A novel multi-epitope recombinant protein elicits an antigen-specific CD8+ T cells response in Trypanosoma cruzi-infected mice

About 6.5 million people worldwide are afflicted by Chagas disease, which is caused by the protozoan parasite Trypanosoma cruzi. The development of a therapeutic vaccine to prevent the progression of Chagasic cardiomyopathy has been proposed as an alternative for antiparasitic chemotherapy. Bioinformatics tools can predict MHC class I CD8 + epitopes for inclusion in a single recombinant protein with the goal to develop a multivalent vaccine. We expressed a novel recombinant protein Tc24-C4.10E harboring ten nonameric CD8 + epitopes and using Tc24-C4 protein as scaffold to evaluate the therapeutic effect in acute T. cruzi infection. T. cruzi-infected mice were immunized with Tc24-C4.10E or Tc24-C4 in a 50-day model of acute infection. Tc24-C4.10E-treated mice showed a decreased parasitemia compared to the Tc24-C4 (non-adjuvant) immunized mice or control group. Moreover, Tc24-C4.10E induced a higher stimulation index of CD8 + T cells producing IFNγ and IL-4 cytokines. These results suggest that the addition of the MHC Class I epitopes to Tc24-C4 can synergize the antigen-specific cellular immune responses, providing proof-of-concept that this approach could lead to the development of a promising vaccine candidate for Chagas disease

<i>Trypanosoma cruzi</i> vaccine candidate antigens Tc24 and TSA-1 recall memory immune response associated with HLA-A and -B supertypes in Chagasic chronic patients from Mexico

<div><p><i>Trypanosoma cruzi</i> antigens TSA-1 and Tc24 have shown promise as vaccine candidates in animal studies. We evaluated here the recall immune response these antigens induce in Chagasic patients, as a first step to test their immunogenicity in humans. We evaluated the <i>in vitro</i> cellular immune response after stimulation with recombinant TSA-1 (rTSA-1) or recombinant Tc24 (rTc24) in mononuclear cells of asymptomatic Chagasic chronic patients (n = 20) compared to healthy volunteers (n = 19) from Yucatan, Mexico. Proliferation assays, intracellular cytokine staining, cytometric bead arrays, and memory T cell immunophenotyping were performed by flow cytometry. Peripheral blood mononuclear cells (PBMC) from Chagasic patients showed significant proliferation after stimulation with rTc24 and presented a phenotype of T effector memory cells (CD45RA^-CCR7^-). These cells also produced IFN-γ and, to a lesser extent IL10, after stimulation with rTSA-1 and rTc24 proteins. Overall, both antigens recalled a broad immune response in some Chagasic patients, confirming that their immune system had been primed against these antigens during natural infection. Analysis of HLA-A and HLA-B allele diversity by PCR-sequencing indicated that HLA-A03 and HLA-B07 were the most frequent supertypes in this Mexican population. Also, there was a significant difference in the frequency of HLA-A01 and HLA-A02 supertypes between Chagasic patients and controls, while the other alleles were evenly distributed. Some aspects of the immune response, such as antigen-induced IFN-γ production by CD4⁺ and CD8⁺ T cells and CD8⁺ proliferation, showed significant association with specific HLA-A supertypes, depending on the antigen considered. In conclusion, our results confirm the ability of both TSA-1 and Tc24 recombinant proteins to recall an immune response induced by the native antigens during natural infection in at least some patients. Our data support the further development of these antigens as therapeutic vaccine against Chagas disease.</p></div

Antigen-specific immune network.

<p>Networks were constructed using Cytoscape to provide an integrated view of Tc24 and TSA-1 recall responses in Chagasic patients and seronegative controls. Circle nodes represent T cell populations and cytokines, with the black circle corresponding to seronegative controls, and the colored circles corresponding to the seropositive Chagasic patients. A larger size of the circle indicates activation, while a smaller size indicates an inhibition/reduction compared to the seronegative controls. Pink colors are associated with a pro-inflammatory Th1 profile, while green is associated with an anti-inflammatory immune profile. Blue reflects non-polarized immune responses. Edges link nodes showing differences between Chagasic patients and controls.</p

Quantification of Th1 and Th2 soluble cytokines.

<p>Th1 and Th2 cytokines were measured in supernatant of cultures after stimulation with TSA-1 (<b>A</b>) or Tc24 (<b>B</b>). Graphs show the concentration in pg/mL comparing seronegative control and Chagasic patients for each cytokine. N.D.: Not detected. Results include 13 Chagasic patients and 7 seronegative controls.</p

Phenotyping of CD4⁺ T memory cells.

<p>PBMC cells from seronegative controls (open circles) and Chagasic patients (black circles) were stimulated with rTc24, and phenotyped as T effector memory (T_EM), T central memory (T_CM) or T naïve (T_NAIVE) cells. Horizontal black lines indicate the mean of the group. *Indicates significant differences between seronegative and Chagasic patients (P <0.05, <i>t</i> test for parametric distribution or U Mann-Whitney for non-parametric distribution). Numbers in parenthesis indicate the sample size for each group.</p

Frequency of HLA-A and HLA-B supertypes in the study population.

<p>Frequency of HLA-A and HLA-B supertypes in the study population.</p

HLA restriction of antigen-specific immune response.

<p>The antigen-specific immune responses were analyzed according to the HLA supertypes of the study participants. Data are presented as mean ± SEM for the induction of CD8⁺ T cell proliferation (<b>A</b>), CD8⁺-IFN- γ ratio (<b>B</b>), and CD4⁺ IFN-γ ratio (<b>C</b>). *Indicates a significant difference in the antigen-specific immune response between HLA supertypes.</p

Antigen-specific proliferation assay.

<p>Stimulation index of CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells populations from seronegative controls (open circles) and Chagasic patients (Black circles), after stimulation with rTc24 (<b>A</b>) or rTSA-1 (<b>B</b>). Horizontal black lines indicate the mean. The dotted horizontal line shows the cut-off value to identify responders/non-responders. *Indicate significant differences in the proportion of responders between seronegative and Chagasic patients (<i>P</i> <0.05, Fisher test). Numbers in parenthesis indicate the sample size for each group.</p

Demographic and clinical characteristics of the study population.

<p>Demographic and clinical characteristics of the study population.</p