Bramwell, Vincent W.; (2003) Particulate delivery systems in vaccine design. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The use of particulate delivery systems in vaccine delivery has been shown to confer significant immunopotentiation, enabling effective delivery of recombinant protein antigens to immune inductive sites and enhancing the efficacy of naked DNA encoding antigenic determinants. This present study looks at lipid and polymer based vehicles for protein and DNA delivery for the purposes of vaccination. Studies incorporate novel delivery vehicles and adjuvants, in vivo biodistribution studies and in vitro immunological studies in an attempt to evaluate candidate vaccine formulations and shed light on immunological mechanisms of action. Liposomes incorporating various additives (bioadhesive agents and absorption enhancers) were used for the delivery of tetanus and diphtheria toxoids (TT and DT). Melittin was shown to function as a novel adjuvant. Small unilamellar vesicles (SUVs) were used to complex plasmid DNA which was then modified by the addition of polymer (poly(lactide) (PLA)). This novel delivery vehicle for plasmid DNA was shown to facilitate markedly enhanced antigen specific splenocyte and T-cell proliferation against transgene encoded antigen (hepatitis surface antigen) following restimulation with the corresponding recombinant antigen when given subcutaneously. Intramuscular, oral and subcutaneous routes were compared. Transgene specific antibody titres following subcutaneous administration of plasmid DNA in this novel delivery vehicle were significantly higher than the liposomal or naked DNA mediated antibody titres by this or any other route tested. In addition, positive results were seen following oral administration. The ability of these formulations to engender protection in a MUC-1 tumour model was also assessed. Biodistribution of microparticles and stimulation of spleen cells in vitro by polymer formulated antigen was investigated. The uptake of fluorescent polystyrene carboxylate microspheres (1.1μm diameter) was studied using histology and fluorescence-activated cell sorting (FACS), following intranasal delivery to BALB/c mice. Irrespective of administration vehicle volume (10 or 50μl), appreciable numbers of fluorescent microspheres were detected within nasal associated lymphoid tissues (NALT) and draining cervical lymph nodes. Spleens removed from mice ten days after fluorescent particle application contained significantly more microspheres if the suspension had been nasally instilled using a 50μl volume (P < 0.05). Appreciable memory (and effector from day 7) responses were detected in mediastinal lymph nodes removed from mice immunised nasally with 50μl volumes of microparticulated or soluble V antigen. Splenic T-cell proliferation results following restimulation with V antigen (from Yersinia pestis determined by [3H]thymidine uptake) corroborated the observed microsphere translocation. Similarly, significant numbers of anti-V IgG secreting cells were only detected in spleens from mice immunised intramuscularly or nasally with microparticles. In separate studies, single cell suspensions of spleen cells from primed and naive animals were cocultured with escalating quantities of soluble TT or TT encapsulated within nanospheres made from poly(lactide-co-glycolide) (PLGA). Polymer formulated antigen was found to elicit increased proliferation of splenocytes from preimmunised mice in comparison to free antigen during coculture at equivalent doses of TT. Cellular proliferation was abolished if B-cells were removed from the splenocyte cultures. Production of IFN-γ and IL-6 was increased, for formulated as compared to free antigen, in microcultures from both naive and pre-immunised animals.

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