Background:  We recently found that SOSIP gp140 envelope glycoproteins based on the subtype A HIV-1 strain KNH1144 form particularly homogenous trimers compared with a prototypic strain (JR-FL, subtype B). Here, we wanted to investigate the molecular determinants for the enhanced trimerization in KNH1144.

Methods:  Using site-directed mutagenesis and molecular cloning, we designed various HIV-1 envelope clones, primarily in JR-FL, expressed them in HEK293T cell lines, and analyzed their expression via native and denaturing polyacrylamide gel electrophoresis. To determine whether the antigenicity of the mutant JR-FL gp140 had been altered by the introduction of the trimer-stabilizing substitutions, we used BIAcore Surface Plasmon Resonance to study the binding of various antibodies to the mutant and wild type gp140.

Results:  We find that specific amino acids in the N-terminal region of gp41 ectodomain (gp41_ECTO) contribute to enhanced oligomerization of cleaved gp140 from KNH1144. Substitutions of those 5 amino acids from the N-terminal region of KNH1144 gp41_ECTO into heterologous Env sequences (eg, JR-FL and Ba‑L) have a similarly beneficial effect on trimer stability. Structural and biophysical studies are underway to determine how the amino acid substitutions in the gp41 N-terminal region increase gp140 trimer stability. SPR analysis shows that the stabilized trimers retain the epitopes for several neutralizing antibodies and related agents (CD4-IgG2, b12, 2G12, 2F5, and 4E10) and the CD4-IgG2 molecule, suggesting that the overall antigenic structure of the gp140 protein has not been adversely impaired by the trimer-stabilizing substitutions. While the neutralizing antibodies bind both the wild type and the stabilized trimers similarly, the modifications to the gp41 N-terminal region cause some non-neutralizing antibodies (PA1, b6, and 17b) to bind less efficiently.

Conclusions:  Modifications of a few selected amino acids in the N-terminal region of gp41 can improve the stability of gp140 trimers, without impairing the exposure of various neutralizing antibody epitopes. This finding is generalizable to multiple HIV-1 genotypes and hence might be useful for neutralizing antibody-based vaccine strategies based on the use of this type of immunogen.