WP1: Successfully completed by integrating wet and dry lab improvements to deliver a state-of-the-art immunopeptidomics pipeline. Key advancements included miniaturized and automated sample preparation, optimized LC-MS/MS data acquisition on a timsTOF mass spectrometer, and AI-powered tool enhancements for timsTOF data. These improvements increased sensitivity, reducing input material requirements 10- to 50-fold while doubling peptide identification rates.
WP2: We applied the immunopeptidomics pipeline to MTB- and AB-infected cells. For MTB, > 200 peptides from 175 antigens were identified by combining experimental and public data re-analysis. For AB, two multidrug-resistant strains were selected based on virulence and intracellular persistence. In vitro infections yielded > 150 MHC I- and > 90 MHC II-presented peptides from over 100 AB proteins. Sixteen antigens from both pathogens were encoded in mRNA LNP vaccines for efficacy testing in mice.
WP3: We developed and characterized mRNA vaccines containing MTB and AB antigens, as well as reporter proteins and model antigens. Fluorescently labeled mRNA LNPs were tested in vitro with human dendritic cells (DCs) and in vivo for biodistribution. Mice were vaccinated via different delivery routes to optimize administration and efficacy. Production of selected N1-methylpseudouridine (1mΨ) mRNAs using Univercells’ Ntensify™ platform has begun. Additionally, an LC-MS assay was developed for LNP lipid and α-GalCer adjuvant quantification, currently being transferred to UGENT’s GMP unit.
WP4: We have produced adjuvanted mRNA-LNPs by including different lipophilic adjuvants such as monophosphoryl-lipid A (MPLA), QS-21, and α-GalCer. Inclusion of α-GalCer resulted in physico-chemically stable mRNA-LNPs, which were subsequently used to vaccinate mice intramuscularly. Increasing dose experiments have been performed to fine-tune α-GalCer adjuvant dosing.
WP5: We have evaluated the protective efficacy of 15 MTB antigens in 8 mRNA vaccines (2/LNP) in an aerosol mouse infection model. The top vaccines were selected for further combination testing. Immunogenicity analysis of all MTB vaccine candidates is ongoing to identify protection determinants. Assay optimization for PBMC-based protective efficacy assessment has begun, including a macrophage infection protocol with BCG. Peripheral blood collection from MTB-exposed patients has also been initiated. For AB, a subcutaneous vaccination model with heat-killed A. baumannii LAC-4 was developed in preparation for protection assays.
WP6: We have initiated translation of the research-grade mRNA-LNP production protocol to a GMP-compliant process, comprising microfluidic mixing of mRNA with lipids and Tangential Flow Filtration (TFF) for formulation and concentration. Key process parameters have been defined. Additionally, an external regulatory consultant has been engaged to support preclinical interactions ahead of the Phase I clinical trial for the lead MTB vaccine.
