BR102016027456A2 - PROCESS FOR OBSERVATION AND USE OF NEOSPORA CANINUM ROP2 PROTEIN FOR USE AS NEOSPOROSIS VACCINE - Google Patents
PROCESS FOR OBSERVATION AND USE OF NEOSPORA CANINUM ROP2 PROTEIN FOR USE AS NEOSPOROSIS VACCINE Download PDFInfo
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Abstract
a presente invenção refere-se ao processo de preparação e utilização de uma vacina recombinante contra a neosporose, causada pelo protozoário n. caninum. devido falta de vacinas comerciais atualmente disponíveis contra a neosporose, a presente invenção descreve o processo de preparação e utilização de uma vacina de subunidade baseado na tecnologia do dna recombinante, cuja formulação contenha o antígenorrop2, sendo este um fragmento de 18kda da roptria 2, pertencente à família quinases. a proteína rrop2 foi produzida em escherichia coli, caracterizada e avaliada como vacina ou componente de formulações vacinais em camundongos ebovinos naturalmente infectados por n. caninumem propriedades combovinos de corte e leiteiros de produção extensiva intensiva. o antígeno vacinal rrop2 induziu um aumento significativo na resposta imune celular e humoral em camundongos e bovinos. a presente invençãose mostra eficaz para a utilização como vacina ou componente de formulações vacinais no controle e prevenção da neosporose.The present invention relates to the process for preparing and using a recombinant neosporosis vaccine caused by protozoan no. caninum. Due to the lack of commercially available vaccines against neosporosis, the present invention describes the process for preparing and using a recombinant DNA technology-based subunit vaccine, the formulation of which contains the antigen-rop2, which is an 18kda fragment of roptria 2, belonging to to the family kinases. rrop2 protein was produced in escherichia coli, characterized and evaluated as a vaccine or component of vaccine formulations in naturally infected n. caninum in intensive beef cattle and intensive production. The rrop2 vaccine antigen induced a significant increase in cellular and humoral immune response in mice and cattle. The present invention is effective for use as a vaccine or component of vaccine formulations in the control and prevention of neosporosis.
Description
(54) Título: PROCESSO DE OBTENÇÃO E UTILIZAÇÃO DA PROTEÍNA ROP2 DE NEOSPORA CANINUM PARA USO COMO VACINA CONTRA NEOSPOROSE (51) Int. Cl.: C12N 15/30; C07K 14/44; A61P 33/02; G01N 33/569 (73) Titular(es): UNIVERSIDADE FEDERAL DE PELOTAS (72) Inventor(es): FÁBIO PEREIRA LEIVAS LEITE; ALCEU GONÇALVES DOS SANTOS JUNIOR; RENAN EUGÊNIO ARAÚJO PIRAINE; LÍVIA BUDZIAREK ESLABÃO (57) Resumo: A presente invenção refere-se ao processo de preparação e utilização de uma vacina recombinante contra a Neosporose, causada pelo protozoário N. caninum. Devido falta de vacinas comerciais atualmente disponíveis contra a Neosporose, a presente invenção descreve o processo de preparação e utilização de uma vacina de subunidade baseado na tecnologia do DNA recombinante, cuja formulação contenha o antígenorROP2, sendo este um fragmento de 18kDa da roptria 2, pertencente à família Quinases. A proteína rROP2 foi produzida em Escherichia coli, caracterizada e avaliada como vacina ou componente de formulações vacinais em camundongos ebovinos naturalmente infectados por N. caninumem propriedades combovinos de corte e leiteiros de produção extensiva intensiva. O antígeno vacinai rROP2 induziu um aumento significativo na resposta imune celular e humoral em camundongos e bovinos. A presente invençãose mostra eficaz para a utilização como vacina ou componente de formulações vacinais no controle e prevenção da Neosporose.(54) Title: PROCESS OF OBTAINING AND USING NEOSPORA CANINUM ROP2 PROTEIN FOR USE AS A VACCINE AGAINST NEOSPOROSIS (51) Int. Cl .: C12N 15/30; C07K 14/44; A61P 33/02; G01N 33/569 (73) Holder (s): FEDERAL UNIVERSITY OF PELOTAS (72) Inventor (s): FÁBIO PEREIRA LEIVAS LEITE; ALCEU GONÇALVES DOS SANTOS JUNIOR; RENAN EUGÊNIO ARAÚJO PIRAINE; LÍVIA BUDZIAREK ESLABÃO (57) Abstract: The present invention relates to the process of preparing and using a recombinant vaccine against Neosporosis, caused by the protozoan N. caninum. Due to the lack of commercial vaccines currently available against Neosporosis, the present invention describes the process of preparing and using a subunit vaccine based on recombinant DNA technology, the formulation of which contains the antigenorROP2, which is an 18kDa fragment from roptria 2, belonging to to the Kinases family. The rROP2 protein was produced in Escherichia coli, characterized and evaluated as a vaccine or component of vaccine formulations in Ebovine mice naturally infected with N. caninum in intensive beef and dairy farming properties. The vaccine antigen rROP2 induced a significant increase in the cellular and humoral immune response in mice and cattle. The present invention shows to be effective for use as a vaccine or component of vaccine formulations in the control and prevention of Neosporosis.
ELISA rROP2ELISA rROP2
1.5-11.5-1
E 1.0ΓΜE 1.0ΓΜ
0.00.5-0.00.5-
¥¥
Controle rROP2 < f F T t »-** A # & φRROP2 control <f F T t »- ** A # & φ
DiasDays
1/111/11
PROCESSO DE OBTENÇÃO E UTILIZAÇÃO DA PROTEÍNA ROP2 DE Neospora caninum PARA USO COMO VACINA CONTRA NEOSPOROSEPROCESS OF OBTAINING AND USING THE ROP2 PROTEIN OF Neospora caninum FOR USE AS A VACCINE AGAINST NEOSPOROSIS
Relatório DescritivoDescriptive Report
CAMPO DA INVENÇÃOFIELD OF THE INVENTION
001 A presente invenção refere-se ao processo de preparação e utilização de uma vacina recombinante contra a Neosporose, causada pelo protozoário Neospora caninum, mais especificamente, a um processo de preparação de uma vacina de subunidade recombinante (ROP2) com potencial protetor, composta pela Roptria 2 de N. caninum expressa em Escherichia coli.001 The present invention relates to the process of preparation and use of a recombinant vaccine against Neosporosis, caused by the protozoan Neospora caninum, more specifically, to a process of preparation of a recombinant subunit vaccine (ROP2) with protective potential, composed by N. caninum Roptria 2 expressed in Escherichia coli.
FUNDAMENTOS DA INVENÇÃOBACKGROUND OF THE INVENTION
002 A neosporose é mundialmente descrita como a principal doença parasitária causadora de aborto em bovinos, responsável por perdas econômicas significativas, conforme M.P. Reichel, M. AlejandraAyanegui-Alcérreca, L.F.P. Gondim, J.T. Ellis, What is the global economicimpact of Neospora caninum in cattle - the billion dollar question., Int. J. Parasitol, v.43 p.133-42, 2013.002 Neosporosis is described worldwide as the main parasitic disease that causes abortion in cattle, responsible for significant economic losses, according to M.P. Reichel, M. AlejandraAyanegui-Alcérreca, L.F.P. Gondim, J.T.Ellis, What is the global economicimpact of Neospora caninum in cattle - the billion dollar question., Int. J. Parasitol, v.43 p.133-42, 2013.
003 A neosporose tem o maior destaque pela infeção em bovinos. Na condição de hospedeiro intermediário, ingerem osooscistos esporulados eliminados por cães, hospedeiro definitivo, (Canis familiaris) parasitados (DUBEY, J. P.; BUXTON, D.; WOUDA, W. Pathogenesis of bovine neosporosis. Journal of comparative pathology, v.134, n.4, p.267-89, 2006), oocisto rompe-se por ação química e libera no intestino quatro esporozoítos, os quais penetram no epitélio passando para fase de taquizoítos (GOODSWEN, S. J.; KENNEDY, P. J.; ELLIS, J. T. A review of the infection, genetics, andevolution of Neospora caninum: From the past to the present. Infection, Genetics and Evolution, v.13, n.1, p.133-150, 2013). A fase aguda é caracterizada pela rápida multiplicação e disseminação via hematógena, atingindo assim vários órgãos, em gestantes o embrião também é infectado (ANDERSON, M.L. et al. Neosporosis in cattle. Animal Reproduction Science, v.60, p.417-431, 2000). O parasito invade células003 Neosporosis has the greatest prominence due to infection in cattle. As an intermediate host, they ingest sporulated osooscysts eliminated by dogs, definitive host, parasitized (Canis familiaris) (DUBEY, JP; BUXTON, D .; WOUDA, W. Pathogenesis of bovine neosporosis. Journal of comparative pathology, v.134, n .4, p.267-89, 2006), the oocyst breaks down by chemical action and releases four sporozoites in the intestine, which penetrate the epithelium passing to the tachyzoite phase (GOODSWEN, SJ; KENNEDY, PJ; ELLIS, JT A review of the infection, genetics, andevolution of Neospora caninum: From the past to the present. Infection, Genetics and Evolution, v.13, n.1, p.133-150, 2013). The acute phase is characterized by rapid multiplication and dissemination via hematogen, thus reaching several organs, in pregnant women the embryo is also infected (ANDERSON, ML et al. Neosporosis in cattle. Animal Reproduction Science, v.60, p.417-431, 2000). The parasite invades cells
Petição 870170057153, de 09/08/2017, pág. 13/24Petition 870170057153, of 08/09/2017, p. 13/24
2/11 como macrófago e linfócitos e usa-se do vacúolo parasitóforo (VP) com blindagem, garantindo assim sua sobrevivência e sua multiplicação. As sucessivas divisões acarretam o rompimento da célula hospedeira, passando a parasitar outras adjacentes. O N. caninum invade determinados tecidos passa a formar cistos teciduais, forma latente, onde ocorre a conversão para bradizoíto, instalando-se assim a fase crônica da doença (INNES, E. A. The host-parasite relationship in pregnant cattle infected with Neospora caninum. Parasitology, v.134, n.13, p.1903-1910, 2007).2/11 as macrophage and lymphocytes and uses the shielded parasitophore vacuole (VP), thus ensuring its survival and multiplication. The successive divisions cause the host cell to rupture, passing to parasite adjacent ones. N. caninum invades certain tissues and starts to form latent tissue cysts, where the conversion to bradyzoite occurs, thus establishing the chronic phase of the disease (INNES, EA The host-parasite relationship in pregnant cattle infected with Neospora caninum. Parasitology , v.134, n.13, p.1903-1910, 2007).
004 O interesse de estudos sobre esse protozoário é motivado pela ausência de tratamentos e medidas profiláticas que possam proteger contra desenvolvimento da doença. No mercado havia sido disponibilizada uma vacina formulada a partir de taquizoítos inativados, no entanto, a vacina produziu resultados contraditórios em países nos quais foi testada; além disso, seu efeito foi associado ao aumento de riso de morte embrionária, motivo pelo qual foi retirada do mercado (Monney T, Hemphill A Vaccines against neosporosis: What can we learn from the past studies? Exp.Parasitol., v.140, p.52-70, 2014).004 The interest in studies on this protozoan is motivated by the absence of treatments and prophylactic measures that can protect against the development of the disease. A vaccine formulated from inactivated tachyzoites had been made available on the market, however, the vaccine produced contradictory results in countries in which it was tested; moreover, its effect was associated with an increased laugh of embryonic death, which is why it was withdrawn from the market (Monney T, Hemphill A Vaccines against neosporosis: What can we learn from the past studies? Exp.Parasitol., v.140, p.52-70, 2014).
005 No setor da bovinocultura são buscados altos índices de eficiência reprodutiva, desde a taxa de concepção à taxas de nascimento, entretanto, animais parasitados por N. caninum são passiveis de aborto, apresentando baixos índices produtivos (ANDREOTTI et al., 2010).005 In the bovine culture sector, high rates of reproductive efficiency are sought, from conception rate to birth rates, however, animals parasitized by N. caninum are susceptible to abortion, with low production rates (ANDREOTTI et al., 2010).
006 Atualmente muito se tem pesquisado sobre antígenos específicos do protozoário, associações de proteínas de diferentes estruturas. Neste cenário, algumas proteínas tem se destacado em seu caráter de proteção ao desafio, entre elas a roptria 2 tem sido amplamente testada (K. DEBACHE, C. GUIONAUD, F. ALAEDDINE, A. HEMPHILL, Intraperitoneal and intra-nasal vaccination of mice with three distinct recombinant Neospora caninum antigens results in differential effects with regard to protection against experimental challenge with Neospora caninum tachyzoites, Parasitology, v.137, p.229, 2010).006 Currently, much has been researched about specific protozoan antigens, associations of proteins of different structures. In this scenario, some proteins have stood out in their character of protection against the challenge, among them roptria 2 has been extensively tested (K. DEBACHE, C. GUIONAUD, F. ALAEDDINE, A. HEMPHILL, Intraperitoneal and intra-nasal vaccination of mice with three distinct recombinant Neospora caninum antigens results in differential effects with regard to protection against experimental challenge with Neospora caninum tachyzoites, Parasitology, v.137, p.229, 2010).
007 As roptrias, localizam-se no complexo apical, estão presentes em todos os estágios de desenvolvimento do parasita, e participam ativamente na invasão do parasita na célula hospedeira, e formação do vacúolo parasitóforo (J.F. Dubremetz,007 Ropters, located in the apical complex, are present in all stages of development of the parasite, and actively participate in the invasion of the parasite in the host cell, and formation of the parasitophore vacuole (J.F. Dubremetz,
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Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction, Cell. Microbiol., v.9 p.841-848, 2007; A.M. Martin, T. Liu, B.C. Lynn, A.P. Sinai, The Toxoplasma gondii parasitophorous vacuole membrane: Transactions across the border, J. Eukaryot. Microbiol., v.54,p.25-28, 2007.Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction, Cell. Microbiol., V.9 p.841-848, 2007; A.M. Martin, T. Liu, B.C. Lynn, A.P. Sinai, The Toxoplasma gondii parasitophorous vacuole membrane: Transactions across the border, J. Eukaryot. Microbiol., V.54, p.25-28, 2007.
008 As vacinas baseadas em rNcROP2 tem mostrado resultados promissores em estudos com desafio, camundongos vacinados com rNcROP2 foram capazes de sobreviver ao desafio, (K. Debache, C. Guionaud, F. Alaeddine, M. Mevissen, A. Hemphill, Vaccination of mice with recombinant NcROP2 antigen reduces mortality and cerebral infection in mice infected with Neospora caninum tachyzoites, Int. J. Parasitol., v.38, p.1455-1463, 2008) a parir desse estudo novas abordagem com a utilização de NcROP2 foram feitas, como co-administração com outras proteinas de N. caninum(K. Debache, F. Alaeddine, C. Guionaud, T. Monney, J. Müller, M. Strohbusch, et al., Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites, Int. J. Parasitol.,v.39 p.1373-1384, 2009). 009 Em outra abordagemde rNcROP2, apenas uma região foi utilizada, e está, fusionada com outros antígenos de N. caninum, formando quimeras (T. Monney, D. Rütti, M. Schorer, K. Debache, D. Grandgirard, S.L. Leib, et al., RecNcMIC3-1-R is a microneme- and rhoptry-based chimeric antigen that protects against acute neosporosis and limits cerebral parasite load in the mouse model for Neospora caninum infection., Vaccine, v.29 p.6967-75, 2011. As quimeras produzidas foram capazes de induzir imunidade protetoranos camundongos quando desafiados.008 Vaccines based on rNcROP2 have shown promising results in challenging studies, mice vaccinated with rNcROP2 were able to survive the challenge, (K. Debache, C. Guionaud, F. Alaeddine, M. Mevissen, A. Hemphill, Vaccination of mice with recombinant NcROP2 antigen reduces mortality and cerebral infection in mice infected with Neospora caninum tachyzoites, Int. J. Parasitol., v.38, p.1455-1463, 2008) from this study new approaches with the use of NcROP2 were made, as coadministration with other N. caninum proteins (K. Debache, F. Alaeddine, C. Guionaud, T. Monney, J. Müller, M. Strohbusch, et al., Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites, Int. J. Parasitol., v.39 p.1373-1384, 2009). 009 In another rNcROP2 approach, only one region was used, and it is, fused with other N. caninum antigens, forming chimeras (T. Monney, D. Rütti, M. Schorer, K. Debache, D. Grandgirard, SL Leib, et al., RecNcMIC3-1-R is a microneme- and rhoptry-based chimeric antigen that protects against acute neosporosis and limits cerebral parasite load in the mouse model for Neospora caninum infection., Vaccine, v.29 p.6967-75, 2011. The chimeras produced were able to induce protective immunity in mice when challenged.
0010 Até o presente, muitas pesquisas em torno de um método preventivo contra neosporose em mamiferos está sendo pesquisado, não existe um produto comercial que assegure a sanidade dos animais. Entre os métodos mais pesquisados estão uso das vacinas recombinantes, e entre os antígenos mais promissores e atualmente utilizados a compor a vacina contra neosporose está a proteína NcROP2 (I. Pastorfernández, D. Arranz-solís, J. Regidor-cerrillo, G. Álvarez-garcía, A. Hemphill, A. García-culebras, et al., A vaccine formulation combining rhoptry proteins NcROP40 and0010 To date, much research on a preventive method against neosporosis in mammals is being researched, there is no commercial product that ensures the health of animals. Among the most researched methods are the use of recombinant vaccines, and among the most promising antigens currently used to compose the vaccine against neosporosis is the protein NcROP2 (I. Pastorfernández, D. Arranz-solís, J. Regidor-cerrillo, G. Álvarez -garcía, A. Hemphill, A. García-culebras, et al., A vaccine formulation combining rhoptry proteins NcROP40 and
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NcROP2 improves pup survival in a pregnant mouse model of neosporosis, Vet. Parasitol., v.207, p.203-215, 2015).NcROP2 improves pup survival in a pregnant mouse model of neosporosis, Vet. Parasitol., V.207, p.203-215, 2015).
0011 Uma das propriedades imunológicas descobertas de NcROP2 é a forte estimulação de resposta Th1, modulação ideal na defesa contra protozoários intracelulares como N. caninum (T. Monney, K. Debache, D. Grandgirard, S.L. Leib, A. Hemphill, Vaccination with the recombinant chimeric antigen recNcMIC3-1-R induces a non-protective Th2-type immune response in the pregnant mouse model for N . caninum infection, Vaccine,v.30 p.6588-6594, 2012).0011 One of the immunological properties discovered by NcROP2 is the strong stimulation of Th1 response, ideal modulation in the defense against intracellular protozoa such as N. caninum (T. Monney, K. Debache, D. Grandgirard, SL Leib, A. Hemphill, Vaccination with the recombinant chimeric antigen recNcMIC3-1-R induces a non-protective Th2-type immune response in the pregnant mouse model for N. caninum infection, Vaccine, v.30 p.6588-6594, 2012).
010 Com a presente invenção, foi possível obter um imunobiológico capaz de induzir uma imunidade protetora, estimulando a geração altos níveis de anticorpos e modulando a resposta imunológica para uma resposta também celular nos animais em experimentação. Além disso, a proteína rROP2 apesar de peptídeo de 18 kDa foi antigênica, e sua expressão em E. coli não interferiu na apresentação para o sistema imune, possibilitando a apresentação de epítopos importantes para uma resposta eficaz e duradoura contra N. caninum.010 With the present invention, it was possible to obtain an immunobiological capable of inducing a protective immunity, stimulating the generation of high levels of antibodies and modulating the immune response to a cellular response in the animals under experimentation. In addition, the rROP2 protein, despite an 18 kDa peptide, was antigenic, and its expression in E. coli did not interfere in the presentation to the immune system, allowing the presentation of important epitopes for an effective and lasting response against N. caninum.
BREVE DESCRIÇÃO DOS DESENHOSBRIEF DESCRIPTION OF THE DRAWINGS
011 A FIGURA 1 representa a sequência de NcROP2, na qual está delimitada a região a ser amplificada e flanqueada pelos sítios de restrição com as enzimas Xhol e Hindlll, respectivamente. A seta em laranja representa toda sequência codificadora de NcROP2, a marcação em azul, compreende a região sintetizada e clonada, 574 1078pb.011 FIGURE 1 represents the NcROP2 sequence, in which the region to be amplified and flanked by the restriction sites with the enzymes Xhol and Hindlll, respectively, is delimited. The orange arrow represents the entire NcROP2 coding sequence, the blue marking, comprises the synthesized and cloned region, 574 1078bp.
012 A FIGURA 2 anexa ilustra a eletroforese em gel de agarose 1% da extração plasmidial da cepa de E. coli Top10 F, na qual 1 representa o plasmídeo pAE-ROP2 (3286pb) e 2 representa o vetor pAE sem inserto (2822 pb) M representa o marcador de massa molecular 100pb (Ludwig).012 The attached FIGURE 2 illustrates the electrophoresis in agarose gel 1% of the plasmid extraction of the E. coli Top10 F strain, in which 1 represents the plasmid pAE-ROP2 (3286bp) and 2 represents the pAE vector without insert (2822 bp) M represents the molecular weight marker 100bp (Ludwig).
013 A FIGURA 3 anexa ilustra a eletroforese em gel de agarose 1% da PCR com iniciadores específicos, na qual 1 representa o produto obtido da técnica de PCR com cerca de 523 pb e M representa o marcador de massa molecular 100pb (Ludwig).013 The attached FIGURE 3 illustrates the electrophoresis in agarose gel 1% of the PCR with specific primers, in which 1 represents the product obtained from the PCR technique with about 523 bp and M represents the molecular weight marker 100 bp (Ludwig).
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014 A FIGURA 4 anexa ilustra o SDS-PAGE 15% da ROP2 concentrada. Na figura, 1 representa a proteína ROP2 concentrada e M - Marcador Page Ruler Prestained Protein Ladder (ThermoScientific).014 The attached FIGURE 4 illustrates the SDS-PAGE 15% of the concentrated ROP2. In the figure, 1 represents the concentrated ROP2 protein and M - Marker Page Ruler Prestained Protein Ladder (ThermoScientific).
015 A FIGURA 5 anexa ilustra o Western blot da ROP2 purificada por cromatografia de afinidade. IgG de camundongo anti-6xhistinida. M - Marcador Prestained SDSPAGE Standards Low Range (BIO-RAD); 1 - ROP2 (18kDa) purificada por cromatografia de afinidade.015 The attached FIGURE 5 illustrates the Western blot of purified ROP2 by affinity chromatography. Mouse anti-6xhistinide IgG. M - Prestained SDSPAGE Standards Low Range Marker (BIO-RAD); 1 - ROP2 (18kDa) purified by affinity chromatography.
016 A FIGURA 6 anexa ilustra o Western blot da ROP2 (18kDa) purificada por cromatografia de afinidade. IgG de bovinos positivos para Neosporose. M - Marcador Marcador Prestained SDS-PAGE Standards Low Range (BIO-RAD); 1 - ROP2 purificada por cromatografia de afinidade.016 The attached FIGURE 6 illustrates the Western blot of ROP2 (18kDa) purified by affinity chromatography. IgG from bovines positive for Neosporosis. M - Marker Marker Prestained SDS-PAGE Standards Low Range (BIO-RAD); 1 - ROP2 purified by affinity chromatography.
017 A FIGURA 7 anexa é um gráfico das leituras obtidas no ELISA utilizando 100ng de ROP2 com os soros camundongos inoculados com ROP2, grupos: ROP2 + adjuvante Hidróxido de Alumínio (15%), solução salina 0,9% + hidróxido de alumínio (15%). Todos os soros foram diluídos 1:100.017 The attached FIGURE 7 is a graph of the readings obtained in the ELISA using 100ng of ROP2 with the mouse sera inoculated with ROP2, groups: ROP2 + adjuvant Aluminum Hydroxide (15%), 0.9% saline solution + aluminum hydroxide (15 %). All sera were diluted 1: 100.
SUMÁRIO DA INVENÇÃOSUMMARY OF THE INVENTION
018 De um modo amplo, a presente invenção trata de um processo de preparação bem como da utilização da ROP2 recombinante, descrita como roptria 2 de N. caninum, em vacinas de subunidade contra Neosporose.Broadly speaking, the present invention deals with a process of preparation as well as the use of recombinant ROP2, described as N. caninum roptria 2, in subunit vaccines against Neosporosis.
019 Deste modo, a invenção provê um processo de preparação da vacina de subunidade recombinante rROP2.Thus, the invention provides a process for preparing the recombinant subunit vaccine rROP2.
020 A invenção provê também uma vacina de subunidade recombinante ROP2.The invention also provides a recombinant ROP2 subunit vaccine.
021 A invenção provê também a utilização da dita vacina de subunidade recombinante ROP2para prevenção e controle da Neosporose em mamíferos.The invention also provides for the use of said recombinant subunit vaccine ROP2 for the prevention and control of Neosporosis in mammals.
022 A invenção provê também a utilização da dita vacina de subunidade recombinante ROP2 para controle em fêmeas prenhes.The invention also provides for the use of said ROP2 recombinant subunit vaccine for control in pregnant females.
023 A invenção provê também a utilização da dita vacina de subunidade recombinante ROP2como método único para imunização dos mamíferos sensíveis a N. caninum.The invention also provides for the use of said recombinant subunit vaccine ROP2 as a unique method for immunizing mammals sensitive to N. caninum.
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024 A invenção provê também a utilização da dita vacina de subunidade recombinante ROP2para manutenção da prenhes, em primíparas e multíparas.The invention also provides for the use of said recombinant subunit vaccine ROP2 for maintenance of pregnant women, in primiparous and multiparous.
025 A invenção provê também a utilização da dita vacina de subunidade recombinante ROP2 para evitar o descarte de animais positivos para Neosporose.The invention also provides for the use of said ROP2 recombinant subunit vaccine to prevent the disposal of animals positive for Neosporosis.
026 A invenção provê também a utilização da dita vacina de subunidade recombinante ROP2para transmissão de anticorpos via colostro.The invention also provides for the use of said recombinant subunit vaccine ROP2 for transmission of antibodies via colostrum.
027 A invenção provê também a utilização da dita vacina de subunidade recombinante ROP para proteger contra transmissão entre animais silvestres e domésticos.The invention also provides for the use of said recombinant subunit vaccine ROP to protect against transmission between wild and domestic animals.
028 No presente relatório e reivindicações, os seguintes termos têm o significado abaixo:028 In this report and claims, the following terms have the meaning below:
029 ROP2 significa uma proteína presente nas glândulas roptrias presentes no complexo apical do protozoário N. caninum, compondo o imunobiológico utilizado na preparação da vacina.029 ROP2 means a protein present in the radial glands present in the apical complex of protozoan N. caninum, composing the immunobiological used in the preparation of the vaccine.
030 A presente invenção trata, pois, da utilização da proteína ROP2 em vacinas de subunidade recombinante com aplicação na imunização contra neosporose, bem como a preparação desta vacina.The present invention therefore deals with the use of the ROP2 protein in recombinant subunit vaccines with application in immunization against neosporosis, as well as the preparation of this vaccine.
031 Sob um aspecto, a invenção trata do processo de preparação de vacina de subunidade recombinante ROP2.031 In one aspect, the invention deals with the process of preparing the ROP2 recombinant subunit vaccine.
032 O processo de preparação da vacina está detalhado abaixo e compreende a etapa de:032 The vaccine preparation process is detailed below and comprises the step of:
1) preparação da vacina1) vaccine preparation
a) sintetizar comercialmente o gene referente a sequência da ROP2 em plasmídeo de armazenamento;a) commercially synthesize the gene referring to the ROP2 sequence in a storage plasmid;
b) amplificar por PCR a região da proteína ROP2 utilizando enzimas de alta fidelidade;b) PCR amplify the ROP2 protein region using high-fidelity enzymes;
c) clonar o dito gene no vetor de expressão escolhido, com auxílio de reação de recombinação sitio específica, obtendo o vetor de expressão-ROP2;c) clone said gene in the chosen expression vector, with the help of specific site recombination reaction, obtaining the expression vector-ROP2;
d) introduzir o vetor de expressão-ROP2 em uma cepa de E. coli a patogênica efetuando transformação por eletroporação;d) introducing the expression vector-ROP2 into a pathogenic E. coli strain performing transformation by electroporation;
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e) induzir a dita cepa de E. coli, que contém o vetor de expressão-ROP2 a expressar a proteína ROP2 no corpo celular, a temperatura entre 28-37°C;e) induce said strain of E. coli, which contains the expression vector-ROP2 to express the protein ROP2 in the cell body, at a temperature between 28-37 ° C;
f) remover o cultivo da dita cepa de E. coli, mantendo apenas o pellet celular;f) remove the cultivation of said E. coli strain, keeping only the cell pellet;
g) processar o pellet celular da dita cepa de E. coli para recuperar a proteína ROP2 através de tampão de lise com agente desnaturante, lisozima e sonicação com ultrassom;g) process the cell pellet of said E. coli strain to recover the ROP2 protein through lysis buffer with denaturing agent, lysozyme and sonication with ultrasound;
h) purificar e concentrar a ROP2 por cromatografia de afinidade e dialisar contra um tampão;h) purify and concentrate ROP2 by affinity chromatography and dialysis against a buffer;
i) recuperar a proteína, armazenando a mesma por congelamento a temperaturas entre -20 e -70 °C.i) recover the protein, storing it by freezing at temperatures between -20 and -70 ° C.
035 Conforme a invenção, cepa de E. coli útil para efetuar a propagação do vetor de expressão compreende Top10F e a cepa de E. coli úteis para efetuar a expressão da proteína recombinante compreendem BL21Star (DE3), BL21(DE3) pLysS, BL21(DE3) pLysE, Over Express C41(DE3) e Rosetta (DE3).035 According to the invention, the E. coli strain useful for propagating the expression vector comprises Top10F and the E. coli strain useful for effecting the recombinant protein expression comprises BL21Star (DE3), BL21 (DE3) pLysS, BL21 ( DE3) pLysE, Over Express C41 (DE3) and Rosetta (DE3).
036 Vetores de expressão úteis são vetores como pET-15b (Novagen), pET-16b (Novagen), pET-19b (Novagen), pREST (Invitrogen) ou pAE (Ramos C. R. et al. A highcopy T7 E. coli expression vector for the production of recombinant proteins with minimal N-terminal His-tagged fusion peptide. Brazilian Journal of Medical and Biological Research, 37, n. 8, p. 1103-1109, 2004).036 Useful expression vectors are vectors such as pET-15b (Novagen), pET-16b (Novagen), pET-19b (Novagen), pREST (Invitrogen) or pAE (Ramos CR et al. A highcopy T7 E. coli expression vector for the production of recombinant proteins with minimal N-terminal His-tagged fusion peptide (Brazilian Journal of Medical and Biological Research, 37, n. 8, p. 1103-1109, 2004).
037 O meio de cultura para expressão em E. coli compreende o meio LB (Luria Bertani), TB (TerrificBroth), 2 x YT (2 x Yeast extract andTryptone) e SB (Super Broth). 038 A Temperatura de expressão varia entre 28 e 39 °C.037 The culture medium for expression in E. coli comprises the medium LB (Luria Bertani), TB (TerrificBroth), 2 x YT (2 x Yeast extract andTryptone) and SB (Super Broth). 038 The expression temperature varies between 28 and 39 ° C.
039 Antibióticos úteis na transformação incluem Ampicilina (25 a 100 qg/mL) (Invitrogen).039 Antibiotics useful in transformation include Ampicillin (25 to 100 qg / mL) (Invitrogen).
040 Indução da produção da proteína recombinante com IPTG (isopropil β-Dtiogalactopiranosídeo) em concentrações que variam de 0,5 a 3 mM no volume final do meio.040 Induction of recombinant protein production with IPTG (isopropyl β-Dtiogalactopyranoside) in concentrations ranging from 0.5 to 3 mM in the final volume of the medium.
041 A seguir as etapas do processo inventivo serão descritas com mais detalhes.041 Next, the steps of the inventive process will be described in more detail.
042 A etapa a) constitui técnica disponível comercialmente e de conhecimento dos especialistas, não necessitando de maior detalhamento. A Figura 1 representa a042 Step a) is a commercially available technique that is known to specialists, and does not require further details. Figure 1 represents the
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8/11 sequência de NcROP2, na qual está delimitada a região a ser amplificada e flanqueada pelos sítios de restrição com as enzimas XhoI e HindIII, respectivamente. A seta em laranja representa toda sequência codificadora de NcROP2 (GenBank), a marcação em azul, compreende a região sintetizada e clonada, 574 - 1078pb.8/11 NcROP2 sequence, in which the region to be amplified and flanked by the restriction sites with the XhoI and HindIII enzymes is delimited, respectively. The orange arrow represents the entire coding sequence for NcROP2 (GenBank), the blue marking, comprising the synthesized and cloned region, 574 - 1078bp.
043 O procedimento utilizado na etapa b) é descrito por Sambrook J. E Russel, W. D. Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 9.16-9.17, 2001. O material genético sintético obtido, gene ROP2 é utilizado em seguida ou armazenado a temperaturas entre -20 e -70°C para utilização posterior.043 The procedure used in step b) is described by Sambrook J. E Russel, W. D. Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 9.16-9.17, 2001. The synthetic genetic material obtained, gene ROP2, is then used or stored at temperatures between -20 and -70 ° C for later use.
044 Na etapa b) de amplificação por PCR, os iniciadores para a amplificação de ROP2 foram desenhados com base na sequência construída no software Vector NTI Advance™ 11 (Thermo Fisher Scientific Inc.). Os seguintes iniciadores foram utilizados na presente invenção: ROP2 senso 5'AGACTCGAGCTGCGACCAGGCCA 3' e ROP2 anti-senso 5'CTCAAGCTTGGCGTGTTAGTCGGG 3', nos quais as sequências sublinhadas referem-se aos sítios das enzimas de restrição Xhol e HindIII respectivamente.044 In step b) of PCR amplification, the primers for ROP2 amplification were designed based on the sequence constructed in the Vector NTI Advance ™ 11 software (Thermo Fisher Scientific Inc.). The following primers were used in the present invention: ROP2 sense 5'AGACTCGAGCTGCGACCAGGCCA 3 'and ROP2 antisense 5'CTCAAGCTTGGCGTGTTAGTCGGG 3', in which the underlined sequences refer to the Xhol and HindIII restriction enzyme sites respectively.
045 Para efetuar a etapa c) de clonagem, o dito gene foi amplificado por PCR em um termociclador Biocycler MJ96G, utilizando o seguinte programa: 95 °C/10min seguido por 35 ciclos de 94 °C/1 min e 58 °C/1 min e 72 °C/1 min, com uma extensão final de 72 °C/5 min. As reações foram realizadas em um volume final de 25 pL, contendo 2,5 pL de tampão 10x, 0,5 pL de dNTP 10 mM, 150 ng de cada iniciador, 0,5 pL (1 unidade) de Taq DNA polymerase, 0,5 pL de MgSO4 50 mM, 18 pL de água Milli-Q e 2 pL de sequência sintética como DNA “template”. Após amplificação por PCR, o gene ROP2 foi submetido a eletroforese em gel de agarose a 1 %.045 To perform cloning step c), said gene was amplified by PCR in a Biocycler MJ96G thermocycler, using the following program: 95 ° C / 10min followed by 35 cycles of 94 ° C / 1 min and 58 ° C / 1 min and 72 ° C / 1 min, with a final extension of 72 ° C / 5 min. The reactions were carried out in a final volume of 25 pL, containing 2.5 pL of 10x buffer, 0.5 pL of 10 mM dNTP, 150 ng of each primer, 0.5 pL (1 unit) of Taq DNA polymerase, 0 , 5 pL of 50 mM MgSO 4 , 18 pL of Milli-Q water and 2 pL of synthetic sequence as template DNA. After PCR amplification, the ROP2 gene was subjected to 1% agarose gel electrophoresis.
046 Para a etapa c) o dito gene amplificado foi submetido a uma reação de recombinação sítio específica, inserindo o gene no vetor de expressão em E. coli pAE. E. coli Top10 F competente foi transformada por eletroporação com o produto da recombinação e semeada em ágar LB low salt contento 100pg/mL de Ampicilina. Os clones recombinantes foram repicados e o pAE-ROP2 foi extraído através de lise alcalina.046 For step c) said amplified gene was subjected to a specific site recombination reaction, inserting the gene into the expression vector in E. coli pAE. E. coli Top10 F competent was transformed by electroporation with the recombination product and seeded on LB low salt agar containing 100pg / mL of Ampicillin. The recombinant clones were picked and pAE-ROP2 was extracted by alkaline lysis.
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047 Para a caracterização do plasmídeo pAE-ROP2 foram realizadas as comparações do plasmídeo pAE-ROP2 com o vetor pAE e a técnica de PCR com iniciadores específicos para o gene ROP2, os resultados foram analisados por eletroforese em gel de agarose. A Figura 2 ilustra a eletroforese em gel de agarose 1% da extração plasmidial da cepa de E. coli Top10 F, na qual,1 representa o vetor pAE sem inserto (2822pb) e 2 representa o plasmídeo pAE-ROP2 (3286pb), M representa o marcador de massa molecular 100pb (Ludwig). A Figura 3 ilustra a eletroforese em gel de agarose 1% da PCR com iniciadores específicos, na qual 1 representa o produto obtido da técnica de PCR com cerca de 523pb e M representa o marcador de massa molecular 100pb (Ludwig).047 For the characterization of plasmid pAE-ROP2, comparisons of plasmid pAE-ROP2 with the pAE vector and the PCR technique with specific primers for the ROP2 gene were performed, the results were analyzed by agarose gel electrophoresis. Figure 2 illustrates the electrophoresis in agarose gel 1% of the plasmid extraction of the E. coli Top10 F strain, in which, 1 represents the pAE vector without insert (2822pb) and 2 represents the plasmid pAE-ROP2 (3286pb), M represents the molecular weight marker 100bp (Ludwig). Figure 3 illustrates the electrophoresis in agarose gel 1% of the PCR with specific primers, in which 1 represents the product obtained from the PCR technique with about 523 bp and M represents the molecular weight marker 100 bp (Ludwig).
048 Na etapa d) E. coli BL21Star (DE3) competente foi transformada por eletroporação com o dito vetor de expressãopAE-ROP2e semeada em meio LB com 100 pg/mL de Ampicilina.048 In step d) competent E. coli BL21Star (DE3) was transformed by electroporation with the said expression vector pAE-ROP2e seeded in LB medium with 100 pg / mL Ampicillin.
049 Então, conforme a etapa e), a expressão da quimera ROP2 foi realizada em meio LB, induzida com IPTG 1 mM (concentração total no meio) durante um período de 2 a 6h, sob agitação de 150 rpm a 28-39 °C. Após este período, a cultura é centrifugada a 15,000 g por 15 min a 4 °C, conforme etapa f).049 Then, according to step e), the expression of the ROP2 chimera was carried out in LB medium, induced with 1 mM IPTG (total concentration in the medium) for a period of 2 to 6 hours, under agitation of 150 rpm at 28-39 ° C . After this period, the culture is centrifuged at 15,000 g for 15 min at 4 ° C, as per step f).
050 O pellet celular foi suspendido em tampão de lise com 8M de ureia e lisozima (100 mg/pL) e incubado por 2-5 h a 4 °C. As células são rompidas através da sonicação por ultrassom e são centrifugadas a 15,000 g por 15 min a 4 °C, etapa g).050 The cell pellet was suspended in lysis buffer with 8M urea and lysozyme (100 mg / pL) and incubated for 2-5 h at 4 ° C. The cells are disrupted by ultrasound sonication and are centrifuged at 15,000 g for 15 min at 4 ° C, step g).
051 A ROP2 presente neste sobrenadante é purificada, etapa h), através da cromatografia de afinidade (6xhis-tag), e dialisada por 24 h em tampão PBS pH 7.4. Após a diálise, a ROP2 é recuperada e armazenada, conforme etapa i).051 The ROP2 present in this supernatant is purified, step h), through affinity chromatography (6xhis-tag), and dialyzed for 24 h in PBS pH 7.4 buffer. After dialysis, ROP2 is recovered and stored, according to step i).
052 A pureza da ROP2 é determinada através de SDS-PAGE e sua concentração pelo método de BCATM (Pierce).052 The purity of ROP2 is determined using SDS-PAGE and its concentration by the BCA TM method (Pierce).
053 A Figura 4ilustra o SDS-PAGE 15% da ROP2 concentrada. Na figura, 1 representa a proteína ROP2 concentrada.053 Figure 4 illustrates the SDS-PAGE 15% of the concentrated ROP2. In the figure, 1 represents the concentrated ROP2 protein.
054 Um segundo aspecto da invenção é a vacina ROP2 obtida pelo processo proposto.054 A second aspect of the invention is the ROP2 vaccine obtained by the proposed process.
055 A caracterização da vacina ROP2 compreende dois aspectos:055 The characterization of the ROP2 vaccine comprises two aspects:
Petição 870170057153, de 09/08/2017, pág. 21/24Petition 870170057153, of 08/09/2017, p. 21/24
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i) Antigenicidade;i) Antigenicity;
ii) Imunogenicidade.ii) Immunogenicity.
056 A antigenicidade da ROP2 foi avaliada mediante Western blot, conforme Sambrook J. e Russel, W. D. citados acima. Os seguintes soros foram utilizados: soro monoclonal anti-6xhistidina (Sigma-Aldrich), soro policlonal de bovinos positivo para N. caninum, previamente definido por RIFI (Reação de Imunofluorescência Indireta). Todos os soros reconheceram uma banda de aproximadamente 18kDa correspondente a ROP2.056 The antigenicity of ROP2 was evaluated using Western blot, according to Sambrook J. and Russel, W. D. mentioned above. The following sera were used: monoclonal anti-6xhistidine serum (Sigma-Aldrich), polyclonal bovine serum positive for N. caninum, previously defined by RIFI (Indirect Immunofluorescence Reaction). All sera recognized a band of approximately 18kDa corresponding to ROP2.
057 A Figuras 5 e 6, anexas ilustra o Western blot da ROP2 purificada por cromatografia de afinidade. IgG de camundongo anti-6xhistinida. M - Marcador Prestained SDS-PAGE Standards Low Range (BIO-RAD); 1 - ROP2 (18kDa) purificada por cromatografia de afinidade. Amostras distribuídas conforme Figura 5; (6) IgG de bovinos positivos para Neosporose.057 The attached Figures 5 and 6 illustrate the Western blot of purified ROP2 by affinity chromatography. Mouse anti-6xhistinide IgG. M - Prestained SDS-PAGE Standards Low Range Marker (BIO-RAD); 1 - ROP2 (18kDa) purified by affinity chromatography. Samples distributed according to Figure 5; (6) IgG from bovines positive for Neosporosis.
058 A imunogenicidade da proteína ROP2 foi avaliada em fêmeas BALB/c (camundongos) com idade entre cinco a sete semanas. Elas foram aleatoriamente separadas e identificadas em dois grupos de 6animais. As inoculações ocorreram nos dias 0 e 14. O grupo um foi inoculado com 25pg da proteína ROP2 emulsificada em dose contendo 15% de Hidróxido de Alumínio. O grupo dois foi inoculado com solução salina 0,9% por via intramuscular em dose contendo 15% de Hidróxido de Alumínio. Coletas de sangue foram realizadas através de punção do plexo venoso retro ocular a cada 7 dias até o dia 60 após a primo vacinação, a última coleta foi realizada no dia 60. Os soros foram estocados a -20 °C.058 The immunogenicity of the ROP2 protein was evaluated in BALB / c females (mice) aged five to seven weeks. They were randomly separated and identified in two groups of 6 animals. The inoculations occurred on days 0 and 14. Group one was inoculated with 25pg of ROP2 protein emulsified in a dose containing 15% Aluminum Hydroxide. Group two was inoculated with 0.9% saline intramuscularly in a dose containing 15% aluminum hydroxide. Blood samples were collected by puncture of the retro ocular venous plexus every 7 days until the 60th day after the first vaccination, the last collection was performed on the 60th day. The serums were stored at -20 ° C.
059 Os soros dos animais foram submetidos a analise em pool para cada coleta por ELISA indireto. Microplacas de 96 cavidades foram sensibilizadas overnight a 4 °C com 100 ng/cavidade de ROP2 diluída em tampão carbonato-bicarbonato pH 9,6. Após três lavagens com PBS-T (PBS com 0,05% Tween 20 e % 5 de leite pó desnatado), 100 pL dos soros diluídos 1:100 em PBS-T em duplicata, foram incubados a 37 °C por 1h. Três novas lavagens com PBS-T foram realizadas, e 100 pL de Anti-IgG de camundongo conjugado à peroxidase (Sigma-Aldrich) foram incubados à 37 °C por 1h. Cinco novas lavagens com PBS-T foram realizadas e as reações foram reveladas com 100 pL oPetição 870170057153, de 09/08/2017, pág. 22/24059 The sera of the animals were submitted to pool analysis for each collection by indirect ELISA. 96-well microplates were sensitized overnight at 4 ° C with 100 ng / well of ROP2 diluted in carbonate-bicarbonate buffer pH 9.6. After three washes with PBS-T (PBS with 0.05% Tween 20 and% 5 skimmed milk powder), 100 pL of the sera diluted 1: 100 in PBS-T in duplicate, were incubated at 37 ° C for 1h. Three new washes with PBS-T were performed, and 100 pL of mouse anti-IgG conjugated to peroxidase (Sigma-Aldrich) were incubated at 37 ° C for 1h. Five new washes with PBS-T were performed and the reactions were revealed with 100 pL oPetition 870170057153, from 08/09/2017, p. 22/24
11/11 phenylenediaminedihydrochloride (Sigma-Aldrich) e peróxido de hidrogênio e incubadas por 15min no escuro. A reação foi finalizada com a adição de 100 pL de ácido sulfúrico a 3%. OD492nm foi determinada em um leitor de ELISA Dynatech MR 700 (DynatechLabs. Inc., Chantilly, VA, USA) após o término da reação. Para a determinação de diferenças estatísticas, foram realizadas as análises de variância (ANOVA) seguido pelo post-tests de Bonferroni das absorbâncias obtidas utilizando o software Graph Pad Prism 6. Foi considerado p < 0,001 como significante nas análises. 060 A Figura 7 é um gráfico que mostra a absorbância referenteaos anticorpos sistêmicos anti-ROP2, determinadas através de ELISA com antígeno ROP2, dos camundongos inoculados com ROP2 + adjuvante Hidróxido de Alumínio (15%); solução salina 0,9% com Hidróxido de Alumínio (15%). Todos os animais receberam duas doses. A curva da Figura 7 ilustra nitidamente absorbância mais elevada para a vacina da invenção aplicada via intramuscular com a presença de adjuvante Hidróxido de Alumínio, em comparação ao controle.11/11 phenylenediaminedihydrochloride (Sigma-Aldrich) and hydrogen peroxide and incubated for 15 minutes in the dark. The reaction was terminated with the addition of 100 pL of 3% sulfuric acid. The 492nm OD was determined in an ELISA reader Dynatech MR 700 (DynatechLabs. Inc., Chantilly, VA, USA) after completion of the reaction. For the determination of statistical differences, analyzes of variance (ANOVA) were performed followed by Bonferroni's post-tests of the absorbances obtained using the Graph Pad Prism 6 software. P <0.001 was considered significant in the analyzes. 060 Figure 7 is a graph showing the absorbance referring to systemic anti-ROP2 antibodies, determined by ELISA with ROP2 antigen, of mice inoculated with ROP2 + adjuvant Aluminum Hydroxide (15%); 0.9% saline solution with Aluminum Hydroxide (15%). All animals received two doses. The curve in Figure 7 clearly illustrates higher absorbance for the vaccine of the invention applied intramuscularly with the presence of Aluminum Hydroxide adjuvant, compared to the control.
Petição 870170057153, de 09/08/2017, pág. 23/24Petition 870170057153, of 08/09/2017, p. 23/24
1/11/1
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| BR102016027456-7A BR102016027456A2 (en) | 2016-11-23 | 2016-11-23 | PROCESS FOR OBSERVATION AND USE OF NEOSPORA CANINUM ROP2 PROTEIN FOR USE AS NEOSPOROSIS VACCINE |
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| BR102016027456-7A BR102016027456A2 (en) | 2016-11-23 | 2016-11-23 | PROCESS FOR OBSERVATION AND USE OF NEOSPORA CANINUM ROP2 PROTEIN FOR USE AS NEOSPOROSIS VACCINE |
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