From classical approaches to new developments in genetic engineering of live attenuated vaccine against cutaneous leishmaniasis: potential and immunization.

Despite the development of a vaccine against cutaneous leishmaniasis in preclinical and clinical studies, we still do not have a safe and effective vaccine for human use. Given this situation, the search for a new prophylactic alternative to control leishmaniasis should be a global priority. A first-generation vaccine strategy-leishmanization, in which live Leishmania major parasites are inoculated into the skin to protect against reinfection, is taking advantage of this situation. Live attenuated Leishmania vaccine candidates are promising alternatives due to their robust protective immune responses. Importantly, they do not cause disease and could provide long-term protection following challenges with a virulent strain. In addition to physical and chemical methods, genetic tools, including the Cre-loxP system, have enabled the selection of safer null mutant live attenuated Leishmania parasites obtained by gene disruption. This was followed by the discovery and introduction of CRISPR/Cas-based gene editing tools, which can be easily and precisely used to modify genes. Here, we briefly review the immunopathology of L. major parasites and then present the classical methods and their limitations for the production of live attenuated vaccines. We then discuss the potential of current genetic engineering tools to generate live attenuated vaccine strains by targeting key genes involved in L. major pathogenesis and then discuss their discovery and implications for immune responses to control leishmaniasis.

Manufacturing and preclinical toxicity of GLP grade gene deleted attenuated Leishmania donovani parasite vaccine.

Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.

Vaccine efficacy induced by virus-like particles containing Leishmania donovani surface glycoprotein GP63.

Leishmania donovani surface glycoprotein 63 (GP63) is a major virulence factor involved in parasite escape and immune evasion. In this study, we generated virus-like particles (VLPs) expressing L. donovani GP63 using the baculovirus expression system. Mice were intramuscularly immunized with GP63-VLPs and challenged with L. donovani promastigotes. GP63-VLP immunization elicited higher levels of L. donovani antigen-specific serum antibodies and enhanced splenic B cell, germinal center B cell, CD4+, and CD8+ T cell responses compared to unimmunized controls. GP63-VLPs inhibited the influx of pro-inflammatory cytokines IFN-γ and IL-6 in the livers, as well as thwarting the development of splenomegaly in immunized mice. Upon L. donovani challenge infection, a drastic reduction in splenic parasite burden was observed in VLP-immunized mice. These results indicate that GP63-VLPs immunization conferred protection against L. donovani challenge infection by inducing humoral and cellular immunity in mice.

Revolutionizing Leishmaniasis Treatment with Cutting Edge Drug Delivery Systems and Nanovaccines: An Updated Review.

Leishmaniasis, one of the most overlooked tropical diseases, is a life-threatening illness caused by the parasite Leishmania donovani that is prevalent in underdeveloped nations. Over 350 million individuals in more than 90 different nations worldwide are at risk of contracting the disease, which has a current fatality rate of 50 000 mortalities each year. The administration of liposomal Amp B, pentavalent antimonials, and miltefosine are still considered integral components of the chemotherapy regimen. Antileishmanial medications fail to treat leishmaniasis because of their numerous drawbacks. These include inadequate effectiveness, toxicity, undesired side effects, drug resistance, treatment duration, and cost. Consequently, there is a need to overcome the limitations of conventional therapeutics. Nanotechnology has demonstrated promising outcomes in addressing these issues because of its small size and distinctive characteristics, such as enhanced bioavailability, lower toxicity, biodegradability, and targeted drug delivery. This review is an effort to highlight the recent progress in various nanodrug delivery systems (nDDSs) over the past five years for treating leishmaniasis. Although the preclinical outcomes of nDDSs have shown promising treatment for leishmaniasis, further research is needed for their clinical translation. Advancement in three primary priority domains─molecular diagnostics, clinical investigation, and knowledge dissemination and standardization─is imperative to propel the leishmaniasis field toward translational outcomes.

PLGA nanoparticle-delivered Leishmania antigen and TLR agonists as a therapeutic vaccine against cutaneous leishmaniasis in BALB/c mice.

Leishmaniasis, caused by Leishmania (L.) species, remains a neglected infection. Therapeutic vaccination presents a promising strategy for its treatment. In this study, we aimed to develop a therapeutic vaccine candidate using Leishmaniaantigens (SLA) and Toll-like receptor (TLR) 7/8 agonist (R848) encapsulated into the poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Moreover, TLR1/2 agonist (Pam3CSK4) was loaded onto the NPs. The therapeutic effects of these NPs were evaluated in L. major-infected BALB/c mice. Footpad swelling, parasite load, cellular and humoral immune responses, and nitric oxide (NO) production were analyzed. The results demonstrated that the PLGA NPs (SLA-R848-Pam3CSK4) therapeutic vaccine effectively stimulated Th1 cell responses, induced humoral responses, promoted NO production, and restricted parasite burden and lesion size.Our findings suggest that vaccination with SLA combined with TLR1/2 and TLR7/8 agonists in PLGA NPs as a therapeutic vaccine confers strong protection againstL. majorinfection. These results represent a novel particulate therapeutic vaccine against Old World cutaneous leishmaniasis.

Leishmania vaccine development: A comprehensive review.

Infectious diseases like leishmaniasis, malaria, HIV, tuberculosis, leprosy and filariasis are responsible for an immense burden on public health systems. Among these, leishmaniasis is under the category I diseases as it is selected by WHO (World Health Organization) on the ground of diversity and complexity. High cost, resistance and toxic effects of Leishmania traditional drugs entail identification and development of therapeutic alternative. Since the natural infection elicits robust immunity, consistence efforts are going on to develop a successful vaccine. Clinical trials have been conducted on vaccines like Leish-F1, F2, and F3 formulated using specific Leishmania antigen epitopes. Current strategies utilize individual or combined antigens from the parasite or its insect vector's salivary gland extract, with or without adjuvant formulation for enhanced efficacy. Promising animal data supports multiple vaccine candidates (Lmcen-/-, LmexCen-/-), with some already in or heading for clinical trials. The crucial challenge in Leishmania vaccine development is to translate the research knowledge into affordable and accessible control tools that refines the outcome for those who are susceptible to infection. This review focuses on recent findings in Leishmania vaccines and highlights difficulties facing vaccine development and implementation.

Intranasal delivery of LaAg vaccine improves immunity of aged mice against visceral Leishmaniasis.

There are no approved vaccines yet for human visceral leishmaniasis (VL), the most severe form of the leishmaniasis clinical manifestations that is fatal in over 95 % of untreated cases. It is well-accepted that immunological changes during aging have deleterious impact on the efficacy of vaccines and response to infections. In this work, we compared the response of young and aged mice to intranasal vaccination with killed Leishmania amazonensis promastigote antigens (LaAg) that were then challenged with L. infantum infection, a species that causes visceral leishmaniasis. Intranasal vaccination with LaAg induced a similar reduction in parasitism and hepatosplenomegaly in both young and aged mice compared to their unvaccinated counterparts. Following infection, there was also a less prominent inflammatory profile particularly in the vaccinated aged group, with lower production of TNF-α and nitrite compared to the respective unvaccinated group. Interestingly, the LaAg intranasal vaccination promoted increased production of IFN-γ that was observed in both young- and aged vaccinated groups. Additionally, CD4+ and CD8+T cells from both vaccinated groups presented decreased expression of the inhibitory receptors PD-1 and KLRG1 compared to their unvaccinated controls. Interestingly, a strong positive correlation was observed between the expression of both inhibitory receptors PD-1 and KLRG1 and parasitism, which was more conspicuous in the unvaccinated-aged mice than in the others. Overall, this study helps define new strategies to improve vaccine effectiveness and provides a perspective for prophylactic alternatives against leishmaniasis.

Interleukin-7 potentiates MAPK10-elicited host-protective vaccine against Leishmania donovani.

Leishmania donovani causes the potentially fatal disease visceral leishmaniasis for which neither a vaccine nor an adjuvant for human use exists. Although interleukin-7 (IL-7) is implicated in CD4+ T-cell response stabilization, its anti-leishmanial function is uncertain. Therefore, we examined whether IL-7 would potentiate the efficacy of Leishmania major-expressed MAPK10 (LmjMAPK10; M10)-elicited anti-leishmanial host-protective response. We observed that aligning with IL-7R expression, IL-7 increased IFN-γ-secreting TH1 cell but reduced IL-4-producing TH2 cells and production of IL-10 and TGF-ß effectuating anti-leishmanial functions in susceptible BALB/c mouse-derived macrophages. Co-culturing IL-7-pre-treated L. donovani-infected macrophages with L. donovani-infected BALB/c-derived T cells induced IFN-γ-dominated TH1 type anti-leishmanial function. IL-7 treatment of L. donovani-infected BALB/c mice significantly reduced splenic and hepatic parasite loads. Co-culturing CD4+ T cells from IL to 7-treated mice with L. donovani-infected macrophages reduced amastigote numbers suggesting IL-7-elicited host-protective effector T cells. Priming BALB/c with M10 + IL-7 reduced the splenic parasite burden more effectively than that was observed in M10-primed mice. An enhanced protection against L. donovani infection was accompanied by enhanced IL-12 and IFN-γ, but suppressed IL-10 and IL-4, response and host-protective TH1 and memory T cells. These results indicate IL-7-induced leishmanial antigen-specific memory T cell response that protects a susceptible host against L. donovani infection.

Leishmaniasis: Epidemiological Report for the Americas. No.12 (December 2023)

This report presents the 2022 epidemiological profile of the Region, as well as describes the analyses of the goals and main indicators of the Plan of Action to Strengthen the Surveillance and Control of Leishmaniasis in the Americas 2017-2022, which at the regional level, were not fully achieved, but when analyzed individually by country, significant progress has been made, demonstrating the commitment and effort of the Ministries of Health with regard to the disease surveillance, patient care and control. Within the framework of the approval of the PAHO's Disease Elimination Initiative, the WHO Roadmap for 2030, and the publication of the cross-cutting resolutions: a) Plan of Action on Entomology and Vector Control 2018-2023 and b) One Health: A Comprehensive Approach to Addressing Health Threats, the leishmaniasis Plan of Action for 2023-2030 was updated, discussed, and agreed upon with endemic countries, specialists, collaborators and partners. With this new Plan, it is expected to improve and strengthen surveillance and control actions to obtain better results, such as expand access and implementation of diagnostic methodologies, and treatment alternatives for affected people living in risk areas, also, execution of actions according to the risk stratification, reduction of vector contact, among others.

Vaccine value profile for leishmaniasis.

Leishmania infections are global, occurring in 98 countries and all World Health Organization (WHO) regions with 600 million to 1 billion people at risk of infection. Visceral leishmaniasis is associated with almost 20,000 reported deaths annually, with children under 5 years of age being at the greatest risk of mortality. Amongst WHO-recognised Neglected Tropical Diseases (NTDs), leishmaniasis is one of the most important in terms of mortality and morbidity. With an increasing global burden of disease and a growing threat from climate change, urbanisation and drug resistance, there remains an imperative to develop leishmaniasis vaccines. New tools to understand correlates of protection and to assess vaccine efficacy are being developed to ease the transition into larger scale efficacy trials or provide alternate routes to licensure. Early indications suggest a diverse portfolio of manufacturers exists in endemic countries with an appetite to develop leishmaniasis vaccines. This Vaccine Value Profile (VVP) provides a high-level, comprehensive assessment of the currently available data to inform the potential public health, economic, and societal value of leishmaniasis vaccines. The leishmaniasis VVP was developed by a working group of subject matter experts from academia, public health groups, policy organizations, and non-profit organizations. All contributors have extensive expertise on various elements of the leishmaniasis VVP and have collectively described the state of knowledge and identified the current gaps. The VVP was developed using only existing and publicly available information.

Protective Humoral Immune Response Induced by Recombinant Virus-like Particle Vaccine Expressing Leishmania donovani Surface Antigen.

To date, Leishmania spp. vaccine studies have mainly focused on cellular immunity induction, which plays a crucial role in host protection. In contrast, vaccine-induced humoral immunity is largely neglected. Virus-like particle (VLP) vaccines generated using the baculovirus expression system are well-known inducers of humoral immunity and would serve as a suitable platform for evaluating humoral immunity-mediated protection against visceral Leishmaniasis. In this study, we investigated the humoral immunity evoked through VLPs expressing the L. donovani promastigote surface antigen (PSA-VLPs) and assessed their contribution to protection in mice. PSA-VLPs vaccines were generated using the baculovirus expression system and used for mouse immunizations. Mice were intramuscularly immunized twice with PSA-VLPs and challenged with L. donovani to confirm vaccine-induced protective immunity. PSA-VLP immunization elicited parasite-specific antibody responses in the sera of mice, which were induced in a dose-dependent manner. B cell, germinal center B cell, and memory B cell responses in the spleen were found to be higher in vaccinated mice compared to unimmunized controls. PSA-VLP immunization diminished the production of pro-inflammatory cytokines IFN-γ and IL-6 in the liver. Overall, the PSA-VLPs conferred protection against L. donovani challenge infection by reducing the total parasite burden within the internal organs. These results suggest that PSA-VLPs induced protective immunity against the L. donovani challenge infection.

Adjuvantation of whole-killed Leishmania vaccine with anti-CD200 and anti-CD300a antibodies potentiates its efficacy and provides protection against wild-type parasites.

One of the major reasons behind the limited success of vaccine candidates against all forms of leishmaniasis is the inability of parasitic antigens to induce robust cell-mediated immunity and immunological memory. Here we find, for the first time, that the adjuvantation of whole-killed Leishmania vaccine (Leishvacc) with anti-CD200 and anti-CD300a antibodies enhances CD4+ T cells mediated immunity in vaccinated mice and provides protection against wild-type parasites. The antibody adjuvantation, either alone or with a TLR4 agonist monophosphoryl A (MPL-A), induced the production of pro-inflammatory cytokines viz., IFN-γ, TNF-α, and IL-2 by antigen experienced CD4+ T cells, and also enhanced their rate of conversion into their memory phenotypes against Leishvacc antigens. The antibody adjuvanted vaccine also promoted the generation of IgG2a-mediated protective humoral immunity in vaccinated mice. Further, the mice vaccinated with antibodies adjuvanted vaccine showed strong resilience against metacyclic forms of L. donovani parasites as we observed reduced clinical features such as splenomegaly, hepatomegaly, granulomatous tissues in the liver, and parasitic load in their spleen. The findings of this study demonstrate that the anti-CD200 and anti-CD300a antibodies have potential to increase the protective efficacy of the whole-killed Leishmania vaccine, and opens up a new gateway to diversify the roles of immune checkpoints in vaccine development against leishmaniasis.

A Tailored Approach to Leishmaniases Vaccination: Comparative Evaluation of the Efficacy and Cross-Protection Capacity of DNA vs. Peptide-Based Vaccines in a Murine Model.

Zoonotic leishmaniases are a worldwide public health problem for which the development of effective vaccines remains a challenge. A vaccine against leishmaniases must be safe and affordable and should induce cross-protection against the different disease-causing species. In this context, the DNA vaccine pHisAK70 has been demonstrated to induce, in a murine model, a resistant phenotype against L. major, L. infantum, and L. amazonensis. Moreover, a chimeric multiepitope peptide, HisDTC, has been obtained by in silico analysis from the histone proteins encoded in the DNA vaccine and has showed its ability to activate a potent CD4+ and CD8+ T-cell protective immune response in mice against L. infantum infection. In the present study, we evaluated the plasmid DNA vaccine pHisAK70 in comparison with the peptide HisDTC (with and without saponin) against L. major and L. infantum infection. Our preliminary results showed that both formulations were able to induce a potent cellular response leading to a decrease in parasite load against L. infantum. In addition, the DNA candidate was able to induce better lesion control in mice against L. major. These preliminary results indicate that both strategies are potentially effective candidates for leishmaniases control. Furthermore, it is important to carry out such comparative studies to elucidate which vaccine candidates are the most appropriate for further development.

Particulate and non-particle adjuvants in Leishmaniasis vaccine designs: A review.

Leishmaniasis is a parasitic disease with different clinical forms caused by protozoan parasites of the genus Leishmania and transmitted by the bite of an infected female sandfly. According to the World Health Organization (WHO), it is the second most common parasitic disease after malaria and it is known that approximately 350 million people are at risk. The disease manifests itself in different clinical forms. In addition to asymptomatic cases, cutaneous leishmaniasis (CL), which creates large lesions on the skin, and visceral leishmaniasis (VL), which causes death if not treated, especially affecting the abdominal organs, are two important clinical forms. When the studies were examined, it was seen that a clinically used vaccine against any form of human leishmaniasis has not been developed yet. In some studies, it was stated that the lack of appropriate adjuvant was responsible for the failure to develop an effective Leishmania vaccine. We can say that strong adjuvants are needed to achieve successful vaccines. In this article, adjuvants and adjuvant candidates used in vaccine studies against leishmaniasis are discussed.

Deletion of MIF gene from live attenuated LdCen-/- parasites enhances protective CD4+ T cell immunity.

Vaccination with live attenuated Leishmania parasites such as centrin deleted Leishmania donovani (LdCen-/-) against visceral leishmaniasis has been reported extensively. The protection induced by LdCen-/- parasites was mediated by both CD4+ and CD8+ T cells. While the host immune mediators of protection are known, parasite determinants that affect the CD4+ and CD8+ T cell populations remain unknown. Parasite encoded inflammatory cytokine MIF has been shown to modulate the T cell differentiation characteristics by altering the inflammation induced apoptosis during contraction phase in experimental infections with Leishmania or Plasmodium. Neutralization of parasite encoded MIF either by antibodies or gene deletion conferred protection in Plasmodium and Leishmania studies. We investigated if the immunogenicity and protection induced by LdCen-/- parasites is affected by deleting MIF genes from this vaccine strain. Our results showed that LdCen-/-MIF-/- immunized group presented higher percentage of CD4+ and CD8+ central memory T cells, increased CD8+ T cell proliferation after challenge compared to LdCen-/- immunization. LdCen-/-MIF-/- immunized group presented elevated production of IFN-γ+ and TNF-α+ CD4+ T cells concomitant with a reduced parasite load in spleen and liver compared to LdCen-/-group following challenge with L. infantum. Our results demonstrate the role of parasite induced factors involved in protection and long-term immunity of vaccines against VL.

Cloning, expression and in vitro validation of chimeric multi epitope vaccine candidate against visceral leishmaniasis infection.

Visceral Leishmaniasis or Kala-Azar is one of the most severe and deadly neglected tropical disease caused by the Leishmania parasite. A few number of vaccines are going through different phases in clinical trial but failing of these vaccines in successive phase trial or less efficacy, urge to develop highly immunogenic and cost-effective treatment to get rid of deadly VL. This study focuses on the development of more potent vaccine candidate against VL. The recombinant vaccine candidate LeiSp was expressed in Pichia pastoris, followed by purification and characterization. The purified protein was also tested for any post-translation modification, which favors being a potent immunogenic candidate. Further, the expression modulation of different pro-inflammatory and anti-inflammatory cytokines was evaluated in THP1 cell lines. A significant upregulation in the expression of pro-inflammatory cytokines while no significant changes were observed in the expression of anti-inflammatory cytokines. The impact of recombinant vaccine protein candidates in infected conditions were determined. Here, upon treatment with chimeric vaccine protein candidate, we observed a considerable recovery in the expression level of pro-inflammatory cytokines, which were downregulated upon infection alone. In addition to this, we found a significant decrease in the expression of anti-inflammatory cytokines, which were upregulated during infection alone. We further validated our findings in infected hPBMCs and observed similar expression modulation of pro-inflammatory and anti-inflammatory cytokines with and without treatment. Thus, the present study indicates that the chimeric LeiSp protein which was designed using bioinformatics approaches shows a potential inductive efficacy for pro-inflammatory cytokines in Leishmania-infected cells.

Live attenuated Leishmania infantum centrin deleted mutant (LiCen-/-) as a novel vaccine candidate: A field study on safety, immunogenicity, and efficacy against canine leishmaniasis.

This study was designed to evaluate the safety, immunogenicity, and efficacy of a single dose of L. infantum (LiCen-/-) live attenuated candidate vaccine against canine leishmaniasis (CanL). Eighteen healthy domestic dogs with no anti-Leishmania antibodies and negative leishmanin skin test (LST) were randomly inoculated intravenously with either L. infantum (LiCen-/-) vaccine candidate in 10 dogs or phosphate-buffered saline (PBS) in 8 dogs. The safety, immunogenicity, and efficacy rate of L. infantum (LiCen-/-) vaccine candidate against CanL were evaluated by different criteria, including clinical manifestations, injection-site lesion, hematology and biochemistry values, anti-Leishmania antibodies using direct agglutination test (DAT), delayed-type hypersensitivity (DTH) using LST, and CD4+ and CD8+ T-cells subsets, as well as by measuring interferon (IFN-γ), interleukin (IL-23), IL-17, and IL-10 cytokines. Spleen aspiration and detection of Leishmania parasite using parasitological examinations (microscopy and culture) were performed in both vaccinated and control groups. Two months after intervention, each dog was challenged intraperitoneally (IP) with wide type (WT) L. infantum. Two-month follow-up post vaccination showed no clinical signs and serious side effects associated with the vaccination. A significant increase was found in the expression of IL-17, CD4+, and CD8+ gene transcripts in PBMCs, as well as increased levels of Th1 cytokines, and reduction of Th2 cytokine. The efficacy of the vaccine candidate was calculated to be 42.85%. While the time window for assessing the vaccine's effectiveness was too limited to draw any real conclusions but the preliminary results showed a moderate efficacy rate due to inoculation a single dose of L. infantum (LiCen-/-) vaccine candidate. Further investigations with more sample sizes and multiple doses of the vaccine candidate using natural challenges in the endemic areas of CanL are recommended.

Evaluation of immunomodulatory potential of recombinant histidyl-tRNA synthetase (rLdHisRS) protein of Leishmania donovani as a vaccine candidate against visceral leishmaniasis.

Visceral leishmaniasis is neglected tropical protozoan disease caused by Leishmania donovani and are associated with high fatality rate in developing countries since prophylactic vaccines are not available. In the present study, we evaluated the immunomodulatory potential of L. donovani histidyl-tRNA synthetase (LdHisRS) and predicted the epitopes using immunoinformatic tools. Histidyl-tRNA synthetase (HisRS) is a class IIa aminoacyl t-RNA synthetase enzyme (aaRS) required for histidine incorporation into proteins during protein synthesis. The recombinant LdHisRS protein (rLdHisRS) was expressed in E coli BL-21cells, and its immunomodulatory role was assessed in J774A.1 murine macrophage and in BALB/c mice, respectively. LdHisRS specifically stimulated and triggered enhance cell proliferation, nitric oxide release and IFN-γ (70%; P < 0.001), and IL-12 (55.37%; P < 0.05) cytokine release in vitro, whereas BALB/c mice immunized with rLdHisRS show higher NO release (80.95%; P<0.001), higher levels of Th1 cytokines IFN-γ (14%; P < 0.05), TNF-α (34.93%; P < 0.001), and IL-12 (28.49%; P < 0.001) and robust IgG (p<0.001) and IgG2a (p<0.001) production. We also identified 20 Helper T-lymphocytes (HTLs), 30 cytotoxic T lymphocytes (CTLs), and 18 B-cell epitopes from HisRS protein of L. donovani. All these epitopes can be further used to make a multiepitope vaccine against L. donovani.

Recombinant endonuclease III protein from Leishmania infantum associated with Th1-type adjuvants is immunogenic and induces protection against visceral leishmaniasis.

Vaccination against visceral leishmaniasis (VL) should be considered as a safe and effective measure to disease control; however, few vaccines are available against canine VL and there is no an approved human vaccine. In this context, in the present study, we evaluated the endonuclease III (ENDO) protein, which was recently showed to be antigenic for human disease, as a vaccine candidate against Leishmania infantum infection. The recombinant protein (rENDO) was administered in BALB/c mice alone or associated with saponin (rENDO/Sap) or micelles (rENDO/Mic) as adjuvants. Controls received saline, saponin or empty micelles. Results showed that both rENDO/Sap and rENDO/Mic compositions induced higher levels of IFN-γ, IL-12, TNF-α, and GM-CSF cytokines, besides nitrite and IgG2a isotype antibodies, before and after challenge infection, which were related to both CD4+ and CD8+ T cell subtypes. The immunological results contributed to significant reductions in the parasite load found in the spleens, livers, bone marrows and draining lymph nodes of the vaccinated animals. In general, mice immunized with rENDO/Mic presented a slightly higher Th1-type cellular and humoral immune response, as compared to those receiving rENDO/Sap. In addition, saponin caused a slight to moderate inflammatory edema in their vaccinated footpads, which was not observed when micelles were used with rENDO. In addition, a preliminary analysis showed that the recombinant protein was immunogenic to human cells cultures, since PBMCs from treated VL patients and healthy subjects showed higher lymphoproliferation and IFN-γ production in the culture supernatants. In conclusion, data suggest that rENDO could be considered as a candidate to be evaluated in future studies as vaccine to protect against VL.

Immunogenicity and protective efficacy of tuzin protein as a vaccine candidate in Leishmania donovani-infected BALB/c mice.

Visceral leishmaniasis (VL) is referred to as the most severe and fatal type of leishmaniasis basically caused by Leishmania donovani and L. infantum. The most effective method for preventing the spread of the disease is vaccination. Till today, there is no promising licensed vaccination for human VL. Hence, investigation for vaccines is necessary to enrich the therapeutic repertoire against leishmaniasis. Tuzin is a rare trans-membrane protein that has been reported in Trypanosoma cruzi with unknown function. However, tuzin is not characterized in Leishmania parasites. In this study, we for the first time demonstrated that tuzin protein was expressed in both stages (promastigote and amastigote) of L. donovani parasites. In-silico studies revealed that tuzin has potent antigenic properties. Therefore, we analyzed the immunogenicity of tuzin protein and immune response in BALB/c mice challenged with the L. donovani parasite. We observed that tuzin-vaccinated mice have significantly reduced parasite burden in the spleen and liver compared with the control. The number of granulomas in the liver was also significantly decreased compared with the control groups. We further measured the IgG2a antibody level, a marker of Th1 immune response in VL, which was significantly higher in the serum of immunized mice when compared with the control. Splenocytes stimulated with soluble Leishmania antigen (SLA) displayed a significant increase in NO and ROS levels compared with the control groups. Tuzin-immunized and parasite-challenged mice exhibit a notable rise in the IFN-γ/IL-10 ratio by significantly suppressing IL-10 expression level, an immunosuppressive cytokine that inhibits leishmanicidal immune function and encourages disease progression. In conclusion, tuzin immunizations substantially increase the protective immune response in L. donovani-challenged mice groups compared with control.