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The aim of this study was to evaluate the efficacy of vaccine using replication-deficient human recombinant Type 5 replication-defective adenoviruses (AdHu5) carrying sequences of the amastigote surface protein 2 (ASP2) (AdASP2) in mice infected with the Trypanosoma cruzi ( T cruzi) Y strain. A total of 16 A/Sn mice female were distributed into four groups, as follows (n = 4 per group): Group 1 - Control Group (CTRL); Group 2 - Infected Group (TC): animals were infected by subcutaneous route with 150 bloodstream trypomastigotes of T cruzi Y strain; Group 3 - Immunized Group (AdASP-2): animals were immunized by intramuscular injection (im) route with 50 µL of AdSP-2 (2 × 10 8 plaque forming units [pfu]/cam) at day 0; Group 4-Immunized and Infected Group (AdASP-2+TC): animals were immunized by im route with 50 µL of ASP-2 (2 × 10 8  pfu/cam) and infected by T cruzi at the same day (day 0). It was observed a significant decrease of nests in the group that was immunized with AdASP-2 and infected on the same day. Tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) gene expressions showed a significant increase in the AdASP-2+TC group when compared to TC group, but it was noted that Cyclooxygenase-2 (Cox-2) was increased in TC group when compared to AdASP-2+TC group. Increase of matrix metalloproteinases-2 (MMP-2) and decrease of MMP-9 immunoexpression in the AdASP-2+TC group was noticed as well. Oxidative DNA damage was present in myocardium for AdASP-2+TC group as a result of 8-hydroxydeoxyguanosine immunoexpression. Taken together, our results highlighted an increased oxidative stress, MMP-2 activity and inflammatory host response promoted by AdASP-2 against T cruzi infection., (© 2018 Wiley Periodicals, Inc.)

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2Kb-restricted cytotoxic and interferon (IFN)γ-producing CD8+ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a+ CD8+ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ+ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas' heart disease.

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.

In earlier studies, we reported that a heterologous prime-boost regimen using recombinant plasmid DNA followed by replication-defective adenovirus vector, both containing Trypanosoma cruzi genes encoding trans-sialidase (TS) and amastigote surface protein (ASP) 2, provided protective immunity against experimental infection with a reticulotropic strain of this human protozoan parasite. Herein, we tested the outcome of genetic vaccination of F1 (CB10XBALB/c) mice challenged with myotropic parasite strains (Brazil and Colombian). Initially, we determined that the coadministration during priming of a DNA plasmid containing the murine IL-12 gene improved the immune response and was essential for protective immunity elicited by the heterologous prime-boost regimen in susceptible male mice against acute lethal infections with these parasites. The prophylactic or therapeutic vaccination of resistant female mice led to a drastic reduction in the number of inflammatory infiltrates in cardiac and skeletal muscles during the chronic phase of infection with either strain. Analysis of the electrocardiographic parameters showed that prophylactic vaccination reduced the frequencies of sinus arrhythmia and atrioventricular block. Our results confirmed that prophylactic vaccination using the TS and ASP-2 genes benefits the host against acute and chronic pathologies caused by T. cruzi and should be further evaluated for the development of a veterinary or human vaccine against Chagas disease.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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-2(a) 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.

A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4(+) and CD8(+) T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4(+) and CD8(+) T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-gamma) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8(+) T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-gamma or IFN-gamma/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-gamma in the presence of highly cytotoxic T cells. Vaccinated IFN-gamma-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-gamma in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy.

Immunisation with Amastigote Surface Protein 2 (asp-2) and trans-sialidase (ts) genes induces protective immunity in highly susceptible A/Sn mice, against infection with parasites of the Y strain of Trypanosoma cruzi. Based on immunological and biological strain variations in T. cruzi parasites, our goal was to validate our vaccination results using different parasite strains. Due to the importance of the CD8(+) T cells in protective immunity, we initially determined which strains expressed the immunodominant H-2K(k)-restricted epitope TEWETGQI. We tested eight strains, four of which elicited immune responses to this epitope (Y, G, Colombian and Colombia). We selected the Colombian and Colombia strains for our studies. A/Sn mice were immunised with different regimens using both T. cruzi genes (asp-2 and ts) simultaneously and subsequently challenged with blood trypomastigotes. Immune responses before the challenge were confirmed by the presence of specific antibodies and peptide-specific T cells. Genetic vaccination did not confer protective immunity against acute infection with a lethal dose of the Colombian strain. In contrast, we observed a drastic reduction in parasitemia and a significant increase in survival, following challenge with an otherwise lethal dose of the Colombia strain. In many surviving animals with late-stage chronic infection, we observed alterations in the heart's electrical conductivity, compared to naive mice. In summary, we concluded that immunity against T. cruzi antigens, similar to viruses and bacteria, may be strain-specific and have a negative impact on vaccine development.

Interference or competition between CD8(+) T cells restricted by distinct MHC-I molecules can be a powerful means to establish an immunodominant response. However, its importance during infections is still questionable. In this study, we describe that following infection of mice with the human pathogen Trypanosoma cruzi, an immunodominant CD8(+) T cell immune response is developed directed to an H-2K(b)-restricted epitope expressed by members of the trans-sialidase family of surface proteins. To determine whether this immunodominance was exerted over other non-H-2K(b)-restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2K(b)-, H-2K(k)-, or H-2K(d)-restricted CD8(+) T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2K(b)-restricted immunodominant epitope. In contrast, H-2K(k)- or H-2K(d)-restricted immune responses were significantly impaired in heterozygote infected mice when compared with homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi Ags. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8(+) T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism.

Protection against protozoan parasite Trypanosoma cruzi has been shown to be dependent on the induction of type 1 immune responses. Replication-deficient human type 5 recombinant adenoviruses have an unsurpassed ability to induce type 1 immune responses. Thus, we constructed two type 5 recombinant adenoviruses encoding parasite antigens trans-sialidase (rAdTS) and amastigote surface protein-2 (rAdASP2). Both antigens were genetically engineered to secrete recombinant products in order to induce both optimal antibody and T cell responses. Immunizations of mice with rAdASP2 and rAdTS induced high levels of serum antibodies specific for their recombinant products. In addition, both recombinant viruses were able to elicit a biased helper T cell type 1 (Th1) cellular immune response and a substantial CD8+ T cell-mediated immune response. Moreover, individual immunization with rAdASP2 or rAdTS induced high levels of protection against a challenge with live parasites. CD8+ T cells mediated, at least in part, such protection. Furthermore, when combined in the same inoculum, rAdTS plus rAdASP2 induced complete protection in all animals tested, even when challenges were performed 14 weeks after the last immunization. Taking together, these results show that recombinant adenoviruses expressing TS and ASP-2 antigens of T. cruzi are interesting candidates for the development of a vaccine against Chagas' disease.

Old yellow enzyme (OYE) is a NAD(P)H flavin oxidoreductase that in Trypanosoma cruzi (TcOYE) catalyzes prostaglandin PGF2alpha synthesis and reduction of some trypanocidal drugs. We performed DNA microarray analysis and it revealed that the levels of transcription of the TcOYE gene were six-fold lower in a T. cruzi population with in vitro-induced resistance to benznidazole (BZ) (17LER) than in the wild-type (17WTS). Further we investigated the TcOYE levels in 15 T. cruzi strains and clones that were either susceptible or naturally resistant to BZ and nifurtimox, or had in vivo-selected resistance to BZ. Northern blot and real-time RT-PCR analyses confirmed our finding that TcOYE transcription levels were lower in 17LER than in 17WTS. In contrast, we detected no differences in TcOYE transcription levels between other T. cruzi samples. All T. cruzi strains contained four copies of TcOYE gene, except 17LER that contained only one. A 42kDa TcOYE protein was detected in all T. cruzi strains tested. The expression of this protein was similar for all samples, with the exception of 17LER for which the protein was nearly seven-fold less expressed. The chromosomal location of the TcOYE gene and the polymorphisms detected in TcOYE nucleotide and amino acid sequences of the T. cruzi strains are associated with the zymodeme but not with drug-resistance phenotype. Our data show that one of the mechanisms conferring in vitro-induced BZ resistance to T. cruzi correlates with deletion of copies of the TcOYE gene. In contrast, the in vivo and natural resistance to BZ are mediated by different mechanisms.

Integrins mediate the lymphocyte migration into an infected tissue, and these cells are essential for controlling the multiplication of many intracellular parasites such as Trypanosoma cruzi, the causative agent of Chagas disease. Here, we explore LFA-1 and VLA-4 roles in the migration of specific CD8+ T cells generated by heterologous prime-boost immunization during experimental infection with T. cruzi. To this end, vaccinated mice were treated with monoclonal anti-LFA-1 and/or anti-VLA-4 to block these molecules. After anti-LFA-1, but not anti-VLA-4 treatment, all vaccinated mice displayed increased blood and tissue parasitemia, and quickly succumbed to infection. In addition, there was an accumulation of specific CD8+ T cells in the spleen and lymph nodes and a decrease in the number of those cells, especially in the heart, suggesting that LFA-1 is important for the output of specific CD8+ T cells from secondary lymphoid organs into infected organs such as the heart. The treatment did not alter CD8+ T cell effector functions such as the production of pro-inflammatory cytokines and granzyme B, and maintained the proliferative capacity after treatment. However, the specific CD8+ T cell direct cytotoxicity was impaired after LFA-1 blockade. Also, these cells expressed higher levels of Fas/CD95 on the surface, suggesting that they are susceptible to programmed cell death by the extrinsic pathway. We conclude that LFA-1 plays an important role in the migration of specific CD8+ T cells and in the direct cytotoxicity of these cells. [ABSTRACT FROM AUTHOR]

The β1i, β2i and β5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8+ T cells and IFN-γ (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi-infected β1i, β2i and β5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8+ effector T cells (CD8+CD44highCD62Llow) specific for the previously characterized immunodominant (VNHRFTLV) H-2Kb-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8+ T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-γ+/TNF+) or single-positive (IFN-γ+) cells specific for the H-2Kb-restricted immunodominant as well as subdominant T. cruzi epitopes were higher in WT mice, whereas TNF single-positive cells prevailed among CD8+ T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8+ T cell responses. [ABSTRACT FROM AUTHOR]

Background: Immunization of mice with the Trypanosoma cruzi trans-sialidase (TS) gene using plasmid DNA, adenoviral vector, and CpG-adjuvanted protein delivery has proven highly immunogenic and provides protection against acute lethal challenge. However, long-term protection induced by TS DNA vaccines has not been reported. The goal of the present work was to test whether the co-administration of a plasmid encoding IL-15 (pIL-15) could improve the duration of protection achieved through genetic vaccination with plasmid encoding TS (pTS) alone. Methodology: We immunized BALB/c mice with pTS in the presence or absence of pIL-15 and studied immune responses [with TS-specific IFN-γ ELISPOT, serum IgG ELISAs, intracellular cytokine staining (IFN-γ, TNF-α, and IL-2), tetramer staining, and CFSE dilution assays] and protection against lethal systemic challenge at 1 to 6 months post vaccination. Mice receiving pTS alone developed robust TS-specific IFN-γ responses and survived a lethal challenge given within the first 3 months following immunization. The addition of pIL-15 to pTS vaccination did not significantly alter T cell responses or protection during this early post-vaccination period. However, mice vaccinated with both pTS and pIL-15 challenged 6 months post-vaccination were significantly more protected against lethal T. cruzi challenges than mice vaccinated with pTS alone (P<0.05). Improved protection correlated with significantly higher numbers of TS-specific IFN-γ producing total and CD8+ T cells detected>6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro re-stimulation. Conclusion: Addition of pIL-15 during genetic vaccination greatly improved long-term T cell survival, memory T cell expansion, and long-term protection against the important human parasite, T. cruzi. Author Summary: Over 11 million individuals are infected with Trypanosoma cruzi, the causative agent of Chagas disease, which kills >50,000 people annually. Although recent vector control efforts and increased use and effectiveness of chemotherapeutic drugs including benznidazole have reduced infection rates and mortality, a safe, effective vaccine is needed. Vaccination with the T. cruzi trans-sialidase (TS) has been used effectively in mice to reduce mortality and chronic disease, however, the establishment of vaccine-induced long-term protective immunity remains elusive. Co-immunization strategies utilizing immune regulators such as interleukin-12 (IL-12) and interleukin-15 (IL-15) can be used to enhance antigen-specific T cell responses and prolong protective immunity. In the present report, we show that genetic vaccination of BALB/c mice with plasmid DNA encoding both TS and IL-15 compared with plasmid DNA encoding TS alone significantly enhanced CD4+ and CD8+ T cell responses including increased TNF-α, IFN-γ, and IL-2 production, and long-term protection against lethal systemic parasite challenge. [ABSTRACT FROM AUTHOR]