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oai:figshare.com:article/33287199 2026-09-23T03:54:07Z category_24610 portal_886 item_type_3 month_year_09_2026

Supporting data for Development of mRNA-based Vaccines Against Pseudomonas aeruginosa and Bordetella pertussis Anqi Xu (13201470) Applied immunology (incl. antibody engineering, xenotransplantation and t-cell therapies) mRNA vaccine Pseudomonas aeruginosa Bordetella pertussis <p dir="ltr"><i>Pseudomonas aeruginosa</i> and <i>Bordetella pertussis</i> are two major respiratory pathogens that pose persistent threats to global public health. <i>P. aeruginosa</i> is a leading cause of multidrug-resistant (MDR) nosocomial infections for which no licensed vaccine exists. Meanwhile, the resurgence of pertussis, despite high acellular vaccine coverage, is linked to the short-lived and Th2-skewed immunity induced by current chemically detoxified pertussis toxoid vaccines. The limitations of traditional recombinant protein and inactivated whole-cell approaches have created an urgent need for next-generation vaccine platforms. These platforms must be capable of presenting complex bacterial antigens in their native conformations and eliciting balanced systemic and mucosal immunity. In this study, an mRNA-lipid nanoparticle platform was developed to create multivalent vaccine candidates targeting the resistance mechanisms and virulence factors of these two pathogens. For <i>P. aeruginosa</i>, mRNA constructs were designed to encode secreted multi-loop tandem antigens derived from the surface-exposed loops of the RND efflux pump outer membrane channels OprM, OprJ, and OprN, as well as the full-length outer membrane protein OpmQ. For <i>B. pertussis</i>, a series of mRNA constructs were systematically engineered to encode genetically detoxified pertussis toxin variants. These variants included truncated catalytic subunits, structural dimers, linear neutralizing epitope tandems, and a self-assembling ferritin nanoparticle displaying PtxA. All constructs were codon-optimized, equipped with clinically validated UTRs and signal peptides, and formulated into LNPs for in vivo delivery.</p><p dir="ltr">The lead <i>P. aeruginosa</i> candidate, Oprj-n-m, induced high antigen-specific IgG titers and potent opsonophagocytic killing activity, yet it failed to confer protection in lethal challenge models. Biochemical and mutational analyses revealed that the Oprj-n-m antigen underwent extensive non-native- and-linked glycosylation during eukaryotic expression, which likely shielded critical neutralizing epitopes. In contrast, mRNA encoding OpmQ elicited antibodies that strongly recognized native proteins in whole-cell lysates and mediated significant opsonophagocytic killing. This candidate ultimately achieved an 80% survival rate in a lethal-dose challenge against a clinical MDR isolate. For pertussis, a bivalent mRNA vaccine combining the linear PT epitope tandem and the ptxA-ferritin nanoparticle induced robust Th1-skewed cellular immunity and high-titer neutralizing antibodies, as demonstrated byELISPOT and the CHO cell toxin neutralization assay. In intranasal challenge models, this bivalent formulation completely prevented body weight loss and reversed pertussis toxin-induced leukocytosis. However, it only modestly reduced respiratory tract bacterial colonization, highlighting the difficulty of establishing sterilizing mucosal immunity via intramuscular mRNA immunization. Furthermore, a prime-pull intranasal booster strategy yielded only weak and variable secretory IgA responses, indicating that conventional LNPs are poorly suited for mucosal delivery.</p><p dir="ltr">In summary, this thesis demonstrates that mRNA-based vaccines can bypass the central bottlenecks of conventional antibacterial vaccine design and elicit functionally superior immune responses against both MDR <i>P. aeruginosa</i> and <i>B. pertussis</i>. The contrasting performance of the Oprj-n-m and Opmq candidates identifies glycosylation-aware antigen engineering as a critical factor for the success of bacterial mRNA vaccines. The optimized Opmq and PT bivalent mRNA candidates represent promising translational leads, and the immunological insights gained provide a conceptual framework for the rational development of next-generation multivalent mRNA vaccines against complex respiratory pathogens.</p> 2026-09-23T03:54:07Z Dataset Dataset 10.25442/hku.33287199.v1 https://figshare.com/articles/dataset/Supporting_data_for_Development_of_mRNA-based_Vaccines_Against_Pseudomonas_aeruginosa_and_Bordetella_pertussis/33287199 CC BY-NC 4.0 Open Access after 2028-08-31