Defective interfering particles (DIPs) are non-infectious particles containing partial viral genome naturally generated during repeated viral replication. DIP has been proven as antiviral that suppresses dengue virus (DENV) infection across various cell types and animal models. However, the practical application of DIPs in therapeutic settings may be limited by challenges in stability and storage. Lyophilization or freeze drying is a well-established technique for stabilizing biological materials, including vaccines. In our study, we investigated the feasibility of applying lyophilisation to enhance the stability of dengue DIPs, utilising techniques commonly used in the preparation of tetravalent dengue vaccines.
Dengue DIPs produced by a stably transduced HEK293T cell line were purified by tangential flow filtration, followed by ceramic hydroxyapatite (CHT) chromatography. The resulting highly purified (>99.9% removal of contaminating protein) and biologically active DENV DIPs were then freeze dried (FD). The FD DIP was resuspended in a Tris-saline buffer containing gelatine-sorbitol stabiliser. We found that the concentration of DIPs was significantly higher in FD DIPs compared to DIPs prepared by standard ultrafiltration method, where DIPs are typically lost during the process.
The stability and integrity of FD DIPs were assessed by determining the host antiviral response and antiviral activity in human fibroblast cells. Our results showed that FD DIPs maintained the ability to activate interferon response in cells, demonstrating that the therapeutic effectiveness of DIPs was preserved, with FD DIPs effectively suppressing DENV infection. The enhanced concentration and stability of FD DIPs presents a viable approach for the storage and transport of DIP based therapies, potentially broadening their application in combating dengue infection.