Periodic Reporting for period 1 - BAXERNA 2.0 (Immunopeptidomics-based Development of Next-Generation Bacterial mRNA Vaccines)
Período documentado: 2023-07-01 hasta 2024-12-31
BAXERNA 2.0 will establish a new vaccine development pipeline based on dramatically improved immunopeptidomics screening
and innovative mRNA vaccine formulation. We will use our powerful new pipeline to develop novel mRNA vaccines against three
bacterial pathogens that can persist within phagocytic cells: Mycobacterium tuberculosis (MTB), Mycobacterium ulcerans (MU), and
Acinetobacter baumannii (AB). MTB and AB are clinically problematic bacteria with alarming levels of antimicrobial resistance (AMR),
while MU is an important neglected tropical disease. Although vaccines are recognized as highly effective tools to mitigate AMR and
tropical diseases, effective vaccine development for these (facultative) intracellular bacteria is held back by a lack of known antigens,
and by current vaccine platforms struggling to elicit the required strong cellular immune responses. We will overcome both limitations
here through two key innovations: (i) novel proteomics and proteomics informatics approaches for immunopeptidomics to allow highly
sensitive discovery and prioritization of bacterial epitopes presented on infected cells; and (ii) novel mRNA vaccines to induce both
humoral and cellular immune responses, with innovative adjuvants to strengthen adaptive immunity, and to modulate innate immunity.
Vaccine production will be done according to GMP standards, and we will pursue novel, low-cost production methods to enable
local production and much-needed improved vaccine stability. We will characterize innate and adaptive immune responses in detail in
human cellular models and mouse infection models. In addition, top vaccine candidates for MTB will be evaluated in unique primate
models, followed by testing of the lead candidate in a first-in-human Phase I clinical trial. Together, we will establish our novel vaccine
development pipeline as a blueprint for world-leading, next-generation bacterial vaccine development.
and innovative mRNA vaccine formulation. We will use our powerful new pipeline to develop novel mRNA vaccines against three
bacterial pathogens that can persist within phagocytic cells: Mycobacterium tuberculosis (MTB), Mycobacterium ulcerans (MU), and
Acinetobacter baumannii (AB). MTB and AB are clinically problematic bacteria with alarming levels of antimicrobial resistance (AMR),
while MU is an important neglected tropical disease. Although vaccines are recognized as highly effective tools to mitigate AMR and
tropical diseases, effective vaccine development for these (facultative) intracellular bacteria is held back by a lack of known antigens,
and by current vaccine platforms struggling to elicit the required strong cellular immune responses. We will overcome both limitations
here through two key innovations: (i) novel proteomics and proteomics informatics approaches for immunopeptidomics to allow highly
sensitive discovery and prioritization of bacterial epitopes presented on infected cells; and (ii) novel mRNA vaccines to induce both
humoral and cellular immune responses, with innovative adjuvants to strengthen adaptive immunity, and to modulate innate immunity.
Vaccine production will be done according to GMP standards, and we will pursue novel, low-cost production methods to enable
local production and much-needed improved vaccine stability. We will characterize innate and adaptive immune responses in detail in
human cellular models and mouse infection models. In addition, top vaccine candidates for MTB will be evaluated in unique primate
models, followed by testing of the lead candidate in a first-in-human Phase I clinical trial. Together, we will establish our novel vaccine
development pipeline as a blueprint for world-leading, next-generation bacterial vaccine development.
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.
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.
A major result from the first reporting period has been the optimization and miniaturization of our immunopeptidomics pipeline. The key need ensure further uptake of our pipeline is access to dissemination platforms to reach other researchers and potential collaborators. In line with this, there are several forthcoming publications detailing our optimized pipeline. One of these manuscripts is currently available as a pre-print: https://www.biorxiv.org/content/10.1101/2024.11.22.624860v1(se abrirá en una nueva ventana).
In reporting period 1, we have also successfully used our immunopeptidomics pipeline to identify bacterial epitopes and antigens for MTB and AB with unprecedented sensitivity. The most promising of these will be evaluated in further preclinical studies in reporting period 2. The top antigens and vaccine candidates for each pathogen will be the subject of upcoming patent applications, which will further increase the impact of these results by facilitating their exploitation through licensing agreements. Identification of partners and financing to take our results to the next development stage (e.g. Phase II trial) will be a key need at the conclusion of the project.
Another important advancement has been the in vivo immune characterization of mRNA-LNPs with different ionizable lipids, with and without α-GalCer (Patent application WO2023209103: Prevention and treatment of infections with intracellular bacteria; https://doi.org/10.1016/j.jconrel.2024.04.052(se abrirá en una nueva ventana)). These results shed light on how LNP components influence immune activation, a critical consideration when selecting which lipids to use in LNPs for vaccines.
In reporting period 1, we have also successfully used our immunopeptidomics pipeline to identify bacterial epitopes and antigens for MTB and AB with unprecedented sensitivity. The most promising of these will be evaluated in further preclinical studies in reporting period 2. The top antigens and vaccine candidates for each pathogen will be the subject of upcoming patent applications, which will further increase the impact of these results by facilitating their exploitation through licensing agreements. Identification of partners and financing to take our results to the next development stage (e.g. Phase II trial) will be a key need at the conclusion of the project.
Another important advancement has been the in vivo immune characterization of mRNA-LNPs with different ionizable lipids, with and without α-GalCer (Patent application WO2023209103: Prevention and treatment of infections with intracellular bacteria; https://doi.org/10.1016/j.jconrel.2024.04.052(se abrirá en una nueva ventana)). These results shed light on how LNP components influence immune activation, a critical consideration when selecting which lipids to use in LNPs for vaccines.