Antigen delivery with poly(propylacrylic acid) conjugation enhances MHC-1 presentation and T-cell activation

Abstract

While many infectious diseases are controlled by vaccine strategies, important limitations continue to motivate the development of better antigen delivery systems. This study focuses on the use of a pH-sensitive polymeric carrier based on poly(propylacrylic acid) (PPAA) to address the need for more potent CD8 cytotoxic T-cell (CTL) responses. An MHC-1/CD8 CTL cell model system with ovalbumin as the protein antigen was used to test whether PPAA could enhance the delivery of ovalbumin into the MHC-1 display pathway. Ovalbumin was conjugated to poly(propylacrylic acid-co-pyridyldisulfide acrylate) (PPAA-PDSA) by disulfide exchange to make reversible conjugates that could be reduced by the glutathione redox system in the cytosol of antigen presenting cells. The PPAA-PDSA ovalbumin conjugates displayed the pH-sensitive membrane disruptive properties of the parent polymer as determined by their hemolysis activities (sharply active at the endosomal pH values of 6-6.5). The polymer-ovalbumin conjugates exhibited strong 22-fold increases in the MHC-1 presentation and ovalbumin-specific CTL activation compared to free ovalbumin. No CTL activation was observed with control conjugates of ovalbumin and poly(methylacrylic acid) (PMAA) that do not display membrane disruptive activies, suggesting that it is the membrane destabilizing properties of the polymer that result in increased MHC-1 display and CTL activation. Further mechanistic studies quantitated the time course of stable intracellular localization of radiolabeled conjugates. 52% of initially internalized PPAA-conjugated ovalbumin remained in the cells after 4 h, compared to less than 10% of ovalbumin or PMAA-ovalbumin. These results showing enhanced cytosolic delivery and MHC-1 presentation for the PPAA-antigen conjugates suggest that they warrant future characterization as a CD8-enhancing vaccine delivery system.

Figure 1

Schematic of chemistry for conjugation of ovalbumin to the PDSA moiety of the PPAA-PDSA polymer. The protein is first modified using Traut’s reagent to convert primary amines to thiols, then the polymer is added and a disulfide exchange reaction occurs, releasing pyridine-2-thione. Reaction progress can be monitored by measuring A₃₄₃ of released pyridine-2-thione. PPAA and PMAA conjugates consisting of 1.7 μg polymer per μg ovalbumin were formed.

Figure 2

pH-dependent hemolysis properties of PPAA polymers and ovalbumin conjugates. Red blood cells were isolated and added to polymer and conjugate solutions (polymer concentrations = 5μg/ml) in 0.1M phosphate buffer at pH values of 7.4 (□), 6.6 (x), and 5.8 (■). Hemolysis is reported as a percentage of complete lysis by Triton X-100. PPAA-PDSA shows high membrane-disruptive activity at pH 5.8 but considerably lower membrane disruption at pH 7.4. The polymer retains its hemolysis capabilities after conjugation to the hydrophilic protein ovalbumin. PMAA-PDSA, however, does not lyse red blood cells at any pH due to the decreased hydrophobicity of its methyl side chain compared with the propyl side chain of PPAA.

Figure 3

CTL activation/MHC-I presentation of PPAA-ovalbumin conjugates. Samples were incubated with RAW macrophages for 6 hrs then removed and B3Z T-cells were added for 16 hrs. Cells were rinsed and incubated 4 hrs with lysis buffer containing chlorophenol red β-D- galactoside, then absorbance of released chlorophenol red was measured at 595 nm. Samples were evaluated in triplicate and errors are reported as +/− one standard deviation. For reference, the maximum possible β-galactosidase production was determined by chemically stimulating the B3Z cells using PMA/ionomycin for 4 hrs, which gave an A₅₉₅ of 0.56. a.) Ovalbumin oncentration=100μg/ml. The PPAA-ovalbumin conjugate shows significantly greater CTL activation than do any of the control samples (p<0.0005). This is likely due to the endosomal disruption provided by PPAA, which allows the protein to more efficiently access the MHC-I pathway in the cytoplasm. However, PMAA conjugation results in low CTL activation, similar to that for free ovalbumin or a PPAA ovalbumin physical mixture (p=0.2). This suggests that the increase in MHC-1 presentation provided by PPAA is due to its endosomal disruptive properties rather than solely to increased uptake due to its larger size compared to free ovalbumin. b) The PPAA-ovalbumin conjugates enhance CTL stimulation is a dose-dependent manner. When the polymer ratio is increased from 1.7 ug/ug ova to 3.2 ug/ug ova, maximal CTL activation is reached at a lower ovalbumin concentration.

Figure 4

Exocytosis of ¹⁴C-ovalbumin after a) 1 min uptake time and b) 15min uptake time. Samples were incubated with RAW macrophages and un-internalized conjugate was removed. Fresh media was added to the cells at various time intervals and the reappearance of ¹⁴C-ovalbumin into the supernatant was measured. After 4 hrs, cells were lysed with 1% Triton X-100 and the radioactivity in the lysate was measured. Samples were evaluated with a minimum of n=3. The amount of ovalbumin initially internalized was determined as a percentage of the total delivered (left-side panels). It can be seen that the amount of ovalbumin taken into the cells in 1 min is similar for all samples (p=0.66) giving a uniform starting point for the exocytosis measurements. However, the preferential accumulation of PPAA-ovalbumin is already noticeable at the 15-min uptake time. The amount of ovalbumin exocytosed at each timepoint was then calculated as a percentage of the amount initially internalized (right-side panels). Most exocytosis occured in the first 30 minutes, and exocytosis rates were similar for all samples. However, the amount of ovalbumin that remained in the cells and was not exocytosed was greatly enhanced for PPAA conjugates. This effect is likely due to the ability of the polymer to disrupt the endosomal membrane and deliver the ovalbumin to the cytosol before exocytosis can occur.

Figure 5

Uptake of ¹⁴C-Ovalbumin-PPAA increases over time. RAW macrophages were incubated with samples at a concentration of 50μg/ml of ovalbumin. The cells were then washed with PBS and lysed using 1% Triton X-100. Radioactivity in the cell media, PBS wash, and cell lysate was measured, and uptake of ¹⁴C-ovalbumin was calculated as the % radioactivity present in the cell lysate compared to the total radioactivity delivered. Experiments were performed in triplicate and error is expressed as +/− one standard deviation. It can be seen that PPAA-conjugated ovalbumin continually accumulates significantly in the cell, whereas the control sample levels remain fairly constant (p>0.09). This is likely due to the ability of PPAA-ovalbumin to escape the endosome before being exocytosed.

Figure 6

Cytotoxicity of PPAA and PMAA polymers and conjugates. Cytotoxicity was determined for both RAW and B3Z cells using the LDH assay. Samples were added to cells for 24 hrs at a concentration of 300 μg/ml polymer, twice the highest concentration used in the MHC-1 presentation assay. The cell supernatant was then combined with LDH reagent and the absorbance at 490 nm was recorded. Percent survival = 1−[(A₄₉₀ of sample − A₄₉₀ of untreated cells control)/(A₄₉₀ of TritonX control − A₄₉₀ of untreated cells control)] × 100%. Samples were evaluated in triplicate and error is expressed as +/− SEM. It can be seen that excessive toxicity was not observed for any of the samples.