*2.1. Induction of Oral Tolerance by B. jararaca Venom Is Dependent on Dose Administed*

Oral tolerance is characterized by low IgG levels in the antiserum of animals after immunization with the antigen previously administered orally. Initially, the induction of oral tolerance by administering *B. jararaca* venom was tested using two conditions and the results are summarized in Figure 1. Group I did not receive venom orally. Briefly, one group of mice orally received a single dose of 1.8 mg of the venom (Group II) and other groups received 1 μg of snake venom on the first, third and fifth day (Groups III and IV). At 12 days after the last low dose of 1 μg, all groups were immunized with snake venom combined with silica adjuvant. Groups I, II and III received venom from *B. jararaca*, while Group IV was challenged with venom from *B. atrox*. Antisera corresponding to 7 days after immunization were analyzed, and no difference in antibody titers between all groups of mice was observed (data not shown). Antisera were then collected on the 35th day after immunization and titers determined (Figure 1), followed by analysis based on ANOVA. According to our results, animals that received high doses of *B. jararaca* venom orally were not tolerized (Group II), with antibody titers being similar to those of mice immunized only (Group I). No statistically significant differences were found between Groups I and II

and Groups I and IV. Interestingly, mice given a low dose of venom orally and immunized with the same antigen (Group III) had the lowest antibody titer showing the induction of oral tolerance with a statistically significant difference compared to other groups. On the other hand, animals that received a low oral dose of *B. jararaca* and were immunized with *B. atrox* venom (Group IV) showed no tolerance induction. Our results suggest that oral tolerance to snake venom appears to be dose-dependent and is specific for the venom of each snake species.

**Figure 1.** Induction of oral tolerance by administration of *B. jararaca* venom evaluated by antibody titer of mouse antiserum after immunization. Group I was not subjected to oral tolerance. The mice were orally tolerized with a single high dose of venom (Group II) or with low doses of venom on the first, third and fifth day (Group III and IV). Twelve days after receiving the last oral dose, Groups I, II and III were immunized with *B. jararaca* venom, and group IV received *B. atrox* venom. Serum antibody titers corresponding to 35 days after immunization were determined by ELISA. Error bars represent the standard deviation of an experiment (*n* = 3). ANOVA with 95% confidence intervals was used to determine significant differences between each group of mice. ns: not significant; \*\*\*\*: *p*-value was <0.0001.
