Defining the multiplicity and type of infection for the production of Zaire Ebola virus-like particles in the insect cell baculovirus expression system

Ebola virus hemorrhagic fever affects thousands of people worldwide with high mortality rates. The Ebola virus has a short incubation time between 2-21 days and death usually occurs within 4-10 days1. Ebola virus disease is characterized by a sudden onset of fever, weakness, headache, diarrhea and vomiting, internal and external bleeding2. In the Filovirus family, Zaire Ebola virus (ZEBOV) is the most aggressive and virulent species, its fatality rates have been reported to be up to 90%3. Even when important advances in vaccine development have occurred, the need of safe and effective vaccines persists4. An alternative is the production of virus-like particles, which are formed by the recombinant virus structural proteins that self-assemble into highly immunogenic structures5. The ZEBOV contains three main structural proteins: the glycoprotein (GP), the viral matrix protein 40 (VP40) and the nucleoprotein (NP). GP induces humoral and cellular responses by itself but when VP40 is co-expressed, the immune response increases in a mouse model6. NP determines the structure of the resulting VLP. To our knowledge, there is no information about the production conditions that result in coexpression and assembly of ZEBOV recombinant proteins. In this work, a multifactorial experimental design was used to evaluate 32 different conditions for the production of the ZEBOV structural proteins utilizing the insect cell-baculovirus expression system technology (BEST). Multiplicity (MOI = 0.1 or 5 ufp/cell) and consecutive times of infection (0 or 6 hours after the first infection) were the principal factors, and the production of each recombinant protein and assembly of VLP were the evaluated responses. We observed that multiplicity of infection had an impact over expression of the recombinant proteins, higher multiplicities increased yield and VLP assembly. In contrast, later times of infection reduced the production of each protein. The initial presence of VP40 resulted in a higher concentration of NP. The conditions where the simultaneous expression of the three structural proteins and where VLP were detected were identified. The highest MOIs for bacVP40 and bacGP were needed. bacNP should be added during the initial infection with an MOI of 0.1, or at 6 hpi at MOI of 5. The obtained ZEBOV-VLPs were similar to native virus. The obtained VLP are a candidate vaccine under evaluation. Research performed thanks to the financial support of PAPIIT-UNAM IT200418 and CONACyT 247101. References: 1. Shuaib F, Gunala R, Musa EO, Mahoney FJ, et al., 2014. Ebola virus disease outbreak-Nigeria, July–September 2014. Morb. Mortal. Wkly. Rep. 63 (39),867–872. 2. Qiu X, Audet J, Wong G. Fernando L, et al., 2013. Sustained protection virus infection following treatment of infected nonhuman primates with ZMAb. Sci. Rep. 3, 3. Richardson JS, Wong G, Pillet S, Schindle S, et al., 2011. Evaluation of different strategies for post-exposure treatment of Ebola virus infection in rodents. J.Bioterror. Biodef. S1, 007 4. Ige, Ohimain E, 2016. Recent advances in the development of vaccines for Ebola virus disease. Virus Research 211: 174-185. 5. Palomares LA, Ramírez OT, 2009. Challenges for the production of virus-like particles in insect cells: The case of rotavirus-like particles. Biochem. Eng. J. 45: 158-167. 6. Wahl-Jensen, V. et al (2005). Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages. Journal of Virology, 79(4), 2413-241

Intraspecific venom variation of Mexican West Coast Rattlesnakes (Crotalus basiliscus) and its implications for antivenom production

14 páginas, 9 figuras, 3 tablasIntraspecific variation in snake venoms has been widely documented worldwide. However, there are few studies on this subject in Mexico. Venom characterization studies provide important data used to predict clinical syndromes, to evaluate the efficacy of antivenoms and, in some cases, to improve immunogenic mixtures in the production of antivenoms. In the present work, we evaluated the intraspecific venom variation of Crotalus basiliscus, a rattlesnake of medical importance and whose venom is used in the immunization of horses to produce one of the Mexican antivenoms. Our results demonstrate that there is variation in biological and biochemical activities among adult venoms and that there is an ontogenetic change from juvenile to adult venoms. Juvenile venoms were more lethal and had higher percentages of crotamine and crotoxin, while adult venoms had higher percentages of snake venom metalloproteases (SVMPs). Additionally, we documented crotoxin-like PLA2 variation in which specimens from Zacatecas, Sinaloa and Michoacán (except 1) lacked the neurotoxin, while the rest of the venoms had it. Finally, we evaluated the efficacy of three lots of Birmex antivenom and all three were able to neutralize the lethality of four representative venoms but were not able to neutralize crotamine. We also observed significant differences in the LD50 values neutralized per vial among the different lots. Based on these results, we recommend including venoms containing crotamine in the production of antivenom for a better immunogenic mixture and to improve the homogeneity of lots.This study was financially supported by DGAPA-PAPIIT (project IN211621), CONACYT (project264255); FORDECYT PRONACE (project 1715618/2020), FORDECYT (project 303045), Clemson University and the National Science Foundation (DEB 1822417) to CLP.Peer reviewe

Biological and proteolytic variation in the venom of Crotalus scutulatus scutulatus from Mexico

Rattlesnake venoms may be classified according to the presence/absence and relative abundance of the neurotoxic phospholipases A2s (PLA2s), such as Mojave toxin, and snake venom metalloproteinases (SVMPs). In Mexico, studies to determine venom variation in Mojave Rattlesnakes (Crotalus scutulatus scutulatus) are limited and little is known about the biological and proteolytic activities in this species. Tissue (34) and venom (29) samples were obtained from C. s. scutulatus from different locations within their distribution in Mexico. Mojave toxin detection was carried out at the genomic (by PCR) and protein (by ELISA) levels for all tissue and venom samples. Biological activity was tested on representative venoms by measuring LD50 and hemorrhagic activity. To determine the approximate amount of SVMPs, 15 venoms were separated by RP-HPLC and variation in protein profile and proteolytic activity was evaluated by SDS-PAGE (n = 28) and Hide Powder Azure proteolytic analysis (n = 27). Three types of venom were identified in Mexico which is comparable to the intraspecific venom diversity observed in the Sonoran Desert of Arizona, USA: Venom Type A ( Type II), with Mojave toxin, highly toxic, lacking hemorrhagic activity, and with scarce proteolytic activity; Type B ( Type I), without Mojave toxin, less toxic than Type A, highly hemorrhagic and proteolytic; and Type A + B, containing Mojave toxin, as toxic as venom Type A, variable in hemorrhagic activity and with intermediate proteolytic activity. We also detected a positive correlation between SVMP abundance and hemorrhagic and proteolytic activities. Although more sampling is necessary, our results suggest that venoms containing Mojave toxin and venom lacking this toxin are distributed in the northwest and southeast portions of the distribution in Mexico, respectively, while an intergradation in the middle of both zones is presentConsejo Nacional de Ciencia y Tecnologia/[221343]/CONACYT/MéxicoUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Comparison of F(ab') versus Fab antivenom for pit viper envenomation: A prospective, blinded, multicenter, randomized clinical trial

BACKGROUND: Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab')2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab')2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. METHODS: We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab')2 with maintenance doses [F(ab')2/F(ab')2], or F(ab')2 with placebo maintenance doses [F(ab')2/placebo], versus Fab with maintenance doses [Fab/Fab]. The primary efficacy endpoint was coagulopathy (platelet count < 150 K/mm(3), fibrinogen level < 150 mg/dL) between end of maintenance dosing and day 8. RESULTS: 121 patients were randomized at 18 clinical sites and received at least one dose of study drug. 114 completed the study. Of these, 11/37 (29.7%) in the Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p < 0.05) in the F(ab')2/F(ab')2 cohort and 2/38 (5.3%, p < 0.05) in the F(ab')2/placebo cohort. The lowest heterologous protein exposure was with F(ab')2/placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. CONCLUSIONS: In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab')2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation

Factors involved in the resilience of incidence and decrease of mortality from scorpion stings in Mexico

International audienceIn Mexico, scorpion sting envenomation (SSE) is a significant public health issue that has engaged the attention of health authorities for more than a century. Rigorously characterized today, scorpion sting incidence is stable around 230 stings per 100,000 population, i.e. 300,000 annual stings treated in Mexican health centers and hospitals. Higher incidence is observed mainly in central and Pacific Mexico. Scorpion populations thrive in populated places, particularly in impoverished areas. Scorpion stings occur in houses. This could explain similar incidence according to gender and age. The number of scorpion stings has remained stable since the mid-2000s. In contrast, mortality, which was over 1500 deaths per year before the 1960s, underwent a dramatic drop after the 1970s, from 500 deaths per year to fewer than 50 annual deaths today. Case fatality rates have shown similar trend. We noted a significantly higher specific mortality in males than in females (0.199 and 0.168 per 100,000 respectively; P < 1.9·10-6). Three causes explained the drop in mortality and case fatality rate, a) ongoing improvement in hospital care, particularly in terms of supportive standardized treatments, b) the use of highly purified immunoglobulin F(ab')2 fragments after 1995 and, c) increasing access to health services for most of the Mexican population. The authors retrace the history of the management of SSE, including the development of antivenoms, in Mexico between 1905 and today

På jagt efter guldhornenes findested

Hunting for the find spots for the golden horns of GallehusIt is common knowledge that the golden horns were found at Gallehus in 1639 and 1734 and, consequently, so early that information on the find spots and finds circumstances is extremely sparse. In 1855, C.C. Rafn reached the conclusion that the horns were discovered in an undeveloped common area in the town (fig. 2), while in 1908, P. Lauridsen believed he had established the precise find spots, which were then marked with commemorative stones (figs. 3 and 4). With the discovery in 1951 of a report from 1734, it became clear that Lauridsen’s locations were incorrect and that the two finds spots are unlikely to have been more than 7 m apart, i.e. significantly less than the distance he concluded (fig. 1). This prompted Professor P.V. Glob to launch an investigation of the area in 1952, aimed at finding possible evidence that could explain these depositions. Glob continued his investigations in 1964, 1969 and 1971-72 (fig. 5). But he never managed to write a concluding report, and this article is an attempt to summarise his findings. The most important of these was the discovery of numerous pits, most of which were not very deep and had a very flat base. Many of them had apparently stood open for a shorter or longer period and they were therefore interpreted as clay pits (figs. 6-8). In 1969, some of these pits were found in an area corresponding to the find spot for one of the golden horns as specified in the 1734 source. Glob therefore believed he had found the actual find spot (figs. 10 and 12). The investigation in 1972 showed that these clay pits lay in the northeastern part of a larger more or less coherent complex of clay pits (fig. 7). Secure dating of these was not possible, but the fact that a house was built in 1832 over the southwesternmost corner of the complex testifies to a considerable age.Sven ThorsenNykøbing Falste

Venomics and biochemical analysis of the black-tailed horned pitviper, Mixcoatlus melanurus, and characterization of Melanurutoxin, a novel crotoxin homolog

11 páginas, 6 figuras y 2 yablas. Se puede consultar información complementaria en: https:// doi.org/10.1016/j.jprot.2020.103865.We report a structural and functional venomics characterization of the black-tailed horned pitviper, Mixcoatlus melanurus. The venom phenotype of this small and elusive pitviper endemic to México comprise peptides and proteins of 16 toxin families whose relative abundance mirror those of neurotoxic (type II) venoms described for some species within genera distributed in Central Asia (Gloydius) and the Americas (Sistrurus, Crotalus, Ophryacus, and Bothriechis). A novel β-neurotoxic heterodimeric PLA2, termed Melanurutoxin was characterized. With a relative abundance of 14.8% of the total M. melanurus venom proteome and a median lethal dose of 0.31 μg/g mouse body weight, Melanurutoxin accounted for 37.8% of the lethality of the whole venom (0.82 μg/g). The low percentage (1.1%) of snake venom metalloproteinases (PIII-SVMPs) and the high content of Melanurutoxin and bradykinin-potentiating peptides (BPP, 16%) found in the type-II venom proteome of M. melanurus correlate with the severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest observed in ex vivo neuromuscular junction experiments and in vivo experimental murine envenoming. Mexican antivenoms manufactured by Birmex and Bioclon showed low neutralization potency per vial (95 LD50s, Birmex; 114 LD50s, Antivipmyn®), and failed to reverse completely the paralysis and the hypotensive effect induced by the black-tailed horned pitviper, Mixcoatlus melanurus. We suggest that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom may be attributed to the use of immunization mixtures that include venom of taxa, C. basiliscus (Birmex) and C. simus (Antivipmyn®), that contain only small amounts of Melanurutoxin-like β-neurotoxic heterodimeric PLA2s. BIOLOGICAL SIGNIFICANCE: This study represents the first proteomics and funcional investigations conducted on the venom of the black-tailed horned, Mixcoatlus melanurus, a pitviper species endemic to México. The venom's features unveiled through combination of bottom-up venomics and ex vivo and in vivo functional assays provided complementary evidence pointing to severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest as the predominant mechanism of murine prey immobilization and death caused by M. melanurus. A novel β-neurotoxic heterodimeric PLA2, coined Melanurutoxin, was identified as a major contributor to the lethality of the whole venom. Our study also showed the inefficacy of two commercial Mexican antivenoms to reverse competely the paralytic and hypotensive effects induced by M. melanurus venom in the murine model. We hypothesize that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom should be ascribed to the use as immunogens of venoms that contain only small amounts of Melanurutoxin-like β-neurotoxic heterodimeric PLA2s.This research was partly funded by grant BFU2017-89103-P (Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain), grants PAPIIT-DGAPA IN207218 and CONACyT-FORjDECYT No. 303045 (“Venenos y Antivenenos”).Peer reviewe