Figure 1 illustrates the electrophoretic profile of the VP1, VP2, and VP3 recombinant structural proteins of TrV-VLPs. The profile was obtained by electrophoresis in a 12.5% polyacrylamide gel (SDS-PAGE), stained with Coomassie blue. The results reveal a protein profile characterised by several proteins with different molecular masses, with a major band around 30 kDa which suggests a higher concentration of this specific protein, corresponding to VP1, VP2 and VP3, respectively.

Serum samples from immunised mice at 15 and 30 days post-immunisation were used and compared with non-immunised mice (negative control) in order to determine the level of anti-Leishmania IgG antibodies, after the primary immunisation in mice by injection of total antigens of L. amazonensis formulated with the different vaccine adjuvants (alum, FIA, and VPs). The antibody profile was assessed against the native antigens of L. amazonensis by ELISA. The results presented in Figure 2 show that an increase in total IgG anti-L. amazonensis antibody levels was observed in all immunised groups 15 days after the first dose when compared to the negative control. All immunised groups (FIA, alum, and VPs) showed similar antibody profiles, with no significant difference between them. However, a statistically significant difference was found when comparing these three groups with the group immunised only with L. amazonensis crude protein extract. Additionally, the total IgG anti-L. amazonensis antibody levels in the immunised groups remained elevated 30 days after the first immunisation compared to the negative control.

Then, the animals were immunised with the second dose of total protein extract of L. amazonensis with the different vaccine adjuvants, and serum samples were obtained 15, 30, 40, and 50 days after the secondary immunisation. The anti-Leishmania IgG antibody profiles during secondary immunisation are shown in Figure 3. It is observed that after 15 days of secondary immunisation, the total IgG anti-Leishmania levels remained more elevated in all immunised groups, when compared to the negative control. The VPs group did not show a significant difference when compared to the group immunised only with the L. amazonensis crude protein extract. The FIA group maintained the highest levels amongst all groups, followed by the group immunised with alum.

Additionally, 30 days post-secondary immunisation, the total IgG anti-Leishmania levels continued to increase in all immunised groups compared to the negative control. A comparison between the group immunised only with the L. amazonensis crude protein extract and the groups immunised with adjuvants (FIA, alum, and VPs) revealed a statistically significant difference. Then, 40 days post-second immunisation, the FIA group presented a higher level compared with negative control, whilst the groups with alum, VPs, and the crude protein extract showed an increase when compared to the negative control (Figure 3). Finally, 50 days post-second immunisation, the FIA group maintained the highest levels, followed by the alum group, the group immunised only with the L. amazonensis crude protein extract, and the VPs group. There was no significant difference between the group immunised with the crude protein extract and the VPs group at this time point.

To characterise the nature and quality of the humoral immune response induced during the immunisation process, the IgG antibody subtypes (IgG1, IgG2a, IgG2b, and IgG3) were determined in immunised mice 60 days post-immunisation. The results presented in Figure 4 show that all immunised groups showed statistically significant differences in total IgG anti-L. amazonensis compared to the negative control. Then, when comparing the group immunised only with the crude protein extract to the other groups, a significant difference was only found for the FIA and alum groups, with no difference observed in the group immunised with VPs.

In turn, the group immunised with VPs did not show significant anti-Leishmania IgG1 antibody levels. In contrast, the groups that received the crude protein extract in combination with FIA or alum presented a significantly higher IgG1 level compared to the negative control and the group immunised only with the crude protein extract (Figure 4). Moreover, the groups immunised with FIA and alum demonstrated statistical significance concerning the anti-Leishmania IgG2a antibodies, whilst the VPs group showed no significant differences when compared to the group immunised only with the crude protein extract and the non-immunised group. Interestingly, it was observed that the FIA, alum, and VPs immunised groups showed the presence of anti-Leishmania IgG2b antibodies compared to the group that received only the crude protein extract. Additionally, these results indicate that the high anti-Leishmania IgG3 antibody levels were measured in the VPs-immunised group, followed by the FIA, alum, and the crude protein extract groups. Then, when comparing the immunised groups, it was observed that the VPs group exhibited a high IgG3 level, with a significant difference when compared to the other groups (FIA, alum, and crude protein extract).

Overall, the analysis of anti-Leishmania IgG subtypes suggests that VPs-immunised mice did not induce a significant IgG2a response, unlike the groups with established adjuvants, such as FIA and alum, which exhibited higher levels of these isotypes. However, mice immunised with VPs together with crude protein extraction of L. amazonensis demonstrated immunoadjuvant potential, with greater IgG3 production, surpassing the other immunised groups (Figure 4).

First, sera from immunised mice 15, 30, and 45 days post-immunisation were used to quantify specific antibodies against chimeric recombinant proteins of T. cruzi by ELISA, in order to evaluate the immunisation process with chimeric recombinant proteins (denominated here IBMP) of T. cruzi, formulated with different vaccine adjuvants (alum, FIA, and VPs). A mixture (Mix) of four recombinant chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) mixed with different vaccine adjuvants was used for immunisation. Figure 5 represents the IgG antibody profile against each recombinant chimeric protein of T. cruzi. The total IgG antibody profiles were against each IBMP antigen: anti-IBMP-8.1 (a), anti-IBMP-8.2 (b), anti-IBMP-8.3 (c) and anti-IBMP-8.4 (d).

The results show (Figure 5) that the groups immunised with adjuvants during the primary immunisation process present statistical disparities when compared to the negative control. The group with only the Mix presents different total IgG antibody profile for each IBMP protein. The anti-IBMP-8.1 (Figure 5a) and anti-IBMP-8.2 (Figure 5b) antibody profiles are higher than the negative control, whilst anti-IBMP-8.3 (Figure 5c) and anti-IBMP-8.4 (Figure 5d) are even more elevated. However, they did not present statistically significant differences at any of the times analysed, or in relation to the negative control.

In this scenario, the association of Mix with FIA showed a difference in total IgG profile when compared to the negative control, with a higher antibody profile observed by inducing the IBMP-8.2 (Figure 5b), IBMP-8.3 (Figure 5c) and IBMP-8.4 proteins (Figure 5d). Only anti-IBMP-8.1 antibodies (Figure 5a) presented lower antibody profiles when compared to the induction of the others; even so, this is the group with the highest antibody profile at all times and proteins in the first dose (Figure 5).

As shown in Figure 5, the immunoadjuvant potential of VPs (VP1, VP2 and VP3) for the induction of total IgG antibodies was evaluated in association with the Mix (Mix + VPs), demonstrating no statistical difference against the IBMP-8.1 (Figure 5a) and IBMP-8.4 proteins (Figure 5d) compared to the negative control. There was a difference in anti-IBMP-8.2 (Figure 5b) in the three analysed times points and anti-IBMP-8.3 (Figure 5c) only for the 15-day time point.

The statistical differences between the groups at the three time points were analysed and it was observed that there was a statistically significant difference in the antibody profiles at 15 days between Mix vs. Mix with FIA; Mix vs. Mix with alum; Mix with FIA vs. Mix with alum; and Mix with FIA vs. Mix with VPs in relation to the IBMP-8.1 protein (Figure 5a). For IBMP-8.2 (Figure 5b), in addition to the difference between Mix vs. Mix with VPs (demonstrated in the graph by the “#” sign), there were differences between: Mix vs. Mix with FIA; Mix with FIA vs. Mix with alum; Mix with FIA vs. Mix with VPs; and Mix with alum vs. Mix with VPs. Taken together, regarding the IBMP-8.3 protein (Figure 5c), there was only no difference between Mix vs. Mix with VPs, and the same occurred for the profile related to IBMP-8.4 (Figure 5d).

In turn, there was no significant difference at 30 days post-immunisation for anti-IBMP-8.1 antibodies (Figure 5a) between the immunised groups Mix vs. Mix with alum; Mix vs. Mix with VPs; and Mix with alum vs. Mix with VPs. Regarding anti-IBMP-8.2 antibodies (Figure 5b), there was no difference when comparing Mix with alum vs. Mix with VPs. In the antibody profiles for the anti-IBMP-8.3 protein (Figure 5c), there was a difference between the groups: Mix vs. Mix with FIA; Mix vs. Mix with alum; Mix with FIA vs. Mix with alum; Mix with FIA vs. Mix with VPs; and Mix with alum vs. Mix with VPs. On the other hand, in the antibody profiles against IBMP-8.4 (Figure 5d), there were differences between: Mix vs. Mix with FIA; Mix with FIA vs. Mix with alum and Mix with FIA vs. Mix with VPs.

In relation to the anti-IBMP-8.1 antibodies (Figure 5a) at 45 days post-immunisation, a difference was only observed between: Mix vs. Mix with FIA; Mix with FIA vs. Mix with alum and Mix with FIA vs. Mix with VPs. However, the scenario changes for anti-IBMP-8.2 antibodies (Figure 5b), presenting a difference only in the comparison of Mix vs. Mix with alum and Mix with alum vs. Mix with VPs. Furthermore, a difference is noted for anti-IBMP-8.3 antibodies (Figure 5c) between: Mix vs. Mix with FIA; Mix vs. Mix with alum; Mix with FIA vs. Mix with alum; and Mix with FIA vs. Mix with VPs. Then, in the analysis related to IBMP-8.4 antibodies (Figure 5d), no difference is observed when comparing Mix vs. Mix with alum; Mix vs. Mix with VPs and Mix with alum vs. Mix with VPs.

Secondary immunisation of the mice was performed after characterising the antibody response of mice immunised with a single dose (Figure 5). In this scenario, the results presented in Figure 6 show that the group immunised with Mix administered without adjuvants shows a considerable increase in total IgG profiles for all proteins and time points after secondary immunisation. Notably, at 45 days post-immunisation, the anti-IBMP-8.3 antibody (Figure 6c) levels increase with time, whilst the anti-IBMP-8.1 (Figure 6a) and anti-IBMP-8.4 (Figure 6d) profiles remain the same. Only the profile of antibodies induced by IBMP-8.2 protein (Figure 6b) decrease at 30 and 45 days.

Immunised mice with Mix with alum showed a significant change for total IgG anti-IBMPs antibodies in the second dose and statistically differs from the first immunisation. The Mix in association with FIA continued to be the group that demonstrated the highest total IgG profile at all times and proteins evaluated. The antibody profiles induced by all proteins increased over time, reaching their highest levels at 45 days. In addition, the association of Mix with VPs, at all times and all immunisations evaluated, presented significant differences compared to the negative control, with an increase in total IgG induced by the IBMP-8.3 protein (Figure 6c), reaching its highest level at 45 days. The lowest antibody profile is reported for anti-IBMP-8.2 antibodies (Figure 6b) at 30 and 45 days, whilst the anti-IBMP-8.1 antibody (Figure 6a) and anti-IBMP-8.4 antibody (Figure 6d) profiles remained similar at all times evaluated.

The statistical differences between the immunised groups at the three time points were also analysed and it was observed that no difference was found for the following immunised groups at 15 days post-immunisation: between Mix with alum vs. Mix with VPs for the anti-IBMP-8.1 antibodies (Figure 6a); between Mix with FIA vs. Mix with alum for anti-IBMP-8.2 antibodies (Figure 6b); between Mix with alum vs. Mix with VPs for the anti-IBMP-8.3 antibodies (Figure 6c); and for anti-IBMP-8.4 antibodies (Figure 6d) between the groups Mix with FIA vs. Mix with alum as presented by anti-IBMP-8.2 antibodies; Mix with FIA vs. Mix with VPs; and Mix with alum vs. Mix with VPs for anti-IBMP-8.1 antibodies.

Next, there was no statistically significant difference for the antibody profiles against IBMP-8.1 (Figure 6a), IBMP-8.3 (Figure 6c) and IBMP-8.4 (Figure 6d) proteins at 30 days post-immunisation between Mix vs. Mix with VPs. However, a difference is observed between all groups for the antibody profiles against the IBMP-8.2 protein (Figure 6b). In turn, no statistically significant difference was found at 45 days post-immunisation for anti-IBMP-8.1 antibodies (Figure 6a) or anti-IBMP-8.4 antibodies (Figure 6d) between Mix vs. Mix with VPs, as well as at 30 days. A difference is observed between all groups for anti-IBMP-8.2 antibodies (Figure 6b) at 45 days post-immunisation, as well as at 30 days. Lastly, a difference is observed only between Mix vs. Mix with alum for anti-IBMP-8.3 antibodies (Figure 6c).

Then, in order to characterise the nature and quality of the humoral immune response induced during the immunisation process with chimeric recombinant proteins of T. cruzi, the IgG antibody subtypes (IgG1, IgG2a, IgG2b, and IgG3) were determined in the immunised mice at 70 days post-second immunisation (Figure 7). Considering that we already know the total IgG profile against each protein separately, at this stage it was decided to present the profile against the Mix of the four proteins in antigenic mixture (Mix-IBMP) to provide a new perspective of the profile and a new approach to interpreting the results.

Therefore, the data presented in Figure 7 show the profiles of the different isotypes of IgG antibodies against chimeric recombinant protein of T. cruzi associated with different adjuvants. The immunised group associated with FIA continues to exhibit the highest IgG profile when compared to the negative control. There is no induction of IgG1, but it is the group that induces the most IgG2a and IgG3, whilst the profile for IgG2b is similar to that of Mix associated with alum. In addition, the total IgG profile for Mix with alum is similar to that observed when Mix was associated with FIA, with 97-times higher levels than the non-immunised mice. Moreover, there was no induction of IgG1, there was little induction of IgG2a with a lower profile compared to the association with FIA and VPs, IgG2b was equal to the association with FIA and IgG3 had a lower profile, but still higher than that observed with VPs. In this context of IgG subtypes, the results demonstrated that there was no induction of IgG1, whilst the IgG2a and IgG3 antibodies induction displayed similar profiles, and IgG2b showed higher levels of antibodies produced. All antibodies produced presented higher profiles than those observed when Mix was administered alone (Mix), mainly IgG2b. VPs was able to induce higher IgG2a levels in relation to alum.

Furthermore, all antibody subclasses produced 70 days after the second dose in association with VPs had a better profile than when Mix was administered alone, especially IgG2b. Mix, whether associated or not with adjuvants, did not produce IgG1 even 70 days after the second dose, including with VPs. After the second dose, Mix alone was capable of inducing levels of total IgG and subclasses at all times and for all proteins, including at 70 days after the second dose. No group showed a statistically significant difference in the IgG1 profile. The highest IgG levels in the second dose were induced by the IBMP-8.2 and IBMP-8.3 proteins. The IgG2b subclass was the most produced in all groups compared to 70 days after the second dose.

The genes of the VPs VP1, VP2 and VP3 of Triatoma virus (TrV) were cloned into pET3d (+), expressed in E. coli BL21 (DE3) and induced with IPTG. The recombinant proteins were purified by size exclusion chromatography and characterised by SDS-PAGE and immunoblotting using rabbit polyclonal serum anti-VP1, anti-VP2, and anti-VP3 antibodies.

L. amazonensis strain was kindly provided by the Institute of Hygiene and Tropical Medicine at the Nova University of Lisbon, Portugal. Promastigote forms of L. amazonensis were cultured for 7 days in RPMI-1640 medium (Roswell Park Memorial Institute 1,640), supplemented with 10% (v/v) inactivated Foetal Bovine Serum (FBS) and 2% (v/v) streptomycin/penicillin antibiotics (100 IU/mL). The cultures were maintained at 27 °C in a BOD incubator (ASP, SP-500) (Limoncu et al., 1997). Maintenance was performed every 2-3 days due to changes in the pH of the medium, detected by the colour change from pink to yellow. After reaching the stationary growth phase with a concentration of 1 × 10⁶ parasites/mL in the Leishmania spp., the culture was centrifuged at 3000 × g and 4 °C for 10 min. The obtained pellets were washed three times with sterile Phosphate-Buffered Saline (PBS), then centrifuged at 3000 × g and 4 °C for 10 min each wash. Pellets containing the parasite were subsequently resuspended in 1 mL of PBS with protease inhibitor, pH 7.0. The content was subjected to six freeze-thaw cycles, followed by mechanical agitation.

T. cruzi recombinant proteins, called IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4, were produced at the Instituto Carlos Chagas (Oswaldo Cruz Foundation, FIOCRUZ, Paraná, Brazil) by synthetic genes optimised for expression in E. coli (Gen-Script, United States). After subcloning into pET28a, the recombinant proteins were purified using affinity chromatography via a 6xHis tag. These recombinant proteins are protected by a patent application at Fiocruz-Brazil (No. 1020180161407) (Santos et al., 2018).

In this work, two immunisation models were studied: the first strategy involved native L. amazonensis antigens and the immunisation schedule is shown in Table 1. The second strategy involved chimeric recombinant antigens of T. cruzi and the immunisation protocol is shown in Table 2. All antigens and proteins were eluted in PBS (pH 7.2) and mixed with each of the adjuvants in a 1:1 (v/v) ratio using a vortex.

All procedures involving animals were conducted in accordance with the guidelines 80 of the National Council for the Control of Animal Experimentation (CONCEA) and international standards of animal welfare. The study was approved by the Animal Ethics Committee of the Federal University of Rio Grande do Norte (CEUA/UFRN), protocols numbers 080/2017 and 041/2023). Female BALB/c mice (20–30 g, 8 weeks old) were maintained under controlled lighting (12 h light/dark) and temperature (22 °C) conditions. The mice were obtained from the Animal Facility of the Health Sciences Centre of the Federal University of Rio Grande do Norte, Brazil. Immunisations were administered subcutaneously using a syringe (Omnican^® 100). After the interventions, the mice received an overdose of xylazine (Syntec, São Paulo, Brazil) and ketamine (Vetnil, São Paulo, Brazil) intraperitoneally, followed by euthanasia by cardiac exsanguination.

Serum samples were initially collected after whole blood clotting by terminal excision of the tail or by cardiac exsanguination after of the euthanasia to determine specific antibodies from immunised mice. Then, samples were maintained at 37 °C for 30 min and then at 5 °C for 1 h. Serum was subsequently obtained by centrifugation at 5,000 rpm for 10 min. The serum samples were fractionated and stored at −20 °C for screening murine antibodies specific anti-L. amazonensis, anti-recombinant T. cruzi proteins (IBMPs proteins), and anti-structural proteins from VP1, VP2, and VP3 by Enzyme Linked Immunosorbent Assay (ELISA).

Next, 96-well microplates (Nunc-Immuno MicroWell, United States) were sensitised with 0.1 M bicarbonate buffer (pH 8.5) with 100 ng/well of L. amazonensis crude protein extract or 50 ng/well of IBMP recombinant proteins of T. cruzi. The plates were sensitised overnight at 5 °C for both protocols (Queiroz et al., 2021a). The microplates were subsequently washed three times with PBS containing 0.05% Tween20 (Promega, United States). Then, the plates were blocked for 1 h at room temperature (25 °C) with 1% (w/v) skim milk in PBS. After five additional washes, sera from immunised mice were added in serial dilutions: 1:25 for total IgG and 1:10 for IgG subtypes (for determining anti-L. amazonensis antibodies), and 1:100 or 1:50 for total IgG and 1:50 for IgG subtypes (for determining anti-recombinant T. cruzi proteins), all in antibody dilution buffer. After five more washes, HRP-conjugated antibodies (rabbit anti-mouse IgG diluted 1:4.000-Sigma-Aldrich, United States; rabbit anti-mouse IgG1 diluted 1:2.000-Caltag Laboratories, United States; rabbit anti-mouse IgG2a diluted 1:2.000-Caltag Laboratories, United States; rabbit anti-mouse IgG2b diluted 1:2.000-Invitrogen, United States, and rabbit anti-mouse IgG3 diluted 1:2.000-Sigma-Aldrich, United States) were added in antibody buffer and incubated for 1 h at room temperature. Subsequent washes were performed, and the plates were incubated for 30 min at room temperature with a substrate solution consisting of 10 mL of citrate buffer (pH 5.0) with 10 mg of o-phenylenediamine dihydrochloride (OPD: Sigma-Aldrich, United States) and 10 μL of hydrogen peroxide 30%. All sera from immunised mice were analysed in duplicate, and the final reaction absorbance at 490 nm was measured using a spectrophotometer (Epoch, BioTek Instruments, Winooski, VT, United States).

Data relating to the differences between the means of the immunised groups and the non-immunised control groups during primary and secondary immunisations, days after immunisations, antigens and antibody profiles were analysed according to Analysis of Variance (ANOVA), followed by the Tukey’s multiple comparison post-test by GraphPad Prism© software version 8.4 (GraphPad Inc., Illinois, Chicago, United States). The significance of differences between means was established when p < 0.05.