Pseudomonas aeruginosa (PA) is one of the most common opportunistic gram-negative pathogens. Compromised immunity or bronchial functions are high-risk factors for nosocomial PA infections. For example, patients with burns, ventilator users, and cystic fibrosis (CF) are suspected to be at high-risk for such infections (Reynolds and Kollef, 2021). Summarized data from 204 countries and territories revealed that PA infections caused up to approximately 79,000 deaths in 2019 (Murray et al., 2022). With the increase in antimicrobial drug applications, PA drug resistance has gradually emerged. In recent years, various multidrug- and pan-drug-resistant PA strains have been isolated, which poses a serious problem (Cillóniz et al., 2019). Effects of conventional antibiotic therapies are becoming increasingly limited. PA vaccine not only reduces PA infections but also decreases drug resistance by reducing the antibiotic use. Several PA vaccines have been tested in clinical trials. However, no PA vaccine has yet been approved for clinical use (Qin et al., 2022).

Flagella are macromolecular nanomachines widely found on the membrane of bacteria, with a diameter of approximately 20 nm and a length ranging from 5 to 20 µm (Chang et al., 2021). It consists of three structural elements: basal body, hook, and filament (Minamino and Imada, 2015). The filament is assembled from monomeric flagellin, which is encoded by fliC gene and is classified into two types (type A and type B) according to its sequence (Beatson et al., 2006). B-type flagellin is well conserved, with a molecular weight of approximately 53 kDa. The A-type flagellum is highly variable, except for its N- and C-terminal domains, resulting in a molecular weight ranging from 45 to 52 kDa. The two types of flagella differ by 35% in their primary sequence, but they share highly similar structures and functions (Spangenberg et al., 1996).

PA has one to three flagella at a single end and is responsible for bacterial motility (Guttenplan and Kearns, 2013). In addition, it also plays key roles in pathogenic processes, such as bacterial adhesion and colonization, biofilm formation, activation of TLR5, and induction of inflammatory responses (Drake and Montie, 1988; Klockgether and Tümmler, 2017). Therefore, flagellin is an essential target for immune therapy against PA infection. Results from animal studies have proven the effective protection of flagellin-targeting vaccines and antibodies (Jurado-Martín et al., 2021). To date, a phase III clinical trial of a purified flagellin vaccine provided partial protection in patients with CF(Döring et al., 2007). The strength of flagellin-based vaccines should be enhanced to provide adequate protection.

Ferritin protein, which is present in almost all organisms, has good biocompatibility and biosafety (Reutovich et al., 2022). Twenty-four ferritin monomers self-assembled into spherical nanoparticles with a diameter of approximately 20 nm, which enhanced the speed and strength of the immune response of antigens when delivering heterogeneous antigens. Therefore, we hypothesized that the application of ferritin nanoparticles to deliver flagellin would enhance immunogenicity.

In this study, we fused the gene encoding type A flagellin (Asn59-Ile335, reFliC) to the N-terminus of ferritin and generated the self-assembled nanoparticle vaccine, reFliC-ferritin (reFliC-FN). We found that the reFliC-FN is of good safety and induced faster and stronger cross-immune protection than reFlic alone.