aureusantigen-specific humoral and cellular immunity in a mouse model

aureusantigen-specific humoral and cellular immunity in a mouse model. == 3.5 Protective effects of OMV-based click vaccine againstS. Keywords:outer membrane vesicles,Staphylococcus aureusvaccine, SpyCatcher-SpyTag, click display, flexible antigen display, multi-targeting vaccine == 1 Introduction == Staphylococcus aureus(S. aureus) is usually a Gram-positive bacterium commonly found as part of the normal flora on the skin of humans BNC375 (1). It usually becomes BNC375 invasive in patients with immunological or Ctsb barrier defects, and has been a leading cause of many infection diseases in the hospital and community (24).S. aureusinfections are notoriously hard to control as an increasing quantity of its clinical isolates exhibits resistance to multiple antibiotics (57). Patients recovered fromS. aureusinfection also show no immunity to subsequent infections (1). To this end, there is a dire need to develop prophylactic vaccines that provide protective immunity againstS. aureus. A variety of surface adhesion proteins and secreted proteins which are implicated inS. aureusimmune evasion have BNC375 been exploited as potential vaccine antigens (1,810a;11). The current antigenic targets mainly focus on virulence factors (10,12,13), capsular polysaccharide (14), iron-regulated proteins (1517) and cell wall-anchored enzymes such as adenosine synthase (18). However, most of the clinical trials for vaccines based on antigens from either a single protein or a certain protein family have failed so far (19,20).S. aureuscan very easily circumvent defense mechanisms of the host immune system by expressing multiple virulence factors and combined function of invasion molecules (21). Therefore, it still demands novel vaccine strategies which combine multiple antigenic components to simultaneously target diverse strategies thatS. aureusdeployed to circumvent host immunity. Outer membrane vesicles (OMVs) are membrane-derived vesicles released spontaneously during growth by many bacteria species (22,23). As natural OMVs obtained from a pathogen present a range of surface antigens in a native conformation, they have been directly explored as vaccine products against parental pathogenic bacteria (24,25). More recently, due to the immunostimulatory, self-adjuvant and ease of genetic manipulation properties, designed OMVs show promise to become a versatile vaccine platform (2628). OMV-based vaccines were initially generated by directly fusing OMV scaffold protein with antigens or routing altered antigens to the OMV lumen (2934). More recently, by combining the SpyCatcher-SpyTag system, the function of OMVs as nanocarriers was further expanded for quick and flexible surface display of recombinant proteins or tumor antigens (3537). The covalent bond formation between SpyCatcher and SpyTag can occur at a range of temperatures and pH values (38). Such a flexible click system should allow quick and multiple antigen attachment to OMVsin vitro, which will enable an effective and multi-targetingS. aureusvaccine platform. In this study, we designed OMVs secreted from a common laboratoryEscherichia coli(E. coli) MG1655 strain with surface uncovered SpyCatcherviatruncated OmpA-SpyCatcher fusions. We show that this designed OMVs can flexibly assemble with numerous SpyTag-fusedS. aureusantigens to generate OMV-based click vaccines. The click vaccine simultaneously induced strong humoral and cellular immunity specific for multiple antigens displayed, which conferred protection againstS. aureuslethal challenge in a mouse model. Our results offered a multipurpose and potent OMV-basedS. aureusvaccine platform. == 2 Materials and methods == == 2.1 Bacterial strains, plasmids and media == Bacteria strains and plasmids used in this study are outlined inSupplementary BNC375 Table 1.E. coliMG1655 wild type (WT) strain was utilized as a parental strain for pointed out isogenic gene deletion and chromosomal modification.E. colistrain BL21(DE3) was utilized for recombinant protein expression. Standard Luria-Bertani (LB) broth with appropriate antibiotics (100 g/ml ampicillin, 50 g/ml kanamycin or 25 g/ml chloramphenicol) was used forE. coliculture. == 2.2 Strain construction == To produce thelpxMdeletion strain, PCR-amplified DNA fragments containing 40 bp homologous arms flankinglpxMopen reading frame together with a selection antibiotic marker were purified. PCR products were then electroporated into MG1655 harboring reddish recombinase expression plasmid pKD46. BNC375 Positive clones were selected by 25 g/ml chloramphenicol and verified by DNA sequencing. Subsequently, the antibiotic selection marker flanked by loxP sites was removed using the helper plasmid p705Cre. The lpxM ompA-spycatcherstrain was further constructed using the comparable recombineering method. AnompA(1-465 bp)-G4S linker-spycatcher-6hisfusion gene was synthesized and used to replace the chromosomalompAgene. Positive clones were selected using 50 g/ml kanamycin and confirmed by DNA sequencing. == 2.3 Plasmid construction == A gene fragment encoding SpyTag (AHIVMVDAYKPTK) was first inserted in.