BR102020006421A2 - USE OF RECOMBINANT CHIMERA IN PURIFIED AND NON-PURIFIED FORMS FOR COMPOSITION OF RECOMBINANT VACCINE AGAINST CASEOUS LYMPHADENITIS - Google Patents

Abstract

In order to develop an efficient and safe vaccine formulation against caseous lymphadenitis, the present invention describes the production of a recombinant chimera obtained from the selection of epitopes from the NanH, PknG, SodC and SpaC proteins of Corynebacterium pseudotuberculosis. The invention also comprises the production of the chimera in a heterologous expression system, using the pAE vector under the action of the inducible T7 promoter, and its use in purified and non-purified form. The invention also includes the association of said vaccine formulation with the saponin adjuvant in order to immunomodulate a more potent immune response. The vaccine formulation refers to a vaccine composition with potential for use against caseous lymphadenitis, since the antigen used in our invention was able to induce the production of specific antibodies.

Description

USE OF RECOMBINANT CHIMERA IN PURIFIED AND NON-PURIFIED FORMS FOR COMPOSITION OF RECOMBINANT VACCINE AGAINST CASEOUS LYMPHADENITIS FUNDAMENTALS OF THE INVENTION FIELD OF THE INVENTION

[001] The present invention relates to the selection of epitopes present in NanH, PknG, SpaC and SodC proteins, deposited in GenBank (NCBI) under accession numbers ADK28179.1, ADK29622.1, ADK29663.1 and ADK28404.1 , respectively. It also demonstrates the construction, synthesis and expression of a recombinant chimera from selected epitopes, and its use in vaccine formulations against caseous lymphadenitis. It also comprises the production of an Escherichia coli bacterin expressing the recombinant chimera and its association, in both purified and unpurified forms, with the saponin adjuvant in order to immunomodulate a more efficient immune response.

DESCRIPTION OF THE STATE OF THE TECHNIQUE

[002] Corynebacterium pseudotuberculosis is a gram-positive and facultative intracellular bacterium capable of infecting numerous species of mammals and developing a chronic disease called caseous lymphadenitis (CL)(WINDSOR, PA Control of Caseous Lymphadenitis, Vet Clin Food Anim, v. 27, p. 193–202, 2011). CL is a globally distributed disease that affects the superficial lymph nodes (cutaneous form) and internal organs (visceral form) of affected animals, causing economic losses by compromising their skin, weight, milk and meat production ( DORELLA, FA; GALA-GARCIA, A.; PINTO, AC; SARROUH, B.; ANTUNES, CA; RIBEIRO, D.; ABURJAILE, FF; FIAUX, KK; GUIMARÃES, LC; SEYFFERT, N.; EL-AOUAR, RA; SILVA, R.; HASSAN, SS; CASTRO, TLP; MARQUES, WS; RAMOS, R.; CARNEIRO, A.; DE SÁ, P.; MIYOSHI, A.; AZEVEDO, V.; SILVA, A. Progression of 'OMICS' methodologies for understanding the pathogenicity of Corynebacterium pseudotuberculosis: the Brazilian experience. Computational and Structural Biotechnology Journal, v. 6, n. 7, 2013).

[003] To date, no commercially available vaccine provides effective protection against CL (COSTA, MP, MCCULLOCH, JA, ALMEIDA, SS, DORELLA, FA, FONSECA, CT, OLIVEIRA, DM, TEIXEIRA, MF, LASKOWSKA, E ., LIPINSKA, B., MEYER, R., PORTELA, RW, OLIVEIRA, SC, MIYOSHI, A., AZEVEDO, V. Molecular characterization of the Corynebacterium pseudotuberculosis hsp60-hsp10 operon, and evaluation of the immune response and efficacy protective induced by hsp60 DNA vaccination in mice, BMC research notes, v. 4, p. 243, 2011). Although there are many vaccines, such as Linfovac, Glanvac and Biodectin, they are primarily intended for use in sheep and goats and provide varying levels of protection (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, p.205–213, 2009). The development of superficial and deep abscesses is also observed in animals vaccinated with them (WILLIAMSON, LH Caseous lymphadenitis in small ruminants, The Veterinary clinics of North America. Food animal practice, v. 17, p. 359–71, 2001) .

[004] Since C. pseudotuberculosis commonly resides intracellularly within abscesses formed in response to infection, most antibiotics cannot penetrate encapsulated abscesses and treatment has little benefit. N. AND VALLE, PS Welfare effects of a disease eradication program for dairy goats, Animal, pp. 1–9, 2016). The absence of an adequate treatment makes prophylaxis based on the development of new vaccine technologies have a prominent place as a disease prevention strategy in herds (GUIMARÃES, ADS, BORGES, F., PAULETTI, RB, SEYFFERT, N., RIBEIRO, D., LAGE, AP, HEINEMANN, MB, MIYOSHI, A., MARIA, A., GOUVEIA, G., FEDERAL, U., GERAIS, DM, AV, U., CARLOS, A., POSTAL, C ., CEP, U., HORIZONTE, B., GERAIS, M. Caseous lymphadenitis: epidemiology, diagnosis, and control. The IIOAB Journal, v. 2, p. 33–43, 2011).

[005] Some strategies already studied in the production of vaccines for CL include: inactivated strains of C. pseudotuberculosis, fractions of bacterial cells containing antigens, recombinant proteins and DNA vaccines (TACHEDJIAN, M., KRYWULT, J., MOORE, RJ, HODGSON, ALM Caseous lymphadenitis vaccine development: site-specific inactivation of the Corynebacterium pseudotuberculosis phospholipase D gene, Vaccine, v. 13, pp. 17850-1792, 1995; CHAPLIN, PJ, ROSE, R. DE, BOYLE, JS, KELLY, J., TENNENT, JM, LEW, AM, SCHEERLINCK, JY, ROSE, RDE, WATERS, PMC & TENNENT, J. A NM Targeting Improves the Efficacy of a DNA Vaccine against Corynebacterium pseudotuberculosis in Sheep. Infect Immun. 4. p. 1–6, 1999; RIBEIRO, D., ROCHA, FDE, LEITE, KM, SOARES, SDE, SILVA, A., PORTELA, RW, MEYER, R., MIYOSHI, A., OLIVEIRA, SC, AZEVEDO, V., DORELLA, FA An iron-acquisition-deficient mutant of Corynebacterium pseudotuberculosis efficiently protects mice against challenge, A cta Veterinaria Scandinavica, v. 45, p. 28, 2014; 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 protected mice against caseous lymphadenitis, BMC Veterinary Research, v. 10, p. 1–8, 2014).

[006] In this context, recombinant subunit vaccines gain prominence mainly due to their higher levels of safety, since they are based on selected and purified antigens, thus excluding the pathogen from the vaccine development process (Mazumder et al., 2011). LILJEQVIST, S. AND STAHL, S. Production of recombinant subunit vaccines: Protein immunogens, live delivery systems and nucleic acid vaccines, Journal of Biotechnology, 73(1), pp. 1–33, 1999; 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, p. 1–8, 2014). However, none of the vaccine prototypes researched so far showed fully satisfactory results, making it necessary to continue and improve the studies.

[007] In this way, structural vaccinology is gaining prominence in the area of vaccine development, since it allows the rational design of target epitopes that could be used as vaccine candidates, through the use of the structure of potential antigens (SERRUTO , D. & RAPPUOLI, R. Post-genomic vaccine development. FEBS Lett. 2006, v. 580, p. 2985–2992, 2006). This tool makes it possible to improve immunogenicity, stability and safety in the production of antigens, in addition to providing information to promote the design of improved antigens (DORMITZER, PR; GRANDI, G.; RAPPUOLI, R. Structural vaccinology starts to deliver. Nature Reviews. Microbiology , v. 10, no. 12, p. 807–813, 2012). Using this knowledge, the production of chimeras appears aiming to maximize the effectiveness and minimize the side effects of the available vaccines. Furthermore, the production of bacterins expressing proteins in the non-purified form eliminates the isolation and purification steps of the recombinant protein, and with this, it reduces the time and costs of production, in addition to not presenting safety risks as they are non-toxic (MOREIRA, C.; DA CUNHA, CEP; MOREIRA, GMSG; MENDONÇA, M.; SALVARANI, FM; MOREIRA, Â.N.; CONCEIÇÃO, FR Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Anaerobe, v. 40 , p. 58-62, 2016), and combine the advantages of producing recombinant vaccines, such as target safety and predictability.

[008] In a previous search in the world's patent filing databases, we found the document PI 1006644-6 A2 as a result, referring to a live vaccine against LC based on an attenuated strain. As already explained, this type of vaccine causes adverse reactions in immunized animals and does not reach sufficient levels of protection, reaffirming the search for new strategies with higher levels of safety.

[009] Our search also included the claim for the protection of a skin test for the diagnosis of subclinical CL in goats and sheep (WO2012149622A1), demonstrating that the search for new diagnoses is also on the rise, since there is a lack of a diagnosis sufficiently sensitive and specific in the detection of the disease in its initial phase.

[010] All other results contained in the state of the art obtained in our search have nothing to do with our invention, they comprise an antigenic peptide against Propionibacterium acnes (EP2430157B1), an oral vaccine for Borrelia (US8821893B2) and a binding agent targeted at DLL4 protein to treat its activity or overproduction (CN102264763B).

[011] However, we identified the existence of the development of some works in the line of structural vaccines, such as the project that aims at the “Production of a rational multi-epitope vaccine against schistosomiasis”. This more elaborate generation of vaccines combines numerous advantageous features that increase the potential for efficacy of a vaccine formulation.

[012] With an emphasis on CL, the research entitled “Construction of a recombinant chimera as a vaccine method for the control of caseous lymphadenitis” is in progress. There are no patent applications filed to date, no publications on this topic and no results obtained in the study have been made available to date. In addition, it is worth mentioning that the composition of our invention is unique and innovative, since the proteins used as a basis for its production, as well as the selected epitopes and their order in the final structure, were established by our group in a pioneering way. .

SUMMARY OF THE INVENTION

[013] The present invention refers to processes of analysis and assembly of gene sequences of epitopes of proteins rNanH, rPknG, rSpaC and rSodC C. pseudotuberculosis in a recombinant chimeric structure. The invention also relates to the synthesis and expression of the chimera in pAE vector, with an IPTG-inducible promoter, in E. coli expression strains from a heterologous expression system. The generated recombinant protein has a molecular weight of approximately 52 kDa and also has a tail containing 6 histidines in the amino-terminal region, in order to facilitate the purification process. The invention further comprises the production of recombinant chimera in unpurified form. More specifically, the invention comprises the association of purified and non-purified recombinant chimera with saponin adjuvant in the production of a vaccine formulation capable of inducing a specific immune response (humoral and cellular) against CL and potentiating the protective effect of recombinant subunit vaccines. However, the formulations are not limited to the use of this class of adjuvant.

DETAILED DESCRIPTION OF THE INVENTION

[014] The present invention is intended for the use of a recombinant chimera in purified and unpurified forms in association with saponin adjuvant in the composition of a vaccine formulation for CL. The invention is described in more detail below:

[015] The presence of MHC II epitopes (H-2-IAb and H-2-IAd alleles), MHC I (H-2-Db, H-2-Dd, H-2-Kb, H-2- Kd, H-2-Kk, H-2-Ld) and linear B cell epitopes (SEQ ID NO:1, NO:2, NO:3, NO:4) present in the NanH protein sequences (SEQ ID NO :5), PknG (SEQ ID NO:6), SodC (SEQ ID NO:7) and SpaC (SEQ ID NO:8) is predicted using ABCpred, BepiPred, TepiTool, NetMHC and NetMHCII software, respectively. The data are used for the screening of epitopes and selection for joining through glycine linkers (simplest amino acid and with the smallest side chain) in the chimera assembly.

[016] The chimera is sent to the Genome company for chemical synthesis and the gene is delivered cloned into the pAE vector (Figure 1). The plasmid containing the recombinant chimeric gene is transformed into different E. coli expression strains in order to obtain the highest level of expression and protein yield.

[017] For expression of the recombinant protein, the synthetic vector containing the CDS of the recombinant chimera (SEQ ID NO:9) is transformed by heat shock into the expression line that obtained the best yield. The induction of expression occurs by the addition of 1 mM of IPTG to the culture maintained under orbital shaking at 37 °C for 3 h. Expression 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, samples containing the recombinant chimera are mixed with buffer (100-mM TrisHCl 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 12% SDS-PAGE gel electrophoresis. Subsequently, transfer to nitrocellulose membrane (GE Healthcare) is performed, which after 2 h is blocked with PBS containing 5% skim milk for 1 h at 37 °C. Then, anti-6Xhistag (Sigma Aldrich) is added to the membrane at a dilution of 1:4000 at 37°C for 1 h. Membranes are washed with 0.05% tween PBS (PBS-T), and incubated with diluted peroxidase-conjugated mouse anti-IgG (Sigma Aldrich) at 1:4000 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 chimera can be confirmed as visualized in Figure 2 by the reactive band with approximate molecular weight of 52 kDa and 491 aa (SEQ ID NO:10).

[018] Purification is performed by affinity chromatography on a sepharose column (HisTrap™; GE Healthcare), loaded with nickel, since the protein has a sequence of 6 histidine amino acids at its amino terminus. Its purity is determined by 12% SDS-PAGE (Figure 3) and the concentration determined by the BCA kit (Pierce).

Expression of the recombinant chimera in the unpurified form

[019] For the production of the bacterin expressing the recombinant chimera, the strain of E. coli BL21 Star (DE3) is used, transformed with the pAE/chimera plasmid previously constructed. Cells are plated on solid LB, containing 100 μg/ml ampicillin, and incubated at 37 °C for 16 h. For expression, a transformed colony is selected to start the pre-inoculum in 50 mL of liquid LB containing 50 μL of ampicillin (100 μg/ml), which is incubated overnight under agitation at 37 °C. This pre-inoculum is scaled up into an Erlenmeyer flask containing 500 mL of liquid LB and 500 μL of ampicillin (100 μg/ml), remaining under agitation for 3 h at 37 °C. Upon reaching an OD600 between 0.4 and 0.6, induction of protein expression is performed by adding 1 mM of IPTG to the culture. After induction for 3 h at 37 °C, 5 eppendorfs containing 1 mL of culture are collected for concentration analysis and other tests. Confirmation of the expression of the recombinant chimera in non-purified form is performed initially by SDS gel (Figure 2) followed by Western blotting (Figure 3).

Inactivation of recombinant E. coli expressing chimera

[020] The culture of E. coli expressing the recombinant chimera is centrifuged at 7,000 g for 10 min and its pellet eluted in 50 mL of sterile PBS (Saline Phosphate Buffer). 0.2% v/v formaldehyde is added to the culture in PBS for inactivation of the bacteria, remaining under agitation in Shaker for 16 h at a temperature of 37 °C (protocol previously established by the group). Then, the culture is centrifuged again (7,000 g for 10 min) and the inactivated cell pellet is washed 3x with PBS to remove all remaining formaldehyde. After washing, it is eluted again in 50 mL of sterile PBS and stored at -4°C until use.

Inactivation confirmation and security reviews

[021] Confirmation of inactivation of the E. coli bacterin expressing the recombinant chimera is performed in three steps. At first, 100 μL of the cell suspension in PBS obtained after treatment with 0.2% v/v formaldehyde is plated in solid LB, to confirm the success of inactivation. Then, 1 mL of this same cell suspension after inactivation is added in liquid LB medium, incubated for 30 days and an aliquot of 100 μl is plated on solid LB medium and incubated for 16 h in an oven at 37 °C to verify growth or not. of remaining colonies and to prove the stability of the inactivation promoted by formaldehyde. In a third step, the plasmid extraction from the recombinant bacterin is performed using the Illustra plasmidPrep Mini Spin kit (GE healthcare), which is used in the transformation via electroporation of electrocompetent E. coli DH5α to verify the inactivation of the resistance gene. The transformation product is plated on solid LB medium containing 100 µg/ml ampicillin. The extracted plasmid is used as a template for amplification of the gene sequence referring to the chimera using specific primers, by means of PCR, to further ensure the safety of the bacterin.

Preparation of Saponin Adjuvant and Vaccine Formulation

[022] 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 PBS-1x, generating a stock solution of 1.5 mg/mL, which is subjected to sterilizing filtration with a 0.22 µm membrane. This should be stored at 4 °C until use. In the vaccine formulation, 5 μL of the stock solution (equivalent to 7.5 μg of saponin) is used associated with the amount of 50 µg of the recombinant chimera for each vaccine dose in a final volume of 300 µL.

[023] The present invention can be better understood through the following examples, however this is not limited to said examples.

BRIEF DESCRIPTION OF THE FIGURES

[024] Figure 1: Figure 1 demonstrates the constructed pAE/chimera vector map.

[025] Figure 2: Figure 2 represents confirmation of the identity of the recombinant protein in purified and unpurified forms expressed by Western blotting with anti-6xhistag monoclonal antibody (Sigma Aldrich). In (1) pre-stained marker (PageRuler™ Prestained Protein Ladder, Thermo Fisher), (2) purified recombinant chimera represented by a reactive band of approximately 52 kDa, (3) chimera in unpurified form represented by a reactive band of approximately 52 kDa.

[026] Figure 3: Figure 3 corresponds to an SDS-PAGE electrophoresis (12%) demonstrating the expression of the recombinant chimera in purified and unpurified form.
In (1) pre-stained marker, (2) purified recombinant chimera, (3) unpurified recombinant chimera.

[027] Figure 4: Figure 4 demonstrates the evaluation of anti-rNanH, anti-rPknG, anti-rSpaC and anti-rSodC total IgG levels in mice immunized with the recombinant chimera in purified and non-purified form 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 1st immunization. Different letters within the same day represent groups with significantly different protection rates (p<0.05).

EXAMPLE EXAMPLE 1 - INDUCTION OF HUMORAL IMMUNE RESPONSE BY RECOMBINANT CHIMERA IN PURIFIED AND UNPURIFIED FORM IN MICE Balb/c

[028] This example illustrates the induction of humoral immune response by recombinant chimera in purified and unpurified forms in Balb/c mice, through detection of the production of specific antibodies by indirect ELISA. The same also allows us to evaluate for which of the proteins (NanH, PknG, SpaC and SodC) a preferential response was induced, which was able to increase the production of antibodies against this vaccine antigen.

[029] For the immunization strategy, 3 groups composed of 10 female Balb/c mice, between 6-8 weeks of age, were inoculated subcutaneously with 50 ng of protein per vaccine dose, in a final volume of 300 μL , according to the immunization schedule described below. The animals were provided by the Central Animal Facility of the Federal University of Pelotas. The conduction of the experiment was approved by the ethics committee in animal experimentation of the aforementioned University (CEEA/UFPel n° 2442).

• - Grupo 1: Solução salina 0,9% (controle negativo);
• - Grupo 2: Quimera recombinante purificada + saponina;
• - Grupo 3: Quimera recombinante não purifica + saponina;

[030] The groups were distributed as follows:

• - Group 1: 0.9% saline solution (negative control);
• - Group 2: Purified recombinant chimera + saponin;
• - Group 3: Recombinant chimera does not purify + saponin;

[031] Immunization was performed following the following protocol:

[032] Day 0: 1st dose of vaccine /Day 21: 2nd dose of vaccine (booster).

[033] The quantification of total IgG levels induced by NanH, PknG, SpaC and SodC proteins was determined by indirect ELISA using sera obtained from animals in groups G1, G2 and G3 through blood collection by means of puncture of the veins of the retro-orbital plexus on days 0, 21 and 42 of the immunization experiment. 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.

[034] For this, 96-well flat-bottomed plates (TPP) were sensitized with 100 μL of the solution containing carbonate bicarbonate buffer pH 9.6 and 1 μg/mL of the recombinant proteins rNanH, rPknG, rSpaC and rSodC and incubated for 18 at 4 °C. Subsequently, the plates were washed 3 times with PBS-T (1X PBS pH 7.4; 0.1% Tween 20) and blocked with 200 μL/well of PBS-T and 5% skim milk for (2 h at 37 °C). ). Afterwards, the plates were washed again with PBS-T and 100 μL/well of mouse sera samples (1:50 in PBS-T) were added in duplicates. After one hour of incubation at 37 °C and 3 washes with PBS-T, 100 μL/well of the antibody conjugated to mouse anti-Total IgG peroxidase (Sigma-Aldrich) at a dilution of 1:5,000 (1 h at 37 °C) was added. Ç). After that, 5 more washes were performed with PBS-T, and 100 μL/well of developer solution [200 pmoles orthophenylenediamine (OPD, Sigma-Aldric) 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 stop solution containing 4N sulfuric acid was added. The absorbance was measured at 492 nm using an ELISA plate reader (Mindray).

[035] Statistical analyzes were performed using the GraphPad Prism software version 6.0 for Windows (GraphPad Software, USA). Differences between the IgG production in the referred experimental groups were verified through the one-way ANOVA, followed by the Tukey post-test. P values less than 0.05 were considered statistically significant.

[036] When evaluating the results obtained in the ELISA, it was concluded that the levels of total IgG specific for the proteins rNanH, rPknG, rSpaC and rSodC (Figure 4) increased progressively over the days for the experimental groups G2 and G3 . On days 0 and 21 there was no significant difference between all groups for the production of anti-rPknG, anti-rSodC and anti-rSpaC antibodies. The G3 group presented a significantly higher production of anti-rNanH total IgG (p< 0.05) when compared to G1 and G2 on days 21 and 42 of the experiment.

[037] As we can see in Figure 4a, the immune response produced for the G2 group occurred preferentially for the rNanH protein, since the levels of specific antibodies produced for this protein were higher than those obtained for rPknG, rSodC and rSpaC. As for the G3 group, the levels of antibodies produced against rNanH and rSpaC proteins were quite similar, making it impossible to point out which a preferential immune response was established.

[038] These results allow us to infer that our formulations have potential for use in vaccines against CL, since they are capable of inducing specific immune responses against antigens of C. pseudotuberculosis, with levels of production of antibodies quite significant and superior to those obtained by other vaccine formulations already researched in other studies (DROPPA-ALMEIDA, D., VIVAS, WLP, SILVA, KKO, REZENDE, AFS, SIMIONATTO, S., MEYER, R., LIMA-VERDE, IB, DELAGOSTIN, O., BORSUK, S. & PADILHA, FF Recombinant CP40 from Corynebacterium pseudotuberculosis confers protection in mice after challenge with a virulent strain Vaccine, v.34, p. 1091–1096, 2016; LEAL, KS; SILVA, MTO; REZENDE, A. de FS; BEZERRA, FSB; BEGNINI, K.; SEIXA, F.; COLLARES, T.; DELLAGOSTIN, O.; PORTELA, RW; AZEVEDO, VA de C.; BORSUK, S. Recombinant M. bovis BCG expressing the PLD protein promotes survival in mice challenged with a C. pseudotuberculosis virulent strain. Vaccine, v.36, p. 3578-3583, 2018).

Claims (7)

”CONSTRUCTION OF RECOMBINANT CHIMERA FOR SUBUNIT VACCINE COMPOSITION AGAINST CASEOUS LYMPHADENITIS”, characterized by having the epitopes (SEQ ID NO:1, NO:2, NO:3, NO:4) present in NanH proteins (SEQ ID NO:5 ), PknG (SEQ ID NO:6), SpaC (as per SEQ ID NO:7) and SodC (SEQ ID NO:8) joined through glycine linkers and assembled into a chimeric gene that will be expressed as a recombinant protein of 491 amino acids and 52 kDa (SEQ ID NO:10). "CONSTRUCTION OF RECOMBINANT CHIMERA FOR SUBUNIT VACCINE COMPOSITION AGAINST CASEOUS LYMPHADENITIS", according to claim 1, characterized by cloning the chimeric gene in vectors for use in prokaryotic or eukaryotic organisms. "CONSTRUCTION OF RECOMBINANT CHIMERA FOR SUBUNIT VACCINE COMPOSITION AGAINST CASEOUS LYMPHADENITIS", according to claim 1, characterized in that it further comprises the chimera in purified and unpurified forms (recombinant bacterin). "FORMULATION OF A SUBUNIT VACCINE AGAINST CASEOUS LYMPHADENITIS COMPOSED OF A RECOMBINANT CHIMERA IN PURIFIED AND NON-PURIFIED FORMS", according to claim 3, characterized by the use of formaldehyde 0.2% v/v in phosphate saline buffer, with a time of exposure of 16 hours to cultivation at 37ºC, for inactivation of E. coli expressing the recombinant chimera in the non-purified form. "FORMULATION OF A SUBUNIT VACCINE AGAINST CASEOUS LYMPHADENITIS COMPOSED OF A RECOMBINANT CHIMERA IN PURIFIED AND NON-PURIFIED FORMS", according to claim 1, characterized by its production in different strains of E. coli expression, not being restricted to this organism . "FORMULATION OF A SUBUNIT VACCINE AGAINST CASEOUS LYMPHADENITIS COMPOSED OF A RECOMBINANT CHIMERA IN PURIFIED AND NON-PURIFIED FORMS", characterized in that it comprises the association of the chimera in purified or non-purified forms, according to any one of the preceding claims, to the saponin vaccine adjuvant , not being restricted to its use. "FORMULATION OF A SUBUNIT VACCINE AGAINST CASEOUS LYMPHADENITIS COMPOSED OF A RECOMBINANT CHIMERA IN PURIFIED AND NON-PURIFIED FORMS", according to any of the preceding claims, characterized in that it is preferably administered subcutaneously, not being restricted to this, in two or more doses containing a range of 50-200 ng of protein, with a minimum interval of 15 days between doses.

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