Influences of glycosylation on antigenicity, immunogenicity, and protective efficacy of ebola virus GP DNA vaccines.
The Ebola virus (EBOV) envelope glycoprotein (GP) is the primary target of protective immunity. Mature GP consists of two disulfide-linked subunits, GP1 and membrane-bound GP2. GP is highly glycosylated with both N- and O-linked carbohydrates. We measured the influences of GP glycosylation on antigenicity, immunogenicity, and protection by testing DNA vaccines comprised of GP genes with deleted N-linked glycosylation sites or with deletions in the central hypervariable mucin region. We showed that mutation of one of the two N-linked GP2 glycosylation sites was highly detrimental to the antigenicity and immunogenicity of GP. Our data indicate that this is likely due to the inability of GP2 and GP1 to dimerize at the cell surface and suggest that glycosylation at this site is required for achieving the conformational integrity of GP2 and GP1. In contrast, mutation of two N-linked sites on GP1, which flank previously defined protective antibody epitopes on GP, may enhance immunogenicity, possibly by unmasking epitopes. We further showed that although deleting the mucin region apparently had no effect on antigenicity in vitro, it negatively impacted the elicitation of protective immunity in mice. In addition, we confirmed the presence of previously identified B-cell and T-cell epitopes in GP but show that when analyzed individually none of them were neither absolutely required nor sufficient for protective immunity to EBOV. Finally, we identified other potential regions of GP that may contain relevant antibody or T-cell epitopes.
Dowling
William
W
U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, MD 21702, USA.
Thompson
Elizabeth
E
Badger
Catherine
C
Mellquist
Jenny L
JL
Garrison
Aura R
AR
Smith
Jeffery M
JM
Paragas
Jason
J
Hogan
Robert J
RJ
Schmaljohn
Connie
C
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D046129:Ebola Vaccines / Q000008:administration & dosage*; D029043:Ebolavirus / Q000235:genetics / Q000276:immunology*; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005260:Female; D017353:Gene Deletion; D006031:Glycosylation; D019142:Hemorrhagic Fever, Ebola / Q000276:immunology* / Q000517:prevention & control*; D007277:Injections, Jet; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009077:Mucins / Q000378:metabolism; D009500:Neutralization Tests; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology; D017354:Point Mutation; D016415:Sequence Alignment; D013045:Species Specificity; D014611:Vaccination; D019444:Vaccines, DNA / Q000008:administration & dosage; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D046129:Ebola Vaccines; D018985:Epitopes, B-Lymphocyte; D018984:Epitopes, T-Lymphocyte; D009077:Mucins; D010455:Peptides; D019444:Vaccines, DNA; D014759:Viral Envelope Proteins; C113189:envelope glycoprotein, Ebola virus