A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term, Protective T Cell Immunity against <i>Trypanosoma cruzi</i>

<div><p>In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against <i>Trypanosoma cruzi</i> infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with <i>T</i>. <i>cruzi</i> at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting <i>T</i>. <i>cruzi</i>. Our data showed that D/P vaccine elicited CD4⁺ (30-38%) and CD8⁺ (22-42%) T cells maintained an effector phenotype up to 180 dpv, and were capable of responding to antigenic stimulus or challenge infection by a rapid expansion (CD8>CD4) with type 1 cytokine (IFNγ⁺ and TFNα⁺) production and cytolytic T lymphocyte (CTL) activity. Subsequently, challenge infection at 120 or 180 dpv, resulted in 2-3-fold lower parasite burden in vaccinated mice than was noted in unvaccinated/infected mice. Co-delivery of IL-12- and GMCSF-encoding expression plasmids provided no significant benefits in enhancing the anti-parasite efficacy of the vaccine-induced T cell immunity. Booster immunization (bi) with recombinant TcG2/TcG4 proteins 3-months after primary vaccine enhanced the protective efficacy, evidenced by an enhanced expansion (1.2-2.8-fold increase) of parasite-specific, type 1 CD4⁺ and CD8⁺ T cells and a potent CTL response capable of providing significantly improved (3-4.5-fold) control of infecting <i>T</i>. <i>cruzi</i>. Further, CD8⁺T cells in vaccinated/bi mice were predominantly of central memory phenotype, and capable of responding to challenge infection 4-6-months post bi by a rapid expansion to a poly-functional effector phenotype, and providing a 1.5-2.3-fold reduction in tissue parasite replication. We conclude that the TcG2/TcG4 D/P vaccine provided long-term anti-<i>T</i>. <i>cruzi</i> T cell immunity, and bi would be an effective strategy to maintain or enhance the vaccine-induced protective immunity against <i>T</i>. <i>cruzi</i> infection and Chagas disease.</p></div

D/P vaccine/bi generated CD8⁺ T_CM cells are capable of responding to challenge infection at 180 days post bi.

<p>Mice were immunized with D/P vaccine followed by a booster dose as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004828#ppat.1004828.g004" target="_blank">Fig 4</a>, and then 180 days post bi, infected with <i>T</i>. <i>cruzi</i>. Mice were harvested 10 days after the challenge infection, and splenocytes were stimulated in the presence or absence of TcG2 and TcG4 recombinant antigens, labeled with fluorescent-conjugated antibodies and analyzed by flow cytometry. <b>(A&B)</b><i>T</i>. <i>cruzi</i>-specific CD4⁺<b><i>(A)</i></b> and CD8⁺<b><i>(B)</i></b> splenic T cell subsets in vaccinated mice infected at 180 days post bi. Insets show <i>T</i>. <i>cruzi</i>-specific, proliferating (Ki67⁺) T cell subsets in mice challenged at 180 days post bi. <b>(C-F)</b> Shown are mean percentage of CD4⁺<b><i>(C&E)</i></b> and CD8⁺<b><i>(D&F)</i></b> T cells that responded to <i>T</i>. <i>cruzi</i> infection by producing IFNγ and/or TNFα cytokines and were proliferative (Ki67⁺, <b><i>C&D</i></b>) with a T effector <b><i>(E&F)</i></b> phenotype in mice challenged at 180 days post bi. <b>(G)</b> The percentage of <i>T</i>. <i>cruzi</i>-specific CD8⁺CD107a⁺ that were IFNγ⁺perforin^- or IFNγ⁺perforin⁺ in mice that were infected at 180 days post bi. <b>(H) Tissue parasite burden</b>. Real time PCR evaluation of <i>Tc18SrDNA</i> level, normalized to murine <i>GAPDH</i>, in spleen, heart and skeletal muscle tissue of vaccinated/bi mice that were infected at 180 days post bi.</p

Two-component D/P vaccine/bi elicited T cells retain the antigen-specific functional profile at 120 days post bi.

<p>Mice were immunized with D/P vaccine followed by a booster dose as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004828#ppat.1004828.g004" target="_blank">Fig 4</a>, and then harvested at 120 days after booster dose. Splenocytes were stimulated in the presence or absence of TcG2 and TcG4 recombinant antigens, labeled with fluorescent-conjugated antibodies, and analyzed by flow cytometry. <b>(A&B)</b><i>Ex vivo</i> percentage of CD4⁺<b><i>(A)</i></b> and CD8⁺<b><i>(B)</i></b> splenic T cell subsets (T_EM: CD44⁺CD62L^-; T_CM: CD44⁺CD62L⁺) at 120 days post bi (Insets: antigen-specific Ki67⁺ T cell subsets). <b>(C-G)</b> Bar graphs of CD4⁺<b><i>(C&E)</i></b> and CD8⁺<b><i>(D&F)</i></b> T cell subsets that proliferated (Ki67⁺, <b><i>C&D</i></b>) or exhibited T_EM<b><i>(E&F)</i></b> phenotype with production of intracellular cytokines (IFN<i>γ</i>, TNFα) in vaccinated mice at 120 days post bi. Shown in <b><i>(G)</i></b> are the percentages of antigen-specific CD8⁺CD107a⁺T cells that were IFN<i>γ</i>⁺perforin^- and IFN<i>γ</i>⁺perforin⁺<b><i>(G)</i></b> in D/P-vaccinated mice at 120 days post bi.</p

D/P vaccine elicited, long-lived T cells exhibited enhanced antigen-specific, poly-functional phenotype after booster immunization (bi).

<p>C57BL/6 mice were immunized at day 0 with V1 (TcG2- and TcG4-encoding plasmids ± IL-12- and GMCSF-expression plasmids), at day 21 with V2 (recombinant TcG2 and TcG4 proteins), and then at day 111 with V3 (booster dose, recombinant TcG2 and TcG4 proteins). Mice were harvested at 14 days after the booster immunization (dbi). Splenocytes were stimulated in the presence or absence of TcG2 and TcG4 recombinant antigens, labeled with fluorescent-conjugated antibodies, and analyzed by flow cytometry. <b>(A&B)</b><i>Ex vivo</i> percentage of CD4⁺<b><i>(A)</i></b> and CD8⁺<b><i>(B)</i></b> splenic T cell subsets (T_EM: CD44⁺CD62L^-; T_CM: CD44⁺CD62L⁺) in vaccinated/bi mice (Insets: antigen-specific Ki67⁺ T cell subsets in vaccinated/bi mice). <b>(C-G)</b> Bar graphs of antigen-specific CD4⁺<b><i>(C&E)</i></b> and CD8⁺<b><i>(D&F)</i></b> T cell subsets with their intracellular cytokine (IFN<i>γ</i>⁺, TNFα⁺) profile that were proliferative (Ki67⁺, <b><i>C&D</i></b>) with T effector <b><i>(E&F)</i></b> phenotype. The percentage of antigen-specific CD8⁺CD107a⁺ T cells that were IFN<i>γ</i>⁺perforin^- and IFN<i>γ</i>⁺Perforin⁺<b><i>(G)</i></b> in vaccinated/bi mice are shown. <b>(H) Cytolytic activity</b>. Splenocytes were <i>in vitro</i> stimulated with recombinant antigens and used as effectors for their ability to lyse EL4 target cells exposed to GFP⁺ recombinant MVA encoding TcG2 <i>(H</i>.<i>a)</i> or TcG4 <i>(H</i>.<i>b)</i> antigens (controls: target cells incubated with empty MVA).</p

Stability and effector phenotype of D/P vaccine elicited T cells in mice.

<p>Mice were vaccinated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004828#ppat.1004828.g001" target="_blank">Fig 1</a> and harvested at 120 days after the 2^nd vaccine dose. <b>(A&B)</b> Splenocytes were <i>ex-vivo</i> labeled with fluorescence-conjugated antibodies and analyzed for CD4⁺<b><i>(A)</i></b> and CD8⁺<b><i>(B)</i></b> T cell subsets (T_EM: CD44⁺CD62L^-; T_CM: CD44⁺CD62L⁺) by flow cytometry. Insets show D/P vaccine-primed T cell subsets that proliferated (Ki67⁺) in response to antigenic stimulus. <b>(C-G)</b> Splenocytes were <i>in vitro</i> stimulated for 48 h in the presence of TcG2 and TcG4 recombinant antigens, and incubated with fluorescent-conjugated antibodies as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004828#ppat.1004828.g001" target="_blank">Fig 1</a>. Shown are bar graphs of antigen-specific CD4⁺<b><i>(C&E)</i></b> and CD8⁺<b><i>(D&F)</i></b> T cell subsets that were proliferative (Ki67⁺, <b><i>C&D</i></b>) with T effector <b><i>(E&F)</i></b> phenotype and produced IFN<i>γ</i> and/or TNFα cytokines. The percentage is shown of antigen-specific CD8⁺CD107a⁺ T cells that were IFN<i>γ</i>⁺perforin^- or IFN<i>γ</i>⁺perforin⁺<b><i>(G)</i></b>.</p

Booster immunization (bi) expanded the parasite-specific recall T cell response that provided superior protection from <i>T</i>. <i>cruzi</i> infection.

<p>Mice were immunized with D/P vaccine followed by a booster dose as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004828#ppat.1004828.g004" target="_blank">Fig 4</a>, and then, 14 days later, challenged with <i>T</i>. <i>cruzi</i>. Mice were harvested at 10 days pi. Splenocytes were <i>in vitro</i> stimulated in the presence or absence of TcG2 and TcG4 recombinant antigens, labeled with fluorescence-conjugated antibodies and analyzed by flow cytometry. <b>(A&B)</b><i>T</i>. <i>cruzi</i>-specific CD4⁺<b><i>(A)</i></b> and CD8⁺<b><i>(B)</i></b> splenic T cell subsets in vaccinated/bi mice at 10 days pi. Insets show antigen-specific T cell subsets that proliferated (Ki67⁺) in response to <i>T</i>. <i>cruzi</i> infection in vaccinated/bi mice. <b>(C-F)</b> Shown are the mean percentage of CD4⁺<b><i>(C&E)</i></b> and CD8⁺<b><i>(D&F)</i></b> T cells that responded to <i>T</i>. <i>cruzi</i> infection with production of IFN<i>γ</i> and/or TNFα cytokines and were proliferative (Ki67⁺, <b><i>C&D</i></b>) with T effector <b><i>(E&F)</i></b> phenotype in vaccinated/bi mice. <b>(G)</b> Percentage of <i>T</i>. <i>cruzi</i>-specific CD8⁺CD107a⁺ that were IFN<i>γ</i>⁺perforin^- or IFN<i>γ</i>⁺perforin⁺ in booster-immunized/infected mice. <b>(H) D/P vaccine/bi was highly effective in controlling tissue parasite burden</b>. Total DNA was isolated from spleen, heart and skeletal muscle (Sk Ms) tissue sections of vaccinated/bi/infected mice, and real time PCR amplification of <i>Tc18SrDNA</i> sequence was performed. Bar graphs show the <i>Tc18SrDNA</i> level normalized to murine <i>GAPDH</i>.</p

Recall of the D/P vaccine elicited, long-lived T cells after challenge infection with <i>T</i>. <i>cruzi</i>.

<p>Mice were vaccinated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004828#ppat.1004828.g002" target="_blank">Fig 2</a>, and at 120 days post-vaccination, challenged with <i>T</i>. <i>cruzi</i> trypomastigotes (10,000 parasites/mouse), as described in Materials and Methods. Mice were harvested at 10 days post-infection (dpi). <b>(A&B)</b> Bar graphs show <i>ex vivo</i> percentage of PE⁺CD4⁺<b><i>(A)</i></b> and FITC⁺CD8⁺<b><i>(B)</i></b> splenic T cell subsets (T_EM: CD44⁺CD62L^-; T_CM: CD44⁺CD62L⁺) in vaccinated/infected mice. Insets show D/P vaccine-primed T cell subsets that proliferated (Ki67⁺) in response to <i>T</i>. <i>cruzi</i> infection (controls: non-vaccinated/infected mice). <b>(C-F)</b> Splenic cells from vaccinated/infected and non-vaccinated/infected mice were <i>in vitro</i> stimulated for 48 h in the presence of TcG2 and TcG4 recombinant antigens, and then labeled with fluorescent-conjugated antibodies, as above. Shown are the mean percentage of CD4⁺<b><i>(C&E)</i></b> and CD8⁺<b><i>(D&F)</i></b> T cells that responded to <i>T</i>. <i>cruzi</i> infection with production of IFN<i>γ</i> and/or TNFα and were proliferative (Ki67⁺, <b><i>C&D</i></b>) with T effector <b><i>(E&F)</i></b> phenotype. <b>(G)</b> The percentage of <i>T</i>. <i>cruzi</i>-specific CD8⁺IFN<i>γ</i>⁺T cells that were CD107a⁺perforin^- or CD107a⁺perforin⁺ are shown (± D/P vaccine). <b>(H) Tissue parasite burden</b>. Mice were challenged at 120 days post D/P vaccination and harvested at 10 dpi. Total DNA was isolated from spleen, heart and skeletal muscle (Sk Ms) tissue sections and submitted to real-time PCR amplification of <i>Tc</i>18SrDNA sequence (normalized to murine <i>GAPDH</i>).</p

Booster-immunized mice expand the recall T cell response capable of controlling <i>T</i>. <i>cruzi</i> infection at 120 days post bi.

<p>Mice were immunized with D/P vaccine followed by a booster dose as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004828#ppat.1004828.g004" target="_blank">Fig 4</a>, and then, at 120 days post bi, infected with <i>T</i>. <i>cruzi</i>. Mice were harvested 10 days after the challenge infection, and splenocytes were stimulated in the presence or absence of TcG2 and TcG4 recombinant antigens, labeled with fluorescent-conjugated antibodies, and analyzed by flow cytometry. <b>(A&B)</b><i>T</i>. <i>cruzi</i>-specific CD4⁺<b><i>(A)</i></b> and CD8⁺<b><i>(B)</i></b> splenic T cell subsets in vaccinated/bi mice infected at 120 days post bi. Insets show <i>T</i>. <i>cruzi</i>-specific, proliferating (Ki67⁺) T cell subsets in vaccinated mice challenged at 120 days post bi. <b>(C-F)</b> Shown are the mean percentage of CD4⁺<b><i>(C&E)</i></b> and CD8⁺<b><i>(D&F)</i></b> T cells that responded to <i>T</i>. <i>cruzi</i> infection by producing IFN<i>γ</i> and/or TNFα cytokines and were proliferative (Ki67⁺, <b><i>C&D</i></b>) with a T effector <b><i>(E&F)</i></b> phenotype in mice challenged at 120 days post bi. <b>(G)</b> The percentage of <i>T</i>. <i>cruzi</i>-specific CD8⁺CD107a⁺ that were IFN<i>γ</i>⁺perforin−^- or IFN<i>γ</i>⁺perforin⁺ in mice that were infected at 120 days post bi. <b>(H) Tissue parasite burden</b>. Bar graphs of <i>Tc18SrDNA</i> levels, normalized to murine <i>GAPDH</i>, were developed by using a real time quantitative PCR approach.</p

Two-component, DNA-prime/protein-boost (D/P) vaccine elicits poly-functional T cell response in mice.

<p>C57BL/6 mice were immunized with an empty vector, cytokines only, or D/P vaccine (V1 dose: TcG2- and TcG4-encoding plasmids ± IL-12- and GMCSF-expression plasmids; and V2 dose: recombinant TcG2 and TcG4 proteins), as detailed in Materials and Methods. <b>(A&B)</b> Splenocytes were obtained at 14 days post-vaccination (dpv) and <i>ex vivo</i> labeled with PE-conjugated anti-CD4, FITC-conjugated anti-CD8, PerCPCy5.5-conjugated anti-CD62L, and APC-conjugated anti-CD44 antibodies. Shown are the PE⁺CD4⁺<b><i>(A)</i></b> and FITC⁺CD8⁺<b><i>(B)</i></b> T cell subsets that were of effector/effector memory (T_EM: CD44⁺CD62L^-) and central memory (T_CM, CD44⁺CD62L⁺) phenotype, determined by flow cytometry. <b>(C-E)</b> Splenocytes were <i>in vitro</i> stimulated for 48 h with TcG2 and TcG4 recombinant antigens and labeled with fluorescent-conjugated antibodies. The mean percentage of PE⁺CD4⁺<b><i>(C)</i></b> and FITC⁺CD8⁺<b><i>(D)</i></b> T cells that were IFN<i>γ</i>⁺ (e-Fluor) and/or TNFα⁺ (Cy5) with or without Ki67⁺ (PerCPCy 5.5) phenotype are shown. <b>(E)</b> The percentage of antigen-specific CD8⁺IFNγ⁺ T cells that were CD107a⁺ (Alexa-Fluor 488) and/or perforin⁺ (APC) was acquired by flow cytometry analysis. In all figures, data are presented as mean ± SD (n = 8/group, triplicate observations per experiment). Significance is presented as *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 (vaccinated versus non-vaccinated or vaccinated/infected versus non-vaccinated/infected).</p

Serological and parasitological analysis of fetuses of guinea pigs infected with <i>T</i>. <i>cruzi</i>.

<p>Female guinea pigs were infected before gestation (IBG) or during gestation (IDG). <b>(A&B)</b> Sera samples of fetuses were obtained close to parturition, and analyzed by an ELISA <b><i>(A)</i></b> and indirect immunofluorescence assay <b>(B)</b>. Bar graphs show the percentage of fetuses in each group that were positive for anti-<i>T</i>. <i>cruzi</i> antibodies. <b>(C)</b> Fetal tissue parasite load was monitored by quantitative PCR as described in Materials and Methods. Control (n = 9); IBG (n = 9); IDG (n = 5) samples were analyzed in triplicate. Data in panel A & B are presented as absolute values, and data in panel C are presented as mean value ± SD. (**P <0.01, IDG vs. IBG).</p