<i>In Vivo</i> and <i>In Vitro</i> Potency of Polyphosphazene Immunoadjuvants with Hepatitis C Virus Antigen and the Role of Their Supramolecular Assembly

Abstract

Two well-defined synthetic polyphosphazene immunoadjuvants, PCPP and PCEP, were studied for their ability to potentiate the immune response to the hepatitis C virus (HCV) E2 glycoprotein antigen <i>in vivo</i>. We report that PCEP induced significantly higher serum neutralization and HCV-specific IgG titers in mice compared to other adjuvants used in the study: PCPP, Alum, and Addavax. PCEP also shifted the response toward the desirable balanced Th1/Th2 immunity, as evaluated by the antibody isotype ratio (IgG2a/IgG1). The <i>in vivo</i> results were analyzed in the context of antigen-adjuvant molecular interactions in the system and <i>in vitro</i> immunostimulatory activity of formulations. Asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) analysis showed that both PCPP and PCEP spontaneously self-assemble with the E2 glycoprotein with the formation of multimeric water-soluble complexes, which demonstrates the role of polyphosphazene macromolecules as vaccine delivery vehicles. Intrinsic <i>in vitro</i> immunostimulatory activity of polyphosphazene adjuvants, which was assessed using a mouse macrophage cell line, revealed comparable activities of both polymers and did not provide an explanation of their <i>in vivo</i> performance. However, PCEP complexes with E2 displayed greater stability against agglomeration and improved <i>in vitro</i> immunostimulatory activity compared to those of PCPP, which is in line with superior <i>in vivo</i> performance of PCEP. The results emphasize the importance of often neglected antigen-polyphosphazene self-assembly mechanisms in formulations, which can provide important insights on their <i>in vivo</i> behavior and facilitate the establishment of a structure-activity relationship for this important class of immunoadjuvants.