Immunogenicity and Serological Cross-Reactivity of Saliva Proteins among Different Tsetse Species

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.     

Tsetse fly saliva biases the immune response to Th2 and induces anti-vector antibodies that are a useful tool for exposure assessment

Tsetse flies (Glossina sp.) are blood-feeding dipteran insects that transmit African trypanosomes, parasites that are responsible for human sleeping sickness and veterinary infections. Increasing attention is being paid to the effects of tsetse fly saliva deposited at the feeding site, which enables the blood-feeding process and putatively promotes parasite transmission. Here we demonstrate that saliva induces strong humoral responses against the major 43-45 kDa protein fraction (tsetse salivary gland proteins 1 and 2 - Tsal1 and Tsal2) in mice and humans and suppresses murine T and B cell responses to heterologous antigen. The saliva-induced immune response is associated with a Th2-biased cytokine profile and the production of mainly IgG1 and IgE antibody isotypes. Functionally, the antibodies raised in mice exposed to tsetse fly bites or induced after experimental saliva immunisation do not affect the fly's blood-feeding efficiency nor its survival. We propose that anti-saliva as well as anti-Tsal1/2 antibody responses can be used in epidemiological studies as a tool to analyze human exposure to tsetse flies.     

Identification of a functional Antigen5-related allergen in the saliva of a blood feeding insect,the tsetse fly

Our previous screening of a Glossina morsitans morsitans λgt11 salivary gland expression library with serum of a tsetse fly exposed rabbit identified a cDNA encoding Tsetse Antigen5 (TAg5, 28.9 kDa), a homologue of Antigen5 sting venom allergens. Recombinant TAg5 was produced in Sf9 cells in order to assess its immunogenic properties in humans. Plasma from a patient that previously exhibited anaphylactic reactions against tsetse fly bites contained circulating anti-TAg5 and anti-saliva IgEs. In a significant proportion of plasma samples of African individuals, TAg5 and saliva binding IgEs (respectively 56 and 65%) can be detected. Saliva, harvested from flies that were subjected to TAg5-specific RNA interference (RNAi), displayed significantly reduced IgE binding potential. Allergenic properties of TAg5 and tsetse fly saliva were further illustrated in immunized mice, using an immediate cutaneous hypersensitivity and passive cutaneous anaphylaxis assay. Collectively, TAg5 was illustrated to be a tsetse fly salivary allergen, demonstrating that Antigen5-related proteins are represented as functional allergens not only in stinging but also in blood feeding insects.     

Identification of a Tsetse Fly Salivary Protein with Dual Inhibitory Action on Human Platelet Aggregation

Background

Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated.

Methodology/Principal Findings

Here we report on the functional characterisation of a 5′nucleotidase-related (5′Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5′Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5′nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5′Nuc/SUMO-fusion protein yielded an active apyrase enzyme with K_m and V_max values of 43±4 µM and 684±49 nmol Pi/min×mg for ATPase and 49±11 µM and 177±37 nmol Pi/min×mg for the ADPase activity. In addition, recombinant 5′Nuc was found to bind to GPIIb/IIIa with an apparent K_D of 92±25 nM. Consistent with these features, 5′Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5′Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process.

Conclusions/Significance

These data show that this 5′nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva.     

Description of a Nanobody-based Competitive Immunoassay to Detect Tsetse Fly Exposure

Background

Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts.

Methodology/Principal Findings

A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%).

Conclusion/Significance

We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.     

The Glossina morsitans tsetse fly saliva: general characteristics and identification of novel salivary proteins

The tsetse fly (Glossina spp.) is an obligate blood-sucking insect that transmits different human-pathogenic and livestock threatening trypanosome species in Africa. To obtain more insight in the tsetse salivary function, some general aspects of the tsetse fly saliva and its composition were studied. Direct pH and protein content measurements revealed a moderately alkaline (pH approximately 8.0) salivary environment with approximately 4.3 microg soluble proteins per gland and a constant representation of the major saliva proteins throughout the blood-feeding cycle. Although major salivary genes are constitutively expressed, upregulation of salivary protein synthesis within 48 h after the blood meal ensures complete protein replenishment from day 3 onwards. Screening of a non-normalised Glossina morsitans morsitans lambdagt11 salivary gland expression library with serum from a saliva-immunized rabbit identified three full-length cDNAs encoding for novel salivary proteins with yet unknown functions: a 8.3 kDa glycine/glutamate-rich protein (G. morsitans morsitans salivary gland protein Gmmsgp1), a 12.0 kDa proline-rich protein (Gmmsgp2), and a 97.4 kDa protein composed of a metallophosphoesterase/5'nucleotidase region with a glutamate/aspartate/asparagines-rich region (Gmmsgp3).     

Identification of major soluble salivary gland proteins in teneral Glossina morsitans morsitans

Salivary glands of tsetse flies (Diptera: Glossinidiae) contain molecules that are involved in preventing blood clotting during feeding as well as molecules thought to be intimately associated with trypanosome development and maturation. Here we present a protein microchemical analysis of the major soluble proteins of the salivary glands of Glossina morsitans morsitans, an important vector of African trypanosomes. Differential solubilization of salivary proteins was followed by reverse-phase, high-performance liquid chromatography (HPLC) and analysis of fractions by 1-D gel electrophoresis to reveal four major proteins. Each protein was subjected to amino acid microanalysis and N-terminal microsequencing. A protein chemical approach using high-resolution 2-D gel electrophoresis and mass spectrometry was also used to identify the salivary proteins. Matrix-assisted, laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry methods were used for peptide mass mapping and sequencing, respectively. Sequence information and peptide mass maps queried against the NCBI non-redundant database confirmed the identity of the first protein as tsetse salivary gland growth factor-1 (TSGF-1). Two proteins with no known function were identified as tsetse salivary gland protein 1 (Tsal 1) and tsetse salivary gland protein 2 (Tsal 2). The fourth protein was identified as Tsetse antigen-5 (TAg-5), which is a member of a large family of anti-haemostatic proteins. The results show that these four proteins are the most abundant soluble gene products present in salivary glands of teneral G. m. morsitans. We discuss the possible functions of these major proteins in cyclical transmission of African trypanosomes.     

Sand Fly Salivary Proteins Induce Strong Cellular Immunity in a Natural Reservoir of Visceral Leishmaniasis with Adverse Consequences for Leishmania

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-γ 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 IgG₂ antibody levels and significant IFN-γ 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-γ 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.     

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

BackgroundPhlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs.ConclusionsThese results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas.     

Using Recombinant Proteins from Lutzomyia longipalpis Saliva to Estimate Human Vector Exposure in Visceral Leishmaniasis Endemic Areas

Background

Leishmania is transmitted by female sand flies and deposited together with saliva, which contains a vast repertoire of pharmacologically active molecules that contribute to the establishment of the infection. The exposure to vector saliva induces an immune response against its components that can be used as a marker of exposure to the vector. Performing large-scale serological studies to detect vector exposure has been limited by the difficulty in obtaining sand fly saliva. Here, we validate the use of two sand fly salivary recombinant proteins as markers for vector exposure.

Methodology/principal findings

ELISA was used to screen human sera, collected in an area endemic for visceral leishmaniasis, against the salivary gland sonicate (SGS) or two recombinant proteins (rLJM11 and rLJM17) from Lutzomyia longipalpis saliva. Antibody levels before and after SGS seroconversion (n = 26) were compared using the Wilcoxon signed rank paired test. Human sera from an area endemic for VL which recognize Lu. longipalpis saliva in ELISA also recognize a combination of rLJM17 and rLJM11. We then extended the analysis to include 40 sera from individuals who were seropositive and 40 seronegative to Lu. longipalpis SGS. Each recombinant protein was able to detect anti-saliva seroconversion, whereas the two proteins combined increased the detection significantly. Additionally, we evaluated the specificity of the anti-Lu. longipalpis response by testing 40 sera positive to Lutzomyia intermedia SGS, and very limited (2/40) cross-reactivity was observed. Receiver-operator characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for the prediction of anti-SGS positivity. These ROC curves evidenced the superior performance of rLJM17+rLJM11. Predicted threshold levels were confirmed for rLJM17+rLJM11 using a large panel of 1,077 serum samples.

Conclusion

Our results show the possibility of substituting Lu. longipalpis SGS for two recombinant proteins, LJM17 and LJM11, in order to probe for vector exposure in individuals residing in endemic areas.     

Cell-free synthesis and immunological characterization of salivary proteins from Chironomus tentans

We have isolated the salivary proteins of the larva of the harlequin fly Chironomus tentans, and characterized its constituents by gel electrophoresis and immunological techniques. The detailed composition of saliva from individual animals is shown to be very variable, but four main protein groups can be defined. The largest, Fraction A, comprises up to five species, with molecular weights of between 820,000 and 700,000 Daltons. It includes at least two distinct antigenic species. This finding is discussed in the context of the known heterogeneity of the 75S RNA fraction which is transcribed in the Balbiani rings 1 and 2. — The other prominent protein classes in isolated saliva range in size from 230,000 down to less than 20,000 Daltons. — We have also employed antiserum against salivary proteins to investigate the products of in vitro translation of salivary gland RNA in the rabbit reticulocyte lysate system. A broad spectrum of polypeptide species is obtained which are immunologically related to salivary components, including species of over 300,000 Daltons. These latter are interpreted as unfinished Fraction A polypeptides resulting from incomplete translation of 75S RNA from BR1 and BR2. Evidence is presented to demonstrate that other salivary proteins, apart from Fraction A, are faithfully translated in the reticulocyte lysate.     

Proteomic Profiling of Cereal Aphid Saliva Reveals Both Ubiquitous and Adaptive Secreted Proteins

The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113.     

Identification of Glossina palpalis gambiensis specific salivary antigens: towards the development of a serologic biomarker of human exposure to tsetse flies in West Africa

The saliva of blood sucking arthropods contains a number of pharmacologically active compounds that induce an antibody response in exposed human individuals. The objectives of the present study were (i) to assess the human IgG response directed against salivary antigens of Glossina palpalis gambiensis, the main vector of Trypanosoma brucei gambiense in West Africa, as a biomarker of human–tsetse contacts; and (ii) to identify specific salivary antigens. Immune reactivity of human plasma collected within active human African trypanosomiasis (HAT) foci (coastal Guinea), historical foci where tsetse flies are still present (South-West Burkina Faso) and a tsetse free area (Bobo-Dioulasso, Burkina Faso), was measured by ELISA against whole saliva extracts. In the active HAT foci and areas where tsetse flies were present in high densities, specific IgG responses were significantly higher (p < 0.0001) to those in Bobo-Dioulasso or in Loropeni, where tsetse flies were either absent or only present at low densities. Furthermore, 2D-electrophoresis combined with mass spectrometry enabled to reveal that several antigens were specifically recognized by plasma from exposed individuals. Among them, four salivary proteins were successfully identified (Ada, 5′Nuc, Ag5 and Tsgf1). These results represent a first attempt to identify Glossina salivary proteins or synthetic peptides to develop a standardized and specific biomarker of tsetse exposure in West Africa.     

Validation of Recombinant Salivary Protein PpSP32 as a Suitable Marker of Human Exposure to Phlebotomus papatasi,the Vector of Leishmania major in Tunisia

BackgroundDuring 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.Conclusions/SignificanceOur 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.     

The salivary secretome of the tsetse fly Glossina pallidipes (Diptera: Glossinidae) infected by salivary gland hypertrophy virus

Background

The competence of the tsetse fly Glossina pallidipes (Diptera; Glossinidae) to acquire salivary gland hypertrophy virus (SGHV), to support virus replication and successfully transmit the virus depends on complex interactions between Glossina and SGHV macromolecules. Critical requisites to SGHV transmission are its replication and secretion of mature virions into the fly''s salivary gland (SG) lumen. However, secretion of host proteins is of equal importance for successful transmission and requires cataloging of G. pallidipes secretome proteins from hypertrophied and non-hypertrophied SGs.

Methodology/Principal Findings

After electrophoretic profiling and in-gel trypsin digestion, saliva proteins were analyzed by nano-LC-MS/MS. MaxQuant/Andromeda search of the MS data against the non-redundant (nr) GenBank database and a G. morsitans morsitans SG EST database, yielded a total of 521 hits, 31 of which were SGHV-encoded. On a false discovery rate limit of 1% and detection threshold of least 2 unique peptides per protein, the analysis resulted in 292 Glossina and 25 SGHV MS-supported proteins. When annotated by the Blast2GO suite, at least one gene ontology (GO) term could be assigned to 89.9% (285/317) of the detected proteins. Five (∼1.8%) Glossina and three (∼12%) SGHV proteins remained without a predicted function after blast searches against the nr database. Sixty-five of the 292 detected Glossina proteins contained an N-terminal signal/secretion peptide sequence. Eight of the SGHV proteins were predicted to be non-structural (NS), and fourteen are known structural (VP) proteins.

Conclusions/Significance

SGHV alters the protein expression pattern in Glossina. The G. pallidipes SG secretome encompasses a spectrum of proteins that may be required during the SGHV infection cycle. These detected proteins have putative interactions with at least 21 of the 25 SGHV-encoded proteins. Our findings opens venues for developing novel SGHV mitigation strategies to block SGHV infections in tsetse production facilities such as using SGHV-specific antibodies and phage display-selected gut epithelia-binding peptides.     

Transgenerational Transmission of the Glossina pallidipes Hytrosavirus Depends on the Presence of a Functional Symbiome

The vertically transmitted endosymbionts (Sodalis glossinidius and Wigglesworthia glossinidia) of the tsetse fly (Diptera: Glossinidae) are known to supplement dietary deficiencies and modulate the reproductive fitness and the defense system of the fly. Some tsetse fly species are also infected with the bacterium, Wolbachia and with the Glossina hytrosavirus (GpSGHV). Laboratory-bred G. pallidipes exhibit chronic asymptomatic and acute symptomatic GpSGHV infection, with the former being the most common in these colonies. However, under as yet undefined conditions, the asymptomatic state can convert to the symptomatic state, leading to detectable salivary gland hypertrophy (SGH⁺) syndrome. In this study, we investigated the interplay between the bacterial symbiome and GpSGHV during development of G. pallidipes by knocking down the symbionts with antibiotic. Intrahaemocoelic injection of GpSGHV led to high virus titre (10⁹ virus copies), but was not accompanied by either the onset of detectable SGH⁺, or release of detectable virus particles into the blood meals during feeding events. When the F₁ generations of GpSGHV-challenged mothers were dissected within 24 h post-eclosion, SGH⁺ was observed to increase from 4.5% in the first larviposition cycle to >95% in the fourth cycle. Despite being sterile, these F₁ SGH⁺ progeny mated readily. Removal of the tsetse symbiome, however, suppressed transgenerational transfer of the virus via milk secretions and blocked the ability of GpSGHV to infect salivary glands of the F₁ progeny. Whereas GpSGHV infects and replicates in salivary glands of developing pupa, the virus is unable to induce SGH⁺ within fully differentiated adult salivary glands. The F₁ SGH⁺ adults are responsible for the GpSGHV-induced colony collapse in tsetse factories. Our data suggest that GpSGHV has co-evolved with the tsetse symbiome and that the symbionts play key roles in the virus transmission from mother to progeny.