The Orf virus (ORFV) is a promising vector platform for the generation of vaccines against infectious diseases and cancer, highlighted by its progression to clinical testing phases. One of the critical steps during GMP manufacturing is the clarification of crude harvest because of the enveloped nature and large size of ORFV. This study presents the first description of ORFV clarification process from a HEK suspension batch process. We examined various filter materials, membrane pore sizes, harvest timings, and nuclease treatments. Employing the Ambr® crossflow system for high-throughput, small-volume experiments, we identified polypropylene-based Sartopure® PP3 filters as ideal. These filters, used in two consecutive stages with reducing pore sizes, significantly enhanced ORFV recovery and addressed scalability challenges. Moreover, we demonstrated that the time of harvest and the use of a nuclease play a decisive role to increase ORFV yields. With these findings, we were able to establish an efficient and scalable clarification process of ORFV derived from a suspension production process, essential for advancing ORFV vaccine manufacturing.

Steric exclusion chromatography (SXC) is a promising purification method for biological macromolecules such as the Orf virus (ORFV) vector. The method’s principle is closely related to conventional polyethylene glycol (PEG) precipitation, repeatedly implementing membranes as porous chromatographic media. In the past decade, several purification tasks with SXC showed exceptionally high yields and a high impurity removal. However, the effect of varying process parameters, on the precipitation success and its limitations to SXC, is not yet well understood. For this reason, the precipitation behavior and SXC adaptation for ORFV were investigated for the PEG/ORFV contact time, the membranes pore size, and the type and concentration of ions. All three parameters influenced the ORFV recoveries significantly. A small pore size and a long contact time induced filtration effects and inhibited a full virus recovery. The application of salts had complex concentration-dependent effects on precipitation and SXC yields, and ranged from a complete prevention of precipitation in the presence of kosmotropic substances to increased efficiencies with Mg²⁺ ions. The latter finding might be useful to reduce PEG concentrations while maintaining high yields. With this knowledge, we hope to clarify several limitations of SXC operations and improve the tool-set for a successful process adaptation.

Prime-2-CoV_Beta is a novel Orf virus (ORFV)-based COVID-19 vaccine candidate expressing both the nucleocapsid and spike proteins of SARS-CoV-2 with the receptor-binding domain (RBD) of the Beta strain. This candidate was shown to be safe and immunogenic in a first-in-human Phase I clinical trial. With the shift in the immune landscape toward the Omicron variant and the widespread vaccine- and/or infection-derived immunity, further pre-clinical research was needed to characterize Prime-2-CoV. Here, we quantified the humoral and cellular response to Prime-2-CoV_Beta in pre-immunized mice and compared the protective efficacy of mono- and bivalent variant-based Prime-2-CoV vaccine candidates in hamsters. Prime-2-CoV_Beta induced robust humoral and cellular immune responses in naïve animals but did not further boost antibody titers in the tested setting when given as repeat booster at short interval. We furthermore showed that Prime-2-CoV_Beta-based mono- and bivalent immunization strategies produced comparable immunogenicity and protection from infection. Our results highlight the potential of the Orf virus as a vaccine platform against SARS-CoV-2 and potentially other infectious viruses.