Protocol design of the experiments was approved by the Comissão de Ética no Uso de Animais of the Universidade Federal do Rio de Janeiro (CEUA protocol IMPPG040-07/16), in agreement with Brazilian laws for animal safety and the guidelines of National Institute of Health (15). Animals were maintained at the Instituto de Microbiologia Paulo de Góes, which is part of Universidade Federal do Rio de Janeiro (UFRJ) facilities, given water and food ad libitum, with a 12 h light/dark cycles and controlled temperature (15). We aimed to reduce any animal suffering to a minimum.

In this investigation, we worked with genetically modified Escherichia coli BL21 and DH5 strains cloned with the pET28b plasmid expressing the NH36 and F3 recombinant proteins. These bacterial clones used in this investigation are considered OGM risk level 1 (CQB 0108/99 IMPG-UFRJ) because they are not associated with disease in adults. We also manipulated non-genetically modified Leishmania parasites for which the CTNBio biosafety level is 2. Following the Brazilian Ministry of Health Biosafety regulations for Biomedical and Microbiology laboratories we performed the following biosafety measures: use of apron, gloves, masks, and protective glasses, decontamination of infected biological material and animal cases before washing and limited access to, security block and signalizing of the risk area. Additionally, we used laminar flow, aseptic chambers, automatic pipetters, and reservoirs with sterilizing solutions. On the day of the experiments with the bacterial clones, no other manipulation of microorganisms occurred. All the non-disposable material and surgery tools are autoclaved. A separated manipulation room with an ultraviolet and exhaust chamber was used when necessary. Carcasses were incinerated.

NH36 is composed of 314 amino acids. The NH36 sequence is deposited in SWISS-PROT (accession code Q8WQX2), EMBL (AY007193), GenBank™ and DDJB (AAG02281.1) data bases (15, 20). F3 is composed of the amino acids 199–314 of NH36. The sequences of the NH36 and F3 were cloned in the pET28b plasmid system, expressed in E. coli Bl21DE3 cells and purified by affinity chromatography (Ni-NTA, Qiagen), as described before (15, 20). The presence or absence of LPS was confirmed using the LAL QCL-1000 kit (Lonza). The levels of LPS were lower than the sensitivity range of the Limulus amebocyte lysate test, which is 0.1–1.0 EU/ml. Therefore, there was no need for endotoxin removal.

Eight-week-old C57BL/6 female mice, obtained from the Fiocruz-Cecal facilities (Rio de Janeiro, Brazil) were vaccinated subcutaneously, with three doses of 100 µg of NH36 or F3 formulated with 100 µg of saponin (SIGMA), at weekly intervals (15). Control mice received only saline. On week 4, mice were challenged with an intravenous injection in the tail vain of 3 × 10⁷ L. (L.) infantum chagasi amastigotes (strain IOC-L 3324) isolated from infected hamsters’ as described before (24, 25). On day 28 after infection, euthanasia was performed and the variation in total body weight and relative liver/body weight were recorded (15). The parasite load in the livers was determined by microscopic observation of Giemsa-stained impression smears. The parasite burden was assessed as LDU values (number of amastigotes per 1,000 of organ cell nuclei/mg of organ weight) (11, 15, 16, 18, 25). Alternatively, the parasite burden was assessed by a limiting dilution assay (LDA) of the aseptically removed liver fragments suspended in a 1/5 serial dilution in Schneider’s medium, and incubated at 26°C. Promastigotes present in the last well containing visible parasites were quantified in a hemocytometer (21). Two-independent experiments were performed with n = 7 mice per treatment in each treatment.

IDR against L. (L.) donovani lysate was measured in the footpads on day 7 after immunization and on day 25 after challenge (15, 26). The right hind footpads were injected intradermally with 10⁷ freeze–thawed L. (L.) donovani promastigotes at the stationary phase (15, 26–28). Before, and at 24 and 48 h after injection the swelling was assessed with a Mitutoyo apparatus. Each animal received only 0.1 ml saline in the left hind footpad as control. Values of the saline control were subtracted from the reaction due to Leishmania antigen, at each measurement (15, 26). Two-independent experiments were performed with n = 6–7 C57BL/6 mice per treatment in each experiment.

Dendritic cells was isolated from spleens of vaccinated mice, 28 days after challenge, following the instructions of the magnetic beads manufacturer (Miltenyi Biotec, USA). Briefly, spleens were collected, shredded, and incubated with collagenase, at 1 mg/ml, and DNAse (Sigma) at 20 µg/ml concentration (29), respectively, for 20 min, at room temperature (Figure S1 in Supplementary Material). After that, the spleens were macerated and the splenocytes passed through a cell mesh. Also an ACK solution was used for erythrocyte lysis. Magnetic microbeads conjugated with anti-mouse CD11c were added to the preparation and then incubated for an additional 30 min. After that, the DCs were purified using MACS magnetic columns (Miltenyi Biotec, Germany) (11) and labeled with anti-mouse CD11c antibody conjugated to fluorescein isothiocyanate (FITC), clone N418 (eBiosciense, USA). Cells were additionally incubated with allophycocyanin (APC)-conjugated to anti-CCR7 (eBioscense) (two experiments with n = 5 mice per treatment for each experiment) (Figure S1 in Supplementary Material). The cytometry analysis was performed using a FACSCalibur apparatus (Becton Dickinson).

The in vitro migration assays used DCs purified from C57BL/6 naïve, L. (L.) infantum chagasi infected and NH36 or F3 vaccinated and infected mice. The DCs (5 × 10⁵ per well) were suspended in RPMI containing 1% fetal calf serum and placed into the upper chamber of Transwell inserts (5 µm pore size, Coaster, Corning). The inserts were further positioned in plates containing RPMI, with or without the addition of 100 ηM CCL19 (R&D Systems). After 2 h of incubation at 37°C, the cells in the lower wells of the inserts were collected and counted in a hemocytometer chamber. For the purity analysis, cells which migrated were stained with anti-mouse CD11c antibody, and analyzed by flow cytometry. Two-independent experiments were performed, each one of them with n = 5–6 mice per treatment. In order to prove that the DCs migration is due to the sensitivity of CCR7 receptors to the CCL19 chemokine gradient, blocking experiments were performed by incubating the DCs obtained from infected or F3sap-vaccinated mice with anti-CCR7 antibody (e Bioscience) at 1/50 dilution for 30 min at 4°C, before the migration assay in transwell plates, as described.

The migration assays in vivo used DCs purified from spleens of L. (L.) infantum chagasi infected and NH36 or F3-vaccinated, or from infected mice. Isolation and purification of DCs were performed using magnetic beads as described for the in vitro assays (Figure S1 in Supplementary Material). Additionally, the DCs were stained with two drops/ml (6 µg/ml) of Hoescht 33342 (Life Technologies), for 20 min at 37°C, protected from light. After that, 10⁶ labeled DCs were injected intravenously into the infected receptors, on day 28 after infection, as described by Ato et al. (11). Spleens of receptors were removed 24 h after the DCs transfer and immersed in paraformaldehyde, followed by cryoprotection in an increasing sucrose gradient (10, 20, and 30%). Cryostat sections (16 µm) were performed and mounted in Fluoromount and observed under a confocal microscope (LeicaTCS SP5). Sections from three mice of each treatment were chosen randomly and analyzed for the presence of labeled DCs in the white pulp (wp) or in the red pulp (rp) or in the MZ. Approximately 1,000 stained DCs were recorded and the differences between the proportions detected in the wp, and the rp, were analyzed by the Fischer exact test for comparison of proportions (https://www.graphpad.com/quickcalcs/contingency2/). Two-independent experiments were performed, each one of them with n = 3 receptor mice per treatment.

Splenic DCs from L. (L.) infantum chagasi-infected mice, and from NH36 or F3-vaccinated and challenged mice, were purified on day 28 after infection. The total amount of purified DCs obtained from the spleens from the different experimental groups varied from 1 to 2.6 × 10⁶. For the immunotherapy experiments, 10⁶ DCs of each group of mice were injected intravenously into L. (L.) infantum chagasi-infected C57BL/6 recipients on day 28 post infection. The parasite loads in spleen and livers, the spleen and liver/corporal relative weights, and the variation in body weights were recorded in the recipient mice 7 days later by LDU. Two-independent experiments were performed with n = 5 mice per treatment in each experiment. As a control, blocking experiments to prove that migration of DC is critical for the protection in mice were performed using DCs obtained from normal, infected, and NH36, and F3-vaccinated and infected mice, that were pre-incubated with anti-CCR7 antibody and further transferred to infected receptors.

Spleens were aseptically removed and splenocyte suspensions were prepared as described before (21). Purified DCs were also obtained. Cells were distributed into 96-well plate (10⁶ cell/well) and incubated with 5 µg/ml NH36 or lysate of stationary phase L. (L.) infantum chagasi promastigotes, or with no addition for 72 h in vitro, at 37°C with 5% CO₂. After that, supernatants were harvested and assayed using the Mouse IFN gamma, TNF-α, and IL10 ELISA Ready-SET-Go! (e-Biosciences, USA). The sensitivity of the assay was established with a range of 10–1,000 pg/ml for TNF-α, 100–1,000 pg/ml for, and of 15–2,000 pg/ml for IFN-γ. Reactions were developed using biotinylated anti-cytokine antibodies, streptavidin (SAv-HRP) enzymatic reagent, and TMB (Zymed, USA). Absorbances were monitored in a BioRad ELISA reader at 655 ηm. Two-independent experiments (n = 5 mice per treatment in each experiment) were performed.

The analysis of the T cell response used splenocytes stimulated in vitro with 5 µg/ml NH36 or lysate of stationary phase L. (L.) infantum chagasi promastigotes or with no addition for 24 h at 37°C with 5% CO₂, as described (21), and with incubation with Brefeldin A (SIGMA). After that, the splenocytes were stained with rat anti-mouse-CD4FITC (clone GK1.5) and -CD8FITC (clone 53–6.7) monoclonal antibodies (eBioscience), fixed, washed, treated with 0.5% saponin-FACS buffer, and stained with IFN-γAPC, IL-2-PerCP-Cy5.5, and TNF-αPE monoclonal antibodies (eBioscience). A total of 100,000 events were acquired. Gating for CD4⁺ ad CD8⁺ T cells was performed using a FACSCalibur apparatus (Becton Dickinson). Data analysis was performed with the Flow-jo program (Treestar, USA). Two-independent experiments were performed with n = 5 mice per treatment in each experiment.

Means were compared using the Kruskal–Wallis and Mann–Whitney non-parametrical tests. Correlation coefficients were established by the Pearson’s two-tailed correlation test (GraphPad Prism 6 software) as described (21). Also compared the survival distribution of individuals in infected controls and vaccinated and challenged mice were compared using the Log-rank (Mantel–Cox) and the Gehan–Breslow–Wilcoxon tests (GraphPad Prism 6 software).

In the present investigation, we aimed to assess whether DCs from vaccinated mice preserve their correct migrating capabilities in response to CCL19 chemokine, and if this fact is related to their normal sustained expression of the CCR7 receptor.

As a source of DCs we used normal, infected, and NH36 and F3-vaccinated and challenged mice. Initially, we confirmed that the vaccine-induced protection in the these mice by assessing the achievement of enhanced IDR responses, reduced liver parasite loads and liver relative weights, and sustained corporal weights (Figure 1). In fact, vaccination with both, the F3 and the NH36 vaccines, significantly increased the IDR after complete immunization (Figures 1A,C), and after challenge (Figures 1B,D) above the levels detected in normal control mice, at all tested times (24 and 48 h after injection). Remarkably, after infection, the F3 vaccine was 74 and 79% stronger than controls, at 24 and 48 h, respectively and 27% stronger (p < 0.0001) than the NH36 vaccine (Figures 1B,D).

IDR values correlated negatively with the liver/body relative weight after immunization (p < 0.0001, R = −0.5768, R² = 0.3327 at 24 h and p < 0.0001, R = −0.5833, R² = 0.3402 at 48 h after injection) and after challenge (p < 0.0001, R = −0.5989, R² = 0.3587 at 24 h and p < 0.0001, R = −0. 6334, R² = 0.4012 at 48 h after injection).

Notably, both vaccines strongly reduced the liver LDU values compared to those of infected mice (p < 0.0001) (Figure 1E). Compared to infected controls, F3 vaccine produced a 98.8% reduction, and the NH36 vaccine, a 97.6% of reduction of the parasite load (p = 0.0318) (Figure 1E). F3 vaccine reduced the LDUs values by 50% in comparison to the NH36 vaccine. The reduction of the parasite load determined by both vaccines was also confirmed by the LDA method (Figure 1F). The F3 vaccine induced 98% (p < 0.079), and the NH36 vaccine only 88% reduction (p < 0.0079) (p < 0.05) of the number of promastigote in liver culture. Additionally, the F3 vaccine reduced the promastigotes counts by 87% (p < 0.05) in comparison to the NH36 vaccine (Figure 1F). The LDU and LDA values for the liver parasite loads were positively correlated (p < 0.026, R = 0.496, R² = 0.2464).

The increases in IDR after infection were strong correlates of protection regarding the liver LDU values. In fact, LDU values were negatively correlated with IDR after immunization, at 24 h (p < 0.0001, R = –0.7875, R² = 0.6202) and at 48 h (p < 0.0001, R = −0.7674, R² = 0.5890) and after challenge, at 24 h (p < 0.0001, R = −0.7796, R² = 0.6077) and at 48 h (p < 0.0001, R = −0.7543, R² = 0.5690).

Increases in liver/body relative weight were also significantly higher in infected control animals than in normal uninfected controls and in F3 and NH36-vaccinated and challenged mice (p < 0.0001 for all comparisons) (Figure 1G). In addition, only infected controls lose corporal weight (Figure 1H) in comparison to normal, F3 and NH36-vaccinated mice (p < 0.0001 for all comparisons). Vaccinated and normal mice showed similar gain in corporal weight. Besides, IDR was positively correlated to corporal weight gain after immunization (p = 0.0009, R = 0.5055, R² = 0.2556, at 24 h and P = 0.0002, R = 0.5504, R² = 0.3030, at 48 h after injection) and after challenge (p < 0.0001, R = 0.5888, R² = 0.3467, at 24 h and p < 0.0001, R = 0.6532, R² = 0.4267, at 48 h after injection).

Once the generation of a protective response was confirmed, we further studied the migrating capabilities of DCs obtained from these vaccinated, naïve controls, and infected mice on day 28 after challenge. In Figure 2, we represent the results as boxes and whiskers. The whiskers show the maximal and minimum values and the top and bottom of the bars show, respectively, the 75th and 25th percentiles. The 75th percentile is the value below which 75% of observations in a group of observations fall. Figure 2A shows that the number of total splenocytes isolated was, as expected, higher in infected mice than in the two vaccinated groups. FACS cytometry analysis of the affinity chromatography purified CD11c⁺ DCs preparations disclosed 90–100% rates of purity. We further observed that the number of DCs purified from total splenocytes was higher in infected and F3-vaccinated, than in normal mice (Figure 2B).

Additionally, none of the DCs migrated toward the medium containing no additions (Figure 2C). In contrast, while DCs of normal controls and of F3 and NH36-vaccinated mice migrated toward the CCL19 gradient, DCs of infected animals did not (p < 0.008 for all comparisons). The maximal number of migrated DCs was 11,496 for the F3, and 9,152 for the NH36 vaccine (Figure 2C). Accordingly, 75% of the migrated DCs count of the F3 vaccine group fall below 8,596, while 75% of the NH36 vaccine counts fall only below 6,130 DCs counts. Therefore, although the differences in the generation of the DC migrating capabilities of the two vaccines were not statistically significant, the maximal values and 75th percentiles suggest the superiority of the F3 vaccine. As a blocking control, we incubated DCs obtained from either infected or F3sap-vaccinated and challenged mice, with anti-CCR7 antibody, in the presence of CCL19 stimulus (Figure 2D). The anti-CCR7 antibody blocked 90% of the in vitro migrating capabilities of the DCs obtained from the F3sap-vaccinated mice.

Our results indicate that protective immunity against the F3 and NH36 antigens, not only restores the DCs migrating dysfunction of mice infected with L. (L.) infantum chagasi, but it also potentiates this capability above the levels found in normal naïve mice (Figure 2). In agreement with the parasitological results, which indicated that the F3 vaccine was the most protective, the superiority of the F3 vaccine is reinforced by the finding of a higher number of purified DCs, higher maximal counts of migrated DCs, and strong inhibition of these migrating capabilities by treatment with anti-CCR7 antibody.

In VL, abnormal DC migration is partially due to the reduced expression of their CCR7 receptor, which is sensitive to the CCL19 chemokine. In order to evaluate whether the restored DCs migrating capabilities of vaccinated mice were related to a preserved CCR7 expression on DCs. With that aim, we analyzed the CCR7 protein expression on the surface of purified splenic DCs using APC-conjugated anti-CCR7 antibody and flow cytometry analysis. Based on our findings (Figure 3), vaccination with F3sap and NH36 upregulated the expression of CCR7 receptor on DCs. CCR7 expression was low in normal mice (1.56%) (Figure 3A), extremely reduced in infected mice (0.37%) (Figure 3B), and much stronger in animals treated with the F3 vaccine (26.06%) (Figure 3C) than in those vaccinated with NH36 (5.29%) (Figure 3D).

Therefore, vaccination with F3 and NH36 stimulates IDR, protects mice from clinical VL, restores and enhances DCs migrating capabilities probably due to the increase in CCR7 expression on DCs. Our findings of the reduced liver parasite load, increased IDR and enhancement of the CCR7 expression highlight the stronger immunogenic effect of the F3 vaccine.

In order to confirm the DC migration in vivo, DCs from infected, or NH36- or F3-vaccinated and L. (L.) infantum chagasi-challenged mice, were stained with Hoescht 33342 in vitro, and further injected into infected mice. Twenty four hours after injection, the spleens of receptors were removed and frozen, and the transferred DCs were observed by fluorescence microscopy in the wp or rp of the frozen sections (Figure 4). Fluorescent DCs were counted in a total of 19–21 sections of each treatment and representative images are shown in Figures 4A–C. Additionally, the records are represented as proportions of DCs from donors distributed either in the white or rps of infected receptor spleens. On day 28 of the infection of recipients, DCs from infected donors remained mostly in the rp (92%) (Figure 4A) and, only minor proportions migrated to the MZ of the wp (8%; p < 0.001). DCs from NH36-vaccinated mice are equally distributed between the white (59%) and the rp (41%; p = 0.1144) (Figure 4B). Remarkably, 84% of DCs from F3-vaccinated mice preferentially migrated to the wp and its MZ (Figure 4C), while only 16% remained in the rp (p < 0.0001).

In order to study the potential use of F3 and NH36 antigen-primed DCs in the immunotherapy of VL we transferred DCs from infected, and from vaccinated and challenged mice, into recipient mice infected with L. (L.) infantum chagasi 28 days before. Seven days after the DCs transfer, the impact on the parasitological and clinical cure was assessed. While infected animals lost 1.54 g of corporal weight, the weight loss was 79% lower in mice that received F3-primed DCs (0.32 g), and 91% lower in mice treated with NH36-primed DCs (0.14 g) (Figure 5A). Additionally, only the F3-primed DCs were able to alter the impact of the infection on mice, by reducing the parasite load in the spleen (p < 0.007) (Figure 5B), the spleen/body relative weight (Figure 5C), the liver/body relative weight (p < 0.05) (Figure 5D), and the liver LDU values (p < 0.05) (Figure 5E). Therefore, the results of immunotherapy with DCs correlate with the results of the preventive vaccination, thus demonstrating that the F3 vaccine is more potent than the NH36 vaccine. The immunotherapeutic effect of F3-primed DCs (Figure 5F), but not of the NH36-primed DCs (not shown) was blocked by pre-incubation with anti-CCR7 antibody, and a 43 and 52% increased parasite load was observed in spleens and livers, respectively of the recipient mice. These results indicate that the increased migrating capabilities of DCs from F3-vaccinated mice, both in vitro and in vivo (Figures 2D, 4C and 5F) are related to the enhanced CCR7 receptor expression (Figure 3), which contributes to the cure of VL.

We assessed the profile of IFN-γ, TNF-α, and IL-10 secretion along the time, in the supernatants of splenocytes of mice vaccinated with F3 or NH36 vaccines and further challenged (Figure 6). We monitored the cytokine secretion of splenocytes in response to NH36 or to the L. (L.) infantum chagasi lysate antigen. On day 15 after challenge, when the parasite load was maximal in livers (Figure 6C), the F3-vaccine enhanced the IFN-γ and TNF-α secretion in response to NH36, five times more than the NH36 vaccine (Figures 6A,D). The F3-vaccine also enhanced by a factor of 1.4–5, the respective IFN-γ and TNF-α levels when compared to infected controls (Figures 6A,D). On the other hand, IL-10 secretion was reduced more by the F3 than by the NH36 vaccine (Figure 6G). The IFN-γ/IL-10 and TNF-α/IL-10 ratios (Figure 6I), that were also higher for the F3 vaccine (10 and 2.9, respectively) than for the NH36 vaccine (6.5 and 1.7, respectively) suggest the superiority of the F3sap vaccine in the induction of a probable Th1 response. The Leishmania lysate promoted a similar response with superiority of the F3 vaccine although with a lower IFN-γ on day 15 and higher IL-10 secretion on day 28 (Figures 6B,G). Globally, the pro-inflammatory cytokine response of splenocytes was maximal (Figures 6A,B,D,E) since the early infection, when vaccine protection was already registered in spleens (Figure 6F). Levels of IFN-γ and TNF-α decreased until day 28, when, in contrast, IL-10 secretion was maximal. This pike in IL-10 secretion is coincident with the maximal parasite load detected in spleens. However, the vaccine efficacy was also long-lasting until day 28, when the parasite load was higher in spleens. In fact, while 90% of the parasite load reduction was induced by the F3 vaccine on day 15, protection was maximal (94%) on day 28 after infection (Figure 6F).

The survival Kaplan–Meier curve analysis represented in Figure 7, confirmed that protection generated by the F3sap vaccine is long-lasting. In fact, while all mice from the infected control group died between day 29 and day 40 after infection, a 100% of mice vaccinated with F3sap survived until euthanasia on day 45, confirming the longevity of the vaccine immune response. In contrast, 80% of NH36-vaccinated mice survived until day 29 and only 40% of them were alive on day 45 (Figure 7).

We also studied the cytokine response in vitro, in the supernatants of DCs obtained from infected, and vaccinated and infected DC donors, on day 28, before transfer to infected mice (Figure 8). Although the total splenocyte cytokine response was lower on day 28, than on day 15 (Figures 6A,B,D,E,G,H), the DCs of F3-vaccinated mice secreted enhanced levels of IFN-γ, in response to NH36 and lysate (Figure 8A) and of TNF-α, in response to lysate only (Figure 8B). No significant differences in IL-10 levels were observed in response to the antigens (Figure 8C).

The Figure S2 in Supplementary Material, summarized the strategy used for the analysis of multifunctional T cell response using a four-color flow cytometry panel to simultaneously analyze multiple cytokines at the single-cell level in splenocytes cultures. After analyzing the total production of each cytokine by CD4⁺ or CD8⁺ cells, the Boolean gates (or combinatorial analysis) tool of the FlowJo program was used to determine the frequencies of the possible seven combinations of cytokine-producing cells.

In correlation with the cytokine secretion to supernatants by splenocytes (Figure 6), the intracellular expression of cytokines by CD4⁺ T lymphocytes in response to NH36, was more intense on day 15 than on day 28 after infection (Figure 9). On both days, the F3sap vaccine was superior to the NH36 formulation enhancing all types of CD4⁺ T cells secreting one cytokine (IL-2⁺, TNF-α⁺, or IFN-γ⁺) (Figures 9A,D,G), the combination of two (IL-2⁺TNF-α⁺, TNF-α⁺IFN-γ⁺, or IL-2⁺IFN-γ⁺) (Figures 9B,E,H), or the three cytokines simultaneously (IL-2⁺TNF-α⁺IFN-γ⁺) (Figure 9C). The only exception was detected in CD4⁺TNF-α⁺ (Figure 9D) and CD4⁺TNF-α⁺IFN-γ⁺ T cells (Figure 9H) which were increased more, in the infected controls than in the F3-vaccinated mice, only on day 15.

Additionally, the global proportions of CD4⁺ T lymphocytes were lower in infected, than in normal mice (Figure 9F), although the F3 vaccine sustained higher CD4⁺ T cells counts than the NH36 vaccine, even despite the advancement of spleen infection on day 28 (Figure 6F).

In contrast to the IFN-γ and TNF-α splenocyte secretion to supernatants (Figure 6), which were slightly higher in response to NH36 than to the lysate (Figure 6), the intracellular expression of cytokines in CD4⁺ T cells was similar, in response to both antigens (Figure 9; Figure S3 in Supplementary Material). In fact, the Leishmania lysate stimulus also increased the frequencies of CD4⁺-secreting T cells, more on day 15 than on day 28 after infection. The F3 vaccine was stronger than the NH36 vaccine for all types of CD4⁺ T cells (Figure S3 in Supplementary Material). Also, and as detected after stimulation with NH36, the frequencies of CD4⁺TNF-α⁺ and CD4⁺TNF-α⁺IFN-α T cells were still higher in infected controls and only increased by the F3-vaccine, on day 28 after infection (Figures S3D,E in Supplementary Material).

Regarding the cytotoxic immunity in response to NH36, the NH36-vaccine was the strongest enhancer of the proportions of CD8⁺IL-2⁺ and CD8⁺TNF-α⁺IFN-α⁺ on day 15 (Figures 10A,E) and of CD8⁺IL-2⁺TNF-α⁺IFN-γ⁺ lymphocytes on day 28 (Figure 10C). In contrast, on day 28, the F3-vaccine was the most potent enhancer of the frequencies of CD8⁺IL-2⁺ (Figure 10A), IFN-γ⁺ (Figure 10G), TNF-α⁺IL-2⁺ (Figure 10B), TNF-α⁺IFN-γ⁺ (Figure 10E), and IL-2⁺IFN-γ⁺-secreting T cells (Figure 10H). The F3-vaccine superiority for TNF-α⁺IL-2⁺ enhancement was already evident on day 15 (Figure 10B).

Additionally, when stimulated with lysate stronger frequencies of CD8⁺IL-2⁺, CD8⁺TNF-α⁺IL-2⁺, CD8⁺TNF-α⁺IFN-γ⁺, and CD8⁺IL-2⁺TNF-α⁺IFN-γ⁺ T cells were observed, on day 15 (Figure S4 in Supplementary Material) in mice vaccinated with the NH36 vaccine. In contrast, the F3-vaccine was superior for the proportions of CD8⁺IFN-γ⁺ and CD8⁺IL-2⁺IFN-γ⁺ T cells, both on days 15 and 28, and of CD8⁺IL-2⁺ and CD8⁺IL-2⁺IFN-γ⁺ T cells, on day 28. Similar to what was detected for CD4⁺ T cell whole frequencies (Figure 9F) the CD8⁺ T cell proportions were reduced in all challenged mice, but in this case, no preventive effective was induced by any of the vaccines (Figure S4F in Supplementary Material).