Immunogenic Profiling Reveals Promising RV-Identified Antigens as Vaccine Candidates Against Klebsiella pneumoniae.

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 IgG2b and IgG2c. 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.