RU2787596C9 - Vaccine to swine influenza a virus - Google Patents

Abstract

FIELD: biotechnology; veterinary medicine.

SUBSTANCE: vaccine for help in prevention of a disease caused by an influenza virus is presented, containing two or more RNA replicon particles (RP), each of which independently encodes one or more neuraminidases (hereinafter – NA), and a pharmaceutically acceptable carrier. In this case, the first particle of alphavirus RNA replicon encodes the first NA, and the second particle of alphavirus RNA replicon encodes the second NA, or the particle of alphavirus RNA replicon encodes two and more NA, provided that an immunogenic composition does not contain either hemagglutinin (hereinafter – HA) of a swine influenza A virus (hereinafter – IAV-S), or a nucleotide sequence encoding HA of IAV-S. The first NA and the second NA contain amino acid sequences, which are 98% or less identical. A method for immunization of a swine against a swine influenza A virus, including administration to the swine of immunologically effective amount of the claimed vaccine, and the use of a vaccine for help in prevention of a disease caused by a swine influenza A virus in swine are also presented.

EFFECT: vaccine to IAV-S, containing and/or encoding a neuraminidase antigen of an influenza A virus in the absence of the corresponding hemagglutinin antigen, can adequately protect a swine from IAV-S infection.

16 cl, 8 tbl, 15 dwg, 6 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under U.S.C. § 119(c) of U.S. Interim Application Serial No. 62/607101, filed December 18, 2017.

FIELD OF THE INVENTION

The present invention relates to vector and/or nucleic acid constructs that encode one or more influenza A virus neuraminidase (NA) antigens. The present invention further relates to influenza A virus vaccines containing these vector and/or nucleic acid constructs. The present invention also relates to methods for preparing and using vaccines alone or in combination with other protective agents.

BACKGROUND OF THE INVENTION

Influenza A viruses (IAVs) pose a significant health burden to humans and animals around the world. A member of the Orthomyyxoviridae family, IAV is classified into various subtypes based on the viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). IAV infects poultry, pigs, horses, cats, dogs, marine mammals (eg whales), bats and humans. Wild waterfowl and shorebirds (ducks, geese, swans and gulls) are natural reservoirs and they can be infested with sixteen different HA subtypes and nine different NA subtypes [Webster et al., Microbiol Rev 56: 152-179 (1992)] . Recently, two new subtypes, H17N10 and H18N11, have been identified in bats [Tong et al., Proc. Natl. Acad. sci. 109 (11): 4269-4274 (2012) and Mehle, Viruses 6 (9): 3438-3449 (2014)]. Interspecific transmission of the virus occurs most frequently between wild and domestic waterfowl and between pigs and humans [Nelson and Vincent, 23(3) Trends in Microbiol. 142-153 (2015)]. Mammals infected with IAV exhibit an acute respiratory illness resulting in airborne transmission of the virus. In waterfowl and poultry, the virus can replicate in both the respiratory and intestinal tracts and spread via the fecal-oral route.

One such IAV, swine influenza A virus (IAV-S), is a serious respiratory pathogen of domestic pigs that has proven to be economically costly, especially for animal production, worldwide [Holtkamp et al., The American Association of Swine Veterinarians Annual Meeting . (2007)]. In addition, the transmission of influenza viruses of swine origin to humans is well documented and represents a serious public health threat, thus providing an even greater incentive to control influenza A virus in the swine herd [Krueger and Gray, Curr Top Microbiol Immunol 370: 201- 225 (2013)].

In response to this problem, many pig producers are now vaccinating their pigs against IAV-S using commercially available vaccines. However, controlling IAV-S with conventional vaccines is difficult because many diverse IAV-S strains co-circulate 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 is due to the genetic structure of the virus. Like other influenza A viruses, IAV-S has genes encoded in eight RNA segments and a genomic replication mechanism that results in frequent mutations. These genetic characteristics allow IAV-S to quickly adapt, including avoiding existing neutralizing antibodies caused by exposure to previous strains. Thus, inactivated IAV-S vaccines that are commercially available on the US market, despite containing up to five different strains of IAV-S, have been found to be deficient due to new strains emerging as a result of continuous antigenic drift. .

Classification of influenza A viruses begins with the HA and NA subtypes, the two main glycoproteins on the surface of the virus. The HA protein mediates attachment and fusion of the virus with host cells. Antibodies that bind to HA block viral attachment, viral replication, and reduce or even prevent disease. Neuraminidase is an enzyme that functions in the final step of the influenza virus replication cycle, cleaving newly formed viral particles from the host cell, thereby allowing new progeny of the virus to spread and infect other cells. Antibodies that bind to NA limit its enzymatic activity and thus may reduce viral shedding and disease severity in certain animal models.

While human influenza A typically has one or two dominant strains circulating throughout the influenza season, many other strains of IAV-S circulate simultaneously, and these strains vary across geographic regions. IAV-S strains have antigenic variability but mostly contain the H1 or H3 HA subtype and the N1 or N2 NA subtype. Within each HA and NA IAV-S subtype, there is additional phylogenetic diversity. There are four predominant H1 phylogenetic clusters in the U.S. pig population (gamma,delta 1,delta 2, pandemic), two predominant H3 clusters (cluster IV and humanoid), two predominant N1 clusters (classic, pandemic), and two predominant N2 clusters (N2-1998 and N2-2002). [Cm.Andersonet al.,Flu and other Respiratory Viruses 7 (Suppl. 4); 42-51 (2013); And Andersonet al.,mSphere 1(6) e00275-16:1-14 (2016)]. There are three main H1 lineages in Europe (Eurasian bird-like H1, Scotland/410440/1994-like H1 and pandemic 2009-like H1), one main H3 lineage (Gent/1/1984-like H3), two main N1 lineages (Eurasian avian N1-like, pandemic 2009 N1-like), two major N2 lineages (Gent/1/1984-N2-like, Scotland/410440/1994-N2-like) and two minor N2 lineages (Italy/4675/2003 N2-like, human-like seasonal N2) [Watson et al., J. Virol., 89: 9920-9931 (2015); DOI: 10.1128/JVI.00840-15].

A number of vector-based strategies have been used over the years for vaccines to protect against certain pathogens. One such vector strategy involves the use of alpha virus (RP) derived RNA particle replicons [Vander Veen, et al. Anim Health Rev. 13(1):1-9. (2012) doi: 10, 1017/S1466252312000011; Kamrud et al., J Gen Virol., 91 (Pt 7): 1723-1727 (2010)], which were developed from several different alpha viruses, including Venezuelan equine encephalitis (VEE) virus [Pushko et al., Virology 239 : 389-401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of Virology 67: 6439-6446 (1993)], and Semliki Forest Virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9: 1356-1361 (1991)]. RP vaccines deliver replication-defective alphavirus RNA replicons to host cells, resulting in the expression of the desired antigenic transgene/transgenes in vivo [Pushko et al., Virology 239 (2): 389-401 (1997)]. RPs have attractive safety and efficacy profiles compared to some conventional vaccine formulations [Vander Veen, et al. Anim Health Rev. 13(1):1-9. (2012)]. The RP platform has been used to encode pathogenic antigens and is the basis for several USDA-licensed swine and poultry vaccines.

As noted above, commercial IAV-S vaccines available for use in domestic pigs often do not protect herds because the 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)]. The conventional platform of inactivated viral antigens is limited by regulatory and manufacturing factors that limit the ability to rapidly renew vaccine strains in response to ongoing viral antigenic drift. When using conventional inactivated IAV-S virus vaccines, the selection of viral strains is based on the properties of the HA antigens. Serological assessment of immune responses similarly focuses on the measurement of antibodies to this HA. The same emphasis on the HA antigen is also true for commercial inactivated influenza virus vaccines for humans as well as other animal species susceptible to influenza (eg, birds, horses, dogs). IAV-S vaccines that induce titers of antibodies that inhibit HA (HI) protect pigs from experimental infection with an antigenically similar strain [Kyriakis et al., Vet Microbiol 144 (1-2): 67-74 (2010)]. However, the relatively rapid genetic drift of HA genes allows the emergence of new strains that are not functionally inhibited by vaccine-induced anti-HA antibodies.

Clinical studies have shown a statistically significant correlation between NA-specific antibody titers and reduced influenza incidence in humans [Memoli et al., MBio 7 (2): e00417-16. (2016); Monto et al., J Infect Dis 212 (8): 1191-1199 (2015); Murphy et al., N Engl. J Med 286 (25): 1329-32 (1972)]. Various studies in inbred mice have shown that NA-specific immunity can protect against challenge [Brett and Johansson, Virology 339 (2): 273-80 (2005); Johansson and Kilbourne, Proc Natl Acad Sci USA 91 (6): 2358-2361 (1994); Kilbourne et al., J Infect Dis 189 (3): 459-461 (2004)]. Hessel et al. [PLoS One 5 (8), e12217 (2010)] showed that a vaccine containing a recombinant poxvirus vector encoding NA IAV-S could partially induce protection in mice against viral challenge, although the NA poxvirus vector was less effective than an equivalent vaccine. coding for the corresponding HA gene. However, it is important to note that the use of mice as an animal model for influenza cannot be considered a reliable indicator of vaccine efficacy because, unlike pigs, birds, and humans, mice do not naturally carry influenza A viruses [Bodewes et al., Expert Rev Vaccines 9 ( 1): 59-72 (2010); Tripp and Tompkins, Curr Top Microbiol Immunol 333: 397-412 (2009); Vander Veen et al., Vaccine 30 (11): 1944-1950 (2012)]. In fact, viral strains must first be adapted for growth in mice because this species is not at risk of infection with the influenza A virus. Thus, pathogenesis and morbidity are presented in terms of weight loss and mortality, which differ from the infection parameters used for the natural host. .

Similar studies were performed in chickens, the natural host species for influenza, using a viral vector vaccine with NA against a challenge infection. NA immunity in chickens only partially protected against infections and was significantly less robust than immunity induced by an equivalent anti-HA c vector vaccine [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)]. Similar results were obtained in another naturally susceptible influenza host, the ferret, which was vaccinated with soluble recombinant NA and/or HA proteins followed by virus challenge [Bosch et al., J Virol 84 (19): 10366-103674 (2010)]. Taken together, these data imply that NA-only immunity may play an additional and/or complementary role to the more important HA immunity. In fact, it appears that in the absence of an influenza A virus vaccine, hemagglutinin is not effective enough to protect against influenza A infection or influenza A virus disease.

One published study tested a pseudorabies virus (PrV) attenuated NA vector vaccine against viral challenge in pigs [Klingbeil et al., Virus Res 199: 20-30 (2015)]. Notably, incorporation of PrV-NA into PrV-HA did not provide a significant improvement over protection induced by PrV-HA alone. The data from this study, however, showed that PrV-NA vaccine, administered alone or with PrV-HA vaccine, induces serum antibodies to the NA protein. However, despite the production of antibodies, pigs vaccinated with NA showed a very modest decrease in the amount of viral replication (statistically significant in only one daily sample: 4 days after infection). As expected, pigs vaccinated exclusively with the PrV-HA vector induced anti-HV antibody responses, but these responses correlated with a greater reduction in virus shedding (statistically significant from day 2-6 post infection). In fact, there have been no published studies so far that indicate that an IAV-S vaccine containing and/or encoding the influenza A neuraminidase antigen in the absence of the corresponding hemagglutinin antigen can adequately protect the pig against IAV-S infection.

The citation of any reference in this specification should not be construed as an admission that such reference is available as "prior art" to the present application.

SUMMARY OF THE INVENTION

Thus, the present invention relates to vector and/or nucleic acid constructs that encode one or more influenza A virus neuraminidase (NA) antigens. These vector and/or nucleic acid constructs can be used in immunogenic compositions containing these vectors. The immunogenic compositions of the present invention can be used in vaccines that help protect a vaccinated individual (e.g., a human, pet, or livestock) from influenza A virus, for example, help prevent influenza A virus infection. In specific embodiments, the immunogenic compositions and vaccines of of the present invention contain one or more NAs that are derived from influenza A virus and contain neither influenza virus hemagglutinin (HA) or an antigenic fragment thereof, nor a nucleotide sequence encoding influenza A virus HA or antigenic fragment thereof.

In certain embodiments, vector and/or nucleic acid constructs are provided that encode one or more human influenza A virus NA antigens. In other embodiments, the vector and/or nucleic acid constructs encode one or more canine influenza A virus NA antigens. In other embodiments, The vectors and/or nucleic acid constructs encode one or more equine influenza A virus NA antigens. In still other embodiments, the vectors and/or nucleic acid constructs encode one or more avian influenza A virus NA antigens. or nucleic acids encode one or more bovine influenza A virus NA antigens. In specific embodiments, the vector and/or nucleic acid constructs encode two to four influenza virus NA antigens.

In specific embodiments, the vector is an alphavirus RNA replicon particle that encodes one or more antigens that are derived from the influenza A virus. In specific embodiments, the alphavirus replicon RNA particle encodes one or more influenza A virus NAs. In related embodiments, the replicon RNA particle alphavirus encodes one or more antigenic fragments of one or more influenza A virus NAs. The present invention also relates to other vector and/or nucleic acid constructs that encode one or more antigens derived from swine influenza A virus (IAV-S). In specific embodiments, the vector and/or nucleic acid constructs may also encode one or more IAV-S neuraminidase (NA) antigens. In related embodiments, the vector and/or nucleic acid constructs may encode one or more antigenic fragments from one or more IAV-S NAs.

In one important aspect, the present invention relates to constructs of vectors and/or nucleic acids that encode one or more neuraminidase (NA) antigens of influenza A viruses (IAV-S). Such vector and/or nucleic acid constructs can be used in immunogenic compositions containing these vectors. The immunogenic compositions of the present invention can be used in vaccines that help protect a vaccinated porcine individual (eg, sow and/or piglet) from IAV-S, for example, help prevent swine influenza virus infection. In specific embodiments, the immunogenic compositions and vaccines containing one or more NAs that are derived from IAV-S of the present invention contain neither the IAV-S HA nor its antigenic fragment nor the nucleotide sequence that encodes the IAV-S HA. or its antigenic fragment. The present invention further relates to combination vaccines for inducing protective immunity against IAV-S and other diseases, eg other swine infectious diseases. The invention also relates to methods for preparing and using the immunogenic compositions and vaccines of the present invention.

In more specific embodiments, the vector is an alphavirus RNA replicon particle that encodes one or more antigens derived from IAV-S. In specific embodiments, the alphavirus RNA replicon particle encodes one or more antigenic fragments of one or more IAV-S NAs. In related embodiments, the alphavirus RNA replicon particle encodes one or more antigenic fragments of one or more NAs that are derived from IAV-S. In specific embodiments, alphavirus RNA replicon particles encoding one or more NAs derived from IAV-S of the present invention contain neither IAV-S HA nor an antigenic fragment thereof, do not encode a nucleotide sequence that encodes IAV-S HA or its antigenic fragment.

In some embodiments, NA IAV-S is derived from the N1-classic phylogenetic cluster. In other embodiments, the alphavirus RNA replicon particle encodes an NA antigenic fragment that is derived from the N1-classical phylogenetic cassette. In other embodiments, NA IAV-S is derived from the N2-1998 phylogenetic cluster. In other embodiments, the alphavirus RNA replicon particle encodes an NA antigenic fragment that is derived from the N2-1998 phylogenetic cassette. In other embodiments, the NA IAV-S is from the N1-pandemic phylogenetic cluster. In other embodiments, the alphavirus RNA replicon particle encodes an NA antigenic fragment that is derived from the N1-pandemic phylogenetic cassette. In other embodiments, the NA IAV-S is from the N2-2002 phylogenetic cluster. In other embodiments, the alphavirus RNA replicon particle encodes an NA antigenic fragment that is derived from the N2-2002 phylogenetic cassette.

In a similar embodiment, the IAV-S NA is from - and/or the alphavirus RNA replicon particle encodes (antigenic fragment) NA from - N1-pandemic (EU) lineage. In a similar embodiment, the IAV-S NA is from - and/or the alphavirus RNA replicon particle encodes (antigenic fragment) NA from - N1-Eurasian Avian lineage.

In a similar embodiment, the IAV-S NA is derived from and/or the alphavirus RNA replicon particle encodes (antigenic fragment) NA from the N2-Gent/1984 lineage. In another similar embodiment, the IAV-S NA is derived from and/or the alphavirus RNA replicon particle encodes (antigenic fragment) NA from the N2-Italy/2003 lineage. In yet another similar embodiment, the IAV-S NA is from - and/or the alphavirus RNA replicon particle encodes (antigenic fragment) NA from the - N2-Scotland/1994 line. In some embodiments of this type, the IAV-S NA is derived from and/or the alphavirus RNA replicon particle encodes (antigenic fragment) NA from the N2-Scotland/1994 lineage (clade 1). In other embodiments of this type, the IAV-S NA is derived from and/or the alphavirus RNA replicon particle encodes (antigenic fragment) NA from the N2-Scotland/1994 lineage (clade 2). In other embodiments of this type, the IAV-S NA is from - and/or the alphavirus RNA replicon particle encodes (antigenic fragment) NA from - A/Swine/Italy/4675/2003, like N2. In other embodiments of this type, the IAV-S NA is from - and/or the alphavirus RNA replicon particle encodes (antigenic fragment) NA from - human influenza, seasonal type, like N2.

In specific embodiments, an IAV-S NA derived from the N1-classic phylogenetic cassette contains an amino acid sequence that has 95% identity, 97% identity, 98% identity or more with the amino acid sequence of SEQ ID NO: 2. In more specific embodiments, implementation this type of NA contains the amino acid sequence of SEQ ID NO: 2. In still more specific embodiments, this type of NA is encoded by the nucleotide sequence of SEQ ID NO: 1. In other embodiments, the NA IAV-S derived from the N1-pandemic phylogenetic cassette contains the amino acid sequence , which has 95% identity, 97% identity, 98% identity or more with the amino acid sequence of SEQ ID NO: 4. In more specific embodiments of this type, the NA contains the amino acid sequence of SEQ ID NO: 4. In even more specific embodiments of this type NA is encoded by the nucleotide sequence of SEQ ID NO: 3. In other embodiments, the IAV-S NA derived from the N2-1998 phylogenetic cassette contains an amino acid sequence that has 92% identity, 94% identity, 97% identity or more with the amino acid sequence SEQ ID NO: 6. In more specific embodiments, this type of NA comprises the amino acid sequence of SEQ ID NO: 6. In even more specific embodiments, this type of NA is encoded by the nucleotide sequence of SEQ ID NO: 5. In other embodiments, IAV-S NA , derived from the N2-2002 phylogenetic cluster, contains an amino acid sequence that has 92% identity, 94% identity, 97% identity or more with the amino acid sequence of SEQ ID NO: 8. In more specific embodiments, this type of NA contains the amino acid sequence of SEQ ID NO: 8. In even more specific embodiments of this type, NA is encoded by the nucleotide sequence of SEQ ID NO: 7.

In specific embodiments, the NA IAV-S derived from the N1-pandemic (EU) lineage contains an amino acid sequence that has 90% identity, 92% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 12. More specific embodiments of this type of NA comprise the amino acid sequence of SEQ ID NO: 12. In even more specific embodiments of this type, NA is encoded by the nucleotide sequence of SEQ ID NO: 11. In related embodiments of this type, NA is encoded by the nucleotide sequence of SEQ ID NO: 23. In in other embodiments, the NA IAV-S derived from the N1 Eurasian Avian lineage contains an amino acid sequence that has 85% identity, 90% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 14. In more specific embodiments of this type NA contains the amino acid sequence of SEQ ID NO: 14. In even more specific embodiments, this type NA is encoded by the nucleotide sequence of SEQ ID NO: 13. In other embodiments, IAV-S NA derived from the N2-Gent/1984 lineage contains the amino acid sequence , which has 90% identity, 92% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 16. In more specific embodiments of this type, the NA contains the amino acid sequence of SEQ ID NO: 16. In even more specific embodiments of this type NA is encoded by the nucleotide sequence of SEQ ID NO: 15. In related embodiments of this type, NA is encoded by the nucleotide sequence of SEQ ID NO: 24. In other embodiments, IAV-S NA derived from the N2-Italy/2003 line contains an amino acid sequence that has 90% identity, 92% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 18. In more specific embodiments, this type of NA contains the amino acid sequence of SEQ ID NO: 18. In even more specific embodiments, this type of NA is encoded nucleotide sequence of SEQ ID NO: 17. In related embodiments of this type, NA is encoded by the nucleotide sequence of SEQ ID NO: 25. In other embodiments, NA IAV-S derived from the N2-Scotland/1994 line (clade 1) includes a sequence that has 90% identity, 92% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 20. In more specific embodiments of this type, the NA comprises the amino acid sequence of SEQ ID NO: 20. In even more specific embodiments of this type NA is encoded by the nucleotide sequence of SEQ ID NO: 19. In related embodiments of this type, NA is encoded by the nucleotide sequence of SEQ ID NO: 26. In other embodiments, IAV-S NA derived from the N2-Scotland/1994 (clade 2) line contains an amino acid sequence that shares 90% identity, 92% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 22. In more specific embodiments of this type, the NA comprises the amino acid sequence of SEQ ID NO: 22. In still more specific embodiments, the implementation this type of NA is encoded by the nucleotide sequence of SEQ ID NO: 21. In related embodiments, this type of NA is encoded by the nucleotide sequence of SEQ ID NO: 27.

In still more specific embodiments, the alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEE) alphavirus replicon particle. In yet more specific embodiments, the VEE alphavirus RNA replicon particle is a TC-83 VEE alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is an alpha Sindbis virus (SIN) replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Semliki Forest alphavirus (SFV) RNA replicon particle. In an alternative embodiment, the naked DNA vector contains a nucleic acid construct that encodes one or more antigens derived from a porcine pathogen. In specific embodiments of this type, the naked DNA vector contains a nucleic acid construct that encodes an NA derived from IAV-S or an antigenic fragment thereof. In other embodiments, the naked RNA vector contains a nucleic acid construct that encodes one or more antigens derived from a porcine pathogen. In particular embodiments of this type, the naked vector contains a nucleic acid construct that encodes an NA derived from IAV-S or an antigenic fragment thereof.

The present invention relates to alphavirus RNA replicon particles that encode two or more IAV-S antigens or antigenic fragments thereof. In specific embodiments, alphavirus RNA replicon particles encoding one or more NAs that are derived from IAV-S of the present invention contain neither IAV-S HA nor an antigenic fragment thereof, nor encode a nucleotide sequence that encodes for IAV-S HA or its antigenic fragment.

In specific embodiments, the alphavirus RNA replicon particles encode two to four or more NA antigens that are derived from IAV-S, or antigenic fragments thereof. In related embodiments, the alphavirus RNA replicon particles encode two to four or more NA antigens that originate from different IAV-S phylogenetic cassettes, or antigenic fragments thereof. In some embodiments, the alphavirus RNA replicon particle encodes an NA derived from the N1-classic phylogenetic cassette, or an antigenic fragment thereof, and an NA derived from the N2-2002 phylogenetic cassette, or an antigenic fragment thereof. In other embodiments, the alphavirus RNA replicon particle encodes an NA derived from the N1-classic phylogenetic cassette, or an antigenic fragment thereof, and an NA derived from the N2-1998 phylogenetic cassette, or an antigenic fragment thereof. In other embodiments, the alphavirus RNA replicon particle encodes an NA derived from the N1-classic phylogenetic cassette, or an antigenic fragment thereof, and an NA derived from the N1-pandemic phylogenetic cassette, or an antigenic fragment thereof.

In some embodiments, the alphavirus RNA replicon particles encode NA derived from the N1-pandemic (EU) lineage, or an antigenic fragment thereof, and NA derived from the N1-Eurasian Avian lineage, or antigenic fragment thereof. In other embodiments, the alphavirus RNA replicon particles encode NA derived from the N2-Gent/1984 lineage, or an antigenic fragment thereof, and NA derived from the N2-Italy/2003 lineage, or antigenic fragment thereof. In other embodiments, the alphavirus RNA replicon particles encode NA derived from the N2-Gent/1984 lineage or an antigenic fragment thereof and NA derived from the N2-Scotland/1994 lineage (clade 1) or an antigenic fragment thereof. In other embodiments, the alphavirus RNA replicon particles encode NA derived from the N2-Gent/1984 lineage or an antigenic fragment thereof and NA derived from the N2-Scotland/1994 lineage (clade 2) or an antigenic fragment thereof. In other embodiments, the alphavirus RNA replicon particles encode NA derived from the N2-Italy/2003 lineage or an antigenic fragment thereof and NA derived from the N2-Scotland/1994 lineage (clade 1) or antigenic fragment thereof. In other embodiments, the alphavirus RNA replicon particles encode NA derived from the N2-Italy/2003 lineage or an antigenic fragment thereof and NA derived from the N2-Scotland/1994 (clade 2) lineage or antigenic fragment thereof. In other embodiments, the NA derived from one of the European N1 lineages, or an antigenic fragment thereof, is encoded by the alphavirus RNA replicon particle, along with the NA derived from one of the European N2 lineages, or an antigenic fragment thereof. Similarly, the present invention relates to alphavirus RNA replicon particles encoding three, four or more NAs, each NA being from a different European lineage. In addition, the present invention relates to alphavirus RNA replicon particles encoding two, three, four or more NAs, each NA being from a different European lineage and/or phylogenetic cluster.

Thus, the present invention further relates to alphavirus RNA replicon particles that encode three or four IAV-S antigens or fragments thereof. In specific embodiments, the immunogenic compositions comprise alphavirus RNA replicon particles that encode three or four IAV-S-derived NA antigens or antigenic fragments thereof. In related embodiments, the immunogenic compositions comprise alphavirus RNA replicon particles that encode three or four NA antigens that originate from different IAV-S phylogenetic cassettes, or antigenic fragments thereof. In some embodiments, the alphavirus RNA replicon particle encodes NA derived from the N1-classic phylogenetic cassette or an antigenic fragment thereof, NA derived from the N2-2002 phylogenetic cassette or antigenic fragment thereof, and NA derived from the N2-1998 phylogenetic cassette, or its antigenic fragment. In other embodiments, the alphavirus RNA replicon particle encodes NA derived from the N1-classic phylogenetic cassette or an antigenic fragment thereof, NA derived from the N2-2002 phylogenetic cassette or antigenic fragment thereof, and NA derived from the N1-pandemic phylogenetic cassette. or its antigenic fragment. In other embodiments, the alphavirus RNA replicon particle encodes NA derived from the N1-classic phylogenetic cassette or an antigenic fragment thereof, NA derived from the N2-1998 phylogenetic cassette or an antigenic fragment thereof, and NA derived from the N1-pandemic phylogenetic cassette. or its antigenic fragment. In other embodiments, the alphavirus RNA replicon particle encodes NA derived from the N2-2002 phylogenetic cassette or an antigenic fragment thereof, NA derived from the N2-1998 phylogenetic cassette or an antigenic fragment thereof, and NA derived from the N1-pandemic phylogenetic cassette. or its antigenic fragment.

In specific embodiments, the alphavirus RNA replicon particles encode four or more IAV-S NA antigens or antigenic fragments thereof. In more specific embodiments, the alphavirus RNA replicon particles encode four or more NA antigens that originate from different IAV-S phylogenetic cassettes, or antigenic fragments thereof. In still more specific embodiments, the alphavirus RNA replicon particle encodes NA derived from the N2-2002 phylogenetic cassette, or an antigenic fragment thereof, NA derived from the N2-1998 phylogenetic cassette, or an antigenic fragment thereof, NA derived from the N1-pandemic phylogenetic cassette. , or its antigenic fragment, and NA derived from the N1-classical phylogenetic cluster, or its antigenic fragment.

In analogous embodiments as above, the alphavirus RNA replicon particle encodes three, four or more NA IAV-S antigens or antigenic fragments derived from the N1-pandemic (EU) IAV lineage, the N1-Eurasian Avian lineage, the NA lineage, the N2 lineage -Gent/1984, lines N2-Italy/2003, lines N2-Scotland/1994 (clade 1) and/or lines N2-Scotland/1994 (clade 2).

As indicated above, all alphavirus RNA replicon particles of the present invention may be components of immunogenic compositions and/or vaccines. Thus, the immunogenic composition and/or vaccine of the present invention may comprise one or more alphavirus RNA replicon particles of the present invention. In some embodiments, the immunogenic composition and/or vaccine comprises only one set of identical alphavirus RNA replicon particles that may encode one or more NAs derived from one or more phylogenetic cassettes and/or one or more lineages, as detailed above.

In a more specific embodiment of this type, the immunogenic composition and/or vaccine comprises replicon RNA particles that encode both NA (or antigenic fragment thereof) derived from the N1-phylogenetic cluster and NA (or antigenic fragment thereof) derived from N2- phylogenetic cluster. In particular, the immunogenic composition and/or vaccine contains an RNA replicon, where the first NA is an N1 phylogenetic cassette selected from the group of N1-classic and N1-pandemic phylogenetic cassettes, and the second NA is an N2 phylogenetic cassette selected from the group, consisting of the phylogenetic cluster N2-1998 and N2-2002.

In further specific embodiments of this type, the immunogenic composition and/or vaccine comprises a replicon RNA, wherein the first NA has an N1 lineage selected from the group consisting of the N1-pandemic (EU) and N1-Eurasian Avian lineage, and the second NA has an N2- phylogenetic lineage selected from the group consisting of N2-Gent/1984, N2-Italy/2003, N2-Scotland 1994 (clade 1) and N2-Scotland 1994 (clade 2) lines.

The present invention further relates to immunogenic compositions and/or vaccines that contain two or more sets of such alphavirus RNA replicon particles. In specific embodiments of this type, one set of alphavirus RNA replicon particles encodes one or more NAs derived from the same phylogenetic cassette and/or lineage, while another set of alphavirus RNA replicon particles encodes one or more NAs derived from a different phylogenetic cassette and/or lineage. lines.

In specific embodiments of this type, the first set of alphavirus RNA replicon particles encode one or more NA antigens that are derived from the N1 phylogenetic cluster and/or lineage or antigenic fragments thereof, and the second set of alphavirus RNA replicon particles encode one or more NA antigens. , which originate from the N2-phylogenetic cluster and/or lineage, or its antigenic fragments. In particular, in the first set, the NA antigen (or fragment thereof) is an N1 phylogenetic cluster selected from the groups consisting of the N1 classic and N1 pandemic phylogenetic cluster, and in the second set, the NA antigen (or antigenic fragment thereof) is is an N2 phylogenetic cluster selected from the group consisting of the N2-1998 and N2-2002 phylogenetic cluster.

In a similar parallel embodiment, in the first set, the NA antigen (or fragment thereof) belongs to an N1 phylogenetic lineage selected from the group consisting of the N1 pandemic (EU) and N1 Eurasian avian lineages, and in the second set, the NA antigen (or its antigenic fragment) belongs to the N2 lineage selected from the group consisting of the N2-Gent/1984, N2-Italy/2003, N2-Scotland 1994 (clade 1) and N2-Scotland 1994 (clade 2) lines.

In other embodiments, the immunogenic composition and/or vaccine comprises one set of alphavirus RNA replicon particles that encode a first antigen, another set of alphavirus RNA replicon particles that encode a second antigen, and a third set of alphavirus RNA replicon particles that encode a third antigen. More specifically, in the immunogenic composition and/or vaccine, the antigens are either NA phylogenetic cluster antigens selected from the groups consisting of the N1-classic, N1-pandemic-, N2-1998- and N2-2002 phylogenetic cluster, or they are NA antigens. from a lineage selected from a group consisting of N1-pandemic (EU), N1-Eurasian Avian, N2-Gent/1984, N2-Italy/2003, N2-Scotland 1994 (clade 1), and N2-Scotland 1994 (clade 2) ).

In particular embodiments of this type, the first set of alphavirus RNA replicon particles encodes an NA derived from the N1-classical phylogenetic cluster, or an antigenic fragment thereof; the second set of alphavirus RNA replicon particles encodes NA derived from the N2-2002 phylogenetic cluster, or an antigenic fragment thereof; and a third set of alphavirus RNA replicon particles encodes NA derived from the N2-1998 phylogenetic cluster, or an antigenic fragment thereof.

In other embodiments, the immunogenic composition and/or vaccine comprises one set of alphavirus RNA replicon particles that encode a first antigen, another set of alphavirus RNA replicon particles that encode a second antigen, a third set of alphavirus RNA replicon particles that encode a third antigen, and a fourth set alphavirus RNA replicon particles that encode a fourth antigen.

More specifically, in the immunogenic composition and/or vaccine, the antigens are either phylogenetic cluster NA antigens selected from the groups consisting of the N1 classic phylogenetic cluster, the N1 pandemic phylogenetic cluster, the N2-1998 phylogenetic cluster, and the N2-2002 phylogenetic cluster, or they are antigens of an NA lineage selected from the group consisting of the N1-pandemic (EU) lineage, the N1-Eurasian Avian lineage, the N2-Gent/1984 lineage, the N2-Italy/2003 lineage, the N2-Scotland 1994 lineage (clade 1), and lines N2-Scotland 1994 (clade 2).

In particular embodiments of this type, the first set of alphavirus RNA replicon particles encodes an NA derived from the classical N1 phylogenetic cluster, or an antigenic fragment thereof; the second set of alphavirus RNA replicon particles encodes NA derived from the N2-2002 phylogenetic cluster, or an antigenic fragment thereof; a third set of alphavirus RNA replicon particles encodes NA derived from the N2 1998 phylogenetic cluster, or an antigenic fragment thereof; and a fourth set of alphavirus RNA replicon particles encodes NA derived from the N1 pandemic phylogenetic cluster or an antigenic fragment thereof.

Thus, the immunogenic compositions/vaccines comprise a plurality of sets (eg 2-10) of alphavirus RNA replicon particles. In particular embodiments of this type, one or more kits comprise alphavirus RNA replicon particles that encode one or more IAV-S NAs derived from one or more phylogenetic clusters (or antigenic fragments thereof) or lineages (or antigenic fragments thereof). In particular embodiments of this type, the immunogenic composition comprises one or more sets of alphavirus RNA replicon particles that encode one or more IAV-S NAs derived from one or more phylogenetic clusters (or antigenic fragments thereof) or lineages (or antigenic fragments thereof), in combination with one or more sets of alphavirus RNA replicon particles that encode one or more non-IAV-S antigens or antigenic fragments thereof. In still more specific embodiments, one or more sets of alphavirus RNA replicon particles encode both one or more IAV-S NAs derived from one or more phylogenetic clusters (or antigenic fragments thereof) or lineages (or antigenic fragments thereof) and encode one or more non-IAV-S antigens or antigenic fragments thereof. In specific embodiments of this type, the alphavirus RNA replicon particle encodes NA derived from the classical N1 phylogenetic cassette or an antigenic fragment thereof, and/or NA derived from the N2-2002 phylogenetic cassette or antigenic fragment thereof, along with one to three antigens or an antigenic fragment thereof. antigenic fragments that are not related to IAV-S.

In specific embodiments, the non-IAV-S antigen is from porcine reproductive and respiratory syndrome (PRRS) virus. In other embodiments, the non-IAV-S antigen is from porcine circovirus (PCV). In other embodiments, the non-IAV-S antigen is from a transmissible gastroenteritis virus (TGE). In other embodiments, the non-IAV-S antigen is from porcine pseudorabies virus (PPRV). In other embodiments, the non-IAV-S antigen is from porcine parvovirus (PPV). In other embodiments, the non-IAV-S antigen is from porcine rotavirus (PRV). In other embodiments, the non-IAV-S antigen is from porcine epidemic diarrhea (PED). In other embodiments, one or more non-IAV-S antigens are from one or more Pasteurella multocida serotypes. In other embodiments, one or more non-IAV-S antigens are from one or more Salmonella ssp serotypes. In other embodiments, the one or more non-IAV-S antigens are from one or more Escherichia coli serotypes. In other embodiments, one or more non-IAV-S antigens are derived from one or more serotypes of Haemophilus parasuis . In other embodiments, the non-IAV-S antigen is from Lawsonia intracellularis . In other embodiments, the non-IAV-S antigen is from Mycoplasma ssp (eg, Mycoplasma hyopneumoniae ). In other embodiments, the non-IAV-S antigen is from Bordetella bronchiseptica . In other embodiments, the non-IAV-S antigen is from Erysipelas ssp . In other embodiments, the non-IAV-S antigen is from Campylobacter ssp . In other embodiments, the non-IAV-S antigen is from Actinobacillus pleuropneumoniae . In other embodiments, the non-IAV-S antigen is from Clostridium perfringens . In other embodiments, the non-IAV-S antigen is from Clostridium difficile .

In more specific embodiments, the alphavirus RNA replicon particles are Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles. In still more specific embodiments, the VEE alphavirus RNA replicon particles are TNK-83 alphavirus VEE RNA replicon particles.

The present invention further relates to combined immunogenic compositions and/or vaccines (multivalent vaccines) which comprise alphavirus RNA replicon particles that encode one or more NAs or antigenic fragments thereof derived from IAV-S as defined above and further comprise one or more modified live/attenuated or killed porcine pathogens. In specific embodiments, the immunogenic compositions comprise one or more modified live/attenuated and/or killed PRRS viruses. In other embodiments, the immunogenic compositions comprise one or more modified live/attenuated and/or killed PCV. In other embodiments, the immunogenic compositions further comprise one or more modified live/attenuated and/or killed TGEs. In other embodiments, the immunogenic compositions comprise one or more modified live/attenuated and/or killed PPRVs. In other embodiments, the immunogenic compositions further comprise one or more modified live/attenuated and/or killed PPVs. In other embodiments, the immunogenic compositions comprise one or more modified live/attenuated and/or killed PRVs. In other embodiments, the immunogenic compositions further comprise one or more modified live/attenuated and/or killed PEDs. In other embodiments, the immunogenic compositions comprise one or more modified live/attenuated and/or killed Pasteurella multocida serotypes. In other embodiments, the immunogenic compositions further comprise one or more modified live/attenuated and/or killed Salmonella ssp serotypes. In other embodiments, the immunogenic compositions comprise one or more modified live/attenuated and/or killed Escherichia coli serotypes. In other embodiments, the immunogenic compositions further comprise one or more modified live/attenuated and/or killed Haemophilus parasuis . In other embodiments, the immunogenic compositions comprise one or more modified live/attenuated and/or killed Lawsonia intracellularis . In other embodiments, the immunogenic compositions further comprise one or more modified live/attenuated and/or killed Mycoplasma ssp . (e.g. Mycoplasma hyopneumoniae ). In other embodiments, the immunogenic compositions also contain one or more modified live/attenuated and/or killed Bordetella bronchiseptica . In other embodiments, the immunogenic compositions further comprise one or more modified live/attenuated and/or killed Erysipelas ssp . In other embodiments, the immunogenic compositions comprise one or more modified live/attenuated and/or killed Campylobacter ssp . In other embodiments, the immunogenic compositions further comprise one or more modified live/attenuated and/or killed Actinobacillus pleuropneumoniae . In other embodiments, the immunogenic compositions comprise one or more modified live/attenuated and/or killed Clostridium perfringens . In other embodiments, the immunogenic compositions further comprise one or more modified live/attenuated and/or killed Clostridium difficile .

In specific embodiments, the nucleic acid construct of the present invention encodes one or more NA antigens derived from IAV-S phylogenetic cassettes, or antigenic fragments thereof. Thus, while alphavirus RNA replicon particles are specifically exemplified below, it should be understood that the present invention further includes other vector and/or nucleic acid constructs.

Thus, in some embodiments, the nucleic acid construct encodes an IAV-S NA that is derived from the N1-classical phylogenetic cluster. In other embodiments, the nucleic acid construct encodes an NA antigenic fragment that is derived from the N1-classical phylogenetic cluster. In other embodiments, NA IAV-S is derived from the N2-1998 phylogenetic cluster. In other embodiments, the alphavirus RNA replicon particle encodes an NA antigenic fragment that is derived from the N2-1998 phylogenetic cassette. In other embodiments, the NA IAV-S is from the N1-pandemic phylogenetic cluster. In other embodiments, the alphavirus RNA replicon particle encodes an NA antigenic fragment that is derived from the N1-pandemic phylogenetic cassette. In other embodiments, the NA IAV-S is from the N2-2002 phylogenetic cluster. In yet another embodiment, the nucleic acid construct encodes an NA antigenic fragment that is derived from the N2-2002 phylogenetic cassette.

In a similar embodiment, NA IAV-S is derived from NA from the N1-pandemic (EU) lineage. In a similar embodiment, NA IAV-S is derived from NA from the N1 Eurasian Avian lineage. In a similar embodiment, NA IAV-S is derived from NA from the N2-Gent/1984 lineage. In another similar embodiment, NA IAV-S is from the N2-Italy/2003 line. In another similar embodiment, NA IAV-S is from the N2-Scotland/1994 line. In some embodiments of this type, the NA IAV-S is from the N2-Scotland/1994 lineage (clade 1). In other embodiments of this type, the NA IAV-S is from the N2-Scotland/1994 lineage (clade 2).

In specific embodiments, an IAV-S NA derived from the N1-classic phylogenetic cassette contains an amino acid sequence that has 95% identity, 97% identity, 98% identity or more with the amino acid sequence of SEQ ID NO: 2. In more specific embodiments, implementation this type of NA contains the amino acid sequence of SEQ ID NO: 2. In other embodiments, an IAV-S NA derived from the N1-pandemic phylogenetic cassette contains an amino acid sequence that has 95% identity, 97% identity, 98% identity or more with the amino acid sequence of SEQ ID NO: 4. In more specific embodiments of this type, the NA contains the amino acid sequence of SEQ ID NO: 4. In other embodiments, the NA IAV-S derived from the N2-1998 phylogenetic cassette contains an amino acid sequence that has 92% identity , 94% identity, 97% identity or more with the amino acid sequence of SEQ ID NO: 6. In more specific embodiments of this type, the NA comprises the amino acid sequence of SEQ ID NO: 6. In other embodiments, the NA IAV-S derived from the N2 phylogenetic cassette -2002 contains an amino acid sequence that has 92% identity, 94% identity, 97% identity or more with the amino acid sequence of SEQ ID NO: 8. In more specific embodiments of this type, NA contains the amino acid sequence of SEQ ID NO: 8.

In specific embodiments, the NA IAV-S derived from the N1-pandemic (EU) lineage comprises an amino acid sequence that has 90% identity, 92% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 12. More specific embodiments of this type, the NA comprises the amino acid sequence of SEQ ID NO: 12. In other embodiments, the IAV-S NA derived from the N1-Eurasian Avian lineage contains an amino acid sequence that is 85% identity, 90% identity, 95% identity, or more with the amino acid sequence of SEQ ID NO: 14. In more specific embodiments, this type of NA comprises the amino acid sequence of SEQ ID NO: 14. In other embodiments, the NA IAV-S derived from the N2-Gent/1984 line includes an amino acid sequence that has 90% identity, 92% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 16. In more specific embodiments of this type, the NA comprises the amino acid sequence of SEQ ID NO: 16. In other embodiments, the IAV-S NA derived from line N2-Italy/2003, contains an amino acid sequence that has 90% identity, 92% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 18. In more specific embodiments, this type of NA contains the amino acid sequence of SEQ ID NO: 18. In other embodiments, the NA IAV-S derived from the N2-Scotland/1994 lineage (clade 1) comprises an amino acid sequence that has 90% identity, 92% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 20. In more specific embodiments of this type, the NA comprises the amino acid sequence of SEQ ID NO: 20. In other embodiments, the NA IAV-S derived from the N2-Scotland /1994 lineage (clade 2) comprises an amino acid sequence that has 90% identity, 92% identity, 95% identity or more with the amino acid sequence of SEQ ID NO: 22. In more specific embodiments of this type, the NA contains the amino acid sequence of SEQ ID NO: 22.

The present invention relates to nucleic acid constructs that encode two or more IAV-S antigens or antigenic fragments thereof. In specific embodiments, the nucleic acid constructs encode two to four or more NA antigens that are derived from IAV-S, or antigenic fragments thereof. In related embodiments, the nucleic acid constructs encode two to four or more NA antigens that originate from different IAV-S phylogenetic cassettes, or antigenic fragments thereof. In some embodiments, the nucleic acid constructs encode an NA derived from the N1-classical phylogenetic cassette, or an antigenic fragment thereof, and an NA derived from the N2-2002 phylogenetic cassette, or an antigenic fragment thereof. In even more specific embodiments, the nucleic acid constructs encode NA derived from the N2-2002 phylogenetic cassette, or an antigenic fragment thereof, NA derived from the N2-1998 phylogenetic cassette, or an antigenic fragment thereof, NA derived from the N1-pandemic phylogenetic cassette. or an antigenic fragment thereof, and an NA derived from the N1-classical phylogenetic cluster, or an antigenic fragment thereof.

In some embodiments, the nucleic acid constructs encode an NA derived from the N1-pandemic (EC) lineage, or an antigenic fragment thereof, and an NA derived from the N1-Eurasian Avian lineage, or an antigenic fragment thereof. In other embodiments, the nucleic acid constructs encode an NA derived from the N2-Gent/1984 lineage, or an antigenic fragment thereof, and an NA derived from the N2-Italy/2003 lineage, or an antigenic fragment thereof. In other embodiments, the nucleic acid constructs encode an NA derived from the N2-Gent/1984 lineage, or an antigenic fragment thereof, and an NA derived from the N2 Scotland/1994 lineage (clade 1), or an antigenic fragment thereof. In other embodiments, the nucleic acid constructs encode an NA derived from the N2-Gent/1984 lineage or an antigenic fragment thereof and an NA derived from the N2-Scotland/1994 lineage (clade 2) or an antigenic fragment thereof. In other embodiments, the nucleic acid constructs encode an NA derived from the N2-Italy/2003 lineage, or an antigenic fragment thereof, and an NA derived from the N2-Scotland/1994 lineage (clade 1), or an antigenic fragment thereof. In other embodiments, the nucleic acid constructs encode an NA derived from the N2-Italy/2003 lineage, or an antigenic fragment thereof, and an NA derived from the N2 Scotland/1994 lineage (clade 2), or an antigenic fragment thereof. In other embodiments, the NA derived from one of the European N1 lineages, or an antigenic fragment thereof, is encoded in the same nucleic acid construct as the NA derived from one of the European N2 lineages, or an antigenic fragment thereof. Similarly, the present invention relates to nucleic acid constructs encoding three, four or more NAs, each NA being from a different European lineage. In addition, the present invention relates to nucleic acid constructs encoding two, three, four or more NAs, with each NA derived from different European lineages and/or phylogenetic clusters.

Thus, the present invention relates to all alphavirus RNA replicon particles of the present invention, naked DNA vectors that contain the nucleic acid constructs of the present invention, naked RNA vectors that contain the nucleic acid constructs of the present invention, to the constructs nucleic acids of the present invention, including synthetic mRNA and RNA replicons, as well as all immunogenic compositions and/or vaccines that contain nucleic acid constructs (e.g. synthetic mRNA, RNA replicons), alphavirus RNA replicon particles, naked vectors RNA and/or "naked" DNA according to the present invention.

The present invention further includes vaccines (multivalent) vaccines comprising the immunogenic compositions of the present invention. In specific embodiments, the implementation of the vaccine is a non-adjuvanted vaccine. In other embodiments, the vaccines contain an adjuvant. In specific embodiments, the implementation of the adjuvant is a biodegradable oil. In specific embodiments of this type, the biodegradable oil is dl-α-tocopheryl acetate (vitamin E acetate). In other embodiments, the adjuvant comprises an oil-in-water emulsion with 2.5%-50% (v/v) mineral oil. In specific embodiments, the adjuvant comprises an oil-in-water emulsion with 2.5% (v/v) mineral oil. In related embodiments, the adjuvant comprises an oil-in-water emulsion with 5% (v/v) mineral oil. In other embodiments, the adjuvant comprises an oil-in-water emulsion with 12.5% (v/v) mineral oil. In other embodiments, the adjuvant comprises an oil-in-water emulsion with 25% (v/v) mineral oil. In other embodiments, the adjuvant comprises an oil-in-water emulsion with 50% (v/v) mineral oil. In more specific embodiments, the adjuvant comprises a mixture of a biodegradable oil with a mineral oil adjuvant. In specific embodiments, the biodegradable oil is dl-α-tocopheryl acetate and the mineral oil is paraffin oil. In more specific embodiments, the biodegradable oil is dl-α-tocopheryl acetate and the mineral oil is a light paraffin oil.

In some embodiments, the implementation of the vaccine helps in the prevention of morbidity caused by IAV-S. In related embodiments, antibodies are induced in a porcine individual when the porcine is immunized with a vaccine. In some embodiments, the pig is a sow. In related embodiments, the vaccine provides protective maternal antibodies to the offspring of the vaccinated sow. In other embodiments, the porcine individual is a piglet. In specific embodiments of this type, the vaccine is administered to the piglet as early as three days of age. In specific embodiments, the vaccine is administered as a booster vaccine. In some embodiments, the implementation of the vaccine is administered as a single dose of the vaccine. In specific embodiments of this type, the vaccine is administered as a booster vaccine. In other embodiments, the vaccine is administered as a multi-dose vaccine. In specific embodiments of this type, the vaccine is administered as a two-dose vaccine.

The present invention also relates to a method of immunizing a pig (eg, a sow or piglet) against a porcine pathogen, for example IAV-S, comprising administering to the pig an immunologically effective amount of the vaccine or polyvalent vaccine of the present invention. In specific embodiments, the implementation of the vaccine is administered by intramuscular injection. In alternative embodiments, the vaccine is administered by subcutaneous injection. In other embodiments, the implementation of the vaccine is administered by intravenous injection. In other embodiments, the implementation of the vaccine is administered by intradermal injection. In other embodiments, the implementation of the vaccine is administered through oral administration. In other embodiments, the implementation of the vaccine is administered via intranasal administration.

Thus, vaccines and polyvalent vaccines can be administered as a primer vaccine and/or as a booster vaccine. In specific embodiments, the implementation of the vaccine of the present invention is administered as a single dose vaccine (single dose), not requiring subsequent administration. In some embodiments, when both a primer vaccine and a booster vaccine are administered, the primer vaccine and the booster vaccine can be administered in the same way. In some embodiments of this type, the primer vaccine and the booster vaccine are administered by the intradermal route. In other embodiments of this type, the primer vaccine and the booster vaccine are administered intramuscularly. In alternative embodiments, when both a primer vaccine and a booster vaccine are administered, the primer vaccine may be administered one way and the booster vaccine the other. In some 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 other embodiments of this type, the primer vaccine may be administered by intramuscular injection and the booster vaccine may be administered by intramuscular injection.

The invention further relates to a method for immunizing a pig (eg, a sow or piglet) against iav-s, comprising injecting the pig with an immunologically effective amount of the vaccines of the invention described above. In specific embodiments, the implementation of the vaccine may include from about 1x10 ⁴ to about 1x10 ¹⁰ rp or higher, for example. In more specific embodiments, the implementation of the vaccine may include from about 1x10 ⁵ to about 1x10 ⁹ rp. In even more specific embodiments, the implementation of the vaccine may include from about 1x10 ⁶ to about 1x10 ⁸ rp.

In specific embodiments, the implementation of the vaccines of the present invention is administered in doses from 0.05 to 3 ml. In more specific embodiments, the implementation of the administered dose is from 0.1 ml to 2 ml. In even more specific embodiments, the administered dose is from 0.2 ml to 1.5 ml. In even more specific embodiments, the administered dose is from 0.3 to 1.0 ml. In even more specific embodiments, the administered dose is between 0.4 ml and 0.8 ml.

These and other aspects of the present invention will be better understood with reference to the following figures and detailed description.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1D show serum neuraminidase (NI) inhibitory antibody responses specific for all four NA strains from the vaccine compositions described in Example 1 below. Serum samples are collected before the primary vaccination (age 3 weeks), before the second vaccination (age 6 weeks) and before challenge (age 9 weeks).

Figures 2A and 2B depict lung injury scores in pigs following administration of the vaccine composition described in Example 1 below and challenge with (A) H1-gamma-N1-classic or (B) H1-delta1-N2-2002A virus infection.

Figures 3A and 3B show viral titers in nasal swabs of pigs following administration of the vaccine composition described in Example 1 below and challenge with (A) H1-gamma-N1-classic or (B) H1-delta1-N2-2002A viral infection.

Figures 4A and 4B show viral titers in swine bronchoalveolar lavage fluid samples following administration of the vaccine compositions described in Example 1 below and challenge with (A) H1-gamma-N1-classic or (B) H1-delta1-N2-2002A viral infection.

Figure 5 shows the responses of serum neuraminidase (NI) inhibitory antibodies specific for the N1-classic strain from the vaccine compositions described in Example 2. Serum samples were collected before the first vaccination (age 3 weeks), before the second vaccination (age 7 weeks), and before infection (age 10 weeks).

Figure 6 shows lung injury scores in pigs after administration of the vaccine composition described in Example 2 and after challenge with H1-gamma-N1-classic virus.

Figure 7 shows the percentage of macroscopic lesion of the lung on the fifth day after infection with the H1N2 virus.

Figure 8 depicts nasal excretion of the pathogen after infection with the H1N2 virus.

The figure 9 shows the titer of inhibition of neuraminidase (NI) for vaccine fraction N ₂₂₀₀₂ after vaccination. Note: DPV=days post-vaccination.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to vaccines and immunogenic compositions that comprise an immunologically effective amount of one or more alphavirus RNA replicon particles that encode one or more neuraminidase (NA) from one or more influenza A virus serotypes. In one aspect of the present invention, vaccines and immunogenic compositions contain neither hemagglutinin (HA) or its antigenic fragment, nor the nucleotide sequence that encodes HA or its antigenic fragment. One advantage of the influenza A vaccine based on multiple NAs and the absence of HAs is that NAs are less affected by antigenic drift than HAs, allowing appropriate vaccines to contain antigens from fewer influenza isolates. This saves manufacturing costs and also increases the time it takes to renew a given influenza vaccine. In addition, since currently marketed influenza A virus vaccines are based on inactivated influenza viruses that generate much higher titers of anti-HA antibodies than anti-NA antibodies in individual animals, the negative impact of maternal antibodies on immunization of the offspring of individual animals should be significantly reduced for an NA-based vaccine.

In one important aspect of the present invention, vaccines and immunogenic compositions are provided that comprise an immunologically effective amount of one or more alphavirus RNA replicon particles that encode one or more neuraminidase (NA) from one or more swine A virus (IAV) phylogenetic cassettes and/or lineages. -S). In one aspect of the present invention, the vaccines and immunogenic compositions contain neither the IAV-S hemagglutinin (HA) or antigenic fragment thereof, nor the nucleotide sequence that encodes the IAV-S HA or antigenic fragment thereof.

As stated above, the IAV-S vaccine, based on few NAs and no HA, allows the respective vaccines to contain antigens from fewer IAV-S isolates. This saves manufacturing costs and also increases the time required to update a given IAV-S vaccine. In addition, since currently commercially available IAV-S vaccines are based on inactivated influenza viruses that generate much higher titers of anti-HA antibodies than anti-NA antibodies in animals, the negative impact of maternal antibodies on piglet immunization should be significantly reduced to vaccines based on neuraminidase.

For a more complete understanding of the invention, the following definitions are provided.

The use of singular terms for convenience of description is not intended to be limiting in any way. Thus, for example, reference to a composition containing a "polypeptide" includes a reference to one or more such polypeptides. In addition, reference to "alphavirus RNA replicon particle" includes reference to a plurality of such alphavirus RNA replicon particles, unless otherwise indicated.

As used herein, the term "about" is used interchangeably with the term "about" and means that the value is within fifty percent of the specified value, i. a composition containing "approximately" 1x10 ⁸ alphavirus RNA replicon particles per milliliter contains 5x10 ⁷ to 1.5x10 ⁸ alphavirus RNA replicon particles per milliliter.

As used herein, the terms "pig" or "pig" are used interchangeably and include all domestic pigs unless otherwise indicated.

As used herein, a "phylogenetic cluster" is a set of influenza virus neuraminidases that have been grouped (on the same branch) into a phylogenetic tree or evolutionary tree that goes back to a similar (homologous) ancestor (see Examples 5). For IAV-S neuraminidases (NA) found in the USA, there are two predominant N1 phylogenetic clusters, N1-pandemic and N1-classic, and two predominant N2 phylogenetic clusters, N2-1998 and N2-2002. The N1-classical phylogenetic cluster contains NAs clustered together with NAs from the classic H1N1 swine flu. The N1-pandemic virus phylogenetic cluster contains NA clustered together with NA derived from the H1N1 pandemic influenza virus. The N2-1998 phylogenetic cluster contains NAs clustered together with NAs from the H3N2 human influenza virus that entered pigs in 1998, while the N2-2002 phylogenetic cluster contains NAs clustered together with NAs from the H3N2 human influenza virus that entered pigs. in 2002. [See Anderson et al. , Influenza and other Respiratory Viruses 7 (Suppl. 4): 42-51 (2013)]. Example 5 presents the methodology for determining the phylogenetic cluster. For US phylogenetic clusters NA IAV-S N1-classic, N1-pandemic, N2-1998, N2-2002, the respective representative neuraminidase have the following respective amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.

As used herein, a "lineage" is a set of influenza virus neuraminidases that have been grouped (on the same branch) in an evolutionary tree that is associated with a similar (homologous) ancestor (see Example 6 below). These groupings have been made for European NAs and are similar, but not equivalent, to the phylogenetic clusters for US viruses. Line definitions can be obtained using readily available software, i.e. MEGA X as described in Example 6 below. For European lines NA IAV-S N1-Pandemic (EU), N1-Eurasian Avian, N2-Gent/1984, N2-Italy/4675/2003, N2-Scotland/1994 clade 1 or N2-Scotland/1994 clade 2 the respective representative neuraminidase have the following corresponding amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and 22.

As used herein, the term "replicon" refers to a modified RNA of the viral genome that lacks one or more elements (e.g., coding elements for structural proteins) that, if present, would allow the parent virus to successfully propagate in cell cultures. . or host animals. In the right cellular context, the replicon will self-replicate and can produce one or more subgenomic RNAs.

As used herein, the term "alphavirus RNA replicon particle", abbreviated as "RP", is an RNA derived RNA replicon packaged in structural proteins, e.g., a capsid and glycoproteins, which are also derived from an alphavirus, e.g., as described by Pushko et al., [Virology 239 (2): 389-401 (1997)]. RP cannot propagate in cell cultures or animal hosts (without a helper plasmid or similar component) because the replicon does not encode the structural components of the alphavirus (eg capsid and glycoproteins).

The term "non-IAV-S" is used to modify terms such as pathogen and/or antigen (or immunogen) to mean that the relevant 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 antigen protein (or immunogen) is not derived from IAV-S.

The terms "derived from", "derived from" and "derived from" are used interchangeably with respect to a given protein antigen and a pathogen, or strain of that pathogen which naturally encodes it, and as used herein means that unmodified and/ or a truncated protein sequence of that given protein antigen is encoded by that pathogen or a strain of that pathogen. The coding sequence within the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may have been genetically manipulated to obtain a modification and/or truncation of the amino acid sequence of the expressed protein antigen relative to the corresponding sequence of that protein antigen in the pathogen or pathogen strain (including naturally attenuated strains) from which it originates.

As used herein, the terms "protection" or "provide protection" or "induce protective immunity", "assistance in disease prevention" and "protective measures" do not require complete protection against any signs of infection. For example, "means of protection" may mean that the protection is sufficient so that after infection, the symptoms of the underlying infection are at least reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms that cause the symptoms are reduced and/ or eliminated. It should be understood that "reduced" as used herein means in relation to the state of the infection, including the molecular state of the infection, and not just the physiological state of the infection.

As used herein, a "vaccine" is a composition that is suitable for use in animals, such as pigs (including, in some embodiments, humans, although in other embodiments it is not specifically intended for humans), containing one or more antigens. , typically in combination with a pharmaceutically acceptable carrier, such as a liquid containing water, which, when administered to an animal, elicits an immune response strong enough to minimally assist in protection against disease resulting from infection with a wild-type microorganism, i. e. strong enough to help prevent a disease and/or prevent, ameliorate, or cure a disease.

As used herein, a polyvalent vaccine is a vaccine that includes two or more different antigens. In a particular embodiment of this type, the multivalent vaccine stimulates the recipient's immune system against two or more different pathogens.

The terms "adjuvant" and "immunostimulant" are used interchangeably herein, and are defined as one or more substances that stimulate the immune system. In this context, an adjuvant is used to enhance the immune response to one or more vaccine antigens/isolates. Thus, "adjuvants" are agents that non-specifically increase the immune response to a particular antigen, thus reducing the amount of antigen needed in any given vaccine and/or the frequency of injections needed to generate an adequate immune response to the antigen of interest. In this context, an adjuvant is used to enhance the immune response to one or more vaccine antigens/isolates.

As used herein, a "non-adjuvanted vaccine" is a vaccine or a multivalent vaccine that does not contain an adjuvant.

As used herein, the term "pharmaceutically acceptable" is used as an adjective meaning that the modified noun is suitable for use in a pharmaceutical formulation. When used, for example, to describe an excipient in a pharmaceutical vaccine, it characterizes the excipient as being compatible with the other ingredients of the composition and not adversely detrimental to the intended recipient animal, such as a pig.

"Parenteral administration" includes subcutaneous injections, submucosal injections, intravenous injections, intramuscular injections, intradermal injections and infusion.

As used herein, the term "antigenic fragment" in relation to a particular protein (for example, a protein antigen) is a fragment of that protein that is antigenic, that is, capable of specifically interacting with an antigen-recognizing molecule of the immune system, such as immunoglobulin (antibody) or T- cellular antigen receptor. For example, the antigenic fragment of neuraminidase IAV-S (NA) is an antigenic fragment of the NA protein. Preferably, the antigenic fragment of the present invention is immunodominant for recognition by an antibody and/or a T cell receptor. In specific embodiments, an antigenic fragment for a given protein antigen is a fragment of that protein that retains at least 25% of the antigenicity of the full length protein. 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. Antigenic fragments can be small, up to 20 amino acids, or, at the other extreme, large fragments that lack only one amino acid from a full-length protein. In specific embodiments, the implementation of the antigenic fragment contains from 25 to 150 amino acid residues. In other embodiments, the implementation of the antigenic fragment contains from 50 to 250 amino acid residues.

As used herein, one amino acid sequence is 100% "identical" or has 100% "identity" with a second amino acid sequence when the amino acid residues of both sequences are identical. Thus, an amino acid sequence is 50% "identical" to a second sequence when 50% of the amino acid residues of the two amino acid sequences are identical. Sequence comparisons are made with the adjacent block of amino acid residues that make up the protein, eg the protein or part of the polypeptide being compared. In a particular embodiment, selected insertions or deletions are taken into account that might otherwise alter the correspondence between two amino acid sequences.

As used herein, percent nucleotide and amino acid sequence identity can be determined using C, MacVector (MacVector, Inc. Cary, NC 27519), NTI Vector (Informax, Inc. MD), Oxford Molecular Group PLC (1996), and the Clustal algorithm. W with default alignment parameters, and with default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or similar default settings. Alternatively, you can use advanced Blast search under default filter conditions, such as using the GCG accumulation program (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) using the default parameters.

As used herein, the term "inactivated" microorganism is used interchangeably with the term "killed" microorganism. For the purposes of the present invention, an "inactivated" microorganism is an organism that is capable of eliciting an immune response in an animal but is not capable of infecting the animal. An antigen of the present invention (eg, inactivated Mycoplasma hyopneumoniae ) can be inactivated with an agent selected from the group consisting of binary ethyleneimine, formalin, beta-propiolactone, thimerosal, or heat. In a specific embodiment, inactivated Mycoplasma hyopneumoniae isolates in combination with the RP of the present invention are inactivated with binary ethyleneimine.

The present invention also relates to vaccines against a variety of porcine pathogens. For example, a coding sequence for a protein antigen or an antigenic fragment thereof, or a combination of such coding sequences for protein antigens useful in a swine vaccine, can be attached to an alphavirus RNA (RP) replicon particle and/or combined in the same RP as the sequence encoding in the NA vaccine derived from IAV-S. Examples of pathogens from which one or more protein antigens or antigenic fragments may be derived 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), Pasteurella multocida with multiple serotypes, Salmonella ssp ., Escherichia coli eg (serotypes K99, K88, 987P or F41), Haemophilus parasuis , Lawsonia intracellularis , Mycoplasma ssp . (e.g. Mycoplasma hyopneumoniae ), Bordetella bronchiseptica , Erysipelas ssp ., Campylobacter ssp ., Actinobacillus pleuropneumoniae , Clostridium perfringens and Clostridium difficile .

In addition, the present invention relates to vaccines containing one or more RPs of the present invention in combination with one or more other vectors encoding several of these porcine antigens (for example, a baculovirus vector encoding the ORF-2 protein from porcine circovirus-2, ( PCV-2) and/or porcine circovirus-3 (PCV-3) and/or inactivated toxoids derived from one or more of these porcine pathogens.In addition, such vaccines may include any alphavirus RNA replicon particle that encodes NA, derived from IAV-S in the vaccine of the present invention together with one or more killed and/or modified (attenuated) live porcine virus isolates and/or porcine bacteria All such polyvalent vaccines are included in the present invention.

Thus, one or more alphavirus RNA (RP) replicon particles that encode one or more NAs derived from IAV-S can be added together with one or more other vectors encoding one or more porcine antigens and/or with one or more killed and/or modified (attenuated) live virus isolates, such as one or more killed or modified live strains of IAS-V, one or more killed and/or modified live PRRS viruses, one or more killed and/or modified live PCV, one or more killed and/or modified live TGE, one or more killed and/or modified live PPRV, one or more killed and/or modified live PPV, one or more killed and/or modified live PRV and one or more killed and/or modified live PEDs. In addition, one or more alphavirus RNA (RP) replicon particles that encode one or more NAs derived from IAV-S can be added together with one or more other vectors encoding one or more porcine antigens and/or added together with one or several killed and/or modified (attenuated) live bacteria that can also infect pigs, including one or more killed and/or modified live Pasteurella multocida (from one or more multiple serotypes), Salmonella ssp., Escherichia coli (from one or more multiple serotypes), Haemophilus parasuis , Lawsonia intracellularis , Mycoplasma ssp . (e.g. Mycoplasma hyopneumoniae ), Bordetella bronchiseptica , Erysipelas ssp ., Campylobacter ssp ., Actinobacillus pleuropneumoniae , Clostridium perfringens and Clostridium difficile .

The alphavirus RNA replicon particles of the present invention can be lyophilized and rehydrated with sterile water. On the other hand, when the alphavirus RNA replicon particles are stored separately but are intended to be mixed with other vaccine components prior to administration, the alphavirus RNA replicon particles can be stored in a stabilizing solution of these components, such as a high sucrose solution.

In one aspect, the vaccine of the present invention may contain an adjuvant. In certain embodiments, the adjuvant is a biodegradable oil. In certain formulations, the biodegradable oil is dl-α-tocopheryl acetate (vitamin E acetate). In other formulations, the adjuvant contains an oil-in-water emulsion of 2.5% to 50% (v/v) mineral oil. In more specific formulations, the adjuvant comprises an oil-in-water emulsion that contains 5 to 25% mineral oil. In related formulations, the adjuvant is a mixture of two components. The first component consists of drops of mineral oil with an approximate average (volume weight) size of about 1 micron, which is stabilized with polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) in water. The first component may be 25 weight percent mineral oil and 1 weight percent polysorbate 80 with a balance of water. The second component may consist of droplets of biodegradable dl-α-tocopheryl acetate with an approximate average (volume weight) size of 400 nm, which is also stabilized by polysorbate 80. Specific formulations include 15 weight percent dl-α-tocopheryl acetate and 6 weight percent polysorbate 80, with the rest of the water. In specific embodiments, the adjuvant is X-SOLVE™ (which is a combination of two component adjuvants: DILUVAC FORTE™, which is based on dl-α-tocopheryl acetate, and MICROSOL™, which is based on light paraffin oil [see, for example, US 8597662] B In related formulations, the adjuvant contains submicron oil droplets and biodegradable oil droplets, the biodegradable oil droplets having an average size that differs from the average mineral oil droplet size [see, for example, US 9084768].

The vaccine of the present invention can be easily administered by any standard route, including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal and/or intraperitoneal vaccination. Those skilled in the art will appreciate that the vaccine composition is formulated appropriately for each type of recipient animal and route of administration.

Thus, the present invention also relates to a method for immunizing a pig against IAV-S and/or other pig pathogens. One such method involves injecting the pig with an immunologically low amount of the vaccine of the present invention so that the pig produces the appropriate anti-IAV-S antibodies.

It should also be understood that this invention is not limited to the specific configurations, process steps, and materials described herein, as such configurations, process steps, and materials may vary slightly. It should also be understood that the terminology used herein is used only to describe specific embodiments and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims and their equivalents.

SEQUENCE TABLE ¹

SEQID NO: Description Type 1 N1-classic (codon-optimized) nucleic acid 2 N1-classic amino acid 3 N1-pandemic (codon-optimized) nucleic acid 4 N1-pandemic amino acid 5 N2-1998 (codon-optimized) nucleic acid 6 N2-1998 amino acid 7 N2-2002 (codon-optimized) nucleic acid 8 N2-2002 amino acid 9 ggcgcgccgcacc nucleic acid 10 ttaattaa nucleic acid eleven N1-pandemic (EU) nucleic acid 12 N1-pandemic (EU) amino acid 13 N1-Eurasian Avian nucleic acid 14 N1-Eurasian Avian amino acid 15 N2-Gent/1984 nucleic acid 16 N2-Gent/1984 amino acid 17 N2-Italy/4675/2003 nucleic acid 18 N2-Italy/4675/2003 amino acid 19 N2-Scotland/1994 (clade 1) nucleic acid 20 N2-Scotland/1994 (clade 1) amino acid 21 N2-Scotland/1994 (clade 2) nucleic acid 22 N2-Scotland/1994 (clade 2) amino acid 23 N1-pandemic (EU) (codon-optimized) nucleic acid 24 N2-Gent/1984 (codon-optimized) nucleic acid 25 N2-Italy/4675/2003 (codon-optimized) nucleic acid 26 N2-Scotland/1994 (clade 1) (codon-optimized) nucleic acid 27 N2-Scotland/1994 (clade 2) (codon-optimized) nucleic acid

¹ Nucleotide sequences are presented in DNA form only, although it should be understood that when a sequence is included in an RNA construct, the corresponding RNA sequence is meant (with uracil, "u" replacing thymidine, "t").

SEQUENCES

N1-classic (SEQ ID NO: 1)

atgaatactaatcaaaggatcattaccattgggacagtctgcatgattgtcggtatcatctcttttgcttcagattggaaacattgtctcactttggattagccattcaattcagaccggatgggagaatcacactgagatgtgcaatcaaagtgtcattacttatgtcaataacacttgggtgaaccggacttatgtgaacattagcaatatcaaga ttgcaactattcaagatgtgactagtatcattttggccggaaattctagtctttgcccggtgtcagggtgggctgtctacagcaaagacaatagcattaggattggatcaaaaggggacattttcgtcattagagagcctttcatctcatgctcacaattggagtgccggaccttctttctgacccaaggggcattgctgaatgacaaacattcaaat ggtaccgtcaaggacaggagtccttatagaaccctgatgagctgccctatcggtgaggccccttcgccatacaactcacggttcgaatctgtcgcatggtcagcatctgcttgtcatgatggaatgggatggcttacaatcgggatcagtggaccggataatggtgctgtcgcagtcttgaaatacaacggaatcattacagatacaatcaa atcttggaggaacaagattcttagaactcaagaatcagagtgtgtctgtatgaacggatcatgttttacagtcttgacagatggcccaagcaatggacaagcctcttacaaaatctttaaggtggaaaaaggaaagattatcaagtcgattgagctggatgcccccaattaccactatgaagaatgctcttgttatccagatactggcaaagtcatgtg tgtctgccgggacaattggcacgcctcaaaccggccatgggtgtcgttcaatcagaatcttgactatcaaattggatacatttgctctggagtctttggtgataaccctagatccaatgatgggaagggcaattgtggcccggtcctttctaatggagcaaatggagtgaaaggtttctcatatcggtatggaaatggtgtgtggat tggtcggaccaagtcaatcaactctcggtcgggttttgagatgatttgggatccgaatggatggactgagacagattcatcattctcgatgaagcaggacattatcgctcttaatgattggtctggatactcgggatcttttgtccaacatccggagcttactggtatgaattgcattaggccttgtttctgggtggaattgatcagagggcaacccaagga aagcactatctgggctagcggttccagcatctcattctgtggcgtcaattcggaaaccgcttcctggtcttggccagacggagctgatctgccattcaccattgacaag

N1-classic (SEQ ID NO: 2)

MNTNQRIITIGTVCMIVGIISLLLQIGNIVSLWISHSIQTGWENHTEMCNQSVITYVNNT

WVNRTYVNISNIKIATIQDVTSIILAGNSSLCPVSGWAVYSKDNSIRIGSKGDIFVIREP

FISCSQLECRTFFLTQGALLNDKHSNGTVKDRSPYRTLMSCPIGEAPSPYNSRFESVAWS

ASACHDGMGWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNKILRTQESECVCMNGSCFT

VLTDGPSNGQASYKIFKVEKGKIIKSIELDAPNYHYEECSCYPDTGKVMCVCRDNWHASN

RPWVSFNQNLDYQIGYICSGVFGDNPRSNDGKGNCGPVLSNGANGVKGFSYRYGNGVWIG

RTKSINSRSGFEMIWDPNGWTETDSSFSMKQDIIALNDWSGYSGSFVQHPELTGMNCIRP

CFWVELIRGQPKESTIWASGSSISFCGVNSETASWSWPDGADLPFTIDK

N1-pandemic (SEQ ID NO: 3)

atgaatcctaaccaaaagatcattaccattggttcggtctgtatgacaattggaatggctaacctgatccttcaaattggaaacattatctcaatctgggtcagccactcaattcaaattggaaatcaatcgcagattgaaacatgcaaccaaagcgtcattacttacgaaaacaacacttgggtgaaccagacctacgtgaacatcag caacaccaacttcgctgctggacagtccgtggtttccgtgaaactggcgggcaactcctctctctgccctgtgagcggatgggctatctactccaaagacaactcagtcagaatcggttccaagggggatgtgtttgtcataagggaaccattcatctcatgctctccccttgaatgcagaaccttcttcttgactcaaggggccttgctaaatga caaacattccaacggaaccattaaagacaggagcccatatcggaccctgatgagctgtcctatcggtgaagtcccctcgccatacaactcaagatttgagtcagtcgcttggtcagcatccgcttgtcatgatggcatcaattggctcaccattggaatttctggcccagacagtggggcagtggctgtgctgaagtacaatggcattataacagac actatcaagtcgtggaggaacaacatattgagaactcaagagtctgaatgtgcatgtgtgaatggttcttgctttaccatcatgaccgatggaccatccgatggacagcctcctacaagatcttcagaatcgaaaagggaaagatcgtcaaatcagtcgaaatgaatgcccctaactaccactatgaggaatgctcctgttatcctgattcct ccgaaatcacgtgcgtgtgcagggataactggcatggctccaatcggccgtgggtgtctttcaaccagaatctggaatatcagatcggatacatttgctccggggtgttcggagacaatccgcgccctaatgataagacaggctcgtgtggtccagtctcgtctaacggagccaacggagtcaaaggattttcattcaaatacggcaatgg agtgtggatagggagaactaagagcatttcctcaagaaaaggtttcgagatgatttgggatccgaatggatggactgggactgacaacaagttctcaatcaagcaagacatcgtgggaatcagcgagtggtcaggatatagcgggtcctttgtgcagcaccccgaactgaccgggctggattgtattagaccttgcttctgggtcgaactcatcagaggg cgacccgaagagaacacaatctggactagcgggagcagcatctccttttgtggtgtgaactcggacactgtgggttggtcttggccagacggtgctgagttgccttttaccattgacaag

N1-pandemic (SEQ ID NO: 4)

MNPNQKIITIGSVCMTIGMANLILQIGNIISIWVSHSIQIGNQSQIETCNQSVITYENNT

WVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREP

FISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWS

ASACHDGINWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFT

IMTDGPSDGQASYKIFRIEKGKIVKSVEMNAPNYHYEECSCYPDSSEITCVCRDNWHGSN

RPWVSFNQNLEYQIGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIG

RTKSISSRKGFEMIWDPNGWTGTDNKFSIKQDIVGISEWSGYSGSFVQHPELTGLDCIRP

CFWVELIRGRPEENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK

N2-1998A (SEQ ID NO: 5)

atgaatccaaaccaaaagataatcacaattggctctgtttctctcactattgccacaatgtgcctccttatgcaaattgccatcctgattactaatgtcacattgcactccaatcagtacgaatgcaactaccccccaaacaaccaagtgatactgtgtgaaccaactatcatcaaaagaaacattactgagattgtgtatctggccaacaccaccatag agaaggaaatctgccccaagctggcagaatacagaaactggtcgaagccgcaatgtaaaattacagggtttgcaccttttttccaaggacaattcgattaggctttctgcgggtggcgacatttgggtaacgagagaaccttatgtgtcatgcgatcctgataagtgttaccagtttgcccttggacaaggaacaacgctcaacaacagacattcaa acgacaccgtgcatgataggaccccttatcgaaccctattgaatgaatgagttgggtattccattccatttggggaccaaacaagtgtgtgcatcgcatggtccagctcatcctgccatgatggacgggcttggcttcatgtctgtattactgggcatgacaacaatgcaactgccagcatcatttacaatggacgccttgtcgatagtattggttcatggtccaaa agaatcctcaggacccaggagtcggaatgcgtgtgcatcaatggaacttgtaccgtggtcatgactgatgggtccgcttcaggaatagctgacactaaaatcctgttcattgaagaggggaaaatcgtgcacattagcccactgctggggtccgctcagcacgtggaggagtgctcctgctatccccgatacccaggtgtcaga tgcatctgtagagacaactggaaaggctccaacagacctgtcgtggatattaatgtgaaggattatagcattgtgtcctcctacgtgtgctccggactggtgggagacacccccagaaaagacgacagatccagctccagcaattgtctgaatcctaacaacgagaaaggggagcatggagtgaaaggctgggcctttgatgatggaaatgac gtgtggatggggaggacaatcaacgagacattacgctcaggttatgaaaccttcaaagtcattgaaggctggtccaaacctaattccaaattgcagataaatcgccaagtcattgttgaaagagatgataggtccggttattctggaattttctctgtcgaaggaaagagctgtatcaatcggtgtttttacgtggagctgatcagagg aaggaaacaggaaactgcagtgtggtggacgtcaaattccattgtggtgttttgtggcacctcaggtacctatggaaccggctcatggcctgatggggcggacatcaatctcatgcctgtg

N2-1998A (SEQ ID NO: 6)

MNPNQKIITIGSVSLTIATMCLLMQIAILITNVTLHSNQYECNYPPNNQVILCEPTIIKR

NITEIVYLANTTIEKEICPKLAEYRNWSKPQCKITGFAPFSKDNSIRLSAGGDIWVTREP

YVSCDPDKCYQFALGQGTTLNNRHSNDTVHDRTPYRTLLMNELGIPFHLGTKQVCIAWSS

SSCHDGRAWLHVCITGHDNNATASIIYNGRLVDSIGSWSKRILRTQESECVCINGTCTVV

MTDGSASGIADTKILFIEEGKIVHISPLLGSAQHVEECSCYPRYPGVRCICRDNWKGSNR

PVVDINVKDYSIVSSYVCSGLVGDTPRKDDRSSSSNCLNPNNEKGEHGVKGWAFDDGNDV

WMGRTINETLRSGYETFKVIEGWSKPNSKLQINRQVIVERDDRSGYSGIFSVEGKSCINR

CFYVELIRGRKQETAVWWTSNSIVVFCGTSGTYGTGSWPDGADINLMPV

N2-2002A (SEQ ID NO: 7)

atgaatccaaatcaaaagatcattactattggatcagtctcactcatcattgccacaatttgtttccttatgcaaattgcaatccttgtcactactgtcacattgcatttcaagcagcatgactacaactccccccaaacaaccaagctactctgtgtgtgaaccaacaatcattgaacggaaaacaactgaaattgtgtatcttactaacaccacca ttgagaaagaagtctgccccaaacttgcagagtaccggaactggtcaaagcctcaatgtaacattactggatttgcaccattttcgaaagacaattctattcggttgtctgctggtggggacatctgggtgactagggaaccttatgtgtcatgcgatcctgacaagtgttaccaatttgcccttggacagggtacaactcttaacaacggacatt cgaataacacagtccatgataggaccccgtatcggacccttcttatgaatgagcttggtgtcccttttcatcttggaaccagacaagtgtgcatggcttggtctagctcatcttgtcacgatgggaaagcatggctgcatgtctgtgtcactggaaatgataacaatgctactgctagcttctacaatggtaggcttgtggattctat tggttcgtggtcgaaaaacattctccggacccaagagtcagaatgcgtctgtatcaatggaacatgtactgtcgtcatgactgatggatccgctagtggaaaagcagataccaaaatcttgttcgtcgaagaggggaagatcgtccatatcagcactctgttgggatctgcacagcacgtcgaggaatgctcctg ttatcctaggtttccgggagtccggtgtgtctgccgggacaactggaaaggatctaatagacccatcgtcgacatcaatgtcaagaattacagcattgtctcttcgtatgtctctgcagtggacttgtcggtgatactcccagagagagcgactcagtctcctcatcttattgcttggatccgaacaatgagaagggtggtcatggggtga aagggtgggcctttgatgatggtaatgacgtgtggatgggaagaacaatcaacgagactttgcgcttgggatatgaaaccttcaaagtcattgaaggctggtccacagctaactccaagtcacagacaaatagacaagtgattgtcgaaaaaggagacaggtcaggatattctgggattttctcagtcgagggaaagaactgcatcaataggtg cttctatgtggagttgattagaggacggaaagaggagacaaaagtctggtggaccagtaactcaattgtcgtgttttgtggcacctcagggacttatggtactggctcttggccggatggtgctgacatcaatctcatgccaatt

N2-2002A (SEQ ID NO: 8)

MNPNQKIITIGSVSLIIATICFLMQIAILVTTVTLHFKQHDYNSPPNNQATLCEPTIIER

KTTEIVYLTNTTIEKEVCPKLAEYRNWSKPQCNITGFAPFSKDNSIRLSAGGDIWVTREP

YVSCDPDKCYQFALGQGTTLNNGHSNNTVHDRTPYRTLLMNELGVPFHLGTRQVCMAWSS

SSCHDGKAWLHVCVTGNNDNNATASFIYNGRLVDSIGSWSKNILRTQESECVCINGTCTVV

MTDGSASGKADTKILFVEEGKIVHISTLLGSAQHVEECSCYPRFPGVRCVCRDNWKGSNR

PIVDINVKNYSIVSSYVCSGLVGDTPRESDSVSSSYCLDPNNEKGGHGVKGWAFDDGNDV

WMGRTINETLRLGYETFKVIEGWSTANSKSQTNRQVIVEKGDRSGYSGIFSVEGKNCINR

CFYVELIRGRKEETKVWWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI

N1-pandemic EU (SEQ ID NO: 11)

atgaatccaaaccaaaagataataaccattggttcggtctgtatgacaattggaatggctaacttaatattacaaattggaaacataatctcaatatggattagccactcaattcaacttgggaatcaaagtcagattgaaacatgcaatcaaagcgtcattacttatgaaaacaacacttgggtaaatcagacatatgttaacatcagcaacaccaactttg ctgctggacagtcagtggtttccgcgaaattagcgggcaattcctccctctgccctgttagtggatgggctatatacagtaaagacaacagtgtaagaatcggttccaagggggatgtgtttgtcataagggaaccattcatatcatgctcccccttagaatgcagaaccttcttcttgactcaaggggccttgctaaatgacaaacattccaat ggaaccattaaagataggagcccatatcgaaccctgatgagctgtcctattggtgaagttccctccatacaactcaagatttgagtcggtcgcttggtcagcaagtgcttgtcacgatggcatcaattggctaacaatcggaatttctggcccagacagtggggcagtggctgtattaaagtacaatggcataataacagacactatcaagagttgg aaaaacaatatattgagaacacaagagtctgaatgtgcatgtgtaaatggttcttgctttaccataatgaccgatggaccaagtgatggacaggcctcatacaagatcttcagaatagaaaagggaaagatagtcaaatcagtcgaaatgaatgcccctaattatcactatgaggaatgctcctgttatcctgattctagtgaaatcacatg tgtgtgcagggataactggcatggctcgaatcgaccgtgggtgtctttcaaccagaatctggaatatcagataggatacatatgcagtgggattttcggagacaatccacgccctaatgataagacaggcagttgtggtccagtatcgtctaatggagcaaatggagtaaaaggattttcattcaaatatggcaatggtgtttggatagggagaact aaaagcattagttcaagaaaaggttttgagatgatttgggatccaaatggatggactgggacagacaaaaacttctcaataaagcaagatatcataggaataaatgagtggtcaggatacagcgggagttttgttcagcatccagaactaacagggctgaattgtataagaccttgcttctgggttgaactaatcagggcgacccaaagagaacacaatctggac tagcgggagcagcatatccttttgtggtgtaaacagtgacactgtgggttggtcttggccagacggtgctgagttgccatttaccattgacaagtaa

N1-pandemic EU (SEQ ID NO: 12)

MNPNQKIITIGSVCMTIGMANLILQIGNIISIWISHSIQLGNQSQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSAKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDSGAVAVL KYNGIITDTIKSWKNNILRTQESECACVNGSCFTIMTDGPSDGQASYKIFRIEKGKIVKSVEMNAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVSSNGANGVKSFKYGNGVWIGFRTKSISSRKGFEMIWDPNGWTGTDKNFSIKQDIIGINEWSGYSGSFVQHPELT GLNCIRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK

N1-Eurasian Avian (SEQ ID NO: 13)

atgaacccaaatcagaagataataatcattagttcaatctgtatgacaaatggaattgctagcttgatattacaaattgggaacataatatcaatatggattagccattcaattcaaattgagaacccaaaccagaccgaccatgcaatcaaagcgttattatttacgaaaacaacacatgggtaaatcaaacgtatgttaacatcagcaacaataattttgtt gttgaacagacagtggtttcaatgaaattagcgggcagttcttctctctgccctgttagtggatgggctatatacagtaaagataacagtgtaagaatcggttccaaaggggatgtgtttgtcataagagccattcatctcatgctcccatttggaatgtagaaccttcttcttaactcaaggggccctactgaatgataaacattctaatggaaccg ttaaagacagaagcccctatcgaaccctgatgagctgtcctattggtgaagtcccctctccatacaactcaaaatttgagtcagttgcttggtcagcaagtgcttgccatgatggcaccagttggttgacaattgggattctggtccagacaatggagcagtggctgtgttgaaatacaatgacataataacagacactatcaagagttggaaa aacaacatattgagaacacaagaatctgaatgtgcatgtttgaatggttcttgctttactgtaatgaccgatggaccaagtaatgggcaggcctcatacaagatcttcaaaatagaaaaggggaaagtagtcaaatcagtcgagttgaatgctcctaattatcactatgaggaatgttcctgttatcctgattctggtgaaatcatatgtgtatg cagggacaattggcatggctcgaatcgaccatgggtgtctttcaatcagaatctggagtatcagataggatacatatgcagtggggttctcggagacaatccgcgccctaatgatagaacaggcagttgtggtccagtatcatctcatggagcaaatggggtaaaagggttttcgtttaaatacggcaatggaatttggatagggagaactaaaagcactatta caaggagtggttttgagatgatttgggacccaaacggatggactggaacagacaataatttctcagtgaagcaagatatcgtaggaataactaactggtcaggatacgcgggagttttgtccaacatccagaattaaccggattggattgtattagaccttgcttctgggttgaactaatcagggagacccaaagagaacacaatctggactagcggaagcagcatatcc ttttgtggtgtaaatagtgacactgtgggttggtcttggccagacggtgctgagttgccatttaccattgacaagtaa

N1-Eurasian Avian (SEQ ID NO: 14)

MNPNQKIIIISSICMTNGIASLILQIGNIISIWISHSIQIENPNQTEPCNQSVIIYENNTWVNQTYVNISNNNFVVEQTVVSMKLAGSSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNGTVKDRSPYRTLMSCPIGEVPSPYNSKFESVAWSASACHDGTSWLTIGISGPDNGAVAVL KYNDIITDTIKSWKNNILRTQESECACLNGSCFTVMTDGPSNGQASYKIFKIEKGKVVKSVELNAPNYHYEECSCYPDSGEIICVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVLGDNPRPNDRTGSCGPVSSHGANGVKSFKYGNGIWIGRTKSTITRSGFEMIWDPNGWTGTDNNFSVKQDIVGITNWSGYSGSFVQHPE LTGLDCIRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK

N2-Gent/1984 (SEQ ID NO: 15)

atgaatccaaatcaaaagataataataacaattggttctgtttctctcactattacaacaatgtgcctcttcttgcagattgccatcctagtaactactataacattgcatttcaagcaatatgaatgcgattcccctgcaaacaaccaagtaataccgtgtgtgaaccaataataatagaaaaaaacataacaaaaatagtgtatttgaccaataccaccatagagaa agaggtatgcccaaaattaggggaatacaggaattggtcaaaaccacaatgcaagatcacaggatttgcacctttttctaaggacaattcaattcggctctctgcgggtggggccatttgggtcacgagagaaccttatgtgtcatgcgaccctaacaagtgttatcaatttgcattaggacagggaaccacattagataacagacattcaaatgacaca atacatgatagaaccccttttagaaccctgttgatgagtgaattaggtgttccattcatttgggaaccagacaagtatgcatagcatggtccagttcaagttgtcacgatgggaaagcttggttgcatgtttgtgtcactgggcatgataaaaatgcaactgctagtttcatttatgacggaaagcttgtagacagcatcagttcatggtccaaaaacat actccggactcaggaatcagaatgtgtttgtatcgatggaatctgtacagtggtgatgactgatggaagtgcttcagggaaagctgatactaagatactatttattgaaaaagggaagatcattcatattagtccattgttgggaagtgctcagcatgtagaagaatgttcctgttaccctagataccctgatgtcaggtgtatttgcaggga taactggaaaggttcaaataggcccatcgtagacataagaatgaaaaattatagcattggttccagttatatgtgctcaggacttgttggcgacacaccccaggaacaatgatgggtctagtaatagcaattgtcggaatcccaataatgaaagaggaaatcatggagtgaaaggttgggcctttgatgatggaaatgacacatggatgggaagaactatcagca aggactcacgcttaggttacgaaaccttcaaagttgttggtggttggtcccaacccaattccaaatcccagataaatagacaagttattgttgacagcgataatagatcaggttactctggtattttctgttgaggggaaagattgcattaataggtgtttttatgtggaactaataagggaaggacaggaaactagagtgtggtggacttcgaacag tattgttgtgttctgtggcacttctggcacctatgggtcaggctcatggcccgatggagcaaacatcaatttcatgcctgtataa

N2-Gent/1984 (SEQ ID NO: 16)

MNPNQKIITIGSVSLTITTMCLFLQIAILVTTITLHFKQYECDSPANNQVIPCEPIIIEKNITKIVYLTNTTIEKEVCPKLGEYRNWSKPQCKITGFAPFSKDNSIRLSAGGAIWVTREPYVSCDPNKCYQFALGQGTTLDNRHSNDTIHDRTTPFRTLLMSELGVPFHLGTRQVCIAWSSSSCHDGKAWLHVCVTGHDKNATAS FIYDGKLVDSISSWSKNILRTQESECVCIDGICTVVMTDGSASGKAADTKILFIEKGKIIHISPLLGSAQHVEECSCYPRYPDVRCICRDNWKGSNRPIVDIRMKNYSIGSSYMCSGLVGDTPRNNDGSSNSNCRNPNNERGNHGVKGWAAFDDGNDTWMGRTISKDSRLGYETFKVVGWSQPNSKSQINRQVIVDSDNRSG YSGIFSVEGKDCINRCFYVELIRGRRQETRVWWTSNSIVVFCGTSGTYGSGSWPDGANINFMPV

N2-Italy/4675/2003 (SEQ ID NO: 17)

atgaattcaaatcaaaagataataataacaattggctctgtttctctcactattgccacactatgcctccttatgcaaattgctatcatggtaactactgtaacatttcatttcaagcagtatgaatacaactcccccgaacaaccaagtaatgttgtgtgtgaaccaacgataattgaaagaaacataacagagacagtgtacatgaccaacaccaccatagtgaaaga aatatgccccaaactagcggaatacagaaattggtcaaaaccgcaatgcaaaattacaggatttgcacctttttcaaaggacaactcaattcggctttccgctggtggggacatctgggtgacaagagaaccttatgtgtcatgcgatcctaacaagtgttatcaatttgcccttgggcagggaacaacgttaaaacaacaggcattcaa atgacacagtacatgatagaaccccttaccgaaccctgttgatgaatgaattgggtgttccattcatttaggaaccaagcaggtttgcatagcttggtccagttcaagttgtcatgatggaaaagcatggttgcatgttgtgtgtaactgggcatgatgaaaatgcaactgccagtttcatttacaacgagagacttgtagatagtattggttcatggtccaaga aaatcctcagaacccaggagtcggaatgcgtttgcataaatgggacttgtacagtggtgatgacagatgggagtgcttcaggtagagctgatactaaaatactattcattgaggaggggaaaatcgttcatgttagccaactgacaggaagtgctcagcatgtagaggagtgctcctgttatccccggtatcctggtgtcagatg tgtttgcagagataattggaaaggctccaataggcccattgtagatataaatgtaaaggatcatagcattgtttccagttatgtgtgctcaggacttgtcggagacacacccagaaaaaacgacagctctagcagtagtaactgcctgaatcctaacaatgaagaagggggtcatggggtgaaaggctgggcctttgatgatgaaaatgactt gtggatgggaagaacgatcagcgaaaagttacgattaggttatgaaaccttcaaggtcattgaaggctggtccaagcctaattccaaattgcagataaataggcaagtaatagttgacaaagataatagatccggttattctggtattttctctgttgaaagtaaaaagttgcatcaatcggtgctttttatgtggagttgataagaggaaggaaacaggaa aatgaagtatggtggacctcaaacagcattgttgtattttgtggcacctcaggtacatatggaacaggctcatggcctgatggggcagacatcaatctcatgcctatatga

N2-Italy/4675/2003 (SEQ ID NO: 18)

MNSNQKIITIGSVSLTIATLCLLMQIAIMVTTVTFHFKQYEYNSPPNNQVMLCEPTIIERNITETVYMTNTTIVKEICPKLAEYRNWSKPQCKITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPNKCYQFALGQGTTLNNRHSNDTVHDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCVTGHDENATA SFIYNERLVDSIGSWSKKILRTQESECVCINGTCTVVMTDGSASGRADTKILFIEEGKIVHVSQLTGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINVKDHSIVSSYVCSGLVGDTPRKNDSSSSSNCLNPNNEEGGHGVKGWAFDDENDLWMGRTISEKLRLGYETFKVIEGWSKPNSKLQINRQVIVDKDNRSGYS GIFSVESKSCINRCFYVELIRGRKQENEVWWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI

N2-Scotland/1994 (clade 1) (SEQ ID NO: 19)

atgaatccaaatcagaagataataacaattggctctgtttctctcgtcattgcaacattatgcttcttaatgcagatggccatcctaataactactgtaaaattacatttcaaacaatatgagtgcggcttccctgcgaacaaccaagtaataacatgtgagccaacagtaatagaaaggaacacaacagagatagtgtacttaactaacaccaccatagagaaagaa acatgccacaaaacagtggaatacaggaattggtcaaagcctcaatgcaaaataacaggctttgcacctttttccaaggacaattcaattcgactttctgctggtggggacatatgggtgacgagggaaccttacgtgtcatgcgagcctggcaaatgttatcagtttgcactcgggcaagggaccactagacaataaacattcaaac gatacaatacatgacagaaccccctatcgaactctattgatgaatgaattgggtgtcccatttcatttagggacaagacaagtgtgtattgcatggtccagctcaagttgttatgatgggaaagcatggttgcatgtctgtatcactggacatgataaaaatgcaactgccagtttcatttacgatggtagacttgtagatagcattggttcatggtctaaaa atatacttagaaccgggaatcagaatgcgtttgcatcaatggggtctgtacagtagtaatgactgatggaagtgcttcgggaagagctgatactaaaatactattcattgaagaagggaaaattgttcatattagcccattagcggggagtgcacagcatgtggagaggagtgctcctgttatccccgatcctggcgtaaggtgtatctgca gagacaactggaaaggctctaacagacccgttgtggatataaatatagaagattatagcattgattccagttatgtgtgttcagggcttgttggcgacacacccagaatcaatgacggatccagtagtagtagctactgccgtgatcctaacaacgaaaaaggaaatcacggagtgaagggctgggcttttgacgatggaaatgatgtgtggatggga agaacgatcaacgaagattcacgctcaggttatgaaacattcaaagtcattggtggttggtccactcctaattccaaattgcagataaataggcaagtaatagttgatagcaacaataggtcaggttattctggtgttttctccgttgaaggcaaaaagctgcatcaatagatgtttctacgtggagttgataagaggaagaaggtcagaagcgcgagtat ggtggacctcaaacagtattgttgtattttgtggcacttcaggtacctatggaacaggctcatggcctgatggagcagacatcaacctcatgcctatatga

N2-Scotland/1994 (clade 1) (SEQ ID NO: 20)

MNPNQKIITIGSVSLVIATLCFLMQMAILITTVKLHFKQYECGFPANNQVITCEPTVIERNTTEIVYLTNTTIEKETCHKTVEYRNWSKPQCKITGFAPFSKDNSIRLSAGGDIWVTREPYVSCEPGKCYQFALGQGTTLDNKHSNDTIHDRTPYRTLLMNELGVPFHLGTRQVCIAWSSSSCYDGKAWLHVCITGHDK NATASFIYDGRLVDSIGSWSKNILRTQESECVCINGVCTVVMTDGSASGRADTKILFIEEGKIVHISPLAGSAQHVEECSCYPRYPGVRCICRDNWKGSNRPVVDINIEDYSIDSSYVCSGLVGDTPRINDGSSSSYCRDPNNEKGNHGVKGWAFDDGNDVWMGRTINEDSRSGYETFKVIGGWSTPNSKLQINRQVIVDSNNRSGYSG VFSVEGKSCINRCFYVELIRGRRSEARVWWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI

N2-Scotland/1994 (clade 2) (SEQ ID NO: 21)

atgaatccaaaccagaaaataataacgatcggttctgtctccttgatcattgcaacaatgtgctttttcatgcaagttgccattctggtaactactgtaacattgcatttcaggcagtgcgaatgcaactcctccgcaaccaaccaaataatgccatgtaaaccaacaaaaatagagaaacataactgaaattgtgtacttaaccaataccaccataa aaacagaggtatgccccaaactagtgaaatacagggattgggcaaaaccacaatgtagaatcacagggttgcaccttttccaaggacaattcgattcggctttctgccggtggggccatttgggtaacgagagaaccctatgtatcatgcgatcttagcaagtgttaccagtttgcgctcggacaggggactacactagacaacagacattcaaatga cacaatacatgatagaactccttatcggaccctattgatgaatgaattgggtgttccatttcatttaggaaccaggcaagtgtgtatagcttggtccagttcaagttgtcacgatggaaaagcatggctgcatgtttgtgtcactgggtatgataaaaatgctactgctagcctcatttatgacggaaggcttgtggacagcatcggttcatggt cccaaaacatcctccggacccaggaatcggaatgtgtttgtataaatggtacttgcacagtggtaatgactgatgggagtgcttcaggaaaagctgataccagaatactatttattgaagaagggaagattattcacattagtccattgacaggaagtgcacagcatgttgaagagtgttcttgttatcctcgataccccggtgtaagatgtg tttgtagagacaactggaagggctctaacagacccgtcgtggatataaatgtaaaagattataaaattaactccagttatgtatgctcaggccttgttggcgatacccagaaacaacgatagatctagcaatagcaactgccaaaatcctaacaaccagagggaatcatggagtgaagggctgggcctttgacgatggaaatgacatatggatgggaagaaccat cagcaatgattcacgtttaggttatgaaactttcaaagttattggtggttggtccaaacccaactccaaagttcagacaaataggcaagtcatagttgacagcgataatagatcaggttattctggcgttttctgttgaaggcaaaagctgcatcaataggtgcttttatgtagagctaataagaggaaggacaggaagctagagtatggtggacttcgaac agtattgttgtgttttgtggtacttcgggtacatatggttcaggctcatggcctgatggggctgacatcaatcttatgcctatataa

N2-Scotland/1994 (clade 2) (SEQ ID NO: 22)

MNPNQKIITIGSVSLIIATMCFFMQVAILVTTVTLHFRQCECNSSATNQIMPCKPTKIERNITEIVYLTNTTIKTEVCPKLVKYRDWAKPQCRITGFAPFSKDNSIRLSAGGAIWVTREPYVSCDLSKCYQFALGQGTTLDNRHSNDTIHDRTPYRTLLMNELGVPFHLGTRQVCIAWSSSSCHDGKAWLHVCVTGYDK NATASLIYDGRLVDSIGSWSQNILRTQESECVCINGTCTVVMTDGSASGKADTRILFIEEGKIIHISPLTGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPVVDINVKDYKINSSYVCSGLVGDTPRNNDRSSNSNCQNPNNQRGNHGVKGWAFDDGNDIWMGRTISNDSRLGYETFKVIGGWSKPNSKVQTNRQVIVDSDNRS GYSGVFSVEGKSCINRCFYVELIRGRRQEARVWWTSNSIVVFCGTSGTYGSGSWPDGADINLMPI

The following examples serve to provide a further understanding of the invention, but are not intended to limit the effective scope of the invention in any way.

EXAMPLES

EXAMPLE 1

EFFICACY OF NON-ADJUVANT POLYVALENT NA-RP VACCINE CONTAINING FOUR SINGLE NA-RP CONSTRUCTS IN WEANED PIGS

INTRODUCTION

RNA viruses have been used as carrier vectors to introduce vaccine antigens that have been genetically integrated into their genomes. However, their use has so far been limited primarily to incorporating viral antigens into an RNA virus and then introducing the virus into a recipient host. The result is the induction of protective antibodies against the inserted viral antigens. Alphavirus RNA replicon particles have been used to encode pathogenic antigens. Such alphavirus replicon platforms have been developed from several different alphaviruses, including Venezuelan equine encephalitis (VEE) virus [Pushko et al., Virology 239: 389-401 (1997)], sindbis virus (SIN) [Bredenbeek et al., Journal of Virology 67: 6439-6446 (1993), the contents of which are hereby incorporated herein in their entirety], and Semliki Forest Virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9: 1356-1611 (1991), the contents of which are hereby incorporated herein. document in its entirety]. In addition, alphavirus RNA replicon particles are the basis for several USDA licensed swine and poultry vaccines. These vaccines include swine epidemic diarrhea vaccine, RNA particles (product code 19U5.P1), swine flu vaccine, RNA (product code 19A5.D0), avian influenza vaccine, RNA (product code 19O5.D0) and a prescription product, RNA particle (Product code 9PP0.00).

A study was conducted to determine the efficacy and immunogenicity of two dose levels of the four-factor non-adjuvanted NA-RP vaccine. The non-adjuvanted vaccine included four RP constructs, each of which individually encoded one distinct NA protein of the current US IAV-S isolate. Together, these NA genes represent two N1 and two N2 phylogenetic clusters that are predominant in the US pig population. The non-adjuvanted vaccine was administered by two intramuscular (IM) vaccinations (1 ml per dose) to weaned pigs seronegative for the vaccine fraction and the control strain. The efficacy of the NA-RP quadruple vaccine was then tested against heterologous N1 (H1N1 virus) and N2 (H1N2 virus) infections.

MATERIALS AND METHODS

Construction of NA-RP antigens :

VEE replicon vectors designed to express neuraminidase (NA) genes were constructed as previously described [see US 9441247 B2; the contents of which are incorporated herein with the following modifications. Vector "pVEK" with a replicon derived from TC-83 [disclosed and described in US 9441247 B2] was digested with restriction enzymes AscI and PacI . A DNA plasmid containing a codon-optimized sequence of the open reading frame of the N1 or N2 genes (Table 1) with a 5' flanking sequence (5'-GGCGCGCCGCACC-3') [SEQ ID NO: 9] and a 3' flanking sequence (5'TTAATTAA-3') [SEQ ID NO: 10] was similarly digested with restriction enzymes AscI and PacI . The synthetic gene cassette was then ligated into the digested pVEK vector and the resulting clones were renamed "pVHV-N1-pandemic", "pVHV-N1-classic", "pVHV-N2-2002" and "pVHV-N2-1998". The vector nomenclature "pVHV" was chosen to refer to pVEK-derived replicon vectors containing transgenic cassettes cloned through the AscI and PacI sites into the pVEK multiple cloning site.

Obtaining particles of replicon RNA TC-83 (RP) was carried out in accordance with previously described methods [US 9441247 B2 and US 8460913 B2; the contents of which are incorporated herein by reference]. Briefly, pVHV replicon vector DNA and helper DNA plasmids were linearized with NotI restriction enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and a "capping" analogue (Promega, Madison, WI). It is important to note that the helper RNA used in production lacks the VEE subgenomic promoter sequence as previously described [Kamrud et al., J Gen Virol. 91 (Pt 7): 1723-1727 (2010)]. Purified RNA for replicon and accessory components was pooled and mixed with Vero cell suspension, electroporated in 4 mm cuvettes, and returned to OptiPro SFM cell culture media (Thermo Fisher, Waltham, MA). After overnight incubation, purified alphavirus RNA replicon particles prepared in PBS with 5% sucrose (w/v) and 1% porcine serum are passed through a 0.22 µm membrane filter and aliquoted for storage. The functional RP titer was determined by immunofluorescence analysis on monolayers of infected Vero cells. Lots of RPs were identified according to the gene encoded by the packaged replicon.

TABLE 1

N1 and N2 genes encoded in NA-RP constructs

Phylogenetic cluster NA gene donor strain GenBank access number N1-classic A/swine/Iowa/A01410307/2014(H1N1) KJ605083 N1-pandemic A/swine/Minnesota/A01483170/2014(H1N1) KP036969 N2-1998 A/swine/Michigan/A02077465/2015(H1N2) KR982634 N2-2002 A/swine/Illinois/A01475495/2014(H1N2) KJ941362

Viruses : Viruses for challenge were obtained from the USDA National Veterinary Services Laboratory.

A/swine/Illinois/A01554351/2015 (H1N1) has the HA gene from the H1-gamma cluster and the NA gene from the N1-classic cluster.

A/swine/Iowa/A02076654/2015 (H1N2) has the HA gene from the H1-delta1 cluster and the NA gene from the N2-2002A cluster.

Viruses were grown in MDCK cell culture. Confluent cells were infected for approximately 48 hours until more than 70% of the cell monolayer was cytopathic. When harvesting the cells, the supernatant was removed from the vessels and clarified by centrifugation before storing the virus at -60° C. or below.

Animals : Weaned piglets were selected from a high health herd based on serological screening to confirm the absence of pre-existing HI or NI antibodies to the vaccine and control strains. These animals were a mix of males and females, approximately three weeks old at the time of primary vaccination.

Vaccination and challenge : The treatment groups are described in Table 2 below. The NA-RP quadruple vaccine is formulated into two dose levels of 10 ⁶ copies per RP/dose (low dose) and 10 ⁷ per RP/dose (high dose) based on an immunofluorescence assay to quantify functional RPs. NA-RP antigens are formulated in a stabilizer consisting of 1% porcine serum and 5% sucrose. The placebo vaccine consisted of the same stabilizer but without the antigen. Non-adjuvanted vaccines were administered to pigs at the age of 3 and 7 weeks im at a dose of 1 ml. Dose levels were back titrated by ELISA test on the remaining vaccine material after vaccination. Serum samples were collected on the day of the first vaccination, the day of the second vaccination and the day of infection.

TABLE 2

Treatment groups

Group Number of pigs Vaccine Number of copies per dose
(1 ml) Control strain 1 8 High dose NA-RP 10 ⁷ H1N1 2 8 Low dose NA-RP 10 ⁶ 3 9 placebo - 4 8 High dose NA-RP 10 ⁷ H1N2 5 8 Low dose NA-RP 10 ⁶ 6 9 placebo -

The virus for infection was administered to pigs 3 weeks after the second vaccination. Materials for infection with H1N1 (H1-gamma-N1-classic) and H1N2 (H1-delta-1b-N2-2002A) viruses were formulated for target doses of ^106.5 TCID50/pig (6 ml volume). Material for infection was administered intratracheally. The dose of virus for infection was confirmed by back-titration of the remaining material for infection. Pigs were collected nasal swabs -1, 1, 3 and 5 days after infection.

The autopsy was performed 5 days after infection (DPC). The pigs were euthanized by a 5 DPC barbiturate overdose under the supervision of a licensed veterinarian. The lungs were harvested and observed to document the surface area of each lobe affected by macroscopic lesions, resulting in a comprehensive percent score of lung injury. Bronchoalveolar lavage and nasal swabs were collected from all pigs to measure virus titers. Lung sections were collected for analysis of microscopic lesions.

Immune response analysis : IAV-S-specific antibodies in porcine serum samples were determined by HI and NI tests. The sera were inactivated by heating for 30-60 minutes at 56°C. For HI, sera were also treated with receptor degrading enzyme and/or kaolin and absorbed onto turkey erythrocytes to remove non-specific agglutinins. The HI test was performed as previously described [Kitikoon et al., Methods Mol Biol 1161: 295-301 (2014)] using turkey erythrocytes. The NI test was performed with minor modifications in the manner previously described [Sandbulte and Eichelberger, Methods Mol Biol 1161: 337-45 (2014)]. Briefly, 2-fold serial dilutions of serum were mixed with expressed protein antigen in equal volumes on fetuin-coated 96-well plates and incubated overnight at 37°C. Horseradish peroxidase-peanut agglutinin conjugate (PNA-HRP) was added for 2 hours at room temperature to bind fetuin molecules lacking sialic acid. The signal was obtained using the TMB substrate and the results were read at 650 nm. The mean optical density (OD) of the negative control (no NA antigen added) was subtracted from all wells. Then the OD values of the test samples were normalized on a scale of 0-100%, where the average OD value for the wells of the positive control (containing NA antigen, but no serum) was determined as 100%. The NI antibody titer was defined as the highest sample dilution that inhibited ≥50% neuraminidase activity.

Pathological examination of the lungs : Macroscopic lesions seen on the outside of all lobes of the lungs (well-demarcated purplish to plum indurations) were recorded on anterior and posterior lung chart grids. Composite scores (percentage of lung lesions) for each pig were calculated depending on the number of meshes with lesions.

Virus isolation : Nasal swabs and bronchoalveolar lavage fluid were serially diluted 10-fold with challenge medium [Dulbeco's minimal maintenance medium (DMEM) supplemented with 0.3% bovine serum albumin, fraction V; 2 mM L-glutamine; 25 µg/ml gentamicin; 2 μg/ml trypsin IX], and 100 μl of each dilution was added to four wells with confluent MDCK cells in a 96-well plate. The plates were incubated at 37° C. with 5% CO ₂ and observed after 72 hours for the presence of infectious virus by hemagglutination tests of supernatants from each well. IAV-S titers were calculated by the Spearman-Karber method and expressed as log10 TCID50 per ml.

RESULTS

Immune responses of pigs vaccinated with polyvalent NA-RP , see figure 1A-1D.

The NA-RP quadruple vaccine, high and low vaccine doses (10 ⁷ and 10 ⁶ RP copies/dose, respectively) induced functional NI antibody titers against all four NA components.

The NI titers induced by the high dose of the vaccine were about 1-2 times higher.

Pigs in the placebo vaccine group remained seronegative.

The effectiveness of the NA-RP quadruple vaccine against infections :

Lung lesions : see figures 2A-2B.

H1N1 virus infection caused over 13% of lung lesions in placebo control pigs. Both high dose and low dose immunized treatment groups had significantly lower lung injury scores at day 5 post H1N1 challenge than the immunologically naive placebo group (p<0.05).

Protection against lung injury was robust regardless of vaccine dose level.

H1N2 viral infection caused only 3.2% of lung lesions in placebo control pigs. Both treatment groups had lower lung injury scores at day 5 post H1N2 challenge than the immunologically naive placebo group, with mean lung injury rates below 1.1% and 1.2%, but differences between vaccinated groups and placebo group were not statistically significant. meaningful.

Nasal virus isolation : see Figures 3A-3B.

Isolation of the H1N1 challenge virus was maximal at day 3 post-challenge (DPC) for all three treatment groups.

At 5 DPC, there was a significant reduction in viral titer in both high and low dose quadruple RP groups compared to placebo control (p<0.05).

Nasal isolation of the H1N2 challenge virus was more delayed, with mean titers in the placebo group not exceeding the lower detection limit of up to 5 DPC. At 5 DPCs, H1N2 viral titers decreased significantly for the vaccinated groups at both dose levels (p<0.05).

Virus in the lungs : see Figures 4A-4B.

The H1N1 virus titer in bronchoalveolar lavage fluid (BALF) collected at 5 DPCs was more than 2 log higher in the placebo vaccine group than in any of the quaternary (4-component) NA-RP vaccinated groups where no detection was found. positive titers. This represented a statistically significant difference for both vaccinated groups (p<0.05).

Viral titers in the H1N2 challenged groups showed a 2-3 log reduction between both vaccinated groups and the placebo vaccine group and were statistically significant (p<0.05).

Brief conclusions

H1N1 infection was a more reliable infection model, with high rates of lung involvement accompanied by nasal and pulmonary viral titers. Groups vaccinated with quadruple NA-RP showed statistically significant protection against the H1N1 virus in terms of severity of lung injury, virus shedding, and virus titer in BALF. Individual animals in the low dose 4-component NA-RP vaccine group that developed lower serum NI titers (<80) were also protected. H1N2 viral infection was a less reliable model because lung involvement was low in the placebo group. However, even in this group, there was significant protection of the vaccinated groups with respect to nasal excretion and virus titer in the lungs. Polyvalent adjuvant-free NA-RP vaccine compositions are immunogenic and show efficacy against challenge with antigenically similar NA strains.

EXAMPLE 2

EFFECTIVENESS OF ADJUVANT FOUR-COMPONENT NA-RP VACCINE AGAINST H1N1 CHALLENGE IN WEANED PIGS WITH N1 ANTIBODY

During primary vaccination

A study was conducted to determine the efficacy and immunogenicity of the adjuvanted NA-RP quadruple vaccine. The adjuvant vaccine included four RP constructs, each of which individually encoded a single, different NA gene from a modern IAV-S isolate in the US. Together, these NA genes represent two N1 phylogenetic clusters (N1-classic and N1-pandemic) and two N2 clusters (N2-1998 and N2-2002). The adjuvant vaccine was administered with two intramuscular (IM) vaccinations (1 ml per dose) to weaned piglets seropositive for the N1 classical antigen at the time of their primary vaccination. The efficacy of the adjuvanted NA-RP quadruple vaccine was tested against heterologous N1 infection (H1N1 virus).

MATERIALS AND METHODS

NA-RP Antigen Construct : Replication Defective RNA Particles (RP) Containing Replicon Alphavirus RNA Expressing the Neuraminidase (NA) Gene Was Prepared As Described in Example 1.

TABLE 3

N1 and N2 genes encoded in NA-RP constructs

Phylogenetic cluster NA gene donor strain GenBank access number N1-classic A/swine/Iowa/A01410307/2014(H1N1) KJ605083 N1-pandemic A/swine/Minnesota/A01483170/2014(H1N1) KP036969 N2-1998 A/swine/Michigan/A02077465/2015(H1N2) KR982634 N2-2002 A/swine/Illinois/A01475495/2014 (H1N2) KJ941362

Viruses : Viruses for challenge were obtained from the USDA National Veterinary Services Laboratory. A/swine/Illinois/A01554351/2015 (H1N1) has the HA gene from the H1-gamma cluster and the NA gene from the N1-classic cluster. Viruses were grown in MDCK cell culture. Confluent cells were infected for approximately 48 hours until more than 70% of the cell monolayer was cytopathic. When harvesting the cells, the supernatant was removed from the vessels and clarified by centrifugation before storing the virus at -0° C. or below.

Animals : Weaned piglets were selected from a high health herd based on serological screening to confirm the absence of pre-existing HI or NI antibodies to the vaccine and control strains. These animals were a mix of males and females, approximately three weeks old at the time of primary vaccination.

Vaccination and challenge : The treatment groups are described in Table 4 below. The NA-RP quadruple vaccine was formulated in 10 ⁶ copies per RP/dose based on immunofluorescence assay to quantify functional RPs. NA-RP antigens are formulated in a stabilizer consisting of 1% porcine serum and 5% sucrose. The placebo vaccine consisted of the same stabilizer but without the antigen. The vaccines were mixed with Xsolve adjuvant (1:1 v/v, 1 ml dose) prior to IM administration to pigs at 3 and 7 weeks of age. Dose levels were back titrated by ELISA test on the remaining vaccine material after vaccination. Serum samples were collected on the day of the first vaccination, the day of the second vaccination and the day of infection.

The day before the first vaccination, all pigs were weighed, pigs in groups positive for antibody to the N1-classic antigen were injected subcutaneously with a 2 ml/kg dose of hyperimmune serum with N1-classic (NI titer 1:2560 to N1-classic antigen).

TABLE 4

Treatment groups

(n=10 pigs per group)

Group N1-classic antibody status Vaccine
(with ^Xsolve® 50) Number of copies per dose (1 ml) strain to infect 1 Positive 4-component NA-RP 10 ⁶ H1N1 2 Negative 4-component NA-RP 10 ⁶ 3 Positive placebo - 4 Negative placebo -

The virus for infection was administered to pigs 3 weeks after the second vaccination. Materials for challenge with the H1N1 virus (H1-gamma-N1-classic) were formulated for target doses of ^106.5 TCID50/pig (6 ml volume). Material for infection was administered intratracheally. The dose of virus for infection was confirmed by back-titration of the remaining material for infection. Pigs were collected nasal swabs -1, 1, 3 and 5 days after infection.

An autopsy was performed 5 days after infection. The pigs were euthanized by a 5 DPC barbiturate overdose under the supervision of a licensed veterinarian. The lungs were harvested and observed to document the surface area of each lobe affected by macroscopic lesions, resulting in a comprehensive percent score of lung injury. Bronchoalveolar lavage and nasal swabs were collected from all pigs to measure virus titers. Lung sections were collected for analysis of microscopic lesions.

Immune response analysis : IAV-S-specific antibodies in porcine serum samples were determined by the NI test. The serum was inactivated by heating for 30-60 minutes at 56°C. The NI test was performed with minor modifications in the manner previously described [Sandbulte and Eichelberger, Methods Mol Biol 1161: 337-45 (2014)]. Briefly, 2-fold serial dilutions of serum were mixed with expressed protein antigen in equal volumes on fetuin-coated 96-well plates and incubated overnight at 37°C. Horseradish peroxidase-peanut agglutinin conjugate (PNA-HRP) was added for 2 hours at room temperature to bind fetuin molecules lacking sialic acid. The signal was obtained using the TMB substrate and the results were read at 650 nm. The mean optical density (OD) of the negative control (no NA antigen added) was subtracted from all wells. Then the OD values of the test samples were normalized on a scale of 0-100%, where the average OD value for the wells of the positive control (containing NA antigen, but no serum) was determined as 100%. The NI antibody titer was defined as the highest sample dilution that inhibited ≥ 50% neuraminidase activity.

Pathological examination of the lungs : Macroscopic lesions seen on the outside of all lobes of the lungs (well-demarcated purplish to plum indurations) were recorded on anterior and posterior lung chart grids. Composite scores (percentage of lung lesions) for each pig were calculated depending on the number of meshes with lesions.

Virus isolation : Nasal swabs and bronchoalveolar lavage fluid were serially diluted 10-fold with challenge medium [Dulbeco's minimal maintenance medium (DMEM) supplemented with 0.3% bovine serum albumin, fraction V; 2 mM L-glutamine; 25 µg/ml gentamicin; 2 μg/ml trypsin IX], and 100 μl of each dilution was added to four wells with confluent MDCK cells in a 96-well plate. The plates were incubated at 37° C. with 5% CO ₂ and observed after 72 hours for the presence of infectious virus by hemagglutination tests of supernatants from each well. IAV-S titers were calculated by the Spearman-Karber method and expressed as log10 TCID50 per ml.

RESULTS

Immune responses of pigs vaccinated with polyvalent NA-RP (cm., figure 5)

Pigs that were passively treated with N1-classic hyperimmune sera one day prior to receiving primary vaccination had a titer of 40-80 at the time of vaccination.

After two vaccinations with 4-component NA-RP vaccine with Xsolve 50, pigs that were negative for N1-classic antigen antibody induced approximately 3-fold higher NI titer compared to N1-classic antigen compared to pigs that were positive. by antibody to the N1-classical antigen during their primary vaccination.

The NI titer of pigs in the N1-classic antigen negative/placebo vaccinated group remained seronegative.

Efficacy of the NA-RP quadruple vaccine against infections , lung lesions ( see figure 6).

The NA-RP 4-component vaccination was very effective in reducing lung damage when pigs were vaccinated without antibody to the N1 classical antigen at the time of their primary vaccination. The percentage of lung involvement was significantly reduced compared to both placebo vaccinated groups.

Pigs vaccinated in the presence of an NI titer to the N1-classical antigen also had significantly reduced lung involvement compared to both placebo vaccinated groups.

Brief conclusions

Quadruple adjuvant vaccination with NA-RP in pigs seropositive and seronegative for the N1-classic antigen demonstrated statistically significant protection against the H1N1 virus in relation to the severity of lung disease compared with placebo vaccinated groups. The N1-classic titer level was reduced, but still positive, when pigs were vaccinated in the presence of passively transferred anti-N1-classic antibodies. In addition, the NA-RP quadruple vaccine used with Xsolve adjuvant was unexpectedly highly immunogenic for the N1 classical antigen when vaccinated in pigs seronegative for the N1 classical antigen.

EXAMPLE 3

EFFECTIVENESS EVALUATION OF A 4-COMPONENT NEURAMINIDASE (NA) VACCINE FORMULATED WITH RP CONSTRUCTS WITH A DUAL NA GENE

The vaccine in this study consisted of two RNA particle constructs, each encoding two NA genes from a modern IAV-S isolate from the United States. One construct has two N1 phylogenetic clusters (N1-classic and N1-pandemic) and the other has two N2 clusters (N2-1998 and N2-2002). Vaccines are formulated in different doses without adjuvant. The aim was to determine the efficacy and immunogenicity of 4-component NA-RP vaccines formulated at various doses against H1N2 virus (N2-2002) infection in weaned pigs seronegative to vaccine fractions.

Materials and Methods

Dual gene (DG) NA-RP antigen design and vaccine composition

Each construct consists of an open reading frame of an alphavirus non-structural protein, a subgenomic promoter followed by one NA glycoprotein gene sequence, an intermediate sequence, a second subgenomic promoter followed by a second NA glycoprotein gene, and finally an alphavirus 3' untranslated region. The NA DG incorporated into a single RP construct was derived from currently circulating swine influenza virus (SIV) isolates in the United States and was used to synthesize NA genes in the pVHV plasmid vector (see Table 5). The pVHV plasmid vector is a derivative of a non-virulent human vaccine strain for Venezuelan equine encephalitis virus (TC-83). Completed replicon plasmids were checked for sequence composition and transcribed into RNA in vitro . Each NA replicon RNA was introduced by electroporation into Vero cells along with an accessory RNA encoding the VEE capsid helper and a glycoprotein sequence causing the packaging of the NA replicon RNA into RPs. Multivalent immunofluorescence assay was used to quantify functional N1- and N2-specific RPs and determine the dose of the vaccine. The test material was serially diluted, added to a monolayer of Vero cells in 48-well plates and incubated at 37°C for 18-24 hours. Cells were fixed and stained with a primary antibody to N1 or N2 followed by a secondary antibody conjugated to FITC. Individual antigen-positive cells were counted and the titer was calculated in units of RP/ml.

TABLE 5

N1 and N2 genes encoded in NA-RP constructs

Design Viral donor and gene position Phylogenetic cluster NA
(access number GENBANK) and gene position N1-DG-RP 5': A/swine/MN/A01483170/2014 (H1N1)
3': A/swine/IA/A01410307/2014 (H1N1) 5'-N1-pandemic (KP036969)-N1-classic (KJ605083)- 3' N2-DG-RP 5': A/swine/MI/A02077465/2015 (H1N2)
3': A/swine/IL/A01475495/2014 (H1N2) 5'- N21998 (KR982634) - N22002 (KJ941362) - 3'

The vaccines are formulated to give four different dose levels ( see Table 6) in a stabilizer consisting of monosodium glutamate, sucrose, PVP, sodium sulfate, and HEPES. The placebo vaccine consisted of phosphate buffered saline (PBS) without any RP constructs.

TABLE 6

Vaccine doses

Vaccine Number of copies of RP per ml N1-DG-RP N2-DG-RP TOTAL Vaccine 1 5.0×10 ⁶ 2.5×10 ⁷ 3.0×10 ⁷ Vaccine 2 5.6×10 ⁵ 2.8×10 ⁶ 3.3×10 ⁶ Vaccine 3 2.2×10 ⁵ 1.1×10 ⁶ 1.3×10 ⁶ Vaccine 4 5.6×10 ⁴ 2.8×10 ⁵ 3.3×10 ⁵

Virus

The challenge virus, A/swine/IL/A01475495/2014 (H1N2), was obtained from the USDA National Veterinary Services Laboratory. The H1N2 isolate possesses the HA gene from the H1-delta1 cluster and the NA gene from the N2-2002 cluster. The virus was grown in MDCK cell culture. Confluent cells were infected for approximately 48 hours until more than 70% of the cell monolayer was cytopathic. When harvesting the cells, the supernatant was removed from the vessels and clarified by centrifugation before storing the virus at -60° C. or below.

Animals

Weaned piglets were selected from a high health herd based on serological screening to confirm the absence of pre-existing neuraminidase inhibitory (NI) antibodies to the vaccine and control strains. These animals were a mix of males and females, approximately three weeks old at the time of primary vaccination.

Vaccination, infection and sample collection; pathological examination of the lungs; virus isolation and neuraminidase inhibition (NI) test .

Carried out as described above.

results

Efficacy of 4-component NA-DG-RP vaccine against challenge

A macroscopic lung lesion is shown in Figure 7. H1N2 challenge resulted in an average of 16.7% lung lesions in placebo controlled pigs. Vaccinated pigs, regardless of dose level, had an average of 3.3% lung disease or less, which was significantly lower than in the placebo group (p<0.0001).

Nasal virus isolation ( see figure 8 )

All pigs were negative for the virus at the time point before challenge. None of the vaccinated pigs shed virus per 1 DPC, while one of the pigs in the placebo group shed 102.5 TCID50/ml of virus. AT 3 DPC, pigs vaccinated with the first two highest formulated doses (vaccine 1 and 2) had significantly less virus in the nasal cavity compared to placebo pigs. At 5 DPC, only pigs vaccinated with the highest vaccine dose (vaccine 1) significantly reduced virus shedding compared to the placebo group.

immune response to vaccination

Neuraminidase inhibition (NI) titers are shown in Figure 9. Vaccines formulated at various dose levels induced seroconversion in all vaccinated pigs (N2-2002 titer >40) and the titer peaked 14 days after the second vaccination. The mean titer found on 14 DPV2 and -1DPC did not differ significantly between the vaccinated groups. However, it appears that the level of NI titer that is detected tends to be related to the vaccine dose (higher dose, higher NI titer). Pigs in the placebo group remained seronegative throughout the sampling period at various time points.

Brief conclusions

H1N2 infection caused high scores of lung injury accompanied by high nasal shedding of the virus. Protection against lung injury was robust regardless of the vaccine dose level tested (lowest dose of N2-DG-RP was 2.5×10 ⁵ RP/ml). The N2-DG-RP construct requires a minimum level of 2.5×10 ⁷ RP/ml to significantly reduce virus shedding after the first 5 days post infection. All pigs had a positive N22002 titer two weeks after the second vaccination. Detected levels of antibodies appear to be dose dependent, with higher levels observed in pigs receiving a higher dose of vaccine.

EXAMPLE 4

EFFECTIVENESS EVALUATION OF A VACCINE WITH A TWO-COMPONENT NEURAMINIDASE (NA) FORMULATED WITH RP CONSTRUCTS WITH A SINGLE NA GENE

A study was conducted to determine the efficacy and immunogenicity of five different adjuvanted bivalent NA-RP vaccines, each of which individually encoded one distinct NA gene of the current IAV-S isolate in the EU. Together, these NA genes represent two N1 phylogenetic lineages and three N2 N2 lineages (see Table 7 below). The adjuvant vaccine was administered to pigs by two intramuscular (IM) vaccinations at 5 and 8 weeks of age (1 ml per dose; 5×10 ⁶ RP/dose). The efficacy of the five adjuvant NA-RP vaccines was individually tested against their respective challenge infection as indicated in Table 8 below.

MATERIALS AND METHODS

NA-RP antigen construction :

Replication of defective RNA particles (RP) containing an alphavirus RNA replicon expressing the neuraminidase (NA) gene was prepared as described in Example 1.

Serological response experiments :

The treatment groups are described in Table 8 below. NA-RP vaccines are formulated at a dose level of 5×10 ⁶ RP/dose in a 1 ml volume with adjuvant (XSolve50). The vaccines were administered IM at a dose of 1 ml to pigs at the age of 5 and 8 weeks. Serum samples were collected two weeks after the last vaccination to quantify NI titers of antibodies against a heterologous influenza strain belonging to the respective lineage and homologous NA protein.

Vaccination and infection :

Nine pigs per group were immunized twice IM at 5 and 8 weeks of age with RP vaccine encoding NA antigens from two different strains adjuvanted with X-Solve50. The placebo group consisted of seven pigs each and were vaccinated with RP encoding foreign antigen with X-solve50. Two weeks after the second vaccination, blood samples were collected from all pigs to quantify the titers of neuraminidase inhibitory (NI) antibodies against a heterologous strain of influenza belonging to the respective lines, and the average NI titers are shown in the table. After collecting blood samples, pigs were infected with a heterologous strain of influenza A, matched by lineage to one of the components of the vaccine. Pigs were sacrificed 3 days post infection, six lung samples were collected from each pig (one from each lobe of the lung) and viral load was determined using pooled lung homogenates. Values are geometric mean virus titer (log10 TCID ₅₀ /ml). Data are analyzed using the Mann-Whitney* U-test. Values are significantly different (P<0.05) from the corresponding unvaccinated group.

TABLE 7

N1 and N2 genes encoded in European NA-RP constructs

Line NA gene donor strain GenBank Access Number ^a 2009 pandemic similar to N1 A/swine/Italy/179057/2015
(H1N1) KU323247.1
ALX30323 Eurasian Avian similar to N1 A/swine/Italy/28762-3/2013(H1N1) KR700532.1
AKJ81669 Gent/1/1984 similar to N2 A/swine/Italy/248147-8/2015(H3N2) KU323318.1
ALX30429 Italy/4675/2003 similar to N2 A/swine/Italy/129452/2015
(H1N2) KU323215.1
ALX30277 Scotland/410440/1994 similar to N2 (clade 1) A/swine/England/61470/2013
(H1N2) KR700793.1
AKJ82042 Scotland/410440/1994 similar to N2 (clade 2) A/swine/Italy/246087/2014(H1N2) KU322976.1
ALX29925

^A) Nucleotide and amino acid sequences.

TABLE 8

Treatment groups and post-vaccination infection outcomes

Treatment group Vaccine component Infection strain NI titers against a strain matching the vaccine component Viral load First Second 1a Pdm N1 and Eurasian Avian N1 Pandemic, similar to H1N1 512 128 2.01±0.90* 1b placebo 16 16 3.64±0.66 2a Pdm N1 and Eurasian Avian N1 Eurasian Avian H1N1 256 32 1.98±086* 2b placebo 16 8 3.23±0.53 3a Gent1984 N2 and Scot1994 N2 (clade 2) Gent1984 similar to H3N2 1024 256 1.63±0.44* 3b placebo 8 32 3.33±0.62 4a Italy4675 N2 and Scot1994 N2 (clade 1) Italy4675 N2 similar to H1N2 2048 1024 1.50±0.00* 4b placebo 4 32 3.14±0.62 5a Italy4675 N2 and Scot1994 N2 (clade 1) Scot1994 N2 similar to H1N2 1024 1024 1.50±0.00* 5b placebo 4 64 4.17±0.62

results

The results of this experiment are shown in Table 8. All five NA-RP vaccines induced functional anti-NI antibody titers in the pig against a heterologous influenza strain belonging to the respective lineage and homologous NA protein.

Lung samples were collected from each pig and viral load determined using pooled lung homogenates. The indicated values are the geometric mean of the virus titer (log 10 TCID50/ml). As can be seen from Table 8, the values of vaccinated pigs are significantly different (P < 0.05) from the corresponding unvaccinated groups, indicating the success of NA-RP vaccines in terms of viral load when challenged with heterologous virus strains.

EXAMPLE 5

DEFINITION OF PHYLOGENETIC CLUSTERS FOR THE USA

Neuraminidase (NA) Cluster Definition for USA

US Cluster N1:

(1) Amino acid identity within N1 clusters

A. N1 _pandemic or N-pandemic is ~97%-100% (mean % difference is 3.12%)

b. N1 _classic or N1-classic is ~95%-100% (average % difference is 4.91%)

(2) Amino acid identity between N1 clusters is ~82% (average % difference is 18.26%).

US Cluster N2:

(1) Amino acid identity within N2 clusters

A. N2 ₁₉₉₈ or N2-1998 is ~94%-100% (mean % difference is 5.94%)

b. N2 ₂₀₀₂ or N2-2002 is ~94%-100% (mean % difference is 6.37%)

(2) Amino acid identity between N2 clusters is ~88% (mean % difference is 11.84%).

Ways:

Full-length non-redundant amino acid sequences from US N1 from H1N1 and H3N1 viruses (about 1550 sequences in total) and US N2 from H1N2 and H3N2 viruses (about 2700 sequences in total), collected from 2000 to present, were downloaded from the Influenza Research Database. Percent amino acid identity within and between clusters for US N1 and N2 data was analyzed using MEGA version 7.0.7 [see, Kumar et al., Bioinformatics 28: 2685-2686 (2012)]. Two sequence alignments using MAFFT version 7.294b Katoh et al. Nucleic Acids Res. 30:3059-3066 (2002); Katoh et al., Molecular Biology and Evolution 30:772-780 (2013 ] and two separate maximum likelihood N1 and N2 phylogenetic trees were obtained in FastTree 2.1.8 [Price et al., PLoS ONE, 5 (3) 2010: e9490]. Depending on the phylogeny, N1 and N2, N1 and N2 were assigned to one of the four previously described NA antigenic lineages, N1 _classic , N1 _pandemic , N2 ₁₉₉₈ , and N2 ₂₀₀₂ [see, Nelson et al., J Virol. 86 (16): 8872-8878 (2012) Anderson et al., Influenza Other Respir Viruses Suppl 4: 42-51 (2013)]. default and p-distance calculation for intra-cluster and inter-cluster diversity with gamma-distributed rate variation (alpha 0.5) and partial deletion set to 95%.

EXAMPLE 6

IDENTIFYING LINES FOR EUROPE

Introduction to the EU-NA part :

Similar to North America, three subtypes of influenza A virus H1N1, H3N2 and H1N2 circulate concurrently among pigs in European pig populations. However, the HA and NA gene lines differ significantly within each subtype [see, Kuntz-Simon and Madec Zoonoses Public Health., 56 (6-7): 310-325 (2009)]. The NA genes of European swine influenza isolates belong to four major and two minor lineages, as established by Watson et al., [J. Virol. 89: 9920-9931 (2015); DOI: 10.1128/JVI.00840-15]. Main lines include:

1. A (H1N1) Pandemic 2009, similar to N1,

2. Eurasian Avian, like N1,

3. A/swine/Gent/1/1984, similar to N2 (Gent/84), and

4. A/swine/Scotland/ 410440/1994, similar to N2 (Scot/94).

Unlike other European main lines, the NA segments of the Scotland/94 line include four major groups of viruses circulating in the European pig population. Small European NA influenza lines include:

1. A/Swine/Italy/4675/2003 similar to N2 and

2. Human seasonal, similar to N2.

Description of the method used to analyze EU NA-N1 and N2 gene segments :

Amino acid sequences of the full-length NA gene from European pig isolates that were collected between 2005 and 2018 were obtained from the NCBI Influenza Virus Resource [see, Ref.32 Watson et al., J. Virol., 89: 9920-9931 (2015 ) ); DOI: 10.1128/JVI.00840-15]. The N1 and N2 sequences were aligned using the Muscle (codon) aligner with the default settings provided in MEGA X [Kumar et al., Molecular Biology and Evolution 35: 1547-1549 (2018)]. The evolutionary history was derived using the maximum likelihood method based on the Whelan and Goldman model [Molecular Biology and Evolution 18: 691-699 (2001)]. The Bootstrap method with 250 repetitions was used for the phylogeny test. A discrete Gamma distribution was used to model evolutionary differences in speed among sites [5 categories (+G, parameter=0.7258)]. The rate change model allowed some sites to be evolutionarily unchanged {[+I], 38.08% of sites}. The analysis included 244 amino acid sequences. All positions with site coverage less than 95% were excluded. That is, less than 5% of alignment gaps, missing data, and ambiguous bases were allowed for each clause. In total, there were 469 positions in the final data set. Evolutionary analysis was performed in MEGA X [Kumar et al., Molecular Biology and Evolution 35: 1547-1549 (2018)]. N1 isolates were assigned either Eurasian Avian-N1-like or pandemic 2009-N1-like lineage, and N2 isolates were assigned Scotland/410440/1994-N2-like lineage (e.g. clade 1 or clade 2), Gent/1/1984- N2-like lineage, Italy/4675/2003-N2-like lineage, or human seasonal N2-like lineage as previously described [Watson et al., J. Virol., 89: 9920-9931 (2015)]. Genetic identity within and between lines was calculated using MEGA X. A representative strain from each of the above lines was then selected as a vaccine candidate.

The following target sequences for the respective lineages and/or clades have the resulting range of variation:

A/swine/Italy/179057/2015 (H1N1) sequence with 10% variability

A/swine/Italy/28762-3/2013 (H1N1) sequence with 15% variability

A/swine/Italy/248147-8/2015 (H3N2) sequence with 10% variability

A/swine/Italy/129452/2015(H1N2) sequence with 10% variability

A/swine/England/61470/2013 (H1N2) sequence with 10% variability

A/swine/Italy/246087/2014 (H1N2) sequence with 10% variability.

The present invention should not be limited in scope to the specific embodiments described herein. In fact, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the above description. Such modifications are intended to be included within the scope of the appended claims.

In addition, it should be understood that all basic sizes or amino acid sizes, as well as molecular weights or molecular weight values given for nucleic acids or polypeptides, are approximate and are provided for description.

--->

SEQUENCE LIST

<110> Intervet, Inc.

Intervet International BV

Mogler, Mark A.

Kitikoon, Pravina

Puttamreddy, Supraja

Strait, Erin

Segers, Ruud A.M.

Nagaraj, Basav

<120> SWINE FLU A VIRUS VACCINE

<130> 24554WO

<150> US 62/607,101

<151> 2017-12-18

<160> 27

<170>PatentIn version 3.5

<210> 1

<211> 1407

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized swine influenza virus sequence

<400> 1

atgaatacta atcaaaggat cattaccatt gggacagtct gcatgattgt cggtatcatc 60

tctcttttgc ttcagattgg aaacattgtc tcactttgga ttagccattc aattcagacc 120

ggatgggaga atcacactga gatgtgcaat caaagtgtca ttacttatgt caataacact 180

tgggtgaacc ggacttatgt gaacattagc aatatcaaga ttgcaactat tcaagatgtg 240

actagtatca ttttggccgg aaattctagt ctttgcccgg tgtcaggggtg ggctgtctac 300

agcaaagaca atagcattag gattggatca aaaggggaca ttttcgtcat tagagagcct 360

ttcatctcat gctcacaatt ggagtgccgg accttctttc tgacccaagg ggcattgctg 420

aatgacaaac attcaaatgg taccgtcaag gacaggagtc cttatagaac cctgatgagc 480

tgccctatcg gtgaggcccc ttcgccatac aactcacggt tcgaatctgt cgcatggtca 540

gcatctgctt gtcatgatgg aatgggatgg cttacaatcg ggatcagtgg accggataat 600

ggtgctgtcg cagtcttgaa atacaacgga atcattacag atacaatcaa atcttggagg 660

aacaagattc ttagaactca agaatcagag tgtgtctgta tgaacggatc atgttttaca 720

gtcttgacag atggcccaag caatggacaa gcctcttaca aaatctttaa ggtggaaaaa 780

ggaaagatta tcaagtcgat tgagctggat gcccccaatt accactatga agaatgctct 840

tgttatccag atactggcaa agtcatgtgt gtctgccggg acaattggca cgcctcaaac 900

cggccatggg tgtcgttcaa tcagaatctt gactatcaaa ttggatacat ttgctctgga 960

gtctttggtg ataaccctag atccaatgat gggaagggca attgtggccc ggtcctttct 1020

aatggagcaa atggagtgaa aggtttctca tatcggtatg gaaatggtgt gtggattggt 1080

1140

actgagacag attcatcatt ctcgatgaag caggacatta tcgctcttaa tgattggtct 1200

ggatactcgg gatcttttgt ccaacatccg gagcttactg gtatgaattg cattaggcct 1260

tgtttctggg tggaattgat cagagggcaa cccaaggaaa gcactatctg ggctagcggt 1320

tccagcatct cattctgtgg cgtcaattcg gaaaccgctt cctggtcttg gccagacgga 1380

gctgatctgc cattcaccat tgacaag 1407

<210> 2

<211> 469

<212> Protein

<213> swine flu virus

<400> 2

Met Asn Thr Asn Gln Arg Ile Ile Thr Ile Gly Thr Val Cys Met Ile

1 5 10 15

Val Gly Ile Ile Ser Leu Leu Leu Gln Ile Gly Asn Ile Val Ser Leu

20 25 30

Trp Ile Ser His Ser Ile Gln Thr Gly Trp Glu Asn His Thr Glu Met

35 40 45

Cys Asn Gln Ser Val Ile Thr Tyr Val Asn Asn Thr Trp Val Asn Arg

50 55 60

Thr Tyr Val Asn Ile Ser Asn Ile Lys Ile Ala Thr Ile Gln Asp Val

65 70 75 80

Thr Ser Ile Ile Leu Ala Gly Asn Ser Ser Leu Cys Pro Val Ser Gly

85 90 95

Trp Ala Val Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly

100 105 110

Asp Ile Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser Gln Leu Glu

115 120 125

Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His

130 135 140

Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser

145 150 155 160

Cys Pro Ile Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser

165 170 175

Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr

180 185 190

Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr

195 200 205

Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Lys Ile Leu

210 215 220

Arg Thr Gln Glu Ser Glu Cys Val Cys Met Asn Gly Ser Cys Phe Thr

225 230 235 240

Val Leu Thr Asp Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe

245 250 255

Lys Val Glu Lys Gly Lys Ile Ile Lys Ser Ile Glu Leu Asp Ala Pro

260 265 270

Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val

275 280 285

Met Cys Val Cys Arg Asp Asn Trp His Ala Ser Asn Arg Pro Trp Val

290 295 300

Ser Phe Asn Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly

305 310 315 320

Val Phe Gly Asp Asn Pro Arg Ser Asn Asp Gly Lys Gly Asn Cys Gly

325 330 335

Pro Val Leu Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Tyr Arg

340 345 350

Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Asn Ser Arg

355 360 365

Ser Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp

370 375 380

Ser Ser Phe Ser Met Lys Gln Asp Ile Ile Ala Leu Asn Asp Trp Ser

385 390 395 400

Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Met Asn

405 410 415

Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Gln Pro Lys

420 425 430

Glu Ser Thr Ile Trp Ala Ser Gly Ser Ser Ile Ser Phe Cys Gly Val

435 440 445

Asn Ser Glu Thr Ala Ser Trp Ser Trp Pro Asp Gly Ala Asp Leu Pro

450 455 460

Phe Thr Ile Asp Lys

465

<210> 3

<211> 1407

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized swine influenza virus sequence

<400> 3

atgaatccta accaaaagat cattaccatt ggttcggtct gtatgacaat tggaatggct 60

aacctgatcc ttcaaattgg aaacattatc tcaatctgggg tcagccactc aattcaaatt 120

ggaaatcaat cgcagattga aacatgcaac caaagcgtca ttacttacga aaacaacact 180

tgggtgaacc agacctacgt gaacatcagc aacaccaact tcgctgctgg acagtccgtg 240

gtttccgtga aactggcggg caactcctct ctctgccctg tgagcggatg ggctatctac 300

tccaaagaca actcagtcag aatcggttcc aagggggatg tgtttgtcat aagggaacca 360

ttcatctcat gctctcccct tgaatgcaga accttcttct tgactcaagg ggccttgcta 420

aatgacaaac attccaacgg aaccattaaa gacaggagcc catatcggac cctgatgagc 480

tgtcctatcg gtgaagtccc ctcgccatac aactcaagat ttgagtcagt cgcttggtca 540

gcatccgctt gtcatgatgg catcaattgg ctcaccattg gaatttctgg cccagacagt 600

ggggcagtgg ctgtgctgaa gtacaatggc attataacag acactatcaa gtcgtggagg 660

aacaacatat tgagaactca agagtctgaa tgtgcatgtg tgaatggttc ttgctttacc 720

atcatgaccg atggaccatc cgatggacag gcctcctaca agatcttcag aatcgaaaag 780

ggaaagatcg tcaaatcagt cgaaatgaat gcccctaact accactatga ggaatgctcc 840

tgttatcctg attcctccga aatcacgtgc gtgtgcaggg ataactggca tggctccaat 900

cggccgtggg tgtctttcaa ccagaatctg gaatatcaga tcggatacat ttgctccggg 960

gtgttcggag acaatccgcg ccctaatgat aagacaggct cgtgtggtcc agtctcgtct 1020

1080

agaactaaga gcatttcctc aagaaaaggt ttcgagatga tttgggatcc gaatggatgg 1140

actgggactg acaacaagtt ctcaatcaag caagacatcg tgggaatcag cgagtggtca 1200

ggatatagcg ggtcctttgt gcagcacccc gaactgaccg ggctggattg tattagacct 1260

tgcttctggg tcgaactcat cagagggcga cccgaagaga acacaatctg gactagcggg 1320

agcagcatct ccttttgtgg tgtgaactcg gacactgtgg gttggtcttg gccagacggt 1380

gctgagttgc cttttaccat tgacaag 1407

<210> 4

<211> 469

<212> Protein

<213> swine flu virus

<400> 4

Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Cys Met Thr

1 5 10 15

Ile Gly Met Ala Asn Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Val Ser His Ser Ile Gln Ile Gly Asn Gln Ser Gln Ile Glu Thr

35 40 45

Cys Asn Gln Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln

50 55 60

Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Ala Ala Gly Gln Ser Val

65 70 75 80

Val Ser Val Lys Leu Ala Gly Asn Ser Ser Leu Cys Pro Val Ser Gly

85 90 95

Trp Ala Ile Tyr Ser Lys Asp Asn Ser Val Arg Ile Gly Ser Lys Gly

100 105 110

Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser Pro Leu Glu

115 120 125

Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His

130 135 140

Ser Asn Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser

145 150 155 160

Cys Pro Ile Gly Glu Val Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser

165 170 175

Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Ile Asn Trp Leu Thr

180 185 190

Ile Gly Ile Ser Gly Pro Asp Ser Gly Ala Val Ala Val Leu Lys Tyr

195 200 205

Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu

210 215 220

Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr

225 230 235 240

Ile Met Thr Asp Gly Pro Ser Asp Gly Gln Ala Ser Tyr Lys Ile Phe

245 250 255

Arg Ile Glu Lys Gly Lys Ile Val Lys Ser Val Glu Met Asn Ala Pro

260 265 270

Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Ser Glu Ile

275 280 285

Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val

290 295 300

Ser Phe Asn Gln Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly

305 310 315 320

Val Phe Gly Asp Asn Pro Arg Pro Asn Asp Lys Thr Gly Ser Cys Gly

325 330 335

Pro Val Ser Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Lys

340 345 350

Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg

355 360 365

Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp

370 375 380

Asn Lys Phe Ser Ile Lys Gln Asp Ile Val Gly Ile Ser Glu Trp Ser

385 390 395 400

Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp

405 410 415

Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Glu

420 425 430

Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val

435 440 445

Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro

450 455 460

Phe Thr Ile Asp Lys

465

<210> 5

<211> 1407

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized swine influenza virus sequence

<400> 5

atgaatccaa accaaaagat aatcacaatt ggctctgttt ctctcactat tgccacaatg 60

tgcctcctta tgcaaattgc catcctgatt actaatgtca cattgcactc caatcagtac 120

gaatgcaact accccccaaa caaccaagtg atactgtgtg aaccaactat catcaaaaga 180

aacattactg agattgtgta tctggccaac accaccatag agaaggaaat ctgccccaag 240

ctggcagaat acagaaactg gtcgaagccg caatgtaaaa ttacagggtt tgcacctttt 300

tccaaggaca attcgattag gctttctgcg ggtggcgaca tttgggtaac gagagaacct 360

tatgtgtcat gcgatcctga taagtgttac cagtttgccc ttggacaagg aacaacgctc 420

aacaacagac attcaaacga caccgtgcat gataggaccc cttatcgaac cctattgatg 480

aatgagttgg gtattccatt ccatttgggg accaaacaag tgtgcatcgc atggtccagc 540

tcatcctgcc atgatggacg ggcttggctt catgtctgta ttactgggca tgacaacaat 600

gcaactgcca gcatcattta caatggacgc cttgtcgata gtattggttc atggtccaaa 660

agaatcctca ggacccagga gtcggaatgc gtgtgcatca atggaacttg taccgtggtc 720

atgactgatg ggtccgcttc aggaatagct gacactaaaa tcctgttcat tgaagagggg 780

aaaatcgtgc acattagccc actgctgggg tccgctcagc acgtggagga gtgctcctgc 840

tatccccgat acccaggtgt cagatgcatc tgtagagaca actggaaagg ctccaacaga 900

cctgtcgtgg atattaatgt gaaggattat agcattgtgt cctcctacgt gtgctccgga 960

ctggtgggag acacccccag aaaagacgac agatccagct ccagcaattg tctgaatcct 1020

1080

tggatgggga ggacaatcaa cgagacatta cgctcaggtt atgaaacctt caaagtcatt 1140

gaaggctggt ccaaacctaa ttccaaattg cagataaatc gccaagtcat tgttgaaaga 1200

gatgataggt ccggttattc tggaattttc tctgtcgaag gaaagagctg tatcaatcgg 1260

tgtttttacg tggagctgat cagaggaagg aaacaggaaa ctgcagtgtg gtggacgtca 1320

aattccattg tggtgttttg tggcacctca ggtacctatg gaaccggctc atggcctgat 1380

ggggcggaca tcaatctcat gcctgtg 1407

<210> 6

<211> 469

<212> Protein

<213> swine flu virus

<400> 6

Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr

1 5 10 15

Ile Ala Thr Met Cys Leu Leu Met Gln Ile Ala Ile Leu Ile Thr Asn

20 25 30

Val Thr Leu His Ser Asn Gln Tyr Glu Cys Asn Tyr Pro Pro Asn Asn

35 40 45

Gln Val Ile Leu Cys Glu Pro Thr Ile Ile Lys Arg Asn Ile Thr Glu

50 55 60

Ile Val Tyr Leu Ala Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Lys

65 70 75 80

Leu Ala Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Lys Ile Thr Gly

85 90 95

Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly

100 105 110

Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Lys

115 120 125

Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asn Asn Arg His

130 135 140

Ser Asn Asp Thr Val His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met

145 150 155 160

Asn Glu Leu Gly Ile Pro Phe His Leu Gly Thr Lys Gln Val Cys Ile

165 170 175

Ala Trp Ser Ser Ser Ser Ser Cys His Asp Gly Arg Ala Trp Leu His Val

180 185 190

Cys Ile Thr Gly His Asp Asn Asn Ala Thr Ala Ser Ile Ile Tyr Asn

195 200 205

Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Lys Arg Ile Leu Arg

210 215 220

Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val

225 230 235 240

Met Thr Asp Gly Ser Ala Ser Gly Ile Ala Asp Thr Lys Ile Leu Phe

245 250 255

Ile Glu Glu Gly Lys Ile Val His Ile Ser Pro Leu Leu Gly Ser Ala

260 265 270

Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg

275 280 285

Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Val Asp

290 295 300

Ile Asn Val Lys Asp Tyr Ser Ile Val Ser Ser Tyr Val Cys Ser Gly

305 310 315 320

Leu Val Gly Asp Thr Pro Arg Lys Asp Asp Arg Ser Ser Ser Ser Asn

325 330 335

Cys Leu Asn Pro Asn Asn Glu Lys Gly Glu His Gly Val Lys Gly Trp

340 345 350

Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Asn Glu

355 360 365

Thr Leu Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Glu Gly Trp Ser

370 375 380

Lys Pro Asn Ser Lys Leu Gln Ile Asn Arg Gln Val Ile Val Glu Arg

385 390 395 400

Asp Asp Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser

405 410 415

Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Gln

420 425 430

Glu Thr Ala Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly

435 440 445

Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile

450 455 460

Asn Leu Met Pro Val

465

<210> 7

<211> 1407

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized swine influenza virus sequence

<400> 7

atgaatccaa atcaaaagat cattactatt ggatcagtct cactcatcat tgccacaatt 60

tgtttcctta tgcaaattgc aatccttgtc actactgtca cattgcattt caagcagcat 120

gactacaact cccccccaaa caaccaagct actctgtgtg aaccaacaat cattgaacgg 180

aaaacaactg aaattgtgta tcttactaac accaccattg agaaagaagt ctgccccaaa 240

cttgcagagt accggaactg gtcaaagcct caatgtaaca ttactggatt tgcaccattt 300

tcgaaagaca attctattcg gttgtctgct ggtggggaca tctgggtgac tagggaacct 360

tatgtgtcat gcgatcctga caagtgttac caatttgccc ttggacaggg tacaactctt 420

aacaacggac attcgaataa cacagtccat gataggaccc cgtatcggac ccttcttatg 480

aatgagcttg gtgtcccttt tcatcttgga accagacaag tgtgcatggc ttggtctagc 540

tcatcttgtc acgatgggaa agcatggctg catgtctgtg tcactggaaa tgataacaat 600

gctactgcta gcttcatcta caatggtagg cttgtggatt ctattggttc gtggtcgaaa 660

aacattctcc ggacccaaga gtcagaatgc gtctgtatca atggaacatg tactgtcgtc 720

atgactgatg gatccgctag tggaaaagca gataccaaaa tcttgttcgt cgaagagggg 780

aagatcgtcc atatcagcac tctgttggga tctgcacagc acgtcgagga atgctcctgt 840

tatcctaggt ttccgggagt ccggtgtgtc tgccgggaca actggaaagg atctaataga 900

cccatcgtcg acatcaatgt caagaattac agcattgtct cttcgtatgt ctgcagtgga 960

cttgtcggtg atactcccag agagagcgac tcagtctcct catcttattg cttggatccg 1020

1080

tggatgggaa gaacaatcaa cgagactttg cgcttgggat atgaaacctt caaagtcatt 1140

gaaggctggt ccacagctaa ctccaagtca cagacaaata gacaagtgat tgtcgaaaaa 1200

ggagacaggt caggatattc tgggattttc tcagtcgagg gaaagaactg catcaatagg 1260

1320

aactcaattg tcgtgttttg tggcacctca gggacttatg gtactggctc ttggccggat 1380

ggtgctgaca tcaatctcat gccaatt 1407

<210> 8

<211> 469

<212> Protein

<213> swine flu virus

<400> 8

Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Ile

1 5 10 15

Ile Ala Thr Ile Cys Phe Leu Met Gln Ile Ala Ile Leu Val Thr Thr

20 25 30

Val Thr Leu His Phe Lys Gln His Asp Tyr Asn Ser Pro Pro Asn Asn

35 40 45

Gln Ala Thr Leu Cys Glu Pro Thr Ile Ile Glu Arg Lys Thr Thr Glu

50 55 60

Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Val Cys Pro Lys

65 70 75 80

Leu Ala Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Asn Ile Thr Gly

85 90 95

Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly

100 105 110

Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Lys

115 120 125

Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asn Asn Gly His

130 135 140

Ser Asn Asn Thr Val His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met

145 150 155 160

Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Arg Gln Val Cys Met

165 170 175

Ala Trp Ser Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val

180 185 190

Cys Val Thr Gly Asn Asp Asn Asn Ala Thr Ala Ser Phe Ile Tyr Asn

195 200 205

Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Lys Asn Ile Leu Arg

210 215 220

Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val

225 230 235 240

Met Thr Asp Gly Ser Ala Ser Gly Lys Ala Asp Thr Lys Ile Leu Phe

245 250 255

Val Glu Glu Gly Lys Ile Val His Ile Ser Thr Leu Leu Gly Ser Ala

260 265 270

Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Phe Pro Gly Val Arg

275 280 285

Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp

290 295 300

Ile Asn Val Lys Asn Tyr Ser Ile Val Ser Ser Tyr Val Cys Ser Gly

305 310 315 320

Leu Val Gly Asp Thr Pro Arg Glu Ser Asp Ser Val Ser Ser Ser Tyr

325 330 335

Cys Leu Asp Pro Asn Asn Glu Lys Gly Gly His Gly Val Lys Gly Trp

340 345 350

Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Asn Glu

355 360 365

Thr Leu Arg Leu Gly Tyr Glu Thr Phe Lys Val Ile Glu Gly Trp Ser

370 375 380

Thr Ala Asn Ser Lys Ser Gln Thr Asn Arg Gln Val Ile Val Glu Lys

385 390 395 400

Gly Asp Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Asn

405 410 415

Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Glu

420 425 430

Glu Thr Lys Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly

435 440 445

Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile

450 455 460

Asn Leu Met Pro Ile

465

<210> 9

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> 5' flanking sequence

<400> 9

ggcgcgccgc acc 13

<210> 10

<211> 8

<212> DNA

<213> Artificial sequence

<220>

<223> 3' flanking sequence

<400> 10

ttaattaa 8

<210> 11

<211> 1410

<212> DNA

<213> swine flu virus

<400> 11

atgaatccaa accaaaagat aataaccatt ggttcggtct gtatgacaat tggaatggct 60

aacttaatat tacaaattgg aaacataatc tcaatatgga ttagccactc aattcaactt 120

gggaatcaaa gtcagattga aacatgcaat caaagcgtca ttacttatga aaacaacact 180

tgggtaaatc agacatatgt taacatcagc aacaccaact ttgctgctgg acagtcagtg 240

gtttccgcga aattagcggg caattcctcc ctctgccctg ttagtggatg ggctatatac 300

agtaaagaca acagtgtaag aatcggttcc aagggggatg tgtttgtcat aagggaacca 360

ttcatatcat gctccccctt agaatgcaga accttcttct tgactcaagg ggccttgcta 420

aatgacaaac attccaatgg aaccattaaa gataggagcc catatcgaac cctgatgagc 480

tgtcctattg gtgaagttcc ctctccatac aactcaagat ttgagtcggt cgcttggtca 540

gcaagtgctt gtcacgatgg catcaattgg ctaacaatcg gaatttctgg cccagacagt 600

ggggcagtgg ctgtattaaa gtacaatggc ataataacag acactatcaa gagttggaaa 660

aacaatatat tgagaacaca agagtctgaa tgtgcatgtg taaatggttc ttgctttacc 720

ataatgaccg atggaccaag tgatggacag gcctcataca agatcttcag aatagaaaag 780

ggaaagatag tcaaatcagt cgaaatgaat gcccctaatt atcactatga ggaatgctcc 840

tgttatcctg attctagtga aatcacatgt gtgtgcaggg ataactggca tggctcgaat 900

cgaccgtggg tgtctttcaa ccagaatctg gaatatcaga taggatacat atgcagtggg 960

attttcggag acaatccacg ccctaatgat aagacaggca gttgtggtcc agtatcgtct 1020

1080

agaactaaaa gcattagttc aagaaaaggt tttgagatga tttgggatcc aaatggatgg 1140

actgggacag acaaaaactt ctcaataaag caagatatca taggaataaa tgagtggtca 1200

ggatacagcg ggagttttgt tcagcatcca gaactaacag ggctgaattg tataagacct 1260

tgcttctggg ttgaactaat cagagggcga cccaaagaga acacaatctg gactagcggg 1320

agcagcatat ccttttgtgg tgtaaacagt gacactgtgg gttggtcttg gccagacggt 1380

gctgagttgc catttaccat tgacaagtaa 1410

<210> 12

<211> 469

<212> Protein

<213> swine flu virus

<400> 12

Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Cys Met Thr

1 5 10 15

Ile Gly Met Ala Asn Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Ile Ser His Ser Ile Gln Leu Gly Asn Gln Ser Gln Ile Glu Thr

35 40 45

Cys Asn Gln Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln

50 55 60

Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Ala Ala Gly Gln Ser Val

65 70 75 80

Val Ser Ala Lys Leu Ala Gly Asn Ser Ser Leu Cys Pro Val Ser Gly

85 90 95

Trp Ala Ile Tyr Ser Lys Asp Asn Ser Val Arg Ile Gly Ser Lys Gly

100 105 110

Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser Pro Leu Glu

115 120 125

Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His

130 135 140

Ser Asn Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser

145 150 155 160

Cys Pro Ile Gly Glu Val Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser

165 170 175

Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Ile Asn Trp Leu Thr

180 185 190

Ile Gly Ile Ser Gly Pro Asp Ser Gly Ala Val Ala Val Leu Lys Tyr

195 200 205

Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Lys Asn Asn Asn Ile Leu

210 215 220

Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr

225 230 235 240

Ile Met Thr Asp Gly Pro Ser Asp Gly Gln Ala Ser Tyr Lys Ile Phe

245 250 255

Arg Ile Glu Lys Gly Lys Ile Val Lys Ser Val Glu Met Asn Ala Pro

260 265 270

Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Ser Glu Ile

275 280 285

Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val

290 295 300

Ser Phe Asn Gln Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly

305 310 315 320

Ile Phe Gly Asp Asn Pro Arg Pro Asn Asp Lys Thr Gly Ser Cys Gly

325 330 335

Pro Val Ser Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Lys

340 345 350

Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg

355 360 365

Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp

370 375 380

Lys Asn Phe Ser Ile Lys Gln Asp Ile Ile Gly Ile Asn Glu Trp Ser

385 390 395 400

Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asn

405 410 415

Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys

420 425 430

Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val

435 440 445

Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro

450 455 460

Phe Thr Ile Asp Lys

465

<210> 13

<211> 1408

<212> DNA

<213> swine flu virus

<400> 13

atgaacccaa atcagaagat aataatcatt agttcaatct gtatgacaaa tggaattgct 60

agcttgatat tacaaattgg gaacataata tcaatatgga ttagccattc aattcaaatt 120

gagaacccaa accagaccga ccatgcaatc aaagcgttat tattacgaa aacaacacat 180

gggtaaatca aacgtatgtt aacatcagca acaataattt tgttgttgaa cagacagtgg 240

tttcaatgaa attagcgggc agttcttctc tctgccctgt tagtggatgg gctatataca 300

gtaaagataa cagtgtaaga atcggttcca aaggggatgt gtttgtcata agagagccat 360

tcatctcatg ctcccatttg gaatgtagaa ccttcttctt aactcaaggg gccctactga 420

atgataaaca ttctaatgga accgttaaag acagaagccc ctatcgaacc ctgatgagct 480

gtcctattgg tgaagtcccc tctccataca actcaaaatt tgagtcagtt gcttggtcag 540

caagtgcttg ccatgatggc accagttggt tgacaattgg gatttctggt ccagacaatg 600

660

acaacatatt gagaacacaa gaatctgaat gtgcatgttt gaatggttct tgctttactg 720

taatgaccga tggaccaagt aatgggcagg cctcatacaa gatcttcaaa atagaaaagg 780

ggaaagtagt caaatcagtc gagttgaatg ctcctaatta tcactatgag gaatgttcct 840

gttatcctga ttctggtgaa atcatatgtg tatgcaggga caattggcat ggctcgaatc 900

gaccatgggt gtctttcaat cagaatctgg agtatcagat aggatacata tgcagtgggg 960

ttctcggaga caatccgcgc cctaatgata gaacaggcag ttgtggtcca gtatcatctc 1020

atggagcaaa tggggtaaaa gggttttcgt ttaaatacgg caatggaatt tggataggga 1080

gaactaaaag cactattaca aggagtggtt ttgagatgat ttgggaccca aacggatgga 1140

ctggaacaga caataatttc tcagtgaagc aagatatcgt aggaataact aactggtcag 1200

gatacgcggg agttttgtcc aacatccaga attaaccgga ttggattgta ttagaccttg 1260

cttctgggtt gaactaatca gagggagacc caaagagaac acaatctgga ctagcggaag 1320

cagcatatcc ttttgtggtg taaatagtga cactgtgggt tggtcttggc cagacggtgc 1380

tgagttgcca tttaccattg acaagtaa 1408

<210> 14

<211> 469

<212> Protein

<213> swine flu virus

<400> 14

Met Asn Pro Asn Gln Lys Ile Ile Ile Ile Ser Ser Ile Cys Met Thr

1 5 10 15

Asn Gly Ile Ala Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Ile Ser His Ser Ile Gln Ile Glu Asn Pro Asn Gln Thr Glu Pro

35 40 45

Cys Asn Gln Ser Val Ile Ile Tyr Glu Asn Asn Thr Trp Val Asn Gln

50 55 60

Thr Tyr Val Asn Ile Ser Asn Asn Asn Phe Val Val Glu Gln Thr Val

65 70 75 80

Val Ser Met Lys Leu Ala Gly Ser Ser Ser Leu Cys Pro Val Ser Gly

85 90 95

Trp Ala Ile Tyr Ser Lys Asp Asn Ser Val Arg Ile Gly Ser Lys Gly

100 105 110

Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu

115 120 125

Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His

130 135 140

Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser

145 150 155 160

Cys Pro Ile Gly Glu Val Pro Ser Pro Tyr Asn Ser Lys Phe Glu Ser

165 170 175

Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Thr Ser Trp Leu Thr

180 185 190

Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr

195 200 205

Asn Asp Ile Ile Thr Asp Thr Ile Lys Ser Trp Lys Asn Asn Asn Ile Leu

210 215 220

Arg Thr Gln Glu Ser Glu Cys Ala Cys Leu Asn Gly Ser Cys Phe Thr

225 230 235 240

Val Met Thr Asp Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe

245 250 255

Lys Ile Glu Lys Gly Lys Val Val Lys Ser Val Glu Leu Asn Ala Pro

260 265 270

Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Gly Glu Ile

275 280 285

Ile Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val

290 295 300

Ser Phe Asn Gln Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly

305 310 315 320

Val Leu Gly Asp Asn Pro Arg Pro Asn Asp Arg Thr Gly Ser Cys Gly

325 330 335

Pro Val Ser Ser His Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Lys

340 345 350

Tyr Gly Asn Gly Ile Trp Ile Gly Arg Thr Lys Ser Thr Ile Thr Arg

355 360 365

Ser Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp

370 375 380

Asn Asn Phe Ser Val Lys Gln Asp Ile Val Gly Ile Thr Asn Trp Ser

385 390 395 400

Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp

405 410 415

Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys

420 425 430

Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val

435 440 445

Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro

450 455 460

Phe Thr Ile Asp Lys

465

<210> 15

<211> 1410

<212> DNA

<213> swine flu virus

<400> 15

atgaatccaa atcaaaagat aataacaatt ggttctgttt ctctcactat tacaacaatg 60

tgcctcttct tgcagattgc catcctagta actactataa cattgcattt caagcaatat 120

gaatgcgatt cccctgcaaa caaccaagta ataccgtgtg aaccaataat aatagaaaaa 180

aacataacaa aaatagtgta tttgaccaat accaccatag agaaagaggt atgcccaaaa 240

ttaggggaat acaggaattg gtcaaaacca caatgcaaga tcacaggatt tgcacctttt 300

tctaaggaca attcaattcg gctctctgcg ggtggggcca tttggggtcac gagagaacct 360

tatgtgtcat gcgaccctaa caagtgttat caatttgcat taggacaggg aaccacatta 420

gataacagac attcaaatga cacaatacat gatagaaccc cttttagaac cctgttgatg 480

agtgaattag gtgttccatt tcatttggga accagacaag tatgcatagc atggtccagt 540

tcaagttgtc acgatgggaa agcttggttg catgtttgtg tcactgggca tgataaaaat 600

gcaactgcta gtttcattta tgacggaaag cttgtagaca gcatcagttc atggtccaaa 660

aacatactcc ggactcagga atcagaatgt gtttgtatcg atggaatctg tacagtggtg 720

atgactgatg gaagtgcttc agggaaagct gatactaaga tactatttat tgaaaaaggg 780

aagatcattc atattagtcc attgttggga agtgctcagc atgtagaaga atgttcctgt 840

taccctagat accctgatgt caggtgtatt tgcagggata actggaaagg ttcaaatagg 900

cccatcgtag acataagaat gaaaaattat agcattggtt ccagttatat gtgctcagga 960

cttgttggcg acacacccag gaacaatgat gggtctagta atagcaattg tcggaatccc 1020

aataatgaaa gaggaaatca tggagtgaaa ggttgggcct ttgatgatgg aaatgacaca 1080

tggatgggaa gaactatcag caaggactca cgcttaggtt acgaaacctt caaagttgtt 1140

ggtggttggt cccaacccaa ttccaaatcc cagataaata gacaagttat tgttgacagc 1200

gataatagat caggttactc tggtattttc tctgttgagg ggaaagattg cattaatagg 1260

1320

aacagtattg ttgtgttctg tggcacttct ggcacctatg ggtcaggctc atggcccgat 1380

ggagcaaaca tcaatttcat gcctgtataa 1410

<210> 16

<211> 469

<212> Protein

<213> swine flu virus

<400> 16

Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr

1 5 10 15

Ile Thr Thr Met Cys Leu Phe Leu Gln Ile Ala Ile Leu Val Thr Thr

20 25 30

Ile Thr Leu His Phe Lys Gln Tyr Glu Cys Asp Ser Pro Ala Asn Asn

35 40 45

Gln Val Ile Pro Cys Glu Pro Ile Ile Ile Glu Lys Asn Ile Thr Lys

50 55 60

Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Val Cys Pro Lys

65 70 75 80

Leu Gly Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Lys Ile Thr Gly

85 90 95

Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly

100 105 110

Ala Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asn Lys

115 120 125

Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asp Asn Arg His

130 135 140

Ser Asn Asp Thr Ile His Asp Arg Thr Pro Phe Arg Thr Leu Leu Met

145 150 155 160

Ser Glu Leu Gly Val Pro Phe His Leu Gly Thr Arg Gln Val Cys Ile

165 170 175

Ala Trp Ser Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val

180 185 190

Cys Val Thr Gly His Asp Lys Asn Ala Thr Ala Ser Phe Ile Tyr Asp

195 200 205

Gly Lys Leu Val Asp Ser Ile Ser Ser Trp Ser Lys Asn Ile Leu Arg

210 215 220

Thr Gln Glu Ser Glu Cys Val Cys Ile Asp Gly Ile Cys Thr Val Val

225 230 235 240

Met Thr Asp Gly Ser Ala Ser Gly Lys Ala Asp Thr Lys Ile Leu Phe

245 250 255

Ile Glu Lys Gly Lys Ile Ile His Ile Ser Pro Leu Leu Gly Ser Ala

260 265 270

Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Asp Val Arg

275 280 285

Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp

290 295 300

Ile Arg Met Lys Asn Tyr Ser Ile Gly Ser Ser Tyr Met Cys Ser Gly

305 310 315 320

Leu Val Gly Asp Thr Pro Arg Asn Asn Asp Gly Ser Ser Asn Ser Asn

325 330 335

Cys Arg Asn Pro Asn Asn Glu Arg Gly Asn His Gly Val Lys Gly Trp

340 345 350

Ala Phe Asp Asp Gly Asn Asp Thr Trp Met Gly Arg Thr Ile Ser Lys

355 360 365

Asp Ser Arg Leu Gly Tyr Glu Thr Phe Lys Val Val Gly Gly Trp Ser

370 375 380

Gln Pro Asn Ser Lys Ser Gln Ile Asn Arg Gln Val Ile Val Asp Ser

385 390 395 400

Asp Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Asp

405 410 415

Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Arg Gln

420 425 430

Glu Thr Arg Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly

435 440 445

Thr Ser Gly Thr Tyr Gly Ser Gly Ser Trp Pro Asp Gly Ala Asn Ile

450 455 460

Asn Phe Met Pro Val

465

<210> 17

<211> 1410

<212> DNA

<213> swine flu virus

<400> 17

atgaattcaa atcaaaagat aataacaatt ggctctgttt ctctcactat tgccacacta 60

tgcctcctta tgcaaattgc tatcatggta actactgtaa catttcattt caagcagtat 120

gaatacaact cccccccgaa caaccaagta atgttgtgtg aaccaacgat aattgaaaga 180

aacataacag agacagtgta catgaccaac accaccatag tgaaagaaat atgccccaaa 240

ctagcggaat acagaaattg gtcaaaaccg caatgcaaaa ttacaggatt tgcacctttt 300

tcaaaggaca actcaattcg gctttccgct ggtggggaca tctggggtgac aagagaacct 360

tatgtgtcat gcgatcctaa caagtgttat caatttgccc ttgggcaggg aacaacgtta 420

aacaacaggc attcaaatga cacagtacat gatagaaccc cttaccgaac cctgttgatg 480

aatgaattgg gtgttccatt tcatttagga accaagcagg tttgcatagc ttggtccagt 540

tcaagttgtc atgatggaaa agcatggttg catgtttgtg taactgggca tgatgaaaat 600

gcaactgcca gtttcattta caacgagaga cttgtagata gtattggttc atggtccaag 660

aaaatcctca gaacccagga gtcggaatgc gtttgcataa atgggacttg tacagtggtg 720

atgacagatg ggagtgcttc aggtagagct gatactaaaa tactattcat tgaggagggg 780

aaaatcgttc atgttagcca actgacagga agtgctcagc atgtagagga gtgctcctgt 840

tatccccggt atcctggtgt cagatgtgtt tgcagagata attggaaagg ctccaatagg 900

cccattgtag atataaatgt aaaggatcat agcattgttt ccagttatgt gtgctcagga 960

cttgtcggag acacacccag aaaaaacgac agctctagca gtagtaactg cctgaatcct 1020

aacaatgaag aagggggtca tggggtgaaa ggctgggcct ttgatgatga aaatgacttg 1080

tggatgggaa gaacgatcag cgaaaagtta cgattaggtt atgaaacctt caaggtcatt 1140

gaaggctggt ccaagcctaa ttccaaattg cagataaata ggcaagtaat agttgacaaa 1200

gataatagat ccggttattc tggtattttc tctgttgaaa gtaaaagttg catcaatcgg 1260

1320

aacagcattg ttgtattttg tggcacctca ggtacatatg gaacaggctc atggcctgat 1380

ggggcagaca tcaatctcat gcctatatga 1410

<210> 18

<211> 469

<212> Protein

<213> swine flu virus

<400> 18

Met Asn Ser Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr

1 5 10 15

Ile Ala Thr Leu Cys Leu Leu Met Gln Ile Ala Ile Met Val Thr Thr

20 25 30

Val Thr Phe His Phe Lys Gln Tyr Glu Tyr Asn Ser Pro Pro Asn Asn

35 40 45

Gln Val Met Leu Cys Glu Pro Thr Ile Ile Glu Arg Asn Ile Thr Glu

50 55 60

Thr Val Tyr Met Thr Asn Thr Thr Ile Val Lys Glu Ile Cys Pro Lys

65 70 75 80

Leu Ala Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Lys Ile Thr Gly

85 90 95

Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly

100 105 110

Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asn Lys

115 120 125

Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asn Asn Arg His

130 135 140

Ser Asn Asp Thr Val His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met

145 150 155 160

Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gln Val Cys Ile

165 170 175

Ala Trp Ser Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val

180 185 190

Cys Val Thr Gly His Asp Glu Asn Ala Thr Ala Ser Phe Ile Tyr Asn

195 200 205

Glu Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Lys Lys Ile Leu Arg

210 215 220

Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val

225 230 235 240

Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Lys Ile Leu Phe

245 250 255

Ile Glu Glu Gly Lys Ile Val His Val Ser Gln Leu Thr Gly Ser Ala

260 265 270

Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg

275 280 285

Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp

290 295 300

Ile Asn Val Lys Asp His Ser Ile Val Ser Ser Tyr Val Cys Ser Gly

305 310 315 320

Leu Val Gly Asp Thr Pro Arg Lys Asn Asp Ser Ser Ser Ser Ser Ser Asn

325 330 335

Cys Leu Asn Pro Asn Asn Glu Glu Gly Gly His Gly Val Lys Gly Trp

340 345 350

Ala Phe Asp Asp Glu Asn Asp Leu Trp Met Gly Arg Thr Ile Ser Glu

355 360 365

Lys Leu Arg Leu Gly Tyr Glu Thr Phe Lys Val Ile Glu Gly Trp Ser

370 375 380

Lys Pro Asn Ser Lys Leu Gln Ile Asn Arg Gln Val Ile Val Asp Lys

385 390 395 400

Asp Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Ser Lys Ser

405 410 415

Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Gln

420 425 430

Glu Asn Glu Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly

435 440 445

Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile

450 455 460

Asn Leu Met Pro Ile

465

<210> 19

<211> 1410

<212> DNA

<213> swine flu virus

<400> 19

atgaatccaa atcagaagat aataacaatt ggctctgttt ctctcgtcat tgcaacatta 60

tgcttcttaa tgcagatggc catcctaata actactgtaa aattacattt caaacaatat 120

gagtgcggct tccctgcgaa caaccaagta ataacatgtg agccaacagt aatagaaagg 180

aacacaacag agatagtgta cttaactaac accaccatag agaaagaaac atgccacaaa 240

acagtggaat acaggaattg gtcaaagcct caatgcaaaa taacaggctt tgcacctttt 300

tccaaggaca attcaattcg actttctgct ggtggggaca tatgggtgac gagggaacct 360

tacgtgtcat gcgagcctgg caaatgttat cagtttgcac tcgggcaagg gaccacacta 420

gacaataaac attcaaacga tacaatacat gacagaaccc cctatcgaac tctattgatg 480

aatgaattgg gtgtcccatt tcatttaggg acaagacaag tgtgtattgc atggtccagc 540

tcaagttgtt atgatgggaa agcatggttg catgtctgta tcactggaca tgataaaaat 600

gcaactgcca gtttcattta cgatggtaga cttgtagata gcattggttc atggtctaaa 660

aatatactta gaacccagga atcagaatgc gtttgcatca atggggtctg tacagtagta 720

atgactgatg gaagtgcttc gggaaagct gatactaaaa tactattcat tgaagaaggg 780

aaaattgttc atattagccc attagcgggg agtgcacagc atgtggagga gtgctcctgt 840

tatccccgat atcctggcgt aaggtgtatc tgcagagaca actggaaagg ctctaacaga 900

cccgttgtgg atataaatat agaagattat agcattgatt ccagttatgt gtgttcaggg 960

cttgttggcg acacacccag aatcaatgac ggatccagta gtagctactg ccgtgatcct 1020

aacaacgaaa aaggaaatca cggagtgaag ggctgggctt ttgacgatgg aaatgatgtg 1080

tggatgggaa gaacgatcaa cgaagattca cgctcaggtt atgaaacatt caaagtcatt 1140

ggtggttggt ccactcctaa ttccaaattg cagataaata ggcaagtaat agttgatagc 1200

aacaataggt caggttattc tggtgttttc tccgttgaag gcaaaagctg catcaataga 1260

1320

aacagtattg ttgtattttg tggcacttca ggtacctatg gaacaggctc atggcctgat 1380

ggagcagaca tcaacctcat gcctatatga 1410

<210> 20

<211> 469

<212> Protein

<213> swine flu virus

<400> 20

Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Val

1 5 10 15

Ile Ala Thr Leu Cys Phe Leu Met Gln Met Ala Ile Leu Ile Thr Thr

20 25 30

Val Lys Leu His Phe Lys Gln Tyr Glu Cys Gly Phe Pro Ala Asn Asn

35 40 45

Gln Val Ile Thr Cys Glu Pro Thr Val Ile Glu Arg Asn Thr Thr Glu

50 55 60

Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Thr Cys His Lys

65 70 75 80

Thr Val Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Lys Ile Thr Gly

85 90 95

Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly

100 105 110

Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Glu Pro Gly Lys

115 120 125

Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asp Asn Lys His

130 135 140

Ser Asn Asp Thr Ile His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met

145 150 155 160

Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Arg Gln Val Cys Ile

165 170 175

Ala Trp Ser Ser Ser Ser Cys Tyr Asp Gly Lys Ala Trp Leu His Val

180 185 190

Cys Ile Thr Gly His Asp Lys Asn Ala Thr Ala Ser Phe Ile Tyr Asp

195 200 205

Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Lys Asn Ile Leu Arg

210 215 220

Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Val Cys Thr Val Val

225 230 235 240

Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Lys Ile Leu Phe

245 250 255

Ile Glu Glu Gly Lys Ile Val His Ile Ser Pro Leu Ala Gly Ser Ala

260 265 270

Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg

275 280 285

Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Val Asp

290 295 300

Ile Asn Ile Glu Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser Gly

305 310 315 320

Leu Val Gly Asp Thr Pro Arg Ile Asn Asp Gly Ser Ser Ser Ser Tyr

325 330 335

Cys Arg Asp Pro Asn Asn Glu Lys Gly Asn His Gly Val Lys Gly Trp

340 345 350

Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Asn Glu

355 360 365

Asp Ser Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser

370 375 380

Thr Pro Asn Ser Lys Leu Gln Ile Asn Arg Gln Val Ile Val Asp Ser

385 390 395 400

Asn Asn Arg Ser Gly Tyr Ser Gly Val Phe Ser Val Glu Gly Lys Ser

405 410 415

Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Arg Ser

420 425 430

Glu Ala Arg Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly

435 440 445

Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile

450 455 460

Asn Leu Met Pro Ile

465

<210> 21

<211> 1410

<212> DNA

<213> swine flu virus

<400> 21

atgaatccaa accagaaaat aataacgatc ggttctgtct ccttgatcat tgcaacaatg 60

tgctttttca tgcaagttgc cattctggta actactgtaa cattgcattt caggcagtgc 120

gaatgcaact cctccgcaac caaccaaata atgccatgta aaccaacaaa aatagagaga 180

aacataactg aaattgtgta cttaaccaat accaccataa aaacagaggt atgccccaaa 240

ctagtgaaat acagggattg ggcaaaacca caatgtagaa tcacaggggtt tgcacctttt 300

tccaaggaca attcgattcg gctttctgcc ggtggggcca tttgggtaac gagagaaccc 360

tatgtatcat gcgatcttag caagtgttac cagtttgcgc tcggacaggg gactacacta 420

gacaacagac attcaaatga cacaatacat gatagaactc cttatcggac cctattgatg 480

aatgaattgg gtgttccatt tcatttagga accaggcaag tgtgtatagc ttggtccagt 540

tcaagttgtc acgatggaaa agcatggctg catgtttgtg tcactgggta tgataaaaat 600

gctactgcta gcctcattta tgacggaagg cttgtggaca gcatcggttc atggtcccaa 660

aacatcctcc ggacccagga atcggaatgt gtttgtataa atggtacttg cacagtggta 720

atgactgatg ggagtgcttc aggaaaagct gataccagaa tactatttat tgaagaaggg 780

aagattattc acattagtcc attgacagga agtgcacagc atgttgaaga gtgttcttgt 840

tatcctcgat accccggtgt aagatgtgtt tgtagagaca actggaaggg ctctaacaga 900

cccgtcgtgg atataaatgt aaaagattat aaaattaact ccagttatgt atgctcaggc 960

cttgttggcg atacacccag aaacaacgat agatctagca atagcaactg ccaaaatcct 1020

aacaaccaga gagggaatca tggagtgaag ggctgggcct ttgacgatgg aaatgacata 1080

tggatgggaa gaaccatcag caatgattca cgtttaggtt atgaaacttt caaagtttatt 1140

ggtggttggt ccaaacccaa ctccaaagtt cagacaaata ggcaagtcat agttgacagc 1200

gataatagat caggttattc tggcgttttc tctgttgaag gcaaaagctg catcaatagg 1260

1320

aacagtattg ttgtgttttg tggtacttcg ggtacatatg gttcaggctc atggcctgat 1380

ggggctgaca tcaatcttat gcctatataa 1410

<210> 22

<211> 469

<212> Protein

<213> swine flu virus

<400> 22

Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Ile

1 5 10 15

Ile Ala Thr Met Cys Phe Phe Met Gln Val Ala Ile Leu Val Thr Thr

20 25 30

Val Thr Leu His Phe Arg Gln Cys Glu Cys Asn Ser Ser Ala Thr Asn

35 40 45

Gln Ile Met Pro Cys Lys Pro Thr Lys Ile Glu Arg Asn Ile Thr Glu

50 55 60

Ile Val Tyr Leu Thr Asn Thr Thr Ile Lys Thr Glu Val Cys Pro Lys

65 70 75 80

Leu Val Lys Tyr Arg Asp Trp Ala Lys Pro Gln Cys Arg Ile Thr Gly

85 90 95

Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly

100 105 110

Ala Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Leu Ser Lys

115 120 125

Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asp Asn Arg His

130 135 140

Ser Asn Asp Thr Ile His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met

145 150 155 160

Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Arg Gln Val Cys Ile

165 170 175

Ala Trp Ser Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val

180 185 190

Cys Val Thr Gly Tyr Asp Lys Asn Ala Thr Ala Ser Leu Ile Tyr Asp

195 200 205

Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile Leu Arg

210 215 220

Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val

225 230 235 240

Met Thr Asp Gly Ser Ala Ser Gly Lys Ala Asp Thr Arg Ile Leu Phe

245 250 255

Ile Glu Glu Gly Lys Ile Ile His Ile Ser Pro Leu Thr Gly Ser Ala

260 265 270

Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg

275 280 285

Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Val Asp

290 295 300

Ile Asn Val Lys Asp Tyr Lys Ile Asn Ser Ser Tyr Val Cys Ser Gly

305 310 315 320

Leu Val Gly Asp Thr Pro Arg Asn Asn Asp Arg Ser Ser Asn Ser Asn

325 330 335

Cys Gln Asn Pro Asn Asn Gln Arg Gly Asn His Gly Val Lys Gly Trp

340 345 350

Ala Phe Asp Asp Gly Asn Asp Ile Trp Met Gly Arg Thr Ile Ser Asn

355 360 365

Asp Ser Arg Leu Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser

370 375 380

Lys Pro Asn Ser Lys Val Gln Thr Asn Arg Gln Val Ile Val Asp Ser

385 390 395 400

Asp Asn Arg Ser Gly Tyr Ser Gly Val Phe Ser Val Glu Gly Lys Ser

405 410 415

Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Arg Gln

420 425 430

Glu Ala Arg Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly

435 440 445

Thr Ser Gly Thr Tyr Gly Ser Gly Ser Trp Pro Asp Gly Ala Asp Ile

450 455 460

Asn Leu Met Pro Ile

465

<210> 23

<211> 1410

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized swine influenza virus sequence

<400> 23

atgaacccta accagaagat tattaccatc ggatccgtgt gcatgaccat cggaatggcc 60

aacctgatcc tgcaaatcgg aaacatcatc tccatctgga ttagccatag cattcagctt 120

ggaaaccaga gccagatcga gacttgtaac cagtccgtga tcacctacga gaacaacacc 180

tgggtcaacc agacttacgt caatatcagc aacaccaact tcgctgcggg gcagtccgtg 240

gtgtccgcaa agctggccgg caacagctcc ctgtgccccg tgtccggatg ggcgatctac 300

tccaaggata acagcgtgcg catcgggtcc aaaggggacg tgttcgtcat ccgcgaacca 360

ttcatttctt gctccccctt ggaatgtcgg accttcttcc tgacccaagg ggcgttgctc 420

aacgacaagc acagcaacgg aaccatcaaa gatcggtcgc cctaccgcac tctgatgtcg 480

tgccctatcg gcgaagtgcc atccccctac aactcacgct tcgagtccgt ggcctggtcc 540

gcttccgcct gccacgatgg aatcaactgg ctcacaatcg gcatctccgg cccggactcg 600

ggagccgtgg ccgtgctgaa gtacaatggt attattactg acactatcaa gtcgtggaag 660

aacaatattc tccggactca agaatctgaa tgcgcctgcg tgaacggttc ctgcttcact 720

atcatgaccg acggcccttc cgatggacag gcctcataca agatcttccg gatcgagaag 780

ggaaagatcg tgaagtccgt cgagatgaac gcaccgaact accattatga ggaatgctcg 840

tgctacccgg actcctcgga aattacttgc gtgtgccgcg acaattggca cgggtccaac 900

aggccctggg tgtccttcaa ccaaaacctg gagtaccaga tcggttacat ctgctccggg 960

atttttggag acaaccctag acctaacgac aagaccggct catgcggacc tgtgtcctcc 1020

aacggagcca acggcgtgaa gggattctcg ttcaaatatg ggaacggcgt ctggataggt 1080

1140

accggaaccg acaagaactt cagcatcaag caggacatta tcggcattaa cgagtggagc 1200

ggatactcgg gcagcttcgt ccagcacccg gaacttacgg gcctcaattg tattaggccc 1260

tgtttttggg tcgagctgat tagagggcgc cccaaggaaa acaccatctg gaccagcggc 1320

tccagcatct cattctgcgg agtgaactcc gacaccgtgg gctggtcgtg gcccgacggt 1380

gccgagctgc cgttcaccat cgataaatga 1410

<210> 24

<211> 1410

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized swine influenza virus sequence

<400> 24

atgaatccta accaaaagat catcaccatc ggctccgtca gcctcactat taccaccatg 60

tgcctgttcc ttcaaattgc catcctcgtg accacgatca ccctgcactt caagcagtac 120

gagtgcgact ccccggccaa caaccaggtc atcccgtgtg aaccgattat cattgagaag 180

aacatcacaa agattgtgta cctgactaac accaccatcg agaaggaagt ctgcccaaaa 240

ttggggagagt accggaactg gtccaagcct caatgcaaaa tcactggatt tgctccgttc 300

agcaaggata actcaatcag actgagcgcg ggtggcgcca tctgggtcac cagggaacct 360

tatgtgtcgt gcgatcccaa caagtgctac cagtttgcgc tgggccaggg caccaccctc 420

gacaaccgcc actccaacga caccatccat gatcgcaccc ctttccgaac cctgctgatg 480

agcgagcttg gcgtgccctt ccacctcgga acgcgccaag tctgtattgc ctggagctcg 540

tcctcctgcc acgatggaaa ggcctggctg catgtctgtg tgactggtca cgacaagaac 600

gccaccgcta gcttcatcta tgacggaaaa ttggtggatt ccatctcctc ctggtctaag 660

aacattctgc ggacccagga atccgaatgc gtgtgcatcg atggcatctg cactgtggtc 720

atgactgacg gatccgcaag cggaaaggcg gacactaaga tcctgtttat cgaaaaggga 780

aagatcattc atatctcgcc gctgctgggg tccgcccagc acgtggaaga gtgcagctgt 840

tacccccggt accccgacgt gcggtgcatt tgtcgggaca actggaaggg tagcaacagg 900

cccatcgtgg acattcgcat gaagaattac tcaatcggat cctcctacat gtgctcaggg 960

ctcgtgggag acacccccag gaacaacgac ggttcctcaa actccaactg tcggaacccg 1020

aacaatgagc gcgggaatca cggagtgaag ggatgggcat tcgacgacgg aaacgacacc 1080

tggatgggtc ggactatcag caaggactcc cgcctgggtt acgaaacttt caaggtcgtg 1140

gggggctggt cgcagccaaa ctcgaagtcg cagattaaca gacaggtcat cgtggactcc 1200

gacaataggt ccggctactc cggaatcttc tcggtcgagg ggaaggattg cattaaccgg 1260

tgcttctacg tggagctgat cagaggcaga cgccaggaaa cccgcgtgtg gtggacctcc 1320

aactcaatcg tggtgttctg cggaacatcc ggcacttacg gcagcggatc ctggcctgat 1380

ggcgccaaca taaacttcat gcccgtgtga 1410

<210> 25

<211> 1410

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized swine influenza virus sequence

<400> 25

atgaacccca accaaaagat cattacaatc ggatcagtgt cacttgtgat cgccaccttg 60

tgtttcctca tgcaaatggc cattctgatc accactgtga agctgcactt caaacagtat 120

gagtgcggat tcccggcgaa caatcaggtc atcacctgtg aacctaccgt gatcgagcgg 180

aacaccaccg agatagtgta cctgaccaat acgactatcg aaaaggaaac ttgccataag 240

accgtcgagt accgaaactg gtcgaagccg cagtgcaaga tcactgggtt cgcaccgttc 300

agcaaggata acagcatccg gctgtccgcc gggggagaca tttgggtcac ccgcgaaccg 360

tacgtgtcgt gcgagcccgg aaagtgctac cagttcgccc tgggccaggg taccaccctg 420

gataacaagc acagcaacga taccatccac gataggaccc cctaccggac tctgctgatg 480

aacgaactgg gcgtgccttt tcacctcggc acgagacaag tctgcattgc ctggtcctca 540

tcgtcctgct acgacggaaa ggcctggctg cacgtctgca tcaccggcca tgacaaaaac 600

gccaccgcgt ccttcatcta cgacggtcgc ctggtggact ccattggatc ctggagcaag 660

aacattctgc ggacccagga atcggaatgc gtgtgtatca acggagtgtg caccgtcgtg 720

atgactgatg gctctgcttc cggccggggcc gacaccaaga tcttgtttat cgaagaaggg 780

aagattgtcc acatttcccc gcttgctgga tccgcgcagc acgtggaaga gtgcagctgt 840

tacccccgct accctggcgt gcggtgcatt tgtcgggaca actggaaggg atccaatagg 900

cccgtggtgg acatcaacat cgaagattac tcgattgact ccagctacgt gtgctccggc 960

ctcgtcgggg acactccccg catcaacgac gggtcgagct cctcctattg ccgggatcca 1020

1080

tggatgggta gaactatcaa cgaggattcc cggagcggat acgagacttt caaggtcatc 1140

ggtggttggt caacccctaa tagcaaactc cagattaaca gacaggtcat cgtggactcg 1200

aacaatcgga gcggctactc cggagtgttc tccgtggaag gaaagtcgtg catcaaccgg 1260

tgcttctacg tggagctgat taggggtcgc cgcagcgaag cccgcgtgtg gtggacttcc 1320

aactcaatcg tggtgttctg cggcacctcc gggacttacg gaaccggatc ctggccggat 1380

ggcgccgaca ttaacctgat gccaatttga 1410

<210> 26

<211> 1410

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized swine influenza virus sequence

<400> 26

atgaacccca accaaaagat cattacaatc ggatcagtgt cacttgtgat cgccaccttg 60

tgtttcctca tgcaaatggc cattctgatc accactgtga agctgcactt caaacagtat 120

gagtgcggat tcccggcgaa caatcaggtc atcacctgtg aacctaccgt gatcgagcgg 180

aacaccaccg agatagtgta cctgaccaat acgactatcg aaaaggaaac ttgccataag 240

accgtcgagt accgaaactg gtcgaagccg cagtgcaaga tcactgggtt cgcaccgttc 300

agcaaggata acagcatccg gctgtccgcc gggggagaca tttgggtcac ccgcgaaccg 360

tacgtgtcgt gcgagcccgg aaagtgctac cagttcgccc tgggccaggg taccaccctg 420

gataacaagc acagcaacga taccatccac gataggaccc cctaccggac tctgctgatg 480

aacgaactgg gcgtgccttt tcacctcggc acgagacaag tctgcattgc ctggtcctca 540

tcgtcctgct acgacggaaa ggcctggctg cacgtctgca tcaccggcca tgacaaaaac 600

gccaccgcgt ccttcatcta cgacggtcgc ctggtggact ccattggatc ctggagcaag 660

aacattctgc ggacccagga atcggaatgc gtgtgtatca acggagtgtg caccgtcgtg 720

atgactgatg gctctgcttc cggccggggcc gacaccaaga tcttgtttat cgaagaaggg 780

aagattgtcc acatttcccc gcttgctgga tccgcgcagc acgtggaaga gtgcagctgt 840

tacccccgct accctggcgt gcggtgcatt tgtcgggaca actggaaggg atccaatagg 900

cccgtggtgg acatcaacat cgaagattac tcgattgact ccagctacgt gtgctccggc 960

ctcgtcgggg acactccccg catcaacgac gggtcgagct cctcctattg ccgggatcca 1020

1080

tggatgggta gaactatcaa cgaggattcc cggagcggat acgagacttt caaggtcatc 1140

ggtggttggt caacccctaa tagcaaactc cagattaaca gacaggtcat cgtggactcg 1200

aacaatcgga gcggctactc cggagtgttc tccgtggaag gaaagtcgtg catcaaccgg 1260

tgcttctacg tggagctgat taggggtcgc cgcagcgaag cccgcgtgtg gtggacttcc 1320

aactcaatcg tggtgttctg cggcacctcc gggacttacg gaaccggatc ctggccggat 1380

ggcgccgaca ttaacctgat gccaatttga 1410

<210> 27

<211> 1410

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized swine influenza virus sequence

<400> 27

atgaacccca accagaagat tatcaccatc ggatccgtgt ctctcatcat cgccaccatg 60

tgcttcttta tgcaagtggc cattctcgtc accactgtga ccctgcactt tcggcagtgc 120

gaatgcaact cctccgctac taaccaaatt atgccgtgca agcctactaa gatcgaacgg 180

aacatcaccg agatcgtgta cttgacgaac accaccatca agaccgaagt ctgcccgaag 240

cttgtgaaat atcgggactg ggcgaagcca caatgtcgca tcaccggttt cgccccgttc 300

agcaaagata acagcattcg gctgtccgcc gggggcgcga tctgggtcac cagggagccg 360

tacgtgtcgt gtgatctgtc caagtgctac caattcgcac tgggacaggg taccaccctc 420

gacaaccgcc actccaacga taccatacat gaccggaccc cttaccgcac tctcttgatg 480

aacgagctgg gagtgccctt ccacctgggt accagacaag tctgcattgc ctggtcctca 540

tcctcctgcc atgacgggaa ggcctggctg catgtctgcg tgactggcta cgataagaac 600

gcgacggcct ccctgatcta cgacggtcgc ctggtggact ccattggggtc gtggagccag 660

aatatcctgc ggactcagga gtcagaatgc gtgtgcatca acggcacttg caccgtggtc 720

atgactgacg gatccgcctc ggggaaggcc gacaccagga tcctgttcat tgaggaggga 780

aagattatcc acatctcgcc tctgactgga tccgcccagc acgtggaaga gtgttcctgc 840

taccctcgat accccggcgt gcgctgtgtg tgtcgggata actggaaggg aagcaaccgg 900

cccgtggtgg acattaacgt gaaggactac aagattaact catcatacgt gtgctccgga 960

ctcgtgggcg atacaccaag aaacaacgac cgcagcagca acagcaattg tcagaacccc 1020

aacaaccagc gcggcaacca cggcgtgaag ggctgggcat tcgacgacgg aaatgacatc 1080

tggatgggga ggactatctc caacgattca cgcctgggct acgaaacctt caaggtcatc 1140

ggcggctggt caaaaccgaa ctccaaggtc cagaccaaca gacaggtcat cgtggattcg 1200

gacaatagaa gcggatacag cggagtgttc agcgtggagg gaaagtcgtg catcaaccgc 1260

1320

aactcgattg tggtgttctg cggtacctcc ggaacctatg ggagcggatc ctggccggac 1380

ggcgctgaca ttaaccttat gccgatctga 1410

<---

Claims (19)

1. A vaccine to help prevent influenza virus disease, comprising two or more replicon RNA(RP) particles each independently encoding one or more neuraminidase (NA), and a pharmaceutically acceptable carrier, where the first alphavirus RNA replicon particle encodes the first neuraminidase (NA) and the second alphavirus RNA replicon particle encodes the second NA, or where the alphavirus RNA replicon particle encodes two or more NAs, with the proviso that the immunogenic composition contains neither swine influenza A virus (IAV-S) hemagglutinin (HA) nor a nucleotide sequence encoding IAV-S hemagglutinin (HA); and where the first NA and the second NA contain amino acid sequences that have an identity of 98% or less. 2. A vaccine to help prevent influenza virus disease according to claim 1, wherein the first NA is from IAV-S from a first phylogenetic cluster and the second NA is from IAV-S from a second phylogenetic cluster, where the first phylogenetic cluster and the second the phylogenetic cluster are different and where the first phylogenetic cluster and the second phylogenetic cluster are independently selected from the group consisting of the N1-classic cluster, the N1-pandemic cluster, the N2-1998 cluster, and the N2-2002 cluster. 3. The vaccine to help prevent influenza virus disease according to claim 2, wherein the first phylogenetic cluster is selected from the group consisting of the N1-classic cluster and the N1-pandemic cluster, and where the second phylogenetic cluster is selected from the group consisting of the N1-classic cluster and the N1-pandemic cluster N2-1998 and cluster N2-2002. 4. The vaccine to help prevent influenza virus disease according to claim 3, wherein the first phylogenetic cluster is the N1-classic cluster and the second phylogenetic cluster is the N2-2002 cluster. 5. A vaccine to help prevent influenza virus disease according to any one of claims 2 to 4, further comprising a third alphavirus RNA replicon particle that encodes a third NA; where the third NA is derived from IAV-S from the third phylogenetic cluster and where the third phylogenetic cluster is different from the first phylogenetic cluster and the second phylogenetic cluster. 6. The vaccine to help prevent influenza virus disease according to claim 5, further comprising a fourth alphavirus RNA replicon particle that encodes a fourth NA, where the fourth NA is derived from IAV-S from a fourth phylogenetic cluster, and where the fourth phylogenetic cluster is different from the first phylogenetic cluster, the second phylogenetic cluster and the third phylogenetic cluster. 7. The vaccine to help prevent influenza virus disease according to claim 6, wherein the first phylogenetic cluster is the N1-classic cluster, the second phylogenetic cluster is the N2-2002 cluster, the third phylogenetic cluster is the N1-pandemic cluster, and the fourth phylogenetic cluster is the N2-1998 cluster. 8. A vaccine to help prevent influenza virus disease according to claim 1, wherein the alphavirus RNA replicon particle encodes two or more NAs, and wherein the first NA is derived from IAV-S from the first line and the second NA is derived from IAV-S from second line; where the first line and the second line are different and where the first line and the second line are independently selected from the group consisting of the N1-pandemic (EU) line, the N1-Eurasian Avian line, the N2-Gent/1984 line, the N2-Italy/4675/2003 line , lines N2-Scotland/1994 (clade 1) and lines N2-Scotland/1994 (clade 2). 9. A vaccine to help prevent disease caused by an influenza virus according to claim 8, wherein the first line is selected from the group consisting of the N1-pandemic (EU) line and the N1-Eurasian Avian line; and where the second lineage is selected from the group consisting of N2-Gent/1984 lineage, N2-Italy/4675/2003 lineage, N2-Scotland/1994 lineage (clade 1) and N2-Scotland/1994 lineage (clade 2). 10. A vaccine to help prevent influenza virus disease according to claim 2, wherein the alphavirus RNA replicon particle encodes two or more NAs, and wherein the first phylogenetic cluster is the N1-classic cluster and the second phylogenetic cluster is the N1-pandemic cluster. . 11. The vaccine to help prevent influenza virus disease according to claim 10, further comprising a second alphavirus RNA replicon particle that encodes a third NA and a fourth NA; where the third NA is from IAV-S from the N2-1998 cluster and the fourth NA is from IAV-S from the N2-2002 cluster. 12. A vaccine to help prevent influenza virus disease according to any one of claims 1 to 11, wherein each alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEEV) RNA replicon particle. 13. A vaccine to help prevent disease caused by an influenza virus according to any one of claims 1-12, which includes an adjuvant selected from the group consisting of a biodegradable oil, an oil-in-water emulsion with 2.5-50% (v/v) mineral oil and biodegradable oil mixed with an oil-in-water emulsion of 2.5-50% (v/v) mineral oil. 14. A vaccine to help prevent influenza virus disease according to claim 13, wherein the biodegradable oil is dl-α-tocopheryl acetate and the mineral oil is paraffin oil. 15. A method of immunizing a pig against swine influenza A virus, comprising administering to the pig an immunologically effective amount of any of the vaccines of claims 1-14. 16. The use of a vaccine according to any one of claims 1 to 14 to help prevent swine influenza A virus disease in pigs.

Images