Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

A bispecific inhibitor of complement and coagulation blocks activation in complementopathy models via a novel mechanism.

Inhibitors of complement and coagulation are present in the saliva of a variety of blood-feeding arthropods that transmit parasitic and viral pathogens. Here, we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits the formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all ß-sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo-electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.

Single-cell RNA sequencing of Trypanosoma brucei from tsetse salivary glands unveils metacyclogenesis and identifies potential transmission blocking antigens.

Tsetse-transmitted African trypanosomes must develop into mammalian-infectious metacyclic cells in the fly's salivary glands (SGs) before transmission to a new host. The molecular mechanisms that underlie this developmental process, known as metacyclogenesis, are poorly understood. Blocking the few metacyclic parasites deposited in saliva from further development in the mammal could prevent disease. To obtain an in-depth perspective of metacyclogenesis, we performed single-cell RNA sequencing (scRNA-seq) from a pool of 2,045 parasites collected from infected tsetse SGs. Our data revealed three major cell clusters that represent the epimastigote, and pre- and mature metacyclic trypanosome developmental stages. Individual cell level data also confirm that the metacyclic pool is diverse, and that each parasite expresses only one of the unique metacyclic variant surface glycoprotein (mVSG) coat protein transcripts identified. Further clustering of cells revealed a dynamic transcriptomic and metabolic landscape reflective of a developmental program leading to infectious metacyclic forms preadapted to survive in the mammalian host environment. We describe the expression profile of proteins that regulate gene expression and that potentially play a role in metacyclogenesis. We also report on a family of nonvariant surface proteins (Fam10) and demonstrate surface localization of one member (named SGM1.7) on mature metacyclic parasites. Vaccination of mice with recombinant SGM1.7 reduced parasitemia early in the infection. Future studies are warranted to investigate Fam10 family proteins as potential trypanosome transmission blocking vaccine antigens. Our experimental approach is translationally relevant for developing strategies to prevent other insect saliva-transmitted parasites from infecting and causing disease in mammalian hosts.

A Composite Recombinant Salivary Proteins Biomarker for Phlebotomus argentipes Provides a Surveillance Tool Postelimination of Visceral Leishmaniasis in India.

Incidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the postelimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Phlebotomus papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after VL elimination.

Immune Response to LinB13, a Lutzomyia Intermedia Salivary Protein Correlates With Disease Severity in Tegumentary Leishmaniasis.

BACKGROUND: We have previously shown that seropositivity to rLinB-13, a salivary protein from Lutzomyia intermedia, predicted sand fly exposure and was associated with increased risk of developing cutaneous leishmaniasis (CL). METHODS: Here, we investigated the cellular immune response to saliva from Lu. intermedia, using rLinB-13 as a surrogate antigen in naturally exposed individuals presenting positive serology to LinB-13. We also investigated the response to rLinB-13 in leishmaniasis patients, displaying active ulcers and positive PCR for Leishmania braziliensis. RESULTS: Peripheral blood mononuclear cells (PBMCs) stimulated in vitro with rLinB-13 secreted elevated levels of interleukin-10 (IL-10), IL-4, IL-1ß, IL-1α, IL-6, and chemokines (CCL3, CCL4, CCL5, and CXCL5). CL and disseminated leishmaniasis (DL) patients displayed a significantly higher immunoglobulin G (IgG) response to rLinB-13 compared with healthy subjects, and anti-rLinB-13 IgG was positively correlated with the number of lesions in DL patients. Positive serology to rLinB-13 was also associated with chemotherapy failure. PBMCs from DL patients stimulated with rLINB-13 secreted significantly higher levels of IL-10 and IL-1ß compared with CL individuals. CONCLUSIONS: In this study, we observed an association between humoral and cellular immune response to the sand fly salivary protein rLinB-13 and disease severity in tegumentary leishmaniasis. This study brings evidence that immunity to rLinB-13 influences disease outcome in L. braziliensis infection and results indicate that positive serology to rLinB-13 IgG can be used as a marker of DL, an emerging and severe form of disease caused by L. braziliensis.

Safety and immunogenicity of a mosquito saliva peptide-based vaccine: a randomised, placebo-controlled, double-blind, phase 1 trial.

BACKGROUND: In animal models, immunity to mosquito salivary proteins protects animals against mosquito-borne disease. These findings provide a rationale to vaccinate against mosquito saliva instead of the pathogen itself. To our knowledge, no vector salivary protein-based vaccine has been tested for safety and immunogenicity in humans. We aimed to assess the safety and immunogenicity of Anopheles gambiae saliva vaccine (AGS-v), a peptide-based vaccine derived from four A gambiae salivary proteins, in humans. METHODS: In this randomised, placebo-controlled, double-blind, phase 1 trial, participants were enrolled at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Participants were eligible if they were healthy adults, aged 18-50 years with no history of severe allergic reactions to mosquito bites. Participants were randomly assigned (1:1:1), using block randomisation and a computer-generated randomisation sequence, to treatment with either 200 nmol of AGS-v vaccine alone, 200 nmol of AGS-v with adjuvant (Montanide ISA 51), or sterile water as placebo. Participants and clinicians were masked to treatment assignment. Participants were given a subcutaneous injection of their allocated treatment at day 0 and day 21, followed by exposure to feeding by an uninfected Aedes aegypti mosquito at day 42 to assess subsequent risk to mosquito bites in a controlled setting. The primary endpoints were safety and immunogenicity at day 42 after the first immunisation. Participants who were given at least one dose of assigned treatment were assessed for the primary endpoints and analysis was by intention to treat. The trial was registered with ClinicalTrials.gov, NCT03055000, and is closed for accrual. FINDINGS: Between Feb 15 and Sept 10, 2017, we enrolled and randomly assigned 49 healthy adult participants to the adjuvanted vaccine (n=17), vaccine alone (n=16), or placebo group (n=16). Five participants did not complete the two-injection regimen with mosquito feeding at day 42, but were included in the safety analyses. No systemic safety concerns were identified; however, one participant in the adjuvanted vaccine group developed a grade 3 erythematous rash at the injection site. Pain, swelling, erythema, and itching were the most commonly reported local symptoms and were significantly increased in the adjuvanted vaccine group compared with both other treatment groups (nine [53%] of 17 participants in the adjuvanted vaccine group, two [13%] of 16 in the vaccine only group, and one [6%] of 16 in the placebo group; p=0·004). By day 42, participants who were given the adjuvanted vaccine had a significant increase in vaccine-specific total IgG antibodies compared with at baseline than did participants who were give vaccine only (absolute difference of log10-fold change of 0·64 [95% CI 0·39 to 0·89]; p=0·0002) and who were given placebo (0·62 [0·34 to 0·91]; p=0·0001). We saw a significant increase in IFN-γ production by peripheral blood mononuclear cells at day 42 in the adjuvanted vaccine group compared with in the placebo group (absolute difference of log10 ratio of vaccine peptide-stimulated vs negative control 0·17 [95% CI 0·061 to 0·27]; p=0·009) but we saw no difference between the IFN-γ production in the vaccine only group compared with the placebo group (0·022 [-0·072 to 0·116]; p=0·63). INTERPRETATION: AGS-v was well tolerated, and, when adjuvanted, immunogenic. These findings suggest that vector-targeted vaccine administration in humans is safe and could be a viable option for the increasing burden of vector-borne disease. FUNDING: Office of the Director and the Division of Intramural Research at the National Institute of Allergy and Infectious Diseases, and National Institutes of Health.

Exploring Lutzomyia longipalpis Sand Fly Vector Competence for Leishmania major Parasites.

Lutzomyia longipalpis sand flies are the major natural vector of Leishmania infantum parasites, responsible for transmission of visceral leishmaniasis in the New World. Several experimental studies have demonstrated the ability of Lu. longipalpis to sustain development of different Leishmania species. However, no study had explored in depth the potential vector competence of Lu. longipalpis for Leishmania species other than L. infantum. Here, we show that Lu. longipalpis is a competent vector of L. major parasites, being able to acquire parasites from active cutaneous leishmaniasis lesions, sustain mature infections, and transmit them to naive hosts, causing disease.

Leishmania infection induces a limited differential gene expression in the sand fly midgut.

BACKGROUND: Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages. RESULTS: The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (< 32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics. CONCLUSION: Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection.

Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from Lu. longipalpis. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized in vitro. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of Leishmania infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis-Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine.

The relapsing fever spirochete Borrelia turicatae persists in the highly oxidative environment of its soft-bodied tick vector.

The relapsing fever spirochete Borrelia turicatae possesses a complex life cycle in its soft-bodied tick vector, Ornithodoros turicata. Spirochetes enter the tick midgut during a blood meal, and, during the following weeks, spirochetes disseminate throughout O. turicata. A population persists in the salivary glands allowing for rapid transmission to the mammalian hosts during tick feeding. Little is known about the physiological environment within the salivary glands acini in which B. turicatae persists. In this study, we examined the salivary gland transcriptome of O. turicata ticks and detected the expression of 57 genes involved in oxidant metabolism or antioxidant defences. We confirmed the expression of five of the most highly expressed genes, including glutathione peroxidase (gpx), thioredoxin peroxidase (tpx), manganese superoxide dismutase (sod-1), copper-zinc superoxide dismutase (sod-2), and catalase (cat) by reverse-transcriptase droplet digital polymerase chain reaction (RT-ddPCR). We also found distinct differences in the expression of these genes when comparing the salivary glands and midguts of unfed O. turicata ticks. Our results indicate that the salivary glands of unfed O. turicata nymphs are highly oxidative environments where reactive oxygen species (ROS) predominate, whereas midgut tissues comprise a primarily nitrosative environment where nitric oxide synthase is highly expressed. Additionally, B. turicatae was found to be hyperresistant to ROS compared with the Lyme disease spirochete Borrelia burgdorferi, suggesting it is uniquely adapted to the highly oxidative environment of O. turicata salivary gland acini.

Mosquito Saliva: The Hope for a Universal Arbovirus Vaccine?

Arthropod-borne viruses (arboviruses) are taxonomically diverse causes of significant morbidity and mortality. In recent decades, important mosquito-borne viruses such as West Nile, chikungunya, dengue, and Zika have re-emerged and spread widely, in some cases pandemically, to cause serious public health emergencies. There are no licensed vaccines against most of these viruses, and vaccine development and use has been complicated by the number of different viruses to protect against, by subtype and strain variation, and by the inability to predict when and where outbreaks will occur. A new approach to preventing arboviral diseases is suggested by the observation that arthropod saliva facilitates transmission of pathogens, including leishmania parasites, Borrelia burgdorferi, and some arboviruses. Viruses carried within mosquito saliva may more easily initiate host infection by taking advantage of the host's innate and adaptive immune responses to saliva. This provides a rationale for creating vaccines against mosquito salivary proteins, rather than against only the virus proteins contained within the saliva. As proof of principle, immunization with sand fly salivary antigens to prevent leishmania infection has shown promising results in animal models. A similar approach using salivary proteins of important vector mosquitoes, such as Aedes aegypti, might protect against multiple mosquito-borne viral infections.

An Inhibitor of the Alternative Pathway of Complement in Saliva of New World Anopheline Mosquitoes.

The complement system present in circulating blood is an effective mechanism of host defense, responsible for the killing of pathogens and the production of potent anaphylatoxins. Inhibitors of the complement system have been described in the saliva of hematophagous arthropods that are involved in the protection of digestive tissues against complement system-mediated damage. In this study, we describe albicin, a novel inhibitor of the alternative pathway of complement from the salivary glands of the malaria vector, Anopheles albimanus The inhibitor was purified from salivary gland homogenates by reverse-phase HPLC and identified by mass spectrometry as a small (13.4-kDa) protein related to the gSG7 protein of Anopheles gambiae and Anopheles stephensi Recombinant albicin was produced in Escherichia coli and found to potently inhibit lysis of rabbit erythrocytes in assays of the alternative pathway while having no inhibitory effect on the classical or lectin pathways. Albicin also inhibited the deposition of complement components on agarose-coated plates, although it could not remove previously bound components. Antisera produced against recombinant albicin recognized both the native and recombinant inhibitors and also blocked their activities in in vitro assays. Using surface plasmon resonance and enzymatic assays, we found that albicin binds and stabilizes the C3-convertase complex (C3bBb) formed on a properdin surface and inhibits the convertase activity of a reconstituted C3bBb complex in solution. The data indicate that albicin specifically recognizes the activated form of the complex, allowing more efficient inhibition by an inhibitor whose quantity is limited.

Overlapping IgG4 Responses to Self- and Environmental Antigens in Endemic Pemphigus Foliaceus.

The etiology of human autoimmune diseases in general remains largely unknown, although the genetic and environmental interplay may be relevant. This applies to the autoimmune diseases of the skin such as the pemphigus phenotypes and others. In this group, there is an endemic form of pemphigus foliaceus (also known as fogo selvagem [FS]) in which the pathogenic IgG4 autoantibody response to the self-antigen desmoglein 1 (Dsg1) cross-reacts with the LJM11 sand fly salivary gland Ag. In this investigation, we dissected the IgG4 autoantibody repertoires used by FS patients in response to endogenous self-Dsg1 and exogenous LJM11 sand fly Ag. Based on analyses of the genetic clonal signatures of these Abs, our results indicate that there is a significant overlap between these two responses, as all identified IgG4 mAbs cross-react to both Dsg1 and LJM11 Ags. Germline H- and L-chain V gene Abs generated according to mutated cross-reactive mAbs preserved their reactivity to both Ags. Our findings suggest that both Dsg1 autoantigen and LJM11 environmental Ag could be the initial antigenic stimulants for the IgG4 autoimmune responses in FS. These results support our hypothesis that LJM11 Ag plays a substantial role in triggering the IgG4 autoantibody development in FS and provide new insights on how noninfectious environmental Ag(s) may drive the generation of autoantibodies in IgG4-related autoimmune diseases.

Properdin-Mediated C5a Production Enhances Stable Binding of Platelets to Granulocytes in Human Whole Blood.

Enhanced levels of platelet/granulocyte aggregates (PGAs) are found in patients suffering from many different inflammatory vascular diseases, and their formation in animal models of vascular disease is associated with increased thromboinflammation and worsened outcomes. The complement system, a part of the innate immune system, influences PGA formation, but the mechanisms for its effects are unknown. In this study, we have defined complement-mediated mechanisms that enhance PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP) using ex vivo flow cytometry assays. We demonstrate that physiological properdin, a positive regulator of complement alternative pathway activity, increases PGA formation when added to TRAP-stimulated blood. All physiological properdin forms increase PGA formation, but properdin tetramers are the most efficient at increasing complement activity and PGA formation. Inhibition of endogenous properdin, either circulating in the blood or produced locally by leukocytes, impairs TRAP-mediated PGA formation to the same level as specific inhibition of either the alternative or classical pathway. Additionally, blocking the interaction of C5a with its cellular receptor prevents properdin-mediated increases in PGA formation. Adding either properdin tetramers or C5a to whole blood increases CD11b expression on granulocytes, and this increase is prevented by blockade of the C5a-C5a receptor axis. Finally, we demonstrate that the effects of properdin on PGA formation are tightly regulated by Factor H. Cumulatively, our data indicate that properdin enhances PGA formation via increased production of C5a, and that inhibition of properdin function has therapeutic potential to limit thromboinflammation in diseases characterized by increased PGA formation.

High-Sensitivity Assays for Plasmodium falciparum Infection by Immuno-Polymerase Chain Reaction Detection of PfIDEh and PfLDH Antigens.

Background: Rapid diagnostic tests based on Plasmodium falciparum histidine-rich protein II (PfHRP-II) and P. falciparum lactate dehydrogenase (PfLDH) antigens are widely deployed for detection of P. falciparum infection; however, these tests often miss cases of low-level parasitemia, and PfHRP-II tests can give false-negative results when P. falciparum strains do not express this antigen. Methods: We screened proteomic data for highly expressed P. falciparum proteins and compared their features to those of PfHRP-II and PfLDH biomarkers. Search criteria included high levels of expression, conservation in all parasite strains, and good correlation of antigen levels with parasitemia and its clearance after drug treatment. Different assay methods were compared for sensitive detection of parasitemia in P. falciparum cultures. Results: Among potential new biomarkers, a P. falciparum homolog of insulin-degrading enzyme (PfIDEh) met our search criteria. Comparative enzyme-linked immunosorbent assays with monoclonal antibodies against PfLDH or PfIDEh showed detection limits of 100-200 parasites/µL and 200-400 parasites/µL, respectively. Detection was dramatically improved by use of real-time immuno-polymerase chain reaction (PCR), to parasitemia limits of 0.02 parasite/µL and 0.78 parasite/µL in PfLDH- and PfIDEh-based assays, respectively. Conclusions: The ability of PfLDH- or PfIDEh-based immuno-PCR assays to detect <1 parasite/µL suggests that improvements of bound antibody sensor technology may greatly increase the sensitivity of malaria rapid diagnostic tests.

New Insights Into the Transmissibility of Leishmania infantum From Dogs to Sand Flies: Experimental Vector-Transmission Reveals Persistent Parasite Depots at Bite Sites.

Canine leishmaniasis (CanL) is a chronic fatal disease of dogs and a major source of human infection through propagation of parasites in vectors. Here, we infected 8 beagles through multiple experimental vector transmissions with Leishmania infantum-infected Lutzomyia longipalpis. CanL clinical signs varied, although live parasites were recovered from all dog spleens. Splenic parasite burdens correlated positively with Leishmania-specific interleukin 10 levels, negatively with Leishmania-specific interferon γ and interleukin 2 levels, and negatively with Leishmania skin test reactivity. A key finding was parasite persistence for 6 months in lesions observed at the bite sites in all dogs. These recrudesced following a second transmission performed at a distal site. Notably, sand flies efficiently acquired parasites after feeding on lesions at the primary bite site. In this study, controlled vector transmissions identify a potentially unappreciated role for skin at infectious bite sites in dogs with CanL, providing a new perspective regarding the mechanism of Leishmania transmissibility to vector sand flies.

Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET) were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep) from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep) shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate)- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized) Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

Inhibition of the classical pathway of the complement system by saliva of Amblyomma cajennense (Acari: Ixodidae).

Inhibition of the complement system during and after haematophagy is of utmost importance for tick success in feeding and tick development. The role of such inhibition is to minimise damage to the intestinal epithelium as well as avoiding inflammation and opsonisation of salivary molecules at the bite site. Despite its importance, the salivary anti-complement activity has been characterised only in species belonging to the Ixodes ricinus complex which saliva is able to inhibit the alternative and lectin pathways. Little is known about this activity in other species of the Ixodidae family. Thus, the aim of this study was to describe the inhibition of the classical pathway of the complement system by the saliva of Amblyomma cajennense at different stages of the haematophagy. The A. cajennense saliva and salivary gland extract (SGE) were able to inhibit the complement classical pathway through haemolytic assays with higher activity observed when saliva was used. The anti-complement activity is present in the salivary glands of starving females and also in females throughout the whole feeding process, with significant higher activity soon after tick detachment. The SGE activity from both females fed on mice or horses had no significant correlation (p > 0.05) with tick body weight. The pH found in the intestinal lumen of A. cajennense was 8.04 ± 0.08 and haemolytic assays performed at pH 8.0 showed activation of the classical pathway similarly to what occurs at pH 7.4. Consequently, inhibition could be necessary to protect the tick enterocytes. Indeed, the inhibition observed by SGE was higher in pH 8.0 in comparison to pH 7.4 reinforcing the role of saliva in protecting the intestinal cells. Further studies should be carried out in order to identify the inhibitor molecule and characterise its inhibition mechanism.

Kv1.3 channel-blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases.

The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7(-) effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.

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.