BRPI1102019A2 - construction process of mutant attenuated lineage of a pathogenic bacterium, vaccine, vaccine vector and use of said vaccine - Google Patents

Abstract

Invention Patent for: "Process of attenuated lineage building process during a pathogenic bacterium, vaccine, vaccine vector and use of said vaccine". The present invention discloses a novel alternative for the prevention of salmonellosis from mutants capable of promoting host immunization. In particular such mutants are null for the HU hupA and / or HuB genes and have been tested for attenuation of virulence and ability to elicit effective and protective immune response against salmonellosis, in particular murine salmonellosis, yielding promising results. Additionally, the present invention is directed to a process of producing this attenuated strain capable of promoting immunization to vaccine vectors and vaccines for the treatment of salmonellosis and their use in combating such infection and possibly other diseases in the case of vectors. multifactorial.

Description

Descriptive Report of the Invention Patent for: "PROCESS OF ATTENUALLY ATTENTED LINE BUILDING OF A PATHOGENIC BACTERIA, VACCINE, VACCINE VECTOR AND USE OF SUCH VACCINE" ·

Field of 工 ηνβηςβο

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. Specifically, such mutants are null for the HU hupA and hupB genes and have been tested for attenuation of virulence and ability to elicit effective and protective immune response against salmonellosis. Fundaments and Background of the Invention Salmonel 1 to

The genus Salmonella sp belongs to the Enterobacteriaceae family and is formed by gram-negative, facultative and generally flagellated anaerobes (Mastroeni and Maskell, 2006). The serological classification of these bacteria is based on the identification of somatic (O), flagellar (H) and capsular (Vi) antigens, the latter when present.

DNA hybridization studies suggest the existence of

two species of Salmonella ： S · enterica and S · bongori. The S · enteric species is subdivided into 7 subgroups, and the vast majority of pathogenic serovarieties for humans are included in the subgroup 工 （Boyd et al. 1996).

Pathogenic S · enterica 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 (Baumler et al., 1998). ). S. en teri ca Typhi is the causative agent of fever

Typhoid, a serious systemic infection in humans (Guzman et al., 2006) · Enteric Choleraesuis causes severe systemic infections, especially in pigs, although it can also cause infections in humans (Mastroeni and Maskell, 2006 ； Salyers and Whitt, 2002). Salmonella enterica Dublin is a serovariety associated mainly with bovines · Like S. enterica Thyphi in humans, Dublin's serovariety is invasive and can cause gastroenteritis and septicemia in these animals (Mastroeni and Maskell, 2006). S. enterica Typhimurium and Enteritidis are common causes of gastroenteritis in humans, but may also be associated with extra-intestinal infections (Mastroeni and Maskell, 2006). In mice, S. enterica Typhimurium and

Enteritidis cause a type of infection very similar to human typhoid. Thus, BALB / c mice are used as a murine model of systemic S · enter! Ca infection. Salmonellosis and disease Salmonellosis is one of the most common infectious diseases.

frequent both in humans and in other animals. S. enteric infection begins with ingestion of contaminated water or food, 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. enter! it persists and proliferates within endothelial reticulum system cell vaciioles and thus can reach different organs and tissues of the host, causing systemic infection (Salyer and Whitt, 2002). These infections still represent a serious public health problem due to their high incidence and severity, especially in underdeveloped areas of the world, where hygiene and basic sanitation conditions are still precarious (Woc-Colburn and Bobak7 2009 ； Boyle et al ., 2007; Coburn et al. 2007 ； Figueroa-Ochoa and Verdugo-Rodrigues ·

2005 ； Graham, 2002) In Brazil salmonellosis is a serious problem of public εβύάβ especially in poorer regions (Fernandes et al. 2006, Ghilardi et al., 2006).

The mean S. enteric infective dose (ID 50) capable of producing clinical or subclinical infections in humans is among 10 5 -10 10 organisms ingested. The infectious process is localized in the ileum, colon and mesenteric lymph nodes following ingestion of contaminated food, with the appearance of symptoms such as diarrhea, vomiting and abdominal pain (reviewed by Darwin and Miller, 1999). Most cases of gastroenteritis occur in children under 10 years of age, and symptoms tend to be more severe in this group and the infection may become systemic.

An important barrier encountered by S. enterica in the infectious process following its passage through the intestinal epithelium is 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. The serovarities of S · enter! Ca, capable of causing

systemic invasion, invade macrophages and then activate virulence mechanisms that allow evasion of phagocyte microbicidal fungi, allowing survival and

replication in the intracellular environment (Mastroeni and Maskell, 2006 ； Salyers and Whitt, 2002 ； Alpuche-Aranda, et al., 1994). Migration of infected macrophages to other organs of the phagocytic monocytic system facilitates the spread of bacteria in the host.

Salmonella and Vaccines

The search for an effective vaccine against typhoid fever is an important chapter in the history of microbiology. Several strategies and vaccine formulations have been tested such as bacterial, subunit vaccines, and live attenuated strains (reviewed by Bueno et al., 2009 ； Galen et al., 2009 ； Guzman et al., 2006). Purified, free or conjugated carrier protein Vi antigen is immunogenic when administered intramuscularly or subcutaneously and has been shown to be effective in some studies (Guzman et al., 2006 ； Strugnell and Wijburg, 2006). However, due to its characteristics and mode of administration, it is unable to induce mucosal level immune system (MALT). Thus, efforts were channeled in the development of vaccines consisting of live attenuated strains, ideal for inducing MALT immune response.

The first live attenuated vaccine developed against typhoid fever and composed of S. enterica Typhi strain

Ty21a. This strain was obtained by chemical mutagenesis of the Ty2 parent strain (Germanier and Furer, 1983). This oral vaccine is safe, but due to its great attenuation, it requires several doses to induce immunity, which is often short-lived (Guzman et al., 2006;

Strugnell and Wijburg, 2006).

Results achieved with the Ty21a strain led several groups to construct new S · enter! Ca vaccine strains, containing delegations in specific target genes that attenuated virulence. Additionally, these strains have a fundamental feature for live vaccines, attenuation stability, since it has been achieved by delegation of one or more genes (reviewed by Guzman et al., 2006). 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 ca Typhi strains, often Ty2 itself (reviewed by Guzman et al., 2006). . Thus, AaroAf AaroCf AaroD S · enterica strains, deficient in the biosynthetic pathway of aromatic amino acids (Tacket et al., 1997), Acya Acrpt mutants unable to express adenylate cyclase and cAMP receptor (Curtiss and Kelly, 1987 ； Tacket et al., 1997),

phoP-phoQ mutants (Hohmann et al., 1996) among others were constructed and demonstrated immunogenic despite attenuation of virulence. Frequently, however, tests on human volunteers have indicated the occurrence of bacteremia and / or the need for multiple doses to induce immunity (Guzman et al., 2006; Strugnell and Wijburg, 2006). In fact, several studies have shown the challenge of adjusting the degree of attenuation of the vaccine strain so as not to compromise the immune response and free of side effects. The ability to invade, survive and proliferate in the

macrophages, polymorphonuclear cells and dendritic cells, together with the availability of attenuated mutants make S. enterica an excellent carrier of antigens to cells of the immune system. Thus attenuated S. enterica 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 S. enterica vaccine strains. These strains were able to induce protective immune response not only against salmonellosis but also against the heterologous donor organism (Cheminay and Hensel, 2007 ； Kwon et al., 2007 ； Loessner et

al., 2007 (Mahoney et al., 2007 - Atkins et al., 2006). A fundamental feature of such strains is, in addition to the reversal of attenuation being very unlikely, the ability to express the heterologous antigen stably and in sufficient amounts to induce the immune system.

At first, cloning and expression of antigens on S ·

Enteric infections were achieved by the use of plasmids carrying antibiotic resistance genes (Cardenas and Clements, 1992). However, some studies have shown that in the absence of selective pressures, such plasmids were unstable and therefore lost after few replication cycles in vivo (Cardenas and Clements, 1992 ； Dunstan et al., 2003) or even during cultivation. in vitro (Everest et al., 1995).

To address such problems, strategies such as using in vivo induced promoters to control antigen expression (Cheminay and Hensel, 2007 ； Marshall et al., 2000), building balanced lethal systems (Curtiss et al., 1990 ； Everest et al ·, 1995) or the integration of the heterologous gene on the S · enter! ca chromosome (Hone et al., 1988; Strugnell et al., 1990 ； Everest et al., 1995) have been described ·

Bacterial nucleoside and target genes: hupA and hupB

Bacteria contain proteins that make up the nucleoid

(n xnucleoid-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 there are 12 proteins and the most studied are HU, FIS, IHF, HNS and DPS (Dorman, 2009 ； Dorman and Kane, 2009 ； Croinin 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 regions supercoiled or distorted in DNA structure (Pinson et al., 1999). Swinger et al. (2004) demonstrated that this protein is capable of inducing and stabilizing curvatures of different angles in DNA. Some studies suggest that HU participates in DNA repair by recombination (Swinger et al., 2004).

There is little data in the literature on the HU and Salmonella protein specifically (Mangan et al., 2011), although there are more reports between this protein and Escherichia coli. Nevertheless, the studies described focused on protein function demonstrating the regulation of pathogenicity factors, but did not investigate the possible

attenuation promoted by the deletion of the hupA or hupB genes and the induction of protection in the murine model.

The Salmonella hup A gene was compared to the E.coli hup A gene by cloning and sequencing, which revealed that the HUa subunits of these bacteria are identical and that the sequences outside the ORFs are highly conserved (Higgins Sc Hillyard, 1988).

These studies reveal that HU protein affects cellular processes such as DNA compacting, replication, recombination, gene regulation, as well as participating in thermosensitive growth, stationary growth phase adaptation, lysogenesis and transposition of the bacteriophage Mu e that is capable of non-specific binding. to DNA (Bi et al., 2009). In S. enter! Ca HU coordinates the expression of genes involved in central metabolism and virulence (Mangan et al., 2011).

The hupB gene has already 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). However, there are controversies about the hupB action, as further studies by another research group have shown that this gene has a positive action on hilA regulation (Schechter et al., 2003).

Berger et al. (2009) constructed a double E.coli AhupAAhupB mutant to verify changes in the gene structure promoted by the absence of the HU protein. From this study, it was possible to observe that the absence of HU causes a spatial rearrangement in the transcription pattern, causing a disturbance of topological homeostasis that is offset by greater accessibility of DNA sites by topoisomerase. The recombinant system λ Red

In enteric bacteria, recombinant procurement using 1inear DNA molecules is a highly rare event, due to the instability of the linear DNA molecule, which once inside the cell is rapidly degraded by nuclease activity of the RecBCD recombination system. To circumvent this difficulty, a system based on the use of bacteriophage λ recombination machinery (ο XRed system) has been proposed.

Datsenko and Wanner (2000) described the construction and use of a bacteriophage-based system for the construction of mutant enteric bacterial strains. This system is made up of accessory plasmids, the plasmid pkD3 being used in the construction of PCR recombinant cassettes (Datsenko and Wanner. 2000).

During the prior search, document US 2004/101531 claims the deletion of the Salmonella cya and crp genes. Although the strategy of the present invention was similar, delegation occurred in other genes, hup A and hupBf ο which corresponded to bacterial attenuation and consequent application of it as a vaccine vector. Therefore, it is noted that an immunization strategy based on genetic isolation HU was not described or even suggested by that document.

Brazilian patent application PI 0006291-0, filed December 27, 2000, on behalf of Intervet International BV, entitled ： Salmonella Genus, Vaccine for the Protection of Animals against Salmonellosis, Use of a Bacteria, and, Process for Preparing a Vaccine "refers to Salmonella bacteria for use as a vaccine. The invention also relates to vaccines based on said bacteria, which are useful for preventing microbial pathogenesis. In addition, said document says regarding the use of such bacteria or the manufacture of such vaccines.2 As can be seen, said PI document

0006291-0 concerns only the attenuation promoted by the delegation of another gene, that is, the recA gene.

U.S. Patent 7,045,122, filed December 15,

November 2001, in the name of AKZO NOBEL NV and entitled ： "Salmonella Vaccine" refers to live attenuated strains of Salmonella and represents a first attenuating mutation, which are not capable of producing functional RecA. The invention also reports that These bacteria are for use in vaccines.

As can be seen, ο said US document

7,045,122 also concerns only the attenuation promoted by the delegation of a gene, that is, the recA gene ·

Internacional International Patent Application WO / 2010/096888, filed on February 25, 2010, on behalf of the State University of Campinas 一 UNICAMP and entitled ： “Vaccines Comprising Attenuated Bloodlines f Process of Building Attenuated Bloodlines, Vaccine Vectors and their Use in the Salmonellosis Treatment "reveals a new alternative for the prevention of salmonellosis from mutants capable of promoting host immunization. In particular such mutants are null for the ΔHF himA and himD genes and have been tested for attenuation of virulence and capacity. trigger an effective and protective immune response against salmonellosis, in particular murine salmonellosis.

As can be seen in said WO / 2010/096888 attenuation is promoted by the deletion of the ihfA or ihfB genes.

US 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 antigen for bacterial periplasmic space or into bacteria.

As can be seen, in said US 2004/101531 the delegation of Salmonella cya and crp genes is described.

The use of live attenuated bacterial strains as a vaccine has historically been associated with effective and persistent immune response induction. The vaccine vector is comprised 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.

Although studies have shown immunization strategies based on gene silencing, in none of the cases has it been revealed and even a process has not been suggested.

allow for an immunization strategy based on genetic silencing of HU.

It is well known that different attenuating mutations confer different biological properties such as immunogenicity. Invention Summary To solve the above mentioned problems, the

The present invention will provide significant advantages over immunization processes based on genetic silencing of HU, enabling an increase in its performance and presenting a more favorable cost / benefit ratio.

The present invention relates in a first aspect to the construction of S. enter! Ca null mutants for the HU (Heat Unstable Protein) encoding genes (hupA and / or hupB) in order to develop attenuated virulence lines, but capable of causing transient infection and effectively inducing the immune system, constituting potential vaccine strains.

And an object of the present invention is an attenuated bacteria comprising at least one mutated HU encoding gene in a pathogenic bacteria.

The present invention also relates to an attenuated bacterial-based vaccine comprising at least one mutated HU gene encoding a bacterium.

pathogenic, capable of inducing and stimulating an immune response in the vertebrate host. Brief Description of Attachments

The structure and operation of the present invention, along with additional advantages thereof may be better understood by reference to the accompanying drawings and the following description:

Appendix I is a 1% agarose gel showing the PCR generated recombinant cassette using the hupA-f and hupA-r primers.

Annex 2a and 2b and a 1% agarose gel showing ο

PCR amplification product of the cat gene present in ST662AhupA: C3. t θ ST662AhupB ： Cd. t, with primers hupADTIf and hupADTIr and hupBDTf and hupBDTr, respectively ；

Annex 3 is a 1% agarose gel showing the cat gene excision of ST662AhupA ： cat and ST662AhupB ： cat for the construction of the double ST662Ah upAAh upB mutant ；

Annex 4 is a 1% agarose gel showing the PCR amplification products of the hupA and hupB gene regions, respectively ；

Annex 5 is a scheme demonstrating the delegation of the hupA and hupB genes and the position of the detection primers,

represented as arrows. Detailed Description of the Invention

Although the present invention may be susceptible to different embodiments, and shown in the drawings and the following detailed discussion, a preferred embodiment is the understanding that the present embodiment should be considered an exemplification of the principles of the invention and is not intended to limit the present invention to the present invention. which has been illustrated and described here.

Annex 1 is a 1% PCR-generated fragment of agarose gel corresponding to the hup A gene recombination cassette for the construction of the mutant strain. From left to right, the samples refer to the 1 kb DNA ladder Molecular Weight Marker (Fermentas) negativo negative control and about 1200 bp hupA gene recombinant cassette comprising ο cat gene and flanking regions composed of FRT (FLP Recognition Targets) sites and regions adjacent to the target genes.

Annexes 2a and 2b are a construct of the S. enterica mutants AhupA and AhupB. 1% agarose gel with PCR products using respectively hupADT and hupBDT primers (detection primers) to confirm mutagenesis. Positive control corresponds to lineage

662ST, with amplification of the entire gene and flanking regions, generating fragment of 396 bp for hup A and 371 bp for hupB, while the mutants have a band of approximately 1.2 kb, equivalent to the cat gene and flanking regions. MPM ： Molecular Weight Marker 1 kb DNA ladder (Fermentation) ； CN ： Negative Control * (no DNA) ； CP: Positive Control (genomic DNA from wild line).

Annex 3 shows the elimination of the chloramphenicol resistance cassette in S. entericaf mutants AhupA and AhupB demonstrating the "scars" of about 100 bp corresponding also to the FRT sites. The hupADTl primers were used for the AhupA and hupBDT mutant strains for the AhupB · MPM mutants. 1 kb DNA ladder molecular marker (Fermentas) ； CN - Negative Control ； CmR - cat. Appendix 4 is a 1% agarose gel showing the

PCR products for hupA and hupB flanking regions, respectively. From left to right ： Molecular weight marker 1 kb DNA ladder (Fermentation) negativo Negative control positivo Positive control for hupA, composed of 662ST wild-type genomic DNA, with fragment referring to the integral hupA gene (important to note that in this case the primers hupADT2 were used, generating

702 bp fragment for hupA) ； 662STAhupA strain, with fragment amplified by hupADT2 primers for cat gene and flanking regions duplo Double mutant strain 6 6 2 S TAh upAAhupB, amplified by hupADT2 primers ； Positive control, composed of genomic DNA from strain wild 662ST, with fragment referring to the hupB integer gene ； 662STAhupBf strain with fragment amplified by hupBOT primers, referring to the cat gene and flanking regions ； Double mutant strain 6 6 2 SΤΔΛupAAhupB, amplified by hupBOT primers. Annex 5 is a demonstrative outline) of the steps of

construction of the AhupA and AhupB mutant strains and position of the hupADT and hupBOT detection primers are shown as arrows. Initially the target gene is whole, shown in white. Subsequently, the target gene is replaced by the cat gene (chloramphenicol acetyl transferase), also shown in blank traces. In the last stage, the chloramphenicol antibiotic resistance gene is deleted, leaving only one "scar" composed of FRT (hatched region) sites.

The pathogenic bacteria according to the present

Invention are bacteria capable of causing disease and / or death in their hosts. Examples of pathogenic bacteria include, without limitation, gram negative bacteria, in particular members of the Enterobacteriaceae, Vibrionaceae, Franci sellaceae,

Legionallales, Pseudomonadacea or Pasteurellaceae, including the genera Salmonella spp., Shigella spp., Escherichia spp., Yersinia spp., And Vibrio spp.

In particular, pathogenic bacteria of the present invention are chosen from species having in their genome at least one HU coding gene, preferably, but not exclusively, S. enterica serovar Typhimurium (662ST). Gene HU Encoder

The HU coding gene of the present invention is a DNA sequence that shows at least 80% homology to at least one sequence chosen from hup A or hupB, which are chosen from at least one genome species. HU coding gene, preferably, but not exclusively, S. enterica serovar Typhimurium (662ST). Attenuated Bacteria

For the purposes of the present invention, attenuated bacteria is understood to be a pathogenic bacterium having at least

least one mutated HU coding gene. By "Silencing" is meant a process of delegating a particular genome sequence of a bacterium. In particular, silencing has been provided by a recombinant cassette.

Recombination Cassette

The recombinant 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 recombinant cassette is composed 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.

Regions of homology with the HU encoding gene

They were selected from the sequence of the Genome S. enterica project database (NC-003197) (Washington University, St. Louis, USA) and chosen so that they were close to the gene initiation and termination codons.

The recombinant cassette further comprises genes

accessories such as antibiotic resistance genes. Transformation and Recombination Process

The process of recombinating the bacteria of the present

Invention is a process that is based on the XRed recombination system. The bacteriophage λ XRed recombinant system includes 3 genes: γ, β and exo (present in plasmid pKD46), which encode the Gam, Bet and Exo proteins respectively.

Gam inhibits the host ExoV system, while Bet and

Exo promote the recombination of linear fragments into the target DNA.

Accessory plasmids contain antibiotic resistance genes (KmR or CmR) flanked by FRT (FLP recombinase recognition targets) sites, forming the recombinant module. Thus, these plasmids are used in the creation of the recombinant cassette.

For generation of the recombinant cassette, approximately 40 bp sequences homologated to the target gene are generated at the ends of that module by PCR. Thus, the recombinant cassette and module is flanked by sequences homologous to the target gene.

For generation of mutants, plasmid ρKD46 is transformed by electroporation into the host lineage and the transformants undergo electroporation with the recombinant cassette. Expression of the γ, β and exo genes inhibits the degradation of the linear strand of DNA, allowing recombination.

from the cassette. This recombination follows the DNA homology conferred by the flanking sequences, such that the recombination will involve homologous sequences.

The recombinants are then selected using the resistance tags carried by the recombinant module. These constructs allow subsequent removal of the resistance cassette by FLP recombinase expressed by a plasmid gene, in the case of plasmid pCP20. In this way, it is possible to construct gene deletions or insertions in enteric bacteria using DNA fragments.

generated by PCR.

In particular, the process of transformation of the present invention comprises the steps of:

a) expression of bacteriophage λ Gam, Bet and Exo proteins present in plasmid pKD46, promoting the

recombination of linear fragments into the target DNA ； and

b) allelic exchange of the HU encoding gene by the recombinant cassette comprising an accessory gene,

c) deletion of the accessory gene present in the recombinant cassette.

Selected bacterial lines for mutagenesis

For this study we selected a virulent S. enteri ca wild strain of the Typhimuirium serovarity (662ST).

The selected samples were stored in 2.5 ol glycerol, according to the protocol described by Sambrook and Russell (2001) and incorporated here by reference, in their entirety.

Sensitivity test of S · enterica Typhimurium lines to antibiotics

The susceptibility of the selected strains was tested for ampicillin, kanamycin, chloramphenicol, streptomycin and tetracycline axitibiotics. For this, the microdilution method was used. In this test it was possible to observe a Concentration

Minimum nibitometry (MIC) of less than 10yg / ml for ampicillin, chloramphenicol, streptomycin and tetracycline antibiotics and MIC in a range of 10-25 pg / ml for kanamycin. This test indicated that ampicillin and chloramphenicol resistance-labeled plasmids can be used.

System used for mutagenesis

Mutagenesis of the hup A and hup B genes was obtained by the λ Red system as described by Datsenko and Wanner (2000) and incorporated herein in its entirety by reference. This system is made up of the strains and plasmids shown in Table 1.

Table 1 - Bacterial strains with their respective plasmids belonging to the λ Red system.

Plasmid Bloodline Reference BT 340 pCP2 0 Datsenko and Wanner, 2000 BW25141 pKD3 Datsenko and Wanner, 2000 BW25141 pKD4 Datsenko and Wanner, 2000 BW25113 pKD46 Datsenko and Wanner, 2000

Plasmid Extraction and Purification

From the stock at -80 ° C a sample was taken from each strain containing the plasmids. BW25113 / pKD46 strain was seeded on ampicillin-containing LB agar medium (10 µg / ml); BW25141 / pKD3 and BT340 / pCP20 were seeded on ampicillin-containing LB agar (100pg / ml) and chloramphenicol (25yg / ml) .

For extraction and purification of plasmids pKD46 and pCP20, the incubation temperature used was 302 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. Primers used to generate the recombination cassettes

Primers for this methodology are composed of 2 continuous parts, the first being a sequence of approximately 40 bases homologous to the target gene and the second 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 pKD3 sequence (AY048742) (Datsenko and Wanner, 2001). Thus, the primers hupA-f, hupA-r, hupB-f and hupB-r, described in Table 2, were designed.

Table 2. Primers used in the construction of the recombination cassette

Primer Sequence (5'-3 ') hupA-f (SEQ ID 1) TAGCAAGCGATAAACACATTGTAAGGATAACTTATGAACAAGGTGT AGGCTGGAGCTGCTTC hupA-r (SEQ ID 2) TTCGATAAAACTGTTCACAGTTATGCGTCTTACTTAACTGCCATAT GAATATCCTCCTTAGTTC hupB-f (SEQ ID 3) GGTGCGATATAAATTATAAAGAGGAAGAGAAGAGTGAATAAAGTGT AGGCTGGAGCTGCTTC hupB-r (SEQ ID 4) CTTTGTCACATCCCCCGAGGGGATCACGCTTAGTTTACCGCCATAT GAATATCCTCCTTAGTTC

PCR Recombination Cassette Construction

For the construction of the recombination cassette the primers described above were used for amplification of a region of plasmid pKD3. Reactions were performed in a final volume of 50 μΐ; containing 20 pmol of each primer, 20 to 30 ng of plasmid DNA, ImM of each dNTP, 2U of Taq DNA polymerase and 2 mM of MgCl2 in appropriate buffer provided with the enzyme.

For the PCR reaction, plasmid DNA was denatured by heating at 942 C for 2 minutes, and amplification performed in 30 cycles consisting of the following steps: (1) denaturation at 94s C for 30 seconds; (2) "ringing" at 562 ° C for 30 seconds; (3) extension at 722 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).

Thus, the hupA-f, hupA-r, hupB-f and hupB-r primers were used to amplify an approximately 1.2 Kbp region (Annex 1) of plasmid pKD3. The amplified region was used to compose the recombination cassette with the hupA and hupB genes, respectively. For illustrative purposes, the PCR generated recombination cassette using the hupA-f and hupA-r primers is shown in Annex 1.

Nucleotide sequencing

2 0 0 nucleotide sequencing was performed

according to standard protocols. The results are presented below.

Overlay sequencing double mutant primers AhupAAhupB with hupBDT "forward" and "reverse": TCGTACTTCGAAGGATTCAGGTGCGATATAAATTATAAAGAGGAAGAGAAGAGTGAATA AAGTGTAGGCTGGAGCTGCTTGGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGA ATAGGAACTAAGGAGGATATTCATATGGCGGTAAACTAAGCGTGATCCCCTCGGGGGAT GTGACAAAGTACAAGGGCGCATCAAC (SEQ ID No. 1) The overlap of bases sequenced from

hupBDT f and r primers (underlined) match the flanking regions of the hupB gene, no longer existing in the double recombinant AhupAAhupBl strain as confirmed by the presence of a pKD3 plasmid scar in place of that gene (Box gray).

Double mutant AhupAAhupB sequenced with hupADT2 f primer:

GGCTCAGGGCAGACCTGCGCGAGGCTGGCGAGAGCA-TATCGGTATAAATTTTCAGCAA TGACACCAGAAAACGTGATTTACGTCTGATTTGTCGTGCCATAAGGCTTCCCTTATGCC CCCCGTCTGGTCTACATTTGGGAGGC-AAAAAAAGTGGCTATCGGTGCGTGTATGCAGG AGAGTGCTTTTCTGGCATTTCC-TCGCACTC-ATGCTTAGCAAGCGATAAACACATTGT AAGGATAACTTATGAACAAGGTGTAGGCT6GAGCTGCTTCGAAGTTCCTATACTTTCTA GAGAATAGGAACTTCGGAATAGGAACTTCATTTAAATGGCGCGCCTTACGCCCCGCCCT GCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCA 2 0 CAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATA ATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAAT CAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAAC CCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTG TAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTT 2-5 GCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCT TTCATTGCCATACGTAATTCCGGATGAGCATTCATCAGGCGGGCAGATG GAATAA GC-GC-ATAAACTTGTGCTTATTTTTCTTTACGTCTTAAAAGGCGTATATCA (SEQ ID No. 2) In this case the sequence is greater as the local of the hupA gene was inserted the c gene at and flanking plasmid regions (-1.2 kb). In the gray box, the sequence corresponding to the flanking region of the Salmonella hupA gene is highlighted, which is soon interrupted by sequences present in plasmid pKD3, corresponding to Pl and the cat gene. The strokes symbolize unidentified bases in sequencing.

Double AhupAAhupB mutant sequenced with hupADT2r primer (complementary and inverted sequence):

CAAACCCTTTAGG-AAATAGGCCAGGTTT-CACCGTAACACG - ACATCTTGCGAATAT ATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTC AGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCAC CGTCTTTCATTGCCATACGTAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGA ATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAAT ATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAAT GTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCC ATTTTAGCTTCCTTAGCTCCTGAAAATCTCGACAACTCAAAAAATACGCCCGGTAGTGA TCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTC 2 0 GCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCT GCGAAGTGATCTTCTGTC-CAGGT AGGCGCGCCGAAGTTCCTAT AC (SEQ ID NO 3).

In this sequencing, only bases referring to plasmid pKD3 were detected, since the beginning and the end of the sequence presented poor quality in the electropherogram and were therefore disregarded. Plasmid alignment regions correspond to a region of the ca t gene (nucleotides 263 to 993 of the plasmid).

Transformation of S. enterica Typhimurium strains with plasmid pKD46

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.

The electroporator (Bio Rad Gene Pulser II Electroporator, Hercules - CA, USA) was adjusted to 1.5 KV, 25 pF and 200 ohms and 0.1 cm cuvettes were used. The samples were electroporated then incubated for 1 hour in SOC medium. Subsequently the cultures were plated on ampicillin-supplied LB agar medium (100 µg / ml) and incubated at 30 ° C overnight.

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). Allelic exchange of hupA and hupB genes by the recombination cassette

For the gene recombination step the 662ST / pKD46 strain was used as described above and the PCR generated recombination cassette.

Competent cells were prepared from a pre-inoculum with 5 ml ampicillin-provided LB medium (100 µg / 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 ImM L-arabinose (Sigma), used to induce expression of γ, β, and exo genes, and this new culture was grown at 30 sC under stirring (150 rpm) until reaching 0.160 D of 0.7. The flasks were cooled in an ice bath for 15 minutes and the cultures were centrifuged for 10 minutes at 5000 g (4e C). The precipitate was resuspended in 4 mL sterile deionized water previously cooled to 4 sC 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 pL and distributed into 90 pL aliquots by the Sambrook and Russell protocol (2,001) and incorporated herein by reference. , in its entirety.

Three tubes containing the aliquots were selected to which 10 pL of the PCR product was added; These were placed on ice for 1 minute. The electroporator was adjusted to 1.5 KV, 25 yF and 200 ohms and the samples electroporated and collected in SOC medium, where they were incubated at 37 ° C for 1 hour and then plated on chloramphenicol LB-agar medium (25ug / mL). Cultures were incubated overnight at 37 ° C. From the grown colonies, some were selected for confirmation by PCR, being named 662STAhupACmR and 662STÁhupBCmR. The selected colonies were stored in 2.5 M glycerol according to the protocol proposed by Sambrook and Russell (2001), and incorporated herein by reference in their entirety. Plasmid pKD46 was then cured.

Elimination of antibiotic resistance cat gene

For elimination of the antibiotic resistance gene, competent cells prepared from 662STAhupACmR and 662STAhupBCmR strains and plasmid pCP20 were used. Electroporation was performed as previously described and transformants were selected on ampicillin LB-agar plates (100 pg / mL). The plates were incubated overnight at 30 ° C. Some of the grown colonies were selected for PCR confirmation and named 662STàhupA and 662STkhupB. 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

Cat gene detection

The cat gene (chloramphenicol acetyl transferase) in S. enterica mutants was initially detected 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://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primers are shown in Table 3.

Table 3 - Sequence of primers used for cat gene detection

Primer String Reference cmDT - f (SEQ ID 5) 5'-GAACTTCGGAATAGGAACTTCA-3 'Mendes et al., Unpublished cmDT - r (SEQ ID 6) 5'-TGTGACGGAAGATCACTTCG-3' Mendes et al., Unpublished

* These primers have already been published.

Reactions were performed in a final volume of 50 pL

containing 20 pmol of each primer, 20 to 30 ng of genomic DNA of each transformant, ImM of each dNTP, 2U of Taq DNA polymerase and 2 mM of MgCl2 in appropriate buffer provided with the enzyme. For the PCR reaction, 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 BC 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.

Detection of mutations by primers external to hupA and hupB genes

Two primer pairs external to the hupA genes and one hupB pair were constructed for mutagenesis detection (Table 4). These were designed with the aid of the Primer 3 software (http://frodo.wi.mit.edu/cgi- bin / primer3 / primer3_www.cgi), based on the sequence of S. enterica Typhimurium LT2 (NC_003197).

Table 4 - Sequence of primers external to 20 hupA and hupB genes

Primers Sequence (5'-3 ') hupADTl-f (SEQ ID 4) AGTGCTTTTCTGGCATTTCC hupADTl-r (SEQ ID 5) ACAGACAAAAGGGG TGATG ■ hupADT2-f (SEQ ID 6) CGACTGCGAAGAACGTGATAGAGATAGTTAATAGTGATAGAGATAGTGATAGAGATAGTTAATAGTAGAG f (SEQ ID 8) TCGTACTTCGAAGGATTCAGG hupEDT-r (SEQ ID 9) GTTGATGCGCCCTTGTACTT

P22 bacteriophage transduction

Selecting electroporation of S. enterica recombinants often selects strains containing incomplete LPS (lipopolysaccharides) as they are more easily transformable but are more sensitive to the immune system and often unable to establish a systemic infection in mice.

To perform the transduction the donor recombinant bacterium was grown in 3 ml LB without antibiotic overnight at 37 ° C. The next day, a 100 μL aliquot of phage was added to 900 μL fresh LB, reaching a density of approximately 109 CFU / mL. This 1 ml aliquot was added to the cultured pre-inoculum with a bacterial density of approximately 109 CFU / mL.

The culture was then again grown in the greenhouse at 37 ° C overnight. That same day, the 662ST wild strain was also grown in 3 ml LB in the greenhouse at 37 ° C overnight. As a control, the bacteriophage was plated on LB Agar for bacterial contamination.

On the third day, 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. To insulate phage and bacteria isolation, 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. Aliquots of phage and receptor wild bacteria

were mixed in the following proportions: 10 μΐ phage + 100 μΐ bacteria; 50 μΐ phage + 100 μΐ bacteria; 50 μΐ of phage + 50 μΐ of bacteria and 10 μΐ of phage + 50 μΐ 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. The following day, the grown colonies were picked for SS, MacConkey with chloramphenicol selective media and again for LB with chloramphenicol and new confirmatory PCR with Cmdt and hupAat or hupBat primers was performed.

Characterization of AhupA, AhupB and AhupAAhupB mutants from S. enterica

In vitro growth curve

Recombinant strains were inoculated in 5 ml LB medium and incubated at 37 ° C overnight. The next day, 500 μL of the pre-inoculum was added to 50 ml of 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 made. in triplicate on LB-agar for next day accounting.

The comparison of the curves displayed by the strains showed some differences in growth. Oral Lethal Dose Determination (LD50) in Mice

For oral LD50 determination, 662ST, 662STAhupA, 662STAhupB and 662STAhupAAhupB strains were grown in LB medium at 37 ° C overnight. The next day, 0.5 mL of these cultures were inoculated into 50 mL of 1% LB-glucose medium and incubated at 37 sC 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). 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 made using an IC800 model needle (INSIGHT, Ribeirão 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. The animals were followed for 30 days after inoculum administration.

Oral LD50 values in BALB / c mice were calculated by the Moving Average Interpolation method (Welkos and O'Brien, 1994). 2 0 Table 5 - LD50 of S. enterica strains orally inoculated in BALB / c mice

DL50 strain (CFU.mL-1) Reference 662ST 1,5 χ IO4 Present invention 662STAhupA 3,2 χ IO7 Present invention 662STAhupB 3,5 χ IOb Present invention 662STAhupAAhupB> IO8 Present invention

It is important to note that from experiment to experiment there is some variation in the LD50 values, as this is an experiment involving several biological variables.

Oral Challenge

The ability to elicit immune response against S. enterica was tested at two times: In the first, the animals were immunized with a single dose of S. enterica and challenged 28 days thereafter. In a second experiment, animals were immunized

orally with two doses of the double mutant strain on days 0 and 14 and challenged with the wild strain on day 30 after the second dose. Subsequent challenge experiments continued for the double mutant strain, as it yielded the most promising results.

The dose (CFU) used was 108 for the double mutant strain, a dose established from previous trials. The animals were challenged with 10 7 CFU doses of ST662 strain. For these experiments 7 female BALB / c mice 6 to 8 weeks old per group were used. The animals were followed for 30 days after challenge and evaluated for survival.

In the 662STAhupAAhupB double immunizations 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 52 and 10 days after being challenged with wild lines.

Some variations in the survival rate of mice with single dose immunization were observed between different experiments.

As can be seen, the present invention enables the use of a live vaccine vector for

development of vaccines against salmonellosis and other diseases, with their own immunogenic properties, as the attenuated strains developed may be employed in the delivery of heterologous antigens to the host immune system.

Additionally, the present invention enables the

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; in addition to using BALB / c 6 to 8 week old females per group. The animals were followed for 30 days after challenge and evaluated for their survival.

In the 662STÁhupAAhupB double immunizations 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.

As can be seen, the present invention enables the use of a live vaccine vector for

development of vaccines against salmonellosis and other diseases, with their own immunogenic properties, as the attenuated strains developed may be employed in the delivery of heterologous antigens to the host immune system.

Additionally, the present invention enables the

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; in addition to use in cancer therapy.

Salmonella 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. enterica 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 lineage (662STAhupAAhupB) still

It has the additional advantage of 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 20 strains, as reacquisition of the lost gene could restore the total virulence of the strain.

The present invention has use as a live vaccine to induce protection against salmonellosis or as potential use as a multifactorial vaccine vector, that is, attenuated strains may express antigens from other pathogens.

Additionally the present invention may be used in cancer therapy. Salmonella 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. enterica 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.

Thus, while only a few embodiments of the present invention have been shown, it will be appreciated that various omissions, substitutions and alterations in the process of constructing the attenuated Salmonella enterica lineage may be made by one of ordinary skill in the art without departing from the spirit and scope of the invention. present invention.

It is expressly provided that all combinations of elements that perform the same function in substantially the same way to achieve the same results are within the scope of the invention. Substitutions of elements from one described embodiment to another are also fully intended and contemplated. One must also understand that the drawings are not necessarily to scale, but that they are only of a conceptual nature. The intent is therefore to be limited as indicated by the scope of the appended claims. References

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Claims (39)

1. Process of constructing mutated attenuated lineage of a pathogenic bacterium characterized by the fact that they comprise the following steps: a) expression of the bacteriophage λ Gam, Bet and Exo proteins present in plasmid pKD46, promoting the recombination of linear fragments into the target DNA ; b) allelic exchange of the HU encoding gene by the recombination cassette comprising an accessory gene; and c) deletion of the accessory gene present in the recombination cassette. Process according to Claim 1, characterized in that the pathogenic bacterium is selected from the group consisting of Enterobacteriaceae, Vibrionaceae, Francisellaceae, Legionallales, Pseudomonadacea or Pasteurellaceae, including the genera Salmonella spp., Shigella spp., Eseheriehia spp. ., Yersinia spp., And Vibrio spp. Process according to claim 2, characterized in that the pathogenic bacterium is still chosen from Salmonella spp., Shigella spp., Eseheriehia spp., Yersinia spp., And Vibrio spp. 4.. Process according to Claim 3, characterized in that the pathogenic bacterium is preferably S. enterica serovar Typhimurium (ST662). A process according to claim 1, characterized in that the HU coding gene is a DNA sequence that has at least 80% homology with at least one sequence chosen from hupA or hupB. Process according to claim 1, characterized in that the recombination cassette comprises at least one sequence capable of silencing at least one HU coding gene in a pathogenic bacterium. Process according to Claim 6, characterized in that the recombination cassette is composed of primers. Process according to claim 7, characterized in that said primers have 2 (two) 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 on the plasmids to be used. Process according to claim 8, characterized in that the regions of homology to the HU coding gene were selected from the sequence of the Genome S. enterica project database (NC_003197) and chosen so that were close to the gene initiation and termination codons. Process according to claim 1, characterized in that for the generation of the recombination cassette, sequences of approximately 40 bp homologous to the target gene are generated at the ends of that module by PCR. Process according to Claim 1, characterized in that the recombination cassette is the module flanked by sequences homologous to the target gene. Process according to claim 1, characterized in that said accessory genes may be antibiotic resistance genes. Process according to Claim 1, characterized in that the antibiotics are selected from the groups comprised of ampicillin, kanamycin, chloramphenicol, streptomycin and tetracycline. Process according to Claim 1, characterized in that for mutant generation, plasmid pKD46 is transformed by electroporation into the host lineage and the transformants are electroporation with the recombination cassette. Process according to claim 1, characterized in that the expression of the γ, β and exo genes inhibits the degradation of the linear strand of DNA, allowing the recombination of the cassette by homologous recombination. Process according to claim 1, characterized in that the recombinants are selected using the resistance marks carried by the recombination module. Process according to Claim 16, characterized in that the resistance cassette is removed by an enzyme. Process according to claim 17, characterized in that said enzyme is preferably FLP recombinase. Process according to claim 17 or 18, characterized in that said recombinase is expressed by a plasmid gene. Process according to claim 1, characterized in that the transformation of the selected strains with the plasmid was performed by electroporation. Process according to claim 1, characterized in that it generates a double mutant lineage. Process according to claim 21, characterized in that said double mutant lineage is comprised of 662STAhupAAhupB. Process according to Claims 1 to 21, characterized in that it still has a lower probability of total reversal of attenuation. Process according to claim 1, characterized in that the primer is composed of a sequence homologous to an HU coding gene. 25. Vaccine vector characterized in that it has at least one knockout HU coding gene and is capable of stimulating an immune response against infection by a pathogenic bacterium. Vaccine vector according to claim 26, characterized in that the immune response is humoral and / or cellular. Vaccine vector according to claim 25, characterized in that it carries antigens consisting of multifactorial vaccine strains. Vaccine as defined in claims 1 to 27, characterized in that it comprises a mutated attenuated strain of a pathogenic bacterium, where the attenuation corresponds to the deletion of one or both of the HU coding genes. Vaccine according to claim 28, characterized in that the pathogenic bacterium is selected from the group comprising Enterobacteriaceae, Vibrionaceae, Francisellaceae, Legionallales, Pseudomonadaeea, Pasteurellaeeae and combinations thereof. Vaccine according to claims 28 and 29, characterized in that the pathogenic bacterium is selected from Salmonella spp., Shigella spp., Eseheriehia spp., Yersinia spp., Vibrio spp and combinations thereof. Vaccine according to claim 30, characterized in that the pathogenic bacterium is preferably S. enteriea, serovar Typhimurium (662ST). Vaccine according to claim 28, characterized in that the HU coding gene is a genomic sequence which has at least 80% homology with at least one sequence chosen from hupA or hupB. Use of the vaccine as defined by claims 28 to 32 characterized in that it is to induce protection against bacterial diseases. Use according to claim 33, characterized in that said bacterial disease is preferably salmonellosis. Use of the vaccine as defined by claims 28 to 32, characterized in that it is a potential use as a multifactorial vaccine vector expressing antigens from other diseases. A method according to claim 1, characterized in that the double mutant AhupAAhupB with the hupBDT primers comprises SEQ ID No. 1. A process according to claim 1, characterized in that the hupADT2f primer-sequenced double mutant AhupAAhupB comprises SEQ ID No. 2. Process according to claim 1, characterized in that the double mutant AhupAAhupB sequenced with the hupADT2r primer comprises SEQ ID No. 3. A method according to claim 1, characterized in that the sequence of primers external to the hupA and hupB genes comprises SEQ ID Nos. 4, 5, 6, 7, 8 and 9.