Immunopeptidomic MHC-I profiling and immunogenicity testing identifies Tcj2 as a new Chagas disease mRNA vaccine candidate

Abstract

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease. Globally 6 to 7 million people are infected by this parasite of which 20-30% will progress to develop Chronic Chagasic Cardiomyopathy (CCC). Despite its high disease burden, no clinically approved vaccine exists for the prevention or treatment of CCC. Developing vaccines that can stimulate T. cruzi-specific CD8+ cytotoxic T cells and eliminate infected cells requires targeting parasitic antigens presented on major histocompatibility complex-I (MHC-I) molecules. We utilized mass spectrometry-based immunopeptidomics to investigate which parasitic peptides are displayed on MHC-I of T. cruzi infected cells. Through duplicate experiments, we identified an array of unique peptides that could be traced back to 17 distinct T. cruzi proteins. Notably, six peptides were derived from Tcj2, a trypanosome chaperone protein and member of the DnaJ (heat shock protein 40) family, showcasing its potential as a viable candidate vaccine antigen with cytotoxic T cell inducing capacity. Upon testing Tcj2 as an mRNA vaccine candidate in mice, we observed a strong memory cytotoxic CD8+ T cell response along with a Th1-skewed humoral antibody response. In vitro co-cultures of T. cruzi infected cells with splenocytes of Tcj2-immunized mice restricted the replication of T. cruzi, demonstrating the protective potential of Tcj2 as a vaccine target. Moreover, antisera from Tcj2-vaccinated mice displayed no cross-reactivity with DnaJ in lysates from mouse and human indicating a decreased likelihood of triggering autoimmune reactions. Our findings highlight how immunopeptidomics can identify new vaccine targets for Chagas disease, with Tcj2 emerging as a promising new mRNA vaccine candidate.

Fig 1. Immunopeptodomics identifies which peptides derived from T. cruzi antigens can be detected by CD8+ cytotoxic T cells (CTLs).

After a T. cruzi trypomastigote invades a host cell, it enters the cytosol and transforms into the amastigote stage. During this process, several parasitic proteins become available for antigen processing by the proteasome followed by peptide presentation on MHC-I. This presentation can activate CTLs which secrete granules and cytokines to clear the infected cells. Immunopeptidomics will help us understand which proteins become available for the antigen processing and presentation machinery. Red encircled is the key event where immunopeptidomics is used to learn information on which peptides are presented. Created with Biorender.com.

Fig 2. Isolation and identification of the peptides presented on MHC-I of T. cruzi infected MC57G fibroblast cells.

A) Schematic overview of the immunopeptidomics workflow. 1: MC57G murine fibroblasts were co-cultured for 48 hours with T. cruzi Tulahuen trypomastigotes. 2: Infected fibroblasts were harvested and lysed using a non-denaturing lysis buffer. 3: Immuno-affinity purification column was prepared by covalently linking MHC-I–specific mAbs (M1/42) to AminoLink Plus resin. 4: Lysed fibroblast material was loaded on the column, allowing the peptide-loaded MHC-I complexes to bind. 5: Four different wash buffers were used to remove the non-specific components. 6: Acetic acid was used to elute the peptide—MHC-I complexes from the column and dissociate the peptide from the MHC-I. 7: A spin filter column separated the peptide fraction from the MHC-I molecules. 8: Peptides were concentrated using a speedvac concentrator. 9: concentrated peptides were analyzed using LC-MS/MS for their sequence. 10: using the T. cruzi proteome from UniProt T. cruzi proteins were identified. Mouse self-proteins were identified using the C57BL/6J proteome. Figure prepared with Biorender.com. B) T. cruzi infected and non-infected MC57G fibroblasts, as well as green monkey kidney (VERO) cells were stained with anti-mouse MHC-I Alexa Fluor 488. Cells were analyzed by flow cytometry and data was reported as median fluorescent intensity (MFI). Mean and standard deviation are shown and were calculated from six technical replicates. Statistical significance: **: p < 0.01. C) Number of MHC-I binding peptides identified from T. cruzi or mouse for each experiment. D) Distribution of the peptide length for each experiment.

Fig 3. List of all T. cruzi peptides presented on MHC-I and their source proteins.

The 24 unique peptides traced back to 17 T. cruzi proteins. The identified heat shock protein DnaJ (protein group 2) is one of 5 DnaJ (heat shock protein 40) proteins described for T. cruzi that is identified in literature as Tcj2.

Fig 4. In vitro evaluation of Tcj2 mRNA showed the translation and antigen presentation of the mRNA construct, as well as the activation of SIINFEKL-specific CD8+ T cells.

DC2.4 cells were transfected with mRNA with Lipofectamine MessengerMAX, or without mRNA (transfection control). After a 24-hour incubation, cells were subjected to analysis. A) Schematic representation of Tcj2 mRNA construct. B) Cell viability measured after transfection. C) Detection of the translated FLAG-tag sequence by intracellular flow cytometry staining using a FLAG-specific antibody. D) Presentation of SIINFEKL on surface MHC-I (H-2K^b) measured by flow cytometry using an antibody specific for the combination of SIINFEKL presented by H-2K^b. E) Cytokines secreted by C57BL/6J “normal” or SIINFEKL specific OT-1 CD8+ T cells after co-culture with transfected cells. DC2.4 cells were transfected for 24 hours and then counted and seeded. Splenocytes were added in a ratio of 1:10 (DC2.4: splenocytes) and the co-culture was incubated for 24 hours before the supernatant was collected and analyzed for cytokines by Luminex. Figure prepared with Biorender.com. From all experiments, mean and standard deviations are shown from triplicate experiments.

Fig 5. Freeze/thawed Tcj2 mRNA LNPs showed desired diameter, polydispersity index and pKa range for immunogenicity studies.

A) Tcj2 mRNA LNPs and empty LNPs were analyzed by Dynamic Light Scattering (DLS), directly after formulation and after storage at -80° C. Diameter in nm (Z-Ave (d.nm)) and polydispersity index (PdI) of LNPs were reported. B) Distribution of the size of the Tcj2 mRNA LNPs and empty LNPs. C) pKa of the surface of Tcj2 mRNA LNPs was analysed though a TNS fluorescence assay.

Fig 6. Tcj2 mRNA LNPs elicited humoral and cellular immune responses in a mouse immunogenicity study.

A) Schematic description of mouse model, study groups and immunization schedule. Figure prepared with Biorender.com. B) SIINFEKL-specific CD8+ T cells measured by SIINFEKL tetramer staining. C) Tcj2-specific IgG, IgG1 and IgG2c was measured using an indirect ELISA coated with rTcj2 protein. The baseline antibody titer was set at 67 and is highlighted by the dotted line. D) Antigen-specific activation of T cells was measured after in vitro restimulation with rTcj2 protein by analyzing CD25 late activation marker expression. E) Cytokines and cytolytic compounds produced by CD8+ T cells after in vitro restimulation with rTcj2 protein. F) Cytokines produced by γδ T cells after in vitro restimulation with rTcj2 protein. For data presented in D, E and F: values from non-stimulated cells were subtracted from rTcj2 protein stimulated cells to obtain antigen-specific cytokine production. Statistical significance: *: p < 0.05, **: p < 0.01, ***: p < 0.001. For panel B, D, E and F the mean and standard deviation are shown, while for C the geometric mean with 95% confidence intervals in shown. Filled symbol shapes represent in vivo study #1, while open symbol shapes represent repeat in vivo study #2.

Fig 7. Tcj2 mRNA LNP immunizations induced antigen-specific cytokine production in central memory and effector memory CD8+ T cells, as well as increase in cytokine producing polyfunctional CD8+ T cells.

A) antigen-specific central memory (CD62high and CD44high) and effector memory (CD62low and CD44high) CD8+ T cells were analyzed for production of granzyme B, perforin, IFN-γ or TNF-α. B) Antigen-specific CD8+ T cells were analyzed for the production of two or more cytolytic enzymes or cytokines using a Boolean combination gate strategy. For data presented in A and B: values from non-stimulated cells were subtracted from rTcj2 protein stimulated cells to obtain antigen-specific cytokine production. Statistical significance: *: p < 0.05, **: p < 0.01. For all panels in this figure the mean and standard deviation are shown. Filled symbol shapes represent in vivo study #1, while open symbol shapes represent repeat in vivo study #2.

Fig 8. Splenocytes from Tcj2 mRNA LNP—Immunized mice decrease T. cruzi infection in vitro.

T. cruzi–infected MC57G mouse fibroblasts were co-cultured with splenocytes from immunized mice for 72 hours. T. cruzi parasites expressed the LacZ gene which encodes for the enzyme β-galactosidase, used to convert a substrate which directly correlated to the parasite load in each well. T. cruzi–infected fibroblasts treated with a high dose of benznidazole were run in parallel and used to calculate the %-reduction of parasite load. Statistical significance: **: p < 0.0001. Mean and standard deviation are shown. Filled symbol shapes represent in vivo study #1, while open symbol shapes represent repeat in vivo study #2.

Fig 9. Flow cytometry fluorophore-conjugated antibodies and viability dye used for the experiment.