Salivary Gland Homogenates Research Articles

Herbivorous Caterpillars Can Utilize Three Mechanisms to Alter Green Leaf Volatile Emission.

Green plants emit green leaf volatiles (GLVs) as a general damage response. These compounds act as signals for the emitter plant, neighboring plants, and even for insects in the ecosystem. However, when oral secretions from certain caterpillars are applied to wounded leaves, GLV emissions are significantly decreased or modified. We examined four caterpillar species representing two lepidopteran families for their capacity to decrease GLV emissions from Zea mays leaf tissue. We also investigated the source of the GLV modifying components in the alimentary tract of the various caterpillars. In Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), and Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae), we found three distinct mechanisms to modify GLV emission: a heat-stable compound in the gut, a heat-labile enzyme in salivary gland homogenate (previously described in Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae), and an isomerase in the salivary gland homogenate, which catalyzes the conversion of (Z)-3-hexenal to (E)-2-hexenal (previously described in M. sexta). These mechanisms employed by caterpillars to suppress or modify GLV emission suggest a counteraction against the induced indirect volatile defenses of a plant and provides further insights into the ecological functions of GLVs.

DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica.

BackgroundThe vector-borne disease leishmaniasis is transmitted to humans by infected female sand flies, which transmits Leishmania parasites together with saliva during blood feeding. In Iran, cutaneous leishmaniasis (CL) is caused by Leishmania (L.) major and L. tropica, and their main vectors are Phlebotomus (Ph.) papatasi and Ph. sergenti, respectively. Previous studies have demonstrated that mice immunized with the salivary gland homogenate (SGH) of Ph. papatasi or subjected to bites from uninfected sand flies are protected against L. major infection.Methods and resultsIn this work we tested the immune response in BALB/c mice to 14 different plasmids coding for the most abundant salivary proteins of Ph. sergenti. The plasmid coding for the salivary protein PsSP9 induced a DTH response in the presence of a significant increase of IFN-γ expression in draining lymph nodes (dLN) as compared to control plasmid and no detectable PsSP9 antibody response. Animals immunized with whole Ph. sergenti SGH developed only a saliva-specific antibody response and no DTH response. Mice immunized with whole Ph. sergenti saliva and challenged intradermally with L. tropica plus Ph. sergenti SGH in their ears, exhibited no protective effect. In contrast, PsSP9-immunized mice showed protection against L. tropica infection resulting in a reduction in nodule size, disease burden and parasite burden compared to controls. Two months post infection, protection was associated with a significant increase in the ratio of IFN-γ to IL-5 expression in the dLN compared to controls.ConclusionThis study demonstrates that while immunity to the whole Ph. sergenti saliva does not induce a protective response against cutaneous leishmaniasis in BALB/c mice, PsSP9, a member of the PpSP15 family of Ph. sergenti salivary proteins, provides protection against L. tropica infection. These results suggest that this family of proteins in Ph. sergenti, Ph. duboscqi and Ph. papatasi may have similar immunogenic and protective properties against different Leishmania species. Indeed, this anti-saliva immunity may act as an adjuvant to accelerate the cell-mediated immune response to co-administered Leishmania antigens, or even cause the activation of infected macrophages to remove parasites more efficiently. These findings highlight the idea of applying arthropod saliva components in vaccination approaches for diseases caused by vector-borne pathogens.

Human antibody reaction against recombinant salivary proteins of Phlebotomus orientalis in Eastern Africa.

BackgroundPhlebotomus orientalis is a vector of Leishmania donovani, the causative agent of life threatening visceral leishmaniasis spread in Eastern Africa. During blood-feeding, sand fly females salivate into the skin of the host. Sand fly saliva contains a large variety of proteins, some of which elicit specific antibody responses in the bitten hosts. To evaluate the exposure to sand fly bites in human populations from disease endemic areas, we tested the antibody reactions of volunteers' sera against recombinant P. orientalis salivary antigens.Methodology/Principal findingsRecombinant proteins derived from sequence data on P. orientalis secreted salivary proteins, were produced using either bacterial (five proteins) or mammalian (four proteins) expression systems and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Using these recombinant proteins, human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure.Conclusions/SignificanceThis is the first report of screening human sera for anti-P. orientalis antibodies using recombinant salivary proteins. The recombinant salivary proteins mYEL1 and mAG5 proved to be valid antigens for screening human sera from both Sudan and Ethiopia for exposure to P. orientalis bites. The utilization of equal amounts of these two proteins significantly increased the capability to detect anti-P. orientalis antibody responses.

The PAGODAS protocol: pediatric assessment group of dengue and Aedes saliva protocol to investigate vector-borne determinants of Aedes-transmitted arboviral infections in Cambodia

BackgroundMosquito-borne arboviruses, like dengue virus, continue to cause significant global morbidity and mortality, particularly in Southeast Asia. When the infectious mosquitoes probe into human skin for a blood meal, they deposit saliva containing a myriad of pharmacologically active compounds, some of which alter the immune response and influence host receptivity to infection, and consequently, the establishment of the virus. Previous reports have highlighted the complexity of mosquito vector-derived factors and immunity in the success of infection. Cumulative evidence from animal models and limited data from humans have identified various vector-derived components, including salivary components, that are co-delivered with the pathogen and play an important role in the dissemination of infection. Much about the roles and effects of these vector-derived factors remain to be discovered.Methods/DesignWe describe a longitudinal, pagoda (community)-based pediatric cohort study to evaluate the burden of dengue virus infection and document the immune responses to salivary proteins of Aedes aegypti, the mosquito vector of dengue, Zika, and chikungunya viruses. The study includes community-based seroprevalence assessments in the peri-urban town of Chbar Mon in Kampong Speu Province, Cambodia. The study aims to recruit 771 children between the ages of 2 and 9 years for a three year period of longitudinal follow-up, including twice per year (rainy and dry season) serosurveillance for dengue seroconversion and Ae. aegypti salivary gland homogenate antibody intensity determinations by ELISA assays. Diagnostic tests for acute dengue, Zika and chikungunya viral infections will be performed by RT-PCR.DiscussionThis study will serve as a foundation for further understanding of mosquito saliva immunity and its impact on Aedes-transmitted arboviral diseases endemic to Cambodia.Trial registrationNCT03534245 registered on 23 May 2018.

Longitudinal monitoring of anti-saliva antibodies as markers of repellent efficacy against Phlebotomus perniciosus and Phlebotomus papatasi in dogs.

A 2-year longitudinal study of enzyme-linked immunosorbent assay (ELISA) antibodies against Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae) sandfly saliva was performed in 32 Beagle dogs treated preventively with an imidacloprid-permethrin topical insecticide in an endemic area in Spain. Dogs were grouped into three sandfly exposure groups according to the time of inclusion in the study. Assays analysed immunoglobulin G (IgG) against salivary gland homogenates (SGH) of both species and recombinant P. papatasi rSP32 and P. perniciosus rSP03B proteins in serum. The dogs were participating in a Leishmania infantum (Kinetoplastida: Trypanosomatidae) vaccine trial and were experimentally infected with the parasite in the second year. No dog acquired natural L. infantum infections during the first year, but most developed anti-saliva antibodies, and median log-transformed optical densities (LODs) were seasonal, mimicking those of local sandflies. This indicates that the repellent efficacy of the insecticide used is below 100%. Multi-level modelling of LODs revealed variability among dogs, autocorrelation and differences according to the salivary antigen and the dog's age. However, dog seroprevalence, estimated using pre-exposure LODs as cut-offs, was relatively low. This, and the fact that dogs did not become naturally infected with L. infantum, would support the efficacy and usefulness of this imidacloprid-permethrin topical insecticide in canine leishmaniasis control.

Seasonal dynamics of canine antibody response to Phlebotomus perniciosus saliva in an endemic area of Leishmania infantum

BackgroundCanine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host-vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas.MethodsIn this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models.ResultsThe dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate.ConclusionsOur results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH- and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas.

Evaluation of the rSP03B sero-strip, a newly proposed rapid test for canine exposure to Phlebotomus perniciosus, vector of Leishmania infantum.

BackgroundCanine leishmaniasis (CanL) is a zoonotic disease, caused by Leishmania infantum and transmitted by Phlebotomus perniciosus in the Mediterranean basin. Previously, an ELISA based on the P. perniciosus salivary protein SP03B was proposed as a valid tool to screen for canine exposure to sand fly bites across regions endemic for CanL. Although this approach is useful in laboratory settings, a practical tool for immediate application in the field is needed. In this study we propose the rSP03B sero-strip, the first immunochromatographic test (ICT) in the field of vector exposure able to rapidly screen dogs living in endemic areas for the presence of P. perniciosus and to aid in the evaluation of vector control programs.Methodology/Principal findingsThe ICT was prepared using the bacterially expressed recombinant protein rSP03B as antigen. For test optimization, pre-immune sera from non-bitten laboratory-bred Beagles were used as negative controls. In order to validate the test, sera from laboratory-bred Beagles experimentally exposed to P. perniciosus bites were used as positive controls. Additionally, all samples were tested by ELISA using whole salivary gland homogenate (SGH) and the rSP03B protein as antigen. An almost perfect degree of agreement was found between the ICT and the SGH-ELISA. Furthermore, the newly proposed rSP03B sero-strip showed a sensitivity of 100% and a specificity of 86.79%.Conclusions/SignificanceWe developed a simple and rapid ICT based on the P. perniciosus rSP03B salivary protein, able to replace the standard ELISA used in previous studies. Our rSP03B sero-strip showed to be highly sensitive and specific in the detection of antibodies (IgG) against P. perniciosus saliva. In the future, this test can be employed during large-scale epidemiological studies of CanL in the Mediterranean area to evaluate the efficacy of vector control programs.

Rhipicephalus appendiculatus ticks transmit Theileria parva from persistently infected cattle in the absence of detectable parasitemia: implications for East Coast fever epidemiology

BackgroundEast Coast fever (ECF) is a devastating disease of cattle and a significant constraint to improvement of livestock production in sub-Saharan Africa. The protozoan parasite causing ECF, Theileria parva, undergoes obligate sexual stage development in its tick vector Rhipicephalus appendiculatus. Tick-borne acquisition and transmission occurs transstadially; larval and nymphal ticks acquire infection while feeding and transmit to cattle when they feed after molting to the next stage. Much of the current knowledge relating to tick-borne acquisition and transmission of T. parva has been derived from studies performed during acute infections where parasitemia is high. In contrast, tick-borne transmission during the low-level persistent infections characteristic of endemic transmission cycles is rarely studied.MethodsCattle were infected with one of two stocks of T. parva (Muguga or Marikebuni). Four months post-infection when parasites were no longer detectable in peripheral blood by PCR, 500 R. appendiculatus nymphs were fed to repletion on each of the cattle. After they molted to the adult stage, 20 or 200 ticks, respectively, were fed on two naïve cattle for each of the parasite stocks. After adult ticks fed to repletion, cattle were tested for T. parva infection by nested PCR and dot blot hybridization.ResultsOnce they had molted to adults the ticks that had fed as nymphs on Muguga and Marikebuni infected cattle successfully transmitted Theileria parva to all naïve cattle, even though T. parva infection was not detectable by nested PCR on salivary gland genomic DNA of a sample of individual ticks. However, a salivary gland homogenate from a single Marikebuni infected tick was able to infect primary bovine lymphocytes. Infection was detected by nested p104 PCR in 3 of 4 calves and detected in all 4 calves by T. parva 18S nested PCR/dot blot hybridization.ConclusionWe show that R. appendiculatus ticks are able to acquire T. parva parasites from infected cattle even in the absence of detectable parasitemia. Although infection was undetectable in a sample of individual ticks, cumulatively as few as 20 ticks were able to transmit T. parva to naïve cattle. These results have important implications for our understanding of T. parva transmission by R. appendiculatus in ECF endemic regions.

Zika virus transmission to mouse ear by mosquito bite: a laboratory model that replicates the natural transmission process

BackgroundZika disease has transformed into a serious global health problem due to the rapid spread of the arbovirus and alarming severity including congenital complications, microcephaly and Guillain-Barré syndrome. Zika virus (ZIKV) is primarily transmitted to humans through the bite of an infective mosquito, with Aedes aegypti being the main vector.MethodsWe successfully developed a ZIKV experimental transmission model by single infectious Ae. aegypti bite to a laboratory mouse using circulating Brazilian strains of both arbovirus and vector. Mosquitoes were orally infected and single Ae. aegypti were allowed to feed on mouse ears 14 days post-infection. Additionally, salivary gland (SG) homogenates from infected mosquitoes were intrathoracically inoculated into naïve Ae. aegypti. Mosquito and mouse tissue samples were cultured in C6/36 cells and processed by quantitative real-time PCR.ResultsA total of 26 Ae. aegypti were allowed to feed individually on mouse ears. Of these, 17 mosquitoes fed, all to full engorgement. The transmission rate of ZIKV by bite from these engorged mosquitoes to mouse ears was 100%. The amount of virus inoculated into the ears by bites ranged from 2 × 102–2.1 × 1010 ZIKV cDNA copies and was positively correlated with ZIKV cDNA quantified from SGs dissected from mosquitoes post-feeding. Replicating ZIKV was confirmed in macerated SGs (2.45 × 107 cDNA copies), mouse ear tissue (1.15 × 103 cDNA copies, and mosquitoes 14 days post-intrathoracic inoculation (1.49 × 107 cDNA copies) by cytopathic effect in C6/36 cell culture and qPCR.ConclusionsOur model illustrates successful transmission of ZIKV by an infectious mosquito bite to a live vertebrate host. This approach offers a comprehensive tool for evaluating the development of infection in and transmission from mosquitoes, and the vertebrate-ZIKV interaction and progression of infection following a natural transmission process.

Humoral Response to Anopheles maculatus and Anopheles sundaicus Salivary Gland Proteins

The salivary gland of female Anopheles mosquito has important role in malaria transmission by salivary proteins that enhancing Plasmodium transmission. The mosquito saliva contains vasomodulatory and immunomodulatory components that inhibit the physiological response of the host. Therefore, the pathogen infects the host without any resistance. The salivary proteins also induce the production of antibody IgG in the host after exposed by the Anopheles mosquito bites repeatedly. This study aims to measured the antibody response of inhabitants living in malaria endemic areas (Kalirejo viilages, Kokap, Kulonprogo) to salivary gland proteins of An. maculatus and An.sundaicus. The level of antibody response was measured by ELISA and analyzed with T test or Anova test for normal distribution of data and Mann Whitney test or Kruskal Wallis test for the data were not normally distributed. The result showed that the level of anti-salivary gland homogenate IgG from sera of people living in malaria endemic area was significantly higher than people living in non-malaria endemic area and negative control (p<0.05). Sera from inhabitants living in Kalirejo village showed that the level of antibody response to both salivary gland homogenate of An.sundaicus and An.maculatus were not significantly different (p<0.05). It was concluded that exposure to An.maculatus and An.sundaicus bites repeatedly could trigger the production of anti-salivary gland proteins IgG antibodies that recognized antigenic proteins from the salivary glands.

Sand fly salivary proteins-parasite-skin interaction

Abstract Leishmaniasis is a vector-borne disease transmitted by infected sand flies. During infection, sand fly salivary proteins are inoculated into the host dermis together with Leishmania parasites where proteins from the extracellular matrix are present. In this context, we hypothesize that sand fly salivary proteins interact with the parasite and with proteins from the extracellular matrix. Our preliminary findings show that proteins from sand fly salivary gland homogenate bind to the surface of promastigote forms of the parasite Leishmania infantum. Using recombinant sand fly salivary proteins we identified two major salivary proteins, LJL143 and LJL04, as the main salivary proteins binding Leishmania parasite surface. Furthermore, by a Surface Plasmon Resonance assay we tested the interaction between salivary recombinant proteins with different extracellular proteins. From all the recombinant proteins tested, only LJL04 interacts with Fibrinogen, Fibronectin, Collagen I, Multimeric VTNC and Laminin. Our plans are to confirm the salivary protein-parasite-extracellular matrix biding in vivo, firstly by exposing Balb/c mice ears to uninfected L. longipalpis bites and by detecting the presence of salivary proteins in the ear of mouse dermis post-sand fly bites. As future direction we are also planning to use L. infantum (transgenic fluorescent parasite)-infected sand flies bites to verify the possible co-localization of parasite and sand fly salivary proteins in the skin. Here, for the first time we show interaction between sand fly salivary proteins and Leishmania parasites and sand fly salivary proteins and extracellular matrix proteins.

Structure of SALO, a leishmaniasis vaccine candidate from the sand fly Lutzomyia longipalpis.

BackgroundImmunity to the sand fly salivary protein SALO (Salivary Anticomplement of Lutzomyia longipalpis) protected hamsters against Leishmania infantum and L. braziliensis infection and, more recently, a vaccine combination of a genetically modified Leishmania with SALO conferred strong protection against L. donovani infection. Because of the importance of SALO as a potential component of a leishmaniasis vaccine, a plan to produce this recombinant protein for future scale manufacturing as well as knowledge of its structural characteristics are needed to move SALO forward for the clinical path.Methodology/Principal findingsRecombinant SALO was expressed as a soluble secreted protein using Pichia pastoris, rSALO(P), with yields of 1g/L and >99% purity as assessed by SEC-MALS and SDS-PAGE. Unlike its native counterpart, rSALO(P) does not inhibit the classical pathway of complement; however, antibodies to rSALO(P) inhibit the anti-complement activity of sand fly salivary gland homogenate. Immunization with rSALO(P) produces a delayed type hypersensitivity response in C57BL/6 mice, suggesting rSALO(P) lacked anti-complement activity but retained its immunogenicity. The structure of rSALO(P) was solved by S-SAD at Cu-Kalpha to 1.94 Å and refined to Rfactor 17%. SALO is ~80% helical, has no appreciable structural similarities to any human protein, and has limited structural similarity in the C-terminus to members of insect odorant binding proteins. SALO has three predicted human CD4+ T cell epitopes on surface exposed helices.Conclusions/SignificanceThe results indicate that SALO as expressed and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing. SALO has a novel structure, is not similar to any human proteins, is immunogenic in rodents, and does not have the anti-complement activity observed in the native salivary protein which are all important attributes to move this vaccine candidate forward to the clinical path.

The recombinant protein rSP03B is a valid antigen for screening dog exposure to Phlebotomus perniciosus across foci of canine leishmaniasis.

The frequency of sandfly-host contacts can be measured by host antibody levels against sandfly salivary proteins. Recombinant salivary proteins are suggested to represent a valid replacement for salivary gland homogenate (SGH); however, it is necessary to prove that such antigens are recognized by antibodies against various populations of the same species. Phlebotomus perniciosus (Diptera: Psychodidae) is the main vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in southwest Europe and is widespread from Portugal to Italy. In this study, sera were sampled from naturally exposed dogs from distant regions, including Campania (southern Italy), Umbria (central Italy) and the metropolitan Lisbon region (Portugal), where P. perniciosus is the unique or principal vector species. Sera were screened for anti-P. perniciosus antibodies using SGH and 43-kDa yellow-related recombinant protein (rSP03B). A robust correlation between antibodies recognizing SGH and rSP03B was detected in all regions, suggesting substantial antigenic cross-reactivity among different P. perniciosus populations. No significant differences in this relationship were detected between regions. Moreover, rSP03B and the native yellow-related protein were shown to share similar antigenic epitopes, as canine immunoglobulin G (IgG) binding to the native protein was inhibited by pre-incubation with the recombinant form. These findings suggest that rSP03B should be regarded as a universal marker of sandfly exposure throughout the geographical distribution of P. perniciosus.

Direct evidence for role of anti-saliva antibodies against salivary gland homogenate of P. argentipes in modulation of protective Th1-immune response against Leishmania donovani

Currently the main concerns regarding control of visceral leishmaniasis (VL) caused by L. donovani are immunosuppression, relating toxicity of anti-leishmanial drug and little development in appropriate vaccine and vector (P. argentipes) control. Reports available from ex-vivo studies reflect significance of vector salivary gland homogenate (SGH) in reverting immunosuppression of infected VL subjects and as such the immunogenic nature of SGH can be a strategy to modulate immune system and anti-leishmanial function to enable immune response to control the disease. Several related studies also identified a better utility of vector anti-saliva antibodies in achieving such effects by an adoptive transfer approach instead of direct stimulation with SGH protein. However, conclusive evidences on VL cases are far beyond satisfactory to suggest role of SGH into modulation of host immune response in VL subjects in India. This study was under taken to make comparison on change in cytokines (TH1 and TH2) response pattern and anti-leishmanial macrophage (Mϕ) function following stimulation of their PBMCS with SGH protein derived from P. argentipes sand fly vector for VL or anti SGH antibodies raised in rabbit.This study reports for the first time that L. donovani sensitized healthy subject demonstrates an up-regulated Interferon-γ (TH1) and down regulate Interleukin-10 (TH2) production following stimulation of their PBMCs by P. argentipes anti-saliva antibodies accompanied with an improvement in anti-leishmanial Mϕ function for nitric oxide (NO) production. Subsequent experiments suggest that P. argentipes based anti-SGH antibodies when used to stimulate LD infected PBMCs in healthy subjects resulted in better clearance of Leishmania amastigotes load compare to SGH protein. Possibly the immunogenic components of anti-saliva an antibody maintains the level of protective cytokine (INF-γ) and seems to restrict the infection by host protection by vector saliva.

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.

Human immune response to salivary proteins of wild-caught Phlebotomus papatasi.

Phlebotomine sand flies are the known vectors of Leishmania parasites. New approaches in vaccination against Leishmania have investigated the possibility of integrating Phlebotomus papatasi salivary proteins to enhance the immune response and protect against the transmission of the infection. The aim of the present study was to screen human immune responses to wild sand fly saliva and evaluate immunogenic salivary proteins. Blood samples were collected from donors in control and sand fly infested areas. Antibodies specific for sand fly antigens in donor plasma were probed using immunoblotting. In addition, recall proliferation capability of peripheral blood mononuclear cells (PBMC) was tested after sand fly salivary homogenates stimulation. The significant immunogenic salivary proteins (SPs) identified by immunoblotting were SP28, SP32, and SP36. A specific proliferative response of PBMC after stimulation with sand fly salivary homogenates was evident in donors that have antibody responses against sand fly salivary proteins. Individuals with antibody recognition to a higher number of salivary proteins (i.e., 3 or more SP bands) showed lower PBMC proliferative responses after in vitro stimulation with salivary gland homogenates (SGH) only in the sand fly infested, leishmaniasis free area. Interestingly, the presence of a humoral immune response to many SP antigens inversely correlates with a strong cell-mediated immune response (CMI). It was also noticed that some other heavily expressed antigens, in sand fly salivary homogenate, lack or have weak humoral immune reactivity in exposed individuals. Therefore, considering these antigens alone as CMI activators, without including the immunodominant humoral immune response proteins, needs future investigation.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Kinetics of Anti-Phlebotomus perniciosus Saliva Antibodies in Experimentally Bitten Mice and Rabbits.

BackgroundSand flies are hematophagous arthropods that act as vectors of Leishmania parasites. When hosts are bitten they develop cellular and humoral responses against sand fly saliva. A positive correlation has been observed between the number of bites and antibody levels indicating that anti-saliva antibody response can be used as marker of exposure to sand flies. Little is known about kinetics of antibodies against Phlebotomus perniciosus salivary gland homogenate (SGH) or recombinant salivary proteins (rSP). This work focused on the study of anti-P. perniciosus saliva antibodies in sera of mice and rabbits that were experimentally exposed to the bites of uninfected sand flies.Methodology/Principal FindingsAnti-saliva antibodies were evaluated by ELISA and Western blot. In addition, antibody levels against two P. perniciosus rSP, apyrase rSP01B and D7 related protein rSP04 were determined in mice sera. Anti-saliva antibody levels increased along the immunizations and correlated with the number of sand fly bites. Anti-SGH antibody levels were detected in sera of mice five weeks after exposure, and persisted for at least three months. Anti-apyrase rSP01B antibodies followed similar kinetic responses than anti-SGH antibodies while rSP04 showed a delayed response and exhibited a greater variability among sera of immunized mice. In rabbits, anti-saliva antibodies appeared after the second week of exposure and IgG antibodies persisted at high levels, even 7 months post-exposure.Conclusions/SignificanceOur results contributed to increase the knowledge on the type of immune response P. perniciosus saliva and individual proteins elicited highlighting the use of rSP01B as an epidemiological marker of exposure. Anti-saliva kinetics in sera of experimentally bitten rabbits were studied for the first time. Results with rabbit model provided useful information for a better understanding of the anti-saliva antibody levels found in wild leporids in the human leishmaniasis focus in the Madrid region, Spain.

Canine Antibodies against Salivary Recombinant Proteins of Phlebotomus perniciosus: A Longitudinal Study in an Endemic Focus of Canine Leishmaniasis.

BackgroundPhlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs.Methodology/Principal FindingsSera from dogs naturally exposed to P. perniciosus bites over two consecutive transmission seasons in a site endemic for canine leishmaniasis (CanL) were tested at different time points by ELISA for the antibodies recognizing whole saliva, single salivary 43 kDa yellow-related recombinant protein (rSP03B), and a combination of two salivary recombinant proteins, 43 kDa yellow-related protein and 35.5 kDa apyrase (rSP01). Dogs were also tested for Leishmania infantum positivity by serology, culture, and PCR and the infection status was evaluated prospectively. We found a significant association between active CanL infection and the amount of anti-P. perniciosus saliva antibodies. Importantly, we detected a high correlation between IgG antibodies recognizing rSP03B protein and the whole salivary antigen. The kinetics of antibody response showed for both a whole saliva and rSP03B a similar pattern that was clearly related to the seasonal abundance of P. perniciosus.ConclusionsThese results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas.

Phlebotomus papatasi exposure cross-protects mice against Leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate

Leishmania parasites are inoculated into host skin together with sand fly saliva and multiple exposures to uninfected sand fly bites protect mice against Leishmania infection. However, sand fly vectors differ in composition of the saliva and therefore the protection elicited by their salivary proteins was shown to be species-specific. On the other hand, the optimal vaccine based on sand fly salivary proteins should be based on conserved salivary proteins conferring cross-reactivity. In the present study we therefore focused on cross-protective properties of saliva from Phlebotomus papatasi and Phlebotomus duboscqi, the two natural vectors of Leishmania major. Two groups of mice exposed to bites of P. papatasi and two control, non-immunized groups were infected with L. major promastigotes along with either P. papatasi or P. duboscqi salivary gland homogenate. All mice were followed for the development of Leishmania lesions, parasite burdens, specific antibodies, and for production of NO, urea, or cytokines by peritoneal macrophages. Protection against Leishmania infection was observed not only in exposed mice challenged with homologous saliva but also in the group challenged with P. duboscqi saliva. Comparing both exposed groups, no significant differences were observed in parasite load, macrophage activity, or in the levels of anti-L. major and anti-P. papatasi/P. duboscqi antibodies. This is the first study showing cross-protection caused by salivary antigens of two Phlebotomus species. The cross-protective effect suggests that the anti-Leishmania vaccine based on P. papatasi salivary proteins might be applicable also in areas where L. major is transmitted by P. duboscqi.