Undirected, constrained and accelerated method for modifying pathogenic bacterial strains for the preparation of live attenuated vaccines and strains resulting therefrom

Abstract

The present invention relates to an undirected, constrained and accelerated method for modifying pathogenic bacterial strains to render them non-pathogenic while preserving their immunogenicity and whose phenotype is stable; it further relates to the bacteria thus obtained and to live attenuated vaccines comprising said bacteria.

Description

[0001] An undirected, constrained, and accelerated process for modifying pathogenic bacterial strains for the preparation of live attenuated vaccines and resulting strains

[0002] The present invention relates to the field of preparation of vaccine composition for the prevention of infections by pathogenic bacteria, in particular live attenuated vaccines, which require the phenotypic modification of pathogenic bacteria.

[0003] More specifically, the present invention relates to a non-directed, constrained, and accelerated method for modifying pathogenic bacterial strains to render them non-pathogenic while preserving their immunogenicity and stable phenotype; it also relates to the bacteria thus obtained and to live attenuated vaccines comprising said bacteria. The method is described as non-directed because it allows the attenuation of bacteria without specifically targeting particular genes; it is constrained because it is conducted in a device that imposes specific, regulated culture conditions; and finally, it is accelerated because it is faster than previously used methods such as cell passage techniques. Live attenuated vaccines consist of attenuated infectious agents: they induce immune protection without triggering an infection that is dangerous to the health of the host to whom they are administered.

[0004] Historically, attenuating pathogenic bacterial strains for vaccines involved empirical methods such as repeated culture or random mutations, often without a full understanding of the underlying genetic modifications.

[0005] Progress has been made with methods such as chemical or physical mutagenesis, allowing for more targeted mitigation but still with uncertainties.

[0006] The advent of genetic engineering has introduced the possibility of precise and targeted genetic modifications. It is now possible to make genetic changes that involve direct intervention on the organism's genome, for example, using techniques such as CRISPR-Cas9, transgenesis, or other biotechnological methods. These techniques allow for the targeted insertion, deletion, or modification of specific genes. Genetically modifying a microorganism to alter its phenotype therefore allows for targeted modifications to its genome, thus offering the possibility of developing strains with specific characteristics. In the context of vaccines, genetic engineering paves the way for the production of more effective vaccines and the creation of genetically modified superior cells, thereby offering new perspectives in the field of immunology.

[0007] However, these techniques often lead to the creation of genetically modified organisms (GMOs), raising ethical and regulatory questions, as well as the risk of environmental contamination. Thus, while powerful, these methods can be limited by safety concerns and public perception, particularly in a prophylactic context (vaccination).

[0008] More recently, the focus has shifted to the phenotypic modification of microorganisms via cell culture. Systems such as the turbidostat have been used to induce phenotypic adaptations through environmental selection. These methods rely on the natural adaptability of microorganisms, thus avoiding direct genetic modifications as in the case of GMOs.

[0009] More specifically, a turbidostat is a continuous culture device that maintains a constant cell concentration in a microbial culture. This is achieved by adjusting the flow rate of the culture medium according to the culture's turbidity. This approach aims to induce phenotypic changes in microorganisms by subjecting them to specific culture conditions, thereby promoting the emergence of desired traits. Modifying the phenotype of microorganisms through cell culture relies on the cells' ability to adapt to their environment in response to selective pressures. This adaptation can lead to changes in the microorganisms' behavior, growth, metabolite production, or other phenotypic characteristics.

[0010] Besides its usefulness for basic microbiology research (studying growth rate, cell cycle duration, etc.), turbidostat culture has also been used to produce bacterial mutants with enhanced growth properties in a specific culture medium, thereby obtaining strains adapted to the production of compounds of interest. However, the preparation of live attenuated vaccines proves to be much more complex than adapting to a culture medium, as it raises several major challenges: reducing the pathogenicity of the strain so that it is not virulent in the vaccine containing it, maintaining the immunogenicity of the strain so that the vaccine is effectively protective, and finally, achieving a stable balance between reducing pathogenicity and maintaining immunogenicity over time to guarantee the safety and efficacy of the vaccines continuously and reproducibly.

[0011] Surprisingly, the applicant succeeded in developing a live attenuated vaccine by culturing pathogenic bacteria in a turbidostat, modifying their phenotype and obtaining bacteria particularly well-suited for use in a live attenuated vaccine. The resulting bacteria have (i) attenuated pathogenicity while retaining (ii) sufficient immunogenicity to induce an immune response in a host to protect against infection, (iii) high growth capacity, and (iv) stable phenotype over time. The turbidostat culture process allows for controlled and stable attenuation of the bacteria, while maintaining high immunogenicity, without resorting to genetic modifications that would classify them as GMOs.Thus, this invention offers a promising solution to the limitations of current methods and paves the way for the production of safer and more effective live attenuated vaccines.

[0012] The present invention relates to several innovative aspects in the field of live attenuated vaccine production from pathogenic bacteria. More specifically, the invention relates to attenuated bacteria obtained directly by a turbidostat culture process, their use in the production of live attenuated vaccines, and the turbidostat culture system adapted to the needs of live attenuated vaccine production.

[0013] In particular, the present invention relates to specific bacterial strains obtained through a turbidostat culture process. These bacterial strains resulting from this process exhibit a set of characteristics including attenuated pathogenicity, while retaining sufficient immunogenicity to induce an immune response, high growth capacity, and phenotypic stability over time. These bacteria represent a significant advance in the field of live attenuated vaccine production, offering the possibility of developing safer and more effective vaccines against a diverse range of pathogens without resorting to genetic modifications that would qualify them as genetically modified organisms (GMOs).

[0014] The invention also aims at the use of these attenuated bacteria for the manufacture of live attenuated vaccines.

[0015] Finally, the invention also relates to a turbidostat culture method and system specifically adapted to the needs of live attenuated vaccine production. This system comprises a series of technical elements, including devices, algorithms, and control methods, designed to maintain optimal culture conditions for the controlled and stable attenuation of pathogenic bacteria. The distinctive feature of this method lies in the periodic introduction of a verification step, during which the attenuation of the bacterial strain's pathogenicity and the maintenance of its immunogenicity are rigorously evaluated. By integrating these elements, the system offers a significant advancement in the production of live attenuated strains using the turbidostat as a tool for genetic and phenotypic modification.

[0016] In summary, the present invention offers new perspectives in the field of live attenuated vaccine production using pathogenic bacteria attenuated by turbidostat culture. It represents a promising solution to the limitations of current methods and contributes to improving vaccine safety and efficacy. The detailed description that follows will elaborate on the features, advantages, and specific applications of each aspect of the invention.

[0017] The present invention thus relates to a method for modifying pathogenic bacteria, by continuous culture of said pathogenic bacteria, comprising the following steps: a) culturing pathogenic bacteria in a culture vessel containing a liquid culture medium under a turbidostat regime; b) regulating the cell concentration of said pathogenic bacteria by adjusting the feed rate of the culture medium; c) taking culture samples at regular time intervals and evaluating the pathogenicity and optionally the immunogenicity of the bacteria in these samples; d) selecting the pathogenic bacteria that have acquired a phenotype with attenuated pathogenicity.

[0018] A turbidostat is a continuous culture device used to maintain the cell density of a microbial culture at a constant level by adjusting the flow rate of the culture medium according to the culture's turbidity. In a typical turbidostat culture system, microorganisms are grown in a reactor where the culture's turbidity is continuously monitored using a spectrophotometer or other optical sensor. When the cell density reaches a predetermined level, the system automatically adjusts the flow rate of the culture medium to maintain this density. This allows the microorganisms to grow continuously in the stationary phase.

[0019] Thus, according to the method of the invention, the turbidostat system is implemented by maintaining a constant cell density in the culture medium while applying an incremental dilution rate to select strains with the fastest growth rate, allowing precise control over growth conditions. This control enables the application of specific environmental selection pressures, leading to desired phenotypic adaptations in bacterial strains and, in particular, ensures stable attenuation of pathogenicity while preserving or improving the immunogenicity of the strains.Unlike natural genetic modifications, the probability of which depends on two factors, natural selection and genetic drift, this constrained system allows strains to evolve towards mutations beneficial to the maintenance of the microorganism; in particular, the selection pressure induced by the increase in the dilution rate forces bacteria to adapt and promotes the loss of genetic material not essential to bacterial multiplication, such as virulence genes, and therefore the generation of non-pathogenic mutants.

[0020] In the turbidostat culture system according to the invention, in addition to the basic functions of the turbidostat, steps for verifying the attenuation of the pathogenicity of the bacterial strain and, optionally, the maintenance of its immunogenicity are regularly integrated.

[0021] There are several types of containers that can be used for turbidostat culture. The type of container to use depends on the number of cells to be cultured.

[0022] Turbidity-controlled culture vessels meet the following conditions:

[0023] - Transparency: the containers must be transparent to allow turbidity measurement.

[0024] - Resistance: the containers must be sufficiently resistant to withstand the growing conditions.

[0025] - Chemical stability: the containers must not react with the culture medium or the cultured cells.

[0026] In addition to the containers, specific accessories are used for turbidostat culture. These accessories include:

[0027] - A turbidity sensor: the turbidity sensor is used to measure the turbidity of the culture medium;

[0028] - A control device: the control device is used to adjust the feed rate of the fresh medium;

[0029] - A temperature control system: the temperature control system is used to maintain the temperature of the culture medium at a constant level;

[0030] - A system for ventilation and regulation of the pC;

[0031] - A pH control system;

[0032] - A regulated agitation system.

[0033] Preferably, devices capable of continuous operation are used.

[0034] According to a preferred embodiment of the method according to the invention, a device is used that prevents the formation of a biofilm and may include at least two containers (also referred to as reactors in what follows). The presence of biofilm prevents the renewal of the bacterial population through the dilution rate of the turbidostat, and therefore prevents the production of mutants. To prevent biofilm formation, a device can be used comprising at least two reactors into which the bacterial culture is transferred at defined intervals (for example, every 48 hours). While one reactor is in use, the other is decontaminated with bactericidal agents, rinsed, and then receives the suspension from the other turbidostat, which is in turn decontaminated, and so on.

[0035] Turbidity-controlled culture is a cell culture technique that maintains cell density at a constant level. This is achieved by measuring the turbidity of the culture medium and automatically adjusting the feed rate of fresh medium.

[0036] The operating parameters for a turbidostat culture are as follows:

[0037] - Target cell density: The target cell density is the cell density at which the culture must be maintained. In the case of the process according to the invention, the target cell density must be sufficiently high for the cells to be able to differentiate. It is evaluated by optical measurement of turbidity and must be within the linearity range of the probe. The bacterial population density must be compatible with the upper and lower limits of the turbidity sensor; it depends on the bacteria cultured and is generally between 10 6 and 109 bacteria per ml, most often between 10 7 and 10 8 bacteria per ml.

[0038] - Feed rate: The feed rate is the rate at which fresh medium is added to the culture medium. This rate is adjusted by a turbidity sensor, which provides an indication of cell density, and the control device.

[0039] The turbidity sensor measures the turbidity of the culture medium. If the turbidity increases, the control device increases the feed rate to add more fresh medium.

[0040] If turbidity decreases, the control device reduces the feed rate to add less fresh medium.

[0041] - Temperature: The temperature of the culture medium can be maintained at a constant level or modified over time. This parameter can increase selection pressure. It will be chosen according to the species of bacteria being cultured.

[0042] - pH: The pH of the culture medium can be maintained at a constant level or modified over time. This parameter can be used to increase selection pressure. It will be chosen according to the species of bacteria being cultured.

[0043] - With possible aeration, the device can be used to cultivate and modify anaerobic bacteria.

[0044] - Culture medium composition: The composition of the culture medium must be adapted to the needs of the cultured cells. It must also allow for turbidity measurement; that is, it must be compatible with the technical specifications of the turbidity probe used (operating range). In particular, it must not be cloudy. For example, for a turbidity sensor measuring at a wavelength of 600 nm, the turbidity from the medium should be less than approximately 0.3 absorbance units, for regulation at approximately 0.5 to 1.0 absorbance units.

[0045] Finally, the culture medium may include compounds that promote genetic and phenotypic modification of bacteria, for example, the presence of biotin affecting capsule production in Streptococcus pneumoniae, or the presence of Mg, SO4 and nicotinic acid for the expression of virulence factors in Bordetella bronchispetica.

[0046] The process according to the invention has the following advantages:

[0047] - stability of attenuation: the process according to the invention guarantees stable attenuation, reducing the risk of reversion to a pathogenic state;

[0048] - stable preservation of immunogenicity: it maintains or improves the immunogenicity of strains, essential for the effectiveness of vaccines;

[0049] - increased safety: by avoiding genetic modifications, the process according to the invention is free from the ethical and regulatory concerns related to GMOs;

[0050] - broad application potential: the process can be applied to a wide variety of pathogenic bacteria, offering a broad range of vaccine applications.

[0051] In the turbidostat culture system according to the invention, step c) includes steps for verifying the attenuation of the bacterial strain's pathogenicity and the maintenance of its immunogenicity at regular time intervals, for example, at least once a week, preferably at least 1 to 5 times a week, and even more preferably at least 1 to 3 times a week. Since the growth rate is specific to each bacterial strain, the sampling frequency can be adapted to the strain used.

[0052] Culture samples can thus be subjected to tests aimed at evaluating the attenuation of the pathogenicity of the bacterial strain.

[0053] Pathogenicity refers to the ability of a bacterium to induce local or systemic functional or metabolic disturbances in a given host. It can be temporary (acute infection) or prolonged (subacute or chronic infection), resolve spontaneously, or be fulminant and fatal without appropriate treatment.

[0054] Pathogenicity depends in particular on virulence, an intrinsic property of the bacterium determined by virulence genes in its genome. Virulence is defined as the ability of a bacterium to enter, multiply, and persist in a normally sterile host site inaccessible to commensal bacterial species (those that live in equilibrium with the host). Virulence is therefore the capacity to cause harm to its host and allows for the assessment of pathogenicity. It can be determined experimentally in the laboratory by evaluating the number of bacteria required to induce lesions, disease, or death in an animal model. The degree of virulence is thus directly linked to the microorganism's ability to trigger disease despite the host's defense mechanisms.The presence or absence of genes encoding virulence factors within the genome of the bacterium in question (bacterial chromosome and mobile genetic elements) is a determining factor in the importance of pathogenic effects and their evolution.

[0055] The attenuation, that is to say the reduction or loss, of the pathogenicity of a pathogenic bacterium is reflected at a minimum by the fact that an infection by this bacterium does not lead to any mortality; preferably, this attenuation is such that the infection by the pathogenic bacterium does not cause any symptoms; this attenuation can be assessed by a test which consists of determining whether a pathogen has been made less virulent or less capable of causing disease.

[0056] There are many different methods for assessing the reduction of pathogenicity. Some of these methods are based on observing and comparing the effects of the wild-type and attenuated pathogen on animals or plants. Other methods are based on in vitro tests, such as cell culture assays or virulence tests.

[0057] - Animal testing: Animals are infected with the pathogen and their reaction is observed. Animal testing is often used to assess the virulence of a pathogen, that is, its ability to cause disease.

[0058] - In vitro tests: The wild and attenuated pathogen are cultured in the laboratory and their effect on cells or tissues is observed and compared.

[0059] - Molecular analyses to detect specific virulence genes.

[0060] The choice of evaluation method depends on the pathogen in question and the desired application.

[0061] In parallel or independently of pathogenicity verification, culture samples may also be subjected to tests aimed at evaluating the maintenance of immunogenicity of the bacterial strain.

[0062] Immunogenicity is the ability of an antigen to elicit an immune response in a host.

[0063] Immunogenicity tests may include measurements of specific antigen production, studies of the induced immune response in animal models, or immunological analyses. They can also be conducted in vivo by inoculating the attenuated bacterial strain into a host and testing the protection conferred by this inoculation against infection with the same bacterial strain but in its unattenuated (pathogenic) form. Advantageously, pathogenicity and the maintenance of immunogenicity are assessed on the same bacterial strain population.

[0064] Indeed, for the production of live attenuated vaccines, it is essential to maintain a balance between reducing the pathogenicity of the bacterial strain and preserving its immunogenicity. However, during the attenuation process, the strain may reach a point where it temporarily loses its ability to induce an adequate immune response, while retaining other desired characteristics (such as loss of pathogenicity). In such situations, it is necessary to be able to revert to a previous subpopulation that exhibits the appropriate phenotype. To this end, the process according to the invention implements a system for the regular collection and preservation of subpopulations, allowing for the storage of culture samples at different stages of the attenuation process.In the event of loss of the adapted phenotype, it is then possible to revert to a previous strain using these preserved samples as a starting point. This ensures greater flexibility and the ability to quickly restore the desired characteristics of the bacterial strain without having to restart the attenuation process from the beginning, which could be costly in terms of time and resources.

[0065] The implementation of a system for preserving bacterial subpopulations is a step that is advantageous to add to the process according to the invention to ensure the stability and reproducibility of the process while preserving the key characteristics of the bacterial strain.

[0066] Thus, according to a particular embodiment, the process according to the invention is characterized in that it includes an additional step following step c) which consists of preserving culture samples at different stages of the attenuation process to allow the restoration of the desired characteristics in the event of loss of said desired characteristics beyond a defined threshold.

[0067] The methods of preserving cell culture are classically known to those skilled in the art; they may, for example, consist of freezing and storage at -80°C.

[0068] The phenotypic selection of bacteria produced by the process according to the invention can also rely on the genetic and phenotypic stability of the strains. This stability specifically aims to prevent the loss of pathogenicity and maintain immunogenicity.

[0069] In summary, the selection criteria, and the means to implement them, applied during step c) can be the following: - good growth capacity; the generation time observed in turbidostat culture is a parameter that cannot be generalized because it is dependent on the strain cultivated and the culture conditions;

[0070] - an in vitro test correlated with pathogenicity; this evaluation is closely linked to the cultured bacterium, examples include, but are not limited to, the evaluation of hemolyticity, infection rate, affinity for an antibody, toxin or other protein known for their pathogenicity, the production of attachment factors, pili, peroxides....;

[0071] - verification of the stability of the non-pathogenicity phenotype in vitro; this is assessed by culturing several generations of the strain modified according to the invention and verifying that the strains obtained always exhibit the same phenotype; more precisely, these cultures are carried out in a liquid medium under optimal temperature, pH, and aeration conditions over several passages (a culture endpoint is used to inoculate a subsequent culture at approximately 1 / 10 ème at l / 100 ème of its volume);

[0072] - verification of in vivo safety; such a test consists of administering the modified bacterium according to the invention to a host and confirming that the latter does not develop any infection; and

[0073] - the verification of the maintenance of sufficient immunogenicity to induce an immune response, i.e. protection against infection by a virulent strain, this test consists of administering the bacterium modified according to the invention to a host, then putting it in contact with a bacterium of the same species but virulent and confirming that the latter does not develop any infection.

[0074] Furthermore, according to a particular embodiment, the bacteria modified according to the process of the invention also have the characteristic of having a reduction or loss, whether partial or complete, of their ability to form biofilms.

[0075] For the purposes of this invention, the term "biofilm" refers to a structured community of bacterial cells adhering to an inert (abiotic) or living (biotic) surface, or adhering to each other, and embedded in a self-produced matrix of extracellular polymeric substances (EPS). This matrix may be composed, without limitation, of polysaccharides, proteins, nucleic acids (RNA / DNA), and / or lipids.

[0076] The expression "ability to create biofilms" (or biofilm-forming capacity) refers to the intrinsic ability of a bacterial population to initiate, develop, or maintain an organized community structure in the form of a biofilm. This capacity encompasses all or part of the physiological and physical phenomena that allow the establishment of a bacterial community, including, in particular, cumulatively or independently: • The initial (reversible then irreversible) adhesion of bacterial cells to a solid support or interface;

[0077] • Cell aggregation and the formation of micro-colonies;

[0078] • The production of an extracellular polymeric matrix (EPS); and / or

[0079] • The structural organization into a complex three-dimensional architecture.

[0080] The loss or reduction (partial or complete) of the ability to form biofilms can be quantified using standardized methods known to those skilled in the art. As a non-limiting example, the ability of bacterial strains to form biofilms on abiotic surfaces can be assessed using a static microplate assay (adapted from the methodology described by O'Toole, 2011, Microtiter Dish Biofilm Formation Assay. JoVE. 47. http: / / www.jove.com / details.php?id=2437, doi: 10.3791 / 2437). This test quantifies the biomass of the biofilm formed on the walls and / or bottom of the wells of a microplate using a colorimetric or metabolic dye such as crystal violet. Applied to pathogenic bacteria, this test might include the following steps:

[0081] 1. Culture and inoculation conditions: The bacterial strains to be tested are initially cultured in a rich medium. For the biofilm assay, the culture is diluted, typically 1:100, in a fresh, biofilm-promoting medium. The composition of the chosen medium will depend on the bacterial strain species. The bacterial suspension is introduced into wells on a microplate and incubated under static (batch) conditions for 4 to 24 hours at the optimal growth temperature (e.g., 37°C).

[0082] 2. Washing and staining: at the end of the incubation period, the biofilm is quantified according to the following steps:

[0083] - Removal of unfixed cells: the medium containing unfixed cells is removed by collecting the medium from the bottom of the wells with a micro-pipette.

[0084] - Washing: the plate is washed using a micropipette. This step is repeated (a total of two washes are recommended) to remove unattached cells and reduce background noise.

[0085] - Addition of the dye: a volume of dye is added to the wells. For example, 125 pL of a 0.1% crystal violet (CV) solution in water is added to each well.

[0086] - Dye incubation: the plate is incubated at room temperature for 10 to 15 minutes.

[0087] - Rinsing: the plate is rinsed 3 to 4 times using a micropipette, then vigorously dried by tapping on absorbent paper to remove excess dye and cells.

[0088] - Drying: the plate is left to dry upside down (for a few hours or overnight).

[0089] 3. Quantification (Absorbance Measurement): For a comparative quantitative evaluation between modified and control strains, the dye retained by the biofilm is solubilized by adding acetic acid (e.g., 125 µL of 30% acetic acid in water) to each well. After incubation for 10–15 minutes at room temperature, the solubilized solution is transferred to a new flat-bottom microplate. When using crystal violet, absorbance is measured using a plate reader at 550 nm (using 30% acetic acid as a blank).

[0090] 4. Interpretation: In the case of the use of crystal violet, a significant reduction in absorbance at 550 nm for the attenuated strain, compared to the parental or wild-type strain, indicates a loss or decrease in the ability to form a biofilm.

[0091] In the context of the invention and according to a preferred embodiment, a strain is considered to have "lost the ability to form biofilms" if it exhibits a reduction of at least 50%, 60%, or 70%, preferably at least 80%, and even more preferably at least 85% or at least 90%, in the biofilm biomass formed compared to the wild-type parental strain. In the case of measurement by crystal violet staining assay, the reduction in the ability to form biofilms is measured by optical densitometry (OD_{570nm} or OD_{595nm}) after crystal violet staining.

[0092] According to a particular embodiment, step c) is carried out by selecting bacteria having:

[0093] - sufficient immunogenicity to induce a sustained immune response; and / or

[0094] - reduced pathogenicity; and / or

[0095] - a stable phenotype.

[0096] Preferably, the selected bacteria exhibit all 3 of these properties.

[0097] Optionally, bacteria will be selected that also have a good growth capacity through metabolic optimization of the strain in the culture medium and / or the inability to form biofilms.

[0098] Pathogenic bacteria that can cause disease in mammals, including humans, include (but are not limited to):

[0099] Gram-positive bacteria

[0100] - Staphylococcus aureus;

[0101] - Streptococcus pneumoniae;

[0102] - Streptococcus pyogenes;

[0103] - Streptococcus suis;

[0104] - Streptococcus spp.;

[0105] - Enterococcus faecalis;

[0106] - Listeria monocytogenes;

[0107] - Mycobacterium tuberculosis; - Mycobacterium leprae;

[0108] - Mycobacterium spp.;

[0109] - Corynebacterium spp.;

[0110] - Clostridium spp.;

[0111] - Haemophilus spp.;

[0112] Gram-negative bacteria

[0113] - Escherichia coll spp. ;

[0114] - Salmonella spp.;

[0115] - Shigella spp.;

[0116] - Yersinia pestis;

[0117] - Pseudomonas aeruginosa;

[0118] - Bordetella bronchispetica;

[0119] - Bordetella spp.

[0120] Intracellular bacteria:

[0121] The process according to the invention implemented with intracellular bacteria requires a prior adaptation step of these bacteria so that they are able to grow in a culture medium.

[0122] - Chlamydia trachomatis;

[0123] - Rickettsia spp.;

[0124] - Coxiella burnetiid;

[0125] - Mycoplasma hyopneumoniae;

[0126] - Mycoplasma hyorhinis.

[0127] Fish pathogens can cause a variety of diseases, ranging from simple, mild infections to serious or even fatal illnesses; these include, but are not limited to:

[0128] - Aeromonas spp.;

[0129] - Pseudomonas aeruginosa;

[0130] - Vibrio spp.;

[0131] - Streptococcus spp.;

[0132] - Salmonella spp.

[0133] - Piscirickettsia spp., for example Piscirickettsia salmonis;

[0134] The pathogenic bacteria of birds include, but are not limited to:

[0135] - Mycoplasma gallisepticum;

[0136] - Mycoplasma synoviae; - Escherichia coli;

[0137] - Salmonella spp.;

[0138] - Chlamydia psittaci;

[0139] - Ornithobacterium rhinotracheale;

[0140] - Pasteu relia multocida ;

[0141] - Haemophilus paragallinarum.

[0142] Preferably, pathogenic bacteria are chosen from:

[0143] - Mycoplasma including Mycoplasma hyopneumoniae and Mycoplasma hyorhinir;

[0144] - the Bordetella;

[0145] - Streptococci including Streptococcus agalactiae and Streptococcus suis;

[0146] - Piscirickettsia including Piscirickettsia salmonis.

[0147] The present invention also relates to an immunogenic bacterium with attenuated pathogenicity that can be obtained according to the process of the invention.

[0148] The present invention further relates to a vaccine comprising an immunogenic bacterium with attenuated pathogenicity according to the invention.

[0149] The vaccine according to the invention may further include pharmaceutically acceptable excipients, for example adjuvants.

[0150] According to one embodiment, the vaccine according to the invention is formulated as a preparation for mucosal administration, such as nasal, pulmonary, oral, rectal, or vaginal administration. Another aspect of the invention relates to an aerosol or spray packaging comprising the vaccine according to the invention.

[0151] Yet another aspect of the invention relates to a nasal dropper packaging comprising the vaccine according to the invention.

[0152] Another aspect of the invention relates to a method of vaccinating a mammal against a pathogenic bacterium, which includes administering to the mammal an induction quantity of the vaccine according to the invention comprising said pathogenic bacterium with attenuated pathogenicity according to the invention.

[0153] Specific use in selected animal species, including but not limited to mammals such as humans, cattle, pigs, sheep, goats, horses, or aquatic organisms including fish (such as salmon, trout, carp, barramundi, tilapia) and crustaceans (such as shrimp), as well as birds such as gallinaceous birds; preferably, these are farm or companion animals. The present invention further relates to a vaccine according to the invention for its use in preventing infectious diseases caused by pathogenic bacteria.

[0154] [Fig. 1] Figure 1 shows the different steps carried out for the preparation of the attenuated bacterial strains. The 100 candidate pathogenic bacterial strains were obtained from limiting cryotube dilutions (10 -3 at 10 -5) directly spread onto blood agar plates, without a reactivation phase. A significant number of colonies were then studied, and for each of the original cryotubes (see Materials and Methods: 10 to 30 colonies were re-isolated, G0 Agar in the diagram). 24 clones were then selected, distributed across the different original samples, and cultured to produce plate G1. Nine 3-hour generations were applied to each clone, and their beta-hemolytic activity was monitored at two time points, G4 and G9. Some clones initially exhibited a chain phenotype, and this was confirmed by microscopy at G9.

[0155] [Fig. 2] Figure 2 is a histogram illustrating the mortality of fish inoculated with pathogenic strains of Streptococcus agalactiae (S. agalactiae “REF” at 10 6 , S. agalactiae REF at 10 7 , S. agalactiae REF at 10 8 , S. agalactiae COMPAR to 10 e, S. agalactiae COMPAR to 10 7 and S. agalactiae COMPAR to 10 8 ) and attenuated strains of Streptococcus agalactiae according to the invention (REFATT1 to 10 6 REFATT1 to 10 7 REFATT1 to 10 8 REFATT2 to 10 6 REFATT2 to 10 7 and REFATT2 at 10 8 ) and the mortality of "negative control" fish.

[0156] [Fig. 3] Figure 3 is a graph representing mortality after challenge with a pathogenic strain of Streptococcus agalactiae on fish that have or have not received inoculation with an attenuated strain of Streptococcus agalactiae according to the invention.

[0157] Example 1 - attenuation of a virulent strain of group B Streptococcus agalactiae (“strain

[0158] REF")

[0159] The Streptococcus agalactiae strain used for this test is a group B Streptococcus agalactiae strain exhibiting hemolytic activity on blood agar.

[0160] Cultures aimed at demonstrating stability were carried out in 24-well microplates (Starlab Int-Cyto one Ref: CC7682-7524).

[0161] This trial consisted of maintaining the bacterial strain in a continuous culture system for a period of 5 months under fixed culture conditions in turbidostat mode and observing the changes on:

[0162] - the length of bacterial chains;

[0163] - the attenuation or loss of the hemolytic character;

[0164] - accelerated growth. Growing conditions.

[0165] The culture conditions utilize a commercial BD TSB culture medium (Bacto Tryptic Soy Broth Soybean-Casein Digest Medium - Ref. 211825) at 30 gppowder-L 1 .

[0166] Composition of the medium:

[0167] Preparation: The powder is diluted with demineralized water. The final pH is adjusted to 7.3 + / - 0.2 upH. The medium is sterilized by autoclaving at 121°C, 15 psi for 15 min.

[0168] The culture temperature is set at 37°C.

[0169] The growth periods of approximately 3 hours for each generation were carried out in an Infors multitron pro shaker type incubator at 350 rpm and 37°C.

[0170] Each well contained 2mL of TSB and was inoculated with 100pL of the previous generation culture.

[0171] The limiting dilutions aimed at isolating the clones were made with Ringer's solution (equivalent to Physiological Water) (Fisher Scientific - Ref 1204-3775).

[0172] The diluted suspensions were then spread on blood agar (BD Biosciences - Ref: 254053 Trypticase Soy Agar II with 5% sheep blood) and incubated for 48 hours at 37°C. These spreads allowed for the isolation of candidate clones and verification of their stability over time by evaluating their beta-hemolytic activity.

[0173] Monitoring progress

[0174] Once a week, a sample is taken in order to obtain samples of intermediate strains stored at -80°C.

[0175] These samples are used to monitor the evolution, which consists of microscopic observation and spreading on blood agar (reference BD: 254053 Trypticase Soy Agar II with 5% sheep blood) which reveals the hemolytic nature.

[0176] Monitoring and adjustment of cultivation parameters

[0177] The turbidity threshold was set at 70 u tU rb during the first two months then was increased to 80 Uturb- From tO at 3 months, the temperature was fixed at 37°C then was decreased to 33°C to increase selection pressure.

[0178] Under these different conditions and after stabilization, the generation time was 21 minutes for the first 4 months and then increased to 31 minutes.

[0179] At the end of the 2 ème Months later, clones exhibiting decreased hemolytic activity are selected to reseed the reactor.

[0180] Cultivation was stopped after 5 months.

[0181] Study of strain attenuation and associated phenotypic stability (particularly through non-hemolytic behavior)

[0182] The stability of the strains selected for their non-hemolytic nature (12 strains) was tested according to the following protocol:

[0183] Culture on blood agar, subculturing of non-hemolytic clones in exponential growth phase (3h) over 5 generations then spreading on blood agar and verification of the maintenance of the non-hemolytic phenotype.

[0184] The absence of hemolytic characteristics has also been verified in the evolved strains.

[0185] Two strains were selected for the stability of the loss of the non-hemolytic phenotype:

[0186] • Attenuated strain 1 (“REFATT1”) obtained from strain REF after 105 days of culture in the turbidostat and demonstrating stable non-hemolytic character

[0187] • Attenuated strain 2 (“REFATT2”) obtained from the REF strain after 140 days of culture in the turbidostat and demonstrating stability of the non-hemolytic character.

[0188] Study of in vivo loss of virulence and associated phenotypic stability

[0189] The virulence of these strains was then tested on fish.

[0190] The trials were carried out with 700 Tilapia (Oreochromis niloticus) fish of 35g which had never been in contact with a pathogen, distributed in 28 tanks (25 fish per tank).

[0191] The fish received the following treatments:

[0192] - Negative control group: uninfected fish;

[0193] - Group 1: fish infected with the S. agalactiae serotype group B “REF” strain (original, unattenuated) tested at 3 concentrations 10 6 10 7 and 10 8 CFU / ml;

[0194] - Group 2: fish infected with the attenuated S. agalactiae strain 1 “REFATT1” tested at 3 concentrations 10 e 10 7 and 10 8 CFU / ml;

[0195] - Group 3: fish infected with the attenuated S. agalactiae 2 strain "REFATT2" tested at 3 concentrations 10 e 10 7 and 10 8 CFU / ml;

[0196] - Group 4: fish infected with another unattenuated group B S.agalactiae strain “COM PAR” tested at 3 concentrations 10 e 10 7 and 10 8 CFU / ml. Each of these conditions was repeated twice, except for the negative control which was repeated four times.

[0197] On day 1, fish in groups 1 to 4 received an intraperitoneal injection of 0.1 ml.

[0198] The results are shown in Figure 2.

[0199] These tests confirm that the two selected attenuated strains have completely lost their pathogenicity.

[0200] Example 2 - attenuation of another virulent strain of Streptococcus agalactiae "SA5009" of group B hemolytic.

[0201] The method for attenuating the Streptoccocus agalactiae SA5009 strain by a turbidostat culture process is the same as that described in Example 1.

[0202] An evolved strain that had lost its hemolytic character was selected.

[0203] In vivo loss of virulence study

[0204] The virulence of the attenuated strain SA5009 / ATT was then tested on fish.

[0205] The trials were carried out with 225 Tilapia (Oreochromis niloticus) fish of 35g which had never been in contact with a pathogen, distributed in 9 tanks (25 fish per tank).

[0206] The fish received the following treatments:

[0207] - Group 1: negative control in which uninfected fish receive 0.1ml of sterile PBS (Negative Control);

[0208] - Group 2: fish infected with the attenuated S. agalactiae strain SA5009 / ATT tested at a concentration of 5.9.10 4 CFU / ml;

[0209] - Group 3: fish infected with the native S. agalactiae strain SA5009 tested at a concentration of 8, 9, 10 4 CFU / ml.

[0210] On day 1, fish from groups 1 to 3 received an intraperitoneal injection.

[0211] These tests are repeated 3 times.

[0212] The results are presented in Table 2: The SA5009 / ATT strain showed no mortality, unlike the unattenuated native strain SA5009, which exhibited a high mortality rate. The SA5009 / ATT strain therefore lost its pathogenicity.

[0213] Evaluation of vaccine protection against the attenuated strain SA5009 / ATT

[0214] Following the previous loss of pathogenicity verification test, the fish from groups 1 and 2 are kept and are subjected to a new test 3 weeks after the administration mentioned in the previous paragraph.

[0215] The groups are therefore still:

[0216] - Group 1: negative control in which uninfected fish receive 0.1ml of sterile PBS (Negative Control);

[0217] - Group 2: fish infected with the attenuated S. agalactiae strain SA5009 / ATT tested at a concentration of 5.9 10 4 CFU / ml.

[0218] Three weeks later, all the fish were infected with the native Streptococcus agalactiae SA5009 pathogenic strain at a dose of 5.6 x 10⁻¹ 4 CFU / fish (10 x LD50) in 0.1ml.

[0219] Mortality is assessed 3 weeks later.

[0220] The results are presented in Table 3 below and in Figure 3:

[0221] The mean mortality rate in the negative control group was 85%, and in the group treated with the attenuated strain, it was 33%. The relative survival rate in the group treated with the attenuated strain was 61%, and the statistical significance between the groups was very high (p < 0.05). <le-10).

[0222] Thus, fish inoculated with the attenuated strain show immunity against infection with a homologous pathogenic strain. In conclusion, the attenuated strains according to the invention provide protection for fish against infection with the wild-type pathogenic strain.

[0223] Example 3 - Verification of the biofilm-generating capacity of another attenuated SA5009 / ATT compared to the native virulent strain of hemolytic group B Streptococcus agalactiae "SA5009" by Crystal Violet test

[0224] This experiment (inspired by the O'Toole publication cited above) aims to compare the biofilm-forming capacity of wild-type and attenuated strains of Streptococcus agalactiae using the crystal violet test, in order to verify whether the attenuated strain has lost its biofilm-forming properties. The biofilm-forming capacity of the attenuated strain SA5009 / ATT of Streptococcus agalactiae was compared to that of the wild-type strain SA5009 according to the quantification protocol described below.

[0225] 1. Preparation of bacterial cultures

[0226] The strains are cultured in Tryptone Soy Broth (TSB). A first culture step is performed by incubating the inoculum in 5 mL of TSB at 25°C with gentle shaking (150 rpm) until an optical density at 600 nm (OD600) of 0.2 is reached. This suspension is then used to inoculate 100 mL of fresh TSB, incubated at 25°C with reduced shaking (130 rpm). The culture is harvested when the final optical density (OD600) reaches a value between 0.8 and 1.0.

[0227] 2. Biofilm Formation

[0228] The harvested night cultures are diluted 1 / 100th in fresh TSB. A 5 ml volume of the diluted suspension is dispensed into 10 ml tubes for each wild-type and attenuated strain. A negative control is prepared using a 5 ml tube of TSB alone. The plate is incubated under static conditions at 37°C for 24 hours.

[0229] 3. Quantification using Crystal Violet

[0230] At the end of incubation, the culture medium is removed with a double wash in water. Staining is performed by adding 5 mL of a 0.1% (w / v) crystal violet solution in distilled water to each of the three tubes, followed by incubation for 10 to 15 minutes at room temperature. After staining, the tubes are rinsed 3 to 4 times with water and dried overnight before visual analysis and optical density (OD) measurement.

[0231] For the measurement of OD, the dye bound to the biofilm is solubilized by the addition of 5ml of 30% acetic acid to each tube, followed by a 15-minute incubation before measuring the optical density at 550 nm (OD550) on an iml sample.

[0232] Visual Observation Results: The incubation in this experiment was static, with no agitation applied. Observations showed that the wild-type strain SA5009 tended to settle to the bottom (likely through self-aggregation), while the attenuated strain SA5009 / ATT remained entirely in suspension. A marked difference was thus observed between the wild-type strain SA5009 and the attenuated strain SA5009 / ATT. This indicates a significant difference in their surface properties or growth characteristics under static conditions.

[0233] Visually, the solution from the wild-type SA5009 strain exhibited a distinct purple coloration, indicating a robust biofilm mass attached to the tube walls, while the solution from the attenuated SA5009 / ATT strain remained clear and colorless, similar to the negative control. OD550 Measurement: The absorbance measurements at 550 nm obtained are as follows:

[0234] The wild-type strain SA5009 exhibited strong biofilm formation (OD550 = 0.116), confirming the visual observation of a distinct purple ring on the tube walls, while the attenuated strain SA5009 / ATT showed negligible biofilm formation (OD550 = 0.015), comparable to the negative control (TSB). The percentage loss of biofilm-forming capacity was calculated based on the OD550 values ​​measured for the wild-type and attenuated strains, correcting the values ​​by removing the background noise from the negative control (0.010). The following values ​​were obtained: native strain SA5009 = 0.106 and attenuated strain SA5009 / ATT = 0.005. Based on these corrected values, the attenuated strain SA5009 / ATT therefore exhibits a biofilm-forming capacity loss of approximately 95.3% compared to the wild-type strain.These OD measurement results confirm that the wild strain possesses strong biofilm formation capabilities, while the attenuated strain SA5009 / ATT has lost this characteristic, which correlates with their respective observed sedimentation and suspension phenotypes.

[0235] These results confirm the significant absence of biofilm formation by the attenuated strain SA5009 / ATT compared to the wild strain SA5009, thus validating the loss of biofilm formation characteristics in the attenuated strain produced according to the process of the invention.

Claims

Demands 1. A method for modifying pathogenic bacteria by continuous culture of said pathogenic bacteria, comprising the following steps: a) culturing pathogenic bacteria in a culture vessel containing a liquid culture medium under a turbidostat regime; b) regulating the cell concentration of pathogenic bacteria by adjusting the feed rate of the culture medium; c) taking culture samples at regular time intervals and evaluating the pathogenicity and optionally the immunogenicity of the bacteria in these samples; d) selecting pathogenic bacteria that have acquired a phenotype with attenuated pathogenicity.

2. A method for modifying pathogenic bacteria according to claim 1, characterized in that it comprises an additional step following step c) which consists of preserving culture samples.

3. A method for modifying pathogenic bacteria according to claim 1 or claim 2, characterized in that step c) is carried out by selecting bacteria having: - maintained immunogenicity; and - reduced pathogenicity; and - a stable phenotype.

4. A method for modifying pathogenic bacteria according to claim 3, characterized in that the selected bacteria also exhibit good growth capacity by metabolic optimization of the strain in the culture medium and / or reduction or partial or complete loss of biofilm formation.

5. A method for modifying pathogenic bacteria according to any one of the preceding claims, characterized in that it is carried out in a device composed of two containers in which the culture is conducted alternately.

6. Immunogenic bacterium with attenuated pathogenicity obtained according to the process of claims 1 to 5.

7. Immunogenic bacterium with attenuated pathogenicity according to claim 6, selected from: - Mycoplasma including Mycoplasma hyopneumoniae and Mycoplasma hyorhinir; - Bordetella; Streptococci including Streptococcus agalactiae and Streptococcus suis; - Piscirickettsia including Piscirickettsia salmonis.

8. Live attenuated vaccine comprising an immunogenic bacterium of attenuated pathogenicity according to claim 5 or claim 7.

9. Vaccine according to claim 8 for its use for the prevention of infectious diseases caused by pathogenic bacteria.

10. Vaccine for its use according to claim 9, for the prevention of infectious diseases caused by pathogenic bacteria in a mammal, an aquatic organism or a bird.

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