Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform

Area of Science:

• Immunology
• Structural Biology
• Bioinformatics

Background:

• Polyclonal antibody responses are crucial for adaptive immunity post-vaccination.
• Understanding antibody epitope interactions is key to vaccine development and therapeutic antibody design.
• The meningococcal factor H binding protein (fHbp) is a target for vaccines against Neisseria meningitidis.

Purpose of the Study:

• To evaluate the PROFILER technology for characterizing monoclonal antibody (mAb) epitopes.
• To identify critical residues within a conformational epitope of fHbp recognized by mAb 12C1.
• To demonstrate PROFILER's capability in pinpointing functional 'hot spots' in antigen-antibody interactions.

Main Methods:

• Construction and screening of an fHbp phage display library against mAb 12C1.
• Massive parallel sequencing of antibody-selected phage inserts.
• Computational analysis of amino acid enrichment to identify key residue stretches.
• Functional validation of identified epitope regions.

Main Results:

• PROFILER identified numerous antibody-selected sequences from the fHbp library.
• A dominant fragment (FrC) was found to largely retain binding to mAb 12C1.
• Two specific amino acid stretches (H248-K254 and S140-G154) were identified as essential for FrC binding to mAb 12C1.
• These identified regions represent critical 'hot spots' within the conformational epitope.

Conclusions:

• The PROFILER technology effectively identifies functional 'hot spots' in complex conformational epitopes.
• PROFILER provides rapid and reliable insights into antigen-antibody interactions.
• This method aids in the functional characterization of epitopes, valuable for vaccine and therapeutic antibody development.