HA stem vaccine for HA antibody-positive targets

Abstract

The present invention relates to a vaccine against influenza virus infection or disease for targets with pre-existing antibodies against the influenza virus HA head domain. The present invention relates to a recombinant vector expressing an HA stem polypeptide, a vaccine comprising the vector, or a host cell together with the vector, the use of the vector, the host cell, or the vaccine, and a method for alleviating influenza virus infection or disease. The recombinant vector can be a nucleic acid such as a eukaryotic expression plasmid or RNA, a virus, or a replicon particle (RP). This vaccination allows for the induction of an early and effective immune response against influenza virus-induced infection or disease without being hindered by pre-existing anti-HA head domain antibodies.

Description

Technical Field

[0001] The present invention relates to the field of vaccinology, and more particularly to vaccines against influenza. In particular, the present invention relates to vaccines against influenza virus infection or disease for targets having existing antibodies against the influenza virus HA protein. The present invention relates to recombinant vectors expressing HA stem polypeptides, vaccines comprising the vector or host cells together with the vector, the use of the vector, host cells or vaccines, and methods for reducing influenza virus infection or disease.

Background Art

[0002] Influenza viruses occur worldwide and infect both humans and animals. The diseases caused are mainly respiratory and have various accompanying symptoms. The pathology varies from mild to lethal, causing much discomfort and economic damage. Furthermore, humans can be infected with common infectious diseases by infected birds or pigs.

[0003] Influenza viruses are enveloped viruses of the Orthomyxoviridae family, possessing a segmented single-stranded negative-sense RNA genome. Influenza viruses A through D are distinct genera within their family, distinguished based on their structural proteins (matrix and nucleoproteins). The most prominent worldwide are influenza A, B, and C viruses. Of these, the genus influenza A includes several virus strains that infect only specific target species, as well as those with a host range of multiple species. Serological differentiation is performed based on expressed viral envelope glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Currently, 18 different HA antigens are known, designated as H1-H18 and 11 NA antigens: N1-N11. Details regarding influenza viruses and the diseases they can induce can be found in well-known handbooks such as: Fields Virology (LWW publ., ISBN: 9781451105636); The Merck veterinary manual (2010, 10th ed., CMKahn edt., ISBN: 091191093X); and Diseases of poultry (2008, 12th ed., Y. Saif ed., Iowa State Univ. Press, ISBN-10: 0813807182). Influenza infections in poultry are also known as avian plague, avian influenza (avian flu, or bird flu).

[0004] The HA protein is the primary antigen of influenza A and B viruses and is central to viral recognition, binding, and entry into host cells. In its natural form, the HA protein is a homotrimer presented on the viral envelope (or "coat") and the membrane of infected host cells. Each monomer has a spherical head domain connected to a transmembrane domain via a stem domain. The head domain results in trimerization and mediates the initial contact with the receptor on the host cell. The stem (or stalk) domain of HA induces fusion with the endosomal membrane of the host cell after viral endocytosis.

[0005] To prevent immature membrane fusion, the HA protein is essentially expressed as an inactive preprotein HA0 (HA-zero) that is post-translationally cleaved into HA1 and HA2 sections by host proteases. The central portion of HA1 forms the head domain of the HA monomer. The major portion of HA2, together with the N-terminal and C-terminal portions of HA1, forms the stem domain. Further HA2 contains a transmembrane domain and a short cytoplasmic domain at its C-terminus.

[0006] Of the HA antigen, the head domain is the immunodominant portion. Consequently, when a target is immunized with a preparation containing the HA head domain or its antigenic portion, such as the full-length HA protein, or even the entire influenza virus preparation, the antibodies produced in the target are primarily directed towards the HA head domain. Since the portion of the HA gene encoding the head domain is sequence-variable, this explains much of the antigenic drift exhibited by (seasonal) variants of the influenza virus. As a result, there is a constant need for novel and updated vaccines against influenza viruses targeting both humans and animals.

[0007] Influenza vaccination is routinely administered in both human and veterinary medicine. The goal is typically to reduce the severity and duration of clinical signs, and preferably also to reduce the amount and duration of viral shedding by the infected host. Several different types of vaccines are available, for example, based on attenuated live viruses, or based on inactivated viruses, viral preparations, such as surfactant extracts (so-called "split" vaccines), or adjuvanted formulations of HA and / or NA protein subunit vaccines. Vaccines may also be based on recombinant products, such as expression plasmids, mRNA, vector viruses, or virus-like particles.

[0008] In veterinary practice, influenza vaccines are available for a variety of animal species. Such vaccinations may be applied incidentally, for example, to poultry or pigs, as emergency vaccinations in outbreak situations. Alternatively, in countries with high pressure for influenza virus infection, vaccinations may be applied routinely to pigs and poultry, as well as to very young offspring.

[0009] Vaccinated target humans or animals may contain (maternally derived) antibodies against influenza, resulting from prior contact with the virus from vaccination or field infection, either in the target itself or its mother. Such pre-existing antibodies in the target are known to interfere with the effectiveness of influenza vaccination. This significantly reduces the effectiveness of influenza vaccination for such antibody-positive targets, leaving them exposed to field infection. To date, no effective solution to this problem has been found.

[0010] In contrast to the HA head domain, the HA stem domain is more conserved among influenza virus strains and, when administered without the HA head domain (i.e., "headless"), can be used to induce broad-spectrum virus-recognizing antibodies. This was already recognized in 1993 (Okuno et al., 1993, J. of Virol., vol. 67, pp. 2552-2558). Since then, many studies have used so-called "headless HA," "mini-HA," or "HA stem" polypeptides as vaccine antigens in attempts to provide a universal influenza vaccine. This has been reviewed, for example, by Krammer and Palese (2013, Curr. Opin. Virol., vol. 3, pp. 521-530) and Ostrowsky et al. (2020, Curr. Opin. Virol., vol. 40, pp. 28-36).

[0011] Much detail is known regarding the structure of the influenza HA protein. Reviews include, for example, Skehel and Wiley (2000, Annu. Rev. Biochem., vol. 69, p. 531-569), Sriwilaijaroen and Suzuki (2012, Proc. Jpn. Acad., Ser. B, vol. 88, p. 226-249), and Russell (2016, Ref. Module in Biomed. Sci., doi: 10.1016 / B978-0-12-801238-3.95721-0). Exemplary graphical representations of various domains and segments of the influenza A HA protein are presented in Figure 1 of Lu et al. (2014, PNAS, vol. 11, p. 125-130).

[0012] International Publication No. 2011 / 123495 (No. 495) describes influenza HA stem domain polypeptides and their use as vaccines. HA stem polypeptides comprise the HA1 N-terminal stem segment, the HA1 C-terminal stem segment, and HA2, sometimes with one or more linker and trimer domains. No. 495 presents several exemplary alignments of HA amino acid sequences in Figures 1 and 2, and in Tables 1-7, exemplary amino acid sequences are described for various domains derived from different HA serotypes of influenza A and B. While No. 495 mentions versions of HA2 with and without transmembrane domains, it does not allow for the disclosure of such constructs in recombinant vectors, nor has it yielded results from any inoculation of stem polypeptides into target humans or animals, much less any vaccination or challenge experiments. No. 495 also does not disclose the use of maternal or other existing anti-influenza HA antibodies as targets. In fact, Issue 495 does not specifically recommend its use in younger target groups, such as human children under 6 months of age (see paragraphs 397, 404, and 405 of Issue 495).

[0013] International Publication No. 2013 / 079473 describes the use of HA stem antigens derived from H1 and H3 influenza viruses as broadly protective vaccine antigens. Several mutations were introduced into HA2 to enhance stability and immunogenicity. Expression constructs were optimized using linker and signal sequences. Trimerization domains and transmembrane (TM) domains were added to the HA stem antigen in some constructs to induce multimerization in the absence of the head domain. Vaccination was performed in specific pathogen-free (SPF) mice using expression plasmids, with occasional booster vaccination using adjuvant-added soluble proteins, and in some cases, induced infection.

[0014] The paper by Impagliazzo et al. (2015, Science, vol.349, pp.1301-1306) replicated the study in international publication 2013 / 079473 by testing soluble HA stem antigen (i.e., without the TM domain) in SPF mice and seronegative macaques.

[0015] Sunwoo et al. (2018, Vaccines, vol. 6, p. 64) used chimeric HA proteins as a vaccine in pigs with maternal antibodies (MDA) against influenza virus. This was to investigate the association between vaccine-enhanced respiratory disease and stem-specific antibodies. The antigens tested consisted of chimeric full-length HA proteins in which the head domain was altered but the stem domain remained constant. These were administered to H1 HA MDA-positive pigs as attenuated live recombinant influenza viruses, inactivated viruses, or viral extracts (split vaccines). Stem-specific antibodies could not be detected after vaccination, but some protection against induced infection was obtained using heterologous prime-boost vaccination regimens. [Prior art documents] [Patent Documents]

[0016] [Patent Document 1] International Publication No. 2011 / 123495 [Patent Document 2] International Publication No. 2013 / 079473 [Non-patent literature]

[0017] [Non-Patent Document 1] Fields Virology(LWW publ.,ISBN:9781451105636);The Merck veterinary manual(2010,10th ed.,CMKahn edt.,ISBN:091191093X) [Non-Patent Document 2] Diseases of poultry(2008,12th ed.,Y.Saif ed.,Iowa State Univ.press,ISBN-10:0813807182).

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Summary of the Invention

Problems to be Solved by the Invention

[0018] An object of the present invention is to provide an influenza vaccine effective against a target having an antibody against the HA head domain of influenza virus at the time of vaccination, corresponding to the needs of the art.

Means for Solving the Problem

[0019] Surprisingly, for vaccination of a target having an antibody against the influenza virus HA head domain, this object can be achieved by using a recombinant vector capable of expressing a polypeptide having a headless HA stem domain with a transmembrane domain and a trimerization domain, and as a result, it has been found that one or more drawbacks of the prior art can be overcome.

[0020] When vaccinating chickens against influenza, the inventors were disappointed to find that little protection was achieved after single vaccination with the soluble HA stem antigen, as described by WO 2013 / 079473 and Impagliazzo et al. (supra). Clearly, the effective and broad protection provided by the HA stem antigen as described in the literature could not be readily reproduced, even in animals without antibodies and even when using adjuvants. How this could be changed was not shown in the literature.

[0021] The HA stem antigen provided good vaccine efficacy in targets with influenza MDA even after single vaccination only when it was delivered by expression from a recombinant vector, provided with a transmembrane domain, and thus expressed on the surface of host cells. This has not been previously disclosed in the art.

[0022] The HA stem domain antigen having a transmembrane domain can be expressed in a target by administration of different types of recombinant vectors, such as expression plasmids, replicon RNAs, recombinant viral vectors, or as replicon particles (RPs). This provides a broad opportunity for use as an influenza vaccine for targets having existing antibodies against the influenza virus HA head domain at the time of vaccination.

[0023] It is unknown how or why vector-expressed and membrane-presented HA stem domains are far more effective as antigens than soluble HA stem antigens in relation to existing HA antibodies. While we do not wish to be bound by any theory or model that could explain these findings, we hypothesize that membrane anchors influence the macromolecular structure of HA stem antigens, resulting in more effective presentation of the stem antigen to the immune system of sequential targets. This would enable the induction of an early and effective immune response at vaccination targets by HA stem domain antigens without interference from existing anti-HA head domain antibodies.

[0024] Accordingly, in one embodiment, the present invention relates to a recombinant vector capable of expressing a recombinant influenza virus hemagglutinin (HA) stem polypeptide in a target for use in mitigating infection or disease caused by influenza virus in a target having antibodies against the influenza virus HA head domain at the time of vaccination, wherein the polypeptide comprises a headless influenza virus HA stem domain, a trimerizing domain, and a transmembrane domain.

[0025] A "vector" is a well-known molecular structure in the field of this invention that transmits genetic information (nucleic acid sequences) to encode a polypeptide via an appropriate signal that enables its expression under appropriate conditions, such as within a host cell. For the purposes of this invention, "expression" refers to the well-known principle of protein expression from genetic information by transcription and / or translation.

[0026] Many types and variants of such vectors are known and can be used for the purposes of this invention, ranging from nucleic acid molecules such as DNA or RNA to more complex structures such as virus-like particles and replicon particles, and even for the replication of recombinant microorganisms such as viruses.

[0027] Depending on the type of vector used, more or less expression signaling needs to be supplied, either in cis (i.e., supplied within the recombinant vector itself) or trans (i.e., supplied from a separate source).

[0028] The “recombinant” vectors of this invention are vectors whose genetic composition does not perfectly match that of their natural counterparts. Therefore, such vectors typically have a molecular composition altered by in vitro manipulation of their genetic information using molecular cloning and recombinant protein expression techniques. The alterations made may help improve or adapt the expression, manipulation, purification, stability, and / or immunological behavior of the vector and / or the protein it expresses. These and other techniques are described in detail in standard texts such as Sambrook & Russell: “Molecular cloning: a laboratory manual” (2001, Cold Spring Harbour Laboratory Press; ISBN: 0879695773); Ausubel et al., in: Current Protocols in Molecular Biology (J. Wiley and Sons Inc, NY, 2003, ISBN: 047150338X); 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).

[0029] Those skilled in the art will have sufficient ability to select and combine the necessary signals into a functional combination so that a recombinant vector for use according to the present invention can "express" the HA stem polypeptide of the present invention under appropriate conditions. Alongside elements that aid in construction and cloning, such as restriction enzyme recognition sites or PCR primers, well-known elements can be selected from one or more of the following: promoters, stop codons, termination signals, polyadenylation signals, 7-methylguanosine (7mG) cap structures, and introns having functional splice donor and acceptor sites.

[0030] "Influenza viruses" are well known in the field of the present invention, and such viruses have characteristic features of their taxonomic group, such as morphological features, genomic features, and biochemical features, as well as biological features such as physiological, immunological, or pathological behavior.

[0031] General information on these viruses is available, for example, from the reference handbook cited herein. Samples of influenza viruses for use in the present invention can be obtained from various sources, for example, as field isolates from humans, or as field isolates from wild or farm animals, or from various laboratories, (depositary) institutions, or (veterinary) universities. Influenza viruses can be readily identified using routine serological, biochemical, or molecular biological tools. Furthermore, a great deal of sequence information on influenza viruses is available digitally in publicly available sequence databases such as NCBI's GenBank and EMBL's EBI. In addition, detailed structural information on HA proteins is available at the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) at www.rcsb.org and the Influenza Research Database at www.fludb.org.

[0032] As is also known in the art, the classification of microorganisms within a particular taxonomic group is based on a combination of their characteristics. Accordingly, the present invention also includes variants of viral species that are subclassified in some way therefrom, such as subspecies, strain, isolate, genotype, variant, subtype, or subgroup.

[0033] Furthermore, while certain viruses of the present invention may now be assigned to a species, it will be apparent to those skilled in the art that this is a taxonomic classification that can change over time, as new insights may lead to reclassification into new or different taxonomic groups. However, since this does not change the virus itself or its antigenic repertoire, but only its scientific name or classification, such reclassified viruses remain within the scope of the present invention.

[0034] Hemagglutinin (HA), also known as hemagglutinin, is a well-known envelope glycoprotein of influenza A or B virus, encoded by the fourth segment of the viral genome. The influenza A HA gene encodes a preprotein approximately 566 amino acids (aa) in size. Without the signal sequence, the mature HA0 protein is approximately 550aa in size, HA1 is approximately 329aa, and the HA2 portion is approximately 221aa.

[0035] In this invention, the naming of the various domains, segments, and sections of the influenza HA protein follows Lu et al. (previously cited). Furthermore, regarding the amino acid numbering of different subtypes of HA proteins, the standard uniform numbering used in this art is based on the so-called "H3 numbering." This is because the HA of the 1968 Hong Kong-derived influenza virus isolate: A / Aichi / 2 / 68(H3N2) was the first to have its crystal structure completely analyzed and subsequently sequenced (Verhoeyen et al., 1980, Nature, vol.286, pp.771-776). The amino acid sequence of the 566aa full-length H3 HA protein is represented by GenBank accession nrAAA43178, thereby applying the H3 numbering system to the mature HA protein and therefore not including the 16aa signal peptide. This approach also forms the basis of the numbering scheme proposed by Burke and Smith (2014, PLoS One 9(11):e112302), which enables the identification of amino acids that are structurally and functionally equivalent across all HA subtypes. The FluDB website (mentioned above) even provides a convenient "HA subtype numbering conversion" tool based on this publication by Burke and Smith. Consequently, the H3 numbering system is used herein to identify specific amino acid residue numbers of HA polypeptides.

[0036] However, when considering the size and sequence of domains, segments, and sections derived from HA proteins, biological variations in HA proteins from various influenza virus strains can be taken into account. For example, some differences arise between the first group of HA proteins (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18) and the second group of HA proteins (H3, H4, H7, H10, H14, and H15). Therefore, in the size notation used herein, "approximately" means that the size may vary by 1 to 5 amino acids before or after the indicated aa residue H3 number.

[0037] Similar indicators apply to the influenza B HA protein, with the size of its code HA0 being approximately 585aa, HA1 approximately 345aa, and HA2 approximately 223.

[0038] HA proteins can be characterized in various ways, for example, biochemically or serologically, all of which are well known to those skilled in the art. Furthermore, from the prior art and common general knowledge, those skilled in the art will know and readily recognize the different domains and regions of HA proteins.

[0039] In the case of influenza A HA, the HA2 section begins after the arginine amino acid near H3 number 329 aa, which is used for proteolytic cleavage of HA0, and is followed by a conserved amino acid sequence named "fusion peptide" containing the amino acid sequence GLFGAIAGFIE (SEQ ID NO: 1), or an aa sequence having at least 90% sequence identity with SEQ ID NO: 1, near H3 numbers 330-340 aa.

[0040] In the stem domain of the HA2 section, the region from HA2 to approximately aa75-90 is the interface between natural stem trimers, and this corresponds to the aa region of H3 numbered 405-420.

[0041] Influenza A HA2 has a transmembrane domain of approximately 27 aa at its C-terminus, with H3 numbering ranging from aa514 to 540, and also has a cytoplasmic domain of approximately 10 aa, with H3 numbering ranging from aa541 to 550.

[0042] A polypeptide refers to a molecular chain of amino acids. Polypeptides can be natural or mature proteins, pre- or pro-proteins, or fragments of proteins. Therefore, proteins, peptides, and oligopeptides are included in the definition of polypeptides in this invention, insofar as they still contain the indicated domain of the influenza virus HA protein.

[0043] The “target” of this invention is any human or animal that can be infected with the influenza virus. The animal may be, for example, a bird, a pig, a dog, a horse, or a weasel.

[0044] The target may be any weight, sex, or age affected by vaccination with a recombinant vector for use according to the present invention. However, it is clearly preferable to treat healthy, uninfected targets and treat them as early as possible to prevent any field infections and their consequences.

[0045] Therefore, recombinant vectors can be used as either a prophylactic or therapeutic measure, or both, because the polypeptides they can express can induce an immune response that can prevent both the establishment and progression of infection by the influenza virus.

[0046] The “use” of the recombinant vector of the present invention relates to human or veterinary medical applications in which the immunological properties of the vector-expressed polypeptide are used. Typically, this includes immunization with the recombinant vector as the active ingredient of a vaccine as described below herein.

[0047] The term “reduce” refers to partially or entirely reducing the establishment or proliferation of a proliferative infection by the influenza virus in susceptible target cells and organs, or in subsequent signs of the disease. This is achieved, for example, by reducing the viral load or shortening the duration of viral replication. This then results in a reduction in the number, intensity, or severity of lesions and associated clinical signs of the disease caused by the viral infection in the target.

[0048] Such reductions in infection or disease can be readily detected, for example, by monitoring the immunological response after vaccination with recombinant vectors for use according to the present invention, and by testing for the appearance of clinical symptoms or mortality after infection of the vaccination target, for example by monitoring disease target signs, clinical scores, serological parameters, or by re-isolation of the infectious pathogen. In animals, these results can be compared to the response to infection induction in pseudovaccinated animals. Various methods for evaluating the symptoms of influenza virus infection and disease are well known in the art.

[0049] "Infection or illness caused by the influenza virus" means infection by the influenza virus, as well as the subsequent well-known symptoms of such infection or illness, and their welfare and economic consequences.

[0050] The protection against influenza virus infection or disease induced by the expression of the HA stem polypeptide for use according to the present invention from a recombinant vector for use according to the present invention results in immunization of the target by the vector and improves health and economic performance. This can be evaluated from parameters such as health, survival rate, growth rate, increased food conversion and egg production, and cost reduction for (veterinary) healthcare.

[0051] The present invention is intended for use in targets that "have antibodies against the influenza virus HA head domain at the time of vaccination." Such existing antibodies interfere with immunization involving the full-length influenza virus HA protein or the antigenic portion of such HA head domain. However, such antibodies, as disclosed herein, do not interfere with, or at least much do not interfere with, immunization by the HA stem polypeptide of the present invention.

[0052] Existing antibodies directed against the HA head domain are typically acquired after infection or immunization with an antigenic preparation containing the HA head domain or a portion thereof, since the head domain is immunodominant. Such preparations may be whole or partial influenza virus preparations containing full-length HA protein, the HA head domain or a portion thereof, or live, inactivated, or recombinant vaccines.

[0053] The target may have acquired such antibodies against the HA head domain after influenza infection or immunization of the target itself. Alternatively, such antibodies can be obtained passively through antibody inoculation or ingestion. If obtained from the target's mother, the antibody is a “maternally derived antibody” (MDA). MDAs are present in several types of targets, whether human or animal.

[0054] In mammals, MDA may originate from the transplacental transfer of antibodies from mother to offspring. Alternatively, or in addition, MDA can be induced by the intake of such antibodies, for example, by the intake of maternal milk-containing antibodies, so-called colostrum. However, the intake of colostrum to obtain passive protection against disease is not limited to younger targets but can also be applied to older targets and cross-species.

[0055] In birds, MDA is present in the yolk, which is incorporated into the abdomen of the unhatched chicken as it matures inside the egg.

[0056] The “antibody” of the present invention relates to any type of immunoglobulin, such as IgA, IgG, IgM, IgD, IgE, or IgY, or a part thereof, such as a single-stranded variable fragment (ScFv) or Fv, F(ab') or F(ab')2 fragment.

[0057] Without immune stimulation or supplementation, the level of antibodies against a target (the so-called "titer") decreases over time due to their limited biological half-lives. This applies to the target, for example, after birth or when the target stops breastfeeding. Therefore, "at the time of vaccination" aims to link the timing of immunization to the titer of anti-HA head domain antibodies in the target of the recombinant vector used according to the present invention. This antibody level can be determined, for example, by taking serum samples from the target before and after vaccination to determine the titer of antibodies against the HA head domain.

[0058] In the present invention, the target antibody level at the time of vaccination is the target antibody titer at a time within ±3 days before or after vaccination.

[0059] However, this does not preclude the actual determination of the existing titer value itself, i.e., the performance of serological tests on serum samples taken before and after vaccination, and / or the analysis and interpretation of the results of those tests, from being performed some considerable time after vaccination, provided that the serum samples are stored under appropriate conditions to maintain sufficiently intact antibodies, for example, at -20°C or lower. Similarly, if the existing titer at the time of vaccination is calculated and fairly accurate data on the decrease in antibody titer over time are available, there is no precludation from levels determined in samples taken at further time before or after vaccination.

[0060] In the present invention, a target "has" antibodies if the titer of anti-HA head domain antibodies in serum from that target exceeds the background level detected in humans or animals of an equivalent target that is naive to influenza virus and influenza antibodies. In animals, this may be, for example, the titer present in the serum of SPF (Specific Pathogen Free) animals of the same age and species.

[0061] In the present invention, "antibody against" the HA head domain refers to an antibody that specifically binds to (i.e., is specific to) a polypeptide containing such an influenza HA head domain, such as an HA subunit antigen or a preparation of the influenza virus. Such specificity can be readily determined by those skilled in the art, for example, in an ELISA, by linear dilution of the antiserum in a test using a coated HA antigen. If the antibody in the antiserum is specific, the test will show a gradual and linear decrease in the detected binding signal.

[0062] The "HA head domain" is known in the art to be the central part of the HA1 section of the HA protein, which can form a 3D spherical structure and will be easily recognized by those skilled in the art. Counting from approximately 550 aa of mature influenza A HA0 protein, the head domain constitutes a polypeptide containing amino acid sequences with H3 numbering from approximately aa number 44 to approximately aa number 274, corresponding to the index given by Lu et al. above. Here again, specific size and aa number variations can occur in different influenza A virus HA proteins; see, for example, Figure 1A in International Publication 2011 / 123495 for influenza A HA sequences of serotypes H1-H16, and also see Table 7 in International Publication 2013 / 079473 for influenza A H1 HA sequences.

[0063] As a result, the "headless" HA stem domain polypeptide of the present invention does not contain the amino acid sequence corresponding to the influenza virus HA head domain as defined above.

[0064] As used herein, the terms “comprises” (and variations such as “comprising,” “comprise,” and “comprised”) are intended to refer to all conceivable elements and any possible combinations relating to the Invention that are covered or included by the sections, paragraphs, claims, etc., of the text in which the terms are used, even if such elements or combinations are not explicitly enumerated, and are not intended to exclude any such elements or combinations.

[0065] Therefore, any such section, paragraph, claim, etc. of text may also relate to one or more embodiments in which the term “comprises” (or a variation thereof) is replaced by terms such as “consist of,” “consisting of,” or “consist essentially of.”

[0066] It is well known in this field that the "influenza virus HA stem domain polypeptide" contains two portions from the HA1 portion of the HA protein and the major portion of the HA2 portion. Stem polypeptides can be identified, for example, by using well-known monoclonal antibodies specific to the HA stem domain of various influenza HA proteins, such as FI6, CR9114, and MEDI8552, all of which are well-known and commercially available.

[0067] Specifically, based on the indicators shown by Lu et al. (see above), the stem domain of the present invention is as follows for the mature influenza virus HA0 protein: -H3 numbering from approximately aa number 1 to approximately aa number 43, the HA1 N-terminal stem segment starts from the first amino acid remaining after the signal sequence is cleaved and continues to the start of the head domain. -H3 numbering from approximately aa number 276 to approximately aa number 329, the HA1 C-terminal stem segment starting after the head domain and extending to the cleavage sites of HA1 and HA2, and -H3 numbering from approximately aa number 330 to approximately aa number 513, starting after the cleavage sites of HA1 and HA2, and extending to the transmembrane domain. Includes.

[0068] As will be described in more detail below, in H3 HA, the length of the N-terminal stem segment of HA1 deviates by more than 5aa, and is approximately 10aa longer. Consequently, in the case of H3, this shifts the "H3 number" of various domains upward by 10aa.

[0069] Since the polypeptide of the present invention is expressed as a single fusion peptide, its components are covalently linked to a single amino acid chain, either directly or by one or more intervening spacer and / or linker amino acid sequences. Further linkage may be achieved by disulfide bonds formed between cysteine ​​amino acids of different portions.

[0070] The components of the stem domain polypeptide may be natural or heterologous, and in this invention, the polypeptide portion is considered "heterologous" if it originates from a different source compared to the HA2 stem domain in the polypeptide of this invention. The use of one or more heterologous elements creates a chimeric version of the polypeptide for this invention, which is within the scope of this invention.

[0071] In the present invention, "derived from" indicates the origin of the polypeptide of the present invention, and therefore its nucleic acid code. These can be isolated from biological sources or prepared recombinantly or synthetically based on sequence information.

[0072] This includes modifying the polypeptide for use in the present invention with respect to the naive HA stem domain, for example, by replacing the native signal sequence with a heterologous signal sequence; altering the basic amino acid that signals HA1-HA2 cleavage; and / or adding a tag to facilitate purification, for example, a 6x histidine tag. In addition, one or more aa linker sequences may be used between the indicated domains constituting the influenza virus HA stem polypeptide of the present invention.

[0073] A “trimerizing domain” is a well-known polypeptide that results in three times the polymerization of the homomeric polypeptide to which it is bound. In this field, various such trimerizing domains are known and available. Examples include the isoleucine zipper 3 domain ("GCN4 domain") of the GCN4 transcription activator from Saccharomyces cerevisiae, and the foldon domain ("foldon domain") of the bacteriophage T4 fibrin protein.

[0074] The trimerized domain may be included in different ways by the recombinant vector for use according to the present invention, for example, before, after, or between the segments and domains constituting the recombinant vector for use according to the present invention.

[0075] A "transmembrane domain" is well known to be a hydrophobic amino acid sequence that can provide adhesion and / or fixation to a lipid bilayer membrane. The transmembrane region bound to the HA stem domain polypeptide of the present invention may be a naive transmembrane domain of the HA2 stem domain used in the recombinant vector for use according to the present invention, or it may be a heterologous transmembrane domain derived from a different influenza virus HA protein or another protein.

[0076] The transmembrane domain may or may not incorporate the cytoplasmic domain of the influenza virus HA protein. [Brief explanation of the drawing]

[0077] [Figure 1] Time-course HI titers (Log2) from pigs vaccinated with an RP vaccine expressing full H1 HA. Piglets were MDA- or MDA+ for H1 HA at the start of the experiment. Details are described in Example 6.1. [Modes for carrying out the invention]

[0078] Details of embodiments and further aspects of the present invention are described below.

[0079] In embodiments of the recombinant vector for use according to the present invention, the disease caused by the influenza virus is caused by either influenza A virus or influenza B virus. Preferably, the disease is caused by influenza A virus.

[0080] In embodiments of recombinant vectors for use according to the present invention, the expressed influenza virus HA stem polypeptide is derived from influenza A virus or influenza B virus. Preferably, the HA stem polypeptide is derived from an influenza A virus HA protein selected from any one of serotypes H1 to H18. More preferably, the HA stem polypeptide is derived from an influenza A virus HA protein selected from any one of serotypes H1, H3, H5, H7, and H9.

[0081] In embodiments of recombinant vectors for use according to the present invention, the amino acid sequence of the expressed HA stem polypeptide is a consensus sequence.

[0082] As is well known, in order to obtain such a consensus sequence, either amino acid sequences or coding nucleotide sequences are compared by aligning several H9 HA stem domain nucleotide sequences using a suitable computer program, for example, and the consensus sequence is derived from that comparison.

[0083] In embodiments of recombinant vectors for use according to the present invention, the general order of the components of the expressed HA stem polypeptide is from the N-terminus to the C-terminus. • HA1 N-terminal stem segment, HA1 C-terminal stem segment, • HA2 external domain, • Transmembrane domain, and • Cytoplasmic domain That is the case.

[0084] In embodiments of recombinant vectors for use according to the present invention, the expressed HA stem polypeptide is • HA1 N-terminal stem segment and HA1 C-terminal stem segment, ·HA1 C-terminal stem segment and HA2 external domain, and • HA2 external domain and transmembrane domain The linker array is included between one or more of the following: In embodiments of recombinant vectors for use according to the present invention, the expressed HA stem polypeptide comprises one or more linkers as described herein. Preferably, the linker amino acid sequence is GGGG (SEQ ID NO: 2).

[0085] In embodiments of recombinant vectors for use according to the present invention, the expressed HA stem polypeptide contains a trimerizing domain. Preferably, the trimerizing domain is a GCN4 domain or a Foldon domain. More preferably, the trimerizing domain is a GCN4 domain. Even more preferably, the GCN4 domain is located inside the HA2 external domain. Even more preferably, the GCN4 domain is located inside the HA2 section at the position of the natural stem-trimer interface as described herein.

[0086] In the present invention, such arrangement of a trimerizing domain within the HA2 external domain may constitute a substitution or insertion, either by replacing or adding to the amino acids of that portion.

[0087] In embodiments of recombinant vectors for use according to the present invention, the expressed influenza virus HA stem polypeptide has an amino acid sequence selected from one of SEQ ID NOs: 4, 6, 8, 10, and 12.

[0088] As those skilled in the art will recognize, the influenza virus HA stem polypeptides of SEQ ID NOs: 4, 6, 8, 10, and 12 all have the same general layout, which is further detailed in Tables 1A and 1B. In short, - The naive HA protein signal sequence 16aa is replaced by the CD5-derived signal sequence 25aa. -The HA head domain is missing, and instead the N-terminal and C-terminal stem domain segments of HA1 are linked by the aa linker of SEQ ID NO: 2, so that the N-terminal segment of HA1 is placed before (i.e., at the N-terminus of) the C-terminal segment of HA1. -To prevent HA1-HA2 cleavage, the arginine residue at H3 numbering residue 329 was replaced with glutamine. -The GCN4 trimerization domain was introduced into the center of the HA2 external domain, and 15 corresponding amino acids were deleted from the stem-trimer interface at that site of HA2, - It includes both the transmembrane domain and the cytoplasmic domain of HA2.

[0089] In the HA stem polypeptides of Sequence IDs 4, 6, 8, 10, and 12, the HA stem domain, transmembrane domain, and cytoplasmic domain segments are specific to H1, H3, H5, H7, and H9, respectively. Furthermore, the H1, H3, and H9 stem domain sequences are consensus sequences determined by aligning H1 and H3 from several recent isolates of influenza A virus, swine influenza virus (SIV) isolates, and H9 from avian influenza virus (AIV) isolates. The H5 HA stem sequence was obtained from AIV strain: H5N1 A / Vietnam / 1203 / 2004 (GenBank: ABW90134), and the H7 HA stem sequence was obtained from AIV strain: H7N9 A / Anhui / 1-YK_RG05 / 2013 (GenBank: AKU41079).

[0090] Furthermore, several aa mutations were introduced to stabilize the polypeptide and / or increase its solubility. These were applied as described in International Publication 2013 / 079473.

[0091] Across the indicators shown in Table 1A, the H7 HA stem polypeptide (SEQ ID NO: 10) has a slight difference compared to SEQ ID NOs: 4, 8, and 12 (H1, H5, and H9 HA stem polypeptides, respectively), in that the HA1 N-terminal stem segment is 1aa longer and the HA2 fragment 2 is 1aa shorter. The H3 HA stem polypeptide (SEQ ID NO: 6) has the same small difference in the HA2 fragment 2, but a more significant difference in the HA1 N-terminal stem segment, which is 10aa longer. This is shown in Table 1B.

[0092] [Table 1]

[0093] [Table 2] In embodiments of recombinant vectors for use according to the present invention, the target is a human. Preferably, the human target is a young, elderly, diseased, or immunocompromised human.

[0094] In embodiments of recombinant vectors for use according to the present invention, the target is an animal. Preferably, the animal is selected from the group consisting of birds, pigs, dogs, horses, or weasels. More preferably: Birds will be selected from chickens, turkeys, ducks, geese, quail, pheasants, partridges, and ostriches; • Pigs are selected from wild or domesticated pigs, wild boars, babirusas, and warthogs; A canine is a dog; • An equine is a horse; • The weasel will be selected from ferrets and minks.

[0095] More preferably, the target is a pig or a chicken.

[0096] In embodiments of the recombinant vector for use according to the present invention, the antibody against the influenza virus HA head domain is a maternal antibody (MDA).

[0097] As described, the recombinant vector for use in accordance with the present invention contains a nucleic acid sequence encoding the HA stem polypeptide, and is therefore capable of expressing the influenza virus HA stem polypeptide of the present invention. In most cases, the encoding nucleic acid is heterogeneous to the vector.

[0098] In embodiments of recombinant vectors for use according to the present invention, the nucleic acid encoding the influenza virus HA stem polypeptide is codon-optimized.

[0099] Codon optimization is well-known and is applied to improve the expression levels of HA stem polypeptides in contexts different from the polypeptide's origin. It involves fitting nucleotide sequences to encode the intended amino acids, but by matching the nucleotide sequence to the codon priority (tRNA repertoire) of the recombinant vector, host cell, or target organism in which the sequence is expressed. Consequently, the nucleotide mutations applied are silent.

[0100] In embodiments of recombinant vectors for use according to the present invention, the HA stem polypeptide is encoded by a nucleic acid sequence that is codon-optimized for the target organism. Preferably, the target organism is selected from humans, birds, pigs, dogs, horses, and weasels.

[0101] In embodiments of recombinant vectors for use according to the present invention, the HA stem polypeptide is encoded by a nucleic acid sequence selected from one of SEQ ID NOs: 3, 5, 7, 9, and 11.

[0102] As those skilled in the art will understand, the nucleotide sequences shown in Sequence IDs 3, 5, 7, 9, and 11 refer to DNA nucleic acids, specifically the "coding strand." In the case of the complementary DNA strand, the "template" strand, the sequences are reverse complements.

[0103] Furthermore, as is obvious, if the recombinant vector for use according to the present invention contains RNA nucleic acid for expressing the HA stem polypeptide, the same coding sequences as SEQ ID NOs. 3, 5, 7, 9, and 11 apply, except that any T is replaced with U.

[0104] Furthermore, sequences 3, 5, 7, 9, and 11, or their corresponding RNA nucleic acids, encode the HA stem polypeptides of sequences 4, 6, 8, 10, and 12, respectively.

[0105] As described, recombinant vectors for use in accordance with the present invention can take various forms, ranging from nucleic acid molecules such as DNA or RNA to more complex structures such as virus-like particles and replicon particles, to the replication of recombinant microorganisms such as viruses.

[0106] Therefore, in embodiments, recombinant vectors for use according to the present invention are selected from nucleic acids, viruses, and replicon particles (RPs).

[0107] In embodiments of recombinant vectors for use according to the present invention, the vector is a nucleic acid.

[0108] In embodiments of recombinant vectors for use according to the present invention, where the vector is a nucleic acid, the nucleic acid is a eukaryotic expression plasmid.

[0109] Typically, eukaryotic expression plasmids of DNA have a suitable signal for the expression of a heterologous gene inserted into the plasmid, under the control of a promoter that is active in eukaryotic cells. The plasmid can then be inserted into a eukaryotic host cell or host organism by some transfection method, e.g., by mechanical means, or by electroporation, using a biochemical carrier, thereby initiating the expression of the heterologous gene insert. Typically, such expression will be transient because the plasmid lacks a signal for stable integration into the host cell's genome. Therefore, such plasmids typically do not transform or immortalize the host or host cell. All these materials and procedures are well known in the art and are described in handbooks. Such eukaryotic expression plasmids are commercially available from various suppliers, e.g., a range of plasmids: pcDNA®, pCR3.1®, pCMV®, pFRT®, pVAX1®, pCI®, Nanoplasmid®, pCAGGS, etc.

[0110] In a preferred embodiment, the eukaryotic expression plasmid is a pFRT (ThemoFisher) or pCAGGS plasmid (Niwa et al., 1991, Gene, vol. 108, p. 193-199).

[0111] Eukaryotic expression plasmids can contain several features for regulation, such as expression and purification. One possible signal is an antibiotic resistance gene, which can be used for selection during the construction and cloning process. However, if the target is intended to be administered to a human or animal, such antibiotic selection is undesirable due to the risk of inducing antibiotic resistance.

[0112] In a preferred embodiment of a recombinant vector for use according to the present invention, where the vector is a nucleic acid and the nucleic acid is a eukaryotic expression plasmid, the plasmid does not contain an antibiotic resistance gene.

[0113] The recombinant vector for use according to the present invention can be delivered to a host cell or target organism in the form of a eukaryotic expression plasmid, which expresses the HA stem polypeptide of the present invention in the host cell. The expression plasmid can be delivered in several ways, for example, by mechanical or chemical means, as raw DNA or encapsulated in a suitable (nanoparticle) carrier such as a protein, polysaccharide, lipid or polymer. Well-known examples of nucleic acid carriers are dendrimers, lipid nanoparticles, cationic polymers and protamines.

[0114] A special form of recombinant vector for use as a eukaryotic expression plasmid according to the present invention is when the plasmid results in the delivery of replicon RNA.

[0115] Therefore, in embodiments of the recombinant vector for use according to the present invention, where the vector is a nucleic acid and the nucleic acid is a eukaryotic expression plasmid, the plasmid encodes replicon RNA.

[0116] "Replicon RNA," also known as self-amplified mRNA, is a self-replicating RNA that, in addition to the nucleic acid encoding the HA stem polypeptide of the present invention, contains elements necessary for RNA replication, such as the replicase gene. However, unlike replicon particles (RP), replicon RNA is not packaged by viral structural proteins, resulting in low efficiency of entry into host cells.

[0117] Expression plasmids encoding replicon RNA can be delivered to host cells in the same way as protein expression plasmids. In this case, the structural viral protein is not supplied together in trans, so the replicon RNA is not packaged with the RP.

[0118] Since replicon RNA involves an amplification step, vaccination with a eukaryotic expression plasmid encoding replicon RNA offers advantages over vaccination with a eukaryotic expression plasmid expressing a protein. Replicase translation causes the replicon RNA to produce a subgenome messenger RNA encoding the HA stem polypeptide. This leads to the high-level expression of the HA stem polypeptide in the host cell and in the target, respectively.

[0119] In preferred embodiments of recombinant vectors for use according to the present invention, the vector is a nucleic acid, the nucleic acid is a eukaryotic expression plasmid, the plasmid encodes a replicon RNA, and the replicon RNA is an alphavirus-based replicon RNA. More preferably, the alphavirus-based replicon RNA is a Venezuelan encephalitis virus (VEEV)-based replicon RNA.

[0120] An example of a eukaryotic expression plasmid encoding VEEV replicon RNA is the pVAX plasmid, which contains VEEV non-structural protein genes 1-4 driven by a eukaryotic promoter, such as the human CMV earliest gene 1 promoter.

[0121] A specific example of such a plasmid is based on the pVAX plasmid (ThermoFisher), e.g., "pVAX-CMV-T7-HHR-VEEV-dPS-Rep," as shown in Sequence ID No. 12. This particular plasmid has 10,709 base pairs, and its composition is as shown in Table 2.

[0122] [Table 3] In alternative embodiments of recombinant vectors for use according to the present invention, where the vector is a nucleic acid, the nucleic acid is an RNA molecule.

[0123] The RNA molecule of the present invention can have different forms and functions, and may be, for example, mRNA or replicon RNA.

[0124] Recombinant vectors for use as RNA molecules according to the present invention can be delivered to target or host cells in various ways, for example by mechanical or chemical means, or they can be encapsulated in a suitable (nanoparticle) carrier such as a protein, polysaccharide, lipid, or polymer, as described. Specific chemical modifications can be applied to stabilize the RNA, for example, to the nucleotide or its backbone, or to incorporate nucleotide analogs.

[0125] In embodiments of recombinant vectors for use according to the present invention, where the vector is a nucleic acid and the nucleic acid is an RNA molecule, the RNA molecule is mRNA.

[0126] mRNA is well known in the art and typically has a 5'7 mG cap and a 3' poly-A tail. mRNA can be delivered to a eukaryotic host organism or host cell by transfection and / or by using a suitable carrier, such as a polymer or cationic lipid.

[0127] In embodiments of recombinant vectors for use according to the present invention, where the vector is a nucleic acid and the nucleic acid is an RNA molecule, the RNA molecule is a replicon RNA.

[0128] Replicon RNA can be produced in vitro using, for example, the pVAX-CMV-T7-HHR-VEEV-dPS-Rep plasmid described above, and then administered to host cells or target organisms using any suitable method.

[0129] Recombinant vectors for the expression and delivery of heterologous antigens in the form of replicated recombinant viral vectors are well known in the art. These provide an efficient method of vaccination because the viral vector replicates and amplifies at the target. The construction and modification of recombinant vector viruses are routinely performed using standard molecular biological techniques.

[0130] Many different virus species have been used over time as recombinant vectors for various human and animal targets.

[0131] Therefore, in embodiments of recombinant vectors for use according to the present invention, the recombinant vector is a virus.

[0132] In embodiments of recombinant vectors for use according to the present invention, where the vector is a virus, the virus is selected from herpesviruses, poxviruses, retroviruses, paramyxoviruses, rhabdoviruses, and adenoviruses.

[0133] For use with human targets, the recombinant vector virus is preferably an adenovirus, rhabdovirus, such as vesicular stomatitis virus, or paramyxovirus, such as measles virus. For use with avian targets, the recombinant vector virus is preferably a herpesvirus, more preferably turkey herpesvirus (HVT) or Marek's disease virus of serotype 1 or 2. For use with swine targets, the recombinant vector virus is preferably a herpesvirus, such as pseudorabies virus (Suid herpesvirus 1).

[0134] Because recombinant vector viruses are relatively large, they can contain not only a single but even multiple inserts of heterologous genes for expression. Examples of recombinant viral vectors expressing and delivering the influenza HA gene are described in International Publication 2012 / 052384 and European Patent No. 19218804.3 for HVT as a vector. An example of Newcastle disease virus (NDV; avian paramyxovirus) as a vector is described in International Publication 2007 / 106882.

[0135] Typically, for the construction of a recombinant viral vector, an expression cassette is inserted into a locus in the vector's genome. Various techniques are available to control the trajectory and orientation of this insertion. For example, by using a suitable adjacent section from the vector's genome to direct the integration of the cassette by a homologous recombination process, or by using a duplicated cosmid, as described in U.S. Patent No. 5,961,982. Alternatively, integration may be performed using CRISPR / Cas technology.

[0136] An "expression cassette" is a nucleic acid fragment containing at least one heterologous gene and a promoter that drives the transcription of that gene, in order to enable the expression of the encoded protein. Termination of transcription can be obtained by a sequence obtained by the genomic insertion site of the cassette, or the expression cassette itself may contain termination signals such as transcription terminators. In such a cassette, both the promoter and terminator must be in close proximity to the gene whose expression they regulate. This is called "operably linked," thereby ensuring effective initiation of transcription, with no other significant sequences intervening between them for their respective terminations. As will be apparent to those skilled in the art, expression cassettes are self-contained expression modules, and therefore their orientation in the vector viral genome is generally not important.

[0137] Recombinant vectors for use according to the present invention can also be delivered to and expressed at targets by virion-like polymer structures. Examples include virus-like particles (VLPs) or replicon particles (RPs). These are known as "single-cycle" infectious particles and contain the features necessary to infect host cells and express the heterologous genes they possess that encode polypeptides, but typically are unable to perform complete viral replication because, as an incorporated safety feature, they lack the viral genome (or the relevant portion thereof) on which they are constructed.

[0138] "RPs" are well-known, and several RPs have been developed as platforms for the expression and delivery of various proteins. The preferred base for RPs is alphaviruses, due to their broad host domain and rapid replication. Of course, some alphaviruses are highly pathogenic in their wild-type form, so appropriate safety measures are needed to mitigate and control infections with such RPs. For reviews, see Kamrud et al., 2010, J. Gen. Virol., vol. 91, pp. 1723-1727, and Vander Veen, et al., 2012, Anim. Health Res. Rev., vol. 13, pp. 1-9.

[0139] Therefore, in embodiments of recombinant vectors for use according to the present invention, the vector is RP. Preferably, the RP is alphavirus RP. More preferably, the alphavirus RP is VEEV RP.

[0140] The preferred alphavirus RP is based on VEEV, which is used as a recombinant vector vaccine for humans, pigs, poultry, and fish. Methods and tools for constructing, testing, and using VEEV-based alphavirus RP are well known and available. See, for example, Pushko et al., 1997, Virology, vol. 239, pp. 389-401, and International Publication No. 2019 / 110481. The preferred VEEV RP technology is SirraVax sm This is RNA particle technology (Harrisvaccine).

[0141] In this embodiment, the pVAX-CMV-T7-HHR-VEEV-dPS-Rep plasmid is used to produce RP: RNA is produced from the plasmid and then transfected into host cells with a helper RNA that transcodes the VEEV structural protein.

[0142] Embodiments of recombinant vectors for use according to the present invention are subject to one or more conditions selected from the following group: - A disease caused by the influenza virus, a disease caused by influenza A virus or influenza B virus; preferably, the disease is caused by influenza A virus; - The expressed HA stem polypeptide is derived from influenza A virus or influenza B virus; preferably, the HA stem polypeptide is derived from influenza A virus HA protein selected from any one of serotypes H1 to H18; more preferably, the HA stem polypeptide is derived from influenza A virus HA protein selected from any one of serotypes H1, H3, H5, H7 and H9; - The expressed HA stem polypeptide is the consensus sequence; -The general order of the components of the expressed HA stem polypeptide is from the N-terminus to the C-terminus: ○HA1 N-terminal stem segment, ○HA1 C-terminal stem segment, ○HA2 external domain, ○ Transmembrane domain, and ○It is a cytoplasmic domain; -The expressed HA stem polypeptide contains a linker sequence between one or more of the following: ○HA1 N-terminal stem segment and HA1 C-terminal stem segment; ○HA1 C-terminal stem segment and HA2 external domain; and ○HA2 external domain and transmembrane domain; - The expressed HA stem polypeptide comprises one or more linkers according to the present invention; preferably, the linker amino acid sequence is GGGG (SEQ ID NO: 2); - The expressed HA stem polypeptide contains a trimerizing domain; preferably, the trimerizing domain is a GCN4 domain or a Foldon domain; more preferably, the trimerizing domain is a GCN4 domain; even more preferably, the GCN4 domain is located inside the HA2 outer domain; even more preferably, the GCN4 domain is located inside the HA2 portion at the position of the natural stem-trimer interface as described herein; - The expressed HA stem polypeptide has the amino acid sequence shown in one of SEQ ID NOs: 4, 6, 8, 10, and 12; - The target is human; preferably, the human target is young, elderly, diseased, or immunocompromised; - The target is an animal; preferably, the animal is selected from the group consisting of birds, pigs, dogs, horses, or weasels; more preferably: Birds will be selected from chickens, turkeys, ducks, geese, quail, pheasants, partridges, and ostriches; ○Pigs are selected from wild or domesticated pigs, wild boars, babirusas, and warthogs; ○ A canine is a dog; ○ An equine is a horse; ○Weasels are selected from ferrets and minks; - The target is a pig or a chicken; - Antibodies against the influenza virus HA head domain are maternal antibodies (MDAs); - The nucleic acid encoding the HA stem polypeptide is codon-optimized; -HA stem polypeptides are encoded by a nucleic acid sequence that is codon-optimized for the target organism; preferably, the target organism is selected from humans, birds, pigs, dogs, horses, and weasels; -HA stem polypeptide is encoded by the nucleic acid sequences shown in SEQ ID NOs: 3, 5, 7, 9 and 11; and - Recombinant vectors for use in accordance with the present invention are selected from nucleic acids, viruses and RPs; preferably, ○ The nucleic acid is a eukaryotic expression plasmid or RNA molecule; ○The virus is selected from herpesviruses, poxviruses, retroviruses, paramyxoviruses, rhabdoviruses and adenoviruses; or ○RP is alphavirus RP.

[0143] In embodiments of recombinant vectors for use according to the present invention, the expressed HA stem polypeptide has the amino acid sequence shown in any of SEQ ID NOs: 4, 6, 8, 10, and 12. The target is a pig or a chicken. The antibody against the influenza virus HA head domain is a maternal antibody. The nucleic acid encoding the HA stem polypeptide is codon-optimized for the target organism. The vector is selected from nucleic acids, viruses, and RPs.

[0144] Recombinant vectors for use according to the present invention can be advantageously used to deliver and express the influenza virus HA stem polypeptide of the present invention to a target, for example, as a method of vaccinating the target. This involves, at one stage, introducing the vector into a suitable host cell. Depending on the type of vector applied, its introduction into the host cell may require a carrier, some transfection method, or may be induced by the vector itself as described above. Nevertheless, once the vector is inside the host cell, the HA stem polypeptide is expressed, thereby the host cell itself infected or transfected with the recombinant vector can be used in the present invention, for example, the infected or transfected host cell can be used for vaccinating a target.

[0145] The “host cell” of the present invention is a cell that enables the expression of the HA stem polypeptide of the present invention after the introduction of the recombinant vector for use according to the present invention into the host cell, for example, by transfection or infection.

[0146] The host cells of the present invention may be primary cells, such as cells of a target organism, or cells maintained in vitro as a suspension, monolayer, or tissue. Typically, primary cells can only undergo a limited number of cell divisions in vitro.

[0147] Alternatively, the host cell may be an immortalized cell, such as one from an established cell line capable of growing and dividing almost indefinitely. Depending on the type of host cell, the expression of the HA stem polypeptide of the present invention may involve more or less a wide range of post-translational processing, such as signal peptide cleavage, disulfide bond formation, glycosylation, and / or lipid modification.

[0148] The primary or immortalized host cells may be of the same or different species as the target of the recombinant vector for use in accordance with the present invention.

[0149] Most of the host cells used are fibroblasts and lymphocytes. When HVT is used as the recombinant viral vector for use in the present invention, the host cells are preferably primary chicken embryo fibroblasts (CEFs) and can be used and stored as described. See, for example, International Publication No. 2019 / 121888.

[0150] The host cells of the present invention are preferably immortalized bird cells. Several immortalized avian cell lines are described, for example, in International Publication No. 97 / 044443 and International Publication No. 98 / 006824. More preferably, the immortalized avian host cells of the present invention are immortalized CEFs. Even more preferably, they are immortalized CEFs such as those disclosed in International Publication No. 2016 / 087560.

[0151] As described, recombinant vectors and host cells of the present invention for use in accordance with the present invention can be advantageously used in vaccines to mitigate infection or disease caused by influenza virus.

[0152] Accordingly, in a further embodiment, the present invention relates to a vaccine for use in mitigating infection or disease caused by influenza virus in a target having antibodies against the influenza virus HA head domain at the time of vaccination, comprising a recombinant vector for use according to the present invention, or a host cell containing the recombinant vector, and a pharmaceutically acceptable carrier.

[0153] It is well known that a "vaccine" is a composition containing an immunologically active compound in a pharmaceutically acceptable carrier. The "immunologically active compound" or "antigen" is a molecule that is recognized by the immune system of the inoculated target and induces a protective immune response from the target's humoral and / or cellular immune system.

[0154] The vaccine for use in accordance with the present invention is a vaccine "against influenza" and, as described above herein, brings about a reduction in the symptoms of infection or disease caused by the influenza virus.

[0155] Specifically, the vaccine for use in accordance with the present invention is effective in targets having existing antibodies against the influenza virus HA head domain, as described above herein.

[0156] A "pharmacoagulably acceptable carrier" is known to be harmless and well-tolerated to the target, while also assisting in the stabilization and administration of the vaccine. Such carriers may, for example, be sterile water or sterile saline. In more complex forms, the carrier may be a buffer that may contain further additives, such as stabilizers or preservatives. Details and examples are found 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).

[0157] In this invention, when the vaccine contains a cell-associated HVT recombinant viral vector, the pharmaceutically acceptable carrier is preferably a mixture of serum and a culture medium containing DMSO. This carrier also provides stabilization of HVT vector-infected host cells during freezing and cryopreservation. The serum may be, for example, fetal or neonatal bovine serum.

[0158] Similarly, if the vaccine for use according to the present invention contains nucleic acid or RP, a pharmaceutically acceptable carrier may be a simple buffer, such as a phosphate buffer containing 5% w / v sucrose.

[0159] Furthermore, to stabilize and / or deliver the recombinant vector for use according to the present invention, additional carriers may be added to encapsulate it with a suitable (nanoparticle) carrier, such as a protein, polysaccharide, lipid, or polymer, as described. Preferably, the additional carrier for the RP of the present invention comprises a nanogel, which is a biodegradable polyacrylic polymer as described in International Publication No. 2012 / 165953.

[0160] The vaccine for use according to the present invention may contain additional immunoactive components. These may function to enhance existing immune defenses or extend them to other pathogens.

[0161] Therefore, in embodiments, the vaccine for use according to the present invention comprises at least one additional immunoactive component.

[0162] Such “additional immune-activating components” may be antigens, immune enhancers, cytokines, further vaccines, or any combination thereof. This offers advantages in terms of cost, efficiency, and welfare. Alternatively, a vaccine for use according to the present invention may be added to the vaccine itself.

[0163] A further advantageous effect of the vaccine for use according to the present invention, which reduces the influenza virus load in the target, is the prevention or reduction of viral shedding by the infected target, and thereby the prevention or reduction of the spread of the influenza virus vertically to offspring, and horizontally within herds or populations and geographical areas. As a result, the use of the vaccine for use according to the present invention leads to a reduction in the prevalence of influenza virus.

[0164] Therefore, further aspects of the present invention are: - Use of vaccines for use in accordance with the present invention to reduce the prevalence of influenza viruses in a population or geographical area, and - Vaccines for use in accordance with the present invention to reduce the prevalence of influenza viruses in populations or geographical areas That is the case.

[0165] A vaccine for use in accordance with the present invention is prepared, for example, by a well-known method as described and illustrated herein, and includes, for example, the step of mixing a recombinant vector or host cell of the present invention for use in accordance with the present invention with a pharmaceutically acceptable carrier.

[0166] Furthermore, various other compounds, such as stabilizers, carriers, adjuvants, diluents, emulsions, etc., can be added to the vaccine for use according to the present invention. Such additives are described in well-known handbooks such as "Remington" and "Veterinary Vaccinology" (both mentioned above).

[0167] Thus, the efficacy of the vaccine for use according to the present invention can be further optimized using routine techniques as needed by those skilled in the art.

[0168] General techniques and considerations applicable to the manufacture of vaccines under well-known standards for pharmaceutical manufacturing are described, for example, in government directives and regulations (Pharmacopoeia, 9th CFR) and well-known handbooks ("Veterinary Vaccineology" and "Remington," both mentioned above). Generally, such vaccines are prepared aseptically and using pharmaceutical-grade excipients.

[0169] Such preparations would incorporate microbiological testing for sterility and the absence of exogenous drugs, and may include in vivo or in vitro studies to confirm efficacy and safety. After completion of testing for quality, quantity, sterility, safety, and efficacy, the vaccine can be released for sale. All of this is well known to those skilled in the art.

[0170] Depending on the route of application of the vaccine for use in accordance with the present invention, it may be necessary to adapt the composition of the vaccine. This is well within the capabilities of those skilled in the art and generally involves fine-tuning the efficacy or safety of the vaccine. This can be done by adapting the dose, quantity, frequency, or route of the vaccine, by using a different form or formulation of the vaccine, or by adapting other components of the vaccine (e.g., stabilizers or adjuvants).

[0171] Preferably, the vaccine for use according to the present invention is formulated as an injectable liquid suitable for injection either intraocularly, intradermally, or parenterally. The injectable liquid may be, for example, a suspension, solution, dispersion, or emulsion.

[0172] In embodiments, the vaccine for use according to the present invention is for parenteral administration, preferably via intramuscular or subcutaneous routes.

[0173] In embodiments, the vaccine for use according to the present invention is for administration via an intradermal route. More preferably, the intradermal route is applied to a porcine target.

[0174] In a further embodiment, the present invention relates to the use of a recombinant vector for use according to the present invention, or a host cell containing the recombinant vector, and / or a vaccine for use according to the present invention, for mitigating infection or disease caused by influenza virus in a target having antibodies against the influenza virus HA head domain at the time of vaccination.

[0175] Similarly, in a further embodiment, the present invention relates to a method for mitigating infection or disease caused by influenza virus in a target having antibodies against the influenza virus HA head domain at the time of vaccination, the method comprising administering to the target a recombinant vector for use according to the present invention, a host cell containing the recombinant vector, and / or a vaccine for use according to the present invention.

[0176] In preferred embodiments of the use of the present invention or the method for mitigating infectious diseases according to the present invention, the vaccine for use according to the present invention further comprises a vaccine containing full-length HA protein.

[0177] In this combination, the vaccine for use according to the present invention can provide early immunity against influenza in the context of MDA, and furthermore, can provide long-term immunity against the influenza virus.

[0178] The volume per target dose of the vaccine for use according to the present invention can be optimized according to the intended route of administration. Intraovo administration is generally administered at a dose of about 0.01 to about 0.5 ml / egg, while parenteral injection is generally performed at a dose of about 0.1 to about 10 ml / target.

[0179] Determining the immunological effective dose of the vaccine for use in accordance with the present invention, or optimizing the volume of vaccine per dose, is both well within the capabilities of those skilled in the art.

[0180] The administration regimen for applying the vaccine to the target organism for use in accordance with the present invention may be a single or multiple doses in an immunologically effective amount, in a manner compatible with the vaccine formulation.

[0181] Preferably, the regimen for administering the vaccine for use according to the present invention is integrated into an existing vaccination schedule of other vaccines the target may require in order to reduce stress on the target and lower labor costs. These other vaccines can be administered concurrently, in parallel, or sequentially in a manner that is appropriate for their authorized use.

[0182] Preferably, the vaccine for use according to the present invention is administered only once as a single injection.

[0183] When the target for treatment by recombinant vectors for use in accordance with the present invention, by host cells containing the vectors, or by vaccines for use in accordance with the present invention is birds, the treatment is preferably applied at a very early stage, such as on the day of incubation ("day 1"), or in the egg, for example, during the first 18 days of embryonic development, all of which are well known in the art.

[0184] The present invention will now be described with reference to the following non-limiting embodiments.

[0185] [Examples] [Example 1] Preparation of Recombinant Structures 1.1. HVT vaccine: The HVT virus vector vaccine was prepared using methods for transfection, recombination, selection, and amplification, essentially as described in International Publication Nos. 2012 / 052384 and International Publication Nos. 2016 / 102647. In HVT, a construct encoding the full H5 HA gene and H5 HA stem (SEQ ID NO: 7) was driven by the PRV gB gene promoter, and the expression cassette was inserted into the Us2 locus of the HVT genome.

[0186] 1.2. Replicant particles VEEV RP was constructed, manufactured, and selected using a split helper system, as described in International Publication No. 2005 / 113782, International Publication No. 2008 / 156829, and Kamrud et al. (2010, J. Gen. Virol., vol. 91, pp. 1723-1727).

[0187] The inserts used in the RP were constructs encoding the H5 HA stem (SEQ ID NO: 7) and the H9 HA stem (SEQ ID NO: 11) for experiments in chickens, and a construct encoding the H1 HA stem (SEQ ID NO: 3) for use in pigs. Details of the use in animal experiments in pigs are described in International Publication No. 2019 / 110481.

[0188] 1.3 Plasmid The VEEV replicon RNA samples had inserts encoding essentially the same HA stems used in the HVT and RP experiments, except that they were delivered using expression plasmids from the pFRT or pVAX1 series as vectors.

[0189] Transformed E. coli K12 containing pFRT or pVAX plasmids was amplified in LB medium. Plasmid DNA was isolated using EndoFree® Plasmid Kits (QIAGEN). Plasmid DNA was eluted in sterile water for injection or TE buffer.

[0190] [Example 2] Vaccination induction experiments in SPF and MDA+ chickens 2.1. Introduction 2.1.1. Purpose The objective was to evaluate various types of influenza vaccines for their ability to provide protection against experimentally induced infection with highly pathogenic avian influenza (HPAI) H5N1 virus in 1-day-old MDA+ or SPF chickens. Induction was performed at 2-week and 3-week pv for SPF chicks and at 4-week or 5-week pv(pv) for MDA+ chicks to determine various aspects of vaccine efficacy in targets with or without existing antibodies and different titers.

[0191] The most reliable parameters for modeling the effectiveness of influenza vaccines are the mortality rate of target animals and the scoring of induced viral replication and shedding. This is described, for example, in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2015, chapter 2.3.4 Avian Influenza.

[0192] 2.1.2. Test Design For this study, we used n=80 (+n=4 reserve) 1-day-old healthy MDA+ and n=55 (+n=4 reserve) 1-day-old healthy SPF laying hens, which were transported to a contract research organization (CRO) on day 0, D8, D14, and D21 of the study. The SPF chicks were negative for antibodies against influenza virus (among other things).

[0193] For this study, n=20 healthy 1-day-old MDA+ chickens and n=10 healthy 1-day-old SPF laying hens were used for blood collection on days 0, 7, 14, and 21, and these were transported to the CRO on days D0, D8, D14, and D21 of the study.

[0194] The chicks were divided into 10 groups and immunized on the day of arrival, as shown in Table 3.

[0195] During the study, the chickens were housed in two separate animal rooms and kept in separate barns for each treatment group (T01-T05, T06-T10).

[0196] On the day of induction, chickens were sampled for serological testing (day 35 of the study). All chickens were inoculated with the induction virus HPAI H5N1 A / turkey / Turkey / 01 / 2005 (clade 2.2.1). For 10 days post-induction infection, chickens were monitored at least twice daily for clinical disease and mortality, and choana swabs were collected once daily on days 1, 2, 3, 5, and 7 post-induction infection (dpci). At dpci 10, the study was terminated by euthanizing all chickens that survived the induction infection.

[0197] The research was completed at D45.

[0198] [Table 4] 2.2. Materials and Methods 2.2.1. Test specimen The HVT vaccine was used as a suspension of infected primary chicken embryo fibroblasts (CEFs). The materials were stored in liquid nitrogen until use, then diluted with commercially available HVT dilution buffer Solvent CA® (MSD Animal Health), and then kept at ambient temperature before being used within one hour of reconstitution.

[0199] The HVT vaccine was administered subcutaneously (sc) into the neck at a dose of 2,000 PFU per 0.2 ml animal dose on days 0, 8, 14, or 21 of the study. One syringe and needle were used per treatment group.

[0200] Test material: HVT-full H5 HA gene insert vector vaccine The HVT vector construct used contains a pseudorabies virus gB gene promoter driving the complete H5 HA gene, and a human CMV early gene 1 promoter, and the NDV F gene, thereby driving the PRV gB prom. The HA gene insert is as described in International Publication 2012 / 052384, and the hCMV IE1 prom+F gene insert is as described in International Publication 2016 / 102647. Each of these promoter+ gene inserts is inserted tailhead into the Us2 locus of the HVT genome. The dual cassette contains a downstream transcription terminator derived from the hCMV-IE1 gene.

[0201] Test material: HVT-H5 HA stem vector vaccine The HVT vector used to deliver and express the H5 HA stem polypeptide is essentially a construct as described in International Publication No. 2012 / 052384, containing a pseudorabies virus gB gene promoter that drives the H5 HA stem polypeptide inserted into the Us2 locus of the HVT genome, encoded by the nucleotide sequence of Sequence ID No. 7, followed by a transcription terminator.

[0202] Inducing virus: HPAI H5N1 strain A / turkey / Turkey / 1 / 2005 The induced virus was kept at -80°C until diluted to approximately 6Log10 EID50 in 0.2 ml (i.e., equivalent to 5Log10 TCID50). It was maintained on ice until use, and the remaining inoculum was back-titrated to determine the actual induced dose administered.

[0203] The induced virus was administered as 0.1 ml intranasal (one nostril) and 0.1 ml intratracheally.

[0204] NB: Working with toxic influenza viruses requires appropriate permits and biosafety precautions.

[0205] 2.2.2 Test animals 135 mixed-sex White Leghorn laying hens, 1 day old, were used in the study, and only healthy animals were transported to the CRO. Chickens that appeared unhealthy upon arrival were excluded from the study (Day 0 of the study). Prior to vaccination, chickens were identified by attaching swift tuck labels to their necks upon acquisition and numbered as shown in Table 3. MDA+: n=80 in the study, n=20 for blood with T=0 and T=7, and n=4 in reserve; SPF: n=55 in the study, n=10 for blood with T=14 and T=21, and n=4 in reserve.

[0206] The MDA+ chicks were descendants of SPF laying chickens vaccinated with an inactivated adjuvant-added AIV vaccine prepared from HPAI H5N1 A / turkey / Turkey / 01 / 2005 (clade 2.2.1). As a result, MDA was homologous to the encoded H5 HA stem polypeptide, creating a worst-case scenario.

[0207] Chickens were immunized at 1 day old on the day of arrival. Unvaccinated chickens in groups T01, T05, and T08 had acclimatization periods of 14, 28, and 35 days, respectively.

[0208] To prevent physical contact, the chickens were housed in separate, enclosed barns under appropriate conditions, with each processing group (maximum n=15) located within a single enclosure.

[0209] 2.2.3. Experimental Procedure General health status was observed and documented at least once a day by a biotechnologist, and a veterinarian was called in when necessary.

[0210] Clinical observations for signs of influenza were performed and recorded daily by animal technicians from the day of inoculation with the induced virus until 10 days post-inoculation. The first observation was performed before inoculation. Clinical findings were assessed using a scoring system from 0 to 3 to increase the severity of signs of depression, rhinorrhea, sneezing, respiration, skin abnormalities, edema, neurological signs, and diarrhea.

[0211] Blood samples for serology (approximately 2 mL per animal) were collected from the wing veins of all chickens on day 20 of the study. Serum was isolated after coagulation and centrifugation (1300 × g for 10 minutes). Serum samples were stored at -20°C.

[0212] Choanaes swab samples were collected from all chickens on days 36, 37, 38, 40, and 42. Samples were collected using cotton swabs (dried rayon tips, Copan 155C). Immediately after sampling, the swabs were stirred in approximately 2 mL of tryptose phosphate buffer supplemented with antibiotics, and the ice was kept melted during transport to the laboratory. Chickens found to be dead were not sampled, and dying chickens were sampled before euthanasia. In the laboratory, the swabs were squeezed to remove the contents, the samples were centrifuged (10 minutes, 1300 × g), and the supernatant was stored at -80°C.

[0213] If a chicken exhibited severe symptoms of depression, respiratory distress, or neurological disorder (score 3) at the first observation, or moderate symptoms of depression, respiratory distress, or neurological disorder (score 2) at the second observation, it was euthanized based on the criteria for the recognition, evaluation, and use of clinical signs as humane endpoints in experimental animals.

[0214] After the end of their lifespan, clinical findings regarding signs of influenza were summarized for each animal and each study day. The median clinical score was calculated for each treatment group and displayed graphically over time. The frequency (number of days), total, and distribution of severity scores were also calculated.

[0215] Hemagglutination inhibition (HI) assays were performed on serum samples using HA antigens homologous to the vaccine antigen and the inducing antigen. Samples were also subjected to specific AIV-H5 HA inhibitory ELISA (ID Screen® Influenza H5 Antibody Competition (IDVet)). HI titers and ELISA scores from serum samples were compiled for each animal and study day. Average titers were calculated for each treatment group.

[0216] Choana eswab samples were used for real-time qPCR of influenza. Ct values ​​were compiled by animal and study day. For each treatment group, the mean Ct value was calculated and displayed graphically over time. In addition, the mean peak Ct value and the number of days until a positive PCR result was obtained were calculated.

[0217] The test was deemed valid when serum samples taken from 1-day-old SPF chicks and unvaccinated SPF chicks did not contain antibodies against AIV-H5. Additionally, unvaccinated induced chickens had to be killed within 10 days of induction.

[0218] 2.3.Results During the experiment, several chicks died before the induction of the reaction. The cause could not be determined, or it was unrelated to the experiment.

[0219] 2.3.1. Induction To determine the induction dose, undiluted induction virus, pre-induction diluted virus, and diluted induction virus returned from the animal facility after induction were subjected to the TCID50 assay. The inoculation dose was calculated using the average of the diluted (pre-administration and post-administration) titers and determined using a TCID50 of 10^3.95 per animal.

[0220] Mortality and morbidity were monitored for 10 days after induction. Induction of HPAI H5N1 was severe and highly effective, as all non-immunized SPF chickens died before induction or on day 2 after induction, or had to be euthanized. Induction of non-immunized AIV MDA+ animals at 4 weeks post-hatch resulted in a 70% mortality rate, indicating that residual MDA titers still provided partial protection to the induced animals. In contrast, at 5 weeks post-hatch, MDA titers were no longer protective.

[0221] 2.3.2. Efficacy of vaccination for survival In SPF animals, HVT-full H5 HA vaccination resulted in 100% survival rates at 2 and 3 weeks of pv in induced animals. Therefore, the onset of immunity (OOI) for HVT-full H5 HA vaccine in SPF animals is less than 2 weeks. The efficacy of HVT-H5 HA stem vaccine in SPF animals was determined only at 3 weeks of pv due to a slight delay in its onset. Since HVT-H5 HA stem provided 60% partial protection, its onset of immunity in SPF animals is more than 3 weeks.

[0222] However, in contrast to the results in SPF animals, in AIV MDA+ animals, HVT-full H5 HA provided only 43% and 46% protection at 4 and 5 weeks, respectively. Surprisingly, the HVT-H5 HA stem vaccine provided 86% and 93% protection in AIV MDA+ animals.

[0223] 2.3.3. Induced Virus Replication Chona swabs were collected from unimmunized (n=5) and immunized (n=10) chickens on days 1, 2, 3, 5, and 7 post-induction days and used to assess induced viral replication by measuring the amount of AIV RNA in the trachea. RT-qPCR results are presented as PCR-equivalent viral titers (EID50 equivalents), including standard deviation (SD). Unimmunized SPF chickens had a high viral replication load in the trachea on day 1 post-induction (mean 10^4.3 EID50 equivalents). In unimmunized AIV MDA+ chickens, titers were 10^3.2 and 10^3.8 EID50 equivalents at 4 and 5 weeks post-hatch, respectively.

[0224] In SPF animals vaccinated with HVT-full H5 HA, the titer of induced viral replication decreased by 2 Log 10 at 2 weeks of PV and by >4 Log 10 at 3 weeks of PV. Vaccination of SPF animals with HVT-H5 HA stem vaccine resulted in only a 1 Log 10 decrease in induced viral replication.

[0225] Vaccination of AIV MDA+ animals, particularly with the HVT-H5 HA stem vaccine, had a strong effect on survival, but only a slight effect on induced viral replication in the trachea. Vaccination of AIV MDA+ animals with HVT-full H5 HA also had only a slight effect, with a reduction in viral load of less than 1 Log 10. The HVT-H5 HA stem vaccine resulted in a reduction of induced viral replication between 1 and 2 Log 10 in both 4 and 5-week pv.

[0226] 2.3.4. Serological results 2.3.4.1. Hemagglutination inhibitor (HI) titer Serum isolated from blood before induction was used in an HI assay using the HA antigen. HI titers were determined using two consecutive assays. As expected, the HVT-H5 HA stem vaccine did not induce antibodies that inhibit hemagglutination, as only antibodies against the head domain can inhibit hemagglutination.

[0227] All SPF hatched crosses (control, T=0) were negative and were the same as unimmunized SPF animals at 2 weeks of PV. HVT-full H5 HA vaccination resulted in seroconversion in 12 out of 14 animals at 2 weeks of PV, with an average HI titer of 10.2. At 3 weeks of PV, HVT-full H5 HA resulted in seroconversion in 13 out of 14 animals, with an average HI titer of 52.8.

[0228] All AIV MDA+ hatched crosses (control, T=0) were positive in the HI test with a mean titer of 46.9. The titer decreased to less than 2 at 4 and 5 weeks post-hatch (control). Surprisingly, HVT-full H5 HA failed to induce any seroconversion in MDA+ chicks at 4 and 5 weeks post-hatch.

[0229] 2.3.4.2.AIV-H5 specific ELISA A commercially available inhibitory ELISA test kit (AIV-H5 ELISA, IDVet) was used to test serum samples obtained from chicks during the experiment, according to the manufacturer's instructions.

[0230] Serum from SPF hatched matings collected before vaccination showed a 0% inhibitory titer. Non-immunized SPF animals (negative controls) showed a background level of 7% inhibition.

[0231] ELISA titers in SPF animals after vaccination with HVT-full H5 HA vaccine slowly increased from 2-3 week pv. Inhibitory titers rose from 35% at 2 week pv to 57% at 3 week pv.

[0232] In contrast to the relatively high antibody titers induced in SPF animals, vaccination of MDA+ animals with HVT-full H5 HA vaccine resulted in only 32% inhibition of pv at 4 weeks and 22% inhibition of pv at 5 weeks. These H5 antibody titers were comparable to the inhibitory anti-H5 titers observed in non-immunized MDA+ animals. Consequently, the HVT-full HA H5 vaccine was severely inhibited by the MDA titer present at the time of vaccination.

[0233] Vaccination of SPF animals with the HVT-H5 HA stem vaccine resulted in a 33% inhibitory titer at 3 weeks of prophylactic exposure (pv). Therefore, the HVT-H5 HA stem vaccine induces a relatively lower titer in SPF animals compared to full HA vector vaccine in SPF chicks.

[0234] Surprisingly, however, MDA+ animals vaccinated with the HVT-H5 HA stem vaccine had inhibitory elisa titers of 41% at 4 weeks PV and 44% at 5 weeks PV. Therefore, these titers were higher at 3 weeks PV compared to HVT-H5 HA stem vaccinated SPF animals and much higher compared to unvaccinated MDA+ controls. Thus, the HVT-H5 HA stem vaccine is not affected by the AIV-H5 MDA titer present at the time of vaccination.

[0235] 2.4. Conclusion HVT-full H5 HA and HVT-H5 HA stem vaccines were evaluated in 1-day-old SPF and AIV MDA-positive chickens. SPF animals were subjected to 2 weeks and 3 weeks of pv for induction of allogeneic H5N1, while AIV MDA-positive animals were subjected to 4 weeks and 5 weeks of pv. Tracheal serological response, mortality, and induced viral replication were evaluated.

[0236] In SPF animals, HVT-full H5 HA vaccination resulted in OOI of less than 2 weeks with 100% protection. Induced viral titers decreased by 2 Log10 at 2 weeks of pv and by >4 Log10 at 3 weeks of pv. 100% protection correlated well with high HI titers and H5 ELISA titers at 2 weeks and 3 weeks of pv. Surprisingly, some chickens had HI titers less than 2, yet they were still protected against HPAI H5N1 induction. Therefore, HI titer does not necessarily correlate with protection.

[0237] The HVT-H5 HA stem vaccine was less effective in SPF chickens compared to the HVT-full H5 HA vaccine. Only 60% of chickens were protected over a 3-week period. Furthermore, the reduction in induced viral replication in the trachea was only 1 log 10.

[0238] In stark contrast to the good protection provided by the HVT-full H5 HA vaccine in SPF animals, this vaccine performed very poorly in AIV MDA+ animals, with protection at 4 weeks and 5 weeks of pv being only 43% and 46%, respectively. This insufficient protection also correlated with a poor serological response and limited effectiveness against induced viral replication in the trachea. Clearly, the HVT-full H5 HA vaccine is severely hindered by high AIV H5 MDA levels.

[0239] Interestingly, while the HVT-H5 HA stem vaccine was poorly effective in SPF animals, it induced 86% and 93% protection at 4 and 5 weeks of PV, respectively, in AIV MDA+ animals. Furthermore, the HVT-H5 HA stem vaccine more efficiently reduced intratracheal viral titers compared to the HVT-full H5 HA vaccine. Efficient protection also correlated with higher antibody titers on induction days in chickens vaccinated with the HVT-H5 HA stem vaccine.

[0240] In conclusion, the HVT-full H5 HA vaccine has an OOI (out of 100 days) of less than 2 weeks in SPF animals, but this is strongly impaired by MDA titers. The HVT-H5 HA stem vaccine has an OOI of more than 3 weeks in SPF animals, but induces a high level of protection in influenza MDA situations at 4 and 5 weeks. Therefore, vaccination with the HA stem polypeptide antigen of the present invention is not affected by existing influenza antibodies.

[0241] [Example 3] Tests in chickens without existing antibodies The experiment described in Example 2 above was performed on SPF chickens and was the first successful experiment by the inventors. It yielded some disappointing results. In the same general setting and performance experiment as described in Example 2 above, the protective efficacy of different HA stem polypeptides against severe influenza virus-induced infection with 3 weeks of pv using heterologous AIV H5N1 strains was tested. As the first experiment, this was performed on target animals without existing influenza antibodies, i.e., SPF chickens. The different types of HA stem polypeptide vaccines tested were purified subunits, two types of HVT vector vaccines, and an RP vaccine.

[0242] 3.1. Materials and Methods Specifically, the vaccine was prepared as follows: - The subunit vaccine contained an H5 HA stem polypeptide with the same amino acid composition as SEQ ID NO: 7, but only up to aa number 272, and therefore lacked the TM and cytoplasmic domains of HA2. For purification purposes, it contained a C-terminal Flag tag / EK cleavage site followed by a triple Strep tag. The subunit was expressed in HEK293T cells, purified, and adjuvanted with standard liquid paraffin mineral oil formulated as a water-in-oil emulsion. The subunit was administered at an animal dose of 4 μg / dose.

[0243] - The HVT vector vaccine contained either the H5 HA stem polypeptide of SEQ ID NO: 7 or the full H5 HA protein.

[0244] -RP was a VEEV-based RP containing the H5 HA stem polypeptide of SEQ ID NO: 7. RP was prepared and purified as essentially described. RP was administered in aqueous buffer at a dose of 1 × 10⁸ RP / animal and adjuvanted 1:1 with XSolve® adjuvant (MSD Animal Health) as an O / W emulsion.

[0245] One of the inventors' intentions was to confirm the protective properties of the HA stem polypeptide described in International Publication No. 2013 / 079473 and the corresponding paper by Impagliazzo (cited above). However, this yielded unexpectedly poor results.

[0246] Similar to Example 2, chickens were vaccinated at 1 day of age via the sc route with 0.2 ml of HVT-full H5 HA, HVT-H5 HA stem, VEEV-H5 HA stem RP, or adjuvant-added HA stem subunit vaccine. A group of 5 chicks remained untreated, and a group of 10 chicks served as unvaccinated induction controls. Serum samples were collected at T=0 (vaccination day) and T=20 (1 day before induction) and used for HI testing and H5-specific ELISA assays. At T=21, animals were induced with a lethal dose of HPAI AIV strain: A / duck / Biddinghuizen / NL / 2016 (H5N8, clade 2.3.4.4), and morbidity and mortality were tracked for 10 days. Tracheal swabs were collected and analyzed using an influenza virus M gene-specific RT-qPCR assay to determine induced viral replication.

[0247] 3.2.Results The results showed that all controls were as expected. All unvaccinated challenged chicks died or were moribund by 2 days post-challenge, and the negative controls were seronegative in HI and ELISA. Furthermore, all chicks vaccinated with the full HA protein showed strong HI titers, and all vaccinations showed ELISA titers above background values, indicating that all vaccines were properly administered.

[0248] The results of the vaccinated group's challenge survival rate 10 days post-challenge were as follows: -HVT-full H5 HA: 100% -HVT-H5 HA stem: 70% -VEEV-H5 HA stem RP: 80% -H5 HA stem soluble subunit: 0% The HVT-full H5 HA vaccine induced strong seroconversion 3 weeks post-vaccination. The strong serological event correlated well with 100% protection of this vaccine in SPF chickens against homologous and heterologous challenge. Interestingly, thus, the full HA protein functioned very well as a vaccine in seronegative animals. When applied to animals with existing influenza antibodies, it was quite contrasting to the results found in Example 2.

[0249] Surprisingly, the HA stem subunit vaccine did not protect chickens from HPAI challenge. Death was somewhat delayed, but all chicks in this group died or were moribund by 4 days p.c. This result was purely contrasting to the positive reports at the time of publication.

[0250] However, when the HA stem polypeptide was given the TM domain, there was significant level of protection both when delivered by a viral vector and when delivered as RP.

[0251] The H5N8 challenge strain used was heterologous to the vaccine as it had 91.7% aa sequence identity.

[0252] 3.2.1. Serology A commercially available inhibitory ELISA test kit (AIV-H5 ELISA, IDVet) was used to test serum collected from chicks during the experiment, following the manufacturer's instructions.

[0253] The serum of SPF animals (hatchlings) before vaccination showed only 4% inhibition, which is within the background level range. The same was true for the titer of unvaccinated controls at 3 weeks of pv, which showed only 2% inhibition, demonstrating the complete absence of anti-H5 HA antibodies in SPF chicks.

[0254] Following vaccination of SPF animals with the HVT-full H5 HA vaccine, an inhibitory titer of 58% was observed in 3-week PV. This correlates very well with the results presented in Example 2.

[0255] Similarly, when SPF chicks were vaccinated with vector vaccines expressing the HA stem polypeptide according to the present invention, inhibitory titers of 32% were obtained with the HVT-H5 HA stem vaccine and 28% with the VEEV-H5 HA stem RP vaccine. These results are consistent with those found in Example 2.

[0256] Finally, when chicks were vaccinated with soluble HA stem antigen as a subunit vaccine, titer was induced with only 9% inhibition in ELISA.

[0257] These ELISA titers are very consistent with the induction and survival results found in this experiment. They also indicate that expressing the HA stem polypeptide of the present invention as a membrane-bound antigen (and therefore having a TM domain) via a recombinant vector vaccine induces a more potent immune response compared to subunit vaccines based on soluble HA stem antigens.

[0258] [Example 4] Extended testing of induced defenses 4.1. Introduction Further experiments, such as those described in Examples 2 and 3 above, re-tested protection against HPAI H5N1 AIV-induced virus in chickens with and without existing influenza antibodies. However, to determine the vaccine's effectiveness under more "field-like" conditions, a contact induction model was used in which vaccinated animals were induced to infection and housed together with vaccinated, uninduced sentinel animals. As a control, unvaccinated animals were also antigen-stimulated and housed together with unvaccinated, uninduced sentinel animals. Induced viral replication was measured via tracheal swabs, and induced viral shedding was tested via cloacal swabs.

[0259] HVT-full H5 HA and HVT-H5 HA stem vaccines were evaluated in 1-day-old chickens that were either SPF or AIV H5 HA MDA+ (offspring of vaccinated SPF chickens, as described above). Half of the vaccinated control SPF animals or the non-vaccinated control SPF animals were subjected to H5N1-induced virus induction for 5 weeks of pv. Half of the AIV MDA+ vaccinated animals or the non-vaccinated animals were subjected to similar induction, but for 6 weeks of pv. Eight hours after induction, sentinel birds were added to the directly induced animal group. Serological response in the trachea, mortality, and induced viral replication, as well as viral shedding in the cloaca, were evaluated.

[0260] Similar levels of protection were found as described in Examples 1 and 2, and the transmission of vaccine-induced viruses for use according to the present invention was also significantly reduced.

[0261] 4.2. Setup Before the start of the experiment, control blood samples were collected from 5 SPF and 10 MDA+ chicken hatchlings to determine antibody status on day 0. Excluding the controls, both the SPF and MDA+ groups had 76 chicks, which were then divided into separate groups based on various treatments: unvaccinated / full HA / HA stem vaccine, induced / uninduced, and sentinel / test animals. At a minimum level, each test group had 6 chicks. The vaccine was administered to the intended group on day 1 at approximately 2,000 PFU / dose in 0.2 ml via the sc pathway. Immediately before induction, blood samples were collected from each animal for the HI test on day 35 (SPF) and day 42 (MDA+). The induced virus was HPAI H5N1 A / turkey / Turkey / 01 / 05 (clade 2.2.1). The administered induction doses were determined to be TCID50 / animal at 10^3.6 and 10^3.2, respectively. Induced animals were not paired with sentinel stimulants until 8 hours after induction. Cloaca swab samples were collected from induced chickens on days 1, 2, 3, 4, 6, and 8. Samples were collected using cotton swabs (dried rayon tips, Copan 155C). Immediately after sampling, the swabs were agitated in approximately 2 mL of tryptose phosphate buffer supplemented with antibiotics, and the ice was allowed to melt during transport to the laboratory. The experiment was concluded two weeks after induction, on days 49 and 56, respectively.

[0262] 4.3.Results 4.3.1.Death The results again showed that all controls were as expected. All unvaccinated challenged chicks died or were moribund by day 2 post-challenge, and the negative controls were seronegative in HI and ELISA. Unvaccinated challenged MDA+ chicks died or had to be euthanized between days 3 and 6 post-challenge, with an average death time of 4.3 days. Thus, MDA levels induced some protection but were not sufficient to withstand a lethal challenge. All chicks vaccinated with the full HA protein showed strong HI titers, and all vaccinations showed ELISA titers above background values, indicating that all vaccines were properly administered.

[0263] All SPF sentinel animals that were not vaccinated and were in contact with challenged animals died or had to be euthanized from day 3 to day 7 post-challenge. Also, all unvaccinated MDA+ sentinel animals in contact with challenged animals died or had to be euthanized from day 6 to day 11 post-challenge. As a result, the transmission of the H5N1 TT05 virus from challenged animals to sentinel animals was very robust.

[0264] Results of the test groups: SPF animals - vaccinated: Survival, reduced transmission to sentinels Direct challenge: -HVT-full H5 HA: 100% 89% -HVT-H5 HA stem: 72% 72% Sentinels: -HVT-full H5 HA: 100% -- -HVT-H5 HA stem: 100% -- MDA+ animals - vaccinated: Direct challenge: -HVT-full H5 HA: 22% 56% -HVT-H5 HA stem: 83% 89% Sentinels: -HVT-full H5 HA: 78% -- -HVT-H5 HA stem: 100% -- In SPF animals, HVT-full H5 HA vaccination provided 100% protection to induced animals at 5 weeks of pv. This result confirmed and was in complete agreement with what had been seen in previous studies.

[0265] However, the HVT-full H5 HA vaccine in MDA+ animals induced by 6 weeks of proliferative disease (PV) only protected 22% of the animals from death. This was considerably worse than the 43% and 46% protection measured in Example 2 with 4 and 5 weeks of PV.

[0266] The protection provided by the HVT-H5 HA stem vaccine against severe induction was highly effective, giving an 83% survival rate at 6 weeks of prophylactic care. These results demonstrate that the vaccine containing the HA stem polypeptide according to the present invention is not interfered with by high levels of existing influenza HA antibodies.

[0267] 4.3.2 Reduction of transmission of induced viruses The induced virus was efficiently transmitted from unvaccinated SPF and MDA+ animals to unvaccinated sentinel SPF and MDA+ animals. Vaccination of SPF animals with HVT-flu H5 HA vaccine reduced the transmission of the induced virus to undetectable levels. In contrast, vaccination of MDA+ animals with HVT-flu H5 HA vaccine reduced transmission to some extent but did not prevent transmission by induction to sentinel animals, resulting in a uniform mortality rate.

[0268] The HVT-H5 HA stem vaccine was able to protect the majority of SPF and MDA+ chickens from induced death. Furthermore, transmission of the induced virus was completely blocked in both SPF and MDA+ animals. Therefore, the use of the HA stem polypeptide according to the present invention in populations with existing influenza antibodies protects most targets from clinical disease and completely blocks viral transmission.

[0269] The results of viral replication in vaccinated animals and sentinel animals showed similar patterns; the HVT-full H5 HA vaccine was effective in SPF animals but not in MDA+ animals, while the HVT-H5 HA stem vaccine showed the opposite result.

[0270] 4.3.3. Serology The HI titer in the MDA+ hatched crosses on day 0 was 159, which was considerably higher than the HI titer in the experiment described in Example 2.

[0271] A commercially available inhibitory ELISA test kit (AIV-H5 ELISA, IDVet) was used to test serum samples obtained from chicks during the experiment, according to the manufacturer's instructions.

[0272] Serum from SPF animals (hatchlings) before vaccination showed 5% inhibition, while serum from unvaccinated SPF animals (negative controls) showed 6% inhibition at background levels.

[0273] Vaccination of SPF animals with HVT-full H5 HA vaccine resulted in an 82% inhibitory titer at 5 weeks of proliferative vaccination (pv). In contrast to these high antibody titers in SPF animals, AIV-H5 MDA+ animals vaccinated with HVT-full H5 HA vaccine produced serum at 6 weeks of pv, showing only 38% inhibition. These H5 antibody titers were only slightly higher than the 26% observed in unvaccinated MDA+ animals, indicating that the HVT-full H5 HA vaccine was severely inhibited by pre-existing AIV-H5 antibody titers on the day of vaccination.

[0274] Vaccination of SPF animals with HVT-H5 HA stem vaccine resulted in a 48% inhibitory titer at 5 weeks of prophylactic exposure (PV). Surprisingly, vaccination of AIV-H5 MDA+ animals with HVT-H5 HA stem vaccine resulted in a 53% serum inhibition at 6 weeks of PV. These H5 antibody titers were higher than those of HVT-H5 HA stem-vaccinated SPF animals at 5 weeks of PV and significantly higher than those of unvaccinated MDA+ animals. Therefore, the HVT-H5 HA stem vaccine is not affected by existing H5 antibodies at the time of vaccination.

[0275] 4.4. Conclusion HVT-full H5 HA and HVT-H5 HA stem vaccines were evaluated in 1-day-old SPF and AIV MDA+ chickens. Half of the vaccinated or unvaccinated SPF control animals were subjected to HPAI H5N1 virus induction with 5 weeks of pv. Half of the vaccinated or unvaccinated AIV MDA+ animals were subjected to induced infection with 6 weeks of pv. Eight hours after induction, the remaining half of the animals were added to the group, and transmission of the induced virus from directly induced animals to sentinel animals was assessed. Serological response in the trachea, mortality, and induced virus replication, as well as viral shedding in cloacae, were evaluated.

[0276] Vaccination of SPF animals with HVT-full H5 HA resulted in 97% seroconversion and 100% protection against induction in the animals. Oral induction viral replication decreased by 2–3 Log10. Viral RNA shedding was also significantly reduced to near-undetectable levels. Based on RT-qPCR results, transmission from directly induced birds to sentinel birds decreased by 89%, while all sentinel birds were protected from clinical disease.

[0277] Full HVT H5 HA vaccination in AIV H5 MDA+ animals did not induce detectable HI titers at 6 weeks of pv. Poor seroconversion correlated with 22% poor protection after induction. Viral RNA shedding from cloacae was significantly reduced, but induced viral replication in the oral cavity decreased by only about 1 Log10. However, this reduction, based on RT-qPCR results, amounted to only a 56% decrease in transmission rate and did not prevent transmission by direct induction in sentinel animals.

[0278] Vaccination of SPF animals with HVT-H5 HA stem reduced oral induced viral replication by 2 Log 10, which was slightly less efficient than with HVT-full H5 HA vaccine. This correlated with a slightly reduced 72% protection against lethal induction. Viral RNA shedding from cloacae from induced animals decreased by approximately 3 Log 10, resulting in a 72% reduction in transmission from directly induced birds to sentinel birds, based on RT-qPCR results.

[0279] Vaccination with HVT-H5 HA stem vaccine in AIV H5 MDA+ animals resulted in 83% protection at 6 weeks of pv, whereas only 22% of birds were protected after vaccination with HVT-full H5 HA vaccine. Furthermore, HVT-H5 HA stem vaccine in AIV H5 MDA+ animals reduced transmission by 89% based on RT-qPCR results.

[0280] In conclusion, the HVT-full H5 HA vaccine is very potent in protecting SPF chickens from the induction of lethal H5N1 while efficiently blocking transmission. However, in AIV MDA+ animals, the protection of the HVT-full H5 HA vaccine was only 22%, and there was still sustained transmission between directly induced animals and sentinel animals, with a mortality rate of 22%. This indicates that the HVT-full H5 HA vaccine is strongly influenced by the AIV H5 MDA titer. As previously observed, the HVT-H5 HA stem vaccine induces a moderate level of protection in SPF animals. However, in AIV H5 MDA+ animals, the HVT-H5 HA stem vaccine yields 83% protection, very efficiently blocking transmission to sentinel animals. Therefore, the HA stem polypeptide-based vaccine according to the present invention does not appear to be affected by existing influenza HA antibodies, and thus this antigen may be a solution to fill the immunological gap of full HA protein-based vaccines when applied to MDA+ targets.

[0281] [Example 5] Trials of HA stem cell combination vaccine In subsequent experiments, the focus was on whether co-vaccination with the HA stem polypeptide and full HA protein of the present invention could improve the immune protection provided by each of them separately. The experimental setup was largely as described in Examples 2-4 above, using AIV H5 HA MDA+ chicks.

[0282] The applied vaccine combination involved administering HVT-full H5 HA in 0.2 ml of an O / W emulsion at a dose of 2000 pfu via the sc pathway to the neck, and VEEV RP, a 1 × 10⁸ dose H5 HA stem polypeptide in 0.2 ml of an O / W emulsion, via the im pathway to the leg using XSolve. Each vaccine was essentially as described in Example 3 above. A group of 40 AIV H5 HA MDA+ laying hens was given the combined vaccine at 1 day of age. A group of 25 chicks served as an unvaccinated MDA+ control. Blood samples were collected at various time points in the experiment up to 8 weeks after vaccination.

[0283] 5.1. Serology using ELISA Serum samples obtained from chicks were tested throughout the experiment using a commercially available inhibitory ELISA test kit (AIV-H5 ELISA, IDVet).

[0284] Serum obtained from MDA+ hatched matings showed high H5 antibody titers with an average inhibition rate of 89%. These MDA titers decreased to 22% and 8% at 6 and 7-8 weeks, respectively, in non-immunized animals.

[0285] The combination vaccine induced very high anti-H5 HA antibody titers in MDA+ animals, scoring >65% inhibition in all chicks and at all time points tested at 6, 7, and 8 weeks of PV. Such high levels of antibody correlated with 100% protection even against heterologous H5 strain influenza infection.

[0286] Such high levels of H5 antibody titers were not achieved by the inventors prior to vaccination of AIVH5 HA MDA+ animals without using HA stem polypeptide vaccine and definitely without using HVT-full H5 HA vaccine.

[0287] [Example 6] Tests in pigs 6.1. Efficacy of SIV H1 RP vaccine in MDA+ piglets and MDA+ piglets.

[0288] To test the effect of MDA on full-length HA in pigs, an RP vaccine expressing the swine influenza virus (SIV) H1 HA protein was administered to young pigs that were both MDA-negative and MDA-positive for H1 SIV.

[0289] To produce MDA+ piglets, healthy, seronegative sows for SIV were vaccinated twice during pregnancy with a gamma-inactivated vaccine of the SIV pandemic H1N1 virus:A / swine / Minnesota / A01483170 / 2014, formulated in an O / W emulsion with XSolve adjuvant. The sow vaccine contained 10^6 TCID50 equivalents / ml.

[0290] Blood samples were collected from all healthy piglets of vaccinated sows at two weeks of age. SIV MDA titers were determined using a standard HI protocol. Based on these results, mixed groups of 10 piglets were formed, each having the same group-average MDA titer of 6.6 Log2HI and equal distributions of individual MDA titers of 4–8 Log2HI.

[0291] Two groups of piglets, one MDA- and one MDA+, were administered the PBS mock vaccine. Two further groups, one MDA- and one MDA+, were administered the VEEV-RP vaccine expressing the full-length H1 HA protein of SIV strain: A / swine / England / 10 / 2010(H1N1) (GenBank: AFR75956). This has 97.5% amino acid sequence identity. The RP was formulated in an O / W emulsion with the XSolve adjuvant.

[0292] Piglets were vaccinated twice: a prime vaccine at 5 weeks of age (Day 1 of the experiment) and a booster vaccine at 8 weeks of age (Day 21 of the experiment). The RP vaccine was administered intramuscularly to the neck at a dose of 5 × 10⁶ particles per 1 ml of animal dose.

[0293] Blood samples were collected several times during the experiment to isolate serum. The results of the HI titration are shown in Figure 1.

[0294] Unvaccinated MDA control animals remained HI-negative throughout the experiment. In unvaccinated MDA+ control animals, MDA titers decreased from approximately 6.6Log2HI at 14 days of age to background levels at approximately 65 days of age and remained so until the end of the experiment (T=56).

[0295] In vaccinated piglets, MDA- animals showed a slight increase in HI titer after prime vaccination, but a very strong increase after booster. The group mean titer reached 9 days after booster immunization was 10.2Log2HI. On the other hand, in MDA+ piglets, HI titer initially decreased somewhat after prime vaccination and increased after booster, but only reached a group mean titer of 6.8, which was much lower than the titer reached in MDA- pigs.

[0296] This demonstrates that, as with chickens, the effectiveness of vaccination with full HA protein is also severely hindered in pigs by the presence of pre-existing anti-HA head antibodies at the time of vaccination.

[0297] 6.2. Experiments using H1-HA stem polypeptide Further vaccination induction experiments in pigs are being prepared to confirm that vaccination with the HA stem polypeptide according to the present invention can overcome the effects of existing HA antibodies and to compare different platforms for delivery and expression. Both MDA- and MDA+ piglets will be vaccinated with H1 HA stem polypeptide and full H1 HA and induced with live H1 SIV. The vaccines are DNA plasmid-expressed VEEV replicon RNA and VEEV RP containing an oily adjuvant.

[0298] SIV H1 MDA+ piglets are produced and divided into groups as described in Section 6.1. Both MDA+ and MDA+ piglets receive two doses of vaccination at 5 and 8 weeks of age. The primary test vaccine is VEEV replicon RNA delivered via pVAX plasmid, either full H1 HA or the H1 HA stem polypeptide described in Sequence ID No. 4. The control is the full H1 HA RP vaccine, which is expected to perform poorly at the MDA+ target, as described in Section 6.1 above.

[0299] The RP vaccine is administered at a dose of 5 x 10^6 particles per animal, while the plasmid vaccine is administered at a dose of 50 μg per animal. Both vaccine types are formulated with XSolve adjuvant in an O / W emulsion and administered intramuscularly to the neck at a dose of 2 ml.

[0300] Induced infection is induced at approximately 11 weeks of age. The inducing virus is A / swine / Minnesota / A01483170 / 2014(H1N1pdm), which is administered intratracheally to each animal at a dose of 1 x 10^6 TCID50 in 5 ml of PBS (10 mM), with necessary containment measures applied at BSL2 level.

[0301] Nasal swabs will be collected from all pigs before induction and for three days after induction to monitor induced viral replication. Clinical signs of infection, such as loss of appetite, shortness of breath (difficulty breathing), fever, cough, and runny nose, will be observed.

[0302] Three days after induction, all animals are sedated and blood is collected for post-mortem examination of lung lesions. Following macroscopic scoring, lung tissue samples are taken for quantitative determination of the induced virus.

[0303] [Example 7] Testing of expression in host cells To investigate the expression of HA stem polypeptides in host cells, a series of experiments were conducted using various forms of polypeptide delivery according to the present invention to host cells. Different staining techniques were applied to visualize the type and location of their expression.

[0304] 7.1.Hela cells In one approach, Hela R19 cells were briefly transfected with an HA stem expression plasmid. The cells were seeded in a 96-well plate and cultured overnight to reach approximately 80% confluence. The following day, a transfection mixture was prepared using 0.1 μg of plasmid DNA, 0.3 μl of FuGENEHD® (Promega) (non-lipid) transfection reagent, and 4.6 μl of OptiMEM® medium (ThermoFisher) per well, and incubated at room temperature for 15 minutes. This was then added to cells in DMEM + 10% v / v serum, without the addition of antibiotics, and incubated overnight. After 24 hours, the cells were fixed with 3.7% formaldehyde containing 1% methanol. This type of fixation ensures that the cell membrane remains intact, and consequently, any observed signals should be expressed on the cell surface. Cells were stained with FI 6 (human anti-HA stem) antibody and secondary goat anti-human IgG Alexa488 antibody (molecular probe, Thermo Fisher) using a standard IFT protocol.

[0305] The results showed that both H1 HA stems and H9 HA stem polypeptides were clearly detectable on the cell surface of HeLa cells, while mock-transfected cells remained negative. This indicates that the HA stem polypeptides are properly expressed and presented on the surface of host cells transfected with the vector according to the present invention.

[0306] 7.2. Vero cells and CHO cells In a similar series of experiments, Vero Ames and CHO-K1 cells were transfected with plasmids expressing the H1 or H9 HA stem polypeptide of the present invention. After transfection and incubation, the cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) and incubated at room temperature for 15 minutes. This type of fixation ensures that the cells remain intact, and as a result, any signals observed must be exposed to the cell surface. Some cells were further treated with PBS containing 0.1% Triton-X100. This permeabilizes the cells, allowing for both intracellular and extracellular staining of the antigen. Next, an IFT assay was performed using FI6 as the primary antibody.

[0307] The results showed that while mock-transfected cells remained negative, H1 and H9 HA stem polypeptides were adequately expressed in both Vero and CHO cells. Permeabilized cells showed staining throughout the cell; however, even without permeabilization, the polypeptides could be clearly detected by the FI6 antibody. This demonstrates the efficient expression of these polypeptides in CHO and Vero host cells. Furthermore, this indicates that H1 and H9 HA stem polypeptides were presented on the cell surface in these types of host cells as well.

[0308] In subsequent experiments, Vero cells were transfected with plasmids expressing the H1, H5-, and H9 HA stem polypeptides of the present invention, or plasmids expressing the full H1, H5, or H9 HA proteins.

[0309] After transfection and incubation, the cells were fixed in formalin / PBS to preserve them intact and stained using an IFT assay with FI6 as the primary antibody.

[0310] The results showed that all Vero cells expressing either the full HA protein or the HA stem polypeptide (although mock cells were negative) showed clear staining on the cell surface. Consequently, the H1, H5, and H9 HA stem polypeptides of the present invention are presented on the cell surface in the same way as the full HA protein.

[0311] [Example 8] Tests using H9 HA stem polypeptides Experiments with H9 HA are being prepared to test further variants of influenza virus and demonstrate the use of different vector types. Both the full HA protein and HA stem polypeptide will be tested in chickens, in both SPF and H9 MDA+, as plasmid-delivered replicon RNA molecules and as RP. Both plasmid-delivered replicon RNA and RP result in the expression of the H9 HA stem polypeptide of SEQ ID NO: 12, which is the consensus sequence of recent H9 AIV isolates, and the modifications described herein: substituted with a 4xG linker, trimerization, and deletion of the head domain including the TM and cytoplasmic domains. The encoding nucleotide (SEQ ID NO: 11) was codon-optimized against the chicken transcription profile, and several stabilizing mutations were applied.

[0312] H9 MDA+ chicks were produced by vaccinating SPF parents with the inactivated H9N2 AIV vaccine. Their H9 amino acid sequence was 97% identical to that of the H9 HA stem polypeptide, thus resulting in near-homonymous MDA. Vaccination was administered at 1 day of age.

[0313] Induction is applied at 4 weeks post-vaccination in SPF chickens and at 5 weeks post-vaccination in MDA+ animals. The induction material is allantoalanthin fluid grown from eggs of LPAI strain A / chicken / Egypt / V1527 / 2018(H9N2), and its H9aa sequence is 94% identical to the sequence of the H9 HA stem polypeptide used. This is administered intranasally at an EID50 of 10^6 per animal in 0.2 ml.

[0314] The exchange is performed in the first week after induction, and clinical signs are monitored for two weeks after induction.

[0315] To compare the effects of MDA on full-length HA, vaccination with full-length H9 HA of similar sequences will be performed using similar plasmids and RP vaccines. In addition, an HVT-H9 HA recombinant viral vector will be subcutaneously administered to an additional group of chicks. This HVT vector contains the HA gene inserted into the HVT genome between the UL44 and UL45 genes and is driven by the PRV gB promoter.

[0316] The replicon RNA vaccine is administered as a plasmid, intramuscularly at a dose of 10 μg / animal in 0.2 ml. One group receives only 1 μg / dose to test the efficacy of the plasmid dose. The plasmid is formulated on a polyacrylic polymer nanogel (20Med Therapeutics) at 2.5 mg / ml in 20 mM HEPES + 5% trehalose. The RP is VEEV RP, and is intramuscularly administered in aqueous buffer at a dose of 10⁸ RP per animal in 0.2 ml of adjuvant-added emulsion.

[0317] Blood samples will be collected from selected animals before, during, and at the end of the experiment to monitor their initial and developing serological status. Animals will also be monitored, and clinical signs will be recorded. Since the only inducer is LPAI, mortality or serious clinical signs are not expected.

Claims

1. A vaccine comprising a recombinant vector capable of expressing recombinant influenza virus hemagglutinin (HA) stem polypeptide in a target, or a host cell containing the recombinant vector, and a pharmaceutically acceptable carrier, wherein the polypeptide comprises a headless influenza virus HA stem domain, a trimerizing domain, and a transmembrane domain, for use in reducing infection or disease caused by influenza virus in a target having antibodies against the influenza virus HA head domain at the time of vaccination by administration of the vaccine.

2. The vaccine for use according to claim 1, characterized in that the expressed recombinant influenza virus hemagglutinin (HA) stem polypeptide has an amino acid sequence selected from one of SEQ ID NOs: 4, 6, 8, 10, and 12.

3. The vaccine according to claim 1 or 2, characterized in that the recombinant vector is selected from nucleic acids, viruses, and replicon particles (RPs).

4. - The nucleic acid is a eukaryotic expression plasmid or RNA molecule. - The virus is selected from herpesviruses, poxviruses, retroviruses, paramyxoviruses, rhabdoviruses and adenoviruses, or - The aforementioned RP is an alphavirus RP. The vaccine according to claim 3, characterized in that

5. Use of the vaccine for use according to any one of claims 1 to 4 for the purpose of reducing infection or disease caused by influenza virus in a non-human target, wherein the non-human target has antibodies against the influenza virus HA head domain at the time of vaccination by administering the vaccine to the non-human target.

6. A method for mitigating an infection or disease caused by an influenza virus in a non-human target, comprising administering to the non-human target a vaccine for use according to any one of claims 1 to 4, wherein at the time of vaccination by administering the vaccine to the non-human target, the non-human target has antibodies against the influenza virus HA head domain.

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