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Title: Recombinant immune complexes as a novel molecular platform for the delivery of mycobacterial antigens
Author: Pepponi, Ilaria
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2012
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This work describes the generation of recombinant immune complexes (RICs) and the evaluation of their vaccine potential in the context of tuberculosis (TB). Novel vaccine candidates based on recombinant immune complexes were synthesised and tested in mice for the immunogenic potential and protective capacity against My- cobacterium tuberculosis infection. Two different approaches were employed to make these molecules: i) utilising a plant expression system which is amenable for expression of complex molecules and ii) utilizing the unique polymeric properties of the Acr (X- crystalline-like) antigen. E. coli recombinant immune complexes (ERICs) were initially generated in the study, and their functionality was demonstrated in vitro by a complement (Clq component) binding ELISA assay. In addition, they bound efficiently to the surface of spleen-derived antigen- presenting cells, suggesting that these molecules could be used as a novel molecu- lar platform for vaccine delivery in vivo. The vaccine potential of ERICs was evaluated by testing their immunogenicity and protective capacity in the mouse model of TB infection. The study demonstrated that ERICs induced strong humoral responses to both Ag85B and Acr, but only a moderate cellular immune response and IFN-y production. Homol- ogous prime-boost subcutaneous immunisation of mice with ERICs conferred little or no protection against M. tuberculosis intranasal challenge; however, some protection (greater than that with BCG alone) was observed when they were used as a booster vaccine in BCG primed mice. This suggests that ERICs could be used as a novel BCG-boosting vaccine candidate against TB infection and also, could be considered for other infections, especially those where protection requires a strong humoral response. 1 • For preparation of plant recombinant immune complexes (RlCs), the ability of Nico- tiana tabacum plants to express a functional TBG65 IgG 1 antibody was initially demon- strated. To express RICs in planta, the DNA construct comprising TBG65y chain se- quence, in frame with Acr or Acr-Ag85B coding sequences, was inserted into two different plant expression vectors, one suitable for transient expression and the other to gener- ate transgenic tobacco plants. Expression of both constructs was obtained and, while proceeding with the lengthy process of generation of stably transformed plants, RICs purification protocols were tested on transiently expressed material. The presence of im- mune complexes in the RICs preparation was verified by Clq-binding ELISA assay, and a preliminary immunisation experiment demonstrated a boosting effect of RICs in BCG- immunised mice. These data complement the current data available on plant RlCs adding important information about their vaccine potential, but further work is now required to optimise production and purification of plant-RICs on a scale suitable for in-depth vaccine studies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available