Destabilising Effect of Class B CpG Adjuvants on Different Proteins and Vaccine Candidates

<b>Background:</b> Adjuvants function by enhancing the breadth, durability, and magnitude of the immune response, but little is known about their impact on vaccine stability. CpG is a widely used adjuvant that is included in several recently approved COVID-19 vaccines using Spike protein, RBD, or whole inactivated virus. <b>Methods</b>: Here, we investigate the in vitro stability of the Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike protein, as well as a number of other proteins formulated with a class B CpG adjuvant. <b>Results</b>: We show that RBD, BSA, and lysozyme proteins are less thermally stable, more aggregation-prone, and more protease-sensitive in the presence of CpG than without it, and that these effects are enhanced with prolonged incubation. For RBD, the effects of CpG are pH-independent but dependent on the salt concentration, with relative destabilisation decreasing with an increasing salt concentration, indicative of an electrostatic component to the interaction between CpG and the protein. The reduced thermal and proteolytic stability found in the presence of CpG is indicative of a preferential interaction of CpG with the unfolded state of the protein relative to its native state. It remains to be determined if these in vitro characteristics are unique to CpG or are also shared by other non-CpG commercial adjuvants, if they are antigen-dependent, and if and how they correlate with the in vivo immunogenicity of an adjuvanted vaccine. <b>Conclusions</b>: It is demonstrated that the CpG adjuvant is critical to enhancing immunogenicity and is a key reason for the success of multiple licensed commercial vaccines. Nonetheless, our work suggests that careful and systematic in vitro formulation studies may be warranted for the development of suitable, stable formulations of CpG-adjuvanted vaccine candidates.