Supporting data for Development of mRNA-based Vaccines Against Pseudomonas aeruginosa and Bordetella pertussis

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>