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Journal of Virology, February 2007, p. 1821-1837, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.02098-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Influences of Glycosylation on Antigenicity, Immunogenicity, and Protective Efficacy of Ebola Virus GP DNA Vaccines{triangledown}

William Dowling ,1,{dagger},{ddagger} Elizabeth Thompson,1,{dagger},§ Catherine Badger,1,{dagger} Jenny L. Mellquist,1,|| Aura R. Garrison,1 Jeffery M. Smith,1 Jason Paragas,1 Robert J. Hogan,2 and Connie Schmaljohn1*

United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland,1 University of Georgia, Athens, Georgia2

Received 25 September 2006/ Accepted 22 November 2006

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.

* Corresponding author. Mailing address: U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, MD 21702. Phone: (301) 619-4103. Fax: (301) 619-2439. E-mail: connie.Schmaljohn{at}amedd.army.mil.

{triangledown} Published ahead of print on 6 December 2006.

{dagger} W.D., E.T., and C.B. contributed equally to this study.

{ddagger} Present address: Chemical and Biological Defense Directorate, Defense Threat Reduction Agency, 8725 John J. Kingman Rd., Ft. Belvoir, VA 22060.

§ Present address: CDER, FDA, 10903 New Hampshire Ave., Silver Spring, MD 20993.

|| Present address: Division of Hematology, CBER, FDA, 29 Lincoln Dr., Bethesda, MD 20892.

Journal of Virology, February 2007, p. 1821-1837, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.02098-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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