WO2025040792A1

WO2025040792A1 - Multivalent vaccine against swine infections

Info

Publication number: WO2025040792A1
Application number: PCT/EP2024/073694
Authority: WO
Inventors: Vicky Fachinger; Preben Mortensen; Stefano Gozio
Original assignee: Ceva Sante Animale SA
Current assignee: Ceva Sante Animale SA
Priority date: 2023-08-24
Filing date: 2024-08-23
Publication date: 2025-02-27
Anticipated expiration: 2026-02-24
Also published as: TW202509058A

Abstract

The present disclosure relates to a multivalent vaccine composition and its use in the protection against Escherichia coli (E. coli), Clostridium spp., porcine parvovirus and Erysipelothrix rhusiopathie (E. rhusiopathie) infections in a pig and its progeny, preferably with a new scheme of administration for passive and active immunization of a pig and its progeny.

Description

MULTIVALENT VACCINE AGAINST SWINE INFECTIONS

TECHNICAL FIELD

BACKGROUND OF THE INVENTION

Pig production accounts for more than one-fourth of total protein consumed worldwide. Infectious diseases impact pig health and productivity of the global swine 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 E. coli, and Clostridium spp.

Sows and gilts are vaccinated against these different pathogens during their lifetime by multiple injections of different vaccines. Vaccine mediated protection declines over time, and thus, the required timing of maximum protection differs for active and passive immunization. Vaccines used to actively protect sows and gilts from diseases such as Erysipelas and Parvovirosis are usually administered before first insemination, usually between 6 and 3 weeks before insemination for gilts and between 3 weeks and one day before insemination for sow and for booster vaccination, usually 1 day to 3 weeks before following insemination in each cycle, or every 6 months by mass vaccination. By these vaccination schedules, it is ensured that protection is achieved during pregnancy because many of the above-mentioned pathogens are associated with reproductive failure and abortions in sows (i.e., protection during pregnancy is required).

Vaccine can also be used for passive immunization. Indeed, maternal immunity at an early age is critical for passive protection of neonatal pigs. Typically, vaccines for passive immunization of piglets against for example neonatal diarrhea caused by Clostridium spp. or E. coli infections are administered twice between 7 and 2 weeks before farrowing and for booster vaccination, usually once between 4 and 2 weeks before farrowing in sows to give piglets protection by passive immunization via colostrum intake. Multivalent vaccines capable of conferring simultaneous immunity to different pathogens or diseases upon one single injection emerge as the most manageable solution to protect animals. Indeed, it is desirable to inject all necessary antigens in a single vaccine, which makes the vaccination procedure less traumatic and painful for the animal, more time efficient and more manageable for the practitioner. Although some combined vaccines have already been successfully developed, immunological interferences might occur between two or more antigen specific immune responses simultaneously induced in vivo.

Moreover, it is needed to elucidate the most appropriate timing for injecting a multivalent vaccine to confer an efficient active and passive immunization protection in a pig and its progeny.

It remains a need to develop a safe and efficient multivalent vaccine that can be administered in a pig to give protection via active immunization and give piglets protection via passive immunization through colostrum intake.

SUMMARY OF THE INVENTION

The inventors showed that a multivalent vaccine confers an efficient protection against E. coli, Clostridium perfringens, Parvovirus and Erysipelas infections administered in a female pig. This multivalent vaccine has no negative effect in vaccinated female pig and protects against Erysipelothrix rhusiopathie, porcine parvovirus, E coli and Clostrida infections by active immunization in a vaccinated female pig as well as to the progeny of the animal through the intake of colostrum.

The inventors thus unexpectedly showed that despite immunological interference, a multivalent vaccine, comprising 9 different antigens/toxoids has a safety profile and serologically detectable efficacy in piglets and is effective in gilts and sows. The demonstration of the efficacy and safety of this multivalent vaccine made in the present application will allow to modify the vaccination protocols of pigs and facilitate greatly the vaccination of pigs by making it less traumatic for the pigs.

Furthermore, in contrast to the classical administration schemes adapted either for passive or active immunization and which require a repeated number of injections in pigs, the inventors in the present application have developed for the first time a new administration scheme for primary vaccination which allows both active and passive immunization at the same time while being highly effective. This highly effective new administration scheme thus allows to reduce the number of injections and facilitate the vaccination in pigs.

The present invention relates to a vaccine composition comprising: i) an Escherichia coli fimbrial adhesin antigen, ii) a Clostridium toxoid, iii) a porcine Parvovirus antigen, preferably an inactivated porcine Parvovirus, more preferably an inactivated porcine parvovirus K22 strain and, iv) an Erysipelothrix rhusiopathiae antigen, preferably an inactivated Erysipelothrix rhusiopathie .

In a preferred embodiment, said 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, more preferably said composition comprises Escherichia coli fimbrial adhesins F4ab, F4ac, F5 and F6 antigens.

In a particular embodiment, said Clostridium toxoid antigen is Clostridium perfringens toxoid antigen, preferably selected from the group consisting of: C. perfringens type A or C alpha toxoid, C. perfringens type A beta 2 toxoid, C. perfringens type C beta 1 toxoid, C. perfringens Type A or C Enterotoxin (CPE) and C. perfringens (all strains) theta toxoid (Perfringolysin, PFO), preferably said composition comprises C. perfringens type A or C alpha toxoid, C. perfringens type A beta 2 toxoid and C. perfringens type C beta 1 toxoid.

In a preferred embodiment, said vaccine composition comprises an adjuvant, preferably aluminum hydroxide adjuvant.

In a more preferred embodiment, the vaccine composition according to the present invention comprises: i) Escherichia coli fimbrial adhesins F4ab, F4ac, F5 and F6 antigens, ii) Clostridium perfringens type A or C alpha toxoid, Clostridium perfringens type A beta 2 toxoid and Clostridium perfringens type C beta 1 toxoid, iii) inactivated porcine Parvovirus, and iv) an inactivated Erysipelothrix rhusiopathie bacteria. In another aspect, the present invention relates to a vaccine composition as described above for use in the protection against Escherichia coli, Clostridium spp., Porcine Parvovirus and Erysipelothrix rhusiopathie infections in a pig and its progeny.

In a preferred embodiment, the present invention relates to a vaccine composition as described above for use in the protection against Escherichia coh, Clostridium spp., porcine Parvovirus and Erysipelothrix rhusiopathie infections in a female pig, preferably a gilt and its progeny wherein said composition is administered to the female pig, preferably the gilt at least two times before insemination and at least one time in the time interval between insemination and farrowing, preferably said composition is administered, at least two times no more than 10 weeks before insemination, preferably no more than 9, 8, 7, 6, 5 weeks before insemination, and preferably with an interval of at least two weeks between each of the two administrations before insemination and at least one time no more than 4 weeks, preferably no more than 3, more preferably no more than 2 weeks before farrowing.

In a more preferred embodiment, said composition is administered at least one time between 10 and 4 weeks, preferably between 9 and 4, between 8 and 4 or more preferably between 7 and 5 weeks before insemination, at least one time between 6 and 1 weeks, preferably between 5 and 1 weeks or between 4 and 1 weeks, more preferably between 3 and 2 weeks before insemination and, at least one time between 6 and 1 weeks, preferably between 5 and 1, between 4 and 1, more preferably between 3 and 2 weeks before farrowing.

In a preferred embodiment, said vaccine composition is administered by intramuscular, intradermal, transdermal, or subcutaneous route, more preferably by intramuscular route. In a particular embodiment, immunologically effective amounts of said Escherichia coli fimbrial adhesin antigen, Clostridium toxoid, porcine parvovirus antigen, and Erysipelothrix rhusiopathie antigen are mixed before administration of the vaccine composition to the female pig.

DETAILED DESCRIPTION OF THE INVENTION

Vaccine composition

The inventors have shown that a multivalent vaccine composition comprising 9 different antigens/toxoids protecting against E.coli, Clostridium spp, porcine Parvovirus (PPV) and Erysipelas infections has a safety profile and serologically detectable efficacy in piglets and is effective in gilts and sows. The present disclosure relates to a vaccine composition comprising: i) an Escherichia coli fimbrial adhesin antigen, ii) a Clostridium toxoid, iii) a porcine parvovirus antigen, and iv) an Erysipelothrix rhusiopathie bacteria antigen.

A vaccine composition is a pharmaceutical composition that is safe to administer to a subject animal, and which elicits an immunological response when administered in a subject against a pathogenic micro-organism, i.e., to induce a successful protection against the microorganism. Vaccine composition comprises molecules with antigenic properties such as immunogenic polypeptides.

According to the present disclosure the vaccine composition comprises a E. coli antigen, a Clostridium toxoid, a porcine parvovirus (PPV) antigen and an Erysipelothrix rhusiopathie antigen.

Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus Escherichia. The key virulence factors of enterotoxigenic E. coli (ETEC) infection are adhesins also called colonization factors and enterotoxins. The fimbrial adhesins allow them to adhere to or colonize the absorptive epithelial cells of the jejunum and ileum.

According to the present disclosure, the vaccine composition comprises at least an E. coli antigen, preferably E. coli fimbrial adhesin antigen, more preferably selected from the group consisting of: F4ab (K88ab, UniProtKB - P02970, last modified on May 25, 2022), F4ac (K88ac, UniProtKB - P14190, last modified on May 25, 2020), F5 (K99, UniProtKB - P18103, last modified on May 25, 2022), F6 (987P, UniProtKB - P21413, last modified on May 25, 2022), F41 (F7, UniProtKB - Pl 1900, last modified on May 25, 2022), F17 (F17a- g, UniProtKB - Q99003, Q47200, Q47033, Q47199, Q9RH92, Q9RH91, Q47341 respectively, last modified on May 25, 2022), F18 (UniProtKB - Q47212, last modified on June 2, 2021), F165 (UniProtKB - Q46685, last modified on May 25, 2022), CS1541(Crotty D. et al. Dev Biol. 2020 Mar 1 ;459(1): 11 -12) and, CS31 A (Song Li et al. Vet Med Sci. 2020 Feb;6(l):69-75).

The common antigenic types of fimbriae associated with pathogenicity are F4ab, F4ac, F5 (K99), F6, F41 (F7) and F18. Infection in neonates is commonly caused by F4ab, F4ac, F5 and F6 strains, whereas postweaning colibacillosis is nearly always due to the F4 and F18 strains (See Vet Rec. 2002 Janl2; 150(2): 35-7).

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

According to the present disclosure, said pharmaceutical composition further comprises at least one Clostridium antigen.

Clotridium spp is a genus of Gram-positive bacteria. This genus includes several significant human pathogens, including the causative agents of botulism and tetanus. The genus includes an important cause of diarrhea, Clostridium difficile. According to the present disclosure, Clostridium can be as non-limiting examples: Clostridium botulinum, Clostridium tetcmi, Clostridium difficile or Clostridium perfringens, preferably Clostridium difficile or Clostridium perfringens, more preferably Clostridium perfringens.

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. C. perfringens is classified into different biotypes designated A through E according to the production of major toxins.

Clostridium perfringens produces the following toxins: alpha toxin also known as Phospholipase C, hemolysin, lecithinase or phosphatidylcholine cholinephosphohydrolase (UniProtKB - Q0TV31, last modified on May 25, 2022), betal toxin (UniProtKB - Q46181, last modified on May 25, 2022), epsilon toxin (UniProtKB - Q02307, last modified on May 25, 2022), iota toxin (NCBI Reference Sequence: WP_003463422.1, last updated on July 29, 2019), beta2 toxin (UniProtKB - 086264, last updated on June 2, 2021), theta toxin also known as perfringolysin O (PFO) or Thiol-activated cytolysin (UniProtKB - P0C2E9, last modified on May 25, 2022), Mu toxin also known as hyaluronoglucosaminidase (UniProtKB - P26831, last modified on May 25, 2022), delta toxin also known as alpha hemolysin (UniProtKB - B8QGZ7, last modified on May 25, 2022), kappa toxin also known as collagenase A (UniProtKB - P43153, last modified on May 25, 2022), lambda toxin (UniProtKB - Q46237, last modified May 25, 2022) Clostridium enterotoxin (CPE) also known as heat-labile enterotoxin B chain (UniProtKB - P01558, last modified on May 25, 2022), and necrotic enteritis B-like toxin (NetB) (UniProtKB - A0A2P0ZHP7, last modified on May 25, 2022).

The term “toxoid” as used herein means inactivated toxin whose toxicity has been inactivated or suppressed either by chemical (e.g., glutaraldehyde), molecular or heat treatment while immunogenicity is maintained. When used during vaccination, an immune response is mounted, and immunological memory is formed against the molecular markers of the toxoid without resulting in toxin-induced illness. Most of the toxoids have the same polypeptide sequence as the toxin as described above from which they derive from. Toxoid can either be isolated from E coll or produced recombinantly by DNA.

The Clostridium toxoid (e.g., Clostridium perfrmgens) according to the present disclosure, can be derived from any naturally toxin encoded by a Clostridium strain. The strain can be selected, among others, from field strains, collection strains or genetically modified strains. The different toxoids can be obtained from the same strain for example for type A alpha and beta2 toxoid or from different strains. Alternatively, the Clostridium perfringens toxoid according to the present disclosure can be obtained by using recombinant DNA method.

The toxoids can be obtained by chemical (e.g., glutaraldehyde), molecular or heat treatment, in particular by chemical treatment, protease cleavage, recombinant DNA methods by making fragments or mutations of the toxins (e.g., point mutations) or by thermal treatment of the corresponding toxins by routinary means known by the skilled in the art. In particular, treatment with EDTA or glutaraldehyde are examples of suitable chemical inactivating agents for use in inactivate bacterial toxoids of the invention. Other chemical inactivating agent is formalin or formaldehyde. The inactivation can be performed using standard methods known to those of skill in the art. In one embodiment, EDTA is preferably used in alpha toxoid preparation and glutaraldehyde used in beta 1 and beta 2 toxoid.

Clostridium perfrmgens type A infections are common causes of enteric diseases in pigs, diarrhea in neonatal piglets and other animal. Clostridium perfringens type A mainly produces alpha toxin with or without beta2 toxin. Clostridium perfringens type C strains cause severe and lethal necrotic enteritis (NE) in newborn piglets and are defined by carrying the two toxin genes cpct (encoding for a-toxin or CPA), cpbl (encoding for pi-toxin).

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

In a preferred embodiment, said vaccine composition comprises at least one antigen selected from the group consisting of: Clostridium perfringens type A or C alpha toxoid, Clostridium perfringens type A beta2 toxoid and Clostridium perfringens type C betal toxoid, more preferably said vaccine composition comprises Clostridium perfringens type A or C alpha toxoid, Clostridium perfringens type A beta2 toxoid and Clostridium perfringens type C betal toxoid.

The vaccine composition according to the present disclosure further comprises a porcine Parvovirus antigen.

Porcine Parvovirus (PPV) is a small ssDNA icosahedral nonenveloped virus with 5 kbp-long genomic DNA, which belongs to the Parvoviridae family, Parvovirinae subfamily and Protoparvovirus genus.

Said PPV can be classified in different genotypes PPV1 to PPV7, which were further classified based on their different characterization as a separate genus within the family Parvoviridae. In a preferred embodiment, the vaccine composition according to the present disclosure comprises PPV1.

According to the present disclosure, the vaccine composition comprises an inactivated or live-attenuated porcine Parvovirus, preferably inactivated porcine Parvovirus.

Any strains of PPV can be used as a source of antigens according to the present disclosure. The strain can be selected, among others, from field strains, collection strains or genetically modified strains. In a particular embodiment, said vaccine composition may comprise any one of parvovirus strain including as non-limiting examples: NADL-2, MSV, Kresse, 143a, 27a or K22. In a preferred embodiment, said vaccine composition comprises inactivated porcine Parvovirus K22 strain.

Said inactivated PPV can be inactivated by any methods known in the art including heat treatment or chemical treatment for example by adding formalin, BEI (binary ethylenimine), or other chemical agents having properties like these agents.

Said vaccine composition may comprise an attenuated live PPV that has been altered, typically by passaging in tissue culture cells, to attenuate its ability to cause disease, but which maintains its ability to protect against disease or infection when administered to animals.

In another embodiment, said vaccine composition may comprise a subunit antigen such as an epitope, a protein, a polysaccharide, a polypeptide or a peptide, or a part of the antigen also called fragment, that can be directly administered to a subject or expressed by viral or bacterial vector administered into said subject. The subunit antigen may also be present in the form of a VLP.

In a preferred embodiment, said subunit antigen may be PPV major structural protein, such as VP1 and/or VP2 capsid proteins. The said subunit antigen may be present in the form of a virus-like particles (VLP) comprising a VP2 protein that assembles into VLP that are similar in size and morphology to the original virion.

The vaccine composition according to the present disclosure further comprises an Erysipelothrix rhusiopathie antigen.

Erysipelothrix rhusiopathie is a Gram-positive, catalase-negative, rod-shaped, non-sporeforming, nonacid-fast, nonmotile bacterium.

According to the present disclosure, said vaccine composition may comprise an E. rhusiopathie antigen selected from the group consisting of: E. rhusiopathie attenuated-live bacteria, E. rhusiopathie inactivated bacteria, also called E. rhusiopathie bacterin, or E. rhusiopathie bacteria subunit antigen including as non-limiting examples: SpaA, cbpB, rspaA, GAPDH, HP0728, HP 1472, CpbA and ERT2T-A antigens.

Any strains of E. rhusiopathiae can be used as a source of antigens according to the present disclosure. In a particular embodiment, said vaccine composition may comprise a E. rhusiopathie bacteria serotype 1 or 2 antigens.

Any strains of E. rhusiopathiae can be used as a source of antigens according to the present disclosure. In a preferred embodiment, said E. rhusiopathiae strain is E. rhusiopathiae IM950 strain.

In a preferred embodiment, said vaccine composition comprises an inactivated Erysipelothrix rhusiopathie bacteria, preferably inactivated Erysipelothrix rhusiopathie serotype 2 bacteria. Said inactivated E. rhusiopathie can be inactivated by any methods known in the art including heat treatment or chemical treatment for example by adding thiomersal, or other chemical agents having properties like these agents. In a preferred embodiment, said vaccine composition comprises: i) E. coli fimbrial adhesin F4ab, F4ac, F5 and F6 antigens, ii) C. perfringens type A or C alpha toxoid, Clostridium perfringens type C beta2 toxoid and C. perfringens type C betal toxoid, iii) inactivated porcine parvovirus, and iv) an inactivated E. rhusiopathie bacteria.

The vaccine composition according to the present disclosure can comprise said antigens as described above in combination with a pharmaceutically acceptable carrier, i.e., a biocompatible medium, i.e., a medium that after administration does not induce significant adverse reactions in the subject animal, capable of presenting the antigen to the immune system of the host animal after administration of the vaccine. Such a pharmaceutically acceptable carrier may for example be a liquid containing water and/or any other biocompatible solvent or a solid carrier such as commonly used to obtain freeze-dried vaccines (based on sugars and/or proteins), optionally comprising immunostimulating agents (adjuvants). Optionally other substances such as stabilisers, viscosity modifiers or other components are added depending on the intended use or required properties of the vaccine.

In a preferred embodiment, the vaccine composition according to the present disclosure may comprise an adjuvant. Adjuvants, as is well known in the art, are nonspecific stimulants of the immune system, which, administered together with the antigen, enhance the immunological response. Conventional adjuvants, well-known in the art are e.g., Freund's Complete and Incomplete adjuvant, aluminum hydroxide, -phosphate or -oxide, silica, Kaolin, Bentonite, oil emulsion such as oil-in-water (O/W), water-in-oil-in-water emulsion (W/O/W), water-in-oil (W/O)), tocopherol-alpha, vitamin E, non-ionic block polymers, muramyl dipeptides, Quill A®, mineral oil, e.g. Bayol® or Markol®; Drakeol®, vegetable oil, and carbomer or combinations thereof. In a preferred embodiment, said adjuvant is aluminum hydroxide. Adjuvants can be incorporated in the vaccine composition as described above to enhance the effectiveness thereof. Alternatively, the adjuvant may be administered before, or after the administration of the vaccine of the invention. Prophylactic uses

In another aspect, the present disclosure relates to the vaccine composition as described above for use in the protection against E. coli, Clostridium spp., porcine parvovirus and E. rhusiopathie infections in a subject in need thereof.

By “protection of an infection” as used herein relates to the protection against a microorganism resulting from the stimulation of the immune response against said microorganism, preventing, ameliorating one or more clinical signs associated with a pathogenic infection or a disorder arising from that infection, for example to prevent or reduce one or more clinical signs resulting from the infection with the pathogen or in particular embodiment, to decrease the mortality associated with said pathogenic infection.

Porcine parvovirus (PPV) infection can cause reproductive failure in naive dams. It is characterized by the occurrence of large numbers of mummified fetuses, an increase in the number of returns to estrus, small litters, failures to farrow, decreased farrowing rate, and rarely abortion. Porcine parvoviral infection (PPV) is endemic in most swine herds and probably the most diagnosed infectious cause of reproductive failure in swine.

Clostridium difficile is a bacterium that causes an infection of the colon. Symptoms can range from diarrhea to life threatening damage to the colon.

Clostridium perfringens type A causes katarrhalic enteritis resulting in creamy-pasty diarrhea (with low mortality) and Clostridium perfringens type C causes haemorrhagic- necrotizing enteritis resulting in peractue, acute or chronic bloody diarrhea with high mortality. Both Infections are oral and occur at birth and the organisms rapidly reach very high numbers within the last part of the small intestine and the large intestine. The betal toxin of C. perfringens type C rapidly destroys the cells lining the small intestine, so that blood is lost into the intestine resulting in death. The alpha and beta 2 toxins of C. perfringens type A cause secretion of fluid in the small intestine, and some inflammation and diarrhea. It may also reach high numbers and cause diarrhea in the suckling pig.

E. rhusiopathiae infection causes the disease known as erysipelas that may affect a wide range of animals. Erysipelas describes an acute bacterial disease seen mainly in growing pigs and characterized clinically for example by septicemias, cutaneous erythema, including characteristic diamond-shaped lesions, septicemia, arthritis, endocarditis, showk or death. E. coli commonly found in the lower intestine of warm-blooded organisms is the causative agent of a wide range of diseases in pigs, which are important causes of death occurring worldwide in suckling and weaned pigs respectively. Two main pathotypes are involved in enteric colibacillosis: enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC). ETEC is the most important pathotype in swine. Escherichia coli infection, in particular enterotoxigenic E. coli (ETEC) infection is responsible for watery diarrhea in farm animals, in particular in newborn, suckling, and as well as in post-weaning piglets and is associated with reduced growth rate, morbidity, and mortality.

As used herein, the term "host" or "subject" is intended for the target individuals in need thereof to whom the immunogenic composition or vaccine of the invention are administered, among other humans, mammals, livestock, or any other animal species susceptible to be vaccinated with the compositions of the invention. Preferably, the mammal is a porcine specie, more preferably is a pig, and more preferably is a female pig.

As used herein, the term "pig" or "swine" is intended for porcine species including, among others, pigs, boars, sows, gilts, and piglets of any age or in any phase of their production cycle. According to the present disclosure, said pig is preferably female pig. Said female pig can be a sow or a gilt. In a preferred embodiment, said pig is a gilt. A gilt is a female pig who has not farrowed. Once a pig has had a litter and is past her first year, she is called a sow.

The inventors determined the most appropriate timing of administration of the multivalent vaccine composition according to the present disclosure for primary vaccination conferring simultaneous immunity to different pathogen by active immunization in vaccinated gilts or sows, preferably gilt and in its progeny via the intake of colostrum.

A primary vaccination refers to the initial series of vaccine doses that are given to subject to establish immunity to a particular disease. The number of doses and the timing of the doses can vary depending on the specific vaccine and the age of the individual.

Booster doses can be thereafter administered to said vaccinated female pig (e g., gilt) that has completed the primary vaccination series when, with time, the immunity and clinical protection has fallen below a rate deemed sufficient in that population.

The present disclosure relates to a vaccine composition as described above for use in the protection against E. coli, Clostridium spp., porcine Parvovirus, and E. rhusiopathie infections in a female pig, preferably a gilt and its progeny wherein said composition is administered for a primary vaccination to said female pig, preferably aid gilt at least two times before insemination and at least one time in the time interval between insemination and farrowing.

According to a preferred embodiment, when said composition is administered to a gilt, the vaccine composition is administered to said gilt as a primary vaccination at least two times before the first insemination and at least one time in the time interval between first insemination and first farrowing.

By “insemination”, it is intended natural or artificial insemination. Artificial insemination is the insertion and delivery of semen into the reproductive canal of a female pig (gilt or sow). The most common method of artificial insemination involves delivering semen to traverse the cervix.

By “farrowing”, it is intended the process of birthing a litter of pigs.

By “weeks before insemination” or “weeks before farrowing”, it is respectively intended the expected natural or artificial insemination date regarding the hormonal reproductive cycle of the female pig and the expected date of delivering the piglets.

In a particular embodiment, said composition is administered for a primary vaccination, at least two times no more than 10 weeks before insemination, preferably no more than 9, 8, 7, 6, 5 weeks before insemination and more preferably with an interval of at least two weeks between each of the two administrations before insemination, and at least one time no more than 4 weeks, preferably no more than 3, more preferably no more than 2 weeks before farrowing.

In a preferred embodiment, said vaccine composition is administered for a primary vaccination: at least two times between 10 and 1 weeks, preferably between 8 and 1 weeks before the insemination, and preferably with an interval of at least two weeks between each of the two administrations before insemination, and at least one time between 6 and 1 weeks, preferably between 4 and 1 before farrowing. Booster doses can be thereafter administered to said vaccinated female pig (e g., gilt) that has completed the primary vaccination series when, with time, the immunity and clinical protection has fallen below a rate deemed sufficient in that population.

In a particular embodiment, said primary vaccination course is followed by a booster vaccination one time between 6 and 1 weeks, preferably between 4 and 1 week, again more preferably between 3 and 2 weeks before each subsequent farrowing.

In a preferred embodiment, said vaccine composition is administered: at least one time between 10 and 4 weeks, preferably between 9 and 4 weeks, more preferably between 8 and 4 weeks before insemination, again more preferably between 7 and 5 weeks before insemination, at least one time between 6 and 1 weeks before insemination, preferably between 5 and 1 weeks, between 4 and 1 weeks before insemination, more preferably between 3 and 2 weeks before insemination and, at least one time between 6 and 1 weeks before farrowing, preferably between 5 and 1, or between 4 and 1 weeks before farrowing, again more preferably between 3 and 2 weeks before farrowing.

In another particular embodiment, the said vaccine composition is administered as a primary vaccination two times before insemination, wherein the two injections are performed within an interval of 2, 3 or 4 weeks, and the second injection is administered at least 1 week before insemination, preferably 2 or 3 weeks before insemination, even more preferably 4 weeks before insemination and said vaccine composition is administered as a booster at least one time between 4 and 1 week before farrowing, preferably 3 and 1 weeks before farrowing, more preferably 2 weeks before farrowing.

In a particular embodiment, said primary vaccination course is followed by a booster vaccination one time between 6 and 1 weeks, preferably between 5 and 1, or between 4 and 1 weeks, again more preferably between 6 and 1 weeks, preferably between 5 and 1, or between 4 and 1 weeks, again more preferably between 3 and 2 weeks before each subsequent farrowing. In a particular embodiment, the present disclosure relates to the use of a vaccine composition as described above in the manufacture of a medicament for the protection against Escherichia coli, Clostridium spp., porcine parvovirus and Erysipelothrix rhusiopathie infections in a female pig, preferably a gilt in need thereof. In another particular embodiment, the present disclosure relates to the use of a vaccine composition as described above in the manufacture of a medicament for the protection against Escherichia coli, Clostridium spp., porcine Parvovirus and Erysipelothrix rhusiopathie infections in a female pig, preferably a gilt and its progeny in need thereof, wherein said composition is administered as described above.

In another particular embodiment, the present disclosure relates to a method for protecting against E. coli, Clostridium spp., porcine Parvovirus and E. rhusiopathie infections a female pig, preferably a gilt in need thereof comprising administering an immunologically effective amount of the vaccine composition as described above in said subject.

In a particular embodiment, said primary vaccination course is followed by a booster vaccination one time between 6 and 1 weeks, preferably between 5 and 1, or between 4 and 1 weeks, again more preferably between 6 and 1 weeks, preferably between 5 and 1, or between 4 and 1 weeks, again more preferably between 3 and 2 weeks before each subsequent farrowing.

An " immunologically effective amount “ as used herein relates to the amount of antigen that is necessary to induce an immune response in subjects to the extent that it decreases an infection or the pathological effects caused by the infection with a wild-type infectious agent, i.e. E. rhusiopathie, E. coli, porcine parvovirus or Clostridium spp., when compared to the pathological effects caused by infection with a wild-type infectious agent in non-immunized pigs. The immunologically effective amount can be established by the skilled person via routine methods commonly known in the art, for instance by administering an experimental challenge infection to vaccinated animals and next determining a target animal’s pathological scoring, feed intake, clinical signs of disease, serological parameters or by measuring re-isolation of the pathogen, followed by comparison of these findings with those observed in field-infected pigs.

The vaccine according to the present invention may be administered by any suitable route of administration, including parenteral administration, e.g., through all routes of injection into or through the skin, e g. intramuscular, intravenous, intraperitoneal, intradermal, submucosal, or subcutaneous, but is typically adapted, i.e. suitable, for intramuscular injection. In a preferred embodiment, said vaccine composition is administered by intramuscular route.

In a particular embodiment, immunologically effective amounts of said Escherichia coli antigen (e.g. E. coli fimbrial adhesins F4ab, F4ac, F5 and F6 antigens), Clostridium perfringens toxoid (e.g. Clostridium perfringens type A alpha toxoid, type A beta 2 toxoid and type C beta 1 toxoid), porcine Parvovirus (e.g. inactivated porcine parvovirus and Erysipelothrix rhusiopathie bacteria (e.g. inactivated E. rhusiopathie bacteria) are mixed before administration of the vaccine composition to the female pig. In a preferred embodiment, the different antigens are mixed no more than 48, preferably no more than 24 hours, more preferably no more than 10 hours before administration, preferably between 1 and 10 hours before administration.

In one embodiment, said vaccine composition according to the present disclosure can be administered in a pig for protecting E. coli, Clostridium spp., porcine Parvovirus and E. rhusiopathie infections at doses well known by one skilled in the art at which protective immunity is obtained, such as those indicated for commercially available vaccines.

The doses can be established by routine experimentation and depends i.e. on the immunogenic properties of the antigen chosen but also on the required level of protection. In a particular embodiment, commercially available vaccines comprising said antigens are mixed before administration to the pig as illustrated in the example of the present application.

FIGURE LEGENDS

Figure 1: Comparison of the conventional (above) and new (below) scheme for vaccines

Figure 2: Mean rectal body temperature after the first vaccination in piglets.

Figure 3: Mean rectal temperature after the second vaccination in piglets.

Figure 4: Antibody titers in blood of piglets at Day 0 and Day 35 after vaccination in piglets.

Figure 5: Rectal body temperatures post vaccination in sows.

Figure 6: Antibody titers in blood and colostrum in sows at different days before and after vaccination.

EXAMPLES

EXAMPLE 1: Safety and efficacy studies in piglets The purpose of the first study was to investigate whether the associated use of two vaccines for either active or passive immunization has any impact on the safety or the level of induced antibodies following vaccination. Piglets were vaccinated at 9 weeks of age. They were selected as free of antibodies against the respective antigens, and because it was not expected that the immunological response of sows or young piglets differs from each other. Furthermore, due to the lower body weight of animals of this age compared to sows, any possible safety reactions would have been observed with higher probability.

The objective of this study was to assess the efficacy and safety of ENTEROPORC Coli AC mixed with a commercial batch PARVORUVAX after vaccination (twice vaccination at an interval of 3 weeks) of piglets. Group 1 (n=10 animals) received a mixture of the vaccines ENTEROPORC Coli AC and PARVORUVAX (IVP), Group 2 (n=10 animals) received ENTEROPORC Coli AC (CPI) and Group 3 received PARVORUVAX (CP2).

All animals were vaccinated twice. Day of first vaccination was study day 0, day of second vaccination was study day 21. Blood samples were taken from the animals at the days of vaccination and 14 days after second vaccination (study day 35) to demonstrate the gain of antibody levels evoked by vaccination. Local reactions at the injection sites were evaluated until 14 days post each vaccination Rectal temperatures were recorded one day before each vaccination, at the day of vaccination (before vaccination), 2, 4 and 6 h after vaccination and afterwards once daily until 7 days post first and 4 days post second vaccination, respectively. General health was checked daily throughout the study. The study was terminated after last blood sampling of the animals (study day 35). Serological response to each of the vaccine antigens was measured before vaccination (study day 0) and 2 weeks after 2nd vaccination (study day 35).

1.1 Material and methods

Animals

For this study 30 piglets are used and first vaccinated at age of 9 weeks +/- 3 days.

Animals of the herd are regularly monitored. The animals were derived from sows which are known to be free of Influenza A viruses, PRRS, Mycoplasma hyopneumoniae, pseudorabies, classical swine fever virus and as well as partially positive for PCV2, E. coli (ETEC) and Pasteurella multocida.

Vaccines For demonstration of the efficacy of the combined vaccine, two registered products ENTEROPORC Coli AC and PARVORUVAX from IDT Biologika GmbH and Ceva Sante Animale were used.

The investigational Veterinary Product (IVP) is ENTEROPORC Coli AC (comprising E. coli fimbrial adhesins F4ab, F4ac, F5 and F6 antigens and Clostridium perfringens toxoid type A alpha toxoid, type A beta 2 toxoid and type C beta 1 toxoid), mixed with PARVORUVAX (comprising an inactivated Erysipelothrix rhusiopathiae bacteria and inactivated porcine Parvovirus). The PARVORUVAX product may also be found in the market with the brand name PARVORUVAC, both products have the same components, only different registered trade names.

CPI and CP2 corresponds respectively to the control ENTEROPORC Coli AC alone and PARVORUVAX alone.

Enteroporc COLI (3 x 10 doses = 67.5 ml) was transferred into a 100 ml sterile Schott flask. Of this, 15 ml Enteroporc COLI suspension was discarded and the remaining 52.5 ml of the ENTEROPORC Coli suspension were used for resuspension the lyophilizate of ENTEROPORC AC (25 doses). Mixing was performed by transferring approximately 5 ml of the ENTEROPORC Coli suspension to the vial containing the ENTEROPORC AC lyophilizate. The vial was gently shaken, and the content retransferred to the sterile Schott flask. This process was repeated once more. From this reconstituted vaccine, 25.0 ml were mixed with 25.0 ml PARVORUVAX (ready to use vaccine) to achieve 50.0 ml of IVP. The final vaccine was stored at 2-8°C until vaccination and used within 2 hours after reconstitution.

Animals were vaccinated by intramuscular route at the right and left side of the neck.

All study animals received 2 x 4 ml of the respective treatment on two days. No misdosing occurred during administration. All animals were observed daily for general health by an Animal Technician to positively confirm if an animal was healthy or not.

Rectal body temperature

Rectal body temperature was measured using a calibrated thermometer on the time points indicated in the schedule of events.

Examination of injection sites Immediately before vaccination and on the days specified in the schedule of events injection sites were examined according to standard procedures. The lesion of the insertion of the injection needle and an area of 0.1 cm around this lesion was not considered as a relevant local reaction and therefore was recorded neither as color change nor as swelling. The size (length and width) of swellings and color changes (redness) was measured by a caliper.

The scoring system described in Table 1 was used for redness and sensible heat at the injection site.

Table 1: Injection site scoring system

- Sample collection and storage

Blood samples (approximately 5-8 ml) were taken from jugular vein without addition of anticoagulant. Serum was prepared from the clotted blood samples. The samples were not heat- inactivated. Until use the sera were stored at -15°C to -25°C. The period between blood sampling and storage did not exceed 24 hours.

- ELISA tests

A 96 well microtiter plate (MTP) was coated with monoclonal antibodies which specifically bind the respective antigens. Antibodies were detected by enzyme-linked anti-pig-IgG antibodies and an enzyme dependent color change by measuring the optical density was measured at 450 and 620 nanometers.

1.2 Results

There was a slight increase in rectal temperature at the day of vaccination in the groups vaccinated with Enteroporc COLI AC alone or Enteroporc COLI AC plus Parvoruvax. The increase in rectal temperature did not differ within these two groups (Figure 2 and 3).

The simultaneous use of Enteroporc COLI AC and Parvoruvax resulted in comparable antibodies against Erysipelas and Parvovirosis compared to Parvoruvax alone (Figure 4). The antibody response against E coli antigens was slightly lower in the associated use group compared to the Enteroporc COLI AC group but still above the antibody titers in gilts that had previously been shown to protect their offspring.

In conclusion, no relevant interference of the mixture Enteroporc COLI AC and PARVORUVAC could be observed following 2 vaccinations of the mixture compared to single use in terms of safety and (serological) efficacy.

EXAMPLE 2: safety and efficacy study in sows

The purpose of the second study was to confirm the piglet results in sows. Sows from Group IVP received mixed vaccine Enteroporc Coli AC and Parvoruvax 6 and 3 weeks before insemination and Enteroporc COLI AC alone 2 weeks before farrowing. The mixing of the vaccines was performed just before vaccination. The vaccine composition and analytical methods used in this study are similar to those used in the first example.

Sows from Group CP received Parvoruvax vaccine at 6 and 3 weeks before insemination.

Rectal temperature was measured at days -1, 0, 0+4h, 0+6h, as well as on the four following days of each vaccination. Local and systemic reactions were examined for 14 days after each vaccination.

Serologiocal response was measured at 14 days before 1st vaccination, at day of 2nd vaccination (study day 21), before insemination (study day 44) and 4 days after farrowing (study day 167). In addition, antibody titers in colostrum were determined.

There was a slight increase in rectal temperature at the day of vaccination in the group vaccinated with Enteroporc COLI AC plus Parvoruvax which was however within the acceptable range given for the two vaccines in the respective SPCs (Figure 5).

No local and no systemic reactions were observed for any of the two vaccinated groups. The simultaneous use of Enteroporc COLI AC and Parvoruvax resulted in comparable antibodies against Erysipelas and Parvovirosis compared to Parvoruvax alone. Unexpectedly, the antibody response against E coli and Clostridia antigens in blood and also colostrum was considerably above the protective antibody titers compared to Enteroporc COLI AC alone (Figure 6). Together, these results indicate that the simultaneous use of 9 different antigens of E.coli, Clostridium spp., Parvovirus and Erisipelothirx rhusiopathie is safe and efficacious in gilts and sows and provides piglets with protection through colostrum intake.

Claims

1. A vaccine composition comprising: i) an Escherichia coli fimbrial adhesin antigen, ii) a Clostridium toxoid, iii) a porcine parvovirus antigen, and iv) an Erysipelothrix rhusiopathie antigen.

2. The vaccine composition of claim 1 wherein said Escherichia coli fimbrial 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 of claim 2 wherein said composition comprises Escherichia coli fimbrial adhesins F4ab, F4ac, F5 and F6 antigens.

4. The vaccine composition according to any one of claims 1 to 3 wherein said Clostridium toxoid is selected from the group consisting of: Clostridium difficile, C. perfringens type A or C alpha toxoid, C. perfringens type A beta 2 toxoid, C. perfringens type C beta 1 toxoid, C. perfringens Type A or C Enterotoxin (CPE) and C. perfringens (all strains) theta toxoid (Perfringolysin, PFO).

5. The vaccine composition according to claim 4 wherein said composition comprises Clostridium perfringens type A or C alpha toxoid, Clostridium perfringens type A beta 2 toxoid and Clostridium perfringens type C beta 1 toxoid.

6. The vaccine composition according to any one of claims 1 to 5 wherein said Erysipelothrix rhusiopathiae antigen is an inactivated Erysipelothrix rhusiopathiae .

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

8. The vaccine composition according to any one of claims 1 to 7 comprising an inactivated porcine parvovirus K22 strain.

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

10. The vaccine composition according to any one of preceding claims comprising: i) Escherichia coli fimbrial adhesins F4ab, F4ac, F5 and F6 antigens, ii) Clostridium perfringens type A alpha toxoid, Clostridium perfringens type A beta2 toxoid and Clostridium perfringens type C betal toxoid, iii) inactivated porcine parvovirus, and iv) an inactivated Erysipelothrix rhusiopathie serotype 2 bacteria.

11. The vaccine composition according to any one of claims 1 to 10 for use in the protection against E. coli. Clostridium spp., Porcine parvovirus and E. rhusiopathie infections in a pig, preferably a female pig and its progeny in need thereof.

12. The vaccine composition according to any one of claims 1 to 10 for use in the protection against E. coh. Clostridium spp., Porcine parvovirus and E. rhusiopathie infections in a female pig, preferably a gilt and its progeny in need thereof wherein said composition is administered to the female pig, preferably the gilt at least two times before insemination and at least one time in the time interval between insemination and farrowing.

13. The vaccine composition for use of claim 12 wherein said composition is administered at least two times no more than 10 weeks before insemination, and preferably with an interval of at least two weeks between each of the two administrations before insemination and at least one time no more than 4 weeks before farrowing, preferably at least one time between 10 and 4 weeks, preferably between 8 and 6 weeks before insemination, at least one time between 6 and 1 weeks, preferably between 5 and 2 weeks before insemination and, at least one time between 6 and 1 weeks, preferably between 4 and 2 weeks before farrowing.

14. The vaccine composition for use according to any one of claims 11 to 13 wherein immunologicaly effective amounts of said Escherichia coli fimbrial adhesin antigen,

Clostridium antigen, Porcine parvovirus antigen, and Erysipelothrix rhusiopathie antigen are mixed before administration of the vaccine composition to the female Pig- 15. The vaccine composition for use according to any one of claims 11 to 14 wherein said vaccine composition is administered by intramuscular route, subcutaneous, intradermal or transdermal route, preferably intramuscular route.