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Unformatted text preview: Ebola Virus, Neutrophils, and Antibody Specificity Z hi-yong Yang et al . ( 1 ) report that the secret- ed form of the Ebola virus glycoprotein (sGP) specifically interacts with neutrophils through the immunoglobulin G (IgG) Fc receptor IIIb (Fc g RIIIb, CD16), which is exclusively ex- pressed on neutrophils. Yang et al . conclude that sGP inhibits early neutrophil activation, which likely affects the host response to [Ebola] infection. The study raised considerable inter- est because it provided at least a partial expla- nation for the high pathogenicity of Ebola virus ( 2, 3 ). The study, however, did not exclude the possibility that the rabbit antibody used to de- tect the binding of Ebola sGP mediated the binding of rabbit IgG-sGP immune complexes to neutrophil Fc g RIIIb through its Fc portion. To clarify this issue, we performed flow cy- tometry to study the putative interaction of sGP with neutrophils with the use of three human monoclonal antibody Fab fragments specific for sGP (Fab KS14, K518, and LS4) ( 4, 5 ) and rabbit IgG F(ab 9 ) 2 fragments to sGP ( 6 ). First, we confirmed that we could detect the binding of sGP-rabbit antiserum immune complexes with neutrophils in flow cytom- etry with the use of a fluorescein isothiocya- nate (FITC)labeled goat antibody to rabbit IgG, as previously shown by Yang et al . ( 7 ). In contrast with this result, however, we could not detect binding of sGP to neutro- phils with the use of three human monoclonal Fab fragments under similar conditions (Fab LS4 shown in Fig. 1A; Fab KS14 and K518 yielded similar results). It is unlikely that this result is due to overlapping binding sites of the Fab fragments and the putative receptor binding site on sGP, because strong binding of all three human Fabs to neutrophil-bound sGP was found in the presence of the rabbit antibody to sGP (Fig. 1B). These data suggest that the binding of Ebola sGP to neutrophils may be mediated by the rabbit IgG used to detect the sGP, rather than by a specific sGP-neutrophil interaction. To investigate whether the rabbit antibody dependent binding of Ebola sGP was mediat- ed by the Fc moiety of the rabbit IgG, we prepared F(ab 9 ) 2 fragments. Rabbit F(ab 9 ) 2 re- tained the same reactivity against sGP as did the whole rabbit IgG to sGP, as measured by en- zyme-linked immunosorbent assay (ELISA) (Fig. 2). The rabbit IgG tested alone gave a weak signal in flow cytometry, which was in- creased significantly in the presence of sGP (Fig. 1C). In contrast, rabbit IgG F(ab 9 ) 2 frag- ments to sGP did not bind to neutrophils either in the absence or presence of sGP (Fig. 1D). Finally, in contrast, to Yang et al ., we did not detect any absorption of sGP by purified neutrophils (Fig. 3), which makes it difficult to predict the presence of any low affinity receptor for sGP on neutrophils....
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