INFLUENZA VIRUS REASSORTMENT

Abstract

Improved methods for the production of reassortant influenza viruses are provided.

Claims

1. A method of preparing an influenza virus, comprising: a) preparing one or more expression construct(s) which comprise(s) coding sequences for expressing at least one segment of an influenza virus genome; b) introducing into a cell which is not 293T one or more expression construct(s) which encode(s) the viral segments of an influenza virus, wherein at least one expression construct is the expression construct prepared in step (a); and c) culturing the cell in order to produce a reassortant influenza virus from the express construct(s) introduced in step (b); wherein steps (a) to (c) are performed in a time period of 124 hours or less.

2. A method of preparing an influenza virus comprising the steps of a) preparing one or more expression construct(s) which comprise(s) coding sequences for expressing at least one segment of an influenza virus genome; b) introducing into a cell one or more expression construct(s) which encode(s) the viral segments of an influenza virus, wherein at least one expression construct is the expression construct prepared in step (a); and c) culturing the cell in order to produce a reassortant influenza virus from the expression construct(s) introduced in step (b); wherein steps (a) to (c) are performed in a time period of 100 hours or less.

3. The method of claim 1, wherein the cell is a non-human cell or a human non-kidney cell.

4. A method of preparing a reassortant influenza virus, comprising: a) providing a synthetic expression construct which comprises coding sequences for expressing at least one segment of an influenza virus genome by (i) synthesising a plurality of overlapping fragments of the synthetic expression construct, wherein the overlapping fragments span the complete synthetic expression construct, and (ii) joining the fragments to provide the synthetic expression construct; b) introducing into a cell which is not 293T one or more expression construct(s) which encode(s) the viral segments required to produce an influenza virus, wherein at least one expression construct is the synthetic expression construct prepared in step (a); and c) culturing the cell in order to produce a reassortant influenza virus from the viral segments introduced in step (b); wherein steps (a) to (c) are performed in a time period of 124 hours or less.

5. The method of claim 4, wherein the cell is a non-human cell or a human non-kidney cell.

6. The method of claim 1, further comprising (d) contacting a cell which is of the same cell type as the cell used in step (c) with the virus produced in step (c) to produce further reassortant influenza virus.

7. A method of preparing an influenza virus, comprising: a) providing a synthetic expression construct which comprises coding sequences for expressing at least one segment of an influenza virus genome by (i) synthesising a plurality of overlapping fragments of the synthetic expression construct, wherein the overlapping fragments span the complete synthetic expression construct, and (ii) joining the fragments to provide the synthetic expression construct; b) introducing into a cell one or more expression construct(s) which encode(s) the viral segments of an influenza virus, wherein at least one expression construct is the synthetic expression construct prepared in step (a); c) culturing the cell in order to produce a reassortant influenza virus from the viral segments introduced in step (b); and d) contacting a cell which is of the same cell type as the cell used in step (c) with the virus produced in step (c) to produce further reassortant influenza virus; wherein steps (a) to (c) are performed in a time period of 124 hours or less.

8. The method of claim 7, wherein the cell used in steps (c) and (d) is not 293T.

9. The method of claim 7, wherein the cell used in steps (c) and (d) is a non-human cell or a human non-kidney cell.

10. The method of claim 1, wherein the synthetic expression construct comprises coding sequences for the HA and/or NA segment.

11. The method of claim 1, wherein the synthetic expression construct is linear.

12. The method of claim 1, wherein the fragments have a length between 61 and 100 nucleotides.

13. The method of claim 12, wherein the fragments have a length between 61 and 74 nucleotides.

14. The method of claim 1, wherein the fragments have an overlap of about 40 nucleotides.

15. The method of claim 1, wherein at least part of the synthetic expression construct obtained in step (a) is amplified.

16. The method of claim 1, wherein the step of providing the synthetic expression construct comprises: (i) synthesising a plurality of overlapping fragments of the synthetic expression construct, wherein the overlapping fragments span the complete synthetic expression construct; (ii) joining the fragments to provide a DNA molecule; (iii) melting the DNA molecule; (iv) re-annealing the DNA in the presence of an agent which excises mismatched nucleotides from the DNA molecule; and (v) amplifying the DNA to produce the synthetic expression construct.

17. The method of claim 1, wherein the reassortant influenza virus is a reassortant influenza A virus.

18. The method of claim 17, wherein the reassortant influenza A virus comprises one or more backbone segments having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NOs 9 to 14.

19. The method of claim 17, wherein the reassortant influenza A virus comprises one or more backbone segments having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NOs 42 to 47.

20. The method of claim 17, wherein the reassortant influenza A virus comprises backbone segments from two or more influenza A strains.

21. The method of claim 17, wherein the reassortant influenza A virus comprises the PB1 segment of SEQ ID NO: 43; the PB2 segment of SEQ ID NO: 44; the PA segment of SEQ ID NO: 9; the NP segment of SEQ ID NO: 45; the M segment of SEQ ID NO: 13; and the NS segment of SEQ ID NO: 14.

22. The method of claim 17, wherein the reassortant influenza A virus comprises the PB1 segment of SEQ ID NO: 18; the PB2 segment of SEQ ID NO: 11; the PA segment of SEQ ID NO: 9; the NP segment of SEQ ID NO: 12; the M segment of SEQ ID NO: 13; and the NS segment of SEQ ID NO: 14.

23. The method of claim 1, wherein the reassortant influenza virus is a reassortant influenza B virus.

24. The method of claim 23, wherein the reassortant influenza B viruses comprises the PA segment 25 of SEQ ID NO: 71, the PB1 segment of SEQ ID NO: 72, the PB2 segment of SEQ ID NO: 73, the NP segment of SEQ ID NO: 74, the NS segment of SEQ ID NO: 76 and the M segment of SEQ ID NO: 75.

25. The method of claim 23, wherein the reassortant influenza B viruses comprises the PA segment of SEQ ID NO: 71, the PB 1 segment of SEQ ID NO: 72, the PB2 segment of SEQ ID NO: 73, the NP segment of SEQ ID NO: 74, the NS segment of SEQ ID NO: 76 and the M segment of SEQ ID NO: 81.

26. A method of preparing an influenza vaccine, comprising: a) contacting a cell with a reassortant influenza virus prepared by the method of claim 1; b) culturing the cell in order to produce an influenza virus; and c) preparing a vaccine from the influenza virus produced in step (b).

27. The method of claim 26, wherein the cell is a human non-kidney cell or a non-human cell.

28. The method of claim 26, wherein the cell used in step (a) is of the same cell type as the cell used to prepare the reassortant influenza virus.

29. The method of claim 26, wherein step (c) involves inactivating the virus.

30. The method of claim 26, wherein the vaccine is a whole virion vaccine.

31. The method of claim 26, wherein the vaccine is a split virion vaccine.

32. The method of claim 26, wherein the vaccine is a surface antigen vaccine.

33. The method of claim 26, wherein the vaccine is a virosomal vaccine.

34. The method of claim 26, wherein the vaccine contains less than 10 ng of residual host cell DNA per dose.

35. A method of preparing a synthetic expression construct which encodes a viral segment from an influenza virus, comprising: a) providing the sequence of at least part of the coding region of the HA or NA segment from an influenza virus; b) identifying the HA and/or NA subtype of the influenza virus from which the coding region is derived; c) providing a UTR sequence from an influenza virus with the same HA or NA subtype as the subtype identified in step (b); and d) preparing a synthetic expression construct which encodes a viral segment comprising the coding sequence and the UTR.

36. The method of claim 1, wherein the cell is a mammalian cell or an avian cell.

37. The method of claim 36, wherein the cell is an MDCK, Vero or PerC6 cell.

38. The method of claim 37, wherein the cell is of the cell line MDCK 33016 (DSM ACC2219).

39. The method of claim 36, wherein the cell grows in suspension.

40. The method of claim 1, wherein the cell grows adherently.

41. A library comprising two or more influenza backbones.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0225] FIG. 1(A), FIG. 1(B), and FIG. 1(C). Method of synthetic gene segment assembly and error correction. FIG. 1(A) Process flow. Time for performance of each step is indicated on the right. FIG. 1(B) Schematic diagram of process. X indicates sites of oligonucleotide synthesis errors. In the circular DNA and final assembled gene diagrams (the bottom two), pKS10 sequences are white, and influenza coding sequences are black. FIG. 1(C) Ethidium bromide stained agarose gel of linear synthetic HA and NA genes, including regulatory elements used for virus rescue. MWmolecular weight marker.

[0226] FIG. 2. Timeline of rescue of synthetic H7N9 influenza viruses from transmission of oligonucleotide sequence information to confirmation of recovered viruses.

[0227] FIG. 3(A), FIG. 3(B), and FIG. 3(C). Performance of synthetic H7N9 reassortant viruses from the simulated pandemic response. FIG. 3(A) Titers of influenza viruses in culture fluid harvested from MDCK-supplemented 293T cells 48 hours (dotted columns) and 72 hours (white columns) after co-transfection with the indicated backbone plasmids and synthetic HA and NA gene constructs. Viral titers were determined by a focus formation assay using MDCK cell monolayers. FIG. 3(B) Replication kinetics of synthetic H7N9 reassortant viruses in MDCK 33016PF suspension cultures. FIG. 3(C) HA yields from synthetic H7N9 viruses in MDCK suspension cultures, determined by RP-HPLC after purification of viruses on sucrose density gradients. The y-axis in FIG. 3(A) and FIG. 3(B) shows infectious units (log10 IU/mL). The y-axis in FIG. 3(C) shows HA yield in g/mL.

[0228] FIG. 4(A) and FIG. 4(B). Effect of MDCK feeder cell addition 24 hours after transfection of MDCK cells on rescue efficiency. Titers of recombinant viruses containing the PR8 backbone with HA and NA segments from either FIG. 4(A) A/WSN/1933 (H1N1) or FIG. 4(B) A/California/04/2009 were measured 72 hours after transfection by a focus formation assay. The dotted column shows the results with additional cells whilst the white column shows the results without additional cells. The y-axis indicates infectious units (log10 IU/mL).

[0229] FIG. 5(A), FIG. 5(B), FIG. 5(C), FIG. 5(D), FIG. 5(E), and FIG. 5(F). Synthetic influenza virus rescue efficiencies. Representative data showing effect of optimized backbones on virus rescue efficiency from transfected cultures of MDCK cells. Detection of influenza viruses in culture fluid harvested at different time points after transfection with the indicated backbone plasmids and synthetic HA and NA constructs, or 24-48 hours after a blind passage using 500 l of the culture fluid on fresh MDCK cell monolayers (Passage 1). Viral titers were determined using a focus formation assay for FIG. 5(A) an H1N1 strain, FIG. 5(B) an H3N2 strain, FIG. 5(C) an attenuated H5N1 strain, FIG. 5(D) a swine origin H3N2v strain, FIG. 5(E) a B/Yamagata lineage strain, and FIG. 5(F) a B/Victoria lineage strain. The y-axis indicates infectious units (log10 IU/mL).

[0230] FIG. 6. Rescue of synthetic H7N9a viruses from either MDCK-supplemented 293T cells or from MDCK cells only. Detection of influenza viruses in culture fluid harvested 48 hours (dotted columns) and 72 hours (white columns) after transfection with the #19 backbone plasmids and synthetic H7 and N9 constructs. Viral titers were determined on MDCK cell monolayers using a focus formation assay. The y-axis indicates infectious units (log10 IU/mL).

[0231] FIG. 7(A), FIG. 7(B), and FIG. 7(C). Replication kinetics of synthetic H7N9 reassortant viruses with alternative NA UTRs in MDCK 33016PF suspension cultures. Replication kinetics of synthetic H7N9 viruses with alternative NA UTRs and different backbones, FIG. 7(A) PR8x, FIG. 7(B) #19, and FIG. 7(C) #21, in MDCK suspension cultures. Starting m.o.i. was 0.001. The x-axis indicates the hours post infection. The y-axis indicates infectious units (log10 IU/mL).

[0232] FIG. 8. HA yield by turkey RBC agglutination by synthetic H7N9 viruses with alternative NA UTRs. The y-axis indicates the HA units.

[0233] FIG. 9(A) and FIG. 9(B) compares 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 FIG. 9(A) a pandemic-like H1 strain (strain 1) or FIG. 9(B) a second pandemic-like strain (strain 2). In FIG. 9(A) and FIG. 9(B), the black bar represents a reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain) as control, the grey bar represents a reassortant virus containing the PR8-X backbone, and the white bar represents a reassortant virus containing the #21 backbone. The y-axis indicates HA yield in g/ml.

[0234] FIG. 10(A) and FIG. 10(B) compares 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 FIG. 10(A) a pre-pandemic H1 strain (strain 1) and FIG. 10(B) a second pre-pandemic H1 strain (strain 2). In FIG. 10(A) and FIG. 10(B), the black bar represents a reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain) as control, the grey bar represents a reassortant virus containing the PR8-X backbone, and the white bar represents a reassortant virus containing the #21 backbone. The y-axis indicates HA yield in g/ml.

[0235] FIG. 11 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 black bar represents a reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain) as control, the grey bar represents a reassortant virus containing the PR8-X backbone, and the white bar represents a reassortant virus containing the #21 backbone. The y-axis indicates HA yield in g/ml.

[0236] FIG. 12(A) and FIG. 12(B) compares virus titers (determined by focus formation assay (FFA); FIG. 12(A)) and HA titers (determined by lectin-capture ELISA; FIG. 12(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. 12(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. 12(B), the black bar represents the reference vaccine strain (derived from WHO-Collaborating Centre-supplied strain), the grey bar represents a reassortant virus containing the PR8-X backbone, and the white bar represents a reassortant virus containing the #21 backbone. The y-axis indicates HA yield in g/ml for pooled egg samples.

[0237] FIG. 13 compares the HA yield of different reassortant influenza B strains in MDCK cells relative to the wild-type (WT) or reverse genetics-derived (RG) B/Brisbane/60/08 strain. The viral segments of the tested influenza B viruses are shown in Table 1. The y-axis indicates the HA yield in g/mL.

[0238] FIG. 14 compares the HA yield of different reassortant influenza B strains in MDCK cells relative to the wild-type (WT) or reverse genetics-derived (RG) B/Panama/45/90 strain. The viral segments of the tested influenza B viruses are shown in Table 1. The y-axis indicates the HA yield in g/mL.

MODES FOR CARRYING OUT THE INVENTION

[0239] Increased Gene Synthesis Speed and Accuracy through Enzymatic Assembly and In Vitro Error Correction.

[0240] A purely enzymatic one-step, isothermal assembly method of gene assembly, previously used to synthesize the entire 16,299 base pair mouse mitochondrial genome from 600 overlapping 60-base oligonucleotides (6), was adapted for the generation of synthetic DNA copies of influenza virus genome segments. The method uses 5 T5 exonuclease (Epicentre), Phusion DNA polymerase (New England Biolabs [NEB]) and Taq DNA ligase (NEB) to join multiple DNA fragments during a brief 50 C. reaction (7). The method was selected to assemble genes for synthetic vaccine seeds because it is rapid and readily automated. All bases of the resulting synthetic genes have their origin in chemically synthesized oligonucleotides. Using current techniques, DNA oligonucleotide synthesis has an error rate of about 1 per 325 bases, typically due to missing bases from failed chemical coupling, and the error rate increases with the length of the oligonucleotide synthesized (6). When DNA copies of the 1.7 kb HA and 1.5 kb NA viral RNA genome segments are synthesized by this technique using oligonucleotides approximately 60 bases in length with 30 bases of overlap between oligonucleotides on opposite strands, only 3% of the synthetic products have the correct sequence. During the mouse mitochondrial genome synthesis, subassemblies were cloned and sequenced, and sets of error-free sequences were selected for subsequent rounds of assembly (6). For the purpose of rapid influenza vaccine seed virus generation, this method of error correction would introduce unacceptable delays.

[0241] The problem of synthesizing DNA copies of HA and NA genome segments with both accuracy and speed was solved by (i) increasing the overlap between oligonucleotides, (ii) introducing an enzymatic error correction step, and (iii) increasing the number of oligonucleotides assembled at once, eliminating the need for stepwise assembly via sub-assemblies (FIG. 1(A) and FIG. 1(B)). Specifically, the length of oligonucleotides was increased to 60-74 bases, and full length genes (including 5 and 3 un-translated regions) were assembled from staggered sets of oligonucleotides that contained all residues of a double-stranded DNA molecule so that, prior to ligation, the full double-stranded gene can be annealed. In practice, a software algorithm generates a set of sequences for oligonucleotides (a maximum of 96 oligonucleotides per HA, NA pair) that meet these criteria. After chemical synthesis of the oligonucleotides, enzymatic isothermal assembly, and PCR amplification, error-containing DNA is removed enzymatically by treating melted and re-annealed DNA with the commercially available ErrASE error correction kit (Novici Biotech), which excises areas of base mismatch in double-stranded DNA molecules before another round of PCR amplification.

[0242] After agarose gel verification of the products' sizes, the control sequences (including Pol I and Pol II promoters and their terminator and polyadenylation signals) needed to generate RNA genome segments and mRNA for virus rescue are added by isothermally coupling the synthetic DNA with a linearized plasmid (pKS10) that contains these regulatory sequences (87). Nucleotide identity between the ends of the linearized plasmid and the 5 and 3 primers used for gene synthesis guide this assembly. The assembled molecule is the substrate for a round of high fidelity PCR amplification using primers outside the transcription control regions.

[0243] After purification and concentration of the amplicons, approximately 10 g of assembled linear DNA cassettes that contain the influenza gene flanked by control sequences are obtained, ready for transfection into the MDCK 33016PF cell line for influenza virus rescue (FIG. 1(C)). The time from receipt of oligonucleotides to a purified HA or NA-encoding DNA cassette ready for transfection is approximately 10 hours. While virus rescue is underway using the enzymatically assembled, error corrected, and amplified DNA, parallel cloning and sequencing verifies the sequence of the assembled genes. Typically, 80-100% of the full-length sequences obtained are correct.

[0244] Optimized Rescue of Influenza Viruses from Synthetic DNA on a Vaccine Manufacturing Cell Line.

[0245] The rescue protocol for synthetic seed virus generation is adapted from a previously described eight-plasmid ambisense system in which each expression plasmid has a cDNA copy of a viral gene segment bounded at the 5 end by a Pol II promoter to drive transcription of messenger RNA and at the 3 end by a human Pol I promoter to drive transcription of negative-stranded influenza RNA genome segments (88). The manufacturing-qualified MDCK 33016PF cell line is a less efficient substrate for transfection and influenza virus rescue by reverse genetics than 293T cells (which are not qualified for vaccine production). Influenza virus reverse genetic rescue has been described using Vero cells (some banks of which are qualified for vaccine production) (89, 90). However, using one cell line for vaccine virus rescue and a different cell line for antigen production would add adventitious agent risk and regulatory and manufacturing complexity. Therefore, we elected to increase the efficiency of reverse genetic DNA rescue in MDCK 33016PF cells so that a single cell line can be used for seed generation and vaccine antigen production. Although Pol I promoters are generally species specific, human Pol I efficiently drives transcription in MDCK 33016PF cells, which are of canine origin.

[0246] One g of each linear synthetic cassette encoding HA or NA is co-transfected into MDCK 33016PF cells together with 1 g of each ambisense plasmid that encodes PA, PB1, PB2, NP, NS, or M and a helper plasmid that encodes the protease TMPRSS2 (91). To increase rescue efficiency, we add cultures of fresh (un-transfected) MDCK 33016PF cells after transfection, which increases the probability of virus recovery, presumably by providing a healthier population of cells in which rescued viruses can further amplify (FIG. 4(A) and FIG. 4(B)). Viruses are detected in cell culture medium within 72 hours after transfection (approximately 24 hours later than after transfection of Vero or 293T cells), using a focus-formation assay in which the medium from the transfected culture is added to a fresh MDCK cell monolayer, and infectious virus is detected by immuno-staining for expressed NP.

[0247] Improved Backbones for Synthetic Virus Rescue.

[0248] A significant increase in rescue efficiency was provided by using improved influenza backbones (sets of genome segments encoding influenza virus proteins other than HA and NA). The initial backbone improvement resulted from using genes from a PR8 variant (designated PR8x) that had been adapted over five passages to growth in MDCK 33016PF cells. Additional improvements resulted from combining backbone genome segments of multiple strains. During pilot manufacturing of influenza vaccines using MDCK 33016PF cells, several human influenza viruses, such as strain 105p30 (an A/New Caledonia/20/1999 (H1N1)-like strain that was passaged 30 times in MDCK 33016PF cells), were adapted to grow efficiently in cultured cells, although not as efficiently as strain PR8x. Synthesized viruses with HA and NA genes from historical H3N2 strains and a backbone (designated #19) composed of NP, PB1, and PB2 genome segments from strain 105p30 and M, NS, and PA genome segments from strain PR8x often outperformed equivalent viruses with entirely PR8x backbones in reverse genetic rescue efficiency and yield of HA (table 1 and FIG. 5(A), FIG. 5(B), FIG. 5(C), FIG. 5(D), FIG. 5(E), and FIG. 5(F)). Similarly, synthesized viruses with HA and NA genes from H1N1 strains and a backbone (designated #21) with the PB1 genome segment of A/California/7/2009 and the other genome segments from strain PR8x often had greater rescue efficiencies and HA yields than equivalent viruses with entirely PR8x backbones (table 1 and FIG. 5(A), FIG. 5(B), FIG. 5(C), FIG. 5(D), FIG. 5(E), and FIG. 5(F)). This finding is consistent with a report that the A/California PB1 genome segment is preferentially found in the reassortant progeny of co-infections of chicken eggs with A/California/7/2009 and a donor strain that has a PR8 backbone (18).

TABLE-US-00001 TABLE 1 Representative data showing virus titers and HA yields (in mass per volume of cell culture medium before purification) from synthetic influenza viruses relative to conventional vaccine viruses (reference strains obtained from the US CDC or the UK National Institute for Biological Standards and Control) in MDCK 33016PF cells. HA yield HA by yield Reference FFA RP- by Best Synthetic H1N1 strain strain titer HPLC ELISA backbone A/Christchurch/16/ NIB74.sup.b 4.9 1.6 2.3 #21 2010.sup.a,b A/Brisbane/10/2010.sup.a wild-type 19 2.1 7.2 #21 A/Brisbane/59/2007 IVR-148 5.5 1.9 2.9 #21 A/Solomon/3/2006 IVR-145 3.4 1.8 5.9 #21 Synthetic H3N2 strain A/Victoria/361/2011.sup.a,b IVR-165.sup.b 2.6 2.5 1.4 PR8x A/Victoria/210/2009.sup.a X187 2.6 2.3 1.7 PR8x A/Wisconsin/15/2009.sup.b X183.sup.b 35 below 15 #19 detection A/Uruguay/716/2007.sup.b X175C.sup.b 2.0 1.3 1.4 #19 Synthetic H5N1 strain A/turkey/Turkey/1/ NIBRG23.sup.b 1.9 1.6 n/a #19 2005.sup.a,b Synthetic H3N2v strain A/Indiana/8/2011.sup.a,b X213.sup.b 1.9 2.3 n/a #21 Synthetic B-Yamagata strain B/Wisconsin/1/2010.sup.a,b wild-type.sup.b 1.7 1.4 1.7 Brisbane B/Brisbane/3/2007 wild-type 0.88 3.5 5.2 #B34 Synthetic B-Victoria strain B/Brisbane/60/2008.sup.a wild-type 0.72 1.8 0.67 Brisbane Data values are normalized and shown as fold-improvement over reference strains, where values of the reference strains are set to 1.0. RP-HPLC or lectin-capture ELISA was used to detect HA antigen directly from the culture medium of virus-infected MDCK cells (m.o.i = 0.001 or 0.0001), unless specified. .sup.arecombinant viruses containing synthetic HA and NA segments .sup.bviruses from culture medium were purified by sucrose-density gradient prior to characterization n/a = data not available because strain-specific anti-sera were not available for ELISA below detection = data not available because the reference strain had undetectable HA levels by RP-HPLC

[0249] Historically, most influenza type B vaccine seeds have been wild type viruses, not reassortants, because wild type influenza B viruses generally provide adequate yields. To use the synthetic procedures for influenza B viruses more readily, two optimized type B backbones that provide consistent rescue of synthetic influenza B viruses were developed (table 1 and FIG. 5(A), FIG. 5(B), FIG. 5(C), FIG. 5(D), FIG. 5(E), and FIG. 5(F)). In the first (designated Brisbane), all backbone genome segments originate from B/Brisbane/60/2008; in the second (designated #B34), the genome segments encoding PA, PB1, PB2, and NP originate from B/Brisbane/60/2008, and those encoding M and NS originate from B/Panama/45/1990.

[0250] Overall, the use of optimized backbones for A strains increased rescue efficiencies up to 1000-fold (as measured by infectious titers obtained after transfection, FIG. 5(A), FIG. 5(B), FIG. 5(C), FIG. 5(D), FIG. 5(E), and FIG. 5(F)) and increased HA yields in research scale infections of MDCK 33016PF cells by 30% to 15-fold, depending on the strain and assay used for HA detection (table 1). In general, yields of HA from these viruses are also increased relative to those from viruses with PR8 backbones when the viruses are propagated in embryonated chicken eggs (table 2). To make use of such strain-specific differences, an optimal synthetic seed generation strategy would combine the HAs and NAs from circulating strains of interest with a panel of alternative backbones to maximize the chances of isolating a high-yielding vaccine virus.

TABLE-US-00002 TABLE 2 Representative data showing virus titers and HA yields (in mass per volume of egg allantoic fluid before purification) from synthetic influenza viruses relative to conventional vaccine viruses (reference strains obtained from the US CDC or the UK National Institute for Biological Standards and Control) in chicken eggs. HA titer by HA yield HA Reference GP-RBC by RP- yield by Best Synthetic strain strains FFA titer agglutination HPLC ELISA backbone A/H1N1/Christchurch/16/2010.sup.b NIB74 3.0 3.5 18 8.4 #21 A/H3N2/Victoria/210/2009.sup.b X187 0.94 1.3 not tested 1.2 PR8x A/H3N2/Victoria/361/2011.sup.b IVR-165 6.4 2.6 not tested 3.4 #21 A/H3N2v/Indiana/8/2011a,.sup.b X213 not tested 3.0 1.6 n/a PR8x B/Yam/Wisconsin/1/2010.sup.a wild-type 4.7 3.4 not tested 3.5 Brisbane B/Vic/Brisbane/60/2008.sup.a wild-type 1.1 0.82 not tested 0.79 Brisbane Data values are normalized and shown as fold-improvement over reference strains, where valuesof the reference strains are set to 1.0. GP-RBC agglutination, RP-HPLC or lectin-capture ELISA was used to detect HA antigen directly from the allantoic fluid of virus-infected chicken eggs, unless specified. .sup.a= recombinant viruses containing synthetic HA and NA genome segments .sup.b= viruses from egg allantoic fluid were purified by sucrose density gradient before characterization n/a = data not available because strain-specific antisera were not available for ELISA not tested = data not available because assay was not performed

[0251] Speed of Synthetic Vaccine Virus Generation in a Simulated Pandemic Response.

[0252] In a timed proof-of-concept test of the synthetic system's first iteration, the virus synthesis group was provided with unidentified HA and NA genome segment sequences by collaborators not directly involved in the synthesis (17). The sequences included complete coding regions but incomplete un-translated regions (UTRs), mimicking the information likely to be available in the early days of a pandemic. Sequence analysis of the HA genome segment showed that it was very closely related (96% nucleotide sequence identity by Blast to GenBank) to a low pathogenicity North American avian H7N3 virus (A/Canada goose/BC/3752/2007), and that the NA genome segment was very closely related (96% nucleotide sequence identity by Blast to GenBank) to a low pathogenicity North American avian H1ON9 virus (A/king eider/Alaska/44397-858/2008). Although our software generates the sequences of the oligonucleotides used for rescue, user intervention is needed when there are ambiguities in the available sequence data. In this case, the unknown terminal UTR sequences were generated based on sequence alignments with a limited number of related full-length H7 sequences and by comparison with consensus UTRs for H7 and N9 genomic segments created from high quality sequence data in GenBank. This analysis revealed heterogeneity in the non-coding regions of NA genes of H7N9 strains (U/C at 1434 in the positive-sense orientation). So, alternative sets of 5 NA oligonucleotides were used to construct two variants of the NA cassettes.

[0253] Oligonucleotide synthesis began at 8:00 am EDT on Monday, Aug. 29, 2011 (FIG. 2). By noon on Friday, September 4, immunostaining of a secondary culture confirmed that the virus had been rescued. The 4 days and 4 hours from start of synthesis to detection of rescued virus included time spent shipping DNA from the oligonucleotide synthesis and gene assembly laboratories in California to the virus rescue laboratory in Massachusetts. When all functions are consolidated in one location, the potential for delays and mishaps due to shipping will be reduced. The original proof-of-concept rescues were conducted using 293T cells; rescue of the strains using MDCK cells, as would be done during an actual pandemic response, slows detection of rescued virus by approximately 24 hours (FIG. 6). The sequences of the HA and NA genome segments of the synthetic H7N9 reassortant viruses from the proof-of-concept exercise were determined following two rounds of virus amplification in MDCK 33016PF cells and were identical to those used to program oligonucleotide synthesis. Two-way hemagglutination inhibition (HI) testing (reciprocal HI assays using antigen from the synthetic and natural strains and ferret sera drawn after synthetic and natural virus infection) (19, 20) demonstrated antigenic identity of the synthetic virus to A/goose/Nebraska/17097-4/2011 (H7N9), which had subsequently been revealed as the wild type virus from which the sequences that were electronically transmitted to the virus synthesis group had been obtained (Table 1).

[0254] The A/goose/Nebraska/17097-4/2011 HA and NA genes were rescued with PR8x, #19, and #21 backbones. Virus rescue was more efficient using the #19 and #21 backbones than the PR8x backbone, based on the titers of viruses harvested 48 and 72 hours after transfection (FIG. 3(A)). To test growth characteristics, the synthetic viruses were amplified once in MDCK 33016 PF monolayers and then used to infect suspension MDCK 33016PF cultures at a multiplicity-of-infection (m.o.i.) of 0.001. Despite differences in the efficiency of virus recovery, viruses exhibited similar growth characteristics, regardless of backbone (FIG. 3(B)). The H7N9a set of viruses (C1434 positive sense NA) achieved infectious titers approximately 10-fold higher than their H7N9b counterparts (U1434 positive sense NA; FIG. 7(A), FIG. 7(B), and FIG. 7(C)). The viruses with the highest infectious yields also produced the most HA per volume of infected MDCK suspension culture (FIG. 3(C)). Thus, the single nucleotide substitution in the 5 NA non-coding region of the genomic RNA strongly influenced both infectious titer and HA yield (FIG. 8). The H7N9a virus with the #19 backbone produced 1.5-fold more HA than a virus with the same HA and NA in the context of the standard PR8x backbone (FIG. 3(C)). This demonstration confirmed the importance of rescuing multiple HA or NA variants with multiple backbones to increase the probability of identifying high yielding vaccine virus strains early in the vaccine seed generation process. Simultaneous rescue of multiple variants is faster and more easily accomplished using the synthetic approach than standard plasmid mutagenesis approaches. This example also indicates the importance for pandemic response of including as complete genome segment sequences as possible in genetic databases and of clearly delineating terminal sequences originating from viral genome segments from those originating from sequencing primers.

[0255] Robustness of the Synthetic Approach to Vaccine Virus Generation.

[0256] By combining gene synthesis, enzymatic error correction, optimized rescue protocols, and optimized backbones, the synthetic approach provides a robust tool to obtain influenza vaccine viruses. To date, the team has not encountered any influenza virus strain that cannot be rescued synthetically. The synthetic process has been used to generate a wide variety of influenza strains, including H1N1 (pre- and post-2009 variants), seasonal H3N2, swine origin H3N2v, B (Yamagata and Victoria lineages), attenuated H5N1, and H7N9 strains (table 3). The robustness of synthetic influenza virus recovery on MDCK cells is in striking contrast to the unreliability of conventional vaccine virus isolation using eggs, particularly for recent H3N2 strains (21).

TABLE-US-00003 TABLE 3 Diversity of synthetic influenza virus strains rescued. SEASONAL SEROTYPE A VIRUSES Backbone Source of synthetic HA NA PR8X #19 #21 A/H1N1/Brisbane/10/2010 + + + A/H1N1/Christchurch/16/2010 (NIB74) + + + A/H1N1/Christchurch/16/2010 NIB74-K170E n/a n/a + A/H1N1/Christchurch/16/2010 NIB74-K171E n/a n/a + A/H1N1/Christchurch/16/2010 NIB74-G172E n/a n/a + A/H1N1/Christchurch/16/2010 NIB74-G173D n/a n/a + A/H3N2/Uruguay/716/2007 + + + A/H3N2/Victoria/210/2009 (X187) + + + A/H3N2/Victoria/361/2011 (CDC E3) + + + A/H3N2/Victoria/361/2011 (WHO E3) + + + A/H3N2/Victoria/361/2011 (MDCK) + + + A/H3N2/Berlin/93/2011 (egg-derived) + + + A/H3N2/Berlin/93/2011 (cell-derived) + + + A/H3N2/Brisbane/402/2011 + + + A/H3N2/Victoria/304/2011 NVD p2/E3 + A/H3N2/Brisbane/256/2011 MDCK P2 + + + A/H3N2/Brisbane/256/2011 P2/E3 + + A/H3N2/South Australia/34/2011 + + A/H3N2/Brisbane/299/2011 (IVR164) + + + A/H3N2/Brisbane/299/2011 (E5) + + + A/H3N2/South Australia/3/2011 + + + A/H3N2/Wisconsin/1/2001 + + + SEASONAL SEROTYPE B VIRUSES Backbone Source of synthetic HA NA Bris #B34 B/Yam/Hubei-Wujiangang/158/2009 + + B/Yam/Wisconsin/1/2010 + + B/Yam/Brisbane/3/2007 + + B/Yam/Jiangsu/10/2003 + + B/Yam/Johannesburg/05/1999 + + B/Yam/Yamanashi/166/1998 + + B/Yam/Yamagata/16/1988 + + B/Yam/Texas/6/2011 + B/Vic/New Hampshire/1/2012 + + B/Vic/Malaysia/2506/2004 + + B/Vic/Brisbane/32/2002 + + B/Vic/Brisbane/60/2008 (cell) + + B/Vic/Brisbane/60/2008 (egg) + n/a B/Vic/Nevada/3/2011 + + PANDEMIC VIRUSES Backbone Source of synthetic HA NA PR8X #19 #21 A/H5N1/Hubei/1/2010 + + + A/H5N1/Egypt/N03072/2010 + + + A/H5N1/Turkey/Turkey/1/2005 + + + A/H7N9/goose/Nebraska/11-017097-4/2011 + + + A/H3N2v/Indiana/8/2011 + + + n/a = not attempted; + = virus recovered in 6 days post-transfection; = virus not recovered by 6 days post-transfection.

[0257] Implications for the Global Strain Change and Pandemic Response Systems.

[0258] The speed, ease, and accuracy with which higher yielding influenza vaccine seeds can be produced using synthetic techniques promises more rapid future pandemic responses and a more reliable supply of better matched seasonal and pandemic influenza vaccines. The potential for propagation of adventitious agents from the human nasal secretions used for original influenza virus isolation will be eliminated when such materials are used only to generate sequence information, not for propagation into viruses used to seed vaccine production bioreactors or eggs. The speed of the technical steps of synthesis and virus rescue is actually a relatively minor component of the potential acceleration of seed generation based on synthetic technology. If the performance of synthetic vaccine viruses is sufficient, much greater time savings will result from the ability of synthetic technology to alleviate the need to ship viruses and clinical specimens between laboratories and use a classic reassortment approach to generate high-yielding vaccine strains.

[0259] Today, the more than 120 National Influenza Centers (NICs) that conduct influenza surveillance periodically ship clinical specimens to WHO Collaborating Centers, where attempts are made to propagate the wild type viruses in MDCK cells. With synthetic vaccine viruses, the system could realize increased efficiency. Sequence data obtained by directly sequencing HA and NA genomic RNAs in clinical specimens at the NICs could be posted on publically accessible websites, where they can be downloaded immediately by manufacturers, public health agencies, and other researchers worldwide. Continuous comparison of the stream of sequence data to databases of sequence and HI data by algorithms now under development could identify those emerging viruses that are most likely to have significant antigenic differences from current vaccine strains. Efficient primary synthetic rescue with a panel of high growth backbones will simultaneously generate the viruses needed for antigenic testing and the best vaccine seed candidates to be used if a virus is found to be antigenically distinct and epidemiologically important.

[0260] Today, vaccine viruses are only shipped from WHO Collaborating Centers or reassortant generating laboratories to manufacturers after they are fully tested, and testing often takes longer than the generation of the vaccine strains. The decentralization of seed generation permitted by these synthetic techniques could allow manufacturers to undertake scale up and process development at risk for strains that they could generate immediately after the NICs post sequences. Carrying out these manufacturing activities simultaneously with seed testing would cut additional weeks from pandemic response times. Libraries of synthetic influenza genes could further accelerate pandemic responses, if the pre-synthesized genes in the libraries match future pandemic strains.

[0261] Growth Characteristics of Reassortant Viruses Containing PR8-X or Canine Adapted PR8-X Backbones

[0262] 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.

[0263] 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.

[0264] 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. 8, FIG. 9(A), FIG. 9(B), FIG. 10(A), FIG. 10(B), and FIG. 11).

[0265] 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 PR8-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.

[0266] 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, FIG. 9(A), and FIG. 9(B)).

[0267] Growth Characteristics of Reassortant Viruses Containing PR8-X, #21 or #21C Backbones

[0268] 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 does 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 HA titres are also determined.

[0269] 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. 5(C), FIG. 5(D), FIG. 5(E), and FIG. 5(F), FIG. 6, FIG. 7(A), FIG. 7(B), and FIG. 7(C)). Reassortant influenza viruses comprising the #21C backbone also show higher HA titres compared with PR8-X reassortants.

[0270] Growth Characteristics of Reassortant Influenza B Viruses

[0271] Reassortant influenza B viruses are produced by reverse genetics which contain the HA and NA proteins from various influenza strains and the other viral segments from B/Brisbane/60/08 and/or B/Panama/45/90. As a control the corresponding wild-type influenza B strain is used. These viruses are cultured either in embyronated chicken eggs or in MDCK cells. The following influenza B strains are used:

TABLE-US-00004 TABLE 4 Backbone segments Antigenic determinants combo # PA PB1 PB2 NP NS M HA NA 1 (WT) Brisbane Brisbane Brisbane Brisbane Brisbane Brisbane Brisbane Brisbane 2 Panama Brisbane Brisbane Brisbane Brisbane Brisbane Brisbane Brisbane 3 Brisbane Panama Brisbane Brisbane Brisbane Brisbane Brisbane Brisbane 4 Brisbane Brisbane Panama Brisbane Brisbane Brisbane Brisbane Brisbane 5 Brisbane Brisbane Brisbane Panama Brisbane Brisbane Brisbane Brisbane 6 Panama Panama Brisbane Brisbane Brisbane Brisbane Brisbane Brisbane 7 Panama Brisbane Panama Brisbane Brisbane Brisbane Brisbane Brisbane 8 Panama Brisbane Brisbane Panama Brisbane Brisbane Brisbane Brisbane 9 Brisbane Panama Panama Brisbane Brisbane Brisbane Brisbane Brisbane 10 Brisbane Panama Brisbane Panama Brisbane Brisbane Brisbane Brisbane 11 Brisbane Brisbane Panama Panama Brisbane Brisbane Brisbane Brisbane 12 Panama Panama Panama Brisbane Brisbane Brisbane Brisbane Brisbane 13 Panama Panama Brisbane Panama Brisbane Brisbane Brisbane Brisbane 14 Panama Brisbane Panama Panama Brisbane Brisbane Brisbane Brisbane 15 Brisbane Panama Panama Panama Brisbane Brisbane Brisbane Brisbane 16 Panama Panama Panama Panama Brisbane Brisbane Brisbane Brisbane 17 Panama Panama Panama Panama Panama Panama Brisbane Brisbane 20 Brisbane Panama Panama Panama Panama Panama Panama Panama 21 Panama Brisbane Panama Panama Panama Panama Panama Panama 22 Panama Panama Brisbane Panama Panama Panama Panama Panama 23 Panama Panama Panama Brisbane Panama Panama Panama Panama 24 Brisbane Brisbane Panama Panama Panama Panama Panama Panama 25 Brisbane Panama Brisbane Panama Panama Panama Panama Panama 26 Brisbane Panama Panama Brisbane Panama Panama Panama Panama 27 Panama Brisbane Brisbane Panama Panama Panama Panama Panama 28 Panama Brisbane Panama Brisbane Panama Panama Panama Panama 29 Panama Panama Brisbane Brisbane Panama Panama Panama Panama 30 Brisbane Brisbane Brisbane Panama Panama Panama Panama Panama 31 Brisbane Brisbane Panama Brisbane Panama Panama Panama Panama 32 Brisbane Panama Brisbane Brisbane Panama Panama Panama Panama 33 Panama Brisbane Brisbane Brisbane Panama Panama Panama Panama 34 Brisbane Brisbane Brisbane Brisbane Panama Panama Panama Panama 35 Brisbane Brisbane Brisbane Brisbane Brisbane Brisbane Panama Panama

[0272] The results indicate that reassortant viruses #2, #9, #30, #31, #32, #33, #34 and #35 grow equally well or even better in the culture host (see FIGS. 13 and 14) than the corresponding wild-type strain. Most of these strains comprise the NP segment from B/Brisbane/60/08 and some (in particular those which grew best) further contain the PB2 segment from B/Brisbane/60/08. All of these viruses also contain viral segments from the BNictoria/2/87-like strain and the B/Yamagata/16/88-like strain at a ratio 7:1, 6:2, 4:4, 3:4 or 1:7.

[0273] 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

[0274] [1] WO2007/002008

[0275] [2] WO2007/124327

[0276] [3] WO2011/012999

[0277] [4] Verity et al. (2012); Influenza Other Respi Viruses;6(2):101-9.

[0278] [5] Zhou et al. (2009) J Virol.;83(19):10309-13

[0279] [6] Gibson et al. (2010); Nature Methods 7, 901-903.

[0280] [7] Gibson et al. (2009) Nature Methods 6, 343-345.

[0281] [8] U.S. Pat. No. 6,576,453

[0282] [9] Yount et al. (2002) J Virol 76:11065-78.

[0283] [10] Kodumal et al. (2004) Proc Natl Acad Sci USA. 101(44):15573-8.

[0284] [11] Yount et al. (2002) J Virol 76:11065-78.

[0285] [12] Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 ed., 1989, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

[0286] [13] WO2010/133964

[0287] [14] WO2009/000891

[0288] [15] Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 ed., 1989, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

[0289] [16] WO2011048560

[0290] [17] Neumann et al. (2005) Proc Natl Acad Sci USA 102: 16825-9

[0291] [18] Kistner et al. (1998) Vaccine 16:960-8.

[0292] [19] Kistner et al. (1999) Dev Biol Stand 98:101-110.

[0293] [20] Bruhl et al. (2000) Vaccine 19:1149-58.

[0294] [21] WO2006/108846.

[0295] [22] Pau et al. (2001) Vaccine 19:2716-21.

[0296] [23] http://www.atcc.org/

[0297] [24] http://locus.umdnj.edu/

[0298] [26] Brands et al. (1999) Dev Biol Stand 98:93-100.

[0299] [27] Halperin et al. (2002) Vaccine 20:1240-7.

[0300] [28] EP-A-1260581 (WO01/64846).

[0301] [29] WO2006/071563.

[0302] [30] WO2005/113758.

[0303] [31] Grachev et al. (1998) Biologicals;26(3):175-93.

[0304] [32] Herlocher et al. (2004) J Infect Dis 190(9):1627-30.

[0305] [33] Le et al. (2005) Nature 437(7062):1108.

[0306] [34] Needleman & Wunsch (1970) J. Mol. Biol. 48, 443-453.

[0307] [35] Rice et al. (2000) Trends Genet 16:276-277.

[0308] [36] Rota et al. (1992) J Gen Virol 73:2737-42.

[0309] [37] GenBank sequence GI:325176.

[0310] [38] McCullers et al. (1999) J Virol 73 :7343-8.

[0311] [39] GenBank sequence GI:325237.

[0312] [40] U.S. Pat. No. 6,468,544.

[0313] [41] WO97/37001

[0314] [42] WO02/28422.

[0315] [43] WO02/067983.

[0316] [44] WO02/074336.

[0317] [45] WO01/21151.

[0318] [46] WO02/097072.

[0319] [47] WO2005/113756.

[0320] [48] Huckriede et al. (2003) Methods Enzymol 373:74-91.

[0321] [49] Vaccines. (eds. Plotkins & Orenstein). 4th edition, 2004, ISBN: 0-7216-9688-0

[0322] [50] Treanor et al. (1996) J Infect Dis 173:1467-70.

[0323] [51] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10.

[0324] [52] Herlocher et al. (2004) J Infect Dis 190(9):1627-30.

[0325] [53] Le et al. (2005) Nature 437(7062):1108.

[0326] [54] WO2008/068631.

[0327] [55] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.

[0328] [56] Banzhoff (2000) Immunology Letters 71:91-96.

[0329] [57] Nony et al. (2001) Vaccine 27:3645-51.

[0330] [58] EP-B-0870508.

[0331] [59] US 5948410.

[0332] [60] WO2007/052163.

[0333] [61] WO2007/052061

[0334] [62] WO90/14837.

[0335] [63] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203.

[0336] [64] Podda (2001) Vaccine 19: 2673-2680.

[0337] [65] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).

[0338] [66] Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan.

[0339] [67] WO2008/043774.

[0340] [68] Allison & Byars (1992) Res Immunol 143:519-25.

[0341] [69] Hariharan et al. (1995) Cancer Res 55:3486-9.

[0342] [70] US-2007/014805.

[0343] [71] US-2007/0191314.

[0344] [72] Suli et al. (2004) Vaccine 22(25-26):3464-9.

[0345] [73] WO95/11700.

[0346] [74] U.S. Pat. No. 6,080,725.

[0347] [75] WO2005/097181.

[0348] [76] WO2006/113373.

[0349] [77] Potter & Oxford (1979) Br Med Bull 35: 69-75.

[0350] [78] Greenbaum et al. (2004) Vaccine 22:2566-77.

[0351] [79] Zurbriggen et al. (2003) Expert Rev Vaccines 2:295-304.

[0352] [80] Piascik (2003) J Am Pharm Assoc (Wash D.C.). 43:728-30.

[0353] [81] Mann et al. (2004) Vaccine 22:2425-9.

[0354] [82] Halperin et al. (1979) Am J Public Health 69:1247-50.

[0355] [83] Herbert et a/. (1979) J Infect Dis 140:234-8.

[0356] [84] Chen et al. (2003) Vaccine 21:2830-6.

[0357] [85] Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30.

[0358] [86] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.

[0359] [87] Suphaphiphat et al. (2010), Virology J. 7, 157.

[0360] [88] Hoffmann et al. (2000) PNAS 97, 6108-6113.

[0361] [89] Nicolson et al. (2005) Vaccine 23, 2943-2952.

[0362] [90] Ozaki et al (2004) J. Virol. 78, 1851-1857.

[0363] [91] Boettcher et al. (2006) J. Virol. 80, 9896-9898.