Obtaining a Salmonella Enteritidis rugosae type and its genetic modifications for use as an avian vaccine.
Patent Information
- Application Number
- BR112019018882
- Authority / Receiving Office
- BR · BR
- Patent Type
- Patents
- Current Assignee / Owner
- Publication Date
- 2026-09-08
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Abstract
Description
1 / 24 Obtaining a rough-type Salmonella Enteritidis and its genetic modifications for use as an avian vaccine. TECHNICAL SECTOR
[0001] The present invention falls within the field of Animal Health. More specifically, the invention relates to the development of an attenuated, rough-phenotype strain of Salmonella enterica serovar Enteritidis (Salmonella Enteritidis or SE), obtained by deleting a gene involved in lipopolysaccharide synthesis. In this context, the use of this strain as a vaccine vector against the Ava-I virus is also included, through a process of insertion and integration into its chromosome of the genes encoding the Ava-I fiber antigen. The SE vector used was genetically modified to function as a vehicle for the expression of the immunodominant genes of the AvA-I virus fiber, with the ultimate goal of stimulating an effective and long-lasting immune response against HCI (inclusion body hepatitis) in poultry.The resulting strain is completely safe because it is free of the twelve genes that encode proteins of the secondary dicyclic GMP messenger signaling pathway, the RpoS sigma factor, and the WaaL protein. Therefore, this strain is proposed as a novel effective and safe recombinant live vaccine against Ava-I virus, suitable for vaccinating avian populations.
[0002] All components of this vaccine were specifically designed and developed with the three priority factors of efficacy, safety, and cost in mind. BACKGROUND OF THE INVENTION
[0003] A 30% growth is expected in the poultry sector over the next 8 to 10 years (APA, 2008), this growth goes hand in hand with successful health programs, which in turn depend on Petition 870250049938, dated 06 / 13 / 2025, page 8 / 100 2 / 24 effective and safe vaccines. HCI / SHP is a disease that causes up to 50% mortality in affected birds and is a latent problem in Peru because, despite vaccination, outbreaks still occur, a problem that is not solved because the technology used to develop vaccines has remained the same for 20 years.
[0004] Recently, genetic manipulation techniques, coupled with the availability of bacterial genomes and a better understanding of pathogenesis mechanisms and the immune response, have enabled new approaches to the development of attenuated bacterial strains as vectors for heterologous genes. Thus, enteropathogenic bacteria such as Monocytogenes, Salmonella spp, Shigella spp, Vibrio cholerae, Yersinia enterocolitica, or Bordetella pertussis have been used as effective vectors for vaccines. Of all of them, Salmonella spp has yielded the most promising results; thus, vaccine vectors based on attenuated strains of Salmonella typhimurium have been used to generate a protective immune response against viral, bacterial, and protozoan pathogens and to perform anti-carcinogenic treatments.Specifically for birds, this type of vaccine has been evaluated as a gene vector for infectious bronchitis virus, avian influenza virus, avian reovirus, and Clostridium perfringens. All these studies agree that these vaccines were able to elicit a long-lasting protective immune response, in addition to being safe by avoiding the risk of causing the disease. Considering the aspects mentioned above, there is strong support for the success that the development of a live recombinant vaccine against AvA-I using an attenuated SE strain as a vector would offer the poultry industry. These advantages can be summarized as follows: (1) severe attenuation through several directed mutagenesis processes via an allelic exchange mechanism that prevents reversion, does not introduce exogenous DNA, and converts the resulting strain into a vector. Petition 870250049938, dated 06 / 13 / 2025, page 9 / 100 3 / 24 fully safe vaccine; (2) the amount of expression of the AvA-I antigen can be regulated by genetic manipulation; (3) it can be administered by oral inoculation; (4) it strongly stimulates the innate and adaptive immune system; and (5) in turn, it would provide protection against SE. Although the development of vaccines using bacterial vectors has been and currently is the subject of numerous investigations, to date there are no commercially licensed vaccines based on SE. Therefore, the development of this vaccine would establish a good precedent in this area of research and development and would have no competition in national and international markets.
[0005] . The strain used in the present invention was isolated and described by (Solano et al, 2002). This study reports a novel selection method based on fluorescence of colonies on calcofluor agar plates to identify Salmonella enteritidis mutants that are defective in biofilm formation. These results not only confirmed the requirement of previously described genes for the modulation of multicellular behavior in Salmonella typhimurium and other species, but also revealed new aspects of the biofilm formation process, such as new genetic elements named as bcsABZC and bcsEFG operons, necessary for the synthesis of a cellulase-hydrolyzable exopolysaccharide. Non-polar mutations of the BcsC and bcsE genes, as well as complementation experiments, demonstrated that both operons are essential for cellulose biosynthesis in LB and ATM media, in both S. enteritidis and S. typhimurium. This study also showed that the biofilm produced by S.Enteritidis is composed of different components, suggesting that the composition and regulation of the biofilm depends on environmental conditions. Furthermore, the results suggest that cellulose is not implicated in the virulence of S. enteritidis, although mutants deficient in the production of this polysaccharide are more sensitive to chlorine treatments, suggesting that the production of... Petition 870250049938, dated 06 / 13 / 2025, page 10 / 100 4 / 24 Cellulose and biofilm formation would be an important factor in the survival of S. enteritidis on surfaces. In a subsequent study, Solano et al (2009, including reference) carried out an ambitious genetic approach in which, in the same strain described above, the 12 genes encoding proteins with diguanylatecyclase enzymatic activity (DGC, proteins with GGDEF domain) were deleted. These enzymes synthesize the secondary messenger c-di-GMP, a molecule whose involvement in important bacterial biological processes such as biofilm formation or virulence has been extensively studied in numerous species. In the specific case of this SE strain, the total depletion of c-di-GMP correlates with the loss of the ability to form biofilms, a slight attenuation in a murine model, and greater sensitivity to extreme environmental conditions.
[0006] . As a result of the genetic approach described above, patent document ES 2324084B1 describes the invention of a method that allows the production of multiple modifications in the chromosome of Gram-negative bacteria and Salmonella strains deficient in C-diGMP synthesis obtained by this method. The invention also refers to two mutant strains of Salmonella enterica obtained by the aforementioned method in which the twelve genes encoding proteins with the GGDEF domain have been deleted (mutant ΔCHII::Km and mutant ΔCHII::Clo) and their use as expression vectors, immunotherapeutic agents and in metabolic studies.
[0007] . García Ona (2011), in the thesis Analysis of an avirulent strain of Salmonella enteritidis for use as a live attenuated vaccine; studied the potential of several derived strains included in document ES 2324084B1 for the development of a vaccine capable of inducing an immune response, decreasing organ colonization and the release of Salmonella in feces on intensive pig farms. In this work it was concluded that the Salmonella strain candidate for development Petition 870250049938, dated 06 / 13 / 2025, p. 11 / 100 5 / 24 of a vaccine would be the strain known as S. Enteritidis 3934ΔCHII*, which has eliminated the 12 genes that encode proteins with the GGDEF domain and carries an additional deletion in a chromosomal region that includes the transcriptional regulator RpoS. This strain is avirulent, has a reduced ability to survive in the environment and, because it does not contain exogenous DNA, would not be classified as a GMO (genetically modified organism). Oral immunization assays using a murine infection model revealed that immunization with a single dose of 10⁷ cfu (Colony Forming Units) of this strain is capable of inducing a protective immune response against Salmonella infection, indicating that this strain may be a suitable candidate for use as a live attenuated vaccine.
[0008] . Latasa et al., 2016 conducted the following study with the aim of constructing a new live attenuated Salmonella vaccine: first, the impact of the absence of cyclic di-GMP (c-di-GMP) on Salmonella virulence was analyzed. As mentioned earlier, c-di-GMP is a secondary intracellular messenger that controls a wide range of bacterial processes, including biofilm formation and the synthesis of virulence factors, as well as the modulation of the host's innate immune response through STING receptors. The results described in this article showed that a multiple mutant of Salmonella, from whose genome the twelve genes encoding diguanylate cyclase proteins were deleted and, as a consequence, cannot synthesize c-di-GMP, exhibits moderate attenuation in a murine systemic infection model. An additional controlled mutation (unlike the S. strain)Enteritidis 3934ΔCHII* (in which the loss of this sigma factor resulted from counter-natural selection) of the rpoS gene resulted in a synergistic attenuating effect, leading to a highly attenuated strain, named ΔCHII. This strain is immunogenic enough to protect mice against a lethal oral attack from a strain. Petition 870250049938, dated 06 / 13 / 2025, p. 12 / 100 6 / 24 virulent strain of S. typhimurium. The immunogenicity of ΔCHIIII depends on both humoral and cellular response activation and is characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-Α, IL-2, IL-17, and IL-10. ΔCHIIII is capable of forming biofilms and does not survive under desiccation conditions, indicating that it can be easily removed from the environment. Furthermore, ΔCHIIII possesses DIVA (Differentiating Infected from Vaccinated Animals) properties, allowing for the differentiation of infected and vaccinated animals. For ease of terminology, the DXII strain is also known as 3934vac.
[0009] . Furthermore, the document by Kong et al., 2011, describes the effect of eliminating the O antigen and core sugars on the virulence and immunogenicity of Salmonella enterica serotype typhimurium. In this study, non-polar deletion mutations were performed on the waaG (rfaG), waaL (rfaI), rfaH, waaJ (rfaJ), wbaP (rfbP), waaL (rfaL), or wzy (rfc) genes in a wild-type S. typhimurium strain. After confirming the LPS structure, several in vitro and in vivo properties of each mutant were analyzed, and it was found that all mutants showed significant attenuation and reduced invasive capacity compared to the wild-type parental strains when administered orally to BALB / c mice. Furthermore, strains carrying a mutation in waaG and waaL were deficient in colonizing Peyer's patches and the liver, a deficiency that was partially compensated for after intranasal administration of the DwaaL mutant.In the context of an attenuated vaccine strain that delivers the pneumococcal PspA antigen, all mutations tested resulted in reduced immune responses against both PspA and Salmonella antigens. These results indicate that irreversible truncation of the outer core is not a viable option for developing a live oral Salmonella vaccine, while a wzy mutant, which retains an O antigen unit, would be suitable for stimulating immunity. Petition 870250049938, dated 06 / 13 / 2025, p. 13 / 100 7 / 24 optimal protection against homologous or heterologous antigens via oral, intranasal, or intraperitoneal routes. GENERAL DESCRIPTION OF THE INVENTION
[0010] The present invention relates to an attenuated Salmonella Enteritidis strain that has been genetically engineered to eliminate the waaL gene and therefore exhibits a rough phenotype. Another aspect of the present invention relates to the aforementioned Salmonella Enteritidis strain mutant in the waaL gene that also carries on its chromosome the genes encoding the AVA-I fiber antigen. This strain, in addition to having a rough phenotype and being avirulent, confers protection against Ava-I. Additionally, the invention comprises the process for obtaining an attenuated Salmonella Enteritidis strain with a rough phenotype and integrating the genes encoding the AVA-I fiber into its chromosome, including the plasmids and methods used to obtain said strain.
[0011] With the aim of reducing toxicity and maintaining the immunogenicity of strains based on the 3934vac gene pool (SE DXIII), one aspect of the present invention relates to the deletion of the waaL gene. Mutation of this gene results in a rough phenotype because the lipopolysaccharide is composed only of the core and lipid A, having lost the ability to synthesize the O antigen. This mutation has the added advantage of allowing differentiation between vaccinated animals and those naturally colonized by wild strains of Salmonella Enteritidis and typhimurium.
[0012] For the generation of rough mutants, an experimental design based on an allelic exchange was used, which gives rise to a thermosensitive plasmid carrying two regions homologous to the adjacent 5' and 3' of the waaL gene. In order to effect the allelic exchange by homologous recombination, two were first cloned into this plasmid. Petition 870250049938, dated 06 / 13 / 2025, p. 14 / 100 8 / 24 fragments of approximately 500 bp flanking waaL in the 3' and 5' regions (AB and CD fragments). The constructed plasmid was introduced into the 3934vac strain by electroporation (the Salmonella strain carrying this plasmid grows at 28°C in the presence of 20 pg / ml of chloramphenicol). After transforming the strain, several clones were selected for plasmid integration into the chromosome by culture at 42°C (a temperature not permissive for plasmid replication). After confirming vector integration into the chromosome by PCR, excision or second recombination was performed. In this second step, the plasmid is lost, generating the desired deletion or restoring the wild-type copy of the gene (the approximate ratio of these genetic events is 50-50%). The second recombination is achieved by cultivation at 30°C in the absence of antibiotics and in the presence of sucrose, since the plasmid carries the sacB lethal gene and the presence of this sugar in the culture medium counterselects the clones that still retain the plasmid.Confirmation of the clones in which the allelic exchange occurred was performed by PCR and subsequent sequencing. It should be noted that the mutants resulting from this procedure do not have antibiotic resistance or traces of exogenous DNA.
[0013] In order to integrate an expression cassette into the chromosome that would allow the production of the fiber antigen in the SE strain, a similar approach to that described previously was performed. In this case, the plasmid contains, in addition to the flanking recombination regions, an expression cassette that is integrated into the chromosome. This expression cassette was cloned into an integrative plasmid containing the AB and CD regions of approximately 500 bp of the Sb13 gene of the ST64B prophage. The cloning was designed so that the AvaI fiber expression cassette is between the two fragments of the defective prophage gene. (The Salmonella strain carrying this plasmid is grown at 28°C in the presence of chloramphenicol 20 pg / ml).Once the plasmid was transformed by electroporation, several clones were subjected to multiple passages of... Petition 870250049938, dated 06 / 13 / 2025, page 15 / 100 9 / 24 growth at 42°C (non-permissive temperature for plasmid replication - plasmid integration occurs thanks to homologous AB and CD regions adjacent to waaL). After PCR verification that the plasmid had been integrated into the chromosome, excision or second recombination was performed at 28°C in the absence of antibiotics and the presence of sucrose. As described above, this counter-selection system allows the identification of clones that lost the plasmid. The final step again consists of selecting by PCR the clones in which the fiber expression cassette insertion occurred in the sb13 gene of the ST64B prophage.
[0014] The resulting strain, to which this invention refers, is a modified mutant strain of Salmonella Enteritidis that carries a deletion of the waaL gene and therefore has a rough phenotype, and expresses Ava-I fiber genes from its own chromosome.
[0015] For the purposes of this invention, Salmonella Enteritidis is an abbreviation for Salmonella enterica serovar Enteritidis.
[0016] Also, for the present invention, it should be considered that PCR is the polymerase chain reaction; and oligo is the primer used in PCR. DETAILED DESCRIPTION OF THE INVENTION Obtaining Attenuated Salmonella Enteritidis (SEA) Design and construction of a system that allows the introduction of any xenoantigen of interest into the Salmonella Enteritidis (SE) chromosome.
[0017] A live vaccine or vaccine vector must be safe and effective, with a fully controlled genotype and phenotype that avoids the risk of reversion to virulence. Furthermore, the strain must maintain a balance between the degree of attenuation and immunogenicity, remaining in the host organism long enough to give rise to a Petition 870250049938, dated 06 / 13 / 2025, page 16 / 100 10 / 24 protective immune response against homologous and / or heterologous antigens. In this case, the avian Salmonella enteritidis (SEA) strain generated is characterized primarily by drastic attenuation in birds. Furthermore, the SEA strain is incapable of forming biofilms and has a very short survival time in the environment, avoiding any risk associated with the period in which vaccinated animals could excrete this strain. Unlike most commercial vaccines (such as 9R), its genotype and phenotype are fully controlled and do not possess antibiotic resistance genes. The safety of live attenuated bacteria has been verified in other models, such as the 9R vaccine, whose reports scientifically rejected a possible reversion to the original virulent form (Okamoto et al., 2010, Brazilian Journal of Poultry Sciences).
[0018] Therefore, the present invention utilizes a Salmonella enteritidis 3934 strain (deposited in the Spanish Collection of Type Cultures (CECT) with accession number CECT 7236), from which, through genetic engineering techniques, the twelve genes encoding diguanylate cyclase enzymes, the rpoS gene, and the waaL gene have been deleted. This latter mutation was performed with the aim of obtaining a vaccine strain of rough phenotype (Salmonella enteritidis 3934vac DwaaL) that confers protection in farm animals. Similarly, another aspect of the present invention comprises a vaccine strain of rough phenotype carrying an expression cassette for Ava-I fiber genes (Salmonella enteritidis 3934vac DwaaL-fiber) that confers immunity against inclusion body hepatitis in poultry. GENERATION OF ROUGH MUTANTS
[0019] In order to reduce toxicity and maintain the immunogenicity of strains based on the genetic background of 3934vac, the waaL gene was deleted. Mutation of this gene results in a rough phenotype because the lipopolysaccharide is composed only of a core and lipid. Petition 870250049938, dated 06 / 13 / 2025, page 17 / 100 11 / 24 A, having lost the ability to synthesize the O antigen. This mutation has the added advantage of allowing differentiation between vaccinated animals and those naturally colonized by wild strains of Salmonella Enteritidis and Typhimurium. Detailed Methodology
[0020] The methodology used is to construct an integrative vector pKO: waaL, integrate this pKO: waaL vector in a first recombination step, and then perform excision of the integrative vector with a second recombination step (Figure 1) 1. Construction of the integrative vector pKO:: waaL
[0021] To construct the integrative plasmid pKO::waaL, two flanking fragments of the waaL gene of 520 bp (oligonucleotides A and B) and 504 bp (oligonucleotides C and D) respectively, are amplified by PCR. The PCR products are purified on gel and cloned independently into conventional cloning vectors, then digested into Notl Xhol (fragment AB) and Xhol Bglll (fragment CD). Both digested fragments are purified from gel and subcloned into the pKO plasmid whose sequence is SEQ ID NO:1 digested to Notl Blglll, giving rise to the pKO::waaL vector. The pKO plasmid carries a chloramphenicol resistance cassette, a thermosensitive origin, a lacZ selection system and a sacB counter-selection system (Figure 2).
[0022] , The PKO:waal construct whose sequence is SEQ ID NO:2 is made from an Escherichia coli strain and verified by PCR, miniprep and digestion.
[0023] , The oligonucleotides used for the construction of the pDEST::waaL vector are presented in Table 1. Sequence (5'-3'). Restriction sites are underlined. Petition 870250049938, dated 06 / 13 / 2025, page 18 / 100 12 / 24 TABLE 1: Oligonucleotides used to perform the deletion of the waaL gene in Salmonella enteritidis 3934 Gene name Oligonucleotides waaL oligo A (SEQ ID NO: 3) oligo B (SEQ ID NO: 4) oligo C (SEQ ID NO: 5) oligo D (SEQ ID NO: 6) 2. Integration of the suicide vector pKO::waaL (first recombination)
[0024] Once the pKO::waaL plasmid (Figure 3) was constructed and verified, it was extracted from the Escherichia coli strain by miniprep and electroporated onto the S. Enteritidis 3934vac strain where its integration was forced by growth at 42°C. This temperature is not permissive for plasmid replication, and plasmid integration occurs thanks to the homologous AB and CD regions adjacent to waaL. Several clones resulting from incubation at 44°C, resistant to chloramphenicol, were tested by PCR with E-F and E-G oligo pairs to verify integration. In the first case, the PCRs should be negative, and in the second case, those clones in which recombination occurred in the AB fragment, the resulting PCR will be 1282 bp. TABLE 2: Oligonucleotides used to verify the integration of the pKO::waaL plasmid. Oligo Name Sequence oligo E SEQ ID NO: 9 oligo F SEQ ID NO: 10 oligo G (hybridized to pKO plasmid) SEQ ID NO: 11 Petition 870250049938, dated 06 / 13 / 2025, page 19 / 100 13 / 24 3. Excision of the integrative vector pKO::waaL (second recombination)
[0025] For the second recombination event to occur and the pKO plasmid to be lost, several integrated clones are cultured in liquid medium at 30°C in the absence of antibiotics. After 24 hours, 5 serial dilutions are placed in solid medium supplemented with 5% sucrose. Only clones that lost the plasmid through a second recombination grow in this medium, which, depending on the fragment in which it occurred, results in the regeneration of the parental genotype or the total deletion of the waaL gene. Both alleles can be differentiated by PCR with E and F oligos (sequence described above).
[0026] In the parental strain 3934vac, PCR with oligonucleotides E and F results in a 2328 bp fragment, the amplicon sequence from the 3934vac genotype is SEQ ID NO: 7, while in clones where the deletion had occurred the fragment has a size of 1106 bp whose amplification sequence is SEQ ID NO: 8
[0027] The 3934vacDwaal or 3934vacR strain can be grown at 37°C in the absence of antibiotics, as it does not contain any selection cassette.
[0028] , The Waa (rfa) region of Salmonella enteritidis 3934vac and the Waa (rfa) region of Salmonella enteritidis 3934vacR can be seen in Figure 4 and Figure 5, respectively.
[0029] , The oligonucleotides used in the verification of DwaaL mutants are: SEQ ID NO: 9 and SEQ ID NO: 10. CONSTRUCTION AND INSERTION OF CASSETTES WITH AVA-I FIBER GENES IN THE 3934VacR STRAIN Construction and insertion of an expression cassette with AvA-l fiber genes on chromosome 3934VacR. Petition 870250049938, dated 06 / 13 / 2025, page 20 / 100 14 / 24
[0030] . The insertion of the genes of interest into the 3934vacR strain is performed at the chromosomal level. This strategy allows for stable expression of heterologous genes, eliminating the problem of plasmid segregation and the need to use antibiotics as selection markers. The steps are summarized in Figure 6. 1. Construction of the pKO integrative vector: sb13-Fiber
[0031] . In this first phase, an integrative plasmid is constructed to integrate the expression cassette into the sb13 gene of the defective ST64B prophage (all SE strains possess it on their chromosome) (Figure 6). Following a similar approach to that described above, the AB (427 bp) and CD (597 bp) regions are amplified with HI and JK oligo pairs. The PCR products are purified on a gel and cloned independently into conventional cloning vectors, to then be digested with SmaI sphI (AB fragment) and spHI SalI (CD fragment). Both digested fragments are purified on a gel and subcloned into the suicide plasmid pKO digested to SmaI SalI, giving rise to the pKO:: sb13 vector (Figure 7) whose sequence is SEQ ID NO: 12
[0032] . Oligos used
[0033] . Fragment AB: oligo H (SEQ ID NO: 13) oligo I (SEQ ID NO: 14)
[0034] . CD of the fragment: oligo J (SEQ ID NO: 15) oligo K (SEQ ID NO: 16)
[0035] . Once the pKO::sb1 plasmid is constructed, the cassette is assembled, which will give rise to the expression of the fiber antigen. This step is performed in several phases: (i) Amplification of<Terminator (43bp) Pr.promotor.RBS (114bp) -clyA (915pb) > from a plasmid Petition 870250049938, dated 06 / 13 / 2025, page 21 / 100 15 / 24 synthetic with L-M oligonucleotides, (ii) amplification of the fiber antigen coding region (1597bp) using the pET28::fiber plasmid with the NO oligonucleotide pair as a template, and (iii) overlapping PCR to fuse two fragments, with the LO external oligos.
[0036] . Oligo L (SEQ ID NO:17)
[0037] . Oligo M (SEQ ID NO:18)
[0038] . Oligo N (SEQ ID NO:19)
[0039] . Oligo O (SEQ ID NO:20)
[0040] . The fiber expression cassette has the sequence SEQ ID NO: 21
[0041] . The resulting fragment (2,702 bp) is purified on a gel and cloned by assembling homologous ends onto the previously spHI-digested pKO::sb13 vector, obtaining the pKO::sb13-Fiber vector whose sequence is SEQ ID NO: 22 2. Integration of the suicide vector pKO::sb13-fiber (first recombination)
[0042] . Once constructed and verified, the pKO::sb13fiber plasmid, which is extracted from the Escherichia coli strain by miniprep and electroporated into the S. Enteritidis 3934vacR (or 3934vac DwaaL) strain, its integration is forced by growth at 42°C. This temperature is not permissive for plasmid replication, and plasmid integration occurs thanks to the AB and CD homologous regions homologous to the sb13 gene. Several clones resulting from incubation at 44°C, resistant to chloramphenicol, are tested by PCR with the P-Q and P-M oligo pairs to verify integration. In the first case, the PCRs should be negative, and in the second case, those clones in which recombination occurred in the AB fragment, the resulting PCR will be 1607 bp.
[0043] . The oligos used were: Oligo P (SEQ ID NO: 23) Petition 870250049938, dated 06 / 13 / 2025, p. 22 / 100 16 / 24 Oligo Q (SEQ ID NO: 24) Oligo M (SEQ ID NO: 25) -Excision of the integrative vector pKO::ab13-fiber (second recombination)
[0044] . For the second recombination event to occur and the pKO plasmid to be lost, leaving the fiber antigen expression cassette inserted into the chromosome, several integrated clones are cultured in liquid medium at 30°C in the absence of antibiotics. After 24 hours, 5 serial dilutions are placed in solid medium supplemented with 5% sucrose. Only the clones that lost the plasmid through a second recombination grow in this medium, which, depending on the fragment in which it occurred, results in the regeneration of the parental genotype or insertion into the sb13 gene of the ST64B defective prophage of the expression cassette responsible for the production of the fiber antigen. These latter clones can be differentiated by PCR, using the P and Q oligos (sequence described above).
[0045] . In the parental strain 3934vacR, PCR with the oligonucleotides P and Q results in a 1314 bp fragment, while in clones where the 3934vacR::FIBRA (AvA) strain was produced, the fiber expression case integration has a size of 3792 bp.
[0046] . The sequence in the gene pool 3934vacR is SEQ ID NO: 25.
[0047] . The sequence sb13 in the gene pool 3934vacR::FIBRA (AvaI) is SEQ ID NO: 26 Detection of fiber protein expression by AAE
[0048] . To confirm the heterologous expression of the fiber antigen, SDS-PAGE and Western blot techniques are used. Heterologous proteins are expressed by culturing the bacteria in medium Petition 870250049938, dated 06 / 13 / 2025, p. 23 / 100 17 / 24 LB, then the proteins are extracted from the cytoplasm, periplasm and segregated. Samples obtained from the expression are separated by SDSPAGE on 10% polyacrylamide gels. The proteins are transferred to nitrocellulose membranes and detected indirectly using IgG-HRP (horseradish peroxidase-linked antibody) as a conjugate, followed by detection using DAB (3,3'-Diaminobenzidine tetrahydrochloride) as a substrate.
[0049] Thus, the results obtained by Western blot satisfactorily validate and corroborate the expression of the fiber antigen in the Salmonella Enteritidis 3934Vac strain under both growth conditions of 37°C and 28°C. Although none of the bands (A, B, and C) exactly correspond to the estimated theoretical molecular weight of the ClyA-fiberAVAI-His fusion (85.4 kDa), band A likely corresponds to the total size of the fusion while bands B and C are products of protein degradation. Where ClyA represents the sequence of the ClyA gene; fiber AVAI represents the sequence of the fiber gene of AVA-I and His represents the sequence of 6 consecutive histidines. Phenotypic study of strains 3934 and derivatives of the rough vaccine.
[0050] In order to study the phenotypes of the different strains, which were plated on Congo Red agar, this allowed for easy differentiation of the Salmonella strain carrying the fiber gene. Unlike wild strains that normally produce cellulose, fimbriae, and other appendages, resulting in the RDAR genotype – red drough and dry – the mutants generated in the present invention have a phenotype known as saw (smooth and white). This phenotype is especially noticeable when the strains produce a truncated lipopolysaccharide, due to the mutation in the waaL gene. The differences are also significant on semi-solid swimming agar. Petition 870250049938, dated 06 / 13 / 2025, p. 24 / 100 18 / 24 although this phenotype may vary depending on the exact composition and moisture content of the culture plates (Figure 8) EVALUATION OF RECOMBINANT VACCINES Immunization and challenge tests: Vaccine inoculum preparation.
[0051] The inoculum is prepared from the RAvA-SE strain that adequately expresses the AvA-l fiber gene. The bacteria are seeded on XLD medium and incubated for 24 hours at 37°C. Typical colonies are transferred to LB medium, and the culture is incubated for 18 hours at 37°C under agitation at 250 rpm (mechanical shaker). Bacterial cells are collected by centrifugation at 5000 g for 10 min and serially diluted in PBS according to the desired concentration. Preparation of AvA-l inoculum for challenge tests.
[0052] The AvA-l isolate used in the study was obtained from an outbreak of HCI (Hepatitis with Inclusion Bodies) on a farm in Chincha, Ilea, Peru, characterized as serotype AvA-l 4 by RFLPPCR. The isolate is inoculated into 10-day-old SPF eggs through the choryantoic membrane (CAM) and incubated for 9 days, after which the livers are collected for homogenization and the viral load is determined by PCR. The median infectious dose of the chicken embryo (DIEP50 / mL) of the inoculum is calculated using the Reed and Muench formula. The homogenized material is stored at -20°C until its use in the challenge. Protection test
[0053] At this point, animal testing will allow for the evaluation of the immunogenicity of the RAvA-SE candidate vaccine. To do this, a similar timetable is established as shown below. The number of groups to work with is determined according to the dose and route of administration. Petition 870250049938, dated 06 / 13 / 2025, page 25 / 100 19 / 24 administration. If more than one candidate vaccine is developed, the same schedule will also be followed. Each treatment group consists of 30 animals; this number is justified according to a schedule that involves sacrificing animals throughout the experiment to assess bacterial colonization in internal organs. The animals referred to are poultry.
[0054] A standardized vaccination schedule model was followed according to the following schedule. TABLE 3: Vaccination schedule Schedule Sample Type Laboratory (ELISA, PCR, Culture) Day 0 7 14 21 28 Date Bird Age 1 day 8 days 15 days 22 days 29 days Vaccine 1st dose X Challenge X Blood serum LMS ELISA XXXXX Organs liver, spleen, feces, Hyssop by LBMG / LMS PCR / Microbiological culture XXXXX Other LMS: Laboratory of Microbiology and Serology LBMG: Laboratory of Molecular Biology and Genomics
[0055] At this point, one-day-old SPF Hyline chickens are used, which are vaccinated according to the scheme mentioned above. After vaccination, the birds are monitored for clinical signs. Each experimental group is kept in separate cages. On day 21, the birds are challenged intramuscularly with 10¹⁰ copies / mL of AvA-l serotype IV, which is equal to 10⁵ DIEPso (median dose of infection). Petition 870250049938, dated 06 / 13 / 2025, p. 26 / 100 20 / 24 in chicken embryo). One group is challenged with AvA-l (intramuscular) and with a wild strain of Salmonella enteritidis (orally, 107 copies / mL). Samples are collected at regular intervals according to the established schedule, which will include feces, cloacal swabs, liver, and spleen for the detection and quantification of RA vA-SE, wild-type SE, and AvA-l. Blood samples are also considered according to the schedule for measuring protective antibodies. Seven days after the challenge, euthanasia is applied to all birds. Determination of excretion and colonization of attenuated Salmonella enteritidis in chickens.
[0056] Animal testing at this point can determine that the attenuated Salmonella enteritidis (MAR) strain provides the characteristics under which it was designed: avirulent, reduced capacity for colonization, persistence and excretion, compared to a wild-type strain.
[0057] The work schedule was developed specifying: treatment groups, route of administration, concentration of bacteria to be used, type of sample to be evaluated, and sampling days. Each treatment consists of 15 animals; this number includes the sacrifice of animals on the days indicated in the schedule to evaluate bacterial colonization in internal organs. One-day-old birds, separated into groups according to treatment, are inoculated with the SEA strain using a sterile 1 mL syringe. The birds are monitored daily for clinical signs, and are fed and watered ad libitum. Appropriate relative humidity and light-dark cycles are maintained. At the end of the experiment, the birds were euthanized by electric shock. Petition 870250049938, dated 06 / 13 / 2025, page 27 / 100 21 / 24
[0058] According to the experiment design, the samples are sent to the laboratory for the respective microbiological, serological and molecular analyses. Microbiological tests for the detection and quantification of Salmonella.
[0059] Allows determining the excretion and tissue colonization of SEA bacteria throughout the experiment, according to the established sample submission schedule.
[0060] Samples of feces, rectal swabs, spleen, and liver from birds in the different groups evaluated are analyzed throughout the experiment according to the established sample submission schedule. The isolation procedure is performed as indicated by the flowchart (Figure 9).
[0061] . The bacterial load in the samples was determined. LIST OF FIGURES
[0062] , Figure 1: Allelic exchange method for the construction of 3934vacR .
[0063] , Figure 2: Integrative plasmid of pKO
[0064] , Figure 3: Integrative plasmid pKO::waaL
[0065] , Figure 4: Waa (rfa) region of Salmonella enteritidis 3934vac
[0066] , Figure 5: Waa (rfa) region of Salmonella enteritidis 3934vacR
[0067] , Figure 6: pKOB::sb13 plasmid
[0068] , Figure 7: pKO B::sb13fiber plasmid
[0069] , Figure 8: Phenotypic differentiation of Salmonella strains on Congo red agar plates (left) and semi-solid agar plates (right). Petition 870250049938, dated 06 / 13 / 2025, page 28 / 100 22 / 24
[0070] , Figure 9: Flowchart for the microbiological detection and quantification of Salmonella. Additionally, TSI: Three-sugar iron agar; LIA: Lysine iron agar.
[0071] , Figure 10: Western blot transfer results of the protein of interest clyA-fibraFAdV-His.
[0072] , Figure 11: SDS-PAGE results of the protein of interest clyA-fibraFAdV-His, with positive controls A and B.
[0073] , Figure 12: Coomassie staining of SDS-PAGE and Western blot of protein extracts obtained after growth in LB medium at 37°C or 28°C. In addition, MW: molecular weight marker, wt: Salmonella Enteritidis 3934, Δ: Salmonella Enteritidis 3934Vac; Δ-fiber: Salmonella enteritidis 3934Vac with ClyAfibraFAdV-His fusion on the chromosome, Δpfiber: Salmonella enteritidis 3934Vac expressing the ClyAfibraFAdV-His fusion from the plasmid. The specific bands obtained in the western blot were indicated (A, B and C).
[0074] , Figure 13: Electrophoretic analysis of PCR products reveals the presence of clones carrying the waaL deletion. For the selection of clones carrying the deletion, the bacterial chromosome was amplified using oligos external to the construct. Furthermore, M = molecular weight marker, wt = wild strain, c = colony. Expected sizes: weight = 2328 bp, Δ waaL = 1112 bp
[0075] , Figure 14: Summary table of oligos used PREFERRED EMBODIMENT OF THE INVENTION: EXAMPLES
[0076] The following examples provided herein serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein.
[0077] , Example 1: Fiber antigen expression in the strain Salmonella Enteritidis 3934 Vac by Western blot. Petition 870250049938, dated 06 / 13 / 2025, page 29 / 100 23 / 24
[0078] , Protein extract
[0079] We planted 1 colony in LB 5 ml of culture medium for the ClyA fiber which has the insertion in the chromosome. Incubate at 37°C at 200 rpm overnight.
[0080] , We centrifuged at 110000 rpm for 5 minutes and added lysis buffer 1 (Tris-HCl 10 mM, EDTA 5 mM, NaCl 50 mM) + 40 μL protease inhibitor (3 g / mL) + 50 μL lysozyme (10 mg / mL), plus SDS loading buffer (50 μL simple 2x buffer + 50 μL 8M urea + 5 μL β-mercaptoethanol in 50 μL of sample) per sample and everything was homogenized and heated to 100 °C for 5 minutes and then on ice for 5 minutes and from there to load 20 μL, took to load. 0.1 Amps for each gel. For 1:30.
[0081] The band of interest for the clyA-fibraFAdV-His protein was observed in the Western Blot results. Ladder markers used were P7709V (175 kDa) on the left and ab 116029 (245 kDa) on the right. (Figure 10)
[0082] Furthermore, when two His+ positive controls are used, but the initials A and B are by weight, A is approximately 60 kDa and B is approximately 30 kDa. These results allow us to conclude that the clyA-FAdV-His fiber fusion is expressed with a molecular weight of approximately 85.4 kDa. (Figure 11)
[0083] , Western blot was performed for the wild strain: Salmonella Enteritidis 3934, Δ: Salmonella Enteritidis 3934Vac; Δ-fiber: Salmonella enteritidis 3934Vac with ClyAfibraFAdV-His fusion on the chromosome, Δ-pfiber: Salmonella enteritidis 3934Vac expressing the ClyAfibraFAdV-His fusion of the plasmid.
[0084] Electrophoretic analysis of the PCR products reveals the presence of clones carrying the waaL deletion (Figure 13) Petition 870250049938, dated 06 / 13 / 2025, p. 30 / 100 24 / 24
[0085] For the purposes of the present invention, the use of the Salmonella enteritidis 3934vac strain in experimental models suggested that it would function in murine and even avian models.
[0086] In our tests, we saw that when inoculated into birds, this does not work well, because the infection develops anyway; In order to achieve immunity, a deletion was made in the waaL gene of Salmonella enteritidis 3934vac, with which immunity was achieved. Likewise, challenge tests with the 3934vac sb13::clyA-fibraFAdV AwaaL strain showed that it confers immunity against Salmonella and avian adenovirus type-1, preventing the appearance of inclusion body hepatitis. Reservoir of microorganisms
[0087] The strains SG-9R sb:ClyA-FIBRA 6His, 3934 vac-mutant Rugoso and 3934 vac-Fibra mutant Rugoso were deposited in the Spanish Collection of Type Cultures (Paterna, Valencia, Spain), following the rules of the Budapest Treaty for the deposit of microorganisms for patent purposes on the following dates and were assigned the following deposit number. Material Deposit Date Accession Number SG-9Rsb: ClyA-FIBRA 6His April 5, 2017 CET 9331 3934 vac-mutant Rough April 5, 2017 CET 9332 3934 vac-Fibra mutant Rough April 5, 2017 CET 9333
[0088] The present invention is not limited to the scope of the microorganisms described in the patent, as they represent a specific illustration of an aspect of the invention. Any microorganism or plasmid that is functionally equivalent to those described in the invention is included in the invention. Petition 870250049938, dated 06 / 13 / 2025, p. 31 / 100
Claims
3 / 3 CLAIMS 1. Mutant strain of Salmonella enteritidis 3934vac, comprising mutations in 12 genes encoding the second messenger C-di-GMP and a deletion in the RPoS gene, with accession number CECT 7236, characterized by comprising a deletion of the waaL gene.
2. Procedure for generating a mutant strain of Salmonella enteritidis 3934 vac, as defined in claim 1, characterized by comprising: • The construction of an integrative vector, wherein for the construction of the integrative vector two flanking fragments to the waaL gene of 520 bp (oligonucleotides A and B) and 504 bp (oligonucleotides C and D), respectively, are amplified by PCR, wherein oligonucleotides A, B, C and D are the sequences SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6; • Purifying and cloning the PCR products separately into pKOB vectors for amplification, to obtain the resulting pKOB::waaL vector for amplification, then enzymatic digestion, purification and ligation into the pDESTINATlON vector; • Transform the resulting pDEST::Waal vector, defined by SEQ ID NO: 2, into Escherichia coli and verify by PCR, miniprep, and digestion, where, once constructed, the pDEST::waaL plasmid, defined by SEQ ID NO: 2, is electroporated into the S. coli strains.enteritidis 3934vac and 3934vac, which contains the pKO::sb13-fiber plasmid, defined by SEQ ID NO: 22, where its integration is forced by growth at 42°C and subsequent excision at 28°C; and • Verify by PCR with oligos external to the construct (waaL E and F) the deletion of the waaL gene by double homologous recombination, where oligos E and F are SEQ ID NO: 9 and SEQ ID NO:
10. Petition 870250049938, dated 06 / 13 / 2025, page 32 / 100 3 / 3.
3. Recombinant avian Salmonella enteritidis vaccine characterized by comprising a mutant strain of Salmonella enteritidis 3934 vac, as defined in claim 1.
4. Mutant strain of Salmonella enteritidis 3934vac, according to claim 1, characterized by comprising the sequences SEQ ID NO: 21 and SEQ ID NO: 26 and a deletion of the waaL gene.
5. Procedure for generating a mutant strain of Salmonella enteritidis 3934vac, comprising a deletion of the waaL gene and the insertion of AVA-I fiber genes, characterized by comprising: • Assembling a strain of Salmonella enteritidis 3934vac, as defined in claim 1; • Inserting, through a first homologous recombination step, a selection cassette, contained in the pKO::sb13-fiber plasmid, defined by SEQ ID NO: 22, wherein the cassette contains, in addition to the antibiotic resistance gene, complementary sequences of the ST64B prophage in its flanking regions; wherein, in the first homologous recombination step, the bacterium is electroporated in the presence of the linear DNA carrying the selection cassette; • Selecting the transforming colonies, after the first homologous recombination step, in LB medium containing the antibiotic; • Confirm the presence of the cassette on the Salmonella enteritidis 3934 chromosome by PCR with the oligos of sequences SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25;• Insert, through a second homologous recombination step, a ClyA-fibraFAdV-His expression cassette, defined by SEQ ID NO: 21, in which the expression cassette replaces the selection cassette, in which the expression cassette comprises the sequence SEQ ID NO: 21; and • Select Salmonella enteritidis 3934vac strains containing the expression of SEQ ID NO: 21 by verifying the expression product of the sequence SEQ ID NO: 21 by Western Blot and SDS-PAGE. Petition 870250049938, dated 06 / 13 / 2025, p. 33 / 100 3 / 3; 6. Procedure according to claim 5, characterized by also comprising: • Constructing an integrative vector, wherein for the construction of the integrative vector two flanking fragments to the waaL gene of 520 bp (oligonucleotides A and B of SEQ ID NO: 3 and SEQ ID NO: 4, respectively) and 504 bp (oligonucleotides C and D of sequences SEQ ID NO: 5 and SEQ ID NO: 6, respectively) are amplified by PCR. • Purifying and cloning the PCR products separately into pKOB vectors for their amplification, to obtain the resulting pKOB::DwaaL vector for its amplification, then enzymatic digestion, purification and ligation into the pDESTINATION vector. • Transforming the resulting pDEST::waaL vector into Escherichia coli and verifying by PCR, miniprep and digestion, wherein, once constructed, the pDEST::waaL plasmid is electroporated into S. coli strains.enteritidis 3934vac and 3934vac containing the expression cassette, defined by SEQ ID NO: 21, where its integration is forced by growth at 42°C and subsequent excision at 28°C. • Verify by PCR with oligos SEQ ID NO: 9 and SEQ ID NO: 10, external to the waaL gene deletion construct by double homologous recombination.
7. Recombinant avian Salmonella enteritidis vaccine characterized by comprising a modified mutant strain of Salmonella enteritidis 3934 vac with an AVAI fiber gene sequence and a waaL gene deletion.
8. Mutant strain of Salmonella enteritidis 3934 vac, characterized by comprising an expression cassette SEQ ID NO: 21 and the waaL deletion which is verified by the presence of the sequence SEQ ID NO:
8.
9. Mutant strain of Salmonella enteritidis 3934 vac, characterized by comprising a sequence SEQ ID NO: 26 and SEQ ID NO:
8. Petition 870250049938, dated 06 / 13 / 2025, p. 34 / 100