Trypanosoma cruzi adjuvants potentiate T cell-mediated immunity induced by a NY-ESO-1 based antitumor vaccine

Abstract

Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4(+) T and CD8(+) T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-γ) production by CD4(+) T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-γ response by CD8(+) T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4(+) T and CD8(+) T cell responses elicited by a specific immunological adjuvant.

Figure 1. T. cruzi derived GIPLs are TLR4 agonists and promote high levels of antigen-specific IgG2c antibodies as well as IFN-γ production by CD4⁺ T cells.

(A) CHO cells control (TLR2⁻/TLR4⁻) or expressing TLR2 (TLR2⁺) or TLR4 (TLR4⁺) were either left untreated (solid gray) or exposed to 100 µg/ml of GIPLs from Trypanosoma cruzi Tulahuen (GTH), Y strain (GY) (black line). MALP-2 (10 ηg/ml) and LPS (200 ηg/ml) were used as positive controls for activation of TLR2⁺ or TLR4⁺, respectively. (B) TLR4⁺ cells were activated with different preparations of GIPLs in the presence of polymyxin B (PB). LPS was used as control. (C) OVA specific immune responses induced by immunization with TLR2 or TLR4 agonists associated with OVA absorbed in alum. Mice were immunized with three doses on days 0, 14 and 28. The production of total IgG, IgG1 and IgG2c were assessed by ELISA using the sera from immunized mice, at day 9 after the second boost. (D) To assess the levels of IFN-γ production by T lymphocytes from vaccinated mice, splenocytes were collected 21 days after the third immunization dose and stimulated with either CD4⁺ T or CD8⁺ T cell epitopes from OVA. The results are representative of two independent experiments yielding similar results. Asterisks indicate that differences were statistically significant, when comparing T cell response from mice receiving different vaccine formulations.

Figure 2. Immunostimulatory and adjuvant activity of TLR9 agonists derived from T. cruzi genome.

(A) PBMCs derived from healthy donors were stimulated with human B-class-like CpG ODNs derived from the T. cruzi genome with four different concentrations (3.0, 1.0, 0.3, and 0.1 µM) and the levels of IFN-α measured in the cell culture supernatants 24 h later. The CpG ODN 2007 was used as positive controls for human B-class ODNs. PBMC experiments were performed in three different donors, yielding similar results. (B) Proinflammatory activity of mouse B class-like CpG motifs was evaluated in inflammatory macrophages from WT (C57BL/6), TLR4 ^−/− and TLR9 ^−/− mice. ODNs were tested at different concentrations (1.5, 0.3 and 0.06 µM) and LPS, as well as CpG ODN 7909 were used as positive controls for TLR4 and TLR9 activation, respectively. IL-12 (p40) was measured in the macrophage culture supernatants 24 h after cellular stimulation. (C) C57BL/6 mice received three immunization doses with alum alone, OVA plus alum or OVA plus alum associated with either CpG ODNs B344, B287, B128 or 7909 (positive control). The levels of OVA-specific total IgG, IgG1 and IgG2c were assessed by ELISA. (D) Amount of IFN-γ secreted by splenocytes after stimulation with OVA derived CD4⁺ T or CD8⁺ T cell epitopes was evaluated in culture supernatants 72 hours post-stimulation. Asterisks indicate that differences were statistically significant, when comparing T cell response from mice receiving different vaccine formulations.

Figure 3. Mapping of immunostimulatory CD4⁺ T and CD8⁺ T cell epitopes present in NY-ESO-1.

(A) C57BL/6 mice were immunized with alum alone, alum plus rNY-ESO-1 associated or not with CpG ODN 7909 to evaluate the immunostimulatory activity of peptides encoding the putative CD4⁺ T and CD8⁺ T cell epitopes from NY-ESO-1. Mice received three immunization doses at day 0, 14 and 28. Splenocytes were harvested 21 days after the last immunization dose, restimulated in vitro with different NY-ESO-1-specific peptides, and the levels of IFN-γ production measured in the cell culture supernatants by ELISA. Asterisks indicate that differences in IFN-γ responses to a specific CD4⁺ T or CD8⁺ T cell peptides (CD8-1, CD8-3 and CD4-3) were statistically significant (p<0.001), when comparing splenocytes from mice receiving the same vaccine formulation, stimulated with CD8-2, CD4-1, CD4-2, or left unstimulated. (B) A schematic illustration shows the sequence and position of immunostimulatory CD4⁺ T and CD8⁺ T cell epitopes selected from NY-ESO-1 to be used in this study. (C) CD4⁺ T and CD8⁺ T lymphocytes were enriched from total spleen cells of immunized mice by magnetic beads. Each subpopulation was restimulated with CD4-3 and CD8-1 peptides and IFN-γ production evaluated by ELISA after 72 hours incubation.

Figure 4. Evaluation of antibody and T cell responses as well as protective immunity elicited by immunization with different formulations containing the tumor-associated NY-ESO-1 antigen.

C57BL/6 mice were subjected to three immunization doses on days 0, 14 and 28. (A) Serum levels of NY-ESO-1-specific total IgG, IgG1 and IgG2c; and (B) IFN-γ production by splenocytes stimulated with NY-ESO-1 CD4⁺ T and CD8⁺ T peptides cells were evaluated by ELISA. (C) Control and immunized mice were challenged with 5×10⁴ B16F10 melanoma cell expressing or not NY-ESO-1. The tumor growth was evaluated every 4 days for 40 days after challenge. Asterisks indicate that differences in IFN-γ responses to NY-ESO-1 CD4⁺ T and CD8⁺ T cell peptide and tumor growth are statistically significant, when comparing mice receiving different vaccine formulations.

Figure 5. Hypernociception induced by TLR agonists and the NY-ESO-1 vaccine formulations.

(A) Different TLR agonists were injected in the footpad of mice and hypernociception evaluated at the indicated time points. (B) Vaccine formulations containing alum; alum plus NY-ESO-1; or alum plus NY-ESO-1 associated with TLR agonists were given to mice that were left untreated or treated with PCM orally, 30 minutes prior injection with different vaccine formulations. Asterisks mean significant difference when comparing PBS group with TLR agonists experimental groups (P<0,05).

Figure 6. Evaluation of antigen-specific immune response after PCM administration.

C57BL/6 mice were subjected to three immunization doses on days 0, 14 and 28. Thirty minutes prior each immunization dose, a group of mice received 10 mg/Kg of PCM by the oral rout. (A) Serum levels of NY-ESO-1-specific total IgG, IgG1 and IgG2c; and (B) IFN-γ responses by splenocytes stimulated with NY-ESO-1 CD4⁺ T and CD8⁺ T cell peptides were evaluated by ELISA. (C) Control and immunized mice were challenged with 5×10⁴ B16F10 melanoma cell expressing or not NY-ESO-1. The tumor growth was evaluated every 4 days for 40 days after challenge. Asterisks indicate that differences in IFN-γ responses to NY-ESO-1 CD4⁺ T and CD8⁺ T cell peptide and curve of tumor growth are statistically significant, when comparing mice receiving different vaccine formulations. (D) The frequency of CD8⁺ T cells NY-ESO-1-specific were evaluated by flow cytometry using as marker FITC CD3, PECy5 CD8 and PE NY-ESO-1 tetramer. Are shown on the left representative dot blots of tetramer staining for each experimental group. Graph on the right shows the average (n = 4) of the percentage of CD3⁺/CD8⁺/NY-ESO-1⁺ for each experimental group.