Bivalent swine influenza virus vaccine

Abstract

The present invention relates to an immunogenic composition comprising: a) a modified live H3 virus of swine influenza, and b) a modified live H1 virus of swine influenza. Furthermore, the present invention relates to methods for immunizing a subject comprising administering to such subject the immunogenic composition of the present invention. Moreover, the present invention relates to methods of treating or preventing clinical signs caused by swine influenza virus in a subject of need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to the present invention.

Claims

1. An immunogenic composition comprising: a) a modified live H3 virus of H3N2 swine influenza, wherein the modified live H3 virus is attenuated by truncating the NS1 protein and the modified live H3 virus comprises a carboxy-terminal truncated NS1 protein consisting of NS1 amino acids 1 through 124, 125, 126, 127 or 128; and b) a modified live H1 virus of H1N1 swine influenza, wherein the modified live H1 virus is attenuated by truncating the NS1 protein and the modified live H3 virus comprises a carboxy-terminal truncated NS1 protein consisting of NS1 amino acids 1 through 124, 125, 126, 127 or 128.

2. The immunogenic composition of claim 1, wherein the H3 and H1 viruses each comprise a carboxy-terminal truncated NS1 protein consisting of NS1 amino acids 1 through 126.

3. The immunogenic composition of claim 1, wherein the H3 virus comprises a protein having 95% or greater sequence identity to SEQ ID NO: 3 or 4.

4. The immunogenic composition of claim 1, wherein the H1 virus comprises a protein having 95% or greater sequence identity to SEQ ID NO: 7 or 8.

5. The immunogenic composition of claim 1, wherein the H3 virus comprises a protein having 95% or greater sequence identity to SEQ ID NO: 3 or 4, and wherein the H1 virus comprises a protein having 95% or greater sequence identity to SEQ ID NO: 7 or 8.

6. The immunogenic composition of claim 1, wherein the modified live H3 and H1 viruses synergistically induce a protective immunize response in an animal against swine influenza.

7. The immunogenic composition of claim 1, wherein the immunogenic composition has between 1 to 4 log.sub.10 of each of the modified live H3 and H1 viruses.

8. A method for inducing protective immunity in an animal against swine influenza comprising: administering to the animal a modified live H3 virus of H3N2 swine influenza and a modified live H1 virus of H1N1 swine influenza, wherein the modified live H3 and H1 viruses are attenuated by truncating the NS1 proteins of the live H3 and H1 viruses and wherein the modified live H3 and H1 viruses each comprise a carboxy-terminal truncated NS1 protein consisting of NS1 amino acids 1 through 124, 125, 126, 127 or 128.

9. The method of claim 8, wherein the modified live H3 and H1 viruses are administered in a single dose.

10. The method of claim 8, wherein the animal is a swine, cattle, cat, or dog.

11. The method of claim 8, wherein the animal is a swine.

12. The method of claim 8, wherein the modified live H3 and H1 viruses are administered intranasally.

13. The method of claim 8, wherein the animal is a pregnant or lactating sow.

14. The method of claim 8, wherein the animal is month old or less.

15. The method of claim 8, wherein the animal is two weeks old or less.

16. The method of claim 8, wherein the animal is one week old or less.

17. The method of claim 8, wherein the animal has circulating maternal antibodies against swine influenza.

18. The method of claim 8, wherein the H3 and H1 viruses each comprise a carboxy-terminal truncated NS1 protein consisting of NS1 amino acids 1 through 126.

19. The method of claim 8, wherein the H3 virus comprises a protein having 95% or greater sequence identity to SEQ ID NO: 3 or 4.

20. The method of claim 8, wherein the H1 virus comprises a protein having 95% or greater sequence identity to SEQ ID NO: 7 or 8.

21. The method of claim 8, wherein the H3 virus comprises a protein having 95% or greater sequence identity to SEQ ID NO: 3 or 4, and wherein the H1 virus comprises a protein having 95% or greater sequence identity to SEQ ID NO: 7 or 8.

22. The method of claim 8, wherein the modified live H3 and H1 viruses synergistically induce a protective immunize response in the animal against swine influenza.

23. The method of claim 8, wherein between 1 to 4 log.sub.10 of each of the modified live H3 and H1 viruses is administered to the animal.

24. The method of claim 8, wherein between 2 to 4 log.sub.10 of each of the modified live H3 and H1 viruses is administered to the animal.

25. The method of claim 8, wherein the H3 virus comprises a polynucleotide having 95% or greater sequence identity to a complementary sequence of SEQ ID NO: 1 or 2.

26. The method of claim 8, wherein the H1 virus comprises a polynucleotide having 95% or greater sequence identity to a complementary sequence of SEQ ID NO: 5 or 6.

Description

DETAILED DESCRIPTION

(1) The invention provides the following exemplary embodiments:

(2) An immunogenic composition comprising: a) a modified live H3 virus of swine influenza, and b) a modified live H1 virus of swine influenza.

(3) The immunogenic composition comprising the above embodiment, wherein the modified live H3 and H1 viruses of swine influenza have a N (neuraminidase) subtype selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9 and N10.

(4) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 virus of swine influenza is H3N2 of swine influenza and/or the modified live H1 virus of swine influenza is H1N1 of swine influenza virus.

(5) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza have one or more mutations in the NS1 gene.

(6) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza have a deletion within the NS1 gene.

(7) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza are attenuated swine influenza viruses.

(8) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza are mutagenized viruses or reassortants.

(9) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza are genetically engineered.

(10) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza have an impaired interferon antagonist phenotype.

(11) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza have one or more mutations at the carboxy terminus of the NS1 protein.

(12) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza have a carboxy-terminal truncated NS1 protein.

(13) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza have a carboxy-terminal truncated NS1 protein comprising NS1 amino acids 1 through 124, 1 through 125, 1 through 126, 1 through 127 or 1 through 128, wherein the amino terminal amino acid is number 1

(14) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza have a carboxy-terminal truncated NS1 protein comprising NS1 amino acids 1 through 126.

(15) The immunogenic composition comprising any of the preceding embodiments, NS1 protein resulting in a deletion of 91, 92, 93 or 94 amino acid residues from the carboxy terminus of NS1.

(16) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza have a NS1 gene or protein from A/Swine/Texas/4199-2/98.

(17) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 virus of swine influenza encodes a carboxy-terminal truncated NS1 protein comprising NS1 amino acids 1 through 124, 1 through 125, 1 through 126, 1 through 127 or 1 through 128, wherein the amino terminal amino acid is number 1.

(18) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 virus of swine influenza encodes a carboxy-terminal truncated NS1 protein comprising NS1 amino acids 1 through 126, wherein the amino terminal amino acid is number 1.

(19) The immunogenic composition comprising any of the preceding embodiments, NS1 protein resulting in a deletion of 91, 92, 93 or 94 amino acid residues from the carboxy terminus of NS1.

(20) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 virus of swine influenza is A/Swine/Texas/4199-2/98.

(21) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 virus of swine influenza is TX/98/del 126.

(22) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 virus of swine influenza contains the HA, NA, PB2, PB1, PA, NP, and M from A/Swine/Texas/4199-2/98 and the NS1-126 gene is from A/Swine/Texas/4199-2/98.

(23) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 virus of swine influenza a) comprises a nucleic acid sequence of gene segments having the NA and HA genes whose cDNA has at least 70% identity to the nucleic acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2, or b) comprises a nucleic acid sequence of gene segments having the NA and HA genes encoding NA and HA proteins having an amino acid sequence with at least 70% identity to the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:4, or c) comprises a NA and HA protein having an amino acid sequence with at least 70% identity to the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:4.

(24) The H1N1 NS1 deletion mutant of Swine Influenza virus of any one of preceding embodiments, wherein the modified live H3 virus of swine influenza a) comprises a nucleic acid sequences of gene segments having the NA and HA genes whose cDNA has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleic acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6, or b) comprises a nucleic sequences of gene segments having the NA and HA genes encoding NA and HA proteins having amino acid sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8, or c) comprises a NA and HA protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8.

(25) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza is a chimeric virus.

(26) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza comprises a carboxy-terminal truncated NS1 of a H3 SIV strain, preferably a carboxy-terminal truncated NS1 of a H3N2 SIV strain.

(27) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza encodes a carboxy-terminal truncated NS1 protein comprising NS1 amino acids 1 through 124, 1 through 125, 1 through 126, 1 through 127 or 1 through 128, wherein the amino terminal amino acid is number 1.

(28) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza encodes a carboxy-terminal truncated NS1 protein comprising NS1 amino acids 1 through 126, wherein the amino terminal amino acid is number 1.

(29) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza encodes a carboxy-terminal truncated NS1 protein resulting in a deletion of 91, 92, 93 or 94 amino acid residues from the carboxy terminus of NS1.

(30) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza is A/Swine/Texas/4199-2/98.

(31) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza comprises the hemagglutinin and neuraminidase gene segments from a H1N1 subtype.

(32) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza comprises the hemagglutinin and neuraminidase gene segments from A/swine/Minnesota/37866/1999.

(33) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza contains HA and NA from A/swine/Minnesota/37866/1999 (H1N1) and PB2, PB1, PA, NP, M from A/Swine/Texas/4199-2/98 (H3N2) and the NS1-126 gene is from A/Swine/Texas/4199-2/98 (H3N2).

(34) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza is a chimeric of A/swine/Minnesota/37866/1999 and TX/98/del 126.

(35) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H1 virus of swine influenza a) comprises a nucleic acid sequence of gene segments having the NA and HA genes whose cDNA has greater than 70% identity to the nucleic acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6, or b) comprises a nucleic sequence of gene segments having the NA and HA genes encoding NA and HA proteins having an amino acid sequence with greater than 70% identity to the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8, or c) comprises a NA and HA protein having an amino acid sequence with greater than 70% identity to the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8.

(36) The H1N1 NS1 deletion mutant of Swine Influenza virus of any one of preceding embodiments, wherein the modified live H1 virus of swine influenza a) comprises a nucleic acid sequences of gene segments having the NA and HA genes whose cDNA has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleic acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6, or b) comprises a nucleic sequences of gene segments having the NA and HA genes encoding NA and HA proteins having amino acid sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8, or c) comprises a NA and HA protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8.

(37) The immunogenic composition comprising any of the preceding embodiments, wherein said immunogenic composition is formulated for a single-dose administration.

(38) The immunogenic composition comprising any of the preceding embodiments, wherein said immunogenic composition is administered intranasal.

(39) The immunogenic composition comprising any of the preceding embodiments, wherein the immunogenic composition is safe for sows during pregnancy and lactation.

(40) The immunogenic composition comprising any of the preceding embodiments, wherein the immunogenic composition is safe for pigs within the first two weeks of age, preferably within the first week of age.

(41) The immunogenic composition comprising any of the preceding embodiments, wherein the immunogenic composition is safe for pigs at day 0 of age.

(42) The immunogenic composition comprising any of the preceding embodiments, wherein said immunogenic composition further comprises a pharmaceutically acceptable carrier.

(43) The immunogenic composition comprising any of the preceding embodiments, wherein the immunogenic composition comprises 2 to 8 log 10 of the modified live H1 virus of swine influenza and/or 2 to 8 log 10 of the modified live H3 virus of swine influenza.

(44) The immunogenic composition comprising any of the preceding embodiments, wherein the immunogenic composition comprises 2 to 6 log 10 of the modified live H1 virus of swine influenza and/or 2 to 6 log 10 of the modified live H3 virus of swine influenza.

(45) The immunogenic composition comprising any of the preceding embodiments, wherein the immunogenic composition comprises 2 to 4 log 10 of the modified live H1 virus of swine influenza and/or 2 to 4 log 10 of the modified live H3 virus of swine influenza.

(46) The immunogenic composition comprising any of the preceding embodiments, wherein said immunogenic composition is a vaccine.

(47) The immunogenic composition comprising any of the preceding embodiments, wherein said immunogenic composition is a bivalent vaccine.

(48) The immunogenic composition comprising any of the preceding embodiments, wherein the immunogenic composition is effective in the treatment and/or prophylaxis of clinical signs caused by SIV in a subject of need.

(49) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza act synergistically together.

(50) The immunogenic composition comprising any of the preceding embodiments, wherein the concentration of the modified live H3 and H1 viruses of swine influenza are reduced compared to the concentration of the H3 virus of swine influenza in a monovalent immunogenic composition and the concentration of the H1 virus of swine influenza in a monovalent immunogenic composition.

(51) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 virus of swine influenza increases the protection against a heterologous challenge.

(52) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 virus of swine influenza increases the protection against a H1 challenge.

(53) The immunogenic composition comprising any of the preceding embodiments, wherein the modified live H3 virus of swine influenza increases the protection against a H1N1 challenge.

(54) A method for immunizing a subject comprising administering to such subject an immunogenic composition of any one of the preceding embodiments.

(55) A method of treating or preventing clinical signs caused by swine influenza virus in a subject of need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of the preceding embodiments.

(56) A method of reducing the viral shedding in a subject of need, in comparison to a subject of a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of the preceding embodiments.

(57) A method of reducing the viral shedding in a subject of need, in comparison to a subject of an immunized control group of the same species immunized with a monovalent modified live H3 or H1 virus of swine influenza, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of the preceding embodiments.

(58) A method of vaccinating a subject having anti-SIV antibodies comprising the step of administering to said animal a single effective dose of an immunogenic composition according to any one of the preceding embodiments.

(59) A method for preventing or reducing early SIV infections in a subject, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of the preceding embodiments.

(60) The method according to any one of the preceding methods, wherein said subject is selected from the group consisting of swine, cattle, cat and dog.

(61) The method according to any one of the preceding methods, wherein the immunogenic composition is administered once.

(62) The method according to any one of the preceding methods, wherein the immunogenic composition is administered at two or more doses.

(63) The method according to any one of the preceding methods, wherein said immunogenic composition is administered intranasal.

(64) A method according to any one of the preceding methods, wherein the subject is Swine influenza virus maternal antibody negative.

(65) The method according to any one of the preceding methods, wherein said immunogenic composition is administered to sows during pregnancy and lactation.

(66) The method according to any one of the preceding methods, wherein the immunogenic composition is administered to pigs within the first month of age.

(67) The method according to any one of the preceding methods, wherein the immunogenic composition is administered to pigs within the first two weeks of age, preferably within the first week of age.

(68) The method according to any one of the preceding methods, wherein the immunogenic composition is administered to pigs at day 1 of age.

(69) The method according to any one of the preceding embodiments, wherein the immunogenic composition is administered to pigs within the first 24 h of age.

(70) The method according to the any one of the preceding methods, wherein the subject is Swine influenza virus maternal antibody positive.

(71) The method according to any one of the preceding methods, wherein the immunogenic composition comprises 2 to 8 log 10 of the modified live H1 virus of swine influenza and/or 2 to 8 log 10 of the modified live H3 virus of swine influenza.

(72) The method according to any one of the preceding methods, wherein the immunogenic composition comprises 2 to 6 log 10 of the modified live H1 virus of swine influenza and/or 2 to 6 log 10 of the modified live H3 virus of swine influenza.

(73) The method according to any one of the preceding methods, wherein the immunogenic composition comprises 2 to 4 log 10 of the modified live H1 virus of swine influenza and/or 2 to 4 log 10 of the modified live H3 virus of swine influenza.

(74) The method according to any one of the preceding methods, wherein said method results in an improvement in an efficacy parameter selected from the group consisting of: a reduction in weight loss, a lower virus load, a reduction in lung lesions, a reduced shedding, a reduced rectal temperature, or combinations thereof, in comparison to a subject of a non-immunized control group of the same species.

(75) The method according to any one of the preceding methods, wherein said method results in an improvement in an efficacy parameter selected from the group consisting of: a reduction in the weight loss, a lower virus load, a reduction in lung lesions, a reduced shedding, a reduced rectal temperature, or combinations thereof, in comparison to a subject of the same species immunized with a monovalent modified live H3 or H1 virus of swine influenza.

(76) The method according to any one of the preceding methods, wherein the treatment or prophylaxis results in shortening of the virus load phase as compared to animals of a non-treated control group of the same species.

(77) The method according to any one of the preceding wherein the treatment or prophylaxis results in a reduction of the shedding from day 5 after challenge or infection.

(78) The method according to any one of the preceding methods, wherein the treatment or prophylaxis results in a reduction of the shedding from day 1 or day 2 after challenge or infection.

(79) The method according to any one of the preceding methods, wherein the subject is immunized with a lower concentration of the immunogenic composition of any one of the preceding embodiments compared to a subject of the same species immunized with a monovalent modified live H3 or H1 virus of swine influenza.

(80) The method according to any one of the preceding methods, wherein the monovalent modified live H3 or H1 virus of swine influenza is a H1N1 NS1 deletion mutant of swine influenza virus.

(81) The method according to the above embodiment, wherein the H1N1 NS1 deletion mutant of swine influenza virus is the H1N1 NS1 deletion mutant of swine influenza virus comprising any one of the preceding embodiments.

(82) The method according to any one of the preceding embodiments, wherein the monovalent modified live H3 or H1 virus of swine influenza is a H3N2 NS1 deletion mutant of swine influenza virus.

(83) The method according to the above embodiment, wherein the H3N2 NS1 deletion mutant of swine influenza virus is the H3N2 NS1 deletion mutant of swine influenza virus comprising any one of the preceding embodiments.

(84) The method according to any one of the preceding embodiments, wherein the modified live H3 and H1 viruses of swine influenza act synergistically together.

(85) The method according to any one of the preceding embodiments, wherein the concentration of the modified live H3 and H1 viruses of swine influenza are reduced compared to the concentration of the H3 virus of swine influenza in a monovalent immunogenic composition and the concentration of the H1 virus of swine influenza in a monovalent immunogenic composition.

(86) The method according to any one of the preceding embodiments, wherein the modified live H3 virus of swine influenza increases the protection against a heterologous challenge.

(87) The method according to any one of the preceding embodiments, wherein the modified live H3 virus of swine influenza increases the protection against a H1 challenge.

(88) The method according to any one of the preceding embodiments, wherein the modified live H3 virus of swine influenza increases the protection against a H1N1 challenge.

(89) An immunogenic composition comprising any one of the preceding embodiments for use in a method for immunizing a subject comprising administering said immunogenic composition to such subject.

(90) The immunogenic composition comprising any one of the preceding embodiments for use in a method of treating or preventing clinical signs caused by swine influenza virus in a subject of need, the method comprising administering to the subject a therapeutically effective amount of said immunogenic composition.

(91) The immunogenic composition comprising any one of the preceding embodiments for use in a method of reducing the viral shedding in a subject of need, in comparison to a subject of a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of said immunogenic composition.

(92) The immunogenic composition comprising any one of the preceding embodiments for use in a method of reducing the viral shedding in a subject of need, in comparison to a subject of an immunized control group of the same species immunized with a monovalent modified live H3 or H1 virus of swine influenza, the method comprising administering to the subject a therapeutically effective amount of said immunogenic composition.

(93) The immunogenic composition comprising any one of the preceding embodiments for use in a method of vaccinating a subject having anti-SIV antibodies comprising the step of administering to said animal a single effective dose of said immunogenic composition.

(94) The immunogenic composition comprising any one of the preceding embodiments for use in a method for preventing or reducing early SIV infections in a subject, the method comprising administering to the subject a therapeutically effective amount of said immunogenic composition.

(95) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein said subject is selected from the list consisting of swine, cattle, cat and dog.

(96) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the immunogenic composition is administered once.

(97) The immunogenic composition comprising any one of the preceding embodiments for use according to of any one of the preceding methods, wherein the immunogenic composition is administered at two or more doses.

(98) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein said immunogenic composition is administered intranasal.

(99) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the subject is Swine influenza virus maternal antibody negative.

(100) The immunogenic composition comprising any one of the preceding embodiments or use according to of any one of the preceding methods, wherein said immunogenic composition is administered to sows during pregnancy and lactation.

(101) The immunogenic composition comprising any one of the preceding embodiments for use according to of any one of the preceding methods, wherein the immunogenic composition is administered to pigs within the first month of age.

(102) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the immunogenic composition is administered to pigs within the first two weeks of age, preferably within the first week of age.

(103) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the immunogenic composition is administered to pigs at day 1 of age.

(104) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the immunogenic composition is administered to pigs within the first 24 h of age.

(105) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the subject is Swine influenza virus maternal antibody positive.

(106) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the immunogenic composition comprises 2 to 8 log 10 of the modified live H1 virus of swine influenza and/or 2 to 8 log 10 of the modified live H3 virus of swine influenza.

(107) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the immunogenic composition comprises 2 to 6 log 10 of the modified live H1 virus of swine influenza and/or 2 to 6 log 10 of the modified live H3 virus of swine influenza.

(108) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the immunogenic composition comprises 2 to 4 log 10 of the modified live H1 virus of swine influenza and/or 2 to 4 log 10 of the modified live H3 virus of swine influenza.

(109) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein said method results in an improvement in an efficacy parameter selected from the group consisting of: a reduction in weight loss, a lower virus load, a reduction in lung lesions, a reduced shedding, a reduced rectal temperature, or combinations thereof, in comparison to a subject of a non-immunized control group of the same species.

(110) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein said method results in an improvement in an efficacy parameter selected from the group consisting of: a reduction in weight loss, a lower virus load, a reduction in lung lesions, a reduced shedding, a reduced rectal temperature, or combinations thereof, in comparison to a subject of the same species immunized with a monovalent modified live H3 or H1 virus of swine influenza.

(111) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the treatment or prophylaxis results in shortening of the virus load phase as compared to animals of a non-treated control group of the same species.

(112) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the treatment or prophylaxis results in a reduction of the shedding from day 5 after challenge or infection.

(113) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the treatment or prophylaxis results in a reduction of the shedding from day 1 or day 2 after challenge or infection.

(114) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding embodiments, wherein the subject is immunized with a lower concentration of the immunogenic composition of any one of the preceding embodiments compared to a subject of the same species immunized with a monovalent modified live H3 or H1 virus of swine influenza.

(115) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the monovalent modified live H3 or H1 virus of swine influenza is a H1N1 NS1 deletion mutant of swine influenza virus.

(116) The immunogenic composition for use according to the above embodiment, wherein the H1N1 NS1 deletion mutant of swine influenza virus is the H1N1 NS1 deletion mutant of swine influenza virus according to any one of the preceding embodiments.

(117) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of preceding methods, wherein the monovalent modified live H3 or H1 virus of swine influenza is a H3N2 NS1 deletion mutant of swine influenza virus.

(118) The immunogenic composition for use according to the above embodiment, wherein the H3N2 NS1 deletion mutant of swine influenza virus is the H3N2 NS1 deletion mutant of swine influenza virus according to any one of the preceding embodiments.

(119) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the modified live H3 and H1 viruses of swine influenza act synergistically together.

(120) The immunogenic composition comprising any one of the preceding embodiments for use according to any one the preceding methods, wherein the concentration of the modified live H3 and H1 viruses of swine influenza are reduced compared to the concentration of the H3 virus of swine influenza in a monovalent immunogenic composition and the concentration of the H1 virus of swine influenza in a monovalent immunogenic composition.

(121) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the modified live H3 virus of swine influenza increases the protection against a heterologous challenge.

(122) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the modified live H3 virus of swine influenza increases the protection against a H1 challenge.

(123) The immunogenic composition comprising any one of the preceding embodiments for use according to any one of the preceding methods, wherein the modified live H3 virus of swine influenza increases the protection against a H1N1 challenge.

(124) The immunogenic composition according to any one of the preceding embodiments for use as a medicament.

(125) The use of an immunogenic composition comprising any one of the preceding embodiments for the treatment and/or prophylaxis of swine influenza virus infections in a subject.

(126) The use of the immunogenic composition comprising any one of the preceding embodiments for the manufacture of a medicament.

(127) A modified live H1 virus of swine influenza.

(128) A modified live H1 virus of swine influenza, wherein the modified live H1 virus of swine influenza has a N (neuraminidase) subtype selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9 and N10.

(129) The modified live H1 virus of swine influenza comprising any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza is H1N1 of swine influenza virus.

(130) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza has one or more mutations in the NS1 gene.

(131) The modified live H1 virus of swine influenza of any one of the embodiments, wherein the modified live H1 virus of swine influenza has a deletion within the NS1 gene.

(132) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza is a chimeric virus.

(133) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza comprises a carboxy-terminal truncated NS1 of a H3 SIV strain, preferably a H3N2 SIV strain.

(134) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza encodes a carboxy-terminal truncated NS1 protein comprising NS1 amino acids 1 through 124, 1 through 125, 1 through 126, 1 through 127 or 1 through 128, wherein the amino terminal amino acid is number 1.

(135) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza encodes a carboxy-terminal truncated NS1 protein comprising NS1 amino acids 1 through 126, wherein the amino terminal amino acid is number 1.

(136) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza encodes a carboxy-terminal truncated NS1 protein resulting in a deletion of 91, 92, 93 or 94 amino acid residues from the carboxy terminus of NS1.

(137) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the NS1 gene or protein of the modified live H1 virus of swine influenza is from A/Swine/Texas/4199-2/98.

(138) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza comprises the hemagglutinin and neuraminidase gene segments from a H1N1 subtype.

(139) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza comprises the hemagglutinin and neuraminidase gene segments from A/swine/Minnesota/37866/1999.

(140) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza contains HA and NA from A/swine/Minnesota/37866/1999 (H1N1) and PB2, PB1, PA, NP, M from A/Swine/Texas/4199-2/98 (H3N2) and the NS1-126 gene is from A/Swine/Texas/4199-2/98 (H3N2).

(141) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza is a chimeric of A/swine/Minnesota/37866/1999 and TX/98/del 126.

(142) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza a) comprises a nucleic acid sequence of gene segments having the NA and HA genes whose cDNA has greater than 70% identity to the nucleic acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6, or b) comprises a nucleic sequence of gene segments having the NA and HA genes encoding NA and HA proteins having an amino acid sequence with greater than 70% identity to the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8, or c) comprises a NA and HA protein having an amino acid sequence with greater than 70% identity to the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8.

(143) The modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the modified live H1 virus of swine influenza a) comprises a nucleic acid sequences of gene segments having the NA and HA genes whose cDNA has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleic acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:6, or b) comprises a nucleic sequences of gene segments having the NA and HA genes encoding NA and HA proteins having amino acid sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8, or c) comprises a NA and HA protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8.

(144) An immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments.

(145) The immunogenic composition of the above embodiment, wherein the immunogenic composition is formulated for a single-dose administration.

(146) The immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is administered intranasal.

(147) The immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is safe for sows during pregnancy and lactation.

(148) The immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is safe for pigs within the first two weeks of age, preferably within the first week of age.

(149) The immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is safe for pigs within the first day of age.

(150) The immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition further comprises a pharmaceutically acceptable carrier.

(151) The immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition comprises 2 to 8 log.sub.10 of the modified live H1 virus of swine influenza.

(152) The immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition comprises 2 to 6 log.sub.10 of the modified live H1 virus of swine influenza.

(153) The immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition comprises 2 to 4 log.sub.10 of the modified live H1 virus of swine influenza.

(154) The immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is a vaccine.

(155) The immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is effective in the treatment and/or prophylaxis of clinical signs caused by SIV in a subject of need.

(156) A method for immunizing a subject comprising administering to such subject an immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments.

(157) A method of treating or preventing clinical signs caused by swine influenza virus in a subject of need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments.

(158) A method of reducing the viral shedding in a subject of need, in comparison to a subject of a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments.

(159) A method of vaccinating a subject having anti-SIV antibodies comprising the step of administering to said animal a single effective dose of an immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments.

(160) A method for preventing or reducing early SIV infections in a subject, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments.

(161) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein said subject is selected from the list consisting of swine, cattle, cat and dog.

(162) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is administered once.

(163) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is administered at two or more doses.

(164) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is administered intranasal.

(165) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the subject is Swine influenza virus maternal antibody negative.

(166) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is administered to sows during pregnancy and lactation.

(167) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is administered to pigs within the first month of age.

(168) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is administered to pigs within the first two weeks of age, preferably within the first week of age.

(169) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is administered to pigs at day 1 or 2 of age.

(170) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition is administered to pigs within the first day of age.

(171) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the subject is Swine influenza virus maternal antibody positive.

(172) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition comprises 2 to 8 log.sub.10 of the modified live H1 virus of swine influenza.

(173) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition comprises 2 to 6 log.sub.10 of the modified live H1 virus of swine influenza.

(174) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the immunogenic composition comprises 2 to 4 log.sub.10 of the modified live H1 virus of swine influenza.

(175) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein said method results in an improvement in an efficacy parameter selected from the group consisting of: a reduction in the weight loss, a lower virus load, a reduction in lung lesions, a reduced shedding, a reduced rectal temperature, or combinations thereof, in comparison to a subject of a non-immunized control group of the same species.

(176) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the treatment or prophylaxis results in shortening of the virus load phase as compared to animals of a non-treated control group of the same species.

(177) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the treatment or prophylaxis results in a reduction of the shedding from day 5 after challenge or infection.

(178) The method of any one of the preceding embodiments comprising the modified live H1 virus of swine influenza of any one of the preceding embodiments, wherein the treatment or prophylaxis results in a reduction of the shedding from day 1 or 2 after challenge or infection.

(179) A method of immunizing a subject, comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus within the first two weeks of age.

(180) A method of treating or preventing clinical signs caused by swine influenza virus in a subject of need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus within the first two weeks of age.

(181) A method of reducing the viral shedding in a subject of need, in comparison to a subject of a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus within the first two weeks of age.

(182) A method of vaccinating a subject having anti-SIV antibodies comprising the step of administering to said animal a single effective dose of an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus within the first two weeks of age.

(183) A method for preventing or reducing early SIV infections in a subject, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus within the first two weeks of age.

(184) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the H3N2 NS1 deletion mutant of swine influenza virus encodes for a carboxy-terminal truncated NS1 protein comprising NS1 amino acids 1 through 124, 1 through 125, 1 through 126, 1 through 127 or 1 through 128, wherein the amino terminal amino acid is number 1.

(185) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the H3N2 NS1 deletion mutant of swine influenza virus encodes for a carboxy-terminal truncated NS1 protein comprising NS1 amino acids 1 through 126, wherein the amino terminal amino acid is number 1.

(186) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the H3N2 NS1 deletion mutant of swine influenza virus encodes for a carboxy-terminal truncated NS1 protein resulting in a deletion of 91, 92, 93 or 94 amino acid residues from the carboxy terminus of NS1.

(187) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the NS1 gene or protein of the H3N2 NS1 deletion mutant swine influenza virus is from A/Swine/Texas/4199-2/98.

(188) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the H3N2 NS1 deletion mutant of swine influenza virus is TX/98/del 126.

(189) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the H3N2 NS1 deletion mutant of swine influenza virus contains the HA, NA, PB2, PB1, PA, NP, and M from A/Swine/Texas/4199-2/98 and the NS1-126 gene is from A/Swine/Texas/4199-2/98.

(190) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the H3N2 NS1 deletion mutant of swine influenza virus is the H3N2 NS1 deletion mutant of swine influenza virus described in WO 2006/083286 A2 designated as TX/98/de1126.

(191) The method of any one the preceding of embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the H3N2 NS1 deletion mutant of swine influenza virus comprises a nucleic acid sequence of gene segments having the NA and HA genes whose cDNA has at least 70% identity to the nucleic acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2, or comprises a nucleic acid sequence of gene segments having the NA and HA genes encoding NA and HA proteins having an amino acid sequence with at least 70% identity to the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:4, or comprises a NA and HA protein having an amino acid sequence with at least 70% identity to the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:4.

(192) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the H3N2 NS1 deletion mutant of swine influenza virus comprises a nucleic acid sequences of gene segments having the NA and HA genes whose cDNA has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleic acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2, or comprises a nucleic sequences of gene segments having the NA and HA genes encoding NA and HA proteins having amino acid sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:4, or comprises a NA and HA protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO:3 or SEQ ID NO:4.

(193) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the subject is selected from the list consisting of swine, cattle, cat and dog.

(194) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the immunogenic composition is administered once.

(195) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the immunogenic composition is administered at two or more doses.

(196) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the immunogenic composition is administered intranasal.

(197) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the subject is Swine influenza virus maternal antibody negative.

(198) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the immunogenic composition is administered to sows during pregnancy and lactation.

(199) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the immunogenic composition is administered to pigs within the first week of age.

(200) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the immunogenic composition is administered to pigs at day 1 or day 2 of age.

(201) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the immunogenic composition is administered to pigs within the first day of age.

(202) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the subject is Swine influenza virus maternal antibody positive.

(203) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the immunogenic composition comprises 2 to 8 log.sub.10 of the H3N2 NS1 deletion mutant of swine influenza virus.

(204) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the immunogenic composition comprises 2 to 6 log.sub.10 of the H3N2 NS1 deletion mutant of swine influenza virus.

(205) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the immunogenic composition comprises 2 to 4 log.sub.in of the H3N2 NS1 deletion mutant of swine influenza virus.

(206) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein said method results in an improvement in an efficacy parameter selected from the group consisting of: a reduction in weight loss, a lower virus load, a reduction in lung lesions, a reduced shedding, a reduced rectal temperature, or combinations thereof, in comparison to a subject of a non-immunized control group of the same species.

(207) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the treatment or prophylaxis results in a reduction of the shedding from day 5 after challenge or infection.

(208) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the treatment or prophylaxis results in a reduction of the shedding from day 1 after challenge or infection.

(209) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the method provides protection against a heterologous challenge.

(210) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus wherein the method provides protection against a H1 challenge.

(211) The method of any one of the preceding embodiments comprising administering to a subject an immunogenic composition comprising a H3N2 NS1 deletion mutant of swine influenza virus, wherein the method provides protection against a H1N1 challenge.

EXAMPLES

(212) The following examples are only intended to illustrate the present invention. They shall not limit the scope of the claims in any way.

Example 1

Methods Used in Propagation of the Modified Live Vaccine Isolates and for Formulation of the Bivalent Vaccine

(213) The propagation of the modified live vaccine isolates can be accomplished using different systems. In this example the materials and methods are described for two different systems that have been evaluated to generate the antigen and ultimately the bivalent vaccine.

Genetic Characteristics of the Modified Live Vaccine Isolates in the Bivalent Vaccine

(214) Using reverse genetics a H1N1 NS1 mutant and a H3N2 NS1 mutant each encoding a carboxy-truncated NS1 protein have been generated. The H3N2 NS1 mutant already has been described in Solorzano et al. 2005 (J Virol 79:7535-7543), Vincent et al 2012 (Journal of Virology 19: 10597 to 10605) and WO 2006/083286 A2.

(215) For H1N1, the hemagglutinin (HA) and neuraminidase (NA) genes from the parent H1N1 (A/swine/Minnesota/37866/1999) were combined with the PB2, PB1, PA, NP, M and NS-126 genes from (A/Swine/Texas/4199-2/98). The generated H1N1 NS1 mutant therefore contained 2 genes from the H1N1 parent and 6 genes from the H3N2 parent.

(216) For H3N2, the HA, NA, PB2, PB1, PA, NP, M and NS-126 genes from A/Swine/Texas/4199-2/98 (H3N2) were combined and the resulting virus was designated TX98 H3N2 NS1 SIV.

Growth of the Modified Live Vaccine Isolates in Embryonated Chicken Eggs and Formulation of the Bivalent Vaccine

(217) Six to seven day embryonated chicken eggs are inoculated by injection of one or the other modified live viruses into the allantoic fluid (allantoic inoculation). Separate pre-labeled sets of eggs are used for growing the H1N1 NS1 isolate and the H3N2 NS1 isolate. After inoculation with the respective virus the eggs are incubated for three to four days then chilled for at least four hours before the virus containing allantoic fluid is harvested. Aliquots are made of the harvested allantoic fluid which contains high concentrations of the respective modified live virus. All of the viruses containing aliquots are stored at or below 40 C. until they are needed for vaccine formulation.

(218) Prior to vaccine formulation an aliquot of each virus congaing allantoic fluid is thawed and titrations are performed to determine the concentration of virus so that plans for vaccine formulations can be made. Once the quantity of virus is determined for the viral stocks the two isolates can be blended together along with phosphate buffered saline directly prior to vaccination to reach the desired inclusion for use in an animal study.

Growth of the Modified Live Vaccine Isolates in Cell Based Tissue Culture and Formulation of the Bivalent Vaccine

(219) A vessel containing a six to eight day old EVERO cell culture is inoculated by addition of one or the other modified live virus into the vessel after a media change and the addition of trypsin containing media. Separate pre-labeled sets of vessels are used for growing the H1N1 isolate and the H3N2 isolate. After inoculation with their respective virus the vessels are incubated for up to seven days then harvested. Aliquots are made of the harvested media that contains high concentrations of the respective modified live virus. All of the virus aliquots are stored at or below 40 C. until they were needed for vaccine formulation.

(220) Prior to vaccine formulation an aliquot of each virus congaing media is thawed and titrations are performed to determine the concentration of virus so that plans for vaccine formulations can be made. Once the quantity of virus is determined for the viral stocks the two isolates can be blended together along with a stabilizer (sucrose gelatin stabilizer) and phosphate buffered saline (only if needed) directly prior to being lyophilized. The volume of each of the virus stocks is controlled in order to reach the desired inclusion level for use in future animal studies. Once blended the bivalent vaccine material is aseptically transferred to sterile glass bottles then the bottles are loaded into a lyophilizer. The lyophilizer is then run through a freeze dry cycle to prepare the vaccine for storage at about 4 C. until use in an animal study.

(221) Directly prior to vaccination the lyophilized bivalent vaccine contained within the glass bottle is rehydrated with an appropriate volume of phosphate buffered saline in order to achieve the desired titer for use in the animal study.

Example #2

Single Dose Intranasal Administration at Various Doses to Pigs 1-6 Days Old Negative for Maternal Immunity Resulting in Reduced Nasal Shedding, Reduced Isolation From Lungs, and Reduced Lung Lesions

(222) Study design considerations, vaccine formulation, dosing information, and efficacy summaries.

Study 1

(223) On study day 0 the piglets (housed in separate rooms by group) were vaccinated with egg propagated vaccine virus at the inclusion levels indicated in Table 1. Only healthy pigs of about one week of age or less were allowed to be enrolled into the study as determined by a health examination prior to vaccination. The pigs were each vaccinated with 2 mL of the respective vaccine by applying 1 mL to each nostril. Feed rations were appropriate for the age and condition of test animals according to facility standard operating procedures. Water was provided ad libitum to the test animals throughout the study.

(224) TABLE-US-00001 TABLE 1 Study 1: Vaccine Inclusion Levels H1N1 Titer H3N2 Titer Group ID (log.sub.10TCID.sub.50/mL) (log.sub.10TCID.sub.50/mL) Higher Dose 7.28 8.10 Lower Dose 6.28 7.10

(225) Prior to challenge the piglets were weaned and moved into challenge rooms, then on or about study day 36 the piglets were challenged with 2 mL of the heterologous H1N2 (A/Swine/North Carolina/001169/2006) SIV isolate at a titer of 7.10 log.sub.10/mL by intratracheal inoculation under anesthesia. Nasal swabs were collected daily from challenge until necropsy and then the lungs were scored for lesions and lung tissue was collected at necropsy. The efficacy of the vaccine was evaluated by its ability to reduce the number of virus isolations in the nasal swabs and from the lung tissues as well as its ability to reduce the lung lesion in the vaccinated groups.

(226) As shown in Table 2 the animals vaccinated at both the higher dose and the lower dose were protected from the heterologous challenge as evidenced by reductions in virus isolation from nasal swabs and from lung tissue, as well as a reduction in lung lesions when comparisons are made to the group of pigs that were challenged after having received no vaccination.

(227) TABLE-US-00002 TABLE 2 Study 1: Vaccine Efficacy Summary From Challenge Through End of Study % Nasal Swab % Lung Tissue Group Average Group Virus Positive Virus Positive Lung Score Higher Dose 32% 0% 9.5% Lower Dose 33% 0% 8.5% No Vaccine 100% 47% 28.2%

(228) The conclusion made from the data generated in this study was that there are reductions in the amount of virus shed from the nasal passages, there are reductions in the virus load in the lungs as well as reductions in the primary efficacy parameter of gross lung lesions, which all taken together indicates that a single intranasal dose at the two inclusion levels used in this study is efficacious in nave pigs vaccinated within one to six days of age.

Study 2

(229) On study day 0 the piglets (housed in separate rooms by group) were vaccinated with reconstituted lyophilized tissue culture (EVERO cell) propagated vaccine virus at the inclusion levels indicated in Table 3. Only healthy pigs of about one week of age or less were allowed to be enrolled into the study as determined by a health examination prior to vaccination. The pigs were each vaccinated with 1 mL of the respective vaccine by applying the full volume to one nostril. Feed rations were appropriate for the age and condition of test animals according to facility standard operating procedures. Water was provided ad libitum to the test animals throughout the study.

(230) TABLE-US-00003 TABLE 3 Study 2: Vaccine Inclusion Levels H1N1 Titer H3N2 Titer Group ID (log.sub.10FAID.sub.50/mL) (log.sub.10FAID.sub.50/mL) Higher Dose 5.7 6.6 Medium Dose 5.1 6.0 Lower Dose 3.7 4.8

(231) Prior to challenge the piglets were weaned and moved into challenge rooms, then on or about study day 25 the piglets were challenged with 2 mL of the heterologous H1N2 (A/Swine/North Carolina/001169/2006) SIV isolate at a titer of 7.96 log.sub.10/mL by intratracheal inoculation under anesthesia. Nasal swabs were collected daily from challenge until necropsy and then the lungs were scored for lesions and lung lavage fluid was collected at necropsy. The efficacy of the vaccine was evaluated by its ability to reduce the number of virus isolations in the nasal swabs and from the lung lavage fluid as well as its ability to reduce the lung lesions in the vaccinated groups.

(232) As shown in Table 4 the animals vaccinated at all of the doses were protected from the heterologous challenge as evidenced by reductions in virus isolation from nasal swabs and from lung lavage fluid, as well as a reduction in lung lesions when comparisons are made to the group of pigs that were challenged after having received no vaccination.

(233) TABLE-US-00004 TABLE 4 Study 2: Vaccine Efficacy Summary From Challenge Through End of Study % Nasal Swab Ever % Lung Lavage Group Average Group Virus Positive Virus Positive Lung Score Higher Dose 45% 0% 10.3% Medium Dose 50% 0% 5.3% Lower Dose 48% 0% 5.8% No Vaccine 100% 10% 18.3%

(234) The conclusion made from the data generated in this study was that there are reductions in the amount of virus shed from the nasal passages, there are reductions in the virus isolation positive lungs as well as reductions in the primary efficacy parameter of gross lung lesions, which all taken together indicates that a single intranasal dose at the three inclusion levels used in this study is efficacious in nave pigs vaccinated within one to six days of age.

Study 3

(235) On study day 0 the piglets (housed in separate rooms by group) were vaccinated with reconstituted lyophilized tissue culture (EVERO cell) propagated vaccine virus at the inclusion levels indicated in Table 5. Only healthy pigs of about one week of age or less were allowed to be enrolled into the study as determined by a health examination prior to vaccination. The pigs were each vaccinated with 1 mL of the respective vaccine by applying the full volume to one nostril. Feed rations were appropriate for the age and condition of test animals according to facility standard operating procedures. Water was provided ad libitum to the test animals throughout the study.

(236) TABLE-US-00005 TABLE 5 Study 3 Vaccine Inclusion Levels H1N1 Titer H3N2 Titer Group ID (log.sub.10FAID.sub.50/mL) (log.sub.10FAID.sub.50/mL) Higher Dose 4.7 5.8 Medium Dose 3.6 4.7 Lower Dose 2.9 3.8

(237) Prior to challenge the piglets were weaned and moved into challenge rooms, then on or about study day 30 the piglets were challenged with 2 mL of the heterologous H3N2 (A/Swine/Nebraska/97901-10/2008) SIV isolate at a titer of 6.09 log.sub.10/mL by intratracheal inoculation under anesthesia. Nasal swabs were collected daily from challenge until necropsy and then the lungs were scored for lesions and lung lavage fluid was collected at necropsy. The efficacy of the vaccine was evaluated by its ability to reduce the number of virus isolations in the nasal swabs and from the lung lavage fluid as well as its ability to reduce the lung lesions in the vaccinated groups.

(238) As shown in Table 6 the animals vaccinated at all of the doses were protected from the heterologous challenge as evidenced by reductions in virus isolation from nasal swabs and from lung lavage fluid, as well as a reduction in lung lesions when comparisons are made to the group of pigs that were challenged after having received no vaccination.

(239) TABLE-US-00006 TABLE 6 Study 3 Vaccine Efficacy Summary From Challenge Through End of Study % Nasal Swab Ever % Lung Lavage Group Average Group Virus Positive Virus Positive Lung Score Higher Dose 46% 0% 2.5% Medium Dose 43% 0% 3.4% Lower Dose 50% 8% 4.5% No Vaccine 96% 65% 9.6%

(240) The conclusion made from the data generated in this study was that there are reductions in the amount of virus shed from the nasal passages, there are reductions in the virus isolation positive lungs as well as reductions in the primary efficacy parameter of gross lung lesions, which all taken together indicates that a single intranasal dose at the three inclusion levels used in this study is efficacious in nave pigs vaccinated within one to six days of age.

Example #3

Single Dose Intranasal Administration to Pigs Three Weeks Old Negative for Maternal Immunity

(241) Study design considerations, vaccine formulation, dosing information, and efficacy summaries.

Study 4

(242) On study day 0 the pigs (housed in separate rooms by challenge group) were vaccinated with egg propagated vaccine virus at the inclusion levels indicated in Table 7. Only healthy pigs of about three weeks of age were allowed to be enrolled into the study as determined by a health examination prior to vaccination. The pigs were each vaccinated with 4 mL of the vaccine by applying 2 mL to each nostril. Feed rations were appropriate for the age and condition of test animals according to facility standard operating procedures. Water was provided ad libitum to the test animals throughout the study.

(243) TABLE-US-00007 TABLE 7 Study 4 Vaccine Inclusion Levels H1N1 Titer H3N2 Titer Group ID (log.sub.10TCID.sub.50/mL) (log.sub.10TCID.sub.50/mL) Bivalent 7.38 7.97

(244) On or about study day 28 the piglets were challenged with either 2 mL of the heterologous H1N1 (A/Swine/Indiana/1726/88) SIV isolate at a titer of 6.63 log.sub.10/mL or 2 mL of the heterologous H3N2 (A/Swine/Nebraska/97901-10/2008) SIV isolate at a titer of 7.58 log.sub.10/mL by intratracheal inoculation under anesthesia. Nasal swabs were collected daily from challenge until necropsy and then the lungs were scored for lesions and lung tissue was collected at necropsy. The efficacy of the vaccine was evaluated by its ability to reduce the number of virus isolations in the nasal swabs and from the lung tissues as well as its ability to reduce the lung lesion in the vaccinated groups.

(245) As shown in Table 8 the vaccinated animals were protected from the heterologous H1N1 challenge as evidenced by reductions in virus isolation from nasal swabs and from lung tissue, as well as a reduction in lung lesions when comparisons are made to the group of pigs that were challenged after having received no vaccination. Similar results (data not shown) were seen for pigs that were vacated intramuscularly with the same vaccine. Also shown in Table 8 the vaccinated animals were protected from the heterologous H3N2 challenge as evidenced by reductions in virus isolation from lung tissue, as well as a reduction in lung lesions when comparisons are made to the group of pigs that were challenged after having received no vaccination. Nasal swabs were collected from the day of challenge (before challenge) through the day of necropsy. Since none of the bivalent vaccinated animals shed virus after the H1N1 challenge and the non-vaccinated animals started shedding at two days post challenge (data not shown) the bivalent vaccine prevented shedding of challenge virus from the vaccinated pigs at two days post challenge.

(246) TABLE-US-00008 TABLE 8 Study 4 Vaccine Efficacy Summary From Challenge Through End of Study % Nasal % Lung Group Challenge Swab Virus Tissue Virus Average Group Virus Positive Positive Lung Score Bivalent H1N1 0% 0% 0.5% No Vaccine Indiana 100% 100% 13.0% Bivalent H3N2 0% 0% 0.8% No Vaccine Nebraska 0% 10% 5.2%

(247) The conclusion made from the data generated in this study was that there are reductions in the amount of virus shed from the nasal passages following an H1N1 challenge and there are reductions in the virus load in the lungs as well as reductions in the primary efficacy parameter of gross lung lesions after challenge with either of the viruses. A single intranasal dose given at the inclusion level used in this study is efficacious in nave pigs vaccinated at three weeks of age.

Example #4

Safety of a Single Dose Intranasal Administration During Pregnancy

(248) Study design considerations, vaccine formulation, dosing information, and safety summaries.

Study 5

(249) On or about study day minus 28 the eight gilts were vaccinated with egg propagated vaccine virus at the inclusion levels indicated in Table 9. Only healthy pregnant gilts were allowed to be enrolled into the study as determined by a health examination prior to vaccination. The gilts were each vaccinated with 2 mL of the vaccine by applying 1 mL to each nostril. Feed rations were appropriate for the age and condition of test animals according to facility standard operating procedures. Water was provided ad libitum to the test animals throughout the study.

(250) TABLE-US-00009 TABLE 9 Study 5 Vaccine Inclusion Levels H1N1 Titer H3N2 Titer Group ID (log.sub.10TCID.sub.50/mL) (log.sub.10TCID.sub.50/mL) Bivalent 6.52 7.71

(251) After vaccination the safety of the vaccine was evaluated by observing and recording clinical signs daily for each of the vaccinated gilts. There were no major clinical signs recorded for any of the gilts between the time that they were vaccinated and when they farrowed. There were however some minor clinical signs recorded for three of the gilts, but none of the signs were observed until the fifth day after vaccination and none of the signs persisted for more than four days.

(252) The conclusion made from the data generated in this study is that the vaccine is safe when administered to gilts at the inclusion levels used in this study when vaccination is done around the ninetieth day of gestation.

Example 5

Single Dose Intranasal Administration to Pigs at 1-8 Days Old Positive for Maternal Immunity

(253) Study design considerations, vaccine formulation, dosing information, and efficacy summaries.

Study 5

(254) On or about study day minus 28, eight gilts were vaccinated with egg propagated vaccine virus at the inclusion level indicated in Table 1 in order to induce maternal immunity. The gilts were each vaccinated with 2 mL of the vaccine by applying 1 mL to each nostril. All of the gilts farrowed prior to study day 0, at which time the piglets were vaccinated with egg propagated vaccine virus at the inclusion level indicated in Table 10. Only healthy pigs of about one week of age or less were allowed to be enrolled into the study as determined by a health examination prior to vaccination. The pigs were each vaccinated with 2 mL of the respective vaccine by applying 1 mL to each nostril. Feed rations were appropriate for the age and condition of test animals according to facility standard operating procedures. Water was provided ad libitum to the test animals throughout the study.

(255) TABLE-US-00010 TABLE 10 Study 5 Vaccine Inclusion Levels H1N1 Titer H3N2 Titer Group ID (log.sub.10TCID.sub.50/mL) (log.sub.10TCID.sub.50/mL) Bivalent Gilts 6.52 7.71 Bivalent Pigs 5.84 7.32

(256) Prior to challenge the piglets were weaned and moved into a challenge room, then on or about study day 42 the piglets were challenged with 2 mL of the heterologous H1N2 (A/Swine/North Carolina/001169/2006) SIV isolate at a titer of 7.61 log.sub.10/mL by intratracheal inoculation under anesthesia. Nasal swabs were collected daily from challenge until necropsy and then the lungs were scored for lesions and lung lavage was collected at necropsy. The efficacy of the vaccine was evaluated by its ability to reduce the number of virus isolations in the nasal swabs and from the lung lavage as well as its ability to reduce the lung lesions in the vaccinated groups.

(257) As shown in Table 11 the animals vaccinated with the bivalent vaccine were protected from the heterologous challenge as evidenced by reductions in virus isolation from nasal swabs and from lung lavage, as well as a reduction in lung lesions when comparisons are made to the group of pigs that were challenged after having received no vaccination.

(258) TABLE-US-00011 TABLE 11 Study 5 Vaccine Efficacy Summary From Challenge Through End of Study % Nasal % Lung Group Swab Virus Tissue Virus Average Group Positive Positive Lung Score Bivalent 38.3% 0% 1.9% No Vaccine 43.3% 60% 4.0%

(259) The conclusion made from the data generated in this study was that there are reductions in the amount of virus shed from the nasal passages, there are reductions in the virus load in the lungs as well as reductions in the primary efficacy parameter of gross lung lesions, which all taken together indicates that a single intranasal dose at the inclusion level used in this study is efficacious in maternal immunity positive pigs vaccinated within one to eight days of age.

Example 6

Advantageous Effect of Single Dose Intranasal Administration of Bivalent Vaccine Over Monovalent Vaccine

(260) Study design considerations, vaccine formulation, dosing information, and efficacy summaries.

Study 6 and Study 1

(261) On study day 0 of the two respective studies the piglets (housed in separate rooms by group) were vaccinated with tissue culture (EVERO cell) propagated vaccine virus at the inclusion levels indicated in Table 12. Only healthy pigs of about one week of age or less were allowed to be enrolled into the studies as determined by a health examination prior to vaccination. In study 1 the pigs were each vaccinated with 1 mL of the monovalent vaccine by applying mL to each nostril. In study 6 the pigs were each vaccinated with 1 mL of the bivalent vaccine by applying the full volume to the left nostril. Feed rations were appropriate for the age and condition of test animals according to facility standard operating procedures. Water was provided ad libitum to the test animals throughout the study.

(262) TABLE-US-00012 TABLE 12 Study 1 and 6 Vaccine Inclusion Levels H1N1 Titer H3N2 Titer Study Group ID (log.sub.10FAID.sub.50/mL) (log.sub.10FAID.sub.50/mL) 1 2 Log 2.17 N/A 3 Log 2.98 N/A 4 Log 3.65 N/A 5 Log 4.99 N/A 6 3 Log 2.81 2.84 4 Log 3.99 4.00 5 Log 4.56 4.63 6 Log 5.48 5.66 N/A = not applicable

(263) Prior to challenge in the two respective studies the piglets were weaned and moved into challenge rooms, then on or about study day 25 the piglets were challenged with 2 mL of the heterologous H1N2 (A/Swine/North Carolina/001169/2006) SIV isolate at a titer of 7.44 log.sub.10/mL in study 6 and 7.92 log.sub.10/mL in study 1 by intratracheal inoculation under anesthesia. Nasal swabs were collected daily from challenge until necropsy and then the lungs were scored for lesions and lung lavage fluid was collected at necropsy. The efficacy of the vaccine was evaluated by its ability to reduce the number of virus isolations in the nasal swabs and from the lung lavage fluid as well as its ability to reduce the lung lesions in the vaccinated groups.

(264) As shown in Table 13 the animals vaccinated with the monovalent vaccine in study 6 required a dose of 4.99 log.sub.10FAID.sub.50/mL of the of the H1N1 vaccine virus before they were protected from the heterologous challenge. Protection is evidenced by a reduction in virus isolations from nasal swabs and from lung lavage fluid, as well as a reduction in lung lesions when comparisons are made to the group of pigs that were challenged after having received no vaccination in that same study.

(265) Also shown in Table 2 the animals in study 1 required a dose of only 2.81 log.sub.10FAID.sub.50/mL of the H1N1 vaccine virus when it was administered in the bivalent vaccine with 2.84 log.sub.10FAID.sub.50/mL of the H3N2 vaccine virus before they were protected from the heterologous challenge. The calculated total dose of antigen included in the protective bivalent vaccine is 3.13 log.sub.10FAID.sub.50/mL when the quantities of each antigen are added together. Protection is evidenced by reductions in virus isolations from nasal swabs and from lung lavage fluid, as well as reductions in lung lesions when comparisons are made to the group of pigs that were challenged after having received no vaccination in that same study.

(266) TABLE-US-00013 TABLE 13 Study 1 and 6 Vaccine Efficacy Summary From Challenge Through End of Study % Nasal % Lung Group Swab Ever Lavage Virus Average Study Group Virus Positive Positive Lung Score 1 5 Log 27% 0% 8.0% 4 Log 13% 0% 11.2% 3 Log 93% 13% 13.1% 2 Log 90% 40% 10.1% No Vaccine 93% 53% 9.4% 6 6 Log 18% 0% 2.44% 5 Log 5% 0% 3.06% 4 Log 14% 0% 5.10% 3 Log 53% 0% 8.50% No Vaccine 100% 14% 11.37%

(267) The experimental data provided herein show that a combination of the H1N1 NS1 and H3N2 deletion mutant of swine influenza virus together is, surprisingly more potent than the aggregate effect of the individual components.

(268) The synergistic effect of the bivalent vaccine of the present invention is very clearly demonstrated by its ability to provide protection when administered at a much lower inclusion level than what is required for the monovalent H1N1 vaccine. The bivalent vaccine of the present invention administered at a total dose of only 3.13 log.sub.10FAID.sub.50/mL provided protection as compared to the monovalent vaccine which showed a similar level of protection after being administered at a dose of 4.99 log.sub.10FAID.sub.50/mL (about 72-times more than the total dose of bivalent vaccine). Further, Vincent et al 2012 (Journal of Virology 19: 10597 to 10605) have shown that 110.sup.6 TCID.sub.50 (correspond to 6 log.sub.10 FAID.sub.50) of the monovalent H3N2 provided protection (about 741-times more than the total dose of bivalent vaccine). Thus, the concentration of the H1 and H3 components in the bivalent vaccine are dramatically reduced compared to the concentrations needed in the monovalent vaccines. This is an unexpected surprising synergistic effect.

(269) There is even further supporting evidence of the synergistic effect of the bivalent vaccine when the amount of challenge virus is considered. The monovalent vaccine even administered at the higher dose provided only a similar level of protection from a challenge of approximately 3-times less of the same challenge isolate.

(270) The conclusion made from the data generated in these studies is that there are reductions in the amount of virus shed from the nasal passages, there are reductions in the virus isolation positive lungs as well as reductions in the primary efficacy parameter of gross lung lesions, at much lower doses of antigen when the vaccine is administered as a bivalent product as compared to when it is administered as a monovalent product. All taken together the data indicates that a single intranasal dose of the bivalent vaccine is more efficacious than the higher dose monovalent vaccine used in nave pigs in these studies.

(271) The synergistic combination of the present invention is advantageous since the chances of adverse effects are reduced. Additionally, a bivalent vaccine containing a lower concentration than the individual components will clearly be easier and cheaper to manufacture as well.

(272) Moreover, there is a further advantageous surprising synergistic effect besides the above described synergistic concentration effect. The H3N2 component synergistically acts with the H1N1 component. The synergistic effect of the bivalent vaccine is very clearly demonstrated by its ability to provide protection from the heterologous H1N2 (A/Swine/North Carolina/001169/2006) SIV isolate. Thus, there is a cross protection effect. This effect is, further, synergistic because the bivalent vaccine of the present invention is administered at a much lower inclusion level than what is required for the monovalent vaccines as described above.

(273) The sequences accompanying this application are hereby incorporated by reference in its entirety:

(274) TABLE-US-00014 NucleotideSequenceofNAofH3N2(A/Swine/Texas/4199-2/98): SEQIDNO:1 1 TATTGGTCTCAGGGAGCAAAAGCAGGAGTAAAGATGAATCCAAATCAAAAGATAATAACG 61 ATTGGCTCTGTTTCTCTCACTATTGCCACAATGTGCTTCCTTATGCAAATTGCCATCCTG 121 GTAACTACTGTAACATTGCATTTCAAGCAATATGAATGCAACTACCCCCCAAACAACCAA 181 GTAATACTGTGTGAACCAACAATAATAGAAAGAAACATAACAGAGATAGTGTATCTGACC 241 AACACCACCATAGAGAAGGAAATATGCCCCAAACTAGCAGAATACAGAAATTGGTCAAAG 301 CCGCAATGTAAAATTACAGGATTTGCACCTTTTTCCAAGGACAATTCGATTAGGCTTTCC 361 GCTGGTGGGGACATTTGGGTGACAAGAGAACCTTATGTGTCATGCGATCCTGACAAGTGT 421 TATCAATTTGCCCTTGGACAGGGAACAACACTAAACAACAGGCATTCAAATGACACAGTA 481 CATGATAGGACCCCTTATCGAACCCTATTGATGAATGAGTTGGGTGTTCCATTTCATTTG 541 GGAACCAAGCAAGTGTGCATAGCATGGTCCAGCTCAAGTTGTCACGATGGAAAAGCATGG 601 CTGCATGTTTGTGTAACTGGGCATGATGAAAATGCAACTGCTAGCTTCATTTACGATGGG 661 AGGCTTGTAGATAGTATTGGTTCATGGTCCAAAAAAATCCTCAGGACCCAGGAGTCGGAA 721 TGCGTTTGTATCAATGGAACTTGTACAGTAGTAATGACTGATGGGAGTGCTTCAGGAAGA 781 GCTGATACTAAAATATTATTCATTGAGGAGGGGAAAATCGTTCATATTAGCCCATTGTTA 841 GGAAGTGCTCAGCATGTCGAGGAGTGCTCCTGTTATCCTCGATATCCTGGTGTCAGATGT 901 GTCTGCAGAGACAACTGGAAAGGCTCCAATAGGCCCATCGTAGATATAAATGTAAAGGAT 961 TATAGCATTGTTTCCAGTTATGTGTGCTCAGGACTTGTTGGAGACACACCCAGAAAAAAC 1021 GACAGATCCAGCAGTAGCAATTGCCTGAATCCTAACAATGAGGAAGGGGGTCATGGAGTG 1081 AAAGGCTGGGCCTTTGATGATGGAAATGACGTGTGGATGGGAAGAACGATCAACGAGAAG 1141 TTACGCTCAGGTTATGAAACCTTCAAAGTCATTGAAGGCTGGTCCAAACCTAACTCCAAA 1201 TTGCAGATAAATAGGCAAGTCATAGTTGACAGAGGTGATAGGTCCGGTTATTCTGGCATT 1261 TTCTCTGTTGAAGGCAAAAGCTGCATCAATCGGTGCTTTTATGTGGAGTTGATAAGGGGA 1321 AGGAAACAGGAAACTGAAGTATGGTGGACCTCAAACAGTATTGTTGTGTTTTGTGGCACC 1381 TCAGGTACATATGGAACAGGCTCATGGCCTGATGGGGCGGACATCAATCTCATGCCTATA 1441 TAAGCTTTCGCAATTTTAGAAAAAAACTCCTTGTTTCTACTAATACGAGACCATAT NucleotideSequenceofHAofH3N2(A/Swine/Texas/4199-2/98): SEQIDNO:2 1 TATTCGTCTCAGGGAGCAAAAGCAGGGGATAATTCTATTAACCATGAAGACTATCATTGC 61 TTTGAGCTACATTTTATGTCTGGTTTTCGCTCAAAAACTTCCCGGAAATGACAACAGCAC 121 AGCAACGCTGTGCCTGGGACACCATGCAGTGCCAAACGGAACCCTAGTGAAAACAATCAC 181 GAATGATCAAATTGAAGTGACTAATGCTACTGAGCTGGTTCAGAGTTCCTCAACAGGTAG 241 AATATGCGACAGTCCTCACCGAATCCTTGATGGAAAAAACTGCACATTGATAGATGCTCT 301 ACTGGGAGACCCTCATTGCGATGGCTTTCAAAATAAGGAATGGGACCTTTTTATTGAACG 361 CAGCAAAGCTTACAGCAACTGTTACCCTTATGATGTGCCGGATTATTCCTCCCTTAGGTC 421 ACTAGTTGCCTCATCAGGCACCCTGGAGTTTACCAATGAAGACTTCAATTGGACTGGGGT 481 CGCTCAGGATGGGGGAAGCTATTCTTGCAAAAGGGGATCTGTTAAAAGTTTCTTTAGTAG 541 ATTGAATTGGTTACACAAATTAGAATACAAATATCCAGCACTGAACGTGACTATGCCAAA 601 CAATGACAAATTTGACAAATTGTACATTTGGGGGGTTCACCACCCGAGCACGGACAGTGA 661 ACAAACCAGCCTGTATGTTCAAGCAATAGGGAGAGTCACAGTCTCTACCAAAAGTAGCCA 721 ACAAACTGTAATCCCGAACATCGGGTCCAGACCCTGGGTGAGGGGCATCTCCAGTAGAAT 781 AAGCATCTATTGGACAATAGTAAAACCGGGAGACATACTTTTGATTAGCAGCACAGGGAA 841 TCTAATTGCTCCTCGGGGTTACTTCAAAATACGAAATGGGAAAAGCTCAATAATGAGGTC 901 AGATGCACCCATTGACAACTGCTATTCTGAATGCATCACTCCAAATGGAAGCATTCCCAA 961 TGACAAACCTTTTCAAAATGTAAATAGGATCACATATGGGGCCTGTCCCAAATATGTTAA 1021 GCAAAAAACCCTGAAATTGGCAACAGGGATGCGGAATGTACCAGAGAAACAAACTAGAGG 1081 CATATTCGGCGCAATCGCAGGTTTCATAGAAAATGGTTGGGAGGGAATGGTAGACGGTTG 1141 GTACGGTTTCAGGCATCAAAATTCTGAGGGCACAGGACAAGCAGCAGATCTTAAAAGCAC 1201 CCAAGCAGCAATCGATCAAGTCAACGGGAAATTGAATAGGTTAATCGAGAAAACGAACGA 1261 GAAATTCCATCAAATCGAAAAAGAATTTTCAGAAGTAGAAGGGAGAATTCAGGACCTCGA 1321 GAAATATGTTGAAGACACTAAAATAGATCTCTGGTCTTACAACGCGGAGCTCCTTGTTGC 1381 CCTGGAGAATCAACATACAATTGATCTAACTGACTCAGAAATGAACAAACTGTTTGAAAA 1441 AACAAGGAAGCAACTGAGGGAAAATGCTGAGGACATGGGCAATGGTTGCTTCAAAATATA 1501 CCACAAATGTGACAATGCCTGCATAGGGTCAATCAGAAATGGAACTTATGACCATGATGT 1561 ATACAGAGACGAAGCATTAAACAACCGGTTCCAGATCAAAGGTGTTGAGCTGAAATCAGG 1621 ATACAAAGATTGGATCCTATGGATTTCCTTTGCCATATCATGCTTTTTGCTTTGTGTTGT 1681 TTTGCTGGGGTTCATCATGTGGGCCTGCCAAAAAGGCAACATTAGGTGCAACATTTGCAT 1741 TTGAGTGCATTAATTAAAAACACCCTTGTTTCTACTAATACGAGACGATAT AminoAcidSequenceofNAofH3N2(A/Swine/Texas/4199-2/98): SEQIDNO:3 MNPNQKIITIGSVSLTIATMCFLMQTAILVTTVTLHFKQYECNYPPNNQVILCEPTIIERNI TEIVYLTNTTIEKEICPKLAEYRNWSKPQCKITGFAPFSKDNSIRLSAGGDIWVTREPYVSC DPDKCYQFALGQGTTLNNRHSNDTVHDRTPYRTLLMNELGVPFHLGTKQVCTAWSSSSCHDG KAWLHVCVTGHDENATASFIYDGRLVDSIGSWSKKILRTQESECVCINGTCTVVMTDGSASG RADTKILFIEEGKIVHISPLLGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINVKDY SIVSSYVCSGLVGDTPRKNDRSSSSNCLNPNNEEGGHGVKGWAFDDGNDVWMGRTINEKLRS GYETFKVIEGWSKPNSKLQINRQVIVDRGDRSGYSGIFSVEGKSCINRCFYVELIRGRKQET EVWWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI AminoAcidSequenceofHAofH3N2(A/Swine/Texas/4199-2/98): SEQIDNO:4 MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTLVKTITNDQIEVTNATELVQSS STGRICDSPHRILDGKNCTLIDALLGDPHCDGFQNKEWDLFIERSKAYSNCYPYDVPDYSSL RSLVASSGTLEFTNEDFNWTGVAQDGGSYSCKRGSVKSFFSRLNWLHKLEYKYPALNVTMPN NDKFDKLYIWGVHHPSTDSEQTSLYVQAIGRVTVSTKSSQQTVIPNIGSRPWVRGISSRISI YWTIVKPGDILLISSTGNLIAPRGYFKIRNGKSSIMRSDAPIDNCYSECITPNGSIPNDKPF QNVNRITYGACPKYVKQKTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQ NSEGTGQAADLKSTQAAIDQVNGKLNRLIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKI DLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRKQLRENAEDMGNGCFKIYHKCDNACIGS IRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQKG NIRCNICI NucleotideSequenceofNAofH1N1 (A/swine/Minnesota/37866/1999): SEQIDNO:5 1 TATTGGTCTCAGGGAGCAAAAGCAGGAGTttaaaatgaatacaaatcaaagaataataac 61 catagggacagtctgtctgatagtcggaatagttagtctattattgcagatagggaatat 121 agtctcattatggataagccattcaattcagactggagaaaaaaaccactctgagatatg 181 caatcaaaacgtcattacatatgaaaataacacatgggtgaaccaaacttatgtaaacat 241 tagcaacaccaacattgctgatggacagggcgtgacttcaataaaactagccggcaattc 301 ctctctttgcccaataagtgggtgggctatatatagcaaagacaatagcataaggattgg 361 ttccaaaggagatatttttgtcataagagaaccattcatttcatgctctcatttggaatg 421 caggactttttttctgacccaaggtgctttgctgaatgacaggcattctaatggaaccgt 481 caaggacaggagcccttatagaaccttaatgagctgcccaattggtgaagctccatctcc 541 gtacaattcaaggttcgaatcagttgcttggtcagcaagtgcatgccatgatggaatggg 601 atggctaacaatcgggatttccggtccagataatggagcagtggcggttttgaaatacaa 661 tggtataataacagatacaataaaaagttggagaaacaaaatactaagaacacaagagtc 721 agaatgtgtttgtataaacggttcatgttttactataatgactgatggcccaagcaatgg 781 gcaggcctcatacaaaatattcaaaatggagaaagggaagattattaagtcagttgagct 841 ggatgcacccaattaccattatgaggaatgctcttgttaccctgatacaggcaaagtgat 901 gtgtgtgtgcagagacaattggcatgcttcgaatcgaccatgggtctctttcgaccggaa 961 tcttgattatcagatagggtacatatgcagtggggttttcggtgataacccgcgttctaa 1021 tgatgggaaaggcaattgtggcccagtactttctaatggagcaaatggagtgaaaggatt 1081 ctcattcagatatggcaatggtgtttggataggaagaactaaaagtatcagttctagaag 1141 tggatttgagatgatttgggatccaaatggatggacggaaactgatagtagtttctctat 1201 aaagcaggatattatagcattaactgactggtcaggatacagtgggagttttgtccaaca 1261 tcctgagttaacaggaatgaactgcataaagccttgtttctgggtagagttaattagagg 1321 acaacccaaggagagcacaatctggactagtggaagcagcatttctttctgtggtgtgga 1381 cagtgaaaccgcaagctggtcatggccagacggagctgatctgccattcaccattgacaa 1441 gtagtctgttcAAAAAACTCCTTGTTTCTACTAATACGAGACCATAT NucleotideSequenceofHAofH1N1 (A/swine/Minnesota/37866/1999): SEQIDNO:6 1 TATTCGTCTCAGGGAGCAAAAGCAGGGGaaaataaaagcaactagaatgaaggcaatact 61 agtagtcttgctatatgcatttacaaccgcaaatgcagacacattatgtataggttatca 121 tgcaaataattcaactgacactgttgacacagtactagaaaagaatgtaacagtaacaca 181 ctctgttaaccttctagaagaaaaacataacgggaaactatgtaaactaagaggagtagc 241 cccattgcatttgggtaaatgtaacattgctggatggatcctgggaaatccagagtgtga 301 atcactattcacagcaagctcatggtcttacattgtggaaacatctaattcagacaatgg 361 gacatgttacccaggagatttcatcaattatgaagagctaagagagcagttgagctcagt 421 gtcatcatttgaaagatttgagatattccccaaggcaagttcatggcccaattatgaaac 481 aagcagaggtgtgacggcagcatgtccttatgctggagcaaacagcttctacagaaattt 541 aatatggctggtaaaaaaaggaaattcatacccaaagctcagcaaatcttatattaacaa 601 taaggagaaggaagtcctcgtgctatggggcattcaccatccatctactagtactgacca 661 acaaagtctctaccagaatgcagatgcctatgtttttgtaggatcatcaaaatacagcaa 721 gaaattcaagccagaaatagcaacaagacccaaagtgagggaccaagcagggagaatgaa 781 ctattactggacactagtagagcctggagacacaataacattcgaagcaaccggaaatct 841 agtggtaccaagatatgccttcgcaatgaagagaggctctggatctggtattatcatttc 901 agatacatcagtccacgattgcaatacgaattgtcaaacacccaaaggtgctataaacac 961 cagtcttccatttcagaacatacatccagtcacaataggagaatgtccgaaatatgtcaa 1021 aagcaaaaaattgagaatggctacaggattaaggaatatcccgtccattcaatctagagg 1081 cctgtttggagccattgctggctttattgaggggggatggacaggaatgatagatggatg 1141 gtacggttatcaccatcaaaatgagcagggatcaggatatgcagccgaccgaaaaagcac 1201 acagagtgccattgacgggatcactaacaaggtaaattctattattgaaaagatgaactc 1261 acaattcacagcagtgggcaaagaattcaaccacctggaaaagagaatagagaatttgaa 1321 cagaaaggttgatgatggttttctggatgtttggacttacaatgccgagctgttggttct 1381 gttggaaaatgaaaggactttggattatcacgattcaaatgtgaagaacctatatgaaaa 1441 agtaagaagccagctaaaaaacaatgccaaggaaattggaaatggctgctttgaatttta 1501 ccacaaatgtgatgacacatgcatggagagcgtcaaaaatgggacttatgattacccaaa 1561 atactcagaagaagcaaaactaaacagagaggagatagatggggtaaagctggaatcaac 1621 agaggtttaccagattttggcgatctattcaactgtcgccagttcattggtactgttagt 1681 ctccctgggggcaatcagcttctggatgtgctccaatgggtctttacagtgcagaatatg 1741 tatttaaaattgggatttcagaggcatgagaAAAACACCCTTGTTTCTACTAATACGAGA 1801 CGATAT AminoAcidSequenceofNAofH1N1 (A/swine/Minnesota/37866/1999): SEQIDNO:7 MNTNQRIITIGTVCLIVGIVSLLLQIGNIVSLWISHSIQTGEKNHSEICNQNVITYENNTWV NQTYVNISNTNIADGQGVTSIKLAGNSSLCPISGWAIYSKDNSIRIGSKGDIFVIREPFISC SHLECRTFFLTQGALLNDRHSNGTVKDRSPYRTLMSCPIGEAPSPYNSRFESVAWSASACHD GMGWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNKILRTQESECVCINGSCFTIMTDGPSN GQASYKIFKMEKGKIIKSVELDAPNYHYEECSCYPDTGKVMCVCRDNWHASNRPWVSFDRNL DYQIGYICSGVFGDNPRSNDGKGNCGPVLSNGANGVKGFSFRYGNGVWIGRTKSISSRSGFE MIWDPNGWTETDSSFSIKQDIIALTDWSGYSGSFVQHPELTGMNCIKPCFWVELIRGQPKES TIWTSGSSISFCGVDSETASWSWPDGADLPFTIDK AminoAcidSequenceofHAofH1N1 (A/swine/Minnesota/37866/1999): SEQIDNO:8 MKAILVVLLYAFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEEKHNGKLCKLR GVAPLHLGKCNIAGWILGNPECESLFTASSWSYIVETSNSDNGTCYPGDFINYEELREQLSS VSSFERFEIFPKASSWPNYETSRGVTAACPYAGANSFYRNLIWLVKKGNSYPKLSKSYINNK EKEVLVLWGIHHPSTSTDQQSLYQNADAYVFVGSSKYSKKFKPEIATRPKVRDQAGRMNYYW TLVEPGDTITFEATGNLVVPRYAFAMKRGSGSGIIISDTSVHDCNTNCQTPKGAINTSLPFQ NIHPVTIGECPKYVKSKKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQN EQGSGYAADRKSTQSAIDGITNKVNSIIEKMNSQFTAVGKEFNHLEKRIENLNRKVDDGFLD VWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDDTCMESV KNGTYDYPKYSEEAKLNREEIDGVKLESTEVYQILAIYSTVASSLVLLVSLGAISFWMCSNG SLQCRICI