Communities in DSpace
Select a community to browse its collections.
Recent Submissions
Harnessing phage display technology for generating fully human IgG antibodies that neutralise elapid neurotoxins
(2019-02-01) Karatt Vellatt, Aneesh; Laustsen, Andreas Hougaard; Masters, Edward W.; Oscoz, Saioa; Pus, Urska; Knudsen, Cecilie; Slavny, Peter; Luther, Alice M.; Leah, Rachael A.; Griffiths, Daniel t.; Olesen, Majken Lindholm; Lomonte, Bruno; Gutiérrez, José María; McCafferty, John
Snakebite constitutes a major health concern in rural, tropical parts of the world, causing mortality, morbidity and suffering to millions of victims. Members of the notorious elapid snake family are known for their potent neurotoxic venom, the clinical manifestation of which is descending paralysis in envenomed victims. The venoms of the two most feared species in their respective geographical regions of sub-Saharan Africa and Southeast Asia, Dendroaspis polylepis and Naja kaouthia, were analysed by toxicovenomics and their medically most important toxins were identified. A diverse panel of single-chain variable fragment (scFv) binders were isolated against two of the most important toxins from these snakes (dendrotoxin 1 and α-cobratoxin) from the IONTAS human naïve antibody library using phage display technology. The most promising binders were converted to human IgG1 format, transiently expressed in HEK293 cells, and tested in vivo in CD-1 mice. Several IgGs showed full protection (>24 hours) at low doses against both toxins and are being further investigated for their ability to cross-neutralize homologous snake venom toxins. These results break new ground as the first report of fully human IgGs capable of neutralizing animal toxins.
Monoclonal human IgGs capable of neutralizing elapid neurotoxins in vivo
(2019-02-01) Pus, Urska; Laustsen, Andreas Hougaard; Karatt Vellatt, Aneesh; Griffiths, Daniel T.; Oscoz, Saioa; Andersen, Mikael Rørdam; Harrison, Robert A.; Casewell, Nicholas R.; Lomonte, Bruno; McCafferty, John; Gutiérrez, José María
The two elapids, the black mamba (Dendroaspis polylepis) and the monocled cobra (Naja kaouthia) are notorious for their bite, which each year causes a substantial share of the severe envenomings that occur in sub-Saharan Africa and Southeast Asia, respectively. Through a combined toxicovenomics and the phage display selection approach, monoclonal fully human IgGs were discovered and assessed for their ability to neutralize medically relevant toxins from the aforementioned snakes in vivo. The discovered monoclonal human IgGs were expressed in mammalian Expi-293 cells and tested in CD-1 mice using two different routes of administration. Initially, IgGs were incubated for 30 min at 37°C together with their target toxins in different molar ratios (mol toxin: mol IgG of 1:3 to 1:8) and then administered either intracerebroventricularly (i.c.v.) (dendrotoxins) using a toxin dose of 0.5 μg or intravenously (i.v.) (α-cobratoxin) using a toxin dose of 4 μg to evaluate the neutralization potential of the IgGs. The survival of mice administered with lethal doses of elapid toxins was substantially prolonged by the monoclonal human IgGs. Hence, we report the discovery of monoclonal fully human IgGs that are able to neutralize snake toxins in vivo. Additionally, one of the tested human IgGs was able to prolong survival both against its cognate toxin (α-cobratoxin) and against whole venom from N. kaouthia. This demonstrates the applicability of the Toxicity Score for identifying medically relevant toxins in a venom and that α-cobratoxin is one of the key toxic components of N. kaouthia venom.
Harnessing phage display technology for discovery of human IgGs targeting clinically relevant toxins from the venom of the Central American coral snake (Micrurus nigrocinctus)
(2019-02-01) Bermúdez Méndez, Erick; Fernández Ulate, Julián; Lomonte Vigliotti, Bruno; Gutiérrez, José María; Laustsen, Andreas Hougaard
Envenomings caused by snakebites represent a serious public health problem in rural tropical areas of Africa, Asia, Latin America, and Oceania. Micrurus nigrocinctus, commonly known as Central American coral snake, is the most abundant and medically important species from the Elapidae family in Central America. The clinically most relevant toxins of M. nigrocinctus venom belong to the phospholipase A2 (PLA2) and three-finger toxin (3FTx) protein families. These toxins induce both myotoxic effects and potent neurotoxic effects that can lead to respiratory arrest due to neuromuscular paralysis. Currently, the only effective treatment available against bites from coral snakes consists of antivenom derived from serum of immunized horses. Although effective in neutralizing toxicity, this antivenom suffers from the drawbacks of having a heterologous nature and therefore being incompatible with the human immune system, difficult to manufacture due to the scarcity of M. nigrocinctus venom, and having a sub-optimally balanced response skewed towards venom components of high immunogenicity, but limited toxicity.
In this project, we harness phage display technology to discover human single-chain variable fragments (scFvs) against key venom toxins of M. nigrocinctus venom. Following identification of high affinity binders employing the IONTAS scFv library, candidate scFv binders will be converted to the IgG format to increase their half-life, providing prolonged systemic protection against the venom. We expect this study will help to pave the way for the development of novel, low-cost antivenoms with improved efficacy and safety against M. nigrocinctus envenomings.
Chapter 208 - Bothrops asper Hemorrhagic Proteinases
(2012) Gutiérrez, José María; Rucavado Romero, Alexandra; Ovadia, Michael; Neil D. Rawlings; Guy Salvesen
This chapter describes the biochemical, structural, and biological characteristics of the hemorrhagic metalloproteinases present in Bothrops asper venom, mainly BaH1, BaH4, and BaP1. These enzymes belong to the M12 family of zinc-dependent metalloproteinases and are responsible for the local and systemic hemorrhagic effects induced by the venom. BaH1 and BaH4 exhibit high hemorrhagic activity, whereas BaP1 shows a milder effect. These proteins hydrolyze several substrates, including type IV collagen, laminin, and fibronectin—key components of the capillary basement membrane. Their proteolytic action leads to blood vessel degradation, muscle necrosis, inflammation, and blister formation in the skin. Structural studies indicate that BaH1 and BaH4 are 64–69 kDa proteins (class III), while BaP1, at 24 kDa, belongs to class I. These enzymes are inhibited by chelating agents and natural antihemorrhagic proteins found in snake and mammalian sera. Research on these metalloproteinases has enhanced the understanding of the pathogenic mechanisms underlying venom-induced tissue damage and contributed to the development of therapeutic strategies complementary to traditional antivenom treatments.
Metalloproteinase inhibitors in snakebite envenomations
(1999-10-25) Gutiérrez, José María; Rucavado Romero, Alexandra; Ovadia, Michael
Pit viper envenomations are characterized by prominent local tissue damage, such as necrosis, hemorrhage and inflammation. These effects are relatively difficult to neutralize with antivenoms because of their rapid onset and development1. When treatment is delayed, as often occurs in tropical regions of the world, patients are at risk of developing permanent sequelae such as tissue loss or dysfunction. Horse- or sheep-derived antivenoms continue to be the mainstay in the treatment of snakebite envenomations, as they effectively neutralize systemically acting venom toxins, and partially decrease the extent of venom-induced local tissue damage. However, there is a need to develop ancillary treatments to inhibit locally acting toxins that could be used in addition to immunotherapy. Metalloproteinases are widely distributed in crotaline and viperine snake venoms2. They play a significant role in local tissue damage by inducing hemorrhage, oedema, myonecrosis, dermonecrosis and inflammation. Inhibitors of venom metalloproteinases, which could be injected directly at the site of venom injection, could offer a means of addressing this problem.