A recombinant Fab neutralizes dengue virus in vitro

Area of Science:

• Virology
• Immunology
• Molecular Biology

Background:

• Dengue virus (DENV) poses a significant global health threat.
• Neutralizing antibodies are crucial for DENV control.
• Protein E contains a key epitope recognized by neutralizing antibodies.

Purpose of the Study:

• To generate and characterize a recombinant antibody fragment (Fab) targeting a neutralizing epitope on DENV protein E.
• To compare the binding affinity and antiviral activity of the Fab with its parent monoclonal antibody (mAb).

Main Methods:

• Cloning hybridoma cells secreting mAb 4E11.
• Amplifying antibody genes (Fd and light chain) via polymerase chain reaction (PCR).
• Cloning genes into pMad phagemid vector, expressing Fab fragments in bacteria, and sequencing.
• Affinity determination using MalE-E(296-400) fusion protein.
• In vitro neutralization assays against different DENV serotypes.

Main Results:

• A recombinant Fab (Fab 4E11) recognizing the DENV protein E (296-400) epitope was successfully produced.
• Fab 4E11 exhibited nanomolar affinity, comparable to the parental mAb 4E11.
• The light chain complementary-determining regions of mAb 4E11 showed homology with other antiviral mAbs.
• Fab 4E11 demonstrated reduced in vitro neutralization capacity (4-8 times lower) compared to mAb 4E11 across DENV serotypes.

Conclusions:

• Recombinant Fab 4E11 retains high binding affinity for the target epitope on dengue virus protein E.
• While effective, the Fab fragment exhibits diminished in vitro antiviral activity compared to the full monoclonal antibody.
• This study provides a foundation for developing antibody-based therapeutics against dengue virus.