Influenza virus reassortment

专利摘要:
New infuenza donor strains for the production of reassortant influenza A viruses are provided.
公开号:AU2013205478A1
申请号:U2013205478
申请日:2013-03-02
公开日:2013-06-06
发明作者:Phillip Ralph Dormitzer；Bjoern Keiner；Peter Mason；Pirada Suphaphiphat；Heidi Trusheim
申请人:Novartis AG；
IPC主号:C12N15-00

专利说明:
INFLUENZA VIRUS REASSORTMENT This patent application claims priority from United States provisional patent application 61/605,922, filed March 2, 2012 and 61/685,766 filed March 23, 2012, the completes contents of which are incorporated herein by reference 5 TECHNICAL FIELD This nivention is in the field of influenza A virus reassortiment, Furthermore, it relates to rmanufacturingvaccines for protecting against influenza A viruses. BACKGROUND ART The rnost effiient protection against influenza infection is vaccination against circulating strains and 10 it is important to produce influenza viruses for vaccine production as quickly as possible. Wild-type influenza viruses often grow to low tires in eggs and cell culure. in order to obtain a better-growing virus strain for vaccine production it is currently common practice to reassort the circulating vaccine strain with a faster-growing high-yield donor strain. This can be achieved by co infecting a culture host with the circulating influenza strain (the vaccine strain) and the high-yield 15 donor strain and selecting for reassortant viruses which contain the hemagglutinin (HA) and neuraminidase (NA) segments from the vaccine strain and the other viral segments (i.e. those encoding PB1, PB2, PA, NP, M, N, NSi and NS 2 ) from the donor strain. Another approach is to reassort the influenza viruses by reverse genetics (see, for example references 1 and 2 Reference 3 reports that a reassortant influenza virus containing a PB 1 gene segment from 20 A/Texas//77, the HA and NA segments from A/New Caledonia/20/99, a modified PA segment derived from A/Puerto Rico/8/34 and the remaining viral segments from A/Puerto Rico/8/34 shows increased growth in ells. There are currently only a limited number of donor strains for reasserting influenza viruses for vaccine manufacture, and the strain most commonly used is the A/Puerto Rico/S/34 (A/PR/8/34) 25 strain. However, reassortant influenza viruses comprising A/PR/8/34 backbone segments do not always grow sufficiently well to ensure efficient vaccine manufacture. Thus, there is a need in the art to provide further and improved donor strains for influenza virus reassortment. SUMMARY OF PREFERRED EMBODIMENTS The inventors have now surprisingly discovered that influenza viruses which comprise backbone 30 segmems from two or more influenza donor strains can grow faster in a culture host compared with reassortant influenza A viruses which contain all backbone segments from the same donor strain. In particular, the inventors have found that influenza viruses which comprise backbone segments derived from two high-yield donor strains can produce higher yield reassortants with target vaccirie relevant HA/NA genes than reassortants made with either of the two original donor strains. 1 In principle, all segments of closely related influenza A viruses can be specifically reassoned to produce viable viruses, but only a small fraction of these viruses will be high-growth reassortants, due to inefficient activities of the resulting viral components. The inventors have provided backbone combinations that produce the high yield strains. Reassortant influenza A viruses comprising 5 backbone segments from two or more influenza donor strains may contain the PB I and the P32 viral segments from the same donor strain, in particular the A/New Caledonia20/19991ike strain, referred to herein as the 105p30 strain. The PB1 and PB2 viral segments may have at least 95% identity or 100% identity with the sequence of SEQ I) NO: 2 and/or SEQ ID NO: 3. Where the reassortant influenza A virus comprises backbone segments fran two or three donor 10 strains, each donor strain may provide more than one of the backbone segments of the reassortant influenza A virus, but one or two of the donor strains can also provide only a single backbone segment. Where the reassortant infuenza A virus comprises backbone segments from two, three, four or five donor strains one or two of the donor strains may provide more than one of the backbone segments 15 of the reassortant influenza A virus, In general the reassortant influenza A virus cannot comprise more than si backbone segments. Accordingly, for example, if one of the donor strains provides five of the viral segments, the reassortant influenza A virus can only comprise backbone segments from a total of two different donor strains. Where a reassortant influenza A virus comprises the PB1 segment from A/Texas/1i/f, it preferably 20 does not comprise the PA, NP or M segment from A/Puerto Rico/8/4 Where a reassortant influenza A virus comprises the PA, NP or M segment from A/Puerto Rico/8/3. it preferably does not comprise the PB I segment from ATexas/1/7. In some embodiments, the invention does not encompass reassortant influenza A viruses which have the PB1 segment from A/Texas/I/7 and the PA. NP and Ni segments from A/Puerto Rico/8/4. The Pf! segment from A/Texas/I may have 25 the sequence of SEQ ID NO: 46 and the PA, NP or Ni segments from A/Puerto Rico/834 may have the sequence of SEQ ID NOs 47; 48 or 49, respectively. The inventors have also discaered that variants of known donor strains can grow to higher viral titres compared to the original donor strain and can therefore be better donor strains for reassorting influenza viruses, Examples of such strains are PR8-X and 105p30. 30 Influenza A virus strains of the invention can grow to higher viral tires in MDCK cells in the same time and under the same growth conditions compared with A/Puerto Rico/8/34 and/or have a higher rescue efficiency compared with reassortant influenza strains that comprise all backbone segments from the same influenza donor strain. Further provided is a reassortant influenza A virus comprising at least one backbone viral segment from such an influenza strain. 35 The invention also provides a reassortant influenza A virus comprising at least one backbone viral segment from a donorstrain, wherein the donor strain is selected from the group consisting of 2 105p30 and PR8-X, When the at least one backbone viral segment is the PA segment it may have a sequence having at least 95% or at least 99% identity with a sequence selected from the group consisting of SEQ ID NOs: 9 and 11 When the at least one backbone viral segment is the R segment, it may have a sequence having at least 95% or at least 99% identity with a sequence 5 selected from the group consisting of SEQ ID NOs 10 and 18. When the at least one backbone viral segment is the PB2 segment, it may have a sequence having at least 95% or at least 99% identity with a sequence selected from the group consisting of or SEQ ID NOs: 1 I and 19. When the at least one backbone viral segment is the M segment it may have a sequence having at least 95% or at least 99% identity with a sequence selected from the group consisting of SEQ ID NOs: 13 and 21. When 10 the at least one backbone viral segment is the NP segment it may have a sequence having at least 95% or at least 99% identity with a sequence selected from the group consisting of SEQ ID NOs: 12 and 20. When the at least one backbone viral segment is the NS segment it may have a sequence having at least 95% or at least 99% identity with a sequence selected from the group consisting of SEQ ID NOs: 14 and 22. 15 In embodiments where the reassortant influenza A virus conprises backbone segments fom at least two influenza donor strains, at least one backbone segment may be derived from a donor strain selected from the group consisting of i05p30 and PR8~X, as discussed in the previous paragraph Preferred reassortant influenza A viruses comprise 1, 2, 3 or 4 vimal segments from the iO5p3O donor strain wherein the PA segment may have at least % identity or 100% identity with SEQ ID N: 20 17, the NP segment may have at least 95% identity or 100% identity with SEQ I) NO: 20, the l segment may have at least 95% identity or 100% identity with SEQ ID NO: 2, and/or the NS segment may have at least 95% identity or 100% identity with SEQ ID NO: 22. In some embodiments such infuenza A viruses may also comprise at least one backbone viral segment from the PR--X donor strain. Where the at least one viral segment is the PA segment it may have at least 25 95% identity or 100% identity with SEQ ID NO: 9. Where the at least one viral segment is the NP segment it may have at least 95% identity or 100% identity with SEQ ID NO: 12. Where the at least one viral segment is the M segment it may have at least 95% identity or 100% identity with SEQ ID NO: 13. Where the at least one viral segment is the NS segment it may have at least 95% identity or 100% identity with SEQ ID NO: 9. The inventors have shown that reassortan influenza A viruses 30 comprising such backbone segments grow well in cell culture. In general a reassortant influenza virus will conain only one of each backbone segment. For example, when the influenza virus comprises the PA segment from 105p3O it will not at the same time comprise the PA segment of PR8-X In preferred embodiments, the virus comprises Wial segments having at least 95% identity or 100% 35 identity with the sequence of (a) the PB2 segment of SEQ ID NO: 19, the PHI segment of SEQ ID: NO 18 and the NS segment of SEQ ID NO: 22; or (b) the PB2 segment of SEQ ID NO: 19, the PHI segment of SEQ ID NO: 18 and the M segment of SEQ ID NO: 21; or (c) the PB2 segment of SEQ 3 ID NO: 19, the PB! segment of SEQ ID NO: IS and the NP segmentof SEQ ID NO: 20; or (d) the PB2 segment of SEQ ID NO 19, the PBI segment of SEQ ID NO 18 and the PA segment of SEQ ID NO 17. These embodiments are preferred because the inventors have found that such reassortant influenza A viruses grow particularly wel in cell culture 5 The invention provides a method of preparing the reassortant influenza A viruses of the invention. These methods comprise steps of (i) introducing into a culture host one or more expression construct(s) which encodes) the viral segments required to produce an influenza A virus wherein the backbone viral segments are from two or more influenza strains; and (ii) culturing the culture host in order to produce reassortant virus and optionally (iii) purifying the virus obtained in step (ii). 10 The method may comprise the steps of (I) introducing into a culture host one or more expression constructs) which encodes) the viral segments required to produce an influenza A virus wherein the backbone viral segments are from two or more influenza strains and the PB 1 and PB2 segments are from the same donor strain; and (ii) cuturing the culture host in order to produce reassortant virus and optionally (iii) purifying the virus obtained in step (ii). 15 Also provided is a method of preparing a reassortant influenza A virus of the invention comprising the steps of (i) introducing into a culture host one or more expression constructs) which encodes) the Aviral segments required to produce an influenza A virus wherein the backbone viral segments are from two or more influenza strains and the HA and the PB! segment are from different influenza strains which have the same infuenza HA subtype; and (ii) culturing the culture host in order to 20 produce reassortant virus and optionally (iii) purifying the virus obtained in step (ii). The invention also provides a method of preparing a reassortant infuenza A virus of the inventon comprising steps of (i) introducing into a culture host one or more expression constructs) which encode(s) the viral segments required to produce an inluenza A virus wherein one or more backbone viral segment(s) is/are from a 105p30 and/or a PR8X influenza stran and wherein at least one viral 25 segment is derived from a second influenza strain; and (ii) culturing the culture host in order to produce reassortant virus and optionally (iii) puriting the virus obtained in step (ii) The methods may further comprise steps of (iv) infecting a culture host with the virus obtained in step (ii) or step (iii); (v) culturing the culture host from step (iv) to produce further virus; and optionaly (vi) purifying the virus obtained in step (v) 30 The invention also provides a method for producing influenza viruses comprising steps of (a) infecting a culure host with a reassortant virus of the invention; (b) culuring the host from step (a) to produce the virus; and optionally (c) purifying the virus obtained in step (b), The invention also provides a method of preparing a vaccine, comprising steps of (d) preparing a virus by the methods of any one of the embodimens described above and (e) preparing vaccine from 35 the virus. 4 In a further embodiment, the invention provides influenza strains PR8-X and 105p30. The invention also encompasses variant H I N influenza virus strains in which the M genorne segment has lysine in the position corresponding to amino acid 95 of SEQ 1D NO: 33 when aligned to SEQ ID NO: 33 using a pairwise alignment algorithm. The variant HiNI influenza vinus strains 5 according to the invention may further have a HA segment which has glycine in the position corresponding to amino acid 225 of SEQ ID NO: 35 when aligned to SEQ iD NO: 35 and/or has asparagine in the position corresponding to anino acid 231 of SEQ ID N): 35 when aligned to SEQ ID NO: 35 using a pairwise alignment algorithm. The variant HNI influenza virus strain may also have a NA segment which has histidine in the position corresponding to amino acid 70 of SEQ ID 10 NO: 31 when aligned to SEQ ID NO: 31 using a pairwise alignment algoritun. The preferred pairwise alignment algorithm is the Needieman-Wunsch global alignment algorithm [4, using default parameters (e.g. with Gap opening penalty = 10.0, and with Gap extension penalty 0.5, using the EBLOSUM62 scoring matrix). This lgorthm is conveniently implemented in the needle tool in the EMBOSS package [5]. 15 The invention provides an expression system comprising one or more expression construct(s) comprising the vRNA encoding segments of an influenza A virus wherein the expression constructs) encodes) the backbone viral segments from two or more influenza donor strains. The expression construct(s) may encode the PB1 and PB2 segments from the same donor strain. The invention also provides an expression system comprising one or more expression construct(s) 20 comprising the vRNA encoding segments of a 105p30 or PRSX strain wherein the expression constructs) comprises) at least one backbone viral segmient from the 105p3O or PR8-X, or strain. The expression construct(s) may further comprise the vRNAs which encode the PB2 .NP, N S, I and PA segments from PR"8X. The invention also provides a host cell comprising the expression systems of the invention. These 25 host cells can express an influenza A virus from the expression constructs in the expression system. Expression constructs which can be used in the expression systems of the invention are also provided.For example the invention provides an expression construct which encodes the backbone segments of the reassortant influenza strains according to the invention on the same construct. Donor strains 30 Influenza donor strains are strains which typically provide the backbone segments in a reassortant influenza virus, even though they may sometimes also provide the HA or NA segment, but not both, of the virus. Usually, however, both the HA and the NA segment in a reassortant influenza virus will be from the vaccine strain. The inventors have surprisingly discovered that reassortant influenza A viruses comprising backbone 35 segments from two or more influenza donor strains can grow to higher tires in cell culture compared 5 with reassortant influenza viruses which contain all backbone segments from the same donor strain. 1he inventors have shown that this effect is due to the presence ofbackbone segments from two donor strains and does not require the presence of viral segments with specific mutations. Particularly good resuls are achieved, however, with influenza A strains in which the M genome 5 segment has lysine in the position corresponding to amino acid 95 of SEQ ID NO:3 when aligned to SEQ ID NO: 33. Reassortant influenza A viruses comprising the PBI and P12 segments from the same influenza strain (for example 105p30) are also advantageous because they showed a better rescue efficiency compared with influenza viruses in which the PBI and PB2 segments are fom different viruses The 10 PB I and PB2 segments of 105p30 have the sequence of SEQ ID NOs I8 and 199 respectively. The inventors have also shown that sonc infuenza virus strains can grow to higher viral litres in MDCK cells in the same time and under the same growth conditions compared with A/Puerto Rico/8/34. Variants of influenza donor strains which are derived frn the donor strains of the invention or other 15 known donor strains such A/PR8/4 (wt PR8) can also be useful as donor strains. These donor strains can grow to higher viral tires (in the same time and under the same growth conditions) compared to the donor strain from which they are derived. For example, the inventors have surprisingly discovered that passaging the A/Pd8/4 influena strain several times in cell culture results in a virus strain (PRS-X) which grows to nmch higher viral titles compared to the original 20 A/PR8/34 strain. Likewise, the inventors have found that massaging the A/New Caledonia/20/1999 strain several times in cells results in a strain i05p30) which grows to even higher viral litres compared to the unpassaged A/New Caledoniai20/ 999 strain in the same time and under the same growth conditions. Donor strain variants of the present invention will typically achieve viral tires whih are at least 10%, at least 20%, at least 50%, at least 100% at least 200%, at least 500% or at 25 least 1000% higher under the same growth conditions and for the same time (for example 12 hours, 24 hours, 48 hours or 72 hours)| compared to the viral litres obtained with the donor strain from which the variant was derived. The segments of PR8-X have the sequences of SEQ IID NOT 11 (PB2), SEQ ID NO: 10 (PB1), SEQ ID NO: 9 (PAN SEQ ID NO: 12 (NP), SEQ ID NO: SM). SEQ ID 14 )EQ ID NO: 15 30 (HA) or SEQ ID NO: 16 (NA) The segments of 105p30 have the sequences of SEQ iiD NO: 19 (PB2), SEQ ID NO: 18 (PB ), SEQ ID NO: 17 (PA), SEQ ID NO: 20 (NP), SEQ ID NO: 21 (M). SEQ ID NO: 22 (N S), SEQ ID NC: 23 (H A) or SEQ ID NO: 24 (NA). Influenza strains which contain one, two, three, four five, six or seven of the segments of the 105p30 35 or P118-X strains can also be used as donor strains.
The invention can be practised with donor strains having a viral segment that has at least about 70%, at least about 75%, at least about t0 at least t least about 90%, at least about 95% or at least about 99% identity to a sequence of SEQ ID NOs i l-i4 or 18- 22. For example, due to the degeneracy of the genetic code, it is possible to have the same polypeptide encoded by several 5 nuclei acids with different sequences. Thus, the invention may be practised with viral segments that encode the same polypeptides as the sequences of SEQ ID NOs 11-44 or 1822. For example, the nucleic acid sequences of SEQ ID NOs 3 and 28 have only 73% identity even though they encode the same Viral protein. The inventiontmay also be pracised with viAl segmens that encode poypetiddes that have at least 10 80%1, at least 8% t least 90%N at least 95% or at leas 99% identity to the poypptdesquen:es enc odedty SEQi 'D~o 41 iorlS - 22. Variations ni the DNA and the amino acid sequence may also stem fom spontaneous mutations which can occur during passaging of the viruses, Such variant influenza strains can also be used in the invention. 15 Rea.;ortat viruses The invention provides reassortant influenza viruses which comprise backbone segments from two or more influenza donor strains. The PB] and PB2 segments may be from the same donor strain. The invention also povides reassortant influenza virses comprising at least one backbone viral segment from an influenza virus strain that can grow to higher viral titles in MDCK cells in the same 20 time and under the same growth conditions compared with A/Puerto Rico/8/34. The invention provides reassortant influenza viruses comprising at least one backbone viral segmera from the donor strains of the inention, e.g a PR8-X or i05p30 strain The reassortant influenza virses of the invention can be reassortants between two, three or more different influenza strains provided that at least one viral segment is derived from a donor strain ofthe invention. 25 Influenza viruses are segmented negative strand RNA virses.nfluenza A nd B viruses have eight segments(NP, M, NS, PA, PB HA and NA) whereas influenza C virus has seven. The reassortant viruses of the invention contain the backbone segments froi two or more donor strains, or at least one (ie. one, two, three four, five or si) backbone viral segment from the donor strains of the invention. The backbone viral segments are those which do not encode H-IA or NA. Thus, backbone 30 segments will typically encode the PBi, PB2 PA, NP, M 1 M N and NS 2 polypeptides of the influenza virus. The reassortant viruses will not typically contain the segments encoding HA and NA from the donor strains even though reassortant viruses which comprise either the IHA or the NA but not both from the danor strains of the invention are also envisioned. When the reassortant viruses of the invention are reassortants comprising the backbone segments 35 from a single donor strain, the reassortant viruses will generally include segments from the donor 7 strain and the vaccine strain in a ratio of 1:7, 26, 3:5, 4:4, 5:3, 6:2 or 7:1 Having a majority of segments from the donor strain, in particular a ratio of 6:2, is typical When the reassortant viruses comprise backbone segments from two donor strains, the reassortant virus will generally include segments from the first donor strain, the seconds donor strain and the vaccine strain in a ratio of 5 1:1:6,1 2:5, 13:4, 1:43, 1:5:2, 1;61. 2:15224, 23,2:4 2, 3:1:2, 3:1, 4:1 4:2:2, 4:3:1, 5:1: 52:1 or 6:1: Preferably ithe reassortant viruses do not contain the HA segment of the donor strain as this encodes the main vaccine antigens of the influenza virus and should therefore come from the vaccine strain. The ressrtn virse 1ofth inventi'-on the-reforerfrbyhv at least thie HAsgetand 10 typial the H NA semnts from the vaccine stain. The inentio also encompasses reassortant viruses which conain virl segmens from more ta one, for example two or three different, donor strains) wherein at least on iral segment peferably not HA. is derived from the PR8-X or 105p30 influenza strains. Such reassortant influenza viruses will typically contain the HA and/or NA segment from a vaccine strain. Where the reassortants 15 contain viral segments from more than one influenza donor strain, the further donor strain(s) can be any donor strain including the donor strains of the invention For example, some of the viral segments may be derived from the APR/8/34 or AA/6/60 (A/Ann Arbor/6/60) influenza strains. Reassortants containing viral segments from the AA/660 strain may be advantageous, for example, where the reassortant virus is to be used in a live attenuated influenza vaccine. 20 The iwention also encompasses reassortants which comprise viral segments from more than one vaccine strain provided that the reassortant comprises a backbone according to the present invention. For example the reassortant infuenza viruses may comprise the HA segment from one donor strain and the NA segment from a different donor strain. The reassortant viruses of the invention can grow to higher viral tires than the wild-type vaccine 25 strain from which some of the viral segment(s) of the reassortant virus are derived in the same time (for example 12 hours, 24u hours 48 hours or 72 hours) and under the same growth conditions. The viral titre can be determined by standard methods known to those of skill in the art. The reassortant viruses of the invention can achieve a viral titre which is at least 10% higher, at least 20% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 500% higher, or at least 30 1000%higher than the viral tire of the wild type vaccine strain in the same tie frame and under the same conditions. The invention is suitable for reassorting pandemic as well as inter-pandemic (seasonal) influenza vaccine strains. The reassartant infuenza strains may contain the infuenza A virus HA subtypes Hi, H2, 113, 1-14, H5, 116, H7, 118, Hig. H111 H 1i, H12, H13 u Hi, Hi5 or H16. They may contain the 35 influenza A virus NA subtypes N3, N2, N3 N4 N, N6, N7, NS or N9. Where the vaccine strain used in the reassortant influenza viruses of the invention is a seasonal influenza strain, the vaccine 8 strain may have a HI or 1-13 subtype. In one aspect of the invenion the vaccine strain is a H1NI or H 3N2 strain. The vaccine strains for use in the invention may also be pandemic strains or potentially pandemic strains. The characteristics of an influenza strain that give it the potential to cause a pandemic 5 outbreak are: (a) it contains a new hemaggiutinin compared to the hemagglutinins in currently circulating human strains, i.e. one that has not been evident in the human population for ovr a decade (eg. H2), or has not previously been seen at all in the human population (eg 15, 116 or 1-19, that have generally been found only in bird populations), such that the human population will be inununologically naIve to the strain's hemagglutinin; (b) it is capable of being transmitted 10 horizontally in the human population; and (c) i is pathogenic to humans. A vaccine strain with H5 hemagglutinin type is preferred where the reassortant virus is used in vaccines for immunizing against pandemic influenza, such as a H5N1 strain. Other possible strains include H5N3, H9N2, H2N2, 17NI and H7N7, and any other emerging potenially pandemic strains. The invention is particularly suitable for producing reassortant viruses for use in vaccine for protecting against 15 potential pandemic virus strains that can or have spread from a non-human animal population to humans, for example a swine-origin HNI influenza strain. le reassortant influenza strain of the invention may comprise the H-A segment and/or the NA segment from an A/CaiifoMia/4/09 strain. Thus, for instance, the HA gene segment may encode a H1 hemaggiutinin which is more closely related to SEQ ID NO: 32 than to SEQ ID NO: 25 (i e. has a 20 higher degree sequence identity when compared to SEQ ID NO: 32 than to SEQ ID NO: 25 using the same algorithm and parameters) SEQ ID NOs: 32 and 25 are 80% intical Similarly, the NA gene may encode a N1 neuraminidase which is more closely related to SEQ ID NO: 27 than to SEQ ID NO: 26. SEQ ID NOs: 27 and 26 are 82% identical. Strains which can be used as vaccine strains include strains which are resistant to antiviral therapy 25 (esg. resistant to oseltamivir [6] and/or zananivir), inuding resistant pandemic strains [7]. The choice of donor strain for use in the methods of the invention can depend on the vaccine strain which is to be reasserted. As reassortants between evolutionary distant strains might not replicate well in cell cukure, it is possible that the donor strain and the vaccine strain have the same HA and/or NA subtype. In other embodiments however, the vaccine strain and the donor strain can have 30 different HA and/r NA subtypes, and this arrangement can facilitate selection for reassortant viruses that contain the HA and/or NA segment from the vaccine strain. Therefore, although the 105p30 and PRS-X strains contain the H1 influenza subtype these donor strains can be used for vaccine strains which do not contain the HI influenza subtype. Reassortants of the donor strains of the invention wherein the IPA and/or NA segment has been 35 changed to another subtype can aiso be used. The HI influenza subtype of the l05p30 or PR8-X strain may be changed, for example, to a H3 or 115 subtype. 9 Thus, the invention encompasses an influenza A virus which comprises one, two, three, four, five, six or seven viral segments from the 105p30 or PR8-X strains of the invention and a HA segment which is not of the I subtype The reassortant donor strains may further comprise an NA segment which is not of the NI subtype Suitable techniques for reasserting the donor strains will be evident 5 to those of skill in the art. The invention also encompasses reassortant donor straws which comprise at least one, at least two, at least three, at least four, at least five, at least six or at least seven viral segments from the 105p30 or PR8-X strains of the invention and a HI HA segment which is derived from a different influenza strain, 10 Reassortant viruses which contain an NS segment that does not encode a functional NS protein are also within the scope of the present invention. NSI knockout mutants are described in reference 8. These NSI--mutant virus strains are particularly suitable for preparing live attenuated influenza vaccines, The 'second influenza strain' used in the methods of the invention is different to the donor strain 15 which is used. Reverse genetics The invention is particularly suitable for producing reassortant influenza virus strains through reverse genetics techniques. In these techniques, the viruses are produced in culture hosts using an expression system. 20 In one aspect, the expression system may encode the PBl and PB2 segments from the same donor strain In this aspect of the invention, the system may encode at least one ( )e. one, two three or four) of the segments NP , NS and/or PA from another influenza donor strain. In another aspect, the system may encode 1 or more (e.g 1, 2, 3, 4, 5 or 6) genome segments from the PR8-X strain, but usually this/these will not include the PRS-X HA segment and usually wil not 25 include the PR8-X NA segment. Thus the system may encode at least one of segments NP, M NS, PA, P1B and/or PB2 (possibly all six) from PRS-X. The system may encode I or more (eg. 1, 2, 3, 4, 5 or 6) genome segments from the 105p30 strain, but usually this/these will not include the 105p3 HA segment and usually will not include the 105p30 NA segment. Thus the system may encode at least one of segments NP, M, NS, PA, P1 30 and/or PB2 (possibly all six) from 105p30 Reverse genetics for influenza A andB viruses can be practised with 12 plasinids to express the four proteins required to initiat tion and transcrption (PB, PB2, PA and nucleoprotein) and all eight viral genome segments. To reduce the number of constructs, however, a plurality of RNA polymerase I transcription cassettes (for viral RNA synthesis can be included on a single plasmid 35 (e g sequences encoding 1 2, 3,4, 5, 6, 7 or all S8 influenza vRNA segments and a plurality of 10 protein-coding regions with RNA polymerase fI promoters on another plasmid (eg sequences encoding 1, 2, 3, 4, 5, 6, 7 or 8 influenza mRNA transcripts) [9]. It is also possible to include one or more influenza vRNA segments under control of a pol 1 promoter and one or more influenza protein coding regions under control of another promoter, in particular a pol II promoter, on the same 5 plasmid. This is preferably done by using hi-directional plasmids Preferred aspects of the reference 9 method involve: (a) PB 1, PB2 and PA mRNA-encoding regions on a single expression construct; and (b) al! 8 vRNA encoding segments on a single expression construct, Including the neuraminidase (NA) and hemaggiutinin (HA) segments on one expression construct and the six other viral segments on another expression construct is particularly preferred as 10 newly emerging influenza virus strains usually have mutations in the NA and/or HA segments. Therefore, the advantage of having the H A and/or NA segments on a separate expression construct is that only the vector comprising the H A and NA sequence needs to be replaced. Thus, in one aspect of the invention the NA and/or HA segments of the vaccine strain may be included on one expression construct and the vRNA encoding segments from the donor strains) of the invention, excluding the 15 HA and/or NA segmentss, are included on a different expression construct. The invention thus provides an expression construct comprising one, two, three, four, five or six vRNA encoding backbone viral segments of a donor strain of the invention. The expression construct may not comprise HA and/or NA viral segments that produce a functional HA an/or NA protein. Known reverse genetics systems involve expressing DNA niolecules which encode desired viral 20 RNA (vRNA) molecules from pol I promoters, bactial RNA polymerase promotersbacteriophage poymerase promoters, etc. As influenza viuses require the presence of viral polymerase to complete the life cycle, systems may also provide these proteins eg the system further comprises DNA molecules that encode viral polymerase proteins such that expression of both types of DNA leads to assembly of a complete infectious virus. It is also possible to supply the viral polymerase as a 25 protein. Where reverse genetics is used for the expression of influenza vRNA, it will be evident to the person skilled in the ar that precise spacing of the sequence elements with reference to each other is important for the polymerase to initiate replication. It is therefore important that the DNA molecule encoding the viral RNA is positioned correctly between the pol 1 promoter and the termination 30 sequence, but this positioning is well within the capabilities of those who work with reverse genetics systems. In order to produce a recombinant virus, a cell must express all segments of the viral genome which are necessary to assemble a virion. DNA clmed into the expression constructs of the present invention preferably provides all of the viral RNA and proteins, but it is also possible to use a helper 35 virus to provide some of the RNA and proteins, although systems which do not use a helper virus are preferred. As the influenza virus is a segmented virus, the viral genome will usually be expressed 1i using more than one expression construct in the methods of the invention. It is also envisioned, however to combine one or more segments or even all segments of the viral genome on a single expression construct In some embodiments an expression construct wil also be included which leadsto expression ofan 5 accessory protein in the host cell For instance, it can be advantageous to express a manniral serine protease (e.g. trypsin) as part of a reverse genetics system. Expression constructs Expression constructs used in the expression systems of the invention may be uni-directional or bi directional expression constructs. Where more than one transgene is used in the methods (whether on 10 the same or different expression constructs) it is possible to use uni-directional and/or hi-directional expression. As influenza viruses require a protein for infectivity, |it is generally preferred to use hi-diretional expression constructs as this reduces the total number of expression constructs required by the host cell. Thus, the method of the invention may utilise at least one bi-directional expression constmut 15 wherein a gene or cDNA is located between an upstream pol U promoter and a downstream non endogenous pol I promoter ranscription of the gene or cDNA from the pol H promoter produces capped positive-sense viral mRNA which can be translated into a protein, while transcription from the non-endogenous pol I promoter produces negative-sense yERNA. 'IThe bi-directional expression construct may be a hi-directional expression vector. 20 Hi-directional expression constructs contain at least two promoters which drive expression in different directions (i.e. both 5' to 3' and 3 to 5') from the same construct. The two promoters can be operably linked to different strands of the same double stranded DNA. Preferably, one of the promoters is a pol i Ipromoter and at least one of the other promoters is a poII promoter This is useful as the pol I promoter can be used to express uncapped vRNAs while the pol H promoter can 25 be used to transcrbe mRNAs which can subsequently be translated into proteins, thus allowing simultaneous expression of RNA and protein from the same construct Where more than one expression construct is used within an expression system, the promoters may be a mixture of endogenous and non-endogenous promoters. The pol I and pol Il promoters used in the expression constructs may be endogenous to an organism 30 from the same taxonomic order from which the host cell is derived. Alternatively, the promoters can be derived from an organism in a different taxonomic order than the host cell. IThe term "order" refers to conventional taxonomic ranking, and examples of orders are primates, rodentia, carivora, marsupialia, cetacean. W. Humans and chimpanzees are in the same taxonomic order (primates), but humans and dogs are in different orders (primates vs. canivora). For example, the human pol I 35 promoter can be used to express viral segments in canine cells (eig. MDCK cells) 12 The expression construct wili typically include an RNA transcription termination sequence. The termination sequence may be an endogenous termination sequence or a termination sequence which is not endogenous to the host cell. Suitable termination sequences will be evident to those of skill in the art and include, but are not limited to, RNA polymerase I transcription termination sequence, 5 RNA polymerase II transcription termination sequence and ribozymes. Furthermore, the expression constructs may contain aone ormore polyadenylation signals for mRNAs, particularly at the end of a gene whose expression is controlled by a pol I promoter. An expression system may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at lst t ten, at lcast eleven or at least twelve expression 10 constructs. An expression construct may be a vector, such as a plasmid or other episomai construct. Such vectors will typicaiiy comprise at least one bacterial and/or eukaryotic origin of replication. Furthermore, the vector may comprise a selectable marker which allows for selection in prokaryotic and/or eukaryotic cells. Examples of such selectable markers are genes conferring resistance to antibiotics, such as 15 ampicillin or kanamvcin. The vector may further comprise one or more multiple cloning sites to facilitate cloning of a DNA sequence. As an alernative, an expression construct may be a linear expression construct. Such linear expression constructs wil typically not contain any amplification and/or selection sequences. However, linear constructs comprising such amplification and/or selection sequences are also within 20 the scope of the present inventing. Reference 10 describes a linear expression construct which describes indiviual linear expression constructs for each viral segment. It is also possible to include more than one, for example two, three four, five or six viral segments on the same linear expression construct. Such a system has been described, for example, in reference 1, Expresion constructs can be generated using methods known in the art. Such methods were 25 described, for example, in reference 12. Where the expression construct is a linear expression construct, it is possible to linearise it before introduction into the host cell utilising a single restriction enzyme site. Alternatively, it is possible to excise the expression construct from a vector using at least two restriction enzyme sites. Furthermore, it is also possible to obtain a linear expression construct by amplifying it using a nucleic acid amplification technique (eg by PCR). 30 The expression constructs used in the systems of the invention may be nounbacterial expression constructs. This means that the construct can drive expression in a eukarvotic cell of viral RNA segments encoded therein, but it does not include components which would be required for propagation of the construct in bacteria. Thus the construct will not include a bacterial origin of replication (on), and usually will not include a bacterial selection marker (eg g. an antibiotic resistance 35 marker) Such expression constructs are described in reference 13 which is incorporated by reference. 13 The expression constructs may be prepared by chemical synthesis. The expression constructs may either be prepared entirely by chemical synthesis or in part Suitable methods for preparing expression constructs by chemical synthesis are described, for example, in reference 13 which is incorporated by reference. 5 The expression constructs of the invention can be introduced into host cells using any technique known to those of skill in the art. For example, expression constructs of the invention can be introduced into host cels by employing electroporation, DEAE-dextran, calcium phosphate precipitation, liposomes, microinjection, or microparticie-bombardment. W 0 .. Th.e culture host for use in -the prs ntnton can be any ukaryotic cell that canprdu ehviu of ilntst 'the inenio ill typically u se a cell lie although, ftor, exam-ple, piaycells' maybe used as an alternative The cell will typically be mammalian. Suitable mammalian cells include, but are not limited to, hamster cattle, primate (including humans and monkeys) and dog cells. Various cell types may be used, such as kidney cells, fibrobiasts, retinal cells, lung cells, etc. Examples of 15 suitable hamster cels are the cell lines having the names BHK21 or HKCC. Suitable monkey cells are eig African green monkey cells, such as kidney cells as in the Vero cell line [14-161, Suitable dog cells are a g. kidney cells as in the CLDK and MDCK cell lines Further suitabe cells include, but are not limited to: C1; 293'1; BHK; MRC 5; PERC6 [17]; FRhL2; WI-38; etc. Suitable'cells are widely available e.g. from the American Type Cel Culture 20 (ATCC) collection [18], from the Cori Cell Repositories [19], or from the European Collection of Cell Culures (ECAC) For example, the ATCC supplies various different Vero cells under catalogue numbers CCL 81, CCL 81.2, CR1. 1586 and CRLN1587, and it supplies MDCK cells under catalogue number CCL 34. PER.C6 is available from the ECACC under deposit number %022940. Preferred cells for use in the invention are MDCK cells [20-22], derived from Madin Darby canine 25 kidney. The original MDCK cells are available from the ATCC as CCL 34 It is preferred that derivatives of MDCK cells are used. Such derivatives were described, foristance, in reference 20 which disloses MDCK cells that were adapted for growth in suspension culture (QMDCK 33016 or '33016-PfW deposited as DSM ACC 2219: see also ref 20). Furthermore, reference 23 discloses MDCK-denived cells that grow in suspension in serum free culture 'iB-702 deposited as FERM BP 30 7449). In some embodiments the MDCK cell line used may be tumorigenic. It is also envisioned to use non-tunmorigenic MCK cells. For example, reference 24 discloses non tumorigenic MDCK cells, including MDCK-S' (ATCC PTA-6500) 'MDCK-SFI0i' (ATCC PTA-6501), 'MDCK SF102' (ATCC PTA-6502) and 'MDCK-SF103 (ATCC Pl A-6503 Reference 25 discloses MDCK cells with high susceptibility to infection, including MDCK.5F1' cels (ATCC CRL 12042). 35 It is possible to use a mixture of more than one cell type to practise the methods of the present invention, However, it is preferred that the methods of the invention are practised with a single cell 14 type eg with monoclonal cells. Preferably, the cells used in the methods of the present invention are from a single cell line. Furthermore, the same cell line may be used for reassorting the virus and for any subsequent propagation of the virus. Preferably, the cells are cultured in the absence of serum, to avoid a common source of contaminants. 5 Wrious serum-free media for eukaryotic cell culture are known to the person skilled in the art (e.g. Iscove's medium, ultra CHO medium (Bit)Whittaker), iX-CELL (JRH Biosciences)). Furthermore, protein-free media may be used (e.g. PFTCHO (JRH Biosciences)). Otherwise, the cells for replication can also be cuhured in the customary serum-containing media (e.g. MEM or DMEM medium with 0.5% to 10% of fetal caf serum). 10 The cells mray be in adherentn co ure or in usesin cnentona resrfnt Traditionally influenza viruses are reassorted by co-infecting a culture host, usually eggs with a donor strain and a vaccine strain. Reassortant viruses are selected by adding antibodies with specificity for the HA and/or NA proteins of the donor strain in order to select for reassortant viruses 15 that contain the vaccine strain's HA and/or NA proteins. Over several passages of this treatment one can select for fast growing reassortant viruses containing the vaccine strain's HA and/or NA segments. The invention is suitable for use in these methods. It can be easier to use vaccine strains with a different HA and/or NA subtype compared to the donor strains) as this facilitates selection for 20 reassortant viruses. It is also possible, however, to use vaccine strains with the same HA and/or NA subtype as the donor strains) and in some aspects of the invention this preferred, In this case, antibodies with preferential specificity for the HA and/or NA proteins of the donor strains) should be available. Virus preparation 25 In one embodiment, the invention provides a method for producing influenza vinses comprising steps of (a) infecting a culture host with a reassortant virus of the invention; (b) culturing the host from step (a) to produce the virus; and optionally (c) purifying the virs obtained in step (b). The culture host may be ells or embryonated hen eggs Where cells are used as a culture host in this aspect of the invention, it is known that cell culture conditions (e.g. temperature cell density, pI 30 value, etc.) are variable over a wide range subject to the cell line and the virus employed and can be adapted to the requirements of the application. The following information therefore merely represents guidelines. As mentioned above, cells are preferably culured in serum-free or protein-fee media. Multiplication of the cells can be conducted in accordance with methods known to those of skill in 35 the art For example, the cells can be cultivated in a perfusion system using ordinary support methods 15 like centrifugation or filtration. Moreover, the cells can be multiplied according to the invention in a fed-batch system before infection. In the context of the present invention, a culture system is referred to as a fed-batch system in which the cells are initially cultured in a batch system and depletion of nutrients (or part of the nutrients) in the medium is compensated by controlled feeding of S concentrated nutrients. It can be advantageous to adjust the pH value of the medium during mnuhiplication of cells before infection to a value between pH 6.6 and pH 7.8 and especially between a value between pH 7.2 and pH 7.3. Culturing of cells preferably occurs at a temperature between 30 and 40"C. When culturing the infected cells (step ii), the cells are preferably cultured at a temperature of between 30 "C and 36"C or between 32 "C and 34 C or at 33 0 C. This is particularly preferred, as it 10 has been shown that incubation of infected cells in this temperature range results in production of a virus that results in improved efficacy when formulated into a vaccine [263. Oxygen partial pressure can be adjusted during culturing before infection preferably at a value between 25% and 95% and especially at a value between 35% and 60%. The values for the oxygen partial pressure stated in the context of the invention are based on saturation of air. Infection of cell 15 occurs at a cell density of preferably about 8-25x1&0 cells/mL in the batch system or preferaly about 5-20x 106 cels/nL in the perfusion system. The ells can be infected with a viral dose (MOT value, "muliplicity of infection; corresponds to the number of virus units per cell at the time of infection) between IY and 10, preferably between 0.0001 and 0.. Virus may be grown on cells in adherent culture or in suspension. Microcarrier cultures can be used. 20 In some embodiments the cells may thus be adapted for growth in suspension. The methods according to the invention also include harvesting and isolation of viruses or the proteins generated by them, During isolation of viruses or proteins, the cells are separated from the culture medium by standard methods like separation, filtration or ultrafiltration. The viruses or the proteins are then concentrated according to methods sufficiently known to those skilled in the art, 25 like gradient centrifugation, filtration, precipitation, chromatography, etc . and then purified. It is also preferred according to the invention that the viruses are inactivated during or after purification. Virus inactivation can occur, for example, by -propiolactone or fonnaldehyde at any point within the purification process. The culture host may be eggs. The current standard method for influenza virus growth for vaccines 30 uses embryonated SPF hen eggs, with virus being purified from the egg contents (allantoic fluid). It is also possible to passage a virus through eggs and subsequently propagate it in cell culture and vice versa, Vaccine The invention utilizes virus produced according to the method to produce vaccines. 35 Vaccines(particularly for influenza virus) are generally based either on live virus or on inactivated virus. Inactivated vaccines may be based on whole visions, 'split' visions, or on purified surface 16 antigens. Antigens can also be presented in the forn of virosomes. The invention can be used for manufacturing any of these types of vaccine. Where an inactivated virus is used, the vaccine may comprise whole vision, split vision, or purified surface antigens (for influenza, including hemaggiutinin and, usually, also including neuraminidase). 5 Chemical means for inactivating a virus include treatment with an effective amount of one or more of the following agents: detergents, formaldehyde, p-propiolactone, methylene blue, psoralen, carboxyful lerene (C60), binary ethy lamine, acety I ethyleneimine, or combinations thereof. Non-chemical methods of viral inactivation are known in the art, such as for example UV light or gamma irradiation 10 Virions can be harvested from virus-containing uids, e.g. allantoic fuid or cell culture superatant, by various methods. For example, a purification process may involve zonal centrifugation using a linear sucrose gradient solution that includes detergent to disrupt the visions. Antigens may then be purified, after optional dution, by diafiltration. Split virions are obtained by treating purified virions with detergents g. ethyl ether, polysorbate 80, 15 deoxycholate, tri-N-butyl phosphate, Triton X-100, Triton Ni0l, cetyltrimethylammonium bromide, Tergitol NP9, etc.) to produce subvirion preparations, including the 'Tween-ether' splitting process. Methods of splitting influenza viruses, for example are well known in the art e.g. see refs. 27-32, etc Splitting of the virus is typically carried out by disrupting or fragmenting whole virus, whether infectious or non-infectious with a disrupting concentration of a splitting agent. The disruption 20 results in a full or partial solubilisation of the virus proteins, altering the integrity of the virus. Preferred splitting agents are non-ionic and ionic (e.g. cationic) surfactants e.g alkylglycosides, alkylthioglycosides, acyl sugars, sulphobetaines, betains, poiyoxyethyleneaikylethers, N,N-dialkyl Glucamides, Hecameg, aikylphenoxy-polyethoxyethanols, NP9, quaternary ammonium compounds, sarcosyl, CTAIs (cetyl trimethyl ammonium bromides), tri-N-butyi phosphate, Cetavion, 25 myristyitrimethylammonium salts, lipofectin, lipofectamine, and DOT-MA, the octyl- or nonyiphenoxy poiyoxyethanois (e.g. the Triton surfactants, such as Triton X-10)0 or Triton N101), polyoxyethylene sorbitan esters (the Tween surfactants), polyoxyethylene ethers, polyoxyethiene esters, etc. One useful splitting procedure uses the consecutive effects of sodium deoxycholate and formaldehyde, and splitting can take place during initial vision purification (e.g. in a sucrose density 30 gradient solution). Thus a splitting process can involve clarification of the viion-containing material (to remove non-virion material), concentration of the harvested virions (e.g. using an adsorption method, such as CaHPO 4 adsorption), separation of whole virions from non-virion material, splitting of visions using a splitting agent in a density gradient centrifugation step (e.g. using a sucrose gradient that contains a splitting agent such as sodium deoxycholate), and then filtration (e.g. 35 ultrafiltration) to remove undesired materials. Split visions can usefully be resuspended in sodium phosphate-buffered isotonic sodium chloride solution. Examples of split influenza vaccines are the BEGRIVACTM FLUARIXTM, FLUZONETM and FLUSHiELDTM products. 17 Purified influenza virus surface antigen vaccines comprise the surface antigens hemagglutinin and, typically, also neuraminidase. Processes for preparing these proteins in purified tnnm are well known in the art. The FLUVIRINTM, AGRIPPALTM and INFLUVACM products are influenza subunit vaccines. 5 Another form of inactivated antigen is the virosome [33] (nucleic acid free viral-like liposomal particles). Virosomes can be prepared by solubilization of virus with a detergent followed by removal of the nucleocapsid and reconstitution of the membrane containing the viral glycoproteins. An alternative method for preparing virosomes involves adding viral membrane glycoproteins to excess amounts of phospholipids, to give liposomes with virai proteins in their membrane. 10 The methods of the invention may also be used to produce live vaccines. Such vaccines are usually prepared by purif'ing virions from virion-containing fluids. For example, the fluids may be clarified by centrifugation. and stabilized with buffer (e.g. containing sucrose, potassium phosphate, and monosodium ghuitamate). Various fos of influenza virus vaccine are currently available (e g. see chapters 17 & 18 of reference 34) Live virus vaccines include Medimmune's FLUMIST product 15 (trivalent live virus vaccine). The virus may be attenuated. The virus may be temperature-sensitive. The virus may be cold-adapted. These three features are particularly useful when using live virus as an antigen. HA is the main immunogen in current inactvated influenza vaccines, and vaccine doses are standardised by reference to HA levels, typically measured by SRID. Existing vaccines typically 20 contain about 1 5pg of HA per strain, although lower doses can be used e.g. for children, or in pandemic situations, or when using an adjuvant. Fractional doses such as % (i' 75g HA per strain), and i have been used, as have higher doses (e0g 3x or 9x doses (35,36]) Thus vaccines may include between 0.1 and i50pg of HA per influenza strain, preferably between 01 and 50.g eg 0.l-2Q0pg. 0.lip~ig, 0.1-Opg, 0.1 -7.5pg, 0.5-5pg, etc. Particular doses include eg' about 45, about 25 30, about 15, about 10, about 7.5, about 5. about|3.8, about3.75, about 1.9, about 15, etc. per strain. For live vaccines, dosing is measured by median tissue culture infectious dose (TCID 0 rather than HA content, and a TCID 0 of between 106 and 1r (preferably between 106 10 per strain is typical. influenza strains wsed with the invention may have a natural HA as found in a wild-type virus, or a 30 modified HA For instance, it is known to modify HA to remove determinants (e.g hyper-basic regions around the HAI/HA2 cleavage site) that cause a virus to be highly pathogenic in avian species The use of reverse genetics faLlitates such modincations. As well as being suitable ft imununizing against inter-pandemic strains, the compositions of the invention are particularly useful for irmunizing against pandemic or potentially-pandemic strains. 35 The invention is suitable for vaccinating humans as well as non-human animals. 18 Other strains whose antigens can usefully be included in the compositions are strains which are resistant to antiviral therapy (eg. resistant to oseltamivir [37] and/or zanamivir), including resistant pandemic strains [38]. Compositions of the invention may include antigen(s) from one or more e l, 2, 3, 4 or more) 5 influenza virus strains, including influenza A virus and/or influenza B virus provided that at least one influenza strain is a reassortant influenza strain of the invention. Compositions wherein at least two, at least three or all of the antigens are from reassortant influenza strains of the invention are also envisioned. Where a vaccine includes more than one strain of influenza, the different strains are typically grown separately and are mixed after the viruses have been harvested and antigens have 10 been prepared. Thus a process of the invention may include the step of mixing antigens from more than one influenza strain. A trivalent vaccine is typical. including antigens from two influenza A virus strains and one influenza B virus strain. A tetravalent vaccine is also useful [39], including antigens from two influenza A virus strains and two influenza B virus strains, or three influenza A virus strains and one influenza B virus strain. 1 5 Pharmaceutical compositions Vaccine compositions manufactured according to the invention are pharmaceutical acceptable. They usually include components in addition to the antigens e.g they typically include one or more pharmaceutical carriers) and/or excipient(s) As described below, adjuvants may also be included. A thorough discussion of such components is available in reference 40 20 Vaccine compositions will generally be in aqueous form. However, some vaccines may be in dry fbn, eg in the form of injectable solids or dried or polymerized preparations on a parch. Vaccine compositions may include preservatives such as thiomersal or 2phenoxyethanoi. It is preferred, however, that the vaccine should be substantially free from (i.e. less than 5pg/T) mercurial material e.g. thiomersal-free [31,41] Vaccines containing no mercury are more preferred. 25 An a-tocopherol succinate can be included as an alternative to mercural compounds [31]. Preservative-free vaccines are particularly preferred. To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCi) is preferred, which may be present at between I and 20 mg/mi. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate 30 dehydrate, magnesium chloride, calcium chlorideetc Vaccine compositions will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will more preferably fall within the range of 290--310 mOsnmkg. Osmolality has previously been reported not to have an impact on pain caused by vaccination I42], but keeping osmolality in this range is nevertheless preferred. 19 Vaccine compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buff a buer; a histidine buffer particularlyy with an aluminum hydroxide adjuvant; or a citrate buffer Buffers will typically be included in the520mM range. The pi of a vaccine composition will generally be between 5.0 and 8, and more typically between 5 6.0 and 80 eg 6.5 and 75, or between 70 and 78. A process of the invention may therefore include a step of adjusting the pH of the bulk vaccine prior to packaging. The vaccine composition is preferably sterile. The vaccine composition is preferably non-pyrogenic egi containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. The vaccine composnion is preferably gluten-free. 10 Vaccine compositions of the invention may include detergent eg. a poiyoxyethyiene sorbitan ester surfactant (known as 'Tweens'), an octoxynol (such as octoxynol-9 (Triton X-100) or t-octy iphenoxypoiyethoxyethanoi). a cetyl trimethyl ammonium bromide (CTAB'), or sodium deoxycholate, particularly for a split or surface antigen vaccine. The detergent may be present only at trace amounts. Thus the vaccine may include less than Img/mIl of each of octoxynol-l0 and 15 polysorbate 80. Other residual components in trace amounts could be antibiotics (eg. neomycin, kanamycin, polymyxin B). A vaccine composition may include material for a single immunisation or may include material for multiple imunisations (ie a 'muitidose' kit) The inclusion of a preservative is preferred in multidose arrangements, As an alternative (or in addition) to including a preservative in multidose 20 compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material Influenza vaccines are typically administered in a dosage volume of about0.5m1, although a half dose( about 0.25m) may be administered to chicken. Compositions and kits are preferably stored at between 2 0 C and 8 0 C. They should not be frozen. 25 They should ideally be kept out of direct light. Host cell DNA Where virus has been isolated and/or grown on a cell line, it is standard practice to minimize the amount of residual cell line DNA in the final vaccine, in order to minimize any potential oncogenic activity of the DNA. 30 Thus a vaccine composition prepared according to the invention preferably contains less than l0ng (preferably less than Ing, and more preferably less than I00pg) of residual host cell DNA per dose although trace amounts of host cell DNA may be present. It is preferred that the average length of any residual host cell DN A is less than 500bp e g. less than 400p, less than 300bp, less than 200bp, less than 100bp, etc, 20 Contaminating DNA can be removed during vaccine preparation using standard purification procedures e.g chromatography, etc. Removal of residual host cell DNA can be enhanced by nuclease treatment e.g. by using a DNase. A convenient method for reducing host cell DNA contamination is disclosed in references 43 & 44, involving a two-step treatment, first using a DNase 5 (e.g. Benzonase), which may be used during viral growth, and then a cationic detergent (e.g. CTAB), which may be used during vision disruption. Treatment with an alkylating agent, such as 9-propiolactone, can also be used to remove host cell DNA, and advantageously may also be used to inactivate virions [45]. Adjuvans 10 Compositions of the invention may advantageously include an adjuvant, which can function to enhance the immune responses (humoral and/or cellular) elicited in a subject who receives the composition. Preferred adjuvants comprise ol-in-water emulsions. Various such adjuvants are known, and they typically include at least one oiH and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and bhiocompatibie. The oil droplets in the 15 emulsion are generally less than 5pm in diameter and ideally have a subaminron diameter, with these small sizes being achieved with a nicrofluhdiser to provide stable emulsions. Droplets with a size less than 220mn are preferred as they can be subjected to filter sterilization. The emulsion can comprise oils such as those from an animal (such as fsh) or vegetable source Sources for vegetable oils include nuts, seeds and grains. Peanut ol, soybean oil, coconut oil, and 20 olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used eg. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like in the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used. 6-10 carbon fatty acid esters of glycerol and 1,2-propanedioi, while not occurring naturally in seed oils, may be 25 prepared by hydrolysis, separation and esterifcation of the appropriate mariaials starting from the nut and seed ols, Fats and oils from mammalian milk are metabolzahle and may therefore be used in the practice of this invention. The procedures for separation, purifiation, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art. Most fish contain metabolizabie oils which may be readily recovered, For example cod liver oil, shark liver oils, and 30 whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are general referred to as terpenoids. Shark liver oil contains a branched, unsaturated terpenoids known as squalene, 2,6,10)5,1 I23Ahexamethyi-2610,14, 182-tetracosahexaene, which is particularly preferred herein. Squalane, the saturated analog to squalene, is also a preferred oil. Fish ois, 35 including squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art Another preferred oil is a-tocopherol (see below). Mixtures of oils can be used. 21 Surfactants can be classified by their nHLB (hydrophile/lipophile balance). Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16. The invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 5 80; copolymers of ethylene oxide (BO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM tradename, such as linear E/PO block copolymers; octoxynois, which can vary in the number of repeating ethoxy (oxy-,2ethanedyl) groups with octoxynol-9 (Triton X-100' or t-.octyiphenoxypolyethoxyethanol) being of particular interest; (octyiphenoxy)poiyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidyicholine (lecithin); nonylphenol 10 ethoxylates, such as the TergitolMA NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Non-ionic surfactants are preferred. Preferred surfactants for including in the emulion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate) 15 lecithin and Triton X-100, Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. A combination of a polyoxyethyiene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanoi (Triton X-100) is also suitable. Another useful combination comprises laureth 9 pius a polyoxyethyiene sorbitan ester and/or an octoxynol. 20 Preferred amounts of surfactants (% by weight) are: polyoxyethyiene sorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1 %; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1 %t, in particular 0.005 to 0.02%; poy oxyethylene ethers (such as laureth 9) 0.1 to 20 %, preferably 0.1 to 10 % and in particular 0.1 to 1% or about 0.5%. 25 Where the vaccine contains a split virus, it is preferred that it contains free surfactant in the aqueous phase. This is advantageous as the free surfactant can exert a 'splitting effect' on he antigen, thereby disrupting any unsplit virions and/or virion aggregates that might otherwise be present. This can improve the safety of split virus vaccines 46]. Preferred emulsions have an average droplets size of lpm eg 750m <5O0nm, <400mn 30 300nm, I250nm, 220nm. 200nm, or smaller These droplet sizes can conveniently be achieved by techniques such as microtluidisation Specific oil-inwater emulsion adjuvants useful with the invention include, but are not limited to: A subinicron emulsion of squalene, Tween 80, and Span 85. The composition of the emulsion by volume can be about 5% squalene, about 0.5% poly sorbate 80 and about 0.5% Span 85. In 35 weight tenns. these ratios become 4 ,3% squalene 0.5% polysorbate80 and 0.48% Span 85. This adjuvant is known as 'MF59' [47-49], as described in more detail in Chapter 10 of ref 50 22and chapter 12 of ref 5i. The MF59 emulsion advantageously includes citrate ions e.g 10mM sodium citrate buffer "An emulsion comprising squalene, a tocopherol, and polysorbate 80. The emulsion may include phosphate buffered saline, These enulsions may have by volume from 2 to 10% 5 squalene, from 2 to 10% tocopherol and from 0.3 to3% polysorbate 80, and the weightratio of squaienettocopheroi is preferably <1 (g. 090) as this can provide a more stable emulsion, Squalene and polysorbate 80 may be present volume ratio of about 5:2 or at a weight ratio of about 11:5. Thus the three componems (squalene, tocopherol, polysorbate 80) may be present at a weight ratio of 1068:1186:485 or around 55:61:25. One such emulsion (AS03') can be 10 made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5g of DL a tocopherol and 5mi squalene), then microfluidising the mixture, the resuming emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250nm, preferably about i8Onm. The emulsion may also include a 3-de-O acylated monophosphoryl lipid A (3d MPL). Another useful emulsion of this type may 15 comprise, per hunan dose, 0.510 ng squalene, 0.511 mg tocopherol and 0.1-4 mg polysorbate 80 [52] e g. in the ratios discussed above. * An emulsion of squalene, a tocopherol, and a Triton detergent g Triton X-100). The emulsion may also include a 3d-MPL (see below). The emulsion may contain a phosphate buffer. 20 An emulsion comprising a polysorbate (e.g polsorbate 80), a Triton detergent (eg Triton X100 and a tocophero . an cheroi sucinate) The emulsion may include these three components at a mass ratio of about 75:11:10 (eg 750ptg/ml polysorbate 80, 110g/ml Triton X-100 and 100pg/ni o.tocopherol succinate) and these concentrations should include any contribution of these components from antigens. The emulsion may also include squalene, 25 The emulsion may also include a 3dMPL (see below). The aqueous phase may contain a phosphate buffer. * An emulsion of squalane, polysorbate 80 and poloxamer 401 ("PiuronicTM L121"). The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the 30 "SAF-" adjuvant [53] (0.05-1% Thr-MDP. 5% squalane, 2.5% Piuronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF" adjuvant [54] (5% squalane, 1.25% Pluronie L121 and 0.2% polysorbate 80) Microfluidisation is preferred. * An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g. polyoxyethyiene (12) cetostearyl ether) and a 35 hydrophobic nonionic surfactant (e.g a sorbhan ester or mannide ester, such as sorbitan monoleate or 'Span 80'). The emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm [55]. The emulsion may also 23 include one or more of: aldito ; a cYoprotectve agent (eg a sugar, such as dodecylmatoside and/or sucrose); and/or an aikylpolyglycoside. The emulsion may include a TLR4 agonist [56]. Such emulsions may be lyophilized. * An emulsion of squalene, poloxamer 105 and AbilCare [57] The final concentration (weight) 5 of these components in adjuvanted vaccines are 5% squalene, 4% poloxamer 105 (piuronic polyoi) and 2% Abi-Care 85 (Bis-PE/PPG-1616 PEG/PPG-1616 dimethicone; caprylic/capric triglyceride) * An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant As described in reference 58 preferred phospholipid components are 10 phosphatidyicholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyeiin and cardiolipin. Submicron droplet sizes are advantageous. o A submicron ol-in-water emulsion of a non-metabolisable oil (such as light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80). Additives may be included, such 15 as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0l00, described in reference 59, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyidioctadecylammonium bromide and/or NN-dioctadecyl--N,N-bis (2-hydroxyethyi)propariediamine * An emulsion in which a saponin (eg QuilA or QS21) and a sterol (e.g a cholesterol) are 20 associated as helical micelles [60] * An emulsion comprising a mineral oll, a non-ionic lipophilic ethoxylated ftity alcohol, and a non-ionic hydrophilic surfactant (e g. an ethoxylated fatty alcohol and/or poiyoxyethyiene polyoxypropylene block copolymer) [61] . * An emulsion comprising a mineral ol, a non-ionic hydrophilic ethoxylated fatly alcohol, and a 25 non-ionic lipophilic surfactant (e.g an ethoxylated fatty alcohol and/or polyoxyethylene polyoxypropylene block copoly mer) [61]. In some embodiments an emulsion may be mixed with antigen extemporaneously, at the time of delivery, and thus the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use. in other embodiment an emulsion is mixed 30 with antigen during manufacture, and thus the composition is packaged in a liquid adjuvanted forn. The antigen will generally be in an aqueous form, such that the vaccine is fmall prepared by mixing two liquids The volume ratio of the two liquids for mixing can vary (eg. between 5:1 and ) but is generally about 1:1. Where concentrations of components are given in the abov descriptions of specific emulsions, these concentrations are typically for an undiluted composition, and the 35 concentration after mixing with antigen solution will thus decrease. 24 Packaging of vaccine compositions Suitable containers for compositons of the invention (or kt components) include vials, syringes (eig. disposable syringes), nasal sprays, etcThese containers should be sterile. Where a composition/component is located in a vial, the vial is preferably made of a glass or plastic 5 material The vial is preferaby sterilized before the composition is added to it. To avoid problems with late-semitive patientsials are preferably sealed with a latex-free stopper, and the absence of latex in all packaging material is preferred. The vial may include a single dose of vaccineor it may include more than one dose (a Multidose , vial) eg. 10 doses. Preferred vials are made of colorless glass. 10 A vial can have a cap (eg a Luer lock) adapted such that a pre-ied syringe can be insered into the cap, the contents of the syringe can be expelled into the vial (eg. to reconstitute lyophilised material therein), and the contents of the vial can be removed hack into the syringe. After removal of the syringe from the vial, a needle can then be attached and the composition can he administered to a patient. The cap is preferably located inside a seal or cover such that the seal or cover has to be 15 removed before the cap can be accessed. A vial may have a cap that permits aseptic removal of its contents, particularly for nmltidose vials. Where a component is packaged into a syringe, the syringe may have a needle attached to it If a needle is not attached, a separate needle may be supplied with the syringe for assembly and use. Such a needle may be sheathed. Safety needles are preferred. 1-inch 23-gauge. 1-inch 25-gauge and 5/8 20 inch 25-gauge needles are typical Syringes may be provided with peel-off labels on which the lot number, influenza season and epiration date of the contents may he printed, to facilitate record keeping. The planger in the syringe preferably has a stopper to prevent the plunger from being accidentally removed during aspiration. The syringes may have a latex rubber cap and/or plunger. Disposable syringes contain a single dose of vaccine. The syringe will generally have a tip cap to seal 25 the tip prior to attachment of a needle, and the tip cap is preferably made of a butl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber shield. Preferred syringes are those marketed under the trade name "Iip-Lok"T. Containers may he marked to show a half-dose volume e.g to facilitate every to children. For instance, a spinge containing a 0.ma a 0.25m1 volume. 30 Where a glass container (e.g. a syringe or a vial) is used, then it is preferred to use a container made from a borosilicate glass rather than from a soda lime glass A kit or composition may be packaged (e.g. in the same box) with a leaflet including details of the vaccine e. instructions for administration, details of the antigens within the vaccine. et. The instructions may also contain warnings e.g. to keep a solution of adrenaline readily available in case 35 of anaphylactic reaction following vaccination. etc. 25 Methods of treatment and adnnistration ofthe vaccine The invention provides a vaccine manufactured according to the invention, These vaccine compositions are suitable for administration to human or non-human animal subjects, such as pigs or birds, and the invention provides a method of raising an immune response in a subject, comprising 5 the step of administering a composition of the invention to the subject. The inventon also provides a composition of the invention for use as a medicament, and provides the use of a composition of the invention for the manufacture of a medicament for raising an immune response in a subject. The immune response raised by these methods and uses will generaly include an antibody response, preferably a protective antibody response. Methods for assessing antibody responses, neutralising 10 capability and protection after influenza virus vaccination are well known in the art Human studies have shown that antibody tigers against hemagglutinin of human influenza virus are correlated with protection (a serum sample hemagglutination-inhibition titer of about 30-40 gives around 50% protection from infection by a homologous virus) [62], Antibody responses are typically measured by hemaggutunation inhibition, by microneutralisation, by single radial immunodiffusion (SRID), 15 and/or by single radial hemolysis (SROH). These assay techniques are well known in the art. Compositions of the invention can be administered in various ways. The most preferred immunization route is by intramuscular injection (e.g. into the arm or leg), but other available routes include subcutaneous injection, intranasal [63-65], oral [66], intradermal [67,68], transcutaneous, transdermal [69], etc. 20 Vaccines prepared according to the invention may be used to treat both children and adults. Influenza vaccines are currently recommended for use in pediatric and adult immunization, from the age of 6 months, Thus a human subject may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred subjects for receiving the vaccines are the elderly (e1g. 50 years old, >60 years old, and preferably >65 years), the young (e.g 5 years old), hospitalized 25 subjects, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, immunodeficient subj sub, subjects who have taken an antiviral compound (e.g an oseltamivir or zanamivir compound; see below) in the 7 days prior to receiving the vaccine, people with egg allergies and people travelling abroad. The vaccines are not suitable solely for these groups, however, and may be used more generally in a population. For pandemic strains, administration to all 30 age groups is preferred. Preferred compositions of the invention satisfy 1, 2 or 3 of the CPMP criteria for efficacy In adults (18-60 years), these criteria are: (i) >70% seroprotection; (2) 40% seroconversion; and/or (3) a GMT increase of 25-fol. in elderly (>60 years), these criteria are: (1) >60% eropotection; (2) 130% seroconversion: and/or (3) a GMT increase of >2-fold. These criteria are based on open 35 label studies with at least 50 patients. 26 Treatment can be by a single dose schedule or a multiple dose schedule. Muliple doses may be used in a prrnary immunisation schedule and/or in a booster nmunisation schedule. In a mulple dose schedule the various doses may be given by the same or different routes eg a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, et. Administration of more than one dose 5 (typically two doses) is particularly useful in immunologically naive patients e g for people who have never received an influenza vaccine before, or for vaccinating against a new HA subtype (as in a pandemic outbreak). Multiple doses will typically be administered at least I week apart (eg about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about X weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). 10 Vaccines produced by the invention may be administered to patients at substanially the same time as (e.g. during the same medical consultation or visit to a healhhcare professional or vaccination centre) other vaccines e.g at substantially the same time as a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a conjugated Hinfluenzae type b vaccine, an inactivated 15 poliovirus vaccine, a hepatitis B virus vaccine, a meningococcal conjugate vaccine (such as a tetravalent A-C-W35 vaccine), a respiratory syncytial virus accine, a pneumococCal conjugate vaccine, etc. Administration at substantially the same time as a pneumococcal vaccine and/or a meningococcal vaccine is particularly useful in elderly patients. Similarly, vaccines of the invention may be administered to patients at substantially the same time as 20 (ega during the same medical consultation or visit to a heaIthcare professional) an antiviral compound, and in particular an antiviral compound active against influenza virus (eg oseltamivir and/or zanamivirW These antivirals include neuraminidase inhibitors, such as a (3R4R.5S)i acetylamino-5-amino-3( 1-ethyipropoxy)-1 -cyciohexene- I-carboxylic acid or 5-(acetyiamino)-4 [(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-giycero-D-galactonon-2-enonic acid, 25 including esters thereof (e.g the ethyl esters) and salts thereof (e g. the phosphate salts). A preferred antiviral is (3R,4R,58S)-4-acetylamino-5~amino~3 (1 -ethylpropoxy)-1 I-cyclohexene- I-carboxylic acid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate (TAMIFLUJTM). General 'The term "comprising" encompasses "including" as well as "consisting" e.g, a composition 30 "comprising" X may consist exclusively of X or may include something additional e.g X + Y. The word "substantially" does not exclude "completely" e.g a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention. The term "about" in relation to a numerical value x is optional and means, for example, xti0%. 35 Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing, Thus components can be mixed in any order Where there are 27 three components then two components can be combined with each other and then the combination may be combined with the third component, etc. The various steps of the methods may be carried out at the Ssame or different tines, in the same or different geographical locatons, egg. countries and by the same or different people or entities. 5 Where anirnal (and particularly bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encephalopathies (SEs) and in partcular free from bovine spongiform encephalopathy (BASE). Overall, it is preferred to culture cells in the total absence of anima-derived materials. Where a compound is administered to the body as part of a composition then that compound may 10 alternatively be replaced by a suitable prodrug. References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences, This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 718 of reference 70. A preferred alignment is 15 determined by the Smith--Waterman homology search algorithm using an affne gap search with a gap open penalty of 12 and a gap extension penaly of 2, BLOSUM matrix of 62. The Smith Waterman homology search algorithm is taught in reference 71. References to a percentage sequence identity between two nuleic acid sequences mean that, when aligned, that percentage of bases are the same in comparing the two sequences. This alignment and 20 the percent homology or sequence identity can be determined using software programs known in the art for example those described in section 7718 of reference 70. A preferred alignment program is GCG Gap (Genetis Computer Group. Wisconsin, Suite Version 10.1), preferably using default parameters, which are as follows: open gap = 3; extend gap = 1, BRIEF DESCRIPTION OF THE DRAWINGS 25 Figure 1 ilustrates virus titers (by Focus-Formation assay FFA); Figure 1A) and iA titers (by Red Blood Cell 1emagglutination assay; Figure 1B) at different times postAinfection of wt PRS and PR8-X viruses grown in MDCK cells. The solid liin Figure lA and hatched columns in Figure 1B represent resuls wih wild-type PRS. The dotted line in Figure IA and empty columns in Figure 1B represent resuls with wild-type PRS-'X The x-axis shows the hours post infection and the y-axis in 30 figures IA and 1B shows the virus titer (U/mi) and HA litre, respectively. Figure 2 illustrates virus titers (by FFA; Figure 2A) and HA titers (by Red Blood Cell Hemagglutination assay; Figure 2B) at different times post-infection of reverse genetics derived PR8 and PR8- viruses grown in MDCK cels. The solid line in Figure 2A and hatched columns in Figure 2B representresults with PR8. The dotted line in Figure 2A and empty columns in Figure 2B 28 represent results with RO-derived PR8-X. The a-axis shows the hours post infection and the y-axis in figures 2A and 2B shows the virus titer (IU/ml) and -A titre, respectively. Figure 3 compares virus tigers (by FFA; Figure 3A) and HA titers (by Red Blood Cell Hemagglutination assay; Figure 3B) at different times post-infection in MDCK cells of reverse 5 genetics-derived 6:2 reassortant viruses made with either PR8 or PRS-X backbone segments which contain the H A and NA segments from PRS-X. The solid line in Figure 3A and hatched columns in Figure 3D represent results with the PRS backbone. The dotted line in Figure 3A and empty columns in Figure 3B represent results with the PR8-X backbone. The x-axis shows the hours post infection and the y-axis in figures 3A and 3B shows the virus titer (lU/mi) and I-IA titre, respectively. 10 Figure 4 compares virus titers by FFA (Figure 4A) and HA tigers (by Red Blood Cell -emagglutination assay; Figure 41) at different times post-infection in MDCK cells of reverse genetics-derived 6:2 reassortant viruses made with either wt PRS or PR8-X backbone segments which contain the HA and NA segments from a pandemic HIl strain (strain ). The solid line in Figure 4A and hatched columns in Figure 4B represent results with the wt PR8 backbone. The 1 dotted ine in Figure 4A and empty columns in Figure 41 represent results with the PRs-X backbone. The x-axis shows the hours post infection and the y-axis in figures 4A and 4B shows the virus titer (I/mi) and HA titre, respectively Figure 5 compares virus titers by a focus-fonnation assay (FFA) (Figure 5A,) and HA titers (Figure 5B) at different times post-infection in MDCK cells of reverse genetics-derived 6:2 reassortant 20 viruses made with either PRS or PR8-X backbone segments which contain the HA and NA segments from 105p30. The solid line in Figure 5A and hatched columns in Figure 5B represent results with the wt PR8 backbone. The dotted line in Figure 5A and empty columns in Figure 5B represent results with the PR8-X backbone. The x-axis shows the hours post infection and the y-axis shows the virus titer (U/mi). 25 Figure 6 illustrates virus titers by a focus-formation assay (FFA) at different times post-infection of wild-type PR8-X and 105p30 viruses (Figure 6A) or reverse genetics-derived PR8-X and 105p30 viruses (Figure 63B) grown in MDCK cells. In Figures 6A and 61B the solid lines represent results with 105pS. The dotted lines represent results with PRSX The x-axis shows the hours post infection and the y-axis in fgures 6A and 613 shows the virus titer (i/nil) and HA litre, respectively. 30 Figure 7 shows the growth characteristics of reassortant viruses containing the backbone segments of the wt PR8 strain (line with triangles) or 105p30 strain (ine with squares) and the HA and NA segments of a pandemic 1 infuenza strain (strain 2). ihe x-axis in figures 7A and 7B indicates the hours postifection. The y-axis in Figure 7A shows the titreLogl0 in FFU per L The y-axis in Figure 7B3 shows the titre logl in virus particles per mL. 35 Figure 8 compares virus tigers by a focus-formation assay (FFA) at differed times post-infection in MDCK cells of reverse genetics-derived 6:2 reassorant viruses made with either 105p30 or PRS-X 29 backbone segments which contain the H A and NA segments from (A) a H i strain (strain 1) or (B) a pandemic HI strain (strain 2). The solid lines represent results with the 105p30 backbone. The dotted lines represent results with the PR-X backbone. The x-axis shows the hours post infection and the y-axis shows the virus titer (IUmi). 5 Figure 9 compares virus titers by a focus-formation assay (FFA) at different times post-infection in MDCK cells of reverse genetics-derived 6:2 reassortant viruses made with either the #17, #19, or PR8-X backbone in combination with the HA and NA segments from (A) a pandemic Hi strain (strain 3) or (B) a H-3 (strain 1). in Figures 9A and 913, the dotted lines with the circle markers represent results with the #17 backbone. The solid lines with diamond markers represent results with 10 the #19 backbone, The dotted lines with square markers represent results with the PR8-X backbone. The x-axis shows the hours post infection and the y-axis shows the virus titer (lU/mi). Figure 10 compares virus titers by a focus-formation assay (FFA) at different times post-infection in MDCK cells of a panel of different reverse genetics-derived 6:2 reassortant viruses made with either the chimeric #19 or PR8-X backbone plus the HA and NA segments from the following strains: (A) a 15 pandemic HI strain (strain 2), (B) a pandemic Hi strain (strain 4), (C) a H1 strain (strain 2), (D) a HI strain (strain 3), or (E) a H3 strain (strain 2). In Figures 10A-E, the solid lines with the triangle markers represent results with the #19 backbone. The dotted lines with square markers represent results with the PR8-X backbone, The x-axis shows the hours post infection and the y-axis shows the virus titer (ili). 20 Figure 11 compares HA yields (by lectin-capture ELISA) at 60hr post-infection in MUCK cells of different 62 reassortant viruses made with either the chimeric #19 (empty colunns) or PIR8-X backbone (solid colunms) plus the HA and NA segments from the following strains: (A) a pandemic 1H1 strain (strain 2, (B) a pandemic Hi strain (stmin 4 (C) a 113 strain (strain 1) or (D) a H3 strain (strain 2). Corresponding 6:2 reassortant viruses made by classical reassortment (classical") wih the 25 wt PR8 backbone were included as control (hatched couimns). The y-axis shows the HA content in pg per ml Figure 12 shows the growth curves of reassortan influenza viruses comprising backbones 17, 18, 19 and 20 (as shown in table 1; line with diamonds, squares, triangles and crosses, respectively), a control comprising the same HA and NA segments from a H3 influenza strain (strain 1) but all 30 backbone segments from PR8-X (line with circles) and the equivalent widtype strain (line with plus sign). The x axis indicates the hours post infection (hpi) and the y-axis shows IU/mL. Figure 13 shows the growth curve of reassortant influenza viruses comprising backbones 17 and 19 (line with diamonds and triangles, respectively) and the HA segments from a H3 influenza strain (strain 3), a control comprising the same -HA and NA segments but all backbonesegments from PR8 35 X (line with plus sign) and the equivalent wiidtype strain (line wth circles 30 Figure 14 shows the results of a FFA (14(A) and 14 (C)) and HA-ELISA (14(B) and 14 (D) assay using reassortant influenza viruses comprising backbone 19 (open box), PR8-X backbone (hatched box) and the wildtype influenza virus (dotted box) . Figures I(A) and 14(B) show the results with a HI influenza strain (strain 2) and figures 14(C) and (D) show the results with a 3 influenza virus 5 strain. The y axis in figures 14(A) and (C) indicates the virus titre in U/nmL and the y axis in figures 14(1) and 14 (Dindicates HA in pg/nL Figure 15 is an alignment of the Mi viral segment of AINew Caledonia2/99 (SEQ ID NO: 33) and 105p30 (SEQ ID NO: 45), MODES FOR CARRY NG OUT THE INVENTION 1Development f'new dor strains In order to provide hiyggowh donor strain, the dono r strain, A/Puerto RiO/8!3 is passaged in. MDCK 33016 cel five mes. Using this method, the inventors were able to obtain the strain PRX which shows improved growth characteristics compared with the original strain, The 105p30 influenza donor strain was provided by isolating an A'New Caledonia/20/1999 influenza 15 virus from a clinical isolate in MDCK 33016 cells and massaging the virus 30 times. The resulting strain has a M segment with lysine in the position corresponding to amino acid 95 of SEQ ID NO: 33 when aligned to SEQ ID NO: 33. Growth characteristis of wt PRS and PR8-X viruses In order to compare the growth characteristics of PRS-X and wt PR8 donor stains, the viral twe of 20 these virus strains is measured in MDCK cells by focus-forming assays and hemaggiutiation assays. Focus-Forming Assays (FF4,) For the FFA, uninfected MUCK cells are plated at a density of I .8x10 4 cells/well in 96 well plates in 100 ul of IMEM with 10% FCS. The next day, medium is aspirated and cells are infected with viruses in a volume of 0pi (viruses diluted in UMEM + 1% CS). The cells are incubated at 37C 25 until the next day. At several time points after infection, the medium is aspirated and the cells washed once with PBS. 50pI of ice-cold 50%/50% acetone-methanol is added to each well followed by incubation at -20 0 C for 30 minutes, The acetone mix is aspirated and the cells washed once with PBST (PBS + 0.1% Tween). 50p of 2% BSA in PBS is added to each well followed by incubation at room temperature 30 (RT) for 30 minutes. '50p of a 1:6000 dilution of anti-NP is added in blocking buffer followed by incubation at RT for I hours. The antibody solution is aspirated and the cells washed three times with pBSTI,, Secondary antibody (goat anti mouse) is added at a dilution 1:2000 in 50p n blocki buffer and the plate is incubated at RT for 1 hours, The antibody solution is aspirated and the cells washed three times with PBST. 50p of KPL True Blue is added to each well and incubated for 10 minutes. 31 The reaction is stopped by aspirating the True-Blue and washing once with d11 2 O. The water is aspirated and the cells are left to dry. The results (Figurei ) show that the PR.-X strain can grow to higher tires in the same time frame compared to the wt PR8 strain from which it is derived. 5 Growth characteristics of reassortant viruses containing PRS-X or wt PRS backbones In order to test the suitability of the PRSX strain as a donor strain for virus reassortment, reassortant viruses are produced by reverse genetics which contain the HA and NA proteins from a pandemic Hi strain and the other viral segments from either PRS-X or PR8. The viral tires of these reassortant viruses are determined by FFA and HA assays as described above. The results are shown in Figure 4. 10 The results indicate that reassonant viruses which contain viral segments from PRS-X grow faster in MDCK cells compared to reassortant viruses containing viral segments from the PR8/34 strain. Growth characteristics of05p3O strain compared with PR8-X MDCK cells are infected with 105p30 and PR8-X at a moi of io- and samples are taken at several time points aner infection. The titre is determined by a FFA assay. The results show that lO5p30 15 grows even faster in MDCK cells compared to P8-X (Figure 6). Growth characteristics of reassortant viruses containing 105p30 or wt PRS backbones in order to test the suitability of the 10 5 p 3 0 strain as a donor strain for virus reassortment, reverse genetics is used to produce reassortant viruses that contain the HA and NA segments from a pandemic HIl influenza strain and the backbone segments either from the 105p3O or the wt PRS 20 strain MDCK eis are infected with the reassortant viruses at a moi of IQ and samples are taken 1 hour, 12 hours 36 hours and 60 hours after infection. The titres are determined either by focus forming assays or by determining the virus parties by real-time detection PCR. The reassortant viruses that contain the backbone segments from the 105p30 strain grow faster than the viruses that are reassorted with the backbone segments of the wt PR8 strain. This shows that the 105p30 strain is 25 a good donor strain for producing fast-growing reassortant viruses (Figure) Rescue of influenza viruses using backbone segments rom two donor strains The rescue efficiency of reassortant influenza viruses containing the HA and NA segments from a H3 influenza virus and backbone segments from the 105p30 and the PR8-X donor strains is tested in MDCK cells. The reassortant infuenza viruses contain backbone segments of the 105p30 and the 30 PR8-X donor strains as indicated in the following table: Table I Backbone # Pil PB2 PA NP M NS PRS-X IPRS-X PRS-X 10p3o 105p30 32 PRS-X PRS-X 3lR- R- 05p30 10p3 PR8-Xn 105p 3 0) 4 jR- PR8-X i05p30 1050 105p30 PR8-X' PR8S-X 105p30 PRtS-X PR8-X' 1 05p30 Ji0p3 6 PRtS-X 105p30 PR8S-X 105p30 PR8-XN 105p30 7PR8S-N 105p3 PRtS-X 1050 105p30 PRt8-X 8 IPR8S-N i05p30 105 p30 PR8S-X PR8S-X IO5p30 - - - --- ------------------ ------------ ---------- 05#0I PRSX MY60 PR8 XXN 12 ~ t- j0p3 PR8-X PR8-X 105p30 PR8-X0 105p3 -mI 13 p0 PR 8-XXR8- 105p30 l~3 10 5p30 P R8- Pt-X 14 105p30 PRS-X 10p30SN PRS-X 105p30 PR 12 10530 PRS-X 100 P PRS-X 105p30 16 105p30 PRS-N SX 105p0 150 PR8-X PR8S-X 17 105p30 P05p30 PR8-X PRtS-X PR8-X 10tS30 18 105p30 105p30 iOP8- PR8-XN P0tS30 10N3 PwtS-N ----- 430 19 105p30 105p30 PRtS~X 105p30 PR8-XN PRtS-X 20 105p-0 10-30 105p30 P8-X PR8-X Reassortant influenza viruses which contain a backbone according to number 3, 4, 10, 11, 14 and 16 20 are rescuable. Influenza viruses which contain backbones number 3, 4, 10, 11i or 16 achieve viral titres of less than 102 I/ni Influenza viruses containing backbone numbers 17 and 18 achieve viral 5 titres between 102 and 106 !U/mL and influenza viruses having backbone umbers 19 and 20 even achieve titres of more than 106 lU/mU These data show that influenza viruses in which the P131 and PB32 segments come from the same influenza donor strain can show a higher rescue efficiency compared with influenza viruses in which these segments come from different influenza donor strains. 3 ------ Isx PR33G Growth characteristics of reassurtant influenza viruses containing backbone segments from two donor stains Reassortant influenza strains are created which contain backbone numbers 17, 18, 19 and 20 (as shown in table I above) and the HA and NA segments from a H3 influenza strain strainn 1. As 5 controls, the equivalent wildtype 113 influenza virus, and a reassortant influenza viruskcomprising the same 1HA and NA segments and all backbone segments from PR8-X are used. Furthermore reassortant influenza strains are produced which contain backbone numbers 1 and 19 and the HA and NA segments from either a second H3 influenza (strain 1) virus or a pandemic Hl influenza virus (strain 3) As controls for the 113 strain, the equivalent wildtype H3 (strain 2) 10 influenza virus, and a reassortant influenza virus comprising the same HA and NA segments and all backbone segments from PR8-X is used. For the pandemic Hi influenza virus a reassortant influenza virus comprising the same HA and NA segments and all backbone segments from PR8-X is used. The reassortant influenza viruses and the control viruses are grown in MDCK cells and the viranitre is measured by FFA at different time points. For the reassortant 113 viruses (strain 1) containing 15 backbones 17, 1|9 and 20, and the H3 influenza viruses (strain 3) containing backbones 17 and 19, the influenza viruses containing backbone segments from two donor strains grow to higher tires compared with the wildtype virus and the reassortant virus which contains backbone segments from only a single donor strain (see figures i1 and 12 For the pandemic Hi infhluenza irus, the reassortant influenza strains containing backbones 17 and 20 19 grow to higher tires compared with the control which contained all backbone segments from PR8X (see figure 9). The data show that reassortant influenza viruses which contain backbone segments from two different donor strains can show improved growth rates compared with reassortant influenza viruses which contain backbone segments from only a single donor strain. 25 [he experiments were also repeated using reassortant inuenza viruses which contain backbone 19 or the backbone segments from PRS-X in combination with the HA and NA segments from four different H1 strains or a 113 strain The results are shown in Figure 10. Reassortant influenza viruses with backbone segments from two different donor strains give higher yields 30 To test whether reassortant influenza viruses containing backbone segments from two different influenza donor strains can also provide higher yields, the HA yield of the reassortant strains is tested bHy AESA. To this end, the same reassortant influenza viruses as described above containing backbone # 19 and the HANA segments of the 113 (strain 2) and Hi influenza strains are used. As controls, the equivalent wildtype influenza viruses and reassortant infuenza viruses comprising the 34 same HA and NA segments and all backbone segments from PR8-X are used. In addition, the viral thres are confirmed with a FFA assay. The results confrm that the reassortant influenza strains which contain backbone segments from two different donor strains can grow to higher yields compared with influenza viruses which contained all 5 backbones from PR8-X (see figures 13 (A) and (C)), Furthermore, reassortant infuenza viruses comprising backbone segments from two donor strains also give higher HA yields (see figures 13(B) and (D)). These data show that reassortant influenza viruses which contain backbone segments from two donor strains give higher yields compared with reassortant influetnza vrsswihconti bl'akbon-e 10 segmentsfo only asigednrtans t 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. 15 REFERENCES [I] W02007/002008 [2] WO2007/124327 [3] W02010/070098 [4] Needieman & Wunsch (1970) J. Mol. Biol. 48, 443-453. [5] Rice et al. (2000) Trends Genet 16:276-277. [6] Herlocher et al. (2004) Jnfect Dis 190(9):1627-30. [7] Le et al. (2005) Nature 437(7062): 1108. [8] US-6468544. [9] Neumann et al, (2005) Proc Nati Acad Sci USA 102: 1682599 [10] W02009/00891 [11] US provisional application no. 61/273.151 [12] Sambrook et al, Molecular Cloning: A Laboratory Manual 2 cd, 1989, Cold Spring Harbor Press, Cold Spring Harbor, N. Y [13] W02011/012999 [14] Kistner et aL (1998) Vaccine 16:960-8. [15] Kistneretal. (1999) Dev Biol Stand 98:101-110. [16] Bruhi et al. (2000) Vaccine 19:1149-58. [17] Paua e (2001) Vaccine 19:2716-21. [1 8] http://www.atcc.org/ [19] http://1ocus.umdnj~edu/ [20] WO97/37000. [21] Brands et al (1999) Dev Biol Stand 98:93-100, [221 Halperin et al, (2002) Vaccine 20:1240-7.
[23] EP-A-1260581 (WO0i/6484+6) [241 WO2006/07 1563 [25] W02005i 13758 [26] WO97/37001 [27] WO02/28422. [28] WO02/067983. [29] WO02/074336. [30] WOOi/21151, [31] WO2/097072. [321 W02005/113756. [331 Huckriede et a. (2003) Methods Enzvmol 373:74-91. [34] Vaccines. (eds. Piotkins & Orenstein). 4th edition, 2004, ISBN: 0-7216-9688-0 [35] Treanor et a/. (1996)JnfectDis 173:1467-70. [36] Keitel et al (1996) Clin Diagn Lab Imunol 3:507-10. [37] Herlocher et al. (2004) Jnfect Dis 190(9):1627-30. [38] Le eta. (2005) Nature 437(7062):1 108. [39] WO2008/06863 1. [401 Gennaro (2000) Remington: The Science and Pracice qi Pharmacy. 20th edkion, ISBN: 0683306472. [41] Banzhoff (2000) Immunoogy Letters 71:91-96. [42] Nony etal. (2001) Vaccine 27:3645-51L [43] EP-B-0870508. [44] US 5948410. [45] W02007/052163. [46] W02007/052061 [47] WO90/14837. [48] Podda & Del Oiudice (2003) Expert Rev Vaccines 2:197-203. [49] Podda (2001) Vaccine 19: 2673-2680. [50] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X). [51] Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan. [52] W02008/043774, [53] Allison & Byars (1 992).Res Immunol 143:519-25. [54] Hariharan etal. (1995) Cancer Res 55:3486-9. [55] US-2007/014805. [56] US-2007/0191314. [57] Sub et a. (2004) Vaccine 22(25-26):3464-9. [58] W095/11700. [59] US patent 6,080,725. [60] W02005/097181. [61] WO02006/1 13373. [62] Potter & Oxford (1979) Br Med Bull 35: 69---75. [63] Greenbaumn et a. (2004) Vaccine 22:2566-77. [64] Zurbriggen et a. (2003) Expert Rev Vaccines 2:295-304, [65] Piascik (2003) JAm Pharm Assoc (Wash DC,). 43:728-30. 36 [66 Mann et al. (2004) Vaccine 22:2425-9. [67] Haiperin et al (1979) Am JPulMic ikath 69:1247-50. [68] Herbert e al. (1979) infectDis 140:234--8. [69] Chen at at (2003) Wcine 21:2830-6 [70] Current Protocols in Moiecuiar Biology (F.M. Ausubel et at, eds, 1987)Supplement 30 [71 Smth & Waterman (1981) Adv. App. Math. 2: 482-489.

权利要求:
Claims (40)
[1] 1. A reassortant influenza A virus comprising backbone segments from two or more donor strains, wherein the PB 1 and the PB2 segments are from the same donor strain.
[2] 2. A reassortant influenza A virus comprising backbone segments from two, three or four donor 5 strains, wherein each donor strain provides more than one backbone segment.
[3] 3. A reassortant influenza A virus comprising backbone segments from two or more donor strains, wherein the PB1 segment is not from the A/Texas/1/77 influenza strain.
[4] 4. A reassortant influenza A virus comprising backbone segments from two or more donor strains, wherein at least the PA, NP, or M segment are not from A/Puerto Rico/8/34. 10
[5] 5. The reassortant influenza A virus of any one of claims 2 to 4, wherein the PB1 and the PB2 segment are from the same donor strain.
[6] 6. The reassortant influenza A virus of any preceding claim wherein the influenza A virus comprises a M genome segment which has lysine in the position corresponding to amino acid 95 of SEQ ID NO: 33 when aligned to SEQ ID NO: 33 using a pairwise alignment algorithm. 15
[7] 7. An influenza donor strain, wherein the donor strain is 105p3O.
[8] 8. An influenza A strain in which the M genome segment has lysine in the position corresponding to amino acid 95 of SEQ ID NO: 33 when aligned to SEQ ID NO: 33 using a pairwise alignment algorithm.
[9] 9. The influenza A strain of claim 8, wherein the influenza A strain is a HiN1 strain. 20
[10] 10. A reassortant influenza A virus comprising at least one backbone viral segment from a donor strain, wherein the donor strain is selected from the group consisting of 105p3O and PR8-X.
[11] 11. The virus of claim 9 wherein the at least one backbone viral segment has a sequence having at least 95% identity with a sequence selected from the group consisting of SEQ ID NOs 11-14 or SEQ ID NOs 18-22. 25
[12] 12. The virus of claim 9 wherein the at least one backbone viral segment has the sequence of SEQ ID NO: 17 or SEQ ID NO: 20.
[13] 13. The virus of any one of claims 9 to 12, wherein the virus comprises backbone segments from two or more donor strains.
[14] 14. The virus of claim 13, wherein the PB1 and the PB2 viral segments are from the same donor 30 strain.
[15] 15. The virus of claim 11, wherein the PB1 and PB2 viral segments have at least 95% identity with the sequences of SEQ ID NOs: 18 and 19. 38
[16] 16. The virus of claim 15, wherein the virus further comprises a viral segment having at least 95% identity with a sequence selected from the group consisting of SEQ ID NOs 17-22.
[17] 17. A method of preparing a reassortant influenza A virus comprising steps of (i) introducing into a culture host one or more expression construct(s) which encode(s) the 5 viral segments required to produce an influenza A virus wherein the backbone viral segments are from two or more influenza strains and the PB 1 and PB2 segment are from the same donor strain; and (ii) culturing the culture host in order to produce reassortant virus.
[18] 18. The method of claim 17 wherein the expression construct(s) encode(s) the backbone segments of 10 two or more influenza donor strains.
[19] 19. A method of preparing a reassortant influenza A virus 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 backbone viral segments are from two or more influenza strains; and 15 (ii) culturing the culture host in order to produce reassortant virus; wherein the expression construct(s) do/does not encode the PB1 segment from the A/Texas/i/77 influenza strain.
[20] 20. A method of preparing a reassortant influenza virus comprising steps of (i) introducing into a culture host one or more expression construct(s) which encode(s) the viral 20 segments required to produce an influenza virus wherein one or more backbone viral segment(s) is/are from a 105p30 and/or a PR8-X influenza strain and wherein at least one viral segment is derived from a second influenza strain; and (ii) culturing the culture host in order to produce reassortant virus.
[21] 21. The method of any one of claims 17 to 20 wherein the at least one expression construct 25 comprises a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs 11-14 and 18-22.
[22] 22. The method of any one of claims 17 to 21, further comprising the step (iii) of purifying the reassortant virus obtained in step (ii).
[23] 23. The method of any one of claims 20 to 22 wherein the at least one viral segment from the second 30 influenza strain is the HA segment.
[24] 24. A method for producing influenza viruses comprising steps of (a) infecting a culture host with the reassortant influenza virus of claims 1-16; (b) culturing the host from step (a) to produce the virus; and optionally (c) purifying the virus obtained in step (b). 39
[25] 25. A method of preparing a vaccine, comprising steps of (a) preparing a virus by the method of claim 24 and (b) preparing vaccine from the virus.
[26] 26. The method of claims 24 or 25, wherein the culture host is an embryonated hen egg.
[27] 27. The method of claims 24 or 25, wherein the culture host is a mammalian cell. 5
[28] 28. The method of claim 27, wherein the cell is an MDCK, Vero or PerC6 cell.
[29] 29. The method of claim 28, wherein the cell grows adherently.
[30] 30. The method of claim 28, wherein the cell grows in suspension.
[31] 31. The method of claim 30, wherein the MDCK cell is cell line MDCK 33016 (DSM ACC2219).
[32] 32. The method of one of claims 25 to 31, wherein step (b) involves inactivating the virus. 10
[33] 33. The method of one of claims 25 to 32, wherein the vaccine is a whole virion vaccine.
[34] 34. The method of one of claims 25 to 32, wherein the vaccine is a split virion vaccine.
[35] 35. The method of any one of claims 25 to 32, wherein the vaccine is a surface antigen vaccine.
[36] 36. The method of any one of claims 25 to 32, wherein the vaccine is a virosomal vaccine.
[37] 37. The method of any one of claims 25 to 36, wherein the vaccine contains less than lOng of 15 residual host cell DNA per dose.
[38] 38. The method of any preceding claim wherein at least one of the influenza strains is of the HI, H2, H5, H7 or H9 subtype.
[39] 39. A 105p3O donor strain which does not comprise the influenza HA HI subtype.
[40] 40. An expression system comprising one or more expression construct(s) comprising the vRNA 20 encoding segments of a105p30 or PR8-X strain wherein the expression construct comprises at least one backbone viral segment from the 105p3O or PR8-X strain. 40

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

HU212924B|1989-05-25|1996-12-30|Chiron Corp|Adjuvant formulation comprising a submicron oil droplet emulsion|

---

WO1995011700A1|1993-10-29|1995-05-04|Pharmos Corp.|Submicron emulsions as vaccine adjuvants|

---

DE19612966B4|1996-04-01|2009-12-10|Novartis Vaccines And Diagnostics Gmbh & Co. Kg|MDCK cells and methods of propagating influenza viruses|

---

DE19612967A1|1996-04-01|1997-10-02|Behringwerke Ag|Process for the propagation of influenza viruses in cell culture, and the influenza viruses obtainable by the process|

---

TW570803B|1997-04-09|2004-01-11|Duphar Int Res|Influenza vaccine|

---

US6080725A|1997-05-20|2000-06-27|Galenica Pharmaceuticals, Inc.|Immunostimulating and vaccine compositions employing saponin analog adjuvants and uses thereof|

---

DK1098961T3|1998-06-12|2008-05-26|Andrej Y Dr Egorov|Genesically attenuated viruses that induce interferon|

---

AR032597A1|1999-09-24|2003-11-19|Smithkline Beecham Biolog|USE OF ANTIGENS OF THE VIRUS OF THE INACTIVATED FLU, IN THE PREPARATION OF VACCINES OF A SINGLE INTRANASAL DOSE; METHOD FOR THE PROPHYLAXIS OF DISEASES OR OF INFLUENCES IN PATIENTS; EQUIPMENT THAT INCLUDES SUCH VACCINES IN SINGLE DOSE AND METHOD FOR MANUFACTURING VACCINES FOR THE FLU FOR APLI|

---

JP4698112B2|2000-03-03|2011-06-08|一般財団法人化学及血清療法研究所|Cells capable of serum-free culture and suspension culture, and method for producing virus for vaccine using the cells|

---

GB0024089D0|2000-10-02|2000-11-15|Smithkline Beecham Biolog|Novel compounds|

---

EP1361890B1|2001-02-23|2011-03-30|GlaxoSmithKline Biologicals s.a.|Influenza vaccine formulations for intradermal delivery|

---

AR032575A1|2001-02-23|2003-11-12|Smithkline Beecham Biolog|USE OF AN ANTIGEN FLU PREPARATION FOR THE MANUFACTURE OF AN INTRADERMIC VACCINE OF THE FLU AND PHARMACEUTICAL CASE THAT INCLUDES SUCH VACCINE|

---

TWI228420B|2001-05-30|2005-03-01|Smithkline Beecham Pharma Gmbh|Novel vaccine composition|

---

MX342639B|2003-05-28|2016-10-07|Wisconsin Alumni Res Found|High titer recombinant influenza viruses for vaccines and gene therapy.|

---

PT1742659E|2004-04-05|2013-06-03|Pah Usa 15 Llc|Microfluidized oil-in-water emulsions and vaccine compositions|

---

WO2005113756A1|2004-05-14|2005-12-01|Glaxosmithkline Biologicals S.A.|Method|

---

CA2566858A1|2004-05-20|2005-12-01|Id Biomedical Corporation|Process for the production of an influenza vaccine|

---

CN103555670B|2004-12-23|2015-08-12|米迪缪尼有限公司|For the non tumorigenic MDCK cell system of virus multiplication|

---

US7691368B2|2005-04-15|2010-04-06|Merial Limited|Vaccine formulations|

---

RU2457250C2|2006-04-19|2012-07-27|Медиммуне, Ллк|Methods and compositions for expressing negative-sense viral rna in canine cells|

---

EP1917352A4|2005-06-21|2009-03-25|Medimmune Vaccines Inc|Methods and compositions for expressing a heterologous protease|

---

US8703095B2|2005-07-07|2014-04-22|Sanofi Pasteur S.A.|Immuno-adjuvant emulsion|

---

DK1951296T4|2005-11-01|2014-09-01|Novartis Vaccines & Diagnostic|Cell-derived viral vaccines with low levels of residual cell DNA|

---

CA2628158C|2005-11-04|2015-12-15|Novartis Vaccines And Diagnostics S.R.L.|Emulsions with free aqueous-phase surfactant as adjuvants for split influenza vaccines|

---

MX2009003325A|2006-10-12|2009-04-09|Glaxosmithkline Biolog Sa|Vaccine comprising an oil in water emulsion adjuvant.|

---

FR2896162B1|2006-01-13|2008-02-15|Sanofi Pasteur Sa|EMULSION OIL IN THERMOREVERSIBLE WATER|

---

CA2647985C|2006-03-31|2014-12-30|Warf-Wisconsin Alumni Research Foundation|High titer recombinant influenza viruses for vaccines|

---

EP2679240A1|2006-12-06|2014-01-01|Novartis AG|Vaccines including antigen from four strains of influenza virus|

---

EP2045323A1|2007-10-05|2009-04-08|Avir Green Hills Biotechnology Research Development Trade Ag|Linear expression constructs for production of influenza virus particles|

---

SG172220A1|2008-12-16|2011-07-28|Baxter Int|Production of influenza vaccines|

---

EP2233152A1|2009-03-24|2010-09-29|Avir Green Hills Biotechnology Research Development Trade Ag|High growth reassortant influenza A virus|

---

CA2769673A1|2009-07-31|2011-02-03|Novartis Ag|Reverse genetics systems|

---

JP2013507990A|2009-10-26|2013-03-07|ダブリュエーアールエフ−ウィスコンシンアラムナイリサーチファウンデーション|High-titer recombinant influenza virus with enhanced replication in Vero cells|

---

JP2014527799A|2011-08-26|2014-10-23|ダブリュエーアールエフ−ウィスコンシン アラムナイ リサーチ ファウンデーション|Influenza virus as a live attenuated vaccine having a mutant PB2 gene segment|JP2016506724A|2013-01-23|2016-03-07|ノバルティス アーゲー|Influenza virus reassembly|

---

US20140274806A1|2013-03-15|2014-09-18|Synthetic Genomics Vaccines|Influenza virus reassortment|

---

MX2015016755A|2013-06-06|2016-04-13|Novartis Ag|Influenza virus reassortment.|

---

RU2605317C1|2015-06-04|2016-12-20|Федеральное государственное автономное образовательное учреждение высшего образования "Новосибирский национальный исследовательский государственный университет" |ADAPTED PANDEMIC INFLUENZA VIRUS STRAINS A/Tomsk/273/2010-MA1, A/Tomsk/273/2010-MA2 AND A/Tomsk/273/2010-MA3 TO ASSESS THE EFFECT OF ANTIVIRAL DRUGS |

---

EP3313439A2|2015-06-26|2018-05-02|Seqirus UK Limited|Antigenically matched influenza vaccines|

---

HUE051783T2|2015-07-07|2021-03-29|Seqirus Uk Ltd|Method for quantifying immunogenic hemagglutinin|

---

WO2017040203A1|2015-08-28|2017-03-09|Yoshihiro Kawaoka|Generation of infectious influenza viruses from virus-like particles|

---

WO2018012498A1|2016-07-15|2018-01-18|一般財団法人阪大微生物病研究会|Reassortant influenza virus production method|

---

JP6826203B2|2017-08-28|2021-02-03|一般財団法人阪大微生物病研究会|Stepwise production method of rear sortant influenza virus|

法律状态:
2014-10-23| FGA| Letters patent sealed or granted (standard patent)|

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2014-11-20| SREP| Specification republished|

---

2017-07-20| PC| Assignment registered|Owner name: SEQIRUS UK LIMITED Free format text: FORMER OWNER(S): NOVARTIS AG |

优先权:

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申请号 | 申请日 | 专利标题

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US201261605922P| true| 2012-03-02|2012-03-02||

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US61/605,922||2012-03-02||

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US201261685766P| true| 2012-03-23|2012-03-23||

---

US61/685,766||2012-03-23||

---

PCT/EP2013/054227|WO2013087945A2|2012-03-02|2013-03-02|Influenza virus reassortment|AU2014203051A| AU2014203051C1|2012-03-02|2014-06-04|Influenza Virus Reassortment|

---

AU2016203397A| AU2016203397A1|2012-03-02|2016-05-24|Influenza Virus Reassortment|