The bacterium Pseudomonas fluorescens 07A produces a protease with potential for industrial application. In order to remedy problems associated with the use of free enzymes and allow its reuse, the protease was immobilized on DEAE Sephacel ® resin via three different strategies based on ionic interaction and covalent bonding. The matrix-bound enzymes were characterized in relation to their activity (pH, temperature and stability), reuse and storage. Immobilization raised the optimum temperature of activity from 37 °C to 50 °C, whereas the pH of highest activity changed from 7.5 to 7.0 or 8.0, depending on the immobilization strategy. Immobilization proved to be efficient for successive reuses, even leading to an increase in the enzymatic activity along the use. The immobilized enzyme also presented greater stability to high temperatures and storage conditions and has potential as a biocatalyst for industrial applications due to its high efficiency, stability and easy recovery.

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: Streptococcus pneumoniae is a major cause of pneumonia and meningitis, resulting in great morbidity and mortality worldwide. The licensed pneumococcal conjugate vaccines, despite reducing the death rates of pneumococcal infections, are expensive and confer protection only against the serotypes included in vaccine formulation. Therefore, the emergence of new circulating serotypes is a non-negligible risk, arguing favorably for the development of new vaccines capable to elicit broad range protection. Thus, in the present work we generated a recombinant influenza virus carrying a pneumococcal surface protein (named X for patent issues), aiming the development of a vaccine able to induce broad range immune response against S. pneumoniae.Objective: This work aims to evaluate the ability of a vaccination protocol using a recombinant influenza virus encoding a S. pneumoniae protein to induce specific anti-pneumococcus humoral immune response in murine model. Methodology:The recombinant influenza virus was constructed by reverse genetics and characterized by PCR, sequencing and titration on MDCK cells. Female C57BL/6 mice (n=6/group, license number LW-9/17) were inoculated twice with recombinant virus and/or recombinant protein or sterile PBS. At previously established time points, blood samples were collected and specific anti-X protein antibodies in serum of immunized mice were assessed by ELISA. The results were submitted to analysis of variance followed by the Tukey multiple comparisons test, with statistical significance ρ<0.05. Results:To date, our results showed that our vaccination protocol has induced high levels of specific anti-X IgG seric antibodies. Moreover, both IgG1 and IgG2c isotype were detected in the sera of immunized mice, with significantly higher titers of IgG2c than IgG1. It is noteworthy that IgG2c is the subclasse of IgG which has the highest ability to mediate the protection against S. pneumoniae by leading the complement deposition on the surface of the bacteria, resulting in bacterial death by IgG-mediated opsonophagocytosis. Conclusion:Overall, our results indicate that immunization with this vaccination protocol was able to induce specific humoral immune response in mice and has a great potential to be used in the development of new vaccines against S. pneumoniae.

Introduction: Streptococcus pneumoniae is a major cause of pneumonia and meningitis, resulting in great mortality worldwide. In addition, secondary pneumococcal infections are the main complication in influenza infected patients, resulting in poor prognosis. The licensed pneumococcal vaccines, despite reducing the death rates, are serotype-specific, becoming non-protective with the circulation of new strains. Thus, to overcome this problem, we generated a recombinant influenza virus carrying a highly immunogenic and conserved pneumococcus surface protein (nicknamed SP protein), aiming the development of a bivalent vaccine against S. pneumoniae and influenza infections.Objective: To evaluate the potential of a vaccination protocol using a recombinant influenza virus encoding the SP protein (Flu-SP) to induce protective immune response against pneumococcus and influenza, in mice. Methodology:The recombinant influenza viruses were constructed by reverse genetics and characterized by PCR, sequencing and titration. Posteriorly, C57BL/6 mice were intranasally immunized with: Flu-SP followed by boost with adjuvanted SP protein (alum); Flu-Control (Flu-CT) and boost with alum; or PBS (two inoculations). Blood samples were collected and serum anti-SP and anti-influenza antibodies were assessed by ELISA. Furthermore, the ability of anti-SP antibodies to bind to different pneumococcal strains was analyzed by flow cytometry. Finally, to evaluate the protective capacity against pneumococcus, the immunized mice were intranasally challenged with a letal dose (5xMLD50) of a highly virulent pneumococcal strain (ATCC 6303). Moreover, to assess the protection against influenza, C57BL/6 mice was inoculated with Flu-SP, Flu-CT or PBS (one dose) and challenged with a letal dose (100xMLD50) of influenza virus (H1N1). The survival was monitored for 10 days. Differences (ρ <0.05) between groups and survival curves were assessed by ANOVA and Log-rank test, respectively. Results:The results showed that our vaccination protocol (primed with Flu-SP and boosted with adjuvanted SP protein) has induced high levels of anti-SP and anti-influenza IgG antibodies. In addition, an efficient binding of anti-SP antibodies to the surface of different pneumococcal strains were observed. After the pneumococcal lethal challenge, our immunization protocol protected almost 65% of vaccinated mice, whereas the animals of the control groups did not present relevant protection rates. Furthermore, immunization with recombinant viruses (Flu-CT or Flu-SP) resulted in 100% protection against a challenge with influenza, whereas all animals inoculated with PBS died. It's known that specific antibodies play a pivotal role in defense against pneumococcal and influenza infections. Therefore, it is possible that the higher anti-pneumococcal and anti-influenza IgG titers induced by immunization might have contributed to the protection from lethal challenges, resulting in a more effective bacterial opsonophagocytosis and virus neutralizing, respectively. Conclusion:In short...