Antibodies from dogs with canine visceral leishmaniasis recognise two ...

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Antibodies from dogs with canine visceral leishmaniasis recognise two proteins from the saliva of Lutzomyia longipalpis. Authors; Authors and affiliations.
Parasitol Res (2007) 100:449–454 DOI 10.1007/s00436-006-0307-8

ORIGINAL PAPER

Antibodies from dogs with canine visceral leishmaniasis recognise two proteins from the saliva of Lutzomyia longipalpis Diana Bahia & Nelder Figueiredo Gontijo & Ileana Rodríguez León & Jonas Perales & Marcos Horácio Pereira & Guilherme Oliveira & Rodrigo Corrêa-Oliveira & Alexandre Barbosa Reis Received: 5 April 2006 / Accepted: 8 August 2006 / Published online: 21 October 2006 # Springer-Verlag 2006

Abstract The saliva of the sand fly Lutzomyia longipalpis, a major vector of Leishmania, exhibits pharmacological and immunomodulatory activities that may facilitate entry and establishment of parasites into the vertebrate host. Salivary gland components of the sand fly are, therefore, potential candidates in the development of a vaccine against human leishmaniasis. With the objective of identifying sand fly saliva proteins that could be used to immunise animals against canine visceral leishmaniasis, we have evaluated anti-saliva antibody reactivity using serum samples collected from dogs

naturally infected with Leishmania chagasi. Two proteins with molecular weights of 28.6 and 47.3 kDa were recognised by dog antibodies in Western blot assays. Protein bands were excised from an SDS-PAGE gel and the sequences determined by mass spectrometry. The proteins were identified as LuLo-D7 and Lulo YELLOW, respectively. The significance of these findings in the context of the development of multicomponent vaccination experiments is discussed.

Introduction D. Bahia : G. Oliveira Laboratório de Parasitologia Celular e Molecular, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil N. F. Gontijo : M. H. Pereira : A. B. Reis Laboratório de Fisiologia de Insetos Hematófagos, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil I. R. León : J. Perales Laboratório de Toxinologia, Departamento de Fisiologia e Farmacodinâmica, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil R. Corrêa-Oliveira : A. B. Reis Laboratório de Imunologia Celular e Molecular, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil A. B. Reis (*) Laboratório de Imunopatologia, Departamento de Arálises Clínicas, Escola de Famácia, Núcleo de Pesquisas em Ciências Biológicas (NUPEB), ICEB II, Morro do Cruzeiro, Universidade Federal de Ouro Preto, CEP 35400-000 Ouro Preto, Minas Gerais, Brazil e-mail: [email protected]

Visceral leishmaniasis (VL) is a zoonosis that is distributed widely throughout the world with an estimated incidence of around 5,000 new human cases per year (World Health Organisation 2000). In Latin America, the major vector of VL is the sand fly Lutzomyia longipalpis, whilst the most important domestic and wild reservoirs are dogs and foxes, respectively (Laison and Shaw 1987). In those endemic areas in which vector colonies and infected dogs are in close contact with humans, and especially with children, the epidemiological impact has been shown to be a major factor. Unfortunately, however, control of the vector or reservoir is difficult to implement and, in most endemic regions, the cost of such a programme would exceed the resources available (Palatnik-de-Sousa et al. 2001). The chemotherapy for dogs infected with canine visceral leishmaniasis (CVL) is not practical since both the rate of relapse and the cost of treatment are very high (Tesh 1995). However, the availability of a vaccine against CVL, even one with a low efficacy rate, would represent a major breakthrough in the control of the disease in many endemic areas. It has long been known that products of the salivary glands of the sand fly facilitate the entry of Leishmania parasites into the macrophages of the host, thus enhancing

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the chance of infection (Lanzaro et al. 1993). Sand fly saliva possesses a number of pharmacological activities including vasodilation, inhibition of platelet aggregation, coagulation and complement inhibition (Sacks and Kamhawi 2001; Cavalcante et al. 2003). Moreover, the saliva exhibits important immunomodulatory activities (Kamhawi et al. 2004), and evidence is available to suggest that the parasites use this activity to facilitate their establishment into the vertebrate host (Gillespie et al. 2000). The extremely potent vasodilator, maxadilan, present in sand fly saliva, is a 7 kDa protein that produces a long-lasting erythema at the bite site (Lerner and Shoemaker 1992). Morris et al. (2001) demonstrated that animals vaccinated with maxadilan, or with salivary gland lysates (SGL), develop antibodies against this salivary protein, thus gaining a degree of protection against infection by Leishmania major. Furthermore, when BALB/c mice were pre-immunised with SGL from L. longipalpis they were less susceptible to infection by Leishmania amazonensis than the untreated controls (Thiakaki et al. 2005). It was also reported that delayed-type hypersensitivity (DTH) response to sand fly salivary protein was responsible for the protective effect against L. major infection in mice (Valenzuela et al. 2001). Some data indicate that factors derived both from the parasite and from the sand fly may contribute to the protective effect (Rogers et al. 2004). Of particular interest is the finding that antibodies are present in human sera that can react against sand fly saliva to produce anti-saliva antibodies and anti-Leishmania DTH response, and this presumably contributes to the development of protective immunity against Leishmania infection (Gomes et al. 2002). It was suggested that components of the salivary glands of the sand fly could be potential candidates for the development of a vaccine against human leishmaniasis (Rogers and Titus 2003). To determine whether such components might also be of value in preventing CVL, we have evaluated anti-saliva antibody reactivity using serum samples from dogs naturally infected with Leishmania chagasi with the objective of identifying saliva proteins that could be used to immunise dogs.

Materials and methods Preparation of saliva from sand flies Intact salivary glands of 3–5 day-old female sand flies were removed under 0.9% saline solution with the aid of a stereoscopic microscope. Dissected salivary lobes (two lobes per insect) were washed in 0.9% saline and transferred to a micro-centrifuge tube containing hypotonic saline (0.7%). The salivary lobes were ruptured by successive pipetting and centrifugation at 10,000×g to

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provide a solution of saliva that was almost entirely free of intracellular contaminants. About 80% of the upper supernatant was aspirated from the micro-centrifuge tube and employed as the saliva preparation in the various experiments. Gel filtration chromatography An aliquot (50 μL) of the saliva preparation was submitted to HPLC analysis (Shimadzu LC-10VP) using a Waters Ultrahydrogel 250 molecular exclusion column (7.8 mm×30 cm) eluted at a rate of 0.8 mL/min with 10 mM HEPES/NaOH buffer (pH 7.4) containing 150 mM NaCl. For the molecular weight estimations, the column was previously calibrated with bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa). Protein gels and Western blots Soluble antigens (9.7 μg; derived from the equivalent of 22.5 salivary glands) were separated, together with SeeBlue™ (Invitrogen, USA) molecular weight markers, on 15% SDS-PAGE gels using standard methods (Sambrook and Russel 2001). The antigens were visualised by staining with Coomassie Blue R-250 (Sigma, St. Louis, MO, USA) and electro-transferred to a nitrocellulose membrane. Western blot experiments were conducted by incubating the nitrocellulose sheets with serum samples derived from dogs (symptomatic and asymptomatic) that had been naturally infected with L. chagasi and from noninfected dogs. Anti-saliva reactive antibodies were identified using anti-dog IgG/alkaline phosphatase antibodies: Reactivity was identified by NBT/BCIP (Sigma) employed according to the manufacturer’s instructions. Peptide sequencing In gel digestion Protein bands, separated on an SDS-PAGE gel, were digested in situ according to a modification of the protocol described by Shevchenko et al. (1996). Briefly, the individual bands were excised, destained and the proteins reduced with 10 mM dithiothreitol (100 μL) for 30 min at room temperature. The solution was discarded and the gel strips treated with 50 mM iodoacetamide solution (100 μL). Proteins were digested for 14 h at 37°C with 10 μL of icecold trypsin solution [20 ng/μL in 50 mM ammonium bicarbonate (pH 8.0)], the peptides extracted, concentrated to 10 μL by vacuum centrifugation and stored at −20°C until required for analysis. Mass spectrometric analysis Peptides were separated by HPLC using a GE healthcare (UK) μRPC C2/C18 column

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(15 cm×300 μm i.d.; 120 Å), sample introduction and mobile phase delivery were performed by a Thermo Finnigan (USA) Surveyor MS pump. The mobile phase was a mixture of 0.1% (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in acetonitrile pA (solvent B), initially at 95:5 (A:B) for 5 min, followed by a linear gradient to 60:40 at 35 min, to 40:60 at 45 min, and to 20:80 at 48 min. The flow rate was 4 μL/min. The peptides that eluted in the column effluent were transferred directly by electrospray into a Thermo Finnigan LCQ Deca XP Plus ion-trap spectrometer for analysis. The data acquired were interpreted manually.

Protein data analysis The determined peptide sequences were submitted to the NCBI non-redundant database using Blastp software (Altschul et al. 1997) (http://www.ncbi.nlm.nih.gov/ BLAST/). ExPASy bioinformatics tools were used to detect signal peptides in the two proteins identified on Western blots (Sigcleave software) and to analyse protein domains (Interproscan software; http://www.ebi.ac.uk/InterProScan).

Results To determine whether the components of the saliva of the sand fly, L. longipalpis, could be considered as potential candidates in developing a vaccine against canine visceral leishmaniasis (CVL), the proteins present in the salivary Fig. 1 Protein profile obtained by molecular exclusion chromatography of a sand fly (L. longipalpis) saliva preparation. A sample of saliva (equivalent to 25 salivary lobes) was chromatographed by HPLC using a Waters Ultrahydrogel 250 molecular exclusion column eluted with 10 mM HEPES/NaOH buffer pH 7.4 containing 150 mM NaCl. The molecular weights of the main peaks eluting at 7.93, 8.94 and 9.64 min were, respectively, 70, 26 and 13.5 kDa

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glands of female insects were analysed by HPLC gel filtration and SDS-PAGE. The chromatographic profile of a preparation of saliva (Fig. 1) exhibited three main proteins bands at retention times of 7.93, 8.94 and 9.64 min with estimated molecular weights of 70, 26 and 13.5 kDa, respectively. The proteins were more efficiently resolved by SDS-PAGE, and six protein bands could be identified on the gel (arrows in Fig. 2) with estimated molecular weights of 84, 62, 47, 30, 20 and 17 kDa. Previously, Valenzuela et al. (2004) employed Tris-glycine gels (4–20%) to separate 30 μg of salivary proteins, and obtained sufficient separation to permit a general proteome analysis of proteins in the molecular weight range 6 to 60 kDa. In addition to these proteomic results, we also observed a major band at 84 kDa in our gels (Fig. 2). Saliva proteins were assayed by Western blot against serum samples derived from dogs who had been naturally infected with L. chagasi and were asymptomatic (AD) or symptomatic (SD), and from non-infected dogs (NID). Two proteins could be clearly observed on the Western blot (arrows on the left in Fig. 3) that were reactive against SD and AD, but not NID, sera. When assayed by colorimetric reaction (data not shown), the SD serum was 10-times more reactive than the AD serum. The proteins that were recognised by dog antibodies exhibited molecular weights of 28.6 and 47.3 kDa, but their sequences showed no homology with any proteins in the Leishmania database, as determined by a BLASTp search. Both proteins possessed signal peptides indicating that they are secreted. The cDNAs of both proteins were previously reported (Charlab et al. 1999; Valenzuela et al. 2004).

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Fig. 3 Soluble antigens from the saliva of the sand fly (L. longipalpis) recognised by antibodies of dogs that had been naturally infected with L. chagasi (SD=symptomatic dogs; AD=asymptomatic dogs) and of non-infected dogs (NID; the control group). Two proteins were identified with molecular weights of 28.6 and 47.3 kDa (arrows on the left), excised from the gel and sequenced. Serum dilutions were: 1:100 for SD and AD; 1:50 for NID

Fig. 2 Soluble antigens from the saliva of the sand fly (L. longipalpis) separated on a 12% SDS-PAGE gel. Six protein bands (arrows on the right) were identified with respective molecular weights of 84, 62, 47, 30, 20, and 17 kDa

The 28.6 kDa protein was identified as LuLo-D7 (NCBI accession number AF420274.1; http://www.ncbi.nlm.nih. gov/entrez/viewer.fcgi?val=AF420274.1), a member of the

D7 subfamily of salivary proteins, the distribution of which is widespread amongst bloodsucking Diptera. Although D7 proteins are among the most abundant salivary proteins in adult female mosquitoes and sand flies, their role in blood feeding remains elusive. The D7 subfamily belongs to a superfamily of proteins, possessing a characteristic fold structure (Graham et al. 2001) adapted to binding small ligands, which contain pheromone binding proteins and associated with pheromone-sensitive neurons and general

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odorant-binding proteins. On this basis, (Arcà et al. 2002) suggested that in bloodsucking insects, the D7-related proteins might play a major anti-haemostatic role by binding agonists of haemostasis. In the search for potential candidates for the development of a vaccine, such a property could represent a very significant characteristic. The second reactive protein (molecular weight 47.3 kDa) identified in sand fly saliva was Lulo YELLOW (NCBI accession number AF132518; http://www.ncbi.nlm.nih.gov/ entrez/viewer.fcgi?val=AF132518). This protein shares significant sequence similarities with Drosophila yellow protein with royal jelly proteins and bee milk protein (Albert et al. 1999). The royal jelly protein domain constitutes ca. 90% of larval jelly proteins and its function is associated with cuticle pigmentation. Moreover, since cuticle pigmentation involves catecholamine metabolism, it is possible that the Lulo YELLOW secretion product may catabolise vasoconstrictor catecholamines. The 47.3 kDa protein possessed a DNA binding homeodomain similar to that shared by a number of homeodomain proteins that act as transcription factors. The homeodomain was first identified in homeotic and segmentation proteins from Drosophila, but is now known to be a common feature in Drosophila engrailed and L. longipalpis proteins, in yeast mating type proteins, in the hepatocyte nuclear factor 1a and in HOX proteins (Mannervik 1999). The domain is well-conserved in many animals, including vertebrates, and functions by binding DNA through a helix-turn-helix (HTH) structure. The HTH motif is characterised by two alpha-helices that make intimate contact with the DNA and are joined by a short turn. The second helix binds to the DNA via a number of hydrogen bonds and hydrophobic interactions that occur between specific side chains and the exposed bases and thymine methyl groups within the major groove of the DNA. The first helix helps to stabilise the bound structure. At present no biological or immunological activities were assigned to these proteins.

Discussion Epidemiologically, CVL is considered to be more important than human VL owing to its higher prevalence and to the fact that both asymptomatic and symptomatic dogs may be reservoirs of the vectors (Molina et al. 1994). In endemic areas in Brazil, many asymptomatic animals were detected with parasites in the skin (Reis et al. 2006), and a total of 414,168 seropositive dogs were identified in the country between 1980 and 1997 with the numbers continuing to increase steadily (Vieira and Coelho 1998). CVL may evolve from asymptomatic cases to a systemic disease, which, in most circumstances, culminates in death.

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Current strategies for the control of VL in humans are based on the elimination of infected dogs, treatment of human cases and vector control (Tesh 1995). However, the impact of the removal of seropositive infected dogs on the reduction in prevalence of human VL in endemic areas is highly controversial (Palatnik-de-Sousa et al. 2001). Such measures remain socially unacceptable, difficult to develop and expensive. Furthermore, they are not particularly effective, most probably because of the difficulty in determining true negative canine incidence and of the seroprevalence maintained by a residual reservoir of Leishmania in the field (Palatnik-de-Sousa et al. 2001). The introduction of new therapeutic strategies to treat CVL-infected dogs is thus an area of great interest not only for the elimination of animal infection, but also as an important tool to impact on the domestic maintenance of the parasite life cycle that has made the dog an important reservoir for this zoonose (Alvar et al. 2004). In this context, the development of a vaccine for dogs would represent a practical and efficient control tool, reducing the dog–sand fly–dog peridomestic transmission cycle that is probably important for the maintenance of transmission to humans (Mohebali et al. 2004). However, the design of a vaccine for parasitic diseases is more difficult than for most bacterial and viral diseases owing to the complexity of the pathogen and its intricate interactions with the vertebrate host. Components of the salivary glands of sand flies were considered to be potential vaccine candidates for preventing human leishmaniasis (Rogers and Titus 2003), and may also be appropriate for use in a vaccine against CVL. In the present study, we have identified two proteins derived from sand fly saliva components that could be considered with respect to their possible use as a recombinant vaccine for the prevention of CVL. The investigation of their immunological activities and their action in an infection process will be the aim of our future studies. DNA cloning and characterisation of the recombinant DNA-derived from the L. longipalpis proteins is currently in hand in our laboratories. Moreover, a recent review (Kubar and Fragaki 2005) has described the potential value of recombinant DNA-derived Leishmania proteins in diagnostics, therapy, and development of vaccines, and has addressed the question of how these proteins can aid in the fight against leishmaniasis. Thus, the results reported in the present study should provide valuable information for the development of multi-component vaccination experiments. Acknowledgements We are grateful to Leila Alves Campus for her technical assistance. This work was supported by the CNPq/BR/Grant: 541521124/98-0, FAPEMIG/BR/Grants: CBS 2222/97 (ABR) and CBB 174/02 (DB); PDTIS (Fiocruz) and FAPERJ (Rede Proteômica do Rio de Janeiro). DB was a recipient of CNPq and FAPESP fellowships. The experiments described in this study were carried out in full accordance with all pertinent laws and codes of practice applicable in Brazil.

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