VACCINE COMPOSITION COMPRISING A DELIVERY SYSTEM FOR A WHOLE INACTIVATED BACTERIUM VIA CATIONIC NANOPARTICLES

Abstract

The invention relates to the field of vaccine compositions. It relates more particularly to a prophylactic vaccine composition for mammals and birds comprising a whole killed bacterium, said bacterium being coated with a cationic agent, in particular cationic nanoparticles.

Description

Title of the invention: VACCINE COMPOSITION COMPRISING A DELIVERY SYSTEM FOR A WHOLE INACTIVATED BACTERIUM VIA CATIONIC NANOPARTICLES

[0001] The invention relates to the field of vaccine compositions. It relates more particularly to a prophylactic vaccine composition for mammals and birds comprising a whole killed bacterium, said bacterium being coated with a cationic agent, in particular cationic nanoparticles.

[0002] technical field

[0003] Bacteria are responsible for numerous diseases. A bacterial infection can be enough to induce a fatal illness, with devastating economic consequences for livestock farms, particularly poultry farms. Escherichia coli is a commensal bacterium of 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 threat to poultry health.

[0004] To prevent the risk of infection of farm animals, prophylactic vaccines have been developed with the aim of immunizing individuals and eliminating the risks of infection and the resulting health consequences.

[0005] Prophylactic vaccination consists of inducing an immune response in a healthy individual who has not yet been exposed to a pathogen, with the aim of activating their immune defenses. To this end, an antigen, such as a pathogen or a fragment of a pathogen, is presented to the individual's immune cells. This presentation allows for the activation of adaptive immune cells, B and T lymphocytes. These cells multiply and produce antibodies that neutralize and eliminate the antigen and / or a cellular response that destroys infected cells. This mechanism induces immunological memory, allowing the individual to be protected upon the next encounter with the pathogen. Prophylactic vaccination thus makes it possible to immunize healthy individuals in order to protect them from future illnesses.

[0006] For this mechanism to be effective, it is essential that the pathogen be identified as an intruder by the individual's immune system so that it develops a protective immune response against the infection.

[0007] The prior art teaches us that there are several types of prophylactic vaccines allowing poultry to be immunized against pathogenic bacteria.

[0008] Patent EP2911688A1 relates to a Salmonella enterica serogroup CI serovar intended for use in protecting poultry against disease resulting from Salmonella enterica infection. This Salmonella enterica serovar is in inactivated form. The serovar is used to manufacture vaccines that may be multivalent. Chicks are vaccinated at 30 hours of age. The serovar is used to formulate a vaccine containing an adjuvant, such as, for example, aluminum hydroxide at approximately 25% v / v.

[0009] Patent EP0256792A2 describes a vaccine for the protection of poultry against colibacillosis infections, comprising as an active ingredient E. coli cells inactivated by ultrasound treatment. The vaccine may contain adjuvants, for example an aluminum compound such as an aluminum hydroxide gel. Inoculation of the vaccine into poultry is preferentially carried out via the cloaca. However, the vaccine can also be administered conventionally, for example, intramuscularly, intravenously, or subcutaneously. The essence of the invention is the implementation of ultrasonic cell membrane disruption in the manufacture of a vaccine against colibacillosis in poultry.

[0010] Although vaccines are marketed to prevent infections caused by bacteria in mammals and birds, these vaccines are not satisfactory because they do not generate total and effective protection. Description of the invention

[0011] The inventors have developed a prophylactic vaccine composition without adjuvants, enabling the immunization of mammals and birds, particularly poultry, against pathogenic bacteria. Specifically, the inventors have developed a novel delivery system in which the coating of at least one whole, killed pathogenic bacterium with cationic nanoparticles enhances the mechanism of cellular endocytosis. This improves the presentation of bacterial antigens to immune cells, thereby activating the immune system more rapidly and effectively. Advantageously, the vaccine composition can be multivalent, thus inducing broad-spectrum protection. The vaccine composition can therefore be used to produce combination vaccines. In particular, the prophylactic vaccine can be used as a treatment against avian salmonellosis or colibacillosis.

[0012] Thus, the invention relates to a prophylactic vaccine composition, particularly for mammals and birds, especially poultry, making it possible to limit the risks of contamination by at least one disease resulting from a bacterial infection, characterized in that said composition comprises a cationic agent such as a cationic nanoparticle consisting of a polysaccharide core, said agent coating the whole inactivated bacterium(s). In a particular embodiment, the invention relates to a multivalent prophylactic vaccine composition intended for the treatment of salmonellosis or colibacillosis, particularly for mammals and birds, more specifically poultry such as laying hens.

[0013] Advantage of the invention

[0014] Quite surprisingly, the inventors have demonstrated that administering a vaccine composition comprising inactivated whole pathogenic bacteria coated with cationic agents, such as cationic nanoparticles, constitutes a novel delivery system for effectively immunizing against a bacterial pathogen. It is known from the prior art that cationic nanoparticles can enhance the endocytosis of immune cells by delivering small antigens (on the order of 5 to 15 nanometers). However, in the present work, the inventors have unexpectedly shown that cationic nanoparticles can also enhance the endocytosis of whole bacterial cells. The size of whole cells is on the order of 1 to 2 microns, meaning that these cells are at least 100 times larger than the antigens.

[0015] The vaccine composition has the advantage of being multivalent, depending on the embodiment of the invention. That is to say, it can comprise at least two strains of bacteria, each of which prevents one infection. Thus, the vaccine composition makes it possible to obtain a combined vaccine.

[0016] Depending on the embodiment of the invention, the vaccine composition also allows for the acquisition of cross-immunity. Indeed, the vaccine composition may include a bacterium that induces immunity against variants of the strain in question.

[0017] Inoculation of the vaccine composition can be carried out in ovo. This approach is innovative: no previously described vaccination strategy has proposed administering a whole inactive bacterium directly into the egg. Here, the combination of whole inactive bacteria and cationic nanoparticles proves to be highly effective in terms of vaccine protection and without any detrimental effect on hatching or on the chick. Moreover, by intervening before hatching, the risks of contamination within the flock (between chicks) are reduced, as is the transmission of the disease within flocks. In particular, the inventors have shown that a vaccine composition comprising 3 different strains of E. coli, administered in ovo to the chick, protects the chick from colibacillosis-type infection at both non-lethal and lethal doses in bacterial challenge experiments. The bacterial load is reduced. and the hatching rate is equivalent to that of unvaccinated eggs.

[0018] On the other hand, an intramuscular injection of a vaccine composition comprising a bacterial strain of Salmonella and cationic nanoparticles (NPL) in the laying hen reduces the bacterial load and the hens lay more eggs than non-immunized hens.

[0019] The vaccine composition contains no adjuvants (other than the nanoparticles themselves), thus avoiding adverse effects. This is advantageous since mineral adjuvants (namely mineral salts such as aluminum salts) remain in the body for a very long time. The nanoparticles act as delivery agents for the killed bacteria to immune cells and induce a protective response against infection.

[0020] In the case of in ovo vaccination, the fact of not introducing molecules likely to disrupt the development of the in ovo chick contributes to the effectiveness of the vaccination approach, the vaccine not disrupting the development of the chick and hatching.

[0021] The vaccine composition can be administered in ovo, but also via mucosal (oral, ocular, nasal) or intramuscular routes. Furthermore, the vaccination approach according to the invention can be implemented in mammals as well as in birds, particularly poultry.

[0022] Description of the implementation methods

[0023] A first object of the present invention relates to a vaccine composition comprising a cationic agent and at least one inactivated bacterium, characterized in that said bacterium is whole and in that said cationic agents cover said bacterium.

[0024] The vaccine composition is intended for mammals and birds.

[0025] For the purposes of this invention, "cationic agent" means any molecule comprising a positive electrical charge, such as a cation.

[0026] In a preferred embodiment of the invention, the cationic agent is a cationic nanoparticle consisting of a polysaccharide core.

[0027] The term "cationic nanoparticle consisting of a cationic polysaccharide core" means a solid nanoparticle (NP) comprising a cationic polysaccharide core. The NP may or may not be cross-linked. Its core may or may not be loaded with an anionic phospholipid. This NP is not surrounded by any phospholipid layer.

[0028] For the purposes of the present invention, nanoparticles are particles having a size range between 1 and 500 nanometers. More preferably, nanoparticles have a size range between 10 and 300 nm, in particular between 30 and 250 nm. They can be made of an organic or inorganic material or a mixture of organic and inorganic compounds. They can also be porous or non-porous, and their surface can be anionic, cationic, neutral (hydrophobic or hydrophilic, or a mixture of all these properties). Furthermore, a nanoparticle according to the invention is advantageously used in solution. Thus, the term nanoparticle also includes particles or molecules that are in a nanoparticulate form in solution, such as chitosan and its derivatives. The solution can be an aqueous solution, a buffer solution, or a serum solution. The inventors have indeed observed that certain linear molecules, such as chitosan, form nanometric coils in solution, which behave like conventional nanoparticles.Chitosan can thus be used in the form of a classic nanoparticle (e.g. Qi et al., Carbohydrate Research, 2004, 339(16), 2693-2700) or as such or as a hydrolysate in solution.

[0029] In a first particular embodiment, the cationic polysaccharide forming the core of the NP is a non-crosslinked polymer obtained by the reaction between a polysaccharide selected from starch, dextran, chitosan, dextrin, and maltodextrin, polyfructoses (inulin), polymannoses, polygalactoses, polygalacto-mannans (guar gum) and at least one cationic ligand selected from a primary, secondary, or tertiary amine or quaternary ammoniums. The core is not loaded with lipids.

[0030] In a second particular embodiment, the cationic polysaccharide forming the core of the NP is a cross-linked polymer obtained by the reaction between a polysaccharide selected from starch, dextran, chitosan, dextrin, and maltodextrin, polyfructoses (inulin), polymannoses, polygalactoses, polygalacto-mannans (guar gum), and at least one cationic ligand selected from a primary, secondary, tertiary amine or quaternary ammoniums, followed by the addition of a cross-linking agent. The cross-linking agent is selected from epichlorohydrin, a dicarboxylic acid, or an acid chloride, such as sebacic acid. The core is not lipid-loaded.

[0031] In a preferred embodiment, the cationic polysaccharide is obtained by the reaction between maltodextrin and glycidyltrimethylammonium, whether the NP is cross-linked or not.

[0032] In a third particular embodiment, the cationic polysaccharide forming the core of the NP is loaded with an anionic phospholipid. This anionic phospholipid can be selected from diacylphosphatidyl glycerol, diacylphosphatidyl serine, or diacylphosphatidyl inositol. In another preferred embodiment, the anionic phospholipid is dipalmitoylphosphatidylglycerol (DPPG). The cationic polysaccharide forming the core of the NP is not cross-linked.

[0033] In a highly preferred embodiment, the NP is a maltodextrin nanoparticle loaded with DPPG.

[0034] In a fourth particular embodiment, the cationic polysaccharide forming the nucleus of the NP is not loaded with lipids and is not cross-linked.

[0035] Among the group of birds, poultry are understood in particular. Poultry, within the meaning of the invention, are domestic birds which serve as a source of eggs or meat and which include commercially important species such as, for example, chickens, laying hens, turkeys, ducks, geese, guinea fowl, pheasants, pigeons and peacocks.

[0036] In a preferred embodiment, the bacterium is chosen from the following group: Salmonella enterica ser. Typhi; Streptococcus Pneumoniae, Haemophilus influenzae type b, Mycobacterium tuberculosis, Extraintestinal pathogenic E.Coli (ExPEC), ente-rotoxigenic E.Coli (ETEC); S. enterica ser., Paratyphi A; Neisseria Gonorheae; Clostridium Difficile; Campylobacter spp: Shigella spp Staphylobactus Aureus, Helicobacter pylori.

[0037] For the purposes of this invention, "whole bacterium" means an unfragmented bacterium in its complete form, in particular one whose cell membrane is intact.

[0038] For the purposes of this invention, "inactivated or inactive bacteria" means a non-living bacterium that has been previously killed but is intact. They can be killed, for example, by treatment with formaldehyde or any other inactivation method known to those skilled in the art.

[0039] For the purposes of this invention, "coated with cationic nanoparticles" means that the nanoparticles coat the surface of the inactivated bacteria. The nanoparticles cover the killed bacteria with a homogeneous layer. The coverage rate can be defined by the weight ratio of bacterial proteins: NPL.

[0040] Good endocytosis efficiency of NPL-coated bacteria, associated with infection prevention, was observed for a bacterial protein:NPL ratio between 1:0.1 and 1:3. In a preferred embodiment, this ratio is between 1:0.01 and 1:10.

[0041] The vaccine composition includes at least one bacterium in a sufficient quantity to induce effective protection to prevent, or at least reduce, bacterial infection.

[0042] By "avoiding infection" is meant in the sense of the invention, that the vaccine composition can protect 100% against the risks of infection or, if it does not totally avoid the risk of infection, then the protection conferred by the vaccine is sufficient so that the individual does not trigger the disease or if he triggers it, the symptoms of the infection are at least reduced and the individual avoids death.

[0043] In one embodiment of the invention, the vaccine composition is prophylactic.

[0044] In another embodiment of the invention, the vaccine composition is intended for birds. In a particular embodiment of the invention, the prophylactic vaccine composition is intended for poultry. In particular, the embryo in the egg.

[0045] By "multivalent vaccine composition" in the sense of the invention, it is understood that the vaccine composition comprises several different bacteria capable of inducing immunity against several diseases associated with the different bacteria.

[0046] In a particular embodiment, the vaccine composition allows for the development of combined vaccines.

[0047] For the purposes of this invention, "combination vaccines" means a vaccine composition comprising several bacteria of different species or families so as to induce, simultaneously, immunity against several different bacteria.

[0048] In one embodiment, the vaccine composition includes at least one whole and inactivated bacterium which enables cross-immunity to be induced in the individual who has been vaccinated.

[0049] For the purposes of this 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.

[0050] Cross-immunity is linked to the phenomenon of cross-reactivity. Antibodies are usually specific to a particular antigen. It is thanks to this specificity that antibodies target and eliminate the antigens they have detected. A mutant bacterium retains common antigens that can be the target of a vaccine-induced response.

[0051] Thus, cross-reactions with closely related bacteria species can occur. A bacterium possesses numerous surface antigens. When an animal is immunized against a bacterium by means of an injection of whole bacteria, it produces antibodies against many bacterial antigens. If two bacteria possess an identical or similar antigen, the individual will have acquired immunity against both bacteria.

[0052] 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.

[0053] The vaccine composition may, for example, be composed of 3 inactivated strains of E. coli mixed with lipidized maltodextrin nanoparticles (LPNPs) in order to prevent colibacillosis.

[0054] In a first particular embodiment, the cationic polysaccharide The core of the NP is a cross-linked polymer obtained by reacting a polysaccharide selected from starch, dextran, dextrin, chitosan, maltodextrin, polyfructoses (inulin), polymannoses, polygalactoses, polygalacto-mannans (guar gum), and at least one cationic ligand selected from a primary, secondary, tertiary, or quaternary amine, followed by the addition of a cross-linking agent. The cross-linking agent is selected from epichlorohydrin, a dicarboxylic acid, or an acid chloride, such as sebacic acid. The core is lipid-free.

[0055] In a preferred embodiment, the cationic polysaccharide is obtained by the reaction between maltodextrin and glycidyltrimethylammonium.

[0056] In a particular embodiment, the cationic polysaccharide forming the core of the NP is loaded with an anionic phospholipid. This anionic phospholipid can be chosen from diacylphosphatidyl glycerol, diacylphosphatidyl serine, or diacylphosphatidyl inositol.

[0057] In another preferred embodiment, the anionic phospholipid is dipal-mitoyIphosphatidyIglycerol (DPPG).

[0058] In a highly preferred embodiment, the NP is a maltodextrin nanoparticle loaded with DPPG.

[0059] In a second particular embodiment of the invention, the polysaccharide forming the core of the NP is a linear, cationic, and non-crosslinked polysaccharide. In this particular embodiment, the cationic polysaccharide forming the core of the NP is a non-crosslinked polymer obtained by the reaction between a polysaccharide selected from starch, dextran, dextrin, and maltodextrin, polyfructoses (inulin), polymannoses, polygalactoses, polygalacto-mannans (guar gum), and at least one cationic ligand selected from a primary, secondary, or tertiary amine or quaternary ammonium compounds. The core is not loaded with lipids.

[0060] Thus, the cationic nanoparticle can be: either in the form of a nanoparticle (I) consisting of a polysaccharide core and a porous cationic core in crosslinked form, or in the form of a nanoparticle (II) consisting of a linear cationic polysaccharide core in non-crosslinked form.

[0061] Thus, according to the different embodiments of the vaccine composition, said composition may comprise:

[0062] - An inactivated and whole bacterium, coated with cationic nanoparticles consisting of a porous polysaccharide core in a cross-linked form.

[0063] - An inactivated and whole bacterium, coated with cationic nanoparticles consisting of a polysaccharide core in non-crosslinked form.

[0064] - At least two bacteria of different strains and / or species or families, said bacteria being inactivated and whole, coated with cationic nanoparticles consisting of a porous polysaccharide core in a cross-linked form.

[0065] - At least two bacteria of different strains and / or species or families, said bacteria being inactivated and whole, covered with cationic nanoparticles consisting of a porous polysaccharide core in non-crosslinked form.

[0066] A second object of the invention relates to a multivalent vaccine composition for mammals and birds comprising at least two different inactivated bacteria characterized in that said bacteria are whole and are covered with cationic nanoparticles consisting of a polysaccharide core for use by intramuscular, mucosal or in ovo administration.

[0067] A third object of the invention relates to a combined vaccine for mammals and birds comprising at least two inactivated bacteria of different species or families characterized in that said bacteria are whole and covered with cationic nanoparticles consisting of a polysaccharide core for use by intramuscular, mucosal or in ovo administration.

[0068] A fourth object of the invention relates to a method for preventing a disease linked to a bacterial infection in mammalian and bird animals, comprising a vaccine composition including at least one inactivated whole pathogenic bacterium coated with cationic nanoparticles consisting of a polysaccharide core and comprising the following steps: - To have cationic nanoparticles consisting of a polysaccharide core which can be in cross-linked or non-cross-linked form and at least one whole bacterium. - Proceed to inactivate the entire bacterium using formaldehyde. - Mix the said cationic nanoparticles with the said inactivated whole bacterium.

[0069] In a particular embodiment, said prevention method comprises a vaccine composition inoculated by mucosal route, injectable and / or administered in ovo.

[0070] In particular embodiments of the invention, the vaccine composition is administered to poultry, namely in ovo, mucosally and orally in chicks and intramuscularly in laying hens. Brief description of the drawings

[0071] [Fig. 1]: [Fig. 1]: Endocytosis of E. coli after NPL coverage. The delivery of whole E. coli bacteria was evaluated in human H292 cells. Fluorescent FITC-E. coli, alone or in association with NPL (ratio 1 / 3 to 1 / 0.05), were incubated with human H292 cells for 4 h, and the percentage of positive cells was measured by flow cytometry with and without trypan blue (TB). The Results represent the mean ± SEM of 3 experiments. Statistical analysis: two-way ANOVA, ** p < 0.01, *** p < 0.001.

[0072] [Fig.2] [Fig.2]: Intracellular delivery by confocal microscopy, with a ratio 1 / 3. The delivery of E. coli bacteria was evaluated in H292 cells. Fluorescent E. coli-FITC associated with NPLs (1 / 3 ratio) were incubated with H292 cells for 4 h, and intracellular localization was observed by confocal microscopy. A representative image was taken. Red: plasma membrane; blue: nuclei; green: E. coli. Scale bar: 1 Opm.

[0073] [Fig.3] [Fig.3]: Diagram of the vaccination protocol for the in ovo vaccination trial. The The commercial vaccine was administered only on the positive control.

[0074] [Fig.4] [Fig.4]: Intestinal permeability test for 8 birds from each group, 6 days after the challenge (Day 20). The results represent the mean ± SD.

[0075] [Fig.5][Fig.5]: Analysis of anti-E. coli slgA in the feces of 8 birds from Each group, 13 days after the challenge (Day 27). The results represent the mean ± SD of the absorbance values ​​obtained by ELISA. Statistical analysis: one-way ANOVA, *p < 0.05.

[0076] [Fig.6] [Fig.6]: Clinical score of liver lesions from 8 birds of each group, 6 days after the challenge (Day 20). The results represent the mean ± SD of the group scores. Statistical analysis: one-way ANOVA, * p < 0.05.

[0077] [Fig.7] [Fig.7]: Diagram of the in ovo vaccination trial protocol. Only the positive group was vaccinated on day 1 with the commercial vaccine (Poulvac).

[0078] [Fig.8] [Fig.8]: The percentage of mortality in each group after the challenge lethal at J14 (n=30).

[0079] [Fig.9] [Fig.9]: Measurement of bacterial load in the air sacs of birds from each group, assessed 2 days after the challenge (Day 16) on 8 birds, by MPN. The results represent the mean ± SD. Statistical analyses were performed using one-way ANOVA, * p < 0.05.

[0080] [Fig. 10] [Fig. 10]: Intestinal permeability of 8 birds from each group 6 days after the challenge (D20). The results represent the mean ± SD. Statistical analyses were performed by one-way ANOVA, * p < 0.05.

[0081] [Fig. 11][Fig. 11]: Intestinal lesion score of 8 birds from each group 6 days after the challenge (D20). Results represent mean ± SD. Statistical analyses were performed by one-way ANOVA, * p < 0.05.

[0082] [Fig. 12] [Fig. 12]: Diagram of the intramuscular vaccination trial protocol.

[0083] [Fig.13][Fig.13]: Egg laying. Top: daily number of eggs laid by the hens in each group after the challenge. Error bars are hidden to clarify the graph. Bottom: Average daily number of eggs laid by hens in each group after the challenge. Statistical analyses: one-way ANOVA * p < 0.05, *** p < 0.001, **** p < 0.0001.

[0084] [Fig. 14] [Fig. 14]: Quantification of bacterial load in the cecum of chickens from each group, measured by qPCR. The results represent the mean ± SEM. Statistical analysis: one-way ANOVA ** p < 0.01, *** p < 0.001.

[0085] [Fig. 15] [Fig. 15]: Quantification of bacterial load in the cecum of the chicken from each group, measured by qPCR. The results represent the mean ± SEM.

[0086] [Fig. 16] [Fig. 16]: Timetable of the in ovo vaccination trial protocol. Only the "positive control" group was vaccinated on day 1 with the commercial vaccine (Poulvac).

[0087] [Fig. 17] [Fig. 17]: Measurement of bacterial infection in the trachea (above) and alveoli (below) of birds in each group, assessed on 8 birds, by most probable number (MPN). The results represent the number of positive and negative birds per group.

[0088] [Fig. 18][Fig. 18]: Analysis of anti-E. coli slgA in the feces of 8 birds from each group after the challenge. The results represent the mean ± ET of the antibody titers. Statistical analysis: One-way ANOVA for each day.

[0089] [Fig. 19] [Fig. 19]: Lung lesion score of 8 birds per group after the challenge. The results represent the average for each group. Statistical analyses were performed for each day using one-way ANOVA. EXAMPLES

[0090] Abbreviations: • NPL: Lipid-based maltodextrin nanoparticles • ID: Intradermal • in: Intranasal • IP: Intraperitoneal • iv: Intravenous

[0091] EXAMPLE 1: Optimization of the formulation with cationic nanoparticles

[0092] The objective of this study is to confirm the efficacy as a delivery system of a composition, based on a nanoparticle and an inactivated whole E. coli strain, to activate immune cells. 1-A Materials and methods:

[0093] A-Vaccine formulations

[0094] Cationic nanoparticles (NPL) are cationic lipid-coated maltodextrin nanoparticles.

[0095] The composition was prepared with an inactivated strain of E. coli mixed with cationic nanoparticles. The E. coli bacteria were inactivated with 0.4% formaldehyde and then purified by centrifugation. The protein quantity was measured by microBCA assay. The composition was prepared by mixing the killed bacteria with an aqueous NPL solution at different weight ratios (100 pg of E. coli protein with 5, 10, 30, 50, 100, or 300 pg of NPL). The size and surface charge of the formulation were characterized by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively (Zetasizer NanoZS, Malvern Analytical, France), to determine whether the nanoparticles covered the surface of the killed bacteria.

[0096] B-Delivery of E. coli

[0097] The ability of NPLs to increase the endocytosis of whole E. coli inactivated by immune cells was evaluated by flow cytometry and confocal microscopy. • E. coli labeling with fluorescein:

[0098] Inactivated bacteria were labeled with fluorescein isothiocyanate (FITC) by mixing 5 mg of E. coli with 50 pg of FITC (1%, Sigma, France) in sodium carbonate buffer at pH 8.3 for 2 h. They were then dialyzed on an 100Da dialysis cassette (Thermofisher, France). Protein content was measured by microBCA assay (Pierce, France). The labeled bacteria were then associated with NPLs at different weight ratios. • Flow cytometry:

[0099] H292 cell lines were seeded in 24-well plates at a rate of 50,000 cells per well until confluence. The cells were then incubated with the equivalent of 1pg of protein alone or in combination with different ratios of NPL 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). To distinguish intracellular bacterial delivery from membrane binding, the cells were incubated with 40 pg / mL of Trypan Blue (TB, Sigma France) to quench external FITC fluorescence. • Confocal Microscopy

[0100] H292 cell lines were seeded in Labtek chambers (Fisher Sci., France) at a rate of 10,000 cells per well until confluence. The cells were then incubated with the equivalent of 1pg of protein alone from whole-cell killed bacteria or associated with NPLs for 4 hours. The cells were washed, and the nuclei were stained by incubating Hoechst 33342 (Sigma, France) at 0.1 pg / mL for 5 minutes at 37°C. The cells were then washed, and the plasma membrane was labeled with AF-633-labeled agglutinin (WGA, Invitrogen France) at 1 pg / mL for 10 minutes at 37°C. The slides were washed again with PBS, fixed with 0.4% formaldehyde for 20 minutes, and mounted for observation under a microscope (LSM 710 Zeiss, France). 1-B Results:

[0101] • Characterization of formulations:

[0102] [Tables 1] Z-average (nm) PDI Zeta potential (mV) E. coli 1420 0.08 -4.6 E. colUWL 1 / 0.05 2451 0.60 -3.6 E. cost^PL 1 / 0.1 1125 0.30 -1.6 E. cost^PL 1 / 0.3 716.9 0.57 -1.1 E. cost^PL 1 / 0.5 1120 0.52 +2.7 E. cost^PL 1 / 1 915.5 0.68 +3.8 E. cost^PL 1 / 3 1010 0.75 +14

[0103] Table 1: Characterization of the size of E. coli / NPL formulations by dynamic light scattering (DLS) and of the zeta potential by electrophoretic light scattering (ELS). Inactivated whole E. coli were mixed with increasing amounts of NPL.

[0104] DLS and ELS analyses showed that the inactivated E. coli had a uniform size of 1.42 pm with a PDI of 0.08, and an anionic surface charge of -4.6 mV. This indicates that the bacterial structure remained intact despite inactivation. When increasing amounts of NPL were added, the overall size did not change, but the zeta potential gradually increased, becoming cationic at a ratio of 1 / 0.5. This indicates that the bacteria were progressively coated by the NPL without any aggregation.

[0105] The delivery of E. coli by NPLs was evaluated by flow cytometry (see [Fig. 1]) on human respiratory tract epithelial cells (H292). Without NPLs, the bacteria were endocytosed by 14% of the cells. Since no difference was observed in the presence of TB, this suggests that the bacteria were indeed endocytosed (inside the cells). When coated with NPLs, the bacteria are taken up by at least 40% of the cells, confirming their potential as a delivery system. Furthermore, delivery was significantly more efficient with a low amount of NPL, and the greatest release was observed with a ratio of 1:0.3 (77%) and 1:0.1 (75.7%). In the presence of TB, 65.6% of the cells were still positive with a ratio of 1 / 0.3 and 63% with a ratio of 1 / 0.1, which confirms that most of the bacteria were inside the cells.

[0106] Intracellular delivery was also confirmed by confocal microscopy, with a ratio of 1 / 3. Approximately 20 to 30 E. coli bacteria (green) were observed in each cell, near the nuclei, confirming intracellular localization. Conclusion:

[0107] The E. coli / NPL vaccine formulation consists of inactivated whole bacteria coated with NPL. Coating the bacteria, even with a small amount of NPL, has a significant impact on the ability of the bacteria to be taken up by cells.

[0108] EXAMPLE 2: In ovo vaccination trial against colibacillosis 2-A Materials and methods:

[0109] A-Preparation of vaccines

[0110] The vaccine consists of three inactivated E. coli strains mixed with lipidized maltodextrin nanoparticles (LNPs). In short, strains O78:K80, O1:K1, and O2:K1 were inactivated with 0.4% formaldehyde, and the protein content was measured by pBCA assay. Finally, 33.3 pg per strain were mixed with the LNPs to obtain 100 pg of protein per vaccine dose.

[0111] B-Animals

[0112] All animal work has been evaluated and approved by the Animal Research Ethics Committee of Imunova Anâlises Biolôgicas, protocol number 06 / 2021.

[0113] For this experiment, 390 fertile eggs were acquired from a commercial incubator and incubated at the Imunova experimental unit. The eggs were randomly divided into 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.

[0114] [Tables2] Group Identification Challenge1 1 Negative control - 2 Positive control Challenge E. coli2 3 Commercial control Vaccinated by mucosal route with Poulvac® E. coli (Zoetis) + challenge2 E. coli 4 Vaccinated in ovo Vaccinated in ovo with NP / 100 pg + challenge E. coli2.

[0115] Table 2: Identification of experimental groups.

[0116] 'All groups, including the negative control, received orally the live attenuated Mass I-H120 vaccine against infectious bronchitis virus (IBV) at a dose 100 times higher than that recommended by the manufacturer, in order to sensitize the animals to a challenge to E. coli.

[0117] 2Infection with 108 CFU A Escherichia coli, at a dose of 100 pL / bird orally. The challenge was confirmed by microbiological recovery of bacteria from the inoculum.

[0118] C-Vaccination

[0119] Animals in group 3 received, on day 1 after hatching, a dose of Poulvac® E. coli live vaccine. Animals in group 4 received, on the 18th day of incubation, an in ovo application of the vaccine with a regulatory vaccine dose of 50 pL. After hatching, the groups of one-day-old broiler chickens were housed in isolators (1.2 m2) and fed ad libitum according to the recommendations for their age.

[0120] D-Challenge

[0121] AJ 10, all animals, including those in the negative control group, received via oral administration of a live attenuated Mass I-H120 vaccine against infectious bronchitis virus (IBV), at a dose 100 times higher than that recommended by the manufacturer.

[0122] At day 14, animals in groups 2, 3 and 4 were infected with 10⁸ CFU AEscherichia coli (strain 19501, a different strain from that used in the vaccine), 100 pL / bird, orally.

[0123] The vaccination protocol is shown in [Fig.3].

[0124] E-Sampling

[0125] Sampling and analysis were carried out on 8 birds, on day 16, day 20 or day 27.

[0126] E-Analysis • Intestinal permeability:

[0127] Intestinal permeability was assessed by oral administration of FITC-Dextran, a non-absorbable fluorescent marker (FITC-Dextran, 3000 to 4000 kDa), and identified in plasma / serum, to monitor gastrointestinal epithelial integrity (Vicuna et al., 2015). • Cytokine expression

[0128] Cytokine expression was assessed by qPCR (IL-1 [3, IFNγ, IL-10, IL-4), using target-specific primers. In this type of analysis, each target-sample combination generates a threshold value, Ct (cycle threshold), which is a relative measure of the target-specific messenger RNA (mRNA) concentration in the sample. This value must be normalized against the expression of a certain reference gene; in this case, the geometric mean of the GAPDH and ACTB genes was used, generating an ACt value (target Ct / Mean Ct of GAPDH+ACTB) (Bustin et al., 2009). In addition to this normalization, the data were also normalized against the mean ACt of the control group, generating an AACt (ACt / Mean Control ACt). For undefined results, the The maximum value of CT (40) was considered and, for analytical purposes, was artificially modified to 41. • Quantification of anti-E slgA. coli:

[0129] The production of secretory IgA specific to E. coli was evaluated by ELISA. In brief, the samples were diluted in 1% casein in PBS. The ELISA plates were coated with LPS of E. coli (field-isolated strain). The plates were then washed three times with 200 qL / well of PBS + 0.05% Tween20 for 5 min / wash. The wells were blocked with 1% casein in PBS. The samples were tested by serial dilution. The plates were washed, and an anti-chicken IgA (BioRad) diluted in 0.1% casein was added. After washing, the assay was developed with a TMB solution (Life Technologies). The absorbance was read at 450 nm. • Detection and quantification of E. coli:

[0130] The detection and quantification of E. coli by Most Probable Number (MPN) was performed according to ISO 7251:2005. E. coli counts were determined using standard microbiological methods (dilution in an enrichment medium followed by plating in a selective / different medium). In short, samples were enriched in buffered peptone water (BPT), then in EC broth, and finally in EMB and MacConkey agar. Samples were serially diluted threefold in BPT 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 tested biochemically and confirmed. • Liver histology:

[0131] 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 assessments and readings were performed under a microscope by an experienced veterinary histopathologist.

[0132] [Tables3] Liver Score 0 Normal Appearance 1 Histologically normal, sometimes with subtle focal or multifocal mononuclear hyperplasia. No necrosis. 2 Possible degenerative liver lesion with varying degrees of vacuolization and / or necrosis. Central or pericentral proliferative reaction involving mononuclear cells. Presence of an interstitial and multifocal neutrophil infiltrate. Hyperemia / hemorrhages. 3 Same lesions as described above, with the addition of fibrinous perihepatitis and / or parenchymal granulomatous reactions.

[0133] Table 3: List of histological parameters for scoring liver lesions. 2-B Results:

[0134] The intestinal permeability of unvaccinated and unchallenged birds (negative control) was 0.26 pg / mL, and 0.31 pg / mL for challenged and unvaccinated birds (positive control). Following vaccination with the commercial vaccine, permeability was 0.26 pg / mL, as in the negative control, confirming the efficacy of this vaccine. Furthermore, when vaccinated in ovo with the VXN-E. coli formulation, all birds exhibited low permeability (0.18 pg / mL), lower than that of the negative control, although not statistically significant.

[0135] Anti-E intestinal A-sIgA. coli (LPS)

[0136] Secretory anti-LPS IgA directed against E. coli was analyzed in feces by ELISA. The absorbance obtained for the negative control birds was approximately 0.05 AU, and 0.095 pg / mL for the positive control birds, indicating that the oral challenge did not induce intestinal IgA secretion. Furthermore, for birds vaccinated mucosally with the commercial vaccine, the absorbance remained at 0.055 AU, as in the negative control, suggesting that this vaccine failed to promote a mucosal humoral response. In contrast, birds vaccinated in ovo with the VXN-E. coli formulation exhibited a significantly higher absorbance of 0.16 AU.

[0137] B-Histopathology of the liver

[0138] The liver lesion score was measured 6 days after the challenge. Birds in the negative control group had a mean score of approximately 1, indicative of mild hyperplasia. In contrast, unvaccinated birds in the positive control group They obtained a mean score of 2.35, suggesting liver damage and necrosis induced by E. coli infection. When vaccinated with the commercial vaccine, the mean lesion score in the birds was 1, as in the negative control group. Birds vaccinated in ovo had a mean lesion score < 1. These results show that both the commercial vaccine and in ovo vaccination protect against E. coli-induced liver damage. Conclusion :

[0139] This first trial indicates that in ovo vaccination with the VXN-E. coli vaccine protects birds against hepatic infection by E. coli and induces the secretion of slgA against the bacterium in the intestine.

[0140] EXAMPLE 3: in ovo vaccination against a lethal challenge to E. coli

[0141] This second trial was identical to the first in ovo trial in terms of timing, animals per group, and treatment, but with a lethal challenge to E. coli. The analyses then focused on the protection provided by the vaccines against bacterial load in representative organs, physiological abnormalities, and subsequent mortality, observed in each group. 3-A. Materials and methods

[0142] A-Challenge

[0143] At J10, all animals, including the negative control, received orally a live attenuated Mass I-H120 vaccine against infectious bronchitis virus (IBV), at a dose 100 times higher than that recommended by the manufacturer.

[0144] At J14, each animal in the determined group was exposed to 4.2xlO12CFU of Es-cherichia coli (strain 19501), at a rate of 100 pL / bird in the air sacs.

[0145] The general schedule is detailed in [Fig.7].

[0146] B-Analysis • Intestinal permeability:

[0147] Intestinal permeability was assessed by oral administration of FITC-Dextran, a non-absorbable fluorescent marker (FITC-Dextran, 3000 to 4000 kDa), and identified in plasma / serum, to monitor gastrointestinal epithelial integrity (Vicuna et al., 2015). • Detection and quantification of E. coli:

[0148] The detection and quantification of E. coli by MPN (most probable number) was carried out according to ISO 7251:2005. E. coli counts were determined using standard microbiological methods (dilution in an enrichment medium followed by plating in a selective / different medium). In short, the samples were enriched in buffered peptone water (BPT), then in EC broth, and finally in EMB and MacConkey agar. The samples were The colonies were serially diluted in triplicate in EPB prior to incubation to allow for quantification using the most probable number technique (Blodgett et al., 2015). For E. coli detection, only the serial dilution step was omitted. The isolated suspect colonies were tested biochemically and confirmed. • Intestinal histology:

[0149] The birds were euthanized, and intestinal samples were collected and fixed according to the method of Rebel et al. (2011). Ileal samples were paraffin-embedded and mounted on slides. All histopathological assessments and readings were performed under a microscope by an experienced veterinary histologist.

[0150] [Tables4] Ileum Score 0 Normal appearance. 1 Epithelial integrity, appropriate presence of inflammatory cells, absence of necrosis, vascular disturbances, abnormal desquamation. Possible lymphoid hyperplasia, with production of primary follicles. 2 Mild to moderate lesions: mucosal and submucosa hyperemia, slight desquamation, superficial epithelial necrosis of the mucosa, moderate heterophilic infiltration of the lamina propria or submucosa, mucosal atrophy or hypertrophy, moderate hemorrhages. Moderate hyperplasia of mucus-producing cells and mononuclear cells. 3 Lesions as above, but of a major degree. Intense heterophilic infiltration, as well as epithelial and submucosa necrosis of the glands, intense mononuclear hyperplasia between the crypts, profuse hemorrhages, and hyperplasia of mucus-producing cells.

[0151] Table 4: List of histological parameters for the evaluation of intestinal lesions (ileum). 3-B. Results

[0152] A-Outbreak

[0153] [Tables5] Outbreak Group (n = 60) Negative Control 81.2% Positive Control Commercial VXN Vaccine in ovo 78.3%

[0154] Table 5: Percentage of outbreak in the in ovo vaccinated group compared to the unvaccinated groups (negative control, positive control, commercial vaccine).

[0155] Hatching was measured in this study to evaluate the safety of the VXNÆ. coli in ovo vaccine. Thus, the hatching rate of vaccinated eggs (n=60) was compared to that of unvaccinated eggs (n=180) before randomization to each group (negative control, positive control, and commercial vaccine). A similar hatching rate was observed between vaccinated (78.3%) and unvaccinated (81.2%) eggs. Therefore, the vaccine formulation is safe as it has no impact on hatching.

[0156] B-Bird survival after lethal challenge

[0157] The E. coli challenge was performed on day 14 with 4.2 x 10¹² CFU directly into the air sacs. Bird survival after the lethal challenge is shown in [Fig. 8]. This high dose impacted 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% for birds vaccinated in ovo with the VXNÆ. coli vaccine, suggesting better protection against infection.

[0158] C-Quantification of E. coli in air sacs by MPN

[0159] Infection was assessed by quantifying bacteria in the air sacs using MPN. The measurement of bacterial load in the air sacs using MPN is shown in [Fig. 9]. The negative control birds had only a low quantity of bacteria in their air sacs, relative to the natural bacterial flora. Both unimmunized and challenged birds had a higher quantity of E. coli (10⁴ MPN / g), confirming the efficacy of the challenge. A significantly higher infection was observed in the air sacs of birds vaccinated with the commercial mucosal vaccine (10⁶ MPN / g), corroborating the survival results. Conversely, as with the survival results, a lower infection was observed in birds vaccinated in ovo with the E. coli / NPE vaccine (3 x 10³ MPN / g), confirming its efficacy in protecting against this bacterial infection.

[0160] D-Intestinal Permeability

[0161] The intestinal permeability of unvaccinated and unchallenged birds was 0.22 pg / mL, and 0.18 pg / mL for challenged and unvaccinated birds. Intestinal permeability is shown in [Fig. 10]. Upon vaccination with the commercial vaccine or in ovo with the VXN / E. coli vaccine, permeability decreased significantly to 1.2 pg / mL, suggesting vaccine-induced protection.

[0162] E-Intestinal histopathology

[0163] The ileal lesion score was measured 6 days after the challenge. The intestinal lesion score is shown in [Fig. 11]. Birds in the negative control group obtained a mean score of less than 1 (0.25), representing a healthy and normal-looking ileum, as expected. In contrast, unvaccinated birds in the positive control group obtained a significantly higher mean score of 1.7, suggesting ileal lesions with vascular disturbances and desquamation, induced by E. coli infection. Surprisingly, when birds were vaccinated with the commercial vaccine, the lesions worsened significantly with a mean score of 2. In contrast, birds vaccinated in ovo had a mean score of 1, suggesting protection against E. coli-induced intestinal lesions. Conclusion:

[0164] This second trial indicates that the in ovo VXN-E. coli vaccine protects birds from mortality induced by lethal E. coli infection. Furthermore, this vaccine reduces the bacterial load in air sacs and intestinal lesions due to infection, confirming the benefit of this vaccine.

[0165] EXAMPLE 4: Intramuscular vaccination trial against Salmonella in laying hens 2-A. Materials and methods:

[0166] A-Preparation of vaccines

[0167] The vaccine is manufactured from an inactivated strain of Salmonella enteritidis mixed with lipid-coated maltodextrin nanoparticles (LPNPs). In short, the Salmonella SE147 strain was inactivated, and the protein content was measured by a pBCA assay. Finally, 200 pg of killed bacteria were mixed with either LPNPs (formulation named "Vaxinano 1") or non-crosslinked LPNPs (formulation named "Vaxinano 2"), at a rate of 200 pg of protein per vaccine dose. The non-crosslinked LPNP is composed of linear cationic maltodextrin with an anionic inner core.

[0168] B-Animals

[0169] For this experiment, a total of 84 LSL chickens (from a commercial breeding farm) were randomly divided into 12 pens (7 birds / pen) as described in Table 7. Serum was collected and tested for antibody titer against Salmonella (performed by DGZ using the Biochek kit).

[0170] [Tables] Group (7 animals / group) Description 1, 5, 9 Vaccinated with saline solution 2, 6, 10 Vaccinated with 200pg of "Vaxinano 1" 3, 7, 11 Vaccinated with 200pg of "Vaxinano 2" 4, 8, 12 Vaccinated with a commercial vaccine (Salenvac)

[0171] Table 6: Identification of experimental groups.

[0172] C-Vaccination

[0173] At W12 age, all chickens were vaccinated intramuscularly in the breast with either 500pL of saline solution, 500pL of the Vaxinano 1 or Vaxinano 2 formulation (containing 200pg of Salmonella protein), or the commercial vaccine. One month later, at W16, the animals received a second dose of the same vaccine formulation.

[0174] D-Challenge

[0175] One month after the boost, all animals were challenged intravenously with 500pL of 1.3xl08 CFU of S. enteritidis SE147.

[0176] The general program is represented [Fig.12].

[0177] E-Direct Debits

[0178] From W20 to W25, the eggs were collected and individually bacteriologically analyzed for Salmonella. At W25, all chickens were euthanized, and the serum and liver were collected and stored at -20°C. The spleen and cecum were bacteriologically analyzed for Salmonella.

[0179] F-Analyses

[0180] Laying: Eggs were collected daily after the challenge (except on Saturday) and stored at 4°C. The number of eggs per group was recorded.

[0181] Infection: Infection was quantified by qPCR in the spleen and cecum of each chicken at W25. 2-B. Results

[0182] A-Laying of eggs

[0183] The average number of eggs laid in each group was counted daily after the test. Egg laying is shown in [Fig. 13]. Hens vaccinated with a sham vaccine laid a low number of eggs after the challenge, averaging 2.6 eggs per day, confirming the bird's infection. In contrast, birds vaccinated with Salenvac laid significantly more eggs than the control group, averaging 4.9 eggs per day (p < 0.001), suggesting protection against the challenge. Similarly, both formulations of Vaxinano enabled hens to lay significantly more eggs, with an average of 4.3 eggs per day for Vaxinano 1 (p < 0.05) and 4.7 for Vaxinano 2 (p < 0.001), suggesting equivalent protection against challenge.

[0184] B-Infection of the cecum and spleen

[0185] The infection was assessed by quantifying the bacterial load in the cecum and spleen.

[0186] Post-challenge infection was assessed by quantifying the bacterial load in the cecum and spleen. This quantification is shown in [Fig. 14]. Chickens that received a saline injection had a significant infection, with a mean infection rate of 400 CFU / g, but with more than 50% of birds above 1000 CFU / g. In contrast, all birds vaccinated with the Vaxinano formulation or with Salenvac had a significantly lower infection rate, with a mean infection rate below the threshold for birds vaccinated with Vaxinano 1. This confirms the strong protection provided by IM vaccination.

[0187] The infection was finally quantified in the spleen. This quantification is shown in [Fig. 15]. Although the infection was lower than in the cecum, 77% of birds vaccinated with the saline solution were still positive in the spleen, whereas 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. Conclusion :

[0188] This trial indicates that the whole inactivated Salmonella / NP vaccine administered intramuscularly protects birds from challenge by S. ente ritidis, regardless of the formulation, which allows the birds to lay significantly more eggs than non-immunized animals.

[0189] EXAMPLE 5: Mucosal vaccination against colibacillosis

[0190] This trial was planned in the same way as the in ovo trial. The analyses then focused on the protection provided by the vaccines against infection in target organs, physiological abnormalities, and mucosal antibody titers. 5-A Materials and methods:

[0191] A-Vaccine preparation

[0192] The vaccine is manufactured from three inactivated strains of E. coli bacteria mixed with lipidized maltodextrin nanoparticles (LNPs). The O78:K80, OLK1, and O2:K1 strains were inactivated with 0.4% formaldehyde, and their protein content was measured by BCA assay. Finally, 33.3 pg of each strain were then mixed with LNPs at a rate of 100 pg of protein per vaccine dose.

[0193] B-Animals

[0194] All work on animals has been evaluated and approved by the Animal Research Ethics Committee of Imunova Anâlises Biolôgicas, protocol number 06 / 2021.

[0195] For this experiment, 150 one-day-old chicks were acquired from a commercial hatchery and randomly sorted into five experimental groups, in individual isolation units within Imunova, and were treated according to the following table:

[0196] [Tables?] Group (30 animals / group) Challenge 1 Negative control Unvaccinated, not challenged Positive control Unvaccinated, challenged 2 Commercial vaccine Poulvac E. coli (mucosal), challenge 2 VXN mucosal vaccine VXN mucosal vaccination, challenge 2 VXN sc vaccination VXN SC vaccination, challenge 2

[0197] Table 7: Identification of experimental groups.

[0198] 'All groups, including the negative control, were infected with the live attenuated Mass I-H120 vaccine against infectious bronchitis virus (IBV), orally, at a dose 100x of that recommended by the manufacturer in order to sensitize the animals to the E. coli challenge.

[0199] 2 Infection with 10⁸ CFU of Escherichia coli, at a dose of 100 pL / bird by route oral. The infection was confirmed by microbiological recovery of bacteria from the inoculum.

[0200] C-Vaccination

[0201] Animals in the 'VXN SC Vaccine', 'VXN Mucosal Vaccine' and 'Commercial Vaccine' (Poulvac® E. coli, Zoetis) groups received primary immunization on day 1 and a second dose on day 12. For the 'VXN mucosal vaccine', administrations were made in the eye, beak and nostril, and for the 'commercial vaccine', doses were administered in drinking water.

[0202] D-Challenge

[0203] The challenge strain was a field isolate that was confirmed to be an APEC by PCR identification of 5 pathogenicity genes (iuaT, iroN, ompC, iss, hly). It was also verified to belong to phylogroup F by the typing method described by Clermont (Clermont et al., 2013). All animals received at day 14 A 100x dose of attenuated IBV vaccine (Massachusetts H-120 strain, Mass® I, Zoetis) was administered. The challenge with E. coli was performed in all groups except the "negative control," and 108 CFU / bird were used.

[0204] The overall schedule of the in ovo vaccination trial protocol is presented in [Fig.16].

[0205] E-Analyses Quantification of anti-E IgA. coli:

[0206] The production of specific anti-E. coli IgA was evaluated by ELISA. In brief, the samples were diluted in 1% casein in PBS. The ELISA plates were coated with LPS of E. coli (field isolate strain). The plates were then washed three times with 200 pL / well of PBS + 0.05% Tween-20 for 5 min / wash. The wells were blocked with 1% casein in PBS. The samples were tested by serial dilution. The plates were washed, and an anti-chicken IgA (Bio-Rad) diluted in 0.1% casein was added. After washing, the assay was developed with a single solution of TMB (Life Technologies). The absorbance was read at 450 nm, and quantification was performed using a proprietary methodology / kit developed by Imunova.

[0207] Quantification and detection of E. coli infection:

[0208] The detection of E. coli by MPN (most probable number) was performed based on ISO 7251:2005. E. coli counts were determined according to standard microbiological methods (dilution in an enrichment medium followed by plating in a selective / differential medium). In short, samples were enriched in buffered peptone water (BPW), followed by EC broth, and finally plating in EMB and MacConkey agar. Samples were serially diluted three times in BPW before incubation to allow quantification by the MPN technique (Blodgett et al., 2015). In the detection of E. coli, only the serial dilution step was omitted. Suspect isolated colonies were biochemically tested and confirmed. Lung histology:

[0209] The birds were euthanized and lung samples were collected and fixed. The samples were embedded in paraffin and mounted on slides. All histopathological assessments and readings were performed under a microscope by an experienced veterinary histopathologist.

[0210] [Tables8] Lung Score 0 Normal Appearance 1 Hyperemia. Parabronchial edema. Luminal parabronchial fibrinous material and endobronchial desquamation. Luminal and mucosal mononuclear and heterophilic infiltration. Ventilation slightly compromised. 2 Hyperemia. Obliterative endobronchitis due to mucofibrinous exudation and, to a lesser extent, infiltration by inflammatory, heterophilic, and mononuclear cells. Secondary bronchial involvement. Atrial elongation. Ventilation reasonably maintained. 3 Hyperemia, perivascular edema. Tertiary or parabronchial bronchial involvement is fairly extensive, with diffuse heterophilic infiltration. The anterior bronchioles show lymphopurulent peribronchitis and endobronchitis. Ventilation reasonably maintained. 4 Hyperemia. The primary bronchus shows fibrino-necrotic / purulent exudation with the remaining desquamated epithelial mucosa showing intense vacuolization.Inflammatory material in the lumen, associated with necrosis products and without significant aeration of the most affected air capillaries. Infiltration by heterophiles, mononuclear cells, including macrophages.

[0211] Table 8: List of histological parameters for the evaluation of 5-B lung disease scores: Results

[0212] Presence of E. coli in the airways (MPN)

[0213] Infection was assessed by quantifying the number of infected birds in the trachea and air sacs using MPN. The measurement of bacterial infection in the trachea and alveoli is shown in [Fig. 17]. Three unchallenged birds were infected in the trachea and seven in the air sacs, probably due to the presence of naturally occurring pathogenic E. coli in the environment. In contrast, unvaccinated challenged birds had more tracheal infections, confirming the effectiveness of the challenge. The same number of birds were infected in the group receiving the commercial vaccine compared to the infected control group, suggesting a lack of protection. However, among the birds vaccinated with the mucosal VXN vaccine, only one bird was infected in the trachea and none in the air sacs. This confirms that the mucosal VXN E. coli vaccine protected the birds from the challenge. Presence of anti-E. coli sIgA in feces

[0214] Secretory anti-LPS IgA of E. coli was analyzed in feces by ELISA. The results of the anti-EcoZz slgA analysis are shown [Fig. 18]. From day 16 to day 28, the antibody titer of the negative control birds was the same as that of the positive control, indicating that the oral challenge did not induce intestinal IgA secretion. However, for birds vaccinated via the mucosal route, both with the VXN E. coli formulation and with the commercial vaccine, a significant increase in antibody titers was observed on day 21. The mucosal vaccine was thus able to induce a humoral response in the intestine. Clinical score in the lungs

[0215] Lung lesion scores were measured from day 16 to day 28. Lesion scores are shown in [Fig. 19]. Although they had the lowest histopathological score from day 16 (score = 2) to day 28 (score = 1.7), the birds in the negative control group exhibited endobronchitis, mucofibrinous exudation, and polymorphonuclear neutrophil infiltration, which is likely related to natural infection ([Fig. 17]). Furthermore, the unvaccinated challenged birds in the positive control group had the highest score, from 3.4 at day 16 to 2.7 at day 28, due to E. coli challenge. In contrast, birds vaccinated mucosally with the VXN-E. coli formulation had a mean lesion score of 2.3 at day 16, lower than the commercial vaccine (score = 2.9).Finally, at day 28, both groups of birds vaccinated via mucosal route had a score comparable to the negative control (1.6 for the commercial vaccine, 1.8 for the VXN vaccine) suggesting protection against lung damage induced by E. coli infection.

Claims

Demands

1. Vaccine composition without adjuvant comprising cationic nanoparticles consisting of a polysaccharide core and at least one inactivated bacterium, characterized in that said bacterium is whole and is coated by said cationic nanoparticles in a bacterial protein:NPL ratio by weight between 1:0.1 and 1:

3.

2. Vaccine composition according to claim 2, wherein said porous cationic polysaccharide core is non-crosslinked and is obtained by the reaction between a polysaccharide selected from starch, dextran, chitosan, dextrin, and maltodextrin, polyfructoses (inulin), polymannoses, polygalactoses, polygalacto-mannans (guar gum) and at least one cationic ligand selected from a primary, secondary or tertiary amine and quaternary ammoniums.

3. Vaccine composition according to claim 2, wherein said porous cationic polysaccharide core is crosslinked and is obtained by the reaction between a polysaccharide selected from starch, dextran, chitosan, dextrin, and maltodextrin, polyfructoses (inulin), polymannoses, polygalactoses, polygalacto-mannans (guar gum) and at least one cationic ligand selected from a primary, secondary or tertiary amine and quaternary ammoniums and then the addition of a crosslinking agent.

4. Composition according to claim 3 wherein said crosslinking agent is selected from epichlorohydrin, a dicarboxylic acid or an acid chloride.

5. Vaccine composition according to any one of claims 3 or 4, wherein said cationic polysaccharide core is obtained by reaction between a maltodextrin and a glycidyltrimethylammonium.

6. Composition according to any one of the preceding claims wherein said cationic polysaccharide core is loaded with anionic phospholipid.

7. Vaccine composition according to claim 1, comprising at least 2 different strains of bacteria.

8. Vaccine composition according to any one of the preceding claims, wherein said bacterium is selected from the following group: Salmonella enterica ser. Typhi; Streptococcus Pneumoniae, Haemophilus influenzae type b, Mycobacterium tuberculosis, Extraintestinal pathogenic E.Coli (ExPEC), enterotoxigenic E.Coli (ETEC); S. enterica ser., Paratyphi A; Neisseria Gonorheae; Clostridium Difficile; Campylobacter spp: Shigella spp; Staphulobactus Aureus, Helicobacter pylori.

9. Vaccine composition as defined according to any one of the preceding claims, for its use in the prevention of diseases related to pathogenic bacteria.

10. Vaccine composition as defined according to claim 9, for its use in the prevention of salmonellosis or colibacillosis.

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