RESUMEN

Objetivos:

Evaluar la capacidad del suero hiperinmune de llama (Lama glama) para neutralizar la letalidad del veneno de la serpiente Bothrops atrox en ratones de laboratorio.

Materiales y métodos:

Se calculó la dosis letal media (DL₅₀) de un pool de venenos de serpientes de Bothrops atrox de Perú, y se midieron los títulos de anticuerpos por ensayo ELISA; así como la potencia de neutralización del suero inmune por el cálculo de la dosis efectiva media (DE₅₀) durante el periodo de inmunización.

Resultados:

La DL₅₀ del veneno fue de 3,96 µg/g, similar a otros trabajos realizados en Bothrops atrox en Perú. Los títulos de anticuerpos contra el veneno se incrementan rápidamente en la llama mostrando una rápida respuesta inmune; sin embargo, la capacidad de neutralización se incrementa más lentamente y requiere de varias dosis y refuerzos de las inmunizaciones alcanzado una DE₅₀ de 3,30 µL/g ratón y una potencia de neutralización 3,6 mg/mL después de 15 inmunizaciones.

Conclusiones:

El suero hiperinmune de llama es capaz de neutralizar la letalidad del veneno de la serpiente Bothrops atrox de Perú en ratones de laboratorio.

Palabras clave:
Bothrops atrox; Lama glama; Neutralización; Sueros Inmunes; Dosis Letal 50; Letalidad

ABSTRACT

Objectives:

To evaluate the capacity of the hyperimmune llama serum (Lama glama) to neutralize the lethal activity of Bothrops atrox venom in laboratory mice.

Materials and methods:

Mean lethal dose (LD₅₀) was calculated from a Bothrops atrox venom sample pool from Peru. The antibody titers were measured by ELISA assay; and the immune serum neutralization potency was measured by calculating the mean effective dose (ED₅₀) during the immunization period.

Results:

The venom’s LD₅₀ was 3.96 μg/g; similar to what was found in other studies about Bothrops atrox carried out in Peru. The titers of antibodies against the venom increased rapidly in the llama, demonstrating a fast immune response; however, the neutralization capacity increased slowly and required several doses and immunization reinforcements, obtaining a ED₅₀ of 3.30 μL/g mouse and a neutralization potency of 3.6 mg/mL after 15 immunizations.

Conclusions:

The hyperimmune llama serum is able to neutralize the lethality of the Bothrops atrox venom from Peru in laboratory mice.

Keywords:
Bothrops atrox; Lama glama; Neutralization; Immune Sera; Lethal Dose 50; Mortality

INTRODUCTION

Ophidism is a syndrome caused by snake venom poisoning, usually species from Viperidae and Elapidae families¹1. Loja OD, Aviles GR, Necochea VY, Vilca VM, Castro TJ. Ofidismo por Bothrops atrox: estudio clínico-epidemiológico. Diagnóstico. 2000;39(5):261-5.^,22. Poggi D. Veneno de serpiente en la industria e investigación farmacológica-Plan estratégico de desarrollo de la bioindustria en el eje Amazonas-Marañón [Internet]. 2002. Disponible en: http://www.iiap.org.pe/upload/publicacion/CD_PEBIAM/documentos/BIO%203/BIO3-A.pdf.
http://www.iiap.org.pe/upload/publicacio... . In Peru, the Bothrops atrox snake is known as the species of greatest medical relevance, responsible for 88%-92% of the ophidism cases in the country ¹1. Loja OD, Aviles GR, Necochea VY, Vilca VM, Castro TJ. Ofidismo por Bothrops atrox: estudio clínico-epidemiológico. Diagnóstico. 2000;39(5):261-5.^,33. Ministerio de Salud. Norma Técnica N° 007-MINSA-DGSP. MINSA 2004. Sobre Prevención y Tratamiento De Accidentes Por Animales Ponzoñosos [Internet]. Lima: MINSA; 2004. Disponible en: http://bvs.minsa.gob.pe/local/dgsp/123_NTPONZONOSOS.pdf.
http://bvs.minsa.gob.pe/local/dgsp/123_N... , causing morbimortality and leaving sequelae, such as amputations or lacerations in the affected parts. Tissue damage is caused by several biological activities from venom’s enzymes, such as the proteolytic (destruction of structural proteins), coagulating, vascular toxic, and nephrotoxic effects ⁴4. Zelanis A, Tashima AK. Unraveling snake venom complexity with 'omics' approaches: challenges and perspectives. Toxicon. 2014;87:131-4. doi: 10.1016/j.toxicon.2014.05.011.
https://doi.org/10.1016/j.toxicon.2014.0... .

The clinical manifestations are characterized by pain, edema, ecchymosis, erythema, and necrosis. In severe cases, vesicles or blisters with serous and hemorrhagic contents may appear, as well as hematemesis and hemorrhagic shock. The only pharmacologically valid treatment for cases of ophidism is the application of passive artificial immunotherapy, by means of the transfer of antivenom IgG antibodies, generally heterologous, of equine origin ⁵5. Mora D. Productividad Antiofídica De Equinos Destinados a la Industria Inmunobiológica en Costa Rica. Nutrición Animal Tropical. 2014;8(1):44-54..

The Instituto Nacional de Salud (INS) produced the polyvalent anti-bothropic serum, a purified solution of specific IgG immunoglobulins obtained from the plasma of hyperimmunized equines with a pool of Bothrops snake venom, specifically Bothrops atrox, Bothrops pictus, Bothrops barnetti, Bothrops braziili and Bothrocophias hyoprora²2. Poggi D. Veneno de serpiente en la industria e investigación farmacológica-Plan estratégico de desarrollo de la bioindustria en el eje Amazonas-Marañón [Internet]. 2002. Disponible en: http://www.iiap.org.pe/upload/publicacion/CD_PEBIAM/documentos/BIO%203/BIO3-A.pdf.
http://www.iiap.org.pe/upload/publicacio... ^,33. Ministerio de Salud. Norma Técnica N° 007-MINSA-DGSP. MINSA 2004. Sobre Prevención y Tratamiento De Accidentes Por Animales Ponzoñosos [Internet]. Lima: MINSA; 2004. Disponible en: http://bvs.minsa.gob.pe/local/dgsp/123_NTPONZONOSOS.pdf.
http://bvs.minsa.gob.pe/local/dgsp/123_N... ^,66. Segura A, Castillo MC, Núñez V, Yarlequé A, Gonçalves LR, Villalta M, et al. Preclinical assessment of the neutralizing capacity of antivenoms produced in six Latin American countries against medically-relevant Bothrops snake venoms. Toxicon. 2010;56(6):980-9. doi: 10.1016/j.toxicon.2010.07.001.
https://doi.org/10.1016/j.toxicon.2010.0... .

Some of the problems associated to the production of the venom antidote are the need to use snakes to obtain the venom, the implicit risk in obtaining the venom, the use of horses to obtain the hyperimmune serum, and the subsequent extraction of the total antibodies which contain the venom neutralizing antibodies and other antibodies from the horse ¹1. Loja OD, Aviles GR, Necochea VY, Vilca VM, Castro TJ. Ofidismo por Bothrops atrox: estudio clínico-epidemiológico. Diagnóstico. 2000;39(5):261-5.^,22. Poggi D. Veneno de serpiente en la industria e investigación farmacológica-Plan estratégico de desarrollo de la bioindustria en el eje Amazonas-Marañón [Internet]. 2002. Disponible en: http://www.iiap.org.pe/upload/publicacion/CD_PEBIAM/documentos/BIO%203/BIO3-A.pdf.
http://www.iiap.org.pe/upload/publicacio... . The equines used often suffer from hepatomegaly, because of the venom immunization. There is some research aimed at improving the physiological and biochemical processes, including testing alternative adjuvants that improve the release and assimilation of the venom within the horse, in order to reduce the negative effects of the inoculations ⁵5. Mora D. Productividad Antiofídica De Equinos Destinados a la Industria Inmunobiológica en Costa Rica. Nutrición Animal Tropical. 2014;8(1):44-54..

In addition, complete equine antibodies contain the constant fraction in the heavy chain, which are recognized by the receptors in various types of cells, such as NK lymphocytes, neutrophils, macrophages, B cells, and mast cells, and by complement factors. Complete equine antibodies can also cause other adverse reactions known as serum sickness, which is mainly characterized by urticaria, cough, nausea and vomiting, tachycardia, and headaches. Additionally, in many cases the patient may develop systemic anaphylaxis with symptoms, such as hypotension, bronchospasm, and angioedema ⁷7. Rodríguez S, Negrin A, Burger M. Efecto adverso por suero antibothrópico. Rev Med Uruguay. 2004; 20:228-32..

The main objective of this study was to determine the effectiveness of the hyperimmune llama serum (Lama glama) as a neutralizer of the lethal activity of the Peruvian snake venom Bothrops atrox; and to provide knowledge regarding new alternatives to biological products, which will contribute to the INS production capacity of antiophidic serums.

MATERIALS AND METHODS

This is an analytical experimental study.

Experimental animals

A male llama (Lama glama) was used to obtain the pre-immune and post-immune serum. The animal was obtained from an agricultural center (SAIS Pachacutec), it was healthy, and passed a period of veterinary evaluation and quarantine of 40 days prior to experimentation. BALB/c mice (Mus musculus) of 17 g to 19 g were used from the biotherium of the National Center of Biological Production of the INS, which were kept in a controlled environment in the laboratory. For mice handling, the researchers followed the Guide for Handling and Care of Laboratory Animals: Mice, from the INS ⁸8. Instituto Nacional de Salud. Guía de Manejo y Cuidado de Animales de Laboratorio: ratón [Internet]. Lima: MINSA, INS; 2008. Disponible en: https://www.ins.gob.pe/insvirtual/images/otrpubs/pdf/GUIA_ANIMALES_RATON.pdf..

Procedures

Obtaining and preserving the venom

Venom samples were obtained by manual extraction, collected in a beaker, and transported to the laboratory via cold chain. They were centrifuged at 5,000 rpm for 20 minutes to remove foreign components, and 2 mL aliquots were taken in glass vials. The vials were frozen at -80 °C overnight, and placed in a freeze-dryer until a homogeneous tablet was formed; the net weight of the freeze-dried venom was registered and stored at -80 °C until use.

Protein electrophoresis by SDS-PAGE

The venom was treated with a protein buffer under reducing conditions for 5 minutes at 100 °C and then underwent electrophoresis by the SDS-PAGE method at 12% for 1 hour at 100 volts. A protein molecular-weight marker was used (Thermo Scientific #26623). The gel was stained with Coomassie blue for 1 hour and then washed with a bleaching solution. For the serum, the conditions of electrophoresis, staining and gel processing were the same as for the venom.

Determination of the average lethal effect

The stock venom solution was prepared at a 1 mg/mL concentration using sterile saline solution as solvent. Mean lethal dose (LD₅₀) values reported in previous studies ¹²12. Ruiz RI, Ruiz LI, Martínez-Vargas AZ, Arruz MS, Gutiérrez JM. Toxicidad y neutralización de venenos ofídicos peruanos de los géneros Bothrops y Lachesis (Serpentes: Viperidae). Rev Biol Trop. 1993;41(3A): 351-7.

13. Ohsaka A. Hemorrhagic, necrotizing and edema-forming effects of snake venoms. In: Handbook of Experimental Pharmacology. Volume 52. Berlin: Springer-Verlag;1979.

14. Barros SF, Friedlanskaia I, Petricevich VL, Kipnis TL. Local inflammation, lethality and cytokine release in mice injected with Bothrops atrox venom. Mediators Inflamm. 1998;7(5):339-46. doi: 10.1080/09629359890866.
https://doi.org/10.1080/09629359890866... ^-1515. Meier J, Theakston RD. Approximate LD50 determinations of snake venoms using eight to ten experimental animals. Toxicon. 1986;24(4):395-401. doi: 10.1016/0041-0101(86)90199-6.
https://doi.org/10.1016/0041-0101(86)901... were taken as a reference; for Bothrops atrox venom, a range from 3 µg/g to 6 µg/g mouse has been reported. Solutions with higher and lower venom concentration were prepared, maintaining a constant dilution factor of 1.22 µg/g mouse. A volume of 0.5 mL of venom solution was inoculated intraperitoneally into each mouse. The number of live/dead (L/D) mice in each group was registered at 24 and 48 hours. The number of dead mice in each box at 48 hours was considered for probit analysis.

Lama glama immunization with Bothrops atrox venom

Immunizations were performed in a stock or with an inoculation sleeve for older animals. Before venom inoculation, the injection area was shaved on the llama’s back side. The injected substance had a 1:1 ratio of venom and the GERBU adjuvant ⁹9. Grubhofer NA. Vaccine Adjuvants Revisited. TOVSJ. 2008;2:63-67.^,1010. Pardon E. A general protocol for the generation of Nanobodies for structural biology. Nat Protoc. 2014;9(3):674-93. doi: 10.1038/nprot.2014.039.
https://doi.org/10.1038/nprot.2014.039... , its total volume was 4 mL (2 mL of venom and 2 mL of the GERBU adjuvant). The venom was inoculated subcutaneously (distributed proportionally in four places on the back and alternating between both lateral parts of the back between each inoculation).

Llama immunization scheme with Bothrops atrox venom

The primary immunization scheme comprised 8 immunizations with total venom from Bothrops atrox plus the GERBU adjuvant in a 1:1 ratio (V/V). The amount of venom inoculated in each immunization dose, as well as the time intervals between doses, are indicated in the supplementary material. After the primary scheme, 7 booster doses were applied, as indicated in Table 1.

Obtaining serum from Lama glama

A blood sample (approx. 20 mL) was taken from the right jugular vein with a 20 mL syringe and an 18-gauge needle. The blood was collected in vacutainer tubes containing a serum separator gel and centrifuged at 3,000 rpm for 5 minutes. The serum was collected in 1.5 mL microcentrifuge tubes, which were then stored at -80°C. Pre-immune serum was obtained before the first immunization; post-immunization serums were collected the same day before each immunization (Table 1).

ELISA for the measurement of Lama glama antibodies against Bothrops atrox venom

The poison was diluted in carbonate buffer (Na₂CO₃ 0.015 M, NaHCO₃ 0.035 M) to a final concentration of 0.5 µg/mL. Then, 100 µL/well of the diluted poison was applied, the plate covered and incubated overnight at 4 °C. The poison solution was removed by inversion. Then, 250 µL of the blocking buffer (AFB or skimmed milk) was added to each well and incubated for 1 hour at room temperature. Titrated sera were diluted from 1/200 to 1/800 in carbonate buffer; as well as the negative control (pre-immune serum) and the blank (antigen-free carbonate buffer). The blocking buffer was removed and washed 5 times, then 100 µL of the titrated serum was added to each well and incubated for 1 hour at room temperature, in duplicate. The serum was removed and washed 5 times; 100 µL of conjugate (anti-Lama-IGG (H+L)-Peroxidase) which was diluted to 1:10,000 in conjugate dilution buffer, was added to each well; it was then incubated for 1 hour at room temperature. Then, the conjugate was removed and washed 5 times; 100 µL of TMB (tetramethyl-benzidine) substrate was added to each well and incubated for 5-15 minutes at room temperature and in the dark. 50 µL of 0.5 M stopping solution (H₂SO₄) was added to each well. The plate was read at a wavelength of 450 nm.

Determination of the lethal effect neutralization

The recommendations from the manual of procedures of the Clodomiro Picado Institute were followed to determine the toxic activities of the venom and its neutralization ¹¹11. Instituto Clodomiro Picado. Determinación de actividades Tóxicas de venenos de serpientes y su neutralización por antivenenos. Manual de métodos de laboratorio [Internet]. Costa Rica: Universidad de Costa Rica, Facultad de Microbiología; 2007. Disponible en: http://www.icp.ucr.ac.cr/sites/default/files/content/Manual%20de%20procedimientos%20determinacion%20actividades%20toxicas%20de%20venenos%20de%20serpientes%20y%20su%20neutralizaci%C3%B3n.pdf.
http://www.icp.ucr.ac.cr/sites/default/f... . Male mice of 17 g to 18 g were used, 5 groups of 6 mice per box and an additional box for controls.

The venom dose was determined by considering 4 times the LD₅₀ value for each gram of mouse to be inoculated (µg venom/g mouse). The venom and antivenom (llama serum) were preincubated at 37 °C for 30 minutes with fixed doses of poison (4DL₅₀) and 5 venom dilutions. The serum volume of each inoculum was determined, assuming that 1 mL of serum should neutralize at least 2.5 mg of venom (anti-bothropic serum-INS). Venom solution (without serum) containing 4DL₅₀ was prepared for control mice. A volume of 0.5 mL of the prepared solution was inoculated intraperitoneally into each mouse. The mice were observed during the following hours. The quantity of L/D animals was registered for each group at 24 and 48 hours after inoculation, the number of dead mice in each box was noted at 48 hours for statistical calculation.

Statistical analysis

Statistical analysis was performed using Stata version 11 and the probit function to determine the LD₅₀; lethality neutralization data was also analyzed to calculate the serum ED₅₀. The differences between the groups were determined with the Chi-square test, considering p values under 0.05 as statistically significant.

Ethical aspects

The Institutional Ethics Committee on Human Beings and the Ethics Committee for the Use of Experimental Animals of the INS approved this study.

RESULTS

The LD₅₀ of the Peruvian Bothrops atrox snake venom pool was of 3.96 μg venom/g mouse at 48 hours, with a lower limit of 3.57 μg/g and an upper limit of 4.38 μg/g, according to its 95% confidence interval. In addition, an R² of 0.99 was found, which indicates a high correlation between the applied dose and the lethal effect of the venom (Figure 1).

Bothrops atrox venom analysis by SDS-PAGE electrophoresis shows a characteristic protein profile that matches the protein profile reported for this species ⁶6. Segura A, Castillo MC, Núñez V, Yarlequé A, Gonçalves LR, Villalta M, et al. Preclinical assessment of the neutralizing capacity of antivenoms produced in six Latin American countries against medically-relevant Bothrops snake venoms. Toxicon. 2010;56(6):980-9. doi: 10.1016/j.toxicon.2010.07.001.
https://doi.org/10.1016/j.toxicon.2010.0... . Four strong bands were observed (more proteins) and at least 6 weak bands; from those 4 with more proteins, 2 bands had a high molecular weight (45- 60 kDa) and 2 bands had a lower molecular weight (10-20 kDa) (Figure 2A). In the serum electrophoresis analysis, we found a band protein profile, which represented heavy and light chains of IgG antibodies (Figure 2B).

Standardization of the ELISA assay for detecting Bothrops atrox venom antibodies against the llama serum showed that the optimal amount of antigen to use is 50 ng, the optimal serum dilution is 1:800 and the ideal dilution of peroxidase conjugated antibody is 1:10,000. Bovine serum albumin (BSA) proved to be a better blocking agent than skimmed milk for the ELISA. The difference in absorbance between pre-immune control serum and immune serum was considered when choosing the optimal assay parameters. The specific antibody titer for Bothrops atrox venom increased rapidly in the serum from the second week after the beginning of the immunizations, reaching a maximum concentration in the fourth week and then remaining constant.

After primary immunization, the expected potency was not achieved, which was similar to the reference serum (equine serum) (2.5 mg/mL); therefore, after this first scheme, 7 immunization boosters of 4 mg of venom in a volume of 2 mL were carried out, until achieving a neutralizing potency equal or higher than 2.5 mg/mL. After the boosters, the ED₅₀ was 3.30 µL/g mouse and the upper and lower limits were 3.86 µL/g mouse and 2.90 µL/g mouse, respectively. This ED₅₀ is equivalent to a venom neutralization potency of 3.6 mg/mL; 1 mL of llama serum is capable of neutralizing 3.6 mg of Bothrops atrox venom. A comparison was made between antibody titers and potencies according to the days elapsed (Figure 3).

DISCUSSION

Although antivenoms produced in horses are the main treatment used in cases of ophidism, there are still some problems associated with the use of these medications, such as the occurrence of adverse reactions in patients ³3. Ministerio de Salud. Norma Técnica N° 007-MINSA-DGSP. MINSA 2004. Sobre Prevención y Tratamiento De Accidentes Por Animales Ponzoñosos [Internet]. Lima: MINSA; 2004. Disponible en: http://bvs.minsa.gob.pe/local/dgsp/123_NTPONZONOSOS.pdf.
http://bvs.minsa.gob.pe/local/dgsp/123_N... . The present study analyzes the llama’s immune response to the venom of the Bothrops atrox snake and the capacity of this hyperimmune serum to neutralize the lethality of the venom, as an approach to evaluate other serum-producing species.

Ruiz et al. ¹²12. Ruiz RI, Ruiz LI, Martínez-Vargas AZ, Arruz MS, Gutiérrez JM. Toxicidad y neutralización de venenos ofídicos peruanos de los géneros Bothrops y Lachesis (Serpentes: Viperidae). Rev Biol Trop. 1993;41(3A): 351-7. determined an intraperitoneal Bothrops atrox LD₅₀ at 48 hours of 2.3 mg/kg. Meanwhile, Ohsaka ¹³13. Ohsaka A. Hemorrhagic, necrotizing and edema-forming effects of snake venoms. In: Handbook of Experimental Pharmacology. Volume 52. Berlin: Springer-Verlag;1979. determined an intraperitoneal LD₅₀ at 48 hours of 3.8 μg/g. Barros et al. ¹⁴14. Barros SF, Friedlanskaia I, Petricevich VL, Kipnis TL. Local inflammation, lethality and cytokine release in mice injected with Bothrops atrox venom. Mediators Inflamm. 1998;7(5):339-46. doi: 10.1080/09629359890866.
https://doi.org/10.1080/09629359890866... determined a LD₅₀ of 6.85 μg/g, and Meier and Theakston ¹⁵15. Meier J, Theakston RD. Approximate LD50 determinations of snake venoms using eight to ten experimental animals. Toxicon. 1986;24(4):395-401. doi: 10.1016/0041-0101(86)90199-6.
https://doi.org/10.1016/0041-0101(86)901... determined a LD₅₀ of 3.95 μg/g under the same conditions. In this study we obtained a LD₅₀ of 3.96 μg/g, a figure similar to those found in the mentioned previous studies; this could be due to the fact that we used the same assay and the biological samples are from the same species. However, some differences could be found due to variations between the populations of snakes studied, since the characteristics of venom from snakes of the same species can vary depending on the age of the specimen, their origin or whether they are found in the wild or in captivity ⁴4. Zelanis A, Tashima AK. Unraveling snake venom complexity with 'omics' approaches: challenges and perspectives. Toxicon. 2014;87:131-4. doi: 10.1016/j.toxicon.2014.05.011.
https://doi.org/10.1016/j.toxicon.2014.0... ^,1616. Sousa LF, Portes-Junior JA, Nicolau CA, Bernardoni JL, Nishiyama-Jr MY, Amazonas DR, et al. Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon. J Proteomics. 2017;159:32-46. doi: 10.1016/j.jprot.2017.03.003.
https://doi.org/10.1016/j.jprot.2017.03.... .

Previous studies ¹⁷17. Olascoaga ME. Estudio del veneno de Bothropspictus: bioquímica, toxicidad, neutralización y efectos biológicos [Tesis doctoral]. Lima: Universidad Nacional Agraria La Molina; 1987. have shown that bothropic venoms are 1.2 to 3.6 times more toxic when inoculated intravenously than intraperitoneally. Previous data also suggest that the subcutaneous route is not recommended for the evaluation of toxic potency ¹⁷17. Olascoaga ME. Estudio del veneno de Bothropspictus: bioquímica, toxicidad, neutralización y efectos biológicos [Tesis doctoral]. Lima: Universidad Nacional Agraria La Molina; 1987.. For these reasons, in this study, inoculation by the intraperitoneal route was used, showing good results in the lethality neutralization test.

The antivenoms produced in horses are evaluated by the same institutions that manufacture them, in our case the INS, but studies are also carried out to verify their effectiveness, their range of action in terms of snake species, and their neutralizing power ¹⁸18. Otero R, Núñez V, Barona J, Díaz A, Saldarriaga M. Características bioquímicas y capacidad neutralizante de cuatro antivenenos polivalentes frente a los efectos farmacológicos y enzimáticos del veneno de Bothrops asper y Porthidium nasutum de Antioquia y Chocó. Latreia; 2002,15(1):5-15.. In our study, the llama hyperimmune serum presented a neutralizing power of 3.6 mg/mL, which means that 1 mL of the llama hyperimmune serum can neutralize 3.6 mg of Bothrops atrox venom.

In this study, the dose-response data were analyzed with the probit model, although other types of possible analyses were found in references, probit was chosen because it was the most suitable for this analysis. Due to the need to obtain objective data, statistics has been used for decades and as it has been advancing, some ways of calculating the LD₅₀ have been devised. For example, in 1985 Meier and Theaicston ¹⁵15. Meier J, Theakston RD. Approximate LD50 determinations of snake venoms using eight to ten experimental animals. Toxicon. 1986;24(4):395-401. doi: 10.1016/0041-0101(86)90199-6.
https://doi.org/10.1016/0041-0101(86)901... proposed a statistical analysis method to reduce the number of experimental mice from 30 to 10 that did not differ significantly in the results and was more desirable for economic and ethical reasons. But this has been later overcome by the probit model, which is particularly useful against dose-response data of this type.

Previous studies have already demonstrated the great importance of the ELISA technique in in vitro assays for analyzing antibodies generated in response to venom immunization, compared to other methods used, as immunodiffusion or hemagglutination ¹⁹19. Sandoval G, Mendoza J, Roldán W, EspinozaY, Solis, H, Yarlequé A. Inmunogenicidad del veneno de Bothrops atrox (Ophidia: Viperidae) y su evaluación por métodos inmunoenzimáticos. Rev Peru Biol; 2011;18(3):335-42..

Simultaneous concordance between the antibody titer and the neutralization capacity during the immunization process was not observed, since the antibodies against Bothrops atrox venom increase rapidly; but the neutralization potency increases slowly, requires more time to reach expected values and also requires the administration of immunization reinforce doses.

Other studies involving llama serum also found no correlation between titer and effectiveness of venom neutralization in preclinical trials ²⁰20. Cook D, Owen T. Analysis of camelid antibodies for antivenom development: Neutralisation of venom-induced pathology. Toxicon. 2010;1;56(3):373-80. doi: 10.1016/j.toxicon.2010.04.005.
https://doi.org/10.1016/j.toxicon.2010.0... . This observation is probably due to the fact that the ELISA assay detects early all the antibodies produced against all the venom antigens, while the neutralization effect depends only on a small group of specific antibodies that neutralize or block the toxicity produced by the toxins or proteins of the venom.

The slow development of the neutralization potency in the llama is understandable, considering that 12 or more immunization doses are required in horses to reach the expected values of venom neutralization potency ²¹21. Espino-Solis G. Los caballos y la producción de antivenenos. [Internet]. Hypatia. 2010; 36(4). Disponible en: https://revistahypatia.org/216.
https://revistahypatia.org/216... .

Harrison et al. ²²22. Harrison RA, Hasson SS, Harmsen M, Laing GD, Conrath K, Theakston RD. Neutralisation of venom-induced haemorrhage by IgG from camels and llamas inmunised with viper venom and also by endogenous, non-IgG components in camelid sera. Toxicon. 2006;47(3):364-8. doi: 10.1016/j.toxicon.2005.10.017.
https://doi.org/10.1016/j.toxicon.2005.1... measured the neutralizing capacity of Lama glama hyperimmune serum against Echis ocellatus venom, and found that an elevated llama antibody titer is produced at 2 weeks; similar to our results, which indicate that it is feasible to prepare bothropic antiserum from llama serum. In addition, they found a yet undetermined component within the llama serum that is not an IgG and that exhibits antihemorrhagic activity. Fernandez et al.²³23. Fernández GP, Segura A, Herrera M, Velasco W, Solano G, Gutiérrez JM, et al. Neutralization of Bothrops mattogrossensis snake venom from Bolivia: experimental evaluation of llama and donkey antivenoms produced by caprylic acid precipitation. Toxicon. 2010; 55(2-3):642-5. doi: 10.1016/j.toxicon.2009.07.031.
https://doi.org/10.1016/j.toxicon.2009.0... found that llama serum is effective against Bothrops mattogrossensis snake venom, which also represents a valuable alternative for antidote manufacture in South America.

Among the limitations of the study, it should be considered that only the immune response to Bothrops atrox snake venom has been evaluated in a single llama and it is recommended to do the analysis in more than one animal; the neutralizing capacity of the llama serum has not been compared to the anti-bothropic serum produced in horses, because the latter is a polyvalent serum and the llama serum is only against Bothrops atrox snake venom. It should be noted that this is one of the few studies that has evaluated the capacity of the llama serum to neutralize the snake venom, and although there are other similar studies with snake venom from the genus Bothrops, such as Bothrops matogrosensis, this is the only known study with the species Bothrops atrox.

In conclusion, the results show that the llama hyperimmune serum can neutralize the venom of Peruvian Bothrops atrox, with a ED₅₀ of 3.30 µL of serum/g mouse and a venom neutralization potency of 3.6 mg/mL, which means that 1 mL of hyperimmune serum can neutralize 3.6 mg of venom. The LD₅₀ of the venom from Peruvian Bothrops atrox was 3.96 µg venom/g mouse and 4 times this amount was used as a challenge dose to test for venom neutralization. The immune response of the llama to the venom is rapid, with high antibody titers from the second immunization week, but several more weeks and additional booster doses are required to achieve the expected venom neutralization potency of this hyperimmune serum. The ELISA test is a good in vitro method for analyzing antibodies generated by immunization with Bothrops atrox venom and allows monitoring the immune response during immunization.

References

Publication Dates

History