Increases in Cellular Immune Responses Due to Positive Effect of CVC1302-Induced Lysosomal Escape in Mice

Abstract

This study found a higher percentage of CD8⁺ T cells in piglets immunized with a CVC1302-adjuvanted inactivated foot-and-mouth disease virus (FMDV) vaccine. We wondered whether the CVC1302-adjuvanted inactivated FMDV vaccine promoted cellular immunity by promoting the antigen cross-presentation efficiency of ovalbumin (OVA) through dendritic cells (DCs), mainly via cytosolic pathways. This was demonstrated by the enhanced levels of lysosomal escape of OVA in the DCs loaded with OVA and CVC1302. The higher levels of ROS and significantly enhanced elevated lysosomal pH levels in the DCs facilitated the lysosomal escape of OVA. Significantly enhanced CTL activity levels was observed in the mice immunized with OVA-CVC1302. Overall, CVC1302 increased the cross-presentation of exogenous antigens and the cross-priming of CD8⁺ T cells by alkalizing the lysosomal pH and facilitating the lysosomal escape of antigens. These studies shed new light on the development of immunopotentiators to improve cellular immunity induced by vaccines.

Keywords: cellular immunity; cross-presentation; cytosolic pathways; lysosomal escape.

Figure 1

CVC1302 treatment led to an enhanced CTL response. (A,B) C57BL/6J mice were immunized with OVA or OVA-CVC1302. Inguinal lymph nodes were sampled from mice at 7 dpi and analyzed via flow cytometry. (A) Representative flow cytometry plot showing CD3⁺ CD8⁺ T cells. (B) Percentages of CD3⁺ CD8⁺ T cells (left) and percentages of CD4⁺ CD8⁺ T cells (right) in mice. (C,D) Piglets were immunized with FMDV or FMDV-CVC1302. Sera were sampled from piglets at 28 dpi. PBMCs were prepared and analyzed using flow cytometry. (C) Representative flow cytometry plot showing CD3⁺ CD8⁺ T cells. (D) Percentages of CD3⁺ CD8⁺ T cells (left) and percentages of CD4⁺ CD8⁺ T cells (right) in piglets. (E,H) SIINFEKL peptide-pulsed target cells were transferred into mice immunized with OVA or OVA-CVC1302 at 7 dpi. The killing rates of the target cells were analyzed using flow cytometry. (E) Representative cytometry plot showing the target cells in the lymph nodes of mice immunized with OVA or OVA-CVC1302. (F) Killing rates in the lymph nodes of mice immunized with OVA or OVA-CVC1302. (G) Representative cytometry plot showing the target cells in the spleens of mice immunized with OVA or OVA-CVC1302. (H) Killing rates in the spleens of mice immunized with OVA or OVA-CVC1302. Data are presented as the mean ± SEM; ** p ≤ 0.05, **** p ≤ 0.0001.

Figure 2

CVC1302 improved antigen cross-presentation by Flt3-L-cultured BMDCs. (A) The cytotoxicity of CVC1302 in Flt3-L-cultured BMDCs. A series of concentrations of CVC1302 were incubated with BMDCs for 12 h, and cellular viability was measured using the CCK-8 assay. (B–I) The activation of BMDCs treated with OVA±CVC1302 for 6 h. The cell surface expression of CD40, CD80, CD86, and MHCI in BMDCs was assessed by flow cytometry. (B) Representative cytometry plot showing the expression of CD40 in BMDCs. (C) Mean fluorescence intensity of CD40 in the BMDCs. (D) Representative cytometry plot showing the expression of CD80 in BMDCs. (E) The mean fluorescence intensity of CD80 in BMDCs. (F) Representative cytometry plot showing the expression of CD86 in BMDCs. (G) The mean fluorescence intensity of CD40 in BMDCs. (H) Representative cytometric plot showing the expression of MHCI in BMDCs. (I) Mean fluorescence intensity of MHCI in the BMDCs. (J) β-galactosidase production by B3Z cells after co-culture with BMDCs pre-treated with OVA±CVC1302 for 24 h. (K) The expression levels of IL-12p70 in BMDCs treated with OVA±CVC1302 for 6 h. Data are presented as the mean ± SEM; * p ≤ 0.01, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

CVC1302 engages in the cytosolic pathway during cross-presentation. BMDCs were pulsed with OVA-FITC±CVC1302, chased at the indicated time points, and immunostained to assess the degree of OVA co-localization with LysoTracker Red DND-99 (lysosome). (A) BMDCs were treated with OVA+CVC1302 for 40 min. (B) BMDCs were treated with OVA for 40 min. (C) BMDCs were treated with OVA+CVC1302 for 60 min. (D) BMDCs were treated with OVA for 60 min. Scale bars: 5 μm.

Figure 4

CVC1302 mediates lysosomal escape via p38α signaling. (A,B) Total proteins were harvested from BMDCs treated with OVA±CVC1302 for 6 h. Next, proteins (A) p38α and P-p38α were activated in BMDCs treated with OVA+CVC1302. BMDCs were stimulated with OVA±CVC1302 as indicated. (B) NOX2 was activated in BMDCs treated with OVA+CVC1302. (C) ROS were activated in BMDCs treated with OVA+CVC1302. (Upper) BMDCs were treated with OVA+CVC1302 for 1 h. (Bottom) BMDCs were treated with OVA+CVC1302 for 3 h. (D,E) CVC1302 interferes with the acidification of lysosomal pH in BMDCs. After BMDCs were treated with OVA±CVC1302 for 6 h, BMDCs were stained with AO or LysoSensor, followed by the measurement of AO or LysoSensor signal with flow cytometric analysis. (D) The MFI of BMDCs stained with AO after OVA±CVC1302 treatment. (E) The MFI of BMDCs stained with LysoSensor after OVA±CVC1302 treatment. Data are presented as the mean ± SEM; ** p ≤ 0.01, *** p ≤ 0.001.