Prophylactic efficacy of TcVac2 against Trypanosoma cruzi in mice

Abstract

Background: Chagas disease is a major health problem in Latin America, and an emerging infectious disease in the US. Previously, we have screened the Trypanosoma cruzi sequence database by a computational/bioinformatics approach, and identified antigens that exhibited the characteristics of vaccine candidates.

Methodology: We investigated the protective efficacy of a multi-component DNA-prime/protein-boost vaccine (TcVac2) constituted of the selected candidates and cytokine (IL-12 and GM-CSF) expression plasmids in a murine model. C57BL/6 mice were immunized with antigen-encoding plasmids plus cytokine adjuvants, followed by recombinant proteins; and two-weeks later, challenged with T. cruzi trypomastigotes. ELISA and flow cytometry were employed to measure humoral (antibody isotypes) and cellular (lymphocyte proliferation, CD4(+) and CD8(+) T cell phenotype and cytokines) responses. Myocardial pathology was evaluated by H&E and Masson's trichrome staining.

Principal findings: TcVac2 induced a strong antigen-specific antibody response (IgG2b>IgG1) and a moderate level of lymphocyte proliferation in mice. Upon challenge infection, TcVac2-vaccinated mice expanded the IgG2b/IgG1 antibodies and elicited a substantial CD8(+) T cell response associated with type 1 cytokines (IFN-gamma and TNF-alpha) that resulted in control of acute parasite burden. During chronic phase, antibody response persisted, splenic activation of CD8(+) T cells and IFN-gamma/TNF-alpha cytokines subsided, and IL-4/IL-10 cytokines became dominant in vaccinated mice. The tissue parasitism, inflammation, and fibrosis in heart and skeletal muscle of TcVac2-vaccinated chronic mice were undetectable by histological techniques. In comparison, mice injected with vector or cytokines only responded to T. cruzi by elicitation of a mixed (type 1/type 2) antibody, T cell and cytokine response, and exhibited persistent parasite burden and immunopathology in the myocardium.

Conclusion: TcVac2-induced activation of type 1 antibody and lymphocyte responses provided resistance to acute T. cruzi infection, and consequently, prevented the evolution of chronic immunopathology associated with parasite persistence in chagasic hearts.

Figure 1. TcVac2 induced antigen-specific antibody response and lymphocyte proliferation in immunized mice.

C57BL/6 mice were immunized with TcVac2 as detailed in Materials and Methods and sera samples were collected after each immunization. (A) Sera samples after last immunization were used to generate titration curves for T. cruzi (TcTL) and antigen-specific (TcG1, TcG2, TcG4) antibody detection in TcVac2-vaccinated mice. (B) T. cruzi specific IgG antibodies (sera dilution, 1∶100) were exponentially increased after each dose of immunization in vaccinated mice. (C–E) Sera levels of parasite- and antigen-specific IgG ( C ), IgG2b ( D ) and IgG1 ( E ) antibodies, measured two-weeks after the last immunization. Sera levels of antibody isotypes in normal mice (NM), and mice injected with vector alone or cytokine (Cyt) adjuvants only are also shown. (F) Spleen cells were obtained 2-weeks after last immunization and in vitro stimulated for 48 h with T. cruzi trypomastigotes lysate (TcTL) or recombinant antigens (TcG1, TcG2 and TcG4). Shown are the extent of lymphocytes proliferation in TcVac2-vaccinated mice and mice given cytokines only (data normalized to control mice given vector alone), measured by an MTT assay. Data (mean ± SD) are representative of three independent experiments (n = 8 mice/group, ^# p<0.05, ^## p<0.01, ^### p<0.001).

Figure 2. Antigen-specific antibodies were enhanced in response to challenge T. cruzi infection in TcVac2-immunized mice.

Mice were vaccinated with TcVac2 and two-weeks after last immunization, infected with T. cruzi (10,000 trypomastigotes / mouse). Sera were collected at day 30 (A,C&E) and day 120 (B,D&F) post-infection. The sera level of T. cruzi- and antigen-specific IgG (A&B), IgG2b (C&D) and IgG1 (E&F) antibodies were measured by an ELISA (n = 8 mice/group, ^# p<0.05, ^## p<0.01, ^### p<0.001). Abbreviations are as in Figure 1.

Figure 3. TcVac2-immunized mice exhibited significant increase in lymphocyte proliferation and type 1 cytokine production in response to acute T. cruzi infection.

Spleen cells from mice sacrificed at day 30 (A–E) and 120 (F–J) pi were in vitro stimulated with TcTL or recombinant antigens for 48 h. The extent of lymphocyte proliferation was determined by an MTT assay (A&F). The IFN-γ (B&G), TNF-α (C&H), IL-4 (D&I), and IL-10 (E&J) levels in cell-free supernatants were measured by an ELISA (n = 8 mice/group, ^# p<0.05, ^## p<0.01, ^### p<0.001).

Figure 4. CD8⁺ T cell subset dominated in splenic T cell population of TcVac2-vaccinated/acutely-infected mice.

Mice were vaccinated and challenged with T. cruzi, and harvested at day 30 (A) and 120 (B) post-infection, corresponding to acute infection phase and chronic disease phase, respectively. Spleen cells were incubated for 30 min with FITC-conjugated anti-CD8 and PE-conjugated anti-CD4 antibodies and CD4⁺ and CD8⁺ T cell subsets monitored by flow cytometry.

Figure 5. Enhanced infiltration of inflammatory infiltrate contributed to control of acute tissue parasite burden in TcVac2-immunized mice.

Mice were vaccinated with TcVac2 or given vector or cytokines (cyt) alone, and challenged with T. cruzi. (A) 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&C) 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 T. cruzi 18SrDNA sequence by traditional PCR (B) or real time PCR (C). Standard deviation was <12% for the data presented in the Figure 5C (^## p<0.01, ^### p<0.001).

Figure 6. Chronic inflammatory infiltrate, tissue fibrosis and parasite burden were arrested inTcVac2-immunized/infected mice.

Heart tissue and skeletal muscle were harvested at 120 dpi (chronic phase) and subjected to H&E (A) or Masson's Trichrome (B) staining (magnification: 20X). The intense blue color (Fig. 6B) shows the collagen deposition (fibrotic area). (C) Real time PCR amplification of T. cruzi 18SrDNA (as in Fig. 5C).