Publication date: Sep 03, 2025
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 h 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 nacEFve 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-γ. 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.