Heterologous expression, protein folding and antibody recognition of a neurotoxin from the Mexican coral snake Micrurus laticorallis

Background The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Method Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). Results The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. Conclusion HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.(AU)

Heterologous expression, protein folding and antibody recognition of a neurotoxin from the Mexican coral snake Micrurus laticorallis

The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Method Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). Results The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. Conclusion HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.

Reconhecimento de frações do veneno de cobras corais brasileiras pelo soro antielapídico / Recognition of venom fractions from Brazilian coral snakes by antielapid serum

Objetivos: Investigar a capacidade do soro antielapídico produzido no Brasil na identificação de frações do veneno de seis espécies, incluindo as que constituem o pool de inoculação: Micrurus brasiliensis, M. corallinus, M. frontalis, M. lemniscatus, M. spixii e M. surinamensis. Métodos: As amostras utilizadas fazem parte do banco de venenos do Centro de Estudos e Pesquisas Biológicas, da Pontifícia Universidade Católica de Goiás. O soro antielapídico foi cedido pela Fundação Ezequiel Dias. As metodologias empregadas foram eletroforese e imunoblotting. Resultados: Foi demonstrada uma variabilidade toxinológica e uma capacidade também variável de reconhecimento desses componentes pelo soro antielapídico. A partir da técnica de western-blotting o soro antielapídico da Fundação Ezequiel Dias foi capaz de reconhecer a maioria, mas não todos os componentes presentes nos venenos analisados. Conclusões: Os resultados sugerem uma eficácia restrita do soro antielapídico já que o mesmo possui limitações quanto as espécies Amazônicas, o que reforça a necessidade de uma revisão dos estudos intra e interespecíficos dos venenos micrúricos.

Aims: To investigate whether the antielapid serum produced in Brazil could identify venom fractions from six species of Micrurus, including those in the inoculation pool: Micrurus brasiliensis, M. corallinus, M. frontalis, M. lemniscatus, M. spixii, and M. surinamensis. Methods: The samples belong to the venom bank of the Center for Biological Studies and Research from the Pontifícia Universidade Católica de Goiás, Brazil. The antielapid serum was granted by the Ezequiel Dias Foundation. Both electrophoresis and immunoblotting methods were used. Results: Variability in venom components and in the ability to recognize such components was demonstrated by the antielapid serum. Based on the western-blotting technique, the antielapid serum from Ezequiel Dias Foundation was able to recognize most, but not all the components present in the analyzed venoms. Conclusions: The results suggest restricted efficacy of the antielapid serum, due to its limitations against species from the Amazon region, reinforcing the need for a review of intraspecific and interspecific studies of Micrurus venoms.

Use of antivenoms for the treatment of envenomation by Elapidae snakes in Guinea, Sub-Saharan Africa

Background In Guinea Elapids are responsible for 20% of envenomations. The associated case fatality rate (CFR) ranged 15-27%, irrespective of treatment. Results We studied 77 neurotoxic envenomations divided in 3 groups: a set of patients that received only traditional or symptomatic treatments, and two other groups that received either 2 or 4 initial vials of Antivipmyn® Africa renewed as necessary. CFR was 27.3%, 15.4% and 17.6%, respectively. Although antivenom treatment was likely to reduce CFR, it didn’t seem to have an obvious clinical benefit for the patients, suggesting a low treatment efficacy. Mean delay to treatment or clinical stages were not significantly different between the patients who recovered and the patients who died, or between groups. Interpretation of these results is complicated by the lack of systematic studies under comparable conditions. Of particular importance is the absence of assisted ventilation, available to patients in all the other clinical studies of neurotoxic envenomation. Conclusion The apparent lack of clinical benefit may have several causes. The hypothesis of a limited therapeutic window, i.e. an insufficient formation of antigen-antibody complexes once toxins are bound to their targets and/or distributed beyond the reach of antivenom, should be explored. .

Use of antivenoms for the treatment of envenomation by Elapidae snakes in Guinea, Sub-Saharan Africa

In Guinea Elapids are responsible for 20% of envenomations. The associated case fatality rate (CFR) ranged 15-27%, irrespective of treatment. Results We studied 77 neurotoxic envenomations divided in 3 groups: a set of patients that received only traditional or symptomatic treatments, and two other groups that received either 2 or 4 initial vials of Antivipmyn® Africa renewed as necessary. CFR was 27.3%, 15.4% and 17.6%, respectively. Although antivenom treatment was likely to reduce CFR, it didn’t seem to have an obvious clinical benefit for the patients, suggesting a low treatment efficacy. Mean delay to treatment or clinical stages were not significantly different between the patients who recovered and the patients who died, or between groups. Interpretation of these results is complicated by the lack of systematic studies under comparable conditions. Of particular importance is the absence of assisted ventilation, available to patients in all the other clinical studies of neurotoxic envenomation. Conclusion The apparent lack of clinical benefit may have several causes. The hypothesis of a limited therapeutic window, i.e. an insufficient formation of antigen-antibody complexes once toxins are bound to their targets and/or distributed beyond the reach of antivenom, should be explored.