Corynebacterium pseudotuberculosis recombinant esterase rcp09720 for use as a subunit vaccine against caseous lymphadenitis
Patent Information
- Application Number
- BR102016016754
- Authority / Receiving Office
- BR · BR
- Patent Type
- Patents
- Current Assignee / Owner
- Publication Date
- 2026-09-15
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Abstract
Description
1 / 19 Recombinant esterase rCP09720 from Corynebacterium pseudotuberculosis for use as a subunit vaccine against caseous lymphadenitis. DESCRIPTIVE REPORT BACKGROUND OF THE INVENTION FIELD OF THE INVENTION
[001] The present invention relates to the cloning, expression and characterization of the recombinant protein rCP09720 from Corynebacterium pseudotuberculosis, a probable esterase deposited in GenBank (NCBI) under accession number WP_013242814. It also demonstrates the use of the rCP09720 protein in vaccine formulations against caseous lymphadenitis, more specifically, the association of this protein with the adjuvant saponin, aiming to assist in the immunomodulation of the immune response. It falls within the international patent classification in the fields of invention A61K 35 / 74, A61K 39 / 00, A61K 39 / 05, A61K 39 / 39. DESCRIPTION OF THE STATE OF THE ART
[002] Caseous lymphadenitis (CL) is a chronic, subclinical, infectious disease affecting sheep and goats worldwide, characterized by the formation of abscesses in superficial and visceral lymph nodes, where the contents of these abscesses are rich in caseous or purulent material containing the microorganism Corynebacterium pseudotuberculosis (GOUVEIA, A. Caseous lymphadenitis. Goats & Bucks, v. 2, n. 5, p. 12-13, 1986), a Gram-positive, facultative anaerobic, pleomorphic, and non-sporulating bacterium (FILGUEIRA, KD, DE PAULA, VV, BATISTA, JS, FEIJÓ, FMC). Deep pyoderma caused by Corynebacterium pseudotuberculosis in agouti (Dasyprocta sp.). Brazilian Animal Science. v. 11, no. 2, pp. 461-464, 2010). Petition 870180146478, dated 10 / 30 / 2018, page 6 / 24 2 / 19
[003] The superficial form of the disease affects the peripheral lymph nodes, causing swelling and abscesses. The visceral form, however, causes systemic complications, as it affects the animal's internal organs (GUIMARÃES, AS, CARMO, FB, PAULETTI, RB, SEYFFERT, N., RIBEIRO, D., LAGE, AP, HEINEMANN, MB, MIYOSHI, A., AZEVEDO, V., GOUVEIA, AMG Caseous lymphadenitis: Epidemiology, diagnosis, and control. IIOAB Journal, v.2, p. 33-43, 2011). These two forms can coexist in asymptomatic infections without showing any clinical signs of infection, which makes diagnosis difficult (WILLIAMSON LH Caseous lymphadenitis in small ruminants. Vet Clin North Am, v. 17, p. 359-371, 2001; ARSENAULT, JO, GIRARD, C., DUBREUIL, P., DAIGNAULT, D., GALARNEAU JR, BOISCLAIR, J., SIMARD, C., BÉLANGER, D. Prevalence of and condemnation of maedi-visna bark, paratuberculosis and caseous lymphadenitis in sheep collected from Quebec, Canada. 67-81, 2003; PATON, MW, WALKER, SB, ROSE, IR, WATT, GF Prevalence of caseous lymphadenitis and usage of caseous lymphadenitis vaccines in sheep flocks. Aust Vet J, v. 81, p. 91-951, 2003).
[004] Aspects related to the clinical signs of CL directly culminate in significant economic losses for sheep and goat farmers, since the presence of abscesses in the superficial lymph nodes affects essential animal functions such as grazing and chewing, in addition to devaluing the animal's skin, depreciating its value by up to 40%, and also affecting wool production (FIGUEIREDO, EAP, SHELTON, M.; PANT, KP Goats skins. In: INTERNATIONAL CONFERENCE ON GOAT PRODUCTION AND DISEASE, Tucson. Proceedings Scottsdale: Dairy Goat Journal, v. 3, p. 488-490, 1982). The visceral form of the disease can lead to more serious consequences for the organism, such as reproductive problems (KRISHNA, L.; KULSHRESTHA, SB; Petition 870180146478, dated 10 / 30 / 2018, page 7 / 24 3 / 19 PALIWAL, OP Epididymo-orchitis in ram due to Corynebacterium ovis. Indian Veterinary Journal, vol. 54, n. 2, p. 517–519, 1977).
[005] Transmission of CL occurs primarily through contamination of cuts or wounds with viable bacteria from abscesses of infected animals; however, this microorganism can even penetrate intact skin (WILLIAMSON, LH Caseous lymphadenitis in small ruminants. Veterinary Clinics of North America - Food Animal Practice, v. 17, p. 359-371, 2001; ELLIS, TM, SUTHERLAND, SS, WILKINSON, FC, MERCY, AR, PATON, MW The role of Corynebacterium pseudotuberculosis lung lesions in the transmission of this bacterium to other sheep. MERCY, AR, ELLIS, TM The spread of Corynebacterium pseudotuberculosis infection to unvaccinated and vaccinated sheep. Aust. Vet., SANTIAGO, LB, PINHEIRO, RR Caseous Lymphadenitis: The State of the Art. Document / Embrapa Goats, 1st ed., 60p. 2007).
[006] Exposure to C. pseudotuberculosis can occur through direct physical contact with the affected animal, or indirectly, through contaminated fomites (BAIRD, GJ, FONTAINE, MC Corynebacterium pseudotuberculosis and its role in ovine caseous lymphadenitis, J. Comp. Pathol., v.137, p.179-210, 2007). Other factors contribute to the spread of infection and can be considered risk factors, such as poor hygiene in facilities, direct contact between infected animals, and shearing.
[007] C. pseudotuberculosis is able to survive for several weeks in the environment and, therefore, becomes a constant source of infection for the Petition 870180146478, dated 10 / 30 / 2018, page 8 / 24 4 / 19 herd (YERUHAM, I., ELAD, D., FRIEDMAN, S., PERL, S. Corynebacterium pseudotuberculosis infection in Israeli dairy cattle. Epidemiol. Infect., v. 131, p. 947-955, 2003). Furthermore, environmental care is often neglected by animal handlers, which contributes to the perpetuation of the disease in the herd (ALVES, FSF, PINHEIRO, RR, PIRES, PC. Caseous lymphadenitis: pathogenesis, diagnosis, control. Sobral: EMBRAPA-CNPC, v.27, 16 p., 1997).
[008] As already mentioned, early diagnosis is quite difficult, and no specific diagnostic test is available, which increases the severity of the disease due to the delay in treatment. To date, there is also no strategy considered effective, since the bacteria are inside a fibrous capsule preventing them from coming into contact with substantial amounts of antibiotics (BARH, D., TIWARI, S., JAIN, N., ALI, A., SANTOS, AR, MISRA, AN, AZEVEDO, V., KUMAR, A. In silico subtractive genomics for target identification in human bacterial pathogens. Drug Dev Res, v. 72, p. 162-177, 2011).
[009] Thus, in order to assist in the prophylaxis of this disease, some vaccines have been produced and are already on the market, among them we can mention Glanvac® (combined toxoid) and LinfoVac® (attenuated vaccine of the strain called 1002 of C. pseudotuberculosis) (DORELLA, FA, PACHECO, LG, SEYFFERT, N., PORTELA, RW, MEYER, R., MIYOSHI, A., AZEVEDO, V. Antigens of Corynebacterium pseudotuberculosis and prospects for vaccine development). These commercial vaccines present some drawbacks such as unsatisfactory protection rates and the need for annual boosters, which makes prophylaxis more expensive (SILVA, JW, DROPPA-ALMEIDA, D., BORSUK, S., AZEVEDO, V., PORTELA, RW, MIYOSHI, A., ROCHA, FS, DORELLA, F. A, VIVAS, WL, PADILHA, FF, HERNÁNDEZ Petition 870180146478, dated 10 / 30 / 2018, page. 5 / 19 MACEDO, M.L., LIMA-VERDE, I.B. Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis. BMC Veterinary Research,v. 10, n. 1, p. 1-8, 2014).
[010] Given the need for a vaccine with greater efficiency and protective potential, several studies have been conducted to identify virulence factors involved in CL. Among them, we can mention toxic cell wall lipids, which mediate the bacteria's resistance to death by phagocytic cells, by preventing its phagocytosis by the host cell's lysosomal enzymes. This virulence factor is associated with the formation of granulomas (HARD, GC. Comparative toxic effect on the surface lipid of Corynebacterium ovis on peritoneal macrophages. Infect. Immun., v.12, p. 1439-1449, 1975; always look) and the exotoxin sphingomyelin-degrading phospholipase D (PLD), cited as responsible for the dissemination of the pathogen within the host through increased local vascular permeability (BATEY, RG. Pathogenesis of caseous lymphadenitis in sheep and goats). Aust. Vet. J., v. 63, p. 269-272, 1986). This is considered the most important virulence factor of the bacterium.
[011] Some vaccines have already been produced using genetic inactivation of phospholipase D (Toxminus). Hodgson et al. (1999) obtained 44% protection in sheep challenged with C. pseudotuberculosis, while Moore et al. (1999), using Toxminus as a live vector expressing a range of vaccine antigen candidates under the action of different promoters, concluded that the expression values presented were sufficient to induce the production of specific antibodies against the recombinant protein after a single dose of the vaccine. However, there are still no concrete results regarding the efficacy of this exotoxin (HODGSON, AL, CARTER, K., TACHEDJIAN, M., KRYWULT, J., CORNER, LA, McCOLL, M., Petition 870180146478, dated 10 / 30 / 2018, page 10 / 24 6 / 19 CAMERON, A. Efficacy of an ovine caseouslymphadenitis vaccine formulated using a genetically inactive form of theCorynebacterium pseudotuberculosis phospholipase D. Vaccine, v. 17, p. 802-808, 1999; MOORE, R.J., ROTHEL, L., KRYWULT, J., RADFORD, A.J., LUND, K.,ADRIAN L.M., HODGSON, A.L.M. Foreign gene expression in C. pseudotuberculosis: development of a live vaccine vector. Vaccine, v. 18, n. 5 / 6, p. 487-497, 1999; ALVES, S.F.S., SANTIAGO, L.B., PINHEIRO, R.R. Linfadenite Caseosa:o Estado da Arte. Documento / Embrapa Caprinos, 1aed., 60p. 2007).
[012] Other strategies have also been adopted seeking better results, including DNA vaccines and recombinant subunit vaccines (DORELLA, FA, PACHECO, LG, SEYFFERT, N., PORTELA, RW, MEYER, R., MIYOSHI, A., AZEVEDO, V. Antigens of Corynebacterium pseudotuberculosis and prospects for vaccine development. Expert review of vaccines, v. 8, n. As an example of the latter, the recombinant protein called CP40 stands out, a serine protease that generated up to 90% protection in mice against challenge with C. pseudotuberculosis, in addition to significant production of the IgG2a isotype, indicative of a Th1 response (SILVA, JW, DROPPA-ALMEIDA, D., BORSUK, S., AZEVEDO, V., PORTELA, RW, MIYOSHI, A., ROCHA, FS, DORELLA, F. A, VIVAS, WL, PADILHA, FF, HERNÁNDEZ-MACEDO, ML, LIMA-VERDE, IB Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis.1-8, 2014). Despite these results, no satisfactory vaccine prototype has yet been developed for use in sheep and goats (BASTOS, LB, LOUREIRO, D., RAYNAL, JY, GUEDES, MT, VALE, VLC, MOURACOSTA, LF, GUIMARÃES, JE, AZEVEDO, V., PORTELA, RW, MEYER, R. Association between haptoglobin and IgM levels and the. Petition 870180146478, dated 10 / 30 / 2018, p. 11 / 24 7 / 19 clinical progression of caseous lymphadenitis in sheep. BMC Veterinary Research, v. 9, p. 1-7, 2013).
[013] Therefore, the search for the characterization and identification of new vaccine targets for C. pseudotuberculosis is constant. In this context, new genome sequencing technologies, target prediction by bioinformatic tools, and comparative genomics and proteomics analyses stand out. Once the genome of a microorganism is comprehensively known, the search for virulence genes, vaccine targets, and components that could be used in diagnostic procedures becomes easier (RUIZ, JC, D'AFONSECA, V., SILVA, A. et al. Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains. Plos One, v. 6, n. 4, 2011).
[014] Another innovative technology that has been gaining ground in the search for potential vaccine targets is reverse vaccinology, which involves sequencing the agent's genome, analyzing its proteins, predicted through bioinformatics and based on hydrophobic or hydrophilic characteristics, determining the probable position of the proteins within the microorganism (SCHATZMAYR, HG New perspectives in viral vaccines. History, Sciences, Health - Manguinhos, v. 10, Supplement 2, p. 655-69, 2003).
[015] In order to obtain this type of information, in 2011, Ruiz and colleagues characterized the genome of two strains of C. pseudotuberculosis (Cp 1002 and CpC231) and compared them with each other and with others already available in databases, and reported several genes and proteins involved in pathogenicity islands of this microorganism (RUIZ, JC, D'AFONSECA, V., SILVA, A. et al. Evidence for evolution of the reducing genome and lateral acquisition of virulence functions in two strains of Corynebacterium pseudotuberculosis). Petition 870180146478, dated 10 / 30 / 2018, p. 12 / 24 8 / 19
[016] Santos and colleagues in 2012 performed pangenomic analyses with the strains 1002, C231, I19, FRC41 and PAT10 of C. pseudotuberculosis. Instead of studying genes in a single genome, this technique makes it possible to study genes present in different strains of the same species. This work presented in silico analyses of these five genomes and collected data to predict the exported proteins common to all of them. The cp1002_RS09720 gene was the target of this analysis (SANTOS, AR, CARNEIRO, A., GALA-GARCÍA, A. et al. The Corynebacterium pseudotuberculosis in silico predicted pan-exoproteome. BMC genomics, v. 13, Suppl. 5, p. S6, 2012).
[017] In January 2014, the genome of the C. pseudotuberculosis 1002 strain, containing the cp1002_RS09720 gene, was deposited in GeneBank under accession number CP001809.2 and identified as a probable Trehalose corynomycolyl transferase B, located in the genome at position 2159222...2160147. However, in August 2015, the genome underwent a re-annotation, receiving the accession number NC_017300.1 and describing that this gene produces an esterase formed by 341 amino acids, becoming known as decp1002_RS09720, under the accession number WP_013242814 GI:503007838 (NCBI Genome. Corynebacterium pseudotuberculosis 1002, complete genome. Available at: http: / / www.ncbi.nlm.nih.gov / nuccore / 340539261. Accessed on: April 5, 2016). This genome also presented the second highest MED score in the analysis of pan-secretomes of strains 1002 and C231 of the same pathogen (REZENDE, AFS, BRUM, AA, REIS, CG, ANGELO, HR, LEAL, KS, SILVA, MTO, SIMIONATTO, S., AZEVEDO, V., SANTOS, A., PORTELA, RWDELLAGOSTIN, O., BORSUK, S. In silico identification of antigenic targets of Corynebacterium pseudotuberculosis and application in immunodiagnosis. J. Med. Microbiol., 2016).
[018] Through a preliminary search in patent filing databases we can conclude that this esterase has not yet been cited in Petition 870180146478, dated 10 / 30 / 2018, p. 13 / 24 9 / 19 no scientific and technological production related to its use, expression, or even its use in recombinant vaccines.
[019] The state of the art existing to date regarding vaccine compositions available for LC is disclosed in Brazilian patents BR PI9903524-3 and BR PI0205673-9. Both describe the use of live attenuated vaccines.
[020] As for documents proposing the use of recombinant proteins in vaccine formulations, we can cite as examples patents BRPI1005625-4 and CA2078801, which claim, respectively, the use of a set of recombinant peptides from C. pseudotuberculosis in a vaccine composition, and the use of a vaccine composed of a phospholipase D mutant.
[021] In this context, due to the fact that live vaccines present numerous disadvantages, such as high cost, side effects, lack of stability regarding their efficiency in different species; and the inventions related to the use of recombinant proteins as vaccine targets have not yet been sufficiently effective to develop a product that can combat CL, it is still necessary to search for new strategies, thus revealing the potential of using the rCP09720 protein as a recombinant subunit vaccine.
[022] However, in the search for a vaccine, several other variables must be taken into account and are considered as important as the choice of targets. For example, the search for new adjuvants, substances added to the vaccine in its final formulation, with the aim of increasing the immune response of the recipient (SCHATZMAYR, HG New perspectives in viral vaccines. History, Sciences, Health Manguinhos, v. 10, Supplement 2, p. 655-69, 2003).
[023] The present invention proposes associating said recombinant protein with saponin adjuvant, which has been used in the formulation of veterinary vaccines, stimulating the Th1 response and the production of Petition 870180146478, dated 10 / 30 / 2018, p. 14 / 24 10 / 19 specific cytotoxic T lymphocytes (RAJPUT ZI, HU, S., XIAO, C., ARIJO, AGAdjuvant effects of saponins on animal immune responses. J Zhejiang Univ Sci B, v. 8, p. 153-161, 2007).
[024] This adjuvant has been tested in some vaccine models in veterinary medicine, and these studies have shown promising results in protection against murine visceral leishmaniasis, bovine respiratory syncytial virus, and foot-and-mouth disease virus. These results suggest that saponins may be a good choice for use as an adjuvant in vaccine models against CL (BASTOS, LB, LOUREIRO, D., RAYNAL, JY, GUEDES, MT, VALE, VLC, MOURA-COSTA, LF, GUIMARÃES, JE, AZEVEDO, V., PORTELA, RW, MEYER, R. Association between haptoglobin and IgM levels and the clinical progression of caseous lymphadenitis in sheep. SUMMARY OF THE INVENTION
[025] The present invention relates to the production and use of the recombinant protein rCP09720 from C. pseudotuberculosis as a vaccine antigen, associated with the saponin adjuvant, aiming to enhance the protective effect of recombinant subunit vaccines for CL. However, the formulations are not limited to the use of this class of adjuvant. The recombinant protein rCP09720, classified as an esterase, has 315 amino acids, a molecular weight of approximately 35 kDa and has a tail composed of 6 histidines, which are used to facilitate the purification process. For its production, a heterologous expression system based on E. coli and the pAE cloning and expression vector were used. ArCP09720 has already demonstrated a protection percentage of up to 40% (Figure 6), not being limited to this value according to the formulation used, in addition to having increased the production of specific anti-rCP09720 antibodies. Petition 870180146478, dated 10 / 30 / 2018, page 15 / 24 11 / 19 Brief Description of the Figures
[026] Figure 1 corresponds to the 1% agarose gel showing the PCR products of the cp1002_RS09720 gene with a size of 933 bp. In (1) 1kB plus molecular weight marker (Invitrogen), (2) band indicating the amplification of cp1002_RS09720 at a size of 933 bp.
[027] Figure 2 shows the characterization of recombinants by enzymatic digestion using BamHI and EcoRI enzymes. In (1) Molecular weight marker 1 Kb plus (Invitrogen); (2) recombinant plasmid pAE / cp1002_RS09720 after digestion. The arrow points to the insert cp1002_RS09720 containing 921 bp.
[028] Figure 3 represents confirmation of the identity of the recombinant protein expressed by Western blotting with anti-6xhistag monoclonal antibody (Sigma Aldrich). In (1) pre-stained marker (PageRuler™ Prestained Protein Ladder, Thermo Fisher), (2) rCP09720 represented by a reactive band of approximately 35 kDa.
[029] Figure 4 shows the constructed pAE / cp1002_RS09720 vector map.
[030] Figure 5 corresponds to an SDS-PAGE electrophoresis (12%) demonstrating the purified protein rCP09720. In (1) pre-stained marker, (2) rCP09720.
[031] Figure 6 represents the survival curve obtained in the immunization assay of mice with the different vaccine compositions and challenge with the virulent strain MIC-6 of Corynebacterium pseudotuberculosis.
[032] Figure 7 shows the evaluation of total IgG (7a), IgG1 (7b) and IgG2a (7c) anti-rCP09720 levels in mice immunized with rCP09720 associated with saponin adjuvant, obtained in the humoral response assay. The results are presented as mean and standard deviation (bars) of the absorbances (nm) found in the indirect ELISA assay for each experimental group. Blood was collected and evaluated on days 0, 21 and 42 after the first immunization. aDifferent letters within the same day Petition 870180146478, dated 10 / 30 / 2018, page 16 / 24 12 / 19 represent groups with significantly different protection rates (p<0.05). DETAILED DESCRIPTION OF THE INVENTION
[033] With the aim of providing new effective antigens in the development of vaccine formulations against caseous lymphadenitis, the present invention proposes the production of a recombinant esterase from C. pseudotuberculosis (rCP09720) and its use in association with saponin adjuvant for the composition of a vaccine formulation.
[034] The present invention is described in more detail as follows: Cloning, expression and characterization of recombinant protein
[035] For the development of the invention, it is necessary to use strains of C. pseudotuberculous 1002 and Mic-6 and Escherichia coli TOP10 and BL21 Star. The C. pseudotuberculous strains are cultivated in “Brain Heart Infusion” (BHI) medium supplemented with 0.5% Tween 80, at 37 °C for 72h under agitation. For cultures in solid medium, 1.5% bacteriological agar is added to the culture medium. The E. coli strains are cultivated in Luria Bertani (LB) medium or LB medium containing 1.5% bacteriological agar for 16h at 37 °C. When necessary, the LB medium should be supplemented with 100μg / mL of ampicillin.
[036] For gene cloning on the pAE plasmid, it is initially necessary to amplify the cp1002_09720 gene (SEQ ID NO:3) via Polymerase Chain Reaction (PCR) using the Forward and Reverse primers as described in SEQ ID NO:4 and SEQ ID NO:5. A restriction site for the HindIII enzyme was added to primer F, while a restriction site for the EcoRI enzyme was added to primer R. For PCR, 50 ng of C. pseudotuberculosis 1002 genomic DNA is required, in addition to 10 μM of each primer and Mastermix (Promega) in a final volume of 50 pL. The PCR product should be visualized on a 1% agarose gel stained with Blue Green (LGC Biotechnology) (Figure 1). The gene Petition 870180146478, dated 10 / 30 / 2018, p. 17 / 24 13 / 19 cp1002_RS09720 has its cloning performed at the BamHIe and EcoRI sites of the pAE plasmid (RAMOS, CR, ABREU, PA, NASCIMENTO, AL, HO, PL A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal Histagged fusion peptide, Braz. J. Med. Biol. Res., v.37, p.1103-1109, 2004). For this purpose, pAEecp1002_RS09720 are digested with the restriction enzymes BamHIeEcoRI (FERMENTAS) and subsequently ligated using the enzyme T4 DNA ligase (FERMENTAS), generating as a product the recombinant plasmid pAE / cp1002_RS09720 (Figure 4) containing the CDS corresponding to the rCP09720 protein (SEQ ID NO:2). The ligation products are transformed by electroporation into competent TOP 10 E. coli cells and cultured in LB medium with 100 μg / mL of ampicillin for 16 h at 37 °C. A rapid screening by colony lysis with phenol chloroform (v / v), followed by digestion with restriction enzymes, is performed for characterization of the recombinant clones (Figure 2).
[037] For recombinant protein expression, the pAE / cp1002_RS09720 vector is transformed by heat shock into the E. coli BL21 Star expression strain. Expression induction occurs by adding 1 mM IPTG to the culture maintained under orbital shaking at 37 °C for 3 h. The identity of the recombinant protein is confirmed by Western blotting (SAMBROOK J., RUSSEL DW Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York, 2001) using peroxidase-conjugated anti-6xhistag monoclonal antibody (Sigma Aldrich). For this purpose, samples containing rCP09720 are mixed with buffer (100-mM Tris-HCl pH 6.8, 100-mM 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) under reducing conditions, heated to 100°C for 10 min, and subjected to electrophoresis on 12% SDS-PAGE gel.Subsequently, the sample is transferred to a nitrocellulose membrane (GE Healthcare), which, after 2 hours, is blocked with PBS containing 5% skim milk for 1 hour. Petition 870180146478, dated 10 / 30 / 2018, page 18 / 24 14 / 19 37°C. Next, anti-6Xhistag (Sigma Aldrich) is added to the membrane at a dilution of 1:4000 at 37°C for 1 h. The membranes are washed with 0.05% PBS-T and incubated with diluted 1:4000 mouse anti-IgG conjugated with peroxidase (Sigma Aldrich) in PBS-T at 37°C for 1 h. Reactive bands are revealed using 3,3'-diaminobenzidine (DAB) and H2O2. The identity of the recombinant esterase rCP09720 (SEQ ID NO:1) can be confirmed, as visualized in Figure 3, by a reactive band of approximately 35 kDa.
[038] Purification is performed by affinity chromatography on a nickel-loaded sepharose column (HisTrap™; GE Healthcare). Its purity is determined by a 12% SDS-PAGE (Figure 5) and the concentration determined by the BCA kit (Pierce). Preparation of Saponin Adjuvant and Vaccine Formulation
[039] To prepare saponin as an adjuvant, 1.5 mg of Saponin for Molecular Biology (Sigma-Aldrich) is weighed, this powder is recovered in 1 mL of 1x PBS, generating a stock solution of 1.5 mg / mL, which is subjected to sterile filtration with a 0.22 nm membrane. This should be stored at 4°C until the time of use. In the vaccine formulation, 5 μL of the stock solution (equivalent to 7.5 μg of saponin) is used in conjunction with 50 ng of the rCP09720 protein for each vaccine dose in a final volume of 300 nL.
[040] The present invention may be better understood by the examples that follow, but it is not limited to those examples. EXAMPLE 1 - EFFECT OF THE RECOMBINANT PROTEIN rCP09720 ASSOCIATED WITH DIFFERENT ADJUVANTS AS A RECOMBINANT SUBUNIT VACCINE AGAINST CASEOUS LYMPHADENITIS - EVALUATION OF THE PROTECTIVE EFFECT Petition 870180146478, dated 10 / 30 / 2018, pp. 19-24 15 / 19
[041] The procedure described in the following example illustrates the performance of an evaluation test of the protective potential of recombinant protein rCP09720 as a vaccine antigen in association with saponin adjuvant, against challenge using a virulent strain of the pathogen C. pseudotuberculosis.
[042] The vaccine formulation consists of 50μg of the rCP09720 esterase per vaccine dose, in a final volume of 300 DL. Its production is as described previously, in combination with the saponin adjuvant, the concentration and preparation method of which have already been reported in the "detailed description of the invention" section. For the immunization and challenge experiment, 3 groups composed of 10 female Balb / c mice, aged 6-8 weeks, susceptible to infection by C. pseudotuberculosis, were used. The animals were provided by the Central Animal Facility of the Federal University of Pelotas. The conduct of the experiment was approved by the ethics committee on animal experimentation of the aforementioned University (CEEA / UFPel No. 2442). The mice were immunized subcutaneously with the recombinant protein CP09720 associated with the saponin adjuvant according to the immunization scheme described below.
[043] The groups were distributed as follows: - Group 1: 0.9% saline solution (negative control); - Group 2: Bacterin (positive control); - Group 3: rCP09720 + saponin; Immunization was carried out following this protocol:
[044] Day 0: 1st dose of vaccine / Day 21: 2nd dose of vaccine (booster) / Day 42: challenge with the MIC-6 strain at a concentration of 10⁴ via intraperitoneal injection. The pathogenic strain of C. pseudotuberculosis MIC-6 was cultured in Brain and Heart Infusion Broth (BHI Broth) or BHI-Agar at 37 °C for 48 h under agitation. The pathogenic strain Mic-6 of C. pseudotuberculosis used in the challenge was subjected to determination Petition 870180146478, dated 10 / 30 / 2018, page 20 / 24 16 / 19 minimum dose required to induce lethal infection in 50% of animals (LD50).
[045] The LD50 was determined by inoculation of serial doses according to the protocol described by SIMMONS, CP, HODGSON, AL, STRUGNELL, RA Attenuation and vaccine potential of aroQ mutants of Corynebacterium pseudotuberculosis, Infect. Immun., v.65, p.3048-3056, 1997. For the calculation of the lethal dose, groups of four mice were infected intraperitoneally with serial doses ranging from 10⁶ to 10¹ colony-forming units (CFU). The LD50 was determined as the number of CFU that led to the death of 50% of the mice according to Reed's method (REED, LJ, MUENCH, H. A simple method of estimating fifty percent endpoints, Am. J. Hygiene, v.27, p.493-497, 1938).
[046] Thus, after the LD50 challenge, the animals were monitored for 30 days, followed by sacrifice of the animals. The protection rate for each group tested was calculated based on the survival of the immunized animals.
[047] For statistical analysis, Fisher's exact test and the logrank test were used to determine significant differences (p < 0.05) in mortality and survival rate, respectively, between the experimental groups.
[048] After this follow-up period, we obtained results showing 40% protection in the group immunized with arCP09720 (Group 3) (Figure 6). In contrast, in the positive control (Bacterin) all animals remained alive, and in the negative control (0.9% saline solution) all died. These data allow us to infer that the use of the recombinant protein rCP09720 in vaccine formulations is effective in protecting against challenge with the virulent strain of C. pseudotuberculosis. Thus, this protein proved promising, as it induced immunological memory. And, since rCP09720 Petition 870180146478, dated 10 / 30 / 2018, pages 21 / 24 17 / 19 can be used both alone and in combination with other proteins; different levels of protection can be achieved, depending on the antigen-adjuvant combination used. EXAMPLE 2 - DArCP09720NA CAPACITY FOR INDUCING A HUMORAL IMMUNE RESPONSE
[049] This example illustrates the determination of the humoral immune response through the detection of specific antibody production by indirect ELISA. It also allows us to assess whether the vaccine formulation containing the recombinant protein rCP09720 is capable of increasing antibody production against this vaccine antigen.
[050] The humoral response, for quantification of total IgG levels and IgG1 and IgG2a isotypes, was determined by indirect ELISA using sera obtained from animals in groups G1 to G3 through blood collection by puncture of the retro-orbital plexus veins on days 0, 21, and 42 of the immunization experiment (as per Example 1). The blood was stored in 1.5 mL microtubes and centrifuged at 3,500 rpm for 15 minutes. The serum was then extracted and used to perform the ELISA.
[051] For this, 96-well flat-bottom plates (TPP) were sensitized with 100 pL of a solution containing carbonate-bicarbonate buffer pH 9.6 and 1 μg / mL of recombinant protein rCP09720 and incubated for 18 ha at 4°C. Subsequently, the plates were washed 3 times with PBS-T (PBS 1X pH 7.4; 0.1% Tween 20) and blocked with 200 pL / well of PBS-T and 5% skim milk for 2 ha at 37°C. After this, the plates were washed again with PBS-T and 100 pL / well of mouse serum samples (1:50 in PBS-T) were added in duplicate. After one hour of incubation at 37°C and 3 washes with PBS-T, 100 pL / well of the antibody conjugated with anti-IgGTotal mouse peroxidase (Sigma-Aldrich) at a 1:5,000 dilution were added. On plates for detection of IgG1 or IgG2a isotypes (Sigma-Aldrich), anti-antibodies were added. Petition 870180146478, dated 10 / 30 / 2018, pages 22 / 24 18 / 19 Mouse IgG1 or anti-IgG2a produced in goats were added at dilutions of 1:5,000 and 1:2,000 respectively, followed by incubation for 1 hour. For the isotype plates, an additional step was performed before the development step, in which each well was supplemented with peroxidase-conjugated goat anti-antibody (Sigma-Aldrich) at a dilution of 1:5,000 (1 hour at 37 °C). After this, 5 more washes were performed with PBS-T, and 100 μL / well of developer solution [200 pmoles orthophenylenediamine (OPD, Sigma-Aldrich) diluted in 50 mL of citrate-phosphate buffer pH 5 and 0.05% H2O2] were added. To stop the reaction, 50 μL / well of the stopping solution containing 4 N sulfuric acid was added. The absorbance was measured at 492 nm using an ELISA plate reader (Mindray).
[052] Statistical analyses were performed using GraphPad Prism version 6.0 for Windows (GraphPad Software, USA). Differences in IgG production in the experimental groups were verified using one-way ANOVA, followed by Tukey's post-hoc test. P-values less than 0.05 were considered statistically significant.
[053] When evaluating the results obtained in the ELISA, it was concluded that the levels of total IgG specific for the recombinant protein CP09720 (Figure 7a) increased progressively over the days for the experimental group G3. This group showed a significantly higher production of total IgG (p < 0.05) when compared to the controls (G1 and G2) on day 42 of the experiment. On the initial days (0 and 21) this difference was not observed.
[054] The data relating to IgG1 (Figure 7b) and IgG2a (Figure 7c) isotypes were very similar to those found for Total IgG. However, the group inoculated with rCP09720 (G3) showed significantly different IgG1 levels on day 21 (p < 0.05) from the other groups, a fact that did not occur for the other isotypes on this day of the experiment. Petition 870180146478, dated 10 / 30 / 2018, pp. 23 / 24 19 / 19
[055] The absorbances found for the two isotypes, IgG1 and IgG2a in group G3 were quite similar to each other, suggesting that the group treated with rCP09720 and saponin presented a mixed immune response mediated by Th1 and Th2 lymphocytes. Th1 cells are involved in immunity against intracellular pathogens, such as C. pseudotuberculosis, and assist in the activation of macrophages and the production of complementary activators, such as IgG2a. Th2 cells, on the other hand, act in immunity against extracellular pathogens and help B cells in the production of neutralizing antibodies (IgG1) (SILVA, JW, DROPPAALMEIDA, D., BORSUK, S., AZEVEDO, V., PORTELA, RW, MIYOSHI, A., ROCHA, FS, DORELLA, F. A, VIVAS, WL, PADILHA, FF, HERNÁNDEZ-MACEDO, ML,LIMA-VERDE, IB. Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis. BMC Veterinary Research., v.10, n.1, p. 1-8, 2014).Thus, it is observed that the vaccine formulation containing the recombinant esterase rCP09720, described in the present invention, was efficient in activating the humoral response and inducing a mixed Th1 / Th2 profile, necessary in combating C. pseudotuberculosis infection. Petition 870180146478, dated 10 / 30 / 2018, p. 24 / 24
Claims
1 / 1 CLAIMS 1. Recombinant esterase rCP09720 from Corynebacterium pseudotuberculosis, characterized by consisting of a recombinant protein comprising the amino acid sequence described in SEQ ID NO:1, produced in a heterologous expression system.
2. Recombinant esterase rCP09720 from Corynebacterium pseudotuberculosis, according to claim 1, characterized by being produced from an expression vector in which the coding sequence described in SEQ ID NO:2 has been inserted.
3. Recombinant esterase rCP09720 from Corynebacterium pseudotuberculosis, according to claims 1 and 2, characterized by comprising a genetically engineered polyhistidine tail inserted at the N-terminal portion.
4. Recombinant esterase rCP09720 from Corynebacterium pseudotuberculosis, according to claims 1 to 3, characterized by comprising 315 amino acids and a molecular mass of 35 kDa.
5. VACCINE FORMULATION AGAINST CASEOUS LYMPHADENITIS, characterized by comprising the association of the purified recombinant esterase rCP09720 from Corynebacterium pseudotuberculosis, as defined in claims 1 to 4, with a physiologically acceptable vaccine adjuvant.
6. VACCINE FORMULATION AGAINST CASEOUS LYMPHADENITIS, according to claim 5, characterized by comprising an amount of 50 to 200 pg of the rCP09720 protein.
7. VACCINE FORMULATION AGAINST CASEOUS LYMPHADENITIS, according to claim 5, characterized by comprising an amount of 5 to 20 pg of saponin as a vaccine adjuvant.
8. VACCINE FORMULATION AGAINST CASEOUS LYMPHADENITIS, according to claims 4 to 7, characterized by comprising a parenteral formulation in a pharmaceutically acceptable vehicle, optionally containing diluent, excipient or stabilizer] Petition 870260075744, dated 07 / 29 / 2026, p. 8 / 9