Individuals co-exposed to sand fly saliva and filarial parasites exhibit altered monocyte function.

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.

The human immune response to saliva of Phlebotomus alexandri, the vector of visceral leishmaniasis in Iraq, and its relationship to sand fly exposure and infection.

BACKGROUND: Sand fly saliva exposure plays an important role in immunity against leishmaniasis where it has mostly been associated with protection. Phlebotomus (Ph.) alexandri transmits Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), in Iraq. Our group recently demonstrated that 20% of Operation Iraqi Freedom (OIF) deployers had asymptomatic VL (AVL) indicative of prior infection by the parasite L. infantum. Little is known about Ph. alexandri saliva, and the human immune response to it has never been investigated. Here, we characterize the humoral and cellular immune response to vector saliva in OIF deployers naturally exposed to bites of Ph. alexandri and characterize their immunological profiles in association to AVL. METHODOLOGY/PRINCIPAL FINDINGS: The humoral response to Ph. alexandri salivary gland homogenate (SGH) showed that 64% of 200 OIF deployers developed an antibody response. To assess the cellular immune response to saliva, we selected a subcohort of subjects based on their post-travel (median 4 months; range 1-22 months) antibody response (SGH Antibody [Ab] positive or negative) as well as their AVL status; ten never-traveled controls were also included. Banked peripheral blood mononuclear cells (PBMC), collected ~10 years after end of deployment, were stimulated with SGH for 96 hours. The levels of IFN- γ, IL-6, IL-10, IL-13 and IL-17 were determined by ELISA. Our findings indicate that OIF deployers mounted a cellular response to SGH where the anti-SGH+ asymptomatic subjects developed the highest cytokine levels. Further, stimulation with SGH produced a mixture of pro-inflammatory and anti-inflammatory cytokines. Contrary to our hypothesis, we observed no correlation between the cellular immune response to Ph. alexandri SGH and prevention from asymptomatic infection with L. infantum. CONCLUSIONS/SIGNIFICANCE: As we found, although all infected deployers demonstrated persistent disease control years after deployment, this did not correlate with anti-saliva systemic cellular response. More exposure to this vector may facilitate transmission of the L. infantum parasite. Since exposure to saliva of Ph. alexandri may alter the human immune response to bites of this vector, this parameter should be taken into consideration when considering the VL risk.

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis.

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Implicating bites from a leishmaniasis sand fly vector in the loss of tolerance in pemphigus.

A possible etiological link between the onset of endemic pemphigus in Tunisia and bites of Phlebotomus papatasi, the vector of zoonotic cutaneous leishmaniasis, has been previously suggested. We hypothesized that the immunodominant P. papatasi salivary protein PpSP32 binds to desmogleins 1 and 3 (Dsg1 and Dsg3), triggering loss of tolerance to these pemphigus target autoantigens. Here, we show using far-Western blot that the recombinant PpSP32 protein (rPpSP32) binds to epidermal proteins with a MW of approximately 170 kDa. Coimmunoprecipitation revealed the interaction of rPpSP32 with either Dsg1 or Dsg3. A specific interaction between PpSP32 and Dsg1 and Dsg3 was further demonstrated by ELISA assays. Finally, mice immunized with rPpSP32 twice per week exhibited significantly increased levels of anti-Dsg1 and -Dsg3 antibodies from day 75 to 120. Such antibodies were specific for Dsg1 and Dsg3 and were not the result of cross-reactivity to PpSP32. In this study, we demonstrated for the first time to our knowledge a specific binding between PpSP32 and Dsg1 and Dsg3, which might underlie the triggering of anti-Dsg antibodies in patients exposed to sand fly bites. We also confirmed the development of specific anti-Dsg1 and -Dsg3 antibodies in vivo after PpSP32 immunization in mice. Collectively, our results provide evidence that environmental factors, such as the exposure to P. papatasi bites, can trigger the development of autoimmune antibodies.

Human immune response to Phlebotomus sergenti salivary gland antigens in a leishmaniasis-endemic focus in Iran.

Salivary proteins specific antibodies have been shown to be useful biomarkers of exposure to sand fly bites. This study aimed to investigate the level, duration, and dynamics of the human immune response against the SGL of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae), and to assess the immunoreactivity of human sera with SGL components in an endemic area of anthroponotic cutaneous leishmaniasis (ACL) in Iran. The study was carried out in 2-phase; longitudinal and cross-sectional. Sand flies were collected monthly from indoors and outdoors. In the longitudinal study, sera from healthy volunteers were collected monthly, and in the cross-sectional study, sera from healthy volunteers and patients with ACL lesion/s, were collected for immunoassay studies. The level of anti-P. sergenti saliva IgG was detected using the ELISA. Immunoreactivity of individual human sera with saliva components was also assessed by western blotting. Phlebotomus sergenti was the predominant sand fly species in the study area. The maximum and minimum percentages of IgG responses were seen in October (66%) and March (29%), respectively. Additionally, the cross-sectional study showed that 59.3% of the healthy volunteers and 80% of the patients were IgG positive. The antibody response against P. sergenti salivary gland was high during the sand fly active season and declined by the end of the activity of the vectors.  Antibody response against the SGL components of P. sergenti was transient and individual-specific. Some individuals shared a strong reaction against certain individual antigens, which could be considered as vector exposure markers for further investigation. LIST OF ABBREVIATIONS: ELISA: Enzyme-Linked Immunosorbent Assay; SDS PAGE: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis; SGL: Salivary Gland Lysate; ACL: Anthroponotic Cutaneous Leishmaniasis; PBS: Phosphate Buffered Saline; BCA: Bicinchoninic Acid; PBS-T: Phosphate Buffered Saline Tween; FBS: Fetal Bovine Serum; HRP: Horseradish Peroxidase; TMB: 3,3',5,5'-Tetramethylbenzidine; PVDF: Polyvinylidene Difluoride; SGA: Salivary Gland Antigens; OD: Optical Density; KDa: Kilodalton; VL: Visceral Leishmaniasis; CL: Cutaneous Leishmaniasis; SGs: Salivary glands.

Phlebotomus papatasi sand fly predicted salivary protein diversity and immune response potential based on in silico prediction in Egypt and Jordan populations.

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.

Exploring the relationship between susceptibility to canine leishmaniosis and anti-Phlebotomus perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in Mallorca, Spain.

BACKGROUND: Canine leishmaniosis caused by Leishmania infantum is a neglected zoonosis transmitted by sand flies like Phlebotomus perniciosus. Clinical signs and disease susceptibility vary according to various factors, including host immune response and breed. In particular, Ibizan hounds appear more resistant. This immunocompetence could be attributed to a more frequent exposure to uninfected sand flies, eliciting a stronger anti-sand fly saliva antibody response. METHODS: This study aimed to investigate the prevalence of anti-P. perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in the Leishmania-endemic area of Mallorca, Spain, and to correlate these antibody levels with clinical, immunological and parasitological parameters. Anti-sand fly saliva IgG was examined in 47 Ibizan hounds and 45 dogs of other breeds using three methods: P. perniciosus whole salivary gland homogenate (SGH) ELISA; recombinant protein rSP03B ELISA; and rSP03B rapid tests (RT). Additionally, diagnostic performance was evaluated between methods. RESULTS: Results indicate significantly higher anti-SGH antibodies (P = 0.0061) and a trend for more positive SGH ELISA and RT results in Ibizan hounds compared to other breeds. General linear model analysis also found breed to be a significant factor in SGH ELISA units and a marginally significant factor in RT result. Although infection rates were similar between groups, Ibizan hounds included significantly more IFN-γ producers (P = 0.0122) and papular dermatitis cases (P < 0.0001). Older age and L. infantum seropositivity were also considered significant factors in sand fly saliva antibody levels according to at least one test. Fair agreement was found between all three tests, with the highest value between SGH and rSP03B RT. CONCLUSIONS: To our knowledge, this is the first study elaborating the relationship between anti-P. perniciosus saliva antibodies and extensive clinical data in dogs in an endemic area. Our results suggest that Ibizan hounds experience a higher frequency of exposure to sand flies and have a stronger cellular immune response to L. infantum infection than other breed dogs. Additional sampling is needed to confirm results, but anti-P. perniciosus saliva antibodies appear to negatively correlate with susceptibility to L. infantum infection and could possibly contribute to the resistance observed in Ibizan hounds.

Site specific microbiome of Leishmania parasite and its cross-talk with immune milieu.

Microbiota consists of commensal, symbiotic and pathogenic microorganisms found in all multicellular organisms. These micro-organisms are found in or on many parts of the body, including the intestinal tract, skin, mouth, and the reproductive tract. This review focuses on interplay of site specific microbiota, vector microbiota along with immune response and severity of Leishmaniasis. Herein, we have reviewed and summarized the counter effect of microbiome post infection with the Leishmania parasite. We have studied skin microbiome along with the gut microbiome of sand-fly which is the vector for transmission of this disease. Our major focus was to understand the skin and gut microbiome during Leishmania infection,their interaction and effect on immunological responses generated during the infection.Moreover, systems biology approach is envisioned to enumerate bacterial species in skin microbiota and Phlebotmus gut microbiota during Leishmania infection.

CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major.

Sand flies are the natural vectors for the Leishmania species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/Leishmania interactions have been limited by the absence of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the Phlebotomus papatasi sand fly, a natural vector for Leishmania major, targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in P. papatasi and were able to generate transmissible null mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with L. major Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for Leishmania parasites.IMPORTANCE Sand flies are the natural vectors of Leishmania parasites. Studies of sand fly/Leishmania interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible null mutant alleles of relish, a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with L. major, showing that the sand fly immune response impacts its vector competence for this pathogen.

Survival Strategies of Leishmania in Hosts

In all major parasite groups, new and surprising evidence is emerging every day about the subtlety, complexity and diversity of avoidance mechanisms from host immune system. In the course of millions of years of evolutionary process, mammalian and sand fly hosts have developed defense systems against Leishmania, but Leishmania has not only escaped from their hosts' defense systems through complex counter-strategies, but has also managed to manipulate them to support their own survival and reproduction. In this study, Leishmania's survival strategies used in the sand fly and mammalian hosts and the mechanisms that underlie these strategies will be summarized.

Antibody response to Phlebotomus perniciosus saliva in cats naturally exposed to phlebotomine sand flies is positively associated with Leishmania infection.

BACKGROUND: Zoonotic leishmaniosis, caused by the protozoan Leishmania infantum, is a public and animal health problem in Asia, Central and South America, the Middle East and the Mediterranean Basin. Several phlebotomine sand fly species from the subgenus Larroussius are vectors of L. infantum. Data from dogs living in endemic areas of leishmaniosis advocate the use of antibody response to phlebotomine sand fly saliva as an epidemiological biomarker for monitoring vector exposure. The aim of this study was to analyse the exposure of cats to phlebotomine sand flies using detection of IgG antibodies to Phlebotomus perniciosus saliva. The association between phlebotomine sand fly exposure and the presence of Leishmania infection was also investigated. RESULTS: IgG antibodies to P. perniciosus saliva were detected in 167 (47.7%) out of 350 cats; higher antibody levels were present in sera collected during the period of phlebotomine sand fly seasonal activity (OR = 19.44, 95% CI: 9.84-38.41). Cats of 12-35 months had higher antibody levels than younger ones (OR = 3.56, 95% CI: 1.39-9.16); this difference was also significant with older cats (for 36-95 months-old, OR = 9.43, 95% CI: 3.62-24.48; for older than 95 months, OR = 9.68, 95% CI: 3.92-23.91). Leishmania spp. DNA was detected in the blood of 24 (6.9%) cats, while antibodies to L. infantum were detected in three (0.9%). Only one cat was positive to Leishmania by both techniques. Cats presenting IgG antibodies to P. perniciosus had a significantly higher risk of being positive for Leishmania infection. CONCLUSIONS: To our knowledge, this is the first study demonstrating anti-sand fly saliva antibodies in cats. The evaluation of the contact of this animal species with the vector is important to the development of prophylactic measures directed to cats, with the aim of reducing the prevalence of infection in an endemic area. Therefore, studies evaluating whether the use of imidacloprid/flumethrin collars reduces the frequency of P. perniciosus bites in cats are needed. It is also important to evaluate if there is a correlation between the number of phlebotomine sand fly bites and IgG antibody levels.

Synthetic peptides as a novel approach for detecting antibodies against sand fly saliva.

BACKGROUND: Hosts repeatedly bitten by sand flies develop antibodies against sand fly saliva and screening of these immunoglobulins can be employed to estimate the risk of Leishmania transmission, to indicate the feeding preferences of sand flies, or to evaluate the effectiveness of vector control campaigns. Previously, antibodies to sand fly saliva were detected using whole salivary gland homogenate (SGH) or recombinant proteins, both of which also have their disadvantages. This is the first study on sand flies where short peptides designed based on salivary antigens were successfully utilized for antibody screening. METHODOLOGY/PRINCIPAL FINDINGS: Specific IgG was studied in hosts naturally exposed to Phlebotomus orientalis, the main vector of Leishmania donovani in East Africa. Four peptides were designed by the commercial program EpiQuest-B, based on the sequences of the two most promising salivary antigens, yellow-related protein and ParSP25-like protein. Short amino acid peptides were synthesised and modified for ELISA experiments. Specific anti-P. orientalis IgG was detected in sera of dogs, goats, and sheep from Ethiopia. The peptide OR24 P2 was shown to be suitable for antibody screening; it correlated positively with SGH and its specificity and sensitivity were comparable or even better than that of previously published recombinant proteins. CONCLUSIONS/SIGNIFICANCE: OR24 P2, the peptide based on salivary antigen of P. orientalis, was shown to be a valuable tool for antibody screening of domestic animals naturally exposed to P. orientalis. We suggest the application of this promising methodology using species-specific short peptides to other sand fly-host combinations.

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

BACKGROUND: The 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 RESULTS: In 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. CONCLUSION: This 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.

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.

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

BACKGROUND: Canine 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. METHODS: In 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. RESULTS: The 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. CONCLUSIONS: Our 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.

BACKGROUND: Canine 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 FINDINGS: The 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/SIGNIFICANCE: We 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.

Exploring new immunological insight on SP15 (∼14 kDa) family protein in saliva of Indian sand-fly (Phlebotomus argentipes) in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is a disease caused by protozoan species of the genus Leishmania and is transmitted through bites from the Phlebotomus sand fly; it is associated with considerable morbidity and mortality in many parts of world, including India. Reports on the protective role played by saliva proteins of Lutozomyia longipalpis, Phlebotomus papatasi and Phlebotomus duboscqi. are available. However, no studies have explored the salivary proteins of P. argentipes, which is the known proven vector for the transmission of VL in the Indian sub-continent. Herein we revealed the presence of two proteins of 14.2 and one protein of 13.6 kDa in Indian strain P. argentipes which is absolute identical to previously reported protein of SP15 family (PagSP01, PagSP02 and PagSP07) of P. argentipes of NIH colony, USA. In an experimental study on P. argentipes from Bihar, India, we demonstrated that a strong humoral and cellular immune response was triggered to reduce the concomitant Leishmania load in groups of immunized mice. The immunized group produced a considerable amount of IgG antibodies, and their splenocytes generated TH1 cytokines (IL-12, IFN-γ) with the support of delayed-type hypersensitivity (DTH) reactivity in such mice at the challenged site. We summarize from our data that some identical proteins to previous from SP15 family protein of 14.2 and 13.6 kDa molecular size, derived from Indian P. argentipes and reported its first time, can also be significant in resolution of VL infection after modulation of host protective T cell response in VL.

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

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

Immunization with LJM11 salivary protein protects against infection with Leishmania braziliensis in the presence of Lutzomyia longipalpis saliva.

Leishmania is transmitted in the presence of sand fly saliva. Protective immunity generated by saliva has encouraged identification of a vector salivary-based vaccine. Previous studies have shown that immunization with LJM11, a salivary protein from Lutzomyia longipalpis, is able to induce a Th1 immune response and protect mice against bites of Leishmania major-infected Lutzomyia longipalpis. Here, we further investigate if immunization with LJM11 recombinant protein is able to confer cross-protection against infection with Leishmania braziliensis associated with salivary gland sonicate (SGS) from Lutzomyia intermedia or Lu. longipalpis. Mice immunized with LJM11 protein exhibited an increased production of anti-LJM11 IgG, IgG1 and IgG2a and a DTH response characterized by an inflammatory infiltrate with the presence of CD4+ IFN-γ+ T cells. LJM11-immunized mice were intradermally infected in the ear with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia SGS. A significant reduction of parasite numbers in the ear and lymph node in the group challenged with L. braziliensis plus Lu. longipalpis SGS was observed, but not when the challenge was performed with L. braziliensis plus Lu. intermedia SGS. A higher specific production of IFN-γ and absence of IL-10 by lymph node cells were only observed in LJM11 immunized mice after infection. After two weeks, a similar frequency of CD4+ IFN-γ+ T cells was detected in LJM11 and BSA groups challenged with L. braziliensis plus Lu. longipalpis SGS, suggesting that early events possibly triggered by immunization are essential for protection against Leishmania infection. Our findings support the specificity of saliva-mediated immune responses and reinforce the importance of identifying cross-protective salivary antigens.

Human cellular and humoral immune responses to Phlebotomus papatasi salivary gland antigens in endemic areas differing in prevalence of Leishmania major infection.

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.