In vitro immune evaluation of adenoviral vector-based platform for infectious diseases

Abstract

New prophylactic vaccine platforms are imperative to combat respiratory infections. The efficacy of T and B memory cell-mediated protection, generated through the adenoviral vector, was tested to assess the effectiveness of the new adenoviral-based platforms for infectious diseases. A combination of adenovirus AdV1 (adjuvant), armed with costimulatory ligands (ICOSL and CD40L), and rRBD (antigen: recombinant nonglycosylated spike protein rRBD) was used to promote the differentiation of T and B lymphocytes. Adenovirus AdV2 (adjuvant), without ligands, in combination with rRBD, served as a control. In vitro T-cell responses to the AdV1+rRBD combination revealed that CD8+ platform-specific T-cells increased (37.2 ± 0.7% vs. 23.1 ± 2.1%), and T-cells acted against SARS-CoV-2 via CD8+TEMRA (50.0 ± 1.3% vs. 36.0 ± 3.2%). Memory B cells were induced after treatment with either AdV1+rRBD (84.1 ± 0.8% vs. 82.3 ± 0.4%) or rRBD (94.6 ± 0.3% vs. 82.3 ± 0.4%). Class-switching from IgM and IgD to isotype IgG following induction with rRBD+Ab was observed. RNA-seq profiling identified gene expression patterns related to T helper cell differentiation that protect against pathogens. The analysis determined signaling pathways controlling the induction of protective immunity, including the MAPK cascade, adipocytokine, cAMP, TNF, and Toll-like receptor signaling pathway. The AdV1+rRBD formulation induced IL-6, IL-8, and TNF. RNA-seq of the VERO E6 cell line showed differences in the apoptosis gene expression stimulated with the platforms vs. mock. In conclusion, AdV1+rRBD effectively generates T and B memory cell-mediated protection, presenting promising results in producing CD8+ platform-specific T cells and isotype-switched IgG memory B cells. The platform induces protective immunity by controlling the Th1, Th2, and Th17 cell differentiation gene expression patterns. Further studies are required to confirm its effectiveness.

Keywords: adenoviral vectors; innate and adaptive immunity; vaccine platform.

Fig. 1

The percentage of the T lymphocyte subpopulations among PBMCs stimulated with the vaccine factors (n = 6) compared with PBMC mock (PBMCs stimulated with LPS at 1.25 μg/ml) after the 24-h treatment at 37°C in 5% CO₂; (A) the percentage of CD4⁺ and CD8⁺ T among CD3⁺; (B) the percentage of central memory (CM), effector memory (EM), effector memory terminally differentiated (EMRA), and CD197⁺CD45RA⁺ among CD4⁺; (C) the heat map analysis of the markers expressed by the subpopulations of CD4⁺CD45RA⁺CD197⁺; the expression of CD95 (T_SCM) after the treatment with AdV1 (left top row) or rRBD+Ab (left bottom row); the expression of NAÏVE (CD95^-) after the treatment with rRBD (right bottom row); (D) CM, EM, EMRA, and CD45RA⁺CD197⁺ among CD8⁺; (E) The heat map analysis of markers expressed by the subpopulations of CD4⁺CD45RA⁺CD197⁺; the expression of CD95 (T_SCM) after the treatment with AdV1 (left top row) or rRBD+Ab (left bottom row); the data are representative of 2–5 different experiments; multiple unpaired t-tests: only comparison with a P-value less than or equal to 0.05 was presented (significant diff. between the means of mock vs. the marked treatments, P ≤ 0.05)

Fig. 2

(A) the CD19⁺ lymphocyte subpopulations induced by the immunogenic factors after the 24-h treatment: CD38^-CD24⁺ (memory cells), CD38⁺CD24⁺ (transitional cells), CD38⁺CD24^-(plasmablasts); (B) the subsets of the memory B cells (CD19⁺ CD24⁺CD38^-): CS, NCS, and NAÏVE; multiple unpaired t-tests: significant differences between the means of mock vs. the marked treatments (P ≤ 0.05). Data are shown as means ± SD of four independent experiments

Fig. 3

The immunoglobulin classes after the treatment with the immunogenic factors; (A) CD27⁺ and IgD^- allow the identification of class-switched B cells; (B) CD27⁺ and IgD⁺ allow the identification of nonclass-switched B cells; (C) CD27^- and IgD⁺ are representative of NAÏVE B cells; data are shown as means ± SD of four independent experiments; multiple unpaired t-tests: significant differences between the means of mock vs. the marked treatments (P ≤ 0.05)

Fig. 4

Effects of the AdV1+rRBD challenge on pro-inflammatory cytokines IL-6, IL-8, and TNF-α production in PBMC; PBMCs were treated with AdV1+rRBD for 24 h, and the supernatants were harvested for flow cytometry analysis; data are presented as means ± CV% (n = 6); TNF – tumor necrosis factor; IL-6 – interleukin 6; IL-8 – interleukin 8; PBMCs’ supernatant treated with LPS (Mock); PBMCs’ supernatant treated with AdV1: mixture of adenovirus 1 (AdV1, Ad5/3-D24 ICOS CD40L) at 100 VP/ml and rRBD at 2.62 μg/ml; Wilcoxon matched-pairs signed rank test: insignificant differences between the means of mock (LPS) vs. AdV1+rRBD (P = 0.25)

Fig. 5

Relative quantification of the CD40 gene of VERO E6; values are given as the cycle threshold (Ct, mean of triplicate samples); normalization factors were calculated as the geometric mean of the expression levels of the most stable reference gene, GAPDH; a control VERO E6 sample was used as the calibrator (= 1); fold gene expression 2^-ΔΔCt was calculated according to the formula: ΔΔCt = ΔCt (sample) ˗ ΔCt (control average) and ΔCt = Ct (gene of interest) – Ct (housekeeping gene); gene expression values from all experiments are displayed in a heatmap indicating the up-regulation (blue) or downregulation (red) of given markers; interpretation of relationship strength between variables: r>1 (strongly positive), 0.5 < r < 1 (moderately positive), 0 < r < 0.5 (weakly positive), r = 0 (none), and negative correlation for the opposite direction; blank results mean the same value of the variable in the rows. the cell expression

Fig. 6

Gene expression changes – log-intensity ratios (differences) versus log-intensity averages (means) (A) and enrichment of up and down regulated genes in KEGG term gene groups (B) in AdV1 IC100 immunized cells; in A NotSig means not significantly expressed vs. mock, Up means expressed at a higher level compared with mock, and Down means expressed at a lower level compared to mock