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.

Immunological characterization of rLdTCP1γ for its prophylactic potential against visceral leishmaniasis in hamster model.

Visceral leishmaniasis (VL) is a chronic tropical disease responsible for devastating epidemics worldwide. Though current treatment relies on drugs, the emergence of resistance, toxic side-effects, and strenuous administration has led to an ineffective remedy. Hence, vaccination remains an alternative and desirable approach for VL control. Though extensive research on anti-leishmanial vaccine candidates has been carried out in past decades, presence of an effective molecule is still missing. In the present study, we have evaluated the immunogenicity and prophylactic potential of a recombinant T-complex protein-1 gamma subunit of L. donovani (rLdTCP1γ), against VL in hamster model. The antigen exhibited in vitro stimulation of lymphoproliferative and NO response in miltefosine and amphotericin B treated hamsters depicting its immunotherapeutic/immunogenic nature. Immunization with rLdTCP1γ revealed a strong protective response against experimental VL as indicated by reduced parasite load in the spleen of immunized group compared to infected control. The immunized animals gained body weight and exhibited significant reduction in the spleen and liver weight as compared to infected controls on days 60, 90, 120 post-challenge. A substantial augmentation of cell-mediated immune response as depicted by an increased lymphocyte proliferation, nitric oxide production, DTH responses and increased levels of IgG2 was observed in rLdTCP1γ immunized hamsters. The Th1 stimulatory potential, imparted by the antigen, was found to be intensified in the presence of adjuvant Bacillus Calmette-Guérin (BCG). The efficacy was further assisted by an upregulated mRNA transcript of Th1 induced cytokines (IL-12, IFN-γ and TNFα) and downregulation of IL-4 and IL-10. The results are thus suggestive of rLdTCP1γ having the potential of a strong vaccine candidate against VL.

Determinants of Innate Immunity in Visceral Leishmaniasis and Their Implication in Vaccine Development.

Leishmaniasis is endemic to the tropical and subtropical regions of the world and is transmitted by the bite of an infected sand fly. The multifaceted interactions between Leishmania, the host innate immune cells, and the adaptive immunity determine the severity of pathogenesis and disease development. Leishmania parasites establish a chronic infection by subversion and attenuation of the microbicidal functions of phagocytic innate immune cells such as neutrophils, macrophages and dendritic cells (DCs). Other innate cells such as inflammatory monocytes, mast cells and NK cells, also contribute to resistance and/or susceptibility to Leishmania infection. In addition to the cytokine/chemokine signals from the innate immune cells, recent studies identified the subtle shifts in the metabolic pathways of the innate cells that activate distinct immune signal cascades. The nexus between metabolic pathways, epigenetic reprogramming and the immune signaling cascades that drive the divergent innate immune responses, remains to be fully understood in Leishmania pathogenesis. Further, development of safe and efficacious vaccines against Leishmaniasis requires a broader understanding of the early interactions between the parasites and innate immune cells. In this review we focus on the current understanding of the specific role of innate immune cells, the metabolomic and epigenetic reprogramming and immune regulation that occurs during visceral leishmaniasis, and the strategies used by the parasite to evade and modulate host immunity. We highlight how such pathways could be exploited in the development of safe and efficacious Leishmania vaccines.

FML/QuilA-Vaccinated Dogs Naturally Infected with Leishmania infantum: Serum Cytokines, Clinicopathological Profile, and Parasitological Parameters.

Dogs are the main reservoir of Leishmania infantum in endemic regions. Canine leishmaniasis, caused by L. infantum, can progress to a chronic disease resulting in death. Vaccines have been developed with a certain degree of success. The pathogenesis of this disease is not completely understood, especially in previously vaccinated dogs. We herein described clinical data, parasite load, serum levels of cytokines, and the reservoir potential in vdogs vaccinated with the fucose-mannose ligand (FML)/QuilA saponin vaccine (Leishmune™) naturally infected (Vi) and compared to vaccinated not infected dogs (Vn). Thirty-four dogs from private owners were divided into two groups: vaccinated/infected and vaccinated/uninfected. Clinical evaluation, hematological and biochemical parameters, and serum levels of cytokines were measured by conventional methods. The parasite burden in the bone marrow was measured by quantitative real-time PCR, and the transmissibility of parasites to sand flies was assessed by xenodiagnosis. Clinical, biochemical, and hematological parameters of vaccinated infected dogs were mostly normal. Vi dogs developed mild disease with low clinical scores. Serum levels of IL-10 were higher in Vi dogs, and a strong correlation was observed in IL-4 levels and the A/G ratio in Vi dogs. These results suggest a role of TH2 response in Vi dogs, although more data is needed to better understand the disease in vaccinated dogs.

Human leishmaniasis vaccines: Use cases, target population and potential global demand.

The development of vaccines against one or all forms of human leishmaniasis remains hampered by a paucity of investment, at least in part resulting from the lack of well-evidenced and agreed estimates of vaccine demand. Starting from the definition of 4 main use cases (prevention of visceral leishmaniasis, prevention of cutaneous leishmaniasis, prevention of post-kala-azar dermal leishmaniasis and treatment of post-kala-azar dermal leishmaniasis), we have estimated the size of each target population, focusing on those endemic countries where incidence levels are sufficiently high to justify decisions to adopt a vaccine. We assumed a dual vaccine delivery strategy, including a wide age-range catch-up campaign before the start of routine immunisation. Vaccine characteristics and delivery parameters reflective of a target product profile and the likely duration of the clinical development effort were considered in forecasting the demand for each of the four indications. Over a period of 10 years, this demand is forecasted to range from 300-830 million doses for a vaccine preventing visceral leishmaniasis and 557-1400 million doses for a vaccine preventing cutaneous leishmaniasis under the different scenarios we simulated. In a scenario with an effective prophylactic visceral leishmaniasis vaccine, demand for use to prevent or treat post-kala-azar dermal leishmaniasis would be more limited (over the 10 years ~160,000 doses for prevention and ~7,000 doses for treatment). Demand would rise to exceed 330,000 doses, however, in the absence of an effective vaccine for visceral leishmaniasis. Because of the sizeable demand and potential for public health impact, a single-indication prophylactic vaccine for visceral or cutaneous leishmaniasis, and even more so a cross-protective prophylactic vaccine could attract the interest of commercial developers. Continuous refinement of these first-of-their kind estimates and confirmation of country willingness and ability to pay will be paramount to inform the decisions of policy makers and developers in relation to a leishmaniasis vaccine. Positive decisions can provide a much-needed contribution towards the achievement of global leishmaniasis control.

Intranasal vaccine from whole Leishmania donovani antigens provides protection and induces specific immune response against visceral leishmaniasis.

Visceral leishmaniasis is a protozoan disease associated with high fatality rate in developing countries. Although the drug pipeline is constantly improving, available treatments are costly and live-threatening side effects are not uncommon. Moreover, an approved vaccine against human leishmaniasis does not exist yet. Using whole antigens from Leishmania donovani promastigotes (LdAg), we investigated the protective potential of a novel adjuvant-free vaccine strategy. Immunization of mice with LdAg via the intradermal or the intranasal route prior to infection decreases the parasitic burden in primary affected internal organs, including the liver, spleen, and bone marrow. Interestingly, the intranasal route is more efficient than the intradermal route, leading to better parasite clearance and remarkable induction of adaptive immune cells, notably the helper and cytotoxic T cells. In vitro restimulation experiments with Leishmania antigens led to significant IFN-γ secretion by splenocytes; therefore, exemplifying specificity of the adaptive immune response. To improve mucosal delivery and the immunogenic aspects of our vaccine strategy, we used polysaccharide-based nanoparticles (NP) that carry the antigens. The NP-LdAg formulation is remarkably taken up by dendritic cells and induces their maturation in vitro, as revealed by the increased expression of CD80, CD86 and MHC II. Intranasal immunization with NP-LdAg does not improve the parasite clearance in our experimental timeline; however, it does increase the percentage of effector and memory T helper cells in the spleen, suggesting a potential induction of long-term memory. Altogether, this study provides a simple and cost-effective vaccine strategy against visceral leishmaniasis based on LdAg administration via the intranasal route, which could be applicable to other parasitic diseases.

Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis.

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen-/-) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen-/- parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen-/- immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen-/- infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen-/- immunized hamsters. This study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial.

Adjuvant effects of TLR agonist gardiquimod admixed with Leishmania vaccine in mice model of visceral leishmaniasis.

Tropical and subtropical areas of the world are affected by leishmaniasis, which is caused by Leishmania spp. It has been categorized as an NTD (neglected tropical disease) because of its negligence. The sand fly of genus Phlebotomus acts as the vector for the transmission of the promastigote form of this protozoan parasite to the mammalian host where it converts to amastigote form in the macrophages. Visceral form of leishmaniasis (VL) is a deadly infection in the endothelial system of the human and other mammals. Only a few chemotherapeutic agents are available for the treatment of this infectious disease whereas no vaccine is available for the control of leishmanial infection. Therefore in the current study, we have tested the effects of gardiquimod (a TLR agonist) as an adjuvant in combination with the formalin-killed antigen of L. donovani as a vaccine. The mice were vaccinated thrice at an interval of 2 weeks and challenged with L. donovani promastigotes after 2 weeks of the last vaccination. We assessed the parasite load, delayed-type hypersensitivity (DTH) responses, humoral and cell-mediated immune response in BALB/c mice before and after challenge infection with L. donovani. Immunized mice were found to have the least parasite load, high DTH response, elevated levels of Th1 cytokines, IgG2a, and nitric oxide than non-immunized and infected control mice. The efficacy of the vaccine was boosted with the use of adjuvant gardiquimod that depicts its potential as an adjuvant in this study. Our study is reporting the adjuvant effects of gardiquimod for the first time. Further studies using other Leishmania species can be performed to signify its role.

Effect of Prophylactic Vaccination with the Membrane-Bound Acid Phosphatase Gene of Leishmania mexicana in the Murine Model of Localized Cutaneous Leishmaniasis.

Leishmaniasis is a disease caused by an intracellular protozoan parasite of the genus Leishmania. Current treatments for leishmaniasis are long, toxic, and expensive and are not available in some endemic regions. Attempts to develop an effective vaccine are feasible, but no vaccine is in active clinical use. In this study, the LmxMBA gene of Leishmania mexicana was selected as a possible vaccine candidate using the reverse vaccinology approach, and the prophylactic effect generated by DNA vaccination with this gene in a murine model of cutaneous leishmaniasis was evaluated. The results showed that prophylactic vaccination with pVAX1::LmxMBA significantly reduced the size of the lesion and the parasitic load on the footpad, compared to the control groups. At a histological level, a smaller number of parasites were evident in the dermis, as well as the absence of connective tissue damage. Mice immunized with plasmid pVAX1::LmxMBA induced immunity characterized by an increase in the IgG2a/IgG1 > 1 ratio and a higher rate of lymphocyte proliferation. In this study, immunization with the plasmid promoted an improvement in the macroscopic and microscopic clinical manifestations of the experimental infection by L. mexicana, with a T helper 1 response characterized by an IgG2a/IgG1 > 1 ratio and high lymphoproliferative response. These findings support immunization with the plasmid pVAX1::LmxMBA as a preventive strategy against cutaneous infection of L. mexicana.

Extracellular vesicles and leishmaniasis: Current knowledge and promising avenues for future development.

Extracellular vesicles (EVs) are small, membrane-bound "delivery trucks" that are present in the extracellular environment, including biological fluids. EVs are capable of inducing changes in the physiological status of neighboring cells through the transfer of key macromolecules, and are thought to play a role in a number of pathological processes. Leishmaniasis, caused by the protozoan parasite Leishmania, is an important example. The biology of Leishmania EVs has been studied in detail, and findings point to their role in exacerbation of disease and potential involvement in the perpetuation of drug resistance. Furthermore, the use of EVs for development of vaccines has been explored, as well as their potential use in a number of fields as biomarkers of disease and drug resistance. Here we discuss the latest findings on EVs, with a particular focus on Leishmania, as well as potential avenues for their future development and clinical applications.

Development of dominant epitope-based vaccines encoding Gp63, Kmp-11 and Amastin against visceral leishmaniasis.

The most dangerous form of leishmaniasis is Visceral leishmaniasis (VL). The elimination of VL depends not only on agent treatments but also on effective vaccines against Leishmania parasites. Epitope-based vaccines composed of alternative short antigenic epitopes have the advantages of MHC epitope easy designing, which has broad application prospects. In a previous study, we analyzed Leishmania Gp63, Kmp-11 and Amastin protein sequence in silico, and found that the amino acid fragments of Gp63 (138-360aa), Kmp-11 (1-91aa) and Amastin (1-72aa) were rich in dominant epitopes. In this study, we used the three amino acid fragments as multi-epitope vaccine candidates to construct DNA and protein vaccines. BALB/c mice were vaccinated with the DNA and protein vaccines by DNA prime-protein boost strategy and challenged with Leishmania promastigotes. To evaluate vaccine immunogenicity and immunoprotection, serum specific antibody titers and cytokines were detected using ELISA, splenic CD3+, CD4+ and CD8+ cells were analyzed by flow cytometry, livers were made into pathological sections to observe pathological changes, and splenic parasitic loads were quantified using qPCR. The results showed that the increased specific IgG titers from vaccinated mice supported the vaccine immunogenicity. The increased cytokines (IFN-γ, IL-12 and TNF-α), splenic CD3+, CD4+ and CD8+ T cells and hepatic granulomas, and the decreased splenic parasitic loads (parasite reduction rates of Gp63, Kmp-11 and Amatin groups were 89%, 86% and 79%, respectively) from immunized mice post-infection were suggested the good immunoprotection of the vaccines. Our study demonstrated that vaccines based on the dominant epitopes of Gp63, Kmp-11 and Amastin with DNA prime-protein boost vaccination strategy showed significant immune effects against Leishmania, especially the Gp63 group showed a nearly 90% parasites reduction rate. This study will provide references for visceral leishmaniasis epitope vaccine design and immune strategy selection.

Safety and immunogenicity of ChAd63-KH vaccine in post-kala-azar dermal leishmaniasis patients in Sudan.

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic, stigmatizing skin condition occurring frequently after apparent clinical cure from visceral leishmaniasis. Given an urgent need for new treatments, we conducted a phase IIa safety and immunogenicity trial of ChAd63-KH vaccine in Sudanese patients with persistent PKDL. LEISH2a (ClinicalTrials.gov: NCT02894008) was an open-label three-phase clinical trial involving sixteen adult and eight adolescent patients with persistent PKDL (median duration, 30 months; range, 6-180 months). Patients received a single intramuscular vaccination of 1 × 1010 viral particles (v.p.; adults only) or 7.5 × 1010 v.p. (adults and adolescents), with primary (safety) and secondary (clinical response and immunogenicity) endpoints evaluated over 42-120 days follow-up. AmBisome was provided to patients with significant remaining disease at their last visit. ChAd63-KH vaccine showed minimal adverse reactions in PKDL patients and induced potent innate and cell-mediated immune responses measured by whole-blood transcriptomics and ELISpot. 7/23 patients (30.4%) monitored to study completion showed >90% clinical improvement, and 5/23 (21.7%) showed partial improvement. A logistic regression model applied to blood transcriptomic data identified immune modules predictive of patients with >90% clinical improvement. A randomized controlled trial to determine whether these clinical responses were vaccine-related and whether ChAd63-KH vaccine has clinical utility is underway.

IFN-γ+ CD4+T cell-driven prophylactic potential of recombinant LDBPK_252400 hypothetical protein of Leishmania donovani against visceral leishmaniasis.

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease causing high morbidity and mortality in developing countries. Vaccination is considered the most effective and powerful tool for blocking transmission and control of diseases. However, no vaccine is available so far in the market for humans. In the present study, we characterized the hypothetical protein LDBPK_252400 of Leishmania donovani (LdHyP) and explored its prophylactic behavior as a potential vaccine candidate against VL. We found reduced hepato-splenomegaly along with more than 50% parasite reduction in spleen and liver after vaccination in mice. Protection in vaccinated mice after the antigen challenge correlated with the stimulation of antigen specific IFN-γ expressing CD4+T cell (~4.6 fold) and CD8+T cells (~2.1 fold) in vaccinated mice in compared to infected mice, even after 2-3 months of immunization. Importantly, antigen-mediated humoral immunity correlated with high antigen specific IgG2/IgG1 responses in vaccinated mice. In vitro re-stimulation of splenocytes with LdHyP enhances the expression of TNF-α, IFN-γ, IL-12 and IL-10 cytokines along with lower IL-4 cytokine and IL-10/IFN-γ ratio in vaccinated mice. Importantly, we observed ~3.5 fold high NO production through activated macrophages validates antigen mediated cellular immunity induction, which is critical in controlling infection progression. These findings suggest that immunization with LdHyP mount a very robust immunity (from IL-10 towards TFN-γ mediated responses) against L. donovani infection and could be explored further as a putative vaccine candidate against VL.

Dendritic cell engineered cTXN as new vaccine prospect against L. donovani.

Dendritic cells (DCs), as antigen-presenting cells, can reportedly be infected withLeishmaniaparasites and hence provide a better option to trigger T-cell primary immune responses and immunological memory. We consistently primed DCs during culture with purified recombinant cytosolic tryparedoxin (rcTXN) and then evaluated the vaccine prospect of presentation of rcTXN against VL in BALB/c mice. We reported earlier the immunogenic properties of cTXN antigen derived fromL. donovani when anti-cTXN antibody was detected in the sera of kala-azar patients. It was observed that cTXN antigen, when used as an immunogen with murine DCs acting as a vehicle, was able to induce complete protection against VL in an infected group of immunized mice. This vaccination triggered splenic macrophages to produce more IL-12 and GM-CSF, and restricted IL-10 release to a minimum in an immunized group of infected animals. Concomitant changes in T-cell responses against cTXN antigen were also noticed, which increased the release of protective cytokine-like IFN-γ under the influence of NF-κß in the indicated vaccinated group of animals. All cTXN-DCs-vaccinated BALB/c mice survived during the experimental period of 120 days. The results obtained in our study suggest that DCs primed with cTXN can be used as a vaccine prospect for the control of visceral leishmaniasis.

A scalable and reproducible manufacturing process for Phlebotomus papatasi salivary protein PpSP15, a vaccine candidate for leishmaniasis.

Cutaneous leishmaniasis is a parasitic and neglected tropical disease transmitted by the bites of sandflies. The emergence of cutaneous leishmaniasis in areas of war, conflict, political instability, and climate change has prompted efforts to develop a preventive vaccine. One vaccine candidate antigen is PpSP15, a 15 kDa salivary antigen from the sandfly Phlebotomus papatasi that facilitates the infection of the Leishmania parasite and has been shown to induce parasite-specific cell-mediated immunity. Previously, we developed a fermentation process for producing recombinant PpSP15 in Pichia pastoris and a two-chromatographic-step purification process at 100 mL scale. Here we expand the process design to the 10 L scale and examine its reproducibility by performing three identical process runs, an essential transition step towards technology transfer for pilot manufacture. The process was able to reproducibly recover 81% of PpSP15 recombinant protein with a yield of 0.75 g/L of fermentation supernatant, a purity level of 97% and with low variance among runs. Additionally, a freeze-thaw stability study indicated that the PpSP15 recombinant protein remains stable after undergoing three freeze-thaw cycles, and an accelerated stability study confirmed its stability at 37 °C for at least one month. A research cell bank for the expression of PpSP15 was generated and fully characterized. Collectively, the cell bank and the production process are ready for technology transfer for future cGMP pilot manufacturing.

Th1 concomitant immune response mediated by IFN-γ protects against sand fly delivered Leishmania infection: Implications for vaccine design.

Leishmaniasis is an unresolved global health problem with a high socio-economic impact. Data generated in mouse models has revealed that the Th1 response, with IL-12, IFN-γ, TNF-α, and IL-2 as prominent cytokines, predominantly controls the disease progression. Premised on these findings, all examined vaccine formulations have been aimed at generating a long-lived memory Th1 response. However, all vaccine formulations with the exception of live Leishmania inoculation (leishmanization) have failed to sufficiently protect against sand fly delivered infection. It has been recently unraveled that sand fly dependent factors may compromise pre-existing Th1 memory. Further scrutinizing the immune response after leishmanization has uncovered the prominent role of early (within hours) and robust IFN-γ production (Th1 concomitant immunity) in controlling the sand fly delivered secondary infection. The response is dependent upon parasite persistence and subclinical ongoing primary infection. The immune correlates of concomitant immunity (Resident Memory T cells and Effector T subsets) mitigate the early effects of sand fly delivered infection and help to control the disease. In this review, we have described the early events after sand fly challenge and the role of Th1 concomitant immunity in the protective immune response in leishmanized resistant mouse model, although leishmanization is under debate for human use. Undoubtedly, the lessons we learn from leishmanization must be further implemented in alternative vaccine approaches.

Boosting immunity to treat parasitic infections: Asaia bacteria expressing a protein from Wolbachia determine M1 macrophage activation and killing of Leishmania protozoans.

Leishmaniases are severe vector-borne diseases affecting humans and animals, caused by Leishmania protozoans. Over one billion people and millions of dogs live in endemic areas for leishmaniases and are at risk of infection. Immune polarization plays a major role in determining the outcome of Leishmania infections: hosts displaying M1-polarized macrophages are protected, while those biased on the M2 side acquire a chronic infection that could develop into a deadly disease. The identification of the factors involved in M1 polarization is essential for the design of therapeutic and prophylactic interventions, including vaccines. Infection by the filarial nematode Dirofilaria immitis could be one of the factors that interfere with leishmaniasis in dogs. Indeed, filarial nematodes induce a partial skew of the immune response towards M1, likely caused by their bacterial endosymbionts, Wolbachia. Here we have examined the potential of AsaiaWSP, a bacterium engineered for the expression of the Wolbachia surface protein (WSP), as an inductor of M1 macrophage activation and Leishmania killing. Macrophages stimulated with AsaiaWSP displayed a strong leishmanicidal activity, comparable to that determined by the choice-drug amphotericin B. Additionally, AsaiaWSP determined the expression of markers of classical macrophage activation, including M1 cytokines, ROS and NO, and an increase in phagocytosis activity. Asaia not expressing WSP also induced macrophage activation, although at a lower extent compared to AsaiaWSP. In summary, the results of the present study confirm the immunostimulating properties of WSP highlighting a potential therapeutic efficacy against Leishmania parasites. Furthermore, Asaia was designed as a delivery system for WSP, thus developing a novel type of immunomodulating agent, worthy of being investigated for immuno-prophylaxis and -therapy of leishmaniases and other diseases that could be subverted by M1 macrophage activation.

Essential Role of Neutrophils in the Protective Immune Response Induced by a Live Attenuated Leishmania Vaccine.

No licensed vaccine exists against visceral leishmaniasis (VL), a disease caused by the Leishmania donovani parasite. We have previously reported both macrophages and dendritic cells play important role in the protection induced by a live attenuated centrin gene-deleted L. donovani (LdCen-/- ) parasite vaccine. The role of neutrophils in orchestrating the initial innate response to pathogens is widely recognized. To investigate the early interaction of LdCen-/- with neutrophils, we immunized mice intradermally in the ear pinna with LdCen-/- Compared with LdWT infection, LdCen-/- parasites induced higher recruitment of neutrophils to the ear dermis and ear draining lymph nodes (dLN) as early as 6-18 h after immunization, which were predominantly proinflammatory in nature. Neutrophils from ear dLN of LdCen-/- -immunized mice exhibited heightened expression of costimulatory molecules and attenuated expression of coinhibitory molecules necessary for higher T cell activation. Further phenotypic characterization revealed heterogeneous neutrophil populations containing Nα and Nß subtypes in the ear dLN. Of the two, the parasitized Nα subset from LdCen-/- -immunized mice exhibited much stronger Ag-specific CD4+ T cell proliferation ex vivo. Adoptive transfer of neutrophils bearing LdCen-/- parasites induced an increased Th1 response in naive mice. Importantly, neutrophil depletion significantly abrogated Ag-specific CD4+ T cell proliferation in LdCen-/- -immunized mice and impaired protection against virulent challenge. Conversely, replenishing of neutrophils significantly restored the LdCen-/- -induced host-protective response. These results suggest that neutrophils are indispensable for protective immunity induced by LdCen-/- parasite vaccine.

Engineering a vector-based pan-Leishmania vaccine for humans: proof of principle.

Leishmaniasis is a spectrum of diseases transmitted by sand fly vectors that deposit Leishmania spp. parasites in the host skin during blood feeding. Currently, available treatment options are limited, associated with high toxicity and emerging resistance. Even though a vaccine for human leishmaniasis is considered an achievable goal, to date we still do not have one available, a consequence (amongst other factors) of a lack of pre-clinical to clinical translatability. Pre-exposure to uninfected sand fly bites or immunization with defined sand fly salivary proteins was shown to negatively impact infection. Still, cross-protection reports are rare and dependent on the phylogenetic proximity of the sand fly species, meaning that the applicability of a sand fly saliva-based vaccine will be limited to a defined geography, one parasite species and one form of leishmaniasis. As a proof of principle of a future vector saliva-based pan-Leishmania vaccine, we engineered through a reverse vaccinology approach that maximizes translation to humans, a fusion protein consisting of immunogenic portions of PdSP15 and LJL143, sand fly salivary proteins demonstrated as potential vaccine candidates against cutaneous and visceral leishmaniasis, respectively. The in silico analysis was validated ex vivo, through T cell proliferation experiments, proving that the fusion protein (administered as a DNA vaccine) maintained the immunogenicity of both PdSP15 and LJL143. Additionally, while no significant effect was detected in the context of L. major transmission by P. duboscqi, this DNA vaccine was defined as partially protective, in the context of L. major transmission by L. longipalpis sand flies. Importantly, a high IFNγ response alone was not enough to confer protection, that mainly correlated with low T cell mediated Leishmania-specific IL-4 and IL-10 responses, and consequently with high pro/anti-inflammatory cytokine ratios. Overall our immunogenicity data suggests that to design a potentially safe vector-based pan-Leishmania vaccine, without geographic restrictions and against all forms of leishmaniasis is an achievable goal. This is why we propose our approach as a proof-of principle, perhaps not only applicable to the anti-Leishmania vector-based vaccines' field, but also to other branches of knowledge that require the design of multi-epitope T cell vaccines with a higher potential for translation.

DDX3 DEAD-box RNA helicase (Hel67) gene disruption impairs infectivity of Leishmania donovani and induces protective immunity against visceral leishmaniasis.

Visceral leishmaniasis (VL) is a vector-borne disease caused by the digenetic protozoan parasite Leishmania donovani complex. So far there is no effective vaccine available against VL. The DDX3 DEAD-box RNA Helicase (Hel67) is 67 kDa protein which is quite essential for RNA metabolism, amastigote differentiation, and infectivity in L. major and L. infantum. To investigate the role of Hel67 in the L. donovani, we created L. donovani deficient in the Hel67. Helicase67 null mutants (LdHel67-/-) were not able to differentiate as axenic amastigotes and were unable to infect the hamster. So, we have analyzed the prophylactic efficacy of the LdHel67-/- null mutant in hamsters. The LdHel67-/- null mutant based candidate vaccine exhibited immunogenic response and a higher degree of protection against L. donovani in comparison to the infected control group. Further, the candidate vaccine displayed antigen-specific delayed-type hypersensitivity (DTH) as well as strong antibody response and NO production which strongly correlates to long term protection of candidate vaccine against the infection. This study confirms the potential of LdHel67-/- null mutant as a safe and protective live attenuated vaccine candidate against visceral leishmaniasis.