A live auxotrophic vaccine confers mucosal immunity and protection against lethal pneumonia caused by Pseudomonas aeruginosa.

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.

A highly-safe live auxotrophic vaccine protecting against disease caused by non-typhoidal Salmonella Typhimurium in mice.

BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) has become an important intestinal pathogen worldwide and is responsible for lethal invasive infections in populations at risk. There is at present an unmet need for preventive vaccines. METHODS: IRTA GN-3728 genome was sequenced by Illumina and d-glutamate and d-glutamate/d-alanine knockout-auxotrophs were constructed. They were characterized using electron microscopy, growth/viability curves, reversion analysis, and motility/agglutination assays. Their potential as vaccine candidates were explored using two BALB/c mouse models for Salmonella infections: a systemic and an intestinal inflammation. Clinical signs/body weight and survival were monitored, mucosal lactoferrin and specific/cross-reactive IgA/IgG were quantified by enzyme-linked-immunosorbent assays and bacterial shedding/burden in fecal/tissues were evaluated. RESULTS: The d-glutamate auxotroph, IRTA ΔmurI, is highly attenuated, immunogenic and fully protective against systemic infection. The IRTA ΔmurI Δalr ΔdadX double auxotroph, constructed to reinforce vaccine safety, showed a higher level of attenuation and was 100% effective against systemic disease. In the intestinal model, it proved to be safe, yielding a low-degree of mucosal inflammation, short-term shedding and undetectable invasiveness in the long-term, while eliciting cross-reactive fecal IgA/serum IgG against clinically relevant multidrug-resistant (MDR) S. Typhimurium strains. It also conferred protection against homologous oral challenge, and protected mice from local and extra-intestinal dissemination caused by one MDR strain responsible for an international outbreak of highly severe human infections. Additionally, oral vaccination promoted extended survival after lethal heterologous infection. CONCLUSION: This study yielded a very safe S. Typhimurium vaccine candidate that could be further refined for mucosal application against disease in humans.

Comparison of the in vivo efficacy of ceftaroline fosamil, vancomycin and daptomycin in a murine model of methicillin-resistant Staphylococcus aureus bacteraemia.

The need for alternative drugs to treat methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia has led to a focus on ceftaroline, for which clinical data remain scarce. Herein, the efficacy of ceftaroline fosamil for the treatment of experimental MRSA bacteraemia was compared with that of approved therapies. Five MRSA strains were tested in an immunocompetent BALB/c bacteraemia model. Serum pharmacokinetics of ceftaroline fosamil were determined using HPLC/MS Q-TOF. Two hours after infection with the MRSA strains, mice were administered 50 mg/kg of ceftaroline fosamil every 6 h, for 24 h. This regimen yielded a T>MIC of 61.5% for an MIC of 1 mg/L and proved efficacious against all strains, including an hVISA strain with non-susceptibility to daptomycin, as indicated by the reduction (mean ± s.d.) in log10 CFU/mL in blood of 2.34 ± 0.33 and log10 CFU/g in kidney of 2.08 ± 0.22. Similarly, treatment with daptomycin yielded a log reduction of 2.30 ± 0.60 in blood and 2.14 ± 0.31 in kidney. The decrease in bacterial density was less accentuated after treatment with vancomycin, which yielded 1.84 ± 0.73 and 1.95 ± 0.32 log reductions in blood and kidney, respectively. The results of the study showed that the efficacy of ceftaroline fosamil against MRSA bacteraemia in mice is not inferior to that of vancomycin and daptomycin, and indicated the potential use of ceftaroline fosamil against difficult-to-treat S. aureus bacteraemia. Considering these promising data, clinical trials should be conducted to ascertain the efficacy of the drug for treating bloodstream infections in humans.

Double Auxotrophy to Improve the Safety of a Live Anti-Pseudomonas aeruginosa 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.

A New Live Auxotrophic Vaccine Induces Cross-Protection against Klebsiella pneumoniae Infections in Mice.

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.