A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing

Area of Science:

• Virology
• Immunology
• Structural Biology

Background:

• Epitope mapping is crucial for designing effective epitope-based vaccines.
• Understanding antibody targets on HIV-1 Envelope (Env) is essential for vaccine development.
• Current methods for mapping neutralizing antibody (NAb) epitopes can be time-consuming.

Purpose of the Study:

• To develop a facile and rapid methodology for epitope mapping of NAbs against HIV-1 Env at single-residue resolution.
• To delineate epitopes for specific NAbs (VRC01, PGT128, PGT151) and assess polyclonal responses.
• To provide a versatile method applicable to other viruses with available reverse genetics or lentiviral display systems.

Main Methods:

• Generation of a library of Cys mutations in the HIV-1 Env glycoprotein.
• Covalent labeling of Cys residues with a Cys-reactive label to mask epitopes.
• Viral neutralization assays using labeled mutant virions in the presence of NAbs.
• Deep sequencing of Env genes from NAb-resistant viruses to identify targeted epitopes.

Main Results:

• Accurate delineation of epitopes for NAbs VRC01, PGT128, and PGT151 at single-residue resolution.
• Methodology successfully mapped epitopes targeted by polyclonal sera from immunized animals and an elite neutralizer.
• Results showed good agreement with previously determined structural epitopes.
• Demonstrated the capability to map multiple epitope specificities within polyclonal responses.

Conclusions:

• The developed methodology provides a rapid and facile approach for high-resolution epitope mapping of NAbs against HIV-1 Env.
• This technique is valuable for understanding antibody responses and advancing epitope-based vaccine design.
• The method's adaptability allows for application to a broader range of viruses.