Fig 1.
Snakebite incidence in Colombia.
The figure is adapted from the event report, epidemiological period XIII, Colombia, 2019 [17]. White dashed lines delimit the Colombian Insular (1), Caribbean (2), Andean (3), Pacific (4), Orinochian (5) and Amazonian (6) natural regions. Snakebite incidence data from the Insular region (San Andrés, Providencia, and Santa Catalina) were "missing or excluded" in the 2019 SIVIGILA-INS event report. The left panel highlights species within the clinically important snake families Viperidae and Elapidae with distribution in the Departments of Nariño and/or Cauca, which can potentially cause snakebite accidents in this region. The list was compiled from Ayerbe and Latorre [22] and Sevilla-Sánchez et al. [21]. 1B. asper includes the lineages B. asper (sensu stricto), B. rhombeatus and B. ayerbei. The map was prepared in the QGIS software, version 3.14.15-Pi, using public domain maps available in QGIS OpenLayers Plugin, and the Geoportals of the Instituto Geográfico Agustín Codazzi-IGAC (https://geoportal.igac.gov.co/contenido/datos-abiertos-cartografia-y-geografia) and the Departamento Administrativo Nacional de Estadística-DANE (https://geoportal.dane.gov.co/servicios/descarga-y-metadatos/descarga-mgn-marco-geoestadistico-nacional/) from Colombia. All maps were used under a CC-BY 4.0 license.
Table 1.
Characteristics of the equine polyvalent antivenoms used in this study.
Table 2.
Reference doses of B. asper venoms considered for the experimental design of this study.
Fig 2.
SDS-PAGE analysis of the polyvalent antivenoms used manufactured by Instituto Nacional de Salud (Colombia) (INS-COL), Laboratorios Probiol S.A, (Colombia) (PROBIOL), Instituto Nacional de Salud de Perú (INS-PERU), Instituto Clodomiro Picado (Costa Rica) (ICP), Centro de Biotecnología de la Unidad de Farmacia de la Universidad Central de Venezuela, (UCV), and Instituto Biológico Argentino S.A.I.C. (BIOL).
Lanes 1a/b or 2a/b, vials used in the experiments. Molecular weight markers (MW) are indicated on the left. Coomassie Brilliant blue-stained bands labeled 1–37 were excised and submitted to tandem mass spectrometry analysis (S1 Table). Bands containing IgG aggregation or degradation products are identified by yellow-filled circles. Red-filled circles denote non-IgG proteins bands.
Fig 3.
Immunocapture capacity of polyvalent antivenoms towards B. asper (sensu stricto) venom from Cauca, Colombia.
Panel A displays the fractionation by reverse-phase HPLC of the venom components. Proteins eluting in each peak (1–14) were assigned using the venomics information reported by Mora-Obando et al. [32]. SVMP, snake venom Zn2+-metalloproteinase and their proteolytic fragments Disintegrin/Cysteine fragments (DC-frag); PLA2-K49/D49, phospholipase types Lys49 and Asp49; SVSP, serine proteinase; CTL, C-type lectin-like; DIS, disintegrin; CRISP, cysteine-rich secretory protein; LAO, L-amino acid oxidase; PDE, phosphodiesterase; SVMPi, SVMP inhibitors; BPP, bradykinin-potentiating-like peptides. Panels B-G represent RP-HPLC fractionations of the non-immunoretained fractions recovered in the flow-through fraction of the affinity columns of immobilized antivenoms INS-COL (B), PROBIOL (C), ICP (D), INS-PERU (E), UCV (F), BIOL (G) incubated with increasing amounts of venom (100–1200 μg). Panels H and I display to chromatographic separations of the venom fraction not retained in the mock matrix control and the naïve equine immunoglobulins control, respectively. The numbers on top of the chromatographic peaks represent the percentage of each fraction.
Fig 4.
Immunocapture capacity of polyvalent antivenoms towards B. rhombeatus venom from Cauca river valley, Colombia.
Panel A displays the fractionation by reverse-phase HPLC of the venom components. Proteins eluting in each peak (1–14) were assigned using the venomics information reported by Mora-Obando et al. [32]. Panels B-G represent RP-HPLC fractionations of the non-immunoretained fractions recovered from the affinity columns of immobilized antivenoms INS-COL (B), PROBIOL (C), ICP (D), INS-PERU (E), UCV (F), BIOL (G) incubated with increasing amounts of venom (100–1200 μg). Panels H and I display to chromatographic separations of the venom fraction not retained in the mock matrix control and the naïve equine immunoglobulins control, respectively. The numbers on top of the chromatographic peaks represent the percentage of each fraction.
Fig 5.
Immunocapture capacity of polyvalent antivenoms towards B. ayerbei venom from Patia river valley, Colombia (A). Panel A displays the fractionation by reverse-phase HPLC of the venom components. Proteins eluting in each peak (1–14) were assigned using the venomics information reported by Mora-Obando et al. [28]. Panels B-G represent RP-HPLC fractionations of the non-immunoretained fractions recovered from the affinity columns of immobilized antivenoms INS-COL (B), PROBIOL (C), ICP (D), INS-PERU (E), UCV (F), BIOL (G) incubated with increasing amounts of venom (100–1200 μg). Panels H and I display to chromatographic separations of the venom fraction not retained in the mock matrix control and the naïve equine immunoglobulins control, respectively. The numbers on top of the chromatographic peaks represent the percentage of each fraction.
Table 3.
Percentages of NOT immunoretained fractions of B. asper venoms by six Latin American antivenoms.
Table 4.
Summary of third generation antivenomics analyses of polyvalent antivenoms against venoms of the three B. asper lineages from south-western Colombia.
Fig 6.
Titration curves of polyvalent antivenoms against the venoms of three lineages of B. asper venoms.
Antivenoms INS-COL (●), PROBIOL (), ICP (○) were serially diluted by a factor of two (starting from a dilution of 1/1000) and tested by ELISA against the following crude venoms: B. asper (sensu stricto) (A), B. rhombeatus (B) and B. ayerbei (C). Equine normal serum was included as negative control (◆). Each point represents the mean ± SD of three independent determinations. Statistically significant differences were observed among the titers of the antivenoms INS-COL vs. PROBIOL and/or ICP against the venoms of B. asper (dilutions 1: 1000, 8000, 16000, 64000), B. rhombeatus (dilutions 1:2000–128000) and B. ayerbei (dilutions 1:1000–16000).
Fig 7.
Comparison of the neutralizing capacity of the antivenoms manufactured by INS-COL (A-C), PROBIOL (D-F), ICP (G-I) towards the hemorrhagic effect caused by the venoms of B. asper (sensu stricto) (●), B. rhombeatus (), B. ayerbei (○). Hemorrhagic lesions were measured as hemorrhagic units (HaU) [55] 2 h after intradermal injection of 10 MHD (challenge dose) mixed with antivenom in the ratios indicated in the figure. Each point represents the mean ± SD of four replicates. In all assays, statistically significant differences (p<0.05) were observed among the ratios 500–1000 μL antivenom/mg venom compared to the positive control.
Fig 8.
Comparison of the neutralizing capacity of the antivenoms manufactured by INS-COL (A-C), PROBIOL (D-F), ICP (G-I) towards the coagulant effect caused by the venoms of B. asper (sensu stricto) (●), B. rhombeatus (), B. ayerbei (○). Clotting time was recorded after mixing and incubating 2 MCD (challenge dose) with different ratios of antivenom, as indicated in the figure, and adding them to human citrated plasma. Each point represents the mean ± SD of replicates of two independent experiments.
Fig 9.
Comparison of the neutralizing capacity of the antivenoms manufactured by INS-COL (A-D), PROBIOL (E-H), ICP (I-L) towards the myotoxic effect caused by the venoms of B. asper (sensu stricto) (A, E, I), B. rhombeatus (B, F, J), B. ayerbei (C, D, G, H, K, L). Muscle damage was measured as plasma creatine kinase activity 3 h after intramuscular injection of 50 μg of venom mixed with antivenom in the ratios indicated in the figure (●). Antivenom control (○). PBS control (---). Each point represents the mean ± SD of four replicates. Statistically significant differences (p<0.05) compared to the venom control are represented by asterisks.
Fig 10.
Comparison of the neutralizing capacity of the antivenom manufactured by INS-COL (A-C), PROBIOL (D-F), ICP (G-I) towards the edematogenic effect caused by the venom of B. asper (sensu stricto) (A, D, G), B. rhombeatus (B, E, H) and B. ayerbei (C, F, I). Edema was measured as the increase in the thickness of footpad compared to the negative control (PBS) and monitored during 6 h after subcutaneous injection of 5 μg of venom (●) mixed with antivenom in the ratios: 1000 μL antivenom/mg venom (○), 500 μL antivenom/mg venom (Δ), 250 μL antivenom/ mg venom (). Each point represents the mean ± SD of four replicates. Statistically significant differences (p<0.05) compared to the venom control are represented by asterisks.
Fig 11.
Proteolytic activity of the venoms of B. asper (sensu stricto), B. rhombeatus, and B. ayerbei (A) and neutralizing capacity of the antivenoms manufactured by INS-COL (●), PROBIOL (○) and ICP () towards B. asper (B), B. rhombeatus (C) and B. ayerbei (D). Neutralization of the proteolytic activity was measured on azocasein, as described in the Material and methods section, 90 min after mixing and incubating 12.5 μg of venom (challenge dose) with antivenom in the ratios indicated in the figure. Each point represents the mean ± SD of three replicates. Statistically significant differences (p<0.05) compared to the venom controls are represented by asterisks.
Fig 12.
Indirect hemolytic effect produced by venoms of B. asper, B. rhombeatus, and B. ayerbei (A) and neutralizing capacity of the antivenoms INS-COL (●), PROBIOL (○) and ICP () towards B. asper (sensu stricto) venom (B) and B. rhombeatus (C). Neutralization of indirect hemolysis was measured on rabbit erythrocytes, as described in the Material and methods section, 1 h after mixing and incubating of 5.1 μg of venom (challenge dose) with antivenom in the ratios indicated in the figure. Each point represents the mean ± SD of three replicates. Statistically significant differences (p<0.05) compared to the venom control are represented by asterisks.
Table 5.
Neutralization of biological activities of B. asper venoms by polyvalent antivenoms INS-COL, PROBIOL and ICP.
Fig 13.
Phylogenetic tree of Bothrops highlighting some species reported by [88], whose venoms have been shown to exhibit remarkable immunoreactivity towards homologous and heterologous antivenoms produced in different Latin American countries using immunization mixtures that include different bothropic venoms.