Polypeptide Sequence Effects on Virus Thermostability in Complex Coacervate Formulations

Abstract

Current vaccine formulations heavily rely on cold chains to avoid degradation during transportation and storage. Vaccines typically degrade when exposed to temperatures outside the 2–8 °C range, leading to waste and logistical challenges, particularly in rural areas. This study investigates the ability of poly(lysine)- and poly(glutamate)-based peptide coacervates to improve the thermal stability of porcine parvovirus (PPV), a model nonenveloped viral vaccine. We hypothesized that both the length and specific amino acid sequence of the peptides forming the coacervates would influence the stability of PPV. Long polypeptides (400–800 mers) provided significant protection, slowing PPV inactivation at 60 °C for up to 7 days by as much as 4 logs (10,000-fold), whereas shorter 48-mer homopolypeptides offered limited stability. Modifying peptide sequences revealed that glutamate-glycine block copolypeptides at larger block sizes improved thermostability, while incorporating alanine residues into lysine block copolypeptides improved stabilization beyond that achieved with long homopolypeptides. The addition of sucrose in the formulations further improved thermostability, while trehalose showed minimal benefit. Although coacervation did not have a significant impact on viral infectivity, in vivo studies leveraging an alum adjuvant demonstrated that PPV released from a coacervate yielded lower antibody responses compared to native virus, indicating the presence of complicating interactions that potentially masked the immunogenic epitopes on the capsid surface or decreased the effectiveness of the adjuvant. Overall, this study showed that coacervate formulations can be adjusted to enhance virus thermal stability; however, further work is necessary to understand how such formulations can provide thermostability without altering the immune response necessary for a successful vaccine. Such design principles would enable the development of formulations that could decrease the vaccine cold-chain dependence and improve vaccine accessibility.