Identification of Immunoreactive Peptides of Toxins to Simultaneously Assess the Neutralization Potency of Antivenoms against Neurotoxicity and Cytotoxicity of Naja atra Venom

Abstract

Assessing the neutralization capability of nonlethal but medically relevant toxins in venom has been a challenging task. Nowadays, neutralization efficacy is evaluated based simply on the survival rates of animals injected with antivenom together with a predefined dose of venom, which can determine potency against neurotoxicity but not validate the capability to neutralize cytotoxin-induced complications. In this study, a high correlation with in-vivo and in-vitro neutralization assays was established using the immunoreactive peptides identified from short-chain neurotoxin and cytotoxin A3. These peptides contain conserved residues associated with toxin activities and a competition assay indicated that these peptides could specifically block the antibody binding to toxin and affect the neutralization potency of antivenom. Moreover, the titers of peptide-specific antibody in antivenoms or mouse antisera were determined by enzyme-linked immunosorbent assay (ELISA) simultaneously, and the results indicated that Taiwanese bivalent antivenom (BAV) and Vietnamese snake antivenom-Naja (SAV-Naja) exhibited superior neutralization potency against the lethal effect of short-chain neurotoxin (sNTX) and cytotoxicity of cardiotoxin/cytotoxin (CTX), respectively. Thus, the reported peptide ELISA shows not only its potential for antivenom prequalification use, but also its capability of justifying the cross-neutralization potency of antivenoms against Naja atra venom toxicity.

Keywords: ELISA; Naja atra; antivenom; assessment; immunoreactive peptide; neutralization.

Figure 1

Cytotoxin exhibits intense cytotoxic effect as compared to other major venom toxins. Here, 1 × 10⁵ HL-60 cells were incubated with toxins of (A) CTX A3, (B) sNTX, and (C) PLA₂ in various concentrations, and the cytotoxicity of toxins was determined. The assay was performed in triplicate and the viability of toxin-treated cells is represented as mean ± SD. The images represent the morphology of cells incubated with 40 μg/mL of (D) CTX A3, (E) sNTX, and (F) PLA₂.

Figure 1

Cytotoxin exhibits intense cytotoxic effect as compared to other major venom toxins. Here, 1 × 10⁵ HL-60 cells were incubated with toxins of (A) CTX A3, (B) sNTX, and (C) PLA₂ in various concentrations, and the cytotoxicity of toxins was determined. The assay was performed in triplicate and the viability of toxin-treated cells is represented as mean ± SD. The images represent the morphology of cells incubated with 40 μg/mL of (D) CTX A3, (E) sNTX, and (F) PLA₂.

Figure 2

The recognition of antivenom toward synthetic peptides derived from sNTX and CTX A3 toxins is shown. The overlapping peptides of (A) sNTX and (B) CTX A3 were coated on the ELISA plate, and the reactivity of peptide-specific antibodies in antivenom of BAV, SAV-Naja, NPAV and DAV was measured. TFF and GIL peptides were used as negative controls in the assay. Significant difference calculated with the two-tailed Student’s t-test is marked by asterisks (peptide vs. TFF (control), **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05). The immunoreactive peptides of sNTX₂₁₋₃₅ and CTX A3_43–57 are marked in yellow in the crystal structure of (C) sNTX (PDB: 1COD) and (D) CTX A3 (PDB: 2BHI), respectively. The part marked in red represents the disulfide bridges of toxin. Images were generated using Cn3D software [37].

Figure 2

The recognition of antivenom toward synthetic peptides derived from sNTX and CTX A3 toxins is shown. The overlapping peptides of (A) sNTX and (B) CTX A3 were coated on the ELISA plate, and the reactivity of peptide-specific antibodies in antivenom of BAV, SAV-Naja, NPAV and DAV was measured. TFF and GIL peptides were used as negative controls in the assay. Significant difference calculated with the two-tailed Student’s t-test is marked by asterisks (peptide vs. TFF (control), **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05). The immunoreactive peptides of sNTX₂₁₋₃₅ and CTX A3_43–57 are marked in yellow in the crystal structure of (C) sNTX (PDB: 1COD) and (D) CTX A3 (PDB: 2BHI), respectively. The part marked in red represents the disulfide bridges of toxin. Images were generated using Cn3D software [37].

Figure 3

Analysis of the peptide-blocking effect on antivenom neutralization potency. Changes in the percentage of (A) animal survival rate (n = 6 per group) and (B) cell viability (1 × 10⁵ cells per assay) revealed that synthetic peptides of sNTX_21–35 and CTX A3_43–57 blocked the binding of antibody with the toxicity sites of toxins, which inhibited antivenom potency against venom toxicities. Significant differences were observed between groups comparing cellular viability by one-way analysis of variance followed Fisher’s least-significant difference test.

Figure 4

Correlation plots of the neutralization potencies and ELISA antibody titers of commercial antivenoms against (A) sNTX_21–35 and (B) CTX A3_43–57 peptides.

Figure 5

In-vitro cell-based neutralization assay. The sigmoid curve of cell viability was obtained by treating cells with a mixture of varying doses of CTX A3 protein together with either a constant amount of BAV (▲), SAV-Naja (○) or NPAV (■). The cells incubated with toxin alone were utilized as controls (●) in the assay. The neutralizing potency of antivenom against cytotoxicity was calculated by the equation described in the experimental section. Results were expressed as mean ± SD from three independent experiments done in triplicate.

Figure 6

Evaluation of the immunogenicity of two species-related cobra venoms in mice. (A) Detection of antibody titers against peptide of sNTX_21–35 in mice immunized with either N. atra or N. kaouthia venom. (B) The survival rate of venom-immunized mice after challenging with N. atra venom (3 × LD₅₀). (C) Correlation of CTX A3_43–57-specific antibody titers with neutralization potency (P_c) of pooled antiserum obtained from in-vitro cell assay. The antibody titer is expressed as mean ± SD.