Adjuvanted Leishmania infantum extracellular vesicles induce protective immunity in experimental visceral leishmaniasis

Adjuvanted Leishmania infantum extracellular vesicles induce protective immunity in experimental visceral leishmaniasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Adjuvanted Leishmania infantum extracellular vesicles induce protective immunity in experimental visceral leishmaniasis Antonia Efstathiou, Maria Agallou, Dimitra K. Toubanaki, Fotis Badounas, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9033802/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract The lack of an effective vaccine remains a critical barrier to controlling visceral leishmaniasis (VL), a lethal parasitic disease with expanding global incidence. Here, we evaluate extracellular vesicles (EVs) released by stationary-phase Leishmania infantum promastigotes as a biologically derived, cell-free vaccine platform when formulated with the clinically relevant oil-in-water adjuvant Addavax. Parasite-derived EVs contained a broad repertoire of immunologically relevant proteins, including GP63, and induced functional maturation of bone marrow–derived dendritic cells in vitro . In vivo , immunization of BALB/c mice with adjuvanted EVs elicited antigen-specific antibody responses and robust Th1-biased cellular immunity, characterized by enhanced T-cell proliferation and increased frequencies of IFN-γ–producing CD4⁺ T cells. Following experimental challenge, vaccinated mice exhibited significantly reduced hepatic and splenic parasite burdens during both acute and chronic infection, accompanied by preserved tissue architecture. Protection correlated with a favorable IFN-γ/IL-10 balance, supporting adjuvanted parasite-derived EVs as a modular, cell-free vaccine strategy for VL. Biological sciences/Immunology Biological sciences/Microbiology Leishmania infantum Extracellular vesicles vaccine visceral leishmaniasis protective immunity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Leishmaniases, a group of vector-borne diseases remain a major public health concern in tropical and subtropical regions worldwide 1 . Among the clinical manifestations, visceral leishmaniasis (VL) represent the most severe form and being almost uniformly fatal if left untreated 2 . Despite ongoing control efforts, VL continues to impose a substantial global burden, accounting for tens of thousands of deaths annually and contributing significantly to disability-adjusted life years lost, with profound socioeconomic consequences in endemic settings 3 , 4 . Current VL control strategies rely primarily on early diagnosis and chemotherapy to reduce the human reservoir, alongside vector control interventions such as indoor residual spraying 5 . Although these measures have reduced disease incidence in some regions, elimination programs have repeatedly failed to achieve sustained success, highlighting intrinsic limitations of existing approaches. These shortcomings underscore the critical need for improved diagnostics, more effective therapeutics, and, most importantly, a prophylactic vaccine capable of inducing durable protective immunity and preventing disease resurgence 5 . The rationale for vaccination against VL is supported by clinical observations showing that individuals who recover from infection often develop long-lasting immunity against reinfection 6 , 7 . Consistent with this concept, a wide range of vaccine platforms—including whole-parasite preparations, recombinant subunit vaccines, viral-vectored constructs, and DNA-based approaches—have been evaluated in preclinical and early clinical studies 8 – 10 . Nevertheless, progress toward a licensed human vaccine has been limited. To date, only the recombinant subunit vaccine LeishF3 has completed a phase I clinical trial, demonstrating acceptable safety but no advancement to licensure 11 . These challenges largely reflect the complex biology of Leishmania parasites, which exhibit a digenetic life cycle, extensive antigenic diversity, and sophisticated immune evasion strategies that complicate vaccine design. Recent advances in nanotechnology and immunology have stimulated interest in extracellular vesicles (EVs), particularly exosomes, as alternative vaccine platforms. Exosomes are nanoscale vesicles generated within multivesicular bodies and released into the extracellular milieu, where they participate in intercellular communication and immune modulation 12 . In oncology, exosomes derived from antigen-loaded dendritic cells have demonstrated the capacity to elicit strong immune responses and, in some cases, clinical benefit 13 – 15 . Similarly, EV-based approaches have shown immunogenic potential against diverse pathogens, including Toxoplasma gondii , Mycobacterium tuberculosis , and Plasmodium species 16 , 17 . Compared with synthetic delivery systems, EVs possess several attributes favorable for vaccine development, including high biocompatibility, stability in biological environments, and efficient antigen delivery 18 , 19 . EVs encapsulate complex molecular cargo composed of proteins, lipids, and nucleic acids that reflect the biological state of their parent cells, potentially providing a multifaceted immunological stimulus 20 . Their endogenous origin is associated with low toxicity, and established purification and scale-up methodologies support their translational potential. However, the immunological effects of EVs are highly context-dependent and influenced by cargo composition, cellular origin, and environmental cues. In the context of leishmaniasis, parasite-derived EVs have been extensively implicated in host–pathogen interactions that promote immune modulation and disease progression. Studies in macrophages and murine models have shown that Leishmania EVs contain virulence-associated molecules, including the metalloprotease GP63, capable of altering host signaling pathways and immune responses 21 – 24 . Conversely, accumulating evidence indicates that EVs derived from specific parasite species or developmental stages can also carry immunogenic components capable of antigen presentation and immune activation 25 . In some contexts, these vesicles exhibit adjuvant-like properties by promoting cytokine and chemokine production and facilitating innate immune cell recruitment 19 . Together, these findings suggest that parasite-derived EVs may exert either pathogenic or protective effects, depending on their molecular composition and the immunological environment in which they are encountered 26 . Notably, Leishmania EVs represent a composite antigenic platform encompassing proteins, lipids, and nucleic acids. Their protein cargo includes several well-characterized immunogens, such as LACK, TSA, KMP11, and LmSTI1, able to induce protection in preclinical studies 27 . Supporting this concept, prior studies demonstrated that dendritic cell–derived exosomes generated following exposure to cutaneous Leishmania parasites enhanced Th1- and Th17-type immunity and reduced parasite burdens 28 . These observations raise the possibility that appropriately formulated parasite-derived EVs could be harnessed to amplify protective immunity rather than promote disease. Effective EV-based vaccination strategies, however, often require adjuvants capable of recruiting and activating dendritic cells and promoting antigen presentation in vivo 29 . Dendritic cells are considered a significant immune population in driving T-cell priming and long-term memory formation 30 . Several adjuvants have been explored in VL vaccine development, including saponins and monophosphoryl lipid A 31 . Addavax, an oil-in-water emulsion that mimics the licensed MF59 adjuvant, has previously been shown by our group to enhance protective immune responses against Leishmania infantum in experimental VL models 32 . MF59-like adjuvants act primarily by inducing local innate immune activation and rapid recruitment of myeloid cells, thereby enhancing antigen uptake and adaptive immune priming 33 , 34 . Based on these considerations, we hypothesized that EVs derived from stationary-phase L. infantum promastigotes, when combined with an appropriate adjuvant, could be redirected from immunomodulatory or pathogenic roles toward the induction of protective Th1-biased immunity. Subsequently, we evaluated the immunogenicity, safety, and efficacy in terms of protection of parasite-derived EVs formulated with Addavax in a murine model of VL. Our findings demonstrate that this cell-free, EV-based vaccine strategy elicits regulated yet robust Th1-type immune responses, characterized by activation of CD4⁺ and CD8⁺ T-cell specific for EVs, and confers significant protection against L. infantum infection in target organs. Together, these findings support the potential of adjuvanted parasite-derived EVs as a promising platform for the development of effective vaccines against visceral leishmaniasis. Results Characterization of Leishmania infantum promastigote-derived EVs Extracellular vesicles (EVs) were isolated from the culture supernatant of L . infantum promastigotes and purified by differential centrifugation followed by ultracentrifugation. The physical and biochemical properties of the isolated vesicles were evaluated in accordance with the MISEV2023 recommendations 35 . EV characterization included assessment of particle size, size distribution, morphology, and enrichment of established exosomal protein markers. Transmission electron microscopy (TEM) revealed rounded, bilayered vesicles with diameters typically below 200 nm, consistent with the morphology of small EVs or exosomes (Fig. 1 A, B). Dynamic light scattering (DLS) analysis showed a unimodal size distribution with a mean diameter of 160 ± 28.1 nm, with Polydispersity Index (PDI) 0.181 which indicates that the isolation method yielded a single dominant vesicle population with minimal aggregation or debris (Fig. 1 C). The zeta potential was − 14.6 ± 4.6 mV, indicating colloidal stability of the vesicle suspension. Western blot analysis confirmed enrichment of EV populations used for immunizations, through the detection of canonical EV markers CD9, CD63, and CD81 (Fig. 1 D), whereas the lack of detectable ALB and CytC signals indicated minimal soluble protein or intracellular contamination (Fig. 1 D). Proteomic analysis of the EV preparations used for subsequent murine immunization identified a total of 1,441 peptides by LC-MS/MS. Among these, several peptides corresponded to previously reported Leishmania antigenic proteins with documented immunogenic potential (Table 1 ). Notably, the EV protein cargo included several well-recognized Leishmania antigenic proteins, including GP63 (isoforms GP63-1, GP63-2, and GP63-4), α- and β-tubulin chains, eukaryotic initiation factors 3, 4A, and 5A, kinesin-like proteins, surface antigen–like proteins, heat shock proteins HSP70, HSP83-1, HSP100, and the putative HSP40/DNAJ, as well as thiol-dependent reductase 1 (TDR1), among others 36 – 41 . These findings indicate that the EV preparations retained key antigenic components relevant for host immune recognition. Collectively, these results demonstrated that the isolated vesicles from L. infantum promastigote cultures exhibited the expected size, morphology, and protein marker profile characteristic of extracellular vesicles. Table 1 Enriched proteins with known antigenic potential of Leishmania infantum promastigotes-derived EVs detected by Mass Spectrometry. Uniprot ID Protein name A4IDN4 Related to elongation factor-2 kinase efk-1b isoform-like protein A4I5B8 Putative surface protein amastin A4IBB3; A4IBB2 Kinetoplastid membrane protein-11 A4HW10; A4HW09 Elongation of fatty acids protein A4HSL2 Surface antigen-like protein Q6RYT3 Tryparedoxin A0A381MG06;A4HU57;A4HY42 Histone H2B A4HVB0;E9AGG5;D1GJ51 Putative surface antigen protein 2 A4HW98; A4HSP4 Histone H4 A4HU18;A4HU19;A4I925 Elongation factor-1 gamma A4HSC2 Actin A4I562 Kinesin-like protein A4I8P2 Thiol-dependent reductase 1 E9AGQ3 Putative elongation factor Tu A4I4I4 ATP-dependent Clp protease subunit, heat shock protein 100 A4I341 Putative heat shock protein DNAJ A4HY48 Kinesin-like protein E9AGP5;A4HX73;E9AGP7 Elongation factor 1-alpha A4I253 Heat shock protein 70-related protein Q6LA77;A4HUF8 Leishmanolysin A4HW62 Enolase A4I5S5;A4I5T0;A4I5S4 Putative heat shock 70-related protein 1, mitochondrial A4HUF6 Leishmanolysin E9AGK7;A0A381MCS3;A4HVG1 Tubulin alpha chain A4HTR1;A0A381MS01; A4HTR0 Tubulin beta chain E9AGQ5 Putative heat shock protein E9AHM9 Heat shock protein 83 − 1 A4ICW8 Elongation factor 2 E9AHH1;A4I412;E9AHH0 Putative heat-shock protein hsp70 EVs induce BMDCs maturation toward a DC1-like phenotype Given that Leishmania -derived EVs may serve as a source of parasite antigens, we next examined their capacity to induce maturation and functional activation of dendritic cells (DCs), which bridge innate and adaptive immune responses through activation of naïve T cells. For this purpose, bone marrow–derived dendritic cells (BMDCs) were stimulated for 24 h with Leishmania EVs, soluble Leishmania antigen (SLA), or lipopolysaccharide (LPS) as a positive control. Flow cytometry revealed that the uptake of EVs by BMDCs significantly enhanced the number of CD40-expressing cells as well as CD40 expression levels per cell as evidenced by MFI values by about 6–fold and 2–fold, respectively, compared to untreated BMDCs (Fig. 2 A, B). Regarding MHCI and MHCII molecules, EVs despite the fact that they did not change the number of MHCI-/II-expressing cells (Fig. 2 A), they significantly upregulated the expression of both co-stimulatory surface molecules according to increased MFI values (Fig. 2 C, D). As expected, LPS stimulation, serving as a positive control stimulus, resulted in maturation of BMDCs as assessed by CD40, MHCI and MHCII molecules expression (Fig. 2 A-D). Importantly, all three surface molecules were found to be expressed at similar levels as in LPS-treated BMDCs indicating also EVs’ probable immunogenicity (Fig. 2 A-D). On the contrary, BMDCs exposure to SLA did not induce their maturation as evidenced by the low expression levels of all three surface molecules that were similar to those detected in untreated BMDCs (Fig. 2 A-D). Cytokine production by BMDCs was also assessed by flow cytometry. EV stimulation resulted in a significant increase in the proportion of IL-12– and TNFα–producing BMDCs (Fig. 2 E, F), along with a detectable increase in IL-10–producing cells (Fig. 2 G). Importantly, the IL-12/IL-10 ratio following EV stimulation was significantly higher than in untreated controls and comparable to that observed following LPS treatment (Fig. 2 H), indicating preferential induction of pro-inflammatory cytokine responses. Taken together, these data indicate that EVs derived from L. infantum promastigote cultures effectively promote BMDC activation toward a DC1-like phenotype. Evaluation of EVs immunization-induced humoral and systemic immune responses The immunogenicity of the proposed vaccine was then evaluated at the level of adaptive immunity. For this purpose, the mice were i.m. vaccinated with EVs alone (ExoVac) or adjuvanted with Addavax (ExoAddaVac) on day 0, followed by a homologous booster dose on day 14. Control groups included non-immunized (PBS) and Addavax-injected mice (Fig. 3 A). Routine hematological analysis performed two weeks after boost injection that ExoAddaVac-immunized mice produced a significantly higher concentration of circulating lymphocytes (LY), whereas the numbers of neutrophils (NE) were reduced compared to control mouse groups (Fig. 3 B). On the contrary, ExoVac-immunized mice exhibited a different profile with significantly elevated numbers of neutrophils (NE) followed by reduced numbers of circulating lymphocytes (LY) (Fig. 3 B). Complementary to the above observations were the histological findings in spleen tissue where vaccinated mice did not show any sign of uncontrolled inflammation (Fig. 3 C). Subsequently, vaccine-induced humoral immune responses were investigated as a surrogate of adaptive immunity activation. As shown in Fig. 3 D, at 2 weeks’ post-boost immunization, adjuvanted (ExoAddaVac) and non-adjuvanted EVs (ExoVac) elicited parasite-specific IgG antibody responses, whereas as it was expected, the other control groups of vaccinated mice were negative for parasite specific antibodies. Importantly, the observed antibody responses were generally evoked to a significantly greater extent in Addavax-adjuvanted group (Fig. 3 D). Analysis of IgG isotypes unveiled that ExoAddaVac promoted both the production of high IgG1 and IgG2a responses, with a bias towards IgG1 antibody isotype profile (Fig. 3 E, F). On the contrary, ExoVac induced the production of IgG1 only and in much lower levels than those detected in ExoAddaVac group (Fig. 3 E, F). Overall, immunization with Addavax-adjuvanted EVs effectively elicited robust humoral and cellular immune responses without inducing sustained inflammatory pathology. EVs immunization induced antigen-specific Th1-type cellular immune responses T cell activation was next evaluated by measuring the proliferative capacity of splenic lymphocytes upon ex vivo stimulation with EVs or SLA. Splenocytes from ExoAddaVac-immunized mice exhibited significantly increased proliferation in response to EV stimulation compared with PBS- and Addavax-treated controls, indicating differentiation of EV-specific lymphocytes (Fig. 3 G). Additionally, ExoAddaVac promoted also the differentiation of parasite-specific spleen cell population as evidenced by increased proliferation levels of spleen cells in the presence of SLA, even though they did not reach statistical significance (Fig. 3 H). On the other hand, spleen cells isolated from ExoVac-immunized mice exhibited increased lymphoproliferative action only in the presence of EVs although at significantly lower levels when compared with ExoAddaVac group (Fig. 3 G, H). Importantly, all groups of mice responded similarly with high proliferation levels when stimulated with ConA, a positive stimulant (Fig. 3 I), indicating that the differences observed were not due to differences in the viability of T cells taken from the different mouse groups. Enhanced proliferation in ExoAddaVac mice was accompanied by significantly increased IL-2 production (Fig. 3 J). Cytokine secretion profiles further supported Th1 polarization. Splenocytes obtained from ExoAddaVac-immunized mice exhibited a significantly and globally increased cytokine secretion compared not only to the control mice groups but also to EVs-immunized mice (ExoVac), which was collectively consistent with the changes in proliferation assays from the same group (Fig. 3 K-N). Importantly, this phenomenon was restricted to EVs stimulation, since in the presence of SLA spleen cells of either ExoVac or ExoAddaVac group did not produced cytokines further supporting the differentiation of EVs-specific effector T cells (Fig. 3 K-N). Specifically, splenocytes obtained from mice vaccinated with adjuvanted EVs showed significantly enhanced production of the pro-inflammatory cytokine IFN-γ in comparison to all other groups tested after stimulation with EVs (Fig. 3 K). On the contrary, EVs stimulation induced similar levels of TNFα production levels in all mice groups irrespective of vaccination regimen (Fig. 3 L). Although IL-4 and IL-10 levels were also increased (Fig. 3 M, N), IFN-γ/IL-10 and IFN-γ/IL-4 ratios were significantly higher in ExoAddaVac-immunized mice, indicating a dominant Th1-biased response (Fig. 3 O). ExoAddaVac immunization induced the differentiation of both antigen-specific CD4 and CD8 T cells To assess in depth, the impact of the vaccine in T cell immune responses, we employed flow cytometry to characterize vaccine-induced T cell subsets following ex vivo EV stimulation. For this purpose, spleen cells were stimulated ex vivo with EVs and antigen-specific populations were determined. Specifically, ExoAddaVac as well as ExoVac immunization led to increased numbers of antigen-specific CD3 + cell population compared to control mouse groups (Fig. 4 A). Analysis of T cell groups unveiled that ExoAddaVac induced a significant increase of activated CD4 + T cells of 6.74% as well as CD8 + T cells to 15.6% compared to naïve/adjuvant-control mice after ex vivo stimulation with EVs (Fig. 4 B, C). In contrast, ExoVac immunization resulted in modest, non-significant increases in CD4⁺ T cells only (Fig. 4 B, C). Cytokine profiling revealed that ExoAddaVac-immunized mice exhibited increased frequencies of IFN-γ-producing CD4 + T and CD8 + T cells, exceeding the proportion of IL-4-producing CD4 + T cells (Fig. 4 E-G), further supporting that EVs immunization induced the differentiation of Th1 mediated immune responses. ExoVac-immunized mice displayed only minimal IFN-γ-producing CD4 + T cells (Fig. 4 E-G). Assessment of biocompatibility and safety of EVs-based vaccine Vaccine safety was evaluated through continuous monitoring of animal behavior and physiology. No morbidity, mortality, or injection-site reactions were observed. Vaccinated mice displayed normal mobility and feeding behavior, comparable to PBS-treated controls. Body weight increased steadily over time in all groups, with no significant differences between vaccinated and control mice (Supplementary Fig. 1). Overall, the immunization regimen was well tolerated. ExoAddaVac immunization conferred protection against L. infantum challenge To verify whether vaccine-induced T cell responses translated into protective immunity against L. infantum infection, mice were immunized with the protocol described above and then challenged with virulent promastigotes of the parasite. As a measure of the systemic effect of the infection, changes in the body weight were determined for 12 weeks following the parasite challenge. In contrast to both control mice groups that lost weight after parasite challenge, both groups of immunized mice slowly regained most of their initial body weight till the end of the study (Supplementary Fig. 2). Additionally, the weights of parasite’s target organs, spleen and liver, were measured and parasite load estimation was conducted in both organs at 4 and 12 weeks post challenge, representing the acute (4 wpc) and the chronic (12 wpc) phase of disease, as a parameter of disease establishment. According to our observations, non-immunized and adjuvant-injected mice possessed heavier livers than ExoVac- and ExoAddaVac-immunized mice at 4 wpc (Fig. 5 A). However, during transition to chronic phase of the disease livers’ weight from the ExoVac group reached in weight those isolated from both control groups, indicating that ExoVac group may failed to restrict parasite load in liver (Fig. 5 A). In agreement with the above observations, immunization with ExoAddaVac significantly reduced the hepatic parasite burden compared to control group during acute phase of disease (Fig. 5 C). At 12 wpc, ExoVac mouse group contained similar numbers of parasite with non-immunized and Addavax-injected mice which were higher than those detected in ExoAddaVac group, reflecting ExoVac’s weakness to restrict parasite replication (Fig. 5 C). Regarding spleen tissues, profound splenomegaly could be easily observed followed by increased weight at chronic phase of disease not only at both control groups but also in the ExoVac-immunized group probably due to parasite establishment (Fig. 5 B). In agreement with this observation, parasite load estimation during chronic phase of disease showed that ExoAddaVac controlled splenic parasite burden significantly better than the ExoVac following L. infantum challenge. Specifically, when compared to the PBS control group, ExoAddaVac-immunized group reduced the parasite burden by 15-fold at acute phase and 5-fold at chronic phase, whereas ExoVac group failed to preserve low parasite burden at chronic phase of disease reaching parasite numbers similar to those detected at PBS and Addavax-injected mice groups (Fig. 5 D). Furthermore, histological analysis of parasite’s major target organs, spleen and liver, revealed that immunization prevented tissue injury caused by the parasite infection (Fig. 5 E). Infected groups showed great haemophagocytosis and multinucleated giant cells (MGCs) formation (Supplementary Fig. 3), whereas vaccinated mice showed less architectural damage and hyperplastic changes between regions of the white pulp (Fig. 5 E). Furthermore, MGCs were absent in vaccinated mice due to the low parasitic load, since forming of MGCs requires chronic inflammatory environment driven by persistent Leishmania infection. Thus, vaccination alone, which does not establish such conditions, was insufficient to induce MGCs in the spleen of mice. Overall, immunization with ExoAddaVac resulted in the most pronounced protective effect. Immune responses following L. infantum challenge Post-challenge recall responses were assessed using splenocytes collected at 4 and 12 wpc. According to results, ExoVac-vaccinated mice exhibited two-fold higher proliferation in response to EVs as compared with both the control groups, as well as the ExoAddaVac mouse group at acute phase of disease (Fig. 6 A). At the same time point, ExoVac-immunized mice exhibited also increased SLA-specific proliferative responses which were not, however, statistically significant (Fig. 6 B). Impressively, the ExoAddaVac mouse group exhibited similar levels of proliferation as both control groups in response to both stimuli at 4 wpc (Fig. 6 A). However, this mouse group presented restoration of cellular responses to EVs as well as to SLA at chronic phase of disease (Fig. 6 A, B). Estimation of cytokines production at 12 wpc revealed that ExoAddaVac splenocytes produced the highest levels of IL-2 and IFN-γ in response to EV stimulation in comparison to the other groups tested (Fig. 6 C, D). Further, TNFα and IL-10 levels were reduced compared with non-immunized controls (Fig. 6 E, F), while IL-4 production was increased but remained lower than IFN-γ levels (Fig. 6 G). To deepen the analysis, the quantification of EVs- and parasite-specific IFN-γ- and IL-10-producing CD4 + and CD8 + T cells was conducted, since the counterbalance of these two cytokines is crucial for the establishment and pathogenesis of VL. Flow cytometric analysis revealed that ExoAddaVac-immunized mice exhibited significantly higher frequencies of IFN-γ–producing CD4⁺ T cells following EV stimulation, as well as increased IFN-γ–producing CD4⁺ and CD8⁺ T cells in response to SLA (Fig. 6 H, K). IL-10–producing CD4⁺ T cell frequencies were comparable across all groups (Fig. 6 I). Elevated IFN-γ/IL-10 ratios in both CD4⁺ and CD8⁺ T cells further supported a dominant protective Th1 response in ExoAddaVac-immunized mice (Fig. 6 J, M). Humoral responses remained elevated post-challenge in ExoAddaVac-immunized mice as compared with ExoVac-immunized mice and control mice groups (Supplementary Fig. 4A). Although IgG1 responses predominated, IgG2a levels were higher in ExoAddaVac mice compared with other groups, consistent with sustained Th1-biased immunity (Supplementary Fig. 4B). (C-G) Levels of IL-2 (C), IFN-γ (D), TNFα (E), IL-10 (F), and IL-4 (G) in culture supernatants after 72 h stimulation. (H and I) Intracellular cytokine staining identifying IFN-γ-producing (H) and IL-4-producing (I) CD4⁺ T cells following EV or SLA stimulation. (J) Ratio of IFN-γ- to IL-10-producing CD4⁺ T cells. (K, L) Intracellular staining of IFN-γ- and IL-4-producing CD8⁺ T cells. (M) Ratio of IFN-γ- to IL-10-producing CD8⁺ T cells. Data representative one independent experiment with n = 4–6 mice per group for lymphoproliferation assay and cytokine production analysis and n = 4–5 mice per group for intracellular cytokine staining. Bars indicate mean ± SEM, with individual mice shown as symbols. Statistical significance was determined using two-way ANOVA with Tukey’s multiple-comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001 Discussion In this work, we show that extracellular vesicles (EVs) released by stationary-phase Leishmania infantum promastigotes can function as a vaccine platform when formulated with the oil-in-water emulsion adjuvant Addavax. In this formulation, parasite-derived EVs elicited a Th1-skewed cellular immune response and were associated with significant protection in a murine model of experimental visceral leishmaniasis (VL). To our knowledge, this study provides the first in vivo evidence that EVs selectively isolated from stationary-phase L. infantum promastigotes can be leveraged as a cell-free vaccine formulation. Efforts to develop vaccines against leishmaniasis have been historically constrained by the parasite’s complex life cycle, antigenic heterogeneity, and capacity to subvert host immune responses. An additional challenge has been the limited availability of adjuvants capable of consistently inducing Th1-biased immunity, which is essential for intracellular parasite clearance 42 . In this context, we hypothesized that parasite-derived EVs could represent an alternative vaccination strategy that may help address some of these challenges. EVs inherently resemble biological liposomes and are well suited for antigen delivery, while retaining native protein conformation, post-translational modifications, and structural integrity—features that are often lost in recombinant or synthetic formulations. Previous studies have shown that liposomal encapsulation of Leishmania antigens enhances immunogenicity relative to soluble antigens 43 , 44 . EVs extend this principle by providing a naturally evolved delivery system that may offer advantages in antigen presentation and manufacturing reproducibility 45 , 46 . Although Leishmania -derived EVs have been extensively associated with parasite virulence, immune evasion, and disease progression 47 – 49 , growing evidence indicates that their immunological effects are highly context dependent. EV composition varies according to parasite species, developmental stage, and environmental conditions, resulting in distinct immunomodulatory properties 50 , 51 . Consistent with this notion, EVs isolated in our study exhibited canonical physicochemical features and a protein repertoire enriched in well-characterized immunogenic and virulence-associated molecules, including GP63, KMP-11, elongation factors, histones, heat shock proteins, and enolase 52 , 53 . Importantly, many of the identified proteins have independently demonstrated protective efficacy as vaccine antigens in experimental models 54 , 55 , supporting the premise that these EVs contain an antigenic repertoire relevant for host immune recognition. The induction of adaptive immunity is critically dependent on early innate immune activation, particularly through dendritic cells (DCs), which orchestrate T-cell priming and differentiation. Here, EV exposure promoted functional maturation of bone marrow–derived DCs, as evidenced by upregulation of MHC class I and II molecules, increased CD40 expression, and production of pro-inflammatory cytokines. Although IL-10 was detectable, its levels remained substantially lower than IL-12, favoring differentiation toward a DC1 phenotype capable of driving Th1 responses. These findings differ from prior reports describing suppressive effects of Leishmania EVs on monocyte-derived DCs 49 , 50 , likely reflecting differences in EV origin, molecular composition, and experimental context. In vivo , immunization with EVs alone resulted in limited adaptive immune activation, consistent with earlier observations that parasite-derived EVs may lack intrinsic immunogenicity and can even facilitate infection establishment 49 . Nonetheless, EV administration increased circulating neutrophil frequencies, a phenomenon previously linked to the phosphatidylserine-rich surfaces and inflammatory properties of Leishmania EVs 48 , 51 . While such responses may promote immune cell recruitment, they may also contribute to immune environments that are less effective at restricting parasite persistence 56 – 59 . To overcome the limited immunogenicity of EVs alone, we employed Addavax, a squalene-based nanoemulsion analogous to MF59, which is known to enhance antigen uptake, recruit antigen-presenting cells, and amplify inflammatory signaling at the injection site 33 , 60 . Addavax-adjuvanted EV vaccination resulted in robust expansion of antigen-specific lymphocytes, with pronounced increases in both CD4⁺ and CD8⁺ T-cell populations. The expansion of CD8⁺ T cells is of interest given their established role in parasite killing, Th1 polarization, and long-term protective memory in VL 61–64 . Cytokine profiling further underscored the protective immune environment induced by adjuvanted EVs. While EVs alone elicited weak IFN-γ responses and a low IFN-γ/IL-10 ratio, EVs formulated with Addavax induced robust IFN-γ production by both CD4⁺ and CD8⁺ T cells, resulting in a cytokine profile consistent with Th1 polarization. Such responses are essential for macrophage activation and intracellular parasite control 65 , 66 . Importantly, although IL-10 was still detectable, it did not override the dominant IFN-γ response, suggesting effective immune regulation rather than pathological suppression. Consistent with these immunological findings, Addavax-adjuvanted EV vaccination conferred significant protection following parasite challenge, particularly in the spleen, whereas hepatic parasite burdens were less affected. This pattern likely reflects the self-resolving nature of liver infection during VL 67 . Histopathological analysis revealed preserved splenic architecture and absence of multinucleated giant cells, which are commonly associated with chronic inflammation and immune dysregulation 68 – 70 . These observations suggest that vaccination promoted parasite control without evidence of overt immunopathology in this experimental setting. Although protective immunity against Leishmania is predominantly T-cell mediated, chronic infection is frequently associated with T-cell exhaustion and anergy 71 . In this regard, sustained antigen-specific T-cell proliferation following challenge represents a key indicator of vaccine efficacy. Ex vivo recall assays demonstrated that Addavax-adjuvanted EV immunization maintained robust T-cell responsiveness during chronic infection, accompanied by preferential IFN-γ production over IL-4 and IL-10. While excessive IFN-γ can contribute to tissue damage, balanced regulation by IL-10 may limit immunopathology while preserving antimicrobial function 72 – 74 . The absence of splenomegaly in vaccinated animals may reflect a balanced cytokine environment associated with effective parasite control. Analysis of humoral immunity revealed that Addavax-adjuvanted EV vaccination predominantly induced parasite-specific IgG1 responses. Although elevated IgG1 titers have traditionally been associated with active VL, emerging evidence suggests that IgG1 responses to EV-associated antigens may contribute to protective immunity through mechanisms distinct from those observed during natural infection 75 . In our study, IgG1 induction correlated with enhanced CD8⁺ T-cell responses, consistent with reports linking early IL-4 production and IgG1-biased environments to effective CD8⁺ T-cell memory formation 76 . In conclusion, while parasite-derived EVs have classically been viewed as mediators of immune evasion and disease progression, our findings demonstrate that appropriate adjuvantation can substantially alter their immunological properties toward protective outcomes. Addavax effectively converted L. infantum EVs into a potent Th1-biased vaccine platform capable of conferring significant protection against visceral infection. Together, these results provide a foundation for further development of EV-based vaccination strategies against VL. However, further optimization of the vaccine platform is warranted, particularly with respect to the functional contribution of EV-associated nucleic acids. EV-encapsulated mRNAs and microRNAs have been shown to modulate host gene expression and immune responses, and elucidating their role in vaccine-induced immunity will be critical for advancing this approach toward clinical application 77 , 78 . Methods Animals and immunization Female BALB/c mice (6–8 weeks old) were used in all experiments. Animals were housed at the Hellenic Pasteur Institute (HPI) under specific pathogen-free (SPF) conditions with controlled temperature (22 ± 2°C), humidity (40–70%), and a 12 h light/dark cycle. All experimental procedures complied with national legislation (Presidential Decree 56/2013) and Directive 2010/63/EU of the European Parliament, following the principles of the 3 + 1R framework. Study protocols were approved by the Official Veterinary Authorities of the Attiki Prefecture (license no. 6381/11-12-2017). Age-matched mice (n = 4–7 per group) were immunized intramuscularly with extracellular vesicles (EVs; 15 µg per mouse per immunization) either alone (ExoVac) or formulated 1:1 with Addavax (ExoAddaVac; InvivoGen), in a total volume of 100 µL (50 µL per hind limb). Immunizations were administered twice at two-week intervals. Control animals received sterile PBS or Addavax alone. Three weeks after the final boost, mice were infected by intravenous injection of 1 × 10⁷ stationary-phase Leishmania infantum promastigotes. Animals were monitored throughout the study, and body weight was recorded every two days. At predefined time points, mice were euthanized by CO₂ inhalation, and blood, spleen, and liver samples were collected for downstream analyses. Leishmania infantum promastigotes culture Leishmania infantum promastigotes (MHOM/GR/2001/GH8 strain) were cultured at 26°C in complete culture medium consisted of RPMI-1640 medium, penicillin (100 U/mL), streptomycin (10 µg/mL), L-glutamine (2 mM), HEPES buffer (10 mM) and 10% (v/v) fetal bovine serum (FBS) for no more than ten in vitro passages. To preserve parasite virulence, promastigotes were periodically passaged in vivo through BALB/c mice by intravenous administration of 1 × 10⁷ stationary-phase parasites. Differentiation of bone marrow–derived dendritic cells Bone marrow–derived dendritic cells (BMDCs) were generated following an established protocol with minor modifications 79 . Bone marrow was harvested from femurs and tibias of BALB/c mice and flushed with RPMI medium. Cells were washed and seeded at 4 × 10⁶ cells in 10 mL complete RPMI supplemented with recombinant murine GM-CSF (20 ng/mL; PeproTech). Cultures were maintained at 37°C in a humidified 5% CO₂ incubator. On day 3, fresh GM-CSF-containing medium was added. At day 6, half of the supernatant was replaced with fresh medium following centrifugation and resuspension of the cells. Semi-adherent and non-adherent cells were collected on day 8. BMDC purity consistently exceeded 75%, as confirmed by CD11c expression. Preparation of soluble Leishmania antigen Soluble Leishmania antigen (SLA) was prepared from stationary-phase promastigote cultures following a previously established protocol 80 . Protein concentrations were quantified using the MicroBCA Protein Assay Kit (Thermo Fisher Scientific). Isolation of L. infantum –derived extracellular vesicles Extracellular vesicles (EVs) were isolated from stationary-phase L. infantum promastigotes cultured for 24 h at 26°C in serum-free Schneider’s Drosophila medium (Biowest) supplemented with L-glutamine (2 mM), HEPES (10 mM), penicillin (100 U/mL), and streptomycin (10 µg/mL). Following incubation, promastigotes were removed by two sequential centrifugation steps at 3,000 × g for 10 min. Supernatants were subsequently filtered twice through 0.45 µm syringe filters to eliminate residual debris. EVs were concentrated using Centricon Plus-70 centrifugal devices with a 100 kDa molecular weight cut-off (Millipore) and further purified by OptiPrep™ density gradient ultracentrifugation at 100,000 × g avg for 16 h at 4°C using an SW40 Ti rotor. Fractions enriched in EVs (fractions 6–7) were pooled, diluted in PBS, and subjected to a second ultracentrifugation step at 100,000 × g avg for 3 h. Final EV pellets were resuspended in 100–200 µL PBS and stored at − 80°C. All procedures were performed using endotoxin-free reagents and consumables. Protein content was measured by MicroBCA Protein Assay Kit. EVs characterization EVs were characterized using dynamic light scattering (DLS), Western blotting, transmission electron microscopy (TEM), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). DLS analysis was performed to estimate EVs size distribution using a Zetasizer NanoS instrument (Malvern Instruments, UK), equipped with a 633 nm He-Ne laser (4.0 mW). Measurements were conducted at 4°C in quartz cuvettes and analyzed using DTS v4.1 software. Western blot analysis was carried out in 12% SDS-PAGE resolving gel and the proteins were next transferred to nitrocellulose membranes using standard protocols. EV protein samples were then incubated at 4°C overnight with monoclonal antibodies against established EV markers: CD9 and CD63 (Santa Cruz Biotechnology, Inc., TX, USA) and CD81 (Affinity Biosciences). To avoid presence of soluble protein or intracellular contamination, albumin and cytochrome c (Santa Cruz Biotechnology, Inc., TX, USA) were used as negative markers respectively. Blot visualization was performed upon incubation with secondary antibody, namely peroxidase-conjugated anti-mouse IgG (Biorad), followed by enhanced chemiluminescence addition (ECL, Pierce) while the image acquisition was achieved by exposure to X-ray film. Transmission electron microscopy was employed to visualize the morphology of isolated EVs. EVs were isolated, fixed, stained and embedded according to previously described protocol 81 . Specifically, EVs were fixed using 2% paraformaldehyde prepared in 0.1M phosphate buffer (pH 7.4) and then adsorbed on formvar/carbon-coated copper grids (200 mesh; Polysciences Europe GmbH). Contrast was achieved with uranyl oxalate, pH = 7.00, before embedding in a mixture of 4% uranyl acetate and 2% methyl cellulose. Finally, EVs were observed using a Jeol JEM2100Plus Transmission Electron Microscope (Jeol, Japan) operated at 120kV and photographed with the Gatan OneView CMOS camera (Gatan Ametek, USA), which is housed at the Centre of Innovative Electron Microscopy Applications and Services of the National and Kapodistrian University of Athens (CEMA-NKUA). Finally, proteomic profiling of EVs cargo was conducted using LC-MS/MS. These data informed the selection of EVs used for immunization studies in animal models. Functional assessment of BMDCs To assess the immunomodulatory effects of L. infantum EVs, BMDCs were incubated with endotoxin-free EVs (5 µg/mL) or SLA (10 µg/mL) for 24 h at a density of 1 × 10⁶ cells/mL. Cells cultured in medium alone or stimulated with lipopolysaccharide (LPS; 1 µg/mL) served as negative and positive controls, respectively. Following incubation, BMDCs were washed in ice cold FACS buffer (PBS supplemented with 3% FBS), blocked with anti-CD16/32 monoclonal antibody (Biolegend) for 10 min at 4°C. Then, cells were stained against CD11c (clone HL3; 1:100 dilution; BD Pharmingen), CD40 (clone 3/23; 1:100 dilution; BD Pharmingen), MHC class I (clone SF1-1.1; 1:100 dilution; BD Pharmingen) or MHC class II (clone 2G9; 1:200 dilution; BD Pharmingen) for 30 min at 4°C, fixed in 2% paraformaldehyde and analyzed by flow cytometry. For intracellular cytokine detection, brefeldin A (10 µg/mL; Cayman, Michigan, USA) was added during the final 4 h of culture. Cells were then surface stained with anti-CD11c antibody, fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Life Sciences) and then stained for IL-12p40/p70 (1:100 dilution; BD Pharmingen), IL-10 (clone JES5-16E3; 1:100 dilution; Biolegend) or TNFα (clone MP6-XT22; 1:100 dilution; Biolegend) for 30 min at 4°C. Data acquisition was performed using a BD FACSCalibur cytometer (Becton-Dickinson, San Jose, CA, USA) and analyzed with FlowJo software version 10.0 (BD Life Sciences). Splenocyte proliferation assay Splenocyte proliferation was assessed using a [³H]-thymidine incorporation assay two weeks after the end of immunization as well as at 4 and 12 weeks post infection. Specifically, aseptically collected spleens from immunized and non-immunized mice (n = 5/group) were minced with a sterile syringe plunger from a 5-mL syringe. The resulting cell suspensions were filtered through a 70 µm cell strainer. Suspensions were centrifuged at 1,300 rpm for 10 min and the resulting cell pellet was resuspended in ACK lysis buffer for 5 min in room temperature in order for RBCs to be lysed. Lysis was stopped by adding complete RPMI and centrifuged as described above. Isolated cells were seeded at 2 × 10⁵ cells/well, and stimulated with SLA (12.5 µg/mL) or EVs (2 µg/mL). Concanavalin A (ConA; 3 µg/mL; Sigma) served as a positive control. After 72 h, [³H]-thymidine (1 µCi; PerkinElmer) was added, and incorporation was measured following an additional 18 h incubation. The radioactivity expressed as mean counts per minute (cpm) using a scintillation counter (Wallac). Results were expressed as Δcpm relative to unstimulated controls. Intracellular cytokine staining Splenic cells were prepared at a density of 1x10 6 cells/well in predetermined time points and stimulated with SLA (25 µg/mL) or EVs (2 µg/mL) for 18 h at 37°C and brefeldin A (10 µg/mL) was added during the final 4 h of incubation. Subsequently, cells were surface-stained using anti-CD3 (clone 17A2), anti-CD4 (clone RM4-5), and anti-CD8 (clone 53 − 6.7) mAbs at a dilution of 1:100, followed by intracellular staining for IFN-γ (clone XMG1.2), IL-4 (clone 11B11), and IL-10 (clone JES5-16E3). All antibodies used were obtained from Biolegend and analysis was conducted on a FACSCalibur system. A representative gating strategy is depicted in Supplementary Fig. 5. Cytokine-producing T-cell frequencies were calculated after subtraction of background values from unstimulated samples. Negative values resulting from the above calculation were set to zero. Cytokine analysis Cytokine concentrations in stimulated spleen cells supernatants, were also determined. For this purpose, spleen cells were treated as described above to induce cytokine production. Cell-free supernatants were collected after 72 h of antigen stimulation by centrifugation, aliquoted, and stored at − 80°C until assayed for cytokine production using Milliplex Mouse Cytokine Magnetic Bead Panel Kit (Millipore, Billerica, MA, USA). Analysis was performed on a Luminex 200 (Thermo Fischer) using xPONENT software (Luminex Corporation) according to the manufacturer’s instructions. ELISA Antigen-specific IgG, IgG1, and IgG2a antibodies were measured in mouse sera by ELISA. Briefly, sera were isolated after blood centrifugation at 4,000 rpm for 5 min. Then, 96-well high-binding plates were coated with SLA (5 µg/mL), blocked with 2% BSA in PBS for 2 h at 37°C, and incubated with diluted serum samples (1:100 dilution) in PBS in duplicates at 37°C for 90 min. Bound antibodies were detected using HRP-IgG (dilution 1:5,000; Biorad) or biotin-conjugated anti-mouse IgG1 (1 µg/mL) or IgG2a (250 ng/mL) (both obtained from Biorad) for 1 h at 37°C followed by TMB substrate development. For IgG1 and IgG2a, plates were further incubated with streptavidin-HRP for 60 min at 37°C. Optical density was measured at 450 nm using an ELISA microplate spectrophotometer (MRX). Parasite burden determination Spleen and hepatic parasitic loads were determined by limiting dilution assay. Briefly, organ homogenates in Schneider’s Insect Medium (Biowest) − 20% FBS at a final concentration of 1 mg/mL. Two hundred microliters of suspension were placed into the first well, and 2-fold serial dilutions were distributed in the 96-well plates. Parasite numbers were calculated based on the highest dilution yielding viable promastigotes and expressed as parasites per gram of tissue after 7 days of incubation at 26°C. Histopathology Spleens and livers were collected at predetermined time points. Then, they were fixed in 10% neutral buffered formalin, paraffin-embedded, sectioned at 4 µm and stained with hematoxylin and eosin for histological examination. Statistical analysis Data are presented as means ± SEM. Statistical comparisons were performed using one- or two-way ANOVA followed by Tukey–Kramer post hoc test to perform multiple comparisons using GraphPad Prism version 6.0 software (GraphPad Software). Differences were considered statistically significant was defined at p < 0.05. In the graphs only the significant differences are noted, where * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001. Data availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 82 partner repository with the dataset identifier PXD074544 and are accessible using the login Username: [email protected] and Password: gSKzgBe53ZHn. All other data supporting the findings of this study are included within the article and its Supplementary Information. Additional relevant data are available from the corresponding author upon reasonable request. Declarations Acknowledgements This work was supported by the EPAnEK Operational Programme (NSFR 2014–2020) co-financed by Greece and the European Union under Grant number (T1EDK-03902). The funder had no role in study design, data collection, data analysis, interpretation writing or submission of the manuscript. Regarding the TEM work, VGG, SH and IT were supported by the Hellenic Foundation for Research and Innovation (HFRI) under the “1st Call for HFRI Research Projects to support Faculty members and Researchers and the procurement of high-cost research equipment” grant no. 2906. Author contributions A.E. performed the EVs isolation and purification. A.E. and D.K.T performed the EVs characterization. I.T. and S.H. performed the TEM analysis of EVs fractions while V.G.G. acquired funding for high-cost research equipment regarding TEM microscopy. M.S. performed the LC-MS/MS analysis. A.E and M.A performed in vivo experiments, and monitoring of the mice. A.E performed splenocyte proliferation assay, intracellular cytokine analysis and parasite burden determination. M.A. performed differentiation and functional assessment of BMDCs, ELISA and cytokine analysis. F.B performed the histological analyses. E.K., A.E. and M.A. designed the studies. M.A. and A.E. analyzed the data and performed the statistical analysis. E.K. supervised experiments and acquired funding. M.A, and A.E. wrote the first draft of the manuscript under the guidance and supervision of E.K., and all authors edited the final version. All authors approved the final draft and take full responsibility for its content. Competing interests The authors declare no competing interests. References World Health, O. 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Journal of immunology 168, 3992–4000 (2002). https://doi.org:10.4049/jimmunol.168.8.3992 Tsagozis, P., Karagouni, E. & Dotsika, E. CD8(+) T cells with parasite-specific cytotoxic activity and a Tc1 profile of cytokine and chemokine secretion develop in experimental visceral leishmaniasis. Parasite immunology 25, 569–579 (2003). https://doi.org:10.1111/j.0141-9838.2004.00672.x Uzonna, J. E., Joyce, K. L. & Scott, P. Low dose Leishmania major promotes a transient T helper cell type 2 response that is down-regulated by interferon gamma-producing CD8 + T cells. The Journal of experimental medicine 199, 1559–1566 (2004). https://doi.org:10.1084/jem.20040172 Engwerda, C. R., Ato, M. & Kaye, P. M. Macrophages, pathology and parasite persistence in experimental visceral leishmaniasis. Trends in parasitology 20, 524–530 (2004). https://doi.org:10.1016/j.pt.2004.08.009 Basu, R., Roy, S. & Walden, P. HLA class I-restricted T cell epitopes of the kinetoplastid membrane protein-11 presented by Leishmania donovani-infected human macrophages. The Journal of infectious diseases 195, 1373–1380 (2007). https://doi.org:10.1086/513439 Murray, H. W. Tissue granuloma structure-function in experimental visceral leishmaniasis. International journal of experimental pathology 82, 249–267 (2001). https://doi.org:10.1046/j.1365-2613.2001.00199.x Carrion, J. et al. Immunohistological features of visceral leishmaniasis in BALB/c mice. Parasite immunology 28, 173–183 (2006). https://doi.org:10.1111/j.1365-3024.2006.00817.x Morimoto, A. et al. Hemophagocytosis induced by Leishmania donovani infection is beneficial to parasite survival within macrophages. PLoS neglected tropical diseases 13, e0007816 (2019). https://doi.org:10.1371/journal.pntd.0007816 da Silva, A. V. A. et al. 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International journal of biological sciences 7, 1093–1100 (2011). https://doi.org:10.7150/ijbs.7.1093 Dey, R. et al. Live attenuated Leishmania donovani p27 gene knockout parasites are nonpathogenic and elicit long-term protective immunity in BALB/c mice. Journal of immunology 190, 2138–2149 (2013). https://doi.org:10.4049/jimmunol.1202801 da Cruz, A. B. et al. Performance of Extracellular Vesicles From Leishmania (Leishmania) infantum for Serological Diagnosis of Human and Canine Visceral Leishmaniasis. Journal of parasitology research 2025, 8355886 (2025). https://doi.org:10.1155/japr/8355886 Stager, S. et al. Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8 + T-cell responses. Nature medicine 9, 1287–1292 (2003). https://doi.org:10.1038/nm933 Ratajczak, J., Wysoczynski, M., Hayek, F., Janowska-Wieczorek, A. & Ratajczak, M. Z. Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication. Leukemia 20, 1487–1495 (2006). https://doi.org:10.1038/sj.leu.2404296 Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature cell biology 9, 654–659 (2007). https://doi.org:10.1038/ncb1596 Lutz, M. B. et al. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. Journal of immunological methods 223, 77–92 (1999). https://doi.org:10.1016/s0022-1759(98)00204-x Agallou, M. et al. Identification of BALB/c Immune Markers Correlated with a Partial Protection to Leishmania infantum after Vaccination with a Rationally Designed Multi-epitope Cysteine Protease A Peptide-Based Nanovaccine. PLoS neglected tropical diseases 11, e0005311 (2017). https://doi.org:10.1371/journal.pntd.0005311 Thery, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current protocols in cell biology Chap. 3 , Unit 3 22 (2006). https://doi.org:10.1002/0471143030.cb0322s30 Perez-Riverol, Y. et al. The PRIDE database at 20 years: 2025 update. Nucleic acids research 53, D543-D553 (2025). https://doi.org:10.1093/nar/gkae1011 Additional Declarations No competing interests reported. Supplementary Files Supplementalinformation.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 16 Apr, 2026 Reviews received at journal 16 Apr, 2026 Reviews received at journal 04 Apr, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers invited by journal 13 Mar, 2026 Editor assigned by journal 13 Mar, 2026 Submission checks completed at journal 05 Mar, 2026 First submitted to journal 04 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9033802","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":606468928,"identity":"56ce380b-2765-4686-ad79-a99c0d3ae13c","order_by":0,"name":"Antonia Efstathiou","email":"","orcid":"","institution":"Pasteur Hellenic Institute","correspondingAuthor":false,"prefix":"","firstName":"Antonia","middleName":"","lastName":"Efstathiou","suffix":""},{"id":606468929,"identity":"3d495cee-b8b0-4449-bbaa-d0c0825d969d","order_by":1,"name":"Maria Agallou","email":"","orcid":"","institution":"Pasteur Hellenic Institute","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Agallou","suffix":""},{"id":606468930,"identity":"e9ff9898-bfe4-43d2-b098-bbf683f04cdb","order_by":2,"name":"Dimitra K. Toubanaki","email":"","orcid":"","institution":"Pasteur Hellenic Institute","correspondingAuthor":false,"prefix":"","firstName":"Dimitra","middleName":"K.","lastName":"Toubanaki","suffix":""},{"id":606468931,"identity":"4b6a1979-52af-4556-a68b-d89010493ad2","order_by":3,"name":"Fotis Badounas","email":"","orcid":"","institution":"Pasteur Hellenic Institute","correspondingAuthor":false,"prefix":"","firstName":"Fotis","middleName":"","lastName":"Badounas","suffix":""},{"id":606468932,"identity":"de32bf0a-5764-49b4-9e6e-c8e209540f78","order_by":4,"name":"Martina Samiotaki","email":"","orcid":"","institution":"Alexander Fleming Biomedical Sciences Research Center","correspondingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Samiotaki","suffix":""},{"id":606468933,"identity":"f0e2403f-b063-4e67-9671-70f720b4a763","order_by":5,"name":"Ioanna Tremi","email":"","orcid":"","institution":"National and Kapodistrian University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Ioanna","middleName":"","lastName":"Tremi","suffix":""},{"id":606468934,"identity":"1c45b100-ef71-4365-a968-e9ecffcb9e2e","order_by":6,"name":"Sophia Havaki","email":"","orcid":"","institution":"National and Kapodistrian University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Sophia","middleName":"","lastName":"Havaki","suffix":""},{"id":606468935,"identity":"a0951717-5754-4971-9baf-810bad1319a2","order_by":7,"name":"Vassilis G. Gorgoulis","email":"","orcid":"","institution":"National and Kapodistrian University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Vassilis","middleName":"G.","lastName":"Gorgoulis","suffix":""},{"id":606468937,"identity":"376bf20c-4771-4dc3-9b4a-082a2bad031e","order_by":8,"name":"Evdokia Karagouni","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIie3Lv2rCQBzA8d9xcC53ukZK9BV+4YYiCH2VZLEP0KWbAeGmxD1vEShU3IRbj+YButQlSx0shRLKCS2BCi4X3YTed/j9GT4APt81xv8OSjcXExb/TryEAMfzyIDret98T5NVj3+9NdaO8HVD3x8dZJgrWeTLWbJeiFWUK5T4ErOJcRCsQILIdFJq8RyIFJPSAItSB7mrep/k0BJeD63FeSdBkUnKm5awG84wRgN06yKBMQ80TGey1OxWhkpGhSGKuMggu38iOzsNy0rX250djfuG6g8XaSOqXez4Bl0CwJ4QoPtu4vP5fP+oH7BkT2tTZpavAAAAAElFTkSuQmCC","orcid":"","institution":"Pasteur Hellenic Institute","correspondingAuthor":true,"prefix":"","firstName":"Evdokia","middleName":"","lastName":"Karagouni","suffix":""}],"badges":[],"createdAt":"2026-03-04 21:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9033802/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9033802/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104753971,"identity":"b59b5879-41ff-4336-b9cf-6d2c01ab9b25","added_by":"auto","created_at":"2026-03-16 21:12:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3505777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLeishmania infantum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e extracellular vesicles (EVs). \u003c/strong\u003e(A, B) Representative transmission electron microscopy (TEM) image of EVs fraction 6 (A) and 7 (B) isolated from \u003cem\u003eLeishmania infantum\u003c/em\u003e promastigotes cultures, showing typical round vesicular structures (scale bar = 200 nm). (C) Representative figure for intensity-weighted size distribution of vesicles determined by dynamic light scattering (DLS). (D) Western blot detection of EV markers CD9, CD63, and CD81 in the pooled EVs fraction 6 and 7 used for mice immunizations.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9033802/v1/92be3b0c665555ce2793d021.png"},{"id":104782604,"identity":"df838cd5-f6ec-408f-a6fb-564e25ffcbec","added_by":"auto","created_at":"2026-03-17 07:57:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5109569,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEVs promote BMDCs maturation in vitro. \u003c/strong\u003e(A) Representative flow cytometry dot plots showing CD40, MHCI, and MHCII expression on BMDCs (gated on CD11c⁺ cells) from BALB/c mice treated for 24 h with EVs, SLA, or LPS, or cultured in medium alone. Numbers indicate the percentage of marker-positive BMDCs. (B-D) Mean fluorescent intensity (MFI) of CD40 (B), MHCI (C), and MHCII (D) on CD11c\u003csup\u003e+\u003c/sup\u003e BMDCs following the indicated treatments. (E-G) Frequencies of IL-12- (E), TNFα- (F), and IL-10- (G) producing CD11c⁺ dendritic cells. (H) Ratio of IL-12- to IL-10-producing CD11c⁺ DCs. Data were compiled from three independent experiments. Bars indicate mean values ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test. Only significant differences are shown. *p\u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9033802/v1/700699bc51c4107592a8ff5f.png"},{"id":104753974,"identity":"dae59d9c-a61e-4799-9790-430ae90c7ee2","added_by":"auto","created_at":"2026-03-16 21:12:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7985723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExoAddaVac induces antigen-specific Th1-biased responses. \u003c/strong\u003e(A) Mice were immunized intramuscularly twice, two weeks apart, with 15 µg EVs alone (ExoVac) or EVs formulated with AddaVax (ExoAddaVac). PBS- and AddaVax-injected mice served as controls. Blood, spleens, and livers were collected two weeks after the booster immunization for immunological and histological analyses. (B) Changes in circulating immune cell populations (NE, neutrophils; LY, lymphocytes; MO, monocytes; EO, eosinophils). (C) Representative H\u0026amp;E-stained spleen (4×) and liver (10×) sections from ExoVac- and ExoAddaVac-immunized mice. (D-F) Parasite-specific IgG (D), IgG1 (E), and IgG2a (F) antibody levels measured by ELISA. (G-I) Lymphoproliferative responses of splenocytes stimulated with EVs (G), SLA (H), or ConA (I), expressed as Δcpm. (J-N) Cytokine concentrations of IL-2 (J), IFN-γ (K), TNFα (L), IL-4 (M), and IL-10 (N) in splenocyte culture supernatants following 72 h stimulation with EVs or SLA. (O) IFN-γ to IL-10 and IFN-γ to IL-4 production levels ratio.Data are representative from one independent experiment with n=2-4 mice per group for hematological analysis, n=4-7 for antibody production detection, lymphoproliferation assay and cytokine production analysis. Each symbol represents an individual mouse; bars show mean ± SEM. Statistical analyses were performed using one- or two-way ANOVA with Tukey’s post-test, as appropriate. Only significant differences are shown. \u0026nbsp;**p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p\u0026lt;0.0001. Scale bars are 400 µm in low magnification and 200 µm in high magnification.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9033802/v1/455e436a690941cd45b591cf.png"},{"id":104753972,"identity":"1c82dafa-8566-4d80-83ee-9b73c9f3c5c3","added_by":"auto","created_at":"2026-03-16 21:12:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1799547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExoAddaVac drives differentiation of EV-specific CD4⁺ and CD8⁺ T cells. \u003c/strong\u003e(A–C) Following two intramuscular immunizations with ExoVac or ExoAddaVac, splenocytes were isolated two weeks after the booster dose and stimulated with EVs for 18 h. Frequencies of EV-specific CD3⁺ (A), CD3⁺CD4⁺ (B), and CD3⁺CD8⁺ (C) T cells are shown. (D–F) Intracellular cytokine staining identifying IFN-γ-producing CD4⁺ (D) and CD8⁺ (E) T cells, and IL-4-producing CD4⁺ T cells (F). Data represent one experiment with n = 4 mice per group. Each symbol represents an individual animal; bars indicate mean ± SEM. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple-comparison test. Only significant differences are shown.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9033802/v1/35b9e90f8f2935514ec5cabc.png"},{"id":104753977,"identity":"a39b2116-7952-47eb-8ebf-2e8523035620","added_by":"auto","created_at":"2026-03-16 21:12:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9706667,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExoAddaVac confers protection against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. infantum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e challenge. \u003c/strong\u003e(A-D) Immunized and control mice were challenged intravenously with L. infantum promastigotes two weeks after the booster dose. Protection was evaluated during the acute (4 weeks post-challenge; 4wpc) and chronic (12 weeks post-challenge; 12 wpc) phases by assessment of liver weight (A), spleen weight (B), and parasite burden in liver (C) and spleen (D). (E) Representative histopathological sections of spleen (20×) and liver (10×) at 4 and 12 weeks post-challenge. White arrows indicate multinucleated giant cells in the spleen, and black arrows indicate hepatic granuloma formation. Data represent one independent experiment with n=4-7 mice per group. Each symbol corresponds to one animal; bars represent mean ± SEM. Statistical comparisons were performed using two-way ANOVA with Tukey’s post-test. Only significant differences are shown. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.001 by two-way ANOVA and Tukey’s multiple comparison tests. Scale bars are 200 µm in both magnifications.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9033802/v1/c044b35f0c40db8ccccf1510.png"},{"id":104783492,"identity":"04cb915f-22e3-4ea3-abb3-63c78d9f2d9d","added_by":"auto","created_at":"2026-03-17 07:59:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1924187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExoAddaVac sustains Th1-biased responses during infection. \u003c/strong\u003e(A-B) Splenocytes isolated at 4 and 12 weeks post-challenge were stimulated with EVs (A) or SLA (B) for 96 h, and lymphoproliferative responses were quantified as Δcpm.\u003c/p\u003e\n\u003cp\u003e(C-G) Levels of IL-2 (C), IFN-γ (D), TNFα (E), IL-10 (F), and IL-4 (G) in culture supernatants after 72 h stimulation. (H and I) Intracellular cytokine staining identifying IFN-γ-producing (H) and IL-4-producing (I) CD4⁺ T cells following EV or SLA stimulation. (J) Ratio of IFN-γ- to IL-10-producing CD4⁺ T cells. (K, L) Intracellular staining of IFN-γ- and IL-4-producing CD8⁺ T cells. (M) Ratio of IFN-γ- to IL-10-producing CD8⁺ T cells. Data representative one independent experiment with n=4-6 mice per group for lymphoproliferation assay and cytokine production analysis and n=4-5 mice per group for intracellular cytokine staining. Bars indicate mean ± SEM, with individual mice shown as symbols. Statistical significance was determined using two-way ANOVA with Tukey’s multiple-comparison test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9033802/v1/f50a775c873ec0f6cc7b4502.png"},{"id":105751810,"identity":"21eab67b-b4f6-4d5d-a16c-2d3072e15e95","added_by":"auto","created_at":"2026-03-30 15:45:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27551525,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9033802/v1/52a942c9-dc15-45cc-b1b8-3d6ace45369a.pdf"},{"id":104753976,"identity":"d58928c1-db0c-44db-8a99-8b832328ffc4","added_by":"auto","created_at":"2026-03-16 21:12:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2414609,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9033802/v1/fd5e08f35081e3b650abc934.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Adjuvanted Leishmania infantum extracellular vesicles induce protective immunity in experimental visceral leishmaniasis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLeishmaniases, a group of vector-borne diseases remain a major public health concern in tropical and subtropical regions worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among the clinical manifestations, visceral leishmaniasis (VL) represent the most severe form and being almost uniformly fatal if left untreated\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Despite ongoing control efforts, VL continues to impose a substantial global burden, accounting for tens of thousands of deaths annually and contributing significantly to disability-adjusted life years lost, with profound socioeconomic consequences in endemic settings\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Current VL control strategies rely primarily on early diagnosis and chemotherapy to reduce the human reservoir, alongside vector control interventions such as indoor residual spraying\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although these measures have reduced disease incidence in some regions, elimination programs have repeatedly failed to achieve sustained success, highlighting intrinsic limitations of existing approaches. These shortcomings underscore the critical need for improved diagnostics, more effective therapeutics, and, most importantly, a prophylactic vaccine capable of inducing durable protective immunity and preventing disease resurgence\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe rationale for vaccination against VL is supported by clinical observations showing that individuals who recover from infection often develop long-lasting immunity against reinfection\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Consistent with this concept, a wide range of vaccine platforms\u0026mdash;including whole-parasite preparations, recombinant subunit vaccines, viral-vectored constructs, and DNA-based approaches\u0026mdash;have been evaluated in preclinical and early clinical studies\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Nevertheless, progress toward a licensed human vaccine has been limited. To date, only the recombinant subunit vaccine LeishF3 has completed a phase I clinical trial, demonstrating acceptable safety but no advancement to licensure\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These challenges largely reflect the complex biology of \u003cem\u003eLeishmania\u003c/em\u003e parasites, which exhibit a digenetic life cycle, extensive antigenic diversity, and sophisticated immune evasion strategies that complicate vaccine design.\u003c/p\u003e \u003cp\u003eRecent advances in nanotechnology and immunology have stimulated interest in extracellular vesicles (EVs), particularly exosomes, as alternative vaccine platforms. Exosomes are nanoscale vesicles generated within multivesicular bodies and released into the extracellular milieu, where they participate in intercellular communication and immune modulation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In oncology, exosomes derived from antigen-loaded dendritic cells have demonstrated the capacity to elicit strong immune responses and, in some cases, clinical benefit\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Similarly, EV-based approaches have shown immunogenic potential against diverse pathogens, including \u003cem\u003eToxoplasma gondii\u003c/em\u003e, \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e, and \u003cem\u003ePlasmodium\u003c/em\u003e species\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Compared with synthetic delivery systems, EVs possess several attributes favorable for vaccine development, including high biocompatibility, stability in biological environments, and efficient antigen delivery\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. EVs encapsulate complex molecular cargo composed of proteins, lipids, and nucleic acids that reflect the biological state of their parent cells, potentially providing a multifaceted immunological stimulus\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Their endogenous origin is associated with low toxicity, and established purification and scale-up methodologies support their translational potential. However, the immunological effects of EVs are highly context-dependent and influenced by cargo composition, cellular origin, and environmental cues.\u003c/p\u003e \u003cp\u003eIn the context of leishmaniasis, parasite-derived EVs have been extensively implicated in host\u0026ndash;pathogen interactions that promote immune modulation and disease progression. Studies in macrophages and murine models have shown that \u003cem\u003eLeishmania\u003c/em\u003e EVs contain virulence-associated molecules, including the metalloprotease GP63, capable of altering host signaling pathways and immune responses\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Conversely, accumulating evidence indicates that EVs derived from specific parasite species or developmental stages can also carry immunogenic components capable of antigen presentation and immune activation\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In some contexts, these vesicles exhibit adjuvant-like properties by promoting cytokine and chemokine production and facilitating innate immune cell recruitment\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Together, these findings suggest that parasite-derived EVs may exert either pathogenic or protective effects, depending on their molecular composition and the immunological environment in which they are encountered\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Notably, \u003cem\u003eLeishmania\u003c/em\u003e EVs represent a composite antigenic platform encompassing proteins, lipids, and nucleic acids. Their protein cargo includes several well-characterized immunogens, such as LACK, TSA, KMP11, and LmSTI1, able to induce protection in preclinical studies\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Supporting this concept, prior studies demonstrated that dendritic cell\u0026ndash;derived exosomes generated following exposure to cutaneous \u003cem\u003eLeishmania\u003c/em\u003e parasites enhanced Th1- and Th17-type immunity and reduced parasite burdens\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. These observations raise the possibility that appropriately formulated parasite-derived EVs could be harnessed to amplify protective immunity rather than promote disease.\u003c/p\u003e \u003cp\u003eEffective EV-based vaccination strategies, however, often require adjuvants capable of recruiting and activating dendritic cells and promoting antigen presentation \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Dendritic cells are considered a significant immune population in driving T-cell priming and long-term memory formation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Several adjuvants have been explored in VL vaccine development, including saponins and monophosphoryl lipid A\u003csup\u003e31\u003c/sup\u003e. Addavax, an oil-in-water emulsion that mimics the licensed MF59 adjuvant, has previously been shown by our group to enhance protective immune responses against \u003cem\u003eLeishmania infantum\u003c/em\u003e in experimental VL models\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. MF59-like adjuvants act primarily by inducing local innate immune activation and rapid recruitment of myeloid cells, thereby enhancing antigen uptake and adaptive immune priming\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on these considerations, we hypothesized that EVs derived from stationary-phase \u003cem\u003eL. infantum\u003c/em\u003e promastigotes, when combined with an appropriate adjuvant, could be redirected from immunomodulatory or pathogenic roles toward the induction of protective Th1-biased immunity. Subsequently, we evaluated the immunogenicity, safety, and efficacy in terms of protection of parasite-derived EVs formulated with Addavax in a murine model of VL. Our findings demonstrate that this cell-free, EV-based vaccine strategy elicits regulated yet robust Th1-type immune responses, characterized by activation of CD4⁺ and CD8⁺ T-cell specific for EVs, and confers significant protection against \u003cem\u003eL. infantum\u003c/em\u003e infection in target organs. Together, these findings support the potential of adjuvanted parasite-derived EVs as a promising platform for the development of effective vaccines against visceral leishmaniasis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of\u003c/b\u003e \u003cb\u003eLeishmania infantum\u003c/b\u003e \u003cb\u003epromastigote-derived EVs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExtracellular vesicles (EVs) were isolated from the culture supernatant of \u003cem\u003eL\u003c/em\u003e. \u003cem\u003einfantum\u003c/em\u003e promastigotes and purified by differential centrifugation followed by ultracentrifugation. The physical and biochemical properties of the isolated vesicles were evaluated in accordance with the MISEV2023 recommendations\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. EV characterization included assessment of particle size, size distribution, morphology, and enrichment of established exosomal protein markers. Transmission electron microscopy (TEM) revealed rounded, bilayered vesicles with diameters typically below 200 nm, consistent with the morphology of small EVs or exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Dynamic light scattering (DLS) analysis showed a unimodal size distribution with a mean diameter of 160\u0026thinsp;\u0026plusmn;\u0026thinsp;28.1 nm, with Polydispersity Index (PDI) 0.181 which indicates that the isolation method yielded a single dominant vesicle population with minimal aggregation or debris (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The zeta potential was \u0026minus;\u0026thinsp;14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 mV, indicating colloidal stability of the vesicle suspension. Western blot analysis confirmed enrichment of EV populations used for immunizations, through the detection of canonical EV markers CD9, CD63, and CD81 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), whereas the lack of detectable ALB and CytC signals indicated minimal soluble protein or intracellular contamination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Proteomic analysis of the EV preparations used for subsequent murine immunization identified a total of 1,441 peptides by LC-MS/MS. Among these, several peptides corresponded to previously reported \u003cem\u003eLeishmania\u003c/em\u003e antigenic proteins with documented immunogenic potential (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, the EV protein cargo included several well-recognized \u003cem\u003eLeishmania\u003c/em\u003e antigenic proteins, including GP63 (isoforms GP63-1, GP63-2, and GP63-4), α- and β-tubulin chains, eukaryotic initiation factors 3, 4A, and 5A, kinesin-like proteins, surface antigen\u0026ndash;like proteins, heat shock proteins HSP70, HSP83-1, HSP100, and the putative HSP40/DNAJ, as well as thiol-dependent reductase 1 (TDR1), among others\u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38 CR39 CR40\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These findings indicate that the EV preparations retained key antigenic components relevant for host immune recognition. Collectively, these results demonstrated that the isolated vesicles from \u003cem\u003eL. infantum\u003c/em\u003e promastigote cultures exhibited the expected size, morphology, and protein marker profile characteristic of extracellular vesicles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEnriched proteins with known antigenic potential of \u003cem\u003eLeishmania infantum\u003c/em\u003e promastigotes-derived EVs detected by Mass Spectrometry.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUniprot ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtein name\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4IDN4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRelated to elongation factor-2 kinase efk-1b isoform-like protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4I5B8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePutative surface protein amastin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4IBB3; A4IBB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKinetoplastid membrane protein-11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4HW10; A4HW09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElongation of fatty acids protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4HSL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurface antigen-like protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ6RYT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTryparedoxin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA0A381MG06;A4HU57;A4HY42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHistone H2B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4HVB0;E9AGG5;D1GJ51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePutative surface antigen protein 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4HW98; A4HSP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHistone H4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4HU18;A4HU19;A4I925\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElongation factor-1 gamma\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4HSC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4I562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKinesin-like protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4I8P2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThiol-dependent reductase 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE9AGQ3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePutative elongation factor Tu\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4I4I4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATP-dependent Clp protease subunit, heat shock protein 100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4I341\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePutative heat shock protein DNAJ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4HY48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKinesin-like protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE9AGP5;A4HX73;E9AGP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElongation factor 1-alpha\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4I253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeat shock protein 70-related protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ6LA77;A4HUF8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeishmanolysin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4HW62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnolase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4I5S5;A4I5T0;A4I5S4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePutative heat shock 70-related protein 1, mitochondrial\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4HUF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeishmanolysin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE9AGK7;A0A381MCS3;A4HVG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTubulin alpha chain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4HTR1;A0A381MS01; A4HTR0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTubulin beta chain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE9AGQ5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePutative heat shock protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE9AHM9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeat shock protein 83\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4ICW8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElongation factor 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE9AHH1;A4I412;E9AHH0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePutative heat-shock protein hsp70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEVs induce BMDCs maturation toward a DC1-like phenotype\u003c/h2\u003e \u003cp\u003eGiven that \u003cem\u003eLeishmania\u003c/em\u003e-derived EVs may serve as a source of parasite antigens, we next examined their capacity to induce maturation and functional activation of dendritic cells (DCs), which bridge innate and adaptive immune responses through activation of na\u0026iuml;ve T cells. For this purpose, bone marrow\u0026ndash;derived dendritic cells (BMDCs) were stimulated for 24 h with \u003cem\u003eLeishmania\u003c/em\u003e EVs, soluble \u003cem\u003eLeishmania\u003c/em\u003e antigen (SLA), or lipopolysaccharide (LPS) as a positive control. Flow cytometry revealed that the uptake of EVs by BMDCs significantly enhanced the number of CD40-expressing cells as well as CD40 expression levels per cell as evidenced by MFI values by about 6\u0026ndash;fold and 2\u0026ndash;fold, respectively, compared to untreated BMDCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Regarding MHCI and MHCII molecules, EVs despite the fact that they did not change the number of MHCI-/II-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), they significantly upregulated the expression of both co-stimulatory surface molecules according to increased MFI values (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). As expected, LPS stimulation, serving as a positive control stimulus, resulted in maturation of BMDCs as assessed by CD40, MHCI and MHCII molecules expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Importantly, all three surface molecules were found to be expressed at similar levels as in LPS-treated BMDCs indicating also EVs\u0026rsquo; probable immunogenicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). On the contrary, BMDCs exposure to SLA did not induce their maturation as evidenced by the low expression levels of all three surface molecules that were similar to those detected in untreated BMDCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Cytokine production by BMDCs was also assessed by flow cytometry. EV stimulation resulted in a significant increase in the proportion of IL-12\u0026ndash; and TNFα\u0026ndash;producing BMDCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F), along with a detectable increase in IL-10\u0026ndash;producing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Importantly, the IL-12/IL-10 ratio following EV stimulation was significantly higher than in untreated controls and comparable to that observed following LPS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), indicating preferential induction of pro-inflammatory cytokine responses. Taken together, these data indicate that EVs derived from \u003cem\u003eL. infantum\u003c/em\u003e promastigote cultures effectively promote BMDC activation toward a DC1-like phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaluation of EVs immunization-induced humoral and systemic immune responses\u003c/h3\u003e\n\u003cp\u003eThe immunogenicity of the proposed vaccine was then evaluated at the level of adaptive immunity. For this purpose, the mice were i.m. vaccinated with EVs alone (ExoVac) or adjuvanted with Addavax (ExoAddaVac) on day 0, followed by a homologous booster dose on day 14. Control groups included non-immunized (PBS) and Addavax-injected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Routine hematological analysis performed two weeks after boost injection that ExoAddaVac-immunized mice produced a significantly higher concentration of circulating lymphocytes (LY), whereas the numbers of neutrophils (NE) were reduced compared to control mouse groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). On the contrary, ExoVac-immunized mice exhibited a different profile with significantly elevated numbers of neutrophils (NE) followed by reduced numbers of circulating lymphocytes (LY) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Complementary to the above observations were the histological findings in spleen tissue where vaccinated mice did not show any sign of uncontrolled inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eSubsequently, vaccine-induced humoral immune responses were investigated as a surrogate of adaptive immunity activation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, at 2 weeks\u0026rsquo; post-boost immunization, adjuvanted (ExoAddaVac) and non-adjuvanted EVs (ExoVac) elicited parasite-specific IgG antibody responses, whereas as it was expected, the other control groups of vaccinated mice were negative for parasite specific antibodies. Importantly, the observed antibody responses were generally evoked to a significantly greater extent in Addavax-adjuvanted group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Analysis of IgG isotypes unveiled that ExoAddaVac promoted both the production of high IgG1 and IgG2a responses, with a bias towards IgG1 antibody isotype profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). On the contrary, ExoVac induced the production of IgG1 only and in much lower levels than those detected in ExoAddaVac group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003eOverall, immunization with Addavax-adjuvanted EVs effectively elicited robust humoral and cellular immune responses without inducing sustained inflammatory pathology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEVs immunization induced antigen-specific Th1-type cellular immune responses\u003c/h3\u003e\n\u003cp\u003eT cell activation was next evaluated by measuring the proliferative capacity of splenic lymphocytes upon \u003cem\u003eex vivo\u003c/em\u003e stimulation with EVs or SLA. Splenocytes from ExoAddaVac-immunized mice exhibited significantly increased proliferation in response to EV stimulation compared with PBS- and Addavax-treated controls, indicating differentiation of EV-specific lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Additionally, ExoAddaVac promoted also the differentiation of parasite-specific spleen cell population as evidenced by increased proliferation levels of spleen cells in the presence of SLA, even though they did not reach statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). On the other hand, spleen cells isolated from ExoVac-immunized mice exhibited increased lymphoproliferative action only in the presence of EVs although at significantly lower levels when compared with ExoAddaVac group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). Importantly, all groups of mice responded similarly with high proliferation levels when stimulated with ConA, a positive stimulant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), indicating that the differences observed were not due to differences in the viability of T cells taken from the different mouse groups. Enhanced proliferation in ExoAddaVac mice was accompanied by significantly increased IL-2 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eCytokine secretion profiles further supported Th1 polarization. Splenocytes obtained from ExoAddaVac-immunized mice exhibited a significantly and globally increased cytokine secretion compared not only to the control mice groups but also to EVs-immunized mice (ExoVac), which was collectively consistent with the changes in proliferation assays from the same group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-N). Importantly, this phenomenon was restricted to EVs stimulation, since in the presence of SLA spleen cells of either ExoVac or ExoAddaVac group did not produced cytokines further supporting the differentiation of EVs-specific effector T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-N). Specifically, splenocytes obtained from mice vaccinated with adjuvanted EVs showed significantly enhanced production of the pro-inflammatory cytokine IFN-γ in comparison to all other groups tested after stimulation with EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). On the contrary, EVs stimulation induced similar levels of TNFα production levels in all mice groups irrespective of vaccination regimen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Although IL-4 and IL-10 levels were also increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM, N), IFN-γ/IL-10 and IFN-γ/IL-4 ratios were significantly higher in ExoAddaVac-immunized mice, indicating a dominant Th1-biased response (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO).\u003c/p\u003e\n\u003ch3\u003eExoAddaVac immunization induced the differentiation of both antigen-specific CD4 and CD8 T cells\u003c/h3\u003e\n\u003cp\u003eTo assess in depth, the impact of the vaccine in T cell immune responses, we employed flow cytometry to characterize vaccine-induced T cell subsets following \u003cem\u003eex vivo\u003c/em\u003e EV stimulation. For this purpose, spleen cells were stimulated \u003cem\u003eex vivo\u003c/em\u003e with EVs and antigen-specific populations were determined. Specifically, ExoAddaVac as well as ExoVac immunization led to increased numbers of antigen-specific CD3\u003csup\u003e+\u003c/sup\u003e cell population compared to control mouse groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Analysis of T cell groups unveiled that ExoAddaVac induced a significant increase of activated CD4\u003csup\u003e+\u003c/sup\u003e T cells of 6.74% as well as CD8\u003csup\u003e+\u003c/sup\u003e T cells to 15.6% compared to na\u0026iuml;ve/adjuvant-control mice after \u003cem\u003eex vivo\u003c/em\u003e stimulation with EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). In contrast, ExoVac immunization resulted in modest, non-significant increases in CD4⁺ T cells only (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Cytokine profiling revealed that ExoAddaVac-immunized mice exhibited increased frequencies of IFN-γ-producing CD4\u003csup\u003e+\u003c/sup\u003e T and CD8\u003csup\u003e+\u003c/sup\u003e T cells, exceeding the proportion of IL-4-producing CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G), further supporting that EVs immunization induced the differentiation of Th1 mediated immune responses. ExoVac-immunized mice displayed only minimal IFN-γ-producing CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAssessment of biocompatibility and safety of EVs-based vaccine\u003c/h3\u003e\n\u003cp\u003eVaccine safety was evaluated through continuous monitoring of animal behavior and physiology. No morbidity, mortality, or injection-site reactions were observed. Vaccinated mice displayed normal mobility and feeding behavior, comparable to PBS-treated controls. Body weight increased steadily over time in all groups, with no significant differences between vaccinated and control mice (Supplementary Fig.\u0026nbsp;1). Overall, the immunization regimen was well tolerated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExoAddaVac immunization conferred protection against\u003c/b\u003e \u003cb\u003eL. infantum\u003c/b\u003e \u003cb\u003echallenge\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo verify whether vaccine-induced T cell responses translated into protective immunity against \u003cem\u003eL. infantum\u003c/em\u003e infection, mice were immunized with the protocol described above and then challenged with virulent promastigotes of the parasite. As a measure of the systemic effect of the infection, changes in the body weight were determined for 12 weeks following the parasite challenge. In contrast to both control mice groups that lost weight after parasite challenge, both groups of immunized mice slowly regained most of their initial body weight till the end of the study (Supplementary Fig.\u0026nbsp;2). Additionally, the weights of parasite\u0026rsquo;s target organs, spleen and liver, were measured and parasite load estimation was conducted in both organs at 4 and 12 weeks post challenge, representing the acute (4 wpc) and the chronic (12 wpc) phase of disease, as a parameter of disease establishment. According to our observations, non-immunized and adjuvant-injected mice possessed heavier livers than ExoVac- and ExoAddaVac-immunized mice at 4 wpc (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, during transition to chronic phase of the disease livers\u0026rsquo; weight from the ExoVac group reached in weight those isolated from both control groups, indicating that ExoVac group may failed to restrict parasite load in liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In agreement with the above observations, immunization with ExoAddaVac significantly reduced the hepatic parasite burden compared to control group during acute phase of disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). At 12 wpc, ExoVac mouse group contained similar numbers of parasite with non-immunized and Addavax-injected mice which were higher than those detected in ExoAddaVac group, reflecting ExoVac\u0026rsquo;s weakness to restrict parasite replication (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Regarding spleen tissues, profound splenomegaly could be easily observed followed by increased weight at chronic phase of disease not only at both control groups but also in the ExoVac-immunized group probably due to parasite establishment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In agreement with this observation, parasite load estimation during chronic phase of disease showed that ExoAddaVac controlled splenic parasite burden significantly better than the ExoVac following \u003cem\u003eL. infantum\u003c/em\u003e challenge. Specifically, when compared to the PBS control group, ExoAddaVac-immunized group reduced the parasite burden by 15-fold at acute phase and 5-fold at chronic phase, whereas ExoVac group failed to preserve low parasite burden at chronic phase of disease reaching parasite numbers similar to those detected at PBS and Addavax-injected mice groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, histological analysis of parasite\u0026rsquo;s major target organs, spleen and liver, revealed that immunization prevented tissue injury caused by the parasite infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Infected groups showed great haemophagocytosis and multinucleated giant cells (MGCs) formation (Supplementary Fig.\u0026nbsp;3), whereas vaccinated mice showed less architectural damage and hyperplastic changes between regions of the white pulp (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, MGCs were absent in vaccinated mice due to the low parasitic load, since forming of MGCs requires chronic inflammatory environment driven by persistent \u003cem\u003eLeishmania\u003c/em\u003e infection. Thus, vaccination alone, which does not establish such conditions, was insufficient to induce MGCs in the spleen of mice. Overall, immunization with ExoAddaVac resulted in the most pronounced protective effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImmune responses following\u003c/b\u003e \u003cb\u003eL. infantum\u003c/b\u003e \u003cb\u003echallenge\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePost-challenge recall responses were assessed using splenocytes collected at 4 and 12 wpc. According to results, ExoVac-vaccinated mice exhibited two-fold higher proliferation in response to EVs as compared with both the control groups, as well as the ExoAddaVac mouse group at acute phase of disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). At the same time point, ExoVac-immunized mice exhibited also increased SLA-specific proliferative responses which were not, however, statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Impressively, the ExoAddaVac mouse group exhibited similar levels of proliferation as both control groups in response to both stimuli at 4 wpc (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). However, this mouse group presented restoration of cellular responses to EVs as well as to SLA at chronic phase of disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Estimation of cytokines production at 12 wpc revealed that ExoAddaVac splenocytes produced the highest levels of IL-2 and IFN-γ in response to EV stimulation in comparison to the other groups tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Further, TNFα and IL-10 levels were reduced compared with non-immunized controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F), while IL-4 production was increased but remained lower than IFN-γ levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). To deepen the analysis, the quantification of EVs- and parasite-specific IFN-γ- and IL-10-producing CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells was conducted, since the counterbalance of these two cytokines is crucial for the establishment and pathogenesis of VL. Flow cytometric analysis revealed that ExoAddaVac-immunized mice exhibited significantly higher frequencies of IFN-γ\u0026ndash;producing CD4⁺ T cells following EV stimulation, as well as increased IFN-γ\u0026ndash;producing CD4⁺ and CD8⁺ T cells in response to SLA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, K). IL-10\u0026ndash;producing CD4⁺ T cell frequencies were comparable across all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Elevated IFN-γ/IL-10 ratios in both CD4⁺ and CD8⁺ T cells further supported a dominant protective Th1 response in ExoAddaVac-immunized mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ, M). Humoral responses remained elevated post-challenge in ExoAddaVac-immunized mice as compared with ExoVac-immunized mice and control mice groups (Supplementary Fig.\u0026nbsp;4A). Although IgG1 responses predominated, IgG2a levels were higher in ExoAddaVac mice compared with other groups, consistent with sustained Th1-biased immunity (Supplementary Fig.\u0026nbsp;4B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(C-G) Levels of IL-2 (C), IFN-γ (D), TNFα (E), IL-10 (F), and IL-4 (G) in culture supernatants after 72 h stimulation. (H and I) Intracellular cytokine staining identifying IFN-γ-producing (H) and IL-4-producing (I) CD4⁺ T cells following EV or SLA stimulation. (J) Ratio of IFN-γ- to IL-10-producing CD4⁺ T cells. (K, L) Intracellular staining of IFN-γ- and IL-4-producing CD8⁺ T cells. (M) Ratio of IFN-γ- to IL-10-producing CD8⁺ T cells. Data representative one independent experiment with n\u0026thinsp;=\u0026thinsp;4\u0026ndash;6 mice per group for lymphoproliferation assay and cytokine production analysis and n\u0026thinsp;=\u0026thinsp;4\u0026ndash;5 mice per group for intracellular cytokine staining. Bars indicate mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, with individual mice shown as symbols. Statistical significance was determined using two-way ANOVA with Tukey\u0026rsquo;s multiple-comparison test. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we show that extracellular vesicles (EVs) released by stationary-phase \u003cem\u003eLeishmania infantum\u003c/em\u003e promastigotes can function as a vaccine platform when formulated with the oil-in-water emulsion adjuvant Addavax. In this formulation, parasite-derived EVs elicited a Th1-skewed cellular immune response and were associated with significant protection in a murine model of experimental visceral leishmaniasis (VL). To our knowledge, this study provides the first \u003cem\u003ein vivo\u003c/em\u003e evidence that EVs selectively isolated from stationary-phase \u003cem\u003eL. infantum\u003c/em\u003e promastigotes can be leveraged as a cell-free vaccine formulation.\u003c/p\u003e \u003cp\u003eEfforts to develop vaccines against leishmaniasis have been historically constrained by the parasite\u0026rsquo;s complex life cycle, antigenic heterogeneity, and capacity to subvert host immune responses. An additional challenge has been the limited availability of adjuvants capable of consistently inducing Th1-biased immunity, which is essential for intracellular parasite clearance\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In this context, we hypothesized that parasite-derived EVs could represent an alternative vaccination strategy that may help address some of these challenges. EVs inherently resemble biological liposomes and are well suited for antigen delivery, while retaining native protein conformation, post-translational modifications, and structural integrity\u0026mdash;features that are often lost in recombinant or synthetic formulations. Previous studies have shown that liposomal encapsulation of \u003cem\u003eLeishmania\u003c/em\u003e antigens enhances immunogenicity relative to soluble antigens\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. EVs extend this principle by providing a naturally evolved delivery system that may offer advantages in antigen presentation and manufacturing reproducibility\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough \u003cem\u003eLeishmania\u003c/em\u003e-derived EVs have been extensively associated with parasite virulence, immune evasion, and disease progression\u003csup\u003e\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, growing evidence indicates that their immunological effects are highly context dependent. EV composition varies according to parasite species, developmental stage, and environmental conditions, resulting in distinct immunomodulatory properties\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Consistent with this notion, EVs isolated in our study exhibited canonical physicochemical features and a protein repertoire enriched in well-characterized immunogenic and virulence-associated molecules, including GP63, KMP-11, elongation factors, histones, heat shock proteins, and enolase\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Importantly, many of the identified proteins have independently demonstrated protective efficacy as vaccine antigens in experimental models\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, supporting the premise that these EVs contain an antigenic repertoire relevant for host immune recognition.\u003c/p\u003e \u003cp\u003eThe induction of adaptive immunity is critically dependent on early innate immune activation, particularly through dendritic cells (DCs), which orchestrate T-cell priming and differentiation. Here, EV exposure promoted functional maturation of bone marrow\u0026ndash;derived DCs, as evidenced by upregulation of MHC class I and II molecules, increased CD40 expression, and production of pro-inflammatory cytokines. Although IL-10 was detectable, its levels remained substantially lower than IL-12, favoring differentiation toward a DC1 phenotype capable of driving Th1 responses. These findings differ from prior reports describing suppressive effects of \u003cem\u003eLeishmania\u003c/em\u003e EVs on monocyte-derived DCs\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, likely reflecting differences in EV origin, molecular composition, and experimental context.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e, immunization with EVs alone resulted in limited adaptive immune activation, consistent with earlier observations that parasite-derived EVs may lack intrinsic immunogenicity and can even facilitate infection establishment\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Nonetheless, EV administration increased circulating neutrophil frequencies, a phenomenon previously linked to the phosphatidylserine-rich surfaces and inflammatory properties of \u003cem\u003eLeishmania\u003c/em\u003e EVs\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. While such responses may promote immune cell recruitment, they may also contribute to immune environments that are less effective at restricting parasite persistence\u003csup\u003e\u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo overcome the limited immunogenicity of EVs alone, we employed Addavax, a squalene-based nanoemulsion analogous to MF59, which is known to enhance antigen uptake, recruit antigen-presenting cells, and amplify inflammatory signaling at the injection site\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Addavax-adjuvanted EV vaccination resulted in robust expansion of antigen-specific lymphocytes, with pronounced increases in both CD4⁺ and CD8⁺ T-cell populations. The expansion of CD8⁺ T cells is of interest given their established role in parasite killing, Th1 polarization, and long-term protective memory in VL\u003csup\u003e61\u0026ndash;64\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCytokine profiling further underscored the protective immune environment induced by adjuvanted EVs. While EVs alone elicited weak IFN-γ responses and a low IFN-γ/IL-10 ratio, EVs formulated with Addavax induced robust IFN-γ production by both CD4⁺ and CD8⁺ T cells, resulting in a cytokine profile consistent with Th1 polarization. Such responses are essential for macrophage activation and intracellular parasite control\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Importantly, although IL-10 was still detectable, it did not override the dominant IFN-γ response, suggesting effective immune regulation rather than pathological suppression.\u003c/p\u003e \u003cp\u003eConsistent with these immunological findings, Addavax-adjuvanted EV vaccination conferred significant protection following parasite challenge, particularly in the spleen, whereas hepatic parasite burdens were less affected. This pattern likely reflects the self-resolving nature of liver infection during VL\u003csup\u003e67\u003c/sup\u003e. Histopathological analysis revealed preserved splenic architecture and absence of multinucleated giant cells, which are commonly associated with chronic inflammation and immune dysregulation\u003csup\u003e\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. These observations suggest that vaccination promoted parasite control without evidence of overt immunopathology in this experimental setting.\u003c/p\u003e \u003cp\u003eAlthough protective immunity against \u003cem\u003eLeishmania\u003c/em\u003e is predominantly T-cell mediated, chronic infection is frequently associated with T-cell exhaustion and anergy\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. In this regard, sustained antigen-specific T-cell proliferation following challenge represents a key indicator of vaccine efficacy. \u003cem\u003eEx vivo\u003c/em\u003e recall assays demonstrated that Addavax-adjuvanted EV immunization maintained robust T-cell responsiveness during chronic infection, accompanied by preferential IFN-γ production over IL-4 and IL-10. While excessive IFN-γ can contribute to tissue damage, balanced regulation by IL-10 may limit immunopathology while preserving antimicrobial function\u003csup\u003e\u003cspan additionalcitationids=\"CR73\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. The absence of splenomegaly in vaccinated animals may reflect a balanced cytokine environment associated with effective parasite control.\u003c/p\u003e \u003cp\u003eAnalysis of humoral immunity revealed that Addavax-adjuvanted EV vaccination predominantly induced parasite-specific IgG1 responses. Although elevated IgG1 titers have traditionally been associated with active VL, emerging evidence suggests that IgG1 responses to EV-associated antigens may contribute to protective immunity through mechanisms distinct from those observed during natural infection\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. In our study, IgG1 induction correlated with enhanced CD8⁺ T-cell responses, consistent with reports linking early IL-4 production and IgG1-biased environments to effective CD8⁺ T-cell memory formation\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, while parasite-derived EVs have classically been viewed as mediators of immune evasion and disease progression, our findings demonstrate that appropriate adjuvantation can substantially alter their immunological properties toward protective outcomes. Addavax effectively converted \u003cem\u003eL. infantum\u003c/em\u003e EVs into a potent Th1-biased vaccine platform capable of conferring significant protection against visceral infection. Together, these results provide a foundation for further development of EV-based vaccination strategies against VL. However, further optimization of the vaccine platform is warranted, particularly with respect to the functional contribution of EV-associated nucleic acids. EV-encapsulated mRNAs and microRNAs have been shown to modulate host gene expression and immune responses, and elucidating their role in vaccine-induced immunity will be critical for advancing this approach toward clinical application\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e,\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and immunization\u003c/h2\u003e \u003cp\u003eFemale BALB/c mice (6\u0026ndash;8 weeks old) were used in all experiments. Animals were housed at the Hellenic Pasteur Institute (HPI) under specific pathogen-free (SPF) conditions with controlled temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C), humidity (40\u0026ndash;70%), and a 12 h light/dark cycle. All experimental procedures complied with national legislation (Presidential Decree 56/2013) and Directive 2010/63/EU of the European Parliament, following the principles of the 3\u0026thinsp;+\u0026thinsp;1R framework. Study protocols were approved by the Official Veterinary Authorities of the Attiki Prefecture (license no. 6381/11-12-2017). Age-matched mice (n\u0026thinsp;=\u0026thinsp;4\u0026ndash;7 per group) were immunized intramuscularly with extracellular vesicles (EVs; 15 \u0026micro;g per mouse per immunization) either alone (ExoVac) or formulated 1:1 with Addavax (ExoAddaVac; InvivoGen), in a total volume of 100 \u0026micro;L (50 \u0026micro;L per hind limb). Immunizations were administered twice at two-week intervals. Control animals received sterile PBS or Addavax alone. Three weeks after the final boost, mice were infected by intravenous injection of 1 \u0026times; 10⁷ stationary-phase \u003cem\u003eLeishmania infantum\u003c/em\u003e promastigotes. Animals were monitored throughout the study, and body weight was recorded every two days. At predefined time points, mice were euthanized by CO₂ inhalation, and blood, spleen, and liver samples were collected for downstream analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLeishmania infantum\u003c/b\u003e \u003cb\u003epromastigotes culture\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eLeishmania infantum\u003c/em\u003e promastigotes (MHOM/GR/2001/GH8 strain) were cultured at 26\u0026deg;C in complete culture medium consisted of RPMI-1640 medium, penicillin (100 U/mL), streptomycin (10 \u0026micro;g/mL), L-glutamine (2 mM), HEPES buffer (10 mM) and 10% (v/v) fetal bovine serum (FBS) for no more than ten in \u003cem\u003evitro\u003c/em\u003e passages. To preserve parasite virulence, promastigotes were periodically passaged \u003cem\u003ein vivo\u003c/em\u003e through BALB/c mice by intravenous administration of 1 \u0026times; 10⁷ stationary-phase parasites.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDifferentiation of bone marrow\u0026ndash;derived dendritic cells\u003c/h2\u003e \u003cp\u003eBone marrow\u0026ndash;derived dendritic cells (BMDCs) were generated following an established protocol with minor modifications\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Bone marrow was harvested from femurs and tibias of BALB/c mice and flushed with RPMI medium. Cells were washed and seeded at 4 \u0026times; 10⁶ cells in 10 mL complete RPMI supplemented with recombinant murine GM-CSF (20 ng/mL; PeproTech). Cultures were maintained at 37\u0026deg;C in a humidified 5% CO₂ incubator. On day 3, fresh GM-CSF-containing medium was added. At day 6, half of the supernatant was replaced with fresh medium following centrifugation and resuspension of the cells. Semi-adherent and non-adherent cells were collected on day 8. BMDC purity consistently exceeded 75%, as confirmed by CD11c expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of soluble\u003c/b\u003e \u003cb\u003eLeishmania\u003c/b\u003e \u003cb\u003eantigen\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSoluble \u003cem\u003eLeishmania\u003c/em\u003e antigen (SLA) was prepared from stationary-phase promastigote cultures following a previously established protocol\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Protein concentrations were quantified using the MicroBCA Protein Assay Kit (Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation of\u003c/b\u003e \u003cb\u003eL. infantum\u003c/b\u003e\u003cb\u003e\u0026ndash;derived extracellular vesicles\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExtracellular vesicles (EVs) were isolated from stationary-phase \u003cem\u003eL. infantum\u003c/em\u003e promastigotes cultured for 24 h at 26\u0026deg;C in serum-free Schneider\u0026rsquo;s Drosophila medium (Biowest) supplemented with L-glutamine (2 mM), HEPES (10 mM), penicillin (100 U/mL), and streptomycin (10 \u0026micro;g/mL). Following incubation, promastigotes were removed by two sequential centrifugation steps at 3,000 \u0026times; g for 10 min. Supernatants were subsequently filtered twice through 0.45 \u0026micro;m syringe filters to eliminate residual debris. EVs were concentrated using Centricon Plus-70 centrifugal devices with a 100 kDa molecular weight cut-off (Millipore) and further purified by OptiPrep\u0026trade; density gradient ultracentrifugation at 100,000 \u0026times; g\u003csub\u003eavg\u003c/sub\u003e for 16 h at 4\u0026deg;C using an SW40 Ti rotor. Fractions enriched in EVs (fractions 6\u0026ndash;7) were pooled, diluted in PBS, and subjected to a second ultracentrifugation step at 100,000 \u0026times; g\u003csub\u003eavg\u003c/sub\u003e for 3 h. Final EV pellets were resuspended in 100\u0026ndash;200 \u0026micro;L PBS and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. All procedures were performed using endotoxin-free reagents and consumables. Protein content was measured by MicroBCA Protein Assay Kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEVs characterization\u003c/h2\u003e \u003cp\u003eEVs were characterized using dynamic light scattering (DLS), Western blotting, transmission electron microscopy (TEM), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). DLS analysis was performed to estimate EVs size distribution using a Zetasizer NanoS instrument (Malvern Instruments, UK), equipped with a 633 nm He-Ne laser (4.0 mW). Measurements were conducted at 4\u0026deg;C in quartz cuvettes and analyzed using DTS v4.1 software. Western blot analysis was carried out in 12% SDS-PAGE resolving gel and the proteins were next transferred to nitrocellulose membranes using standard protocols. EV protein samples were then incubated at 4\u0026deg;C overnight with monoclonal antibodies against established EV markers: CD9 and CD63 (Santa Cruz Biotechnology, Inc., TX, USA) and CD81 (Affinity Biosciences). To avoid presence of soluble protein or intracellular contamination, albumin and cytochrome c (Santa Cruz Biotechnology, Inc., TX, USA) were used as negative markers respectively. Blot visualization was performed upon incubation with secondary antibody, namely peroxidase-conjugated anti-mouse IgG (Biorad), followed by enhanced chemiluminescence addition (ECL, Pierce) while the image acquisition was achieved by exposure to X-ray film. Transmission electron microscopy was employed to visualize the morphology of isolated EVs. EVs were isolated, fixed, stained and embedded according to previously described protocol\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. Specifically, EVs were fixed using 2% paraformaldehyde prepared in 0.1M phosphate buffer (pH 7.4) and then adsorbed on formvar/carbon-coated copper grids (200 mesh; Polysciences Europe GmbH). Contrast was achieved with uranyl oxalate, pH\u0026thinsp;=\u0026thinsp;7.00, before embedding in a mixture of 4% uranyl acetate and 2% methyl cellulose. Finally, EVs were observed using a Jeol JEM2100Plus Transmission Electron Microscope (Jeol, Japan) operated at 120kV and photographed with the Gatan OneView CMOS camera (Gatan Ametek, USA), which is housed at the Centre of Innovative Electron Microscopy Applications and Services of the National and Kapodistrian University of Athens (CEMA-NKUA). Finally, proteomic profiling of EVs cargo was conducted using LC-MS/MS. These data informed the selection of EVs used for immunization studies in animal models.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFunctional assessment of BMDCs\u003c/h2\u003e \u003cp\u003eTo assess the immunomodulatory effects of \u003cem\u003eL. infantum\u003c/em\u003e EVs, BMDCs were incubated with endotoxin-free EVs (5 \u0026micro;g/mL) or SLA (10 \u0026micro;g/mL) for 24 h at a density of 1 \u0026times; 10⁶ cells/mL. Cells cultured in medium alone or stimulated with lipopolysaccharide (LPS; 1 \u0026micro;g/mL) served as negative and positive controls, respectively. Following incubation, BMDCs were washed in ice cold FACS buffer (PBS supplemented with 3% FBS), blocked with anti-CD16/32 monoclonal antibody (Biolegend) for 10 min at 4\u0026deg;C. Then, cells were stained against CD11c (clone HL3; 1:100 dilution; BD Pharmingen), CD40 (clone 3/23; 1:100 dilution; BD Pharmingen), MHC class I (clone SF1-1.1; 1:100 dilution; BD Pharmingen) or MHC class II (clone 2G9; 1:200 dilution; BD Pharmingen) for 30 min at 4\u0026deg;C, fixed in 2% paraformaldehyde and analyzed by flow cytometry. For intracellular cytokine detection, brefeldin A (10 \u0026micro;g/mL; Cayman, Michigan, USA) was added during the final 4 h of culture. Cells were then surface stained with anti-CD11c antibody, fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Life Sciences) and then stained for IL-12p40/p70 (1:100 dilution; BD Pharmingen), IL-10 (clone JES5-16E3; 1:100 dilution; Biolegend) or TNFα (clone MP6-XT22; 1:100 dilution; Biolegend) for 30 min at 4\u0026deg;C. Data acquisition was performed using a BD FACSCalibur cytometer (Becton-Dickinson, San Jose, CA, USA) and analyzed with FlowJo software version 10.0 (BD Life Sciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSplenocyte proliferation assay\u003c/h2\u003e \u003cp\u003eSplenocyte proliferation was assessed using a [\u0026sup3;H]-thymidine incorporation assay two weeks after the end of immunization as well as at 4 and 12 weeks post infection. Specifically, aseptically collected spleens from immunized and non-immunized mice (n\u0026thinsp;=\u0026thinsp;5/group) were minced with a sterile syringe plunger from a 5-mL syringe. The resulting cell suspensions were filtered through a 70 \u0026micro;m cell strainer. Suspensions were centrifuged at 1,300 rpm for 10 min and the resulting cell pellet was resuspended in ACK lysis buffer for 5 min in room temperature in order for RBCs to be lysed. Lysis was stopped by adding complete RPMI and centrifuged as described above. Isolated cells were seeded at 2 \u0026times; 10⁵ cells/well, and stimulated with SLA (12.5 \u0026micro;g/mL) or EVs (2 \u0026micro;g/mL). Concanavalin A (ConA; 3 \u0026micro;g/mL; Sigma) served as a positive control. After 72 h, [\u0026sup3;H]-thymidine (1 \u0026micro;Ci; PerkinElmer) was added, and incorporation was measured following an additional 18 h incubation. The radioactivity expressed as mean counts per minute (cpm) using a scintillation counter (Wallac). Results were expressed as Δcpm relative to unstimulated controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular cytokine staining\u003c/h2\u003e \u003cp\u003eSplenic cells were prepared at a density of 1x10\u003csup\u003e6\u003c/sup\u003e cells/well in predetermined time points and stimulated with SLA (25 \u0026micro;g/mL) or EVs (2 \u0026micro;g/mL) for 18 h at 37\u0026deg;C and brefeldin A (10 \u0026micro;g/mL) was added during the final 4 h of incubation. Subsequently, cells were surface-stained using anti-CD3 (clone 17A2), anti-CD4 (clone RM4-5), and anti-CD8 (clone 53\u0026thinsp;\u0026minus;\u0026thinsp;6.7) mAbs at a dilution of 1:100, followed by intracellular staining for IFN-γ (clone XMG1.2), IL-4 (clone 11B11), and IL-10 (clone JES5-16E3). All antibodies used were obtained from Biolegend and analysis was conducted on a FACSCalibur system. A representative gating strategy is depicted in Supplementary Fig.\u0026nbsp;5. Cytokine-producing T-cell frequencies were calculated after subtraction of background values from unstimulated samples. Negative values resulting from the above calculation were set to zero.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCytokine analysis\u003c/h2\u003e \u003cp\u003eCytokine concentrations in stimulated spleen cells supernatants, were also determined. For this purpose, spleen cells were treated as described above to induce cytokine production. Cell-free supernatants were collected after 72 h of antigen stimulation by centrifugation, aliquoted, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until assayed for cytokine production using Milliplex Mouse Cytokine Magnetic Bead Panel Kit (Millipore, Billerica, MA, USA). Analysis was performed on a Luminex 200 (Thermo Fischer) using xPONENT software (Luminex Corporation) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eAntigen-specific IgG, IgG1, and IgG2a antibodies were measured in mouse sera by ELISA. Briefly, sera were isolated after blood centrifugation at 4,000 rpm for 5 min. Then, 96-well high-binding plates were coated with SLA (5 \u0026micro;g/mL), blocked with 2% BSA in PBS for 2 h at 37\u0026deg;C, and incubated with diluted serum samples (1:100 dilution) in PBS in duplicates at 37\u0026deg;C for 90 min. Bound antibodies were detected using HRP-IgG (dilution 1:5,000; Biorad) or biotin-conjugated anti-mouse IgG1 (1 \u0026micro;g/mL) or IgG2a (250 ng/mL) (both obtained from Biorad) for 1 h at 37\u0026deg;C followed by TMB substrate development. For IgG1 and IgG2a, plates were further incubated with streptavidin-HRP for 60 min at 37\u0026deg;C. Optical density was measured at 450 nm using an ELISA microplate spectrophotometer (MRX).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eParasite burden determination\u003c/h2\u003e \u003cp\u003eSpleen and hepatic parasitic loads were determined by limiting dilution assay. Briefly, organ homogenates in Schneider\u0026rsquo;s Insect Medium (Biowest)\u0026thinsp;\u0026minus;\u0026thinsp;20% FBS at a final concentration of 1 mg/mL. Two hundred microliters of suspension were placed into the first well, and 2-fold serial dilutions were distributed in the 96-well plates. Parasite numbers were calculated based on the highest dilution yielding viable promastigotes and expressed as parasites per gram of tissue after 7 days of incubation at 26\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eSpleens and livers were collected at predetermined time points. Then, they were fixed in 10% neutral buffered formalin, paraffin-embedded, sectioned at 4 \u0026micro;m and stained with hematoxylin and eosin for histological examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical comparisons were performed using one- or two-way ANOVA followed by Tukey\u0026ndash;Kramer post hoc test to perform multiple comparisons using GraphPad Prism version 6.0 software (GraphPad Software). Differences were considered statistically significant was defined at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. In the graphs only the significant differences are noted, where * for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and **** for \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e partner repository with the dataset identifier PXD074544 and are accessible using the login Username: [email protected] and Password: gSKzgBe53ZHn. All other data supporting the findings of this study are included within the article and its Supplementary Information. Additional relevant data are available from the corresponding author upon reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the EPAnEK Operational Programme (NSFR 2014\u0026ndash;2020) co-financed by Greece and the European Union under Grant number (T1EDK-03902).\u0026nbsp;The funder had no role in study design, data collection, data analysis, interpretation writing or submission of the manuscript. Regarding the TEM work, VGG, SH and IT were supported by the Hellenic Foundation for Research and Innovation (HFRI) under the \u0026ldquo;1st Call for HFRI Research Projects to support Faculty members and Researchers and the procurement of high-cost research equipment\u0026rdquo; grant no. 2906.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.E. performed the EVs isolation and purification. A.E. and D.K.T performed the EVs characterization. \u0026nbsp;I.T. and S.H. \u0026nbsp;performed the TEM analysis of EVs fractions while V.G.G. acquired funding for high-cost research equipment regarding TEM microscopy. M.S. performed the LC-MS/MS analysis. A.E and M.A performed \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eexperiments, and monitoring of the mice. A.E performed splenocyte proliferation assay, intracellular cytokine analysis and parasite burden determination. M.A. performed differentiation and functional assessment of BMDCs, ELISA and cytokine analysis. F.B performed the histological analyses. E.K., A.E. and M.A. designed the studies. M.A. and A.E. analyzed the data and performed the statistical analysis. E.K. supervised experiments and acquired funding. M.A, and A.E. wrote the first draft of the manuscript under the guidance and supervision of E.K., and all authors edited the final version. All authors approved the final draft and take full responsibility for its content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health, O. 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Here, we evaluate extracellular vesicles (EVs) released by stationary-phase \u003cem\u003eLeishmania infantum\u003c/em\u003e promastigotes as a biologically derived, cell-free vaccine platform when formulated with the clinically relevant oil-in-water adjuvant Addavax. Parasite-derived EVs contained a broad repertoire of immunologically relevant proteins, including GP63, and induced functional maturation of bone marrow\u0026ndash;derived dendritic cells \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eIn vivo\u003c/em\u003e, immunization of BALB/c mice with adjuvanted EVs elicited antigen-specific antibody responses and robust Th1-biased cellular immunity, characterized by enhanced T-cell proliferation and increased frequencies of IFN-γ\u0026ndash;producing CD4⁺ T cells. Following experimental challenge, vaccinated mice exhibited significantly reduced hepatic and splenic parasite burdens during both acute and chronic infection, accompanied by preserved tissue architecture. Protection correlated with a favorable IFN-γ/IL-10 balance, supporting adjuvanted parasite-derived EVs as a modular, cell-free vaccine strategy for VL.\u003c/p\u003e","manuscriptTitle":"Adjuvanted Leishmania infantum extracellular vesicles induce protective immunity in experimental visceral leishmaniasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 21:12:43","doi":"10.21203/rs.3.rs-9033802/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-16T15:08:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-16T14:52:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-04T15:07:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1671651393288290221566975725519539367","date":"2026-03-17T14:52:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282027033478710281637679907755780338712","date":"2026-03-16T05:45:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-13T16:55:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-13T16:50:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-05T09:13:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2026-03-04T20:57:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ada68a58-2e7d-44c9-a571-5aa4d994b1a3","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":64534986,"name":"Biological sciences/Immunology"},{"id":64534987,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-16T15:25:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 21:12:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9033802","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9033802","identity":"rs-9033802","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}