# Patent

## Title
Method for rapid screening of bacterial transformants and novel simian adenovirus proteins

## Abstract

Chimpanzee serotype C68 proteins, peptides, and polypeptide are provided. Also provided are novel adenoviruses derived from these proteins, as well as compositions containing these proteins and methods of using same for immunization and therapy. Further, a rapid method for screening recombinant transformants using a visually detectable method is described.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a US National Phase of Application No. PCT/US02/19735, filed Jun. 20, 2002, which claims the benefit under 35 USC 119(e) of US Provisional Appln. No. 60/300,501, filed on Jun. 22, 2001 and US Provisional Appln. No. 60/385,632, filed on Jun. 4, 2002.
BACKGROUND OF THE INVENTION
Recombinant adenoviruses have been described for gene therapy and vaccine uses.
Adenoviruses have a characteristic morphology with an icosahedral capsid consisting of three major proteins, hexon (II), penton base (III) and a knobbed fibre (IV), along with a number of other minor proteins, VI, VIII, IX, IIIa and IVa2 [W. C. Russell, J. Gen Virol., 81:2573-2604 (Nov. 2000)]. The virus genome is a linear, double-stranded DNA with a terminal protein attached covalently to the 5′ termini, which have inverted terminal repeats (ITRs). The virus DNA is intimately associated with the highly basic protein VII and a small peptide termed mu. Another protein, V, is packaged with this DNA-protein complex and provides a structural link to the capsid via protein VI. The virus also contains a virus-encoded protease, which is necessary for processing of some of the structural proteins to produce mature infectious virus.
There continues to be a need for recombinant viral vectors and improved methods for making these vectors.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method for rapid screening of bacterial transformants. The method involves engineering a recombinant shuttle vector comprising a nucleic acid cassette containing a transgene and a nucleic acid sequence encoding prokaryotic green fluorescent protein (GFP) operably linked to regulatory sequences which permit its expression in a host cell. Thereafter, host cells are transfected with the shuttle vector and screened for expression of GFP. The absence of green color (i.e., white) is indicative of a cell carrying the recombinant virus. Expression of GFP is readily detected by the green color when activated by fluorescent light, and indicates the presence of parent virus (i.e., absence of recombinant).
In another aspect, the invention provides capsid proteins of C68, isolated from other C68 proteins, and characterized by the amino acids provided herein.
In still another embodiment, the invention provides adenoviral vectors and non-viral targeting proteins derived from the C68 capsid proteins, termed herein C68-derived constructs.
Yet other advantages of the present invention will be readily apparent from the following detailed description of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 summarizes the genetic organization of the chimpanzee adenovirus C68 genome. In FIG. 1A the genome of the C68 chimpanzee adenovirus is schematically represented by the box at the top. The inverted terminal repeats are shaded black and the early regions are shaded gray. The arrowheads above the box indicate the direction of expression of the early genes. The line below the box represents the division of the genome into 100 map units. The arrows below the line represent the five late gene regions and the proteins encoded in each region. The numbers below the box or arrows indicate the start (promoter or initiation codon) and end (canonical PolyA signal) for each region. * represents the E2A late promoter. FIG. 1B illustrates the PstI clones; FIG. 1C illustrates the BamHI clones. FIG. 1D illustrates the HindIII clones. For parts 1B-1D, the unshaded regions indicate that a fragment was cloned into a plasmid vector, while the shaded regions indicate that the restriction fragment was not cloned. For each section the fragment name, alphabetical with A being the largest fragment, and the fragment size are listed above the box and the fragment end points are listed below the box.
FIG. 2 provides a sequence alignment of the C68 hexon protein [aa 131 to 441 of SEQ ID NO:16] with Ad4 [SEQ ID NO:34], Ad16 [SEQ ID NO:35], Ad3 [SEQ ID:NO:36], Ad7 [SEQ ID NO:37], and Ad2 [SEQ ID NO:38]. The deduced amino acid sequences of highly similar human adenovirus hexons were compared with the C68 chimpanzee adenovirus using CLUSTAL X. Serotypes and subgroups are indicated on the left margin, followed by the residue number. The numbering refers to the amino acid position with respect to the start of translation. Amino acids are shaded with respect to C68 to highlight sequence similarities (gray) and identities (black). The seven hypervariable regions within loop domains DE1 and FG1 are labeled along the bottom and correspond to the following Ad2 sequences in the alignment: HVR1, 137-188; HVR2, 194-204; HVR3, 222-229; HVR4, 258-271; HVR5, 278-294; HVR6, 316-327; and HVR7, 433-465 of SEQ ID NO:16. The GenBank accession numbers for the sequences shown are as follow: AAD03657 (Ad4), S37216 (Ad16), S39298 (Ad3), AAD03663 (Ad7), and NP040525 (Ad2).
FIG. 3 provides an alignment of the amino acid sequences of the fiber knob domains of chimpanzee C68 (Pan-9) [amino acids 247 to 425 of SEQ ID NO: 27] and the human adenovirus serotypes 2 [SEQ ID NO: 39] and 5 [SEQ ID NO:40].
FIG. 4 provides an alignment of the amino acid sequences of the L1 and a portion of the L2 loops of the capsid hexon on the human adenovirus serotype 5 [SEQ ID NO:41]and chimpanzee C68 (Pan-9) [amino acids 125 to 443 of SEQ ID NO:16] adenovirus sequences. The intervening conserved region is part of the pedestal domain conserved between adenovirus serotypes.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel adenovirus capsid proteins derived from the unique sequences of chimpanzee adenovirus C68. The capsid proteins of the invention are useful for a variety of purposes, including non-viral targeted delivery to cells and for creating recombinant viral vectors. These proteins and viral vectors are useful for delivery of heterologous molecules to target cells.
The invention further provides a novel method for rapid screening of bacterial transformants obtained during production of the novel adenoviral capsids of the invention, and during production of a variety of other viral or non-viral constructs. In this method, at least the shuttle vector is engineered to contain a marker gene, e.g., green fluorescent protein (GFP), gene under the control of a suitable promoter. The transformed cells are screened for expression of marker. In the case of GFP, white colonies are recombinants while green colonies are residual parental plasmid.
I. Novel Adenovirus Capsid Proteins
In one aspect, the invention provides unique C68 adenoviral capsid proteins, including the C68 hexon region, the C68 penton region, and the C68 fiber region, and fragments thereof. Suitably, these capsid proteins can be substantially pure, i.e., are free of other proteins. Preferably, these proteins are at least 10% homogeneous, more preferably 60% homogeneous, and most preferably 95% homogeneous.
In addition, the invention provides unique C68-derived capsid proteins. As used herein, a C68-derived capsid protein includes any C68 capsid protein or a fragment thereof including, without limitation, a polypeptide, peptide or a consecutive sequence of at least 8 amino acid residues unique to a C68 capsid protein and which is free of other proteins. A C68-derived capsid protein also includes a capsid protein that contains a C68 capsid protein or fragment thereof as defined above, including, without limitation, a chimeric capsid protein, a fusion protein, an artificial capsid protein, a synthetic capsid protein, and a recombinant capsid proteins, without limitation to means of generating these proteins. Suitably, these C68-derived capsid proteins contain one or more C68 regions or fragments thereof (e.g., a hexon) in combination with capsid regions or fragments thereof of different adenoviral serotypes, or of non-adenoviral sources, as described herein. These C68-derived capsid proteins may be used in non-viral targeting of useful molecules to cells, or for-production of viral vectors, as described herein.
A “modification of a capsid protein associated with altered tropism” as used herein includes an altered capsid protein, i.e, a penton, hexon or fiber protein region, or fragment thereof, such as the knob domain of the fiber region, or a polynucleotide encoding same, such that specificity is altered.
In one embodiment, the amino acid sequences of the C68 penton protein are provided








                MMRRAYPEGPPPSYESVMQQAMAAAAHQPPLEAPYVPPRYLAPT



 


EGRNSIRYSELAPLYDTTRLYLVDNKSADIASLNYQNDHSNFLTTVVQNNDFTPTEAS


 


TQTINFDERSRWGGQLKTIMHTNMPNVNEFMYSNKFKARVMVSRKTPNGVTVTEDYDG


 


SQDELKYEWVEFELPEGNFSVTMTIDLMNNAIIDNYLAVGRQNGVLESDIGVKFDTRN


 


FRLGWDPVTELVMPGVYTNEAFHPDIVLLPGCGVDFTESRLSNLLGIRKRQPFQEGFQ


 


IMYEDLEGGNIPALLDVDAYEKSKEDAAAEATAAVATASTEVRGDNFASAAAVAAAEA


 


AETESKIVIQPVEKDSKNRSYNVLPDKINTAYRSWYLAYNYGDPEKGVRSWTLLTTSD


 


VTCGVEQVYWSLPDMMQDPVTFRSTRQVSNYPVVGAELLPVYSKSFFNEQAVYSQQLR


 


AFTSLTHVFNRFPENQILVRPPAPTITTVSENVPALTDHGTLPLRSSIRGVQRVTVTD


 


ARRRTCPYVYKALGIVAPRVLSSRTF.






Suitably, this penton protein, or unique fragments thereof, may be utilized for a variety of purposes. Examples of suitable fragments include the C68 penton having N-terminal and/or C-terminal truncations of about 50, 100, 150, or 200 amino acids, based upon the amino acid numbering provided above and in SEQ ID NO:12. Other suitable fragments include shorter internal, C-terminal, or N-terminal fragments. Further, the penton protein may be modified for a variety of purposes known to those of skill in the art.
The sequences of the C68 hexon are provided in SEQ ID NO:16:








            MATPSMLPQWAYMHIAGQDASEYLSPGLVQFARATDTYFSLGNK



 


FRNPTVAPTHDVTTDRSQRLTLRFVPVDREDNTYSYKVRYTLAVGDNRVLDMASTYFD


 


IRGVLDRGPSFKPYSGTAYNSLAPKGAPNTCQWTYKADGETATEKTYTYGNAPVQGIN


 


ITKDGIQLGTDTDDQPIYADKTYQPEPQVGDAEWHDITGTDEKYGGRALKPDTKMKPC


 


YGSFAKPTNKEGGQANVKTGTGTTKEYDIDMAFFDNRSAAAAGLAPEIVLYTENVDLE


 


TPDTHIVYKAGTODSSSSINLGQQAMPNRPNYIGFRDNFIGLMYYNSTGNMGVLAGQA


 


SQLNAVVDLQDRNTELSYQLLLDSLGDRTRYFSMWNQAVDSYDPDVRIIENHGVEDEL


 


PNYCEPLDAVGRTDTYQGIKANGTDQTTWTKDDSVNDANEIGKGNPFAMEINIQANLW


 


RNFLYANVALYLPDSYKYTPANVTLPTNTNTYDYMNGRVVAPSLVDSYINIGARWSLD


 


PMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKSLLLLPGSYTYEWNF


 


RKDVNMILQSSLGNDLRTDGASISFTSINLYATFFPMAHNTASTLEAMLRNDTNDQSF


 


NDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWSFTRLKTKETPSLGSGFDPYFVY


 


SGSIPYLDGTFYLNHTFKKVSITFDSSVSWPGNDRLLTPNEFEIKRTVDGEGYNVAQC


 


NMTKDWFLVQMLAHYNIGYQGFYVPEGYKDRMYSFFRNFQPMSRQVVDEVNYKDYQAV


 


TLAYQHNNSGFVGYLAPTMRQGQPYPAXYPYPLIGKSAVTSVTQKKFLCDRVMWRIPF


 


SSNFNSNGALTDLGQNMLYANSAHALDMNFEVDPMOESTLLYVVFEVFDVVRVHQPHR


 


GVIEAVYXRTPFSAGNATT.






Suitably, this hexon protein, or unique fragments thereof, may be utilized for a variety of purposes. Examples of suitable fragments include the C68 hexon having N-terminal and/or C-terminal truncations of about 50, 100, 150, 200, 300, 400, or 500 amino acids, based upon the amino acid numbering provided above and in SEQ ID NO:16. Other suitable fragments include shorter internal, C-terminal, or N-terminal fragments. For example, one suitable fragment the loop region (domain) of the hexon protein, designated DE1 and FG1, or a hypervariable region thereof. Such fragments include the regions spanning amino acid residues about 125 to 443; about 138 to 441, or smaller fragments, such as those spanning about residue 138 to residue 163; about 170 to about 176; about 195 to about 203; about 233 to about 246; about 253 to about 264; about 287 to about 297; and about 404 to about 430 of C68, with reference to SEQ ID NO:16. Other suitable fragments may be readily identified by one of skill in the art. Further, the hexon protein may be modified for a variety of purposes known to those of skill in the art.
In one example, it may be desirable to generate an adenovirus having an altered hexon protein utilizing the C68 hexon protein sequences of the invention. One suitable method for altering hexon proteins is described in U.S. Pat. No. 5,922,315, which is incorporated by reference. In this method, at least one loop region of the adenovirus hexon is changed with at least one loop region of another adenovirus serotype. Thus, at least one loop region of such an altered adenovirus hexon protein is a C68 hexon loop region. In one embodiment, a loop region of the C68 hexon protein is replaced by a loop region from another adenovirus serotype. In another embodiment, the loop region of the C68 hexon is used to replace a loop region from another adenovirus serotype. Suitable adenovirus serotypes may be readily selected from among human and non-human serotypes, as described herein. Where non-human adenoviruses are selected, the serotypes are preferably selected from non-human primates. However, the selection of a suitable serotype is not a limitation of the present invention. Still other uses for the C68 hexon protein sequences of the invention will be readily apparent to those of skill in the art.
The sequences of the C68 fiber protein are: SEQ ID NO:27:








      MSKKRVRVDDDFDPVYPYDADNAPTVPPINPPFVSSDGFQEKPL



 


GVLSLRLADPVTTKNGEITLKLGEGVDLDSSGKLISNTATKAAAPLSFSNNTISLNMD


 


PFYTKDGKLSLQVSPPLNILRTSILNTLALGFGSGLGLRGSALAVQLVSPLTFDTDGN


 


IKLTLDRGLHVTTGDAIESNISWAKGLKFEDGAIATNIGNGLEFGSSSTETGVDDAY


 


PIQVKLGSGLSFDSTGAIMAGNKEDDKLTLWTTPDPSPNCQILAENDAKLTLCLTKCG


 


SQILATVSVLVVGSGNLNPITGTVSSAQVFLRFDANGVLLTEHSTLKKYWGYRQGDSI


 


DGTPYTNAVGFMPNLKAYPKSQSSTTKNNIVGQVYMNGDVSKPMLLTITLNGTDDSNS


 


TYSMSFSYTWTNGSYVGATFGANSYTESYIAQE.






Suitably, this fiber protein, or unique fragments thereof, may be utilized for a variety of purposes. One suitable fragment is the fiber knob, which spans about amino acids 247 to 425 of SEQ ID NO: 27. Examples of other suitable fragments include the C68 fiber having N-terminal and/or C-terminal truncations of about 50, 100, 150, or 200 amino acids, based upon the amino acid numbering provided above and in SEQ ID NO:27. Still other suitable fragments include internal fragments. Further, the fiber protein may be modified using a variety of techniques known to those of skill in the art.
The amino acid sequences of other useful gene products of C68 are provided in SEQ ID Nos. 1-11, 13-15, 17-26, and 28-38 of the attached sequence listing. More particularly, these sequences are as follows.














Ad C68



Ad C68 - CDS,
SEQ


Regions
With ref to SEQ ID NO: 33.
ID NO:


















E1a
  11 kDa
 578 . . . 649, 1236 . . . 1469
1



28.2 kDa
 578 . . . 1142, 1236 . . . 1444
2



24.8 kDa
 578 . . . 1049, 1236 . . . 1444
3


E1b
20.5 kDa
 1603 . . . 2163
4



54.7 kDa
 1908 . . . 3404
5



18.5 kDa
 1908 . . . 2200, 3188 . . . 3404
6



10.1 kDa
 1908 . . . 2170, 3306 . . . 3324
7


IX
Hexon-associated
 3489 . . . 3917
8



protein - pIX


IVa2
Maturation protein -
Complement (3976 . . . 5309,
9



pIVa2
 5588 . . . 5600)


L1
21.9 kDa
17858 . . . 8460
10



42.9 kDa
10825 . . . 12000
11


L2
Penton - pIII
13888 . . . 15492
12



Major core protein -
15493 . . . 16098
13



pVIII



Minor Core Protein -
16120 . . . 17190
14



pV


L3
Hexon-associated
17442 . . . 18215
15



protein - pVI



Hexon - pII
18322 . . . 21123
16


E2a
DNA-Binding Protein
Complement
17



Endopeptidase
(21835 . . . 23376)


L4
Virion
Complement (25529 . . . 
18



morphogenesis-
25862, 26032 . . . 26366)



associated protein



24.3 kDa



Hexon-associated
26446 . . . 27129
19



protein - pVIII


E3
11.6 kDa
27130 . . . 27450
20



  16 kDa
(27404 . . . 27477, 27666 . . . 
21




28032)



19.3 kDa
28014 . . . 28544
22



22.3
28572 . . . 29186
23



 9.9 kDa
30722 . . . 30997
24



15.6 kDa
31003 . . . 31434
25



14.7 kDa
31427 . . . 31834
26


L5
Fiber - pIV
32137 . . . 33414
27


E4
ORF7-like protein
Complement(33521 . . . >33772)
28



Orf6 - 33 kDa
Complement (33769 . . . 34674)
29



Orf4 - 13.2 kDa
Complement (34580 . . . 34945)
30



Orf3 - 12.8 kDa
Complement (34955 . . . 35308)
31



Orf2 - 14.2 kDa
Complement (35305 . . . 35694)
32









Thus, the invention provides unique C68 proteins, peptides and fragments thereof, which are produced recombinantly or by other methods. Suitably, such fragments are at least 8 amino acids in length. However, fragments of other desired lengths are readily utilized. In addition, the invention encompasses such modifications as may be introduced to enhance yield and/or expression of a C68 protein or fragment, construction of a fusion molecule in which all or a fragment of the C68 protein or fragment is fused (either directly or via a linker) with a fusion partner to enhance. Other suitable modifications include, without limitation, truncation of a coding region (e.g., a protein or enzyme) to eliminate a pre- or pro-protein ordinarily cleaved to produce the mature protein or enzyme and/or mutation of a coding region to provide a secretable gene product. Still other modifications will be readily apparent to one of skill in the art. The invention further encompasses proteins having at least about 95% to 99% identity to the C68 proteins provided herein.
The term “substantial homology” or “substantial similarity,” when referring to a protein or fragment thereof, indicates that, when optimally aligned with appropriate amino acid insertions or deletions with another protein, there is nucleotide sequence identity in at least about 95 to 99% of the aligned sequences.
The term “percent sequence identity” or “identical” in the context of proteins or fragments thereof refers to the amino acids in the two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over the full length of a protein, enzyme, polypeptide, peptide, or other fragment of at least about 200 to 500 amino acids, is desired. However, identity among smaller fragments, e.g. of at least about 8 amino acids, usually at least about 20 to 24 amino acids, at least about 28 to 32 amino acids, at least about 50 or more amino acids, may also be desired.
Identity is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art. As described herein, alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs, such as “Clustal W”, accessible through Web Servers on the internet. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure amino acid sequence identity, including those contained in the programs described above. Generally, these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another-algorithm or computer program that provides at least the level of identity or alignment as that provided by the referenced algorithms and programs.
As described herein, the C68-derived capsid proteins of the invention are particularly well suited for use in applications in which the neutralizing antibodies diminish the effectiveness of other Ad serotype based targeting proteins and vectors, as well as other viral vectors. The C68-derived constructs of the invention are particularly advantageous in readministration for repeat gene therapy or for boosting immune response (vaccine titers).
Also provided by the present invention are artificial adenoviral capsid proteins, which involve modifications and chimeric capsids constructed using the C68 adenoviral capsid proteins of the invention. Such artificial capsid proteins can be constructed using the amino acid sequences of the chimp C68 Ad hexon of the invention. Because the hexon protein is the determinant for serotype of an adenovirus, such artificial hexon proteins would result in adenoviruses having artificial serotypes. Other artificial capsid proteins can also be constructed using the chimp Ad penton sequences and/or fiber sequences of the invention and/or fragments thereof.
In one embodiment, a chimeric C68 capsid is constructed using C68 hexon and C68 fiber and a penton from another adenovirus. Alternatively, a chimeric C68 capsid comprises a C68 hexon and a fiber and penton from one or more different adenoviruses. Another chimeric adenovirus capsid comprises the C68 fiber and a penton and a hexon from one or more different different adenovirus serotypes. Yet another chimeric adenovirus capsid comprises the C68 penton and a fiber and hexon from one or more different adenovirus serotypes. Suitably, for such chimeric and artificial capsids constructed from C68 proteins, the non-C68 adenovirus components may be readily selected from other adenovirus serotypes.
Under certain circumstances, it may be desirable to use one or more of the C68-derived capsid proteins or a fragment thereof to generate an antibody. The term “an antibody,” as used herein, refers to an immunoglobulin molecule which is able to specifically bind to an epitope. The antibodies in the present invention exist in a variety of forms including, for example, high affinity polyclonal antibodies, monoclonal antibodies, synthetic antibodies, chimeric antibodies, recombinant antibodies and humanized antibodies. Such antibodies originate from immunoglobulin classes IgG, IgM, IgA, IgD and IgE. Such antibodies may be generated using any of a number of methods know in the art. Suitable antibodies may be generated by well-known conventional techniques, e.g. Kohler and Milstein and the many known modifications thereof. Similarly desirable high titer antibodies are generated by applying known recombinant techniques to the monoclonal or polyclonal antibodies developed to these antigens [see, e.g., PCT Patent Application No. PCT/GB85/00392; British Patent Application Publication No. GB2188638A; Amit et al., 1986 Science, 233:747-753; Queen et al., 1989 Proc. Nat'l. Acad. Sci. USA, 86:10029-10033; PCT Patent Application No. PCT/WO9007861; and Riechmann et al., Nature, 332:323-327 (1988); Huse et al, 1988a Science, 246:1275-1281]. Alternatively, antibodies can be produced by manipulating the complementarity determining regions of animal or human antibodies to the antigen of this invention. See, e.g., E. Mark and Padlin, “Humanization of Monoclonal Antibodies”, Chapter 4, The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology of Monoclonal Antibodies, Springer-Verlag (June, 1994); Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Bird et al., 1988, Science 242:423-426.
Alternatively, one or more of the C68 capsid proteins of the invention are assembled as multi-antigenic complexes [see, e.g., European Patent Application 0339695, published Nov. 2, 1989] and employed to elicit high titer antibodies. Further provided by the present invention are anti-idiotype antibodies (Ab2) and anti-anti-idiotype antibodies (Ab3). See, e.g., M. Wettendorff et al., “Modulation of anti-tumor immunity by anti-idiotypic antibodies.” In Idiotypic Network and Diseases, ed. by J. Cerny and J. Hiernaux, 1990 J. Am. Soc. Microbiol., Washington DC: pp. 203-229]. These anti-idiotype and anti-anti-idiotype antibodies are produced using techniques well known to those of skill in the art. These antibodies may be used for a variety of purposes, including diagnostic and clinical methods and kits.
Under certain circumstances, it may be desirable to introduce a detectable label or a tag onto a C68 antibody or other construct of the invention. As used herein, a detectable label is a molecule which is capable, alone or upon interaction with another molecule, of providing a detectable signal. Most desirably, the label is detectable visually, e.g. by fluorescence, for ready use in immunohistochemical analyses or immunofluorescent microscopy. For example, suitable labels include fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), coriphosphine-O (CPO) or tandem dyes, PE-cyanin-5 (PC5), and PE-Texas Red (ECD). All of these fluorescent dyes are commercially available, and their uses known to the art. Other useful labels include a colloidal gold label. Still other useful labels include radioactive compounds or elements. Additionally, labels include a variety of enzyme systems that operate to reveal a colorimetric signal in an assay, e.g., glucose oxidase (which uses glucose as a substrate) releases peroxide as a product which in the presence of peroxidase and a hydrogen donor such as tetramethyl benzidine (TMB) produces an oxidized TMB that is seen as a blue color. Other examples include horseradish peroxidase (HRP) or alkaline phosphatase (AP), and hexokinase in conjunction with glucose-6-phosphate dehydrogenase which reacts with ATP, glucose, and NAD+ to yield, among other products, NADH that is detected as increased absorbance at 340 nm wavelength. Other label systems that are utilized in the methods of this invention are detectable by other means, e.g., colored latex microparticles [Bangs Laboratories, Indiana] in which a dye is embedded are used in place of enzymes to form conjugates with the target sequences provide a visual signal indicative of the presence of the resulting complex in applicable assays.
Methods for coupling or associating the label with a desired molecule are similarly conventional and known to those of skill in the art. Known methods of label attachment are described [see, for example, Handbook of Fluorescent probes and Research Chemicals, 6th Ed., R. P. M. Haugland, Molecular Probes, Inc., Eugene, Oreg., 1996; Pierce Catalog and Handbook, Life Science and Analytical Research Products, Pierce Chemical Company, Rockford, Ill., 1994/1995]. Thus, selection of the label and coupling methods do not limit this invention.
The C68-derived proteins, peptides, and fragments described herein can be produced by any suitable means, including chemical synthesis, or other synthetic means, or by recombinant production and conventional genetic engineering methodologies. For example, peptides can be synthesized by the well known solid phase peptide synthesis methods (Merrifield, J. Am. Chem. Soc., 85:2149 (1962); Stewart and Young, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp. 27-62). These and other suitable production methods are within the knowledge of those of skill in the art and are not a limitation of the present invention.
Alternatively, suitable methods for recombinant production can be used. Selection of suitable expression systems, including expression vectors and host cells for protein expression and/or viral packaging is within the ability of one of skill in the art-and is not a limitation of the present invention. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.).
Nucleic acid sequences for the C68 genome, which is 36521 bp in length, may be obtained using information available in U.S. Pat. No. 6,083,716 and from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 (Pan-9). This sequences is also available from GenBank. Other chimpanzee adenovirus sequences are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, and other sources. Desirable chimpanzee strains Pan 5 [ATCC VR-591], Pan 6 [ATCC VR-592], and Pan 7 [ATCC VR-593]. Another particularly desirable chimpanzee adenovirus strain is chimpanzee adenovirus strain Bertha or C1 [ATCC Accession No. VR-20]. The sequence of the C1 serotype, and the location of the adenovirus genes E1a, E1b, E2a, E2b, E3, E4, L1; L2, L3, L4 and L5 are provided in U.S. Pat. No. 6,083,716, which is incorporated by reference herein. Optionally, non-chimpanzee simian adenoviral sequences may be used. Such non-chimpanzee adenovirus include those obtained from baboon adenovirus strains [e.g., ATCC VR-275], adenovirus strains isolated from rhesus monkeys [e.g., ATCC VR-209, ATCC VR-275, ATCC VR-353, ATCC VR-355], and adenovirus strains isolated from African green monkeys [e.g., ATCC VR-541; ATCC VR-941; ATCC VR-942; ATCC VR-943]. Alternatively, one may readily select from among the at least 51 different human serotypes, including, without limitation, human adenovirus serotypes 1, 2, 3, 4, 5, 12, 35, 37, and 40, and other, non-human primate adenovirus serotypes. Further, the sequences of these and other suitable serotypes are available from a variety of databases including, e.g., PubMed and GenBank [see, for example, U.S. Pat. No. 5,240,846]. Selection of an appropriate adenovirus is not a limitation of the present invention.
The invention further provides molecules useful for production of the C68 and C68-derived proteins of the invention, including such molecules which carry polynucleotides including DNA sequences. Thus, the invention further encompasses the nucleic acid sequences encoding the C68-derived constructs of the invention, and molecules and host cells useful in expression thereof, including suitable DNA molecules and vectors, which can be any suitable genetic element as defined herein. Preferably, these vectors are DNA-based (e.g., plasmids) or viral vectors.
In one embodiment, the C68-derived capsid proteins and other C68 adenovirus proteins described herein are used for non-viral, protein-based delivery of genes, proteins, and other desirable diagnostic, therapeutic and immunogenic molecules. A desired molecule for delivery to a target call may be associated with a C68-derived capsid protein or other protein by any suitable means, including, e.g., covalent or non-covalent binding. For example, the C68 penton protein may be readily utilized for such a purpose by production of a fusion protein using the C68 penton sequences of SEQ ID NO:12 in a manner analogous to that described in Medina-Kauwe LK, et al, Gene Ther. 2001 May; 8(10):795-803 and Medina-Kauwe LK, et al, Gene Ther. 2001 Dec; 8(23): 1753-1761. Alternatively, the amino acid sequences of C68 protein IX may be utilized for targeting vectors by associating the protein IX with a ligand that binds to a cell surface receptor, as described in U.S. patent application Ser. No. 20010047081. Suitable ligands include a CD40 antigen, an RGD-containing or polylysine-containing sequence, and the like. Still other C68 proteins may be used for used for these and similar purposes.
Further, the C68 adenovirus proteins of the invention are particularly well suited for use in producing viral vectors in C68-derived capsids. Suitably, these adenoviruses are pseudotyped such that a nucleic acid molecule carrying adenovirus ITRs from a non-C68 serotype and a minigene are packaged in a C68-derived adenoviral capsid of the invention. Alternatively, adenoviruses may be generated which contain at least the 5′ ITRs or the 3′ ITRs from C68, in a C68-derived capsid protein. The adenoviral vectors described herein may contain adenoviral sequences derived from one, more than one adenoviral strain. In yet another alternative, other C68 elements described herein may be utilized in production of recombinant vectors, or other desirable constructs.
The C68 proteins of the invention are useful for a variety of purposes, including construction of recombinant viruses. The C68-derived capsid proteins of the invention are useful in producing hybrid vectors, including, hybrid C68-adeno-associated viruses, Epstein-Barr virus, and retroviruses [Caplen et al, Gene Ther. 6: 454-459 (1999); Tan et al, J Virol., 73:7582-7589 (1999)]. Such viruses include C68-derived capsids which encapsidated vectors with adeno-associated virus (AAV) ITRs [Lieber et al, J Virol, 73:9314-9324 (1999), Recchia et al, Proc Natl Acad Sci USA, 96:2615-2620 (1999); or lentivirus ITRs (Zheng et al, Nat Biotech, 18:176-180 (2000), using Maloney leukemia virus long terminal repeats).
In a particularly desirable embodiment, the C68-derived capsid proteins, and optionally, the other C68 sequences described herein, are used to produce recombinant adenoviruses and pseudotyped adenoviruses. However, it will be readily understood that the C68-derived capsid proteins and other novel C68 sequences can be utilized for a variety of purposes, including production of other types of viral vectors (such as, e.g., hybrid vectors) carrying the therapeutic and immunogenic transgenes described below. Additionally, it will be readily understood that viral vectors carrying the unique C68 proteins and other sequences of the invention can be utilized for targeting and/or delivery of other types of molecules, including proteins, chemical molecules and other moieties useful for diagnostic, therapeutic and/or immunization purposes.
II. Recombinant Adenoviral Vectors
The compositions of this invention include vectors that deliver a heterologous molecule to cells, either for therapeutic or vaccine purposes. As used herein, a vector may include any genetic element including, without limitation, a cosmid, episome, plasmid, or a virus. In a particularly preferred embodiment, these vectors are viral vectors having capsid proteins derived from the C68 proteins of the invention. Alternatively, these vectors may contain other C68 sequences of the invention. These viral vectors suitably contain a minigene. By “minigene” is meant the combination of a selected heterologous gene and the other regulatory elements necessary to drive translation, transcription and/or expression of the gene product in a host cell.
Typically, an adenoviral vector is designed such that the minigene is flanked on its 5′ end and/or its 3′ end by adenoviral sequences which include, at a minimum, the cis-elements necessary for replication and virion encapsidation. Thus, in one embodiment, the vector contains adenoviral sequences encompassing at least the 5′ end of the adenoviral genome, i.e., the 5′ inverted terminal repeat sequences (which functions as origins of replication) and the native 5′ packaging enhancer domains (that contain sequences necessary for packaging linear Ad genomes and enhancer elements for the E1 promoter). The vector is also provided with the cis-acting 3′ ITRs. Suitably, the minigene is located between the 5′ adenoviral elements and the 3′ adenoviral elements. An adenoviral vector of the invention may also contain additional adenoviral sequences. For example, the minigene may be located in the site of such as the site of a functional E1 deletion or functional E3 deletion, among others that may be selected. Alternatively, the minigene may be inserted into an existing gene region to disrupt the function of that region, if desired.
The term “functionally deleted” or “functional deletion” means that a sufficient amount of the gene region is removed or otherwise damaged, e.g., by mutation or modification, so that the gene region is no longer capable of producing functional products of gene expression. If desired, the entire gene region may be removed.
Suitably, these adenoviral vectors of the invention contain one or more adenoviral elements derived from C68. In one embodiment, the vectors contain adenoviral ITRs from an adenoviral serotype which differs from C68. Alternatively, C68 ITRs may be utilized in a viral vector of the invention in which the capsid is not naturally occurring, but contains one or more C68 proteins, or fragments thereof. The selection of the serotype of the ITRs and the serotype of any other adenoviral sequences present in vector is not a limitation of the present invention. A variety of adenovirus strains are described herein.
The viral sequences, helper viruses, if needed, and recombinant viral particles, and other vector components and sequences employed in the construction of the vectors described herein are obtained as described above. See, e.g., U.S. Pat. No. 5,240,846. The DNA sequences of the adenovirus sequences are employed to construct vectors and cell lines useful in the preparation of such vectors. See, e.g., U.S. Pat. No. 6,083,716.
Modifications of the nucleic acid sequences forming the vectors of this invention, including sequence deletions, insertions, and other mutations may be generated using standard molecular biological techniques and are within the scope of this invention.
A. The “Minigene”
The methods employed for the selection of the transgene, the cloning and construction of the “minigene” and its insertion into the viral vector are within the skill in the art given the teachings provided herein.
1. The Transgene
The transgene is a nucleic acid sequence, heterologous to the vector sequences flanking the transgene, which encodes a polypeptide, protein, or other product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a host cell.
The composition of the transgene sequence will depend upon the use to which the resulting vector will be put. For example, one type of transgene sequence includes a reporter sequence, which upon expression produces a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc. These coding sequences, when associated with regulatory elements which drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for beta-galactosidase activity. Where the transgene is GFP or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.
However, desirably, the transgene is a non-marker sequence encoding a product which is useful in biology and medicine, such as proteins, peptides, RNA, enzymes, or catalytic RNAs. Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNAs, and antisense RNAs. One example of a useful RNA sequence is a sequence which extinguishes expression of a targeted nucleic acid sequence in the treated animal.
The transgene may be used for treatment, e.g., of genetic deficiencies, as a cancer therapeutic or vaccine, for induction of an immune response, and/or for prophylactic vaccine purposes. As used herein, induction of an immune response refers to the ability of a molecule (e.g., a gene product) to induce a T cell and/or a humoral immune response to the molecule. The invention further includes using multiple transgenes, e.g., to correct or ameliorate a condition caused by a multi-subunit protein. In certain situations, a different transgene may be used to encode each subunit of a protein, or to encode different peptides or proteins. This is desirable when the size of the DNA encoding the protein subunit is large, e.g., for an immunoglobulin, the platelet-derived growth factor, or a dystrophin protein. In order for the cell to produce the multi-subunit protein, a cell is infected with the recombinant virus containing each of the different subunits. Alternatively, different subunits of a protein may be encoded by the same transgene. In this case, a single transgene includes the DNA encoding each of the subunits, with the DNA for each subunit separated by an internal ribozyme entry site (IRES). This is desirable when the size of the DNA encoding each of the subunits is small, e.g., the total size of the DNA encoding the subunits and the IRES is less than five kilobases. As an alternative to an IRES, the DNA may be separated by sequences encoding a 2A peptide, which self-cleaves in a post-translational event. See, e.g., M. L. Donnelly, et al, J. Gen. Virol., 78(Pt 1): 13-21 (Jan 1997); Furler, S., et al, Gene Ther., 8(11):864-873 (June 2001); Klump H., et al., Gene Ther., 8(10):811-817 (May 2001). 2A peptide is significantly smaller than an IRES, making it well suited for use when space is a limiting factor. However, the selected transgene may encode any biologically active product or other product, e.g., a product desirable for study.
Suitable transgenes may be readily selected by one of skill in the art. The selection of the transgene is not considered to be a limitation of this invention.
2. Regulatory Elements
In addition to the major elements identified above for the minigene, the vector also includes conventional control elements necessary which are operably linked to the transgene in a manner that permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter [Invitrogen].
Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. For example, inducible promoters include the zinc-inducible sheep metallothionine (MT) promoter and the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter. Other inducible systems include the T7 polymerase promoter system [WO 98/10088]; the ecdysone insect promoter [No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)], the tetracycline-repressible system [Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], the tetracycline-inducible system [Gossen et al, Science, 268:1766-1769 (1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518 (1998)]. Other systems include the FK506 dimer, VP16 or p65 using castradiol, diphenol murislerone, the RU486-inducible system [Wang el al, Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)] and the rapamycin-inducible system [Magari et al, J Clin. Invest., 100:2865-2872 (1997)]. The effectiveness of some inducible promoters increases over time. In such cases one can enhance the effectiveness of such systems by inserting multiple repressors in tandem, e.g., TetR linked to a TetR by an IRES. Alternatively, one can wait at least 3 days before screening for the desired function. Once can enhance expression of desired proteins by known means to enhance the effectiveness of this system. For example, using the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
In another embodiment, the native promoter for the transgene will be used. The native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.
Another embodiment of the transgene includes a transgene operably linked to a tissue-specific promoter. For instance, if expression in skeletal muscle is desired, a promoter active in muscle should be used. These include the promoters from genes encoding skeletal β-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, as well as synthetic muscle promoters with activities higher than naturally occurring promoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Examples of promoters that are tissue-specific are known for liver (albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell receptor chain), neuronal such as neuron-specific enolase (NSE) promoter (Andersen el al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene (Piccioli et al., Neuron, 15:373-84 (1995)), among others.
Optionally, vectors carrying transgenes encoding therapeutically useful or immunogenic products may also include selectable markers or reporter genes may include sequences encoding geneticin, hygromicin or purimycin resistance, among others. Such selectable reporters or marker genes (preferably located outside the viral genome to be packaged into a viral particle) can be used to signal the presence of the plasmids in bacterial cells, such as ampicillin resistance. Other components of the vector may include an origin of replication. Selection of these and other promoters and vector elements are conventional and many such sequences are available [see, e.g., Sambrook et al, and references cited therein].
These vectors are generated using the techniques and sequences provided herein, in conjunction with techniques known to those of skill in the art. Such techniques include conventional cloning techniques of cDNA such as those described in texts [Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.], use of overlapping oligonucleotide sequences of the adenovirus genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.
III. Production of the Recombinant Viral Particle
In one embodiment, the chimpanzee adenoviral plasmids (or other vectors) are used to produce recombinant adenoviral particles. In one embodiment, the recombinant adenoviruses are functionally deleted in the E1a or E1b genes, and optionally bearing other mutations, e.g., temperature-sensitive mutations or deletions in other genes. In other embodiments, it is desirable to retain an intact E1and/or E1b region in the recombinant adenoviruses. Such an intact E1 region may be located in its native location in the adenoviral genome or placed in the site of a deletion in the native adenoviral genome (e.g., in the E3 region).
In the construction of useful chimpanzee adenovirus vectors for delivery of a gene to the human (or other mammalian) cell, a range of adenovirus nucleic acid sequences can be employed in the vectors. For example, all or a portion of the adenovirus delayed early gene E3 may be eliminated from the C68 adenovirus sequence which forms a part of the recombinant virus. The function of adenovirus E3 is believed to be irrelevant to the function and production of the recombinant virus particle. Adenovirus vectors may also be constructed having a deletion of at least the ORF6 region of the E4 gene, and more desirably because of the redundancy in the function of this region, the entire E4 region. Still another vector of this invention contains a deletion in the delayed early gene E2a. Deletions may also be made in any of the late genes L1 through L5 of the chimpanzee adenovirus genome. Similarly, deletions in the intermediate genes IX and IVa2 may be useful for some purposes. Other deletions may be made in the other structural or non-structural adenovirus genes. The above discussed deletions may be used individually, i.e., an adenovirus sequence for use in the present invention may contain deletions in only a single region. Alternatively, deletions of entire genes or portions thereof effective to destroy their biological activity may be used in any combination. For example, in one exemplary vector, the adenovirus sequence may have deletions of the E1 genes and the E4 gene, or of the E1, E2a and E3 genes, or of the E1 and E3 genes, or of E1, E2a and E4 genes, with or without deletion of E3, and so on. As discussed above, such deletions may be used in combination with other mutations, such as temperature-sensitive mutations, to achieve a desired result.
An adenoviral vector lacking any essential adenoviral sequences (e.g., E1a, E1b, E2a, E2b, E4 ORF6, L1, L2, L3, L4 and L5) may be cultured in the presence of the missing adenoviral gene products which are required for viral infectivity and propagation of an adenoviral particle. These helper functions may be provided by culturing the adenoviral vector in the presence of one or more helper constructs (e.g., a plasmid or virus) or a packaging host cell. See, for example, the techniques described for preparation of a “minimal” human Ad vector in International Patent Application WO96/13597, published May 9, 1996, and incorporated herein by reference.
1. Helper Viruses
Thus, depending upon the chimpanzee adenovirus gene content of the viral vectors employed to carry the minigene, a helper adenovirus or non-replicating virus fragment may be necessary to provide sufficient chimpanzee adenovirus gene sequences necessary to produce an infective recombinant viral particle containing the minigene. Useful helper viruses contain selected adenovirus gene sequences not present in the adenovirus vector construct and/or not expressed by the packaging cell line in which the vector is transfected. In one embodiment, the helper virus is replication-defective and contains a variety of adenovirus genes in addition to the sequences described above. Such a helper virus is desirably used in combination with an E1-expressing cell line.
Helper viruses may also be formed into poly-cation conjugates as described in Wu et al, J. Biol. Chem., 264:16985-16987 (1989); K. J. Fisher and J. M. Wilson, Biochem. J., 299:49 (Apr. 1, 1994). Helper virus may optionally contain a second reporter minigene. A number of such reporter genes are known to the art. The presence of a reporter gene on the helper virus which is different from the transgene on the adenovirus vector allows both the Ad vector and the helper virus to be independently monitored. This second reporter is used to enable separation between the resulting recombinant virus and the helper virus upon purification.
2. Complementation Cell Lines
To generate recombinant chimpanzee adenoviruses (Ad) deleted in any of the genes described above, the function of the deleted gene region, if essential to the replication and infectivity of the virus, must be supplied to the recombinant virus by a helper virus or cell line, i.e., a complementation or packaging cell line. In many circumstances, a cell line expressing the human E1 can be used to transcomplement the chimp Ad vector. This is particularly advantageous because, due to the diversity between the chimp Ad sequences of the invention and the human AdE1 sequences found in currently available packaging cells, the use of the current human E1-containing cells prevents the generation of replication-competent adenoviruses during the replication and production process. However, in certain circumstances, it will be desirable to utilize a cell line which expresses the E1 gene products can be utilized for production of an E1-deleted chimpanzee adenovirus. Such cell lines have been described. See, e.g., U.S. Pat. No. 6,083,716.
If desired, one may utilize the sequences provided herein to generate a packaging cell or cell line that expresses, at a minimum, the adenovirus E1 gene under the transcriptional control of a promoter for expression in a selected parent cell line. Inducible or constitutive promoters may be employed for this purpose. Examples of such promoters are described in detail elsewhere in this specification. A parent cell is selected for the generation of a novel cell line expressing any desired Ad gene. Without limitation, such a parent cell line may be HeLa [ATCC Accession No. CCL 2], A549 [ATCC Accession No. CCL 185], KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] and WI-38 [CCL 75] cells, among others. These cell lines are all available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209. Other suitable parent cell lines may be obtained from other sources.
Such E1-expressing cell lines are useful in the generation of recombinant chimpanzee adenovirus E1 deleted vectors. Additionally, or alternatively, the invention provides cell lines that express one or more chimpanzee adenoviral gene products, e.g., E1a, E1b, E2a, and/or E4ORF6, can be constructed using essentially the same procedures for use in the generation of recombinant chimpanzee viral vectors. Such cell lines can be utilized to transcomplement adenovirus vectors deleted in the essential genes that encode those products, or to provide helper functions necessary for packaging of a helper-dependent virus (e.g., adeno-associated virus). The preparation of a host cell according to this invention involves techniques such as assembly of selected DNA sequences. This assembly may be accomplished utilizing conventional techniques. Such techniques include cDNA and genomic cloning, which are well known and are described in Sambrook et al., cited above, use of overlapping oligonucleotide sequences of the adenovirus genomes, combined with polymerase chain reaction, synthetic methods, and any other suitable methods which provide the desired nucleotide sequence.
In still another alternative, the essential adenoviral gene products are provided in trans by the adenoviral vector and/or helper virus. In such an instance, a suitable host cell can be selected from any biological organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including, insect cells, yeast cells and mammalian cells. Particularly desirable host cells are selected from among any mammalian species, including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2, 293 cells (which express functional adenoviral E1), Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived-from mammals including human, monkey, mouse, rat, rabbit, and hamster. The selection of the mammalian species providing the cells is not a limitation of this invention; nor is the type of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.
3. Assembly of Viral Particle and Transfection of a Cell Line
Generally, when delivering the vector comprising the minigene by transfection, the vector is delivered in an amount from about 5 μg to about 100 μg DNA, and preferably about 10 to about 50 μg DNA to about 1×104 cells to about 1×1013 cells, and preferably about 105 cells. However, the relative amounts of vector DNA to host cells may be adjusted, taking into consideration such factors as the selected vector, the delivery method and the host cells selected.
The vector may be any vector known in the art or disclosed above, including naked DNA, a plasmid, phage, transposon, cosmids, viruses, etc. Introduction into the host cell of the vector may be achieved by any means known in the art or as disclosed above, including transfection, and infection. One or more of the adenoviral genes may be stably integrated into the genome of the host cell, stably expressed as episomes, or expressed transiently. The gene products may all be expressed transiently, on an episome or stably integrated, or some of the gene products may be expressed stably while others are expressed transiently. Furthermore, the promoters for each of the adenoviral genes may be selected independently from a constitutive promoter, an inducible promoter or a native adenoviral promoter. The promoters may be regulated by a specific physiological state of the organism or cell (i.e., by the differentiation state or in replicating or quiescent cells) or by exogenously-added factors, for example.
Introduction of the molecules (as plasmids or viruses) into the host cell may also be accomplished using techniques known to the skilled artisan and as discussed throughout the specification. In preferred embodiment, standard transfection techniques are used, e.g., CaPO4 transfection or electroporation.
Assembly of the selected DNA sequences of the adenovirus (as well as the transgene and other vector elements into various intermediate plasmids, and the use of the plasmids and vectors to produce a recombinant viral particle are all achieved using conventional techniques. Such techniques include conventional cloning techniques of cDNA such as those described in texts [Sambrook et al, cited above], use of overlapping oligonucleotide sequences of the adenovirus genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence. Standard transfection and co-transfection techniques are employed, e.g., CaPO4 precipitation techniques. Other conventional methods employed include homologous recombination of the viral genomes, plaquing of viruses in agar overlay, methods of measuring signal generation, and the like.
For example, following the construction and assembly of the desired minigene-containing viral vector, the vector is transfected in vitro in the presence of a helper virus into the packaging cell line. Homologous recombination occurs between the helper and the vector sequences, which permits the adenovirus-transgene sequences in the vector to be replicated and packaged into virion capsids, resulting in the recombinant viral vector particles. The current method for producing such virus particles is transfection-based. However, the invention is not limited to such methods.
The resulting recombinant chimpanzee adenoviruses are useful in transferring a selected transgene to a selected cell. In in vivo experiments with the recombinant virus grown in the packaging cell lines, the E1-deleted recombinant chimpanzee adenoviral vectors of the invention demonstrate utility in transferring a transgene to a non-chimpanzee, preferably a human, cell.
IV. Use of Non-Viral C68 Proteins and C68-derived Adenoviruses
The recombinant adenovirus vectors of the invention are useful for gene transfer to a human or non-chimpanzee veterinary patient in vitro, ex vivo, and in vivo. In addition, a variety of C68 proteins described herein are useful in non-viral targeting of transgenes, proteins, chemical molecules, and other moieties or molecules to cells. Suitable methods of delivery and dosing regimens are readily determined based upon the targeted molecule and targeting protein. Examples of suitable genes and sources of proteins for protein-mediated delivery are provided in the sections below relating to viral delivery of therapeutic and immunogenic molecules. While the discussion below focuses on viral vectors, it will be appreciated that the C68-derived proteins of the invention may be formulated as described herein for the C68-derived viral vectors and the same routes of administration and regimens may be utilized.
The recombinant adenovirus vectors described herein can be used as expression vectors for the production of the products encoded by the heterologous genes in vitro. For example, the recombinant adenoviruses containing a gene inserted into the location of an E1 deletion may be transfected into an E1-expressing cell line as described above. Alternatively, replication-competent adenoviruses may be used in another selected cell line. The transfected cells are then cultured in the conventional manner, allowing the recombinant adenovirus to express the gene product from the promoter. The gene product may then be recovered from the culture medium by known conventional methods of protein isolation and recovery from culture.
A C68-derived vector or C68-derived protein of the invention provides an efficient gene transfer vehicle that can deliver a selected transgene or other molecule to a selected host cell in vivo or ex vivo even where the organism has neutralizing antibodies to one or more AAV serotypes. In one embodiment, the rAAV and the cells are mixed ex vivo; the infected cells are cultured using conventional methodologies; and the transduced cells are re-infused into the patient. These compositions are particularly well suited to gene delivery for therapeutic purposes and for immunization, including inducing protective immunity.
More commonly, the C68-derived vectors and C68-derived proteins of the invention will be utilized for delivery of therapeutic or immunogenic molecules, as described below. It will be readily understood for both applications, that the C68-derived constructs of the invention are useful for use in regimens involving single administrations, as well as in regimens involving repeat delivery of adenoviral vectors or non-viral targeted delivery, or repeat delivery of the transgene or other molecule to the cells.
Such regimens typically involve delivery of a series of viral vectors in which the viral capsids are alternated. The viral capsids may be changed for each subsequent administration, or after a pre-selected number of administrations of a particular serotype capsid (e.g., one, two, three, four or more). For example, a regimen may involve delivery of a rAd with a C68-derived capsid and delivery with a rAd with another human or non-human primate adenovirus serotype. Optionally, these regimens may involve administration of rAd with capsids of other non-human primate adenoviruses, human adenoviruses, or artificial serotypes such as are described herein. Alternativley, the regimens involve administration of C68-derived proteins for non-viral targeting with repeat administrations of C68-derived proteins, or with other protein-based delivery systems. Each phase of these regimens can involve administration of a series of injections (or other delivery routes) with a single C68-derived construct followed by a series with another Ad serotype construct. Alternatively, the C68-derived vectors and proteins of the invention may be utilized in regimens involving other non-adenoviral-mediated delivery systems, including other viral systems, non-viral delivery systems, protein, peptides, and other biologically active molecules.
The following sections will focus on exemplary molecules which may be delivered via the adenoviral vectors of the invention.
A. Ad-Mediated Delivery of Therapeutic Molecules
In one embodiment, the above-described C68-derived constructs are administered to humans according to published methods for gene therapy. A C68-derived construct bearing a transgene can be administered to a patient, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle. A suitable vehicle includes sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose.
The C68-derived adenoviral vectors are administered in sufficient amounts to transduce the target cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the retina and other intraocular delivery methods, direct delivery to the liver, intranasal, intravenous, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral and other parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the transgene or the condition. The route of administration primarily will depend on the nature of the condition being treated.
Dosages of the viral vector will depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and may thus vary among patients. For example, a therapeutically effective adult human or veterinary dosage of the viral vector is generally in the range of from about 100 μL to about 100 mL of a carrier containing concentrations of from about 1×106 to about 1×1015 particles, about 1×1011 to 1×1013 particles, or about 1×109 to 1×1012 particles virus. Dosages will range depending upon the size of the animal and the route of administration. For example, a suitable human or veterinary dosage (for about an 80 kg animal) for intramuscular injection is in the range of about 1×109 to about 5×1012 particles per mL, for a single site. Optionally, multiple sites of administration may be delivered. In another example, a suitable human or veterinary dosage may be in the range of about 1×1011 to about 1×1015 particles for an oral formulation.
When C68 proteins of the invention are utilized for targeted delivery, suitable dosage ranges, a therapeutically effective adult human or veterinary dosage of the construct is generally in the range of from about 100 μL to about 100 mL of a carrier containing concentrations of from about 0.01 μg to about 100 mg protein, about 0.1 μg to about 10 mg, about 1 μg to about 1 mg protein. Dosages will range depending upon the size of the animal and the route of administration. Routes of administration may be readily selected from any suitable route including, without limitation, the routes described above.
One of skill in the art may adjust these doses, depending the route of administration, and the therapeutic or vaccinal application for which the C68-derived construct is employed. The levels of expression of the transgene, or for an immunogen, the level of circulating antibody, can be monitored to determine the frequency of dosage administration. Yet other methods for determining the timing of frequency of administration will be readily apparent to one of skill in the art.
An optional method step involves the co-administration to the patient, either concurrently with, or before or after administration of the C68-derived construct, of a suitable amount of a short acting immune modulator. The selected immune modulator is defined herein as an agent capable of inhibiting the formation of neutralizing antibodies directed against the recombinant vector of this invention or capable of inhibiting cytolytic T lymphocyte (CTL) elimination of the vector. The immune modulator may interfere with the interactions between the T helper subsets (TH1 or TH2) and B cells to inhibit neutralizing antibody formation. Alternatively, the immune modulator may inhibit the interaction between TH1 cells and CTLs to reduce the occurrence of CTL elimination of the vector. A variety of useful immune modulators and dosages for use of same are disclosed, for example, in Yang et al., J. Virol., 70(9) (Sept., 1996); International Patent Application No. WO96/12406, published May 2, 1996; and International Patent Application No. PCT/US96/03035, all incorporated herein by reference.
1. Therapeutic Transgenes
Useful therapeutic products encoded by the transgene include hormones and growth and differentiation factors including, without limitation, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth honnone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermnal growth factor (EGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor superfamily, including TGF, activins, inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1-15, any one of the heregluin/neuregulin/ARIA/neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, any one of the family of semaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.
Other useful transgene products include proteins that regulate the immune system including, without limitation, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-1 through IL- 18, monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors and, interferons, and, stem cell factor, flk-2/flt3 ligand. Gene products produced by the immune system are also useful in the invention. These include, without limitations, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules, as well as engineered immunoglobulins and MHC molecules. Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2 and CD59.
Still other useful gene products include any one of the receptors for the hormones, growth factors, cytokines, lymphokines, regulatory proteins and immune system proteins. The invention encompasses receptors for cholesterol regulation, including the low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, the very low density lipoprotein (VLDL) receptor, and the scavenger receptor. The invention also encompasses gene products such as members of the steroid hormone receptor superfamily including glucocorticoid receptors and estrogen receptors, Vitamin D receptors and other nuclear receptors. In addition, useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins, interferon regulation factor (IRF-1), Wilms tumor protein, ETS-binding protein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.
Other useful gene products include, carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase. fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor VIII, factor IX, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, a cystic fibrosis transmembrane regulator (CFTR) sequence, and a dystrophin cDNA sequence.
Other useful gene products include non-naturally occurring polypeptides, such as chimeric or hybrid polypeptides having a non-naturally. occurring amino acid sequence containing insertions, deletions or amino acid substitutions. For example, single-chain engineered immunoglobulins could be useful in certain immunocompromised patients. Other types of non-naturally occurring gene sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, which could be used to reduce overexpression of a target.
Reduction and/or modulation of expression of a gene are particularly desirable for treatment of hyperproliferative conditions characterized by hyperproliferating cells, as are cancers and psoriasis. Target polypeptides include those polypeptides which are produced exclusively or at higher levels in hyperproliferative cells as compared to normal cells. Target antigens include polypeptides encoded by oncogenes such as myb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu, trk and EGRF. In addition to oncogene products as target antigens, target polypeptides for anti-cancer treatments and protective regimens include variable regions of antibodies made by B cell lymphomas and variable regions of T cell receptors of T cell lymphomas which, in some embodiments, are also used as target antigens for autoimmune disease. Other tumor-associated polypeptides can be used as target polypeptides such as polypeptides which are found at higher levels in tumor cells including the polypeptide recognized by monoclonal antibody 17-1A and folate binding polypeptides. Such target polypeptides and their ligands are also useful in forming fusion partners with a C68 protein of the invention.
Other suitable therapeutic polypeptides and proteins include those which may be useful for treating individuals suffering from autoimmune diseases and disorders by conferring a broad based protective immune response against targets that are associated with autoimmunity including cell receptors and cells which produce self-directed antibodies. T cell mediated autoimmune diseases include Rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease and ulcerative colitis. Each of these diseases is characterized by T cell receptors (TCRs) that bind to endogenous antigens and initiate the inflammatory cascade associated with autoirnmune diseases.
The C68-derived constructs of the invention are particularly well suited for therapeutic regimens in which multiple deliveries of transgenes is desired, e.g., in regimens involving redelivery of the same transgene or in combination regimens involving delivery of other transgenes. Such regimens may involve administration of a C68-derived construct, followed by re-administration with a vector from the same serotype adenovirus. Particularly desirable regimens involve administration of a C68-derived construct of the invention, in which the serotype of the viral vector delivered in the first administration differs from the serotype of the viral vector utilized in one or more of the subsequent administrations. For example, a therapeutic regimen involves administration of a C68-derived vector and repeat administration with one or more adenoviral vectors of the same or different serotypes. In another example, a therapeutic regimen involves administration of an adenoviral vector followed by repeat administration with a C68-derived vector of the invention which differs from the serotype of the first delivered adenoviral vector, and optionally further administration with another vector which is the same or, preferably, differs from the serotype of the vector in the prior administration steps. These regimens are not limited to delivery of adenoviral vectors constructed using the C68-derived capsids of the invention. Rather, these regimens can readily utilize constructs, including non-viral targeting proteins and viral vectors, from other adenoviral serotypes, including, without limitation, other chimpanzee adenoviral serotypes (e.g., C1, etc), other non-human primate adenoviral serotypes, or human adenoviral serotypes, in combination with one or more of the C68-derived constructs of the invention. Examples of such chimpanzee, other non-human primate and human adenoviral serotypes are discussed elsewhere in this document. Further, these therapeutic regimens may involve either simultaneous or sequential delivery of C68-derived constructs of the invention in combination with non-adenoviral vectors, non-viral vectors, and/or a variety of other therapeutically useful compounds or molecules. The present invention is not limited to these therapeutic regimens, a variety of which will be readily apparent to one of skill in the art.
B. Ad-Mediated Delivery of Immunogenic Transgenes
The C68-derived constructs of the invention, including viral vectors and proteins, may also be employed as immunogenic compositions. As used herein, an immunogenic composition is a composition to which a humoral (e.g., antibody) or cellular (e.g., a cytotoxic T cell) response is mounted to a transgene product delivered by the immunogenic composition following delivery to a mammal, and preferably a primate. The present invention provides a recombinant C68-derived Ad that can contain in any of its adenovirus sequence deletions a gene encoding a desired immunogen, or a C68 protein capable of targeting an immunogenic molecule. The C68-derived adenovirus is well suited for use as a live recombinant virus vaccine in different animal species compared to an adenovirus of human origin, but is not limited to such a use. The recombinant adenoviruses and C68 proteins can be used as prophylactic or therapeutic vaccines against any pathogen for which the antigen(s) crucial for induction of an immune response and able to limit the spread of the pathogen has been identified and for which the cDNA is available.
Such vaccinal (or other immunogenic) compositions are formulated in a suitable delivery vehicle, as described above. Generally, doses for the immunogenic compositions are in the range defined above for therapeutic compositions. The levels of immunity of the selected gene can be monitored to determine the need, if any, for boosters. Following an assessment of antibody titers in the serum, optional booster immunizations may be desired.
Optionally, a vaccinal composition of the invention may be formulated to contain other components, including, e.g. adjuvants, stabilizers, pH adjusters, preservatives and the like. Such components are well known to those of skill in the vaccine art. Examples of suitable adjuvants include, without limitation, liposomes, alum, monophosphoryl lipid A, and any biologically active factor, such as cytokine, an interleukin, a chemokine, a ligands, and optimally combinations thereof. Certain of these biologically active factors can be expressed in vivo, e.g., via a polynucleotide, plasmid or viral vector. For example, such an adjuvant can be administered with a priming DNA vaccine encoding an antigen to enhance the antigen-specific immune response compared with the immune response generated upon priming with a DNA vaccine encoding the antigen only.
The recombinant adenoviruses are administered in a “an immunogenic amount”, that is, an amount of recombinant adenovirus that is effective in a route of administration to transfect the desired cells and provide sufficient levels of expression of the selected gene to induce an immune response. Where protective immunity is provided, the recombinant adenoviruses are considered to be vaccine compositions useful in preventing infection and/or recurrent disease.
Alternatively, or in addition, the vectors of the invention may contain, or C68-capsid or other protein can be utilized to target a transgene encoding a peptide, polypeptide or protein which induces an immune response to a selected immunogen. The C68-derived viruses of this invention are expected to be highly efficacious at inducing cytolytic T cells and antibodies to the inserted heterologous antigenic protein expressed by the vector.
1. Immunogenic Transgenes
For example, immunogens may be selected from a variety of viral families. Example of desirable viral families against which an immune response would be desirable include, the picornavirus family, which includes the genera rhinoviruses, which are responsible for about 50% of cases of the common cold; the genera enteroviruses, which include polioviruses, coxsackieviruses, echoviruses, and human enteroviruses such as hepatitis A virus; and the genera apthoviruses, which are responsible for foot and mouth diseases, primarily in non-human animals. Within the picornavirus family of viruses, target antigens include the VP1, VP2, VP3, VP4, and VPG. Another viral family includes the calcivirus family, which encompasses the Norwalk group of viruses, which are an important causative agent of epidemic gastroenteritis. Still another viral family desirable for use in targeting antigens for inducing immune responses in humans and non-human animals is the togavirus family, which includes the genera alphavirus, which include Sindbis viruses, RossRiver virus, and Venezuelan, Eastern & Western Equine encephalitis, and rubivirus, including Rubella virus. The flaviviridae family includes dengue, yellow fever, Japanese encephalitis, St. Louis encephalitis and tick borne encephalitis viruses. Other target antigens may be generated from the Hepatitis C or the coronavirus family, which includes a number of non-human viruses such as infectious bronchitis virus (poultry), porcine transmissible gastroenteric virus (pig), porcine hemagglutinating encephalomyelitis virus (pig), feline infectious peritonitis virus (cats), feline enteric coronavirus (cat), canine coronavirus (dog), and human respiratory coronaviruses, which may cause the common cold and/or non-A, B or C hepatitis. Within the coronavirus family, target antigens include the E1 (also called M or matrix protein), E2 (also called S or Spike protein), E3 (also called HE or hemagglutin-elterose) glycoprotein (not present in all coronaviruses), or N (nucleocapsid). Still other antigens may be targeted against the rhabdovirus family, which includes the genera vesiculovirus (e.g., Vesicular Stomatitis Virus), and the general lyssavirus (e.g., rabies). Within the rhabdovirus family, suitable antigens may be derived from the G protein or the N protein. The family filoviridae, which includes hemorrhagic fever viruses such as Marburg and Ebola virus may be a suitable source of antigens. The paramyxovirus family includes parainfluenza Virus Type 1, parainfluenza Virus Type 3, bovine parainfluenza Virus Type 3, rubulavirus (mumps virus), parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastle disease virus (chickens), rinderpest, morbillivirus, which includes measles and canine distemper, and pneumovirus, which includes respiratory syncytial virus. The influenza virus is classified within the family orthomyxovirus and is a suitable source of antigen (e.g., the HA protein, the N1 protein). The bunyavirus family includes the genera-bunyavirus (California encephalitis, La Crosse), phlebovirus (Rift Valley Fever), hantavirus (puremala is a hemahagin fever virus), nairovirus (Nairobi sheep disease) and various unassigned bungaviruses. The arenavirus family provides a source of antigens against LCM and Lassa fever virus. The reovirus family includes the genera reovirus, rotavirus (which causes acute gastroenteritis in children), orbiviruses, and cultivirus (Colorado Tick fever, Lebombo (humans), equine encephalosis, blue tongue).
The retrovirus family includes the sub-family oncorivirinal which encompasses such human and veterinary diseases as feline leukemia virus, HTLVI and HTLVII, lentivirinal (which includes human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus, and spumavirinal). Among the lentiviruses, many suitable antigens have been described and can readily be selected. Examples of suitable HIV and SIV antigens include, without limitation the gag, pol, Vif, Vpx, VPR, Env, Tat, Nef, and Rev proteins, as well as various fragments thereof. For example, suitable fragments of the Env protein may include any of its subunits such as the. gp120, gp160, gp41, or smaller fragments thereof, e.g., of at least about 8 amino acids in length. Similarly, fragments of the tat protein may be selected. [See, U.S. Pat. No. 5,891,994 and U.S. Pat. No. 6,193,981.] See, also, the HIV and SIV proteins described in D. H. Barouch et al, J. Virol., 75(5):2462-2467 (March 2001), and R. R. Amara, et al, Science, 292:69-74 (6 April 2001). In another example, the HIV and/or SIV immunogenic proteins or peptides may be used to form fusion proteins or other immunogenic molecules. See, e.g., the HIV-1 Tat and/or Nef fusion proteins and immunization regimens described in WO 01/54719, published Aug. 2, 2001, and WO 99/16884, published Apr. 8, 1999. The invention is not limited to the HIV and/or SIV immunogenic proteins or peptides described herein. In addition, a variety of modifications to these proteins have been described or could readily be made by one of skill in the art. See, e.g., the modified gag protein that is described in U.S. Pat. No. 5,972,596. Further, any desired HIV and/or SIV immunogens may be delivered alone or in combination. Such combinations may include expression from a single vector or from multiple vectors. Optionally, another combination may involve delivery of one or more expressed immunogens with delivery of one or more of the immunogens in protein form. Such combinations are discussed in more detail below.
The papovavirus family includes the sub-family polyomaviruses (BKU and JCU viruses) and the sub-family papillomavirus (associated with cancers or malignant progression of papilloma). The adenovirus family includes viruses (EX, AD7, ARD, O.B.) which cause respiratory disease and/or enteritis. The parvovirus family feline parvovirus (feline enteritis), feline panleucopeniavirus, canine parvovirus, and porcine parvovirus. The herpesvirus family includes the sub-family alphaherpesvirinae, which encompasses the genera simplexvirus (HSVI, HSVII), varicellovirus (pseudorabies, varicella zoster) and the sub-family betaherpesvirinae, which includes the genera cytomegalovirus (HCMV, muromegalovirus) and the sub-family gammaherpesvirinae, which includes the genera lymphocryptovirus, EBV (Burkitts lymphoma), infectious rhinotracheitis, Marek's disease virus, and rhadinovirus. The poxvirus family includes the sub-family chordopoxvirinae, which encompasses the genera orthopoxvirus (Variola (Smallpox) and Vaccinia (Cowpox)), parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, and the sub-family entomopoxvirinae. The hepadnavirus family includes the Hepatitis B virus. One unclassified virus which may be suitable source of antigens is the Hepatitis delta virus. Still other viral sources may include avian infectious bursal disease virus and porcine respiratory and reproductive syndrome virus. The alphavirus family includes equine arteritis virus and various Encephalitis viruses.
The present invention may also encompass immunogens which are useful to immunize a human or non-human animal against other pathogens including bacteria, fungi, parasitic microorganisms or multicellular parasites which infect human and non-human vertebrates, or from a cancer cell or tumor cell. Examples of bacterial pathogens include pathogenic gram-positive cocci include pneumococci; staphylococci; and streptococci. Pathogenic gram-negative cocci include meningococcus; gonococcus. Pathogenic enteric gram-negative bacilli include enterobacteriaceae; pseudomonas, acinetobacteria and eikenella; melioidosis; salmonella; shigella; haemophilus; moraxella; H. ducreyi (which causes chancroid); brucella; Franisella tularensis (which causes tularemia); yersinia (pasteurella); streptobacillus moniliformis and spirillum; Gram-positive bacilli include listeria monocytogenes; erysipelothrix rhusiopathiae; Corynebacterium diphtheria (diphtheria); cholera; B. anthracis (anthrax); donovanosis (granuloma inguinale); and bartonellosis. Diseases caused by pathogenic anaerobic bacteria include tetanus; botulism; other clostridia; tuberculosis; leprosy; and other mycobacteria. Pathogenic spirochetal diseases include syphilis; treponematoses: yaws, pinta and endemic syphilis; and leptospirosis. Other infections caused by higher pathogen bacteria and pathogenic fungi include actinomycosis; nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, and mucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis, torulopsosis, mycetoma and chromomycosis; and dermatophytosis. Rickettsial infections include Typhus fever, Rocky Mountain spotted fever, Q fever, and Rickettsialpox. Examples of mycoplasma and chlamydial infections include: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydial infections. Pathogenic eukaryotes encompass pathogenic protozoans and helminths and infections produced thereby include: amebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans; Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworrn) infections.
Many of these organisms and/or toxins produced thereby have been identified by the Centers for Disease Control [(CDC), Department of Heath and Human Services, USA], as agents which have potential for use in biological attacks. For example, some of these biological agents, include, Bacillus anthracis (anthrax), Clostridium botulinum and its toxin (botulism), Yersinia pestis (plague), variola major (smallpox), Francisella tularensis (tularemia), and viral hemorrhagic fever, all of which are currently classified as Category A agents; Coxiella burnetti (Q fever); Brucella species (brucellosis), Burkholderia mallei (glanders), Ricinus communis and its toxin (ricin toxin), Closiridium perfringens and its toxin (epsilon toxin), Staphylococcus species and their toxins (enterotoxin B), all of which are currently classified as Category B agents; and Nipan virus, multidrug-resistant tuberculosis, yellow fever, tickborne hemorrhagic fever viruses, tickborne encephalitis viruses, and hantaviruses, which are currently classified as Category C agents. In addition, other organisms, which are so classified or differently classified, may be identified and/or used for such a purpose in the future. It will be readily understood that the viral vectors and other constructs described herein are useful to deliver antigens from these organisms, viruses, their toxins or other by-products, which will prevent and/or treat infection or other adverse reactions with these biological agents.
Administration of the vectors and proteins of the invention to deliver immunogens against the variable region of the T cells elicit an immune response including CTLs to eliminate those T cells. In RA, several specific variable regions of TCRs which are involved in the disease have been characterized. These TCRs include V-3, V-14, V-17 and Vα-17. Thus, delivery of a nucleic acid sequence that encodes at least one of these polypeptides will elicit an immune response that will target T cells involved in RA. In MS, several specific variable regions of TCRs which are involved in the disease have been characterized. These TCRs include V-7 and Vα-10. Thus, delivery of a nucleic acid sequence that encodes at least one of these polypeptides will elicit an immune response that will target T cells involved in MS. In scleroderma, several specific variable regions of TCRs which are involved in the disease have been characterized. These TCRs include V-6, V-8, V-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12. Thus, delivery of a recombinant chimpanzee adenovirus that encodes at least one of these polypeptides will elicit an immune response that will target T cells involved in scleroderma.
C. Ad-Mediated Delivery Methods
The therapeutic levels, or levels of immunity, of the selected gene can be monitored to determine the need, if any, for boosters. Following an assessment of CD8+T cell response, or optionally, antibody titers, in the serum, optional booster immunizations may be desired. Optionally, the C68-derived constructs of the invention may be delivered in a single administration or in various combination regimens, e.g., in combination with a regimen or course of treatment involving other active ingredients or in a prime-boost regimen. A variety of such regimens have been described in the art and may be readily selected.
For example, prime-boost regimens may involve the administration of a DNA (e.g., plasmid) based vector to prime the immune system to second, booster, administration with a traditional antigen, such as a protein or a recombinant virus carrying the sequences encoding such an antigen. See, e.g., WO 00/11140, published Mar. 2, 2000, incorporated by reference. Alternatively, an immunization regimen may involve the administration of a recombinant chimpanzee adenoviral vector of the invention to boost the immune response to a vector (either viral or DNA-based) carrying an antigen, or a protein. In still another alternative, an immunization regimen involves administration of a protein followed by booster with a vector encoding the antigen.
In one embodiment, the invention provides a method of priming and boosting an immune response to a selected antigen by delivering a plasmid DNA vector carrying said antigen, followed by boosting with a recombinant chimpanzee adenoviral vector of the invention. In one embodiment, the prime-boost regimen involves the expression of multiproteins from the prime and/or the boost vehicle. See, e.g., R. R. Amara, Science, 292:69-74 (6 Apr. 2001) which describes a multiprotein regimen for expression of protein subunits useful for generating an immune response against HIV and SIV. For example, a DNA prime may deliver the Gag, Pol, Vif, VPX and Vpr and Env, Tat, and Rev from a single transcript. Alternatively, the SIV Gag, Pol and HIV-1 Env is delivered in a recombinant adenovirus construct of the invention. Still other regimens are described in WO 99/16884 and WO 01/54719.
However, the prime-boost regimens are not limited to immunization for HIV or to delivery of these antigens. For example, priming may involve delivering with a first chimp vector of the invention followed by boosting with a second chimp vector, or with a composition containing the antigen itself in protein form. In one example, the prime-boost regimen can provide a protective immune response to the virus, bacteria or other organism from which the antigen is derived. In another desired embodiment, the prime-boost regimen provides a therapeutic effect that can be measured using convention assays for detection of the presence of the condition for which therapy is being administered.
The priming composition may be administered at various sites in the body in a dose dependent manner, which depends on the antigen to which the desired immune response is being targeted. The invention is not limited to the amount or situs of injection(s) or to the pharmaceutical carrier. Rather, the regimen may involve a priming and/or boosting step, each of which may include a single dose or dosage that is administered hourly, daily, weekly or monthly, or yearly. As an example, the mammals may receive one or two doses containing between about 10 μg to about 50 μg of plasmid in carrier. A desirable amount of a DNA composition ranges between about 1 μg to about 10,000 μg of the DNA vector. Dosages may vary from about 1 μg to 1000 μg DNA per kg of subject body weight. The amount or site of delivery is desirably selected based upon the identity and condition of the mammal.
The dosage unit of the vector suitable for delivery of the antigen to the mammal is described herein. The vector is prepared for administration by being suspended or dissolved in a pharmaceutically or physiologically acceptable carrier such as isotonic saline; isotonic salts solution or other formulations that will be apparent to those skilled in such administration. The appropriate carrier will be evident to those skilled in the art and will depend in large part upon the route of administration. The compositions of the invention may be administered to a mammal according to the routes described above, in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using micelles, gels and liposomes. Optionally, the priming step of this invention also includes administering with the priming composition, a suitable amount of an adjuvant, such as are defined herein.
Preferably, a boosting composition is administered about 2 to about 27 weeks after administering the priming composition to the mammalian subject. The administration of the boosting composition is accomplished using an effective amount of a boosting composition containing or capable of delivering the same antigen as administered by the priming DNA vaccine. The boosting composition may be composed of a recombinant viral vector derived from the same viral source (e.g., adenoviral sequences of the invention) or from another source. Alternatively, the “boosting composition” can be a composition containing the same antigen as encoded in the priming DNA vaccine, but in the form of a protein or peptide, which composition induces an immune response in the host. In another embodiment, the boosting composition contains a DNA sequence encoding the antigen under the control of a regulatory sequence directing its expression in a mammalian cell, e.g., vectors such as well-known bacterial or viral vectors. The primary requirements of the boosting composition are that the antigen of the composition is the same antigen, or a cross-reactive antigen, as that encoded by the priming composition.
In another embodiment, the chimpanzee adenoviral vectors and C68 targeting proteins of the invention are also well suited for use in a variety of other immunization and therapeutic regimens. Such regimens may involve delivery of C68 constructs of the invention simultaneously or sequentially with Ad constructs of different serotype capsids, regimens in which C68-derived constructs of the invention are delivered simultaneously or sequentially with non-Ad vectors, regimens in which the adenoviral vectors of the invention are delivered simultaneously or sequentially with proteins, peptides, and/or other biologically useful therapeutic or immunogenic compounds. Such uses will be readily apparent to one of skill in the art.
V. Method for Rapid Screening of Bacterial Transformants
An elegant selection method is provided by the present invention, which permits the rapid screening of constructs produced by homologous recombination or direct cloning methods. As used herein, these constructs are preferably viruses, but may include other types of vectors, such as a cosmid, episome, plasmid, or other genetic element that delivers a heterologous molecule to cells.
In one desired embodiment, the method utilizes the gene encoding green fluorescent protein (GFP), to provide a green-white selection method in which the presence of a recombinant is detected by the absence of GFP expression (i.e., the recombinants are observed as white in a green background). Alternatively, the method may utilize another suitable marker genes, including, without limitation, other fluorescent proteins and luciferase.
In one example, the method is used for production of a recombinant construct from homologous recombination of co-transected vectors into a selected host cell. As used herein, a host cell may be readily selected from an biological organism, including prokaryotic and eukaryotic cells, such as those discussed in the section related to production of a recombinant viral particle. Selection of the host cell is not a limitation of the present invention.
Suitably, each of the vectors contains the marker gene (e.g., GFP) under the control of a promoter that directs expression thereof in a host cell. Alternatively, each of the parental vectors may contain a different marker gene that allows them to be distinguished not only from the recombinant construct produced, but also from each other. Preferably, where prokaryotic GFP is utilized, it is under the control of a prokaryotic promoter such as the promoter from lacZ. However, other suitable prokaryotic or non-prokaryotic promoters may be readily selected from among the promoters described herein and known to those of skill in the art. Advantageously, the GFP protein is placed in the portion of the vectors that are eliminated during homologous recombination and thus, the GFP protein is absent from the recombinant vector produced. In this manner, the presence of unrecombined parental vectors are readily detected under a phase contrast fluorescent microscope (or other suitable detection means) as expressing the marker gene and the recombinant constructs lack expression of the marker. In the methods in which both parent vectors utilize GFP, the recombinant appears as white in a background of green.
In another example, the method is used for production of a recombinant construct involving homologous recombination, in which the host cell stably contains at least one of the parental constructs to be utilized for production of the recombinant construct. In this embodiment, the host cell can be subjected to a single transfection. In still other embodiments, the method of the invention may be utilized for triple transfections. As with the double transfection described above, the parental constructs may contain the same marker gene or may contain different marker genes.
In another example, the method of the invention is used from production of a recombinant construct by direct cloning. Suitably, in this embodiment, the marker gene is present is that portion of the parent construct which is deleted during the cloning process. For example, the marker gene expression cassette (i.e., the gene, promoter, and any other necessary regulatory sequences) is engineered into the E1- or E3-region of an adenoviral vector, into which a transgene or minigene cassette will be cloned. The success of direct cloning into the target region can be readily detected by the absence of marker gene expression.
Optionally, the method of the invention can be readily assembled in the form of a kit which is available in a commercially useful format for production of recombinant constructs, e.g., recombinant adenoviruses. Typically, such kits will include plasmid backbones containing a desired viral genome containing a marker gene inserted at a point upstream or downstream of the recombination site, as appropriate, or a plasmid backbone containing the marker gene inserted at the splice site for direct cloning of a heterologous gene. Such a kit can further include appropriate culture media, host cells, a test control, instructions, and other suitable materials.
In the examples below, this method is used in production of adenoviruses. However, it will be readily understood that this method may be readily adapted for use in generating other types of adenoviral, or non-adenoviral viral vectors.
The following examples are provided to illustrate the invention and do not limit the scope thereof. One skilled in the art will appreciate that although specific reagents and conditions are outlined in the following examples, modifications can be made which are meant to be encompassed by the spirit and scope of the invention.
EXAMPLE 1
Creation of an E1 Deleted Vector Based on Chimpanzee Adenovirus C68 Using Green-white Selection of Recombinants
A replication defective version of C68 was isolated for use in gene transfer. The classic strategy of creating a recombinant with E1 deleted, by homologous recombination in an E1 expressing cell line was pursued. The first step was creation of a plasmid containing m.u. 0 through 1.3 followed by addition of a minigene expressing enhanced green fluorescent protein (GFP) from a CMV promoter and C68 sequence spanning 9-16.7 m.u. This linearized plasmid was cotransfected into an E1 expressing cell line with Ssp I-digested C68 plasmid (SspI cuts at 3.6 m.u. leaving 4644 bp for homologous recombination). Experiments were initially conducted with 293 cells which harbor E1 from human Ad5 with the hope that this would suffice for transcomplementation. Indeed, plaques formed which represented the desired recombinant. The resulting vector was called C68-CMV-GFP.
The strategy for generating recombinants was modified to enable efficient and rapid isolation of recombinants. First, the alkaline phosphatase DNA in the initial shuttle vector was replaced with a prokaryotic GFP gene driven by the prokaryotic promoter from lacZ. This allowed efficient screening of bacterial transformations when attempting to incorporate a desired eukaryotic RNA pol II transcriptional unit into the shuttle vector. The resulting transformation can be screened for expression of GFP; white colonies are recombinants while green colonies are residual parental plasmid.
A green-white selection has been used to screen the products of cotransfection for the isolation of human Ad5 recombinants (A. R. Davis et al, Gene Thera., 5:1148-1152 (1998)). In the present system, and in contrast to Davis, the initial shuttle vector was revised to include extended 3′ sequences from 9 to 26 MU. This vector was cotransfected with viral DNA from the original C68-CMV-GFP isolate that had been restricted with Xba I, which cuts at MU 16.5 allowing for 9.5 Kb of overlap for homologous recombination. The resulting plaques were screened under a phase contrast fluorescent microscope for non-fluorescing isolates that represent the desired recombinants. This greatly simplified screening in comparison to the standard methods based on structure or transgene expression. Thus, this method may be readily adapted for use in generating other types of adenoviral, or non-adenoviral viral vectors.
A. Shuttle Plasmid
To construct a plasmid shuttle vector for creation of recombinant C68 virus, the plasmid pSP72 (Promega, Madison, Wis.) was modified by digestion with Bgl. II followed by filling-in of the ends with Klenow enzyme (Boehringer Mannheim, Indianapolis, Ind.) and ligation with a synthetic 12 bp Pac I linker (New England Biolabs, Beverly, Mass.) to yield pSP72-Pac. A 456 bp Pac I/SnaB I fragment spanning map unit (m.u. or MU) 0-1.3 of the C68 genome was isolated from the pNEB-BamE plasmid containing BamHI E fragment of the C68 genome and cloned into Pac I and EcoR V treated pSP72-Pac to yield pSP-C68-MU 0-1.3. A minigene cassette consisting of the cytomegalovirus early promoter driving lacZ with a SV40 poly A signal was separated from pCMVβ (Clontech, Palo Alto, Calif.) as a 4.5 kb EcoRI/SalI fragment and ligated to pSP-C68-MU 0-1.3 restricted with the same set of enzymes, resulting in pSP-C68-MU 0-1.3-CMVLacZ.
For the initial step in the isolation of the 9-16.7 MU region of C68, both pGEM-3Z (Promega, Madison, Mich.) and pBS-C68-BamF were double-digested with BamHI and Sph I enzymes. Then the 293 bp fragment from pBS-C68-BamF was ligated with pGEM-3Z backbone to form pGEM-C68-MU 9-9.8. A 2.4 kb fragment including the C68 MU 9.8-16.7 was obtained from the pBS-C68 BamHB clone after XbaI digestion, filling in reaction and subsequent BamHI treatment and cloned into BamHI/SmaI double digested pGEM-C68-MU 9-9.8 to generate pGEM-C68-MU 9-16.7. The C68 9-16.7 m.u. region was isolated from pGEM-C68-MU 9-16.7 by digestion with EcoRI, filling in of the ends with Klenow enzyme (Boehringer Mannheim, Indianapolis, Ind.), ligation of a synthetic 12 bp HindIII linker (NEB) and then digestion with HindIII. This 2.7 kb fragment spanning the C68 MU 9-16.7 was cloned into the HindIII site of pSP-C68-MU 0-1.3-CMVlacZ to form the final shuttle plasmid pC68-CMV-LacZ. In addition, an 820 bp alkaline phosphatase (AP) cDNA fragment was isolated from pAdCMVALP (K. J. Fisher, et al., J. Virol., 70:520-532 (1996)) and exchanged for lacZ at Not I sites of pC68-CMV-lacZ, resulting in pC68-CMV-AP.
B. Construction of Recombinant Virus
To create the E1-deleted recombinant C68-CMVEGFP vector, a pC68-CMV-EGFP shuttle plasmid was first constructed by replacing the lacZ transgene in pC68-CMV-lacZ with the enhanced green fluorescent protein (EGFP) gene. The replacement cloning process was carried out as the follows. An additional NotI restriction site was introduced into the 5′ end of the EGFP coding sequence in the pEGFP-1 (Clontech, Palo Alto, Calif.) by BamHI digestion, filling in reaction and ligation of a 8 bp synthetic NotI linker (NEB). After NotI restriction of both constructs, the EGFP sequence was isolated from the modified pEGFP-1 and used to replace the lacZ gene in the pC68-CMV-lacZ. The pC68-CMVEGFP construct (3 μg) was co-transfected with Ssp I-digested C68 genomic DNA (1 μg) into 293 cells for homologous recombination as previously described (G. Gao, et al, J. Virol, 70:8934-8943 (1996)). Green plaques visualized by fluorescent microscopy were isolated for 2 rounds of plaque purification, expansion and purification by CsCl gradient sedimentation (G. Gao, et al, cited above).
The invention provides a uniquely modified version of the green/white selection process (A. R. Davis, et al., Gene Thera., 5:1148-1152 (1998)). The present example illustrates use of this method for construction of recombinant C68 vectors. A 7.2 kb fragment spanning 9 to 36 MU was isolated from the pBSC68-BamB plasmid by treatment with AgeI and BsiwI restriction enddnucleases and cloned into Asp718 and AgeI sites of pC68-CMV-AP shuttle plasmid, resulting in a new plasmid called pC68CMV-AP-MU36. A further modification was made to remove 26 to 36 m.u. from pC68CMV-AP-MU36 by Eco47III and NruI digestions. The new-shuttle plasmid called pC68CMV-AP-MU26 has a shorter region for homologous recombination (i.e., 16.7-26 MU) 3′ to the minigene. To make a recombinant C68 vector, alkaline phosphatase (AP) is replaced with the gene of interest. The resulting pC68CMV-Nugene-MU26 construct is co-transfected with Xba I (16.5 MU) restricted C68-CMVGFP viral DNA into 293 cells, followed by top agar overlay. The recombinant virus plaques (white) are generated through the homologous recombination in the region of 16.7-26 MU which is shared between pC68CMV-Nugene construct and C68 viral backbone; the recombinants which form white plaques are selected from green plaques of uncut C68-CMVGFP virus.
The green/white selection mechanism was also introduced to the process of cloning of the gene of interest into the pC68 shuttle plasmid. The AP gene in both pC68CMV-AP-MU36 and pC68CMV-AP-MU26 was replaced with a cassette of prokaryotic GFP gene driven by the lacZ promoter isolated from pGFPMU31 (Clontech, Palo Alto, Calif.). Thus, white colonies of bacterial transformants will contain the recombinant plasmid. This green/white selection process for bacterial colonies circumvented the need for making and characterizing large numbers of minipreped DNAs and so further enhanced the efficiency in creating recombinant C68 vectors.
EXAMPLE 2
Chimpanzee C68 Virus Stock and Replication
Examples 3 through 5 which follow provide additional characterization of the chimpanzee C68. It will be appreciated by one of skill in the art that this information can be readily used in the construction of novel recombinant chimpanzee adenoviral constructs.
The C68 virus stock was obtained from ATCC (Rockville, Md.) and propagated in 293 cells (ATCC) cultured in DMEM (Sigma, St. Louis, Mo.) supplemented with 10% fetal calf serum (FCS; Sigma or Hyclone, Logan, Utah) and 1% Penicillin-Streptomycin (Sigma). Infection of 293 cells was carried out in DMEM supplemented with 2% FCS for the first 24 hours, after which FCS was added to bring the final concentration to 10%. Infected cells were harvested when 100% of the cells exhibited virus-induced cytopathic effect (CPE), collected, and concentrated by centrifugation. Cell pellets were resuspended in 10 mM Tris (pH 8.0), and lysed by 3 cycles of freezing and thawing. Virus preparations were obtained following 2 ultra centrifuge steps on cesium chloride density gradients and stocks of virus were diluted to 1×1012 particles/ml in 10 mM Tris/100 mM NaCl/50% glycerol and stored at −70° C.
EXAMPLE 3
Cloning and Sequencing of Viral Genomic DNA
Genomic DNA was isolated from the purified virus preparation following standard methods and digested with a panel of 16 restriction enzymes following the manufacturer's recommendations. Except as noted, all restriction and modifying enzymes were obtained from Boehringer Mannheim, Indianapolis, Ind. Genomic DNA was digested with BamHI, PstI, SalI, HindIII or XbaI and the fragments were subcloned into plasmids (K. L. Berkner and P. A. Sharp, Nucl. Acids Res., 11:6003-20 (1983)). After deproteination, synthetic 10 bp PacI linkers (New England Biolabs, Beverly, Mass.) were double digested with PacI and BamHI, or PstI.
The PstI, BamHI and HindIII clones generated from C68 are illustrated in FIG. 1, parts C, D and E, respectively. The fragments indicated by the shaded boxes were not cloned, but the sequence of the entire genome has been determined through sequencing overlapping clones and viral DNA directly (unshaded boxes). The cloned fragments and insert sizes are described in Table 1. In the following table, pBS =pBluescript SK+clone; pNEB=pNEB 193 clone; pBR=pBR322 clone; No prefix=fragment not cloned







TABLE 1







C68 plasmid clones and insert sizes













Insert
Frag-
Frag-
5′ End
3′ End



Size
ment
ment
Map
Map


Construct Name
(base pairs)
5′ End
3′ End
Unit
Unit















Pst-I Fragments







C68-Pst-A
6768
24784
31551
67.9%
86.4%


pBS:C68-Pst-B
6713
4838
11550
13.2%
31.6%


pBS:C68-Pst-C
5228
14811
20038
40.6%
54.9%


pBS:C68-Pst-D
2739
12072
14810
33.1%
40.6%


pBS:C68-Pst-E
2647
20039
22685
54.9%
32.1%


pBS:C68-Pst-F
1951
32046
33996
87.8%
93.1%


pNEB:C68-Pst-G
1874
1
1874
0.0%
5.1%


pBS:C68-Pst-H
1690
23094
24783
63.2%
67.9%


pBS:C68-Pst-I
1343
33997
35339
93.1%
96.8%


pNEB:C68-Pst-J
1180
35340
36519
96.8%
100.0%


pBS:C68-Pst-K
1111
2763
3873
7.6%
10.6%


pBS:C68-Pst-L
964
3874
4837
10.6%
13.2%


pBS:C68-Pst-M
888
1875
2762
5.1%
7.6%


pBS:C68-Pst-N
408
22686
23093
62.1%
63.2%


C68-Pst-O
380
31666
32045
86.7%
87.7%


pBS:C68-Pst-P
285
11551
11835
31.6%
32.4%


C68-Pst-Q
236
11836
12071
32.4%
33.1%


pBS:C68-Pst-R
114
31552
31665
86.4%
86.7%


BamHI Fragments


C68-Bam-A
16684
19836
36519
54.3%
100.0%


pBS:C68-Bam-B
8858
3582
12439
9.8%
34.1%


pBS:C68-Bam-C
4410
12440
16849
34.1%
46.1%


pBS:C68-Bam-D
2986
16850
19835
46.1%
54.3%


pNEB:C68-Bam-E
2041
1
2041
0.0%
5.6%


pBS:C68-Bam-F
1540
2042
3581
5.6%
9.8%


HindIII Fragments


pBR:C68-Hind-B
9150
23471
32620
64.3%
89.3%









Chimpanzee adenovirus, C68, was obtained from ATCC and propagated in human 293 cells. Viral genomic DNA was isolated from purified virions using established procedures (A. R. Davis, et al., Gene Thera., 5:1148-1152 (1998)) and digested with a panel of restriction enzymes; the data were consistent with previous studies (data not shown) (G. R. Kitchingman, Gene, 20:205-210 (1982); Q. Li and G. Wadell, Arch Virol. 101:65-77 (1998); R. Wigand, et al., Intervirology. 30:1-9 (1989)). Restriction fragments spanning the entire genome of C68 were subcloned into plasmids. A schematic drawing of the C68 genome is shown in FIG. 1A, and the Pst-I, BamHI and HindIII fragments that were cloned into plasmid vectors are indicated by the unshaded boxes, in FIGS. 1B, 1C, and 1D, respectively. The cloned fragments, fragment sizes and genomic position are also listed in Table 1. Both plasmid clones and genomic DNA were used as templates for sequencing. The genome was sequenced by primer walking in both directions and each base was included in an average of approximately four reactions.
The C68 genome is 36521 bp in length [see, U.S. Pat. No. 6,083,716]. Preliminary comparison with GenBank sequences indicated varying degrees of similarity with other human and animal adenoviruses along the entire length of the viral genome. Regions with homology to all of the previously described adenoviral genetic units, early regions 1-4 and the major late genes, were found in the C68 genome (FIG. 1A). DNA homology between C68 and the human adenoviruses that have been completely sequenced, Ad2 (NC001405), Ad5 (NC001405), Ad12 (NC001460), Ad17 (NC002067) and Ad4O (NC01464), was used to order the clones. The open reading frames (ORF) were determined and the genes were identified based on homology to other human adenoviruses. All of the major adenoviral early and late genes are present in C68. The inverted terminal repeats (ITR=s) are 130 bp in length.
EXAMPLE 4
Analysis of C68 Sequence
The complete nucleotide sequence of every member of the Mastadenovirus genus accessible from GenBank, including isolates from different species, were screened for identity to C68. The Ad4 minigenome was assembled from the following GenBank sequences: Left-hand ITR (J01964); E1A region (M14918); DNA pol and pTP (X74508, 74672); VA RNA-I, II (U10682); 52, 55K (U52535); pVII (U70921); hexon (X84646); endoprotease (M16692); DNA-binding protein (M12407); fiber (X76547); right-hand ITR (J01965). The Ad7 composite genome was created from the following sequence data: Mu 3-21 (X03000); VA RNA-I, II, pTP & 52, 55K (U52574); penton (AD001675); pVI, hexon and endoprotease (AF065065); DNA-binding protein (K02530); E3 and fiber region (AF104384); right-hand ITR (V00037).
The amino acid sequence alignment was generated with Clustal X, edited with Jalview (available on the world wide web at ebi.ac.uk/ ̃michele/jalview), and analyzed with Boxshade (available on the world wide web at ch.embnet.org/software/BOX_form.html). Publicly available hexon protein sequences from all human adenovirus serotypes were initially aligned to identify the set showing the highest homology to C68.
The nucleotide sequence and predicted amino acid sequences of all significant open reading frames in the C68 genome were compared to known DNA and protein sequences. The nucleotide sequence of C68 was compared to sequences of Ad2, 4, 5, 7, 12, 17 and 40. In agreement with previous restriction analysis (Kitchingman, cited above) C68 is most similar to human Ad4 (subgroup E).
The E1A region of C68 extends from the TATA box at nt 480 to the poly A addition site at 1521. The consensus splice donor and acceptor sites are in the analogous position of the human Ad counterparts, and the 28.2K and 24.8K proteins are similar in size to the human Ad proteins. The ORF for the smallest E1A protein of C68 is predicted to encode 101 residues as opposed to approximately 60 amino acids for other adenoviruses. There is a TTA codon at residue 60 for C68 where other adenoviruses often have a TGA stop codon. The first 60 residues of C68 E1A 100R protein have 85% identity to the Ad4 homolog.
The C68 genome encodes genes for the four E 1B proteins, 20.5K, 54.7K, 10.1K and 18.5K as well as pIX. All five C68 encoded proteins are similar in size to that of other Ad E1B and pIX proteins. The Ad4 homolog of the E1B 21 K protein has only 142 amino acids, where C68 has 186 residues and other human adenoviruses have 163-178 residues. The C68 and Ad4 proteins share 95% identity over the first 134 aa, then the similarity ends and the Ad4 protein terminates at 142 amino acids.
The C68 genome encodes homologs of the E2A 55K DNA binding protein and the Iva2 maturation protein, as well as the E2B terminal protein and the DNA polymerase. All of the E2 region proteins are similar in size to their human Ad counterparts, and the E2B proteins are particularly well conserved. The C68 E2B 123.6K DNA polymerase is predicted to be 1124 residues, while Ad4 is predicted to have 1193 although the other human adenoviruses have smaller polymerases. Residues 1-71 of the Ad4 polymerase have no similarity to any other Ad polymerase, and it is possible that this protein actually initiates at an internal ATG codon. From amino acids 72-1193, Ad4 and C68 polymerases have 96% amino acid identity.
The E3 regions of human adenoviruses sequenced so far exhibit considerable sequence and coding capacity variability. Ad40 has five E3 region genes, Ad12 has six, C68 and Ad5 have seven, Ad38 has eight and Ad3 as well as Ad7 (subgroup B human adenoviruses) have nine putative E3 region genes. The Ad4 E3 region has not yet been sequenced. In comparison with the E3 region of Ad35, all 7 E3 gene homologs were identified in the C68 genome (C. F. Basler and M. S. Horwitz, Virology, 215: 165-177 (1996)).
The C68 E4 region has 6 ORFs and each is homologous to proteins in the human Ad5, 12 and 40 E4 region. The E4 nomenclature is confusing because the ORF2 homologs of C68, Ad12 and Ad40 are approximately 130 residues, while in Ad5 there are two ORFs encoding proteins of 64 and 67 residues with homology, respectively, to the amino and carboxy terminal ends of the larger ORF2 proteins. ORF5 has been omitted in our nomenclature because the 5th ORF in the E4 region is homologous to the widely studied ORF6 protein of human Ad5.
The major late promoter and the tri-partite leader sequences of the C68 genome were located. ORFs with the potential to encode the 15 major late proteins were located. All of the C68 late proteins are similar in size to their human Ad counterparts. The percent amino acid identity between chimpanzee and human Ad late proteins varies considerably. The C68 fiber protein is predicted to have 90% amino acid identity with the Ad4 protein, but much less similarity to the other human Ad fiber proteins. The CAR binding site in the fiber knob is present in C68.
EXAMPLE 5
Virus Neutralizing Antibody Assays
Several studies were performed to determine if there is cross-reactivity between type specific antisera of C68 and human adenovirus. The neutralizing activity of sera was tested as follows. Panels of sera from normal human subjects (N=50), rhesus monkeys (N=52) and chimpanzees (N=20) were evaluated for neutralizing antibodies against Ad5 and C68 based vectors using 293 cells as an indicator cell line. Sera collected from individual humans, rhesus monkeys, or chimpanzees were inactivated at 56 C for 30 minutes. A serial dilution of each sample (1:10, 1:20, 1:40, 1:80, 1:160, 1:320 in 100 μl of DMEM containing 10% FCS) was added to equal amounts of H5.010CMVEGFP (1000 PFU/well) or C68CMVEGFP virus and incubated at 4 C for two hrs. One hundred and fifty microliters of the mixture were transferred onto 2×10 293 cells in 96 well flat bottom plates. Control wells were infected with equal amounts of virus (without addition of serum). Samples were incubated at 37 C in 5% CO2 for 48 hrs and examined under a fluorescent microscope. Sample dilutions that showed>50% reduction of green-fluorescent foci as compared to infected controls were scored positive for neutralizing antibodies.
As expected, approximately 35% of normal human subjects demonstrated neutralizing antibody against Ad5, a frequency much higher than observed in sera of rhesus monkeys and chimpanzee. Neutralizing antibody to C68 was observed in 80% of chimpanzee and only 2% of normal human subjects or rhesus monkeys. Titers of neutralizing antibodies in the non-target species were generally low.
To further evaluate cross-reactivity of C68 with human adenovirus vectors, mice were immunized with 2×107 plaque forming units (pfu) of Ad 2, 4, 5, 7 and 12 as well as C68. Sera were harvested 2 weeks later and tested for antibodies that neutralized either Ad5 or C68 vectors. Neutralizing antibody to Ad5 vector was only detected in animals immunized with Ad5. Importantly, the only animals with neutralizing antibody to C68 vector were those inmmunized with C68 vector; none of the human serotypes tested, including Ad4, generated antibodies in mice that neutralized C68 in vitro.
Important to the utility of C68 vector in human trials is the absence of neutralizing antibody in the human population. In our study, a screen of 50 normal human subjects failed to detect any significant neutralizing antibodies (>1:10) using the same assay that showed neutralizing antibodies in>50% of chimpanzees. Furthermore, sera of mice immunized with multiple human Ad serotypes including Ad4, did not neutralize infection with C68.
EXAMPLE 6
Structural Analysis of Hexon Proteins
The absence of neutralizing antibodies between C68 and human serotypes compelled us to more carefully evaluate structural differences in the regions of hexon presumed to harbor type specific epitopes. Previous studies have suggested that these epitopes are located within the 7 hypervariable regions of hexon determined by Crawford-Miksza and Schnurr (J. Virol, 70:1836-1844 (1996)). A comparison of the amino acid sequences of hexon proteins between C68 and several human adenoviruses is shown in FIG. 3. Indeed, C68 is substantially dissimilar in significant regions of these hypervariable sequences.
EXAMPLE 7
Construction of C68-Derived Capsid Containing a Human Fiber Gene
To generate a C68-derived vector with an altered tropism, the chimeric fiber gene construct containing the Ad5 fiber knob fused to the C68 tail and shaft is incorporated into a plasmid carrying the C68 genome. For the precise replacement of the wild-type C68 fiber gene, a plasmid carrying the green fluorescent protein driven by a CMV prormoter is used for modification of C68 fiber. The resulting transfer vector contains a CMV promoter driven green fluorescent protein (GFP) expression cassette inserted into the E3 region, the chimeric C68/Ad5 fiber gene, and E4. This transfer vector was used for incorporation of GFP cassette and modified fiber gene into the backbone of an E3 deleted C68 infectious plasmid via homologous recombination in E. coli. The viral genome was released from the plasmid by Pacd digestion and used to transfect 293 cells. The chimeric C68-derived virus is produced about 3 weeks following transfection using techniques described herein. Similar techniques can be readily utilized to construct other C68-derived capsids.
All publications cited in this specification are incorporated herein by reference. While the invention has been described with reference to a particularly preferred embodiment, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

## Claims

1. A recombinant virus comprising an adenovirus capsid, which capsid comprises one or more fragments of the C68 hexon protein having the amino acid sequence of SEQ ID NO: 16 fused to a heterologous adenovirus hexon peptide, said capsid encapsidating a molecule for delivery to a target cell, wherein the molecule comprises an adenovirus 5′ inverted terminal repeat sequence (ITRs), a minigene, and an adenovirus 3′ ITR, wherein said one or more fragments of the C68 hexon protein consists of an amino acid sequence selected from the group consisting of:
(a) amino acids 125 to 443 of SEQ ID NO:16;
(b) amino acids 131 to 441 of SEQ ID NO:16;
(c) amino acids 138 to 44l of SEQ ID NO:16;
(d) amino acids 138 to 163 of SEQ ID NO:16;
(e) amino acids 170 to 176 of SEQ ID NO:16;
(f) amino acids 195 to 203 of SEQ ID NO:16;
(g) amino acids 233 to 246 of SEQ ID NO:16;
(h) amino acids 253 to 264 of SEQ ID NO:16;
(i) amino acids 287 to 297 of SEQ ID NO:16; and
(j) amino acids 404 to 430 of SEQ ID NO:16.

2. The recombinant virus according to claim 1, wherein the capsid further comprises a fiber protein, a penton protein, or both a fiber protein and a penton protein from an adenovirus other than C68.

3. The recombinant virus according to claim 1, wherein the C68 fragment is a loop region of the hexon.

4. The recombinant adenovirus according to claim 3, wherein at least one loop region of the C68 hexon protein is substituted with a loop region from a heterologous adenovirus.

5. The recombinant adenovirus according to claim 2, wherein the penton and fiber are from a human adenovirus.

6. The recombinant adenovirus according to claim 2, wherein the penton is from C68 and the fiber is from a different adenovirus.

7. The recombinant adenovirus according to claim 2, wherein the fiber is from C68 and the penton is from a different adenovirus.

8. The recombinant adenovirus according to 1, wherein the adenovirus ITRs are from a serotype heterologous to C68.

9. A pharmaceutical composition comprising a physiologically acceptable carrier and a recombinant virus comprising an adenoviral capsid according to claim 1, said capsid encapsidating a molecule for delivery to a target cell.

10. A recombinant virus comprising an adenovirus capsid, which capsid comprises one or more fragments of the C68 hexon protein of SEQ ID NO:16 fused to a heterologous adenovirus hexon peptide, said capsid encapsidating a molecule for delivery to a target cell, wherein the molecule comprises an adenovirus 5′ inverted terminal repeat sequence (ITRs), a minigene, and an adenovirus 3′ ITR,
said fragment selected from the group consisting of: SEQ ID NO:16 having a 50 amino acid N-terminal or C-terminal truncation; SEQ ID NO:16 having a 100 amino acid N-terminal or C-terminal truncation; SEQ ID NO:16 having a 150 amino acid N-terminal or C-terminal truncation; SEQ ID NO:16 having a 200 amino acid N-terminal or C-terminal truncation.