INFLUENZA VIRUS REASSORTMENT

Abstract

New influenza donor strains for the production of reassortant influenza A viruses are provided.

Claims

1-24. (canceled)

25. A reassortant influenza A virus comprising an HA segment, an NA segment and backbone segments PA, PB1, PB2, NP, NS and M, wherein the backbone segments are from two or more donor strains, wherein (a) the HA segment and the PB1 segment are from different influenza A strains with the same influenza virus HA subtype, (b) wherein the HA segment and the PB1 segment are from different influenza A strains with different influenza virus HA subtypes, wherein the PB1 segment is not from an influenza virus with a H3 HA subtype and/or wherein the HA segment is not from an influenza virus with a H1 or H5 HA subtype, or (c) at least one backbone segment is from the A/California/07/09 influenza strain.

26. The reassortant influenza A virus of claim 25, wherein the HA segment and the PB1 segment are from a H1 influenza strain.

27. The reassortant influenza A virus of claim 25, wherein the reassortant influenza A virus comprises (b) and the PB1 segment is from a H1 virus and/or wherein the HA segment is from a H3 influenza vims.

28. The reassortant influenza A virus of claim 25, wherein the reassortant influenza A virus comprises (c) and the at least one backbone segment is the PB1 segment.

29. The reassortant influenza A virus of claim 28, wherein the PB1 segment has at least 95%, at least 99% identity, or 100% identity with the sequence of SEQ ID NO: 16.

30. The reassortant influenza A virus of claim 25, wherein the reassortant influenza A virus comprises (c) and the HA segment is from a H1 influenza strain.

31. The reassortant influenza A virus of claim 25, wherein the PB1 segment and the PB2 segment are from the same donor strain.

32. The reassortant influenza A virus of claim 25, wherein the segments are selected from the group consisting of: a) the PA segment having at least 95% or 99% identity to the sequence of SEQ ID NO: 1; b) the PB2 segment having at least 95% or 99% identity to the sequence of SEQ ID NO: 3; c) the M segment having at least 95% or 99% identity to the sequence of SEQ ID NO: 5; d) the NP segment having at least 95% or 99% identity to the sequence of SEQ ID NO: 4; and/or e) the NS segment having at least 95% or 99% identity to the sequence of SEQ ID NO: 6.

33. The reassortant influenza A virus of claim 32, wherein the PA segment has 95% identity to the sequence of SEQ ID NO: 1, the PB2 segment has 95% identity to the sequence of SEQ ID NO: 3, the M segment has 95% identity to the sequence of SEQ ID NO: 5, the NP segment has 95% identity to the sequence of SEQ ID NO: 4 and the NS segment has 95% identity to the sequence of SEQ ID NO: 6.

34. The reassortant influenza A virus of claim 33, wherein the PA segment has the sequence of SEQ ID NO: 1, the PB2 segment has the sequence of SEQ ID NO: 3, the M segment has the sequence of SEQ ID NO: 5, the NP segment has the sequence of SEQ ID NO: 4 and the NS segment has the sequence of SEQ ID NO: 6.

35. The reassortant influenza A virus of claim 25 comprising backbone segments (i) from two, three or four donor strains, wherein each donor strain provides more than one backbone segment, (ii) from two or more donor strains, wherein the PB1 segment is not from the A/Texas/1/77 influenza strain, or (iii) from two or more donor strains, wherein at least the PA, NP, or M segment are not from A/Puerto Rico/8/34.

36. The reassortant influenza A virus of claim 25, wherein at least one of the backbone segments is selected from the group consisting of: a) the PB2 segment which has lysine in the position corresponding to amino acid 389 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm; and/or b) the PB2 segment which has asparagine in the position corresponding to amino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm; and/or c) the PA segment which has lysine in the position corresponding to amino acid 327 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm; and/or d) the PA segment which has aspartic acid in the position corresponding to amino acid 444 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm; and/or e) the PA segment which has aspartic acid in the position corresponding to amino acid 675 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm; and/or f) the NP segment which has threonine in the position corresponding to amino acid 27 of SEQ ID NO: 4 when aligned to SEQ ID NO: 4 using a pairwise alignment algorithm; and/or g) the NP segment which has asparagine in the position corresponding to amino acid 375 of SEQ ID NO: 4 when aligned to SEQ ID NO: 4, using a pairwise alignment algorithm.

37. The reassortant influenza A strain of claim 36, wherein (i) the PB2 segment has lysine in the position corresponding to amino acid 389 of SEQ ID NO: 3 and asparagine in the position corresponding to amino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm, (ii) the PA segment has lysine in the position corresponding to amino acid 327: aspartic acid in the position corresponding to amino acid 444 of SEQ ID NO: 1 and aspartic acid in the position corresponding to amino acid 675 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm, or (iii) the NP genome segment has threonine in the position corresponding to amino acid 27 of SEQ ID NO: 4 and asparagine in the position corresponding to amino acid 375 when aligned to SEQ ID NO: 4. using a pairwise alignment algorithm, or (iv) the influenza A strain is a H1 strain.

38. The reassortant influenza A strain of claim 25, wherein the PB2 segment has lysine in the position corresponding to amino acid 389 of SEQ ID NO: 3 and asparagine in the position corresponding to amino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm, PA genome segment has lysine in the position corresponding to amino acid 327; aspartic acid in the position corresponding to amino acid 444 of SEQ ID NO: 1 and aspartic acid in the position corresponding to amino acid 675 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm, and an NP genome segment has threonine in the position corresponding to amino acid 27 of SEQ ID NO: 4 and asparagine in the position corresponding to amino acid 375 when aligned to SEQ ID NO: 4, using a pairwise alignment algorithm

39. A method of preparing a reassortant influenza A virus of claim 25 comprising steps of (i) introducing into a culture host one or more expression construct(s) which encode(s) the viral segments required to produce an influenza A virus wherein the expression construct(s) encode the backbone segments from two or more donor strains and wherein the HA and PB1 genome segments are from different influenza strains which have the same influenza HA subtype; and (ii) culturing the culture host in order to produce the reassortant influenza A virus of claim 25, wherein the reassortant influenza A virus comprises (a).

40. The method of claim 39, wherein (1) the expression construct(s) do/does not encode the PB1 segment from the A/Texas/1/77 influenza strain, (2) the at least one expression construct comprises a sequence having at least 90% or 100% identity with the sequence of SEQ ID NO: 22, (3) the expression construct(s) further comprise(s) one or more of the sequences having at least 90% identity or 100% identity with the sequences of SEQ ID Nos.: 9 and/or 11 to 14, or (4) further comprising the step (iii) of purifying the reassortant virus obtained in step (ii).

41. A method of preparing a reassortant influenza A virus of claim 28 comprising steps of (i) introducing into a culture host one or more expression construct(s) which encode(s) the viral segments required to produce an influenza A virus wherein the expression construct(s) encode the backbone segments from two or more donor strains and wherein the PB1 backbone viral segment is from A/California/07/09; and (ii) culturing the culture host in order to produce the reassortant influenza A virus of claim 28.

42. The method of claim 39, wherein the expression construct(s) are (3) and comprise(s) all of the sequences having at least 90% identity or 100% identity with the sequences of SEQ ID Nos.: 9 and 11 to 14.

43. The method of claim 40, wherein the HA segment is from a H1 influenza virus.

44. A method for producing influenza viruses comprising steps of (a) infecting a culture host with the reassortant influenza virus of claim 25; (b) culturing the host from step (a) to produce the virus; and optionally (c) purifying the virus produced in step (b).

45. A method of preparing a vaccine, comprising the steps of (a) preparing a virus by the method of claim 44 and (b) preparing a vaccine from the virus.

46. The method of claim 44, wherein the culture host is an embryonated hen egg.

47. The method of claim 44, wherein the culture host is a mammalian cell, optionally an MDCK (such as MDCK 33016 (DSM ACC2219)), Vero or PerC6 cell.

48. The method of claim 47, wherein the cell grows adherently or in suspension.

49. The method of claim 45, wherein step (b) involves inactivating the virus.

50. The method of claim 45, wherein the vaccine is a whole virion vaccine, a split virion vaccine. a surface antigen vaccine, or a virosomal vaccine.

51. The method of claim 45, wherein the vaccine contains less than 10 ng of residual host cell DNA per dose.

52. The method of claim 45, wherein at least one of the influenza strains is of the H1, H2, H5, H7 or H9 subtype.

53. An expression system comprising one or more expression construct(s) comprising the vRNA encoding segments of an influenza A virus wherein the expression construct(s) encode(s) the backbone viral segments from two or more influenza donor strains, wherein (i) the HA and PB1 segments are from two different influenza strains with the same influenza HA subtype, (ii) the HA and PB1 segments are from two different influenza strains with different influenza virus HA subtypes, wherein the expression construct(s) do(es) not encode the PB1 segment from an influenza virus with a H3 HA subtype and/or wherein the expression construct(s) do(es) not encode the HA segment from an influenza virus with a H1 or H5 HA subtype. or (iii) wherein the PB1 segment is from A/California/07/09.

54. The expression system of claim 53, wherein (a) the expression construct(s) may further comprise the vRNAs which encode the PB2, NP, NS, M and PA segments from PR8-X,(b) wherein the at least one expression construct comprises a sequence having at least 90%, at least 95%, at least 99% or 100% identity with the sequence of SEQ ID NO: 22, (c) the expression construct(s) further comprise(s) one or more of the sequences having at least 90%, at least 95%, at least 99% or 100% identity with the sequences of SEQ ID Nos.: 9, and/or 11 to 14. or (d) the expression construct(s) comprise(s) all of the sequences having at least 90%, at least 95%, at least 99% or 100% identity with the sequences of SEQ ID Nos.: 9 and 11 to 14.

55. A host cell comprising the expression system of claim 53.

56. The host cell of claim 55, wherein the host cell is a mammalian cell, optionally an MDCK, Vero or PerC6 cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0167] FIG. 1(A) and FIG. 1(B) compare the HA content (determined by lectin-capture ELISA) of sucrose gradient-purified viruses harvested at 60 h post-infection from MDCK cell cultures infected with reverse genetics-derived 6:2 reassortants containing either the PR8-X or #21 backbone with the HA and NA segments from a pandemic-like H1 strain (strain 1; FIG. 1(A)) or a second pandemic-like strain (strain 2; FIG. 1(B)). In FIG. 1(A) and FIG. 1(B), the white bar represents a reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain) as control, the dotted bar represents a reassortant virus containing the PR8-X backbone, and the checked bar represents a reassortant virus containing the #21 backbone. The y-axis indicates HA yield in g/ml.

[0168] FIG. 2(A) and FIG. 2(B) compare the HA content (determined by a lectin-capture ELISA) of unpurified viruses harvested at 60 h post-infection from MDCK cell cultures infected with reverse genetics-derived 6:2 reassortants containing either the PR8-X or #21 backbone with the HA and NA segments from a pre-pandemic H1 strain (strain 1; FIG. 2(A)) and a second pre-pandemic H1 strain (strain 2; FIG. 2(B)). In FIG. 2(A) and FIG. 2(B), the white bar represents a reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain) as control, the dotted bar represents a reassortant virus containing the PR8-X backbone, and the checked bar represents a reassortant virus containing the #21 backbone. The y-axis indicates HA yield in g/ml.

[0169] FIG. 3 compares the HA yield (determined by HPLC) of sucrose-purified viruses harvested at 60 h post-infection from MDCK cell cultures infected with reverse genetics-derived 6:2 reassortants containing either the PR8-X or #21 backbone with the HA and NA segments from an H3 strain (strain 1). The white bar represents a reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain) as control, the dotted bar represents a reassortant virus containing the PR8-X backbone, and the checked bar represents a reassortant virus containing the #21 backbone. The y-axis indicates HA yield in g/ml.

[0170] FIG. 4(A) and FIG. 4(B) compare virus titers (determined by focus formation assay (FFA); FIG. 4(A)) and HA titers (determined by lectin-capture ELISA;

[0171] FIG. 4(B)) of viruses harvested from embyronated chicken eggs at 60 h post-infection with a reference vaccine strain or reverse genetics-derived 6:2 reassortant viruses made with either the PR8-X or #21 backbone and the HA and NA segments from a pandemic-like H1 strain (strain 2). In FIG. 4(A), the individual dots represent data from single eggs. The line represents the average of the individual data points. The y-axis indicates infectious units/ml. In FIG. 4(B), the white bar represents the reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain), the dotted bar represents a reassortant virus containing the PR8-X backbone, and the checked bar represents a reassortant virus containing the #21 backbone. The y-axis indicates HA yield in g/ml for pooled egg samples

[0172] FIG. 5(A) and FIG. 5(B) compare virus titers (determined by FFA; FIG. 5(A)) and HA titers (determined by lectin-capture ELISA; FIG. 5(B)) from viruses harvested at 60 h post-infection from MDCK cells infected with a reference vaccine strain or reverse genetics-derived 6:2 reassortant viruses made with either the #21 or #21C backbone and the HA and NA segments from a pandemic-like H1 strain (strain 2). In both figures, the white bar represents a reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain) as control, the dotted bar represents a reassortant virus made with the #21 backbone, and the checked bar represents a reassortant virus made with a modified #21 backbone (#21C) containing two canine-adapted mutations (R389K, T559N) in the PR8-X PB2 segment that comprises the backbone. The y-axis in FIG. 5(A) and FIG. 5(B) indicates infectious units/ml and HA yield in g/ml, respectively.

[0173] FIG. 6 compares virus titers (determined by FFA) from viruses harvested at 60 h post-infection from MDCK cells infected with reverse genetics-derived 6:2 reassortant viruses made with either the PR8-X, #21 or #21C backbone and the HA and NA segments from a different pandemic-like H1 strain (strain 1). The white bar represents the PR8-X backbone, the dotted bar represents the #21 backbone, and the checked bar represents the #21C backbone containing two canine-adapted mutations (R389K, T559N) in the PR8-X PB2 segment that comprises the backbone. The y-axis indicates infectious units/ml.

[0174] FIG. 7 compares HA titers (determined by red blood cell hemagglutination assay) from viruses harvested at 60 h post-infection from embryonated chicken eggs infected with a reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain) or reverse genetics-derived 6:2 reassortant viruses containing either the PR8-X or #21C backbone and the HA and NA segments from a pandemic-like H1 strain (strain 1). The individual dots represent data from a single egg. The line represents the average of the individual data points. The y-axis indicates HA units.

[0175] FIG. 8(A) and FIG. 8(B) compare infectious titers (determined by FFA) of viruses harvested at different time points post-infection of MDCK cells infected with reverse genetics-derived 6:2 reassortants made with either a PR8-X backbone or a modified PR8-X backbone containing canine-adapted polymerase mutations and the HA and NA segments from a pandemic-like H1 strain (strain 1). In FIG. 8(A), the dotted line with triangle markers indicates the PR8-X backbone and the solid line with square markers indicates a modified PR8-X backbone PRS-X(cPA) containing three canine-adapted mutations (E327K, N444D, and N675D) in the PR8-X PA segment. In FIG. 8(B), the dotted line with triangle markers indicates the PR8-X backbone and the solid line with open circle markers indicates a modified PR8-X backbone PR8-X(cNP) containing two canine-adapted mutations (A27T, E375N) in the PR8-X NP segment. In both figures, the x-axis indicates hours post-infection and the y-axis indicates infectious units/ml.

[0176] FIG. 9(A) and FIG. 9(B) compare infectious titers (determined by FFA; FIG. 9(A)) and HA titers (determined by red blood cell hemagglutination assay; FIG. 9(B)) of virus harvested at different times post-infection from MDCK cells infected with a reference vaccine strain or reverse genetics-derived 6:2 reassortant viruses made with either the PR8-X or modified PR8-X backbones containing canine-adapted mutations and the HA and NA segments from an H3 strain (strain 2). In FIG. 9(A), the dotted line with x markers indicates the reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain), the dotted line with triangle markers indicates the PR8-X backbone, the solid line with square markers indicates a modified PR8-X backbone PR8-X (cPA) containing three canine-adapted mutations (E327K, N444D, and N675D) in the PR8-X PA segment, and the solid line with open circle markers indicates a modified PR8-X backbone PR8-X (cNP) containing two canine-adapted mutations a in the PR8-X NP segment. The y-axis represents infectious units/ml and the x-axis represents hours post-infection. In FIG. 9(B), the white bar indicates the reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain), the dotted bar indicates the PR8-X backbone, the checked bar indicates the PRB-X(cPA) backbone and the cross-hatched bar indicates the PR8-X(cNP) backbone. The y-axis represents HA units from the 60 h post-infection time-point.

[0177] FIG. 10(A), FIG. 10(B), and FIG. 10(C) compare the HA content (determined by lectin-capture ELISA) of sucrose gradient-purified viruses harvested at 60 h post-infection from MDCK cell cultures infected with reverse genetics-derived 6:2 reassortants containing either the PR8-X or #21 backbone with the HA and NA segments from an H3 (strain 2; FIG. 10(A)) or a second H3 strain (strain 3; FIG. 10(B)) or a third H3 strain (strain 4; FIG. 10(C)). In FIG. 10(A) and FIG. 10(B), the white bar represents a reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain) as control, the dotted bar represents a reassortant virus containing the PR8-X backbone, and the checked bar represents a reassortant virus containing the #21 backbone. The y-axis indicates HA yield in g/ml.

MODES FOR CARRYING OUT THE INVENTION

Development of New Donor Strains

[0178] In order to provide high-growth donor strains, the inventors found that a reassortant influenza virus comprising the PB1 segment of A/California/07/09 and all other backbone segments from PR8-X shows improved growth characteristics compared with reassortant influenza viruses which contain all backbone segments from PR8-X. This influenza backbone is referred to as #21.

Focus-Forming Assays (FFA)

[0179] For the FFA, uninfected MDCK cells are plated at a density of 1.8104 cells/well in 96 well plates in 100 pi of DMEM with 10% FCS. The next day, medium is aspirated and cells are infected with viruses in a volume of 50 pi (viruses diluted in DMEM+1% FCS). The cells are incubated at 37 C. until the next day.

[0180] At several time points after infection, the medium is aspirated and the cells washed once with PBS. 50 l of ice-cold 50%/50% acetone-methanol is added to each well followed by incubation at 20 C. for 30 minutes. The acetone mix is aspirated and the cells washed once with PBST (PBS+0.1% Tween). 50 l of 2% BSA in PBS is added to each well followed by incubation at room temperature (RT) for 30 minutes. 50 l of a 1:6000 dilution of anti-NP is added in blocking buffer followed by incubation at RT for 1 hours. The antibody solution is aspirated and the cells washed three times with PBST. Secondary antibody (goat anti mouse) is added at a dilution 1:2000 in 50 l blocking buffer and the plate is incubated at RT for 1 hours. The antibody solution is aspirated and the cells washed three times with PBST. 50 l of KPL True Blue is added to each well and incubated for 10 minutes. The reaction is stopped by aspirating the True-Blue and washing once with dH.sub.2O. The water is aspirated and the cells are left to dry.

Growth Characteristics of Reassortant Viruses Containing PR8-X or #21 Backbones

[0181] In order to test the suitability of the #21 strain as a donor strain for virus reassortment, reassortant influenza viruses are produced by reverse genetics which contain the HA and NA proteins from various influenza strains (including zoonotic, seasonal, and pandemic-like strains) and the other viral segments from either PR8-X or the #21 backbone. The HA content, HA yield and the viral titres of these reassortant viruses are determined. As a control a reference vaccine strain which does not contain any backbone segments from PR8-X or A/California/07/09 is used. These viruses are cultured either in embyronated chicken eggs or in MDCK cells.

[0182] The results indicate that reassortant viruses which contain the #21 backbone consistently give higher viral titres and HA yields compared with the control virus and the virus which contains all backbone segments from PR8-X in both eggs and cell culture. This difference is due to the PB1 segment because this is the only difference between #21 reassortants and PR8-X reassortants (see FIG. 1(A), FIG. 1(B), FIG. 2(A), FIG. 2(B), FIG. 3, FIG. 4(A), and FIG. 4(B)).

Growth Characteristics of Reassortant Viruses Containing PR8-X or Canine Adapted PR8-X Backbones

[0183] In order to test the effect of canine-adapted mutations on the growth characteristics of PR8-X, the inventors introduce mutations into the PA segment (E327K, N444D, and N675D), or the NP segment (A27T, E375N) of PR8-X. These backbones are referred to as PR8-X(cPA) and PR8-X(cNP), respectively. Reassortant influenza viruses are produced containing the PRB-X(cPA) and PR8-X(cNP) backbones and the HA and NA segments of a pandemic-like H1 influenza strain (strain 1) or a H3 influenza strain (strain 2). As a control a reference vaccine strain which does not contain any backbone segments from PR8-X is used. The reassortant influenza viruses are cultured in MDCK cells.

[0184] The results show that reassortant influenza viruses which contain canine-adapted backbone segments consistently grow to higher viral titres compared with reassortant influenza viruses which contain unmodified PR8-X backbone segments (see FIG. 8(A), FIG. 8(B), FIG. 9(A), and FIG. 9(B)).

Growth Characteristics of Reassortant Viruses Containing PR8-X, #21 or #21C Backbones

[0185] In order to test whether canine-adapted mutations in the backbone segments improve the growth characteristics of the #21 backbone, the inventors modify the #21 backbone by introducing mutations into the PR8-X PB2 segment (R389K, T559N). This backbone is referred to as #21C. Reassortant influenza viruses are produced by reverse genetics which contain the HA and NA proteins from two different pandemic-like H1 strains (strains 1 and 2) and the other viral segments from either PR8-X, the #21 backbone or the #21C backbone. As a control a reference vaccine strain which docs not contain any backbone segments from PR8-X or A/California/07/09 is used. These viruses are cultured in MDCK cells. The virus yield of these reassortant viruses is determined. For reassortant influenza viruses containing the HA and NA segments from the pandemic-like H1 strain (strain 1) and the PR8-X or #21C backbones the FIA titres are also determined.

[0186] The results show that reassortant influenza viruses which contain the #21C backbone consistently grow to higher viral titres compared with reassortant influenza viruses which contain only PR8-X backbone segments or the #21 backbone (see FIG. 5(A), FIG. 5(B), FIG. 6, and FIG. 7). Reassortant influenza viruses comprising the #21C backbone also show higher HA titres compared with PR8-X reassortants.

[0187] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

[0188] [1] WO2007/002008 [0189] [2] WO20071124327 [0190] [3] WO2010/070098 [0191] [4] Needleman & Wunsch (1970) J. Mol. Biol. 48, 443-453. [0192] [5] Rice et al. (2000) Trends Genet 16:276-277. [0193] [6] U.S. Pat. No. 6,468,544. [0194] [7] Herloeher et al. (2004) J Infect Dis 190(9): 1627-30. [0195] [8] Le et al. (2005) Nature 437(7062): 1108. [0196] [9] Neumann et al. (2005) Proc Natl Acad Sci USA 102: 16825-9 [0197] [10] WO2010/133964 [0198] [11] WO2009/000891 [0199] [12] U.S. provisional application No. 61/273,151 [0200] [13] Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed., 1989, Cold Spring Harbor Press, Cold Spring Harbor, N. Y [0201] [14] WO201 1/012999 [0202] [15] Kistner et al. (1998) Vaccine 16:960-8. [0203] [16] Kistner et al. (1999) Dev Biol Stand 98: 101-1 10. [0204] [17] Briilil et al. (2000) Vaccine 19: 1149-58. [0205] [18] Pau et al. (2001) Vaccine 19:2716-21. [0206] [19] http://www.atcc. org/ [0207] [20] http://locus.umdnj.edu/ [0208] [21] WO97/37000. [0209] [22] Brands et al. (1999) Dev Biol Stand 98:93-100. [0210] [23] Halperin et al. (2002) Vaccine 20: 1240-7. [24] EP-A-1260581 (WOO 1/64846) [0211] [25] WO2006/071563 [0212] [26] WO2005/113758 [0213] [27] WO97/37001 [0214] [28] WO02/28422. [0215] [29] WO02/067983. [0216] [30] WO02/074336. [0217] [31] WOOI/21 151. [0218] [32] WO02/097072. [0219] [33] WO2005/113756. [0220] [34] Huckriede et al. (2003) Methods Enzymol 373:74-91. [0221] [35] Vaccines, (eds. Plotkins & Orenstein). 4th edition, 2004, ISBN: 0-7216-9688-0 [0222] [36] Treanor et al. (1996) J Infect Dis 173: 1467-70. [0223] [37] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10. [0224] [38] Herlocher et al. (2004) J Infect Dis 190(9): 1627-30. [0225] [39] Le et al. (2005) Nature 437(7062): 1108. [0226] [40] WO2008/068631. [0227] [41] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. [0228] [42] Banzhoff (2000) Immunology Letters 71:91-96. [0229] [43] Nony et al. (2001) Vaccine 27:3645-5 1. [0230] [44] EP-B-0870508. [0231] [45] U.S. Pat. No. 5,948,410. [0232] [46] WO2007/052163. [0233] [47] WO2007/052061 [0234] [48] WO90/14837. [0235] [49] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203. [0236] [50] Podda (2001) Vaccine 19: 2673-2680. [0237] [51]Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X). [0238] [52] Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan. [0239] [53] WO2008/043774. [0240] [54] Allison & Byars (1992) Res Immunol 143:519-25. [0241] [55] Hariharan et al. (1995) Cancer Res 55:3486-9. [0242] [56] US-2007/014805. [0243] [57] US-2007/0191314. [0244] [58] S li et al. (2004) Vaccine 22(25-26):3464-9. [0245] [59] WO95/11700. [0246] [60] U.S. Pat. No. 6,080,725. [0247] [61]WO2005/097181. [0248] [62] WO2006/113373. [0249] [63] Potter & Oxford (1979) Br Med Bull 35:69 75. [0250] [64] Greenbaum et al. (2004) Vaccine 22:2566-77. [0251] [65] Zurbriggen et al. (2003) Expert Rev Vaccines 2:295-304. [0252] [66] Piascik (2003) J Am Pharr Assoc (Wash DC). 43:728-30. [0253] [67] Mann et al. (2004) Vaccine 22:2425-9. [0254] [68] Halperin et al. (1979) Am J Public Health 69:1247-50. [0255] [69] Herbert et al. (1979) J Infect Dis 140:234-8. [0256] [70] Chen et al. (2003) Vaccine 21:2830-6. [0257] [71]Current Protocols in Molecular Biology (F. M. Ausubel et al, cds., 1987) Supplement 30. [0258] [72] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489