**1. Introduction**

Snake venoms are composed of a high diversity of proteins and peptides with biological activities, allowing these animals to defend themselves and immobilize their prey [1]. The composition of snake venoms among species displays high variability, both in qualitative and quantitative aspects and complexity [2]. Accidents with snakebite envenoming cause local and systemic effects and represent a public health problem in developing countries, where they reach lower socio-economic segments and kill >100,000 people each year [3]. The primary treatment for the systemic effects of snake envenoming is the intravenous administration of antivenom against specific venoms. Antivenoms specifically neutralize the venoms used in their production and those of related species, which means that antivenoms are produced regionally depending on demand [3]. Indeed, there is a crisis related to the supply of antivenoms, especially in sub-Saharan Africa and parts of Asia; the development of new treatments for patients with snakebite envenoming should be promoted on the basis of recent scientific knowledge related to snake venoms [3]. Recently, several studies have reported that antivenom serum antibodies, generated against specific snake venoms, are cross-reactive with venoms from other species, considering homologous and heterologous snake venoms [4–7].

**Citation:** Tsuruta, L.R.; Moro, A.M.; Tambourgi, D.V.; Sant'Anna, O.A. Oral Tolerance Induction by *Bothrops jararaca* Venom in a Murine Model and Cross-Reactivity with Toxins of Other Snake Venoms. *Toxins* **2021**, *13*, 865. https://doi.org/10.3390/ toxins13120865

Received: 15 October 2021 Accepted: 30 November 2021 Published: 3 December 2021

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**<sup>\*</sup>** Correspondence: lilian.tsuruta@butantan.gov.br

Most snakebites in Brazil occur because of the genus *Bothrops* and are considered a serious public health problem. *Bothrops* venom components mainly cause local damage and systemic effects targeting blood hemostasis, endothelial microcirculation, extracellular matrix, and the cardiovascular system [1,8].

Oral tolerance is the induction of peripheral immune tolerance by the oral administration of the antigen and is characterized by the inhibition of the specific immune response to this antigen due to prior oral exposure [9–14]. It is a natural and continuous process driven by external antigens. It has a unique immunological importance, as it develops unresponsiveness to ingested food and potential insults from the environment to maintain host homeostasis by protecting against food allergies and colitis caused by autoimmunity [12,13,15]. The gut is regularly exposed to multiple types of antigens, and the associated immune system has specialized immune cells and lymph nodes to balance responses to commensal bacteria (microbiome), innocuous antigens, and harmful microorganisms [11]. Depending on the properties of the antigen, such as size and solubility, the orally administrated antigen that reaches the intestinal epithelium is transported by different routes and can lead to the induction of tolerance or immunity [14]. The oral tolerance induction mechanism has been extensively studied using animal models, mainly for food allergens [11]. It involves multiple factors, and it is known that the dose of the administered antigen and the consumption time are decisive. Administration of a single high dose of antigen leads to the mechanisms of anergy or depletion, whereas exposure to multiple low doses favors the development of regulatory T cells [11,16]. Anergy induction means obtaining antigen-unresponsive T cells, while depletion induction refers to apoptosis of antigen-specific T cells [14]. Previous studies have shown that genetic and environmental factors are involved in the induction of oral tolerance, demonstrating that this characteristic is a process under the influence of multiple factors [17–19].

Oral tolerance induction by the administration of one kind of antigen/allergen has been extensively investigated, as has been the mechanism of this process involving immune cells and pathways [11,13,20]. This process has not been explored by the administration of a complex mixture of proteins. Snake envenomation by the oral route does not occur in nature; instead, snakes inject their venoms when there is a dangerous situation and/or they need to defend themselves. Oral antigen application of this kind to induce oral tolerance represents a novel experimental approach. To our knowledge, there is no report of oral tolerance induction using *B. jararaca* venom as an antigen. We propose a method for inducing oral tolerance to *B. jararaca* venom in mice, followed by evaluation of serum crossreactivity with the toxins from other *Bothrops* species compared to the serum generated by the immunization process.

#### **2. Results**
