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

Abstract

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

Description

Title of the invention: Undirected, constrained, and accelerated method for modifying pathogenic bacterial strains for the preparation of live attenuated vaccine and resulting strains

[0001] 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.

[0002] More particularly, 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 allows for the imposition of specific, regulated culture conditions; and finally, it is accelerated because it is faster than previously used methods such as cell passage techniques.

[0003] Live attenuated vaccines consist of attenuated infectious agents: they induce immune protection without triggering an infection dangerous to the health of the host to whom they are administered.

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

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

[0006] The advent of genetic engineering has introduced the possibility of precise and targeted genetic modifications. It is thus possible to carry out genetic modifications that involve direct intervention on the organism's genome, for example, using techniques such as CRISPR-Cas9, transgenesis, or other biotechnological methods. These techniques make it possible to insert, delete, or modify specific genes in a targeted manner. Genetic modification of a microorganism with the aim of altering its phenotype therefore makes it possible to introduce targeted modifications into the microorganism's 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 superior genetically modified cells, thus 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, although powerful, these methods can be limited by safety concerns and public perception, particularly in a prophylactic context (vaccination).

[0008] More recently, the focus has been on 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, the 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 turbidity of the culture. This approach aims to induce phenotypic changes in microorganisms by subjecting them to specific culture conditions, thereby promoting the emergence of desired characteristics. Modifying the phenotype of microorganisms through cell culture relies on the ability of cells to adapt to their environment in response to selective pressures. This adaptation can lead to changes in the behavior, growth, production of metabolites, or other phenotypic characteristics of the microorganisms.

[0010] Besides its interest for basic research in microbiology (study of growth rate, cell cycle duration, etc.), turbidostat culture has also been used to produce bacterial mutants with improved growth properties in a specific culture medium to obtain strains adapted to the production of compounds of interest.

[0011] However, the preparation of live attenuated vaccines proves to be much more complex than adaptation to a culture medium in that it raises several major challenges: reducing the pathogenicity of the strain so that it is not virulent in the vaccine which will contain it, maintaining the immunogenicity of the strain so that the vaccine is effectively protective, and finally achieving this balance between reducing pathogenicity and maintaining immunogenicity in a stable way over time to guarantee the safety and efficacy of the vaccines in a continuous and reproducible manner.

[0012] Surprisingly, the applicant succeeded in developing a live attenuated vaccine by a turbidostat culture process of pathogenic bacteria by modifying their phenotype and obtaining bacteria particularly adapted to be used 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 strong 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.

[0013] The present invention thus relates to several innovative aspects in the field of the production of live attenuated vaccines 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.

[0014] 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).

[0015] The invention also relates to the use of these attenuated bacteria for the manufacture of live attenuated vaccines.

[0016] Finally, the invention also relates to a turbidostat culture method and system specifically adapted to the needs of producing live attenuated vaccines. 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 advance in the production of live attenuated strains using the turbidostat as a tool for genetic and phenotypic modification.

[0017] 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 explain in detail the features, advantages, and specific applications of each aspect of the invention.

[0018] The present invention thus relates to a method for modifying pathogenic bacteria by continuous culture of said pathogenic bacteria, comprising the following steps:

[0019] a) cultivate pathogenic bacteria in a culture vessel containing a liquid culture medium under a turbidostat regime;

[0020] b) regulate the cellular concentration of said pathogenic bacteria by adjusting the feed rate of the culture medium;

[0021] c) take culture samples at regular time intervals and evaluate the pathogenicity and optionally the immunogenicity of the bacteria in these samples;

[0022] d) select pathogenic bacteria having acquired a phenotype with attenuated pathogenicity.

[0023] 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 turbidity of the culture. In a conventional turbidostat culture system, microorganisms are cultured in a reactor where the turbidity of the culture is continuously monitored using a spectrophotometer or other optical sensor. When the cell density reaches a predefined level, the system automatically adjusts the flow rate of the culture medium to maintain this density constant. This allows the microorganisms to grow continuously in the stationary phase.

[0024] 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 toward 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.

[0025] 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 integrated in a regular manner.

[0026] There are several types of containers that can be used for turbidostat culture. The type of container to use depends on the quantity of cells to be cultured.

[0027] The turbidostat culture vessels meet the following conditions:

[0028] - Transparency: the containers must be transparent to allow measurement of turbidity.

[0029] - Resistance: the containers must be sufficiently resistant to withstand the growing conditions.

[0030] - Chemical stability: the containers must not react with the culture medium or cultured cells.

[0031] In addition to the containers, specific accessories are used for turbidostat culture. These accessories include:

[0032] - A turbidity sensor: the turbidity sensor is used to measure turbidity of the growing environment;

[0033] - A control device: the control device is used to adjust the flow rate feeding the fresh environment;

[0034] - A temperature control system: the temperature control system is used to maintain the temperature of the culture medium at a constant level;

[0035] - A system for aeration and regulation of pO2;

[0036] - A pH control system;

[0037] - A regulated agitation system.

[0038] Preferably, devices capable of continuous operation are used.

[0039] 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). Indeed, the presence of biofilm prevents the renewal of the bacterial population by the turbidostat dilution rate, and therefore prevents the production of mutants. To prevent biofilm formation, a device may be used that includes 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 agents. bactericidal, rinsed, then receives the suspension from the other turbidostat which is in turn decontaminated, and so on.

[0040] 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 the fresh medium.

[0041] The operating parameters for a turbidostat culture are as follows:

[0042] - Target cell density: the target cell density is the cell density at which The culture must be maintained. In the case of the method according to the invention, the target cell density must be sufficiently high to allow the cells to differentiate. It is assessed by optical turbidity measurement and must fall 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 10⁶ and 10⁹ bacteria per ml, most often between 10⁷ and 10⁸ bacteria per ml.

[0043] - Feed rate: the feed rate is the flow rate at which the fresh medium is added to the culture medium. This speed is adjusted by means of a turbidity sensor which gives an indication of cell density and the control device.

[0044] 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.

[0045] If the turbidity decreases, the control device reduces the feed rate to add less fresh medium.

[0046] - Temperature: the temperature of the culture medium can be maintained at a The level can be constant or modified over time. This parameter can increase selection pressure. It will be chosen according to the species of bacteria being cultured.

[0047] - pH: the pH of the culture medium can be maintained at a constant level or be This parameter, which changes over time, can increase selection pressure. It will be chosen based on the species of bacteria being cultured.

[0048] - With possible ventilation, the device can be used to cultivate and modify anaerobic bacteria.

[0049] - Composition of the culture medium: the composition of the culture medium must be adapted to the needs of the cultured cells. It must also be such that it allows for turbidity measurement, that is, it must be compatible with the technical characteristics of the turbidity probe used (operating range); in particular, it must not be cloudy. For example, for a turbidity sensor measuring in the At a wavelength of 600 nm, the turbidity from the medium should be less than about 0.3 absorbance units, for regulation to about 0.5 to 1.0 absorbance units.

[0050] Finally, the culture medium may include compounds promoting 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.

[0051] The method according to the invention has the following advantages:

[0052] - stability of attenuation: the method according to the invention guarantees attenuation stable, reducing the risk of reversion to a pathogenic state;

[0053] - stable preservation of immunogenicity: it maintains or improves immunogenicity strains, essential for the effectiveness of vaccines;

[0054] - Increased safety: by avoiding genetic modifications, the process according the invention is free from the ethical and regulatory concerns linked to GMOs;

[0055] - Wide application potential: the process can be applied to a wide variety of pathogenic bacteria, offering a wide range of vaccine applications.

[0056] 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.

[0057] Culture samples can thus be subjected to tests aimed at evaluating the attenuation of the pathogenicity of the bacterial strain.

[0058] Pathogenicity refers to the ability of a bacterium to induce local or systemic functional or metabolic disturbances in a given host. It may be temporary (acute infection) or prolonged (subacute or chronic infection), resolve spontaneously, or be fulminant and fatal in the absence of appropriate treatment.

[0059] Pathogenicity depends in particular on virulence, which is an intrinsic property of the bacterium determined by virulence genes in the bacterial genome. It is defined as the ability of a bacterium to enter, multiply, and persist in a site of the host that is normally sterile and inaccessible to commensal bacterial species (those that live in equilibrium with the host). Virulence is therefore the ability to cause harm to its host and allows for the assessment of its pathogenicity. It can be determined experimentally in the laboratory by evaluating the number of bacteria required to induce lesions, illness, or death in an animal. model. The degree of virulence is therefore directly linked to the microorganism's ability to trigger a 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 severity of the pathogenic effects and their evolution.

[0060] 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 evaluated by a test which consists of determining whether a pathogenic agent has been made less virulent or less capable of causing disease.

[0061] There are many different methods for assessing the reduction of pathogenicity. Some of these methods are based on observation and comparison of 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 tests or virulence tests.

[0062] - Animal testing: The 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.

[0063] - In vitro tests: The wild and attenuated pathogen are cultured in the laboratory and their effect on cells or tissues is observed and compared.

[0064] - molecular analyses to detect specific virulence genes.

[0065] The choice of evaluation method depends on the pathogen in question and the desired application.

[0066] In parallel or independently of the verification of pathogenicity, culture samples may also be subjected to tests aimed at evaluating the maintenance of the immunogenicity of the bacterial strain.

[0067] Immunogenicity is the ability of an antigen to elicit an immune response in a host.

[0068] Immunogenicity tests may include measurements of specific antigen production, studies of the induced immune response in animal models, or immunological analyses. They may 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.

[0069] Advantageously, the verification of pathogenicity and the verification of the maintenance of immunogenicity are evaluated on the same bacterial strain population.

[0070] Indeed, for the production of live attenuated vaccines, it is essential to maintain a balance between reducing the pathogenicity of the bacterial strain and maintaining 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, for example). 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.

[0071] 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.

[0072] Thus, according to a particular embodiment, the process according to the invention is characterized in that it comprises 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 case of loss beyond a defined threshold.

[0073] 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.

[0074] 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 aims in particular at the loss of pathogenicity and the maintenance of immunogenicity.

[0075] In summary, the selection criteria, and the means for implementing them, applied during step c) can be as follows:

[0076] - good growth capacity; the generation time observed in culture in turbidostat is a parameter that cannot be generalized because it is dependent on the strain cultivated and the culture conditions;

[0077] - an in vitro test correlated with pathogenicity; this assessment is closely linked Examples of tests performed on cultured bacteria include, but are not limited to, the evaluation of hemolytic characteristics, infection rates, affinity for antibodies, toxins or other proteins known for their pathogenicity, and the production of attachment factors, pili, peroxides....

[0078] - verification of the stability of the non-pathogenicity phenotype in vitro; this is evaluated by culturing several generations of a modified strain 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 (the end of a culture is used to inoculate a subsequent culture at approximately 1 / 10th to 1 / 100th of its volume);

[0079] - in vivo safety verification, such a test consists of administering the bacterium modified according to the invention to a host and to confirm that the latter does not develop any infection;

[0080] - verification of the maintenance of sufficient immunogenicity to induce a immune response, i.e. protection against infection by a virulent strain, this test consists of administering the modified bacterium 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.

[0081] According to a particular embodiment, step c) is carried out by selecting bacteria having:

[0082] - sufficient immunogenicity to induce a sustained immune response; and / or

[0083] - reduced pathogenicity; and / or

[0084] - a stable phenotype.

[0085] Preferably, the selected bacteria exhibit all 3 of these properties.

[0086] Optionally, bacteria will be selected that also have a good growth capacity by metabolic optimization of the strain in the culture medium.

[0087] Pathogenic bacteria that can cause disease in mammals, including humans, include (but are not limited to): Gram-positive bacteria

[0088] - Staphylococcus aureus;

[0089] - Streptococcus pneumoniae;

[0090] - Streptococcus pyogenes;

[0091] - Streptococcus suis;

[0092] - Streptococcus spp. ;

[0093] - Enterococcus faecalis;

[0094] - Listeria monocytogenes.

[0095] - Mycobacterium tuberculosis;

[0096] - Mycobacterium leprae;

[0097] - Mycobacterium spp. ;

[0098] - Corynebacterium spp. ;

[0099] - Clostridium spp. ;

[0100] - Haemophilus spp. ; Gram-negative bacteria

[0101] - Escherichia coli spp. ;

[0102] - Salmonella spp. ;

[0103] - Shigella spp. ;

[0104] - Yersinia pestis;

[0105] - Pseudomonas aeruginosa;

[0106] - Bordetella bronchispetica;

[0107] - Bordetella spp. Intracellular bacteria#:

[0108] The process according to the invention implemented with intracellular bacteria requires a prior step of adapting these bacteria so that they are able to grow in a culture medium.

[0109] - Chlamydia trachomatis;

[0110] - Rickettsia spp. ; [YES] - Coxiella burnetiid;

[0112] - Mycoplasma hyopneumoniae;

[0113] - Mycoplasma hyorhinis.

[0114] Fish pathogenic bacteria can cause a variety of diseases, ranging from simple benign infections to severe or even fatal illness; these include, but are not limited to:

[0115] - Aeromonas spp. ;

[0116] -Pseudomonas aeruginosa;

[0117] - Vibrio spp. ;

[0118] - Streptococcus spp. ;

[0119] - Salmonella spp.

[0120] - Piscirickettsia spp., for example Piscirickettsia salmonis;

[0121] The pathogenic bacteria of birds are more particularly (without limitation):

[0122] - Mycoplasma gallisepticum;

[0123] - Mycoplasma synoviae;

[0124] - Escherichia coli;

[0125] - Salmonella spp. ;

[0126] - Chlamydia psittaci;

[0127] - Omithobacterium rhinotracheale;

[0128] - Pasteurella multocida;

[0129] - Haemophilus paragallinarum.

[0130] Preferably, the pathogenic bacteria are chosen from:

[0131] - the Mycoplasma including Mycoplasma hyopneumoniae and Mycoplasma hyorhinir;

[0132] - the Bordetella;

[0133] - Streptococci including Streptococcus agalactiae and Streptococcus suis;

[0134] - Piscirickettsia including Piscirickettsia salmonis.

[0135] The present invention also relates to an immunogenic bacterium with attenuated pathogenicity that can be obtained according to the process of the invention.

[0136] The present invention further relates to a vaccine comprising an immunogenic bacterium with attenuated pathogenicity according to the invention.

[0137] The vaccine according to the invention may further include pharmaceutically acceptable excipients, for example adjuvants.

[0138] 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 package comprising the vaccine according to the invention.

[0139] Yet another aspect of the invention relates to a nasal dropper packaging comprising the vaccine according to the invention.

[0140] 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.

[0141] 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 livestock or companion animals.

[0142] The present invention further relates to a vaccine according to the invention for its use in the prevention of infectious diseases caused by pathogenic bacteria.

[0143] [Fig. 1] Fig. 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 (103 to 105) directly The colonies were spread onto blood agar plates without undergoing 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, GO Agar in the diagram). Twenty-four 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.

[0144] [Fig.2] Fig.2 is a histogram illustrating the mortality of inoculated fish with pathogenic strains of Streptococcus agalactiae (S. agalactiae “REF” to 106, S. agalactiae REF to 107, S. agalactiae REF to 108, S. agalactiae COMPAR to 106, S. agalactiae COMPAR to 107 and S. agalactiae COMPAR to 108) and attenuated strains of Streptococcus agalactiae according to the invention (REFATT1 to 106, REFATT1 to 107, REFATT1 to 108, REFATT2 to 106, REFATT2 to 107 and REFATT2 to 108) and “negative control” fish mortality.

[0145] [Fig.3] Fig.3 is a graph representing mortality after challenge with a pathogenic strain of Streptococcus agalactiae on fish that have or have not received an inoculation of an attenuated strain of Streptococcus agalactiae according to the invention.

[0146] Example 1 - attenuation of a virulent strain of group B Streptococcus agalactiae (“REF strain”)

[0147] The Streptococcus agalactiae strain used for this test is a group B Streptococcus agalactiae strain exhibiting hemolytic characteristics on blood agar.

[0148] Cultures aimed at demonstrating stability were carried out in 24-well microplates (Starlab Int-Cyto one Ref: CC7682-7524).

[0149] This test 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:

[0150] - the length of bacterial chains;

[0151] - the attenuation or loss of hemolytic character;

[0152] - the acceleration of growth. Growing conditions.

[0153] The culture conditions employ a commercial TSB culture medium from BD (Bacto Tryptic Soy Broth Soybean-Casein Digest Medium - Ref. 211825) at 30 gppowder*L.

[0154] Composition of the medium:

[0155] [Tables 1] Component Concentration [gL 1 ] Pancreatic Digest of casein 17 Papaic Digest of soybean 3 Dextrose 2.5 Dipotassium Phosphate 2.5 Sodium chloride 5

[0156] 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.

[0157] The culture temperature is set at 37°C.

[0158] Growths of approximately 3 hours for each generation were carried out in an Infors multitron pro shaker type incubator at 350 rpm and 37°C.

[0159] Each well contained 2mL of TSB and was inoculated with 100uL of the previous generation culture.

[0160] The limiting dilutions aimed at isolating the clones were made with Ringer's solution (physiological water equivalent) (Fisher Scientific - Ref 1204-3775).

[0161] 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 the isolation of candidate clones and verification of their stability over time, by evaluating their beta-hemolytic activity. Monitoring progress

[0162] Once a week, a sample is taken in order to obtain samples of intermediate strains stored at -80°C.

[0163] 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. Monitoring and adjustment of cultivation parameters

[0164] The turbidity threshold was set at 70 uturb for the first two months and then was increased to 80 uturb.

[0165] From t0 to 3 months, the temperature was fixed at 37°C and then decreased to 33°C to increase selection pressure.

[0166] Under these different conditions and after stabilization, the generation time was 21 minutes for the first 4 months and then increased to 31 minutes.

[0167] At the end of the 2nd month, clones exhibiting a decrease in hemolytic activity are selected to reseed the reactor.

[0168] The culture was stopped at 5 months.

[0169] Study of strain attenuation and associated phenotypic stability (in particular by the non-hemolytic character)

[0170] The stability of the strains selected for their non-hemolytic nature (12 strains) was tested according to the following protocol:

[0171] 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.

[0172] The absence of hemolytic character has also been verified on the evolved strains.

[0173] 2 strains were selected for the stability of the loss of the non-phenotype hemolytic: • Attenuated strain 1 (“REFATT1”) obtained from strain REF after 105 days of culture in the turbidostat and demonstrating stable non-hemolytic behavior • 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.

[0174] In vivo loss of virulence study and associated phenotypic stability

[0175] The virulence of these strains was then tested on fish.

[0176] The tests were carried out with 700 Tilapia (Oreochromis niloticus) fish of 35g that had never been in contact with a pathogen, distributed in 28 tanks (25 fish per tank).

[0177] The fish received the following treatments:

[0178] - Negative control group: uninfected fish;

[0179] - Group 1: Fish infected with the S. agalactiae strain serotype group B REF » (original, unattenuated) tested at 3 concentrations 106, 107 and 108 CFU / ml;

[0180] - Group 2: Fish infected with the attenuated S. agalactiae strain 1 “REFATT 1” tested at 3 concentrations 106, 107 and 108 CFU / ml;

[0181] - Group 3: Fish infected with the attenuated S. agalactiae 2 strain "REFATT2" tested at 3 concentrations 106, 107 and 108 CFU / ml;

[0182] - Group 4: fish infected with another strain of S.agalactiae of group B COMPAR » unattenuated tested at 3 concentrations 106, 107 and 108 CFU / ml.

[0183] Each of these conditions was repeated twice, except for the negative control which was repeated four times.

[0184] On day 1, fish in groups 1 to 4 received an intraperitoneal injection of 0.1 ml.

[0185] The results are presented in [Fig.2].

[0186] These tests confirm that the two selected attenuated strains have completely lost their pathogenicity.

[0187] Example 2 - attenuation of another virulent strain of Streptococcus agalactiae _ «SA2009» of group B hemolytic.

[0188] The method for attenuating the Streptoccocus agalactiae SA5009 strain by a turbidostat culture process is the same as that described in Example 1.

[0189] An evolved strain having lost its hemolytic character was selected. In vivo loss of virulence study

[0190] The virulence of the attenuated strain SA5009 / ATT was then tested on fish.

[0191] The tests 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).

[0192] The fish received the following treatments:

[0193] - Group 1: negative control in which uninfected fish receive 0.1 ml of PBS sterile (Negative Control);

[0194] - Group 2: Fish infected with the attenuated S. agalactiae strain SA2009 / ATT tested at a concentration of 5.9.104 CFU / ml;

[0195] - Group 3: Fish infected with the native S. agalactiae strain SA5009 1 tested at the concentration of 8.9.104 CFU / ml.

[0196] On day 1, fish in groups 1 to 3 received an intraperitoneal injection.

[0197] These tests are repeated 3 times.

[0198] The results are presented in Table 2:

[0199] [Tables2] Negative control (NC) = PBS SA5009 / ATT SA5009 native Alive 25 25 25 25 25 25 1 0 3 Dead 0 0 0 0 0 0 24 25 22 Mean bedridden mortality 0.00 0.00% 23.67% Standard deviation 0.00 0.00 1.53 Mortality 0% 0% 0% 0% 0% 0% 96% 100% 88% Mean 0.00% 0.00% 94.67 + 6.11%

[0200] The SA5009 / ATT strain thus showed no mortality, unlike the unattenuated native strain SA5009, which exhibits a high mortality rate. The SA5009 / ATT strain has therefore lost its pathogenicity.

[0201] Evaluation of vaccine protection against the attenuated strain SA2009 / ATT

[0202] Following the previous loss of pathogenicity verification test, the fish from groups 1 and 2 are kept and are retested 3 weeks after the administration mentioned in the previous paragraph.

[0203] The groups are therefore still:

[0204] - Group 1: negative control in which uninfected fish receive 0.1 ml of PBS sterile (Negative Control);

[0205] - Group 2: Fish infected with the attenuated S. agalactiae strain SA2009 / ATT tested at a concentration of 5.9 x 104 CFU / rnl.

[0206] Three weeks later, all fish are infected with the pathogenic strain of Streptococcus agalactiae SA5009native at a dose of 5.6x104 CFU / fish (10 x LD50) in 0.1ml.

[0207] Mortality is assessed 3 weeks later.

[0208] The results are presented in Table 3 below and in [Fig.3]:

[0209] [Tables3] Negative controls (NC) SA5009 / ATT Alive 3 3 5 17 17 16 Dead 22 22 20 8 8 9 Mean bedridden mortality 21.33 8.33 Standard deviation 1.15 0.58 Mortality 88% 88% 80% 32% 32% 36% Mean 85.33 ± 4.62% 33.33 + 2.31% Relative survival percentage: 6 0.94%

[0210] 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).

[0211] Thus, fish inoculated with the attenuated strain show immunity against infection with a homologous pathogenic strain.

[0212] In conclusion, the attenuated strains according to the invention allow the protection of fish against infections with the wild pathogenic strain.

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 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.

5. 5. Immunogenic bacterium with attenuated pathogenicity obtained according to the process of claims 1 to 4.

6. Immunogenic bacterium with attenuated pathogenicity according to claim 5, selected from: - Mycoplasma including Mycoplasma hyopneumoniae and Mycoplasma hyorhinir; - Bordetella; - Streptococcus including Streptococcus agalactiae and Streptococcus suis; - Piscirickettsia including Piscirickettsia salmonis.

7. 7. Live attenuated vaccine comprising an immunogenic bacterium of attenuated pathogenicity according to claim 5 or claim 6.

8. 8. Vaccine according to claim 7 for its use in the prevention of infectious diseases caused by pathogenic bacteria.

9. 9. Vaccine for its use according to claim 8, for the prevention of infectious diseases caused by pathogenic bacteria in a mammal, an aquatic organism or a bird.

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