Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits

Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30-35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody's neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization. Key Contribution:The humanization of a mouse antibody V-domains able to neutralize Loxosceles intermedia venom paves the way for a new generation of anti-venom therapy. This study highlights the importance of understanding the antibody's mechanism of neutralization for appropriate design of innovative antidotes. Likewise, preserving favorable physico-chemical properties and antigen-binding activity is a challenge yet to be further addressed when considering pre-clinical trials.

Section: Discussionmentioning

confidence: 99%

Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential

Karim-Silva

Becker-Finco

Jiacomini

et al. 2020

“…In fact, a synthetic chimera containing sequences of PLDs from L. intermedia and L. laeta venoms, and sequences of a LALP and hyaluronidase isoforms from L. intermedia venom was recently constructed. This fusion molecule stimulated the production of polyclonal antibodies which recognized venoms of the different species of Loxosceles, and blocked the activities of PLDs, metalloproteases and hyaluronidases present in the venoms [107].…”

Section: A New Generation Of Anti-loxoscelic Sera and Vaccines Using mentioning

confidence: 99%

“…This would allow PLDs recognition of different species of Loxosceles spiders, and also the possibility of using monoclonal antibodies for sandwich ELISA tests. Such methods, based on the search for PLD antigens in samples collected from skin lesions of victims, could be directed to these toxins by the high biological conservation of these molecules in the different venoms [5,6] and for their high determinant antigenic properties [107].…”

Section: Diagnosis Of Loxoscelism Based On the Identification Of Pldsmentioning

confidence: 99%

Forty Years of the Description of Brown Spider Venom Phospholipases-D

Gremski

Justa

Silva

et al. 2020

Spiders of the genus Loxosceles, popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all Loxosceles species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from Loxosceles venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field.

“…Similarly, a major knowledge gap exists regarding the ability of available antivenoms to neutralize fibrinogenolytic effects of Loxosceles venoms. Most studies on Loxosceles antivenom, either using crude venom or recombinant toxins, focus on the necrotizing effects of the venom rather than coagulotoxic effects [25][26][27][28][29]. This is reflected in the antivenom production, with the immunizing mixture typically being recombinant spider sphingomyelinase D toxin, not crude (whole) venom due to the impracticality of collecting large quantities of spider venom.…”

Section: Introductionmentioning

confidence: 99%

“…One study showed a polyvalent serum from rabbits using a combination of SMase D toxins, hyaluronidases, and LALPs. The authors suggested that this serum was able to prevent fibrinogen alpha-chain digestion using 100 µL of serum to target 3 µL of L. intermedia venom [29]. This high serum:antivenom ratio indicates poor neutralization capacity of this antivenom.…”

Section: Introductionmentioning

confidence: 99%

A Web of Coagulotoxicity: Failure of Antivenom to Neutralize the Destructive (Non-Clotting) Fibrinogenolytic Activity of Loxosceles and Sicarius Spider Venoms

Grashof

Zdenek

Dobson

et al. 2020

Envenomations are complex medical emergencies that can have a range of symptoms and sequelae. The only specific, scientifically-validated treatment for envenomation is antivenom administration, which is designed to alleviate venom effects. A paucity of efficacy testing exists for numerous antivenoms worldwide, and understanding venom effects and venom potency can help identify antivenom improvement options. Some spider venoms can produce debilitating injuries or even death, yet have been largely neglected in venom and antivenom studies because of the low venom yields. Coagulation disturbances have been particularly under studied due to difficulties in working with blood and the coagulation cascade. These circumstances have resulted in suboptimal spider bite treatment for medically significant spider genera such as Loxosceles and Sicarius. This study identifies and quantifies the anticoagulant effects produced by venoms of three Loxoscles species (L. reclusa, L. boneti, and L. laeta) and that of Sicarius terrosus. We showed that the venoms of all studied species are able to cleave the fibrinogen Aα-chain with varying degrees of potency, with L. reclusa and S. terrosus venom cleaving the Aα-chain most rapidly. Thromboelastography analysis revealed that only L. reclusa venom is able to reduce clot strength, thereby presumably causing anticoagulant effects in the patient. Using the same thromboelastography assays, antivenom efficacy tests revealed that the commonly used Loxoscles-specific SMase D recombinant based antivenom failed to neutralize the anticoagulant effects produced by Loxosceles venom. This study demonstrates the fibrinogenolytic activity of Loxosceles and Sicarius venom and the neutralization failure of Loxosceles antivenom, thus providing impetus for antivenom improvement.