5 research outputs found
Antigen Localization Influences the Magnitude and Kinetics of Endogenous Adaptive Immune Response to Recombinant Salmonella Vaccines
The use of recombinant attenuated Salmonella vaccine (RASV) strains is a promising strategy for presenting heterologous antigens to the mammalian immune system to induce both cellular and humoral immune responses. However, studies on RASV development differ on where heterologous antigens are expressed and localized within the bacterium, and it is unclear how antigen localization modulates the immune response. Previously, we exploited the plasmid-encoded toxin (Pet) autotransporter system for accumulation of heterologous antigens in cell culture supernatant. In the present study, this Pet system was used to express early secretory antigen 6 (ESAT-6), an immunodominant and diagnostic antigen from Mycobacterium tuberculosis, in Salmonella enterica serovar Typhimurium strain SL3261. Three strains were generated, whereby ESAT-6 was expressed as a cytoplasmic (SL3261/cyto), surface-bound (SL3261/surf), or secreted (SL3261/sec) antigen. Using these RASVs, the relationship between antigen localization and immunogenicity in infected C57BL/6 mice was systematically examined. Using purified antigen and specific tetramers, we showed that mice infected with the SL3261/surf or SL3261/sec strain generated large numbers of Th1 CD4+ ESAT-6+ splenic T cells compared to those of mice infected with SL3261/cyto. While all mice showed ESAT-6-specific antibody responses when infected with SL3261/surf or SL3261/sec, peak total serum IgG antibody titers were reached more rapidly in mice that received SL3261/sec. Thus, how antigen is localized after production within bacteria has a more marked effect on the antibody response than on the CD4+ T cell response, which might influence the chosen strategy to localize recombinant antigen in RASVs
Optimising the autotransporter system for secretion and display of heterologous proteins on GMMA
The Pet Autotransporter protein was engineered and recently proposed as recombinant protein production (RPP) system. This system allows targeting the protein of interest in the culture supernatant fraction. The reduction of diversity and quantity of process impurities and size and number of downstream steps required, increase the overall process robustness and speed-up the process development time for RPP.
In the context of this study the platform was investigated for the production of a “difficult” E.coli protein with commercial relevance, C1275. Pet autotransporter was suitable for the production and one step-purification of a protein with comparable purity, thermal stability and immunogenicity to that produced using conventional technology. Additionally, the autotransporter platform was tested for the first time in scaled-up production conditions. The system was compatible with fermentation and the scaled-up conditions resulted in high yield of antigen production even though a further system optimization would be required for a one step-purification process
Sequencing a piece of history:complete genome sequence of the original Escherichia coli strain
In 1885, Theodor Escherich first described the Bacillus coli commune, which was subsequently renamed Escherichia coli. We report the complete genome sequence of this original strain (NCTC 86). The 5 144 392 bp circular chromosome encodes the genes for 4805 proteins, which include antigens, virulence factors, antimicrobial-resistance factors and secretion systems, of a commensal organism from the pre-antibiotic era. It is located in the E. coli A subgroup and is closely related to E. coli K-12 MG1655. E. coli strain NCTC 86 and the non-pathogenic K-12, C, B and HS strains share a common backbone that is largely co-linear. The exception is a large 2 803 932 bp inversion that spans the replication terminus from gmhB to clpB. Comparison with E. coli K-12 reveals 41 regions of difference (577 351 bp) distributed across the chromosome. For example, and contrary to current dogma, E. coli NCTC 86 includes a nine gene sil locus that encodes a silver-resistance efflux pump acquired before the current widespread use of silver nanoparticles as an antibacterial agent, possibly resulting from the widespread use of silver utensils and currency in Germany in the 1800s. In summary, phylogenetic comparisons with other E. coli strains confirmed that the original strain isolated by Escherich is most closely related to the non-pathogenic commensal strains. It is more distant from the root than the pathogenic organisms E. coli 042 and O157 : H7; therefore, it is not an ancestral state for the species