Single-Domain Antibodies That Specifically Recognize Intact Capsids of Multiple Foot-and-Mouth Disease Serotype O Strains

Figures

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  Figure 1. Structure of published VHHs or clonally related to published VHHs. (a) The mature VHH protein sequences of 8 VHHs as produced in yeast. The three VHHs clonally related to M170 are shown below the M170 sequence, where dots represent their identity in relation to M170. M81, which is clonally related to M210, is similarly shown below the M210 sequence. The IMGT system [43] was used for alignment and numbering of VHHs as well as defining the different complementarity-determining regions (CDRs) and framework regions (FRs). The Kabat numbering scheme is given for reference. Amino acids were classified and color-coded based on their physicochemical properties. The VP1 and VP3 residues within 4 Å distance of a M170 residue based on the cryo-EM structure of M170 complexed to O/BY/CHA/2010 are indicated [34]. The M170 residue numbering used by Dong et al. [34] is given for reference as well. (b) Cartoon presentation of M170 complexed to O/BY/CHA/2010 (PDB: 7DST). VP1, VP2, VP3, and VP4 of a single protomer are shown in addition to the VP1 C-terminal of 20 amino acids of an adjacent protomer (VP1′), which is close to the M170 binding site. The left panel shows the side chains of the two…

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  Figure 2. FMDV particle and strain specificities of VHHs. DAS-ELISAs were performed using a matrix of 18 VHHs and 15 FMDV strains representing 5 serotypes. Antigens from each strain were used in ELISA either untreated (146S; blue) or after conversion into 12S by heating for 1 h at 56 °C (red). Most antigens were BEI-inactivated and used at 1 µg/mL 146S. For the strain Asia1/BAR/8/2009, the crude culture supernatant of FMDV-infected BHK21 cells was used without BEI inactivation at a 5-fold dilution with unknown antigen concentration. VHHs are color-coded by their clonal group. Control VHHs isolated and characterized previously are underlined. The VHHs are arranged from top to bottom according to their clonal group and their particle and strain specificity. The ELISA with the strain Asia1/BAR/8/2009 that was not BEI-inactivated was performed separately within the high-containment unit of WBVR and resulted in a higher background absorbance value. This is most evident with M918F, Nb45F, and Nb205F, which already have a high background absorbance.

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  Figure 3. Sequence divergence of FMDV serotype O capsid encoding P1 region. A phylogenetic tree of P1 nucleotide sequences was generated using the maximum likelihood method and 1000 cycles for bootstrap sampling. Bootstrap values are indicated next to branches. For each sequence, the FMDV strain name, database accession number, and topotype/lineage are indicated and separated by dashes. Strains used to analyze antigenicity are boxed. Branches are color-coded according to their topotype, using orange for all 4 East African (EA) topotypes.

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  Figure 4. FMDV particle specificity of VHHs in DAS-ELISA. FMDV antigens of three serotype O strains, as indicated on the right, were titrated in DAS-ELISAs using various VHHs either as untreated FMDV antigen (146S) or after heating at 56 °C for 1 h (12S). VHHs are color-coded by their clonal group. Control VHHs isolated and characterized previously are underlined.

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  Figure 5. ELISA analysis of VHH specificity for FMDV particles fractionated by SDGs. BEI-inactivated antigens from the three FMDV strains indicated on the right were used for SDG fractionation. SDGs were layered with a 1:1 mixture of untreated and heated FMDV antigens to ensure the presence of both 146S and 12S. SDG fractions were analyzed by the measurement of absorbance at 254 nm to identify the 146S peak and by DAS-ELISAs using the broadly reactive VHHs M3F and M8F to identify all peaks. A further 10 ELISAs were performed using different 146S-specific VHHs. The names of control VHHs isolated and characterized previously are underlined. The M81F ELISA of O1/Manisa/TUR/69 fractions 1–10 is also shown on an extended secondary axis to visualize low levels of 12S particles that were detected.

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  Figure 6. Epitope binning of VHHs by competition ELISAs using FMDV strain O1/Manisa/TUR/69. A red–blue coloring is used to visualize differences in percentage inhibition. The VHHs are color-coded by their clonal group. The control VHHs isolated previously are underlined. Antigenic sites are indicated by boxing. Antigenic sites identified previously by competition ELISAs using VHHs [26], isolation of mAb escape mutants [49], or cryo-EM analysis of antibody–FMDV complexes [9,34] are indicated on the right.

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  Figure 7. Capsid specificity and interference of 12S particles in capsid quantification in DAS-ELISAs using M907F or M916F. Untreated antigens consisting predominantly of 146S and 12S particles generated by heating three FMDV strains, as indicated on the right, were titrated in DAS-ELISAs with M907F or M916F, as indicated on top. Another titration was performed with a 3-fold dilution series of untreated FMDV particles to which a constant amount of 4 µg/mL 12S particles was added to measure the interference of 12S particles. The mean and standard deviation of triplicate measurements are presented.