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 pendent 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 25-30nm 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 (DC 2.4) 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 1.5h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4h 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 90min post injection, ova-micelle conjugates were associated with 28% and 55% of dendritic cells and macrophages, respectively. After 24h, 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.

Keywords: CD8(+) T cell response; Nanoparticles; Polymer micelles; Subunit vaccine; pH-responsive.

Figure 1. Conjugation of ova to polymer carrier via a reducible disulfide linkage

a) pH-responsive carrier transitions from micelle to unimer as a function of pH. HP-PDB was incubated in phosphate buffers ranging from pH 7.4 (physiologic) to 5.8 (endosomal) and particle size analyzed by dynamic light scattering, 1 mg/mL polymer. Data are from a single experiment run in triplicate with error bars representing the standard deviation. b) Fluorescence image of non-reducing SDS-PAGE validating protein-polymer conjugation via a reducible disulfide linkage, 2.8 μg ova-AF488/lane: native ova (Ova) (1), pH-responsive conjugate at 20:1 polymer:ova molar ratio [Ova-(HP-PDB)] (2), conjugate + 20mM TCEP (3), physical mixture of ova and polymer [Ova+(H-PDB)] (4), mixture + 20mM TCEP (5), non pH-responsive control conjugate [Ova-(HP-MMA)] (6), non pH-responsive control conjugate + 20mM TCEP (7). c) Hemolytic activity of diblock copolymer with [Ova-(HP-PDB)] and without (HP-PDB) antigen conjugation, of mixture control polymer (H-PDB), and of non pH-responsive control polymer (HP-MMA), at a polymer concentration of 40 μg/mL. Values are normalized relative to a positive control, 1% v/v Triton X-100, and data represent a single experiment conducted in triplicate ± SD.

Figure 2. Antigen conjugation enhances CTL activation/MHC-I presentation in vitro

DC2.4 cells were stimulated with free protein, physical mixture, or conjugates (10 or 100 μg/mL ova) for 4 h and subsequently co-cultured with B3Z T-cells for 24 h. Cells were rinsed, incubated 24 h with lysis buffer containing chlorophenol red β-D-galactoside, and the absorbance of released chlorophenol red measured at 570 nm. Data represent a representative experiment performed in quadruplicate, mean ± SD. One-way ANOVA followed by Tukey’s Multiple Comparison Test was used for statistical analysis, * = p <0.05.

Figure 3. Conjugation enhances antigen accumulation in DC 2.4s

a,b) DC 2.4s were pulsed with ova or active conjugate (2.5 μg/mL ³H-ova) for 4 h. After various chase periods cells were homogenized and cytosolic and vesicular components separated by ultracentrifugation. Ova content was determined by radioactivity measurements and is plotted in terms of μmol ova. Data represent n = 3 ± SD. c) DC2.4s were pulsed with treatment groups (2.5 μg/mL ³H-ova) for 4 h. After various chase times cells were lysed to measure the intracellular radioactivity. Exocytosis of ³H-ova represents the decrease in intracellular radioactivity in cells post chase relative to radioactivity in cell lysates after the 4 h pulse period. Data from a representative experiment conducted in triplicate ± SD is shown.

Figure 4. Conjugation enhances antigen delivery to lymph node APCs

C57Bl/6 mice were immunized with free ova, mixture, or conjugates in the dorsal part of the foot and draining popliteal lymph nodes isolated 90 min and 24 h post injection. a,b) Uptake of fluorescent ova in total lymphocyte populations as measured by flow cytometry. One-way ANOVA followed by Tukey’s Multiple Comparison Test was used for statistical analysis at a level of p < 0.05 with * indicating significance as compared to Ova-(HP-PDB). c,d) Uptake of ova in individual lymphocyte cell subtypes. Data shown are from two independent experiments with n = 6 mice per group, mean ± SEM. * = p < 0.05, one-way ANOVA followed by Tukey’s Multiple Comparison Test.

Figure 5. Conjugation enhances antigen-specific CD8⁺ IFN-γ⁺ T cell and antibody responses

Splenocytes were isolated on day 29 from mice immunized twice, 3 weeks apart, with free ova, physical mixture, and pH-responsive conjugate. a) Cells were plated and tested for IFN-γ in vitro recall responses by incubating with CD8⁺ T cell epitope ova_257-264 (SINFEKL). CD8⁺ T cells were identified by ICS based on CD8 expression, and the percentage of these cells expressing IFN-γ determined. Means ± SEM (n = 7–15) shown are pooled from three independent experiments. Representative flow cytometry dot plots of CD8⁺ IFN-γ⁺ T cells from individual mice. b) Ova-specific IgG1 (black bars) and IgG2c (white bars) antibody titres were measured on day 28 in sera from immunized mice. Data shown are pooled from four independent experiments and represent the mean of the reciprocal dilution ± SEM (n = 7–15). One-way ANOVA followed by Tukey’s Multiple Comparison Test was used for all statistical analyses. *p < 0.05.

Scheme 1. Nanoparticle vaccine based on pH-responsive polymers for antigen delivery

Amphiphilic diblock copolymers were synthesized by controlled Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization and consist of a neutral, hydrophilic corona segment with pendant pyridyl disulfide groups for reversible antigen conjugation, and a hydrophobic, pH-responsive core that drives endosomal membrane destabilization. The carrier self-assembles into 25–30 nm micelles under aqueous conditions.