WO2016090440A1 - Vecteur vaginal comprenant des mutants de salmonella enterica typhimurium atténués pour le gène fis, vaccin comprenant ledit vecteur et utilisation dudit vecteur - Google Patents

Vecteur vaginal comprenant des mutants de salmonella enterica typhimurium atténués pour le gène fis, vaccin comprenant ledit vecteur et utilisation dudit vecteur Download PDF

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Publication number
WO2016090440A1
WO2016090440A1 PCT/BR2015/000137 BR2015000137W WO2016090440A1 WO 2016090440 A1 WO2016090440 A1 WO 2016090440A1 BR 2015000137 W BR2015000137 W BR 2015000137W WO 2016090440 A1 WO2016090440 A1 WO 2016090440A1
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vaccine
vector
fis
mutants
strains
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PCT/BR2015/000137
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English (en)
Portuguese (pt)
Inventor
Marcelon BROCCHI
Guilherme Paiez MILANEZ
Aline Ferreira de Oliveira PEREIRA
Maria Cristina Roque Antunes BARREIRA
Luciana Pereira RUAS
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Universidade Estadual De Campinas - Unicamp
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Publication of WO2016090440A1 publication Critical patent/WO2016090440A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/42—Salmonella

Definitions

  • the present invention relates to the development of vaccine vector comprising S. enteral Typhimurium or attenuated 4, [5 ⁇ , 12: i: deletion of the fis gene encoding global regulatory protein.
  • the Fis protein regulates the expression of several others, including proteins related to bacterial pathogenicity, and when the fis gene is deleted, a unique attenuation pattern is generated that can be altered by combining with other mutations, inducing different types of responses. of the host.
  • the present invention relates to a vaccine comprising said vaccine strain and its use as a vaccine for the protection of humans and animals such as poultry and swine.
  • the first live attenuated vaccine developed against typhoid is the S. enteric Typhi Ty21a strain obtained by chemical mutagenesis of the Ty2 parent strain. This oral vaccine is safe, but because of its great attenuation, it requires several doses to induce immunity, which is often short-lived.
  • the Ty21a strain is the live attenuated typhoid vaccine licensed for human use, marketed under the name Vivitof by Bernabiotech Ltd of Switzerland.
  • S. enteral Typhi Ty21 stimulated the construction of new S. enteral vaccine lines.
  • Such strains show attenuation stability, since it was achieved by deletion of one or more specific virulence-related genes, which is fundamental for their use as live vaccines.
  • These mutants were initially constructed in S. enterica Typhimurium due to the availability of the murine model of infection and promising mutations were transferred to S. enterica Typhi strains. Therefore, S.
  • enteral AaroA, AaroC, AaroD strains deficient in the biosynthetic pathway of aromatic amino acids, mutants Acya Acrp, unable to express adenylate cyclase and cAMP receptor, among others, were constructed and demonstrated to be immunogenic, despite the fact that attenuation of virulence.
  • bacteremia and / or the need for multiple doses to induce immunity in human volunteers are frequent.
  • Another focus of studies is on the development of vaccines for the control of salmonellosis in birds and other animals.
  • strains The construction of these strains is achieved by the introduction of mutations in genes associated with pathogenicity, biosynthetic pathways or global regulatory genes, which result in attenuation of virulence without, however, compromising the in vivo colonization capacity and consequently , of induction of the immune system effectively.
  • Such strains can be genetically engineered to express heterologous antigens, allowing the development of multifactor vaccine formulations.
  • Enteric S. is complex and involves both transcriptional and post-transcriptional regulation processes.
  • Attenuated S. enteral Typhimurium strains have already been developed by constructing null mutants for Host Integration Factor A (ihfA) or ihf as seen in patent documents WO2010096888 and BR200902944. Data demonstrate that such strains are able to confer protection against salmonellosis in the murine model of infection.
  • Integration host factor (IHF) protein is a bacterial nucleoid protein that causes DNA to bend about 180 °, and this activity can induce significant topological changes in DNA by modulating the expression of various genes, as well as other events. cell phones. IHF expression is increased at the stationary phase of growth and several genes are under its positive control, including virulence genes in S. enterica Typhimurium. Following this same reasoning, null mutants for the Fis protein (Factor for inversion) were obtained. It is a nucleoid protein described as the most abundant in the exponential phase of growth in E. coli, with a drastic decrease in its concentration during the stationary phase. This drastic variation in concentration suggests an important function of Fis in the transition from the exponential phase to the stationary phase of growth. In fact, Fis's most relevant biological function is the activation of the expression of genes involved in translation machinery, such as RNA operons and translation factors.
  • Microarray data indicated an important role of Fis in the expression of genes associated with pathogenicity as well as maintenance genes in S. enterica Typhimurium.
  • US 8,101,168 describes attenuated enterobacteria comprising an attenuating mutation in the fnr gene, and optionally further comprising a heterologous nucleic acid encoding a foreign antigen.
  • this document relates to a different gene from that proposed in the present invention, as will be set forth below to achieve a similar attenuation phenotype.
  • the US document mentioned does not demonstrate data confirming that said mutant is capable of inducing an appropriate immunogenic response to protect immunized animals against challenge with wild bacteria.
  • US 8,703,153 already refers to an attenuated Salmonella vaccine vector comprising one or more heterologous polynucleotides encoding chlamydial immunogenic peptides.
  • This document refers to two deletions, one for Pathogen Island 2 (SPI2) genes and one for an aromatic amino acid biosynthesis pathway gene. These two deletions lead to attenuated lineage.
  • SPI2 Pathogen Island 2
  • WO 2011/079361 describes an enteric Salmonella Gallinarum mutant, which is defective in the cobS and cbiA genes (SGCobSCbiA), which are associated with bacterial production of cobalamin under anaerobic conditions for use as vaccines.
  • the present invention achieves these and other objects by means of a vaccine vector comprising an attenuated mutant of the enteric Salmonella Typhimurium serovars and 4, [5], 12: i: - by silencing / deletion of the fis gene and further comprising the nucleotide sequence of Seq Id no 01.
  • the present invention relates to the vaccine comprising said vaccine vector.
  • the present invention also relates to the use of the vaccine vector for preparing said vaccine.
  • Figure 1 is the Contig assembled from the sequencing of the amplification product generated with FisDT primers for the two mutated strains.
  • the S. enteral homology region is underlined with gray text and the FRT sites are highlighted in gray.
  • the coding sequence for Chloramphenicol Acetyl Transferase (CAT) protein is underlined.
  • Figure 3 refers to the percentage survival of immunized animals after challenge with wild S. enterica strains.
  • BALB / c mice (8 per group) that were immunized on days 0 and 14 with the LGBM01A / ' s (A) or LGBM02A // s (B) mutants.
  • the challenge was made on the 28th day after the first immunization, with 10 5 CFU of wild S. enterica strains.
  • the present invention relates to a vaccine vector comprising an attenuated enteric Salmonella Typhimurium serovar mutant or
  • the present invention relates to a vaccine comprising said vaccine vector and the use of the vaccine for the protection of humans and animals such as poultry and swine.
  • enteral being: S. enteral Typhimurium LGB 01 or and S. enteral I 4,5,12: i LGBM02. These strains are pathogenic in the murine model of infection.
  • the S. enteral mutants LGBM0 / s, LGB 02A / S, objects of the present invention were constructed using the ⁇ Red system, based on the bacteriophage recombination system ⁇ . For this, specific primers were designed for deletion of the fis gene (FisF: Seq. Id 02) and (FisR: Seq. Id. 03).
  • the generated fragments were used to transform cells of said LGBM01 and LGBM02 strains by electroporation, both containing plasmid pKD46 (previously inserted, also by electroporation).
  • the resulting mutants were selected in culture medium plus chloramphenicol, confirmed by PCR using specific deletion detection primers (FisDT_F: Seq. Id 04) and (FisDT _R: Seq. Id 05) and used as donors for Generalized transduction using the P22HT phage to pass mutations to wild cells of the same lineage.
  • Isolated mutants following transduction, were tested for P22 in Mintgreen culture medium (which detects cell lysis due to P22 phage) and only colonies negative for phage were maintained. These were sequenced (Seq Id no. 01) and stored at a temperature of -80 ° C.
  • mice Groups of 8 BALB / c mice were infected with different doses of the enteric Salmonella LGBM01 (Typhimurium) and LGBM02 (I 4, [5], 12: i) strains in 200 ⁇ l bacterial suspension orally, as a means of determining mortality rates and then comparing them to those found in the groups of animals inoculated with the mutant strains.
  • Figure 2 shows the percentage of animals that survived the inoculum. The data shown here are representative of two experiments with similar results.
  • BALB / c mice (8 per group) were immunized with two doses of attenuated strains (approximately 10 7 CFU) on days 0 and 14 and challenged with a lethal dose of S. enteric (10 4 CFU). , 28 days after the first dose. All animals immunized with LGBMO / s and challenged with LGBM01 or LGBM02 survived the challenge, whereas in animals immunized with c with LGBM02A // s, survival rates of 100 and 75% were observed after challenge with LGBM02 and LGBM01, respectively. , as illustrated in Figure 3.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cell Biology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La mise au point de vaccins vivants atténués contre la salmonellose s'est accrue ces dernières années en raison de l'absence de formulations vaccinales totalement efficaces permettant une lutte plus efficace contre les diverses sérovariétés de S. enterica. Le développement d'une lignée sûre et capable d'induire une réponse immunitaire efficace implique l'obtention de divers mutants atténués pouvant être testés cliniquement ou présentant des caractéristiques uniques susceptibles d'être combinées à d'autres mutations spécifiques, produisant des lignées de plus en plus sûres et efficaces. La présente invention concerne un vecteur vaccinal comprenant des mutants pour le gène fis, dont la protéine résultante de son expression, Fis, s'associe au nucléoïde bactérien, régulant l'expression de différents gènes, dont beaucoup sont liés au processus de pathogénicité bactérienne. L'invention concerne également un vaccin comprenant lesdites lignées mutantes, produisant une réponse capable d'induire une protection efficace contre la salmonellose.
PCT/BR2015/000137 2014-12-08 2015-09-04 Vecteur vaginal comprenant des mutants de salmonella enterica typhimurium atténués pour le gène fis, vaccin comprenant ledit vecteur et utilisation dudit vecteur WO2016090440A1 (fr)

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BRBR1020140306382 2014-12-08
BR102014030638-2A BR102014030638B1 (pt) 2014-12-08 2014-12-08 Uso da linhagem salmonella enterica typhimurium para preparar vacina contra salmonelose

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004056973A2 (fr) * 2002-12-19 2004-07-08 E.I. Du Pont De Nemours And Company Technique de genie chromosomique
BRPI0902944A2 (pt) * 2009-02-27 2011-07-05 Unicamp vacinas compreendendo linhagens atenuadas, processo de construção de linhagens atenuadas, vetores vacinais e seu uso no tratamento da salmonelose
BRPI1003750A2 (pt) * 2010-07-22 2012-04-10 Univ Sao Paulo microrganismos recombinantes, métodos de preparação de linhagens vacinais, antìgenos, composições vacinais vetorizadas, seus usos, anticorpos, kit de diagnóstico e métodos de tratamento e/ou profilaxia
BRPI1102019A2 (pt) * 2011-04-19 2013-06-25 Unicamp processo de construÇço de linhagem atenuada mutante de uma bactÉria patogÊnica, vacina, vetor vacinal e uso da referida vacina

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004056973A2 (fr) * 2002-12-19 2004-07-08 E.I. Du Pont De Nemours And Company Technique de genie chromosomique
BRPI0902944A2 (pt) * 2009-02-27 2011-07-05 Unicamp vacinas compreendendo linhagens atenuadas, processo de construção de linhagens atenuadas, vetores vacinais e seu uso no tratamento da salmonelose
BRPI1003750A2 (pt) * 2010-07-22 2012-04-10 Univ Sao Paulo microrganismos recombinantes, métodos de preparação de linhagens vacinais, antìgenos, composições vacinais vetorizadas, seus usos, anticorpos, kit de diagnóstico e métodos de tratamento e/ou profilaxia
BRPI1102019A2 (pt) * 2011-04-19 2013-06-25 Unicamp processo de construÇço de linhagem atenuada mutante de uma bactÉria patogÊnica, vacina, vetor vacinal e uso da referida vacina

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MORAES, M. H.: "Construção de linhagens atenuadas de Salmonella entérica Enteritidis : avaliação do potencial imunogênico e protetor.", DISSERTAÇÃO - UNIVERSIDADE ESTADUAL DE CAMPINAS - INSTITUTO DE BIOLOGIA, 2012, Campinas, SP., pages 62 f., Retrieved from the Internet <URL:http://www.bibliotecadigital.unicamp.br/document/?code=000876696&fd=y> *
OSUNA, R ET AL.: "Sequence, regulation, and functions of fis in Salmonella typhimurium.", J BACTERIOL., vol. 177, no. 8, 1995, pages 2021 - 2032, ISSN: 1098-5530 *
SOYER, Y. ET AL.: "Salmonella entérica Serotype 4,5,12:i:-, an Emerging Salmonella Serotype That Represents Multiple Distinct Clones.", J. CLIN. MICROBIOL., vol. 47, no. 11, 2009, pages 3546 - 3556, ISSN: 1098-660X *
WILSON, R. L. ET AL.: "Fis, a DNA nucleoid-associated protein, is involved in Salmonella typhimurium SPI-1 invasion gene expression.", MOL MICROBIOL., vol. 39, no. 1, 2001, pages 79 - 88 *
ZHANG, H. ET AL.: "Fis is essential for the stability of linear plasmid pBSSBI and affects the motility of Salmonella entérica serovar Typhi.", PLOS ONE., vol. 7, no. 7, 2012, pages e37462 *

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BR102014030638B1 (pt) 2022-12-06
BR102014030638A8 (pt) 2021-03-23

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