Archaeosome adjuvant overcomes tolerance to tumor-associated melanoma antigens inducing protective CD8 T cell responses

Abstract

Vesicles comprised of the ether glycerolipids of the archaeon Methanobrevibacter smithii (archaeosomes) are potent adjuvants for evoking CD8(+) T cell responses. We therefore explored the ability of archaeosomes to overcome immunologic tolerance to self-antigens. Priming and boosting of mice with archaeosome-antigen evoked comparable CD8(+) T cell response and tumor protection to an alternate boosting strategy utilizing live bacterial vectors for antigen delivery. Vaccination with melanoma antigenic peptides TRP(181-189) and Gp100(25-33) delivered in archaeosomes resulted in IFN-γ producing antigen-specific CD8(+) T cells with strong cytolytic capability and protection against subcutaneous B16 melanoma. Targeting responses against multiple antigens afforded prolonged median survival against melanoma challenge. Entrapment of multiple peptides within the same vesicle or admixed formulations were both effective at evoking CD8(+) T cells against each antigen. Melanoma-antigen archaeosome formulations also afforded therapeutic protection against established B16 tumors when combined with depletion of T-regulatory cells. Overall, we demonstrate that archaeosome adjuvants constitute an effective choice for formulating cancer vaccines.

Figure 1

CD8⁺ T cell response after heterologous prime-boost with archaeosomes and live vectors. Mice were vaccinated (subcutaneously) with Ms-OVA (20 μg OVA entrapped in Ms archaeosomes, 94.1 nm average size, 38 μg/mg loading), or 10⁴ CFU of live LM-OVA or BCG-OVA. Prime-boost regimens involved the same Ms-OVA vaccine given on days 0 and 30, or heterologous boost on day 30 with the BCG-OVA or LM-OVA vector. The CD8⁺ T cell response was evaluated based on the percentage of OVA_257–264 tetramer positive cells in the blood (a, b) and in vivo CTL response (c). (a) Representative scatter plots showing tetramer positive cells on day 7 after a single injection (top panel) or after prime-boost on day 37 (bottom panel). The square gate indicates percentage of tetramer positive cells in the blood of immunized mice. (b, c), Mean ± SD of 5 mice per group. *Response was statistically significant from single dose group by student's t-test (P < .05).

Figure 2

Tumor protection following heterologous prime-boost vaccination. C57BL/6J mice were vaccinated subcutaneously with a single dose of Ms-OVA (20 μg OVA, 38 μg/mg lipid loading, 94.1 nm average archaeosome size), LM-OVA (10⁴ CFU), or BCG-OVA (10⁴ CFU) or with a prime-boost regimen as indicated. Mice were challenged with subcutaneous B16-OVA tumors 4 weeks post vaccination. Survival plots are based on euthanizing animals upon reaching a maximum tumor size of 300 mm² (n = 5/group). Survival curves for vaccinated groups were significantly different from naïve group by log-rank test (**P < .01, ***P < .001).

Figure 3

CD8⁺ T cell response and tumor protection induced by Ms-TRP vaccine. C57BL/6J mice were immunized in (a) with 1, 10, or 30 μg TRP peptide entrapped in Ms archaeosomes on days 0 and 21. At 5 weeks, representative mice (n = 2 per group) were euthanized, the spleen cells were stimulated with IL-2 (0.1 ng/mL) and peptide (5 μg/mL) for 48 h, and the frequency of IFN-gamma secreting cells was enumerated by ELISPOT. Mean ± SD (n = 3) of IFN-gamma secreting cells per 10⁶ spleen cells is indicated. Spleen cells from mice immunized with 10 μg TRP-peptide per injection were also cultured for 5 days with antigen to generate CTL effectors. The ability of effectors to kill peptide specific (EL-4-TRP) versus nonspecific (EL-4) targets was evaluated in an in-vitro CTL assay (b). Mean killing ± SD of 2 mice per group at different effector : target ratio is indicated for Naïve, PBS-TRP, and Ms-TRP vaccinated mice (b). In another group of representative mice vaccinated with 20 μg Ms-TRP, in vivo CTL response was evaluated on day 7 and day 28. Mean ± SD of n = 4 mice per group is indicated (c). Finally, groups of naïve (n = 12), Ms-TRP vaccinated (n = 12), and TRP-PBS (n = 4) vaccinated mice were challenged with B16 tumors at 6 weeks. Survival was monitored based on a maximum tumor size of 300 mm². Tumor survival data are presented as an aggregate from 3 different experiments conducted, and TRP dose was 15–30 μg/injection. Loading was 19 μg peptide/mg lipid, and average archaeosome size was 99 nm. Survival with Ms-TRP is significantly different (P < .001) by log-rank test relative to naïve group.

Figure 4

CD8⁺ T cell response and tumor protection induced by Ms-Gp100 vaccine. Mice were immunized with 1, 10, or 30 μg Gp100 peptide entrapped in Ms archaeosomes at days 0 and 21. At 5 weeks, representative mice (n = 2 per group) were euthanized, the spleen cells were stimulated with IL-2 (0.1 ng/mL) and peptide (25 μg/ml) for 48 h and the frequency of IFN-gamma secreting cells was enumerated by ELISPOT. Mean ± SD (n = 3) of IFN-gamma secreting cells per 10⁶ spleen cells is indicated. (a) At 5 weeks, in vitro CTL assay was also carried out on spleen cells of representative mice (n = 2 per group) immunized with 10 μg Ms-Gp100 (b). Mean Killing ± SD of 2 mice per group at different effector : target ratio is indicated (b). In another group of representative mice vaccinated with 20 μg Ms-Gp100, in vivo CTL response was evaluated on day 7 and day 28. Mean ± SD of n = 4 mice per group is indicated (c). Finally, response to subcutaneous B16 tumor challenge was evaluated at 6 weeks in groups of naïve (n = 12), Ms-Gp100 vaccinated (n = 12), and PBS-Gp100 (n = 4) vaccinated mice. Survival was monitored based on a maximum tumor size of 300 mm². Tumor survival data are presented as an aggregate from 3 different experiments conducted, and Gp100 peptide vaccination dose ranged from 25 to 30 μg/injection. Loading was 112 μg peptide/mg archaeosomes and average size 94.3 nm. Survival for Ms-Gp100 group is significantly different (P < .05) from naïve mice by log-rank test.

Figure 5

CD8⁺ T cell response and tumor protection induced by coentrapped melanoma peptide-archaeosome vaccine. Mice were vaccinated (days 0 and 21) with 25 μg of peptides (coentrapped 5 μg TRP and 20 μg Gp100) or an equivalent admixed formulation of Ms-TRP and Ms-Gp100. In vitro CTL response of spleen effectors from representative mice (n = 2) was evaluated at 5 weeks (a, b) on TRP and Gp100 specific targets. Mean ± SD of triplicate cultures of effectors: targets at various ratios are indicated. At 6 weeks mice were challenged subcutaneously with B16 melanoma, and survival (n = 4/group) was evaluated based on a maximum tumor size of 300 mm² (c). Loadings were 13 μg Gp100 and 3 μg TRP/mg archaeosomes for the coentrapped vaccine used in (a, c), and 60 μg Gp100/mg lipid and 30 μg TRP/mg archaeosomes for the admixed used in (b, c). Archaeosome size ranged from 104 to 110 nm. Survival for the vaccinated groups was significantly different compared to naïve animals by log-rank test (*P < .05; **P < .01).

Figure 6

Optimized induction of CD8⁺ T cell response to dual melanoma antigen vaccine. Mice were vaccinated (days 0 and 21) with 30 μg of individual peptide-archaeosome vaccines or in an admixed formulation containing 30 μg of each peptide. At 4 weeks, representative mice (n = 3 per group) were euthanized, the spleen cells were stimulated with IL-2 (0.1 ng/mL) and each peptide (5–25 μg/mL) for 48 h, and the frequency of IFN-gamma secreting cells was enumerated by ELISPOT. Mean ± SD (n = 3) of IFN-gamma secreting cells per 10⁶ spleen cells is indicated for TRP-peptide (a) and Gp100 peptide (b) stimulation. The percentage of tetramer-specific CD8⁺ T cells was enumerated in the blood on day 28. Representative plots for the various groups are shown (c), and the Mean ± SD (n = 3) of the response is indicated within each panel. In vitro CTL response in representative mice (n = 3/group) vaccinated with the admixed formulation was evaluated on day 28 against EL-4, EL-4-TRP, and EL-4-Gp100 targets (d). Mean ± SD at 100 : 1 effector : target ratio is indicated. At 4 or 6 weeks postvaccination, mice (n = 5 per group) were challenged with B16 melanoma (e). Survival curves for the vaccinated groups were significantly different from naïve by log-rank test (P < .05). Archaeosome loadings were 40 μg Gp100/mg and 20 μg TRP/mg lipid. Archaeosome size ranged from 110 to 117 nm.

Figure 7

Therapeutic tumor protection by the dual melanoma antigen-archaeosome vaccine. Mice were injected with 10⁵ B16 melanoma cells subcutaneously on day 0. Vaccination with 30 μg of each peptide in PBS (PBS-admixed) or as mixture of Ms-Gp100 and Ms-TRP (Ms-admixed) was carried out on days 1 and 21 posttumor challenge. One group of mice received the anti-CD25 antibody injection (100 μg), intraperitoneally on day 1 posttumor challenge. Mean tumor size ± SD (n = 5/group) over time is indicated for all groups (a). Animals that received the Ms-admixed vaccine and anti-CD25 antibody showed significantly slower tumor progression over time based on one-way ANOVA Bonferronis Post-test compared to the naïve group (P < .01). Tumor survival (b) is based on animals reaching a maximum tumor size of 300 mm². Survival for the Ms-Admixed plus anti-CD25 antibody group was also significantly longer (P < .01) relative to the naïve group (n = 5 mice/group) based on Log rank test. The admixed group contained 2.2 mg of lipid and 60 μg of peptide (30 μg of each peptide).