OVERCOMING ANTIBODY-INTERFERENCE IN AVIANS

The present invention relates to the field of vaccination of avians; more specifically the invention relates to a recombinant protein for use in a method to protect an avian that possesses antibodies reactive with the antigen in said protein. In particular the invention relates to a recombinant protein, a recombinant vector, and a vaccine for use in said method. Further the invention relates to a use and a method for the treatment of avians by administration of the protein, the vector or the vaccine.

As a nutritious and affordable source of protein, avian meat and eggs are a prominent part of the diet of most of the world's human population. The main species of poultry bred for such economic purposes are chickens, turkeys, ducks and geese. To raise these birds in the large numbers that are required, while maintaining their health and well-being, the poultry industry is keen to optimise management conditions, and to provide good veterinary care. A vital part of this strategy is the prophylactic protection by vaccination against a wide variety of avian pathogens that may cause infection and disease, with sometimes devastating effects on animal well-being and the economy of operation. For many years a wide variety of vaccines have been commercially available against most of the viral-, bacterial-, and parasitic diseases that may affect avians of economic relevance. Such vaccines can be of different types, such as live attenuated, inactivated, subunit, nucleic acid, viral vector, etcetera.

Especially for the poultry that is produced in very large numbers, i.e. meat-type birds (broilers), it is common practice to protect young birds as early as possible. However the active vaccination of very young animals with an immature immune system is often not very successful. Therefore an effective work-around is by the vaccination of their mothers before and during their egg-laying period. The maternal antibodies generated by the hens are transferred to the egg with the yolk, which is internalised by the developing chick. This way the chicks can be passively protected by these maternally derived antibodies (MDAs) against a variety of pathogens, already at their day of hatch. However most of the MDAs have worn off again in 3 weeks' time due to biological degradation, therefore an active vaccination of the growing chicks themselves must also be provided to induce a proper immune-protection after the first weeks. At that time some MDAs may still be present in the birds.

A similar situation of vaccination in the context of pre-existing antibodies, arises in the case of birds of older age which have antibodies induced by a prior vaccination that slowly wear off, so that a booster vaccination is required, in order to restore antibody titres to protective levels.

An important veterinary and scientific puzzle arises in deciding when a vaccination can be given to birds that already possess antibodies which are reactive with an antigen that is comprised in the vaccine to be used. Clearly, vaccinating when antibody titres have almost gone is too late as that leaves a gap period between the drop of those antibody titres below protective level, and the onset of protection from the active immunisation. In this gap period the birds are vulnerable to infection and disease.

However, vaccinating when the birds still have considerably high titres of circulating antibodies, is too early, as that often affects the efficacy of the vaccination; this happens probably because those antibodies in some way bind to and sequester the vaccine antigens, which may speed up their degradation and/or prevent them from inducing a proper immune-response. This last phenomenon is called ‘antibody-interference', a variant of which is where this regards MDA: ‘M DA- interfere nee'. This is a well-known problem for the effective vaccination against the main pathogens that affect the poultry industry worldwide. Examples of these main pathogens are: infectious bursal disease virus (IBDV; a.k.a. Gumboro disease virus), Infectious Bronchitis virus (IBV), Newcastle disease virus (NDV), and Avian Influenza virus (AIV; a.k.a. fowl plague virus); the last two are even notifiable diseases of the OIE [World Organisation for Animal Health]

For these diseases antibody-interference is well-known to diminish vaccination efficacy which leaves the birds vulnerable to field infection, especially when they are kept in close proximity, and/or in areas with a high prevalence of an avian pathogen.

Overtime, many different approaches have been tried to overcome antibody-interference, in order to prevent a gap in protection, and to optimise the vaccination of seropositive avians. The more straightforward attempts for overcoming antibody-interference included adaptations to the vaccine to increase antigen dose, and/or to use (stronger) adjuvants. Also more virulent, c.q. less attenuated, strains of live vaccine-pathogens have been tried in a hope those could break-through higher titres of antibodies and thus could be administered at an earlier time. As these methods are not generally satisfactory, more complicated approaches were tested.

For the active vaccination of young birds against IBDV, one method involves the monitoring of their MDA levels by serological testing of a sample of the birds to determine the optimal date for the vaccination. However the result is that effective active vaccination can only be applied at 2 - 3 weeks of age, and a protection gap for many of the birds is inevitable because of the variations in a large flock. Alternatively, ‘complexed IBDV vaccines' (live attenuated vaccine-virus bound by antibodies) have been administered at early age, whereby the antigen is only released at a later time. Also viral vector systems have been used, for example using a fowl-pox-, or an avian herpes virus, as a vector for expressing the main IBDV antigen, the viral protein 2 (VP2). This was reviewed by MOIIer et al. (2012, Avian Pathol., vol. 41, p. 133-139).

For NDV, different approaches in vaccination have been applied, but antibody-interference is still a problem today; for a review see: Dimitrov et al. (2017, Vet. Microbiol., vol. 206, p. 126 - 136).

Even the use of recombinant vector vaccines may suffer from antibody-interference, for example in the case the antibodies react with the vector virus itself and/or with the antigen it expresses, see: Hu et al. (2020, Vaccines, vol. 14, p. 222, doi: 10.3390). For NDV as vector, solutions considered were e.g. to change the serological profile of the NDV vector (Steglich et al., 2013, PLoS One, vol. 8, e72530), or to select a strain of NDV that allegedly is less inhibited by anti-NDV antibodies (EP 2998315).

For IBV, MDA are well-known to interfere with vaccination of 1 day old chicks, see: Terregino et al., 2008 (Avian Pathol., vol. 37, p. 487-493).

For AIV, the relevance of effective vaccination even extends beyond the veterinary field, as this virus can give rise to zoonotic infections of humans with pandemic potential. Overtime many different approaches of using classical- or recombinant AIV vaccines have been tried, with varying levels of success, see: D. Swayne, 2009 (Comp. Imm. Microbiol and Inf. Dis., vol. 32, p. 351 - 363). However, and similar to the situation for several other vaccines, dealing with the interference by AlV-reactive antibodies still remains to be a problem (Murr et al., 2020, Avian Dis., vol. 64, p. 427 - 436). Consequently, in spite of the many different approaches tested in the field of avian vaccination, there still is a pressing need for an effective way to overcome the negative effect that pre-existing antibodies in the target animals have on the efficacy of vaccination with an antigen to which these antibodies can bind.

Shrestha et al. (2018, Vaccines, vol. 6, p. 75, doi: 10.3390) reviewed options for improving the vaccination of avian targets, by the selective targeting of an antigen to antigen-presenting cells (APCs). A wide variety of ways are described to achieve such targeting, e.g.: by using ligands, antibodies, nanoparticles, viral vectors, or cell-penetrating peptides. No method for overcoming antibody-interference in avians is described or suggested.

WO 2017/055235 describes antigen-targeting to antigen-presenting cells (APCs), but employs antigen-internalisation. The treatments described are exclusively for mammalians, specifically cats and dogs, and are aimed at the reduction of allergies. Antibody interference is not discussed.

Jauregui et al. (2017, Res. Vet. Sci., vol. 111 , p. 55 - 62) describe the targeting of AIV HA antigen to dendritic cells in chickens. Purified H5 HA antigen was chemically conjugated to a mouse monoclonal antibody directed against one domain of Dec-205. This conjugate was used to vaccinate chickens of 21 weeks of age. As all chickens employed were seronegative foranti-HA antibodies (see Jauregui, Figure 7, day 0), thus Jaurequi et al., do not describe or suggest overcoming antibody interference in seropositive avians.

It is therefore an object of the present invention to overcome one or more disadvantages in the prior art, by providing an effective way of overcoming the negative effect of antibody-interference on the vaccination of avian targets.

Surprisingly it was found that this object can be met, and consequently one or more disadvantages of the prior art can be overcome, by providing a method for the protection of avians that possess antibodies reactive with an antigen that is comprised in the vaccine to be administered, namely by targeting the antigen to APCs of the avian.

In the experiments as described hereinafter in detail, chickens with high or medium antibody levels received either targeted or untargeted vaccine. The results show a spectacular difference in the vaccination efficacy, in favour of the targeted antigen. On the contrary, untargeted vaccine and a classic control vaccine hardly produced any response in the seropositive avians. Consequently, this method of protecting seropositive avians was able to effectively overcome the negative effect of antibody- interference on vaccination, and is unexpectedly efficacious, even with only a single dose, and even in very young birds.

Consequently, the inventors were properly surprised to find that this antigen-targeting, particularly in the context of pre-existing antibodies, on the one hand worked well even in immune-immature avians, and on the other hand did not give rise to any vaccine-enhanced disease, or vaccine-induced immune- disturbance, such as by overstimulation of the immune-system, auto-immunity, or induction of tolerance. This method to protect an avian is equally applicable when not using the targeted antigen directly, but as a recombinant vector, for example a DNA plasmid, an RNA molecule, or a vector virus, that expresses the recombinant protein.

In addition, because this favourable effect is considered to be caused by the targeting and thus is independent of the antigen that is employed, it is perfectly conceivable that this method will also be equally successful using a different antigen. This method thus enables the protection against a variety of avian pathogens for which the vaccination normally suffers from antibody-interference, such as e.g. NDV, IBDV, AIV and others.

It is not known exactly how or why this method of vaccination can break-through high antibody levels and still induce such an effective protective immune response. Although the inventors do not want to be bound by any theory or model that might explain these findings, they speculate that it is the targeting of the antigen to the APC, which in some way reduces clearing of the antigen by the pre-existing antibodies against it.

The success of the use of antigen targeting to APCs for the vaccination of avians with pre-existing antibodies against the vaccine antigen, was in no way predictable from any prior publication. This mainly because the mechanism of how antibody-interference works (blocking, masking, crosslinking, neutralising etc. of the vaccine antigen) is still not well understood today. This is especially true for antibody- interference in avians as that is a poorly studied animal system.

Further, while the first studies on antigen targeting were described already in the 1980's, these were aimed at human cancer therapy. Later a more general use in (predominantly human-) vaccination was considered. This is reviewed by Keler et al. (2007, Oncogene, vol. 26, p. 3758 - 3767).

Also, in some instances (maternal) antibodies have been responsible for enhancing viral diseases by antibody-dependent enhancement, this is called: vaccine-enhanced disease. This effect has been observed for a variety of viruses such as Lentiviruses and Dengue virus (Huisman et al., 2009, Vaccine, vol. 27, p. 505 - 512), and most recently for SARS-CoV-2 (Lee et al., 2020, Nat. Microbiol., vol. 5, p. 1185 - 1191). It was therefore a genuine concern that targeted vaccination could evoke such unwanted effects upon subsequent contact with the corresponding pathogen.

In addition, a translation from the mammalian- to the avian situation is far from straightforward as there is only little information on the functioning of the avian immune system, as compared to that in mammals/humans. Also the general review of Shrestha et al. (supra) does not enable particular methods, or take away any of the hesitations a skilled person would have in employing antigen-targeting. This because it could be feared to give rise to a type of immune-disturbance, and/or require a matured immune system.

Combined, this lack of information, and potential for complications made the use of antigen targeting to APCs an unlikely option for the vaccination of avians that have high levels of circulating antibodies reactive with the antigen in the vaccine. In addition, the choice of this method of vaccination for young avians was particularly uncertain, as the immune system of an avian at hatch is not yet matured so that it was unpredictable whether its APCs did already display suitable target proteins at their surface, and were matured enough to be able to transfer the binding to such surface protein into a productive stimulation of the animals' immune-system.

Therefore in one aspect the invention relates to a recombinant protein comprising an antigen and a binding domain that is capable of binding to a cell surface protein on an avian antigen-presenting cell (APC), for use in a method to protect an avian that possesses antibodies reactive with said antigen, against a pathogen from which said antigen was derived.

A “recombinant protein” is a protein of which the amino acid sequence is man-made and artificial. For the invention the recombinant protein can be obtained via molecular cloning- and recombinant protein expression techniques. After expression the protein can be isolated from the expression system, processed and purified when desired, and can subsequently be formulated into a composition suitable for use in the method to protect of the invention. Alternatively, the recombinant protein can be expressed and delivered via a recombinant vector, e.g. a DNA plasmid, an RNA molecule, or a viral vector, as described below.

Such techniques are well-known in the art and are disclosed in great detail in standard text-books like Sambrook & Russell: “Molecular cloning: a laboratory manual” (2001 , Cold Spring Harbour Laboratory Press; ISBN: 0879695773); and: Ausubel et al., in: Current Protocols in Molecular Biology (J. Wiley and Sons Inc, NY, 2003, ISBN: 047150338X).

For the invention, the term “protein” incorporates similar terms such ‘peptides', ‘oligopeptides' and ‘polypeptides'.

The recombinant protein for use according to the invention is a fusion protein, composed of polypeptides from different origins, such as the antigen and a binding domain, both as defined for the invention, and optionally one or more peptides such as linkers, markers, etcetera, all connected in one amino acid chain.

The term “comprising” (as well as variations such as “comprises”, “comprise”, and “comprised”) as used herein, intends to refer to all elements, and in any possible combination conceivable for the invention, that are covered by or included in the text section, paragraph, claim, etc., in which this term is used, even if such elements or combinations are not explicitly recited; and not to the exclusion of any of such element(s) or combinations.

Therefore any such text section, paragraph, claim, etc., can therefore also relate to one or more embodiment(s) wherein the term “comprising” (or its variants) is replaced by terms such as “consisting of, “consists of, or “consist essentially of.

An “antigen” is commonly known as a molecule that can interact with elements of the immune system such as antibodies and lymphocytes, which interaction may give rise to a humoral- and/or cellular immune response.

The sections of the antigen that are recognised by the immune system are called ‘epitopes', which can be of linear or three-dimensional type. A 3D epitope is typically formed by the folding of a larger protein. A linear epitope needs to be of sufficient size e.g. at least 5 amino acids, either on its own or by being connected to a carrier molecule, e.g. by being comprised in a recombinant protein for use according to the invention.

The antigen is a polypeptide, thus: an antigenic polypeptide, contains at least one epitope, and is “derived” from a pathogen. For the invention ‘derived' refers to the way 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 encoding the recombinant protein for use according to the invention.

For the invention, the antigen selected may thus be the whole or a part of a protein from a pathogen, wherein the pathogen is selected from a virus, a bacterium, a parasite, and a fungus.

The antigen can be derived from the natural sequence of an antigen from a pathogen, or can be an assembly, for example: have an amino acid sequence that is a consensus from several homologues of the antigen to be expressed, such as e.g. the same type of protein but derived from a variant of the pathogen, such as a different species, serotype, subtype, strain, isolate, etcetera. As is well-known, to obtain such a consensus sequence, either amino acid- or encoding nucleotide sequences can be compared and a consensus sequence can be derived from that comparison; for example by aligning several H9 HA nucleotide sequences using an appropriate computer program.

The antigen for the invention can also be a chimeric antigen, consisting of assembled parts from different antigens, biologically related or not. Further the sequence encoding the antigen can be subjected to ‘codon optimisation', as is described below.

For the invention the antigen is selected from proteins that can generate a protective immune response against the pathogen from which the antigen was derived. For example, selected from: the VP2 protein from IBDV; the fusion (F)- or the hemagglutinin-neuraminidase (HN) protein of NDV; the spike protein from infectious bronchitis virus (IBV); and the HA- or the neuraminidase (NA) protein of AIV.

A “binding domain” for the invention is derived from the antigen-binding site of an immunoglobulin molecule and can be a part of an antibody comprising one or more of the complementarity-determining regions, for example can be a ‘single chain variable fragment' (scFv) polypeptide.

For the invention, the binding domain “is capable of binding”. This refers to a binding that is specific, i.e. with sufficient avidity, to differ from any non-specific- or background binding. The difference between specific- and non-specific binding is well-known to the skilled artisan and can readily be distinguished for example in an in vitro binding assay, by diluting-out either the binding domain or the ligand; any nonspecific binding is typically lost rapidly, e.g. at 1:10 or 1:100 dilution, while specific binding remains even with higher dilutions.

An “APC” is well known to be a cell of the lymphoid system that is capable of processing antigenic molecules and presenting (parts of) those molecules to the immune system of a human or animal. This presentation induces a cascade of reactions leading to the immune-maturation and -stimulation that is at the basis of a protective immune-response. APCs are e.g. B-lymphocytes, dendritic cells, macrophages, and natural killer cells. A “cell surface protein on an avian APC” is a protein that is attached to- or anchored in the external side of the cell-membrane of an APC. These proteins play a role in the APC's functions in detecting and signalling. Many of the cell-surface proteins on APCs are members of the immunoglobulin superfamily of proteins. Examples of APC surface protein are e.g. CD83 and CD11c proteins. The ‘CD' notation refers to ‘cluster of differentiation', which is an international protocol for the classification and identification of surface proteins on cells of the lymphoid system.

An “avian” for the invention is any animal of the taxonomic Class Aves that is of economic- or of (veterinary) medical relevance. For example: chicken, turkey, duck, goose, quail, guinea fowl, partridge, pheasant, pigeon, falcon, and ostrich.

The terms “for use in a method to protect an avian” refer to the medical use of the recombinant protein for use according to the invention and as defined herein. The use can be of the protein directly or can be of the protein indirectly via expression from a recombinant vector.

For the invention the “method” applied refers to vaccination.

The term “protect” refers to the effect of the method for the invention, namely to a protective immune response that is induced by the method, namely by vaccination. Such an immune response protects the vaccinated avian against infection and/or disease caused by the pathogen from which the antigen (present in the recombinant polypeptide for use according to the invention) was derived.

The method to protect regards a reduction, in whole or in part, of the establishment or the proliferation of a productive infection by the pathogen in cells and organs of a susceptible avian, or of the subsequent signs of disease. This is achieved for example by reducing the pathogen's load or shortening the duration of the pathogen's replication. In turn this leads to a reduction in the avian of the number, the intensity, or the severity of lesions and associated clinical signs of disease, that can be caused by the infection with the pathogen.

Such reduction of infection or disease can readily be detected, for instance by monitoring the immunological response following vaccination with the recombinant protein for use according to the invention, and by testing the appearance of clinical symptoms or mortality after a (challenge) infection of vaccinated avians, e.g. by monitoring the avian' s signs of disease, clinical scores, serological parameters, or by re-isolation of the infecting pathogen. These results can be compared to a response to a similar infection in mock-vaccinated avians. Several ways to assess infection and symptoms of disease for the main avian pathogens are well-known in the art.

The protection against infection or disease by the method for the invention, provides immunised avians with an improvement of health, welfare, and economic performance. This can for instance be assessed from parameters such as an increase of well-being, survival, growth rate, feed conversion, and production of eggs, as well as reduced costs for (veterinary) health care.

The avian to be protected by the method of the invention, “possesses antibodies”. This applies to the moment in time when the method of the invention is applied: the time of vaccination. Whether an avian indeed has such antibodies can readily be determined e.g. by taking a blood sample from the avian around the time of vaccination and determining the titre of the antibodies against the antigen using standard serological methods. This does however not require that the determination itself of the value of that pre-existing titre, i.e. the performance of the serological test on a serum sample taken around the time of vaccination and/or the analysis and interpretation of the results of that test, is done at that time. Similarly, this does not prevent that the pre-existing titre at the time of vaccination is calculated and extrapolated from the level determined in a sample taken some time before the vaccination.

For the invention an avian “possesses” antibodies against an antigen when the titre of the antibodies reactive with that antigen in serum from that avian is above a background level. Such a background level is typically the level as present in a comparable avian that is naive for the antigen or pathogen of interest. For the invention, this background level can conveniently be taken e.g. from the titre present in the serum of an SPF (specific pathogen free) avian of the same age and species.

The pre-existing antibodies can result from a passive transfer, as is typically the case with antibodies that were obtained from the mother via the egg-yolk. Such a seropositive avian would be called ‘MDA positive', or ‘MDA+'. This applies to avians of very young age, e.g. from day of hatch (i.e. 1 day old), to about 3 weeks of age. Alternatively, the pre-existing antibodies can result from an active immunisation that the avian to be protected received earlier, and which resulted in the generation of antibodies; this applies to avians from about 3 weeks of age.

The terms “reactive with”, or its synonym: ‘specific for', describe the capability of the pre-existing antibodies to interact with the antigen comprised in the recombinant polypeptide for use according to the invention, by a specific immune recognition. Similar terms are also ‘can bind to', ‘can recognise', etcetera, for as far as those refer to specific binding.

The unexpected advantageous effect of the present invention is prominent in the case that the pre-existing antibodies (in the avian to be protected) are reactive with the antigen that is comprised in the recombinant protein of the invention. In that situation an antibody-interference would normally occur which would reduce the efficacy of the protection.

The terms “a pathogen from which said antigen was derived” serve to indicate that the pathogen against which the method for the invention intends to protect, contains the antigen as defined above. This includes also homologues of the antigen and/or variants of the pathogen.

As the skilled person will understand, the match between the antigen in the recombinant protein for use according to the invention, and the pathogen against which the avian is to be protected, forms the basis for the protective immune response induced.

Details of embodiments and of further aspects of the invention will be described below.

In an embodiment of the recombinant protein for use according to the invention, the avian APC is selected from: a B-lymphocyte, a dendritic cell, a macrophage, and a natural killer cell. Each of these cell-types can clearly be distinguished using standard serological- and biochemical methods, for example using determination based on proteins with CD designation, as described below.

In a preferred embodiment of the recombinant protein for use according to the invention, the avian APC is a dendritic cell.

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

All these proteins are well-known in this field and are surface proteins on APCs: CD11c is a transmembrane protein on dendritic cells and some other APCs, which plays a role in the activation of neutrophils. A CD11 c-specific scFv comprises the amino acid sequence of SEQ ID NO: 18.

Dec-205 is an endocytic receptor on dendritic cells and lymphocytes. An example of a chicken Dec-205 is presented in GenBank accession number: AJ574899. A Dec-205-specific scFv comprises the amino acid sequence of SEQ ID NO: 19.

CD83 is a surface glycoprotein which belongs to the immunoglobulin superfamily. It is predominantly expressed on dendritic cells, and to a lesser extent also on lymphocytes and macrophages. It is a well-known marker for mature dendritic cells. An example of an avian CD83 is the protein presented in GenBank accession number XP_040519591.

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

In an embodiment of the recombinant protein for use according to the invention, the binding domain comprises the antigen binding site of an antibody.

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

As is well-known, an scFv is the smallest part of an immunoglobulin which retains one complete antigen binding domain but lacks the Fc part. An scFv is a single peptide which is itself a fusion construct, comprising one variable light chain (vL), a linker, and one variable heavy chain (vH). The order of these elements can be vL-linker-vH, or vH-linker-vL. In both cases the variable chains are oriented (relative to each other) as tail-to-head, whereby the c-terminal side is the tail.

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

The linker sequence of an scFv provides a flexible region so that the two variable chains can orient themselves to form an antigen binding domain. In a preferred embodiment the linker sequence of the scFv comprises Glycine, and Serine or Threonine amino acids, and is from 10 to 50 amino acids long. In a more preferred embodiment the linker sequence ofthe scFv comprises the amino acid sequence (Gly4-Ser)4, as presented in SEQ ID NO: 1.

The specificities of the two variable chains of an scFv can be both for the same- or each for a different antigen. In a preferred embodiment, the two variable chains have the same specificity. In an embodiment, the scFv is specific for CD83, in other words: is a CD83-scFv. Preferably, the scFv is specific for CD83 on an avian dendritic cell; more preferably the scFv comprises the amino acid sequence of SEQ ID NO: 2.

In embodiments of the binding domain, the scFv can be present two or more times.

In an embodiment of the recombinant protein for use according to the invention, the pathogen is pathogenic to avians. More preferably the pathogen is a virus. Even more preferably the virus is an RNA virus. Still more preferably the RNA virus is selected from: IBDV, NDV, IBV, and AIV. Still even more preferably the pathogen is selected from: IBDV, NDV, and AIV. Most preferably the pathogen is AIV.

In an 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. Still more preferably the antigen is selected from an AIV HA protein of H5, H7 or H9 type.

All these viral protein antigens are well-known in this field, and many versions of their encoding 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. ACP50708.1 ; NDV F: GenBank acc.nr. AAK55550.1 ; NDV HN: GenBank acc.nr. MH614933.1 ; IBDV VP2: GenBank acc.nr. KX827589.1 ; and IBV spike: GenBank acc.nr. AAA66578.1.

In addition, detailed information on HA proteins is available in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) at: www.rcsb.org, and in the Influenza Research Database at: www.fludb.org.

In an embodiment of the recombinant protein for use according to the invention wherein the antigen is selected from an AIV HA protein, the antigen contains only the ectodomain of the HA protein. This can prevent attachment to the cell-membrane of cells used for the expression of the recombinant protein for use according to the invention.

The ectodomain of a mature AIV HA protein comprises the N-terminal part -without the signal sequence- and the central part of the HA protein, thus comprising the HA1 and HA2 domains, but not the transmembrane- and cytoplasmic domains; typically these last two sections together form the C-terminal 35 - 40 amino acids of an HA.

In an embodiment of the recombinant protein for use according to the invention wherein the antigen is the ectodomain of an AIV HA protein of the H5, H7, or H9 type, the antigen comprises a protein having the amino acid sequence selected from: SEQ ID NO's: 3, 4, and 5.

In an embodiment of the recombinant protein for use according to the invention wherein the antigen is an ectodomain from an AIV HA protein, the antigen also comprises a trimerization domain.

Such a trimerization domain can compensate for the loss of the transmembrane- and cytoplasmic domains of HA, and restore the ability to form a homo-trimer and resemble its natural 3D shape. Further it improves the solubility and stability of the recombinant protein of the invention with an HA-ectodomain antigen.

For the invention a trimerization domain is a peptide and can be one of several known to be suitable for this function, for example: the isoleucine zipper 3 domain of the GCN4 transcriptional activator from Saccharomyces cerevisiae, or the Foldon domain of the bacteriophage T4 fibritin protein (‘Foldon').

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

In an embodiment of the recombinant protein for use according to the invention wherein the antigen is an ectodomain of an AIV HA protein and the antigen also comprises a trimerization domain, the trimerization domain is situated at the C-terminal side (downstream) of the HA ectodomain.

In a preferred embodiment the HA ectodomain and the trimerization domain are placed in the recombinant protein for use according to the invention, without an intervening amino acid.

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

In the recombinant protein for use according to the invention, the antigen and the binding domain can be placed in two orientations relative to each other, with either the antigen or the binding domain nearer to the N-terminal end of the recombinant protein for use according to the invention. In this regard, the trimerization domain that can be employed when the antigen is selected to be an HA ectodomain, is considered as part of the antigen.

In an embodiment of the recombinant protein for use according to the invention, the antigen is situated in said recombinant protein at the N-terminal side (upstream) of the binding domain.

In an alternate embodiment, the binding domain is situated in said recombinant protein at the N- terminal (upstream) side of the antigen.

In an embodiment, the recombinant protein for use according to the invention comprises a linker that is situated in-between the antigen and the binding domain, or in-between the binding domain and the antigen, depending on their mutual orientation. Preferably said linker is between 1 and 30 amino acids in size. More preferably the linker contains Glycine and Serine amino acids. Even more preferably said linker comprises the amino acid sequence of SEQ ID NO: 8.

Therefore, in an embodiment, the recombinant protein for use according to the invention comprises, one of the combinations selected from: an AIV H5 HA ectodomain, a trimerization domain, a linker, and a CD83-scFv; an AIV H7 HA ectodomain, a trimerization domain, a linker, and a CD83-scFv; and an AIV H9 HA ectodomain, a trimerization domain, a linker, and a CD83-scFv; wherein the indicated elements are presented in N- to C-terminal direction. In a preferred embodiment, the AIV HA ectodomain is selected from SEQ ID NO's: 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.

For the purposes of expressing, harvesting, quantifying, and (optionally) purifying the recombinant protein for use according to the invention, the recombinant protein may also comprise one or more peptides that function as a biochemical- or serological marker (or tag). The markers may be the same or different. The markers can be placed at different locations in the recombinant protein.

Well-known markers are: affinity tags such as a Maltose binding protein (MBP)- or Histidine (His)- tag; epitope tags such as Myc-, Ctag-, V5- or Flag-tag; or fluorescent protein tags such as a GFP or YFP, or a part thereof; all well-known in the art.

The marker can be used for detection and quantification purposes, e.g. for detection or binding with specific antibodies, e.g. in an IFT or an ELISA. Purification can be done e.g. using immune- or metal affinity chromatography.

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

A “Ctag”, comprises SEQ ID NO: 9, and is the C-terminus of a-synuclein protein, which is known to cause aggregates found in neurological disorders such Parkinson's disease. When used, the Ctag is preferably comprised in the C-terminus of a recombinant protein for the invention. Ctag purification by immuno-affinity chromatography is sometimes more effective than His-tag purification, e.g. in case there is disturbance from protein in the culture of the expression system.

A V5 tag is derived from Simian virus 5. Preferably the V5 tag comprises the amino acid sequence of SEQ ID NO: 10.

In an 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 2 or more from a Ctag, a His tag and a V5 tag.

For the expression of the recombinant protein for use according to the invention some further adaptations can be made when desired. Such fine-tuning or optimisation is routine and is well-known to the skilled artisan. For example, depending on how the protein is to be expressed by host cells of an expression system: inside the cells, on their surface, or secreted to their exterior. In the last two cases a signal sequence can be provided at the N-terminal side, which signal functions well in the cells of the expression system to be used. An example is the use the ‘Drosophila melanogaster immunoglobulin heavy chain binding protein' (BIP) signal sequence, to enable secretion when expressing in S2 cells.

In an embodiment the recombinant protein for use according to the invention comprises a signal sequence; preferably the signal sequence is a BIP signal sequence; more preferably the BIP signal sequence comprises the amino acid sequence of SEQ ID NO: 11.

In the course of the construction process of the nucleic acid that is to provide express of the recombinant protein for use according to the invention, one or more restriction enzyme (RE) sites may be used. When those RE sites are located in the coding region of the recombinant protein, their remaining nucleotides will translate into a few amino acids which are then located in-between some of the elements that make up the recombinant protein for use according to the invention.

For example, one construct used for the invention, employed RE sites Kpnl and Pad to subclone the H9 HA ectodomain-Foldon element, and used RE sites Notl and Xbal to subclone the CD83-scFv at the C-terminal side of the HA antigen-Foldon and the linker of SEQ ID NO: 8.

As a result one version of a recombinant protein for use according to the invention comprises the amino acid sequence of SEQ ID NO: 12, the details of which are described in Table 1.

Table 1 : Composition of SEQ ID NO: 12

A control construct, without the linker and the CD83-scFv was prepared. This construct lacked the region of SEQ ID NO: 12 from aa 545 - 802, and comprised the amino acid sequence of SEQ ID NO: 13.

Similar constructs to SEQ ID NOs: 12 and 13 can readily be made using one of the other HA antigen sequences: a H5 HA- or H7 HA ectodomain, for example as presented in SEQ ID NOs: 4 and 5 respectively.

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

For the invention, it can readily be established whether the pre-existing antibodies are maternally derived or not: practically, only chicks of less than 2 - 4 weeks of age will have MDAs. Also, MDAs consists mainly of IgY, which is a functional homolog of mammalian IgG, but differs structurally: IgY has 4 heavy chain constant domains, compared to three in IgG. In an embodiment of the recombinant protein for use according to the invention, the avian to be protected is a poultry. More preferably the poultry is selected from: chicken, turkey, duck, and goose. Even more preferred, the poultry is a chicken.

For the invention, the avian may be of any type, breed, or variety, such as: layers, breeders, broilers, combination breeds, or parental lines of any of such breeds. Preferred poultry types are selected from: broiler, breeder, and layer. More preferred are broiler- and layer type poultry. Most preferred are broiler poultry.

As described, the present invention provides a recombinant protein for use in a method to protect seropositive avians against pathogens. This method can advantageously be applied either in older birds where the pre-existing antibodies are the result of a prior active vaccination, or in young birds where the pre-existing antibodies are MDA.

Therefore, in an embodiment of the recombinant protein for use according to the invention, the avian to be protected is less than 4 weeks of age; preferably less than 3 weeks of age; more preferably less than 2 week of age; even more preferably less than 1 week of age, still even more preferably is 1 day old (i.e. at day of hatch). In an embodiment the avian to be protected is at about 18 days of embryonic development (i.e. in ovo).

In an alternate embodiment of the recombinant protein for use according to the invention, the avian to be protected is 2 weeks or more of age.

As described, the recombinant protein for use according to the invention can equally well be applied by an indirect use, namely by expressing the recombinant protein from a recombinant vector, e.g. a DNA plasmid, an RNA molecule, or a viral vector.

Therefore in a further aspect the invention relates to a recombinant vector capable of expressing the recombinant protein for use according to the invention, for use in a method to protect an avian that possesses antibodies reactive with an antigen that is comprised in the recombinant protein expressed by said recombinant vector, against a pathogen from which said antigen was derived.

A “vector” is well-known in the field of the invention as a molecular structure that carries genetic information (a nucleic acid sequence) for encoding a polypeptide, with appropriate signals to allow its expression under suitable conditions, such as in a host cell. For the invention ‘expression' regards the well-known principle of the expression of protein from genetic information by way of transcription and/or translation.

Many types and variants of such a vector are known and can be used for the invention, ranging from nucleic acid molecules like DNA or RNA, to more complex structures such as virus-like particles and replicon particles, up to replicating recombinant micro-organisms such as a virus. The recombinant vector for use according to the invention is a “recombinant”, as it has a molecular makeup that was changed by manipulation in vitro of its genetic information. The changes made can serve to provide for, to improve, orto adapt the replication, expression, manipulation, purification, stability and/or the immunological behaviour of the vector and/or of the protein it expresses. These, and other techniques are explained in great detail in standard text-books like Sambrook & Russell, and Ausubel et al., both supra, and: C. Dieffenbach & G. Dveksler: “PCR primers: a laboratory manual” (CSHL Press, ISBN 0879696540); and “PCR protocols”, by: J. Bartlett and D. Stirling (Humana press, ISBN: 0896036421).

Depending on the type of vector employed more or less signals need to be provided for the replication and expression, either in cis (i.e. provided within the recombinant vector itself) or in trans (i.e. provided from a separate source), this is all well-known.

The skilled person is well equipped to select and combine the required signals into operational combinations to make the recombinant vector for use according to the invention “capable of expressing” the recombinant protein for use according to the invention under appropriate conditions. Next to elements to assist with the 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, stop codon, termination signal, polyadenylation signal, 7-methylguanosine (7mG) cap structure, and an intron with functional splice donor- and -acceptor sites.

In embodiments of the recombinant vector for use according to the invention, the features of the recombinant protein, the use, the method, the protection, the avian, the antibodies, the antigen, and the pathogen, are all as embodied herein.

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

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

Therefore, in an embodiment of the recombinant vector for use according to the invention, the vector comprises the nucleotide sequence of SEQ ID NO: 14.

The control protein of SEQ ID NO: 13 is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 15.

Both SEQ ID NOs: 14 and 15 have been codon optimised towards the codon-usage table of D. melanogaster S2 cells, to optimise the expression in these cells. Details as described hereinbelow.

As described, the recombinant vector for use according to the invention can have several different forms.

Therefore in an embodiment, the recombinant vector for use according to the invention is selected from a nucleic acid, a virus, and a replicon particle (RP). For the invention, the nucleic acid can be a DNA or an RNA, can be single or double stranded, and can be natural or synthetic in origin.

In an embodiment of the recombinant vector for use according to the invention wherein the vector is a nucleic acid, the nucleic acid is a eukaryotic expression plasmid.

A “eukaryotic expression plasmid”, usually of DNA, has the appropriate signals for expression of a heterologous gene that is inserted into the plasmid, under the operational control of a promoter that is active in a eukaryotic cell. The plasmid can then be inserted into a eukaryotic host cell or host organism by some method of transfection, e.g. using a biochemical substance as carrier, by mechanical means, or by electroporation, and will provide for the expression of the heterologous gene insert. Typically such expression will be transient, as the plasmid lacks signals for stable integration into the genome of a host cell; consequently such a plasmid will typically not transform or immortalise the host or the host cell. All these materials and procedures are well known in the art and are described in handbooks.

Such eukaryotic expression plasmids are commercially available from a variety of suppliers, for example the plasmid series: pcDNA™, pCR3.1 ™, pCMV™, pFRT™, pVAX1 ™, pCI™, Nanoplasmid™, pCAGGS etc..

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).

A eukaryotic expression plasmid can comprise 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 when intended for administration to a human or animal target, such antibiotic selection is not desired for fear of generating antibiotic resistance.

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

The recombinant vector for use according to the invention, in the form of a eukaryotic expression plasmid, can be delivered to a host cell or target organisms, where it will express the HA stem polypeptide for the invention in the host cell. Delivery of the expression plasmid can be in several ways, e.g. by mechanical or chemical means, as naked DNA, or encapsulated with an appropriate (nanoparticulate) carrier, such as a protein, polysaccharide, lipid or a polymer. Well-known examples of nucleic acid carriers are dendrimers, lipid nanoparticles, cationic polymers and protamine.

A special form of the recombinant vector for use according to the invention, as a eukaryotic expression plasmid, is when the plasmid provides for the delivery of replicon RNA.

Therefore in an embodiment of the recombinant vector for use according to the invention, wherein the vector is a nucleic acid, and the nucleic acid is a eukaryotic expression plasmid, the plasmid encodes a replicon RNA. A “replicon RNA”, is a self-replicating RNA which contains, in addition to the nucleic acid encoding the recombinant polypeptide for the invention, elements necessary for RNA replication, such as a replicase gene. However, unlike a replicon particle (RP), a replicon RNA is not packaged by viral structural proteins and is thus less efficient at entering host cells on its own.

The replicon RNA-encoding plasmid can be delivered to a host cell in the same way as a protein- expressing plasmid.

Vaccination with a eukaryotic expression plasmid encoding replicon RNA provides an advantage over vaccination with a eukaryotic expression plasmid expressing protein, because the replicon RNA provides for an amplification step: the translation of replicase makes the replicon RNA produce sub genomic messenger RNA encoding the recombinant protein for use according to the invention. This results in the expression of high amounts of the recombinant protein in the host cell, respectively in the target avian.

In a preferred embodiment of the recombinant vector for use according to the 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.

An example of a eukaryotic expression plasmid encoding a VEEV replicon RNA is e.g. a pVAX plasmid (Thermo Fisher Scientific), comprising VEEV non-structural protein genes 1 - 4, driven by a eukaryotic promoter such as a human CMV immediate early gene 1 promoter.

In an alternate embodiment of the recombinant vector for use according to the invention wherein the vector is a nucleic acid, the nucleic acid is an RNA molecule.

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

A recombinant vector for use according to the invention, as an RNA molecule can be delivered to the avian or to a host cell in different ways, e.g. by mechanical or chemical means, or encapsulated with an appropriate (nanoparticulate) carrier, such as a protein, polysaccharide, lipid or a polymer, as described herein. To stabilise the RNA nucleotide-analogues can be incorporated, or certain chemical modifications may be applied, e.g. to the nucleotides or to their backbone.

In an embodiment of the recombinant vector for use according to the invention wherein the vector is a nucleic acid and the nucleic acid is an RNA molecule, the RNA molecule is an mRNA.

An “mRNA” (messenger RNA) is well-known in the art, and typically has a 5' 7-methylGuanosine (7mG) cap and a 3' poly-A tail. An mRNA can be delivered to a eukaryotic host organism or host cell by way of transfection and/or by using an appropriate carrier, e.g. a polymer or a cationic lipid. In an embodiment of the recombinant vector for use according to the invention wherein the vector is a nucleic acid and the nucleic acid is an RNA molecule, the RNA molecule is a replicon RNA.

The replicon RNA can be produced in vitro e.g. using a pVAX plasmid as described herein, and then be administered to a host cell or a target organism, using any suitable method.

Recombinant vectors for the expression and delivery of a heterologous protein in the form of replicating recombinant virus vectors, are well-known in the art. These provide for an efficient method of vaccination, as the viral vector replicates and amplifies in the target avian. Assembly and modification of a recombinant vector virus is routine and can be done using standard molecular biological techniques.

Therefore in an embodiment of the recombinant vector for use according to the invention, the recombinant vector is a virus.

For the invention, the viral vector is a virus that replicates in an avian. Many different virus species have been used overtime as recombinant vector for avians.

In an embodiment of the recombinant vector for use according to the invention wherein the vector is a virus, the virus is selected from a Herpesvirus, a Poxvirus, a Paramyxovirus, and an Adenovirus.

Examples of suitable vector viruses that can be used as vector for avians are well known in the art and are e.g. of a Herpesvirus: a Herpesvirus of Turkeys (HVT), or a Marek's disease virus (MDV) of serotype 1 or 2; of a poxvirus: fowl poxvirus; of a Paramyxovirus: NDV; and of an Adenovirus: fowl Adenovirus.

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

Examples of recombinant viral vectors expressing and delivering an Influenza HA gene are described: for HVT as vector in WO 2012/052384 and EP19218804.3. For NDV as vector, an example is described in: WO 2007/106882.

For the construction of a recombinant viral vector, typically an expression cassette is inserted into a locus in the vector's genome. Different techniques are available to control the locus and the orientation of that insertion. 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, e.g. by using overlapping Cosmids as described in US 5,961,982. Alternatively the integration may be done by using the CRISPR/Cas technology.

An ‘expression cassette' is a nucleic acid fragment comprising at least one heterologous gene and one promoter to drive the transcription of that gene, to enable the expression of the encoded protein. The termination of the transcription may be provided by sequences provided by the genomic insertion site of the cassette, or the expression cassette can itself comprise a termination signal, such as a transcription terminator. In such a cassette, both the promoter and the terminator need to be in close proximity to the gene of which they regulate the expression; this is termed being ‘operatively linked', whereby no significant other sequences are present between them that would intervene with an effective start-, respectively termination of the transcription. As will be apparent to a skilled person, an expression cassette is a self-contained expression module, therefore the orientation of its reading direction relative to the vector virus genome is generally not critical.

Other than the use of a virus as a vector for use according to the invention, the recombinant vector for use according to the invention can also be delivered and expressed to an avian by way of a macro- molecular structure that resembles a virion. Examples are virus-like particle (VLPs), or replicon particles (RPs). Known as ‘single cycle' infectious particles, these structures contain features necessary to infect a host cell, and express the heterologous gene it carries, however, they will typically not be capable of full viral replication, for lack of (relevant parts of) the viral genome from which they were constructed. This serves as a built-in safety feature,

“RPs” are well-known, and several RPs have been developed as a platform for the expression and delivery of a variety of proteins. Favourable basis for an RP is an Alphavirus, because of its broad host-range and rapid replication. Of course appropriate safety measures need to be taken to attenuate and control the infection of such RPs, as some Alphaviruses are highly pathogenic in their wildtype form. For a review, 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.).

Therefore in an embodiment of the recombinant vector for use according to the invention, the vector is an RP. Preferably the RP is an Alphavirus RP. More preferably the Alphavirus RP is a VEEV RP.

Preferred Alphavirus RPs are based on VEEV, which have been applied as recombinant vector vaccine for human, swine, poultry, and fish. Methods and tools to construct, test, and use VEEV-based Alphavirus RPs are well-known and available, see for example: Pushko et al. (1997, Virology, vol. 239, p. 389-401), and: WO 2019/110481. Preferred VEEV RP technology is the SirraVax ^sm RNA Particle technology (Harris vaccine).

The RNA for an RP can conveniently be produced in vitro: a DNA plasmid is used to translate a gene into RNA, which is harvested and transfected into a host cell together with helper RNA encoding in trans the VEEV structural proteins.

As described, the recombinant vector for use according to the invention can advantageously be used to deliver and express the recombinant protein for use according to the invention to an avian, e.g. as a way to vaccinate that target. This involves at some stage the administration of that vector to an avian, for example in the case the vector is a nucleic acid such as a DNA expression plasmid or an RNA molecule.

Also, the vector may be introduced into a host cell in vitro, for the amplification of the vector and/or the expression of the recombinant protein, after which the host cell (with the vector and/or the protein) is administered to the avian; for example in the case the vector is a viral vector, e.g. an HVT. Still further, the vector may be introduced into a cell of a recombinant expression system for expression of the recombinant protein, and the protein be harvested from that cell culture, and used to vaccinate an avian as described above. Also, the host cell itself, infected or transfected with the recombinant vector for use according to the invention and containing and/or expressing the recombinant protein for use according to the invention, can be used for the method to protect for the invention, e.g. as the infected or transfected host cell may itself be used for the vaccination of an avian.

Depending on the type of vector applied, that introduction into a host cell may require a carrier, some method of transfection, or may be guided by the vector itself, as described herein.

Therefore in a further aspect the invention regards a host cell kept in vitro, said host cell comprising the recombinant protein for use according to the invention and/or the recombinant vector for use according to the invention.

A “host cell” for the invention, is a cell that allows the expression of the recombinant protein for use according to the invention, and/or allows the replication of the recombinant vector for use according to the invention.

A host cell for the invention can be a primary cell kept in vitro, and can be e.g. in a suspension, in a monolayer, or in a tissue.

Alternatively, the host cell can be an immortalised cell kept in vitro, for example a cell from an established cell-line, which can grow and divide almost indefinitely. Depending on the type of the host cell, the expression of the HA stem polypeptide for the invention will include more or less extensive post- translational processing, such as e.g. signal peptide cleavage, disulphide bond formation, glycosylation, and/or lipid modification.

The primary- and the immortalised host cell can be of the same- or from a different species. Also one or both can be of the same or of a different species as the avian that is the subject of the method to protect for the invention.

Much used host cells are fibroblasts and lymphocytes. In case of the use of HVT as recombinant vector virus for the invention, the host cells are preferably primary chicken embryo fibroblasts (CEF's), which can be used and stored as described, see e.g. WO 2019/121888.

In an embodiment of the host cell for the invention, said host cell is preferably an immortalised avian cell. Several immortalised avian cell-lines have been described, for example in WO 97/044443 and WO 98/006824; more preferably the immortalised avian host cell for the invention is an immortalised CEF; even more preferably an immortalised CEF as disclosed in WO 2016/087560.

In an embodiment of the host cell for the invention, said host cell is preferably a cell of a recombinant expression system. Examples of cells from expression systems are e.g. cells from bacteria, yeasts, insects, avians, or mammalians

Cells from bacterial expression systems are e.g. cells from the genera Escherichia, Bacillus, Salmonella, Caulobacter, or Lactobacillus. Cells from yeast expression systems are e.g. cells from Saccharomyces cerevisiae or Pichia pastores.

Cells from insect cell expression systems are e.g. cells from Drosophila melanogaster, e.g. Schneider 2 (S2) cells, or cells for use in the Baculovirus-insect cell expression system: from Spodoptera frugiperda, e.g. Sf21 or Sf9 cells; or from Trichoplusia ni, e.g. High Five™ cells.

Cells from mammalian expression system are e.g. cells from hamsters, e.g. Chinese hamster ovary (CHO) cells.

All these cell-lines and their corresponding use in a recombinant expression system are well known in the art and can be employed using routine techniques and materials.

In an embodiment of the recombinant vector for use according to the invention, the nucleic acid that encodes the recombinant protein for use according to the invention, is codon optimised.

Codon optimisation is well-known and is applied to improve the expression level of a gene in an expression system, which is typically a context that differs from that of the origin of the gene. The optimization involves the adaptation of the nucleotide sequence to encode the intended amino acids, but in a way that corresponds to the codon preference (the tRNA repertoire) of the recombinant vector, of the host cell, or of the target organism in which the sequence will be expressed. Consequently, the nucleotide mutations applied are silent.

Therefore in an 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 optimised towards the avian organism that is intended to be protected by the method to protect for the invention. Preferably the codon optimisation is towards a poultry. More preferably the codon optimisation is towards a poultry selected from: a chicken, a turkey, a duck, and a goose.

In an 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 optimised towards a cell of a recombinant expression system, preferably towards a cell from a bacterium, yeast, insect, avian, or mammalian. More preferably the nucleic acid is optimised towards an insect cell; even more preferably towards a Drosophila Schneider 2 (S2) cell.

The recombinant protein for use and the recombinant vector for use, both according to the invention, can also be characterised by other wording to suit specific jurisdictions.

Therefore, in a further aspect the invention regards the use of the recombinant protein for use according to the invention, or of the recombinant vector for use according to the invention, for the manufacture of a vaccine to protect an avian against a pathogen, whereby the antigen that is comprised in said recombinant protein or that is comprised in the recombinant protein expressed by said recombinant vector, was derived from said pathogen, characterised in that said avian possess antibodies reactive with said antigen.

In embodiments of the recombinant protein, or of the recombinant vector, both for the manufacture of a vaccine according to the invention, the features of the recombinant protein, the recombinant vector, the protection, the avian, the pathogen, the antigen, and the antibodies, are all as embodied herein.

A “vaccine” is well-known to be a composition comprising at least one compound that can induce a protective immunological effect, in a pharmaceutically acceptable carrier. The ‘immunologically active compound' for the present invention is the recombinant protein for use, or the recombinant vector for use, both according to the invention.

The manufacture of a vaccine for the invention can be done using routine methods and procedures all well known in the art. General techniques and considerations that apply to the manufacture of vaccines under well-known standards for pharmaceutical production are described for instance in governmental directives and regulations (Pharmacopoeia, 9CFR) and in well-known handbooks like “Veterinary vaccinology” and: “Remington” (both supra). Commonly such vaccines are prepared sterile and are prepared using excipients of pharmaceutical quality grade.

Such a manufacture will incorporate microbiological tests for sterility, and absence of extraneous agents, and may include studies in vivo or in vitro for confirming efficacy and safety. After completion of the testing for quality, quantity, sterility, safety and efficacy, the vaccine can be released for sale. All these are well-known to a skilled person.

For example when the recombinant protein for use according to the invention is produced by way of a recombinant expression system, the protein can be harvested from the expression system culture, e.g. as a whole culture. Alternatively the harvest can be as a part of such culture, e.g. the supernatant or the cell- pellet after centrifugation of the cell culture, or a filtrate or retentate after filtration. The supernatant can be obtained after gravity settling of the culture, e.g. by standing overnight or by centrifugation; the filtrate is what passes through the filter upon filtration.

As described, the recombinant protein, and the recombinant vector, both for use according to the invention, achieve their advantageous effect in protecting an avian, through a vaccine comprising said recombinant protein and/or said recombinant vector.

Therefore, in a further aspect the invention regards a vaccine comprising the recombinant protein for use according to the invention, or comprising the recombinant vector for use according to the invention, and a pharmaceutically acceptable carrier, for use in a method to protect an avian that possess antibodies reactive with the antigen that is comprised in said recombinant protein or that is comprised in the recombinant protein expressed by said recombinant vector, against a pathogen from which said antigen was derived. In embodiment of the vaccine for use according to the invention, the features of the recombinant protein, the recombinant vector, the use, the method, the protection, the avian, the antibodies, the antigen, and the pathogen, are all as embodied herein.

A “pharmaceutically acceptable carrier” is well-known to aid in the stabilisation and the administration of a vaccine, while being relatively harmless and well-tolerated by the vaccinee. Such a carrier can for instance be water or a physiological salt solution. In a more complex form the carrier can e.g. be a buffer, which can comprise further additives, such as a stabiliser or a preservant. Details and examples are for instance described in well-known handbooks such as: “Remington: the science and practice of pharmacy” (2000, Lippincott, USA, ISBN: 683306472), and: “Veterinary vaccinology” (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681).

When the vaccine according to the invention comprises a recombinant vector that is a replicating virus, then the pharmaceutically acceptable carrier is preferably a composition stabilising that virus, or the host cell in which that virus is contained. Examples are several viral vaccine diluents, and stabilisers for frozen or freeze-dried storage, typically comprising e.g. a sugar, an amino acid, a physiological buffer (e.g. saline, PBS, or 50 mM HEPES), and often a bulky compound such as an albumin, a polymer etc. For example, when the vaccine comprises a recombinant HVT vector, such a vaccine is typically marketed as a cell-associated product. In that case the pharmaceutically acceptable carrier is preferably a mixture of culture medium, about 10 % serum, and about 6% DMSO. This carrier also provides for the stabilisation of the HVT-infected host cells during freezing and frozen storage. The serum can be any serum routinely used for cell culturing such as foetal- or new-born calf serum.

When the vaccine according to the invention comprises a recombinant vector for use according to the invention that is a nucleic acid or an RP, the pharmaceutically acceptable carrier can be a simple buffer, e.g. a phosphate buffer with 5 % w/v sucrose.

Further, an additional carrier can be added to stabilise and/or deliver the recombinant vector for a use in the invention, e.g. to encapsulate the recombinant vector according to the invention that is a nucleic acid or an RP with an appropriate (nanoparticulate) carrier, such as a protein, polysaccharide, lipid or a polymer. Preferably the additional carrier for a recombinant vector according to the invention that is an RP, comprises a nanogel that is a biodegradable polyacrylic polymer as described in WO 2012/165953.

Evidently, the recombinant vector or the in vitro host cell comprising such a vector, both for the invention, can be employed herein alive (i.e. replicative), or dead (non-replicative, or inactivated). In turn, only a part of the recombinant vector or the host cell, both for the invention, can be used for example as a: pellet, supernatant, concentrate, dialysate, extract, sonicate, lysate or as a fraction of a composition, e.g. a culture, comprising the vector and/or the host cell. All this is well-known to the skilled person.

When the vaccine for use according to the invention comprises the recombinant protein for use according to the invention, the vaccine can comprise an adjuvant to stimulate the immune response induced. Therefore, in an embodiment, the vaccine for use according to the invention comprises an adjuvant.

An “adjuvant” is a well-known vaccine ingredient that stimulates the immune response of a target in a non-specific manner. 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 sulphate, Carbopol™, pyran, Saponin, such as: Quil A™, or Q-vac™. Saponin and vaccine components may be combined in an ISCOM™. Furthermore, peptides such as muramyl dipeptides, dimethylglycine, and tuftsin. Also, aluminium salts, such as aluminium-phosphate or an aluminium-hydroxide which is available for example as: Alhydrogel™ (Brenntag Biosector), Rehydragel™ (Reheis), and Rehsorptar™ (Armour Pharmaceutical).

A much-used adjuvant is an oil, e.g. a mineral oil such as a light (white) mineral (paraffin) oil; or a non-mineral oil such as: squalene; squalane; vegetable oils or derivatives thereof, e.g. ethyl-oleate. Also combination products such as ISA™ (Seppic), or DiluvacForte™ and Xsolve™ (both MSD Animal Health) can advantageously be used.

A handbook on adjuvants and their uses and effects is: “Vaccine adjuvants” (Methods in molecular medicine, vol. 42, D. O'Hagan ed., 2000, Humana press, NJ, ISBN: 0896037355).

The adjuvant can be comprised in the vaccine for use according to the invention, in several ways. When the adjuvant comprises an oil, the vaccine can be provided in aqueous form, and can be formulated as an emulsion with the oil, in different ways: as a water-in-oil (W/O), an oil-in-water (O/W), or as a double emulsion, either W/O/W or O/W/O.

An “emulsion” is a mixture of at least two immiscible liquids, whereby one is dispersed in another. Typically the droplets of the dispersed phase are very small, in the range of micrometres or less. Procedures and equipment for the preparation of an emulsion at any scale are well-known in the art. To stabilise an emulsion, one or more emulsifiers can be used.

An “emulsifier” is a molecule with amphiphilic properties, having both a hydrophobic- and a hydrophilic side. Many emulsifiers are known in the art with their various properties. Most are readily available commercially, and in several degrees of purity. Common emulsifiers for vaccines are sorbitan monooleate (Span® 80) and polyoxyethylene-sorbitan-monooleate (polysorbate 80, or Tween® 80).

A well-known way to characterise the properties of (mixtures of) emulsifiers is the HLB number (hydrophile-lipophile balance; Griffin, 1949, J. Soc. Cosm. Chem., vol. 1, p. 311-326). Typically an emulsifier or emulsifier mixture with HLB number below 10 favours W/O emulsions, while an emulsifier (mixture) with HLB number of 10-16 will favour O/W emulsions.

Also an emulsion-stabiliser can be added; examples are benzyl alcohol, and triethanolamine.

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

Examples of light liquid paraffin oils are: Drakeol® 6VR (Penreco), Marcol® 52 (Exxon Mobile), and Klearol® (Sonneborn). In a preferred embodiment of the vaccine for use according to the invention, wherein the vaccine comprises an adjuvant, and the adjuvant comprises an oil, the vaccine is formulated as a water-in-oil emulsion.

In other wordings and for specific jurisdictions, further aspects of the invention can be defined as follows:

In a further aspect the invention regards the use of the recombinant protein for use according to the invention, or of the recombinant vector for use according to the invention, or of the vaccine for use according to the invention, to protect an avian against a pathogen, whereby the antigen that is comprised in said recombinant protein or that is comprised in the recombinant protein expressed by said recombinant vector, was derived from said pathogen, characterised in that said avian possess antibodies reactive with said antigen.

In an embodiment of the use according to the invention, the use comprises the administration to an avian of the recombinant protein, the recombinant vector, or the vaccine, all for the invention.

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

In a further aspect the invention regards a method for protecting an avian against a pathogen, the method comprising the step of administering to said avian the vaccine for use according to the invention, whereby the antigen that is comprised in said vaccine was derived from said pathogen, and whereby said avian possess antibodies reactive with said antigen.

A vaccine for use according to the invention is typically prepared in a form that is suitable for administration to an avian, and that matches with a desired route of application, and with the desired effect.

Depending on the route of application of the vaccine for use according to the invention, it may be necessary to adapt the vaccine's composition. This is well within the capabilities of a skilled person, and generally involves the fine-tuning of the efficacy or the safety of the vaccine. This can be done by adapting the vaccine dose, quantity, frequency, route, by using the vaccine in another form or formulation, or by adapting one of the excipients of the vaccine (e.g. a stabiliser or an adjuvant).

The vaccine according to the invention in principle can be given to an avian by different routes of administration, and at different points in their lifetime; specifically the vaccine can be administered to an avian of any age that possess antibodies reactive with the antigen in the recombinant protein for use according to the invention. When the administration is to be administered as early as possible, it can be administered at the day of hatch (“day one”), or even in ovo, e.g. at about 18 days of embryonic development, all well-known in the art.

Equipment for automated injection of a vaccine into a fertilized egg at industrial scale, is available commercially. This provides the earliest possible protection, while minimising labour costs. Different in ovo inoculation routes are known, such as into the yolk sac, the embryo, or the allantoic fluid cavity; these can be optimised routinely, when required.

The vaccine for use according to the invention can be formulated as an injectable liquid, suitable for injection, either in ovo, or parenteral.

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

In an embodiment, the vaccine for use according to the invention is administered by parenteral route. Preferably the parenteral route is by intramuscular- or subcutaneous route.

The exact amount of the recombinant protein or of the recombinant vector, both for the invention, is not critical and can readily be established by comparing the protective effects of different amounts.

Also, when the vaccine for use according to the invention comprises a viral vector, this can replicate in the vaccinated avian and only needs to be administered in an amount that is enough to establish a productive infection in the avian.

For example, when the viral vector for use according to the invention is a recombinant HVT, a suitable inoculum dose is between 1x10 ^Λ 1 and 1x10 ^Λ 5 plaque forming units (pfu) of the HVT for the invention per animal dose; preferably between 1x10 ^Λ 2 and 1x10 ^Λ 4 pfu/dose, even more preferably between 500 and 5000 pfu/dose; most preferably between about 1000 and about 3000 pfu/dose.

Methods to count viral particles of the HVT for the invention are well-known.

When the HVT vector for use according to the invention is cell-associated, these amounts of the HVT are comprised in infected host cells.

The volume per animal dose of the vaccine for use according to the invention can be optimised according to the intended route of application: in ovo inoculation is commonly given in a volume of 0.01 to 0.5 ml/egg, and parenteral injection in an avian is commonly given in a volume of 0.1 to 1 ml/bird.

Determination of what is an immunologically effective amount of the vaccine according to the invention, or the optimisation of the vaccine's volume per animal dose, are both well within the capabilities of the skilled artisan.

The dosing regimen for applying the vaccine for use according to the invention to an avian can be in single or multiple doses, in a manner compatible with the formulation of the vaccine, and in such an amount as will be immunologically effective.

Preferably, the regimen for the administration of a vaccine for use according to the invention is integrated into existing vaccination schedules of other vaccines that the target avian may require, in order to reduce stress to the animals and to reduce labour costs. These other vaccines can be administered in a simultaneous, concurrent or sequential fashion, in a manner compatible with their registered use.

SEQUENCE LISTING

<110> Intervet International BV <120> Overcoming antibody-interference in avians <130> 25260

<160> 21

<170> Patentln version 3.5 210 1 211 20 <212> PRT

<213> Artificial Sequence 220

<223> Linkerfor scFv constructs <400> 1

Gly Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15

Gly Gly Gly Ser 20 210 2 <211> 248

<212> PRT

<213> Artificial Sequence 220

<223> CD83-scFv

<400> 2

Asp lle ValMet ThrGlnSerPro Ser Ser LeuAla Val SerVal Gly 1 5 10 15 Gln LysValThr Met SerCys Thr Ser Ser GlnVal Leu Leu His Ser 20 25 30

Pro Asn Gln LysAsn Tyr Leu Ala Trp Tyr GlnGln Lys Pro Gly Gln 35 40 45

Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser ThrArg Glu Ser Gly Val 50 55 60 Pro AspArg Phe ThrGly Ser Gly Ser Gly ThrAsp Phe Thr Leu Thr 65 70 75 80 lle Ser SerValGln Ala Glu Asp LeuAla ValTyr Tyr Cys Gln Gln 85 90 95

His Tyr SerThr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110

LysGly Gly Gly Gly Ser Gly Gly Gly Gly SerGly Gly Gly Gly Ser 115 120 125

Gly Gly Gly Gly SerGlu Val Gln Leu Gln Gln SerGly Pro Glu Leu 130 135 140

Val Lys Pro Gly Ala Ser Val Lys lle Ser Cys LysAla Ser Gly Tyr 145 150 155 160

Thr Phe ThrAsp TyrTyr IleAsn TrpVal LysGln Ser His Gly Lys 165 170 175

Ser Leu Glu Trp lle Gly Asp He Asn Pro ThrAsn Gly Asp Ser Thr 180 185 190

Tyr SerGln Lys Phe Lys Gly LysAla Thr Leu ThrValAsp Lys Ser 195 200 205

Ser SerThrAla TyrMet Glu Leu Arg Ser Leu Thr Ser Glu Val Ser 210 215 220

AlaValTyrTyrCysAla Arg Asp TyrAla Met Asp Tyr Trp Gly Gln 225 230 235 240

Gly Thr SerVal ThrVal Ser Ser 245

<210> 3

<211> 491

<212> PRT

<213> Artificial Sequence 220

<223> H9 HA ectodomain consensus <400> 3

Asp Lys lle Cys lle Gly His Gln Ser Thr Asn Ser Thr Glu Thr Val 1 5 10 15

Asp Thr Leu The Glu Thr Asn Val Pro Val The His Ala Lys Glu Leu 20 25 30

Leu His Thr Glu His Asn Gly Met Leu Cys Ala Thr Asn Leu Gly His 35 40 45

Pro Leu lle Leu Asp Thr Cys Thr lle Glu Gly Leu lle Tyr Gly Asn 50 55 60

Pro Ser Cys Asp Leu Leu Leu Gly Gly Arg Glu Trp Ser Tyr lle Val 65 70 75 80

Glu Arg Pro Ser Ala Val Asn Gly Thr Cys Tyr Pro Gly Asn Val Glu 85 90 95

Asn Leu Glu Glu Leu Arg Thr Leu Phe Ser Ser Ser Ser Ser Tyr Gln 100 105 110

Arg lle Gln lle Phe Pro Asp Thr lle Trp Asn Val Thr Tyr Thr Gly 115 120 125

Thr Ser Lys Ser Cys Ser Asp Ser Phe Tyr Arg Asn Met Arg Trp Leu 130 135 140

Thr Gln Lys Ser Gly Leu Tyr Pro Val Gln Asp Ala Gln Tyr Thr Asn 145 150 155 160

Asn Arg Gly Lys Asp lle Leu Phe Val Trp Gly lle His His Pro Pro 165 170 175

Thr Asp Thr Ala Gln Thr Asn Leu Tyr Thr Arg Thr Asp Thr Thr Thr 180 185 190 Ser Val Thr Thr Glu Asn Leu Asp Arg Thr Phe Lys Pro Val lle Gly 195 200 205

Pro Arg Pro Leu Val Asn Gly Leu lle Gly Arg lle Asn Tyr Tyr Trp 210 215 220

Ser Val Leu Lys Pro Gly Gln Thr Leu Arg Val Arg Ser Asn Gly Asn 225 230 235 240

Leu lle Ala Pro Trp Tyr Gly His Val Leu Ser Gly Glu Ser His Gly 245 250 255

Arg lle Leu Lys Thr Asp Leu Asn Ser Gly Asn Cys Val Val Gln Cys 260 265 270 Gln Thr Glu Lys Gly Gly Leu Asn Ser Thr Leu Pro Phe His Asn lle 275 280 285

Ser Lys Tyr Ala Phe Gly Asn Cys Pro Lys Tyr lle Gly Val Lys Ser 290 295 300

Leu Lys Leu Ala lle Gly Leu Arg Asn Val Pro Ala Arg Ser Ser Arg 305 310 315 320

Gly Leu Phe Gly Ala lle Ala Gly Phe lle Glu Gly Gly Trp Pro Gly 325 330 335

Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln Gly Val 340 345 350

Gly Met Ala Ala Asp Arg Asp Ser Thr Gln Lys Ala Val Asp Lys lle 355 360 365

Thr Ser Lys Val Asn Asn lle Val Asp Lys Met Asn Lys Gln Tyr Glu 370 375 380 lle lle Asp His Glu Phe Ser Glu Val Glu Thr Arg Leu Asn Met lle 385 390 395 400

Asn Asn Lys lle Asp Asp Gln lle Gln Asp Val Trp Ala Tyr Asn Ala 405 410 415 Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr Leu Asp Glu His Asp 420 425 430

Ala Asn Val Asn Asn Leu Tyr Asn Lys Val Lys Arg Ala Leu Gly Ser 435 440 445

Asn Ala Met Glu Asp Gly Lys Gly Cys Phe Glu Leu Tyr His Lys Cys 450 455 460

Asp Asp Gln Cys Met Glu Thr lle Arg Asn Gly Thr Tyr Asn Arg Arg 465 470 475 480

Lys Tyr Lys Glu Glu Ser Arg Leu Glu Arg Gln 485 490

<210> 4

<211> 511

<212> PRT

<213> Artificial Sequence 220

<223> H5 HA ectodomain , modified polybasic cleavage site <400> 4

Asp Gln lle Cys lle Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 1 5 10 15

Asp Thr lle Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp lle 20 25 30

Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys 35 40 45

Pro Leu lle Leu Lys Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 50 55 60

Pro Met Cys Asp Glu Phe lle Arg Val Pro Glu Trp Ser Tyr lle Val 65 70 75 80

Glu Arg Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Ser Leu Asn 85 90 95 Asp TyrGlu Glu Leu Lys His Leu Leu SerArg lle Asn His Phe Glu 100 105 110

Lys lle Leu lle lle Pro Lys Ser Ser Trp Pro Asn His Glu Thr Ser 115 120 125

Leu Gly Val SerAla Ala Cys Pro Tyr Gln Gly Thr Pro Ser Phe Phe 130 135 140

Arg AsnValValTrp Leu lle Lys LysAsn AspAla Tyr Pro Thr lle 145 150 155 160

Lys lle SerTyrAsn Asn ThrAsn Arg Glu Asp Leu Leu lle Met Trp 165 170 175

Gly lle His His SerAsn Asn Ala Glu Glu GlnThrAsn Leu Tyr Lys 180 185 190

Asn Pro ThrThrTyr lle SerVal Gly Thr Ser Thr Leu Asn Gln Arg 195 200 205

LeuVal Pro Lys lle Ala ThrArg Ser GlnValAsn Gly GlnArg Gly 210 215 220

Arg Met Asp Phe Phe Trp Thr lle Leu Lys Pro Asn Asp Ala lle His 225 230 235 240

Phe Glu SerAsn Gly Asn Phe lle Ala Pro Glu TyrAla Tyr Lys lle 245 250 255

Val Lys LysGly Asp Ser Thr lle Met Lys SerGlu Val Glu Tyr Gly 260 265 270

His CysAsn Thr LysCys Gln Thr Pro Val Gly Ala lle Asn Ser Ser 275 280 285

Met Pro Phe HisAsn lle His Pro Leu Thr lle Gly Glu Cys Pro Lys 290 295 300 Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 305 310 315 320

Pro Gln Gly Glu Thr Arg Gly Leu Phe Gly Ala lle Ala Gly Phe lle 325 330 335

Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His 340 345 350

Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln 355 360 365

Lys Ala lle Asp Gly Val Thr Asn Lys Val Asn Ser lle lle Asp Lys 370 375 380

Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu 385 390 395 400

Arg Arg lle Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp 405 410 415

Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg 420 425 430

Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val 435 440 445

Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe 450 455 460

Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn 465 470 475 480

Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg 485 490 495

Glu Glu lle Ser Gly Val Lys Leu Glu Ser lle Gly Thr Tyr Gln 500 505 510

<210> 5

<211> 507 <212> PRT

<213> Artificial Sequence 220

<223> H7 HA ectodomain, modified polybasic cleavage site <400> 5

Asp Lys lle Cys Leu Gly His His Ala Val Ser Asn Gly Thr Lys Val 1 5 10 15

Asn Thr Leu Thr Glu Arg Gly Val Glu Val Val Asn Ala Thr Glu Thr 20 25 30

Val Glu Arg Thr Asn Thr Pro Arg lle Cys Ser Lys Gly Lys Arg Thr 35 40 45

Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr lle Thr Gly Pro Pro 50 55 60 Gln Cys Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu lle lle Glu Arg 65 70 75 80

Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn Glu Glu 85 90 95

Ala Leu Arg Gln lle Leu Arg Glu Ser Gly Gly lle Asp Lys Glu Pro 100 105 110

Met Gly Phe Thr Tyr Asn Gly lle Arg Thr Asn Gly Val Thr Ser Ala 115 120 125

Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp Leu Leu 130 135 140

Ser Asn Thr Asp Asn Ala Ala Phe Pro Gln Met Thr Lys Ser Tyr Lys 145 150 155 160

Asn Thr Lys Glu Ser Pro Ala lle lle Val Trp Gly lle His His Ser 165 170 175

Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn Lys Leu 180 185 190 ValThrValGly SerSer Asn Tyr Gln GlnSerPhe ValPro SerPro 195 200 205

Gly Ala Arg ProGln Val Asn Gly GlnSerGly Arg lle Asp Phe His 210 215 220 Trp Leu lle Leu AsnProAsn AspThrValThrPhe SerPhe Asn Gly 225 230 235 240

Ala Phe lle AlaProAsp ArgAlaSerPhe Leu Arg Gly LysSerMet 245 250 255

Gly lle Gln Ser ArgVal GlnValAsp Ala Asn Cys Glu Gly Asp Cys 260 265 270 TyrHisSerGly GlyThrlle lle SerAsn LeuPro Phe GlnAsn lle 275 280 285

AspSer ArgAla ValGly Lys CysPro Arg TyrVal Lys Gln Arg Ser 290 295 300

Leu Leu Leu AlaThrGly Met LysAsnValProGlu ValPro LysGly 305 310 315 320

ArgGly Leu Phe Gly Ala lle Ala Gly Phe lle Glu Asn GlyTrp Glu 325 330 335

Gly Leu lleAsp GlyTrp TyrGly Phe Arg HisGln Asn Ala Gln Gly 340 345 350

Glu GlyThrAla Ala AspTyrLysSerThrGlnSerAla lleAsp Gln 355 360 365 lleThrGly Lys Leu Asn Arg Leu lle Ala LysThrAsn Gln Gln Phe 370 375 380

Lys Leu lle Asp Asn Glu Phe Asn GluVal Glu LysGln lle Gly Asn 385 390 395 400 Val lle Asn Trp ThrArgAsp Ser lle Thr GluVal TrpSerTyrAsn 405 410 415

Ala Glu Leu Leu ValAla Met Glu Asn Gln HisThr lle Asp Leu Ala 420 425 430

Asp SerGlu Met Asp Lys LeuTyrGlu ArgVal Lys Arg Gln Leu Arg 435 440 445

GluAsnAla Glu Glu Asp GlyThrGly Cys Phe Glu lle Phe His Lys 450 455 460

CysAspAspAsp CysMet AlaSer lle ArgAsnAsnThrTyrAsp His 465 470 475 480

Arg LysTyr ArgGlu Glu Ala Met GlnAsn Arg lle Gln lleAspPro 485 490 495

Val Lys LeuSerSerGly TyrLysAspVal lle 500 505 210 6 <211> 29

<212> PRT

<213> Antificial Sequence 220

<223> Foldon,tnimenisation domain <400> 6

Gly SerGlyTyr llePro Glu AlaPro ArgAsp Gly Gln AlaTyrVal 1 5 10 15

Arg LysAsp Gly GluTrpVal Leu LeuSerThrPhe Leu 20 25

<210> 7

<211> 520

<212> PRT

<213> Antificial Sequence 220

<223> H9 HA ectodomain + Foldon 400 7

Asp LyslleCyslleGly HisGlnSerThrAsnSerThrGluThrVal 1 5 10 15

AspThrLeuThrGluThrAsnValProValThrHisAla LysGlu Leu 20 25 30

LeuHisThrGluHisAsnGlyMet LeuCysAlaThrAsn LeuGly His 35 40 45 Pro Leu lle LeuAspThrCysThrlleGluGly Leu lleTyrGlyAsn 50 55 60 Pro SerCysAsp Leu Leu LeuGlyGly Arg GluTrp SerTyrlleVal 65 70 75 80

Glu Arg Pro SerAlaValAsnGlyThrCysTyrProGlyAsnValGlu 85 90 95

Asn LeuGluGlu Leu ArgThrLeu PheSerSerSerSerSerTyrGln 100 105 110

Arg lle Gln llePheProAspThrlleTrpAsnValThrTyrThrGly 115 120 125 ThrSer LysSerCysSerAspSerPheTyr ArgAsnMet ArgTrp Leu 130 135 140 ThrGln LysSerGly LeuTyrProValGlnAspAlaGlnTyrThrAsn 145 150 155 160

Asn Arg Gly LysAsp lle LeuPheValTrp Gly lleHisHisPro Pro 165 170 175 ThrAspThrAlaGlnThrAsn LeuTyrThr ArgThrAspThrThrThr 180 185 190 SerValThrThrGluAsn LeuAsp Arg ThrPhe LysProVallleGly 195 200 205 Pro Arg Pro Leu Val Asn Gly Leu lle Gly Arg lle Asn Tyr Tyr Trp 210 215 220

Ser Val Leu Lys Pro Gly Gln Thr Leu Arg Val Arg Ser Asn Gly Asn 225 230 235 240

Leu lle Ala Pro Trp Tyr Gly His Val Leu Ser Gly Glu Ser His Gly 245 250 255

Arg lle Leu Lys Thr Asp Leu Asn Ser Gly Asn Cys Val Val Gln Cys 260 265 270 Gln Thr Glu Lys Gly Gly Leu Asn Ser Thr Leu Pro Phe His Asn lle 275 280 285

Ser Lys Tyr Ala Phe Gly Asn Cys Pro Lys Tyr lle Gly Val Lys Ser 290 295 300

Leu Lys Leu Ala lle Gly Leu Arg Asn Val Pro Ala Arg Ser Ser Arg 305 310 315 320

Gly Leu Phe Gly Ala lle Ala Gly Phe lle Glu Gly Gly Trp Pro Gly 325 330 335

Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln Gly Val 340 345 350

Gly Met Ala Ala Asp Arg Asp Ser Thr Gln Lys Ala Val Asp Lys lle 355 360 365

Thr Ser Lys Val Asn Asn lle Val Asp Lys Met Asn Lys Gln Tyr Glu 370 375 380 lle lle Asp His Glu Phe Ser Glu Val Glu Thr Arg Leu Asn Met lle 385 390 395 400

Asn Asn Lys lle Asp Asp Gln lle Gln Asp Val Trp Ala Tyr Asn Ala 405 410 415

Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr Leu Asp Glu His Asp 420 425 430 AlaAsnValAsn Asn Leu TyrAsn LysVal LysArg Ala Leu Gly Ser 435 440 445

AsnAla Met Glu Asp Gly Lys Gly Cys Phe Glu Leu Tyr His Lys Cys 450 455 460

AspAsp Gln CysMet Glu Thr lle Arg Asn Gly ThrTyrAsn ArgArg 465 470 475 480

Lys Tyr LysGlu Glu Ser Arg Leu GluArg GlnGly Ser Gly Tyr lle 485 490 495

Pro GluAla Pro Arg Asp Gly Gln Ala TyrValArg LysAsp Gly Glu 500 505 510

TrpVal Leu Leu SerThr Phe Leu 515 520 210 8 <211> 5

<212> PRT

<213> Artificial Sequence 220

<223> Linker <400> 8

Gly SerGly SerGly 1 5

<210> 9

<211> 4

<212> PRT

<213> Artificial Sequence 220

<223> Ctag

<400> 9

Glu Pro GluAla 1 210 10 <211> 14

<212> PRT

<213> Artificial Sequence 220

<223> V5 tag <400> 10

Gly Lys Pro lle Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10

<210> 11 <211> 18 <212> PRT

<213> Artificial Sequence 220

<223> BIP signal sequence <400> 11

Met Lys Leu Cys lle Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15

Leu Gly

<210> 12 <211> 822 <212> PRT

<213> Artificial Sequence 220

<223> Full recombinant protein <400> 12

Met Lys Leu Cys lle Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15

Leu Gly Gly Thr Gly Asp Lys lle Cys lle Gly His Gln Ser Thr Asn 20 25 30

Ser Thr Glu Thr Val Asp Thr Leu Thr Glu Thr Asn Val Pro Val Thr His Ala Lys Glu Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala 50 55 60

Thr Asn Leu Gly His Pro Leu lle Leu Asp Thr Cys Thr lle Glu Gly 65 70 75 80

Leu lle Tyr Gly Asn Pro Ser Cys Asp Leu Leu Leu Gly Gly Arg Glu 85 90 95

Trp Ser Tyr lle Val Glu Arg Pro Ser Ala Val Asn Gly Thr Cys Tyr 100 105 110

Pro Gly Asn Val Glu Asn Leu Glu Glu Leu Arg Thr Leu Phe Ser Ser 115 120 125

Ser Ser Ser Tyr Gln Arg lle Gln lle Phe Pro Asp Thr lle Trp Asn 130 135 140

Val Thr Tyr Thr Gly Thr Ser Lys Ser Cys Ser Asp Ser Phe Tyr Arg 145 150 155 160

Asn Met Arg Trp Leu Thr Gln Lys Ser Gly Leu Tyr Pro Val Gln Asp 165 170 175

Ala Gln Tyr Thr Asn Asn Arg Gly Lys Asp lle Leu Phe Val Trp Gly 180 185 190 lle His His Pro Pro Thr Asp Thr Ala Gln Thr Asn Leu Tyr Thr Arg 195 200 205

Thr Asp Thr Thr Thr Ser Val Thr Thr Glu Asn Leu Asp Arg Thr Phe 210 215 220

Lys Pro Val lle Gly Pro Arg Pro Leu Val Asn Gly Leu lle Gly Arg 225 230 235 240 lle Asn Tyr Tyr Trp Ser Val Leu Lys Pro Gly Gln Thr Leu Arg Val 245 250 255

Arg Ser Asn Gly Asn Leu lle Ala Pro Trp Tyr Gly His Val Leu Ser 260 265 270 Gly Glu Ser His Gly Arg lle Leu Lys Thr Asp Leu Asn Ser Gly Asn 275 280 285

Cys Val Val Gln Cys GlnThr Glu Lys Gly Gly Leu Asn SerThr Leu 290 295 300 Pro Phe His Asn lleSer Lys Tyr Ala Phe Gly Asn Cys Pro Lys Tyr 305 310 315 320 lle Gly Val Lys Ser Leu Lys Leu Ala lle Gly Leu Arg Asn Val Pro 325 330 335

Ala Arg SerSer Arg Gly Leu Phe Gly Ala lle Ala Gly Phe lle Glu 340 345 350

Gly Gly Trp Pro Gly Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser 355 360 365

Asn Asp Gln Gly Val Gly Met Ala Ala Asp Arg Asp SerThrGln Lys 370 375 380

Ala Val Asp Lys lleThrSer Lys Val Asn Asn lle Val Asp Lys Met 385 390 395 400

Asn Lys GlnTyr Glu lle lle Asp His Glu Phe Ser Glu Val Glu Thr 405 410 415

Arg Leu Asn Met lle Asn Asn Lys lle Asp Asp Gln lle Gln Asp Val 420 425 430 Trp Ala Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr 435 440 445

Leu Asp Glu His Asp Ala Asn Val Asn Asn Leu Tyr Asn Lys Val Lys 450 455 460

Arg Ala Leu Gly Ser Asn Ala Met Glu Asp Gly Lys Gly Cys Phe Glu 465 470 475 480 Leu Tyr His Lys Cys Asp Asp Gln Cys Met Glu Thr lleArg Asn Gly 485 490 495

Thr Tyr Asn Arg Arg Lys Tyr Lys Glu Glu Ser Arg Leu Glu Arg Gln 500 505 510

Gly Ser Gly Tyr llePro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val 515 520 525

Arg Lys Asp Gly Glu Trp Val Leu Leu SerThr Phe Leu Leu lle Lys 530 535 540

Gly Ser Gly Ser Gly Ala Ala Ala Asp lle Val Met ThrGlnSerPro 545 550 555 560 SerSer Leu Ala Val Ser Val Gly Gln Lys Val Thr Met Ser Cys Thr 565 570 575 SerSerGln Val Leu Leu His SerPro Asn Gln Lys Asn Tyr Leu Ala 580 585 590 TrpTyrGln Gln Lys Pro Gly GlnSerPro Lys Leu Leu Val Tyr Phe 595 600 605

Ala SerThr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 610 615 620 Ser Gly Thr Asp Phe Thr Leu Thrlle SerSer Val Gln Ala Glu Asp 625 630 635 640

Leu Ala Val TyrTyr Cys Gln Gln His TyrSerThrPro Leu Thr Phe 645 650 655

Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly 660 665 670

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln 675 680 685

Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys 690 695 700 lleSer Cys Lys Ala Ser Gly TyrThr Phe Thr Asp TyrTyrlle Asn 705 710 715 720 Trp Val Lys GlnSer His Gly Lys Ser Leu Glu Trp lle Gly Asp lle 725 730 735

Asn ProThr Asn Gly Asp SerThrTyrSerGln Lys Phe Lys Gly Lys 740 745 750

Ala Thr Leu Thr Val Asp Lys SerSer SerThr Ala Tyr Met Glu Leu 755 760 765

Arg Ser Leu ThrSer Glu Val Ser Ala Val TyrTyr Cys Ala Arg Asp 770 775 780 Tyr Ala Met Asp TyrTrp Gly Gln Gly ThrSer Val Thr Val SerSer 785 790 795 800 Ser Arg Gly Lys Pro lle Pro Asn Pro Leu Leu Gly Leu Asp SerThr 805 810 815

His His His His His His 820

<210> 13

<211> 564

<212> PRT

<213> Antificial Sequence <220>

<223> Control recombinant pnotein <400> 13

Met Lys Leu Cys lle Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15

Leu Gly Gly Thr Gly Asp Lys lle Cys lle Gly His Gln SerThr Asn 20 25 30 SerThr Glu Thr Val Asp Thr Leu Thr Glu Thr Asn Val Pro Val Thr His Ala Lys Glu Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala 50 55 60

Thr Asn Leu Gly His Pro Leu lle Leu Asp Thr Cys Thr lle Glu Gly 65 70 75 80

Leu lle Tyr Gly Asn Pro Ser Cys Asp Leu Leu Leu Gly Gly Arg Glu 85 90 95

Trp Ser Tyr lle Val Glu Arg Pro Ser Ala Val Asn Gly Thr Cys Tyr 100 105 110

Pro Gly Asn Val Glu Asn Leu Glu Glu Leu Arg Thr Leu Phe Ser Ser 115 120 125

Ser Ser Ser Tyr Gln Arg lle Gln lle Phe Pro Asp Thr lle Trp Asn 130 135 140

Val Thr Tyr Thr Gly Thr Ser Lys Ser Cys Ser Asp Ser Phe Tyr Arg 145 150 155 160

Asn Met Arg Trp Leu Thr Gln Lys Ser Gly Leu Tyr Pro Val Gln Asp 165 170 175

Ala Gln Tyr Thr Asn Asn Arg Gly Lys Asp lle Leu Phe Val Trp Gly 180 185 190 lle His His Pro Pro Thr Asp Thr Ala Gln Thr Asn Leu Tyr Thr Arg 195 200 205

Thr Asp Thr Thr Thr Ser Val Thr Thr Glu Asn Leu Asp Arg Thr Phe 210 215 220

Lys Pro Val lle Gly Pro Arg Pro Leu Val Asn Gly Leu lle Gly Arg 225 230 235 240 lle Asn Tyr Tyr Trp Ser Val Leu Lys Pro Gly Gln Thr Leu Arg Val 245 250 255 Arg Ser Asn Gly Asn Leu lle Ala Pro Trp Tyr Gly His Val Leu Ser 260 265 270

Gly Glu Ser His Gly Arg lle Leu Lys Thr Asp Leu Asn Ser Gly Asn 275 280 285

Cys Val Val Gln Cys Gln Thr Glu Lys Gly Gly Leu Asn Ser Thr Leu 290 295 300

Pro Phe His Asn lle Ser Lys Tyr Ala Phe Gly Asn Cys Pro Lys Tyr 305 310 315 320 lle Gly Val Lys Ser Leu Lys Leu Ala lle Gly Leu Arg Asn Val Pro 325 330 335

Ala Arg Ser Ser Arg Gly Leu Phe Gly Ala lle Ala Gly Phe lle Glu 340 345 350

Gly Gly Trp Pro Gly Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser 355 360 365

Asn Asp Gln Gly Val Gly Met Ala Ala Asp Arg Asp Ser Thr Gln Lys 370 375 380

Ala Val Asp Lys lle Thr Ser Lys Val Asn Asn lle Val Asp Lys Met 385 390 395 400

Asn Lys Gln Tyr Glu lle lle Asp His Glu Phe Ser Glu Val Glu Thr 405 410 415

Arg Leu Asn Met lle Asn Asn Lys lle Asp Asp Gln lle Gln Asp Val 420 425 430

Trp Ala Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr 435 440 445

Leu Asp Glu His Asp Ala Asn Val Asn Asn Leu Tyr Asn Lys Val Lys 450 455 460

Arg Ala Leu Gly Ser Asn Ala Met Glu Asp Gly Lys Gly Cys Phe Glu 465 470 475 480 Leu Tyr His LysCysAsp Asp Gln Cys Met Glu Thr lle Arg Asn Gly 485 490 495

Thr TyrAsnArg Arg Lys Tyr Lys Glu Glu SerArg Leu Glu Arg Gln 500 505 510

Gly Ser Gly Tyr lle Pro Glu Ala Pro Arg Asp Gly Gln Ala TyrVal 515 520 525

Arg LysAsp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Leu lle Lys 530 535 540

Gly Lys Pro lle Pro Asn Pro Leu Leu Gly LeuAsp Ser Thr His His 545 550 555 560

His His His His

<210> 14

<211> 2466

<212> DNA

<213> Artificial Sequence 220

<223> Nucleotide sequence encoding SEQ ID NO:12

<400> 14 atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct cgggggtacc 60 ggggataaga tctgcatcgg ccaccagagc accaacagca ccgagaccgt ggataccctg 120 accgagacca acgtgccagt gacccacgcc aaggagctgc tgcacaccga gcacaacgga 180 atgctgtgcg ccaccaatct gggccacccc ctgatcctgg atacctgcac catcgagggc 240 ctgatctacg gcaaccccag ctgcgatctg ctgctgggag gacgcgagtg gtcctacatt 300 gtggagcgcc ccagcgccgt gaacggaacc tgctatccag gcaacgtgga gaacctggag 360 gagctgcgca ccctgttcag cagctcgagc agctaccagc gcatccagat cttccccgat 420 accatctgga acgtgaccta caccggcacc agcaagagct gcagcgatag cttctaccgc 480 aacatgcgct ggctgaccca gaagtccggc ctgtacccag tgcaggatgc ccagtacacc 540 aacaatcgcg gcaaggacat cctgttcgtg tggggcatcc accacccccc aaccgatacc 600 gcccagacca atctgtacac ccgcaccgat accaccacca gcgtgaccac cgagaatctg 660 gatcgcacct tcaagcccgt gatcggccca cgcccactcg tgaatggact gatcggccgc 720 atcaactact attggagcgt gctgaagccc ggccagaccc tgcgcgtgcg cagcaatgga 780 aatctgatcg ccccgtggta cggccacgtg ctgagcggag agagccacgg ccgcattctg 840 aagaccgatc tgaacagcgg caactgcgtg gtgcagtgcc agaccgagaa gggcggcctg 900 aatagcaccc tgcccttcca caacatctcg aagtacgcct tcggaaactg ccccaagtac 960 atcggcgtga agtccctgaa gctggccatc ggcctgcgca atgtgccagc ccgcagtagt 1020 cgcggactgt tcggagccat tgccggcttc attgagggcg gctggccagg actggtggcc 1080 ggatggtacg gattccagca cagcaacgat cagggcgtgg gaatggccgc cgatcgcgat 1140 agtacccaga aggccgtgga taagatcacc tccaaagtga acaacatcgt ggacaagatg 1200 aacaagcagt acgagatcat cgaccacgag ttcagcgagg tggagacccg cctgaacatg 1260 atcaacaaca agatcgacga ccagatccag gatgtgtggg cctacaacgc cgagctgctg 1320 gtgctgctgg agaaccagaa gaccctggac gagcacgatg ccaacgtgaa caatctgtat 1380 aacaaagtga agcgcgccct gggcagcaac gccatggagg atggaaaggg atgcttcgag 1440 ctgtaccaca agtgcgacga tcagtgcatg gagaccatccgcaacggcac ctacaaccgc 1500 cgcaagtaca aggaggagag ccgcctggag cgccagggca gcggctacat cccagaggcc 1560 ccacgcgacg gacaggccta tgtgcgcaag gatggcgagt gggtgctgct gagcaccttc 1620 ctgttaatta agggttctgg ctctggtgcg gccgcggata tcgtgatgac ccagtcgcca 1680 agcagtctgg ctgtgtccgt gggacagaaa gtgaccatga gctgcaccag cagccaggtg 1740 ctgctgcaca gccccaacca gaagaattac ctggcctggt atcagcagaa gcccggccaa 1800 agtccgaagc tgctggtcta ctttgccagc acacgcgaga gcggagtgcc agatcgtttt 1860 accggaagcg gcagcggcac cgatttcacc ctgacaatta gtagcgtgca ggccgaggat 1920 ctggccgtgt attactgcca gcagcactac agcaccccgc tgacatttgg cgccggaacg 1980 aagctggaac tgaaaggcgg aggtggtagt ggtggcggag gatcaggtgg tggtggttct 2040 ggcggtggtg gaagtgaagt gcaactgcag cagagcggcc cagagctggt caaaccaggt 2100 gccagcgtga agatcagctg caaggccagc ggatacacct tcaccgatta ctacatcaac 2160 tgggtcaagc agagccacgg caagagcctg gaatggatcg gcgatatcaa ccccaccaac 2220 ggcgatagca cctacagcca gaagttcaag ggcaaagcca cgctgaccgt ggataagagt 2280 agcagcaccg cctacatgga actgcgcagc ctgacaagcg aagtgtccgc cgtgtactat 2340 tgcgcccgtg attacgccat ggattactgg ggacagggca ccagtgtgac cgttagtagt 2400 tctagaggta agcctatccc taaccctctc ctcggtctcg attctacgca tcatcaccat 2460 caccat 2466

<210> 15

<211> 1692

<212> DNA

<213> Artificial Sequence <220>

<223> Nuceotide sequence encoding SEQ ID NO: 13 <400> 15 atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct cgggggtacc 60 ggggataaga tctgcatcgg ccaccagagc accaacagca ccgagaccgt ggataccctg 120 accgagacca acgtgccagt gacccacgcc aaggagctgc tgcacaccga gcacaacgga 180 atgctgtgcg ccaccaatct gggccacccc ctgatcctgg atacctgcac catcgagggc 240 ctgatctacg gcaaccccag ctgcgatctg ctgctgggag gacgcgagtg gtcctacatt 300 gtggagcgcc ccagcgccgt gaacggaacc tgctatccag gcaacgtgga gaacctggag 360 gagctgcgca ccctgttcag cagctcgagc agctaccagc gcatccagat cttccccgat 420 accatctgga acgtgaccta caccggcacc agcaagagct gcagcgatag cttctaccgc 480 aacatgcgct ggctgaccca gaagtccggc ctgtacccag tgcaggatgc ccagtacacc 540 aacaatcgcg gcaaggacat cctgttcgtg tggggcatcc accacccccc aaccgatacc 600 gcccagacca atctgtacac ccgcaccgat accaccacca gcgtgaccac cgagaatctg 660 gatcgcacct tcaagcccgt gatcggccca cgcccactcg tgaatggact gatcggccgc 720 atcaactact attggagcgt gctgaagccc ggccagaccc tgcgcgtgcg cagcaatgga 780 aatctgatcg ccccgtggta cggccacgtg ctgagcggag agagccacgg ccgcattctg 840 aagaccgatc tgaacagcgg caactgcgtg gtgcagtgcc agaccgagaa gggcggcctg 900 aatagcaccc tgcccttcca caacatctcg aagtacgcct tcggaaactg ccccaagtac 960 atcggcgtga agtccctgaa gctggccatc ggcctgcgca atgtgccagc ccgcagtagt 1020 cgcggactgttcggagccattgccggcttcattgagggcggctggccagg actggtggcc 1080 ggatggtacg gattccagcacagcaacgat cagggcgtgggaatggccgccgatcgcgat 1140 agtacccagaaggccgtggataagatcacctccaaagtgaacaacatcgt ggacaagatg 1200 aacaagcagt acgagatcat cgaccacgagttcagcgaggtggagacccg cctgaacatg 1260 atcaacaacaagatcgacgaccagatccag gatgtgtggg cctacaacgc cgagctgctg 1320 gtgctgctgg agaaccagaagaccctggacgagcacgatg ccaacgtgaa caatctgtat 1380 aacaaagtgaagcgcgccctgggcagcaacgccatggagg atggaaaggg atgcttcgag 1440 ctgtaccacaagtgcgacgatcagtgcatggagaccatccgcaacggcac ctacaaccgc 1500 cgcaagtacaaggaggagag ccgcctggagcgccagggcagcggctacat cccagaggcc 1560 ccacgcgacggacaggcctatgtgcgcaaggatggcgagtgggtgctgct gagcaccttc 1620 ctgttaattaagggtaagcctatccctaaccctctcctcggtctcgattctacgcatcat 1680 caccatcaccat 1692

<210> 16 <211> 246

<212> PRT

<213> ArtificialSequence 220

<223> CDllc-scFv <400> 16

GluValGln LeuGln GlnSerGly ProGlu LeuVal LysProGlyAla 1 5 10 15

SerValLysMet SerCys LysAlaSerGly TyrThrPheThrAsnTyr 20 25 30

ValLeuHisTrpValLysGlnLysProGly GlnGly LeuGluTrp lle 35 40 45

GlyTyrlleAsnProTyrAsnAspGlyThr LysPheAsnGlu LysPhe 50 55 60

LysGly LysAlaThrLeuThrSerAsp Thr SerSer SerThrAlaPhe 65 70 75 80 Met Glu LeuSerSer LeuThrSerGluAsp SerAla ValTyrTyrCys 85 90 95

Ala Arg Gly Asp Asn Leu Arg Pro TyrTyrPheAspTyrTrpGly Gln 100 105 110

GlyThrThr LeuThrValSerSer Gly Gly Gly Gly SerGly Gly Gly 115 120 125

Gly SerGly Gly Gly Gly SerGly Gly Gly Gly SerGln lleVal Leu 130 135 140 ThrHisSerProAla lle MetSer Ala SerProGly Glu LysValThr 145 150 155 160

MetThrCysSerAlaSerSerSer ValSerPhe Met TyrTrpTyrGln 165 170 175 Gln LysProGly SerSerPro Arg Leu Leu LeuTyrAspThrSerSer 180 185 190

LeuSerSerGly ValProVal Arg PheSerGly SerGly SerGlyThr 195 200 205 SerTyrSer LeuThrlle SerArg Met Glu Ala Glu Asp Ala AlaThr 210 215 220 TyrTyrCysGln GlnTrp Ser Arg TyrPro ProThrPhe Gly Gly Gly 225 230 235 240 ThrLys Leu Glu lle Lys 245

<210> 17

<211> 247

<212> PRT

<213> Antificial Sequence 220

<223> Dec205-SCFv 400 17

Glu lle Val LeuThrGlnSerProAla Leu Met Ala Ala SerPro Gly 1 5 10 15

Glu LysValThrlleThr CysSerValSerSerSer lleSerSerGly 20 25 30

Asn Phe HisTrpTyrGln Gln LysSerGly ThrSerPro Lys LeuTrp 35 40 45 lleTyrGlyThrSerAsn Leu AlaSerGly ValProVal Arg Phe Ser 50 55 60

Gly SerGly SerGlyThrSerTyrSer LeuThrlleSerSerMet Glu 65 70 75 80

Ala GluAspAla AlaThrTyrTyrCysGln GlnTrp SerSerTyrPro 85 90 95

PheThrPheGly SerGly ThrLys Leu Glu lle Lys Gly Gly Gly Gly 100 105 110 SerGly Gly Gly Gly Ser Gly Gly Gly Gly SerGly Gly Gly Gly Ser 115 120 125

GluValGln Leu ValGlu SerGly Gly Asp LeuVal LysProGly Gly 130 135 140 SerLeu Lys LeuSerCys Ala AlaSerGly PheThrPhe SerSerTyr 145 150 155 160

Gly Met SerTrpVal Arg GlnThrProAsp Lys Arg Leu GluTrpVal 165 170 175

AlaThrlle SerSerGly Gly SerTyrThrTyrTyrProAspSerVal 180 185 190

Lys Gly Arg PheThrlle Ser ArgAspAsn Ala LysAsn lle LeuTyr 195 200 205 Leu Gln Met SerSer Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 210 215 220

Ala Arg Leu Ser Thr Trp Asp Trp Tyr Phe Asp Val Trp Gly Thr Gly 225 230 235 240

Thr Thr Val Thr Val Ser Ser 245

<210> 18 <211> 470

<212> PRT

<213> Artificial Sequence 220

<223> NDV F antigen, consensus <400> 18

Ala Leu Asp Gly Arg Pro Leu Ala Ala Ala Gly lle Val Val Thr Gly 1 5 10 15

Asp Lys Ala Val Asn Val Tyr Thr Ser Ser Gln Thr Gly Ser lle lle 20 25 30

Val Lys Leu Leu Pro Asn Met Pro Lys Asp Lys Glu Ala Cys Ala Arg 35 40 45

Ala Pro Leu Glu Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro 50 55 60

Leu Gly Asp Ser lle Arg Lys lle Gln Gly Ser Val Ala Thr Ser Gly 65 70 75 80

Ser Gly Ser Gly Ser Arg Phe lle Gly Ala Val lle Gly Ser lle Ala 85 90 95

Leu Gly Val Ala Thr Ala Ala Gln lle Thr Ala Ala Ala Ala Leu lle 100 105 110 Gln Ala Asn Gln Asn Ala Ala Asn lle Leu Arg Leu Lys Glu Ser lle 115 120 125 Ala Ala Thr Asn Glu Ala Val His Glu Val Thr Asn Gly Leu Ser Gln 130 135 140

Leu Ser Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe 145 150 155 160

Asn Asn Thr Ala Arg Glu Leu Asp Cys lle Lys lle Thr Gln Gln Val 165 170 175

Gly Val Glu Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gly 180 185 190

Pro Gln lle Thr Ser Pro Ala Leu Thr Gln Leu Thr lle Gln Ala Leu 195 200 205

Tyr Asn Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly 210 215 220

Val Gly Asn Asn Gln Leu Ser Ser Leu lle Gly Ser Gly Leu lle Thr 225 230 235 240

Gly Tyr Pro lle Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly lle Gln 245 250 255

Val Asn Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr 260 265 270

Leu Glu Thr Leu Ser Val Ser Thr Thr Lys Gly Phe Ala Ser Ala Leu 275 280 285

Val Pro Lys Val Val Thr Gln Val Gly Ser Val lle Glu Glu Leu Asp 290 295 300

Thr Ser Tyr Cys lle Glu Ser Asp Leu Asp Leu Tyr Cys Thr Arg lle 305 310 315 320

Val Thr Phe Pro Met Ser Pro Gly lle Tyr Ser Cys Leu Ser Gly Asn 325 330 335

Thr Ser Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Thr Thr Pro 340 345 350 Tyr Met Ala Leu Lys Gly Ser Val lle Ala Asn Cys Lys lle Thr Thr 355 360 365

Cys Arg Cys Ala Asp Pro Pro Gly lle lle Ser Gln Asn Tyr Gly Glu 370 375 380

Ala Val Ser Leu lle Asp Arg His Ser Cys Asn Val Leu Ser Leu Asp 385 390 395 400

Gly lle Thr Leu Arg Leu Ser Gly Glu Phe Asp Ala Thr Tyr Leu Lys 405 410 415

Asn lle Ser lle Leu Asp Ser Gln Val lle Val Thr Gly Asn Leu Asp 420 425 430 lle Ser Thr Glu Leu Gly Asn Val Asn Asn Ser lle Ser Asn Ala Leu 435 440 445

Asp Lys Leu Thr Glu Ser Asn Ser Lys Leu Asp Lys Val Asn Val Arg 450 455 460

Leu Thr Ser Thr Ser Ala 465 470

<210> 19

<211> 525

<212> PRT

<213> Artificial Sequence 220

<223> NDV HN antigen, consensus <400> 19

Ser Met Gly Thr Ser Thr Pro Arg Asp Leu Thr Gly lle Ser lle Ala 1 5 10 15 lle Ser Lys Thr Glu Asp Lys Val Thr Ser Leu Leu Ser Ser Ser Gln 20 25 30

Asp Val lle Asp Arg lle Tyr Lys Gln Val Ala Leu Glu Ser Pro Leu 35 40 45 Ala Leu LeuAsn ThrGlu Ser lle lle Met AsnAla lle Thr Ser Leu

50 55 60

Ser TyrGln lleAsn Gly Ala Ala AsnAsn SerGly Cys Gly Ala Pro 65 70 75 80

Val HisAsp Pro Asp Tyr lle Gly Gly lle Gly Lys Glu Leu lle Val 85 90 95

AspAsp Thr SerAsp Val Thr Ser Phe Tyr Pro SerAla Tyr Gln Glu 100 105 110

His LeuAsn Phe lle Pro Ala Pro ThrThr Gly Ser Gly Cys ThrArg 115 120 125 lle Pro Ser Phe Asp Met Ser Thr Thr His TyrCys Tyr Thr HisAsn 130 135 140

Val lle Leu SerGly Cys Arg Asp His Ser His Ser His Gln Tyr Leu 145 150 155 160

Ala Leu Gly Val Leu Arg Thr SerAla Thr Gly Lys Val Phe Phe Ser 165 170 175

Thr LeuArg Ser lle Asn Leu Asp Asp Thr GlnAsn Arg Lys Ser Cys 180 185 190

SerVal SerAla Thr Pro Leu Gly CysAsp lle Leu Cys Ser LysVal 195 200 205

Thr Glu ThrGlu Glu Glu Asp Tyr Lys SerValThr Pro Thr Ser Met 210 215 220

Val HisGly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu LysAsp Leu 225 230 235 240

Asp ThrThrAla Leu Phe LysAsp TrpVal AlaAsn Tyr Pro Gly Val 245 250 255 Gly Gly Gly Ser Phe Val Asp Glu Arg Val Trp Phe Pro Val TyrGly 260 265 270

Gly Leu Lys Pro Asn Ser Pro SerAsp ThrAla Gln Glu Gly Lys Tyr 275 280 285

Val lle Tyr LysArg Tyr Asn Asp Thr Cys Pro Asp Glu GlnAsp Tyr 290 295 300 Gln lle Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly Gly 305 310 315 320

LysArg ValGln Gln Ala lle Leu Ser lle LysVal Ser Thr Ser Leu 325 330 335

Gly GluAsp Pro Met Leu Thr lle Pro Pro Asn Thr lle Thr Leu Met 340 345 350

Gly Ala Glu Gly Arg lle Leu ThrVal Gly Thr Ser His Phe Leu Tyr 355 360 365 GlnArg Gly Ser SerTyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr 370 375 380 lle SerAsn LysThrAla Thr Leu His Ser Pro TyrThr Phe Asn Ala 385 390 395 400

Phe ThrArg Pro Gly Ser Val Pro Cys GlnAla SerAla Arg Cys Pro 405 410 415

Asn SerCys lle ThrGly Val Tyr ThrAsp Pro Tyr Pro Leu lle Phe 420 425 430

HisArg Asn HisThr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Asp 435 440 445

Gly GlnAlaArg Leu Asn Pro Val SerAla Val Phe Asp Asp lle Ser 450 455 460

Arg SerArg ValThrArg Val Ser Ser Ser SerThr Lys Ala Ala Tyr 465 470 475 480 Thr ThrSerThr Cys Phe Lys Val Val Lys Thr Asn Lys ThrTyr Cys 485 490 495

Leu Ser lle Ala Glu lle Ser Asn Thr Leu Phe Gly Glu Phe Arg lle 500 505 510

Val Pro Leu Leu Val Glu lle Leu Lys Asp Asn Arg Ala 515 520 525 210 20 <211> 444

<212> PRT

<213> Infectious bunsal disease vinus <400> 20 Gln Gln lle Val Pro Phe lle Arg Ser Leu Leu Met Pro ThrThr Gly 1 5 10 15 Pro Ala Ser llePro Asp Asp Thr Leu Glu Lys His Thr Leu Arg Ser 20 25 30

Glu ThrSerThrTyr Asn Leu Thr Val Gly Asp Thr Gly Ser Gly Leu 35 40 45 lle Val Phe Phe Pro Gly Phe Pro Gly Ser lle Val Gly Ala His Tyr 50 55 60 Thr Leu Gln Ser Asn Gly Asn Tyr Lys Phe Asp Gln Met Leu Leu Thr 65 70 75 80

Ala Gln Asn Leu Pro Ala SerTyr Asn Tyr Cys Arg Leu Val Ser Arg 85 90 95 Ser Leu Thr Val Arg SerSerThr Leu Pro Gly Gly Val Tyr Ala Leu 100 105 110

Asn Gly Thr lle Asn Ala Val Thr Phe Gln Gly Ser Leu Ser Glu Leu 115 120 125 Thr Asp Val SerTyr Asn Gly Leu Met Ser Ala Thr Ala Asn lle Asn 130 135 140

Asp Lys lle Gly Asn Val Leu Val Gly Glu Gly Val Thr Val Leu Ser 145 150 155 160

Leu Pro Thr Ser Tyr Asp Leu Gly Tyr Val Arg Leu Gly Asp Pro lle 165 170 175

Pro Ala lle Gly Leu Asp Pro Lys Met Val Ala Thr Cys Asp Ser Ser 180 185 190

Asp Arg Pro Arg Val Tyr Thr lle Thr Ala Ala Asp Asp Tyr Gln Phe 195 200 205

Ser Ser Gln Tyr Gln Ala Gly Gly Val Thr lle Thr Leu Phe Ser Ala 210 215 22Q

Asn lle Asp Ala lle Thr Ser Leu Ser lle Gly Gly Glu Leu Val Phe 225 230 235 240 Gln Thr Ser Val Gln Gly Leu lle Leu Gly Ala Thr lle Tyr Leu lle 245 250 255

Gly Phe Asp Gly Thr Ala Val lle Thr Arg Ala Val Ala Ala Asp Asn 260 265 270

Gly Leu Thr Ala Gly Thr Asp Asn Leu Met Pro Phe Asn lle Val lle 275 280 285

Pro Thr Ser Glu lle Thr Gln Pro lle Thr Ser lle Lys Leu Glu lle 290 295 300

Val Thr Ser Lys Ser Gly Gly Gln Ala Gly Asp Gln Met Ser Trp Ser 305 310 315 320

Ala Ser Gly Ser Leu Ala Val Thr lle His Gly Gly Asn Tyr Pro Gly 325 330 335

Ala Leu Arg Pro Val Thr Leu Val Ala Tyr Glu Arg Val Ala Thr Gly 340 345 350 SerValValThrValAla Gly Val SerAsn Phe Glu Leu lle Pro Asn 355 360 365

Pro Glu Leu Ala LysAsn Leu Val Thr Glu TyrGly Arg Phe Asp Pro 370 375 380

Gly Ala Met Asn TyrThr Lys Leu lle Leu Ser Glu Arg Asp Arg Leu 385 390 395 400

Gly lle Lys ThrValTrp Pro Thr Arg Glu Tyr ThrAsp Phe Arg Glu 405 410 415

Tyr Phe Met Glu ValAla Asp Leu Asn Ser Pro Leu Lys lleAla Gly 420 425 430

Ala Phe Gly Phe LysAsp lle lle Arg Ala lleArg 435 440

<210> 21 <211> 1093

<212> PRT

<213> Infectious Bronchitis virus <400> 21

Met LeuVal Lys Ser Leu Phe Leu Val Thr lle Leu Cys Ala Leu Cys 1 5 10 15

SerAlaAsn Leu Phe Asp SerAsp AsnAsn TyrVal Tyr Tyr Tyr Gln 20 25 30

SerAla PheArg Pro Pro Asn Gly Trp His Leu Gln Gly Gly Ala Tyr 35 40 45

AlaValValAsn SerThr Asn Tyr ThrAsn AsnAla Gly SerAla His 50 55 60

Val CysThrVal Gly Val lle Lys AspVal TyrAsn Gln SerValAla 65 70 75 80

Ser lle Ala Met ThrAla Pro Leu Gln Gly Met Ala Trp Ser Lys Ser 85 90 95 Gln Phe CysSerAla His CysAsn PheSerGlu lleThrVal Phe Val 100 105 110 ThrHisCysTyrSerSerGly SerGly SerCysPro lleThrGly Met 115 120 125 llePro ArgAsp His lle Arg lle SerAla Met LysAsn Gly Ser Leu 130 135 140

PheTyrAsn LeuThrValSerValSer LysTyrProAsn Phe LysSer 145 150 155 160

Phe Gln CysValAsn Asn PheThrSerValTyrLeu Asn Gly Asp Leu 165 170 175

Val PheThrSerAsn LysThrThrAsp ValThrSerAla Gly ValTyr 180 185 190

Phe LysAla Gly Gly ProValAsnTyrSer lle Met Lys Glu Phe Lys 195 200 205

Val LeuAlaTyrPhe Val Asn GlyThrAla GlnAsp Val lle Leu Cys 210 215 220

AspAsnSerPro LysGly Leu Leu Ala Cys GlnTyrAsnThrGly Asn 225 230 235 240

PheSerAsp Gly PheTyrPro PheThrAsn SerThr Leu Val Arg Glu 245 250 255

Lys Phe lle ValTyr Arg GluSerSerVal AsnThrThr Leu Ala Leu 260 265 270 ThrAsn PheThrPheThrAsn ValSerAsn Ala GlnProAsnSerGly 275 280 285

Gly ValAsnThrPhe His LeuTyrGlnThrGlnThrAla GlnSerGly 290 295 300 Tyr Tyr Asn Phe Asn Leu Ser Phe Leu Ser Gln Phe Val Tyr Lys Ala 305 310 315 320

Ser Asp Phe Met Tyr Gly Ser Tyr His Pro Ser Cys Ser Phe Arg Pro 325 330 335

Glu Thr lle Asn Ser Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu 340 345 350

Thr Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly 355 360 365

Lys Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Lys Gly Pro Met Ala Cys 370 375 380

Lys Gly Val Tyr Ser Gly Glu Leu Ser Thr Asn Phe Glu Cys Gly Leu 385 390 395 400

Leu Val Tyr Val Thr Lys Ser Asp Gly Ser Arg lle Gln Thr Arg Thr 405 410 415

Glu Pro Leu Val Leu Thr Gln Tyr Asn Tyr Asn Asn lle Thr Leu Asp 420 425 430

Lys Cys Val Ala Tyr Asn lle Tyr Gly Arg Val Gly Gln Gly Phe lle 435 440 445

Thr Asn Val Thr Asp Ser Ala Ala Asn Phe Ser Tyr Leu Ala Asp Gly 450 455 460

Gly Leu Ala lle Leu Asp Thr Ser Gly Ala lle Asp Val Phe Val Val 465 470 475 480 Gln Gly lle Tyr Gly Leu Asn Tyr Tyr Lys Val Asn Pro Cys Glu Asp 485 490 495

Val Asn Gln Gln Phe Val Val Ser Gly Gly Asn lle Val Gly lle Leu 500 505 510

Thr Ser Arg Asn Glu Thr Gly Ser Glu Gln Val Glu Asn Gln Phe Tyr 515 520 525 Val Lys Leu Thr Asn Ser Ser His Arg Arg Arg Arg Ser lle Gly Gln 530 535 540

Asn Val Thr Ser Cys Pro Tyr Val Ser Tyr Gly Arg Phe Cys lle Glu 545 550 555 560

Pro Asp Gly Ser Leu Lys Met lle Val Pro Glu Glu Leu Lys Gln Phe 565 570 575

Val Ala Pro Leu Leu Asr lle Thr Glu Ser Val Leu lle Pro Asr Ser 580 585 590

Phe Asn Leu Thr Val Thr Asp Glu Tyr lle Gln Thr Arg Met Asp Lys 595 600 605

Val Gln lle Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Leu Glu Cys 610 615 620

Arg Lys Leu Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn lle Leu Ser 625 630 635 640

Val Val Asn Ser Val Ser Gln Lys Glu Asp Met Glu Leu Leu Ser Phe 645 650 655

Tyr Ser Ser Thr Lys Pro Lys Gly Tyr Asp Thr Pro Val Leu Ser Asn 660 665 670

Val Ser Thr Gly Glu Phe Asn lle Ser Leu Leu Leu Lys Pro Pro Ser 675 680 685

Ser Pro Ser Gly Arg Ser Phe lle Glu Asp Leu Leu Phe Thr Ser Val 690 695 700

Glu Thr Val Gly Leu Pro Thr Asp Ala Glu Tyr Lys Lys Cys Thr Ala 705 710 715 720

Gly Pro Leu Gly Thr Leu Lys Asp Leu lle Cys Ala Arg Glu Tyr Asn 725 730 735 Gly Leu Leu Val Leu Pro Pro lle lle Thr Ala Asp Met Gln Thr Met 740 745 750

Tyr Thr Ala Ser Leu Val Gly Ala Met Ala Phe Gly Gly lle Thr Ser 755 760 765

Ala Ala Ala lle Pro Phe Ala Thr Gln lle Gln Ala Arg lle Asn His 770 775 780

Leu Gly lle Thr Gln Ser Leu Leu Met Lys Asn Gln Glu Lys lle Ala 785 790 795 800

Ala Ser Phe Asn Lys Ala lle Gly His Met Gln Glu Gly Phe Arg Ser 805 810 815

Thr Ser Leu Ala Leu Gln Gln lle Gln Asp Val Val Asn Lys Gln Ser 820 825 830

Ala lle Leu Thr Glu Thr Met Asn Ser Leu Asn Lys Asn Phe Gly Ala 835 840 845 lle Thr Ser Val lle Gln Asp lle Tyr Ala Pro Pro Asp Ala lle Gln 850 855 860

Ala Asp Ala Gln Val Asp Arg Leu lle Thr Gly Arg Leu Ser Ser Leu 865 870 875 880

Ser Val Leu Ala Ser Ala Lys Gln Ser Glu Tyr lle Arg Val SerGln 885 890 895 Gln Arg Glu Leu Ala Thr Gln Lys lle Asn Glu Cys Val Lys Ser Gln 900 905 910

Ser Asn Arg Tyr Gly Phe Cys Gly Ser Gly Arg His Val Leu Ser lle 915 920 925

Pro Gln Asn Ala Pro Asn Gly lle Val Phe lle His Phe Thr Tyr Thr 930 935 940

Pro Glu Ser Phe Val Asn Val Thr Ala lle Val Gly Phe Cys Val Asn 945 950 955 960 Pro Ala Asn Ala SerGln Tyr Ala lle Val Pro Ala Asn Gly Arg Gly 965 970 975 lle Phe lle Gln Val Asn Gly ThrTyrTyr lleThr Ala Arg Asp Met 980 985 990

Tyr Met Pro Arg Asp lleThr Ala Gly Asp lle Val Thr Leu ThrSer 995 1000 1005

Cys Gln Ala Asn Tyr Val Asn Val Asn Lys Thr Val lleThrThr 1010 1015 1020

Phe Val Glu Asp Asp Asp Phe Asp Phe Asp Asp Glu Leu Ser Lys 1025 1030 1035 TrpTrp Asn Asp Thr Lys His Gln Leu Pro Asp Phe Asp Asp Phe 1040 1045 1050

Asn Tyr Thr Val Pro lle Leu Asn lleSer Gly Glu lle Asp Tyr 1055 1060 1065 lle Gln Gly Val lle Gln Gly Leu Asn Asp Ser Leu lle Asn Leu 1070 1075 1080

Glu Glu Leu Serlle lle Lys ThrTyrlle 1085 1090

The invention is described herein in various aspects and embodiments. It should be understood that any combination of these is considered to be within the scope of the invention. However merely for conciseness, not every possible combination is outlined herein in full.

The invention will now be further described by the following, non-limiting, examples. EXAMPLES

Example 1: Generating AIV-MDA positive chicken

1.1. Introduction

In order to be able to test vaccination of seropositive chicken, an animal model resembling the actual situation in the field was created. Specifically, AIV MDA positive offspring was generated, by repeated vaccination of parental hens intramuscularly, using an inactivated-adjuvated vaccine. Aim was to reach HI titres in the offspring that resembled those in the field: at least between 5 and 7 Log2.

1.2. Materials and methods

SPF White Leghorn layer chickens were vaccinated to generate MDA positive hatchlings. All chickens were housed in isolation rooms with floor pens. All chickens were given food and water ad libitum for the duration of the experiment, and were kept under veterinary surveillance.

1.2.1. Preparation of vaccine for MDA generation:

An inactivated AIV vaccine was made by propagating an avian influenza A virus of H9N2 subtype in 10- day old embryonated SPF chicken eggs. Specifically this was AIV strain: A/Chicken/Pakistan- /UDL-01/2008 (‘UDL-01 '), see: GenBank: ACP50708.1 , and: Iqbal et al. (2009, PLoS One, vol. 4: e5788). At 72 hours post infection, eggs were refrigerated at 4°C, and virus was obtained by harvesting the allantoic fluid, which was cleared by centrifugation at 3.000 rpm for 20 minutes. Virus was titrated by plaque assay or TCID50 on Madin-Darby canine kidney (MDCK) cells.

The virus was inactivated chemically using 0.1 % Beta-propiolactone, after which three blind passages were performed 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. Next the inactivated virus was adjuvated with a liquid light paraffin oil, and formulated into a water-in-oil emulsion. The resulting vaccine had a titre of 1040 haemagglutination units (HAU) /ml.

1.2.2. Vaccination of hens and generation of MDA+ hatchlings

A group of 40 SPF White Leghorn layer hens of 17 weeks old, were used. The chickens were marked individually. They were immunised with 0.5 ml of the inactivated-adjuvated H9N2 virus vaccine, at 520 HAU/dose, which was administered i.m. in the leg. The first dose of vaccine was given at 17 weeks of age (T=0), followed by second and third doses at 20 weeks of age (T=3 weeks post first dose) and 41 weeks of age (T=24 weeks post first dose), respectively.

Blood samples were collected from the wing vein of the hens at day 0 and at weeks 5, 11 , 18, 29 and 36 after the first dose, for serological monitoring of the developing anti-AIV HI titre. Five SPF roosters were included in the group for fertilization, but these were not part of the actual study.

Fertilized eggs were collected from 36 weeks after the first vaccination dose. These were set to incubate until hatch. 10 hatchlings were sacrificed at day old (D0) to determine their level of MDA. Their hatchmates were used in the MDA-vaccination experiment. 1.2.3. HI assay

For HI assays, International guidance was complied with (WHO 676 global influenza surveillance network: manual for the laboratory diagnosis and virological surveillance of influenza. 153 (2011)). In short: two-fold serial dilutions of the sera were prepared by mixing 25 μl of serum with 25 μl PBS. Next, 4 HA units of influenza virus was added to the diluted serum and incubated at 37 °C for 1 hour. Finally, 50 μl of 1 % chicken red blood cells were added to the serum-virus mixture and incubated at room temperature for 45 minutes. HI titres were expressed as the reciprocal of the highest dilution of antiserum that caused a total inhibition of the 4 units of virus hemagglutination activity.

The virus used in the HI assays was AIV H9N2 of strain UDL-01.

1.3. Results

The results of the hyper-immunisation of the mother hens to generate AIV MDA+ offspring, are depicted in Figure 1. HI titrations were done with the homologous UDL-01 strain.

As a drop in HI titres was observed in the hen's sera at 18 weeks post start, a third vaccination was given. This resulted in very high HI titres in the hens, which remained at that level until the last sampling point.

Fertilized eggs were collected at 36 weeks post start (53 weeks of age), when the average (n=10) HI titre of the hens was 4096 (12 Log2).

As is clear from these results, and indicated in Figure 1 , there were significant differences between the HI titres at T=11 and T=18 weeks (p<0.05), and between T=18 and T=29 (p<0.001).

The HI titres induced by the MDA in the (unvaccinated) offspring from these hens was measured, at day of hatch and overtime: at day 1, 7, 14, 21, 28, 35, 42, 56, 70 and 84 post hatch. Results are depicted in Figure 2. HI titrations were done with the homologous UDL-01 strain.

At day 1 , the HI titres in the chicks averaged (n=10): 588 (9.2 Log2). This titre had dropped slightly (not significant) at day 7 of age, but was more than halved at day 14 of age, at 181 (7.5 Log2), and rapidly declined further after that: at day 35 the average (n=10) HI titre was 16 (4 Log2), and no HI titre was detectable anymore at day 42 of age.

The International standard for protection from AIV mortality as defined by the OIE (www. oie.int/fileadmin/Home/eng/Health_standards/tahm/3.03.04_Al. pdf) is at an HI titre of 32 (5 Log2). The hatchlings used experimentally here were found to still have an HI titre around this value at 28 days of age, but these chicks started off far above normal MDA levels. Therefore additional active vaccination is normally required.

For confirmation the antibody titres of the hatchlings were also tested by ELISA, to assure the antibodies measured were directed at AIV H9 HA. A commercial kit was used according to the manufacturer's instructions: ID Screen® Influenza H9 Indirect kit (ID Vet), which is an indirect ELISA. The ELISA scores found, closely matched the pattern of the HI scores. This confirmed that the HI titres detected in the hatchlings originated from antibodies specific for AIV H9 HA. Example 2: Preparation of vaccines for MDA+ avians

2.1. Introduction

Three vaccines were used in the vaccination of the seropositive avians.

The positive control was a classic 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, strain Clone 30.

The HA proteins of AIV H9N2 strains UDL-01 and UAE have 94 % amino acid identity when aligned over their full length.

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

Further, two variants of a recombinant HA antigen-based vaccine were used: one version untargeted, and one targeted to CD83 by a fusion to a CD83-scFv. This last version is a recombinant protein for use according to the invention.

2.2. Materials and methods

2.2.1. Preparation of HA antigen expression constructs

A mouse hybridoma producing antibodies against chicken CD83 (GenBank acc. nr. XM_040663657.1) was used to obtain the vL and vH chain sequences. Synthetic cDNA containing the vL and vH sequences were joined by (Gly ₄ Ser) ₄ linker peptide sequence and manufactured commercially by Geneart (Thermo Fisher Scientific). The vH-Linker-vL cDNA was then cloned into a D. melanogaster expression vector: pMT-BIP-V5-His™ (Version A, Thermo Fisher Scientific) using the Notl and Xbal 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. Further, multiple cloning sites, a V5 epitope for recombinant protein detection, and a 6xHis tag for recombinant protein purification are provided in this plasmid.

The resultant vector named pMT-BIP-CD83-scFv-V5-His was used to insert the ectodomain of an H9 HA gene that lacked the HA gene signal peptide and the TM domain. A 29 amino acid trimerization Foldon sequence was added from the trimeric protein fibritin from bacteriophage T4, using Kpnl and Pad restriction sites. This plasmid comprised the nucleotide sequence of SEQ ID NO: 14, under the operational control of the MT promoter.

The H9 HA used in this study was synthetically produced incorporating consensus sequence of HA of H9N2 viruses derived from analysis of over 2000 H9 HA sequences from the public databases of G1 -like H9 virus lineage using the Minimum Sphere Consensus (MScon) method (Kim et al., 2015, abstracts from German Conference on Bioinformatics, Dortmund, September 27th-30th 2015, poster 20: PeerJ Preprints 3:e1350v1), which is also closely related to the COBRA technique (Giles et al., 2011, Vaccine, vol. 29, p. 3043 - 3054).

This synthetic HA has 98 % amino acid sequence identity to the HA ectodomain of the H9N2 virus of strain UDL-01 (GenBank accession number: ACP50708.1, HA1: aa 19-338 and HA2: aa 339- 560), which would qualify it as homologous, and is codon optimised towards S2 cells.

The H9 HA-Foldon antigen without CD83 targeting signal was prepared in a similar way, to provide plasmid pMT-BIP-H9HA-Foldon-V5-His. This plasmid comprised the nucleotide sequence of SEQ ID NO: 15, under the operational control of the MT promoter.

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 foetal bovine serum and grown at 28 °C. The cells were passaged once a week by centrifuging at 1200 rpm for 10 minutes, and resuspending in fresh complete S2 cell medium.

Recombinant proteins were produced and purified using the Drosophila Expression System (DES®, Life Technologies). In short: the plasmids pMT-BIP-rH9HA-V5-His and pMT-BIP-rH9HA-CD83-scFv-V5-His, were each co-transfected into S2 cells using calcium phosphate transfection. Prior to the transfection, S2 cells at 1x10 ^Λ 6/mL had been pre-seeded in 5 mL of complete S2 cell growth medium for 6 to 16 hours at 28 °C. A transfection solution was prepared by adding 60 μL 2 M CaCl ₂ , 32 μg of expression plasmid DNA, 1.5 μg of a hygromycin B resistance plasmid (pCoHYGRO, Life Technologies), and sterile water to bring the total volume up to 500 μL. The transfection solution was slowly added to the equal volume of 2x Hepes buffered saline (HBS) and incubated at room temperature for 30 minutes. The resulting solution was slowly added dropwise to the pre-seeded S2 cells and incubated for 24 hours at 28 °C. At 24 hours post transfection, the transfection medium was replaced with fresh complete S2 cell medium and the cells were incubated for 3 more days at 28 °C.

Stable S2 transfectants were generated by antibiotic selection: complete growth medium containing hygromycin B at 250 μg/mL was added every week for at least 4 weeks.

Next a single cell clone was obtained via limiting dilution (Zitzmann et al., 2010, Biotechnol. Reports, vol. 19, e00272). In short: 2 ^c 10 ^Λ 3 S2 transfected cells were mixed with gamma-irradiated 10 ^Λ 6 parental S2 cells as feeder cells. 100 μL of this mixture of cells was seeded into each well of 96-well plates. The single clones within each well became clearly visible after 4 weeks of incubation at 28 °C. About 10-15 single clones were screened per plasmid construct. The single clones expressing the highest amount of recombinant protein were selected by indirect ELISA for H9 HA protein.

2.2.3. Expression and purification of recombinant antigens

The selected transfected S2 cell clones were then cultured at larger scale. In short: single clones expressing high amounts of the HA recombinant proteins were grown in 2 litre roller bottles (Corning) containing 400 mL of Ex-Cell® 420 serum-free medium (Merck GmbH Life Science) for expression and purification. The metallothionein promoter in the plasmids used was induced by adding CuSO ₄ to a final concentration of 500 μM. After 4 days post induction, the cell supernatants were harvested via centrifugation at 1200 rpm for 20 minutes and dialysed to remove the excess copper ions. In total, about 2 litres of protein expression supernatants were collected and filtered through 0.22 μM filter stericup (Merck GmbH Life Science) before purification.

The use of the His tag allowed purification of the recombinant proteins by metal-affinity column chromatography. In short: the dialysed and filtered supernatants containing recombinant proteins were loaded on 10 mL Profinity™ IMAC uncharged resin column (Bio-Rad) and washed with 5 column volumes of wash buffer. Copper-bound proteins were then eluted with elution buffers containing increasing concentration of imidazole (25, 50, 100 or 500 mM). The purified proteins were analysed using SDS- PAGE on 10 % PAA gels, followed by Coomassie Blue staining. The protein fractions were combined and concentrated using 15 mL Amicon Ultra-15™ Centrifugal Filter column (3 kDa MWCO, Merck GmbH Life Science) by centrifuging at 4600 rpm for 30 minutes. The concentration of the purified proteins was determined using a Pierce BCA Protein Assay Kit™ (Life Technologies) according to the manufacturer's instructions.

The H9 HA activity of the recombinant proteins produced was confirmed using a haemagglutination assay. Briefly, 35 μg of the recombinant protein was serially diluted 2-fold in PBS in V- bottom 96-well plates. 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 hour, tipped 90° in a biosafety cabinet to visualise haemagglutination, and scored.

2.2.4. Preparation of vaccine emulsions

The recombinant HA antigen vaccines were formulated as water-in-oil emulsions, with light liquid paraffin oil (Marcol® 52) as adjuvant, and contained Polysorbate 80 (Tween® 80) and Sorbitan mono- oleate (Span® 80) as emulsifiers. The water:oil weight-ratio of the vaccines was 45:55. All vaccines were stored at 4 °C until use.

The recombinant HA vaccines contained per dose of 0.2 ml: 35 μg of the untargeted HA antigen, or 49 μg of the targeted antigen. This difference was to provide equimolar amounts, compensating for the addition of the scFv

Example 3: Vaccination of seropositive birds

3.1. Introduction

As the protection against AIV infection and disease is essentially serologically determined, and the main AlV-neutralising antibodies are those against the HA antigen, therefore serological testing for anti-HA antibody development, i.e. HI titre determination, is an excellent predictor of in vivo protection from AIV.

The hatchlings generated as described in Example 1 , were used in a vaccination experiment: one group was vaccinated at day 1 , these had a very high average MDA HI titre of 588 (9.2 Log2), called the MDA++ group. One other group was only vaccinated at day 14 of age, when MDA levels had decreased somewhat, these had a medium average MDA HI titre of 181 (7.5 Log2), called the MDA+ group.

This approach allowed the testing and comparison of a ‘worst case' and an ‘average case' of antibody interference, respectively, on the efficacy of vaccination with targeted or untargeted HA antigen. For comparison a classic inactivated H9N2 vaccine was included. Also a non-vaccinated group of chicks was included in the study to follow the natural decline of the anti-AIV H9 HA MDA levels.

3.2. Materials and methods

3.2.1. Animals, sampling and vaccinations

The AIV H9 HA MDA positive chicks used, were obtained as described in Example 1. The vaccines used were as described in Example 2.

To avoid incoming environmental pathogens, the birds were housed in positive pressure isolation rooms with high-efficiency particulate air (HEPA) filtered air inflow.

After hatching, only chicks that appeared healthy and normal were used. These were assigned to the groups as they came to hand, and were individually numbered. Daily clinical observations were made to monitor health and performance. Each test group had 10 animals.

All vaccines were at ambient temperature at use, and were mixed strongly just before use to ensure homogeneity.

All chicks received only a single vaccination, either on day 1 or on day 14 of age. The administration was via the standard route for these types of vaccines: subcutaneous (sc). A volume of 0.25 ml/dose was used for the Nobilis® vaccine as that is the registered dose; for the recombinant HA antigen vaccines, 0.2 ml/dose was given.

Nobilis Influenza H9N2+ND vaccine was given at day 1 to MDA++. The H9HA-Foldon and the H9HA-Foldon-CD83-scFv vaccines were given both to the ‘MDA++' chicks at day 1 , and to the ‘MDA+' chicks that were then 14 days of age.

Blood samples were collected once every week up to week 6 post start of the experiment, and once every two weeks for weeks 8, 10 and 12 post start, to determine the serological responses induced by the vaccinations.

Blood samples at days 1 and 7 were collected after euthanisation; samples from day 14 onwards were taken from the wing vein. Volumes collected were 2-3 ml, as permitted by the weight of the animals. Blood samples were left at ambient temperature to clot, and serum was separated by centrifugation. The serum samples were heat-inactivated for 30 min. at 56 °C, and stored at -20 °C until use.

3.3. Results

The results of the HI titrations with the serum samples taken during this experiment from the MDA++ and the MDA+ chicks, are presented in Figures 3 and 4 respectively. The non-vaccinated controls showed a level of HI titre, and a pattern of degradation, as described in Example 1 , and Figure 2.

The positive controls were MDA++ chicks receiving whole inactivated virus vaccine (‘Nobilis Influenza H9N2+ND') at day one of age. In spite of this vaccination, their HI titres steadily declined, and no vaccination response could be detected. This is remarkable, as the MDA and the HA antigen in the classic vaccine were heterologous: the MDA were induced against an HA antigen that closely resembled the H9 HA of strain UDL-01 , while the Nobilis vaccine contained a heterologous H9 HA antigen, namely that from strain UAE, which has 94 % amino acid identity to the UDL-01 H9 HA protein. Consequently one would expect a lesser level of antibody interference because of this difference between the HA antigens. Apparently however, the HI levels in the MDA++ chicks were so high that they even interfered with the efficacy of a heterologous H9 HA vaccine.

The vaccinations with targeted (Ή9HA Foldon-CD83-scFv') and untargeted (Ή9HA Foldon') HA antigen showed spectacular differences in the HI titres they induced, both in the MDA++ and in the MDA+ chicks.

The HI titres in the chicks vaccinated with untargeted HA antigen steadily decreased and did not show any significant rise in HI titre at any time point after vaccination, neither in the MDA++ nor in the MDA+ chicks.

However the targeted HA antigen induced very high HI titres. In the MDA++ group there was an initial decline from the very high starting value (9.7 Log2), but the HI titres showed a strong and steady increase after that: visible from 4 weeks post vaccination (p.v.), reaching significance at 5 weeks p.v., and strongly increasing to 9.7 log2 at 12 weeks p.v.. This demonstrates that this vaccine can be applied at day 1 of age, even in the context of very high levels of homologous MDA, and is still capable of inducing a strong protection against AIV infection and disease.

In the MDA+ group the HI titres from the targeted HA vaccine showed a rapid induction of high HI titres, already from 1 week after vaccination. The HI titre reached an average of 1835 (10.8 Log2), from 4 weeks after vaccination.

In both test groups, the targeted vaccine was the only one capable of inducing significantly increased HI titres. Also, the lowest HI titre measured in the targeted vaccine groups was 6.2 and 6.9 Log2 in the MDA++ and MDA+ groups respectively. This indicates that all chicks receiving this type of vaccine, remained well above the 5 Log2 threshold for protection, for the duration of the experiment.

This rapid onset of immunity, and long duration, perfectly compensate for the drop-off in MDA level, without leaving a gap in the protection.

Again indirect ELISA was performed on the sera, which confirmed that all antibodies were H9 HA specific. Example 4: Tarqetinq of non-HA antiqens

Experiments essentially similar to those described above are in preparation for recombinant proteins for use according to the invention, but comprising another antigen than AIV HA. These are: AIV HN, NDV F, NDV HN, IBDV VP2, and IBV spike. In short: hens can be vaccinated with a suitable vaccine against one of these pathogens: AIV, NDV, IBDV, or IBV; such vaccines are generally available.

The hens can be given 2 or 3 vaccinations, starting before onset of lay, and continuing during their laying period. The specific antibody titres reached in the hens can be checked to be sufficiently high. Then eggs can be collected and hatched, and the chicks can be checked for having sufficiently high MDA levels for the pathogen to be studied.

Vaccines can be prepared comprising a recombinant protein for use according to the invention, as described above, for example by constructing an expression plasmid, comprising a nucleotide sequence encoding one of the antigens to be tested. Also a binding domain will be comprised, e.g. an scFv, directed at an avian APC's surface protein, such as CD83, CD11c, or Dec-205. Similar constructs but without a binding domain can be prepared to serve as control, to assess the effect of the targeting of the antigen to the APC.

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

An example of an CD83-scFv is a peptide comprising the amino acid sequence of SEQ ID NO: 2. An example of an scFv specific for CD11 c or Dec-205, is a peptide comprising an amino acid sequence as presented in SEQ ID NO: 16 or 17, respectively.

Examples of antigens to be expressed comprise an amino acid sequence selected from:

- for AIV H5 HA: SEQ ID NO: 4;

- for AIV H7 HA: SEQ ID NO: 5;

- for NDV F: SEQ ID NO: 18;

- for NDV HN: SEQ ID NO: 19;

- for IBDV VP2: SEQ ID NO: 20, and

- for IBV spike: SEQ ID NO: 21.

The corresponding nucleic acids, encoding these antigens are preferably codon optimised towards the codon usage table of S2 cells. In the expression construct, additional elements can be added when desired, such as a signal sequence, a linker, and one or more tags, in order to facilitate expression, secretion, and purification,.

Next, the chicks with the specific MDAs will be vaccinated with these recombinant proteins, and their serology will be monitored overtime.

Because for these pathogens the levels of specific antibodies are known that correlate with in vivo protection, therefore serological testing of antibody levels at various times after vaccination suffices to get a good impression on the efficacy of targeted vaccination in avians seropositive for the antigen from these pathogens.

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. AXY66755.1. Selected were 511 aa of the HA ectodomain: HA1 : 17-340 and HA2: 346-530. The polybasic cleavage sequence was modified: from PLR to PQG, and the number of arginines was reduced.

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. Selected was the HA ectodomain of 507 amino acids: HA1: 19-339 and HA2: 1-186, with a modified polybasic cleavage sequence: from PKR to PKG.

The NDV F sequence of SEQ ID NO: 18, is the consensus sequence from over 1200 F amino acid sequences from avian avulavirus 1 sequences in public databases, using the MScon technique as described herein. The consensus F protein has 98.5 % amino acid similarity to the closest natural relative: avian orthoavulavirus 1 F protein, GenBank acc. nr. AHX74055.1. The F protein ectodomain was selected from aa. 31 - 500.

The NDV HN sequence of SEQ ID NO: 19, is a consensus sequence, starting from the HN protein from Avian orthoavulavirus 1 of GenBank acc. nr. AXK59828.1 , combined with a number of HN sequences from the public databases, using the MScon technique as described herein. Amino acids 47 - 571 from the HN were selected.

The IBDV VP2 protein of SEQ ID NO: 20, represents aa 9 - 452 from IBDV VP2 protein of GenBank acc. nr. AMA19770.1.

The IBV spike protein of SEQ ID NO: 21, represents aa 1 - 1096 from IBV spike protein of GenBank acc. nr. ARS22410.1. The spike protein was stabilised by making two amino acid substitutions: Q859P and L860P.

LEGEND TO THE FIGURES

Figure 1

Presentation of the results of antibody titres in hyper-immunised mother hens, in order to generate AIV MDA+ offspring. Details are described in Example 1.

The vertical axis presents the average (n=10) HI titres measured by HI assay in the serum of mother hens, after immunisation with inactivated-adjuvated AIV H9N2 virus vaccine (UDL 01/08). The time points are indicated on the horizontal axis, in weeks post start of the experiment (day 0 = 17 weeks of age). Vaccinations are indicated by arrows, at 0, 3, and 24 weeks post start.

Fertilized eggs were collected after 36 weeks post start; the box indicates the average (n=10) HI titre in the hen's serum at the time of laying the eggs that were used in the follow up experiment: HI = 12 Log2 (4096).

Data are presented as mean (columns) and standard deviation (error bars). The asterisks represent a significant difference between the HI antibody titres at 11 and 18 weeks post start of the experiment, and at 18 and 29 weeks post start, whereby: * = p<0.05, and *** = p<0.001.

Figure 2

Presentation of the results of the MDA derived HI titres in unvaccinated offspring from the triple vaccinated hens. Details are described in Example 1.

Anti-H9 HA MDA titres were measured by HI assay in serum samples from day 1 , 7, 14, 21 , 28, 35, 42, 56, 70 and 84 post hatch. HI titres are expressed as the reciprocal of the highest dilution of the serum causing the total inhibition of 4 HA units of virus hemagglutination activity. Data are presented as mean ± SD, and were analysed by one-way ANOVA followed by Tukey's multiple comparison test. Statistically significant differences are shown as: **** = p<0.0001.

The dotted horizontal line indicates the minimal protective level of the HI titre of 32 (5 Log2).

Figure 3

Presentation of the results of the HI titres in the chicks vaccinated at day 1 of age, having high levels of MDA (MDA++). Details are described in Example 3.

The vertical axis indicates HI titres, and the horizontal axis the days post vaccination. NB: There is a gap in the vertical axis to be able to display the very high HI titres found.

Groups of MDA++ chicks (n=10) were vaccinated at day 1 of age with one of three vaccines: whole inactivated virus vaccine (‘Nobilis Influenza H9N2+ND'); untargeted HA antigen (Ή9HA Foldon'); or CD83 targeted HA antigen (Ή9HA Foldon-CD83-scFv'). As controls, one group of MDA++ chicks remained unvaccinated.

Anti-H9 HA antibody titres were measured by HI assay, using the UDL-01 virus in the HI assay.

The data are presented as mean (columns) and SD (error bars). Statistically significant differences are indicated with asterisks, whereby: *** = p<0.001 and * = p<0.1. Figure 4

Presentation of the results of the HI titres of the chicks vaccinated at day 14 of age, having medium levels of MDA (MDA+). Details are described in Example 3. Similar presentation as for Figure 3, except that there was no group vaccinated with the Nobilis vaccine.