We followed the National Institutes of Health guidelines for housing and care of laboratory animals. All animal experiments were conducted in accordance with the protocols that were approved by the Institutional Animal Care and Use Committee (protocol number 08-05-029) at the University of Texas Medical Branch at Galveston. All experiments were conducted in ABSL2/BSL2-approved laboratory and all personnel have received appropriate ABSL2/BSL2 training.

The sequences for cDNAs for TcG2 and TcG4 are deposited in the GenBank (AY727915 and AY727917, respectively). The full-length cDNAs for the two antigens were previously cloned into pCDNA3.1 eukaryotic expression plasmid and the recombinant plasmids were purified by anion exchange chromatography by using a Qiagen Endo-free maxi prep kit (Qiagen, Chatsworth, CA) (9, 10). A CMV promoter-based NTC9385R-MCS nanoplasmid (Nature technology, Lincoln, NE) was utilized for sub-cloning the full-length genes encoding for TcG2 and TcG4 (18). The recombinant nanoplasmids NTC9385R-TcG2 and NTC9385R-TcG4 were sequenced to confirm the orientation and open reading frame, and purified by using the HyperGRO fermentation process (18).

Mouse myoblast C2C12 cells (ATCC CRL-1772) were propagated in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM sodium pyruvate. Trypomastigotes of T. cruzi SylvioX10/4 strain were amplified in vitro in C2C12 cells (19).

C57BL/6 mice (6-week-old) were purchased from Jackson laboratory (Bar Harbor, ME), and randomly distributed in four groups: (1) mock treatment/mock infection; (2) mock treatment/T. cruzi infection; (3) two doses of p2/4 followed by T. cruzi infection; and (4) two doses of nano2/4 followed by T. cruzi infection. The size of the backbone of NTC9385R nanoplasmid is approximately half of the size of backbone of eukaryotic expression plasmid pcDNA3.1. To compensate for the differences in backbone size and deliver similar amounts of antigen-encoding DNA, the vaccine referred as p2/4 was constituted of 25 µg each of pCDNA3.1-TcG2 and pCDNA3.1-TcG4 and the vaccine referred as nano2/4 was constituted of 12 µg each of NTC9385R-TcG2 and NTC9385R-TcG4. Both vaccines were constituted in 50 µl PBS and delivered by intramuscular injection in the hind thighs in two doses at 21 days interval. Mice in groups 2, 3, and 4 were challenged with T. cruzi trypomastigotes (10,000 parasites per mouse) via intraperitoneal route at 21 days after the 2^nd immunization and euthanized on day 10 and day 21 post-infection (pi). The rational for choosing 10 days and 21 days pi was to check the efficacy of the vaccine against parasite dissemination and replication, respectively, based on our previous published studies (20).

To examine the vaccine efficacy against re-exposure, some mice in groups 2, 3, and 4 were re-challenged on day 21 after first infection and euthanized 7 days after the second parasite exposure. Mice were daily monitored for mortality during the study period. Sera and splenocytes were obtained for evaluating the cytokines and T cell responses in vaccinated and non-vaccinated mice after infection and reinfection. Heart, skeletal muscle, and spleen tissues were collected to monitor the parasite burden and histopathology. The schematic timeline of immunization, infection and re-challenge is presented in Figures 1A and 4A .

To evaluate the parasite burden at various stages of infection and reinfection, total DNA was purified from spleen, heart and skeletal muscles tissues by standard procedures. Total DNA (50 ng) was used as template and added to the reaction mix consisting SYBR Green Supermix (Bio-Rad) and oligonucleotides specific for Tc18SrDNA (forward, 5′-TTTTGGGCAACAGCAGGTCT-3′; reverse, 5′-CTGCGCCTACGAGACATTCC-3′; amplicon size: 199 bp) or murine Gapdh (forward, 5′-AACTTTGGCATTGTGGAAGG-3′; reverse, 5′-ACACATTGGGGGTAGGAACA-3′; amplicon size: 223 bp), and a real-time quantitative PCR was performed for 40 cycles on an iCycler thermal cycler. The threshold cycle (C_T ) values for Tc18SrDNA were calculated and normalized to Gapdh reference cDNA. The relative burden of T. cruzi in each sample was calculated by the following formula: 2 − Δ C t [ 2 ( − Δ C t   s a m p l e ) / 2 ( − Δ C t   o f   c o n t r o l ) ] as previously described by us (21).

Heart and skeletal muscle tissue sections were fixed by incubation in 10% buffered formalin for 24 h and dehydrated in absolute alcohol. Tissue samples were then cleared in xylene and embedded in paraffin. Slides containing 5-micron paraffin-embedded tissue sections were stained with hematoxylin (stains nuclei blue) and eosin (stains extracellular matrix and cytoplasm pink). The H&E stained tissue slides were imaged at 20 × magnification by using an Olympus BX-15 microscope (Center Valley, PA) equipped with digital camera and Simple PCI software (v.6.0, Compix, Sewickley, PA). The inflammatory infiltrate and tissue damage were scored as described previously (22). Briefly, scoring was defined as (0) - absent/none, (1) - focal or mild with ≤1 foci, (2) - moderate with ≥2 inflammatory foci, (3) - extensive with generalized coalescing of inflammatory foci or disseminated inflammation, (4) - severe with diffused inflammation, interstitial edema, and loss of tissue integrity (22). For each group, data were captured from at least three mice, two slides per tissue, and ten microscopic fields per slide, and presented as mean value ± standard error mean (SEM).

Spleen samples were macerated, and single-cell suspensions of spleen cells were washed with flow cytometry staining buffer (00-4222-26, eBioscience, San Diego, CA) and incubated for 10 min with Fc Block (anti-CD16/CD32; BD Pharmingen). Splenocytes (5 × 10⁴/50 ml) were incubated in dark with the fluorochrome-conjugated monoclonal antibodies to surface molecules (concentration determined by titration) for 30 min at 4°C, washed twice in cold staining buffer, fixed and permeabilized by incubation with fixation and permeabilization solution (BD Biosciences, San Jose, CA) for 20 min, and washed with perm wash buffer (BD Biosciences) (23). For analyzing intracellular molecules, cells were stained with monoclonal antibodies against cytokine (IFN-γ) or cytotoxic molecules perforin (PFN) and granzyme B (GZB) for 30 min, washed, and resuspended in staining buffer. All samples were visualized and analyzed using the 10-color BD LSRII Fortessa flow cytometer. As controls, tubes containing unstained cells and cells incubated with isotype matched IgG, live/dead stain and FMO controls were included. Data were acquired and analyzed by using a FlowJo software (v.10.5.3; TreeStar, San Carlo, CA) (18). All antibodies are listed in Table S1 .

To establish parameters for distinguishing T cell subpopulations, we generated concatenated fcs file from all samples of the four groups. The concatenated file was filtered to remove dead/doublet cells and select on an average 1 × 10⁵ of CD3⁺ T live cells per sample by FlowJo Downsample platform. The resultant cumulative dataset was analyzed by FlowSOM FlowJo plugin, with the grid dimensions set to 7 × 7 that created a minimum spanning tree with 49 nodes ( Figure S1A ) distributed to 10 metaclusters named P0–P9 ( Figure S1B ) (24, 25). Heatmap ( Figure S1C ) was constructed to capture the identity of the T cell metaclusters based on the expression levels of the five surface markers, presented as relative median fluorescence intensity (MFI) in Figure S1D . We also utilized cumulative dataset to generate common t-distributed stochastic neighbor embedding (t-SNE) ( Figure S1E ) using parameters: iteration = 1,000, perplexity = 30, and learning rate (eta) = 4900 that allows reduction of dimensionality and is well suited for the visualization of high-dimensional datasets (26, 27).

To identify the changes in T cell subsets in treatment-specific manner, fcs files were individually filtered to remove dead/doublet cells and select 1 × 10⁵ of CD3⁺ T cells. Parameters defined in cumulative FlowSOM metacluster map ( Figure S1B ) were then applied to individual files to identify treatment- and time-specific changes in T cell subsets. Likewise, parameters defined in cumulative t-SNE map ( Figure S1E ) were applied to generate treatment- and time-specific t-SNE maps.

T effector/effector memory (T_EM) and T central memory (T_CM) sub-populations were further examined for median fluorescent intensity for intracellular IFN-g cytokine and PFN and GZB cytotoxic activity markers.

Serum levels of cytokines, including TNF-α, IFN-γ, IL-1β, and IL-6, were measured by using sandwich ELISA kits according to the instructions provided by the manufacturer (Invitrogen).

All datasets were acquired from a minimum of five mice per group (minimum of duplicate observations per mouse per experiment) and managed in GraphPad Prism5 software. Data are presented as mean value ± SEM. Significance was analyzed by Student’s t test (comparison of 2 groups) and one-way analysis of variance (ANOVA) with Tukey’s post-hoc test or Kruskal–Wallis H/Dunn’s post-hoc test (comparison of multiple groups). Unless specified otherwise, significance is annotated in figures as ^* T. cruzi vs. control, ^{^} T. cruzi vs. p2/4.Tc, ^# T. cruzi vs. nano2/4.Tc, and ^&p2/4.Tc vs. nano2/4.Tc, and one, two, and three symbol characters were used to represent the p-values of ≤0.05, ≤0.01, and ≤0.001, respectively.

Schematics of immunization and challenge protocol is presented in Figure 1A . Briefly, mice were immunized at 21 days interval with two doses of p2/4 or nano2/4 encoding TcG2 and TcG4. Mice were challenged on day 42 and euthanized on day 10 and day 21 post-infection corresponding to the early and acute parasitemic phase, respectively.

We first determined the nanovaccine efficacy by qPCR evaluation of tissue parasite burden. Mice infected with T. cruzi exhibited very high levels of parasite burden in heart and skeletal tissue and lower levels of parasite burden in spleen at day 10 and day 21 post-infection ( Figures 1B–D ). In comparison, mice given prophylactic p2/4 or nano2/4 exhibited a significant decline in heart, skeletal, and splenic tissue levels of parasite burden at 10 days (101–597 fold, 31–159 fold, and 46–103 fold decline, respectively) as well as at 21 days (10–37 fold, 10–44 fold, and 50–120 fold, respectively) post-infection ( Figures 1B–D , all ^{^,#} p <0.01). The nano2/4 exhibited better efficacy in controlling tissue dissemination and replication of the parasite than was noted with p2/4 vaccine ( Figures 1B–D , ^&p < 0.05).

The histological examination of the tissue sections revealed extensive inflammatory infiltrate in the heart (histology score: 2.59) and skeletal muscle (histology score: 3.33) of infected (vs. non-infected) mice at 21 days pi ( Figures 1E.a–d, 1F ). While myocardial inflammation was more diffused, skeletal tissues exhibited diffused as well as localized large foci of inflammatory infiltrate in infected mice ( Figures 1E.c, d ). Importantly, p2/4 and nano2/4 immunization led to notable control of infiltrating cells in the heart tissue (histology score: 0.31-0.4, ^{^,#} p < 0.001) and skeletal muscle (histology score: 1.02–1.48, ^{^,#} p < 0.001) ( Figures 1E, compare c and d with e–h, 1F ). Slightly better efficacy of nano2/4 (compared to p2/4) was observed in reducing the skeletal tissue inflammatory infiltrate (^&p < 0.01) and preserving the tissue integrity in infected mice. The uninfected control mice exhibited no detectable parasite DNA and inflammatory infiltrate in their tissues ( Figures 1B–F ). These results suggest that the TcG2/TcG4 based subunit vaccine (nano2/4>p2/4) offered protection from T. cruzi infection and acute parasitemic phase and consequently reduced the tissue inflammatory infiltrate in infected mice.

We enumerated the T cell profile in mice at 10 days pi to examine if vaccine triggers the T cell activity immediately after infection and at 21 days pi to examine if nanovaccine enhances the potency of T cell activity against replicating parasite. For this, splenocytes of all groups of mice were analyzed by flow cytometry as detailed in Materials and Methods.

The group-specific tSNE plots of the ten metaclusters at the beginning of acute phase (i.e., 10 days pi) are presented in Figures 2A–D , and mean values (± SEM) of these T cell subsets are presented in Figure 2E and Table S2A . These data showed that CD4⁻CD8⁻ double negative (DN) T cells of naïve (P9: 0.76%–3.46%), T_EM (P4: 0.25%–0.92%), or T_CM (P6: 1.18%–3.12%) phenotypes constituted a small proportion of the total T cells in all the treatment groups. The non-vaccinated/infected (vs. vaccinated/infected) mice exhibited higher levels of premature CD4⁺T_o subset (P1) and a decline in the CD4⁺ T naïve (T_N) subset (P2), while immunized mice exhibited significant increase in CD4⁺T_EM subset (P0, nano2/4.Tc > p2/4.Tc). Regarding CD8⁺T cells, we observed two metaclusters of naive cells, including CD62L^hiCD44^lo (P8) subset that exhibited true naive phenotype and CD62L^intermediateCD44^low (P3) subset that was categorized as activated naïve subset ( T N L ) . We observed no major changes in the frequencies of the CD8⁺ T cell subsets in any of the infected (vs. non-infected) groups of mice at 10 days pi. At a functional level, the IFN-γ expression was increased in CD4⁺T_EM (P0) subset in vaccinated/infected (nano2/4.Tc > p2/4.Tc) mice ( Figure 2F and Table S3A ). The PFN production was increased in all of the T_EM and T_CM subsets in infected mice (nano2/4.Tc > p2/4.Tc or Tc only, Figure 2G and Table S3A ). Except for the GZB-expressing DN and CD8⁺ T_EM populations, the maximal increase in GZB expression was also noted in nano2/4.Tc mice (vs. p2/4.Tc or Tc only, Figure 2H and Table S3A ). These results suggest that nano2/4 was most effective in eliciting an early T cell response to T. cruzi infection evidenced by a) transition of the CD4⁺T cells into effector phenotype, b) IFN-γ production by CD4⁺T_EM subset; and c) production of cytolytic molecules (PFN/GZB) by T cell subsets of T_EM and T_CM phenotypes in nano2/4.Tc (vs. p2/4.Tc or Tc only) mice.

To capture the T cell profile in vaccinated and non-vaccinated mice during parasite replication phase, we defined and visualized the abundance of ten T cell meta-clusters in each group at 21 days pi ( Figures 3A–D and Table S2B ). These data showed no major change in the frequencies of DN T subsets (P9, P6, P9) and a decline in the frequencies of CD4⁺ and CD8⁺ naïve T cells (P2 and P8) in all of the infected groups (vs. non-infected/control group) ( Figure 3E ). Further, CD4⁺T_EM subset (P0) was highest in p2/4.Tc mice while the C D 8 + T N L and CD8⁺T_EM subsets (P3 & P5) were highest in nano2/4.Tc mice ( Figure 3E ). At a functional level, we observed significant increase in IFN-γ production by all T_EM and T_CM subsets in vaccinated/infected mice (nano2/4.Tc > p2/4.Tc) while only T_EM subsets produced IFN-γ in non-vaccinated/infected mice ( Figure 3F and Table S3B ). Likewise, PFN production was enhanced in all T_EM and T_CM subsets of vaccinated/infected mice (nano2/4.Tc > p2/4.Tc), while fewer T cell subsets produced low levels of PFN in non-vaccinated/infected mice ( Figure 3G and Table S3B ). The GZB expression was noticeably enhanced in CD8⁺T_EM subset only in vaccinated/infected mice ( Figure 3H and Table S3B ). These data suggest that without the help of a vaccine, host responds to T. cruzi by delayed and sub-par T cell activation and low levels of production of effector molecules. Prophylactic immunization with nano2/4 (more effective than p2/4) enhanced the differentiation of naïve T cells toward CD4⁺ and CD8⁺ T_EM and T_CM phenotypes and increased the production of IFN-γ that is critical for induction of innate/adaptive immunity and of cytolytic molecules (PFN, GZB) that are required for controlling the intracellular pathogen.

Comparative analysis of T cell profile at 21 days vs. 10 days pi is presented in Figure S2 . Though present at minimal frequencies, DN T_N subset decreased, DN T_EM increased and DN T_CM subset was not changed in infected mice ( Figure S2A ). Further, CD4⁺T_o subset (P1) was decreased in non-vaccinated/infected mice, CD4⁺T_N subset (P2) was decreased in vaccinated/infected mice (nano2/4.Tc > p2/4.Tc), and CD4⁺ T_EM population (P0) was increased by 44.9% in p2/4.Tc and decreased by 14.5% in nano2/4.Tc mice at 21 days (vs. 10 days) pi. With respect to CD8⁺T cells, the CD8⁺T_N subset was decreased and the C D 8 + T N L (178-644%) and CD8⁺T_EM (994%–2390%) subsets were increased in all infected groups at 21 days (vs. 10 days) pi, and maximal changes were noted in nano2/4-immunized mice. The CD8⁺T_CM subset was increased in non-vaccinated/infected mice only at 21 days (vs. 10 days) pi ( Figure S2A ). When comparing the functional response, the IFN-γ expression level was increased in all of the T_EM and T_CM subsets in infected mice at 21 days (vs. 10 days) pi, and CD8⁺T_EM and T_CM subsets exhibited maximal increase in IFN-γ production in nano2/4.Tc mice ( Figure S2B ). The PFN production was increased in all the T_EM and T_CM subsets in vaccinated/infected mice while fewer effector T cell subsets exhibited increase in PFN production in non-vaccinated/infected mice at 21 days (vs. 10 days) pi ( Figure S2C ). In comparison, GZB production was generally decreased in all infected groups, while only CD8⁺T_EM subset exhibited increaser in GZB levels in vaccinated/infected groups at 21 days (vs. 10 days) pi ( Figure S2D ). These our results suggest that at the peak of acute infection, maximum proliferation of CD8⁺ T_EM takes place in nano2/4-immunized mice, while CD4⁺T_EM subset was still expanding in p2/4-immunized mice. The observation of a decline in T cell expansion in nanovaccine immunized mice, when comparing 21 days vs. 10 days pi ( Figure S2A ), indicates that the proliferation time of effector phenotypes of both CD4⁺ and CD8⁺ T cells occurred early on and this vaccine composition reduced the time required for better resolution of infection. Overall, nano2/4 was more effective than p2/4 in signaling activation of CD4⁺ and CD8⁺ T cells at a faster rate, and in stimulating the production of high amounts of the intracellular effector molecules.

To determine if vaccine- and T. cruzi-induced immunity is protective against re-infection, we challenged mice 21 days after the 2^nd immunization with p2/4 or nano2/4, and then re-challenged with T. cruzi 21 days after the first exposure. Mice were monitored daily and surviving mice were euthanized 7 days after 2^nd infection to examine tissue pathology ( Figure 4A ).

Upon re-infection with T. cruzi, non-vaccinated/infected mice exhibited clinical features of sickness (hunched posture, ruffled fur, lethargic), and 59% (10 out of 17) of non-vaccinated/infected mice succumbed within 7 days after re-infection ( Figure 4B ). The surviving non-vaccinated/re-infected mice exhibited very high levels of tissue parasite burden in heart, skeletal muscle, and splenic tissues that were 2-fold, 4-fold, and 1.5-fold higher than was observed after 1^st infection (compare Figures 4C–E with Figures 1B–D ). No mortality was observed in re-infected mice that had received prophylactic p2/4 or nano2/4 vaccine as was also noted in mock-infected/mock-vaccinated control mice ( Figure 4B ). Parasites were almost undetectable in tissues of immunized mice after re-challenge with T. cruzi. We noted 44- to 65-fold, 93- to 102-fold, and 501- to 643-fold lower levels of parasite burden in heart, skeletal muscle and splenic tissue of mice given prophylactic p2/4 or nano2/4 compared to what was observed in non-vaccinated/infected mice after re-challenge infection ( Figures 4C–E ). Furthermore, tissue parasite burden in vaccinated mice after re-challenge was even lower than that noted after 1^st exposure (compare Figures 4C–E with Figures 1B–D ).

Extensive and large foci of tissue inflammatory infiltrate were observed in heart (histology score: 2.92) and skeletal muscle (histology score: 3.51) tissues of mice at 7 days post re-infection ( Figures 4F.c, d, G ). Tissue inflammatory foci and tissue degeneration were more pronounced after re-infection in non-vaccinated mice (compare Figures 4F.c, d with Figures 1E.c, d ). The p2/4- and nano2/4-immunized mice showed noteworthy decline in the inflammatory infiltrate in the heart (histology score: 0.74-0.96, ^{^} p < 0.001) and skeletal muscle (histology score: 1.14–1.5, ^{^} p < 0.001) tissue as compared to what was observed in non-vaccinated/infected mice ( Figures 4F, compare c and d with e–h, 4G ). Together, these results suggest that a) T. cruzi-induced immunity offered no protection from repeat exposure to the same pathogen isolate in this murine model; b) TcG2- and TcG4-based vaccines offer significantly better control of tissue parasite burden after re-infection than was observed after 1^st infection; and c) vaccine-induced parasite control was associated with pronounced decline in tissue inflammatory pathology and tissue injuries.

As above, we first defined and visualized the abundance of 10 meta-clusters of T cells in vaccinated and non-vaccinated mice at 7 days post reinfection by t-SNE plots ( Figures 5A–D ). The mean values (± SEM) of the T cell subsets are presented in Figure 5E and Table S2C , and functional profile of the T cell subsets are presented in Figures 5F–H and Table S3C . Comparative analysis of T cell profile at 7 days after re-infection vs. 21 days post 1^st infection is presented in Figure S3 .

Upon re-challenge, mice reverted to T cell effector phenotypes as was observed at early stage of 1^st infection (i.e., 10 days pi in Figure 2 ). The DN T subsets (P9, P4, and P6) largely remained non-responsive and CD8⁺ naïve subset (P3) contracted in all infected (vs. control) groups, and CD4⁺T_o were primarily enhanced in non-vaccinated/infected mice ( Figure 5E and Table S2C ). The CD4⁺T_EM subset (P0) was increased in all infected (vs. control) groups (p2/4.Tc = nano2/4.Tc > Tc only, Figure 5E and Table S2C ), and maximal percentage increase in CD4⁺T_EM subset at 7 days after re-challenge (vs. 21 days after 1^st infection) was observed in nano2/4.Tc mice ( Figure S3A ). The CD8⁺T_EM subset (P5), though in contraction phase when compared to that observed at 21 days after 1^st infection ( Figure S3A ), remained significantly higher in nano2/4.Tc mice compared to other groups of infected mice ( Figure 5E ). Functional analysis of T cell subsets after reinfection showed that the IFN-γ production was practically absent in DN T cell subsets and CD8⁺T_CM subpopulation, and maintained at higher levels in CD4⁺T_EM and CD8⁺T_EM subsets in vaccinated (nano2/4.Tc> p2/4.Tc) mice but not in non-vaccinated/infected mice ( Figure 5F ). Overall intensities of PFN and GZB in all of the T_EM and T_CM subsets were very high after re-infection in vaccinated mice (nano2/4.Tc > p2/4.Tc) while non-vaccinated/infected mice continued to produce none-to-low levels of PFN/GZB after reinfection ( Figures 5G, H ). When comparing the effector molecule levels after re-challenge with that observed at 21 days post 1^st infection, the intensity of IFN-γ production was increased or maintained at high levels in CD4⁺ and CD8⁺ T_EM subsets (P0 & P5) in vaccinated groups ( Figure S3B ). The PFN expression was maintained at high levels in all T_EM and T_CM subsets in vaccinated/infected mice. With respect to GZB production, while CD8⁺T_EM (P5) were contracting, CD4⁺T_EM and CD8⁺T_CM (P0 & P7) subsets exhibited increased GZB expression in vaccinated/infected mice ( Figure S3D ). All the effector molecules exhibited a downward trend in non-vaccinated/infected mice after re-challenge ( Figures S3B–D ). Overall, these results suggest that upon re-challenge, vaccinated mice were able to enhance or maintain the CD4⁺ and CD8⁺ T_EM sub-populations that produced high levels of effector molecules (nano2/4>p2/4). In comparison, non-vaccinated/infected mice responded to re-challenge with no increase in effector T cell populations and an overall decline in their production of effector/cytotoxic molecules.

Finally, we measured plasma levels of TNF-α, IFN-γ, IL-6, and IL-1β cytokines by an ELISA to determine if systemic secretion of cytokines offers an indication of anti-parasite protective immunity after infection and re-infection. We noted a potent increase in plasma levels of inflammatory cytokines in all infected groups of mice. The p2/4- and nano2/4-immunized mice elicited high plasma levels of TNF-α and IFN-γ at 10 days pi that were maintained in high range at 21 days pi and at 7 days after re-infection ( Figures 6A–C ). The TNF-α levels in non-vaccinated/infected mice were comparable to vaccinated/infected mice at 10 days pi, lowest at 21 days pi and spiked again in response to re-infection. Specifically, plasma IFN-γ levels were increased by 1.84-fold, 4.3- to 5.7-fold, and >50-fold in vaccinated/infected (vs. non-vaccinated/infected) mice at day 10 and day 21 pi and 7 days after re-infection, respectively (^p < 0.05, Figures 6A–C ). The plasma levels of IFN-γ in non-vaccinated/infected were significantly induced, albeit at lower levels than in vaccinated/infected mice, at 10 days pi and after that very low levels of IFN-γ were detectable during the acute parasitemic phase (i.e., 21 days pi) as well as after re-infection in non-vaccinated/infected mice ( Figures 6A–C ). The increase in TNF-α + IFN-γ was maximal in vaccinated mice (nano2/4.Tc ≥ p2/4.Tc > Tc only) and correlated with the control of parasite burden at all stages of infection and re-infection. The IL-6 levels were higher in p2/4.Tc group as compared to that noted in nano2/4.Tc group at all stages of infection and reinfection. In non-vaccinated/infected mice, IL-6 levels were high at 10 days pi and then it remained at low levels at 21 days pi and 7 days after reinfection. No detectable increase in IL-1β was observed in plasma of any of the infected groups of mice (data not shown). These results, along with the parasite burden data presented in Figures 1 and 4 , suggest that while T. cruzi infection induces systemic cytokine response, it is not maintained and fails to control parasite dissemination and replication as well as fails to respond to and provide protection from re-infection. Immunization with p2/4 and nano2/4 elicited sustained TNF-α– and IFN-g–dominated systemic immunity that correlated with control of parasite dissemination and replication as well as protection from repeat infections.