Subdominant/Cryptic CD8 T Cell Epitopes Contribute to Resistance against Experimental Infection with a Human Protozoan Parasite

During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8+ T-Cell Response: Reversal by Adenoviral Vaccine

MHC class Ia-restricted CD8+ T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8+ T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8+ T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8+ T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8+ T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8+ cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8+ T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination

Swimming Against the Current: Genetic Vaccination Against Trypanosoma Cruzi Infection in Mice

Vaccines have had an unquestionable impact on public health during the last century. The most likely reason for the success of vaccines is the robust protective properties of specific antibodies. However, antibodies exert a strong selective pressure and many microorganisms, such as the obligatory intracellular parasite Trypanosoma Cruzi , have been selected to survive in their presence. Although the host develops a strong immune response to T. cruzi, they do not clear the infection and instead progress to the chronic phase of the disease. Parasite persistence during the chronic phase of infection is now considered the main factor contributing to the chronic symptoms of the disease. Based on this finding, containment of parasite growth and survival may be one method to avoid the immunopathology of the chronic phase. In this context, vaccinologists have looked over the past 20 years for other immune effector mechanisms that could eliminate these antibody-resistant pathogens. We and others have tested the hypothesis that non-antibody-mediated cellular immune responses (CD4+ Th1 and CD8+ Tc1 cells) to specific parasite antigens/genes expressed by T. cruzi could indeed be used for the purpose of vaccination. This hypothesis was confirmed in different mouse models, indicating a possible path for vaccine development

CD8(+)-T-Cell-Dependent Control of Trypanosoma cruzi Infection in a Highly Susceptible Mouse Strain after Immunization with Recombinant Proteins Based on Amastigote Surface Protein 2

We previously described that DNA vaccination with the gene encoding amastigote surface protein 2 (ASP-2) protects approximately 65% of highly susceptible A/Sn mice against the lethal Trypanosoma cruzi infection. Here, we explored the possibility that bacterial recombinant proteins of ASP-2 could be used to improve the efficacy of vaccinations. Initially, we compared the protective efficacy of vaccination regimens using either a plasmid DNA, a recombinant protein, or both sequentially (DNA priming and protein boosting). Survival after the challenge was not statistically different among the three mouse groups and ranged from 53.5 to 75%. The fact that immunization with a recombinant protein alone induced protective immunity revealed the possibility that this strategy could be pursued for vaccination. We investigated this possibility by using six different recombinant proteins representing distinct portions of ASP-2. The vaccination of mice with glutathione S-transferase fusion proteins representing amino acids 261 to 500 or 261 to 380 of ASP-2 in the presence of the adjuvants alum and CpG oligodeoxynucleotide 1826 provided remarkable immunity, consistently protecting 100% of the A/Sn mice. Immunity was completely reversed by the in vivo depletion of CD8(+) T cells, but not CD4(+) T cells, and was associated with the presence of CD8(+) T cells specific for an epitope located between amino acids 320 and 327 of ASP-2. We concluded that a relatively simple formulation consisting of a recombinant protein with a selected portion of ASP-2, alum, and CpG oligodeoxynucleotide 1826 might be used to cross-prime strong CD8(+)-T-cell-dependent protective immunity against T. cruzi infection

CD8 T cell dependence of protective immunity of A/Sn mice immunized with <i>asp-2</i> using the heterologous DNA prime-adenovirus boost vaccination regimen.

<p>A/Sn mice were immunized as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022011#pone-0022011-g005" target="_blank">Fig. 5</a>. pIgSPTAWETGQA/AdTAWTEGQA had significantly lower parasitemia (<i>P</i><0.01) than pcDNA3/Adβ-gal-injected animals. Before and after challenge, mice were treated as described in Methods section with rat IgG (control) or anti-CD8 MAb. The parasitemia for each mouse group is represented as mean ± SD (n = 6). Asterisks denote that mice from groups immunized with pIgSPCl.9/AdASP-2 or pIgSPTAWETGQA/AdTAWTEGQA and treated with Rat IgG had significantly lower parasitemia (<i>P</i><0.01) than vaccinated mice treated with anti-CD8 (Panels A and B). Panels C and D represent Kaplan-Meier curves for survival of the mouse groups immunized and challenged as described above (n = 6). Mice immunized with pIgSPCl.9/AdASP-2 or pIgSPTAWETGQA/AdTAWETGQA and treated with Rat IgG survived significantly longer than vaccinated animals treated with anti-CD8 (<i>P</i><0.01 in both cases).</p

Trypomastigote-induced parasitemia and mortality in A/Sn mice immunized with <i>asp-2</i> using the heterologous DNA prime-adenovirus boost vaccination regimen.

<p>A/Sn mice were immunized as depicted in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022011#pone-0022011-g005" target="_blank">Fig. 5</a>. Two weeks after the final immunizing dose, mice were challenged i.p. (Panels A and B) or s.c. (Panels C and D) with 150 bloodstream trypomastigotes. Parasitemia for each mouse group is represented as mean ± SD (n = 10 or 11). Asterisks denote that mice from groups immunized with pIgSPCl.9/AdASP-2 or pIgSPTAWETGQA/AdTAWTEGQA had significantly lower parasitemia (<i>P</i><0.01) than pcDNA3/Adβ-gal-injected animals. Panels B and D represent Kaplan-Meier curves for survival of the mouse groups immunized and challenged as described above (n = 10 or 11). Mice immunized with pIgSPCl.9/AdASP-2 survived significantly longer than animals immunized with pIgSPTAWETGQA/AdTAWETGQA or pcDNA3/Adβ-gal (<i>P</i> = 0.01 or <i>P</i><0.01, respectively). Mice immunized with pIgSPTAWETGQA/AdTAWETGQA survived significantly longer than pcDNA3/Adβ-gal-injected animals (<i>P</i><0.01). Results are representative of two pooled experiments. No animals died after the 40^th day.</p

Genetic vectors used in the study.

<p>Genetic vectors used in the study.</p

Structure and localization of Amastigote Surface Protein-2 (ASP-2).

<p>A- Schematic view of the primary structure of <i>T. cruzi</i> ASP-2. B- HeLa cells were infected for 48 h with trypomastigotes of the Y strain. After fixation, indirect immunofluorescence or DAPI staining were performed as described using MAb K22 and imaged using fluorescence microscopy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022011#pone.0022011-Claser1" target="_blank">[39]</a>. Bar, 14 µM.</p

CD8 immune responses in A/Sn mice immunized with <i>asp-2</i> using the heterologous DNA prime-adenovirus boost vaccination regimen.

<p>A/Sn mice were primed i.m. with 100 µg of plasmids pcDNA3, pIgSPCl.9 or pIgSPTAWETGQA. Three weeks later, these mice were boosted i.m. with 2×10⁸ pfu Adβ-gal, AdASP-2 or AdTAWETGQA. A- Two weeks after the last dose, splenic cells were re-stimulated <i>in vitro</i> in the presence of medium only or the indicated peptides at a final concentration of 10 µM. The number of splenic IFN-γ spot forming cells (SFC) was estimated by <i>ex vivo</i> ELISPOT assay. B- <i>In vivo</i> cytotoxic activity was estimated by injecting each mouse with syngeneic CFSE-labeled splenic cells coated with or without 2 µM of the indicated peptide. Results are expressed as mean ± SD of 4 mice per group and are representative of experiments performed at least twice with similar results. Asterisks denote that the number of SFC or <i>in vivo</i> cytotoxicity were significantly higher when compared to SFC found in naïve or pcDNA3/Adβ-gal injected mice (<i>P</i><0.01). C- Fourteen days after the last dose, these mice had their splenic cells cultured in the presence of anti-CD28 and Medium or the indicated peptides. After 12 h, cells were stained for CD8, IFN-γ and TNF-α. Examples of splenic CD8⁺ cells from immunized mice. Representative analyses (medians) are shown from four mice performed per experiment.</p