Development of solid matrix-antibody-antigen (SMAA) complexes as multivalent subunit vaccines
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In the course of the work presented in this thesis, the construction of solid matrix-antibody-antigen (SMAA) complexes as vaccines was further developed. In particular, it was demonstrated that it is feasible to assemble SMAA complexes using a short oligopeptide tag (Pk) attached to the C-termini of antigens and a Pk tag- specific mAb SV5-P-k. In order to facilitate the purification of recombinant proteins for immunization purposes, a second affinity tag was attached to the antigen N-termini. Initially, the N-terminal tag was 26-kDa-large thrombin-removable glutathione S-transferase (GST), which permitted first-step purification on immobilized glutathione. However, because of problems with protein insolubility and the proteolytical removal of GST from the hybrid proteins, the GST domain was substituted by an N-terminal 12-amino acid-long tag (His) containing an array of 6 histidines. The His tag was small and thus did not require removal prior to immunization, and allowed purification of His-linked proteins on a nickel-affinity column. Moreover, it was possible to preform nickel-affinity chromatography under protein denaturing conditions, which allowed purification of insoluble or aggregated proteins. In addition, novel prokaryotic expression vectors were constructed for a single-cloning-step addition of these N- and C-terminal tags to proteins of interest. These vectors were used to individually express all non-glycosylated products encoded by the simian immunodeficiency virus (SIV) in E. coli. The SIV envelope glycoprotein gp160 with the Pk tag attached to its C-terminus was expressed in insect cells and first-step purified on a lentile lectin column. Following the first purification step on either nickel or lentile columns, all SIV proteins were purified and successfully incorporated into SMAA complexes using anti-Pk tag mAb SV5-P-k. Thus, efficient purification protocols were developed, which purified recombinant proteins via two different affinity tags attached to their N- and C-termini and isolated predominantly full-size proteins. As a stage in achieving the goal of human multivalent vaccines, the SV5-P-k mAb was humanized and is currently being expressed in Chinese hamster ovary cells.
Thesis, PhD Doctor of Philosophy
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