Neutral polymer micelle carriers with pH-responsive, endosome-releasing activity modulate antigen trafficking to enhance CD8+ T cell responses

Abstract

Synthetic subunit vaccines need to induce CD8+ cytotoxic T-cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8+ cytotoxic T-cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8+ T-cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendant pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25–30 nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5 h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4 h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8+ T cell responses (0.4 % IFN-γ+ of CD8+) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells in the draining lymph nodes. As early as 90 min post injection ova-micelle conjugates were associated with 28% and 55% of dendritic cells and macrophages, respectively. After 24 h, conjugates preferentially associated with dendritic cells, affording 30-, 3-, and 3-fold enhancements in uptake relative to free protein, physical mixture, and the non pH-responsive conjugate controls, respectively. These results demonstrate the potential of pH-responsive polymeric micelles for use in vaccine applications that rely on CD8+ T cell activation.

Fatty Acid-Mimetic Micelles for Dual Delivery of Antigens and Imidazoquinoline Adjuvants

“…The final composition of the first block consisted of 92 mole % PEGMA along with 8 mole % PDSM which has been found to be a sufficient amount of pyridyl disulfide moieties for antigen conjugation by other nanocarriers that have been used for in vivo applications. 43, 45, 60 …”

Modulating the immune system through nanotechnology

“…Keller et al showed how pH-responsive micelles significantly enhance cytotoxic T lymphocyte responses, in comparison to micelles without these properties [117]. This effect was achieved because the forming polymers are protonated at endosomal pH which allows them to interact with the membrane and disrupt the endosome [117,118]. The same tendency was reported in the case of pH-sensitive liposomes, cationic liposomes and bioreducible linkages [119–122].…”

“…This endosomal escape can be promoted by the disruption of the endosome membrane, as discussed by several authors [115,116]. Keller et al showed how pH-responsive micelles significantly enhance cytotoxic T lymphocyte responses, in comparison to micelles without these properties [117]. This effect was achieved because the forming polymers are protonated at endosomal pH which allows them to interact with the membrane and disrupt the endosome [117,118].…”

Synthetic nanovaccines for immunotherapy

“…The proton sponge effect is observed with nanoparticles that have a high buffering capacity, as the influx of ions and water into the endosomes leads to high osmotic pressure, and eventually rupture of the organelle [46]. Keller et al have also reported the use of a pH-sensitive polymer micelle nanoparticle for cytosolic delivery of antigens, and demonstrated higher concentrations of antigen-specific cytotoxic T cells compared to controls [47]. …”