BR102019027574A2 - recombinant chimeric proteins containing bartonella henselae antigens - Google Patents

Abstract

The present invention falls within the technical sectors A61K 39/02 (bacterial antigens), C12N 15/09 (recombinant DNA technology), C12N 15/10 (processes for purification, isolation or purification of DNA or RNA), C12N 15/ 31(genes encoding microbial proteins), C12N 15/66 (general methods for inserting a gene into a vector to form a recombinant vector using cleavage or ligation), C12N 15/70(vectors or expression systems specially adapted to E. coli), C12N 15/74 (vectors or expression systems specially adapted to prokaryotes in addition to E. coli) and G01N 33/53 (immunoassay). The invention relates to the construction of three synthetic chimeric proteins from immunogenic epitopes of different Bartonella henselae antigens. These chimeras are produced in heterologous expression systems and serve for the development of diagnostic tests and recombinant vaccines to control human and animal bartonellosis.

Description

RECOMBINANT CHIMERIC PROTEINS CONTAINING BARTONELLA HENSELAE ANTIGENS FIELD OF THE INVENTION

[001] The present invention, internationally classified in the patent classifications A61K 39/02 (bacterial antigens), C12N 15/09 (recombinant DNA technology), C12N 15/10 (processes for purification, isolation or purification of DNA or RNA), C12N 15/31(genes encoding microbial proteins), C12N 15/66 (general methods for inserting a gene into a vector to form a recombinant vector using cleavage or ligation), C12N 15/70(vectors or systems of expression specially adapted to E. coli), C12N 15/74 (vectors or expression systems specially adapted to prokaryotes in addition to E. coli) and G01N 33/53 (Immunoassay) refer to the construction of synthetic chimeric genes for protein production chimeric, containing immunogenic epitopes of different proteins of Bartonella henselae. These chimeric proteins will be used as antigens in diagnostic tests for bartonellosis caused by B. henselae, better known as Cat Scratch Disease.

DESCRIPTION OF TECHNICAL STATUS

[002] Bartonella henselae is a bacterium that belongs to the Rhizobiales family, is a fastidious gram-negative α-Proteobacteria, facultatively intracellular and can infect the erythrocytes and endothelial cells of their hosts (GIL, H., ESCUDERO, R. , PONS, I., RODRIGUEZ-VARGAS, A., GARCÍA, A., RODRIGUEZ-MORENO, I., et al. Distribution of Bartonella henselae variants in patients, reservoir hosts and vectors in Spain. Plos One., v. 8 , p. 1-10, 2013). Cats (Felis catus) are considered primary reservoirs for B. henselae, however, dogs (Canis familiaris) have also been identified as reservoirs, while humans are considered accidental hosts (BREI-TSCHWERDT, E. Bartonellosis, One Health and all creatures great and small. Vet. Dermatol., v. 28, p. 96-e21, 2017). Within the genus Bartonella, there are around 22 species related to diseases in mammals (PENNISI, MG, MAR-SILIO, F., HARTMANN, K., et al. Bartonella species infection in cats - ABCD guidesline on prevention and managment. J . Fel. Med. Surg., v.15, p. 563-569, 2013), and B. henselae is considered one of the most medically important species because it infects both cats and humans (BREITSCHWERDT, E. Bartonellosis, One Health and all creatures great and small. Vet. Dermatol., v. 28, p. 96-e21, 2017).

[003] The transmission of B. henselae to humans occurs through bites or scratches from infected cats or infected with fleas containing the bacteria in their feces, or directly through contaminated blood. In humans, B. henselae is the etiologic agent of Cat Scratch Disease (CSD), which is an often self-limiting infection in immunocompetent individuals (REGIER, Y, O'ROURKE, F., KEMPF, VAJ Bartonella spp. - the chance to establish One Health Concepts in veterinary and human medicine. Parasites Vectors., v. 9, p. 1-12, 2016), and its main symptom is regional lymphadenopathy (GIL, H., ESCUDERO, R., PONS, I., RODRIGUEZ-VARGAS, M., GARCIA, E., RODRIGUEZ-MORENO, I., et al Distribution of Bartonella henselae variants in patients, reservoir hosts and vectors in Spain. 8, p. 1-10, 2013). Other symptoms may involve fever, headache, lesions on the skin and mucous membranes near the site of inoculation and splenomegaly. Endocar-ditis, oculoglandular involvement (Parinaud's Syndrome), encephalopathies, neuroretinitis and osteomyelitis are described as complications of the infection. In immunocompromised individuals, chronic infections can occur, leading to angioproliferative diseases such as bacillary angiomatosis and hepatic peliosis, which can be fatal if not treated (REGIER, Y, O'ROURKE, F., KEMPF, VAJ Bartonella spp. - a chance to establish One Health Concepts in Veterinary and Human Medicine. Parasites Vectors., v. 9, p. 1-12, 2016).

[004] The species of Bartonella have a worldwide distribution, being more pronounced in areas where conditions are more favorable for arthropod vectors, mainly fleas (PENNISI, MG, MARSILIO, F. , HARTMANN , K., et al. Bartonella species infection in cats - ABCD guidessline on prevention and management. J. Fel. Med. Surg., v.15, p. 563-569, 2013). In areas endemic for the presence of fleas, the seroprevalence levels of Bartonella spp. in cats they can be greater than 90%, and bacteremia levels greater than 50%. Initially, B. henselae was isolated from an HIV-positive individual, and later from cats around the world. Currently, bacteremia by B. henselae has been reported in different species, such as cows, horses, marine mammals, small terrestrial mammals and sea turtles, making the epidemiology of this species more complex than initially described (BREITSCHWERDT, E. Bartonellosis, One Health and all creatures great and small. Vet. Dermatol., v. 28, p. 96-e21, 2017). In Brazil, epidemiological studies have shown the presence of Bartonella species. The detection of this pathogen ranges from cats to human beings, in the most diverse Brazilian states. Interestingly, recent studies in Brazil have shown the presence of bacteremia by Bartonella spp. in asymptomatic blood donors, reinforcing the need to assess the transmission of Bartonella by blood (PITASSI, LHU, DINIZ, PPVP, SCORPIO, DG, DRUMMOND, MR , LANIA, BG, BARJAS-CASTRO, ML, et al. Bartonella spp. Bacteremia in blood donors from Campinas, Brazil. Plos Negl. Trop. Dis., v. 9, p. 1-2, 2015; VIEIRA-DAMIANI, G., DINIZ, PPV, PITASSI, LHU, SOWY, S., SCORPIO , DG, LANIA, BG, DRUMMOND, MR, SOARES, TCB, BARJAS-CASTRO, ML, BREITSCHWERDT, EB, NICHOLSON, WL, OLD, PENF Bartonella clarridgeiae bacteremia detected in asymptomatic blood donor. J. Clin. Microb., v. 53, p. 352-356, 2015).

[005] An accurate microbiological diagnosis of bartonellosis can be a great challenge, especially in patients with chronic and long-term infection. Conventional isolation techniques, ELISA (Enzyme Linked Immuno Sorbent Assay), Western Blotting or Immunofluorescence for antibody detection and Polymerase Chain Reaction (PCR) for bacterial DNA amplification have shown diagnostic limitations (BREITSCHWERDT, E. Bartonellosis, One Health and all creatures great and small. Vet. Dermatol., v. 28, p. 96-e21, 2017). Furthermore, the serological detection of B. henselae is also difficult and is hampered by the low sensitivity and specificity of the assays used in the routine laboratory (OTSUYAMA, K. , TSUNEOKA, Η., KONDOU, Κ. , et al. Development of a Highly specific IgM enzyme-linked immunosorbent assay for Bartonella henselae using n-lauroyl-sar-cosine-insoluble proteins for serogianosis of cat scratch diaseae. J. Clin. Microbiol., v. 54, p. 1958-1964, 2016). In serological tests, detection is done by indirect immunofluorescence using whole cell antigens co-cultured on Vero cells, which have good sensitivity, but are expensive and can cross-react. In the ELISA method, whole cell antigens have been used, however, in addition to their low sensitivity, they have little specificity. In this sense, the use of purified proteins could improve the sensitivity and specificity of these tests (FERRARA, F., DI NIRO, R ., D'ANGELO, S., BUSETTI, M., MAR-CARI, R., NOT, T., SBLATTERO, D. Development of an enzyme-linked immunosorbent assay for Bartonella henselae infection detection Letters Applied Microbiol., v 59, p. 253-262, 2014). Some studies with recombinant proteins have been published. In these, proteins known to be antigenic, such as GroEL and 17-kDa (FERRARA, F., DI NIRO, R., D'ANGELO, S., BUSETTI, M. , MARCARI, R. , NOT, T., SBLATTERO, D. Development of an enzyme-linked immunosorbent assay for Bartonella henselae infection detection. Letters Applied Microbiol., v. 59, p. 253-262, 2014), Pap31 (ANGKASEKWINAI, N. , ATKINS, EH, ROMERO, S., GRIECO, J. , CHUNG CHAO, C., CHING, WM An evaluation study of enzyme-linked immunosorbent assay (ELISA) using recombinant protein Pap31 for detection of antibodies against Bartonella hacilliformis infection among the Peruvian population. Am. J. Trop. Med. ., v. 90, p. 690-696, 2014) and P26 (WERNER, JA , FENG, S., CHOMEL, B. , HODZIC, E., HASTEN, RW, BARTHOLD, S. P-26 based serodiagnosis for Bartonella spp. infection in cats. Comparative Med., v. 58, p. 375-380, 2008) have been produced recombinantly, and have been tested in serological assays, mainly ELISA. However, in all assays the proteins were used in isolation, and, in general, they presented low sensitivity, which indicates that the use of purified proteins as antigens can improve the sensitivity and specificity of diagnostic tests (FERRARA, F. , DI NIRO, R., D'ANGELO, S., BUSETTI, M., MARCARI, R., NOT, T., SBLATTERO, D. Development of an enzyme-linked immunosorbent assay for Bartonella henselae infection detection Letters Applied Microbiol ., v. 59, p. 253-262, 2014) as well as could be used in vaccines.

[006] Thus, the development of inputs for serological diagnosis and production of vaccines for infection by B. henselae is of considerable interest, both in human and veterinary medicine, contributing to the correct treatment of diseases caused by B. henselae , as well as for the real knowledge of the epidemiology of the infection. In this context, the need to develop chimeras as alternatives for the diagnosis and treatment of infection is reinforced, as these chimeric proteins are made up of different antigens that make up the bacterial proteins, and this could make the tests more sensitive and specific, and would be possible obtain an effective response in the administration of vaccines, since antibodies can be produced against the different chimeric antigens. Based on this, our proposal is to use in silico analysis to identify antigenic proteins capable of composing an effective serological test and vaccines, based on the use of these recombinant proteins in the form of chimeras.

SUMMARY OF THE INVENTION

[007] In this context, the events described below, present the invention related to chimeric proteins based on different combinations of epitopes of different antigens of B. henselae, aiming at the use of these proteins as inputs in the development of diagnostic tests.

[008] The first objective of the present invention deals with the construction of DNA sequences for chemical synthesis of chimeric genes that encode epitopes of different antigens of B. henselae. Another objective is the production of recombinant chimeric proteins in a heterologous expression system for their use as an input in the diagnosis of bartonellosis.

DETAILED DESCRIPTION OF THE INVENTION

[009] For convenience the meaning of some terms used in the specifications are described below.

[010] Q1: refers to the nucleotide sequence (SEQ ID N° 1) or amino acid sequence (SEQ ID N°4) of chimera 1, composed of portions of the GroEL, 17kDa and P26 proteins.

[011] Q2: refers to the nucleotide sequence (SEQ ID N°2) or amino acid sequence (SEQ ID N°5) of chimera 2, composed of portions of the proteins BadA, Pap31 and GroEL.

[012] Q3: refers to the nucleotide sequence (SEQ ID N° 3) or amino acid sequence (SEQ ID N°6) of chimera 3, composed of portions of the proteins OMP89, OMP43 and P26.

[013] In one aspect, the invention deals with the construction of synthetic DNA sequences that encode chimeric proteins composed of epitopes of different antigens of B. henselae. Chimera construction and cloning steps are detailed below.

[014] Step 1: In the present invention the identification of antigens was based on the in silico analysis of B. henselae antigens previously described in the literature. The online predictors of B cell antigens used were BepiPred-2.0: Sequential B-Cell Epitope Predictor and Immune Epitope Database and Analysis Resource.

[015] Step 2: The nucleotide sequences encoding epitopes involved with the stimulation of B lymphocytes were used to build the chimeric genes Q1 (SEQ ID N° 1), Q2 (SEQ ID N° 2) and Q3 (SEQ ID N° 3) and these synthesized by the company GO Genone®. All genes were cloned into pAE plasmids that have the following characteristics: origin of replication in Escherichia coli, multiple cloning site, antibiotic ampicillin resistance gene and phage T7 promoter, which is recognized by E. coli RNA polymerase. Furthermore, this vector allows the expression of recombinant proteins fused to a tail of six histidine residues in its amino-terminal portion. Among the sequences of each epitope, "linkers" containing 3x the amino acid glycine and 1x the amino acid serine were used. The initiation codon was the amino acid methionine. All sequences were digested with restriction enzymes BamHI (N-terminal region) and KpnI (C-terminal region).

[016] Under a second aspect, the invention deals with the obtainment and characterization of recombinant chimeric proteins, composed of epitopes of different antigens of B. henselae. The production steps and characterization of the chimeric proteins are detailed below.

[017] For expression of the chimeric proteins Q1 (SEQ ID No. 4), Q2 (SEQ ID No. 5) and Q3 (SEQ ID No. 3) fused to a six histidine tail, the recombinant vectors pAE/Q1, pAE /Q2 and pAE/Q3 were used to transform E. coli BL21(DE3) Star by heat shock (15 min at 4°C, 1 min at 2°C and 2 min at 4°C). A recombinant clone for each construct was used to inoculate 10 mL of LB containing 100 µg.mL-1 of ampicillin and cultured under 180 rpm shaking for 12 - 20 h at 37 °C. This culture was used to inoculate 500 mL of LB with 100 µg.mL-1 of ampicillin, which was incubated at 37 °C and shaking at 180 rpm until the exponential growth phase (OD600 between 0.6 and 0.8), when then the expression of the chimeras was induced with 1 mM of IPTG (isopro-pilthio-β-D-galactoside) for 3 h. After this period, the culture was fractionated in 250 mL tubes, centrifuged at 6,000 xg for 15 min at 4o C. The pellet was suspended in a solubilization buffer (8 M urea, 0.1 M Tris-base, 0 0.3 M NaCl and 5 mM imidazole, pH 8.0) and incubated in an orbital shaker at 60 rpm for 18 hours at room temperature. The cells were then sonicated (3 cycles of 30 s, 20 kHz), centrifuged (10,000 χ g for 30 min at 4 °C) and the supernatant collected and filtered on a 0.8 μΜ membrane (Mil-lipore). The chimeric proteins were purified by affinity chromatography using Ni2+ Sepharose HisTrap columns and dialyzed slowly using a cellulose membrane for dialysis (14 kDa pores) (Sigma). Dialysis was performed at 4°C against buffer containing 100 mM Tris-base and 300 mM Nacl in decreasing concentrations of urea. Finally, the protein was dialyzed against phosphate-buffered saline (PBS) (pH 7.2) overnight at 4 °C and stored at -20 °C. The concentration of purified proteins was determined using BCA Protein Assay Kit (Pierce, USA), with a standard curve for bovine serum albumin (BSA).

[018] Step 3: Recombinant proteins were subjected to Western Blot (SAMBROOK, J.; RUSSEL, DW Molecular cloning: a laboratory manual. 2001. Cold Spring Harbor Laboratory Press, 3rd Ed.), with anti-histidine monoclonal antibody ( Sigma-Aldrich, USA) to verify its expression. The chimeras were subjected to 10% SDS-PAGE and electrotransferred to Hybond™ ECL™ nitrocellulose membranes (Amersham Biosciences). Membranes were blocked with PBST plus 5% skimmed milk powder at 4°C, 12 - 20 h and, after this period, washed 3 times with PBST. Subsequently, the membranes were incubated for 1 h at room temperature with peroxidase-conjugated anti-histidine MAb at a dilution of 1:10000. After 3 washes with PBST, reactions were revealed with diaminobenzidine (DAB) and H2O2. The results can be seen in Figure 1.

[019] Step 4: Recombinant proteins were subjected to Western Blot (SAMBROOK, J.; RUSSEL, DW Molecular cloning: a laboratory manual. 2001. Cold Spring Harbor Laboratory Press, 3rd Ed.), with sera from individuals naturally infected with B. henselae, to verify its antigenicity. The protocol described above (Step 3) was also used in this step. However, the secondary antibody used was the MAb anti-human IgG conjugated with peroxidase at a dilution of 1:10000. The results can be seen in Figure 2.

[020] Step 5: For the use of these chimeric proteins in serological diagnosis, it is necessary that standardized control sera be used for reaction with the recombinant chimeras to establish the cutoff point of each evaluated protein, as well as specificity and sensitivity, which will result in the accuracy of the test developed for each chimera.

[021] Step 6: In another aspect, the invention deals with the use of chimeric proteins as recombinant vaccines (subunits, vectored and genetic) against bartonellosis. The steps of experimental evaluation of this application would be the immunization of rats with recombinant chimeric vaccines, evaluation of the humoral and cellular immune response and even the immunoprotective capacity of the vaccines, through challenge and sterility tests.
Brief description of the figures
Figure 1 illustrates the expression of recombinant chimeric proteins Q1, Q2 and Q3, produced in E. coli, by Western blotting using anti-histidine monoclonal antibody. M: Pre-Stained Protein Molecular Weight Marker (Ludwig); 1: rQ1(37 kDa) protein; 2: rQ2 protein (35 kDa); 3: rQ3 protein (37 kDa).
Figure 2 illustrates the antigenic characterization of the recombinant chimeras by Western blot with human serum naturally infected with B. henselae. M: Pre-Stained Protein Molecular Weight Marker (Ludwig); 1: rQ1(37 kDa) protein; 2: rQ2 protein (35 kDa); 3: rQ3 protein (37 kDa).

Claims (5)

Recombinant chimeric proteins containing Bartonella henselae antigens characterized by nucleotide sequences (SEQ ID N° 1, SEQ ID N° 2 and SEQ ID N° 3) present in the GroEL, 17kDa, P26, BadA, Pap31, OMP43 and OMP89 proteins of Bartonella henselae ; Recombinant vectors containing nucleotide sequences according to claim 1, characterized in that they are used for cloning or expression of chimeric proteins (SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6) in prokaryotes, eukaryotes or mammals, but not by them limited; Process for the production of recombinant chimeric proteins according to claim 1, characterized in that it uses a recombinant expression vector in Escherichia coli, followed or not by the purification of the proteins; Recombinant chimeric proteins obtained according to claims 1, 2 and 3 characterized by being used individually or in combination in serological diagnostic tests for bartonellosis; Recombinant chimeric proteins obtained according to claims 1, 2 and 3 characterized by composing individual or combined vaccine formulations to induce a specific immune response against Bartonella henselae.

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