Immune response profiles from humans experimentally exposed to Phlebotomus duboscqi bites.

Introduction: Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease. Methods: To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. Results: A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Discussion: Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

PpSP32, the Phlebotomus papatasi immunodominant salivary protein, exerts immunomodulatory effects on human monocytes, macrophages, and lymphocytes.

BACKGROUND: The saliva of sand flies, vectors of Leishmania parasites, contains several components that exert pharmacological activity facilitating the acquisition of blood by the insect and contributing to the establishment of infection. Previously, we demonstrated that PpSP32 is the immunodominant salivary antigen in humans exposed to Phlebotomus papatasi bites and validated its usefulness as a predictive biomarker of disease. PpSP32, whose functions are little known to date, is an intriguing protein due to its involvement in the etiopathogenesis of pemphigus, an auto-immune disease. Herein, we aimed to better decipher its role through the screening of several immunomodulatory activity either on lymphocytes or on monocytes/macrophages. METHODS: Peripheral mononuclear cells from healthy volunteers were stimulated with anti-CD3/anti-CD28 antibodies, phytohemagglutinin, phorbol 12-myristate 13-acetate/ionomycin, or lipopolysaccharide in the presence of increasing doses of PpSP32. Cell proliferation was measured after the addition of tritiated thymidine. Monocyte activation was tested by analyzing the expression of CD86 and HLA-DR molecules by flow cytometry. Cytokine production was analyzed in culture supernatants by ELISA. THP-1-derived macrophages were stimulated with LPS in the presence of increasing doses of PpSP32, and cytokine production was analyzed in culture supernatants by ELISA and multiplex technique. The effect of PpSP32 on NF-kB signaling was tested by Western blot. The anti-inflammatory activity of PpSP32 was assessed in vivo in an experimental inflammatory model of carrageenan-induced paw edema in rats. RESULTS: Our data showed that PpSP32 down-modulated the expression of activation markers in LPS-stimulated monocytes and THP1-derived macrophages. This protein negatively modulated the secretion of Th1 and Th2 cytokines by human lymphocytes as well as pro-inflammatory cytokines by monocytes, and THP1-derived macrophages. PpSP32 treatment led to a dose-dependent reduction of IκB phosphorylation. When PpSP32 was injected into the paw of carrageenan-injected rats, edema was significantly reduced. CONCLUSIONS: Our data indicates that PpSP32 induces a potent immunomodulatory effect on monocytes and THP-1-derived macrophages. This inhibition could be mediated, among others, by the modulation of the NF-kB signaling pathway. The anti-inflammatory activity of PpSP32 was confirmed in vivo in the carrageenan-induced paw edema model in rats.

Protective Efficacy in a Hamster Model of a Multivalent Vaccine for Human Visceral Leishmaniasis (MuLeVaClin) Consisting of the KMP11, LEISH-F3+, and LJL143 Antigens in Virosomes, Plus GLA-SE Adjuvant.

Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, fatal if untreated. Vaccination is the most cost-effective approach to disease control; however, to date, no vaccines against human VL have been made available. This work examines the efficacy of a novel vaccine consisting of the Leishmania membrane protein KMP11, LEISH-F3+ (a recombinant fusion protein, composed of epitopes of the parasite proteins nucleoside hydrolase, sterol-24-c-methyltransferase, and cysteine protease B), and the sand fly salivary protein LJL143, in two dose ratios. The inclusion of the TLR4 agonist GLA-SE as an adjuvant, and the use of virosomes (VS) as a delivery system, are also examined. In a hamster model of VL, the vaccine elicited antigen-specific immune responses prior to infection with Leishmania infantum. Of note, the responses were greater when higher doses of KMP11 and LEISH-F3+ proteins were administered along with the GLA-SE adjuvant and/or when delivered within VS. Remarkably, hamsters immunized with the complete combination (i.e., all antigens in VS + GLA-SE) showed significantly lower parasite burdens in the spleen compared to those in control animals. This protection was underpinned by a more intense, specific humoral response against the KMP11, LEISH-F3+, and LJL143 antigens in vaccinated animals, but a significantly less intense antibody response to the pool of soluble Leishmania antigens (SLA). Overall, these results indicate that this innovative vaccine formulation confers protection against L. infantum infection, supporting the advancement of the vaccine formulation into process development and manufacturing and the conduction of toxicity studies towards future phase I human clinical trials.

Sandfly Fever Sicilian Virus-Leishmania major co-infection modulates innate inflammatory response favoring myeloid cell infections and skin hyperinflammation.

BACKGROUND: The leishmaniases are a group of sandfly-transmitted diseases caused by species of the protozoan parasite, Leishmania. With an annual incidence of 1 million cases, 1 billion people living in Leishmania-endemic regions, and nearly 30,000 deaths each year, leishmaniasis is a major global public health concern. While phlebotomine sandflies are well-known as vectors of Leishmania, they are also the vectors of various phleboviruses, including Sandfly Fever Sicilian Virus (SFSV). Cutaneous leishmaniasis (CL), caused by Leishmania major (L. major), among other species, results in development of skin lesions on the infected host. Importantly, there exists much variation in the clinical manifestation between individuals. We propose that phleboviruses, vectored by and found in the same sandfly guts as Leishmania, may be a factor in determining CL severity. It was reported by our group that Leishmania exosomes are released into the gut of the sandfly vector and co-inoculated during blood meals, where they exacerbate CL skin lesions. We hypothesized that, when taking a blood meal, the sandfly vector infects the host with Leishmania parasites and exosomes as well as phleboviruses, and that this viral co-infection results in a modulation of leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: In vitro, we observed modulation by SFSV in MAP kinase signaling as well as in the IRF3 pathway that resulted in a pro-inflammatory phenotype. Additionally, we found that SFSV and L. major co-infection resulted in an exacerbation of leishmaniasis in vivo, and by using endosomal (Toll-like receptor) TLR3, and MAVS knock-out mice, deduced that SFSV's hyperinflammatory effect was TLR3- and MAVS-dependent. Critically, we observed that L. major and SFSV co-infected C57BL/6 mice demonstrated significantly higher parasite burden than mice solely infected with L. major. Furthermore, viral presence increased leukocyte influx in vivo. This influx was accompanied by elevated total extracellular vesicle numbers. Interestingly, L. major displayed higher infectiveness with coincident phleboviral infection compared to L. major infection alone. CONCLUSION/SIGNIFICANCE: Overall our work represents novel findings that contribute towards understanding the causal mechanisms governing cutaneous leishmaniasis pathology. Better comprehension of the potential role of viral co-infection could lead to treatment regimens with enhanced effectiveness.

A second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen-/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization results in protection and an immune response comparable to leishmanization. LmCen-/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

Leishmania: A Maestro in Epigenetic Manipulation of Macrophage Inflammasomes.

Epigenetic manipulation of host cells by intracellular pathogens has become increasingly evident. Lecoeur et al. show us how Leishmania amazonensis inhibits macrophage inflammasomes by modifying histone H3 activation marks on NF-κB-associated gene promoters that increase the expression of inhibitors and downmodulates activators of this pathway.

Phlebotomus papatasi Yellow-Related and Apyrase Salivary Proteins Are Candidates for Vaccination against Human Cutaneous Leishmaniasis.

Nowadays, there is no available vaccine for human leishmaniasis. Animal experiments demonstrate that pre-exposure to sand fly saliva confers protection against leishmaniasis. Our preceding work in humans indicates that Phlebotomus papatasi saliva induces the production of IL-10 by CD8+ T lymphocytes. The neutralization of IL-10 enhanced the activation of a T-cell CD4+ population-producing IFN-γ. Herein, we used a biochemical and functional genomics approach to identify the sand fly salivary components that are responsible for the activation of the T helper type 1 immune response in humans, therefore constituting potential vaccine candidates against leishmaniasis. Fractionated P. papatasi salivary extracts were first tested on T lymphocytes of immune donors. We confirmed that the CD4+ lymphocytes proliferate and produce IFN-γ in response to stimulation with the proteins of molecular weight >30 kDa. Peripheral blood mononuclear cells from immune donors were transfected with plasmids coding for the most abundant proteins from the P. papatasi salivary gland cDNA library. Our result showed that the "yellow related proteins," PPTSP42 and PPTSP44, and "apyrase," PPTSP36, are the proteins responsible for the aforementioned cellular immune response and IFN-γ production. Strikingly, PPTSP44 triggered the highest level of lymphocyte proliferation and IFN-γ production. Multiplex cytokine analysis confirmed the T helper type 1-polarized response induced by these proteins. Importantly, recombinant PPTSP44 validated the results observed with the DNA plasmid, further supporting that PPTSP44 constitutes a promising vaccine candidate against human leishmaniasis.

A defined subunit vaccine that protects against vector-borne visceral leishmaniasis.

Vaccine development for vector-borne pathogens may be accelerated through the use of relevant challenge models, as has been the case for malaria. Because of the demonstrated biological importance of vector-derived molecules in establishing natural infections, incorporating natural challenge models into vaccine development strategies may increase the accuracy of predicting efficacy under field conditions. Until recently, however, there was no natural challenge model available for the evaluation of vaccine candidates against visceral leishmaniasis. We previously demonstrated that a candidate vaccine against visceral leishmaniasis containing the antigen LEISH-F3 could provide protection in preclinical models and induce potent T-cell responses in human volunteers. In the present study, we describe a next generation candidate, LEISH-F3+, generated by adding a third antigen to the LEISH-F3 di-fusion protein. The rationale for adding a third component, derived from cysteine protease (CPB), was based on previously demonstrated protection achieved with this antigen, as well as on recognition by human T cells from individuals with latent infection. Prophylactic immunization with LEISH-F3+formulated with glucopyranosyl lipid A adjuvant in stable emulsion significantly reduced both Leishmania infantum and L. donovani burdens in needle challenge mouse models of infection. Importantly, the data obtained in these infection models were validated by the ability of LEISH-F3+/glucopyranosyl lipid A adjuvant in stable emulsion to induce significant protection in hamsters, a model of both infection and disease, following challenge by L. donovani-infected Lutzomyia longipalpis sand flies, a natural vector. This is an important demonstration of vaccine protection against visceral leishmaniasis using a natural challenge model.

A Listeria monocytogenes-based vaccine that secretes sand fly salivary protein LJM11 confers long-term protection against vector-transmitted Leishmania major.

Cutaneous leishmaniasis is a sand fly-transmitted disease characterized by skin ulcers that carry significant scarring and social stigmatization. Over the past years, there has been cumulative evidence that immunity to specific sand fly salivary proteins confers a significant level of protection against leishmaniasis. In this study, we used an attenuated strain of Listeria monocytogenes as a vaccine expression system for LJM11, a sand fly salivary protein identified as a good vaccine candidate. We observed that mice were best protected against an intradermal needle challenge with Leishmania major and sand fly saliva when vaccinated intravenously. However, this protection was short-lived. Importantly, groups of vaccinated mice were protected long term when challenged with infected sand flies. Protection correlated with smaller lesion size, fewer scars, and better parasite control between 2 and 6 weeks postchallenge compared to the control group of mice vaccinated with the parent L. monocytogenes strain not expressing LJM11. Moreover, protection correlated with high numbers of CD4(+), gamma interferon-positive (IFN-γ(+)), tumor necrosis factor alpha-positive/negative (TNF-α(+/-)), interleukin-10-negative (IL-10(-)) cells and low numbers of CD4(+) IFN-γ(+/-) TNF-α(-) IL-10(+) T cells at 2 weeks postchallenge. Overall, our data indicate that delivery of LJM11 by Listeria is a promising vaccination strategy against cutaneous leishmaniasis inducing long-term protection against ulcer formation following a natural challenge with infected sand flies.

Enhanced protective efficacy of nonpathogenic recombinant leishmania tarentolae expressing cysteine proteinases combined with a sand fly salivary antigen.

BACKGROUND: Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. CONCLUSION/SIGNIFICANCE: The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis.

Genetic polymorphisms of Echinococcus granulosus sensu stricto in the Middle East.

Echinococcus granulosus sensu stricto is a cosmopolitan parasite causing cystic echinococcosis in humans and livestock. Recent molecular phylogeographic studies suggested the rapid dispersal of the parasite by the anthropogenic movement of domestic animal hosts. In the present study, genetic polymorphism of E. granulosus s. s. in the Middle East, where the domestication started, was investigated to validate the dispersal history of the parasite. Thirty-five and 26 hydatid cysts were collected from Iran and Jordan, respectively, and mitochondrial cytochrome c oxidase subunit I (cox1) gene was sequenced. Chinese and Peruvian specimens were also analyzed for comparison. Haplotype network analysis demonstrated the existence of a common haplotype EG01 in all populations. Although EG01 and its one-step neighbors were the majority in all regions, most of the neighboring haplotypes were unique in each locality. Haplotype diversity was high but nucleotide diversity was low in Iran, Jordan and China. Both diversities were lowest and only a few haplotypes were found in Peru. Neutrality indices were significantly negative in Iran, Jordan and China, and positive but not significant in Peru. Pairwise fixation index was significant for all pairwise comparisons, indicating genetic differentiation among populations. These results suggest a evolutionary history of E. granulosus s. s. in which a genetic subgroup including EG01 was selected at the dawn of domestication, and then it was rapidly dispersed worldwide through the diffusion of stock raising. To approach the origin of the ancestral strain, extensive sampling is needed in many endemic regions. To evaluate the hypothetical evolutionary scenario, further study is needed to analyze specimens from diverse host species in wider regions.

Vaccines to combat the neglected tropical diseases.

The neglected tropical diseases (NTDs) represent a group of parasitic and related infectious diseases such as amebiasis, Chagas disease, cysticercosis, echinococcosis, hookworm, leishmaniasis, and schistosomiasis. Together, these conditions are considered the most common infections in low- and middle-income countries, where they produce a level of global disability and human suffering equivalent to better known conditions such as human immunodeficiency virus/acquired immunodeficiency syndrome and malaria. Despite their global public health importance, progress on developing vaccines for NTD pathogens has lagged because of some key technical hurdles and the fact that these infections occur almost exclusively in the world's poorest people living below the World Bank poverty line. In the absence of financial incentives for new products, the multinational pharmaceutical companies have not embarked on substantive research and development programs for the neglected tropical disease vaccines. Here, we review the current status of scientific and technical progress in the development of new neglected tropical disease vaccines, highlighting the successes that have been achieved (cysticercosis and echinococcosis) and identifying the challenges and opportunities for development of new vaccines for NTDs. Also highlighted are the contributions being made by non-profit product development partnerships that are working to overcome some of the economic challenges in vaccine manufacture, clinical testing, and global access.

Characterization of a Leishmania stage-specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulence.

Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27 kDa protein-encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co-immunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene-deleted parasites (Ldp27(-/-)) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27(-/-) parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis.

In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies.

Infection with the obligate intracellular protozoan Leishmania is thought to be initiated by direct parasitization of macrophages, but the early events following transmission to the skin by vector sand flies have been difficult to examine directly. Using dynamic intravital microscopy and flow cytometry, we observed a rapid and sustained neutrophilic infiltrate at localized sand fly bite sites. Invading neutrophils efficiently captured Leishmania major (L.m.) parasites early after sand fly transmission or needle inoculation, but phagocytosed L.m. remained viable and infected neutrophils efficiently initiated infection. Furthermore, neutrophil depletion reduced, rather than enhanced, the ability of parasites to establish productive infections. Thus, L.m. appears to have evolved to both evade and exploit the innate host response to sand fly bite in order to establish and promote disease.

Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis.

BACKGROUND: Immune responses to sandfly saliva have been shown to protect animals against Leishmania infection. Yet very little is known about the molecular characteristics of salivary proteins from different sandflies, particularly from vectors transmitting visceral leishmaniasis, the fatal form of the disease. Further knowledge of the repertoire of these salivary proteins will give us insights into the molecular evolution of these proteins and will help us select relevant antigens for the development of a vector based anti-Leishmania vaccine. RESULTS: Two salivary gland cDNA libraries from female sandflies Phlebotomus argentipes and P. perniciosus were constructed, sequenced and proteomic analysis of the salivary proteins was performed. The majority of the sequenced transcripts from the two cDNA libraries coded for secreted proteins. In this analysis we identified transcripts coding for protein families not previously described in sandflies. A comparative sandfly salivary transcriptome analysis was performed by using these two cDNA libraries and two other sandfly salivary gland cDNA libraries from P. ariasi and Lutzomyia longipalpis, also vectors of visceral leishmaniasis. Full-length secreted proteins from each sandfly library were compared using a stand-alone version of BLAST, creating formatted protein databases of each sandfly library. Related groups of proteins from each sandfly species were combined into defined families of proteins. With this comparison, we identified families of salivary proteins common among all of the sandflies studied, proteins to be genus specific and proteins that appear to be species specific. The common proteins included apyrase, yellow-related protein, antigen-5, PpSP15 and PpSP32-related protein, a 33-kDa protein, D7-related protein, a 39- and a 16.1- kDa protein and an endonuclease-like protein. Some of these families contained multiple members, including PPSP15-like, yellow proteins and D7-related proteins suggesting gene expansion in these proteins. CONCLUSION: This comprehensive analysis allows us the identification of genus- specific proteins, species-specific proteins and, more importantly, proteins common among these different sandflies. These results give us insights into the repertoire of salivary proteins that are potential candidates for a vector-based vaccine.