Luis Giovani de Oliveira Guardalini
@butantan.gov.br
Laboratório de Biotecnologia Viral
Instituto Butantan
Scopus Publications
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Scopus Publications
Jaci Leme, Luis Giovani Oliveira Guardalini, Thaissa Consoni Bernardino, Renato Mancini Astray, Aldo Tonso, Eutimio Gustavo Fernández Núñez, and Soraia Attie Calil Jorge
Springer Science and Business Media LLC
Luis Giovani Oliveira Guardalini, Rafaela Morais Rangel, Jaci Leme, Thaissa Consoni Bernardino, Suellen Regina da Silveira, Aldo Tonso, Soraia Attie Calil Jorge, and Eutimio Gustavo Fernández Núñez
Wiley
AbstractBACKGROUNDThis study aimed to establish chemometric models using Raman spectroscopy data for biochemical monitoring of rabies Virus‐Like Particles (VLP) production based on baculovirus/insect cell system. The models were developed using fresh and stored samples from the initial development stages (Schott culture flasks). The following modeling techniques were assessed: partial least squares (PLS) and artificial neural networks (ANN). The effects of spectral filtering approaches, spectral ranges (400–1850 cm−1; 100–3425 cm−1), and sample cryopreservation were also considered. The applicability of the models was evaluated using experimental data from assays carried out in a benchtop bioreactor.RESULTSThe results showed that the prediction capacity of the chemometric models was negatively impacted when samples from rabies VLP production were cryopreserved. Further studies are needed to confirm the maximum storage time for samples (< 4 months) without a significant difference in model predictions compared to those from an in line database. The dilution of the sample should be kept constant throughout the rabies VLP development stages. A nonlinear correlation was observed between dilution and the predicted values of biochemical parameters from Raman spectral data. The choice of spectral filtering has a major impact on the prediction accuracy of chemometric models.CONCLUSIONThe optimal filtering approach should be individually optimized for each biochemical parameter. The ANN models were significantly more suitable for biochemical monitoring than the PLS approach. The 400–1850 cm−1 Raman shift range is recommended for biochemical monitoring of rabies VLP using a baculovirus/insect cell platform when samples are cell‐free. © 2023 Society of Chemical Industry (SCI).
Renata Gois de Mello, Thaissa Consoni Bernardino, Luis Giovani Oliveira Guardalini, Renato Mancini Astray, Marta Maria Antoniazzi, Simone Gonçalves Silva Jared, Eutimio Gustavo Fernández Núñez, and Soraia Attie Calil Jorge
Frontiers Media SA
Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines.Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins’ conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy.Results and discussion: Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.
Luis Giovani Oliveira Guardalini, Jaci Leme, Paulo Eduardo da Silva Cavalcante, Renata Gois de Mello, Thaissa Consoni Bernardino, Simone Gonçalves Silva Jared, Marta Maria Antoniazzi, Renato Mancini Astray, Aldo Tonso, Eutimio Gustavo Fernández Núñez,et al.
Springer Science and Business Media LLC
Luis Giovani Oliveira Guardalini, Paulo Eduardo da Silva Cavalcante, Jaci Leme, Renata Gois de Mello, Thaissa Consoni Bernardino, Simone Gonçalves Silva Jared, Marta Maria Antoniazzi, Renato Mancini Astray, Aldo Tonso, Eutimio Gustavo Fernández Núñez,et al.
MDPI AG
This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM’s multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.
Marcia Regina Dezan, Luis Giovani O. Guardalini, Elaine Pessoa, Ingrid Helena Ribeiro, Valeria Brito Oliveira, Fabio Luz, Denise Rossite Novac, António Gallucci, Silvia Bonifácio, Francisco Gomes,et al.
Wiley
Molecular tests designed to detect the presence of active RHD gene among D– donors have been successfully applied in people of European ancestry, but not in admixed populations with a considerable frequency of RHD*Ψ. Our goal was to evaluate the performance of a molecular screening tool for identifying active RHD alleles among Brazilian blood donors classified as D– C+ and/or E+.