Browsing by Author "Pinheiro, Ricardo"
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- Development of a new mRNA vaccine platform for tuberculosisPublication . Matarazzo, Laura; Taina‑González, Laura; Pinheiro, Ricardo; Pires, David; Fuente, Maria de la; Bettencourt, Paulo J. G.
- Development of a new mRNA vaccine platform for tuberculosisPublication . Matarazzo, Laura; Taina‑González, Laura; Pinheiro, Ricardo; Pires, David; de la Fuente, María; Bettencourt, Paulo J. G.Background Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is the frst cause of death by an infectious disease worldwide, killed 1.6 million people in 2021. Bacillus Calmette-Guerin (BCG) is the only approved vaccine for TB to date. However, while BCG is efective in preventing severe forms in children, its efcacy in adults is inconsistent and it does not prevent transmission, highlighting the need for new vaccine development [1]. The recent success of COVID-19 vaccines raised the interest for mRNA-based vaccines, as they are efective, safe and easy to produce. This project aims to develop a new mRNA vaccine platform for TB, based on mRNA coding for antigenic peptides from BCG and M.tb identifed by immunopeptidomics [2], and formulated with a patented technology of lipid nanoemulsions (NE) (WO2019138139A1), adapted for efcient intracellular delivery of mRNA [3]. Materials and methods We tested diferent prototypes of NE-mRNA formulations, coding for EGFP, in vitro. Human alveolar basal epithelial cells (A549), human monocytic cells (THP-1), and primary human monocyte-derived macrophages, were transfected with NE-mRNA formulations. Transfection efciency was assessed by measuring the percentage of transfected cells, and the intensity of GFP fuorescence. The cytotoxicity of the formulations was evaluated using AlamarBlue, and by 7-AAD viability staining. Results In vitro preliminary data using EGFP-mRNA-NE formulations indicate that NE formulations can efciently deliver mRNA and induce expression of the encoded protein in diferent cell types, with low cytotoxicity. Conclusions The NE technology presented here is safe, stable, and can efciently deliver mRNA to various cell types. Selected NE formulations will be used as a carrier for a new vaccine candidate against TB, based on mRNA encoding relevant antigenic peptides. These will be tested in mice for safety, immunogenicity, efcacy and dose optimization in order to generate an efective and sustained humoral and cellular immune response against TB. The mRNA vaccines are rapid and relatively simple to produce. The vaccine platform described here could be adapted to develop vaccines against other infectious diseases, particularly to quickly respond to emerging pathogens.
- Identification of peptides presented by major histocompatibility complex in macrophages through immunopeptidomicsPublication . Mateus, Hugo; Pinheiro, Ricardo; Santos, Hugo M.; Bettencourt, Paulo J. G.
- Implementation of a pre-Good Laboratory Practice management system for academic researchPublication . Pinheiro, Ricardo; Abreu, Cloé; Bettencourt, Paulo J. G.
- Implementation of a pre‑Good Laboratory Practice management system for academic researchPublication . Pinheiro, Ricardo; Abreu, Cloé; Bettencourt, Paulo J. G.The implementation of quality control procedures, at an academiclaboratory, relies on a system that flows information to scientists, staff,and students in a clear and accountable manner.The organization and implementation of new methodologies, in a newlaboratory, implies the definition of a work culture and structure frominception to completion. Establishing and maintaining a new workphilosophy is demanding and requires constant and close supervisionof all laboratory actions. Particularly, when the methods are innovativeand require a significant change of work culture from users.By establishing a system that standardizes common laboratory protocolsto facilitate training while simultaneously tracking progress, wesuccessfully implemented a pre-Good Laboratory Practices (pre-GLP)facility at the Faculty of Medicine of the Universidade Católica Portuguesa(FM).The pre-GLP system is an adaption of the system adopted by theJenner Institute, University of Oxford. Briefly, the new users aretrained on Standard Operations Procedures (SOP), provided by acompetent user. Once training is successfully completed, the user isapproved and qualified as competent user. All training actions arerecorded in the researcher’s internal record. The internal records areinternally verified by the laboratory manager, and laboratory director,and externally audited.The SOPs are regularly updated and improved to reflect any significantupdates on procedures, equipment, and reagents. UpdatedSOP´s are reassessed and follow the pipeline of approval. Implementationof this laboratory management system is a step forwardin quality assurance and standardization of methodologies towardsgood laboratorial practices, increased health, and safety, and qualitydata production.Finally, the implementation of this quality assurance method at theFM, provides an additional layer of health and safety protection forusers, simultaneously assuring reproducibility and reliability of protocolsacross the campus.
- Mass spectrometry‑based identification of peptides presented by major histocompatibility complex in macrophagesPublication . Mateus, Hugo; Pinheiro, Ricardo; Santos, Hugo M.; Bettencourt, Paulo J. G.Immunopeptidomics is a field of research that has progressed in thelast years due to advances in sophisticated analytical techniquesbased on mass spectrometry and bioinformatics. The ability to identifymolecules to the extent of a single ion led to a step forward inimmunopeptidomics. Mass spectrometry enables the identificationof thousands of peptide sequences in a single sample, thus providinglarge-scale reliable information. The immunopeptidome is the entire group of peptides presented by the major histocompatibility complexClass-I (MHC-I), at the surface of all nucleated cells and Class II, at thesurface of professional antigen presenting cells. The MHC-bound peptidesare recognized by T cells and constitute the immunological synapse,leading to the initiation of the adaptive immune response. Underpathological conditions, peptides originating from the proteolysis ofpathogen proteins are presented to the cells of the host immune systemvia MHC. Thus, the identification of pathogen peptides throughimmunopeptidomics is an unbiased method for understanding thegeneration of adaptive immune responses against pathogens.Here we describe the establishment of a new mass spectrometrybasedimmunopeptidomics platform for peptide identification inphysiological and pathological conditions. Using the macrophage cellline with THP-1, with a known HLA-type, we were able to identify atotal of 2913 unique MHC-I bound peptides. The peptide length distribution,NetMHCpan-4.1 rank affinity, and best match HLA bindingallele for each peptide will be presented.Finally, identifying MHC-I and MHC-II peptides under physiologicaland pathological conditions could uncover the most relevant peptidesable to stimulate the right type of T-cell response for vaccine designand development.
- New mRNA - nanoemulsions vaccine platform: application for Tuberculosis vaccine developmentPublication . Matarazzo, Laura; Taina‑González, Laura; Pinheiro, Ricardo; Pires, David; Fuente, María de la; Bettencourt, Paulo J. G.Background: Tuberculosis (TB) is a deadly infectious disease caused by the airborne bacterium Mycobacterium Tuberculosis (M.tb), killing 1.6 million people every year, especially in low- and middle-income countries. The only licensed TB vaccine, a live attenuated strain of M. bovis (BCG), has variable efficacy, does not prevent transmission and is not safe in immunocompromised patients, particularly AIDS patients, who are at high risk of developing TB disease. Thus, the development of an efficient, safe and cost-effective tuberculosis vaccine remains a research priority. mRNA vaccine technology, which proved its potential during the COVID-19 pandemic, could represent a valuable alternative to conventional vaccines against TB. Objectives: We aim to establish a novel vaccine platform against TB, combining antigen identification by immunopeptidomics, mRNA design and production using advanced techniques, and a patented technology of lipid nanoemulsion (NE) adapted to efficiently deliver mRNA to target cells. Methods: Using a prototype mRNA encoding the fluorescent protein EGFP, we assessed in vitro the safety and transfection efficiency of different NE formulations on human alveolar basal epithelial cells (A549) and primary human monocyte-derived macrophages and dendritic cells. Results: Here we show that NEs can efficiently deliver mRNA to different cell types without significant cytotoxicity, and the formulation can be tailored to increase uptake by specific cells relevant for vaccination applications. Conclusion: Lipid NEs appear to be safe, easily adaptable and efficient transporters for mRNA. We will use selected NE formulations as carriers for mRNA vaccine candidates encoding relevant antigenic peptides from M.tb and other Mycobacterium species. Safety and immunogenicity studies will be performed in mice and the vaccination schedule will be optimized to generate a sustained humoral and cellular immune response against TB. This vaccine platform could be adapted to develop vaccines against other infectious diseases, particularly to quickly respond to emerging pathogens.