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Background: In Mali, cutaneous leishmaniasis (CL) and filariasis are co-endemic. Previous studies in animal models of infection have shown that sand fly saliva enhance infectivity of Leishmania parasites in naïve hosts while saliva-specific adaptive immune responses may protect against cutaneous and visceral leishmaniasis. In contrast, the human immune response to Phlebotomus duboscqi (Pd) saliva, the principal sand fly vector in Mali, was found to be dichotomously polarized with some individuals having a Th1-dominated response and others having a Th2-biased response. We hypothesized that co-infection with filarial parasites may be an underlying factor that modulates the immune response to Pd saliva in endemic regions., Methodology/principal Findings: To understand which cell types may be responsible for polarizing human responses to sand fly saliva, we investigated the effect of salivary glands (SG) of Pd on human monocytes. To this end, elutriated monocytes were cultured in vitro, alone, or with SG, microfilariae antigen (MF ag) of Brugia malayi, or LPS, a positive control. The mRNA expression of genes involved in inflammatory or regulatory responses was then measured as were cytokines and chemokines associated with these responses. Monocytes of individuals who were not exposed to sand fly bites (mainly North American controls) significantly upregulated the production of IL-6 and CCL4; cytokines that enhance leishmania parasite establishment, in response to SG from Pd or other vector species. This selective inflammatory response was lost in individuals that were exposed to sand fly bites which was not changed by co-infection with filarial parasites. Furthermore, infection with filarial parasites resulted in upregulation of CCL22, a type-2 associated chemokine, both at the mRNA levels and by its observed effect on the frequency of recruited monocytes., Conclusions/significance: Together, our data suggest that SG or recombinant salivary proteins from Pd alter human monocyte function by upregulating selective inflammatory cytokines., Competing Interests: The authors have declared that no competing interests exist.

Phlebotomus papatasi sand flies inject their hosts with a myriad of pharmacologically active salivary proteins to assist with blood feeding and to modulate host defenses. In addition, salivary proteins can influence cutaneous leishmaniasis disease outcome, highlighting the potential of the salivary components to be used as a vaccine. Variability of vaccine targets in natural populations influences antigen choice for vaccine development. Therefore, the objective of this study was to investigate the variability in the predicted protein sequences of nine of the most abundantly expressed salivary proteins from field populations, testing the hypothesis that salivary proteins appropriate to target for vaccination strategies will be possible. PpSP12, PpSP14, PpSP28, PpSP29, PpSP30, PpSP32, PpSP36, PpSP42, and PpSP44 mature cDNAs from field collected P. papatasi from three distinct ecotopes in the Middle East and North Africa were amplified, sequenced, and in silico translated to assess the predicted amino acid variability. Two of the predicted sequences, PpSP12 and PpSP14, demonstrated low genetic variability across the three geographic isolated sand fly populations, with conserved multiple predicted MHCII epitope binding sites suggestive of their potential application in vaccination approaches. The other seven predicted salivary proteins revealed greater allelic variation across the same sand fly populations, possibly precluding their use as vaccine targets., Competing Interests: The authors declare they have no competing interests.

Cutaneous leishmaniasisis a vector-borne disease transmitted by Leishmania infected sand flies. PpSP15 is an immunogenic salivary protein from the sand fly Phlebotomus papatasi. Immunization with PpSP15 was shown to protect against Leishmania major infection. Lactococcus lactis is a safe non-pathogenic delivery system that can be used to express antigens in situ. Here, the codon-optimized Ppsp15-egfp gene was cloned in pNZ8121 vector downstream of the PrtP signal peptide that is responsible for expression and secretion of the protein on the cell wall. Expression of PpSP15-EGFP recombinant protein was monitored by immunofluorescence, flow cytometry and Western blot. Also, expression of protein in cell wall compartment was verified using whole cell ELISA, Western blot and TEM microscopy. BALB/c mice were immunized three times with recombinant L. lactis-PpSP15-EGFPcwa, and the immune responses were followed up, at short-term (ST, 2 weeks) and long-term (LT, 6 months) periods. BALB/c mice were challenged with L. major plus P. papatasi Salivary Gland Homogenate. Evaluation of footpad thickness and parasite burden showed a delay in the development of the disease and significantly decreased parasite numbers in PpSP15 vaccinated animals as compared to control group. In addition, immunized mice showed Th1 type immune responses. Importantly, immunization with L. lactis-PpSP15-EGFPcwa stimulated the long-term memory in mice which lasted for at least 6 months., Competing Interests: The authors have declared that no competing interests exist.

Background: Phlebotomus 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 Findings: Recombinant 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/significance: This 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., Competing Interests: The authors have declared that no competing interests exist.

Background: Aedes mosquitoes severely affect the health and wellbeing of human populations by transmitting infectious diseases. In French Polynesia, Aedes aegypti is the main vector of dengue, chikungunya and Zika, and Aedes polynesiensis the primary vector of Bancroftian filariasis and a secondary vector of arboviruses. Tools for assessing the risk of disease transmission or for measuring the efficacy of vector control programmes are scarce. A promising approach to quantify the human-vector contact relies on the detection and the quantification of antibodies directed against mosquito salivary proteins., Methodology/principal Findings: An ELISA test was developed to detect and quantify the presence of immunoglobulin G (IgG) directed against proteins from salivary gland extracts (SGE) of Ae. aegypti and Ae. polynesiensis in human populations exposed to either species, through a cross-sectional study. In Tahiti and Moorea islands where Ae. aegypti and Ae. polynesiensis are present, the test revealed that 98% and 68% of individuals have developed IgG directed against Ae. aegypti and Ae. polynesiensis SGE, respectively. By comparison, ELISA tests conducted on a cohort of people from metropolitan France, not exposed to these Aedes mosquitoes, indicated that 97% of individuals had no IgG directed against SGE of either mosquito species. The analysis of additional cohorts representing different entomological Aedes contexts showed no ELISA IgG cross-reactivity between Ae. aegypti and Ae. polynesiensis SGE., Conclusions/significance: The IgG response to salivary gland extracts seems to be a valid and specific biomarker of human exposure to the bites of Ae. aegypti and Ae. polynesiensis. This new immuno-epidemiological tool will enhance our understanding of people exposure to mosquito bites, facilitate the identification of areas where disease transmission risk is high and permit to evaluate the efficacy of novel vector control strategies in Pacific islands and other tropical settings., Competing Interests: The authors have declared that no competing interests exist.

Mosquito saliva is a very complex concoction of >100 proteins, many of which have unknown functions. The effects of mosquito saliva proteins injected into our skin during blood feeding have been studied mainly in mouse models of injection or biting, with many of these systems producing results that may not be relevant to human disease. Here, we describe the numerous effects that mosquito bites have on human immune cells in mice engrafted with human hematopoietic stem cells. We used flow cytometry and multiplex cytokine bead array assays, with detailed statistical analyses, to detect small but significant variations in immune cell functions after 4 mosquitoes fed on humanized mice footpads. After preliminary analyses, at different early times after biting, we focused on assessing innate immune and subsequent cellular responses at 6 hours, 24 hours and 7 days after mosquito bites. We detected both Th1 and Th2 human immune responses, and delayed effects on cytokine levels in the blood, and immune cell compositions in the skin and bone marrow, up to 7 days post-bites. These are the first measurements of this kind, with human immune responses in whole animals, bitten by living mosquitoes, versus previous studies using incomplete mouse models and salivary gland extracts or needle injected saliva. The results have major implications for the study of hematophagous insect saliva, its effects on the human immune system, with or without pathogen transmission, and the possibility of determining which of these proteins to target for vaccination, in attempts to block transmission of numerous tropical diseases., Competing Interests: The authors have declared that no competing interests exist.

Background: Sand fly saliva compounds are able to elicit specific immune responses that have a significant role in Leishmania parasite establishment and disease outcome. Characterizing anti-saliva immune responses in individuals living in well defined leishmaniasis endemic areas would provide valuable insights regarding their effect on parasite transmission and establishment in humans., Methodology/principal Findings: We explored the cellular and humoral immune responses to Phlebotomus (P.) papatasi salivary gland extracts (SGE) in individuals living in cutaneous leishmaniasis (CL) old or emerging foci (OF, EF). OF was characterized by a higher infection prevalence as assessed by higher proportions of leishmanin skin test (LST) positive individuals compared to EF. Subjects were further subdivided into healed, asymptomatic or naïve groups. We showed anti-SGE proliferation in less than 30% of the individuals, regardless of the immune status, in both foci. IFN-γ production was higher in OF and only observed in immune individuals from OF and naïve subjects from EF. Although IL-10 was not detected, addition of anti-human IL-10 antibodies revealed an increase in proliferation and IFN-γ production only in individuals from OF. The percentage of seropositive individuals was similar in immune and naïves groups but was significantly higher in OF. No correlation was observed between anti-saliva immune responses and LST response. High anti-SGE-IgG responses were associated with an increased risk of developing ZCL. No differences were observed for anti-SGE humoral or cellular responses among naïve individuals who converted or not their LST response or developed or not ZCL after the transmission season., Conclusions/significance: These data suggest that individuals living in an old focus characterized by a frequent exposure to sand fly bites and a high prevalence of infection, develop higher anti-saliva IgG responses and IFN-γ levels and a skew towards a Th2-type cellular response, probably in favor of parasite establishment, compared to those living in an emerging focus.

We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway's antiviral role., Competing Interests: The authors have declared that no competing interests exist.

Background: Arboviral diseases are an important public health concerns. Vector control remains the sole strategy to fight against these diseases. Because of the important limits of methods currently used to assess human exposure to Aedes mosquito bites, much effort is being devoted to develop new indicators. Recent studies have reported that human antibody (Ab) responses to Aedes aegypti Nterm-34kDa salivary peptide represent a promising biomarker tool to evaluate the human-Aedes contact. The present study aims investigate whether such biomarker could be used for assessing the efficacy of vector control against Aedes., Methodology/principal Findings: Specific human IgG response to the Nterm-34kDa peptide was assessed from 102 individuals living in urban area of Saint-Denis at La Reunion Island, Indian Ocean, before and after the implementation of vector control against Aedes mosquitoes. IgG response decreased after 2 weeks (P < 0.0001), and remained low for 4 weeks post-intervention (P = 0.0002). The specific IgG decrease was associated with the decline of Aedes mosquito density, as estimated by entomological parameters and closely correlated to vector control implementation and was not associated with the use of individual protection, daily commuting outside of the house, sex and age. Our findings indicate a probable short-term decrease of human exposure to Aedes bites just after vector control implementation., Conclusion/significance: Results provided in the present study indicate that IgG Ab response to Aedes aegypti Nterm-34kDa salivary peptide could be a relevant short-time indicator for evaluating the efficacy of vector control interventions against Aedes species., Competing Interests: The authors have declared that no competing interests exist.

Background: During a blood meal, female sand flies, vectors of Leishmania parasites, inject saliva into the host skin. Sand fly saliva is composed of a large variety of components that exert different pharmacological activities facilitating the acquisition of blood by the insect. Importantly, proteins present in saliva are able to elicit the production of specific anti-saliva antibodies, which can be used as markers for exposure to vector bites. Serological tests using total sand fly salivary gland extracts are challenging due to the difficulty of obtaining reproducible salivary gland preparations. Previously, we demonstrated that PpSP32 is the immunodominant salivary antigen in humans exposed to Phlebotomus papatasi bites and established that humans exposed to P. perniciosus bites do not recognize it., Methodology/principal Findings: Herein, we have validated, in a large cohort of 522 individuals, the use of the Phlebotomus papatasi recombinant salivary protein PpSP32 (rPpSP32) as an alternative method for testing exposure to the bite of this sand fly. We also demonstrated that screening for total anti-rPpSP32 IgG antibodies is sufficient, being comparable in efficacy to the screening for IgG2, IgG4 and IgE antibodies against rPpSP32. Additionally, sera obtained from dogs immunized with saliva of P. perniciosus, a sympatric and widely distributed sand fly in Tunisia, did not recognize rPpSP32 demonstrating its suitability as a marker of exposure to P. papatasi saliva., Conclusions/significance: Our data indicate that rPpSP32 constitutes a useful epidemiological tool to monitor the spatial distribution of P. papatasi in a particular region, to direct control measures against zoonotic cutaneous leishmaniasis, to assess the efficiency of vector control interventions and perhaps to assess the risk of contracting the disease.

Tsetse are vectors of pathogenic trypanosomes, agents of human and animal trypanosomiasis in Africa. Components of tsetse saliva (sialome) are introduced into the mammalian host bite site during the blood feeding process and are important for tsetse's ability to feed efficiently, but can also influence disease transmission and serve as biomarkers for host exposure. We compared the sialome components from four tsetse species in two subgenera: subgenus Morsitans: Glossina morsitans morsitans (Gmm) and Glossina pallidipes (Gpd), and subgenus Palpalis: Glossina palpalis gambiensis (Gpg) and Glossina fuscipes fuscipes (Gff), and evaluated their immunogenicity and serological cross reactivity by an immunoblot approach utilizing antibodies from experimental mice challenged with uninfected flies. The protein and immune profiles of sialome components varied with fly species in the same subgenus displaying greater similarity and cross reactivity. Sera obtained from cattle from disease endemic areas of Africa displayed an immunogenicity profile reflective of tsetse species distribution. We analyzed the sialome fractions of Gmm by LC-MS/MS, and identified TAg5, Tsal1/Tsal2, and Sgp3 as major immunogenic proteins, and the 5'-nucleotidase family as well as four members of the Adenosine Deaminase Growth Factor (ADGF) family as the major non-immunogenic proteins. Within the ADGF family, we identified four closely related proteins (TSGF-1, TSGF-2, ADGF-3 and ADGF-4), all of which are expressed in tsetse salivary glands. We describe the tsetse species-specific expression profiles and genomic localization of these proteins. Using a passive-immunity approach, we evaluated the effects of rec-TSGF (TSGF-1 and TSGF-2) polyclonal antibodies on tsetse fitness parameters. Limited exposure of tsetse to mice with circulating anti-TSGF antibodies resulted in a slight detriment to their blood feeding ability as reflected by compromised digestion, lower weight gain and less total lipid reserves although these results were not statistically significant. Long-term exposure studies of tsetse flies to antibodies corresponding to the ADGF family of proteins are warranted to evaluate the role of this conserved family in fly biology.

A meta-analysis of the effects of vector saliva on the immune response and progression of vector-transmitted disease, specifically with regard to pathology, infection level, and host cytokine levels was conducted. Infection in the absence or presence of saliva in naïve mice was compared. In addition, infection in mice pre-exposed to uninfected vector saliva was compared to infection in unexposed mice. To control for differences in vector and pathogen species, mouse strain, and experimental design, a random effects model was used to compare the ratio of the natural log of the experimental to the control means of the studies. Saliva was demonstrated to enhance pathology, infection level, and the production of Th2 cytokines (IL-4 and IL-10) in naïve mice. This effect was observed across vector/pathogen pairings, whether natural or unnatural, and with single salivary proteins used as a proxy for whole saliva. Saliva pre-exposure was determined to result in less severe leishmaniasis pathology when compared with unexposed mice infected either in the presence or absence of sand fly saliva. The results of further analyses were not significant, but demonstrated trends toward protection and IFN-γ elevation for pre-exposed mice.

Background: Tsetse flies are obligate blood-feeding insects that transmit African trypanosomes responsible for human sleeping sickness and nagana in livestock. The tsetse salivary proteome contains a highly immunogenic family of the endonuclease-like Tsal proteins. In this study, a recombinant version of Tsal1 (rTsal1) was evaluated in an indirect ELISA to quantify the contact with total Glossina morsitans morsitans saliva, and thus the tsetse fly bite exposure., Methodology/principal Findings: Mice and pigs were experimentally exposed to different G. m. morsitans exposure regimens, followed by a long-term follow-up of the specific antibody responses against total tsetse fly saliva and rTsal1. In mice, a single tsetse fly bite was sufficient to induce detectable IgG antibody responses with an estimated half-life of 36-40 days. Specific antibody responses could be detected for more than a year after initial exposure, and a single bite was sufficient to boost anti-saliva immunity. Also, plasmas collected from tsetse-exposed pigs displayed increased anti-rTsal1 and anti-saliva IgG levels that correlated with the exposure intensity. A strong correlation between the detection of anti-rTsal1 and anti-saliva responses was recorded. The ELISA test performance and intra-laboratory repeatability was adequate in the two tested animal models. Cross-reactivity of the mouse IgGs induced by exposure to different Glossina species (G. m. morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and other hematophagous insects (Stomoxys calcitrans and Tabanus yao) was evaluated., Conclusion: This study illustrates the potential use of rTsal1 from G. m. morsitans as a sensitive biomarker of exposure to a broad range of Glossina species. We propose that the detection of anti-rTsal1 IgGs could be a promising serological indicator of tsetse fly presence that will be a valuable tool to monitor the impact of tsetse control efforts on the African continent.

Background: Salivary proteins of Triatoma infestans elicit humoral immune responses in their vertebrate hosts. These immune responses indicate exposure to triatomines and thus can be a useful epidemiological tool to estimate triatomine infestation. In the present study, we analyzed antibody responses of guinea pigs to salivary antigens of different developmental stages of four T. infestans strains originating from domestic and/or peridomestic habitats in Argentina, Bolivia, Chile and Peru. We aimed to identify developmental stage- and strain-specific salivary antigens as potential markers of T. infestans exposure., Methodology and Principal Findings: In SDS-PAGE analysis of salivary proteins of T. infestans the banding pattern differed between developmental stages and strains of triatomines. Phenograms constructed from the salivary profiles separated nymphal instars, especially the 5th instar, from adults. To analyze the influence of stage- and strain-specific differences in T. infestans saliva on the antibody response of guinea pigs, twenty-one guinea pigs were exposed to 5th instar nymphs and/or adults of different T. infestans strains. Western blot analyses using sera of exposed guinea pigs revealed stage- and strain-specific variations in the humoral response of animals. In total, 27 and 17 different salivary proteins reacted with guinea pig sera using IgG and IgM antibodies, respectively. Despite all variations of recognized salivary antigens, an antigen of 35 kDa reacted with sera of almost all challenged guinea pigs., Conclusion: Salivary antigens are increasingly considered as an epidemiological tool to measure exposure to hematophagous arthropods, but developmental stage- and strain-specific variations in the saliva composition and the respective differences of immunogenicity are often neglected. Thus, the development of a triatomine exposure marker for surveillance studies after triatomine control campaigns requires detailed investigations. Our study resulted in the identification of a potential antigen as useful marker of T. infestans exposure.

Background: The analysis of humoral responses directed against the saliva of blood-sucking arthropods was shown to provide epidemiological biomarkers of human exposure to vector-borne diseases. However, the use of whole saliva as antigen presents several limitations such as problems of mass production, reproducibility and specificity. The aim of this study was to design a specific biomarker of exposure to tsetse flies based on the in silico analysis of three Glossina salivary proteins (Ada, Ag5 and Tsgf1) previously shown to be specifically recognized by plasma from exposed individuals., Methodology/principal Findings: Synthetic peptides were designed by combining several linear epitope prediction methods and Blast analysis. The most specific peptides were then tested by indirect ELISA on a bank of 160 plasma samples from tsetse infested areas and tsetse free areas. Anti-Tsgf118-43 specific IgG levels were low in all three control populations (from rural Africa, urban Africa and Europe) and were significantly higher (p<0.0001) in the two populations exposed to tsetse flies (Guinean HAT foci, and South West Burkina Faso). A positive correlation was also found between Anti-Tsgf118-43 IgG levels and the risk of being infected by Trypanosoma brucei gambiense in the sleeping sickness foci of Guinea., Conclusion/significance: The Tsgf118-43 peptide is a suitable and promising candidate to develop a standardize immunoassay allowing large scale monitoring of human exposure to tsetse flies in West Africa. This could provide a new surveillance indicator for tsetse control interventions by HAT control programs.

Background: Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG) homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins., Methods and Findings: A cDNA library from the Salivary Glands (SGs) of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11--coding for a 4.5-kDa protein--induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells., Conclusions: We identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis.

Background: Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is highly prevalent in Tunisia and is transmitted by a hematophagous vector Phlebotomus papatasi (P. papatasi). While probing for a blood meal, the sand fly injects saliva into the host's skin, which contains a variety of compounds that are highly immunogenic. We recently showed that the presence of anti-saliva antibodies was associated with an enhanced risk for leishmaniasis and identified the immunodominant salivary protein of Phlebotomus papatasi as a protein of approximately 30 kDa., Methodology/principal Findings: We cloned and expressed in mammalian cells two salivary proteins PpSP30 and PpSP32 with predicted molecular weights close to 30 kDa from the Tunisian strain of P. papatasi. The two recombinant salivary proteins were purified by two-step HPLC (High-Performance Liquid Chromatography) and tested if these proteins correspond to the immunodominant antigen of 30 kDa previously shown to be recognized by human sera from endemic areas for ZCL and exposed naturally to P. papatasi bites. While recombinant PpSP30 (rPpSP30) was poorly recognized by human sera from endemic areas for ZCL, rPpSP32 was strongly recognized by the tested sera. The binding of human IgG antibodies to native PpSP32 was inhibited by the addition of rPpSP32. Consistently, experiments in mice showed that PpSP32 induced the highest levels of antibodies compared to other P. papatasi salivary molecules while PpSP30 did not induce any detectable levels of antibodies., Conclusions: Our findings demonstrate that PpSP32 is the immunodominant target of the antibody response to P. papatasi saliva. They also indicate that the recombinant form of PpSP32 is similar to the native one and represents a good candidate for large scale testing of human exposure to P. papatasi bites and perhaps for assessing the risk of contracting the disease.

Background: Phlebotomine sand flies are blood-sucking insects transmitting Leishmania parasites. In bitten hosts, sand fly saliva elicits specific immune response and the humoral immunity was shown to reflect the intensity of sand fly exposure. Thus, anti-saliva antibodies were suggested as the potential risk marker of Leishmania transmission. In this study, we examined the long-term kinetics and persistence of anti-Phlebotomus papatasi saliva antibody response in BALB/c and C57BL/6 mice. We also tested the reactivity of mice sera with P. papatasi salivary antigens and with the recombinant proteins., Methodology/principal Findings: Sera of BALB/c and C57BL/6 mice experimentally bitten by Phlebotomus papatasi were tested by ELISA for the presence of anti-saliva IgE, IgG and its subclasses. We detected a significant increase of specific IgG and IgG1 in both mice strains and IgG2b in BALB/c mice that positively correlated with the number of blood-fed P. papatasi females. Using western blot and mass spectrometry we identified the major P. papatasi antigens as Yellow-related proteins, D7-related proteins, antigen 5-related proteins and SP-15-like proteins. We therefore tested the reactivity of mice sera with four P. papatasi recombinant proteins coding for most of these potential antigens (PpSP44, PpSP42, PpSP30, and PpSP28). Each mouse serum reacted with at least one of the recombinant protein tested, although none of the recombinant proteins were recognized by all sera., Conclusions: Our data confirmed the concept of using anti-sand fly saliva antibodies as a marker of sand fly exposure in Phlebotomus papatasi-mice model. As screening of specific antibodies is limited by the availability of salivary gland homogenate, utilization of recombinant proteins in such studies would be beneficial. Our present work demonstrates the feasibility of this implementation. A combination of recombinant salivary proteins is recommended for evaluation of intensity of sand fly exposure in endemic areas and for estimation of risk of Leishmania transmission.

Background: Mosquito salivary proteins (MSPs) modulate the host immune response, leading to enhancement of arboviral infections. Identification of proteins in saliva responsible for immunomodulation and counteracting their effects on host immune response is a potential strategy to protect against arboviral disease. We selected a member of the D7 protein family, which are among the most abundant and immunogenic in mosquito saliva, as a vaccine candidate with the aim of neutralizing effects on the mammalian immune response normally elicited by mosquito saliva components during arbovirus transmission., Methodology/principal Findings: We identified D7 salivary proteins of Culex tarsalis, a West Nile virus (WNV) vector in North America, and expressed 36 kDa recombinant D7 (rD7) protein for use as a vaccine. Vaccinated mice exhibited enhanced interferon-γ and decreased interleukin-10 expression after uninfected mosquito bite; however, we found unexpectedly that rD7 vaccination resulted in enhanced pathogenesis from mosquito-transmitted WNV infection. Passive transfer of vaccinated mice sera to naïve mice also resulted in increased mortality rates from subsequent mosquito-transmitted WNV infection, implicating the humoral immune response to the vaccine in enhancement of viral pathogenesis. Vaccinated mice showed decreases in interferon-γ and increases in splenocytes producing the regulatory cytokine IL-10 after WNV infection by mosquito bite., Conclusions/significance: Vector saliva vaccines have successfully protected against other blood-feeding arthropod-transmitted diseases. Nevertheless, the rD7 salivary protein vaccine was not a good candidate for protection against WNV disease since immunized mice infected via an infected mosquito bite exhibited enhanced mortality. Selection of salivary protein vaccines on the bases of abundance and immunogenicity does not predict efficacy.

Background: Much effort is being devoted for developing new indicators to evaluate the human exposure to Aedes mosquito bites and the risk of arbovirus transmission. Human antibody (Ab) responses to mosquito salivary components could represent a promising tool for evaluating the human-vector contact., Methodology/principal Findings: To develop a specific biomarker of human exposure to Aedes aegypti bites, we measured IgG Ab response to Ae. aegypti Nterm-34 kDa salivary peptide in exposed children in 7 villages of Southern Benin (West Africa). Results showed that specific IgG response presented high inter-individual heterogeneity between villages. IgG response was associated with rainfall and IgG level increased from dry (low exposure) to rainy (high exposure) seasons. These findings indicate that IgG Ab to Nterm-34 kDa salivary peptide may represent a reliable biomarker to detect variation in human exposure to Ae. aegypti bites., Conclusion/significance: This preliminary study highlights the potential use of Ab response to this salivary peptide for evaluating human exposure to Ae. aegypti. This biomarker could represent a new promising tool for assessing the risk of arbovirus transmission and for evaluating the efficacy of vector control interventions.

Background: Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species., Methodology/principal Findings: Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins., Conclusions: Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.

Background: Pruritic scabies lesions caused by Sarcoptes scabiei burrowing in the stratum corneum of human skin facilitate opportunistic bacterial infections. Emerging resistance to current therapeutics emphasizes the need to identify novel targets for protective intervention. We have characterized several protein families located in the mite gut as crucial factors for host-parasite interactions. Among these multiple proteins inhibit human complement, presumably to avoid complement-mediated damage of gut epithelial cells. Peritrophins are major components of the peritrophic matrix often found in the gut of arthropods. We hypothesized that a peritrophin, if abundant in the scabies mite gut, could be an activator of complement., Methodology/principal Findings: A novel full length scabies mite peritrophin (SsPTP1) was identified in a cDNA library from scabies mites. The amino acid sequence revealed four putative chitin binding domains (CBD). Recombinant expression of one CBD of the highly repetitive SsPTP1 sequence as TSP-hexaHis-fusion protein resulted in soluble protein, which demonstrated chitin binding activity in affinity chromatography assays. Antibodies against a recombinant SsPTP1 fragment were used to immunohistochemically localize native SsPTP1 in the mite gut and in fecal pellets within the upper epidermis, co-localizing with serum components such as host IgG and complement. Enzymatic deglycosylation confirmed strong N- and O-glycosylation of the native peritrophin. Serum incubation followed by immunoblotting with a monoclonal antibody against mannan binding lectin (MBL), the recognition molecule of the lectin pathway of human complement activation, indicated that MBL may specifically bind to glycosylated SsPTP1., Conclusions/significance: This study adds a new aspect to the accumulating evidence that complement plays a major role in scabies mite biology. It identifies a novel peritrophin localized in the mite gut as a potential target of the lectin pathway of the complement cascade. These initial findings indicate a novel role of scabies mite peritrophins in triggering a host innate immune response within the mite gut.

Background: Sand flies deliver Leishmania parasites to a host alongside salivary molecules that affect infection outcomes. Though some proteins are immunogenic and have potential as markers of vector exposure, their identity and vector specificity remain elusive., Methodology/principal Findings: We screened human, dog, and fox sera from endemic areas of visceral leishmaniasis to identify potential markers of specific exposure to saliva of Lutzomyia longipalpis. Human and dog sera were further tested against additional sand fly species. Recombinant proteins of nine transcripts encoding secreted salivary molecules of Lu. longipalpis were produced, purified, and tested for antigenicity and specificity. Use of recombinant proteins corresponding to immunogenic molecules in Lu. longipalpis saliva identified LJM17 and LJM11 as potential markers of exposure. LJM17 was recognized by human, dog, and fox sera; LJM11 by humans and dogs. Notably, LJM17 and LJM11 were specifically recognized by humans exposed to Lu. longipalpis but not by individuals exposed to Lu. intermedia., Conclusions/significance: Salivary recombinant proteins are of value as markers of vector exposure. In humans, LJM17 and LJM11 emerged as potential markers of specific exposure to Lu. longipalpis, the vector of Leishmania infantum chagasi in Latin America. In dogs, LJM17, LJM11, LJL13, LJL23, and LJL143 emerged as potential markers of sand fly exposure. Testing these recombinant proteins in large scale studies will validate their usefulness as specific markers of Lu. longipalpis exposure in humans and of sand fly exposure in dogs.

Background: Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans., Methodology/principal Findings: T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI) database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6) in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia., Conclusions/significance: The recombinant rTiSP14.6 is a suitable and promising epidemiological marker for detecting the presence of small numbers of different species of triatomines and could be developed for use as a new tool in surveillance programs, especially to corroborate vector elimination in Chagas disease vector control campaigns.

Background: Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects biologically active salivary components that favorably change the environment at the feeding site. Exposure to bites or to salivary proteins results in immunity specific to these components. Mice immunized with Phlebotomus papatasi salivary gland homogenate (SGH) or pre-exposed to uninfected bites were protected against Leishmania major infection delivered by needle inoculation with SGH or by infected sand fly bites. Immunization with individual salivary proteins of two sand fly species protected mice from L. major infection. Here, we analyze the immune response to distinct salivary proteins from P. papatasi that produced contrasting outcomes of L. major infection., Methodology/principal Findings: DNA immunization with distinct DTH-inducing salivary proteins from P. papatasi modulates L. major infection. PpSP15-immunized mice (PpSP15-mice) show lasting protection while PpSP44-immunized mice (PpSP44-mice) aggravate the infection, suggesting that immunization with these distinct molecules alters the course of anti-Leishmania immunity. Two weeks post-infection, 31.5% of CD4(+) T cells produced IFN-gamma in PpSP15-mice compared to 7.1% in PpSP44-mice. Moreover, IL-4-producing cells were 3-fold higher in PpSP44-mice. At an earlier time point of two hours after challenge with SGH and L. major, the expression profile of PpSP15-mice showed over 3-fold higher IFN-gamma and IL-12-Rbeta2 and 20-fold lower IL-4 expression relative to PpSP44-mice, suggesting that salivary proteins differentially prime anti-Leishmania immunity. This immune response is inducible by sand fly bites where PpSP15-mice showed a 3-fold higher IFN-gamma and a 5-fold lower IL-4 expression compared with PpSP44-mice., Conclusions/significance: Immunization with two salivary proteins from P. papatasi, PpSP15 and PpSP44, produced distinct immune profiles that correlated with resistance or susceptibility to Leishmania infection. The demonstration for the first time that immunity to a defined salivary protein (PpSP44) results in disease enhancement stresses the importance of the proper selection of vector-based vaccine candidates.