Swine influenza A virus vaccine containing two different RNA replicon particles

Abstract

The present invention relates to an immunogenic composition comprising first and second RNA replicon particles. The first RNA replicon particle comprises a nucleic acid construct comprising first and second nucleic acid sequences encoding first and second hemagglutinin (HA) antigens of swine influenza A virus (IAV-S). The first HA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and the second HA antigen is of the A(H1N1)pdm09 (pdm09) lineage. The second RNA replicon particle comprises a nucleic acid construct comprising third and fourth nucleic acid sequences encoding third and fourth HA antigens of IAV-S. The third HA antigen is of the A / swine / Scotland / 410440 / 1994-like H1 hu N2 (Scot / 94) lineage, and the fourth HA antigen is Eurasian avian-like H1 av In another embodiment, the present invention relates to a vaccine comprising the immunogenic composition, which can be used against influenza A virus infection. Methods for producing the vaccine and uses of the vaccine are further provided.

Description

[Technical Field]

[0001] The present invention relates to an immunogenic composition comprising first and second RNA replicon particles. The first RNA replicon particle comprises a nucleic acid construct comprising first and second nucleic acid sequences encoding first and second hemagglutinin (HA) antigens of swine influenza A virus (IAV-S). The first HA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and the second HA antigen is of the A(H1N1)pdm09 (pdm09) lineage. The second RNA replicon particle comprises a nucleic acid construct comprising third and fourth nucleic acid sequences encoding third and fourth HA antigens of IAV-S. The third HA antigen is of the A / swine / Scotland / 410440 / 1994-like H1 hu It is of the N2(Scot / 94) lineage, and the fourth HA antigen is Eurasian avian-like H1 av It is of the N1(EA) lineage. In other embodiments, the present invention relates to a vaccine comprising an immunogenic composition that can be used against influenza A virus infection. Methods for producing the vaccine and uses of the vaccine are further provided. [Background technology]

[0002] Influenza A virus (IAV) poses a significant burden on human and animal health worldwide. IAV is classified into different subtypes based on its viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). IAV infects poultry, pigs, horses, cats, dogs, marine mammals (e.g., whales), bats, and humans. Wild waterfowl and wild birds (ducks, geese, swans, and seagulls) are natural carriers, and they may be infected with 16 different HA and 9 different NA subtypes [Webster et al., Microbiol Rev 56:152-179 (1992)].

[0003] Swine influenza A virus (IAV-S) is a serious respiratory pathogen in domestic pigs worldwide, proving to be economically costly, particularly for the livestock industry [Holtkamp et al., The American Association of Swine Veterinarians Annual Meeting (2007)]. It is characterized by the sudden onset of respiratory illness, usually accompanied by loss of appetite, lethargy, and fever. In addition to the clinical complications associated with IAV-S in production animals, there have been reports of pigs being involved in the transmission of influenza viruses to humans [Myers KP, Olsen CW, Gray GC. Clin Infect Dis 2007;44(8):1084-8, Krueger and Gray, Curr Top Microbiol Immunol 370:201-225 (2013)], which poses a significant public health threat and provides a stronger incentive for IAV control in pig populations.

[0004] In response to this problem, many pig farmers now vaccinate their pigs using commercially available vaccines to prevent IAV-S. However, controlling IAV-S with conventional vaccines is difficult because many diverse IAV-S strains circulate simultaneously in the field and continue to evolve [Gao et al., J Gen Virol 98(8):2001-2010(2017)]. The diversity and variability of IAV-S are caused by the virus's genetic structure. Like other influenza A viruses, IAV-S has genes encoded in eight segments of RNA and a genome replication mechanism that introduces frequent mutations. These genetic features allow IAV-S to adapt rapidly, for example, to evade existing neutralizing antibodies induced by exposure to previous strains. As a result, inactivated viral IAV-S vaccines commercially available in the US market have proven insufficient, even though they contain up to five different IAV-S strains, because the emergence of new strains occurs as a result of continuous antigenic continuum and / or antigenic discontinuum mutations.

[0005] The classification of influenza A viruses begins with the subtype classification of two major glycoproteins on the viral surface, HA and NA. The HA protein mediates the attachment and fusion of the virus to host cells. Neuraminidase is an enzyme that functions in the final stage of the influenza virus replication cycle by cleaving newly formed viral particles from host cells, thereby enabling the new progeny to spread to and infect other cells. Recent studies have shown that NA immunity can only play a supplementary and / or complementary role to the more important HA immunity [Nayak et al., J Virol 84(5):2408-2420(2010); Pavlova et al., Vaccine 27(5):773-785(2009); Sylte et al., Vaccine 25(19):3763-72(2007)]. In fact, in the absence of hemagglutinin antigen, the efficacy of neuraminidase influenza A virus vaccine appears to be insufficient for protection against influenza A infection or influenza A virus-induced illness.

[0006] While human influenza A typically has one or two dominant strains that circulate globally during a given influenza season, many more strains of IAV-S circulate simultaneously, and these strains differ geographically. Similarly, IAV-S strains are also antigenically variable, but primarily include H1 or H3 subtypes of HA and N1 or N2 subtypes of NA. Within each HA and NA subtype of IAV-S, there is further phylogenetic diversity.

[0007] In the US pig population, there are four dominant phylogenetic clusters of H1 (gamma, delta 1, delta 2, and pandemic), two dominant clusters of H3 (cluster IV and human-like), two dominant clusters of N1 (classic and pandemic), and two dominant clusters of N2 (N2-1998 and N2-2002) [Anderson et al., Influenza and other Respiratory Viruses 7(Suppl.4);42-51(2013); and Anderson et al., mSphere 1(6)e00275-16:1-14(2016)].

[0008] In Europe, there are three major strains of H1 (Eurasian-avian-like H1, Scotland / 410440 / 1994-like H1, and pandemic 2009-like H1), one major strain of H3 (Gent / 1 / 1984-like H3), two major strains of N1 (Eurasian Avian-like N1, pandemic 2009-like N1), two major strains of N2 (Gent / 1 / 1984-like N2, Scotland / 410440 / 1994-like N2), and two minor strains of N2 (Italy / 4675 / 2003-like N2, human seasonal-like N2) [Watson et al., J. Virol., 89:9920-9931 (2015); doi:10.1128 / JVI.00840-15].

[0009] Vaccination against IAV-S is the best option for reducing clinical complications in pigs and decreasing the chances of further genetic reassortment and the spread of zoonotic diseases from pigs to humans. Until recently, the only vaccine available for widespread use was an inactivated vaccine prepared from influenza viruses grown in embryogenerated eggs, but their supply has been limited, mainly due to a shortage of specific pathogen-free eggs, and the need for a new approach to influenza vaccination is well recognized.

[0010] In conventional inactivated virus IAV-S vaccines, the selection of virus strains is based on HA antigen characteristics. IAV-S vaccines that induce HA inhibition (HI) antibody titers protect pigs from experimental infection with antigenically similar strains [Kyriakis et al., Vet Microbiol 144(1-2):67-74(2010)]. However, the relatively rapid genetic drift of the HA gene gives rise to new strains that are not functionally inhibited by vaccine-induced HA antibodies.

[0011] As a result, commercially available vaccines often do not protect against all strains contemporaneously circulating in the field and are antigenically mismatched, and thus provide only limited protection against heterosubtype challenges and do not protect against novel and emerging virus subtypes / clusters, [Lee et al., Can J Vet Res 71(3):207-12(2007); Vincent et al., Vaccine 28(15):2782-2787(2010)]. Therefore, such vaccines must be periodically updated to match the currently circulating strains.

[0012] Therefore, there is a need in the art to develop novel IAV-S vaccines that are safe, effective, and can be rapidly modified to antigenically match emerging strains.

[0013] Because most viruses, such as influenza viruses, have a relatively simple structure, the use of a single antigen from their antigenic profile may be sufficient to generate a protective immune response. Such subunit vaccines can be produced by extraction from the virus or its culture, or by recombinant expression of specific antigens. Alternatively, viral antigens can be delivered to the target animal by live recombinant carrier microorganisms that act as vectors and expressed inside. The vector may be live attenuated or non-live. Many vector-based strategies have been used in vaccines for many years to protect against specific pathogens.

[0014] Variations of the use of viral vector vaccines are the use of replicon particle-based vaccines [see RP; Lundstrom, 2014, Vaccines, vol. 6, p. 2392-2415]. These are virus-like particles but contain a defective viral genome, typically a heterologous gene. These replicon particles typically enter the target animal host cells without the ability to form new particles and contain RNA packaged into the particles so that they can perform one round of viral genome amplification (i.e., they are capsidated). Since replicon particles lack the necessary structural protein coding sequence(s), they do not proliferate from infected cells. Thus, they are more similar to wild-type viruses (e.g., from the perspective of tropism) than other replicon vaccines such as naked RNA vaccines or vaccines containing RNA released from DNA plasmids.

[0015] The genome of RP typically expresses a heterologous gene encoding an immunoprotective antigen. The most widely used and most extensively studied are alphavirus RNA replicon particles [Vander Veen et al., 2012, Anim. Health Res. Rev., vol. 13, p. 1-9; and: Kamrud et al., 2010, J. Gen. Virol., vol. 91, p. 1723-1727], which are therefore preferred for practical reasons and were developed from the viral genome by replacing the structural protein gene with a heterologous gene. The resulting RNA, called a replicon, can direct its own replication and express high levels of heterologous genes when introduced into the cytoplasm of host cells. Since these replicons lack the alphavirus structural protein gene, they cannot form virions and spread to adjacent cells. However, replicons can be efficiently packaged into virus replicon particles (RP) by introducing them into cells where the structural proteins are provided in trans [Pushko et al., 1997, Virology, vol. 239, p. 389-401].

[0016] Furthermore, alphavirus RPs are considered to be somewhat stronger immunostimulants than other RPs based on other viruses, such as bunyaviruses, that are known in the art. Several alphavirus species, such as Venezuelan encephalitis virus (VEEV) [Pushko et al., 1997, Virology, vol.239, p.389-401], Sindbis virus [Bredenbeek et al., 1993, J. of Virol., vol.67, p.6439-6446], and Semryki forest virus [Liljestrom & Garoff, 1991, Biotechnology (NY), vol.9, p.1356-1361], have been used to develop RP vaccines.

[0017] RP vaccines can induce mucosal and systemic immune responses after immunization of target animals [Davis et al., 2002, IUBMB Life, vol. 53, pp. 209-211]. VEE-based RP vaccines are also the basis for several USDA-approved vaccines, including swine epidemic diarrhea vaccine, RNA (product code 19U5.P1), swine influenza vaccine, RNA (product code 19A5.D0), avian influenza vaccine, RNA (product code 19O5.D0), and formulation product, RNA particles (product code 9PP0.00).

[0018] Because RP vector systems can be easily manipulated at the molecular level, vaccines can be rapidly manufactured to respond to emerging viral subtypes.

[0019] Therefore, there is a continuing demand for novel vaccines that provide broad protection against circulating IAV-S strains, particularly against the four major IAV-S strains circulating in Europe: EurAsianAvian H1N1, Gent84 H3N2, Scot / 94 H1N2, and pandemic2009 H1N1, and that can be adapted to rapidly respond to emerging viral subtypes and antigenic continuum mutations.

[0020] However, RP vector systems, such as alphavirus replicon platforms, do not allow for the insertion of any desired number of antigens to achieve the broadest possible protection, such as the insertion of all NA and HA genes of the four major epidemic IAV-S strains into the replicon vector. Alphavirus vector platforms are typically three-component systems consisting of RNA containing non-structural genes from which the relevant packaging signals and structural proteins have been removed and replaced with heterologous gene sequences. Two helper RNAs contain viral structural proteins that lack packaging signals. Systems based on these three-component replicons are limited by the volume of the viral capsid [Nanda K. et al., Vol.390(2), 2009, 368-373]. This inherent limitation of RP vector systems makes it difficult to meet the ongoing demand for vaccines that provide broad protection against most or all epidemic IAV-S strains. [Prior art documents] [Non-patent literature]

[0021] [Non-Patent Document 1] Webster et al., Microbiol Rev 56:152-179(1992) [Non-Patent Document 2] Holtkamp et al.,The American Association of Swine Veterinarians Annual Meeting(2007) [Non-Patent Document 3] Myers KP, Olsen CW, Gray GC. Clin Infect Dis 2007;44(8):1084-8 [Non-Patent Document 4] Krueger and Gray,Curr Top Microbiol Immunol 370:201-225(2013) [Non-Patent Document 5] Gao et al.,J Gen Virol 98(8):2001-2010(2017)

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[0022] In a first aspect of the present invention, it was surprisingly found that in cases where two or more swine influenza A virus hemagglutinin (IAV-S HA) antigens are inserted, the location of the gene encoding the HA antigen within the viral genome of the RNA replicon particle significantly influences the level of induced immunity. [Means for solving the problem]

[0023] Thus, the present invention provides a nucleic acid construct encoding a combination of two IAV-S HA antigens from different lineages in a specific order. These nucleic acid constructs can be used in RNA replicon particles. These RNA replicon particles of the present invention can be used in an immunogenic composition for providing a vaccine for use in preventing diseases caused by swine influenza A virus (IAV-S) in vaccine recipients (e.g., humans, companion animals or livestock, particularly pigs).

[0024] In a first embodiment of the present invention, the nucleic acid construct comprises a combination of IAV-S HA antigens of the Scot / 94 lineage and the Eurasian avian-like (EA) lineage, with the IAV-S HA of the Scot / 94 lineage being placed first (in the 5' to 3' order of the nucleic acid sequence) and the IAV-S HA of the EA lineage being placed second. The term "in the 5' to 3' direction" is also known as "in the downstream direction" and is known in the art. Together with the term "in this order", it serves to indicate the relative orientation that the elements to be joined subsequently need to have with respect to each other in order to function with the host cell's gene expression machinery (i.e., so that the RP according to the present invention containing the nucleic acid construct can be replicated and expressed). As will be understood by those skilled in the art, in this case, this direction relates to the nucleic acid strand of the genome that is the "coding strand". Genes can be present in a sequential order from 5' to 3', i.e., there are no intervening genes for expression into the proteins present in the construct. In that case, the nucleic acid construct typically comprises, in the 5' to 3' order, a backbone viral non-structural protein open reading frame, a subgenomic promoter and the subsequent first HA antigen gene sequence, an intervening sequence, a second subgenomic promoter sequence and the subsequent second HA antigen gene, and finally a backbone viral 3' untranslated region.

[0025] Thus, the present invention provides, in the 5' to 3' order of the nucleic acid sequence: A / swine / Scotland / 410440 / 1994-like H1 huThe first nucleic acid sequence encoding the first hemagglutinin (HA) antigen of IAV-S of the N2(Scot / 94) lineage, and Eurasian avian H1 av The present invention provides a nucleic acid construct comprising a second nucleic acid sequence encoding a second HA antigen of the N1(EA) lineage IAV-S.

[0026] In a second embodiment of the present invention, the nucleic acid construct comprises a combination of Gent / 84 and pdm09-like IAV-S HA antigens, with the Gent / 84 IAV-S HA positioned first (in 5' to 3' order of the nucleic acid sequence) and the pdm09 IAV-S HA positioned second. Thus, the present invention is expressed in 5' to 3' order of the nucleic acid sequence: The first nucleic acid sequence encoding the first HA antigen of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage of IAV-S, and The present invention provides a nucleic acid construct comprising a second nucleic acid sequence encoding a second HA antigen of the A(H1N1)pdm09(pdm09) lineage of IAV-S.

[0027] In a second aspect of the present invention, it was surprisingly found that swine influenza A virus hemagglutinin (IAV-S HA) of specific strains of four major epidemic IAV-S lineages could provide improved immunity against IAV-S compared to other strains. In particular, it was found that specific combinations of IAV-S HA could provide improved immunity. Thus, such combinations of IAV-S HA can be usefully used in nucleic acid constructs that can be contained in RNA replicon particles. These RNA replicon particles can be used as immunogenic compositions to provide vaccines that aid in defense against IAV-S in vaccinating subjects (e.g., humans, companion animals, or livestock, especially pigs) and, for example, aid in defense against IAV-S virus infection.

[0028] Accordingly, the present invention further provides nucleic acid constructs that encode combinations of two IAV-S HA antigens of a particular strain as defined herein.

[0029] In a first embodiment, the present invention provides a nucleic acid construct comprising first and second nucleic acid sequences. The first nucleic acid sequence is H1 from strain A / swine / Italy / 3033-1 / 2015(H1N2), similar to A / swine / Scotland / 410440 / 1994. hu The first HA antigen of IAV-S of the N2(Scot / 94) lineage is encoded, and then, The second nucleic acid sequence is a Eurasian avian-like H1 from strain A / swine / Italy / 28762-3 / 2013(H1N1). av It encodes the second HA antigen of IAV-S of the N1(EA) lineage.

[0030] In a second embodiment, the present invention provides a nucleic acid construct for use in the prevention or treatment of a disease caused by swine influenza A virus in a subject, wherein the nucleic acid construct comprises first and second nucleic acid sequences. The first nucleic acid sequence encodes the first hemagglutinin (HA) antigen of the A / swine / Gent / 1 / 1984-like H3N2(Gent / 84) lineage of swine influenza A virus (IAV-S) derived from strain A / swine / Italy / 240849 / 2015(H3N2), and, The second nucleic acid sequence encodes the second HA antigen of IAV-S from the A(H1N1)pdm09(pdm09) lineage derived from strain A / swine / England / 373 / 2010(H1N1).

[0031] In another important embodiment, RNA replicon particles containing the nucleic acid construct of the present invention are provided. Thus, RNA replicon particles may contain the nucleic acid construct according to the first or second embodiment.

[0032] Any combination of embodiments of the first and second embodiments described herein is included in the present invention. Accordingly, the present invention further provides nucleic acid constructs in which the IAV-S HA antigen is arranged in a specific order as defined in the first embodiment, and the IAV-S antigen is derived from a specific strain as defined in the second embodiment.

[0033] In another important aspect, the present invention provides RNA replicon particles comprising nucleic acid constructs described herein.

[0034] In another important aspect, the present invention provides immunogenic compositions comprising RNA replicon particles as described herein.

[0035] In another important aspect, the present invention provides an immunogenic composition comprising a combination of RNA replicon particles, the combination comprising a first RNA replicon particle comprising a nucleic acid construct according to a first embodiment and a second RNA replicon particle comprising a nucleic acid construct according to a second embodiment.

[0036] Further embodiments of the present invention relate to vaccines comprising the immunogenic composition described herein.

[0037] In another important embodiment, the vaccine of the present invention may be used to prevent or treat diseases caused by swine influenza A virus in a subject.

[0038] In another important embodiment, the present invention provides a method for immunizing pigs against swine influenza A virus, the method comprising administering an immunologically effective dose of the vaccine of the present invention to the pigs.

[0039] In a third aspect, it was surprisingly found that a combination of two RNA replicon particles, each containing nucleic acid constructs encoding the first and second HA antigens of different strains of IAV-S, could provide improved immunity against IAV-S.

[0040] Accordingly, the present invention further provides an immunogenic composition comprising first and second RNA replicon particles, wherein the first RNA replicon particle comprises a nucleic acid construct comprising first and second nucleic acid sequences encoding first and second HA antigens of IAV-S, where, The first HA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and, The second HA antigen is of the A(H1N1)pdm09(pdm09) lineage. The second RNA replicon particle comprises a nucleic acid construct containing third and fourth nucleic acid sequences encoding the third and fourth HA antigens of IAV-S, where, The third HA antigen is A / swine / Scotland / 410440 / 1994-like H1 hu It belongs to the N2 (Scot / 94) system, and, The fourth HA antigen is Eurasian avian-like H1 av It belongs to the N1(EA) system.

[0041] The present invention includes embodiments of the third embodiment described herein and any combination of embodiments of the first and second embodiments. Accordingly, the present invention further provides replicon particles according to the third embodiment, wherein the nucleic acid construct encodes an IAV-S HA antigen, which is arranged in a specific order as defined in the first embodiment, and / or the IAV-S antigen is derived from a specific strain as defined in the second embodiment.

[0042] In a fourth aspect, it was surprisingly found that nucleic acid constructs containing specific combinations of the three different lineages of IAV-S neuraminidase (NA) antigens described herein could be used to provide immunity against all four major epidemic IAV-S lineages.

[0043] Therefore, the present invention further provides nucleic acid constructs comprising first, second, and third nucleic acid sequences encoding the first, second, and third NA antigens of IAV-S, wherein, The first NA antigen is of the A / swine / Scotland / 410440 / 1994-like H1 hu N2(Scot / 94) lineage, and the second NA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and the third NA antigen is selected from the A(H1N1)pdm09 (pdm09) lineage or the Eurasian avian-like H1 av N1(EA) lineage.

[0044] In another important embodiment, the present invention provides an RNA replicon particle comprising a nucleic acid construct described in the fourth aspect.

[0045] In another important embodiment, the present invention provides an immunogenic composition comprising the RNA replicon particle described in the fourth aspect.

[0046] A further embodiment of the present invention relates to a vaccine comprising the immunogenic composition described in the fourth aspect.

[0047] In another important embodiment, the vaccine described in the fourth aspect can be used for the prevention or treatment of diseases caused by porcine influenza A virus in a subject.

[0048] In another important embodiment, the present invention provides a method for immunizing pigs against porcine influenza A virus, the method comprising administering an immunologically effective amount of the vaccine described in the fourth aspect to a pig.

[0049] In another important aspect, the present invention provides an immunogenic composition comprising a combination of RNA replicon particles, the combination comprising a first and a second RNA replicon particle according to the third aspect and a third RNA replicon particle comprising a nucleic acid construct according to the fourth embodiment.

[0050] The present invention includes embodiments of the fourth embodiment described herein and any combination of embodiments of the first, second, and / or third embodiments. Accordingly, the present invention further provides replicon particles according to the third embodiment, wherein the nucleic acid construct encodes an IAV-S HA antigen, which is arranged in a specific order as defined in the first embodiment, and / or the IAV-S antigen is derived from a specific strain as defined in the second embodiment in combination with the replicon particles according to the fourth embodiment. [Brief explanation of the drawing]

[0051] [Figure 1] Hemagglutination inhibition (HI) antibody titer induced by a single-gene RNA particle encoding one HA antigen of the EurAsianAvian lineage IAV-S. [Figure 2] HI antibody titer induced by a single-gene RNA particle encoding one HA antigen of the Scot 1994 strain IAV-S. [Figure 3] HI antibody titer induced by a single-gene RNA particle encoding one HA antigen of the Pdm 2009 strain IAV-S. [Figure 4] HI antibody titer induced by a single-gene RNA particle encoding one HA antigen of the Gent 1984 strain IAV-S. [Figure 5] HI antibody titers induced by dual-gene RNA particles encoding one HA antigen from EurAsianAvian (EUHA1-2, EUHA1-3, and EUHA1-5) and another from the Scot1994 (EUHA1-15 or EUHA1-17) lineage IAV-S strain in different combinations. [Figure 6] HI antibody titers induced by dual-gene RNA particles encoding one HA antigen from pandemic (EUHA1-11) and another from Gent1984 (EUHA3-4), or one HA antigen from Scot1994 (EUH1-15, EUHA1-17) and another from the EurAsianAvian (EUHA1-3 and EUHA1-5) lineage IAS strains, at two different positions. [Figure 7] Neuraminidase inhibitor (NI) antibody titer induced by a single-gene RNA particle encoding one NA antigen of the EurAsianAvian (EA) lineage IAV-S. [Figure 8] Antibody titer (NI) induced by a single-gene RNA particle encoding one NA antigen of the Pdm09 lineage IAV-S. [Figure 9] Antibody titer (NI) induced by a single-gene RNA particle encoding one NA antigen of the Scot / 94 lineage IAV-S. [Figure 10] Antibody titer (NI) induced by a single-gene RNA particle encoding one NA antigen of the Gent / 84 lineage IAV-S. [Figure 11] NI antibody titers induced by dual-gene RNA particles encoding one NA antigen of EurAsianAvian (EUNA1-2) and another NA of Gent1984 (EUNA2-7) at different positions, or by triple-gene RNA particles encoding one NA antigen each of the IAS strains of the EurAsianAvian (EUNA1-2), Gent1984 (EUNA2-7), and Scot1994 (EUHNA2-6) lineages. [Figure 12] NI antibody titers induced by dual-gene RNA particles encoding one NA antigen of EurAsianAvian (EUNA1-2) and another NA of Gent1984 (EUNA2-7) at different positions, or by triple-gene RNA particles encoding one NA antigen each of the IAV-S strains of EurAsianAvian (EUNA1-2), Gent1984 (EUNA2-7), and Scot1994 (EUHNA2-6) or Pdm09 (EUNA1-4) lineages. [Figure 13A] This shows the results of the evaluation of the vaccine efficacy of the multivalent IAV-S vaccine. [Figure 13B] This shows the results of the evaluation of the vaccine efficacy of the multivalent IAV-S vaccine. [Figure 13C] This shows the results of the evaluation of the vaccine efficacy of the multivalent IAV-S vaccine. [Figure 13D] This shows the results of the evaluation of the vaccine efficacy of the multivalent IAV-S vaccine. [Figure 14A] The results of the evaluation of vaccine efficacy after ID administration are shown. [Figure 14B] The results of the evaluation of vaccine efficacy after ID administration are shown. [Modes for carrying out the invention]

[0052] Definitions of terms: To fully understand the present invention, the following definitions are provided.

[0053] Nucleic acid constructs are typically artificially constructed segments of nucleic acids (e.g., DNA, RNA, mRNA) intended for transplantation into target cells.

[0054] The use of singular terms for explanatory purposes is not intended to be limiting. Therefore, for example, a reference to a composition containing a "polypeptide" includes references to one or more such polypeptides. Furthermore, a reference to an "alphaviral RNA replicon particle" includes references to multiple such alphaviral RNA replicon particles unless otherwise specified.

[0055] As used herein, the term “approximately” is used interchangeably with the term “about” and means that the value is within 50% of the value indicated, i.e., approximately 1 × 10 per milliliter. 8 A composition containing alphavirus RNA replicon particles is 5 × 10 per milliliter. 7 pieces~1.5×10 8 This means it contains individual alphavirus RNA replicon particles.

[0056] As used herein, the terms “pig,” “swine,” and “porcine” are interchangeable and include all domesticated pig breeds unless otherwise specified.

[0057] As used herein, a “phylogenetic cluster” is a set of influenza virus antigens, such as hemagglutinin (HA) or neuraminidase (NA), that are grouped together (on the same branch) in a phylogenetic or evolutionary tree rooted in similar (congeneral) ancestors. For IAV-S neuraminidase and hemagglutinin discovered in the United States, the dominant phylogenetic clusters are described in [Anderson et al., Influenza and other Respiratory Viruses 7(Suppl.4):42-51(2013)].

[0058] As used herein, “lineage” refers to a set of influenza virus hemagglutinins grouped together (on the same branch) in a phylogenetic or evolutionary tree rooted in similar (congeneral) ancestors. These groupings have been made for European hemagglutinins and neuraminidases and are similar to, but not identical to, the phylogenetic clusters of US viruses. Lineage determination can be obtained by phylogenetic analysis of the HA or NA sequence in question using a pre-established reference sequence, with readily available software, namely Clustal Omega [Sievers F., et al., (2011) Mol.Syst.Biol.7:539] or a web-accessible annotation tool for H1 HA sequences [Anderson TK, et al., mSphere, 2016;1(6):e00275-16].

[0059] Regarding IAV-S hemagglutinin (HA) discovered in Europe, there are four dominant strains, as described in [Watson et al., J. Virol. 89:9920-9931 (2015)], corresponding to three H1 HA branches and one H3 HA branch [Anderson et al., unpublished] described in Anderson et al., mSphere 1(6):e00275-16 (2016). Until 1979, when a genetically distinct avian H1N1 virus called "Eurasian avian-like swine H1N1" (EA) was isolated from pigs in Belgium and Germany, European pigs were infected only with viruses of the CS strain. The EA lineage continued to circulate among European pigs and, since its emergence, has recombined with seasonally occurring human viruses, resulting in three distinct viral subtypes in Europe: (i) Eurasian avian-like H1avN1 (EA or branch 1C.2.), (ii) A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84 or branch 3.1970.1), and (iii) A / swine / Scotland / 410440 / 1994-like H1huN2 (Scot / 94 or branch 1B.1). Since April 2009, a novel H1N1 IAV virus of swine origin, named (iv) A(H1N1)pdm09 or branch 1A.3.3.2, has spread throughout the human population. In the context of this invention, these four lineages are therefore referred to as "EA," "Gent / 84," "Scot / 94," and "pdm09."

[0060] As used herein, the term "replicon" refers to a modified RNA viral genome that lacks one or more elements (e.g., coding sequences of structural proteins) that, if present, would enable the successful replication of the parent virus in a cell culture or animal host. In a suitable cellular environment, a replicon may amplify itself and produce one or more subgenomic RNA species.

[0061] As used herein, the term “RNA replicon particle,” abbreviated as “RP,” means an RNA replicon packaged in structural proteins, such as capsids and glycoproteins, which may be derived from alphaviruses, e.g., alphaviral RNA replicon particles described by Pushko et al., [Virology 239(2):389-401 (1997)], but may also be from Sindbis virus [Bredenbeek et al., 1993, J. of Virol., vol. 67, p. 6439-6446], and Semlik Forest virus [Liljestrom & Garoff, 1991, Biotechnology (NY), vol. 9, p. 1356-1361]. RPs cannot grow in cell cultures or animal hosts (without a helper plasmid or similar component) because the replicon does not encode alphaviral structural components (e.g., capsids and glycoproteins). Preferably, the RNA RP of the present invention is an alphavirus RNA RP.

[0062] The term "non-IAV-S" is used to modify terms such as pathogen and / or antigen (or immunogen) to indicate that the respective pathogen and / or antigen (or immunogen) is neither an IAV-S pathogen nor an IAV-S antigen (or immunogen), and that a non-IAV-S protein antigen (or immunogen) is not derived from IAV-S.

[0063] The term “derived from ~” is used herein to indicate that the unmodified and / or cleaved amino acid sequence of a given protein antigen is encoded by the pathogen or a strain of that pathogen. The coding sequence may be genetically engineered within the nucleic acid construct of the present invention for a pathogen-derived protein antigen, thereby resulting in modification and / or cleavage of the amino acid sequence of the protein antigen expressed relative to the corresponding sequence of the protein antigen in the derived pathogen or strain of the pathogen (including naturally attenuated strains).

[0064] Where used herein, the terms “treat” or “to treat,” “prevent” or “to prevent,” “protect” or “to provide protection” or “to induce protective immunity,” “to help prevent disease,” and “to support protection” do not necessarily mean complete protection from the signs of infection. For example, “for use in prevention” may mean that the protection provided is sufficient to at least reduce the symptoms of the underlying infection after antigen challenge and / or to reduce and / or eliminate one or more of the underlying cellular, physiological, or biochemical causes or mechanisms that cause the symptoms. Where used in this context, “reduced” is understood to mean the state of infection, including not only the physiological state of the infection but also the molecular state of the infection. Thus, the terms “prevention of disease” or “treatment” encompass prophylactic treatment for viral infection or disorders resulting from infection.

[0065] As used herein, “vaccine” is a composition suitable for application to animals, such as pigs (including humans in certain embodiments, but not specifically for humans in other embodiments), and typically comprises one or more antigens combined with a pharmaceutically acceptable carrier, such as a liquid containing water, which, upon administration to an animal, induces an immune response strong enough to help minimize protection from disease resulting from infection by wild-type microorganisms, i.e., strong enough to help prevent disease, and / or prevent, improve or treat disease.

[0066] As used herein, a polyvalent vaccine is a vaccine containing two or more different antigens. In certain embodiments of this type, the polyvalent vaccine stimulates the recipient's immune system against two or more different pathogens.

[0067] The terms “adjuvant” and “immunostimulant” are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this regard, an adjuvant is used to enhance the immune response to one or more vaccine antigens / isolates. Thus, an “adjuvant” is a drug that nonspecifically increases the immune response to a particular antigen and therefore reduces the amount of antigen required for any given vaccine, and / or the frequency of injections required, in order to produce an appropriate immune response to the antigen of interest. In this regard, an adjuvant is used to enhance the immune response to one or more vaccine antigens / isolates.

[0068] As used herein, "adjuvant-free vaccine" refers to a vaccine that does not contain an adjuvant or a polyvalent vaccine.

[0069] As used herein, the term “pharmaceutically acceptable” is used adjectivally to mean that the modified noun is suitable for medicinal use. For example, when used to describe an excipient in a medicinal vaccine, it means that the excipient is compatible with the other components of the composition and is not adversely harmful to the intended recipient animal, such as a pig.

[0070] Parenteral administration includes subcutaneous injection, submucosal injection, intravenous injection, intramuscular injection, intradermal injection, and infusion.

[0071] The hemagglutinin and neuraminidase antigens of IAV-S may be associated with the complete, i.e., full-length protein specified by the sequence defined herein, or with an antigenic fragment thereof, which may be equally suitable for inducing a suitable immunological response as commonly known in the field of influenza vaccines (see, for example, PLOS ONE Research Article "An Influenza A / H1N1 / 2009 Hemagglutinin Vaccine Produced in Escherichia coli," Jose M. Aguilar-Yanez et al. July 22, 2010; https: / / doi.org / 10.1371 / journal.pone.0011694; Vaccines (Basel) "Optimal Use of Vaccines for Control of Influenza A Virus in Swine," Matthew R. Sandbulte et al. 2015 Marc 3(1)22-73).

[0072] Generally, an antigenic fragment of a particular protein (e.g., a protein antigen) is antigenic, that is, a fragment of that protein that can specifically interact with an antigen-recognizing molecule of the immune system, such as an immunoglobulin (antibody) or a T cell antigen receptor. For example, an antigenic fragment of IAV-S hemagglutinin (HA) is an antigenic fragment of the HA protein, that is, it performs the function of an immunogenic epitope. Preferably, the antigenic fragment of the present invention is immunodominant for antibody and / or T cell receptor recognition. In certain embodiments, an antigenic fragment relating to an antigen of a given protein is a fragment of that protein that retains at least 25% of the antigenicity of the full-length protein (i.e., the ability to induce an antibody equivalent to that established by the HI or NI inhibition assay described below). In preferred embodiments, the antigenic fragment retains at least 50% of the antigenicity of the full-length protein. In more preferred embodiments, the antigenic fragment retains at least 75% of the antigenicity of the full-length protein. The antigenic fragment can be as small as about 20 amino acids, or conversely, it can be a large fragment with a defect as small as a single amino acid from the full-length protein. In certain embodiments, the antigenic fragment contains 25 to 150 amino acid residues. In other embodiments, the antigenic fragment contains 50 to 250 amino acid residues.

[0073] As used herein, if the amino acid residues of both sequences are identical, one amino acid sequence is 100% "identical" to the second amino acid sequence, or has 100% "sequence identity." Therefore, if 50% of the amino acid residues of two amino acid sequences are identical, the amino acid sequences are 50% "identical" to the second amino acid sequence. Sequence comparison is performed over a contiguous block of amino acid residues contained in a given protein, e.g., a protein, or a portion of the polypeptide being compared. In certain embodiments, selected deletions or insertions that may otherwise alter the correspondence between the two amino acid sequences are considered.

[0074] Where used herein, the identity percentage of nucleotide and amino acid sequences can be determined using Clustal Omega, a web-based multiplex sequence alignment program with default parameters [Sievers and Higgins, Protein Sci. 2018 Jan;27(1):135-145 2018]. The identity percentage value is a single numerical score determined for each pair of sequences being aligned. It measures the number of identical residues ("matches") with respect to the length of the alignment. After Clustal Omega, other programs that can be used to determine the identity percentage of nucleotide and amino acid sequences include C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996), and the Clustal W algorithm, which has default alignment parameters and default identity parameters. Alternatively, Advanced Blast search can be used under default filter conditions, for example, by using a GCG (Genetics Computer Group, GCG Package Program Manual, Version 7, Madison, Wisconsin) pile-up program with default parameters. Detailed Description of the Invention

[0075] A first embodiment of a first aspect of the present invention relates to a first nucleic acid construct comprising a combination of at least first and second nucleic acid sequences encoding a hemagglutinin (HA) antigen in a specific order. The first HA antigen encoded by the first nucleic acid sequence in the 5' to 3' direction of the nucleic acid construct is of the Scot / 94 lineage. The second HA antigen encoded by the second nucleic acid sequence in the 5' to 3' direction of the nucleic acid construct is of the EA lineage.

[0076] The first HA antigen of the Scot / 94 lineage may be from any strain, for example, strain A / swine / Italy / 3033-1 / 2015(H1N2) or A / swine / France / 35-140041(H1N2). In a preferred embodiment, the first HA antigen of the Scot / 94 lineage is derived from strain A / swine / Italy / 3033-1 / 2015(H1N2).

[0077] More preferably, the first HA antigen comprises, and more preferably consists of, an amino acid sequence having at least 85%, at least 87%, at least 89%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0078] The second HA antigen of the EA lineage may be derived from any strain, for example, strains A / swine / Denmark / 101048-2 / 2011(H1N1), A / swine / Italy / 28762-3 / 2013(H1N1), or A / swine / France / 44-120070 / 2012(H1N1). In a preferred embodiment, the second HA antigen of the EA lineage is derived from strain A / swine / Italy / 28762-3 / 2013(H1N1).

[0079] More preferably, the second HA antigen comprises, and more preferably consists of, the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0080] A second embodiment of the present invention relates to a second nucleic acid construct comprising a combination of at least first and second nucleic acid sequences encoding a hemagglutinin (HA) antigen in a specific order. The first HA antigen encoded by the first nucleic acid sequence in the 5' to 3' direction of the nucleic acid construct is of the Gent / 84 lineage. The second HA antigen encoded by the second nucleic acid sequence in the 5' to 3' direction of the nucleic acid construct is of the pdm09 lineage.

[0081] The first HA antigen of the Gent / 84 lineage may be from any strain, for example, strain A / swine / Italy / 240849 / 2015(H3N2). In a preferred embodiment, the first HA antigen of the Gent / 84 lineage is derived from strain A / swine / Italy / 240849 / 2015(H3N2).

[0082] More preferably, the first HA antigen comprises, and more preferably consists of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9.

[0083] The second HA antigen of the pdm09 lineage may be from any strain, for example, strain A / swine / England / 373 / 2010(H1N1). In a preferred embodiment, the second HA of the EA lineage is derived from strain A / swine / England / 373 / 2010(H1N1).

[0084] More preferably, the second HA antigen comprises, and more preferably consists of, the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity. More preferably, the first HA antigen comprises, or an amino acid sequence having at least 95%, preferably at least 96%, and more preferably at least 97%, 98%, or 99% sequence identity.

[0085] In the first embodiment of the second aspect, a nucleic acid construct comprising first and second nucleic acid sequences is provided. The first nucleic acid sequence is H1 from strain A / swine / Italy / 3033-1 / 2015(H1N2), similar to A / swine / Scotland / 410440 / 1994. hu The first HA antigen of IAV-S of the N2(Scot / 94) lineage is encoded, and then, The second nucleic acid sequence is a Eurasian avian-like H1 from strain A / swine / Italy / 28762-3 / 2013(H1N1). av It encodes the second HA antigen of IAV-S of the N1(EA) lineage.

[0086] Preferably, the amino acid sequence of the first HA antigen of the Scot / 94 lineage IAV-S derived from strain A / swine / Italy / 3033-1 / 2015(H1N2) comprises, and more preferably consists of, the sequence of SEQ ID NO: 3, or an amino acid sequence having at least 85%, preferably at least 90%, sequence identity. The amino acid identity is more preferably at least 91%, 92%, more preferably at least 93%, 94%, 95%, 96%, 97%, 98%, or even 99% or more.

[0087] Preferably, the amino acid sequence of the second HA antigen of IAV-S of the EA lineage derived from strain A / swine / Italy / 28762-3 / 2013(H1N1) comprises, and more preferably consists of, the sequence of SEQ ID NO: 6, or an amino acid sequence having at least 90%, preferably at least 93%, sequence identity. The amino acid identity is more preferably at least 94%, 95%, more preferably at least 96%, 97%, 98%, or even 99% or more.

[0088] In a second embodiment of the second aspect, a nucleic acid construct is provided for use in the prevention or treatment of a disease caused by swine influenza A virus in a subject, wherein the nucleic acid construct comprises first and second nucleic acid sequences. The first nucleic acid sequence encodes the first hemagglutinin (HA) antigen of the A / swine / Gent / 1 / 1984-like H3N2(Gent / 84) lineage of swine influenza A virus (IAV-S) derived from strain A / swine / Italy / 240849 / 2015(H3N2), and, The second nucleic acid sequence encodes the second HA antigen of IAV-S from the A(H1N1)pdm09(pdm09) lineage derived from strain A / swine / England / 373 / 2010(H1N1).

[0089] Preferably, the amino acid sequence of the first HA antigen of the Gent / 84 lineage IAV-S derived from strain A / swine / Italy / 240849 / 2015(H3N2) includes, and more preferably consists of, the sequence of SEQ ID NO: 9, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity. The amino acid identity is preferably at least 96%, 97%, more preferably at least 98%, or even 99% or more.

[0090] Preferably, the amino acid sequence of the second HA antigen of the IAV-S of the pdm09 lineage derived from strain A / swine / England / 373 / 2010(H1N1) includes, and more preferably consists of, the sequence of SEQ ID NO: 12, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity. The amino acid identity is preferably at least 96%, 97%, more preferably at least 98%, or even 99% or more.

[0091] The nucleic acid constructs according to the first and / or second embodiments of the first and / or second aspects may be contained in an expression cassette incorporating a nucleic acid sequence encoding the hemagglutinin (HA) antigen as a heterogene, together with a transcriptional and / or expression regulatory nucleic acid sequence, such as an alphavirus subgenome promoter sequence, which is suitable for the expression of the HA antigen. Such an expression cassette can be prepared using well-known techniques, by incorporating a heterogeneous nucleic acid sequence encoding the HA antigen into a vector such as a DNA vector or an RNA vector. The vector may be a viral replicon backbone, such as an RNA replicon particle (RP), and is preferably an alphavirus RNA replicon particle.

[0092] Accordingly, the first and second embodiments of the present invention further provide RNA RPs, preferably alphaviral RNA RPs, comprising the nucleotide construct according to the first embodiment. Furthermore, the present invention further provides RNA, preferably alphaviral RNA RPs, comprising the nucleotide construct according to the second embodiment.

[0093] Alphaviral RNA replicon particles (RPs) are well known as non-transmissible, single-cycle, or non-replicating virus-like particle vectors. Their genomes can encode one or more heterologous genes from their 26S subgenome promoter. RPs can replicate within target cells without producing offspring, thus delivering and expressing heterologous antigens(s) to the target animal's immune system. Alphaviral RNA RPs may be based on the human-Venezuelan encephalitis vaccine (VEEV) TC-83 strain.

[0094] RP expression systems for heterologous expression of antigens are available in the art, including, for example, commercially available RP vector-based platforms for vaccine production, such as the VEE virus-based Alphavaccine Platform System, and SEQUIVITY® Technology available from MSD / Merck Animal Health, USA. In a more preferred embodiment, the RNA replicon particle is a Venezuelan encephalitis (VEE) alphavirus-based RNA replicon particle.

[0095] For example, a viral HA antigen gene(s) can then be expressed from a (26S-alphavirus) subgenome promoter, and the transcribed replicon RNA can be packaged into a replicon plasmid by expression of a structural protein by a packaging cell line, or by simultaneous transfection of the replicon RNA and one or more "helper" RNAs encoding the structural protein into a suitable host cell. The generation of VEE TC-83 RNA replicon particles is described, for example, in U.S. Patents 9,441,247 and 8,460,913. Briefly, the HA or NA gene was de novo synthesized using sequences from an SIV strain (DNA 2.0). Two HA or three NA genes were cloned into a replicon vector plasmid using a tandem unidirectional expression cassette, and the sequences were verified to ensure that no mutations were introduced during the cloning process. RNA was generated by in vitro transcription of linearized replicon plasmid DNA using T7 RNA polymerase, as previously described [Kamrud et al., Virology. 2007;360(2):376-387]. RP was generated by co-electroporating HA or NA replicon RNA and structural gene helper RNA into Vero cells, followed by particle recovery [Hooper et al., Vaccine. 2009;28(2):494-511].

[0096] Common molecular biology techniques, including cloning, transfection, recombination, selection, and amplification, are described in great detail in standard textbooks 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; 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].

[0097] The nucleic acid constructs of the present invention can be used in immunogenic compositions comprising the nucleic acid constructs. Preferably, the immunogenic composition comprises one or more replicon particles comprising the nucleic acid constructs of the present invention. Therefore, the replicon particles of the present invention can be used in immunogenic compositions, such as vaccines, comprising the replicon particles. The immunogenic composition or vaccine may consist of replicon particles or may comprise replicon particles in combination with additional components such as carriers or adjuvants. The immunogenic compositions of the present invention can be used in vaccines for the prevention of diseases caused by swine influenza A virus (IAV-S) in a target population.

[0098] Accordingly, in first and / or second embodiments, the present invention further provides an immunogenic composition comprising or comprising an RNA RP comprising a nucleic acid construct according to the first embodiment. Alternatively, the present invention further provides an immunogenic composition comprising or comprising an RNA RP comprising a nucleic acid construct according to the second embodiment.

[0099] In preferred embodiments of the first and / or second embodiments, the present invention provides an immunogenic composition comprising a first RNA RP comprising a nucleotide construct according to the first embodiment and a second RNA RP comprising a nucleotide construct according to the second embodiment. The present invention can demonstrate that an immunogenic composition comprising a combination of replicon particles according to the first and second embodiments provides broad protection against existing IAV-S strains, and therefore such an immunogenic composition can be usefully used as a vaccine to aid in protection against IAV-S infection in vaccination targets, e.g., pigs (e.g., sows or piglets), i.e., to aid in prevention or treatment.

[0100] Therefore, in a preferred embodiment, the present invention provides an immunogenic composition such as a vaccine comprising first and second RNA replicon particles. (i) The first RNA replicon particle, preferably the alphavirus RNA replicon particle, comprises a nucleic acid construct comprising first and second nucleic acid sequences encoding first and second hemagglutinin (HA) antigens of swine influenza A virus (IAV-S), wherein, The first HA antigen is from the Gent / 84 lineage, and, The second HA antigen is of the pdm09 lineage. (ii) The second RNA replicon particle, preferably the alphavirus RNA replicon particle, comprises a nucleic acid construct containing third and fourth nucleic acid sequences encoding the third and fourth HA antigens of IAV-S, wherein, The third HA antigen is from Scot / 94, and, The fourth HA antigen belongs to the EA lineage.

[0101] In a particularly preferred embodiment, the present invention provides an immunogenic composition such as a vaccine comprising first and second RNA replicon particles. (i) The first RNA replicon particle, preferably an alphavirus RNA replicon particle, In order from 5' to 3' of the nucleic acid sequence, A first nucleic acid sequence encoding the first HA antigen of the Scot / 94 lineage IAV-S and a second nucleic acid sequence encoding the second HA antigen of the EA lineage IAV-S, A first nucleic acid construct comprising, (ii) The second RNA replicon particle, preferably the alphavirus RNA replicon particle, has the nucleic acid sequence in the order from 5' to 3', The third nucleic acid sequence encoding the third HA antigen of Gent / 84 lineage IAV-S, The fourth nucleic acid sequence encoding the fourth HA antigen of the pdm09 lineage IAV-S, It includes a second nucleic acid construct that contains [the specified component].

[0102] In a particularly preferred embodiment, the present invention provides an immunogenic composition such as a vaccine comprising first and second RNA replicon particles. (i) The first RNA replicon particle, preferably an alphavirus RNA replicon particle, In order from 5' to 3' of the nucleic acid sequence, A first nucleic acid sequence encoding the first HA antigen of the Scot / 94 lineage IAV-S and a second nucleic acid sequence encoding the second HA antigen of the EA lineage IAV-S, A first nucleic acid construct comprising, (ii) The second RNA replicon particle, preferably the alphavirus RNA replicon particle, has the nucleic acid sequence in the order from 5' to 3', The third nucleic acid sequence encoding the third HA antigen of Gent / 84 lineage IAV-S, The fourth nucleic acid sequence encoding the fourth HA antigen of the pdm09 lineage IAV-S, It includes a second nucleic acid construct that contains [the specified component].

[0103] Therefore, in a third aspect, the present invention provides an immunogenic composition such as a vaccine comprising first and second RNA replicon particles, (i) The first RNA replicon particle, preferably the alphavirus RNA replicon particle, comprises a nucleic acid construct comprising first and second nucleic acid sequences encoding the first and second hemagglutinin (HA) antigens of swine influenza A virus (IAV-S) in 5' to 3' order of nucleic acid sequence, where, The first HA antigen encoded by the first nucleic acid sequence is of the Gent / 84 lineage derived from strain A / swine / Italy / 240849 / 2015(H3N2), preferably the one of sequence number 9, or an amino acid sequence having at least 90% sequence identity thereto, The second HA antigen encoded by the first nucleic acid sequence is from the pdm09 lineage derived from strain A / swine / England / 373 / 2010(H1N1), preferably the sequence of SEQ ID NO: 12, or an amino acid sequence having at least 95% sequence identity thereto. (ii) The second RNA replicon particle, preferably the alphavirus RNA replicon particle, comprises a nucleic acid construct comprising third and fourth nucleic acid sequences encoding the third and fourth HA antigens of IAV-S in 5' to 3' order of nucleic acid sequence, where, The third HA antigen encoded by the third nucleic acid sequence is of the Scot / 94 lineage derived from strain A / swine / Italy / 3033-1 / 2015 (H1N2), preferably the one from SEQ ID NO: 3, or an amino acid sequence having at least 85% sequence identity thereto, The fourth HA antigen encoded by the fourth nucleic acid sequence is from the EA lineage derived from strain A / swine / Italy / 28762-3 / 2013(H1N1), preferably the one from sequence number 6, or an amino acid sequence having at least 90% sequence identity thereto.

[0104] The nucleic acid constructs, immunogenic compositions, and replicon particles of the third embodiment are as described above with respect to the first and second embodiments of the present invention. Accordingly, the present invention further comprises any combination of embodiments of the third embodiment described herein and embodiments of the first and second embodiments. Accordingly, the present invention further provides replicon particles according to the third embodiment, wherein the nucleic acid construct encodes the IAV-S HA antigen, which is arranged in a specific order as defined in the first embodiment, and / or the IAV-S antigen is derived from a specific strain as defined in the second embodiment.

[0105] The immunogenic composition can be adapted for the simultaneous or sequential administration of the first and second RNA replicon particles as described above, i.e., the simultaneous or sequential administration of RNA RPs containing nucleic acid constructs according to the first and second embodiments. Preferably, the immunogenic composition is adapted for the simultaneous administration of the first and second RNA replicon particles. Therefore, in a preferred embodiment, the immunogenic composition comprises the first and second RNA replicon particles in unit dosage forms.

[0106] In a more preferred embodiment, the immunogenic composition may comprise one or more additional RNA replicon particles. Such additional RNA RPs may comprise nucleic acid constructs encoding one or more additional antigens. For example, an additional RNA RP may comprise a nucleic acid construct encoding one or more neuraminidase (NA) antigens of IAV-S. In a particular embodiment, the nucleic acid construct encodes two or three, preferably three, NA antigens of IAV-S, or immunogenic fragments thereof.

[0107] In a particularly preferred embodiment, the additional RNA RP further comprises nucleic acid constructs comprising first, second, and third nucleic acid sequences encoding the first, second, and third NA antigens of IAV-S, where, The first NA antigen is of the Scot / 94 lineage. The second NA antigen is of the Gent / 84 lineage, and, The third NA antigen is selected from the pdm09 or EA lineage.

[0108] Therefore, in a fourth aspect of the present invention, a nucleic acid construct is further provided comprising first, second, and third nucleic acid sequences encoding first, second, and third NA antigens of IAV-S, where, The first NA antigen encoded by the first nucleic acid sequence is of the Scot / 94 lineage. The second NA antigen encoded by the second nucleic acid sequence is from the Gent / 84 lineage, and, The third NA antigen, encoded by the third nucleic acid sequence, is selected from the pdm09 or EA lineage.

[0109] Preferably, the amino acid sequence of the first NA antigen of the Scot / 94 lineage IAV-S is derived from strain A / swine / England / 61470 / 2013(H1N2). The amino acid sequence of the first NA antigen preferably includes, and more preferably consists of, the sequence of SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity. The amino acid identity is preferably at least 96%, 97%, more preferably at least 98%, or even 99% or more.

[0110] Preferably, the amino acid sequence of the second NA antigen of the Gent / 84 lineage IAV-S is derived from strain A / swine / Italy / 248147-8 / 2015(H3N2). The amino acid sequence of the second NA antigen preferably includes, and more preferably consists of, the sequence of SEQ ID NO: 18 or an amino acid sequence having at least 90% sequence identity. The amino acid identity is preferably at least 96%, 97%, more preferably at least 98%, or even 99% or more.

[0111] Preferably, the amino acid sequence of the third NA antigen of the pdm09 lineage IAV-S is derived from strain A / swine / England / 373 / 2010(H1N1) or A / swine / Italy / 179057 / 2015(H1N1), preferably from strain A / swine / Italy / 179057 / 2015(H1N1). The amino acid sequence of the third NA antigen preferably includes, and more preferably consists of, the sequence of SEQ ID NO: 21 or an amino acid sequence having at least 90% sequence identity. The amino acid identity is preferably at least 96%, 97%, more preferably at least 98%, or even 99% or more.

[0112] Alternatively, the amino acid sequence of the third NA antigen of the EA lineage IAV-S is derived from strain A / swine / Italy / 28762-3 / 2013(H1N1). The amino acid sequence of the third NA antigen preferably includes, and more preferably consists of, the sequence of SEQ ID NO: 24 or an amino acid sequence having at least 90% sequence identity. The amino acid identity is preferably at least 96%, 97%, more preferably at least 98%, or even 99% or more.

[0113] RNA replicon particles, preferably alphaviral RNA replicon particles, comprising nucleic acid constructs containing first, second, and third nucleic acid sequences encoding first, second, and third neuraminidase (NA) antigens of swine influenza A virus (IAV-S), are further provided, where, The first NA antigen is of the Scot / 94 lineage. The second NA antigen is of the Gent / 84 lineage, and, The third NA antigen is selected from the pdm09 or EA lineage.

[0114] Replicon particles comprising nucleic acid constructs according to the fourth aspect can be used alone or in combination with replicon particles according to the first, second and / or third aspects of the present invention as described herein, and are usefully used in combination with replicon particles comprising hemagglutinin antigens according to the first, second and / or third aspects of the present invention.

[0115] The replicon particles in this fourth embodiment are not particularly limited, but are preferably replicon particles such as alphavirus replicon particles, most preferably Venezuelan encephalitis virus (VEEV) alphavirus RNA replicon particles as described in the first, second and / or third embodiments.

[0116] In a more preferred embodiment, the present invention provides an immunogenic composition such as a vaccine comprising at least first, second, and third RNA replicon particles. The first RNA replicon particle comprises a nucleic acid construct containing first and second nucleic acid sequences encoding the first and second HA antigens of IAV-S in 5' to 3' order of nucleic acid sequence, where, The first HA antigen is from the Scot / 94 lineage, and, The second HA antigen is of the EA lineage, The second RNA replicon particle comprises a nucleic acid construct containing third and fourth nucleic acid sequences encoding the third and fourth HA antigens of IAV-S in 5' to 3' order of nucleic acid sequence, where, The third HA antigen is from the Gent / 84 lineage, and, The fourth HA antigen is of the pdm09 lineage, and, The third RNA replicon particle comprises a nucleic acid construct containing first, second, and third nucleic acid sequences encoding the first, second, and third NA antigens of IAV-S, where, The first NA antigen is of the Scot / 94 lineage. The second NA antigen is of the Gent / 84 lineage, and, The third NA antigen is selected from the pdm09 or EA lineage.

[0117] The immunogenic compositions described above, such as vaccines, can be usefully used as vaccines to supplement protection against IAV-S infection in vaccinated animals such as pigs (e.g., sows or piglets).

[0118] The immunogenic composition can be adapted to the simultaneous or sequential administration of the first, second, and third RNA replicon particles as described above, i.e., the simultaneous or sequential administration of RNA RP containing nucleic acid constructs according to the first, second, and / or third embodiments in combination with nucleic acid constructs according to the fourth embodiment. Preferably, the immunogenic composition is adapted to the simultaneous administration of the first, second, and third RNA replicon particles. Therefore, in a preferred embodiment, the immunogenic composition contains the first, second, and third RNA replicon particles in unit dosage forms.

[0119] The present invention also provides vaccines against multiple porcine pathogens. For example, coding sequences of protein antigens or antigenic fragments thereof, or combinations of such protein antigen coding sequences useful for a porcine vaccine, can be added to RNA replicon particles (RPs) as described herein, and / or combined with the same RPs that encode HA or NA derived from IAV-S in the vaccine. Examples of pathogens that can be the source of one or more protein antigens or antigenic fragments include: Porcine Reproductive and Respiratory Syndrome (PRRS), Porcine Circovirus (PCV), Infectious Gastroenteritis Virus (TGE), Porcine Pseudorabies (PPRV), Porcine Parvovirus (PPV), Porcine Rotavirus (PRV), Porcine Epidemic Diarrhea Virus (PED), Pasteurella multocida (multiple serotypes), Salmonella, Escherichia coli (e.g., serotypes K99, K88, 987P, or F41), Haemophilus parasuis, Lawsonia intracellularis, Mycoplasma (e.g., Mycoplasma hyopneumoniae), Bacillus bronchoseptica, Erysipelas ssp., Campylobacter, and Actinobacillus pluprneumoniensis. This includes Clostridium pleuropneumoniae, Clostridium perfringens, and Clostridium difficile.

[0120] Furthermore, the present invention provides a vaccine comprising one or more RPs of the present invention in combination with one or more other vectors encoding one or more of these porcine antigens (e.g., porcine circovirus-2 (PCV-2) and / or porcine circovirus-3 (PCV-3), and / or baculovirus vectors encoding ORF-2 proteins derived from inactivated toxoids of one or more of these porcine pathogens). Furthermore, such a vaccine may also contain any RNA replicon particles encoding HA and / or NA derived from IAV-S in the vaccine of the present invention, together with one or more dead and / or modified (attenuated) live porcine virus isolates and / or porcine bacteria.

[0121] Therefore, one or more RNA RPs encoding one or more HAs and / or NAs derived from IAV-S can be added together with one or more other vectors encoding one or more porcine antigens and / or one or more dead and / or modified (attenuated) live virus isolates, such as one or more dead or modified live IAS-V strains, one or more dead and / or modified live PRRS viruses, one or more dead and / or modified live PCVs, one or more dead and / or modified live TGEs, one or more dead and / or modified live PPRVs, one or more dead and / or modified live PPVs, one or more dead and / or modified live PRVs, and one or more dead and / or modified live PEDs. Furthermore, one or more alphaviral RNA replicon particles (RPs) encoding one or more HA or NA derived from IAV-S can be added together with one or more other vectors encoding one or more porcine antigens, and / or one or more dead and / or modified (attenuated) live bacteria that can also infect pigs, e.g., one or more dead and / or modified (one or more serotypes) live Pasteurella multocida, Salmonella, Escherichia coli (one or more serotypes), Haemophilus parasuis, Lawsonia intracellularis, Mycoplasma (e.g., Mycoplasma hyopneumoniae), Bacillus bronchoseptica, Erysipelas ssp., Campylobacter, Actinobacillus pleuropneumoniae, Clostridium perfringens It can be added together with *Clostridium perfringens* and *Clostridium difficile*.

[0122] Accordingly, the present invention also comprises all RNA replicon particles of the present invention, naked DNA vectors containing nucleic acid constructs of the present invention, naked RNA vectors containing nucleic acid constructs of the present invention, nucleic acid constructs of the present invention containing synthetic messenger RNA, and RNA replicons, as well as all immunogenic compositions and / or vaccines comprising nucleic acid constructs of the present invention (e.g., synthetic messenger RNA, RNA replicons), alphavirus RNA replicon particles, naked RNA vectors, and / or naked DNA vectors.

[0123] The immunogenic composition of the present invention can be used as a vaccine, which may be an adjuvant-free vaccine or an adjuvant-containing vaccine. Accordingly, the present invention further includes a vaccine (polyvalent) containing the immunogenic composition of the present invention. In certain embodiments, the vaccine is an adjuvant-free vaccine. In other embodiments, the vaccine contains an adjuvant. Adjuvants suitable for use in the vaccine of the present invention are not particularly limited and may include one or more adjuvants selected from the group consisting of biodegradable oil, an oil-in-water emulsion containing 2.5-50% (v / v) mineral oil, and biodegradable oil mixed with an oil-in-water emulsion containing 2.5-50% (v / v) mineral oil.

[0124] In certain embodiments, the adjuvant is a biodegradable oil. In these particular embodiments, the biodegradable oil is dl-α-tocopheryl acetate (vitamin E acetate). In other embodiments, the adjuvant comprises an oil-in-water emulsion containing 2.5% to 50% (v / v) mineral oil. In specific embodiments, the adjuvant comprises an oil-in-water emulsion containing 2.5% (v / v) mineral oil. In related embodiments, the adjuvant comprises an oil-in-water emulsion containing 5% (v / v) mineral oil. In other embodiments, the adjuvant comprises an oil-in-water emulsion containing 12.5% ​​(v / v) mineral oil. In yet another embodiment, the adjuvant comprises an oil-in-water emulsion containing 25% (v / v) mineral oil. In yet another embodiment, the adjuvant comprises an oil-in-water emulsion containing 50% (v / v) mineral oil. In a more specific embodiment, the adjuvant comprises a mixture of biodegradable oil and mineral oil adjuvant. In specific embodiments, the biodegradable oil is dl-α-tocopheryl acetate and the mineral oil is liquid paraffin. In a more specific embodiment, the biodegradable oil is dl-α-tocopheryl acetate, and the mineral oil is light liquid paraffin.

[0125] In the relevant formulations, the adjuvant is a mixture of two components. The first component consists of mineral oil droplets with an average (weighed volume) size of approximately 1 μm, which are stabilized with polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) in water. The first component may contain 25% by weight of mineral oil and 1% by weight of polysorbate 80, with the remainder being water. The second component may consist of droplets of biodegradable dl-α-tocopheryl acetate with an average (weighed volume) size of approximately 400 nm, which are also stabilized with polysorbate 80. A particular formulation contains 15% by weight of dl-α-tocopheryl acetate and 6% by weight of polysorbate 80, with the remainder being water. In certain embodiments, the adjuvant is X-SOLVE®, which is a combination of two component adjuvants: DILUVAC FORTE® based on dl-α-tocopheryl acetate and MICROSOL® based on light liquid paraffin [see, for example, U.S. Patent No. 8,597,662]. In the relevant formulations, the adjuvant contains submicrometer-sized oil droplets and biodegradable oil droplets, the biodegradable oil droplets having an average size different from the average size of the mineral oil droplets [see, for example, U.S. Patent No. 9,084,768].

[0126] In certain embodiments, the vaccine helps prevent disease caused by IAV-S. In related embodiments, antibodies are induced in pig subjects when pigs are immunized with the vaccine. In certain embodiments, the pig subjects are sows. In related embodiments, the vaccine provides maternal protective antibodies to the offspring of vaccinated sows. In other embodiments, the pig subjects are piglets. In certain embodiments of this type, the vaccine is administered to piglets at an early stage, as early as 3 days of age. In certain embodiments, the vaccine is administered as a booster vaccine. In certain embodiments, the vaccine is administered as a single-dose vaccine. In certain embodiments of this type, the vaccine is administered as a booster vaccine. In yet other embodiments, the vaccine is administered as a multi-dose vaccine. In certain embodiments of this type, the vaccine is administered as a two-dose vaccine.

[0127] The present invention also provides a method for immunizing pigs (e.g., sows or piglets) with a porcine pathogen, such as IAV-S, comprising administering an immunologically effective dose of the vaccine or polyvalent vaccine of the present invention to the pigs. In certain embodiments, the vaccine is administered by intramuscular injection. In alternative embodiments, the vaccine is administered by subcutaneous injection. In other embodiments, the vaccine is administered by intravenous injection. In yet another embodiment, the vaccine is administered by intradermal injection. In yet another embodiment, the vaccine is administered orally. In yet another embodiment, the vaccine is administered by intranasal administration. A preferred method is intradermal administration. Another preferred method is intramuscular administration.

[0128] Therefore, the vaccines and polyvalent vaccines of the present invention can be administered as primer vaccines and / or booster vaccines. In certain embodiments, the vaccines of the present invention are administered as a single-shot vaccine (one dose), and no further administration is required. In certain embodiments, when both the primer vaccine and the booster vaccine are administered, the primer vaccine and the booster vaccine can be administered via the same route.

[0129] In certain embodiments of this type, both the primer vaccine and the booster vaccine are administered by intradermal injection. In other embodiments of this type, both the primer vaccine and the booster vaccine are administered by intramuscular injection. In alternative embodiments, when both the primer vaccine and the booster vaccine are administered, the primer vaccine may be administered via one route and the booster vaccine via another. In certain embodiments of this type, the primer vaccine may be administered by intradermal injection and the booster vaccine may be administered orally. In related embodiments of this type, the primer vaccine may be administered by intramuscular injection and the booster vaccine may be administered orally. In other embodiments of this type, the primer vaccine may be administered by intramuscular injection and the booster vaccine may be administered by intradermal injection. In yet another embodiment of this type, the primer vaccine may be administered by intradermal injection and the booster vaccine may be administered by intramuscular injection. Those skilled in the art will understand that vaccine compositions are preferably appropriately formulated for each type of recipient animal and route of administration.

[0130] The present invention further provides a method for immunizing pigs with IAV-S, the method comprising administering an immunologically effective amount of the vaccine of the present invention to the pigs. The method preferably comprises intradermal administration of the vaccine. The present invention further provides a method for immunizing pigs (e.g., sows or piglets) with IAV-S, the method comprising injecting the pigs with an immunologically effective amount of the vaccine of the present invention described above, so that the pigs produce appropriate IAV-S antibodies. In certain embodiments, the vaccine is, for example, about 1 × 10⁻⁶ 4 ~Approx. 1×10 10 It may contain more than 1 RP. In a more specific embodiment, the vaccine may contain approximately 1 × 10 5 ~Approx. 1×10 9 It may contain RPs. In a more specific embodiment, the vaccine may contain approximately 1 × 10⁶ vaccines. 6 ~Approx. 1×10 8 It can contain individual RPs.

[0131] In certain embodiments, the vaccine of the present invention is administered at a dosage of 0.05 mL to 3 mL. In more specific embodiments, the dosage administered is 0.1 mL to 2 mL. In even more specific embodiments, the dosage administered is 0.2 mL to 1.5 mL. In even more specific embodiments, the dosage administered is 0.3 to 1.0 mL. In even more specific embodiments, the dosage administered is 0.4 mL to 0.8 mL.

[0132] Thus, in a first aspect, the present invention provides the following embodiments; [1] A nucleic acid construct for use in preventing a disease caused by swine influenza A virus (IAV-S) in a subject, in the order from 5' to 3' of the nucleic acid sequence, A / swine / Scotland / 410440 / 1994-like H1 hu a first nucleic acid sequence encoding the first hemagglutinin (HA) antigen of IAV-S of the N2(Scot / 94) lineage, and Eurasian avian-like H1 av a second nucleic acid sequence encoding the second HA antigen of IAV-S of the N1(EA) lineage, a nucleic acid construct comprising.

[0133] [2] The nucleic acid construct for use according to [1], wherein the first HA antigen is derived from the strain A / swine / Italy / 3033-1 / 2015 (H1N2).

[0134] [3] The nucleic acid construct for use according to [1] or [2], wherein the first HA antigen encoded by the first nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 85% sequence identity thereto.

[0135] [4] The nucleic acid construct for use according to any one of [1] to [3], wherein the second HA antigen is derived from the strain A / swine / Italy / 28762-3 / 2013 (H1N1).

[0136] [5] A nucleic acid construct for use according to any one of [1] to [4], wherein the second HA antigen encoded by the second nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90% sequence identity with respect to that sequence.

[0137] [6] A nucleic acid construct for use in the prevention of disease caused by swine influenza A virus (IAV-S) in a subject, wherein the nucleic acid sequence is in the order of 5' to 3', The first nucleic acid sequence encoding the first HA antigen of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage of IAV-S, and A nucleic acid construct comprising a second nucleic acid sequence encoding the second HA antigen of IAV-S of the A(H1N1)pdm09(pdm09) lineage.

[0138] [7] A nucleic acid construct for use as described in [6], wherein the first HA antigen is derived from strain A / swine / Italy / 240849 / 2015(H3N2).

[0139] [8] A nucleic acid construct for use according to [6] or [7], wherein the first HA antigen encoded by the first nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 90% sequence identity thereto.

[0140] [9] A nucleic acid construct for use as described in any one of [6] to [8], wherein the second HA antigen is derived from strain A / swine / England / 373 / 2010(H1N1).

[0141]

[10] A nucleic acid construct for use according to any one of [6] to [9], wherein the second HA antigen encoded by the second nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 95% sequence identity with respect to that sequence.

[0142] RNA replicon particles containing a nucleic acid construct described in any one of

[11] [1] to [5].

[0143] RNA replicon particles containing the nucleic acid construct described in any one of

[12] [6] to

[10] .

[0144]

[13] An alphavirus RNA replicon particle, as described in

[15] or

[16] .

[0145]

[14] The RNA replicon particle described in

[13] is a Venezuelan encephalitis (VEE) alphavirus RNA replicon particle.

[0146] An immunogenic composition containing RNA replicon particles as described in any one of

[15] ,

[11] , to

[14] .

[0147] The immunogenic composition according to

[15] , comprising the RNA replicon particles described in

[16] ,

[11] , and

[12] .

[0148] The immunogenic composition according to

[16] , which is suitable for the simultaneous administration of alphavirus RNA replicon particles as described in

[17] ,

[11] and

[12] .

[0149] A vaccine comprising an immunogenic composition described in any one of

[18] ,

[15] , or

[17] .

[0150]

[19] The adjuvant-free vaccine described in

[18] .

[0151]

[20] The vaccine according to

[18] , comprising an adjuvant selected from the group consisting of biodegradable oil, an oil-in-water emulsion containing 2.5-50% (v / v) mineral oil, and biodegradable oil mixed with an oil-in-water emulsion containing 2.5-50% (v / v) mineral oil.

[0152]

[21] A vaccine described in any one of

[18] to

[20] for use in preventing disease caused by swine influenza A virus in the target population.

[0153] A method of immunizing pigs against swine influenza A virus, the method comprising administering to the pig an immunologically effective amount of the vaccine according to any one of

[18] to

[20] .

[0154]

[23] In the 5' to 3' order of the nucleic acid sequence, A / swine / Scotland / 410440 / 1994-like H1 hu A first nucleic acid sequence encoding the first HA antigen of swine influenza A virus (IAV-S) of the N2(Scot / 94) strain, and Eurasian avian-like H1 av A nucleic acid construct comprising a second nucleic acid sequence encoding the second HA antigen of IAV-S of the N1(EA) strain.

[0155]

[24] In the 5' to 3' order of the nucleic acid sequence, A first nucleic acid sequence encoding the first HA antigen of IAV-S of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) strain, and A nucleic acid construct comprising a second nucleic acid sequence encoding the second HA antigen of IAV-S of the A(H1N1)pdm09 (pdm09) strain.

[0156] In a second aspect, the present invention provides the following embodiments; [1] A nucleic acid construct for use in preventing a disease caused by swine influenza A virus in a subject, the nucleic acid construct comprising a first and a second nucleic acid sequence, The first nucleic acid sequence encodes the first HA antigen of IAV-S of the A / swine / Scotland / 410440 / 1994-like H1 derived from the strain A / swine / Italy / 3033-1 / 2015 (H1N2), and hu The first nucleic acid sequence encodes the first HA antigen of IAV-S of the N2(Scot / 94) strain, and The second nucleic acid sequence encodes the second HA antigen of IAV-S of the Eurasian avian-like H1 derived from the strain A / swine / Italy / 28762-3 / 2013 (H1N1), av A nucleic acid construct encoding the second HA antigen of IAV-S of the N1(EA) strain.

[0157] [2] A nucleic acid construct for use according to [1], wherein the first HA antigen encoded by the first nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 85% sequence identity with respect to that sequence.

[0158] [3] A nucleic acid construct for use according to [1] or [2], wherein the second HA antigen encoded by the second nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90% sequence identity with respect to that sequence.

[0159] [4] A nucleic acid construct for use in the prevention of disease caused by swine influenza A virus in a subject, comprising a first and a second nucleic acid sequence, The first nucleic acid sequence encodes the first hemagglutinin (HA) antigen of the A / swine / Gent / 1 / 1984-like H3N2(Gent / 84) lineage of swine influenza A virus (IAV-S) derived from strain A / swine / Italy / 240849 / 2015(H3N2), and, A nucleic acid construct in which the second nucleic acid sequence encodes the second HA antigen of IAV-S from the A(H1N1)pdm09(pdm09) lineage derived from strain A / swine / England / 373 / 2010(H1N1).

[0160] [5] A nucleic acid construct for use according to [4], wherein the first HA antigen encoded by the first nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 95% sequence identity with respect to that sequence.

[0161] [6] A nucleic acid construct for use according to [4] or [5], wherein the second HA antigen encoded by the second nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 95% sequence identity with respect to that sequence.

[0162] RNA replicon particles containing a nucleotide construct described in any one of [7][1] to [3].

[0163] RNA replicon particles containing the nucleotide construct described in any one of [8][4]~[6].

[0164] [9] An RNA replicon particle described in [7] or [8], which is an alphavirus RNA replicon particle.

[0165]

[10] The RNA replicon particle described in [9], which is a Venezuelan encephalitis virus (VEEV) alphavirus RNA replicon particle.

[0166] An immunogenic composition containing RNA replicon particles as described in any one of

[11] [7] to

[10] .

[0167] The immunogenic composition according to

[11] , comprising the RNA replicon particles described in

[12] [7] and [8].

[0168] A vaccine comprising the immunogenic composition described in

[13]

[12] .

[0169]

[14] The adjuvant-free vaccine described in

[13] .

[0170]

[15] The vaccine according to

[13] , comprising an adjuvant selected from the group consisting of biodegradable oil, an oil-in-water emulsion containing 2.5-50% (v / v) mineral oil, and biodegradable oil mixed with an oil-in-water emulsion containing 2.5-50% (v / v) mineral oil.

[0171]

[16] A vaccine described in any one of

[13] to

[15] for use in preventing disease caused by swine influenza A virus in the target population.

[0172]

[17] A method for immunizing a pig against swine influenza A virus, comprising administering an immunologically effective dose of any one of the vaccines described in

[14] to

[16] to the pig.

[0173]

[18] comprising the first and second nucleic acid sequences, The first nucleic acid sequence encodes the first hemagglutinin (HA) antigen of the A / swine / Gent / 1 / 1984-like H3N2(Gent / 84) lineage of swine influenza A virus (IAV-S) derived from strain A / swine / Italy / 240849 / 2015(H3N2), and, A nucleic acid construct in which the second nucleic acid sequence encodes the second HA antigen of IAV-S from the A(H1N1)pdm09(pdm09) lineage derived from strain A / swine / England / 373 / 2010(H1N1).

[0174]

[19] comprising the first and second nucleic acid sequences, The first nucleic acid sequence is similar to H1 from strain A / swine / Italy / 3033-1 / 2015(H1N2) and A / swine / Scotland / 410440 / 1994. hu The first HA antigen of IAV-S of the N2(Scot / 94) lineage is encoded, and then, The second nucleic acid sequence is a Eurasian avian-like H1N1 derived from strain A / swine / Italy / 28762-3 / 2013(H1N1). av A nucleic acid construct that encodes the second HA antigen of IAV-S of the N1(EA) lineage.

[0175] In a third aspect, the present invention provides the following embodiments; [1] An immunogenic composition for use in the prevention of disease caused by swine influenza A virus in a subject, comprising first and second RNA replicon particles, The first RNA replicon particle comprises a nucleic acid construct containing first and second nucleic acid sequences encoding the first and second hemagglutinin (HA) antigens of swine influenza A virus (IAV-S), wherein, The first HA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and, The second HA antigen is of the A(H1N1)pdm09(pdm09) lineage. The second RNA replicon particle comprises a nucleic acid construct containing third and fourth nucleic acid sequences encoding the third and fourth HA antigens of IAV-S, where, The third HA antigen is A / swine / Scotland / 410440 / 1994-like H1 hu It belongs to the N2 (Scot / 94) system, and, The fourth HA antigen is Eurasian avian-like H1 av An immunogenic composition of the N1(EA) class.

[0176] [2] An immunogenic composition for use as described in [1], wherein the first HA antigen is derived from strain A / swine / Italy / 240849 / 2015(H3N2).

[0177] [3] An immunogenic composition for use according to [1] or [2], wherein the first HA antigen encoded by the first nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 90% sequence identity with respect to that sequence.

[0178] [4] An immunogenic composition for use according to any one of the preceding [1] to [3], wherein the second HA antigen is derived from strain A / swine / England / 373 / 2010(H1N1).

[0179] [5] An immunogenic composition for use according to any one of the preceding [1] to [4], wherein the second HA antigen encoded by the second nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 12 or amino acids having at least 95% sequence identity therewith.

[0180] [6] An immunogenic composition for use according to any one of the preceding [1] to [5], wherein the third HA antigen is derived from strain A / swine / Italy / 3033-1 / 2015(H1N2).

[0181] [7] An immunogenic composition for use according to any one of the preceding [1] to [6], wherein the third HA antigen encoded by the third nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 85% sequence identity with respect to that sequence.

[0182] [8] An immunogenic composition for use according to any one of the preceding [1] to [7], wherein the fourth HA antigen is derived from strain A / swine / Italy / 28762-3 / 2013(H1N1).

[0183] [9] An immunogenic composition for use according to any one of the preceding [1] to [8], wherein the fourth HA antigen encoded by the fourth nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90% sequence identity with respect to that sequence.

[0184]

[10] An immunogenic composition for use according to any one of the preceding [1] to [9], which is suitable for the simultaneous administration of a first and a second RNA replicon particle.

[0185]

[11] Further containing a third RNA replicon particle, The third RNA replicon particle comprises nucleic acid constructs containing first, second, and third nucleic acid sequences encoding the first, second, and third neuraminidase (NA) antigens of IAV-S, wherein, The first NA antigen is A / swine / Scotland / 410440 / 1994-like H1 hu It belongs to the N2 (Scot / 94) system. The second NA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and, The third NA antigen is the A(H1N1)pdm09(pdm09) lineage or Eurasian avian-like H1 av An immunogenic composition for use as described in any one of the preceding [1] to

[10] , selected from the N1(EA) lineage.

[0186]

[12] An immunogenic composition for use according to any one of the preceding [1] to

[11] , wherein the RNA replicon particle is an alphavirus RNA replicon particle.

[0187]

[13] An immunogenic composition for use as described in

[12] , comprising Venezuelan encephalitis virus (VEEV) alphavirus RNA replicon particles.

[0188]

[14] A vaccine comprising an immunogenic composition described in any one of the preceding [1] to

[13] .

[0189]

[15] The adjuvant-free vaccine described in

[14] .

[0190]

[16] The vaccine according to

[14] , comprising an adjuvant selected from the group consisting of biodegradable oil, an oil-in-water emulsion containing 2.5-50% (v / v) mineral oil, and biodegradable oil mixed with an oil-in-water emulsion containing 2.5-50% (v / v) mineral oil.

[0191]

[17] A vaccine described in any one of

[14] to

[16] for use in preventing disease caused by swine influenza A virus in the target population.

[0192]

[18] A method for immunizing a pig against swine influenza A virus, comprising administering an immunologically effective dose of any one of the vaccines described in

[14] to

[16] to the pig.

[0193]

[19] An immunogenic composition comprising first and second RNA replicon particles, The first RNA replicon particle comprises a nucleic acid construct containing first and second nucleic acid sequences encoding the first and second hemagglutinin (HA) antigens of swine influenza A virus (IAV-S), where, The first HA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and, The second HA antigen is of the A(H1N1)pdm09(pdm09) lineage. The second RNA replicon particle comprises a nucleic acid construct containing third and fourth nucleic acid sequences encoding the third and fourth HA antigens of IAV-S, where, The third HA antigen is A / swine / Scotland / 410440 / 1994-like H1 hu It belongs to the N2 (Scot / 94) system, and, The fourth HA antigen is Eurasian avian-like H1 av An immunogenic composition of the N1(EA) class.

[0194]

[20] An immunogenic composition comprising first, second and third RNA replicon particles, The first RNA replicon particle comprises a nucleic acid construct containing first and second nucleic acid sequences encoding the first and second hemagglutinin (HA) antigens of swine influenza A virus (IAV-S), where, The first HA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and, The second HA antigen is of the A(H1N1)pdm09(pdm09) lineage. The second RNA replicon particle comprises a nucleic acid construct containing third and fourth nucleic acid sequences encoding the third and fourth HA antigens of IAV-S, where, The third HA antigen is A / swine / Scotland / 410440 / 1994-like H1 hu It belongs to the N2 (Scot / 94) system, and, The fourth HA antigen is Eurasian avian-like H1 av It belongs to the N1(EA) system. The third RNA replicon particle comprises a nucleic acid construct containing first, second, and third nucleic acid sequences encoding the first, second, and third neuraminidase (NA) antigens of IAV-S, where, The first NA antigen is A / swine / Scotland / 410440 / 1994-like H1 huIt belongs to the N2 (Scot / 94) system. The second NA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and, The third NA antigen is the A(H1N1)pdm09(pdm09) lineage or Eurasian avian-like H1 av An immunogenic composition selected from the N1(EA) lineage.

[0195] In a fourth aspect, the present invention provides the following embodiments; [1] A nucleic acid construct for use in the prevention of disease caused by swine influenza A virus in a subject, comprising first, second, and third nucleic acid sequences encoding first, second, and third neuraminidase (NA) antigens of swine influenza A virus (IAV-S), wherein, The first NA antigen is A / swine / Scotland / 410440 / 1994-like H1 hu It belongs to the N2 (Scot / 94) system. The second NA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and, The third NA antigen is the A(H1N1)pdm09(pdm09) lineage or Eurasian avian-like H1 av Nucleic acid constructs selected from the N1(EA) lineage.

[0196] [2] A nucleic acid construct for use as described in [1], wherein the first NA antigen is derived from strain A / swine / England / 61470 / 2013(H1N2).

[0197] [3] A nucleic acid construct for use according to [1] or [2], wherein the first NA antigen encoded by the first nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity therewith.

[0198] [4] A nucleic acid construct for use as described in any one of [1] to [3], wherein the second NA antigen is derived from strain A / swine / Italy / 248147-8 / 2015(H3N2).

[0199] [5] A nucleic acid construct for use according to any one of [1] to [4], wherein the second NA antigen encoded by the second nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 90% sequence identity with respect to that sequence.

[0200] [6] A nucleic acid construct for use as described in any one of [1] to [5], wherein the third NA antigen is derived from strain A / swine / England / 373 / 2010(H1N1) or A / swine / Italy / 179057 / 2015(H1N1).

[0201] [7] A nucleic acid construct for use as described in any one of [1] to [6], wherein the third NA antigen is derived from strain A / swine / Italy / 28762-3 / 2013(H1N1).

[0202] [8] A nucleic acid construct for use according to any one of [1] to [7], wherein the third NA antigen encoded by the third nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 90% sequence identity with respect to that sequence.

[0203] RNA replicon particles containing the nucleic acid construct described in any one of [9][1] to [8].

[0204]

[10] The RNA replicon particle described in [9], which is an alphavirus RNA replicon particle.

[0205]

[11] The RNA replicon particle described in [9] or

[10] is a Venezuelan encephalitis virus (VEEV) alphavirus RNA replicon particle.

[0206] An immunogenic composition containing RNA replicon particles as described in any one of

[12] [9] to

[11] .

[0207]

[13] An immunogenic composition comprising first, second, and third RNA replicon particles, where the first RNA replicon particle comprises a nucleic acid construct comprising first and second nucleic acid sequences encoding the first and second hemagglutinin (HA) antigens of porcine influenza A virus (IAV-S), wherein the first HA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and the second HA antigen is of the A(H1N1)pdm09 (pdm09) lineage, the second RNA replicon particle comprises a nucleic acid construct comprising third and fourth nucleic acid sequences encoding the third and fourth HA antigens of IAV-S, wherein the third HA antigen is of the A / swine / Scotland / 410440 / 1994-like H1 hu N2 (Scot / 94) lineage, and the fourth HA antigen is of the Eurasian avian-like H1 av N1 (EA) lineage, and the third RNA replicon particle is the RNA replicon particle according to any one of [9] to

[11] , an immunogenic composition.

[0208]

[14] A vaccine comprising the immunogenic composition according to

[12] or

[13] .

[0209]

[15] The vaccine according to

[14] , which is an adjuvant-free vaccine.

[0210]

[16] The vaccine according to

[14] , comprising an adjuvant selected from the group consisting of biodegradable oil, an oil-in-water emulsion containing 2.5 to 50% (v / v) mineral oil, and a biodegradable oil mixed with an oil-in-water emulsion containing 2.5 to 50% (v / v) mineral oil.

[0211]

[17] A vaccine according to any one of

[14] to

[16] for use in preventing a disease caused by swine influenza A virus in a subject.

[0212]

[18] A method of immunizing a pig against swine influenza A virus, the method comprising administering to the pig an immunologically effective amount of a vaccine according to any one of

[14] to

[16] .

[0213]

[19] A nucleic acid construct comprising first, second and third nucleic acid sequences encoding the first, second and third neuraminidase (NA) antigens of swine influenza A virus (IAV-S), wherein the first NA antigen is of the A / swine / Scotland / 410440 / 1994-like H1 hu N2(Scot / 94) lineage, the second NA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and the third NA antigen is selected from the A(H1N1)pdm09 (pdm09) lineage or the Eurasian avian-like H1 av N(EA) lineage, a nucleic acid construct.

[0214] The following examples serve to provide a further understanding of the present invention, but are not meant to limit the effective scope of the present invention in any way.

[0215] [Example] Materials and Methods [[ID=(E)]]Preparation of alphavirus RNA RP vaccine Production of single HA or NA gene replicon particles (RP). A VEE repliconvector designed to express a hemagglutinin (HA) or neuraminidase (NA) gene was constructed as previously described with the following modifications [see U.S. Patent No. 9,441,247B2, which is incorporated herein by reference]. The TC-83-derived repliconvector "pVEK" [disclosed and described in U.S. Patent No. 9,441,247B2] was digested with the restriction enzymes Ascl and Pad. A DNA plasmid containing codon-optimized open reading frame sequences of the HA or NA gene (Tables 1a and 1b) having a 5'-flanking sequence (5'-GGCGCGCCGCACC-3') and a 3'-flanking sequence (5'-TTAATTAA-3') was similarly digested with the restriction enzymes Ascl and Pad. The synthetic gene cassette was then ligated into the digested pVEK vector, and the resulting clones were renamed "pVHV" for each RP code. The vector name "pVHV" was chosen to refer to a pVEK-derived replica vector containing a transgene cassette cloned via the Ascl and Pad sites of the pVEK multicloning site.

[0216] The TC-83 RNA replicon particles (RPs) were prepared according to previously described methods [U.S. Patents 9,441,247B2 and 8,460,913B2, the contents of which are incorporated herein by reference]. Briefly, the pVHV replicon vector DNA and helper DNA plasmid were linearized with Not1 restriction enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and cap analogues (Promega, Madison, Wl). Importantly, the helper RNA used in the preparation lacked the VEE subgenome promoter sequence, as previously described [Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727(2010)]. Purified RNA for the replicon and helper components was combined and mixed with a Vero cell suspension, electroporated into a 4 mm cuvette, and returned to OptiPro SFM cell culture medium (Thermo Fisher, Waltham, MA). After overnight incubation, alphaviral RNA replicon particles were purified, formulated with phosphate-buffered saline containing 5% sucrose (w / v) and 1% porcine serum, filtered through a 0.22 micron membrane filter, and aliquoted for storage. The titer of functional RP was determined by immunofluorescence assay on an infected Vero cell monolayer. Batch of RP was identified according to the gene encoded in the packaged replicon (Tables 1a and 1b).

[0217] Production of multiple HA or NA gene replicon particles (RPs). A VEE repliconvector used to express the HA or NA gene was constructed as previously described with the following modifications [see U.S. Patent No. 9,441,247B2, which is incorporated herein by reference]. The TC-83-derived repliconvector "pVEK" [disclosed and described in U.S. Patent No. 9,441,247B2] was digested with restriction enzymes AscI and PacI. Selected open reading frame sequences were codon-optimized and synthesized for the dual-gene HA and NA constructs using flanking AscI and PacI sites. Furthermore, the intermediate sequence between the two synthetic HA or NA open reading frames consisted of 47 nucleotides of non-coding heterologous sequences, as well as a second copy of the native TC-83 subgenome (sg)RNA promoter and 5' untranslated sgRNA region sequences. These dual-gene constructs were referred to as "pVDG" to distinguish them from the parent vector having a single sgRNA promoter sequence. For the triple-gene NA construct, a pVDG-based construct containing two NA genes was further modified as follows: A third selected NA open reading frame was codon-optimized and synthesized using flanking PacI and SphI sites for directional cloning to the pVDG vector downstream of the two existing NA genes. The novel synthetic construct also contained 50 nucleotides of heterologous non-coding sequences and a third copy of the native TC-83 sgRNA promoter and the 5' untranslated sgRNA region sequence up to 5' of the third NA gene sequence. The 3' region from the third NA gene sequence consisted of the 3' untranslated region of TC-83 up to the corresponding SphI site of the parental pVDG vector. The triple-gene vector was referred to as "pVTG" to distinguish it from the related vectors pVEK, pVHV, and pVDG.

[0218] Using the sequences of HA (Table 1a: EUHA1-3, EUHA1-2, EUHA1-5, EUHA1-15, EUHA1-17, EUHA1-8, EUHA1-11, and HA3-4) or NA (Table 1b: EUNA1-2, EUN1-4, EUN2-6, and EUN2-7) genes selected from Examples 1 and 3, multiple HA or NA genes were synthesized in plasmid vectors pVDG or pVTG as described above.

[0219] The TC-83 RNA replicon particles (RPs) were prepared according to previously described methods [U.S. Patents 9,441,247B2 and 8,460,913B2, the contents of which are incorporated herein by reference]. Briefly, the pVDG or pVTG replicon vector DNA and helper DNA plasmids were linearized with NotI restriction enzymes prior to in vitro transcription using MegaScript T7 RNA polymerase and cap analogues. Importantly, the helper RNA used in the preparation lacked the VEE subgenome promoter sequence, as previously described [Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727(2010)]. Purified RNAs for the replicon and helper components were combined and mixed with a suspension of Vero cells, electroporated into 4 mm cuvettes, and then returned to serum-free culture medium. After overnight incubation, alphavirus RNA replicon particles were purified from cells and culture medium by passing the suspension through a depth filter, washing with phosphate-buffered saline containing 5% sucrose (w / v), and finally eluting the retained RP with 200 mM Na2SO4 + 5% sucrose (w / v) buffer. Alternatively, cells and culture medium were centrifuged in the presence of prepared Cellufine Sulfate® resin, washed with phosphate-buffered saline containing 5% sucrose (w / v), and then eluted with 200 mM Na2SO4 + 5% sucrose (w / v) buffer. The eluted RP was passed through a 0.22 micron membrane filter and aliquoted for storage. The titer of functional RP was determined by immunofluorescence assay on a monolayer of infected Vero cells.

[0220] The following replicon particles were constructed and used in the experiments.

[0221]

Table 1

[0222]

Table 2

[0223] Unless otherwise indicated in the examples or figures, the following strains and lines were used for the HI assay.

[0224]

Table 3

[0225]

Table 4

[0226]

Table 5

[0227]

Table 6

[0228] General test design Healthy pigs at about 5 weeks of age that are seronegative or have low antibodies against SIV (3 pigs per vaccine), a single or multiple RNA particle vaccines encoding HA or NA genes, 5 - 10×10 per pig once 6 It should be noted that in the original text, "了吧" in line 40 seems to be an incorrect character. I have translated it as it is in the provided text. If this is a known error or specific term, it may need to be corrected according to the actual situation.Intramuscular vaccination was then administered with Xsolve50 adjuvant. Each vaccination was repeated at approximately 8 weeks of age, and blood samples were collected at approximately 9 weeks of age. Antigen-specific antibody levels were quantified using either a hemagglutination inhibition (HI) assay or a neuraminidase inhibition (NI) assay.

[0229] Hemagglutination inhibition (HI) assay: All serum samples were heat-inactivated at 56°C for 30 minutes, followed by treatment with 0.25% periodate, then 0.75% glycerol, and adsorbed with 2.6% chicken erythrocytes to remove nonspecific agglutinin. For HI antibody titration, a series of dilutions of the pre-treated serum were incubated for 1 hour with 8 erythrocyte agglutination units of the SIV strain listed in Table 1c or Table 1d as the HA antigen. The mixture was then incubated with 0.2% chicken erythrocytes at room temperature for 1 hour, and the plates were read for inhibition of agglutination. The reciprocal of the highest serum dilution that completely inhibited erythrocyte agglutination was assigned as the HI titer and expressed as a log-based binary value.

[0230] Serum neuraminidase (NA) inhibition (NI) assay: SIV strains of Vero cells expressing NA antigens, obtained by electroporating replicon RNA encoding the genes of each NA (Table 1e and 1f), were used as the source of NA antigens. The enzymatic activity of these NAs was quantified by sialic acid cleavage from fetuin on a 96-well plate during an overnight incubation at 37°C. Peanut agglutinin-horseradish peroxidase conjugate (PNA-HRP) was then added at room temperature for 2 hours to conjugate to the sialic acid-removed fetuin molecule. A signal was obtained using a 3,3',5,5'-tetramethylbenzidine (TMB) substrate and read at 450 nm. The test antigens were titrated to determine the dilution that yielded 70% of the maximum signal. Equal volumes of NA antigen were added to a series of serum dilutions in fetuin-coated wells during an overnight incubation at 37°C. Optical density (OD) values ​​were normalized relative to values ​​from positive control wells without serum. Neuraminidase inhibitory titer was defined as the reciprocal of the interpolated serum dilution having an absorbance equivalent to 50% inhibition compared to the control, and was expressed as a log-based binary value.

[0231] The correlation between neuraminidase antibody titers and hemagglutinin antibody titers and vaccine-induced protection against SIV-A is described below: [Hobson D. et al., J Hyg (Lond) 70, 767-777 (1972); Ohmit SE, et al., J. Infect. Dis 204, 1879-1885 (2011); Walz L, et al., J Virol. 2018; 92 (17): e01006-18. (2018). Therefore, the serological results of the Hi and Ni inhibitory assays described in the following examples indicate prevention of disease caused by SIV-A.

[0232] [Example 1] The following tests were performed to determine the hemagglutination inhibition (HI) antibody titers induced by RPs encoding single HA antigens from the EurAsianAvian (EA), Gent / 84, Scot / 94, and pdm09 strains, as well as their protection and cross-protection against alphaviral RNA RPs encoding single HA antigens.

[0233] Five-week-old pigs (three pigs per group) were vaccinated with each RNA particle along with the XSolve50 adjuvant using Prime-Boost Resimen at approximately three-week intervals. Serum was collected 1–2 weeks after booster vaccination to determine the titer of influenza antigen-specific hemagglutination inhibitory antibody, a correlate of protection against influenza. The HI assay measures the maximum serum dilution that prevents hemagglutination of red blood cells induced by the influenza virus. The reciprocal of this dilution was defined as the HI titer on a Log 2 basis. The reported values ​​are the average of three animals. The detection limit of this assay is 4 (dotted line in the figure), and therefore titers less than 4 are reported as 3 in the figure.

[0234] The results of the HI experiment are shown in Figures 1 to 4. The following conclusions can be drawn.

[0235] Figure 1: RP of EA strains EUHA1-3 showed the highest antigen-specific HI antibody titers against almost all IAS EA antigens tested, followed by EUH1-5 and EUH1-2. Furthermore, cross-reactivity titers against several Scot / 94 and pdm09 HA antigens were observed. None of the tested strains showed cross-reactivity titers against the Gent / 84 IAS antigen (all HI titers were less than 4).

[0236] Figure 2: The RP of strain EUHA1-15 showed the highest antigen-specific HI antibody titer against almost all Scot / 94 antigens tested, followed by EUH1-17, and thus performed best against Scot / 94 antigens of branches 2 and 3. The RP of strain EUHA1-8 showed the highest antigen-specific HI antibody titer against the Scot / 94 antigen of branch 1 tested. Furthermore, cross-reactivity titers against several EA and pdm09 HA IAS antigens could be observed. None of the strains tested showed cross-reactivity titers against the Gent / 84 IAS antigen (all HI titers were less than 4).

[0237] Figure 3: The RP of the Pdm09 strain EUHA1-11 showed the highest antigen-specific HI antibody titer against almost all pdm09 antigens. Furthermore, cross-reactivity titers against most EA and Scot / 94HA antigens were observed. None of the tested strains showed cross-reactivity titer against the Gent / 84 IAS antigen (all HI titers were less than 4).

[0238] Figure 4: The RP of the Gent / 84 strain EUHA3-4 showed the highest antigen-specific HI antibody titer against all Gent / 84 antigens tested. No significant cross-reactivity titers of EA, Scot / 94, and pdm09 antigens against HA antigens were observed.

[0239] [Example 2] Hemagglutination inhibitor (HI) antibody titers induced by RP encoding bivalent HA antigens 1) HA antigens of the pdm09 and Gent / 84 lineages, or 2) HA antigen of EA and Scot / 94 antigen To determine the serological efficacy of alphaviral RNA RP encoding a combined dual eHA antigen, we conducted studies using the design described in Example 1.

[0240] The results of the HI assay are shown in Figure 5. The following conclusions can be drawn.

[0241] It could be observed that not all combinations tested induced a strong serological response. Furthermore, surprisingly, it could be observed that the order of genes in the viral genome of the replicon particles was important in inducing a serological response.

[0242] • Combinations of HA antigens from lineage Pdm09 and Gent / 84: Only the combination of Gent / 84 positioned first and pdm09 positioned second in the viral genome of the replicon particle induced a strong serological response. Conversely, the order of Pdm09 positioned first and Gent / 84 positioned second in the viral genome of the replicon particle resulted in a much weaker serological response to the Gent / 84 HA antigen and a very weak serological response to the Pdm09 HA antigen.

[0243] • Combinations of HA antigens from lineage EA and Scot / 94: Not all combinations tested induced a strong serological response. The combination of strains EUHA1-17 from Scot / 94 and EUHA1-3 from EA showed the best serological response (highest HI titers for IAS antigens from both lineages).

[0244] Furthermore, only the combination of Scot / 94 first and EA second-placed in the replicon RNA of the replicon particle induced a strong serological response. Conversely, no significant serological response to the EA HA antigen was observed when EA was first and Scot / 94 second-placed in the replicon RNA of the replicon particle.

[0245] Among the various combinations tested, the strain combinations EUHA3-4+EUHA1-11 and EUHA1-17+EUHA1-3 induce the best immunity, as measured by HI titer. Therefore, these combinations are useful for use in formulations that combine two replicon particles, namely, a first RNA replicon particle encoding the EUHA3-4+EUHA1-11 strain in this order and a second RNA replicon particle encoding the EUHA1-17+EUHA1-3 strain in this order.

[0246] As a result, surprisingly, it was demonstrated that the location of the HA gene within the RNA replicon particle and / or a specific combination of HA antigens can determine the level of induced immunity, which is measured as HI titer.

[0247] [Example 3] Neuraminidase inhibitor (NI) antibody titers induced by RP encoding a single NA antigen The following tests were performed to determine the serological efficacy of alphaviral RNA RPs encoding single NA antigens from the EurAsianAvian (EA), Gent / 84, Scot / 94, and pdm09 strains.

[0248] Five-week-old pigs (3 pigs per group) were vaccinated with each RNA replicon particle along with the XSolve50 adjuvant using a prime-boost regimen at approximately three-week intervals. Serum was collected 1–2 weeks after booster vaccination to determine influenza antigen-specific neuraminidase inhibitory (NI) antibody titers. NI titers were measured using a lectin (peanut agglutinin)-based assay as described above, and the NI titer was defined as the reciprocal of the maximum serum dilution that inhibited NA activity by at least 50% compared to the control well. The detection limit of this assay was 2 (dotted line in the figure).

[0249] The results of the NI experiment are shown in Figures 7 to 10. The following conclusions can be drawn.

[0250] Figure 7: The RP of the EA strain EUNA1-2 showed the highest antigen-specific NI antibody titer against almost all IAS EA antigens tested. Furthermore, cross-reactivity against several Scot / 94, pdm09, and Gent / 84 NA antigens was observed.

[0251] Figure 8: The RP of strains EUNA1-4 showed the highest antigen-specific NI antibody titer against most of the pdm09 antigens tested, but the observed NI titer levels were lower compared to those achieved by the RP of the EA lineage. Furthermore, cross-reactivity titers against EA, Scot / 94, and Gent / 84 NA IAS antigens could be observed. The difference in titers measured among the tested strains was small.

[0252] Figure 9: The RP of strain EUNA2-6 of the Scot / 94 lineage showed the highest antigen-specific NI antibody titer against all tested Scot / 94 antigens. Furthermore, high levels of cross-reactivity to EA, pdm09, and Gent / 84 NA antigens were observed for strain EUNA2-6.

[0253] Figure 10: The RP strain EUNA2-7 of the Gent / 84 lineage showed high antigen-specific NI antibody titers against all Gent / 84 antigens tested, and also demonstrated significant cross-protection against NA antigens for EA, Scot / 94, and pdm09 antigens.

[0254] [Example 4] NI antibody titers induced by RP encoding bilayer or trilayer NA antigens To determine the serological efficacy of alphaviral RNA RPs encoding bilingual or trilingual NA antigens, RPs encoding NA antigens from the following strains were designed and prepared, and tested using the design described in Example 3. 1) NA antigens of the EA and Gent / 84 lineage, or 2) NA antigens of EA, Gent / 84, and Scot / 94 antigens

[0255] The results of the NI experiment are shown in Figure 11. The following conclusions can be drawn.

[0256] We were able to demonstrate that all tested combinations induced a serological response regardless of gene order. Therefore, surprisingly, in contrast to the observations using HA antigen (see Example 2 above), we were able to observe that the order of NA genes in the viral genome of the replicon particles is not important for inducing a serological response.

[0257] [Example 5] NI antibody titers induced by RP encoding bilingual and trilingual NA antigens The results shown in Figures 7-10 reveal that the combination of strains from the EA lineage, Gent / 84 lineage, and Scot / 94 lineage should provide the best protection against IAS, exhibiting the best protection and cross-protection against all four lineages. Therefore, the best candidate for testing such cross-protection is the combination of strain EUNA2-6 from the Scot / 94 lineage and strain EUNA2-7 from the Gent / 84 lineage, which may then be further combined with strains from either the EA lineage, e.g., strain EUNA1-2, or the pdm09 lineage, e.g., strain EUNA1-4. As a result, these combinations of strains were tested for their serological responses.

[0258] therefore, 1) NA antigens of the EA and Gent / 84 lineages 2) NA antigens of Scot / 94, Gent / 84 and EA antigens, or 3) NA antigens of Scot / 94, Gent / 84, and pdm09 antigens To determine the protection against alphaviral RNA RPs encoding dual and triple NA antigens, we investigated using the design described in Example 3.

[0259] The results are shown in Figure 12.

[0260] In contrast to the results observed for HA antigens, combinations of NA antigens derived from only three lineages are sufficient to induce a serological response to all four IAS lineages.

[0261] Regardless of the gene order of the RNA replicon particles, we have already achieved a weak serological response to all four IAS lineages using combinations of NA antigens derived from only two lineages.

[0262] The best serological response was achieved by combining either the pdm09 or EA lineage NA antigen with a combination of Scot / 94 and Gent / 84 NA antigens.

[0263] [Table 7]

[0264] [Example 6] Evaluation of the vaccine efficacy of the multivalent IAV-S vaccine A study was conducted to determine the immunogenicity and efficacy of a multivalent IAV-S vaccine containing two dual HA RPs (EUSIV-T8 RP encoding EUHA1-17 and EUHA1-3 antigens, and EUSIV-K RP encoding EUH3-4 and EUH1-11 antigens, Tables 1a and 2) and one triple NA construct (EUSIV-R encoding EUN2-6, EUN1-2, and EUN2-5 antigens, Tables 1b and 2). The adjuvant-containing vaccine was administered to five pigs in two intramuscular (IM) vaccinations at 5 and 8 weeks of age (2 mL per dose; 3 × 5 × 10⁶ per dose). 6(RP, vaccination). An equal number of unvaccinated and unvaccinated individuals were administered phosphate-buffered saline containing adjuvant. The immunogenicity of the vaccine was measured by quantifying the HI and NI titers in serum samples collected before experimental infection at 10 weeks of age. The efficacy of the vaccine was tested against Gent / 84[A / swine / Belgium / 113 / 2013(H3N2)] challenge infection via the intratracheal route at 10 weeks of age (day 32 of the study). The vaccine efficacy against IAV-S infection-induced fever, i.e., elevated rectal temperature and pulmonary lesions, was measured 3 days after infection.

[0265] The results of this experiment are shown in Figures 13A, 13B, 13C, and 13D. The multivalent IAV-S vaccine induced functional HI titers against heterologous IAV-S strains belonging to all four lineages (Figure 13A) and NI titers against allologous NA antigens of all three lineages (Figure 13B). Furthermore, the multivalent IAV-S vaccine protected pigs from experimentally induced rectal temperature elevation, fever (Figure 13C), and lesions (Figure 13D). These results demonstrate that the tested multivalent IAV-S possessed both immunogenicity and efficacy.

[0266] [Example 7] Evaluation of vaccine efficacy after ID administration A study was conducted to determine the serological efficacy of a polyvalent IAV-S vaccine containing two dual HA RPs (EUSIV-T8 RP encoding EUHA1-17 and EUHA1-3 antigens, and EUSIV-K RP encoding EUH3-4 and EUH1-11 antigens, Tables 1a and 2) and one triple NA construct (EUSIV-R encoding EUN2-6, EUN1-2, and EUN2-5 antigens, Tables 1b and 2). The adjuvant-containing vaccine was administered to three pigs at 5 and 8 weeks of age in two intradermal (ID) vaccinations using an IDAL® needleless syringe (200 μL per dose; 3 × 3 × 10⁶ doses per dose). 6(RP, vaccination). An equal number of unvaccinated and unvaccinated animals were administered phosphate-buffered saline containing an adjuvant. The immunogenicity of the vaccine was measured by quantifying the HI and NI titers in serum samples collected at 10 weeks of age.

[0267] The results of this experiment are shown in Figures 14A and 14B. The multivalent IAV-S vaccine induced functional HI titers in heterologous IAV-S strains belonging to three of the four strains tested (Figure 14A), and induced NI titers in two of the three allogeneic NA antigens tested (Figure 14B). These results demonstrate the effectiveness of intradermal application of the multivalent IAV-S vaccine.

Claims

1. An immunogenic composition for use in the prevention of disease caused by swine influenza A virus in a target, comprising first and second alphavirus RNA replicon particles, The first RNA replicon particle comprises a nucleic acid construct, the nucleic acid construct comprising first and second nucleic acid sequences encoding first and second hemagglutinin (HA) antigens of swine influenza A virus (IAV-S) in the 5' to 3' direction, where, The first HA antigen is of the A / swine / Gent / 1 / 1984-like H3N2 (Gent / 84) lineage, and, The second HA antigen is of the A(H1N1)pdm09(pdm09) lineage, The second RNA replicon particle comprises a nucleic acid construct, the nucleic acid construct comprising third and fourth nucleic acid sequences encoding the third and fourth HA antigens of IAV-S in the 5' to 3' direction, where, The third HA antigen is of the A / swine / Scottland / 410440 / 1994-like H1huN2 (Scott / 94) lineage, and, The fourth HA antigen is of the Eurasian avian-like H1avN1 (EA) lineage, and here, The RNA replicon particle is an alphavirus RNA replicon particle derived from Venezuelan equine encephalitis virus (VEEV). Immunogenic composition.

2. The immunogenic composition for use according to claim 1, wherein the first HA antigen is derived from strain A / swine / Italy / 240849 / 2015 (H3N2).

3. The immunogenic composition for use according to claim 1 or 2, wherein the first HA antigen encoded by the first nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 90% sequence identity with respect to that sequence.

4. An immunogenic composition for use according to any one of claims 1 to 3, wherein the second HA antigen is derived from strain A / swine / England / 373 / 2010 (H1N1).

5. The immunogenic composition for use according to any one of claims 1 to 4, wherein the second HA antigen encoded by the second nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 12 or amino acids having at least 95% sequence identity therewith.

6. An immunogenic composition for use according to any one of claims 1 to 5, wherein the third HA antigen is derived from strain A / swine / Italy / 3033-1 / 2015 (H1N2).

7. The immunogenic composition for use according to any one of claims 1 to 6, wherein the third HA antigen encoded by the third nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 85% sequence identity with respect to that sequence.

8. An immunogenic composition for use according to any one of claims 1 to 7, wherein the fourth HA antigen is derived from strain A / swine / 28762-3 / 2013 (H1N1).

9. The immunogenic composition for use according to any one of claims 1 to 8, wherein the fourth HA antigen encoded by the fourth nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90% sequence identity with respect to that sequence.

10. An immunogenic composition for use according to any one of claims 1 to 9, which is suitable for the simultaneous administration of the first and second RNA replicon particles.

11. A vaccine comprising the immunogenic composition according to any one of claims 1 to 10.

12. The vaccine according to claim 11, which is an adjuvant-free vaccine.

13. The vaccine according to claim 11, comprising an adjuvant selected from the group consisting of biodegradable oil, an oil-in-water emulsion containing 2.5 to 50% (v / v) mineral oil, and biodegradable oil mixed with an oil-in-water emulsion containing 2.5 to 50% (v / v) mineral oil.

14. A vaccine according to any one of claims 11 to 13, for use in preventing diseases caused by swine influenza A virus in a target population.

15. A method for immunizing a pig against swine influenza A virus, comprising administering an immunologically effective amount of the vaccine described in any one of claims 11 to 13 to the pig.

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