Isolation and characterization of Hc-targeting chimeric heavy chain antibodies neutralizing botulinum neurotoxin type B

Abstract

Background: Botulinum neurotoxin (BoNT) produced by Clostridium botulinum is one of the most potent known toxins. Moreover, BoNT is classified as one of the most important biological warfare agents that threatens the biosafety of the world. Currently, the approved treatment for botulism in humans is the use of polyvalent horse serum antitoxins. However, they are greatly limited because of insufficient supply and adverse reactions. Thus, treatment of human botulism requires the development of effective toxin-neutralizing antibodies. Considering their advantages, neutralizing nanobodies will play an increasing role as BoNTs therapeutics.

Methods: Herein, neutralizing nanobodies binding to the heavy chain (Hc) domain of BoNT/B (BHc) were screened from a phage display library. Then, BoNT/B-specific clones were identified and fused with the human Fc fragment (hFc) to form chimeric heavy chain antibodies. Finally, the affinity, specificity, and neutralizing activity of antibodies against BoNT/B in vivo were evaluated.

Results: The B5-hFc, B9-hFc and B12-hFc antibodies demonstrated high affinity for BHc in the nanomolar range. The three antibodies were proven to have potent neutralizing activity against BoNT/B in vivo.

Conclusion: The results demonstrate that inhibiting toxin binding to the host receptor is an efficient strategy and the three antibodies could be used as candidates for the further development of drugs to prevent and treat botulism.

Keywords: Hc domain; botulinum neurotoxin type B; heavy chain antibody; nanobody; neutralizing antibody; phage display library.

Figure 1

ELISA was used to detect the binding activity against BHc antigen produced in camel serum. When the ratio of (OD value of positive serum - OD value of blank) to (OD value of negative control - OD value of blank) was more than 2, the well was usually defined as positive and the maximum serum dilution in the positive wells represented the antibody titer.

Figure 2

Positive clones were identified using Phage-ELISA. (A) Identification of single clones after the second round of screening; (B) Identification of single clones after the third round of screening. “A” represents clones binding to the BHc antigen; “B” represents clones binding to the BSA antigen. The darker the red color, the higher the OD492nm/630nm value.

Figure 3

Analysis of diverse purified heavy chain antibodies using SDS-PAGE. (A) SDS-PAGE under reducing conditions. Lane 1, B5-hFc; lane 2, B9-hFc; lane 3, B12-hFc, lane 4, B14-hFc, lane 5, B20-hFc; M, protein markers. (B) SDS-PAGE under non-reducing conditions. Lane 1, B5-hFc; lane 2, B9-hFc; lane 3, B12-hFc, lane 4, B14-hFc, lane 5, B20-hFc; M, protein markers.

Figure 4

Specificity of the B5-hFc, B9-hFc, B12-hFc, B14-hFc and B20-hFc. The binding between the antibodies and different antigens. The concentration of antigens and antibodies were all 2 μg/mL.

Figure 5

Competitive binding analysis of five antibodies with BHc using BLI. This figure illustrates the dynamic kinetic process of competitive binding among various antibodies to BHc. Antibodies were immobilized on the biosensor for determination. The antibodies included are B5-hFc (A), B9-hFc (B), B12-hFc (C), B14-hFc (D), and B20-hFc (E). As detailed in the legend, stages of the assay were demarcated by slashes “/”, specifically denoting the “Loading”, “Association”, and “Re-association” phases. (F) delineates the competitive interaction amongst the five antibodies and BHc. A “√” signifies that the respective second antibody competitively binds to BHc in the presence of a first antibody, whereas an “x” denotes the absence of competitive binding between the first and second antibodies.

Figure 6

Evaluation of antibody neutralization efficiency. Each mouse was injected 500 mL mixture, which means that the five antibodies at descending dosages of 2 μg, 1 μg, 0.5 μg, 0.25 μg, 0.125 μμg, and 0.0625 μg were seperately mixed with 20 × LD50 BoNT/B. The five antibodies are B5-hFc (A), B9-hFc (B), B12-hFc (C), B14-hFc (D), and B20-hFc (E). The figure presents the survival rate of mice against BoNT/B in different concentrations of antibodies.