A camelid antibody candidate for development of a therapeutic agent against Hemiscorpius lepturus envenomation

Abstract

Hemiscorpius lepturus scorpionism poses one of the most dangerous health problems in many parts of the world. The common therapy consists of using antivenom antibody fragments derived from a polyclonal immune response raised in horses. However, this immunotherapy creates serious side effects, including anaphylactic shock sometimes even leading to death. Thus, many efforts have been made to introduce new replacement therapeutics that cause less adverse reactions. One of the most attractive approaches to replacing the available therapy is offered by single-domain antibody fragments, or nanobodies (Nbs). We immunized dromedaries with H. lepturus toxin and identified a functional recombinant Nb (referred to as F7Nb) against heminecrolysin (HNc), the major known hemolytic and dermonecrotic fraction of H. lepturus venom. This Nb was retrieved from the immune library by phage display selection. The in vitro neutralization tests indicated that 17.5 nmol of the F7Nb can inhibit 45% of the hemolytic activity of 1 EC100 (7.5 μg/ml) of HNc. The in vivo neutralization tests demonstrated that F7Nb had good antihemolytic and antidermonecrotic effects against HNc in all tested mice. Surprisingly, F7Nb (8.75 nmol) neutralized 1 LD100 of HNc (10 μg) via an intracerebroventricular route or 1 LD100 (80 μg) via a subcutaneous route. All of the control mice died. Hence, this Nb is a potential leading novel candidate for treating H. lepturus scorpionism in the near future.

Keywords: HCAb; heavy-chain antibody; nanobody; phage display; scorpionism.

Figure 1.

A) HNc purification by gel filtration chromatography. Seven fractions were separated from Iranian H. lepturus venom on a Sephadex G-50 superfine column (1.6×100 cm) equilibrated with 20 mM ammonium acetate (pH 8.5). B) HNc purification by HPLC. Analytical C18 HPLC runs with the P5 eluting at 38.26 min, identified as HNc.

Figure 2.

SDS-PAGE analysis of the hemolytic fraction (P5). P5 fraction purity was analyzed by 15% SDS-PAGE under reducing conditions and after Coomassie blue staining. M, protein size marker. P5, P5-HPLC fraction (HNc), ∼33 kDa band.

Figure 3.

Immune response of camels immunized with H. lepturus venom determined as follows. A) ELISA: an increasing immune response over time was observed for H. lepturus venom and HNc fraction in pooled serum of both camels. All experiments were applied in triplicate; each bar represents the mean ± sd; 0.01 < P < 0.05 vs. negative control of each group. B) Western blot: lane 1: preimmune serum; lanes 2 and 3: pooled immune sera of camels. C) The SDS-PAGE pattern of purified camel HCAbs: the purified IgG1, IgG2, and IgG3 subtractions from 1 camel, separated by SDS-PAGE before and after reduction.

Figure 4.

A) Phage ELISA showing that the enrichment of antigen-specific Nbs occurred mainly during rounds 3 and 4 of panning with HNc. B) The predicted amino acid sequences of the F7 and H39 Nbs.

Figure 5.

A) SDS-PAGE analysis of 2 HNc-specific Nbs. The 15 kDa bands have been identified as Nbs. B) Western blot analysis of HNc Nbs. The 15 kDa bands were identified as His-tagged Nbs by an anti-His tag antibody.

Figure 6.

In vitro hemolytic test of HNc. The minimum concentration of HNc that showed maximum hemolytic activity was 7.5 μg/ml.

Figure 7.

Hemolytic inhibition of HNc by F7Nb. A) F7Nb (17.5 nmol; 250 μg) inactivated 45% hemolytic activity of 1 EC₁₀₀ HNc. B) The remaining percentage of hemolytic activity of 1 EC₁₀₀ of HNc, when preincubated with 17.5 nmol of F7Nb (or 250 μg) commercial polyclonal antibody in comparison with the negative (PBS) and positive (Triton X-100) controls.

Figure 8.

Antidermonecrotic assay: A) F7Nb 125 μg mixed with HNc 4 μg, (B) ×1 PBS as the negative control, (C) HNc (4 μg) injected intradermally alone as the positive control.