola viruses that are known to be pathogenic for humans. Some protective mAbs were also effective therapeutically when administered to mice 2 days after exposure to lethal Ebola virus. The identification of protective mAbs has important implications for developing vaccines and therapies for Ebola virus.

Ebola viruses cause acute, lethal hemorrhagic fevers for which no vaccines or treatments currently exist. Knowledge about the immune mechanisms mediating protection is limited. The membrane-anchored glycoprotein (GP) is the only viral protein known to be on the surfaces of virions and infected cells and is presumed to be responsible for receptor binding and fusion of the virus with host cells. As a result, Ebola GP may be an important target of protective antibodies. However, the contribution of antibodies to Ebola GP in disease resistance is unclear. Negligible serum titers of neutralizing antibodies in convalescent patients, together with inconsistent results in achieving protection through experimental transfers of immune sera to animals (1, 2), have led to suggestions that antibodies to Ebola GP cannot confer protection to Ebola virus (3).

The role of antibodies to GP in protection is further confounded by the observation that Ebola GP occurs in several forms. Transcriptional editing of the GP mRNA is required for production of the virion-associated GP (4, 5). Proteolytic processing of GP results in two products, GP1 and membrane-bound GP2, that covalently associate to form a monomer of the GP spike found on the surfaces of virions (6). GP1 is also released from infected cells in a soluble form (7). The unedited GP mRNA encodes a secreted glycoprotein (sGP) that is synthesized in abundance early in infection (4, 5, 8). sGP and GP1 are identical in their first 295 NH2-terminal amino acids, whereas the remaining COOH-terminal 69 amino acids of sGP and 206 amino acids of GP1 are encoded by different reading frames. It has been suggested that secreted GP1 or sGP may effectively bind antibodies that might otherwise be protective (5,7).

This study identified protective GP-specific mAbs (9–11) that were classified into five groups on the basis of competitive binding assays (12–14). Individual mAbs in these five groups were protective against Ebola challenge when administered prophylactically or therapeutically (Table 1). Three of the epitopes bound by protective mAbs are linear sequences on GP1, whereas the other two are conformational epitopes shared between GP1 and sGP (Table 2). Ten out of 14 mAbs identified in these five competition groups protected BALB/c mice from a lethal challenge with mouse-adapted Ebola Zaire virus when 100 μg of purified mAb (15) was administered 24 hours before challenge (Table 1). Similar results were observed in a second mouse strain (C57BL/6, Table 1). Protection from Ebola challenge decreased when the mAb dose was lowered to 50 or 25 μg (Table 1). For the most effective mAbs, the amount required for protection was within an acceptable human therapeutic dose of 3 to 5 mg/kg.

Table 1

Protective efficacy of Ebola GP mAbs.

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Table 2

Epitopes bound by Ebola GP mAbs.

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Some of the mAbs were effective even when administered up to 2 days after challenge (Table 1), after substantial viral replication had occurred (16). None of the mAbs were protective when 100 μg was administered 3 days after challenge, when there are high viral titers (16) and possibly irreversible damage of cells and organs.

The ability of the mAbs to inhibit plaque formation by Ebola virus (17, 18), a standard assay of virus neutralization, did not always predict their protective efficacy. None of the protective mAbs inhibited plaque formation in the absence of complement. In the presence of complement, only mAbs in competition groups 2 and 4 neutralized the virus (80% at 6.25 μg/ml). Monoclonal antibody 12B5-1-1 (group 3) did not reduce the number of plaques, but did reduce plaque size, suggesting that it restricted subsequent infection of adjacent cells.

When the mAbs were tested for reactivity with the Ebola viruses that are human pathogens, mAbs in groups 1, 2, and 3 bound to the two Zaire isolates that have caused the most devastating outbreaks, but did not bind to the Ivory Coast or Sudan viruses (Table 2). Monoclonal antibodies in these three groups immunoprecipitated GP, but not sGP, from supernatants of cell cultures infected with either Ebola Zaire virus or Ebola GP replicons (Fig. 1) and reacted only with GP1 in Western blots (19). The sequences bound by these mAbs were identified by means of synthetic peptides immobilized on membranes and were confirmed with soluble peptides in competition enzyme-linked immunosorbent assays (ELISAs) (Table 2). These protective mAbs bound linear epitopes within a region of 106 amino acids in the COOH-terminal portion of GP1. This region is poorly conserved among Ebola viruses and is not shared with sGP. The epitopes bound by mAbs in groups 1 and 2 are separated by only three amino acids (Table 2).

Figure 1

Immunoprecipitation (14) of35S-labeled Ebola GPs from supernatants of Vero cells infected with (A) Ebola GP replicons or (B) Ebola Zaire 1995 virus, with either the group 1 mAb 13F6-1-2 (lane 1) or the group 4 mAb 13C6-1-1 (lane 2). Both preparations contained secreted GP1 and sGP. Disulfide-linked GP1 and GP2constitute the spikes on the virions that are also present in the Ebola-infected preparation (B). The immunoprecipitation of GPs with 13F6-1-2 was identical to that observed with mAbs in groups 1, 2, and 3. Monoclonal antibodies in groups 4 and 5 had reactivities identical to that of mAb 13C6-1-1. GP proteins were resolved under reducing conditions on an 11% SDS-polyacrylamide gel.

In contrast, mAbs in groups 4 and 5 immunoprecipitated both GP and sGP from supernatants of infected cells (Fig. 1) but did not bind GP on Western blots under reducing conditions (19). These epitopes are therefore discontinuous or require a specific conformation for binding and are located within the NH2-terminal 295 amino acids that are identical between sGP and GP1. Monoclonal antibodies in group 4 effectively blocked the binding of mAbs in group 5 (12), but reciprocal competition was observed only at high concentrations of unlabeled group 5 mAbs (19). All of the mAbs in groups 4 and 5 bound to the Ebola Zaire and Ivory Coast viruses. Furthermore, mAbs in group 4, but not group 5, also bound to Ebola Sudan (Table 2).

These results suggest that it is possible to elicit by vaccination, or produce for therapeutic use, antibodies protective against all Ebola viruses that are pathogenic for humans. Moreover, the idea that an antibody's reactivity with both sGP and GP would render it ineffectual in protection (5, 7) is not supported.

Nonprotective mAbs were identified that bound competitively with protective mAbs in groups 1, 4, and 5 (Table 1). All of the antibodies that were completely protective were of the immunoglobulin G2a (IgG2a) subclasses, whereas the competing nonprotective mAbs in groups 1 and 4 were of the IgG1 or IgG3 subclass. Furthermore, the group 3 mAb (12B5-1-1), which was only partially protective, was IgG1. Thus, antibody subclass may be an important factor in protection. Murine IgG2a binds complement more effectively than IgG1 or IgG3 and varies in its affinity for different Fc receptors (20). The subclass of the antibody may therefore affect the ability of the mAbs to resolve Ebola infections either by lysing infected cells through the classical complement pathway or by binding Fc receptors on cellular effectors of antibody-dependent cell-mediated cytotoxicity.

Alternatively, the affinity of an antibody for its epitope, possibly influenced by posttranslational modifications such as glycosylation, may be an important determinant of protective efficacy. For instance, although group 5 consisted of three IgG2a mAbs, only 6D3-1-1 (Table 1) was protective. This mAb bound to Ebola virus at 10-fold lower concentrations than the two nonprotective mAbs (13, 19). In addition, the protective mAb in group 1 was more effective than the nonprotective mAb in competition assays (19), suggesting that protective mAbs may have higher affinities for the epitope than nonprotective mAbs.

Monoclonal antibodies to Ebola GP protect immunocompetent animals from lethal Ebola challenge, demonstrating that antibodies are a feasible option for the design of safe and standardized treatments for Ebola infections. However, antibody specificity and the ability to neutralize the Ebola virus in vitro cannot be used as sole predictors of protective efficacy. Protection may depend on the proper specificity, isotype, and/or affinity of the antibody. These observations may explain conflicting data and interpretations regarding the role of antibodies in protection from Ebola virus. Furthermore, the induction of antibodies should not be overlooked when designing vaccines and therapies for Ebola virus.

  • * To whom correspondence should be addressed. E-mail: marykate.hart{at}


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