Comprehensive Functional Analysis of N-Linked Glycans on Ebola Virus GP1

Lennemann, Nicholas J.; Rhein, Bethany A.; Ndungo, Esther; Chandran, Kartik; Qiu, Xiangguo; Maury, Wendy

Removal of N-linked glycans enhances antiserum sensitivity. Our studies indicated that the most deglycosylated EBOV GP1 mutants were expressed at WT levels and provided the highest levels of transduction into Vero cells and peritoneal macrophages. Nonetheless, the conservation of the glycan sites, particularly in the GP1 core sequences, across the species suggested that positive selection for these sites was occurring. Therefore, our deglycosylated EBOV GPs were evaluated for their sensitivity to antiserum neutralization, since deglycosylation may increase the exposure of potential EBOV GP epitopes. Purified anti-EBOV IgG from convalescent cynomolgus macaques that were vaccinated prior to challenge with Ebola virus (a gift from John M. Dye, USAMRIID) was incubated with VSV pseudovirions that were normalized for the amount of matrix protein. Little to no enhancement of antiserum sensitivity was observed with a mutant lacking three glycans in the GP core; however, the removal of six glycans from the glycan cap increased the neutralization sensitivity more than 4-fold ( Fig. 6A and C) . In contrast, VSV pseudotyped with the Marburg virus GP was not neutralized by anti-EBOV IgG. Complete deglycosylation of the GP1 core (7G) led to a further increase in antiserum sensitivity ( Fig. 6B and C) . Since the MLD is a major target of neutralizing antibodies (7-9), we anticipated that the removal of N-glycans from this domain would enhance anti-EBOV IgG sensitivity. Surprisingly, GPm8G was no more sensitive to neutralization than WT GP (Fig. 6B) , indicating that NGS in the MLD had little to no positive or negative effect on neutralization by the pooled anti-EBOV IgG. Consistent with this, removal of all N-linked glycans on GP1 (7Gm8G) did not enhance antibody neutralization beyond that observed with deglycosylation of GP1 core (7G). We found similar but less pronounced neutralization sensitivity with pooled sera collected from mice surviving challenge with mouse-adapted EBOV (see Fig. S8 in the supplemental material).