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1.
J. venom. anim. toxins incl. trop. dis ; 28: e20210080, 2022. graf, tab
Artigo em Inglês | VETINDEX | ID: biblio-1395757

Resumo

Background: A new pit viper, Protobothrops kelomohy, has been recently discovered in northern and northwestern Thailand. Envenoming by the other Protobothrops species across several Asian countries has been a serious health problem since their venom is highly hematotoxic. However, the management of P. kelomohy bites is required as no specific antivenom is available. This study aimed to investigate the biochemical properties and proteomes of P. kelomohy venom (PKV), including the cross-neutralization to its lethality with antivenoms available in Thailand. Methods: PKV was evaluated for its neutralizing capacity (ER50), lethality (LD50), procoagulant and hemorrhagic effects with three monovalent antivenoms (TAAV, DSAV, and CRAV) and one polyvalent (HPAV) hematotoxic antivenom. The enzymatic activities were examined in comparison with venoms of Trimeresurus albolabris (TAV), Daboia siamensis (DSV), Calloselasma rhodostoma (CRV). Molecular mass was separated on SDS-PAGE, then the specific proteins were determined by western blotting. The venom protein classification was analyzed using mass spectrometry-based proteomics. Results: Intravenous LD50 of PKV was 0.67 µg/g. ER50 of HPAV, DSAV and TAAV neutralize PKV at 1.02, 0.36 and 0.12 mg/mL, respectively. PKV exhibited procoagulant effect with a minimal coagulation dose of 12.5 ± 0.016 µg/mL and hemorrhagic effect with a minimal hemorrhagic dose of 1.20 ± 0.71 µg/mouse. HPAV was significantly effective in neutralizing procoagulant and hemorrhagic effects of PKV than those of TAAV, DSAV and CRAV. All enzymatic activities among four venoms exhibited significant differences. PKV proteome revealed eleven classes of putative snake venom proteins, predominantly metalloproteinase (40.85%), serine protease (29.93%), and phospholipase A2 (15.49%). Conclusions: Enzymatic activities of PKV are similarly related to other viperid venoms in this study by quantitatively hematotoxic properties. Three major venom toxins were responsible for coagulopathy in PKV envenomation. The antivenom HPAV was considered effective in neutralizing the lethality, procoagulant and hemorrhagic effects of PKV.(AU)


Assuntos
Animais , Venenos de Víboras/análise , Fenômenos Bioquímicos/fisiologia , Proteômica/métodos , Tailândia , Antivenenos/análise
2.
Acta sci. vet. (Impr.) ; 50: Pub.1860-2022. tab
Artigo em Português | VETINDEX | ID: biblio-1458535

Resumo

Background: Scorpionism is a worldwide medical issue, especially relevant in the tropical and subtropical countries. Tityusserrulatus is the species responsible for most cases in Brazil. Antivenom administration to victims is the sole specific therapyobtained from donor animals. Most of these donors suffer with symptoms of the poisoning, debilitating their health andreducing their life expectancy. The aim of the present research was to evaluate whether the immunogens prepared fromthe crude and detoxified venom of T. serrulatus promoted different changes in fractionated sheep plasma proteins, duringa scorpion antivenom serum production.Materials, Methods & Results: Twelve sheep, healthy, mean weight of 30 kg, were distributed into 3 groups (n = 4): G1(control), G2 (crude venom) and G3 (detoxified venom). The adopted immunization protocol (first cycle) had 6 doses, 3using Freund’s adjuvant, with a 21-day interval between each one (day 0, 22 and 43), and 3 doses with no adjuvant (booster)and 0.2 mg of antigen (reinforcement), spaced 3 days between each other (day 50, 53 and 56). Group control (G1) received6 immunizations with phosphate buffered saline (PBS) associated with Freund’s adjuvant (1:1), while the other 2 groupsreceived 0.5 mg of venom (G2) and detoxified venom (G3), respectively, diluted in PBS, associated with the Freund adjuvant. The boosters were 1/3 of the initial dose, diluted only PBS. At baseline (T0) and at 24 and 48 h after immunization,all animals underwent clinical examinations. Blood samples were collected at day 0, 22, 43, 53 and 56 for proteinogramanalysis. Total protein, albumin and globulins fractions were measured. Plasma albumin concentration at T0 ranged from3.41-4.86 g/dL, with a mean value of 4.12 g/dL. There was no statistical difference between...


Assuntos
Animais , Antivenenos , Ovinos/imunologia , Proteínas Sanguíneas/análise , Venenos de Escorpião/imunologia , Escorpiões , Soros Imunes
3.
J. venom. anim. toxins incl. trop. dis ; 27: e20210051, 2021. tab, graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1356458

Resumo

King Cobra (Ophiophagus hannah) has a significant place in many cultures, and is a medically important venomous snake in the world. Envenomation by this snake is highly lethal, manifested mainly by neurotoxicity and local tissue damage. King Cobra may be part of a larger species complex, and is widely distributed across Southeast Asia, southern China, northern and eastern regions as well as the Western Ghats of India, indicating potential geographical variation in venom composition. There is, however, only one species-specific King Cobra antivenom available worldwide that is produced in Thailand, using venom from the snake of Thai origin. Issues relating to the management of King Cobra envenomation (e.g., variation in the composition and toxicity of the venom, limited availability and efficacy of antivenom), and challenges faced in the research of venom (in particular proteomics), are rarely addressed. This article reviews the natural history and sociocultural importance of King Cobra, cases of snakebite envenomation caused by this species, current practice of management (preclinical and clinical), and major toxinological studies of the venom with a focus on venom proteomics, toxicity and neutralization. Unfortunately, epidemiological data of King Cobra bite is scarce, and venom proteomes reported in various studies revealed marked discrepancies in details. Challenges, such as inconsistency in snake venom sampling, varying methodology of proteomic analysis, lack of mechanistic and antivenomic studies, and controversy surrounding antivenom use in treating King Cobra envenomation are herein discussed. Future directions are proposed, including the effort to establish a standard, comprehensive Pan-Asian proteomic database of King Cobra venom, from which the venom variation can be determined. Research should be undertaken to characterize the toxin antigenicity, and to develop an antivenom with improved efficacy and wider geographical utility. The endeavors are aligned with the WHO´s roadmap that aims to reduce the disease burden of snakebite by 50% before 2030.(AU)


Assuntos
Animais , Intoxicação , Mordeduras de Serpentes , Serpentes , Antivenenos , Proteoma , Venenos Elapídicos , História Natural
4.
J. venom. anim. toxins incl. trop. dis ; 27: e20200066, 2021. graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1154773

Resumo

In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.(AU)


Assuntos
Animais , Venenos de Serpentes , Antivenenos , Bothrops , Serina Proteases , Peptídeos
5.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 27: e20200177, 2021. tab, graf
Artigo em Inglês | VETINDEX | ID: vti-31990

Resumo

The western Russell's viper (Daboia russelii) is widely distributed in South Asia, and geographical venom variation is anticipated among distant populations. Antivenoms used for Russell's viper envenomation are, however, raised typically against snakes from Southern India. The present study investigated and compared the venom proteomes of D. russelii from Sri Lanka (DrSL) and India (DrI), the immunorecognition of Indian VINS Polyvalent Antivenom (VPAV) and its efficacy in neutralizing the venom toxicity. Methods: The venoms of DrSL and DrI were decomplexed with C18 high-performance liquid chromatography and SDS-polyacrylamide gel electrophoresis under reducing conditions. The proteins fractionated were identified through nano-ESI-liquid chromatography-tandem mass spectrometry (LCMS/MS). The immunological studies were conducted with enzyme-linked immunosorbent assay. The neutralization of the venom procoagulant effect was evaluated in citrated human plasma. The neutralization of the venom lethality was assessed in vivo in mice adopting the WHO protocol. Results: DrSL and DrI venom proteomes showed comparable major protein families, with phospholipases A2 (PLA2) being the most abundant (> 60% of total venom proteins) and diverse (six protein forms identified). Both venoms were highly procoagulant and lethal (intravenous median lethal dose in mice, LD50 = 0.24 and 0.32 µg/g, for DrSL and DrI, respectively), while lacking hemorrhagic and anticoagulant activities. VPAV was immunoreactive toward DrSL and DrI venoms, indicating conserved protein antigenicity in the venoms. The high molecular weight venom proteins were, however, more effectively immunorecognized than small ones. VPAV was able to neutralize the coagulopathic and lethal effects of the venoms moderately. Conclusion: Considering that a large amount of venom can be injected by Russell's viper during envenomation, the potency of antivenom can be further improved for optimal neutralization and effective treatment. Region-specific venoms and key toxins may be incorporated into the immunization procedure during antivenom production.(AU)


Assuntos
Animais , Venenos/toxicidade , Antivenenos/biossíntese , Daboia , Proteômica , Localizações Geográficas
6.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 27: e20200066, 2021. graf
Artigo em Inglês | VETINDEX | ID: vti-31986

Resumo

In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.(AU)


Assuntos
Animais , Venenos de Serpentes , Antivenenos , Bothrops , Serina Proteases , Peptídeos
7.
J. venom. anim. toxins incl. trop. dis ; 27: e20200177, 2021. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1250255

Resumo

The western Russell's viper (Daboia russelii) is widely distributed in South Asia, and geographical venom variation is anticipated among distant populations. Antivenoms used for Russell's viper envenomation are, however, raised typically against snakes from Southern India. The present study investigated and compared the venom proteomes of D. russelii from Sri Lanka (DrSL) and India (DrI), the immunorecognition of Indian VINS Polyvalent Antivenom (VPAV) and its efficacy in neutralizing the venom toxicity. Methods: The venoms of DrSL and DrI were decomplexed with C18 high-performance liquid chromatography and SDS-polyacrylamide gel electrophoresis under reducing conditions. The proteins fractionated were identified through nano-ESI-liquid chromatography-tandem mass spectrometry (LCMS/MS). The immunological studies were conducted with enzyme-linked immunosorbent assay. The neutralization of the venom procoagulant effect was evaluated in citrated human plasma. The neutralization of the venom lethality was assessed in vivo in mice adopting the WHO protocol. Results: DrSL and DrI venom proteomes showed comparable major protein families, with phospholipases A2 (PLA2) being the most abundant (> 60% of total venom proteins) and diverse (six protein forms identified). Both venoms were highly procoagulant and lethal (intravenous median lethal dose in mice, LD50 = 0.24 and 0.32 µg/g, for DrSL and DrI, respectively), while lacking hemorrhagic and anticoagulant activities. VPAV was immunoreactive toward DrSL and DrI venoms, indicating conserved protein antigenicity in the venoms. The high molecular weight venom proteins were, however, more effectively immunorecognized than small ones. VPAV was able to neutralize the coagulopathic and lethal effects of the venoms moderately. Conclusion: Considering that a large amount of venom can be injected by Russell's viper during envenomation, the potency of antivenom can be further improved for optimal neutralization and effective treatment. Region-specific venoms and key toxins may be incorporated into the immunization procedure during antivenom production.(AU)


Assuntos
Animais , Venenos/toxicidade , Antivenenos/biossíntese , Daboia , Proteômica , Localizações Geográficas
8.
J. venom. anim. toxins incl. trop. dis ; 26: e20200025, 2020. tab, graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1135152

Resumo

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)


Assuntos
Espectrometria de Massas , Antivenenos , Cromatografia , Corrente Jusante , Plasma , Imunoterapia
9.
J. venom. anim. toxins incl. trop. dis ; 26: e20200056, 2020. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1135145

Resumo

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)


Assuntos
Animais , Venenos de Serpentes , Antivenenos , Galinhas , Trimeresurus , Anticorpos , Bacteriófagos
10.
J. venom. anim. toxins incl. trop. dis ; 26: e20190053, 2020. graf, mapas, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1091020

Resumo

Abstract Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.(AU)


Assuntos
Animais , Antivenenos , Crotalus , Venenos de Crotalídeos/análise , Distribuição Animal
11.
J. venom. anim. toxins incl. trop. dis ; 26: e20200059, 2020. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1143217

Resumo

Acute kidney injury (AKI) is a frequent complication of snakebite envenomation, which is still little known in sub-Saharan Africa. This study aims to describe the clinical, biological and ultrasonographic aspects of AKI following severe snakebite envenomation managed in the intensive care unit. Method: A prospective observational survey was performed in Benin over a period of 18 months. All patients suffering severe snakebite envenomation (SBE) were included. The diagnosis of AKI was made using the KDIGO criteria. Kidney ultrasound exam was performed in all patients to assess internal bleeding and morphological and structural abnormalities of the kidneys. Results: Fifty-one cases of severe SBE were included. All patients presented inflammatory syndrome and showed abnormal WBCT whereas bleeding was found in 46 of them (90%). The median time to hospital presentation was three days. The majority of patients were male (M/F sex ratio = 1.55) and the median age was 26. Sixteen patients (31%) showed AKI according to the KDIGO criteria. Severe AKI (KDIGO stage 2 and 3) was observed in three patients, including one stage 2 and two stage 3. Kidney ultrasound revealed three cases of kidney capsular hematoma (6%), two cases of kidney hypertrophy (3%), three cases of kidney injury (4%), two stage 1 KDIGO and one stage 2 KDIGO. Only one patient benefited from hemodialysis. All patients showing AKI recovered without sequels. The median duration of hospital stays was four days. Seven patients died (14%) including four among the 16 AKI patients. Antivenom has been administered to 41 patients (80%). The comparison between patients without and with AKI did not show any significant difference except gender (p = 10-2). Conclusion: AKI is a common complication of severe snakebite envenomation. Resulting from inflammatory and hemorrhagic disorders, AKI may prove to be a short-term life-threatening factor.(AU)


Assuntos
Animais , Venenos de Víboras , Técnicas de Laboratório Clínico , Insuficiência Renal , Antivenenos , Fatores Biológicos
12.
J. venom. anim. toxins incl. trop. dis ; 26: e20190099, 2020. tab, graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1135151

Resumo

The production of antivenom from immunized animals is an established treatment for snakebites; however, antibody phage display technology may have the capacity to delivery results more quickly and with a better match to local need. Naja oxiana, the Iranian cobra, is a medically important species, responsible for a significant number of deaths annually. This study was designed as proof of principle to determine whether recombinant antibodies with the capacity to neutralize cobra venom could be isolated by phage display. Methods: Toxic fractions from cobra venom were prepared by chromatography and used as targets in phage display to isolate recombinant antibodies from a human scFv library. Candidate antibodies were expressed in E. coli HB2151 and purified by IMAC chromatography. The selected clones were analyzed in in vivo and in vitro experiments. Results: Venom toxicity was contained in two fractions. Around a hundred phage clones were isolated against each fraction, those showing the best promise were G12F3 and G1F4. While all chosen clones showed low but detectable neutralizing effect against Naja oxiana venom, clone G12F3 could inhibit PLA2 activity. Conclusion: Therefore, phage display is believed to have a good potential as an approach to the development of snake antivenom.(AU)


Assuntos
Animais , Mordeduras de Serpentes , Bacteriófagos/isolamento & purificação , Antivenenos , Venenos Elapídicos/síntese química , Anticorpos , Técnicas In Vitro
13.
J. venom. anim. toxins incl. trop. dis ; 26: e20200043, 2020. tab
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1135129

Resumo

Trimeresurus stejnegeri stejnegeri bite induces tissue swelling, pain, thrombocytopenia, rhabdomyolysis, and acute renal failure. However, the incidence of coagulopathy, factors associated with wound necrosis, and the appropriate management of this condition have not been well characterized yet. Materials: This study included patients bitten by T. s. stejnegeri that were admitted to the study hospitals from 2001 to 2016. Patient characteristics, laboratory data, and management approaches were compared in victims with and without wound necrosis. Results: A total of 185 patients were evaluated: three patients (1.6%) were asymptomatic; whereas tissue swelling and pain, local ecchymosis, wound necrosis, coagulopathy, thrombocytopenia, rhabdomyolysis, and renal impairment were present in 182, 53, 13, 15, 10, 1, and 3 patients, respectively. One patient died from coagulopathy and hemorrhagic shock. Antivenom was administered to all envenomed patients at a median time of 1.8 h after the bite. The median total dose of antivenom was five vials. Chi-square analysis showed that bitten fingers, using cold packs during first aid, presence of bullae or blisters, lymphangitis or lymphadenitis, local numbness and suspected infection to be significantly associated with wound necrosis. After adjustment using a multivariate logistic regression model, only cold packs as first aid, bulla or blister formation, and wound infection remained significant. Conclusions: The main effects of T. s. stejnegeri envenomation are tissue swelling, pain, and local ecchymosis. We do not recommend the use of cold packs during first aid to reduce wound pain, as this may be a risk factor for wound necrosis. In addition, patients with bulla or blister formation should be carefully examined for subsequent wound necrosis. Antiplatelet use may worsen systemic bleeding. No severe rhabdomyolysis or renal failure was observed in this large case series, we therefore considered that they were not prominent effects of T. s. stejnegeri bite.(AU)


Assuntos
Animais , Trombocitopenia , Mordeduras e Picadas , Antivenenos , Fatores de Risco , Trimeresurus , Venenos de Crotalídeos , Necrose , Ferimentos e Lesões
14.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 26: e20200025, 2020. tab, graf
Artigo em Inglês | VETINDEX | ID: vti-32211

Resumo

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)


Assuntos
Antivenenos , Corrente Jusante , Imunoterapia , Cromatografia por Troca Iônica , Espectrometria de Massas
15.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 26: e20200056, 2020. tab, graf
Artigo em Inglês | VETINDEX | ID: vti-32273

Resumo

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)


Assuntos
Animais , Galinhas/imunologia , Venenos de Serpentes , Trimeresurus/imunologia , Antivenenos/análise , Antivenenos/imunologia
16.
J. venom. anim. toxins incl. trop. dis ; 26: e20190048, 2020. ilus, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1056677

Resumo

The Eastern Russell's viper, Daboia siamensis, is a WHO Category 1 medically important venomous snake. It has a wide but disjunct distribution in Southeast Asia. The specific antivenom, D. siamensis Monovalent Antivenom (DsMAV-Thailand) is produced in Thailand but not available in Indonesia, where a heterologous trivalent antivenom, Serum Anti Bisa Ular (SABU), is used instead. This study aimed to investigate the geographical venom variation of D. siamensis from Thailand (Ds-Thailand) and Indonesia (Ds-Indonesia), and the immunorecognition of the venom proteins by antivenoms. Methods: The venom proteins were decomplexed with reverse-phase high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by in-solution tryptic digestion, nano-liquid chromatography-tandem mass spectrometry and protein identification. The efficacies of DsMAV-Thailand and SABU in binding the various venom fractions were assessed using an enzyme-linked immunosorbent assay optimized for immunorecognition profiling. Results: The two most abundant protein families in Ds-Thailand venom are phospholipase A2 (PLA2) and Kunitz-type serine protease inhibitor (KSPI). Those abundant in Ds-Indonesia venom are PLA2 and serine protease. KSPI and vascular endothelial growth factor were detected in Ds-Thailand venom, whereas L-amino acid oxidase and disintegrin were present in Ds-Indonesia venom. Common proteins shared between the two included snaclecs, serine proteases, metalloproteinases, phosphodiesterases, 5'nucleotidases and nerve growth factors at varying abundances. DsMAV-Thailand exhibited strong immunorecognition of the major protein fractions in both venoms, but low immunoreactivity toward the low molecular weight proteins e.g. KSPI and disintegrins. On the other hand, SABU was virtually ineffective in binding all fractionated venom proteins. Conclusion: D. siamensis venoms from Thailand and Indonesia varied geographically in the protein subtypes and abundances. The venoms, nevertheless, shared conserved antigenicity that allowed effective immunorecognition by DsMAV-Thailand but not by SABU, consistent with the neutralization efficacy of the antivenoms. A specific, appropriate antivenom is needed in Indonesia to treat Russell's viper envenomation.(AU)


Assuntos
Animais , Antivenenos , Cromatografia Líquida de Alta Pressão , Daboia , Proteômica , Eletroforese em Gel de Poliacrilamida , Fosfolipases A2
17.
J. venom. anim. toxins incl. trop. dis ; 26: e20190103, 2020. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1101265

Resumo

The Brazil's lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. Methods: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. Results: The venom proteome of the Brazil's lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome. Third generation antivenomics profile of the Brazilian pentabothropic antivenom showed paraspecific immunoreactivity against all the toxin classes of B. brazili venom, with maximal binding capacity of 132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom's F(ab')2 antibodies bind B. brazili venom toxins. Conclusion: The proteomics outcome contribute to a deeper insight into the spectrum of toxins present in the venom of the Brazil's lancehead, and rationalize the pathophysiology underlying this snake bite envenomings. The comparative qualitative and quantitative immunorecognition profile of the Brazilian pentabothropic antivenom toward the venom toxins of B. brazili and B. jararaca (the reference venom for assessing the bothropic antivenom's potency in Brazil), provides clues about the proper use of the Brazilian antibothropic polyvalent antivenom in the treatment of bites by the Brazil's lancehead.(AU)


Assuntos
Animais , Oxirredutases , Mordeduras de Serpentes , Venenos de Serpentes , Mordeduras e Picadas , Antivenenos , Bothrops , Proteoma
18.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 26: e20190048, Jan. 31, 2020. tab, graf
Artigo em Inglês | VETINDEX | ID: vti-25640

Resumo

Background The Eastern Russells viper, Daboia siamensis, is a WHO Category 1 medically important venomous snake. It has a wide but disjunct distribution in Southeast Asia. The specific antivenom, D. siamensis Monovalent Antivenom (DsMAV-Thailand) is produced in Thailand but not available in Indonesia, where a heterologous trivalent antivenom, Serum Anti Bisa Ular (SABU), is used instead. This study aimed to investigate the geographical venom variation of D. siamensis from Thailand (Ds-Thailand) and Indonesia (Ds-Indonesia), and the immunorecognition of the venom proteins by antivenoms. Methods: The venom proteins were decomplexed with reverse-phase high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by in-solution tryptic digestion, nano-liquid chromatography-tandem mass spectrometry and protein identification. The efficacies of DsMAV-Thailand and SABU in binding the various venom fractions were assessed using an enzyme-linked immunosorbent assay optimized for immunorecognition profiling. Results: The two most abundant protein families in Ds-Thailand venom are phospholipase A2 (PLA2) and Kunitz-type serine protease inhibitor (KSPI). Those abundant in Ds-Indonesia venom are PLA2 and serine protease. KSPI and vascular endothelial growth factor were detected in Ds-Thailand venom, whereas L-amino acid oxidase and disintegrin were present in Ds-Indonesia venom. Common proteins shared between the two included snaclecs, serine proteases, metalloproteinases, phosphodiesterases, 5nucleotidases and nerve growth factors at varying abundances. DsMAV-Thailand exhibited strong immunorecognition of the major protein fractions in both venoms, but low immunoreactivity toward the low molecular weight proteins e.g. KSPI and disintegrins. On the other hand, SABU was virtually ineffective in binding all fractionated venom proteins. Conclusion: D. siamensis venoms from Thailand and Indonesia varied...(AU)


Assuntos
Animais , Proteômica , Venenos de Víboras/antagonistas & inibidores , Antivenenos , Fosfolipases A2 , Inibidores de Serinopeptidase do Tipo Kazal
19.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 26: e20190103, Apr. 17, 2020. tab, graf
Artigo em Inglês | VETINDEX | ID: vti-25949

Resumo

Background: The Brazils lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. Methods: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. Results: The venom proteome of the Brazils lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome...(AU)


Assuntos
Animais , Bothrops , Venenos de Crotalídeos/análise , Venenos de Crotalídeos/química , Venenos de Crotalídeos/antagonistas & inibidores , Antivenenos
20.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 26: e20190053, Apr. 6, 2020. ilus, mapas, graf
Artigo em Inglês | VETINDEX | ID: vti-25941

Resumo

Background:Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods:The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results:The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion:These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.(AU)


Assuntos
Animais , Crotalus/anatomia & histologia , Crotalus/classificação , Crotalus/genética , Venenos de Crotalídeos/antagonistas & inibidores , Antivenenos
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