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Variability in antivenom neutralization of Mexican viperid snake venoms [1]

['Alid Guadarrama-Martínez', 'Departamento De Medicina Molecular Y Bioprocesos', 'Instituto De Biotecnología', 'Universidad Nacional Autónoma De México', 'Cuernavaca', 'Morelos', 'Edgar Neri-Castro', 'Facultad De Ciencias Biológicas', 'Universidad Juárez Del Estado De Durango', 'Gómez Palacio']

Date: 2024-05

Neutralization of lethality.

Sixty-two neutralizations of the lethal activity were performed (Tables 3 and S1), and their specific neutralization values were classified into 5 categories according to the mg of antivenom required to neutralize the lethal activity of 1 mg of venom (mgAV/mgV). The categories were established as follows: well neutralized (ranging from 1.0 to 8.9 mgAV/mgV), medium neutralized (ranging from 9.0 to 16.9 mgAV/mgV), poorly neutralized (ranging from 17.0 to 24.9 mgAV/mgV), very poorly neutralized (above 25 mgAV/mgV), and not neutralized (no neutralization of lethality, even with the highest amount of antivenom tested). Among the venoms tested, the best neutralized were those from B. asper and C. basiliscus. All eight lots tested achieved good neutralization of B. asper venom, while the venom of C. basiliscus was well neutralized by 7 lots, with B3 showing medium neutralization.

The three lots of Antivipmyn and the two lots of Inoserp exhibited medium neutralization of C. atrox venom. In comparison, two lots of Birmex poorly neutralized it, and B3 showed very poor neutralization. The venom of C. mictlantecuhtli was found to be very poorly neutralized by B1, B3, and I2; poorly neutralized by A3 and B2; medium neutralized by A2 and I1, and only A1 showed good neutralization. Regarding A. bilineatus venom, all three batches of Birmex showed very poor neutralization, the two batches of Inoserp poorly neutralized it, and two batches of Antivipmyn exhibited good neutralization, while A1 showed medium neutralization. On the other hand, no neutralization was observed by A2 or I2 for C. tzotzilorum venom. However, the remaining two lots of Antivipmyn showed good neutralization, and all three lots of Birmex medium neutralized it, whereas I1 poorly neutralized it. Interestingly, none of the eight tested lots could neutralize M. nummifer venom. Lastly, O. sphenophrys and P. yucatanicum venom were evaluated against one product batch (A1, B1, and I1). The first venom was medium neutralized by A1 but poorly neutralized by B1 and I1. However, none of these three batches could neutralize the lethality of P. yucatanicum (Table 3).

Regarding the Antivipmyn batches, out of the 23 neutralization tests conducted, 11 (48%) exhibited good neutralization, 6 (26%) showed medium neutralization, one (4%) was classified as bad neutralization, and 5 (22%) did not neutralize even with the highest amount of antivenom tested. For Birmex, which also underwent 23 neutralization tests, 5 (22%) demonstrated good neutralization, 4 (17%) exhibited medium neutralization, another 4 (17%) displayed bad neutralization, 6 (26%) showed very bad neutralization, and in 4 (17%) there was no neutralization observed. In the case of Inoserp, as only 2 lots were tested, a total of 16 trials were conducted, where 4 (25%) showed good neutralization, 3 (19%) displayed medium neutralization, 4 (25%) demonstrated bad neutralization, only one (6%) showed very bad neutralization, and in 4 (25%) there was no neutralization observed.

Theoretically, antivenoms would show better neutralization towards the venoms used as immunogens; however, this was only the case for Birmex. For example, Antivipmyn has a better specific neutralization of C. basiliscus venom than that of C. mictlantecuhtli; such cross-reactivity could be explained by the antigenic similarity of these venoms [36]. Inoserp, on the other hand, although it includes C. atrox venom in its immunization mixture, its specific neutralization is not very different from that of the venoms not used in its preparation. In addition, antivenoms can neutralize (generally with lesser efficacy) some venoms not used as immunogens in their preparation. This cross-neutralization is due to the similarity between the toxins in the venom of phylogenetically related snakes [59]. Previous work has reported the immunogenic deficiency of C. atrox venom, finding that antibodies produced from this immunogen have lower recognition by the homologous venom compared to those generated from the venom of other species of the genus [60]. Additionally, specific neutralization values of Birmex against C. atrox venom of 12.8 mgAV/mgV have been obtained [51], which represents double the neutralizing potency for Birmex lot 3 analyzed in the present work, highlighting the variation between lots of the same antivenom.

The venoms not neutralized by the antivenoms were M. nummifer and P. yucatanicum. Similarly, two of the eight batches failed to neutralize the lethality of C. tzotzilorum venom. Although there are no reports of neutralization trials for either P. yucatanicum or C. tzotzilorum venoms, the results are contrasting for the case of M. nummifer. Neutralization of M. nummifer venom by Antivipmyn injecting five LD 50 intraperitoneally has been reported [61]; however, in another study, the lethality was not neutralized by Antivipmyn nor Birmex when challenged with two or three LD 50 injected intravenously [43]. This difference could be explained by the variability in the neutralizing potency of the antivenoms when comparing different batches. In the case of Porthidium, the neutralization of P. nasutum venom from Colombia by Antivipmyn, whose specific neutralization is 8.3 mgAV/mgV, has been reported [33].

We observed a wide variation in the neutralizing potency of the antivenom lots, clear examples being the tests against the venom of Ophryacus, Cerrophidion, and Porthidium. This could be due to various reasons, including the possibility that the plasma mix arises from different horses, changes in both the immunization mix, and in the final protein concentration of each vial may result in a different final product. For this reason, we recommend the use of constant immunogenic mixes to maintain consistent neutralization results.

In Mexico, antivenoms are regulated by the Pharmacopoeia of the United Mexican States (FEUM) and the Official Mexican Standard NOM-036-SSA2-2012, which on a per-vial basis establishes "a neutralizing capacity of not less than 790 LD 50 per mouse of Crotalus sp. venom and not less than 780 LD 50 per mouse of Bothrops sp. venom" [62]. To compare our results with the specifications required by NOM-036-SSA2-2012, the neutralizing potencies are also expressed as neutralized LD 50 per vial (LD 50 /vial) (Fig 6). In this aspect, the analyzed batches of Antivipmyn and Birmex comply with the standard when challenged against the venoms used in their manufacture. The tested batches of Inoserp, which is not currently licensed under NOM-036-SSA2-2012, would not have met the minimum neutralization threshold using C. atrox venom.

Although species-specific C. atrox neutralization is not mandated by the FEUM, C. atrox venom has been broadly studied because of its medical importance in both northern Mexico and the United States [63], and its neutralization is an important basis for comparison across antivenom products. All batches of Inoserp and Antivipmyn showed a medium neutralization of C. atrox venom on a mgAV/mgV basis, but this is not reflected in the number of neutralized LD 50 , which ranges from 102 to 178 LD 50 /vial. On the other hand, two batches of Birmex neutralized it poorly, and B3 neutralized it very poorly; however, on average, they neutralized more than twice the LD 50 per vial compared to the other products. In summary, none of the eight batches tested neutralized more than 350 LD 50 of C. atrox venom, putting them well below the 790 LD 50 standard of NOM-036-SSA2-2012. However, this standard does not define the species of the genus Crotalus from which venom should be neutralized. The choice of test species used by manufacturers and regulators is essential to releasing commercial lots. However, this leaves the question of whether the standards by genus-level potency support a broad clinical efficacy.

Although preclinical analyses are required to verify the safety and evaluate the effectiveness of an antivenom [64], these studies have physiological limitations because the venom and antivenom injection protocols do not correspond to a real scenario of envenoming. There is even the possibility that the organic response to envenoming/treatment on the part of murine models may differ from that developed by human patients [65]. Although the use of animal models is currently the best tool for analyzing the symptoms associated with poisoning, it is imperative to avoid simplistic extrapolations of these tests to the clinical situation [64].

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[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0012152

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