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 focused on developing in vitro 2D and 3D models for assessing 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 (S-His), nucleocapsid-His and adjuvants: human adenovirus five 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 the potency of biologics in vitro revealed a significant increase in the percentage of CD4+ TCM, CD4+ TEMRA, and CD4+ TEM lymphocyte subpopulations involved in memory cell generation after 24 hours of treatment. Prolonging the exposure for 7 days, a significant increase in CD4+ cells was observed when PBMCs were treated with AdV1 (56.00 ± 0.26% vs. 48.17 ± 1.10%). In contrast, a decrease in CD8+ cells was observed in those treated with AdV1 (37.93 ± 0.35%) compared to AdV1+S-His+N-His (38.47 ± 0.38%) versus the untreated group (44.63 ± 1.07%). A decrease in EMRA was noted when PBMCs were treated with AdV1+S-His+N-His (2.97 ± 0.23% vs. 4.50 ± 0.35%). Moreover, it was pointed out that PBMCs treated with AdV1 alone or in combination with S-His and N-His showed an elevated number of naïve CD4+/CD8+ and SCM CD4+/CD8+ cells. No changes in the number of EMRA CD4+ subpopulations were detected when PBMCs were treated with AdV2 compared with untreated ones (4.27 ± 0.06% vs. 4.50 ± 0.35%). Analysis of the humoral response induced by AdV1, AdV2, S-His, N-His, AdV1+S-His+N-His, and AdV2+S-His+N-His showed that AdV1 alone (4.17 ± 0.25% vs. 3.17 ± 0.06%) and in combination with S-His and N-His (3.87 ± 0.25 vs. 3.17 ± 0.06%) slightly increased the number of CD19+ cells. RNA-Seq analysis of PBMC cells in the 3D model revealed gene overexpression, including FGFR4, associated with the Rap1 pathway in samples 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 AdV showed increased levels of IFN-?. The safety and biodistribution studies of the vaccine platform demonstrated that a 30-day exposure did not impact mice's survival or organ morphology. Exploring the CD40 pathway notably reveals its significant impact on immune cell populations, suggesting potential therapeutic avenues. Overall design: Comparative gene expression profiling analysis of RNA-seq data for PBMC and Calu-3 cells untreated and treated with AdV1 (100 VP/mL), S-His, and N-His.