Characterization and Enhanced Processing of Soluble, Oligomeric GP140 Envelope Glycoproteins Derived from Human Immunodeficiency Virus Type-1 Primary Isolates
Abstract
HIV-1 first interacts with host cells through its envelope glycoprotein (Env), the major target of neutralizing antibodies. The biologically relevant form of Env is an oligomer, both in its presentation to the immune system and in virus entry. Also, native Env is cleaved into its gp120 and gp41 non-covalently associated subunits. Env-based vaccines have thus far failed to efficiently generate broadly cross-reactive, neutralizing antibodies towards primary isolates. Possible explanations for their failure are either they were derived from laboratory-adapted strains or they have not preserved the conformational structures necessary to elicit broadly reactive responses. Producing a broadly effective Env-based vaccine is also potentially complicated by the existence of multiple HIV-1 genotypes. Here, I have constructed a panel of truncated env genes from primary isolates of several different HIV-1 clades. Recombinant vaccinia viruses expressing these genes produce a secreted Env known as gp140. These gp140s were characterized by sucrose density gradient centrifugation and size exclusion chromatography analyses to determine oligomeric status and degree of processing. While most processed gp140s dissociated to monomeric forms, there was evidence that certain isolates could retain gp120 in an oligomer. A large scale purification scheme was developed using lentil lectin affinity and size exclusion chromatographies to prepare gp140 oligomers. Pre- and post-production processing enhancers (furin and plasmin) were examined as means to bolster the amount of processed gp140, and reducible crosslinkers were employed to analyze the ability of processed gp140 to maintain oligomeric forms. The antigenic properties of the gp140s, both cleaved vs. uncleaved, and crosslinked vs. non-crosslinked, were analyzed by immunoprecipitation with a panel of well-characterized human and mouse antibodies.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2001
- Accession Number
- ADA421053
Entities
People
- Agnes Y. Jones-trower
Organizations
- Uniformed Services University of the Health Sciences