Vaccine compositions comprising system for delivery of inactivated complete bacteria via cationic polysaccharide nanoparticles without any adjuvant

Abstract

The present invention relates to the field of vaccine compositions. The invention more particularly relates to a prophylactic vaccine composition comprising killed intact bacteria intended for use in mammals and birds, said bacteria being wrapped with a cationic agent, in particular cationic nanoparticles.

Description

[0001] The present invention relates to the field of vaccine compositions. More particularly, the present invention relates to prophylactic vaccine compositions intended for use in mammals and birds comprising killed whole bacteria coated with a cationic agent, in particular cationic nanoparticles. Technical Field

[0002] Bacteria are responsible for many diseases. A single bacterial infection can be enough to induce fatal illness, with devastating economic consequences for farms, particularly poultry farms. Escherichia coli is a commensal bacterium found in the digestive tracts of animals and humans. It is the most common bacterium. It is found in the environment and intestinal flora of both animals and humans. Other pathogens, such as Salmonella enterica, which causes salmonellosis, also pose a significant challenge to poultry health.

[0003] To prevent infection risks in farm animals, preventive vaccines have been developed with the goal of immunizing the individual and avoiding the risk of infection and the resulting health consequences.

[0004] Preventive vaccination consists of inducing an immune response in a healthy individual who has not yet come into contact with a pathogen to activate the individual's immune defense. To achieve this, antigens (such as pathogens or pathogen fragments) are presented to the individual's immune cells. This presentation activates adaptive immune cells, i.e., B lymphocytes and T lymphocytes. They proliferate and produce antibodies that neutralize and eliminate antigens and / or destroy the cellular response of infected cells. This mechanism induces immune memory, thereby allowing the individual to be protected the next time the pathogen is encountered. Therefore, preventive vaccination makes it possible to immunize healthy individuals to protect them from the effects of future diseases.

[0005] For this mechanism to be effective, it is important that the pathogen is recognized as an invader by the individual's immune system, allowing them to develop a protective immune response against the infection.

[0006] The prior art teaches that there are several types of prophylactic vaccines for immunizing poultry against pathogenic bacteria.

[0007] Patent EP2911688A1 relates to a serovar of Salmonella enterica serogroup C1, which is used to protect poultry from conditions caused by Salmonella enterica infection. This serovar is inactivated. It is used to manufacture a vaccine that can be multivalent. Chicks are vaccinated at 30 hours of age. This serovar is used to formulate a vaccine containing an adjuvant, such as, for example, approximately 25% v / v aluminum hydroxide.

[0008] Patent EP0256792A2 discloses a vaccine for protecting poultry from colibacillosis. The vaccine contains as an active ingredient E. coli cells inactivated by ultrasonic treatment to destroy the bacteria. The vaccine may contain an adjuvant, for example, an aluminum compound such as aluminum hydroxide gel. Poultry are preferably vaccinated via the cloaca. However, the vaccine can also be administered in conventional ways, such as by intramuscular, intravenous, or subcutaneous injection. The essence of this invention is the use of ultrasonic cell membrane disruption in the manufacture of a vaccine against colibacillosis in poultry.

[0009] Patent WO2022 / 008848 discloses a method for preparing a vaccine composition from at least one freeze-dried antigen, the method comprising the following steps:

[0010] - providing an aqueous solution comprising cationic nanoparticles consisting of a cationic polysaccharide core;

[0011] - Adding the freeze-dried antigen to the aqueous solution

[0012] - Incubating the resulting composition at room temperature.

[0013] The method according to this document comprises the presence of a partial or whole pathogen extract, which may contain proteins, polysaccharides and lipids. The antigen according to this document is a complex protein extract obtained from the whole pathogen.

[0014] Patent application US2021 / 093705 discloses a nanoparticle composition for use as a vaccine against Salmonella Enteritidis in poultry. The vaccine composition comprises highly immunogenic protein antigens (outer membrane protein (OMP) or intact antigen protein (KAg) and flagellin) extracted from killed Salmonella Enteritidis. These antigens are entrapped inside polyanhydride or chitosan nanoparticles.

[0015] Patent application WO2021 / 021778 discloses a composition comprising a polyacrylic acid mucosal adjuvant and / or an inactivated antigen from respiratory or enteric bacteria or viruses. Additionally, the composition may comprise a cell suspension comprising non-cationic polyacrylic acid particles having a size range of 250 nm to 10 microns.

[0016] Anthony Pavic et al. describe studies on the development of an autologous trivalent inactivated vaccine. The trivalent vaccine was produced from equal amounts of cell suspension (3×108 CFU / mL), combined with aluminum hydroxide adjuvant, and administered intramuscularly into the breast of hens aged 12 to 17 weeks.

[0017] The accessibility of vaccination campaigns is often limited by the cost of the vaccine composition dosage. Indeed, current vaccines are often expensive, which limits their widespread use, particularly in agriculture where the number of individuals to be treated is considerable. Although vaccines are marketed to prevent infections caused by bacteria in mammals and poultry, these vaccines are unsatisfactory because they do not provide fully effective protection at an affordable cost. Furthermore, the compositions proposed in the prior art contain all vaccine adjuvants, which are essential for their effectiveness, but whose side effects have been widely documented. Summary of the Invention

[0018] The present inventors have developed a non-adjuvanted prophylactic vaccine composition for immunizing mammals and birds, particularly poultry, against pathogens. In particular, the present inventors have developed a new delivery system in which cationic nanoparticles (NPs) are used to encapsulate at least the entire pathogenic bacteria that have been killed, thereby enhancing the cellular uptake mechanism. This has the effect of improving the mechanism for presenting bacterial antigens to immune cells and, therefore, activating the immune system faster and more effectively. Advantageously, the vaccine composition can be multivalent in order to induce broad-spectrum protection. Therefore, the vaccine composition can be used to produce a combination vaccine.

[0019] Endocytosis is a mechanism by which extracellular material enters the cell via invagination of the plasma membrane and subsequently forms isolated vesicles within the cytoplasm. When endocytosis is performed by specialized immune cells (polymorphonuclear neutrophils, macrophages, dendritic cells), it is called phagocytosis.

[0020] Preventive vaccines can be used to treat salmonellosis, colibacillosis, campylobacteriosis or any other bacterial infection in poultry.

[0021] The present invention thus relates to an adjuvant-free prophylactic vaccine composition, in particular for mammals and birds, and more particularly for poultry, comprising cationic nanoparticles consisting of a polysaccharide core and at least one inactivated bacterium, characterized in that the bacterium is intact and encapsulated by the cationic nanoparticles. This composition limits the risk of contamination by at least one disease-causing infection by a pathogenic bacterium. In a particular embodiment, the present invention relates to a multivalent prophylactic vaccine composition intended for the treatment of salmonellosis or colibacillosis, in particular for mammals and birds, more particularly for poultry, such as laying hens, broilers, turkeys, ducks, guinea fowl, ostriches, emus, quail, etc.

[0022] The present invention also relates to the use of the vaccine composition for preventing bacterial infections, in particular colibacillosis, salmonellosis and campylobacteriosis.

[0023] Advantages of the invention

[0024] Surprisingly, the present inventors have demonstrated that administration of vaccine compositions comprising inactivated whole pathogenic bacteria encapsulated with cationic agents, such as, for example, cationic nanoparticles, constitutes a new antigen delivery system for effective immunization against pathogens of bacterial origin.

[0025] It is known from the prior art that cationic nanoparticles have the ability to enhance the phenomenon of endocytosis in immune cells by delivering small antigens, such as protein antigens from fragmented pathogens, with a size of about 5 to 15 nanometers. In this configuration, a large number of nanoparticles is required to internalize each element of the fragmented bacterial cell. These delivery systems use cationic nanoparticles combined with a full or partial extract of the fragmented pathogen, in which case the full or partial extract of the fragmented pathogen is contained in the core of the nanoparticle; this "antigen in the nanoparticle core" configuration requires the use of more nanoparticles (by weight) than antigen, preferably 10 to 100 times as many nanoparticles as antigen. This multiple can be even higher when using, for example, PLGA nanoparticles or liposomes. This prior art delivery system is not capable of delivering intact bacteria; it can only deliver small antigens with a size of about 5 to 15 nanometers, while the minimum size of an intact cell is about 1 to 10 micrometers.

[0026] In this work, the inventors unexpectedly demonstrated that cationic nanoparticles allow the phagocytosis of intact bacterial cells. In other words, the inventors have developed a delivery system that can deliver at least 100 times more bacteria than protein antigens. This is therefore a new delivery system in which bacteria are coated with positively charged nanoparticles according to the present invention, allowing them to interact with cell membranes and enter immune cells through phagocytosis. In fact, this combination mimics the process by which viruses enter cells through the regulation of the ionic charge provided by the nanoparticles. By coating intact bacteria with nanoparticles according to the present invention, the "nanoparticle-coated intact bacteria" combination is positively charged. This positive charge promotes interaction with the cell membrane and allows the intact bacteria-nanoparticle complex to enter through phagocytosis. This method is made possible by the specific combination developed by the applicant, namely nanoparticles that coat intact, inactive bacteria. Combinations of nanoparticle coatings of intact bacteria have not been described before.

[0027] This new delivery system offers the innovative advantage of allowing intact bacteria to enter cells via the vaccine composition according to the invention. Advantageously, a smaller amount of nanoparticles can be used than the amount of bacterial protein by weight; in fact, the amount of NPs can be 1 / 3 to 1 / 100 of the amount of bacterial protein.

[0028] Therefore, it is interesting that even small amounts of nanoparticles are sufficient to enable entry of intact bacterial cells via phagocytosis and achieve effective immunization.

[0029] Reducing the number of nanoparticles used in vaccination significantly reduces the cost of producing vaccine compositions, making vaccination against pathogens accessible on a large scale, especially in areas where cost is a practical limitation to vaccination campaigns. One example is poultry farming using in ovo vaccination.

[0030] Furthermore, an advantage of the vaccine composition according to embodiments of the present invention is that the vaccine composition can be multivalent. In other words, the vaccine composition can contain at least two bacterial strains, each of which is responsible for preventing infection. Thus, the present invention makes it easy to obtain a combination vaccine.

[0031] According to an embodiment of the invention, the vaccine composition also enables cross immunity to be obtained.In fact, the vaccine composition may comprise bacteria that induce immunity against variants of the strain in question.

[0032] An interesting application of this technology is in ovo vaccination. This approach is innovative: none of the previously described vaccine strategies propose administering inactive whole bacteria directly into eggs. Here, a combination of inactive whole bacteria partially coated with cationic nanoparticles was shown to be highly effective in vaccine protection without adverse effects on hatching or on chicks. In addition, by intervening before hatching, the risk of contamination within the farm (between chickens) is reduced, as is the spread of disease within the farm. In particular, the inventors have demonstrated that a vaccine composition comprising three different strains of E. coli administered in ovo to chicks protects chicks from E. coli disease-type infection at non-lethal as well as lethal doses in bacterial challenge experiments. The bacterial load was reduced, and the hatching rate was comparable to that of unvaccinated eggs.

[0033] On the other hand, in another application, intramuscular injection of a vaccine composition comprising a Salmonella bacterial strain and cationic nanoparticles (LNPs) in laying hens reduced bacterial loads, and the hens laid more eggs than non-immunized hens.

[0034] The vaccine composition does not contain any adjuvants, which avoids undesirable effects. This is advantageous because mineral adjuvants (i.e., mineral salts, such as aluminum salts) remain in the body for a very long time. The nanoparticles act as a delivery agent for killed bacteria to immune cells and induce a protective response against infection.

[0035] In the case of in ovo vaccination, the fact that no molecules are introduced that could disrupt chick development in ovo contributes to the efficacy of the vaccine approach, as the vaccine will not disrupt chick development or hatching.

[0036] The vaccine composition can be administered in ovo, or can be administered transmucosally (orally, ocularly, nasally) or intramuscularly. In addition, the vaccine method according to the present invention can be implemented in mammals as well as birds, especially in poultry.

[0037] Thus, the composition according to the invention provides a simple, easy to prepare and inexpensive formulation which can be administered in particular in ovo. Furthermore, the fact that the composition comprises whole and inactivated bacteria also has the advantage that antigen characterisation is easier than with partial or whole antigen extracts. DETAILED DESCRIPTION

[0038] A first object of the present invention relates to an adjuvant-free vaccine composition comprising cationic nanoparticles consisting of a polysaccharide core and at least one inactivated bacterium, characterized in that the bacterium is intact and encapsulated by the cationic nanoparticles.

[0039] In a specific embodiment, the vaccine composition is intended for use in mammals and birds.

[0040] In another specific embodiment, the cationic nanoparticles encapsulate the bacteria at a bacterial protein:NP weight ratio greater than or equal to 1. In a preferred embodiment, the bacterial protein:NP weight ratio is greater than or equal to 2, preferably greater than 5, and even more preferably greater than 10, or even 50 or 500.

[0041] "Cationic nanoparticles composed of a cationic polysaccharide core" refers to solid nanoparticles (NPs) comprising a cationic polysaccharide core. The NPs may be cross-linked or non-cross-linked. Their cores may or may not be loaded with anionic phospholipids. The NPs are not surrounded by any phospholipid layer.

[0042] For the purposes of the present invention, cationic nanoparticles are particles having a size range between 1 nm and 500 nm. More preferably, polysaccharide nanoparticles have a size range between 10 nm and 300 nm, in particular between 20 nm and 250 nm. In addition, the nanoparticles according to the present invention are advantageously used in solution. Therefore, the term nanoparticles also includes particles or molecules in the form of nanoparticles in solution, such as, for example, chitosan and its derivatives. The solution can be an aqueous solution, a buffer solution or a serum solution. The inventors have found that certain linear molecules such as chitosan form nanocoils in solution, which behave similarly to conventional nanoparticles. Therefore, chitosan can be used as conventional nanoparticles (e.g., Qi et al., Carbohydrate Research, 2004, 339 (16), 2693-2700) or as such or as a hydrolyzate in solution.

[0043] In a first specific embodiment, the cationic polysaccharide forming the core of the nanoparticle (NP) is a non-crosslinked polymer obtained by reacting a polysaccharide selected from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum) with at least one cationic ligand selected from primary, secondary or tertiary amines or quaternary ammoniums. The core is not loaded with lipids. In other words, in this embodiment, the nanoparticle is a cationic nanoparticle consisting of a non-crosslinked and lipid-free polysaccharide core consisting of (i) a polysaccharide selected from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum), and (ii) at least one cationic ligand selected from primary, secondary or tertiary amines or quaternary ammoniums.

[0044] In a second specific embodiment, the cationic polysaccharide forming the core of the nanoparticle (NP) is a cross-linked polymer obtained by reacting a polysaccharide selected from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum) with at least one cationic ligand selected from primary, secondary or tertiary amines or quaternary ammonium groups, followed by the addition of a cross-linking agent. The cross-linking agent is selected from epichlorohydrin, dicarboxylic acids or acid chlorides, such as sebacic acid. The core is not loaded with lipids. In other words, in this embodiment, the nanoparticles are cationic nanoparticles consisting of a cross-linked, non-lipid-loaded polysaccharide core consisting of: (i) a polysaccharide selected from starch, dextran, chitosan, dextrin, maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum), (ii) at least one cationic ligand selected from primary, secondary or tertiary amines or quaternary ammonium, and (iii) a cross-linking agent selected from epichlorohydrin, dicarboxylic acids or acid chlorides, such as sebacic acid.

[0045] In a third embodiment, the nanoparticles (NPs) are nanoparticles consisting of a phospholipid-loaded, non-crosslinked cationic polysaccharide core. In other words, in this embodiment, the nanoparticles are cationic nanoparticles consisting of a non-crosslinked polysaccharide core loaded with anionic phospholipids, the polysaccharide core consisting of (i) a polysaccharide selected from starch, dextran, chitosan, dextrin, maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum), (ii) at least one cationic ligand selected from primary, secondary or tertiary amines or quaternary ammoniums, and (iii) an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine or diacylphosphatidylinositol.

[0046] In a fourth embodiment of the invention, the nanoparticles (NP) are cationic nanoparticles consisting of a cross-linked polysaccharide core loaded with anionic phospholipids, which polysaccharide core consists of: (i) a polysaccharide selected from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum), (ii) at least one cationic ligand selected from primary, secondary or tertiary amines or quaternary ammonium, (iii) a cross-linking agent selected from epichlorohydrin, dicarboxylic acids or acid chlorides, such as sebacic acid, and (iv) an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine or diacylphosphatidylinositol.

[0047] In a preferred embodiment applicable to all four types of nanoparticles (NPs) described above, the cationic polysaccharide is based on maltodextrin; the cationic polysaccharide is obtained by the reaction between maltodextrin and glycidyltrimethylammonium, whether the NPs are cross-linked or lipidated. In other words, the cationic polysaccharide core comprises maltodextrin and glycidyltrimethylammonium.

[0048] In a preferred embodiment suitable for nanoparticles (NPs) having a phospholipid-loaded core, the NPs are DPPG-loaded cationic polysaccharide nanoparticles, whether or not the NPs are cross-linked.

[0049] Avian populations are particularly understood to mean poultry. For the purposes of the present invention, poultry are poultry that serve as a source of eggs or meat and which comprise commercially important species such as, for example, chickens, laying hens, turkeys, ducks, geese, guinea fowl, pheasants, pigeons and peacocks.

[0050] In a preferred embodiment, the bacteria are selected from the group consisting of: Salmonella enterica serotype Typhi; Streptococcus pneumoniae; Haemophilus influenzae type b; Mycobacterium tuberculosis; Extratestinal pathogenic E. coli (ExPEC); Enterotoxigenic E. coli (ETEC); Salmonella enterica serotype Paratyphi A (S. enterica ser.; Paratyphi A); Neisseria gonorrhoeae; Clostridium difficile; Campylobacter spp.; Shigella spp.; Staphylobacter aureus; Helicobacter pylori. pylori).

[0051] For the purposes of the present invention, "intact bacteria" means bacteria that are in their intact form and are not fragmented, in particular bacteria whose cell membrane is intact. In other words, this means that the membrane of the bacteria remains intact. In contrast, for the purposes of the present invention, bacteria with damaged, fragmented or ruptured membranes are not considered intact bacteria.

[0052] For the purposes of the present invention, "cationic agent" means an agent having a positive charge, such as cationic nanoparticles.

[0053] For the purposes of the present invention, "inactivated or inactive bacteria" means non-living bacteria that have been previously killed but are still intact. Intact means bacteria with an unchanged membrane. They can be killed, for example, by treatment with formaldehyde or any other inactivation method known to those skilled in the art.

[0054] For the purposes of this invention, "coated with cationic nanoparticles" means that the nanoparticles coat the surface of the inactivated bacteria. The nanoparticles coat the killed bacteria in a uniform layer. The coating efficiency can be defined by the weight ratio of bacterial protein:NP. In a preferred embodiment, the weight ratio of bacterial protein:NP is greater than or equal to 2, preferably greater than 5, and even more preferably greater than 10, or even 50 or 500.

[0055] For bacterial protein (dry weight) between 1:0.01 and 1:10 and more particularly between 1:0.01 and 1:3: the weight ratio of NP, it is observed that the good effect of the bacterium of NP parcel associated with infection prevention.This is that bacterial weight is about 100 times of nanoparticle weight.In a specific embodiment, this ratio is greater than 1, and particularly, the amount of bacterial protein by weight is at least equal to the amount of NP, and can be up to 100 times (ratio is between 1:1 and 1:0.01) of the amount of NP. In an even more preferred embodiment, the amount of bacterial protein by weight is 2 to 100 times (ratio is between 1:0.5 and 1:0.01) of the amount of NP. In an even more preferred embodiment, the amount of bacterial protein by weight is 10 to 100 times (ratio is between 1:0.01 and 1:0.1) of the amount of NP. In a specific embodiment, this ratio can be between 1:0.1 and 1:10, or even between 1:0.1 and 1:3.

[0056] The vaccine composition comprises at least one bacterium to induce effective protection against bacterial infection or at least reduce bacterial infection.

[0057] For the purposes of this invention, "protection against infection" means that the vaccine composition provides 100% protection against the risk of infection, or, if the vaccine composition does not completely protect against the risk of infection, the protection conferred by the vaccine is sufficient for the individual not to develop disease, or, if disease develops, to at least reduce the symptoms of infection and avoid the individual's death. Protection against infection means preventing the spread of infection on the farm.

[0058] In one embodiment of the invention, the vaccine composition is prophylactic.

[0059] For the purposes of the present invention, a "prophylactic vaccine composition" is taken to mean a vaccine composition that enables the induction of an immune response in a healthy individual that has not yet come into contact with a pathogen, with the aim of activating the individual's immune defenses and preparing the immune system to react against future infections.

[0060] In another embodiment of the invention, the vaccine composition is intended for use in poultry.In a specific embodiment of the invention, the prophylactic vaccine composition is intended for use in poultry, particularly embryos in eggs.

[0061] For the purpose of infection, a "multivalent vaccine composition" means that the vaccine composition comprises several different bacteria, making it possible to induce immunity against several diseases associated with the different bacteria.

[0062] In a specific embodiment, the vaccine composition can be used to produce a combination vaccine.

[0063] For the purposes of the present invention, a "combination vaccine" means a vaccine composition comprising several bacteria of different species or families in order to induce immunity against several different bacteria at the same time.

[0064] In one embodiment, the vaccine composition comprises at least one whole and inactivated bacterium to induce cross immunity in the vaccinated individual.

[0065] For the purposes of the present invention, "cross immunity" means acquired immunity against a bacterial pathogen that confers immunity against another bacterial pathogen of a different species, strain, or family that is not part of the vaccine composition.

[0066] Cross-immunity is related to the phenomenon of cross-reactivity. Antibodies are typically specific for a particular antigen. This specificity allows antibodies to target and eliminate the antigen they have detected. Mutated bacteria retain common antigens that can serve as targets for vaccine-induced responses.

[0067] Therefore, cross-reactions with bacteria from closely related species can occur. Bacteria have many surface antigens. When an animal is immunized against a bacterium by injecting it with whole bacteria, it produces antibodies against many of the bacterial antigens. If two bacteria have the same or similar antigens, the individual will have acquired immunity to both bacteria.

[0068] In another embodiment of the invention, the vaccine composition is multivalent and comprises at least 2 different strains of bacteria of different species or families, said bacteria being whole and inactivated.

[0069] A vaccine composition may, for example, consist of three inactivated E. coli strains mixed with lipidated maltodextrin nanoparticles (NPs) to prevent colibacillosis.

[0070] Thus, according to various embodiments of the vaccine composition, the composition may comprise:

[0071] - Intact and inactivated bacteria encapsulated with cationic nanoparticles consisting of a porous polysaccharide core in cross-linked form loaded with anionic phospholipids.

[0072] - Intact and inactivated bacteria encapsulated with cationic nanoparticles consisting of a porous polysaccharide core in cross-linked form without lipid loading.

[0073] - Intact and inactivated bacteria encapsulated with cationic nanoparticles consisting of a polysaccharide core in non-crosslinked form loaded with anionic phospholipids.

[0074] - Intact and inactivated bacteria encapsulated with cationic nanoparticles consisting of a polysaccharide core in a non-crosslinked, non-lipid-loaded form.

[0075] - at least two bacteria of different strains and / or species or families, said bacteria being inactivated and intact bacteria encapsulated with cationic nanoparticles consisting of a porous polysaccharide core in cross-linked form loaded with phospholipids.

[0076] - at least two bacteria of different strains and / or species or families, said bacteria being inactivated and intact bacteria encapsulated with cationic nanoparticles consisting of a polysaccharide core in cross-linked form that is not loaded with lipids.

[0077] - at least two bacteria of different strains and / or species or families, said bacteria being inactivated and intact bacteria encapsulated with cationic nanoparticles consisting of a porous polysaccharide core in non-crosslinked form loaded with phospholipids.

[0078] - at least two bacteria of different strains and / or species or families, said bacteria being inactivated and intact bacteria encapsulated with cationic nanoparticles consisting of a non-lipid-loaded polysaccharide core in non-crosslinked form.

[0079] A second object of the present invention relates to a vaccine composition as defined above, comprising at least two different inactivated bacteria, characterized in that the bacteria are intact and encapsulated by cationic nanoparticles consisting of a polysaccharide core. This vaccine composition is used in a form suitable for intramuscular, transmucosal or in ovo administration. Thus, the composition is multivalent and can be used to obtain a combination vaccine.

[0080] For the purposes of the present invention, "different inactivated bacteria" refers to different strains and / or species or families of bacteria.

[0081] A third object of the invention relates to the use of the vaccine composition as defined previously for preventing bacterial infections in mammals or birds.

[0082] In a particular embodiment of the invention, the bacterial infection is salmonellosis, colibacillosis or campylobacteriosis.

[0083] In a preferred embodiment, the use is for the in ovo prevention of bacterial infections in poultry embryos (by in ovo administration).

[0084] The present invention also relates to a method for preventing diseases associated with bacterial infection, which is intended for use in mammals and poultry, comprising a vaccine composition comprising at least one inactivated whole pathogen encapsulated by cationic nanoparticles consisting of a polysaccharide core, and comprising the following steps:

[0085] - Cationic nanoparticles consisting of a polysaccharide core and at least one intact bacterium.

[0086] - The whole bacteria were inactivated with formaldehyde.

[0087] - mixing the cationic nanoparticles with the inactivated whole bacteria to obtain the vaccine composition.

[0088] - administering said vaccine composition to said animal.

[0089] In a specific embodiment, the mixing of cationic nanoparticles with the inactivated whole bacteria is performed such that the nanoparticles encapsulate the bacteria at a bacterial protein:NP weight ratio greater than or equal to 1. In a preferred embodiment, the bacterial protein:NP weight ratio is greater than or equal to 5.

[0090] In the vaccine composition, the cationic nanoparticles may be selected from:

[0091] - cationic nanoparticles consisting of a non-crosslinked, non-lipid-loaded polysaccharide core consisting of (i) a polysaccharide chosen from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannans (guar gum), and (ii) at least one cationic ligand chosen from primary, secondary or tertiary amines or quaternary ammoniums;

[0092] - cationic nanoparticles consisting of a cross-linked polysaccharide core not loaded with lipids, the polysaccharide core consisting of (i) a polysaccharide chosen from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannans (guar gum), (ii) at least one cationic ligand chosen from primary, secondary or tertiary amines or quaternary ammoniums, and (iii) a cross-linking agent chosen from epichlorohydrin, dicarboxylic acids or acid chlorides, such as sebacic acid;

[0093] - cationic nanoparticles consisting of a non-crosslinked polysaccharide core loaded with anionic phospholipids, the polysaccharide core consisting of: (i) a polysaccharide chosen from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannans (guar gum), (ii) at least one cationic ligand chosen from primary, secondary or tertiary amines or quaternary ammoniums, and (iii) anionic phospholipids chosen from diacylphosphatidylglycerol, diacylphosphatidylserine or diacylphosphatidylinositol;

[0094] - Cationic nanoparticles consisting of a cross-linked polysaccharide core loaded with anionic phospholipids, the polysaccharide core consisting of: (i) a polysaccharide chosen from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum), (ii) at least one cationic ligand chosen from primary, secondary or tertiary amines or quaternary ammonium, (iii) a cross-linking agent chosen from epichlorohydrin, dicarboxylic acids or acid chlorides, such as sebacic acid, and (iv) an anionic phospholipid chosen from diacylphosphatidylglycerol, diacylphosphatidylserine or diacylphosphatidylinositol.

[0095] In a preferred embodiment, the cationic polysaccharide is obtained by the reaction between maltodextrin and glycidyltrimethylammonium, whether or not the NPs are cross-linked.

[0096] In a specific embodiment, the prophylactic method comprises administering the vaccine composition transmucosally, by injection, and / or in ovo.

[0097] In a specific embodiment of the invention, the vaccine composition is administered in poultry, namely in ovo, transmucosally and orally in chicks and intramuscularly in laying hens. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 : E. coli uptake after LNP packaging. Delivery of intact E. coli bacteria was evaluated in human H292 cells. Fluorescent FITC-E. coli alone or in combination with LNPs (ratios 1:3 to 1:0.05) were incubated with human H292 cells for 4 hours, and the percentage of positive cells was measured by flow cytometry with or without trypan blue (TB). The results represent the mean ± SEM of 3 experiments. Statistical analysis: Two-way ANOVA, **p<0.01, ***p<<0.001.

[0099] Figure 2 Intracellular delivery at a 1:3 ratio by confocal microscopy. Delivery of E. coli bacteria was evaluated in H292 cells. Fluorescent FITC-E. coli conjugated to LNPs (1:3 ratio) was incubated with H292 cells for 4 hours, and intracellular localization was observed by confocal microscopy. Representative images were captured. Red: plasma membrane; blue: nucleus; green: E. coli. Scale bar: 10 μm.

[0100] Figure 3 : Schematic representation of the vaccine protocol for the in ovo vaccination trial. Commercial vaccine was administered only in the positive control.

[0101] Figure 4 : Intestinal permeability test of 8 birds per group 6 days after challenge (D20). The results are expressed as mean ± SD.

[0102] Figure 5 : Analysis of anti-E. coli sIgA in feces from 8 birds per group 13 days after challenge (D27). Results represent the mean ± SD of absorbance values ​​obtained by ELISA. Statistical analysis: One-way Anova, *p < 0.05.

[0103] Figure 6 : Clinical scores of liver lesions in 8 birds per group 6 days after challenge (D20). Results are mean ± SD of group scores. Statistical analysis: One-way Anova, *p < 0.05.

[0104] Figure 7 : Schematic diagram of the in ovo vaccination trial protocol. On D1, only the positive group was vaccinated with a commercial vaccine (Poulvac).

[0105] Figure 8 : Percent mortality in each group after lethal challenge on D14 (n=30).

[0106] Figure 9 : Measurement of bacterial load in the air sac of 8 birds from each group, assessed by MPN, 2 days after challenge (D16). Results represent mean ± SD. Statistical analysis was performed by one-way ANOVA, *p < 0.05.

[0107] Figure 10 : Intestinal permeability of 8 birds from each group 6 days after challenge (D20). Results represent mean ± SD. Statistical analysis was performed by one-way ANOVA, *p < 0.05.

[0108] Figure 11 : Intestinal lesion scores of 8 birds from each group 6 days after challenge (D20). Results are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA, *p < 0.05.

[0109] Figure 12 : Schematic representation of the intramuscular vaccination trial protocol.

[0110] Figure 13 : Egg production. Top: Daily number of eggs laid by hens in each group after challenge. Error bars are hidden to improve clarity. Bottom: Mean daily number of eggs laid by hens in each group after challenge. Statistical analysis: One-way Anova, *p < 0.05, ***p < 0.001, ****p < 0.0001.

[0111] Figure 14 : Quantification of bacterial load in the cecum of chickens from each group measured by qPCR. Results represent mean ± SEM. Statistical analysis: One-way ANOVA, **p < 0.01, ***p < 0.001.

[0112] Figure 15 : Quantification of bacterial load in the cecum of chickens from each group measured by qPCR. Results represent mean ± SEM.

[0113] Figure 16 : Schedule of the in ovo vaccination trial protocol. On D1, only the "positive control" group was vaccinated with a commercial vaccine (Poulvac).

[0114] Figure 17 : Measurement of bacterial infection in the trachea (above) and alveoli (below) of birds in each group evaluated by the Most Probable Number (MPN) on 8 birds. The results represent the number of positive and negative birds in each group.

[0115] Figure 18 : Analysis of anti-E. coli sIgA in feces from 8 birds per group after challenge. Results represent mean ± SD of antibody titers. Statistical analysis: One-way Anova for each day.

[0116] Figure 19 Figure 2: Lung lesion scores of 8 birds per group after challenge. Results represent the mean of each group. Statistical analysis was performed daily by one-way ANOVA.

[0117] Figure 20 Evaluation of intact E. coli bacterial delivery in H292 cells. Fluorescent FITC-E. coli, alone or in combination with NPs, were incubated with H292 cells for 4 hours, and the percentage of positive cells was measured by flow cytometry. Results represent the mean ± SD of two experiments.

[0118] Figure 21 Evaluation of intact E. coli bacterial delivery in THP-1 cells. Fluorescent FITC-E. coli, alone or in combination with NPs, were incubated with THP-1 cells for 4 hours, and the percentage of positive cells was measured by flow cytometry. Results represent the mean ± SD of two experiments.

[0119] Example

[0120] abbreviation:

[0121] LNP: cross-linked lipidated maltodextrin nanoparticles

[0122] id: intradermal

[0123] ·in: intranasal

[0124] ip: intraperitoneal

[0125] ·im: intramuscular

[0126] Example 1: Optimizing Formulations Using Cationic Nanoparticles

[0127] The aim of this study was to confirm the efficacy of a nanoparticle and inactivated whole E. coli strain based composition as a delivery system for activating immune cells.

[0128] 1-A Materials and Methods:

[0129] A-Vaccine Preparation

[0130] Cationic nanoparticles (LNPs) are cationic lipidated maltodextrin nanoparticles.

[0131] The composition was prepared using an inactivated E. coli strain mixed with cationic nanoparticles. The E. coli bacteria were inactivated with 0.4% formaldehyde and then purified by centrifugation. The protein content was measured by micro-BCA assay. The composition was prepared by mixing the killed bacteria with an aqueous solution of LNPs in different weight ratios (100 μg of E. coli protein with 5 μg, 10 μg, 30 μg, 50 μg, 100 μg or 300 μg of LNPs). The size and surface charge of the preparation were characterized by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) (Zetasizer NanoZS, Malvern Analytical, France) to observe whether the nanoparticles wrapped the surface of the killed bacteria.

[0132] B- Delivery of E. coli

[0133] The ability of LNPs to increase the uptake of killed, intact E. coli by immune cells was assessed by flow cytometry and confocal microscopy.

[0134] · Labeling E. coli with fluorescein:

[0135] Inactivated bacteria were labeled with fluorescein isothiocyanate (FITC) by mixing 5 mg of E. coli with 50 μg of FITC (1%, Sigma, France) in sodium carbonate buffer at pH 8.3 for 2 hours. They were then dialyzed on a 10 kDa dialysis cassette (Thermo Fisher, France). Protein content was measured by micro-BCA assay (Pierce, France). The labeled bacteria were then conjugated to LNPs at different weight ratios.

[0136] Flow cytometry:

[0137] H292 cell lines were seeded in 24-well plates at 50,000 cells / well until confluent. The cells were then incubated for 4 hours with 1 μg of protein alone or in combination with LNPs at different ratios. The cells were then washed with phosphate-buffered saline (PBS), harvested with trypsin, and analyzed by flow cytometry on an Attune Nxt (Thermo Fisher, France). To distinguish intracellular bacterial delivery from membrane attachment, cells were incubated with 40 μg / mL trypan blue (TB, Sigma France) to quench external FITC fluorescence.

[0138] Confocal microscopy

[0139] H292 cell line is seeded in Labtek chamber (Fisher Sci., France) with 10,000 cells / well until confluence.Then the cells are incubated for 4 hours with respect to 1 μg of the protein alone or the protein combined with LNP from the complete bacteria killed.The cells are washed and the nuclei are stained by incubating Hoechst 33342 (Sigma, France) for 5 minutes at 37°C with 0.1 μg / mL.Then the cells are washed and the plasma membrane is stained for 10 minutes at 37°C with 1 μg / mL of AF-633-labeled lectin (WGA, Invitrogen France).The slides are washed again with PBS, fixed with 0.4% formaldehyde for 20 minutes, and sealed for microscopic observation (LSM 710 Zeiss, France).

[0140] 1-B Results:

[0141] Characterization of the formulation:

[0142]

[0143] Table 1: Characterization of the size of E. coli / LNP formulations by dynamic light scattering (DLS) and the zeta potential of E. coli / LNP formulations by electrophoretic light scattering (ELS) Inactivated whole E. coli were mixed with increasing amounts of LNPs.

[0144] DLS and ELS analysis revealed that the inactivated E. coli had a uniform size of 1.42 μm, a PDI of 0.08, and a negative surface charge of -4.6 mV. This indicates that despite inactivation, the bacterial structure remained intact. When increasing amounts of LNPs were added, the overall size did not change, but the zeta potential gradually increased, becoming cationic starting from a 1:0.5 ratio. This indicates that the bacteria were gradually encapsulated by the LNPs without any aggregation.

[0145] By flow cytometry (see Figure 1 ) E. coli delivery via LNP was evaluated on human airway epithelial cells (H292). In the absence of LNP, the bacteria were endocytosed by 14% of the cells. Since no difference was observed in the presence of TB, this indicates that the bacteria were actually endocytosed (inside the cells). When wrapped with LNP, the bacteria were absorbed by at least 40% of the cells, thereby confirming their potential as a delivery system. In addition, delivery with a small amount of LNP was significantly more efficient, and the highest release was observed at a ratio of 1:0.3 (77%) and 1:0.1 (75.7%). In the presence of TB, at a ratio of 1:0.3, 65.6% of the cells were still positive, and at a ratio of 1:0.1, 63% of the cells were still positive, thereby confirming that most of the bacteria were intracellular.

[0146] Intracellular delivery was also confirmed by confocal microscopy at a ratio of 1:3. Approximately 20 to 30 E. coli bacteria (green) were observed close to the nucleus per cell, confirming intracellular localization.

[0147] in conclusion:

[0148] The E. coli / LNP vaccine formulation consists of inactivated whole bacteria encapsulated by LNPs. Even small amounts of LNPs encapsulating the bacteria have a significant impact on their ability to be taken up by cells.

[0149] Example 2: In ovo vaccination trial against Escherichia coli disease

[0150] 2-A Materials and Methods:

[0151] A-Vaccine Preparation

[0152] The vaccine consists of three inactivated E. coli strains mixed with lipidated maltodextrin nanoparticles (LNPs). Briefly, strains O78:K80, O1:K1, and O2:K1 were inactivated with 0.4% formaldehyde, and protein content was measured using the μBCA assay. Finally, 33.3 μg of each strain was mixed with LNPs to obtain 100 μg of protein per vaccine dose.

[0153] B-animals

[0154] All animal work has been reviewed and approved by the Animal Research Ethics Committee of Imunova Análises Biológicas, protocol number 06 / 2021.

[0155] For this experiment, 390 fertilized eggs were obtained from commercial incubators and incubated in an Imunova experimental unit. The eggs were randomly assigned to seven different experimental groups and placed in an industrial hatchery with precise temperature and humidity control for 21 days. The groups used in this test consisted of 30 animals and are identified in Table 1.

[0156]

[0157] Table 2: Identification of experimental groups.

[0158] All groups, including the negative control, received orally attenuated live Mass I-H120 infectious bronchitis virus (IBV) vaccine at a dose 100 times the manufacturer's recommended dose to sensitize the animals to E. coli challenge.

[0159] 2 The animals were orally challenged with 10<8 > CFU of E. coli at a dose of 100 μL / bird. The challenge was confirmed by microbial recovery of bacteria from the inoculum.

[0160] C-Vaccination

[0161] The animals in group 3 received a dose of live E. coli vaccine. On day 18 of incubation, group 4 animals received in ovo administration of the vaccine, with a controlled vaccine dose of 50 μL. After hatching, groups of one-day-old broilers were housed in isolators (1.2 m 2 ) and fed ad libitum as recommended for their age.

[0162] D-Attack

[0163] On D10, all animals, including those in the negative control group, received orally attenuated live Mass I-H120 infectious bronchitis virus (IBV) vaccine at a dose 100 times the manufacturer's recommended dose.

[0164] In D14, use 10 8 Animals in Groups 2, 3, and 4 were orally infected with CFU of E. coli (strain 19501, a different strain than that used in the vaccine) at 100 μL / bird.

[0165] Vaccine regimens are described in Figure 3 middle.

[0166] E-Sampling

[0167] Eight birds were sampled and analyzed on D16, D20, or D27.

[0168] F-Analysis

[0169] Intestinal permeability:

[0170] Intestinal permeability was assessed by oral administration of FITC-dextran, a non-absorbable fluorescent marker (FITC-dextran, 3000 kDa to 4000 kDa) and detected in plasma / serum to monitor gastrointestinal epithelial integrity ( et al., 2015 ).

[0171] Cytokine expression

[0172] Cytokine expression was assessed by qPCR (IL-1β, IFNγ, IL-10, IL-4) using specific primers for each target. In this type of analysis, a threshold value is generated for each combination of target and sample. The Ct (threshold cycle number) is a relative measure of the concentration of target-specific messenger RNA (mRNA) in a sample. This value must be normalized based on the expression of a reference gene, in this case the geometric mean of the GAPDH and ACTB genes, thereby generating a ΔCt value (target Ct / average GAPDH+ACTB Ct) (Bustin et al., 2009). In addition to this normalization, the data were normalized to the average ΔCt of the control group, thereby generating a ΔΔCt (Δct / average ΔCt control). For undefined results, the maximum CT value (40) was considered, and for analysis purposes, this value was manually modified to 41.

[0173] Quantification of anti-E. coli sIgA:

[0174] The production of E. coli-specific secretory IgA was assessed by ELISA. Briefly, samples were diluted in 1% casein in PBS. ELISA plates were coated with E. coli LPS (field isolate). The plates were then washed three times with 200 μL / well of PBS + 0.05% Tween 20 for 5 minutes each wash. The wells were blocked with 1% casein in PBS. The samples were tested in serial dilutions. The plates were washed and anti-chicken IgA (BioRad) diluted in 0.1% casein was added. After washing, the assay was developed with TMB (Life Technologies) solution. The absorbance was read at 450 nm.

[0175] Detection and quantification of Escherichia coli:

[0176] Detection and quantification of E. coli were performed by MPN (most probable number) according to standard ISO 7251:2005. E. coli counts were determined according to standard microbiological methods (dilution in enrichment medium and then plating in selective / different media). Briefly, the sample was enriched in buffered peptone water (BPW), then enriched in EC broth, and finally enriched in EMB and MacConkey agar. Before incubation, the sample was serially diluted in triplicate in EPB to enable quantification by the most probable number technique (Blodgett et al., 2015). For E. coli detection, only the serial dilution step was omitted. Suspicious colonies isolated were subjected to biochemical testing and confirmation.

[0177] Liver histology:

[0178] The birds were euthanized and liver samples were collected and fixed according to the method of Rebel et al. (2011). The samples were embedded in paraffin and mounted on slides. All histopathological evaluations and readings were performed under a microscope by an experienced veterinary histopathologist.

[0179]

[0180] Table 3: List of histological parameters used to score liver injury.

[0181] 2-B Results:

[0182] The intestinal permeability of the unchallenged and unvaccinated birds (negative control) was 0.26 μg / mL, and the intestinal permeability of the challenged and unvaccinated birds (positive control) was 0.31 μg / mL. When vaccinated with the commercial vaccine, the permeability was 0.26 μg / mL, as was the negative control, confirming the efficacy of the vaccine. In addition, when vaccinated in ovo with the VXN-E. coli formulation, all birds showed low permeability (0.18 μg / mL), which was lower than the permeability of the negative control, although not significantly so.

[0183] Enteroanti-E. coli A-sIgA (LPS)

[0184] Feces were analyzed for anti-LPS secretory IgA against E. coli by ELISA. Negative control birds achieved an absorbance of approximately 0.05 AU, while positive control birds achieved 0.095 μg / mL, indicating that oral challenge did not induce intestinal IgA secretion. Furthermore, for birds vaccinated mucosally with the commercial vaccine, the OD remained at 0.055 AU, similar to the negative control, indicating that the vaccine failed to stimulate a mucosal humoral response. In contrast, birds vaccinated in ovo with the VXN-E. coli formulation displayed a significantly higher OD of 0.16 AU.

[0185] B-Histopathology of the liver

[0186] Liver lesion scores were measured 6 days after challenge. Birds in the negative control group had an average score of approximately 1, indicating discrete hyperplasia. In contrast, the unvaccinated birds in the positive control group had an average score of 2.35, indicating liver damage and necrosis induced by E. coli infection. When vaccinated with the commercial vaccine, the average lesion score of the birds was 1, the same as in the negative control group. Birds vaccinated in ovo had an average lesion score of <1. These results show that commercial vaccines and in ovo vaccination prevent E. coli-induced liver damage.

[0187] in conclusion:

[0188] This first trial showed that in ovo vaccination with the VXN-E. coli vaccine protected birds against E. coli liver infection and induced sIgA secretion in the intestine against the bacteria.

[0189] Example 3: In ovo vaccination against lethal E. coli challenge

[0190] This second trial was identical to the first in ovo trial in terms of schedule, animals per group, and treatments, but with a lethal E. coli challenge. Analysis then focused on the protection provided by the vaccine against bacterial loads in representative organs, physiological abnormalities, and subsequent mortality observed in each group.

[0191] 3-A. Materials and Methods

[0192] A - Attack

[0193] On D10, all animals, including the negative control group, received orally attenuated live MassI-H120 infectious bronchitis virus (IBV) vaccine at a dose 100 times the manufacturer's recommended dose.

[0194] In D14, with 4.2x10 12 Each animal in a defined group was challenged with CFU of E. coli (strain 19501) in the air pouch at a rate of 100 μL / bird.

[0195] Figure 7 The general timetable is described in detail in .

[0196] B-Analysis

[0197] Intestinal permeability:

[0198] Intestinal permeability was assessed by oral administration of FITC-dextran, a non-absorbable fluorescent marker (FITC-dextran, 3000 kDa to 4000 kDa) and detected in plasma / serum to monitor gastrointestinal epithelial integrity ( et al., 2015 ).

[0199] Detection and quantification of Escherichia coli:

[0200] Detection and quantification of E. coli were performed by MPN (most probable number) according to standard ISO 7251:2005. E. coli counts were determined according to standard microbiological methods (dilution in enrichment medium and then plating in selective / different media). Briefly, the sample was enriched in buffered peptone water (BPW), then enriched in EC broth, and finally enriched in EMB and MacConkey agar. Before incubation, the sample was serially diluted in triplicate in EPB to enable quantification by the most probable number technique (Blodgett et al., 2015). For E. coli detection, only the serial dilution step was omitted. Suspicious colonies isolated were subjected to biochemical testing and confirmation.

[0201] Intestinal histology:

[0202] Birds were euthanized and intestinal samples were collected and fixed according to the method of Rebel et al. (2011). Ileal samples were embedded in paraffin and mounted on slides. All histopathological evaluations and readings were performed microscopically by an experienced veterinary histopathologist.

[0203]

[0204] Table 4: List of histological parameters used to evaluate intestinal lesions (ileum).

[0205] 3-B. Results

[0206] A- hatching rate

[0207]

[0208] Table 5: Percentage of chicks in the in ovo vaccinated groups relative to the chicks in the non-vaccinated groups (negative control, positive control, commercial vaccine).

[0209] In this study, hatchability was measured to assess the safety of the in ovo VXN / E. coli vaccine. Therefore, prior to randomization into groups (negative control, positive control, and commercial vaccine), the hatchability of vaccinated eggs (n=60) was compared with that of unvaccinated eggs (n=180). A similar percentage of chicks was observed between vaccinated eggs (78.3%) and unvaccinated eggs (81.2%). Therefore, the vaccine formulation was safe, as it had no effect on hatchability.

[0210] B - Bird survival after lethal challenge

[0211] At D14, 4.2×10 12 CFU and challenged with E. coli directly in the air sac. Bird survival after lethal challenge is depicted in Figure 8 This high dose had an impact on bird survival, with a 26% mortality rate observed in unvaccinated birds. Furthermore, mortality increased to 36% in birds vaccinated with the commercial mucosal vaccine, indicating that the vaccine did not induce protection against lethal E. coli infection. On the other hand, for birds vaccinated in ovo with the VXN / E. coli vaccine, mortality was only 10%, indicating better protection against infection.

[0212] C-Quantification of E. coli in air sacs by MPN

[0213] Infection was assessed by quantifying bacteria in the air pouch by MPN. The bacterial load in the air pouch, as measured by MPN, was Figure 9 The negative control birds showed only a small number of bacteria in the air sac relative to the natural bacterial flora. The non-immunized challenged birds had higher levels of E. coli (10 4 MPN / g), which confirmed the efficacy of the challenge. 6 MPN / g) were significantly higher in the air sacs of birds, confirming the survival results. However, similar to the survival results, the infection rate was significantly higher in birds treated with the E. coli / LNP vaccine (3×10 3 MPN / g) in ovo vaccinated birds, confirming the efficacy of the vaccine in preventing infection with this bacterium.

[0214] D-Intestinal Permeability

[0215] The intestinal permeability of the unchallenged and unvaccinated birds was 0.22 μg / mL and the intestinal permeability of the challenged and unvaccinated birds was 0.18 μg / mL. Figure 10 When vaccinated in ovo with the commercial vaccine or with the VXN / E. coli vaccine, the permeability was significantly reduced to 1.2 μg / mL, indicating vaccine-induced protection.

[0216] E-Intestinal Histopathology

[0217] Ileal lesion scores were measured 6 days after challenge. Figure 11As expected, birds in the negative control group received an average score of less than 1 (0.25), indicating a healthy ileum with a normal appearance. In contrast, non-vaccinated birds in the positive control group had a significantly higher average score of 1.7, indicating ileal lesions induced by E. coli infection, as well as vascular conditions and desquamation. Surprisingly, when the birds were vaccinated with the commercial vaccine, the lesions were significantly worse, with an average score of 2. In contrast, birds vaccinated in ovo had an average score of 1, indicating protection against E. coli-induced intestinal lesions.

[0218] in conclusion:

[0219] This second trial showed that the in ovo VXN-E. coli vaccine protected birds from mortality induced by a lethal E. coli infection. Furthermore, the vaccine reduced bacterial loads in the air sac and intestinal lesions caused by infection, confirming the vaccine's value.

[0220] Example 4: Intramuscular Salmonella vaccination trial in laying hens

[0221] 2-A. Materials and Methods

[0222] A-Vaccine Preparation

[0223] The vaccine is made from an inactivated strain of Salmonella Enteritidis mixed with lipidated maltodextrin nanoparticles (LNPs). Briefly, Salmonella strain SE147 was inactivated and protein content was measured by μBCA assay. Finally, 200 μg of killed bacteria were mixed with either LNPs (a formulation called "Vaxinano 1") or non-crosslinked LNPs (a formulation called "Vaxinano 2") at a ratio of 200 μg protein per dose of vaccine. Non-crosslinked LNPs are composed of linear cationic maltodextrins with an anionic core.

[0224] B-animals

[0225] For this experiment, a total of 84 LSL chickens (from a commercial breeder farm) were randomly assigned to 12 pens (7 birds / pen) as described in Table 7. Sera were collected and tested for Salmonella antibody titers (performed by DGZ using the BioChek kit).

[0226]

[0227] Table 6: Identification of experimental groups.

[0228] C-Vaccination

[0229] At W12, all chickens were vaccinated IM in the breast with 500 μL saline solution or 500 μL Vaxinano 1 or Vaxinano 2 formulations (containing 200 μg Salmonella protein) or commercial vaccine. One month later, at W16, the animals received a second dose of the same vaccine formulation.

[0230] D-Attack

[0231] One month after booster immunization, 500 μL of 1.3x10 8 All animals were challenged intravenously with CFU of Salmonella enterica SE147.

[0232] The general timetable is depicted in Figure 12 middle.

[0233] E-Sample

[0234] From W20 to W25, eggs were collected and bacteriologically analyzed for Salmonella. At W25, all chickens were euthanized, and serum and liver were collected and stored at -20°C. Spleen and cecum were bacteriologically analyzed for Salmonella.

[0235] F-Analysis

[0236] Egg production: Eggs were collected daily after challenge (except Saturday) and stored at 4° C. The number of eggs per group is reported.

[0237] Infection: Infection was quantified by qPCR in the spleen and cecum of each chicken at W25.

[0238] 2-B. Results

[0239] A-Egg Production

[0240] The average number of eggs laid in each group was counted every day after the test. Figure 13 Hens vaccinated with the mock vaccine laid a low number of eggs after challenge, an average of 2.6 eggs per day, confirming infection of the birds. In contrast, birds vaccinated with Salenvac laid significantly more eggs than the control group, an average of 4.9 eggs per day (p<0.001), indicating protection against challenge. Similarly, both Vaxinano formulations enabled hens to lay significantly more eggs, an average of 4.3 eggs per day for Vaxinano 1 (p<0.05) and an average of 4.7 eggs per day for Vaxinano 2 (p<0.001), indicating comparable protection against challenge.

[0241] B-Infection of the cecum and spleen

[0242] Infection was assessed by quantification of bacterial loads in the cecum and spleen.

[0243] Post-challenge infection was assessed by quantification of bacterial loads in the cecum and spleen. Figure 14 Chickens injected with saline had significant infection, with an average infection of 400 CFU / g, but more than 50% of the birds had infections above 1000 CFU / g. In contrast, all birds vaccinated with the Vaxinano formulation or with Salenvac had significantly lower infection, with average infection below the threshold for birds vaccinated with Vaxinano 1. This confirms the strong protection provided by IM vaccination.

[0244] Finally, the infection was quantified in the spleen. This quantification is depicted in Figure 15 Despite infection below the cecum, 77% of birds vaccinated with saline solution remained positive in the spleen, compared to only 35% of birds vaccinated with Vaxinano 1, 36% of birds vaccinated with Vaxinano 2, and 35% of birds vaccinated with the commercial vaccine, confirming the protection provided by IM vaccination.

[0245] in conclusion:

[0246] This trial demonstrated that killed whole Salmonella / NP vaccines administered intramuscularly protected birds against Salmonella enterica challenge, regardless of formulation, and enabled the birds to lay significantly more eggs than non-immunized animals.

[0247] Example 5: Mucosal vaccination against Escherichia coli disease

[0248] The trial was planned in the same manner as the in ovo trial. Analysis then focused on the protection provided by the vaccine against target organ infection, physiological abnormalities and mucosal antibody titers.

[0249] 5-A Materials and Methods:

[0250] A-Vaccine Preparation

[0251] The vaccine is made from three strains of inactivated Escherichia coli bacteria mixed with lipidated maltodextrin nanoparticles (LNPs). Strains O78:K80, O1:K1, and O2:K1 were inactivated with 0.4% formaldehyde, and protein content was measured by BCA assay. Finally, 33.3 μg of each strain was then mixed with LNPs at a ratio of 100 μg protein per vaccine dose.

[0252] B-animals

[0253] All animal work has been reviewed and approved by the Animal Research Ethics Committee of Imunova Análises Biológicas, protocol number 06 / 2021.

[0254] For this experiment, 150-day-old chickens were obtained from a commercial hatchery and randomly divided into five experimental groups, placed in individual isolating units within Imunova, and treated according to the following table:

[0255]

[0256] Table 7: Identification of experimental groups.

[0257] All groups, including the negative control, were orally infected with live attenuated Mass I-H120 infectious bronchitis virus (IBV) vaccine at a dose 100x the manufacturer's recommended dose to sensitize the animals to E. coli challenge.

[0258] 2 The birds were orally infected with 10<8 > CFU of E. coli at a dose of 100 μL / bird. Infection was confirmed by microbial recovery of bacteria from the inoculum.

[0259] C-Vaccination

[0260] 'VXN vaccine SC', 'VXN mucosal vaccine' and 'commercial vaccine' groups ( coli, Zoetis) received a primary immunization on D1 and a second dose on D12. For the "VXN mucosal vaccine," the doses were administered in the eyes, beak, and nostrils, while for the "commercial vaccine," the doses were administered in the drinking water.

[0261] D-Attack

[0262] The challenge strain was a field isolate that was confirmed to be APEC by PCR identification of five pathogenicity genes (iuaT, iroN, ompC, iss, hly). It was also confirmed to belong to the genetic lineage F by the typing method described by Cleremont (Cleremont et al., 2013). All animals received a 100x dose of attenuated IBV vaccine (Massachusetts H-120 strain, I, Zoetis). E. coli challenge was performed in all groups except the "negative control" and 108 CFU / bird was used.

[0263] The overall protocol timeline for the in ovo vaccination trial is shown in Figure 16 middle.

[0264] E-Analysis

[0265] Quantification of anti-E. coli IgA:

[0266] The production of specific anti-E. coli IgA was assessed by ELISA. Briefly, samples were diluted in 1% casein in PBS. ELISA plates were coated with E. coli LPS (field isolate). The plates were then washed three times with 200 μL / well of PBS + 0.05% Tween-20 for 5 minutes each wash. The wells were blocked with 1% casein in PBS. The samples were tested by serial dilution. The plates were washed and anti-chicken IgA (Bio-Rad) diluted in 0.1% casein was added. After washing, the test was developed with a single solution of TMB (Life Technologies). The absorbance was read at 450 nm and quantified using a proprietary method / kit developed by Imunova.

[0267] Quantification and Detection of E. coli Infection:

[0268] Based on standard ISO 7251:2005, the detection of E. coli was performed by MPN (most probable number). E. coli counts were determined according to standard microbiological methods (dilution in enrichment medium and then plating in selective / differential medium). In brief, the sample was enriched in buffered peptone water (BPW), then enriched in EC broth, and finally plated on EMB and MacConkey agar. Before incubation, the sample was serially diluted in triplicate in BPW to enable quantification by MPN technology (Blodgett et al., 2015). In the detection of E. coli, only the serial dilution step was omitted. Suspicious isolated colonies were subjected to biochemical testing and confirmation.

[0269] Lung Histology:

[0270] Birds were euthanized and lung samples were removed and fixed. Samples were embedded in paraffin and mounted on glass slides. All histopathological evaluations and readings were performed microscopically by an experienced veterinary histopathologist.

[0271]

[0272]

[0273] Table 8: List of histological parameters used to assess lung injury scores

[0274] 5-B: Results

[0275] Presence of E. coli in the airways (MPN)

[0276] Infection was assessed by quantifying the number of birds infected per MPN in the trachea and air sacs. Bacterial infection in the trachea and alveoli was measured as Figure 17 As shown. Three unchallenged birds were infected in the trachea, and seven were infected in the air sacs, likely due to the presence of naturally occurring pathogenic E. coli in the environment. In contrast, unvaccinated, challenged birds had more infections in the trachea, confirming the efficacy of the challenge. The same number of birds in the group receiving the commercial vaccine were infected relative to the infected control group, indicating a lack of protection. However, among birds vaccinated with the VXN mucosal vaccine, only one bird was infected in the trachea, and no birds were infected in the air sacs. This confirms that the mucosally administered VXN E. coli vaccine protects birds from the effects of challenge.

[0277] Presence of anti-E. coli sIgA in feces

[0278] The feces were analyzed for E. coli anti-LPS secretory IgA by ELISA. The results of the anti-E. coli sIgA analysis are depicted in Figure 18 From D16 to D28, the antibody titers of birds in the negative control were the same as those in the positive control, indicating that oral challenge does not induce intestinal IgA secretion. However, a significant increase in antibody titers was observed on D21 for both birds vaccinated with the VXN E. coli formulation and with the commercial vaccine mucosally. Therefore, mucosal vaccines are able to induce humoral responses in the intestine.

[0279] Pulmonary clinical score

[0280] Lung lesion scores were measured from D16 to D28. Lesion scores are depicted on Figure 19 Although they had the lowest histopathological scores from D16 (score = 2) to D28 (score = 1.7), birds in the negative control group showed bronchial mucositis, mucofibrinous exudate and polynuclear neutrophil infiltration, which may be related to natural infection ( Figure 17 ). In addition, due to the E. coli challenge, the non-vaccinated challenged birds in the positive control group had the highest scores, 3.4 at D16 and 2.7 at D28. In contrast, the birds vaccinated mucosally with the VXN-E. coli formulation had an average lesion score of 2.3 at D16, which was lower than the commercial vaccine (score = 2.9). Finally, on D28, both groups of mucosally vaccinated birds had scores comparable to the negative control (1.6 for the commercial vaccine and 1.8 for the VXN vaccine), indicating protection against lung lesions induced by E. coli infection.

[0281] Example 6: Optimizing formulations using different cationic particles

[0282] Studies were conducted on inactivated whole E. coli strains to compare formulations prepared with different maltodextrin-based particles, measuring their in vitro uptake by immune cells.

[0283] 6-A Materials and Methods:

[0284] A-Vaccine Preparation

[0285] Vaccine formulations were prepared in which one of the inactivated E. coli strains used in the vaccine (11101) was mixed with maltodextrin (NP+) or lipidated nanoparticles (LNP), as well as cationized but non-crosslinked single (NP+NR) or lipidated maltodextrin (LNP-NR). The nanoparticles partially or highly partially encapsulated the bacteria.

[0286] B-particle synthesis and characterization

[0287] NP+ is a nanoparticle synthesized by cationic and cross-linked maltodextrin. More specifically, the synthesis consists of maltodextrin (Roquette, France) dissolved in a 2M NaOH solution under magnetic stirring and room temperature. Epichlorohydrin (Merck group, France) as a cross-linking agent and glycidyl trimethylammonium (GTMA, Merck group, France) as a cationizing agent are then added. The resulting gel is then neutralized with acetic acid and ground in an ultrahigh pressure homogenizer (LM20, Microfluidics, France). The crushed material is then purified by tangential flow filtration (AKTA flux 6, GE Healthcare, France) via a 750kDa membrane (GE Healthcare, France) to obtain purified NP+. These NP+ can encapsulate antigens from various pathogens (viruses, bacteria or parasites) and deliver them to immune cells (1).

[0288] Similarly, LNP is NP+, in which a core of anionic phospholipids (DPPG) has been added. More specifically, a solution of dipalmitoylphosphatidylglycerol (PPG, Lipoid, Germany) is dissolved in Solutol and then injected into the NP+ solution at a mass percentage of 70% under stirring. Phospholipids are incorporated into the core of the nanoparticles to form LNPs. These LNPs can also encapsulate antigens from various pathogens (viruses, bacteria or parasites) and deliver them to immune cells (2-4).

[0289] Finally, NP+NR and LNP-NR are their respective equivalents, synthesized according to the same synthetic protocol but without the cross-linker, thus forming linear cationic polymers.

[0290] The particles were characterized according to their size by dynamic light scattering (DLS) and according to their surface charge (or zeta potential) by electrophoretic light scattering (ELS) using a Zetasizer Nano ZS (Malvern, France).

[0291] C-Inactivation of E. coli bacteria

[0292] Bacteria were inactivated with 0.4% formaldehyde and then purified by centrifugation. The protein content of intact bacteria was measured by micro-BCA assay. Formulations were prepared by mixing the killed bacteria with an aqueous solution of particles at different weight ratios (100 μg of E. coli protein with 1 μg, 5 μg, 10 μg, 30 μg, 50 μg, or 100 μg of particles).

[0293] D- Delivery of E. coli into cells via particles

[0294] The ability of particles to enhance phagocytosis-mediated uptake of inactivated intact E. coli by epithelial cells and entry into macrophages was assessed by flow cytometry.

[0295] Labeling proteins with fluorescein:

[0296] Inactivated E. coli were labeled with fluorescein isothiocyanate (FITC) by mixing 5 mg of E. coli with 50 μg of FITC (1% w / w, Merck, France) in sodium carbonate buffer at pH 8.3 for 2 hours. They were then dialyzed on a 10 kDa dialysis cassette (Thermo Fisher, France). The protein content of intact bacteria was measured by microBCA assay (Pierce, France). The labeled bacteria were then associated with particles at different weight ratios.

[0297] E-Flow Cytometry:

[0298] The H292 cell line was seeded in a 24-well plate at 50,000 cells / well and treated after 3 days of culture. The THP-1 cell line was seeded in a 24-well plate at 100,000 cells / well and differentiated into macrophages with 20 ng / mL of PMA for 24 hours. After replacing the culture medium, the cells were incubated with 1 μg of killed bacteria (alone or in combination with particles) for 4 hours. The cells were then washed with phosphate-buffered saline (PBS), harvested with trypsin, and analyzed by flow cytometry on an Attune Nxt (ThermoFisher, France).

[0299] 6-B Results:

[0300] Particle characterization

[0301] Z-average value (nm) Quantity (nm) Zeta potential (mV) <![CDATA[NP + ]]> 92±7 33±4 36±5 <![CDATA[NP + NR]]> 50±3 17±3 32±2 LNP 83±15 36±5 39±2 LNP-NR 54±12 23±9 38±3

[0302] Table 9: Characterization of the size (Z-average value and number) of the different particles and their surface charge (zeta potential).

[0303] After the synthesis of the different particles, the physicochemical properties of the particles were analyzed (Table 9). NP+ had a diameter of 33 nm and a surface charge of 36 mV, and LNP had a diameter of 36 nm and a surface charge of 39 mV, indicating that the phospholipids were not associated with the particles on the surface but within the maltodextrin structure of the particles.

[0304] NP+NR had a diameter of 17 nm and a surface charge of 32 mV, and LNP-NR had a diameter of 23 nm and a surface charge of 38 mV, again indicating association of phospholipids within the maltodextrin structure.

[0305] In the absence of a cross-linker, the resulting particles appear to have a smaller diameter. Indeed, in the absence of a cross-linker, maltodextrin in solution should remain primarily linear, but it can also fold on itself through hydrophobic bonds to form the nanoparticles detected during analysis.

[0306] Phagocytosis of Escherichia coli by cells after association with particles

[0307] The delivery of particles to Escherichia coli was first evaluated on respiratory epithelial cells (H292). In the absence of particles, the bacteria were not endocytosed by the cells ( Figure 2 When bacteria were coated with particles, their uptake was greatly increased, reaching 20%-50% positive cells regardless of the particle type used. Furthermore, improved phagocytosis was observed as early as a 1:0.01 ratio, demonstrating that a small number of particles partially encapsulating bacteria is sufficient to improve intracellular delivery.

[0308] Similarly, the phagocytosis of E. coli by the particles was evaluated on differentiated macrophages (THP-1). In the absence of particles, bacteria were phagocytosed by 0.2% of the cells ( Figure 3 When bacteria were coated with particles, their phagocytosis increased to 4%-15% of positive cells, depending on the ratio. Furthermore, improved phagocytosis was observed from a 1:0.01 ratio, again demonstrating that a small number of particles coating bacteria is sufficient to improve bacterial delivery.

[0309] in conclusion

[0310] Bacterial adhesion and uptake by respiratory epithelial cells and macrophages can be achieved by single (NP + ) or lipidated (LNP) cationized maltodextrin nanoparticles and by single (NP+ The enhancement was equivalent between the NPs. Low doses of particles (1 / 10 to 1 / 100 of the bacteria in mass %) were sufficient to improve this delivery to cells.

Claims

1. An adjuvant-free vaccine composition comprising cationic nanoparticles composed of a polysaccharide core and at least one inactivated bacterium, characterized in that The bacteria are intact, and the cationic nanoparticles encapsulate the bacteria.

2. The vaccine composition of claim 1 , wherein the cationic nanoparticles encapsulate the bacteria at a bacterial protein:NP ratio (by weight) greater than or equal to 1.

3. The vaccine composition of claim 2, wherein the bacterial protein:NP ratio is greater than or equal to 5 (by weight).

4. The vaccine composition according to one of claims 1 to 3, wherein the nanoparticles are cationic nanoparticles consisting of a non-lipid loaded cross-linked polysaccharide core consisting of: (i) a polysaccharide selected from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannans (guar gum), (ii) at least one cationic ligand selected from primary, secondary or tertiary amines or quaternary ammonium, and (iii) a cross-linking agent selected from epichlorohydrin, dicarboxylic acids or acid chlorides, such as sebacic acid.

5. The vaccine composition according to one of claims 1 to 3, wherein the nanoparticles are cationic nanoparticles consisting of a non-crosslinked, non-lipid-loaded polysaccharide core consisting of: (i) a polysaccharide selected from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannans (guar gum), and (ii) at least one cationic ligand selected from primary, secondary or tertiary amines or quaternary ammonium.

6. The vaccine composition according to claim 1 , wherein the nanoparticles are cationic nanoparticles consisting of a phospholipid-loaded cross-linked polysaccharide core consisting of: (i) a polysaccharide selected from starch, dextran, chitosan, dextrin, maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum), (ii) at least one cationic ligand selected from primary, secondary or tertiary amines or quaternary ammonium, (iii) a cross-linking agent selected from epichlorohydrin, dicarboxylic acids or acid chlorides, such as sebacic acid, and (iv) an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine or diacylphosphatidylinositol.

7. The vaccine composition according to one of claims 1 to 3, wherein the nanoparticles are cationic nanoparticles consisting of a phospholipid-loaded non-crosslinked polysaccharide core consisting of: (i) a polysaccharide selected from starch, dextran, chitosan, dextrin, maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum), (ii) at least one cationic ligand selected from primary, secondary or tertiary amines or quaternary ammonium, and (iii) an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine or diacylphosphatidylinositol.

8. The vaccine composition according to one of claims 4 to 7, wherein the cationic polysaccharide core comprises maltodextrin and glycidyltrimethylammonium.

9. The composition according to one of claims 6 or 7, wherein the cationic polysaccharide core is loaded with an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine or diacylphosphatidylinositol.

10. Vaccine composition according to one of the preceding claims, comprising at least 2 different strains of bacteria.

11. The vaccine composition according to claim 1 , wherein the bacteria is selected from the group consisting of: Salmonella enterica serotype Typhi; Streptococcus pneumoniae; Haemophilus influenzae type b; Mycobacterium tuberculosis; extraintestinal pathogenic Escherichia coli (ExPEC); enterotoxigenic Escherichia coli (ETEC); Salmonella enterica serotype Paratyphi A; Neisseria gonorrhoeae; Clostridium difficile; Campylobacter spp.; Shigella spp.; Staphylococcus aureus; Helicobacter pylori.

12. Vaccine composition according to one of the preceding claims, which is used in a form suitable for intramuscular, transmucosal or in ovo administration.

13. Vaccine composition according to one of the preceding claims for use in preventing bacterial infections in mammals or birds.

14. Vaccine composition according to one of the preceding claims for use in the prevention of salmonellosis, colibacillosis or campylobacteriosis.

15. A composition according to claim 14 for use in ovo prevention of bacterial infection in poultry embryos.

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