WO2026003181A1

WO2026003181A1 - Outer membrane vesicles from avian pathogenic escherichia coli and aerosol vaccine comprising the same

Info

Publication number: WO2026003181A1
Application number: PCT/EP2025/068091
Authority: WO
Inventors: Fabio ANTENUCCI; Anders Miki Bojesen
Original assignee: Københavns Universitet
Current assignee: Københavns Universitet
Priority date: 2024-06-26
Filing date: 2025-06-26
Publication date: 2026-01-02
Anticipated expiration: 2026-12-26

Abstract

The present invention relates to an avian pathogenic Escherichia coli (APEC) strain and a method for obtaining outer membrane vesicles (OMV) therefrom. The OMVs may be obtained in high concentrations suitable for use in aerosol vaccine preparations.

Description

Outer membrane vesicles from avian pathogenic Escherichia coli and aerosol vaccine comprising the same

Technical field of the invention

The present invention relates to an avian pathogenic Escherichia coli (APEC) strain and a method for obtaining outer membrane vesicles (OMV) therefrom. The OMVs may be obtained in high concentrations suitable for use in aerosol vaccine preparations.

Background of the invention

Avian pathogenic Escherichia coli (APEC) are the single most important cause of disease and mortality in chickens, with APEC infections accounting for up to 30% mortality within normal chicken populations. Bacterial infections affect all age groups ranging from day- old chickens to adult hens and cocks although with age-associated frequencies. However, from time to time the frequency of E. coli disease increases and resembles regular outbreaks that are detrimental to animal welfare as well as the economy of the farmer. To avoid more widespread outbreaks, it is common practice to put down entire populations to avoid disease from spreading in situations where sick animals cannot be isolated.

E. coli infections in poultry have traditionally been addressed by antimicrobial treatment. Unfortunately, increasing frequencies of antimicrobial resistant strains have led to treatment failure and policies discouraging antimicrobial usage have recently shifted focus towards prevention. Accordingly, there is a large animal welfare and economical incitement to efficiently prevent disease in large poultry, e.g. chicken, populations. Moreover, the zoonotic risk of transferring antimicrobial resistant strains from animals to humans is concerning. Indeed, there is increasing evidence that some types of E.coli from chickens appear as a main cause of urinary tract infections in humans.

To prevent E. co//-associated disease and reduce antimicrobial use most chickens reared for the purpose of egg-laying and breeding are vaccinated. Several vaccines are commercially available. However, the protective capacity is of varying quality due to the antigenic variation among E. coli strains that cause morbidity and mortality in poultry. Particularly one commercial vaccine termed "Poulvac® E. coli" (herein "Poulvac"), which is a live-attenuated aroA-mutant of an E. coli serogroup 078 strain, has been available in the EU since 2013 and is widely used. Yet, an important limitation of this vaccine is the limited protection against E. coli strains belonging to different serotypes. To account for this limitation, a popular strategy is the use of autogenous or farm-specific vaccines, which consists of killed bacterial isolates as an add-on to Poulvac. Autogenous vaccines have the advantage of being based on isolates from the farm of origin, which specifically can provide an important protective advantage when there is a sudden outbreak, against which commercially available vaccines may not adequately protect due to the heterogeneity of the avian pathogenic E. coli outbreak strains. A commonly recognized drawback to the autogenous approach is the limited protective breadth of killed bacterial strains, which therefore requires the autogenous vaccines of being frequently updated to reflect the present pathological threat on the farm. The autogenous vaccines are used either alone or more commonly in combination with a live-attenuated vaccine like Poulvac. The combination treatment can provide an important means of reducing recurring E. coli infections at farm level.

Unfortunately, the combination of the two different modes of prevention to diminish the risk of morbidity and mortality is resource demanding due to the constant surveillance needed to secure a relevant autogenous vaccine content and because the autogenous vaccine needs to be injected into each individual animal to induce a protective effect with no ability for mass-vaccination, while still having to vaccinate all birds with the live- attenuated vaccine.

Therefore, there is a pressing unmet need for an efficient vaccine for protecting poultry against disease caused by APEC. In particular, the vaccine should be suitable for use in an industrial setting where large populations of poultry are in need of protection.

Hence, it would be advantageous to provide a vaccine against avian pathogenic E. coli (APEC) that efficiently protect poultry against disease and is suitable for mass vaccination without the need for handling individual animals.

Moreover, it would be advantageous to provide a vaccine that can be produced at low cost and deliver broader spectrum protection against multiple serotypes of pathogens.

Summary of the invention

The present invention relates to the provision of a genetically engineered avian pathogenic E. coli (APEC) strain and a method using the same for preparation of vaccine compositions for prevention of disease in poultry caused by clinically relevant APEC. The active pharmaceutical ingredient (API) of the vaccine compositions is outer membrane vesicles (OMVs) harbouring a set of immunological determinants from the parent bacterium E. coli STI 17/078: H4 (strain E44). Subunit vaccines prepared from outer membrane proteins, whole-cell proteins, flagellin, pilus proteins, or LPS have been developed and tested widely but in general none of the prototypes are industrially viable options. Obstacles include prohibitive high costs of production, lack of batch-to-batch consistency, low production yield, insufficient protective effect and inability to perform mass vaccination.

Herein is described a derivative strain of the parent strain E. coli ST117/O78:H4 (strain E44) which has been genetically engineered to produce larger amounts of OMVs by inactivation of the exbDl gene. The hypervesiculating derivative strain (E44A) enables quick production of OMVs, thereby significantly cutting the cost for industrial scale batches of API for preparation of vaccines. The API itself does not contain any live bacteria and is therefore a safe option compared to vaccines based on live-attenuated bacteria, which have the risk of reverting to wildtype pathogenicity. The vaccines provided herein can be administered as an aerosol which markedly reduce the handling time associated with performing the vaccination and improve induction of the respiratory immune system.

Thus, an object of the present invention relates to the provision of means for producing on an industrial scale an efficient vaccine for protecting poultry against disease caused by APEC.

Another object of the present invention relates to provision of a low-cost vaccine suitable for mass vaccination of poultry against disease caused by APEC.

Thus, an aspect of the present invention relates to a hypervesiculating Avian Pathogenic Escherichia coli (APEC) strain, wherein said hypervesiculating APEC strain is a derivative strain of a parental APEC strain in which the exbDl gene has been inactivated.

Another aspect of the present invention relates to a method for producing a preparation comprising bacterial outer membrane vesicles (OMVs), said method comprising the steps of:

(i) providing an APEC strain as described herein;

(ii) culturing said APEC strain in a growth medium; and

(iii) recovering OMVs secreted by said APEC strain by tangential flow filtration (TFF), thereby obtaining said preparation.

Yet another aspect of the present invention relates to a preparation obtained by a method as described herein. A further aspect of the present invention relates to a vaccine or immunogenic composition comprising a preparation as described herein.

A still further aspect of the present invention relates to a vaccine or immunogenic composition as described herein for use in the prevention, inhibition or treatment of a disease caused by an APEC infection.

An even further aspect of the present invention relates to a kit comprising:

- a vaccine or immunogenic composition as described herein;

- a container; and

- optionally instructions for use.

Another aspect of the present invention relates to a hypervesiculating APEC strain as described herein for preparation of a vaccine or immunogenic composition.

Brief description of the figures

Figure 1 shows visualization of OMVs by Cryo-TEM. The E44, DH23 and DH36 are wildtype (wt) strains. E44A is mutant strain of E44 wherein the exbDl gene is inactivated.

Figure 2 shows quantification of OMVs from E44 and E44A using NTA. (A) Amount of particles pr. ml of OMV preparation. Size histograms of OMV diameter for OMVs produced by (B) the E44A strain and (C) the E44 strain.

Figure 3 shows OMV-specific serum IgY titers depending on the route and dose of vaccination.

Figure 4 shows (A) histological presentation of lung specimens from chickens vaccinated with different doses of OMVs. (B) Lung-body weight ratios for chickens in each vaccination group.

Figure 5 shows (A) Lung and airsac lesion scores and (B) CFU E. coli E44 per gram lung tissue.

Figure 6 shows the average normalised velocity over all animals for each group of animals.

Figure 7 shows representative images of (A) HD-11 cells not exposed to APEC, (B) HD-11 cells exposed to APEC, and (C) HD-11 cells HD-11 cells exposed to APEC and added IgY. Detailed description of the invention

Definitions

Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

Outer membrane vesicles (OMVs)

In the present context, the term "outer membrane vesicles (OMVs)'' refers to vesicles released from the outer membranes Gram-negative bacteria. OMVs are secreted from the surface of the bacteria. Structurally, OMVs are bilayered lipid membrane nanostructures that contain various biomolecules, such as DNA, RIMA proteins, endotoxins and virulence molecules. Accordingly, OMVs shed from pathogenic bacteria may be used for presenting immunological determinants, in their natural conformation, as part of a vaccine formulation.

Hypervesiculating

In the present context, the term "hypervesiculation" refers to an increased production of outer membrane vesicles (OMVs) by Gram-negative bacteria. Hypervesiculation may be induced by genetic mutations. The term "hypervesiculating" is used for a derivative strain with reference to a parental strain, i.e. a hypervesiculating strain is a derivative strain that has been genetically modified from its parental strain to gain the ability of increased OMV production.

The mutation may alter the content of OMVs produced and/or the amount of OMVs secreted by the hypervesiculating strain.

Avian Pathogenic Escherichia coli (APEC) strain

In the present context, the term "Avian Pathogenic Escherichia coli (APEC) strain" refers to a subset of Escherichia coli E. coli) strains that specifically affect avian species, including chickens. APEC strains can colonize the respiratory tract and cause severe respiratory and systemic disease in avian species. This may manifest itself as pneumonia, septicaemia, air-sacculitis, cellulitis and or other localized infections following on from colonization of the lungs, liver or other organs.

E. coli is a remarkably diverse species of bacteria, encompassing a wide range of strains that vary significantly in their genetic makeup, behaviour, and impact on human and veterinary health. While many E. coli strains are harmless and play a beneficial role in the intestinal microbiota, others are pathogenic and can cause serious illnesses, including foodborne outbreaks, urinary tract infections, and neonatal meningitis. These pathogenic strains are classified into distinct pathotypes based on their virulence factors and disease mechanisms, such as Shiga toxin-producing E. coli (STEC) and enterotoxigenic E. coli (ETEC). The genetic adaptability of E. coli, driven by horizontal gene transfer and environmental pressures, contributes to its vast strain diversity, making it both a valuable model organism in research and a significant public health concern.

APEC strains possess various virulence factors that contribute to their pathogenicity. These factors include adhesins, toxins, and invasion proteins that allow them to adhere to host tissues, evade immune responses, and cause tissue damage. Accordingly, given the diversity among E.coli strains and their distinct set of virulence factor, it is not possible to apply learnings from one strain directly to another unrelated strain.

Tangential flow filtration (TFF)

In the present context, the term "tangential flow filtration (TFF)'' refers to filtration method for separating and purifying biological substances in which a feed solution comprising the target substance is passed tangentially across a filter membrane. Smaller substances are passed through as a permeate (or filtrate) and larger substances and impurities are retained on the feed side of the membrane as a retentate.

Tangential flow filtration is also referred to as crossflow filtration.

Average size

In the present context, the term "average size" refers to the average value of the particle diameter. Particle diameter can be determined by nano particle tracking analysis (NTA) using a NS300 device (Malvern Panalytical) equipped with an sCMOS camera and Blue405 nm laser.

Median size

In the present context, the term "median size" refers to the median particle size, i.e. the particle diameter where half of the population falls below this value. Also referred to as d50.

Vaccine

In the present context, the term "vaccine" refers to a biological preparation that provides active acquired immunity to a particular infectious or malignant disease. Herein, such a biological preparation comprises at least outer membrane vesicles generated and obtained by the method described herein. The vaccine can be either preventive or therapeutic. Immunogenic composition

In the present context, the term "immunogenic composition" refers to any preparation comprising one or more immunogenic components which may be used as part of a vaccine or as a component in the preparation of a vaccine.

Excipient

In the present context, the term "excipient" refers to a natural or synthetic substance formulated alongside the active substance (an ingredient that is not the active substance) of a medication, included for the purpose of stabilization, bulking, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, enhancing solubility, adjusting tonicity, mitigating injection site discomfort, depressing the freezing point, or enhancing stability.

Aerosol

In the present context, the term "aerosol" refers to a suspension of fine solid or liquid particles in air or another gas. The liquid or solid particles in an aerosol typically have diameters of less than 5 pm or in some case less than 1 pm.

Aerosols may be administrated via a nebulizer or to a larger population of subjects, such as avian subjects, via a centralised means from which aerosols are distributed into the air of the location of the subjects.

Carrier

In the present context, the term "carrier" refers to refers to any solvents, dispersion media, vehicles, coatings, diluents, isotonic agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for active substances, such as OMVs, is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

Derivative strain

In the present context, the term "derivative strain" refers to a microorganism that is a second generation derived from a parental strain. A derivative strain may be developed by mutagenesis, wherein one or more mutations are introduced into the genome of the parental strain. The one or more mutations are preferably introduced by genetic engineering. Mutation

In the present context, the term "mutation" refers to an alteration in the nucleotide sequence of the genome of an organism resulting in changes in the phenotype of said organism, wherein the alteration may be a deletion of a nucleotide, a substitution of a nucleotide by another nucleotide, an insertion of a nucleotide, or a frameshift.

A deletion is to be understood as a genetic mutation resulting in the removal of one or more nucleotides of a nucleotide sequence of the genome of an organism; an insertion is to be understood as the addition of one or more nucleotides to the nucleotide sequence; a substitution (or point mutation) is to be understood as a genetic mutation where a nucleotide of a nucleotide sequence is substituted by another nucleotide; a frameshift is to be understood as a genetic mutation caused by a insertion or deletion of a number of nucleotides in a nucleotide sequence that is not divisible by three, therefore changing the reading frame and resulting in a completely different translation from the original reading frame; an introduction of a stop codon is to be understood as a point mutation in the DNA sequence resulting in a premature stop codon; an inhibition of substrate binding of the encoded protein is to be understood as any mutation in the nucleotide sequence that leads to a change in the protein sequence responsible for preventing binding of a substrate to its catalytic site of the protein.

Furthermore, a knockout mutant is to be understood as genetic mutation resulting in the removal or deletion of a gene, such as an entire gene or an entire open reading frame from the genome of an organism.

In the present description and claims the conventional one-letter code for nucleotides is used following the analogous principles as described for amino acids nomenclature supra.

Variant or variant strain

In the present context, the term "variant" or "variant strain" refers to a strain which is functionally equivalent to a strain of the invention, e.g. having substantially the same properties (e.g. regarding the ability to produce and/or secrete outer membrane vesicles). Such variants, which may be identified by further genetic engineering using conventional techniques, are a part of the present invention.

Sequence identity

In the present context, the term "sequence identity" is here defined as the sequence identity between proteins at the amino acid level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned.

To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes {e.g. gaps may be introduced in the sequence of a first amino acid sequence for optimal alignment with a second amino acid sequence). The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100).

In one embodiment, the two sequences are the same length. In another embodiment, the two sequences are of different length and gaps are seen as different positions.

One may manually align the sequences and count the number of identical amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the XBLAST program of (Altschul et al. 1990). BLAST protein searches may be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of the invention.

To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized. Alternatively, PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules. When utilising the XBLAST and Gapped BLAST programs, the default parameters of the respective programs may be used. See http://www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment.

The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.

About Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, concentrations, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%).

A hvoervesiculatinq APEC strain for production of a poultry vaccine

APEC is one of the most economically damaging diseases affecting the poultry industry. It causes clinical conditions such as a pneumonia, septicaemia, air-sacculitis, cellulitis, leading to reduced productivity, slower growth rates, increased mortality rates, increased use of antimicrobials, reduced animal welfare, and compromised meat quality.

It is difficult to develop a universal vaccine against APEC because the strains are heterogeneous meaning that that different isolates rarely harbour the same combinations of virulence factors. On top of this overall challenge comes considerations on cost, delivery method and timing of vaccination, all of which needs to be optimised to provide an economically viable and effective vaccine for use in the poultry industry.

Outer membrane vesicles (OMVs) are nanoscale, spherical structures naturally released by Gram-negative bacteria, and they have emerged as a promising platform for vaccine development. These vesicles carry a rich array of bacterial surface antigens, including proteins, lipopolysaccharides, and other immunogenic components, making them ideal for mimicking the pathogen and stimulating a strong immune response.

However, scaling up the production of OMVs for industrial vaccine manufacturing presents several significant challenges. OMVs are naturally secreted in low quantities by Gramnegative bacteria, and while various methods exist to enhance their yield, such as genetic modifications or chemical induction, these often compromise the structural integrity or immunogenic properties of the vesicles. Additionally, the downstream purification of OMVs is technically demanding due to their nanoscale size and the need to remove contaminants like proteases and cell debris while preserving antigenic components. Ensuring batch-to- batch consistency and scalability without altering the vesicle's composition or efficacy remains a major hurdle, limiting the widespread industrial application of OMV-based vaccines. Accordingly, obtaining a system founded on an E.coli production strain satisfying all these criteria for efficient vaccine production is far from trivial. This challenge is compounded by the fact that the E.coli species is exceptionally diverse and previous learnings and optimization are not easily transferred between strains.

Herein are provided a hypervesiculating mutant APEC strain from which large amounts of immunogenic outer membrane vesicles (OMVs) can efficiently be harvested. The mutant APEC strain enables cost efficient production of highly immunogenic compositions that may be used as APEC vaccines or as components for preparation of APEC vaccines. The process for generating the OMVs is inexpensive and the OMVs can readily be mass-administered to the avian subjects as an aerosol in a non-labour intensive manner.

Surprisingly, inactivation of the exbDl gene in a parental APEC strain leads to a viable derivative strain that produce large amounts of outer membrane vesicles, i.e. it is hypervesiculating.

Thus, an aspect of the present invention relates to a hypervesiculating Avian Pathogenic Escherichia coli (APEC) strain as described herein, wherein said hypervesiculating APEC strain is a derivative strain of a parental APEC strain in which the exbDl gene has been inactivated.

It is to be understood that the hypervesiculating APEC strain described herein comprises also mutants and variant strains thereof which are essentially functionally equivalent, i.e. possess the hypervesiculating due to genetic engineering of the exbDl gene. Thus, derivative strains of the hypervesiculating APEC strain wherein parts of the genome that are not related to the vesiculating property of the bacterium has been mutated are considered to be essentially functionally equivalent.

Thus, an embodiment of the present invention relates to a hypervesiculating Avian Pathogenic Escherichia coli (APEC) strain, wherein said hypervesiculating APEC strain, or a mutant or variant thereof, is a derivative strain of a parental APEC strain in which the exbDl gene has been inactivated.

It is demonstrated herein that the strain E44/ST117/078: H4 presents a particular favourable parental strain from which a hypervesiculating strain can be generated. The parental strain is a clinically relevant strain that has previously caused a major international outbreak. Thus, an embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the parental strain is of the sequence type (ST)/ST117/ serotype 078 :H4.

Another embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the parental strain comprises a genome deposited in DDBJ/ENA/GenBank under the accession number LXWV00000000.

A further embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the parental strain is E44/ST117/O78:H4.

A still further embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein said hypervesiculating APEC strain has increased production of OMVs compared to said parental APEC strain

Herein it has been found that inactivation of the exbDl gene can lead to a hypervesiculating strain. The nucleic acid sequence of the exbDl gene is generally highly conserved across E. coli species and produces a protein comprising a single transmembrane domain flanked by domains on the cytoplasmic side and in the periplasm. Inactivation of the exbDl gene may be accomplished by any conventional means, such as inter-gene mutations and deletion of part or all of the gene.

Thus, an embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the exbDl gene of the parental strain comprises:

(i) a nucleic acid sequence according to SEQ ID NO: 1, or

(ii) a nucleic acid sequence with at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity, to SEQ ID NO: 1.

Another embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the exbDl gene of the parental strain comprises:

(i) a nucleic acid sequence according to SEQ ID NO: 1, or

(ii) a nucleic acid sequence with at least 70% sequence identity, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, to SEQ ID NO: 1. Another embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein said hypervesiculating APEC strain is genetically distinct from said parental APEC strain.

Yet another embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein said exbDl gene comprises one or more mutations compared to the exbDl gene of the parental APEC strain.

A further embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein said one or more mutations are deletion(s), substitution(s), insertion(s) and/or frame shifts.

A preferred embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the exbDl gene has at least partially deleted or fully deleted, preferably the exbDl gene has been fully deleted.

Inactivation can be accomplished by insertion of a means for screening into or in place of the exbDl gene, such as insertion of a resistance gene. Insertion of a resistance gene may be performed together with deletion of all or part of the exbDl gene. Mutants and variant strains with hypervesiculating properties derived from the hypervesiculating strain described herein may be identified via further screening through a pool of genetically engineered strains. Screening may be based on selection through a resistance gene, such as a kanamycin resistance gene aphA-3).

Therefore, an embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the exbDl gene is at least partially replaced or fully replaced with an exogenous gene.

Another embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the exogenous gene is a resistance gene.

A further embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the exogenous gene is a kanamycin resistance gene.

Herein has been identified a genetically engineered hypervesiculating AEPC strain which allowed both selection of the relevant strain and highly increased vesiculation properties. Thus, an embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the exbDl gene is at least partially replaced or fully replaced with a nucleic acid comprising a sequence according to SEQ ID NO:2.

Another embodiment of the present invention relates to the hypervesiculating APEC strain as described herein, wherein the APEC strain is deposited as DSM35056 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D- 38124 Braunschweig, Germany, by University of Copenhagen, Copenhagen, Denmark on 19 June 2024.

For successful commercialization of avian vaccines, it is a necessity that the production cost is reduced as much as possible as profit margins in the industry are tight. Poultry vaccines based on OMVs as the antigenic component are promising because the raw material generally is inexpensive, easier to disperse than live cells or recombinant proteins, and has long shelf-life due to the high lipid content. However, development of OMV-based vaccines has been halted due to the unavailability of an efficient and cost- effective process for production and purification of OMVs in amounts sufficient to obtain the required immunogenicity for generating a relevant clinical response. In particular, Gram-negative wild type (wt) strains do not produce any significant amount of OMVs sufficient for establishing a commercial production line based thereupon. In contrast, the hypervesiculating APEC strain provided herein may be used for production of large quantities of immunogenic outer membrane vesicles (OMVs). Utilized in a process where the resulting OMVs are recovered by tangential flow filtration (TFF), the resulting method is a cost-efficient way to produce immunogenic compositions comprising high concentrations of OMVs.

TFF, also known as crossflow filtration, is a rapid and efficient method for separating and purifying biomolecules. TFF involves passing the feed solution tangentially across a filter membrane under positive pressure relative to the permeate side, and it works particularly well for feed solutions comprising a high proportion of small particles.

TFF presents several advantages over other filtration and separation techniques. Unlike dead-end filtration where the feed passes directly through the membrane, TFF prevents filter cake formation and allows continuous operation. Moreover, the increased liquid removal rate of TFF prevents filter cake buildup and therefore efficient removal of impurities. As opposed to ultracentrifugation, which has been the main method for OMV extraction, TFF is a gentle extraction method that allows higher batch-to-batch reproducibility and better uniformity of the OMVs. Together, this makes TFF suitable for industrial scale production as high purity OMVs can be obtained without regularly pausing operation.

The hypervesiculating APEC strain in combination with TFF present a new and beneficial method of producing a high-quality immunogenic component for subsequent preparation of a poultry vaccine.

Therefore, an aspect of the present invention relates to use of a hypervesiculating APEC strain as described herein for preparation of a vaccine or immunogenic composition.

(i) providing an APEC strain as described herein;

(ii) culturing said APEC strain in a growth medium; and

It has been found that high concentrations of OMVs can be obtained with the method described herein, i.e. up to more than an order of magnitude higher concentrations than for a corresponding method utilizing the wild-type APEC strain. Importantly, the present method is fast which contributes to keeping the OMV production cost low in an industrial setting.

As a non-limiting example, the present method may handle a bioreactor with a starting volume of 4L to a composition with at least 10¹² OMV particles/ml over a period of approx.

4 hours of culturing followed by approx. 3 hours of recovering OMVs by TFF. The ability to recover such high concentration of OMVs in a greatly reduced time is facilitated by the combination of the hypervesiculating mutant APEC strain and the use of TFF.

Accordingly, an embodiment of the present invention relates to the method as described herein, wherein said preparation comprises OMVs in a concentration of at least IxlO¹² particles/ml, such as at least 2xl0¹² particles/ml, such as at least 5xl0¹² particles/ml, such as at least 8xl0¹² particles/ml, such as at least IxlO¹³ particles/ml.

Another embodiment of the present invention relates to the method as described herein, wherein the APEC strain is cultured no longer than about 6 hours, such as no longer than about 5 hours, preferably no longer than about 4 hours. Yet another embodiment of the present invention relates to the method as described herein, wherein the APEC strain is cultured for a period of time in the range of about 3 to about 8 hours, such as about 3.5 hours to about 6 hours, preferably about 4 hours to about 5 hours.

A further embodiment of the present invention relates to the method as described herein, wherein said TFF is performed for about 8 hours or less, such as about 6 hours or less, such as about 5 hours or less, such as about 4 hours or less, such as about 3 hours or less.

A still further embodiment of the present invention relates to the method as described herein, wherein said TFF is performed for a period of time in the range of about 3 to about 8 hours, such as about 3 to about 6 hours, such as about 3.5 hours to about 5.5 hours, preferably about 3 hours to about 5 hours.

There are several types of TFF systems, including cassette-based and hollow fiber modules. Cassette systems are modular and scalable, making them ideal for both laboratory and industrial applications. The filter materials used in TFF membranes vary depending on the application, with cellulose-based membranes (such as regenerated cellulose) and polyethersulfone (PES) being among the most prevalent due to their chemical compatibility and low protein binding. Typical pore sizes are expressed in terms of molecular weight cut-off (MWCO), and ranges from 1 kDa to 1000 kDa, depending on whether the goal is to retain small peptides, proteins, or larger biomolecules like viruses. For recovery of OMVs, membranes with pore sizes that are 3-6 times smaller than the vesicles diameter may be advantageous as it allows reliable retention of the OMVs while allowing smaller protein and solute to pass through.

An embodiment of the present invention to the method as described herein, wherein said TFF is cassette-based TFF.

Another embodiment of the present invention to the method as described herein, wherein the membranes utilized in said TFF is cellulose-based membranes or polyethersulfone (PES) membranes, preferably cellulose-based membranes.

A further embodiment of the present invention to the method as described herein, wherein the molecular weight cut-off (MWCO) of the membranes utilized in said TFF is in the range of about 100 kDa to about 1000 kDa, such as in the range of about 200 kDa to about 750 kDa, preferably in the range of about 250 kDa to about 500 kDa.

A still further embodiment of the present invention to the method as described herein, wherein the molecular weight cut-off (MWCO) of the membranes utilized in said TFF is about 300 kDa.

An even further embodiment of the present invention to the method as described herein, wherein the pore size of the membranes utilized in said TFF is in the range of about 10 nm to about 50 nm, such as about 15 nm to about 30 nm.

Production simplicity and speed are critical for industrial-scale biomanufacturing. Overall, faster production means higher throughput, which in turn enables manufacturers to respond to market needs promptly. The present method is simple in the sense that no further purification step(s) is required to recover the OMVs in concentrations and purity sufficient to further process the composition into a final vaccine product. Wild type strains cannot be utilised for commercial production of OMVs because their output is so poor that that it is not possible to achieve clinically relevant amounts of OMVs, The challenges with industrial production are further compounded by other residual particles also being present in the production line, e.g. protein complexes, bacterial debris and polymer residues from downstream purification, that needed to be separated from the active ingredient - the OMVs. Therefore, it is critical for designing an efficient production method that the amount OMVs is significantly above the baseline level of residual particles, to avoid excessive purification and clogging of filters.

Importantly, the method provided herein is capable of handling larger volumes of sample and obtaining high concentrations of OMVs over a short duration of time. In contrast, a conventional technique such as ultracentrifugation, is not suited for large scale production because it requires long run times at high speed to separate biomolecules, it is typically only suitable for small sample volumes lending itself poorly to upscaling, and it is insufficient to obtain the desired concentration and purity, thereby necessitating additional downstream steps, such as chromatography. In practice, processing high volumes of sample, e.g. 4L, to recover OMVs with a conventional method, such as ultracentrifugation, would take days.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the sample volume of step (ii) is at least about IL, such as at least about 2L, such as at least about 3L, such as at least about 4L. Another embodiment of the present invention relates to the method as described herein, wherein the sample volume of step (ii) is in the range of about IL to about 10L, such as about 2L to about 8L, such as about 3L to about 6L, such as about 4L to about 5L.

It is to be understood that by "the sample volume of step (ii)" is meant the APEC strain in the growth medium.

However, it is contemplated herein that the method is suitable for even larger scale industrial production, such as volumes of up to 50L, or even 1000-20000L. The large culture volumes can quickly be converted to immunogenic compositions comprising high concentrations of OMVs without any excessive steps of recovering the OMVs.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the sample volume of step (ii) is in the range of about 50L to about 20000L, such as about 100L to about 10000L, such as about 200L to about 5000L, such as about 500L to about 1000L.

Another embodiment of the present invention relates to the method as described herein, wherein the sample volume of step (ii) is at least about 50L, such as at least about 100L, such as at least about 500L, such as at least about 1000L, such as at least about 10000L.

The hypervesiculating strain provided herein enables fast and efficient recovery of large amounts of immunogenic OMVs, suitable for clinical use. In particular, the reduced culture time and recovery (purification) time is only possible with the use of the hypervesiculating APEC strain provided herein.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the sample volume in step (ii) is at least about IL, such as at least about 4L, wherein the APEC strain is cultured no longer than about 6 hours, no longer than about 4 hours, and wherein TFF is performed for about 8 hours or less, such as about 6 hours or less, such as about 3 hours or less.

Another embodiment of the present invention relates to the method as described herein, wherein the sample volume in step (ii) is at least about IL, such as at least about 4L, wherein the APEC strain is cultured no longer than about 6 hours, no longer than about 4 hours, wherein TFF is performed for about 8 hours or less, such as about 6 hours or less, such as about 3 hours or less, and wherein said preparation comprises OMVs in a concentration of at least IxlO¹² particles/ml.

Yet another embodiment of the present invention relates to the method as described herein, wherein the sample volume in step (ii) is at least about 50L, such as at least about 100L, such as at least about 500L, such as at least about 1000L, such as at least about 10000L, wherein the APEC strain is cultured no longer than about 6 hours, no longer than about 4 hours, and wherein TFF is performed for about 8 hours or less, such as about 6 hours or less, such as about 3 hours or less.

Still another embodiment of the present invention relates to the method as described herein, wherein the sample volume in step (ii) is at least about 50L, such as at least about 100L, such as at least about 500L, such as at least about 1000L, such as at least about 10000L, wherein the APEC strain is cultured no longer than about 6 hours, no longer than about 4 hours, wherein TFF is performed for about 8 hours or less, such as about 6 hours or less, such as about 3 hours or less, and wherein said preparation comprises OMVs in a concentration of at least IxlO¹² particles/ml.

Yet embodiment of the present invention relates to the method as described herein, wherein said method does not comprise any further steps of recovering or purifying said secreted OMVs.

A further embodiment of the present invention relates to the method as described herein, wherein said method does not comprise a step of ultracentrifugation.

In some variants of the method, it can be advantageous to include a pre-filtration step of the cultured APEC strain prior to the recovering step. The purpose is to create a crude OMV extract that can more easily be processed by TFF without clogging of the system during operation. Thus, an embodiment of the present invention relates to the method as described herein, wherein the composition comprising the cultured APEC strain is pre-filtered to produce cell-free crude OMV extracts prior to said recovering step.

Another embodiment of the present invention relates to the method as described herein, wherein the filter size during pre-filtering is in the range of about 0.2 pm to about 0.7 pm, such as about 0.3 pm to about 0.6 pm, such as about 0.35 pm to about 5.5 pm, such as about 0.4 pm to about 0.5 pm, preferably about 0.45 pm.

The hypervesiculating APEC strain may be cultured in any suitable growth medium, including, but not limited to, LB medium, BHI medium, TSB, and M9 minimal medium. Preferably, the hypervesiculating APEC strain is cultured in a growth medium that promotes vesiculation and retain the native properties of the secreted outer membrane vesicles.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein said growth medium is selected from the group consisting of brain-heart infusion (BHI) medium, and Tryptic Soy Broth (TSB) medium.

Another embodiment of the present invention relates to the method as described herein, wherein the average size of the OMVs is in the range of about 60 nm to about 125 nm, such as about 80 nm to about 120 nm, such as about 85 nm to about 115 nm, such as about 90 nm to about 110 nm, such as about 95 nm to about 105 nm, such as about 98 nm to about 102 nm.

Yet another embodiment of the present invention relates to the method as described herein, wherein the median size (d50) of the OMVs is in the range of about 40 nm to about 100 nm, such as about 50 nm to about 90 nm, such as about 55 nm to about 85 nm, such as about 60 nm to about 80 nm, such as about 65 nm to about 75 nm.

The OMV preparation obtained from the present method may be used in an immunogenic composition or as a component in a vaccine.

Thus, an aspect of the present invention relates to a preparation obtained by a method as described herein.

Another aspect of the present invention relates to a vaccine or immunogenic composition comprising a preparation as described herein. Surprisingly, it was found that the obtained OMVs retained their structure and integrity upon transformation to aerosol form. An aerosol of the OMV preparation was readily prepared without any further need for additives in the composition. Often aerosol formulations include stabilising sugars, such as sucrose, trehalose, mannitol, sorbitol or inulin. Sugars are typically included to preserve the structure and activity of biological material during storage, such as storage in the form of dry powder in systems for inhalation delivery. The ability to form a stabile aerosol without the need of further additives is advantageous as it enables delivery of the OMVs by the respiratory route and reduce the cost of the final product as inclusion of further expensive additives can be negated. Without being bound by theory, it is contemplated that the high purity of OMVs produced by the method provided herein may contribute to the improved stability of the OMVs when transformed to aerosol form. In particular, the absence of residual particles may promote successful transformation of individual OMVs to aerosol form.

Accordingly, an embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein said vaccine or immunogenic composition is in aerosol form.

Another embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein said vaccine or immunogenic composition does not comprise any stabilizing excipient.

A further embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein said stabilizing excipient is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

For some variants of the present invention, it may be beneficial to add an excipient to the vaccine or immunogenic composition to achieve some supportive effect, such as enhanced thermostability to facilitate long-term storage if needed or facilitate transport to remote regions, or alternatively to improve particle dispersion.

Accordingly, an embodiment of the present invention relates to the vaccine or immunogenic composition as described herein further comprising an excipient to promote thermostability. Another embodiment of the present invention relates to the vaccine or immunogenic composition as described herein further comprising a cellulosic excipient.

The immunogenic composition or vaccine may be provided in a pharmaceutically acceptable carrier. This include, but is not limited to, any conventional solvents, diluents buffers, and suspensions that would conventionally be used for formulation of an immunogenic composition or vaccine.

Therefore, an embodiment of the present invention relates to the vaccine or immunogenic composition as described herein further comprising one or more pharmaceutical acceptable carriers.

APEC infections pose significant challenges for avian populations due to their detrimental impact on poultry health and production. Traditional control methods using antibiotics have led to an increased prevalence of antibiotic-resistant APEC strains, and there are increasing concerns that APEC could be a potential foodborne zoonotic pathogen that could present a risk to not only poultry but also humans. This worry about zoonotic transfer particularly relates to extra-intestinal infections in humans. Efficient vaccines to prevent and/or combat APEC infections are consequently highly sought after.

The vaccine or immunogenic composition may be used for preventing and/or treating outbreaks of disease caused by APEC infections. It may conveniently be administered as an aerosol which significantly reduces the handling of the animals and their welfare compared to competing vaccines that are administered by a different route that requires handling of individual animals, such as intramuscular administration.

Thus, an aspect of the present invention relates to a vaccine or immunogenic composition as described herein for use in the prevention, inhibition or treatment of a disease caused by an APEC infection.

An embodiment of the present invention relates to the vaccine or immunogenic composition for use as described herein, wherein said vaccine or immunogenic composition is administered via the respiratory route.

Another embodiment of the present invention relates to vaccine or immunogenic composition for use as described herein, wherein said vaccine or immunogenic composition is administered as an aerosol. The aerosol may be delivered to the avian subject via any conventional route. Aerosol formulations may be administered to poultry using spray vaccination. This technique is suitable for delivering vaccines that target primarily the respiratory system. The process involves spraying a mist of vaccine aerosol particles above the birds, ensuring even and complete coverage. Alternatively, administration may be facilitated through vaccination chambers or inline automatic sprayers, both of which offer an even mist of aerosol particles.

Therefore, an embodiment of the present invention relates to the vaccine or immunogenic composition for use as described herein, wherein said vaccine or immunogenic composition is administered with a nebulizer system.

Another embodiment of the present invention relates to the vaccine or immunogenic composition for use as described herein, wherein said vaccine or immunogenic composition is administered as a spray vaccination.

The vaccine or immunogenic composition presented herein may be beneficial for any avian subject that are in the risk of getting exposed to pathogenic APEC strains. Preferably, the avian subject is a domesticated bird since these subjects often are many individual animals assembled and therefore the risk of disease spread is significantly increase - thus these avian populations benefit the most from vaccination.

Accordingly, an embodiment of the present invention relates to the vaccine or immunogenic composition for use as described herein, wherein said vaccine or immunogenic composition is administered to an avian subject.

Another embodiment of the present invention relates to the vaccine or immunogenic composition for use as described herein, wherein said avian subject is a domesticated bird.

A further embodiment of the present invention relates to the vaccine or immunogenic composition for use as described herein, wherein said avian subject is selected from the group consisting of chicken, turkey, goose, duck, partridge, pheasant, squab, guinea fowl, ostrich, emu, and rhea.

The administered dosage can be adjusted depending on the avian subject and particularly in of the weight and age of the avian subject. Thus, an embodiment of the present invention relates to the vaccine or immunogenic composition for use as described herein, wherein the concentration of vaccine or immunogenic composition administrated to each avian subject is in the range of about 0.1 pg to about 4000 pg, such as about 20 pg to about 4000 pg, such as about 25 pg to about 1000 pg, such as about 30 pg to about 200 pg, such as about 35 pg to about 100 pg, such as about 35 pg to about 50 pg.

Another embodiment of the present invention relates to the vaccine or immunogenic composition for use as described herein, wherein the concentration of vaccine or immunogenic composition administrated to each avian subject is in the range of about 0.1 pg to about 50 pg, such as about 1 pg to about 25 pg, about 2 pg to about 10 pg, such as about 3 pg to about 5 pg.

It is to be understood that in the present context, the term "concentration" refers to the total protein concentration present in the formulation. This measurement serves as a practical and consistent proxy for quantifying the amount of OMVs, since these vesicles are composed largely of membrane-associated and luminal proteins. This approach may be useful because direct enumeration of vesicles may not always correlate linearly with immunogenic content, whereas total protein provides a more standardized metric for dosing and quality control during vaccine development and production.

The vaccine or immunogenic composition may conveniently be provided as a kit comprising the active ingredient contained within a suitable container. Preferably, the container is capable of administrating the vaccine or immunogenic composition as an aerosol or can be coupled to a centralised means for distributing the vaccine or immunogenic composition as an aerosol.

Herein are provided for the first time a preparation that can be administered as a vaccine to avian subjects in aerosol form without any need for costly excipients. The preparation can be produced in a cost-efficient manner from a novel mutant bacterial strain.

Thus, an aspect of the present invention relates to a preparation comprising outer membrane vesicles (OMVs) in aerosol form, wherein said OMVs are derived from the APEC strain deposited as DSM35056 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by University of Copenhagen, Copenhagen, Denmark on 19 June 2024.

Therefore, an aspect of the present invention relates to a kit comprising: a vaccine or immunogenic composition as described herein; a container; and optionally instructions for use.

An embodiment of the present invention relates to the kit as described herein, wherein the container is a nebulizer.

The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the hypervesiculating APEC strain and all its features, which may readily be part of the corresponding immunogenic composition or vaccine or preparation and the method for producing a preparation comprising OMVs. Embodiments and features of the present invention are also outlined in the following items.

Items

XI. A hypervesiculating Avian Pathogenic Escherichia coli (APEC) strain, wherein said hypervesiculating APEC strain is a derivative strain of a parental APEC strain in which the exbDl gene has been inactivated.

X2. The hypervesiculating APEC strain according to item XI, wherein the parental strain is of the sequence type (ST)/ST117/serotype 078: H4.

X3. The hypervesiculating APEC strain according to any one of items XI or X2, wherein the parental strain comprises a genome deposited in DDBJ/ENA/GenBank under the accession number LXWV00000000.

X4. The hypervesiculating APEC strain according to any one of the preceding items, wherein the parental strain is E44/ST117/078: H4.

X5. The hypervesiculating APEC strain according to any one of the preceding items, wherein said hypervesiculating APEC strain has increased production of OMVs compared to said parental APEC strain. X6. The hypervesiculating APEC strain according to any one of the preceding items, wherein the exbDl gene of the parental strain comprises:

(i) a nucleic acid sequence according to SEQ ID NO: 1, or

X7. The hypervesiculating APEC strain according to any one of the preceding items, wherein said hypervesiculating APEC strain is genetically distinct from said parental APEC strain.

X8. The hypervesiculating APEC strain according to any one of the preceding items, wherein said exbDl gene comprises one or more mutations compared to the exbDl gene of the parental APEC strain.

X9. The hypervesiculating APEC strain according to item X8, wherein said one or more mutations are deletion(s), substitution(s), insertion(s) and/or frame shifts.

X10. The hypervesiculating APEC strain according to any one of items X8 or X9, wherein the exbDl gene has at least partially deleted or fully deleted, preferably the exbDl gene has been fully deleted.

XI 1. The hypervesiculating APEC strain according to any one of the preceding items, wherein the exbDl gene is at least partially replaced or fully replaced with an exogenous gene.

X12. The hypervesiculating APEC strain according to item Xll, wherein the exogenous gene is a resistance gene.

X13. The hypervesiculating APEC strain according to any one of items Xll or X12, wherein the exogenous gene is a kanamycin resistance gene.

X14. The hypervesiculating APEC strain according to any one of the preceding items, wherein the exbDl gene is at least partially replaced or fully replaced with a nucleic acid comprising a sequence according to SEQ ID NO:2.

X15. The hypervesiculating APEC strain according to any one of the preceding items, wherein the APEC strain is deposited as DSM35056 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by University of Copenhagen, Copenhagen, Denmark on 19 June 2024.

Yl. A method for producing a preparation comprising bacterial outer membrane vesicles (OMVs), said method comprising the steps of:

(i) providing an APEC strain according to any one of items X1-X15;

(ii) culturing said APEC strain in a growth medium; and

Y2. The method according to item Yl, wherein said preparation comprises OMVs in a concentration of at least IxlO¹² particles/ml, such as at least 2xl0¹² particles/ml, such as at least 5xl0¹² particles/ml, such as at least 8xl0¹² particles/ml, such as at least IxlO¹³ particles/ml.

Y3. The method according to any one of items Yl or Y2, wherein the APEC strain is cultured no longer than about 6 hours, such as no longer than about 5 hours, preferably no longer than about 4 hours.

Y4. The method according to any one of items Y1-Y3, wherein the APEC strain is cultured for a period of time in the range of about 3 to about 8 hours, such as about 3.5 hours to about 6 hours, preferably about 4 hours to about 5 hours.

Y5. The method according to any one of items Y1-Y4, wherein said TFF is performed for about 8 hours or less, such as about 6 hours or less, such as about 5 hours or less, such as about 4 hours or less, such as about 3 hours or less.

Y6. The method according to any one of items Y1-Y5, wherein said TFF is performed for a period of time in the range of about 3 to about 8 hours, such as about 3 to about 6 hours, such as about 3.5 hours to about 5.5 hours, preferably about 3 hours to about 5 hours.

Y7. The method according to any one of items Y1-Y6, wherein the sample volume of step (ii) is at least about IL, such as at least about 2L, such as at least about 3L, such as at least about 4L. Y8. The method according to any one of items Y1-Y7, wherein the volume of the sample volume of step (ii) is in the range of about IL to about 10L, such as about 2L to about 8L, such as about 3L to about 6L, such as about 4L to about 5L.

Y9. The method according to any one of items Y1-Y8, wherein the sample volume in step (ii) is at least about IL, such as at least about 4L, wherein the APEC strain is cultured no longer than about 6 hours, no longer than about 4 hours, and wherein TFF is performed for about 8 hours or less, such as about 6 hours or less, such as about 3 hours or less.

Y10. The method according to any one of items Y1-Y9, wherein the sample volume in step (ii) is at least about IL, such as at least about 4L, wherein the APEC strain is cultured no longer than about 6 hours, no longer than about 4 hours, wherein TFF is performed for about 8 hours or less, such as about 6 hours or less, such as about 3 hours or less, and wherein said preparation comprises OMVs in a concentration of at least IxlO¹² particles/ml.

Yll. The method according to any one of items Y1-Y10, wherein said method does not comprise any further steps of recovering or purifying said secreted OMVs.

Y12. The method according to any one of items Yl-Yll, wherein said method does not comprise a step of ultracentrifugation.

Y13. The method according to any one of items Y1-Y12, wherein the composition comprising the cultured APEC strain is pre-filtered to produce cell-free crude OMV extracts prior to said recovering step.

Y14. The method according to item Y13, wherein the filter size during pre-filtering is in the range of about 0.2 pm to about 0.7 pm, such as about 0.3 pm to about 0.6 pm, such as about 0.35 pm to about 5.5 pm, such as about 0.4 pm to about 0.5 pm, preferably about 0.45 pm.

Y15. The method according to any one of items Y1-Y14, wherein said growth medium is selected from the group consisting of brain-heart infusion (BHI) medium, and Tryptic Soy Broth (TSB) medium. Y16. The method according to any one of items Y1-Y15, wherein the average size of the OMVs is in the range of about 60 nm to about 125 nm, such as about 80 nm to about 120 nm, such as about 85 nm to about 115 nm, such as about 90 nm to about 110 nm, such as about 95 nm to about 105 nm, such as about 98 nm to about 102 nm.

Y17. The method according to any one of items Y1-Y16, wherein the median size (d50) of the OMVs is in the range of about 40 nm to about 100 nm, such as about 50 nm to about 90 nm, such as about 55 nm to about 85 nm, such as about 60 nm to about 80 nm, such as about 65 nm to about 75 nm.

Y18. The method according to any one of items Y16 or Y17, wherein the average size or median size (d50) of the OMVs are measured by nano particle tracking analysis (NTA).

Y19 The method according to item Y18, wherein NTA is performed using a NS300 device (Malvern Panalytical) equipped with an sCMOS camera and Blue405 nm laser.

Y20. The method according to any one of items Y1-Y19, wherein said preparation is essentially free from cell debris.

Zl. A preparation obtained by a method according to any one of items Y1-Y20.

QI. A vaccine or immunogenic composition comprising a preparation according to item Zl.

Q2. The vaccine or immunogenic composition according to item QI, wherein said vaccine or immunogenic composition is in aerosol form.

Q3. The vaccine or immunogenic composition according to any one of items QI or Q2, wherein said vaccine or immunogenic composition does not comprise any stabilizing excipient.

Q4. The vaccine or immunogenic composition according to item Q3, wherein said stabilizing excipient is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

Q5. The vaccine or immunogenic composition according to any one of items Q1-Q4 further comprising one or more pharmaceutical acceptable carriers. Tl. A vaccine or immunogenic composition according to any one of items Q1-Q5 for use in the prevention, inhibition or treatment of a disease caused by an APEC infection.

T2. The vaccine or immunogenic composition for use according to item Tl, wherein said vaccine or immunogenic composition is administered via the respiratory route.

T3. The vaccine or immunogenic composition for use according to any one of items Tl or T2, wherein said vaccine or immunogenic composition is administered as an aerosol.

T4. The vaccine or immunogenic composition for use according to any one of items T1-T3, wherein said vaccine or immunogenic composition is administered with a nebulizer system.

T5. The vaccine or immunogenic composition for use according to any one of items T1-T4, wherein said vaccine or immunogenic composition is administered as a spray vaccination.

T6. The vaccine or immunogenic composition for use according to any one of items T1-T5, wherein said vaccine or immunogenic composition is administered to an avian subject.

T7. The vaccine or immunogenic composition for use according to item T6, wherein said avian subject is a domesticated bird.

T8. The vaccine or immunogenic composition for use according to any one of items T6 or T7, wherein said avian subject is selected from the group consisting of chicken, turkey, goose, duck, partridge, pheasant, squab, guinea fowl, ostrich, emu, and rhea.

T9. The vaccine or immunogenic composition for use according to any one of items T1-T8, wherein the concentration of vaccine or immunogenic composition administrated to each avian subject is in the range of about 0.1 pg to about 4000 pg, such as about 20 pg to about 4000 pg, such as about 25 pg to about 1000 pg, such as about 30 pg to about 200 pg, such as about 35 pg to about 100 pg, such as about 35 pg to about 50 pg.

Rl. A preparation comprising outer membrane vesicles (OMVs) in aerosol form, wherein said OMVs are derived from the APEC strain deposited as DSM35056 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D- 38124 Braunschweig, Germany, by University of Copenhagen, Copenhagen, Denmark on 19 June 2024. Ul. A kit comprising:

- a vaccine or immunogenic composition according to any one of items Q1-Q5;

- a container; and

- optionally instructions for use.

U2. The kit according to item Ul, wherein the container is a nebulizer.

Pl. Use of a hypervesiculating APEC strain according to any one of items X1-X15 for preparation of a vaccine or immunogenic composition.

The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1: Construction of hypervesiculating mutant E. coli strain

The purpose of this example was to investigate if a hypervesiculating E.coli strain could be produced by genetically engineering of a selection of APEC strains.

Method

Bacterial strain construction

The candidate E. coli strains were constructed by targeted mutagenesis using homologous recombination via natural transformation. Briefly, the exbDl gene was replaced by a kanamycin resistance gene to impair the expression of exbDl. A total of three E. coli wildtype strains (E44, DH23, DH36) were included. All strains originate from diseased chickens and have been implicated in larger disease outbreaks. Subsequent to the mutagenesis attempts, the resulting bacterial strains were selected on kanamycin containing agar plates. Strains able of propagating on the selective medium are suspected to be mutants expressing the kanamycin resistance gene. Correct gene insertion was confirmed by genome sequencing and polymerase chain reaction (PCR).

No positive clones were isolated from the DH23 (ST95/O1 :H7) and DH36 (STI 17/053 :H4) isolates subjected to the experimental protocol described above. Subsequent attempts at mutagenizing these strains were made by using different experimental combinations of one or more of the following parameters: Growth conditions (pre-electroporation); administered amplicon concentration; amplicon design (length of flanking loci); electroporation conditions; recovery time (post electroporation); recovery medium formulation (post electroporation) and kanamycin concentration (overnight incubation). To compare the effect of the exbDl deletion, a gualitative and guantitative assessment of the wildtype strains and corresponding mutants was performed.

Culturing bacteria and harvesting OMVs

A single colony of Escherichia coli ST117 O78:H4 (E44) AexbDl: :apha-3 was sub-cultured in brain-heart infusion medium (BHI) and incubated overnight (ON) under aerobic conditions (37 °C/160 rpm). On the following day, a 7 L fermenter flask containing 5 L preheated BHI broth was inoculated with 50 mL ON culture (1% v/v), followed by culturing (37 °C) under peristaltic agitation (5 Hz) and active aeration (10 PSI) until reaching ODeoo = 4. The total culturing time in the fermenter flask was 4 hours.

Cultures were then extracted, mixed with 80 g/L of diatomaceous earth filtration aid additive and filtered through single use vacuum filtration units (0.45 pm filter pore size; <100 mbar pressure) to produce cell-free crude OMV extracts.

Crude OMV extracts were kept refrigerated (4 °C), subjected to three subseguent steps of buffer exchange (PBS 1: 1, progressively reduced volume) and finally concentrated (66x) by recirculation through a TFF crossflow cassette system (300 kDa filter pore size) powered by a peristaltic pump and articulated by platinum-cured silicone tubing. Recirculation through the TFF crossflow cassette system was repeated for 3 hours.

The resulting OMV isolates were then aliguoted and stored at -20 °C.

Protein content

SDS-PAGE was used to assess the protein content and guality. Sample buffer and reducing agent were mixed and 5 pl of OMV samples were added. The reaction mixture was then boiled at 100°C for 10 min and loaded into 10%-12% SDS-gel wells along with a reference protein ladder (10-250 kDa). The gel was subjected to electrophoresis at 100 V for 10 min following this at 150 V for 1 hour. The protein bands were visualized using Coomassie Blue.

Visualization of OMV morphology

To assess the OMVs visually, Cryo-Transmission Electron Microscopy (Cryo-TEM) was applied. A hydrophilized lacey carbon 300 mesh copper grid (Ted Pella Inc.) was loaded with 3 pl of OMV solution and blotted using blot force 2, blot and drain times 5.5 and 0 s, temperature 4°C and relative humidity 100% (FEI Vitrobot IV). Subseguently, the sample was mounted into a cryo holder for direct observation at -180°C in a Tecnai G2 20 transmission electron microscope (FEI) at 200 kV. The imaging was conducted using a FEI Eagle camera 4 k x 4 k at variable nominal magnifications.

Quantification of size and concentration of OMVs

The OMV concentration and size distribution was assessed by Nano particle Tracking Analysis (NTA). The OMV batches were quantified using a NS300 device (Malvern Panalytical) equipped with an sCMOS camera and Blue405 nm laser. The concentration and particle size distribution (diameter) of the isolated OMVs were detected in accordance with the manufacturer's instructions. Briefly, 1 ml of the diluted sample (1: 10,000) in filtered PBS was loaded into a sample chamber via a syringe pump. The camera level of 16 and detection threshold of five were applied to capture the video for 60 s, five times, with the particles per frame of 20-100 at 24.5°C. Data analysis was performed using NTA 3.4 Build 3.4.003 software. The reported results were an average of five 60 s reads.

Protein concentration

The protein concentration was assessed by Qubit analysis. The isolated OMVs were quantitated by Qubit® 2.0 fluorometer provided with a Qubit™ assay kit containing three standards (Invitrogen) according to the manufacturer's instructions. Briefly, the Qubit reagent was diluted at 1:200 in the Qubit buffer provided in the kit to prepare the Qubit working solution. OMV samples were diluted at 1 :5 in the filtered PBS to fall within the calibration curve range. The amount of 10 pl and 1 pl of the standards (each) and the diluted OMV sample were added to 190 and 199 pl of working solution respectively. The assay tubes containing the standard and sample were subsequently vortexed and incubated at room temperature for 15 min. The assay tubes were then inserted into the fluorometer chamber for the data analysis. The curve-fitting algorithm was used to determine protein concentration based on the relationship between the three standards applied in calibration and the sample dilution factor.

Results

No viable kanamycin resistant clones could be isolated from DH23 or DH36 despite several mutagenesis attempts employing modified protocol parameters. DH23 and DH36 are APEC strains as E44 but they were not viable upon deletion of the exbDl gene.

The wildtype strains (E44, DH23, DH36) produced only few OMVs (Fig. 1A and Fig. 1C-D). In particular, the wt E44 strain hardly produced any OMVs visible by TEM, and many of the visible particles appeared to be residual particles different from the OMVs (Fig. 1A). In contrast, the mutated E44 strain (E44A) secreted high amounts of OMVs, i.e. it was hypervesiculating (Fig. IB, arrow indicates OMV). The E44A strain produced more than 25 times more OMVs compared to the wt E44 strain (Fig. 2A), 1.17xl0¹³ OMVs/ml vs. 4.41X10¹¹ OMVs/ml, respectively. The morphology of the OMVs produced by the E44 and E44A strains were comparable (Fig. 1A-B) and had a similar size distribution (Fig. 2B-C), with OMVs of E44 having an average diameter of 100 nm ± 30 nm and E44A having an average diameter of 98 nm ± 13.7 nm. While the sample from the wt E44 strain contained a significant portion of irregular, non-spherical particles, the E44A contained almost exclusively OMVs.

Two OMV isolates (LS2; LS3), independently produced according to the protocol described herein, showed the following total protein concentration: 1) LS2: 2.5 mg/mL; LS3: 3.34 mg/mL.

Conclusion

This example demonstrates that a functional deletion of exbDl in an APEC strain will not necessarily lead to a hypervesiculating or viable mutant strain despite application of a wide array of mutagenesis conditions. The E44A strain created herein has an approx. x25 increase in vesiculation compared to the WT, with low amounts of residual particles. The OMVs produced by the mutant are highly similar to the vesicles produced by the parent wildtype bacterium.

Example 2: Administration of OMVs - aerosol vs intramuscularly

The purpose of this example was to evaluate the efficiency with which vaccination with OMVs obtained from the E44A strain could protect birds when subsequently challenged with the wt E44 strain.

Method

To investigate the ability of OMVs to induce protective immunity via vaccination by respiratory (aerosol) and intramuscular (IM) route, vaccination-challenge experiments were performed in duplicate. The birds in each experiment originated from the same commercial Ross308 breeder and had not previously been vaccinated against E. coli. A total of eight group were included in the experiments (Table 1).

Vaccination by intramuscular injection was done into the superficial pectoral muscle. Aerosol vaccination was done by keeping the chickens in a vaccination chamber while aerosolizing specific concentrations of OMVs using an Omron ultra nebulizer to reach the desired exposure. All birds were vaccinated twice four weeks apart commencing at 22 weeks of age. Two different vaccine doses were used for the aerosol vaccination, while one dose was used for the IM vaccination.

The birds were observed daily for potential adverse effects originating from the vaccination. Serum samples from all birds were obtained before vaccination (To), after first vaccination (Ti), and after the second vaccination (T2), two week after the 2^nd vaccination (but before challenge). The level of OMV specific IgY antibodies were assessed by ELISA.

Two weeks after the 2^nd vaccination, all birds (vaccinated and unvaccinated controls) were challenged with the E. coli E44 strain (wt) via intratracheal inoculation.

Results

No chickens showed signs of adverse reactions after vaccination, independent of dose. All vaccinated birds, independent of route and dose, had a significantly higher vaccine-specific antibody titer than the mock-vaccinated controls (Figure 3).

Following intratracheal challenge with E44 a mortality rate of 28% was observed in the unvaccinated group. No mortality was observed in any of the vaccinated groups independent of vaccination route or dose.

Conclusion

This example demonstrates that OMVs induce a highly significant increase in serum IgY titer following two vaccinations independent of route or dose when compared to vaccination with buffer. Moreover, the results from the bacterial challenge study indicated that vaccination with OMVs induced protective immunity independent of route or dose of vaccination. Thus, OMVs can be efficiently administrated as an aerosol vaccination.

Example 3: OMVs vs. commercially available vaccine The purpose of this example was to compare the efficiency of the OMVs with a commercially available vaccine.

Method

A vaccination-challenge experiment in commercial Ross308 breeders (22 weeks old) was performed to investigate if the OMVs had the ability to stimulate formation of OMV-specific IgY antibodies following a low vaccination dose by the aerosol route. The birds had not previously been vaccinated against E. coli. A total of six groups were included in the experiment (Table 2).

Table 2. Aerosol vaccination with OMVs at low and medium doses, and vaccination with Pou Iva c E. coli®.

Aerosol vaccination was done by keeping the chickens in a vaccination chamber while aerosolizing specific concentrations of OMVs using an Omron ultra nebulizer to reach the desired exposure. All birds were vaccinated twice four weeks apart commencing at 22 weeks of age. Two different vaccine doses (Low: 37.5 ug or Medium: 150 ug) were used for the aerosol vaccination. The Poulvac vaccination was done via the drinking water according to the recommendations of the manufacturer.

The birds were observed daily for potential adverse effects originating from the vaccination. Serum samples from all birds were obtained before vaccination (To) and two weeks after the 2^nd vaccination (T2). Two weeks after the 2^nd vaccination, birds (vaccinated and unvaccinated controls) were challenged with the E. coli E44 strain via intratracheal inoculation. The daily egg yield per group was monitored seven days before and two days after challenge, respectively.

Results

No chickens showed signs of adverse reactions after vaccination, independent of dose or type of vaccine. Data are presented in Table 3. The two unvaccinated, non-challenged control groups (A and E) had significantly lower lesion score compared to the unvaccinated yet E44 challenged group (B) (p<0.001). The aerosol unvaccinated control birds (group B) had a significantly higher lesion score (combined lung and airsac score)(P<0.05) compared to the aerosol vaccinated and E44 challenged birds (37.5 ug OMV) (group C). The birds which were aerosol vaccinated with a medium dose (37.5 ug OMV) and E44 challenged (group D) had even lower lesion score. The PoulVac vaccinated birds did not have a significantly different lung and airsac lesions score than unvaccinated control birds.

The egg-laying numbers are included seven days before the challenge and two days after challenge. In Table 3 is reported the egg laying yield after challenge in respect to the initial egg laying yield before challenge (/.e. percentual yield). Results are calculated on the basis of number of eggs laid by the number of hens in the group, with significance of the result given in parenthesis. Quantification of the egg yield showed that groups B (unvaccinated and challenged) and F (Poulvac vaccinated and challenged) had a significant lower egg yield after challenge. In contrast, the OMV vaccinated birds (groups C and D) showed no significant drop in egg yield.

Table 3. Accumulated lung and airsac scores and egg lay yield of chickens in different vaccination schemes (Groups A-F).

Conclusion

This example demonstrates that aerosol vaccination using 37.5 ug OMV induced a significantly lower lung and airsac lesion score than unvaccinated controls. The lesions in the PoulVac vaccinated birds were comparable to the unvaccinated controls indicating that Poulvac vaccination did not protect from lesions. Thus, the aerosol vaccination provides a superior protective effect compared to a commercially available vaccine. Importantly, the OMV vaccinated birds were more resistant to the E. coli challenge with respect to egg yield.

Example 4: Tolerance to OMVs in day-old broiler chickens

The purpose of this example was to examine the tolerance of day-old broiler chickens to the aerosol vaccination. Method

Day-old Ross308 chicken were vaccinated with three different doses of OMVs (Table 4) to investigate a possible dose-response effect following aerosol exposure to OMVs. Chickens were monitored daily for clinical sign a least three times daily. At termination of the trial (day 21), the chickens were necropsied and histological examination of the lungs were performed. The chickens were weighed at days 2, 5, 13 and 21, respectively. The lungs were also weighed at necropsy to permit registration of the lung-body weight ratio.

Table 4. Dose-response study in day-old chicks.

Results

No chickens showed any signs of adverse reactions after vaccination, independent of dose. The lung-body weight ratio was significantly lower in vaccinated chickens compared to the unvaccinated controls. Histology on lungs from five randomly selected birds per group showed no signs of inflammation or cell infiltration (Fig. 4A). Moreover, the lung-body ratio for each chicken group showed that the aerosol vaccination was well tolerated with no adverse effects, even at very high doses (Fig. 4B).

Conclusion

This example demonstrates that day-old chickens tolerated aerosol administered OMVs very well, even at 100X the dose anticipated as the standard vaccination dose. No indications of inflammation or cell infiltration was observed on histological examinations. The lung-body weight ratio was significantly smaller for all vaccinated groups compared to the non-vaccinated control, which supports the histological evaluation, as there were no signs of inflammation. Based hereon it can be concluded that the aerosol vaccination is safe to use for immunization of day-old chickens.

Example 5: Protective effect of OMVs in day-old broiler chickens

The purpose of this example was to evaluate the effect of multiple administrations of the aerosol vaccine.

Method To investigate the protective effect of one or two vaccinations with OMVs in day-old chickens, 20 chickens were included in four groups (Table 5). The chicks were vaccinated at the day of hatch and, for groups A, B and D, again four weeks later. Two weeks after the second vaccination, all groups, except group A, were challenged with E. coli E44. All birds were euthanized two days PI and subjected to necropsy. Lesion scoring was made on lungs and airsacs and quantitative bacterial culture was performed on lung tissue.

Groups A, B and D were monitored by GoPro cameras to obtain data on movement/activity level as a proxy for the well-being of the chicken during the trial. The activity data was analysed using DeepLapCut software using standard conditions.

Table 5. Aerosol vaccination with OMVs once or twice in day-old chicks

Results

Chickens vaccinated twice had a highly significant (p=0.008) lower lung and airsac lesion score compared to the non-vaccinated and challenged chicks (Fig. 5A) and chicks only vaccinated once (data not shown). The double vaccinated chicks also had a highly significant lower number of E. coli E44 per gram lung compared to the non-vaccinated controls (Fig. 5B).

The activity recording revealed a significantly lower activity of non-vaccinated E. coli challenged chicks compared to two times vaccinated and challenged chicks and nonvaccinated and non-challenged chicks, respectively (Figure 6).

Conclusion

This example demonstrates that OMV vaccination at hatch and four weeks later provides great protection compared to non-vaccinated chickens.

Example 6: Protective effect of OMVs in day-old broiler chickens

The purpose of this example was to examine the growth inhibitory effect of OMV-specific antibodies on a broad selection of Avian Pathogenic E. coli (APEC) strains. Method

Six APEC strains (Table 6) were investigated to evaluate the inhibitory effect of OMV- specific antibodies. Inhibition of bacterial growth was investigated by adding a 1 : 10 dilution of yolk-derived IgY antibodies extracted from eggs from 26-weeks old Ross308 breeders to the bacterial growth medium. The yolk-derived IgY antibodies originated from two groups of chickens: 1) two times OMV vaccinated chickens and 2) non-vaccinated control chickens. The antibody dilutions were added in equal amounts to the bacterial growth medium (Tryptic Soy Broth) and bacterial growth was compared.

The growth experiments were performed in triplicate using a Bioscreen analyzer measuring medium transparency (ODeoo) every 15 min for 20 hours.

Table 6. APEC strains investigated for growth inhibition.

Results

All APEC strains showed reduced growth of at least 17% and up to 26% (table 6) (p<0.05) when OMV-specific antibodies from vaccinated chickens were added to the growth medium as compared to the growth of the same strain in medium added antibodies from nonvaccinated control chickens.

Conclusion

This example demonstrates that OMV-specific antibodies have a growth inhibitory effect on APEC strains belonging to different multilocus sequence typing (MLST) and serotypes, respectively. Based hereon, it can be concluded that the vaccine-induced antibodies have the capacity to inhibit growth of a broad selection of APECs dominating in the field.

Example 7: Cell protection ability by OMV-specific antibodies

The purpose of this example was to examine the cell protective effect of OMV-specific antibodies against four Avian Pathogenic E. coli (APEC) strains.

Method To investigate whether OMV-specific antibodies were able of protecting avian macrophagelike cells (HD11), confluent cell cultures of HD11 cells were exposed to four different APEC strains (Table 7, Figure 7) in cell culture medium added yolk-derived antibodies from adult Ross308 chickens. The yolk-derived IgY antibodies originated from two groups of chickens: 1) two times OMV vaccinated chickens and 2) non-vaccinated control chickens.

APEC strains can impact negatively on HD11 cells, which will release lactate dehydrogenase (LDH) from damaged or lysed cells. The LDH assay was used for assessment of cell protection. The LDH assay using Nitro-Blue Tetrazolium (NBT) dye is a colorimetric method where LDH converts lactate into pyruvate, generating NADH, which in turn reduces NBT into a blue formazan product that can be quantified by spectrophotometry. The formation of LDH is thus a proxy for cell protection, which was compared with or without OMV-specific antibodies. All cell experiments were performed in triplicate.

Results

Addition of OMV-specific antibodies from vaccinated chickens to the HD11 cells resulted in a 36%-84% (p<0.05) reduction of LDH release with respect to the baseline LDH release from HD11 cells to which were added antibodies from non-vaccinated birds.

Conclusion

The example demonstrates that OMV-specific antibodies have a cell protective effect following exposure of HD11 cells to APEC strains belonging to different MLST types and serotypes, respectively. Based hereon, it can be concluded that the vaccine-induced antibodies have the capacity to inhibit the cell damaging effect of APECs dominating in the field.

References

• Altschul et a/. (1990), J. Mol. Biol., 215, 403-410

• Ronco et al. (2016), Genome Announc., e00768-16 Deposits and Expert Solution

The applicant requests that a sample of the deposited microorganism stated in Table 8 below may only be made available to an expert, until the date on which the patent is granted.

The applicant requests that the availability of the deposited microorganism referred to in Rule 33 EPC shall be effected only by the issue of a sample to an independent expert nominated by the requester (Rule 32(1) EPC). If an expert solution has been requested, restrictions concerning the furnishing of samples apply.

The deposit was made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D- 38124 Braunschweig, Germany.

The Budapest Treaty provides that any restriction of public access to samples of deposited biological material must be irrevocably removed as of the date of grant of the relevant patent.

Table 8. Deposited strain made at a depositary institution.

Claims

1. A hypervesiculating Avian Pathogenic Escherichia coli (APEC) strain, deposited as DSM35056 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by University of Copenhagen, Copenhagen, Denmark on 19 June 2024.

2. A method for producing a preparation comprising bacterial outer membrane vesicles (OMVs), said method comprising the steps of:

(i) providing an APEC strain according to claim 1;

(ii) culturing said APEC strain in a growth medium; and

3. The method according to claim 2, wherein the APEC strain is cultured for a period of time in the range of about 3 to about 8 hours, such as about 3.5 hours to about 6 hours, preferably about 4 hours to about 5 hours.

4. The method according to any one of claims 2 or 3, wherein said preparation comprises OMVs in a concentration of at least IxlO¹² particles/ml, such as at least 2xl0¹² particles/ml, such as at least 5xl0¹² particles/ml, such as at least 8xl0¹² particles/ml, such as at least IxlO¹³ particles/ml.

5. The method according to any one of claims 2-4, wherein the sample volume in step (ii) is at least about 50L, such as at least about 100L, such as at least about 500L, such as at least about 1000L, such as at least about 10000L, wherein the APEC strain is cultured no longer than about 6 hours, no longer than about 4 hours, and wherein TFF is performed for about 8 hours or less, such as about 6 hours or less, such as about 3 hours or less.

6. The method according to any one of claims 2-5, wherein said method does not comprise any further steps of recovering or purifying said secreted OMVs.

7. The method according to any one of claims 2-6, wherein said method does not comprise a step of ultracentrifugation.

8. The method according to any one of claims 2-7, wherein said preparation is essentially free from cell debris.

9. A preparation obtained by a method according to any one of claims 2-8.

10. A vaccine or immunogenic composition comprising a preparation according to claim 9.

11. The vaccine or immunogenic composition according to claim 10, wherein said vaccine or immunogenic composition is in aerosol form.

12. The vaccine or immunogenic composition according to any one of claims 10 or 11, wherein said vaccine or immunogenic composition does not comprise any stabilizing excipient.

13. The vaccine or immunogenic composition according to claim 12, wherein said stabilizing excipient is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

14. A vaccine or immunogenic composition according to any one of claims 10-13 for use in the prevention, inhibition or treatment of a disease caused by an APEC infection.

15. The vaccine or immunogenic composition for use according to claim 14, wherein said vaccine or immunogenic composition is administered via the respiratory route.

16. The vaccine or immunogenic composition for use according to claim 15, wherein said vaccine or immunogenic composition is administered as an aerosol.

17. The vaccine or immunogenic composition for use according to any one of claims 15 or 16, wherein said vaccine or immunogenic composition is administered to an avian subject.

18. A preparation comprising outer membrane vesicles (OMVs) in aerosol form, wherein said OMVs are derived from the APEC strain deposited as DSM35056 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D- 38124 Braunschweig, Germany, by University of Copenhagen, Copenhagen, Denmark on 19 June 2024.

19. A kit comprising:

- a vaccine or immunogenic composition according to any one of claims 10-13, or a preparation according to claim 18;

- a container; and optionally instructions for use.

20. Use of a hypervesiculating APEC strain according to claim 1 for preparation of a vaccine or immunogenic composition.