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Journal of Virology, October 2003, p. 11244-11259, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11244-11259.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Purification, Characterization, and Immunogenicity of a Soluble Trimeric Envelope Protein Containing a Partial Deletion of the V2 Loop Derived from SF162, an R5-Tropic Human Immunodeficiency Virus Type 1 Isolate

Indresh K. Srivastava,1* Leonidas Stamatatos,2 Elaine Kan,1 Michael Vajdy,1 Ying Lian,1 Susan Hilt,1 Loic Martin,3 Claudio Vita,3 Ping Zhu,4 Kenneth H. Roux,4 Lucia Vojtech,2 David C. Montefiori,5 John Donnelly,1 Jeffrey B. Ulmer,1 and Susan W. Barnett1*

Vaccines Research, Chiron Corporation, Emeryville, California 94608,1 Department of Pathobiology, University of Washington and Seattle Biomedical Research Institute, Seattle, Washington 98103,2 Department of Protein Engineering and Research, CEA Saclay, Gif-sur-Yvette, France,3 Department of Biological Science and Structural Biology Program, Florida State University, Tallahassee, Florida 32306,4 Department of Surgery, Duke University Medical Center, Durham, North Carolina 277105

Received 21 March 2003/ Accepted 14 July 2003

The envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the major target of neutralizing antibody responses and is likely to be a critical component of an effective vaccine against AIDS. Although monomeric HIV envelope subunit vaccines (gp120) have induced high-titer antibody responses and neutralizing antibodies against laboratory-adapted HIV-1 strains, they have failed to induce neutralizing antibodies against diverse heterologous primary HIV isolates. Most probably, the reason for this failure is that the antigenic structure(s) of these previously used immunogens does not mimic that of the functional HIV envelope, which is a trimer, and thus these immunogens do not elicit high titers of relevant functional antibodies. We recently reported that an Env glycoprotein immunogen (o-gp140SF162{Delta}V2) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162, when used in a DNA priming-protein boosting vaccine regimen in rhesus macaques, induced neutralizing antibodies against heterologous subtype B primary isolates as well as protection to the vaccinated animals upon challenge with pathogenic SHIVSF162P4 virus. Here we describe the purification of this protein to homogeneity, its characterization as trimer, and its ability to induce primary isolate-neutralizing responses in rhesus macaques. Optimal mutations in the primary and secondary protease cleavage sites of the env gene were identified that resulted in the stable secretion of a trimeric Env glycoprotein in mammalian cell cultures. We determined the molecular mass and hydrodynamic radius (Rh) using a triple detector analysis (TDA) system. The molecular mass of the oligomer was found to be 324 kDa, close to the expected Mw of a HIV envelope trimer protein (330 kDa), and the hydrodynamic radius was 7.27 nm. Negative staining electron microscopy of o-gp140SF162{Delta}V2 showed that it is a trimer with considerable structural flexibility and supported the data obtained by TDA. The structural integrity of the purified trimeric protein was also confirmed by determinations of its ability to bind the HIV receptor, CD4, and its ability to bind a panel of well-characterized neutralizing monoclonal antibodies. No deleterious effect of V2 loop deletion was observed on the structure and conformation of the protein, and several critical neutralization epitopes were preserved and well exposed on the purified o-gp140SF162{Delta}V2 protein. In an intranasal priming and intramuscular boosting regimen, this protein induced high titers of functional antibodies, which neutralized the vaccine strain, i.e., SF162. These results highlight a potential role for the trimeric o-gp140SF162{Delta}V2 Env immunogen in a successful HIV vaccine.

* Corresponding author. Mailing address: Vaccines Research, Chiron Corporation, 4560 Horton St., Emeryville, CA 94608. Phone for Indresh K. Srivastava: (510) 923-5485. Phone for Susan W. Barnett: (510) 923-7565. Fax: (510) 923 2586. E-mail for Indresh K. Srivastava: Indresh_Srivastava{at}Chiron.com. E-mail for Susan W. Barnett: Susan_Barnett{at}Chiron.com.

Journal of Virology, October 2003, p. 11244-11259, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11244-11259.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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