Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals

Abstract

Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

Keywords: Antibody repertoire analysis; next-generation sequencing; ricin A chain; yeast surface display.

Figure 1.

Overview of the experimental approach. (A) Mouse is immunized at footpad with ricin A chain, and peripheral lymphoid organs, including bone marrow, spleen, and draining lymph node are isolated after 3 booster immunizations. (B) Total cells in bone marrow and spleen are collected, and V_H and V_L mRNA are reverse transcribed and amplified, which are used to construct scFv libraries, respectively. These are selected against ricin A chain using yeast surface display to isolate high-affinity binders. V_H and V_L cDNAs are also sequenced on an Illumina platform. (C) CD138⁺ antibody secreting cells are isolated from draining lymph node, and processed through our high-throughput V_H:V_L pairing platform.

Figure 2.

Selection of libraries constructed from bone marrow and spleen antibody repertoires. Cells are doubly stained with chicken anti-c-Myc IgY/GaC-488 for scFv surface expression and biotinylated ricin A chain/SA-633 for antigen binding. (A) Bivariate plots are shown for bone marrow antibody library stained with 100 nM ricin A chain before selection, after MACS, after 1st FACS, after 2nd FACS, and after 3rd FACS, with x axis being surface expression, and y axis being antigen binding. (B) Bivariate plots are shown for spleen antibody library stained with 100 nM ricin A chain before and after each round of selection. For both libraries, cells in the upper-right quadrant (both express scFv on the surface and bind ricin A chain) are sought. Cells falling within strict FACS sort gates designed to ensure enrichment of clones showing increased binding to ricin A chain are collected for the next round of selection.

Figure 3.

Characteristics of bone marrow and spleen antibody repertoires from the same mouse immunized with ricin A chain. (A) Germ-line V_H gene usage in bone marrow and spleen repertoires. (B) Germ-line V_L gene usage in the 2 repertoires. (C) CDRH3 length distribution in the 2 repertoires. Green denotes bone marrow repertoire, and blue denotes spleen repertoire.

Figure 4.

Characteristics of CD138⁺ antibody-secreting cells repertoire in draining lymph node from mouse immunized with ricin A chain. (A) The frequency of each unique antibody clone in the repertoire, which is calculated as the percentage of its read counts in the read counts of all clones, is shown with its rank, which is ordered by the number of read counts of each unique clone. Clones with only 1 read are removed, and CDRH3 nucleotide sequences are clustered to 96% identity. Inset shows the distribution of the top 10 most abundant clones in the repertoire. (B) Germ-line V gene family usage in the same repertoire is shown. (C) CDRH3 length (calculated as amino acid length) distribution of antibodies in the repertoire is shown as the percentage of antibodies that have the denoted CDRH3 length in the whole repertoire.

Figure 5.

Sequence comparison between yeast surface display and next-generation sequencing discovered antibodies. (A), (B) V_H alignments of BM1, BM3, BM17, SP19 and RAM1.5 and of SP1 and RAM1.4 showed mutations through the complete V_H sequences, indicating they are somatic variants. (C), (D) V_L alignments of BM1/BM3/BM17, SP19 and RAM1.5 and of SP1 and RAM1.4 also showed mutations. Mutations are indicated by different colors.