Crotoxin (Crtx), the main toxin in the venom of Crotalus durissus terrificus snake, is a heterodimer with a basic subunit, CB, and an acidic subunit, CA. CB is a phospholipase A2 that depends on CA to specifically bind to the cell membrane. This toxin acts in the central nervous system (CNS) causing chronic seizure effects and other cytotoxic effects. Here, we report its action on glutamate release in rat cerebral cortex synaptosomes. Aiming at a better understanding of the mechanism of action of Crtx, calcium channel blockers were used and internalization studies were performed in cerebellar granule neurons. Our results show that Crtx induces calcium-dependent glutamate release via N and P/Q calcium channels. In addition, the CB subunit of Crtx is shown to be internalized. This internalization does not depend on the presence of CA subunit neither on the PLA2 activity of CB. A correlation between CB internalization and glutamate release remains to be established.
Introduction: Seasonal influenza viruses infect 5-15% of the human population each year, resulting in approximately 500,000 deaths worldwide. Influenza A viruses have two glycoproteins anchored on the viral envelope: haemagglutinin (HA) and neuraminidase (NA). The SARS-CoV-2 is a β-coronavirus that was discovered in December 2019 in Wuhan, China. SARS-CoV-2 is now responsible for an ongoing outbreak of atypical pneumonia that has affected people worldwide. Coronavirus spike (S) glycoprotein promotes entry into cells and comprises two functional subunits responsible for binding the host cell receptor: S1 subunit, which contains the receptor-binding domain (RBD) and S2 subunit, responsible for fusion between the viral and cellular membranes. As the S glycoprotein is surface-exposed and mediates entry into host cells, it is the main target of neutralizing antibodies upon infection and is the focus of therapeutics and vaccine design.Objective: In this work we aimed to develop a bivalent vaccine against SARS-Cov-2 and seasonal flu using recombinant influenza virus with an impaired capacity to multiply. Methodology:To do so we utilized eight plasmids to driven reverse genetics to generate a recombinant influenza virus carrying only the first 169 and the last 178 nucleotides of NA sequence. We used two different portions of S protein that are known to be immunogenic (RBD and RBD-SD1) and incorporated inside NA sequence to allow expression of this protein in the surface of the virus (169RBD and 169RBD-SD1). In parallel we also generated a recombinant influenza virus in which the expression of the RBD-SD1 domain of S protein is secreted in the cell (166RBD-SD1). Following Balb/c or C57BL/6 mice were immunized with two doses containing 105 PFU of 169RBD, 169RBD-SD1 or 166RBD-SD1 recombinant virus. To evaluate the immune response elicited by vaccination, we investigated the presence of specific IgG antibodies in mice sera and the production of IFN-g by splenocytes stimulated with recombinant RBD protein or RBD peptides.Results: Immunofluorescence assays using antibodies against RBD and HA confirmed the generation of the recombinant influenza virus expressing SARS-CoV-2 RBD and RBD-SD1 domains. Immunization assays demonstrated that mice immunized with 169RBD, 169RBD-SD1 and 166RBD-SD1 recombinat virus produced only timid levels of anti-RBD IgG antibodies but high levels of anti-HA antibodies. Splenocytes from immunized mice stimulated with RBD or peptides from RBD protein were able to generate strikingly high levels of IFN-g, detected by ELISA and ELISPOT. Conclusion:Immunization of mice with recombinant influenza virus expressing RBD and RBD-SD1 generated low levels of IgG antibodies but induced high levels of IFN-g. We are currently evaluating if immunization with the recombinant influenza virus expressing RBD and RBD-SD1 are capable to protect mice against a challenged with SARS-Cov-2 virus.
Introduction:Influenza is an important public health problem due to its high transmissibility, the ability to cause serious illness and pandemic potential. Immunization is the main strategy to reduce the impact of influenza infections. However, the current vaccines are unable to confer broad range protection against other FLU isolates and subtypes. Therefore, is important to seek alternative vaccine approaches able to induce the heterosubtypic and long-term memory responses. In this context, the cytokine IL-Z (encoded) is an important target in the study of new vaccine adjuvants, since it is associated with homeostasis and cell survival.Objective: This work aims to evaluate the role of IL-Z cytokine in infection and immunization against influenza virus in murine model. Methodology:To this end, by using the plasmid driven reverse genetics techniques, we generated a defective recombinant influenza virus (FluIL-Z) encoding the murine IL-Z sequence. After generation of the viral particles, the stocks were produced by cloning in cell culture and characterized by PCR, ELISA, sequencing and titration. Therefore, this virus was used for intranasal immunization of mice (License number: LW-7/17) and evaluated in the co-infection with a H1N1 wild-type virus and in the heterosubtipic protection after challange with H3N2 virus. Results:Our results demonstrate that the FluIL-Z is completely safe to mice and was able to induce the expression of high levels of IL-Z cytokine in the cell culture supernatant. In mice infected with the construct, the IL-Z cytokine peak occurs in 24h and 48h in the lungs and bronchoalveolar lavage, respectively. In addition, mice co-infected with wild-type virus and recombinant virus showed better recovery from the disease and mortality is lower in this group. Finally, FluIL-Z conferred long-term heterosubtypic protection to animals previously immunized and challenged after 30 and 75 days with an influenza virus of another subtype. Conclusion:In conclusion, our results suggest that FluIL-Z is a promising tool and may support the development of new and more effective immunization strategies against influenza in the future.
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