Isolation and characterization of foot and mouth disease virus-specific antibodies by combining high-throughput single cell sequencing, B cell culture, and antibody repertoire analysis

Abstract

Foot and mouth disease virus (FMDV) causes a highly contagious disease in cattle with the potential of causing severe economic impact. Current vaccines are predominantly serotype-specific with short-lived protection. As vaccine driven protection correlates with neutralising antibodies, a more comprehensive understanding of the B cell response could present opportunities to improve vaccine design.

This research project established a high-throughput single-cell antibody technique sequencing method for cattle, ensuring accurately paired heavy and light chain sequences. Subsequently, this method was combined with whole antibody repertoire sequences from the same cattle sequentially vaccinated for the identification of FMDV-specific antibody sequences. Additionally, a B cell culture system was also employed for the enrichment and identifiction of FMDV-specific B cells.

The qualitative analysis of the cattle antibody response has faced challenges stemming from the complexities of identifying paired heavy and light chain (VH:VL) sequences within mixed cellular populations. To overcome this hurdle, B cells were sorted in bulk and thereafter used to generate 5' rapid droplet-based encapsulation of single cells. This enabled the generation of high-throughput antibody barcoded cDNA using Chromium 10x Genomics platform for the production of accurately paired heavy and light chain sequences. This resulted in close to 5,000 accurately paired heavy and light chain sequences from single B cells.

To identify FMDV-specific antibody sequences, parallel analysis of paired single cell sequences of heavy and light chains were analysed alongside the antibody repertoire sequence data from the same animals collected during the same vaccination study. Sequence annotation and subsequent clustering revealed unique kinetics in individual antibody clusters within the whole repertoire, with some aligning with ELISpot data showing serotype specific reactivity occurring and reoccurring after each immunization. Thirteen single cell sequenced antibodies belonging to these clusters were selected. These antibodies were expressed as cattle IgG1 in mammalian cells, and were then assessed for their FMDV-specific binding via ELISA. Remarkably, over 75% of these rAbs demonstrated binding, with two of the rAbs being mono- and bi- serotype specific, while seven rAbs exhibited binding to three of the distinct FMDV serotypes used in the initial sequential immunization.

In parallel, I developed an in vitro cattle B cell culture and proliferation system, successfully increasing B cell frequency from ~35% in isolated PBMCs to ~89% after a 9-day culture period. This cell culture system proved instrumental in the identification and characterization of FMDV-specific B cells from vaccinated cattle. Significantly, this methodology marked the first successful identification of FMDV-specific B cells using extracellular viral capsid staining. These identified cells were subsequently sorted and integrated into the pre-established 10x Genomics pipeline.

Overall, this research has established a high-throughput single cell antigen-specific antibody recovery pipeline in cattle, providing a foundation for exploring antibody responses at high resolution between vaccinated and infected cattle. In addition, the study has presented alternative methods for identifying FMDV-specific B cells and the capability to culture and perform extracellular staining. These significant advances will contribute greatly to much needed reagents, not only benefiting cattle and FMDV research but also extending its applicability to a range of veterinary animals and disease models. It holds the potential to enhance our understanding of what makes a protective antibody response following vaccination or infection across various disease contexts.