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Clinical and Vaccine Immunology, August 2008, p. 1158-1164, Vol. 15, No. 8
1071-412X/08/$08.00+0     doi:10.1128/CVI.00144-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Previously Unrecognized Vaccine Candidates Control Trypanosoma cruzi Infection and Immunopathology in Mice

Vandanajay Bhatia¹ and Nisha Jain Garg^1,2,3*

Departments of Microbiology and Immunology,¹ Pathology, University of Texas Medical Branch, Galveston, Texas,² Center for Biodefense and Emerging Infectious Diseases and Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas³

Received 25 April 2008/ Returned for modification 28 May 2008/ Accepted 4 June 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Trypanosoma cruzi is the etiologic agent of Chagas' disease, a major health problem in Latin America and an emerging infectious disease in the United States. Previously, we screened a T. cruzi sequence database by a computational-bioinformatic approach and identified antigens that exhibited the characteristics of good vaccine candidates. In this study, we tested the vaccine efficacy of three of the putative candidate antigens against T. cruzi infection and disease in a mouse model. C57BL/6 mice vaccinated with T. cruzi G1 (TcG1)-, TcG2-, or TcG4-encoding plasmids and cytokine (interleukin-12 and granulocyte-macrophage colony-stimulating factor) expression plasmids elicited a strong Th1-type antibody response dominated by immunoglobulin G2b (IgG2b)/IgG1 isotypes. The dominant IgG2b/IgG1 antibody response was maintained after a challenge infection and was associated with 50 to 90% control of the acute-phase tissue parasite burden and an almost undetectable level of tissue parasites during the chronic phase, as determined by a sensitive T. cruzi 18S rRNA gene-specific real-time PCR approach. Splenocytes from vaccinated-and-infected mice, compared to unvaccinated-and-infected mice, exhibited decreased (50% lower) proliferation and gamma interferon (IFN-) production when stimulated in vitro with T. cruzi antigens, thus suggesting that protection from challenge infection was not provided by an active T-cell response. Subsequently, the serum and cardiac levels of IFN- and tumor necrosis factor alpha and infiltration of inflammatory infiltrate in the heart were decreased in vaccinated mice during the course of infection and chronic disease development. Taken together, these results demonstrate the identification of novel vaccine candidates that provided protection from T. cruzi-induced immunopathology in experimental mice.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Trypanosoma cruzi is the causative agent of Chagas' disease in humans, which is a major health problem in Latin America and is considered an emerging infectious disease in the United States (19, 20). Infection with T. cruzi results in an acute parasitemia that is generally associated with mild illness and followed by an intermediate phase wherein infected individuals remain serologically positive but exhibit no clinical symptoms. After several years, 30 to 40% of infected individuals develop the clinical form of Chagas' disease, which results in >50,000 congestive heart failure-related deaths of young adults in areas of endemicity every year (19, 20). No vaccines are available.

A variety of experimental animal models have been used to identify the effector mechanisms required for the control of T. cruzi infection (reviewed in reference 35). These studies attributed potential roles to all of the components of the immune system, i.e., granulocytes, natural killer cells, the action of lytic antibodies, and CD4⁺ and CD8⁺ T-cell subsets, in the control of T. cruzi infection. Others have suggested that the persistent activation of CD8⁺ T cells and proinflammatory cytokines (tumor necrosis factor alpha [TNF-] and gamma interferon [IFN-]) contribute to immunopathology and tissue damage, the hallmarks of chronic Chagas' disease (9). It can be deduced from these studies that a finely tuned, regulated activation of the immune system capable of controlling T. cruzi infection and not having adverse effects on the host cellular components would be essential to prevent the progression of chronic Chagas' disease.

Efforts toward subunit vaccine development against T. cruzi have mainly focused on antigens that are expressed on the plasma membrane of the parasite, attached by a glycosylphosphatidylinositol (GPI) anchor. GPI proteins are considered good antigenic targets because they are abundantly expressed in the infective and intracellular stages of T. cruzi (36) and were shown to be recognized by both the humoral and cellular arms of the immune system in infected hosts (14, 22). Subsequently, several defined T. cruzi GPI-anchored proteins were tested as vaccine candidates. Recombinant GPI proteins, e.g., GP90, GP56, and GP82 (18, 29, 30), and DNA expression plasmids encoding GPI proteins, e.g., ASP-1, ASP-2, TSA-1, and trans-sialidase (TS) (8, 11, 13), were demonstrated to elicit various degrees of resistance to T. cruzi infection in different animal models.

A majority of the protective candidate antigens identified so far belong to the TS gene family of T. cruzi. The attempts to enhance the protective efficacy by codelivery of TS family members as a multiantigen vaccine with or without cytokine adjuvants (13) or by a DNA prime-protein boost approach (34) have not been successful. The limited protective efficacy of vaccine cocktails was attributed to the fact that TS family members with shared epitopes represent only a minor proportion of all of the possible target molecules in T. cruzi, and additional candidate antigens will be required to elicit an immune response of sufficient magnitude to efficiently control T. cruzi infection.

We have previously conducted an in silico analysis of a T. cruzi sequence database to identify putative vaccine candidates. The selection strategy was designed to disregard TS family members and select candidate antigens that exhibit the characteristics of GPI-anchored or secreted proteins (2). Of the 19 selected sequences, 8 (T. cruzi G1 [TcG1] to TcG8) were phylogenetically conserved in diverse T. cruzi strains and expressed in the infective and intracellular mammalian stages of T. cruzi (2). TcG1-, TcG2-, and TcG4-encoded antigens were expressed on the plasma membrane of the mammalian stages of T. cruzi (trypomastigote/amastigote) and elicited significant levels of antiparasite lytic antibody responses in mice (2), thus forming the basis for testing of their vaccine efficacy in this study. Our data show that the three antigens (TcG1, TcG2, and TcG4), delivered as a DNA vaccine, elicited effective immunity that provided resistance to acute T. cruzi infection in a murine model. Sterile immunity was not achieved; however, vaccinated mice exhibited moderate to no cardiac immunopathology and tissue damage. These results validate the applicability of a rational computational approach in the identification of novel vaccine candidates and demonstrate that vaccines capable of controlling the tissue parasite burden below a threshold level will be effective in preventing the chronic pathology of the heart in Chagas' disease.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Parasites and mice. Trypomastigotes of T. cruzi (Sylvio X 10/4 strain) were maintained and propagated by continuous in vitro passage in C2C12 cells. C57BL/6 female mice (6 to 8 week old) were obtained from Harlan Labs (Indianapolis, IN). Animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of Texas Medical Branch Animal Care and Use Committee.

T. cruzi genes and plasmid construction. TcG1-encoded protein (accession no. AY727914) is 76% identical to the Leishmania donovani 23-kDa cell surface protein (accession no. X86551) (5) and also showed significant homology to the sequence encoding a 40S ribosomal protein (accession no. XM_811404.1), identified by the T. cruzi CL Brenner sequencing project (10). TcG2 (accession no. AY727915) exhibits 100% homology to a CL Brenner sequence (accession no. XM_806323) suggested to encode a hypothetical protein (291 amino acids) (10). TcG4 (accession no. AY727917) encodes a small protein (92 amino acids) that is not functionally characterized. TcG4 exhibits 99% homology to a CL Brenner sequence (accession no. XM_816508) suggested to encode a hypothetical protein (10).

The cDNAs for TcG1, TcG2, and TcG4 were cloned into eukaryotic expression plasmid pCDNA 3.1 (2) for vaccination purposes. The eukaryotic expression plasmids encoding the murine cytokine interleukin-12 (IL-12) (pcDNA3.msp35 and pcDNA3.msp40) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (pCMVI.GM-CSF) were previously described (13). Recombinant plasmids were transformed into Escherichia coli DH5 competent cells grown in L broth containing 100 µg/ml ampicillin and purified by anion-exchange chromatography with the Qiagen maxi prep kit (Qiagen, Chatsworth, CA) according to the manufacturer's specifications.

DNA immunization and challenge infection. C57BL/6 mice were injected in the quadriceps muscle thrice at 2-week intervals with antigen-encoding plasmids (pCDNA3.TcG1, pCDNA3.TcG2, and pCDNA3.TcG4, individually or in combination; 25 µg per DNA/mouse) and cytokine-encoding plasmids (pcDNA3.msp35, pcDNA3.msp40 [IL-12], and pCMVI.GM-CSF; 25 µg per plasmid DNA/mouse). Two weeks after the last immunization, mice were challenged with culture-derived T. cruzi trypomastigotes (2.5 x 10⁴/mouse, intraperitoneally) and sacrificed at days 30, 75, and 120 postinfection (p.i.), corresponding to the acute phase of peak parasitemia, the intermediate phase of immune control of parasites, and the chronic phase of disease development, respectively. Serum and tissue samples (heart and skeletal muscle) were harvested and stored at –80°C until further use.

Antibody response. Serum immunoglobulin G (IgG) antibody response in immunized mice was monitored by use of an enzyme-linked immunosorbent assay (ELISA) (2). Briefly, 96-well U-bottom plates were coated with T. cruzi lysate (5 x 10⁵ parasite equivalents/well) and blocked with 1% nonfat dry milk (NFDM). Plates were then sequentially incubated with test sera (1:50 dilution in 0.5% NFDM, 100 µl/well) and horseradish peroxidase-conjugated goat anti-mouse IgG antibody, color was developed with 100 µl/well Sure Blue TMB substrate (KPL, MD), and the ensuing antibody response was monitored at 650 nm with an automated microplate reader (Molecular Devices). To identify the antibody subtypes, plates were coated and incubated with sera as described above and then incubated with a biotin-conjugated goat anti-mouse IgG1, IgG2a, or IgG2b secondary antibody, followed by horseradish peroxidase-streptavidin. All antibodies were from Southern Biotech and were used at a 1:5,000 dilution in phosphate-buffered saline-0.05% Tween 20-0.5% NFDM.

Cytokine detection. T. cruzi antigenic lysate was prepared by subjecting parasites (10⁹/ml phosphate-buffered saline; 70% amastigotes, 30% trypomastigotes) to six cycles of repeated freezing and thawing, followed by sonication in an ice-cold water bath for 30 min. Single-cell splenocyte preparation from immunized and control mice was suspended at 2 x 10⁶/ml RPMI-5% fetal bovine serum in 24-well plates. Cells were incubated with 25 µg/ml T. cruzi antigenic lysate, and culture supernatants were collected at 48 h for the measurement of IFN- and TNF- with optEIA ELISA kits (Pharmingen, San Diego, CA) according to the manufacturer's specifications. Splenocytes incubated with 1 µg/ml concavalin A were used as positive controls. Serum levels of cytokines were also determined by ELISA.

To measure cytokine levels in tissues, we performed real-time reverse transcription (RT)-PCR. Total RNA from tissue samples (50 mg each) was isolated by guanidinium thiocyanate-phenol-chloroform extraction (6), treated with DNase (Ambion) to remove contaminating DNA, and analyzed for quality and quantity with SPECTRAmax PLUS 384 and for integrity with an Agilent 2100 Bioanalyzer. Total RNA (2.5 µg) was reverse transcribed with 2.5 U of Moloney murine leukemia virus reverse transcriptase (New England BioLabs) and 1 µM poly(dT)₁₈ oligonucleotide, and the first-strand cDNA was used as a template in a real-time PCR on an iCycler thermal cycler (Bio-Rad) with Sybr green Supermix (Bio-Rad) and oligonucleotides for murine cytokines (26). The threshold cycle (C_T) values for each target gene were normalized to β-actin gene expression, and the relative expression level of each target gene in infected mice was calculated with the formula n-fold change = , where C_T represents C_T (infected sample) – C_T (control). To better visualize the mRNA levels in all groups, bar graphs were generated by plotting the values on the y axis (16).

Tissue parasite burden and histopathology. Tissues (50 mg) were subjected to proteinase K lysis, and total DNA was isolated by phenol-chloroform extraction and ethanol precipitation. Total DNA (100 ng) was used as the template in a real-time PCR (as described above) with oligonucleotides specific for a sequence encoding the T. cruzi 18S rRNA (forward, 5'-TAGTCATATGCTTGTTTC-3'; reverse, 5'-GCAACAGCATTAATATACGC-3'). C_T values for the T. cruzi-specific signal were normalized to murine β-actin gene DNA levels.

For histological studies, heart and skeletal muscle sections were fixed in 10% buffered formalin for 24 h, dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin. Five-micrometer tissue sections were stained by hematoxylin and eosin and evaluated by light microscopy. Tissues were scored 0 to 4 in blind studies, according to the extent of inflammation and tissue damage, from normal to total wall involvement (15).

Statistical analysis. Data are expressed as means ± standard deviations (SD) and were derived from at least triplicate observations per sample (six or more animals per group). Results were analyzed for significant differences by using analysis-of-variance procedures and Student t tests. The level of significance was accepted as P < 0.05.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The development of an antibody response induced by the intramuscular immunization of mice with DNA vaccines was determined by use of an ELISA. T. cruzi-specific antibodies became detectable after a second immunization and were enhanced after a third dose (Fig. 1). We detected a substantial level of parasite-specific IgG, IgG1, and IgG2b antibodies in mice immunized with TcG1-, TcG2-, and TcG4-encoding plasmids (individually and in combination, P < 0.01). The antigen-induced antibody response was predominantly of the Th1 type, with IgG2b/IgG1 ratios of >1 (Fig. 1B). Delivery of the three antigen-encoding plasmids in a mixture induced a par level of the IgG and IgG1 responses and an up to 50% higher level of the IgG2b response than was noted in mice immunized with individual antigen-encoding plasmids (P < 0.01). Control mice immunized with plasmid vector alone or cytokine plasmids only exhibited no parasite-specific antibodies (Fig. 1; data not shown).

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FIG. 1. Vaccination with TcG1-, TcG2-, and TcG4-encoding plasmids induced a Th1 antibody response in mice. C57BL/6 mice were immunized with antigen-encoding plasmids plus IL-12 and GM-CSF expression plasmids thrice at 2-week intervals. Mix, mice immunized with a mixture of the three antigen-encoding plasmids. Normal mouse serum (NMS) and sera from mice injected with the empty plasmid (Vector) were used as controls. Shown are the serum levels of IgG (A) and IgG2b and IgG1 (B) antibody responses measured 2 weeks after the last immunization. Data (mean ± SD) are representative of three independent experiments. *, P < 0.05; **, P < 0.01.

Upon challenge infection with T. cruzi, vaccinated mice continued to exhibit an approximately two- to threefold higher level of parasite-specific IgG antibodies compared to unvaccinated controls (Fig. 2A, P < 0.01). A maximal antibody response was noted in TcG2-immunized mice (Fig. 2A). After 30 days p.i. (dpi), the overall IgG response appeared to be at par between vaccinated-and-infected and unvaccinated-and-infected mice (data not shown). The IgG2b/IgG1 levels in sera of immunized mice were higher than that noted in unvaccinated-and-infected mice at all stages of infection and disease development (Fig. 2B and C, P < 0.01). Mice immunized with a TcG1-encoding plasmid exhibited an early increase in the IgG2b/IgG1 ratio beginning at 30 dpi, while mice immunized with TcG2- and TcG4-encoding plasmids exhibited a substantial increase in the IgG2b/IgG1 ratio by day 75 p.i. (Fig. 2B and C). T. cruzi-specific IgG2a antibodies were detectable at days 75 and 120 p.i. and were not statistically significantly different among different groups (data not shown). We did not observe an inhibitory or competitive effect on antibody response elicitation in mice immunized with the mixture of antigen-encoding plasmids. Together, the results presented in Fig. 1 and 2 suggest that immunization with DNA vaccines resulted in the elicitation of a strong, parasite-specific, Th1-type antibody response that persisted after challenge T. cruzi infection.

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FIG. 2. Mice primed with antigen-encoding plasmid vaccines maintained a strong parasite-specific antibody response after a challenge infection with T. cruzi. Mice were vaccinated as described in the legend to Fig. 1 and infected with T. cruzi (2.5 x 104 parasites/mouse). The serum levels of T. cruzi-specific IgG, IgG2b, and IgG1 antibodies were measured during the acute (30 dpi, panel A), intermediate (75 dpi, panel B), and chronic (120 dpi, panel C) phases of infection and disease development. Data (mean ± SD) are representative of three independent experiments. Abbreviations are as in Fig. 1. None, unvaccinated mice challenged with T. cruzi; ND, not determined; *, P < 0.05; **, P < 0.01 (vaccinated-and-infected versus unvaccinated-and-infected mice).

In contrast to the antibody response, splenocyte activation and cytokine production were not enhanced in vaccinated-and-infected mice. Splenocytes were stimulated in vitro with antigenic lysate, and cytokine secretion was assessed via an ELISA. Splenocytes from unvaccinated mice (or mice injected with an empty plasmid vector), harvested at 30 days after a challenge infection, exhibited strong IFN- (Fig. 3A) and TNF- (Fig. 3B) production (P < 0.01). In comparison, IFN- production was decreased by >50% in splenocytes of TcG2- and TcG4-immunized and infected mice (Fig. 3A; P < 0.01, vaccinated versus unvaccinated). Splenocytes from vaccinated-and-infected mice exhibited a rate of TNF- production similar to that noted in infected controls (Fig. 3B). No cytokine release was observed when splenocytes from normal mice were incubated with T. cruzi antigenic lysate or when splenocytes from infected-and-immunized mice were incubated with nonspecific antigens (Fig. 3A and B, white bars). Incubation with ConA resulted in a substantial increase in cytokine levels in splenocytes from all animal groups (data not shown). These results suggest that splenocyte activation and proliferation and Th1 cytokine production were not the predominant protective responses elicited in TcG1-, TcG2-, and TcG4-immunized and infected mice.

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FIG. 3. Splenocyte production of cytokines in vaccinated mice. Mice were vaccinated with antigen-encoding plasmids and challenged with T. cruzi as described in the legend to Fig. 1. Splenocytes from vaccinated-and-infected and unvaccinated-and-infected mice were stimulated in vitro for 48 h with antigenic lysate (gray bars, 30 dpi) or no antigen (open bars), and the levels of IFN- (A) and TNF- (B) expression in cell-free supernatants were measured with an ELISA. Data (mean ± SD) are representative of three independent experiments. Abbreviations are as in Fig. 1 and 2. **, P < 0.01 (vaccinated-and-infected versus unvaccinated-and-infected mice).

We monitored the parasite burden in the heart tissue of vaccinated-and-infected mice by a sensitive real-time PCR with primers specific for the T. cruzi 18S rRNA gene (Fig. 4). Mice immunized with TcG1, TcG2, and TcG4 exhibited lower acute-phase (30 dpi) tissue parasite burdens of 66%, 50%, and 90%, respectively, compared to that detected in infected mice that were not vaccinated or were injected with the plasmid vector only (P < 0.01). Vaccinated mice continued to exhibit a 58 to 90% lower tissue parasite burden than did unvaccinated control mice during the intermediate stage (75 dpi, P < 0.05). At the chronic stage (120 dpi), a parasite-specific signal was undetectable in the heart tissue of TcG4-immunized mice and was detected at a very low level in mice vaccinated with TcG1- and TcG2-encoding plasmids (<10% of unvaccinated-and-infected controls) (Fig. 4). Infected control mice exhibited the consistent presence of tissue parasites. These results suggest that TcG1-, TcG2-, and TcG4-vaccinated mice were better equipped to control an acute-phase parasite burden that resulted in a substantially decreased persistence of parasites in tissue during the chronic phase.

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FIG. 4. Mice vaccinated with antigen-encoding plasmids exhibited substantial control of the tissue parasite burden. C57BL/6 mice were vaccinated with TcG1-, TcG2-, and TcG4-encoding plasmids plus cytokine adjuvants and challenged with T. cruzi. Heart total DNA was used as the template in a real-time PCR for amplification of the T. cruzi 18S rRNA-encoding sequence. The T. cruzi rRNA gene signal was normalized to the murine β-actin gene, and the data represent mean values obtained from two independent experiments with heart tissue of at least three mice per experiment. Unvaccinated mice (none) or mice injected with plasmid DNA (vector) before a challenge infection were used as positive controls. Specificity of the PCR for the T. cruzi rRNA gene was confirmed by the detection of no signal when total DNA isolated from normal mice was used as the template. The SD for all of the data points was <10%. *, P < 0.05; **, P < 0.01 (vaccinated-and-infected versus unvaccinated-and-infected mice).

To determine if T. cruzi-induced immunopathology is controlled in vaccinated mice, we assessed the serum and cardiac levels of the IFN- and TNF- cytokines and the proinflammatory infiltrate in the hearts of vaccinated-and-infected mice. Unvaccinated mice challenged with T. cruzi exhibited substantially high serum (Fig. 5A) and cardiac (Fig. 5B and C) levels of IFN- and TNF- at 30 dpi that persisted at moderate levels at days 75 and 120 p.i. (P < 0.01). In comparison, mice immunized with TcG1-, TcG2-, and TcG4-encoding plasmids exhibited significantly decreased serum and myocardial levels of inflammatory cytokines. We noted that the TNF- level in the serum from vaccinated-and-infected mice was 46 to 73% lower than in unvaccinated-and-infected mice (Fig. 5A, P < 0.05). IFN- in the sera of vaccinated-and-infected mice was almost undetectable after the acute phase (data not shown). The IFN- (Fig. 5B) and TNF- (Fig. 5C) mRNA expression in the myocardia of TcG1-, TcG2-, and TcG4-immunized mice was 50 to 80% less compared to that detected in unvaccinated-and-infected mice (Fig. 5B and C, P < 0.01).

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FIG. 5. Inflammatory cytokine response is decreased in vaccinated mice. Mice were vaccinated with antigen-encoding plasmids and challenged with T. cruzi. (A) Serum levels of TNF- at days 30 (gray bars), 75 (checkered bars), and 120 (dotted bars) p.i. (B and C) Myocardial levels of mRNAs for IFN- (B) and TNF- (C) were determined by real-time RT-PCR as described in Materials and Methods. Shown are the RT-PCR values for the cytokines normalized to β-actin gene expression. The data represent mean values obtained from two independent experiments with heart tissue of at least three mice per experiment. The SD for all of the data points in panels B and C was <10%. *, P < 0.05; **, P < 0.01 (vaccinated-and-infected versus unvaccinated-and-infected mice).

Histopathological analysis of tissue sections from vaccinated-and-infected mice showed an extensive infiltration of inflammatory cells in the heart (Fig. 6) and skeletal muscle (data not shown) during the acute phase. Irrespective of the immunization conditions, mice in all groups exhibited moderate-to-high inflammatory responses at 30 dpi (Fig. 6). The extent of inflammation and associated tissue damage in heart tissue and skeletal muscle during the chronic phase of infection was remarkably reduced in mice immunized with T. cruzi antigen-encoding plasmids (Fig. 6). The reduction of the extent of cardiac inflammation was in the order TcG4 < TcG1 < TcG2. In comparison, extensive moderate levels of cardiac and skeletal inflammation and tissue necrosis, the hallmarks of Chagas' disease, persisted in chronically infected animals that were not vaccinated or were injected with the empty vector only (Fig. 6D). These results demonstrate that the DNA vaccines used in this study were effective in controlling the tissue parasite burden and, consequently, the associated immunopathology in chronic Chagas' disease.

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FIG. 6. Control of chronic tissue inflammation in vaccinated mice. C57BL/6 mice were immunized with antigen-encoding plasmids and infected with T. cruzi. Shown are hematoxylin-and-eosin stained heart tissue sections obtained during the acute (30 dpi, panels C, E, G, and I) and chronic (120 dpi, panels D, F, H, and J) phases of infection and disease development. Tissue sections from age-matched normal mice are shown in panels A and B.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present study was performed to test the protective efficacy of three antigens (TcG1, TcG2, and TcG4) that we have previously identified, by computational analysis of a T. cruzi sequence database, as putative vaccine candidates. The three genes were found to be conserved among several clinically relevant T. cruzi strains and expressed as plasma surface proteins in infective trypomastigote and intracellular amastigote stages. The TcG1-, TcG2-, and TcG4-specific antibodies exhibited lytic activity against the infective trypomastigote form, suggesting their potential utility as vaccine candidates (2).

We immunized mice with TcG1-, TcG2-, and TcG4-encoding plasmids along with cytokine expression plasmids. The IL-12 and GM-CSF cytokines were chosen as adjuvants because we and others have found that codelivery of these cytokines enhances the magnitude and characteristics of the immune response elicited by DNA vaccines (reviewed in reference 12). GM-CSF was chosen because it is known to enhance the antigen presentation capability of dendritic and other antigen-presenting cells and to facilitate B- and T-cell-mediated immunity. IL-12 is considered to be the key cytokine involved in directing the immune responses to type 1. Accordingly, mice immunized with TcG1-, TcG2-, and TcG4-encoding plasmids elicited a strong parasite-specific antibody response that remained intact after a challenge infection with T. cruzi (Fig. 1 and 2). The levels of both the IgG2b and IgG1 antibody isotypes were significantly elevated in immunized mice, and the antibody response was primarily of the Th1 type, with IgG2b/IgG1 ratios being >1 during the acute phase of infection and chronic disease development. Studies have shown that the lytic antibodies involved in parasite clearance upon T. cruzi infection are of the IgG1 and IgG2 isotypes (4, 24). Others have shown in experimental models that the control of T. cruzi infection by preimmunization with a KMP11-encoding plasmid was associated with elicitation of the IgG2a isotype (23), while the IgG1 and IgG2b isotypes were found to be the predominant antibody isotypes when mice were immunized with the complement-reactive protein and TS antigens (8, 31). We have shown that TcG1-, TcG2-, and TcG4-specific antibodies are lytic in nature and efficiently killed trypomastigotes in a complement-dependent manner (2). Subsequently, all vaccinated mice exhibited significant control of the tissue parasite burden during the acute phase. Mice immunized with TcG4 and TcG1 exhibited better control of T. cruzi infection than did TcG2-immunized mice (Fig. 4), and this protection was correlated with the persistence of a high ratio of serum levels of IgG2b/IgG1 antibodies. At the intermediate and chronic stages, the parasite burden determined by a highly sensitive real-time RT-PCR approach was almost undetectable in immunized-and-infected mice. We surmise that elicitation of a strong IgG2b and IgG1 lytic antibody response by DNA vaccines encoding TcG1, TcG2, and TcG4 contributed to the effective control of T. cruzi infection.

An assortment of studies with either knockout mice or infected animals treated with antibodies to deplete specific immune molecules have shown that, besides the T. cruzi-specific lytic antibody response, efficient control of acute parasitemia also requires concerted activities of macrophages, T helper cells, and cytotoxic T lymphocytes (reviewed in reference 35). The IFN- and TNF- cytokines are believed to be the key activators of macrophage NO production and oxidative burst for parasite clearance in the early stages of T. cruzi infection (17). CD4⁺ Th1 helper cells stimulate type 1 cytokine production and have been implicated in the induction of protective immunity, while CD8⁺ T cells are considered important for the killing of infected cells (3, 28). Interestingly, in this study, the analysis of splenocyte proliferation and cytokine production in mice immunized with TcG1-, TcG2-, or TcG4-encoding plasmids showed a substantially reduced proinflammatory response compared to that detected in unvaccinated-and-infected control mice at all stages of infection and disease development (Fig. 3). It was particularly surprising that, even at the acute stage (30 dpi), IFN- production by splenocytes incubated in vitro with T. cruzi antigenic lysate was significantly lower in immunized mice compared to infected controls. Despite a compromised cytokine profile, all groups of vaccinated mice were better equipped to control the tissue parasite burden throughout the course of infection and disease development. We surmise that the lytic antibody response elicited by immunization with TcG1-, TcG2-, and TcG4-encoding plasmids was sufficiently strong to control the acute phase of infection and the tissue parasite burden in vaccinated mice. This notion is supported by other studies in which mice were immunized with an avirulent CL14 clone of T. cruzi and challenged with a virulent CL strain of T. cruzi. The latter cited elevated IgG1 and IgG2b isotype antibody levels and diminished IFN- levels, as well as substantially low parasitemia and a decreased percentage of mortality, in comparison to control infected mice that were not primed with an avirulent strain (21, 25, 32).

A major criticism of vaccine development against T. cruzi has been that elicitation of stronger, potent immune responses by vaccine may exacerbate immune pathology. The persistence of inflammatory responses associated with tissue necrosis and cell death are hallmarks of chronic Chagas' disease. It has been suggested that CD8⁺ T cells, the dominant resident immune cells in the heart, have a toxic effect on the host, as many of the CD8⁺ T cells express granzyme A, which results in nonspecific bystander cell death and tissue necrosis (27). Others have shown that the frequency of IFN--producing T cells specific for T. cruzi correlate with the severity of chronic disease in chagasic humans and experimental animals (7, 33). Circulating levels of IFN- and its production by peripheral blood mononuclear cells stimulated in vitro with T. cruzi antigen have been implicated as risk factors in identifying asymptomatic, seropositive patients at risk of developing symptomatic chronic cardiomyopathy (7). In this study, we indeed observed, irrespective of vaccination status, a substantially high infiltration of mononuclear cells in the heart and skeletal muscle in response to a challenge infection with T. cruzi. However, as the chronic phase developed, mice vaccinated with antigen-encoding plasmids exhibited minimal immunopathology. This was evidenced by substantially lower serum and cardiac levels of inflammatory cytokines and minimal to no detection of parasite 18S rRNA gene-specific signal and inflammatory infiltrate in the heart tissue of vaccinated-and-infected mice during the chronic disease phase (Fig. 4 and 6). Similar observations were made with mice immunized with a nonpathogenic strain (CL14 or TCC) and challenged with a virulent strain (CL or Tulahuen) of T. cruzi (1, 21). Substantially high levels of inflammatory infiltrate in the heart, skeletal muscle, and intestinal tissue were noted in response to a challenge infection. As the chronic phase developed, the tissue parasite burden and associated inflammation were marginal in mice immunized with an attenuated strain while those directly challenged with a virulent strain maintained severe symptoms of Chagas' disease (1, 21). Experimental studies with mice suggest a clear correlation of the acute-phase parasite burden, parasite persistence, and the severity of cardiomyopathy symptoms during the chronic phase (37). On the basis of these studies and our data, we conclude that the strong type 1 antibody response elicited in TcG1-, TcG2-, and TcG4-immunized mice (Fig. 1 and 2) prevented parasite replication and propagation in the host (Fig. 4). Though complete parasite clearance was likely not achieved, vaccinated mice controlled the parasite burden below a threshold level, resulting in limited to no activation and infiltration of inflammatory cells in the heart and absence of tissue destruction during the chronic phase (Fig. 6).

In summary, we have demonstrated that vaccination of mice with antigen-encoding plasmids provided resistance to T. cruzi infection and chronic inflammatory pathology of the heart in the order TcG4 < TcG1 < TcG2. The elicitation of a strong IgG2b/IgG1 antibody response in immunized mice contributed to parasite control. Subsequently, splenic production of inflammatory cytokines and cardiac infiltration of inflammatory infiltrate were reduced in vaccinated mice. Our results validate the utility of a bioinformatic-computational approach to the identification of a potent vaccine candidates against T. cruzi.

 
This study was supported in part by grants from the American Heart Association (0160074Y) and the National Institutes of Health (AI072538) to N.J.G. V.B. was supported by a postdoctoral fellowship from the Sealy Center of Vaccine Development at the University of Texas Medical Branch.

We thank Mardelle Susman for editing the manuscript.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, 3.142C Medical Research Building, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1070. Phone: (409) 747-6865. Fax: (409) 747-6869. E-mail: nigarg{at}utmb.edu

Published ahead of print on 11 June 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Clinical and Vaccine Immunology, August 2008, p. 1158-1164, Vol. 15, No. 8
1071-412X/08/$08.00+0     doi:10.1128/CVI.00144-08
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