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Journal of Virology, May 2009, p. 5077-5086, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02600-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Enhanced Exposure of the CD4-Binding Site to Neutralizing Antibodies by Structural Design of a Membrane-Anchored Human Immunodeficiency Virus Type 1 gp120 Domain{triangledown}

Lan Wu,1 Tongqing Zhou,1 Zhi-yong Yang,1 Krisha Svehla,1 Sijy O'Dell,1 Mark K. Louder,1 Ling Xu,1 John R. Mascola,1 Dennis R. Burton,2 James A. Hoxie,3 Robert W. Doms,4 Peter D. Kwong,1 and Gary J. Nabel1*

Vaccine Research Center, NIAID, National Institutes of Health, Bldg. 40, 40 Convent Drive, Bethesda, Maryland 20892-3005,1 Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, IMM-2, La Jolla, California 92037,2 Department of Medicine, University of Pennsylvania, 356 Biomedical Research Bldg. II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104,3 Department of Microbiology, University of Pennsylvania, Room 225 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, Pennsylvania 19104-60764

Received 16 December 2008/ Accepted 24 February 2009

The broadly neutralizing antibody immunoglobulin G1 (IgG1) b12 binds to a conformationally conserved surface on the outer domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) glycoprotein. To develop outer domain proteins (ODs) that could be recognized selectively by CD4-binding-site (CD4-BS) antibodies, membrane-anchored ODs were generated from an HIV-1 clade B virus, TA1 R3A, which was highly sensitive to neutralization by the IgG1 b12 antibody. A 231-residue fragment of gp120 (residues 252 to 482) linked to transmembrane regions from CD4 showed b12 binding comparable to that of the native Env spike as measured by flow cytometry. Truncation of the β20-β21 hairpin (residues 422 to 436 to Gly-Gly) improved overall protein expression. Replacement of the immunodominant central 20 amino acids of the V3 loop (residues 302 to 323) with a basic hexapeptide (NTRGRR) increased b12 reactivity further. Surface calculations indicated that the ratio of b12 epitope to exposed immunogenic surface in the optimized OD increased to over 30%. This OD variant [OD(GSL)({Delta}β20-21)(hCD4-TM)] was recognized by b12 and another CD4-BS-reactive antibody, b13, but not by eight other CD4-BS antibodies with limited neutralization potency. Furthermore, optimized membrane-anchored OD selectively absorbed neutralizing activity from complex antisera and b12. Structurally designed membrane-anchored ODs represent candidate immunogens to elicit or to allow the detection of broadly neutralizing antibodies to the conserved site of CD4 binding on HIV-1 gp120.

* Corresponding author. Mailing address: Vaccine Research Center, NIAID, National Institutes of Health, Bldg. 40, Room 4502, MSC-3005, 40 Convent Drive, Bethesda, MD 20892-3005. Phone: (301) 496-1852. Fax: (301) 480-0274. E-mail: gnabel{at}nih.gov

{triangledown} Published ahead of print on 4 March 2009.

Journal of Virology, May 2009, p. 5077-5086, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02600-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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