Overcoming antibody interference in birds

Abstract

The present invention provides recombinant proteins, and recombinant vectors expressing the proteins, that can be used to vaccinate seropositive birds, whereby antibodies in the birds target specific for the antigen contained in the recombinant protein. The antigen is targeted to avian antigen-presenting cells (APCs) by also including in the recombinant protein a domain capable of binding to cell surface proteins on those APCs. It has been found that this type of vaccine can safely overcome the adverse effects of antibody interference, even after a single dose, in very young birds, and in situations where antibody levels are very high.

Description

Technical Field

[0001] The present invention relates to the field of avian vaccination. More specifically, the present invention relates to a recombinant protein for use in a method of protecting birds, which recombinant protein has an antibody reactive with an antigen in the protein. Specifically, the present invention relates to a recombinant protein, a recombinant vector, and a vaccine for use in the method. Further, the present invention relates to uses and methods for the treatment of birds by administration of the protein, vector or vaccine.

Background Art

[0002] As a protein source with high nutritional value and an affordable price, avian meat and eggs are an important part of the diet of most human populations in the world. The main species of poultry raised for such economic purposes are chickens, turkeys, ducks and geese. In order to raise the large numbers of birds required while maintaining their health and desirable living conditions, the poultry industry is eager to optimize management conditions and provide good veterinary care. An important part of this strategy is preventive protection by vaccination against the various avian pathogens that can cause infectious diseases and disorders, which can have a devastating impact on animal health and business economics. Over the years, a variety of vaccines have been commercially available for most of the viral, bacterial and parasitic diseases that can affect economically relevant birds. Such vaccines can be of different types, such as live attenuated, inactivated, subunit, nucleic acid, virus vector, etc.

[0003] In particular, for poultry produced in very large quantities, namely broilers, it is common practice to protect young birds as early as possible. However, active vaccination of very young animals with immature immune systems is often unsuccessful. Therefore, an effective alternative is vaccination of the mother before and during the laying period. Maternal antibodies produced by the hen are transferred to the yolk-containing egg and internalized by the developing chick. In this way, the chick can be passively protected by these maternal antibodies (MDAs) against various pathogens as early as the hatching day. However, since most MDAs lose their effectiveness again after three weeks due to biological degradation, active vaccination of the growing chick itself is also necessary to induce adequate immunoprotection after the first few weeks. At that point, some MDAs may still be present in the bird.

[0004] A similar situation with vaccination in relation to existing antibodies occurs in older birds that have antibodies induced by previous vaccinations, and because the effect of those antibodies gradually diminishes, a booster vaccination is required to restore antibody titers to protective levels.

[0005] A significant veterinary and scientific challenge arises in determining when to vaccinate birds that already possess antibodies that are reactive to the antigens contained in the vaccine being administered. Vaccination when antibody titers are nearly gone is clearly too late, as it leaves a gap period between the decline in those antibody titers and the onset of protection from active immunization. During this gap period, the birds are vulnerable to infection and disease.

[0006] However, vaccination is premature when birds still have fairly high circulating antibody titers, because in many cases this affects the effectiveness of the vaccination, likely because those antibodies may somehow bind to and capture the vaccine antigen, thereby accelerating its degradation and / or preventing the vaccine antigen from inducing a proper immune response. This last phenomenon is called "antibody interference," and its alternative notation in relation to MDA is "MDA interference." This is a well-known issue regarding effective vaccination against major pathogens affecting the poultry industry worldwide. Examples of these major pathogens include infectious bursal disease virus (IBDV, also known as gumboro disease virus), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and avian influenza virus (AIV, also known as avian plague virus), the latter two of which are indeed notifiable diseases by the OIE [World Organisation for Animal Health].

[0007] In these diseases, antibody interference is well known to reduce the effectiveness of vaccination, making birds vulnerable to field infections, especially when they are raised in close proximity to and / or in areas with high prevalence of avian pathogens.

[0008] Over the years, many different approaches have been attempted to overcome antibody interference in order to prevent protection gaps and optimize vaccination of seropositive birds. More direct attempts to overcome antibody interference have included adapting vaccines by increasing antigen doses and / or using (more potent) adjuvants. More pathogenic or unattenuated live vaccine pathogen strains have also been attempted in the hope that they can overcome higher antibody titers and thus be administered earlier. As these methods have generally been unsatisfactory, more complex approaches have been attempted.

[0009] Regarding the proactive vaccination of young birds against IBDV, one method involves monitoring MDA levels by serological testing of bird samples to determine the optimal timing for vaccination. However, this results in effective proactive vaccination only being applicable at 2-3 weeks of age, and due to variability in large flocks, protective gaps are unavoidable for many birds. Alternatively, a "combined IBDV vaccine" (a live attenuated vaccine virus conjugated with antibodies) is administered at a young age, in which case the antigen is released only at a later time. Furthermore, viral vector systems are used, for example, using fowlpox virus or avian herpesvirus as a vector to express viral protein 2 (VP2), the main IBDV antigen. This is reviewed in Muller et al. (2012, Avian Pathol., vol.41, pp.133-139).

[0010] Regarding NDV, various approaches have been applied to vaccination, but antibody interference remains a problem today. For a review, please refer to Dimitrov et al. (2017, Vet. Microbiol. vol. 206, pp. 126-136).

[0011] Even with recombinant vector vaccines, antibody interference can occur, for example, if antibodies react with the vector virus itself and / or the antigens it expresses. See Hu et al. (2020, Vaccines, vol.14, p.222, doi:10.3390). Possible solutions for NDV as a vector include, for example, altering the serological profile of the NDV vector (Steglich et al., 2013, PLoS One, vol.8, e72530) or selecting an NDV strain that is less inhibited by anti-NDV antibodies (European Patent Application Publication No. 2998315).

[0012] Regarding IBV, it is well known that MDA interferes with the vaccination of 1-day-old chicks. See Terregino et al., 2008 (Avian Pathol., vol.37, pp.487-493).

[0013] Regarding AIV, the relevance of effective vaccination extends beyond the veterinary field, as this virus can cause zoonotic diseases in humans with pandemic potential. Over the years, many different approaches using classical or recombinant AIV vaccines have been attempted, with varying levels of success. See D. Swayne, 2009 (Comp. Imm. Microbiol. and Inf. Dis., vol. 32, pp. 351-363). However, as with some other vaccine situations, dealing with interference by AIV-reactive antibodies remains a problem (Murr et al., 2020, Avian Dis., vol. 64, pp. 427-436).

[0014] As a result, despite the many different approaches tried in the field of avian vaccination, there remains an urgent need for effective methods to overcome the negative effects that existing antibodies in target animals have on the effectiveness of vaccination with antigens to which these antibodies can bind.

[0015] Shrestha et al. (2018, Vaccines, vol.6, p.75, doi:10.3390) review options for improving avian targeting vaccination by selectively targeting antigens to antigen-presenting cells (APCs). A wide variety of methods for achieving such targeting are described, for example, using ligands, antibodies, nanoparticles, viral vectors, or cell-permeable peptides. Methods for overcoming antibody interference in birds are neither described nor suggested.

[0016] International Publication No. 2017 / 055235 describes antigen targeting to antigen-presenting cells (APCs), but uses antigen internalization. The described treatments are for mammals, particularly cats and dogs, and aim to reduce allergies. Antibody interference is not mentioned.

[0017] Jauregui et al. (2017, Res.Vet.Sci., vol.111, pp.55-62) described the targeting of AIV HA antigen to dendritic cells in chickens. Purified H5 HA antigen was chemically conjugated to a mouse monoclonal antibody against one domain of Dec-205. This conjugate was used to vaccinate 21-week-old chickens. Since all chickens used were seronegative for anti-HA antibodies (see Jauregui, Figure 7, Day 0), Jauregui et al. neither described nor suggested overcoming antibody interference in seropositive birds. [Prior art documents] [Patent Documents]

[0018] [Patent Document 1] European Patent Application Publication No. 2998315 [Patent Document 2] International Publication No. 2017 / 055235 [Non-patent literature]

[0019] [Non-Patent Document 1] Muller et al.,2012,Avian Pathol.,vol.41,p.133-139 [Non-Patent Document 2] Dimitrov et al.,2017,Vet.Microbiol.vol.206,p.126-136 [Non-Patent Document 3] Hu et al.,2020,Vaccines,vol.14,p.222,doi:10.3390 [Non-Patent Document 4] Steglich et al., 2013, PLoS One, vol. 8, e72530 [Non-Patent Document 5] Terregino et al., 2008, Avian Pathol., vol. 37, p. 487-493 [Non-Patent Document 6] D. Swayne, 2009, Comp. Imm. Microbiol. and Inf. Dis., vol. 32, p. 351-363 [Non-Patent Document 7] Murr et al., 2020, Avian Dis., vol. 64, p. 427-436 [Non-Patent Document 8] Shrestha et al., 2018, Vaccines, vol. 6, p. 75, doi:10.3390 [Non-Patent Document 9] [[ID=XX]]Jauregui et al., 2017, Res. Vet. Sci., vol. 111, p. 55-62 [Summary of the Invention]

[0020] Therefore, an object of the present invention is to overcome one or more drawbacks in the prior art by providing an effective method for overcoming the adverse effects of antibody interference in avian target vaccination.

[0021] Surprisingly, this object can be achieved by providing a method for protecting birds that possess antibodies reactive to the antigen contained in the administered vaccine, i.e., by targeting the antigen to the avian APCs, and as a result, it has been found that one or more drawbacks of the prior art can be overcome.

[0022] Note: There seems to be a mistake in the original text where the ID for "Jauregui et al., 2017, Res. Vet. Sci., vol. 111, p. 55-62" is "ID=XX" instead of a proper 7-digit tag. I've translated it as best as possible with the given text.In the experiments detailed below, chickens with high or moderate antibody levels were administered either a targeted vaccine or a non-targeted vaccine. The results showed a significant difference in the effectiveness of vaccination favoring the targeted antigen. In contrast, non-targeted vaccines and classical control vaccines elicited little to no response in seropositive birds. Consequently, this method of protecting seropositive birds can effectively overcome the negative effects of antibody interference in vaccination and is unexpectedly effective, even with a single dose and in very young birds.

[0023] As a result, to our great surprise, the inventors found that this antigen targeting, particularly in relation to existing antibodies, functions well even in immunomature birds, and does not cause vaccine-enhancing diseases or vaccine-induced immunodeficiency due to excessive stimulation of the immune system, autoimmunity, or induction of tolerance.

[0024] This method for protecting birds is equally applicable when the target antigen is not used directly, but rather as a recombinant vector expressing recombinant protein, such as a DNA plasmid, RNA molecule, or vector virus.

[0025] Furthermore, since this favorable effect is thought to be due to targeting and therefore independent of the antigen used, it is quite possible that this method would be equally successful even when using different antigens. Thus, this method enables protection against various avian pathogens, such as NDV, IBDV, and AIV, against which vaccination is normally subjected to antibody interference.

[0026] It is not precisely known how or why this vaccination method can overcome high antibody levels and still induce such an effective protective immune response. While the inventors do not wish to be bound by any theory or model that could explain these findings, they hypothesize that it is due to the targeting of the antigen to APC, which in some way reduces the removal of the antigen by existing antibodies against APC.

[0027] The success of using antigen targeting to APCs in avian vaccination of birds with existing antibodies against vaccine antigens could never have been predicted in any previous publication. This is mainly because the mechanisms by which antibody interference acts (blocking, masking, cross-linking, neutralization, etc. of vaccine antigens) are still not well understood. This is especially true for antibody interference in birds, as they are an understudied animal system.

[0028] Furthermore, while the first studies on antigen targeting were already described in 1980, these were aimed at the treatment of human cancer. Later, its more general use in (primarily human) vaccination was considered. This is outlined by Keler et al. (2007, Oncogene, vol.26, pp.3758-3767).

[0029] Furthermore, in some cases, (maternal) antibodies are involved in the enhancement of viral diseases through antibody-dependent enhancement, a phenomenon known as vaccine-enhanced disease. This effect has been observed with various viruses, including lentiviruses and dengue viruses (Huisman et al., 2009, Vaccine, vol.27, pp.505-512), and more recently with SARS-CoV-2 (Lee et al., 2020, Nat. Microbiol., vol.5, pp.1185-1191). Therefore, there has been genuine concern that targeted vaccination could lead to such undesirable effects upon subsequent contact with the corresponding pathogen.

[0030] Furthermore, the transition from mammalian to avian situations is by no means easy, as there is little information available on the function of the avian immune system compared to mammals / humans. Also, the general review by Shrestha et al. (mentioned above) does not enable certain methods or alleviate all the hesitations that those skilled in the art might have when using antigen targeting, as it may cause a kind of immunodeficiency and / or require a mature immune system.

[0031] Furthermore, due to this lack of information and the potential for complications, the use of antigen targeting to APCs became an unsuitable option for vaccinating birds that have high levels of circulating antibodies that are reactive to the antigens in the vaccine. In addition, the immune system of birds at hatching is not yet mature, and as a result, it was unpredictable whether their APCs were mature enough to already present the appropriate target protein on their surface and translate binding to such surface proteins into productive stimulation of the animal's immune system, making the selection of this vaccination method for young birds particularly uncertain.

[0032] Accordingly, in one embodiment, the present invention relates to a recombinant protein comprising an antigen and a binding domain capable of binding to a cell surface protein on an avian antigen-presenting cell (APC), for use in a method of protecting birds possessing antibodies that are reactive with the antigen from a pathogen from which the antigen originates.

[0033] A "recombinant protein" is a protein whose amino acid sequence is artificially constructed. In the present invention, recombinant proteins can be obtained by molecular cloning techniques and recombinant protein expression techniques. After expression, the protein can be isolated from the expression system, processed and purified as necessary, and then formulated into a composition suitable for use in the protective methods of the present invention. Alternatively, recombinant proteins can be expressed and delivered via recombinant vectors, such as DNA plasmids, RNA molecules, or viral vectors, as described below.

[0034] Such techniques are well-known in this field and are disclosed in great detail in standard textbooks such as Sambrook and Russell's "Molecular cloning: a laboratory manual" (2001, Cold Spring Harbour Laboratory Press; ISBN: 0879695773) and Ausubel et al.'s "Current Protocols in Molecular Biology" (J. Wiley and Sons Inc, NY, 2003, ISBN: 047150338X). In relation to the present invention, the term "protein" incorporates similar terms such as "peptide," "oligopeptide," and "polypeptide."

[0035] The recombinant protein for use according to the present invention is a fusion protein composed of polypeptides of different origins, for example, all derived from antigens and binding domains as defined in the present invention, and may also be composed of one or more peptides such as linkers and markers, all linked together in a single amino acid chain.

[0036] As used herein, the term “comprising” (and variations such as “comprises,” “comprise,” and “comprised”) is intended to refer to all conceivable elements and all possible combinations relating to the Invention, even if such elements or combinations are not expressly described, as are covered or included in the text sections, paragraphs, claims, etc., in which the term is used, and is not intended to exclude any such (one or more) elements or combinations.

[0037] Therefore, any such text section, paragraph, claim, etc. may also relate to one or more embodiments in which the term “comprising” (or a variation thereof) is replaced by terms such as “consisting of,” “consists of,” or “consist essentially of.”

[0038] Antigens are generally known as molecules that can interact with components of the immune system, such as antibodies and lymphocytes, and this interaction can result in humoral and / or cellular immune responses.

[0039] The portion of the antigen recognized by the immune system is called an "epitope," which can be linear or three-dimensional. 3D epitopes are typically formed by the folding of larger proteins. Linear epitopes need to be of sufficient size, for example, consisting of at least five amino acids, either on their own or by being linked to a carrier molecule, for example, by being included in recombinant proteins for use according to the present invention.

[0040] An antigen is a polypeptide, i.e., an antigenic polypeptide comprises at least one epitope and "originates" from a pathogen. In the present invention, "originates" typically refers to a state in which a coding sequence for a particular antigen is selected by analysis of the genetic information of the pathogen and its protein repertoire. The selected sequence is then recombined into a construct encoding a recombinant protein for use according to the present invention.

[0041] Therefore, in the present invention, the selected antigen may be all or part of a protein derived from a pathogen, and the pathogen may be selected from viruses, bacteria, parasites, and fungi.

[0042] Antigens may originate from the native sequences of pathogen-derived antigens or from aggregates, for example, a consensus from several homologs of the expressed antigen, such as the same type of protein, but with amino acid sequences derived from variants of the pathogen, such as different species, serotypes, subtypes, strains, or isolates. As is well known, to obtain such a consensus sequence, either amino acid sequences or coding nucleotide sequences can be compared, and from this comparison, for example, the consensus sequence can be derived by aligning several H9 HA nucleotide sequences using a suitable computer program.

[0043] The antigen of the present invention may also be a chimeric antigen consisting of aggregate portions derived from different antigens, whether or not they are biologically related. Furthermore, the sequence encoding the antigen may be subjected to "codon optimization" as described below.

[0044] In the present invention, the antigen is selected from proteins capable of eliciting a protective immune response against the pathogen from which the antigen originates. For example, it is selected from the VP2 protein derived from IBDV, the fusion (F)- or hemagglutinin-neuraminidase (HN) protein of NDV, the spike protein derived from infectious bronchitis virus (IBV), and the HA- or neuraminidase (NA) protein of AIV.

[0045] The "binding domain" of the present invention may be a part of an antibody that originates from the antigen-binding site of an immunoglobulin molecule and includes one or more complementarity-determining regions, and may be, for example, a "single-strand variable fragment" (scFv) polypeptide.

[0046] In this invention, the binding domain is "capable of binding." This refers to specific binding, i.e., binding that has sufficient affinity to be distinct from any nonspecific binding or background binding. The difference between specific and nonspecific binding is well known to those skilled in the art and can be easily distinguished, for example, in an in vitro binding assay by diluting either the binding domain or the ligand. Nonspecific binding is typically rapidly lost by dilution, for example, 1:10 or 1:100, while specific binding remains even at higher dilution ratios.

[0047] APCs are well known to be lymphoid cells that can process antigenic molecules and present (some of) those molecules to the human or animal immune system. This presentation induces a cascade of reactions that lead to immunomaturation and stimulation, which underlie the protective immune response. Examples of APCs include B lymphocytes, dendritic cells, macrophages, and natural killer cells.

[0048] "Cell surface proteins on avian APCs" are proteins attached to or fixed to the outside of the cell membrane of APCs. These proteins play a role in the function of APCs in detection and signal transduction. Many cell surface proteins on APCs are members of the immunoglobulin superfamily of proteins. Examples of APC surface proteins are, for example, CD83 and CD11c proteins. The notation "CD" refers to the "differentiation cluster," an international protocol for the classification and identification of surface proteins on lymphoid cells.

[0049] In this invention, "birds" refers to any animal of the taxonomic class Aves that has economic or (veterinary) medical relevance. For example, chickens, turkeys, ducks, geese, quail, guinea fowl, partridges, pheasants, pigeons, falcons, and ostriches.

[0050] The term "for use in methods of protecting birds" refers to the medical use of the recombinant protein as defined herein for use according to the present invention. This use may be a direct use of the protein or an indirect use of the protein via expression from a recombinant vector.

[0051] In this invention, the "method" to be applied refers to vaccination.

[0052] The term "protect" refers to the effect of the method of the present invention, namely the protective immune response induced by the method, i.e., by vaccination. Such an immune response protects vaccinated birds from infection and / or disease caused by pathogens from which the antigen (the antigen present in the recombinant polypeptide for use according to the present invention) is derived.

[0053] The methods of protection relate to the establishment or reduction of proliferative infection by the pathogen in the cells and organs of susceptible birds, in whole or in part, or to the reduction of subsequent signs of the disease. This is achieved, for example, by reducing the pathogen load or shortening the pathogen replication period, which results in a reduction in the frequency, intensity, or severity of the lesions and associated disease clinical signs that may be caused by pathogen infection in birds.

[0054] Such reductions in infection or disease can be readily detected, for example, by monitoring the immunological response after vaccination with recombinant proteins for use according to the present invention, by examining the clinical symptoms or mortality status after (attack) infection in vaccinated birds, by monitoring disease signs, clinical scores, serological parameters in birds, or by re-isolation of the infectious pathogen. These results can be compared to responses to similar infections in shamvaccinated birds. Several methods for evaluating the symptoms of infection and disease of major avian pathogens are well known in the art.

[0055] Protection from infection or disease by the method of the present invention provides improved health, welfare, and economic performance in immunized birds. This can be evaluated by parameters such as increased living conditions, survival rate, growth rate, feed efficiency, and egg production, as well as cost reductions for (veterinary) healthcare.

[0056] Birds protected by the method of the present invention “possess antibodies.” This applies at the time the method of the present invention is applied, i.e., at the time of vaccination. Whether birds actually possess such antibodies can be easily determined, for example, by taking blood samples from the birds before and after vaccination and determining the titer of antibodies against the antigen using standard serological methods. However, this does not require that the determination of the existing titer value itself, i.e., the performance of serological tests on serum samples taken before and after vaccination, and / or the analysis and interpretation of the results of such tests, be performed at that time. Similarly, this does not prevent the calculation and extrapolation of the existing titer at the time of vaccination from the level measured in samples taken some time before vaccination.

[0057] In the present invention, a bird "possesses" antibodies against an antigen if the titer of antibodies reacting to the antigen in its avian serum exceeds a background level. Such a background level is typically the level present in equivalent birds that are naive to the antigen or pathogen of interest. In the present invention, this background level can conveniently be obtained, for example, from the titer present in the serum of SPF (Specific Pathogen-Free) birds of the same age and species.

[0058] Existing antibodies can arise from passive transfer, typically in the case of antibodies obtained from the mother via the yolk. Such seropositive birds are called "MDA-positive" or "MDA+". This applies to very young birds, for example, from the day of hatching (i.e., 1 day old) to about 3 weeks old. Alternatively, existing antibodies can arise from active immunization that the protected bird had previously received, which resulted in antibody production; this applies to birds from about 3 weeks old onward.

[0059] The terms "reactive with" or its synonym "specific to" describe the ability of an existing antibody to interact with the antigen contained in the recombinant polypeptide for use according to the present invention by specific immunorecognition. Similar terms also include "able to bind" and "able to recognize," insofar as they refer to specific binding.

[0060] An unexpected and advantageous effect of the present invention is particularly evident when existing antibodies (in the protected birds) are reactive with the antigens contained in the recombinant protein of the present invention. In that situation, antibody interference usually occurs, which reduces the effectiveness of protection.

[0061] The term "pathogen from which the antigen is derived" serves to indicate that the pathogen that the method of the present invention seeks to protect includes the antigen defined above. This also includes homologs of the antigen and / or variants of the pathogen.

[0062] As those skilled in the art will understand, the correspondence between the antigen in the recombinant protein for use according to the present invention and the pathogen to which the birds are to be protected forms the basis of the induced protective immune response. [Modes for carrying out the invention]

[0063] The details of embodiments and further aspects of the present invention are described below.

[0064] In one embodiment of the recombinant protein for use according to the present invention, the avian APC is selected from B lymphocytes, dendritic cells, macrophages, and natural killer cells.

[0065] Each of these cell types can be clearly distinguished using standard serological and biochemical methods, for example, by protein-based determinations that specify CD as described below.

[0066] In preferred embodiments of the recombinant protein for use according to the present invention, the avian APC is a dendritic cell.

[0067] In one embodiment of the recombinant protein for use according to the present invention, the cell surface protein on the avian APC is selected from differentiation cluster 83 (CD83), differentiation cluster 11c (CD11c), and dendritic cell receptor for endocytosis-205 (Dec205).

[0068] All of these proteins are well-known in this field, being surface proteins on APCs, and CD11c is a transmembrane protein on dendritic cells and several other APCs, playing a role in neutrophil activation. CD11c-specific scFv contains the amino acid sequence of SEQ ID NO: 18.

[0069] Dec-205 is an endocytosis receptor on dendritic cells and lymphocytes. An example of chicken Dec-205 is shown in GenBank accession number: AJ574899. A Dec-205-specific scFv contains the amino acid sequence of SEQ ID NO: 19.

[0070] CD83 is a surface glycoprotein belonging to the immunoglobulin superfamily. It is primarily expressed on dendritic cells, and to a lesser extent on lymphocytes and macrophages. It is a well-known marker for mature dendritic cells. An example of avian CD83 is the protein shown with GenBank accession number XP_040519591.

[0071] In a preferred embodiment of the recombinant protein for use according to the present invention, the cell surface protein is CD83.

[0072] In one embodiment of a recombinant protein for use according to the present invention, the binding domain includes the antigen-binding site of the antibody.

[0073] In preferred embodiments of recombinant proteins for use according to the present invention, the binding domain is a single-stranded variable fragment (scFv).

[0074] As is well known, scFv is the smallest portion of immunoglobulin that possesses one complete antigen-binding domain but lacks an Fc moiety. scFv is a single peptide that is itself a fusion construct, comprising one variable light chain (vL), a linker, and one variable heavy chain (vH). The order of these elements can be vL-linker-vH or vH-linker-vL. In either case, the variable chains are oriented in a tail-to-head configuration (relative to each other), so that the c-terminal end becomes the tail.

[0075] In a preferred embodiment, the order of elements in scFv is vH-linker-vL.

[0076] The linker sequence of the scFv provides a flexible region that allows the two variable chains to orient themselves to form an antigen-binding domain. In a preferred embodiment, the linker sequence of the scFv comprises the amino acids glycine and serine or threonine and is 10 to 50 amino acids long. In a more preferred embodiment, the linker sequence of the scFv comprises the amino acid sequence (Gly4-Ser)4 shown in SEQ ID NO: 1.

[0077] The specificity of the two variable chains of scFv may be that both are directed towards the same antigen, or they may be directed towards different antigens. In a preferred embodiment, the two variable chains have the same specificity.

[0078] In one embodiment, scFv is specific to CD83, or in other words, CD83-scFv. Preferably, scFv is specific to CD83 on avian dendritic cells, and more preferably, scFv contains the amino acid sequence of SEQ ID NO: 2.

[0079] In embodiments of the binding domain, there may be two or more scFv instances.

[0080] In one embodiment of the recombinant protein for use according to the present invention, the pathogen is pathogenic to birds. More preferably, the pathogen is a virus. Even more preferably, the virus is an RNA virus. Even more preferably, the RNA virus is selected from IBDV, NDV, IBV, and AIV. Even more preferably, the pathogen is selected from IBDV, NDV, and AIV. Most preferably, the pathogen is AIV.

[0081] In one embodiment of the recombinant protein for use according to the present invention, the antigen is selected from IBDV VP2 protein, NDV F protein, NDV HN protein, IBV spike protein, AIV HA protein, and AIV NA protein. More preferably, the antigen is selected from one of AIV HA protein and AIV NA protein. Even more preferably, the antigen is AIV HA protein. Even more preferably, the antigen is selected from H5, H7, or H9 type AIV HA protein.

[0082] All of these viral protein antigens are well known in this field, and many versions of their coding sequences are readily available digitally in public sequence databases such as NCBI's GenBank and EMBL's EBI. Examples include AIV H9 HA: GenBank acc.nr.ACP 50708.1, NDV F: GenBank acc.nr.AAK 55550.1, NDV HN: GenBank acc.nr.MH 614933.1, IBDV VP2: GenBank acc.nr.KX 827589.1, and IBV spike: GenBank acc.nr.AAA 66578.1.

[0083] Furthermore, more detailed information on HA proteins is available at www.rcsb.org, the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB), and www.fludb.org, the influenza research database.

[0084] In one embodiment of the recombinant protein for use according to the present invention, the antigen is selected from the AIV HA protein, and the antigen comprises only the external domain of the HA protein. This prevents adhesion to the cell membrane of the cells used to express the recombinant protein for use according to the present invention.

[0085] The external domain of a mature AIV HA protein includes an N-terminal portion without a signal sequence and a central portion of the HA protein, and therefore contains the HA1 and HA2 domains, but does not contain a transmembrane domain or cytoplasmic domain. Typically, these last two portions combine to form the C-terminal 35-40 amino acids of HA.

[0086] In one embodiment of the recombinant protein for use according to the present invention, the antigen is the external domain of an H5, H7, or H9 type AIV HA protein, and the antigen comprises a protein having an amino acid sequence selected from SEQ ID NOs: 3, 4, and 5.

[0087] In one embodiment of the recombinant protein for use according to the present invention, the antigen is an ectodomain derived from the AIV HA protein, and the antigen also includes a trimerizing domain.

[0088] Such trimerizing domains can compensate for the loss of the transmembrane and cytoplasmic domains of HA, form homotrimers, and restore the ability to resemble its native 3D shape. Furthermore, they improve the solubility and stability of the recombinant protein of the present invention having the HA-ectodomain antigen.

[0089] In the present invention, the trimerizing domain is a peptide and may be one of several known to be suitable for this function, for example, the isoleucine zipper 3 domain of the GCN4 transcription activator derived from Saccharomyces cerevisiae, or the Foldon domain ("Foldon") of the bacteriophage T4 fibrin protein.

[0090] In a preferred embodiment, the trimerizing domain is Foldon, and more preferably, Foldon comprises the amino acid sequence of SEQ ID NO: 6.

[0091] In one embodiment of a recombinant protein for use according to the present invention, the antigen is the external domain of the AIV HA protein, and the antigen also includes a trimerizing domain, the trimerizing domain is located on the C-terminal (downstream) side of the HA external domain.

[0092] In preferred embodiments, the HA external domain and the trimerization domain are positioned in the recombinant protein for use according to the present invention without intervening amino acids.

[0093] In a preferred embodiment, the antigen comprising the AIV H9 HA external domain and Foldon comprises the amino acid sequence of SEQ ID NO: 7.

[0094] In recombinant proteins for use according to the present invention, the antigen and the binding domain may be positioned in two orientations relative to each other, such that either the antigen or the binding domain is closer to the N-terminus of the recombinant protein for use according to the present invention. In this regard, the trimerizing domain, which may be used when the antigen is selected to be an HA extradomain, is considered part of the antigen.

[0095] In one embodiment of the recombinant protein for use according to the present invention, the antigen is located in the recombinant protein at the N-terminal (upstream) end of the binding domain.

[0096] In an alternative embodiment, the binding domain is located within the recombinant protein at the N-terminal (upstream) end of the antigen.

[0097] In one embodiment, the recombinant protein for use according to the present invention includes a linker located between the antigen and the binding domain, or between the binding domain and the antigen, depending on their mutual orientation. Preferably, the linker is 1 to 30 amino acids in size. More preferably, the linker contains the amino acids glycine and serine. Even more preferably, the linker contains the amino acid sequence of SEQ ID NO: 8.

[0098] Therefore, in one embodiment, the recombinant protein for use according to the present invention is -AIV H5 HA external domain, trimerization domain, linker and CD83-scFv, -AIV H7 HA external domain, trimerization domain, linker and CD83-scFv, and -AIV H9 HA external domain, trimerization domain, linker and CD83-scFv, (Here, the elements are presented from the N-terminus to the C-terminus.) Includes one of the combinations selected from.

[0099] In a preferred embodiment, the AIV HA external domain is selected from SEQ ID NOs: 3, 4, and 5, the trimer domain is SEQ ID NO: 6, the linker is SEQ ID NO: 8, and CD83-scFv is SEQ ID NO: 2.

[0100] For the purpose of expressing, isolating, quantifying, and (optionally) purifying recombinant proteins for use according to the present invention, the recombinant proteins may also contain one or more peptides that function as biochemical or serological markers (or tags). The markers may be the same or different. The markers may be located at different positions within the recombinant protein.

[0101] Well-known markers include affinity tags such as maltose-binding protein (MBP) tags or histidine (His) tags, epitope tags such as Myc tags, Ctag tags, V5 tags or Flag tags, or fluorescent protein tags such as GFP or YFP, or parts thereof, all of which are well-known in the art.

[0102] The markers may be used for detection and quantification purposes, for example, for detection or binding to specific antibodies, in IFT or ELISA. Purification can be performed, for example, using immunoassay or metal affinity chromatography.

[0103] The His tag typically has 4 to 10 histidine molecules. Preferably, the His tag is a 6×histidine tag, i.e., it has 6 consecutive histidine molecules.

[0104] "Ctag" is the C-terminus of the α-synuclein protein, which includes SEQ ID NO: 9 and is known to cause aggregates seen in neurological disorders such as Parkinson's disease. When used, Ctag is preferably included in the C-terminus of the recombinant protein of the present invention. Purification of Ctag by immunoaffinity chromatography is sometimes more effective than purification of His tag, for example, when there is protein disturbance in the culture of the expression system.

[0105] The V5 tag is derived from Simian virus 5. Preferably, the V5 tag contains the amino acid sequence of SEQ ID NO: 10.

[0106] In one embodiment, the recombinant protein for use according to the present invention comprises a marker peptide. More preferably, the marker peptide is one or more selected from Ctag, His tag, and V5 tag. Even more preferably, the recombinant protein comprises two or more from Ctag, His tag, and V5 tag.

[0107] For the expression of recombinant proteins for use according to the present invention, several further adjustments may be made as needed. Such fine-tuning or optimizations are standard and well known to those skilled in the art. For example, it depends on how the protein is expressed by the host cells of the expression system, i.e., whether it is secreted inside the cell, on the cell surface, or outside the cell. In the last two cases, a signal sequence can be provided on the N-terminal side, and this signal functions well in the cells of the expression system used. One example is the use of the "Drosophila melanogaster immunoglobulin heavy chain binding protein" (BIP) signal sequence to enable secretion when expressed in S2 cells.

[0108] In one embodiment, the recombinant protein for use according to the present invention comprises a signal sequence, preferably a BIP signal sequence, and more preferably a BIP signal sequence comprising the amino acid sequence of SEQ ID NO: 11.

[0109] In the process of constructing nucleic acids that result in the expression of recombinant proteins for use according to the present invention, one or more restriction enzyme (RE) sites may be used. If those RE sites are located in the coding region of the recombinant protein, their remaining nucleotides are translated into several amino acids, which are then located among some of the elements that make up the recombinant protein for use according to the present invention.

[0110] For example, one construct used in the present invention subcloned the H9 HA external domain-Foldon element using RE sites KpnI and PacI, and subcloned the HA antigen-Foldon and the C-terminal CD83-scFv of the linker of SEQ ID NO: 8 using RE sites NotI and XbaI.

[0111] As a result, one version of the recombinant protein for use according to the present invention comprises the amino acid sequence of SEQ ID NO: 12, the details of which are described in Table 1. [Table 1] A control construct without the linker and CD83-scFv was prepared. This construct lacked the amino acid region 545-802 of SEQ ID NO: 12 and contained the amino acid sequence of SEQ ID NO: 13.

[0112] Constructs similar to sequence numbers 12 and 13 can be readily constructed using other HA antigen sequences, namely, one of the H5 HA extradomain or H7 HA extradomain shown in sequence numbers 4 and 5, respectively.

[0113] In one embodiment of the recombinant protein for use according to the present invention, the antibody that reacts with the antigen is a maternal antibody.

[0114] In this invention, it is possible to easily determine whether existing antibodies are of maternal origin or not, and in fact, only chicks less than 2-4 weeks old possess MDA. Furthermore, MDA mainly consists of IgY, which is a functional homolog of mammalian IgG, but is structurally different, as IgY has four heavy chain constant domains compared to the three of IgG.

[0115] In one embodiment of the recombinant protein for use according to the present invention, the protected bird is poultry. More preferably, the poultry is selected from chickens, turkeys, ducks, and geese. Even more preferably, the poultry is chicken.

[0116] In the present invention, the birds may be of any type, breed, or subspecies, for example, laying hens, breeding hens, broilers, hybrids, or parent lines of any of such breeds. Preferred types of poultry are selected from broilers, breeding hens, and laying hens. More preferred are broiler-type and laying-type poultry. Broiler poultry are the most preferred.

[0117] As described, the present invention provides a recombinant protein for use in a method of protecting serologically positive birds from pathogens. This method can be advantageously applied to either older birds whose existing antibodies are the result of previous active vaccination, or to younger birds whose existing antibodies are MDA.

[0118] Therefore, in one embodiment of the recombinant protein for use according to the present invention, the protected bird is less than 4 weeks old, preferably less than 3 weeks old, more preferably less than 2 weeks old, even more preferably less than 1 week old, and even more preferably 1 day old (i.e., the day of hatching). In one embodiment, the protected bird is about 18 days old from embryonic development (i.e., still in the egg).

[0119] In alternative embodiments of recombinant proteins for use according to the present invention, the protected birds are at least two weeks old.

[0120] As described, recombinant proteins for use according to the present invention can also be successfully applied by indirect use, i.e., by expressing recombinant proteins from recombinant vectors, such as DNA plasmids, RNA molecules, or viral vectors.

[0121] Accordingly, in a further embodiment, the present invention relates to a recombinant vector capable of expressing a recombinant protein for use according to the present invention, for use in a manner that protects birds possessing antibodies reactive to an antigen contained in the recombinant protein expressed by the recombinant vector from a pathogen from which the antigen originates.

[0122] A "vector" is a well-known molecular structure in the field of this invention that carries genetic information (nucleic acid sequences) for encoding a polypeptide, along with appropriate signals that enable its expression under appropriate conditions, for example, within a host cell. In relation to this invention, "expression" refers to the well-known principle of expressing a protein from genetic information by transcription and / or translation.

[0123] Many types and variations of such vectors are known, ranging from nucleic acid molecules such as DNA or RNA to more complex structures such as virus-like particles and replicon particles, and even to the replication of recombinant microorganisms such as viruses, and can be used in the present invention.

[0124] Recombinant vectors for use according to the present invention are “recombinant” because they have a molecular structure altered by in vitro manipulation of their genetic information. The alterations made may result in, improve, or adapt the replication, expression, manipulation, purification, stability, and / or immunological behavior of the vector and / or the protein it expresses. These and other techniques are described in great detail in the standard textbooks of Sambrook & Russell and Ausubel et al. (both cited above), “PCR primers: a laboratory manual” by C. Dieffenbach & G. Dveksler (CSHL Press, ISBN 0879696540), and “PCR protocols” by J. Bartlett and D. Stirling (Humana press, ISBN: 0896036421).

[0125] Depending on the type of vector used, it is necessary to provide more or less signal for replication and expression, either in cis (i.e., provided within the recombinant vector itself) or trans (i.e., provided from a separate source), and this is all well known.

[0126] Those skilled in the art have the knowledge to select the necessary signals and combine them into usable combinations so that a recombinant vector for use according to the present invention can "express" a recombinant protein for use according to the present invention under appropriate conditions. Elements to aid in construction and cloning, such as restriction enzyme recognition sites or PCR primers, can be selected from one or more of the following: promoters, stop codons, termination signals, polyadenylation signals, 7-methylguanosine (7mG) cap structures, and introns having functional splice donor and acceptor sites.

[0127] In embodiments of recombinant vectors for use according to the present invention, the characteristics, uses, methods, protection, birds, antibodies, antigens, and pathogens of recombinant proteins are all as embodied herein.

[0128] In one embodiment of the recombinant vector for use according to the present invention, the recombinant protein it expresses comprises the amino acid sequence of SEQ ID NO: 12.

[0129] The nucleotide sequence used for the expression of the amino acid sequence of SEQ ID NO: 12 includes the nucleotide sequence of SEQ ID NO: 14.

[0130] Therefore, in one embodiment of a recombinant vector for use according to the present invention, the vector comprises the nucleotide sequence of SEQ ID NO: 14.

[0131] The control protein of SEQ ID NO: 13 is encoded by a nucleotide sequence that includes the nucleotide sequence of SEQ ID NO: 15.

[0132] Both Sequence IDs 14 and 15 have been codon-optimized against the codon usage frequency table of Drosophila melanogaster (D. melanogaster) S2 cells to optimize expression in these cells. Further details are provided below.

[0133] As described, recombinant vectors for use according to the present invention may have several different forms.

[0134] Therefore, in one embodiment, the recombinant vector for use according to the present invention is selected from nucleic acids, viruses, and replicon particles (RPs).

[0135] In this invention, nucleic acids may be DNA or RNA, may be single-stranded or double-stranded, and may be of natural or synthetic origin.

[0136] In one embodiment of a recombinant vector for use according to the present invention, where the vector is a nucleic acid, the nucleic acid is a eukaryotic expression plasmid.

[0137] A typical DNA "eukaryotic expression plasmid" has a suitable signal for the expression of a heterologous gene inserted into the plasmid, under the control of an active promoter in eukaryotic cells. The plasmid can then be inserted into a eukaryotic host cell or host organism by some transfection method, for example, by using a biochemical carrier, by mechanical means, or by electroporation, resulting in the expression of the heterologous gene insertion. Typically, such expression is transient because the plasmid lacks a signal for stable integration into the host cell's genome; therefore, such plasmids typically do not transform or immortalize the host or host cell. All these materials and procedures are well known in the art and are described in handbooks.

[0138] Such eukaryotic expression plasmids are commercially available from various suppliers, including a series of plasmids such as pcDNA(trademark), pCR3.1(trademark), pCMV(trademark), pFRT(trademark), pVAX1(trademark), pCI(trademark), Nanoplasmid(trademark), and pCAGGS.

[0139] In a preferred embodiment, the eukaryotic expression plasmid is either a pFRT plasmid (Thermo Fisher Scientific) or a pCAGGS plasmid (Niwa et al., 1991, Gene, vol. 108, pp. 193-199).

[0140] Eukaryotic expression plasmids can contain several features for regulation, such as expression and purification. One possible signal is an antibiotic resistance gene, which can be used for selection during the construction and cloning process. However, when intended for administration to human or animal targets, selecting such antibiotics is undesirable due to the risk of inducing antibiotic resistance.

[0141] In a preferred embodiment of the recombinant vector for use according to the present invention, where the vector is a nucleic acid and the nucleic acid is a eukaryotic expression plasmid, the plasmid does not contain an antibiotic resistance gene.

[0142] The recombinant vector for use according to the present invention can be delivered to a host cell or target organism in the form of a eukaryotic expression plasmid, which expresses the HA stem polypeptide of the present invention in the host cell. Delivery of the expression plasmid can be by several means, for example, mechanical or chemical means, as naked DNA, or encapsulated in a suitable (nanoparticle) carrier such as a protein, polysaccharide, lipid, or polymer. Well-known examples of nucleic acid carriers are dendrimers, lipid nanoparticles, cationic polymers, and protamines.

[0143] A particular form of recombinant vector for use according to the present invention, as a eukaryotic expression plasmid, is when the plasmid enables the delivery of replicon RNA.

[0144] Therefore, in one embodiment of a recombinant vector for use according to the present invention, where the vector is a nucleic acid and the nucleic acid is a eukaryotic expression plasmid, the plasmid encodes replicon RNA.

[0145] "Replicon RNA" is a self-replicating RNA that, in addition to the nucleic acid encoding the recombinant polypeptide of the present invention, contains elements necessary for RNA replication, such as replicase genes. However, unlike replicon particles (RPs), replicon RNA is not packaged by viral structural proteins, and therefore its efficiency in entering host cells on its own is low.

[0146] Plasmids encoding replicon RNA can be delivered to host cells in the same way as protein expression plasmids.

[0147] Vaccination with eukaryotic expression plasmids encoding replicon RNA offers advantages over vaccination with eukaryotic expression plasmid-expressed proteins because the replicon RNA amplifies the process; that is, through replicase translation, the replicon RNA produces subgenomic messenger RNA encoding recombinant proteins for use according to the present invention. This results in the expression of large amounts of recombinant proteins in both host cells and target birds.

[0148] In a preferred embodiment of the recombinant vector for use according to the present invention, the vector is a nucleic acid, the nucleic acid is a eukaryotic expression plasmid, and the plasmid encodes a replicon RNA, the replicon RNA is an alphavirus-based replicon RNA, and more preferably the alphavirus-based replicon RNA is a Venezuelan encephalitis virus (VEEV)-based replicon RNA.

[0149] An example of a eukaryotic expression plasmid encoding VEEV replicon RNA is the pVAX plasmid (Thermo Fisher Scientific), which contains VEEV non-structural protein genes 1-4 and is driven by a eukaryotic promoter, such as the human CMV pre-early gene 1 promoter.

[0150] In alternative embodiments of recombinant vectors for use according to the present invention, where the vector is a nucleic acid, the nucleic acid is an RNA molecule.

[0151] The RNA molecule of the present invention may have different forms and functions, and may be, for example, mRNA or replicon RNA.

[0152] Recombinant vectors as RNA molecules for use according to the present invention can be delivered to birds or host cells in various ways, for example, by mechanical or chemical means, or encapsulated with a suitable (nanoparticle) carrier such as a protein, polysaccharide, lipid, or polymer, as described herein. For stabilization, RNA nucleotide analogs or specific chemical modifications can be incorporated into or applied to the nucleotide or its backbone, for example.

[0153] In one embodiment of a recombinant vector for use according to the present invention, where the vector is a nucleic acid and the nucleic acid is an RNA molecule, the RNA molecule is mRNA.

[0154] mRNA (messenger RNA) is well known in the art and typically has a 5'7-methylguanosine (7mG) cap and a 3' polyA tail. mRNA can be delivered to a eukaryotic host organism or host cell by transfection and / or by using a suitable carrier, such as a polymer or cationic lipid.

[0155] In one embodiment of a recombinant vector for use according to the present invention, where the vector is a nucleic acid and the nucleic acid is an RNA molecule, the RNA molecule is a replicon RNA.

[0156] Replicon RNA can be produced in vitro, for example, using the pVAX plasmid described herein, and then administered to host cells or target organisms using any suitable method.

[0157] Recombinant vectors in the form of replicated recombinant viral vectors for the expression and delivery of heterologous proteins are well known in the art. These provide an efficient method of vaccination because the viral vector replicates and amplifies in target birds. The construction and modification of recombinant vector viruses are standard and can be carried out using standard molecular biological techniques.

[0158] Therefore, in one embodiment of the recombinant vector for use according to the present invention, the recombinant vector is a virus.

[0159] In the present invention, the viral vector is a virus that replicates in birds. Many different virus species have been used for a long time as recombinant vectors for birds.

[0160] In one embodiment of a recombinant vector for use according to the present invention, where the vector is a virus, the virus is selected from herpesviruses, poxviruses, paramyxoviruses, and adenoviruses.

[0161] Examples of suitable vector viruses that can be used as vectors for birds are well known in the art, for example, herpesviruses include turkey herpesvirus (HVT) or serotype 1 or 2 Marek's disease virus (MDV), poxviruses include fowlpox virus, paramyxoviruses include NDV, and adenoviruses include poachian adenovirus.

[0162] In a preferred embodiment of the recombinant vector for use according to the present invention, where the vector is a virus and the virus is a herpesvirus, the herpesvirus is selected from HVT, MDV1, and MDV2.

[0163] Examples of recombinant viral vectors expressing and delivering the influenza HA gene are described in International Publication No. 2012 / 052384 and European Patent No. 19218804.3 for HVT as a vector. An example of NDV as a vector is described in International Publication No. 2007 / 106882.

[0164] Typically, for the construction of a recombinant viral vector, an expression cassette is inserted into a locus within the vector's genome. Various techniques are available to control the locus and the orientation of its insertion. For example, by directing the cassette's insertion by a homologous recombination process using duplicate cosmids, for example, using a suitable adjacent section from the vector's genome, as described in U.S. Patent No. 5,961,982. Alternatively, this insertion may be performed using CRISPR / Cas technology.

[0165] An "expression cassette" is a nucleic acid fragment containing at least one heterologous gene and one promoter that drives the transcription of that gene, in order to enable the expression of an encoded protein. Termination of transcription may be brought about by a sequence provided by the genomic insertion site of the cassette, or the expression cassette itself may contain termination signals such as transcription terminators. In such a cassette, both the promoter and terminator must be in close proximity to the gene they regulate the expression of, this is called "operably linked," so that there are no other significant sequences between them that intervene in the effective initiation or termination of transcription, respectively. As will be apparent to those skilled in the art, an expression cassette is a self-contained expression module, and therefore the orientation of its read direction relative to the vector viral genome is generally not important.

[0166] In addition to the use of viruses as vectors for use according to the present invention, recombinant vectors for use according to the present invention can also be delivered to and expressed in birds by virion-like macromolecular structures. Examples include virus-like particles (VLPs) or replicon particles (RPs). These structures, known as "single-cycle" infectious particles, contain the features necessary to infect host cells and express the heterologous genes they possess, but they are usually unable to perform complete viral replication because they lack the viral genome (or the relevant portion thereof) from which they were constructed. This functions as an integrated safety feature.

[0167] "RPs" are well-known, and several RPs have been developed as platforms for the expression and delivery of various proteins. Alphaviruses are preferred as basic raw materials for RPs due to their broad host range and rapid replication. Of course, some alphaviruses are highly pathogenic in their wild form, so appropriate safety measures are necessary to weaken and control infections caused by such RPs. For reviews, see Kamrud et al. (2010, J. Gen. Virol., vol. 91, pp. 1723-1727) and Vander Veen et al. (2012, Anim. Health Res. Rev., vol. 13, pp. 1-9).

[0168] Therefore, in one embodiment of the recombinant vector for use according to the present invention, the vector is RP. Preferably, the RP is alphavirus RP. More preferably, the alphavirus RP is VEEV RP.

[0169] The preferred alphavirus RP is based on VEEV, which is used as a recombinant vector vaccine for humans, pigs, poultry, and fish. Methods and tools for constructing, testing, and using VEEV-based alphavirus RP are well known and available; see, for example, Pushko et al. (1997, Virology, vol. 239, pp. 389-401) and International Publication No. 2019 / 110481. The preferred VEEV RP technology is SirraVax. smThis is RNA particle technology (Harris vaccine).

[0170] RNA for RP can be easily prepared in vitro as follows: a DNA plasmid is used to translate the gene into RNA, which is then recovered and transfected into host cells along with a helper RNA that transcodes the VEEV structural protein.

[0171] As described, the recombinant vectors for use according to the present invention can be advantageously used to deliver and express recombinant proteins for use according to the present invention in birds, for example, as a method of vaccinating their targets. This involves, at one stage, administering the vector to birds, for example, in which case the vector is a nucleic acid such as a DNA expression plasmid or an RNA molecule.

[0172] The vector may also be introduced into host cells in vitro for vector amplification and / or recombinant protein expression, after which the host cells (containing the vector and / or protein) are administered to birds, for example, in which case the vector is a viral vector, e.g., HVT.

[0173] Furthermore, the vector can be introduced into cells of a recombinant expression system for the expression of recombinant protein, the protein can be recovered from the cell culture and used to vaccinate birds as described above. In addition, host cells themselves that are infected with or transfected with the recombinant vector for use according to the present invention and that contain and / or express the recombinant protein for use according to the present invention can be used in the protective method of the present invention, for example, the infected or transfected host cells themselves can be used for vaccinating birds.

[0174] Depending on the type of vector applied, its introduction into host cells may require a carrier or some transfection method, as described herein, or it may be induced by the vector itself.

[0175] Accordingly, in a further embodiment, the present invention relates to a host cell maintained in vitro, wherein the host cell comprises a recombinant protein for use according to the present invention and / or a recombinant vector for use according to the present invention.

[0176] The “host cell” of the present invention is a cell that enables the expression of recombinant proteins for use according to the present invention and / or enables the replication of recombinant vectors for use according to the present invention.

[0177] The host cells of the present invention may be primary cells maintained in vitro, and may be maintained, for example, in a suspension, a monolayer, or a tissue.

[0178] Alternatively, the host cell may be an immortalized cell maintained in vitro, such as a cell derived from an established cell line capable of growing and dividing almost indefinitely. Depending on the type of host cell, the expression of the HA stem polypeptide of the present invention may involve more or less a wide range of post-translational processing, such as signal peptide cleavage, disulfide bond formation, glycosylation, and / or lipid modification.

[0179] The primary cells and the immortalized host cells may be of the same or different species. Furthermore, one or both may be of the same or different species as the bird to which the method protected by this invention is applied.

[0180] Most of the host cells used are fibroblasts and lymphocytes. When HVT is used as the recombinant vector virus of the present invention, the host cells are preferably primary chicken embryo fibroblasts (CEFs), which can be used and stored as described; see, for example, International Publication No. 2019 / 121888.

[0181] In one embodiment of the host cell of the present invention, the host cell is preferably an immortalized avian cell. Several immortalized avian cell lines are described, for example, in International Publication No. 97 / 044443 and International Publication No. 98 / 006824, and more preferably the immortalized avian host cell of the present invention is an immortalized CEF, and even more preferably the immortalized CEF disclosed in International Publication No. 2016 / 087560.

[0182] In one embodiment of the host cell of the present invention, the host cell is preferably a cell from a recombinant expression system. Examples of cells derived from an expression system include, for example, cells derived from bacteria, yeast, insects, birds, or mammals.

[0183] Cells derived from bacterial expression systems include, for example, cells from the genera Escherichia, Bacillus, Salmonella, Caulobacter, or Lactobacillus.

[0184] Cells derived from yeast expression systems include, for example, cells derived from Saccharomyces cerevisiae or Pichia pastores.

[0185] Cells derived from insect cell expression systems include, for example, cells from Drosophila melanogaster, such as Schneider 2 (S2) cells, or cells for use in baculovirus-insect cell expression systems, namely cells from Spodoptera frugiperda, such as Sf21 or Sf9 cells, or cells from Trichoplusia ni, such as High Five® cells.

[0186] Cells derived from mammalian expression systems include, for example, hamster-derived cells, such as Chinese hamster ovary (CHO) cells.

[0187] The corresponding uses of all these cell lines and recombinant expression systems are well known in the art and can be used using standard techniques and materials.

[0188] In one embodiment of the recombinant vector for use according to the present invention, the nucleic acid encoding the recombinant protein for use according to the present invention is codon-optimized.

[0189] Codon optimization is well-known and is applied to improve the level of gene expression in an expression system, which is typically a different situation from that of gene origin. Optimization involves fitting a nucleotide sequence to encode a desired amino acid, but in a sense, this corresponds to the codon priority (tRNA repertoire) of the recombinant vector, host cell, or target organism in which the sequence is expressed. As a result, the nucleotide mutations applied are silent.

[0190] Accordingly, in one embodiment of the recombinant vector for use according to the present invention, the recombinant protein for use according to the present invention is encoded by a nucleic acid sequence that is codon-optimized for avian organisms, which are intended to be protected by a method for protecting the present invention. Preferably, the codon optimization is for poultry. More preferably, the codon optimization is for poultry selected from chickens, turkeys, ducks, and geese.

[0191] In one embodiment of the recombinant vector for use according to the present invention, the recombinant protein for use according to the present invention is encoded by a nucleic acid sequence that is codon-optimized for cells of a recombinant expression system, preferably cells derived from bacteria, yeast, insects, birds, or mammals. More preferably, the nucleic acid is optimized for insect cells, and even more preferably for Drosophila Schneider 2(S2) cells.

[0192] The recombinant proteins for use and recombinant vectors for use according to the present invention may also be characterized by other expressions to suit a particular jurisdiction.

[0193] Accordingly, in a further embodiment, the present invention relates to the use of recombinant proteins for use according to the present invention or the use of recombinant vectors for use according to the present invention for the production of vaccines for protecting birds from pathogens, characterized in that the antigen contained in the recombinant protein or contained in the recombinant protein expressed by the recombinant vector is derived from the pathogen, and the birds possess antibodies that are reactive to the antigen.

[0194] In any embodiment of the recombinant protein or recombinant vector for the production of vaccines according to the present invention, the characteristics of the recombinant protein, recombinant vector, protection, avian, pathogen, antigen and antibody are all as described herein.

[0195] It is well known that a "vaccine" is a composition containing at least one compound capable of inducing a protective immunological effect within a pharmaceutically acceptable carrier. The "immunologically active compound" of the present invention is a recombinant protein for use according to the present invention or a recombinant vector for use according to the present invention.

[0196] The vaccine for the present invention can be manufactured using standard methods and procedures well known in the art. General techniques and considerations applicable to the manufacture of vaccines under well known standards for the manufacture of pharmaceuticals are described, for example, in government directives and regulations (Pharmacopoeia, 9th CFR) and in well known handbooks such as "Veterinary Vaccineology" and "Remington" (both mentioned above). Generally, such vaccines are prepared aseptically and using pharmaceutical-grade excipients.

[0197] Such manufacturing processes may include microbiological testing for sterility and the absence of exogenous drugs, and may include in vivo or in vitro experiments to confirm efficacy and safety. After testing for quality, quantity, sterility, safety, and efficacy is complete, the vaccine can be marketed. All of this is well known to those skilled in the art.

[0198] For example, if a recombinant protein for use according to the present invention is produced by a recombinant expression system, the protein can be recovered from the expression system culture, for example, as the whole culture. Alternatively, the recovered material may be a portion of such a culture, for example, the supernatant or cell pellet after centrifugation of the cell culture, or the filtrate or residue after filtration. The supernatant can be obtained after the culture has settled by gravity, for example, by leaving it overnight, or by centrifugation, and the filtrate is what passes through the filter during filtration.

[0199] As described, the recombinant proteins for use according to the present invention and the recombinant vectors for use according to the present invention achieve their beneficial effects in bird conservation through vaccines containing the recombinant proteins and / or the recombinant vectors.

[0200] Accordingly, in a further embodiment, the present invention relates to vaccines and pharmaceutically acceptable carriers comprising recombinant proteins for use according to the present invention or recombinant vectors for use according to the present invention, for use in a manner that protects birds possessing antibodies reactive to antigens contained in the recombinant protein or expressed by the recombinant vector from pathogens from which the antigens originate.

[0201] In embodiments of the vaccine for use according to the present invention, the characteristics of the recombinant protein, recombinant vector, use, method, protection, avian, antibody, antigen and pathogen are all as embodied herein.

[0202] "Pharmacologically acceptable carriers" are well known to aid in the stabilization and administration of vaccines, and moreover, are relatively harmless and well-tolerated by vaccinators. Such carriers may be, for example, water or physiological saline solutions. In more complex forms, carriers may be buffers that may contain further additives such as stabilizers or preservatives. Details and examples are described in well-known handbooks such as "Remington: The Science and Practice of Pharmacy" (2000, Lippincott, USA, ISBN: 683306472) and "Veterinary Vaccinology" (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681).

[0203] If the vaccine according to the present invention contains a recombinant vector which is a replicating virus, the pharmaceutically acceptable carrier is preferably a composition that stabilizes the virus or the host cells containing the virus. Examples include several viral vaccine diluents and stabilizers for freeze- or lyophilization storage, typically containing, for example, sugars, amino acids, physiological buffers (e.g., saline, PBS, or 50 mM HEPES), and often bulky compounds such as albumin or polymers. For example, if the vaccine contains a recombinant HVT vector, such vaccines are typically marketed as cell-related products. In that case, the pharmaceutically acceptable carrier is preferably a mixture of culture medium containing about 10% serum and about 6% DMSO. This carrier also provides stabilization of HVT-infected host cells during freezing and cryopreservation. The serum may be any serum routinely used in cell culture, such as fetal or neonatal calf serum.

[0204] If the vaccine according to the present invention comprises a recombinant vector for use according to the present invention, which is a nucleic acid or RP, a pharmaceutically acceptable carrier may be a simple buffer, for example, a phosphate buffer containing 5% w / v sucrose.

[0205] Furthermore, additional carriers may be added to stabilize and / or deliver the recombinant vector for use in the present invention, for example, to encapsulate the recombinant vector according to the present invention, which is a nucleic acid or RP, with a suitable (nanoparticle) carrier such as a protein, polysaccharide, lipid, or polymer. Preferably, the additional carrier for the recombinant vector according to the present invention, which is an RP, includes a nanogel, which is a biodegradable polyacrylic polymer, as described in International Publication No. 2012 / 165953.

[0206] Clearly, recombinant vectors or in vitro host cells containing such vectors can be used herein in either a living (i.e., replicated) or dead (non-replicating or inactivated) state. Then, only a portion of the recombinant vector or host cells can be used herein, for example, as a pellet, supernatant, concentrate, dialysate, extract, sonication solution, lysate, or as part of a composition containing the vector and / or host cells, such as a culture. This is all well known to those skilled in the art.

[0207] If the vaccine for use according to the present invention comprises a recombinant protein for use according to the present invention, the vaccine may contain an adjuvant for stimulating an induced immune response.

[0208] Therefore, in one embodiment, the vaccine for use according to the present invention includes an adjuvant.

[0209] An "adjuvant" is a well-known vaccine component that nonspecifically stimulates the immune response of a target. Many different adjuvants are known in the art. Examples of adjuvants include complete or incomplete Freund's adjuvants, vitamin E or alpha-tocopherol, nonionic block polymers and polyamines, e.g., dextran sulfate, Carbopol®, pyran, saponins, e.g., Quil A® or Q-vac®. Saponins and vaccine components can be combined in ISCOM®. Furthermore, peptides such as muramyl dipeptide, dimethylglycine, and tuftosine are also included. In addition, aluminum salts such as aluminum phosphate or aluminum hydroxide, available as Alhydrogel® (Brenntag Biosector), Rehydragel® (Reheis), and Rehsorptar® (Armour Pharmaceutical).

[0210] Widely used adjuvants include oils, such as mineral oils, such as light (white) mineral (paraffin) oils, or non-mineral oils, such as squalene, squalane, vegetable oils, or their derivatives, such as ethyl oleate. Combination products such as ISA® (Seppic) or DiluvacForte® and Xsolve® (both from MSD Animal Health) can also be advantageously used.

[0211] A handbook on adjuvants, their use, and effects is "Vaccine adjuvants" (Methods in molecular medicine, vol. 42, D. O'Hagan ed., 2000, Humana press, NJ, ISBN: 0896037355).

[0212] Adjuvants may be included in vaccines for use according to the present invention in several embodiments. If the adjuvant contains oil, the vaccine may be provided in aqueous form, or it may be formulated in different embodiments as an emulsion with oil, namely as a water-in-oil (W / O), oil-in-water (O / W), or as a double emulsion of either W / O / W or O / W / O.

[0213] An emulsion is a mixture of at least two immiscible liquids, in which one is dispersed in the other. Typically, droplets of the dispersed phase are very small, ranging from a micrometer to less than a meter in size.

[0214] Procedures and apparatus for preparing emulsions on any scale are well known in the art. One or more emulsifiers can be used to stabilize the emulsion.

[0215] An emulsifier is an amphiphilic molecule that possesses both hydrophobic and hydrophilic properties. Many emulsifiers are known in the art for their various properties. Most are readily available commercially and in several purities. Common emulsifiers for vaccines are sorbitan monooleate (Span® 80) and polyoxyethylene sorbitan monooleate (Polysorbate 80, or Tween® 80).

[0216] A well-known method for characterizing the properties of emulsifiers (or mixtures thereof) is the HLB number (hydrophile-lipophile balance; Griffin, 1949, J.Soc.Cosm.Chem., vol.1, pp.311-326). Typically, emulsifiers or mixtures of emulsifiers with an HLB number of less than 10 are suitable for W / O emulsions, while emulsifiers (or mixtures) with an HLB number of 10-16 are suitable for O / W emulsions.

[0217] Emulsion stabilizers can also be added, examples of which include benzyl alcohol and triethanolamine.

[0218] In a preferred embodiment of the vaccine for use according to the present invention, the vaccine comprises an adjuvant, wherein the adjuvant comprises an oil. More preferably, the oil comprises a mineral oil. Even more preferably, the mineral oil comprises a light (or white) liquid paraffinic oil.

[0219] Examples of light liquid paraffin oils include Drakeol® 6 VR (Penreco), Marcol® 52 (Exxon Mobile), and Klearol® (Sonneborn).

[0220] In a preferred embodiment of the vaccine for use according to the present invention, in which the vaccine contains an adjuvant and the adjuvant contains an oil, the vaccine is formulated as a water-in-oil emulsion.

[0221] In other expressions and in specific jurisdictions, further embodiments of the present invention may be defined as follows:

[0222] In a further embodiment, the present invention relates to the use of a recombinant protein for use according to the present invention, a recombinant vector for use according to the present invention, or a vaccine for use according to the present invention for protecting birds from pathogens, characterized in that the antigen contained in the recombinant protein or expressed by the recombinant vector is derived from the pathogen, and the birds possess antibodies that are reactive to the antigen.

[0223] In one embodiment of use according to the present invention, such use includes the administration of all recombinant proteins, recombinant vectors, or vaccines of the present invention to birds.

[0224] In embodiments of use according to the present invention, the characteristics of recombinant proteins, recombinant vectors, vaccines, uses, methods, protection, birds, pathogens, antigens, and antibodies are all as embodied herein.

[0225] In a further embodiment, the present invention relates to a method for protecting birds from a pathogen, comprising the step of administering a vaccine for use according to the present invention to the birds, wherein the antigen contained in the vaccine is derived from the pathogen, and the birds possess antibodies that are reactive to the antigen.

[0226] The vaccines for use according to the present invention are typically suitable for administration to birds and are prepared in a form that matches the desired route of application and desired effect.

[0227] Depending on the route of application of the vaccine for use according to the present invention, it may be necessary to adapt the composition of the vaccine. This is well within the capabilities of those skilled in the art and involves overall fine-tuning of the vaccine's efficacy or safety. This can be done by adapting the vaccine's dose, volume, frequency, or route; by using a different form or formulation of the vaccine; or by adapting one of the vaccine's excipients (e.g., stabilizers or adjuvants).

[0228] In principle, the vaccine according to the present invention can be administered to birds at different points in their lifespan via different routes of administration. Specifically, the vaccine can be administered to birds of any age that possess antibodies reactive to the antigen in the recombinant protein for use according to the present invention.

[0229] When administration is carried out as early as possible, it can be administered on the day of hatching ("day 1") or immediately within the egg, for example, around 18 days after embryonic development, and all of these are well known in the art.

[0230] Devices for automated vaccine injection into fertilized eggs on an industrial scale are commercially available. This provides the earliest possible protection while minimizing labor costs. Different intraocular inoculation routes are known, including into the yolk sac, embryo, or allantois cavity, and these can be systematically optimized as needed.

[0231] The vaccine for use according to the present invention can be formulated as an injectable solution suitable for either intraovo or parenteral injection.

[0232] In one embodiment, the vaccine for use according to the present invention is formulated as a liquid selected from suspensions, solutions, dispersions, and emulsions.

[0233] In one embodiment, the vaccine for use according to the present invention is administered by a parenteral route. Preferably, the parenteral route is an intramuscular or subcutaneous route.

[0234] The exact amount of recombinant protein or recombinant vector is not important in this invention and can be easily determined by comparing the protective effects of different amounts.

[0235] Furthermore, if the vaccine for use according to the present invention contains a viral vector, it can replicate in vaccinated birds and only needs to be administered in an amount sufficient to establish a reproductive infection in the birds.

[0236] For example, if the viral vector for use according to the present invention is recombinant HVT, a suitable inoculation dose is 1 × 10¹ to 1 × 10⁵ plaque-forming units (pfu) of the HVT of the present invention per animal dose, preferably 1 × 10² to 1 × 10⁴ pfu / dose, more preferably 500 to 5000 pfu / dose, and most preferably about 1000 to about 3000 pfu / dose. Methods for counting viral particles of the HVT of the present invention are well known.

[0237] If the HVT vector for use according to the present invention is cell-associated, these amounts of HVT are contained within the infected host cells.

[0238] The volume per animal dose of the vaccine for use according to the present invention can be optimized according to the intended route of application, with intraocular administration generally given at a volume of 0.01 to 0.5 ml / egg and parenteral injection in birds generally given at a volume of 0.1 to 1 ml / bird.

[0239] Determining the immunological effective dose of the vaccine according to the present invention, or optimizing the volume of vaccine per animal dose, is both well within the capabilities of those skilled in the art.

[0240] The administration regimen for applying the vaccine for use according to the present invention to birds may be a single or multiple dose, may be in a form compatible with the vaccine formulation, and may be an amount that would be immunologically effective.

[0241] Preferably, the regimen for administering the vaccine for use according to the present invention is integrated into an existing vaccination schedule for other vaccines that the target birds may require, in order to reduce stress on the animals and lower labor costs. These other vaccines can be administered simultaneously, in parallel, or sequentially, in a manner that is appropriate to their registered use.

[0242] TIFF0007900480000002.tif197131TIFF0007900480000003.tif191131TIFF0007900480000004.tif184130TIFF0007900480000005.tif195130TIFF0007900480000006.tif188130TIFF0007900480000007.tif180130TIFF0007900480000008.tif194130TIFF0007900480000009.tif196130TIFF0007900480000010.tif182131TIFF0007900480000011.tif193130TIFF0007900480000012.tif188130TIFF0007900480000013.tif195129TIFF0007900480000014.tif181132TIFF0007900480000015.tif188129TIFF0007900480000016.tif196129TIFF0007900480000017.tif183131TIFF0007900480000018.tif195130TIFF0007900480000019.tif188130TIFF0007900480000020.tif182130TIFF0007900480000021.tif197129TIFF0007900480000022.tif189130TIFF0007900480000023.tif194130TIFF0007900480000024.tif193131TIFF0007900480000025.tif192129TIFF0007900480000026.tif185130TIFF0007900480000027.tif190129TIFF0007900480000028.tif187129TIFF0007900480000029.tif193133TIFF0007900480000030.tif188130TIFF0007900480000031.tif182129TIFF0007900480000032.tif196130TIFF0007900480000033.tif188131TIFF0007900480000034.tif191130TIFF0007900480000035.tif191131TIFF0007900480000036.tif182129TIFF0007900480000037.tif196130TIFF0007900480000038.tif181130TIFF0007900480000039.tif197130 The present invention is described herein in various aspects and embodiments. Naturally, any combination of these is considered to be within the scope of the invention. However, for the sake of brevity, this specification does not fully outline all possible combinations.

[0243] The present invention will now be further explained by the following non-limiting embodiments.

[0244] [Examples] [Example 1] Production of AIV-MDA-positive chickens 1.1. Introduction To enable testing of serologically positive chicken vaccination, an animal model simulating real-world conditions was created. Specifically, AIV MDA-positive offspring were produced by repeatedly vaccinating hens intramuscularly with an inactivated adjuvant-containing vaccine. The goal was to achieve HI titers in the offspring that were similar to those observed in the field, at least 5–7Log2.

[0245] 1.2. Materials and Methods SPF White Leghorn laying chickens were vaccinated to produce MDA-positive hatched chicks. All chickens were housed in an isolation room and kept on the floor. All chickens were given free access to feed and water throughout the experiment and were raised under veterinary supervision.

[0246] 1.2.1. Preparation of vaccines for MDA production: An inactivated AIV vaccine was prepared by culturing the H9N2 subtype of avian influenza A virus in 10-day-old embryonic SPF chicken eggs. Specifically, this was AIV strain:A / chicken / Pakistan- / UDL-01 / 2008 ("UDL-01"), see GenBank:ACP50708.1 and:Iqbal et al. (2009, PLoS One, vol.4:e 5788). 72 hours after infection, the eggs were refrigerated at 4°C, and the allantoic fluid was collected and removed by centrifugation at 3,000 rpm for 20 minutes to obtain the virus. The virus was titrated by a plaque assay against Maidin Darby canine kidney (MDCK) cells or by TCID50.

[0247] The virus was chemically inactivated using 0.1% beta-propiolactone, and then blinded through three passages in 10-day-old embryonic SPF chicken eggs to confirm inactivation. The inactivated virus was then concentrated by ultracentrifugation at 27,000 rpm for 2 hours at 4°C. The inactivated virus was then adjuvanted with liquid light paraffin oil and formulated into a water-in-oil emulsion. The resulting vaccine had a titer of 1040 hemagglutination units (HAU) / ml.

[0248] 1.2.2. Vaccination of hens and production of MDA+ hatched chicks A flock of 40 17-week-old SPF White Leghorn laying hens was used. The chickens were individually labeled. They were immunized with 0.5 ml of inactivated adjuvant-added H9N2 virus vaccine at a dose of 520 HAU / dose, administered via intraleg injection. The first dose of the vaccine was administered at 17 weeks of age (T=0), followed by the second and third doses at 20 weeks of age (T=3 weeks after the first dose) and 41 weeks of age (T=24 weeks after the first dose), respectively.

[0249] For serological monitoring of anti-AIV HI titers during development, blood samples were collected from the wing veins of hens on day 0 and at weeks 5, 11, 18, 29, and 36 after the first administration. Five SPF roosters were included in the group for fertilization, but these were not part of the actual experiment.

[0250] Fertilized eggs were collected 36 weeks after the first vaccination. These were incubated until hatching. Ten hatched chicks were euthanized on day 1 (D0), and their MDA levels were measured. These hatched chicks were then used in an MDA vaccination experiment.

[0251] 1.2.3. HI Assay The HI assay followed international guidelines (WHO 676 global influenza surveillance network: manual for the laboratory diagnosis and virological surveillance of influenza. 153 (2011)). Briefly, serial dilutions of serum were prepared by mixing 25 μl of serum with 25 μl of PBS. Next, 4 HA units of influenza virus were added to the diluted serum and incubated at 37°C for 1 hour. Finally, 50 μl of 1% chicken erythrocytes were added to the serum-virus mixture and incubated at room temperature for 45 minutes. The HI titer was expressed as the reciprocal of the highest dilution of antiserum that completely inhibited the hemagglutination activity of 4 units of viral hemagglutination.

[0252] The virus used in the HI assay was AIV H9N2 strain UDL-01.

[0253] 1.3.Results Figure 1 shows the results of hyperimmunization of hens that produce AIV MDA+ offspring. HI titration was performed using the homologous UDL-01 strain.

[0254] A third vaccination was administered 18 weeks after the start of the program when a decrease in HI titer in the hens' serum was observed. This resulted in very high HI titers in the hens, which were maintained until the final sampling point.

[0255] When the average HI titer of hens (n=10) was 4096 (12 Log2), fertilized eggs were retrieved at 36 weeks (53 weeks old) after the start of the breeding program.

[0256] These results clearly show, as shown in Figure 1, that there were significant differences in HI values ​​between week T=11 and week T=18 (p<0.05) and between week T=18 and week T=29 (p<0.001).

[0257] The HI titer induced by MDA in the offspring of these hens (unvaccinated) was measured on the day of hatching and over time, i.e., on days 1, 7, 14, 21, 28, 35, 42, 56, 70, and 84 post-hatch. The results are shown in Figure 2. HI titration was performed using homologous UDL-01 strain.

[0258] On day 1, the average HI titer of the chicks (n=10) was 588 (9.2 Log2). This titer decreased slightly (non-significantly) at 7 days of age, but fell below half at 181 (7.5 Log2) at 14 days of age, and then decreased even more rapidly, with the average (n=10) HI titer being 16 (4 Log2) at 35 days of age, and the HI titer no longer being detectable at 42 days of age.

[0259] The international standard for protection against AIV mortality, as defined by the OIE (www.oie.int / fileadmin / Home / eng / Health_standards / tahm / 3.03.04_AI.pdf), is an HI titer of 32 (5 Log2). While the hatched chicks used experimentally here were found to still have HI titers near this value at 28 days of age, these chicks started far above normal MDA levels. Therefore, additional active vaccination is usually required.

[0260] For confirmation, antibody titers in hatched chicks were also tested by ELISA to ensure that the measured antibodies were directed towards AIV H9 HA. A commercially available kit, the ID Screen® Influenza H9 Indirect kit (ID Vet), an indirect ELISA, was used according to the manufacturer's instructions. The resulting ELISA scores closely matched the HI score patterns. This confirmed that the HI titers detected in hatched chicks originated from antibodies specific to AIV H9 HA.

[0261] [Example 2] Preparation of MDA+ vaccines for birds 2.1. Introduction Three types of vaccines were used to vaccinate serologically positive birds.

[0262] The positive control was the classic inactivated whole virus vaccine: Nobilis® influenza H9N2+ND (MSD Animal Health). This commercially available vaccine contains inactivated AIV subtype H9N2, strain A / chicken / UAE / 415 / 99 ("UAE") and inactivated Newcastle disease virus, 30 clones.

[0263] The HA proteins of AIV H9N2 strain UDL-01 and UAE exhibit 94% amino acid identity when aligned across their entire length.

[0264] The NDV component in the inactivated vaccine was not considered to have a significant effect on the efficacy of AIV vaccination.

[0265] Furthermore, two variants of the recombinant HA antigen-based vaccine were used: one version was a non-targeted variant, and the other was a variant that targeted CD83 by fusion to CD83-scFv. This last version is a recombinant protein for use according to the present invention.

[0266] 2.2. Materials and Methods 2.2.1. Preparation of HA antigen expression constructs The vL and vH chain sequences were obtained using mouse hybridomas that produced antibodies against chicken CD83 (GenBank registration number XM_040663657.1). Synthetic cDNA containing the vL and vH sequences was ligated with a (Gly4Ser)4 linker peptide sequence and commercially manufactured by Geneart (Thermo Fisher Scientific). The vH-linker-vL cDNA was then cloned into the Drosophila melanogaster (D. melanogaster) expression vector: pMT-BIP-V5-His® (version A, Thermo Fisher Scientific) using NotI and XbaI restriction sites. This vector provides the Drosophila melanogaster (D. melanogaster) metallothionein (MT) promoter and Drosophila melanogaster (D. melanogaster) immunoglobulin heavy chain binding protein (BIP) secretion signal for expression and secretion in S2 cells. Furthermore, this plasmid is equipped with a multi-cloning site, a V5 epitope for recombinant protein detection, and a 6xHis tag for recombinant protein purification.

[0267] Using the resulting vector, named pMT-BIP-CD83-scFv-V5-His, the HA gene signal peptide and the external domain of the H9 HA gene lacking the TM domain were inserted. A 29-amino acid trimerized Foldon sequence was added from the bacteriophage T4-derived trimerized protein fibrin using KpnI and PacI restriction sites. This plasmid contained the nucleotide sequence of SEQ ID NO: 14 under the control of the MT promoter.

[0268] The H9 HA used in this experiment was synthetically produced by incorporating the consensus sequence of H9N2 virus HA obtained from the analysis of over 2000 H9 HA sequences from a publicly available database of G1-like H9 virus strains, using the minimal sphere consensus (MScon) method (Kim et al., 2015, abstracts from German Conference on Bioinformatics, Dortmund, September 27th-30th 2015, poster 20: PeerJ PrePrints 3: e1350v1), which is closely related to COBRA technology (Giles et al., 2011, Vaccine, vol.29, p.3043-3054).

[0269] This synthetic HA has 98% amino acid sequence identity with the HA external domain of the H9N2 virus from strain UDL-01 (GenBank accession number: ACP 50708.1, HA1: aa 19-338 and HA2: aa 339-560), which is considered homologous, and it is codon-optimized for S2 cells.

[0270] H9 HA-Foldon antigen lacking a CD83 targeting signal was prepared by a similar method to obtain the plasmid pMT-BIP-H9 HA-Foldon-V5-His. This plasmid contained the nucleotide sequence of SEQ ID NO: 15 under the control of the MT promoter.

[0271] 2.2.2. Preparation and Selection of Recombinant Insect Cells S2 cells (Thermo Fisher Scientific) were maintained in Schneider's insect medium (Merck GmbH Life Science) supplemented with 10% v / v fetal bovine serum and grown at 28°C. Cells were subcultured weekly by centrifugation at 1200 rpm for 10 minutes and resuspended in fresh complete S2 cell medium.

[0272] Recombinant proteins were prepared and purified using the Drosophila Expression System (DES®, Life Technologies). In short, plasmids pMT-BIP-rH9 HA-V5-His and pMT-BIP-rH9 HA-CD83-scFv-V5-His were simultaneously transfected into S2 cells using calcium phosphate transfection. Prior to transfection, 1 × 10^6 / mL of S2 cells were pre-seed in 5 mL of complete S2 cell growth medium at 28°C for 6 to 16 hours. The transfection solution was prepared by adding 60 μL of 2 M CaCl2, 32 μg of expression plasmid DNA, 1.5 μg of hygromycin B-resistant plasmid (pCoHYGRO, Life Technologies), and sterile water to a total volume of 500 μL. The transfection solution was slowly added to an equal volume of 2 × Hepes-buffered saline (HBS) and incubated at room temperature for 30 minutes. The resulting solution was slowly added dropwise to pre-seed S2 cells and incubated at 28°C for 24 hours. 24 hours after transfection, the transfection medium was replaced with fresh complete S2 cell medium, and the cells were incubated at 28°C for a further 3 days.

[0273] Stable S2 transimplants were created by antibiotic selection; specifically, complete growth medium containing 250 μg / mL of hygromycin B was added weekly for at least 4 weeks.

[0274] Next, single-cell clones were obtained via limiting dilution (Zitzmann et al., 2010, Biotechnol. Reports, vol. 19, e00272). Briefly, 2 × 10^3 S2 transfected cells were mixed with 10^6 gamma-irradiated parental S2 cells as feeder cells. 100 μL of this cell mixture was seeded into each well of a 96-well plate. Single clones in each well became clearly visible after 4 weeks of incubation at 28°C. Approximately 10–15 single clones were screened for each plasmid construct. Single clones expressing the maximum amount of recombinant protein were selected by indirect ELISA for the H9 HA protein.

[0275] 2.2.3. Expression and Purification of Recombinant Antigens Next, the selected transfected S2 cell clones were cultured on a large scale. In short, a single clone expressing a large amount of recombinant HA protein was grown in a 2-liter roller bottle (Corning) containing 400 mL of Ex-Cell® 420 serum-free medium (Merck GmbH Life Science) for expression and purification. The metallothionein promoter in the plasmid used was induced by adding CuSO4 to a final concentration of 500 μM. Four days after induction, the cell supernatant was collected by centrifugation at 1200 rpm for 20 minutes and dialyzed to remove excess copper ions. A total of approximately 2 liters of protein expression supernatant was collected and filtered through a 0.22 μM filter Stericup (Merck GmbH Life Science) before purification.

[0276] The use of His-tag enabled the purification of recombinant proteins by metal affinity column chromatography. Briefly, the dialysis and filtration supernatant containing the recombinant protein was loaded onto a 10 mL Profinity™ IMAC Uncharged Resin column (Bio-Rad) and washed with 5 column volumes of wash buffer. Subsequently, the copper-bound protein was eluted with elution buffer containing increasing concentrations of imidazole (25, 50, 100 or 500 mM). The purified protein was analyzed using SDS-PAGE on a 10% PAA gel, followed by Coomassie Blue staining. The protein fractions were combined and concentrated by centrifugation at 4600 rpm for 30 minutes using a 15 mL Amicon Ultra-15™ Centrifugal Filter column (3 kDa MWCO, Merck GmbH Life Science). The concentration of the purified protein was determined using the Pierce BCA Protein Assay Kit™ (Life Technologies) according to the manufacturer's instructions.

[0277] The H9 HA activity of the produced recombinant protein was confirmed using a hemagglutination assay. Briefly, 35 μg of the recombinant protein was serially diluted 2-fold with PBS in a V-bottom 96-well plate. Chicken red blood cells were diluted to 1% with PBS and added to each well. Subsequently, the plate was incubated at 4 °C for 1 hour, tilted 90° in a biosafety cabinet to visualize hemagglutination, and scored.

[0278] 2.2.4. Preparation of vaccine emulsion The recombinant HA antigen vaccine was formulated as a water-in-oil emulsion using light liquid paraffin oil (Marcol® 52) as an adjuvant and containing polysorbate 80 (Tween® 80) and sorbitan monooleate (Span® 80) as emulsifiers. The water:oil weight ratio of the vaccine was 45:55. All vaccines were stored at 4 °C until use.

[0279] The recombinant HA vaccine contained 35 μg of non-targeted HA antigen or 49 μg of targeted antigen per 0.2 ml dose. This difference was to provide an equimolar amount to compensate for the addition of scFv.

[0280] [Example 3] Vaccination of seropositive birds 3.1. Introduction Since protection against AIV infection and disease is essentially serologically determined and the main AIV neutralizing antibodies are against the HA antigen, serological testing for the expression of anti-HA antibodies, i.e., determining the HI titer, is an excellent indicator for predicting in vivo protection against AIV.

[0281] The hatched chicks prepared as described in Example 1 were used in the vaccination experiment. That is, one group was vaccinated on the first day, and these had a very high average MDA HI titer of 588 (9.2 Log2) and were called the MDA++ group. Another group was vaccinated only at 14 days of age with a somewhat reduced MDA level, and these had a moderate average MDA HI titer of 181 (7.5 Log2) and were called the MDA+ group.

[0282] This approach enabled testing and comparison of antibody interference with the effectiveness of vaccination with targeted HA antigen or non-targeted HA antigen for the "worst-case" and "average-case" scenarios, respectively. For comparison, a classical inactivated H9N2 vaccine was included. Also, a group of unvaccinated chicks was included in the experiment to track the natural decline in anti-AIV H9 HA MDA levels.

[0283] 3.2. Materials and methods 3.2.1. Animals, sampling and vaccination The AIV H9 HA MDA-positive chicks used were obtained as described in Example 1. The vaccines used were as described in Example 2.

[0284] To prevent the entry of environmental pathogens, the birds were housed in a positive-pressure isolation chamber where air filtered through high-efficiency particulate air (HEPA) filters flowed in.

[0285] Only chicks that appeared healthy and normal after hatching were used. These were assigned to groups upon acquisition and individually numbered. Daily clinical observations were conducted to monitor health and performance. Each test group consisted of 10 animals.

[0286] All vaccines were at ambient temperature at the time of use and were thoroughly mixed immediately before use to ensure homogeneity.

[0287] All chicks received a single dose of vaccine on either day 1 or day 14. Administration was subcutaneous (sc), the standard route for these types of vaccines. For the Nobilis® vaccine, the approved dose of 0.25 ml / dose was used, and for the recombinant HA antigen vaccine, 0.2 ml / dose was administered.

[0288] On day 1, the Nobilis influenza H9N2+ND vaccine was administered to MDA++ chicks. The H9HA-Foldon and H9HA-Foldon-CD83-scFv vaccines were given to both MDA++ chicks on day 1 and MDA+ chicks that were 14 days old at that time.

[0289] Blood samples were collected once a week for the first six weeks after the start of the experiment, and then every two weeks during weeks 8, 10, and 12, to examine the serological response induced by vaccination.

[0290] Blood samples were collected after euthanasia on day 1 and day 7, and samples from day 14 onward were collected from the wing veins. The volume collected was 2-3 ml, depending on the amount the animal's body weight could tolerate. The blood samples were allowed to coagulate at ambient temperature, and the serum was separated by centrifugation. The serum samples were heat-inactivated at 56°C for 30 minutes and stored at -20°C until use.

[0291] 3.3.Results The results of HI titrations using serum samples collected from MDA++ chicks and MDA+ chicks during this experiment are shown in Figures 3 and 4, respectively.

[0292] The unvaccinated control group exhibited HI titer levels and degradation patterns as described in Example 1 and Figure 2.

[0293] The positive control group consisted of MDA++ chicks that received the whole-inactivated virus vaccine ("Nobilis influenza H9N2+ND") on day 1 of age. Despite this vaccination, their HI titers steadily decreased, and no vaccination response was detected. It is noteworthy that the MDA and HA antigens in the classical vaccine were heterologous; MDA was induced against an HA antigen very similar to the H9 HA of the UDL-01 strain, while the Nobilis vaccine contained a heterologous H9 HA antigen, derived from the UAE strain, which has 94% amino acid identity with the UDL-01 H9 HA protein. Consequently, lower levels of antibody interference would be expected due to this difference between HA antigens. However, clearly, the HI levels in MDA++ chicks were very high, even interfering with the efficacy of the heterologous H9 HA vaccine.

[0294] Vaccination with the targeted HA antigen ("H9 HA Foldon-CD83-scFv") and the non-targeted HA antigen ("H9 HA Foldon") showed significant differences in the HI titers induced by these antigens in both MDA++ and MDA+ chicks.

[0295] The HI titer of chicks vaccinated with non-targeted HA antigen steadily decreased, and neither MDA++ nor MDA+ chicks showed a significant increase in HI titer at any point after vaccination.

[0296] However, the target HA antigen induced a very high HI titer. In the MDA++ group, there was an initial decrease from a very high starting value (9.7 Log2), but thereafter, the HI titer showed a steady and substantial increase, which was evident from 4 weeks post-vaccination (p.v.), reached significance at 5 weeks p.v., and steadily increased to 9.7 Log2 at 12 weeks p.v. This indicates that this vaccine can be applied on day 1 of age even in a situation where the level of homologous MDA is very high, and still can induce strong protection against AIV infection and disease.

[0297] In the MDA+ group, the HI titer from the target HA vaccine already showed a rapid induction of a high HI titer 1 week after vaccination. 4 weeks after vaccination, the HI titer reached an average of 1835 (10.8 Log2).

[0298] In both test groups, the target vaccine was the only one that could induce a significantly increased HI titer. Also, the lowest HI titers measured in the target vaccine groups were 6.2 and 6.9 Log2 in the MDA++ group and MDA+ group, respectively. This indicates that all chicks receiving this type of vaccine remained far above the threshold of 5 Log2 for protection throughout the experimental period.

[0299] This rapid onset of immunity and long duration fully compensate for the decline in MDA level without leaving a gap in protection.

[0300] Indirect ELISA was performed again on the sera to confirm that all antibodies were H9 HA-specific.

[0301] [Example 4] Targeting of non-HA antigens Experiments essentially similar to those described above are being prepared for recombinant proteins for use according to the present invention, however, recombinant proteins containing antigens other than AIV HA. These are AIV HN, NDV F, NDV HN, IBDV VP2, and IBV spike. Briefly, hens can be vaccinated with a suitable vaccine against one of these pathogens: AIV, NDV, IBDV, or IBV, and such vaccines are generally available.

[0302] Hens can receive two or three vaccinations, starting before egg-laying begins and continuing throughout the egg-laying period. The specific antibody titers achieved in the hens can be confirmed to be sufficiently high. The eggs can then be collected and hatched, and the chicks can be confirmed to have sufficiently high MDA levels against the pathogen to be investigated.

[0303] For example, a vaccine containing a recombinant protein for use according to the present invention can be prepared as described above by constructing an expression plasmid containing a nucleotide sequence encoding one of the antigens to be tested. It also includes a binding domain, such as an scFv directed to a surface protein of an avian APC, such as CD83, CD11c, or Dec-205. To evaluate the effect of antigen targeting on APCs, a similar construct, but without the binding domain, can be prepared to serve as a control.

[0304] Plasmids can be transfected into S2 cells as described, selected, amplified, and used to express antigens (with or without targeted signals). Recombinant proteins can then be recovered.

[0305] An example of CD83-scFv is a peptide containing the amino acid sequence of SEQ ID NO: 2.

[0306] Examples of scFv specific to CD11c or Dec-205 are peptides containing the amino acid sequences shown in SEQ ID NOs: 16 or 17, respectively.

[0307] Examples of expressed antigens include amino acid sequences selected from the following:

[0308] -For AIV H5 HA, see Sequence ID 4. -For AIV H7 HA, see Sequence ID No. 5. -NDV F is sequence number 18. -NDV HN is sequence number 19. -For IBDV VP2, see Sequence ID 20, and - For IBV spikes, see Sequence ID 21.

[0309] The corresponding nucleic acids encoding these antigens are preferably codon-optimized with respect to the codon usage frequency table of S2 cells. The expression construct may optionally include additional elements such as signal sequences, linkers, and one or more tags to facilitate expression, secretion, and purification.

[0310] Next, chicks with specific MDAs are vaccinated with these recombinant proteins, and their serology is monitored over time.

[0311] Since specific antibody levels correlated with in vivo protection are known for these pathogens, serological testing of antibody levels at various time points after vaccination is sufficient to obtain a favorable impression of the effectiveness of targeted vaccination in seropositive birds against antigens derived from these pathogens.

[0312] The H5 HA sequence of Sequence ID No. 4 was derived from the HA of AIV isolate: A / duck / Egypt / SS19 / 2017, H5N8, GenBank acc.nr.AXY 66755.1. 511 amino acids from the HA external domain: HA1: 17-340 and HA2: 346-530 were selected. The polybasic cleavage sequence was modified from PLR to PQG, reducing the number of arginine molecules.

[0313] The H7 HA sequence of Sequence ID No. 5 was derived from the HA of AIV isolate: A / chicken / Jiangxi / JX4 / 2017, H7N9, GenBank acc.nr.ARG44105.1. The HA external domains consisting of 507 amino acids, HA1:19-339 and HA2:1-186, which have a polybasic cleavage sequence modified from PKR to PKG, were selected.

[0314] The NDV F sequence of Sequence ID No. 18 is a consensus sequence derived from over 1200 F amino acid sequences from avian abrasive virus 1 sequences in public databases using the MScon technique described herein. The consensus F protein has 98.5% amino acid similarity to its closest natural relative: avian abrasive virus 1 F protein, GenBank acc.nr.AHX 74055.1. The F protein ectodomain was selected from aa.31-500.

[0315] The NDV HN sequence of Sequence ID No. 19 is a consensus sequence starting from the HN protein from Avian orthoavulavirus 1, GenBank acc.nr.AXK 59828.1, combined with several HN sequences from public databases using the MScon technique described herein. Amino acids 47-571 were selected from the HN.

[0316] The IBDV VP2 protein of sequence number 20 represents amino acids 9-452 of the IBDV VP2 protein from GenBank acc.nr.AMA 19770.1.

[0317] The IBV spike protein of SEQ ID NO: 21 represents amino acids 1-1096 of the IBV spike protein from GenBank acc.nr.ARS22410.1. The spike protein was stabilized by two amino acid substitutions: Q859P and L860P. [Brief explanation of the drawing]

[0318] [Figure 1] Presentation of antibody titer results in hyperimmunized hens to produce AIV MDA+ offspring. Details are described in Example 1.

[0319] The vertical axis shows the mean (n=10) HI titer measured by HI assay in the serum of hen immunized with inactivated adjuvanted AIV H9N2 virus vaccine (UDL 01 / 08). The horizontal axis shows the time point in weeks after the start of the experiment (day 0 = 17 weeks old). Vaccination is indicated by arrows at weeks 0, 3, and 24 after the start of the experiment.

[0320] The fertilized eggs were collected 36 weeks after the start of the study, and the box shows the mean (n=10) HI titer in the serum of laying hens used in the follow-up experiment: HI=12 Log2(4096).

[0321] The data is shown as the mean (column) and standard deviation (error bars). An asterisk indicates a significant difference in HI antibody titers at 11 and 18 weeks after the start of the experiment, and at 18 and 29 weeks after the start, where * = p < 0.05 and *** = p < 0.001. [Figure 2] Presentation of MDA-derived HI titer results in unvaccinated offspring from hens that received three vaccinations. Details are described in Example 1.

[0322] Anti-H9 HA MDA titers were measured by HI assays on serum samples at 1, 7, 14, 21, 28, 35, 42, 56, 70, and 84 days post-hatch. HI titers are expressed as the reciprocal of the highest dilution of serum that completely suppressed viral hemagglutination activity at 4 HA units. Data are presented as mean ± SD and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Statistically significant differences are indicated as **** = p < 0.0001.

[0323] The horizontal dotted line indicates the minimum protection level for an HI titer of 32 (5 Log2). [Figure 3] Presentation of HI titer results in chicks vaccinated on day 1 of age with high levels of MDA (MDA++). Details are described in Example 3.

[0324] The vertical axis shows the HI titer, and the horizontal axis shows the number of days after vaccination. NB: There is a gap in the vertical axis, allowing for the display of extremely high HI titers found.

[0325] A group of MDA++ chicks (n=10) were vaccinated on day 1 of age with one of three vaccines: a whole-inactivated virus vaccine ("Nobilis Influenza H9N2+ND"), a non-targeted HA antigen ("H9 HA Foldon"), or a CD83-targeted HA antigen ("H9 HA Foldon-CD83-scFv"). One group of MDA++ chicks was not vaccinated as a control.

[0326] Anti-H9 HA antibody titers were measured by an HI assay using the UDL-01 virus.

[0327] The data is shown as the mean (column) and standard deviation (error bars). Statistically significant differences are indicated by an asterisk, in which case *** = p < 0.001 and * = p < 0.1. [Figure 4]Presentation of HI titer results for chicks with moderate levels of MDA (MDA+) vaccinated on day 14. Details are described in Example 3.

[0328] The presentation is the same as in Figure 3, except that there was no group that received the Nobilis vaccine. This disclosure provides, in one aspect, the following: [Item 1] A recombinant protein comprising an antigen and a binding domain capable of binding to a cell surface protein on an avian antigen-presenting cell (APC), for use in a method for protecting birds possessing antibodies reactive with the antigen from a pathogen from which the antigen originates. [Item 2] Recombinant protein for use as described in item 1, characterized in that the avian APC is a dendritic cell. [Item 3] A recombinant protein for use according to item 1 or 2, characterized in that the cell surface protein is CD83. [Item 4] A recombinant protein for use according to any one of items 1 to 3, characterized in that the binding domain is a single-stranded variable fragment (scFv). [Item 5] Recombinant protein for use according to any one of items 1 to 4, characterized in that the antigen is selected from infectious bursal disease virus (IBDV) viral protein 2 (VP2), Newcastle disease virus (NDV) fusion (F) protein, NDV hemagglutinin-neuraminidase (HN) protein, infectious bronchitis virus (IBV) spike protein, avian influenza virus (AIV) hemagglutinin (HA) protein, and AIV neuraminidase (NA) protein. [Item 6] A recombinant protein for use according to any one of items 1 to 5, characterized in that the antigen comprises an amino acid sequence selected from SEQ ID NOs: 7, 8, and 9. [Item 7] A recombinant vector capable of expressing a recombinant protein described in any one of items 1 to 6, for use in a method of protecting birds possessing antibodies reactive to an antigen contained in the recombinant protein expressed by the recombinant vector from a pathogen from which the antigen originates. [Item 8] Use of a recombinant protein as described in any one of items 1 to 6 or a recombinant vector as described in item 7 for the manufacture of a vaccine for protecting birds from a pathogen, characterized in that the antigen contained in the recombinant protein or contained in the recombinant protein expressed by the recombinant vector is derived from the pathogen, and the birds possess antibodies that are reactive to the antigen. [Item 9] A vaccine comprising a recombinant protein as described in any one of items 1 to 6 or a recombinant vector as described in item 7, and a pharmaceutically acceptable carrier, for use in a manner that protects birds possessing antibodies reactive to an antigen contained in the recombinant protein or a recombinant protein expressed by the recombinant vector from a pathogen from which the antigen originates. [Item 10] A vaccine for use as described in item 9, characterized by containing an adjuvant. [Item 11] Use of a recombinant protein described in any one of items 1 to 6, a recombinant vector described in item 7, or a vaccine described in item 9 or 10 for the protection of birds from a pathogen, characterized in that the antigen contained in the recombinant protein or contained in the recombinant protein expressed by the recombinant vector is derived from the pathogen, and the birds possess antibodies that are reactive to the antigen. [Item 12] A method for protecting birds from a pathogen, comprising the step of administering a vaccine described in item 9 or 10 to the birds, wherein the antigen contained in the vaccine is derived from the pathogen, and the birds possess antibodies that are reactive to the antigen.

Claims

1. A composition for use in a method for protecting birds possessing antibodies reactive with an antigen from a pathogen from which the antigen originates, comprising the antigen and a recombinant protein comprising a binding domain capable of binding to a cell surface protein on an avian antigen-presenting cell (APC), characterized in that the avian APC is a dendritic cell, the cell surface protein is CD83, and the binding domain is a single-stranded variable fragment (scFv).

2. The composition according to claim 1, characterized in that the antigen is selected from infectious bursal disease virus (IBDV) viral protein 2 (VP2), Newcastle disease virus (NDV) fusion (F) protein, NDV hemagglutinin-neuraminidase (HN) protein, infectious bronchitis virus (IBV) spike protein, avian influenza virus (AIV) hemagglutinin (HA) protein, and AIV neuraminidase (NA) protein.

3. The composition according to claim 1, characterized in that the antigen comprises an amino acid sequence selected from SEQ ID NOs: 7, 8, and 9.

4. A composition comprising a recombinant vector capable of expressing the recombinant protein described in claim 1, for use in a method of protecting birds possessing antibodies reactive to an antigen contained in the recombinant protein expressed by the recombinant vector from a pathogen from which the antigen originates.

5. A use of the recombinant protein according to claim 1 or the recombinant vector according to claim 4 for the production of a vaccine for protecting birds from a pathogen, characterized in that the antigen contained in the recombinant protein or contained in the recombinant protein expressed by the recombinant vector is derived from the pathogen, and the birds possess antibodies that are reactive to the antigen.

6. A vaccine comprising a recombinant protein according to claim 1 or a recombinant vector according to claim 4 and a pharmaceutically acceptable carrier, for use in a method of protecting birds having antibodies that are reactive to an antigen contained in the recombinant protein or a recombinant protein expressed by the recombinant vector from a pathogen from which the antigen originates.

7. A vaccine for use according to claim 6, characterized by containing an adjuvant.

8. A use of the recombinant protein according to claim 1 or the recombinant vector according to claim 4 for protecting birds from a pathogen, characterized in that the antigen contained in the recombinant protein or the recombinant protein expressed by the recombinant vector is derived from the pathogen, and the birds possess antibodies that are reactive to the antigen.

9. A method for protecting birds from a pathogen, comprising the step of administering the vaccine described in claim 6 to the birds, wherein the antigen contained in the vaccine is derived from the pathogen, and the birds possess antibodies that are reactive to the antigen.

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