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A human type 5 adenovirus-based Trypanosoma cruzi therapeutic vaccine re-programs immune response and reverses chronic cardiomyopathy.

Pereira IR, Vilar-Pereira G, Marques V, da Silva AA, Caetano B, Moreira OC, Machado AV, Bruna-Romero O, Rodrigues MM, Gazzinelli RT, Lannes-Vieira J - PLoS Pathog. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia das Interações, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
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.

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rAdVax immunotherapy recovered the injured heart tissue of chronically T. cruzi-infected mice.(A) Chronically Colombian T. cruzi strain-infected mice were evaluated for heart injury markers pre-therapy (120 dpi) or primed-boosted with 2 × 108 plaque-forming units (PFU) of rAdCtrl or a mixture of 108 PFU of each adenovirus vaccine preparation (rAdASP2+rAdTS; rAdVax). Mortality was recorded until 230 dpi (110 days post-therapy; dpt), when the surviving mice were analyzed for heart injury markers. (B) Kaplan-Meier curve representing the percentages of surviving mice (14–20 mice/group in two independent experiments). (C) Relative spleen weight (mg of spleen/g of body) and quantitative immunohistochemical staining (IHS) data for T. cruzi parasitism (nests/100 microscopic fields) in the heart tissue of chronically infected mice (120 and 230 dpi, respectively, pre- and post-therapy). (D) IHS showing fibronectin (FN)-stained areas in representative cardiac tissue sections of noninfected (NI) controls and chronically T. cruzi-infected mice pre- (120 dpi) and post-therapy (230 dpi; 110 dpt) with rAdVax. (E) Quantification of the FN-stained area (%) and connexin 43 (Cx43)-containing gap junction distances detected using IHS staining of heart tissue sections of NI controls or T. cruzi-infected mice pre- and post-therapy with rAdVax. (F) Evaluation of CK-MB activity in the serum of NI controls and T. cruzi-infected mice pre- and post-therapy with rAdVax. The data are presented as the means ± SD. ** P <0.01 and ***P <0.001, experimental groups compared with NI controls. #P <0.05 and ##P <0.01, rAdVax-immunized compared with pre-therapy T. cruzi-infected mice.
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ppat.1004594.g003: rAdVax immunotherapy recovered the injured heart tissue of chronically T. cruzi-infected mice.(A) Chronically Colombian T. cruzi strain-infected mice were evaluated for heart injury markers pre-therapy (120 dpi) or primed-boosted with 2 × 108 plaque-forming units (PFU) of rAdCtrl or a mixture of 108 PFU of each adenovirus vaccine preparation (rAdASP2+rAdTS; rAdVax). Mortality was recorded until 230 dpi (110 days post-therapy; dpt), when the surviving mice were analyzed for heart injury markers. (B) Kaplan-Meier curve representing the percentages of surviving mice (14–20 mice/group in two independent experiments). (C) Relative spleen weight (mg of spleen/g of body) and quantitative immunohistochemical staining (IHS) data for T. cruzi parasitism (nests/100 microscopic fields) in the heart tissue of chronically infected mice (120 and 230 dpi, respectively, pre- and post-therapy). (D) IHS showing fibronectin (FN)-stained areas in representative cardiac tissue sections of noninfected (NI) controls and chronically T. cruzi-infected mice pre- (120 dpi) and post-therapy (230 dpi; 110 dpt) with rAdVax. (E) Quantification of the FN-stained area (%) and connexin 43 (Cx43)-containing gap junction distances detected using IHS staining of heart tissue sections of NI controls or T. cruzi-infected mice pre- and post-therapy with rAdVax. (F) Evaluation of CK-MB activity in the serum of NI controls and T. cruzi-infected mice pre- and post-therapy with rAdVax. The data are presented as the means ± SD. ** P <0.01 and ***P <0.001, experimental groups compared with NI controls. #P <0.05 and ##P <0.01, rAdVax-immunized compared with pre-therapy T. cruzi-infected mice.

Mentions: The chronic infection of C57BL/6 mice with the Colombian strain of T. cruzi induces CCC, which is characterized by heart injury with connexin-43 (Cx43) disorganization and fibronectin (FN) deposition in the cardiac tissue and increased CK-MB activity in the serum [5, 18, 20]. Therefore, we tested the capacity of the homologous prime-boost rAdVax immunotherapy to reverse heart injury. To this end, C57BL/6 mice were infected and all mice were analyzed at 120 dpi (pre-therapy), when groups were formed, and the prime-boost rAdVax immunization protocol was initiated. A group of T. cruzi-infected mice was euthanized and the tissues were collected (pre-therapy). At 160 dpi and 40 days post-therapy, all mice were analyzed for electrical alterations, boosted and analyzed at 230 dpi, 110 days post-therapy (Fig. 3A). No difference in survival rate was observed in T. cruzi-infected mice that received saline or rAdCtrl and all mice in these groups were dead at 200 dpi (Fig. 3B). At 230 dpi, 87% of rAdVax-immunized mice survived (13/15), compared with 0% of rAdCtrl-inoculated (0/14) and saline-injected (0/14) mice (Fig. 3B). The surviving mice were analyzed at 230 dpi (110 days post-therapy) for heart electrical abnormalities, sacrificed and analyzed for cardiac tissue alterations compared with mice sacrificed at 120 dpi (pre-therapy). In post-therapy rAdVax-inoculated mice (at 230 dpi), there was a significant decrease in T. cruzi-induced splenomegaly (P < 0.01). Similarly to pre-therapy (120 dpi) mice, low heart parasitism persisted in rAdVax-immunized mice (Fig. 3C). In addition, pre-therapy and post-therapy parasites were rarely detected in the circulating blood (110 ± 70.2 × 103 trypomastigotes/mL in pre-therapy vs. 22.8 ± 9.5 × 103 trypomastigotes/mL in rAdVax; P > 0.05). Nevertheless, immunotherapy with rAdVax significantly reduced FN deposition in the cardiac tissue (Fig. 3D and 3E). Furthermore, Cx43 disorganization in the cardiac intercalary discs revealed as enhanced distance of Cx43-stained junctions, a marker of cardiomyocyte injury [21], was significantly reversed in mice treated with rAdVax compared with the pre-therapy condition (Fig. 3E). In addition, levels of CK-MB activity in the serum were lower in post-therapy mice compared with pre-therapy mice (Fig. 3F). Thus, these data support that immunotherapy with rAdVax during chronic T. cruzi infection ameliorated electrical abnormalities and recovered heart tissue injury.


A human type 5 adenovirus-based Trypanosoma cruzi therapeutic vaccine re-programs immune response and reverses chronic cardiomyopathy.

Pereira IR, Vilar-Pereira G, Marques V, da Silva AA, Caetano B, Moreira OC, Machado AV, Bruna-Romero O, Rodrigues MM, Gazzinelli RT, Lannes-Vieira J - PLoS Pathog. (2015)

rAdVax immunotherapy recovered the injured heart tissue of chronically T. cruzi-infected mice.(A) Chronically Colombian T. cruzi strain-infected mice were evaluated for heart injury markers pre-therapy (120 dpi) or primed-boosted with 2 × 108 plaque-forming units (PFU) of rAdCtrl or a mixture of 108 PFU of each adenovirus vaccine preparation (rAdASP2+rAdTS; rAdVax). Mortality was recorded until 230 dpi (110 days post-therapy; dpt), when the surviving mice were analyzed for heart injury markers. (B) Kaplan-Meier curve representing the percentages of surviving mice (14–20 mice/group in two independent experiments). (C) Relative spleen weight (mg of spleen/g of body) and quantitative immunohistochemical staining (IHS) data for T. cruzi parasitism (nests/100 microscopic fields) in the heart tissue of chronically infected mice (120 and 230 dpi, respectively, pre- and post-therapy). (D) IHS showing fibronectin (FN)-stained areas in representative cardiac tissue sections of noninfected (NI) controls and chronically T. cruzi-infected mice pre- (120 dpi) and post-therapy (230 dpi; 110 dpt) with rAdVax. (E) Quantification of the FN-stained area (%) and connexin 43 (Cx43)-containing gap junction distances detected using IHS staining of heart tissue sections of NI controls or T. cruzi-infected mice pre- and post-therapy with rAdVax. (F) Evaluation of CK-MB activity in the serum of NI controls and T. cruzi-infected mice pre- and post-therapy with rAdVax. The data are presented as the means ± SD. ** P <0.01 and ***P <0.001, experimental groups compared with NI controls. #P <0.05 and ##P <0.01, rAdVax-immunized compared with pre-therapy T. cruzi-infected mice.
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ppat.1004594.g003: rAdVax immunotherapy recovered the injured heart tissue of chronically T. cruzi-infected mice.(A) Chronically Colombian T. cruzi strain-infected mice were evaluated for heart injury markers pre-therapy (120 dpi) or primed-boosted with 2 × 108 plaque-forming units (PFU) of rAdCtrl or a mixture of 108 PFU of each adenovirus vaccine preparation (rAdASP2+rAdTS; rAdVax). Mortality was recorded until 230 dpi (110 days post-therapy; dpt), when the surviving mice were analyzed for heart injury markers. (B) Kaplan-Meier curve representing the percentages of surviving mice (14–20 mice/group in two independent experiments). (C) Relative spleen weight (mg of spleen/g of body) and quantitative immunohistochemical staining (IHS) data for T. cruzi parasitism (nests/100 microscopic fields) in the heart tissue of chronically infected mice (120 and 230 dpi, respectively, pre- and post-therapy). (D) IHS showing fibronectin (FN)-stained areas in representative cardiac tissue sections of noninfected (NI) controls and chronically T. cruzi-infected mice pre- (120 dpi) and post-therapy (230 dpi; 110 dpt) with rAdVax. (E) Quantification of the FN-stained area (%) and connexin 43 (Cx43)-containing gap junction distances detected using IHS staining of heart tissue sections of NI controls or T. cruzi-infected mice pre- and post-therapy with rAdVax. (F) Evaluation of CK-MB activity in the serum of NI controls and T. cruzi-infected mice pre- and post-therapy with rAdVax. The data are presented as the means ± SD. ** P <0.01 and ***P <0.001, experimental groups compared with NI controls. #P <0.05 and ##P <0.01, rAdVax-immunized compared with pre-therapy T. cruzi-infected mice.
Mentions: The chronic infection of C57BL/6 mice with the Colombian strain of T. cruzi induces CCC, which is characterized by heart injury with connexin-43 (Cx43) disorganization and fibronectin (FN) deposition in the cardiac tissue and increased CK-MB activity in the serum [5, 18, 20]. Therefore, we tested the capacity of the homologous prime-boost rAdVax immunotherapy to reverse heart injury. To this end, C57BL/6 mice were infected and all mice were analyzed at 120 dpi (pre-therapy), when groups were formed, and the prime-boost rAdVax immunization protocol was initiated. A group of T. cruzi-infected mice was euthanized and the tissues were collected (pre-therapy). At 160 dpi and 40 days post-therapy, all mice were analyzed for electrical alterations, boosted and analyzed at 230 dpi, 110 days post-therapy (Fig. 3A). No difference in survival rate was observed in T. cruzi-infected mice that received saline or rAdCtrl and all mice in these groups were dead at 200 dpi (Fig. 3B). At 230 dpi, 87% of rAdVax-immunized mice survived (13/15), compared with 0% of rAdCtrl-inoculated (0/14) and saline-injected (0/14) mice (Fig. 3B). The surviving mice were analyzed at 230 dpi (110 days post-therapy) for heart electrical abnormalities, sacrificed and analyzed for cardiac tissue alterations compared with mice sacrificed at 120 dpi (pre-therapy). In post-therapy rAdVax-inoculated mice (at 230 dpi), there was a significant decrease in T. cruzi-induced splenomegaly (P < 0.01). Similarly to pre-therapy (120 dpi) mice, low heart parasitism persisted in rAdVax-immunized mice (Fig. 3C). In addition, pre-therapy and post-therapy parasites were rarely detected in the circulating blood (110 ± 70.2 × 103 trypomastigotes/mL in pre-therapy vs. 22.8 ± 9.5 × 103 trypomastigotes/mL in rAdVax; P > 0.05). Nevertheless, immunotherapy with rAdVax significantly reduced FN deposition in the cardiac tissue (Fig. 3D and 3E). Furthermore, Cx43 disorganization in the cardiac intercalary discs revealed as enhanced distance of Cx43-stained junctions, a marker of cardiomyocyte injury [21], was significantly reversed in mice treated with rAdVax compared with the pre-therapy condition (Fig. 3E). In addition, levels of CK-MB activity in the serum were lower in post-therapy mice compared with pre-therapy mice (Fig. 3F). Thus, these data support that immunotherapy with rAdVax during chronic T. cruzi infection ameliorated electrical abnormalities and recovered heart tissue injury.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia das Interações, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus