A vaccine for protecting piglets against swine influenza A virus infection

Abstract

The present invention relates to the use of a vaccine based on an alphavirus RNA replicon particle (αRP) vector encoding an antigen of IAV-S for the passive immunization of piglets against pathogenic infection with swine influenza viruses.

Description

[Technical field]

[0001] General Field of the Invention The present invention relates to swine influenza virus type A (IAV-S) vaccines that can be used for passive immunization of progeny piglets through active immunization of sows. [Background technology]

[0002] 2. Background of the Invention Influenza A viruses (IAV) pose a significant burden to human and animal health worldwide. IAVs are classified into different subtypes based on their viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). IAVs infect poultry, pigs, horses, cats, dogs, marine mammals (e.g., whales), bats, and humans. Wild waterfowl and shore birds (ducks, geese, swans, and gulls) are the natural hosts, which can be infected with 16 different HA and 9 different NA subtypes (Webster et al., Microbiol Rev 56:152-179, 1992).

[0003] Influenza A virus in swine (IAV-S) is a significant respiratory pathogen of domestic pigs, which has been found to cause significant economic losses worldwide, especially in the livestock industry (Holtkamp et al., The American Association of Swine Veterinarians Annual Meeting, 2007). It is characterized by the sudden onset of respiratory disease, usually accompanied by anorexia, lethargy and fever. In addition to the clinical complications associated with IAV-S in production animals, there are published reports implicating pigs in the transmission of influenza viruses to humans (Myers et al., Clin Infect Dis, 2007;44(8),1084-8; Krueger and Gray, Curr Top Microbiol Immunol 370:201-225, 2013), which represents a significant public health threat and provides further motivation to suppress IAV-S in swine herds.

[0004] To address this issue, many pig farmers are now vaccinating pigs against IAV-S using commercially available vaccines. However, it is difficult to suppress IAV-S with regular vaccines because many diverse IAV-S strains simultaneously circulate in the field and continue to evolve (Gao et al., J Gen Virol 98(8), 2001-2010, 2017). The diversity and mutability of IAV-S are caused by the genetic structure of the virus. Similar to other influenza A viruses, IAV-S has genes encoded on eight RNA segments and a genome replication mechanism that introduces frequent mutations. These genetic features allow IAV-S to rapidly adapt, including evading pre-existing neutralizing antibodies induced by exposure to previous strains.

[0005] Classification of influenza A viruses begins with subtyping of the two major glycoproteins on the surface of the virus, HA and NA. The HA protein mediates viral attachment and fusion to the host cell. Neuraminidase is an enzyme that functions in the final step of the influenza virus replication cycle by cleaving the newly formed virus particle from the host cell, allowing new progeny viruses to spread and infect other cells.

[0006] While human influenza A usually has one or two predominant strains that circulate worldwide during a particular influenza season, many more strains of IAV-S co-circulate and these strains vary by geographic region. Similarly, IAV-S strains are antigenically diverse but primarily comprise HA subtypes H1 or H3, and NA subtypes N1 or N2. Within each IAV-S HA and NA subtype, further phylogenetic diversity exists.

[0007] In the US swine population, there are four major phylogenetic clusters of H1 (gamma, delta 1, delta 2, pandemic), two major clusters of H3 (cluster IV and human-like), two major clusters of N1 (classical, pandemic), and two major phylogenetic clusters of N2 (N2-1998 and N2-2002) (see 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 lineages of H1 (Eurasiatori-like, Scotland / 410440 / 1994-like H1 and pandemic), one major lineage of H3 (Gent / 1 / 1984-like H3), two major lineages of N1, two major lineages of N2, and two minor lineages of N2 (Watson et al., J. Virol., 89:9920-9931 (2015); doi:10.1128 / JVI.00840-15).

[0009] As a result of the continuous emergence of mutant IAV-S strains, commercially available whole virus inactivated vaccines often do not protect against emerging viral subtypes / clusters and provide only limited protection against heterosubtypic challenges because their antigens do not match all current strains circulating in the field (Lee et al., Can J Vet Res 71(3), 207-12, 2007; Vincent et al., Vaccine 28(15), 2782-2787, 2010). Therefore, such vaccines need to be regularly updated to match currently circulating strains.

[0010] A common strategy to prevent piglets from infectious diseases involves vaccinating pregnant sows and / or piglet progeny with whole virus inactivated vaccines before farrowing. Vaccinated pregnant sows transfer their protection to the piglets in colostrum and subsequent milk (passive immunity). In fact, the piglet's passive immunity can come from its dam or from another sow that was vaccinated before farrowing. Colostrum contains a set of immune system components that help the piglet survive until it acquires its own active immunity. The antibody levels in colostrum are 60 times higher than those in milk. Approximately 65-90% of these antibodies consist of the IgG type, which provides systemic protection. The absorption of these IgG immunoglobulins by the piglets is not selective, they are present in the plasma 2 hours after colostrum intake and peak at 12 hours. The antibody profile of the colostrum and the passive protection induced in the piglets depend on the antigens to which the mother is exposed and the time period between antigen exposure and farrowing. During lactation, IgG immunoglobulins are gradually replaced by IgA, which functions to protect the piglet's intestinal mucosa. After about 36 hours, the intestine stops absorbing antibodies and cells, and the piglet continues to receive passive mucosal IgA antibodies from the milk in the gastrointestinal tract.

[0011] In addition to whole virus inactivated vaccines, several other types of vaccines have been described in the art for use in pigs (reviewed by Opriessnig et al., Porcine Health Management, 2021, 7, 1). Broadly, these can be classified into whole pathogen live attenuated vaccines, chimeric (live attenuated or inactivated) vaccines, subunit vaccines, mammalian live virus vector (replicating or replication-deficient) vaccines, and nucleic acid-based vaccines (DNA plasmid or mRNA).

[0012] WO 2019 / 121513 and WO 2019 / 110481 describe the use of replication-deficient alphavirus RNA replicon particle vectors for active vaccination of piglets against IAV-S.

[0013] It is an object of the present invention to develop novel methods and vaccines for pigs that are safe, effective against pathogenic challenge with IAV-S, and can be rapidly modified to be antigenically compatible with emerging strains. Summary of the Invention

[0014] Overview of the Invention In order to achieve the objectives of the present invention, there is provided a vaccine comprising an immunogen and a pharma- ceutically acceptable carrier, the vaccine being for use in a method for protecting piglets against pathogenic infection by porcine influenza A virus (IAV-S) by passive immunization of the piglets via ingestion of colostrum or milk from a sow that has been actively immunized with the vaccine, wherein the immunogen is an alphavirus RNA replicon particle (αRP) vector encoding an antigen of IAV-S.

[0015] Until weaning, protection of piglets against local pathogens, such as porcine epidemic diarrhea virus (PEDV), E. coli, Clostridium, parvovirus, PCV2, rotavirus, TGE virus, is mainly due to the immunity conferred by milk (milk immunity). This immunity depends on immune activation at the induction site in the sow and migration of activated B cells to the mammary gland with local production of secretory IgA. In sows, immune activation occurs in the GALT (gut-associated lymphoid tissue) by lymphocyte recirculation between the intestine and the mammary gland, and in the BALT (bronchus-associated lymphoid tissue) by B cell recirculation between the respiratory system and the mammary gland.

[0016] For optimal milk immunity in the local mucosa of pigs, the immunizing antigen in the vaccine must stimulate local immunity, in addition to systemic immunity, mainly by acting on the lymphoid tissue of the intestine ("Peyer's patches"). This leads to the production of IgA just below the superficial layer of mucosal tissue, for example in the intestine and airways. IgA passes through the cells of the mucosa and binds to another molecule called the secretory component. It is therefore called secretory IgA and is composed of two IgA antibody molecules linked together by the secretory component. This combination enhances their potency, making them resistant to digestion by intestinal enzymes and more easily absorbed by mucus. Since mucus coats the entire inner lining of the intestine and airways, secretory IgA acts as a shield against potentially pathogenic infections.

[0017] The present invention arose from the unexpected finding that, in contrast to experiments showing that an αRP vector-based vaccine did not confer passive protection in piglets against pathogenic infection with PEDV, a porcine pathogen that replicates locally in mucosal tissues similar to IAV-S (Example 3), active vaccination of sows with an αRP IAV-S vector-based vaccine did confer passive protection in the sow's piglets (Example 2). [Brief description of the drawings]

[0018] [Figure 1] IAV-S hemagglutination inhibition (HI) titers in gilt colostrum, sow serum, and piglet serum. Eurasian avian (France / 53-130065) lineage IAV-S strain-specific HI titers (log2) measured in sow colostrum collected at farrowing (n=3), sow serum collected at farrowing (n=3), and piglet serum collected at 1 week of age (n=18–27). [Diagram 2]Weighted lung lesion scores and mean ± SD of piglets measured 3 days after challenge. Mean ± SD was based on 14 (IAV-S RP+ColiClos IM), 16 (IAV-S RP XSolve50 IM), 16 (IAV-S RP XSolve50 ID) and 15 (PBS+ColiClos IM) animals from 3 litters, respectively. Three gilts per group were vaccinated 6 and 2 weeks before farrowing, progeny were infected at 4WOA and necropsied 3 days after infection to assess lung lesion scores. [Diagram 3] IAV-S titers and means ± SD in piglet lung tissue taken 3 days after challenge. Means ± SD were based on 14 (IAV-S RP+ColiClos IM), 16 (IAV-S RP XSolve50 IM), 16 (IAV-S RP XSolve50 ID) and 15 (PBS+ColiClos IM) animals from 3 litters, respectively. Three gilts per group were vaccinated 6 and 2 weeks before farrowing, progeny were infected 4WOA and dissected 3 days after infection to measure viral load in the lungs. [Figure 4] PEDV neutralizing antibody titers in sera of sows vaccinated with RP-PEDV or placebo. PEDV neutralizing titers or serum neutralizing titers were measured as FFN titers using an FFN assay against cell culture adapted PEDV strain USA / Colorado / 2013. DPV = days post vaccination, DPF = days post farrowing. Values ​​below the dotted line are considered to have no PEDV neutralizing activity. [Diagram 5] PEDV neutralizing antibody titers in serum samples from pigs born to sows vaccinated with RP-PEDV or placebo. PEDV neutralizing titers or serum neutralizing titers as FFN titers were measured in pig sera collected on the day of challenge (3–5 days of age) using an FFN assay against cell culture-adapted PEDV strain USA / Colorado / 2013. [Figure 6]Pig mortality due to PEDV challenge. Three to five day old pigs born to sows vaccinated with RP-PEDV or placebo were inoculated intragastrically with 106 TCID50 of the PEDV / Colorado / 2013 challenge strain in a total volume of 3 mL per pig. Mortality of infected pigs per litter was recorded up to 14 days after challenge. Mortality of pigs per litter is shown in the figure.

[0019] definition A vaccine is a composition suitable for administration to an animal containing an immunologically effective amount of one or more antigens of an infectious agent, typically combined with a pharma- ceutically acceptable carrier, which, upon administration to an animal, induces an immune response that protects the animal against pathogenic infection by the infectious agent.

[0020] Protecting against pathogenic infection by an infectious agent means obtaining protective immunity in an animal, i.e., helping to prevent, ameliorate or cure the deleterious effects caused by infection with that infectious agent, for example by reducing the number or duration of viral replication in the animal, or by diminishing or reducing the number, intensity or severity of tissue lesions, or by preventing one or more clinical signs.

[0021] By pig or swine is meant any animal of the family Suidae, in particular of the genus Sus, such as wild or domestic pig, Sus scrofa, babirusa or warthog.

[0022] Piglets are the young offspring of a sow.

[0023] A sow is an adult female pig that has already given birth to a litter of piglets.

[0024] A gilt is a young female pig that has not yet given birth to a litter.

[0025] Colostrum is the first milk produced by a sow or gilt with each litter of piglets.

[0026] As used herein, active immunization is the stimulation of the pig's immune system following exposure of the pig's body to a foreign antigen, resulting in the production of antibodies and immune cells. The antigen can be in the form of an infectious agent, an inactivated form of the infectious agent, or an immunogenic component of the infectious agent.

[0027] Passive immunization as used herein refers to the transfer of immunity (antibodies and / or immune cells) of the sow to the newborn piglet progeny via ingestion of colostrum and preferably subsequent milk. In practice, passive immunity of the piglet progeny can be obtained from its dam or from another sow that has been vaccinated prior to farrowing.

[0028] Alphaviruses are a genus of RNA viruses belonging to the family Togaviridae and are small, spherical, enveloped, positive-sense ssRNA viruses (Fields Virology: Emerging Viruses, Authors: Howley, Knipe and Whelan, ISBN / ISSN 9781975112547, 2020).

[0029] The term alphavirus RNA replicon particle (αRP) as used herein refers to an alphavirus-like particle that contains a modified RNA viral genome that lacks one or more coding sequences for structural proteins that, if present, would allow successful propagation of the parent virus in cell culture or animal host that is also packaged within viral structural proteins (e.g., capsid and glycoproteins) derived from alphaviruses, as described, for example, by Pushko et al. (Virology 239, 389-401, 1997). Thus, αRP can enter animal host cells and undergo one round of viral genome amplification without being able to form new particles. Replicon particles do not propagate from infected cells because they lack the necessary structural protein coding sequences (see also Vander Veen et al., 2012, Anim. Health. Res. Rev., vol. 13, p. 1-9; and Kamrud et al., 2010, J. Gen. Virol. 91, 1723-1727).

[0030] Several alphavirus species have been used to develop alphaRP-based vaccines, such as Venezuelan equine encephalitis virus (VEEV) (Pushko et al., 1997, Virology 239, 389-401), Sindbis virus (Bredenbeek et al., 1993, J. of Virol. 67, 6439-6446) and Semliki Forest virus (Liljestrom and Garoff, 1991, Biotechnology 9, 1356-1361).

[0031] The αRP vector contains a heterologous nucleic acid molecule encoding an antigen of interest inserted into the viral genome. Transcription and translation of the antigen-encoding nucleic acid molecule contained within the αRP vector results in expression of the antigen in cells infected with the αRP vector without producing progeny, thus delivering and expressing the heterologous antigen to the immune system of the infected animal. The nucleic acid molecule can contain a complete gene or open reading frame (ORF) encoding a complete protein, or it can be a fragment thereof encoding a portion of the protein. The nucleic acid molecule encoding the antigen can be transcribed and expressed from an alphavirus subgenomic promoter, such as the 26S-alphavirus subgenomic promoter. The transcribed replicon RNA can be packaged into the RP by expression of the structural proteins by a packaging cell line, or by co-transfection of the replicon RNA with one or more "helper" RNAs encoding the structural proteins into a suitable host cell.

[0032] An αRP vector may contain two or more heterologous nucleic acid molecules encoding the same or different antigens of interest inserted into its genome. This may be accomplished in several ways. For example, such an αRP vector may encode a polycistronic reading frame or may encode separate genes, for example, by using one or more additional copies of a subgenomic promoter that allows for the expression of separate, additional proteins.

[0033] Some αRP vectors are already available, such as the commercially available VEEV-based αRP vector described below, or the vectors can be constructed using well-known techniques by incorporating a heterologous nucleic acid molecule encoding an antigen into the viral replicon backbone.

[0034] As used herein, an antigen of IAV-S is a protein or fragment thereof capable of eliciting an immune response in pigs that protects the animal against pathogenic infection with IAV-S.

[0035] The IAV genome is composed of eight segments that code for at least 12 proteins. Three of these proteins, namely the viral hemagglutinin (HA), neuraminidase (NA) and matrix 2 (M2) proteins, are incorporated into the viral envelope. Vaccines based on HA and / or NA can confer protective immunity in pigs. The HA protein mediates the binding of influenza virions to host cells. The function of the IAV-S NA protein is to cleave sialic acid on the host cell, allowing the newly formed virions to be efficiently released from the infected cell. The transmembrane matrix 2 (M2) protein is a proton-selective ion channel and is required for efficient uncoating of influenza A viruses (see reviews by Sandbulte et al., Vaccines 2015, 3, 22-73; doi:10.3390 and Aguilar-Yanez et al., PLOS ONE 2010; doi.org / 10.1371 / journal.pone.0011694).

[0036] The amino acid and nucleotide sequences of IAV-S protein antigens can be readily obtained from publicly available literature and sequence databases, such as NCBI (Influenza Database), Influenza Research Database (IRD) or GenBank (GB). The IAV-S antigens used in the present invention can be of any IAV-S (sub)type, phylogenetic clade, cluster, lineage, strain, etc.

[0037] The term phylogenetic as used herein refers to a set of influenza virus hemagglutinins or neuraminidases grouped together (on the same branch) in an evolutionary tree that traces back to a similar (homologous) ancestor. These groupings have been made for European hemagglutinins and neuraminidases and are similar, but not equivalent, to the phylogenetic clusters for viruses in the United States. Determination of lineage can be achieved by phylogenetic analysis of the HA or NA sequence in question using pre-established reference sequences using readily available software, i.e., Clustal Omega (Sievers et al., 2011, Mol. Syst. Biol. 7, 539) or a web-accessible annotation tool for H1 HA sequences (Anderson et al., mSphere, 2016, 1(6):e00275-16). In Europe, there are three major lineages of H1 (Eurasiatori-like H1, Scotland / 410440 / 1994-like H1 and pandemic 2009-like H1), one major lineage of H3 (Gent / 1 / 1984-like H3), two major lineages of N1 (Eurasiatori-like N1 and pandemic 2009-like N1), two major lineages of N2 (Gent / 1 / 1984-like N2 and Scotland / 410440 / 1994-like N2), and two minor lineages of N2 (Italy / 4675 / 2003-like N2 and human seasonal-like N2). Rules for IAV-S classification, and tools to evaluate classification, are described by Watson et al. (J. Virol., 89, 9920-9931, 2015, doi:10.1128 / JVI.00840-15), https: / / journals.asm.org / doi / 10.1128 / mSphere.00275-16 , and https: / / www.fludb.org / brc / h1CladeClassifier.spg?method=ShowCleanInputPage&decorator= It is described in influenza.

[0038] A pharma- ceutically acceptable carrier is a biocompatible medium, i.e., a medium that can present an antigen to the animal's immune system after administration of a composition containing the carrier and that does not induce significant adverse reactions in the treated subject. Such pharma-ceutically acceptable carriers can be liquids, including, for example, water and / or any other biocompatible solvent, or solid carriers, including, for example, solid carriers commonly used to obtain lyophilized vaccines (based on sugars and / or proteins), optionally including adjuvants.

[0039] Adjuvants are compounds or compositions that can provide a non-specific stimulation to the immune system of an animal. Adjuvants are commonly used in vaccines based on inactivated or subunit antigens. A wide variety of adjuvant types and compositions exist, such as aluminum salts, e.g. aluminum hydroxide or aluminum phosphate, liposomes, glucans, alginates, bacterial components, e.g. cell wall components, mineral or non-mineral oil and water emulsions, synthetic adjuvants, e.g. non-ionic block polymers, polyamines, e.g. dextran sulfate, Carbopol™, pyran, and saponins, e.g. Quil A™ or Q-vac™. Saponins and vaccine components can be combined with ISCOM™. Furthermore, peptides, e.g. muramyl dipeptide, dimethylglycine, tuftsin, are often used as adjuvants. Similarly, combination products, such as ISA™ composition (Seppic, France), can be used. More detailed information on adjuvants, their use and effects can be found in Handbook: Vaccine adjuvants (Methods in molecular medicine, vol. 42, edited by D. O'Hagan, 2000, Humana press, NJ, ISBN: 0896037355). For a detailed review of adjuvants used in pigs, see Charerntantanakul W., Vaccine, 2020; 38(43), 6659-81.

[0040] Further detailed embodiments of the present invention In one embodiment of a vaccine for use according to the invention, the αRP is Venezuelan Equine Encephalitis (VEE) αRP. In a more specific embodiment, the αRP is TC-83 VEE αRP. The production of VEE TC-83 αRP is described, for example, in US 9,441,247 and US 8,460,913. The VEE-based αRP vaccine is also the basis of several vaccines licensed by the USDA, including: Porcine 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 Pharmaceutical Product, RNA Particle (product code 9PP0.00).

[0041] In a preferred embodiment of the vaccine for said use, the sow is a sow or a gilt.

[0042] In yet another embodiment, sows are vaccinated twice. Vaccinating sows twice can increase the level of antibodies that ultimately reach the piglets via ingestion of colostrum, and such a strategy does not cause significant problems in the routine practice of raising adult animals. In such a two-vaccination regimen, the first vaccination is typically administered 5-8 weeks before the expected farrowing date, and is boosted with a second vaccination 3-4 weeks later.

[0043] In yet another preferred embodiment, sows are immunized during gestation.

[0044] It is expected that sows can be vaccinated moderately during pregnancy in order to keep antibody levels reasonably high (continuously), but it has been shown to be useful to vaccinate the animals (at least once) during pregnancy. In particular, it has been shown to be useful to vaccinate pregnant sows in the period 5-8 weeks before farrowing, followed by a booster vaccination, particularly in the period 2-6 weeks before the expected farrowing date, in either case typically at least 1-3 weeks before the expected farrowing date.

[0045] For sows that have already been vaccinated during a previous pregnancy, one revaccination can be administered 1 to 4 weeks before the expected farrowing date.

[0046] In another preferred embodiment, the IAV-S antigen encoded by the αRP vector in the vaccine for said use is an HA antigen. The examples herein demonstrate that the IAV-S HA antigen can be advantageously used in the vaccine to induce a strong hemagglutinin-inhibiting (HI) antibody response in sows by active vaccination, resulting in high levels of these antibodies in the serum and colostrum of pregnant sows. These HI antibodies are passively transferred to their progeny piglets, resulting in high levels of (systemic) HI antibody titers in the piglet serum. Importantly, maternally acquired antibodies can also induce local protection in the piglet's lungs (milk immunity), as demonstrated by a reduction in both viral load and lung lesions in the lungs.

[0047] In the context of the present invention, any IAV-S HA antigen can be used, such as those disclosed in the art, e.g. those disclosed in publicly available sequence databases (NCBI (Influenza Database), Influenza Research Database (IRD) or GenBank (GB)) that provide a large number of amino acid sequences of IAV-S HA antigens and nucleotide sequences encoding such HA antigens.

[0048] Of particular interest are HA antigens from any of the four major lineages of IAV-S HA. These lineages are designated Scot / 94 H1N2, EurAsianAvian H1N1, Gent1984 H3N2 and Pandemic 2009 H1N1 (Watson et al., J. Virol., 89, 9920-9931, 2015, doi:10.1128 / JVI.00840-15). Vaccines for said uses may include HA antigens from any of these lineages.

[0049] The HA antigen of the Scot / 94 lineage can be from any strain, for example from the A / swine / Italy / 3033-1 / 2015 (H1N2) or A / swine / France / 35-140041 (H1N2) strains. In a preferred embodiment, the HA antigen of the Scot / 94 lineage is from the A / swine / Italy / 3033-1 / 2015 (H1N2) strain. In another preferred embodiment, the HA antigen comprises an amino acid sequence having at least 90%, preferably at least 93%, more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to the HA antigen of the Scot / 94 reference strain A / swine / Italy / 3033-1 / 2015 (H1N2):GB# ALX30160.1.

[0050] The HA antigen of the EA lineage can be from any strain, for example from the A / swine / Denmark / 101048-2 / 2011 (H1N1) strain, the A / swine / Italy / 28762-3 / 2013 (H1N1) strain or the A / swine / France / 44-120070 / 2012 (H1N1) strain. In a preferred embodiment, the HA antigen of the EA lineage is from the A / swine / Italy / 28762-3 / 2013 (H1N1) strain. In another preferred embodiment, the HA antigen comprises an amino acid sequence having at least 90%, preferably at least 93%, more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to the HA antigen of the EA reference strain A / swine / Italy / 28762-3 / 2013 (H1N1):GB# AKJ81667.1.

[0051] The HA antigen of the Gent / 84 lineage can be of any strain, preferably of the A / swine / Italy / 240849 / 2015(H3N2) strain. In another preferred embodiment, the HA antigen comprises an amino acid sequence having at least 90%, preferably at least 93%, more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to the HA antigen of the Gent / 84 reference strain A / swine / Italy / 240849 / 2015(H3N2):GB# ALX30415.1.

[0052] The HA antigen of the pdm09 lineage can be of any strain, with the A / Swine / England / 373 / 2010 (H1N1) strain being preferred. In another preferred embodiment, the HA antigen comprises an amino acid sequence having at least 90%, preferably at least 93%, more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to the HA antigen of the pdm09 reference strain A / Swine / England / 373 / 2010 (H1N1):GB# AFR76205.1.

[0053] In particularly preferred embodiments, the IAV-S HA antigen used in the present invention is any of the HA antigens listed in Table 1 (infra).

[0054] In one embodiment, the present invention also provides a vaccine for said use, characterized in that it comprises at least one αRP vector encoding two or more distinct IAV-S antigens, in particular distinct HA antigens, more preferably HA antigens from different lineages, and even more preferably said distinct HA antigens are of IAV-S strains belonging to lineages selected from the group of Scot / 94 H1N2, EurAsianAvian H1N1, Gent1984 H3N2 and pandemic 2009 H1N1.

[0055] In a more detailed form of this embodiment, the vaccine comprises a first αRP vector encoding the HA protein of an IAV-S strain belonging to two lineages selected from Scot / 94 H1N2, EurAsianAvian H1N1, Gent1984 H3N2 and pandemic 2009 H1N1, and a second αRP vector encoding the HA protein of an IAV-S strain belonging to the other two lineages of that group. In particular, the vaccine comprises a first αRP vector encoding the HA protein of an IAV-S strain belonging to lineages Scot / 94 H1N2 and EurAsianAvian H1N1, and a second αRP vector encoding the HA protein of an IAV-S strain belonging to lineages Gent1984 H3N2 and pandemic 2009 H1N1.

[0056] In yet another advantageous embodiment of the vaccine for use according to the invention, the pharma- ceutically acceptable carrier comprises an oil and water emulsion adjuvant. The examples demonstrate that the presence of an oil and water emulsion adjuvant as a pharma- ceutically acceptable carrier for the antigen in the vaccine has a positive effect on the production of HI antibody titers in both sows and their piglet progeny.

[0057] The emulsion may be a water-in-oil (W / O) emulsion, where the oil is the continuous external phase. Preferably, the emulsion is an oil-in-water (O / W) emulsion, where the oil is the dispersed internal phase. Such emulsions can be formed and maintained stable using standard emulsification techniques by selecting the appropriate type and concentration of emulsifiers ("Remington: the science and practice of pharmacy", 2000, Lippincot, USA, ISBN: 683306472, and Veterinary Vaccinology, edited by Pastoret et al., 1997, Elsevier, Amsterdam, ISBN 0444819681).

[0058] The oil and water emulsion adjuvant, when in the form of a preferred O / W emulsion, facilitates mixing of the emulsion with the αRP vector, which can be achieved, for example, by mixing an aqueous or lyophilized composition containing the αRP vector with an O / W emulsion, in which case simple shaking by hand for about 1 minute is sufficient to adequately mix the two aqueous compositions.

[0059] In more particular embodiments, the oil and water emulsion adjuvant may comprise a biodegradable mineral oil or a non-mineral oil. Preferably, the mineral oil is liquid paraffin oil (e.g., commonly available as Drakeol® 6VR (Penreco), Marcol® 52 (Exxon Mobile) and Klearol® (Sonneborn), CAS number: 8042-47-5). Preferably, the biodegradable non-mineral oil is selected from the group consisting of squalane, squalene, vitamin E, vitamin E-acetate, oleate and ethyl oleate, with vitamin E-acetate being most preferred.

[0060] In yet another embodiment, the oil and water emulsion adjuvant comprises two or more of a biodegradable mineral oil and / or a non-mineral oil, preferably the oil and water emulsion adjuvant comprises liquid paraffin oil and vitamin E-acetate.

[0061] Examples of adjuvants useful in vaccines for said use include the following proprietary O / W adjuvants: (micro)Diluvac Forte™ (based on a W / O emulsion of dl-α-tocopheryl acetate), XSolve™ (a combination of two O / W emulsion adjuvant components: Diluvac Forte (see EP 382.271), which is based on vitamin E acetate, and Microsol™ (see WO 2009 / 144.088), which is based on liquid paraffin oil), SVEA™ (a W / O emulsion of squalane and vitamin E-acetate, WO 2018 / 115.435) and ImpranFLEX™ (a water-in-oil adjuvant).

[0062] Vaccines for use according to the invention comprise an immunologically effective amount of an oil emulsion adjuvant that enhances the immune response elicited by the antigen of the vaccine. In one embodiment, the vaccine comprises the oil emulsion adjuvant in an amount of about 10%-90% v / v of the vaccine. More preferably, the vaccine comprises the oil adjuvant in an amount of about 20%-80% v / v, 30-70% v / v or even 40-60% v / v of the vaccine. Most preferably, the vaccine comprises the oil adjuvant in an amount of about 50% v / v of the vaccine.

[0063] The vaccine for said use comprises an immunologically effective amount of the αRP vector such that piglet progeny are protected against pathogenic infection with IAV-S. In one embodiment, the vaccine comprises about 1×10 ∧ 3~about 1×10 ∧ In a more particular embodiment, the vaccine comprises about 1×10 ∧ 4~about 1×10 ∧ 10 RP. In even more particular embodiments, the vaccine contains about 1×10 ∧ 5~about 1×10 ∧ Contains 9 RP.

[0064] In another embodiment, the vaccine for said use is administered in a dose of 0.05ml to 3ml. In a more particular embodiment, the administered dose is 0.1ml to 2ml. In yet more particular embodiments, the administered dose is 0.2 to 2ml.

[0065] The invention also provides vaccines for said use against multiple swine pathogens, for example the vaccine may comprise additional inactivated, attenuated or subunit antigens other than the IAV-S viral and / or bacterial swine pathogens. Examples of such swine pathogens include porcine reproductive and respiratory syndrome virus (PRRS), porcine circovirus (PCV), transmissible gastroenteritis virus (TGE), porcine pseudorabies virus (PPRV), porcine parvovirus (PPV), porcine rotavirus (PRV), porcine epidemic diarrhea virus (PED), multiple serotypes of Pasteurella multocida, Salmonella spp., Escherichia coli (e.g., serotypes K99, K88, 987P or F41), Haemophilus parasuis, Lawsonia intracellularis, Mycoplasma spp. [e.g., Mycoplasma hyopneumoniae, Mycoplasma spp.] ... hyopneumoniae), Bordetella bronchiseptica, Erysipelas spp., Campylobacter spp., Actinobacillus pleuropneumoniae, Clostridium perfringens and Clostridium difficile.

[0066] In particularly preferred embodiments, the vaccine further comprises antigens for E. coli and Clostridium perfringens.

[0067] In another embodiment of the vaccine for use according to the invention, the vaccine is administered by a route common for vaccination of pigs, in particular by parenteral administration, including subcutaneous, intramuscular and intradermal injection, with intramuscular injection being preferred.

[0068] In another aspect, the invention provides the use of an alphavirus RNA replicon particle (αRP) vector encoding an antigen of IAV-S for the manufacture of a vaccine to protect the progeny of piglets from pathogenic infection by IAV-S by passive immunization of the piglets through active immunization of the sow. All of the features of this aspect are as outlined above.

[0069] In yet another aspect, the present invention provides a method for passively immunizing the progeny of piglets from a sow against pathogenic infection by IAV-S by actively immunizing the sow with a vaccine comprising an alphavirus RNA replicon particle (αRP) vector encoding an antigen of IAV-S, all of the features of this aspect being as outlined above.

[0070] The invention will now be further illustrated by the following specific examples.

[0071] Working Example Example 1. Production of an IAV-S vaccine based on the HA αRP vector 1. Construction of multi-HA gene αRP vector The VEE replicon vectors used to express the HA and NA genes were constructed as previously described (US9,441,247, US8,460,913, WO 2019 / 121513 and WO2019110481) with the following modifications. The TC-83-derived replicon vector "pVEK" was digested with the restriction enzymes AscI and PacI. For the double-gene HA constructs, the selected open reading frame sequences were codon-optimized and synthesized with flanking AscI and PacI sites. Furthermore, the intervening sequence between the two synthetic HA open reading frames consisted of 47 nucleotides of non-coding heterologous sequence and a second copy of the native TC-83 subgenomic (sg)RNA promoter and 5' untranslated sgRNA region sequence. These double-gene constructs were named "pVDG" to distinguish them from the parent vectors with a single sgRNA promoter sequence.

[0072] The following replicon particles were constructed: [Table 1]

[0073] TIFF2024536600000002.tif101163

[0074] The αRP vector EUSIV-K and RP EUSIV-T8 were used to determine the immunogenicity and efficacy of a multivalent IAV-S vaccine containing two dual-HA RPs.

[0075] 2. Production of vaccines based on αRP vectors RP particles dissolved in PBS 0.01M containing phenol red of batch numbers RP EUSIV-K and RP EUSIV-T8 were mixed 1:1 (v / v) with X-Solve® adjuvant (an O / W emulsion of 21% v / v of the non-metabolizable oil Marcol® 52 and 1.25% v / v of the metabolizable oil Vitamin E acetate, available from MSD Animal Health, Boxmeer, The Netherlands) or Micro Diluvac Forte® adjuvant (an O / W emulsion of 7.5% of the metabolizable oil Vitamin E acetate) in Porcilis® ColiClos vaccine (available from MSD Animal Health, Boxmeer, The Netherlands). The Porcilis® ColiClos vaccine contains multiple Escherichia coli (E. coli) antigens (F4ab / F4ac / F5 / F6 fimbria adhesins) and the Clostridium perfringens (C. perfringens) LT toxoid antigen.

[0076] Example 2 Milk / passive immune protection of piglets against influenza viruses This study evaluated milk immune protection in piglets against influenza A virus induced by multigene IAV-S HA RNA particles mixed with 50% (v / v) Xsolve adjuvant or Porcilis® ColiClos vaccine (microDiluvac Forte® adjuvant).

[0077] Study design Twelve healthy pregnant gilts (not yet vaccinated against Clostridium perfringens type C and / or E. coli and with no / low antibody levels against IAV-S) expected to give birth to 8–10 piglets were purchased from a suitable commercial farm. Gilts were vaccinated intradermally (id) or intramuscularly (im) 6 and 2 weeks before farrowing (5 per RP). *10 6 RP / dose):Table 1. Blood samples were collected from all gilts 6 and 2 weeks prior to farrowing (denoted as ~WPF for ~weeks prior to farrowing) and on the day of farrowing to measure antigen-specific HI titers. Colostrum samples were also collected on the day of farrowing and milk samples were collected 3 days after farrowing.

[0078] Piglets: Blood samples were collected from all piglets at approximately 1 week of age (WOA) (90 pigs) and on the day of challenge (88 pigs) to measure antigen-specific HI titers. When piglets reached approximately 4 WOA, they were challenged with IAV-S challenge strain A / swine / France / 53-130065 / 2013 (H1N1) (10 per pig in 5 mL PBS). 5 They were inoculated intratracheally (it) with 1000 TCID50 of 1000 ng / litter. Up to 10 piglets per litter were challenged. Viral shedding was measured by nasal swabs taken from the pigs on the day of challenge (i.e., day 0) and 1, 2 and 3 days after challenge. Some piglets (24) were necropsied 24 hours after challenge and the rest (61) were necropsied 72 hours after challenge. Lung lesions were recorded at necropsy and six lung samples were taken from each pig (one each from the left cranial, middle and caudal lobes and one each from three corresponding right lung lobes) to measure the viral load in the lungs. [Table 2]

[0079] Experimental procedure 1. IAV-S HI antibody titers in serum and colostrum The two dual HA gene RPs were tested for their in vivo function by monitoring the HI titers in the sera of vaccinated pigs. Representative IAV-S strains from four IAV-S lineages with more than 85% amino acid identity to the HA-RP were used for the HI test. In Table 2, next to the lineage and strain name, the clade classification and amino acid identity (%) of the IAV-S strains used for the quantification of the HI titers to each vaccine component are listed. IAV-S-specific antibodies in pig serum samples were measured by HI test by standard methods. Briefly, the serum was pretreated with periodate to remove non-specific inhibitors. The pretreated serum was then serially diluted two-fold and incubated with influenza virus strains (Table 2). After the incubation step, chicken red blood cells were added, incubated, and the hemagglutination inhibition of the plates was measured. The reciprocal of the highest serum dilution that completely inhibited hemagglutination was assigned as the HI titer and expressed as a log base 2 value. [Table 3]

[0080] 2. Antibody titers against Escherichia coli in serum and colostrum Antibody titers in serum against E. coli antigens in the vaccine (K88ab, K88ac, K99, 987P and LT types) were measured by ELISA according to standard procedures.

[0081] 3. Antibody titers against Clostridium perfringens in serum and colostrum Serum antibody titers against Clostridium perfringens type C beta-toxin were measured by ELISA according to standard procedures.

[0082] 4. Virus quantification in clinical samples Nasal swabs and lung tissues were tested for infectious titers by serial dilution of the original nasal swab and 20% lung tissue homogenate samples. Two lung homogenates per animal were tested, one prepared by pooling equal amounts of samples taken from the left cranial, median and caudal lobes, and the other prepared from the corresponding right lung lobe. Briefly, samples were serially diluted 10-fold in IAV-S infection medium. Each dilution was inoculated onto three replicate wells of a confluent MDCK monolayer. Plates were incubated at 37°C, 5% CO2 for 4 days, and the presence of virus in the supernatant was detected by hemagglutination. IAV-S titers were calculated as log 1 / mL using the Spearman-Karber method. 10 TCID 50 It was calculated as:

[0083] 5. Macroscopic Lung Assessment Macroscopic lung lesions indicative of swine influenza were scored based on the percentage of abnormal lung tissue per lobe, and a weighted score was assigned to each of the seven lobes according to the relative weight of the lobe.

[0084] result 1. IAV-S hemagglutination inhibition titers in serum, colostrum and milk of gilts Blood samples were taken from all gilts 6 and 2 weeks before farrowing (WPF) and on the day of farrowing (FD) to measure antigen-specific HI titers. Colostrum samples were taken on the day of farrowing and milk samples were taken 3 days after farrowing. The tested IAV-S vaccines (based on EUSIV-T8 and EUSIV-K) induced HI titers against all representative heterologous strains from each lineage in serum, colostrum and milk. (IAV-S HI titers in gilt serum were 8log2EurAsian, 9log2Gent84, 11log2Pandemic, 5log2Scot94 Clu1 and 7log2 vs Scot94 Clu2). The measured mean HI titers and standard deviations per group for EU IAV-S strains in gilts are shown in Figure 1. In general, the IAV-S vaccine induced higher HI titers against all four IAV-S strains when mixed with XSolve50 adjuvant than when mixed with Porcilis® ColiClos adjuvant. Furthermore, administration of the IAV-S vaccine by the intramuscular route was observed to induce higher HI titers than when administered by the intramuscular route.

[0085] 2. IAV-S hemagglutination inhibition titers in piglet serum Blood samples were taken from all piglets at approximately 1 week of age and on the day of challenge (approximately 4 WOA). HI titers against all representative heterologous strains from each line in the progeny were measured at approximately 1 week of age and on the day of challenge. As with IAV-S HI titers in gilt serum, the progeny of gilts vaccinated with IAV-S vaccine Xsolve50 adjuvant showed the highest HI titers against all lines among all three test groups, followed by the group receiving IAV-S vaccine mixed with Porcilis® Coliclos vaccine by intramuscular route and the group receiving IAV-S vaccine mixed with Xsolve50 adjuvant by intradermal route. Furthermore, antibody titers in serum collected at 1 week of age were higher than those in serum collected at 4 WOA. A summary of IAV-S HI antibody titers (EA lineage) measured at 1 WOA is shown in Figure 1.

[0086] 3. Antibody titers in piglets against E. coli and Clostridium perfringens type C in serum Piglet serum samples were tested for antibody titers against five E. coli antigens and Clostridium perfringens at approximately 1 week of age and the day of challenge in groups 1 and 4 (which used the IAV-S vaccine combined with Porcilis® ColiClos or the Porcilis® ColiClos vaccine alone, respectively). Blood samples were taken from all piglets at approximately 1 WOA and 4 WOA (the day of challenge).

[0087] Serum samples from gilts administered Porcilis® ColiClos vaccine alone or in combination with IAV-S vaccine showed the highest antibody titers against all E. coli types when compared to other experimental groups. In general, no significant differences in measured E. coli antibody titers were observed between groups administered Porcilis® ColiClos alone and in combination with IAV-S vaccine. Furthermore, antibody titers in sera collected at 1 week of age were higher than those in sera collected at 4 WOA.

[0088] Serum samples from gilts receiving Porcilis® ColiClos vaccine alone or in combination with IAV-S vaccine showed the highest antibody titers against all E. coli types when compared to other experimental groups. In general, no significant differences were observed in measured C. perfringens antibody titers between groups receiving Porcilis® ColiClos alone and in combination with IAV-S vaccine. Furthermore, antibody titers in sera collected at 1 week of age were higher than those in sera collected at 4 WOA.

[0089] 4. Protection against pulmonary lesions in piglets following IAV-S challenge Four-week-old progeny of vaccinated gilts were infected intratracheally with IAV-S EA lineage A / Pig / France / 53-130065 / 2013 (H1N1) strain and dissected 1 or 3 days after infection to evaluate lung lesions induced by IAV-S infection. Lung lesion scores evaluated 3 days after infection are summarized in Figure 2. The data showed that all progeny of gilts administered the IAV-S vaccine alone or in combination with the Porcilis® ColiClos vaccine had lower LLS compared to groups that did not receive the IAV-S vaccine. The progeny in the group in which the IAV-S vaccine was administered by the intramuscular route with XSolve50 adjuvant (Group 2) showed the lowest lung lesion scores, followed by the groups in which the IAV-S vaccine was administered by the intradermal route with Xsolve50 adjuvant (Group 3) or the groups in which the IAV-S vaccine was administered with Porcilis® ColiClos (Group 1).

[0090] 5. Reduction of viral load in piglet lungs after IAV-S challenge The measured titer data obtained from lung homogenates 3 days after infection are summarized in Figure 3. The data showed that all progeny of gilts administered the IAV-S vaccine alone or in combination with the Porcilis® ColiClos vaccine had lower virus levels in lung homogenates compared to groups that did not receive the IAV-S vaccine. The progeny of the group administered the IAV-S vaccine by intramuscular route with XSolve50 adjuvant (group 2) showed the lowest virus load in lung homogenates, followed by the groups administered the IAV-S vaccine by intradermal route with Xsolve50 adjuvant (group 3) or with Porcilis® ColiClos (group 1).

[0091] conclusion IAV-S RP (EUSIV-T8 and EUSIV-K) vaccines induced high serum and colostrum HI titers against all tested representative heterologous IAV-S strains from each lineage. A direct correlation between HI titers in gilt serum, colostrum and piglet serum was observed. IAV-S vaccine induced higher HI titers against all four IAV-S strains when mixed with XSolve50 adjuvant instead of Porcilis® ColiClos and applied. The magnitude of antibody titers induced by Porcilis® ColiClos against all five Escherichia coli types and Clostridium β-toxin was not affected when it was applied in combination with IAV-S RP vaccine. The intramuscular route of application of IAV-S vaccine induced higher HI titers (in both serum and colostrum) against all four strains compared to the intradermal route. HI titers measured in piglet serum correlated with reduced lung lesions and viral load.

[0092] Example 3 Milk / passive immune protection of piglets against porcine epidemic diarrhea virus (PEDV) This experiment evaluated the lacteal / passive immune protection of piglets against PEDV induced by vaccination with RNA particles encoding the PEDV spike glycoprotein gene in Xsolve50 adjuvant.

[0093] Study design Twenty-four serologically and clinically PED-free sows were vaccinated intramuscularly at 8, 4 and 2 weeks prior to farrowing with RNA particles (lyophilized T9 RNA-based vaccine containing an alphavirus RNA RP based on a replicon construct derived from VEEV strain TC-83) encoding the PEDV spike glycoprotein gene. This was constructed to contain the coding sequence for the spike protein of US pathogenic PEDV strain AH2012 (see GenBank accession number KC210145). 1 mL per dose was mixed with X-Solve at 50% at time of use. Pigs were vaccinated at 3-5 days of age with CO / 2013 strain (10 per pig). 6 I took on the IT challenge at TCID50. [Table 4]

[0094] result The vaccine induced moderate (80-160) serum PED neutralization (PED SN) titers after three vaccinations (Figure 4). FFN (fluorescent focus neutralization) assays are used to measure PEDV serum neutralization titers. Piglets born to vaccinated sows had PED maternal antibodies (80-640) against the PED challenge strain at prechallenge (Figure 5).

[0095] conclusion However, despite the generation of SN antibodies in sows and their transfer to piglets, the vaccine did not protect suckling piglets from PED-induced mortality (Table 5 and Figure 6). [Table 5]

[0096] Example 4 Milk / passive immune protection of piglets against influenza viruses In full correspondence with Example 2, this experiment evaluated piglet milk immune protection against influenza A virus induced by multigene IAV-S HA RNA particles mixed with 50% (v / v) Xsolve adjuvant by vaccinating multiparous sows and demonstrating passive protection, but in this case against another IAV-S challenge strain, namely A / Pig / Belgium / 113 / 2013 (H3N2), at 6 WOA. [Table 6]

[0097] Figure 7 shows the weighted lung lesion scores (LLS) and mean ± SD of piglets measured 1 or 3 days after challenge. Mean ± SD was based on 15 animals belonging to 6 litters each. Six sows per group were vaccinated 6 and 2 weeks before farrowing or served as unvaccinated controls. The progeny were infected with A / swine / Belgium / 113 / 2013 (H3N2) IAV-S at 6 WOA and sacrificed 1 or 3 days after infection to assess the lung lesion scores. [Table 7]

[0098] Figure 8 shows IAV-S titers and means ± SD in lung tissue of piglets taken 1 or 3 days after challenge. Means ± SD were based on 15 animals per group, each belonging to 6 litters. Six sows per group were vaccinated 6 and 2 weeks before farrowing or served as unvaccinated controls. The progeny were infected with A / swine / Belgium / 113 / 2013 (H3N2) IAV-S at 6 WOA and sacrificed 1 or 3 days after infection to measure viral load in the lungs.

[0099] It is clear that a good immune response and protection against the challenge strain is induced.

Claims

1. 1. A vaccine comprising an immunogen and a pharmaceutically acceptable carrier, the vaccine for use in a method of protecting piglets against pathogenic infection with swine influenza A virus (IAV-S) by passive immunization of the piglets via ingestion of colostrum or milk from a sow that has been actively immunized with the vaccine; The vaccine, wherein the immunogen is an alphavirus RNA replicon particle (αRP) vector encoding an antigen of IAV-S.

2. 2. The vaccine for use according to claim 1, wherein the sow is a sow or a gilt.

3. The vaccine for use according to claim 1, wherein the antigen is the IAV-S hemagglutinin (HA) protein.

4. The vaccine for use according to claim 1, wherein the αRP vector encodes two or more distinct IAV-S antigens.

5. 2. The vaccine for use according to claim 1, wherein the vaccine comprises two or more separate αRP vectors encoding separate IAV-S antigens.

6. 2. The vaccine for use according to claim 1, wherein the antigen is of an IAV-S strain belonging to a lineage selected from the group consisting of Scot / 94 H1N2, EurAsianAvian H1N1, Gent1984 H3N2 and pandemic 2009 H1N1.

7. 7. The vaccine for use according to claim 6, wherein the vaccine comprises a first αRP vector encoding HA antigens of IAV-S strains of two lineages selected from Scot / 94 H1N2, EurAsianAvian H1N1, Gent1984 H3N2 and pandemic 2009 H1N1, and a second αRP vector encoding HA antigens of IAV-S strains of the other two lineages of that group.

8. 8. The vaccine for use according to claim 7, wherein the vaccine comprises a first αRP vector encoding HA antigens of IAV-S strains of lineages Scot / 94 H1N2 and EurAsianAvian H1N1, and a second αRP vector encoding HA antigens of IAV-S strains of lineages Gent1984 H3N2 and pandemic 2009 H1N1.

9. 2. The vaccine for use according to claim 1, wherein the pharmaceutically acceptable carrier comprises an oil and water emulsion adjuvant.

10. 2. The vaccine for use according to claim 1, wherein the vaccine is administered intramuscularly.

11. 2. The vaccine for use according to claim 1, wherein the vaccine further comprises an antigen of E. coli.

12. 2. The vaccine for use according to claim 1, wherein the vaccine further comprises an antigen of Clostridium perfringens.

13. Use of an alphavirus RNA replicon particle (αRP) vector encoding an antigen of IAV-S for the production of a vaccine that protects the progeny of a sow's piglets against pathogenic infection with IAV-S by passive immunization of the piglets through active immunization of the sow.

14. 1. A method for passively immunizing piglet progeny of a sow against pathogenic infection with IAV-S, the method comprising: actively immunizing sows with a vaccine containing an alphavirus RNA replicon particle (αRP) vector encoding an antigen of IAV-S; method.

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