Sand fly salivary proteins induce strong cellular immunity in a natural reservoir of visceral leishmaniasis with adverse consequences for Leishmania.
ABSTRACT: Immunity to a sand fly salivary protein protects against visceral leishmaniasis (VL) in hamsters. This protection was associated with the development of cellular immunity in the form of a delayed-type hypersensitivity response and the presence of IFN-gamma at the site of sand fly bites. To date, there are no data available regarding the cellular immune response to sand fly saliva in dogs, the main reservoirs of VL in Latin America, and its role in protection from this fatal disease. Two of 35 salivary proteins from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG(2) antibody levels and significant IFN-gamma production following in vitro stimulation of PBMC with salivary gland homogenate (SGH). Upon challenge with uninfected or infected flies, immunized dogs developed a cellular response at the bite site characterized by lymphocytic infiltration and IFN-gamma and IL-12 expression. Additionally, SGH-stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. Certain sand fly salivary proteins are potent immunogens obligatorily co-deposited with Leishmania parasites during transmission. Their inclusion in an anti-Leishmania vaccine would exploit anti-saliva immunity following an infective sand fly bite and set the stage for a protective anti-Leishmania immune response.
Project description: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.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.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.
Project description:BACKGROUND: Canine Visceral Leishmaniasis (CVL) is a zoonotic disease caused by Leishmania infantum, transmitted by the bite of Lutzomyia longipalpis sand flies. Dogs are the main domestic reservoir of the parasite. The establishment of an experimental model that partially reproduces natural infection in dogs is very important to test vaccine candidates, mainly regarding those that use salivary proteins from the vector and new therapeutical approaches. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we describe an experimental infection in dogs, using intradermal injection of Leishmania infantum plus salivary gland homogenate (SGH) of Lutzomyia longipalpis. Thirty-five dogs were infected with 1×10(7) parasites combined with five pairs of Lutzomyia longipalpis salivary glands and followed for 450 days after infection and clinical, immunological and parasitological parameters were evaluated. Two hundred and ten days after infection we observed that 31,4% of dogs did not display detectable levels of anti-Leishmania antibodies but all presented different numbers of parasites in the lymph nodes. Animals with a positive xenodiagnosis had at least 3,35×10(5) parasites in their lymph nodes. An increase of IFN-? and IL-10 levels was detected during infection. Twenty two percent of dogs developed symptoms of CVL during infection. CONCLUSION: The infection model described here shows some degree of similarity when compared with naturally infected dogs opening new perspectives for the study of CVL using an experimental model that employs the combination of parasites and sand fly saliva both present during natural transmission.
Project description:Visceral leishmaniasis (VL) is a fatal disease for humans, and no vaccine is currently available. Sand fly salivary proteins have been associated with protection against cutaneous leishmaniasis. To test whether vector salivary proteins can protect against VL, a hamster model was developed involving intradermal inoculation in the ears of 100,000 Leishmania infantum chagasi parasites together with Lutzomyia longipalpis saliva to mimic natural transmission by sand flies. Hamsters developed classical signs of VL rapidly, culminating in a fatal outcome 5-6 months postinfection. Saliva had no effect on the course of infection in this model. Immunization with 16 DNA plasmids coding for salivary proteins of Lu. longipalpis resulted in the identification of LJM19, a novel 11-kDa protein, that protected hamsters against the fatal outcome of VL. LJM19-immunized hamsters maintained a low parasite load that correlated with an overall high IFN-gamma/TGF-beta ratio and inducible NOS expression in the spleen and liver up to 5 months postinfection. Importantly, a delayed-type hypersensitivity response with high expression of IFN-gamma was also noted in the skin of LJM19-immunized hamsters 48 h after exposure to uninfected sand fly bites. Induction of IFN-gamma at the site of bite could partly explain the protection observed in the viscera of LJM19-immunized hamsters through direct parasite killing and/or priming of anti-Leishmania immunity. We have shown that immunity to a defined salivary protein (LJM19) confers powerful protection against the fatal outcome of a parasitic disease, which reinforces the concept of using components of arthropod saliva in vaccine strategies against vector-borne diseases.
Project description:<h4>Background</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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-? 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.<h4>Conclusions</h4>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.
Project description:Leishmania parasites are transmitted to their vertebrate hosts by infected phlebotomine sand fly bites. Sand fly saliva is known to enhance Leishmania infection, while immunity to the saliva protects against infection as determined by coinoculation of parasites with vector salivary gland homogenates (SGHs) or by infected sand fly bites (Kamhawi, S., Y. Belkaid, G. Modi, E. Rowton, and D. Sacks. 2000. Science. 290:1351-1354). We have now characterized nine salivary proteins of Phlebotomus papatasi, the vector of Leishmania major. One of these salivary proteins, extracted from SDS gels and having an apparent mol wt of 15 kD, was able to protect vaccinated mice challenged with parasites plus SGH. A DNA vaccine containing the cDNA for the predominant 15-kD protein (named SP15) provided this same protection. Protection lasted at least 3 mo after immunization. The vaccine produced both intense humoral and delayed-type hypersensitivity (DTH) reactions. B cell-deficient mice immunized with the SP15 plasmid vaccine successfully controlled Leishmania infection when injected with Leishmania plus SGH. These results indicate that DTH response against saliva provides most or all of the protective effects of this vaccine and that salivary gland proteins or their cDNAs are viable vaccine targets against leishmaniasis.
Project description:Leishmania vaccines that protect against needle challenge fail against the potency of a Leishmania-infected sand fly transmission. Here, we demonstrate that intradermal immunization of mice with 500 ng of the sand fly salivary recombinant protein LJM11 (rLJM11) from Lutzomyia longipalpis, in the absence of adjuvant, induces long-lasting immunity that results in ulcer-free protection against Leishmania major delivered by vector bites. This protection is antibody independent and abrogated by depletion of CD4(+) T cells. Two weeks after challenge, early induction of IFN-γ specifically to rLJM11 correlates to diminished parasite replication in protected animals. At this time point, Leishmania-specific induction of IFN-γ in these mice is low in comparison with its high level in non-protected controls. We hypothesize that early control of parasites in a T-cell helper type 1 environment induced by immunity to LJM11 permits the slow development of Leishmania-specific immunity in the absence of open ulcers. Leishmania-specific immunity observed 5 weeks after infection in rLJM11-immunized mice shows a twofold increase over controls in the percentage of IFN-γ-producing CD4(+) T cells. We propose LJM11 as an immunomodulator that drives an efficient and controlled protective immune response to a sand fly-transmitted Leishmania somewhat mimicking "leishmanization"-induced protective immunity but without its associated lesions.
Project description: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.
Project description:BACKGROUND:In endemic areas of zoonotic leishmaniosis caused by L. infantum, early detection of Leishmania infection in dogs is essential to control the dissemination of the parasite to humans. The aim of this study was to evaluate the serological and/or molecular diagnostic performance of minimally and non-invasive samples (conjunctiva cells (CS) and peripheral blood (PB)) for monitoring Leishmania infection/exposure to Phlebotomus perniciosus salivary antigens in dogs at the beginning and the end of sand fly seasonal activity (May and October, respectively) and to assess associated risks factors. METHODS:A total of 208 sheltered dogs from endemic areas of leishmaniosis were screened. Leishmania DNA detection in PB on filter paper and CS was performed by nested-PCR (nPCR), while the detection of anti-Leishmania antibodies was performed using IFAT and ELISA. The exposure to P. perniciosus salivary antigens (SGH, rSP01 and rSP03B?+?rSP01) was measured by ELISA. RESULTS:Ninety-seven (46.6%) and 116 (55.8%) of the 208 dogs were positive to Leishmania antibodies or DNA by at least one test at the beginning and end of the sand fly season, respectively. IFAT and ELISA presented a substantial agreement in the serodiagnosis of leishmaniosis. Discrepant PB nPCR results were obtained between sampling points. Leishmania DNA was detected in CS of 72 dogs at the end of the phlebotomine season. The presence of antibodies to the parasite measured by ELISA was significantly higher in dogs presenting clinical signs compatible with leishmaniosis at both sampling points. Phlebotomus perniciosus salivary antibodies were detected in 179 (86.1%) and 198 (95.2%) of the screened dogs at the beginning and end of the phlebotomine season, respectively. CONCLUSIONS:The association between ELISA positivity and clinical signs suggests its usefulness to confirm a clinical suspicion. CS nPCR seems to be an effective and non-invasive method for assessing early exposure to the parasite. PB nPCR should not be used as the sole diagnostic tool to monitor Leishmania infection. The correlation between the levels of antibodies to P. perniciosus saliva and Leishmania antibodies suggests the use of a humoral response to sand fly salivary antigens as biomarkers of L. infantum infection.
Project description:BACKGROUND:Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. METHODOLOGY/PRINCIPAL FINDINGS:Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. CONCLUSIONS/SIGNIFICANCE:Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.
Project description:The anti-inflammatory properties of sand fly saliva favor the establishment of the Leishmania infantum infection. In contrast, an antibody response against Lutzomyia longipalpis saliva is often associated with a protective cell-mediated response against canine visceral leishmaniasis. Genetic studies may demonstrate to what extent the ability to secrete anti-saliva antibodies depends on genetic or environmental factors. However, the genetic basis of canine antibody response against sand fly saliva has not been assessed. The aim of this study was to identify chromosomal regions associated with the anti-Lu. longipalpis salivary IgG response in 189 dogs resident in endemic areas in order to provide information for prophylactic strategies. Dogs were classified into five groups based on serological and parasitological diagnosis and clinical evaluation. Anti-salivary gland homogenate (SGH) IgG levels were assessed by Enzyme-Linked Immunosorbent Assay (ELISA). Genomic DNA was isolated from blood samples and genotyped using a SNP chip with 173,662 single nucleotide polymorphism (SNP) markers. The following linear regression model was fitted: IgG level = mean + origin + sex + age + use of a repellent collar, and the residuals were assumed as pseudo-phenotypes for the association test between phenotypes and genotypes (GWA). A component of variance model that takes into account polygenic and sample structure effects (EMMAX) was employed for GWA. Phenotypic findings indicated that anti-SGH IgG levels remained higher in exposed and subclinically infected dogs than in severely diseased dogs even in regression model residuals. Five associated markers were identified on chromosomes 2, 20 and 31. The mapped genes included CD180 (RP105) and MITF related to the rapid activation of B lymphocytes and differentiation into antibody-secreting plasma cells. The findings pointed to chromosomal segments useful for functional confirmation studies and a search for adjuvant molecules of the anti-saliva response.