Expression and characterisation of recombinant oligomeric envelope glycoproteins derived from primary isolates of HIV-1.
The production, purification and characterisation of recombinant gp140 oligomeric envelope glycoproteins derived from six primary isolates of HIV-1 (covering clades A, B, C, D, F and O) are described. Using a Chinese hamster ovary cell expression system, expression levels of between 0.1 and 1 mg/l cell-conditioned culture media were obtained, and purified to >95% by affinity chromatography. A, B, D, F and O clade gp 140s were found to be multimeric, bind to a panel of defined env-specific monoclonal antibodies and interact with CD4 and CXCR4, demonstrating correct folding. Their immunogenicity was confirmed by the generation of high-titre anti-gp140 antibodies in rabbits. The C clade gp140 was incorrectly folded and poorly antigenic. Despite the presence of an unmodified gp120/41 cleavage site, only the B clade gp140 showed significant processing to gp120 and gp41. Each gp140 has a specific pattern of oligomerisation, and varies in its resistance to reducing agents and salt concentration. The binding of gp140 to soluble and cell-surface CD4 and CXCR4 is related to the degree of oligomerisation. The C1 and C5 regions, CD4 binding domain and the epitope defined by the 2G12 monoclonal antibody were well exposed, but the C-terminal region of the extracellular domain of gp41 appears to be occluded by oligomerisation. These reagents have potential as immunogens for use in vaccine development.
Jeffs
S A
SA
Division of Retrovirology, NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK. simon.jeffs@imperial.ac.uk
Goriup
S
S
Kebble
B
B
Crane
D
D
Bolgiano
B
B
Sattentau
Q
Q
Jones
S
S
Holmes
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015686:Gene Products, env / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D015488:HIV Antigens / Q000737:chemistry / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D015497:HIV-1 / Q000302:isolation & purification; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D021122:Protein Subunits; D011817:Rabbits; D019718:Receptors, CXCR4 / Q000276:immunology / Q000378:metabolism; D054299:env Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D015686:Gene Products, env; D015488:HIV Antigens; D015699:HIV Envelope Protein gp120; D007106:Immune Sera; D021122:Protein Subunits; D019718:Receptors, CXCR4; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1