This study evaluated a subunit vaccine candidate, L-PaF, combined with novel lipid A mimetic adjuvants produced by the Bacterial Enzymatic Combinatorial Chemistry (BECC) platform. L-PaF is a genetic fusion of Pseudomonas aeruginosa type III secretion system proteins PcrV and PopB with LTA1, the active moiety of the dmLT enterotoxin. Two BECC adjuvant scaffolds were tested: BECC438 and BECC470. Each was prepared as a biologically derived mixture (BECC438b, BECC470b) and as a high-purity chemically synthesized form (BECC438s, BECC470s). The BECC molecules are designed to selectively engage the TLR4/MD-2 complex as partial agonists to enhance immune responses while limiting excessive inflammation.
Vaccine formulations used a squalene-based oil-in-water emulsion (ME). The emulsion was prepared using polysorbate 80 and squalene with histidine buffer and sucrose, processed by high-speed mixing and microfluidization to produce a 4X ME. BECC adjuvants were solubilized (0.5 mg/mL) and combined with ME, then mixed with L-PaF to achieve the final dosing formulation. Each vaccine dose contained 1 μg of L-PaF and 0.5 μg of BECC adjuvant in 30 μL. Vaccinations were administered on days 0, 14, and 28.
Female C57BL/6J mice (6–8 weeks) were used. Groups received the formulations either intranasally (IN) or intramuscularly (IM). Blood sampling occurred on days 0, 28, 42, and 56. On day 56 five mice per group were necropsied for pre-challenge immunology; another five per group were challenged intranasally with the clinical Pa strain mPA08-31 (4 × 10^7 CFU in 30 μL) on day 56. Lungs were harvested two days after challenge for CFU enumeration. Animal procedures were performed under institutional IACUC oversight.
Antigen-specific serum IgG and IgA against recombinant PcrV and PopB were measured by ELISA. Intranasal vaccination with BECC438 formulations (both synthetic and biologic) induced robust antigen-specific IgG and IgA responses. IgG subclass profiling showed a mixed IgG1/IgG3 profile consistent with combined Th2 and Th1 features, although IgG1 remained the predominant subclass across groups. Overall, IN delivery with BECC438s produced the most pronounced mucosal and systemic antibody responses compared with BECC470 formulations and IM administration.
Opsonophagocytic killing (OPK) assays were performed using heat-inactivated sera collected post-immunization (day 42). The assay combined mPA08-31, J774A.1 murine macrophages, and diluted sera to measure bactericidal activity without exogenous complement. Vaccination with L-PaF/ME/BECC438 formulations generated OPK activity, supporting the functional capacity of antibody responses induced by these adjuvanted vaccines.
Protection was assessed by enumerating lung CFU two days post-intranasal challenge. Intranasal immunization with BECC438s conferred the greatest reduction in lung bacterial burden compared with the other adjuvant formulations and IM routes. The data indicate that mucosal delivery of L-PaF with BECC438s provided superior local protection in this pulmonary infection model.
Pre-challenge lung and spleen cells were stimulated ex vivo with PcrV or PopB to profile cytokine responses; post-challenge pro-inflammatory responses were also assessed (methods truncated in source). Serum antibody titers and cytokine responses measured either before or after challenge were reported to correlate with bacterial clearance, suggesting mechanistic links between humoral and cellular immunity and protection. Specific cytokine assay results and full post-challenge cytokine details were not completely reported in the provided source text.
In this murine pulmonary model, BECC438 formulations—particularly the chemically synthesized BECC438s given intranasally—elicited stronger mucosal and systemic antibody responses, greater functional OPK activity, and superior reduction in lung bacterial burden than BECC470 formulations. While BECC470 has shown beneficial performance in other preclinical settings (for example, in aged mice in prior reports cited by the authors), BECC470 performed less favorably than BECC438 in the L‑PaF/ME pulmonary vaccine context described here.
The BECC approach offers both biologically derived (heterogeneous) and chemically synthesized (high-purity) lipid A analogues. The synthetic versions aim to remove congener species that could impact selective TLR4 engagement and reduce reactogenicity. The authors note that BECC-derived adjuvants can enhance dendritic cell activation and antigen presentation while maintaining a favorable safety profile in rodent studies cited previously. Given the observed superiority of BECC438s with IN delivery, the authors propose BECC438 as a promising adjuvant candidate for mucosal next-generation vaccines targeting multidrug-resistant Pa.
The source text indicates further studies are warranted to evaluate translational potential in humans. Specific details on some post-challenge cytokine measurements and longer-term safety or durability of protection were not fully reported in the provided excerpt. Future work should expand on cytokine profiling, dose optimization, safety across age groups, and translation to clinical trials to determine whether BECC438s-adjuvanted mucosal vaccines can protect at-risk human populations against Pseudomonas aeruginosa.