WO2007020017A1

WO2007020017A1 - Composition for bird immunization administered by aerosol

Info

Publication number: WO2007020017A1
Application number: PCT/EP2006/007965
Authority: WO
Inventors: José Francisco RODRIGUEZ AGUIRRE; Ana Maria Ona Blanco; Juan José BERNAL MUNOZ; Juan Ramon Rodriguez Fernandez-Alba
Original assignee: Bionostra, S.L.
Priority date: 2005-08-12
Filing date: 2006-08-11
Publication date: 2007-02-22

Abstract

The present invention relates to a pharmaceutical composition comprising empty virus-like particles (VLPs) and, administered via aerosol, producing a protective immune response in birds. The invention likewise describes the incorporation of an adjuvant in the vaccine formulation of the invention which significantly increases the resistance of birds to the IBDV infection.

Description

COMPOSITION FOR BIRD IMMUNIZATION ADMINISTERED BY AEROSOL

Field of the Invention

The invention, in general, relates to pharmaceutical compositions administered by aerosol and particularly, to aerosol formulations for the administration of immunogens by inhalation, for prophylactic or therapeutic treatment of avian infections .

Background of the Invention

The field of animal breeding is currently subjected to a strong commercial pressure directed towards the maximum reduction in production costs. Due to this situation, only those farms provided with modern installations allowing animal breeding with very high density populations are economically viable. This type of installation, especially developed in bird exploitations, facilitates the occurrence and rapid spreading of epidemic outbreaks, with the subsequent sanitary and economic risk that this entails. Due to this reason, one of the priority objectives of this area of poultry farming is focused on obtaining new vaccines which, apart from inducing a high protection degree, are safe and can be applied massively.

For most of the avian infectious diseases of interest, the vaccines in use are based on the use of an attenuated (live vaccines) or an inactivated (inactivated vaccines) version of the pathogen causing the disease as an lmmunogen. In the case of live vaccines, its application is carried out by different procedures; by means of drinking water, eye drops or by sprinkling. Their major drawback is that, since they are formed by live microorganisms, they can cause diseases, mostly when administered to stressed birds or birds affected by another type of concurrent infections. On the other hand, inactivated vaccines have the advantage of being a non-infectious material, therefore of great biosafety, nevertheless, their mam drawback is that their effectiveness is almost exclusively limited to their administration by a parenteral route, with the subsequent increased cost entailing the individual handling of birds during the vaccination process.

Due to all this, an inactivated vaccine administered by aerosol would allow: i) completely eliminating the biosafety risks associated to the use of live vaccines and 11) immunizing massively without the need for a complex and expensive handling of birds .

An aerosol is a system which allows dispersing colloidal liquid or solid particles in a gas (e.g. air), said particles being small enough to have a slow fall and capable of remaining stable in air for sufficiently long periods of time. When the particles in suspension, with a size (diameter) comprised between 2 and 5 μm, are breathed in by the animals, they are deposited in the respiratory tract at the level of the bronchi and can reach the alveoli. The vaccination by aerosol is capable of inducing a high immune response of mucous membranes. Several cases of attenuated live vaccine administration by aerosol have been described; nevertheless, bird immunization by means of the administration by aerosol of inactivated antigens, viral subunits or empty viral capsids or virus-like particles (VLPs) has not been described to date. Assays related to the administration by aerosol of subunit vaccines have not provided an effective protection (CW. Purdy et al . , Current Microbiology, (1998), Vol. 37, page 5). VLPs constitute an alternative to the use of attenuated live vaccines and of recombinant subunit vaccines. VLPs are obtained by the self- assembly of the subunits forming the viral capsid and mimic the structure and antigenic properties of the native virion although they lack genetic material so they are incapable of replicating.

Infectious bursal disease (IBD), or Gumboro disease, is an acute-course viral disease which mainly affects industrially fattened chickens (broilers) in a growth stage (3-8 weeks). This disease is caused by the infectious bursal disease virus (IBDV), also known as the Gumboro disease virus, having a very pronounced tropism by the lymphoid cells located in the bursa of Fabricius, giving rise to a selective elimination of the B- lymphocyte precursors present in the bursa of Fabricius, which leads to an immunosuppression of the affected animals. The severity of the disease depends both on the degree of virulence of the viral strain and on the immunological state of the infected animal and its clinical consequences range between establishing a transitory immunosuppression, which essentially means a reduction in growth rates and a decrease in the capacity of response against infection and vaccination against other avian pathogens, and a severe immunosuppression leading to the death of a high percentage of the infected animals. Therefore, the infection by IBDV causes high mortality rates due to two causes: i) the infective IBDV cycle itself, and ii) opportunist infections of other pathogens affecting immunodepressed animals. The immunosuppressant effect of IBDV also decreases the response of the animals to vaccines against other avian pathogens.

The economic importance of the disease mainly lies in these two aspects: on the one hand, the high mortality produced by several IBDV strains in chickens of 3 weeks of age or even more and, on the other hand, the second clinical indication of the disease consisting of a prolonged immunosuppression of infected birds in early ages. The main aftereffects associated to said immunosuppression are: gangrenous dermatitis, anemia- hepatitis syndrome with inclusion bodies, E. coli infections and failures in the efficacy of other vaccinations such as those against Newcastle disease and infectious bronchitis.

As a general rule, the outbreaks of this disease affect virtually all the animals present in the affected farms. IBD is currently the most important viral disease for the poultry industry, its distribution scope is worldwide and IBD outbreaks have been reported in all producing countries with a high mortality (50-75%) produced by very virulent strains of the virus . The IBDV virions are extraordinarily resistant to the environmental conditions and persist in the environment for a period of at least 4 months. The virus is transmitted through water, food or excrements, but there is no vertical transmission through eggs and there are no chronic carriers of this disease. Therefore, the success in IBD control lies in the application of strict hygiene and installation disinfection programs together with the preventive vaccination of breeders and offspring. The immunization of breeding birds is especially important for the transmission of passive immunity to the offspring; nevertheless, the presence of said passive immunity can interfere in the efficacy of the offspring vaccination.

The vaccination programs against IBDV involve a double action: i) vaccination of laying hens; and ii) vaccination of broilers. The laying hens receive an inactivated vaccine dose at 16 or 20 weeks of age, followed by one or more vaccinations with an attenuated live vaccine at different time intervals. The basic purpose of this vaccination is the transmission to descendants of maternal antibodies capable of neutralizing the ineffectiveness of the virus for the first 3-4 weeks of the life of the chicken through the yolk sac. From 2 weeks of life onwards, the chickens are vaccinated (with one or several doses) by means of attenuated live vaccine administration in drinking water .

IBDV is the prototype of the Avibirnavirus genus of the Birnavindae family, a double-stranded RNA virus (dsRNA) . The IBDV virions have a size of approximately 70 nm (60-65 nm) , do not have lipid coating and have an icosahedral symmetry. The genome has an encoding capacity for five proteins VPl, VP2, VP3, VP4 and VP5. The capsid is formed by the VP2 protein whereas the VP3 and VPl polypeptides (the latter has RNA polymerase activity) are found associated to the RNA of the virion.

The serological response to the virus is mainly directed to the VP2 protein. In fact, most monoclonal antibodies capable of neutralizing the ineffectiveness of IBDV recognize conformational epitopes in the VP2 protein and are therefore neutralizing antibodies.

Vaccines containing attenuated live IBDV (US 5,632,989) and vaccines with inactivated IBDV capable of producing protection against field strains have been described Likewise, recombinant subunit vaccines containing the VP2 protein of IBDV expressed in various systems, for example, bacteria, yeasts, virus, etc, normally in the form of fusion proteins have been described (US 5,605,792, US 5,605,827). On the other hand, the possibility that VP2 forms VLPs in combination with the entire or part of the VP3 of IBDV is known (US 5,788,970, Hu et al., 1999, Biotechnology and Bioengineering, 63(6), 721-729; Wang et al., 2000, Biotechnology and Bioengineering, 67(1), 104-111; Martmez-Torrecuadrada et al., 2000a, Clin. Diagn. Lab. Immunol., 7 (4 ): 645-651 ; Martinez-Torrecuadrada et al., 2000b, Virology 278:322-331; Cheng et al . , 2001 Biotechnol . Prog., 17, 318-325) . Likewise, the obtaining and use of VLPs of IBDV as vaccines have been described, see, for example, US patent 5,788,970, patent publications WO2004087900, WO2005/071068, WO2005/071069 and WO2005/105834 , Spanish patent application P200501733, etc.

It has now been observed surprisingly, that the administration by aerosol of vaccines consisting of VLPs of IBDV immunize and protect 1 day old chickens against the infection caused by IBDV. Likewise, the inventors have observed that incorporating an adjuvant in the vaccine formulation significantly increases the resistance of chickens to the IBDV infection .

Brief Description of the Figures Figure 1 is a bar diagram showing the survival of the animals in different treatment groups in day 31, after subjecting the different groups of chickens to an experimental challenge (for more information, see the Example accompanying this description) . Figure 2 is a bar diagram showing the weight evolution of the animals of different experimental groups of animals throughout the assay; the weight of the animals was measured on days 7, 14, 21 and 31 (for more information, see the Example accompanying this description) . Figure 3 is a bar diagram showing animal growth after subjecting them to an experimental challenge (for more information, see the Example accompanying this description) .

Figure 4 is a bar diagram showing the bursa of Fabπcius/body weight ratio index of the animals surviving the experimental challenge (for more information, see the Example accompanying this description) .

Figure 5 is a bar diagram showing antibody titles at different times post-vaccination and post-challenge. Tl, PBS diluent; T2, 50 μg of VLPs in PBS; T3, 50 μg of VLPs + CPGS in PBS; and T4, 100 μg of VLPs in PBS;

Detailed Description of the Invention

In one aspect, the invention relates to a formulation for an aerosol, hereinafter formulation of the invention, comprising empty virus-like particles (VLPs) producing a protective immune response in the birds to which it is administered in a liquid. In a particular embodiment, the formulation of the invention is a pharmaceutical formulation for the administration to birds, by inhalation, of said VLPs producing a protective immune response in the birds to which it is administered, comprising VLPs, typically in a pharmaceutically effective amount, and a pharmaceutically acceptable liquid for the administration of said VLPs by inhalation.

As used herein, the term "VLPs producing a protective immune response in birds to which it is administered", hereinafter called, for simplicity, "protective VLPs", refers to protein structures formed by the assembly of subunits forming the viral capsid or fragments thereof, mimicking the antigenic structure and properties of the native virion although they lack genetic material, having the ability to induce or produce a protective immune response in the birds to which said protective VLPs are administered. Generally, said protective VLPs come from viruses capable of infecting birds, for example, avian viral pathogens. Illustrative and non-limiting examples of avian viral pathogens include IBDV, Marek, bird flu, Newcastle, encephalomyelitis, rhmotracheitis, reovirus. Optionally, said protective VLPs can include one or more heterologous peptides that contain one or more antigens of one or more avian pathogens for the purpose of producing a protective immune response in the birds to which said protective VLPs against the avian pathogens, the antigens or antigenic determinants of which are present in said protective VLPs, are administered. In this way, it is possible to simultaneously immunize or protect birds against one, two or more avian pathogens. Bacterium, fungi, parasites, etc. are found amongst said avian pathogens. Illustrative, non- 1uniting examples of avian pathogens generally include the pathogens causing bird flu, rhmotracheitis, coccidiosis, Marek, Newcastle, encephalomyelitis, reovirus, salmonellosis, etc. In a particular embodiment, said protective VLPs are VLPs formed by the assembly of one or more structural proteins of the IBDV capsid or of fragments thereof capable of assembling and forming VLPs producing a protective immune response in the birds to which they are administered. Illustrative, non-limitmg examples of VLPs potentially useful for immunizing birds include the VLPs of IBDV described, for example, in US 5,788,970, Hu et al., 1999, Biotechnology and Bioengineering, 63(6), 721-729; Wang et al., 2000, Biotechnology and Bioengineering, 67(1), 104-111; Martmez-Torrecuadrada et al . , 2000a, Clin. Diagn. Lab. Immunol., 7 (4 ): 645-651 ; Martmez-Torrecuadrada et al., 2000b, Virology 278:322-331; Cheng et al . , 2001 Biotechnol. Prog., 17, 318-325, and especially, the VLPs described in the international patent publications WO2004087900, WO2005/071068 , WO2005/071069, WO2005/105834, and in the Spanish patent application P200501733. By way of illustration, these patent publications describe: - VLPs formed by the assembly of pVP2 , VP2, VP3 and VPl of IBDV, called "complete VLPs" in WO2004087900;

VLPs formed by the assembly of VP2 and VP3 of IBDV, called

VLPs (-VP4) in WO2005/071068 ;

VLPs formed by the assembly of fragments of VP2 of IBDV, called VLPs-pVP2* in WO2005/105834 ;

VLPs of IBDV further comprising an antigen of an avian infectious agent different from IBDV, such as those called

CVLPs in WO2005/071069 or CVLPs (pVP2*) in Spanish patent application P200501733. The term "complete VLPs" refers to empty virus-like particles derived from IBDV which are formed by the self- assembly of all the proteins present in purified and infective IBDV virions, specifically, the pVP2, VP2, VP3 and VPl proteins of IBDV. The term "VLPs (-VP4)" refers to empty virus-like particles derived from IBDV and formed by the self-assembly of only, pVP2 and VP3 of IBDV, therefore they lack VP4 of IBDV, and, for this reason, they are called VLPs (-VP4) .

The term "VLPs-pVP2*" refers to chimeric empty virus-like particles derived from IBDV as a consequence of the expression of the IBDV pVP2 protein, or a fragment of said protein which is capable of self-assembling and forming said virus-like particles, genetically modified to include a nucleotide sequence encoding a heterologous polypeptide comprising a polypeptide of interest.

The term "CVLPs" refers to empty virus-like particles derived from IBDV which are formed by the proteins pVP2 and VP3 of IBDV, the latter genetically modified to include the nucleotide sequence encoding a heterologous polypeptide comprising a polypeptide of interest. The resulting CVLPs are formed by the assembly of (i) pVP2 proteins of IBDV and (ii) fusion proteins comprising a region A formed by the VP3 protein of IBDV bound to a region B formed by a heterologous polypeptide comprising a polypeptide of interest. Therefore, in a particular embodiment, the formulation of the invention is useful for protecting birds against IBDV. Illustrative, non-limiting examples of birds which can be infected by said virus include chickens, turkeys, geese, ganders, pheasants, quails, ostriches etc. In a particular and preferred embodiment, the formulation of the invention is useful for immunizing chickens via aerosol by inhalation against the infection caused by IBDV.

The liquid in which said protective VLPs are found can be any liquid in which said protective VLPs can be dispersed, preferably, a pharmaceutically acceptable liquid, typically a suitable solvent for pharmaceutical use; by way of a non- limiting illustration, said liquid can be a polar fluid, including a protic fluid, such as water or an aqueous liquid medium, for example, phosphate buffered saline (PBS) solution or alternately, a non-aqueous liquid medium. In a particular embodiment, the formulation of the invention comprises a dispersion of said protective VLPs in PBS, or any other pharmaceutically acceptable aqueous solvent.

The term "aerosol", as defined herein, refers to a formulation which can be supplied in the form of a solid and/or liquid suspension (or dispersion) in a gas. Although virtually any gas can be used, in practice the use of pharmaceutically acceptable gases, for example, air, etc., is preferred. The aerosols can be prepared by conventional methods known by persons skilled in the art. By way of illustration, the aerosols can be prepared from suspensions of said protective VLPs in a liquid using a conventional device, such as a nebulizer which atomizes said suspensions. In the case of a nebulized aerosol, the dispersion essentially comprises said VLPs producing a protective immune response in the birds to which they are administered essentially moistened or humidified in air.

The formulation of the invention can also contain an adjuvant for the purpose of enhancing the immune response against the antigen or antigens administered to the birds. Virtually any adjuvant that can be administered by aerosol can be used. Advantageously, said adjuvant is an adjuvant enhancing the response in mucous membranes. By way of a non-limiting illustration, said adjuvant can be an immunomodulating agent, e.g., an oligodeoxynucleotide (ODN), preferably, an ODN comprising one or more cytosine-guanine trinucleotides (CpG) , an enterotoxin, etc.

The formulation of the invention can also contain, if necessary, other pharmaceutically acceptable auxiliary substances or compounds in addition to the protective VLPs and the liquid, such as co-solvents, additives for stabilizing the solution, e.g., pharmaceutically acceptable preservatives, pharmaceutically acceptable acids, bases or buffers for adjusting the pH, surfactants, etc. Likewise, it is possible to add metal chelating agents to stabilize the suspension or to adjust the quality of the aerosol. The solubility and/or stability of VLPs in the liquid of the formulation of the invention can be increased by adding additional substances, for example, amino acids such as aspartic acid, glutamic acid etc. These pharmaceutically acceptable substances which can be used in the formulation of the invention are generally known by the persons skilled in the art and are usually used in the manufacture of formulations for aerosols. Additional information on these substances can be found in Galenic or animal health treatises, for example, in the book "Tratado de Farmacia Galemca", by C. Fauli i Trillo, 10 Edicion, 1993, Luzan 5, S. A. de Ediciones .

The amount of protective VLPs present in the formulation of the invention can vary within a wide range. Preferably, said range is 10-150 micrograms. Generally, said amount will be an amount such that, when the aerosol is produced, each bird receives a therapeutically effective amount of protective VLPs In the sense used in this description, the expression "therapeutically effective amount" refers to the amount of protective VLPs calculated to produce the desired effect and it is generally determined, amongst other factors, by the characteristic features of the VLPs used and the immunization effect to be achieved.

The formulation of the invention is useful for administering a reagent (lmmunogen) capable of producing a protective immune response in the birds to which it is administered, specifically, the previously defined protective VLPs. Assays carried out by the inventors (see Example) have shown that the formulation of the invention serves to carry immunogens of avian pathogens to mucous membranes and to the respiratory tract of birds to which they are administered.

The formulation of the invention can be supplied in the form of a suspension of said protective VLPs and a liquid in a gas (aerosol) . In a particular embodiment, said protective VLPs are VLPs of IBDV, as defined previously, including, optionally, one or more heterologous peptides containing one or more antigens of one or more avian pathogens. By way of a non- 1uniting illustration, m a specific embodiment, said VLPs are VLPs of IBDV formed by the assembly of fusion proteins comprising a fragment of the pVP2 protein of IBDV (residues 1- 456) bound to a heterologous peptide (his-tag) formed by 28 amino acid residues including 6 consecutive histidme residues, whose amino acid sequence is set forth in SEQ ID NO: 1 obtained according to the process described in the Spanish patent application P200501733; said liquid is PBS, and an ODN of 20 nucleotides containing various CpG (SEQ ID NO: 2) motifs is used as an adjuvant. Said VLPs can be administered to birds in widely variable amounts; nevertheless, in a particular embodiment, the VLP dose administered to each bird is 50 μg whereas in another particular embodiment it is 100 μg/bird. The formulation of the invention can be prepared by conventional methods, mixing the protective VLPs with the liquid and adding, if that is the case, the desired adjuvants and/or auxiliary substances.

The formulation of the invention is administered by inhalation in the form of aerosol (suspension of said protective VLPs and a liquid in a gas), forming an additional aspect of this invention. The features of said protective VLPs, liquid and gas, have been mentioned previously. Said aerosol can be obtained by conventional methods. By way of illustration, it can be prepared from a suspension of protective VLPs in a liquid, for example, water or an aqueous solution such as PBS, using a nebulizer. Briefly, the protective VLP doses to be administered are resuspended in an aqueous solvent (diluent), e.g., PBS, in the suitable amounts. The available dose per bird is generally calculated on the basis of the concentration of the protective

VLPs, the volume and the nebulization time. Any suitable conventional nebulizer which allows obtaining mhalable drops

(particles) of a suitable size can be used for the nebulization.

As it is known, a nebulizer is an apparatus capable of generating very fine liquid drops for their inhalation. Within this apparatus, the dispersion containing the protective VLPs is atomized in a plurality of drops by known methods, for example, by using compressed air, ultrasound, etc. The drops (or particles) generated by the nebulizer have a size such that it is allowed for them to be inhaled by the birds. In a particular embodiment, said drops (particles) have a particle size comprised between 1 and 5 μm. Said particles are inhaled by the birds and they develop a humoral response against the administered antigen. If desired, the formulation of the invention as well as the suspension of said protective VLPs and a liquid in a gas (aerosol of the invention) can include one or more antigens of one or more avian pathogens, separated from said VLPs; said antigens can be in the form of pharmaceutical formulations or compositions; nevertheless, said antigens (pharmaceutical formulations or compositions) must be suitable for their administration to the birds by inhalation by means of aerosol.

In another aspect, the invention relates to the use of said VLPs producing a protective immune response in the birds to which they are administered (protective VLPs) in the manufacture of an aerosol formulation for the immunization of birds against at least one avian infectious agent. In a particular embodiment, said avian infectious agent is a pathogenic bird virus such as, for example, IBDV, Marek, bird flu, Newcastle, encephalomyelitis, rhmotracheitis reovirus, etc. In another aspect, said formulation for aerosol comprises two or more antigens of two or more avian pathogens capable of inducing a protective immune response in the bird to which it is administered; generally, one of said antigens will be the viral antigen forming the protective VLPs and the others can be included in the VLP structure, either adhered or separated.

In another aspect, the invention relates to the use of said VLPs producing a protective immune response in the birds to which it is administered in the manufacture of a pharmaceutical composition for its administration as an aerosol. In a particular embodiment, said pharmaceutical composition is a vaccine useful for immunizing birds against one or more avian infectious agents. Generally, said pharmaceutical composition is administered to the bird by inhalation after the formation of the aerosol by means of nebulization . In a particular embodiment, said avian infectious agent is a pathogenic bird virus such as, for example, IBDV. In another aspect, said pharmaceutical composition administered as aerosol comprises two or more avian pathogens capable of inducing a protective immune response in the bird to which it is administered; generally, one of said antigens will be the viral antigen forming the protective VLPs and the others can be included in the VLP structure, either adhered or separated as mentioned previously.

In another aspect, the invention relates to a method for producing a protective immune response in a bird comprising the administration, in the form of aerosol, of a therapeutically effective amount of protective VLPs to said bird by inhalation. A way to immunize chickens by means of inhalation of VLPs of IBDV in an aerosol generated by nebulization of an aerosol formulation provided by this invention is described in a detailed manner in the Example accompanying this description.

The following example illustrates the invention and must not be considered as limiting the scope thereof.

EXAMPLE

Immunization of birds by means of a subunit vaccine against infectious bursal disease virus (IBDV) administered by aerosol

The tolerance and clinical and anatomopathological effectiveness of an experimental vaccine based on VLPs of IBDV applied by aerosol to chickens has been studied and compared with a non-vaccinated control group (placebo group) .

I. MATERIAL AND METHODS A. Experimental groups

Animals and groups

One-day old White-Leghorn chickens (mdistinctively males and females) obtained from the incubation of SPF eggs (Spafas or Lhoman) were used. Three treatment groups plus a control group without treatment were formed, each group formed by 27 animals assigned by weight and randomly to the different treatments. All the animals subjected to the treatments were free of antibodies against IBDV and Salmonella spp.

In order to form the experimentation group, only those animals that were viable were selected, i.e., those having a homogeneous weight (the weights furthest from the average were rejected) and not showing clinical signs before the application of the experimental subunit vaccine based on VLPs of IBDV. The animals which were ill during the experimental process due to causes that were not attributable to the administered treatment were excluded, weighed and subjected to an autopsy in order to determine any possible reaction with the VLPs. The animals were sacrificed by means of the intravenous administration of 150 mg/kg w.v. of sodium pentothal. Animal Control and isolation units

The birth, breeding of chickens and the accommodation of the animals during the experimental part were carried out in isolation units belonging to the Animal Health Laboratory of Barcelona of the Department of Agriculture, Cattle Raising and Fishing of Catalonia (Departament d 'Agricultura, Ramaderia i Pesca de the Generalitat de Catalunya (DARP) ) . The installation has 4 isolated habitats (A1-A4) which have a filtered air system at negative pressure BSL3 (Biosafety Level 3) and it is equipped with a waste treatment system by autoclave. The different groups of 27 birds were housed in independent habitats with an approximate space of 0.1 rrr per animal.

The premises have a ventilation system by filtered air and an environmental temperature control system by heat pump which allows maintaining an approximate room temperature of 22°C. Furthermore, during the first days of life of the chickens, a heating lamp system was installed in each compartment for the purpose of providing localized areas with a focal temperature of approximately 35°C.

Acelima tization

Treatments prior to the test were not applied and the animal acclimatization was carried out in the same installations in which the chickens were incubated and born.

B. Assay substances

Experimental subunit vaccine (hereinafter VLPs): Protein structures based on VLPs of IBDV formed by the assembly of fusion proteins comprising a fragment of the pVP2 protein of IBDV (residues 1-456) bound to a heterologous peptide (his- tag) formed by 28 residues of amino acids among which 6 consecutive histidine residues are included; the "his-tag" ammo acid sequence is set forth in SEQ ID NO: 1; said VLPs have been obtained according to the process described in the Spanish patent application P200501733. Adjuvant : oligo CpG [oligonucleotide of 20 nucleotides (SEQ ID NO: 2), supplied by Bonsai Technologies Group (reference number BHSPF50) ] .

Diluent for the aerosol route (resuspension medium composition) : sterile PBS (phosphate buffered saline) . Diluent for the placebo group: sterile PBS.

Viral strain used in the challenge: K357/88 strain of IBDV

(Mardassi H. et al., 2004, "A Very Virulent Genotype of

Infectious Bursal Disease Virus Predominantly Associated with Recurrent Infectious Bursal Disease Outbreaks in Tunisian

Vaccinated Flocks", Avian Diseases Vol. 48, No. 4, pp 829-

840) .

C . Treatment Dose and Administration Several VLP doses were administered with the aim of determining their efficacy at different doses, with or without ad] uvant :

Group 1: Placebo dose, 4 ml of PBS

Group 2: Simple dose, 50 μg/bird of VLPs in PBS (approximately 1,250 μg in 4 ml of PBS)

Group 3: Double dose, 100 μg/bird of VLPs xn PBS

(approximately 2,500 μg in 4 ml of PBS)

Group 4: Ad^uvanted simple dose, 50 μg/bird of VLPs + CpG in

PBS (approximately 1,250 μg + CpGs until a total volume of 4 ml in PBS) .

The different doses were resuspended with the diluent, until a total volume of 4 ml, for 27 birds. The calculation of the dose available per bird was calculated on the basis of the concentration of the product, the volume and the nebulization time.

The control group received a placebo treatment formed by the diluent of the vaccine administered in the same way as the rest of the experimental groups.

For the nebulization, an apparatus was used to produce aerosols, formed by a methacrylate chamber of 49 x 33.2 x 33.2 cm (0.054 m³) , with a perforation in one side in which a MICROMIST® (HUDSON Respiratory Care Inc.) nebulizer was applied and an upper opening between the opposite end in the upper side of the chamber with a surface of 6.64 cm² (0.2 x 33.2 cm) to release the overpressure produced by the nebulizer.

The standard MICROMIST® nebulizer provides a particle with an average diameter of 3.6 μm (99.4% of particles under 5 μm) and a volume ratio of 0.25 to 0.30 ml/minute. The nebulizer was connected to a pressure of approximately 2.76xlO⁵ Pa (40 psi) which, under these conditions, allowed obtaining an aerosol with a particle size of 1.9 μm (within the interval of 1 to 5 μm) . The average nebulization time was 8 minutes with a volume of 4 ml (amount adjusted in order to obtain a dose calculated over 27 animals) . Once the dose in the deposit of the nebulizer was ran out, all the exits were closed for a few more minutes to assure maximum exposure to the product.

For safety and efficacy reasons, the chamber was placed inside each isolation unit so that the access of each group of animals to a single treatment was guaranteed.

D. Study design

The study design is summarized in Tables 1 (chronology test) and 2 (experimental groups) .

Table 1 Test chronology

Study day Action to be carried out

Day 0 Birth of chicks, weighing, distribution of the experimental groups and taking of blood sample

Day 1 Application of the experimental product

Day 21 Experimental challenge (infection with IBDV)

Day 31 End of the test, necropsies and taking of samples .

Table 2 Experimental groups i _^ ., , „ „ TreatmentChallengeTreatment

Description Dose/bird Regimen Route

^" Day (IBDV) Animals

Group 1 ζ⁸^ Sⁱngle _{Aerosol Day χ Day 2}\ 27

Diluent dose

50 μg of

Single

Group 2 VLPs in ^y Aerosol Day 1 Day 21 27

PBS ^dOSe

100 μg of

Group 3 VLPs m ^^{ng e} Aerosol Day 1 Day 21 27 dose

PBS

50 μg of

_ . VLPs + Single , „_ „_

Group 4 ^y Aerosol Day 1 Day 21 27

CpG in dose

PBS

At one day of age, the weight of all the animals was obtained and distributed randomly m the different experimental groups.

Adverse Reactions

Detection of adverse reactions :

After applying the treatments, all the possible adverse reactions were recorded 12, 24, 48 and 72 hours after the treatment by means of a clinical follow-up table.

E. Evaluation of the Results

Tolerance assessment

Effects: The zootechnical and clinical parameters were recorded for the measurement of the effects caused by the vaccination, and thus the tolerance to said vaccination could be evaluated.

(a) Zootechnical parameters

The weight of each animal was recorded on days 7, 14, 21 and 31.

The evaluation of the zootechnical parameters was carried out by means of the comparison of the average weights of the animals and the average daily weight gain. (b) Clinical parameters

The general tolerance was assessed 12, 24 and 48 hours after each treatment and any indication of the existence of any type of secondary effect (behavioural changes, especially respiratory, digestive or nervous alterations) was taken into account.

The alterations were evaluated by means of a score system detailed in Table 3.

Table 3 Assessment of the clinical symptomatology during the vaccination

Score Observed signs

0 Nothing outstanding

1 Bristly feathers

2 Bristly feathers and/or slight apathy

3 Bristly feathers and apathy

Efficacy assessment

1. Effects: The zootechnical , clinical, serological and pathological parameters were recorded for the measurement of the effects caused by the administration of IBDV to the chickens (VLPs) .

(a) Zootechnical parameters : The weight of all the animals on the day of the experimental challenge (day 21), of all the animals dead or sacrificed during the experimental infection and of all the surviving animals 4 days after the infection was recorded. The consumption of feed in each treatment group during the experimental infection period was also recorded.

The evaluation of zootechnical parameters was carried out by the comparison of the average weights of the animals, the average daily weight gain and the consumption of feed.

(b) Clinical parameters

The presence of clinical signs was evaluated 12, 24, 48 and 72 hours after the challenge with the IBDV. The observed alterations were evaluated by means of a score system detailed in Table 4.

Table 4

Assessment of the clinical signs during the experimental infection

Score Observed signs

0 Nothing outstanding

1 Bristly feathers

2 Bristly feathers and/or apathy

Bristly feathers, apathy, digestive

3 affectation (diarrhea) .

Severe symptoms of disease. Two or more these signs: intense apathy, trembling, immobility,

4 severe dehydration, hypothermia (Humanitarian sacrifice recommended) .

5 Spontaneous death.

The evaluation criteria were the comparison of the averages of the detected signs and the mortality percentage between the different treatment groups.

(c) Serological parameters

The immunization assays have been carried out in poultries Determination of the specific antibodies present in serum from the animals was carried out by an ELISA test. The antibody response of the vaccine was compared to a standard antiserum and the non-vaccinated control animals. The evaluation of the results obtained with this test was carried out by comparing the antibody titles means among each treatment group. Serum samples from ten animals were collected at different times: previous to vaccine treatment (1 day-old), 16 days post- vaccmation, the same day of the challenge and when the test finished (10 days later) . In general, at least 10 blood samples per group were collected. Table 5 Treatments

* The final volume was adjusted to 4 mL in all vials.

(d) Pathological parameters

The necropsy of all the animals dead or sacrificed during the experimental infection was carried out. The macroscopic lesions or findings were evaluated by means of a score system for each studied organ (see Tables 6 and 7) . The bursa of Fabricius and the spleen were weighed during the necropsy carried out on the tenth post-infection day (pid) .

Table 6

Assessment of the macroscopic lesions in the bursa of Fabricius

Score Observed signs

Nothing outstanding

Increase in size, swelling

Yellowish discolouration, presence of exudate

Atrophy and/or interfolicular hemorrhages (to extensive hemorrhage)

The assessment of microscopic lesions was carried out blindly using the criteria defined by Sharma et al . (1989) [Sharma JM, Dohms JE & Metz AL. "Comparative pathogenesis of serotype 1 and variant serotype 1 isolates of infectious bursal disease virus and their effect on humoral and cellular immune competence of specific-pathogen-free chickens. Avian Dis. 1989 Jan-Mar; 33(1) : 112-24] on the lymphoid depletion degree of the bursa of Fabricius (Table 7) . Table 7 Assessment of the macroscopic lesions in the bursa of Fabricius

Lesion degree Criteria

0 Less than 5% of lymphoid follicles affected

1 5-25% of lymphoid follicles affected

2 25-50% of lymphoid follicles affected

3 50-75% of lymphoid follicles affected

4 75- up to less than 100% of lymphoid follicles affected

5 100% of lymphoid follicles affected

For the comparisons between groups, the averages of the macroscopic and microscopic lesions detected in each organ and the sum of all the lesions between the different treatment groups were taken. The weight of the bursa of Fabricius and the spleen was corrected with respect to the weight of the animal expressed as a percentage (weight of organ x 100/live weight) to compare the averages between the different treatment groups.

F. Sample treatment

Sampling

Serological and microbiological controls were carried out for the purpose of detecting the presence of antibodies against IBDV and the possible existence of animals carrying Salmonella spp pathogenic strains. Necropsy samples of animals were taken out and those sacrificed were taken in order to detect any other concurrent pathology.

The occurrence of any positive result to the specified tests or the additional tests was used as an assessment for determining the continuity or end of the test. Samples were taken at the end of the test to assess the vaccine efficacy based on the weight score and the histological lesion degree.

Faeces for the isolation of Salmonella spp. Faeces samples were taken from the cubicles of 5-7 day old chickens. A minimum sample of 10 animals per cubicle was collected with the object of detecting a minimum prevalence of 25% of animals infected by Salmonella spp. (Thrusfield M. (1990) Veterinary epidemiology. Ed. Butterworths & Co. (Publishers) Ltd. ) .

Isolation of Salmonella spp.

The samples (faeces, blind content) were mixed with 20% Peptone Water (5 mL of peptone water per each gram of water) . A direct seeding of the mixture was carried out in XLT4 medium (xylose-lysine-Tergitol 4 medium) (24 hours incubation at 42°C) and the mixture was also incubated for 24 hours at 37°C. Subsequently, the suspension was seeded in XLT4 agar plates and was incubated for 24 hours at 37°C. The presence of black colour colonies on a violet medium was taken as a positive isolation, and the presence of white colonies with a dark centre and yellow medium was considered to be a doubtful isolation. In the latter case, a re-seeding of the doubtful colony was carried out m the XLT4 medium and, subsequently, it was biochemically identified by the Vitek system (Biomerieux) .

Necropsy

At the end of the study, all the surviving animals and all the animals suffering from intense stress were sacrificed (m the case of experimental infection, the animals incapable of feeding themselves) were sacrificed.

The necropsy was carried out on all the dead or sacrificed animals, any possible lesion observed being noted. The sacrifices were carried out by means of the intravenous administration of 150 mg/kg w.v. of sodium pentothal per animal, followed by a bleeding by incision of the atlanto-occipital joint vessels (experimental procedure approved by the Ethics Committee of the IRTA (Agroalimentary Research and Technology Institute of Catalonia) ) Blind samples were taken in the animals that died before the challenge for the isolation of Salmonella sp.

Samples of the bursa of Fabricius were taken in the animals that died or were sacrificed after the challenge with IBDV.

Additionally, samples were taken for the diagnosis of other infections that were not expected initially in the experience and of which there was no indication.

G. Evaluation Criteria

Criteria used to evaluate the tolerance to the product: Absence of clinical signs after the vaccination, No affectation of the growth of the animals with respect to the control (daily weight gam) .

Criteria used to determine the product efficacy: After the challenge with IBDV:

(a) Lower growth reduction

(b) Lower clinical affectation

(c) Lower degree or score of macroscopic lesions (d) Increased bursa of Fabricius Index in the vaccinated animals (e) Reduction of the degree of macroscopic lesion in the bursa of Fabricius 4 days after the challenge. After the sacrifice, the bursas were withdrawn to carry out the macroscopic assessment of the affectation of the bursa of Fabricius.

H. Data analysis

Before the challenge, the antibody levels were established as the mam variable but after the challenge, the main variables were the mortality percentage, the degree of microscopic lesion of the bursa of Fabricius and the daily weight gain after the IBDV challenge. The analyzed secondary variables were the weight of the animals on the different days, the bursa of Fabricius index and the spleen with respect to the live weight of the animal .

All the quantitative data (weight, weight gain, lesions, index, etc.) were analyzed by means of the analysis of variance.

The comparison between the treatments was carried out by means of the Tukey test and Dunnett' s C test (according to the homogeneity of the variances) .

All the qualitative data (lesions, mortality, etc.) were analyzed by means of non-parametric tests such as the χ² test, Fisher's statistical test, the Mantel-Haenszel stratified analysis or the Kruskall-Wallis H test.

The analysis of vaccine efficacy was carried out by studying cohorts (vaccine efficacy = (1-relative risk) xlOO) of the data of mortality and lesions (Dean et al. (1995) . Epi info, Version 6: A word-processing, database and statistics program for public health on IBM-compatible microcomputers. Centers for disease control and prevention. Atlanta, Georgia, USA) .

A 5% significance level was applied to all the tests.

II. RESULTS

Survival

After subjecting the chickens treated with VLPs to the experimental challenge, animal survival m the different treatment groups on day 31 was analyzed. As can be observed m Figure 1, the greatest survival Index was obtained in the animals belonging to Group 4, the treatment of which consisted of administering 50 (g of VLPs plus an adjuvant (CpGs) . In this Group, 21 of the 27 animals forming this Group survived, a much greater number than that obtained in Group 2 in which, although the amount of VLPs administered was the same as the one in Group 4, the absence of adjuvant meant that only 6 of the 27 animals survived. The results obtained with Group 2 are not different from those obtained with the control Group (Group 1).

In Group 3, in spite of doubling the concentration of VLPs administered (100 (g) , the survival results did not reach those of Group 4, showing that the presence of adjuvant is a decisive element in the survival response of the animal against the infection by IBDV.

Weight evolution

The weight evolution of the animals was determined throughout the process in all the groups in order to evaluate the efficacy of VLPs. As shown in Figure 2, the weight was measured on days 7, 14, 21 and 31. During the study period, the weight of the animals in the different groups increased progressively, without a considerable weight difference being observed between the different treatments. Nevertheless, the results obtained on day 31 show that while groups 1, 2 and 3 maintained a similar weight to the one on the day before the challenge, the animals of Group 4 continued to increase their weight, showing the efficacy of the treatment comprising the administration of VLPs together with an adjuvant.

Evolution of growth after the challenge The growth of the animals was evaluated after subjecting them to the experimental challenge. As can be observed in Figure 3, the animals of Group 4, immunized with 50 μg of VLPs plus the adjuvant, continued to grow after the experimental challenge. In contrast, the animals of Groups 1 and 2 did not have a positive growth.

Bursa of Fabricius/Total Weight of Survivors Index

The bursa of Fabπcius/body weight of the animals surviving the experimental challenge index can be observed in Figure 4. Said figure shows that the animals immunized with 50 μg of VLPs plus the adjuvant (Group 4) have the highest Index. These data are related to the results shown in Figure 1, in which Group 4 has the highest number of surviving animals (21 out of 27 animals) .

Inmunization Assay

Immunological data showed here have been carried out using exclusively serum samples and applying a commercial detection system of anti-IBDV antibodies (IDEXX). The serological samples are used, in general, to study immune responses of humoral type. Nevertheless, the data presented herewith clearly define a high degree of protection possibly due to an excellent immune response at the respiratory and cellular mucosal level what represents a success in the design and application of vaccines against IBDV. This explains why antibody levels present in serum

(Table 8 and Figure 5) are not totally correlated with protection to the challenge against the virus. This lack of correlation shows, without doubts, the importance of a suitable protective cellular response against IBDV, and confirms the efficacy of the administration route used in this invention for immunizing birds. Nevertheless, the administration of the vaccine herein disclosed has a clear and important effect in the production of anti-IBDV antibodies in serum.

Table 8

Serological results expressed as antibody title according to ELISA test ^(1> at different times post-vaccination and postinfection.

(1) Antibody titles obtained by ELISA IDEXX test, calculated according to the reference positive control serum.

(a) Values with different superindex show stadistic differences (p<0.05) among treatments. III. DISCUSSION

In view of the results obtained in the experimental study, the efficacy of the experimental product (VLPs) administered to the animals in order to combat an infection by IBDV is shown. Said experimental product consists of the administration of VLPs of IBDV by means of nebulization, obtaining a greater effectiveness when the experimental product is administered in combination with an adjuvant such as CpG.

Claims

1. An aerosol formulation comprising virus-like particles (VLPs) producing a protective immune response in birds to which it is administered in a liquid.

2. A formulation according to claim 1, wherein said VLPs are administered to the birds as an aerosol, by inhalation.

3. A formulation according to claim 1 or 2, wherein said VLPs are from viruses capable of infecting birds.

4. A formulation according to claim 3, wherein said virus capable of infecting birds is the infectious bursal disease virus (IBDV) .

5. A formulation according to any of the previous claims, wherein said VLPs comprise one or more heterologous peptides containing one or more antigens of one or more avian pathogens .

6. A formulation according to claim 5, wherein said heterologous peptides contain one or more antigens of an avian pathogen selected from the avian pathogens causing bird flu, rhinotracheitis , coccidiosis and combinations thereof.

7. A formulation according to any of the previous claims, wherein said VLPs are VLPs formed by the assembly of one or more structural proteins of the IBDV capsid or of fragments thereof capable of assembling and forming VLPs producing a protective immune response in the birds to which they are administered.

8. A formulation according to claim 7, wherein said VLPs of IBDV are selected from:

VLPs formed by the assembly of VP2, VP3 and VPl of IBDV

[complete VLPs];

VLPs formed by the assembly of VP2 and VP3 of IBDV [VLPs (-

VP4 ) ] ; - VLPs formed by the assembly of fragments of VP2 of IBDV

[VLPs-pVP2*] ;

VLPs of IBDV further comprising an antigen of an avian infectious agent different from IBDV [CVLPs or

CVLPs (pVP2*) ] .

9. A formulation according to any of the previous claims, wherein said liquid is a pharmaceutically acceptable liquid.

10. A formulation according to claim 9, wherein said pharmaceutically acceptable liquid is a polar fluid selected from water, an aqueous liquid medium and a non-aqueous liquid medium.

11. A formulation according to claim 10, wherein said pharmaceutically acceptable liquid is phosphate buffered saline

(PBS) solution.

12. A formulation according to any of the previous claims, further comprising an adjuvant.

13. A formulation according to claim 12, wherein said adjuvant is an adjuvant enhancing the response in mucous membranes .

14. A formulation according to claim 13, wherein said adjuvant is an immunomodulator, e.g., an oligodeoxynucleotide

(ODS), preferably an ODS comprising one or more cytosme-guanine (CpG) dmucleotides, or an enterotoxm.

15. A formulation according to any of the previous claims, further comprising one or more antigens of one or more avian pathogens, separated from said VLPs, suitable for their administration to birds by inhalation by means of aerosol.

16. A VLPs suspension comprising empty virus-like particles (VLPs) producing a protective immune response in birds to which it is administered and a liquid in a gas.

17. A suspension according to claim 16, further comprising one or more antigens of one or more avian pathogens, separated from said VLPs, suitable for their administration to birds by inhalation by means of aerosol.

18. Use of said VLPs producing a protective immune response in birds to which they are administered in the manufacture of an aerosol formulation for the immunization of birds against at least one avian infectious agent.

19. Use according to claim 18, wherein said avian infectious agent is a pathogenic bird virus.

20. Use according to claim 19, wherein said pathogenic bird virus is infectious bursal disease virus (IBDV) .

21. Use according to any of claims 19 or 20, wherein said aerosol formulation comprises two or more antigens of two or more avian pathogens capable of inducing a protective immune response in the bird to which it is administered.

22. Use of said VLPs producing a protective immune response in birds to which it is administered in the manufacture of a pharmaceutical composition for its administration as an aerosol.

23. Use according to claim 22, wherein said pharmaceutical composition is a vaccine useful for immunizing birds against one or more avian infectious agents.

24. Use according to claim 23, wherein said avian infectious agent is a pathogenic bird virus.

25. Use according to claim 24, wherein said pathogenic bird virus is infectious bursal disease virus (IBDV) .

26. Use according to any of claims 22 to 25, wherein said aerosol formulation comprises two or more antigens of two or more avian pathogens capable of inducing a protective immune response in the bird to which it is administered.