WO2012142684A1 - Procédé de construction de lignée atténuée mutante d'une bactérie pathogène, vaccin, vecteur vaccinal et utilisation dudit vaccin - Google Patents

Procédé de construction de lignée atténuée mutante d'une bactérie pathogène, vaccin, vecteur vaccinal et utilisation dudit vaccin Download PDF

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WO2012142684A1
WO2012142684A1 PCT/BR2012/000059 BR2012000059W WO2012142684A1 WO 2012142684 A1 WO2012142684 A1 WO 2012142684A1 BR 2012000059 W BR2012000059 W BR 2012000059W WO 2012142684 A1 WO2012142684 A1 WO 2012142684A1
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process according
vaccine
gene
spp
recombination
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Marcelo Brocchi
Luciane Benedita Duarte PIVETTA
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Universidade Estadual De Campinas- Unicamp
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Patent Descriptive Report for: "Process for the attenuated lineage building process of a pathogenic bacterium, vaccine, vaccine vector and use of said vaccine".
  • the present invention relates to a process of producing attenuated mutant strains capable of promoting immunization to vaccine vectors and vaccines for the treatment of salmonellosis and their use in combating such infection.
  • mutants are null for HU hupA and hupB genes and have been tested for attenuation of virulence and ability to elicit effective and protective immune response against salmonellosis.
  • the genus Salmonella sp belongs to the Enterobacteriaceae family and is made up of gram-negative, facultative anaerobic bacilli usually flagellated (Mastroeni and Maskell, 2006).
  • the serological classification of this bacterium is based on the identification of somatic (O), flagellar (H) and capsular (Vi) antigens, the latter when present.
  • S. enterica S. bongori. THE S. enteric species is subdivided into 7 subgroups, and the great majority of pathogenic serovegions for humans are included in subgroup I (Boyd et al. 1996).
  • Pathogenic S. enteric serovarities can cause infections in mammals with varying degrees of severity, from gastroenteritis located in the intestinal mucosa to severe systemic infections, depending on the bacterial serovarity and type of host involved (B ⁇ umler et al., 1998).
  • S. enterica Typhi is the causative agent of typhoid fever, a serious systemic infection in man (Guzman et al., 2006).
  • S. enterica Choleraesuis causes severe systemic infections, especially in pigs, although it can also cause infections in humans (Mastroeni and Maskell, 2006; Salyers and Whitt, 2002).
  • Enteric Salmonella Dublin is a serovarity associated mainly with cattle. Like S. enterica Thyphi in humans, serovar Dublin is invasive and can cause gastroenteritis and septicemia in these animals (Mastroeni and Maskell, 2006).
  • enterica Typhimurium and Enteritidis are common causes of gastroenteritis in humans, but may also be associated with extra-intestinal infections (Mastroeni and Maskell, 2006).
  • S. enterica Typhimurium and Enteritidis cause a type of infection very similar to human typhoid fever.
  • BALB / c mice are used as the murine model of systemic S. enteric infection.
  • Salmonellosis is one of the most common infectious diseases in both humans and animals.
  • S. enteric infection begins with ingestion of contaminated water or foods, particularly poultry and pork foods. These microorganisms are facultative intracellular pathogens and, once ingested, have the ability to adhere to and invade intestinal mucosal cells, preferably M cells (Jones et al., 1994). Once the intestinal mucosa is exceeded, S. enterica invades, persists and proliferates within endothelial reticulum cell vacuoles and can thus reach different organs and tissues of the host, causing systemic infection (Salyer and Whitt, 2002).
  • the average infective dose (DI5 0) of S. enterica capable of producing clinical or subclinical infections in humans is between 10 5 -10 10 ingested organisms.
  • the infectious process is localized to the ileum, colon, and mesenteric lymph nodes after ingestion of contaminated food, with symptoms such as diarrhea, vomiting, and abdominal pain appearing (reviewed by Darwin and Miller, 1999).
  • Most cases of gastroenteritis occur in children under 10 years of age, and the symptoms tend to be more severe in this group and the infection may become systemic.
  • S. enterica An important barrier encountered by S. enterica in the infectious process following its passage through the intestinal epithelium is the submucosa macrophages (Mastroeni and Maskell, 2006; Salyers and Whitt, 2002). Macrophages detect and internalize the bacterial pathogen in order to eliminate it from the host.
  • S. enteric serovarieties capable of causing systemic infection, invade macrophages and then activate virulence mechanisms that allow evasion of phagocyte microbicidal functions, allowing survival and replication in the intracellular environment (Mastroeni and Maskell, 2006; Salyers and Hitt, 2002; Alpuche-Aranda, et al., 1994). Migration of infected macrophages to other organs of the phagocytic monocytic system facilitates the spread of the bacteria into the host.
  • MALT mucosal associated lymphoid tissue
  • the first live attenuated vaccine developed against typhoid fever is the S. enteral Typhi Ty21a strain. This strain was obtained by chemical mutagenesis of the Ty2 parental lineage (Germanier and Furer, 1983). This oral vaccine is safe, but because of its great attenuation, it requires several doses to induce immunity, which is often short-lived (Guzman et al., 2006; Strugnell and Wijburg, 2006).
  • enteral AaroA, AaroC, AaroD strains deficient in the aromatic amino acid biosynthetic pathway (Tacket et al., 1997), Acya Accrp mutants, unable to express adenylate cyclase and the cAMP receptor (Curtiss and Kelly, 1987 ; Tacket et al., 1997), phoP-phoQ mutants (Hohmann et al., 1996) among others, were constructed and demonstrated to be immunogenic despite attenuation of virulence.
  • S. enterica an excellent carrier of antigens to cells of the immune system.
  • attenuated S. enteric strains can be genetically engineered to express heterologous antigens by constructing multifactorial vaccine strains. Different antigens derived from other bacteria, viruses, fungi, parasites and even ⁇ Mammalian Cells were expressed in vaccine strains of S. enterica.
  • strains were able to induce protective immune response not only against salmonellosis but also against the heterologous antigen donor organism (Cheminay and Hensel, 2007; Kwon et al., 2007; Loessner et al., 2007; Mahoney et al., 2007; Atkins et al., 2006).
  • An A fundamental feature of such strains is, besides being unlikely to reverse attenuation, the ability to express the heterologous antigen stably and in sufficient quantities to induce the immune system.
  • Bacterial nucleoid and target genes hupA and hupB
  • Bacteria contain proteins that make up the bacterial nucleoid ("nucleoid-associates proteins", Nap) together with chromosomal DNA. Unlike eukaryotic histones, bacterial proteins do not appear to form nucleosome-like complexes with DNA (Thanbichler et al., 2005). Within this group are 12 proteins and the most studied are HU, FIS, IHF, HNS and DPS (Dorman, 2009; Dorman and Kane, 2009; Cróin ⁇ n and Dorman et al, 2007; Dorman et al., 2006; Drlica and Rouviere-Yaniv, 1987).
  • HU is a heterodimeric protein encoded by two genes, namely hupA and hupB. This protein does not appear to recognize any specific DNA binding sequence, but has great affinity for supercoiled or distorted DNA structure regions (Pinson et al., 1999). Swinger et al. (2004) demonstrated that this protein is capable of inducing and stabilizing bends from different angles in DNA. Some studies suggest that HU participates in DNA repair by recombination (Swinger et al., 2004).
  • the Salmonella hupA gene was compared to the E.coli hupA gene by cloning and sequencing, which revealed that the HUa subunits of these bacteria are identical and that sequences outside the ORFs are highly conserved (Higgins & Hillyard, 1988).
  • hupB gene has been described as a negative modulator in hilA expression, which is a crucial gene in the expression of invasive Salmonella phenotype (Fahlen et al., 2000).
  • hupB has been described as a negative modulator in hilA expression, which is a crucial gene in the expression of invasive Salmonella phenotype (Fahlen et al., 2000).
  • hupB has a positive action on hilA regulation (Schechter et al., 2003).
  • Datsenko and Wanner described the construction and use of a bacteriophage ⁇ -based system for the construction of mutant strains of enteric bacteria.
  • This system is composed of accessory plasmids, the plasmid pkD3 being used in the construction of PCR recombination cassettes (Datsenko and Wanner. 2000).
  • said PI 0006291-0 relates only to attenuation promoted by deletion of another gene, that is, the recA gene.
  • 7,045,122 also concerns only the attenuation promoted by deletion of a gene, that is, the recA gene.
  • US Patent Document 2004/101531 describes vaccines and immunogenic compositions that use live attenuated pathogenic bacteria, such as Salmonella, to deliver ectopic antigens to the mucosal immune system of vertebrate animals. Attenuated pathogenic bacteria are prepared to secrete the antigen into the bacteria's periplasmic space or into the bacteria.
  • the use of live attenuated bacterial strains as a vaccine has historically been associated with the induction of effective and persistent immune response.
  • the vaccine vector consists of a bacterium that expresses antigens (proteins) from other species of microorganisms obtained by the recombinant DNA method. When presented to the host (target organism) immune system, these microorganisms stimulate the production of antibodies against the disease of interest without causing serious symptoms or damage to the host.
  • the present invention will provide significant advantages over the immunization processes based on gene silencing of HU, enabling an increase of its performance and presenting a more favorable cost / benefit ratio.
  • the present invention relates in a first aspect to the construction of S. enteral null mutants for the HU (Heat Unstable Protein) encoding genes (hupA and / or hupB) in order to develop attenuated but virulence lines. cause transient infection and effectively induce the immune system, constituting potential vaccine strains.
  • HU Heat Unstable Protein
  • An object of the present invention is an attenuated bacterium comprising at least one mutated HU coding gene in a pathogenic bacterium.
  • the present invention also relates to an attenuated bacterial-based vaccine comprising at least one mutant HU encoding gene in a pathogenic bacterium capable of inducing and stimulating an immune response. in the vertebrate host.
  • Figure 1 is a 1% agarose gel showing the PCR generated recombination cassette using the hupA-f and hupA-r primers;
  • Figures 2a and 2b are a 1% agarose gel showing the PCR amplification product of the cat gene present in ST662AhupA: cat and ST662AhupB: cat, with primers hupADTlf and hupADTlr and hupBDTf and hupBDTr, respectively;
  • Figure 3 is a 1% agarose gel demonstrating cat gene excision of the ST662AhupA: cat and ST662AhupB: cat strains for the construction of the double ST662AhupA ⁇ hupB mutant;
  • Figure 4 is a 1% agarose gel demonstrating PCR amplification products of the hupA and hupB gene regions, respectively;
  • Figure 5 is a schematic showing the deletion of the hupA and hupB genes and the position of the detection primers, represented as arrows.
  • Figure 1 is a 1% Agarose Gel with the PCR generated fragment corresponding to the hupA gene recombination cassette for the construction of the mutant strain. From left to right, samples refer to the Molecular Weight Marker 1 kb DNA ladder (Fermentas); negative control and the hupA gene recombination cassette, about 1200 bp, comprising the cat gene and flanking regions composed of FRT (FLP Recognition Targets) sites and regions adjacent to the target genes.
  • FRT FLP Recognition Targets
  • Figures 2a and 2b is a construction of the S. enteral mutants AhupA and AhupB. 1% agarose gel with PCR products using hupAD and hupBOT (detection primers) respectively to confirm mutagenesis.
  • the positive control corresponds to the wild line 662ST, with amplification of the intact gene and regions flanking machines, generating a fragment of 396 bp for hupA and 371 bp for hupB, while the mutants have a bandwidth of approximately 1.2 kb, equivalent to the cat gene and flanking regions.
  • MPM Molecular Weight Marker 1 kb DNA ladder (Fermentas); CN: Negative Control (without DNA); CP: Positive Control (wild line genomic DNA).
  • Figure 3 shows the elimination of the chloramphenicol resistance cassette in S. entérica mutants ⁇ and AhupB, demonstrating the "scars" of about 100 bp, corresponding also to the FRT sites.
  • the hupAO 1 primers were used for AhupA mutant strains and hupBOT primers for AhupB mutants.
  • MPM Molecular Weight Marker 1 kb DNA ladder (Fermentas); CN - Negative Control; CmR - cat.
  • Figure 4 is a 1% agarose gel demonstrating PCR products for hupA and hupB flanking regions, respectively. From left to right: Molecular Weight Marker 1 kb DNA ladder (Fermentas); Negative control; Positive control for hupA, consisting of genomic DNA from the wild line 662ST, with fragment referring to the intact hupA gene (important to note that in this case the hupADT2 primers were used, generating a 702 bp fragment for hupA); Lineage 662STA.hupA, with fragment amplified by hupAD 2 primers, referring to the cat gene and flanking regions; Mutant double line 662STAhupAAhupB, amplified by hupADT2 primers; Positive control, consisting of genomic DNA from the wild line 662ST, with fragment referring to the intact hupB gene; 662STAhupB strain, with fragment amplified by hupBOT primers, referring to the cat gene and flanking regions
  • FIG. 5 is a schematic diagram showing the construction steps of the AhupA and AhupB mutant strains and position of the hupAOT and hupBOT detection primers, represented as arrows. Initially the target gene is intact, shown in white. Subsequently, the target gene is replaced by the cat gene (chloramphenicol acetyl transferase), also shown in white. In the last stage, the chloramphenicol antibiotic resistance gene is eliminated, leaving only one "scar” composed of FRT (hatched region) sites.
  • cat gene chloramphenicol acetyl transferase
  • Pathogenic bacteria are bacteria capable of causing disease and / or death in their hosts.
  • pathogenic bacteria include, without limitation, gram negative bacteria, in particular members of the Enterobacteriaceae, Vibrionaceae, Francisellaceae,
  • Legionallales, Pseudomonadacea or Pasteurellaceae including the genera Salmonella spp., Shigella spp., Escherichia spp., Yersinia spp., And Vibrio spp.
  • pathogenic bacteria of the present invention are chosen from species having in their genome at least one HU encoding gene, preferably, but not exclusively, S. enterica serovar Typhimurium (662ST).
  • the HU coding gene of the present invention is a DNA sequence having at least 80% homology to at least one sequence chosen from hupA or hupB, which are chosen from species having at least one coding gene in their genome.
  • HU preferably, but not exclusively, S. enterica serovar Typhimurium (662ST).
  • Attenuated bacteria is understood to be a pathogenic bacterium having at least one mutated HU coding gene.
  • stressing is meant a process of deletion of a particular sequence of the genome of a bacterium. In particular, silencing was provided by a recombination cassette.
  • the recombination cassette according to the present invention is a cassette comprising at least one sequence capable of silencing at least one HU encoding gene in a pathogenic bacterium.
  • the recombination cassette consists of primers, which have 2 continuous parts, the first being a sequence of approximately 40 bases homologous to the target gene and the second approximately 20 bases homologous to a region present in the plasmids to be used.
  • the homology regions with the HU coding gene were selected from the sequence of the Genome S. enteral project database (NC_003197) (Washington University, St. Louis, USA) and chosen so that they were close to the initiation codons. and gene termination.
  • the recombination cassette further comprises accessory genes, such as antibiotic resistance genes.
  • the bacterial recombination process of the present invention is a process based on the recombination Red.
  • the bacteriophage ⁇ XRed recombination system includes 3 genes: ⁇ , ⁇ and exo (present in plasmid pKD46), which encode the proteins Gam, Bet and Exo respectively.
  • Accessory plasmids contain antibiotic resistance genes (Km R or Cm R ) flanked by FRP (FLP recombinase recognition targets) sites, forming the recombination module. Thus, these plasmids are used in the creation of the recombination cassette.
  • sequences of approximately 40 bp homologous to the target gene are generated at the ends of this module by PCR.
  • the recombination cassette is the module flanked by sequences homologous to the target gene.
  • plasmid p D46 is transformed by electroporation into the host lineage and transformants undergo electroporation with the recombination cassette.
  • the expression of the ⁇ , ⁇ and exo genes inhibits the degradation of the linear strand of DNA, allowing the recombination of the cassette.
  • This recombination follows DNA homology conferred by flanking sequences such that recombination will involve homologous sequences.
  • the recombinants are then selected using the resistance tags carried by the recombination module.
  • Such constructs allow subsequent removal of the resistance cassette by FLP recombinase expressed by a plasmid gene, in this case the plasmid pCP20.
  • FLP recombinase expressed by a plasmid gene, in this case the plasmid pCP20.
  • the transformation process of the present invention comprises the steps of:
  • the selected samples were stored in glycerol 2.5 M, according to the protocol described by Sambrook and Russell (2001) and incorporated herein by reference in its entirety.
  • the susceptibility of the selected strains was tested for the antibiotics ampicillin, kanamycin, chloramphenicol, streptomycin and tetracycline. For this, the microdilution method was used.
  • MIC Minimum Inhibitory Concentration
  • mutagenesis of the hupA and hupB genes was obtained by the ⁇ Red system as described by Datsenko and anner (2000) and incorporated herein in its entirety by reference. This system consists of the strains and plasmids shown in Table 1.
  • Table 1 Bacterial strains with their respective plasmids belonging to the ⁇ Red system.
  • BW25113 / pKD46 strain was seeded on LB agar medium containing ampicillin (100ug / ml); BW25141 / pKD3 and BT340 / pCP20 were seeded on LB agar medium containing ampicillin (100ug / mL) and chloramphenicol (25 ⁇ g / mL).
  • the incubation temperature used was 30 ° C, since such plasmids contain thermosensitive origin of replication. After extraction, they were subjected to 0.8% agarose gel electrophoresis according to the protocol of Sambrook and Russell (2001), incorporated herein by reference in its entirety.
  • the primers for this methodology are composed of 2 continuous parts, the first one being a sequence of approximately 40 bases homologous to the target gene and the second 20 bases homologous to a region present in plasmid pKD3.
  • the region used to amplify the sequence contained in the plasmid was selected using the Primer 3 program (http: // frodo. Wi .mit. Edu / cgi-bin / primer3 / primer3_www. Cgi), based on the plasmid sequence pKD3 (AY048742) (Datsenko and Wanner, 2001).
  • the hupA-f, hupA-r, hupB-f, and hupB-r primers, described in Table 2 were designed.
  • the primers described above were used for amplification of a region of plasmid pKD3. Reactions were performed in a final volume of 50 ⁇ l containing 20 pmol of each primer, 20 to 30 ng of plasmid DNA, 1 ⁇ M of each dNTP, 2U Taq DNA polymerase and 2 mM MgCl 2 in appropriate buffer provided with the enzyme.
  • plasmid DNA was denatured by heating at 94 ° C for 2 minutes, and amplification performed in 30 cycles consisting of the following steps: (1) denaturation at 94 ° C for 30 seconds; (2) "ringing" at 56 ° C for 30 seconds; (3) extension at 72 ° C for 1 minute and 30 seconds. A final extension was performed for 5 minutes.
  • the PCR product was analyzed by 1% agarose gel electrophoresis (Sambrook and Russell, 2001).
  • the hupA-f, hupA-r, hupB-f and hupB-r primers were used to amplify an approximately 1.2 Kbp region (Figure 1) of plasmid pKD3.
  • the amplified region was used to compose the recombination cassette with the hupA and hupB genes, respectively.
  • the PCR generated recombination cassette using the hupA-f and hupA-r primers is shown in Figure 1.
  • Double mutant AhupAAhupB sequenced with hupADT2f primer Double mutant AhupAAhupB sequenced with hupADT2f primer:
  • sequence is longer, because at the hupA gene site was inserted the cat gene and flanking plasmid regions ( ⁇ 1.2 kb).
  • sequence corresponding to the flanking region of the Salmonella hupA gene is highlighted, which is shortly thereafter interrupted by sequences present in plasmid pKD3, corresponding to PI and cat.
  • the strokes symbolize unidentified bases in sequencing.
  • Double mutant ⁇ hupAAhupB sequenced with hupADT2r primer (complementary and inverted sequence):
  • Plasmid alignment regions correspond to a region of the cat gene
  • Transformation of the selected strains with plasmid pKD46 was performed by electroporation following the protocol described in Ausubel et al. (2007), incorporated herein by reference in its entirety.
  • Electroporator Bio Rad. Gene Pulser Electroporator
  • the presence of the plasmid was evaluated by analysis of the plasmid profile by agarose gel electrophoresis.
  • the selected strains were stored in glycerol 2.5 M according to protocol proposed by Sambrook and Russell (2001).
  • Competent cells were prepared from a pre-inoculum with 5 ml ampicillin-containing LB medium (100 pg / ml), which was incubated overnight at 30 ° C. From this culture 0.5 ml were inoculated in 50 ml LB medium with the same antibiotic and 1mM L-arabinose (Sigma), used as inducer of ⁇ , ⁇ , and exo gene expression, and this new culture was grown at 30 °. C under agitation (150 rpm) until it reaches ⁇ . ⁇ of 0.7. The flasks were cooled in an ice bath for 15 minutes, the cultures were centrifuged for 10 minutes at 5000 g (4 ° C).
  • the precipitate was resuspended in 4 mL sterile deionized water previously cooled to 4 ° C and centrifuged again under the same conditions. This wash step was repeated three times using 10% glycerol and the pellet formed after the last wash was resuspended at 400 L and distributed in 90 ⁇ L aliquots by the protocol of Sambrook and Russell (2001) and incorporated herein by reference. in its entirety.
  • Three tubes were selected containing the aliquots to which 10 ⁇ L of the PCR product was added; These were placed on ice for 1 minute.
  • the electroporator has been set to 1.5 KV, 25 ⁇ iF and 200 ohms and The samples were electroporated and collected in SOC medium, where they were incubated at 37 ° C for 1 hour and then plated on LB-agar medium with chloramphenicol (25 g / mL). Cultures were incubated overnight at 37 ° C. From the grown colonies, some were selected for PCR confirmation, being named 662ST ⁇ hupACm R and 662STAhupBCm R. The selected colonies were stored in 2.5 M glycerol according to protocol proposed by Sambrook and Russell (2001), and incorporated herein by reference in their entirety. Then plasmid pKD46 was cured.
  • competent cells prepared from 662STAhupACm R and 662STAhupSCm R strains and plasmid pCP20 were used. Electroporation was performed as previously described and the transformants were selected on ampicillin LB-agar plates (100 ⁇ g / mL). The plates were incubated overnight at 30 ° C. Some of the grown colonies were selected for PCR confirmation and named 662STàhupA and 662STAhupB. These strains were stored in 2.5M glycerol as previously described by Sambrook and Russell (2001) and incorporated herein by reference in their entirety. Detection and characterization of hupA and hupB mutations by PCR
  • a pair of primers internal to this gene were designed based on the sequence of plasmid pKD3 (AY048742) (Datsenko and Wanner, 2001) and constituent of the recombination cassette. These were designed with the Primer 3 software (http: 111rodo. I .mit. Edu / cgi-bin / primer3 / primer3_www. Cgi). Primers are shown in Table 3.
  • Reactions were performed in 50 ⁇ l final volume containing 20 pmol of each primer, 20 to 30 ng of genomic DNA of each transformant, 1mM of each dNTP, 2U of Taq DNA polymerase and 2 mM of MgCl 2 in appropriate buffer provided with the enzyme.
  • genomic DNA was denatured by heating at 94 ° C for 2 minutes and amplification performed in 35 cycles consisting of the following steps: (1) denaturation at 94 ° C for 30 seconds; (2) "ringing" at 55 ° C for 30 seconds; (3) extension at 72 ° C for 1 minute. A final extension was performed for 5 minutes.
  • the PCR product was analyzed by 1% agarose gel electrophoresis using the protocol described by Sambrook and Russell (2001), incorporated herein by reference in its entirety.
  • the donor recombinant bacterium was grown in 3 ml LB without antibiotic overnight at 37 ° C. The next day, a 100 ⁇ 1 aliquot of phage was added to 900 ⁇ fresh LB, reaching a density of approximately 10 9 CFU / mL. This 1 ml aliquot was added to the cultured pre-inoculum with a bacterial density of approximately 10 9 CFU / mL.
  • the culture was then re-grown in the greenhouse at 37 ° C overnight. That same day, the 662ST wild strain was also grown in 3 ml of LB in the greenhouse at 37 ° C overnight. As a control, the bacteriophage was plated on LB Agar for bacterial contamination.
  • the culture with donor bacteria and phage was centrifuged at 5000 g for 10 minutes at 4 ° C to allow the bacteria to settle away from the phage.
  • the supernatant was carefully filtered and stored in a new container. As a control the supernatant was streaked on LB Agar to verify that there was no trace of bacteria.
  • phage and receptor wild bacteria were mixed, following the following proportions: 10 ⁇ phage + 100 ⁇ bacteria; 50 ⁇ phage + 100 ⁇ bacteria; 50 ⁇ of phage + 50 ⁇ of bacteria and 10 ⁇ of phage + 50 ⁇ of bacteria.
  • Containers containing phage and bacteria were incubated at 37 ° C for 20 minutes for adsorption. After this period, aliquots were plated on LB Agar with chloramphenicol for selection of transduced bacteria.
  • Recombinant strains were inoculated in 5 ml LB medium and incubated at 37 ° C overnight. The next day, 500 ⁇ ] _. from the pre-inoculum was added to 50 ml 1% LB glucose medium and the new culture incubated at 37 ° C under shaking (150 rpm), aliquots were taken every hour for five hours and serial dilutions were plated in triplicate over LB -agar for next day accounting.
  • 662ST, 662STAhupA, 662STAhupB and 662STbhupAàhupB strains were grown in LB medium at 37 ° C overnight. In day Next, 0.5 mL of these cultures were inoculated into 50 mL of 1% LB-glucose medium and incubated at 37 ° C with shaking (150rpm) until they reached the desired titer. Cells were pelleted by centrifugation (5000g for 5 min) and washed in the same volume of saline phosphate buffer (PBS, pH 7.4). This step was repeated a second time and the pellet resuspended in PBS (same initial volume).
  • PBS saline phosphate buffer
  • Serial dilutions were prepared in PBS and used to infect 6 to 8 week old female BALB / c mice. Dilutions were plated on LB-agar medium for CFU determination. The oral inoculum was performed using a model IC800 gavage needle (INSIGHT, Ribeir ⁇ o Preto-SP, Brazil).
  • the amount of inoculated bacteria varied according to each group, from 10 2 to 10 9 CFU / mL, using three mice per dilution. Animals were followed for 30 days after inoculum administration.
  • the ability to elicit immune response against S. enteric was tested at two times: In the first, the animals were immunized with a single dose of S. enteric and challenged 28 days after it.
  • CFU dose used was 10 8 for the double mutant strain, a dose established from previous trials. Animals were challenged with 10 7 CFU doses of ST662 strain.
  • mice were used.
  • BALB / c females 6 to 8 weeks old per group.
  • the animals were followed for 30 days after challenge and evaluated for their survival.
  • mice with single dose immunization all animals survived the challenge with the wild strain while the single immunization group showed an 85% rate after 30 days of experiment. Placebo animals inoculated with PBS died between days 5 and 10 after being challenged with wild lines. Some variations in the survival rate of mice with single dose immunization were observed between different experiments.
  • the present invention enables the use of a live vaccine vector for the development of vaccines against salmonellosis and other diseases having their own immunogenic properties, since the developed attenuated strains may be employed in the delivery of heterologous antigens to the immune system. of the host.
  • the present invention enables use as a live vaccine to induce protection against salmonellosis; potential use as a multifactorial vaccine vector, ie attenuated strains may express antigens from other diseases; beyond use in cancer therapy.
  • Enteric salmonella has been used as a biological agent in the treatment of some types of solid tumors. This is because this bacterium presents tropism for tumor masses. Treatment of different tumors with S. enteric has resulted in a decrease or even disappearance of the tumor mass. However, for this type of employment, lineage must be attenuated. Thus, the developed mutant strains of the present invention have the potential to be used in tumor therapy. However, specific studies are needed to verify this potential and to indicate other modifications in S. enterica strains that may be necessary.
  • the double mutant strain (662S AhupAAhupB) still has the added advantage of a lower probability of reversal of attenuation to pathogenicity levels of the wild strain, since two genes were knocked out. This advantage is not present in single mutated strains, as reacquisition of the lost gene could restore the total virulence of the strand.
  • the present invention has use as a live vaccine to induce protection against salmonellosis or as potential use as a multifactorial vaccine vector, i.e. attenuated may express antigens from other pathogens.
  • the present invention may be used in cancer therapy.
  • Salihonella enterica has been used as a biological agent in the treatment of some types of solid tumors. This is because this bacterium presents tropism for tumor masses. Treatment of different tumors with S. enteric has resulted in a decrease or even disappearance of the tumor mass. However, for this type of employment, lineage must be attenuated. Thus, the developed mutant strains of the present invention have the potential to be used in tumor therapy.
  • Salmonella from pathogenesis to therapeutics. J Bacteriol. 189: 1489-95.
  • Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infect Immun. 55: 3035-43.
  • IHF integration host factor
  • phoP / phoQ-deleted Salmonella typhi (Ty800) is a safe and immunogenic single-dose typhoid fever vaccine in volunteers. J Infect Dis. 173: 1408-14.
  • Salmonella. typhimurium initiates murine infection by penetrating and destroying ephitelial specialists M cells of the Peyer's patches. J. Exp. Med. 180: 15-23.

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Abstract

La présente invention concerne une nouvelle alternative pour la prévention de la salmonellose, à partir de mutants capables de promouvoir l'immunisation de l'hôte. Plus particulièrement, ces mutants sont nuls pour les gènes hupA et/ou hupB de HU et ont été testés en termes d'atténuation de virulence et de capacité à provoquer une réponse immunitaire efficace et protectrice contre la salmonellose, notamment la salmonellose murine, donnant ainsi lieu à des résultats prometteurs. Par ailleurs, la présente invention concerne un procédé de production de cette lignée atténuée, capable de promouvoir une immunisation, des vecteurs vaccinaux et des vaccins pour le traitement de la salmonellose, ainsi que leur utilisation pour lutter contre cette infection et éventuellement contre d'autres maladies, lorsqu'il s'agit de vecteurs multifactoriels.
PCT/BR2012/000059 2011-04-19 2012-03-07 Procédé de construction de lignée atténuée mutante d'une bactérie pathogène, vaccin, vecteur vaccinal et utilisation dudit vaccin WO2012142684A1 (fr)

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BR102014030638B1 (pt) * 2014-12-08 2022-12-06 Universidade Estadual De Campinas - Unicamp Uso da linhagem salmonella enterica typhimurium para preparar vacina contra salmonelose

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016113322A1 (fr) * 2015-01-14 2016-07-21 Centre National De La Recherche Scientifique Souches de vibrio cholerae génétiquement modifiées et utilisation de ces dernières

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