*2.2. Characterization of Antisera from Orally Tolerized Animals Reveals Specificity for Other Snake Venoms*

To characterize the specificity and cross-reactivity of the antiserum after induction of oral tolerance with *B. jararaca* venom, another experiment was carried out with induction of oral tolerance by administering three doses of 1 μg of the *B. jararaca* venom (Groups I and III). Eight days after the last dose, all groups of mice were immunized intraperitoneally with snake venom combined to adjuvant. Groups I and II were immunized with *B. jararaca*, while Groups III and IV received *B. jararacussu* venom. Antiserum was collected 12, 25 and 45 days after immunization to analyze the specificity of response to *B. jararaca* and *B. jararacussu* venoms. The specificity of antiserum for the venom of *B. jararaca*, using this venom as the antigen is shown in Figure 2, while Figure 3 shows the specificity for the venom of *B. jararacussu.* Our results showed that Group I, orally tolerized and immunized with *B. jararaca* venom, was tolerized for snake venom in view of the low IgG level in all analyzed antisera (Figures 2A and 3A). Group III was tolerized orally and immunized with *B. jararacussu* venom and the antibody titer increased on the 45th day after venom injection when specificity for *B. jararaca* venom was evaluated (Figure 2C), while the same phenomenon was observed on day 25 for the specificity of *B. jararacussu* venom (Figure 3C). Antiserum from groups of mice immunized with snake venom only (Groups II and IV) had high antibody titers for both snake venoms 25 days after immunization

(Figures 2B,D and 3B,D). Furthermore, antisera from mice immunized with *B. jararacussu* venom (Group IV) showed reactivity with *B. jararaca* venom (Figure 2B) and antisera from mice immunized with *B. jararaca* venom (Group II) showed reactivity with *B. jararacussu* venom (Figure 3B). In the case of specificity for *B. jararacussu* venom, mice immunized with *B. jararaca* venom (Group II) showed a reduction in antibody titer 45 days after immunization (Figure 3B).

**Figure 2.** Antiserum specificity for *B. jararaca* venom from mice subjected or not to oral tolerance followed by immunization with snake venom. Microplate wells were coated with 1 μg of *B. jararaca* venom, and serial dilutions of antiserum of Groups I–IV corresponding to 12, 25 and 45 days after snake venom immunization were applied; antibody titer was determined by ELISA. (**A**) Group I: mice tolerized with *B. jararaca* venom and immunized with the same snake venom; (**B**) Group II: mice immunized with *B. jararaca* venom; (**C**) Group III: mice tolerized with *B. jararaca* venom and immunized with *B. jararacussu* venom; (**D**) Group IV: mice immunized with *B. jararacussu* venom. The mean and standard deviation (*n* = 5) of each antibody titer obtained were plotted. ANOVA with 95% confidence intervals was used to determine significant differences between each group of mice group. ns: not significant; \*: *p*-value was 0.01–0.05; \*\*: *p*-value was 0.001–0.01; \*\*\*\*: *p*-value was <0.0001.

IgG1 and IgG2a titers were determined for antisera obtained 45 days after immunization for Groups I, II, III and IV (Figure 4). Orally tolerized mice had lower antibody titers than immunized mice and no difference between IgG1 and IgG2a titers was observed. IgG1 antibody titers from mice immunized with snake venoms (*B. jararaca* or *B. jararacussu* venom) were higher than IgG2a titers. IgE was not found in any of the four groups (data not shown).

**Figure 3.** Antiserum specificity for *B. jararacussu* venom from mice subjected or not to oral tolerance followed by immunization with snake venom. Microplate wells were coated with 1 μg of *B. jararacussu* venom, and serial dilutions of antiserum of Groups I–IV corresponding to 12, 25 and 45 days after snake venom immunization were applied; antibody titer was determined by ELISA. (**A**) Group I: mice tolerized with *B. jararaca* venom and immunized with the same snake venom; (**B**) Group II: mice immunized with *B. jararaca* venom; (**C**) Group III: mice tolerized with *B. jararaca* venom and immunized with *B. jararacussu* venom; (**D**) Group IV: mice immunized with *B. jararacussu* venom. The mean and standard deviation (*n* = 5) of each antibody titer obtained were plotted. ANOVA with 95% confidence intervals was used to determine significant differences between each mice group. ns: not significant; \*: *p*-value was 0.01–0.05; \*\*: *p*-value was 0.001–0.01; \*\*\*: *p*-value was 0.0001–0.001; \*\*\*\*: *p*-value was <0.0001.

Next, we evaluated the reactivity of antisera from mice subjected to oral tolerance with venoms of some *Bothrops* species and *Bitis anetans* by Western blotting. Protein profiles of the snake venoms under non-reducing conditions are shown in Figure 5; venoms differed in composition and band intensity.

Snake venoms subjected to electrophoresis were transferred to PVDF membranes, the membrane was incubated with a pool of antisera corresponding to 45 days after the immunization of Groups I, II, III and IV and revealed by chemiluminescent reagent (Figure 6). Antisera from orally tolerized animals showed lower reactivity with snake venom proteins compared to antisera obtained from mice immunized with snake venom. Antisera of mice tolerized with *B. jararaca* venom and immunized with the same snake venom (Group I) recognized some proteins from the venom of *B. jararaca*, and a protein band from the venom of *B. alternatus*, *B. jararacussu* and *B. atrox amazonia* (Figure 6A). On the other hand, the antisera from mice immunized with the venom of *B. jararaca* recognized venom proteins of the *Bothrops* species analyzed (*Bothrops jararaca*, *B. alternatus*, *B. jararacussu* and *B. atrox amazonia*), and a wide range of reactivity was found for the venoms of *B. jararaca* and *B. alternatus* (Figure 6B). Interestingly, some protein bands recognized by both tolerized and non-tolerized mouse antisera (Groups I and II) were very similar (Figure 6A,B). When antisera from mice orally tolerized or not and immunized with *B. jararacussu* venom (Groups III and IV) were evaluated, at least one protein of the snake venom was recognized by antisera from Group III (Figure 6C), and antisera from immunized mice (Group IV) showed higher reactivity with *B. jararacussu* and *B. jararaca* venoms and low reactivity with other snake venoms (Figure 6D). The reactivity of Groups III and IV antisera was tested for *Bitis anetans* venom, and antisera from tolerized or not mice showed reactivity with this venom. Each antiserum recognized a distinct protein (Figure 6C,D). Our results showed that antisera from mice tolerized orally with *B. jararaca* venom and immunized with snake venoms had reactivity with venoms from other species at different levels. Interestingly, antisera from orally tolerized mice demonstrated cross-reactivity with different venom epitopes in comparison to the antisera of immunized animals.

**Figure 4.** IgG1 and IgG2a antibody titers of groups of mice subjected to oral tolerance induction with *B. jararaca* venom followed by immunization. Microplate wells were coated with 1 μg of *B. jararaca* venom, and serial dilutions of Groups I-IV antiserum, as previously described in "Section 4", corresponding to 45 days after immunization with snake venom, were applied; antibody titer was determined by ELISA using mouse anti-IgG1 or anti-IgG2a antibodies conjugated to HRP. Error bars represent the standard deviation of one experiment (*n* = 5). 2-way ANOVA with 95% confidence intervals was used to determine significant difference between IgG1 and IgG2a antibody titers for each group. ns: not signigficant; \*\*\*\*: *p*-value was < 0.0001.

**Figure 5.** Electrophoretic profiles of snake venoms under non-reducing conditions. 12% SDS-PAGE of 5 μg of each snake venom. Coomassie blue staining. M: Rainbow marker high range (Cytiva); snake venom from 1: *B. jararaca*; 2: *B. alternatus*; 3: *B. jararacussu*; 4: *B. atrox amazonia*; 5: *Bitis anetans*.

**Figure 6.** Analysis of the reactivity of the pool of antisera from animals subjected or not to oral tolerance induction with *B. jararaca* venom followed by immunization with snake venom. Snake venoms were subjected to electrophoresis and transferred to PVDF membrane according to Figure 5. The membrane was incubated with a pool of antisera corresponding to 45 days after immunization. (**A**): 1/400 dilution of Group I antisera; (**B**): 1/1000 dilution of Group II antisera; (**C**): 1/400 dilution of Group III antisera; (**D**) 1/1500 dilution of Group IV antisera. Peroxidase-conjugated anti-mouse IgG was then added, and the membrane was revealed with ECL reagent.

#### **3. Discussion**

Oral tolerance is an immunological process in which the specific immune response is inhibited by prior oral administration of antigen. The induction of this process can be assessed after antigen immunization and measured by determination of antibody titer or by the decrease in allergic symptoms after allergen challenge [14].

The ability of the immune system to adapt to dietary antigens and commensal bacteria is important and prevents the development of food allergies, celiac disease, and autoimmune diseases [20]. Most studies related to oral tolerance have been carried out in animal models to establish the safe dose and duration of the process and to understand the immune cells and pathways involved, because of the risk of testing in humans [20]. These studies are of crucial importance for understanding the mechanisms involved and can promote strategies for the development of natural oral tolerance and prevent intoxication, allergies, and autoimmune diseases.

There is a report of daily oral administration for 14 days of *Crotalus durissus* terrificus snake venom (200 μg/kg) in male Swiss mice (17–21 g) that induced tolerance to the antinociceptive effect, and no antibody titers were measurable after prolonged treatment [21]. The expected effect was obtained by administering a dose of venom higher than that in our study, and mice were not immunized after receiving the oral dose. The cited study had a different purpose in relation to the present work and a different approach was taken.

Most incidences of snakebites in Brazil, considering all regions, are related to *Bothrops* species [22] and, therefore, constituted the objective of this study. They are also extensively investigated because they are responsible for more fatality cases in Central and South America than other groups of snakes [1]. So far, oral tolerance by administering *B. jararaca* venom in animal models has not been published and we have successfully established a protocol in BALB/c mice. The antibody titers were measured in the antisera after immunization with snake venoms to understand the mechanism involved.

We observed that mice receiving a single dose of 1.8 mg of *B. jararaca* venom orally did not develop tolerance and that the antibody titer was similar to that of the group of mice that was only immunized (Figure 1). These results are in agreement with those found in the literature for other antigens [11,16]. We have shown that repeated exposure to a dose of 1 μg of *B. jararaca* venom induced tolerance (Figure 1). We also found that tolerance was more effectively induced when animals received the same snake venom during oral administration and immunization (Figures 1–3), showing that it is specific for one type of antigen, even when the antiserum showed cross-reactivity with venom from other snakes.

There was no reference concerning an immunization protocol with snake venom after oral administration, and we established that immunization would be performed about 7 days after the last dose. In the first protocol, mice were immunized 12 days after the last dose, while in the second protocol it was 8 days, and under both conditions, the oral tolerance induction was verified by low antibody titer. We think that the immunization protocol could be applied in this period of time to induce oral tolerance with *B. jararaca* venom.

In the first protocol of oral tolerance induction, mice received a low dose of *B. jararaca* venom and were immunized with *B. atrox* venom, and no tolerance induction was observed (Figure 1). On the basis of this result, we excluded this condition in the next protocol of oral tolerance induction and challenged the immunization with the venom of another *Bothrops* species (*B. jararacussu*). Partial oral tolerance induction was observed (Figures 2 and 3) with *B. jararacussu* venom immunization, showing that this tolerance can be induced with another *Bothrops* venom. Variations in venom composition and biological activities of Brazilian snakes from *Bothrops* genus were observed [23] and these differences could be influenced on oral tolerance induction when immunization with heterologous venom was applied. Further studies involving different conditions for establishing oral tolerance induction with *B. jararaca* venom and other *Bothrops* species could elucidate these observations.

We also performed immunoblot analysis of the venom of different species of *Bothrops* incubating with antisera from animals tolerized with administration of *B. jararaca* venom or only immunized with snake venom. The epitopes recognized by each antiserum were clearly different (Figure 6), showing that the reactivity profile of antisera in relation to venom components changed according to the protocol used to induce tolerance. To see if proteins in snake venom from species other than *Bothrops* are recognized by antisera from mice orally tolerized or not, reactivity with *Bitis anetans* venom was evaluated. Antisera from mice partially tolerized (Group III) and from animals immunized with *B. jararacussu* venom (Group IV) were tested and each antiserum recognized different proteins in *Bitis anetans* venom, showing that reactivity with other snake venoms could be explored in future studies.

The application of oral tolerance has advantages because it is non invasive and uses a simple route. New knowledge and in-depth understanding of this process could contribute to the application of oral tolerance in prophylaxis and treatment of diseases [20].

Most of the studies related to oral tolerance induction involve the administration of one antigen [11,13,20]. The present work is an experimental study of oral tolerance induction developed with *B. jararaca* venom that is composed of a complex of proteins with biological activity. This process does not happen in the nature, and thus, the observation of oral tolerance induction represented a novel experimental approach. To demonstrate oral tolerance induction in our approach, the immune response was evaluated by the determination of the antibody titer, and then, the epitopes recognized by antiserum from

orally tolerized or not tolerized mice were compared. Our results showed that it is possible to induce oral tolerance by administration of the complex mixture of the proteins, such as *B. jararaca* venom. Investigations related to the modulation of immune response and the mechanisms involved in this process were not part of the scope of the present work. The administration of a single high dose of *B. jararaca* venom was not lethal to the animals but did not induce oral tolerance. Further experimental studies should be conducted with other snake venoms to confirm this phenomenon as this approach would be used to develop vaccines by oral administration. The protocol established for oral tolerance induction should also be applied to other snake venoms in future studies together with the investigation of the mechanisms involved in this process. Therefore, the present work opened new perspectives to explore the process of oral tolerance induction.

#### **4. Materials and Methods**

#### *4.1. Animals*

BALB/c mice, female, aged 2–4 months, were used and maintained at the animal facilities of the Immunochemistry Laboratory, Butantan Institute, and they were caged and handled according to the International Animal Welfare Recommendations and in line with the guidelines for the use of animals in biomedical research [24]. Ethics Committee on Animal Use of the Butantan Institute approved the experiment protocol (Protocol IBU 454/08; 9 April 2008).
