Comparing Traditional and Toxin-Oriented Approaches towards Antivenom Production against Bitis arietans Snake Venom

Abstract

Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom ("traditional approach") and immunization with selected key toxins isolated from the snake venom ("toxin oriented" approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa.

Keywords: Bitis; Bitis arietans; antibody; antivenom; snake venom; sub-Saharan Africa.

Figure 1

Profile of B. arietans venom obtained by molecular size exclusion chromatography and electrophoretic profiling. (a) Venom samples (100 µg) were fractioned in a Shim-Pack DIOL-300 column at a flow of 0.5 mL/min of disodium phosphate buffer. Absorbance was measured at 280 nm. (b) Fractions corresponding to crude venom (1 µg) and peaks 1 to 7 (2 µg) were submitted to 12.5% SDS-PAGE in non-reducing conditions and silver stained.

Figure 2

Western blot analysis showing the recognition of obtained peaks from B. arietans venom after size exclusion chromatography with monovalent anti-B. arietans (a) and polyvalent anti-B. nasicornis + B. rhinoceros antivenoms (b). B. arietans venom (8 µg) or peaks (3 µg) samples were submitted to 12.5% SDS-PAGE in non-reducing conditions under a current of 100 V and transferred to nitrocellulose membranes. Primary antibodies were diluted 1:1000 in PBS containing 0.1% BSA. The detection antibody (anti-horse conjugated with alkaline phosphatase) was diluted 1:7500 in PBS containing 0.1% BSA. Antibodies were incubated for 1 h at room temperature.

Figure 3

Progression of the immune response. (a) Balb/C mice (n = 7/group) were immunized with B. arietans venom (1.0 µg/animal) or with samples from peaks 2, 3, or 5 (0.5 µg/animal). Animals received 6 subcutaneous inoculations at intervals of 15 days. Before immunizations, blood was collected, and plasma was submitted to titration by ELISA. Plates were sensitized with 1 µg/well of B. arietans venom. Experimental antibodies were serially diluted from 1:1000 to 1:512,000 in PBS containing 0.1% BSA. Titers from the 6th immunization were compared between groups. (b) Samples from the 6th immunization were submitted for antibody affinity determination by ELISA. Antibodies were diluted 1:1000 in PBS containing 0.1% BSA. The affinity score was determined as the KSCN molarity necessary to displace 50% of the antibodies bound at KSCN 0 M. The detection antibody (anti-mouse IgG conjugated with peroxidase) was diluted 1:5000 in PBS containing 0.1% BSA. Experiments were performed in duplicate. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s post-test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Progression of the immune response. (a) Balb/C mice (n = 7/group) were immunized with B. arietans venom (1.0 µg/animal) or with samples from peaks 2, 3, or 5 (0.5 µg/animal). Animals received 6 subcutaneous inoculations at intervals of 15 days. Before immunizations, blood was collected, and plasma was submitted to titration by ELISA. Plates were sensitized with 1 µg/well of B. arietans venom. Experimental antibodies were serially diluted from 1:1000 to 1:512,000 in PBS containing 0.1% BSA. Titers from the 6th immunization were compared between groups. (b) Samples from the 6th immunization were submitted for antibody affinity determination by ELISA. Antibodies were diluted 1:1000 in PBS containing 0.1% BSA. The affinity score was determined as the KSCN molarity necessary to displace 50% of the antibodies bound at KSCN 0 M. The detection antibody (anti-mouse IgG conjugated with peroxidase) was diluted 1:5000 in PBS containing 0.1% BSA. Experiments were performed in duplicate. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s post-test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Recognition of protein bands present in Bitis spp. venoms by experimental antivenoms. Samples of B. arietans, B. nasicornis, and B. rhinoceros venom (5 µg) were submitted to 12.5% SDS-PAGE in non-reducing conditions and transferred to nitrocellulose membranes. Experimental antivenoms Anti-Ba (a), Anti-P2 (b), Anti-P3 (c), and Anti-P5 (d) were diluted 1:500 in PBS containing 0.1% BSA. The detection antibody (anti-mouse conjugated with alkaline phosphatase) was diluted 1:5000 in PBS containing 0.1% BSA. Antibodies were incubated for 1 h at room temperature.

Figure 5

Cross-reaction with different Bitis spp. venoms. Plates were sensitized with 1 µg/well of antigen. Experimental antivenoms were serially diluted from 1:500 to 1:256,000 in PBS containing 0.1% BSA. Detection antibodies (anti-mouse IgG conjugated with peroxidase) were diluted 1:5000 in PBS containing 0.1% BSA. Absorbance at 420 nm was recorded. Titers are expressed as ELISA units/mL. Experiments were performed in duplicate. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s post-test, *** p < 0.001.