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.

A specific Leishmania infantum polyepitope vaccine triggers Th1-type immune response and protects against experimental visceral leishmaniasis.

The development of an immunogenic, effective, and safe vaccine is essential as an alternative for disease control. The present study aimed to evaluate the immunogenicity and efficacy potential of a polyepitope T-cell antigen candidate against visceral leishmaniasis in a murine model. BALB/c mice were immunized with three doses subcutaneously with Poly-T Leish alone or adjuvanted with Saponin plus Monophosphoryl lipid A, with 15-day intervals between doses, and challenged with 107 stationary-phase Leishmania infantum promastigotes via tail vein. Immunogenicity and parasitism in spleen and liver of immunized mice were evaluated 45 days post-challenge. Our results revealed that the immunization with Poly-T Leish and Poly-T Leish/SM increases the percentage of specific T (CD4+ and CD8+) lymphocytes proliferation in vitro after antigen-specific stimulation. Also, Poly-T Leish and Poly-T Leish/SM groups showed a high percentage of IFN-γ and TNF-α-producing T cells, meanwhile, the Poly-T Leish/SM group also showed an increased percentage of multifunctional T cells producing double and triple-positive (IFN-γ+TNF-α+IL-2+) cytokines. The immunization with Poly-T Leish or Poly-T Leish/SM stimulated a decreased IL-4 and IL-10 compared to the Saline and adjuvant group. Poly-T Leish/SM immunized mice exhibit a noteworthy reduction in the parasite burden (spleen and liver) through real-time PCR (96%). Moreover, we observed higher nitrite secretion in 120-hour stimulated-culture supernatant using Griess method. We demonstrated that the Poly-T Leish/SM candidate was potentially immunogenic, providing enhancement of protective immune mechanisms, and conferred protection reducing parasitism. Our candidate was considered potential against visceral leishmaniasis, and eventually, could be tested in phase I and II clinical trials in dogs.

Immunoprophylaxis using polypeptide chimera vaccines plus adjuvant system promote Th1 response controlling the spleen parasitism in hamster model of visceral leishmaniasis.

In recent years, several advances have been observed in vaccinology especially for neglected tropical diseases (NTDs). One of the tools employed is epitope prediction by immunoinformatic approaches that reduce the time and cost to develop a vaccine. In this scenario, immunoinformatics is being more often used to develop vaccines for NTDs, in particular visceral leishmaniasis (VL) which is proven not to have an effective vaccine yet. Based on that, in a previous study, two predicted T-cell multi-epitope chimera vaccines were experimentally validated in BALB/c mice to evaluate the immunogenicity, central and effector memory and protection against VL. Considering the results obtained in the mouse model, we assessed the immune response of these chimeras inMesocricetus auratushamster, which displays, experimentally, similar pathological status to human and dog VL disease. Our findings indicate that both chimeras lead to a dominant Th1 response profile, inducing a strong cellular response by increasing the production of IFN-γ and TNF-α cytokines associated with a decrease in IL-10. Also, the chimeras reduced the spleen parasite load and the weight a correlation between protector immunological mechanisms and consistent reduction of the parasitic load was observed. Our results demonstrate that both chimeras were immunogenic and corroborate with findings in the mouse model. Therefore, we reinforce the use of the hamster as a pre-clinical model in vaccination trials for canine and human VL and the importance of immunoinformatic to identify epitopes to design vaccines for this important neglected disease.

Screening Novel Vaccine Candidates for Leishmania Donovani by Combining Differential Proteomics and Immunoinformatics Analysis.

Visceral leishmaniasis (VL), also known as kala-azar, is the most dangerous form of leishmaniasis. Currently no effective vaccine is available for clinical use. Since the pathogenicity of different Leishmania strains is inconsistent, the differentially expressed proteins in Leishmania strains may play an important role as virulence factors in pathogenesis. Therefore, effective vaccine candidate targets may exist in the differentially expressed proteins. In this study, we used differential proteomics analysis to find the differentially expressed proteins in two Leishmania donovani strains, and combined with immunoinformatics analysis to find new vaccine candidates. The differentially expressed proteins from L. DD8 (low virulent) and L. 9044 (virulent) strains were analyzed by LC-MS/MS, and preliminarily screened by antigenicity, allergenicity and homology evaluation. The binding peptides of MHC II, IFN-γ and MHC I from differentially expressed proteins were then predicted and calculated for the second screening. IFN-γ/IL-10 ratios and conserved domain prediction were performed to choose more desirable differentially expressed proteins. Finally, the 3D structures of three vaccine candidate proteins were produced and submitted for molecular dynamics simulation and molecular docking interaction with TLR4/MD2. The results showed that 396 differentially expressed proteins were identified by LC-MS/MS, and 155 differentially expressed proteins were selected through antigenicity, allergenicity and homology evaluation. Finally, 16 proteins whose percentages of MHC II, IFN-γ and MHC I binding peptides were greater than those of control groups (TSA, LmSTI1, LeIF, Leish-111f) were considered to be suitable vaccine candidates. Among the 16 candidates, amino acid permease, amastin-like protein and the hypothetical protein (XP_003865405.1) simultaneously had the large ratios of IFN-γ/IL-10 and high percentages of MHC II, IFN-γ and MHC I, which should be focused on. In conclusion, our comprehensive work provided a methodological basis to screen new vaccine candidates for a better intervention against VL and associated diseases.

Leishmania tarentolae as Potential Live Vaccine Co-Expressing Distinct Salivary Gland Proteins Against Experimental Cutaneous Leishmaniasis in BALB/c Mice Model.

Leishmaniasis is a neglected vector-borne disease caused by Leishmania parasites transmitted through the infected sand flies bite. Current treatments are limited, partly due to their high cost and significant adverse effects, and no human vaccine is yet available. Sand flies saliva has been examined for their potential application as an anti-Leishmania vaccine. The salivary protein, PpSP15, was the first protective vaccine candidate against L. major. Additionally, PsSP9 was already introduced as a highly immunogenic salivary protein against L. tropica. Herein, we aimed to develop an effective multivalent live vaccine to control Cutaneous Leishmaniasis induced by two main species, L. major and L. tropica. Hence, the two above-mentioned salivary proteins using T2A linker were incorporated inside the L. tarentolae genome as a safe live vector. Then, the immunogenicity and protective effects of recombinant L. tarentolae co-expressing PpSP15 and PsSP9 were evaluated in pre-treated BALB/c mice with CpG against L. major and L. tropica. Following the cytokine assays, parasite burden and antibody assessment at different time-points at pre and post-infection, promising protective Th1 immunity was obtained in vaccinated mice with recombinant L. tarentolae co-expressing PpSP15 and PsSP9. This is the first study demonstrating the potency of a safe live vaccine based on the combination of different salivary proteins against the infectious challenge with two different species of Leishmania.

Estimating the global demand curve for a leishmaniasis vaccine: A generalisable approach based on global burden of disease estimates.

BACKGROUND: A pressing need exists to develop vaccines for neglected diseases, including leishmaniasis. However, the development of new vaccines is dependent on their value to two key players-vaccine developers and manufacturers who need to have confidence in the global demand in order to commit to research and production; and governments (or other international funders) who need to signal demand based on the potential public health benefits of the vaccine in their local context, as well as its affordability. A detailed global epidemiological analysis is rarely available before a vaccine enters a market due to lack of resources as well as insufficient global data necessary for such an analysis. Our study seeks to bridge this information gap by providing a generalisable approach to estimating the commercial and public health value of a vaccine in development relying primarily on publicly available Global Burden of Disease (GBD) data. This simplified approach is easily replicable and can be used to guide discussions and investments into vaccines and other health technologies where evidence constraints exist. The approach is demonstrated through the estimation of the demand curve for a future leishmaniasis vaccine. METHODOLOGY/PRINCIPAL FINDINGS: We project the ability to pay over the period 2030-2040 for a vaccine preventing cutaneous and visceral leishmaniasis (CL / VL), using an illustrative set of countries which account for most of the global disease burden. First, based on previous work on vaccine demand projections in these countries and CL / VL GBD-reported incidence rates, we project the potential long-term impact of the vaccine on disability-adjusted life years (DALYs) averted as a result of reduced incidence. Then, we apply an economic framework to our estimates to determine vaccine affordability based on the abilities to pay of governments and global funders, leading to estimates of the demand and market size. Based on our estimates, the maximum ability-to-pay of a leishmaniasis vaccine (per course, including delivery costs), given the current estimates of incidence and population at risk, is higher than $5 for 25-30% of the countries considered, with the average value-based maximum price, weighted by quantity demanded, being $5.7-6 [$0.3 - $34.5], and total demand of over 560 million courses. CONCLUSION/SIGNIFICANCE: Our results demonstrate that both the quantity of vaccines estimated to be required by the countries considered as well as their ability-to-pay could make a vaccine for leishmaniasis commercially attractive to potential manufacturers. The methodology used can be equally applied to other technology developments targeting health in developing countries.

Development of an immunogen containing CD4+/CD8+ T-cell epitopes for the prophylaxis of tegumentary leishmaniasis.

Tegumentary leishmaniasis (TL) is a disease of high severity and incidence in Brazil, and Leishmania braziliensis is its main etiological agent. The inefficiency of control measures, such as high toxicity and costs of current treatments and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present work developed a gene encoding multiple T-cell (CD4+/CD8+) epitope, derived from conserved proteins found in Leishmania species and associated with TL, to generate a chimeric protein (rMEP/TL) and compose a vaccine formulation. For this, six T-cell epitopes were selected by immunoinformatics approaches from proteins present in the amastigote stage and associated with host-parasite interactions. The following formulations were then tested in an L. braziliensis murine infection model: rMEP/TL in saline or associated with MPLA-PHAD®. Our data revealed that, after immunization (three doses; 14-day intervals) and subsequent challenging, rMEP/TL and rMEP/TL + MPLA-vaccinated mice showed an increased production of key immunological biomarkers of protection, such as IgG2a, IgG2a/IgG1, NO, CD4+, and CD8+ T-cells with IFN-γ and TNF-α production, associated with a reduction in CD4+IL-10+ and CD8+IL-10+ T-cells. Vaccines also induced the development of central (CD44highCD62Lhigh) and effector (CD44highCD62Llow) memory of CD4+ and CD8+ T-cells. These findings, associated with the observation of lower rates of parasite burdens in the vaccinated groups, when compared to the control groups, suggest that immunization with rMEP/TL and, preferably, associated with an adjuvant, may be considered an effective tool to prevent TL. KEY POINTS: • Rational design approaches for vaccine development. • Central and effector memory of CD4+ and CD8+ T-cells. • Vaccine comprised of rMEP/TL plus MPLA as an effective tool to prevent TL.