Utilizing synthetic biology strategies for the development of attenuated vaccines targeting Staphylococcus aureus and Pseudomonas aeruginosa

Abstract

Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are opportunistic pathogens that infect various tissues and are potentially fatal. These two pathogens pose a significant burden on society and the economy. Current treatments for S. aureus and P. aeruginosa infections primarily depend on antibiotics, leading to the prevalence of multidrug-resistant strains. In addition to novel antibiotics, developing vaccines against these pathogens may help to control their prevalence. Despite the urgent need for such vaccines, none are commercially available to date. Previous efforts to develop S. aureus and P. aeruginosa vaccines mainly focused on using a single antigen or combinations of simple antigens or inactivated whole cells. Therefore, we explore whether attenuated vaccines delivering abundant and active components would improve protective efficacy. Primary infection with adenosine synthase A (AdsA) defect S. aureus has been shown to provide mice with protection against subsequent S. aureus re-infection. Based on this finding, we utilized a synthetic biology approach to design attenuated S. aureus vaccines and constructed two vaccine candidates: Newman ΔadsA ΔCPv2, and Newman ΔadsA ΔCPv2 ΔsaePQRS, the latter of which bears deletion of the saeRS two-component system. Similarly, two attenuated P. aeruginosa vaccine candidates, PAO1 Δasd and PAO1 ΔoprF, were constructed by removing life-essential components or virulence factors. The growth capacity and morphology of the engineered bacterial vaccine candidates were characterized. Their safety and protective efficacy were then validated in animal models. Immune responses during vaccination and against wild-type strain infections were also investigated. The S. aureus vaccine candidate Newman ΔasdA ΔCPv2 ΔsaePQRS displayed a shortened lag phase and significant morphological changes under scanning electron microscope (SEM) compared with the parental strain. In contrast, Newman ΔasdA ΔCPv2 exhibited identical growth and morphology to the wild-type strain. Both candidates demonstrated reduced virulence compared with the wildtype. Newman ΔasdA ΔCPv2 induced a Th17 response and high titer of IgG, while Newman ΔasdA ΔCPv2 ΔsaePQRS stimulated a Th1 response and low IgG production. Both candidates successfully protected mice from lethal S. aureus peritonitis, with Newman ΔasdA ΔCPv2 ΔsaePQRS additionally alleviating skin infection. A mixed Th1, Th2 and Th17 immune response was observed in Newman ΔadsA ΔCPv2 ΔsaePQRS after S. aureus peritoneal infection. The growth of P. aeruginosa vaccine candidates PAO1 Δasd and PAO1 ΔoprF was impaired, particularly in the auxotrophic PAO1 Δasd. Morphological changes were observed under SEM for both candidates. They demonstrated decreased virulence compared with the wild-type strain. Both PAO1 Δasd and PAO1 ΔoprF stimulated significant humoral and cellular immune response after vaccination. Mice vaccinated with these candidates exhibited a mixed Th1/Th2/Th17 response following exposure to P. aeruginosa and survived lethal peritonitis. Additionally, PAO1 ΔoprF provided mice with protection against pneumonia. In summary, our findings on S. aureus and P. aeruginosa attenuated vaccines demonstrate that applying synthetic biology strategies on vaccine development is both practical and promising. Furthermore, attenuated bacterial vaccines with active components can effectively induce potent immune responses and improve protection efficacy.