INTRODUCTION

Ophidism is a syndrome caused by snake venom poisoning, usually species from Viperidae and Elapidae families ^{1 , 2}. 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 ^{1 , 3}, 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 ⁴.

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 ⁵.

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 hyoprora ^{2 , 3 , 6}.

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 ^{1 , 2}. 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 ⁵.

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 ⁷.

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.

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).

Figure 1 Determination of Bothrops atrox venom mean lethal dose (LD50). Different amounts of venom were evaluated, from 2.5 to 6.75 µg of venom per gram of mouse in groups of 12 mice. The venom solutions were inoculated intraperitoneal and the mice were counted alive and dead after 48 hours. 

Bothrops atrox venom analysis by SDS-PAGE electrophoresis shows a characteristic protein profile that matches the protein profile reported for this species ⁶. 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).

Figure 2 SDS-PAGE electrophoresis of Bothrops atrox venom and llama serum. A) Snake venom protein profile of Bothrops atrox. Lane 1: protein molecular weight marker (Termo Scientific #26623). Lane 2: Bothrops atrox total venom. B) Llama serum electrophoresis (Lama glama). Lane 1: protein molecular weight marker (Thermo Scientific #26623). Lane 2: llama pre-immune serum. Track 3: llama immune serum. 

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).

Figure 3 Measurement of antibodies against Bothrops atrox venom by ELISA and neutralization of the lethal effect in mice. The antibodies were measured in the llama serum after the immunizations by ELISA assay (values in left Y axis, black color line), and the potency of lethality neutralization of the llama hyperimmune serum was analyzed, in laboratory mice (values in right Y axis, red color line). 

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 ³. 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. ¹² determined an intraperitoneal Bothrops atrox LD₅₀ at 48 hours of 2.3 mg/kg. Meanwhile, Ohsaka ¹³ determined an intraperitoneal LD₅₀ at 48 hours of 3.8 μg/g. Barros et al. ¹⁴ determined a LD₅₀ of 6.85 μg/g, and Meier and Theakston ¹⁵ 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 ^{4 , 16}.

Previous studies ¹⁷ 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 ¹⁷. 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 ¹⁸. 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 ¹⁵ 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 ¹⁹.

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 ²⁰. 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 ²¹.

Harrison et al. ²² 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. ²³ 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.