BALB/c and C57BL/6 female mice (6-8 weeks old) used in all experimental procedures were obtained from the Centro de Medicina Comparada (CMC) of Universidad Nacional del Litoral (UNL), Argentina. All protocols for animal studies were approved by the Advisory Committee of Ethics and Research Safety (CAESI) of the Facultad de Bioquímica y Ciencias Biológicas, UNL, Argentina, according to international ethical guidelines for biomedical research involving animals (CAESI 2019-7/19).

Tulahuen blood trypomastigotes were obtained from the blood of Cb1-infected mice, as previously reported (25, 43). Cb1 is an outbreeding albino strain that is bred in the animal facilities of the Centro de Investigación y Producción de Reactivos Biológicos (CIPReB-FCM-UNR), following protocols for animal studies approved by the Institutional Bioethics and Biosecurity Committees (Resolution N 3913/2008). Dm28c trypomastigotes were obtained in vitro from VERO cells cultured in complete RPMI 1640 medium (GibcoTM) supplemented with 10% fetal bovine serum (Internegocios SA) and 2% penicillin (100 mg/mL) and streptomycin (100 U/mL) (Gibco TM).

Parasites from RA strain (47) were maintained by weekly intraperitoneal inoculation of three weeks-old male CF1 mice (1 × 10⁵ parasites/mouse). RA bloodstream forms (Tp) were obtained from whole blood at the peak of parasitemia 7 days post-infection. RA strain was exclusively used to infect BALB/c male mice according to protocols CD N° 04/2015 approved by the University of Buenos Aires´s Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) in accordance with the Council for International Organizations of Medical Sciences (CIOMS) and International Council for Laboratory Animal Science (ICLAS) international ethical guidelines for biomedical research involving animals.

BALB/c male mice were immunized and challenged with RA parasites as described in Supplementary Figure 7 .

BALB/c mice (n=5-10/group) were immunized with three subcutaneous doses, one every two weeks, containing 10 µg of a trans-sialidase antigen (TSf), (genBank AJ276679.1), prepared as previously described (26), with 3 µl of ISPA as adjuvant (group TSf-ISPA). ISPA is a cage-like particle adjuvant prepared as previously described, using dipalmitoylphosphatidylcholine (DPPC), cholesterol (CHOL), stearylamine (STEA), tocopherol (TOCOP), and Quillaja Saponaria (QuilA) (28).

The 5FU (Sigma Aldrich) was administered at a dose of 50mg/kg, a dosage previously described to selectively deplete MDSCs in several mice studies (29, 48) and even during T. cruzi infection (21, 22). BALB/c or C57BL/6 mice were treated with 5FU one day before TSf-ISPA vaccine (group 5FU TSf-ISPA) or both before each dose of TSf-ISPA vaccine and 7 days after each TSf-ISPA immunization, in a protocol termed double 5FU TSf-ISPA (group double 5FU TSf-ISPA). (According to the experiment, when the double 5FU TSf-ISPA group of mice was sacrificed two days or seven days after the third immunization dose, they did, in fact, receive the 5FU dose at day 7 post immunization during the two initial immunizations but not after the third immunization ( Supplementary Figure 1 ).

Control groups were immunized with phosphate-buffered saline (PBS) solution, following the same protocol (group PBS).

Several independent groups have already shown that 5FU administration at the dose employed does not affect other immune populations (22, 29, 48).

Additionally, we have already reported that TSf administration alone or ISPA administration alone does not generate protective capacity against T. cruzi (26).

Mouse blood was obtained following the method previously described (49). Subsequently, the level of plasmatic IgG total antibodies (IgGt) anti-TSf was evaluated by ELISA 7 days after the third TSf-ISPA dose, as we previously described (26). Delayed-type hypersensitivity (DTH) test was performed 5 days after the third TSf-ISPA dose by intradermal (i.d.) challenge with 5 μg of TSf in the right hind footpad, as we previously described (26).

Gr-1+ cells were purified by magnetic cell sorting from spleens of immunized BALB/c mice using the Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi Biotec), according to the manufacturer’s instructions. On day 7 post-immunization (post-imm.), TSf-ISPA immunized mice were sacrificed, spleens were aseptically removed, and splenocytes were harvested as we previously described (25). After purification, the purity of Gr-1+ purified cells was higher than 90%, as evaluated by flow cytometry using an anti-Gr-1 antibody (BD-Biosciences).

Bone-marrow-derived dendritic cells (BMDCs) were obtained from healthy BALB/c mice. Briefly, femurs were removed from mice, and bone marrow cells were collected by rinsing RPMI 1640 (GibcoTM) medium inside the bone. The purified cells were cultured in a specific medium for dendritic cell differentiation: RPMI 1640 medium (Gibco TM), 10% fetal bovine serum (Internegocios SA), 2% penicillin (100 mg/mL), streptomycin (100 U/mL) (GibcoTM), and 30% J588-GM cell line supernatant (50). Cells were seeded in 24-well plates (Nunc) (0.75x10⁶ cells/well) at 37°C and 5% CO₂. On days 3 and 5, fresh medium was added. On day 8, BMDCs were harvested and used for flow cytometry analysis and cultures.

The acquisition of cells compatible with Bone Marrow-Derived Dendritic Cells (BMDCs) was controlled by assessing the expression of costimulatory molecules in CD11c+ cells stimulated with Lipopolysaccharide (LPS) from Escherichia coli (ThermoFisher). For this purpose, 1x10⁵ BMDCs were cultured in 96 well plates (Nunc) in RPMI 1640 medium (Gibco TM), 10% fetal bovine serum (Internegocios SA), 2% penicillin (100 mg/mL), streptomycin (100 U/mL) for 24 h in the presence or absence of LPS (1µg/ml). Flow cytometry using anti-CD11c (Miltenyi Biotec), anti-CD80 (BD-Biosciences), and anti-MHCII (Miltenyi Biotec) antibodies in appropriate combinations of fluorophores confirmed the obtention of CD11c+ cells with the capacity to increase the expression of costimulatory markers in the presence of LPS, as previously described by (40).

Spleen cells from untreated BALB/c mice were labeled with 10 μM of 5,6-carboxifluorescein diacetate succinimidyl ester (CFSE) (Molecular probes, Eugene, OR), following the manufacturer’s instructions. The CFSE label was verified by flow cytometry.

For the suppression assay, 1x10⁵ Gr-1+ cells were cultured with 1x10⁵ BMDCs alone or in the additional presence of 1x10⁵ CFSE-stained splenocytes in 96-well plates (Nunc) in supplemented RPMI medium (RPMI 1640 medium (GibcoTm), 10% fetal bovine serum (Internegocios), 2% penicillin (100 mg/mL), streptomycin (100 U/mL) (GibcoTM). LPS (1µg/ml, ThermoFisher) was added to cultures containing BMDCs and MDSCs. LPS (1µg/ml, ThermoFisher) plus concanavalin A (ConA, Sigma) (5 µg/ml) were added to wells containing BMDCs, MDSCs, and CFSE-stained splenocytes. To complete the total number of cells per well, BMDCs were added instead of MDSCs in the control cultures without purified Gr-1+ cells. After 72 hours at 37°C and 5% CO₂, cells were harvested, and the suppressor activity of MDSCs was evaluated by flow cytometry using anti-CD11c (Miltenyi Biotec), anti-CD80 (BD-Biosciences), anti-MHCII (Miltenyi Biotec), and anti-CD4 (BD-Biosciences) antibodies conjugated to fluorophores. Flow cytometry was performed in a similar manner as described below.

The kinetic analysis of the CD11b+ Gr-1+ MDSC population was performed by flow cytometry. Mice treated with TSf-ISPA, 5FU TSf-ISPA, and double 5FU TSf-ISPA were sacrificed on days 2, 7, and 15 post-third TSf-ISPA dose, and spleen cells were obtained and counted as previously described (25). Additionally, on day 7 post-imm., lymph nodes were also collected.

For flow cytometry, 1x10⁶ cells were first incubated with anti-FcγIII/II receptor antibody (Biolegends), and then stained for 40 minutes at 4°C with appropriate combinations of the following antibodies conjugated to fluorophores from BD-Biosciences: anti-CD11b, anti-Gr-1, anti-Ly6G, anti-Ly6C, anti-CD4, anti-CD8α, anti-CD44, and anti-CD11c. Samples were acquired on a BD AccuriTM C6 plus (BD-Biosciences) cytometer, and data analysis was performed using FlowJo software.

After T. cruzi infection, analysis of the MDSC subtypes (G-MDSCs and M-MDSCs) was assessed. Mice immunized with 5FU TSf-ISPA or double 5FU TSf-ISPA were infected with T. cruzi 15 days after the last immunization dose and were sacrificed at day 21 post-infection. Splenocytes were harvested as previously described (25), and MDSC subtypes were studied by flow cytometry. PBS-treated mice were also infected, but in many cases did not survive until day 21 post-infection.

Splenocytes from mice sacrificed at day 7 post-immunization were also used for cell culture. After red blood cell lysis with NH₄Cl, splenocytes were re-suspended in RPMI 1640 medium (Gibco), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin (Gibco), and 0.4 mM 2-mercaptoethanol. Cells (0.5 × 10⁶ cells/mL) were cultured in 48-well plates (Nunc) in supplemented RPMI. Splenocytes were stimulated with 5 µg/mL TSf. After 72 hours at 37°C and 5% CO₂, cells were incubated with monensin (BD-Pharmingen) according to the manufacturer’s instructions. Four hours later, supernatants were collected, and cells were washed twice with PBS, incubated with anti-FcγRIII/II receptor antibody for 30 minutes, and stained with anti-CD8 FITC and anti-CD4 APC for 30 minutes. Cells were then washed and stained for intracellular IFN-γ using PE-labeled anti-IFN-γ antibody with the BD-Pharmingen intracellular staining kit, according to the manufacturer’s instructions. Samples were acquired on an Accuri C6 Plus flow cytometer (BD-Biosciences) and analyzed using FlowJo software.

Additionally, IFN-γ levels in culture supernatants were measured using ELISA kits from BD Biosciences, according to the manufacturer’s instructions.

For infection, different mouse hosts, parasite strains, and route of infection were assessed. BALB/c mice were challenged i.p. with 2000 or 3000 bloodstream trypomastigotes of the Tulahuen strain or with 20000 cultured trypomastigotes of the Dm28c strain, as detailed in the results, 15 days after the last TSf-ISPA immunization. C57BL/6 mice were i.p. infected with 1000 bloodstream trypomastigotes of the Tulahuen strain or with 20000 cultured trypomastigotes of the Dm28c strain, 15 days after the last TSf-ISPA immunization. Additionally, BALB/c and C57BL/6 mice were challenged with 2500 bloodstream trypomastigotes of the Tulahuen strain via i.d. injection in the footpad 15 days post-TSf-ISPA immunization.

Parasitemia was monitored at day 20 or 21 post-infection (p.i.) by examining 5 µl of blood directly under a microscope (Leica Microsystems), as previously described (26). Survival was recorded until day 21 post-infection in the case of flow cytometry studies or until day 35 p.i. according to the experiment.

Normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was evaluated with the Levene test. For comparisons between two groups, a t-test was applied when normality was not rejected, while ANOVA was used for comparisons involving more than two groups. In cases where ANOVA results were significant, Fisher’s test was employed to assess differences between columns. When normality was rejected, the Mann-Whitney test was used for two-group comparisons, and the Kruskal-Wallis test for multiple groups, with Dunn’s test for post-hoc comparisons. Depending on prior knowledge of how the variable was expected to change, one- or two-tailed tests were used in both the t-test and Mann-Whitney test. Survival curves were evaluated using the Mantel-Cox (Log-rank) test. Except for the Levene tests, which were performed using Python with the Pandas and SciPy libraries, all analyses were conducted using GraphPad Prism (GraphPad, California, USA). Statistical significance was indicated by an asterisk (*) for p < 0.05 between the specified groups. Supplementary Table 2 provides the mean ± SD, exact p-values, and the tests used in each case.

To confirm that the Gr-1+ splenocytes that increase during the immunization phase exert suppressive capacity, in vitro assays were performed. Gr-1+ splenocytes from TSf-ISPA-immunized mice were purified by magnetic cell sorting and cultured with immature bone marrow-derived dendritic cells (BMDCs) alone or in the additional presence of splenocytes stained with CFSE. LPS was added to favor the maturation of BMDCs (50), and the suppressive capacity of the potential cells resembling MDSCs (as has been previously reported) (54). Additionally, LPS + ConA were used to analyze the suppressive capacity of purified Gr-1+ cells over CD4+ lymphocyte proliferation.

Figure 1A shows the gating strategy used to analyze the expression of MHCII and CD80 by CD11chigh+ BMDCs in the presence or absence of purified Gr-1+ cells. A marked decrease in the percentage of MHCII^high and CD80^high cells within CD11chigh+ cells was observed when those cells were cocultured with Gr-1+ purified cells from immunized mice ( Figures 1B, C ). In the same line, a clear and significant decrease in the mean fluorescence intensity (MFI) of MHCII and CD80 within CD11chigh+ cells were observed when those cells were cocultured with Gr-1+ purified cells ( Figures 1B, C ).

Additionally, CFSE-labeled splenocytes were incorporated into the wells containing BMDCs and Gr-1+ purified cells to analyze the proliferation of CD4+ lymphocytes. Figure 2A shows the gating strategy employed, Figure 2B displays representative histograms, and Figure 2C displays the percentage of proliferating CFSE+ cells within CD4+ splenocytes. Results obtained showed that the proliferation of ConA-stimulated CD4+ lymphocytes was suppressed in the presence of Gr-1+ purified cells from TSf-ISPA-immunized mice.

Taken together, these results provide evidence supporting that the Gr-1+ cells that increase during the TSf-ISPA immunization phase include MDSCs with suppressive capacity.

Although several studies have shown that immunization can elicit MDSCs with the ability to limit the efficacy of vaccine candidates (27), to our knowledge, the analysis of the kinetics of MDSCs has not been addressed yet. Thus, considering that a better understanding of the alterations of MDSCs at this stage may favor rational vaccine design, a deeper study of the MDSC dynamics during TSf-ISPA immunization was conducted.

Figure 3A shows representative dot plots of the expression of CD11b and Gr-1 markers in splenocytes at 2, 7, and 15 days post-imm. with TSf-ISPA. As shown in Figure 3B , the absolute number of CD11b+ Gr-1+ splenocytes as well as the percentage of those cells ( Supplementary Figure 2A ) were early increased at day 2 after the last immunization dose, then remained increased at day 7 post-imm., and finally decreased by day 15 post-imm. This result suggests that TSf-ISPA immunization generate a marked increase of MDSCs that decrease by day 15 post-imm.

In addition, we have previously reported that i.p. pretreatment with 5FU before TSf-ISPA immunization increased the protective capacity of the formulation (25). The selective effect of the dose of 5FU employed to target MDSCs has been reported previously, and even in the context of T. cruzi infection (21, 22, 29, 48).

In this study, we seek to elucidate how 5FU administration modifies the kinetics of CD11b+ Gr-1+ MDSCs during TSf-ISPA immunization. Figure 4B , Supplementary Figure 2B shows that 5FU treatment was clearly effective, avoiding an early increase of CD11b+ Gr-1+ cells at day 2 post TSf-ISPA immunization. However, it was noteworthy that this effect was transitory and both the absolute number ( Figure 4B ) and percentage ( Supplementary Figure 2B ) of CD11b+ Gr-1+ splenocytes increased markedly by day 7 post-imm., likely as a rebound effect, as has also been observed in other infection processes (55), a fact that could be associated with a strong immune effector response elicited in the early absence of MDSCs. Then, at day 15 post-imm., the level of CD11b+ Gr-1+ cells slightly decreased, yet it remained significantly higher compared to the levels observed in control mice ( Figure 4 , Supplementary Figure 2B ).

The knowledge obtained regarding the kinetics of MDSCs after 5FU treatment opened the possibility to rationally design new protocols to improve the TSf-ISPA vaccine candidate. Thus, to attempt to tackle the increase of MDSCs observed at day 7 post-imm., a second dose of 5FU was administered at day 7 post-imm., in a protocol termed double 5FU TSf-ISPA. As shown in Figure 5A , Supplementary Figure 2C , the absolute number and percentage of CD11b+ Gr+1+ splenocytes were close to basal levels at day 2 post-imm. Subsequently, there was a significant increase at day 7 post-imm. However, in this case, because of the second dose of 5FU, CD11b+ Gr-1+ cells finally decreased to almost basal levels at day 15 post-imm ( Figure 5A , Supplementary Figure 2C ).

To compare all the protocols simultaneously, a ratio was calculated by dividing the absolute number and percentage of MDSCs in each mouse from every group by the mean values of the control group treated with PBS.

It is interesting to note that at day 2 post-imm. both groups of mice treated with 5FU before TSf-ISPA immunization decreased the absolute number ( Figure 5B ) and percentage ( Supplementary Figure 2D ) of MDSCs compared to TSf-ISPA vaccinated mice. Then, at day 7 post-imm., all groups of treated mice generated an increase of MDSCs compared to control mice, with a ratio similar or higher than 2 regarding the absolute number and percentage of MDSCs ( Figure 5B , Supplementary Figure 2D ). Finally, the second dose of 5FU at day 7 post-imm. caused a marked modification in the dynamics of MDSCs in double 5FU TSf-ISPA versus 5FU TSf-ISPA mice, which received only one dose of the drug. In this sense, a significant decrease in the percentage and absolute number of CD11b+ Gr-1+ splenocytes in double 5FU TSf-ISPA mice was observed compared to 5FU TSf-ISPA mice, strongly suggesting that the second dose is necessary to effectively decrease the expansion of those cells ( Figure 5 , Supplementary Figure 2D ). Both subtypes of MDSCs, CD11b+Ly6G+Ly6C+/low (G-MDSCs) and CD11b+Ly6G-Ly6C+ (M-MDSCs) were increased at day 7 post-imm in 5 FU TSf-ISPA and double 5FU TSf-ISPA vaccinated mice ( Supplementary Figure 3 ), in line with the increases registered measuring CD11b and Gr-1 markers.

According to these results, a challenge with a very high dose of parasites was employed to assess whether the decrease of MDSCs obtained with 2 doses of 5FU was able to improve the protective capacity of the TSf-ISPA vaccine candidate. Since we have previously reported that mice vaccinated with 5FU TSf-ISPA survived 100% against an i.p. challenge with 1500 parasites (25), a high lethal dose of 2000 and 3000 parasites was used in this study. As shown in Figure 6A , when 2000 parasites were used, the survival of the 5FU TSf-ISPA group was nearly 20%, whereas double 5FU TSf-ISPA mice showed a higher protective capacity and still remained at 100% survival. In addition, a dose of 3000 parasites was strong enough to cause 100% mortality in 5FU-TSf-ISPA mice, whereas double 5FU TSf-ISPA showed 71% survival ( Figure 6B ). Additionally, both parasitemia studies also supported the results observed regarding survival, and double 5FU TSf-ISPA treated mice showed lower levels of parasitemia compared to mice that received the 5FU-TSf-ISPA vaccine. As expected, PBS-treated control mice were unable to withstand such a high dose of parasites and had 100% mortality even before day 20 of infection.

Taken together, these results strongly support that two doses of 5FU to deplete MDSCs during the immunization phase are able to markedly potentiate the TSf-ISPA vaccine candidate, even when high lethal doses of parasites are used.

It has been reported that MDSC increases during immunization may limit the efficacy of vaccination, likely by suppressing dendritic cells and the elicitation of a more potent effector response (27, 54). To analyze whether the better protective capacity observed with 2 doses of 5FU was the consequence of a better effector immune response, several immune parameters were analyzed at day 7 post-imm. in both groups of mice treated with 5FU: 5FU TSf-ISPA mice and double 5FU TSf-ISPA.

Both groups of 5FU-vaccinated mice generated significant levels of anti-TSf IgG antibodies ( Figure 7A ) and specific cellular response ( Figure 7B ) compared with control PBS-treated mice. In addition, although no difference in antibody levels was observed at day 7 post-imm., double 5FU TSf-ISPA mice showed a significant increase in the DTH response compared to 5FU TSf-ISPA mice ( Figure 7B ).

Vaccinated mice were sacrificed at day 7 after the last immunization dose to analyze quantitative changes in several splenic immune populations. Figures 8A, B shows the gating strategy used to analyze the changes in CD4+, CD8+, CD11c+ CD11c+CD8α+ cells ( Supplementary Figure 4 shows the gating strategy used to analyze CD4+CD44+ and CD8+CD44+ cells). Interestingly, double 5FU-treated mice elicited the highest increase in the absolute number of CD4+, CD4+CD44+, CD8+, CD8+CD44+ cells ( Figures 8C–F ), and a trend supporting the elicitation of a higher increase of CD11c+ and CD11c+CD8α+ splenocytes ( Figures 8G–J ). (Percentages of all analyzed populations are shown in Supplementary Figure 4 ). In line with these results, cultured splenocytes from mice immunized with double 5FU TSf-ISPA showed a higher percentage of CD4+IFN-γ+ cells and a trend toward a similar increase in CD8+IFN-γ+ cells after culture with TSf, compared to splenocytes from 5FU TSf-ISPA mice ( Figures 8K, L ). Additionally, culture supernatants from double 5FU TSf-ISPA mice exhibited higher levels of IFN-γ than those from 5FU TSf-ISPA mice, as measured by ELISA ( Figure 8M ).

Supporting the analysis performed in the spleen, very similar results were observed when the absolute number of lymph node cells from 5FU TSf-ISPA versus double 5FU TSf-ISPA vaccinated mice was analyzed. In all cases, double 5FU TSf-ISPA mice showed higher numbers of CD4+, CD4+CD44+, CD8+, CD8+CD44+, CD11c+, and CD11c+CD8α+ ( Figures 9A–F ), also supporting that double administration of 5FU potentiates the effector response caused by the TSf-ISPA vaccine.

Taken together, these results support the notion that the immunization protocol based on two doses of 5FU not only decreased MDSCs during immunization but also generated an increase in the absolute number of several effector immune populations in the spleen and lymph nodes of vaccinated mice, correlating with the higher protection observed when the double 5FU TSf-ISPA protocol was compared to 5FU TSf-ISPA, based on one dose of 5FU.

Moreover, we have previously shown that TSf-ISPA vaccinated mice (without 5FU) already have higher survival than control mice, correlating with lower levels of MDSCs after T. cruzi infection (26). To also analyze whether the protective effect of two doses of 5FU during immunization was evidenced by a decrease in MDSCs after infection, an additional assay was performed. In addition, and similarly to our previous study, both subtypes of MDSCs were measured G-MDSCs (CD11b+, Ly6G+ Ly6C+/low), and M-MDSCs, (CD11b+ Ly6G- Ly6C+). In accordance with all the results described herein, double 5FU TSf-ISPA vaccinated and infected mice showed a lower absolute number of G-MDSCs and M-MDSCs splenocytes as compared to 5FU TSf-ISPA vaccinated and infected mice ( Figure 10 ). Supplementary Figures 5A, B show the percentages of G-MDSCs and M-MDSCs. Thus, taken together, these results support that rational control of MDSC dynamics during immunization caused an increased effector response, and then, after infection, TSf-ISPA vaccinated mice that received a double dose of 5FU showed lower levels of MDSCs correlating with lower parasitemia and better survival after T. cruzi infection.

To date, very few studies have assessed the protective capacity of vaccine candidates against T. cruzi from different DTUs. Given the genetic diversity of the parasite, this may constitute an important aspect to be considered when evaluating the usefulness of a vaccine candidate. Taking into consideration the high level of protection obtained by the double 5FU TSf-ISPA vaccine in the model of BALB/c mice i.p. infected with the Tulahuen T. cruzi strain, the protective capacity of the formulation was also assessed in several different preclinical models.

To initiate this type of analysis, we first assessed the immune response generated by the double 5FU TSf-ISPA vaccine in C57BL/6 mice and then evaluated the protective capacity of the formulation against i.p. Tulahuen T. cruzi infection. The vaccine protocol was able to elicit significant levels of anti-TSf IgG antibodies ( Figure 11A ) and a strong DTH response ( Figure 11B ), in line with the results obtained in the model of BALB/c mice immunization. Thus, the results described herein support the capacity of the vaccine protocol to elicit immune responses in both strains of mice, BALB/c and C57BL/6.

C57BL/6 mice were then challenged with 1000 T. cruzi of Tulahuen strain (DTU VI) via the i.p. route. This dose did not cause mortality in control PBS-treated mice nor in vaccinated mice. Regarding parasitemia, there was a trend supporting that double 5FU TSf-ISPA vaccinated mice developed a lower level of parasitemia ( Figure 12A ). To analyze whether better protection with lower parasitemia correlated with lower levels of MDSCs after infection, mice were sacrificed at day 21 post-infection. A significant decrease in the number of both subtypes of MDSCs (G-MDSCs and M-MDSCs) was observed in double 5FU TSf-ISPA vaccinated mice, as compared to PBS-treated and infected mice ( Figures 12B, C ). Supplementary Figure 6 displays that the percentage of MDSCs was also lower in vaccinated mice. Therefore, once again, these results are also in accordance with our previously published results and other results described herein, in which vaccinated mice were protected against T. cruzi correlating with lower levels of spleen MDSCs (25, 26).

To analyze whether the vaccine formulation has protective capacity against additional DTUs, i.p. infection with Dm28c (DTU I) was performed. This strain does not commonly cause mortality during acute infection and generates lower parasitemia levels. After the immunization protocol, both strains BALB/c and C57BL/6 mice were i.p. infected with 20000 cultured trypomastigotes. As expected, there was no difference in the percentage of survival, given that control and infected BALB/c and C57BL/6 mice survived 100% after infection. However, results obtained showed that parasitemia levels were significantly reduced in vaccinated BALB/c mice at day 14 post-infection ( Figure 13B ), as compared to infected PBS-treated mice, and showed a similar trend at day 12 post-infection in BALB/c mice, and at day 12 and 14 post-infection in vaccinated and control C57BL/6 mice ( Figures 13A, C, D ).

Then, to analyze a model of infection resembling a natural route of T. cruzi transmission, i.d. infection was accomplished in BALB/c and C57BL/6 mice with the Tulahuen strain, which generates a more evident effect. After i.d. infection with 2500 trypomastigotes, double 5FU TSf-ISPA vaccinated BALB/c mice showed lower parasitemia levels at day 21 post-infection ( Figure 14A ) and a clear increase in survival ( Figure 14B ) compared to PBS-treated and infected mice.

In addition, a clear trend suggesting protection was also observed in C57BL/6 mice at day 21 post-infection of double 5FU TSf-ISPA compared to PBS-control mice ( Figure 14C ). Thus, taken together, these results provide strong evidence supporting that the double 5FU TSf-ISPA vaccine can confer protection in several different preclinical models, including different genetic backgrounds of the host, different DTUs of parasites, and different doses and routes of T. cruzi infection.