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Journal of Virology, March 2002, p. 2835-2847, Vol. 76, No. 6
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.6.2835-2847.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of Oligomeric Envelope Glycoprotein from a Primary R5 Subtype B Human Immunodeficiency Virus

Indresh K. Srivastava,1* Leonidas Stamatatos,2,{dagger} Harold Legg,1 Elaine Kan,1 Anne Fong,1 Stephen R. Coates,1 Louisa Leung,1 Mark Wininger,1 John J. Donnelly,1 Jeffrey B. Ulmer,1 and Susan W. Barnett1*

Department of Immunology and Infectious Diseases, Chiron Corporation, Emeryville, California 94608,1 Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York 10021-63992

Received 9 May 2001/ Accepted 13 December 2001

Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1US4. We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (Rh) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smaller Rh of the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.

* Corresponding author. Mailing address: Immunology and Infectious Diseases, Chiron Corporation, m/s 4.3, 4560 Horton St., Emeryville, CA 94608. Phone: (510) 923-5485 (for Indresh K. Srivastava) or (510) 923-7565 (for Susan W. Barnett). Fax: (510) 923-2586. E-mail: Indresh_Srivastava{at}chiron.com or Susan_Barnett{at}chiron.com.

{dagger} Present address: Seattle Biomedical Research Institute, Seattle, Wash.

Journal of Virology, March 2002, p. 2835-2847, Vol. 76, No. 6
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.6.2835-2847.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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