Utilizing fluorescence spectroscopy to investigate the tertiary structure of antigens adsorbed onto and eluted from aluminum-containing adjuvants

Jennifer Ann Maguire, Purdue University

Abstract

Since their discovery in 1926 by Glenny et al., aluminum-containing adjuvants have been used in vaccines to safely potentiate the immune response and are still the only adjuvants approved by the Food and Drug Administration for human use. The mechanism(s) of immunopotentiation have yet to be fully elucidated although adsorption of the antigen by aluminum-containing adjuvants is a frequently employed formulation strategy. Few researchers have studied the structural stability of adsorbed antigens due to the lack of suitable techniques for examining proteins adsorbed to particles in a physiologically relevant manner. In this study, steady-state intrinsic protein fluorescence spectroscopy was employed to investigate the tertiary structure of adsorbed antigens. A long-pass cut-on filter effectively eliminated the light scattering caused by turbid adjuvant suspensions, allowing standard right angle sampling geometry to be utilized. The results suggest that while antigens may be perturbed when adsorbed onto charged, hydrophilic aluminum-containing adjuvants, this perturbation differs from the global denaturation induced by chaotropic agents. It is well understood that electrostatically adsorbed antigens elute readily in interstitial fluid after subcutaneous or intramuscular administration of vaccines. This study shows that antigens refold when eluted from the surface. Such findings imply that the theory of increased antigen immunogenicity via destabilization by adsorption onto the adjuvant surface is not a major mechanism of immunopotentiation. The current work also shows that the degree of perturbation experienced by ligand exchange antigens can be lessened by pretreating aluminum-containing adjuvant with phosphate anion to reduce the adsorptive coefficient and thus the strength of binding. Of the two ligand exchange antigens investigated, the smaller antigen was more sensitive to changes in the strength of binding. Many researchers have noted that elution becomes more difficult with time. One explanation of this observation is that antigens continue to unfold on the surface of aluminum-containing adjuvants during storage. No additional changes in the tertiary structure of electrostatically adsorbed proteins were detected in model vaccines aged at 4°C for up to 16 weeks, suggesting that most of the perturbation occurs upon initial adsorption. This work demonstrates that the use of a long-pass cut-on filter in fluorescence spectroscopy facilitates the investigation of important questions related to the formulation of vaccines with aluminum-containing adjuvants.

Degree

Ph.D.

Advisors

Hem, Purdue University.

Subject Area

Pharmacy sciences|Immunology

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