Third Generation Antivenomics: Pushing the Limits of the In Vitro Preclinical Assessment of Antivenoms

Abstract

Second generation antivenomics is a translational venomics approach designed to complement in vivo preclinical neutralization assays. It provides qualitative and quantitative information on the set of homologous and heterologous venom proteins presenting antivenom-recognized epitopes and those exhibiting impaired immunoreactivity. In a situation of worrying antivenom shortage in many tropical and sub-tropical regions with high snakebite mortality and morbidity rates, such knowledge has the potential to facilitate the optimal deployment of currently existing antivenoms and to aid in the rational design of novel broad specificity antidotes. The aim of the present work was to expand the analytical capability of the immunoaffinity second-generation antivenomics platform, endowing it with the ability to determine the maximal binding capacity of an antivenom toward the different toxins present in a venom, and to quantify the fraction of venom-specific antibodies present in a given antivenom. The application of this new platform, termed third generation (3G) antivenomics, in the preclinical evaluation of antivenoms is illustrated in this paper for the case of antivenom EchiTAb-Plus-ICP^® reactivity towards the toxins of homologous (B. arietans) and heterologous (N. melanoleuca) venoms.

Keywords: antivenom; preclinical assessment of antivenom; size-exclusion analysis of IgG-toxin complexes; snake venom; third generation antivenomics.

Figure 1

Reverse-phase HPLC profiles of 100 µg of B. arietans (Ghana) venom toxins (panel a). Toxins eluting in the different chromatographic peaks were reported in [26,27]. DISI, disintegrin; Kun, Kunitz-type inhibitor; PLA₂, phospholipase A₂; SerPro, serine proteinase; CTL, C-type lectin-like protein; PI- and PIII-SVMP, snake enom metalloproteinase of class PI and PIII, respectively. Panels b–i, not immunoretained venom fraction in 300 µL of immobilized (8 mg) EchiTAb-Plus-ICP^® antivenom immunoaffinity columns incubated with increasing amounts (100–1500 μg), respectively, of venom proteins. Panels j and k, not immunoretained and retained venom fractions in a mock column run in parallel to the immunoaffinity columns as matrix control; panel l, specificity control, venom proteins retained in a control IgG column.

Figure 2

Graphical representation of the binding capacity of the different toxin classes of B. arietans (Ghana) venom in 300 µL of immobilized (8 mg) EchiTAb-Plus-ICP^® antivenom immunoaffinity columns as a function of the amounts of incubated venom proteins (100–1500 μg). The maximal binding capacity of EchiTAb-Plus-ICP^® for toxin “I”, defined as the ratio (µg toxin “i”/mg antivenom) that saturates the affinity column (Rsat“i”), was determined in a series of antivenomic experiments where a set of identical affinity columns are incubated with increasing amounts of venom, and plotting the amount of µg of toxin “i” immunoretained in the columns against the total amount of toxin“i”, (t_“i”)_TOT, contained in the incubated venom samples. (t_“i”)_TOT (in µg) = ((% of toxin“i” in the venom) × µg incubated venom)/100, and the relative abundance (%) of toxin “i” corresponds to the % of the total chromatographic area.

Figure 3

Reverse-phase HPLC profiles of 100 mg of N. melanoleuca (Ghana) venom toxins (panel a). Toxins eluting in the different chromatographic peaks has been reported in [33] and unpublished results. 3FTx, three-finger toxin; PLA₂, phospholipase A₂; CRISP, cysteine-rich secretory protein; PIII-SVMP, snake venom metalloproteinase of class PIII; PDE, phosphodiesterase; LAO, L-amino acid oxidase. Panels b–h, not immunoretained venom fraction in 300 µL of immobilized (8 mg) EchiTAb-Plus-ICP^® antivenom immunoaffinity columns incubated with increasing amounts (100–1200 μg), respectively, of venom proteins.

Figure 4

Graphical representation of the binding capacity of the different toxin classes of N. melanoleuca (Ghana) venom in 300 µL of immobilized (8 mg) EchiTAb-Plus-ICP^® antivenom immunoaffinity columns as a function of the amounts of incubated venom proteins (100–1200 μg). The maximal binding capacity of EchiTAb-Plus-ICP^® for forest cobra venom toxins was calculated as described in the legend of Figure 2.

Figure 5

Size-exclusion chromatographic approach used to quantify the fraction of EchiTAb-Plus-ICP^® IgG molecules toxin capable of forming stable complexes with B. arietans (Ghana) venom toxins. Panel (A) displays SEC elution profiles of different amounts of B. arietans venom proteins used to construct the calibration standard curve displayed in panel (B). Zone 1 includes venom components that overlap with antivenom IgGs, and antigen-antibody complexes. Zone 2 is the region of the chromatogram where free venom proteins are eluted. Panel (C) displays superposition of SEC profiles of reaction mixtures comprising a constant amount of antivenom (300 µg) incubated with increasing amounts (12.75–306 µg) of B. arietans venom proteins. Increment of zone 1 area was attributed to antivenom-venom complex formation. and calculated using the Equation (Area venom complexed with antivenom IgG) = (total area zone-1) − (area 300µg IgG) − (area zone-1 control venom curve). Panel (D) displays the calculated area attributed to antivenom-venom complexes as a function of the amount of venom in the reaction mixture. Blue line, sum of the zone 1 areas of the standard curves of venom and antivenom; Magenta line, area of zone 1 of the chromatographic profiles of venom-antivenom mixtures. The maximal increment in area was then used to calculate, using the calibration curve plotted in (B), the maximal binding capacity of a defined amount of EchiTAb-Plus-ICP^® antivenom.