Vaccine compositions comprising a system for delivery of inactivated whole bacteria via cationic polysaccharide nanoparticles without any adjuvant - Patent Application 20070122999

Abstract

The present invention relates to the field of vaccine compositions, and more particularly to prophylactic vaccine compositions for mammals and birds comprising killed whole bacteria, which are coated with cationic agents, in particular cationic nanoparticles.

Description

[Technical Field]

[0001] The present invention relates to the field of vaccine compositions, and more particularly to prophylactic vaccine compositions for mammals and birds comprising killed whole bacteria, which are coated with cationic agents, in particular cationic nanoparticles. [Background technology]

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

[0003] To prevent the risk of infection in livestock, prophylactic vaccines have been developed with the aim of immunizing individuals and avoiding the risk of infection and the resulting health consequences.

[0004] Prophylactic vaccination involves inducing an immune response to activate immune defenses in healthy individuals who have not yet come into contact with a pathogen. To achieve this, an antigen, such as a pathogen or a pathogen fragment, is presented to the individual's immune cells. This presentation activates adaptive immune cells, B lymphocytes, and T lymphocytes, which expand and produce antibodies that neutralize and eliminate the antigen and / or cellular responses that destroy infected cells. This mechanism induces immunological memory, allowing the individual to be protected the next time the pathogen is encountered. Thus, prophylactic vaccination allows healthy individuals to be immunized to protect them from future disease.

[0005] For this mechanism to be effective, it is essential that the pathogen be identified as an invader by the individual's immune system so that a protective immune response against infection can be developed.

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

[0007] Patent document 1 relates to a serotype of Salmonella enterica serogroup C1 for use in protecting poultry against disorders resulting from Salmonella enterica infection. This Salmonella enterica serotype is in an inactivated form. The serotype is used to produce a vaccine that may be multivalent. Chicks are vaccinated at 30 hours of age. This serotype is used to formulate a vaccine containing an adjuvant, such as about 25% v / v aluminum hydroxide.

[0008] Patent Document 2 discloses a vaccine for protecting poultry against colibacillosis infection, which contains as an active ingredient Escherichia 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 inoculated with the vaccine via the cloaca. However, the vaccine can also be administered in a conventional manner, for example, intramuscularly, intravenously, or subcutaneously. The essence of the present invention is the use of ultrasonic cell membrane disruption in the production of a vaccine against colibacillosis in poultry.

[0009] Patent document 3 discloses a process for preparing a vaccine composition from at least one freeze-dried antigen, which process comprises: - providing an aqueous solution comprising cationic nanoparticles consisting of a cationic polysaccharide core; - adding the lyophilized antigen to the aqueous solution; - incubating the resulting composition at room temperature.

[0010] The process according to this document involves the presence of a partial or total 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.

[0011] Patent Document 4 discloses a nanoparticle composition for use as a vaccine against Salmonella enteritidis in poultry. The vaccine composition contains highly immunogenic protein antigens (outer membrane proteins (OMPs) or whole antigen proteins (KAg)) and flagellar proteins extracted from killed Salmonella enteritidis. These antigens are entrapped inside polyanhydride or chitosan nanoparticles.

[0012] Patent Document 5 discloses a composition comprising a polyacrylic acid mucosal adjuvant and / or an inactivated antigen derived from a respiratory or intestinal bacterium or virus. In addition, the composition may include a cell suspension containing non-cationic polyacrylic acid particles ranging in size from 250 nm to 10 microns.

[0013] Anthony Pavic and colleagues described a study on the development of an autologous trivalent inactivated vaccine. The trivalent vaccine was produced from an equal volume cell suspension (3 x 108 CFU / mL), combined with aluminum hydroxide adjuvant, and administered intramuscularly to the breast of 12- to 17-week-old hens.

[0014] The availability of vaccination campaigns is often limited by the cost of the dose of the vaccine composition. Indeed, current vaccines are often expensive, which limits their widespread use, especially in agriculture, where the number of individuals to be treated is considerable. Vaccines are commercially available to prevent infections caused by bacteria in mammals and birds, but these vaccines are unsatisfactory because they do not provide complete, effective protection at an affordable cost. In addition, the compositions proposed in the prior art contain all vaccine adjuvants that are necessary for their effectiveness, but whose side effects have been widely reported. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] European Patent No. 2911688(A1) [Patent Document 2] European Patent No. 0256792(A2) [Patent Document 3] International Publication No. 2022 / 008848 [Patent Document 4] U.S. Patent Application Publication No. 2021 / 093705 [Patent Document 5] International Publication No. 2021 / 021778 Summary of the Invention

[0016] Disclosure of the Invention The present inventors have developed an adjuvant-free prophylactic vaccine composition for immunizing mammals and birds, particularly poultry, against pathogenic bacteria. In particular, the present inventors have developed a novel delivery system in which at least killed whole pathogenic bacteria are coated with cationic nanoparticles (NPs), enhancing the cellular uptake mechanism. This has the effect of improving the mechanism for presenting bacterial antigens to immune cells and thus activating the immune system more rapidly and effectively. Advantageously, the vaccine composition can be multivalent to induce broad-spectrum protection. Therefore, the vaccine composition can be used to produce combination vaccines.

[0017] Endocytosis is the mechanism by which extracellular material enters cells by invagination of the plasma membrane followed by the formation of isolated vesicles in the cytoplasm. When endocytosis is carried out by specialized immune cells (polymorphonuclear neutrophils, macrophages, dendritic cells), it is referred to as phagocytosis.

[0018] The prophylactic vaccine can be used to treat avian salmonellosis, colibacillosis, campylobacteriosis, or any other bacterial infection.

[0019] Thus, the present invention relates to an adjuvant-free prophylactic vaccine composition, particularly for mammals and birds, more particularly for poultry, comprising cationic nanoparticles consisting of a polysaccharide core and at least one inactivated bacterium, wherein the bacterium is intact and the cationic nanoparticles coat the bacterium. This composition limits the risk of contamination with at least one disease resulting from infection with a pathogenic bacterium. In a particular embodiment, the present invention relates to a multivalent prophylactic vaccine composition, particularly for mammals and birds, more particularly for poultry, such as layer hens, broilers, turkeys, ducks, guinea fowl, ostriches, emus, quail, etc., intended for the treatment of salmonellosis or colibacillosis.

[0020] The present invention also relates to the use of the vaccine composition for the prevention of bacterial infections, in particular colibacillosis, salmonellosis and campylobacteriosis.

[0021] Advantages of the Invention Surprisingly, the present inventors have demonstrated that administration of a vaccine composition comprising inactivated whole pathogenic bacteria coated with a cationic agent, such as, for example, cationic nanoparticles, constitutes a novel antigen delivery system for effective immunization against pathogens of bacterial origin.

[0022] Cationic nanoparticles are known from the prior art to have the ability to enhance immune cell endocytosis by delivering small antigens, such as protein antigens derived from fragmented pathogens, with sizes on the order of 5-15 nanometers. In this configuration, a significant amount of nanoparticles is required to internalize each element of the fragmented bacterial cells. These delivery systems use cationic nanoparticles combined with a whole or partial extract of fragmented pathogenic bacteria, which is contained in the core of the nanoparticles. This "antigen in nanoparticle core" configuration requires the use of more nanoparticles (by weight) than antigen, preferably 10-100 times more nanoparticles than antigen. This can be even higher when using, for example, PLGA nanoparticles or liposomes. This prior art delivery system cannot deliver whole bacteria; it can only deliver small antigens, with sizes on the order of 5-15 nanometers, whereas whole cells have a minimum size of about 1-10 microns.

[0023] In this study, the inventors unexpectedly demonstrated that cationic nanoparticles enable the phenomenon of phagocytosis of whole bacterial cells. In other words, the inventors have developed a delivery system capable of delivering bacteria at least 100 times larger than protein antigens. Therefore, this is a new delivery system in which bacteria are coated with positively charged nanoparticles according to the present invention, allowing the bacteria to interact with the cell membrane and enter immune cells via phagocytosis. In fact, this combination mimics the process by which viruses enter cells by modulating the ionic charge provided by the nanoparticles. By coating whole bacteria with nanoparticles according to the present invention, the "whole bacterium-coating nanoparticle" combination becomes positively charged. This positive charge promotes interaction with the cell membrane, allowing the whole bacterium-nanoparticle complex to enter via phagocytosis. This process is made possible by the specific combination developed by the applicants, namely, nanoparticles coating whole inactive bacteria. The combination of whole bacteria coated with nanoparticles has not been described before.

[0024] This novel delivery system offers the innovative advantage of allowing the whole bacteria to enter the cell via the vaccine composition according to the invention. Advantageously, a smaller amount of nanoparticles can be used relative to the weight of bacterial protein; in fact, the amount of NPs can be 3-100 times less than the amount of bacterial protein.

[0025] Interestingly, therefore, even small amounts of nanoparticles are sufficient to allow phagocytosis of whole bacterial cells and effective immunization.

[0026] Reducing the number of nanoparticles used for vaccination significantly reduces the cost of producing the vaccine composition, making vaccination against pathogenic bacteria accessible on a large scale, especially in areas where cost is a real limitation for vaccination campaigns. An example is poultry farming with in ovo vaccination.

[0027] Furthermore, one advantage of the vaccine composition according to the present invention is that it can be multivalent. In other words, it can contain at least two bacterial strains, each involved in preventing infection. Thus, the present invention facilitates the creation of combination vaccines.

[0028] According to an embodiment of the present invention, the vaccine composition also makes it possible to obtain cross-immunity: in fact, the vaccine composition may contain bacteria that induce immunity against variants of the strain in question.

[0029] One targeted application of this technology is in ovo vaccination. This approach is innovative: previously described vaccine strategies do not propose administering inactive whole bacteria directly into eggs. Here, the combination of inactive whole bacteria partially coated with cationic nanoparticles has proven highly effective in terms of vaccine protection and has no adverse effects on hatching or chicks. Furthermore, pre-hatch intervention reduces the risk of intra-farm (chick-to-chick) contamination and reduces intra-farm disease transmission. In particular, the inventors have shown that a vaccine composition containing three different strains of E. coli administered in ovo to chicks can protect chicks from colibacillosis-type infections at non-lethal and even lethal doses in bacterial challenge experiments. Bacterial load is reduced, and hatch rates are comparable to those of unvaccinated eggs.

[0030] Meanwhile, in another application, intramuscular injection of a vaccine composition comprising a Salmonella bacterial strain and cationic nanoparticles (LNPs) in laying hens reduces the bacterial load and the hens lay more eggs than non-immunized hens.

[0031] The vaccine composition does not contain any adjuvants, thereby avoiding undesirable effects. This is advantageous because inorganic adjuvants (i.e., inorganic salts such as aluminum salts) remain in the body for a very long time. The nanoparticles act as delivery agents for killed bacteria to immune cells, inducing a protective response against infection.

[0032] In the case of in ovo vaccination, the fact that no molecules are introduced that may interfere with chick development in ovo contributes to the effectiveness of the vaccine approach, as the vaccine does not interfere with chick development or hatching.

[0033] The vaccine composition can be administered in ovo, but also mucosally (oral, ocular, nasal) or intramuscularly. Furthermore, the vaccine approach according to the invention can be carried out in mammals as well as in birds, especially poultry.

[0034] 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 inactivated bacteria also has the advantage that it is a simpler antigen to characterise than partial or whole antigen extracts. DETAILED DESCRIPTION OF THE INVENTION

[0035] 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, wherein the bacterium is intact and the cationic nanoparticles coat the bacterium.

[0036] In particular embodiments, the vaccine composition is directed to mammals and birds.

[0037] In another particular embodiment, the cationic nanoparticles coat the bacteria at a bacterial protein:NP weight ratio of 1 or more. In preferred embodiments, the bacterial protein:NP weight ratio is 2 or more, preferably greater than 5, even more preferably greater than 10, or even 50 or 500.

[0038] "Cationic nanoparticles consisting of a cationic polysaccharide core" refers to solid nanoparticles (NPs) that contain a cationic polysaccharide core. The NPs may or may not be crosslinked. The core may or may not be filled with anionic phospholipids. The NPs are not surrounded by any phospholipid layer.

[0039] For the purposes of the present invention, cationic nanoparticles are particles having a size range of 1 to 500 nanometers. More preferentially, polysaccharide nanoparticles have a size range of 10 to 300 nm, especially 20 to 250 nm. Furthermore, the nanoparticles according to the present invention are advantageously used in solution. The term nanoparticles therefore also includes particles or molecules that are in nanoparticle form in solution, such as chitosan and its derivatives. The solution may be an aqueous solution, a buffer solution, or a serum solution. The inventors have found that certain linear molecules, such as chitosan, form nanometric coils in solution, which behave like 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.

[0040] In a first particular embodiment, the cationic polysaccharide forming the core of the nanoparticle (NP) is a non-crosslinked polymer obtained by reaction between a polysaccharide selected from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum) and at least one cationic ligand selected from primary, secondary, or tertiary amines or quaternary ammonium. 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 non-lipid-loaded 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 ammonium.

[0041] In a second particular embodiment, the cationic polysaccharide forming the core of the nanoparticle (NP) is a crosslinked polymer obtained by reaction between a polysaccharide selected from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactomannan (guar gum) and at least one cationic ligand selected from primary, secondary, or tertiary amines or quaternary ammonium, followed by addition of a crosslinking agent. The crosslinking 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 crosslinked, non-lipid-filled 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 amine, or quaternary ammonium, and (iii) a crosslinker selected from epichlorohydrin, a dicarboxylic acid, or an acid chloride, e.g., sebacic acid.

[0042] 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 an anionic 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 amine, or quaternary ammonium, and (iii) an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine, or diacylphosphatidylinositol.

[0043] In a fourth embodiment of the present invention, the nanoparticles (NPs) are cationic nanoparticles consisting of an anionic phospholipid-filled cross-linked polysaccharide core consisting 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 amine, or quaternary ammonium, (iii) a cross-linker selected from epichlorohydrin, a dicarboxylic acid, or an acid chloride, e.g., sebacic acid, and (iv) an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine, or diacylphosphatidylinositol.

[0044] In a preferred embodiment applicable to all four types of nanoparticles (NPs) described hereinabove, the cationic polysaccharide is maltodextrin-based, 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.

[0045] In a preferred embodiment applicable to nanoparticles (NPs) with a phospholipid-filled core, the NPs are DPPG-filled cationic polysaccharide nanoparticles, whether the NPs are crosslinked or not.

[0046] Birds are understood to mean in particular poultry, which for the purposes of the present invention are domesticated birds that serve as a source of eggs or meat and include commercially important species such as chickens, laying hens, turkeys, ducks, geese, guinea fowl, pheasants, pigeons, and peacocks.

[0047] In a preferred embodiment, the bacterium is selected from the following group: Salmonella typhi, Streptococcus pneumoniae, Haemophilus influenzae b, Mycobacterium tuberculosis, Extraintestinal pathogenic E. coli (ExPEC), enterotoxigenic E. coli (ETEC), Salmonella paratyphi A, Neisseria gonorrhoeae, Clostridium difficile, Campylobacter spp., Shigella spp., Staphylobacter aureus, Helicobacter pylori.

[0048] For the purposes of the present invention, "whole bacteria" refers to bacteria in their entirety, in particular with intact cell membranes, and not fragmented. In other words, this means that the bacterial membrane remains unchanged. In contrast, bacteria with damaged, fragmented, or damaged membranes cannot be considered whole bacteria for the purposes of the present invention.

[0049] For purposes of the present invention, "cationic agent" means an agent that has a positive charge, such as a cationic nanoparticle.

[0050] For purposes of the present invention, "inactivated or inactive bacteria" means non-viable bacteria that have been previously killed but are still intact. By intact, we mean bacteria whose membranes are not altered. They can be killed, for example, by treatment with formaldehyde or any other inactivation method known to those skilled in the art.

[0051] For the purposes of the present invention, "coated with cationic nanoparticles" means that the nanoparticles coat the surface of the inactivated bacteria. The nanoparticles cover the dead bacteria with a homogeneous layer. The coverage can be defined by the weight ratio of bacterial protein:NP. In preferred embodiments, the weight ratio of bacterial protein:NP is 2 or more, preferably greater than 5, even more preferably greater than 10, or even 50 or 500.

[0052] Good efficacy of NP-coated bacteria in preventing infection was observed for bacterial protein:NP weight ratios (dry weight) of 1:0.01 to 1:10, more specifically 1:0.01 to 1:3. This corresponds to approximately 100 times less nanoparticles by weight than bacteria. In particular embodiments, this ratio is greater than 1, particularly where the bacterial protein weight is at least equal to, and up to 100 times greater than, the NP weight (ratio of 1:1 to 1:0.01). In even more preferred embodiments, the bacterial protein weight is 2 to 100 times greater than the NP weight (ratio of 1:0.5 to 1:0.01). In even more preferred embodiments, the bacterial protein weight is 10 to 100 times greater than the NP weight (ratio of 1:0.01 to 1:0.1). In particular embodiments, this ratio can be 1:0.1 to 1:10, or even 1:0.1 to 1:3.

[0053] The vaccine composition comprises at least one bacterium to induce effective protection against or at least reduce bacterial infection.

[0054] For the purposes of the present invention, "avoiding infection" means that the vaccine composition may provide 100% protection against the risk of infection, or if it does not completely avoid the risk of infection, that the protection conferred by the vaccine is sufficient so that the individual does not develop the disease, or if it does develop, the symptoms of the infection are at least reduced and the individual avoids death. Avoiding infection also means preventing the infection from spreading within the farm.

[0055] In one embodiment of the present invention, the vaccine composition is prophylactic.

[0056] For the purposes of the present invention, a "prophylactic vaccine composition" means a vaccine composition that makes it possible to induce an immune response in a healthy individual that has not yet come into contact with a pathogen, with the aim of activating its immune defenses and preparing the immune system to react against future infections.

[0057] In another embodiment of the invention, the vaccine composition is directed to avian species. In a particular embodiment of the invention, the prophylactic vaccine composition is directed to poultry, particularly embryos in eggs.

[0058] For purposes of infection, a "multivalent vaccine composition" means that the vaccine composition comprises several different bacteria that can induce immunity against several diseases associated with the different bacteria.

[0059] In certain embodiments, the vaccine compositions may be used to produce combination vaccines.

[0060] For the purposes of the present invention, a "combination vaccine" means a vaccine composition that contains several bacteria of different species or families so as to simultaneously induce immunity against several different bacteria.

[0061] In one embodiment, the vaccine composition comprises at least one whole inactivated bacterium to induce cross-immunity in the vaccinated individual.

[0062] For purposes of the present invention, "cross-immunity" means acquired immunity to a bacterial pathogen that confers immunity to another bacterial pathogen of a different species, strain, or family that is not part of the vaccine composition.

[0063] Cross-immunity is associated with the phenomenon of cross-reactivity. Antibodies are usually specific for a particular antigen. This specificity allows them to target and eliminate the antigen they detect. Mutant bacteria preserve common antigens that can be targeted by vaccine-induced responses.

[0064] Therefore, cross-reactivity can occur between closely related species of bacteria. Bacteria have multiple surface antigens. When an animal is immunized against a bacterium by injection of whole bacteria, it produces antibodies to multiple bacterial antigens. If two bacteria have the same or similar antigens, the individual will have acquired immunity to both bacteria.

[0065] In another embodiment of the invention, the vaccine composition is multivalent and comprises at least two different strains of bacteria of different species or families, the bacteria being whole and inactivated.

[0066] A vaccine composition may consist, for example, of three inactivated E. coli strains mixed with lipidated maltodextrin nanoparticles (NPs) to prevent colibacillosis.

[0067] Thus, according to various embodiments of the vaccine composition, the composition may comprise: - Completely inactivated bacteria coated with cationic nanoparticles consisting of a cross-linked porous polysaccharide core filled with anionic phospholipids. - Completely inactivated bacteria coated with cationic nanoparticles consisting of a non-lipid-filled, cross-linked porous polysaccharide core. - Completely inactivated bacteria coated with cationic nanoparticles consisting of a non-crosslinked polysaccharide core filled with anionic phospholipids. - Completely inactivated bacteria coated with cationic nanoparticles consisting of a non-crosslinked, non-lipid-filled polysaccharide core. - at least two bacteria of different strains and / or species or families, which have been inactivated and coated entirely with cationic nanoparticles consisting of a cross-linked porous polysaccharide core filled with phospholipids. - at least two bacteria of different strains and / or species or families, which have been inactivated and entirely coated with cationic nanoparticles consisting of a non-lipid-filled polysaccharide core in cross-linked form. - at least two bacteria of different strains and / or species or families, which have been inactivated and entirely coated with cationic nanoparticles consisting of a porous polysaccharide core in a non-crosslinked form, filled with phospholipids. - at least two bacteria of different strains and / or species or families, which have been inactivated and entirely coated with cationic nanoparticles consisting of a non-lipid-filled polysaccharide core in a non-crosslinked form.

[0068] 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 coated with cationic nanoparticles consisting of a polysaccharide core, in a form suitable for intramuscular, mucosal or in ovo administration. This composition is therefore multivalent and can be used to obtain a combination vaccine.

[0069] For the purposes of the present invention, "different inactivated bacteria" means bacteria of different strains and / or species or families.

[0070] A third object of the invention relates to the use of a vaccine composition as defined above for preventing bacterial infections in mammals or birds.

[0071] In particular embodiments of the invention, the bacterial infection is salmonellosis, colibacillosis, or campylobacteriosis.

[0072] In a preferred embodiment, the use is applied to the prevention of bacterial infections in poultry embryos in ovo (by in ovo administration).

[0073] The present invention also relates to a method for preventing diseases associated with bacterial infections in mammals and birds, comprising a vaccine composition comprising at least one inactivated whole pathogenic bacterium coated with cationic nanoparticles consisting of a polysaccharide core, the method comprising the steps of: - having cationic nanoparticles consisting of a polysaccharide core and at least one whole bacterium. - Inactivating all bacteria with formaldehyde. - mixing the cationic nanoparticles with the inactivated whole bacteria to obtain the vaccine composition. - administering said vaccine composition to said animal.

[0074] In certain embodiments, mixing cationic nanoparticles with the inactivated whole bacteria is performed such that the nanoparticles coat the bacteria at a weight ratio of bacterial protein:NP of 1 or greater, in a preferred embodiment, the weight ratio of bacterial protein:NP is 5 or greater.

[0075] In vaccine compositions, the cationic nanoparticles may be selected from: - cationic nanoparticles consisting of a non-crosslinked, non-lipid-filled 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 amine, or quaternary ammonium, - cationic nanoparticles consisting of a non-lipid-filled cross-linked polysaccharide core, consisting 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, and (iii) a cross-linker selected from epichlorohydrin, dicarboxylic acids, or acid chlorides, for example, sebacic acid; - cationic nanoparticles consisting of an anionic phospholipid-filled non-crosslinked polysaccharide core consisting 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 amine, or quaternary ammonium, and (iii) an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine, or diacylphosphatidylinositol, Cationic nanoparticles consisting of an anionic phospholipid-filled crosslinked polysaccharide core consisting of (i) a polysaccharide selected from starch, dextran, chitosan, dextrin and maltodextrin, polyfructose (inulin), polymannose, polygalactose, polygalactosemannan (guar gum), (ii) at least one cationic ligand selected from primary, secondary, or tertiary amine, or quaternary ammonium, (iii) a crosslinker selected from epichlorohydrin, a dicarboxylic acid, or an acid chloride, e.g., sebacic acid, and (iv) an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine, or diacylphosphatidylinositol.

[0076] In a preferred embodiment, the cationic polysaccharide is obtained by reaction between maltodextrin and glycidyltrimethylammonium, whether the NPs are cross-linked or not.

[0077] In certain embodiments, the prophylactic method comprises a vaccine composition that is administered mucosally, by injection, and / or in ovo.

[0078] In particular embodiments of the present invention, the vaccine composition is administered to poultry, i.e., chicks in ovo, mucosally, and orally, and to laying hens intramuscularly. [Brief explanation of the drawings]

[0079] [Figure 1] : E. coli uptake after cell coating with LNPs. Delivery of whole E. coli bacteria was evaluated in human H292 cells. Fluorescent FITC-E. coli, alone or in combination with LNPs (ratios of 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). Results represent the mean ± SEM of three experiments. Statistical analysis: two-way ANOVA, **p<0.01, ***p<0.001. [Figure 2]Intracellular delivery by confocal microscopy at a ratio of 1:3. Delivery of E. coli bacteria was evaluated in H292 cells. Fluorescent FITC-E. coli associated with LNPs (1:3 ratio) was incubated with H292 cells for 4 hours, and intracellular localization was observed by confocal microscopy. Representative images were taken. Red: plasma membrane, blue: nucleus, green: E. coli. Scale bar: 10 μm. [Figure 3] : Schematic diagram of the vaccine protocol for the in ovo vaccination study. The commercial vaccine was administered only to the positive control. [Figure 4] : Intestinal permeability test for 8 birds in each group 6 days after challenge (D20). Results represent the mean ± SD. [Figure 5] : Analysis of anti-E. coli sIgA in the faeces of 8 birds from each 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. [Figure 6] : Clinical scores of liver lesions from 8 birds in each group 6 days after challenge (D20). Results represent the mean ± SD of group scores. Statistical analysis: One-way Anova, *p<0.05. [Figure 7] : Schematic diagram of the in ovo vaccination study protocol. Only the positive group was vaccinated with the commercial vaccine (Poulvac) on D1. [Figure 8] : Percentage of death in each group after lethal challenge at D14 (n=30). [Figure 9] : Measurement of bacterial load in the air sacs of birds from each group assessed 2 days after challenge (D16) in 8 birds by MPN. Results represent mean ± SD. Statistical analysis was performed by one-way ANOVA, *p<0.05. [Figure 10] : Intestinal permeability of 8 birds from each group 6 days after challenge (D20). Results represent the mean ± SD. Statistical analysis was performed by one-way ANOVA, *p<0.05. [Figure 11]: Intestinal lesion scores of 8 birds from each group 6 days after challenge (D20). Results represent the mean ± SD. Statistical analysis was performed by one-way ANOVA, *p<0.05. [Figure 12] : Schematic diagram of the intramuscular vaccination trial protocol. [Figure 13] : Egg production. Top: Number of eggs laid per day by hens in each group after challenge. Error bars have been hidden for clarity of the graph. Bottom: Mean number of eggs laid per day by hens in each group after challenge. Statistical analysis: One-way Anova *p<0.05, ***p<0.001, ****p<0.0001. [Figure 14] Quantification of bacterial load in the ceca of chickens from each group, measured by qPCR. Results represent mean ± SEM. Statistical analysis: One-way ANOVA, **p<0.01, ***p<0.001. [Figure 15] Quantification of bacterial load in the ceca of chickens from each group, measured by qPCR. Results represent the mean ± SEM. [Figure 16] : Schedule of in ovo vaccination study protocol. Only the "positive control" group was vaccinated with the commercial vaccine (Poulvac) on D1. [Figure 17] : Measurement of bacterial infection in the trachea (top) and alveoli (bottom) of birds in each group, assessed on 8 birds by most probable number (MPN). Results represent the number of positive and negative birds per group. [Figure 18] : Analysis of anti-E. coli sIgA in the faeces of 8 birds from each group after challenge. Results represent the mean ± SD of antibody titers. Statistical analysis: One-way Anova for each day. [Figure 19] : Lung lesion scores of 8 birds per group after challenge. Results represent the mean for each group. Statistical analysis was performed for each day by one-way ANOVA. [Figure 20]: Delivery of whole E. coli bacteria was evaluated in H292 cells. Fluorescent FITC-E. coli, alone or in combination with NPs, was 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. [Figure 21] : Delivery of whole E. coli bacteria was evaluated in THP-1 cells. Fluorescent FITC-E. coli, alone or in combination with NP, was 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. [Example]

[0080] Abbreviation: LNP: Crosslinked lipidated maltodextrin nanoparticle ·id:Intradermal ·in:Intranasal ·ip: Intraperitoneal, intraperitoneal im: Intramuscular

[0081] Example 1: Optimization of formulations using cationic nanoparticles The objective of this study was to confirm the effectiveness of a composition based on nanoparticles and an inactivated whole E. coli strain as a delivery system for activating immune cells.

[0082] 1-A Materials and Methods: A-Vaccine preparation Cationic nanoparticles (LNPs) are cationic lipidated maltodextrin nanoparticles.

[0083] Compositions were prepared using inactivated E. coli strains mixed with cationic nanoparticles. The E. coli bacteria were inactivated with 0.4% formaldehyde and then purified by centrifugation. Protein content was measured by microBCA assay. Compositions were prepared by mixing killed bacteria with an aqueous solution of LNPs at different weight ratios (100 μg of E. coli protein to 5, 10, 30, 50, 100, or 300 μg of LNPs). The size and surface charge of the formulations were characterized by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively (Zetasizer NanoZS, Malvern Analytical, France), to observe whether the nanoparticles coated the surface of the killed bacteria.

[0084] B-E. coli delivery The ability of LNPs to increase the uptake of inactivated whole E. coli by immune cells was assessed by flow cytometry and confocal microscopy.

[0085] Fluorescein labeling of E. coli 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 h. They were then dialyzed in a 10 kDa dialysis cassette (Thermo Fisher, France). Protein content was measured by microBCA assay (Pierce, France). The labeled bacteria were then associated with LNPs at different weight ratios.

[0086] Flow cytometry: H292 cell line was seeded at 50,000 cells per well in 24-well plates until confluent. Cells were then incubated with the equivalent of 1 μg of protein alone or in combination with different ratios of LNP for 4 hours. 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.

[0087] Confocal microscopy H292 cell line was seeded at 10,000 cells per well in a Labtek chamber (Fisher Sci., France) until confluent. Cells were then incubated for 4 hours with the equivalent of 1 μg of protein from killed whole bacteria alone or in combination with LNP. Cells were washed, and nuclei were stained by incubating with 0.1 μg / mL Hoechst 33342 (Sigma, France) for 5 minutes at 37°C. Cells were then washed, and plasma membranes were stained with 1 μg / mL AF-633-labeled agglutinin (WGA, Invitrogen, France) for 10 minutes at 37°C. Slides were washed again with PBS, fixed with 0.4% formaldehyde for 20 minutes, and mounted for microscopy (LSM 710 Zeiss, France).

[0088] 1-B result: Formulation characterization:

[0089] [Table 1]

[0090] DLS and ELS analyses showed that the inactivated E. coli had a uniform size of 1.42 μm with a PDI of 0.08 and an anionic surface charge of -4.6 mV, indicating that the bacterial structure remained intact despite inactivation. When increasing amounts of LNP were added, the overall size did not change, but the zeta potential gradually increased and became cationic from a ratio of 1:0.5. This indicates that the bacteria were gradually coated with LNPs without any aggregation.

[0091] E. coli delivery by LNPs was evaluated on human airway epithelial cells (H292) by flow cytometry (see Figure 1). Without LNPs, bacteria were endocytosed by 14% of cells. No difference was observed in the presence of TB, suggesting that bacteria were indeed endocytosed (inside the cells). When coated with LNPs, bacteria were taken up by at least 40% of cells, confirming their potential as a delivery system. Furthermore, delivery was significantly more efficient with lower amounts of LNPs, with the highest release observed at ratios of 1:0.3 (77%) and 1:0.1 (75.7%). In the presence of TB, 65.6% of cells remained positive at a ratio of 1:0.3, and 63% remained positive at a ratio of 1:0.1, confirming that most of the bacteria were inside the cells.

[0092] Intracellular delivery was also confirmed by confocal microscopy at a 1:3 ratio. Approximately 20–30 E. coli bacteria (green) were observed in each cell near the nucleus, confirming intracellular localization.

[0093] Conclusion: The E. coli / LNP vaccine formulation consists of inactivated whole bacteria coated with LNP. Coating the bacteria with even small amounts of LNP has a significant effect on the bacteria's ability to be taken up by cells.

[0094] Example 2: In ovo vaccination trial against colibacillosis 2-A Materials and Methods: A - Vaccine preparation 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 the protein content was measured by μBCA assay. Finally, 33.3 μg per strain was mixed with LNPs to obtain 100 μg of protein per vaccine dose.

[0095] B-Animal All animal studies have been reviewed and approved by the Imunova Analises Biologicas Animal Research Ethics Committee, protocol number 06 / 2021.

[0096] In this study, 390 fertilized eggs were obtained from a commercial incubator 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 study consisted of 30 animals and are identified in Table 1.

[0097] [Table 2]

[0098] C-Vaccination Animals in group 3 received a single dose of live Poulvac® E. coli vaccine on day 1 after hatch. On day 18 of incubation, animals in group 4 received an in ovo application of vaccine at a regulated vaccine dose of 50 μL. After hatch, groups of 1-day-old broilers were housed in isolators (1.2 m 2 ) and fed ad libitum as recommended for their age.

[0099] D-Challenge On D10, all animals, including those in the negative control group, received orally a live attenuated Mass I-H120 infectious bronchitis virus (IBV) vaccine at a dose 100-fold higher than that recommended by the manufacturer.

[0100] On D14, animals in groups 2, 3, and 4 were treated with 100 μL / bird at 10 8 The animals were orally infected with CFU of Escherichia coli (strain 19501, a different strain from that used in the vaccine).

[0101] The vaccination protocol is shown in Figure 3.

[0102] E-sampling Samples and analyses were performed on eight birds on D16, D20, or D27.

[0103] F-analysis ·Intestinal permeability: Intestinal permeability was assessed by oral administration of FITC-dextran (FITC-dextran, 3000–4000 kDa), a non-absorbable fluorescent marker, identified in plasma / serum to monitor the integrity of the gastrointestinal epithelium ( Vicuna et al., 2015 ).

[0104] Cytokine expression Cytokine expression was assessed by qPCR (IL-1β, IFNγ, IL-10, IL-4) using primers specific for each target. In this type of analysis, each target-sample combination generates a threshold value. The Ct (threshold cycle) is a relative measure of the concentration of target-specific messenger RNA (mRNA) in the sample. This value must be normalized based on the expression of a specific reference gene; in this case, the geometric mean of the GAPDH and ACTB genes was used to generate a ΔCt value (target Ct / mean GAPDH + ACTB Ct) (Bustin et al., 2009). In addition to this normalization, data were also normalized to the mean ΔCt of the control group to generate a ΔΔCt (Δct / mean ΔCt control). For undefined results, the maximum Ct value (40) was considered and artificially corrected to 41 for analytical purposes.

[0105] Quantification of anti-E. coli sIgA: E. coli -specific secretory IgA production was assessed by ELISA. Briefly, samples were diluted in 1% casein in PBS. ELISA plates were coated with E. coli LPS (field isolate). Plates were then washed three times with 200 μL / well of PBS + 0.05% Tween 20 for 5 min / wash. Wells were blocked with 1% casein in PBS. Samples were tested in serial dilutions. Plates were washed, and anti-chicken IgA (BioRad) diluted in 0.1% casein was added. After washing, the assay was revealed with a solution of TMB (Life Technologies). Absorbance was read at 450 nm.

[0106] Detection and quantification of E. coli: Detection and quantification of E. coli by MPN (Most Probable Number) was performed according to the ISO 7251:2005 standard. E. coli counts were determined according to standard microbiological methods (dilution in enrichment medium followed by plating in selective / differential media). Briefly, samples were enriched in buffered peptone water (BPW), then in EC broth, and finally in EMB and MacConkey agar. Samples were serially diluted in triplicate in EPB before incubation to allow quantification by the most probable number technique (Blodgett et al., 2015). For E. coli detection, only the serial dilution step was omitted. Isolated suspect colonies were biochemically tested and confirmed.

[0107] Liver histology: Birds were euthanized and liver samples were collected and fixed according to the method of Rebel et al. (2011). Samples were embedded in paraffin and mounted on slides. All histopathological evaluations and readings were performed under a microscope by an experienced veterinary histopathologist.

[0108] [Table 3]

[0109] 2-B Results: Intestinal permeability in unchallenged, unvaccinated birds (negative control) was 0.26 μg / mL, and in challenged, unvaccinated birds (positive control) was 0.31 μg / mL. When vaccinated with the commercial vaccine, permeability was 0.26 μg / mL for the negative control, confirming the efficacy of this vaccine. Furthermore, when vaccinated in ovo with the VXN-E. coli formulation, all birds showed low permeability (0.18 μg / mL), which was not significantly lower than that of the negative control.

[0110] Intestinal anti-E. coli A-sIgA (LPS) Anti-LPS secretory IgA against E. coli was analyzed in feces by ELISA. The absorbance obtained for the negative control birds was approximately 0.05 AU and for the positive control birds was 0.095 μg / mL, indicating that oral challenge did not induce intestinal IgA secretion. Furthermore, for birds mucosally vaccinated with the commercial vaccine, the OD remained at 0.055 AU, similar to the negative control, suggesting that the vaccine failed to promote a mucosal humoral response. In contrast, birds vaccinated in ovo with the VXN-E. coli formulation showed a significantly higher OD of 0.16 AU.

[0111] B-Liver histopathology Liver lesion scores were measured 6 days after challenge. Birds in the negative control group had an average score of approximately 1, representing discrete hyperplasia. In contrast, unvaccinated birds in the positive control group had an average score of 2.35, suggesting liver damage and necrosis induced by E. coli infection. When vaccinated with the commercial vaccine, the average bird lesion score was 1, similar to the negative control group. Birds vaccinated in ovo had an average lesion score <1. These results indicate that the commercial vaccine and in ovo vaccination protect against E. coli-induced liver damage.

[0112] Conclusion: This first study demonstrates that in ovo vaccination with a VXN-E. coli vaccine protects birds against E. coli liver infection and induces sIgA secretion against bacteria in the intestine.

[0113] Example 3: In ovo vaccination against lethal E. coli challenge This second study was identical to the first in ovo study in terms of schedule, animals per group, and treatment, but involved a lethal E. coli challenge. Analysis then focused on the protection provided by the vaccine against bacterial load in representative organs, physiological abnormalities, and subsequent death observed in each group.

[0114] 3-A. Materials and Methods A-Challenge On D10, all animals, including the negative control group, received orally a live attenuated Mass I-H120 infectious bronchitis virus (IBV) vaccine at a dose 100-fold higher than the dose recommended by the manufacturer.

[0115] On D14, each animal in the determined group was administered 4.2 x 10 12 The air sacs were challenged with CFU of Escherichia coli (strain 19501).

[0116] A typical schedule is detailed in Figure 7.

[0117] B-Analysis ·Intestinal permeability: Intestinal permeability was assessed by oral administration of FITC-dextran (FITC-dextran, 3000–4000 kDa), a non-absorbable fluorescent marker, identified in plasma / serum to monitor the integrity of the gastrointestinal epithelium ( Vicuna et al., 2015 ).

[0118] Detection and quantification of E. coli: Detection and quantification of E. coli by MPN (Most Probable Number) was performed according to ISO 7251:2005. E. coli counts were determined according to standard microbiological methods (dilution in enrichment medium followed by plating in selective / differential media). Briefly, samples were enriched in buffered peptone water (BPW), then in EC broth, and finally in EMB and MacConkey agar. Samples were serially diluted in triplicate in EPB before incubation to allow quantification by the most probable number technique (Blodgett et al., 2015). For E. coli detection, only the serial dilution step was omitted. Isolated suspect colonies were biochemically tested and confirmed.

[0119] Intestinal histology: 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 under a microscope by an experienced veterinary histopathologist.

[0120] [Table 4]

[0121] 3-B.Results A-Hatch rate

[0122] [Table 5]

[0123] In this study, hatch rates were measured to evaluate the safety of the in ovo VXN / E. coli vaccine. Therefore, before randomization into each group (negative control, positive control, and commercial vaccine), the hatch rates of vaccinated eggs (n=60) were compared with those of unvaccinated eggs (n=180). Similar percentages of hatch were observed between vaccinated eggs (78.3%) and unvaccinated eggs (81.2%). Consequently, the vaccine formulation is safe because it has no effect on hatch rates.

[0124] B - Bird survival after lethal challenge E. coli challenge, 4.2 x 10 12 The test was performed on D14 using CFU directly in the air sac. Bird survival after lethal challenge is shown in Figure 8. This high dose had an impact on bird survival, with 26% mortality observed in unvaccinated birds. Furthermore, mortality increased to 36% in birds vaccinated with the commercial mucosal vaccine, suggesting that it did not induce protection against lethal E. coli infection. In contrast, mortality was only 10% in birds vaccinated in ovo with the VXN / E. coli vaccine, suggesting better protection against infection.

[0125] Quantification of E. coli in air sacs by C-MPN Infection was assessed by MPN by quantifying bacteria in the air sacs. Measurement of bacterial load in the air sacs by MPN is shown in Figure 9. Negative control birds showed low levels of bacteria in the air sacs compared to the natural flora. Non-immunized challenge birds showed higher levels of E. coli (10 4 MPN / g, which confirms the effectiveness of the challenge. 6 A significantly higher infection was observed in the air sacs of birds vaccinated with the E. coli / LNP vaccine (3 × 10 MPN / g), confirming the survival results. However, similar to the survival results, the E. coli / LNP vaccine (3 × 10 3 Lower infections were observed in birds vaccinated in ovo with 1000 ng / g of IgG1 (MPN / g), confirming its effectiveness in protecting against this bacterial infection.

[0126] D-intestinal permeability Intestinal permeability in unchallenged, unvaccinated birds was 0.22 μg / mL and in challenged, unvaccinated birds was 0.18 μg / mL. Intestinal permeability is shown in Figure 10. When vaccinated in ovo with the commercial vaccine or the VXN / E. coli vaccine, permeability was significantly reduced to 1.2 μg / mL, suggesting vaccine-induced protection.

[0127] E-Intestinal histopathology Ileal lesion scores were measured 6 days after challenge. The intestinal lesion scores are shown in Figure 11. Birds in the negative control group obtained an average score of less than 1 (0.25), indicating healthy ileums with a normal appearance, as expected. In contrast, unvaccinated birds in the positive control group had a significantly higher average score of 1.7, suggesting ileal lesions with vascular damage and desquamation induced by E. coli infection. Surprisingly, when birds were vaccinated with the commercial vaccine, the lesions were significantly worse, with an average score of 2. In contrast, in ovo vaccinated birds had an average score of 1, suggesting protection against E. coli-induced intestinal lesions.

[0128] Conclusion: This second study demonstrates that the in ovo VXN-E. coli vaccine protects birds from mortality induced by lethal E. coli infection. Furthermore, the vaccine reduces bacterial load in the air sac and intestinal lesions caused by the infection, confirming the value of the vaccine.

[0129] Example 4: Intramuscular Salmonella vaccination trial for laying hens 2-A. Materials and Methods: A - Vaccine preparation The vaccine is produced from an inactivated strain of Salmonella enteritidis mixed with lipidated maltodextrin nanoparticles (LNPs). Briefly, Salmonella strain SE147 was inactivated, and the protein content was measured by μBCA assay. Finally, 200 μg of killed sterilized Salmonella enteritidis was mixed with either LNPs (formulation designated "Vaxinano1") or non-crosslinked LNPs (formulation designated "Vaxinano2") at a ratio of 200 μg per vaccine dose. Non-crosslinked LNPs are composed of linear cationic maltodextrin with an anionic inner core.

[0130] B-Animal For this experiment, a total of 84 LSL chickens (from a commercial farm) were randomly assigned to 12 pens (7 birds / pen) as described in Table 7. Serum was collected and tested for Salmonella antibody titers (performed by DGZ using a BioChek kit).

[0131] [Table 6]

[0132] C-Vaccination At W12 weeks of age, all chickens were vaccinated IM in the chest with either 500 μL of saline solution, or 500 μL of Vaxinano1 or Vaxinano2 formulations (containing 200 μg of Salmonella protein), or a commercial vaccine. One month later, at W16, the animals received a second dose of the same vaccine formulation.

[0133] D-Challenge One month after the booster immunization, all animals received 500 μL of 1.3 × 10 8 were challenged intravenously with CFU of S. enteritidis SE147.

[0134] A typical schedule is shown in Figure 12.

[0135] E-Sample From W20 to W25, eggs were collected and individually analyzed bacteriologically for Salmonella. At W25, all chickens were euthanized, and serum and livers were collected and stored at -20°C. Spleens and ceca were analyzed bacteriologically for Salmonella.

[0136] F-analysis Egg production: Eggs were collected daily after challenge (except Saturday) and stored at 4° C. The number of eggs per group was reported.

[0137] Infection: Infection was quantified by qPCR in the spleen and cecum of each chicken at W25.

[0138] 2-B.Results A-Spawning The average number of eggs laid for each group was counted each day after the challenge. Egg production is shown in Figure 13. Hens vaccinated with the dummy vaccine laid a small number of eggs, an average of 2.6 per day after challenge, confirming bird infection. 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), suggesting protection against challenge. Similarly, both Vaxinano formulations enabled hens to lay significantly more eggs, an average of 4.3 eggs per day for Vaxinano1 (p<0.05) and 4.7 eggs for Vaxinano2 (p<0.001), suggesting equivalent protection against challenge.

[0139] B - Infection of the cecum and spleen Infection was assessed by quantifying the bacterial load in the cecum and spleen.

[0140] Post-challenge infection was assessed by quantification of bacterial load in the cecum and spleen. This quantification is shown in Figure 14. Saline-injected chickens had significant infection with an average infection of 400 CFU / g, with over 50% of birds exceeding 1000 CFU / g. In contrast, all birds vaccinated with either the Vaxinano formulation or Salenvac had significantly lower infection, with the average infection being below the threshold for birds vaccinated with Vaxinano1. This confirms the strong protection offered by IM vaccination.

[0141] Infection was finally quantified in the spleen and is shown in Figure 15. Although infection was lower than in the cecum, 77% of birds vaccinated with saline solution were still positive in the spleen compared to only 35% of birds vaccinated with Vaxinano1, 36% of birds vaccinated with Vaxinano2, and 35% of birds vaccinated with the commercial vaccine, confirming the protection offered by IM vaccination.

[0142] Conclusion: This study demonstrates that an intramuscularly administered inactivated whole Salmonella / NP vaccine protects birds against challenge with S. enteritidis and enables the birds to produce significantly more eggs than non-immunized animals, regardless of the formulation.

[0143] Example 5: Mucosal vaccination against colibacillosis This study was designed in the same manner as the in ovo study. Analysis then focused on the protection provided by the vaccine against infection in target organs, physiological abnormalities, and mucosal antibody titers.

[0144] 5-A Materials and Methods: A - Vaccine preparation The vaccine is made from three strains of inactivated E. coli bacteria mixed with lipidated maltodextrin nanoparticles (LNPs). Strains O78:K80, O1:K1, and O2:K1 were inactivated with 0.4% formaldehyde, and their protein content was measured by BCA assay. Finally, 33.3 μg of each strain was mixed with LNPs at a ratio of 100 μg of protein per vaccine dose.

[0145] B-Animal All animal studies have been reviewed and approved by the Imunova Analises Biologicas Animal Research Ethics Committee, protocol number 06 / 2021.

[0146] For this study, 150-day-old chicks were obtained from a commercial hatchery, randomized into five test groups in individual isolation units within Imunova, and treated according to the table below.

[0147] [Table 7]

[0148] C-Vaccination Animals in the "VXN Vaccine SC," "VXN Mucosal Vaccine," and "Commercial Vaccine" groups (Poulvac® E. coli, Zoetis) received a primary immunization on D1 and a second dose on D12. For the "VXN Mucosal Vaccine," doses were administered to the eyes, beak, and nostrils, whereas for the "Commercial Vaccine," doses were administered in the drinking water.

[0149] D-Challenge The challenge strain was a field isolate confirmed as APEC by PCR identification of five virulence genes (iuaT, iroN, ompC, iss, hly). It was also confirmed to belong to phylogenetic group F by the typing method described by Clermont (Clermont et al., 2013). All animals received a 100x dose of attenuated IBV vaccine (Massachusetts H-120 strain, Mass®I, Zoetis) on D14. E. coli challenge was performed in all groups except the "negative control" using 108 CFU / bird.

[0150] The overall protocol schedule for the in ovo vaccination trial is shown in FIG.

[0151] E-analysis Quantification of anti-E. coli IgA: 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). Plates were then washed three times with 200 μL / well of PBS + 0.05% Tween-20 for 5 min / wash. Wells were blocked with 1% casein in PBS. Samples were tested by serial dilution. Plates were washed, and anti-chicken IgA (Bio-Rad) diluted in 0.1% casein was added. After washing, the test was revealed with a single solution of TMB (Life Technologies). Absorbance was read at 450 nm, and quantification was performed using a proprietary method / kit developed by Imunova.

[0152] Quantification and detection of E. coli infection: E. coli detection by MPN (Most Probable Number) was performed according to ISO 7251:2005. E. coli counts were determined according to standard microbiological methods (dilution in enrichment medium followed by plating on selective / confirmatory media). Briefly, samples were concentrated in buffered peptone water (BPW), then in EC broth, and finally plated on EMB and MacConkey agar. Samples were serially diluted in triplicate in BPW before incubation to allow quantification by the MPN technique (Blodgett et al., 2015). For E. coli detection, only the serial dilution step was omitted. Suspect isolated colonies were biochemically tested and confirmed.

[0153] Lung histology: Birds were euthanized and lung samples were collected and fixed. Samples were embedded in paraffin and mounted on slides. All histopathological evaluations and readings were performed under a microscope by an experienced veterinary histopathologist.

[0154] [Table 8]

[0155] 5-B: Results Presence of E. coli in the respiratory tract (MPN) Infection was assessed by quantifying the number of birds infected in the trachea and air sacs per MPN. Measurements of bacterial infection in the trachea and alveoli are shown in Figure 17. Three unchallenged birds were infected in the trachea and seven in the air sacs, likely due to the presence of naturally occurring pathogenic E. coli in the environment. In contrast, unvaccinated challenged birds were more infected in the trachea, confirming the effectiveness of the challenge. A similar number of birds were infected in the group receiving the commercial vaccine compared to the infected control group, suggesting a lack of protection. However, from birds vaccinated with the VXN mucosal vaccine, only one bird was infected in the trachea and none in the air sacs. This confirms that the mucosally administered VXN E. coli vaccine protected birds from challenge.

[0156] Presence of anti-Escherichia coli sIgA in feces E. coli anti-LPS secretory IgA was analyzed in feces by ELISA. The results of the anti-E. coli sIgA analysis are shown in Figure 18. From D16 to D28, the antibody titers of the birds in the negative control were the same as those in the positive control, indicating that oral challenge did not induce intestinal IgA secretion. However, for mucosally vaccinated birds, a significant increase in antibody titers was observed at D21 for both the VXN E. coli formulation and the commercial vaccine. Thus, the mucosal vaccine could induce a humoral response in the intestine.

[0157] Pulmonary Clinical Score Lung lesion scores were measured from D16 to D28. The lesion scores are shown in Figure 19. Although they had the lowest histopathological scores from D16 (score = 2) to D28 (score = 1.7), birds in the negative control group exhibited bronchial epitheliitis, mucofibrinous exudates, and polymorphonuclear neutrophil infiltration, likely associated with natural infection (Figure 17). Furthermore, unvaccinated challenged birds in the positive control group had the highest scores of 3.4 at D16 and 2.7 at D28 due to E. coli challenge. In contrast, birds mucosally vaccinated with the VXN-E. coli formulation had an average lesion score of 2.3 at D16, lower than the commercial vaccine (score = 2.9). Finally, at 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), suggesting protection against lung lesions induced by E. coli infection.

[0158] Example 6: Optimization of formulations using different cationic particles To compare formulations prepared with different maltodextrin-based particles, studies were performed on inactivated whole E. coli strains and to measure their uptake by immune cells in vitro.

[0159] 6-A Materials and Methods: A-Vaccine preparation Vaccine formulations were made herein using one of the inactivated E. coli strains used in vaccine (11101) mixed with maltodextrin (NP+) or lipidated nanoparticles (LNP), as well as cationized but uncrosslinked (NP+NR) or lipidated maltodextrin (LNP-NR). The nanoparticles partially to very partially coat the bacteria.

[0160] B-particle synthesis and characterization NP+ are nanoparticles synthesized from cationic cross-linked maltodextrin. More specifically, the synthesis consisted of dissolving maltodextrin (Roquette, France) in 2M NaOH solution under magnetic stirring and at room temperature. Epichlorohydrin (Merck Group, France) was then added as a cross-linking agent, along with glycidyl trimethylammonium (GTMA, Merck Group, France) as a cationizing agent. The resulting gel was then neutralized with acetic acid and ground in an ultra-high-pressure homogenizer (LM20, Microfluidics, France). The ground material was then purified by tangential flow filtration (AKTA flux6, GE Healthcare, France) through a 750 kDa 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).

[0161] Similarly, LNPs are NPs+ to which an anionic phospholipid (DPPG) core has been added. More specifically, a solution of dipalmitoyl phosphatidylglycerol (DPPG, Lipoid, Germany) is dissolved in solutol and then injected into the NP+ solution at a mass percentage of 70% while stirring. The phospholipid is incorporated into the nanoparticle core, thus forming the LNPs. These LNPs can also encapsulate antigens from various pathogens (viruses, bacteria, or parasites) and deliver them to immune cells (2-4).

[0162] Finally, NP+NR and LNP-NR are their respective equivalents, synthesized according to the same synthetic scheme, but without the crosslinker, thus forming linear cationic polymers.

[0163] 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).

[0164] C - Inactivation of E. coli bacteria Bacteria were inactivated with 0.4% formaldehyde and then purified by centrifugation. Total bacterial protein content was measured by microBCA assay. Formulations were made by mixing killed bacteria with aqueous particle solutions at different weight ratios (100 μg E. coli protein to 1, 5, 10, 30, 50, or 100 μg particles).

[0165] Delivery of E. coli into cells by D-particles The ability of the particles to enhance phagocytosis-mediated uptake of inactivated whole E. coli by epithelial cells and into macrophages was assessed by flow cytometry.

[0166] Labeling proteins with fluorescein: 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 h. They were then dialyzed in a 10 kDa dialysis cassette (Thermo Fisher, France). The protein content of the total bacteria was measured by a microBCA assay (Pierce, France). The labeled bacteria were then associated with particles at different weight ratios.

[0167] E-Flow Cytometry: H292 cell lines were seeded at 50,000 cells per well in 24-well plates and treated after 3 days of culture. THP-1 cell lines were seeded at 100,000 cells per well in 24-well plates and differentiated into macrophages with 20 ng / mL PMA for 24 hours. After changing the culture medium, cells were incubated with 1 μg equivalent of killed bacteria, either alone or in combination with particles, for 4 hours. Cells were then washed with phosphate-buffered saline (PBS), harvested with trypsin, and analyzed by flow cytometry on an Attune Nxt (Thermo Fisher, France).

[0168] 6-B Results: Particle characterization

[0169] [Table 9]

[0170] The physicochemical characteristics of the different particles were analyzed after their synthesis (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 associated with the particles within the maltodextrin structure of the particles, rather than on their surface.

[0171] NP+NR had a diameter of 17 nm with a surface charge of 32 mV, and LNP-NR had a diameter of 23 nm with a surface charge of 38 mV, similarly indicating that phospholipids were associated within the maltodextrin structure.

[0172] In the absence of a cross-linking agent, the resulting particles appeared to have a smaller diameter. Indeed, in the absence of a cross-linking agent, maltodextrin in solution should remain primarily linear, but can also fold back on itself through hydrophobic bonds, thus forming nanoparticles that are detected during analysis.

[0173] Phagocytosis of Escherichia coli by cells after association with particles Particle-mediated delivery of E. coli was first evaluated in respiratory epithelial cells (H292). In the absence of particles, bacteria were not endocytosed by the cells (Figure 2). When they were coated with particles, their uptake was greatly increased, reaching 20–50% of positive cells, regardless of the particle used. Furthermore, improved phagocytosis could be observed as early as a 1:0.01 ratio, demonstrating that a small amount of particles, which partially coats the bacteria, is sufficient to improve intracellular delivery.

[0174] Similarly, particle-mediated phagocytosis of E. coli was evaluated in differentiated macrophages (THP-1). In the absence of particles, bacteria were phagocytosed by 0.2% of the cells (Figure 3). When they were coated with particles, their phagocytosis increased to 4–15% of positive cells, depending on the ratio. Furthermore, improved phagocytosis could be observed from a ratio of 1:0.01, again demonstrating that a small amount of particles coating the bacteria is sufficient to improve bacterial delivery.

[0175] conclusion Bacterial adhesion and uptake by respiratory epithelial cells and macrophages is mediated by a single (NP + ) or lipidated (LNP) cationized maltodextrin nanoparticles, as well as single (NP +This can be enhanced equally between NPs by either non-crosslinked (NR) or lipidated (LNP-NR) cationic maltodextrin. Low particle doses (10-100 times less particles than bacteria by mass%) are sufficient to improve this delivery to cells.

Claims

1. 1. An adjuvant-free vaccine composition comprising cationic nanoparticles consisting of a polysaccharide core and at least one inactivated bacterium, wherein the bacterium is whole and the cationic nanoparticles coat the bacterium.

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

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

4. 4. The vaccine composition of any one of claims 1 to 3, wherein the nanoparticles are cationic nanoparticles consisting of a non-lipid-filled cross-linked polysaccharide core consisting 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 amine, or quaternary ammonium, and (iii) a cross-linking agent selected from epichlorohydrin, a dicarboxylic acid, or an acid chloride, e.g., sebacic acid.

5. 4. The vaccine composition of claim 1, wherein the nanoparticles are cationic nanoparticles consisting of a non-crosslinked, non-lipid-filled 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 amine, or quaternary ammonium.

6. 4. The vaccine composition of claim 1, wherein the nanoparticles are cationic nanoparticles consisting of a phospholipid-filled 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 amine, or quaternary ammonium, (iii) a cross-linking agent selected from epichlorohydrin, a dicarboxylic acid, or an acid chloride, such as sebacic acid, and (iv) an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine, or diacylphosphatidylinositol.

7. 4. The vaccine composition of claim 1, wherein the nanoparticles are cationic nanoparticles consisting of a phospholipid-filled, 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 amine, or quaternary ammonium, and (iii) an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine, or diacylphosphatidylinositol.

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

9. 8. The composition of claim 6 or 7, wherein the cationic polysaccharide core is filled with an anionic phospholipid selected from diacylphosphatidylglycerol, diacylphosphatidylserine, or diacylphosphatidylinositol.

10. The vaccine composition according to any one of claims 1 to 9, comprising at least two different bacterial strains.

11. 11. The vaccine composition of any one of claims 1 to 10, wherein the bacteria is selected from the following group: Salmonella typhi, Streptococcus pneumoniae, Haemophilus influenzae b, Mycobacterium tuberculosis, extraintestinal pathogenic Escherichia coli (ExPEC), enterotoxigenic Escherichia coli (ETEC), Salmonella paratyphi A, Neisseria gonorrhoeae, Clostridium difficile, Campylobacter spp., Shigella spp., Staphylococcus aureus, Helicobacter pylori.

12. A vaccine composition according to any one of claims 1 to 11 for use in a form suitable for intramuscular, mucosal or in ovo administration.

13. A vaccine composition according to any one of claims 1 to 12 for use in the prevention of bacterial infections in mammals or birds.

14. A vaccine composition according to any one of claims 1 to 13 for use in the prevention of salmonellosis, colibacillosis or campylobacteriosis.

15. 15. The composition of claim 14 for use in preventing bacterial infection in poultry embryos in ovo.

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