Characterization of the antibody diversity of glycoprotein E2 domain B/C in pestivirus by using high-throughput...

Area of Science:

• Immunology
• Virology
• Biotechnology

Background:

• Classical swine fever virus (CSFV) poses a significant threat to swine populations.
• The E2 protein's B/C domain is a key target for neutralizing antibodies.
• Developing effective diagnostic tools and vaccines requires understanding CSFV antigenicity.

Purpose of the Study:

• To identify and characterize monoclonal antibodies (mAbs) targeting the CSFV E2 protein's B/C domain.
• To leverage microfluidic single B cell technology for accelerated antibody discovery.
• To investigate potential pan-pestivirus epitopes within the CSFV E2 B/C domain.

Main Methods:

• Utilized a customized immunization strategy combined with a high-throughput, microfluidic-based single B cell platform.
• Isolated 4241 antigen-specific plasma cells and performed next-generation sequencing to obtain 82 paired antibody sequences.
• Conducted binding assays on 81 mAbs to assess their reactivity against CSFV E2 protein variants and related pestiviruses.

Main Results:

• Identified 38 mAbs that bind to the CSFV E2 B/C domain, with 14 specific to genotype 2 QZ07-E2.
• Discovered 24 mAbs reactive with both genotype 2 QZ07-E2 and genotype 1 C-E2, including 8 with cross-reactivity against other pestiviruses.
• Phylogenetic analysis of CDR sequences correlated with binding profiles, suggesting three distinct antibody clusters.

Conclusions:

• The study identified novel, broadly reactive pan-pestivirus epitopes in the CSFV E2 B/C domain.
• Microfluidics integrated with high-throughput sequencing significantly accelerates antibody discovery.
• This approach provides a robust platform for developing diagnostic assays and next-generation marker vaccines against pestiviruses.