Pseudomonas aeruginosa is one of the leading causes of nosocomial pneumonia and its associated mortality. Moreover, extensively drug-resistant high-risk clones are globally widespread, presenting a major challenge to the healthcare systems. Despite this, no vaccine is available against this high-concerning pathogen. Here we tested immunogenicity and protective efficacy of an experimental live vaccine against P. aeruginosa pneumonia, consisting of an auxotrophic strain which lacks the key enzyme involved in D-glutamate biosynthesis, a structural component of the bacterial cell wall. As the amounts of free D-glutamate in vivo are trace substances in most cases, blockage of the cell wall synthesis occurs, compromising the growth of this strain, but not its immunogenic properties. Indeed, when delivered intranasally, this vaccine stimulated production of systemic and mucosal antibodies, induced effector memory, central memory and IL-17A-producing CD4 + T cells, and recruited neutrophils and mononuclear phagocytes into the airway mucosa. A significant improvement in mice survival after lung infection caused by ExoU-producing PAO1 and PA14 strains was observed. Nearly one third of the mice infected with the XDR high-risk clone ST235 were also protected. These findings highlight the potential of this vaccine for the control of acute pneumonia caused by this bacterial pathogen.

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The development of a whole-cell vaccine from bacteria auxotrophic for D-amino acids present in the bacterial cell wall is considered a promising strategy for providing protection against bacterial infections. Here, we constructed a prototype vaccine, consisting of a glutamate racemase-deficient mutant, for preventing Klebsiella pneumoniae infections. The deletion mutant lacks the murI gene and requires exogenous addition of D-glutamate for growth. The results showed that the K. pneumoniae ΔmurI strain is attenuated and includes a favourable combination of antigens for inducing a robust immune response and conferring an adequate level of cross-protection against systemic infections caused by K. pneumoniae strains, including some hypervirulent serotypes with elevated production of capsule polysaccharide as well as multiresistant K. pneumoniae strains. The auxotroph also induced specific production of IL-17A and IFN-γ. The rapid elimination of the strain from the blood of mice without causing disease suggests a high level of safety for administration as a vaccine.

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen that causes serious infections in the respiratory tract of immunocompromised or critically ill patients, and it is also a significant source of bacteremia. Treatment of these infections can be complicated due to the emergence of multidrug-resistant P. aeruginosa strains worldwide. Hence, the development of prophylactic vaccines is a priority for at-risk patients. We have previously developed a vaccine candidate with a single auxotrophy for D-glutamate, PAO1 ΔmurI, which protects against sepsis and acute pneumonia caused by P. aeruginosa. Given the paramount importance of safety in the development of live attenuated vaccines, we have improved the safety of the vaccine candidate by reducing the probability of a reversion to virulence by the inclusion of an additional auxotrophy for D-alanine. Single and double auxotrophs behaved in a similar manner in relation to the attenuation level, immunogenicity and protective efficacy, but the double auxotroph has the advantage of being more stable and safer as a candidate vaccine against respiratory infections caused by P. aeruginosa.

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