title: Purification and characterization of oligomeric envelope glycoprotein from a primary R5 subtype B human immunodeficiency virus.
abstract: 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-1(US4). 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 (R(h)) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smaller R(h) 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.
authors.affiliation: Department of Immunology and Infectious Diseases, Chiron Corporation, Emeryville, California 94608, USA. Indresh_Srivastava@chiron.com
authors.forename: Indresh K
authors.initials: IK
authors.lastname: Srivastava
authors.forename: Leonidas
authors.initials: L
authors.lastname: Stamatatos
authors.forename: Harold
authors.initials: H
authors.lastname: Legg
authors.forename: Elaine
authors.initials: E
authors.lastname: Kan
authors.forename: Anne
authors.initials: A
authors.lastname: Fong
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Coates
authors.forename: Louisa
authors.initials: L
authors.lastname: Leung
authors.forename: Mark
authors.initials: M
authors.lastname: Wininger
authors.forename: John J
authors.initials: JJ
authors.lastname: Donnelly
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Ulmer
authors.forename: Susan W
authors.initials: SW
authors.lastname: Barnett
mesh_terms: D016915:AIDS Vaccines / Q000008:administration & dosage / Q000276:immunology; D000818:Animals; D000915:Antibody Affinity; D015704:CD4 Antigens / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D019281:Dimerization; D015686:Gene Products, env / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D015483:HIV Antibodies / Q000097:blood*; D015497:HIV-1 / Q000145:classification* / Q000276:immunology; D006801:Humans; D007114:Immunization; D009500:Neutralization Tests; D009844:Oligosaccharides / Q000032:analysis; D011487:Protein Conformation; D011817:Rabbits
chemical_list: D016915:AIDS Vaccines; D015704:CD4 Antigens; D015686:Gene Products, env; D015483:HIV Antibodies; D009844:Oligosaccharides