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16 Purification, Structural Characterization, and Immunogenicity of HIV-1 Envelope Oligomer from the Primary R5 Subtype B Strains
I. Srivastava*, A. Fong, E. Kan, H. Legg, S. Hilt, Y. Lian, K. Matsuoka, E. Calderon, M. Wininger, J. Ulmer, and S. Barnett
Chiron Corp., Emeryville, CA, USA
Considerable efforts to develop an effective vaccine for HIV have been directed toward the generation of cellular, humoral, and mucosal immune responses. For the induction of humoral responses, the focus has been on the HIV envelope glycoprotein with special consideration given to different structural forms and conformations. A major emphasis of our work has been toward the evaluation and comparison of secreted monomeric (gp120) and oligomeric (o-gp140) forms of the HIV-1 envelope proteins for their relative abilities to induce functional neutralizing antibody responses. We have derived stable CHO cell lines expressing high levels of gp120 and o-gp140 envelope proteins from the primary (NSI; R5) subtype B strain, HIV-1US4 and HIV-1SF162. These cell lines were adapted to low serum and grown in perfusion bioreactors for the high levels of protein expression. We have developed a very simple, effective, and efficient purification strategy to purify oligomers and monomers to near homogeneity. To address structural issues regarding the oligomer, we were able to demonstrate that the purified oligomers were trimers using a combination of 3 detectors measuring intrinsic viscosity, light scattering, and refractive index. The calculated molecular mass for the oligomer was 474 kD, close to the predicted molecular mass of a trimer (420 kD). The hydrodynamic radius (Rhw) of o-gp140 was determined to be 8.40 nm. To assess native conformation, we performed a detailed characterization of the glycosylation profile of ogp140 and its ability to bind CD4. First, NANase followed by O-glycosidase and PNGF digestions indicate ogp140 contained predominantly N-linked oligosaccharides. Based on carbohydrate profiling and sequencing analyses, it had 2 kinds of N-linked oligosaccharides: a terminal mannose type and a complex carbohydrate type. These observations are similar to those made by us and by others for gp120. Second, purified o-gp140 protein readily bound soluble CD4. Furthermore, purified oligomer markedly enhanced the induction of neutralizing antibody responses in rabbits and primates suggesting its potential as an immunogen.
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