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BR-102016016938-B1 - Recombinant Corynebacterium pseudotuberculosis Antigen RCP00660 and its use in immunoassay and vaccine formulation against caseous lymphadenitis.

BR102016016938B1BR 102016016938 B1BR102016016938 B1BR 102016016938B1BR-102016016938-B1

Abstract

Recombinant Antigen rCP00660 of Corynebacterium pseudotuberculosis and its use in immunoassays and vaccine formulation against caseous lymphadenitis. Technical sector: A61K 39/00, A61K 39/02, A61K 39/05. Commercially available vaccines for caseous lymphadenitis (CL) have limitations, such as local and systemic adverse reactions and partial protection. Immunoassays available for CL have disadvantages such as low sensitivity and specificity and inability to distinguish between previously exposed animals and those still harboring the pathogen. Thus, the present invention relates to the production process of the recombinant antigen rCP00660 of Corynebacterium pseudotuberculosis, from the partial cloning of the cp1002_RS00660 gene in the pAE vector in cloning and expression strains of Escherichia coli. The protein produced has a size of approximately 23.5 kDa, possessing a 6-amino acid histidine tail, which facilitates its purification process. The invention also addresses the use of this antigen in a vaccine formulation against CL containing saponin, which generated a protection rate of 30%. An indirect ELISA for CL diagnosis demonstrated that this antigen has 77.5% specificity and 95% sensitivity. Given the few recombinant antigens of C. pseudotuberculosis available, rCP00660 presents itself as a real alternative to be used for industrial purposes.

Inventors

  • Sibele Borsuk
  • MARINA CARDOSO DE FREITAS
  • ANDRÉA DE FÁTIMA SILVA REZENDE
  • THAÍS FARIAS COLLARES
  • FRANCISCO SILVESTRE BRILHANTE BEZERRA
  • MARA THAIS DE OLIVEIRA SILVA
  • ÂNGELA SENA LOPES
  • VASCO ARISTON DE CARVALHO AZEVEDO
  • ANDERSON RODRIGUES DOS SANTOS
  • RICARDO WAGNER DIAS PORTELA
  • RODRIGO ANDRADE SCHUCH
  • KARINA PEREIRA LUDUVICO

Assignees

  • UNIVERSIDADE FEDERAL DE PELOTAS

Dates

Publication Date
20260317
Application Date
20160721

Claims (3)

  1. 1. Recombinant subunit vaccine characterized by comprising in its composition the recombinant protein rCP00660 (as per SEQ ID NO:1) containing a tail with 6 histidine amino acids in the amino-terminal region, having a total of 217 amino acids and an approximate molecular size of 23.5 kDa in association with a vaccine adjuvant, aiming at the immunoprophylaxis of caseous lymphadenitis.
  2. 2. Recombinant subunit vaccine according to claim 1, characterized by comprising an amount between 20 and 200 μg of the rCP00660 protein as antigen.
  3. 3. Recombinant subunit vaccine according to claims 1 and 2, characterized by comprising an amount between 1 and 10 μg of saponin as a vaccine adjuvant.

Description

DESCRIPTIVE REPORT FUNDAMENTALS OF THE INVENTION FIELD OF THE INVENTION [001] The present invention falls within the area of the development of recombinant bacterial antigens for use as immunobiologicals, whether for vaccine purposes or for diagnostic tests. It involves the production of the recombinant antigen rCP00660 from the partial cloning of the cp1002_RS00660 gene of C. pseudotuberculosis in the pAE vector, using a heterologous expression system (in Escherichia coli). The invention also addresses the use of rCP00660 in a vaccine formulation and an immunoassay for caseous lymphadenitis, a disease of veterinary importance that has a significant economic impact on sheep and goat farming. It falls within the international patent classification in the fields of invention A61K 39/00, A61K 39/02, A61K 39/05. DESCRIPTION OF THE STATE OF THE ART [002] Sheep and goat farming is an important productive activity developed in almost all of Brazil. The Northeast and South regions stand out, concentrating the largest number of small ruminants (PORTO, L.L., DA, M.A., SALUM, W.B. et al. Characterization of sheep and goat farming cut in the Central North Baiano region. Revista Brasileira de Gestao e Desenvolvimento Regional, v. 9, n. 1, p. 281-296, 2013). According to data collected in the last Municipal Livestock Survey, in 2014, Brazil had 17,614,454 head of sheep and 8,851,879 head of goats, with the Northeast region having the largest number of animals, with approximately 57.5% sheep and 91.6% goats, followed by the South region in second place, with approximately 29.3% and 3.5% sheep and goats, respectively (INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA. Pesquisa Pecuária Municipal. Available at: http://www.sidra.ibge.gov.br/bda/pecua [Accessed on May 17, 2016].IBGE, 2016). [003] Despite this large number of animals, the productive indices of Brazilian sheep and goat farming have been low, mainly due to inadequate management practices, poor sanitary conditions, and lack of technology, which is reflected in high morbidity rates (ALENCAR, S.P., MOTA, R.A., COELHO, M.C.O.C. et al. Sanitary profile of goat and sheep herds in the hinterland of Pernambuco. Ciência Animal Brasileira, v. 11, n. 1, p. 131-140, 2010; SANTOS, T.C., PENA-ALFARO, C.E., FIGUEIREDO, S.M. Sanitary and management aspects in goat and sheep farming in the microregion of Patos, Semi-Arid region of Paraíba. Ciência Animal Brasileira, v. 12, n. 2, p. 206-212, 2012; PORTO, L.L., DA, M.A., SALUM, W.B. et al. Characterization of sheep and goat meat production in the North-Central region of Bahia. Brazilian Journal of Management and Regional Development, v. 9, n. 1, p. 281-296, 2013). [004] Given this scenario, health problems such as caseous lymphadenitis (CL) are common and have a high prevalence. It is estimated that in Brazil, most sheep and goat herds are infected and that the clinical prevalence exceeds 30% of the animals (FARIA, G.A., MORAIS, O.R., GUIMARÃES, P.H.S. Analysis of sheep and goat farming in the North and Northeast of Minas Gerais. 1st ed., Belo Horizonte: SEBRAEMG, FAEMG, EMATER. 2004; CARMO, F.B., GUIMARÃES, A.S., PAULETTI, R.B. et al. Prevalence of antibodies against caseous lymphadenitis in commercial sheep farms in the Federal District, Brazil. Archives of the Biological Institute, v. 79, n. 2, p. 293-296, 2012). In fact, a serological survey conducted in the state of Minas Gerais on sheep and goats showed prevalences exceeding 70% in the herds studied (GUIMARÃES, A.S.; SEYFFERT, N.; PORTELA, R.W.D. et al. Caseous lymphadenitis in sheep flocks of the state of Minas Gerais, Brazil: Prevalence and management surveys. Small Ruminant Research, v.87, p.86-91, 2009; SEYFFERT, N.; GUIMARÃES, A.S.; PACHECO, L.G.C. et al. High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted proteins-based ELISA. Research in Veterinary Science, v.88, n.1, p.50-55, 2010). [005] Thus, the large number of sheep and goats in the country, associated with the high prevalence of LC, results in direct and significant economic losses, caused by skin depreciation, carcass condemnation, reduced weight gain in animals, milk production, and reproductive rates, or even the culling of sick animals (ARSENAULT, J., GIRARD, C., DUBREUIL, P. et al. Prevalence of and carcass condemnation from maedi-visna, paratuberculosis and caseous lymphadenitis in culled sheep from Quebec, Canada. Preventive Veterinary Medicine, v. 59, p. 67-81, 2003; GUIMARÃES, A.D.S., BORGES, F., PAULETTI, R.B. et al. Caseous lymphadenitis: epidemiology, diagnosis and control. The IIOAB Journal, v. 2, n. 2, p. 33-43, 2011; WINDSOR, P.A. Control of caseous lymphadenitis. The Veterinary clinics of North America, vol. 27, no. 1, p. 193-202, 2011). [006] CL is a chronic disease with a global distribution that affects sheep and goats. Its etiological agent is the Gram-positive bacterium Corynebacterium pseudotuberculosis, a facultative in