Complementing cell lines

Abstract

A packaging cell line that complements recombinant adenoviruses based on serotypes from subgroup B, preferably adenovirus type 35. The cell line is preferably derived from primary, diploid human cells that are transformed by adenovirus E1 sequences either operatively linked on one DNA molecule or located on two separate DNA molecules, the sequences being operatively linked to regulatory sequences enabling transcription and translation of encoded proteins. Also disclosed is a cell line derived from PER.C6 that expresses functional Ad35 E1B sequences. The Ad35-E1B sequences are driven by the E1B promoter or a heterologous promoter and terminated by a heterologous poly-adenylation signal. The cell lines are useful for producing recombinant adenoviruses designed for gene therapy and vaccination. The cell lines can also be used for producing human recombinant therapeutic proteins such as human growth factors and human antibodies. Also, the cell lines are useful for producing human viruses other than adenovirus such as influenza virus, herpes simplex virus, rotavirus, and measles virus.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 11/165,697, filed Jun. 24, 2005, which is a continuation of U.S. patent application Ser. No. 10/002,750, filed Nov. 15, 2001, now U.S. Pat. No. 6,974,695, issued Dec. 13, 2005, which is a continuation-in-part of application Ser. No. 09/713,678, filed Nov. 15, 2000, now U.S. Pat. No. 6,492,169, issued Dec. 10, 2002, the contents of each of which are hereby incorporated herein by this reference.

TECHNICAL FIELD

[0002] The invention relates to the field of biotechnology generally and, more specifically, to adenoviral-based complementing cell lines.

SEQUENCE LISTING

[0003] Pursuant to 37 C.F.R. 1.821(e), applicants request that the compliant computer readable form Sequence Listing already submitted in the incorporated patent application U.S. Ser. No. 10/002,750, filed Nov. 15, 2001 be used for this patent application. The paper copy of the "Sequence Listing" in this application is identical to the computer readable copy filed for the patent application U.S. Ser. No. 10/002,750, filed Nov. 15, 2001.

BACKGROUND

[0004] Typically, vector and packaging cells have to be adapted to one another so that they have all the necessary elements, but they do not have overlapping elements that lead to replication-competent virus by recombination. Therefore, the sequences necessary for proper transcription of the packaging construct may be heterologous regulatory sequences derived from, for example, other human adenovirus (Ad) serotypes, nonhuman adenoviruses, other viruses like, but not limited to, SV40, hepatitis B virus (HBV), Rous Sarcoma Virus (RSV), cytomegalovirus (CMV), etc., or from higher eukaryotes such as mammals. In general, these sequences include a promoter, enhancer and poly-adenylation sequences.

[0005] PER.C6 is an example of a cell line devoid of sequence overlap between the packaging construct and the adenoviral vector (Fallaux et al., 1998). The PER.C6 cell line was deposited under ECACC deposit number 96022940 under the provisions of the Budapest Treaty with the Centre for Applied Microbiology and Research Authority (European Collection of Animal Cell Cultures), Porton Down, Salisbury, Wiltshire SP4, OJG, United Kingdom, an International Depository Authority, on Feb. 29, 1996. Recombinant viruses based on subgroup C adenoviruses, such as Ad5 and Ad2, can be propagated efficiently on these packaging cells. Generation and propagation of adenoviruses from other serotypes, like subgroup B viruses, has proven to be more difficult on PER.C6 cells. However, as described in EP Appln. 00201738.2, recombinant viruses based on subgroup B virus Ad35 can be made by co-transfection of an expression construct containing the Ad35 early region-1 sequences (Ad35-E1). Furthermore, Ad35-based viruses that are deleted for E1A sequences were shown to replicate efficiently on PER.C6 cells. Thus, the E1A proteins of Ad5 complement Ad35-E1A functions, whereas, at least part of the E1B functions of Ad35 are necessary. This serotype specificity in E1B functions was recently also described for Ad7 recombinant viruses. In an attempt to generate recombinant adenoviruses derived from subgroup B virus Ad7, Abrahamsen et al. (1997) were not able to generate E1-deleted viruses on 293 cells without contamination of wild-type (wt) Ad7. Viruses that were picked after plaque purification on 293-ORF6 cells (Brough et al., 1996) were shown to have incorporated Ad7-E1B sequences by nonhomologous recombination. Thus, efficient propagation of Ad7 recombinant viruses proved possible only in the presence of Ad7-E1B expression and Ad5-E4-ORF6 expression. The E1B proteins are known to interact with cellular, as well as viral, proteins (Bridge et al., 1993; White, 1995). Possibly, the complex formed between the E1B-55K protein and E4-ORF6 which is necessary to increase mRNA export of viral proteins and to inhibit export of most cellular mRNAs, is critical and in some way serotype-specific.

DISCLOSURE OF THE INVENTION

[0006] The invention provides new packaging cell lines capable of complementing recombinant adenoviruses based on serotypes other than subgroup C viruses, such as serotypes from subgroup B like adenovirus type 35.

[0007] In one aspect, the invention provides packaging cell lines capable of complementing recombinant adenovirus based on a serotype of subgroup B, preferably of serotype 35. With the terms "based on or derived from an adenovirus" is meant that it utilizes nucleic acid corresponding to nucleic acid found in the serotype. The utilized nucleic acid may be derived by PCR cloning or other methods known in the art.

[0008] In one aspect, the new packaging cells are derived from primary, diploid human cells such as, but not limited to, primary human retinoblasts, primary human embryonic kidney cells or primary human amniocytes. Transfection of primary cells or derivatives thereof with the adenovirus E1A gene alone can induce unlimited proliferation (immortalization), but does not result in complete transformation. However, expression of E1A in most cases results in induction of programmed cell death (apoptosis), and occasionally immortalization is obtained (Jochemsen et al., 1987). Co-expression of the E1B gene is required to prevent induction of apoptosis and for complete morphological transformation to occur (reviewed in White, 1995). Therefore, in one aspect of the invention, primary human cells or derivatives thereof are transformed by expression of adenovirus E1 proteins of a subgroup other than subgroup C, preferably subgroup B, more preferably adenovirus type 35. The combined activity of the E1A and E1B proteins establishes indefinite growth of the cells and enables complementation of recombinant adenoviruses.

[0009] The complete morphological transformation of primary cells by adenovirus E1 genes is the result of the combined activities of the proteins encoded by the E1A and E1B regions. The roles of the different E1 proteins in lytic infection and in transformation have been studied extensively (reviewed in Zantema and van der Eb, 1995; White, 1995, 1996). The adenovirus E1A proteins are essential for transformation of primary cells. The E1A proteins exert this effect through direct interaction with a number of cellular proteins that are involved in regulation of transcription. These include the pRB family of proteins, p300/CBP and TATA binding protein. In addition to this E1A increases the level of p53 protein in the cells. In the absence of adenovirus E1B activity the rise in p53 levels leads to the induction of apoptosis. Both proteins encoded by the E1B region counteract the induction of apoptosis although by different mechanisms. E1B-21K seems to counteract apoptosis in a manner similar to Bcl-2 via interaction with the effector proteins downstream in the apoptosis pathway (Han et al., 1996), whereas E1B-55K functions through direct interaction with p53. Importantly, the molecular mechanism by which the E1B-55K proteins of Ad2 and 5 (subgroup C) and Ad12 (subgroup A) function in the ability to neutralize p53 may differ. Whereas Ad5 E1B-55K binds p53 strongly and the complex localizes to the cytoplasm, Ad12 E1B-55K binds p53 weakly and both proteins are localized in the nucleus (Zantema et al., 1985; Grand et al., 1999). Both proteins, however, inhibit the transactivation of other genes by p53 (Yew and Berk, 1992).

[0010] In rodent cells, the activity of E1A together with either E1B-21K or 55K is sufficient for full transformation although expression of both E1B proteins together is twice as efficient (Rao et al., 1992). In human cells however, the activity of the E1B-55K protein seems to be more important given the observation that E1B-55K is indispensable for the establishment of transformed cells (Gallimore, 1986).

[0011] Example 6 hereof describes the generation of pIG270. In this construct, the Ad35-E1 genes are expressed from the hPGK promoter and transcription is terminated by the HBVpA. The hPGK promoter constitutes a HincII-EcoRI fragment of the promoter sequence described by Singer-Sam et al. (1984). The HBVpA is located in a BamHI-BglII fragment of the Hepatitis B virus genome (Simonsen and Levinson, 1983; see also Genbank HBV-AF090841). As mentioned before, the promoter and polyadenylation sequences of the E1 expression constructs described in this invention may be derived from other sources without departing from the invention. Also, other functional fragments of the hPGK and HBVpA sequences mentioned herein may be used.

[0012] The functionality of pIG270 was shown by transformation of primary Baby Rat Kidney cells (BRK). Comparison with an equivalent Ad5-E1 expression construct taught that Ad35-E1 genes were less efficient in transforming these cells. The same has been found for the E1 genes of Ad12 (Bernards et al., 1982).

[0013] It is unclear which E1 protein(s) determine(s) the difference in transformation efficiency of E1 sequences observed for adenoviruses from different subgroups. In the case of Ad12, transfection studies with chimeric E1A/E1B genes suggested that the efficiency of transformation of BRK cells was determined by the E1A proteins (Bernards et al., 1982). The E1B-55K protein is shown infra to contain serotype-specific functions necessary for complementation of E1-deleted adenoviruses. If these functions are related to the regulation of mRNA distribution or another late viral function, it is unlikely that these are involved in the transformation efficiency.

[0014] Analysis of functional domains in the Ad2 or Ad5 E1B-55K proteins using insertion mutants have revealed that functions related to viral replication, late protein synthesis and host protein shut-off are not confined to specific domains but are distributed along the protein (Yew et al., 1990). Using the same set of mutants, the domains important for interaction with p53 and E4-Orf6 were found to be more restricted. In addition to one common binding region (amino acids 262 to 326), p53 binding was affected by mutations at aa 180 and E4-Orf6 binding was affected by mutations at aa 143 (Yew and Berk, 1992; Rubenwolf et al., 1997).

[0015] Altogether these results indicate that it is difficult to separate the E1B-55K functions related to transformation (p53 binding) and late protein synthesis (Orf6 binding).

[0016] The invention discloses new E1 constructs that combine the high efficiency of transformation of one serotype with the serotype-specific complementation function of another serotype. These new constructs are used to transform primary human embryonic retinoblast cells and human amniocytes.

[0017] In another aspect of the invention, the transforming E1 sequences are derived from different serotypes. As disclosed in European Patent application 00201738.2, Ad35E1 sequences are capable of transforming Baby Rat Kidney (BRK) cells, albeit with a lower efficiency than that seen with Ad5-E1 sequences. This was also observed for E1 sequences from Ad12 (Bernards et al., 1982). Therefore, in this aspect of the invention, primary diploid human cells or derivatives thereof are transformed with chimeric E1 construct that consists of part of the E1 sequences of a serotype that enables efficient transformation of primary human cells or derivatives thereof and part of the E1 sequences of another serotype which E1 sequences provide the serotype-specific E1B function(s) that enable(s) efficient propagation of E1-deleted viruses of that serotype. In a preferred embodiment of this aspect of the invention, the E1A region is derived from a subgroup C adenovirus like, but not limited to, Ad5, and the E1B coding sequences are derived from an alternative adenovirus, more particularly from an adenovirus of subgroup B, even more particularly from adenovirus type 35. E1B-21K coding sequences may also be chimeric comprising both subgroup C and subgroup B coding sequences. Preferably, all or most of E1B-21K comprises subgroup C coding sequences. In a more preferred embodiment, the E1A coding sequences and the E1B-21K coding sequences are derived from a subgroup C adenovirus, like, but not limited to, Ad5. In one embodiment the cell further comprises E1B-55k coding sequences that are, preferably, as far as not overlapping with the 21K coding sequences derived from an adenovirus of subgroup B, more particularly from adenovirus type 35. In an even more preferred embodiment, all E1 coding sequences are derived from a subgroup C adenovirus, like but not limited to Ad5, except for at least the part of the E1B-55K coding sequences that are necessary for serotype-specific complementation of an alternative adenovirus subgroup, more particularly adenovirus subgroup B, even more particular adenovirus type 35. The invention also provides a packaging cell line wherein the primary, diploid human cells or derivatives thereof have been transformed with a chimeric adenovirus E1 construct comprising part of a first adenovirus E1 coding sequence of a first adenovirus serotype that enables efficient transformation of primary human cells and derivatives thereof; and part of a second adenovirus E1 coding sequence of a second adenovirus serotype, wherein the second adenovirus E1 coding sequence provides the serotype-specific adenovirus E1B function(s) that enable(s) efficient propagation of recombinant adenovirus E1-deleted viruses of the second adenovirus serotype. Preferably, the first adenovirus serotype is a subgroup C adenovirus and the second adenovirus serotype is a subgroup B adenovirus, more particular adenovirus type 35. In one embodiment the packing cell line of the invention comprises bovine adenovirus E1B-55k. Such a bovine E1B-55k expressing cell line is particularly suited for obtaining high yields of a complemented bovine recombinant adenovirus.

[0018] The primary diploid human cells or derivatives thereof are transformed by adenovirus E1 sequences, either operatively linked on one DNA molecule or located on two separate DNA molecules. In the latter case, one DNA molecule carries at least part of the E1 sequences of the serotype-enabling efficient transformation and the second DNA molecule carries at least part of the sequences necessary for serotype-specific complementation. Also provided is a hybrid construct including E1-sequences of the serotype enabling efficient transformation and E1-sequences of another serotype necessary for serotype-specific complementation. The sequences providing serotype specific complementation may of course also contain further activities contributing to transformation. Preferably, the sequences enabling efficient transformation comprise E1A. Preferably, the sequences and the sequences necessary for serotype specific complementation preferably comprise E1B sequences. More preferably, the sequences enabling efficient transforming comprise E1A and E1B-21K sequences and the sequences necessary for serotype specific complementation comprise E1B-55K sequences. Also provided are cells transformed by such hybrid construct. Such cells can favorably be used for the propagation of recombinant E1-deleted adenovirus of another serotype. Of course, it is also possible to provide both functions of E1 sequences on separate constructs. In all aspects, the sequences are operatively linked to regulatory sequences enabling transcription and translation of the encoded proteins. Preferably, a packaging cell of the invention further comprises a DNA encoding at least E4-orf6 of an adenovirus of subgroup B, preferably adenovirus serotype 35. Preferably, the E4-orf6 is derived from the other serotype. Preferably, the cell comprises E1B-55K and E4-orf6 of the same serotype as the recombinant vector to be propagated/complemented or otherwise produced.

[0019] In another aspect of the invention, new packaging cells are described that are derived from PER.C6 (ECACC deposit number 96022940; Fallaux et al., 1998) and contain Ad35-E1 sequences integrated into their genome. These Ad35-E1 sequences are present in a functional expression cassette, but preferably do not contain sequences overlapping with sequences present in the recombinant viral vector. Preferably, the functional expression cassette consists of a heterologous promoter and poly-adenylation signal functionally linked to Ad35-E1 sequences. More specifically, the Ad35-E1 coding sequences are functionally linked to the human phosphoglycerate gene promoter (hPGK) and hepatitis B virus poly-adenylation signal (HBV-pA). Preferably, Ad35-E1 coding sequences comprise the coding regions of the E1A proteins and the E1B promoter sequences linked to E1B coding sequences up to and including the stop codon of the E1B 55K protein. More preferably, the Ad35-E1 sequences comprise nucleotide 468 to nucleotide 3400 of the Ad35 wt sequence. To be able to select for transfected cells, a dominant selection marker like, but not limited to, the neo.sup.r gene has to be incorporated on the expression vector or the Ad35 expression vector is cotransfected with a separate expression vector mediating expression of the selection marker. In both cases, the selection marker becomes integrated in the cellular genome. Other Ad5-E1 transformed cell lines like 293 (Graham et al., 1977) and 911 (Fallaux et al., 1996) or established human cell lines like A549 cells may be used without departing from the present invention.

[0020] In another aspect of the invention, PER.C6-derived cells are described that express functional Ad35-E1B sequences. In one embodiment, the Ad35-E1B coding sequences are driven by the E1B promoter and terminated by a heterologous poly-adenylation signal like, but not limited to, the HBVpA. In a preferred embodiment, the Ad35-E1B coding sequences are driven by a heterologous promoter like, but not limited to, the hPGK promoter or Elongation Factor-1.alpha. (EF-1.alpha.) promoter and terminated by a heterologous pA signal like, but not limited to, the HBVpA. These Ad35-E1B sequences preferably comprise the coding regions of the E1B-21K and the E1B-55K proteins located between nucleotides 1611 and 3400 of the wild-type (wt) Ad35 sequence. More preferably, the Ad35-E1B sequences comprise nucleotides 1550 to 3400 of the wt Ad35 sequence. In an even more preferred embodiment, the E1B sequences comprise the coding sequences of the E1B-55K gene located between nucleotides 1916 and 3400 of the wt Ad35 sequence. In an even more preferred embodiment a packaging cell line or a cell line of the invention lacks a functional coding sequence for E1B 21k. Such cell lines, in general, produce significantly more recombinant adenovirus than E1B 21K positive cell lines.

[0021] The invention further provides a method for complementing a recombinant adenovirus comprising providing a packaging cell line or a cell line according to the invention, with the recombinant adenovirus and culturing the cell to allow for complementation. In a preferred embodiment the method further comprises harvesting complemented recombinant adenovirus. Preferably, the recombinant adenovirus is derived from adenovirus subgroup B. More preferably, the recombinant adenovirus is derived from adenovirus serotype 35.

[0022] In another aspect, the invention provides a recombinant adenovirus obtained by a method of the invention or with a packaging cell of the invention. Such an adenovirus can be obtained essentially free from contaminating wild type adenovirus, or replication competent adenovirus. Such recombinant adenovirus preparations are very suited for administration of therapeutic sequences to somatic tissues in vivo in for instance a gene therapeutic setting. Preferred are recombinant adenoviruses comprising a deletion of nucleic acid encoding at least one E1-region protein. Preferably, such adenovirus further comprises a deletion of nucleic acid encoding at least one E3-region protein. Preferably, such adenovirus further comprises a deletion of nucleic acid encoding at least one E4-region protein. Preferably, such adenovirus further comprises a deletion of nucleic acid encoding at least one E4-Orf6 protein. For this reason, the invention also provides the use of a recombinant adenovirus of the invention for the preparation of a medicament.

[0023] With the term E1B-55K protein as used herein, is meant the protein encoded by the E1B-region in an adenovirus serotype having a similar function in the serotype as provided by the E1B-55K protein Ad5.

[0024] With the term E1B-21K protein as used herein, is meant the protein enclosed by the E1B-region in an adenovirus serotype having a similar function in the serotype as provided by the E1B-19K protein of Ad5. The same terminology applies for the sequences encoding these proteins. When referring to Ad35-E1 sequences from a specified nucleotide to nucleotide 3400 is meant "up to and including nucleotide 3400."

[0025] The cell lines of this invention are useful for, among other things, producing recombinant adenoviruses designed for gene therapy and vaccination. The cell lines, being derived from cells of human origin, are also useful for the production of human recombinant therapeutic proteins like, but not limited to human growth factors, human antibodies. In addition the cell lines are useful for the production of human viruses other than adenovirus like, but not limited to, influenza virus, herpes simplex virus, rotavirus, measles virus.

[0026] A preferred derivative of primary, diploid human cells is the PER.C6 cell line (ECACC deposit number 960022940).

[0027] It is within the skills of the artisan to provide for proteins having a similar function in kind as the adenovirus E1 protein referred to in this document. For instance a functional part may be provided and/or a derivative may be provided with a similar function in kind, not necessarily in amount.

[0028] Such parts and derivatives are considered to be part of the invention, in as far as similar transforming/complementing and/or serotype specificity function is provided in kind, not necessarily in amount.

BRIEF DESCRIPTION OF THE FIGURES

[0029] FIG. 1: Bar graph showing the percentage of serum samples positive for neutralization for each human wt adenovirus tested (see, Example 1 for description of the neutralization assay).

[0030] FIG. 2: Graph showing absence of correlation between the VP/CCID50 ratio and the percentage of neutralization.

[0031] FIG. 3: Bar graph presenting the percentage sera samples that show neutralizing activity to a selection of adenovirus serotypes. Sera were derived from healthy volunteers from Belgium and the UK.

[0032] FIG. 4: Bar graph presenting the percentage sera samples that show neutralizing activity to adenovirus serotypes 5, 11, 26, 34, 35, 48 and 49. Sera were derived from five different locations in Europe and the United States.

[0033] FIG. 5: Map of pAdApt35IP1.

[0034] FIG. 6: Schematic representation of the steps undertaken to construct pWE.Ad35.pIX-rITR.

[0035] FIG. 7: Map of pWE.Ad35.pIX-rITR.

[0036] FIG. 8: Map of pRSV.Ad35-E1.

[0037] FIG. 9 Map of pPGKneopA

[0038] FIG. 10: Map of pRSV-Pneo.

[0039] FIG. 11: Map of pRSVhbv.Neo.

[0040] FIG. 12: Map of pIG.E1A.E1B.

[0041] FIG. 13: Map of pIG135.

[0042] FIG. 14: Map of pIG270.

[0043] FIG. 15: Map of pBr.Ad35.leftITR-pIX.

[0044] FIG. 16: Map of pBr.Ad35.leftITR-pIXdE1A.

[0045] FIG. 17: Map of pBr.Ad35.d21K.

[0046] FIG. 18: Map of pBr.Ad35.d55K1.

[0047] FIG. 19: Map of pBr.Ad35DdSM.

[0048] FIG. 20: Schematic representation of Ad35-E1A/E1B deletion constructs.

[0049] FIG. 21: Map of pIG.35BL.

[0050] FIG. 22: Map of pRSVneo4.

[0051] FIG. 23: Map of pIG35Bneo.

[0052] FIG. 24: Map of pIG35.55K.

[0053] FIG. 25: Map of pIG535.

[0054] FIG. 26: Map of pIG635.

[0055] FIG. 27: Map of pIG735.

[0056] FIG. 28: Map of pCC271.

[0057] FIG. 29: Map of pCC535s.

[0058] FIG. 30: Map of pCR535E1B.

[0059] FIG. 31: Map of pCC2155s.

[0060] FIG. 32: Map of pCC536s.

[0061] FIG. 33: Map of pIG536.

[0062] FIG. 34: Map of pBr.Ad35.PRn.

[0063] FIG. 35: Map of pBr.Ad35.PRn.DELTA.E3.

[0064] FIG. 36: Map of pWE.Ad35.pIX-rITR.DELTA.E3.

[0065] FIG. 37: Alignment of E1B-21K amino acid sequences in pCC536s (SEQ ID NO:45), wtAd5 (SEQ ID NO:46) and wtAd35 (SEQ ID NO:47) (A) and E1B-55K amino acid sequences in pCC536s (SEQ ID NO:48), wtAd5 (SEQ ID NO:49) and wtAd35 (SEQ ID NO:50) (B).

[0066] The invention is further explained by the use of the following illustrative examples.

DETAILED DESCRIPTION OF THE INVENTION

Examples

Example 1

A High Throughput Assay for the Detection of Neutralizing Activity in Human Serum

[0067] To enable screening of a large amount of human sera for the presence of neutralizing antibodies against all adenovirus serotypes, an automated 96-wells assay was developed.

Human Sera

[0068] A panel of 100 individuals was selected. Volunteers (50% male, 50% female) were healthy individuals between ages 20 and 60 years old with no restriction for race. All volunteers signed an informed consent form. People professionally involved in adenovirus research were excluded.

[0069] Approximately 60 ml blood was drawn in dry tubes. Within two hours after sampling, the blood was centrifuged at 2500 rpm for 10 minutes. Approximately 30 ml serum was transferred to polypropylene tubes and stored frozen at -20.degree. C. until further use.

[0070] Serum was thawed and heat-inactivated at 56.degree. C. for 10 minutes and then aliquoted to prevent repeated cycles of freeze/thawing. Part was used to make five steps of twofold dilutions in medium (DMEM, Gibco BRL) in a quantity large enough to fill out approximately 70 96-well plates. Aliquots of undiluted and diluted sera were pipetted in deep well plates (96-well format) and using a programmed platemate dispensed in 100 .mu.l aliquots into 96-well plates. The plates were loaded with eight different sera in duplo (100 .mu.l/well) according to the scheme below:

TABLE-US-00001 S1/2 S1/4 S1/8 S1/16 S1/32 S5/2 S5/4 S5/8 S5/16 S5/32 -- -- S1/2 S1/4 S1/8 S1/16 S1/32 S5/2 S5/4 S5/8 S5/16 S5/32 -- -- S2/2 S2/4 S2/8 S2/16 S2/32 S6/2 S6/4 S6/8 S6/16 S6/32 -- -- S2/2 S2/4 S2/8 S2/16 S2/32 S6/2 S6/4 S6/8 S6/16 S6/32 -- -- S3/2 S3/4 S3/8 S3/16 S3/32 S7/2 S7/4 S7/8 S7/16 S7/32 -- -- S3/2 S3/4 S3/8 S3/16 S3/32 S7/2 S7/4 S7/8 S7/16 S7/32 -- -- S4/2 S4/4 S3/8 S3/16 S3/32 S8/2 S8/4 S8/8 S8/16 S8/32 -- -- S4/2 S4/4 S3/8 S3/16 S3/32 S8/2 S8/4 S8/8 S8/16 S8/32 -- --

Where S1/2 to S8/2 in columns 1 and 6 represent 1.times. diluted sera and Sx/4, Sx/8, Sx/16 and Sx/32 the two-fold serial dilutions. The last plates also contained four wells filled with 100 .mu.l fetal calf serum as a negative control. Plates were kept at -20.degree. C. until further use.

Preparation of Human Adenovirus Stocks

[0071] Prototypes of all known human adenoviruses were inoculated on T25 flasks seeded with PER.C6 cells (Fallaux et al., 1998) and harvested upon full CPE. After freeze/thawing, 1 to 2 ml of the crude lysates were used to inoculate a T80 flask with PER.C6 and virus was harvested at full CPE. The timeframe between inoculation and occurrence of CPE, as well as the amount of virus needed to re-infect a new culture, differed between serotypes. Adenovirus stocks were prepared by freeze/thawing and used to inoculate 3 to 4 T175 cm.sup.2 three-layer flasks with PER.C6 cells. Upon occurrence of CPE, cells were harvested by tapping the flask, pelleted and virus was isolated and purified by a two-step CsCl gradient as follows. Cell pellets were dissolved in 50 ml 10 mM NaPO.sub.4 buffer (pH 7.2) and frozen at -20.degree. C. After thawing at 37.degree. C., 5.6 ml sodium deoxycholate (5% w/v) was added. The solution was mixed gently and incubated for 5 to 15 minutes at 37.degree. C. to completely lyse the cells. After homogenizing the solution, 1875 .mu.l 1 M MgCl.sub.2 was added. After the addition of 375 .mu.L DNAse (10 mg/ml), the solution was incubated for 30 minutes at 37.degree. C. Cell debris was removed by centrifugation at 1880.times.g for 30 minutes at RT without brake. The supernatant was subsequently purified from proteins by extraction with FREON (3.times.). The cleared supernatant was loaded on a 1 M Tris/HCl buffered cesium chloride block gradient (range: 1.2/1.4 g/ml) and centrifuged at 21000 rpm for 2.5 hours at 10.degree. C. The virus band is isolated after which a second purification using a 1 M Tris/HCl buffered continues gradient of 1.33 g/ml of cesium chloride was performed. The virus was then centrifuged for 17 hours at 55000 rpm at 10.degree. C. The virus band is isolated and sucrose (50% w/v) is added to a final concentration of 1%. Excess cesium chloride is removed by dialysis (three times 1 hour at RT) in dialysis slides (Slide-a-lizer, cut off 10000 kDa, Pierce, USA) against 1.5 liter PBS supplemented with CaCl.sub.2 (0.9 mM), MgCl.sub.2 (0.5 mM) and an increasing concentration of sucrose (1, 2, 5%). After dialysis, the virus is removed from the slide-a-lizer after which it is aliquoted in portions of 25 and 100 .mu.l upon which the virus is stored at -85.degree. C.

[0072] To determine the number of virus particles per milliliter, 50 .mu.l of the virus batch is run on a high-pressure liquid chromatograph (HPLC) as described by Shabram et al (1997). Viruses were eluted using a NaCl gradient ranging from 0 to 600 mM. As depicted in Table I, the NaCl concentration by which the viruses were eluted differed significantly among serotypes.

[0073] Most human adenoviruses replicated well on PER.C6 cells with a few exceptions. Adenovirus type 8 and 40 were grown on 911-E4 cells (He et al., 1998). Purified stocks contained between 5.times.10.sup.10 and 5.times.10.sup.12 virus particles/ml (VP/ml; see table I).

Titration of Purified human adenovirus stocks

[0074] Adenoviruses were titrated on PER.C6 cells to determine the amount of virus necessary to obtain full CPE in five days, the length of the neutralization assay. Hereto, 100 .mu.l medium was dispensed into each well of 96-well plates. Twenty-five .mu.l of adenovirus stocks pre-diluted 10.sup.4, 10.sup.5, 10.sup.6 or 10.sup.7 times were added to column 2 of a 96-well plate and mixed by pipetting up and down 10 times. Then 25 .mu.l was brought from column 2 to column 3 and again mixed. This was repeated until column 11, after which 25 .mu.l from column 11 was discarded. This way, serial dilutions in steps of five were obtained starting off from a pre-diluted stock. Then 3.times.10.sup.4 PER.C6 cells (ECACC deposit number 96022940) were added in a 100 .mu.l volume and the plates were incubated at 37.degree. C., 5% CO.sub.2 for five or six days. CPE was monitored microscopically. The method of Reed and Muensch was used to calculate the cell culture-inhibiting dose 50% (CCID50).

[0075] In parallel, identical plates were set up that were analyzed using the MTT assay (Promega). In this assay, living cells are quantified by colorimetric staining. Hereto, 20 .mu.l MTT (7.5 mgr/ml in PBS) was added to the wells and incubated at 37.degree. C., 5% CO.sub.2 for two hours. The supernatant was removed and 100 .mu.l of a 20:1 isopropanol/triton-X100 solution was added to the wells. The plates were put on a 96-well shaker for 3 to 5 minutes to solubilize the precipitated staining. Absorbance was measured at 540 nm and at 690 nm (background). By this assay, wells with proceeding CPE or full CPE can be distinguished.

Neutralization Assay

[0076] Ninety-six-well plates with diluted human serum samples were thawed at 37.degree. C., 5% CO.sub.2. Adenovirus stocks diluted to 200 CCID50 per 50 .mu.l were prepared and 50 .mu.l aliquots were added to columns 1 to 11 of the plates with serum. Plates were incubated for 1 hour at 37.degree. C., 5% CO.sub.2. Then, 50 .mu.l PER.C6 cells at 6.times.10.sup.5/ml were dispensed in all wells and incubated for one day at 37.degree. C., 5% CO.sub.2. Supernatant was removed using fresh pipette tips for each row and 200 .mu.l fresh medium was added to all wells to avoid toxic effects of the serum. Plates were incubated for another four days at 37.degree. C., 5% CO.sub.2. In addition, parallel control plates were set up in duplo, with diluted positive control sera generated in rabbits and specific for each serotype to be tested in rows A and B and with negative control serum (FCS) in rows C and D. Also, in each of the rows E-H, a titration was performed as described above with steps of five times dilutions starting with 200 CCID50 of each virus to be tested. On day 5, one of the control plates was analyzed microscopically and with the MTT assay. The experimental titer was calculated from the control titration plate observed microscopically. If CPE was found to be complete, i.e., the first dilution in the control titration experiment analyzed by MTT shows clear cell death, all assay plates were processed. If not, the assay was allowed to proceed for one or more days until full CPE was apparent, after which all plates were processed. In most cases, the assay was terminated at day 5. For Ad1, 5, 33, 39, 42 and 43 the assay was left for six days and for Ad2 for eight days.

[0077] A serum sample is regarded as "non-neutralizing" when, at the highest serum concentration, a maximum protection of 40% is seen compared to controls without serum.

[0078] The results of the analysis of 44 prototype adenoviruses against serum from 100 healthy volunteers are shown in FIG. 1. As expected, the percentage of serum samples that contained neutralizing antibodies to Ad2 and Ad5 was very high. This was also true for most of the lower numbered adenoviruses. Surprisingly, none of the serum samples contained neutralizing antibodies to Ad35. Also, the number of individuals with neutralizing antibody titers to the serotypes 26, 34 and 48 was very low. Therefore, recombinant E1-deleted adenoviruses based on Ad35 or one of the other above-mentioned serotypes have an important advantage compared to recombinant vectors based on Ad5 with respect to clearance of the viruses by neutralizing antibodies.

[0079] Also, Ad5-based vectors that have parts of the capsid proteins involved in immunogenic response of the host replaced by the corresponding parts of the capsid proteins of Ad35 or one of the other serotypes will be less, or even not, neutralized by the vast majority of human sera.

[0080] As can be seen in Table I, the VP/CCID50 ratio calculated from the virus particles per ml and the CCID50 obtained for each virus in the experiments was highly variable and ranged from 0.4 to 5 log. This is probably caused by different infection efficiencies of PER.C6 cells and by differences in replication efficiency of the viruses. Furthermore, differences in batch qualities may play a role. A high VP/CCID50 ratio means that more viruses were put in the wells to obtain CPE in five days. As a consequence, the outcome of the neutralization study might be biased since more inactive virus particles could shield the antibodies. To check whether this phenomenon had taken place, the VP/CCID50 ratio was plotted against the percentage of serum samples found positive in the assay (FIG. 2). The graph clearly shows that there is no negative correlation between the amount of viruses in the assay and neutralization in serum.

Example 2

The Prevalence of Neutralizing Activity (NA) to Ad35 is Low in Human Sera from Different Geographic Locations

[0081] In Example 1, the analysis of neutralizing activity ("NA") in human sera from one location in Belgium was described. Strikingly, of a panel of 44 adenovirus serotypes tested, one serotype, Ad35, was not neutralized in any of the 100 sera assayed. In addition, a few serotypes, Ad26, Ad34 and Ad48 were found to be neutralized in 8%, or less, of the sera tested. This analysis was further extended to other serotypes of adenovirus not previously tested and, using a selection of serotypes from the first screen, was also extended to sera from different geographic locations.

[0082] Hereto, adenoviruses were propagated, purified and tested for neutralization in the CPE-inhibition assay as described in Example 1. Using the sera from the same batch as in Example 1, adenovirus serotypes 7B, 11, 14, 18 and 44/1876 were tested for neutralization. These viruses were found to be neutralized in, respectively, 59, 13, 30, 98 and 54% of the sera. Thus, of this series, Ad11 is neutralized with a relatively low frequency.

[0083] Since it is known that the frequency of isolation of adenovirus serotypes from human tissue, as well as the prevalence of NA to adenovirus serotypes, may differ on different geographic locations, we further tested a selection of the adenovirus serotypes against sera from different places. Human sera were obtained from two additional places in Europe (Bristol, UK and Leiden, NL) and from two places in the United States (Stanford, Calif. and Great Neck, N.Y.). Adenoviruses that were found to be neutralized in 20% or less of the sera in the first screen, as well as Ad2, Ad5, Ad27, Ad30, Ad38, Ad43, were tested for neutralization in sera from the UK. The results of these experiments are presented in FIG. 3. Adenovirus serotypes 2 and 5 were again neutralized in a high percentage of human sera. Furthermore, some of the serotypes that were neutralized in a low percentage of sera in the first screen are neutralized in a higher percentage of sera from the UK, for example, Ad26 (7% vs. 30%), Ad28 (13% vs. 50%), Ad34 (5% vs. 27%) and Ad48 (8% vs. 32%). Neutralizing activity against Ad11 and Ad49 that were found in a relatively low percentage of sera in the first screen, are found in an even lower percentage of sera in this second screen (13% vs. 5% and 20% vs. 11%, respectively). Serotype Ad35 that was not neutralized in any of the sera in the first screen, was now found to be neutralized in a low percentage (8%) of sera from the UK. The prevalence of NA in human sera from the UK is the lowest to serotypes Ad11 and Ad35.

[0084] For further analysis, sera was obtained from two locations in the US (Stanford, Calif. and Great Neck, N.Y.) and from The Netherlands (Leiden). FIG. 4 presents an overview of data obtained with these sera and the previous data. Not all viruses were tested in all sera, except for Ad5, Ad11 and Ad35. The overall conclusion from this comprehensive screen of human sera is that the prevalence of neutralizing activity to Ad35 is the lowest of all serotypes throughout the western countries: on average 7% of the human sera contain neutralizing activity (5 different locations). Another B-group adenovirus, Ad11 is also neutralized in a low percentage of human sera (average 11% in sera from five different locations). Adenovirus type 5 is neutralized in 56% of the human sera obtained from five different locations. Although not tested in all sera, D-group serotype 49 is also neutralized with relatively low frequency in samples from Europe and from one location of the US (average 14%).

[0085] In the herein described neutralization experiments, a serum is judged non-neutralizing when, in the well with the highest serum concentration, the maximum protection of CPE is 40% compared to the controls without serum. The protection is calculated as follows:

1 % protection = OD corresponding well - OD virus control OD non - infected control - OD virus control .times. 100 % ##EQU00001##

[0086] As described in Example 1, the serum is plated in five different dilutions ranging from 4.times. to 64.times. diluted. Therefore, it is possible to distinguish between low titers (i.e., neutralization only in the highest serum concentrations) and high titers of NA (i.e., also neutralization in wells with the lowest serum concentration). Of the human sera used in our screen that were found to contain neutralizing activity to Ad5, 70% turned out to have high titers, whereas, of the sera that contained NA to Ad35, only 15% had high titers. Of the sera that were positive for NA to Ad11, only 8% had high titers. For Ad49, this was 5%. Therefore, not only is the frequency of NA to Ad35, Ad11 and Ad49 much lower as compared to Ad5, but of the sera that do contain NA to these viruses, the vast majority have low titers. Adenoviral vectors based on Ad11, Ad35 or Ad49 have, therefore, a clear advantage over Ad5-based vectors when used as gene therapy vehicles or vaccination vectors in vivo or in any application where infection efficiency is hampered by neutralizing activity.

[0087] In the following examples, the construction of a vector system for the generation of safe, RCA-free Ad35-based vectors is described.

Example 3

Sequence of the Human Adenovirus Type 35

[0088] Ad35 viruses were propagated on PER.C6 cells and DNA was isolated as follows: To 100 .mu.l of virus stock (Ad35: 3.26.times.10.sup.12 VP/ml), 10 .mu.l 10.times.DNAse buffer (130 mM Tris-HCl pH 7.5; 1.2 M CaCl.sub.2; 50 mM MgCl.sub.2) was added. After addition of 10 .mu.l 10 mgr/ml DNAse I (Roche Diagnostics), the mixture was incubated for 1 hour at 37.degree. C. Following addition of 2.5 .mu.l 0.5M EDTA, 3.2 .mu.l 20% SDS and 1.5 .mu.l ProteinaseK (Roche Diagnostics; 20 mgr/ml), samples were incubated at 50.degree. C. for 1 hour. Next, the viral DNA was isolated using the GENECLEAN spin kit (BIO 101 Inc.) according to the manufacturer's instructions. DNA was eluted from the spin column with 25 .mu.l sterile MilliQ water. The total sequence was generated by Qiagen Sequence Services (Qiagen GmbH, Germany). Total viral DNA was sheared by sonification and the ends of the DNA were made blunt by T4 DNA polymerase. Sheared blunt fragments were size fractionated on agarose gels and gel slices corresponding to DNA fragments of 1.8 to 2.2 kb were obtained. DNA was purified from the gel slices by the QIAquick gel extraction protocol and subcloned into a shotgun library of pUC19 plasmid cloning vectors. An array of clones in 96-well plates covering the target DNA 8 (+/-2) times was used to generate the total sequence. Sequencing was performed on Perkin-Elmer 9700 thermocyclers using Big Dye Terminator chemistry and AmpliTaq FS DNA polymerase followed by purification of sequencing reactions using QIAGEN DyeEx 96 technology. Sequencing reaction products were then subjected to automated separation and detection of fragments on ABI 377 XL 96 lane sequencers. Initial sequence results were used to generate a contiguous sequence and gaps were filled in by primer walking reads on the target DNA or by direct sequencing of PCR products. The ends of the virus turned out to be absent in the shotgun library, most probably due to cloning difficulties resulting from the amino acids of pTP that remain bound to the ITR sequences after proteinase K digestion of the viral DNA. Additional sequence runs on viral DNA solved most of the sequence in those regions, however, it was difficult to obtain a clear sequence of the most terminal nucleotides. At the 5' end the sequence portion obtained was 5'-CCAATAATATACCT-3' (SEQ ID NO:1) while at the 3' end, the obtained sequence portion was 5'-AGGTATATTATTGATGATGGG-3' (SEQ ID NO:2). Most human adenoviruses have a terminal sequence 5'-CATCATCAATAATATACC-3' (SEQ ID NO:3). In addition, a clone representing the 3' end of the Ad35 DNA obtained after cloning the terminal 7 kb Ad35 EcoRI fragment into pBr322 also turned out to have the typical CATCATCAATAAT . . . sequence. Therefore, Ad35 may have the typical end sequence and the differences obtained in sequencing directly on the viral DNA are due to artifacts correlated with run-off sequence runs and the presence of residual amino acids of pTP.

[0089] The total sequence of Ad35 with corrected terminal sequences is given in SEQ ID NO:44. Based sequence homology with Ad5 (Genbank # M72360) and Ad7 (partial sequence Genbank # X03000) and on the location of open reading frames, the organization of the virus is identical to the general organization of most human adenoviruses, especially the subgroup B viruses. The total length of the genome is 34,794 basepairs.

Example 4

Construction of a Plasmid-Based Vector System to Generate Recombinant Ad35-Based Viruses

[0090] A functional plasmid-based vector system to generate recombinant adenoviral vectors comprises the following components: [0091] 1. An adapter plasmid comprising a left ITR and packaging sequences derived from Ad35 and at least one restriction site for insertion of a heterologous expression cassette and lacking E1 sequences. Furthermore, the adapter plasmid contains Ad35 sequences 3' from the E1B coding region including the pIX promoter and coding sequences enough to mediate homologous recombination of the adapter plasmid with a second nucleic acid molecule. [0092] 2. A second nucleic acid molecule, comprising sequences homologous to the adapter plasmid, and Ad35 sequences necessary for the replication and packaging of the recombinant virus, that is, early, intermediate and late genes that are not present in the packaging cell. [0093] 3. A packaging cell providing at least functional E1 proteins capable of complementing the E1 function of Ad35.

[0094] Other methods for generating recombinant adenoviruses on complementing packaging cells are known in the art and may be applied to Ad35 viruses without departing from the invention. As an example, the construction of a plasmid-based system, as outlined above, is described in detail below.

1) Construction of Ad35 adapter plasmids

[0095] The adapter plasmid pAdApt (described in International Patent Publication WO99/55132) was first modified to obtain adapter plasmids that contain extended polylinkers and that have convenient unique restriction sites flanking the left ITR and the adenovirus sequence at the 3' end to enable liberation of the adenovirus insert from plasmid vector sequences. Construction of these plasmids is described below in detail:

[0096] Adapter plasmid pAdApt was digested with SalI and treated with Shrimp Alkaline Phosphatase to reduce religation. A linker, composed of the following two phosphorylated and annealed oligos: ExSalPacF 5'-TCG ATG GCA AAC AGC TAT TAT GGG TAT TAT GGG TTC GAA TTA ATT AA-3' (SEQ ID NO:4) and ExSalPacR 5'-TCG ATT AAT TAA TTC GAA CCC ATA ATA CCC ATA ATA GCT GTT TGC CA-3' (SEQ ID NO:5) was directly ligated into the digested construct, thereby replacing the SalI restriction site by Pi-PspI, SwaI and PacI. This construct was designated pADAPT+ExSalPac linker. Furthermore, part of the left ITR of pAdApt was amplified by PCR using the following primers: PCLIPMSF: 5'-CCC CAA TTG GTC GAC CAT CAT CAA TAA TAT ACC TTA TTT TGG-3' (SEQ ID NO:6) and pCLIPBSRGI: 5'-GCG AAA ATT GTC ACT TCC TGT G-3' (SEQ ID NO:7). The amplified fragment was digested with MunI and BsrGI and cloned into pAd5/Clip (described in International Patent Application WO99/55132), which was partially digested with EcoRI and after purification digested with BsrGI, thereby re-inserting the left ITR and packaging signal. After restriction enzyme analysis, the construct was digested with ScaI and SgrAI and an 800 bp fragment was isolated from gel and ligated into ScaI/SgrAI digested pADAPT+ExSalPac linker. The resulting construct, designated pIPspSalAdapt, was digested with SalI, dephosphorylated, and ligated to the phosphorylated ExSalPacF/ExSalPacR double-stranded linker previously mentioned. A clone in which the PacI site was closest to the ITR was identified by restriction analysis and sequences were confirmed by sequence analysis. This novel pAdApt construct, termed pIPspAdapt, thus harbors two ExSalPac linkers containing recognition sequences for PacI, PI-PspI and BstBI, which surround the adenoviral part of the adenoviral adapter construct, and which can be used to linearize the plasmid DNA prior to co-transfection with adenoviral helper fragments.

[0097] In order to further increase transgene cloning permutations, a number of polylinker variants were constructed based on pIPspAdapt. For this purpose, pIPspAdapt was first digested with EcoRI and dephosphorylated. A linker composed of the following two phosphorylated and annealed oligos: Ecolinker+: 5'-AAT TCG GCG CGC CGT CGA CGA TAT CGA TAG CGG CCG C-3' (SEQ ID NO:8) and Ecolinker: 5'-AAT TGC GGC CGC TAT CGA TAT CGT CGA CGG CGC GCC G-3' (SEQ ID NO:9) was ligated into this construct, thereby creating restriction sites for AscI, SalI, EcoRV, ClaI and NotI. Both orientations of this linker were obtained, and sequences were confirmed by restriction analysis and sequence analysis. The plasmid containing the polylinker in the order 5' HindIII, KpnI, AgeI, EcoRI, AscI, SalI, EcoRV, ClaI, NotI, NheI, HpaI, BamHI and XbaI was termed pIPspAdapt1, while the plasmid containing the polylinker in the order HindIII, KpnI, AgeI, NotI, ClaI, EcoRV, SalI, AscI, EcoRI, NheI, HpaI, BamHI and XbaI was termed pIPspAdapt2.

[0098] To facilitate the cloning of other sense or antisense constructs, a linker composed of the following two oligonucleotides was designed to reverse the polylinker of pIPspAdapt: HindXba+5'-AGC TCT AGA GGA TCC GTT AAC GCT AGC GAA TTC ACC GGT ACC AAG CTT A-3' (SEQ ID NO:10); HindXba-5'-CTA GTA AGC TTG GTA CCG GTG AAT TCG CTA GCG TTA ACG GAT CCT CTA G-3' (SEQ ID NO:11). This linker was ligated into HindIII/XbaI digested pIPspAdapt and the correct construct was isolated. Confirmation was done by restriction enzyme analysis and sequencing. This new construct, pIPspAdaptA, was digested with EcoRI and the previously mentioned Ecolinker was ligated into this construct. Both orientations of this linker were obtained, resulting in pIPspAdapt3, which contains the polylinker in the order XbaI, BamHI, HpaI, NheI, EcoRI, AscI, SalI, EcoRV, ClaI, NotI, AgeI, KpnI and HindIII. All sequences were confirmed by restriction enzyme analysis and sequencing.

[0099] Adapter plasmids based on Ad35 were then constructed as follows:

[0100] The left ITR and packaging sequence corresponding to Ad35 wt sequences nucleotides 1 to 464 (SEQ ID NO:44) were amplified by PCR on wt Ad35 DNA using the following primers: Primer 35F1: 5'-CGG AAT TCT TAA TTA ATC GAC ATC ATC AAT AAT ATA CCT TAT AG-3' (SEQ ID NO:12); Primer 35R2: 5'-GGT GGT CCT AGG CTG ACA CCT ACG TAA AAA CAG-3' (SEQ ID NO:13). Amplification introduces a PacI site at the 5' end and an AvrII site at the 3' end of the sequence.

[0101] For the amplification, Platinum Pfx DNA polymerase enzyme (LTI) was used according to manufacturer's instructions, but with primers at 0.6 .mu.M and with DMSO added to a final concentration of 3%. Amplification program was as follows: 2 minutes at 94.degree. C. (30 seconds 94.degree. C., 30 seconds at 56.degree. C., 1 minute at 68.degree. C.) for 30 cycles, followed by 10 minutes at 68.degree. C.

[0102] The PCR product was purified using a PCR purification kit (LTI) according to the manufacturer's instructions and digested with PacI and AvrII. The digested fragment was then purified from gel using the GENECLEAN kit (BIO 101, Inc.). The Ad5-based adapter plasmid pIPspAdApt-3 was digested with AvrII and then partially with PacI and the 5762 bp fragment was isolated in an LMP agarose gel slice and ligated with the above-mentioned PCR fragment digested with the same enzymes and transformed into electrocompetent DH10B cells (LTI). The resulting clone is designated pIPspAdApt3-Ad351ITR.

[0103] In parallel, a second piece of Ad35 DNA was amplified using the following primers: 35F3: 5'-TGG TGG AGA TCT GGT GAG TAT TGG GAA AAC-3' (SEQ ID NO:14); 35R4: 5'-CGG AAT TCT TAA TTA AGG GAA ATG CAA ATC TGT GAG G-3' (SEQ ID NO:15).

[0104] The sequence of this fragment corresponds to nucleotides 3401 to 4669 of wt Ad35 (SEQ ID NO:44) and contains 1.3 kb of sequences starting directly 3' from the E1B-55k coding sequence. Amplification and purification were done as previously described herein for the fragment containing the left ITR and packaging sequence. The PCR fragment was then digested with PacI and subcloned into pNEB193 vector (New England Biolabs) digested with SmaI and PacI. The integrity of the sequence of the resulting clone was checked by sequence analysis. pNEB/Ad35pF3R4 was then digested with BglII and PacI and the Ad35 insert was isolated from gel using the QIAExII kit (Qiagen). pIPspAdApt3-Ad351ITR was digested with BglII and then partially with PacI. The 3624 bp fragment (containing vector sequences, the Ad35 ITR and packaging sequences as well as the CMV promoter, multiple cloning region and polyA signal) was also isolated using the QIAExII kit (Qiagen). Both fragments were ligated and transformed into competent DH10B cells (LTI). The resulting clone, pAdApt35IP3, has the expression cassette from pIPspAdApt3 but contains the Ad35 left ITR and packaging sequences and a second fragment corresponding to nucleotides 3401 to 4669 from Ad35. A second version of the Ad35 adapter plasmid having the multiple cloning site in the opposite orientation was made as follows:

[0105] pIPspAdapt1 was digested with NdeI and BglII and the 0.7 kbp band containing part of the CMV promoter, the MCS and SV40 polyA was isolated and inserted in the corresponding sites of pAdApt35IP3 generating pAdApt35IP1 (FIG. 5).

[0106] pAdApt35.LacZ and pAdApt35.Luc adapter plasmids were then generated by inserting the transgenes from pcDNA.LacZ (digested with KpnI and BamHI) and pAdApt.Luc (digested with HindIII and BamHI) into the corresponding sites in pAdApt35IP1. The generation of pcDNA.LacZ and pAdApt.Luc is described in International Patent Publication WO99/55132.

2) Construction of Cosmid pWE.Ad35.pIX-rITR

[0107] FIG. 6 presents the various steps undertaken to construct the cosmid clone containing Ad35 sequences from bp 3401 to 34794 (end of the right ITR) that are described in detail below.

[0108] A first PCR fragment (pIX-NdeI) was generated using the following primer set:

TABLE-US-00002 35F5: (SEQ ID NO: 16) 5'-CGG AAT TCG CGG CCG CGG TGA GTA TTG GGA AAA C- 3' 35R6: (SEQ ID NO: 17) 5'-CGC CAG ATC GTC TAC AGA ACA G-3'

[0109] DNA polymerase Pwo (Roche) was used according to manufacturer's instructions, however, with an end concentration of 0.6 .mu.M of both primers and using 50 ngr wt Ad35 DNA as template. Amplification was done as follows: 2 minutes at 94.degree. C., 30 cycles of 30 seconds at 94.degree. C., 30 seconds at 65.degree. C. and 1 minute 45 seconds at 72.degree. C., followed by 8 minutes at 68.degree. C. To enable cloning in the TA cloning vector PCR2.1, a last incubation with 1 unit superTaq polymerase (HT Biotechnology LTD) for 10 minutes at 72.degree. C. was performed.

[0110] The 3370 bp amplified fragment contains Ad35 sequences from bp 3401 to 6772 with a NotI site added to the 5' end. Fragments were purified using the PCR purification kit (LTI).

[0111] A second PCR fragment (NdeI-rITR) was generated using the following primers: 35F7: 5'-GAA TGC TGG CTT CAG TTG TAA TC-3' (SEQ ID NO:18); 35R8: 5'-CGG AAT TCG CGG CCG CAT TTA AAT CAT CAT CAA TAA TAT ACC-3' (SEQ ID NO:19).

[0112] Amplification was done with pfx DNA polymerase (LTI) according to manufacturer's instructions but with 0.6 .mu.M of both primers and 3% DMSO using 10 ngr. of wt Ad35 DNA as template. The program was as follows: 3 minutes at 94.degree. C. and five cycles of 30 seconds at 94.degree. C., 45 seconds at 40.degree. C., 2 minutes 45 seconds at 68.degree. C. followed by 25 cycles of 30 seconds at 94.degree. C., 30 seconds at 60.degree. C., 2 minutes 45 seconds at 68.degree. C. To enable cloning in the TA-cloning vector PCR2.1, a last incubation with 1 unit superTaq polymerase for 10 minutes at 72.degree. C. was performed. The 1.6 kb amplified fragment ranging from nucleotides 33178 to the end of the right ITR of Ad35, was purified using the PCR purification kit (LTI).

[0113] Both purified PCR fragments were ligated into the PCR2.1 vector of the TA-cloning kit (Invitrogen) and transformed into STBL-2 competent cells (LTI). Clones containing the expected insert were sequenced to confirm correct amplification. Next, both fragments were excised from the vector by digestion with NotI and NdeI and purified from gel using the GENECLEAN kit (BIO 101, Inc.). Cosmid vector pWE15 (Clonetech) was digested with NotI, dephosphorylated and also purified from gel. These three fragments were ligated and transformed into STBL2 competent cells (LTI). One of the correct clones that contained both PCR fragments was then digested with NdeI, and the linear fragment was purified from gel using the GENECLEAN kit. Ad35 wt DNA was digested with NdeI and the 26.6 kb fragment was purified from LMP gel using agarase enzyme (Roche) according to the manufacturer's instructions. These fragments were ligated together and packaged using .lamda.1 phage packaging extracts (Stratagene) according to the manufacturer's protocol. After infection into STBL-2 cells, colonies were grown on plates and analyzed for presence of the complete insert. One clone with the large fragment inserted in the correct orientation and having the correct restriction patterns after independent digestions with three enzymes (NcoI, PvuII and ScaI) was selected. This clone is designated pWE.Ad35.pIX-rITR. It contains the Ad35 sequences from bp 3401 to the end and is flanked by NotI sites (FIG. 7).

3) Generation of Ad35-Based Recombinant Viruses on PER.C6

[0114] Wild type Ad35 virus can be grown on PER.C6 packaging cells to very high titers. However, whether the Ad5-E1 region that is present in PER.C6 is able to complement E1-deleted Ad35 recombinant viruses is unknown. To test this, PER.C6 cells were cotransfected with the above-described adapter plasmid pAdApt35.LacZ and the large backbone fragment pWE.Ad35.pIX-rITR. First, pAdApt35.LacZ was digested with PacI and pWE.Ad35.pIX-rITR was digested with NotI. Without further purification, 4 .mu.gr of each construct was mixed with DMEM (LTI) and transfected into PER.C6 cells, seeded at a density of 5.times.10.sup.6 cells in a T25 flask the day before, using Lipofectamin (LTI) according to the manufacturer's instructions. As a positive control, 6 .mu.gr of PacI digested pWE.Ad35.pIX-rITR DNA was cotransfected with a 6.7 kb NheI fragment isolated from Ad35 wt DNA containing the left end of the viral genome including the E1 region. The next day, medium (DMEM with 10% FBS and 10 mM MgCl.sub.2) was refreshed and cells were further incubated. At day 2 following the transfection, cells were trypsinized and transferred to T80 flasks. The positive control flask showed CPE at five days following transfection, showing that the pWE.Ad35.pIX-rITR construct is functional, at least in the presence of Ad35-E1 proteins. The transfection with the Ad35 LacZ adapter plasmid and pWE.Ad35.pIX-rITR did not give rise to CPE. These cells were harvested in the medium at day 10 and freeze/thawed once to release virus from the cells. 4 ml of the harvested material was added to a T80 flask with PER.C6 cells (at 80% confluency) and incubated for another five days. This harvest/re-infection was repeated two times but there was no evidence for virus associated CPE.

[0115] From this experiment, it seems that the Ad5-E1 proteins are not, or not well enough, capable of complementing Ad35 recombinant viruses. However, it may be that the sequence overlap of the adapter plasmid and the pWE.Ad35.pIX-rITR backbone plasmid is not large enough to efficiently recombine and give rise to a recombinant virus genome. The positive control transfection was done with a 6.7 kb left end fragment and, therefore, the sequence overlap was about 3.5 kb. The adapter plasmid and the pWE.Ad35.pIX-rITR fragment have a sequence overlap of 1.3 kb. To check whether the sequence overlap of 1.3 kb is too small for efficient homologous recombination, a co-transfection was done with PacI digested pWE.Ad35.pIX-rITR and a PCR fragment of Ad35 wt DNA generated with the above-mentioned 35F1 and 35R4 using the same procedures as previously described herein. The PCR fragment thus contains left end sequences up to bp 4669 and, therefore, has the same overlap sequences with pWE.Ad35.pIX-rITR as the adapter plasmid pAdApt35.LacZ, but has Ad35-E1 sequences. Following PCR column purification, the DNA was digested with SalI to remove possible intact template sequences. A transfection with the digested PCR product alone served as a negative control. Four days after the transfection, CPE occurred in the cells transfected with the PCR product and the Ad35 pIX-rITR fragment, and not in the negative control. This result shows that a 1.3 kb overlapping sequence is sufficient to generate viruses in the presence of Ad35-E1 proteins. From these experiments, we conclude that the presence of at least one of the Ad35-E1 proteins is necessary to generate recombinant Ad35 based vectors from plasmid DNA on Ad5 complementing cell lines.

Example 5

1) Construction of Ad35-E1 Expression Plasmids

[0116] Since Ad5-E1 proteins in PER.C6 are incapable of complementing Ad35 recombinant viruses efficiently, Ad35-E1 proteins have to be expressed in Ad5 complementing cells (e.g., PER.C6). Alternatively, a new packaging cell line expressing Ad35-E1 proteins has to be made, starting from either diploid primary human cells or established cell lines not expressing adenovirus E1 proteins. To address the first possibility, the Ad35-E1 region was cloned in expression plasmids as described below.

[0117] First, the Ad35-E1 region from bp 468 to bp 3400 was amplified from wt Ad35 DNA using the following primer set: 35F11: 5'-GGG GTA CCG AAT TCT CGC TAG GGT ATT TAT ACC-3' (SEQ ID NO:20); 35F10: 5'-GCT CTA GAC CTG CAG GTT AGT CAG TTT CTT CTC CAC TG-3' (SEQ ID NO:21).

[0118] This PCR introduces a KpnI and EcoRI site at the 5' end and an SbfI and XbaI site at the 3' end.

[0119] Amplification on 5 ngr. template DNA was done with Pwo DNA polymerase (Roche) using the manufacturer's instructions, however, with both primers at a final concentration of 0.6 .mu.M. The program was as follows: 2 minutes at 94.degree. C., five cycles of 30 seconds at 94.degree. C., 30 seconds at 56.degree. C. and 2 minutes at 72.degree. C., followed by 25 cycles of 30 seconds at 94.degree. C., 30 seconds at 60.degree. C. and 2 minutes at 72.degree. C., followed by 10 minutes at 72.degree. C. PCR product was purified by a PCR purification kit (LTI) and digested with KpnI and XbaI. The digested PCR fragment was then ligated to the expression vector pRSVhbvNeo (see below) also digested with KpnI and XbaI. Ligations were transformed into competent STBL-2 cells (LTI) according to manufacturer's instructions and colonies were analyzed for the correct insertion of Ad35-E1 sequences into the polylinker in between the RSV promoter and HBV polyA.

[0120] The resulting clone was designated pRSV.Ad35-E1 (FIG. 8). The Ad35 sequences in pRSV.Ad35-E1 were checked by sequence analysis.

[0121] pRSVhbvNeo was generated as follows: pRc-RSV (Invitrogen) was digested with PvuII, dephosphorylated with TSAP enzyme (LTI), and the 3 kb vector fragment was isolated in low melting point agarose (LMP). Plasmid pPGKneopA (FIG. 9; described in International Patent Application WO96/35798) was digested with SspI completely to linearize the plasmid and facilitate partial digestion with PvuII. Following the partial digestion with PvuII, the resulting fragments were separated on a LMP agarose gel and the 2245 bp PvuII fragment, containing the PGK promoter, neomycin-resistance gene and HBVpolyA, was isolated. Both isolated fragments were ligated to give the expression vector pRSV-pNeo that now has the original SV40prom-neo-SV40polyA expression cassette replaced by a PGKprom-neo-HBVpolyA cassette (FIG. 10). This plasmid was further modified to replace the BGHpA with the HBVpA as follows: pRSVpNeo was linearized with ScaI and further digested with XbaI. The 1145 bp fragment, containing part of the Amp gene and the RSV promoter sequences and polylinker sequence, was isolated from gel using the GENECLEAN kit (Bio Inc. 101). Next, pRSVpNeo was linearized with ScaI and further digested partially with EcoRI and the 3704 bp fragment containing the PGKneo cassette and the vector sequences were isolated from gel as above. A third fragment, containing the HBV polyA sequence flanked by XbaI and EcoRI at the 5' and 3' end, respectively, was then generated by PCR amplification on pRSVpNeo using the following primer set: HBV-F: 5'-GGC TCT AGA GAT CCT TCG CGG GAC GTC-3' (SEQ ID NO:22) and HBV-R: 5'-GGC GAA TTC ACT GCC TTC CAC CAA GC-3' (SEQ ID NO:23).

[0122] Amplification was done with Elongase enzyme (LTI) according to the manufacturer's instructions with the following conditions: 30 seconds at 94.degree. C., then five cycles of 45 seconds at 94.degree. C., 1 minute at 42.degree. C. and 1 minute at 68.degree. C., followed by 30 cycles of 45 seconds at 94.degree. C., 1 minute at 65.degree. C. and 1 minute at 68.degree. C., followed by 10 minutes at 68.degree. C. The 625 bp PCR fragment was then purified using the Qiaquick PCR purification kit, digested with EcoRI and XbaI and purified from gel using the GENECLEAN kit. The three isolated fragments were ligated and transformed into DH5.alpha. competent cells (LTI) to give the construct pRSVhbvNeo (FIG. 11). In this construct, the transcription regulatory regions of the RSV expression cassette and the neomycin selection marker are modified to reduce overlap with adenoviral vectors that often contain CMV and SV40 transcription regulatory sequences.

2) Generation of Ad35 Recombinant Viruses on PER.C6 Cells Cotransfected with an Ad35-E1 Expression Construct

[0123] PER.C6 cells were seeded at a density of 5.times.10.sup.6 cells in a T25 flask and, the next day, transfected with a DNA mixture containing:

[0124] 1 .mu.g pAdApt35.LacZ digested with PacI

[0125] 5 .mu.g pRSV.Ad35E1 undigested

[0126] 2 .mu.g pWE.Ad35.pIX-rITR digested with NotI

[0127] Transfection was done using Lipofectamine according to the manufacturer's instructions. Five hours after addition of the transfection mixture to the cells, medium was removed and replaced by fresh medium. After two days, cells were transferred to T80 flasks and further cultured. One week post-transfection, 1 ml of the medium was added to A549 cells and, the following day, cells were stained for LacZ expression. Blue cells were clearly visible after two hours of staining indicating that recombinant LacZ expressing viruses were produced. The cells were further cultured, but no clear appearance of CPE was noted. However, after 12 days, clumps of cells appeared in the monolayer and 18 days following transfection, cells were detached. Cells and medium were then harvested, freeze-thawed once, and 1 ml of the crude lysate was used to infect PER.C6 cells in a six-well plate. Two days after infection, cells were stained for LacZ activity. After two hours, 15% of the cells were stained blue. To test for the presence of wt and/or replicating competent viruses, A549 cells were infected with these viruses and further cultured. No signs of CPE were found indicating the absence of replication-competent viruses. These experiments show that recombinant AdApt35.LacZ viruses were made on PER.C6 cells cotransfected with an Ad35-E1 expression construct.

[0128] Ad35 recombinant viruses escape neutralization in human serum containing neutralizing activity to Ad5 viruses.

[0129] The AdApt35.LacZ viruses were then used to investigate infection in the presence of serum that contains neutralizing activity to Ad5 viruses. Purified Ad5-based LacZ virus served as a positive control for NA. Hereto, PER.C6 cells were seeded in a 24-well plate at a density of 2.times.10.sup.5 cells/well. The next day, a human serum sample with high neutralizing activity to Ad5 was diluted in culture medium in five steps of five times dilutions. 0.5 ml of diluted serum was then mixed with 4.times.10.sup.6 virus particles AdApt5.LacZ virus in 0.5 ml medium and after 30 minutes of incubation at 37.degree. C., 0.5 ml of the mixture was added to PER.C6 cells in duplicate. For the AdApt35.LacZ viruses, 0.5 ml of the diluted serum samples were mixed with 0.5 ml crude lysate containing AdApt35.LacZ virus and, after incubation, 0.5 ml of this mixture was added to PER.C6 cells in duplo. Virus samples incubated in medium without serum were used as positive controls for infection. After two hours of infection at 37.degree. C., medium was added to reach a final volume of 1 ml and cells were further incubated. Two days after infection, cells were stained for LacZ activity. The results are shown in Table II. From these results, it is clear that whereas AdApt5.LacZ viruses are efficiently neutralized, AdApt35.LacZ viruses remain infectious irrespective of the presence of human serum. This proves that recombinant Ad35-based viruses escape neutralization in human sera that contain NA to Ad5-based viruses.

Example 6

Generation of Cell Lines Capable of Complementing E1-deleted Ad35 Viruses

[0130] Generation of pIG135 and pIG270

[0131] Construct pIG.E1A.E1B (FIG. 12) contains E1 region sequences of Ad5 corresponding to nucleotides 459 to 3510 of the wt Ad5 sequence (Genbank accession number M72360) operatively linked to the human phosphoglycerate kinase promoter ("PGK") and the Hepatitis B Virus polyA sequences. The generation of this construct is described in International Patent Application No. WO97/00326. The E1 sequences of Ad5 were replaced by corresponding sequences of Ad35 as follows. pRSV.Ad35-E1 (described in Example 5) was digested with EcoRI and Sse83871 and the 3 kb fragment corresponding to the Ad35-E1 sequences was isolated from gel. Construct pIG.E1A.E1B was digested with Sse83871 completely and partially with EcoRI. The 4.2 kb fragment corresponding to vector sequences without the Ad5-E1 region but retaining the PGK promoter were separated from other fragments on LMP agarose gel and the correct band was excised from gel. Both obtained fragments were ligated resulting in pIG.Ad35-E1.

[0132] This vector was further modified to remove the LacZ sequences present in the pUC19 vector backbone. Hereto, the vector was digested with BsaAI and BstXI and the large fragment was isolated from gel. A double stranded oligo was prepared by annealing the following two oligos: BB1: 5'-GTG CCT AGG CCA CGG GG-3' (SEQ ID NO:24) and BB2: 5'-GTG GCC TAG GCA C-3' (SEQ ID NO:25).

[0133] Ligation of the oligo and the vector fragment resulted in construct pIG135 (FIG. 13). Correct insertion of the oligo restores the BsaAI and BstXI sites and introduces a unique AvrII site. Next, we introduced a unique site at the 3' end of the Ad35-E1 expression cassette in pIG135. Hereto, the construct was digested with SapI and the 3' protruding ends were made blunt by treatment with T4 DNA polymerase. The thus treated linear plasmid was further digested with BsrGI and the large vector-containing fragment was isolated from gel. To restore the 3' end of the HBVpolyA sequence and to introduce a unique site, a PCR fragment was generated using the following primers: 270F: 5'-CAC CTC TGC CTA ATC ATC TC-3' (SEQ ID NO:26) and 270R: 5'-GCT CTA GAA ATT CCA CTG CCT TCC ACC-3' (SEQ ID NO:27).

[0134] The PCR was performed on pIG.Ad35.E1 DNA using Pwo polymerase (Roche) according to the manufacturer's instructions. The obtained PCR product was digested with BsrGI and dephosphorylated using Tsap enzyme (LTI), the latter to prevent insert dimerization on the BsrGI site. The PCR fragment and the vector fragment were ligated to yield construct pIG270 (FIG. 14).

Ad35-E1 Sequences are Capable of Transforming Rat Primary Cells

[0135] Newborn WAG/RIJ rats were sacrificed at one week of gestation and kidneys were isolated. After careful removal of the capsule, kidneys were disintegrated into a single cell suspension by multiple rounds of incubation in trypsin/EDTA (LTI) at 37.degree. C. and collection of floating cells in cold PBS containing 1% FBS. When most of the kidney was trypsinized, all cells were re-suspended in DMEM supplemented with 10% FBS and filtered through a sterile cheesecloth. Baby Rat Kidney (BRK) cells obtained from one kidney were plated in five dishes (Greiner, 6 cm). When a confluency of 70 to 80% was reached, the cells were transfected with 1 or 5 .mu.gr DNA/dish using the CaPO.sub.4 precipitation kit (LTI) according to the manufacturer's instructions. The following constructs were used in separate transfections: pIG.E1A.E1B (expressing the Ad5-E1 region), pRSV.Ad35-E1, pIG.Ad35-E1 and pIG270 (expressing the Ad35-E1 region). Cells were incubated at 37.degree. C., 5% CO.sub.2 until foci of transformed cells appeared. Table III shows the number of foci that resulted from several transfection experiments using circular or linear DNA. As expected, the Ad5-E1 region efficiently transformed BRK cells. Foci also appeared in the Ad35-E1 transfected cell layer although with lower efficiency. The Ad35 transformed foci appeared at a later time point: .about.two weeks post transfection compared with seven to ten days for Ad5-E1. These experiments clearly show that the E1 genes of the B group virus Ad35 are capable of transforming primary rodent cells. This proves the functionality of the Ad35-E1 expression constructs and confirms earlier findings of the transforming capacity of the B-group viruses Ad3 and Ad7 (Dijkema, 1979). To test whether the cells in the foci were really transformed, a few foci were picked and expanded. From the seven picked foci, at least five turned out to grow as established cell lines.

Generation of New Packaging Cells Derived from Primary Human Amniocytes

[0136] Amniotic fluid obtained after amniocentesis was centrifuged and cells were re-suspended in AmnioMax medium (LTI) and cultured in tissue culture flasks at 37.degree. C. and 10% CO.sub.2. When cells were growing nicely (approximately one cell division/24 hours), the medium was replaced with a 1: 1 mixture of AmnioMax complete medium and DMEM low glucose medium (LTI) supplemented with Glutamax I (end concentration 4 mM, LTI) and glucose (end concentration 4.5 gr/L, LTI) and 10% FBS (LTI). For transfection .about.5.times.10.sup.5 cells were plated in 10 cm tissue culture dishes. The day after, cells were transfected with 20 .mu.gr of circular pIG270/dish using the CaPO.sub.4 transfection kit (LTI) according to manufacturer's instructions and cells were incubated overnight with the DNA precipitate. The following day, cells were washed four times with PBS to remove the precipitate and further incubated for over three weeks until foci of transformed cells appeared. Once a week the medium was replaced by fresh medium. Other transfection agents like, but not limited to, LipofectAmine (LTI) or PEI (Polyethylenimine, high molecular weight, water-free, Aldrich) were used. Of these three agents, PEI reached the best transfection efficiency on primary human amniocytes: .about.1% blue cells 48 hours.

Following Transfection of pAdApt35. LacZ

[0137] Foci are isolated as follows. The medium is removed and replaced by PBS after which foci are isolated by gently scraping the cells using a 50 to 200 .mu.l Gilson pipette with a disposable filter tip. Cells contained in .about.10 .mu.ml PBS were brought in a 96-well plate containing 15 .mu.l trypsin/EDTA (LTI) and a single cell suspension was obtained by pipetting up and down and a short incubation at room temperature. After addition of 200 .mu.l of the above described 1:1 mixture of AmnioMax complete medium and DMEM with supplements and 10% FBS, cells were further incubated. Clones that continued to grow were expanded and analyzed for their ability to complement growth of E1-deleted adenoviral vectors of different sub-groups, specifically ones derived from B-group viruses, and more specifically from Ad35 or Ad11.

Generation of New Packaging Cell Lines from HER Cells

[0138] HER cells are isolated and cultured in DMEM medium supplemented with 10% FBS (LTI). The day before transfection, .about.5.times.10.sup.5 cells are plated in 6 cm dishes and cultured overnight at 37.degree. C. and 10% CO.sub.2. Transfection is done using the CaPO.sub.4 precipitation kit (LTI) according to the manufacturer's instructions. Each dish is transfected with 8 to 10 .mu.mgr pIG270 DNA, either as a circular plasmid or as a purified fragment. To obtain the purified fragment, pIG270 was digested with AvrII and XbaI and the 4 kb fragment corresponding to the Ad35-E1 expression cassette was isolated from gel by agarase treatment (Roche). The following day, the precipitate is washed away carefully by four washes with sterile PBS. Then fresh medium is added and transfected cells are further cultured until foci of transformed cells appear. When large enough (>100 cells), foci are picked and brought into 96 wells as described above. Clones of transformed HER cells that continue to grow, are expanded and tested for their ability to complement growth of E1-deleted adenoviral vectors of different sub-groups, specifically ones derived from B-group viruses, and more specifically from Ad35 or Ad11.

New Packaging Cell Lines Derived from PER.C6

[0139] As described in Example 5, it is possible to generate and grow Ad35-E1-deleted viruses on PER.C6 cells with cotransfection of an Ad35-E1 expression construct, e.g., pRSV.Ad35.E1. However, large-scale production of recombinant adenoviruses using this method is cumbersome because, for each amplification step, a transfection of the Ad35-E1 construct is needed. In addition, this method increases the risk of non-homologous recombination between the plasmid and the virus genome with high chances of generation of recombinant viruses that incorporate E1 sequences resulting in replication-competent viruses. To avoid this, the expression of Ad35-E1 proteins in PER.C6 has to be mediated by integrated copies of the expression plasmid in the genome. Since PER.C6 cells are already transformed and express Ad5-E1 proteins, addition of extra Ad35-E1 expression may be toxic for the cells. However, it is not impossible to stably transfect transformed cells with E1 proteins since Ad5-E1-expressing A549 cells have been generated.

[0140] In an attempt to generate recombinant adenoviruses derived from subgroup B virus Ad7, Abrahamsen et al. (1997) were not able to generate E1-deleted viruses on 293 cells without contamination of wt Ad7. Viruses that were picked after plaque purification on 293-ORF6 cells (Brough et al., 1996) were shown to have incorporated Ad7-E1B sequences by nonhomologous recombination. Thus, efficient propagation of Ad7 recombinant viruses proved possible only in the presence of Ad7-E1B expression and Ad5-E4-ORF6 expression. The E1B proteins are known to interact with cellular as well as viral proteins (Bridge et al., 1993; White, 1995). Possibly, the complex formed between the E1B-55K protein and E4-ORF6 which is necessary to increase mRNA export of viral proteins and to inhibit export of most cellular mRNAs is critical and, in some way, serotype-specific. The above experiments suggest that the E1A proteins of Ad5 are capable of complementing an Ad7-E1A deletion and that Ad7-E1B expression in adenovirus packaging cells on itself is not enough to generate a stable complementing cell line. To test whether one or both of the Ad35-E1B proteins is/are the limiting factor in efficient Ad35 vector propagation on PER.C6 cells, we have generated an Ad35 adapter plasmid that does contain the E1B promoter and E1B sequences but lacks the promoter and the coding region for E1A. Hereto, the left end of wt Ad35 DNA was amplified using the primers 35F1 and 35R4 (both described in Example 4) with Pwo DNA polymerase (Roche) according to the manufacturer's instructions. The 4.6 kb PCR product was purified using the PCR purification kit (LTI) and digested with SnaBI and ApaI enzymes. The resulting 4.2 kb fragment was then purified from gel using the QIAExII kit (Qiagen). Next, pAdApt35IP1 (Example 4) was digested with SnaBI and ApaI and the 2.6 kb vector-containing fragment was isolated from gel using the GENECLEAN kit (BIO 101, Inc). Both isolated fragments were ligated to give pBr/Ad35.leftITR-pIX (FIG. 15). Correct amplification during PCR was verified by a functionality test as follows: The DNA was digested with BstBI to liberate the Ad35 insert from vector sequences and 4 .mu.g of this DNA was cotransfected with 4 .mu.g of NotI digested pWE/Ad35.pIX-rITR (Example 4) into PER.C6 cells. The transfected cells were passaged to T80 flasks at day 2 and again two days later CPE had formed showing that the new pBr/Ad35.leftITR-pIX construct contains functional E1 sequences. The pBr/Ad35.leftITR-pIX construct was then further modified as follows. The DNA was digested with SnaBI and HindIII and the 5' HindIII overhang was filled in using Klenow enzyme. Religation of the digested DNA and transformation into competent cells (LTI) gave construct pBr/Ad35.leftITR-pIX.DELTA.DE1A (FIG. 16). This latter construct contains the left end 4.6 kb of Ad35 except for E1A sequences between bp 450 and 1341 (numbering according to SEQ ID NO:44) and thus lacks the E1A promoter and most of the E1A coding sequences. pBr/Ad35.leftITR-pIX.DELTA.DE1A was then digested with BstBI and 2 .mu.g of this construct was cotransfected with 6 .mu.mgr of NotI digested pWE/Ad35.pIX-rITR (Example 4) into PER.C6 cells. One week following transfection, full CPE had formed in the transfected flasks.

[0141] This experiment shows that the Ad35-E1A proteins are functionally complemented by Ad5-E1A expression in PER.C6 cells and that at least one of the Ad35-E1B proteins cannot be complemented by Ad5-E1 expression in PER.C6. It further shows that it is possible to make a complementing cell line for Ad35-E1-deleted viruses by expressing Ad35-E1B proteins in PER.C6. Stable expression of Ad35-E1B sequences from integrated copies in the genome of PER.C6 cells may be driven by the E1B promoter and terminated by a heterologous poly-adenylation signal like, but not limited to, the HBVpA. The heterologous pA signal is necessary to avoid overlap between the E1B insert and the recombinant vector, since the natural E1B termination is located in the pIX transcription unit that has to be present on the adenoviral vector. Alternatively, the E1B sequences may be driven by a heterologous promoter like, but not limited to, the human PGK promoter or by an inducible promoter like, but not limited to, the 7xtetO promoter (Gossen and Bujard, 1992). Also, in these cases, the transcription termination is mediated by a heterologous pA sequence, e.g., the HBV pA. The Ad35-E1B sequences at least comprise one of the coding regions of the E1B-21K and the E1B-55K proteins located between nucleotides 1611 and 3400 of the wt Ad35 sequence. The insert may also include part of the Ad35-E1B sequences between nucleotides 1550 and 1611 of the wt Ad35 sequence (SEQ ID NO:44).

Example 7

Ad35-Based Viruses Deleted for E1A and E1B-21K Genes Efficiently Propagate on Ad5 Complementing Cell Lines

[0142] The generation of Ad35-based viruses that are deleted for E1A and retain the full E1B region is described in Example 6 of this application. Such viruses can be generated and propagated on the Ad5 complementing cell line PER.C6. The E1B region comprises partially overlapping coding sequences for the two major proteins 21K and 55K (Bos et al., 1981). Whereas, during productive wt adenoviral infection, both 21K and 55K are involved in counteracting the apoptose-inducing effects of E1A proteins, the E1B-55K protein has been suggested to have additional functions during the late phase of virus infection. These include the accumulation of viral mRNAs, the control of late viral gene expression and the shutoff of most host mRNAs at the level of mRNA transport (Babiss et al., 1984, 1985; Pilder et al., 1986). A complex formed between E1B-55K and the ORF6 protein encoded by the adenovirus early region 4 (Leppard and Shenk, 1989; Bridge and Ketner, 1990) exerts at least part of these functions.

[0143] To analyze which of the E1B proteins is required for propagation of Ad35-E1A-deleted recombinant viruses on PER.C6 packaging cells, the E1B region in construct pBr.Ad35.leftITR-pIX.DELTA.E1A (see Example 6 and FIG. 16) was further deleted. A first construct, pBr.Ad35.DELTA.21K, retains the full E1B-55K sequence and is deleted for E1A and E1B-21K. Hereto, pBr.Ad35.leftITR-pIX.DELTA.E1A was digested with NcoI and BspE1 and the 5 KB vector fragment was isolated from agarose gel using the GENECLEAN kit (BIO 101, Inc.) according to the manufacturer's instructions. Then a PCR fragment was generated with pBr.Ad35.leftITR-pIX.DELTA.E1A as template DNA using the following primers: 35D21: 5'-TTA GAT CCA TGG ATC CCG CAG ACT C-3' (SEQ ID NO:28) and 35B3: 5'-CCT CAG CCC CAT TTC CAG-3' (SEQ ID NO:29). Amplification was done using Pwo DNA polymerase (Roche) according to manufacturer's recommendations with the addition of DMSO (final concentration 3%) in the reaction mixture. The PCR program was as follows: 94.degree. C. for 2 minutes, then 30 cycles of 94.degree. C. for 30 seconds, 58.degree. C. for 30 seconds and 72.degree. C. for 45 seconds and a final step at 68.degree. C. for 8 minutes to ensure blunt ends.

[0144] This PCR amplifies Ad35-E1B sequences from nucl. 1908 to 2528 (sequence Ad35, SEQ ID NO:44) and introduces an NcoI site at the start codon of the E1B-55K coding sequence (bold in primer 35D21). The 620 bp PCR fragment was purified using the PCR purification kit (Qiagen) and then digested with NcoI and BspEI, purified from agarose gel as above and ligated to the above-described NcoI/BspEI digested vector fragment to give pBr.Ad35.DELTA.21K (FIG. 17).

[0145] Since the coding regions of the 21K and 55K proteins overlap, it is only possible to delete part of the 55K coding sequences while retaining 21K. Hereto, pBr.Ad35.leftITR-pIX.DELTA.E1A was digested with BglII and the vector fragment was religated to give pBr.Ad35.DELTA.55K1 (FIG. 18). This deletion removes E1B coding sequences from nucl. 2261 to 3330 (Ad35 sequence in SEQ ID NO:44). In this construct the N-terminal 115 amino acids are retained and become fused to 21 additional amino acids out of the proper reading frame before a stop codon is encountered. The 21K coding region is intact in construct pBr.Ad35.DELTA.55K1.

[0146] A third construct that has a deletion of E1A, 21K and most of the 55K sequences was generated as follows. pBr.Ad35.leftITR-pIX (FIG. 15) was digested with SnaBI and MfeI (isoschizomer of MunI) and the 5' overhang resulting from the MfeI digestion was filled in using Klenow enzyme. The 4.4 kb vector fragment was isolated from gel using the GENECLEAN kit (BIO 101, Inc.) according to the manufacturer's instructions and religated to give construct pBr.Ad35ASM (FIG. 19). In this construct, the Ad35 sequences between nucl. 453 and 2804 are deleted. Thus, 596 nucl. of the 3' end of E1b-55K are retained. A further deletion of 55K sequences was made in construct pBr.Ad35.DELTA.E1A..DELTA.E1B by digestion of pBr.Ad35.leftITR-pIX with SnaBI and BglII, Klenow treatment to fill in the BglII cohesive ends, and religation. FIG. 20 shows a schematic representation of the above-mentioned constructs.

[0147] To test whether Ad35-based viruses can be generated with these constructs, each of the constructs was cotransfected with NotI digested pWE.Ad35pIX-rITR (see, Example 4) onto PER.C6 cells. Hereto, the respective fragments were PCR amplified using primers 35F1 and 35R4 (see, Example 4). This PCR amplification was done since some of the constructs were difficult to isolate in large enough quantities. In this way, equal quality of the different adapter fragments was ensured. For the amplification, Pwo DNA polymerase (Roche) was used according to the manufacturer's instructions but with DMSO (3% final concentration) added to the PCR mixture. Of each template .about.50 ng DNA was used. The conditions for the PCR were as follows: 94.degree. C. for 2 minutes, then five cycles of 94.degree. C. for 30 seconds, 48.degree. C. for 45 seconds and 72.degree. C. for 4 minutes, followed by 25 cycles of 94.degree. C. for 30 seconds, 60.degree. C. for 30 seconds and 72.degree. C. for 4 minutes and a final step at 68.degree. C. for 8 minutes.

[0148] PCR fragments were generated from pBr.Ad35leftITR-pIX, pBr.Ad35.leftITR-pIX.DELTA.E1A, pBr.Ad35A21K, pBr.Ad35.DELTA.55K1, pBr.Ad35.DELTA.SM and pBr.Ad35.DELTA.E1A.DELTA.E1B. All fragments were using the PCR purification kit (Qiagen) according to manufacturer's instructions and final concentrations were estimated on EtBr stained agarose gel using the Eagle Eye II Still Video system and EagleSight software (Stratagene) with the SmartLadder molecular weight marker (Eurogentec) as reference.

[0149] PER.C6 cells were seeded at a density of 2.5.times.10.sup.6 cells in a T25 culturing flask in DMEM containing 10% fetal calf serum (FCS) and 10 mM MgSO.sub.4 and cultured in a humidified stove at 37.degree. C., 10% CO.sub.2. The next day, 3 mg of each of the PCR fragments was cotransfected with 5 .mu.gr NotI digested pWE.Ad35pIX-rITR using LipofectAmine (GIBCO, Life Technologies Inc.) according to the manufacturer's instructions. Two days after the transfection, all cells were passed to a T80 flask and further cultured. Cultures were then monitored for the appearance of CPE. In line with the outcome of previous experiments described in Examples 4 and 6, pBr.Ad35.leftITR-pIX and pBr.Ad35.leftITR-pIX.DELTA.E1A showed almost full CPE within one week following transfection. Of the fragments with different E1B deletions, only pBr.Ad35.DELTA.21K showed CPE at the same time as the above two fragments. Constructs pBr.Ad35.DELTA.55K1, pBr.Ad35.DELTA.SM and pBr.Ad35.DELTA.E1A.DELTA.E1B did not give CPE at all, not even after harvesting by freeze-thawing and re-infection of the crude lysate onto fresh PER.C6 cells.

[0150] From these experiments, it can be concluded that Ad35-E1B-55K, and not E1B-21K, is necessary for generation and propagation of Ad35-based viruses on Ad5 complementing cell lines. Therefore, Ad35-based viruses having a deletion of the E1A and E1B-21K genes and having the E1B-55K gene or a functional fragment thereof, can be grown on Ad5 complementing cell lines. Alternatively, Ad35-based viruses can be grown on PER.C6 cells that stably express the full E1B region or the E1B-55K gene, or a functional fragment thereof. The Ad35-E1B-55K gene, or functional parts thereof, may be expressed from a heterologous promoter like, but not limited to, the human PGK promoter, the human cytomegalovirus immediate early promoter (CMV), Rous sarcoma virus promoter, etc., and terminated by a heterologous poly adenylation sequence (pA) like, but not limited to, the hepatitis B virus poly adenylation sequence (HBVpA) and the Simian Virus 40 poly adenylation sequence (SV40pA), etc. As nonlimiting examples, PER.C6 cells that express the Ad35-E1B region driven by the E1B promoter and HBVpA, PER.C6 cells that express the Ad35-E1B region driven by the human PGK promoter and HBVpA and PER.C6 cells that express a functional fragment of Ad35-E1B-55K driven by the human PGK promoter and HBVpA are described below.

[0151] We describe the generation of two expression constructs, pIG.35BS and pIG.35BL, that both carry the Ad35-E1B genes and a neomycin selection marker. The two constructs differ in the length of the fragment containing the E1B promoter. In 35BL, the promoter fragment is longer and includes the 3' end of the E1A region (103 nucl. coding sequence and pA). The E1B region is terminated by the HBVpolyA and the neo.sup.r gene is driven by a hPGK promoter/HBVpA cassette.

[0152] pIG.35BL was made as follows. Construct pRSV.Ad35E1 (described in Example 5, FIG. 8) was digested with NruI and HindIII and the protruding ends were filled in by Klenow treatment. The 7 kb vector fragment was separated from the smaller fragment on gel and isolated using the GENECLEAN kit (BIO 101, Inc.). After religation of the DNA and transformation into competent STBL2 cells (Gibco, LTI), correct clones were isolated. pIG.35BL (FIG. 21) contains 273 nucl. upstream of the start site of the E1B-21K coding region.

[0153] pIG.35BS was made in the same way as pIG.35BL except that pRSV.Ad35E1 was digested with NruI and HpaI (both enzymes leave blunt ends), resulting in a shorter fragment upstream of the coding region of E1B-21K: 97 nucleotides.

[0154] To generate Ad35-E1B expressing cells, PER.C6 cells were seeded in 10 cm dishes at 1.times.10.sup.6 cells/dish. Two days later, cells were transfected with ScaI linearized constructs. Four dishes were transfected with 1 .mu.g and four with 2 .mu.g DNA (total of 16 dishes; Lipofectamine (Gibco, LTI), no carrier DNA used) according to the manufacturer's instructions. The next day, transfected cells received G418-containing medium (0.75 mg/ml). Control transfections using LacZ expression constructs (2 .mu.g) were stained after 48 hours and showed a transfection efficiency of .about.25%. Four days following addition of selection medium, untransfected cells started to die and again, three days later, clones were becoming visible. A week later, the first clones were picked. Transfection with 1 .mu.g resulted in less and also, initially, smaller clones (total .about.20 clones/dish against >50 clones/dish for the transfection with 2 .mu.g DNA). The positive control transfection using 2 .mu.g pcDNA3 (Invitrogen) resulted in .about.50 clones.

[0155] In total, 120 clones were picked and 107 were successfully established (55 from pIG35BS and 52 from pIG35BL).

Generation of pIG35Bneo

[0156] pIG35Bneo is an Ad35-E1B expression plasmid from which the E1B genes are expressed from a heterologous promoter (hPGK) and that also contains a neomycin resistance expression cassette. To avoid instability of the plasmid due to recombination events on homologous sequences, the RSV promoter drives the neo.sup.r gene. To achieve this, construct pRSVhbv.Neo (described in Example 5, FIG. 11) was digested with ScaI and BamHI and protruding ends were filled in using Klenow enzyme. The 1070 bp fragment containing part of the Ampicilin gene and the RSV promoter was isolated from gel using the GENECLEAN kit (BIO 101, Inc.). Next, pRSVhbvNeo was digested with ScaI and EcoRI, blunted with Klenow and the 3.2 kb fragment containing the neo gene, HBVpA, vector and part of the Ampicilin gene was isolated as above. The two fragments were then ligated to give pRSVneo4 (FIG. 22). Construct pIG270 (FIG. 14, described in Example 6) was then digested with EcoRI and NcoI and sticky ends were blunted with Klenow enzyme. The vector-containing fragment was isolated from gel as described above and religated to give pIG270delE1A. This construct was digested with AvrII and XbaI and protruding ends were filled in using Klenow enzyme. The 2.9 kb fragment containing the hPGK promoter and Ad35-E1B sequences was isolated from gel as above. Next, pRSVneo4 was digested with BglII, blunted with Klenow enzyme, dephosphorylated and isolated from gel. The blunted AvrII/XbaI Ad35-E1B fragment was then ligated with the above prepared pRSVneo4 vector fragment and resulting clones were analyzed. One clone that contained both expression cassettes in the same orientation was chosen and named pIG35Bneo (FIG. 23). Detailed analysis of this clone revealed that an extra BglII site was present, probably due to an incomplete Klenow reaction (BglII site at nucl. 2949 in FIG. 23).

Generation of pIG35.55K

[0157] Construct pIG35.55K is similar to pIG35Bneo, however, it lacks the coding region of Ad35-E1B-21K. Hereto, both the E1A and E1B-21K sequences are first deleted from pIG270 as follows:

[0158] Construct pIG270 is digested with EcoRI, treated with Klenow enzyme and purified using a PCR purification kit (Qiagen) according to the manufacturer's instructions. The recovered DNA is then digested with AgeI and the .about.5 kb vector fragment was isolated from gel as above. Next, Ad35-E1B-55K sequences are amplified by PCR on pIG270 template DNA using the following primers: 35D21: 5'-TTA GAT CCA TGG ATC CCG CAG ACT C-3' (SEQ ID NO:28) and 35B3: 5'-CCT CAG CCC CAT TTC CAG-3' (SEQ ID NO:29). The conditions used for the amplification are as previously described. The PCR fragment is purified using the PCR purification kit (Qiagen) and digested with NcoI. Following Klenow treatment to fill in the protruding ends, the DNA is further digested with AgeI and again column purified. The thus treated PCR fragment is then cloned into the above prepared EcoRI/AgeI digested vector fragment to give pIG270..DELTA.E1A.DELTA.21K. The last steps to obtain pIG35.55K (FIG. 24) are equivalent to the last steps described above for the generation of pIG35Bneo, starting with pIG270..DELTA.E1AA21K instead of pIG270..DELTA.E1A.

[0159] pIG35.55K is then linearized with ScaI and used to transfect PER.C6 cells as described above. Clones that are resistant to G418 selection are picked and analyzed for their ability to complement the propagation of E1-deleted Ad35 viruses.

Example 8

New Packaging Cell Lines for the Generation and Propagation of E1-Deleted Ad35-Based Vectors Derived from Primary Human Cells

[0160] The complete morphological transformation of primary cells by adenovirus E1 genes is the result of the combined activities of the proteins encoded by the E1A and E1B regions. The roles of the different E1 proteins in lytic infection and in transformation have been studied extensively (reviewed in Zantema and van der Eb, 1995; White, 1995, 1996). The adenovirus E1A proteins are essential for transformation of primary cells. The E1A proteins exert this effect through direct interaction with a number of cellular proteins that are involved in regulation of transcription. These include the pRB family of proteins, p300/CBP and TATA binding protein. In addition to this, E1A increases the level of p53 protein in the cells. In the absence of adenovirus E1B activity, the rise in p53 levels leads to the induction of apoptosis. Both proteins encoded by the E1B region counteract the induction of apoptosis, although by different mechanisms. E1B-21K seems to counteract apoptosis in a manner similar to Bcl-2 via interaction with the effector proteins downstream in the apoptosis pathway (Han et al., 1996), whereas E1B-55K functions through direct interaction with p53. Importantly, the molecular mechanism by which the E1B-55K proteins of Ad2 and 5 (subgroup C) and Ad12 (subgroup A) function in the ability to neutralize p53 may differ. Whereas Ad5 E1B-55K binds p53 strongly and the complex localizes to the cytoplasm, Ad12-E1B-55K binds p53 weakly and both proteins are localized in the nucleus (Zantema et al., 1985; Grand et al., 1999). Both proteins, however, inhibit the transactivation of other genes by p53 (Yew and Berk, 1992).

[0161] In rodent cells, the activity of E1A, together with either E1B-21K or 55K, is sufficient for full transformation, although expression of both E1B proteins together is twice as efficient (Rao et al., 1992). In human cells, however, the activity of the E1B-55K protein seems to be more important, given the observation that E1B-55K is indispensable for the establishment of transformed cells (Gallimore, 1986).

[0162] Example 6 hereof describes the generation of pIG270. In this construct, the Ad35-E1 genes are expressed from the hPGK promoter and transcription is terminated by the HBVpA. The hPGK promoter constitutes a HincII-EcoRI fragment of the promoter sequence described by Singer-Sam et al. (1984). The HBVpA is located in a BamHI-BglII fragment of the Hepatitis B virus genome (Simonsen and Levinson, 1983; see also Genbank HBV-AF090841). As mentioned before, the promoter and polyadenylation sequences of the E1 expression constructs described in this invention may be derived from other sources without departing from the invention. Also, other functional fragments of the hPGK and HBVpA sequences mentioned above may be used.

[0163] The functionality of pIG270 was shown by transformation of primary Baby Rat Kidney cells (BRK). Comparison with an equivalent Ad5-E1 expression construct taught that Ad35-E1 genes were less efficient in transforming these cells. The same has been found for the E1 genes of Ad12 (Bernards et al., 1982).

[0164] It is unclear which E1 protein(s) determine(s) the difference in transformation efficiency of E1 sequences observed for adenoviruses from different subgroups. In the case of Ad12, transfection studies with chimeric E1A/E1B genes suggested that the efficiency of transformation of BRK cells was determined by the E1A proteins (Bernards et al., 1982). The E1B-55K protein is shown infra to contain serotype-specific functions necessary for complementation of E1-deleted adenoviruses. If these functions are related to the regulation of mRNA distribution or another late viral function, it is unlikely that these are involved in the transformation efficiency.

[0165] Analysis of functional domains in the Ad2 or Ad5-E1B-55K proteins using insertion mutants have revealed that functions related to viral replication, late protein synthesis and host protein shut-off are not confined to specific domains but are distributed along the protein (Yew et al., 1990). Using the same set of mutants, the domains important for interaction with p53 and E4-Orf6 were found to be more restricted. In addition to one common binding region (amino acids 262 to 326), p53 binding was affected by mutations at aa 180 and E4-Orf6 binding was affected by mutations at aa 143 (Yew and Berk, 1992; Rubenwolf et al., 1997).

[0166] Altogether, these results indicate that it is difficult to separate the E1B-55K functions related to transformation (p53 binding) and late protein synthesis (Orf6 binding).

[0167] The invention discloses new E1 constructs that combine the high efficiency of transformation of one serotype with the serotype-specific complementation function of another serotype. These new constructs are used to transform primary human embryonic retinoblast cells and human amniocytes.

The Generation of pIG535, pIG635 and pIG735

[0168] Construct pIG535 contains the Ad5-E1A region and E1B promoter sequences linked to the Ad35-E1B sequences. Hereto, pIG270 (FIG. 14; see Example 6) was digested with EcoRI and NcoI. The 5.3 kb vector fragment was then isolated from gel using the GENECLEAN kit (BIO Inc. 101) according to the instructions of the manufacturer. Next, construct pIG.E1A.E1B (FIG. 12; see Example 6) was digested with EcoRI and XbaI and the resulting 890 bp fragment was isolated as above. A third fragment was generated by PCR amplification on pIG.E1A.E1B using the following primers: 5E1A-F: 5'-GAG ACG CCC GAC ATC ACC TG-3' (SEQ ID NO:30) and 5E1B-R: 5'-CAA GCC TCC ATG GGG TCA GAT GTA AC-3' (SEQ ID NO:31). The following PCR program was used: 94.degree. C. for 2 minutes followed by 30 cycles of 94.degree. C. for 30 seconds, 60.degree. C. for 30 seconds and 72.degree. C. for 1 minute, and a final step at 72.degree. C. for 10 minutes to ensure blunt ends.

[0169] The resulting 400 bp PCR fragment was digested with XbaI and NcoI. After gel isolation as above, the three fragments were ligated and transformed into STBL-2 bacteria. One colony containing all three fragments in the correct order was selected and designated pIG535 (FIG. 25).

[0170] Construct pIG635 contains the Ad5-E1A and a chimeric Ad5-Ad35-E1B region such that the 21K sequence is essentially from Ad5 and linked to the Ad35-E1B-55K sequences as far as not overlapping with the 21K sequences. First, part of the Ad5-E1 sequences are amplified by PCR using pIG.E1A.E1B as template and the following primers: 5AK: 5'-GAG CGA AGA AAC CCA TCT GAG-3' (SEQ ID NO:32) and 2155R: 5'-GGT CCA GGC CGG CTC TCG G-3' (SEQ ID NO:33). Amplification is accomplished with Pwo DNA polymerase (Roche) according to manufacturer's instructions. The 210 bp fragments are then purified from the primer sequences using the PCR purification kit (Qiagen).

[0171] A second PCR fragment is amplified from pIG270 DNA as described above but with the following primers: 2155F: 5'-CCG AGA GCC GGC CTG GAC-3' (SEQ ID NO:34) and 35F10: 5'-GCT CTA GAC CTG CAG GTT AGT CAG TTT CTT CTC CAC TG-3' (SEQ ID NO:35).

[0172] The 1.3 kb amplified fragment is purified as above and mixed in a 1:1 molar ratio with the first PCR fragment. The mixture is then first subjected to a PCR reaction without the addition of primers using Pwo DNA polymerase and the following program: 94.degree. C. for 2 minutes and then five cycles of 94.degree. C. for 30 seconds, 60.degree. C. for 30 seconds, 72.degree. C. for 90 seconds. Subsequently, primers 5AK and 35F10 are added at 0.6 .mu.m concentration after which a last PCR amplifies a 1.5 kb fragment. Hereto, temperature was set as follows: 94.degree. C. for 2 minutes, then 30 cycles of 94.degree. C. for 30 seconds, 60.degree. C. for 30 seconds and 72.degree. C. for 90 seconds, followed by a final step at 72.degree. C. for 10 minutes to ensure blunt ends. The resulting product is purified using the PCR purification kit (Qiagen) as above and digested with KpnI and SbfI (isoschizomer of Sse83871). The digested DNA is then isolated from gel using the GENECLEAN kit (BIO Inc., 101). Construct pIG.E1A.E1B is digested with KpnI and SbfI and the vector-containing fragment is isolated from gel as above. This fragment is ligated to the above prepared final PCR product and the ligation mixture is transformed into STBL-2 cells (Gibco, LTI) according to manufacturer's instructions. This gives construct pIG635 (FIG. 26).

[0173] In construct pIG735, the border between Ad5 derived sequences and Ad35 derived sequences is located more 3' than in construct pIG635. First, a BspEI site is introduced in the Ad5 sequence of construct pIG.E1A.E1B without changing the amino acid sequence. Hereto, Ad5 sequences from pIG.E1A.E1B are amplified using the following PCR primers:

[0174] 5AK: see above (SEQ ID NO:32), and Bsp-R: 5'-GCT CTA GAC CTG CAG GGT AGC AAC AAT TCC GGA TAT TTA CAA G-3' (SEQ ID NO:36). Amplification is accomplished using Pwo DNA polymerase (Roche) according to the manufacturer's instruction. The following PCR program is used: 94.degree. C. for 2 minutes followed by 30 cycles of 94.degree. C. for 30 seconds, 60.degree. C. for 30 seconds and 72.degree. C. for 30 seconds, and a final step at 72.degree. C. for 10 minutes to ensure blunt ends. The resulting 0.6 kb fragment is purified as above and digested with KpnI and SbfI and ligated to the above described KpnI/SbfI digested pIG.E1A.E1B vector fragment. Selection of colonies after transformation of STBL-2 bacteria (Life Techn. Inc.) gives construct pIG.E1A55K. pIG.E1A55K is then digested with SbfI and partially with BspEI. The 6.4 kb SbfI-partial BspEI digested vector fragment is then isolated from gel using the GENECLEAN kit (BIO 101, Inc.). Next, pIG270 is digested with BspEI and SbfI and the resulting 915 bp fragment is isolated from gel as above. This fragment is then ligated to the above prepared SbfI/partial BspEI digested pIG.E1.DELTA.55K vector fragment and transformed into STBL-2 competent cells. This gives construct pIG735 (FIG. 27). Clones are analyzed by restriction enzyme digestion and sequencing to ensure correct ligation of the fragments. Constructs pIG535, pIG635 and pIG735 can be used to generate complementing cell lines from primary human cells as described in Example 6.

Example 9

PER.C6-Based Complementing Cell Lines for E1-Deleted Ad35 Viruses

[0175] PER.C6 cells were seeded in 10 cm culture dishes at a density of 3.times.10.sup.6 cells/dish in DMEM (Gibco BRL) complemented with FBS (Gibco BRL) up to 10% and 10 mM MgCl.sub.2 (4.9 M stock solution, Sigma). Two days later, nine dishes were transfected with 1 .mu.g ScaI linearized pIG35.55K DNA (see Example 7) and nine dishes were transfected with 1.5 .mu.g ScaI linearized pIG35.55K DNA. Separate control dishes were transfected with 1 or 1.5 .mu.g ScaI linearized pAdApt35.LacZ to monitor transfection efficiency and with 1 or 1.5 .mu.g ScaI linearized pcDNA.nlsLacZ. pcDNA.nlsLacZ is a pcDNA3-based plasmid (Invitrogen) with the nlsLacZ gene (Bonnerot et al., 1987) driven by the CMV promoter. pcDNA.nlsLacZ also contains a neo.sup.r expression cassette. As a negative control one extra dish was transfected with linearized pAdApt35.LacZ, a construct that lacks the neo.sup.r selection gene. All transfections were performed with the LipofectAmine transfection kit (Invitrogen/Life Technologies) according to manufacturers' instructions using 5 ml LipofectAmine reagent/.mu.g DNA. Cells were incubated for 4 hours with the transfection mixture after which the medium was replaced with PER.C6 culture medium. The next day medium was replaced with culture medium containing 0.5 mg/ml G418 (Gibco BRL) except in the two dishes that were transfected with 1 or 1.5 .mu.g pAdApt35.LacZ. These latter dishes were used to monitor LacZ expression two days following transfection. After X-gal staining of these cultures transfection efficiency was estimated at approximately 40% with slightly more blue cells in the dish transfected with 1.5 .mu.g DNA. Selection medium was refreshed twice weekly in the remaining transfected dishes. Within two weeks following first addition of selection medium most cells in the negative control dish (transfected with 1.5 .mu.g pAdApt35.LacZ) were dead. In the dishes transfected with pcDNA.nlsLacZ cell clones were becoming visible. Since the cells transfected with pIG35.55K seemed to be more resistant to G418, the concentration was raised to 0.75 mg/ml three weeks following transfection. Three days and seven days later a total of 196 cell clones were picked from the dishes transfected with pIG35.55K and seeded in separate wells of 96-well plates.

[0176] Cells remaining after colony picking of two 10 cm dishes of the transfection with 1 .mu.g pIG35.55K DNA were trypsinized, pooled and expanded to give pool PER55K(1.0) The same was done for two dishes of the 1.5 .mu.g transfection. The PER55K(1.0) cell pool was expanded and seeded in four T25 flasks at a density of 3.5.times.10.sup.6 cells/flask for transfection to test virus generation. In addition, three T25 flasks with parental PER.C6 cells were seeded at the same density. pAdApt35.eGFP (an adapter plasmid containing the green fluorescent protein as marker gene; see Example 4) was digested with PacI to liberate the adenoviral sequences from the plasmid backbone. pWE.Ad35.pIX-rITR (see, Example 4) was digested with NotI to liberate the adenoviral sequences from the cosmid backbone. Two flasks with PER.C6 cells and two flasks with PER55K(1.0) cells were transfected with 2 .mu.g digested pAdApt35.eGFP and 6 .mu.g digested pWE.Ad35.pIX-rITR each. One flask of each cell line was transfected with 8 .mu.g pAdApt35.LacZ to monitor transfection efficiency. The remaining flask with PER55K(1.0) cells served as a negative control and was treated as the others but did not receive the transfection mixture. All transfections were performed with LipofectAmine (Invitrogen/Life Techn.) according to manufacturers' instructions using for each transfection a total of 8 .mu.g DNA and 40 .mu.l LipofectAmine reagent. The transfection mixture was removed after 4 hours incubation and fresh culture medium was added. Transfections were done the day after seeding of the cells and again two days later cells in the T25 flasks were transferred to a T80 flask except for the LacZ control transfections. These were stained with X-gal solution after mild fixation. After five hours incubation with staining solution, the percentage of blue cells was estimated at approximately 90% in both flasks showing that transfection went well for both cell lines. Four days following the passage to the T80 flasks the transfected PER55K(1.0) cultures showed starting CPE (cytopathogenic effect, indicative of virus replication) with approximately 100 events/flask. The untransfected PER55K(1.0) cells were grown confluent with no evidence of CPE. In the transfected PER.C6 cultures only three CPE events were visible in the confluent monolayer of cells. Again three days later, the transfected PER55K(1.0) cultures showed full CPE, with all cells rounded and detached in clumbs. In contrast, in the PER.C6 cultures the few events of CPE had not progressed and cells were still in monolayer. This confirms earlier observations that generation of E1-deleted Ad35-based viruses on PER.C6 is very inefficient. Also the untransfected PER55K(1.0) cultures showed, as expected, a confluent monolayer with no CPE. The cells and medium in the PER55K(1.0) flasks with full CPE were harvested and subjected to two freeze/thaw cycles after which the cell debris was removed by centrifugation at 3000 rpm for 10 minutes in a table centrifuge. One of the resulting crude lysates was used to infect a fresh culture of PER55K(1.0) cells in a T175 flask (1.5 ml/flask). Cells and medium were harvested at full CPE four days later. This shows that infectious virus had formed in the initial transfections. GFP expression was confirmed by fluorescent microscopy of A549 cells infected with the crude lysate. The crude lysate was then used to analyze complementation of this E1-deleted Ad35.AdApt.eGFP virus in the individual clones as described below.

[0177] The above-described clones that were picked from the pIG35.55K transfected PER.C6 cells were expanded and were functionally tested for the ability to sustain replication of Ad35.AdApt.eGFP. Hereto, the clones were seeded at two densities in six-well plates and one day later infected with 15 ml of the above described crude lysate. CPE was monitored the day after. Of the 146 clones tested in this way 19 gave full CPE at day 2 or 3 and 68 gave full CPE at day 5 or 6. The remaining clones had only partial CPE or showed a few non-progressing events. The latter were indistinguishable from PER.C6 cells that were taken along as a negative control.

[0178] Based on these results a selection of 24 clones was made that were further screened for the ability to generate recombinant E1-deleted viruses following transfection of the pAdApt35.GFP adapter plasmid and the large pWE.Ad35.pIX-rITR cosmid clone. Hereto, clones were plated in T25 flasks and transfected with 2 .mu.g of the adapter and 6 .mu.g of the backbone plasmid using LipofectAmine as described above. Two days following the transfection, cells were transferred to T80 flasks to prevent overconfluency of the cultures. Of the 24 clones, five gave full CPE three days after passage to T80 and another 13 clones gave progressing to full CPE the day after. The remaining six clones showed no CPE or only starting. In comparison: routine generation of E1-deleted Ad5 vectors on PER.C6 cells generally results in full CPE four to six days after transfer to T80 flasks.

[0179] This shows that the new clones efficiently complement E1-deleted adenovirus vectors. One of the clones (clone #16) described above was used to generate and produce multiple batches of E1 and E1/E3-deleted Ad35 viruses containing different transgenes. Hereto, virus in crude lysates resulting from transfections as described above, but using different adapter plasmids, were plaque purified on the new cell line. Single plaques were tested for transgene activity and then amplified for medium scale production in four to eight triple layer flasks (3.times.175 cm/flask). Cells were harvested at full CPE and the virus was released and purified as routinely done for adenoviruses and described in Example 1. The extraction step with freon to remove cellular debris was, however, replaced by a centrifugation step. Thus after incubation with DnaseI, the cell debris was centrifugated in conical 50 ml tubes (Greiner) at 8000 rpm in a table top centrifuge (Beckman Coulter Allegra 21R with fixed angle rotor) for 30 minutes at 4.degree. C. This step is repeated in a fresh 50 ml tube until the supernatant was clear (usually one time). The amount of virus particles was determined by HPLC (Shabram et al., 1997). Table IV presents the yields after downstream processing of medium scale productions of E1- and E1/E3-deleted Ad35 viruses on triple layer flasks with PER55K clone #16 cells. The amount of purified virus particles is comparable with the yields of Ad5-based vectors on PER.C6 cells.

[0180] We conclude that we have generated multiple cell lines that efficiently complement fully E1-deleted Ad35-based vectors. Thus, Ad35 E1B-55K expression in an Ad5 complementing cell line facilitates replication of Ad35 vectors.

Example 10

New Complementing Cell Lines from Primary Cells

[0181] Example 8 described the generation of construct pIG535, a hybrid Ad5E1A-Ad35 E1B expression plasmid. pCC536s and pIG536 are also hybrid Ad5-Ad35 E1 constructs but with the E1A region, E1B promoter and most of the E1B-19K gene derived from Ad5 and most of the E1B-55K gene derived from Ad35. Constructs pCC536s and pIG536 differ only in the heterologous poly adenylation sequence that terminates the E1B transcript: pIG536 has the HBV pA sequence and pCC536s has a synthetic pA sequence (SpA). The SpA sequence consists of the upstream sequence element (USE) of the human C2 complement gene (Moreira et al., 1995) and the synthetic pA sequence (SPA) described by Levitt et al., 1989.

[0182] The synthetic polyA sequence is build up using the following oligos: C2SPA-1: 5'-CCC TGC AGG GAC TTG ACT CAT GCT TGT TTC ACT TTC ACA TGG AAT TTC CCA GTT ATG AAA TTA ATA AAG-3' (SEQ ID NO:37) and C2SPA-2: 5'-GTC TAG ACA CAC AAA AAA CCA ACA CAC TAT TGC AAT GAA AAT AAA TTT CCT TTA TTA ATT TCA TAA CTG-3' (SEQ ID NO:38). Oligonucleotides were mixed at 10 .mu.M concentration in 1.times. annealing buffer (10 mM Tris HCl pH 7.5, 100 mM NaCl, 1 mM EDTA) and, using a PCR machine, the solution was heated to 94.degree. C. for 5 minutes and then cooled down to 65.degree. C. at 0.5.degree. C./second and after incubation at 65.degree. C. for 5 minutes further cooled down to 20.degree. C. at 0.05.degree. C./second. Subsequently, 10 .mu.l 2 mM dNTPs, 0.5 .mu.l 1 M MgCl2 and 3 .mu.l Klenow fragment (New England Biolabs) was added to 87 .mu.l of the annealed sample and the mixture was incubated at room temperature for 30 minutes. One .mu.l of the annealed and Klenow treated sample was then amplified using the following primers: C2for: 5'-CGG GAT CCC CTG CAG GGA CTT GAC-3' (SEQ ID NO:39) and SPArev: 5'-TTG CGA CTT AAG TCT AGA CAC ACA AAA AAC C-3' (SEQ ID NO:40) using Pwo DNA polymerase (Roche) according to manufacturer's instructions but with addition of DMSO (Sigma) to a final concentration of 3%. The PCR program was set at 94.degree. C. for 2 minutes, followed by 30 cycles of (94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds and 72.degree. C. for 20 seconds). Where in this document PCR programs are described "means time in minutes" and "means time in seconds." The amplified DNA was then purified using the QIAquick PCR purification kit (Qiagen) and digested with XbaI and SbfI. The digested product was then again purified with the PCR purification kit to remove the small digested ends. Construct pIG270 was also digested with XbaI and SbfI (isoschizomer of Sse8387I) and the resulting 5.9 kb vector containing fragment was isolated from gel using the GeneClean II kit (BIO 101, Inc). The treated vector and PCR insert were then ligated to give pCC271 (FIG. 28). pCC271 thus contains the PGK promoter, the Ad35 E1 region (nucl. 468 to and including 3400 from Ad35 sequence in Example 3 and SEQ ID NO:44) and the synthetic pA (SpA). The synthetic pA sequence was then also cloned into the construct pIG535 as follows.

[0183] pIG535 was digested with EcoRI, PstI and ScaI (All enzymes from New England Biolabs digested in NEB buffer 3) and the 3 kb insert corresponding to chimeric Ad5-Ad35 E1 region was purified using the GeneClean II kit (BIO 101, Inc.). Construct pCC271 was digested with EcoRI and PstI and the 3 kb vector fragment containing the SpA and PGK promoter was isolated as above. Both isolated fragments were ligated and transformed into STBL-2 competent cells (Invitrogen/LifeTechnologies) to give pCC535s (FIG. 29). pCC535s contains the same Ad5-Ad35 E1 sequences as pIG535 however, a different pA sequence.

[0184] For the construction of pCC536s, a subclone was made with the new hybrid E1B sequences. Hereto, Ad5 E1A/E1B21K sequences were amplified using the primers 5AK: 5'-GAG CGA AGA AAC CCA TCT GAG-3' (SEQ ID NO:32) and 2155R: 5'-GGT CCA GGC CGG CTC TCG G-3' (SEQ ID NO:33) with pIG.E1A.E1B (see, Example 6 and FIG. 12) as template DNA using Pwo DNA polymerase (Roche) according to manufacturers instructions and in addition a final concentration of 3% DMSO. The program was set at: 94.degree. C. for 2 minutes followed by 30 cycles of (94.degree. C. for 30 seconds, 58.degree. C. for 30 seconds and 72.degree. C. for 30 seconds) and ended with 68.degree. C. for 8 minutes. This resulted in a 210 bp fragment corresponding to nucl. 2022 to 2233 of the Ad5 sequence. A second PCR was performed on pCC271 with primers 2155F: 5'-CCG AGA GCC GGC CTG GAC C-3' (SEQ ID NO:41) and 35F10: 5'-GCT CTA GAC CTG CAG GTT AGT CAG TTT CTT CTC CAC TG-3' (SEQ ID NO:21).

[0185] The same PCR program was used but now with an elongation time of 90 seconds. The resulting 1.3 kb fragment corresponds to nucl. 2112 to 3400 of the Ad35 sequence with an SbfI site at the 3' end. Note that primers 2155F (SEQ ID NO:41) and 2155R (SEQ ID NO:33) are fully complementary allowing assembly of the two fragments as follows:

[0186] Both PCR fragments were purified from gel using the Qiagen gel extraction kit. Aliquots of the purified samples were then mixed in equimolar ratio and used as template for an assembly PCR amplification with primers 5AK (SEQ ID NO:32) and 35F10 (SEQ ID NO:21) with Pwo DNA polymerase as above using the program settings: 94.degree. C. for 2 minutes, and five cycles of (94.degree. C. for 30 seconds, 60.degree. C. for 30 seconds and 72.degree. C. for 2 minutes) followed by 25 cycles of (94.degree. C. for 30 seconds, 58.degree. C. for 30 seconds and 72.degree. C. for 90 seconds). The resulting 1.5 kb fragment was purified from gel using the QIAquick gel extraction kit (Qiagen), ligated to the pCR-Script/Amp cloning vector (Stratagene) and transformed into DH5a competent cells (Invitrogen/Life Technologies) resulting in pCR535E1B (FIG. 30). This construct was checked by restriction analysis and sequencing to confirm correct amplification of target sequences.

[0187] pCR535E1B was then digested with NotI and protruding ends were made blunt with Klenow fragment. The DNA was then purified using the QIAquick PCR purification kit (Qiagen) and eluted DNA was digested with PstI. The 1.5 kb fragment containing the chimeric E1 sequences from the pCR535E1B vector was purified from gel using the GeneClean II kit (BIO 101, Inc.). This fragment was ligated to vector pCC535s digested with PvuII and PstI, and transformed into STBL-2 competent cells (Invitrogen/Life Technologies) to give pCC2155s (FIG. 31). To complete the pCC536s construct Ad5-E1 sequences were then cloned into the pCC2155s subclone. Hereto, pIG.E1A.E1B was digested with EcoRI and KpnI and the 1.6 kb fragment corresponding to Ad5 E1A and Ad5 E1B 21K (nucl. 459 to 2048 of the Ad5 sequence) was isolated from gel using the GeneClean kit. pCC2155s was digested with EcoRI and KpnI and the vector containing fragment was also gel purified. Ligation of both isolated fragments and transformation into DH10B electrocompetent cells (Invitrogen/Life Technologies) resulted in pCC536s (FIG. 32). The hybrid E1B sequences are shown in FIG. 37 in more detail. FIG. 37A shows an alignment of protein sequences of E1B-21K in the pCC536s construct with wild type (wt) Ad35 and Ad5 sequences. As can be seen most of the E1B-21K protein in pCC536s is derived from Ad5 except for the C-terminal six amino acids that are identical to Ad35 E1B-21K. FIG. 37B shows the same alignment for the E1B-55K proteins. In this case the N-terminal amino acids of pCC536s are identical to Ad5 up to aa 65. The remainder is identical to Ad35 E1B-55K. Obviously, different hybrid E1B-55K constructs can be designed using the general method outlined above without departing from the invention.

[0188] Construct pIG536 was made by replacing a fragment with the SpA in pCC536s with the corresponding fragment from pIG270 (Example 6, FIG. 14) containing the HBVpA. Hereto, pIG270 was digested with BamHI and BglI and the 1.8 kb insert was isolated from gel using the GeneClean II kit (BIO 101, Inc.). pCC536s was digested with the same enzymes and the 4.8 kb vector containing fragment was purified from gel as above. Ligation of both isolated fragments and transformation into STBL-2 competent cells (Invitrogen/Life Technologies) gave construct pIG536 (FIG. 33).

[0189] The generated E1 constructs were tested in primary baby rat kidney (BRK) cells as described in Example 6. The results (Table V) confirm earlier observations that Ad5-E1 genes more efficiently transform primary BRK cells than Ad35 E1 genes. The chimeric Ad5-Ad35 E1 expression constructs, pCC535s and pCC536s, produced more transformed colonies than the full Ad35 E1 constructs, pIG270 and pCC271. Furthermore, the use of a synthetic poly adenylation sequence in pCC535s resulted in slightly more foci compared to the HBVpA variant pIG535.

[0190] Human embryonic retinoblast (HER) cells were isolated from the eyes of aborted fetuses of 18 and 21 weeks of age. The eyes were brought in a 6 cm dish with PBS and cleared from outside tissue. An incision was made to reach the inner side and the gray cell layer at the inner back of the eyes containing the retinoblasts, was scraped off. This layer was transferred to a 14 ml tube in 2 ml of PBS and tissue was allowed to sediment after which the PBS was removed. 2 ml trypsin (0.25%, no EDTA, GibcoBRL) was added and incubated for 5 minutes at 37.degree. C. with occasional swirling. Tissue pieces were allowed to sediment and 1 ml trypsin with cells was transferred to a new tube. To this tube 4 ml culture medium (DMEM with 10% FCS) was added and the tube was stored on ice. The remaining tissue pieces in trypsin were brought in a 6 cm dish and cut into smaller pieces. These were, after addition of 2 ml fresh trypsin, again incubated in a 14 ml tube at 37.degree. C. with occasionally swirling. Then this mixture was added to the first isolated cells in culture medium and the total was centrifugated at 1000 rpm in a table top centrifuge. Supernatant was removed and cells were resuspended in 10 ml of culture medium. The isolated HER cells were plated in two 6 cm dishes and incubated at 37.degree. C./10% CO.sub.2. Upon 90% confluency cultures were split 1:3 and further incubated. This procedure was repeated until enough dishes were obtained to be used for transfection and further culturing. Transfections were performed at different passage numbers using the CaPO.sub.4 cotransfection kit (Invitrogen/Life Technologies) according to the manufacturer's instructions. For each dish (50 to 70% confluency) 20 .mu.g DNA was used. Initial transfections were performed with pIG.E1A.E1B, an Ad5-E1 expression construct, and with pIG535, the hybrid Ad5-E1A/Ad35-E1B expression construct. Two to three weeks following transfection transformed foci became visible in the pIG.E1A.E1B transfected dishes. On average, 15 to 20 foci/dish were found in the dishes that were transfected with pIG.E1A.E1B. Over 30 clones were picked and transferred to 96-well plates. Upon confluency cells were passaged to larger culture plates or flasks and finally viable frozen in ampoules in liqN.sub.2 from a T175 flask. All picked clones were established in this way. Transformed foci appeared much later in the dishes that were transfected with pIG535, the first around five weeks following transfection. On average, three to four clones were found per dish. A total of 46 clones were picked from seven weeks to three months after transfections of which 14 were viable and could be passaged multiple times. Of these, two clones (clone #45 and #75) were grown up to a T175 flask and viable frozen in ampoules in liqN.sub.2.

[0191] Primary HER cells were also transfected with constructs pCC535s and pCC536s. Transfection of pCC535s let to an average of two clones/dish and a total of 50 clones were picked. Of these picked clones two could be established. From the transfection with pCCS36s, at least one clone could be established.

[0192] The above-described experiments show that primary HER cells can be transformed with hybrid Ad5-Ad35 E1 sequences. The efficiency of transformation was lower than obtained with the complete Ad5 E1 region. We then tested whether the new cell lines could complement recombinant Ad35-based E1-deleted vectors. Hereto, the clone #45 that was obtained from the pIG535 transfection was seeded in T25 flasks at a density of 7.times.10.sup.6 cells/flask and infected with Ad35.AdApt.eGFP virus (see Example 9) at a multiplicity of infection (moi) of 5 and 25 virus particles/cell. Full CPE was seen at days 4 and 5 for the moi 25 and 5, respectively. As a comparison parallel cultures of clone #45 cells that were infected with Ad5.AdApt.eGFP viruses gave full CPE at days 7 and 8 for moi 25 and 5, respectively. The initial infection efficiency was comparable for Ad5 and Ad35 viruses, .about.80% (moi=5) and .about.95% (moi=25) of the cells were infected with GFP virus one day following infection as measured by fluorescence microscopy. Cells from clone #75 were seeded in a six-well plate at a density of 2.times.10.sup.6 cells/well and infected with Ad35.AdApt.eGFP or Ad5.AdApt.eGFP at moi 5 (VP/cell). Again initial infection efficiency was comparable for both viruses. Full CPE was observed at day 4 in case of Ad35.AdApt.eGFP infection whereas Ad5.AdApt.eGFP infected clone #75 cells gave full CPE on day 7. The difference in replication efficiency on Ad35 complementing cells between Ad35 and Ad5 recombinant vectors is even more clear when virus is generated by plasmid transfection. This is exemplified by the following transfection experiment. Clone #45 cells were seeded in T25 flasks at a density of 3.5.times.10.sup.6 cells and transfected three days later using LipofectAmine reagent (Invitrogen/Life Technologies) according to manufacturers instructions and described above. 2 .mu.g pAdApt35.eGFP adapter plasmid digested with PacI was cotransfected with 6 .mu.g pWE.Ad35.pIX-ITR or pWE.Ad35.pIX-rITR.DELTA.E3 backbone cosmid digested with NotI. 2 .mu.g pAdApt.eGFP (Ad5 adapter plasmid, described in WO 00/70071) digested with PacI was cotransfected with 6 .mu.g pWE.Ad5.AflII-rITRsp (Ad5 backbone plasmid, described in WO 00/70071) also digested with PacI. One T25 was not transfected and served as a negative control. One day later transfection efficiencies were monitored by fluorescent microscopy and estimated at 10 to 15% in all eGFP transfections. Three days following transfection cells were transferred to T80 flasks and further incubated at 37.degree. C./10% CO.sub.2. Again three days later CPE events were becoming visible in the cultures transfected with the pAdApt35.eGFP and the pWE.Ad35pIX-rITR+ or -E3. The transfections with the E3-deleted backbone contained more green fluorescent cells and more CPE events. The transfection with Ad5 plasmids showed only around 20% green fluorescent cells, of which most were dying, and no CPE events. Two days later this difference had become bigger since cultures transfected with the pAdApt35.eGFP and the pWE.Ad35pIX-ITR.DELTA.E3 clearly showed 80% CPE and cultures transfected with the pAdApt35.eGFP and the pWE.Ad35pIX-rITR constructs showed progressing CPE events. The Ad5 transfected culture did not show any progression. Table VI summarizes these results.

[0193] We conclude that the new complementing cell lines described above efficiently sustain replication of E1-deleted Ad35-based viruses and that the generation and replication of E1-deleted Ad5-based viruses is less efficient. Apparently, also Ad35-E1B55K proteins do not form a functional complex with Ad5-E4Orf6 proteins. Thus the serotype specificity for complementation is now also shown for recombinant Ad5 vectors on Ad35 packaging cells.

Example 11

Generation of pWE.Ad.pIX-rITR.DELTA.E3

[0194] The early region-3 of human adenoviruses contains multiple coding regions for proteins that interfere with the host immune response to adenoviral infection. When adenoviral vectors are used as vaccine carrier such interference is unwanted. Therefore, we constructed an Ad35 backbone cosmid lacking the E3 region.

[0195] Hereto, construct pBr.Ad35.PRn (FIG. 34; described in Example 13 in publication EP 1 054 064 A1) was digested with StuI and MluI and the 17.3 kb vector fragment was purified from low melting point (LMP) gel using agarase enzyme (Roche) according to manufacturers instructions. Next, a PCR fragment was generated on pBr.Ad35.PRn using primers: 35E3for: 5'-AAT GAC TAA TGC AGG TGC GC-3' (SEQ ID NO:42) and 35E3rev: 5'-CGA CGC GTT GTA GTC GTT GAG CTT CTA G-3' (SEQ ID NO:43). For the amplification Pwo DNA polymerase (Roche) was used according to manufacturers instructions and program set at: 94.degree. C. for 2 minutes, 30 cycles of (94.degree. C. for 30 seconds, 58.degree. C. for 30 seconds and 72.degree. C. for 1 minute) and a final incubation at 68.degree. C. for 8 minutes. The 833 bp PCR product was purified using the QIAquick PCR purification kit (Qiagen) and digested with MluI and StuI. The digested DNA was purified from gel using the QIAquick gel extraction kit (Qiagen). Both isolated fragments were ligated and transformed into DH5a competent cells (Invitrogen/Life Technologies) to give pBr.Ad35.PRn.DELTA.E3 (FIG. 35). The plasmid was checked by restriction analysis and sequencing of the PCR amplified insert. The E3 deletion was then cloned into the pWE.Ad35.pIX-rITR cosmid backbone. Hereto, pWE.Ad35.pIX-rITR (see Example 4 and FIG. 7) was digested with PacI and the DNA was purified by precipitation with isopropanol and washing with 70% EtOH. Following resuspension in milliQ water, the DNA was digested with SwaI and the 22.8 kb vector containing fragment was purified from LMP gel using agarase enzyme as above. Construct pBr.Ad35.PRn.DELTA.E3 was digested with PacI and SwaI in the same manner and the 16.6 kb fragment was also isolated using agarase enzyme. Both isolated fragments were ligated using 0.5 to 0.6 .mu.g of each fragment. Ligated fragments were then packaged using .lamda.-phage packaging extracts (Stratagene) according to the manufacturer's instructions and mixed with STBL-2 cells. Bacteria were plated on LB+Amp plates and resulting colonies were analyzed for the presence of the correct construct. This gave construct pWE.Ad35.pIX-rITR.DELTA.E3 (FIG. 36). The E3 deletion extends from nucl. 27648 to 30320 of the Ad35 sequence (Example 3) and thus spans a 2.6 kb region.

[0196] Cotransfection of NotI digested pWE.Ad35.pIX-rITR.DELTA.E3 and pIPsp-1 digested pAdApt35.eGFP onto PER55-clone #16 cells (see Example 9) as described above gave rise to GFP expressing Ad35-based viruses. Upon isolation of viral DNA from these viruses, PCR amplification of the E3 region showed that the viruses were deleted for 2.6 kb of E3 sequences as expected.

TABLE-US-00003 TABLE I Sero- Elution log.sub.10 VP/ type [NaCl] mM VP/ml CCID50 CCID50 ratio 1 597 8.66 .times. 10.sup.10 5.00 .times. 10.sup.7 3.2 2 574 1.04 .times. 10.sup.12 .sup. 3.66 .times. 10.sup.11 0.4 3 131 1.19 .times. 10.sup.11 1.28 .times. 10.sup.7 4.0 4 260 4.84 .times. 10.sup.11 2.50 .times. 10.sup.8 3.3 5 533 5.40 .times. 10.sup.11 .sup. 1.12 .times. 10.sup.10 1.7 6 477 1.05 .times. 10.sup.12 .sup. 2.14 .times. 10.sup.10 1.7 7 328 1.68 .times. 10.sup.12 2.73 .times. 10.sup.9 2.4 9 379 4.99 .times. 10.sup.11 3.75 .times. 10.sup.7 4.1 10 387 8.32 .times. 10.sup.12 1.12 .times. 10.sup.9 3.9 12 305 3.64 .times. 10.sup.11 1.46 .times. 10.sup.7 4.4 13 231 4.37 .times. 10.sup.12 7.31 .times. 10.sup.8 3.8 15 443 5.33 .times. 10.sup.12 1.25 .times. 10.sup.9 3.6 16 312 1.75 .times. 10.sup.12 5.59 .times. 10.sup.8 3.5 17 478 1.39 .times. 10.sup.12 1.45 .times. 10.sup.9 3.0 19 430 8.44 .times. 10.sup.11 8.55 .times. 10.sup.7 4.0 20 156 1.41 .times. 10.sup.11 1.68 .times. 10.sup.7 3.9 21 437 3.21 .times. 10.sup.11 1.12 .times. 10.sup.8 3.5 22 365 1.43 .times. 10.sup.12 5.59 .times. 10.sup.7 3.4 23 132 2.33 .times. 10.sup.11 1.57 .times. 10.sup.7 4.2 24 405 5.12 .times. 10.sup.12 4.27 .times. 10.sup.8 4.1 25 405 7.24 .times. 10.sup.11 5.59 .times. 10.sup.7 4.1 26 356 1.13 .times. 10.sup.12 1.12 .times. 10.sup.8 4.0 27 342 2.00 .times. 10.sup.12 1.28 .times. 10.sup.8 4.2 28 347 2.77 .times. 10.sup.12 5.00 .times. 10.sup.7 4.7 29 386 2.78 .times. 10.sup.11 2.00 .times. 10.sup.7 4.1 30 409 1.33 .times. 10.sup.12 5.59 .times. 10.sup.8 3.4 31 303 8.48 .times. 10.sup.10 2.19 .times. 10.sup.7 3.6 33 302 1.02 .times. 10.sup.12 1.12 .times. 10.sup.7 5.0 34 425 1.08 .times. 10.sup.12 .sup. 1.63 .times. 10.sup.11 0.8 35 446 3.26 .times. 10.sup.12 .sup. 1.25 .times. 10.sup.11 1.4 36 325 9.26 .times. 10.sup.12 3.62 .times. 10.sup.9 3.4 37 257 5.86 .times. 10.sup.12 2.8 .times. 10.sup.9 3.3 38 337 3.61 .times. 10.sup.12 5.59 .times. 10.sup.7 4.8 39 241 3.34 .times. 10.sup.11 1.17 .times. 10.sup.7 4.5 42 370 1.95 .times. 10.sup.12 1.12 .times. 10.sup.8 4.2 43 284 2.42 .times. 10.sup.12 1.81 .times. 10.sup.8 4.1 44 295 8.45 .times. 10.sup.11 2.00 .times. 10.sup.7 4.6 45 283 5.20 .times. 10.sup.11 2.99 .times. 10.sup.7 4.2 46 282 9.73 .times. 10.sup.12 2.50 .times. 10.sup.8 4.6 47 271 5.69 .times. 10.sup.11 3.42 .times. 10.sup.7 4.2 48 264 1.68 .times. 10.sup.12 9.56 .times. 10.sup.8 3.3 49 332 2.20 .times. 10.sup.12 8.55 .times. 10.sup.7 4.4 50 459 7.38 .times. 10.sup.12 2.80 .times. 10.sup.9 3.4 51 450 8.41 .times. 10.sup.11 1.88 .times. 10.sup.8 3.7

[0197] Legend to Table I: All human adenoviruses used in the neutralization experiments were produced on PER.C6 cells (Fallaux et al., 1998) and purified on CsCl as described in Example 1. The NaCl concentration at which the different serotypes eluted from the HPLC column is shown. Virus particles/ml (VP/ml) were calculated from an Ad5 standard. The titer in the experiment (CCID50) was determined on PER.C6 cells as described in Example 1 by titrations performed in parallel with the neutralization experiment. The CCID50 is shown for the 44 viruses used in this study and reflects the dilution of the virus needed to obtain CPE in 50% of the wells after five days. The ratio of VP/CCID50 is depicted in log.sub.10 and is a measurement of the infectivity of the different batches on PER.C6 cells.

TABLE-US-00004 TABLE II AdApt35.LacZ viruses escape neutralization by human serum. Human serum dilution Virus no serum 10x 50x 250x 1250x 6250x AdApt5.LacZ 100% 0% 0% 1% 40% 80% moi: 5 VP/cell AdApt35.LacZ 100% 100% 100% 100% 100% 100% 250 .mu.l crude lysate

TABLE-US-00005 TABLE III The numbers of foci obtained with the different E1 expression constructs in BRK transformation experiments. Average # of foci/dish: Construct 1 .mu.gr 5 .mu.gr Experiment 1 pIG.E1A.E1B nd 60 pIG.E1A.E1B nd 35 pRSVAd35E1 0 3 pIG.Ad35.E1 3 7 Experiment 2 pIG.E1A.E1B 37 nd pIG.Ad35.E1 nd 2 Experiment 3 pIG.E1A.E1B nd 140 pIG.Ad35.E1 nd 20 pIG270 nd 30

TABLE-US-00006 TABLE IV Yields of E1- and E1/E3-deleted Ad35 viruses on clone #16 cells produced on triple layer flasks. Scale Total # of Virus Virus (T175III flasks) Particles after DSP VP/cell Ad35.AdApt.eGFP 4 7.5 .times. 10.sup.11 2500 Ad35..DELTA.E3.AdApt.empty 8 2 .times. 10.sup.12 3300 Ad35..DELTA.E3.AdApt.LacZ 8 3.8 .times. 10.sup.11 600 Ad35..DELTA.E3.AdApt.MV-F 4 8.8 .times. 10.sup.11 2900 Ad35..DELTA.E3.AdApt.MV-H 8 2.6 .times. 10.sup.12 4250

TABLE-US-00007 TABLE V Transformation efficiencies on BRK cells with different Ad-E1 expression constructs. Construct Transfected DNA (.mu.g) # foci per dish Experiment 1 pIG.E1A.E1B 5 44 pIG270 5 0 pCC271 5 0 pIG535 5 1 pCC535s 5 2.5 Experiment 2 pIG.E1A.E1B 4 15 pCC271 4 0 pCC535s 4 3 pCC536s 4 3

TABLE-US-00008 TABLE VI Generation of recombinant Ad35 viruses on the new established complementing cell line clone #45. Transfected constructs Day 1 Day 3 Day 6 Day 8 GFP Expression x pAdApt35.eGFP + pWE.Ad35.pIX-rITR 15% 20% 30% 50% pAdApt35.eGFP + pWE.Ad35.pIX-rITR.DELTA.E3 10% 25% 40-50% 100% pAdApt5.eGFP + pWE.Ad5.AflII-rITR 15% 25% 20% 20% untransfected 0% 0% 0% 0% CPE events x pAdApt35.eGFP + pWE.Ad35.pIX-rITR 0 0 1 several pAdApt35.eGFP + pWE.Ad35.pIX-rITR.DELTA.E3 0 0 several 80% pAdApt5.eGFP + pWE.Ad5.AflII-rITR 0 0 0 0 untransfected 0 0 0 0

REFERENCES

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Sequence CWU 1

1

50114DNAadenoviridaemisc_feature(1)..(14)/note="5'end" 1ccaataatat acct 14221DNAadenoviridaemisc_feature(1)..(21)/note="3'end" 2aggtatatta ttgatgatgg g 21318DNAadenoviridaemisc_feature(1)..(18)/note="Terminal sequence" 3catcatcaat aatatacc 18447DNAArtificial SequenceDescription of Artificial Sequence oligo ExSalPacF 4tcgatggcaa acagctatta tgggtattat gggttcgaat taattaa 47547DNAArtificial SequenceDescription of Artificial Sequence oligo ExSalPacR 5tcgattaatt aattcgaacc cataataccc ataatagctg tttgcca 47642DNAArtificial SequenceDescription of Artificial Sequence primer PCLIPMSF 6ccccaattgg tcgaccatca tcaataatat accttatttt gg 42722DNAArtificial SequenceDescription of Artificial Sequence primer pCLIPBSRGI 7gcgaaaattg tcacttcctg tg 22837DNAArtificial SequenceDescription of Artificial Sequence oligo Ecolinker+ 8aattcggcgc gccgtcgacg atatcgatag cggccgc 37937DNAArtificial SequenceDescription of Artificial Sequence oligo Ecolinker- 9aattgcggcc gctatcgata tcgtcgacgg cgcgccg 371049DNAArtificial SequenceDescription of Artificial Sequence oligonucleotide HindXba+ 10agctctagag gatccgttaa cgctagcgaa ttcaccggta ccaagctta 491149DNAArtificial SequenceDescription of Artificial Sequence oligonucleotide HindXba- 11ctagtaagct tggtaccggt gaattcgcta gcgttaacgg atcctctag 491244DNAArtificial SequenceDescription of Artificial Sequence primer 35F1 12cggaattctt aattaatcga catcatcaat aatatacctt atag 441333DNAArtificial SequenceDescription of Artificial Sequence primer 35R2 13ggtggtccta ggctgacacc tacgtaaaaa cag 331430DNAArtificial SequenceDescription of Artificial Sequence primer 335F3 14tggtggagat ctggtgagta ttgggaaaac 301537DNAArtificial SequenceDescription of Artificial Sequence primer 435R4 15cggaattctt aattaaggga aatgcaaatc tgtgagg 371634DNAArtificial SequenceDescription of Artificial Sequence primer 535F5 16cggaattcgc ggccgcggtg agtattggga aaac 341722DNAArtificial SequenceDescription of Artificial Sequence primer 635R6 17cgccagatcg tctacagaac ag 221823DNAArtificial SequenceDescription of Artificial Sequence primer 735F7 18gaatgctggc ttcagttgta atc 231942DNAArtificial SequenceDescription of Artificial Sequence primer 835R8 19cggaattcgc ggccgcattt aaatcatcat caataatata cc 422033DNAArtificial SequenceDescription of Artificial Sequence primer 135F11 20ggggtaccga attctcgcta gggtatttat acc 332138DNAArtificial SequenceDescription of Artificial Sequence primer 235F10 21gctctagacc tgcaggttag tcagtttctt ctccactg 382227DNAArtificial SequenceDescription of Artificial Sequence primer 3HBV-F 22ggctctagag atccttcgcg ggacgtc 272326DNAArtificial SequenceDescription of Artificial Sequence primer 4HBV-R 23ggcgaattca ctgccttcca ccaagc 262417DNAArtificial SequenceDescription of Artificial Sequence oligonucleotide 1BB1 24gtgcctaggc cacgggg 172513DNAArtificial SequenceDescription of Artificial Sequence oligonucleotide 2BB2 25gtggcctagg cac 132620DNAArtificial SequenceDescription of Artificial Sequence primer 3270F 26cacctctgcc taatcatctc 202727DNAArtificial SequenceDescription of Artificial Sequence primer 4270R 27gctctagaaa ttccactgcc ttccacc 272825DNAArtificial SequenceDescription of Artificial Sequence primer 135D21/535D21 28ttagatccat ggatcccgca gactc 252918DNAArtificial SequenceDescription of Artificial Sequence primer 235B3/635B3 29cctcagcccc atttccag 183020DNAArtificial SequenceDescription of Artificial Sequence primer 15E1A-F 30gagacgcccg acatcacctg 203126DNAArtificial SequenceDescription of Artificial Sequence primer 25E1B-R 31caagcctcca tggggtcaga tgtaac 263221DNAArtificial SequenceDescription of Artificial Sequence primer 45AK/5AK 32gagcgaagaa acccatctga g 213319DNAArtificial SequenceDescription of Artificial Sequence primer 52155R/2155R 33ggtccaggcc ggctctcgg 193418DNAArtificial SequenceDescription of Artificial Sequence primer 62155F 34ccgagagccg gcctggac 183538DNAArtificial SequenceDescription of Artificial Sequence primer 735F10/35F10 35gctctagacc tgcaggttag tcagtttctt ctccactg 383643DNAArtificial SequenceDescription of Artificial Sequence primer Bsp-R 36gctctagacc tgcagggtag caacaattcc ggatatttac aag 433769DNAArtificial SequenceDescription of Artificial Sequence oligonucleotide C2SPA-1 37ccctgcaggg acttgactca tgcttgtttc actttcacat ggaatttccc agttatgaaa 60ttaataaag 693869DNAArtificial SequenceDescription of Artificial Sequence oligonucleotide C2SPA-2 38gtctagacac acaaaaaacc aacacactat tgcaatgaaa ataaatttcc tttattaatt 60tcataactg 693924DNAArtificial SequenceDescription of Artificial Sequence primer C2for 39cgggatcccc tgcagggact tgac 244031DNAArtificial SequenceDescription of Artificial Sequence primer SPArev 40ttgcgactta agtctagaca cacaaaaaac c 314119DNAArtificial SequenceDescription of Artificial Sequence primer 2155F 41ccgagagccg gcctggacc 194220DNAArtificial SequenceDescription of Artificial Sequence primer 35E3for 42aatgactaat gcaggtgcgc 204328DNAArtificial SequenceDescription of Artificial Sequence primer 35E3rev 43cgacgcgttg tagtcgttga gcttctag 284434794DNAadenoviridaemisc_feature(1)..(34794)/note="Nucleic acid sequence of Ad 35" 44catcatcaat aatatacctt atagatggaa tggtgccaat atgtaaatga ggtgatttta 60aaaagtgtgg gccgtgtggt gattggctgt ggggttaacg gttaaaaggg gcggcgcggc 120cgtgggaaaa tgacgtttta tgggggtgga gtttttttgc aagttgtcgc gggaaatgtt 180acgcataaaa aggcttcttt tctcacggaa ctacttagtt ttcccacggt atttaacagg 240aaatgaggta gttttgaccg gatgcaagtg aaaattgctg attttcgcgc gaaaactgaa 300tgaggaagtg tttttctgaa taatgtggta tttatggcag ggtggagtat ttgttcaggg 360ccaggtagac tttgacccat tacgtggagg tttcgattac cgtgtttttt acctgaattt 420ccgcgtaccg tgtcaaagtc ttctgttttt acgtaggtgt cagctgatcg ctagggtatt 480tatacctcag ggtttgtgtc aagaggccac tcttgagtgc cagcgagaag agttttctcc 540tctgcgccgg cagtttaata ataaaaaaat gagagatttg cgatttctgc ctcaggaaat 600aatctctgct gagactggaa atgaaatatt ggagcttgtg gtgcacgccc tgatgggaga 660cgatccggag ccacctgtgc agctttttga gcctcctacg cttcaggaac tgtatgattt 720agaggtagag ggatcggagg attctaatga ggaagctgtg aatggctttt ttaccgattc 780tatgctttta gctgctaatg aaggattaga attagatccg cctttggaca ctttcaatac 840tccaggggtg attgtggaaa gcggtacagg tgtaagaaaa ttacctgatt tgagttccgt 900ggactgtgat ttgcactgct atgaagacgg gtttcctccg agtgatgagg aggaccatga 960aaaggagcag tccatgcaga ctgcagcggg tgagggagtg aaggctgcca atgttggttt 1020tcagttggat tgcccggagc ttcctggaca tggctgtaag tcttgtgaat ttcacaggaa 1080aaatactgga gtaaaggaac tgttatgttc gctttgttat atgagaacgc actgccactt 1140tatttacagt aagtgtgttt aagttaaaat ttaaaggaat atgctgtttt tcacatgtat 1200attgagtgtg agttttgtgc ttcttattat aggtcctgtg tctgatgctg atgaatcacc 1260atctcctgat tctactacct cacctcctga tattcaagca cctgttcctg tggacgtgcg 1320caagcccatt cctgtgaagc ttaagcctgg gaaacgtcca gcagtggaga aacttgagga 1380cttgttacag ggtggggacg gacctttgga cttgagtaca cggaaacgtc caagacaata 1440agtgttccat atccgtgttt acttaaggtg acgtcaatat ttgtgtgaga gtgcaatgta 1500ataaaaatat gttaactgtt cactggtttt tattgctttt tgggcgggga ctcaggtata 1560taagtagaag cagacctgtg tggttagctc ataggagctg gctttcatcc atggaggttt 1620gggccatttt ggaagacctt aggaagacta ggcaactgtt agagagcgct tcggacggag 1680tctccggttt ttggagattc tggttcgcta gtgaattagc tagggtagtt tttaggataa 1740aacaggacta taaacaagaa tttgaaaagt tgttggtaga ttgcccagga ctttttgaag 1800ctcttaattt gggccatcag gttcacttta aagaaaaagt tttatcagtt ttagactttt 1860caaccccagg tagaactgct gctgctgtgg cttttcttac ttttatatta gataaatgga 1920tcccgcagac tcatttcagc aggggatacg ttttggattt catagccaca gcattgtgga 1980gaacatggaa ggttcgcaag atgaggacaa tcttaggtta ctggccagtg cagcctttgg 2040gtgtagcggg aatcctgagg catccaccgg tcatgccagc ggttctggag gaggaacagc 2100aagaggacaa cccgagagcc ggcctggacc ctccagtgga ggaggcggag tagctgactt 2160gtctcctgaa ctgcaacggg tgcttactgg atctacgtcc actggacggg ataggggcgt 2220taagagggag agggcatcca gtggtactga tgctagatct gagttggctt taagtttaat 2280gagtcgcaga cgtcctgaaa ccatttggtg gcatgaggtt cagaaagagg gaagggatga 2340agtttctgta ttgcaggaga aatattcact ggaacaggtg aaaacatgtt ggttggagcc 2400agaggatgat tgggcggtgg ccattaaaaa ttatgccaag atagctttga ggcctgataa 2460acagtataag atcagtagac ggattaatat ccggaatgct tgttacatat ctggaaatgg 2520ggctgaggtg gtaatagata ctcaagacaa gacagttatt agatgctgca tgatggatat 2580gtggcctgga gtagtcggta tggaagcagt cacttttgta aatgttaagt ttaggggaga 2640tggttataat ggaatagtgt ttatggccaa taccaaactt atattgcatg gttgtagctt 2700ttttggtttc aacaatacct gtgtagatgc ctggggacag gttagtgtac gggggtgtag 2760tttctatgcg tgttggattg ccacagctgg cagaaccaag agtcaattgt ctctgaagaa 2820atgcatattc caaagatgta acctgggcat tctgaatgaa ggcgaagcaa gggtccgtca 2880ctgcgcttct acagatactg gatgttttat tttaattaag ggaaatgcca gcgtaaagca 2940taacatgatt tgtggtgctt ccgatgagag gccttatcaa atgctcactt gtgctggtgg 3000gcattgtaat atgctggcta ctgtgcatat tgtttcccat caacgcaaaa aatggcctgt 3060ttttgatcac aatgtgttga ccaagtgcac catgcatgca ggtgggcgta gaggaatgtt 3120tatgccttac cagtgtaaca tgaatcatgt gaaagtgttg ttggaaccag atgccttttc 3180cagaatgagc ctaacaggaa tctttgacat gaacacgcaa atctggaaga tcctgaggta 3240tgatgatacg agatcgaggg tgcgcgcatg cgaatgcgga ggcaagcatg ccaggttcca 3300gccggtgtgt gtagatgtga ccgaagatct cagaccggat catttggtta ttgcccgcac 3360tggagcagag ttcggatcca gtggagaaga aactgactaa ggtgagtatt gggaaaactt 3420tggggtggga ttttcagatg gacagattga gtaaaaattt gttttttctg tcttgcagct 3480gacatgagtg gaaatgcttc ttttaagggg ggagtcttca gcccttatct gacagggcgt 3540ctcccatcct gggcaggagt tcgtcagaat gttatgggat ctactgtgga tggaagaccc 3600gttcaacccg ccaattcttc aacgctgacc tatgctactt taagttcttc acctttggac 3660gcagctgcag ccgctgccgc cgcctctgtc gccgctaaca ctgtgcttgg aatgggttac 3720tatggaagca tcgtggctaa ttccacttcc tctaataacc cttctacact gactcaggac 3780aagttacttg tccttttggc ccagctggag gctttgaccc aacgtctggg tgaactttct 3840cagcaggtgg ccgagttgcg agtacaaact gagtctgctg tcggcacggc aaagtctaaa 3900taaaaaaaat tccagaatca atgaataaat aaacgagctt gttgttgatt taaaatcaag 3960tgtttttatt tcatttttcg cgcacggtat gccctggacc accgatctcg atcattgaga 4020actcggtgga ttttttccag aatcctatag aggtgggatt gaatgtttag atacatgggc 4080attaggccgt ctttggggtg gagatagctc cattgaaggg attcatgctc cggggtagtg 4140ttgtaaatca cccagtcata acaaggtcgc agtgcatggt gttgcacaat atcttttaga 4200agtaggctga ttgccacaga taagcccttg gtgtaggtgt ttacaaaccg gttgagctgg 4260gaggggtgca ttcgaggtga aattatgtgc attttggatt ggatttttaa gttggcaata 4320ttgccgccaa gatcccgtct tgggttcatg ttatgaagga ctaccaagac ggtgtatccg 4380gtacatttag gaaatttatc gtgcagcttg gatggaaaag cgtggaaaaa tttggagaca 4440cccttgtgtc ctccgagatt ttccatgcac tcatccatga taatagcaat ggggccgtgg 4500gcagcggcgc gggcaaacac gttccgtggg tctgacacat catagttatg ttcctgagtt 4560aaatcatcat aagccatttt aatgaatttg gggcggagcg taccagattg gggtatgaat 4620gttccttcgg gccccggagc atagttcccc tcacagattt gcatttccca agctttcagt 4680tctgagggtg gaatcatgtc cacctggggg gctatgaaga acaccgtttc gggggcgggg 4740gtgattagtt gggatgatag caagtttctg agcaattgag atttgccaca tccggtgggg 4800ccataaataa ttccgattac aggttgcagg tggtagttta gggaacggca actgccgtct 4860tctcgaagca agggggccac ctcgttcatc atttccctta catgcatatt ttcccgcacc 4920aaatccatta ggaggcgctc tcctcctagt gatagaagtt cttgtagtga ggaaaagttt 4980ttcagcggtt ttagaccgtc agccatgggc attttggaaa gagtttgctg caaaagttct 5040agtctgttcc acagttcagt gatgtgttct atggcatctc gatccagcag acctcctcgt 5100ttcgcgggtt tggacggctc ctggagtagg gtatgagacg atgggcgtcc agcgctgcca 5160gggttcggtc cttccagggt ctcagtgttc gagtcagggt tgtttccgtc acagtgaagg 5220ggtgtgcgcc tgcttgggcg cttgccaggg tgcgcttcag actcattctg ctggtggaga 5280acttctgtcg cttggcgccc tgtatgtcgg ccaagtagca gtttaccatg agttcgtagt 5340tgagcgcctc ggctgcgtgg cctttggcgc ggagcttacc tttggaagtt ttcttgcata 5400ccgggcagta taggcatttc agcgcataca gcttgggcgc aaggaaaatg gattctgggg 5460agtatgcatc cgcgccgcag gaggcgcaaa cagtttcaca ttccaccagc caggttaaat 5520ccggttcatt ggggtcaaaa acaagttttc cgccatattt tttgatgcgt ttcttacctt 5580tggtctccat aagttcgtgt cctcgttgag tgacaaacag gctgtccgta tctccgtaga 5640ctgattttac aggcctcttc tccagtggag tgcctcggtc ttcttcgtac aggaactctg 5700accactctga tacaaaggcg cgcgtccagg ccagcacaaa ggaggctatg tgggaggggt 5760agcgatcgtt gtcaaccagg gggtccacct tttccaaagt atgcaaacac atgtcaccct 5820cttcaacatc caggaatgtg attggcttgt aggtgtattt cacgtgacct ggggtccccg 5880ctgggggggt ataaaagggg gcggttcttt gctcttcctc actgtcttcc ggatcgctgt 5940ccaggaacgt cagctgttgg ggtaggtatt ccctctcgaa ggcgggcatg acctctgcac 6000tcaggttgtc agtttctaag aacgaggagg atttgatatt gacagtgccg gttgagatgc 6060ctttcatgag gttttcgtcc atttggtcag aaaacacaat ttttttattg tcaagtttgg 6120tggcaaatga tccatacagg gcgttggata aaagtttggc aatggatcgc atggtttggt 6180tcttttcctt gtccgcgcgc tctttggcgg cgatgttgag ttggacatac tcgcgtgcca 6240ggcacttcca ttcggggaag atagttgtta attcatctgg cacgattctc acttgccacc 6300ctcgattatg caaggtaatt aaatccacac tggtggccac ctcgcctcga aggggttcat 6360tggtccaaca gagcctacct cctttcctag aacagaaagg gggaagtggg tctagcataa 6420gttcatcggg agggtctgca tccatggtaa agattcccgg aagtaaatcc ttatcaaaat 6480agctgatggg agtggggtca tctaaggcca tttgccattc tcgagctgcc agtgcgcgct 6540catatgggtt aaggggactg ccccagggca tgggatgggt gagagcagag gcatacatgc 6600cacagatgtc atagacgtag atgggatcct caaagatgcc tatgtaggtt ggatagcatc 6660gcccccctct gatacttgct cgcacatagt catatagttc atgtgatggc gctagcagcc 6720ccggacccaa gttggtgcga ttgggttttt ctgttctgta gacgatctgg cgaaagatgg 6780cgtgagaatt ggaagagatg gtgggtcttt gaaaaatgtt gaaatgggca tgaggtagac 6840ctacagagtc tctgacaaag tgggcataag attcttgaag cttggttacc agttcggcgg 6900tgacaagtac gtctagggcg cagtagtcaa gtgtttcttg aatgatgtca taacctggtt 6960ggtttttctt ttcccacagt tcgcggttga gaaggtattc ttcgcgatcc ttccagtact 7020cttctagcgg aaacccgtct ttgtctgcac ggtaagatcc tagcatgtag aactgattaa 7080ctgccttgta agggcagcag cccttctcta cgggtagaga gtatgcttga gcagcttttc 7140gtagcgaagc gtgagtaagg gcaaaggtgt ctctgaccat gactttgaga aattggtatt 7200tgaagtccat gtcgtcacag gctccctgtt cccagagttg gaagtctacc cgtttcttgt 7260aggcggggtt gggcaaagcg aaagtaacat cattgaagag aatcttaccg gctctgggca 7320taaaattgcg agtgatgcgg aaaggctgtg gtacttccgc tcgattgttg atcacctggg 7380cagctaggac gatttcgtcg aaaccgttga tgttgtgtcc tacgatgtat aattctatga 7440aacgcggcgt gcctctgacg tgaggtagct tactgagctc atcaaaggtt aggtctgtgg 7500ggtcagataa ggcgtagtgt tcgagagccc attcgtgcag gtgaggattt gcatgtagga 7560atgatgacca aagatctacc gccagtgctg tttgtaactg gtcccgatac tgacgaaaat 7620gccggccaat tgccattttt tctggagtga cacagtagaa ggttctgggg tcttgttgcc 7680atcgatccca cttgagttta atggctagat cgtgggccat gttgacgaga cgctcttctc 7740ctgagagttt catgaccagc atgaaaggaa ctagttgttt gccaaaggat cccatccagg 7800tgtaagtttc cacatcgtag gtcaggaaga gtctttctgt gcgaggatga gagccgatcg 7860ggaagaactg gatttcctgc caccagttgg aggattggct gttgatgtga tggaagtaga 7920agtttctgcg gcgcgccgag cattcgtgtt tgtgcttgta cagacggccg cagtagtcgc 7980agcgttgcac gggttgtatc tcgtgaatga gctgtacctg gcttcccttg acgagaaatt 8040tcagtgggaa gccgaggcct ggcgattgta tctcgtgctc ttctatattc gctgtatcgg 8100cctgttcatc ttctgtttcg atggtggtca tgctgacgag cccccgcggg aggcaagtcc 8160agacctcggc gcgggagggg cggagctgaa ggacgagagc gcgcaggctg gagctgtcca 8220gagtcctgag acgctgcgga ctcaggttag taggtaggga cagaagatta acttgcatga 8280tcttttccag ggcgtgcggg aggttcagat ggtacttgat ttccacaggt tcgtttgtag 8340agacgtcaat ggcttgcagg gttccgtgtc ctttgggcgc cactaccgta cctttgtttt 8400ttcttttgat cggtggtggc tctcttgctt cttgcatgct cagaagcggt gacggggacg 8460cgcgccgggc ggcagcggtt gttccggacc cgggggcatg gctggtagtg gcacgtcggc 8520gccgcgcacg ggcaggttct ggtattgcgc tctgagaaga cttgcgtgcg ccaccacgcg 8580tcgattgacg tcttgtatct gacgtctctg ggtgaaagct accggccccg tgagcttgaa 8640cctgaaagag agttcaacag aatcaatttc ggtatcgtta acggcagctt gtctcagtat 8700ttcttgtacg tcaccagagt tgtcctggta ggcgatctcc gccatgaact gctcgatttc 8760ttcctcctga agatctccgc gacccgctct ttcgacggtg gccgcgaggt cattggagat 8820acggcccatg agttgggaga atgcattcat gcccgcctcg ttccagacgc ggctgtaaac 8880cacggccccc tcggagtctc ttgcgcgcat caccacctga gcgaggttaa gctccacgtg 8940tctggtgaag accgcatagt tgcataggcg ctgaaaaagg tagttgagtg tggtggcaat 9000gtgttcggcg acgaagaaat acatgatcca tcgtctcagc ggcatttcgc taacatcgcc 9060cagagcttcc aagcgctcca tggcctcgta gaagtccacg gcaaaattaa aaaactggga 9120gtttcgcgcg gacacggtca attcctcctc gagaagacgg

atgagttcgg ctatggtggc 9180ccgtacttcg cgttcgaagg ctcccgggat ctcttcttcc tcttctatct cttcttccac 9240taacatctct tcttcgtctt caggcggggg cggagggggc acgcggcgac gtcgacggcg 9300cacgggcaaa cggtcgatga atcgttcaat gacctctccg cggcggcggc gcatggtttc 9360agtgacggcg cggccgttct cgcgcggtcg cagagtaaaa acaccgccgc gcatctcctt 9420aaagtggtga ctgggaggtt ctccgtttgg gagggagagg gcgctgatta tacattttat 9480taattggccc gtagggactg cgcgcagaga tctgatcgtg tcaagatcca cgggatctga 9540aaacctttcg acgaaagcgt ctaaccagtc acagtcacaa ggtaggctga gtacggcttc 9600ttgtgggcgg gggtggttat gtgttcggtc tgggtcttct gtttcttctt catctcggga 9660aggtgagacg atgctgctgg tgatgaaatt aaagtaggca gttctaagac ggcggatggt 9720ggcgaggagc accaggtctt tgggtccggc ttgctggata cgcaggcgat tggccattcc 9780ccaagcatta tcctgacatc tagcaagatc tttgtagtag tcttgcatga gccgttctac 9840gggcacttct tcctcacccg ttctgccatg catacgtgtg agtccaaatc cgcgcattgg 9900ttgtaccagt gccaagtcag ctacgactct ttcggcgagg atggcttgct gtacttgggt 9960aagggtggct tgaaagtcat caaaatccac aaagcggtgg taagcccctg tattaatggt 10020gtaagcacag ttggccatga ctgaccagtt aactgtctgg tgaccagggc gcacgagctc 10080ggtgtattta aggcgcgaat aggcgcgggt gtcaaagatg taatcgttgc aggtgcgcac 10140cagatactgg taccctataa gaaaatgcgg cggtggttgg cggtagagag gccatcgttc 10200tgtagctgga gcgccagggg cgaggtcttc caacataagg cggtgatagc cgtagatgta 10260cctggacatc caggtgattc ctgcggcggt agtagaagcc cgaggaaact cgcgtacgcg 10320gttccaaatg ttgcgtagcg gcatgaagta gttcattgta ggcacggttt gaccagtgag 10380gcgcgcgcag tcattgatgc tctatagaca cggagaaaat gaaagcgttc agcgactcga 10440ctccgtagcc tggaggaacg tgaacgggtt gggtcgcggt gtaccccggt tcgagacttg 10500tactcgagcc ggccggagcc gcggctaacg tggtattggc actcccgtct cgacccagcc 10560tacaaaaatc caggatacgg aatcgagtcg ttttgctggt ttccgaatgg cagggaagtg 10620agtcctattt tttttttttt tttgccgctc agatgcatcc cgtgctgcga cagatgcgcc 10680cccaacaaca gcccccctcg cagcagcagc agcagcaacc acaaaaggct gtccctgcaa 10740ctactgcaac tgccgccgtg agcggtgcgg gacagcccgc ctatgatctg gacttggaag 10800agggcgaagg actggcacgt ctaggtgcgc cttcgcccga gcggcatccg cgagttcaac 10860tgaaaaaaga ttctcgcgag gcgtatgtgc cccaacagaa cctatttaga gacagaagcg 10920gcgaggagcc ggaggagatg cgagcttccc gctttaacgc gggtcgtgag ctgcgtcacg 10980gtttggaccg aagacgagtg ttgcgagacg aggatttcga agttgatgaa gtgacaggga 11040tcagtcctgc cagggcacac gtggctgcag ccaaccttgt atcggcttac gagcagacag 11100taaaggaaga gcgtaacttc caaaagtctt ttaataatca tgtgcgaacc ctgattgccc 11160gcgaagaagt tacccttggt ttgatgcatt tgtgggattt gatggaagct atcattcaga 11220accctactag caaacctctg accgcccagc tgtttctggt ggtgcaacac agcagagaca 11280atgaggcttt cagagaggcg ctgctgaaca tcaccgaacc cgaggggaga tggttgtatg 11340atcttatcaa cattctacag agtatcatag tgcaggagcg gagcctgggc ctggccgaga 11400aggtagctgc catcaattac tcggttttga gcttgggaaa atattacgct cgcaaaatct 11460acaagactcc atacgttccc atagacaagg aggtgaagat agatgggttc tacatgcgca 11520tgacgctcaa ggtcttgacc ctgagcgatg atcttggggt gtatcgcaat gacagaatgc 11580atcgcgcggt tagcgccagc aggaggcgcg agttaagcga cagggaactg atgcacagtt 11640tgcaaagagc tctgactgga gctggaaccg agggtgagaa ttacttcgac atgggagctg 11700acttgcagtg gcagcctagt cgcagggctc tgagcgccgc gacggcagga tgtgagcttc 11760cttacataga agaggcggat gaaggcgagg aggaagaggg cgagtacttg gaagactgat 11820ggcacaaccc gtgttttttg ctagatggaa cagcaagcac cggatcccgc aatgcgggcg 11880gcgctgcaga gccagccgtc cggcattaac tcctcggacg attggaccca ggccatgcaa 11940cgtatcatgg cgttgacgac tcgcaacccc gaagccttta gacagcaacc ccaggccaac 12000cgtctatcgg ccatcatgga agctgtagtg ccttcccgat ctaatcccac tcatgagaag 12060gtcctggcca tcgtgaacgc gttggtggag aacaaagcta ttcgtccaga tgaggccgga 12120ctggtataca acgctctctt agaacgcgtg gctcgctaca acagtagcaa tgtgcaaacc 12180aatttggacc gtatgataac agatgtacgc gaagccgtgt ctcagcgcga aaggttccag 12240cgtgatgcca acctgggttc gctggtggcg ttaaatgctt tcttgagtac tcagcctgct 12300aatgtgccgc gtggtcaaca ggattatact aactttttaa gtgctttgag actgatggta 12360tcagaagtac ctcagagcga agtgtatcag tccggtcctg attacttctt tcagactagc 12420agacagggct tgcagacggt aaatctgagc caagctttta aaaaccttaa aggtttgtgg 12480ggagtgcatg ccccggtagg agaaagagca accgtgtcta gcttgttaac tccgaactcc 12540cgcctgttat tactgttggt agctcctttc accgacagcg gtagcatcga ccgtaattcc 12600tatttgggtt acctactaaa cctgtatcgc gaagccatag ggcaaagtca ggtggacgag 12660cagacctatc aagaaattac ccaagtcagt cgcgctttgg gacaggaaga cactggcagt 12720ttggaagcca ctctgaactt cttgcttacc aatcggtctc aaaagatccc tcctcaatat 12780gctcttactg cggaggagga gaggatcctt agatatgtgc agcagagcgt gggattgttt 12840ctgatgcaag agggggcaac tccgactgca gcactggaca tgacagcgcg aaatatggag 12900cccagcatgt atgccagtaa ccgacctttc attaacaaac tgctggacta cttgcacaga 12960gctgccgcta tgaactctga ttatttcacc aatgccatct taaacccgca ctggctgccc 13020ccacctggtt tctacacggg cgaatatgac atgcccgacc ctaatgacgg atttctgtgg 13080gacgacgtgg acagcgatgt tttttcacct ctttctgatc atcgcacgtg gaaaaaggaa 13140ggcggtgata gaatgcattc ttctgcatcg ctgtccgggg tcatgggtgc taccgcggct 13200gagcccgagt ctgcaagtcc ttttcctagt ctaccctttt ctctacacag tgtacgtagc 13260agcgaagtgg gtagaataag tcgcccgagt ttaatgggcg aagaggagta cctaaacgat 13320tccttgctca gaccggcaag agaaaaaaat ttcccaaaca atggaataga aagtttggtg 13380gataaaatga gtagatggaa gacttatgct caggatcaca gagacgagcc tgggatcatg 13440gggactacaa gtagagcgag ccgtagacgc cagcgccatg acagacagag gggtcttgtg 13500tgggacgatg aggattcggc cgatgatagc agcgtgttgg acttgggtgg gagaggaagg 13560ggcaacccgt ttgctcattt gcgccctcgc ttgggtggta tgttgtgaaa aaaaataaaa 13620aagaaaaact caccaaggcc atggcgacga gcgtacgttc gttcttcttt attatctgtg 13680tctagtataa tgaggcgagt cgtgctaggc ggagcggtgg tgtatccgga gggtcctcct 13740ccttcgtacg agagcgtgat gcagcagcag caggcgacgg cggtgatgca atccccactg 13800gaggctccct ttgtgcctcc gcgatacctg gcacctacgg agggcagaaa cagcattcgt 13860tactcggaac tggcacctca gtacgatacc accaggttgt atctggtgga caacaagtcg 13920gcggacattg cttctctgaa ctatcagaat gaccacagca acttcttgac cacggtggtg 13980cagaacaatg actttacccc tacggaagcc agcacccaga ccattaactt tgatgaacga 14040tcgcggtggg gcggtcagct aaagaccatc atgcatacta acatgccaaa cgtgaacgag 14100tatatgttta gtaacaagtt caaagcgcgt gtgatggtgt ccagaaaacc tcccgacggt 14160gctgcagttg gggatactta tgatcacaag caggatattt tggaatatga gtggttcgag 14220tttactttgc cagaaggcaa cttttcagtt actatgacta ttgatttgat gaacaatgcc 14280atcatagata attacttgaa agtgggtaga cagaatggag tgcttgaaag tgacattggt 14340gttaagttcg acaccaggaa cttcaagctg ggatgggatc ccgaaaccaa gttgatcatg 14400cctggagtgt atacgtatga agccttccat cctgacattg tcttactgcc tggctgcgga 14460gtggatttta ccgagagtcg tttgagcaac cttcttggta tcagaaaaaa acagccattt 14520caagagggtt ttaagatttt gtatgaagat ttagaaggtg gtaatattcc ggccctcttg 14580gatgtagatg cctatgagaa cagtaagaaa gaacaaaaag ccaaaataga agctgctaca 14640gctgctgcag aagctaaggc aaacatagtt gccagcgact ctacaagggt tgctaacgct 14700ggagaggtca gaggagacaa ttttgcgcca acacctgttc cgactgcaga atcattattg 14760gccgatgtgt ctgaaggaac ggacgtgaaa ctcactattc aacctgtaga aaaagatagt 14820aagaatagaa gctataatgt gttggaagac aaaatcaaca cagcctatcg cagttggtat 14880ctttcgtaca attatggcga tcccgaaaaa ggagtgcgtt cctggacatt gctcaccacc 14940tcagatgtca cctgcggagc agagcaggtt tactggtcgc ttccagacat gatgaaggat 15000cctgtcactt tccgctccac tagacaagtc agtaactacc ctgtggtggg tgcagagctt 15060atgcccgtct tctcaaagag cttctacaac gaacaagctg tgtactccca gcagctccgc 15120cagtccacct cgcttacgca cgtcttcaac cgctttcctg agaaccagat tttaatccgt 15180ccgccggcgc ccaccattac caccgtcagt gaaaacgttc ctgctctcac agatcacggg 15240accctgccgt tgcgcagcag tatccgggga gtccaacgtg tgaccgttac tgacgccaga 15300cgccgcacct gtccctacgt gtacaaggca ctgggcatag tcgcaccgcg cgtcctttca 15360agccgcactt tctaaaaaaa aaaaatgtcc attcttatct cgcccagtaa taacaccggt 15420tggggtctgc gcgctccaag caagatgtac ggaggcgcac gcaaacgttc tacccaacat 15480cccgtgcgtg ttcgcggaca ttttcgcgct ccatggggtg ccctcaaggg ccgcactcgc 15540gttcgaacca ccgtcgatga tgtaatcgat caggtggttg ccgacgcccg taattatact 15600cctactgcgc ctacatctac tgtggatgca gttattgaca gtgtagtggc tgacgctcgc 15660aactatgctc gacgtaagag ccggcgaagg cgcattgcca gacgccaccg agctaccact 15720gccatgcgag ccgcaagagc tctgctacga agagctagac gcgtggggcg aagagccatg 15780cttagggcgg ccagacgtgc agcttcgggc gccagcgccg gcaggtcccg caggcaagca 15840gccgctgtcg cagcggcgac tattgccgac atggcccaat cgcgaagagg caatgtatac 15900tgggtgcgtg acgctgccac cggtcaacgt gtacccgtgc gcacccgtcc ccctcgcact 15960tagaagatac tgagcagtct ccgatgttgt gtcccagcgg cgaggatgtc caagcgcaaa 16020tacaaggaag aaatgctgca ggttatcgca cctgaagtct acggccaacc gttgaaggat 16080gaaaaaaaac cccgcaaaat caagcgggtt aaaaaggaca aaaaagaaga ggaagatggc 16140gatgatgggc tggcggagtt tgtgcgcgag tttgccccac ggcgacgcgt gcaatggcgt 16200gggcgcaaag ttcgacatgt gttgagacct ggaacttcgg tggtctttac acccggcgag 16260cgttcaagcg ctacttttaa gcgttcctat gatgaggtgt acggggatga tgatattctt 16320gagcaggcgg ctgaccgatt aggcgagttt gcttatggca agcgtagtag aataacttcc 16380aaggatgaga cagtgtcaat acccttggat catggaaatc ccacccctag tcttaaaccg 16440gtcactttgc agcaagtgtt acccgtaact ccgcgaacag gtgttaaacg cgaaggtgaa 16500gatttgtatc ccactatgca actgatggta cccaaacgcc agaagttgga ggacgttttg 16560gagaaagtaa aagtggatcc agatattcaa cctgaggtta aagtgagacc cattaagcag 16620gtagcgcctg gtctgggggt acaaactgta gacattaaga ttcccactga aagtatggaa 16680gtgcaaactg aacccgcaaa gcctactgcc acctccactg aagtgcaaac ggatccatgg 16740atgcccatgc ctattacaac tgacgccgcc ggtcccactc gaagatcccg acgaaagtac 16800ggtccagcaa gtctgttgat gcccaattat gttgtacacc catctattat tcctactcct 16860ggttaccgag gcactcgcta ctatcgcagc cgaaacagta cctcccgccg tcgccgcaag 16920acacctgcaa atcgcagtcg tcgccgtaga cgcacaagca aaccgactcc cggcgccctg 16980gtgcggcaag tgtaccgcaa tggtagtgcg gaacctttga cactgccgcg tgcgcgttac 17040catccgagta tcatcactta atcaatgttg ccgctgcctc cttgcagata tggccctcac 17100ttgtcgcctt cgcgttccca tcactggtta ccgaggaaga aactcgcgcc gtagaagagg 17160gatgttggga cgcggaatgc gacgctacag gcgacggcgt gctatccgca agcaattgcg 17220gggtggtttt ttaccagcct taattccaat tatcgctgct gcaattggcg cgataccagg 17280catagcttcc gtggcggttc aggcctcgca acgacattga cattggaaaa aaaacgtata 17340aataaaaaaa aatacaatgg actctgacac tcctggtcct gtgactatgt tttcttagag 17400atggaagaca tcaatttttc atccttggct ccgcgacacg gcacgaagcc gtacatgggc 17460acctggagcg acatcggcac gagccaactg aacgggggcg ccttcaattg gagcagtatc 17520tggagcgggc ttaaaaattt tggctcaacc ataaaaacat acgggaacaa agcttggaac 17580agcagtacag gacaggcgct tagaaataaa cttaaagacc agaacttcca acaaaaagta 17640gtcgatggga tagcttccgg catcaatgga gtggtagatt tggctaacca ggctgtgcag 17700aaaaagataa acagtcgttt ggacccgccg ccagcaaccc caggtgaaat gcaagtggag 17760gaagaaattc ctccgccaga aaaacgaggc gacaagcgtc cgcgtcccga tttggaagag 17820acgctggtga cgcgcgtaga tgaaccgcct tcttatgagg aagcaacgaa gcttggaatg 17880cccaccacta gaccgatagc cccaatggcc accggggtga tgaaaccttc tcagttgcat 17940cgacccgtca ccttggattt gccccctccc cctgctgcta ctgctgtacc cgcttctaag 18000cctgtcgctg ccccgaaacc agtcgccgta gccaggtcac gtcccggggg cgctcctcgt 18060ccaaatgcgc actggcaaaa tactctgaac agcatcgtgg gtctaggcgt gcaaagtgta 18120aaacgccgtc gctgctttta attaaatatg gagtagcgct taacttgcct atctgtgtat 18180atgtgtcatt acacgccgtc acagcagcag aggaaaaaag gaagaggtcg tgcgtcgacg 18240ctgagttact ttcaagatgg ccaccccatc gatgctgccc caatgggcat acatgcacat 18300cgccggacag gatgcttcgg agtacctgag tccgggtctg gtgcagttcg cccgcgccac 18360agacacctac ttcaatctgg gaaataagtt tagaaatccc accgtagcgc cgacccacga 18420tgtgaccacc gaccgtagcc agcggctcat gttgcgcttc gtgcccgttg accgggagga 18480caatacatac tcttacaaag tgcggtacac cctggccgtg ggcgacaaca gagtgctgga 18540tatggccagc acgttctttg acattagggg cgtgttggac agaggtccca gtttcaaacc 18600ctattctggt acggcttaca actctctggc tcctaaaggc gctccaaatg catctcaatg 18660gattgcaaaa ggcgtaccaa ctgcagcagc cgcaggcaat ggtgaagaag aacatgaaac 18720agaggagaaa actgctactt acacttttgc caatgctcct gtaaaagccg aggctcaaat 18780tacaaaagag ggcttaccaa taggtttgga gatttcagct gaaaacgaat ctaaacccat 18840ctatgcagat aaactttatc agccagaacc tcaagtggga gatgaaactt ggactgacct 18900agacggaaaa accgaagagt atggaggcag ggctctaaag cctactacta acatgaaacc 18960ctgttacggg tcctatgcga agcctactaa tttaaaaggt ggtcaggcaa aaccgaaaaa 19020ctcggaaccg tcgagtgaaa aaattgaata tgatattgac atggaatttt ttgataactc 19080atcgcaaaga acaaacttca gtcctaaaat tgtcatgtat gcagaaaatg taggtttgga 19140aacgccagac actcatgtag tgtacaaacc tggaacagaa gacacaagtt ccgaagctaa 19200tttgggacaa cagtctatgc ccaacagacc caactacatt ggcttcagag ataactttat 19260tggactcatg tactataaca gtactggtaa catgggggtg ctggctggtc aagcgtctca 19320gttaaatgca gtggttgact tgcaggacag aaacacagaa ctttcttacc aactcttgct 19380tgactctctg ggcgacagaa ccagatactt tagcatgtgg aatcaggctg tggacagtta 19440tgatcctgat gtacgtgtta ttgaaaatca tggtgtggaa gatgaacttc ccaactattg 19500ttttccactg gacggcatag gtgttccaac aaccagttac aaatcaatag ttccaaatgg 19560agaagataat aataattgga aagaacctga agtaaatgga acaagtgaga tcggacaggg 19620taatttgttt gccatggaaa ttaaccttca agccaatcta tggcgaagtt tcctttattc 19680caatgtggct ctgtatctcc cagactcgta caaatacacc ccgtccaatg tcactcttcc 19740agaaaacaaa aacacctacg actacatgaa cgggcgggtg gtgccgccat ctctagtaga 19800cacctatgtg aacattggtg ccaggtggtc tctggatgcc atggacaatg tcaacccatt 19860caaccaccac cgtaacgctg gcttgcgtta ccgatctatg cttctgggta acggacgtta 19920tgtgcctttc cacatacaag tgcctcaaaa attcttcgct gttaaaaacc tgctgcttct 19980cccaggctcc tacacttatg agtggaactt taggaaggat gtgaacatgg ttctacagag 20040ttccctcggt aacgacctgc gggtagatgg cgccagcatc agtttcacga gcatcaacct 20100ctatgctact tttttcccca tggctcacaa caccgcttcc acccttgaag ccatgctgcg 20160gaatgacacc aatgatcagt cattcaacga ctacctatct gcagctaaca tgctctaccc 20220cattcctgcc aatgcaacca atattcccat ttccattcct tctcgcaact gggcggcttt 20280cagaggctgg tcatttacca gactgaaaac caaagaaact ccctctttgg ggtctggatt 20340tgacccctac tttgtctatt ctggttctat tccctacctg gatggtacct tctacctgaa 20400ccacactttt aagaaggttt ccatcatgtt tgactcttca gtgagctggc ctggaaatga 20460caggttacta tctcctaacg aatttgaaat aaagcgcact gtggatggcg aaggctacaa 20520cgtagcccaa tgcaacatga ccaaagactg gttcttggta cagatgctcg ccaactacaa 20580catcggctat cagggcttct acattccaga aggatacaaa gatcgcatgt attcattttt 20640cagaaacttc cagcccatga gcaggcaggt ggttgatgag gtcaattaca aagacttcaa 20700ggccgtcgcc ataccctacc aacacaacaa ctctggcttt gtgggttaca tggctccgac 20760catgcgccaa ggtcaaccct atcccgctaa ctatccctat ccactcattg gaacaactgc 20820cgtaaatagt gttacgcaga aaaagttctt gtgtgacaga accatgtggc gcataccgtt 20880ctcgagcaac ttcatgtcta tgggggccct tacagacttg ggacagaata tgctctatgc 20940caactcagct catgctctgg acatgacctt tgaggtggat cccatggatg agcccaccct 21000gctttatctt ctcttcgaag ttttcgacgt ggtcagagtg catcagccac accgcggcat 21060catcgaggca gtctacctgc gtacaccgtt ctcggccggt aacgctacca cgtaagaagc 21120ttcttgcttc ttgcaaatag cagctgcaac catggcctgc ggatcccaaa acggctccag 21180cgagcaagag ctcagagcca ttgtccaaga cctgggttgc ggaccctatt ttttgggaac 21240ctacgataag cgcttcccgg ggttcatggc ccccgataag ctcgcctgtg ccattgtaaa 21300tacggccgga cgtgagacgg ggggagagca ctggttggct ttcggttgga acccacgttc 21360taacacctgc tacctttttg atccttttgg attctcggat gatcgtctca aacagattta 21420ccagtttgaa tatgagggtc tcctgcgccg cagcgctctt gctaccaagg accgctgtat 21480tacgctggaa aaatctaccc agaccgtgca gggcccccgt tctgccgcct gcggactttt 21540ctgctgcatg ttccttcacg cctttgtgca ctggcctgac cgtcccatgg acggaaaccc 21600caccatgaaa ttgctaactg gagtgccaaa caacatgctt cattctccta aagtccagcc 21660caccctgtgt gacaatcaaa aagcactcta ccattttctt aatacccatt cgccttattt 21720tcgctctcat cgtacacaca tcgaaagggc cactgcgttc gaccgtatgg atgttcaata 21780atgactcatg taaacaacgt gttcaataaa catcacttta tttttttaca tgtatcaagg 21840ctctggatta cttatttatt tacaagtcga atgggttctg acgagaatca gaatgacccg 21900caggcagtga tacgttgcgg aactgatact tgggttgcca cttgaattcg ggaatcacca 21960acttgggaac cggtatatcg ggcaggatgt cactccacag ctttctggtc agctgcaaag 22020ctccaagcag gtcaggagcc gaaatcttga aatcacaatt aggaccagtg ctctgagcgc 22080gagagttgcg gtacaccgga ttgcagcact gaaacaccat cagcgacgga tgtctcacgc 22140ttgccagcac ggtgggatct gcaatcatgc ccacatccag atcttcagca ttggcaatgc 22200tgaacggggt catcttgcag gtctgcctac ccatggcggg cacccaatta ggcttgtggt 22260tgcaatcgca gtgcaggggg atcagtatca tcttggcctg atcctgtctg attcctggat 22320acacggctct catgaaagca tcatattgct tgaaagcctg ctgggcttta ctaccctcgg 22380tataaaacat cccgcaggac ctgctcgaaa actggttagc tgcacagccg gcatcattca 22440cacagcagcg ggcgtcattg ttggctattt gcaccacact tctgccccag cggttttggg 22500tgattttggt tcgctcggga ttctccttta aggctcgttg tccgttctcg ctggccacat 22560ccatctcgat aatctgctcc ttctgaatca taatattgcc atgcaggcac ttcagcttgc 22620cctcataatc attgcagcca tgaggccaca acgcacagcc tgtacattcc caattatggt 22680gggcgatctg agaaaaagaa tgtatcattc cctgcagaaa tcttcccatc atcgtgctca 22740gtgtcttgtg actagtgaaa gttaactgga tgcctcggtg ctcttcgttt acgtactggt 22800gacagatgcg cttgtattgt tcgtgttgct caggcattag tttaaaacag gttctaagtt 22860cgttatccag cctgtacttc tccatcagca gacacatcac ttccatgcct ttctcccaag 22920cagacaccag gggcaagcta atcggattct taacagtgca ggcagcagct cctttagcca 22980gagggtcatc tttagcgatc ttctcaatgc ttcttttgcc atccttctca acgatgcgca 23040cgggcgggta gctgaaaccc actgctacaa gttgcgcctc ttctctttct tcttcgctgt 23100cttgactgat gtcttgcatg gggatatgtt tggtcttcct tggcttcttt ttggggggta 23160tcggaggagg aggactgtcg ctccgttccg gagacaggga ggattgtgac gtttcgctca 23220ccattaccaa ctgactgtcg gtagaagaac ctgaccccac acggcgacag gtgtttttct 23280tcgggggcag aggtggaggc gattgcgaag ggctgcggtc cgacctggaa ggcggatgac 23340tggcagaacc ccttccgcgt tcgggggtgt gctccctgtg gcggtcgctt aactgatttc 23400cttcgcggct ggccattgtg ttctcctagg cagagaaaca acagacatgg aaactcagcc 23460attgctgtca acatcgccac gagtgccatc acatctcgtc ctcagcgacg aggaaaagga 23520gcagagctta agcattccac cgcccagtcc tgccaccacc tctaccctag aagataagga 23580ggtcgacgca tctcatgaca tgcagaataa aaaagcgaaa gagtctgaga cagacatcga 23640gcaagacccg ggctatgtga caccggtgga acacgaggaa gagttgaaac gctttctaga 23700gagagaggat gaaaactgcc caaaacagcg agcagataac tatcaccaag atgctggaaa 23760tagggatcag aacaccgact acctcatagg gcttgacggg gaagacgcgc tccttaaaca 23820tctagcaaga cagtcgctca tagtcaagga tgcattattg gacagaactg aagtgcccat 23880cagtgtggaa gagctcagct gcgcctacga gcttaacctt ttttcacctc gtactccccc 23940caaacgtcag ccaaacggca cctgcgagcc aaatcctcgc ttaaactttt atccagcttt 24000tgctgtgcca gaagtactgg ctacctatca catctttttt aaaaatcaaa aaattccagt 24060ctcctgccgc gctaatcgca cccgcgccga tgccctactc aatctgggac ctggttcacg 24120cttacctgat atagcttcct tggaagaggt tccaaagatc ttcgagggtc tgggcaataa 24180tgagactcgg gccgcaaatg ctctgcaaaa gggagaaaat

ggcatggatg agcatcacag 24240cgttctggtg gaattggaag gcgataatgc cagactcgca gtactcaagc gaagcgtcga 24300ggtcacacac ttcgcatatc ccgctgtcaa cctgccccct aaagtcatga cggcggtcat 24360ggaccagtta ctcattaagc gcgcaagtcc cctttcagaa gacatgcatg acccagatgc 24420ctgtgatgag ggtaaaccag tggtcagtga tgagcagcta acccgatggc tgggcaccga 24480ctctccccgg gatttggaag agcgtcgcaa gcttatgatg gccgtggtgc tggttaccgt 24540agaactagag tgtctccgac gtttctttac cgattcagaa accttgcgca aactcgaaga 24600gaatctgcac tacactttta gacacggctt tgtgcggcag gcatgcaaga tatctaacgt 24660ggaactcacc aacctggttt cctacatggg tattctgcat gagaatcgcc taggacaaag 24720cgtgctgcac agcaccctta agggggaagc ccgccgtgat tacatccgcg attgtgtcta 24780tctctacctg tgccacacgt ggcaaaccgg catgggtgta tggcagcaat gtttagaaga 24840acagaacttg aaagagcttg acaagctctt acagaaatct cttaaggttc tgtggacagg 24900gttcgacgag cgcaccgtcg cttccgacct ggcagacctc atcttcccag agcgtctcag 24960ggttactttg cgaaacggat tgcctgactt tatgagccag agcatgctta acaattttcg 25020ctctttcatc ctggaacgct ccggtatcct gcccgccacc tgctgcgcac tgccctccga 25080ctttgtgcct ctcacctacc gcgagtgccc cccgccgcta tggagtcact gctacctgtt 25140ccgtctggcc aactatctct cctaccactc ggatgtgatc gaggatgtga gcggagacgg 25200cttgctggag tgccactgcc gctgcaatct gtgcacgccc caccggtccc tagcttgcaa 25260cccccagttg atgagcgaaa cccagataat aggcaccttt gaattgcaag gccccagcag 25320ccaaggcgat gggtcttctc ctgggcaaag tttaaaactg accccgggac tgtggacctc 25380cgcctacttg cgcaagtttg ctccggaaga ttaccacccc tatgaaatca agttctatga 25440ggaccaatca cagcctccaa aggccgaact ttcggcttgc gtcatcaccc agggggcaat 25500tctggcccaa ttgcaagcca tccaaaaatc ccgccaagaa tttctactga aaaagggtaa 25560gggggtctac cttgaccccc agaccggcga ggaactcaac acaaggttcc ctcaggatgt 25620cccaacgacg agaaaacaag aagttgaagg tgcagccgcc gcccccagaa gatatggagg 25680aagattggga cagtcaggca gaggaggcgg aggaggacag tctggaggac agtctggagg 25740aagacagttt ggaggaggaa aacgaggagg cagaggaggt ggaagaagta accgccgaca 25800aacagttatc ctcggctgcg gagacaagca acagcgctac catctccgct ccgagtcgag 25860gaacccggcg gcgtcccagc agtagatggg acgagaccgg acgcttcccg aacccaacca 25920gcgcttccaa gaccggtaag aaggatcggc agggatacaa gtcctggcgg gggcataaga 25980atgccatcat ctcctgcttg catgagtgcg ggggcaacat atccttcacg cggcgctact 26040tgctattcca ccatggggtg aactttccgc gcaatgtttt gcattactac cgtcacctcc 26100acagccccta ctatagccag caaatcccga cagtctcgac agataaagac agcggcggcg 26160acctccaaca gaaaaccagc agcggcagtt agaaaataca caacaagtgc agcaacagga 26220ggattaaaga ttacagccaa cgagccagcg caaacccgag agttaagaaa tcggatcttt 26280ccaaccctgt atgccatctt ccagcagagt cggggtcaag agcaggaact gaaaataaaa 26340aaccgatctc tgcgttcgct caccagaagt tgtttgtatc acaagagcga agatcaactt 26400cagcgcactc tcgaggacgc cgaggctctc ttcaacaagt actgcgcgct gactcttaaa 26460gagtaggcag cgaccgcgct tattcaaaaa aggcgggaat tacatcatcc tcgacatgag 26520taaagaaatt cccacgcctt acatgtggag ttatcaaccc caaatgggat tggcagcagg 26580cgcctcccag gactactcca cccgcatgaa ttggctcagc gccgggcctt ctatgatttc 26640tcgagttaat gatatacgcg cctaccgaaa ccaaatactt ttggaacagt cagctcttac 26700caccacgccc cgccaacacc ttaatcccag aaattggccc gccgccctag tgtaccagga 26760aagtcccgct cccaccactg tattacttcc tcgagacgcc caggccgaag tccaaatgac 26820taatgcaggt gcgcagttag ctggcggctc caccctatgt cgtcacaggc ctcggcataa 26880tataaaacgc ctgatgatca gaggccgagg tatccagctc aacgacgagt cggtgagctc 26940tccgcttggt ctacgaccag acggaatctt tcagattgcc ggctgcggga gatcttcctt 27000cacccctcgt caggctgttc tgactttgga aagttcgtct tcgcaacccc gctcgggcgg 27060aatcgggacc gttcaatttg tagaggagtt tactccctct gtctacttca accccttctc 27120cggatctcct gggcactacc cggacgagtt cataccgaac ttcgacgcga ttagcgagtc 27180agtggacggc tacgattgat gtctggtgac gcggctgagc tatctcggct gcgacatcta 27240gaccactgcc gccgctttcg ctgctttgcc cgggaactta ttgagttcat ctacttcgaa 27300ctccccaagg atcaccctca aggtccggcc cacggagtgc ggattactat cgaaggcaaa 27360atagactctc gcctgcaacg aattttctcc cagcggcccg tgctgatcga gcgagaccag 27420ggaaacacca cggtttccat ctactgcatt tgtaatcacc ccggattgca tgaaagcctt 27480tgctgtctta tgtgtactga gtttaataaa aactgaatta agactctcct acggactgcc 27540gcttcttcaa cccggatttt acaaccagaa gaacaaaact tttcctgtcg tccaggactc 27600tgttaacttc acctttccta ctcacaaact agaagctcaa cgactacacc gcttttccag 27660aagcattttc cctactaata ctactttcaa aaccggaggt gagctccacg gtctccctac 27720agaaaaccct tgggtggaag cgggccttgt agtactagga attcttgcgg gtgggcttgt 27780gattattctt tgctacctat acacaccttg cttcactttc ctagtggtgt tgtggtattg 27840gtttaaaaaa tggggcccat actagtcttg cttgttttac tttcgctttt ggaaccgggt 27900tctgccaatt acgatccatg tctagacttt gacccagaaa actgcacact tacttttgca 27960cccgacacaa gccgcatctg tggagttctt attaagtgcg gatgggaatg caggtccgtt 28020gaaattacac acaataacaa aacctggaac aataccttat ccaccacatg ggagccagga 28080gttcccgagt ggtacactgt ctctgtccga ggtcctgacg gttccatccg cattagtaac 28140aacactttca ttttttctga aatgtgcgat ctggccatgt tcatgagcaa acagtattct 28200ctatggcctc ctagcaagga caacatcgta acgttctcca ttgcttattg cttgtgcgct 28260tgccttctta ctgctttact gtgcgtatgc atacacctgc ttgtaaccac tcgcatcaaa 28320aacgccaata acaaagaaaa aatgccttaa cctctttctg tttacagaca tggcttctct 28380tacatctctc atatttgtca gcattgtcac tgccgctcac ggacaaacag tcgtctctat 28440cccactagga cataattaca ctctcatagg acccccaatc acttcagagg tcatctggac 28500caaactggga agcgttgatt actttgatat aatctgtaac aaaacaaaac caataatagt 28560aacttgcaac atacaaaatc ttacattgat taatgttagc aaagtttaca gcggttacta 28620ttatggttat gacagataca gtagtcaata tagaaattac ttggttcgtg ttacccagtt 28680gaaaaccacg aaaatgccaa atatggcaaa gattcgatcc gatgacaatt ctctagaaac 28740ttttacatct cccaccacac ccgacgaaaa aaacatccca gattcaatga ttgcaattgt 28800tgcagcggtg gcagtggtga tggcactaat aataatatgc atgcttttat atgcttgtcg 28860ctacaaaaag tttcatccta aaaaacaaga tctcctacta aggcttaaca tttaatttct 28920ttttatacag ccatggtttc cactaccaca ttccttatgc ttactagtct cgcaactctg 28980acttctgctc gctcacacct cactgtaact ataggctcaa actgcacact aaaaggacct 29040caaggtggtc atgtcttttg gtggagaata tatgacaatg gatggtttac aaaaccatgt 29100gaccaacctg gtagattttt ctgcaacggc agagacctaa ccattatcaa cgtgacagca 29160aatgacaaag gcttctatta tggaaccgac tataaaagta gtttagatta taacattatt 29220gtactgccat ctaccactcc agcaccccgc acaactactt tctctagcag cagtgtcgct 29280aacaatacaa tttccaatcc aacctttgcc gcgcttttaa aacgcactgt gaataattct 29340acaacttcac atacaacaat ttccacttca acaatcagca tcatcgctgc agtgacaatt 29400ggaatatcta ttcttgtttt taccataacc tactacgcct gctgctatag aaaagacaaa 29460cataaaggtg atccattact tagatttgat atttaatttg ttcttttttt ttatttacag 29520tatggtgaac accaatcatg gtacctagaa atttcttctt caccatactc atctgtgctt 29580ttaatgtttg cgctactttc acagcagtag ccacagcaac cccagactgt ataggagcat 29640ttgcttccta tgcacttttt gcttttgtta cttgcatctg cgtatgtagc atagtctgcc 29700tggttattaa ttttttccaa cttctagact ggatccttgt gcgaattgcc tacctgcgcc 29760accatcccga ataccgcaac caaaatatcg cggcacttct tagactcatc taaaaccatg 29820caggctatac taccaatatt tttgcttcta ttgcttccct acgctgtctc aaccccagct 29880gcctatagta ctccaccaga acaccttaga aaatgcaaat tccaacaacc gtggtcattt 29940cttgcttgct atcgagaaaa atcagaaatc cccccaaatt taataatgat tgctggaata 30000attaatataa tctgttgcac cataatttca tttttgatat accccctatt tgattttggc 30060tggaatgctc ccaatgcaca tgatcatcca caagacccag aggaacacat tcccccacaa 30120aacatgcaac atccaatagc gctaatagat tacgaaagtg aaccacaacc cccactactc 30180cctgctatta gttacttcaa cctaaccggc ggagatgact gaaacactca ccacctccaa 30240ttccgccgag gatctgctcg atatggacgg ccgcgtctca gaacaacgac ttgcccaact 30300acgcatccgc cagcagcagg aacgcgtggc caaagagctc agagatgtca tccaaattca 30360ccaatgcaaa aaaggcatat tctgtttggt aaaacaagcc aagatatcct acgagatcac 30420cgctactgac catcgcctct cttacgaact tggcccccaa cgacaaaaat ttacctgcat 30480ggtgggaatc aaccccatag ttatcaccca acaaagtgga gatactaagg gttgcattca 30540ctgctcctgc gattccatcg agtgcaccta caccctgctg aagaccctat gcggcctaag 30600agacctgcta ccaatgaatt aaaaaaaaat gattaataaa aaatcactta cttgaaatca 30660gcaataaggt ctctgttgaa attttctccc agcagcacct cacttccctc ttcccaactc 30720tggtattcta aaccccgttc agcggcatac tttctccata ctttaaaggg gatgtcaaat 30780tttagctcct ctcctgtacc cacaatcttc atgtctttct tcccagatga ccaagagagt 30840ccggctcagt gactccttca accctgtcta cccctatgaa gatgaaagca cctcccaaca 30900cccctttata aacccagggt ttatttcccc aaatggcttc acacaaagcc cagacggagt 30960tcttacttta aaatgtttaa ccccactaac aaccacaggc ggatctctac agctaaaagt 31020gggaggggga cttacagtgg atgacactga tggtacctta caagaaaaca tacgtgctac 31080agcacccatt actaaaaata atcactctgt agaactatcc attggaaatg gattagaaac 31140tcaaaacaat aaactatgtg ccaaattggg aaatgggtta aaatttaaca acggtgacat 31200ttgtataaag gatagtatta acaccttatg gactggaata aaccctccac ctaactgtca 31260aattgtggaa aacactaata caaatgatgg caaacttact ttagtattag taaaaaatgg 31320agggcttgtt aatggctacg tgtctctagt tggtgtatca gacactgtga accaaatgtt 31380cacacaaaag acagcaaaca tccaattaag attatatttt gactcttctg gaaatctatt 31440aactgaggaa tcagacttaa aaattccact taaaaataaa tcttctacag cgaccagtga 31500aactgtagcc agcagcaaag cctttatgcc aagtactaca gcttatccct tcaacaccac 31560tactagggat agtgaaaact acattcatgg aatatgttac tacatgacta gttatgatag 31620aagtctattt cccttgaaca tttctataat gctaaacagc cgtatgattt cttccaatgt 31680tgcctatgcc atacaatttg aatggaatct aaatgcaagt gaatctccag aaagcaacat 31740agctacgctg accacatccc cctttttctt ttcttacatt acagaagacg acaactaaaa 31800taaagtttaa gtgtttttat ttaaaatcac aaaattcgag tagttatttt gcctccacct 31860tcccatttga cagaatacac caatctctcc ccacgcacag ctttaaacat ttggatacca 31920ttagagatag acattgtttt agattccaca ttccaaacag tttcagagcg agccaatctg 31980gggtcagtga tagataaaaa tccatcgcga tagtctttta aagcgctttc acagtccaac 32040tgctgcggat gcgactccgg agtttggatc acggtcatct ggaagaagaa cgatgggaat 32100cataatccga aaacggtatc ggacgattgt gtctcatcaa acccacaagc agccgctgtc 32160tgcgtcgctc cgtgcgactg ctgtttatgg gatcagggtc cacagtttcc tgaagcatga 32220ttttaatagc ccttaacatc aactttctgg tgcgatgcgc gcagcaacgc attctgattt 32280cactcaaatc tttgcagtag gtacaacaca ttattacaat attgtttaat aaaccataat 32340taaaagcgct ccagccaaaa ctcatatctg atataatcgc ccctgcatga ccatcatacc 32400aaagtttaat ataaattaaa tgacgttccc tcaaaaacac actacccaca tacatgatct 32460cttttggcat gtgcatatta acaatctgtc tgtaccatgg acaacgttgg ttaatcatgc 32520aacccaatat aaccttccgg aaccacactg ccaacaccgc tcccccagcc atgcattgaa 32580gtgaaccctg ctgattacaa tgacaatgaa gaacccaatt ctctcgaccg tgaatcactt 32640gagaatgaaa aatatctata gtggcacaac atagacataa atgcatgcat cttctcataa 32700tttttaactc ctcaggattt agaaacatat cccagggaat aggaagctct tgcagaacag 32760taaagctggc agaacaagga agaccacgaa cacaacttac actatgcata gtcatagtat 32820cacaatctgg caacagcggg tggtcttcag tcatagaagc tcgggtttca ttttcctcac 32880aacgtggtaa ctgggctctg gtgtaagggt gatgtctggc gcatgatgtc gagcgtgcgc 32940gcaaccttgt cataatggag ttgcttcctg acattctcgt attttgtata gcaaaacgcg 33000gccctggcag aacacactct tcttcgcctt ctatcctgcc gcttagcgtg ttccgtgtga 33060tagttcaagt acagccacac tcttaagttg gtcaaaagaa tgctggcttc agttgtaatc 33120aaaactccat cgcatctaat tgttctgagg aaatcatcca cggtagcata tgcaaatccc 33180aaccaagcaa tgcaactgga ttgcgtttca agcaggagag gagagggaag agacggaaga 33240accatgttaa tttttattcc aaacgatctc gcagtacttc aaattgtaga tcgcgcagat 33300ggcatctctc gcccccactg tgttggtgaa aaagcacagc taaatcaaaa gaaatgcgat 33360tttcaaggtg ctcaacggtg gcttccaaca aagcctccac gcgcacatcc aagaacaaaa 33420gaataccaaa agaaggagca ttttctaact cctcaatcat catattacat tcctgcacca 33480ttcccagata attttcagct ttccagcctt gaattattcg tgtcagttct tgtggtaaat 33540ccaatccaca cattacaaac aggtcccgga gggcgccctc caccaccatt cttaaacaca 33600ccctcataat gacaaaatat cttgctcctg tgtcacctgt agcgaattga gaatggcaac 33660atcaattgac atgcccttgg ctctaagttc ttctttaagt tctagttgta aaaactctct 33720catattatca ccaaactgct tagccagaag ccccccggga acaagagcag gggacgctac 33780agtgcagtac aagcgcagac ctccccaatt ggctccagca aaaacaagat tggaataagc 33840atattgggaa ccaccagtaa tatcatcgaa gttgctggaa atataatcag gcagagtttc 33900ttgtagaaat tgaataaaag aaaaatttgc caaaaaaaca ttcaaaacct ctgggatgca 33960aatgcaatag gttaccgcgc tgcgctccaa cattgttagt tttgaattag tctgcaaaaa 34020taaaaaaaaa acaagcgtca tatcatagta gcctgacgaa caggtggata aatcagtctt 34080tccatcacaa gacaagccac agggtctcca gctcgaccct cgtaaaacct gtcatcgtga 34140ttaaacaaca gcaccgaaag ttcctcgcgg tgaccagcat gaataagtct tgatgaagca 34200tacaatccag acatgttagc atcagttaag gagaaaaaac agccaacata gcctttgggt 34260ataattatgc ttaatcgtaa gtatagcaaa gccacccctc gcggatacaa agtaaaaggc 34320acaggagaat aaaaaatata attatttctc tgctgctgtt taggcaacgt cgcccccggt 34380ccctctaaat acacatacaa agcctcatca gccatggctt accagagaaa gtacagcggg 34440cacacaaacc acaagctcta aagtcactct ccaacctstc cacaatatat atacacaagc 34500cctaaactga cgtaatggga ctaaagtgta aaaaatcccg ccaaacccaa cacacacccc 34560gaaactgcgt caccagggaa aagtacagtt tcacttccgc aatcccaaca agcgtcactt 34620cctctttctc acggtacgtc acatcccatt aacttacaac gtcattttcc cacggccgcg 34680ccgccccttt taaccgttaa ccccacagcc aatcaccaca cggcccacac tttttaaaat 34740cacctcattt acatattggc accattccat ctataaggta tattattgat gatg 3479445180PRTadenoviridaeSITE(1)..(180)/note="pCC536s E1B-21K sequence" 45Met Glu Ala Trp Glu Cys Leu Glu Asp Phe Ser Ala Val Arg Asn Leu 1 5 10 15Leu Glu Gln Ser Ser Asn Ser Thr Ser Trp Phe Trp Arg Phe Leu Trp 20 25 30Gly Ser Ser Gln Ala Lys Leu Val Cys Arg Ile Lys Glu Asp Tyr Lys 35 40 45Trp Glu Phe Glu Glu Leu Leu Lys Ser Cys Gly Glu Leu Phe Asp Ser 50 55 60Leu Asn Leu Gly His Gln Ala Leu Phe Gln Glu Lys Val Ile Lys Thr 65 70 75 80Leu Asp Phe Ser Thr Pro Gly Arg Ala Ala Ala Ala Val Ala Phe Leu 85 90 95Ser Phe Ile Lys Asp Lys Trp Ser Glu Glu Thr His Leu Ser Gly Gly 100 105 110Tyr Leu Leu Asp Phe Leu Ala Met His Leu Trp Arg Ala Val Val Arg 115 120 125His Lys Asn Arg Leu Leu Leu Leu Ser Ser Val Arg Pro Ala Ile Ile 130 135 140Pro Thr Glu Glu Gln Gln Gln Gln Gln Glu Glu Ala Arg Arg Arg Arg145 150 155 160Gln Glu Gln Ser Pro Trp Asn Pro Arg Ala Gly Leu Asp Pro Pro Val 165 170 175Glu Glu Ala Glu 18046176PRTadenoviridaeSITE(1)..(176)/note="Ad5. E1B-21K sequence" 46Met Glu Ala Trp Glu Cys Leu Glu Asp Phe Ser Ala Val Arg Asn Leu 1 5 10 15Leu Glu Gln Ser Ser Asn Ser Thr Ser Trp Phe Trp Arg Phe Leu Trp 20 25 30Gly Ser Ser Gln Ala Lys Leu Val Cys Arg Ile Lys Glu Asp Tyr Lys 35 40 45Trp Glu Phe Glu Glu Leu Leu Lys Ser Cys Gly Glu Leu Phe Asp Ser 50 55 60Leu Asn Leu Gly His Gln Ala Leu Phe Gln Glu Lys Val Ile Lys Thr 65 70 75 80Leu Asp Phe Ser Thr Pro Gly Arg Ala Ala Ala Ala Val Ala Phe Leu 85 90 95Ser Phe Ile Lys Asp Lys Trp Ser Glu Glu Thr His Leu Ser Gly Gly 100 105 110Tyr Leu Leu Asp Phe Leu Ala Met His Leu Trp Arg Ala Val Val Arg 115 120 125His Lys Asn Arg Leu Leu Leu Leu Ser Ser Val Arg Pro Ala Ile Ile 130 135 140Pro Thr Glu Glu Gln Gln Gln Gln Gln Glu Glu Ala Arg Arg Arg Arg145 150 155 160Gln Glu Gln Ser Pro Trp Asn Pro Arg Ala Gly Leu Asp Pro Arg Glu 165 170 17547180PRTadenoviridaeSITE(1)..(180)/note="Ad35.E1B-21K sequence" 47Met Glu Val Trp Ala Ile Leu Glu Asp Leu Arg Lys Thr Arg Gln Leu 1 5 10 15Leu Glu Ser Ala Ser Asp Gly Val Ser Gly Phe Trp Arg Phe Trp Phe 20 25 30Ala Ser Glu Leu Ala Arg Val Val Phe Arg Ile Lys Gln Asp Tyr Lys 35 40 45Gln Glu Phe Glu Lys Leu Leu Val Asp Cys Pro Gly Leu Phe Glu Ala 50 55 60Leu Asn Leu Gly His Gln Val His Phe Lys Glu Lys Val Leu Ser Val 65 70 75 80Leu Asp Phe Ser Thr Pro Gly Arg Thr Ala Ala Ala Val Ala Phe Leu 85 90 95Thr Phe Ile Leu Asp Lys Trp Ile Pro Gln Thr His Phe Ser Arg Gly 100 105 110Tyr Val Leu Asp Phe Ile Ala Thr Ala Leu Trp Arg Thr Trp Lys Val 115 120 125Arg Lys Met Arg Thr Ile Leu Gly Tyr Trp Pro Val Gln Pro Leu Gly 130 135 140Val Ala Gly Ile Leu Arg His Pro Pro Val Met Pro Ala Val Leu Glu145 150 155 160Glu Glu Gln Gln Glu Asp Asn Pro Arg Ala Gly Leu Asp Pro Pro Val 165 170 175Glu Glu Ala Glu 18048494PRTadenoviridaeSITE(1)..(494)/note="pCC536s E1B-55K sequence" 48Met Glu Arg Arg Asn Pro Ser Glu Arg Gly Val Pro Ala Gly Phe Ser 1 5 10 15Gly His Ala Ser Val Glu Ser Gly Cys Glu Thr Gln Glu Ser Pro Ala 20 25 30Thr Val Val Phe Arg Pro Pro Gly Asp Asn Thr Asp Gly Gly Ala Ala 35 40 45Ala Ala Ala Gly Gly Ser Gln Ala Ala Ala Ala Gly Ala Glu Pro Met 50 55 60Glu Pro Glu Ser Arg Pro Gly Pro Ser Ser Gly Gly Gly Gly Val Ala 65 70 75 80Asp Leu Ser Pro Glu Leu Gln Arg Val Leu Thr Gly Ser Thr Ser Thr 85 90 95Gly Arg Asp Arg Gly Val Lys Arg Glu Arg Ala Ser Ser Gly Thr Asp 100 105 110Ala Arg Ser Glu Leu Ala Leu Ser Leu Met Ser Arg Arg Arg Pro Glu 115 120 125Thr Ile Trp Trp His Glu Val Gln Lys Glu Gly Arg Asp Glu Val Ser 130 135 140Val Leu Gln Glu Lys Tyr Ser Leu Glu Gln Val Lys Thr Cys Trp Leu145 150

155 160Glu Pro Glu Asp Asp Trp Ala Val Ala Ile Lys Asn Tyr Ala Lys Ile 165 170 175Ala Leu Arg Pro Asp Lys Gln Tyr Lys Ile Ser Arg Arg Ile Asn Ile 180 185 190Arg Asn Ala Cys Tyr Ile Ser Gly Asn Gly Ala Glu Val Val Ile Asp 195 200 205Thr Gln Asp Lys Thr Val Ile Arg Cys Cys Met Met Asp Met Trp Pro 210 215 220Gly Val Val Gly Met Glu Ala Val Thr Phe Val Asn Val Lys Phe Arg225 230 235 240Gly Asp Gly Tyr Asn Gly Ile Val Phe Met Ala Asn Thr Lys Leu Ile 245 250 255Leu His Gly Cys Ser Phe Phe Gly Phe Asn Asn Thr Cys Val Asp Ala 260 265 270Trp Gly Gln Val Ser Val Arg Gly Cys Ser Phe Tyr Ala Cys Trp Ile 275 280 285Ala Thr Ala Gly Arg Thr Lys Ser Gln Leu Ser Leu Lys Lys Cys Ile 290 295 300Phe Gln Arg Cys Asn Leu Gly Ile Leu Asn Glu Gly Glu Ala Arg Val305 310 315 320Arg His Cys Ala Ser Thr Asp Thr Gly Cys Phe Ile Leu Ile Lys Gly 325 330 335Asn Ala Ser Val Lys His Asn Met Ile Cys Gly Ala Ser Asp Glu Arg 340 345 350Pro Tyr Gln Met Leu Thr Cys Ala Gly Gly His Cys Asn Met Leu Ala 355 360 365Thr Val His Ile Val Ser His Gln Arg Lys Lys Trp Pro Val Phe Asp 370 375 380His Asn Val Leu Thr Lys Cys Thr Met His Ala Gly Gly Arg Arg Gly385 390 395 400Met Phe Met Pro Tyr Gln Cys Asn Met Asn His Val Lys Val Leu Leu 405 410 415Glu Pro Asp Ala Phe Ser Arg Met Ser Leu Thr Gly Ile Phe Asp Met 420 425 430Asn Thr Gln Ile Trp Lys Ile Leu Arg Tyr Asp Asp Thr Arg Ser Arg 435 440 445Val Arg Ala Cys Glu Cys Gly Gly Lys His Ala Arg Phe Gln Pro Val 450 455 460Cys Val Asp Val Thr Glu Asp Leu Arg Pro Asp His Leu Val Ile Ala465 470 475 480Arg Thr Gly Ala Glu Phe Gly Ser Ser Gly Glu Glu Thr Asp 485 49049494PRTadenoviridaeSITE(1)..(494)/note="Ad35. E1B-55K sequence" 49Met Asp Pro Ala Asp Ser Phe Gln Gln Gly Ile Arg Phe Gly Phe His 1 5 10 15Ser His Ser Ile Val Glu Asn Met Glu Gly Ser Gln Asp Glu Asp Asn 20 25 30Leu Arg Leu Leu Ala Ser Ala Ala Phe Gly Cys Ser Gly Asn Pro Glu 35 40 45Ala Ser Thr Gly His Ala Ser Gly Ser Gly Gly Gly Thr Ala Arg Gly 50 55 60Gln Pro Glu Ser Arg Pro Gly Pro Ser Ser Gly Gly Gly Gly Val Ala 65 70 75 80Asp Leu Ser Pro Glu Leu Gln Arg Val Leu Thr Gly Ser Thr Ser Thr 85 90 95Gly Arg Asp Arg Gly Val Lys Arg Glu Arg Ala Ser Ser Gly Thr Asp 100 105 110Ala Arg Ser Glu Leu Ala Leu Ser Leu Met Ser Arg Arg Arg Pro Glu 115 120 125Thr Ile Trp Trp His Glu Val Gln Lys Glu Gly Arg Asp Glu Val Ser 130 135 140Val Leu Gln Glu Lys Tyr Ser Leu Glu Gln Val Lys Thr Cys Trp Leu145 150 155 160Glu Pro Glu Asp Asp Trp Ala Val Ala Ile Lys Asn Tyr Ala Lys Ile 165 170 175Ala Leu Arg Pro Asp Lys Gln Tyr Lys Ile Ser Arg Arg Ile Asn Ile 180 185 190Arg Asn Ala Cys Tyr Ile Ser Gly Asn Gly Ala Glu Val Val Ile Asp 195 200 205Thr Gln Asp Lys Thr Val Ile Arg Cys Cys Met Met Asp Met Trp Pro 210 215 220Gly Val Val Gly Met Glu Ala Val Thr Phe Val Asn Val Lys Phe Arg225 230 235 240Gly Asp Gly Tyr Asn Gly Ile Val Phe Met Ala Asn Thr Lys Leu Ile 245 250 255Leu His Gly Cys Ser Phe Phe Gly Phe Asn Asn Thr Cys Val Asp Ala 260 265 270Trp Gly Gln Val Ser Val Arg Gly Cys Ser Phe Tyr Ala Cys Trp Ile 275 280 285Ala Thr Ala Gly Arg Thr Lys Ser Gln Leu Ser Leu Lys Lys Cys Ile 290 295 300Phe Gln Arg Cys Asn Leu Gly Ile Leu Asn Glu Gly Glu Ala Arg Val305 310 315 320Arg His Cys Ala Ser Thr Asp Thr Gly Cys Phe Ile Leu Ile Lys Gly 325 330 335Asn Ala Ser Val Lys His Asn Met Ile Cys Gly Ala Ser Asp Glu Arg 340 345 350Pro Tyr Gln Met Leu Thr Cys Ala Gly Gly His Cys Asn Met Leu Ala 355 360 365Thr Val His Ile Val Ser His Gln Arg Lys Lys Trp Pro Val Phe Asp 370 375 380His Asn Val Leu Thr Lys Cys Thr Met His Ala Gly Gly Arg Arg Gly385 390 395 400Met Phe Met Pro Tyr Gln Cys Asn Met Asn His Val Lys Val Leu Leu 405 410 415Glu Pro Asp Ala Phe Ser Arg Met Ser Leu Thr Gly Ile Phe Asp Met 420 425 430Asn Thr Gln Ile Trp Lys Ile Leu Arg Tyr Asp Asp Thr Arg Ser Arg 435 440 445Val Arg Ala Cys Glu Cys Gly Gly Lys His Ala Arg Phe Gln Pro Val 450 455 460Cys Val Asp Val Thr Glu Asp Leu Arg Pro Asp His Leu Val Ile Ala465 470 475 480Arg Thr Gly Ala Glu Phe Gly Ser Ser Gly Glu Glu Thr Asp 485 49050496PRTadenoviridaeSITE(1)..(496)/note="Ad5. E1B-55K sequence" 50Met Glu Arg Arg Asn Pro Ser Glu Arg Gly Val Pro Ala Gly Phe Ser 1 5 10 15Gly His Ala Ser Val Glu Ser Gly Cys Glu Thr Gln Glu Ser Pro Ala 20 25 30Thr Val Val Phe Arg Pro Pro Gly Asp Asn Thr Asp Gly Gly Ala Ala 35 40 45Ala Ala Ala Gly Gly Ser Gln Ala Ala Ala Ala Gly Ala Glu Pro Met 50 55 60Glu Pro Glu Ser Arg Pro Gly Pro Ser Gly Met Asn Val Val Gln Val 65 70 75 80Ala Glu Leu Tyr Pro Glu Leu Arg Arg Ile Leu Thr Ile Thr Glu Asp 85 90 95Gly Gln Gly Leu Lys Gly Val Lys Arg Glu Arg Gly Ala Cys Glu Ala 100 105 110Thr Glu Glu Ala Arg Asn Leu Ala Phe Ser Leu Met Thr Arg His Arg 115 120 125Pro Glu Cys Ile Thr Phe Gln Gln Ile Lys Asp Asn Cys Ala Asn Glu 130 135 140Leu Asp Leu Leu Ala Gln Lys Tyr Ser Ile Glu Gln Leu Thr Thr Tyr145 150 155 160Trp Leu Gln Pro Gly Asp Asp Phe Glu Glu Ala Ile Arg Val Tyr Ala 165 170 175Lys Val Ala Leu Arg Pro Asp Cys Lys Tyr Lys Ile Ser Lys Leu Val 180 185 190Asn Ile Arg Asn Cys Cys Tyr Ile Ser Gly Asn Gly Ala Glu Val Glu 195 200 205Ile Asp Thr Glu Asp Arg Val Ala Phe Arg Cys Ser Met Ile Asn Met 210 215 220Trp Pro Gly Val Leu Gly Met Asp Gly Val Val Ile Met Asn Val Arg225 230 235 240Phe Thr Gly Pro Asn Phe Ser Gly Thr Val Phe Leu Ala Asn Thr Asn 245 250 255Leu Ile Leu His Gly Val Ser Phe Tyr Gly Phe Asn Asn Thr Cys Val 260 265 270Glu Ala Trp Thr Asp Val Arg Val Arg Gly Cys Ala Phe Tyr Cys Cys 275 280 285Trp Lys Gly Val Val Cys Arg Pro Lys Ser Arg Ala Ser Ile Lys Lys 290 295 300Cys Leu Phe Glu Arg Cys Thr Leu Gly Ile Leu Ser Glu Gly Asn Ser305 310 315 320Arg Val Arg His Asn Val Ala Ser Asp Cys Gly Cys Phe Met Leu Val 325 330 335Lys Ser Val Ala Val Ile Lys His Asn Met Val Cys Gly Asn Cys Glu 340 345 350Asp Arg Ala Ser Gln Met Leu Thr Cys Ser Asp Gly Asn Cys His Leu 355 360 365Leu Lys Thr Ile His Val Ala Ser His Ser Arg Lys Ala Trp Pro Val 370 375 380Phe Glu His Asn Ile Leu Thr Arg Cys Ser Leu His Leu Gly Asn Arg385 390 395 400Arg Gly Val Phe Leu Pro Tyr Gln Cys Asn Leu Ser His Thr Lys Ile 405 410 415Leu Leu Glu Pro Glu Ser Met Ser Lys Val Asn Leu Asn Gly Val Phe 420 425 430Asp Met Thr Met Lys Ile Trp Lys Val Leu Arg Tyr Asp Glu Thr Arg 435 440 445Thr Arg Cys Arg Pro Cys Glu Cys Gly Gly Lys His Ile Arg Asn Gln 450 455 460Pro Val Met Leu Asp Val Thr Glu Glu Leu Arg Pro Asp His Leu Val465 470 475 480Leu Ala Cys Thr Arg Ala Glu Phe Gly Ser Ser Asp Glu Asp Thr Asp 485 490 495

Claims

1.-37. (canceled)

38. An adenoviral packaging cell line permissive for replication of an E1A/E1B deficient adenovirus vector, wherein said adenoviral packaging cell line comprises an adenovirus E1A coding sequence and an adenovirus E1B coding sequence each operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, and wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated into said adenoviral packaging cell line and are operably linked to identical promoters.

39. The adenoviral packaging cell line of claim 38, wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated at different sites in said adenoviral packaging cell line.

40. The adenoviral packaging cell of claim 38, wherein said promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter is a retrovirus promoter.

41. The adenoviral packaging cell of claim 38, wherein said adenovirus E1A coding sequence comprises the E1A coding sequence of GenBank Accession Number M73260.

42. The adenoviral packaging cell of claim 38, wherein said adenovirus E1B coding sequence comprises the E1B coding sequence of GenBank Accession Number M73260.

43. The adenoviral packaging cell line of claim 39, wherein said packaging cell line is of human origin.

44. The adenoviral packaging cell line of claim 43, wherein said adenoviral packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production.

45. An adenoviral packaging cell line comprising a first expression vector and a second expression vector stably integrated into the genome of said packaging cell line, wherein said first expression vector comprises adenovirus E1A coding sequences, operably linked to a non-adenoviral heterologous promoter, and said second expression vector comprises adenovirus E1B coding sequences operably linked to a non-adenoviral heterologous promoter.

46. A method of producing an adenovirus packaging cell line permissive for replication of an E1A/E1B deficient adenovirus vector, the method comprising: introducing into a cell line permissive for adenovirus replication, nucleic acid comprising (i) an adenovirus E1A coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter and (ii) an adenovirus E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, and wherein the nucleic acid comprising the adenovirus E1A coding sequence and the nucleic acid comprising the adenovirus E1B coding sequence are present on separate vectors.

47. The method according to claim 46, wherein one of the separate vectors comprises a retroviral nucleotide sequence.

48. The method according to claim 46, wherein each of the separate vectors comprises a retroviral nucleotide sequence.

49. An adenoviral packaging cell line permissive for replication of an E1A/E1B deficient adenovirus vector, wherein said adenoviral packaging cell line comprises an adenovirus E1A coding sequence, each and an adenovirus E1B coding sequence operably linked to a promoter that lacks substantial sequence identity to a native adenovirus E1A or E1B promoter, and wherein said adenovirus EA coding sequence and said adenovirus E1B coding sequence are stably integrated into the adenoviral packaging cell line and are operable linked to different heterologous promoters.

50. The adenoviral packaging cell line of claim 49, wherein said adenovirus E1A coding sequence and said adenovirus E1B sequence are stable integrated at different sites in said adenoviral packaging cell line.

51. The adenoviral packaging cell line of claim 49, wherein said heterologous promoters that lacks substantial sequence identity to a native adenovirus E1A or E1B promoter are retrovirus promoters.

52. The adenoviral packaging cell of claim 49, wherein said adenovirus EA coding sequence comprises the E1A coding sequence of GenBank Accession Number M73260.

53. The adenoviral packaging cell of claim 49, wherein said adenovirus E1B coding sequence comprises the E1B coding sequence of GenBank Accession Number M73260.

54. The adenoviral packaging cell line of claim 50, wherein said adenoviral packaging cell line is of human origin.

55. The adenoviral packaging cell line of claim 50, wherein said adenoviral packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production.

56. A method of producing an adenoviral packaging cell line permissive for replication of an E1A/E1B deficient adenovirus vector, the method comprising: introducing into a cell line permissive for adenovirus replication, nucleic acid comprising (i) an adenovirus E1A coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter and (ii) an adenovirus E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, wherein the nucleic acid comprising the adenovirus E1A coding sequence and the nucleic acid comprising the adenovirus E1B coding sequence are present on separate vectors and the promoters operably linked to the E1A and E1B coding sequences are different.

57. The method according to claim 56, wherein one of the separate vectors comprises a retroviral nucleotide sequence.

58. The method according to claim 56, wherein each of the separate vectors comprises a retroviral nucleotide sequence.

59. An adenoviral vector substantially free of replication competent adenovirus, produced by a packaging cell line permissive for replication of an E1A/E1B deficient adenoviral vector, wherein said packaging cell line comprises an adenoviral E1A coding sequence and an adenoviral E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter, and wherein said adenoviral E1A coding sequence and said adenoviral E1B coding sequence are stably integrated into said packaging cell line and are operably linked to different heterologous promoters.

60. The adenoviral vector of claim 59, wherein said adenoviral E1A coding sequence and said adenoviral E1B sequence are stable integrated at different sites in said packaging cell line.

61. The adenoviral vector of claim 59, wherein said packaging cell line is of human origin.

62. The adenoviral vector of claim 61, wherein said adenoviral packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production.

63. The adenoviral vector of claim 59, wherein the promoter in said adenoviral packaging cell line that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter is a retrovirus promoter.

64. The adenoviral vector of claim 59, wherein said adenoviral E1A coding sequence in said packaging cell line comprises the E1A coding sequence of GenBank Accession Number M73260.

65. The adenoviral vector of claim 59, wherein said adenoviral E1B coding sequence in said packaging cell line comprises the E1B coding sequence of GenBank Accession Number M73260.

66. The adenoviral vector according to claim 59, wherein said packaging cell line comprises a first expression vector and a second expression vector stably integrated into said packaging cell line's genome, wherein said first expression vector comprises adenoviral E1A coding sequences, operably linked to a non-adenoviral heterologous promoter, and said second expression vector comprises adenoviral E1B coding sequences operably linked to a non-adenoviral heterologous promoter.

67. The adenoviral vector according to claim 66, wherein said first expression vector comprises a retroviral nucleotide sequence.

68. The adenoviral vector according to claim 66, wherein said first and second expression vectors each comprise a retroviral nucleotide sequence.

69. The adenoviral vector according to claim 59, wherein said adenoviral vector is replication defective.

70. A composition comprising the adenoviral vector of claim 59, together with a pharmaceutically acceptable excipient.

71. An adenoviral vector substantially free of replication competent adenovirus, produced by a packaging cell line permissive for replication of an E1A/E1B deficient adenoviral vector, wherein said packaging cell line comprises an adenoviral E1A coding sequence and an adenoviral E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter, and wherein said adenoviral E1A coding sequence and said adenoviral E1B coding sequence are stably integrated into said packaging cell line and are operably linked to identical promoters.

72. The adenoviral vector of claim 71, wherein said adenoviral E1A coding sequence and said adenoviral E1B sequence are stable integrated at different sites in said packaging cell line.

73. The adenoviral vector of claim 71, wherein said packaging cell line is of human origin.

74. The adenoviral vector of claim 73, wherein said packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production.

75. The adenoviral vector of claim 71, wherein the promoter in said packaging cell line that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter is a retrovirus promoter.

76. The adenoviral vector of claim 71, wherein said adenoviral E1A coding sequence in said packaging cell line comprises the E1A coding sequence of GenBank Accession Number M73260.

77. The adenoviral vector of claim 71, wherein said adenoviral E1B coding sequence in said packaging cell line comprises the E1B coding sequence of GenBank Accession Number M73260.

78. The adenoviral vector according to claim 71, wherein said adenoviral vector is replication defective.

79. A composition comprising the adenoviral vector of claim 71, together with a pharmaceutically acceptable excipient.

80. A cell comprising an adenovirus E1A coding sequence and an adenovirus E1B coding sequence each operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, and wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated into said packaging cell line and are operably linked to identical promoters.

81. A method of producing a modified cell line, the method comprising: introducing into a first cell line permissive for adenovirus replication, nucleic acid comprising (i) an adenovirus E1A coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter and (ii) an adenovirus E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, and wherein the nucleic acid comprising the adenovirus E1A coding sequence and the nucleic acid comprising the adenovirus E1B coding sequence are present on separate vectors.

82. A cell line comprising an adenovirus E1A coding sequence, each and an adenovirus E1B coding sequence operably linked to a promoter that lacks substantial sequence identity to a native adenovirus E1A or E1B promoter, and wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated into the cell line and are operable linked to different heterologous promoters.

83. A method of producing a modified cell line, the method comprising: introducing into a first cell line permissive for adenovirus replication, nucleic acid comprising (i) an adenovirus E1A coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter and (ii) an adenovirus E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, wherein the nucleic acid comprising the adenovirus E1A coding sequence and the nucleic acid comprising the adenovirus E1B coding sequence are present on separate vectors and the promoters operably linked to the E1A and E1B coding sequences are different.

84. A system comprising an adenoviral vector substantially free of wild-type replication competent adenovirus, and a packaging cell line permissive for replication of an E1A/E1B deficient adenoviral vector, wherein said packaging cell line comprises an adenoviral E1A coding sequence and an adenoviral E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter, and wherein said adenoviral E1A coding sequence and said adenoviral E1B coding sequence are stably integrated into said packaging cell line and are operably linked to different heterologous promoters.

85. The system of claim 84, wherein said adenoviral E1A coding sequence and said adenoviral E1B sequence are stable integrated at different sites in said packaging cell line.

86. The system of claim 84, wherein said packaging cell line is of human origin.

87. The system of claim 86, wherein said packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production.

88. The system of claim 84, wherein the promoter in said packaging cell line that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter is a retrovirus promoter.

89. The system of claim 84, wherein said adenoviral E1A coding sequence in said packaging cell line comprises the E1A coding sequence of GenBank Accession Number M73260.

90. The system of claim 84, wherein said adenoviral E1B coding sequence in said packaging cell line comprises the E1B coding sequence of GenBank Accession Number M73260.

91. The system according to claim 84, wherein said packaging cell line comprises a first expression vector and a second expression vector stably integrated into said packaging cell line's genome, wherein said first expression vector comprises adenoviral E1A coding sequences, operably linked to a non-adenoviral heterologous promoter, and said second expression vector comprises adenoviral E1B coding sequences operably linked to a non-adenoviral heterologous promoter.

92. The system according to claim 91, wherein said first expression vector comprises a retroviral nucleotide sequence.

93. The system according to claim 91, wherein said first and second expression vectors each comprise a retroviral nucleotide sequence.

94. The system according to claim 84, wherein said adenoviral vector is replication defective.

95. The system according to claim 84, wherein no wild type replication competent adenovirus is detected following 18 cycles of infection.

96. A system comprising an adenoviral vector substantially free of wild type replication competent adenovirus, and a packaging cell line permissive for replication of an E1A/E1B deficient adenoviral vector, wherein said packaging cell line comprises an adenoviral E1A coding sequence and an adenoviral E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter, and wherein said adenoviral E1A coding sequence and said adenoviral E1B coding sequence are stably integrated into said packaging cell line and are operably linked to identical promoters.

97. The system of claim 96, wherein said adenoviral E1A coding sequence and said adenoviral E1B sequence are stable integrated at different sites in said packaging cell line.

98. The system of claim 96, wherein said packaging cell line is of human origin.

99. The system of claim 98, wherein said packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production.

100. The system of claim 96, wherein the promoter in said packaging cell line that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter is a retrovirus promoter.

101. The system of claim 96, wherein said adenoviral E1A coding sequence in said packaging cell line comprises the E1A coding sequence of GenBank Accession Number M73260.

102. The system of claim 96, wherein said adenoviral E1B coding sequence in said packaging cell line comprises the E1B coding sequence of GenBank Accession Number M73260.

103. The system according to claim 96, wherein said adenoviral vector is replication defective.

104. The system according to claim 96, wherein no wild type replication competent adenovirus is detected following 18 cycles of infection.