Multivalent vaccine against porcine infection

Abstract

The present disclosure relates to multivalent vaccine compositions and their use in protecting pigs and progeny thereof from infection with E. coli, Clostridium spp, porcine parvovirus, and E. rhusiopathie, preferably with a new administration regimen for passive and active immunization of pigs and progeny thereof, as well as their use in protecting pigs and progeny thereof from infection with E. coli, Clostridium spp, porcine parvovirus, and E. rhusiopathie.

Description

Technical Field

[0001] This disclosure relates to the use of multivalent vaccine compositions and their use in protecting pigs and their offspring from infection with E. coli, Clostridium spp., porcine parvovirus and E. rhusiopathiae, preferably using novel administration regimens for passive and active immunization of pigs and their offspring. Background Technology

[0002] Pigs account for more than a quarter of the world's total protein consumption. Infectious diseases affect the health of pigs and the productivity of the global pig industry. The most common pathogens in sows and their offspring include parvovirus, Erysipelas rhusiopathiae, Leptospira, Salmonella, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, PRRSV, PCV2, influenza A virus, pathogenic Escherichia coli, and Clostridium.

[0003] Sows and gilts are vaccinated against different pathogens multiple times throughout their lives through various vaccinations. Vaccine-mediated protection declines over time, and therefore, the timing required for maximum protection differs for active and passive immunization. Vaccines for active protection of sows and gilts against diseases such as erysipelas and parvovirus are typically administered before the first insemination, usually between 6 and 3 weeks prior to insemination for gilts, and for sows, booster vaccinations are administered between 3 weeks and 1 day prior to insemination, usually 1 day to 3 weeks prior to the next insemination in each cycle, or every 6 months via mass vaccination. These vaccination schedules ensure protection during gestation, as many of the aforementioned pathogens are associated with reproductive failure and abortion in sows (i.e., protection during gestation is necessary).

[0004] Vaccines can also be used for passive immunization. In fact, early maternal immunization is crucial for the passive protection of newborn piglets. Typically, vaccines used for passive immunization of piglets, for example, against newborn diarrhea caused by Clostridium or Escherichia coli infection, are administered twice, between 7 weeks and 2 weeks before farrowing, and as a booster, usually once between 4 weeks and 2 weeks before farrowing, to provide passive immunization to piglets through colostrum intake.

[0005] Multivalent vaccines, capable of conferring simultaneous immunity against different pathogens or diseases in a single injection, have become the most manageable solution for protecting animals. In fact, the expectation is to inject all the necessary antigens in a single vaccine, making the vaccination procedure less traumatic and painful for animals, more time-efficient, and easier for practitioners to manage. Although some combination vaccines have been successfully developed, immune interference can occur between the two or more antigen-specific immune responses induced simultaneously in vivo.

[0006] In addition, it is necessary to clarify the most appropriate timing for administering multivalent vaccines to confer effective active and passive immune protection in pigs and their offspring.

[0007] There is still a need to develop a safe and effective multivalent vaccine that can be administered to pigs to provide protection through active immunization and to piglets through passive immunization via colostrum intake. Summary of the Invention

[0008] The inventors have demonstrated that a multivalent vaccine provides effective protection against Escherichia coli, Clostridium perfringens, parvovirus, and erysipelas infections administered to female pigs. This multivalent vaccine has no negative effects in vaccinated female pigs and prevents swine erysipelas, swine parvovirus, Escherichia coli, and Clostridium infections through active immunization of vaccinated female pigs and active immunization of offspring through colostrum intake.

[0009] Therefore, the inventors have unexpectedly demonstrated that, despite immune interference, a multivalent vaccine containing nine different antigens / toxins exhibits safety characteristics and serologically detectable efficacy in piglets, and is effective in gilts and sows. The demonstrated efficacy and safety of this multivalent vaccine prepared in this application will allow for modifications to swine vaccination programs and significantly promote swine vaccination by making them less invasive.

[0010] Furthermore, unlike classic administration regimens suitable for passive or active immunization that require repeated injections in pigs, the inventors of this application have developed for the first time a novel administration regimen for primary vaccination that allows for simultaneous active and passive immunization with high efficacy. Therefore, this highly efficient new administration regimen allows for a reduction in the number of injections and promotes vaccination in pigs.

[0011] This invention relates to a vaccine composition comprising:

[0012] i) Escherichia coli fimbriae adhesin antigen,

[0013] ii) Clostridium toxoids,

[0014] iii) Porcine parvovirus antigen, preferably inactivated porcine parvovirus, more preferably inactivated porcine parvovirus strain K22, and

[0015] iv) Erysipelothrix rhusiopathiae antigen, preferably inactivated Erysipelothrix rhusiopathiae.

[0016] In a preferred embodiment, the Escherichia coli fimbrial adhesin antigen is selected from the group consisting of: F4ab, F4ac, F5, F6, F41, F17, F18, F165, CS1541 and CS31A antigens, preferably F4ab, F4ac, F5 and F6 antigens, and more preferably the composition comprises Escherichia coli fimbrial adhesin F4ab, F4ac, F5 and F6 antigens.

[0017] In a particular embodiment, the Clostridium toxoid antigen is a Clostridium perfringens toxoid antigen, preferably selected from the group consisting of: Clostridium perfringens α-toxin of type A or C, Clostridium perfringens β2-toxin of type A, Clostridium perfringens β1-toxin of type C, Clostridium perfringens enterotoxin (CPE) of type A or C, and Clostridium perfringens (all strains) θ-toxin (clostridium perfringens lysin, PFO). Preferably, the composition comprises Clostridium perfringens α-toxin of type A or C, Clostridium perfringens β2-toxin of type A, and Clostridium perfringens β1-toxin of type C.

[0018] In a preferred embodiment, the vaccine composition comprises an adjuvant, preferably an aluminum hydroxide adjuvant.

[0019] In a more preferred embodiment, the vaccine composition according to the present invention comprises:

[0020] i) Escherichia coli fimbrial adhesins F4ab, F4ac, F5, and F6 antigens.

[0021] ii) Clostridium perfringens type A or C α-toxin, Clostridium perfringens type A β2-toxin, and Clostridium perfringens type C β1-toxin,

[0022] iii) Inactivated porcine parvovirus, and

[0023] iv) Inactivated Erysipelothrix rhusiopathiae bacteria.

[0024] In another aspect, the present invention relates to a vaccine composition as described above, for use in protecting pigs and their offspring from infection by Escherichia coli, Clostridium, porcine parvovirus and Erysipelothrix rhusiopathiae.

[0025] In a preferred embodiment, the present invention relates to a vaccine composition as described above for protecting female pigs, preferably gilts, and their offspring from infection by *Escherichia coli*, *Clostridium*, porcine parvovirus, and *Erysipelothrix rhusiopathiae*, wherein the composition is administered to female pigs, preferably gilts, at least twice before insemination and at least once during the interval between insemination and farrowing, preferably no more than 10 weeks before insemination, preferably no more than 9 weeks, 8 weeks, 7 weeks, 6 weeks, or 5 weeks before insemination, and preferably at least twice within an interval of at least 2 weeks between each of the two administrations before insemination, and at least once before farrowing no more than 4 weeks, preferably no more than 3 weeks, more preferably no more than 2 weeks.

[0026] In a more preferred embodiment, the composition is applied at least once between 10 and 4 weeks, preferably between 9 and 4 weeks, 8 and 4 weeks, or more preferably between 7 and 5 weeks, before insemination; at least once between 6 and 1 week, preferably between 5 and 1 week, or between 4 and 1 week, or more preferably between 3 and 2 weeks, before insemination; and at least once between 6 and 1 week, preferably between 5 and 1 week, 4 and 1 week, or more preferably between 3 and 2 weeks, before parturition.

[0027] In a preferred embodiment, the vaccine composition is administered via intramuscular, intradermal, transdermal, or subcutaneous routes, more preferably via intramuscular routes. In a particular embodiment, prior to administering the vaccine composition to female pigs, immunologically effective amounts of the *Escherichia coli* fimbriae adhesin antigen, *Clostridium* toxoid, porcine parvovirus antigen, and *Erysipelothrix rubrum* antigen are mixed. Detailed Implementation

[0028] vaccine composition

[0029] The inventors have demonstrated that a multivalent vaccine composition containing nine different antigens / toxins for protection against Escherichia coli, Clostridium, porcine parvovirus (PPV), and erysipelas has safety characteristics and serologically detectable efficacy in piglets, and is effective in gilts and sows.

[0030] This disclosure relates to a vaccine composition comprising:

[0031] i) Escherichia coli fimbriae adhesin antigen,

[0032] ii) Clostridium toxoids,

[0033] iii) Porcine parvovirus antigen, and

[0034] iv) Erysipelothrix rhusiopathiae bacterial antigen.

[0035] A vaccine composition is a pharmaceutical composition that is safely administered to test animals and, when administered to human subjects, elicits an immune response against a pathogenic microorganism, i.e., induces successful protection against the microorganism. Vaccine compositions contain molecules with antigenic properties, such as immunogenic peptides.

[0036] According to this disclosure, the vaccine composition comprises Escherichia coli antigen, Clostridium toxoid, porcine parvovirus (PPV) antigen, and Erysipelothrix rubrum antigen.

[0037] Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped bacterium belonging to the genus Escherichia. The key virulence factor in enterotoxigenic Escherichia coli (ETEC) infection is adhesin, also known as a colonization factor or enterotoxin. Pili adhesins allow them to adhere to or colonize the absorptive epithelial cells of the jejunum and ileum.

[0038] According to this disclosure, the vaccine composition comprises at least *E. coli* antigen, preferably *E. coli* fimbriae adhesin antigen, more preferably selected from the group consisting of: F4ab (K88ab, UniProtKB-P02970, last modified May 25, 2022), F4ac (K88ac, UniProtKB-P14190, last modified May 25, 2020), F5 (K99, UniProtKB-P18103, last modified May 25, 2022), and F6 (987P, UniProtKB-P21413, last modified May 25, 2022). F41 (F7, UniProtKB-P11900, last modified May 25, 2022), F17 (F17a-g, respectively UniProtKB-Q99003, Q47200, Q47033, Q47199, Q9RH92, Q9RH91, Q47341, last modified May 25, 2022), F18 (UniProtKB-Q47212, last modified June 2, 2021), F165 (UniProtKB-Q46685, last modified May 25, 2022), CS1541 (Crotty) D. et al., Dev Biol. 2020, 1 Mar; 459(1):11-12) and CS31A (Song Li et al., Vet Med Sci. 2020, 6(1):69-75).

[0039] Common antigenic types of fimbriae associated with pathogenicity are F4ab, F4ac, F5 (K99), F6, F41 (F7), and F18. Infections in newborn piglets are usually caused by strains of F4ab, F4ac, F5, and F6, while post-weaning colibacillosis is almost always caused by strains of F4 and F18 (see Vet Rec. Jan 12, 2002; 150(2): 35-7).

[0040] In a preferred embodiment, the vaccine composition comprises one or more Escherichia coli antigens selected from fimbrial adhesins F4ab (K88ab), F4ac (K88ac), F5 (K99), F6 (987P), F41 (F7), and F18. In a more preferred embodiment, the vaccine composition according to this disclosure comprises Escherichia coli F4ab (K88ab), F4ac (K88ac), and F5 (K99) and F6 (987P) antigens.

[0041] According to this disclosure, the pharmaceutical composition further comprises at least one Clostridium antigen.

[0042] Clostridium is a genus of Gram-positive bacteria. This genus includes several important human pathogens, including those causing botulism and tetanus. It includes Clostridium difficile, a significant cause of diarrhea. According to this disclosure, the genus Clostridium can be used as a non-limiting example: Clostridium botulinum, Clostridium tetani, Clostridium difficile, or Clostridium perfringens, preferably Clostridium difficile or Clostridium perfringens, more preferably Clostridium perfringens.

[0043] *Clostridium perfringens* (formerly known as *Bacillus aerogenes capsulatus*, *Bacillus perfringens*, *Bacillus welchii*, or *Clostridium welchii*) is a Gram-positive, spore-forming, anaerobic rod-shaped bacterium. Based on the production of its main toxin, *Clostridium perfringens* is classified into different biotypes, named A through E.

[0044] Clostridium perfringens produces the following toxins: α-toxin, also known as phospholipase C, hemolysin, lecithinase, or phosphatidylcholine phosphohydrolase (UniProtKB-Q0TV31, last modified May 25, 2022), β1-toxin (UniProtKB-Q46181, last modified May 25, 2022), ε-toxin (UniProtKB-Q02307, last modified May 25, 2022), ι-toxin (NCBI reference sequence: WP_003463422.1, last updated July 29, 2019), β2-toxin (UniProtKB-O86264, last updated June 2, 2021), and θ-toxin, also known as Clostridium perfringens lysin O (PFO) or thiol-activated cytolysin (UniProtKB-P0C2E9, last modified May 2022). May 25, 2022), Mu toxin, also known as hyaluronidase glycosidase (UniProtKB-P26831, last modified on May 25, 2022), δ toxin, also known as α-hemolysin (UniProtKB-B8QGZ7, last modified on May 25, 2022), κ toxin, also known as collagenase A (UniProtKB-P43153, last modified on May 25, 2022), λ toxin (UniProtKB-Q46237, last modified on May 25, 2022), Clostridium enterotoxin (CPE), also known as heat-labile enterotoxin B chain (UniProtKB-P01558, last modified on May 25, 2022), and necrotizing enteritis B-like toxin (NetB) (UniProtKB-A0A2P0ZHP7, last modified on May 25, 2022).

[0045] As used herein, the term "toxoid" refers to an inactivated toxin whose toxicity has been inactivated or inhibited by chemical (e.g., glutaraldehyde), molecular, or thermal treatment while retaining its immunogenicity. When used during vaccination, it generates an immune response and forms immune memory against molecular markers of the toxoid without causing toxin-induced disease. Most toxoids have the same polypeptide sequence as the aforementioned toxins from which they are derived. Toxoids can be isolated from *E. coli* or produced through DNA recombination.

[0046] The Clostridium toxoids (e.g., Clostridium perfringens) according to this disclosure can be derived from any naturally occurring toxin encoded by a Clostridium strain. The strain can be selected from wild-type strains, collected strains, or genetically modified strains, etc. Different toxoids can be obtained from the same strain, such as type A α and β2 toxoids, or from different strains. Alternatively, the Clostridium perfringens toxoids according to this disclosure can be obtained using recombinant DNA methods.

[0047] Toxoids can be obtained by chemical (e.g., glutaraldehyde), molecular, or thermal treatment, particularly by chemical treatment, protease cleavage, methods for preparing fragments of the toxin or recombinant DNA with mutations (e.g., point mutations), or by thermal treatment of the corresponding toxin using conventional methods known to those skilled in the art. In particular, treatment with EDTA or glutaraldehyde is an example of a suitable chemical inactivator for inactivating the bacterial toxoids of the present invention. Other chemical inactivators are formalin or formaldehyde. Inactivation can be performed using standard methods known to those skilled in the art. In one embodiment, EDTA is preferably used for the preparation of α-toxins, and glutaraldehyde is used for β1 and β2 toxins.

[0048] Clostridium perfringens type A infection is a common cause of intestinal disease in pigs, diarrhea in newborn piglets, and other animals. Clostridium perfringens type A primarily produces alpha toxin, with or without β2 toxin. Clostridium perfringens type C strains cause severe and fatal necrotic enteritis (NE) in newborn piglets and are defined by carrying two toxin genes: cpa (encoding alpha toxin or CPA) and cpb1 (encoding β1 toxin).

[0049] In a preferred embodiment, the vaccine composition according to this disclosure comprises Clostridium perfringens antigen type A and / or Clostridium perfringens antigen type C, preferably Clostridium perfringens toxoid type A and / or Clostridium perfringens toxoid type C.

[0050] In a preferred embodiment, the vaccine composition comprises at least one antigen selected from the group consisting of: Clostridium perfringens type A or C α-toxin, Clostridium perfringens type A β2-toxin, and Clostridium perfringens type C β1-toxin; more preferably, the vaccine composition comprises Clostridium perfringens type A or C α-toxin, Clostridium perfringens type A β2-toxin, and Clostridium perfringens type C β1-toxin.

[0051] The vaccine composition according to this disclosure also contains porcine parvovirus antigen.

[0052] Porcine parvovirus (PPV) is a small, non-enveloped ssDNA icosahedral virus with a genomic DNA length of 5 kbp. It belongs to the family Picornaviridae, subfamily Parvovirinae, and genus Proparvovirus.

[0053] The PPVs can be classified into different genotypes, PPV1 to PPV7, which are further classified into separate genera within the Parvoviridae family based on their different characteristics. In a preferred embodiment, the vaccine composition according to this disclosure comprises PPV1.

[0054] According to this disclosure, the vaccine composition comprises inactivated porcine parvovirus or live attenuated porcine parvovirus, preferably inactivated porcine parvovirus.

[0055] Any PPV strain can be used as an antigen source according to this disclosure. The strain can be selected from wild-type strains, collected strains, or genetically modified strains, etc. In a particular embodiment, the vaccine composition may comprise any of the parvovirus strains, including, as non-limiting examples, NADL-2, MSV, Kresse, 143a, 27a, or K22. In a preferred embodiment, the vaccine composition comprises an inactivated porcine parvovirus strain K22.

[0056] The inactivated PPV can be inactivated by any method known in the art, including heat treatment or chemical treatment, such as by adding formalin, BEI (diethyleneimine), or other chemical reagents with similar properties.

[0057] The vaccine composition may contain an attenuated live PPV, which has typically been modified by passage in tissue culture cells to reduce its ability to cause disease, but retains its ability to protect against disease or infection when administered to animals.

[0058] In another embodiment, the vaccine composition may comprise a subunit antigen, such as an epitope, protein, polysaccharide, polypeptide, or peptide, or a portion of an antigen (also called a fragment), which may be administered directly to a subject or expressed by a viral or bacterial vector administered to the subject. The subunit antigen may also be present in the form of a VLP.

[0059] In a preferred embodiment, the subunit antigen may be a major structural protein of the PPV, such as VP1 and / or VP2 capsid proteins. The subunit antigen may exist in the form of a virus-like particle (VLP) containing the VP2 protein, which assembles into a VLP similar in size and morphology to the original viral particle.

[0060] The vaccine composition according to this disclosure also contains erysipelas antigen.

[0061] Erysipelothrix rhusiopathiae is a Gram-positive, catalase-negative, rod-shaped, non-spore-forming, non-acid-fast, and non-motile bacterium.

[0062] According to this disclosure, the vaccine composition may comprise *Erysipelothrix purpureus* antigen, selected from the group consisting of: live attenuated *Erysipelothrix purpureus* bacteria, inactivated *Erysipelothrix purpureus* bacteria, also known as *Erysipelothrix purpureus* vaccine, or *Erysipelothrix purpureus* bacterial subunit antigen, including, as non-limiting examples, SpaA, cbpB, rspaA, GAPDH, HP0728, HP1472, CpbA, and ERT2T-A antigens.

[0063] Any strain of *Erysipelothrix purpureus* can be used as an antigen source according to this disclosure. In a particular embodiment, the vaccine composition may contain *Erysipelothrix purpureus* bacterial serotype 1 or 2 antigen.

[0064] Any strain of *Erysipelothrix purpureus* can be used as an antigen source according to this disclosure. In a preferred embodiment, the *Erysipelothrix purpureus* strain is *Erysipelothrix purpureus* strain IM950.

[0065] In a preferred embodiment, the vaccine composition comprises inactivated *Erysipelothrix purpureus* bacteria, preferably inactivated *Erysipelothrix purpureus* serotype 2 bacteria. The inactivated *Erysipelothrix purpureus* can be inactivated by any method known in the art, including heat treatment or chemical treatment, such as by adding thimerosal or other chemical agents with similar properties.

[0066] In a preferred embodiment, the vaccine composition comprises:

[0067] i) Escherichia coli fimbrial adhesins F4ab, F4ac, F5, and F6 antigens.

[0068] ii) Clostridium perfringens type A or C α-toxin, Clostridium perfringens type C β2-toxin, and Clostridium perfringens type C β1-toxin,

[0069] iii) Inactivated porcine parvovirus, and

[0070] iv) Inactivated Erysipelothrix rhusiopathiae bacteria.

[0071] The vaccine composition according to this disclosure may comprise the antigen as described above, combined with a pharmaceutically acceptable carrier, i.e., a biocompatible medium, which is a medium that does not induce significant adverse reactions in test animals after administration, and is capable of presenting the antigen to the host animal's immune system after vaccine administration. Such a pharmaceutically acceptable carrier may be a liquid or solid carrier, for example, containing water and / or any other biocompatible solvent, such as those commonly used to obtain freeze-dried vaccines (based on sugars and / or proteins), optionally containing an immunostimulant (adjuvant). Optionally, other substances, such as stabilizers, viscosity modifiers, or other components, may be added depending on the intended use or desired properties of the vaccine.

[0072] In a preferred embodiment, the vaccine composition according to this disclosure may include an adjuvant. Adjuvants well known in the art are nonspecific stimulants of the immune system that, when administered with an antigen, enhance the immune response. Conventional adjuvants well known in the art include, for example, Freund's complete and incomplete adjuvants, aluminum hydroxide, aluminum phosphate or alumina, silica, kaolin, bentonite, oil emulsions such as oil-in-water (O / W), water-in-oil-in-water (W / O / W), water-in-oil (W / O), tocopherol-α, vitamin E, nonionic block polymers, muramyl dipeptides, Quill A. ® Mineral oils such as Bayol ® or Markol ® Drakeol ® Vegetable oils and carbomer, or combinations thereof. In a preferred embodiment, the adjuvant is aluminum hydroxide. The adjuvant may be incorporated into the vaccine composition described above to enhance its efficacy. Alternatively, the adjuvant may be administered before or after the administration of the vaccine of the present invention.

[0073] Preventive use

[0074] On the other hand, this disclosure relates to a vaccine composition as described above for use in protecting subjects in need from infection by Escherichia coli, Clostridium, porcine parvovirus and Erysipelothrix rhusiopathiae.

[0075] As used herein, “protection against infection” refers to protection against a microorganism resulting from stimulation of an immune response against the microorganism, preventing or improving one or more clinical signs associated with a pathogenic infection or the condition caused by such infection, such as preventing or reducing one or more clinical signs caused by a pathogen infection, or, in a particular embodiment, reducing mortality associated with the pathogenic infection.

[0076] Porcine parvovirus (PPV) infection can cause reproductive failure in primiparous sows. It is characterized by a large number of mummified fetuses, increased frequency of multiple estrus cycles, fewer litters, littering failure, decreased farrowing rate, and very few abortions. Porcine parvovirus infection (PPV) is endemic in most pig herds and is likely the most confirmed cause of reproductive failure in pigs.

[0077] Clostridium difficile is a bacterium that causes colon infections. Symptoms can range from diarrhea to life-threatening damage to the colon.

[0078] Clostridium perfringens type A causes catarrhal enteritis, leading to a creamy, pasty diarrhea (low mortality), while Clostridium perfringens type C causes hemorrhagic necrotizing enteritis, leading to hyperacute, acute, or chronic hemorrhagic diarrhea with a high mortality rate. Both infections are oral and occur at birth, with the organism rapidly reaching very high numbers in the final parts of the small and large intestines. The β1 toxin of Clostridium perfringens type C rapidly destroys the cells lining the small intestine, causing blood to leak into the intestine and leading to death. The α and β2 toxins of Clostridium perfringens type A cause fluid secretion in the small intestine, as well as some inflammation and diarrhea. It can also reach high numbers and cause diarrhea in suckling pigs.

[0079] Erysipelas infection causes a disease known as erysipelas, which can affect a wide range of animals. Erysipelas describes an acute bacterial disease that is primarily seen in growing pigs and is clinically characterized by, for example, septicemia, skin erythema including characteristic rhomboid lesions, arthritis, endocarditis, shock, or death.

[0080] Escherichia coli, commonly found in the lower intestine of warm-blooded organisms, is a pathogen of a wide range of diseases in pigs, which are major causes of death in suckling and weaned pigs worldwide. Two main pathogenic serotypes are involved in enterococcosis: enterotoxigenic Escherichia coli (ETEC) and enteropathogenic Escherichia coli (EPEC). ETEC is the most important pathogenic serotype in pigs. Escherichia coli infection, particularly enterotoxigenic ETEC infection, is a cause of watery diarrhea in farm animals, especially newborn piglets, suckling piglets, and post-weaning piglets, and is associated with reduced growth rate, morbidity, and mortality.

[0081] As used herein, the terms "host" or "subject" are intended for a target individual in need of being administered the immunogenic composition or vaccine of the present invention, particularly a human, mammal, livestock, or any other animal species readily immunized with the composition of the present invention. Preferably, the mammal is a pig species, more preferably a pig, and even more preferably a female pig.

[0082] As used herein, the term "pig" is intended to refer to the pig species, including pigs of any age or at any stage of their reproductive cycle, boars, sows, gilts, and piglets. According to this disclosure, the pig is preferably a female pig. The female pig can be a sow or a gilt. In a preferred embodiment, the pig is a gilt. A gilt is a female pig that has not yet given birth. Once a pig has had one litter and is past its first year, it is referred to as a sow.

[0083] The inventors have determined the most suitable timing of administration of the multivalent vaccine composition according to this disclosure for primary vaccination, thereby conferring simultaneous immunity against different pathogens through active immunization via colostrum intake in vaccinated gilts or sows (preferably gilts) and their offspring.

[0084] Primary vaccination refers to the initial series of vaccine doses given to a subject to establish immunity against a specific disease. The number of doses and the timing of administration can vary depending on the specific vaccine and the individual's age.

[0085] Subsequently, when immunity and clinical protection have declined to a level considered adequate in the population over time, a booster dose may be administered to the vaccinated female pigs (e.g., gilts) that have completed the initial vaccination series.

[0086] This disclosure relates to a vaccine composition as described above for protecting female pigs, preferably gilts, and their offspring from infection with Escherichia coli, Clostridium, porcine parvovirus, and Erysipelothrix rhusiopathiae, wherein the composition is administered to the female pigs, preferably gilts, at least twice before insemination and at least once during the time interval between insemination and farrowing for primary vaccination.

[0087] According to a preferred embodiment, when the composition is administered to gilts, the vaccine composition is administered as a primary vaccination to the gilts at least twice before the first insemination and at least once during the time interval between the first insemination and the first farrowing.

[0088] The term "insemination" refers to natural or artificial insemination. Artificial insemination involves inserting and delivering semen into the reproductive tract of a female pig (a gilt or sow). The most common method of artificial insemination involves delivering semen through the cervix.

[0089] The term "birthing" refers to the process of producing a litter of pigs.

[0090] The terms "weeks before insemination" and "weeks before farrowing" refer to the expected date of natural or artificial insemination and the expected date of farrowing of piglets, respectively, which are related to the hormonal reproductive cycle of female pigs.

[0091] In a particular embodiment, the composition is administered at least twice, preferably at least 9, 8, 7, 6, or 5 weeks before fertilization and more preferably at least two weeks between each administration before fertilization, and at least once, no more than 4 weeks before birth, preferably no more than 3 weeks and more preferably no more than 2 weeks before birth, for the purpose of initial vaccination.

[0092] In a preferred embodiment, the vaccine composition is administered for the initial vaccination:

[0093] - At least twice, between 10 weeks and 1 week before insemination, preferably between 8 weeks and 1 week, and preferably within at least two weeks between each of the two applications prior to insemination.

[0094] - and at least once between 6 weeks and 1 week before giving birth, preferably between 4 weeks and 1 week before giving birth.

[0095] Subsequently, when immunity and clinical protection have declined to a level considered adequate in the population over time, a booster dose may be administered to the vaccinated female pigs (e.g., gilts) that have completed the initial vaccination series.

[0096] In a particular implementation, the initial vaccination process is followed by a booster vaccination between 6 weeks and 1 week, preferably between 4 weeks and 1 week, and even more preferably between 3 weeks and 2 weeks before each subsequent birth.

[0097] In a preferred embodiment, the vaccine composition is administered as follows:

[0098] - At least once between 10 and 4 weeks before insemination, preferably between 9 and 4 weeks, more preferably between 8 and 4 weeks, and even more preferably between 7 and 5 weeks.

[0099] - At least once between 6 weeks and 1 week before insemination, preferably between 5 weeks and 1 week, between 4 weeks and 1 week before insemination, more preferably between 3 weeks and 2 weeks before insemination, and

[0100] - At least once between 6 weeks and 1 week before farrowing, preferably between 5 weeks and 1 week or 4 weeks and 1 week before farrowing, and even more preferably between 3 weeks and 2 weeks before farrowing.

[0101] In a particular implementation, the initial vaccination process is followed by a booster vaccination between 6 weeks and 1 week, preferably between 4 weeks and 1 week, and even more preferably between 3 weeks and 2 weeks before each subsequent birth.

[0102] In another specific embodiment, the vaccine composition is administered twice as a primary vaccination prior to fertilization, wherein the two injections are given at intervals of 2, 3, or 4 weeks, and the second injection is administered at least 1 week prior to fertilization, preferably 2 or 3 weeks prior to fertilization, or even more preferably 4 weeks prior to fertilization, and the vaccine composition is administered at least once as a booster between 4 and 1 week prior to farrowing, preferably between 3 and 1 week prior to farrowing, and more preferably 2 weeks prior to farrowing.

[0103] In a particular implementation, the initial vaccination process is followed by a booster vaccination between 6 and 1 week, preferably between 5 and 1 week, or between 4 and 1 week, more preferably between 6 and 1 week, preferably between 5 and 1 week, or between 4 and 1 week, and more preferably between 3 and 2 weeks before each subsequent birth.

[0104] In a particular embodiment, this disclosure relates to the use of the vaccine composition described above in the manufacture of a medicament for protecting female pigs, preferably gilts, from infection with *Escherichia coli*, *Clostridium*, porcine parvovirus, and *Erysipelothrix rhusiopathiae*. In another particular embodiment, this disclosure relates to the use of the vaccine composition described above in the manufacture of a medicament for protecting female pigs, preferably gilts, and their offspring from infection with *Escherichia coli*, *Clostridium*, porcine parvovirus, and *Erysipelothrix rhusiopathiae*, wherein the composition is administered as described above.

[0105] In another particular embodiment, this disclosure relates to a method for protecting female pigs, preferably gilts, from infection with Escherichia coli, Clostridium, porcine parvovirus, and Erysipelothrix rhusiopathiae, the method comprising administering an immunologically effective amount of the vaccine composition as described above to the subject.

[0106] In a particular implementation, the initial vaccination process is followed by a booster vaccination between 6 and 1 week, preferably between 5 and 1 week, or between 4 and 1 week, more preferably between 6 and 1 week, preferably between 5 and 1 week, or between 4 and 1 week, and more preferably between 3 and 2 weeks before each subsequent birth.

[0107] As used herein, “immunely effective dose” refers to the amount of antigen necessary to induce an immune response in a subject, to the extent that it reduces the infection or pathological effect induced by wild-type infectious agents (i.e., *Erysipelothrix rhusiopathiae*, *Escherichia coli*, porcine parvovirus, or *Clostridium*) compared to the pathological effect induced by infection with wild-type infectious agents in unimmunized pigs. Immunoeffective doses can be determined by a technician using conventional methods known in the art, such as by administering an experimental challenge infection to vaccinated animals and subsequently determining the target animal’s pathological score, feed intake, clinical signs of disease, serological parameters, or by measuring the re-isolation of the pathogen and then comparing these findings with those observed in field-infected pigs.

[0108] The vaccine according to the invention can be administered via any suitable route of administration, including parenteral administration, such as by injection or through the skin, such as intramuscular, intravenous, intraperitoneal, intradermal, submucosal, or subcutaneous routes, but is generally suitable (i.e., appropriate) for intramuscular injection. In a preferred embodiment, the vaccine composition is administered via the intramuscular route.

[0109] In a particular embodiment, immunologically effective amounts of the *E. coli* antigens (e.g., *E. coli* fimbriae adhesins F4ab, F4ac, F5, and F6 antigens), *Clostridium perfringens* toxoids (e.g., *Clostridium perfringens* type A α-toxin, type A β2-toxin, and type C β1-toxin), porcine parvovirus (e.g., inactivated porcine parvovirus), and *Erysipelothrix rhusiopathiae* bacteria (e.g., inactivated *Erysipelothrix rhusiopathiae* bacteria) are mixed before administering the vaccine composition to female pigs. In a preferred embodiment, the different antigens are mixed no more than 48 hours, preferably no more than 24 hours, more preferably no more than 10 hours, and most preferably between 1 hour and 10 hours before administration.

[0110] In one embodiment, the vaccine composition according to this disclosure can be administered to pigs at doses known to those skilled in the art to protect against infection by *Escherichia coli*, *Clostridium*, porcine parvovirus, and *Erysipelothrix rhusiopathiae*, at which protective immunity is obtained, as indicated for those commercially available vaccines.

[0111] The dosage can be determined through routine experiments and depends on both the immunogenic properties of the selected antigen and the desired level of protection. In a particular embodiment, as illustrated in the examples of this application, a commercially available vaccine containing the antigen is mixed before administration to pigs. Attached Figure Description

[0112] Figure 1 Comparison of standard vaccine regimens (top) and new regimens (bottom)

[0113] Figure 2 The average rectal temperature of piglets after their first vaccination.

[0114] Figure 3 : Average rectal temperature in piglets after the second vaccination.

[0115] Figure 4 Antibody titers in the blood of piglets on day 0 and day 35 after vaccination.

[0116] Figure 5 Rectal body temperature of sows after vaccination.

[0117] Figure 6 Antibody titers in sow blood and colostrum at different times before and after vaccination.

[0118] Example

[0119] Example 1: Safety and efficacy study in piglets

[0120] The aim of the first study was to investigate whether the associated use of the two vaccines for active or passive immunization had any effect on the safety or level of antibodies induced after vaccination. Piglets were vaccinated at 9 weeks of age. They were selected to be free of antibodies against the corresponding antigens, and because it was not expected that the immune responses of the sows or piglets would differ from each other. Furthermore, since animals at this age have a lower body weight compared to sows, any potential safety reactions would be observed with a higher probability.

[0121] The aim of this study was to evaluate the efficacy and safety of ENTEROPORC Coli AC mixed with commercial batch PARVORUVAX in piglets after vaccination (two vaccinations at 3-week intervals). Group 1 (n=10 animals) received a mixture of ENTEROPORC Coli AC and PARVORUVAX (IVP), Group 2 (n=10 animals) received ENTEROPORC Coli AC (CP1), and Group 3 received PARVORUVAX (CP2).

[0122] All animals received two vaccinations. The first vaccination was administered on day 0 of the study, and the second vaccination on day 21. Blood samples were collected from the animals on the day of vaccination and 14 days after the second vaccination (day 35 of the study) to demonstrate an increase in antibody levels induced by vaccination. Local reactions at the injection site were assessed until 14 days after each vaccination. Rectal temperatures were recorded one day before each vaccination, on the day of vaccination (before vaccination), 2 hours, 4 hours, and 6 hours after vaccination, and daily thereafter until 7 days after the first vaccination and 4 days after the second vaccination. General health was checked daily throughout the study. The study was terminated after the last blood sampling of the animals (day 35 of the study). Serological responses to each vaccine antigen in the vaccine antigens were measured before vaccination (day 0 of the study) and 2 weeks after the second vaccination (day 35 of the study).

[0123] 1.1 Materials and Methods

[0124] - animal

[0125] For this study, 30 piglets were used, and the first vaccination was given at 9 weeks of age + / - 3 days.

[0126] Animals in the herd are regularly monitored. Animals are derived from sows known to be free of influenza A virus, PRRS, Mycoplasma hyopneumoniae, pseudorabies, classical swine fever virus, and partially positive for PCV2, Escherichia coli (ETEC), and Pasteurella multocida.

[0127] - vaccine

[0128] To demonstrate the efficacy of the combination vaccine, two registered products, ENTEROPORC Coli AC and PARVORUVAX, from IDT Biologika GmbH and Ceva SantéAnimale, were used.

[0129] The investigational veterinary product (IVP) is ENTEROPORC Coli AC (containing E. coli fimbriae adhesins F4ab, F4ac, F5, and F6 antigens, as well as Clostridium perfringens type A alpha toxin, type A β2 toxin, and type C β1 toxin), mixed with PARVORUVAX (containing inactivated erysipelas bacteria and inactivated porcine parvovirus). PARVORUVAX is also available on the market under the trademark PARVORUVAC; both products have the same components but are registered under different trademarks.

[0130] CP1 and CP2 correspond to the individual controls ENTEROPORC Coli AC and PARVORUVAX, respectively.

[0131] Transfer Enteroporc COLI (3 x 10 doses = 67.5 ml) to a 100 ml sterile Schott flask. Discard 15 ml of the Enteroporc COLI suspension, and use the remaining 52.5 ml of Enteroporc Coli suspension to resuspend the lyophilized Enteroporc AC (25 doses). Mix by transferring approximately 5 ml of the Enteroporc Coli suspension to a vial containing the lyophilized Enteroporc AC. Gently shake the vial and transfer the contents back to a sterile Schott flask. Repeat this process. From this reconstituted vaccine, mix 25.0 ml with 25.0 ml of PARVORUVAX (ready-to-use vaccine) to obtain 50.0 ml of IVP. The final vaccine should be stored at 2°C–8°C until inoculation and used within 2 hours after reconstitution.

[0132] The animals were vaccinated via intramuscular routes on the right and left sides of the neck.

[0133] All study animals received the appropriate treatment of 2 × 4 ml over two days. No dosing unevenness occurred during administration. Animal technicians observed the overall health of all animals daily to confirm their health.

[0134] - Rectal temperature

[0135] Measure rectal temperature using a calibrated thermometer at the times indicated in the event timeline.

[0136] - Examination of the injection site

[0137] Injection sites are examined according to standard procedures immediately prior to vaccination and on the days specified in the event schedule. Damage from needle insertion and an area of ​​0.1 cm around the lesion are not considered relevant local reactions and are therefore not recorded as color changes or swelling. The size (length and width) of swelling and color changes (redness) are measured using calipers.

[0138] For redness and heat at the injection site, use the scoring system described in Table 1.

[0139]

[0140] Sample collection and storage

[0141] Collect a blood sample (approximately 5-8 ml) from the jugular vein, without adding anticoagulants. Prepare serum from the clotted blood sample. The sample is not heat-inactivated. Store the serum at -15°C to -25°C until use. The time between blood sampling and storage should not exceed 24 hours.

[0142] - ELISA test

[0143] 96-well microtiter plates (MTP) were coated with monoclonal antibodies that specifically bind to the corresponding antigens. Antibodies were detected by enzyme-linked anti-pig IgG antibodies, and enzyme-dependent color changes were measured by measuring optical density at 450 nm and 620 nm.

[0144] 1.2 Results

[0145] In the groups vaccinated with Enteroporc COLI AC alone or Enteroporc COLI AC plus Parvoruvax, rectal temperature was slightly elevated on the day of vaccination. The increase in rectal temperature was not different between the two groups. Figure 2 and Figure 3 ).

[0146] Compared to Parvoruvax alone, the combined use of Enteroporc COLI AC and Parvoruvax produced comparable antibodies against erysipelas and parvovirus disease. Figure 4 Compared to the Enteropoc COLI AC group, the antibody response against E. coli antigens was slightly lower in the combination therapy group, but still higher than the antibody titers previously shown to protect their offspring in gilts.

[0147] In summary, regarding safety and (serological) efficacy, no interference with the mixture's Enteroporc COLI AC and PARVORUVAC was observed after two vaccinations following the use of the mixture, compared to a single application.

[0148] Example 2: Safety and efficacy study in sows

[0149] The purpose of the second study was to confirm the results in sows. Sows from the IVP group received a mixed vaccine, Enteroporc Coli AC and Parvoruvax, at 6 weeks and 3 weeks prior to insemination, and received EnteroporcCOLI AC alone 2 weeks prior to farrowing. The vaccine mixing was performed precisely before vaccination. The vaccine compositions and analytical methods used in this study were similar to those used in the first embodiment.

[0150] Sows from the CP group received Parvoruvax vaccine 6 and 3 weeks before insemination.

[0151] Rectal temperature was measured on day -1, day 0, day 0 + 4 hours, day 0 + 6 hours, and the following four days after each vaccination. Local and systemic reactions were monitored for 14 days after each vaccination.

[0152] Serological responses were measured 14 days before the first vaccination, on the day of the second vaccination (day 21 of the study), before insemination (day 44 of the study), and 4 days postpartum (day 167 of the study). In addition, antibody titers in colostrum were determined.

[0153] In the group vaccinated with Enteroporc COLI AC plus Parvoruvax, a slight increase in rectal temperature was observed on the day of vaccination; however, this was within the acceptable range given in the respective SPCs for both vaccines. Figure 5 ).

[0154] No local or systemic reactions were observed in either of the two vaccination groups. Concomitant administration of Enteroporc COLI AC and Parvoruvax produced comparable antibody responses against erysipelas and parvovirus compared to Parvoruvax alone. Surprisingly, antibody responses against E. coli and Clostridium antigens in blood and colostrum were significantly higher than protective antibody titers compared to Enteroporc COLI AC alone. Figure 6 In summary, these results demonstrate that the simultaneous use of nine different antigens of Escherichia coli, Clostridium, parvovirus, and Erysipelothrix rhusiopathiae is safe and effective in both gilts and sows, and provides protection for piglets through colostrum intake.

Claims

1. A vaccine composition comprising: i) Escherichia coli fimbriae adhesin antigen, ii) Clostridium toxoids, iii) Porcine parvovirus antigen, and iv) Erysipelothrix rhusiopathiae antigen.

2. The vaccine composition according to claim 1, wherein the Escherichia coli fimbriae adhesin antigen is selected from the group consisting of: F4ab, F4ac, F5, F6, F41, F17, F18, F165, CS1541 and CS31A antigens.

3. The vaccine composition according to claim 2, wherein the composition comprises Escherichia coli fimbriae adhesins F4ab, F4ac, F5 and F6 antigens.

4. The vaccine composition according to any one of claims 1 to 3, wherein the Clostridium toxoid is selected from the group consisting of: Clostridium difficile, Clostridium perfringens type A or C α toxin, Clostridium perfringens type A β2 toxin, Clostridium perfringens type C β1 toxin, Clostridium perfringens type A or C enterotoxin (CPE), and Clostridium perfringens (all strains) θ toxin (clostridium perfringens lysin, PFO).

5. The vaccine composition according to claim 4, wherein the composition comprises Clostridium perfringens type A or C α-toxin, Clostridium perfringens type A β2-toxin, and Clostridium perfringens type C β1-toxin.

6. The vaccine composition according to any one of claims 1 to 5, wherein the erysipelas antigen is inactivated erysipelas.

7. The vaccine composition according to any one of claims 1 to 6, wherein the porcine parvovirus antigen is inactivated porcine parvovirus.

8. The vaccine composition according to any one of claims 1 to 7, wherein the vaccine composition comprises an inactivated porcine parvovirus strain K22.

9. The vaccine composition according to any one of the preceding claims, wherein the vaccine composition comprises an adjuvant, preferably an aluminum hydroxide adjuvant.

10. The vaccine composition according to any one of the preceding claims, wherein the vaccine composition comprises: i) Escherichia coli fimbrial adhesins F4ab, F4ac, F5, and F6 antigens. ii) Clostridium perfringens type A α-toxin, Clostridium perfringens type A β2-toxin, and Clostridium perfringens type C β1-toxin, iii) Inactivated porcine parvovirus, and iv) Inactivated serotype 2 of Erysipelothrix rhusiopathiae.

11. The vaccine composition according to any one of claims 1 to 10, wherein the vaccine composition is used to protect pigs, preferably female pigs and their offspring, from infection with Escherichia coli, Clostridium, porcine parvovirus and Erysipelothrix rhusiopathiae.

12. The vaccine composition according to any one of claims 1 to 10, wherein the vaccine composition is used to protect female pigs, preferably gilts and their offspring, from infection with *Escherichia coli*, *Clostridium*, porcine parvovirus and *Erysipelothrix rhusiopathiae*, wherein the composition is administered to the female pigs, preferably gilts, at least twice before insemination and at least once during the time interval between insemination and farrowing.

13. The vaccine composition for the said use according to claim 12, wherein the composition is administered at least twice, preferably within a period of at least two weeks between each of the two administrations prior to insemination, and at least once, preferably no more than four weeks prior to farrowing, as follows: - At least once between 10 and 4 weeks, preferably between 8 and 6 weeks, before insemination. - At least once between 6 weeks and 1 week before insemination, preferably between 5 weeks and 2 weeks, and - At least once between 6 weeks and 1 week before farrowing, preferably between 4 weeks and 2 weeks.

14. The vaccine composition for the purpose according to any one of claims 11 to 13, wherein, prior to administering the vaccine composition to the female pig, an immunologically effective amount of the *Escherichia coli* fimbriae adhesin antigen, *Clostridium* antigen, porcine parvovirus antigen, and *Erysipelothrix rhusiopathiae* antigen is mixed.

15. The vaccine composition for the said use according to any one of claims 11 to 14, wherein the vaccine composition is administered via an intramuscular, subcutaneous, intradermal, or transdermal route, preferably an intramuscular route.

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