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

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

Functional profile of vaccine-induced T cells during chronic chagasic disease.

<p>Mice were vaccinated and infected as in Fig. 5, and harvested at day 120 post-infection. (<b>A</b>) Splenic frequency of CD4⁺ and CD8⁺ T cells. (<b>B &C</b>) Splenocytes were stimulated as in Fig. 5. (<b>B&E</b>) Shown are percentages of CD4⁺T cells that were IL-10⁺ (B) and IL-4⁺ (C); and the percentages of CD8⁺T cells that were IL-10⁺ (D) or Ki67⁺IFN-γ⁺ (E), measured by flow cytometry.</p

Chronic inflammatory infiltrate, tissue fibrosis and parasite burden were arrested inTcVac2-immunized/infected mice.

<p>Heart tissue and skeletal muscle were harvested at 120 dpi (chronic phase) and subjected to H&E (<b>A</b>) or Masson's Trichrome (<b>B</b>) staining (magnification: 20X). The intense blue color (Fig. 6B) shows the collagen deposition (fibrotic area). (C) Real time PCR amplification of <i>T. cruzi</i> 18SrDNA (as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000797#pntd-0000797-g005" target="_blank">Fig. 5C</a>).</p

Enhanced infiltration of inflammatory infiltrate contributed to control of acute tissue parasite burden in TcVac2-immunized mice.

<p>Mice were vaccinated with TcVac2 or given vector or cytokines (cyt) alone, and challenged with <i>T. cruzi</i>. (<b>A</b>) Shown are H&E staining (blue-nuclear and pink-muscle/cytoplasm/keratin) of heart tissue and skeletal muscle (Sk Ms) sections at day 30 pi (magnification: 20X). Arrows mark parasite nests in panels b & d. (<b>B&C</b>) Total DNA from Heart (Hrt), Skeletal Muscle (Sk Ms), Spleen (Spl), Liver (Liv) and Kidney (Kid) was isolated and used as a template for the amplification of <i>T. cruzi</i> 18SrDNA sequence by traditional PCR (<b>B</b>) or real time PCR (<b>C</b>). Standard deviation was <12% for the data presented in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000797#pntd-0000797-g005" target="_blank">Figure 5C</a> (^##<i>p</i><0.01, ^###<i>p</i><0.001).</p

Antigen-specific antibodies were enhanced in response to challenge <i>T. cruzi</i> infection in TcVac2-immunized mice.

<p>Mice were vaccinated with TcVac2 and two-weeks after last immunization, infected with <i>T. cruzi</i> (10,000 trypomastigotes / mouse). Sera were collected at day 30 (<b>A,C&E</b>) and day 120 (<b>B,D&F</b>) post-infection. The sera level of <i>T. cruzi</i>- and antigen-specific IgG (<b>A&B</b>), IgG2b (<b>C&D</b>) and IgG1 (<b>E&F</b>) antibodies were measured by an ELISA (n = 8 mice/group, ^#<i>p</i><0.05, ^##<i>p</i><0.01,^###<i>p</i><0.001). Abbreviations are as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000797#pntd-0000797-g001" target="_blank">Figure 1</a>.</p

Splenic T cell functional profile in TcVac3-immunized mice infected with <i>T. cruzi.</i>

<p>Mice were immunized with empty vector or TcVac3 (± cytokine adjuvants), and infected with <i>T. cruzi</i>, as detailed in Materials and Methods. Splenocytes were obtained 2-weeks after vaccination or at day 30 (acute) and 120 (chronic) post-infection, and <i>in vitro</i> stimulated for 48 h with TcG2 and TcG4 recombinant antigens or <i>T. cruzi</i> lysate (TcTL). The cell free supernatants were used to measure IFN-γ, TNF-α, IL-4, and IL-10 levels by an ELISA (A). Cells were labeled with appropriate fluorescent-tagged antibodies, and flow cytometry was performed to gate for proliferating (Ki67+) and non-proliferating (Ki67-) CD8+ T cells producing cytokines (B). Data (mean ± SD) are representative of three independent experiments (n = 4/group). ND = Not detectable. Statistical analysis was done using ANOVA with post hoc Tukey’s test. The level of significance between control versus vaccinated (TcVac3 or TcVac3+cytokine adjuvants) is shown by *and between TcVac3 versus TcVac3+cytokine adjuvants by # (*^,#p<0.05; **^,##p<0.01; ***^,###p<0.000).</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

TcVac3 Induced Control of <i>Trypanosoma cruzi</i> Infection and Chronic Myocarditis in Mice

<div><p>We characterized the immune responses elicited by a DNA-prime/MVA-boost vaccine (TcVac3) constituted of antigenic candidates (TcG2 and TcG4), shown to be recognized by B and T cell responses in <i>Trypanosoma cruzi</i> (<i>Tc</i>) infected multiple hosts. C57BL/6 mice immunized with TcVac3 elicited a strong antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1); and a robust antigen- and <i>Tc</i>-specific CD8⁺T cell response with type-1 cytokine (IFN-γ⁺TNF-α>IL-4⁺IL-10) and cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺Perforin⁺) phenotype. The vaccine-induced effector T cells significantly expanded upon challenge infection and provided >92% control of <i>T. cruzi</i>. Co-delivery of IL-12 and GMCSF cytokine adjuvants didn’t enhance the TcVac3-induced resistance to <i>T. cruzi</i>. In chronic phase, vaccinated/infected mice exhibited a significant decline (up to 70%) in IFN-γ⁺CD8⁺T cells, a predominance of immunoregulatory IL-10⁺/CD4⁺T and IL10⁺/CD8⁺T cells, and presented undetectable tissue parasitism, inflammatory infiltrate, and fibrosis in vaccinated/infected mice. In comparison, control mice responded to challenge infection by a low antibody response, mixed cytokine profile, and consistent activation of pro-inflammatory CD8⁺T cells associated with parasite persistence and pathologic damage in the heart. We conclude that TcVac3 elicited type-1 effector T cell immunity that effectively controlled <i>T. cruzi</i> infection, and subsequently, predominance of anti-inflammatory responses prevented chronic inflammation and myocarditis in chagasic mice.</p> </div

TcVac3-immunized mice responded to challenge <i>T. cruzi</i> infection by a strong expansion of parasite- and antigen-specific antibody response.

<p>Mice were immunized as in Fig. 1, and challenged with <i>T. cruzi</i> (10,000/mouse). The sera levels of antigen-specific (<b>A&C</b>) and TcTL-specific (<b>B&D</b>) IgG, IgG2b and IgG1 antibodies were measured at day 30 (A&B) and 120 (C&D) post-infection by an ELISA. (<b>E–H</b>). Shown are antigen-specific (E&G) and TcTL-specific (F&H) percent avidity of the antibodies induced in vaccinated/infected mice at day 30 (E&F) and 120 (G&H) pi. (<b>I&J</b>) Trypanolytic activity of sera antibodies induced in vaccinated/infected mice at day 30 (I) and 120 (J) pi.</p