An immunogenic or vaccine composition comprising a live attenuated avian metapneumovirus (AMPV) for use in the prevention and / or treatment of an ampv infection

Abstract

An immunogenic or vaccine composition comprising a live attenuated avian metapneumovirus (aMPV) for use in the prevention and / or treatment of an aMPV infection The present invention refers to an immunogenic or vaccine composition comprising a live attenuated avian metapneumovirus (aMPV) for use in the prevention and / or treatment of an aMPV infection, wherein said use comprises administering the composition to laying birds, or for use in active immunization of laying birds.

Description

[0001] DESCRIPTION

[0002] An immunogenic or vaccine composition comprising a live attenuated avian metapneumovirus (aMPV) for use in the prevention and / or treatment of an aMPV infection

[0003] This application claims the benefit of Spanish Patent Application P202430254 filed on 05 April 2024.

[0004] The present invention belongs to the field of infections caused by avian metapneumovirus (aMPV). More particularly, the present invention relates to immunogenic and vaccine compositions comprising a live attenuated aMPV for use in the prevention and / or treatment of said infections in laying birds.

[0005] BACKGROUND ART

[0006] At the end of the 1970s, a new and acute respiratory affection appeared in turkeys in South Africa, showing nasal and ocular discharge besides slight sinusitis on the infraorbital sinuses, described as Turkey Rhinotracheitis. During the same decade, an unknown respiratory disease was reported in chickens in South Africa, causing respiratory symptoms and facial edema. It was called SHS, which stands for Swollen Head Syndrome.

[0007] When the disease appeared in Europe, it was suggested that the virus belonged to the Paramyxoviridae family according to different properties. Still, since it did not haemagglutinate red blood cells, the virus was classified in the subfamily of Pneumovirinae, which lacks haemagglutinin and neuraminidase glycoproteins. Because that virus was the first and the only pneumovirus described to affect birds, it used to be identified as avian pneumovirus.

[0008] The avian pneumovirus or avian metapneumovirus (aMPV) infection is characterized by respiratory and reproductive disorders. Avian metapneumovirus (aMPV) principally causes an acute highly contagious upper respiratory tract infection combined with reproductive disorders, primarily of turkeys, chickens and ducks. aMPV is a single-stranded, non-segmented, negative-sense RNA virus belonging to the family Pneumoviridae, and the genus Metapneumovirus. The virus is pleomorphic (80 to 200 nm) or spherical. The viral genome in a nucleocapsid is about 14 kilo-bases with a helical symmetry. Moreover, the aMPV characteristics differ from mammalian pneumoviruses at the molecular level. As aMPV has no hemagglutination or neuraminidase activity in G attachment protein, it could be distinguished from other paramyxoviruses. Avian metapneumovirus has 4 subtypes A, B, C and D, according to the nucleotide and amino acids sequences of the G attachment gene, and the serological properties. All 4 subtypes are circulating in many countries all over the world. Subtypes A and B of aMPV were first recognized in the 1980s and they are prevalent, especially in Europe. Subtype C of the virus was demonstrated in turkey flocks of the USA and wild avian populations. Moreover, subtype C was detected in breeder flocks of ducks in France and China. In Europe, a second distinctive lineage of subtype C has been found. In the USA, though strains of aMPV are classified as subgroup C, a novel subgroup showed a higher genetic similarity to human metapneumovirus than avian types. Later, subtype C was distributed in France and South Korea in avian species. Also, in France and other countries, subtype D of aMPV was isolated from turkeys. Subtypes A, B, and D of the virus are nearly similar. Recent sequence data suggest that additional subgroups may exist in gulls and parakeets.

[0009] Avian metapneumovirus (aMPV) is one of the most relevant respiratory pathogens responsible for causing infections of the upper respiratory tract and may induce respiratory disorders. aMPV is capable of affecting the reproductive system of birds, directly impacting shell quality and decreasing egg production. Consequently, aMVP infections cause severe economic losses in poultry farming systems and disorders related to animal welfare and zootechnical losses.

[0010] Meat and egg-type birds are susceptible to aMPV infection. Turkeys and chickens are the natural hosts of aMPV subtypes A and B. However, all aMPV subtypes are adapted to Galliformes, particularly turkeys. Subtype C of duck origin is well adapted to ducks. Despite the tropism of subtype C for ducks, chickens could be infected by this subtype. Turkeys are susceptible and transmit all aMPV subtypes except for subtype C of the duck lineage. Antibodies to aMPV have also been detected in non-vaccinated birds, apparently healthy Pekin and Muscovy duck. Subtype C has also been isolated in wild waterfowl. The virus was also isolated in wild geese. In the USA, aMPV has been detected in pheasants and Guinea fowls developing swollen head signs. Ostriches have also been found to be seropositive for aMPV.

[0011] All age groups can get infected with aMPV. Broilers are more susceptible to the virus infection than layers and breeders. Turkeys are susceptible for TRT infection at 3 to 12 weeks of age. The aMPV tends to affect birds older than 26 days of age.

[0012] Infection with aMPV is most likely horizontal (airborne) via aerosol or dust particles. The virus is highly infectious and characterized by a rapid spread. Transmission of aMPV occurs through direct contact between susceptible birds as well as through aerosolized respiratory secretions, and infected, contaminated objects are the main routes of aMPV transmission. Wild and migratory free-living birds are another important means of aMPV transmission. Therefore, free living birds may act as virus reservoirs for commercial poultry flocks.

[0013] The disease can cause significant economic losses for the poultry industry, that are exacerbated by secondary pathogens. The disease has global distribution in poultry-producing regions, with only Oceania and Canada reported to be free of aMPV infection. Infection with aMPV can occur from a very young age and is characterised by clinical signs such as snicking, rales, sneezing, nasal discharge, foaming conjunctivitis, swelling of the infraorbital sinuses and submandibular oedema. Secondary adventitious agents can dramatically exacerbate such clinical signs.

[0014] Regarding the pathogenesis of aMPV, it is affected by several factors. The virus primarily targets the upper respiratory tract epithelium, leading to inflammation and damage to the respiratory tract. Virus replication induces ciliostasis and possibly loss of cilia, which favours the respiratory disease caused by aMPV, allowing secondary pathogens to invade the host. After the respiratory infection of the bird with aMPV, the virus will likely spread in layers and breeders from the respiratory tract to the reproductive organs. Accordingly, besides the respiratory manifestations, aMPV-affected adult layers may exhibit a 10-40% decrease in egg production and changes (abnormalities) in egg quality (including eggshell). Although SHS was primarily reported in broiler chickens, it is also present in laying hens that in addition to respiratory clinical signs associated with an aMPV infection involve about a 10-40% drop in egg production as well as abnormalities in egg quality.

[0015] Infected birds show a respiratory disease with severe respiratory clinical signs, particularly showing unilateral and / or bilateral swelling of infraorbital sinuses facial edema, and accumulation of discharge in nostrils, sinuses, and eyes. Snicking, sneezing, periorbital and submandibular edema, and watery frothy to mucopurulent nasal and ocular discharges are the upper respiratory clinical signs that are mostly observed, especially in young birds. Coughing, gasping, dyspnea, and rales can also be detected in the later stages, especially with complications. Regarding general clinical signs, they include depression, anorexia, ruffled feathers, and reduced growth. In the case of chickens with SHS, they may show cerebral disorientation, torticollis and opisthotonos as main nervous clinical signs.

[0016] Infection with aMPV is generally characterized by high morbidity (40-100%) and low mortality (1-5%) rates depending on the presence of many factors, including the age of birds, secondary infections, and constitution of the affected flock. In severe cases, aMPV infection can progress to secondary bacterial infections, exacerbating the respiratory clinical signs and leading to increased mortality rates. In cases with concomitant secondary bacterial infection, the mortality rate reaches about 90%. Egg production is frequently affected. Serological evidence suggests aMPV is widespread throughout the world and of considerable economic importance.

[0017] In view of all the above, preventing and controlling aMPV infections in poultry requires a multifaceted approach, including biosecurity measures, vaccination, management practices and surveillance monitoring.

[0018] Regarding prevention and control, vaccines strategies are crucial. Currently, two types of vaccine are implemented, i.e., live attenuated vaccines, and inactivated vaccines. Additionally, live recombinant vaccines based on a fowlpox vector expressing the F protein of aMPV have been investigated in turkeys. Furthermore, DNA vaccines with bacterial vectors encoding various aMPV proteins have also been tested; more recently, genetically attenuated aMPV vaccines produced by reverse genetics have been disclosed.

[0019] Inactivated vaccines usually contain aMPV subtypes A and B, only healthy birds, known to be sensitised by previous exposure to aMPV, should be vaccinated. Typically, intramuscular vaccination of breeder turkeys with aMPV inactivated vaccine is performed at 30 weeks of age after priming with a live attenuated vaccine at 7 days of age. However, the usual vaccination program in turkey's vaccination includes the administration of an inactivated aMPV vaccine 4-6 weeks of age following the last vaccination with a live vaccine up to 28 weeks of age but avoiding the last 4 weeks before the laying period. The usual protocol vaccinations for broiler breeders and layers consist of administering a live aMPV vaccine as first dose followed by a second dose with an inactivated aMPV vaccine.

[0020] Available live attenuated vaccines generally comprise aMPV subtype A or subtype B isolated in Europe, also from subtype C where the aMPV was isolated in the USA. Live attenuated aMPV vaccines are intended for use in young birds, not during laying period, to induce an active immune response that will help to prevent the respiratory disease caused by aMPV. Additionally, live attenuated aMPV vaccines are also used in turkeys to produce a primary response and always administered prior to vaccination near to point-of-lay based exclusively on inactivated vaccines.

[0021] Live attenuated aMPV vaccines are usually applied several times by coarse spray, in the drinking water, or by oculonasal administration. Different vaccination protocols have been disclosed for aMPV vaccines. A first protocol is based on the administration of a first dose of a live attenuated vaccine from one day of age until seven days of age. A second administration protocol is based on a first dose of a live attenuated vaccine around 3 to 6 weeks of age or after 6 weeks of age. Repeated administrations are also disclosed. The rationale for doing repeated vaccinations is linked to several issues, first because of the difficulties of inducing a prolonged antibody response lasting for the whole life of the vaccinated bird. Second, due to the need of avoiding vaccination in young animals when they are vaccinated against haemorrhagic enteritis (HEV) which usually occurs at around 28 days of age, to avoid interference with maternally derived antibodies (MDAs) to HEV (WOAH Terrestrial Manual 2022, Chapter 3.3.15, Turkey Rhinotracheitis (Avian Metapneumovirus Infections)). Interference with anti-aMPV MDAs has also been reported.

[0022] To date, the use of live attenuated vaccines involves the risk of causing mild to severe clinical signs of the disease they are designed to protect against because the viral strain preserves some degree of virulence and / or can revert to virulence. In the case of live attenuated aMPV vaccines, the main concern is that vaccine-induced infection could lead to a reduction in egg production and affect egg quality, since it is widely known that an aMPV infection is capable of affecting the reproductive system of birds, directly resulting in a decreased egg production and an increase of abnormal eggs (Sugiyama et al. 2006). Additionally, live vaccines may potentially spread to unvaccinated birds, posing a risk of disease transmission within the flock.

[0023] In view of the safety concerns mentioned above, live attenuated aMPV vaccines are not used in laying birds, or in birds close to the laying period, or more importantly, within the laying period. Instead, inactivated aMPV vaccines are currently used in particular in animals that have been previously primed by a live attenuated vaccine or by natural exposure to the field virus during rearing. The usual strategy is to administer the inactivated vaccine at least 4-6 weeks after the administration of a live attenuated vaccine, up to 28 weeks of age, avoiding the 4 last weeks before the laying period. The preferred routes for the inactivated vaccines are intramuscular in the leg muscle, avoiding proximity to joints, tendons or major blood vessels or the subcutaneous route. Used in this way the inactivated vaccine should produce a good antibody response that will protect birds against respiratory and reproductive clinical signs during the period of lay.

[0024] On the one hand, inactivated vaccines, while being safer than live attenuated vaccines, present a big handling handicap due to their administration method, which typically involves individual injection (intramuscular) contrary to the spray or drinking water administration of live attenuated vaccines. The process of injection can cause stress to the birds and may result in temporary discomfort, muscle damage, or injection-site reactions. Handling stress and associated health issues can potentially impact egg production, egg quality, and overall welfare of laying birds. Moreover, the administration of inactivated vaccines requires skilled personnel and specialized equipment to perform the correct intramuscular injection, adding to the operational complexity and cost of vaccination programs. On the other hand, the live attenuated vaccines while administered by more convenient routes (coarse spray, in the drinking water, or by oculonasal) have safety use concerns, avoiding its particular use in laying birds and during the laying period.

[0025] In view of all the problems, it is apparent from the above that there is a need in the art to provide safer and effective vaccines and improved vaccination strategies that can be used to enhance immunity of laying birds against aMPV without any adverse effects, particularly concerning egg performance, particularly during the laying period.

[0026] SUMMARY OF THE INVENTION

[0027] A first aspect of the present invention is directed to an immunogenic or vaccine composition comprising a live attenuated avian metapneumovirus (aMPV) for use in the prevention and / or treatment of an aMPV infection, wherein said use comprises administering the composition to laying birds. This aspect may be reformulated as an immunogenic or vaccine composition comprising a live attenuated aMPV for use in the preparation of a medicament for the prevention and / or treatment of an aMPV infection, wherein said use comprises administering the composition to laying birds. The invention also contemplates a method for the prevention and / or treatment of an aMPV infection, said method comprising administering an immunogenic or vaccine composition comprising a live attenuated aMPV to laying birds.

[0028] The inventors have surprisingly found that administering an immunogenic or vaccine composition comprising a live attenuated aMPV to laying birds provides effective immunization against an aMPV infection without adverse effects. In particular, as shown in the examples below, it has been demonstrated that laying birds vaccinated with the compositions of the present invention do not show any drop in performance regarding egg production and egg quality. Also shown in the examples below is the efficacy of the compositions of the present invention for preventing and / or treating an aMPV infection. The immunogenic or vaccine composition comprising a live attenuated aMPV has further demonstrated to elicit a robust immune response against an aMPV infection from at least 3 weeks after administration and until at least 9 weeks after administration. The composition of the present invention comprising a live attenuated aMPV can be an immunogenic or a vaccine composition.

[0029] The lack of side effects of the immunogenic and vaccine compositions comprising the live attenuated aMPV when administered during the laying period enables effective immunisation by oculonasal or oral routes during this particular period. The present invention thus provides significant advantages for poultry farming systems in terms of ease of handling, animal welfare and general benefits in egg production and egg quality. Moreover, as shown in the examples below, the efficacy of the immunogenic or vaccine composition of the invention in one-day-old chickens, which represents the worst-case scenario, is not influenced by maternally derived antibodies (MDA).

[0030] In a second aspect, the invention is directed to an immunogenic or vaccine composition comprising a live attenuated aMPV for use in active immunization of laying birds against aMPV. This can be reworded as an immunogenic or vaccine composition comprising a live attenuated aMPV for use in the preparation of a medicament for active immunization of laying birds against aMPV. The invention also contemplates a method for the active immunization of laying birds against aMPV, said method comprising administering an immunogenic or vaccine composition comprising a live attenuated aMPV to the laying birds. This second aspect is also directed to an immunogenic or vaccine composition comprising a live attenuated aMPV for use in active immunization of laying birds against an aMPV infection. This can be reworded as an immunogenic or vaccine composition comprising a live attenuated aMPV for use in the preparation of a medicament for active immunization of laying birds against an aMPV infection. The invention also contemplates a method for the active immunization of laying birds against an aMPV infection, said method comprising administering an immunogenic or vaccine composition comprising a live attenuated aMPV to the laying birds

[0031] A third aspect of the invention refers to an immunogenic or vaccine composition comprising a live attenuated aMPV for use in the prevention and / or treatment of an aMPV infection, or for use in active immunization of laying birds, in combination therapy with a further avian vaccine, wherein said use comprises administering the composition comprising a live attenuated aMPV to laying birds. The further avian vaccine may be a different aMPV vaccine or a vaccine against a totally different avian pathogen. When the further vaccine is a vaccine against a different avian pathogen (besides aMPV), the combination therapy may be a therapy for treating both, the infection caused by an aMPV and the infection caused by the other avian pathogen.

[0032] A fourth aspect of the present invention refers to a vaccination kit comprising an immunogenic or vaccine composition comprising a live attenuated aMPV for use in the prevention and / or treatment of an aMPV infection, wherein said use comprises administering the composition to laying birds. A fifth aspect refers to a vaccination kit comprising an immunogenic or vaccine composition comprising a live attenuated aMPV for use in active immunization of laying birds against aMPV. This fifth aspect also refers to a vaccination kit comprising an immunogenic or vaccine composition comprising a live attenuated aMPV for use in active immunization of laying birds against an aMPV infection.

[0033] A sixth aspect of the invention refers to an immunogenic or vaccine composition comprising a live attenuated aMPV and further comprising pharmaceutically or veterinary acceptable excipients and / or carriers. Further aspects of the invention refer to vaccination kits comprising the immunogenic or vaccine composition of the sixth aspect, as well as said composition and vaccination kits for use in the prevention and / or treatment of an aMPV infection or for use in active immunization of laying birds, in particular wherein said use comprises administering the immunogenic or vaccine composition of the sixth aspect or vaccination kits containing the same to laying birds.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : Number of tracheal rings with abnormal ciliary activity per chick represented on the ordinates for the negative MDA groups (Groups A and C) represented on the abscissas, between days 10 and 11 postchallenge. Each circle represents a different animal. Group A: animals negative to MDA that received a live attenuated aMPV vaccine. Group C: animals negative to MDA mock vaccinated. The dotted line represents the threshold to consider animals affected with respiratory clinical signs (> 1 ring with abnormal ciliary activity).

[0035] Figure 2: Number of tracheal rings with abnormal ciliary activity per chick represented on the ordinates for the positive MDA groups (Groups B and D) represented on the abscissas, between days 10 and 11 postchallenge. Group B: animals positive to MDA that received a live attenuated aMPV vaccine. Group D: animals positive to MDA mock vaccinated. The dotted line represents the threshold to consider animals affected with respiratory clinical signs (> 1 ring with abnormal ciliary activity).

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] Definitions

[0038] In the present description, as well as in the claims, the singular forms "a", "an" and "the" include the plural reference unless the context clearly indicates otherwise. The ranges defined by the terms "between ... and ..." or by the terms "from ...to...” include also the two ends thereof. The term "about" refers to a deviation of plus / minus 10 %, preferably plus / minus 5 %. Further, unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series.

[0039] As used herein, the conjunctive term "and / or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and / or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or."

[0040] In present invention, the term "immunogenic composition” refers to a preparation or composition that is administered to stimulate the subject's immune response against a specific infectious disease, in particular, a composition for the prevention and / or treatment of an aMPV infection. The "immunogenic composition” is meant to elicit an immunological response in the subject of a cellular or antibody-mediated type. The immunological response may be protective.

[0041] In the present invention, the term "vaccine” or "vaccine composition”, refers to a composition that establishes or improves immunity to a particular disease by inducing an adaptive immune response including an immunological memory response. A vaccine typically contains an antigen that resembles a disease-causing microorganism or a part thereof (e.g. a polypeptide). Vaccines can be prophylactic or therapeutic. The term "vaccine composition”, as also used herein, refers to an immunogenic composition of the invention complemented by pharmaceutically acceptable excipients and / or carriers, that when administered to a subject, elicits, or is able to elicit directly or indirectly, an immune response in the subject. Particularly, the vaccines of the present invention elicit an immunological response in the subject of a cellular or antibody- mediated type upon administration to the subject, more particularly the immunological response is protective. In particular, the vaccine is for the prevention and / or treatment of an aMPV infection. The vaccine may be a "combination vaccine”. The term "combination vaccine” means that the vaccine contains various antigens in a single preparation. The vaccine composition of the invention can be also administered in combination with other vaccine compositions. More particularly, the vaccine compositions of the present invention can further comprise at least one antigen against another avian pathogen selected from the group consisting of Infectious Bronchitis Virus (IBV), Egg Drop Syndrome virus (EDS virus), Newcastle disease virus (NDV), coccidia, Eimeria spp., infectious bursal disease virus (IBDV), and combinations thereof.

[0042] The term "immunological response” or "immune response”, as used herein, unless otherwise indicated, means an immune reaction to an antigen or composition that develops in the vaccinated subject of an innate, humoral and / or a cellular immune response to an antigen present in the composition of interest. Thus, the response elicits a rise in corresponding antibody titers and / or an activation of cell-mediated immunity. Preferably, the immune reaction that is elicited provides protective immunity that prevents and / or reduces an aMPV infection and / or of a disease caused by an aMPV, and / or prevents and / or reduces clinical signs associated to an aMPV infection.

[0043] The term "active immunization of laying birds” as used herein refers to induction of an immune response in laying hens after exposure to the immunogenic or vaccine composition comprising a live attenuated aMPV and, in particular, an immune response that prevents and / or reduces an aMPV infection and / or of a disease caused by aMPV, and / or prevents and / or reduces clinical signs associated to an aMPV infection. The term "active immunization of laying birds” also refers to induction of an immune response in laying hens after exposure to the immunogenic or vaccine composition comprising a live attenuated aMPV and, in particular, an immune response that prevents and / or reduces an aMPV infection and / or of a disease caused by aMPV, and / or prevents and / or reduces clinical signs associated to an aMPV infection during the laying period. Active immunization contrasts with passive immunization, where in the later the antibodies are transferred from one subject to another subject, providing immediate but temporary protection. In contrast, active immunization means that the subject's immune system is actively engaged in generating an immune response to confer immunity after receiving the immunogenic or vaccine composition of the present invention. In the sense of the present invention, the active immunization of the laying birds against an aMPV infection with the immunogenic and vaccine composition comprising a live attenuated aMPV is during the laying period.

[0044] The term "effective immunizing dose" or "immunologically effective amount”, as used herein, unless otherwise indicated, means an amount that induces an immune response in vaccinated subjects. In particular, it means the amount of the live attenuated aMPV or antigen contained therein sufficient to elicit an immune response in vaccinated subjects with the immunogenic or vaccine compositions of the present invention, and further provides immunological protection to the birds that is greater than the inherent immunity of non-immunized birds and / or non-treated control group of the same birds. As used herein, the terms "immunize" or "immunisation” and "vaccinate" or "vaccination” are synonymous and are used interchangeably. The effective immunizing dose may be administered either in a single dose or as part of a series, and it is effective for treatment and / or prevention of an aMPV infection and / or the diseases or disorders caused by said infection, including the clinical signs associated to an aMPV infection. The effective immunizing dose varies depending upon the health and physical condition of the subject to be treated; age; the capacity of the subject's immune system to produce antibodies from any of the immunological classes, such as immunoglobulins A, D, E, G or M, the proliferation of B and T lymphocytes, the provision of activation, growth and differentiation signals to immunological cells, expansion of helper T cells, suppressor T cells, and / or cytotoxic T cell and / or y, 5-T cell populations; the degree of protection desired; the formulation of the vaccine; the veterinarian's assessment of the medical situation, and other relevant factors.

[0045] The expression "pharmaceutically acceptable excipients and / or carriers" refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition, including the immunogenic and vaccine compositions of the present invention. It must also be suitable for use in contact with the tissue or organ of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit / risk ratio. In the sense of the present invention pharmaceutically acceptable encompasses veterinary acceptable excipients and / or carriers and / or adjuvants.

[0046] By "antigen" or "immunogen" is meant a substance that induces a specific immune response in a subject. The antigen or the immunogen may comprise a whole organism, a killed organism, a live or attenuated organism; a subunit or partial fragment of an organism; a recombinant vector containing an insert with immunogenic properties; a fragment of DNA capable of inducing an immune response upon presentation to a subject; a polypeptide, a protein or a fragment thereof, an epitope, or any combination thereof. In particular, in the sense of the present invention, the "antigen” or "immunogen” induces an immune response against an aMPV infection in the subject in need thereof. More particularly, the "antigen” or "immunogen” induces an immune response against an aMPV infection in laying birds. In particular, in the sense of the present invention, the "antigen” or "immunogen” is a live attenuated aMPV.

[0047] The term "live attenuated" strain and "live attenuated” virus as used herein, unless otherwise indicated, means a virus or a strain, particularly an aMPV virus or an aMPV strain, that is altered from an original wildtype virus or strain in such a way that its ability to infect a host, replicate within a host, be packaged, infect the host again, or a combination thereof, is reduced. In the sense of the present invention "live attenuated strain” and "live attenuated virus” are used interchangeably. The live attenuated virus or strain has been selected for its reduced pathogenicity in the host. Attenuation of virulent virus or strains of aMPV can be achieved by a number of means known by the skilled person, such as attenuation by repeated passage through a foreign host, for example passage through chicken embryos to produce embryo-adapted lines. Other non-limiting means for attenuation of virus are chemical mutagenesis and irradiation methods. The virus or strain being attenuated is called the "parental virus”, "parent virus”, "parent strain” or "parental strain”.

[0048] The term "subject" as used herein refers to domesticated animals, livestock and avian or bird species. The term "avian", or "bird" is understood to encompass all avian species prone or susceptible to an aMPV infection. This term extends to include domesticated species that are kept and raised specifically for egg and meat production. In one embodiment, the terms "avian" or "bird" extends to commercially important or farmed bird species, such as poultry.

[0049] The term "domesticated bird(s)", as used herein, unless otherwise indicated, includes chickens, turkeys, ducks, game birds (including, but not limited to, quail, pheasants, guinea fowl, and geese) and ratites (including, but not limited to, ostrich). Preferably, the domesticated birds are poultry. By the term "poultry” it is understood as any domesticated bird raised commercially or domestically for production of meat, eggs, and feathers. Poultry includes chicken, turkey, goose, duck, Rock Cornish hens, pheasant, squab and guinea fowl.

[0050] The term "laying birds” refers to female domestic birds which have reached laying maturity and are kept for the production of eggs, particularly eggs for human consumption. The term "laying hens” or "laying chickens” is meant for adult female chickens (Gallus domesticus), that are primarily kept for production of eggs. Laying hens lay eggs more often than other breeds of chicken, such as broiler chickens intended to produce meat. In order for layers to produce an above average amount of eggs, they need to have a high level of nutrition to boost egg production and ensure that they are large, heavy, tasty and of good quality. Between 18 to 19 weeks of age, layers begin to produce approximately one egg every 22 hours, and they reach the peak of egg production around 30 weeks of age. Once the layers reach 72 to 78 weeks of age, they start to lay less, and are they are usually rotated out. In the sense of the present invention, the term "laying birds” may be interchangeably used with the expression "birds during the laying period”. In the sense of the present invention, the term "laying birds”, "laying hens”, or "laying chickens” refers to laying birds of at least 16 weeks of age, particularly to laying birds between 16 weeks of age and 70 weeks of age, and more particularly to laying birds between 16 weeks of age and 60 weeks of age.

[0051] The term "treatment”, as used herein, unless otherwise indicated, refers to any type of therapy, including combination therapy, with the immunogenic or vaccine composition for the use of the invention, which is aimed at terminating, preventing, ameliorating or reducing the susceptibility to an infection or clinical condition or disease caused thereby, including complete clearing of the infection, reducing the infection, curing of the disease or condition, as well as ameliorating or alleviating said disease or condition and reducing the clinical signs associated to said disease or condition. In a preferred embodiment, the term treatment relates to prophylactic treatment. Thus, "treatment,” "treating,” and their equivalent terms refer to obtaining a desired pharmacologic or physiologic effect, covering any treatment of a pathological condition or disorder in a subject, including domesticated birds. The effect may be prophylactic in terms of completely or partially preventing an infection or disorder or clinical sign caused by said infection and / or may be therapeutic in terms of a partial or complete clearing of the infection or curing a disorder and / or adverse effect attributable to the disorder caused by said infection. That is, "treatment” includes (1) preventing the infection or disorder from occurring or recurring in a subject, (2) inhibiting the infection or disorder, such as arresting its development, (3) stopping or terminating the infection or disorder or, at least, clinical signs associated therewith, so that the subject no longer suffers from the infection or disorder or their clinical signs, such as causing regression of the infection or disorder or its clinical signs, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the disorder, or clinical signs associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a clinical sign parameter, reduction of the pathogen load, the pathogen shedding, or reduction in pathogen transmission. In the sense of the present invention, the infection, disease, condition or disorder is associated to an aMPV infection. The invention also includes other disorders, conditions, diseases and infections associated to a further avian pathogen, wherein the further avian pathogen is selected from the group consisting of Infectious Bronchitis Virus (IBV), Egg Drop Syndrome virus (EDS virus), Newcastle disease virus (NDV), coccidia, Eimeria spp., infectious bursal disease virus (IBDV), and combinations thereof. The term "prevention” or "reduction" or "preventing" or "reducing", respectively, as used herein, means, but is not limited to, a process of prophylaxis in which a subject is exposed to the immunogenic or vaccine composition for the use of the present invention prior to the induction or onset of the infection or disease process, and wherein said immunogenic or vaccine composition, when administered to said subject, elicits or is able to elicit an immune response in said subject against an aMPV infection. Altogether, such prophylaxis results without limitation, in preventing, decreasing, reducing or ameliorating the risk or the severity of the clinical signs caused by an aMPV infection. More specifically, the term "prevention" or "preventing", as used herein, means generally a process of prophylaxis in which a subject is exposed to the immunogenic or vaccine composition for the use of the present invention prior to the induction or onset of a disease caused by aMPV, such as avian rhinotracheitis and swollen head syndrome.

[0052] Herein, "reduction of clinical signs associated with an aMPV infection" means, but is not limited to, reducing the number of infected subjects in a group, reducing or eliminating the number of subjects exhibiting clinical signs of an aMPV infection, or reducing the severity of any clinical signs that are present in the subjects, in comparison either to wild-type infected subjects or to experimentally infected non-immunized, i.e., nontreated, subjects. Particularly, the immunogenic or vaccine compositions for use according to the present invention, reduces the detrimental effect caused by an aMPV infection on the respiratory tract. More particularly, the detrimental effect caused by an aMPV infection is on the ciliary activity of affected birds. Also, in the sense of the present invention, the immunogenic or vaccine compositions for use according to the present invention prevents and / or reduces at least one clinical sign associated to an aMPV infection. Said clinical signs may be general clinical signs, respiratory clinical signs and reproductive clinical signs. The term "general clinical signs” comprises at least one clinical sign selected from the group consisting of depression, anorexia, ruffled feathers, head shaking, head scratching, nervous signs, apathy, cerebral disorientation, torticollis, opisthotonos, reduced growth and mortality. The term "respiratory clinical signs” comprises at least one clinical sign selected from the group consisting of dyspnea, ocular discharge, nasal discharge, swollen infra-orbital sinuses, respiratory distress and conjunctivitis. The term "reproductive clinical signs” comprises at least one clinical sign selected from the group consisting of drop in egg production, drop in egg quality, egg abnormalities such as egg size abnormalities, egg shell abnormalities (thin shell eggs, soft shell eggs, shell-less eggs, rough or corrugated shell eggs, small eggs, abnormal calcareous deposits, eggs with dimples, depressions or ridging, blood-stained eggs, and misshapen eggs), egg yolk abnormalities (blood spots, pale yolk, mottled yolk, discolored yolk (blue-green), rubbery yolk, cheesy yolk, and pasty yolk), egg albumen abnormalities (thin albumen, watery albumen, and off-color albumen (green or pink)). Preferably these clinical signs are reduced in subjects receiving the immunogenic or vaccine composition according to the present invention by at least 10 percent (%) in comparison to subjects not receiving the composition and may become infected. More preferably, clinical signs are reduced in subjects receiving the immunogenic or vaccine composition according to the present invention by at least 20 percent (%), preferably by at least 30 percent (%), more preferably by at least 40 percent (%),even more preferably by at least 50 percent (%), even more preferably by at least 60 percent (%), even more preferably by at least 70 percent (%), even more preferably by at least 80 percent (%), even more preferably by at least 90 percent (%), and even more preferably by at least 100 percent (%) in comparison to subjects not receiving the composition and may become infected.

[0053] The term "adjuvant” is herein understood as a substance used to enhance the immune response. The word adjuvant is derived from Latin: adjuve, meaning "to help." Many classes of compounds have been described as adjuvants including mineral salts, microbial products, emulsions, saponins, cytokines, polymers, microparticles, and liposomes. A variety of compounds with adjuvant properties currently exist, and they exert their functions through different mechanisms of action. Based on their mechanisms of action, the adjuvants can be divided into delivery systems and immunostimulants (immune potentiator). Delivery system adjuvants can function as carriers to which antigens can be associated, also create local proinflammatory response that recruit innate immune response cells to the site of the injection. The role of the immunostimulant is to activate innate response through pattern-recognition receptors (PRRs) or directly (i.e. cytokines). In general, activation of PRRs by their agonists induces "Antigen Presenting Cells" (APC) activation / maturation and cytokine / chemokine production that ultimately leads to adaptive immune responses. Thus, "Immunostimulant" as used herein is a compound that stimulates the immune system by inducing activation or increasing activity of any of its components. The stimulation derives from the direct or indirect stimulatory effect of the immunostimulant upon the cells of the immune system itself. Immunostimulants may activate the immune response through pattern-recognition receptors (PRRs) or directly. Immunostimulants can be natural or synthetic compounds. Immunostimulants may be given by themselves to activate nonspecific defence mechanisms, or they may be administered with a vaccine composition to activate nonspecific defence mechanisms as well as heightening a specific immune response. Immunostimulants can be combined with antigens and other adjuvants.

[0054] The composition

[0055] The present invention generally refers, in the first aspect, to an immunogenic or vaccine composition comprising a live attenuated avian metapneumovirus (aMPV) for use in the prevention and / or treatment of an aMPV infection, wherein said use comprises administering the composition to laying birds. In a second aspect, the invention provides an immunogenic or vaccine composition comprising a live attenuated aMPV for use in active immunization of laying birds against aMPV. The composition may be regarded as an immunogenic or vaccine composition and therefore, the present invention refers to an immunogenic or vaccine composition comprising a live attenuated aMPV. In one embodiment the composition for use according to the first or second aspects is an immunogenic composition. In another embodiment the composition for use according to the first or second aspects is a vaccine composition. All embodiments disclosed below may be applied to both, the immunogenic and the vaccine compositions.

[0056] As outline above, current vaccination protocols, in particular related to vaccines based on live attenuated viruses, specifically exclude the laying period when administering the vaccine. Recommended vaccination schedules usually also avoid administering the vaccines during the weeks before laying period. Therefore, up to date, laying hens are not to be vaccinated. This is related to concerns about vaccine-induced infection leading to a reduction in egg production and egg quality, even if otherwise producing no more than mild clinical signs. However, avoiding the laying period may lead to inefficient immunization and, consequently, aMPV infection during this important production period. The present invention solves this problem by providing an immunogenic or vaccine composition comprising a live attenuated aMPV with an administration regime that may include the laying period which has demonstrated effective immunisation of the vaccinated birds and lack of side effects, in particular, related to egg production and egg quality.

[0057] As previously described, attenuation of viral strains can be achieved by a number of means which are well known to the skilled person, including serial passaging the parent strain in a foreign host. In one embodiment, the live attenuated aMPV has been obtained by an attenuation process that comprises at least 1 passage, at least 2 passages, at least 3 passages, at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages, at least 11 passages, at least 12 passages, at least 13 passages, at least 14 passages, at least 15 passages, at least 16 passages, at least 17 passages, at least 18 passages, at least 19 passages, at least 20 passages, at least 21 passages, at least 22 passages, at least 23 passages, at least 24 passages, or at least 25 passages through foreign host cells. In a particular embodiment, serial passages are carried out in VERO cells (African green monkey kidney cells), in DF-1 cells (chicken embryo fibroblast cells), in primary chicken embryo fibroblast cells, in primary turkey embryo fibroblast cells, in tracheal organ cultures from turkeys or chickens, and / or any combination thereof. In a particular embodiment, at least serial passages are carried out in embryo cell lines and / or in tracheal organ cultures form chickens or turkeys. In a particular embodiment, at least serial passages are carried out in VERO cells. In more particular embodiments, at least 3 or at least 5 serial passages are carried out in embryo cell lines. In more particular embodiments, at least 3 or at least 5 serial passages are carried out in tracheal organ cultures. In another particular embodiment, at least 5 or at least 8 passages are carried out in VERO cell lines.

[0058] In a more particular embodiment, the aMPV has been attenuated by (I) serial passages in embryonating eggs, tracheal organ cultures or in chicken embryo fibroblasts, or by alternate passages using a combination thereof; and (II) subsequent serial passages in VERO cells. In a more particular embodiment, the aMPV has been attenuated by (I) alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblasts; followed by (II) serial passages in VERO cells. In this particular embodiment, the aMPV has been attenuated by (I) at least 1 alternate passage in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 1 serial passage in Vero cells. In another embodiment, the aMPV has been attenuated by (I) at least 2 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 2 serial passages in Vero cells. In another embodiment, the aMPV has been attenuated by (I) at least 3 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 3 serial passages in Vero cells. In another embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 5 serial passages in Vero cells. In another embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 8 serial passages in Vero cells. In a further embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 10 serial passages in Vero cells. In a further embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 15 serial passages in Vero cells. In a further embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 18 serial passages in Vero cells. In a further embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 19 serial passages in Vero cells. In a further embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 20 serial passages in Vero cells. In a further embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 21 serial passages in Vero cells. In a further embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 22 serial passages in Vero cells. In a further embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 23 serial passages in Vero cells. In a further embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 24 serial passages in Vero cells. In a further embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblast; followed by (II) at least 25 serial passages in Vero cells. In a more particular embodiment, the aMPV has been attenuated by (I) at least 5 alternate passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblasts; followed by (II) at least 18 serial passages in VERO cells. In this particular embodiment, the alternate passages in step (I) may comprise any combination of passages in embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblasts. In this particular embodiment, the step (i) is selected from 1 passage in embryo tracheal organ tissues of turkeys and 4 serial passages in SPF chicken embryo fibroblasts, or vice versa; 2 serial passages in embryo tracheal organ tissues of turkeys and 3 serial passages in SPF chicken embryo fibroblasts, or vice versa; or alternating 1 passage in embryo tracheal organ tissues of turkeys and 1 passage in SPF chicken embryo fibroblasts (up to 5 passages altogether). In another particular embodiment, the obtained aMPV attenuated according to (i) is further attenuated by serial passages in VERO cells. More particularly, at least 18 serial passages in VERO cells are carried out.

[0059] In some embodiments, the aMPV is selected from the group consisting of aMPV subtype A, aMPV subtype B, aMPV subtype C, aMPV subtype D, and combinations thereof. In a particular embodiment, the immunogenic or vaccine composition comprises a live attenuated aMPV subtype B. In a more particular embodiment, the composition comprises a live attenuated aMPV subtype B strain JFB24. aMPV subtype B strain JFB24, originates from a wild-type strain isolated from a poultry farm located in Spain in 1988. The wild-type isolate was attenuated by serial passages in Vero cells resulting in the live attenuated aMPV strain JFB24, which was deposited by HIPRA SCIENTIFIC, S.L.U. (Avda. La Selva, 135, 17170, Amer, Girona, Spain), according to the Budapest Treaty, under the Accession Number CNCM I-6069 on 22 March 2024 in the Collection Nationale de Cultures de Microorganismes (CNCM) located at Institut Pasteur, 25, Rue de Docteur Roux, F-75724 Paris, France. This strain deposited as CNCM I-6069 has a reduced pathogenicity when administered to the subject.

[0060] In one embodiment, the immunogenic or vaccine composition for use according to the first or second aspects comprises an immunologically effective amount of a live attenuated aMPV as antigen. In one embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of at least 101 0CCID50 (cell culture 50% infective dose). In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of at least 101 8CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of at least 102 0CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of at least 103 0CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of at least 104 0CCID50 In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of at least 105 0CCID50 In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 101 0to 108 0CCID50 In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 101 8to 107 0CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 101 8to 106 0CCID50. In a particular embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 101 8to 105 4CCID50. In a particular embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 103 0to 105 4CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 101 8to 105 0CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 101 8to 104 0CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 101 8to 103 0CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 101 8to 102 5CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 101 8to 102 0CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 1030CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 102 0CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per dose equivalent to a titre of about 101 8CCID50.

[0061] In another embodiment, the immunogenic or vaccine composition of the invention is a freeze-dried lyophilizate. In other embodiments, the immunogenic or vaccine composition according to the present invention comprises an immunologically effective amount of a live attenuated aMPV as antigen, wherein the amount of antigen per unit of freeze-dried lyophilizate is equivalent to a titre of at least 103 0CCID50 (cell culture 50% infective dose). In the sense of the present invention, a unit of a freeze-dried lyophilizate may be, for example, a tablet or a vial. In another embodiment, the immunologically effective amount is an amount of antigen per unit of freeze-dried lyophilizate equivalent to a titre of at least 104 0CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per unit of freeze-dried lyophilizate equivalent to a titre of at least 104 8CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per unit of freeze-dried lyophilizate equivalent to a titre of at least 105 0CCID50 In another embodiment, the immunologically effective amount is an amount of antigen per unit of freeze-dried lyophilizate equivalent to a titre of at least 106 0CCID50 In another embodiment, the immunologically effective amount is an amount of antigen per unit of freeze-dried lyophilizate equivalent to a titre of about 103 0to 1010 0CCID50. In another embodiment, the immunologically effective amount is an amount of antigen per unit of freeze-dried lyophilizate equivalent to a titre of about 104 0to 109 5CCID50. In a particular embodiment, the immunologically effective amount is an amount of antigen per unit of freeze- dried lyophilizate equivalent to a titre of about 1048to 109 4CCID50. In one embodiment, the immunogenic or vaccine composition for use according to the first or second aspects further comprises pharmaceutically acceptable excipients and / or carriers. In other embodiments, the immunogenic or vaccine composition may further comprise an adjuvant. As mentioned above "pharmaceutically” acceptable excipients and / or carriers encompass veterinary acceptable excipients and / or carriers. The pharmaceutically acceptable excipients and / or carriers may be any compound or combination of compounds facilitating the administration and / or delivery of the live attenuated aMPV.

[0062] Pharmaceutically or veterinary acceptable carriers or vehicles are well known to the one skilled in the art. Suitable excipients for the immunogenic or vaccine composition as defined in the present invention may be found, for example, in the book Handbook of pharmaceutical excipients, R.C. Rowe, P.J. Sheskey and M.E. Quinn, editors, Pharmaceutical Press, sixth edition, 2009.

[0063] Appropriate pharmaceutically acceptable excipients include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, suspending agents, stabilizing agents, and / or oils. Excipients such as coloring agents, coating agents, sweetening, and flavouring agents can be present in the composition, according to the judgment of the formulator. Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn-starch, powdered sugar, water, saline, sucrose, mannose, trehalose, sodium glutamate, glycerol, olive, sesame and soybean oils, and non-ionic and ionic polymers such as polyoxyethylenesorbitan monooleate (Tween), hyaluronic acid and combinations thereof. Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof. Exemplary binding excipients include, but are not limited to, starch (e.g., corn-starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminium silicate (Veegum), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof. Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, ascorbyl oleate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate. Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

[0064] The immunogenic or vaccine compositions for use according to the first or second aspects can be presented in any dosage form, for example, solid or liquid, and can be administered by any suitable route, for example, oral, oculonasal, parenteral, rectal, intravaginal, intravesical, topical, intranasal or sublingual route, for which they will include the pharmaceutically acceptable excipients necessary for the formulation of the desired dosage form, for example, topical formulations (ointment, creams, lipogel, hydrogel, etc.), eye drops, aerosol sprays, oral solutions, oral suspensions, injectable solutions, injectable suspensions, injectable emulsions, osmotic pumps, etc. The immunogenic or vaccine compositions for use according to the invention comprising the live attenuated aMPV can be formulated in liquid suspensions or solid forms. Liquid preparations can be solutions for suspension or emulsion in aqueous solutions (water, saline or phosphate- buffered saline (PBS)), non-aqueous solutions or both (aqueous suspensions, oil emulsions, water-in-oil emulsions or oil-in-water emulsions, microemulsions, nanoemulsions or liposomes). In the sense of the present invention, the immunogenic or vaccine composition can be in a form of a freeze-dried lyophilizate or can be reconstituted in a suspension or in drinking water. In a preferred aspect of the invention, the immunogenic or vaccine composition for use according to the invention is a suspension for oculonasal administration. In other preferred aspect of the invention, the immunogenic or vaccine composition for use according to the invention is a suspension for use in drinking water. In some embodiments, the immunogenic or vaccine composition according to the first or second aspects is in a form appropriate for administration by oculonasal route, in particular, administration via spray. In the sense of the present invention, spray also refers to coarse spray, meaning that an increase in water volume is done to create a sufficient number of droplets to be sprayed onto the animals. Appropriate devices well known to the skilled person may be used for spray administration. In a particular embodiment, the immunogenic or vaccine composition is administered with a device providing at least a droplet size equal or above 100 m. In a preferred embodiment, the immunogenic or vaccine composition is administered with a device providing at least a droplet size equal or above 120 pm. In another embodiment, the immunogenic or vaccine composition is administered with a device providing at least a droplet size between 120 pm and 190 pm, or between 120 pm and 180 pm, or between 120 pm and 175 pm. In one embodiment, the volume of water for preparing one dose of the immunogenic or vaccine composition to be administered via spray is between about 0.1 and 30 ml, between about 0.1 and 25 ml, between about 0.1 and 20 ml, between about 0.1 and 15 ml, between about 0.1 and 10 ml, between about 0.1 and 9 ml, between about 0.1 and 8 ml, between about 0.1 and 7 ml, between about 0.1 and 6 ml, between about 0.1 and 5 ml, between about 0.1 and 4 ml, between about 0.1 and 3 ml, between about 0.1 and 2 ml, in particular between about 0.1 and 1 ml. In a particular embodiment, the volume of water for preparing one dose of the immunogenic or vaccine composition to be administered via spray is between about 0.1 and 0.8 ml, or between about 0.1 and 0.6 ml, or between about 0.1 and 0.5 ml, or between about 0.1 and 0.4 ml, or between about 0.1 and 0.3 ml.

[0065] In other embodiments, the immunogenic or vaccine composition is in a form appropriate for administration by oral route, in particular, administration via drinking water. In a particular embodiment, the volume of water for preparing one dose of the immunogenic or vaccine composition to be administered via drinking water is that amount of water which can be ingested within a time period of about 2 hours at most, keeping in mind the age of the birds. For avoidance of doubt, water intake the day before administering the immunogenic or vaccine composition of the invention can be measured in order to calculate the time period necessary for water consumption. In a particular embodiment, the volume of water for preparing one dose of the immunogenic or vaccine composition to be administered via drinking water is between about 1 ml and about 500 ml, between about 1 ml and about 400 ml, between about 1 ml and about 300 ml, between about 1 ml and 200 ml, between about 1 ml and about 100 ml, between about 1 ml and about 50 ml, between about 1 ml and about 40 ml, between about 1 ml and about 37 ml, between about 1 ml and about 35 ml, between about 1 ml and about 30 ml, between about 1 ml and about 25 ml, between about 1 ml and about 20 ml, between about 1 ml and about 15 ml between about 1 ml and about 10 ml, in particular between about 2 ml and about 10 ml, also depending on the age of the subject. In one embodiment, the volume of water for preparing one dose of immunogenic or vaccine composition to be administered via drinking water for birds of one day of age is about 10 ml. In another embodiment, the volume of water for preparing one dose of immunogenic or vaccine composition to be administered via drinking water for birds between 7 and 14 days of age is about 2.8 ml.

[0066] In particular embodiments, the pharmaceutically acceptable excipients and / or carriers may include wetting agents, dispersant agents, emulsifying agents, buffer agents (for example phosphate buffer), stabilizing agents such as carbohydrates (for example glucose, sucrose, mannitol, sorbitol, starch or dextrans) or proteins (for example, albumin, casein, bovine serum or skimmed milk).

[0067] In one embodiment, the pharmaceutically or veterinary acceptable excipients and / or carriers are selected from the group consisting of dextran 70, sucrose, gelatine, NZ amine, sorbitol, potassium dihydrogen phosphate, dipotassium phosphate, and combinations thereof.

[0068] In more particular embodiments, the immunogenic or vaccine composition comprises, in addition to live attenuated aMPV, dextran 70 and a further compound selected from the group consisting of sucrose, gelatine, NZ amine, sorbitol, potassium dihydrogen phosphate, dipotassium phosphate, and combinations thereof. In a more particular embodiment, the immunogenic or vaccine composition comprises a live attenuated aMPV, dextran 70, sucrose, gelatine, NZ amine, sorbitol, potassium dihydrogen phosphate, and dipotassium phosphate. In another embodiment, the immunogenic or vaccine composition of the invention, in addition to the live attenuated aMPV, further comprises from about 50 mg (milligram) to 80 mg of dextran 70, from about 30 mg to 50 mg of sucrose, from about 30 mg to 50 mg of gelatine, from about 20 mg to 35 mg of NZ amine, from about 50 mg to 85 mg of sorbitol, from about 0.05 mg to 0.50 mg of potassium dihydrogen phosphate, and from about 0.50 mg to 2 mg of dipotassium phosphate per unit of freeze-dried lyophilizate.

[0069] In a further embodiment, the immunogenic or vaccine composition for use according to the first or second aspects is a freeze-dried composition, in particular, is a freeze-dried lyophilizate.

[0070] In an embodiment, the immunogenic or vaccine composition for use according to the present invention is further conveniently diluted in a vehicle prior to administration. Said vehicle can be a diluent or a solvent. Said diluent or solvent is preferably an aqueous solution. The diluent or solvent may be selected from any physiological medium, preferably an aqueous solution comprising a salt selected from disodium phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, and mixtures thereof, or water in order to have the immunogenic or vaccine compositions for use comprising the immunologically effective amount of a live attenuated aMPV mentioned above per dose. The aqueous solution may further comprise a sugar compound, such as dextrose, sucrose or saccharose, a nitrogen source such as peptone (e.g., NZ amine) or a casein hydrolysate (e.g., Hy-case amino). In one embodiment, the immunogenic and vaccine composition for use according to the present invention is diluted in clean water prior to be administered to animals.

[0071] Dose volumes can be determined by the skilled artisan from the teachings of the present invention in conjunction with the common knowledge in the art, without any undue experimentation. In an embodiment, when the composition of the invention is administered by spray, the immunogenic or vaccine composition for use according to the first or second aspects is diluted in a volume of diluent or solvent per unit of freeze- dried lyophilizate between about 0.1 L and about 100 L, between about 0.1 L and about 50 L, between about 0.1 L and about 10 L, or between about 0.14 L and about 10 L. In another embodiment, when the composition of the invention is administered by drinking water, the immunogenic or vaccine composition for use according to the first or second aspects is diluted in a volume of diluent or solvent per unit of freeze- dried lyophilizate between about 1 L and about 500 L, between about 1 L and about 400 L, between about 1 L and about 370 L, between 1 L and about 300 L, between about 1 L and about 250 L, between about 1 L and about 200 L , between about 1 L and about 150 L, or between about 1 L and about 100 L.

[0072] The immunogenic or vaccine composition for use according to the first or second aspects may also comprise one or more adjuvants. Non-limiting suitable adjuvants are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units, (3) an oil in water emulsion, (4) cationic lipids containing a quaternary ammonium salt, (5) cytokines, (6) aluminium hydroxide or aluminium phosphate or (7) any combinations or mixtures thereof.

[0073] Among the type (1) adjuvant polymers, preference is given to polymers of crosslinked acrylic or methacrylic acid, especially crosslinked by polyalkenyl ethers of sugars or polyalcohols. These compounds are known under the name carbomer. Products sold under the name Carbopol (BF Goodrich, Ohio, USA).

[0074] The mixture with the adjuvant may be carried out extemporaneously or preferably contemporaneously with administration of the preparation or shortly before administration of the preparation. For instance, the composition-adjuvant mixture is prepared before administration. The time spanning between contacting the composition-adjuvant and its administration is advantageously enough to allow the mixture to form a complex. In some examples, composition-adjuvant mixture is prepared between about 10 and about 60 minutes prior to administration, preferably about 30 minutes before administration.

[0075] The immunogenic or vaccine composition for use according to the first or second aspects can include further immunogenic active components such as a further immunogen, antigen or epitope of another avian pathogen (parasite, bacteria or virus). Active immunogens, antigens or epitopes against other avian pathogens are disclosed under the section "Combination therapy” below and may be used as further immunogenic active components in the immunogenic or vaccine composition disclosed herein.

[0076] In a particular embodiment, the immunogenic or vaccine compositions for use according to the first or second aspects, further comprises at least one antigen against another avian pathogen. In a particular embodiment, the immunogenic or vaccine compositions for use according to the first or second aspects, further comprises at least one antigen against another avian pathogen selected from the group consisting of the Marek's disease virus (MDV) (e.g., serotypes 1 and 2, advantageously 1), Turkey Herpesvirus (HVT), Newcastle disease virus (NDV), paramyxoviruses other than Newcastle disease (PMV2 to PMV7), infectious bronchitis virus (I BV), infectious anaemia virus or chicken anaemia virus (CAV), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV, Gumboro disease), encephalomyelitis virus or avian encephalomyelitis virus (AEV or avian leukosis virus ALV), virus of haemorrhagic enteritis of turkeys (HEV), pneumovirosis virus (TRTV), fowl plague virus (avian influenza), chicken hydropericarditis virus, avian reoviruses, coccidia, egg drop syndrome virus (EDS), fowl pox, inclusion body hepatitis (adenovirus), lymphoproliferative disease virus (LDV), reticuloendotheliosis virus (REV), rotavirus enteritis, and turkey rhinotracheitis virus, Clostridium spp., Eimeria spp., Escherichia coli, Mycoplasma gallinarum, Mycoplasma gallisepticum, Haemophilus avium, Pasteurella gallinarum, Pasteurella multocida gallicida, Salmonella spp., and combinations thereof.

[0077] In a more particular embodiment, the immunogenic or vaccine compositions for use according to the first or second aspects, further comprises at least one antigen against another avian pathogen selected from the group consisting of Infectious Bronchitis Virus (IBV), Egg Drop Syndrome virus (EDS virus), Newcastle disease virus (NDV), coccidia, Eimeria spp., infectious bursal disease virus (IBDV), and combinations thereof.

[0078] Administration

[0079] The immunogenic or vaccine composition comprising a live attenuated aMPV for use according to the first and second aspects of the present invention may be administered to laying birds without causing side effects, as shown in Example 2 below. As mentioned above "laying birds” is herein understood as birds during the laying period. Thus, the first aspect of the invention may be worded as an immunogenic or vaccine composition comprising a live attenuated aMPV for use in the prevention and / or treatment of an aMPV infection, wherein said use comprises administering the composition to birds during the laying period. Similarly, the second aspect may be worded as an immunogenic or vaccine composition comprising a live attenuated aMPV for use in active immunization of birds against aMPV during the laying period. All embodiments and examples mentioned above for the immunogenic or vaccine composition are applicable to the administration regime.

[0080] In one embodiment, the birds (subjects) are domesticated birds. In another embodiments, the birds are poultry birds. In another embodiment, the birds are selected from the group consisting of: chicken, pullet, pheasant, turkey, goose, duck, broiler, quail, pigeon, swan, and ostrich. In a particular embodiment the birds are selected from chicken and turkeys. Since the composition may be administered to laying birds, in particular embodiments the birds are selected from chicken hens and turkey hens (i.e. female chicken and female turkeys) that have reached laying maturity and are kept for the production of eggs, particularly eggs for human consumption.

[0081] In one embodiment of the first or second aspects, the immunogenic or vaccine composition is administered to chicken hens during the laying period. Hens typically reach sexual maturity and begin laying eggs at around 18 to 20 weeks of age, the typical laying age of a hen is anywhere between 20 and 24 weeks of age. Production of eggs before 20 weeks (5 months) in any hen is not advisable because the egg size will be small, although this can vary depending on the breed and environmental factors. Once they start laying, hens usually continue to produce eggs for about 12 to 14 months, with peak production occurring in the first six months. After this period, egg production gradually declines, and hens may eventually stop laying altogether.

[0082] In one embodiment, the immunogenic or vaccine composition for use according to the first or second aspects is administered to chicken hens of at least 16 weeks of age. In other embodiments, it is administered to chicken hens of at least 17 weeks of age, of at least 18 weeks of age, of at least 19 weeks of age, of at least 20 weeks of age, of at least 21 weeks of age, of at least 22 weeks of age, of at least 23 weeks of age, or of at least 24 weeks of age. There is no particular age above which the composition cannot be administered. In particular embodiments of the first or second aspects, the immunogenic or vaccine composition is administered to chicken hens from the age of 16 weeks to the age of 20 months, in particular from the age of 18 weeks to the age of 20 months, particularly from the age of 20 weeks to the age of 20 months, particularly from the age of 20 weeks to the age of 24 months, more particularly from the age of 20 weeks to the age of 36 months, and more particularly from the age of 20 weeks to the age of 48 months.

[0083] The above age ranges are indicative of the laying period for chicken hens. However, the immunogenic or vaccine composition for use according to the present invention is not solely indicated for chickens. It is also very useful for immunizing other birds that are susceptible to aMPV infections, for example, turkeys. Thus, in one embodiment, the immunogenic or vaccine composition for use according to the first or second aspects is administered to turkey hens of at least 26 weeks of age. In other embodiments, it is administered to turkey hens of at least 28 weeks of age, of at least 29 weeks of age, of at least 30 weeks of age, of at least 31 weeks of age, of at least 32 weeks of age, of at least 33 weeks of age, or of at least 34 weeks of age. In particular embodiments of the first or second aspects, the immunogenic or vaccine composition is administered to turkey hens from the age of 29 weeks to the age of 26 months, in particular from the age of 30 weeks to the age of 26 months, particularly from the age of 30 weeks to the age of 30 months, more particularly from the age of 30 weeks to the age of 36 months, and more particularly form the age of 30 weeks to the age of 48 months.

[0084] In a particular embodiment, the immunogenic or vaccine composition for use according to the first or second aspects is preferably administered by oculonasal route or oral route. When using oculonasal route it is conveniently administered via spray. When the oral route is used, it is conveniently administered via the drinking water. Thus, in other particular embodiments, the immunogenic or vaccine composition for use according to the first or second aspects is administered by oculonasal route via spray or by oral route via drinking water.

[0085] In some embodiments, the administration regime of the immunogenic or vaccine composition for use according to the first or second aspects comprises a first administration of the composition to birds of at least 1 day of age or at least 7 days of age. Oculonasal route is usually employed for new-born birds. Oral route is usually employed for birds of at least 5 to at least 7 days of age, in particular birds of at least 7 days of age.

[0086] In particular embodiments, the administration regime may comprise a first administration of the immunogenic or vaccine composition to birds of at least 1 day of age by oculonasal route or at least 7 days of age by drinking water. This first dose may be preferably administered to birds up to 2 weeks of age. In a particular embodiment, the administration regime comprises administering a first dose of the immunogenic or vaccine composition to birds from 1 day to 7 days of age. In other embodiments the administration regime comprises administering a first dose of the immunogenic or vaccine composition to birds from 1 day to 2 weeks of age. In particular embodiments, one or more doses are administered to the birds after the first dose. In a particular embodiment, a second dose is administered 9 weeks after the first dose. In particular embodiments, a further single dose is administered to the birds every 9 weeks after the first single dose. In particular embodiments, the further single doses every 9 weeks are administered spanning part or all of the laying period. This maintenance doses improve long-term immunization, in particular, during the laying period.

[0087] In particular embodiments of the first or second aspects of the invention, the administration regime comprises: (i) administering a first single dose of the immunogenic or vaccine composition to birds from 1 day to 14 days of age, and (ii) administering a further single dose of the immunogenic or vaccine composition once every nine weeks after the first dose. In other particular embodiments of the first or second aspects, the administration regime comprises: (I) administering a first single dose of the immunogenic or vaccine composition to birds from 1 day to 10 days of age, and (ii) administering a further single dose of the immunogenic or vaccine composition once every nine weeks after the first dose. In a more particular embodiment of the first or second aspects, the administration regime comprises: (i) administering a first single dose of the immunogenic or vaccine composition by oculonasal route via spray to birds of at least 1 day of age or by drinking water to birds of at least 7 days of age, and (ii) administering a further single dose of the immunogenic or vaccine composition once every nine weeks after the first dose. In step (ii) as defined above one or more further doses may be administered after the first dose (every 9 weeks). In particular embodiments, two further doses are administered. In other particular embodiments, three, four, or five further doses are administered after the first dose every 9 weeks. In other embodiments, more than five further doses are administered. The further doses are preferably administered by oculonasal route or oral route, for example via spray or drinking water, respectively. With these further doses the immunogenic and vaccine compositions of the present invention provide long-term immunization during the whole laying period.

[0088] An advantage of the present invention is that there is no need to administer an inactivated aMPV vaccine during the laying period as suggested by the state of the art, which in turn needs to be administered by injection to each bird individually. Instead, effective immunization during the laying period is achieved administering the immunogenic and vaccine composition comprising the live attenuated aMPV of the present invention by oculonasal or oral route, which greatly simplifies the vaccination protocols. Thus, one additional advantage of the compositions of the present invention is that mass vaccination of laying birds can be performed with said immunogenic or vaccine compositions. This is particularly relevant, for example, in poultry farming systems containing hundreds or thousands of birds. Thus, in another embodiment of the first or second aspects, the use does not comprise administering an inactivated aMPV vaccine. In another embodiment of the first or second aspects, the administration regime does not include an inactivated aMPV vaccine. Thus, in another embodiment of the first or second aspects, the use does not comprise administering an inactivated aMPV vaccine during the laying period. In another embodiment of the first or second aspects, the administration regime does not include an inactivated aMPV vaccine during the laying period. In a further embodiment of the first or second aspects, the use is for mass vaccination of laying birds. Said mass vaccination can be carried out either by spray or drinking water administration.

[0089] Prevention and / or treatment of an aMPV infection

[0090] The present invention encompasses an immunogenic or vaccine composition comprising a live attenuated avian metapneumovirus (aMPV) for use in the prevention and / or treatment of an aMPV infection. As shown in examples section (see Example 1), the immunogenic or vaccine compositions comprising a live attenuated aMPV showed an onset of immunization of 3 weeks post administration and a duration of immunity of at least 9 weeks post-administration. Accordingly, birds actively immunized against aMPV with the immunogenic or vaccine composition of the present invention are effectively protected. The composition comprising a live attenuated aMPV is thus an immunogenic composition that may be used as a vaccine, since it conferred protection against an aMPV infection. The prevention and / or reduction of an aMPV infection results in an improvement in comparison to an infected non-immunized or non-treated control group of the same domesticated birds. All embodiments and examples mentioned in the sections above or below for the immunogenic or vaccine composition and the administration are applicable to the prevention and / or treatment of an aMPV infection.

[0091] In one embodiment, the immunogenic or vaccine composition for use according to the first or second aspects elicits an immune response against an aMPV infection from at least 3 weeks after administration. In another embodiment, the immunogenic or vaccine composition for use according to the present invention elicits an immune response against an aMPV infection until at least 9 weeks after administration. In another embodiment, the immunogenic or vaccine composition for use according to the first or second aspects elicits an effective immune response against an aMPV infection in the presence of Maternal Derived Antibodies (MDAs). In another embodiment the immunogenic or vaccine composition for use according to the first or second aspects is administered in the presence of MDAs. In another embodiment, the immunogenic or vaccine composition for use according to the first or second aspects elicits an effective immune response against an aMPV infection in the presence of Maternal Derived Antibodies (MDAs) against aMPV. In another embodiment the immunogenic or vaccine composition for use according to the first or second aspects is administered in the presence of MDAs against aMPV.

[0092] In one embodiment, the immunogenic or vaccine composition for use according to the present invention is for use in the prevention and / or reduction of mortality caused by an aMPV infection. In another embodiment, the immunogenic or vaccine composition for use according to the present invention is for use in the prevention and / or reduction of morbidity caused by an aMPV infection.

[0093] In one embodiment, the immunogenic or vaccine composition for use according to the present invention reduces the detrimental effect caused by an aMPV infection on the respiratory tract. In a particular embodiment, it is for reducing the detrimental effect caused by aMPV on the ciliary activity. In one embodiment, the immunogenic or vaccine composition for use according to the present invention is for use in the prevention and / or treatment of a disease caused by an aMPV infection. In particular embodiments, the disease is selected from the group consisting of avian rhinotracheitis and swollen head syndrome. In some embodiments, the immunogenic or vaccine composition for use according to the present invention is for preventing and / or treating one or more of the clinical signs caused by an aMPV infection. In a particular embodiment, the immunogenic or vaccine composition for use according to the present invention prevents and / or reduces at least one clinical sign associated to an aMPV infection. In another particular embodiment, the immunogenic or vaccine composition for use according to the present invention prevents and / or reduces at least one clinical sign associated to an aMPV infection selected from the group consisting of general clinical signs, respiratory clinical signs, reproductive clinical signs, and combinations thereof. The general clinical signs may be selected from the group consisting of depression, anorexia, ruffled feathers, head shaking, head scratching, nervous signs, apathy, cerebral disorientation, torticollis, opisthotonos, reduced growth, mortality, and combinations thereof. The respiratory clinical signs may be selected from the group consisting of dyspnea, ocular discharge, nasal discharge, swollen infra-orbital sinuses, respiratory distress, conjunctivitis, and combinations thereof. The reproductive clinical signs may be selected from the group consisting of drop in egg production, drop in egg quality, egg abnormalities such as egg size abnormalities, egg shell abnormalities (thin shell eggs, soft shell eggs, shell-less eggs, rough or corrugated shell eggs, small eggs, abnormal calcareous deposits, eggs with dimples, depressions or ridging, blood-stained eggs, and misshapen eggs), egg yolk abnormalities (blood spots, pale yolk, mottled yolk, discolored yolk (bluegreen), rubbery yolk, cheesy yolk, and pasty yolk), egg albumen abnormalities (thin albumen, watery albumen, off-color albumen (green or pink)), and combinations thereof.

[0094] In a particular embodiment, the immunogenic or vaccine composition for use according to the present invention is for preventing egg drop production and / or egg drop quality when administered to laying birds. More in particular, the immunogenic or vaccine composition for use according to the present invention prevents and / or reduces a detrimental effect on an aMPV infection in egg production and / or egg quality. More particularly, the immunogenic or vaccine composition for use of the present invention prevents and / or reduces at least one egg abnormality associated to an aMPV infection, such as egg size, egg shell abnormalities, egg yolk abnormalities and / or egg albumen abnormalities. In another embodiment, egg abnormalities that can be prevented or reduced by administering the immunogenic or vaccine composition above to laying birds (i.e. to hens during the laying period) are selected from the group consisting of thin shell eggs, soft shell eggs, shell-less eggs, rough or corrugated shell eggs, small eggs, abnormal calcareous deposits, eggs with dimples, depressions or ridging, blood-stained eggs, and misshapen eggs. In another embodiment, egg yolk quality parameters that may be prevented from dropping when administering the immunogenic or vaccine composition comprising a live attenuated aMPV during the laying period are selected form the group consisting of blood spots, pale yolk, mottled yolk, discolored yolk (blue-green), rubbery yolk, cheesy yolk, and pasty yolk. In another embodiment, egg albumen quality parameters that may be prevented from dropping when administering the immunogenic or vaccine composition comprising a live attenuated aMPV to birds during the laying period are selected from the group consisting of thin albumen, watery albumen, and off-color albumen (green or pink).

[0095] In other particular embodiments the immunogenic or vaccine composition for use according to the invention prevents and / or reduces a drop in egg production (number of eggs laid).

[0096] In one embodiment, the aMPV causing the infection is selected from the group consisting of an aMPV subtype A, an aMPV subtype B, an aMPV subtype C, an aMPV subtype D, and combinations thereof. In a particular embodiment, the aMPV causing the infection comprises aMPV subtype A. In another particular embodiment, the aMPV causing the infection comprises aMPV subtype B. In another embodiment, the aMPV causing the infection consists of aMPV subtype B.

[0097] Combination therapy

[0098] A third aspect of the present invention refers to an immunogenic or vaccine composition comprising a live attenuated avian metapneumovirus (aMPV) for use in the prevention and / or treatment of an avian metapneumovirus (aMPV) infection, or for use in active immunization of laying birds, in combination therapy with a further avian vaccine, wherein said use comprises administering the immunogenic or vaccine composition comprising a live attenuated aMPV to laying birds. All embodiments and examples mentioned in the sections above for the immunogenic or vaccine composition, the administration and the prevention and / or treatment of an aMPV infection are applicable to the combination therapy.

[0099] In one embodiment of the third aspect, the further avian vaccine is another vaccine for the prevention and / or treatment of an aMPV infection. Said other aMPV vaccine may contain a live attenuated aMPV, an inactivated aMPV or it may be a recombinant vaccine. In one embodiment of the third aspect, the combined therapy may comprise administering the immunogenic or vaccine composition comprising the live attenuated aMPV as defined above and the other aMPV vaccine concomitantly or separately within a therapeutically effective interval. In another embodiment of the third aspect, the combined therapy may comprise admixing the immunogenic or vaccine composition comprising the live attenuated aMPV as defined above and the other aMPV vaccine prior to use and administering the admixture.

[0100] In some embodiments, the immunogenic or vaccine composition for use according to the third aspect is for the prevention and / or treatment of an aMPV infection and of an infection caused by a different avian pathogen. In said embodiments the combination therapy comprises administering the immunogenic or vaccine composition comprising the live attenuated aMPV as disclosed above and another avian vaccine composition, which comprises at least one immunogen, antigen or epitope of another avian pathogen. Such an immunogen, antigen or epitope may be of bacterial, parasitic or viral origin and may be an inactivated or attenuated form of such bacteria, parasite or virus.

[0101] In some embodiments of the third aspect, the combined therapy comprises administering the immunogenic or vaccine composition comprising a live attenuated aMPV as defined above and the other avian vaccine (for a different pathogen) concomitantly or separately within a therapeutically effective interval. In another embodiment, the combined therapy may comprise admixing the immunogenic or vaccine composition comprising a live attenuated aMPV as defined above and the other avian vaccine for the different pathogen prior to use and administering the admixture.

[0102] The additional avian vaccine may comprise at least one immunogen, antigen or epitope of another avian pathogen, which may be without limitation the pathogen in inactivated or attenuated form, or it may be a recombinant vaccine. The other avian pathogen may be selected from viruses, bacteria, or pathogens selected from the group consisting of the Marek's disease virus (MDV) (e.g., serotypes 1 and 2, advantageously 1), Turkey Herpesvirus (HVT), Newcastle disease virus (NDV), paramyxoviruses other than Newcastle disease (PMV2 to PMV7), infectious bronchitis virus (I BV), infectious anaemia virus or chicken anaemia virus (CAV), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV, Gumboro disease), encephalomyelitis virus or avian encephalomyelitis virus (AEV or avian leukosis virus ALV), virus of haemorrhagic enteritis of turkeys (HEV), pneumovirosis virus (TRTV), fowl plague virus (avian influenza), chicken hydropericarditis virus, avian reoviruses, coccidia, egg drop syndrome virus (EDS), fowl pox, inclusion body hepatitis (adenovirus), lymphoproliferative disease virus (LDV), reticuloendotheliosis virus (REV), rotavirus enteritis, and turkey rhinotracheitis virus, Clostridium spp., Eimeria spp., Escherichia coli, Mycoplasma gallinarum, Mycoplasma gallisepticum, Haemophilus avium, Pasteurella gallinarum, Pasteurella multocida gallicida, Salmonella spp., and combinations thereof.

[0103] In one embodiment of the third aspect, the additional avian vaccine comprises at least one immunogen, antigen or epitope selected from the group consisting of I BV (Infectious Bronchitis Virus), EDS virus (Egg Drop Syndrome), NDV (Newcastle disease virus), and combinations thereof. In another embodiment of the third aspect, the additional avian vaccine comprises at least one immunogen, antigen or epitope of infectious bursal disease virus (IBDV, Gumboro disease). In another particular embodiment of the third aspect of the invention, the additional avian vaccine comprises at least one immunogen, antigen or epitope of coccidia. More particular, the avian vaccine comprises at least one immunogen, antigen or epitope of Eimeria spp.

[0104] Safety and efficacy data demonstrate that the immunogenic or vaccine composition for use according to the third aspect can be mixed with an additional vaccine comprising at least one immunogen, antigen or epitope of aMPV for the prevention and / or treatment of an aMPV infection, or alternatively with at least one immunogen, antigen or epitope of a different avian pathogen for the prevention and / or treatment of an infection caused by said other avian pathogen

[0105] The combined therapy of the third aspect may be, for example, with an additional vaccine comprising an IBV antigen, an EDS virus antigen, a NDV antigen, a coccidia antigen, an Eimeria spp. antigen, an I BDV antigen, and / or combinations thereof. In one embodiment, the immunogenic or vaccine composition for use according to the third aspect may be used in combination with a vaccine comprising an IBV antigen, an EDS virus antigen, a NDV antigen, a coccidia antigen, an Eimeria spp. antigen and / or an I BDV antigen, for the prevention and / or treatment of both an aMPV infection and an IBV infection, an EDS virus infection, a NDV infection, a coccidia infection, an Eimeria spp infection or an IBDV infection. In one embodiment, the immunogenic or vaccine composition for use according to the third aspect may be used in combination with a vaccine comprising an IBV immunogen, an EDS virus immunogen, a NDV immunogen, a coccidia immunogen, an Eimeria spp. immunogen and / or an IBDV immunogen, and then provides immunogenic and / or protective immune response against an aMPV infection and an IBV infection, an EDS virus infection, a NDV infection, a coccidia infection, an Eimeria spp infection or an IBDV infection. Vaccines against IBV, EDS, NDV, coccidia, Eimeria spp., and / or IBDV are known in the prior art and commercially available, for example, GUMBOHATCH (Laboratories HIPRA S.A., Avda. La Selva 135, 17170 Amer, Girona, Spain), CEVAC TRANSMUNE (Ceva Animal Health, S.A., 08028 Barcelona, Spain), EVANT (Laboratories HIPRA S.A., Avda. La Selva 135, 17170 Amer, Girona, Spain), EVALON (Laboratories HIPRA S.A., Avda. La Selva 135, 17170 Amer, Girona, Spain), Paracox-5 (Merck Sharp & Dohme Animal Health, S.L., 37188 Carbajosa de La Sagrada, Salamanca, Spain), Nobilis IB 4-91 (Intervet International B.V., Wim de Kbrverstraat 35, 5831 AN Boxmeer, The Netherlands), Nobilis IB+ND+EDS (Merck Sharp & Dohme Animal Health, S.L., 37188 Carbajosa de La Sagrada Salamanca, Spain), CEVAC MASS L (Ceva Animal Health, S.A. Avda. Diagonal 609-615, 08028 Barcelona, Spain), AVISAN MULTI (Laboratories HIPRA S.A., Avda. La Selva 135, 17170 Amer, Girona, Spain), or BRONIPRA-ND (Laboratories HIPRA S.A., Avda. La Selva 135, 17170 Amer, Girona, Spain).

[0106] In an embodiment of third aspect, the immunogenic or vaccine composition comprising the live attenuated aMPV as defined above and the other avian vaccine for the same or a different pathogen are in separate containers, optionally in the same package, and optionally with instructions for admixture and / or administration according to a particular schedule. In particular embodiments the other avian vaccine is a vaccine comprising an IBV immunogen, antigen or epitope, an EDS virus immunogen, antigen or epitope, a NDV immunogen, antigen or epitope, a coccidia immunogen, antigen or epitope, an Eimeria spp. immunogen, antigen or epitope, or an IBDV immunogen, antigen or epitope.

[0107] Vaccination kits A fourth aspect of the present invention refers to a vaccination kit comprising an immunogenic or vaccine composition comprising a live attenuated aMPV for use in the prevention and / or treatment of an aMPV infection, wherein said use comprises administering the immunogenic or vaccine composition to laying birds, or for use in active immunization of laying birds. A fifth aspect refers to a vaccination kit comprising an immunogenic or vaccine composition comprising a live attenuated aMPV for use in active immunization of laying birds against an aMPV infection.

[0108] The vaccination kits for use according to the fourth and fifth aspects may comprise instructions for the administration regime according to any of the embodiments described above under the section "Administration”. Additionally, all the embodiments defined above under the section "The composition” may be applied to the vaccination kits for use according to the fourth or fifth aspects. Moreover, all embodiments defined above under the section "Prevention and / or treatment of aMPV” may be applied to the vaccination kits for use according to the fourth or fifth aspects.

[0109] In a particular embodiment of the fourth or fifth aspects, the vaccination kit comprises: (a) at least one vial comprising the immunogenic or vaccine composition comprising a live attenuated aMPV, and (b) a leaflet with instructions for use in preventing and / or treating an aMPV infection, or for active immunization of laying birds. The instructions preferably comprise instructions for administering the immunogenic or vaccine composition to laying birds. In a particular embodiment of the fourth or fifth aspects, the vaccination kit comprises: (a) at least one vial with a freeze-dried lyophilizate comprising the immunogenic or vaccine composition comprising a live attenuated aMPV, and (b) a leaflet with instructions for the administration of the composition via spray or via drinking water to laying birds. The instructions may also comprise indications to the administration regime, for example, for administering a first dose of the immunogenic or vaccine composition comprising a live attenuated aMPV to chicks during the first days of life and administering a further single dose every 9 weeks after the first single dose, for example, during a time spanning part or all of the laying period. More particularly the instructions comprise indications to administer a first dose of the immunogenic or vaccine composition comprising a live attenuated aMPV to chicks of 1 day of age by spraying or of 7 days of age via drinking water and a further single dose every 9 weeks after the first single dose, for example, during a time spanning part or all of the laying period.

[0110] In one embodiment of the fourth or fifth aspects, the vaccination kit comprises a vial with one dose or multiple doses of the immunogenic or vaccine composition comprising the live attenuated aMPV as described above. In another embodiment of the fourth or fifth aspects, the vaccination kit comprises a vial containing at least 1 ,000 doses, at least 2,000 doses, at least 5,000 doses, or at least 10,000 doses of the immunogenic or vaccine composition comprising the live attenuated aMPV as defined above. In one embodiment of the fourth or fifth aspects, the vaccination kit comprises an immunologically effective amount of an aMPV as antigen. In one embodiment the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of at least 101 0CCID50 (cell culture 50% infective dose). In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of at least 101 8CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of at least 102 0CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of at least 103 0CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of at least 104 0CCID50 In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of at least 105 0CCID50 In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 101 0to 108 0CCID50 In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 101 8to 107 0CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 101 8to 1060CCID50. In a particular embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 101 8to 105 4CCID50. In a particular embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 103 0to 105 4CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 101 8to 1050CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 101 8to 104 0CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 101 8to 1030CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 101 8to 102 5CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 101 8to 1020CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 103 0CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 102 0CCID50. In another embodiment, the vaccination kit comprises an immunologically effective amount of antigen per dose equivalent to a titre of about 101 8CCID50. In another embodiment, the vaccination kit, in addition to the live attenuated aMPV as antigen, further comprises dextran 70, and optionally a further compound selected from the group consisting of sucrose, gelatine, NZ amine, sorbitol, potassium dihydrogen phosphate, dipotassium phosphate, and combinations thereof. In a more particular embodiment, the vaccination kit further comprises dextran 70, sucrose, gelatine, NZ amine, sorbitol, potassium dihydrogen phosphate, and dipotassium phosphate.. In one embodiment of the fourth or fifth aspects, the vaccination kit comprises at least one vial comprising an immunogenic or vaccine composition comprising a live attenuated aMPV. Preferably, the immunogenic or vaccine composition in the at least one vial is a freeze-dried lyophilizate. In one embodiment, each vial comprises an amount of live attenuated aMPV as antigen equivalent to a titre of at least 103 0CCID50 (cell culture 50% infective dose). In another embodiment, each vial comprises an amount of antigen equivalent to a titre of at least 104 0CCID50. In another embodiment, each vial comprises an amount of antigen equivalent to a titre of at least 104 8CCID50. In another embodiment, each vial comprises an amount of antigen equivalent to a titre of at least 105 0CCID50 In another embodiment, each vial comprises an amount of antigen equivalent to a titre of at least 106 0CCID50 In another embodiment, each vial comprises an amount of antigen equivalent to a titre of about 103 0to 10100CCID50. In another embodiment, each vial comprises an amount of antigen equivalent to a titre of about 104 0to 109 5CCID50. In a particular embodiment, each vial comprises an amount of antigen equivalent to a titre of about 104 8to 1094CCID50. In preferred embodiments, in addition to the live attenuated aMPV as antigen, each vial further comprises dextran 70. In more particular embodiments, each vial further comprises dextran 70, and a further compound selected from the group consisting of sucrose, gelatine, NZ amine, sorbitol, potassium dihydrogen phosphate, dipotassium phosphate, and combinations thereof. In a more particular embodiment, each vial further comprises dextran 70, sucrose, gelatine, NZ amine, sorbitol, potassium dihydrogen phosphate, and dipotassium phosphate. In another particular embodiment, each vial further comprises from about 50 mg (milligram) to 80 mg of dextran 70, from about 30 mg to 50 mg of sucrose, from about 30 mg to 50 mg of gelatine, from about 20 mg to 35 mg of NZ amine, from about 50 mg to 85 mg of sorbitol, from about 0.05 mg to 0.50 mg of potassium dihydrogen phosphate, and from about 0.50 mg to 2 mg of dipotassium phosphate.

[0111] In a particular embodiment, the aMPV is aMPV subtype B strain JFB24. In another embodiment the aMPV is the aMPV subtype B strain JFB24 deposited under the Budapest Treaty by HIPRA SCIENTIFIC, S.L.U. (Avda. La Selva, 135, 17170 Amer, Girona, Spain) in the Collection Nationale de Cultures de Microorganismes (CNCM) located at Institut Pasteur, 25, Rue de Docteur Roux, F-75724 Paris, France under the Accession Number CNCM I-6069 on 22 March 2024.

[0112] In another embodiment of the fourth or fifth aspects, the vaccination kit further comprises a vial comprising an additional immunogenic or vaccine composition comprising at least one antigen against another avian pathogen. In a particular embodiment, the additional immunogenic or vaccine composition comprising at least one antigen against another avian pathogen are selected from the group consisting of Infectious Bronchitis Virus (IBV), Egg Drop Syndrome virus (EDS virus), Newcastle disease virus (NDV), coccidia, Eimeria spp., infectious bursal disease virus (IBDV), and combinations thereof. All embodiments referring to the further avian pathogen as described above under the sections "The composition” or "Combination therapy” also apply to the kit for use according to the fourth or fifth aspects.

[0113] EXAMPLES

[0114] EXAMPLE 1 : Efficacy study of a live attenuated vaccine composition against avian metapneumovirus (aMPV) administered by oculonasal route in one-day-old chickens

[0115] The aim of this study was to assess the efficacy of a live attenuated vaccine composition against avian metapneumovirus (aMPV) in one-day-old chicks when it is administered as a single dose via spray against an aMPV virulent strain. The efficacy study included animals with and without maternally-derived antibodies (MDA) against aMPV.

[0116] A total of sixty SPF chicks {Gallus gallus) of one-day-old MDA negative against aMPV and sixty-six one- day-old commercial chicks MDA positive against aMPV were enrolled in this study. The animals were randomly allocated to four treatment groups (A to D) as follows:

[0117] The immunological status was checked for all groups in order to confirm whether they were MDA positive or negative according to the assigned group.

[0118] Groups A and B were vaccinated with a single dose of a live attenuated aMPV vaccine composition by oculonasal route via spray on day 0 of the study (DO). Groups C and D received a single dose of a mock-up vaccine composition of clean, fresh, antiseptic and disinfectant free water by the same route and method on day 0 (DO). The animals in groups A and B received a vaccine composition based on a freeze-dried lyophlizate of live attenuated aMPV strain JFB24, formulated in a composition comprising from 50 mg to 80 mg of dextran 70, from 30 mg to 50 mg of sucrose, from 30 mg to 50 mg of gelatin, from 20 mg to 35 mg of NZ amine, from 50 mg to 85 mg of sorbitol, from 0.05 mg to 0.50 mg of potassium dihydrogen phosphate, and from 0.50 to 2 mg of dipotassium phosphate. This formulation was reconstituted with clean, fresh, antiseptic and disinfectant free water to achieve a final concentration of 101 8CCIDso / ml. The vial was gently mixed until the lyophilizate was completely dissolved.

[0119] The live attenuated aMPV strain was obtained from the commercial vaccine HIPRAVIAR-SHS (Laboratories HIPRA, S.A., Avda. La Selva, 135, 17170 Amer, Girona, Spain), which also corresponds to the aMPV strain JFB24 deposited by HIPRA SCIENTIFIC, S.L.U. (Avda. La Selva, 135, 17170, Amer, Girona, Spain) in the Collection Nationale de Cultures de Microorganismes, CNCM (Institute Pasteur, Rue de Docteur Roux 25, F-75724 Paris, France) under the accession number CNCM I-6069 on 22 March 2024. This live attenuated aMPV strain JFB24 was attenuated by serial passages alternatively through embryo tracheal organ tissues of turkeys and SPF chicken embryo fibroblasts, followed by serial passages in Vero cells. The freeze-dried lyophilizate was obtained by standard procedures of freeze-drying process of the antigenic phase (aMPV strain JFB24) with the freeze-drying excipients based on dextran 70, sucrose, gelatin, NZ amine, sorbitol, potassium dihydrogen phosphate and dipotassium phosphate.

[0120] The vaccine composition was sprayed evenly over the laying birds at a distance of 30-40 cm and ensuring a droplet size of at least 120 pm. Each dose of the vaccine was 0.140 mL per animal. The vaccine composition was administered via oculonasal route using a spray applicator (Spray Gun, Giordano Poultry Plast S.p.a) or any other spray applicator that allowed to spray under the same condition and droplet size of at least 120 pm.

[0121] On Day 21 of the study (D21), three weeks after vaccination, all animals were challenged with a virulent strain of avian metapneumovirus (aMPV), by oculonasal route. Each animal was challenged with 105 0CD50 (ciliostatic dose 50) of a virulent aMPV isolate in a total volume of 0.1 ml per bird. The administration was performed via oculonasal route (eye and nostril drop), namely, 0.025 ml was administered to each eye and 0.025 ml was administered to each nostril using a micropipette and sterile disposable tips.

[0122] The efficacy of the vaccine composition was based on the tracheal ciliary activity to determine the presence or absence of respiratory clinical signs in the chicks (i.e. affected or not-affected respiratory clinical signs). The efficacy of the vaccine was demonstrated if the proportion of chicks with respiratory clinical signs (affected) was significantly lower in vaccinated chicks than in mock-up vaccinated group. Animals in Group D (MDA positive, mock-up vaccinated) were weekly monitored to assess the decay of MDAs against aMPV. At three weeks post-vaccination, at the time when all animals of the study were challenged, MDAs had completely disappeared or had reached low levels in this control group.

[0123] General clinical signs were daily observed during the whole study, until 14 days post-challenge. No relevant clinical signs were observed in all groups and no significant differences were observed between groups. Body weight gain at different time points along the study was also evaluated and no significant differences were observed between groups, after challenge.

[0124] The main parameter to assess efficacy was the presence of respiratory clinical signs during the whole study. After challenge and until day 14 post-challenge, animals were monitored even more toughly. Between days 10 and 11 post-challenge, 8 animals from each group were humanely euthanized to evaluate the presence or absence of respiratory clinical signs after challenge with the virulent aMPV strain. Evaluation of chicks as having or not respiratory clinical signs was performed based on the tracheal ciliary activity assessment. Necropsy of all euthanized animals (at days 7, 10, 11 and 14 post-challenge) was performed in order to evaluate macroscopic lesions, related to the experimental infection, examining especially at the upper respiratory tract. No lesions were observed in animals necropsied post-challenge.

[0125] Serology was also evaluated to confirm that control animals were still seronegative before challenge.

[0126] Overall, the following parameters were evaluated in the study:

[0127] General clinical signs, daily post-challenge.

[0128] Body weight gain, post-challenge.

[0129] Macroscopic lesions, after challenge, especially at the upper respiratory tract.

[0130] Respiratory clinical signs, after challenge based on the tracheal ciliary activity assessment of animals euthanized between days 10 and 11 post-challenge.

[0131] Serology: ELISA titers and percentage of seroconverted animals were determined.

[0132] The main parameter assessed for the efficacy was the respiratory clinical signs based on the tracheal ciliary activity. The number of chicks affected with any respiratory clinical sign was recorded for each group.

[0133] Eight animals per group were humanely euthanized between 10- and 11-days post-challenge corresponding to day 31 (D31) and day 32 (D32) of the study, respectively, to evaluate the presence or absence of respiratory clinical signs based on the tracheal ciliary activity assessment. For each animal, the ciliary movement of the ten tracheal rings was evaluated. The tracheas were placed in Hank's minimum essential medium immediately after euthanizing the animals. Ten tracheal rings per animal were cut and placed in said medium at 37°C. The level of tracheal activity was determined by the percentage of beating cilia in each ring. The percentage of beating cilia in each ring was expressed as 4 (<25% beating cilia), 3 (25 to 50% beating cilia), 2 (50 to 75% beating cilia), 1 (75 to 99% beating cilia) or 0 (all cilia beating). A chick was considered affected or not affected of respiratory clinical signs based on the evaluation of the ciliary activity of 10 tracheal rings. For each ring, the ciliary activity was considered as normal when at least 50 % of the ciliary movement of the ring was vigorous. Each chick was considered not affected if at least 9 out of 10 rings showed normal ciliary activity. In contrast, a chick was considered affected if more than one ring showed not normal ciliary activity.

[0134] The results of this study demonstrated that all animals in the mock-up vaccinated groups (Group C and D) presented an affectation in the ciliary activity after challenge (8 out of 8 animals). On the contrary, vaccinated animals were protected from challenge regarding the respiratory clinical signs. Only 2 out of 8 animals in Group A (vaccinated MDA negative) and 1 out of 8 animals in Group B (vaccinated MDA positive) had some degree of affectation in their ciliary activity (Figures 1 and 2). The proportion of not vaccinated chickens (Groups C and D) that showed respiratory clinical signs was significantly higher from the proportion observed in the vaccinated ones (Groups A and B), either in presence or absence of MDA. Hence, the results clearly demonstrated that the live attenuated aMPV vaccine composition of the present invention is efficacious in protecting chicks against virulent challenge of aMPV. The results also demonstrated that the MDAs against aMPV did not interfere in the efficacy of a live attenuated aMPV vaccine.

[0135] Although not all vaccinated birds seroconverted to avian metapneumovirus (aMPV) after the administration of the vaccine composition, it is clear that animals vaccinated at one day of age were protected from a virulent challenge of aMPV, either in presence and absence of maternal derived antibodies (MDA) against aMPV based on the protection observed for the respiratory clinical signs assessed. The vaccine composition also showed an onset of immunization of 3 weeks post vaccination and a duration of immunity of at least 9 weeks post-vaccination. Accordingly, birds actively immunized against aMPV with the vaccine compositions of the invention are effectively protected in the worst-case scenario, at one-day of age. The vaccine composition demonstrated to be effective in reducing the detrimental effects caused by virulent avian metapneumovirus (aMPV) on the ciliary activity, manifested as a respiratory clinical sign.

[0136] EXAMPLE 2: Safety study of a live attenuated vaccine composition against avian metapneumovirus (aMPV) in laving hens

[0137] The aim of this study was to assess the safety of a vaccine composition comprising a live attenuated aMPV when it is administered to laying birds during the laying period (laying hens). A total of fifteen SPF (specific pathogen free) laying birds (Gallus gallus) seronegative to antibodies against aMPV were enrolled in this study. The laying birds enrolled in this study were 26 weeks of age, and they were vaccinated at the peak of egg production, typically around 30 weeks of age, which is the most stressful period for hens within the lay period, so it represented the worst-case scenario. Thus, four weeks after enrolment, at day 0 (DO) of the study, when the animals had 30 weeks of age, were vaccinated with one dose of a live attenuated aMPV vaccine composition by spray administration.

[0138] The animals received a vaccine composition based on a freeze-dried lyophlizate containing 107 23CCID50 (50% cell culture infectious dose) of the live attenuated aMPV strain JFB24, formulated in a composition comprising from 50 mg to 80 mg of dextran 70, from 30 mg to 50 mg of sucrose, from 30 mg to 50 mg of gelatin, from 20 mg to 35 m of NZ Amine, from 50 mg to 85 mg of sorbitol, from 0.05 mg to 0.50 mg of potassium dihydrogen phosphate, and from 0.50 mg to 2 mg of dipotassium phosphate. This formulation was reconstituted with clean, fresh, antiseptic and disinfectant free water to achieve a final concentration of 105 4CCIDso / ml to be administered to the animals. The vial was gently mixed until the freeze-dried lyophilizate was completely dissolved.

[0139] The vaccine composition was sprayed evenly over the laying birds at a distance of 30-40 cm and ensuring a droplet size of at least 120 pm. The vaccine composition was administered via the oculonasal route using a spray applicator (Spray Gun, Giordano Poultry Plast S.p.a) or any other spray applicator that allowed to spray under the same conditions and droplet size of at least 120 pm. For spray administration the volume of one dose of vaccine was 1 ml per animal.

[0140] The criteria for considering the vaccine composition to be safe to be used during laying period were: no animal should show abnormal systemic reactions or clinical signs, no animal should die from causes attributable to the vaccine, and no decrease in egg production or egg quality should be observed.

[0141] Animals were monitored individually on a daily basis for clinical signs, including general clinical signs (such as depression), respiratory clinical signs and nervous clinical signs, mortality, egg production and egg quality from 4 weeks before vaccination to 4 weeks after vaccination. All eggs laid produced by the vaccinated laying birds were collected at several timepoints post-vaccination (on days 4, 7, 11 , 14, 21 , and 28 postvaccination), the eggs laid were checked for albumen and yolk quality. Vaccine virus detection in eggcontent was also assessed by molecular methods (qRT-PCR). At the end of the study (day 28 postvaccination), animals were humanely euthanized and then necropsied to evaluate macroscopic lesions, with special attention to the reproductive tract. Serology of all the animals was performed with an ELISA assay to check the level of antibodies against aMPV just before vaccination, confirming that all animals were negative to anti-aMPV antibodies.

[0142] The results of the study demonstrated that no clinical signs or local reactions attributable to the administration of the vaccine composition were observed during the entire study period, thus indicating that the vaccine did not cause adverse reactions on the health status of the vaccinated laying hens during the laying period.

[0143] Egg production and egg quality parameters were obtained from daily records as percentage in respect to one egg per hen and day, assuming one egg / hen per day as the 100% of egg production. Percentage of weekly total, normal and abnormal egg were also recorded for each animal. Pre-vacci nation and postvaccination egg quality parameters were compared. The quality of the eggs was considered as normal if no abnormalities were observed. The common eggshell abnormalities assessed were: thin shell eggs, soft shell eggs, shell-less eggs, rough or corrugated shell eggs, small egg, abnormal calcareous deposits, eggs with dimples, depressions or ridging, blood-stained eggs or misshapen eggs.

[0144] The mean percentage of normal egg production of the laying hens was evaluated prior and after vaccination. The mean percentage of normal egg production 4 weeks prior to vaccination was 81 %. The results revealed an increase in normal egg production from 4 weeks prior to vaccination to one week prior to vaccination, from 70% to 94% of normal eggs, respectively, confirming that the hens were at the peak of lay at the time of vaccination. Following vaccination, egg production remained stable, with no statistically significant differences observed between the pre-vacci nation and post-vaccination periods. From 81 % of normal egg production in the 4-week pre-vacci nation period to 84% normal egg production in the 4-week postvaccination period.

[0145] Regarding the egg quality, the percentage of abnormal eggs remained also stable at 2% in both 4 weeks before and after vaccination. The percentage of egg production per week is shown in Tables 1 and 2.

[0146] These findings confirmed that a live attenuated aMPV vaccine composition when administered to laying hens did not negatively impact on their health status. More importantly, the vaccine composition did not affect the egg production and egg quality of the eggs laid produced by vaccinated laying hens.

[0147] Table 1: Mean percentage of normal and abnormal egg production during the 4 weeks prior to vaccination.

[0148] The eggs were also assessed for albumen or yolk quality parameters on days 4, 7, 11 , 14, 21 , and 28 postvaccination. The albumen and yolk quality of the eggs were monitored for the following yolk quality disorders: (1) blood spots, (2) pale yolk, (3) mottled yolk, (4) discolored yolk (blue-green), and (5) rubbery, cheesy or pasty yolk; and for the following common albumen quality problems: (1) thin, watery albumen, and (2) offcolor albumen (green or pink). The proportion of eggs presenting albumen or yolk quality problems is shown in Table 3.

[0149] No alterations on albumen and / or yolk quality were observed after vaccination. Thus, the results of the study confirmed that a vaccine composition comprising a live attenuated aMPV when administered to laying hens did not impact negatively on the albumen and yolk quality of the eggs laid produced by vaccinated laying hens during the laying period.

[0150] Table 3 Proportion of eggs presenting albumen or yolk quality disorders on days 4, 7, 11, 14, 21 and 28 of the study (post-vaccination). aMPV vaccine virus detection in egg content (albumen and yolk) was also assessed on days 4, 7, 11 , 14, 21 , and 28 post-vaccination by qRT-PCR. The results of the study demonstrated that the aMPV vaccine virus was not detected in any of the assessed eggs at any timepoint (Table 4), thus confirming that the live attenuated aMPV vaccine virus was not spread to the laid eggs. This feature is relevant considering that the vaccine composition comprises a live attenuated virus, thus guarantying the safety of this novel attenuated aMPV vaccine composition when it is administered to laying hens, including the laying period, by the fact that the vaccine virus is not transferred to the laid eggs of vaccinated laying birds.

[0151] Table 4: Proportion of eggs with a positive detection of the vaccine virus.

[0152] At day 28 post-vaccination all animals were humanely euthanized and necropsied for assessing eventual clinical lesions at the reproductive tract. The study showed no relevant lesions in the reproductive tract for any vaccinated laying hen. All vaccinated laying hens presented an active and functional reproductive tract, well developed left oviduct and ovary with mature follicles. Thus, no reproductive disorders associated with vaccination were observed.

[0153] Overall, the results of this study demonstrate that one dose of a vaccine composition comprising a live attenuated aMPV is safe to be administered in laying birds during the laying period. No clinical signs, mortality, alterations in the egg production and egg quality, no affectation in the albumen and yolk quality, and no lesions in the reproductive tract were observed. Moreover, no vaccine aMPV virus was detected in the egg content of the eggs laid produced by the vaccinated laying hens. Thus, confirming the safety of the live attenuated aMPV compositions of the invention when administered in laying birds.

[0154] Although not all vaccinated laying hens seroconverted to avian metapneumovirus (aMPV), it is clear that animals vaccinated at one day of age (Example 1), corresponding to the worst-case scenario, with the same live attenuated aMPV vaccine composition were protected from a virulent challenge of aMPV, either in presence and absence of maternal derived antibodies (MDA) against aMPV. Thus, future laying hens derived from said one-day old chickens are also effectively protected. The vaccine composition also showed an onset of immunization of 3 weeks post vaccination and a duration of immunity of at least 9 weeks postvaccination.

[0155] Accordingly, laying hens immunized against aMPV with the vaccine compositions of the invention are effectively protected, the vaccine composition demonstrated to be effective in reducing the detrimental effects caused by virulent avian metapneumovirus (aMPV) on the ciliary activity, manifested as a respiratory clinical sign. Thus, the compositions of the invention are particularly useful for vaccinating laying birds during the laying period.

[0156] REFERENCES CITED IN THE APPLICATION

[0157] WOAH Terrestrial Manual 2022, Chapter 3.3.15, Turkey Rhinotracheitis (Avian Metapneumovirus Infections)

[0158] Sugiyama, Mikl et al. 2006. "Drop of Egg Production in Chickens by Experimental Infection with an Avian Metapneumovirus Strain PLE8T1 Derived from Swollen Head Syndrome and the Application to Evaluate Vaccine.” Journal of Veterinary Medical Science 68(8): 783-87. Handbook of pharmaceutical excipients, R.C. Rowe, P.J. Sheskey and M.E. Quinn, editors, Pharmaceutical Press, sixth edition, 2009

Claims

CLAIMS1. An immunogenic or vaccine composition comprising a live attenuated avian metapneumovirus (aMPV) for use in the prevention and / or treatment of an aMPV infection, wherein said use comprises administering the composition to laying birds.

2. An immunogenic or vaccine composition comprising a live attenuated aMPV for use in active immunization of laying birds against aMPV.

3. The immunogenic or vaccine composition for use according to any one of claim 1 to 2, wherein the birds are selected from the group consisting of chicken, pullet, pheasant, turkey, goose, duck, broiler, quail, pigeon, swan, and ostrich.

4. The immunogenic or vaccine composition according to any one of claims 1 to 3, wherein the birds are poultry birds, in particular, the birds are selected from chicken hens and turkey hens.

5. The immunogenic or vaccine composition for use according to any one of claims 1 to 4, wherein said use comprises administering the composition to chicken hens of at least 18 weeks of age or to turkey hens of at least 29 weeks of age.

6. The immunogenic or vaccine composition according to claim 5, wherein said use comprises administering the composition to chicken hens of at least 20 weeks of age or to turkey hens of at least 30 weeks of age.

7. The immunogenic or vaccine composition for use according to any one of claims 1 to 6, wherein said use comprises a first administration of the composition to birds of at least 1 day of age or at least 7 days of age.

8. The immunogenic or vaccine composition for use according to claim 7, wherein said use comprises a first administration of the composition to birds of at least 1 day of age by oculonasal route or at least 7 days of age by drinking water.

9. The immunogenic or vaccine composition for use according to any one of claims 7 or 8, wherein said first administration comprises a single dose of the composition administered to birds of at least 1 day of age or at least 7 days of age.

10. The immunogenic or vaccine composition for use according to claim 9, wherein after the first single dose a further single dose of the composition is administered to the birds every 9 weeks.

11. The immunogenic or vaccine composition for use according to any one of claims 1 to 10, wherein the composition is administered by oculonasal route or oral route.

12. The immunogenic or vaccine composition for use according to claim 11 , wherein the composition is administered by oculonasal route via spray or by oral route via drinking water.

13. The immunogenic or vaccine composition for use according to any one of claims 1 to 12, wherein said use comprises the following administration regime:I) administering the composition by oculonasal route via spray to birds of at least 1 day of age or by drinking water to birds of at least 7 days of age, and ii) administering the composition once every nine weeks after the first administration.

14. The immunogenic or vaccine composition for use according to any one of claims 1 to 13 wherein said use does not comprise administering an inactivated aMPV vaccine.

15. The immunogenic or vaccine composition for use according to any one of claims 1 to 14, wherein said composition elicits an immune response against an aMPV infection from at least 3 weeks after administration until at least 9 weeks after administration.

16. The immunogenic or vaccine composition for use according to any one of claims 1 to 15, wherein said composition reduces the detrimental effect caused by an aMPV infection on the respiratory tract.17 The immunogenic or vaccine composition for use according to claim 16, wherein the detrimental effect caused by an aMPV infection is on the ciliary activity.

18. The immunogenic or vaccine composition for use according to any one of claims 1 to 17, wherein said use is for the prevention and / or treatment of a disease caused by an aMPV infection.

19. The immunogenic or vaccine composition for use according to claim 18, wherein the disease is selected from the group consisting of avian rhinotracheitis and swollen head syndrome.

20. The immunogenic or vaccine composition for use according to any one of claims 1 to 19, wherein said composition prevents and / or reduces at least one clinical sign associated to an aMPV infection selected from the group consisting of depression, anorexia, ruffled feathers, head shaking, head scratching, nervous signs, apathy, cerebral disorientation, torticollis, opisthotonos, reduced growth, mortality, dyspnea, oculardischarge, nasal discharge, swollen infra-orbital sinuses, respiratory distress, conjunctivitis, drop in egg production, drop in egg quality, egg abnormalities such as egg size abnormalities, egg shell abnormalities, egg yolk abnormalities, rubbery yolk, cheesy yolk, pasty yolk, egg albumen abnormalities, and combinations thereof.21 . The immunogenic or vaccine composition for use according to any one of claims 1 to 20, wherein said composition prevents or reduces the detrimental effect of an aMPV infection in egg production and / or egg quality.

22. The immunogenic or vaccine composition for use according to claim 21 , wherein said composition prevents and / or reduces at least one egg abnormality associated to an aMPV infection selected from the group consisting of thin shell eggs, soft shell eggs, shell-less eggs, rough or corrugated shell eggs, small eggs, abnormal calcareous deposits, eggs with dimples, depressions or ridging, blood-stained eggs, and misshapen eggs.

23. The immunogenic or vaccine composition for use according to claim 21 , wherein said composition prevents and / or reduces the detrimental effect caused by an aMPV infection in at least one egg yolk quality parameter selected from the group consisting of blood spots, pale yolk, mottled yolk, discolored yolk (bluegreen), rubbery yolk, cheesy yolk, and pasty yolk.

24. The immunogenic or vaccine composition for use according to claim 21 , wherein said composition prevents and / or reduces the detrimental effect caused by an aMPV infection in at least one egg albumen quality parameter selected from the group consisting of thin albumen, watery albumen, and off-color albumen (green or pink).

25. The immunogenic or vaccine composition for use according to any one of claims 1 to 24, wherein the aMPV is aMPV subtype B.

26. The immunogenic or vaccine composition for use according to any one of claims 1 to 25, wherein the aMPV has been attenuated byI) serial passages, in embryonating eggs, tracheal organ cultures or in chicken embryo fibroblasts, or by alternate passages using a combination thereof; andII) subsequent serial passages in VERO cells.

27. The immunogenic or vaccine composition for use according to any one of claims 1 to 26, wherein the composition comprises an amount of antigen per dose equivalent to a titre of at least 101 0CCID50.

28. The immunogenic or vaccine composition for use according to claim 27, wherein the composition comprises an amount of antigen per dose equivalent to a titre of at least 101 8.

29. The immunogenic or vaccine composition for use according to any one of claims 1 to 26, wherein the composition comprises an amount of antigen per dose equivalent to a titre of about 101 0to about 108 0CCID5030. The immunogenic or vaccine composition for use according to any one of claims 1 to 29, wherein aMPV is aMPV subtype B strain JFB24 deposited under the Budapest Treaty in the Collection Nationale de Cultures de Microorganismes (CNCM) under the Accession Number CNCM I-6069.31 . The immunogenic or vaccine composition for use according to any one of claims 1 to 30, wherein the composition further comprises a pharmaceutically acceptable excipient and / or a carrier selected from the group consisting of dextran 70, sucrose, gelatine, NZ amine, sorbitol, potassium dihydrogen phosphate, dipotassium phosphate, and combinations thereof.

32. The immunogenic or vaccine composition for use according to any one of claims 1 to 31, wherein the composition is a freeze-dried lyophilizate.

33. The immunogenic or vaccine composition for use according to any one of claims 1 to 32, wherein the immunogenic or vaccine composition further comprises at least one antigen against another avian pathogen.

34. The immunogenic or vaccine composition for use according to claim 33, wherein the other avian pathogen is selected from the group consisting of Infectious Bronchitis Virus (IBV), Egg Drop Syndrome virus (EDS virus), Newcastle disease virus (NDV), coccidia, Eimeria spp., infectious bursal disease virus (IBDV), and combinations thereof.

35. A vaccination kit comprising an immunogenic or vaccine composition as defined in any one of the preceding claims for use in the prevention and / or treatment of an avian metapneumovirus (aMPV) infection, wherein said use comprises administering the composition to laying birds.

36. The vaccination kit for use according to claim 35, wherein the kit comprises: a) at least one vial with a freeze-dried lyophilizate comprising the immunogenic or vaccine composition as defined in any one of claims 1 to 34, and b) a leaflet with instructions for the administration of the composition via spray or via drinking water.

37. The vaccination kit for use according to claim 36, wherein the kit comprises a vial with one dose or multiple doses of the immunogenic or vaccine composition as defined in any of claims 1 to 34.

38. The vaccination kit for use according to claim 37, wherein the kit comprises a vial with at least 1,000 doses, at least 2,000 doses, at least 5,000 doses, or at least 10,000 doses of the immunogenic or vaccine composition as defined in any of claims 1 to 34.

39. The vaccination kit for use according to claim 38, wherein the kit further comprises a vial comprising an additional immunogenic or vaccine composition comprising at least one antigen against another avian pathogen.

40. The vaccination kit for use according to claim 39, wherein the other avian pathogen is selected from the group consisting of Infectious Bronchitis Virus (IBV), Egg Drop Syndrome virus (EDS virus), Newcastle disease virus (NDV), coccidia, Eimeria spp., infectious bursal disease virus (IBDV), and combinations thereof.41 . An immunogenic or vaccine composition for use according to any one of claim 1 to 34 or a vaccination kit for use according to any one of claims 35 to 40, in combination therapy with a further avian vaccine.

42. The immunogenic or vaccine composition or the vaccination kit for use according to claim 41 , wherein the further avian vaccine comprises at least one antigen against another avian pathogen selected from the group consisting of Infectious Bronchitis Virus (IBV), Egg Drop Syndrome virus (EDS virus), Newcastle disease virus (NDV), coccidia, Eimeria spp., infectious bursal disease virus (IBDV), and combinations thereof.

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