Anti-Metalloprotease P-I Single-Domain Antibodies: Tools for Next-Generation Snakebite Antivenoms

Abstract

In order to address the global antivenom crisis, novel antivenoms need to present high therapeutic efficacy, broad neutralization ability against systemic and local damage, sufficient safety, and cost-effectiveness. Due to biological characteristics of camelid single-domain antibodies (VHH) such as high affinity, their ability to penetrate dense tissues, and facility for genetic manipulation, their application in antivenoms has expanded considerably. VHHs that are active against the metalloprotease BjussuMP-II from the snake Bothrops jararacussu were selected. After isolation of BjussuMP-II, a camelid was immunized with the purified toxin in order to construct the recombinant phage library. Following a round of biopanning, 52% of the selected clones were able to recognize BjussuMP-II in an ELISA assay. After sequencing, seven sequence profiles were identified. One selected clone (VHH61) showed cross-reactivity to B. brazili venom, but did not recognize the Crotalus and Lachesis genera, indicating specificity for the Bothrops genus. Through in vitro tests, the capacity to neutralize the toxicity triggered by BjussuMP-II was observed. Circular dichroism spectroscopy indicated a robust secondary structure for VHH61, and the calculated melting temperature (T _M) for the clone was 56.4°C. In silico analysis, through molecular docking of anti-BjussuMP-II VHHs with metalloprotease, revealed their potential interaction with amino acids present in regions critical for the toxin's conformation and stability. The findings suggest that anti-BjussuMP-II VHHs may be beneficial in the development of next-generation antivenoms.

Figure 1

Purification and characterization of BjussuMP-II. (a) Chromatographic profile of B. jararacussu venom using cation exchange chromatography. Fraction 4 was selected for presenting a relative molecular mass of 24 kDa in electrophoretic analysis on a 12.5% polyacrylamide gel. (b) Polyacrylamide gel electrophoresis (12.5%) of the fraction corresponding to the metalloprotease isolated from B. jararacussu venom (1) and of the purified metalloprotease BjussuMP-II, with an approximate molecular mass of 24 kDa (2). (c) Mass spectrum of BjussuMP-II in AXIMA TOF2. The spectrum measures the mass of the protein BjussuMP-II, at 23,423.40 Da. (d) Electrophoretic analysis of the fibrinogenolytic activity of B. jararacussu venom and BjussuMP-II at different incubation times, for 30 min (6), 1 h (7), 3 h (8), and 6 h (9). The negative control corresponds to fibrinogen (5).

Figure 2

Identification of anti-BjussuMP-II VHHs. (a) Monitoring of the camelid immune response after administration of BjussuMP-II. Results obtained in an ELISA assay, demonstrating the absorbances obtained in different dilutions of the serum collected on the following days: 0 (∗), 14 (■), 21 (▲), and 28 (•). (b) Analysis of VHHs against the toxin BjussuMP-II after biopanning. The cut-off line corresponds to the average of the cut-off values calculated for each of the 47 clones. (c) Multiple alignments among clones selected against BjussuMP-II, demonstrating CDRs and FRs. Letters highlighted in red represent the cysteines present in all clones. The gray shaded areas represent the CDRs. Amino acids underlined in the FR2 region represent the characteristic amino acids of VHHs. (∗) represents identical amino acids; (:) represents strongly similar amino acids; (.) represents weakly similar amino acids.

Figure 3

Evaluation of anti-BjussuMP-II VHHs' immunoreactivity by ELISA assay. (a): Absorbances obtained in ELISA performed on plates sensitized with 10 μg/mL of BjussuMP-II and anti-MP-II VHHs in blocking solution (10 μg/mL). PC: positive control with serum from the immunized animal. NC: uncorrelated VHH negative control (anti-Hanta 36 VHH). Columns marked with an asterisk (∗) represent statistical difference in relation to the sample cut-off (^∗∗p < 0.01 and ^∗∗∗p < 0.001). Columns marked with hash (#) represent statistical difference between the nanobodies and the negative control (^###p < 0.001). (b) Absorbances obtained in an ELISA assay performed on a plate sensitized with 1 μg/well of snake venoms of the genera Bothrops, Crotalus, and Lachesis, using a 1 : 1 ratio (p/p) of anti-BjussuMP-II VHH61. The positive control, shown as a striped bar, stands out above the average cut-off value, represented by a dashed line. The specificity of anti-BjussuMP-II VHH61 for the crude venom of B. jararacussu is observed. PC: positive control that corresponds to the absorbance of wells showing the interaction of anti-BjussuMP-II VHH61 with BjussuMP-II from B. jararacussu. NC: negative control corresponding to the absorbance of wells containing BjussuMP-II with uncorrelated anti-Hanta36 VHH obtained from Lama glama, specific for hantavirus.

Figure 4

Analysis of inhibition of proteolytic activity and cytotoxicity of BjussuMP-II by anti-BjussuMP-II VHHs, and circular dichroism of anti-BjussuMP-II VHH61. (a) Proteolytic activity of BjussuMP-II on casein after exposure to a preincubated solution containing the toxin and the VHHs (anti-BjussuMP-II VHH47 and anti-BjussuMP-II VHH61) in different molar ratios. NC: negative control consisting of casein and buffer. PC: casein incubated with 10 μg of BjussuMP-II. Columns marked with an asterisk (∗) represent a statistical difference in relation to the positive control (^∗p < 0.001). (b) Analysis of the inhibition of LDH release by tEND cells against exposure to BjussuMP-II by anti-BjussuMP-II VHH61. Cytotoxicity estimated by LDH release after 48 h of exposure to a preincubated solution containing BjussuMP-II and anti-BjussuMP-II VHH61 at a 1 : 5 ratio (w/w). NC: negative control consisting of untreated cells. Triton: cells treated with Triton X-100 at a concentration of 0.1%. Columns marked with an asterisk (∗) represent a statistical difference in relation to the negative control (^∗∗∗p < 0.001 and ^∗∗p < 0.01). Columns marked with a pound sign (#) represent a statistical difference between BjussuMP-II and BjussuMP-II+anti-BjussuMP-II VHH 61 (^#p < 0.05). (c) Analysis of anti-BjussuMP-II VHH61 circular dichroism spectra collected in the 191-260 nm interval and thermal denaturation curve obtained at a wavelength of 202 nm during heating from 20 to 90°C. The measurements were done in a JASCO J-815 spectropolarimeter.

Figure 5

Molecular docking results showing the binding sites of VHHs (VHH47, VHH61, and VHH64) on the surface of BjussuMP-II. Cartoon representations of the BjussuMP-II VHH interaction structures (side view) covered by a translucent electronic surface, where the α-chains of BjussuMP-II are shown as blue ribbons and the VHH is in red. The active site of BjussuMP-II (His144, His148, His154, and Glu145) is represented in yellow. (a) Anti-BjussuMP-II VHH47 and BjussuMP-II. (b) Anti-BjussuMP-II VHH61 and BjussuMP-II. (c) Anti-BjussuMP-II VHH64 and BjussuMP-II. The interaction sites have been magnified to show the hydrogen bonds that formed between the amino acid residues.