Evaluation of Calcium Phosphate Nanoparticles Mineralized with Proteins and Peptides for Use as Adjuvants in Protein and Nucleic Acid Vaccines

Abstract

Subunit and inactivated vaccines are a safer but less immunogenic alternative to live attenuated vaccines. Adjuvants are often added to subunit and inactivated vaccine formulations to boost immune responses. Aluminum mineral adjuvants are the most commonly used adjuvants in human vaccines and are strong stimulators of antibody-mediated immune response. However, aluminum adjuvants elicit weak or absent cell-mediated T_H1 and cytotoxic CD8 T-cell responses. As an alternative adjuvant, calcium phosphate (CaP) is an ideal material due to its biocompatibility and biodegradability. Additionally, CaP has been used as an adjuvant in approved human vaccines in several European countries. When CaP is precipitated in the absence of a capping agent, large polydisperse and polymorphous micron-sized particles are formed. Particle size has been shown to be an important parameter for vaccine antigen carriers and adjuvants, and particles in the nanometer size range are of particular interest due to their unique cellular uptake and biodistribution properties. CaP binding dodecapeptides selected by cell surface display biopanning were used in <italic>E. coli</italic> thioredoxin A derivatives (TrxA::CaP) and thioredoxin A derivative-ovalbumin fusion proteins (TrxA::CaP-OVA) to mineralize sub-100nm CaP nanoparticles (NPs). Addition of CaP NP adjuvant to TrxA::CaP-OVA vaccine formulations significantly increased the population of antigen specific splenic effector-memory CD8+ T-cells in immunized mice after challenge with WSN-OVA_I, an OVA expressing influenza A strain. Due to autoimmunity concerns, the thioredoxin A derivative CaP mineralization agents were replaced with a disulfide-constrained cyclic peptide containing an identified CaP binding motif (cPN38). Addition of cyclic peptide mineralized CaP NPs to OVA vaccine formulations, increased sensitization of antigen-specific IFN-gamma (T_H1 cytokine) secreting splenocytes in immunized mice. As compared to protein antigen vaccines, nucleic acid vaccines containing pDNA or mRNA encoding genes are advantageous due to their ease of production, inherently immunogenicity, and ability to target the endogenous MHC-I loading pathway. When added to OVA pDNA vaccines, CaP NPs increased antigen-specific humoral and splenocyte IFN-gamma responses. Conversely, CaP NPs had no adjuvant effect in OVA mRNA vaccines. Increased cell-mediated (T_H1) and cytotoxic (CD8) T-cell responses were observed when CaP NP adjuvant was added to OVA protein and pDNA vaccine formulations. As an alternative adjuvant to aluminum compounds, CaP NP adjuvants may be effective in vaccines against intracellular pathogens in which an antibody-mediated immune response alone is insufficient for protective immunity.