Increased functional stability and homogeneity of viral envelope spikes through directed evolution

Area of Science:

• Virology
• Immunology
• Biochemistry

Background:

• The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is labile and heterogeneous, often eliciting non-neutralizing antibodies.
• This instability and heterogeneity pose challenges for developing effective HIV-1 vaccines targeting the Env trimer.

Purpose of the Study:

• To overcome the inherent instability and heterogeneity of the primary HIV-1 Env spike.
• To develop methods for selecting and generating more stable Env variants for potential immunogen development.

Main Methods:

• Directed evolution involving iterative cycles of Env destabilization and replication to select for enhanced stability.
• Isolation and characterization of stable Env variants with specific mutations.
• Assessment of trimer stability, homogeneity, resistance to proteolysis and detergent, and reactivity with neutralizing antibodies.

Main Results:

• Two distinct pools of stable Env variants were selected, with seven specific mutations identified as key to increased trimer stability, particularly in gp41 and gp120.
• A combined variant exhibited superior homogeneity, stability, resistance to proteolysis and detergent, and reduced heterogeneity.
• Stabilizing mutations increased the proportion of gp140 in a trimeric conformation and showed potential to stabilize Env spikes from multiple HIV-1 clades.

Conclusions:

• Directed evolution is an effective strategy for generating hyper-stable HIV-1 Env trimers.
• These stabilizing mutations enhance Env homogeneity and stability, offering a promising avenue for developing more effective HIV-1 vaccine immunogens.
• The identified stabilizing mutations are applicable across different HIV-1 clades, suggesting broad utility.