Outer membrane vesicles (OMVs) are nanostructures derived from the outer membrane of Gram-negative bacteria. We previously demonstrated that vaccination with endotoxin-free OMVs isolated from an Acinetobacter baumannii strain lacking lipooligosaccharide (LOS) biosynthesis, due to a mutation in lpxD, provides full protection in a murine sepsis model. The present study characterizes the protein content of highly-purified OMVs isolated from LOS-replete and LOS-deficient strains. Four purification methods were evaluated to obtain highly purified OMV preparations: ultracentrifugation, size exclusion chromatography (SEC), ultracentrifugation followed by SEC, and Optiprep™. OMVs from each method were characterized using nanoparticle tracking analysis and electron microscopy. OMVs from LOS-deficient and LOS-replete strains purified using the Optiprep™ method were subjected to LC-MS/MS analysis to determine protein content. Significant differences in protein composition between OMVs from LOS-deficient and LOS-replete strains were found. Computational analyses using Bepipred 3.0 and SEMA 2.0 indicated that the lack of LOS led to the overexpression of immunogenic proteins found in LOS-containing OMVs and the presence of immune-stimulating proteins absent in LOS-replete OMVs. These findings have important implications for developing OMV-based vaccines against A. baumannii, using both LOS-containing and LOS-free OMVs preparations.

The emergence and global dissemination of bacterial strains from numerous species with resistance to multiple antibiotic classes has increased in recent years, both in the healthcare and the community setting

The antigenic diversity of Klebsiella pneumoniae surface polysaccharides, O- and K-antigen, represents a significant challenge for developing broadly-acting vaccines. Here we report the development of Kp-OMV4, a multivalent outer membrane vesicle (OMV) vaccine incorporating the four predominant K. pneumoniae O-antigen serotypes responsible for most invasive disease worldwide. Kp-OMV4 elicits robust humoral and cellular immune responses, generating high-titer antibodies against both surface polysaccharides and conserved outer membrane proteins. Vaccine-induced antibodies display complement-mediated bactericidal activity against hypervirulent and multidrug-resistant clinical strains. In a murine sepsis model, immunization confers protection against lethal challenge and significantly reduces post-infection bacterial burden. Importantly, immune sera broadly recognize diverse clinical isolates, including strains expressing O- and K-antigen serotypes not included in the vaccine, supporting potential for broad strain coverage. This study indicates that the multicomponent nature of OMVs can be leveraged to overcome bacterial antigenic diversity and enable the development of vaccines with broad strain coverage.

Multidrug-resistant Klebsiella pneumoniae is an increasing global threat, and the limited availability of new antibiotics highlights vaccination as a promising strategy for infection prevention. Although different vaccine candidates have been evaluated, none is currently approved, mainly due to capsular heterogeneity among strains. Protein-based vaccines may overcome this limitation by targeting conserved epitopes. In this study, we assessed the immunogenicity of five outer membrane proteins (ChiP, LamB, RafY, OmpW, PagP) previously selected by reverse vaccinology (RV), comparing them with the well-characterized antigens OmpA and OmpK36. Mice were immunized intramuscularly with three doses of purified recombinant proteins, and antibody responses were analyzed by ELISA. All antigens elicited high, booster-induced IgG levels, with PagP slightly being less immunogenic. Regarding IgG subclasses, IgG₁ predominated, followed by IgG_2b and IgG_2c. Cross-reactivity was evaluated against six K. pneumoniae strains representing different clonal groups, and recognition by sera from previously infected mice was also examined. OmpA, ChiP and LamB showed the broadest cross-reactivity, while OmpA and LamB were most strongly recognized after infection. Overall, OmpA, LamB, ChiP and RafY emerged as the most promising vaccine candidates, although further optimization, such as a multicomponent vaccine, may be required. This work also highlights the importance of experimentally validating RV-selected antigens, as computational predictions alone do not ensure immunogenicity or in vivo relevance.