Recombinant subunit vaccines : protein immunogens

Abstract: Recombinant techniques provide valuable tools for thedevelopment of modern subunit vaccines. In this thesisdifferent systems for production of recombinant subunitvaccines are presented. The strategies investigated includeprotein immunogens, live bacterial vectors and nucleicacids.An expression system for recombinant protein production wasdeveloped forSalmonella typhimurium, and when investigating theproduction ofPlasmodium falciparum-derived antigens, yield andquality were comparable to those obtained forE. coli.S. typhimuriumwas further investigated as a livebacterial vaccine vector, comparing the effect on the immuneresponses of expressing a malarial antigen as surface-displayedor periplasmically located. The bacterial vector carrying thesurface-exposed antigen was able to induce antibody responses,upon immunisation of mice, of the same magnitude as thebacterium with periplasmic location of the antigen, althoughwith approximately tenfold lower level of expression, thusdemonstrating beneficial effects of using surface display ofthe foreign antigen.Aiming for a bacterial live vector for subunit vaccinedelivery with increased persistence at mucosal sites, differentadhesion molecules were investigated. The characteristics ofthe cholera toxin B subunit (CTB) with N-, C-, or dual fusionswere studied. Three different fusion proteins were constructed,and expressed inE. coli. All fusion proteins were able to pentamerise,and GM1-binding studies showed significant binding ablilty forthe single-fusion CTB proteins. The CTB or bacterially derivedfibronectin-binding domains (FNBDs), were included inexpression systems for non-pathogenic staphylococci, previouslyextensively investigated in the context of vaccine delivery. Onthese staphylococcal cells, CTB and the FNBDs were expressed ina surface displayed fashion. In whole-cell assays, the CTBmoiety and the FNBDs were confirmed on the surface of the livebacteria, displayed in a functional form, with retainedcapacity to bind GM1 and fibronectin, respectively. Suchbacteria will be of future interest for vaccine delivery.In a DNA vaccine vector, the influence of a secretion signalpreceding the gene encoding the antigen was investigated. Twoplasmid DNA vaccine vectors encoding a malarial antigen wereconstructed, one containing and the other one lacking signalsequence, and upon immunisation of mice, the levels ofantibodies elicited to the antigen were not affected by thedifference in cellular targeting. Interestingly, looking at IgGsubclasses, as correlates of different Th subsets, apredominant IgG1 response was evoked by the DNA vector encodeda secreted antigen, suggesting a dominating Th2 type ofresponse, whereas animals injected with the DNA vector encodeda intracellular antigen demonstrated a more equal distributionof the two IgG subclasses studied, proposing a mixed Thprofile.Taken together, the recombinant strategies investigated havedemonstrated to provide a variety of possibilities in thedesign anddelivery modes in vaccination, and the futureexpansion of the use of recombinant techniques can beenvisioned for the construction of purpose-designed recombinantsubunit vaccines.Key words: albumin binding protein, CTB, DNA vaccine,fibronectin binding domain, GM1 binding, live bacterial vaccinevector,Plasmodium falciparum,Salmonella typhimurium, signal sequence, staphylococcalprotein A,Staphylococcus carnosus,Staphylococcus xylosus