BR102013013069B1 - method, kit for immunodiagnostic test of canine visceral leishmaniasis and vaccine - Google Patents

Abstract

METHOD, KIT FOR IMMUNODIAGNOSIS TEST OF CANINE VISCERAL LEISHMANIASIS AND VACCINE This technology deals with the selection, by the phage display technique, of six peptides that were highly specific and reactive in the identification of sera from dogs with visceral leishmaniasis. More particularly, the technology describes a serodiagnostic method, diagnostic kits and vaccine for symptomatic and asymptomatic visceral leishmaniasis, using as marker antigens the bacteriophage clones that were selected expressing the peptides of interest, as well as such synthesized peptides, being used isolated or associates. Furthermore, the technology comprises preventive and/or therapeutic vaccine compositions containing these markers.

Description

The present technology deals with the selection, using the phage display technique, of six peptides that were highly specific and reactive in the identification of sera from dogs with visceral leishmaniasis. More particularly, the technology describes a serodiagnostic method, diagnostic kits and vaccine for symptomatic and asymptomatic visceral leishmaniasis, using as marker antigens the bacteriophage clones that were selected expressing the peptides of interest, as well as such synthesized peptides, using isolated or associates. Furthermore, the technology comprises preventive and/or therapeutic vaccine compositions containing these markers.

With the advance, in the last decades, in the field of biotechnology, the more refined identification of antigens has been showing itself as a new perspective for the development of new biotechnological products, such as those aimed at the laboratory diagnosis of several diseases. Among such techniques, the phage display presents itself as a promising methodology.

Phage display technology is based on the expression of peptides, proteins or antibody fragments associated with proteins present on the external surface of recombinant bacteriophages (=phages). The nucleotide sequence coding for the inserted peptides is genetically fused to a coding sequence for some bacteriophage proteins, resulting in a hybrid product, which is then exposed on the outer surface of the viral particles (Smith, 1985).

This technology has been used in several works to research new antigens, so that in the form of bacteriophage clones expressing the peptides of interest, or in the form of individual peptides synthetically produced, they can be tested in the laboratory diagnosis of various diseases, such as in viral infections, cancer and autoimmune diseases; as well as in the production of recombinant vaccines (Bastien et al., 1997; Manoutcharian et al., 1999; Manoutcharian et al., 2001; Noya et al., 2003; Manoutcharian et al., 2004; Manoutcharian, 2005; Morales et al., al., 2008; Sartorius et al., 2008).

The identification of reactive peptides on the surface of phages allows their use in a global way, also using the structure of the phage itself, or their synthetic construction, followed by their use in laboratory tests, aiming at laboratory diagnosis and/or the development of vaccines against the various diseases.

Leishmaniasis is an endemic disease in several countries around the world. It is estimated that around 350 million people are exposed to the risk of infection and that 12 million are clinically affected by the disease. According to surveys by the World Health Organization (WHO), around 1.5 to 2.0 million new cases of the disease occur each year, of which around 1.0 to 1.5 million correspond to cases of tegumentary leishmaniasis (TL) and around 500,000 cases of Visceral leishmaniasis (VL) are reported (WHO, 2003). The severity of the disease in the mammalian host, which includes humans and dogs, ranges from a single skin lesion, at the site of the bite and which normally presents spontaneous cure, to the visceral form of the disease, which can be fatal in the acute form and if not treated. The dog is considered the main domestic reservoir of the parasite and, therefore, it is the main source of transmission between the vector and the mammalian host (Graimaldi & Tesh, 1993).

The clinical and laboratory diagnosis of leishmaniasis is based on epidemiological evidence, clinical examination and parasitological confirmation of the presence of the parasite (Tesh, 1995). Histological analyzes using fragments collected from biopsies and/or aspirates are necessary to confirm the diagnosis of the disease (Tafuri et al., 2001). The parasitological test, with microscopic identification of the parasite, is necessary for the definitive diagnosis of the disease; however, it is considered an invasive method. Other techniques, such as immunohistochemistry and polymerase chain reaction (PCR) are used, however, they are not practical to be used in field analyzes and require specialized technical training (Tafuri et al., 2001 and Tafuri et al. , 2004).

Serological tests used in the laboratory diagnosis of leishmaniasis aim at detecting antibodies and/or antigens specific to the parasites. The most used techniques are indirect immunofluorescence reaction (IFAT), enzyme-linked immunosorbent assay (ELISA), direct agglutination test (DAT) and Western-Blot (Tavares et al., 2003; Da Silva et al. ., 2006). Some antigens have been and are being tested for the serodiagnosis of leishmaniasis (Carvalho et al., 2002; Da Costa et al., 2003; Boarino et al., 2005; Silveira et al., 2006; Ferreira et al., 2007 ); however, such molecules have variable sensitivity and/or specificity and still need further studies, so that there is still no antigen, whether in the form of recombinant protein, synthetic peptide or other antigenic form, that is considered the gold standard for diagnosis of the disease.

The immunological diagnosis of canine visceral leishmaniasis (CVL) is hampered by factors such as the variable specificity and/or sensitivity of the antigens used. The occurrence of a high number of false-positive results is described in healthy animals, however, residing in endemic areas of the disease, as well as in animals with other diseases, such as Chagas disease, babesiosis, toxoplasmosis, rickettsiosis and erlichiosis. In addition, there is great difficulty in correctly interpreting serological tests that present false-negative results, such as in infected animals, but asymptomatic or with low anti-Leishmania antibody titers. In addition to this, there is the fact that vaccines against CVL commercially available in Brazil can induce the production of high levels of antibodies specific to the parasites in vaccinated animals, causing them to be confused with infected animals in the serological screenings performed. Thus, such animals end up not being differentiated from those actually infected by Leishmania.

The antigens currently used in the serological diagnosis of CVL are, in general, complete proteins, with regions common to proteins of other parasites, which can generate cross-reactions between different species. The use of small peptides as antigens tends to provide more defined results and, therefore, more efficient diagnosis of different diseases. In the case of CVL, there is clearly a need for a constant search for new antigens that can promote a more sensitive and specific immunological diagnosis for symptomatic and asymptomatic disease.

In the state of the art, several patent documents dealing with the use of proteins and peptides in the immunodiagnosis of leishmaniasis and as a vaccine against this disease were found.

The document PI0804859-2 refers to two synthetic antigenic peptides (epitopes/mimotopes), selected and synthesized by the techniques of phage display and spot synthesis, and to the process of obtaining protein polymer obtained through the conjugation of these peptides. The polymeric antigen formed by these synthetic peptides was able to induce protection against Visceral Leishmaniasis and offer specific diagnosis for its detection.

US5834592 describes polypeptides that contain at least an immunogenic portion of one or more Leishmania antigens and the use of these polypeptides in the prevention, treatment and diagnosis of leishmaniasis.

US5674503 describes peptides capable of inducing an immune response against leishmaniasis.

The document WO2012/019268 describes a vaccine and immunodiagnostic kit for leishmaniasis, developed through the identification, production and selection of new antigens through proteomic analysis, bioinformatics, peptide synthesis and enzyme immunoassays.

However, none of the peptides presented in the patent documents found in the searches are similar to those of the present technology.

The innovative points of this technology are based on the development and identification of new markers for the immunological diagnosis of symptomatic and asymptomatic canine visceral leishmaniasis and with probable vaccine and/or therapeutic application through the application and improvement of a more refined phage display technique, unprecedented for research on antigens of biotechnological interest in leishmaniasis.

In the present technology, new antigens of simple, rapid production, with high final yield were identified, using the phage display technique, which are capable of identifying animals that are infected by the parasite that causes leishmaniasis, even those with low titers of specific antibodies (in the case, mainly, of asymptomatic animals). In addition, the antigens do not allow cross-reaction with animals carrying diseases other than VL, and still do not show positivity in animals vaccinated with commercially available vaccines. Such antigens will allow the specific immunological and laboratory identification only of animals carrying the Leishmania parasite. These antigens may also serve as vaccine immunogens, preventing the disease and its spread in dogs, which behave as important reservoirs of the parasites.

DETAILED DESCRIPTION OF THE TECHNOLOGY

In the present invention, the phage display technique was improved with an unprecedented methodology based on negative and positive selection protocols of bacteriophage clones aimed at selecting those expressing peptides of interest for the serodiagnosis of CVL. The selection of high-affinity molecules in relation to a specific target receptor, adhered to a solid surface, was carried out by consecutive selection steps called “bio-panning cycles.” Conceptually, the number of bio-panning cycles performed in phage display determines the degree of enrichment of phages that bind to a target receptor. A population of phage clones with high affinity for a given receptor can be obtained by performing 3 to 5 cycles of bio-pannings (Crameri &Suter , 1993) Methodologically, bio-panning cycles can be adapted according to the research group that dominates such technology. For example, in our case, IgG molecules from animal sera to be investigated are fused on the surface of microspheres (called "beads") and such hybrids are subjected to processes of negative and positive uptake (selection) in relation to the phage clones present in the used recombinant library.

As reported, a new phage display method for the selection of new serodiagnostic markers of canine visceral leishmaniasis was developed in this invention. Initially, the negative selection process was used. It was based on the exposure of a recombinant library of phage clones against microspheres incorporated with IgG molecules purified from serum samples from healthy dogs and, later, from animals infected by Trypanosoma cruzi. Bacteriophage clones that did not bind to these microspheres were retrieved and, in the following process, a "positive selection" was performed, based on the exposure of such clones to microspheres incorporated in IgG molecules purified from sera from dogs with symptomatic and VL asymptomatic. Those clones that remained adhered to such microspheres incorporated with these IgGs were recovered, amplified and sequenced. Such antigens, still incorporated in bacteriophage clones, were tested in the serological diagnosis of CVL in ELISA experiments and those phage clones expressing the peptides of interest that obtained the best results were selected and tested against a large serological panel, in order to be used in the development of a serological diagnostic kit for symptomatic and asymptomatic CVL.

The method for immunodiagnosis of leishmaniasis can be performed using ELISA, Western-Blot, dot-blot, immunodiffusion and immunochromatography and comprises the following steps: a) exposing the antibodies of a sample to one or more peptides or to phages expressing a or more such peptides, attached to a solid support or a carrier and the peptides consist of the amino acid sequences of SEQ ID Nos 1, 2, 3, 4, 5 and 6; b) placing the antibodies from step “a” in contact with a secondary antibody or a protein, conjugated to an enzyme or a marker that binds to the antibodies from step “a”; c) detecting the anti-Leshmania antibodies in the above-mentioned sample by detecting the secondary antibody or protein specifically bound to said anti-Leishmania antibody.

Considering the different immunodiagnostic tests, peptides or phages used as antigens may be linked to a solid support or to a carrier such as a gold particle. The detection of contaminated sera may be through the use of secondary antibodies of the IgG, IgM, IgA or IgE type or through protein of the type A or G. These may be conjugated to an enzyme or a marker, the enzymes being selected from the group comprising alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. Markers may be selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents. Secondary antibodies can be detected by fluorescence, immunoluminescence, absorbance or radioisotope techniques.

The present technology may be presented in the form of an immunodiagnostic kit for leishmaniasis, which basically will comprise: - at least one of the peptides consisting of the sequences SEQ ID Nos 1, 2, 3, 4, 5 and 6, isolated or expressed in phages; - a secondary antibody or protein, where the secondary antibody or protein is conjugated to an enzyme or marker; and - a reagent for detecting said enzyme or label.

Furthermore, peptides of sequences SEQ ID Nos 1, 2, 3, 4, 5 and 6, isolated or expressed in phages, together with pharmacologically acceptable excipients, can be used in the preparation of vaccine compositions.

The technology can be better understood through the non-limiting examples that follow.

EXAMPLE 1 - Serum samples

The following serum samples were used to perform the bio-panning cycles and ELISA assays: sera from dogs with symptomatic (n=13) and asymptomatic (n=10) VL; sera from healthy dogs (n=13); sera from animals vaccinated with the Leish-Tec® vaccine (n=13) and sera from dogs infected with T. cruzi (n=13). For these experiments, the ELISA technique was used because it is considered less invasive, easy to perform and automated, allowing studies and application in a large number of samples.

EXAMPLE 2 - Purification of IgGs antibodies by coupling to G-protein microspheres

The purification of IgG class immunoglobulins from the serum pools listed above was performed by coupling them to G-protein-conjugated magnetic microspheres. For this purpose, microsphere particles were washed in 0.1 M MES buffer pH 5.0 and were added to them: (a) 300 μL of the pool of sera from dogs with symptomatic VL, for a final volume of 500 μL; (b) 195 μL of the pool of sera from dogs with asymptomatic VL, for a final volume of 260 μL; (c) 375 μL from the pool of sera from healthy dogs, for a final volume of 500 μL, or (d) 150 μL from the pool of sera from dogs with Chagas disease, for a final volume of 200 μL. The ratio of antibodies to the amount of microspheres, in all cases, was 1:1. Then, an incubation was carried out under constant agitation and at a temperature of 20 to 28°C. The microspheres adsorbed with the antibodies were washed 3 times in 0.1 M MES buffer pH 5.0, in order to remove non-adhered IgG antibodies. To complete the covalent binding between the microspheres and the antibodies, the "beads-antibody" system was washed with 0.2M triethanolamine buffer pH 8.2 and resuspended in covalent binding buffer (20 mM dimethylpimelinidate/HCl in triethanolamine buffer), under stirring constant and at RT. The neutralization of the reaction was done by incubating the system with 50 mM Tris buffer pH 7.5, at RT. Then, the incorporated microspheres were washed in TBS-T 0.1% Tween 20 buffer and blocked with blocking solution (5% BSA in TBS-T 0.05% Tween 20) for 1 h at 37°C, then resuspended in TBS buffer. To certify the coupling, the beads covered by the IgGs were incubated for 1 h and at 37°C with the anti-dog IgG antibody (dilution 1:5,000). After incubation, the beads were washed with 0.1% TBS-Tween and the reaction was revealed by the addition of tetramethylbenzidine (TMB) substrate. The reaction was then stopped by the addition of 2N sulfuric acid and the absorbance was read at 450 nm in a microplate reader (Titertek Multiskan Plus, Flow Laboratories, USA).

EXAMPLE 3 - Bio-panning cycles

To perform negative and positive selections in biopanning cycles, a recombinant library containing peptides fused to filamentous phage proteins (library Ph.D.-C7C, New England, BioLabs®) was used. It was diluted in TBS-Tween 0.1% to select the ligands in the microspheres incorporated by the purified IgG antibodies.

The 1st bio-panning cycle started with negative selection. For this purpose, the bacteriophage library was incubated with the microspheres coupled with the IgGs of healthy dogs and then the microspheres were precipitated by magnetic attraction to the Dynal Biotech support (n° 12020), with the supernatant containing the clones that did not if adhered to the IgGs, transferred to a new tube containing the microspheres coupled with the IgGs from the T. cruzi group. This procedure was then performed 3 times with the IgG group from healthy animals and with the IgG from the T. cruzi group. Only phage clones that did not adhere to such antibodies were recovered.

For positive selection, the phage supernatant collected previously was transferred to a new tube containing the microspheres coupled with the IgGs of dogs with asymptomatic VL, being incubated for 30 min and at RT. This procedure was performed twice. The microspheres containing the beads-antibody system precipitated in the bio-panning tubes of the IgGs of asymptomatic dogs, plus the phages of interest adhered, were washed 10 times with 0.1% TBS-Tween 20 and the selected phages were eluted in 0.2 glycine buffer M pH 2.0, such solution being neutralized by the addition of Tris-base pH 9.0. Subsequently, such phage clones were transferred to a new tube containing the microspheres coupled with the IgGs of dogs with symptomatic VL, and this whole process was repeated 3 times. The microspheres containing the beads-antibody system precipitated in the tubes of the IgGs bio-pannings of asymptomatic and symptomatic dogs, plus the adhered phages of interest, were processed as reported above. Phage clones were recovered for further titration.

For the 2nd cycle of selection, the entire process described for the 1st cycle was repeated, however, the starting point was the use of phage clones recovered at the end of the 1st cycle. This procedure was repeated in the 3rd bio-panning cycle, in which the starting material for the selections was the clones recovered after the end of the 2nd bio-panning cycle.

EXAMPLE 4 - Selection of phages and identification of peptide sequences after bio-pannings cycles with negative and positive selections by phage display

After purification of IgG antibodies from different serum pools, and the negative and positive selection processes, the phage clones, recovered in the different cycles of bio-pannings, were titrated and the results are shown in Table 1. the amount of retrieved phage ("output") was lower than the amount used at the beginning of the positive selection ("input"), indicating that there was a significant restriction on the number of recovered phage clones, thus making them with a high potential to be specifically recognized by antibodies present in serum samples from dogs with symptomatic and asymptomatic VL. Table 1: Titration of phage clones recovered after biopanning cycles in the positive selection processes by immunoaffinity using IgGs from asymptomatic dogs and, later, symptomatic for LV. Titrations are expressed as colony forming units per ml_ (PFU/ml).

The phage clones recovered after the 3rd selection cycle were cultivated in plates containing LB agar culture medium plus ampicillin (100 mg/mL), to obtain individual clones and their subsequent identification. The plates were striated and incubated at 37°C for 16 h. Afterwards, 96 isolated colonies were transferred individually to a cell culture plate (Nunc) with 96 wells, containing liquid LB medium plus ampicillin and were incubated for 16 h at 37°C, under constant agitation. The next day, the recovered clones had their DNA concentration estimated using the NanoDrop device (260 nm). Afterwards, they were tested in a direct ELISA using 1x109 individual phages in each well of the plate and with their direct reaction against a pool of sera from dogs with VL and a pool of sera from healthy dogs. The clones listed in Table 2 were those that showed greater ability to discriminate between the pool of LV sera in relation to samples from healthy dogs. Thus, these clones, with probable potential for the serodiagnosis of CVL, had their DNA amplified and purified and they were sequenced in order to obtain the amino acid sequences of the peptides of interest. The result of the analyzes is shown in Table 2. Table 2: Sequence of the phage clones expressing the peptides of interest that were selected for serodiagnosis of CVL.

Phage clones of interest with the amino acid sequences of the peptides

EXAMPLE 5 - ELISA using phage clones of interest

Subsequently, the 18 selected phage clones (which had their sequences determined) and the exogenous peptides recognized (probably responsible for the high recognition against the LVC serum samples) were tested individually against a panel of individualized sera, 10 containing 13 samples of each group, namely: animals with symptomatic and asymptomatic VL; sera from non-LV (healthy) animals; sera from healthy animals vaccinated with the Leish-Tec® vaccine and sera from animals infected with Trypanosoma cruzi. Such tests aimed to obtain the individual results of each phage clone.

The results of the means and standard deviations of the absorbances of each group, in relation to the analyzed phage clone, are shown in Table 3. Positive and negative controls on each plate were used. Furthermore, in parallel, a wild phage clone, that is, one not expressing any exogenous peptide ("wild" phage) and a phage clone expressing a peptide not related to Leishmania antigens ("random" clone), were used as controls of the experiment. Table 3: Results of the means and standard deviations of the evaluated groups: dogs with VL, healthy animals, vaccinated with the Leish-Tec® vaccine or infected with T. cruzi (Chagas' disease) against the recognition of individualized phage clones (1x109 by well). Thirteen serum samples from each evaluated group were used.

It can be seen that all clones evaluated were able to be recognized, with higher absorbance values, by the LVC group in relation to the other evaluated groups. Thus, the 18 clones evaluated showed greater sensitivity and specificity against antibodies present in serum samples from dogs with VL, compared to antibodies present in serum samples from the other groups evaluated, which allowed us to infer that such clones are relevant for use in the serodiagnosis of CVL.

Subsequently, with a view to increasing the reliability of the results in order to allow greater discrimination by the diagnostic antigens against different groups of animals, new samples of sera from dogs vaccinated with the commercial Leishmune® vaccine and from dogs infected with Erlichia canis, an important canine infection that induces the production of antibodies that cross-react with Leishmania proteins, were inserted into serological tests using the 18 selected phage clones. This time, the objective was to increase the specificity of diagnostic antigens. In analyzing the results, it was observed that, of the 18 pre-selected phage clones, only 6 maintained a high capacity to differentiate serum samples from dogs with VL in relation to other serum samples (Table 4). In this sense, such clones were selected and present themselves as new serological markers to compose an immunological kit for the sensitive and specific laboratory diagnosis of symptomatic and asymptomatic CVL, either in the form of the phage clones themselves expressing the peptides of interest, or as such peptides synthesized in their soluble form. Table 4: Results of the means and standard deviations of the evaluated groups: dogs vaccinated with the commercial Leishmune® vaccine and dogs infected with Erlichia canis against the recognition of individualized phage clones (1x109 per well). Thirteen serum samples from the Erlichia canis group and 13 samples from dogs vaccinated with Leishmune® were used.

Claims (14)

1. Method for immunodiagnostic test of canine visceral leishmaniasis characterized by comprising the following steps: a. Expose antibodies from a sample to one or more peptides or to phages expressing one or more such peptides, attached to a solid support or a carrier and the peptides consist of the amino acid sequences SEQ ID Nos 1, 2, 3, 4, 5 and 6; B. Putting the antibodies from step “a” in contact with a secondary antibody or a protein, conjugated to an enzyme or a marker that binds to the antibodies from step “a”; ç. Detect anti-Leshmania antibodies in the above-mentioned sample by detecting the secondary antibody or protein specifically bound to said anti-Leishmania antibody. 2. Method for immunodiagnostic test of canine visceral leishmaniasis, according to claim 1, step "a", characterized in that the carrier is a gold particle. 3. Method for immunodiagnostic test of canine visceral leishmaniasis, according to claim 1, step "b", characterized in that the secondary antibody is selected from a group consisting of IgG, IgM, IgA, IgE and subclasses thereof. 4. Method for immunodiagnostic test of canine visceral leishmaniasis, according to claim 1, step "b", characterized in that the protein is selected from a group consisting of Protein A and Protein G. 5. Method for immunodiagnostic test of canine visceral leishmaniasis, according to claim 1, step "b", characterized in that the enzyme is selected from the group consisting of alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase . 6. Method for immunodiagnostic test of canine visceral leishmaniasis, according to claim 1, step "b", characterized in that the marker is selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents. 7. Method for immunodiagnostic test of canine visceral leishmaniasis, according to claim 1, step "c", characterized in that anti-Leshmania antibody detection is selected from the group consisting of fluorescence, immunoluminescence, absorbance or radioisotope detection. 8. Kit for immunodiagnostic test of canine visceral leishmaniasis, characterized in that it comprises at least one of the peptides consisting of the sequences SEQ ID Nos 1, 2, 3, 4, 5 and 6, isolated or expressed in phages; and a secondary antibody or protein, where the secondary antibody or protein is conjugated to an enzyme or marker; and a reagent for detecting said enzyme or label. 9. Kit for immunodiagnostic test of canine visceral leishmaniasis, according to claim 8, characterized in that the recombinant proteins or peptides are linked to a solid support or carrier. 10. Kit for immunodiagnostic test of canine visceral leishmaniasis, according to claim 8, characterized in that the carrier consists of gold particles. 11. Kit for immunodiagnostic test of canine visceral leishmaniasis, according to claim 8, characterized in that the secondary antibody is selected from the group consisting of IgG, IgM, IgA, IgE and subclasses thereof. 12. Kit for immunodiagnostic test of canine visceral leishmaniasis, according to claim 8, characterized in that the protein is selected from the group consisting of protein A and protein G. 13. Kit for immunodiagnostic test of canine visceral leishmaniasis, according to claim 8, characterized in that said marker is selected from the group consisting of enzymes, redioisotopes, biotin, chromophores, fluorophores and chemiluminescents. 14. Kit for immunodiagnostic test of canine visceral leishmaniasis, according to claim 8, characterized in that the enzyme is selected from the group comprising alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.