Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies against Loxosceles Spider Venom

Abstract

Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen’s formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.

Prospective Use of Brown Spider Venom Toxins as Therapeutic and Biotechnological Inputs

“…In addition, pre-incubation of this venom with anti-rMEPLox IgGs reduced 60% of the hyaluronidase activity of venom (Lima et al, 2018). Another chimeric protein (LgRec1ALP1) which included antigenic regions of a PLD (LgRec1) and a metalloprotease (LgALP1) of L. gaucho venom was also evaluated as antigen (Calabria et al, 2019). Previous incubation of anti-LgRec1ALP IgGs with the venoms of L. gaucho, L. intermedia, L. laeta reduced the dermonecrotic effects of these venoms in 100, 79 and 68%, respectively (Calabria et al, 2019).…”

“…Another chimeric protein (LgRec1ALP1) which included antigenic regions of a PLD (LgRec1) and a metalloprotease (LgALP1) of L. gaucho venom was also evaluated as antigen (Calabria et al, 2019). Previous incubation of anti-LgRec1ALP IgGs with the venoms of L. gaucho, L. intermedia, L. laeta reduced the dermonecrotic effects of these venoms in 100, 79 and 68%, respectively (Calabria et al, 2019). In vitro, those IgGs reduced the platelet aggregation and the proteolytic action of metalloproteases of the three venoms (Calabria et al, 2019).…”

“…General data acquired from venom gland transcriptomes and venom proteomes elucidated the toxins' expression profile of many Loxosceles species (Machado et al, 2005;Fernandes-Pedrosa et al, 2008;Dos Santos et al, 2009;Gremski et al, 2010;Trevisan-Silva et al, 2017;Calabria et al, 2019). In addition, these screening efforts allowed the description of coding sequences of less expressed toxins, which enabled the production of these molecules as recombinant proteins (Sade et al, 2012;Ferrer et al, 2013;Graeff et al, 2019;Justa et al, 2020;De-Bona et al, 2021).…”

“…However, the production of other recombinant proteins, i.e., hyaluronidases, TCTP, ICK peptides, allergen and serpin, was only possible after the initial descriptions of their coding sequences (Sade et al, 2012;Ferrer et al, 2013;Matsubara et al, 2017;Graeff et al, 2019;Justa et al, 2020). Recombinant chimeric proteins consisting of Loxosceles toxins' epitopes were also produced (Mendes et al, 2013;Lima et al, 2018;Souza et al, 2018;Calabria et al, 2019). The expression model differs for each recombinant Loxosceles toxin since some of them are produced as soluble and active proteins by prokaryotic expression systems, while others require a more complex model to achieve the desired production protocol (Boia-ferreira et al, 2019;Graeff et al, 2019;Gremski et al, 2020;Justa et al, 2020;De-Bona et al, 2021).…”

Arthropod Venom Components and Their Potential Usage

“…An example of this alternative in the present special issue is the production of recombinant hybrid toxin/immunogen. Taking phospholipase D from the spider Loxoceles as toxin moiety, a chimeric hybrid was produced by Calabria and colleagues to raise protective antibodies in Loxoceles antivenom therapy [21]. Last but not least, the use of arthropod toxins as bioinsecticide is continuously showed to be a promising application of this classes of animal venom.…”