Trypanosoma cruzi as an effective cancer antigen delivery vector

Abstract

One of the main challenges in cancer research is the development of vaccines that induce effective and long-lived protective immunity against tumors. Significant progress has been made in identifying members of the cancer testis antigen family as potential vaccine candidates. However, an ideal form for antigen delivery that induces robust and sustainable antigen-specific T-cell responses, and in particular of CD8(+) T lymphocytes, remains to be developed. Here we report the use of a recombinant nonpathogenic clone of Trypanosoma cruzi as a vaccine vector to induce vigorous and long-term T cell-mediated immunity. The rationale for using the highly attenuated T. cruzi clone was (i) the ability of the parasite to persist in host tissues and therefore to induce a long-term antigen-specific immune response; (ii) the existence of intrinsic parasite agonists for Toll-like receptors and consequent induction of highly polarized T helper cell type 1 responses; and (iii) the parasite replication in the host cell cytoplasm, leading to direct antigen presentation through the endogenous pathway and consequent induction of antigen-specific CD8(+) T cells. Importantly, we found that parasites expressing a cancer testis antigen (NY-ESO-1) were able to elicit human antigen-specific T-cell responses in vitro and solid protection against melanoma in a mouse model. Furthermore, in a therapeutic protocol, the parasites expressing NY-ESO-1 delayed the rate of tumor development in mice. We conclude that the T. cruzi vector is highly efficient in inducing T cell-mediated immunity and protection against cancer cells. More broadly, this strategy could be used to elicit a long-term T cell-mediated immunity and used for prophylaxis or therapy of chronic infectious diseases.

Fig. 1.

Expression of recombinant NY-ESO-1 by stably transfected T. cruzi parasites. (A) Genomic DNA from WT and transgenic parasites was digested with BamHI (B) or SacI (S), submitted to Southern blot, and hybridized with NY-ESO-1 or amastin probes. (B) Western blot analysis was used to detect NY-ESO-1 expression, in metacyclic lysates (lanes 1–3 and 5) or supernatants (lane 4) of parasite cultures, using anti-NY-ESO-1 or anti-His tag mAbs. (C) NY-ESO-1 expression by the four different stages of the CL-14-NY-ESO-1His(+) was analyzed by immunofluorescence. Parasites stained with anti-NY-ESO-1 mAb (green), or DAPI for DNA, were visualized by differential interference contrast (DIC).

Fig. 2.

Priming with transgenic parasites elicits IFN-γ–producing T cells specific for NY-ESO-1. (A) A melanoma cell line (SK-MEL-149) that does not express NY-ESO-1 was infected with either WT or transgenic parasites and expression of rNY-ESO-1 detected by immunohistochemistry. SK-MEL-52 cells were used as positive control. (B) CD8⁺ T as well as CD4⁺ T cells from healthy donors (NC235 and NC236) were cocultured with autologous APCs infected with WT or transgenic parasites expressing different forms of rNY-ESO-1. Induction of antigen-specific T cells was analyzed by quantifying the number of IFN-γ–producing cells by ELISPOT after restimulation with autologous EBV-B cells pulsed with peptides or infected with rFowlpox virus, as indicated. (C) The frequency of NY-ESO-1–specific CD8⁺ T cells was determined by flow cytometry. Percentages indicate the frequency of CD8⁺ T cells stained with NY-ESO-1 tetramer.

Fig. 3.

Antigen-specific humoral and cellular responses and complete protection induced by immunization with transgenic parasites expressing NY-ESO-1. (A) ELISA plates coated with rNY-ESO-1 were used to quantify the levels of NY-ESO-1–specific total IgG, IgG1, and IgG2c isotypes present in sera of control and immunized mice; Western blot membranes containing rNY-ESO-1 were individually incubated with sera of mice immunized with each of the three transgenic or WT parasites, or the anti-NY-ESO-1 mAb. (B) Splenocytes from control or immunized mice were cultured in the presence or absence of NY-ESO-1 peptides encoding epitopes specific for CD4⁺ T or CD8⁺ T cells. As positive control we used a T. cruzi–derived immunodominant epitope named TSKB18. IFN-γ production was measured at 72 h after stimulation by a sandwich ELISA. Control and immunized mice were challenged with 5 × 10⁴ B16F10 melanoma cells expressing or not NY-ESO-1 (n = 4). Tumor growth (C) and survival (D) were measured for 90 d (n = 6).

Fig. 4.

Therapeutic protocols using transgenic parasites delay the growth of tumor cells expressing NY-ESO-1. 1 × 10⁶ cells of the fibrosarcoma CMS5a (A and B) expressing or not rNY-ESO-1 were used to challenge BALB/c mice (n = 6). Mice were treated with three doses of 10⁷ metacyclic forms of WT (CL-14) or transgenic parasites given 5 d apart (arrows), starting at day 5 after challenge. Tumor growth (A) and survival (B) were monitored for 40 and 90 d, respectively.

Fig. 5.

MyD88- and IL-12–dependent induction of IFN-γ–producing T cells and protective immunity elicited by immunization with CL-14 expressing NY-ESO-1. (A) Splenocytes from vaccinated WT (C57BL/6) and KO mice (MyD88^−/−, IL-12^−/−, iNOs^−/−, and CD8^−/−) were restimulated with T CD4⁺- or T CD8⁺-specific peptides or with rNY-ESO-1 protein (n = 4). IFN-γ production was measured by ELISA at 72 h after stimulation. (B) Before restimulation, the splenocytes were stained with anti-CD3, anti-CD8, and NY-ESO-1 or TSKB20 tetramers and analyzed by flow cytometry. Representative dot blots and a graph summarizing the percentage of double-positive cells are shown at Left and Right, respectively. (C) Immunized C57BL/6 and KO mice were challenged with 5 × 10⁴ of the B16F10 melanoma cells expressing the NY-ESO-1 and monitored 40 d for tumor growth (n = 6).