Search

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.

ABSTRACTRecently, 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 vitrostimulation, 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.

In the present work we investigated the role of killed Propionibacterium acnesor 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. cruziinfection. In this study we describe that both P. acnesand 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 vitroIFN‐γ synthesis by CD4+T cells. The most important fact was that treatment with P. acnesor its soluble polysaccharide fraction in the presence of p154/13 significantly reduced the peak of parasitemia observed 7 to 8 days after T. cruzichallenge. These data suggest that P. acnesor its soluble polysaccharide fraction may improve the protective potential of a DNA vaccine against experimental T. cruziinfection.

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.

ABSTRACTWe 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 cruziinfection. 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. cruziinfection.