Two different expression systems were investigated for the production of an 80 amino acid polypeptide, M3, from the C-terminus of the Plasmodium falciparum blood stage antigen Pf155/RESA in an attenuated Salmonella typhimurium vaccine strain. Upon expression, the malarial polypeptide was targeted either to the periplasm as a soluble fusion protein containing two IgG-binding domains (ZZ) from the staphylococcal protein A or, to the bacterial surface as an insert within a chimeric outer membrane protein A (OmpA) derived from Escherichia coli and Shigella dysenteriae. Both the ZZM3 and the OmpAM3 proteins were stably expressed in the periplasm or on the surface of Salmonella, respectively. The ZZ expression system yielded 10–100 times more malarial immunogen than did the OmpA system. Live recombinant Salmonella expressing ZZM3 or OmpAM3 were used to immunize mice intraperitoneally. Both the ZZM3 and OmpAM3 genes persisted for up to three weeks in bacteria isolated from different lymphoid organs. Bacteria expressing ZZM3 induced antibodies to M3, ZZ and to the Pf155/RESA antigen whereas, bacteria producing OmpAM3 induced similar levels of antibodies reactive with M3 but not with Pf155/RESA. Both recombinants induced a memory response of antibodies reactive with both M3 and Pf155/RESA. The high levels of M3 produced by the ZZ expression system make it suitable for the expression of heterologous antigens in Salmonella. Nevertheless, in spite of the quantitative difference in M3 expression, the ZZ and OmpA constructs elicited comparable immune responses to M3.
Recombinant techniques provide valuable tools for thedevelopment of modern subunit vaccines. In this thesisdifferent systems for production of recombinant subunitvaccines are presented. The strateg ...
Abstract Subunit vaccines, both purified native components of pathogens and vaccines developed and produced by recombinant techniques, are now dominating in the strive for an ideal vaccine. Subunit vaccines are highly attractive alternatives to the classical vaccines consisting of live attenuated or killed whole bacteria or viruses.
The possibility of improving the antibody responses to a model streptococcal antigen, administered by intranasal immunization as surface-displayed on the food-grade bacterium Staphylococcus carnosus, by co-exposure of a peptide (CTBp) comprising amino acids 50-75 of the cholera toxin B subunit, was investigated. It was found that the introduction of the CTBp into the chimeric surface proteins, containing a serum albumin binding protein (ABP) from streptococcal protein G as model antigen, significantly increased serum IgG responses upon intranasal immunization. Similarly, elicited local IgA responses were also found to be improved. Furthermore, it was demonstrated that live delivery of the staphylococci was required to obtain this effect, since UV-irradiated or heat-killed bacteria exposing the same chimeric surface proteins did not show increased anti-ABP IgG responses.
Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare‐codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor‐made product‐specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial‐scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning‐in‐place procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.
