fimmu-11-00655 May 7, 2020 Time: 13:9 # 1 ORIGINAL RESEARCH published: 07 May 2020 doi: 10.3389/fimmu.2020.00655 Development of Nanobodies Against Hemorrhagic and Myotoxic Components of Bothrops atrox Snake Venom Henri Bailon Calderon1*, Verónica Olga Yaniro Coronel1,2, Omar Alberto Cáceres Rey1, Elizabeth Gaby Colque Alave1, Walter Jhon Leiva Duran1, Carlos Padilla Rojas1, Harrison Montejo Arevalo1, David García Neyra1, Marco Galarza Pérez1, César Bonilla3, Benigno Tintaya3, Giulia Ricciardi4, Natalia Smiejkowska4, Ema Romão4, Cécile Vincke4, Juan Lévano1, Mary Celys1, Bruno Lomonte5 and Serge Muyldermans4 Edited by: 1 Abdul Qader Abbady, Laboratorio de Referencia Nacional de Biotecnología y Biología Molecular, Centro Nacional de Salud Pública, Instituto 2 Atomic Energy Commission of Syria, Nacional de Salud, Lima, Peru, Laboratorio de Biología Molecular, Universidad Nacional Mayor de San Marcos, Lima, Peru, 3 4 Syria Centro Nacional de Producción de Biológicos (INS), Lima, Peru, Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium, 5 Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jose, Reviewed by: Costa Rica Wayne Robert Thomas, The University of Western Australia, Australia Snake envenoming is a globally neglected public health problem. Antivenoms produced Peter Timmerman, using animal hyperimmune plasma remain the standard therapy for snakebites. Although Pepscan, Netherlands effective against systemic effects, conventional antivenoms have limited efficacy against *Correspondence: Henri Bailon Calderon local tissue damage. In addition, potential hypersensitivity reactions, high costs for [email protected]; animal maintenance, and difficulties in obtaining batch-to-batch homogeneity are [email protected] some of the factors that have motivated the search for innovative and improved Specialty section: therapeutic products against such envenoming. In this study, we have developed a set of This article was submitted to nanobodies (recombinant single-domain antigen-binding fragments from camelid heavy Vaccines and Molecular Therapeutics, a section of the journal chain-only antibodies) against Bothrops atrox snake venom hemorrhagic and myotoxic Frontiers in Immunology components. An immune library was constructed after immunizing a Lama glama with Received: 03 December 2019 whole venom of B. atrox, from which nanobodies were selected by phage display Accepted: 23 March 2020 using partially purified hemorrhagic and myotoxic proteins. Biopanning selections Published: 07 May 2020 retrieved 18 and eight different nanobodies against the hemorrhagic and the myotoxic Citation: Bailon Calderon H, Yaniro proteins, respectively. In vivo assays in mice showed that five nanobodies inhibited the Coronel VO, Cáceres Rey OA, Colque hemorrhagic activity of the proteins; three neutralized the hemorrhagic activity of whole Alave EG, Leiva Duran WJ, Padilla Rojas C, Montejo Arevalo H, García B. atrox venom, while four nanobodies inhibited the myotoxic protein. A mixture of the Neyra D, Galarza Pérez M, Bonilla C, anti-hemorrhagic and anti-myotoxic nanobodies neutralized the local tissue hemorrhage Tintaya B, Ricciardi G, and myonecrosis induced by the whole venom, although the nanobody mixture failed Smiejkowska N, Romão E, Vincke C, Lévano J, Celys M, Lomonte B and to prevent the venom lethality. Nevertheless, our results demonstrate the efficacy and Muyldermans S (2020) Development usefulness of these nanobodies to neutralize important pathologies of the venom, of Nanobodies Against Hemorrhagic and Myotoxic Components highlighting their potential as innovative therapeutic agents against envenoming by B. of Bothrops atrox Snake Venom. atrox, a viperid species causing many casualties in South America. Front. Immunol. 11:655. doi: 10.3389/fimmu.2020.00655 Keywords: nanobodies, snake, venom, myotoxic, hemorrhagic, neutralization, Bothrops atrox, viperidae Frontiers in Immunology| www.frontiersin.org 1 May 2020| Volume 11| Article 655 fimmu-11-00655 May 7, 2020 Time: 13:9 # 2 Bailon Calderon et al. Nanobodies Against Bothrops atrox Venom INTRODUCTION levels in microorganisms, such as E. coli, thereby reducing the production cost. Snakebite envenoming is an important public health problem Bothrops atrox snake venom has been characterized in worldwide, especially in tropical and subtropical countries. previous studies (16) including proteomic analysis, which have Around the world, 5 million snakebites occur each year, affecting determined the predominant presence of metalloproteinases of mainly the rural populations and causing an estimated 100,000 the SVMP-I and SVMP-III classes in addition to other protein deaths (1). Victims that survive snake envenoming may suffer types, such as phospholipase A2 (PLA2) and PLA2-like homologs, from permanent physical sequelae, such as amputation of the serine proteinases, and disintegrins, among others (17). affected body parts, as well as psychological sequela, such SVMPs are relevant toxins of Bothrops spp. venoms since as depression, which can reduce their productive capacities many display a potent hemorrhagic effect, especially those of the and affect normal life. Moreover, most snake envenoming SVMP-III class (18, 19). On the other hand, basic PLA2 and accidents in developing countries occur in remote areas, often PLA2-like proteins induce a strong myotoxic effect leading to far away from health services, or where antivenoms are local necrosis (20, 21). Together, these two types of toxins are unavailable, which decreases the chances for effective treatment mainly responsible for the local tissue damage that may develop and lifesaving of the victims. in severe envenoming by Bothrops species (3). Venomous snakes around the world include the families Currently, there are few countries in Latin America that Viperidae, Elapidae, Atractaspididae, and Colubridae (2), the produce antivenoms. In Peru, the National Institute of Health former two being the most medically relevant. Within the produces a polyvalent antivenom in equines, which is the only Viperidae family, the genus Bothrops is responsible for the most effective treatment against snake bites. The antivenom is obtained snake envenoming in Central and South America, causing high after successive subcutaneous injection of equines every 8 days morbidity and mortality (3). with the venoms of several snake species. Blood is collected Antivenom administration is the only effective treatment for after a period of 3 months and plasma is processed to obtain snake envenoming. Currently, antivenom production is based the IgG fraction. on the immunization of equines or ovine with snake venoms It has been calculated that probably less than 30% of the total according to the species responsible for the accidents in a antibodies from an equine antivenom are effectively neutralizing region or country. the toxins of the venom (22). Moreover, among important Hyperimmune plasma is obtained and used for purification potential adverse effects of the antivenoms produced in equines of whole IgG antibodies (150 kDa) or for obtaining antibody are vasculitis, arthritis, and renal failure, which may originate fragments, such as F(ab’)2 (100 kDa) or Fab (50 kDa), for the from the formation of immunocomplexes between antivenom antivenom formulation (4). antibodies and the anti-horse antibodies that accumulate in blood Besides conventional antibodies, camelids and some shark vessels, joints, and glomeruli (22). species produce naturally a unique type of antibody that is There is an urgent need to reduce the costs and to increase composed of heavy chains only, referred to as heavy-chain- the efficiency of current antivenoms. In this context, many only antibodies (HCAbs) (5). The antigen recognition of researchers have proposed novel alternatives to the common these functional HCAbs is comprised in the variable region use of equine antibodies, such as recombinant antibodies from of their heavy chain [abbreviated as VHH and referred to human or camelid VHH (23). as Nanobody (Nb)]. In this study, we have constructed a camelid (Lama glama) Nanobodys are small proteins of approximately 15 kDa; immune VHH library to retrieve Nbs against B. atrox venom they are the smallest intact antigen-binding fragment (6) that using a phage display strategy. Several Nbs directed against retains the specificity and affinity of the original HCAb in hemorrhagic and myotoxic components were cloned and recognizing the antigen (7,8). The Nbs have promising potential recombinantly expressed in E. coli. Their ability to neutralize as therapeutic (9–11) and diagnostic tools (12). Their third hemorrhage and myotoxicity was screened in preincubation-type antigen-binding loop, or CDR3 (complementarity determining assays in mice, to reveal their potential as eventual therapeutic region), is longer than that of VH domains of conventional agents against snakebite envenoming, particularly against the antibodies, and this prolonged loop interacts preferentially with local tissue pathology induced by venom SVMPs and PLA2. cavities or concave surfaces, such as the active site of enzymes (13). While large clefts or grooves on the surface of antigens are less likely to interact with the flat surface of the paratope MATERIALS AND METHODS of classical antibodies, they are frequently observed to associate with the convex paratope formed mainly by the CDR3 of camelid Ethics Statement VHHs (14). Furthermore, the substitution of conserved large Experiments involving animals were carried