pH and ROS Responsiveness of Polymersome Nanovaccines for Antigen and Adjuvant Codelivery: An In Vitro and In Vivo Comparison

The antitumor immunity can be enhanced through the synchronized codelivery of antigens and immunostimulatory adjuvants to antigen-presenting cells, particularly dendritic cells (DCs), using nanovaccines (NVs). To study the influence of intracellular vaccine cargo release kinetics on the T cell activating capacities of DCs, we compared stimuli-responsive to nonresponsive polymersome NVs. To do so, we employed “AND gate” multiresponsive (MR) amphiphilic block copolymers that decompose only in response to the combination of chemical cues present in the environment of the intracellular compartments in antigen cross-presenting DCs: low pH and high reactive oxygen species (ROS) levels. After being unmasked by ROS, pH-responsive side chains are exposed and can undergo a charge shift within a relevant pH window of the intracellular compartments in antigen cross-presenting DCs. NVs containing the model antigen Ovalbumin (OVA) and the iNKT cell activating adjuvant α-Galactosylceramide (α-Galcer) were fabricated using microfluidics self-assembly. The MR NVs outperformed the nonresponsive NV in vitro, inducing enhanced classical- and cross-presentation of the OVA by DCs, effectively activating CD8+, CD4+ T cells, and iNKT cells. Interestingly, in vivo, the nonresponsive NVs outperformed the responsive vaccines. These differences in polymersome vaccine performance are likely linked to the kinetics of cargo release, highlighting the crucial chemical requirements for successful cancer nanovaccines

More native antigen in live than in apoptotic donor cells: improved antigen crosspresentation by DC.

<p>A, B, 30.10⁶ live (zVAD treated) or apoptotic (γ-irradiated) B16 cells (A) or L-OVA cells (B) were lysed for protein extraction. Lysates from B16 cells were analysed using anti-gp100, anti-TRP2 and anti-actin antibodies (A). Lysates from L-OVA cells were analysed using anti-OVA and anti-actin antibodies antibodies (B). Anti-actin antibodies were used as controls. C, DC were cultured with different numbers of live (filled triangle) or apoptotic (open triangle) L-OVA cells or live L cells (filled square). DC were then purified by magnetic sorting using anti-CD11c microbeads and cultured with B3Z-T cells hybridoma cells for 18 h. Activation after recognition of the SIINFEKL-K^b complex was detected by optical density measurement at 560 nm after addition of the CPRG substrate. The significance of differences between series of results was assessed using a two-tailed unpaired t test. **p<0.01, *p<0,05, n.s. not significant. Mean ± SEM, representative of two independent experiments.</p

DC cytokine profile after culture with live or apoptotic B16 cells.

<p>DC were cultured alone (DC) or with live (zVAD treated) or apoptotic (γ-irradiated) B16 cells for 24 h and stimulated or not with LPS and IFNγ. As controls, B16 zVAD and B16γ cells were also cultured alone. DC were then assessed for their ability to produce IL-12p70 (A) or IL-10 (B) by ELISA. In the upper panels, data from one out of three independent experiments are shown. In the bottom panels, for LPS and IFNγ stimulation, relative IL-12p70 and IL-10 productions are expressed as a percentage of the cytokine production obtained for DC alone. Mean percentage values±SEM are shown. The significance of differences between series of results was assessed using a paired t test (n = 3, 3 independent experiments).</p

DC maturation after culture with live or apoptotic B16 cells.

<p>DC were cultured with live (zVAD treated) or apoptotic (γ-irradiated) B16 cells for 24 h in the presence of culture medium or LPS and IFNγ. The expression of maturation molecules was then tested by flow cytometry. One representative experiment out of three independent experiments is shown.</p