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 particularly to a recombinant protein for use in a method for protecting avian species, the recombinant protein having an antibody reactive with an antigen in the protein. In particular, the present invention relates to recombinant proteins, recombinant vectors and vaccines for use in the method. Furthermore, the present invention relates to uses and methods for the treatment of avian species by administration of the protein, vector or vaccine. [Background technology]

[0002] As a nutritious and affordable source of protein, avian meat and eggs are an important part of the diet of most human populations in the world. The main species of poultry kept for such economic purposes are chickens, turkeys, ducks and geese. To raise the required large numbers of these birds while maintaining their health and desirable living conditions, the poultry industry is keen to optimize management conditions and provide good veterinary care. An important part of this strategy is preventive protection by vaccination against a wide variety of avian pathogens that can cause infections and diseases, which can have devastating effects on animal health and business economics. Over the years, a wide variety of vaccines have been commercially available against 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 and viral vector.

[0003] Especially for poultry that are produced in great numbers, namely meat birds (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 not very successful. An effective workaround is therefore by vaccination of the mother before and during the laying season. Maternal antibodies produced by the hen are transferred to the yolk-bearing eggs and internalized by the developing chicks. In this way, already on the day of hatching, the chicks can be passively protected by these maternally derived antibodies (MDA) against various pathogens. However, active vaccination of the growing chicks themselves must also be performed to induce adequate immune protection after the first few weeks, since most of the MDA becomes ineffective again in three weeks due to biological degradation. At that point, some MDA may still be present in the bird.

[0004] A similar situation of vaccination in the context of pre-existing antibodies occurs in the case of older birds which have antibodies induced by a previous vaccination, the effect of which gradually fades and therefore requires a booster vaccination to restore antibody titers to protective levels.

[0005] An important veterinary and scientific challenge arises in determining when a vaccination can be given to birds that already possess antibodies reactive with the antigens contained in the vaccine being used. Vaccination when antibody titers are nearly depleted is clearly too late as it leaves a gap period between the decline of those titers and the onset of protection from active immunization. During this gap period, the bird is vulnerable to infection and disease.

[0006] However, vaccination is too early when birds still have fairly high titers of circulating antibodies, as this often affects the efficacy of vaccination, possibly because those antibodies may somehow bind and capture the vaccine antigens, thereby accelerating their degradation and / or preventing them from inducing an appropriate immune response. This last phenomenon is called "antibody interference" and its different notation for MDA is "MDA interference". This is a well-known problem for effective vaccination against the main pathogens affecting the poultry industry worldwide. Examples of these main pathogens are 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 fowl plague virus), the last two of which are indeed notifiable diseases of the OIE [International Organization for Animal Health].

[0007] For these diseases, antibody interference is well known to reduce the effectiveness of vaccination, making birds vulnerable to field infection, especially when birds are kept in close proximity 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 gaps in protection and optimize vaccination of seropositive birds. More direct attempts to overcome antibody interference have included adaptations to the vaccine, increasing the antigen dose and / or using (stronger) adjuvants. More virulent or non-attenuated live vaccine pathogen strains have also been attempted in the hope that they can break through the higher titers of antibodies and thus be administered at earlier time points. These methods have generally been unsatisfactory, so more complex approaches have been tried.

[0009] For active vaccination of young birds against IBDV, one method involves monitoring MDA levels by serological testing of bird samples to determine the optimal date for vaccination. However, as a result, effective active vaccination can only be applied at 2-3 weeks of age, and protection gaps are inevitable in many birds due to variability in large flocks. Alternatively, "conjugated IBDV vaccines" (live attenuated vaccine viruses conjugated with antibodies) have been administered at a young age, whereby antigens are released only at later times. Also, viral vector systems have been used, for example those using fowlpox virus or avian herpes virus as vectors to express viral protein 2 (VP2), the main IBDV antigen. This is reviewed in Muller et al. (2012, Avian Pathol., vol. 41, p. 133-139).

[0010] For NDV, various approaches in vaccination have been applied, but antibody interference remains a problem today, for a review see Dimitrov et al. (2017, Vet. Microbiol. vol. 206, p. 126-136).

[0011] Even the use of recombinant vector vaccines can suffer from antibody interference, for example, if the 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). For NDV as a vector, possible solutions have been, for example, to change the serological profile of the NDV vector (Steglich et al., 2013, PLoS One, vol. 8, e72530) or to select NDV strains that are less inhibited by anti-NDV antibodies (European Patent Application Publication No. 2998315).

[0012] For IBV, MDA is well known to prevent vaccination of one-day-old chicks, see Terregino et al., 2008 (Avian Pathol., vol. 37, p. 487-493).

[0013] For AIV, the relevance of effective vaccination extends even beyond the veterinary field, since this virus can cause human zoonotic diseases 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, p. 351-363). However, as with some other vaccine situations, addressing interference by AIV-reactive antibodies remains problematic (Murr et al., 2020, Avian Dis., vol. 64, p. 427-436).

[0014] As a result, despite the many different approaches that have been tried in the field of avian vaccination, there remains a pressing need for effective methods to overcome the negative effect that pre-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 vaccination of avian targets by selective targeting of antigens to antigen-presenting cells (APCs). A wide variety of methods have been described to achieve such targeting, for example by using ligands, antibodies, nanoparticles, viral vectors, or cell-penetrating peptides. No methods have been described or suggested for overcoming antibody interference in birds.

[0016] WO2017 / 055235 describes antigen targeting to antigen-presenting cells (APCs), but uses antigen internalization. The described treatment is for mammals, especially cats and dogs, and is aimed at alleviating allergies. Antibody interference is not mentioned.

[0017] Jauregui et al. (2017, Res. Vet. Sci., vol. 111, p. 55-62) describe 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. All chickens used were seronegative for anti-HA antibodies (see Jauregui, Figure 7, day 0), so Jaurequi et al. do not describe or suggest 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] Jauregui et al.,2017,Res.Vet.Sci.,vol.111,p.55-62 Summary of the Invention

[0020] It is therefore an object of the present invention to overcome one or more of the shortcomings in the prior art by providing an effective method for overcoming the adverse effects of antibody interference in vaccination of avian targets.

[0021] Surprisingly, it has been found that this objective can be met, thereby overcoming one or more of the shortcomings of the prior art, by providing a method for protecting birds that possess antibodies that react to an antigen contained in an administered vaccine, i.e., by targeting the antigen to avian APCs.

[0022] In the experiment described in detail below, chickens with high or medium antibody levels were administered either targeted or non-targeted vaccines. The results show a significant difference in the effectiveness of vaccination in favor of targeted antigens. In contrast, non-targeted vaccines and classical control vaccines produce little response in seropositive birds. As a result, this method of protecting seropositive birds can effectively overcome the negative effect of antibody interference in vaccination, and is unexpectedly effective even in single doses and in very young birds.

[0023] As a result, the inventors have found, to their complete surprise, that this antigen targeting, especially in relation to existing antibodies, on the one hand works well even in immunologically immature birds, and on the other hand, does not cause vaccine-enhanced diseases or vaccine-induced immune disorders, such as by overstimulation 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 as a recombinant vector expressing a recombinant protein, such as a DNA plasmid, an RNA molecule, or a vector virus.

[0025] Moreover, since this positive effect appears to occur by targeting and is therefore independent of the antigen used, it is entirely conceivable that this method will be equally successful using different antigens, thus allowing protection against a range of avian pathogens for which vaccination is normally subject to antibody interference, e.g., NDV, IBDV, AIV, etc.

[0026] It is not known exactly how or why this vaccination method is able to break through high antibody levels and still induce such an effective protective immune response. Although the inventors do not wish to be bound by any theory or model that may explain these findings, the inventors speculate that it is the targeting of antigens to APCs that somehow reduces their clearance by pre-existing antibodies against APCs.

[0027] The successful use of antigen targeting to APCs in the vaccination of birds with pre-existing antibodies against vaccine antigens could never have been predicted by any prior publications, mainly because the mechanisms by which antibody interference works (blocking, masking, cross-linking, neutralization of vaccine antigens, etc.) are still poorly understood to this day. This is especially true for antibody interference in birds, as this is a poorly studied animal system.

[0028] Moreover, the first studies on antigen targeting were already described in 1980, but these were aimed at the treatment of human cancers. Later, a more general use in (mainly human) vaccination was considered, as reviewed by Keler et al. (2007, Oncogene, vol. 26, p. 3758-3767).

[0029] Also, in some instances, (maternally derived) antibodies are involved in enhancing viral disease through antibody-dependent enhancement, which is called vaccine-enhanced disease. This effect has been observed with various viruses, such as lentiviruses and dengue viruses (Huisman et al., 2009, Vaccine, vol. 27, p. 505-512), and more recently with SARS-CoV-2 (Lee et al., 2020, Nat. Microbiol., vol. 5, p. 1185-1191). Therefore, there was a real concern that targeted vaccination could result in such undesirable effects upon subsequent contact with the corresponding pathogen.

[0030] Moreover, there is little information on the functioning of the avian immune system compared to that of mammals / humans, so the translation from the mammalian to the avian situation is far from straightforward, and the general review by Shrestha et al., supra, does not enable specific methods or remove all the hesitations that a person skilled in the art may have when using antigen targeting, due to concerns that it may cause a type of immune disorder and / or require a mature immune system.

[0031] Combined, this lack of information and potential complications made the use of antigen targeting to APCs an unlikely option for vaccination of birds that have high levels of circulating antibodies reactive with the antigens in the vaccine. Furthermore, the choice of this vaccination method for young birds was particularly uncertain, since the immune system of birds at hatch is still immature and, as a result, it was unpredictable whether their APCs would already present the appropriate target protein on their surface and would be sufficiently mature to transfer binding to such surface proteins into productive stimulation of the animal's immune system.

[0032] Thus, in one aspect, the 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 for protecting an avian animal bearing antibodies reactive with the antigen from a pathogen from which the antigen is derived.

[0033] A "recombinant protein" is a protein whose amino acid sequence has been artificially created. In the present invention, recombinant proteins can be obtained by molecular cloning and recombinant protein expression techniques. After expression, the protein can be isolated from the expression system, treated and purified as necessary, and then formulated into a composition suitable for use in the method for protection 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 the art and are disclosed in great detail in standard textbooks such as "Molecular cloning: a laboratory manual" by Sambrook and Russell (2001, Cold Spring Harbour Laboratory Press; ISBN: 0879695773) and Current Protocols in Molecular Biology by Ausubel et al. (J. Wiley and Sons Inc, NY, 2003, ISBN: 047150338X). For purposes of the present invention, the term "protein" incorporates similar terms such as "peptide," "oligopeptide," and "polypeptide."

[0035] A recombinant protein for use according to the invention is a fusion protein, composed of polypeptides from different origins, e.g. both antigen and binding domains as defined in the present invention, and optionally also composed of one or more peptides such as linkers, markers, etc., all linked in one amino acid chain.

[0036] As used herein, the term "comprising" (and variations such as "comprises," "comprise," and "comprised") is intended to refer to all possible elements and every possible combination of the invention covered or included by the text section, paragraph, claim, etc. in which the term is used, even if such element or combination is not explicitly recited, and is not intended to exclude any such element or combination.

[0037] Thus, any such text section, paragraph, claim, etc. may also relate to one or more embodiments in which the term "comprising" (or variations thereof) is replaced with terms such as "consisting of," "consists of," or "consist essentially of."

[0038] An "antigen" is commonly known as a molecule capable of interacting with elements of the immune system, such as antibodies and lymphocytes, which interaction can result in a humoral and / or cellular immune response.

[0039] The part of an antigen that is 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. A linear epitope must be of sufficient size, e.g., at least 5 amino acids, to be included in a recombinant protein for use according to the invention, either by itself or by being linked to a carrier molecule.

[0040] Antigens are polypeptides, i.e., antigenic polypeptides contain at least one epitope and are "derived" from a pathogen. In the context of the present invention, "derived" refers to a situation in which the coding sequence for a particular antigen is selected, typically by analysis of the genetic information of the pathogen and its protein repertoire. The selected sequence is then recombined into a construct that encodes a recombinant protein for use according to the present invention.

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

[0042] The antigen may be derived from the native sequence of the antigen from the pathogen or may be a collection, e.g., a consensus from several homologs of the expressed antigen, e.g., proteins of the same type, but with amino acid sequences derived from variants of different species, serotypes, subtypes, strains, isolates, etc. of the pathogen. As is well known, to obtain such a consensus sequence, either the amino acid sequences or the coding nucleotide sequences can be compared, from which a consensus sequence can be derived, e.g., by aligning several H9 HA nucleotide sequences using a suitable computer program.

[0043] The antigens of the present invention may also be chimeric antigens, consisting of a collection of moieties from different antigens, whether biologically related or not. Furthermore, the sequences encoding the antigens may be subjected to "codon optimization", as described below.

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

[0045] A "binding domain" of the present invention is derived from the antigen binding site of an immunoglobulin molecule and may be a portion of an antibody comprising one or more complementarity determining regions, for example a "single chain variable fragment" (scFv) polypeptide.

[0046] In the present invention, a binding domain is "capable of binding". This refers to specific binding, i.e. binding with sufficient affinity to be distinct from any non-specific or background binding. The difference between specific and non-specific 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, where non-specific binding is typically quickly lost, for example, by diluting 1:10 or 1:100, while specific binding remains even at higher dilutions.

[0047] It is well known that "APC" are cells of the lymphoid system that are capable of processing antigenic molecules and presenting (parts of) these molecules to the immune system of a human or animal. This presentation induces a cascade of reactions that result in immune maturation and stimulation that are the basis of a protective immune response. APCs are, for example, B lymphocytes, dendritic cells, macrophages and natural killer cells.

[0048] "Cell surface proteins on avian APCs" are proteins that are attached or anchored to the outside of the cell membrane of APCs. These proteins play a role in the function of APCs in sensing and signaling. Many of the 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 "CD" designation refers to "Cluster of Differentiation," an international protocol for the classification and identification of surface proteins on cells of the lymphoid system.

[0049] An "avian" according to the present invention is any animal of the taxonomic class Aves that is of economic or (veterinary) medical relevance, such as chickens, turkeys, ducks, geese, quails, guinea fowl, partridges, pheasants, pigeons, peregrine falcons and ostriches.

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

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

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

[0053] The methods of protection involve, in whole or in part, reducing the establishment or proliferation of a productive infection by a pathogen in cells and organs of a susceptible bird, or reducing subsequent signs of disease, for example by reducing the burden of the pathogen or shortening the replication period of the pathogen, which in turn results in a reduction in the number, intensity, or severity of lesions and associated clinical signs of disease that may be caused by infection by the pathogen in the bird.

[0054] Such a reduction in infection or disease can be easily detected, for example, by monitoring the immunological response after vaccination with a recombinant protein for use according to the invention, and by examining the clinical symptoms or mortality profile of vaccinated birds after (challenge) infection, for example by monitoring the birds' disease signs, clinical scores, serological parameters, or by re-isolation of the infectious agent. These results can be compared with the response to a similar infection in mock-vaccinated birds. Several methods for assessing infection and disease symptoms of major avian pathogens are well known in the art.

[0055] Protection from infection or disease by the methods of the invention provides improved health, welfare and economic performance to immunized birds, which can be assessed, for example, from parameters such as increased living conditions, survival rate, growth rate, feed efficiency and egg production, as well as reduced costs for (veterinary) health care.

[0056] Birds protected by the method of the invention "carry antibodies". This applies at the time when the method of the invention is applied, i.e. at the time of vaccination. Whether a bird actually has such antibodies can be easily determined, for example, by taking blood samples from the bird 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 value of that pre-existing titer 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 that test, take place at that time. Likewise, this does not prevent the calculation and extrapolation of the pre-existing titer at the time of vaccination from the levels measured in samples taken some time before vaccination.

[0057] In the present case, a bird "harbors" an antibody to an antigen if the titer of the antibody reactive to that antigen in serum from that bird is above a background level. Such background level is typically the level present in a comparable bird that is naive to the antigen or pathogen of interest. In the present case, this background level can conveniently be obtained, for example, from the titers present in the serum of SPF (specific pathogen free) birds of the same age and species.

[0058] Pre-existing antibodies may result from passive transfer, as is the case for antibodies obtained from the mother, typically via egg yolk. Such seropositive birds are called "MDA positive" or "MDA+". This applies to birds at a very young age, for example from the day of hatch (i.e. 1 day old) to about 3 weeks of age. Alternatively, pre-existing antibodies may result from an active immunization previously received by the bird to be protected, which resulted in the production of antibodies, and applies to birds from about 3 weeks of age onwards.

[0059] The term "reactive with" or its synonym "specific for" refers to the ability of a pre-existing antibody to interact with an antigen comprised in a recombinant polypeptide for use according to the present invention by specific immune recognition. Similar terms are also "capable of binding," "capable of recognizing," etc., insofar as they refer to specific binding.

[0060] The unexpected advantageous effect of the present invention is evident when pre-existing antibodies (in the bird to be protected) are reactive with the antigens contained in the recombinant protein of the present invention, a situation in which antibody interference would normally occur, reducing the effectiveness of protection.

[0061] The term "pathogen from which the antigen is derived" serves to indicate that the pathogen against which the method of the invention is intended to protect comprises an antigen as defined above, including antigen homologues and / or pathogen variants.

[0062] As one skilled in the art will appreciate, the match between the antigen in the recombinant protein for use according to the invention and the pathogen against which birds are to be protected forms the basis of the protective immune response that is induced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] Details of embodiments and further aspects of the invention are described below.

[0064] In one embodiment of the recombinant protein for use according to the invention, the avian APCs are 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, using protein-based determinations designated CD as described below.

[0066] In a preferred embodiment of the recombinant protein for use according to the present invention, the avian APCs are dendritic cells.

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

[0068] All of these proteins are well known in the art and are surface proteins on APCs, with CD11c being a transmembrane protein on dendritic cells and some other APCs that plays a role in neutrophil activation. The CD11c-specific scFv comprises the amino acid sequence of SEQ ID NO:18.

[0069] Dec-205 is an endocytic receptor on dendritic cells and lymphocytes. An example of chicken Dec-205 is shown in GenBank Accession No. AJ574899. Dec-205 specific scFv comprises the amino acid sequence of SEQ ID NO:19.

[0070] CD83 is a surface glycoprotein belonging to the immunoglobulin superfamily. It is expressed mainly on dendritic cells and to a lesser extent on lymphocytes and macrophages. It is a well-known marker of mature dendritic cells. An example of avian CD83 is the protein shown in GenBank Accession No. 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 the recombinant protein for use according to the invention, the binding domain comprises an antigen-binding site of an antibody.

[0073] In a preferred embodiment of the recombinant protein for use according to the invention, the binding domain is a single chain variable fragment (scFv).

[0074] As is well known, scFv is the smallest part of an immunoglobulin that retains one complete antigen-binding domain but lacks the Fc portion. The scFv is a single peptide that is itself a fusion construct, and contains 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 fashion (relative to each other), so that the c-terminal side is the tail.

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

[0076] The linker sequence of the scFv provides a flexible region so that the two variable chains can orient themselves to form the 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 shown in SEQ ID NO: 1 (Gly 4 -Ser) 4 Includes.

[0077] The specificity of the two variable chains of the scFv may be for both the same antigen or for each different antigen, in a preferred embodiment the two variable chains have the same specificity.

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

[0079] In embodiments of the binding domain, the scFv may be present more than once.

[0080] In one embodiment of the recombinant protein for use according to the invention, the pathogen is pathogenic to avian species. 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 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 an AIV HA protein. Even more preferably, the antigen is selected from AIV HA protein of H5, H7 or H9 type.

[0082] All these viral protein antigens are well known in the art 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 are 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] Further information about the HA protein is available at the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) at www.rcsb.org and at the Influenza Research Database at www.fludb.org.

[0084] In one embodiment of the recombinant protein for use according to the invention, where the antigen is selected from the AIV HA protein, the antigen comprises only the ectodomain of the HA protein, which prevents attachment to the cell membrane of the cell used to express the recombinant protein for use according to the invention.

[0085] The ectodomain of the mature AIV HA protein includes the N-terminal portion without the signal sequence and the central portion of the HA protein, thus including the HA1 and HA2 domains, but not the transmembrane and cytoplasmic domains; typically, these last two portions together form the C-terminal 35-40 amino acids of HA.

[0086] In one embodiment of a recombinant protein for use according to the invention, wherein the antigen is the ectodomain of an AIV HA protein of H5, H7, or H9 type, 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 invention, where the antigen is an ectodomain from the AIV HA protein, the antigen also comprises a trimerization domain.

[0088] Such a trimerization domain can compensate for the loss of the transmembrane and cytoplasmic domains of HA, restoring the ability to form homotrimers and resemble its native 3D shape, and further improving the solubility and stability of the recombinant protein of the invention carrying the HA-ectodomain antigen.

[0089] In the present case, the trimerization domain is a peptide and can be one of several known to be suitable for this function, such as the isoleucine zipper 3 domain of the GCN4 transcriptional activator from Saccharomyces cerevisiae, or the Foldon domain of the bacteriophage T4 fibritin protein ("Foldon").

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

[0091] In one embodiment of a recombinant protein for use according to the invention, where the antigen is the ectodomain of the AIV HA protein and the antigen also comprises a trimerization domain, the trimerization domain is located C-terminal (downstream) to the HA ectodomain.

[0092] In a preferred embodiment, the HA ectodomain and trimerization domain are arranged in a recombinant protein for use according to the invention, without any intervening amino acids.

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

[0094] In a recombinant protein for use according to the invention, the antigen and the binding domain may be arranged in two orientations relative to each other, with either the antigen or the binding domain being closer to the N-terminus of the recombinant protein for use according to the invention. In this regard, the trimerization domain, which may be used when the antigen is selected to be the HA ectodomain, is considered to be part of the antigen.

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

[0096] In an alternative embodiment, the binding domain is located in the recombinant protein N-terminally (upstream) of the antigen.

[0097] In one embodiment, the recombinant protein for use according to the invention comprises 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 comprises the amino acids glycine and serine. Even more preferably, the linker comprises the amino acid sequence of SEQ ID NO:8.

[0098] Thus, in one embodiment the recombinant protein for use according to the invention comprises: - AIV H5 HA ectodomain, a trimerization domain, a linker and a CD83-scFv, - AIV H7 HA ectodomain, trimerization domain, linker and CD83-scFv, and - AIV H9 HA ectodomain, a trimerization domain, a linker and a CD83-scFv, (wherein the elements shown are presented from N-terminus to C-terminus) The combination includes one selected from the following:

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

[0100] For the purposes of expressing, harvesting, quantitating and (optionally) purifying the recombinant protein for use according to the invention, the recombinant protein 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 in the recombinant protein.

[0101] Well-known markers are 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 portions thereof, all well known in the art.

[0102] Markers can be used for detection and quantification purposes, e.g., for detection or binding with specific antibodies, e.g., in IFT or ELISA. Purification can be performed, e.g., using immuno- or metal affinity chromatography.

[0103] A His tag typically has 4 to 10 histidines. Preferably, the His tag is a 6x histidine tag, i.e. has 6 consecutive histidines.

[0104] "Ctag" comprises SEQ ID NO:9 and is the C-terminus of the alpha-synuclein protein known to cause the aggregates seen in neurological disorders such as Parkinson's disease. When used, the Ctag is preferably included at the C-terminus of the recombinant protein of the invention. Purification of the Ctag by immunoaffinity chromatography is sometimes more effective than purification of the 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 comprises the amino acid sequence of SEQ ID NO:10.

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

[0107] For the expression of recombinant proteins for use according to the invention, some further adjustments can be made as necessary. Such fine-tuning or optimization is routine and well known to those skilled in the art. For example, it depends on how the protein is expressed by the host cell of the expression system, i.e., whether it is secreted inside the cell, on the surface of the cell, or outside the cell. In the last two cases, a signal sequence can be provided on the N-terminus, which works 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 allow secretion when expressed in S2 cells.

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

[0109] During the process of constructing a nucleic acid that results in the expression of a recombinant protein for use according to the invention, one or more restriction enzyme (RE) sites may be used: if these RE sites are located in the coding region of a recombinant protein, their remaining nucleotides will be translated into several amino acids, which will then be located between some of the elements that make up the recombinant protein for use according to the invention.

[0110] For example, one construct used in the present invention subcloned the H9 HA ectodomain-Foldon element using RE sites KpnI and PacI, and the CD83-scFv C-terminal to the HA antigen-Foldon and 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 set out in Table 1. [Table 1] A control construct was prepared that did not contain a linker and CD83-scFv, which lacked the region from amino acids 545 to 802 of SEQ ID NO:12 and contained the amino acid sequence of SEQ ID NO:13.

[0112] Constructs similar to SEQ ID NOs: 12 and 13 can readily be made using other HA antigen sequences, i.e., for example, one of the H5 HA ectodomain or H7 HA ectodomains shown in SEQ ID NOs: 4 and 5, respectively.

[0113] In one embodiment of the recombinant protein for use according to the invention, the antibodies reactive with the antigen are maternally derived antibodies.

[0114] In the present invention, it is easy to ascertain whether existing antibodies are maternally derived, and in fact, only chicks under 2-4 weeks of age have MDA. Also, MDA consists mainly of IgY, which is the functional homologue of mammalian IgG, but is structurally different, with IgY having four heavy chain constant domains compared to IgG's three.

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

[0116] In the present invention, the bird may be of any type, breed, or subspecies, for example, layer, breeder, broiler, hybrid, or parent line of any of such breeds. Preferred poultry types are selected from broilers, breeders, and layers. More preferred are broiler-type and layer-type poultry. Broiler poultry are most preferred.

[0117] As described, the present invention provides recombinant proteins for use in a method for protecting seropositive birds against pathogens, which can be advantageously applied to either older birds whose pre-existing antibodies are the result of previous active vaccination, or to young birds whose pre-existing antibodies are MDA.

[0118] Thus, in one embodiment of the recombinant protein for use according to the present invention, the protected avian 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, even more preferably 1 day old (i.e. day of hatch).In one embodiment, the protected avian is about day 18 of embryonic development (i.e. in ovo).

[0119] In an alternative embodiment of the recombinant protein for use according to the present invention, the protected birds are over 2 weeks old.

[0120] As mentioned, recombinant proteins for use according to the invention can equally well be applied by indirect use, i.e. by expressing the recombinant protein from a recombinant vector, e.g. a DNA plasmid, an RNA molecule or a viral vector.

[0121] Thus, in a further aspect, the present invention relates to a recombinant vector capable of expressing a recombinant protein for use according to the invention, for use in a method for protecting birds carrying antibodies reactive with an antigen comprised in the recombinant protein expressed by the recombinant vector, from a pathogen from which the antigen is derived.

[0122] A "vector" is well known in the art as a molecular structure that carries genetic information (nucleic acid sequence) for encoding a polypeptide, together with appropriate signals that allow its expression under appropriate conditions, e.g., in a host cell. In the context of the present invention, "expression" refers to the well-known principle of expressing a protein from the genetic information by transcription and / or translation.

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

[0124] A recombinant vector for use according to the invention is "recombinant" since it has a molecular constitution that has been altered by in vitro manipulation of its genetic information. The alterations made may serve to effect, improve or adapt the replication, expression, manipulation, purification, stability and / or immunological behaviour of the vector and / or the protein it expresses. These and other techniques are explained in great detail in the standard textbooks by Sambrook & Russell and Ausubel et al., both cited above, and in "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, more or less signals must be provided for replication and expression, either in cis (i.e., provided within the recombinant vector itself) or in trans (i.e., provided from a separate source), all of which are well known.

[0126] The skilled artisan has the knowledge to select and combine the necessary signals into a usable combination so that a recombinant vector for use according to the invention is "capable of expressing" a recombinant protein for use according to the invention under appropriate conditions. Next to elements that aid in construction and cloning, such as restriction enzyme recognition sites or PCR primers, well-known elements can be selected from one or more of a promoter, a stop codon, a termination signal, a polyadenylation signal, a 7-methylguanosine (7mG) cap structure, and an intron with functional splice donor and acceptor sites.

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

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

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

[0130] Thus, 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 comprising the nucleotide sequence of SEQ ID NO:15.

[0132] Both SEQ ID NOs: 14 and 15 have been codon-optimized against the codon usage table of D. melanogaster S2 cells to optimize expression in these cells, as described in more detail below.

[0133] As noted, recombinant vectors for use according to the present invention can have a number of different forms.

[0134] Thus, in one embodiment, the recombinant vector for use according to the invention is selected from a nucleic acid, a virus and a replicon particle (RP).

[0135] In the present invention, a nucleic acid can be DNA or RNA, can be single-stranded or double-stranded, and can be of natural or synthetic origin.

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

[0137] A "eukaryotic expression plasmid" of DNA usually has the appropriate signals for the expression of a heterologous gene inserted into the plasmid under the operational control of a promoter active in eukaryotic cells. The plasmid can then be inserted into a eukaryotic host cell or host organism by any transfection method, for example by using biochemicals as carriers, by mechanical means, or by electroporation, resulting in the expression of the heterologous gene insert. Typically, such expression is transient, since the plasmid lacks signals for stable integration into the genome of the host cell, and 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 a variety of suppliers, for example the series of plasmids: pcDNA™, pCR3.1™, pCMV™, pFRT™, pVAX1™, pCI™, Nanoplasmid™, pCAGGS, etc.

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

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

[0141] In a preferred embodiment of the recombinant vector for use according to the 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 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 invention in the host cell. The delivery of the expression plasmid can be in several ways, for example by 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 special form of a recombinant vector for use according to the invention as a eukaryotic expression plasmid is where the plasmid allows the delivery of a replicon RNA.

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

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

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

[0147] Vaccination with a eukaryotic expression plasmid encoding a replicon RNA offers an advantage over vaccination with a eukaryotic expression plasmid expressing a protein, since the replicon RNA provides an amplification step, i.e., by translation of the replicase, it produces a subgenomic messenger RNA that codes for the recombinant protein for use according to the invention, which results in the expression of large amounts of the recombinant protein in the host cell and in the target bird, respectively.

[0148] In a preferred embodiment of a recombinant vector for use according to the present invention, wherein 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, more preferably, the alphavirus-based replicon RNA is a Venezuelan Equine Encephalitis Virus (VEEV)-based replicon RNA.

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

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

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

[0152] Recombinant vectors as RNA molecules for use according to the invention can be delivered to the bird or host cell in various ways, for example by mechanical or chemical means, or can be encapsulated in suitable (nanoparticle) carriers such as proteins, polysaccharides, lipids or polymers, as described herein. For stabilization, RNA nucleotide analogues or specific chemical modifications can be incorporated or applied, for example, to the nucleotides or their backbones.

[0153] In one embodiment of a recombinant vector for use according to the 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' poly-A tail. mRNA can be delivered to a eukaryotic host organism or host cell by transfection and / or by using appropriate carriers, such as polymers or cationic lipids.

[0155] In one embodiment of the recombinant vector for use according to the 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 a host cell or target organism using any suitable method.

[0157] Recombinant vectors in the form of replicating recombinant viral vectors for the expression and delivery of heterologous proteins are well known in the art. They provide an efficient method of vaccination since the viral vectors replicate and amplify in the target avian species. The construction and modification of recombinant vector viruses is routine and can be performed using standard molecular biology techniques.

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

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

[0160] In one embodiment of the recombinant vector for use according to the invention, where the vector is a virus, the virus is selected from a herpesvirus, a poxvirus, a paramyxovirus, and an adenovirus.

[0161] Examples of suitable vector viruses that can be used as avian vectors are well known in the art, e.g., an example of a herpesvirus is herpesvirus of turkeys (HVT) or Marek's disease virus (MDV) of serotype 1 or 2, an example of a poxvirus is fowlpox virus, an example of a paramyxovirus is NDV, and an example of an adenovirus is avian adenovirus.

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

[0163] Examples of recombinant viral vectors expressing and delivering influenza HA genes are described for HVT as a vector in WO 2012 / 052384 and EP 19218804.3, and for NDV as a vector in WO 2007 / 106882.

[0164] For the construction of recombinant viral vectors, typically, an expression cassette is inserted into a locus in the genome of the vector. To control the locus and the orientation of its insertion, various techniques are available. For example, by using the appropriate flanking sections from the genome of the vector to direct the integration of the cassette by a homologous recombination process, for example, by using overlapping Cosmids as described in U.S. Pat. No. 5,961,982. Alternatively, this integration can be performed using CRISPR / Cas technology.

[0165] An "expression cassette" is a nucleic acid fragment that contains at least one heterologous gene and one promoter that drives the transcription of the gene to allow expression of the encoded protein. The termination of transcription can be provided by a sequence provided by the genomic insertion site of the cassette, or the expression cassette itself can contain a termination signal, such as a transcription terminator. In such a cassette, both the promoter and the terminator must be adjacent to the gene whose expression they regulate, which is called "operably linked", so that there is no significant other sequence between them that interferes with the effective initiation, termination, respectively, of transcription. As will be apparent to those skilled in the art, an expression cassette is a self-contained expression module, and therefore its reading direction orientation relative to the vector virus genome is generally not important.

[0166] Besides the use of viruses as vectors for use according to the invention, recombinant vectors for use according to the invention can also be delivered and expressed in avian species by polymeric structures that resemble virions. Examples are virus-like particles (VLPs) or replicon particles (RPs). These structures, known as "single-cycle" infectious particles, contain the necessary characteristics to infect a host cell and express the heterologous gene it carries, but are usually incapable of complete viral replication due to the absence of (the relevant part of) the viral genome from which they were constructed. This serves as a built-in safety feature.

[0167] "RPs" are well known, and several RPs have been developed as platforms for the expression and delivery of various proteins. The preferred base material for RPs is alphaviruses due to their broad host range and rapid replication. Of course, some alphaviruses are highly pathogenic in their wild-type form, so appropriate safety measures must be taken to attenuate and control the infection of such RPs. For reviews, see Kamrud et al. (2010, J. Gen. Virol., vol. 91, p. 1723-1727) and Vander Veen et al. (2012, Anim. Health Res. Rev., vol. 13, p. 1-9).

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

[0169] A preferred alphavirus RP is based on VEEV, which has been applied 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, p. 389-401) and WO 2019 / 110481. A preferred VEEV RP technology is SirraVax. smRNA Particle technology (Harris vaccine).

[0170] RNA for RP can be conveniently produced in vitro by using a DNA plasmid to translate genes into RNA that is recovered and transfected into host cells along with helper RNAs encoding the VEEV structural proteins in trans.

[0171] As described, recombinant vectors for use according to the invention may be advantageously used to deliver and express recombinant proteins for use according to the invention to avian species, for example as a method of vaccinating the target, which at one stage involves administering the vector to the avian species, for example where the vector is a nucleic acid such as a DNA expression plasmid or an RNA molecule.

[0172] The vector may also be introduced into a host cell in vitro for amplification of the vector and / or expression of the recombinant protein, and then the host cell (containing the vector and / or protein) is administered to the avian species, e.g., where the vector is a viral vector, e.g., HVT.

[0173] Furthermore, the vector can be introduced into cells of a recombinant expression system for expression of the recombinant protein and the protein can be recovered from the cell culture and used to vaccinate avian species as described above. Also, the host cell infected or transfected with a recombinant vector for use according to the invention and containing and / or expressing a recombinant protein for use according to the invention can itself be used in the method of protection of the invention, e.g. the infected or transfected host cell can itself be used to vaccinate avian species.

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

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

[0176] A "host cell" according to the invention is a cell that allows for the expression of a recombinant protein for use according to the invention and / or allows for the replication of a recombinant vector for use according to the invention.

[0177] Host cells of the invention can be primary cells maintained in vitro, for example in suspension, in monolayers, or in tissues.

[0178] Alternatively, the host cells can be immortalized cells maintained in vitro, e.g., cells from an established cell line that are capable of growing and dividing almost indefinitely. Depending on the type of host cell, expression of the HA stem polypeptides of the invention will involve more or less extensive post-translational processing, e.g., signal peptide cleavage, disulfide bond formation, glycosylation, and / or lipid modification.

[0179] The primary cell and the immortalized host cell may be of the same or different species, and one or both may be of the same or different species as the avian species that is the subject of the method of protection of the invention.

[0180] Most of the host cells used are fibroblasts and lymphocytes.When using HVT as the recombinant vector virus of the present invention, the host cell is preferably primary chicken embryo fibroblast (CEF), which can be used and stored as described, for example, see WO2019 / 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 WO 97 / 044443 and WO 98 / 006824, more preferably, the immortalized avian host cell of the present invention is an immortalized CEF, even more preferably, the immortalized CEF disclosed in WO 2016 / 087560.

[0182] In one embodiment of a host cell of the invention, said host cell is preferably a cell of a recombinant expression system. Examples of cells from expression systems are, for example, cells of bacterial, yeast, insect, avian or mammalian origin.

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

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

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

[0186] Cells from a mammalian expression system are, for example, cells from hamsters, such as Chinese hamster ovary (CHO) cells.

[0187] All of these cell lines and their corresponding use in recombinant expression systems are well known in the art and can be used using routine techniques and materials.

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

[0189] Codon optimization is well known and is applied to improve the expression level of genes in expression systems, which are typically in a different context to that of the gene's origin. Optimization involves adapting a nucleotide sequence to code for an amino acid of interest, which in a sense corresponds to the codon preference (tRNA repertoire) of the recombinant vector, host cell, or target organism in which the sequence is expressed. As a result, the applied nucleotide mutations are silent.

[0190] Thus, in one embodiment of the recombinant vector for use according to the invention, the recombinant protein for use according to the invention is encoded by a nucleic acid sequence that is codon-optimized for the avian organism intended to be protected by the method for protecting the invention. Preferably, the codon-optimization is for poultry. More preferably, the codon-optimization is for poultry selected from chicken, turkey, duck and goose.

[0191] In one embodiment of a recombinant vector for use according to the invention, the recombinant protein for use according to the invention is encoded by a nucleic acid sequence that is codon-optimized for a cell of a recombinant expression system, preferably a cell of bacterial, yeast, insect, avian or mammalian origin. More preferably, the nucleic acid is optimized for insect cells, even more preferably for Drosophila Schneider 2 (S2) cells.

[0192] Both the recombinant proteins for use according to the invention and the recombinant vectors for use may also be characterized by other terms as appropriate for a particular jurisdiction.

[0193] Thus, in a further aspect, the present invention relates to the use of a recombinant protein for use according to the invention or a recombinant vector for use according to the invention for the manufacture of a vaccine for protecting birds against a pathogen, whereby an antigen comprised in said recombinant protein or contained in a recombinant protein expressed by said recombinant vector is derived from said pathogen and said bird possesses antibodies reactive with said antigen.

[0194] In any of the embodiments of recombinant proteins or recombinant vectors for the manufacture of a vaccine according to the invention, the recombinant protein, recombinant vector, protection, avian, pathogen, antigen and antibody characteristics are all as embodied herein.

[0195] It is well known that a "vaccine" is a composition comprising at least one compound capable of inducing a protective immunological effect in a pharma- ceutically acceptable carrier. An "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 manufacture of vaccines for the present invention can be carried out using routine methods and procedures well known in the art. General techniques and considerations that apply to the manufacture of vaccines under well-known standards for pharmaceutical manufacturing are described, for example, in government directives and regulations (pharmacopoeias, 9 CFR) and well-known handbooks such as "Veterinary vaccinology" and "Remington" (both supra). Generally, such vaccines are prepared sterile and using pharmaceutical grade excipients.

[0197] Such manufacture incorporates microbiological testing for sterility and absence of adventitious agents, 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 may be sold, all of which are well known to those skilled in the art.

[0198] For example, when a recombinant protein for use according to the invention is produced by a recombinant expression system, the protein may be harvested from the expression system culture, e.g., as the whole culture. Alternatively, the harvest may be a portion of such a culture, e.g., the supernatant or cell pellet after centrifugation of a cell culture, or the filtrate or retentate after filtration. The supernatant may be obtained after the culture has settled by gravity, e.g., by leaving it overnight, or by centrifugation, and the filtrate is that which passes through the filter during filtration.

[0199] As described, the recombinant proteins for use according to the invention and the recombinant vectors for use according to the invention achieve their beneficial effects in the protection of avian species via vaccines comprising said recombinant proteins and / or said recombinant vectors.

[0200] Thus, in a further aspect, the present invention relates to a vaccine comprising a recombinant protein for use according to the invention or comprising a recombinant vector for use according to the invention and a pharma- ceutically acceptable carrier for use in a method for protecting birds bearing antibodies reactive with an antigen comprised in the recombinant protein or contained in a recombinant protein expressed by the recombinant vector, from a pathogen from which the antigen is derived.

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

[0202] "Pharmaceutically acceptable carriers" are well known to aid in the stabilization and administration of vaccines, and are moreover relatively harmless and well tolerated by the vaccinator. Such carriers may be, for example, water or physiological salt solutions. In more complex forms, the carriers may be, for example, buffers which may contain further additives such as stabilizers or preservatives. Details and examples thereof are described in well-known handbooks such as, for example, "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] When the vaccine according to the present invention comprises a recombinant vector that is a replicating virus, the pharma- ceutically acceptable carrier is preferably a composition that stabilizes the virus or the host cell in which the virus is contained. Examples include some virus vaccine diluents and stabilizers for frozen or lyophilized storage, which typically include, for example, sugars, amino acids, physiological buffers (e.g., saline, PBS, or 50 mM HEPES), and often bulky compounds such as albumin, polymers, etc. For example, when the vaccine comprises a recombinant HVT vector, such vaccines are typically commercially available as cell-associated products. In that case, the pharma-ceutically acceptable carrier is preferably a mixture of about 10% serum and about 6% DMSO in culture medium. This carrier also provides stabilization of HVT-infected host cells during freezing and cryopreservation. The serum can be any serum routinely used for cell culture, such as fetal or newborn calf serum.

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

[0205] Furthermore, additional carriers can be added to stabilise and / or deliver the recombinant vector for use in the present invention, e.g. to encapsulate the recombinant vector according to the present invention, which is a nucleic acid or a RP, in a suitable (nanoparticulate) carrier such as a protein, polysaccharide, lipid or polymer. Preferably, additional carriers for recombinant vectors according to the present invention, which are RPs, include nanogels, which are biodegradable polyacrylic polymers as described in WO 2012 / 165953.

[0206] Obviously, either recombinant vectors or in vitro host cells comprising such vectors can be used herein in the present invention either viable (i.e. replicating) or dead (non-replicating or inactivated). Then, either recombinant vectors or only parts of the host cells can be used herein in the present invention, e.g. as pellets, supernatants, concentrates, dialysates, extracts, sonicates, lysates, or as part of a composition comprising vectors and / or host cells, e.g. a culture. All this is well known to the skilled artisan.

[0207] Where a vaccine for use according to the invention comprises a recombinant protein for use according to the invention, the vaccine may comprise an adjuvant to stimulate the induced immune response.

[0208] Thus, in one embodiment, a vaccine for use according to the invention comprises an adjuvant.

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

[0210] Widely used adjuvants are oils, such as mineral oils, e.g., light (white) mineral (paraffin) oils, or non-mineral oils, e.g., squalene, squalane, vegetable oils or their derivatives, e.g., ethyl oleate. Also advantageously used are combination products such as ISA™ (Seppic) or DiluvacForte™ and Xsolve™ (both MSD Animal Health).

[0211] A handbook on adjuvants and 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 invention in several aspects: if the adjuvant comprises an oil, the vaccine may be provided in aqueous form or may be formulated in different ways as an emulsion with oil, i.e. as water-in-oil (W / O), oil-in-water (O / W) or as a double emulsion, either W / O / W or O / W / O.

[0213] An "emulsion" is a mixture of at least two immiscible liquids, whereby one is dispersed in the other. Typically, the droplets of the dispersed phase are very small, in the submicrometer range.

[0214] Procedures and equipment for preparing emulsions at any scale are well known in the art. One or more emulsifying agents can be used to stabilize the emulsion.

[0215] "Emulsifiers" are molecules that are amphipathic, possessing both hydrophobic and hydrophilic sides. Many emulsifiers are known in the art for their various properties. Most are readily available commercially and come in several degrees of purity. Common emulsifiers for vaccines are sorbitan monooleate (Span® 80) and polyoxyethylene-sorbitan monooleate (Polysorbate 80, or Tween® 80).

[0216] A well-known way to characterize the properties of emulsifiers (mixtures of emulsifiers) is the HLB number (hydrophile-lipophile balance; Griffin, 1949, J. Soc. Cosm. Chem., vol. 1, p. 311-326). Typically, emulsifiers or emulsifier mixtures with HLB numbers below 10 are favored for W / O emulsions, while emulsifiers (mixtures) with HLB numbers between 10 and 16 are favored for O / W emulsions.

[0217] Emulsion stabilizers may also be added, examples of which are benzyl alcohol, triethanolamine.

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

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

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

[0221] In other terms and in particular jurisdictions, further aspects of the present invention can be defined as follows.

[0222] In a further aspect, the present invention relates to the use of a recombinant protein for use according to the invention, or a recombinant vector for use according to the invention, or a vaccine for use according to the invention, for protecting birds against a pathogen, whereby an antigen comprised in said recombinant protein or contained in a recombinant protein expressed by said recombinant vector is derived from said pathogen and said bird possesses antibodies reactive with said antigen.

[0223] In one embodiment of the use according to the invention, the use comprises the administration of any recombinant protein, recombinant vector or vaccine of the invention to an avian species.

[0224] In embodiments of the use according to the invention, the recombinant protein, recombinant vector, vaccine, use, method, protection, avian, pathogen, antigen and antibody features are all as embodied herein.

[0225] In a further aspect, the present invention relates to a method for protecting an avian animal against a pathogen comprising the step of administering to said avian animal a vaccine for use according to the invention, wherein an antigen contained in said vaccine is derived from said pathogen, and wherein said avian animal possesses antibodies reactive with said antigen.

[0226] Vaccines for use according to the invention will typically be prepared in a form suitable for administration to birds, suited to the desired route of application and desired effect.

[0227] Depending on the application route of the vaccine for use according to the invention, it may be necessary to adapt the composition of the vaccine. This is well within the capabilities of the skilled artisan and generally involves fine-tuning the efficacy or safety of the vaccine. This can be done by adapting the dose, amount, frequency, route of the vaccine, by using a different form or formulation of the vaccine, or by adapting one of the excipients of the vaccine (e.g. stabilizer or adjuvant).

[0228] The vaccine according to the invention can in principle be given to birds by different routes of administration and at different times in their life span, in particular the vaccine can be administered to birds of any age that possess antibodies reactive with the antigen in the recombinant protein for use according to the invention.

[0229] If administered as early as possible, it can be administered on the day of hatch ("day 1"), or right in ovo, for example, at about day 18 of embryonic development, all of which are well known in the art.

[0230] Equipment for automated injection of vaccines into fertilized eggs on an industrial scale is commercially available. This provides the earliest possible protection while minimizing labor costs. Different in ovo vaccination routes are known, such as into the yolk sac, embryo, or allantoic cavity, and these can be routinely optimized as required.

[0231] Vaccines for use according to the invention may be formulated as injectable solutions suitable for injection either in ovo or parenterally.

[0232] In one embodiment, a vaccine for use according to the invention is formulated as a liquid selected from a suspension, a solution, a dispersion, and an emulsion.

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

[0234] The exact amount of either the recombinant protein or the recombinant vector is not critical to the present invention and can be readily established by comparing the protective effects of different amounts.

[0235] Also, where a vaccine for use according to the invention comprises a viral vector, this is capable of replicating in the vaccinated bird and need only be administered in an amount sufficient to establish a productive infection in the bird.

[0236] For example, when the viral vector for use according to the present invention is a recombinant HVT, a suitable inoculation dose is 1x10^1 to 1x10^5 plaque forming units (pfu) of the HVT of the present invention per animal dose, preferably 1x10^2 to 1x10^4 pfu / dose, even 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] Where an HVT vector for use according to the invention is cell-associated, these amounts of HVT are contained within infected host cells.

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

[0239] Determining an immunologically effective amount of a vaccine according to the invention, or optimizing the volume of vaccine per animal dose, is both well within the capabilities of one skilled in the art.

[0240] The administration regimen for administering the vaccine to birds for use according to the present invention may be single or multiple doses, in a manner compatible with the formulation of the vaccine, and in an amount that will be immunologically effective.

[0241] Preferably, the regimen for administration of the vaccine for use according to the invention is integrated into existing vaccination schedules of other vaccines that the target bird may require, in order to reduce stress on the animals and reduce labour costs. These other vaccines may be administered simultaneously, in parallel or sequentially in a manner compatible with their registered use.

[0242] TIFF2024525697000003.tif197131TIFF2024525697000004.tif191131TIFF2024525697000005.tif184130TIFF2024525697000006.tif195130TIFF2024525697000007.tif188130TIFF2024525697000008.tif180130TIFF2024525697000009.tif194130TIFF2024525697000010.tif196130TIFF2024525697000011.tif182131TIFF2024525697000012.tif193130TIFF2024525697000013.tif188130TIFF2024525697000014.tif195129TIFF2024525697000015.tif181132TIFF2024525697000016.tif188129TIFF2024525697000017.tif196129TIFF2024525697000018.tif183131TIFF2024525697000019.tif195130TIFF2024525697000020.tif188130TIFF2024525697000021.tif182130TIFF2024525697000022.tif197129TIFF2024525697000023.tif189130TIFF2024525697000024.tif194130TIFF2024525697000025.tif193131TIFF2024525697000026.tif192129TIFF2024525697000027.tif185130TIFF2024525697000028.tif190129TIFF2024525697000029.tif187129TIFF2024525697000030.tif193133TIFF2024525697000031.tif188130TIFF2024525697000032.tif182129TIFF2024525697000033.tif196130TIFF2024525697000034.tif188131TIFF2024525697000035.tif191130TIFF2024525697000036.tif191131TIFF2024525697000037.tif182129TIFF2024525697000038.tif196130TIFF2024525697000039.tif181130TIFF2024525697000040.tif197130The invention is described herein in various aspects and embodiments. It will be appreciated that any combination of these is considered to be within the scope of the invention. However, merely for the sake of brevity, this specification does not provide an exhaustive overview of all possible combinations.

[0243] The invention will now be further illustrated by the following non-limiting examples.

[0244] [Example] [Example 1] Generation of AIV-MDA positive chickens Introduction To be able to test vaccination of seropositive chickens, we created an animal model that resembles the actual situation in the field. Specifically, AIV MDA-positive progeny were generated by repeated intramuscular vaccination of parent hens with an inactivated adjuvanted vaccine. The aim was to reach HI titers in the progeny similar to those in the field, of at least 5-7 Log2.

[0245] 1.2. Materials and Methods SPF White Leghorn egg-laying chickens were vaccinated to generate MDA-positive hatchlings. All chickens were housed in an isolation room and kept on floors. All chickens were provided with food and water ad libitum during the study and were kept under veterinary supervision.

[0246] 1.2.1. Preparation of the vaccine for MDA production: Inactivated AIV vaccine was produced by propagating avian influenza A virus of the H9N2 subtype in 10-day-old embryonated SPF chicken eggs. Specifically, this is 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, eggs were refrigerated at 4°C and virus was obtained by harvesting allantoic fluid, which was removed by centrifugation at 3.000 rpm for 20 minutes. Virus was titrated by plaque assay or TCID50 on Madin-Darby canine kidney (MDCK) cells.

[0247] The virus was chemically inactivated using 0.1% beta-propiolactone, followed by three blind passages in 10-day-old embryonated SPF chicken eggs to confirm inactivation. The inactivated virus harvest 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+ hatchlings A flock of 40 SPF White Leghorn layer chickens at 17 weeks of age was used. The chickens were individually labeled. They were immunized with 0.5 ml of 520 HAU / dose of inactivated adjuvanted H9N2 virus vaccine administered im in the leg. The first dose of 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 taken from the wing vein of the hens on day 0 and at weeks 5, 11, 18, 29 and 36 after the first dose. Five SPF roosters were included in the groups for insemination but were not part of the actual experiment.

[0250] Fertilized eggs were collected from 36 weeks after the first vaccination dose. They were set to incubate until hatching. Ten hatchlings were sacrificed on day 1 of life (D0) to measure their MDA levels. The hatchlings were used for the MDA vaccination experiment.

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

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

[0253] 1.3.Results The results of hyperimmunization of dams giving rise to AIV MDA+ progeny are shown in Figure 1. HI titrations were performed with the homologous UDL-01 strain.

[0254] A third vaccination was administered 18 weeks after initiation, when a decline in HI titers in the serum of hens was observed, which resulted in very high HI titers in hens that were maintained at that level until the last sampling point.

[0255] Fertilized eggs were collected 36 weeks after initiation (53 weeks of age) when the mean (n=10) HI titer of hens was 4096 (12 Log2).

[0256] It is evident from these results, and as shown in Figure 1, there were significant differences in HI titers between T = 11 weeks and T = 18 weeks (p < 0.05) and between T = 18 weeks and T = 29 weeks (p < 0.001).

[0257] MDA-induced HI titers in the (unvaccinated) progeny of these hens were measured at hatch and over time, i.e., 1, 7, 14, 21, 28, 35, 42, 56, 70 and 84 days after hatch. The results are shown in Figure 2. HI titers were performed with the homologous UDL-01 strain.

[0258] On day 1, the HI titer in chicks averaged (n=10) 588 (9.2 Log2). This titer declined slightly (non-significantly) at 7 days of age, but more than halved to 181 (7.5 Log2) at 14 days of age, then declined more rapidly; by day 35, the mean (n=10) HI titer was 16 (4 Log2), and by day 42, HI titers were no longer detectable.

[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). Hatchlings used experimentally here were found to still have HI titers around this value at 28 days of age, but these chicks started out well above normal MDA levels. Therefore, additional active vaccination is usually required.

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

[0261] [Example 2] Preparation of vaccines for MDA+ birds 2.1. Introduction Three vaccines were used for vaccination of seropositive birds.

[0262] The positive control was the classical inactivated whole virus vaccine: Nobilis® Influenza H9N2+ND (MSD Animal Health). This commercial vaccine contains inactivated AIV of subtype H9N2, strain A / chicken / UAE / 415 / 99 ("UAE") and inactivated Newcastle Disease Virus, clone 30 strain.

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

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

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

[0266] 2.2. Materials and Methods 2.2.1. Preparation of HA antigen expression construct A mouse hybridoma producing an antibody against chicken CD83 (GenBank accession number XM_040663657.1) was used to obtain the vL and vH chain sequences. Synthetic cDNA containing the vL and vH sequences was synthesized (Gly 4 Ser) 4 The vH-linker-vL cDNA was then cloned into the D. melanogaster expression vector pMT-BIP-V5-His™ (version A, Thermo Fisher Scientific) using the NotI and XbaI restriction sites. This vector provides the D. melanogaster metallothionein (MT) promoter and the D. melanogaster immunoglobulin heavy chain binding protein (BIP) secretion signal for expression and secretion in S2 cells. In addition, the plasmid is provided with a multiple cloning site, a V5 epitope for recombinant protein detection, and a 6xHis tag for recombinant protein purification.

[0267] The resulting vector, designated pMT-BIP-CD83-scFv-V5-His, was used to insert the ectodomain of the H9 HA gene lacking the HA gene signal peptide and TM domain. The KpnI and PacI restriction sites were used to add the 29 amino acid trimerization Foldon sequence from the trimeric protein fibritin from bacteriophage T4. This plasmid contained the nucleotide sequence of SEQ ID NO: 14 under the operational control of the MT promoter.

[0268] The H9 HA used in this experiment was synthetically produced by incorporating the consensus sequence of the HA of H9N2 viruses obtained from the analysis of over 2000 H9 HA sequences from a public database of G1-like H9 virus lineages using the Minimum Spherical 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 also closely related to the 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 ectodomain of the H9N2 virus of the UDL-01 strain (GenBank accession number: ACP 50708.1, HA1: aa 19-338 and HA2: aa 339-560), which was found to be homologous, and has been codon-optimized for S2 cells.

[0270] The H9 HA-Foldon antigen without the CD83 targeting signal was prepared in a similar manner, resulting in the plasmid pMT-BIP-H9 HA-Foldon-V5-His, which contained the nucleotide sequence of SEQ ID NO: 15 under the operational control of the MT promoter.

[0271] 2.2.2. Generation 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 passaged once a week by centrifugation at 1200 rpm for 10 min and resuspended in fresh complete S2 cell medium.

[0272] Recombinant proteins were produced and purified using the Drosophila Expression System (DES®, Life Technologies). Briefly, plasmids pMT-BIP-rH9 HA-V5-His and pMT-BIP-rH9 HA-CD83-scFv-V5-His were co-transfected into S2 cells, respectively, using calcium phosphate transfection. Prior to transfection, 1×10^6 / mL S2 cells were pre-seeded in 5 mL of complete S2 cell growth medium at 28°C for 6 to 16 hours. 60 μL of 2M CaCl 2 The transfection solution was prepared by adding 32 μg of expression plasmid DNA, 1.5 μg of hygromycin B resistance 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 min. The resulting solution was slowly added dropwise to pre-seeded S2 cells and incubated at 28° C. for 24 h. 24 h after transfection, the transfection medium was replaced with fresh complete S2 cell medium, and the cells were incubated at 28° C. for an additional 3 days.

[0273] Stable S2 transfectants were generated by antibiotic selection, ie, complete growth medium containing 250 μg / mL hygromycin B was added weekly for at least 4 weeks.

[0274] Single cell clones were then obtained via limiting dilution (Zitzmann et al., 2010, Biotechnol. Reports, vol. 19, e00272). Briefly, 2x10^3 S2 transfected cells were mixed with 10^6 gamma-irradiated parental S2 cells as feeder cells. 100μL of this cell mixture was seeded into each well of a 96-well plate. Single clones in each well were clearly visible after 4 weeks of incubation at 28℃. Approximately 10-15 single clones were screened for each plasmid construct. The single clone expressing the highest amount of recombinant protein was selected by indirect ELISA for H9 HA protein.

[0275] 2.2.3. Expression and purification of recombinant antigens Selected transfected S2 cell clones were then cultured on a large scale. Briefly, a single clone expressing a large amount of HA recombinant 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 used plasmid was transformed into a cytoplasmic ... 4 The cells were induced by adding 500 μM of 100% ethanol to a final concentration of 500 μM. Four days after induction, the cell supernatant was harvested by centrifugation at 1200 rpm for 20 min and dialyzed to remove excess copper ions. A total of approximately 2 liters of protein expression supernatant was harvested and filtered through a 0.22 μM filter Stericup (Merck GmbH Life Science) before purification.

[0276] The use of the His tag allowed purification of the recombinant protein by metal affinity column chromatography. Briefly, the dialyzed and filtered 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. The copper-binding protein was then 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. Protein fractions were combined and concentrated by centrifugation at 4600 rpm for 30 min 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 proteins was confirmed using a hemagglutination assay. Briefly, 35 μg of recombinant proteins were serially diluted 2-fold in PBS in a V-bottom 96-well plate. Chicken red blood cells were diluted to 1% in PBS and added to each well. The plates were then incubated at 4°C for 1 h and tilted 90° in a biosafety cabinet to visualize and score hemagglutination.

[0278] 2.2.4. Preparation of vaccine emulsion The recombinant HA antigen vaccine was formulated as a water-in-oil emulsion with light liquid paraffin oil (Marcol® 52) as the adjuvant and contained 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, the difference being to provide equimolar amounts to compensate for the addition of scFv.

[0280] [Example 3] Vaccination of seropositive birds Introduction Because protection against AIV infection and disease is essentially serologically determined and the majority of AIV neutralizing antibodies are directed against the HA antigen, serological testing for the development of anti-HA antibodies, i.e., HI titers, is an excellent predictor of in vivo protection from AIV.

[0281] Hatchlings generated as described in Example 1 were used in the vaccination experiment: one group was vaccinated on day 1, they had a very high mean MDA HI titer of 588 (9.2 Log2) and were designated the MDA++ group, the other group was vaccinated only at 14 days of age when MDA levels had somewhat decreased, they had a moderate mean MDA HI titer of 181 (7.5 Log2) and were designated the MDA+ group.

[0282] This approach allowed testing and comparison of "worst case" and "average case" antibody interference with the efficacy of vaccination with targeted or non-targeted HA antigens, respectively. For comparison, a classical inactivated H9N2 vaccine was included. Also, a group of unvaccinated chicks was included in the experiment to follow the natural decline of 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 vaccine used was as described in Example 2.

[0284] To avoid the introduction of environmental pathogens, birds were housed in positive pressure isolation rooms with inflowing high efficiency particulate air (HEPA) filtered air.

[0285] After hatching, only healthy and normal appearing chicks were used. They were assigned to groups upon receipt and individually numbered. Daily clinical observations were performed to monitor health and performance. Each test group had 10 animals.

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

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

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

[0289] Blood samples were taken weekly for the first 6 weeks after the start of the study and then biweekly for the 8th, 10th and 12th weeks to examine the serological responses induced by the vaccination.

[0290] Blood samples were taken on days 1 and 7 after euthanasia, and from day 14 onwards from the wing vein. The volume collected was 2-3 ml, depending on what the animal's weight would allow. Blood samples were left to clot at ambient temperature and serum was separated by centrifugation. Serum samples were heat inactivated at 56°C for 30 min and stored at -20°C until use.

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

[0292] Non-vaccinated controls showed HI titer levels and degradation patterns as described in Example 1 and FIG.

[0293] Positive controls were MDA++ chicks that received a whole inactivated virus vaccine ("Nobilis Influenza H9N2+ND") on day 1 of age. Despite this vaccination, their HI titers steadily declined and no vaccination response was detectable. This was noteworthy, since the MDA and HA antigens in the classical vaccine were heterologous, and MDA was induced against an HA antigen that closely resembled the H9 HA of the UDL-01 strain, whereas the Nobilis vaccine contained a heterologous H9 HA antigen, i.e., from the UAE strain, which has 94% amino acid identity with the UDL-01 H9 HA protein. As a result, a lower level of antibody interference would be expected due to this difference between the HA antigens. However, apparently, the HI levels in MDA++ chicks were very high and even hindered the efficacy of the heterologous H9 HA vaccine.

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

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

[0296] However, the targeted HA antigen induced very high HI titers. In the MDA++ group, there was an initial decline from a very high starting value (9.7 Log2), but then a robust and steady increase in HI titers was evident from 4 weeks post vaccination (pv), reaching significance at 5 weeks pv, and robustly increasing to 9.7 Log2 at 12 weeks pv. This indicates that the vaccine can be applied at day 1 of age, even in the setting of very high levels of homologous MDA, and still induce strong protection against AIV infection and disease.

[0297] In the MDA+ group, HI titers from the targeted HA vaccine showed rapid induction of high HI titers already 1 week after vaccination, reaching a mean HI titer of 1835 (10.8 Log2) by 4 weeks after vaccination.

[0298] In both test groups, the targeted vaccine was the only one capable of inducing significantly increased HI titers, and the lowest HI titers measured in the targeted vaccine groups were 6.2 and 6.9 Log2 in the MDA++ and MDA+ groups, respectively, indicating that all chicks receiving this type of vaccine remained well above the 5 Log2 threshold for protection throughout the duration of the experiment.

[0299] This rapid onset and long duration of immunity fully compensates for the decline in MDA levels, leaving no gaps in protection.

[0300] Again, indirect ELISA was performed on the sera to confirm that all antibodies were H9 HA specific.

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

[0302] Hens can be vaccinated two or three times, beginning before lay begins and continuing through the laying period. The specific antibody titers achieved in the hens can be confirmed as being sufficiently high. Eggs can then be collected and hatched, and chicks can be confirmed for having sufficiently high MDA levels against the pathogen being 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. Also included is a binding domain, e.g., a scFv directed against a surface protein of avian APC, such as CD83, CD11c or Dec-205. To evaluate the effect of targeting the antigen to APC, a similar construct, but without the binding domain, can be prepared to serve as a control.

[0304] The plasmids can be transfected into S2 cells as described, selected, amplified, and used to express the antigen (with or without a targeting signal). The recombinant protein can then be harvested.

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

[0306] An example of an scFv specific for CD11c or Dec-205 is a peptide comprising the amino acid sequence shown in SEQ ID NO: 16 or 17, respectively.

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

[0308] - SEQ ID NO: 4 for AIV H5 HA; - SEQ ID NO: 5 for AIV H7 HA, - SEQ ID NO: 18 for NDV F; - SEQ ID NO: 19 for NDV HN; - SEQ ID NO: 20 for IBDV VP2, and - SEQ ID NO: 21 for IBV spike.

[0309] The corresponding nucleic acids encoding these antigens are preferably codon-optimized with respect to the codon usage table of S2 cells. In the expression constructs, additional elements such as signal sequences, linkers, and one or more tags can be added as needed to facilitate expression, secretion, and purification.

[0310] Chicks with specific MDA are then vaccinated with these recombinant proteins and their serology monitored over time.

[0311] For these pathogens, the levels of specific antibodies that correlate with in vivo protection are known, so serological testing of antibody levels at various time points after vaccination is sufficient to get a good impression of the effectiveness of targeted vaccination in birds that are seropositive for antigens derived from these pathogens.

[0312] The H5 HA sequence of SEQ 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 of the HA ectodomain were selected: HA1: 17-340 and HA2: 346-530. The polybasic cleavage sequence was modified from PLR to PQG and the number of arginines was reduced.

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

[0314] The NDV F sequence of SEQ ID NO: 18 is a consensus sequence from over 1200 F amino acid sequences from avian orthoabulvirus 1 sequences in public databases using the MScon technology described herein. The consensus F protein has 98.5% amino acid similarity with the closest natural counterpart: avian orthoabulvirus 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 SEQ 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 technology described herein. Amino acids 47-571 from HN were selected.

[0316] The IBDV VP2 protein of SEQ ID NO: 20 represents amino acids 9 to 452 of the IBDV VP2 protein of GenBank acc.nr.AMA 19770.1.

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

[0318] [Figure 1] A presentation of antibody titer results in dams hyperimmunized to generate AIV MDA+ offspring. Details are provided in Example 1.

[0319] The vertical axis shows the mean (n=10) HI titers measured by HI assay in sera of dams after immunization with inactivated adjuvanted AIV H9N2 virus vaccine (UDL 01 / 08). The horizontal axis shows time points in weeks after the start of the experiment (day 0=17 weeks of age). Vaccinations are indicated by arrows at 0, 3, and 24 weeks after the start.

[0320] Fertilized eggs were collected 36 weeks after initiation and the boxes indicate the mean (n=10) HI titers in serum of laying hens used in follow-up experiments: HI=12 Log2 (4096).

[0321] Data are presented as means (columns) and standard deviations (error bars). Asterisks indicate significant differences in HI antibody titers at weeks 11 and 18, and weeks 18 and 29 after the start of the study, *=p<0.05 and ***=p<0.001. [Diagram 2] Presentation of MDA-derived HI titer results in unvaccinated progeny from hens vaccinated three times, details are provided in Example 1.

[0322] Anti-H9 HA MDA titers were measured by HI assay of serum samples on days 1, 7, 14, 21, 28, 35, 42, 56, 70 and 84 post-hatch. HI titers are expressed as the reciprocal of the highest dilution of serum that completely inhibited viral hemagglutination activity of 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 of an HI titer of 32 (5 Log2). [Diagram 3] Presentation of HI titer results in chicks vaccinated on day 1 of age with high levels of MDA (MDA++), details are provided in Example 3.

[0324] The vertical axis shows the HI titers and the horizontal axis shows the days post-vaccination. NB: There are gaps in the vertical axis to allow for the very high HI titers found to be displayed.

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

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

[0327] Data are presented as mean (columns) and SD (error bars). Statistically significant differences are indicated by asterisks, where ***=p<0.001 and *=p<0.1. [Figure 4]Presentation of HI titer results for chicks with intermediate levels of MDA (MDA+) vaccinated on day 14. Details are provided in Example 3.

[0328] Presentation similar to Fig. 3 , except that no group received the nobilis vaccine.

Claims

1. 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 an avian having antibodies reactive with said antigen from a pathogen from which said antigen is derived.

2. The recombinant protein for use according to claim 1, characterized in that the avian APCs are dendritic cells.

3. 2. The recombinant protein for use according to claim 1, characterized in that the cell surface protein is CD83.

4. 2. A recombinant protein for use according to claim 1, characterized in that the binding domain is a single chain variable fragment (scFv).

5. 2. The recombinant protein for use 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.

6. 2. The recombinant protein for use according to claim 1, characterized in that the antigen comprises an amino acid sequence selected from SEQ ID NOs: 7, 8 and 9.

7. A recombinant vector capable of expressing the recombinant protein of claim 1, for use in a method for protecting birds having antibodies reactive with an antigen contained in the recombinant protein expressed by the recombinant vector from a pathogen from which the antigen is derived.

8. Use of a recombinant protein according to claim 1 or a recombinant vector according to claim 7 for the manufacture of a vaccine for protecting birds from a pathogen, characterized in that an antigen contained in the recombinant protein or contained in a recombinant protein expressed by the recombinant vector is derived from the pathogen, and the bird possesses antibodies reactive with the antigen.

9. 10. A vaccine comprising the recombinant protein of claim 1 or the recombinant vector of claim 7 and a pharmaceutically acceptable carrier, for use in a method for protecting birds that have antibodies reactive with an antigen contained in the recombinant protein or contained in a recombinant protein expressed by the recombinant vector from a pathogen from which the antigen is derived.

10. A vaccine for use according to claim 9, characterized in that it comprises an adjuvant.

11. Use of the recombinant protein of claim 1 or the recombinant vector of claim 7 for protecting birds from a pathogen, characterized in that an antigen contained in the recombinant protein or contained in the recombinant protein expressed by the recombinant vector is derived from the pathogen, and the bird possesses antibodies reactive with the antigen.

12. A method for protecting birds from pathogens, comprising administering the vaccine of claim 9 to the birds, wherein an antigen contained in the vaccine is derived from the pathogen, and the birds possess antibodies reactive with the antigen.

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