Variable-Loop-Deleted Variants of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Can Be Stabilized by an Intermolecular Disulfide Bond between the gp120 and gp41 Subunits

Abstract

We have described an oligomeric gp140 envelope glycoprotein from human immunodeficiency virus type 1 that is stabilized by an intermolecular disulfide bond between gp120 and the gp41 ectodomain . In this protein, the protease cleavage site between gp120 and gp41 is fully utilized. Here we report the characterization of gp140 variants that have deletions in the first, second, and/or third variable loop (V1, V2, and V3 loops). The SOS disulfide bond formed efficiently in gp140s containing a single loop deletion or a combination deletion of the V1 and V2 loops. However, deletion of all three variable loops prevented formation of the SOS disulfide bond. Some variableloop-deleted gp140s were not fully processed to their gp120 and gp41 constituents even when the furin protease was cotransfected. The exposure of the gp120-gp41 cleavage site is probably affected in these proteins, even though the disabling change is in a region of gp120 distal from the cleavage site. Antigenic characterization of the variable-loop-deleted SOS gp140 proteins revealed that deletion of the variable loops uncovers cryptic, conserved neutralization epitopes near the coreceptor-binding site on gp120. These modified, disulfide-stabilized glycoproteins might be useful as immunogens.

Oligomeric and Conformational Properties of a Proteolytically Mature, Disulfide-Stabilized Human Immunodeficiency Virus Type 1 gp140 Envelope Glycoprotein

“…The pPPI4 eukaryotic expression vectors encoding SOS and uncleaved forms of HIV-1 JR-FL gp140 have been described previously (4,72). The SOS gp140 protein contains cysteine substitutions at residues A501 in the C5 region of gp120 and T605 in gp41 (4,57). In gp140 UNC , the sequence KRRV-VQREKRAV at the junction between gp120 and gp41 ECTO has been replaced with a hexameric Leu-Arg motif to prevent scission of gp140 into gp120 and gp41 ECTO (4).…”

“…In gp140 UNC , the sequence KRRV-VQREKRAV at the junction between gp120 and gp41 ECTO has been replaced with a hexameric Leu-Arg motif to prevent scission of gp140 into gp120 and gp41 ECTO (4). Plasmids encoding variable-loop-deleted forms of HIV-1 JR-FL SOS gp140 have been described (57). In these constructs, the tripeptide GAG is used to replace V1 loop sequences (D133-K155) and V2 loop sequences (F159-I194), alone or in combination.…”

“…We have taken an alternative approach to the problem of gp120-gp41 instability, which is to retain the cleavage site but to introduce a disulfide bond between the gp120 and gp41 ECTO subunits (4,57). Properly positioned, this intermolecular disulfide bond forms efficiently during envelope glycoprotein (Env) synthesis, allowing the secretion of gp140 proteins that are proteolytically processed but in which the association between the gp120 and gp41 ECTO subunits is maintained by the disulfide bond.…”

Role of Neutralizing Antibodies in Protective Immunity Against HIV

“…Several approaches have been tried with reasonable success. 88,[155][156][157][158][159][160][161][162][163][164][165][166] Yang and colleagues used GCN4-stabilized oligomers to demonstrate that antibodies induced by oligomeric gp140 were more effective at neutralizing heterologous primary isolates, compared to antibodies elicited by the corresponding monomeric gp120 protein. 167 Earl and colleagues made similar observations in rhesus macaques using a trimeric Env protein from SIV.…”

Aiming to induce broadly reactive neutralizing antibody responses with HIV-1 vaccine candidates

“…Additional efforts to target conserved neutralization epitopes, such as those in the MPER of gp41 and perhaps elsewhere on the gp140 molecule, focus on breaking tolerance and preserving non-pathogenic autoreactive B cell clones. Other future prospects include various combinations of these new Env design concepts, some of which are in early stages of development [193,194]. …”