José Ronnie Vasconcelos

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

ABSTRACT 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.

Importance of CD8 T cell-mediated immune response during intracellular parasitic infections and its implications for the development of effective vaccines

Obligatory intracellular parasites such as Plasmodium sp, Trypanosoma cruzi, Toxoplasma gondii and Leishmania sp are responsible for the infection of hundreds of millions of individuals every year. These parasites can deliver antigens to the host cell cytoplasm that are presented through MHC class I molecules to protective CD8 T cells. The in vivo priming conditions of specific CD8 T cells during natural infection are largely unknown and remain as an area that has been poorly explored. The antiparasitic mechanisms mediated by CD8 T cells include both interferon-gamma-dependent and -independent pathways. The fact that CD8 T cells are potent inhibitors of parasitic development prompted many investigators to explore whether induction of these T cells can be a feasible strategy for the development of effective subunit vaccines against these parasitic diseases. Studies performed on experimental models supported the hypothesis that CD8 T cells induced by recombinant viral vectors or DNA vaccines could serve as the basis for human vaccination. Regimens of immunization consisting of two different vectors (heterologous prime-boost) are much more efficient in terms of expansion of protective CD8 T lymphocytes than immunization with a single vector. The results obtained using experimental models have led to clinical vaccination trials that are currently underway.

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.

Heterologous Plasmid DNA Prime-Recombinant Human Adenovirus 5 Boost Vaccination Generates a Stable Pool of Protective Long-Lived CD8+ T Effector Memory Cells Specific for a Human Parasite, Trypanosoma cruzi

ABSTRACT Recently, we described a heterologous prime-boost strategy using plasmid DNA followed by replication-defective human recombinant adenovirus type 5 as a powerful strategy to elicit long-lived CD8+ T-cell-mediated protective immunity against experimental systemic infection of mice with a human intracellular protozoan parasite, Trypanosoma cruzi. In the present study, we further characterized the protective long-lived CD8+ T cells. We compared several functional and phenotypic aspects of specific CD8+ T cells present 14 or 98 days after the last immunizing dose and found the following: (i) the numbers of specific cells were similar, as determined by multimer staining or by determining the number of gamma interferon (IFN-γ)-secreting cells by enzyme-linked immunospot (ELISPOT) assay; (ii) these cells were equally cytotoxic in vivo; (iii) following in vitro stimulation, a slight decline in the frequency of multifunctional cells (CD107a+ IFN-γ+ or CD107a+ IFN-γ+ tumor necrosis factor alpha positive [TNF-α+]) was paralleled by a significant increase of CD107a singly positive cells after 98 days; (iv) the expression of several surface markers was identical, except for the reexpression of CD127 after 98 days; (v) the use of genetically deficient mice revealed a role for interleukin-12 (IL-12)/IL-23, but not IFN-γ, in the maintenance of these memory cells; and (vi) subsequent immunizations with an unrelated virus or a plasmid vaccine or the depletion of CD4+ T cells did not significantly erode the number or function of these CD8+ T cells during the 15-week period. From these results, we concluded that heterologous plasmid DNA prime-adenovirus boost vaccination generated a stable pool of functional protective long-lived CD8+ T cells with an effector memory phenotype.

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.

Adjuvant Effect of the Propionibacterium acnes and Its Purified Soluble Polysaccharide on the Immunization with Plasmidial DNA Containing a Trypanosoma cruzi Gene

In the present work we investigated the role of killed Propionibacterium acnes or a soluble polysaccharide extracted from bacterium cell wall in modulated experimental immunization with plasmidial DNA. We used a plasmid, p154/13, containing a gene‐encoding catalytic domain of Trypanosoma cruzi (T. cruzi) trans‐sialidase. As previously described, immunization of BALB/c mice with p154/13 elicited humoral, cell‐mediated and protective immune responses against T. cruzi infection. In this study we describe that both P. acnes and its soluble polysaccharide fraction have the ability to modulate the immune response elicited by p154/13. Treatment with these adjuvants enhanced specific trans‐sialidase Th1 immune response, as revealed by a lower IgG1/IgG2a ratio and stronger in vitro IFN‐γ synthesis by CD4+ T cells. The most important fact was that treatment with P. acnes or its soluble polysaccharide fraction in the presence of p154/13 significantly reduced the peak of parasitemia observed 7 to 8 days after T. cruzi challenge. These data suggest that P. acnes or its soluble polysaccharide fraction may improve the protective potential of a DNA vaccine against experimental T. cruzi infection.

A DNA-priming protein-boosting regimen significantly improves type 1 immune response but not protective immunity to Trypanosoma cruzi infection in a highly susceptible mouse strain.

BALB/c or C57Bl/6 mice immunized with plasmids containing Trypanosoma cruzi genes developed specific immune responses and protective immunity against lethal parasitic infection. In contrast, in the highly susceptible mouse strain A/Sn, DNA vaccination reduced the peak parasitemia but promoted limited mouse survival after challenge. In the present study, we tested whether the immunogenicity and protective efficacy of vaccination could be improved by combining DNA and recombinant protein immunization regimens. A/Sn mice immunized with plasmid p154/13 which harbours the gene encoding Trypanosoma cruzi trans‐sialidase developed a predominant type 1 immune response. In contrast, immunization with the recombinant Trypanosoma cruzi trans‐sialidase protein adsorbed to alum generated a typical type 2 immune response. Simultaneous administration of both p154/13 and recombinant Trypanosoma cruzi trans‐sialidase protein also led to a predominant type 2 immune response. Sequential immunization consisting of two priming doses of p154/13 followed by booster injections with recombinant Trypanosoma cruzi trans‐sialidase protein significantly improved specific type 1 immune response, as revealed by a drastic reduction of the serum IgG1/IgG2a ratio and by an increase in the in vitro interferon‐gamma secretion by CD4 T cells. Our observations confirm and extend previous data showing that a DNA‐priming protein‐boosting regimen might be a general strategy to enhance type 1 immune response to DNA vaccines. Upon challenge with Trypanosoma cruzi, no improvement in protective immunity was observed in mice immunized with the DNA‐priming protein‐boosting regimen when compared to animals that received DNA only. Therefore, our results suggest that in this experimental model there is no correlation between the magnitude of type 1 immune response and protective immunity against Trypanosoma cruzi infection.

Re-circulation of lymphocytes mediated by sphingosine-1-phosphate receptor-1 contributes to resistance against experimental infection with the protozoan parasite Trypanosoma cruzi

T-cell mediated immune responses are critical for acquired immunity against infection by the intracellular protozoan parasite Trypanosoma cruzi. Despite its importance, it is currently unknown where protective T cells are primed and whether they need to re-circulate in order to exert their anti-parasitic effector functions. Here, we show that after subcutaneous challenge, CD11c(+)-dependent specific CD8(+) T-cell immune response to immunodominant parasite epitopes arises almost simultaneously in the draining lymph node (LN) and the spleen. However, until day 10 after infection, we observed a clear upregulation of activation markers only on the surface of CD11C(+)PDCA1(+) cells present in the LN and not in the spleen. Therefore, we hypothesized that CD8(+) T cells re-circulated rapidly from the LN to the spleen. We investigated this phenomenon by administering FTY720 to T. cruzi-infected mice to prevent egress of T cells from the LN by interfering specifically with signalling through sphingosine-1-phosphate receptor-1. In T. cruzi-infected mice receiving FTY720, CD8 T-cell immune responses were higher in the draining LN and significantly reduced in their spleen. Most importantly, FTY720 increased susceptibility to infection, as indicated by elevated parasitemia and accelerated mortality. Similarly, administration of FTY720 to mice genetically vaccinated with an immunodominant parasite antigen significantly reduced their protective immunity, as observed by the parasitemia and survival of vaccinated mice. We concluded that re-circulation of lymphocytes mediated by sphingosine-1-phosphate receptor-1 greatly contributes to acquired and vaccine-induced protective immunity against experimental infection with a human protozoan parasite.

CD8(+) T cell-mediated immunity during Trypanosoma cruzi infection: a path for vaccine development?

MHC-restricted CD8+ T cells are important during infection with the intracellular protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. Experimental studies performed in the past 25 years have elucidated a number of features related to the immune response mediated by these T cells, which are important for establishing the parasite/host equilibrium leading to chronic infection. CD8+ T cells are specific for highly immunodominant antigens expressed by members of the trans-sialidase family. After infection, their activation is delayed, and the cells display a high proliferative activity associated with high apoptotic rates. Although they participate in parasite control and elimination, they are unable to clear the infection due to their low fitness, allowing the parasite to establish the chronic phase when these cells then play an active role in the induction of heart immunopathology. Vaccination with a number of subunit recombinant vaccines aimed at eliciting specific CD8+ T cells can reverse this path, thereby generating a productive immune response that will lead to the control of infection, reduction of symptoms, and reduction of disease transmission. Due to these attributes, activation of CD8+ T lymphocytes may constitute a path for the development of a veterinarian or human vaccine.

Adenovirus Vector-Induced CD8(+) T Effector Memory Cell Differentiation and Recirculation, But Not Proliferation, Are Important for Protective Immunity Against Experimental Trypanosoma cruzi Infection

Heterologous prime-boost vaccination using plasmid DNA followed by replication-defective adenovirus vector generates a large number of specific CD8⁺ T effector memory (TEM) cells that provide long-term immunity against a variety of pathogens. In the present study, we initially characterized the frequency, phenotype, and function of these T cells in vaccinated mice that were subjected to infectious challenge with the human protozoan parasite Trypanosoma cruzi. We observed that the frequency of the specific CD8⁺ T cells in the spleens of the vaccinated mice increased after challenge. Specific TEM cells differentiated into cells with a KLRG1(High) CD27(Low) CD43(Low) CD183(Low)T-bet(High) Eomes(Low) phenotype and capable to produce simultaneously the antiparasitic mediators IFNγ and TNF. Using the gzmBCreERT2/ROSA26EYFP transgenic mouse line, in which the cells that express Granzyme B after immunization, are indelibly labeled with enhanced yellow fluorescent protein, we confirmed that CD8⁺ T cells present after challenge were indeed TEM cells that had been induced by vaccination. Subsequently, we observed that the in vivo increase in the frequency of the specific CD8⁺ T cells was not because of an anamnestic immune response. Most importantly, after challenge, the increase in the frequency of specific cells and the protective immunity they mediate were insensitive to treatment with the cytostatic toxic agent hydroxyurea. We have previously described that the administration of the drug FTY720, which reduces lymphocyte recirculation, severely impairs protective immunity, and our evidence supports the model that when large amounts of antigen-experienced CD8⁺ TEM cells are present after heterologous prime-boost vaccination, differentiation, and recirculation, rather than proliferation, are key for the resultant protective immunity.