View All Abstracts for Session 24
124 Production, Characterization, and Oligomeric Stabilization of Recombinant Disulfide Linked (SOS) HIV-1 Envelope Glycoproteins
N. Schülke*1, M. Vesanen2, R. Sanders2, A. Villa1, D. Anselma1, J. Binley3, P. Maddon1, J. Moore2, and W. Olson1
1Progenics Pharmaceuticals Inc., Tarrytown, NY, USA; 2Weill Med. Coll., Cornell Univ., New York, NY, USA; and 3The Scripps Res. Inst., La Jolla, CA, USA
Background: The native functional unit of the HIV-1 envelope glycoprotein (env) on the surface of infectious viral particles is a trimer of gp120-gp41 heterodimers. Native trimers represent attractive immunogens but are difficult to produce in recombinant form since the noncovalent interactions between gp120 and gp41 are weak, as are those between gp41 subunits in the prefusion conformation. We have stabilized the gp120-gp41 interaction by introducing a disulfide bond between gp120 and gp41, and we have demonstrated that the resulting molecule mimics virion-associated env.
Methods: In this study, we compared the antigenic and oligomeric properties of a series of SOS gp140 glycoproteins produced using transient and stable mammalian expression systems. A novel "Blue Native" gel electrophoresis method was used to rapidly explore the oligomeric properties of purified and unpurified SOS gp140s as a function of time and handling conditions. BIAcore, ELISA, and other methods were used to examine the reactivities of the SOS gp140s with neutralizing and nonneutralizing antibodies as well as with CD4 and coreceptor.
Results: Differences in oligomeric stability were observed between full-length and variable loop-deleted SOS gp140s and between SOS gp140s expressed transiently and stably in different cell types. In addition, temperature and storage conditions had profound effects on the oligomeric stability of a given SOS gp140, and the oligomeric state of env influenced its binding to antibodies and receptors.
Conclusions: SOS gp140 oligomers can be generated and preserved via appropriate utilizations of env modifications, expression systems, and protein handling conditions. These findings provide insights into the noncovalent forces that stabilize oligomeric env and suggest a path to producing purified recombinant SOS gp140 subunit vaccines that mimic the virion-associated structure.
Contact Author about this Abstract
