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.

本研究报道了一系列临床前研究，旨在构建体外（in vitro）二维（2D）与三维（3D）模型，以评估用于预防传染病的免疫原性因子。本研究使用人类外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMC）与Calu-3细胞系（支气管上皮细胞）构建2D与3D模型。实验所用肽段包括Spike-S1-His（S-His）、核衣壳蛋白-His（nucleocapsid-His）；佐剂则包括基于5型人腺病毒的病毒载体AdV-D24-ICOSL-CD40L（携带有可诱导共刺激分子ICOSL与CD40配体CD40L），以及缺失上述转基因的载体AdV5/3，选择此类载体是因其可与抗原呈递细胞（Antigen-Presenting Cells, APC）实现高效的初始相互作用。体外检测生物制剂效力的实验结果显示，处理24小时后，参与记忆细胞生成的CD4+ 中央记忆T细胞（TCM）、CD4+ 终末分化记忆T细胞（TEMRA）及CD4+ 效应记忆T细胞（TEM）淋巴细胞亚群的占比显著升高。将暴露时间延长至7天，经AdV1处理的PBMC中CD4+细胞占比显著升高（56.00 ± 0.26% vs. 48.17 ± 1.10%）。与之相反，与未处理组（44.63 ± 1.07%）及AdV1+S-His+N-His处理组（38.47 ± 0.38%）相比，AdV1单独处理组的CD8+细胞占比出现下降（37.93 ± 0.35%）。经AdV1+S-His+N-His处理的PBMC中，EMRA细胞占比出现下降（2.97 ± 0.23% vs. 4.50 ± 0.35%）。此外，研究发现经AdV1单独处理或联合S-His、N-His处理的PBMC中，初始型CD4+/CD8+及干细胞记忆T细胞（SCM）CD4+/CD8+细胞的数量均有所升高。与未处理组（4.27 ± 0.06% vs. 4.50 ± 0.35%）相比，经AdV2处理的PBMC中EMRA CD4+亚群的数量未出现显著变化。对AdV1、AdV2、S-His、N-His、AdV1+S-His+N-His及AdV2+S-His+N-His诱导的体液免疫应答进行分析后发现，AdV1单独处理组（4.17 ± 0.25% vs. 3.17 ± 0.06%）及AdV1联合S-His、N-His处理组（3.87 ± 0.25 vs. 3.17 ± 0.06%）的CD19+细胞数量均出现轻度升高。对3D模型中的PBMC进行RNA测序（RNA-Seq）分析后发现，经AdV1+S-His+N-His处理的样本中，包括成纤维细胞生长因子受体4（FGFR4）在内的多个基因出现过表达，且这些基因与Rap1通路相关。由此证实了本研究提出的疫苗平台对淋巴细胞分化的调控作用，且该样本的细胞因子谱分析结果显示，白细胞介素-10（IL-10）、白细胞介素-12p70（IL-12p70）及白细胞介素-8（IL-8）的水平显著升高。所有经腺病毒载体（AdV）处理的样本中，干扰素-γ（IFN-γ）的水平均出现升高。对该疫苗平台进行的安全性与生物分布研究表明，30天暴露并未对小鼠的存活率或器官形态造成影响。对CD40通路的研究进一步揭示了其对免疫细胞群的显著调控作用，为相关治疗策略的开发提供了潜在方向。实验整体设计：对未处理及经AdV1（100 病毒颗粒/毫升，VP/mL）、S-His、N-His处理的PBMC与Calu-3细胞的RNA测序数据进行比较基因表达谱分析。