THE GENERATION AND CHARACTERIZATION OF GLYCOSYLATION MUTANTS OF THE EBOLA VIRUS GLYCOPROTEIN (GP)

Abstract

The protective immunity needed to prevent Ebola virus (EBOV) infection has not been completely elucidated. The only surface glycoprotein of EBOV, GP, is believed to be both a target of protective antibody responses and the attachment protein necessary for infection. Currently it is thought that both strong cellular and humoral immune responses are required to afford protection. We have generated a series of N-linked glycosylation (NLG) and O-linked glycosylation (OLG) mutants of the EBOV GP to investigate whether glycosylation in this immunodominant protein is critical for expression and/or immunogenicity of GP. NLG mutants of EBOV GP (strain Zaire) were generated by overlapping extension PCR. Individual NLG sites were deleted in either the GP₁ portion of GP, the GP2 portion of GP, or both by changing the consensus gene sequence of asparagine-X-serine/threonine such that alanine was in the third position. OLG mutants were made by serial deletions in the C-terminal mucin-like domain of GP₁ by overlapping extension PCR. These mutated GP genes were then cloned into an expression plasmid under the control of a CMV promoter and assayed for gene expression. All of the GP NLG and OLG mutants expressed in transiently transfected cells. Changes in molecular mass of the mutated GP could be seen by SDS-PAGE analysis indicating changes in glycosylation. Immunoprecipitation analysis with a panel of monoclonal antibodies (mAbs) revealed that both the NLG and OLG mutants maintained antigenic determinants recognized by the mAbs. Groups of 10 mice were vaccinated three times with DNA encoding the various glycosylation mutants at 3-week intervals using a gene gun. ELISA titers against purified, irradiated EBOV were measured after each vaccination, as were pre- and post-challenge neutralizing antibody responses. Vaccinated and control mice were challenged with a mouse-adapted strain of EBOV and observed for 28 days. Varying ELISA titers and neutralizing antibody titers were observed in mice vaccinated with the DNA constructs. The absence of one of the NLG sites in the GP2 portion of GP resulted in decreased antibody responses and reduced protection after challenge with EBOV. Overall, the OLG mutants did not induce as high of antibody responses as the NLG mutants and were less protective. This study indicates that glycosylation may play an important role in the design of vaccines and for EBOV infection.