Development of an in vitro method to assess the immunogenicity of biologics in the prevention of infectious diseases

We present a series of preclinical studies focusing on developing in vitro 2D and 3D models for assessing the immunogenic factors in preventing infectious diseases. Human peripheral blood mononuclear cells (PBMC) and Calu-3 cell lines (bronchial epithelial cells) were used to develop 2D and 3D models. Peptides: Spike-S1-His, nucleocapsid-His and adjuvants: human adenovirus 5 serotype-based viral vector (AdV-D24-ICOSL-CD40L), armed with inducible co-stimulator (ICOSL) and CD40 ligand (CD40L), and a vector lacking these transgenes (AdV5/3) were used due to their effective initial interaction with antigen-presenting cells (APC). Studying biologics’ potency in vitro showed a significant increase in the percentage of CD4 ⁺ _TCM , CD4 ⁺ _TEMRA , and CD4 ⁺ _TEM lymphocyte subpopulations involved in memory cell generation after 24-h treatment. Prolonging the exposure for 7 days significantly increased the number of CD4 ⁺ T and CD19 ⁺ B lymphocytes. RNA-Seq analysis of PBMC cells in the 3D model demonstrated gene overexpression (including FGFR4) associated with the Rap1 pathway in the sample exposed to AdV1+S-His+N-His. Thus, the proposed platform's impact on lymphocyte differentiation was confirmed, and cytokine profile analysis in this sample revealed elevated levels of IL-10, IL-12p70, and IL-8. All samples exposed to AdV1 showed increased levels of IFN-γ. Safety studies of the vaccine platform demonstrated that a 30-day exposure did not impact mice's survival or organ morphology. The safety and biodistribution of the biologics were confirmed in in vivo studies. The research resulted in the development of a method providing a reliable assessment of immunogenic factors under in vitro conditions. By establishing a 3D in vitro model using PBMCs and Calu-3 cells, the research shed light on the dynamics of the immune responses to novel adenovirus-based vaccine platforms. The study identifies critical factors influencing immune reactions, including inflammation, immune cell activation, and regulatory responses, providing insights into the virus-host dynamics. Exploring the CD40 pathway notably reveals its significant impact on immune cell populations, suggesting potential therapeutic avenues. The findings underscore the importance of extended culture times and the need for further research into the mechanistic role of the CD40 pathway.