DEVELOPMENT OF AN ANALYTICAL PLATFORM TO DIRECTLY CHARACTERIZE ALUM ADJUVANTED VACCINES

Adjuvants are molecules that boost the potency and the longevity of the immune response to vaccine antigens, with little or no increase of toxicity or reactogenicity. The adjuvant activity of aluminum-containing compounds was first discovered in 1926 when an alum-precipitated diphtheria vaccine showed improved antigenic properties compared to the standard diphtheria vaccine. Since then, aluminum containing compounds have been routinely used in the formulation of vaccines due to their good record of safety, low cost and compatability with various antigenclasses. Despite its extensive use, the immune mechanism of action of aluminum is not yet completely understood . This is in part due to the complexity of developing effective analytical and qualitative tools to characterize the antigen in aluminium adjuvanted vaccines without performing any preliminary treatment to desorb the antigen from the aluminium. Most current methods used to assess the antigen content or purity in formulated vaccines containing aluminum adjuvant complexes, require the desorption of antigens from model aluminum-containing formulations. The aim of this project was to develop an analytical tool to directly quantify and assess both the content and purity of formulated aluminium-adsorbed antigens, whitout any prelimary sample manipulation (e.g antigen desorption). As an adjuvant model system, we selected three recombinant proteins (Neisseria adhesin A (NadA), Neisserial Heparin Binding Antigen (NHBA), factor H binding protein (fHbp)) as antigens, and aluminum hydroxide (AH) as an adjuvant system. The three recombinant proteins were selected due to the substantial quantity of literature regarding their structure and immunological activities, meaning that they are well characterized. In addition, their adhesion rate to aluminium is well known. Capillary electrophoresis techniques showed promising results in detecting and quantifying the antigenic components in a single run. The physical-chemical characteristics, however, of the adjuvanted components impacted the robustness of the separation In addition, other critical issuea related to: (i) aluminium sedimentation, which caused inhomogeneity in the final sample; (ii) poor reproducibility regarding samples with proteins adsorbed to aluminium, presumably due to varying aluminium populations, were found. Although aluminiumsedimentation might be overcome by simple practices such as continuous stirring of the sample, the presence of poor reproducibility remained unsolved. The study of multivalent (three recombinant proteins) and monovalent (only NHBA protein) formulations with alum through the In vitro relative potency immunoassay (IVRP) and the hydroxyl radical foot printing (HRF) mass spectrometry showed the peculiar behaviour of NHBA at different alum concentrations, in particular for monovalent formulations. In particular, the IVRP results showed that the relative potency of NHBA increases according to the concentration of alum. As the assay does not discriminate between the percentage of adsorption of the antigen, this was taken to suggest a possible 'rearrangement' of the protein to maximize its adhesion to aluminium when present at low concentrations. This was confirmed by the HRF studies, which monitored the methionine residues that formed part of the known epitope: it was observed that they were less exposed to the solvent (hence less oxidized) at lower concentrations of aluminium. This confirmed that in the presence of low concentrations of aluminium, , the protein tends to change its 'structure' to maximize adhesion, probably leading it to assume a spatial conformation in which epitopes are less accessible to the antibodies used in the IVRP assa