Recombinant Anti-C. difficile TcdA VHH Single Domain Antibody (A26.8)

Figure 1 In vitro neutralizing activity and in vivo expression of A20.1 and A26.8 VHH antibodies.

Dot blot analysis of B. longum-produced A20.1 and A26.8 VHH antibodies binding to TcdA immobilized on a PVDF membrane in quantities from 35 to 140 ng. Bovine serum albumin (BSA) immobilized in quantities from 50 to 200 ng was used as a control.

Shkoporov, A. N., Khokhlova, E. V., Savochkin, K. A., Kafarskaia, L. I., & Efimov, B. A. (2015). Production of biologically active scFv and VHH antibody fragments in Bifidobacterium longum. FEMS microbiology letters, 362(12).

Figure 2 In vitro neutralizing activity and in vivo expression of A20.1 and A26.8 VHH antibodies.

Dose-effect curve of TcdA toxicity on CHO-K1 cell line in the presence or absence of conditioned media from B. longum 44B [pESH100/VHH-A20.1] (BL-A20.1) and B. longum 44B [pESH100/VHH-A26.8] (BL-A20.1). Conditioned media were applied on the CHO-K1 cells prior to the treatment with TcdA toxin (5.5 to 1400 ng/ml). 24 hours after treatment the percentage of rounded cells was manually calculated using phase contrast microscopy

Shkoporov, A. N., Khokhlova, E. V., Savochkin, K. A., Kafarskaia, L. I., & Efimov, B. A. (2015). Production of biologically active scFv and VHH antibody fragments in Bifidobacterium longum. FEMS microbiology letters, 362(12).

Figure 3 In vitro neutralizing activity and in vivo expression of A20.1 and A26.8 VHH antibodies.

In vivo expression of A20.1 and A26.8 antibodies in (C57/BL6 x DBA/2)F1 mice after intragastric administration of transformed B. longum strains (BL-D2E7, BL-A20.1, and BL-A26.8) and untransformed B. longum 44B (BL) assayed using semiquantitative ELISA (Materials and Methods section for details); control +, culture supernatant from strain BL-A26.8; control -, empty medium.

Shkoporov, A. N., Khokhlova, E. V., Savochkin, K. A., Kafarskaia, L. I., & Efimov, B. A. (2015). Production of biologically active scFv and VHH antibody fragments in Bifidobacterium longum. FEMS microbiology letters, 362(12).

Figure 4 Antitoxin VHHs recognize conformational (A4.2, A5.1, A20.1, and A26.8) and linear (A19.2) epitopes.

ELISA demonstrates that purified anti-TcdA VHHs recognize both native toxins and recombinant RBD fragments. Wells were coated with equimolar concentrations of the proteins shown on the x axis. VHHs were added at 2 g/ml. The B5.2 VHH bound TcdA.

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 5 Antitoxin VHHs recognize conformational (A4.2, A5.1, A20.1, and A26.8) and linear (A19.2) epitopes.

ELISA illustrating the binding of various concentrations of purified VHHs to immobilized TcdA, respectively.

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 6 Antitoxin VHHs recognize conformational (A4.2, A5.1, A20.1, and A26.8) and linear (A19.2) epitopes.

ELISA on TcdA treated with various temperatures for 30 min before probing with VHHs. The dotted line represents the TcdA midpoint thermal unfolding temperature (Tm) of
55 °C. At treatment temperatures above the TcdA Tm, binding of four out of five TcdA-specific VHHs was abolished. A19.2 bound TcdA exposed to temperatures above its Tm, confirming that denatured TcdA was coated in the wells and that A19.2 recognized a linear epitope.

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 7 VHHs bind TcdA with high affinity.

SPR sensorgrams of TcdA-specific VHHs A26.8 binding to immobilized TcdA.

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 8 Potent neutralization of TcdA-induced cell rounding in vitro.

Summary of neutralization assays. The final concentration of VHHs in each assay well was 1000 nM, and VHHs were added as singles, pairs, or triplet combinations. White barsrepresent single VHHs or PBS control; gray barsrepresent paired combinations, and black barsrepresent triplet combinations. Percent inhibition of cell rounding is relative to cells receiving TcdA only. Combinations of VHHs (i.e. pairs and triples) increased toxin neutralizing efficacy.

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 9 Potent neutralization of TcdA-induced cell rounding in vitro.

Summary of neutralization assays. The final concentration of VHHs in each assay well was 10 nM, and VHHs were added as singles, pairs, or triplet combinations. White barsrepresent single VHHs or PBS control; gray barsrepresent paired combinations, and black barsrepresent triplet combinations. Percent inhibition of cell rounding is relative to cells receiving TcdA only. Combinations of VHHs (i.e. pairs and triples) increased toxin neutralizing efficacy.

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 10 Potent neutralization of TcdA-induced cell rounding in vitro.

Summary of neutralization assays. The final concentration of VHHs in each assay well was 0.1 nM, and VHHs were added as singles, pairs, or triplet combinations. White barsrepresent single VHHs or PBS control; gray barsrepresent paired combinations, and black barsrepresent triplet combinations. Percent inhibition of cell rounding is relative to cells receiving TcdA only. Combinations of VHHs (i.e. pairs and triples) increased toxin neutralizing efficacy.

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 11 Anti-TcdA VHHs recognize overlapping and nonoverlapping epitopes.

SPR co-injection experiments were used to determine whether pairs of VHHs could bind TcdA simultaneously. The sensorgrams of all of the possible paired combinations of A26.8, in both orientations, are shown.

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 12 Anti-TcdA VHHs recognize overlapping and nonoverlapping epitopes.

SPR co-injection experiments were used to determine whether pairs of VHHs could bind TcdA simultaneously. The sensorgrams of all of the possible paired combinations of A26.8, in both orientations, are shown.

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 13 Mixtures of A4.2, A5.1, and A26.8 VHHs show binding at overlapping TcdA epitopes.

The three VHHs suspected of sharing an overlapping epitope were further analyzed by SPR by injecting the VHHs alone (A4.2, A5.1, or A26.8) or as a triplet mixture (Mix) over immobilized TcdA.

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 14 TcdA-binding trisaccharides do not inhibit VHH binding.

SPR illustrating the responses generated from A26.8 binding TcdA (red), CD-grease binding TcdA (black), and co-injection of A26.8 and CD-grease (light green).

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.

Figure 15 Pre-bound VHHs do not impair trisaccharide binding to TcdA.

SPR illustrate the injection of A26.8 (a,solid and dashed lines), followed by injection of A26.8 (b, dashed line) or co-injection of A26.8 CD-grease (b, solid line), and finally injection of buffer (c, solid and dashed lines).

Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.